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The effect of predator netting on clam recruitment in Baynes Sound, B.C. with a special focus on the… Munroe, Daphne Marie 2006

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T H E EFFECT OF P R E D A T O R N E T T I N G O N C L A M R E C R U I T M E N T IN B A Y N E S SOUND, B C W I T H A SPECIAL FOCUS O N T H E R E S P O N S E O F T H E M A N I L A C L A M (VENERUPIS PHILIPPINARUM)  by DAPHNE MARIE MUNROE B . S c . H o n s . , S i m o n Fraser U n i v e r s i t y , 2000  A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF D O C T O R OF PHILOSOPHY  in T H E F A C U L T Y OF G R A D U A T E  STUDIES  ( A n i m a l Science)  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A S E P T E M B E R 2006  © Daphne M a r i e M u n r o e , 2006  OF  Abstract Passive and active forces determine the patterns o f settlement o f invertebrate larvae. Research efforts into larval settlement have been dominated by attached and conspicuous species i n hard substrate environments. Here, data o n early recruitment patterns o f a mobile bivalve species f r o m a soft-sediment habitat is p r o v i d e d . In particular, h o w intertidal c l a m aquaculture netting influences the distribution o f settling pediveliger larvae was investigated. E a r l y recruitment patterns o f M a n i l a c l a m larvae (Venerupis philippinarum) were examined i n relation to predator netting used in farming clams i n B r i t i s h C o l u m b i a . A method for sampling recent settlers from intertidal sediments was developed, proven effective and e m p l o y e d to sample settled clams (<600 u m shell length) f r o m four sites in B a y n e s Sound, o n the eastern side o f V a n c o u v e r Island, B . C . in 2003 and 2004. Paired netted and non-netted plots were compared for number o f early recruits. Plots w i t h the netting and high density o f adult clams experienced lower levels o f settlement. Settlement varied annually w i t h 2003 experienceing an order o f magnitude less recruitment than 2004. In addition, laboratory tests were run using flumes to examine the retention o f competent c l a m larvae w i t h i n flumes w i t h netting o n the bottom. N o difference in the retention o f c l a m larvae was observed due to netting o r sediment treatments. Sediment properties (sediment grain size, organic carbon and inorganic carbon) were also compared between netted and non-netted plots. N o difference was seen in the sediment properties measured except for slightly higher levels o f organic carbon beneath nets; this was l i k e l y due to the higher number o f adult clams beneath the nets. T h e netting buffers temperature at the sediment surface during tidal exposure by up to 3°C, the b i o l o g i c a l relevance o f this remains untested. N o increase i n sedimentation was measured beneath netting; however, decreased bivalve settlement beneath netting was observed but only i n the year w h e n overall settlement was high. T h i s decrease in recruitment was not supported by the flume trials; however these were run at ii  one velocity. Trials at different velocities may produce different results. These field observations are an important contribution to understanding larval settlement o f mobile species in a soft-sediment habitat.  Table of Contents ABSTRACT  H  TABLE O F CONTENTS  IV  LIST O F T A B L E S  VI  LIST O F FIGURES ACKNOWLEDGEMENTS  DEDICATION C H A P T E R 1: I N T R O D U C T I O N  INTRODUCTION LARVAL BIOLOGY  Spawning and Fertilization Larval Development Settlement and Metamorphosis  VH XI  Xffl 1  1 2  2 4 7  JUVENILE DISPERSAL LARVAL ECOLOGY  .10 17  Biological Factors Chemical Factors Physical Factors  19 22 26  History Factors Influencing Settlement Patterns  17 19  FLUID MOTION  28  SHELLFISH AQUACULTURE  32  Benthic Boundary Layer Reynolds Number Turbulence  Manila Clam Aquaculture Clam Culture in British Columbia Predator Netting  THESIS OUTLINE REFERENCES  C H A P T E R 2: S A M P L I N G R E C E N T L Y S E T T L E D C L A M S F R O M S E D I M E N T S  INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES C H A P T E R 3: T H E E F F E C T O F N E T T I N G O N E S T E R T I D A L S E D I M E N T A T I O N  INTRODUCTION MATERIALS AND METHODS Site Clam Populations. Sediment Grain Size Carbon Temperature  RESULTS  Clam Populations. Sediment Grain Size Carbon  28 30 31 33 35 36  38 40 56  56 57 59 61 63 64 66  66 68 68 70 70 72 72  73  73 75 76 iv  Temperature  77  DISCUSSION  79  Clam Populations. Sediment Grain Size Carbon Temperature  79 80 81 81  CONCLUSIONS  82  REFERENCES  83  C H A P T E R 4: B I V A L V E R E C R U I T M E N T T O C U L T U R E P L O T S  86  INTRODUCTION  86  MATERIALS AND METHODS RESULTS  89 91  DISCUSSION  97  CONCLUSIONS  102  REFERENCES  104 Ill  C H A P T E R 5: S E T T L E M E N T O F L A R V A E I N E X P E R I M E N T A L F L U M E S INTRODUCTION MATERIALS AND METHODS RESULTS  Ill 113 117  DISCUSSION  120  CONCLUSIONS REFERENCES  123 124  A  C H A P T E R 6: C O N C L U S I O N S A N D G E N E R A L D I S C U S S I O N  128  A P P E N D I X 1: S U M M A R Y T A B L E O F J U V E N I L E B I V A L V E D I S P E R S A L R E S E A R C H  131  REFERENCES APPENDLX 1  134  A P P E N D I X 2: C O M P A R I S O N O F M E T H O D S F O R T H E D E T E R M I N A T I O N O F C A R B O N E S ENTERTEDAL SEDIMENTS  137  INTRODUCTION  137  MATERIALS AND METHODS  139  Sample Collection and Preparation: Acid-Burn: LOI: CHN:  139 140 141 142  RESULTS  143  DISCUSSION  148  CONCLUSIONS  149  REFERENCES APPENDLX 2  150  A P P E N D L X 3: L A R V A L S E T T L E M E N T D A T A F R O M 2002  152  A P P E N D I X 4: F I E L D S I T E V E L O C I T Y M E A S U R E M E N T S  156  A P P E N D I X 5: C O N S I D E R A T I O N O F T U R B U L E N C E I N C A L I B R A T I O N O F P L A S T E R B L O C K S U S E D FOR FLOW MEASUREMENT  158  INTRODUCTION  158  MATERIALS AND METHODS  159  RESULTS  161  DISCUSSION  162  REFERENCES APPENDLX 5  163  v  List of Tables  Table 1-1.  Table 2-1.  Current velocities reported to cause byssal drift in post metamorphic bivalves Grain size components, percentage by dry weight, of each sediment type. The size category >2000um contains both granule+ and broken shell  Table 3-1. Table 3-2.  Table 4-1.  Table 4-2.  Table 5-1.  Table 5- 2.  Table 5-3.  Site characteristics (tidal height is reported at meters above chart datum)  Table A2-1. Table A2-2.  Table A2-3.  59 69  Results of tests of assumptions for paired T-test. Normality tested on the distribution of the difference between pairs, correlation calculated for linear regression of pairs Results of A N O V A test of factors influencing Venerupis philippinarum settlement  71 93  Results of linear regression of Venerupis philippinarum biomass versus larval settlement  94  Lengths of Venerupis philippinarum larvae (nm ± SD) used for each trial and source batch; n = 20 for each measure  115  Summary statistics from A N O V A test for percentage of Venerupis philippinarum larvae leaving the system during the trial  117  Summary statistics from A N O V A test for proportion of Venerupis philippinarum larvae leaving in the last 30 minutes of the trial  Table Al-1.  15  Summary of research on juvenile bivalve dispersal  120 131-  Means and standard errors for each test for carbon analysis method and each value measured  145  Significance values for multiple comparisons of means for each comparison of test type for organic carbon values  146  Significance values for multiple comparisons of means for each comparison of test type for inorganic carbon values  147  List of Figures Figure 1-1.  The general life cycle of marine bivalves  Figure 1-2.  General diagram of the trochophore and veliger larvae of marine bivalves. (A) trochophore larva; (B) veliger larva with velum extended. Not drawn to  Figure 1-3.  Figure 1-4.  Figure 1-5.  Figure 1-6.  scale  5  Modes of post-larval dispersal. A. Byssus drift; long byssus threads carry the bivalve through the water column. B. Climbing (from Yankson, 1986); the animal uses its ciliated foot and strong byssus to climb walls. Side branches of byssus are used to hold the animal while it probes with its foot. C. Drifting by foot protrusion (from Sorlin, 1988); the animal begins in a normal feeding position, works its way to the surface then protrudes its foot to act as a sail  13  Graphic representation of the flows in the Benthic Boundary Layer. Longer arrows represent faster flows; grey at the bottom represents the surface. Flow increases with distance from the surface and eventually reaches a rate equivalent to the free-stream  29  Annual production of mollusc aquaculture by mass shown with open squares and on left axis. Number of molluscan species in production worldwide shown with solid grey circles and on right axis. DatafromF A O 2005  32  Global molluscan production by mass contribution by country. Country labels are listed on the right. For each year shown, the top eight countries are graphed, the rest of the countries for that year are pooled in "rest of world" category. DatafromF A O 2005  34  Figure 1- 7. Comparison of Manila clam (Venerupis philippinarum) production from capture fishery versus aquaculture. Capture fishery is shown with grey bars and aquaculture shown in black. DatafromF A O 2005 Figure 1-8.  Figure 2-1.  Figure 3-1.  Figure 3-2.  3  35  Location of Baynes Sound on Vancouver Island, Canada. Inset left shows location of Vancouver Island in relation to Canada  38  Means and standard deviation for numbers of clams (Venerupis philippinarum) per sample for the three sediment types. The dashed line indicates the expected number of clams per sample (58.8) based on number of larvae placed in each tank. N = 3 for each treatment  60  Map of beach sampling sites within Baynes Sound. Each beach is marked with number and labelled with site name. Inset top right: Location of Vancouver Island within Canada. Inset bottom left: Location of Baynes Sound on Vancouver Island, British Columbia, Canada  69  Lengthfrequencies(count) of Venerupis philippinarum (>5mm) from each site, 2003 in left column and 2004 in right. Clams measured from netted plots represented by black bars, clams from non-netted plots represented by open bars. Shell length in mm plotted along the horizontal axis, frequency on the vertical axis 74  Figure 3-3.  Mean number of Venerupis philippinarum (>5mm shell length) per m from sites in 2003 (left) and 2004 (right). Netted samples represented with hatched bars, non-netted plots represented with grey bars. Error bars represent 95% confidence interval. For each bar, n=16 75  Figure 3-4.  Mean number of Nuttalia obscurata (>5mm shell length) per m from sites in 2003 (left) and 2004 (right). Netted samples represented with hatched bars, non-netted plots represented with grey bars. Error bars represent 95% confidence interval. For each bar, n=16 75  Figure 3-5.  Percent silt (<0.063 mm grain size) content of samples from each site and plot. Datafrom2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, n=6 except for Beach 4 netted plot in 2004 where n=5. Error bars represent 95% confidence interval .76  Figure 3-6.  Percent gravel (>2mm grain size) content of samples from each site and plot. Datafrom2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, n=6. Error bars represent 95% confidence interval 76  Figure 3-7.  Percent inorganic carbon content of samplesfromeach site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, n=6 except for Beachl netted plot in 2004 (marked above bar with N=5) where n=5. Error bars represent 95% confidence interval  2  2  77  Figure 3-8.  Percent organic carbon content of samples from each site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, .77 n=6. Error bars represent 95% confidence interval  Figure 3-9.  Daily temperature measurements at the sediment/air interface, x-axis shows hour of the day. Grey boxes connected with dashed line show the temperature at netted plots, black triangles show non-netted plots and tidal exposure is shown with the shaded grey vertical band. Left column shows the new moon, center shows first quarter moon and right shows the full moon .78  Figure 4-1.  Average rates of Venerupis philippinarum settlement per m for each beach site (net and no-net combined). Grey circles and black line shows the average settlement rate for all sites combined. 10/11 August, 25/26 August and 7/8 September in 2003 and 10/11 August and 25/56 August in 2004 are considered "pre-settlement" 91  Figure 4-2.  Average Venerupis philippinarum early recruits per m for 2003 (upper panels) and 2004 (lower panels) counted on non-netted (left panels) and netted plots (right panels). Sample date shown on x-axis, error bars represent 95% confidence interval. Upper panels are shown with a finer scale than lower panels to allow data to be viewed more clearly  2  2  92  Figure 4-3.  Relationship of early recruit (<0.5mm shell length) density to Venerupis philippinarum biomass (shell length >5mm). Each data point represents the average early recruit density for each beach. Data from 2003 are counts from date 4, thus representing peak initial settlement and are shown with triangle markers. Data from 2004 are counts from date 3, representing initial peak settlement and are shown with square markers. White markers indicate non-netted plots and black markers indicate netted plot values. The R value is shown on the graph next to the corresponding trend line 94 2  Figure 4-4.  Average Venerupis philippinarum early recruit length for each beach and plot within beach on the post-settlement date in 2003 (date 4 - shown on left) and 2004 (date 3 - shown on right). Hatched bars represent netted plots, black bars represent non-netted plots. Error bars represent 95% confidence interval. The number directly below each bar represents the sample size 95  Figure 4-5.  Shell lengthfrequenciesof Venerupis philippinarum early recruits from 2004 samples. Date 3 shown with circles and solid line, date 4 shown with squares and hatched line. Non-netted plots are shown in the left column and netted plots shown on the right. Beach 3 and Beach 4 are shown with a smaller y-axis because of lower recruitment overall to those sites. Difference between the peaks of the solid line versus the hatched line is considered to represent growth of that cohort from date 3 to date 4  96  Figure 5-1.  Top view of flume dimensions. Flow is from left to right. Acoustic Doppler measurements were made at the position marked by the X . Bottom treatments were applied between the straws and the outflow 113  Figure 5-2.  Larval batch spawning and splitting dates and resulting groups used in each trial  114  Figure 5-3.  Percentage of total Venerupis philippinarum larvae input at time zero that left the system by the end of the trial (75 minutes). Error bars represent ± standard deviation (n=5 for each bar). Percentage of polystyrene spheres leaving the system is shown on the right side of the chart 118  Figure 5-4.  Proportion of all polystyrene spheres exiting the system shown by time collected. Error bars represent ± standard deviation  Figure 5-5.  118  Proportion of Venerupis philippinarum larvae (or beads in case of trial 7) exiting the flume over time. White band indicates proportion leaving in the first 15 minutes; the dotted band indicates the proportion leaving in the second 15 minutes and so on. Trial number and treatment listed along the xaxis. ** Trial 7 was conducted with polystyrene beads; all other trials shown were conducted with larvae 119  Figure A2-1. Mean values of organic carbon obtained from each test performed with 95% confidence intervals. N for each test is listed along the x-axis. Letters next to the data points indicate means that are not significantly different (based on non-parametric multiple comparison)  144  Figure A2-2. Mean values of inorganic carbon obtained from each test performed with 95% confidence intervals. N for each test is listed along the x-axis. Letters next to the data points indicate means that are not significantly different (based on non-parametric multiple comparison)  145  Figure A2-3.  Figure A2-4.  Result of multiple comparison of error variances for organic carbon values. Variances connected with underline represent variances that were not significantly different  146  Result of multiple comparison of error variances for inorganic carbon values. Variances connected with underline represent variances that were not significantly different  147  Figure A3-1.  Density of settled Venerupis philippinarum larvae per m in 2002 at Beach 1 site. Netted plot is shown with the black hatched line, non-netted plot is shown in the grey solid line. Error bars represent the 95% confidence interval. For each point, n=24 except the October data where n=12 153  Figure A3-2.  Lengths of clams (Venerupis philippinarum) sampled in 2002. Netted plots shown with hatched bars and non-netted plots shown with grey bars. Error bars represent 95% confidence interval 154  Figure A3-3.  Length frequency graphs for Venerupis philippinarum early recruits collected in 2002 on Beach 1. Solid black line shows the length frequency for August 19*, the open dotted line shows September 5 . The top panel shows data from the neted plot, the bottom panel shows the non-netted plot  155  Positioning of the clod card in the sediment. A small plastic spike attached to the bottom of the clod allows it to be inserted into the sediment to keep it in place  157  Velocity measurements from field sites as estimated by clod card dissolution. Beach 1 shown in white bars, Beach 2 in light grey bars, Beach 3 in dark grey bars and Beach 4 in black bars. Bars with hatch marks represent netted plots, bars without hatching represents non-netted plots. Error bars show the 95% confidence interval, n=6 for each bar. Relationship for turbulent calibration is shown in Appendix 5  157  2  th  Figure A4-1.  Figure A4-2.  Figure A5-1. Figure A5-2.  Dimensions of clod cards used Graph of percentage mass lost from blocks over 24 hour period at flows from 0 - 4 cm/sec. Turbulent data points shown with black squares and solid trendline (R =0.7085), laminar datapoints shown with grey triangles and dashed trendline (R =0.8291)  160  2  2  161  Acknowledgements This thesis c o u l d not have been completed without help from many people. Thank y o u to D r . W i l l i a m P e n n e l l and B r i a n K i n g z e t t , w h o made time to meet w i t h me i n the early stages and encouraged me to take this research o n . T o m y supervisor, D r . R. Scott M c K i n l e y , thank y o u for the freedom to pursue avenues that I w o u l d otherwise not have been able to explore. A n d to the rest o f m y committee: D r . N e i l B o u r n e , D r . Douglas B r i g h t and D r . M u r r a y Isman, thank y o u for your guidance and support throughout. M o s t considerably, I a m forever awestruck and grateful f o r the encouragement and love provided me by m y best friend, S h a w n M a s o n , w h o continues to sustain me through the thickest and thinnest times. The co-operation o f the shellfish growers o f Baynes Sound was essential to completion o f this w o r k . In particular, thank y o u to Odyssesy Shellfish and M a c ' s Oysters for their contributions o f thoughts and advice, use o f sites and transport to and from. In particular, thanks to D w a y n e Johnson and R o b M a r s h a l l for w a l k i n g the beach and sharing their insight.  Thanks  also to T a y l o r Shellfish f o r donation o f larvae. I o w e enormous gratitude to the staff at M a l a s p i n a U n i v e r s i t y - C o l l e g e ( M U C ) , w h o provided me w i t h a second home for the duration o f a majority o f m y w o r k . I am especially grateful to Jennifer D a w s o n - C o a t e s , G o r d o n E d m o n d s o n and S i m o n Y u a n for their hands o n help w i t h absolutely everything, to D o n Tillapaugh and D r . Pennelope Barnes for always saying yes w h e n I needed lab space o r equipment use, and especially to the fleet o f M U C undergrads w h o toiled w i t h me in the field and lab: K e r r y Bates, E d i t h B i l l i n g t o n , A m y H o a r e , Sabrina H a l v o r s e n , H e i d i Lydersen, A m b e r P e r k o v i c h , S o l i e l Switzer, B r e n d o n C a m p b e l l and C a m e r o n Robinson. T o m y altruistic friend, H e i d i L o w , thank y o u for g i v i n g up so many weeks o f your summer vacations to spend time o n beaches at l o w tide getting stinky and muddy. A n d to xi  H e i d i ' s parents, V e r o n i c a and B i l l Phaneuf, thanks for p r o v i d i n g us w i t h " f i e l d a c c o m m o d a t i o n " and glorious meals w h i l e we w o r k e d . A n enormous number o f people helped improve chapter drafts w i t h generous comments and important suggestions for improvement o f drafts o f chapters. Thanks to D r . Stefanie Duff, Robert M a r s h a l l , D r . Chris Pearse, D r . L o u i s G o s s e l i n , D r . K e v i n Butterworth, D r . T e r r i Sutherland, D r . N e i l B o u r n e , James H i l l and D r . W i l l i a m P e n n e l l w h o all brandished the red pen at some time o n m y behalf. Temporary space and use o f various laboratory equiptment was shared generously by D r . M a t t h e w D o d d o f R o y a l R o a d s U n i v e r s i t y and D r . R a y Lett o f the M i n i s t r y o f E n e r g y , M i n e s and Petroleum Resources. M a u r e e n S o o n o f the U n i v e r s i t y o f B r i t i s h C o l u m b i a gladly provided me w i t h guidance and patiently answered questions regarding carbon analysis and D r . C a r l S c h w a r z offered statistical help w i t h some tests. T o the G o r g i n g Dragons, I o w e many hours o f mental, p h y s i c a l and emotional escape that proved invaluable in maintaining balance through the years o f w o r k . T o m y parents, Deborah and K e n n e t h M u n r o e , thanks for p r o v i d i n g me with a foundation o f tenacity, patience and the genetics to handle it a l l . T h i s research was supported by student scholarships from the N a t u r a l Science and Engineering Research C o u n c i l ( N S E R C ) and was conducted in B a y n e s Sound and at the Center for S h e llf ish Research at M a l a s p i n a U n i v e r s i t y - C o l l e g e .  Dedication  Tor my <Dad. I miss you andwill Cove you always.  "So don't you sit upon the shoreline find say you're satisfied Choose to chance the rapids Jinddare to dance the tide."  C H A P T E R 1 : Introduction  Introduction S u r v i v a l and early settlement patterns o f larval bivalves in the natural environment are poorly understood, particularly for mobile species like clams ( E c k m a n , 1990). Research efforts into larval settlement have been dominated by attached and conspicuous species in hard substrate environments ( U n d e r w o o d , 2000) and polychaete settlement (Qian, 1999). In this thesis, I provide data currently lacking in the literature, o n early recruitment patterns o f a mobile bivalve species f r o m a soft-sediment habitat. Determination o f a suitable settlement location is influenced b y m a n y factors: chemical cues f r o m c o n - s p e c i f i c s o r predators ( P a w l i k , 1992a; Steinberg et a l . , 2002), bacterial films ( W i e c z o r e k and T o d d , 1998), physical properties o f the sediment, light, temperature, salinity and hydrodynamics (Orton, 1937; B a y n e , 1964a, b; M e a d o w s and C a m p b e l l , 1972; B u t m a n , 1987; A b l e s o n and D e n n y , 1997). Intertidal shellfish farming practices have the potential to alter settlement patterns. A t most farm sites i n B r i t i s h C o l u m b i a , c l a m farming involves covering portions o f the intertidal lease area w i t h predator netting. The netting is intended to provide protection f r o m predation by fish, birds, crabs and other bivalve predators (Anderson, 1982; T o b a et a l . , 1992). Netting placed o n beaches has been shown to stabilize sediment and increase sedimentation (Spencer et a l . , 1996). Evidence o f increased sedimentation indicates that the nets influence local hydrodynamic processes. This alteration o f f l o w c o u l d direct settlement o f larvae and thereby increase the local bivalve population (Heath et a l . , 1992; B e a l and K r a u s , 2002). The purpose o f this research w a s to determine i f there is an effect o n recruitment o f the M a n i l a c l a m (Venerupisphilippinarum  - A . A d a m s and R e e v e , 1850) by intertidal predator  netting used in culture o f clams i n B r i t i s h C o l u m b i a . In addressing this question I was first interested i n c o n f i r m i n g appropriate and accurate sampling methods for m a k i n g quantitative  1  measures o f early bivalve settlers in soft-sediments. Secondly, I examined whether the nets were creating a small-scale depositional environment. T o assess this, I measured levels o f silt and organic carbon (among other parameters) at netted and non-netted plots. I was then able to ask the question: A r e bivalve larvae recruiting in higher numbers to netted o r non-netted plots? F i n a l l y , in an effort to better understand the observed patterns o f recruitment to field sites, I carried out controlled flume experiments designed to examine settlement patterns o f c l a m larvae in relation to netted and non-netted bottom types. T h i s introductory chapter discusses the importance o f carrying out this research, provides evidence from the literature illustrating w h y this area needs to be addressed and offers an introduction to the questions answered in subsequent chapters.  Larval Biology The general life cycle o f free spawning marine bivalves involves t w o distinct modes o f life; pelagic and benthic. T h e life cycle (Figure 1-1) begins w i t h release o f gametes into the water c o l u m n v i a the exhalent siphon o f the adult. In the early stages o f life, the trochophore and veliger stages, the animals are pelagic and can s w i m w e a k l y . T h r o u g h the process o f settlement and metamorphosis, the pelagic larva transforms into a sedentary benthic juvenile and adult f o r m .  S p a w n i n g and Fertilization S p a w n i n g in most marine bivalves involves the release o f a large number o f eggs and sperm through their siphon into the water. The M a n i l a c l a m , Venerupis philippinarum are oviparous (gametes released into the water) and dioecious (separate sexes) ( H e l m and B o u r n e , 2004). There are a number o f stimuli that trigger gamete release including temperature (Podniesinski and M c A l i c e 1986; Devauchelle, 1990; B a r b e r and B l a k e , 1991; T h o m p s o n et a l . ,  2  1996), physical agitation (Seed and Suchaneck, 1992), salinity, tide, solar or lunar phase (Devauchelle, 1990; M o r g a n , 1995), algae blooms (Starr et a l . , 1990) and the presence o f other gametes (Galtsoff, 1964; T h o m p s o n et a l . , 1996). The M a n i l a c l a m w i l l t y p i c a l l y undergo one or two spawning events per season (one large spawning in July f o l l o w e d by continuous female spawning through the summer was observed in Washington - H o l l a n d , 1972); the latter o f the two being the largest (Ponurovsky and Y a k o v l e v , 1992).  0'  o  D-hlnged larva Pedlvellger larva  Trochophore  Pelagic Gametes released Settlement and rnetamorphosls  Figure 1-1: The general life cycle of marine bivalves.  A mature female (shell length >35 m m ) can produce 5-8 m i l l i o n eggs in a single spawning depending upon condition and time o f the year; in general, larger clams w i l l produce more eggs (Utting and Spencer, 1991).  Venerupis philippinarum releases eggs that are initially  pear-shaped but become round after a brief period in seawater ( H e l m and B o u r n e , 2004). Before fertilization can take place, shed gametes must encounter one another in the water  c o l u m n , a process that can be hindered by advection and dilution (Pennington 1985; D e n n y and Shibata 1989; L e v i t a n , 1995). Adaptations exist to m a x i m i z e the encounter rate o f gametes such as synchronous spawning (Galtsoff, 1964; L e v i t a n , 1995; T h o m p s o n et a l . , 1996), sperm chemotaxis ( M i l l e r et a l . , 1994; L e v i t a n , 1995) and dense aggregations o f adults (Levitan et al. 1992; L e v i t a n , 1995; M a n n and Evans 1998). Once sperm and egg meet and successful fertilization occurs, meiosis is completed in the egg ( G o s l i n g , 2003). The fertilised egg undergoes spiral cleavage and eventually becomes a ciliated, motile, trochophore larva ( K a s y a n o v et a l . , 1998). T i m e required for the embryo to develop is dependant o n the species and water temperature ( H e l m and B o u r n e , 2004).  L a r v a l Development W i t h i n roughly 24 hours o f fertilization, most embryos become trochophore larvae (Figure 1-2) ( G o s l i n g , 2003). The trochophore is ciliated, w i t h 1 to 3 rows o f c i l i a around the middle called the prototroch (Kasyanov et a l , 1998). These rows o f c i l i a are used for s w i m m i n g (in a spiral pattern) and although trochophores have been reported to have developed a mouth (Galtsoff, 1964; K a s y a n o v , et a l . , 1998), they are not believed to feed until later stages. Anterior to the prototroch is the pretrochal region w h i c h contains the apical plate and apical tuft o f c i l i a at the top that performs sensory functions. B e l o w the prototroch is the posttrochal region, in some groups this area contains another c r o w n o f c i l i a called the telotroch (Raven, 1958).  4  Figure 1-2: General diagram of the trochophore and veliger larvae of marine bivalves. (A) trochophore larva; (B) veliger larva with velum extended. Not drawn to scale. The trochophore becomes a veliger larva (Figure 1-2), which is more complex and has more fully developed organs than the trochophore (Raven, 1958; Kasyanov et al., 1998). The prototroch becomes the velum of the veliger, which remains ciliated and is still the swimming and feeding organ (Raven, 1958). The velum is attached to the mantle by two pairs of velar retractor muscles and can be extended or retracted by the larvae. Cilia around the periphery of the velum beat to cause swimming in a sprial motion and create water currents for collection and transfer of food particles to the mouth located in the ventral part of the velum (Kasyanov et al., 1998; Gosling, 2003). Veligers feed on many different species of phytoplankton (Bayne, 1965; Paulay et al., 1985), bacteria, detritus and dissolved organic matter (Olson and Olson, 1989; Baldwin and Newell, 1991; Lutz and Kennish, 1992; Boidron-Metairon, 1995). The apical plate remains in the center of the velum and when the veliger is swimming, contact with the apical flagelium causes the velum to be retracted (Kasyanov et al., 1998). The posttrochal region becomes the body of the bivalve and a shell develops, the shell is at first dshaped and the larvae are called straight-hinge or D-hinge larvae. The shell continues to grow and starts to take on a typical bivalve shape; however the shell is primarily composed of aragonite and is thin and transparent (Kennedy et al., 1996).  5  Larval development takes place in the water column (Thorson, 1946) and although the larvae can swim, they do so slowly (approximately 1-10 mm/s - Mileikovsky, 1973, Chia and Buckland-Nicks 1984, also summary table of multi-species speeds provided by Kennedy et al., 1996 pg. 382). Larval development can take from three to five weeks depending upon species food and temperature (Gosling, 2003) (larval development in V. philippinarum is approximately three weeks at 25°C - Quayle and Bourne, 1972; Helm and Pellizzato 1990). Thus, for the duration of larval development, swimming speeds cannot overcome water currents and the animals are primarily passively distributed (Young, 1995). Larvae exhibit some control over horizontal advection by moving up and down in the water column (into different flow regimes) in response to various cues (Carriker, 1951; Morgan, 1995; Shanks, 1995; Young, 1995; Carriker 2001); this vertical migration is also believed to be used additionally as predator avoidance (Gosling, 2003). Mortality has been estimated to be extremely high during pelagic larval development (Thorson, 1946; Morgan, 1995; Gosling, 2003). The cause of this loss remains unclear. Johnson and Shanks (2003) have shown that predatory losses may be much lower than previously believed. The authors made in situ measurements on predation rates on near-natural plankton assemblages in Oregon and Washington and found that observed predation rates were lower than has been formerly assumed and may only be of importance when specific predators are present. As the veliger develops it begins to form a foot, and like the velum, pedal retractor muscles are developed which allow it to be extended beyond the shell (Carriker, 2001). At the stage when the larva has both a velum and a foot, it is called a pediveliger (Carriker, 1956). As a pediveliger the animal can use both the velum for swimming and the foot for crawling and periodically lands to crawl and test the substrate with its foot (Kasyanov et al., 1998; Zardus and Mattel, 2002). The gills also begin to develop at this stage, but are not used in feeding until 6  after metamorphosis. The anterior adductor muscle is developed early and in the latter veliger stages the posterior adductor is developed. The digestive system is well developed by the late veliger stage. A pigmented eye spot forms in the middle o f the shell in some species. A t this point the larva is considered "competent", or ready to metamorphose.  Settlement and Metamorphosis Metamorphosis involves shedding or breakdown of the pelagic larval structures and the development o f benthic adult structures (Raven, 1958). Generally for bivalves this includes loss of the velum and associated musculature, development of labial palps (the apical plate becomes incorporated in the labial palps - Hickman and Gruffydd, 1971), gill growth, and dissoconch formation along the outer edge of the larval shell. The dissochonch is the adult shell; it is thicker and stronger and composed of calcite instead o f aragonite (Ansell, 1962; Zardus and Martel, 2002). During metamorphosis, feeding slows for some species because o f the change in primary feeding mechanisms from velar feeding to suspension feeding with the gills (Baker and Mann, 1994). It was noted by Quayle (1952) that changes at metamorphosis are much more dramatic for fixed species than for burrowing bivalve species. Clams are seen as a group that has undergone less evolutionary specialization, and therefore metamorphosis involves less change. Interestingly, this group also has the longest period o f metamorphosis (Ansell, 1962), the loss o f the velum happens rapidly, while growth of the foot, dissochonch and gills develops slowly (Ansell, 1962). Siphons develop during metamorphosis, with the exhalent siphon first in most species (Quayle, 1952; Caddy, 1969). During the period before the siphons can be used to create feeding currents, some species use cilia on the foot to draw water into the mantle for feeding (Reid et al., 1992), a process called pedal feeding.  7  S h e l l length at metamorphosis varies little between species; most species metamorphose at around a shell length o f 250 p m . The geoduck (Panopea abruptd) is one o f the largest burrowing clams i n the w o r l d and is larger at metamorphosis ( 3 5 0 - 4 0 0 m m - G o o d w i n and Pease, 1989) than most other bivalves. M o s t species are able to delay metamorphosis i f suitable habitats are not available at the time that competence is reached ( M a z z a r e l l i , 1922 i n Y o u n g , 1990; T h o r s o n , 1946; B a y n e , 1965). This delay a l l o w s more time to search for appropriate settlement sites; however, the longer metamorphosis is delayed, the less discriminating the larva becomes among habitats (Bayne, 1965). Larvae i n cooler water o r i n water w i t h optimal salinity are able to delay for a longer period o f time (Bayne, 1965). T h e P a c i f i c oyster, Crassostrea gigas, has been seen to delay metamorphosis up to 30 days ( C o o n et a l . , 1990), the blue mussel, Mytilus edulis, can delay metamorphosis between 12 and 45 days ( B a y n e , 1965), and the geoduck P. abrupta can delay up to six weeks ( K i n g , 1986). D u r i n g the p e r i o d o f delay, degeneration o f the v e l u m may begin and s w i m m i n g ability o f the larvae weakens, leading to decreased feeding in larvae w h o have delayed f o r some time. C o l l e t et al. (1999) compared post-metamorphic growth rates o f juvenile P a c i f i c oysters that had delayed metamorphosis to those that had not delayed and found that the oysters that did not delay metamorphosis grew faster. Metamorphosis c a n be induced i n m a n y species once the larvae become competent, through the use o f chemical inducers. C o o n and B o n a r (1985) showed that oyster larvae (C. gigas) c o u l d be induced to settle and metamorphose by L - 3 , 4 - D i h y d r o x y p h e n y l a l a n i n e ( L D O P A ) at a concentration o f 2.5 x 1 0 " M ; prolonged exposure o r higher concentrations had 5  negative effects. The same study also found that oyster larvae c o u l d be made to metamorphose without settlement w i t h epinephrine o r norepinephrine at a concentration o f l O ^ M . T a n and W o n g (1995) also showed that the oyster, Crassostrea belcheri, settled and metamorphosed w i t h exposure to gamma-amino butyric acid ( G A B A ) .  Gastropods have been induced to 8  metamorphose with increased concentrations of potassium ions (K ) (Pechenik and Heyman, +  1987). Urrutia et al. (2004) demonstrated that Ruditapesphilippinarum  could be induced to  metamorphose using treatment with acetylcholine, carbamylcholine and serotonin but not catecholamines and L-DOPA, suggesting differences in metamorphic triggers between clams versus oysters, scallops or mussels. Chemical cues are present in the environment that are also involved in settlement and metamorphosis; these will be discussed in greater detail in a subsequent section ("Factors Influencing Settlement Patterns: Chemical"). During metamorphosis the feeding mechanism is in transition and decreased feeding or no feeding is possible until metamorphosis is complete. This process may take days or weeks depending on the species. Consequently, the pediveliger must rely on stored energy reserves to sustain them through metamorphosis. It has been proposed (Rodriguez et al., 1990) that stored food reserves are in the form of protein. Others postulate that stored reserves are lipids (Bayne, 1965). Whyte et al. (1992) believed that either type of reserve can be utilized and that the quality and level of food reserve available is dependant on the quality of the larval diet long before metamorphosis. Still others have related the quality of food provided during conditioning of the brood stock prior to spawning to the metamorphic food reserves available. Reid et al. (1992) showed that some species can use pedal feeding during the metamorphic transition to supplement food reserves. For bivalve larvae to be able to test surfaces and eventually settle, they must travel through the benthic boundary layer and make contact with the surface. Directed swimming or sinking has been shown to aid in concentrating competent larvae near the bottom, increasing the likelihood of contact with the surface (Butman, 1987; Eckman, 1990; Gross, et al. 1992; Eckman, et al. 1994). In a study using video observations in a flume, Finelli and Wethey (2003) were able to document a novel behavior of Crassostrea virginica larvae whereby the larvae contacted the bottom of the flume using abrupt, accelerated downward swimming that the 9  authors termed " d i v e - b o m b i n g " . H y d r o d y n a m i c s i n the near-bed region have also been demonstrated to influence the number o f competent larvae reaching the surface (see later section "Factors Influencing L a r v a l Settlement: P h y s i c a l " ) . U l t i m a t e l y , control over contact with the surface results from a combination o f active forces by larvae and passive forces o f water motion (Butman, 1987; U n d e r w o o d and K e o u g h , 2001). L a r v a l settlement is often used interchangeably w i t h the term recruitment although the two terms refer to different processes ( K e o u g h and D o w n e s , 1982). Settlement involves the larval contact with a suitable settlement site, subsequent attachement (permanent attachement i n the case o f attached speces like oysters or non-permanent in the case o f mobile species like clams) and metamorphosis. Recruitment is the survival o f the settled larvae to an arbitraty point in time and can therefore be influenced by factors like predation, differential settlement and immigration/emigration ( K e o u g h and D o w n e s , 1982; Olafsson et a l . , 1994)  Juvenile Dispersal A l t h o u g h both pre-metamorphic and post-metamorphic events influence recruitment ( K e o u g h and D o w n e s , 1982; W o o d i n , 1991), evidence n o w indicates that the initial dispersal as veliger larvae m a y be as important as subsequent dispersals carried out as post-metamorphic early juveniles (Palmer, 1988; B a k e r and M a n n , 1997; A r m o n i e s , 1996). The presence o f post-metamorphic bivalves in the water c o l u m n w a s evident early in the previous century (Nelson, 1928; S u l l i v a n 1948; B a g g e r m a n , 1953; B a y n e , 1964a) but few explanations were presented for h o w these bivalves entered or maintained their occupancy i n the water c o l u m n without a velum. O n e theory, offered by N e l s o n (1928), was that air bubbles found w i t h i n the shells o f sampled M. edulis increased the buoyancy o f the animal and allowed it to drift. N e l s o n also observed that these bubbles originated from the g i l l area, suggesting the bubble contained o x y g e n much like a s w i m bladder o f a fish.  10  A n o t h e r theory o n the floating behaviour seen in post-metamorphic mussels was suggested b y B a y n e (1964a). H e observed the migration o f early M. edulis juveniles in the M e n a i Strait, N o r t h W a l e s and postulated that young mussels extended their long ciliated foot to act as a sail to catch passing currents. A l t h o u g h both N e l s o n (1928) and B a y n e (1964a) noted young mytilids hanging from the water surface b y thin threads, the link between the threads and entrance into the water c o l u m n was not made at the time. Post-metamorphic juveniles o f M. edulis have n o w been s h o w n to enter the water c o l u m n b y secreting a long byssus thread (Lane et a l . , 1985). T h i s mode o f dispersal was termed "byssus d r i f t i n g " b y Sigurdsson et al. (1976) w h o tested and found byssus drift to occur in 22 species o f bivalves. M a n y authors have observed byssus drift behaviour (See A p p e n d i x 1 for a summary table o f juvenile dispersal research). T h e mechanism o f byssus drifting is secretion o f a long thin " b y s s u s " thread into a passing current. T h e increased viscous drag o n the thread allows the small bivalves to be lifted b y the current and carried (Figure 1 - 3 A ) , m u c h like the flight o f young spiders o n w e b strands. W a n g and X u (1997) studied drifting o f the larval bivalve Sinonovacula constricta and found it could produce a byssal thread 50 times its body length from a small pore in the base o f the foot and calculated that the thread increased the viscous drag 6.7 times the drag o n the body alone. B y s s u s drift is easily overlooked b y observers because the threads are very thin and transparent ( Y a n k s o n , 1986) and small bivalves are sensitive and w i l l release the threads when disturbed ( B e u k e m a and de V l a s , 1989). C o m p o s i t i o n o f these postlarval byssus threads is poorly understood (Baker and M a n n , 1997). Y o n g e (1962) identified the presence o f byssal attachment structures in adult bivalves as a neotenous condition. A l t h o u g h the adult byssus may be a derived f o r m o f postlarval byssus threads, it is apparent that the structure o f postlarval and adult byssus differs (Lane et a l . , 1985; M o n t a u d o u i n , 1997). Scanning electron microscope images taken b y L a n e et al. (1985) o f both attachment and drift byssus o f postlarval M. edulis,  11  show distinct differences. T h e two types o f threads are similar i n diameter, however the drift byssus is considerably longer (up to 11cm long) and mono filamentous compared to the fibrous attachment threads. These differences in structure m a y constitute evidence o f functional differences (attachement versus drift). T h e postlarval drifting threads have been described as "transparent, elastic, and non-sclerotized" by Y a n k s o n , 1986. M a n y studies indicate that the threads may be mucous-derived (Prezant and Chalermwat, 1984; B e u k e m a and de V l a s , 1989; C a c e r e s - M a r t i n e z et a l . , 1994). D r i f t i n g gastropod molluscs have also been reported to use mucous for drifting ( M a r t e l and C h i a , 1991; O l i v i e r , 1996). Sigurdsson et al. (1976) was able to stain drift byssus w i t h A l c i a n blue w h i c h indicates it contains acid mucopolysaccarides. It appears that there is a variety o f forms o f byssus i n postlarval bivalves. In two closely related cockle species, Cerastoderma edule and Cerastoderma  glaucum,  Y a n k s o n (1986) revealed differences in the f o r m o f byssus and h o w it is used by these t w o species. T h e threads o f C. glaucum were thicker w i t h more forked side branches and appeared stronger than those o f C. edule. T h e more forked, stronger threads o f C. glaucum facilitated c l i m b i n g o f walls i n the w a y a r o c k climber does, o n a single byssus w i t h tufts o f side branches where the a n i m a l stops to provide support w h i l e it probes w i t h its foot (Figure 1 - 3 B ) . M o n t a u d o u i n (1997) carried out a similar study comparing the different functions o f postlarval byssus in C. edule and R. philippinarum, t w o less closely related species. It was found that the byssus o f the c l a m (R. philippinarum)  was better at adhesion w h i l e the cockle (C. edule) byssus  was more effective for suspension and drift.  12  Figure 1-3: Modes of post-larval dispersal. A. Byssus drift; long byssus threads carry the bivalve through the water column. B. Climbing (from Yankson, 1986); the animal uses its ciliated foot and strong byssus to climb walls. Side branches of byssus are used to hold the animal while it probes with its foot. C. Drifting by foot protrusion (from Sorlin, 1988); the animal begins in a normal feeding position, works its way to the surface then protrudes its foot to act as a sail.  The number o f different species that carry out byssus drift is extensive and the body size o f animals using drift is variable. Byssus drifters have been recorded as small as 0.25mm (M.edulis- Newell, 1994) to as large as 18.8mm (Pecten maximus - Beaumont and Barnes, 1992); however, scallops o f 18.8 mm shell length are also capable o f active swimming and therefore this observation o f drift in animals o f that size may be due to swimming and not byssus drift. Calculation of drag on the byssus in relation to the size of the animal should allow for prediction o f the upper limit of the shell length possible for byssus drifting using a single byssus thread. I have come across no literature that goes through such a mathematical exercise. A large range o f species performing byssus drift has been documented (see Appendix 1 for a summary).  In addition to byssal drifting, other methods of post-larval dispersal have been noted in the literature. Mucous drifting (Prezant and Chalermwat, 1984; Beukema and de Vlas, 1989; Caceres-Martinez et al., 1994), where mucous is used similarly to byssus described above, to catch passing currents and facilitate entrance into the water column. Another method, also  13  already mentioned above, is c l i m b i n g ( R y g g , 1970; B o o z e r and M i r k e s , 1979; Y a n k s o n , 1986; A r m o n i e s , 1994a) i n w h i c h the foot is used to p u l l the animal up the w a l l and byssus is used to support the animal as it probes w i t h the foot to determine direction. D r i f t i n g v i a foot protrusion, first discussed by B a y n e (1964a), was revisited by S o r l i n (1988) and documented to occur i n Macoma balthica.  Sorlin (1988) provides an extensive description and diagram (Figure 1 - 3 C )  o f the method o f foot protrusion and s w e l l i n g o f the foot and successive drifting o f M  balthica,  however, he also notes the use o f byssal threads and does not test whether the foot o r the threads are more important in drifting. S i m p l y opening the valves has also been reported as a means o f drifting or reducing f a l l velocity ( O l i v i e r et a l . , 1996; M o n t a u d o u i n , 1997). C r a w l i n g has been noted extensively as a method o f postlarval m o b i l i t y ( B r a f i e l d and N e w e l l , 1961; R y g g , 1970; B o o z e r and M i r k e s , 1979; Y a n k s o n , 1986; A h n et a l . , 1993; C a c e r e s - M a r t i n e z et a l . , 1994; M o n t a u d o u n , 1997). C r a w l i n g i n young postlarvae is carried out by extension o f the long foot by the heavy covering o f c i l i a and mucous o n the sole o f the foot.  Pedal c r a w l i n g by bivalves  offers a smaller range dispersal than drifting and therefore is u n l i k e l y as the means for long range dispersal.  S i m p l e bedload transport has also been observed to cause dispersal o f recently settled bivalves (Emerson and Grant, 1991; Roegner et a l . , 1995; Turner et a l . , 1997) and juvenile brooding bivalves (Sellmer, 1967; C o m m i t o et a l . , 1995), although unlike other forms o f dispersal already discussed, this is p r i m a r i l y a passive process. W h i l e the process o f byssal drift is seen to occur at lower current velocities (Table 1-1), bedload transport takes place at relatively higher velocities (Emerson and Grant, 1991; Roegner et a l . , 1995). In a study carried out in N o v a Scotia, analysis o f sediment trap contents showed Mya arenaria ranging f r o m 8 - 1 5 m m in length being carried w i t h sediments at both exposed and sheltered sites (Emerson and Grant, 1991).  14  Table 1-1: Current velocities reported to cause byssal c rift in post metamorphic bivalves. Species studied Current velocity Reference Sigurdsson et al., 1976  1 cm/sec.  Mytilus edulis, Abra alba  Prezant and Chalermwat, 1984  10-20 cm/sec.  Corbicula fluminea  Laneetal., 1985  0.1 cm/sec.  Mytilus edulis  Sorlin, 1988  5 cm/sec.  Macoma balthica  Cummings et al., 1995  6 cm/sec.  Macomona lilliana  Montaudouin, 1997  10-24 cm/sec.  Wang andXu, 1997  lcm/sec.  Sinonovacula constricta  Hiddink et al., 2002  0.2 cm/sec.  Macoma balthica  Cerastoderma edule, Ruditapes philippinarum  Directional movement could be accomplished by byssal drift preferentially o n an ebb or f l o o d tide depending o n the intended direction (onshore/offshore).  O l i v i e r et a l . (1996) reported  Abra alba drifting o n the peak f l o o d tide resulting i n net movement towards adult conspecifics. C u m m i n g s et a l . (1995) provided evidence o f drift in the N e w Zealand species Macomona lilliana, and found that it drifted most often o n ebb tides. Post-settlement migrations o f five species in the W a d d e n Sea were examined by A r m o n i e s (1996) and each was seen to move differently. M. balthica and Ensis americanus both initially settled at mean l o w tide, M. balthica then migrated to the upper intertidal w h i l e E. americanus m o v e d to the subtidal. M. edulis initially settled in areas near adults and later m o v e d laterally in the intertidal. C. edule was seen to distribute evenly after patchy settlement and M. arenaria simply m o v e d randomly. Differences in the resulting movement o f these five bivalve species indicate that active processes are at w o r k in determining their final patterns o f distribution. Byssus drifting bivalves have also been reported to show d i e l patterns i n t i m i n g o f drift ( A r m o n i e s , 1994a; H i d d i n k et a l . , 2002). S a m p l i n g o f drifting juveniles i n the W a d d e n Sea by A r m o n i e s (1994a) showed C. edule, M. balthica and E. americanus were more abundant i n plankton samples taken in the dark than in light. H i d d i n k et al. (2002) also found postlarvae M.  15  balthica migrated through the water c o l u m n at night, and suggested that this behaviour helps w i t h predator avoidance although the authors were unable to detect postlarvae i n the stomach contents o f pelagic fish predators. Palmer (1988) also noted that meio fauna were more likely to enter the water c o l u m n at night when risks o f pelagic predation b y visual predators decreased. A ten year study by W i l l i a m s and Porter (1971) revealed annual patterns in the occurrence o f post metamorphic bivalves i n plankton samples, some species being found regularly during the summer, others were found during winter months. The directionality, distances involved, duration and t i m i n g i n post settlement dispersals leads to what some consider active migrational movement (Bayne, 1964a; A r m o n i e s , 1994a,b; B e u k e m a and de V l a s , 1989). M i g r a t i o n happens as a result o f an organism choosing one habitat over another based o n advantages that the n e w habitat offers ( H i d d i n k et a l . , 2001). O n e o f the more extensively studied postlarval migrations is that o f M. balthica i n the Wadden Sea, Netherlands. F i e l d sampling o n tidal flats in K o n i g s h a f e n showed that M. balthica initially settled i n the lower intertidal, f o l l o w e d by a pelagic summer migration into the upper intertidal, then winter migrations seaward again to areas o f adult populations ( A r m o n i e s , 1994a,b). It was also observed that growth rate for post-settlement larvae was higher i n the upper intertidal compared to the lower intertidal. B e u k e m a and de V l a s (1989) confirmed the same migrational pattern o f M  balthica in the W a d d e n Sea. T h e y also noted that the lower intertidal offers better  adult survival, lower parasite infection and higher growth. H o w e v e r , this area is p o o r l y suited as a nursery for juveniles since higher epifaunal predation, unsuitable sediments and increased exposure make growth rate and survival o f juveniles l o w . T h e authors attribute the success o f M. balthica in the W a d d e n Sea to its adaptive migration pattern. A second postlarval migration that has been identified in bivalves is that o f M edulis in the M e n a i Strait (Bayne, 1964a). S a m p l i n g was carried out to track the various life stages o f M. edulis f r o m planktonic larvae to late plantigrades. B a y n e reported that the larvae settled o n 16  filamentous algae in early June, then migrated to adult mussel beds as platigrades ( 1 . 0 - 1 . 5 m m in length) two months later. H e notes that a short growth period before the young mytilids enter into direct competition w i t h adults for resources is advantageous. A large body o f evidence n o w indicates that in the typical bivalve life cycle there are two stages o f dispersal. The first is as pelagic larvae and the second by recently metamorphosed juveniles. Evidence shows this second stage o f dispersal is carried out w o r l d w i d e by a variety o f species in a variety o f habitats.  L a r v a l Ecology The majority o f marine species have a pelagic larval phase. U n l i k e terrestrial insects, marine species experience dispersal during the larval stage rather than as adults. A s dispersing zooplankton, larvae are under the control o f many factors that influence their distribution and survival. The study o f factors that control distribution and abundance o f larvae is the field o f larval ecology. M a r i n e larval ecology is a relatively young discipline o w i n g partly to difficulties in observing and studying larvae in the field ( Y o u n g , 1990).  History The f o l l o w i n g derives from the comprehensive review by Y o u n g (1990) documenting origins and history o f larval ecology. Initial recognition o f larvae as transitional forms l i n k i n g embryo and adult life stages can be attributed to John V a u g h n T h o m p s o n in the early 1800's. This discovery was essentially the birth o f the field o f larval ecology, because without an understanding o f larval taxonomy the field could not exist. T h r o u g h the remainder o f the nineteenth century, many more key discoveries o f larval linkages between mysterious larval forms and adults were made. These important connections laid the foundation necessary for larval ecology to begin.  One o f the earliest contributions in the field o f larval ecology came from E d w a r d Forbes in 1844 w h e n he noted that molluscs were appearing in areas o f benthos previously uninhabited and connected this appearance w i t h recruitment o f larvae. D u r i n g the early twentieth century, oyster culturists began field studies o n larval ecology. O f particular note, Julius N e l s o n made observations o f oyster larvae in the field and was able to witness larval migrations and abundance patterns. H i s son, T h u r l o w N e l s o n , f o l l o w e d the w o r k o f his father and documented predation by ctenophores o n larval oysters in 1925. Predation in the plankton continues today as an important but understudied element. The concept o f delay o f metamorphosis has proven to be o f central importance in larval ecology and is accurately attributed to Guiseppe M a z z a r i l l i in 1922 and Theodore Mortensen in 1921 but is often misattributed to a later paper by Douglas P. W i l s o n in 1932. M e t a m o r p h i c delay led to t h i n k i n g that larvae had more control over resultant distributions than previously believed. V i c t o r L o o s a n o f f studied specific invertebrate populations for an extended period and was able to determine that populations resulting from planktonic larvae show extreme temporal variability. In addition, L o o s a n o f f made significant contributions to larval s p a w n i n g and nutrition. D u r i n g the Second W o r l d W a r , emphasis shifted f r o m aquaculture driven research to patterns o f fouling organisms. P a u l V i s s c h e r was among the scientists o f the time investigating the problem and made important contributions concerning searching behaviour o f competent larvae and larval responses to light. Another major contributor to larval ecology was Gunnar T h o r s o n whose meticulous field w o r k provided a basis o n w h i c h thorough reviews were created. H e also developed many essential field apparati that are indispensable today, for example larval traps and in situ rearing chambers. This is not intended to be an extensive review o f a l l contributions to the field but a brief synopsis o f some o f the key advances in the field. F o r a detailed examination o f the development o f the field o f larval ecology, one should consult Y o u n g ' s (1990) thorough review. 18  Factors Influencing Settlement Patterns Factors influencing larval settlement patterns vary w i d e l y and can be classed as either b i o l o g i c a l or physical. Often, factors overlap and specific influences become difficult to separate and especially difficult to study (Butman, 1987; B u t m a n and Grasle, 1992; U n d e r w o o d and K e o u g h , 2 0 0 1 ; C r i m a l d i et a l . , 2 0 0 2 ; Pernet et a l . , 2003). In addition, differential survival versus differential settlement obscures what factors play the largest roles ( W o o d i n , 1976). Nevertheless, I w i l l outline briefly the physical and b i o l o g i c a l factors that have been demonstrated to influence settlement; I have included chemical factors as a separate sub-section w i t h i n b i o l o g i c a l factors as this field is emerging as heavily influential in larval settlement and warrants a separate summary.  B i o l o g i c a l Factors The microscopic nature and expansive distribution o f most planktotrophic larvae makes tracking and determining the interactions and influences o f certain b i o l o g i c a l factors challenging for a given larval cohort ( U n d e r w o o d and K e o u g h , 2001). Nonetheless, many b i o l o g i c a l factors have been identified as having importance to the distribution and settlement patterns o f invertebrate species (Scheltema, 1974). These factors include fertilization success (Levitan, 1995), predation (Thorson, 1946), adult filter feeders ( W o o d i n , 1976), larval nutrition ( B o i d r o n M e t a i r o n , 1995), and larval behaviour. L a r v a l supply is the original source o f variability o f larvae arriving at settlement sites. This supply is initially determined by fertilization success and later m o d i f i e d by other processes like predation and larval behaviour. L e v i t a n (1995) summarises factors that establish fertilization success and notes that in  free-spawning  species fertilization can be l o w , but  numerous adaptations c o u l d be selected for a l l o w i n g increased fertilization success rate. M o s t  19  studies o n fertilization success have been conducted in the laboratory. M o d e l s o f field situations have been based o n data collected in laboratory measurements (Denny and Shibata, 1989). Pennington (1985) was the first to conduct in situ fertilization experiments w i t h the urchin Strongylocentrotus droebachiensis, and revealed the importance o f dilution i n realistic fertilization success. A f t e r fertilization, significant losses o f larvae in the planktonic stage, a phenomenon termed b y T h o r s o n (1946) "wastage", can also strongly m o d i f y larval supply. It is believed that the primary source o f this "wastage" is predation ( N e l s o n , 1925; Thorson, 1946; M o r g a n , 1995; G o s l i n g , 2003). One o f the earliest records o f predatory losses o f larvae was made b y N e l s o n (1925) w h o observed larval oysters in the guts o f ctenophores. It is estimated that pelagic predators consume greater quantities o f larvae than their benthic counterparts ( M o r g a n , 1995); however, Johnson and Shanks (2003) made in situ observations o n larval predation b y planktonic predators and found that predatory losses m a y be m u c h lower than p r e v i o u s l y estimated. W i t h limited relevant data f r o m field based research, the question o f the impact o f predation o n pelagic larval stages remains. One group o f benthic organisms that has received recent attention for their potential to shape larval settlement patterns is adult filter feeders. T h e y have been considered to influence recruitment by direct filtration o f larvae from the water c o l u m n ( W o o d i n , 1976; W i l l i a m s , 1980; M a u r e r , 1983; A m b r o s e , 1984; H i n e s et a l . , 1989; A n d r e and Rosenberg, 1991; B o r s a and M i l l e t , 1992; M i t c h e l l , 1992; A n d r e et a l . , 1993; Olafsson et a l . , 1994, B e u k e m a and Cadee, 1996; Lehane and Davenport, 2004) or b y changing near bed f l o w patterns ( N o w e l l and Jumars, 1984; E r t m a n and Jumars, 1988; Lindegarth et a l . , 2002). Reduced larval settlement has been associated w i t h high adult filter feeder densities i n populations o f V. philippinarum ( W i l l i a m s , 1980), M. arenaria (Hines et a l . , 1989) and C. edule (Andre and Rosenberg, 1991; A n d r e et a l . , 1993). Other studies; however, have shown no overall effect o f adult filter feeding populations  o n settlement o f larvae (Maurer, 1983; H u n t et a l . , 1987; E r t m a n and Jumars, 1988; H i n e s , et al. 1989; Thrush et a l . , 1996). T h i s topic remains unclear o n the resulting influence o f filter feeding populations o n larval settlement patterns. S u r v i v a l through the planktonic phase can also be influenced by larval nutrition (this does not apply to lecithotrophic larvae that are supplied by y o l k and do not feed w h i l e planktonic - L e v i n and Bridges, 1995). Starvation is a risk i f larvae are subjected to extended periods o f f o o d limitation; however, food sources are patchy in distribution and larvae are able to survive for extended periods without food ( O l s o n and O l s o n , 1989; B o i d r o n - M e t a i r o n , 1995). Phytoplankton, dissolved organic matter, bacteria and detritus a l l contribute to the diets o f larvae (Olson and O l s o n , 1989; B a l d w i n and N e w e l l , 1991; L u t z and K e n n i s h , 1992; B o i d r o n M e t a i r o n , 1995). A l t h o u g h they enjoy a diverse food source, limitations in t i m i n g and quality o f f o o d have been shown to reduce the number o f larvae surviving metamorphosis because o f compromised food reserves ( H o l l a n d and Spencer, 1973; M c E d w a r d and Q i a n , 2 0 0 1 ; Pernet et al., 2006). B e h a v i o u r a l responses o f larvae to various cues lead to larval ability to influence their o w n settlement patterns thus m a k i n g "active c h o i c e s " in settlement ( K e o u g h and D o w n e s , 1982).  Larvae s w i m m i n g up and d o w n in the water c o l u m n can result in differential dispersal,  particularly i f the water is stratified and water in separate strata are m o v i n g independently (Forward, 1988). The influence o f large-scale circulation is summarized b e l o w in the section titled " P h y s i c a l Factors". C h e m i c a l cues in the environment have a strong influence on larval behaviour resulting in reactions to b i o f i l m s and gregarious settlement. These and other chemical factors involved in larval settlement w i l l be summarised in the f o l l o w i n g section.  21  C h e m i c a l Factors C h e m i c a l s have been isolated that influence settlement and metamorphosis in certain larval organisms ( P a w l i k , 1992a). Some chemicals inhibit settlement o f larvae; these are often referred to as anti-fouling chemicals (Dobretsov et a l . 2006). Others encourage settlement o f certain species o f larvae (Steinberg et a l . , 2002). The chemical nature o f inhibitory and inductive settlement cues is inherently different. Inhibitory settlement cues w o u l d be most effective when associated directly w i t h the surface (Steinberg et a l . , 2002). This w a y , chemicals w o u l d not be lost to the ov erl yi n g water wasting metabolic production energy. There is no need for an inhibitory c h e m i c a l to act in the overlying water, since there is no need to prevent settlement o f a larva that is passing by the surface. Therefore, inhibitory chemicals tend to be non-polar, surface-associated metabolites. Inducers, o n the other hand, should be diffusible and operate i n the ov erl yi n g water, encouraging settlement o f larva that w o u l d otherwise pass by the surface. Therefore, inducers tend to be water-soluble, primary metabolic compounds. P r i m a r y metabolites w o u l d be more effective as inducers because organisms are pre-adapted to have an affinity for primary metabolites since they are nutrient or internal signal type molecules (Steinberg et a l . , 2002). The anti-fouling properties o f the red alga, Delicea pulchra, have been w e l l studied (Steinberg et a l , 2001). It produces a variety o f nonpolar halogenated fiiranones that are held in surface vesicles able to release the chemicals onto the plant surface (Steinberg et a l . , 2001). Tests with the fiiranones produced by the red algae at relevant concentrations have shown that the fiiranones are effective i n preventing settlement o f c o - o c c u r i n g larvae o n the algal surface ( D e N y s et a l . , 1998). Sponge metabolites have also been shown to have inhibitory effects (Lee et a l . , 2001). The morphology o f the sponge provides channels and voids where diffused metabolites w o u l d be trapped and the effect o f the chemical inhibitor w o u l d be kept near the diffusing organism.  22  Bacterially derived inhibitory chemicals have been identified, indicating that some anti-fouling characteristics o f organisms m a y be the result o f surface bio f i l m s (Lee et a l . , 2 0 0 1 ; Steinberg et a l . , 2001). Peptides appear to be important settlement induction chemicals (Steinberg et a l . , 2001). One example o f this is a dipeptide molecule from oyster shells that induces settlement behaviour in larval oysters. This molecule has not been characterised, but the tripeptide g l y c i n e - g l y c i n e arginine ( G G R ) has the same effect as oyster conditioned water and a similar molecular weight ( T a m b u r r i et a l . , 1996). Other water soluble peptide-based examples exist for various larvae like sand dollars, barnacles, abalone, nudibranchs, and tube w o r m s (Steinberg et a l . , 2001). Carbohydrates have received increased attention as c h e m i c a l inducers o f settlement and metamorphosis o f larvae. T h e urchin Holopneustes purpurascens, has been s h o w n to metamorphose in response to a sugar derivative from a red algal host species. T h e urchin d i d not respond to extracts from a host kelp species that d i d not contain the sugar inducer ( W i l l i a m s o n , et a l . , 2000). Research b y P a w l i k and associates ( P a w l i k , 1992a; P a w l i k and B u t m a n , 1993) demonstrated the importance o f a fatty a c i d cue i n settlement and gregarious behaviour o f the tube w o r m Phragmatopoma lapidus californica.  T h e fatty acid was isolated f r o m sand used by  adults o f the species i n b u i l d i n g their tubes. B i o films have been k n o w n for some time as an important factor in settlement o f many larval species (Scheltema, 1974; B o n a r et a l . , 1986; Tamburri et a l . , 1992; W i e c z o r e k and T o d d , 1998; H a d f i e l d and P a u l , 2 0 0 1 ; H u a n g and H a d f i e l d , 2003). T h e composition o f the b i o f i l m i n the field is difficult, i f not impossible, to re-create i n the laboratory (Zhao et a l . , 2003). M o s t experimental w o r k o n the influence o f bacterial colonies o n a surface o n settling larvae has used bacterial monocultures. T h i s method provides valuable information about the influence o f the bacteria o n settlement, but has limited ecological relevance to films i n the field that are complex 23  polycultures and may themselves be c h e m i c a l l y influenced by the host surface (Steinberg et a l . , 2001). The bio films produce inductive chemicals that facilitate larval settlement and responses o f larvae are varied (reviewed by H a d f i e l d and P a u l , 2001). L a r v a l responses to b i o f i l m s have been shown to change as the bio f i l m ages ( K e o u g h and R a i m o n d i , 1996; W i e c z o r e k et a l . , 1995) and as the density o f the bacteria changes (Huang and H a d f i e l d , 2003). Some species settle e x c l u s i v e l y o n another plant or animal species, this is called associative settlement (Crisp, 1974). A good example o f this is metamorphosis o f the nudibranch (Phestilla sibogae) larvae in response to cues from its coral (Parties) prey. The nudibranch responds to waterborne chemical inducers from coral specifically, resulting in the juvenile nudibranch l i v i n g o n an abundant f o o d source (Hadfield and P a u l , 2001). Some other examples o f associative settlement are abalone larvae settling o n crustose coralline algae o n w h i c h it feeds, blue crab settlement o n seagrasses, a sacoglossan that settles in response to a cue from its algal food , and opisthobranch molluscs that settle in response to prey (Hadfield and P a u l , 2001 and references therein). Gregariousness, or settlement in the presence o f conspecifics ( C o l e and K n i g h t - J o n e s , 1939), is seen in a variety o f marine organisms and is thought to be related to chemical cues presented by adults or juveniles. Tube worms (Toonen and P a w l i k , 1996) barnacles ( K n i g h t Jones, 1953), sand dollars ( H i g h s m i t h , 1982), and oysters (Cole and K n i g h t - J o n e s , 1939) have all been reported to exhibit gregariousness. Because o f their c o m m e r c i a l significance, extensive research has been done concerning oyster settlement and metamorphosis. It has been suggested that oyster larvae respond to surface chemicals from bio f i l m e d oyster shells (Bonar et a l , 1986), water conditioned w i t h adult oysters (Crisp, 1967), a m m o n i a released at oyster beds (also thought to be related to the m i c r o b i a l films o n the oyster) and a peptide similar to G G R (Tamburri et a l . , 1996). The larvae may respond to one or a l l cues at different stages o f development. 24  A c t i o n o f chemical inducers o n settlement and metamorphosis can be m i m i c k e d by neurotransmitter compounds. G A B A , o r y-amino butyric acid, induces metamorphosis in abalone ( M o r s e , 1991) and oysters (Tan and W o n g , 1995). C o o n and B o n a r (1985) demonstrated that competent oyster larvae (C. gigas) could be forced to settle and metamorphose by L - 3 , 4 - D i h y d r o x y p h e n y l a l a n i n e ( L - D O P A ) at a concentration o f 2.5 x 1 0 " M , 5  and forced to metamorphose without settlement using epinephrine or norepinephrine at a concentration o f l O ^ M . These neurotransmitter m i m i c s have not been p r o v e n to be associated w i t h natural settlement in any o f the organisms tested; however, involvement o f these compounds in settlement and metamorphosis a l l o w s the neuroactive pathways to be investigated in more detail. Little is k n o w n about the mechanisms by w h i c h larvae interpret chemical signals that induce or inhibit settlement and metamorphosis. P a w l i k (1992b) notes two reasons for the difficulty in study o f the chemosensory organs o f larvae. First, the size o f the larval body and the fine structure makes neurophysio logical examination difficult. Second, repeat experimentation is often impossible since sensory organs to be tested are lost or m o d i f i e d at metamorphosis w h i c h occurs directly after sensing the signal chemical(s). Development in this area o f research has been slow, and it is not yet proven that sensory organ stimulation is required for settlement induction. In some cases, direct exposure o f larvae to ions, neuroactive agents or electrical impulses has stimulated metamorphosis ( P a w l i k , 1992b). E v i d e n c e exists, however, in favour o f the theory that chemoreception is involved in settlement choice in larvae. K n i g h t - J o n e s (1953) showed that the antennules o f barnacle cyprids functioned in that way. The barnacle larvae use brush-like discs o n their antennules to walk over the surface, and these discs m a y sense the adult c h e m i c a l cue, arthropodin (Nott and Foster, 1969).  25  Studies on nudibranch larval sensory organs have proposed that the chemoreceptive organs are located between the velar lobes (Bonar, 1978; C h i a and K o s s , 1982), an area called the apical sensory system. T h i s was confirmed by H a d f i e l d et al. (2000) in an experiment where apical cells o f the larvae (Phestilla sibogae) were irradiated. Once irradiated, the nudibranch larvae were unable to respond to cues that induce metamorphosis in larvae that have a fully functioning apical sensory system. A p p l i c a t i o n o f K  +  and C s ions to the disabled larvae still +  caused metamorphosis, indicating that these ions operated downstream o f the initial c h e m i c a l inducer. F o r most other organisms studied, c i l i a are proposed to be i n v o l v e d in the chemo sensory activities o f the larvae.  P h y s i c a l Factors M a n y p h y s i c a l factors play a role in settlement and ultimate distribution o f invertebrate larvae. Some o f these factors include light (Thorson, 1946), gravity (Bayne, 1964b), w i n d (Bertness et a l . , 1996), upwelling (Roughgarden et a l . , 1988; W i n g et a l . , 1995), hydrodynamics (Butman, 1987; B o x s h a l l , 2000), surface contour ( E c k m a n , 1990), and geochemistry (Butman and Grassle, 1992; E n g s t r o m and M a r i n e l l i , 2005). M a n y larvae are k n o w n to be differentially light and gravity responsive through their larval life span. Planktotrophic larvae are k n o w n to be photopositive ( s w i m towards light) when they are young and become increasingly photonegative as they near metamorphosis. This behaviour is believed to increase feeding opportunities w h e n y o u n g , and improve settlement opportunities w h e n competent (Thorson, 1946). S i m i l a r l y , during the majority o f life in the plankton, larvae are geonegative (move away from the direction o f the p u l l o f gravity) and w h e n competent, become geopositive again increasing contact w i t h surfaces (Bayne, 1964b). Large scale oceanographic circulation patterns c a n dictate larval dispersal and arrival at settlement sites (Carriker, 1951). The magnitude o f this influence is dependant o n duration o f 26  the larvae in the water c o l u m n w h i c h is typically longer for planktotrophic larvae ( U n d e r w o o d and K e o u g h , 2001). Currents driven b y u p w e l l i n g along the coast o f C a l i f o r n i a have been shown ( W i n g et a l . , 1995) to influence distribution o f settling crabs {Cancer sp.). Bertness et al. (1996) were able to connect distribution o f settling barnacle larvae (Semibalanus balanoides) and overall w i n d - d r i v e n circulation in a bay i n R h o d e Island. Shanks and B r i n k (2005), on the other hand, studied the movement o f bivalve larvae (Tellina sp., Mulina lateralis, Spisula solidissima and Ensis directus) v i a u p w e l l i n g and d o w n w e l l i n g currents o n the coast o f N o r t h C a r o l i n a and found that movement o f larvae was dependant o n vertical distribution o f larvae and the species, not the u p w e l l i n g or d o w n w e l l i n g currents. M a n y authors have identified bottom roughness and related changes to the near-surface hydrodynamics as factors influencing settling larvae (Crisp, 1955, 1974; W i l l i a m s , 1978, 1980; N o w e l l and Jumars, 1984; Wethey, 1986; E r t m a n and Jumars, 1988; Gallagher et a l . , 1983; E c k m a n , 1990; Snelgrove et a l . , 1993; H a r v e y et a l , 1995; Gregoire et a l . , 1996; A b l e s o n and D e n n y , 1997; K o h l e r , et a l . , 1999; B o x s h a l l , 2 0 0 0 ; P e c h , at a l . , 2 0 0 2 ; C r i m a l d i et a l . , 2002). In an experiment using needles to m i m i c the effect o f a n i m a l tubes o n f l o w , E c k m a n (1979) found increased recruitment o f a tanaid shrimp and a sabellid polychaete near the needles. Gallagher et al. (1983) carried out a similar experiment using sticks instead o f needles and found that they facilitated larval settlement i n a number o f species. A n d w h i l e t r y i n g to make observations o n j u v e n i l e cockles (Cardium edule), B a g g e r m a n (1953) used iron gauze screens placed perpendicular to intertidal f l o w to create a "current shadow" that captured cockle settlers in the W a d d e n S e a indicating that the turbulent w a k e created b y the screen led to increases i n recruitment. Slight increases in bottom irregularity (approximately 1.5 mm) have been measured and result in changes in small scale turbulence structure near the bottom (Hendriks et a l . , 2006). Turbulence structure has been highlighted b y A b l e s o n and D e n n y (1997) as influencing 2 7  settlement in a number o f ways. The authors note that flow can be a settlement cue unto itself, it can help to mediate other settlement cues in the water by distributing them, and it can help place larvae in p h y s i c a l contact w i t h a surface. The influence o f turbulence has been confirmed by other authors who have also observed changes in settlement o f larvae resulting from altered turbulence structures (Crisp, 1955; B o x s h a l l , 2 0 0 0 ; Pernet, et a l . , 2 0 0 3 ; Fuchs et a l . , 2004).  Fluid Motion The m o t i o n o f water relative to surfaces and organisms, affects a l l marine biota and in particular w i t h reference to this thesis, it affects bivalve gametes, larvae and juveniles. A s such, it is appropriate to highlight some aspects o f fluid dynamics o f relevance to the dispersing and/or settling larvae.  Benthic B o u n d a r y L a y e r A s a fluid moves past a solid, there is a gradient o f velocity created. A t the surface o f the solid, the " n o - s l i p " condition dictates that the fluid in contact w i t h the solid is stationary ( V o g e l , 1994). F r i c t i o n o f the fluid (shear) away from the solid creates a gradient o f velocity, eventually terminating in the "free-stream" or m a x i m u m velocity. The area under the influence o f friction is called the benthic boundary layer ( B B L ) (Figure 1-4) and the thickness o f this layer varies according to a number o f parameters (bottom roughness, temperature, water velocity). T h i s variability and differing research approaches leads to a number o f different definitions for the outer limit and an arbitrary nature depending o n the scale o f interest (Boudreau and Jorgensen, 2001). V o g e l (1994) sums up the t y p i c a l b i o l o g i c a l understanding o f the B B L w i t h this statement: "(most biologists) have a f u z z y notion that it's a distinct region rather than a distinct notion that it is a f u z z y r e g i o n . "  28  I:  Bottom  ;  Figure 1-4: Graphic representation of the flows in the Benthic Boundary Layer. Longer arrows represent faster flows; grey at the bottom represents the surface. Flow increases with distance from the surface and eventually reaches a rate equivalent to the free-stream.  Because hydrodynamic changes in the B B L are o f such a fine-scale, certain challenges and hindrances exist f o r investigators. Recent advances i n instrumentation and technology have a l l o w e d high resolution measurements to be made within the B B L ( K h a l i l i et a l . , 2001). C r i m a l d i et a l . (2002) employed a specialised instrument called a laser-Doppler anemometer ( L D A ) to analyse f l o w and help m o d e l larval f l u x to the bottom in turbulence. T h e L D A uses D o p p l e r scatter from three laser beams to make detailed measurements o f flows i n the B B L . Lasers are non-invasive and thus the sampling artifacts that constrain other systems are eliminated. F l u m e s are also an excellent method f o r study o f the B B L and other f l o w related phenomena because they a l l o w control and manipulation o f any number o f variables ( K h a l i l i at a l . , 2001). Other researchers have used flumes to test patterns o f larval settlement i n realistic f l o w s ( B u t m a n , 1987; Snelgrove et a l . , 1993, 1999; Gregoire et a l . , 1996; B o x s h a l l , 2 0 0 0 ; F i n e l l i and Wethey, 2003). B o t t o m roughness is k n o w n to affect friction above the bottom and the B B L characteristics (Dade et a l . , 2 0 0 1 ; Hendricks et a l . , 2006) and dispersing and settling larvae  interact strongly w i t h the B B L (Jumars et a l . , 2001). The influence o f bottom roughness and turbulence o n larval settlement patterns was discussed above in section "Factors Influencing Settlement Patterns: P h y s i c a l Factors".  Reynolds Number R e y n o l d s number is a unitless measure o f the ratio o f viscous and inertial forces. T h i s relationship was established by Osborne R e y n o l d s in the late 1800's w h i l e w o r k i n g w i t h f l o w in pipes and attempting to understand the laws concerning the change in f l o w f r o m laminar to turbulent ( V o g e l , 1994). The formula for the R e y n o l d s number (Re) is shown here:  H  v  Where /?=density, ^ c h a r a c t e r i s t i c length (greatest length o f the solid in the direction o f the f l o w ) , f/=velocity, w=dynamic viscosity, and v=kinematic viscosity. The ratio o f density and /  viscosity (v: the kinematic viscosity) becomes an important part o f this relationship. W h e n R e is l o w , kinematic viscosity is high and f l o w s are laminar; w h e n R e is h i g h , kinematic viscosity is l o w and turbulence develops. The R e y n o l d s number can be used to predict transitions from laminar to turbulent f l o w , and is also useful in scaling experimental parameters to accurately reflect a system o f interest. W h e n the characteristic length (/) is s m a l l , as is the case for bivalve larvae, R e is s m a l l and laminar viscous forces dominate ( V o g e l , 1994). L i f e at l o w R e is very different than f r o m what w e find n o r m a l , domination o f viscosity and virtual elimination o f inertial forces makes movement an entirely different matter for bivalve larvae and other plankton. W i t h essentially no inertia to carry it along, the s w i m m i n g larvae that stops actively s w i m m i n g stops m o v i n g altogether.  30  Turbulence Turbulence is ubiquitous i n the marine environment (Denny, 1988). I n a clever metaphor, V o g e l (1994) compared viscous forces to "groupiness" and inertial forces to " i n d i v i d u a l i t y " and in this manner, laminar f l o w w o u l d be an orderly march w h i l e turbulence w o u l d be a randomly strolling c r o w d . Turbulence results when water velocity is high enough for the inertial forces to overcome the viscous forces ( V o g e l , 1994). In turbulent f l o w , inertial forces dominate and viscous forces oppose it. Turbulence causes m i x i n g and makes c h e m i c a l signals unpredictable i n space and time (Weissberg et a l . , 2002). Crisp (1965) predicted that turbulent f l o w over a surface w o u l d effectively dilute any water-soluble cue released a small distance f r o m the surface. Thus, any c h e m i c a l cue released from a surface w o u l d o n l y be held i n sufficient quantities in the boundary layer, and in natural conditions that boundary layer w o u l d be approximately the same depth as the larvae itself. Despite this prediction, Tamburri and colleagues (1996) demonstrated that oyster larvae {Crassostrea virginica) were able to detect chemical cues in f l o w i n g conditions. The authors used both adult oyster conditioned water and the artificial settlement inducer, g l y c y l - g l y c y l - L arginine, in f l o w i n g water to test i f oyster larvae reacted differently to the cue i n f l o w . The larvae demonstrated the same behaviour in f l o w i n g conditions as had been observed previously in still water. In a test using polychaete larvae {P. lapidosa californica), P a w l i k and B u t m a n (1993) investigated the effects o f f l o w speed and turbulence o n settlement. Larvae and passive larvalm i m i c s were passed over metamorphosis inducing sand and non-inducive sand at various f l u i d velocities. Intermediate velocities were the most effective for settlement; the larvae s w a m away at l o w flows and were swept away from the surface at high flows.  31  Shellfish A q u a c u l t u r e Shellfish farming has a long history, but it was not until the later part o f the 1 9 century th  that modern methods were developed ( G o s l i n g , 2003). M o r e recently, the number o f species and the amount o f production has g r o w n enormously. Figure 1-5 shows the production o f molluscs from aquaculture (millions o f tonnes) and the number o f species in production per year since 1970 ( F A O , 2005). Average annual g r o w t h in the w o r l d w i d e production o f clams and cockles alone i n 2002 was 1 4 . 1 % , g r o w i n g from 2.63 m i l l i o n tonnes i n 2 0 0 0 , to 3.43 m i l l i o n tonnes in 2002 ( F A O , 2004).  14  n  12 73  s  Global Trends in Molluscan Aquaculture  70 60  - o — Production - © - - - N u m b e r of s p e c i e s  ©®  8 >  fe©®'*  10  50  8  40  "O O  «,  o cu  5,®®®-®©®-®-®  30  CL W CD  + 20  .O  E 3  10  2  1969  1974  1979  1984  1989  1994  1999  2004  Year Figure 1-5: Annual production of mollusc aquaculture by mass shown with open squares and on left axis. Number of molluscan species in production worldwide shown with solid grey circles and on right axis. Data from FAO 2005.  B i v a l v e s are particularly attractive for culture purposes; they feed o n naturally produced algae and need very little husbandry once they are outplanted (Folke & K a u t s k y 1989, 1992; C r a w f o r d et a l , 2003). M a n y parts o f the w o r l d still rely o n collection o f w i l d settled juveniles as a source for production; however advancing hatchery technology is m a k i n g production o f 32  hatchery spawned and raised seed animals more c o m m o n l y available. Hatcheries have the advantage o f being able to produce seed at all times o f the year and in large, reliable quantities ( H e l m and B o u r n e , 2004). A l t h o u g h many countries practice shellfish aquaculture, the global production is heavily dominated b y C h i n a . Figure 1-6 shows the proportional contribution by mass (top eight countries each year only shown) f r o m 1970 through 2004. C h i n a ' s o v e r w h e l m i n g dominance o f w o r l d molluscan production began in the early 1990's; in 2004 C h i n a produced 10.4 m i l l i o n tonnes o f molluscs b y farming, equalling 7 9 % o f the global production that year ( F A O , 2005). In comparison, Canada produced 30,000 tonnes o f molluscs accounting for 0 . 3 % o f the global total i n 2 0 0 4 ( F A O , 2005).  M a n i l a C l a m Aquaculture M a n i l a clams (V. philippinarum)  are native to Japan but were accidentally introduced to  B r i t i s h C o l u m b i a in the 1930's along w i t h oyster seed shipments f r o m Japan (Bourne, 1982). The clams were first observed in L a d y s m i t h Harbour, B r i t i s h C o l u m b i a and were misidentified as Paphia bifurcata (Quayle, 1938). T h e clams q u i c k l y spread through the southern portion o f B r i t i s h C o l u m b i a and into Puget Sound (Quayle, 1964). T h e M a n i l a c l a m soon supported a strong recreational and c o m m e r c i a l fishery (Quayle and B o u r n e , 1972) and i n 1985, became the basis o f a culture industry.  33  Global Molluscan Production by Country  1970  1975  1980  1985  1990  1995  2000  2004  Figure 1-6: G l o b a l molluscan production by mass contribution by country. Country labels are listed on the right. For each year shown, the top eight countries are graphed, the rest o f the countries for that year are pooled in "rest o f w o r l d " category. Data from F A O 2005.  A l t h o u g h the M a n i l a c l a m is native to J a p a n and other parts o f southeast A s i a , it has b e e n i n t r o d u c e d (both a c c i d e n t a l l y a n d i n t e n t i o n a l l y ) to m a n y parts o f the w o r l d . M a n i l a c l a m s w e r e i n t e n t i o n a l l y i n t r o d u c e d to the H a w a i i a n Islands i n the early 1 9 0 0 ' s a n d i n F r a n c e i n 1972 ( G o u l l e t q u e r , 1997). Subsequent i n t r o d u c t i o n s have o c c u r r e d throughout E u r o p e and i n m o s t locations n a t u r a l l y r e c r u i t i n g p o p u l a t i o n s have b e c o m e established ( G o u l l e t q u e r , 1997). T h e M a n i l a c l a m b e l o n g s to the class B i v a l v i a , subclass H e t e r o d o n t a , order V e n e r o i d a , f a m i l y V e n e r i d a e ( C o a n et a l . , 2 0 0 0 ) . W o r l d w i d e d i s t r i b u t i o n and c o m m e r c i a l importance o f the species has l e d to extensive t a x o n o m i c c o n f u s i o n . T h i s species has b e e n referred to u s i n g 34 different s c i e n t i f i c (genus and species) n a m e s i n the literature a n d has o v e r 2 2 c o m m o n names ( P o n u r o v s k y a n d Y a k o v l e v , 1 9 9 2 ; G o u l l e t q u e r , 1997).  34  The M a n i l a c l a m is a high value w o r l d crop. In 2004, w o r l d w i d e production o f the M a n i l a c l a m was 2.9 m i l l i o n tonnes, valued at 2.2 m i l l i o n U S $ ( F A O , 2005). T h e capture fishery was once the o n l y source o f M a n i l a clams, but aquaculture production has been rapidly increasing since the m i d - 1 9 8 0 ' s and q u i c k l y outpaced the capture fishery. M a n i l a c l a m aquaculture today produces nearly 50 times the amount produced by the capture fishery (Figure 1-7). 2,500  World Production Tapes philippinarum 2,000  o  ? 1,500 CO CD  c c o  H Capture Fishery • Aquaculture  •= 1,000  500  1950  1955  1960  1965  1970  1975  1980  1985  1990  1995  2000  Figure 1-7: Comparison of Manila clam (Venerupis philippinarum) production from capture fishery versus aquaculture. Capture fishery is shown with grey bars and aquaculture shown in black. Data from FAO 2005.  C l a m Culture i n B r i t i s h C o l u m b i a In B r i t i s h C o l u m b i a , shellfish aquaculture production totalled 9,324 tonnes w i t h a value o f $15 m i l l i o n in 2 0 0 4 ; 1 7 % o f this production (1,528 tonnes) and nearly h a l f o f the value ($7 m i l l i o n C d n $) were farmed clams (farmgate values - Statistics Canada, 2005). C l a m farming i n B r i t i s h C o l u m b i a has been traced to pre-European contact when aboriginal people manipulated 35  and managed intertidal areas to make " c l a m gardens" as a consistent f o o d source (Harper et a l . , 2006). Today, c l a m farming exists in a different, more industrial f o r m . Farmers in B r i t i s h C o l u m b i a t y p i c a l l y obtain seed clams from hatcheries. In the hatchery, broodstock adults are conditioned then spawned to obtain larvae. The larvae are fed cultured algae and raised i n the hatchery until they metamorphose. Once metamorphosed, the small seed can be m o v e d to a nursery system where it is g r o w n to a suitable size for planting out o n intertidal plots. These nursery systems come i n a variety o f forms; for a summary see H e l m and B o u r n e , 2004. Y o u n g clams ( 5 - 1 0 m m in shell length) are spread o n intertidal plots at a density o f roughly 4 0 0 - 6 0 0 individuals per meter . The details o f seed size and density are often 2  site specific and each grower w i l l make slight adjustments based o n what is best suited to the beach where culture takes place. A t most farms, these plots are protected by predator nets (see next section for more detail). The clams take from t w o to four years to reach marketable size o n most beaches and once that size is reached, d i g g i n g crews visit the beach and harvest using hand-rakes.  Predator Netting D u r i n g grow-out, intertidal clams are vulnerable to predators such as m o o n snails (eg. Euspira lewisii - T o b a et a l . , 1992) crabs (eg. Cancer productus - Quayle and B o u r n e , 1972) and d i v i n g ducks (eg. Melanitta deglani, M. perspincillata  andM. nigra - B o u r n e , 1984).  Efforts to reduce mortality by predation were undertaken i n the early 1980's by researchers at the U n i v e r s i t y o f Washington w h o tested the efficacy o f nets placed o n the surface o f the sediment (Anderson, 1982). The netting proved effective at reducing predatory losses o f clams and the use o f nets soon became c o m m o n practice for c l a m farmers (Toba et a l . , 1992). U s e o f nets has since been tested and proven effective i n estuaries in N W Spain (Cigarria and Fernandez, 2000) and M a i n e , U S A (Beal and K r a u s , 2002).  In addition to protecting clams from predation, nets have the potential to stabilize sediments, interfere w i t h local hydrodynamic processes, and increase silt and organic matter deposition (Spencer et a l . , 1996). Stabilization o f beach sediments may lead to a more favourable habitat for the clams and consequently a larger, healthier population.  Alternatively,  it could lead to changes in sediment characteristics that are important to the benthos, and nutrient and gas exchange at the sediment-water interface ( D r i s c o l l , 1975, B a r t o l i et a l . , 2001). N u g u e s et al. (1996) noted small changes in the benthic c o m m u n i t y associated w i t h increases in organic carbon and silt content o f sediments beneath intertidal oyster trestles in the R i v e r E x e estuary, E n g l a n d . The trestles altered water f l o w in the area around the cultivation site resulting in a decrease in the depth o f the oxygenated sediment layer and reduced abundance o f benthos below culture structures. The most c o m m o n l y used nets in B r i t i s h C o l u m b i a at the time o f w r i t i n g this thesis are plastic w i t h a mesh size o f 1 - 2 c m aperture although some farmers use heavier cotton nets. The nets are secured by p i n n i n g d o w n the edge with rebar staples pushed into the ground, digging the edges into the sediment or placing rocks around the periphery. The nets are left in place until harvesting; w h e n the nets are removed for digging (this c a n be done in one day) then they are replaced (Toba et a l . , 1992). Depending o n the farm location and time o f year, the nets can become fouled w i t h algae. W h e n fouling is heavy, the clams b e l o w can be at risk o f anoxia, so in those cases the nets are removed and de-fouled before replacement (Toba et a l . , 1992). The Baynes Sound region o f B r i t i s h C o l u m b i a is the source o f roughly 5 0 % o f the farmed shellfish products in B r i t i s h C o l u m b i a . The study sites chosen for research described in this thesis are located in this region. The intertidal area o f B a y n e s Sound is approximately 1,530 hectares; it was estimated that 3 2 % o f that area (493 ha) was under tenure for beach culture and 5 % (approximately 76 ha.) was covered w i t h c l a m netting in 2002 ( M i n i s t r y o f Sustainable Resource Management, 2002).  37  Figure 1-8: Location of Baynes Sound on Vancouver Island, Canada. Inset left shows location of Vancouver Island in relation to Canada.  Thesis Outline Some settling larvae can actively select settlement locations o n a small scale and in some cases turbulence influences that selection. S m a l l changes to the roughness o f the sediment can influence turbulence in near-bottom flows. Predator netting applied to the surface o f intertidal c l a m farms presents a unique scenario to test, in a natural environment, whether distribution o f recently settled larvae is affected by netting. G i v e n recent increases in c l a m farming w o r l d w i d e , it is important to understand the potential influences o f predator netting o n settling larvae. A s a prerequisite to sampling recently settled larvae in the field, I first evaluated a field sampling method to establish the recovery rate and ensure that differences in sediment grain size w o u l d not bias estimates o f recruits. C h a p t e r 2 describes the field sampling method devised and the evaluation o f the method in a laboratory trial using four different sediment types.  38  I f netting influences intertidal turbulence, then it follows that the sediments beneath the nets should also display changes beneath netting compared to adjacent plots without netting. Based o n results o f other research I predicted that the netting w o u l d interrupt intertidal f l o w such that it w o u l d create a more depositional environment i n w h i c h larvae could potentially settle out i n higher numbers. In Chapter 3 I examine the influences o f c l a m netting o n sediment grain size, organic and inorganic carbon content as w e l l as e x a m i n i n g temperature at netted versus non-netted plots. Evidence f r o m other research ( G l o c k , 1978; M i t c h e l l , 1992) suggests that clams (>5 mm) recruit at a higher rate to intertidal areas covered w i t h netting. I propose that this higher observed rate o f recruitment o f adult clams c o u l d be explained b y greater deposition o f larvae i f the larvae are acting as passive particles. T o test this hypothesis, I measured the density o f early recruits o n netted and non-netted plots over t w o years.  Chapter 4 describes recruitment rate o f  recently settled larval clams to plots w i t h netting and without to determine i f there is a pattern related to nets o n farmed beaches. The preceding chapters describe results o f sampling from field sites where there is limited ability to control certain variables such as predation rate, desiccation, larval influx etc. Observed patterns o f early recruits (described i n Chapter 4) were potentially confounded w i t h biotic factors also measured at the field sites. T o test the influence o f netting o n settlement o f clams in the absence o f biotic factors, laboratory flume trials were used. In  Chapter 5 I  describe the results o f laboratory flume trials comparing settlement o f competent, hatcheryraised larvae i n flumes w i t h sediment and nets used as crossed treatment factors. In c o n c l u s i o n ,  Chapter 6 provides a summary o f the results, a b r i e f discussion and  recommendations for future research.  39  References A b e l s o n , A . , and M . Denny.  1997. Settlement o f marine organisms in f l o w . A n n u a l R e v i e w o f  E c o l o g y and Systematics, 2 8 : 3 1 7 - 3 3 9 . 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Induction o f metamorphosis i n the sea urchin Holopneustespurpurascens b y a metabolite c o m p l e x f r o m the algal host Deliseapulchra. B i o l o g i c a l B u l l e t i n , 198: 3 3 2 - 3 4 5 .  54  W i n g , S . R . , L . W . B o t s f o r d , J . L . Largier, and L . E . M o r g a n . 1995. Spatial structure o f relaxation events and crab settlement i n the northern C a l i f o r n i a u p w e l l i n g system M a r i n e E c o l o g y Progress Series, 128: 199-211. W o o d i n , S . A . 1976. A d u l t - l a r v a l interactions in dense infaunal assemblages: Patterns o f abundance. Journal o f M a r i n e Research. 3 4 : 2 5 - 4 1 . W o o d i n , S . A . 1991. Recruitment o f infauna: positive or negative cues? A m e r i c a n Zoologist, 31: 797-807. Y a n k s o n , K . 1986. Observations o f byssus systems in the spat o f Cerastoderma glaucum and C. edule. Journal o f the M a r i n e B i o l o g i c a l A s s o c i a t i o n o f U . K . , 6 6 : 2 7 7 - 2 9 2 . Y o n g e , C . M . 1962. O n the primitive significance o f the byssus i n the B i v a l v i a and its effects in evolution. Journal o f the M a r i n e B i o l o g i c a l A s s o c i a t i o n o f U . K . , 4 2 : 113-125. Y o u n g , C . M . 1990. L a r v a l e c o l o g y o f marine invertebrates - A sesquicentennial history. O p h e l i a , 3 2 : 1-48. Y o u n g , C . M . 1995. B e h a v i o r and locomotion during the dispersal phase o f larval life. In: L . M c E d w a r d (ed) E c o l o g y o f M a r i n e Invertebrate Larvae, p p . 2 5 0 - 2 7 0 . Zardus, J . D . , and A . L . M a t t e l . 2002. P h y l u m m o l l u s c a : b i v a l v i a . In: C . M . Y o u n g (ed.), Atlas o f marine invertebrate larvae. A c a d e m i c Press, San D i e g o C a l i f o r n i a , p p . 2 8 9 - 3 2 9 . Z h a o , B . , S . Z h a n g and P. Y . Q i a n . 2003. L a r v a l settlement o f the silver o r goldlip pearl oyster Pinctada maxima (Jameson) in response to natural b i o f i l m s and c h e m i c a l cues. Aquaculture, 2 2 0 : 8 8 3 - 9 0 1 .  55  CHAPTER 2: Sampling Recently Settled Clams from Sediments* Introduction The Manila clam (Venerupis philippinarum, A. Adams and Reeve, 1850) is of major importance to both the wild fishery and aquaculture industry worldwide. In British Columbia, Canada, this species is non-native and is thought to have been introduced along with Pacific oyster seed from Japan in the 1930s (Bourne, 1982). Since its introduction, V. philippinarum has become an important species economically and is the basis of the current clam culture industry (Jones et al., 1993). Conditions in British Columbia are favorable for the Manila clam and it has become well established throughout the southern coastline (Quayle, 1974). In bivalves, a pelagic larval stage is followed by metamorphosis, during which the swimming organ or velum is lost, and the bivalve transforms into the benthic or epibenthic juvenile form. The post-metamorphic juvenile stage of the Manila clam is found in the upper layers of the sediment; however, sampling juveniles from these environments presents many problems. The clams are similar in colour and size to the fine sediments. Consequently, few studies have focussed on this life stage. When sampling has been carried out and early juveniles in the sediment are counted, (Jones, 1974; Glock, 1978; Williams, 1980) the work is extremely time consuming and prone to error. Rarely is reference made in published studies of bivalve settlement to the accuracy of the sorting and counting methods used. A simple, consistent and effective method for separation of post-settlement bivalves from the sediments would allow for more studies to be carried out and more insight gained into recruitment patterns of the Manila clam and other valuable clam species.  " A version of this chapter has been published in: Munroe, D. M . , D. Bright and S. McKinley. 2004. Separation of recently settled Manila clams (Tapes philippinarum A. Adams and Reeve, 1850) from three sediment types using sucrose density solution. Journal of Shellfish Research, 23: 89-92. 56  The density o f juvenile post-settlement clams and cockles was estimated to be 1.036 1.076 g/mL in a study b y M o n t a u d o u i n (1997), and 1.1 g/mL in a study by Jonsson et al. (1991). M i n e r a l s are denser, typically w i t h a specific weight o f 2.5 g/mL and higher (Denny, 1993). Density gradients o f silica sols have been used to separate lighter meiofauna from higher density sediment fractions (Burgess, 2 0 0 1 ; Schwinghamer, 1981; N i c h o l s , 1979). It has been shown that high density sucrose solutions can also be used to separate meiofauna from muddy organic sediment ( H e i p et a l . , 1974). The technique explored herein involves wet serving to isolate the size fraction o f the sediment containing the bivalves, then a l l o w i n g that size fraction to settle through a high density sucrose solution (1.9 g/mL) to isolate the meifauna and a l l o w for easier counting o f the bivalves. This technique was tested using 3 different sediment types to determine efficacy o f the method o n various sediments. The juvenile clams were expected to float i n a solution w i t h a density o f 1.9 g/mL. H o w e v e r , the high concentration o f sucrose increases the osmotic pressure on the animal cells causing them to dehydrate, thereby increasing the density o f the animals and causing them to sink (Price et a l . , 1978; B o w e n et a l . , 1972). A l t h o u g h the juveniles become more dense and sink, they do so s l o w l y in relation to the higher density mineral components o f the sample that are found in the top layer o f the sediment once it has settled out. This process o f isopycnic sedimentation at one solute density to separate particles o f different densities is called "rho spectrometry" b y Price et al. (1977).  Materials and  Methods  Three different sediment types were obtained from intertidal beaches north o f N a n a i m o , B r i t i s h C o l u m b i a . N i n e tanks were prepared w i t h each o f the three different sediment types (treatments) randomly assigned to three tanks per sediment type. The sediment types were cobble/mud, cobble/sand/shell and mud/sand according to their properties, and a subsample 57  (approximately 20 c m ) o f each sediment type was analysed for grain size components, b y wet 3  sieving using methods adapted from K o m a r (1998). Organic material was not removed prior to wet sieving. B r i e f l y , the sediment was wet-sieved, dried at 60°C ( B o y d and Tucker, 1992) for 30 minutes, and weighed. Sediments used in the experiment were autoclaved prior to placement i n each aquarium. E a c h aquarium was filled to a depth o f 5 c m (surface area o f each tank was 800 c m ) with the 2  assigned sediment type, then f i l l e d w i t h 15 L seawater (filtered to 1 u m and sterilized with U V radiation). A l l aquaria were aerated and warmed to 20°C before addition o f competent, hatchery reared M a n i l a c l a m larvae. A total o f 2400 clams were added to each o f the nine tanks. Tanks were maintained at 20°C and clams were fed a combination o f 50:50 Chaetoceros meullerii and Isochrysis spp (Tahitian strain) at a combined concentration o f 20,000 - 30,000 cells/ m L (Jones et a l . , 1993). C l a m s were left in the tanks for 11 days to settle and metamorphose. The tanks were then drained to simulate l o w tide s a m p l i n g conditions, and core samples o f the sediment were taken. F o u r sediment cores were taken from each o f the nine tanks (36 samples total). A small corer made o f P V C pipe (5 c m internal diameter) was inserted 1 c m into the sediment and a thin metal lid was s l i d under the pipe to prevent the sediment from falling out. Samples were placed i n a plastic sample bag, labelled and frozen for later counting. Freezing was chosen as a method o f sample preservation to ensure consistency between lab methods and field methods described i n Chapter 4. F o r enumeration, samples were thawed then placed i n 0 . 0 1 % phloxine B dye for at least 20 minutes ( W i l l i a m s , 1978). Samples were then washed through a series o f sieves; the  fraction  o f sediment from 125 - 500 [im was placed into a high density sucrose solution (1.9 g/mL) in 30 m L test tubes. Tubes were inverted to m i x the sediments, to avoid particle-particle interactions (Price et a l . , 1977) then left to settle out b y gravity ( m i n i m u m o f 25 minutes). T h e top layer o f sediment was pipetted o f f the surface o f the settled sediments in the test tube. T h e amount o f  sediment pipetted off the top was approximately 1 - 2 mL which is less than 10% of the original core sample volume. The stain coloured the tissue of the clams and made them highly visible when viewed under a microscope and the number of clams in each sample was counted at 60X magnification. Statistical analysis of variance was calculated using JMP statistical software.  Results The grain size for each sediment type is shown below in Table 2-1. The cobble/mud sediment contained the largest fraction of the >2000 pm size category with 82% by weight. The cobble/sand/shell also contained mostly >2000 pm sized components; however, it should be noted that in this case over half of this size category was comprised of broken shell while the >2000 pm fraction in the cobble/mud was entirely small cobble. The mud/sand sediment was mostly comprised of medium sand (70% dry weight); with coarse sand and fine sand making up another 18% dry weight. The components >2000 pm were only broken shell in the mud/sand sediment.  T A B L E 2-1: Grain size components, percentage by dry weight, of each sediment type. The size category >2000um contains both granule+ and broken shell.  Size class Shell Granule + Very Coarse Sand Coarse Sand Medium Sand Fine Sand Silt  Size Range (u.m) (>2000) (>2000) (1000-2000) (500-1000) (125-500) (75-125) (<75 )  cobble/mud ~ 82 3 4 7 2 2  % overall weight cobble/sand/shell 43 30 8 7 9 2 1  mud/sand 4 3 11 70 7 5_  2400 clams were added to each tank and each tank had a sediment surface area of 800 cm on which to settle; therefore, the expected average density of clams in each tank was 3 2  59  clams/cm . The surface area o f each core was 19.7 c m , so the expected average number of 2  2  clams per core was 58.8. Means and 95% confidence limits for number o f clams recovered from coring the three sediment types is shown in Figure 2-1. Comparison o f mean numbers o f clams per core among the three sediment types was done to test if all means were equal regardless o f sediment type. Mean values for clams per core were 60.0 for cobble/sand/shell, 53.1 for cobble/mud, and 57.9 for mud/sand. Based on analysis o f variance, there was no significant difference in the number of juvenile clams recovered from each of the different sediment types and the expected value 58.8 (n =3, prob. ranged from 0.70 to 0.91). N o r was there a significant difference between treatments (n =3, prob. = 0.90). Individual t-tests were also run to determine i f the recovery o f clams from coring of any o f the experimental units (9 tanks) departed significantly from the expected value based on larval seeding density. There was no statistically significant difference between the observed and expected values for any of the experimental units (n=4, prob. ranged from 0.30 to 0.75).  80 70 60 k O  o  a. JS  , am  50 40  O c  30  2  20  co 0)  Ifllll^pl^^  10 0  Cobble/Mud  Cobble/Sand/Shell  Mud/Sand  Figure 2-1: Means and standard deviation for numbers of clams (Venerupis philippinarum)  per sample for the three sediment types. The dashed line indicates the expected number of clams per sample (58.8) based on number of larvae placed in each tank. N= 3 for each treatment.  60  Discussion A n a l y s i s o f grain size components (Table 2-1) shows that there were large differences between the compositions o f the three sediment types used in this experiment. This was important since I was testing the accuracy o f the sampling methods for extraction and counting o f post-settlement juveniles i n different types o f sediments. F o r example, the mud/sand sediment was comprised o f 7 0 % m e d i u m sand, w h i c h means 7 0 % o f the entire sample was the same size class in w h i c h the bivalves are found; therefore initial p h y s i c a l separation w i t h sieving w o u l d only eliminate a small v o l u m e o f sediment f r o m the sample. T h i s has the potential to lead to difficulty and inaccuracy in extraction o f the clams f r o m this large sediment fraction. In this experiment, sample c o m p o s i t i o n o f 7 0 % m e d i u m sand d i d not create additional inaccuracies in counts: The numbers o f clams counted per core did not differ in the three types o f sediment. Further, for a l l three sediment types, the number o f clams counted in each sample d i d not differ from the value expected per sample based o n the number o f clams placed in tanks initially. This means that w i t h this method there was nearly 1 0 0 % recovery o f bivalves from sediment regardless o f sediment type. Individual tanks w i t h i n each sediment type were tested to ensure that mean numbers o f clams counted per sample d i d not differ statistically from one tank to the next. Large standard errors were seen in data from some individual tanks. These were overcome w h e n a l l samples o f each sediment type were analyzed together. T h i s m a y be interpreted as a result o f the patchy settlement o f c l a m larvae ( W i l l i a m s , 1980), especially i n coarser and heterogeneous substrates, and implies that the sampling effort may need to be increased for such sediment types. The recovery o f 1 0 0 % o f the bivalves that were placed i n tanks also suggests 1 0 0 % survival from the time o f introduction o f the larvae to the time o f recovery o f post-settlement clams. S u r v i v a l rates through metamorphosis for V. philippinarum  in a hatchery generally vary 61  from 50 to 90% depending on larval quality (Utting and Spencer, 1991). The recovery of an estimated 100% of added larvae (had the entire sediment surface been sampled) is probably due to the relatively short duration of the study. Some of the post-settlement clams, in fact, may have been non-viable or dead at the time of sampling, but freezing and subsequent staining would not distinguish recently deceased clams from live ones. In other circumstances, it might be expected that some mortality would occur prior to settlement, so a failure to account for 100% of the introduced larvae might not be attributable to the sorting techniques in other studies. Separation and counting of live clams prior to freezing was not attempted leaving some question about whether separation as described herein would be equally effective for live specimens or those preserved using other methods such as formalin fixation. Schwinghamer (1981) conducted tests on live separation of benthos from mud and sediments using centrifugation in sorbitol and Percoll and found it to be an effective separation method that allowed for proper identification and observation of sampled benthos. In a study by Burgess (2001), density separation of meiofauna from sediment was carried out using Ludox®. Sediment samples were mixed with Ludox® then centrifuged to separate the meifauna. Using this method, Burgess was able to recover 95.9% of the bivalves in the sample. Jonge and Bouwman (1977) also found use of density separation of nematodes and copepods from sediment and detritus to be more effective and accurate than hand-sorting decantation methods. Both Burgeses (2001), and Jonge and Bouwman (1977) noted that a potential shortcoming of the density separation method is that animals may attach to sediments and therefore sink with them. Post-metamorphic bivalve juveniles have the ability to attach to larger sediments using a byssus. In this study, the sediment fractions larger than 500 um were not examined to look for attached juveniles; however, recovery was estimated at 100% in the size fraction examined, so few, if any, clams were likely to have been found in larger fractions. It is  62  possible that if there were any clams attached to sediments by byssal threads, that the threads were released when the sediment samples were frozen.  Conclusions  Use of the sucrose-density separation method described here is effective for counting newly settled juvenile clams from sediment. Use of the high density sucrose solution to isolate the lower density animals increases sampling efficiency by decreasing the time to sort through sediment; which in turn increases sampling accuracy since less physical and psychological variance is introduced (Price et al., 1977). This decrease in sorting time would be especially important for sediments like the mud/sand sediment used here, where sieving would result in retention of the majority of the sediments along with the bivalves and, thus, hand sorting and counting would be quite tedious and prone to error. These results show that these methods can be used in the field with the confidence to count recently settled clams in sediment samples involving a variety o f sediment types.  63  References  Bourne, N. 1982. Distribution, reproduction and growth of Manila clam, Tapes philippinarum (Adams and Reeve), in British Columbia. Journal of Shellfish Research, 2: 47-54. Bowen, R.A., J.M. St. Onge, J.B. Colton, and C A . Price. 1972. Density-gradient centrifugation as an aid to sorting planktonic organisms, I. Gradient Materials. Marine Biology, 14: 242247. Boyd, C. E., and C. S. Tucker. 1992. Water quality and pond soil analysis for aquaculture. Alabama Aqricultural Experiment Station, Auburn University. 183 pp. Burgess, R. 2001. An improved protocol for separating meiofaunafromsediments using colloidal silica sols. Marine Ecology Progress Series, 214:161-165. Denny, M.W. 1993. Air and water The biology and physics of life's media. Princeton University Press, Princeton New Jersey, 342 pp. Glock, J.W. 1978. Growth, recovery and movement of Manila clams, Venerupis japonica, planted under protective devices and on open beaches at Squaxin Island, Washington. Unpublished M. S. Thesis, Univ. Washington, Seattle. 69 pp. Heip, C , N. Smol, and W. Hautekiet. 1974. A rapid method of extracting meiobenthic nematodes and copepodsfrommud and detritus. Marine Biology, 28: 79-81. Jones, CR. 1974. Initial mortality and growth of hatchery-reared Manila clams, Venerupis japonica, planted in Puget Sound, Washington beaches. Unpublished M.S. thesis, Univ. Washington, Seattle. 90 pp. Jones, G. G., C L . Sanford, and B. L. Jones. 1993. Manila clam hatchery and nursery methods. Innovative Aquaculture Products Ltd. and Science Council of British Columbia. 73 pp. Jonge, V. N. de, and L. A. Bouwman. 1977. A simple density separation technique for quantitative isolation of meiobenthos using the colloidal silica Ludox®. Marine Biology, 42: 143-148. Jonsson, R., C. Andre and M. Lindegarth. 1991. Swimming behaviour of marine bivalve larvae in a flume boundary-layer flow: evidence for near bottom confinement. Marine Ecology Progress Series, 79: 67-76. Komar, P.D. 1998. Beach processes and sedimentation Second edition. Prentice Hall, New Jersey. 544 pp. Montaudouin, X de. 1997. Potential of bivalves' secondary settlement differs with species: a comparison between cockle (Cerastoderma edule) and clam (Ruditapes philippinarum) juvenile resuspension. Marine Biology, 128: 639-648.  64  N i c h o l s , J . A . 1979. A simple floatation technique for separating meibenthic nematodes from fine-grained sediments. Transactions o f the A m e r i c a n M i c r o s c o p i c Society, 9 8 : 127-130. P r i c e , C . A . , J . M . St. O n g e - B u r n s , J . B . C o u l t o n and J . E . Joyce. 1977. A u t o m a t i c sorting o f zooplankton b y isopycnic sedimentation i n gradients o f silica: Performance o f "rho spectrometer". M a r i n e B i o l o g y , 4 2 : 2 2 5 - 2 3 1 . P r i c e , C . A . , E . M . Reardon and R . R . L . G u i l l a r d . 1978. C o l l e c t i o n o f dinoflagellates and other marine microalgae by centrifugation in density gradients o f a m o d i f i e d silica s o l . L i m n o l o g y and Oceanography, 2 3 : 5 4 8 - 5 5 3 . Quayle, D . B . 1974. T h e intertidal bivalves o f B r i t i s h C o l u m b i a . B r i t i s h C o l u m b i a P r o v i n c i a l M u s e u m . H a n d b o o k N o . 17. Schwinghamer, P. 1981. Extraction o f l i v i n g meiofauna from marine sediments b y cetrifugation i n a s i l i c a sol-sorbitol mixture. Canadian Journal o f Fisheries and A q u a t i c Sciences, 3 8 : 4 7 6 - 4 7 8 . Utting, S . D . , and B . E. Spencer. 1991. T h e hatchery culture o f bivalve mollusc larvae and juveniles. L a b . L e a f l . N o . 6 8 . , M A F F F i s h . R e s . Lowenstoft. 31 p p . W i l l i a m s , J . G . 1978. T h e influence o f adults o n the settlement, growth, and survival o f spat i n the c o m m e r c i a l l y important clam, Tapes japonica Deshayes. U n p u b l i s h e d P h D . Thesis, U n i v . W a s h i n g t o n , Seattle. 59 pp. W i l l i a m s , J . G . 1980. G r o w t h and survival i n n e w l y settled spat o f the M a n i l a c l a m , Tapes japonica. Fisheries B u l l e t i n , 7 7 : 8 9 1 - 9 0 0 .  65  CHAPTER 3: The effect of netting on intertidal sedimentation " Introduction Although clam culture dates back several centuries in China (Pillay, 1993), recent developments in hatchery technology and grow-out methods have increased production substantially. Average annual growth in the worldwide production of clams and cockles in 2002 was 14.1%, increasing from 2.63 million tonnes in 2000, to 3.43 million tonnes in 2002 (FAO, 2004). In British Columbia, shellfish aquaculture production totalled 9,324 tonnes in 2004; 17% of this production consisted of farmed clams (Statistics Canada, 2005). Baynes Sound, located between Denman Island and Vancouver Island in southern British Columbia, Canada, is a highly productive area with large gravel and sand intertidal zones, ideal for clam culture. For almost a century, shellfish have been an important part of the local economy, and nearly one half of the cultured clams and oysters grown in British Columbia come from farms in this region (Ministry of Sustainable Resource Management, 2002). Of the total intertidal area in Baynes Sound, it was estimated that 32% was under tenure for beach culture and 4.9% was covered with clam netting in 2002 (Ministry of Sustainable Resource Management, 2002). The swift growth of this industry has increased awareness of possible risk posed to the environment by shellfish aquaculture such as local phytoplankton depletion (Ogilvie et al., 2000; Zhou et al., 2006), increased biodeposition (Dahlback and Gunnarsson, 1981; Baudinet et al., 1990; Bartoli, et al., 2001; Jie et al., 2001) and ecosystem changes (Inglis and Gust, 2003; Beadman et a l , 2004). Grow-out of clams is performed on suitable intertidal areas. Intertidal soft-bottom ecosystems are dynamic and subject to a wide range of environmental conditions that create habitats for a vast diversity of species (Lenihan and Micheli, 2001). Although, many * A version of this chapter has been submitted for publication in: Munroe, D. M . and R. S. McKinley. (2006) Commercial Manila clam (Tapesphilippinarum) tenures in British Columbia, Canada: the effects of anti-predator netting on intertidal sediment characteristics. Estuarine, Coastal and Shelf Science. Submitted May 2006. 66  studies have examined the environmental impacts o f bivalve culture, most have focused on oyster and mussel cultivation (Dahlback and Gunnarsson, 1981; Kaspar et al., 1985; Castel et al., 1989; Baudinet et al., 1990; Chamberlain et al., 2001; Caldow et a l , 2003; Crawford et al., 2003; Mazouni, 2004; Harstein and Stevens, 2005) while fewer have examined how clam culture affects intertidal systems (Spencer et al., 1996; Bartoli et al., 2001; J i e et al., 2001). During grow-out, intertidal clams are vulnerable to predators such as moon snails (eg. Euspira lewisii - Toba et al., 1992) crabs (eg. Cancer productus - Quayle and Bourne, 1972) and diving ducks (eg. Melanitta deglani, M. perspincillata andM. nigra - Bourne, 1984). To reduce mortality by predation, nets are placed on the surface of the sediment over the small seed clams (Spencer et a l , 1992). This practice significantly decreased mortality o f small clams (10.4-34 mm shell length) in estuaries in N W Spain (Cigarria and Fernandez, 2000), and in Maine, U S A , Beal and Kraus (2002) found that netting enhanced collection o f wild Mya arenaria spat, resulting from decreased predation and/or increased spatfall. Nets were also proven effective protection from crab and fish predation on beaches in Washington (Anderson, 1982). In addition to protecting clams from predation, nets have the potential to stabilize sediments, interfere with local hydrodynamic processes, and increase silt and organic matter deposition. This could lead to changes in sediment characteristics that are important to the benthos, and nutrient and gas exchange at the sediment-water interface (Driscoll, 1975, Bartoli et al., 2001). Nugues et al. (1996) noted small changes in the benthic community associated with increases in organic carbon and silt content of sediments beneath intertidal oyster trestles in the River Exe estuary, England. The trestles altered water flow in the immediate area around the cultivation site resulting in a decrease in the depth of the oxygenated sediment layer and reduced abundance o f benthos below culture structures. Netting applied to the sediment surface could alter the bottom roughness and alter the sediment properties below. I propose that the netting 67  could cause increased levels o f silt and organic matter beneath the netting and in this chapter I examine these effects and test this prediction.  Materials and Methods  Site F o u r active M a n i l a c l a m (Venerupisphilippinarum)  aquaculture sites were selected  w i t h i n the B a y n e s Sound area o n the east coast o f V a n c o u v e r Island, B C , Canada (Figure 3-1 sites are called B e a c h 1-4 as labelled i n the figure). These beaches are t y p i c a l l y seeded w i t h clams ( 5 - 1 0 m m shell length at a density o f approximately 4 0 0 individuals per m ) each year and 2  are continuously harvested as stock grows into harvestable sizes (>38 m m shell length), although practices vary slightly in relation to beach and operational circumstances. A t each beach, measurements were made o n paired netted and non-netted plots located directly adjacent to one another. Adjacent paired plots were used to m i n i m i s e the influence o f beach to beach variability. A t a l l four beaches, the nets (mesh size 2 c m x 2 cm) were positioned b y shellfish growers as part o f regular farm practice prior to initiation o f sampling and in a l l cases the netting had been i n place for at least 1 year p r i o r to initiation o f sampling. I n B r i t i s h C o l u m b i a it is c o m m o n for netting to be left i n place year round unless significant fouling requires nets to be removed. N o n e o f the nets used i n this study experienced notable f o u l i n g . Experimental plots were treated as part o f regular f a r m practices as outlined above and the beaches used were representative o f the farmed plots in B a y n e s Sound. E a c h o f the four beaches used was approximately 0.2 hectares (thus each plot was approximately 0.1 hectare) and had a l o w slope w i t h sandy/cobble beach substrate. N o samples were taken w i t h i n 1 meter o f the edges o f the designated beach area to m i n i m i z e edge effects. Characteristics including latitude and longitude, tidal height, slope and aspect o f each site are  68  summarised in Table 3 - 1 . A l l sampling (described in subsequent sections) was done during daytime l o w tide (spring tides) in 2003 and repeated again in 2004.  V  1  V  Den man  \  Island Vancouver  vancoiPer~-^X. IsJand  y  Island  -  ^—^—^  w v ^rr,-  ',/*N  \i_r[Vancouver  Baynes Sound  ^"  |  Figure 3-1: Map of beach sampling sites within Baynes Sound. Each beach is marked with number and labelled with site name. Inset top right: Location of Vancouver Island within Canada. Inset bottom left: Location of Baynes Sound on Vancouver Island, British Columbia, Canada.  Table 3-1: Site characteristics (tidal height is reported at meters above chart datum). Character  Beach 1  Beach 2  Beach 3  Beach 4  Latitude  N49°30'55.2"  N49°31'16.1"  N49°27'30.9"  N49°27'24.6  Longitude  W124°49'28.1"  W124°49'31.2"  W124°44'50.1"  W124°44'37.0"  T i d a l Height (m)  2  2.4  1.8  2.5  Slope  1.0%  1.3%  1.3%  2.2 %  Aspect  Southeast  Northeast  Northeast  North  M  69  Clam Populations C l a m populations were sampled at each beach, and each plot w i t h i n each site o n 10-13 A u g u s t 2003 and 2 6 - 2 9 August 2004. Sixteen large core (15 c m diameter by 15 c m depth) samples were randomly taken at each plot. A l l cores were sieved to 1 m m in situ and clams retained o n the sieve were counted and shell length measured to the nearest m m using vernier callipers. The majority o f clams observed were V. philippinarum  although small numbers o f  Nuttalia obscurata ( V a r n i s h clam) were also observed; these t w o species w i l l be reported here since they were the only t w o species to occur in high enough densities to be examined. C l a m density (mean individuals/m ) was calculated for each plot in both years (n=16). 2  These mean densities were used in paired t-tests (netted and non-netted plot o n each beach was paired) to compare V. philippinarum  and N. obscurata density between netted and non-netted  plots. N o r m a l i t y o f the difference between the paired data and correlation o f the pairs was tested; results o f tests are shown in Table 3 - 2 . Density differences o f Venerupis philippinarum were n o r m a l l y distributed (p=0.64), however, N. obscurata differences were not normal (p=0.016) therefore a log-transformation was done to normalize the data (p=0.055). L e n g t h frequency  o f V. philippinarum  (the dominant species) was tabulated and tests for normality  showed that the data were not n o r m a l l y distributed therefore non-parametric tests were used to compare the length distribution by beach (4 groups, therefore a K r u s k a l W a l l i s test was used), net (2 groups, tested w i t h K o l m o g o r o v - Smirnov test) and year (2 groups, tested w i t h K o l m o g o r o v - S m i r n o v test).  Sediment Grain Size W i t h i n each plot, six core samples (5 c m diameter by 1 c m depth) were taken o n 7-8 September 2003 and 9 - 1 0 September 2004 then stored frozen in labelled plastic bags. F o r analysis, samples were thawed then dried i n a d r y i n g o v e n f o r 2 4 hours at 60°C and  70  subsequently homogenized by hand w i t h a mortar and pestle to break up particles aggregated by the d r y i n g process. D r i e d , homogenised samples were placed i n the top o f a sieve stack sequentially containing sieves w i t h mesh 2 m m , 1 m m , 0 . 5 m m , 0 . 2 5 m m , 0 . 1 2 5 m m , 0 . 0 6 3 m m , and a bottom p a n , f o l l o w i n g the geometric W e n t w o r t h grade (Buchanan, 1984). The stack was placed in a n automated sieve shaker and agitated f o r 15 minutes. Sediment retained o n each sieve was weighed o n an analytical balance.  Table 3-2: Results of tests of assumptions for paired T-test. Normality tested on the distribution of the difference between pairs, correlation calculated for linear regression of pairs. N  N o r m a l i t y test result (p)**  Correlation ( R )  8  0.64  0.002  Nuttalia obsurata density*  8  0.055  0.703  % Silt  8  0.507  0.512  % Gravel  8  0.825  0.087  % Inorganic carbon  8  0.452  0.356  % Organic carbon  8  0.553  0.813  Pair Tested  Venerupis philippinarum density  2  *data were normalised with log transformation ** Shapiro - Wilk test  D a t a f r o m the six cores were pooled to avoid pseudoreplication (Hurlburt, 1984) resulting in t w o paired values (one f r o m the netted plot, the other f r o m the non-netted plot) f o r % silt and % gravel for each combination o f beach and year (n=8). The mean o f the six cores from each plot was used in a paired T-test to compare the percentage silt (<0.063mm) and percentage gravel (>2mm) o n netted and non-netted plots. F o r proper application o f a paired T test, the difference o f the t w o paired values must be normally distributed and the pairs must be correlated. Results o f tests for normality o f the difference o f pairs and correlation ( R ) are listed 2  in Table 3 - 2 .  71  Carbon Cores were taken in the same manner described for sediment grain size above. T h a w e d samples were dried in a d r y i n g oven for 24 hours at 60°C then m i l l e d to <0.063mm in a S w i n g M i l l (Rocklabs L i m i t e d , A u c k l a n d , N e w Zealand). The methods described below for measurement o f organic and inorganic carbon were tested to ensure accuracy w h e n employed o n intertidal sediments. The methods were compared to t w o other c o m m o n l y used carbon assessment methods and the results o f these tests can be found i n A p p e n d i x 2. T o measure inorganic carbon, the m i l l e d sample w a s stirred to ensure complete m i x i n g and a 30 m g subsample placed i n sample tubes i n a coulometer. The air lines o f the coulometer system were purged for 1 minute to eliminate contamination by atmospheric CO2. Subsequently, the sample w a s injected w i t h 2 0 % H C L w i t h a C M 5 1 3 0 A c i d i f i c a t i o n M o d u l e and the CO2 gas evolved from the sample titrated using a C M 5 0 1 4 Coulometer, U I C Inc. ( H u f f m a n , 1977). Once the titration endpoint was reached, the concentration o f inorganic carbon i n the sample w a s recorded. Measurement o f total carbon was achieved by flash combustion o f a 2 0 m g sample o f m i l l e d sediment prior to elemental analysis using a C a r l o E r b a N A - 1 5 0 0 A n a l y z e r f o l l o w i n g the methods outlined in V e r a r d o et a l . (1990). Organic carbon w a s calculated by subtracting inorganic carbon from total carbon values obtained above. Comparisons o f organic and inorganic carbon at netted and non-netted plots were again tested using pooled data in a paired T-test as described in the previous sections. Results o f normality and correlation tests are listed in Table 3 - 2 .  Temperature Temperature was monitored at each plot using B o x c a r ® Tidbit temperature loggers. E a c h logger was attached to a 10 c m long spike that was pushed into the sediment at each plot 7 2  leaving the data logger flush w i t h the sediment surface. O n netted plots they were placed flush w i t h the sediment surface beneath the netting 2 meters from edges o f the net to avoid possible edge effects. The loggers recorded temperature every minute for one h a l f o f a lunar cycle (2 weeks, n e w m o o n through f u l l moon) in early September 2005.  Results  Clam Populations L e n g t h frequencies for netted and non-netted plots are shown i n Figure 3 - 2 . It should be noted that the population o f V. philippinarum  i n both netted and non-netted plots derives from  both seeded clams (initially seeded beneath netting but m a y m o v e from nets to the adjacent n o n netted plot) and w i l d settlement from previous years. C l a m s (V. philippinarum  > 5 m m shell  length) from the netted sites measured 32 m m o n average (±8 m m S . D . ) . Non-netted plots contained slightly smaller clams o n average (mean=22 m m ±10 m m S . D . ) and demonstrated a more even length frequency distribution over size classes w i t h the exception o f B e a c h 2 i n 2004, w h i c h shows a h i g h frequency o f s m a l l clams. L e n g t h frequency distribution differed between netted and non-netted plots (pO.OOOOl), between beaches (p=<0.0001), and between years (p=0.004) at B o n f e r r o n i adjusted a=0.0167 (a=0.05 adjusted for 3 tests). Density o f Venerupis philippinarum  was significantly higher (p=0.001) in netted plots,  although at B e a c h 1 in 2003 and B e a c h 2 in 2004, there appears to be little difference (Figure 3 3). I failed to detect a difference (p=0.108) i n the density o f Nuttalia obscurata (>5mm shell length) between netted and non-netted plots (Figure 3-4).  73  Beach 1 2004  Beach 1 2003  • No Net • Netted  • No Net • Netted  Pp,,,P, ,«n|ll.[J,X  ill  l  18-1l  • No Net • Netted  16 14  J  Beach 2 2004  Beach 2 2003  20  ll  20 25 30 Size Class (mm)  20 25 : Size Class (mm)  §10  • M  20  20 18  Beach 3 2003  • No Net • Netted  16 14  16 14  • No Net • Netted  Beach 3 2004  &12 §10 • 8  110 • 8 *  20 16  &12  .III  6 4 2 0  lilt,  20 25 30 Size Class (mm)  25 30 Size Class (mm)  Anita  x) limn j, Hi 20  20  20 25 30 Size Class (mm)  25 30 Size Class (mm)  Beach 42004  Beach 4 2003  18  • No Net • Netted  16 14  taUi  : 1111! 11:  • No Net • Netted &12  £12 §10  §10  i .  ,TI,M  , .mil  20 25 30 Size Class (mm)  10  y  20 25 30 Size Class (mm)  Hi  Figure 3-2: Length frequencies (count) of Venerupis philippinarum (>5mm) from each site, 2003 in left column and 2004 in right. Clams measured from netted plots represented by black bars, clams from nonnetted plots represented by open bars. Shell length in mm plotted along the horizontal axis,frequencyon the vertical axis.  74  Figure 3-3: Mean number of Venerupis philippinarum (>5mm shell length) per m from sites in 2003 (left) and 2004 (right). Netted samples represented with hatched bars, non-netted plots represented with grey bars. Error bars represent 95% confidence interval. For each bar, n=16. 2  400 350  6 300 | 250 | 200  5 150 |  jafeu et  50 0  | NoNst  Nat  Beach 1  j NoNet  Beach 2  et  | NoNet  Beach 3  et  | No Net  Beach 4  Net  | NoNet  Beach 1  Net  \  ND  Beach 2  Net  Net  | NoNet  Beach 3  Net  | NoNet  Beach 4  Figure 3-4: Mean number of Nuttalia obscurata (>5mm shell length) per m from sites in 2003 (left) and 2004 (right). Netted samples represented with hatched bars, non-netted plots represented with grey bars. Error bars represent 95% confidence interval. For each bar, n= 16.  Sediment Grain  Size  Percentage o f silt (grain size <0.063mm) f r o m sediment samples for each site and plot is shown in Figure 3 - 5 . There was no significant difference detected between percentage o f silt between netted and non-netted plots (p=0.129). Percentage o f gravel (grain size >2mm) from samples at each site and plot is shown in Figure 3 - 6 ; percentage gravel w a s not significantly different between netted and non-netted plots (p=0.723).  75  25 2  (0  w  # 1  # 1  0.5  0.5  Net  I NoNet Beach 1  et  | NoNet  et  | NjNet  Beach 3  Beach 2  et  | NoNet  Beach 4  et  | NoNet  Beach 1  Net  | isbNet  Beach 2  Net  | to Net  Beach 3  Net  | NoNet  Beach 4  Figure 3-5: Percent silt (<0.063 mm grain size) content of samples from each site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, n=6 except for Beach 4 netted plot in 2004 where n=5. Error bars represent 9 5 % confidence interval.  2003  iC  50  o 40 j.30  20  m  Nohfet  Net  | NoNet Beach 2  Net  | No Met  Beach 3  Net  | NoNet  Be.ach 4  Figure 3-6: Percent gravel (>2mm grain size) content of samples from each site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, n=6. Error bars represent 9 5 % confidence interval.  Carbon The percentage o f inorganic carbon in sediments at each site and plot are shown in Figure 3 - 7 . Inorganic carbon was not significantly different between netted and non-netted plots at a=0.05 (p=0.07); however organic carbon (Figure 3-8) was significantly (p=0.014) higher at netted plots.  76  2004 £ 12  s ,  3  o  1  o 0.8  C 0.8 « ff 0.6  10.6 |  0.4  SS 0.2  J±L  Jfl et  | NoNet  Beach 1  et  | NoNet  Beach 2  krt  f N°Net  Beach 3  fit  | NoNet  Beach 4  Figure 3-7: Percent inorganic carbon content of samples from each site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, n=6 except for Beachl netted plot in 2004 (marked above bar with N=5) where n=5. Error bars represent 95% confidence interval.  a  0 6  S o.s  2. 0.5  I. < > • <  | 0.4 O 03 £ 02 0.1 kit  | NoNet  Beach 1  Net  | NoNet Beach 2  krt  | NoNet  Beach 3  kit  | NoNet  Net  No Net  Net  No Net  Net  No Net  Net  No Net  Beach 4  Figure 3-8: Percent organic carbon content of samples from each site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent nonnetted plots. For each bar, n=6. Error bars represent 95% confidence interval.  Temperature Temperature was measured over one tidal cycle during September 2005. Graphs o f temperatures measured over 24 hours o n the new m o o n , first quarter m o o n and f u l l m o o n (September 2, 9 and 16, respectively) at each beach and plot are shown in Figure 3 - 9 . The presence o f netting appeared to have no effect o n the water temperature. H o w e v e r during periods w h e n these plots were exposed by l o w tide the temperature at the sediment/air interface appeared to be affected by the presence o f netting. These periods correspond to times w h e n the tidal level drops below approximately 2 . 5 m (shown w i t h shaded vertical bar). The temperature at the sediment/air interface during the l o w tide events either drops during night time l o w tides, or increases during daytime l o w tides. The plots without netting experience w i d e r temperature extremes during l o w tides compared to plots w i t h nets.  77  40 35 30 25 20 15 10 5 40 35 30 25 20 15 10 5 40 35 30 25 20 15 10 5 40 35 30 25 20 15 10 5 —i—i—i—i—i—i—i—i  0:00  —i oo  4:00  •  •.  Full Moon - September 16 2005  First Quarter - September 9 2005  New Moon - September 2 2005  Beach 4  Beach 3  ID  —A—NoNet Netted  Beach 2  Beach 1  40 35 30 25 20 15 10 5 40 35 30 25 20 15 10 5 40 35 30 25 20 15 10 5 40 35 30 25 20 15 10 5  Beach 4  —*—NoNet - - - Q - - - Netted  Beach 2  1—i i i r  8:00 12:66 16:00 20:00  Iw^wlfvclfP?—  —  1—1—1  0:00  4:00  8:00  12:00 16:00 20:00  0:00  4:00  1  1  1  8:00 TitoO "1*6:00 20:00  Figure 3-9: Daily temperature measurements at the sediment/air interface, x-axis shows hour of the day. Grey boxes connected with dashed line show the temperature at netted plots, black triangles show non-netted plots and tidal exposure is shown with the shaded grey vertical band. Left column shows the new moon, center shows first quarter moon and right shows the full moon.  Discussion  Clam Populations The appearance o f greater densities o f large clams beneath nets may be the result o f higher survival due to the presence o f nets. Alternatively, the nets may provide substrate stabilisation or offer better feeding opportunities leading to faster growth or less emigration from beneath nets. These alternate hypotheses were not examined here. The length  frequency  distribution appears to indicate that there is a difference i n the population structure o f Venerupis philippinarum  between the netted and non-netted plots at B e a c h 2 i n 2 0 0 4 ; however, there was  no significant difference i n density o f V. philippinarum  (> 5 m m ) measured. The netted plot  contains mostly larger clams between 35 and 40 m m shell length, while the non-netted plot contains mostly smaller clams between 10 and 15 m m shell length. This may be a result o f movement o f seeded clams from adjacent areas either b y passive (for example b u l k transport) o r active (for example competition avoidance) mechanisms. V a r n i s h clams (N. obscurata > 5 m m shell length) appeared i n higher density at t w o o f the beaches o n non-netted plots. Currently the V a r n i s h c l a m is not a valued c o m m e r c i a l product i n B r i t i s h C o l u m b i a ; however, it has recently begun to be harvested from beaches. H i g h e r densities observed outside o f netted plots may be due to recent intense harvesting o f those clams o n netted plots that, unlike the M a n i l a c l a m , are not replaced b y seeding. The M a n i l a c l a m , Venerupis philippinarum,  is a suspension feeder and feeding m a y  cause biodeposition at sites o f high population density. Jie et al. (2001) calculated the mean clearance rate o f V. philippinarum  at 0.90 ± 0.34 L.hr" per individual and biodeposition rate at 1  0.06 g.hr" per individual (shell length not reported) and found that biodeposition rates at farm 1  sites (high population density) tended to be higher than n o n - f a r m sites. In contrast, K a n a y a et al. (2005) found no effect o f either M a n i l a clams or the facultative deposit feeder N. olivacia o n surface nitrogen, carbon or silt content to estuarine sediments i n Japan. T h i s study demonstrated  79  significantly higher densities o f V. philippinarum  (>5mm shell length) in the netted plots  compared to non-netted plots possibly leading to increased biodeposition at these plots. A t t w o sites, I found higher densities o f N. obscurata (>5mm shell length). N. obscurata is both a suspension feeder and deposit feeder and therefore has the potential to contribute to biodeposition o r reduction in carbon o n these non-netted plots depending o n the mode o f feeding.  Sediment Grain Size Placement o f netting o n intertidal plots has the potential to obstruct o v e r l y i n g water f l o w and thus cause sediment deposition and an increase i n silt beneath netting. In addition, higher densities o f filter feeding clams beneath netted plots may also alter sediment characteristics through biodeposition (Jie et a l . , 2001). H o w e v e r , no difference i n % silt or % gravel content i n samples f r o m netted and non-netted plots was observed i n this study. In contrast, M o j i c a and N e l s o n (1993) observed higher levels o f % silt i n sediments for the hard c l a m (Mercenaria mercenaria) at an intertidal g r o w - o u t site compared with t w o adjacent control sites; however, the authors only reported o n a single farm site that utilised an alternative farming method where the clams were placed in net bags o n the beach, rather than large nets over the substrate as was used here. In a manipulative experiment e x a m i n i n g the effects o f intertidal V. philippinarum  culture  in E n g l a n d , Spencer et a l . (1996) observed a four fold increase i n sedimentation o n netted plots compared to non-netted plots. In their study, sedimentation rate was measured using sediment traps. In addition to sedimentation rate, Spencer et a l . (1996) also measured the percentage o f silt i n sediments and found significantly lower silt (<63um) in the treatment plot with netting and clams compared to adjacent control plots, seemingly in contrast to the results seen for sedimentation. Goulletquer et al. (1999) also observed increased silt levels at a c l a m farm site using netting to protect V. philippinarum  f a r m plots i n France. T h e authors account the 80  significantly higher levels o f silt o n the netted plot to the increased sedimentation caused by netting.  Carbon I observed no significant effect o f netting or beach site o n inorganic carbon; however organic carbon was significantly higher in netted plots. Goulletquer et al. (1999) observed only a slightly significant difference in organic carbon i n sediments due to netting ataV. philippinarum rearing site in France. A d d i t i o n a l l y , D e G r a v e et a l . (1998) reported no increase in organic carbon at an intertidal site in Ireland where trestles were used for intertidal oyster seed production. Conversely, M o j i c a and N e l s o n (1993) and Spencer et a l . (1996) observed increased organic carbon i n samples taken at c l a m farm sites (both o n M. mercenaria bag culture and netted V. philippinarum culture) compared to adjacent control sites. H o w e v e r , the observed increase was attributed to high levels o f Enteromorpha f o u l i n g o n nets over the p e r i o d o f study. It appears that impacts o f intertidal farm practices o n carbon i n sediments are dependant o n site specific ecology and oceanography. F o r the locations observed here, it is l i k e l y that the elevated density o f V. philippinarum in netted plots contributed to increased organic carbon levels there through biodeposition (Jie et a l . , 2001). It is also possible that populations ofN. obscurata found in t w o o f the four non-netted plots (approximately 250 individuals per m ) 2  could have contributed to reduced organic carbon levels there b y deposit feeding. T h e factor o f netting is confounded w i t h the distribution o f c l a m populations and because c l a m density was not manipulated and crossed w i t h netting, I was unable to separate the influence o f the t w o .  Temperature There was no difference in water temperature between netted and non-netted plots during tidal i m m e r s i o n ; however I did observe a difference in temperature at the sediment/air interface between netted and non-netted plots during l o w tide events. T h e netted plots showed " b u f f e r e d " temperature changes during l o w tides b y up to 3°C. D u r i n g night l o w tides, the sediment/air 81  interface temperature reduction was less in netted plots compared to non-netted plots, and during day l o w tides, the sediment/air interface temperature increase was also lower at netted plots. This is likely due to water retention by nets thus creating insulation at the sediment/air interface. T o m y knowledge, this is the first observation o f such an effect o f netting in the literature.  Conclusions B a s e d o n this study, it appears that netting and c l a m f a r m i n g , as it is currently practiced in B a y n e s Sound B r i t i s h C o l u m b i a , has limited effects o n the sediment. I was unable to detect a significant impact o f netting o n levels o f silt, gravel or inorganic carbon in sediments. I did observe elevated levels o f organic carbon in netted plots relative to non-netted plots although this is likely due to distribution o f c l a m populations in the plots. The netting provides a temperature buffer during tidal exposure by up to 3°C but this effect is u n l i k e l y to be b i o l o g i c a l l y significant as most organisms are adapted to tolerate extreme temperature  fluxes  during tidal exposure. This study reports results that are inconsistent w i t h those reported elsewhere particularly w i t h reference to the effect o f netting o n siltation ( M o j i c a and N e l s o n , 1993; Spencer et a l . , 1996; D e G r a v e et a l . , 1998; Goulletquer et a l . , 1999). These discrepancies highlight the importance o f understanding the specific effects o f aquaculture practices w i t h i n the ecosystem in w h i c h they are being applied.  82  References A n d e r s o n , G . J . 1982. Intertidal culture o f the M a n i l a c l a m , Tapes philippinarum,  using large  netting enclosures in Puget Sound, Washington. M S c . Thesis, U n i v e r s i t y o f Washington, Seattle. 100pp. B a r t o l i , M . , D . N i z z o l i , P. V i a r o l i , E . T u r o l l a , G . Castaldelli, E . A . Fano, and R. R o s s i . 2 0 0 1 . Impact o f Tapes philippinarum farming o n nutrient dynamics and benthic respiration in the S a c c a d i G o r o . Hydrobiologia. 455:203-212. Baudinet, D . , E . A l l i o t , B . Berland, C . Grenz, M . P l a n t e - C u n y , R. Plante, and C . Salen-Picard. 1990 Incidence o f mussel culture o n biogeochemical fluxes at the sediment-water interface. H y d r o b i o l o g i a  2 0 7 : 187-196.  B e a d m a n , H . A . , M . J . K a i s e r , M . G a l a n i d i , R. Shucksmith, and R. I. W i l l o w s . 2004.  Changes  in species richness w i t h stocking density o f marine bivalves. Journal o f A p p l i e d E c o l o g y , 41:464-475. B e a l , B . F . , and G . M . K r a u s . 2002. 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Castel, J . , P. L a b o u r g , V . Escaravage, I. A u b y and M . E. G a r c i a . 1989. Influence o f seagrass beds and oyster parks o n the abundance and biomass patterns o f m e i o - and macrobenthos in tidal flats. Estuarine, Coastal and S h e l f Science, 2 8 : 7 1 - 8 5 . C h a m b e r l a i n , J . , T . F. Fernandes, P. R e a d , T . D . N i c k e l l and I. M . D a v i e s . 2 0 0 1 . Impacts o f biodeposits from suspended mussel (Mytilus edulis L.) culture o n the surrounding surficial sediments. I C E S Journal o f M a r i n e Science, 5 8 : 4 1 1 - 4 1 6 . C i g a r r i a , J , and J . M Fernandez. 2 0 0 0 . Management o f M a n i l a c l a m beds I. Influence o f seed size, type o f substratum and protection o n initial mortality. Aquaculture. 182:173-182. C r a w f o r d , C . M . , C . K . A . M a c l e o d , and I . M . M i t c h e l l . 2003. benthic environment. Aquaculture. D a h l b a c k , B . , and L . A . H . Gunnarsson.  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Effects o f different feeding habits o f three bivalve species o n sediment characteristics and benthic diatom abundance. M a r i n e E c o l o g y Progress Series, 2 9 9 : 6 7 - 7 8 . Kaspar, H . F., P . A . G i l l e s p i e , I. C . B o y e r , and A . L . M a c K e n z i e . 1985. Effects o f mussel aquaculture o n the nitrogen cycle and benthic communities i n K e n e p u r u Sound, M a r l b o r o u g h Sounds, N e w Zealand. M a r i n e B i o l o g y , 8 5 : 1 2 7 - 1 3 6 . L e n i h a n , H . S., and F. M i c h e l i . 2 0 0 1 . Soft-sediment communities. In: Bertness, M . D., S . D . Gaines and M . E . H a y (Eds.). M a r i n e c o m m u n i t y ecology. Sinauer Associates Inc., Sunderland Massachusetts, U S A . pp. 2 5 3 - 2 8 7 . M a z o u n i , N . 2004. Influence o f suspended oyster cultures o n nitrogen regeneration i n a coastal lagoon (Thau, France). M a r i n e E c o l o g y Progress Series, 2 7 6 : 1 0 3 - 1 1 3 . M i n i s t r y o f Sustainable Resource Management, B r i t i s h C o l u m b i a , Coast and M a r i n e P l a n n i n g B r a n c h . 2002. The Baynes Sound coastal p l a n for shellfish aquaculture. B r i t i s h C o l u m b i a M i n i s t r y o f Sustainable Resource Management. V i c t o r i a , Canada. 79 pp.  84  M o j i c a , R., and W . G . N e l s o n . 1993. Environmental effects o f a hard c l a m {Mercenaria mercenaria) aquaculture site in the Indian R i v e r L a g o o n , F l o r i d a . Aquaculture.  113:313-  329. Nugues, M . M . , M . J. K a i s e r , B . E . Spencer, and D . B . Edwards. 1996. Benthic c o m m u n i t y changes associated w i t h intertidal oyster cultivation. Aquaculture Research, 2 7 : 913-924. O g i l v i e , S. C , A . H . R o s s , and D . R. Schiel. 2000. Phytoplankton biomass associated with mussel farms in B e a t r i x B a y , N e w Zealand. Aquaculture 181: 7 1 - 8 0 . P i l l a y , T . V . R . 1993. Aquaculture: Principles and Practices. F i s h i n g N e w s B o o k s , O x f o r d . 575 pp. Quayle, D . B . and N . Bourne. 1972. The shellfisheries o f B r i t i s h C o l u m b i a . Fisheries Research B o a r d o f Canada B u l l e t i n , 179: 70 pp. Spencer, B . E. D . B . Edwards, and P. F. M i l l i c a n . 1992. Protecting M a n i l a c l a m (Tapes philippinarum) beds w i t h plastic netting. Aquaculture. 105: 2 5 1 - 2 6 8 . Spencer, B . E . , M . J . K a i s e r , and D . B . Edwards. 1996. The effect o f M a n i l a c l a m cultivation o n an intertidal benthic c o m m u n i t y : the early cultivation phase. Aquaculture Research, 2 7 : 261-276. Statistics Canada. 2005. Aquaculture statistics 2004. Catalogue no. 2 3 - 2 2 2 - X I E . M i n i s t r y o f Industry, Ottawa, Ontario, Canada. T o b a , D. R., D. S. Thompson, K. K . C h e w , G . J . A n d e r s o n and M . B . M i l l e r . 1992. Guide to M a n i l a C l a m Culture in Washington. Washington Sea Grant Program, U n i v e r s i t y o f Washington. 80 pp. V e r a r d o , D . J . , P. N . F r o e l i c h , and A . M c l n t y r e . 1990. Determination o f organic carbon and nitrogen in marine sediments using the Carlo E r b a N A - 1 5 0 0 A n a l y z e r . Deep Sea Research, 3 7 : 157-165. Z h o u , Y . , H . Y a n g , T. Z h a n g , P. Q i n , X . X u , and F. Z h a n g . 2006. Density-dependent effects o n seston dynamics and rates o f filtering and biodeposition o f the suspension-cultured scallop Chlamys farreri in a eutrophic bay (northern C h i n a ) : A n experimental study in s e m i - i n situ f l o w - t h r o u g h systems. Journal o f M a r i n e Systems, 5 9 : 1 4 3 - 158.  85  CHAPTER 4: Bivalve recruitment to culture plots* Introduction Supply-side ecology has been highlighted as an important determinant o f ecological communities ( L e w i n , 1986; Roughgarden et a l . , 1988; U n d e r w o o d and Fairweather, 1989; Y o u n g , 1990) and emphasis has been placed o n factors that determine settlement patterns o f larval life stages (Gaines and Roughgarden, 1985; K e o u g h , 1998; O l i v i e r et a l , 2 0 0 0 ; T o o n e n and P a w l i k , 2 0 0 1 ; H u a n g and H a d f i e l d , 2003). M a n y benthic invertebrates have c o m p l e x life histories i n v o l v i n g a pelagic larval stage that terminates w i t h settlement and metamorphosis into the benthic juvenile f o r m (Thorson, 1950). T o better understand larval i n f l u x as a factor i n c o m m u n i t y dynamics, it is important to also understand the conditions that dictate larval settlement and recruitment (Hadfield, 1998; C r i m a l d i et a l . , 2002). Current understanding maintains that both behaviour and p h y s i c a l processes influence recruitment patterns o f marine invertebrates (Butman, 1987; B u t m a n and Grasle, 1992; U n d e r w o o d and K e o u g h , 2 0 0 1 ; C r i m a l d i et a l . , 2002; Pernet et a l . , 2003). Large scale observations o f larval settlement tend to support the hypothesis that larvae are largely distributed passively ( B o u s f i e l d , 1955; Gaines and Bertness, 1992; B o r s a and M i l l e t , 1992; N o d a , 2004), w h i l e small scale observations tend to indicate behavioural control over settlement (Meadows and C a m p b e l l , 1972; W o o d i n , 1976; P a w l i k , 1992; R i t t s c h o f et a l . , 1998). D u e to substantial time investment, relatively f e w studies have examined initial settlement patterns to soft bottom intertidal systems in situ compared to rocky intertidal o r systems i n v o l v i n g attached juvenile and adult stages (Baggerman, 1953; Ishii et a l . , 2001), although some exceptions exist ( W i l l i a m s , 1978, 1980; Chicharo and C h i c h a r o , 2001b; * A version o f this chapter has been submitted for publication i n : M u n r o e , D . M . , and R . S . M c K i n l e y , 2006. Effect o f predator netting o n recruitment and growth o f w i l d c l a m (Tapes philippinarum) spat o n soft-bottom intertidal plots i n B r i t i s h C o l u m b i a , Canada. M a r i n e B i o l o g y - submitted M a y 2006. 86  Fraschetti et a l . , 2003). In addition, once sampled and counted, recently settled bivalves are extremely difficult to identify and f e w reliable taxonomic resources exist (Quayle, 1 9 5 1 ; L o o s a n o f f et a l . , 1966, Ishii, et a l . , 2001). Because o f the inherent challenges, the bulk o f larval behaviour research has been accomplished i n lab settings w h i c h can be argued to have limited relevance to the natural environment (Butman et a l . , 1988; E n g s t r o m and M a r i n e l l i , 2005). Settlement o f individual larvae is not synchronous and is therefore effectively not measurable o n the population level at any one point i n time w i t h the tools at our disposal currently. Measurement o f settlement is most often estimated by measuring recruitment, a process that incorporates settlement and post-settlement events like immigration, emigration and mortality ( K e o u g h and D o w n e s , 1982; Olafsson et a l . , 1994). A s time elapses between settlement and measurement o f recruitment, potential for erroneous estimation o f settlement grows (Pomerat and Reiner, 1942). T h e larvae o f the M a n i l a c l a m  (Venerupis philippinarum)  settle at approximately 220 p m ( H e l m and B o u r n e , 2004). Here, I have attempted to measure recruitment o f  V. philippinarum at a postlarval size (200-600 um) that w o u l d most closely  reflect the magnitude o f settlers to the benthos (Butman, 1987) and w i l l thus c a l l these "early recruits" i n an effort to reflect the discrepancy between settlement and recruitment. It has been suggested by many authors (Crisp, 1955, 1974; W i l l i a m s , 1978, 1980; N o w e l l and Jumars, 1984; E r t m a n and Jumars, 1988; E c k m a n , 1990; H a r v e y et a l , 1995; A b l e s o n and D e n n y , 1997; B o x s h a l l , 2 0 0 0 ; P e c h , at a l . , 2 0 0 2 ; C r i m a l d i et a l . , 2002) that sediment surface rugosity and related alteration o f near-bottom f l o w s can influence transmission o f chemical cues, physical contact with the bottom and ultimately retention o f settled larvae. In an experiment where needles were stuck into sediments to m i m i c the effect o f a n i m a l tubes o n f l o w , E c k m a n (1979) found increased recruitment o f a tanaid shrimp and a sabellid polychaete in the immediate vicinity o f the needles. In a similar experiment where sticks were used i n place o f needles, Gallagher et al. (1983) found that sticks facilitated larval settlement i n a number o f  87  species. T h e Japanese have been reported to use a variety o f methods to slow water currents and increase recruitment o f M a n i l a clams such as bamboo fences and p l a c i n g sticks vertically throughout the tidal flats (a procedure called "brushing") (Cahn, 1951). A n d finally, iron gauze screens placed perpendicular to intertidal f l o w created a "current shadow" o n study plots in the W a d d e n Sea w h i c h resulted in elevated levels o f cockle (Cardium edule) settlement (Baggerman, 1953). The practice o f c l a m farming i n many countries w o r l d w i d e involves applying large nets to intertidal surfaces suitable for c l a m production to protect valuable crops o f cultured clams f r o m predation (Spencer et a l . , 1992; T o b a et a l . , 1992; Spencer, 2002). U s e o f these nets has the potential to alter f l o w patterns near the sediment surface and thus influence recruitment patterns o f shellfish larvae to those areas (Heath et a l . , 1992; B e a l and K r a u s , 2002). A d u l t filter feeders have also been predicted to m o d i f y larval settlement through direct filtration  o f the potential recruits from the water c o l u m n ( W o o d i n , 1976; Lehane and Davenport,  2004) o r alteration o f near bed f l o w patterns ( N o w e l l and Jumars, 1984; E r t m a n and Jumars, 1988; Lindegarth et a l . , 2002). Correlation o f decreased larval settlement w i t h high adult filter feeder densities has been observed for populations o f V. philippinarum  ( W i l l i a m s , 1980), Mya  arenaria (Hines et a l . , 1989) and Cerastoderma edule (Andre and Rosenberg, 1991; A n d r e et a l . , 1993). H o w e v e r , other studies have shown no overall effect o f adult filter feeding populations o n settlement o f larvae (Maurer, 1983; Hunt et a l . , 1987; Thrush et a l . , 1996). In this study, I measured early recruitment o f c l a m larvae (V. philippinarum)  to farmed  plots w i t h and without netting and at differing levels o f c l a m (>5 m m shell length) density. It was m y intention to (1) determine i f netting alters recruitment patterns o f early post-larvae, and (2) examine the relationship between filter feeder density and early recruitment levels in the field.  88  Materials and Methods F o u r active M a n i l a c l a m (V. philippinarum)  aquaculture sites were selected within  Baynes Sound o n the east coast o f V a n c o u v e r Island, B C , Canada (Figure 3 - 1 ) . Baynes Sound is a highly productive area w i t h large gravel/sand intertidal regions ideal for c l a m culture. These sampling sites w i l l be referred to as beaches w i t h numbers assigned as shown in Figure 3 - 1 . E a c h beach was considered a b l o c k and netting was applied as a treatment to one half o f the beach (each beach was approximately 0.2 hectares). A t all four beaches, the nets had been applied by shellfish growers as part o f regular farm practice prior to initiation o f sampling. T h e beaches were large, l o w energy and l o w slope w i t h sand/cobble beach substrate, typical o f the Baynes Sound area. Characteristics such as latitude and longitude, tidal height, slope and aspect o f each site are listed in Table 3 - 1 . A t each plot (to w h i c h net o r no-net was applied) within each beach, sixteen s m a l l core samples (5 c m diameter by 1 c m depth) were taken at randomly determined locations by hand w i t h a plastic corer. S a m p l i n g was repeated in this manner o n four different daytime l o w tides (spring tides) each year between A u g u s t and October o f 2003 and 2004 to coincide w i t h peak V. philippinarum  larval settlement ( N e i l B o u r n e , D F O N a n a i m o , B r i t i s h C o l u m b i a , Canada,  personal c o m m u n i c a t i o n ; W i l l i a m s , 1978). E a c h sampling event was carried out over t w o consecutive days and the sampling dates in both 2003 and 2004 were as f o l l o w s : 10/11 August, 25/26 A u g u s t , 7/8 September, and 7/8 October. A l l samples were stored frozen (-20°C), then each was enumerated for recently settled clams (200-600 p m shell length) f o l l o w i n g the methods outlined i n M u n r o e et al. (2004). A l l clams counted were photographed using a Zeiss Stemi S V 11 dissecting microscope ( C a r l Zeiss Inc., Oberkochen, Germany) w i t h a C o o l S n a p Pro digital camera ( M e d i a Cybernetics Inc., Silver Springs, M a r y l a n d , U S A ) attached. These  89  images were used to measure shell length w i t h ImagePro Plus 4.5.1 image processing software ( M e d i a Cybernetics Inc., Silver Springs, M a r y l a n d , U S A ) . Sixteen samples from each plot were counted to compensate for the patchy nature o f larval settlement ( M u u s , 1973; H a l l et a l . , 1992) and the small size required for sample cores. These sixteen samples were averaged to avoid pseudoreplication (Hurlbert, 1984) resulting in one value for mean settlement for each combination o f beach-net-year-date (n=63 due to loss o f one data point prior to testing). The data were log-transformed to normalise the data prior to analysis. N o r m a l i t y was assessed using the S h a p i r o - W i l k test and homogeneity o f variances was assessed with L e v e n e ' s test. Settlement rates were compared using a univariate A N O V A w i t h a split-plot i n time model using S P S S statistical software. Sample dates prior to settlement ( l o w settlement rates) were considered "pre-settlement"; these were 10/11 August, 25/26 August, and 7/8 September in 2003 and 10/11 August and 25/26 A u g u s t i n 2004. Dates after the peak o f settlement were considered "post-settlement". Settlement trends demonstrating peak settlement events are shown in Figure 4 - 1 . In the A N O V A m o d e l , netting and year were considered fixed factors, settlement (pre-settlement and post-settlement as described above) was considered a covariate and beach was a block (therefore a random factor). Species identification at such early developmental stages is challenging and f e w keys exist to aid in identification ( L o o s a n o f f et a l . , 1966; LePennec, 1980; G o o d s e l l et a l . , 1992; Sakai and S e k i g u c h i , 1992; Evseev et a l . , 2001). D u e to time constraints, I did not identify a l l animals to species. H o w e v e r , random samples were examined for species composition and it was verified that > 9 5 % o f the clams were V. philippinarum. the beaches sampled were V. philippinarum,  The predominant populations o n  and sampling coincided w i t h k n o w n settlement  periods for this species, therefore it is highly probable that counts reflect the early recruitment o f  V. philippinarum. 90  Pre-Set | 2004  20000 18000  E i o  I Beach 1  16000  • Beach 2  14000  • Beach 3  12000 1  • Beach 4  10000  re  o  « o>  > <  8000 6000 4000 2000  10/11 Aug  Figure 4-1: Average rates of Venerupis philippinarum recruitment per m for each beach site (net and no-net combined). Grey circles and black line shows the average settlement rate for all sites combined. 10/11 August, 25/26 August and 7/8 September in 2003 and 10/11 August and 25/56 August in 2004 are considered "pre-settlement". 2  Results Density (early recruits/m ) o f early recruits was measured f o r each treatment plot at each 2  beach (Figure 4 - 2 ) . In 2003 the density ranged f r o m 509 - 748 early recruits/m and in 2004 2  from 4,396 - 5,720 early recruits/m ( 9 5 % C.I. for each year, a l l beaches, netted and non-netted 2  combined). Results o f the S h a p i r o - W i l k s test showed the post-settlement data were normal (p=0.296) however the pre-settlement data were not normal ( p - 0 . 0 1 ) and L e v e n e ' s test showed marginal equality o f variances (p=0.059). A n a l y s i s o f variance showed that year (p<0.001) was a significant factor i n determining recruitment, and netting w a s not significant at a=0.05 (p=0.061). The interaction o f year and net w a s also significant (p=0.037) indicating that in each year the effect o f netting o n settlement changed (Table 4 - 1 ) . T h e covariate, settlement status, was also highly significant in the m o d e l (p<0.00001).  91  Non-Netted Plots 2003  5000 A  E 4000  co  B Beach 1  E 4000  • Beach 2  ID  Beach 4  • Beach 2  £ 3000  Beach 4  a 2000  o 2000  u>  o £ >  E  o  < t  ^ 1000  10/11 Aug. 40000  H Beach 1 • Beach 3  Beach 3 £ 3000  Netted Plots 2003  5000  n  25/26 Aug.  7/8 Sept.  1000  10/11 Aug.  7/8 Oct. 40000 i  Non-Netted Plots 2004  25/26 Aug.  7/8 Sept.  7/8 Oct.  Netted Plots 2004  35000 f= 30000  IS Beach 1  o)  • Beach 2  g 25000 o  S Beach 3  £ >. 20000 •c ra  • Beach 4  0) <B 15000 o>  £  § 10000 5000 10/11 Aug.  25/26 Aug.  7/8 Sept  10/11 Aug.  7/8 Oct.  25/26 Aug.  7/8 Sept.  7/8 Oct  Sample Date Figure 4-2: Average Venerupis philippinarum early recruits per m for 2003 (upper panels) and 2004 (lower panels) counted on non-netted (left panels) and netted plots (right panels). Sample date shown on x-axis, error bars represent 95% confidence interval. Upper panels are shown with i finer scale than lower panels to allow data to be viewed more clearly. 2  Table 4-1: Results of A N O V A test of factors influencing Venerupis philippinarum settlement. Source  d.f.  SS  F  P  Beach  3  0.344  2.40  0.246  Net  1  0.394  8.25  0.064  Settlement  1  3.182  162.3  0.00000  Year  1  1.770  67.19  0.0001  Year x Net  1  0.191  7.15  0.037  Error  46  0.902  There were significantly more V. philippinarum  (>5 m m shell length) in netted plots  compared to non-netted plots and the length frequency structure o f the populations varied among plots and years. V. philippinarum  populations (clams w i t h shell length >5 m m ) were  sampled and measured for each plot in both 2003 and 2004 (see Chapter 3 for methods and results). I used length frequency data from the c l a m survey to estimate biomass densities o f filter feeders and compared early recruits to biomass density u s i n g linear regression (Figure 4 3). O n non-netted plots there was l o w biomass and a negative correlation between biomass and early recruit density; however i n netted plots there was higher biomass and a slightly positive relationship (Table 4 - 2 ) . There was no significant difference i n mean lengths o f early recruits found between beaches ( p O . O O O O l ) , years ( p O . O O O O l ) o r between netted and non-netted plots (p=0.0002) (Figure 4 - 4 ) . O n average, the early recruits counted measured 3 1 0 p m ( ± 9 6 , n=5465) i n length. A n a l y s i s o f length frequency distributions i n 2004 showed a larval settlement peak (mode) at a size o f 223 p m ( ± 6 9 , n=2828) o n date 3 w h i l e o n date 4 the peak has shifted to 382 nm ( ± 8 1 , n=1316) (Figure 4 - 5 ) . There were 30 days between date 3 and 4 and I therefore estimate growth rate o f early settlers at 5.25 p m shell length per day. H o w e v e r , I was unable to estimate immigration, emigration and mortality here, and therefore estimates o f growth may be  93  inaccurate. This estimated growth rate remained consistent between all 4 sites and d i d not differ w i t h the presence o f netting (Figure 4 - 5 ) . I was unable to estimate growth rate for 2003 because settlement occurred o n date 4 and no subsequent samples were taken.  35000  CM  A2003-NO Net  •  30000  • 2004-No Net A2003-Netted  25000 -  • 2004-Netted  E Early Recrulit Density  a  \  20000  Q  \  R = 0.2433 2  15000 -  a  10000  \  R •  R = 0.1606  5000  2  R = Q.4194 2  • •  n U  i <3  XA  i  I  1000  i  2000  3000  1  4000  A  l  i  5000  6000  1  7000  Tapes philippinamm Biomass (g/m ) 2  Figure 4-3: Relationship of early recruit (<0.5mm shell length) density to Venerupis philippinarum biomass (shell length >5mm). Each data point represents the average early recruit density for each beach. Data from 2003 are counts from date 4, thus representing peak initial settlement and are shown with triangle markers. Data from 2004 are counts from date 3, representing initial peak settlement and are shown with square markers. White markers indicate non-netted plots and black markers indicate netted plot values. The R value is shown on the graph next to the corresponding trend line. 2  Table 4-2: Results of linear regression of Venerupis philippinarum biomass versus larval settlement. Year 2003  2004  Net  R  Netted  0.419  0.4  NoNet  0.161  -3.4  Netted  0.58  1.5  NoNet  0.243  -16.6  2  Slope ( B )  94  Figure 4-4: Average Venerupis philippinarum early recruit length for each beach and plot within beach on the post-settlement date in 2003 (date 4 - shown on left) and 2004 (date 3 - shown on right). Hatched bars represent netted plots, black bars represent non-netted plots. Error bars represent 95% confidence interval. The number directly below each bar represents the sample size.  No Net  Net  Figure 4-5: Shell length frequencies of Venerupis philippinarum early recruits from 2004 samples. Date 3 shown with circles and solid line, date 4 shown with squares and hatched line. Non-netted plots are shown in the left column and netted plots shown on the right. Beach 3 and Beach 4 are shown with a smaller y-axis because of lower recruitment overall to those sites. Difference between the peaks of the solid line versus the hatched line is considered to represent growth of that cohort from date 3 to date 4.  96  Discussion M o d e l l i n g o f f l o w has been used to make predictions about larval supply to sites i n an effort to account for spatial variation in levels o f settlement (Borsa and M i l l e t , 1992; Chicharo and C h i c h a r o , 2001a; Lundquist et a l . , 2 0 0 4 ; A r n o l d et a l . , 2005). I observed higher densities o f early recruits at beaches 1 and 2 versus beaches 3 and 4. A report by H a y and C o m p a n y (2003) described the circulation patterns o f Baynes Sound in detail in an effort to determine shellfish carrying capacity. T h i s report indicated that flushing at the south end o f D e n m a n Island is greater than in the north end, therefore retention o f larvae at northern beaches (1 and 2) should be greater than at southern beaches (3 and 4), consistent w i t h m y observations. A l s o mean and rms (root mean square) currents from September 2002 (Hay and C o m p a n y , 2003) show that there is i n f l o w from the N o r t h and South end o f D e n m a n Island that may hold larvae and a l l o w greater settlement in B a y n e s Sound in general; perhaps resulting in higher recruitment there compared to other areas o f the coast (other areas were not measured in the present study). The Baynes Sound region contains higher population densities than other locations w h i c h w o u l d produce a greater local larval input m a k i n g this theory difficult to test. O v e r a l l settlement densities o f early recruits observed at these study beaches was on average slightly lower than densities observed in both A r i a k e Sound, Japan (Ishii et a l . , 2001) and Puget Sound, Washington ( W i l l i a m s , 1980). I observed settlement o f new recruits at densities o f 509 - 748 early recruits/m in 2003 and 4,396 - 5,720 early recruits/m in 2004 2  2  ( 9 5 % C.I.). In A r i a k e Sound, Japan, where the M a n i l a c l a m is w i t h i n its native range and is c o m m e r c i a l l y cultivated, Ishii et al. (2001) observed settlement rates o f 13,000 - 27,000 early recruits/m at two tidal flats. In Puget Sound, W a s h i n g t o n , where, as in B r i t i s h C o l u m b i a , the 2  M a n i l a c l a m was introduced nearly 80 years ago (Bourne, 1982), W i l l i a m s (1980) reported density o f M a n i l a c l a m settlement at 18,600 - 31,200 early recruits/m . I observed notably 2  97  higher densities o f settlers than C h i c a r o and C h i c a r o (2001b) from the R i a F o r m o s a L a g o o n i n Portugal. A f t e r observing densities o f early settlers for one year at one site, they reported 197 early recruits/m during the peak settlement event. 2  O u r results demonstrated that year had the largest effect o n variability in level o f settlement. T h e covariate, settlement status was also highly significant in the m o d e l (p>0.00001). G i v e n that settlement status represents whether settlement has occurred o r not, it is expected it to be important and thus included it as a covariate. A n n u a l variability was remarkable w i t h settlement density i n 2004 being a n order o f magnitude higher than i n 2003. W i l l i a m s (1978) also noted large annual variation i n settlement rates o f M a n i l a clams i n Puget Sound, Washington; he reported a large set i n 1976 but essentially none i n 1977. In a 7 year study o n Ruditapes philippinarum,  Musculista senhousia, and Nuttalia olivacea populations,  M i y a w a k i and S e k i g u c h i (1999) also reported large fluctuation i n annual rate o f early settlers to tidal flats in Japan. I also observed a marginally significant effect o f netting o n density o f early recruits, netted plots receiving fewer settlers than non-netted plots. A s noted earlier, this effect m a y be due to the interference o f netting w i t h benthic boundary layer flows carrying competent larvae to settlement sites. Such interference w i t h f l o w s c o u l d potentially also contribute to increased sedimentation and higher organic carbon beneath netting ( M o j i c a and N e l s o n , 1993; Spencer et a l . , 1996). In laboratory flume trials, B u t m a n et a l . (1988) tested settlement o f M. mercenaria larvae in still and f l o w i n g water and found greater retention o f larvae in still water o n glass beads compared w i t h m u d suggesting a preference for substrates w i t h lower organic matter. These results were, however, not supported i n further tests (Snelgrove et a l . , 1998). N o s h o and C h e w (1972) saw no difference i n distribution o f recently settled V. philippinarum  recruits (shell  length >0.5 m m , therefore larger than those observed in the present study) to plots w i t h different combinations o f sand, gravel and shell. I tested sediments from beaches used here for levels o f  98  silt and percent organic matter (see Chapter 3). These results showed that netting d i d not change the levels o f either on the study beaches and therefore conclude that silt and percent organic matter are unlikely to account for the settlement patterns observed here. N e t t i n g may increase turbulence at the sediment-water interface and alteration o f turbulence structure is k n o w n to influence larval settlement patterns ( A b l e s o n and D e n n y , 1997; B o x s h a l l , 2000). Fuchs et al. (2004) studied the sinking behaviour o f the gastropod larvae Ilyanassa obsoleta in a variety o f turbulent conditions. Their results demonstrated that the snail reacted to increased turbulence b y ceasing to s w i m and therefore may use turbulence as a cue for settlement. Pernet et a l . (2003) studied settlement o f mussel larvae (Mytilus spp.) i n downwellers and also found a positive correlation between turbulence and settlement. Pearce et al. (1998) observed the opposite effect w i t h scallop (Placopecten magellanicus) larval settlement; i n laboratory mesocosms, scallop larvae showed lower settlement w i t h increased turbulence. Here, I observed higher density o f recruits to non-netted plots where turbulence may be lower. Constant motion o f the nets as tidal f l o w s pass over them m a y create a more frequently disturbed sediment environment beneath the netting. It has recently been demonstrated that larvae o f some species are less l i k e l y to settle i n sediments that have been disturbed ( W o o d i n et a l . , 1995, 1998). M a r i n e l l i and W o o d i n (2004) used polychaete (Capitella sp.) and bivalve (M. mercenaria) early juveniles to test their response to disturbed and undisturbed sediments. Their tests showed that early juveniles avoided disturbed sediment and they attribute this to s m a l l scale geochemical changes associated w i t h sediment disturbance. In this study, I did not make small-scale measurements o f geochemical properties o f study sites; however, it is possible that the motion o f the netting during tidal f l u x could agitate the sediment b e l o w and create "disturbed" patches that are less suitable for settlement. Alternatively, it is possible that net m o t i o n causes smothering o r otherwise k i l l s early recruits thus influencing post-settlement  99  survival, I did not collect data concerning survival and therefore cannot evaluate these two possibilities. Further research to investigate this possibility is warranted. The interaction o f netting and year was also significant in the A N O V A m o d e l (p=0.037). H i g h e r recruitment observed in 2004 correlated w i t h a greater difference between numbers o f early recruits counted at netted and non-netted plots. T h i s is possibly a result o f differential survival at netted versus non-netted plots in years w i t h higher competition pressure (higher recruitment years). It may also be related to condition o f the larvae at the time o f settlement. Presumably, in a high recruitment year, larval survival in the plankton is high (evidenced by large numbers surviving to settlement) indicating adequate f o o d supply and generally better environmental conditions. If the larvae are arriving at settlement sites in better condition, they m a y be more selective in choice o f settlement site (Rittschof et a l . , 1984; Snelgorve et a l . , 1993). H i g h e r numbers o f filter feeders beneath netting o n the study beaches m a y lower the number o f recruits at those sites. M a n y studies have suggested that the presence o f filter feeders can decrease recruitment o f larvae through direct filtration o f the larvae from the o v e r l y i n g water ( W o o d i n , 1976; W i l l i a m s , 1980; M a u r e r , 1983; A m b r o s e , 1984; H i n e s et a l . , 1989; A n d r e and Rosenberg, 1991; B o r s a and M i l l e t , 1992; M i t c h e l l , 1992; A n d r e et a l . , 1993; Olafsson et a l . , 1994, B e u k e m a and Cadee, 1996; Lehane and Davenport, 2004).  W i l l i a m s (1980)  measured settlement o f c l a m larvae in relation to c l a m populations and found that dense populations decreased, but d i d not prevent larval settlement. H o w e v e r , results o f many field studies investigating the effect o f suspension feeders o n larval settlement have failed to support the hypothesis that there is a negative relationship between filter feeder density and larval settlement ( M a u r e r 1983; H u n t et a l , 1987; E r t m a n and Jumars, 1988; H i n e s , et al. 1989; Thrush et a l . , 1996). I found that at l o w filter feeder densities, here associated w i t h non-netted plots, there was a negative correlation between biomass and larval settlement. H o w e v e r , these data showed that w i t h higher density c l a m populations (around 450/m ), there is no effect, or even an 2  100  increase in larval settlement w i t h increases in c l a m biomass. D u e to the nature o f the sites I examined and limited replication, I lack sufficient data from intermediate levels o f c l a m biomass. Nevertheless, this is a yet unresolved result and may explain some o f the discrepancies in the literature. It is possible that at l o w densities o f filter feeders there is a decrease in larval settlement as predicted ( W o o d i n , 1976), but once filter feeder densities reach a certain threshold, the effect o n larval settlement changes and larvae are no longer hindered from settling or are even entrained as proposed by E r t m a n and Jumars (1988). Neither the presence o f netting nor extremely high c l a m density prevented the recruitment o f bivalve larvae completely, and in plots w i t h both nets and high numbers o f clams there remained a strong level o f recruitment o f bivalves. M i y a w a k i and S e k i g u c h i (2000) found that density o f early recruits did not influence the success o f that cohort for survival; however, they d i d find a positive relationship between density o f early recruits and density o f clams o f that cohort. In other words, a large settlement did not necessarily mean that cohort would survive, but w h e n it did survive it tended to g r o w into a higher density group. Thus, I can not expect that higher recruitment to non-netted plots corresponds w i t h increased survival o f that cohort compared to netted plots. A c c o r d i n g to M i y a w a k i and S e k i g u c h i (2000), the success o f the cohort is most closely related to "unpredictable environmental disturbances". In this study I d i d not measure survival o f the early recruits beyond the four sample dates discussed and therefore can not make predictions o n survival rates through the periods o f high mortality noted by others (Gosselin and Q i a n , 1997; Stoner, 1990; M i y a w a k i and S e k i g u c h i , 2 0 0 0 ; N a b u et a l . , 2005). C l a m length is often omitted in studies concerning settlement and recruitment meaning that those studies cannot be accurately placed in the context o f early post-larval development or compared to other research. H i g h levels o f mortality immediately f o l l o w i n g larval settlement can q u i c k l y change recruitment patterns (Stoner, 1990; G o s s e l i n and Q i a n , 1997; Snelgrove, 101  1999; M i y a w a k i and S e k i g u c h i , 2 0 0 0 ; N a b u et a l . , 2005). The early recruits considered here a l l measured w i t h i n the range o f early post-settlement (mean length = 310 pm) in an effort to measure recruitment as close i n size to settlement and m i n i m i z e in influence o f predation. G r o w t h rate o f early settlers has rarely been measured in the field. I used length frequency o f early settlers to estimate growth rate in the field post-settlement. B a s e d o n the difference between the peaks from subsequent sampling dates o f the length frequency plot, I estimated a growth rate o f 5 pm/day averaged over the first 30 days o f benthic life. This rate can be compared w i t h growth under optimal hatchery conditions o f 10 - 12 pm/day (Utting and Spencer, 1991) for the same postsettlement stage. D a t a f r o m B e a c h 1 in 2002 also allowed estimation o f g r o w t h rates under field conditions in the same manner noted above (see A p p e n d i x 3 for summary o f 2002 data). F r o m 2002 data, larval settlement occurred approximately one m o n t h earlier and the growth rate was estimated to be 7.6 pm/day.  Conclusions These results demonstrated that intertidal recruitment o f clams varies both spatially and annually. I observed an order o f magnitude difference in density o f settlers between 2003 and 2004. I also noted a large amount o f variability in t i m i n g and density o f settlement in one year compared to another; density also varied spatially. C o l l e c t i v e l y , this highlights the importance for future studies concerning settlement patterns o f bivalves to utilize adequate replicate sites, multiple years and replicate samples to ensure an accurate reflection o f the overall patterns o f settlement. The presence o f nets and high densities o f clams correlated w i t h lower settlement o f bivalve larvae. Because c l a m abundances were not manipulated it is impossible to separate the effect o f the nets and the filter feeders, however, it appears that both lead to lower levels o f settlement to intertidal plots. These results confirmed that increasing densities o f c l a m biomass 102  correlated w i t h lower densities o f early recruits, however that relationship d i d not hold at higher densities suggesting that the relationship is more complicated than first predicted, particularly in dense populations. Netting and filter feeders d i d not have any effect o n the t i m i n g o f settlement, the size at settlement or the growth rate observed for early recruits indicating that these environments were not unsuitable for settlement. It continues to be important to observe larval settlement patterns in situ and understand the factors that influence recruitment success. Here, I demonstrated that annual and spatial variability are high in bivalve larval settlement patterns, but that netting and c l a m populations also alter the level o f recruitment. 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Bourget, and D . Rittschof. 2000. Barnacle settlement: field experiments o n the influence o f larval supply, tidal level, b i o f i l m quality and age o n Balanus amphitrite cyprids. M a r i n e E c o l o g y Progress Series, 199: 185-204. P a w l i k , J . R. 1992. C h e m i c a l ecology o f the settlement o f benthic marine invertebrates. Oceanography and M a r i n e B i o l o g y A n n u a l R e v i e w , 3 0 : 2 7 3 - 3 3 5 . Pearce, C . M . , S . M . Gallager, J . C . M a n u e l , D . A . M a n n i n g , R . K . O ' D o r , and E . Bouget. 1998. Effect o f thermoclines and turbulence o n larval settlement and spat recruitment o f giant scallop, Placopecten magellanicus, i n 9.5 m deep laboratory mesocosms. M a r i n e E c o l o g y Progress Series, 165: 145-215. P e c h , D . , P. L . A r d i s s o n , and E . Bourget. 2002. Settlement o f a tropical marine epibenthic assemblage o n artificial panels: Influence o f substratum heterogeneity and c o m p l e x i t y scales. Estuarine, Coastal and S h e l f Sciences, 5 5 : 7 4 3 - 7 5 0 . 108  Pemet, F., R. Tremblay and E. Bourget. 2003. Settlement success, spatial pattern and behavior o f mussel larvae Mytilus spp. in experimental ' d o w n w e l l i n g ' systems o f varying velocity and turbulence. M a r i n e E c o l o g y Progress Series, 2 6 0 : 1 2 5 - 1 4 0 . Pomerat, C . M . , and E. R. Reiner. 1942. The influence o f surface angle and o f light o n the attachment o f barnacles and other sedentary organisms. B i o l o g i c a l B u l l e t i n , 82: 14-25. Quayle, D . B . 1951. Structure and biology o f the larvae and spat o f Venerupis pullustra (Montagu). Transactions o f the R o y a l Society o f E d i n b u r g h , 6 2 : 2 5 5 - 2 9 7 . Rittschof, D., E. S. B r a n s c o m b and J . D . C o s t l o w . 1984. Settlement and behavior in relation to f l o w and surface in larval barnacles, Balanus amphitrite D a r w i n . Journal o f Experimental M a r i n e B i o l o g y and E c o l o g y . 8 2 : 1 3 1 - 1 4 6 . Rittschof, D., R. B . F o r w a r d , G . C a n n o n , J . M . W e l c h , M . M c C l a r y , E. R. H o l m , A . S . Clare, S. C o n o v a , L. M . M c K e l v e y , P. B r y a n , and C . L . V a n D o v e r . 1998. Cues and context: L a r v a l responses to physical and c h e m i c a l cues. B i o f o u l i n g , 12: 3 1 - 4 4 . Roughgarden, J . , S . Gaines, and H . Possingham. 1988. Recruitment dynamics in complex life cycles. Science, 2 4 1 : 1460-1466. Sakai, A . , and S e k i g u c h i , H . 1992. Identification o f planktonic late-stage larval and settled bivalves in a tidal flat. B u l l e t i n o f the Japanese Society Fisheries and Oceanography, 56: 410-425. Snelgrove, P. V . R  1999. Getting to the bottom o f marine biodiversity: Sedimentary habitats.  B i o s c i e n c e , 4 9 : 129-138. Snelgrove, P. V . R , C . A . B u t m a n , and J . P. Grassle. 1993. H y d r o d y n a m i c enhancement o f larval settlement in the bivalve Mulinia lareralis (Say) and the polychaete Capitella sp. I in microdepositional environments. Journal o f E x p e r i m e n t a l M a r i n e B i o l o g y and Ecology., 168:71-109. Snelgrove, P. V . R , J . P. Grassle, and C . A . B u t m a n . 1998. Sediment choice by settling larvae o f the bivalve, Spisula solidissima ( D i l l w y n ) , in f l o w and still water. Journal o f Experimental M a r i n e B i o l o g y and E c o l o g y , 2 3 : 171-190. Spencer, B . E., 2002. M o l l u s c a n shellfish f a r m i n g . B l a c k w e l l P u b l i s h i n g L T D . O x f o r d , E n g l a n d . 272 pp. Spencer, B . E. D . B . Edwards, and P. F. M i l l i c a n . 1992. Protecting M a n i l a c l a m (Tapes philippinarum) beds w i t h plastic netting. Aquaculture, 105: 2 5 1 - 2 6 8 . Spencer, B . E . , M . J . K a i s e r , and D . B . E d w a r d s . 1996. The effect o f M a n i l a c l a m cultivation o n an intertidal benthic c o m m u n i t y : the early cultivation phase. Aquaculture Research, 2 7 : 261-276.  109  Stoner, D . S . , 1990. Recruitment o f a tropical ascidian: relative importance o f pre-settlement versus post-settlement processes. E c o l o g y , 7 1 : 1 6 8 2 - 1 6 9 0 . Thorson, G . 1950. Reproductive and larval ecology o f marine bottom invertebrates. B i o l o g i c a l R e v i e w s , 2 5 : 1-45. Thrush, S . F., J . E . Hewitt, R. D . Pridmore, and V . J . C u m m i n g s . 1996. Adult/juvenile interactions o f infaunal bivalves: contrasting outcomes in different habitats. M a r i n e E c o l o g y Progress Series, 132: 8 3 - 9 2 . T o b a , D . R., D . S. T h o m p s o n , K . K . C h e w , G . J . A n d e r s o n , and M . B . M i l l e r . 1992. Guide to M a n i l a C l a m Culture in Washington. Washington Sea Grant Publications 9 2 - 0 0 1 . U n i v e r s i t y o f Washington. 80 p p . T o o n e n , R . J . and J . R . P a w l i k . 2 0 0 1 . Settlement o f the gregarious tube w o r m Hydroides dianthus (Polychaeta : Serpulidae). I. Gregarious and nongregarious settlement. M a r i n e E c o l o g y Progress Series, 2 2 4 : 103-114. U n d e r w o o d , A . J . , and P. G . Fairweather. 1989. Supply-side ecology and benthic marine assemblages. Trends in E c o l o g y and E v o l u t i o n , 4: 16-20. U n d e r w o o d , A . , and K . K e o u g h . 2 0 0 1 . Supply-side ecology: The nature and consequences o f variations in recruitment o f intertidal organisms. In: M . Bertness, S . Gaines, and M . H a y (Eds.) M a r i n e C o m m u n i t y E c o l o g y . Sinnauer Associates Inc. Sunderland, Massachusetts, pp. 183-200. Utting, S . D . , and B . E . Spencer. 1991. The hatchery culture o f bivalve m o l l u s c larvae and juveniles. L a b . L e a f l . N o . 6 8 . , M A F F F i s h . R e s . Lowenstoft. 31pp. W i l l i a m s , J . G . 1978. The influence o f adults on the settlement, growth and survival o f spat i n the c o m m e r c i a l l y important clams, Tapes japonica (Deshayes). P h D . Thesis, U n i v e r s i t y o f Washington. W i l l i a m s , J . G . 1980. The influence o f adults o n the settlement o f spat o f the c l a m , Tapes japonica. Journal o f M a r i n e Research, 3 8 : 7 2 9 - 7 4 1 . W o o d i n , S . A . 1976. A d u l t - l a r v a l interactions in dense infaunal assemblages: Patterns o f abundance. Journal o f M a r i n e Research, 3 4 : 2 5 - 4 1 . W o o d i n , S . A . , S . M . L i n d s a y and D . S . Wethey. 1995. Process-specific recruitment cues i n marine sedimentary systems. B i o l o g i c a l B u l l e t i n , 189: 4 9 - 5 8 . W o o d i n , S . A . , R . L . M a r i n e l l i and S . M . L i n d s a y . 1998. Process-specific cues for recruitment i n sedimentary environments: G e o c h e m i c a l signals? Journal o f M a r i n e Research, 56: 5 3 5 558. Y o u n g , C . M . 1990. L a r v a l e c o l o g y o f marine invertebrates - A sesquicentennial history. O p h e l i a , 3 2 : 1-48.  110  CHAPTER 5: Settlement of larvae in experimental flumes'  Introduction Settlement patterns o f invertebrate larvae are a basis o f marine c o m m u n i t y dynamics (Gaines and Roughgarden, 1985; H a d f i e l d , 1998). T h e cues involved in larval settlement are poorly understood and have become the focus o f many studies (reviewed b y : B u t m a n , 1987; P a w l i k , 1992; Q i a n , 1999). M a n y factors, both b i o l o g i c a l and p h y s i c a l , have been shown to influence settlement i n a variety o f species including cues f r o m conspecifics (Knight-Jones, 1953; P a w l i k and B u t m a n , 1993; T o o n e n and P a w l i k , 1994; Turner et a l , 1994), prey ( W i l l i a m s o n , et a l . , 2 0 0 0 ; H a d f i e l d and P a u l , 2001), sediment chemistry (Butman et a l . , 1988; E n g s t r o m and M a r i n e l l i ; 2005), p h y s i c a l relief o f the bottom (Wethey, 1986; Gregoire et a l . , 1996; K o h l e r , et a l , 1999) and turbulence structure o f the water c o l u m n (Crisp, 1955; A b l e s o n and D e n n y , 1997; B o x s h a l l , 2 0 0 0 ; Pernet, et a l . , 2 0 0 3 ; F u c h s et a l . , 2004). F l o w structure was highlighted by A b l e s o n and D e n n y (1997) as influencing settlement in a number o f ways. The authors note that flow can be a settlement cue unto itself, it can help to mediate other settlement cues in the water by distributing them, and it can help place larvae i n p h y s i c a l contact with a surface. H i g h resolution measurements made w i t h i n the benthic boundary layer above smooth and rough bottoms have shown that even small changes in roughness height (1.5 m m ) can invoke distinct differences i n small scale turbulence structure (Shafi and A n t o n i a , 1997; P o g g i et a l . , 2 0 0 3 ; H e n d r i k s et a l . , 2006). Others have shown differences in larval settlement in relation to roughness elements that interrupt and alter f l o w (Baggerman, 1953; E c k m a n , 1979; Gallagher et a l . , 1983; Snelgrove et a l . , 1993). * A version o f this chapter has been submitted for publication as: M u n r o e , D . M . , and R. S . M c K i n l e y . Settlement o f M a n i l a c l a m (Tapes philippinarum, A d a m s and R e e v e , 1850) larvae to netted and non-netted sediments in a flume. Journal o f Sh el l f i sh Research, submitted: M a y 2006.  Ill  Intertidal c l a m farming involves placement o f large nets over the substrate to protect clams from predation; these nets have the potential to alter tidal f l o w near the sediment surface and the benthic boundary layer, in turn possibly affecting recruitment patterns o f shellfish larvae (Heath et a l . , 1992; B e a l and K r a u s , 2002). Aquaculture production o f the M a n i l a c l a m (Venerupisphilippinarum) has g r o w n rapidly from 315,000 metric tonnes in 1990 to 1,694,000 metric tonnes in 2000, while the capture fishery decreased from 84,000 metric tonnes to 57,000 metric tonnes ( F A O , 2004). C l a m farming is globally widespread and g r o w i n g rapidly. M u c h o f the industry practices the use o f nets for protection o f highly valued seed clams laid out o n beaches (Spencer et a l . , 1992; T o b a et a l . , 1992; Spencer, 2002), however, little is k n o w n o f the contribution o f w i l d settlement in addition to seeded clams o n farmed beaches m a k i n g the question o f potential influence o f nets o n larval settlement important and timely. In this experiment, I investigated the influence o f bottom roughness elements; c l a m netting and sediments; on retention o f V. philippinarum larvae in flume flows. A l t h o u g h direct observation o f settlement patterns in natural settings can also y i e l d useful information ( W i l l i a m s , 1980; Ishii et a l . , 2001) use o f a flume allows for greater control over variables o f interest ( M u s c h e n h e i m et a l . , 1986). In the flume I was able to generate a laminar (based on calculated R e y n o l d s numbers, summarised in the materials and methods section) and w e l l characterised f l o w environment as w e l l as eliminating the potential influence o f adults or predators. I predicted that retention o f larvae w o u l d be higher in flume treatments w i t h no netting applied to the bottom based o n the pattern observed in the field (described in the previous chapter). This prediction was tested in replicated flume trials w i t h netting and sediment bottom treatments.  112  Materials and Methods F o u r fibreglass flumes were used in this study, a l l measured 0.47 m w i d e , 0.25 m deep (water depth at 0.20 m) and 4.9 m long w i t h a w a l l o f d r i n k i n g straws ( 5 m m diameter x 2 0 0 m m length) at the i n f l o w end to entrain water f l o w and a standpipe at the outflow (Figure 5-1). F o r each trial, each flume contained a different bottom type based o n two factors (net and sediment) w i t h two levels each (present and not present) f u l l y crossed; the resulting bottom types were none (control), sediment only, net only, and sediment and net. Sediment used was pea-sized gravel (15 m m diameter) combined in a 50:50 ratio w i t h filter sand (2 m m diameter) to m i m i c the heterogeneity o f field sediments. A l l gravel and sand was washed thoroughly with dechlorinated freshwater to ensure that it was clean and free o f fine sediments. The nets used in the trial were cotton netting (mesh opening size 2 c m x 2 cm) used for predator protection o n c l a m farms, cut to fit the bottom o f the flume and sewn around the edge w i t h lead line to sink the edges o f the net (this is c o m m o n farming practice). The net type used is o n l y one o f a variety employed o n beaches in B r i t i s h C o l u m b i a .  3.0 m  0.3 m  Outflow  0.2 m  A.  V  o  T  Straws  4.9 m  Figure 5-1: Top view of flume dimensions. Flow is from left to right. Acoustic Doppler measurements were made at the position marked by the X . Bottom treatments were applied between the straws and the outflow.  113  T w o separate batches o f V. philippinarum  larvae were used in the trials. Larvae were  reared at a c o m m e r c i a l hatchery facility ( T a y l o r S h e l l f i s h Farms, K o n a , H a w a i i , U S A ) f o r 10 days before being sent to the experimental facility at the Centre for Shellfish Research at M a l a s p i n a U n i v e r s i t y in N a n a i m o , B r i t i s h C o l u m b i a , Canada. U p o n arrival, each batch w a s split into three groups and placed into aerated static larval rearing tanks, fed at a rate o f 15,000 algae cells per m L (50:50 combination o f Isochrysis galbana (Tahitian strain) and Chaetoceros muellerii) twice daily and water was changed every 2.5 days. Splitting o f the batch a l l o w e d each group to be delayed slightly to facilitate a 4 day lag between trial runs (the time necessary to "re-set" the flumes). A diagram o f the batch splitting, delay period and resulting trial that the group was used in is shown in Figure 5 - 2 . The larvae were observed daily until the presence o f pediveligers was confirmed indicating competence to metamorphose. Once the larvae were competent they were siphoned from the larval tanks, length measurements were made and they were then counted and randomly assigned to a flume f o r the trial (see Table 5-1 f o r summary o f larval lengths used in trials).  Figure 5-2: Larval batch spawning and splitting dates and resulting groups used in each trial. 114  Table 5-1: Lengths of Venerupis philippinarum larvae (um ± SD) used for each trial and source batch; n = 20 for each measure.  Trial  Batch Number  Larval Length (pm)  1  1  196(±14)  2  1  211(±13)  3  1  226(±12)  4  2  198(±15)  5  2  216 (±14)  6  2  224 (±13)  Each flume was set up with bottom treatment randomly assigned 48 hours prior to running the trial with larvae. The flumes were filled completely with filtered sea water and a small amount of algae (C. muellerii) was added to each. The flumes were left for 48 hours to establish biofilms on the surfaces as biofilms have been shown to be important in settlement of many invertebrate larvae (Scheltema, 1974; Hadfield and Paul, 2001). During each trial, larvae were introduced into the inflow end of each flume on the treatment side of the straws across the width of the flume at 3 cm depth. Water flow was driven by submersible pumps in an 40 L tub located beneath the outflow of the flume and flow was held steady at 0.3 L/sec (free-stream velocity of 0.45 cm/sec, depth averaged fluid velocity of 0.35 cm/sec). A 105 pm screen was used at the outflow end of the flume to catch any animals exiting the flume. Approximately 100,000 larvae were introduced into each flume for each trial (equal to 44,500/m : twice the maximum settlement density observed in the field for this 2  species: (Williams, 1980; Ishii et al., 2001)). The screens at the downstream end were exchanged every 15 minutes and washed into a beaker. Larval concentration in the beaker was  115  counted using a Sedgewick Rafter (1 m L ) counting slide and the number o f animals was calculated from the concentration and volume in the beaker. A separate trial was also conducted using polystyrene microshperes (manufactured by A l f a Aesar, Parkridge Massachusetts, U S A , 75.0 u m diameter) as larval controls (Butman et a l . , 1988; Ertman and Jumars, 1988). The microspheres were used and counted in the same manner as for the larvae listed above. D u r i n g each trial a l l four flumes were used w i t h a different bottom type in each. The trial was repeated six times, each time the troughs were emptied, the bottom treatments were removed and all components (including sediments) were cleaned. O n each replicate trial, bottom treatment was randomly re-assigned to flumes. F l o w in each o f the flumes was p r o f i l e d using a Sontek A c o u s t i c D o p p l e r V e l o c i m e t e r (Sontek/YSI Inc., San D i e g o , C a l i f o r n i a , U S A ) mounted above each flume. Measurements were made at steps o f 1 c m from the bottom up to a depth o f 10 c m . A t each height above the bottom, 15 measurements were taken at 10 H z over a period o f 120 seconds. E a c h flume was characterised in this manner and one randomly selected flume was characterised under a l l four o f the bottom treatments. The calculated R e y n o l d s number (Re) for the length o f the flume (distance f r o m the leading edge o f the treatment section to the measurement point) was R e = l 2 , 0 0 0 - 15,000. The R e y n o l d s number for the channel (using the flume w i d t h as the length value) was R e = l , 8 8 0 - 2 , 3 5 0 . The Reynolds number for open channel flow using the depth averaged f l u i d velocity and the hydraulic radius as the characteristic length is R e f = 302 indicating that the flow in the flumes were laminar ( K h a l i l i et a l , 2001). The boundary layer R e y n o l d s number, as calculated using the free stream velocity and the B L thickness was Reb = 252 indicating that the boundary layer flow is also laminar ( K h a l i l i et a l . , 2001).  116  Results  I compared the percentage of larvae leaving the system during the entire trial (number leaving/number input x 100) using A N O V A with trial as a block and netting and sediment as factors. The data were tested for normality using the Shapiro-Wilk test, and homogeneity of variance was tested using Levene's test statistic. The results of the A N O V A are shown in Table 5-2. Trial was significant in the percentage of larvae that left the system (p=0.001) while neither netting nor sediment (p=0.603, p=0.391 respectively) were significant. This can be seen in Figure 5-2 where the percentage of larvae leaving the system varies greatly between trials; however, in any given trial there is no consistent difference between treatments.  Table 5- 2: Summary statistics from A N O V A test for percentage of  Venerupis philippinarum larvae  leaving the system during the trial.  Source  df  SS  F  P  Trial  5  11770  38.76  0.001  Net  1  18.7  0.31  0.603  Sediment  1  111.5  0.80  0.391  Net x Sediment  1  201.4  1.45  0.256  Error  10  138.9  Figure 5-3 also shows the results of the trial using polystyrene spheres. On average, 57% (±2.2 S.D.) of the spheres input left the system over the course of the trial (75 minutes total). The majority of the spheres that left the system (approximately 97% ±1.2% 95% confidence interval) did so within the first 45 minutes of the trial (Figure 5-4). This was in contrast to the larvae which showed approximately 73% (±5% 95% confidence interval) of leavers exiting the flume in the first 45 minutes (Figure 5-5 - note lower proportion of darkest bands for trial 7 using beads). This discrepancy indicates the larvae were leaving the flume in a  117  manner inconsistent with inert particles. A portion of the larvae were "leaving later" than would be predicted by particle motion in the flows created.  100 • Control S3 Net • Net & Sediment • Sediment  3  4 Trial Number  Polystyrene Spheres  Figure 5-3: Percentage of total Venerupis philippinarum larvae input at time zero that left the system by the end of the trial (75 minutes). Error bars represent ± standard deviation (n=5 for each bar). Percentage of polystyrene spheres leaving the system is shown on the right side of the chart.  Figure 5-4: Proportion of all polystyrene spheres exiting the system shown by time collected. Error bars represent ± standard deviation.  118  100% 90% 80%  I  nmllll  70%  • 75 min.  60%  E) 60 min.  50%  • 45 min.  40%  • 30 min. • 15 min.  30% 20% 10% 0%  Treatment and Trial Number Figure 5-5: Proportion of Venerupis philippinarum larvae (or beads in case of trial 7) exiting the flume over time. White band indicates proportion leaving in the first 15 minutes; the dotted band indicates the proportion leaving in the second 15 minutes and so on. Trial number and treatment listed along the xaxis. ** Trial 7 was conducted with polystyrene beads; all other trials shown were conducted with larvae.  Based on this "leaving later" behaviour, I tested the relative proportion of larvae leaving in the last 30 minutes of the trial (larvae leaving in last 30 minutes/larvae leaving the system in entire trial) using A N O V A with trial as a block and netting and sediment as factors. The data were tested for normality using the Shapiro-Wilk test, and homogeneity of variance was tested using Levene's test statistic. Results are shown in Table 5-3. In this case none of the factors were significant in influencing the proportion of larvae leaving the system later in the trial.  119  Summary statisticsfromANOVA test for proportion of Venerupis philippinarum larvae leaving in the last 30 minutes of the trial.  Table 5-3:  Source  df  SS  F  P  Trial  5  1078.4  3.26  0.11  Net  1  0.0126  0.00  0.99  Sediment  1  137.4  1.30  0.281  Net x Sediment  1  194.4  1.83  0.205  Error  10  1059.6  Discussion  Larvae retained within the flumes varied from one trial to the next. Because it was necessary to clean the flumes, re-set the bottom treatments and allow biofilms to establish between trials, there was a delay of about 2.5 days between trials. Although efforts were made to slow development of larvae for trials to be run later (reduced feeding for brief periods and reduced water temperature) as the larvae aged, they became less likely to exit the flume. In flume trials with larvae of the bivalve Mulinia lateralis, Snelgorve et al. (1993) also saw reduced swimming ability with larval age that they note may increase "hydrodynamic trapping" of older larvae. In flume experiments with Mya arenaria larva, Snelgrove et al. (1999) saw a large influence of "run" in their trials demonstrating decreased selectivity at older larval age. In work with barnacle settlement, Rittschof et al. (1984) noted that younger larvae were more discriminating in settlement habitat than older cyprids. Despite this, I was still able to test the effects of the treatments by using trial as a blocking factor. Neither sediment nor netting appeared to have an impact on the number of larvae retained in the flumes. This is contrary to the original hypothesis, however evidence from field surveys described in Chapter 4 indicates that in years when overall recruitment is low, netting has limited influence on small-scale recruitment patterns.  120  Water velocity (free stream = 0.45 cm/sec) used here was l o w compared to average f l o w s o n intertidal V. philippinarum  farm plots i n B r i t i s h C o l u m b i a ( 4 - 8 cm/sec averaged over  24 hours o n spring tide, A p p e n d i x 4). Oscillatory intertidal f l o w s are difficult to match and poorly estimated w i t h unidirectional single speed flows in a flume (Petersen and Hastings, 2001) and field f l o w s have numerous additional sources o f turbulence (Hendriks et a l . , 2006). A novel approach to this p r o b l e m was presented by A s m u s et a l . (1995) w h o placed stationary flume channels in the shallow intertidal and allowed naturally generated tidal f l o w to drive the currents w i t h i n it. L o w e r unidirectional f l o w s were used for this experiment because it has been predicted that larvae may settle o n slack tides (Gross et a l . , 1992) therefore I used lower flows, replicating those closer to slack tide. In addition, lower flows a l l o w e d for longer exploration time for the larvae w i t h i n the flume. It is possible that differences in the larval retention may be observed i f trials were carried out under conditions o f higher f l o w that might demonstrate greater interaction between the netting and the benthic boundary layer. A s f l o w rate increases, the influence o f roughness elements o n the bottom w o u l d also l i k e l y increase. It has been suggested b y other authors ( K o h l e r , 1999; P a w l i k and B u t m a n , 1993) that laminar benthic boundary layer f l o w s may lead to decreased encounter rate o f larvae w i t h the sediment surface because turbulence is a mechanism that helps to place larvae in contact w i t h the bottom ( C r i m a l d i et a l . , 2002). Since each trial was r u n f o r a duration o f five times the total time required for a particle to travel in bulk f l o w to the outflow o f the flume, and some larvae d i d not pass through the flume in this t i m e , I suggest that the larvae were able to reach and remain in contact with the bottom. Further, the Rouse number ( R o = fall velocity/(von K a m a n n ' s constant x shear velocity) can be used to indicate the probability o f a larvae to control its position i n the water c o l u m n b y s w i m m i n g . F o r R o > 0.75 the larvae can control their vertical position by s w i m m i n g , while at R o < 0.75 turbulence dominates the larval position (Gross et a l , 1992). Here, I observed s i n k i n g rates o f dead larvae o f 0.17 cm/sec and the shear 121  velocity was 0.16 cm/sec therefore the R o u s e number for trials was 2.69 indicating that the larvae were able to control their vertical position through s w i m m i n g . In addition, recent evidence from observations w i t h oyster larvae has s h o w n that bivalve larvae m a y have control over vertical position in the water c o l u m n i n a greater range o f velocities than previously predicted ( F i n e l l i and Wethey, 2003). The t i m i n g o f exit from the flumes differed between larvae and polystyrene beads showing that the larvae were not acting as inert particles. This has been observed in other studies i n v o l v i n g larval settlement patterns in f l o w . B u t m a n et a l . (1988) demonstrated that bivalve larvae {Mercenaria mercenaria) did not distribute in the same manner as inert particles in settlement tests. Gregoire et al. (1996) compared the patterns o f settlement o f inert particles and dead larvae to those seen for w i l d caught spat and found differences. In flume tests w i t h settlement o f abalone (Haliotis rufescens), B o x s h a l l (2000) used dead larvae and empty shells as larval m i m i c s and found live larvae settled i n a different pattern than larval m i m i c s .  Conversely,  H a r v e y et al. (1995) reported that larval settlement patterns in the field and distribution o f inert particles under controlled conditions showed no difference and that passive process alone could explain the settlement o f bivalve larvae onto filamentous structures. The polystyrene beads showed that most particles m o v i n g through the flume w i l l exit w i t h i n 45 minutes, therefore by examining the patterns o f larvae exiting after 45 minutes I can more effectively focus o n the portion o f the animals that are remaining i n the flume for a period before exiting the flume (represented by the black and dark grey bands i n Figure 5-5). Results o f this comparison showed no significant effect o f any o f the factors o n the proportion o f larvae leaving the system i n the last 30 minutes. T h i s result may differ w h e n f l o w rates are increased leading to greater effects o f the roughness elements. In field trials described i n Chapter 4, it was observed that i n years w h e n larval settlement rates are high, netting appears to depress settlement o f V. philippinarum  larvae to intertidal areas covered w i t h netting. 122  Conclusions A t the f l o w rates tested i n the flumes used here, I was unable to detect differences in retention o f V. philippinarum  larvae in relation to either sediment or netting applied to the  bottom o f the flumes. Possibly, V. philippinarum  is a poor candidate for these types o f trials  because the competent period for this species can be prolonged i f conditions are unfavourable and retention i n the flume is not necessarily an indication o f intention to remain until settlement. H o w e v e r , this species is part o f an important fishery and an even more important culture industry w o r l d w i d e , therefore understanding the effects o f farm practices like netting, o n larval settlement and recruitment are crucial i n ensuring a sustainable and efficient industry in the future.  123  References  A b e l s o n , A . , and M . Denny.  1997. Settlement o f marine organisms in f l o w . A n n u a l R e v i e w o f  E c o l o g y and Systematics, 2 8 : 3 1 7 - 3 3 9 . A s m u s , H . , R. M . A s m u s , and G . B . Z u b i l l a g a . 1995. D o mussel beds intensify the phosphorus exchange between sediment and tidal waters? Ophelia, 4 1 : 3 7 - 5 5 . Baggerman, B . 1953. Spatfall and transport o f Cardium edule. A r c h i v e s Neerlandaises do Zoologie, 10: 315-342. B e a l , B . F . , and G . M . K r a u s . 2002. 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P., J . K . T h o m p s o n , J . H . R o s m a n , R. J . L o w e , and J . R. Koseff. 2002. H y d r o d y n a m i c s o f larval settlement: The influence o f turbulent stress events at potential recruitment sites. L i m n o l o g y and Oceanography,  47:1137-1151.  C r i s p , D . J . 1955. The behaviour o f barnacle cyprids in relation to water movement over a surface. Journal o f E x p e r i m e n t a l B i o l o g y , 3 2 : 5 6 9 - 5 9 0 . E c k m a n , J . E . 1979. Small-scale patterns and processes in a soft-substratum, intertidal community. Journal o f M a r i n e Research, 3 7 : 4 3 7 - 4 5 7 . E n g s t r o m , S . J . , and R. L . M a r i n e l l i . 2 0 0 5 . Recruitment responses o f benthic infauna to manipulated sediment geochemical properties i n natural flows. Journal o f M a r i n e Research, 6 3 : 4 0 7 - 4 3 6 . Ertman, S . C , and P. A . Jumars. 1988. Effects o f bivalve siphonal currents o n the settlement o f inert particles and larvae. 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Settlement success, spatial pattern and behavior o f mussel larvae Mytilus spp. i n experimental ' d o w n w e l l i n g ' systems o f varying velocity and turbulence. M a r i n e E c o l o g y Progress Series, 2 6 0 : 1 2 5 - 1 4 0 . Petersen, J . E., and A . Hastings. 2 0 0 1 . D i m e n s i o n a l Approaches to S c a l i n g Experimental Ecosystems: D e s i g n i n g Mousetraps to Catch Elephants. The A m e r i c a n Naturalist, 157: 324-333. P o g g i , D . , A . Porporato, and L . R i d o l f i . 2003. A n a l y s i s o f the small-scale structure o f turbulence o n smooth and rough walls. Physics o f F l u i d s , 15: 3 5 - 4 6 . Q i a n , P. Y . 1999. L a r v a l settlement o f polychaetes. H y d r o b i o l o g i a , 4 0 2 : 2 3 9 - 2 5 3 . Rittschof, D . , E . S . B r a n s c o m b , and J . D . C o s t l o w . 1984. Settlement and behavior i n relation to f l o w and surface in larval barnacles, Balanus amphitrite D a r w i n . J . E x p . M a r . B i o l . 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M o l l u s c a n shellfish f a r m i n g . B l a c k w e l l P u b l i s h i n g L T D . O x f o r d , England. 272 p p .  126  Spencer, B . E . , D . B . E d w a r d s , and P . F. M i l l i c a n . 1992. Protecting M a n i l a c l a m (Tapes philippinarum) beds w i t h plastic netting. Aquaculture, 105: 2 5 1 - 2 6 8 . T o b a , D . R., D . S. T h o m p s o n , K . K . C h e w , G . J . A n d e r s o n , and M . B . M i l l e r . 1992. Guide to M a n i l a C l a m Culture i n Washington. Washington Sea Grant Publications 9 2 - 0 0 1 . U n i v e r s i t y o f Washington. 80 p p . Toonen, R. J . , and J . R. P a w l i k . 1994. Foundations o f gregariousness. Nature, 3 7 0 : 5 1 1 - 5 1 2 . Turner, E . J . , R. K . Z i m m e r - F a u s t , M . A . Palmer, M . L u c h e n b a c h , and N . D . Pentcheff. 1994. Settlement o f oyster Crassostrea virginica larvae: effects o f water f l o w and a watersoluble c h e m i c a l cue. L i m n o l o g y and Oceanography, 3 9 : 1 5 7 9 - 1 5 9 3 . Wethey, D . S . 1986. R a n k i n g o f settlement cues b y barnacle larvae: influence o f surface contour. B u l l e t i n o f M a r i n e Science, 3 9 : 3 9 3 - 4 0 0 . W i l l i a m s , J . G . 1980. T h e influence o f adults o n the settlement o f spat o f the c l a m , Tapes japonica. Journal o f M a r i n e Research, 3 8 : 7 2 9 - 7 4 1 . W i l l i a m s o n , J . E . , R. D e N y s , N . K u m a r , D . G . Carson, and P . D . Steinberg. 2 0 0 0 . Induction o f metamorphosis in the sea urchin Holopneustespurpurascens b y a metabolite c o m p l e x from the algal host Deliseapulchra. B i o l o g i c a l B u l l e t i n , 198: 3 3 2 - 3 4 5 .  127  C H A P T E R 6: Conclusions and General Discussion  Density separation o f newly settled bivalves from sediments is simple and effective for field sampling. I was able to prove the accuracy of this method in various sediment types allowing us the awareness that results o f field sampling are an accurate reflection of the magnitude o f early settlement. Beyond that, these methods are easily applicable for other researchers or even shellfish growers interested in tracking larval settlement on their beaches. Results o f field measurements of sediment properties indicated that the netting is not altering the sediment grain size distribution or carbon levels substantially on the study sites. This does not mean however, that differences do not exist as I measured only a small sub-set o f possible parameters. It is likely that the influence o f netting on sediment properties is site specific and depends largely on local hydrography. I did not measure biofilms or sediment geochemical properties that could alter the sediment environment. The motion of the netting on the sediment surface may create areas o f frequent disturbance that might host different populations of biofilms or change sediment chemistry. Further investigation of this possibility could illuminate driving forces in succession o f biofilms. A n unexpected result o f field measurements was the "buffering" o f low tide sediment/air interface temperature by netting. These results were not explored in depth but may be of significance to the metabolic rates and stress levels of animals beneath the netting particularly at temperature extremes. This may prove to be a worthwhile question to explore further in future research as it may pertain to optimal growth o f farmed animals within those plots. I observed large annual and spatial variation in settlement o f larval clams. Plots with netting and dense populations o f adult bivalves had depressed levels o f early recruits in 2004 when settlement was high. A n interesting trend appeared at higher populations o f adult clams. A s adult population increased, early recruits initially decreased then levelled off or possibly  128  increased. Larger populations o f filter feeders m a y actually facilitate settlement o f larvae by disruption o f near bed f l o w s w i t h siphon currents. Questions surrounding the influence o f filter feeding benthos o n larval delivery and settlement patterns are interesting and as yet, inconclusive. W h y smaller populations tend to decrease larval settlement while larger populations do not should be studied further to gain insight in this area. The presence o f adult filter feeders at study plots made isolating the influence o f netting alone impossible. A controlled experiment where adults and predators were removed was run in laboratory flumes to try to isolate the effect o f netting o n settlement o f larvae. The experiment showed no change in retention o f larvae w i t h i n f l u m e s as a result o f netting. T h i s might be because the animals are poorly suited to this type o f trial because they do not cement permanently and take a long time searching for suitable settlement sites. In addition, the water velocity used in the trial may have been too l o w to generate a turbulence structure that would influence settlement. Repeated trials at a variety o f water velocities c o u l d be useful in determining i f this is the case. A l t h o u g h laboratory experiments are desirable for the control over variables and the accuracy o f the observations that can be made w h e n dealing w i t h invertebrate larvae,  field-based  research is also important. A s was the case here, field experimentations are often confounded w i t h uncontrolled factors; however, laboratory-based experiments often lack the k i n d o f complexity found in natural systems. It is therefore important to partner field and laboratory research in an attempt to understand the patterns o f larval settlement in both a controlled and relevant manner. T h i s thesis described a combination o f laboratory and field experimentation and highlites some o f the discrepancies that exist. Nonetheless, these types o f experiments are more relevant and informative than either field or laboratory w o r k can be w h e n used exclusively.  129  E a r l y recruitment patterns measured here d i d not appear to be random, nor did they f o l l o w the pattern predicted by deposition o f silt and passive particles. T h i s indicates an active role in habitat selection by these early settlers that should be investigated further. The majority o f the contributions to the field o f larval settlement involve observations o n attached settlers o n hard substrates or conspicous species like polychaetes. This thesis described field settlement patterns o f a mobile species i n soft-sediments and is an important addition to this f i e l d o f literature. Further studies, like this one, w i l l a l l o w paradigms and hypothesis that have been established and tested for attached and conspicous species in the field and laboratory to be applied and tested o n this less-examined group. The trend in settlement patterns observed at field sites c o u l d be used by farmers to increase w i l d recruitment to culture plots. T i m i n g harvest o f adults and temporary removal o f nets to coincide w i t h larval settlement could lead to higher recruitment, particularly in years w h e n larval settlement is high. The relationship between early recruits and the recruitment into adult populations is u n k n o w n however. Research focussed o n determining survival and migration o f early recruits is crucial to connecting larval recruitment w i t h adult populations. W i t h o u t this connection, patterns o f early recruitment cannot be used to understand population dynamics.  130  Appendix 1: Summary table of juvenile bivalve dispersal research Reference Ahn et al., 1993 Amyot and Downing, 1998  Lab/Field  Species observed  Shell length  Drift mechanism  Location  Season studied  both  Mercenaria mercenaria  0.2 mm - 0.65 mm  crawling  Flax Pond, New York, USA  September 1989  52 -67 mm  active processes  Lac de l'Achigan Quebec, Canada  Spring summer 1988, 1989,1990  Wadden Sea, Netherlands  June September 1991  Wadden Sea, Netherlands  Summer 19901992  field  Elliptio complanata  field  0.7-4 mm M.balthica, Ensis directus, Cerastoderma edule, Macoma 0.5-3.5 mm C. edule, 1-5 byssus balthica, Mytilus edulis, Venerupis pullustra, mm E. directus Mya arenaria  Armonies, 1994(a)  both  balthica, l-18mm£. Cerastoderma edule, Macoma balthica, Ensis byssus, climbing americanus, l-2mmM. americanus, Mytilus edulis, Venerupis edulis, V. pullustra & M. pullustra, Mya arenaria arenaria  Armonies, 1994(b)  field  Macoma balthica, Cerastoderma edule  Armonies, 1996  field  Baggerman, 1953  field  Armonies, 1992  0.5-4mm C. edule & M.  > 0.5 mm  Cerastoderma edule, Macoma balthica, Ensis 0.125 - 2 mm americanus, Mytilus edulis, Venerupis pullustra, Mya arenaria Cardium edule, Mya arenaria, Mytilus edulis, 0.4- 11.0 mm Macoma balthica, Petricola pholadiformis  movement noted  Wadden Sea, Netherlands  active and passive processes  Wadden Sea, Netherlands  bedload transport  Wadden Sea, Netherlands  pelagic drift (possibly byssus)  Chesapeake Bay, Virginia, USA  foot protrusion  Menai Strait, North Wales  June - August 1992 May - August 1993 A p r i l August 1994 Summer 1950 July - August 1990 and May September 1991-1993 April - July 1963  Baker and Mann, 1997  field  Anadara transversa, Geukensia demissa, 0.25 - 0.5 mm Tellina agilis  Bayne, 1964  both  Mytilus edulis  Beaumont and Barnes, 1992  lab  Pecten maximus, Aequipecten opercularis 6.4-13.0 mm A. opercularis  byssus  field  Macoma balthica  0.5 - 5 mm  byssus  Wadden Sea, Netherlands  both  Macoma balthica  to 10 mm field, 2-9 mm lab  byssus, mucous  Wadden Sea, Netherlands  winter  field  Musculium partumeium, Corbiculafluminea 1.2-6.2 mm  crawling  Savannah River, USA  January - July 1976  Beukema, 1993 Beukema and de Vlas, 1989 Boozer and Mirkes, 1979  0.25 -3 mm  3.7-18.8 mm P. maximus,  Drift mechanism oriented crawling, short distances  Location  Season studied  Reference  Lab/Field  Species observed  Shell length  Brafield and Newell, 1961  field  Macoma balthica  adults  CaceresMartinez et al., 1994  both  Mytilus galloprovincalis  0.25-2 mm  crawling, mucous drift  Ria do Vigo, Spain  1991 - 1993  Commito et al., 1995  field  Gemma gemma (brooding)  0.2 - 2.2 mm  active drift  Assateague Island, Virginia, USA  October 1983 and January 1984  Cummings et al., 1993  lab  Macomona lilliana  1-2 mm  byssus  New Zealand  field  Macomona lilliana, Arthritica bifurca, Auatrovenus stuchburyi, Mactra ovata, < 3 mm Mytilus sp., Nucula hartvigiana, Paphies australis, Hiatula siliquens, Zenatia acinaces  movement noted  Manakau Harbour, New Zealand  October & December 1991  field  Mya arenaria  8-15 mm  bedload transport  both  Macoma balthica  0.5 - 7 mm  byssus  Eastern Passage, Nova Scotia Canada Groninger Wad, The Netherlands  Highsmith, 1985  lab  Transemlla trantilla (brooding)  > 0.5 mm  floating  June 1988April 1989 December 1999 -March 2001 May to August 1982 July to August 1984  Lane et al., 1985  lab  Mytilus edulis  to 2 mm  byssus  Martel and Chia, 1991  field  Montaudouin, 1997  lab  Nelson, 1928  field  Mytilus edulis  0.35 - 0.94 mm  air bubble  Newell, 1994  field  Mytilus edulis  0.25-2 mm  byssus  field  Macomona lilliana, Austravenus stuchburyi to 4 mm  active processes  field  Abra alba, Cultelluspellucidus, Mysella bidentata, Tellina fabula  byssus, valves  Cummings et al., 1995 Emerson and Grant, 1991 Hiddink et al., 2002  Norkko et al., 2001 Olivier et al 1996  Musculus sp, Lasaea sp, Transemlla trantilla - brooders, Mytilus sp, Hiatella arctica, unk. 0.5 - 0.95 mm Clam - non-brooders 1.4-2.1 mm C. edule, 0.4-5.1 mm R. Cerastoderma edule, Ruditapes philippinarum philippinarum  byssus  Whitstable, U.K.  Friday Harbour, Washington, USA  Bamfield, BC Canada  Summer 1989  Frenchman Bay, Maine, USA Mt. Desert Narrows, Maine, USA Manakau Harbour, New Zealand  August 1924 and 1927  English Channel  June 1992  byssus, valves  1985 - 1991 February 1997  Lab/Field  Species observed  Shell length  Drift mechanism  Location  Season studied  lab  Corbicula fluminea  7 - 14 mm  byssus, mucous  Mississippi, USA  March 1984  lab  Mya arenaria  0.24 - 0.29 mm  bedload transport  Rygg, 1970  lab  Cerastoderma edule, Cerastoderma glaucum  young juveniles  crawling, climbing  Sellmer, 1967  field  Gemma gemma (brooding)  0.33-0.51 mm  passive processes  Sigurdsson et al, 1976  both  Nucula tenuis, Mytilus edulis, Modiolus sp., Musculus marmoratus, Heteranomia squamula, Turtonia minuta, Montacuta ferruginosa, Montacuta substriata, Cardium echinatum, Venerupis pullastra, Mactra corallina, Spisula sp., Tellina tenuis, Gari fervensis, Abra alba, Donax vittatus,Cultellus pellucidus, Ensis sp.,Hiatella arctica, Hiatella gallicana, Mya truncata, Corbula gibba  Sorlin, 1988  lab  Macoma balthica  Sugawara et al., 1953  lab  Ruditapes philippinarum  Sullivan, 1948  field  Gemma gemma (brooding)  0.35 mm  passive processes  field  Macomona lilliana, Austrovenus stuchburyi  to 4 mm  passive processes  lab  Sinonovacula constricta  0.55 - 0.90 mm  byssus, climbing  Williams and Porter, 1971  field  Ensis directus, Tagelus divisus, Solemya velum, Solen viridis, Dondax variabilis, Petricola pholadiformis, Spisula raveneli  1.7-46.0 mm  water expulsion, kicking with foot, bedload transport  Yankson, 1986  lab  Cerastoderma edule, Cerastoderma glaucum  8 -10.3 mm  byssus, climbing  Reference Prezant and Chalermwat, 1984 Roegner et al., 1995  Turner et al, 1997 Wang and X U , 1997  Table A l - 1 : Summary o f research on juvenile bivalve dispersal.  Trondheimsfj orden, Norway Union Beach, New Jersey, USA  1967-1968 1955-1958  byssus  4 - 1 4 mm  byssus, foot protrusion  Wadden Sea, Netherlands  byssus  Japan  Winter/spring 1982  Malpeque Bay, P.E.I, Canada Manakau Harbour, New Zealand Ronhai County, Fujian, China  February 1994  Eastern North Carolina, USA  1957 - 1966  References A p p e n d i x 1 A h n , I., G . L o p e z and R. M a l o u f . 1993. Effects o f the gem c l a m Gemma gemma o n early postsettlment emigration, growth and survival o f the hard c l a m Mercenaria mercenaria. M a r i n e E c o l o g y Progress Series, 9 9 : 6 1 - 7 0 . A m y o t , J . , and J . A . D o w n i n g . 1998. L o c o m o t i o n in Elliptio complanata ( M o l l u s c a : Unionidae): a reproductive function? Freshwater B i o l o g y , 3 9 : 3 5 1 - 3 5 8 . A r m o n i e s , W . 1992. M i g r a t o r y rhythms o f drifting juvenile molluscs in t i d a l waters o f the W a d d e n Sea. M a r i n e E c o l o g y Progress Series, 8 3 : 197-206. A r m o n i e s , W . 1994(a). D r i f t i n g meio - and macrobenthic invertebrates o n tidal flats in K o n i g s h a f e n : a review. Helgolander Meeresuntersuchungen, 4 8 : 2 9 9 - 3 2 0 . A r m o n i e s , W . 1994(b). Turnover o f postlarval bivalves i n sediments o f tidal flats in K o n i g s h a f e n ( G e r m a n W a d d e n Sea). Helgolander Meeresuntersuchungen, 4 8 : 2 9 1 - 2 9 7 . A r m o n i e s , W . 1996. Changes i n distribution patterns o f 0-group bivalves in the W a d d e n S e a : byssus-drifting releases juveniles f r o m the constraints o f hydrography. Journal o f Sea Research, 3 5 : 3 2 3 - 3 3 4 . B a g g e r m a n , B . 1953. Spatfall and transport o f Cardium edule L . A r c h i v e s Neerlandaises de Zoologie,  10:315-342.  B a k e r , P., and R. M a n n . 1997. T h e postlarval phase o f bivalve m o l l u s c s : a review o f functional ecology and n e w records o f postlarval drifting o f Chesapeake B a y bivalves. B u l l e t i n o f M a r i n e Science 6 1 : 4 0 9 - 4 3 0 . B a y n e , B . L . 1964. Primary and secondary settlement in Mytilus edulis L . ( M o l l u s c a ) . Journal o f Animal Ecology, 33: 513-523. Beaumont, A . R., and D . A . Barnes. 1992. Aspects o f veliger larval growth and byssus drifting o f the spat o f Pecten maximus and Aequipecten (Chlamys) opercularis.  I C E S Journal o f  M a r i n e Science, 4 9 : 4 1 7 - 4 2 3 . B e u k e m a , J . J . 1993. Successive changes in distribution patterns as an adaptive strategy in the bivalve Macoma balthica ( L ) i n the W a d d e n Sea. Helgolander Meersuntersuchungen, 4 7 : 287-304. B e u k e m a , J . J . , and J . de V l a s . 1989. Tidal-current transport o f thread-drifting postlarval juveniles o f the bivalve Macoma balthica from the W a d d e n Sea to the N o r t h Sea. M a r i n e E c o l o g y Progress Series, 5 2 : 193-200. B o o z e r , A . C , and P. E . M i r k e s . 1979. Observations o n the fingernail c l a m , Musculium partumeium (Pisidiidae), and its association w i t h the introduced A s i a t i c c l a m , Corbicula fluminea. T h e Nautilus 9 3 : 7 3 - 8 3 .  1 3 4  B r a f i e l d , A . E., and G . E . N e w e l l . 1961. T h e behaviour o f Macoma balthica ( L . ) . Journal o f the M a r i n e B i o l o g i c a l A s s o c i a t i o n o f U . K . , 4 1 : 8 1 - 8 7 . C a c e r e s - M a r t i n e z , J . , J . A . F. R o b l e d o , A . Figueras. 1994. Settlement and post-larvae behaviour of Mytilus galloprovincialis: F i e l d and laboratory experiments. M a r i n e E c o l o g y Progress Series, 112: 107-117. C o m m i t o , J . A . , C . A . Currier, L . R . K a n e , K . A . R e i n s e l , and I. M . U l m . 1995. Dispersal dynamics o f the bivalve Gemma gemma in a patchy environment. E c o l o g i c a l M o n o g r a p h s , 65:1-20. C u m m i n g s , V . J . , R. D . Pridmore and S . F. Thrush. 1993. Emergence and floating behaviours o f post-settlement juveniles o f Maomona liliana ( B i v a l v i a : Tellinacea). M a r i n e B e h a v i o u r Physiology, 24: 25-32. C u m m i n g s , V . J . , R. D . Pridmore, S . F. Thrush, and J . E . H e w i t t . 1995. Post-settlement movement by intertidal benthic macro invertebrates: D o c o m m o n N e w Zealand species drift in the water c o l u m n ? N e w Zealand Journal o f M a r i n e and Freshwater Research, 2 9 : 5 9 - 6 7 . E m e r s o n , C . W . , and J . Grant. 1991. The control o f soft-shell c l a m (Mya arenaria) recruitment o n intertidal sandflats by bedload sediment transport. L i m n o l o g y and Oceanography, 3 6 : 1288-1300. H i d d i n k , J . G . , R. P. K o c k , and W . J . W o l f f . 2002. A c t i v e pelagic migrations o f the bivalve Macoma balthica are dangerous. M a r i n e B i o l o g y , 140: 1149-1156. H i g h s m i t h , R. C . 1985. Floating and algal rafting as potential dispersal mechanisms in brooding invertebrates. M a r i n e E c o l o g y Progress Series, 2 5 : 169-179. L a n e , D . J . W . , A . R. Beamont, and J . R. Hunter. 1985. Byssus drifting and the drifting threads o f the young post-larval mussel Mytilus edulis. M a r i n e B i o l o g y , 8 4 : 3 0 1 - 3 0 8 . M a r t e l , A . , and F. C h i a . 1991. D r i f t i n g and dispersal o f small bivalves and gastropods w i t h direct development. Journal o f Experimental M a r i n e B i o l o g y and E c o l o g y , 150: 131-147. M o n t a u d o u i n , X de. 1997. Potential o f bivalves' secondary settlement differs w i t h species: a comparison between c o c k l e (Cerastoderma edule) and c l a m (Ruditapes philippinarum) juvenile resuspension. M a r i n e B i o l o g y , 128: 6 3 9 - 6 4 8 . N e l s o n , T . C . 1928. Pelagic dissoconchs o f the c o m m o n mussel, Mytilus edulis, with observations o n the behaviour o f the larvae o f a l l i e d genera. B i o l o g i c a l B u l l e t i n , 5 5 : 180192. N e w e l l , C . R. 1994. Estuary-scale dispersal o f post-larval mussels, Mytilus edulis, among eelgrass (Zostera marina) meadows and subsequent recruitment to planted live and mussel shell cultch. Journal o f Sh e llf ish Research, 1 3 : 2 8 1 .  135  N o r k k o , A . , V . J . C u m m i n g s , S. F. Thrush, and J . E. H e w i t t and T. H u m e . 2 0 0 1 . L o c a l dispersal o f juvenile bivalves: implications for sandflat ecology. M a r i n e E c o l o g y Progress Series, 2 1 2 : 131-144. O l i v i e r , F., C . V a l l e t , J . D a u v i n , and C . Retiere. 1996. D r i f t i n g in post-larvae and juveniles in an Abra alba ( W o o d ) community o f the eastern part o f the bay o f Seine (English Channell). Journal o f Experimental M a r i n e B i o l o g y and E c o l o g y , 199: 8 9 - 1 0 9 . Prezant, R. S . , and K . Chalermwat. 1984. Floatation o f the bivalve Corbicula fluminea as a means o f dispersal. Science, 2 2 5 : 1491-1493. Roegner, C , C . A n d r e , M . Lindegarth, J . E c k m a n , and J . Grant. 1995. Transport o f recently settled soft-shell clams (Mya arenaria L.) in laboratory flume f l o w . Journal o f Experimental M a r i n e B i o l o g y and E c o l o g y , 187: 13-26. R y g g , B . 1970. Studies o n Cerastoderma edule (L.) and Cerastoderma glaucum (Poiret). Sarsia, 4 3 : 6 5 - 8 0 . Sellmer, G . P. 1967. Functional morphology and ecological life history o f the g e m c l a m , Gemma gemma (Eulamellibranchia: Veneridae). M a l a c o l o g i a , 5 : 137-223. Sigurdsson, J . B . , C. W . T i t m a n , and P. A . Davies. 1976. The dispersal o f young post-larval bivalve molluscs by byssus threads. Nature, 2 6 2 : 3 8 6 - 3 8 7 . S o r l i n , T.  1988. Floating behaviour in the tellinid bivalve Macoma balthica (L.).  Oecologia,  77: 2 7 3 - 2 7 7 . Sugawara, K . , and M . Tamaga. 1953. Floatation by drifting threads in Ruditapesphilippinarum in laboratory. B u l l e t i n o f the Japanese Society o f Fisheries and Oceanography, 18: 8 1 - 8 2 . (in Japanese). S u l l i v a n , C . M . 1948. B i v a l v e larvae o f M a l p e q u e B a y , P.E.I. Fisheries Research B o a r d o f Canada B u l l e t i n , 7 7 . 59pp. Turner, S. J . , J . Grant, R. D . P r i d m o r e , J . E. Hewitt, M . R. W i l k i n s o n , T. M . H u m e , and D . J M o r r i s e y . 1997. B e d l o a d and water-column transport and colonization processes by postsettlement benthic macro fauna: D o e s infaunal density matter? Journal o f Experimental M a r i n e B i o l o g y and E c o l o g y 2 1 6 : 5 1 - 7 5 . W a n g , W . X . , and Z . Z . X u . 1997. L a r v a l s w i m m i n g and postlarval drifting behaviour in the infaunal bivalve Sinonovacula constricta. M a r i n e E c o l o g y Progress Series, 148: 7 1 - 8 1 . W i l l i a m s , A . B . , and H . J. Porter. 1971. A ten-year study o f meroplankton in N o r t h C a r o l i n a estuaries: occurrence o f postmetamorphal bivalves. Chesapeake Science, 12: 2 6 - 3 2 . Y a n k s o n , K . 1986. Observations o f byssus systems in the spat o f Cerastoderma glaucum and C. edule. Journal o f the M a r i n e B i o l o g i c a l A s s o c i a t i o n o f U . K . , 6 6 : 2 7 7 - 2 9 2 .  136  Appendix 2: Comparison of methods for the determination of carbon in intertidal sediments. Introduction Classification o f habitats is essential to understanding behaviours and life histories o f the benthos that live i n the sediment. Determination o f organic and inorganic carbon concentrations in sediments is a n important aspect o f this classification and has been used as a measurement t o o l and classification scheme for many years. Despite years o f use as a descriptor o f sediment, separation o f organic and inorganic carbon fractions remains challenging and the methods employed are varied. Frequently used methods differ in the amount o f time to carry them out, their expense and accuracy.  Three o f the most c o m m o n methods include: •  A c i d leaching o f the sample to dissolve the inorganic carbonates f o l l o w e d b y dry combustion at 500°C - 600°C to determine the residual organic carbon fraction (referred to here as " a c i d - b u r n " ) (Gibbs, 1977).  •  L o s s o n ignition at 500°C - 600°C to oxidise the organic matter to carbon dioxide and ash. T h e n a second loss o n ignition at 900°C - 1000°C to oxidise the inorganic fraction, or carbonate, into carbon dioxide (referred to here as " L O I " ) ( L u c z a k et a l . , 1997).  •  Determination o f inorganic carbon by coulometry ( H u f f m a n , 1977) and elemental analysis by flash combustion to determine total carbon. T h e n subtraction o f inorganic from total carbon to give organic carbon (referred to here as " C H N " ) (Verardo et a l . , 1990). Other methods exist, however the three above are those most c o m m o n l y used.  The  literature contains many examples o f conflicting evidence supporting and c r i t i c i z i n g the use o f both acid-burn and L O I ( W e l i k y et a l . , 1983; T u n g & Tanner, 2 0 0 3 ; and B i s u t t i et a l . , 2004).  137  The L O I method is inexpensive, easy to carry out and w i d e l y used, however it has limitations. L u c z a k et a l . (1997) and H e i r i et a l . (2001) noted that use o f this method involves great variability in temperature, duration o f combustion and amount o f sample used. These discrepancies in methodology produce results that make comparison between studies difficult. M a n y authors ( e.g. W e l i c k y et a l . 1983, Y a m a m u r o & K a y a n n e , 1995; 1983; B i s u t t i et a l . , 2004) point out that the combustion temperatures o f organic and inorganic forms o f carbon overlap and therefore one can not achieve f u l l separation o f the two forms using ignition methods. C l a y particles can also lead to large inaccuracies w h e n using this method; the loss o f associated structural water upon combustion can lead to overestimation o f carbon ( B a r i l l e - B o y e r et a l . , 2003). The acid-burn method is slightly more complicated compared to L O I , yet is still an inexpensive and simple method. Nevertheless, it also has shortcomings that have been noted in the literature (Byers et a l . , 1978). F r o e l i c h (1980) and W e l i k y et al. (1983) explain that upon acidification, acid-soluble organic carbon compounds w i l l be dissolved along w i t h carbonate dissolution producing errors. A c i d pre-treatment can also alter the ratios o f elements that may be o f interest such at hydrogen, nitrogen and o x y g e n (Lohse et a l . , 2 0 0 0 ; R y b a & Burgess, 2002) leading to problems w i t h subsequent analysis for those compounds. The acidification treatment is f o l l o w e d by three repetitions o f rinsing, centrifuging then decanting to remove the acid. W i t h a greater number o f steps comes increased human error and possible sample loss o n decanting (Hedges and Stern, 1984). It is generally agreed that the C H N elemental analysis is an accurate method for analysis o f total carbon (Verardo et a l . , 1990; L e o n g & Tanner, 1999). The most obvious drawback o f this procedure is the expense o f the equipment involved in the analysis. It has also been noted that this method may lead to inaccurate results w h e n samples w i t h high carbonate content are being analysed ( W e l i k y et a l . , 1983; B i s u t t i et a l , 2004). This error comes f r o m the subtraction 138  o f two large values (total carbon and inorganic carbon) to determine a small value (organic carbon). F o r a researcher seeking an inexpensive method for analysing carbon in sediment samples it is difficult to k n o w w h i c h o f L O I or acid-burn is more accurate or i f either is adequate. Further, no comparison has been made among these three methods to determine performance w h e n analysing intertidal or gravel/shell/sand substrates. A n d finally, w h e n w o r k i n g w i t h w h o l e intertidal samples that include small rocks and shell fragments, it is unclear whether these samples must be m i l l e d to get an accurate value for carbon. In this experiment, I address these uncertainties by comparing the performance o f L O I and acid-burn for measuring both organic and inorganic carbon from m i l l e d and u n m i l l e d intertidal sediment samples.  Materials and Methods  Sample Collection and Preparation: Intertidal sediment was collected from a c l a m beach near L a d y s m i t h B r i t i s h C o l u m b i a , Canada ( N 48°59'18.8", W 123°45'33.2") o n 19 January 2005 during an evening l o w tide event. Sediment from the m i d - t i d a l range was excavated from a grid 2 0 c m by 2 0 c m to a depth o f approximately 2 c m . The sediment consisted p r i m a r i l y o f m i x e d sand, gravel and broken shell. The excavated sediment was placed in t w o large plastic sealable bags ( Z i p l o c k brand), labelled and returned to the lab. The sample was kept in refrigeration for two days before being dried in a drying oven (Precision oven by Thermo E l e c t r o n C o r p . ) . The sediment was spread out on a metal pan and dried in the o v e n at 60°C for 48 hours. Once dried, the sediment was fully homogenised by hand in a large mortar and pestle. The homogenised, dry sediment (roughly 80 g) was split into two portions. Representative separation was achieved by t a k i n g 1 scoop and p l a c i n g it in the first portion, the second scoop into the second portion, the third scoop into the first portion  139  again, the fourth scoop into the second portion and so o n until the sample was completely split. F r o m the first portion, 10, 2 g subsamples were taken. E a c h o f these subsamples was randomly assigned to either acid-burn or L O I (described in detail below) for analysis (5, 2 g samples were analysed using each method). The second portion o f the sample was m i l l e d to < 6 3 p m in a R o c k l a b s S w i n g M i l l ( M o d e l # R o c k l a b s C H 2 ) . The m i l l e d sample was stirred to ensure complete m i x i n g then was subsampled into 15 2 g subsamples that were again randomly assigned to acid-burn, L O I , or C H N (again 5 subsamples were analysed w i t h each method). The analysis o f sediment v i a a c i d burn and L O I was repeated w i t h these m i l l e d samples to examine the effect o f m i l l i n g on the value o f carbon and the precision o f measurement.  Acid-Burn: E a c h o f the 2 g subsamples from both the m i l l e d and u n m i l l e d portions (10 subsamples total) were analysed as follows. E a c h subsample was re-dried in the d r y i n g o v e n at 60°C for 2 4 hours, left in a glass desiccator to c o o l to r o o m temperature then weighed o n an analytical balance (Denver Instruments A P X - 2 0 0 ) to obtain Wtj. E a c h subsample was then placed in a 50 m L glass beaker in a fume hood. T o each beaker, 1 0 % H C 1 was added dropwise. A s the carbonate present reacted w i t h the acid, effervescence was apparent. The acid must be added s l o w l y and be dilute (no stronger than 1 0 % ) for this step to be effective (Froelich, 1980). D r o p s o f acid continued to be added until no more effervescence could be observed; this acidification process took approximately 8 hours to complete. Once a c i d i f i e d , the subsamples and acid were washed w i t h distilled water into 5 0 m L centrifuge tubes and centrifuged for 10 minutes at 3500 r p m at r o o m temperature in a Thermo I E C Centra C L 3 refrigerated centrifuge. The supernatant was decanted, then more distilled water was added (roughly 4 0 m L ) to w a s h the acid f r o m the subsample. The subsample and  distilled water was again centrifuged for 10 minutes. T h i s rinsing process was repeated three times then the subsamples were washed w i t h distilled water into p r e - m u f f l e d f o i l pans for drying. T h e y were dried at 60°C for 24 hours, and then weighed again to obtain Wt2. The difference between Wtj.AB and Wt2-AB gave the mass o f carbonate lost in acidification (see equation 1 below). Equation 1:  Wtj.AB - Wt2-AB = Wtc co3-AB a  A f t e r drying and w e i g h i n g , the subsamples were ready for determination o f organic carbon by ashing. The dry subsamples were placed in ceramic crucibles. E a c h crucible was p r e - m u f f l e d at 600°C for 2 hours before adding the subsample to ensure there was no residual carbon o n the crucible. The crucible and subsample were placed in a muffle furnace (Barnstead Thermolyne 114300) at 550°C for 4 hours, then into a glass desiccator to c o o l to r o o m temperature. A f t e r c o o l i n g , the final weight o f the subsample was obtained (Wtf-AB). The difference between Wt2-AB and Wtf-AB gave the mass o f organic carbon lost during m u f f l i n g (see equation 2 below). E q u a t i o n 2:  W t - A B - Wtf.AB = Wtor .c-AB 2  g  LOI: E a c h o f the 2 g subsamples from both m i l l e d and u n m i l l e d portions (10 subsamples total) were analysed as f o l l o w s . E a c h subsample was re-dried in the drying oven at 60°C for 24 hours, left in a glass desiccator to c o o l to r o o m temperature then w e i g h e d o n an analytical balance (Denver Instruments A P X - 2 0 0 ) to obtain Wtj-LoiE a c h subsample was put into a labelled crucible that had been p r e - m u f f l e d at 600°C for 2 hours to remove residual carbon. The crucibles and subsamples were muffled in a Barnstead Thermolyne 114300 furnace at 550°C for 4 hours. A f t e r m u f f l i n g the crucibles were left to c o o l in a glass desiccator until they reached r o o m temperature. Once cooled, the subsamples were re-  141  weighed to obtain \Vt2.L01. The difference between W t j . o i and Wt2-Loi gave the mass o f organic L  carbon lost during m u f f l i n g at 550°C (see equation 3 below). Equation 3:  Wtj.Loi - Wt -LOi = W t 2  o r g  . C-LOI  The crucibles and subsamples were put back into the furnace and m u f f l e d again at 950°C for 2 hours. T h e y were again cooled i n a glass desiccator to r o o m temperature and final weights taken ( W t . i ) . f  LO  The difference between \Vt2-L01and W t f . i gave the mass o f carbonate lost during L O  m u f f l i n g at 950°C (see equation 4 below). Equation 4:  Wt -Loi - W t . o i = Wt co3-LOi 2  f  L  Ca  CHN: The C H N analysis required only a s m a l l amount o f sample for analysis; therefore each 2g subsample was further subsampled for each step in the C H N analysis. It is impossible to carry out this method w i t h u n m i l l e d samples because o f the s m a l l amount o f sample is used, therefore only m i l l e d sediments were analysed by C H N . First, approximately 30 m g o f m i l l e d sediment w a s w e i g h e d out o n a M e t t l e r H 2 0 analytical balance. T h e samples were weighed into glass w e i g h i n g vials then placed in sample tubes. T h e air in the lines o f the coulometer system was purged for at least 1 minute to eliminate contamination by atmospheric CO2. T h e sample w a s injected w i t h 2 0 % H C L w i t h a C M 5 1 3 0 A c i d i f i c a t i o n M o d u l e and the C 0 gas e v o l v e d from the sample w a s carried to and titrated by a 2  C M 5 0 1 4 Coulometer, U I C Inc. This device has been shown to be accurate and reliable for measurement o f CO2 by H u f f m a n (1977). Once the titration endpoint was reached, the coulometer reading showed the amount o f inorganic carbon i n the sample. Measurement o f total carbon was achieved by flash combustion and elemental analysis using a Carlo E r b a N A - 1 5 0 0 A n a l y z e r f o l l o w i n g the methods outlined i n V e r a r d o et a l . , 1990. A p p r o x i m a t e l y 2 0 m g o f m i l l e d sediment was measured into each t i n c u p (pressed cups, 8x5 142  m m ) using a spatula and weighed o n a Mettler Toledo M T 5 M i c r o b a l a n c e for elemental analysis. F r o m this step a value for total carbon was obtained. T o calculate a value for organic carbon (OCCHN), the inorganic carbon value (ICCHN) from coulometric analysis was subtracted from the total carbon (TCCHN) value from elemental analysis (see equation 5 below). Equation 5:  TCCHN - ICCHN= OCCHN  Results E a c h o f the three methods outlined in the introduction were used to analyze the same homogeneous sediment sample for organic and inorganic carbon. Results o f those tests are used to compare L O I and acid-burn w i t h C H N ( C H N is assumed here to accurately measure carbon levels) for the accuracy o f analysis o f carbon from intertidal gravel/sand/shell samples. R e s u l t i n g organic carbon values (expressed in % by weight) from the three methods are presented in figure A 2 - 1 (mean value from each test and sample type w i t h 9 5 % confidence interval) and summarized in table A 2 - 1 . R e s u l t i n g inorganic carbon values are presented in figure A 2 - 2 (mean value from each test and sample type w i t h 9 5 % confidence interval) and summarized in table A 2 - 1 . A n a l y s i s o f variance determined that in both the organic and inorganic trials the means were not equal (F=84.9 and F = l 14.2 respectively).  L e v e n e ' s test for equality o f error variances  also determined that variances were not equal for organic and inorganic measurements (F=4.036 and F=6.541 respectively), therefore the assumption o f equal variances was violated and n o n parametric tests were used for multiple comparisons.  143  Organic Carbon For Each Test Type  o ro  a,b,c 3  O  o  ro cn i O o in  2  1  0 5  5  LOI  CHN  Acid Burn Milled Acid-Burn  LOl-Mlled  Test Used Figure A 2 - 1 : Mean values of organic carbon obtained from each test performed with 95% confidence intervals. N for each test is listed along the x-axis. Letters next to the data points indicate means that are not significantly different (based on non-parametric multiple comparison).  Because means were not equal, multiple c o m p a r i s o n o f means was carried out using a Tamhane non-parametric comparison to determine w h i c h means differed. F o r the values o f organic carbon, the acid-burn mean value did not differ significantly from any o f the other means, l i k e l y due to the large variance in the data. T h e means f o r a c i d - b u r n - m i l l e d and L O I m i l l e d were not significantly different (p=1.00) but differed from C H N and L O I (p<0.0001, p<0.013 respectively). Significant differences are shown i n figure A 2 - 1 , means w i t h the same letter next to it are not significantly different from each other.  144  Inorganic C a r b o n F o r E a c h T e s t T y p e 301  c o n  20-  i_  ro O o 'c co  O)  10-  i o _c O  b  in o> 5  5  Acid Burn Mlled  5  5  LOI  CHN  LOI-Mlled  Acid-Burn  Test Used Figure A2-2: Mean values of inorganic carbon obtained from each test performed with 95% confidence intervals. N for each test is listed along the x-axis. Letters next to the data points indicate means that are not significantly different (based on non-parametric multiple comparison).  Table A2-1: Means and standard errors for each test for carbon analysis method and each value measured. Inorganic Carbon Organic Carbon Test Mean SE N Mean SE N 1.252 19.355 3.094 0.334 5 4 Acid-Burn 0.564 5 14.299 5.014 0.097 Acid-Bum-Milled 5 0.039 5 1.449 1.036 0.049 CHN 5 4.895 0.753 0.258 5 3.239 5 LOI 0.087 5 5.031 5.067 0.096 5 LOI-Milled  F o r the inorganic carbon means, acid-burn and a c i d - b u r n - m i l l e d showed no significant difference (p=0.112) but both means differed significantly from the rest o f the means. The mean inorganic carbon value for C H N and L O I - m i l l e d were significantly different (p<0.0001) but both showed no significant difference w h e n compared to the mean for L O I (p=0.097; p= 1.000 respectively). Significant differences are s h o w n in figure A 2 - 2 as means w i t h different letter labels.  145  Variances were determined to be unequal, therefore I compared variances using multiple comparison o f the variances according to L e v y (1975). Results o f the multiple comparison o f variances for the organic carbon values showed that the error variance for all tests except C H N were not significantly different, while the variance o f C H N was not significantly different f r o m the a c i d - b u r n - m i l l e d and L O I - m i l l e d variance (table A 2 - 2 ) . Figure A 2 - 3 shows the results o f the multiple comparison for organic carbon values, the underline connects variances that were not significantly different.  Table A2-2: Significance values for multiple comparisons of means for each comparison of test type for organic carbon values. LOI LOI-Milled CHN Acid-Burn Acid-Burn-Milled 0.068* 1.000* 0.074* 0.076* Acid-Burn 0.012 1.000 0.000 0.074* Acid-Burn-Milled 0.000 0.008 0.000 0.076* CHN 0.011 0.012 0.008 1.000* LOI 0.011 0.000 1.000 0.068* LOI-Milled *SE=0.2702 for these comparisons SE=0.2548 for the remaining comparisons  a acid-burn  a LOI  a acid-burn-mill  a LOI-Mil  a CHN  Figure A2-3: Result of multiple comparison of error variances for organic carbon values. Variances connected with underline represent variances that were not significantly different.  F o r the inorganic carbon values, the error variance for acid-burn, L O I and a c i d - b u r n m i l l e d showed no significant difference. T h e variance for L O I - m i l l e d and C H N also showed no significant difference. Table A 2 - 3 and figure A 2 - 4 shows the results o f the multiple comparison for inorganic carbon values, the underline connects variances that were not significantly different.  146  Table A2-3: Significance values for multiple comparisons of means for each comparison of test type for Acid-Burn Acid-Burn Acid-Bum-Milled CHN LOI LOI-Milled  3 s«9 :  0.112 0.001 0.000 0.003  Acid-Burn-Milled 0.112 0.000 0.000 0.001  CHN 0.001 0.000 0.097 0.000  LOI 0.000 0.000 0.097  LOI-Milled 0.003 0.001 0.000 1.000  1.000  SE=0.9924 for all comparisons  q acid-burn  a LOI  a acid-burn-mill  a LOI-Mil  Q CHN  Figure A2-4: Result of multiple comparison of error variances for inorganic carbon values. Variances connected with underline represent variances that were not significantly different.  The L O I and acid-burn methods presented essentially the same mean level o f organic carbon for the sample analysed and had comparable variance. B o t h methods use m u f f l i n g to determine the amount o f organic carbon present so this result shows that pre-treatment w i t h acid does not significantly affect the level o f organic carbon measured. L O I and acid-burn both overestimated the amount o f organic carbon present b y nearly three times compared to C H N . The amount o f organic carbon appeared to increase after m i l l i n g b y the same amount i n both the L O I - m i l l e d and a c i d - b u r n - m i l l e d tests. T h i s is possibly due to contamination o n handling during m i l l i n g , or more likely caused b y increased surface area a l l o w i n g for more o f the sample to be oxidised o n m u f f l i n g . The acid-burn method showed significantly higher values for inorganic carbon measurements compared to both C H N and L O I , however both L O I and acid-burn methods overestimated the amount o f inorganic carbon present in the samples compared to C H N . L O I overestimated the level o f inorganic carbon b y approximately three times compared to C H N , w h i l e acid-burn overestimated b y nearly 12 times. M i l l i n g greatly decreased the error variance for L O I measurement o f inorganic carbon i n the sample. 147  A l t h o u g h comparably inexpensive to run, acid-burn demanded a greater time investment compared to L O I .  Furthermore, the steps involved in a c i d i f y i n g , washing and decanting the  sample were tedious and I found that this resulted in a greater potential for error such as loss o n decanting. L O I is a simple and straightforward method to carry out. It involves less handling o f the sample and fewer transfers from one container to another thus decreasing the opportunity for false sample loss. One drawback o f L O I is that there are no widely accepted temperatures and length o f time for each step ( H e i r i et a l . , 2001).  Discussion W h e n comparison o f samples from several sites is the goal o f analysis, both L O I and acid-burn w o u l d provide sufficient data for relative comparison. In comparison studies the overestimation o f carbon levels should remain constant across samples and therefore not impact the end comparison o f one group o f samples to another. H o w e v e r , in cases where quantitative analysis is being carried out and the specific levels o f carbon are o f interest, both o f these methods w o u l d lead to false overestimation and possible incorrect conclusions. The values estimated for inorganic carbon by acid-burn are o f greatest concern as they largely overestimated the levels in this study w h e n compared to the more accurate (and costly) C H N analysis. This study makes it possible to assess the most appropriate method for analysis o f intertidal sediments and provides an estimation o f the errors inherent in each method. In this case, L O I was both easier to carry out and more accurate when measuring inorganic carbon. In the future, investigators with a particular interest in inorganic carbon from intertidal samples should avoid the use o f acid-burn and interpret previous data collected in this manner appropriately. Past research using a c i d - b u r n to determine carbon levels for intertidal sediments are likely to have estimations o f organic carbon that are comparable to research using L O I , 148  however the estimations for inorganic carbon are l i k e l y a far greater overestimation than i f L O I had been used to measure carbon.  Conclusions The overall performance o f the two methods ( L O I and acid-burn) was comparable w h e n measuring organic carbon and preformed best o n the u n m i l l e d sample in that case. H o w e v e r , when measuring inorganic carbon, L O I performed better than acid-burn. In cases when elemental analysis is not affordable, intertidal samples consisting o f gravel/shell/sand substrates should be analysed using L O I o n homogenised samples. M i l l i n g can be done to increase precision; however that additional step does not change the accuracy o f inorganic carbon measurement. It is cautioned that m i l l i n g may produce overestimations o f organic carbon. I advise that investigators analysing levels o f organic and inorganic carbon in intertidal sediments initially compare reference sample L O I determinations w i t h levels determined by elemental analysis to quantify the level o f overestimation that L O I yields. In cases where comparisons are being made and the actual carbon values are not the goal, this overestimation becomes less o f a concern.  149  References A p p e n d i x 2 B a r i l l e - B o y e r , A . , L. B a r i l l e , H . M a s s e , D . Razet, and M . H e r a l , 2003. Correction for particulate organic matter as estimated by loss o n ignition in estuarine ecosystems. Estuarine Coastal and S h e l f Sciences, 5 8 : 147-153. B i s u t t i , I., I. H i l k e , and M . Raessler, 2004. Determination o f total organic carbon - an o v e r v i e w o f current methods. Trends in A n a l y t i c a l Chemistry, 2 3 : 7 1 6 - 7 2 6 . B y e r s , S . C , E. L. M i l l s , and P. L. Stewart, 1978. A comparison o f methods o f determining organic carbon in marine sediments, w i t h suggestions for a standard method. Hydrobiologia, 58: 43-47. F r o e l i c h , P. N , 1980. A n a l y s i s o f organic carbon in marine sediments. L i m n o l o g y and Oceanography, 2 5 : 5 6 4 - 5 7 2 . G i b b s , R. J . 1977, Effects o f combustion temperature and time, and o f the oxidation agent used in organic carbon and nitrogen analysis o f marine sediments and dissolved organic material. Journal o f Sedimentary Petrology, 4 7 : 5 4 7 - 5 5 0 . Hedges, J . L , and J . H . Stern, 1984. C a r b o n and nitrogen determinations o f carbonate-containing solids. L i m n o l o g y and Oceanography, 2 9 : 6 5 7 - 6 6 3 . H e i r i , O., A . F. Lotter, and G . L e m c k e , 2 0 0 1 . L o s s o n ignition as a method for estimation organic and carbonate content in sediments: reproducibility and comparability o f results. Journal o f P a l e o l i m n o l o g y , 2 5 : 101-110. H u f f m a n , E. W . D , 1977. Performance o f a new automatic carbon dioxide coulometer. M i c r o c h e m i s t r y Journal, 2 2 : 5 6 7 - 5 7 3 . L e o n g , L. S . , and P. A . Tanner, 1999. C o m p a r i s o n o f methods for determination o f organic carbon in marine sediment. M a r i n e P o l l u t i o n B u l l e t i n , 3 8 : 8 7 5 - 8 7 9 . L e v y , K . J , 1975. A n e m p i r i c a l comparison o f several multiple range tests for variances. Journal o f the A m e r i c a n Statistical A s s o c i a t i o n , 7 0 : 180-183. L o h s e , L., R. T. Kloosterhuis, H . C . de Stiger, W . Helder, W . V . Raaphorst, and T. C . E. V a n W e e r i n g , 2000. Carbonate removal by acidification causes loss o f nitrogenous compounds in continental margin sediments. M a r i n e Chemistry, 6 9 : 1 9 3 - 2 0 1 . L u c z a k , C , M . Janquin, and A . K u p k a , 1997. S i m p l e standard procedure for the routine determination o f organic matter in marine sediment. H y d r o b i o l o g i a , 3 4 5 : 87-94. R y b a , S. A . , and R. M . Burgess, 2002. Effects o f sample preparation o n the measurement o f organic carbon, hydrogen, nitrogen, sulphur and o x y g e n concentrations in marine sediments. Chemosphere, 4 8 : 139-147. T u n g , J . W . T., and P. A . Tanner, 2003. Instrumental determination o f organic carbon in marine sediments. M a r i n e Chemistry, 8 0 : 161-170. 150  V e r a r d o , D . J . , P. N . F r o e l i c h , and A . M c l n t y r e , 1990. Determination o f organic carbon and nitrogen in marine sediments using the Carlo E r b a N A - 1 5 0 0 A n a l y z e r . Deep Sea Research, 3 7 : 157-165. W e l i k y , K., E. Suess, C . A . Ungerer, P. J . M u l l e r , and K . Fisher, 1983. Problems w i t h the accurate carbon measurements in marine sediments and particulate matter in seawater: A new approach. L i m n o l o g y and Oceanography, 2 8 : 1252-1259. Y a m a m u r o , M . , and H . K a y a n n e , 1995. R a p i d direct determination o f organic carbon and nitrogen in carbonate-bearing sediments w i t h a Y a n a c o M T - 5 C H N analyzer. L i m n o l o g y and Oceanography, 4 0 : 1001-1005.  151  A p p e n d i x 3 : L a r v a l s e t t l e m e n t d a t a f r o m 2002 In addition to larval s a m p l i n g i n 2003 and 2004 reported in Chapter 4 , samples were taken f r o m B e a c h 1 only in 2002 and counted for recently settled Venerupis philippinarum.  The  methods o f sampling and counting were the same as reported previously i n Chapter 4. The f o l l o w i n g is a b r i e f summary o f the results o f the 2002 counts. Counts o f V. philippinarum  early recruits were made at the netted and non-netted plot at  B e a c h 1 over 4 sampling events in 2 0 0 2 . T h e dates o f sampling were A u g u s t 8 , A u g u s t 1 9 , t h  September 5  t h  th  and October 1 0 . T w e n t y - f o u r cores were taken at each plot o n each sampling th  date, except October w h e n only twelve were taken. These cores were frozen a n d later counted for the number o f n e w l y settled c l a m larvae. The results o f these counts are shown i n Figure A 3 - 1 . T h e first s a m p l i n g date showed very f e w settlers, f o l l o w e d b y a larger set o n the three subsequent s a m p l i n g dates. I tested the mean number o f early recruits per m at netted and n o n 2  netted plots using A N O V A for each sample date separately. T h e first and third sample date (August 8 and September 5 ) showed no significant difference between early recruit density at t h  th  netted versus non-netted plots (p=0.25, n=48; p=0.173, n=49 respectively). T h e second and fourth sample dates (August 1 9 and October 10 ) d i d s h o w a significant difference between th  th  netted and non-netted early recruit density (p=0.018, n=44; p=0.023, n=24 respectively). L e n g t h measurements were also made o n the clams counted f r o m the samples taken in 2002. A v e r a g e lengths for each sample date for netted and non-netted plots are shown in Figure A 3 - 2 . O v e r a l l sample dates c o m b i n e d , average early recruit length for the netted plot was 290 p m ( ± 78 p m standard deviation) and for the non-netted plot was 2 9 9 u m ( ± 8 1 p m S . D . ) . E a r l y recruit length showed little difference between the netted and non-netted plots. A n a l y s i s o f variance was performed o n length measurements f o r each sample date separately to compare mean length o f clams at netted versus non-netted plots. There was no significant difference  152  between shell lengths at netted and non-netted plots (Date 1: p=0.67, n=20; Date 2: p=0.34, n=91; Date 3 : p=0.29, n = l 10; Date 4: p - 0 . 1 4 , n=56).  7000  Early Recruit Density Beach 1 2002  6000 5000 A  -®—NoNet ••— N e t  |  CO C  4000  J : 3000 co 2000 1000  Aug. 8  Aug. 19  Sept. 5  Oct. 10  S a m p l e Date Figure A 3 - 1 : Density of settled Venerupis philippinarum larvae per m in 2002 at Beach 1 site. Netted plot is shown with the black hatched line, non-netted plot is shown in the grey solid line. Error bars represent the 9 5 % confidence interval. For each point, n=24 except the October data where n=12. 2  L e n g t h frequency o f the larvae between the second and third sample dates (August 1 9  th  -  September 5 ) a l l o w us to examine growth as I did in Chapter 4. D a t a from both plots shows a th  peak at length 250 u m o n the second sampling date, that peak shifts to 380 p m o n the next sampling date (Figure A 3 - 3 ) . Based o n the difference in shell length and time elapsed, the growth rate was approximately 7.6 um/day. This estimate is based o n the assumption that the group measured on date 2 is the same cohort that was measured o n the third date. This does not account for immigration/emigration and mortality losses and therefore is a generalised estimate o f growth rate.  153  450  Early Recruit L e n g t h B e a c h 1 2002  • Net • NoNet  400 350 E 3  cn c 0) CO  300 250  Sfef  200 150 -\ 100 50 0 Aug. 8  Aug. 19 Sept. 5 Sample Date  Oct. 10  Figure A3-2: Lengths of Venerupis philippinarum clams sampled in 2002. Netted plots shown with hatched bars and non-netted plots shown with grey bars. Error bars represent 95% confidence interval.  154  Length Frequency Beach 1 Net 2002  200  250  300  350  400  450  500  550  600  Shell Length (um) A3-3: Length frequency graphs for Venerupis philippinarum early recruits collected in 2002 Beach 1. Solid black line shows the length frequency for August 19 , the open dotted line shows September 5 . The top panel shows data from the neted plot, the bottom panel shows the non-netted plot. Figure  th  th  A p p e n d i x 4: F i e l d site v e l o c i t y m e a s u r e m e n t s C l o d cards were used to estimate the water velocity at each field site. E a c h c l o d card was made f o l l o w i n g the methods outlined in A p p e n d i x 5. A spike 3 c m in length attached to the bottom o f each plaster c l o d allowed it to be anchored in the sediment (Figure A 4 - 1 ) . E a c h c l o d was dried in a drying oven and weighed before deployment in the field. A f t e r deployment the clods were again dried and re-weighed to determine mass lost during deployment. The mass lost can be converted to an estimate o f velocity using controlled calibration in the laboratory.  I  calibrated the clods using both laminar and turbulent f l o w regimes (see A p p e n d i x 5 for the calibration). S i x clods were deployed at each plot within each beach for a period o f 24 hours.  An  initial deployment was carried out o n August 2 4 2004 however an overnight storm occurred t h  and those data were not used. A second deployment o n A u g u s t 3 0 2006 w a s used to generate t h  the data shown in Figure A 4 - 2 . This figure shows the estimated velocities based o n the turbulent calibration relationship. V e l o c i t y estimates ranged f r o m three to eight cm/s. B e a c h 4 showed the slowest velocity and B e a c h 2 showed the highest. These measurements were made o n a spring tide and therefore are l i k e l y to be a slight overestimation o f average intertidal velocities for these locations.  156  Figure A4-1: Positioning of the clod card in the sediment. A small plastic spike attached to the bottom of the clod allows it to be inserted into the sediment to keep it in place.  Velocity predicted by Clod Cards  10  Based on turbulence calibration  9 8 7 « E 3  6  5  No  Yes Beach 1  No  Yes Beach 2  No Beach 3  Figure A4-2: Velocity measurements from field sites as estimated by clod card dissolution. Beach 1 shown in white bars, Beach 2 in light grey bars, Beach 3 in dark grey bars and Beach 4 in black bars. Bars with hatch marks represent netted plots, bars without hatching represents non-netted plots. Error bars show the 95% confidence interval, n=6 for each bar. Relationship for turbulent calibration is shown in Appendix 5.  157  Appendix 5: Consideration of turbulence in calibration of plaster blocks used for flow measurement.  Introduction Measurement o f rate o f water f l o w is important to the study o f marine ecological systems, particularly those i n v o l v i n g settlement o f invertebrate larvae ( B u t m a n , 1990). A simple and inexpensive method f o r assessing relative rate o f water movement was first described by M u u s (1968). The rate o f dissolution o f plaster balls in seawater is dependant o n the rate o f water f l o w past the plaster surface. M u u s placed plaster balls o f k n o w n d r y mass into f l o w i n g water where a portion o f the b a l l w o u l d dissolve. T h e balls were then removed from the water, dried and weighed again to determine the amount o f dissolution. This technique has been used by many investigators to determine water motion in the field (see Porter et al. (2000) for references listed therein). The preparation o f plaster blocks, or c l o d cards as they were called by D o t y (1971), involves casting plaster o f paris (gypsum) i n ice cube trays (Doty, 1971). A l t h o u g h a radially symmetrical shape is preferable for dissolution trials (Denny, 1988), the efficiency and ease that is a l l o w e d b y using ice cube trays makes this a desirable cast. T h e success o f this method is dependant o n accurate laboratory calibration prior to placement o f cards into the field. Calibration involves measurement o f dissolution o f the clods under k n o w n f l o w rates at the same temperature and salinity as those that w i l l be encountered in the field. Rate o f dissolution increases w i t h temperature (Denny, 1988), therefore, temperature must be matched i n the laboratory calibrations to what w i l l be seen the  field.  A version o f this appendix was published i n : M u n r o e , D . , and S . M c K i n l e y . (2005) Consideration o f turbulence in calibration o f plaster blocks used for f l o w measurement. Aquaculture Canada 2004 Proceedings o f Contributed Papers. A A C Spec. P u b l . N o . 9. C.I. Henrdry Editor. 158  One p r o b l e m w i t h this method that is rarely adequately accounted for by investigators is the influence o f steady f l o w versus turbulent f l o w (Porter et a l . , 2000). In m a n y calibrations, f l o w is steady and smooth, while f l o w s in the field are l i k e l y to be more turbulent or m i x e d . Turbulent f l o w leads to a greater exchange between a surface and the o v e r l y i n g water (Denny, 1988) and thus greater dissolution o f the c l o d card. Without consideration o f this effect velocity o f water w o u l d be overestimated w h e n steady f l o w calibrations are used to evaluate turbulent f i e l d flows (Porter, et a l . , 2000). In this experiment, both turbulent and steady f l o w calibrations were carried out at overall f l o w rates ranging from 0 cm/sec to 4 cm/sec to determine the difference in dissolution rate created by the difference in f l o w structure.  Materials and Methods C l o d cards were prepared f o l l o w i n g the methods used b y T h o m p s o n and G l e n n (1994). Plaster o f Paris dry m i x (produced by D A P Inc. 2002) was m i x e d 2 parts plaster to 1 part clean c o l d water. The liquid was transferred into ice cube trays w i t h a baster then the side o f the tray was tapped to remove air bubbles. Plastic c o c k t a i l swords were inserted; handle d o w n , into each cast to be used later for attachment in the field and for labelling. The plastic swords were used to avoid metal that may corrode and break apart in the field and thus affect the final weight o f the c l o d card. The c l o d cards were allowed to harden for at least 30 minutes before removal from the tray. A f t e r removal f r o m the tray they were dried in a drying o v e n for 68 hours at 30 °C. P r i o r to initiation o f the experiment, each dry block was pre-weighed and labelled w i t h a unique number. E a c h block was 4.4 c m by 3.6 c m at the base and 3.0 c m by 1.9 c m at the top and had a height o f 2.6 c m . The final dimensions o f the c l o d cards are shown in figure A 5 - 1 . The weight o f each card was 25.39 g (standard error = 0.1 Og). F l u m e tanks were set up w i t h  bulk f l o w o f 1, 2, 3 , or 4 cm/s. T w o tanks were used, each was 250 c m long, one was 40.5 c m wide w h i l e the other was 35 c m w i d e and both were filled to a depth o f 10.5 c m . Water supplied to each flume was from a re-circulating system that contains roughly 7 6 0 0 L o f filtered salt water, therefore saturation as the blocks dissolved was not a concern. In each flume, one end contained turbulence w h i l e the other flowed smoothly. L a m i n a r f l o w s were achieved by p l a c i n g a large honeycomb shaped m a n i f o l d (holes 0.5 c m diameter, 15 c m long) w i t h i n the f l o w to constrain it. B o t h turbulent and laminar flows were confirmed by using dye to visualize the streaklines ( V o g e l , 1996). A t each bulk f l o w rate 4 blocks were placed in each type o f f l o w for 24 hours. S t i l l water (0 cm/s) calibrations were carried out w i t h 1 b l o c k suspended in a 20 L tank w i t h no i n f l o w or outflow for 24 hours. S t i l l water calibrations were repeated 4 times w i t h water replacement each time. A l l trials were run at a temperature o f 13°C. A f t e r 24 hours in the water the blocks were retrieved and again placed in a drying oven for 68 hours at 30 °C. After drying, each b l o c k was weighed a second time to determine mass lost over 24 hours.  4.4 c m  Figure A5-1: Dimensions of clod cards used.  160  Results The dissolution o f the blocks showed a linear relationship between percent mass lost and water velocity for both laminar and turbulent flows (figure A 5 - 2 ) . T h e slope o f the graph o f percentage mass lost and water velocity was 1.65 f o r laminar flows (R =0.83) and 2.74 for 2  turbulent flows (R =0.71). T h e dissolution rate o f b l o c k s i n turbulent f l o w was significantly 2  higher than in laminar f l o w at the same b u l k f l o w rate (P<0.0001). T h e slope o f the turbulent f l o w relationship is greater than that o f the laminar f l o w and therefore the difference i n percent mass lost between laminar and turbulent flows is enhanced at higher f l o w rates. T h e variability o f the data points around the linear relationship is greater for turbulent f l o w than for laminar f l o w (lower R ) . 2  37  m  R = 0.708 5 2  •  m  "'" •  .o  A  R = 0.8291 2  • turbulent A laminar  1.5  2  4.5  2.5  Velocity (cm/sec) Figure A5-2: Graph of percentage mass lost from blocks over 24 hour period at flows from 0 - 4 cm/sec. Turbulent data points shown with black squares and solid trendline (R =0.7085), laminar datapoints shown with grey triangles and dashed trendline (R =0.8291). 2  2  161  Discussion D i s s o l u t i o n o f plaster blocks has been used extensively as a method for field estimation for f l o w rates (Peticrew and K a l f f , 1 9 9 1 ; K o m a t s u and K a w a i , 1992). Calibration o f the blocks prior to placement in the field is crucial to the success o f the method. Calibration has been p r i m a r i l y carried out under smooth f l o w conditions and the observed relationships between dissolution and f l o w are good in most studies (Porter et a l . , 2000). H o w e v e r , one factor that is often overlooked is the type o f f l o w being investigated and laminar calibrations are c o m m o n l y applied to turbulent flows in the field ( K o m a t s u and K a w a i , 1992; Porter et a l , 2000). In this experiment, I found that dissolution o f the blocks increased w i t h increasing water f l o w in a linear relationship, as expected. I also found that the rate o f increase was higher w h e n the f l o w was turbulent versus when it was smooth. The difference in dissolution rates between laminar and turbulent f l o w s must be considered w h e n calibrations are carried out. In cases where calibrations are carried out in a laminar environment and field measurements are made in a turbulent environment, the water velocity w i l l be overestimated due to this increase in dissolution. The degree o f overestimation is increased at higher bulk f l o w rates. Another result o f this experiment was that in turbulent flows there was greater variability in the relationship between f l o w and dissolution. T h i s is also important to note when carrying out calibrations that w i l l be used to measure turbulent flows in the  field.  O v e r a l l , this method o f f l o w measurement is effective and easy to use. I f the intended outcome is comparison o f sites w i t h similar turbidity, then an appropriate calibration w i l l lead to reliable results. H o w e v e r , i f sites to be examined show differences in the level o f turbulence, then the effect o f the turbulence o n the dissolution must be considered.  162  References A p p e n d i x 5 B u t m a n , C . A . 1990. Sediment-trap experiments o n the importance o f hydrodynamical processes in distributing settling invertebrate larvae i n near-bottom waters. Journal o f Experimental M a r i n e B i o l o g y and E c o l o g y ,  134: 3 7 - 8 8 .  D e n n y , M . W . 1988. Biology and the Mechanics of the wave-swept environment. Princeton U n i v e r s i t y Press, Princeton, N e w Jersey. 329 pp. D o t y , M . S . 1971. Measurement o f water movement i n reference to benthic algal growth. B o t a n i c a M a r i n a , 14: 3 2 - 3 5 . K o m a t s u , T., and H . K a w a i . 1992. Measurement o f time-averaged intensity o f water motion w i t h plaster balls. Journal o f Oceanography, 4 8 : 3 5 3 - 3 6 5 . M u u s , B . 1968. A field method for "exposure" by means o f plaster balls. A preliminary account. Sarsia, 3 4 : 6 1 - 6 8 . Petticrew, E . L., and J . K a l f f . 1991. Calibration o f a gypsum source for freshwater flow measurements. Canadian Journal o f Fisheries and A q u a t i c Sciences, 4 8 : 1244-1249. Porter, E . T., L . P . Sanford, and S . E . Suttles. 2000. G y p s u m dissolution is not a universal integrator o f 'water m o t i o n ' . L i m n o l o g y and Oceanography, 4 5 : 145-158. T h o m p s o n , T . L., and E . P. G l e n n . 1994. Plaster standards to measure water motion. L i m n o l o g y and Oceanography, 3 9 : 1768-1779. V o g e l , S . 1996. Life in moving fluids. Princeton U n i v e r s i t y Press, N e w Jersey. 467 p.  163  

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