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Salinity and the physiology of three chironomid species which inhabit saline lakes Sargent, Randall Wayne 1978

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SALINITY AND THE PHYSIOLOGY OF THREE CHIRONOMID SPECIES WHICH INHABIT SALINE LAKES by RANDALL WAYNE SARGENT B.Sc. (Honours), University of B r i t i s h Columbia,  1975  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October, 1978 @  Randall Wayne Sargent, 1978  In p r e s e n t i n g t h i s  thesis  in p a r t i a l  f u l f i l m e n t o f the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, the L i b r a r y s h a l l I  make i t  freely available  f u r t h e r agree t h a t p e r m i s s i o n  for  I agree  r e f e r e n c e and  f o r e x t e n s i v e copying o f t h i s  study. thesis  f o r s c h o l a r l y purposes may be granted by the Head of my Department by h i s of  this  representatives.  It  written permission.  Department of  2075 Wesbrook Place Vancouver, Canada V6T 1W5  or  is understood that copying o r p u b l i c a t i o n  thesis f o r f i n a n c i a l gain s h a l l  The U n i v e r s i t y o f B r i t i s h  that  Columbia  not be allowed without my  ii  ABSTRACT This thesis deals with the importance of s a l i n i t y to the d i s t r i b u t i o n of three chironomid  species of the genus  Chironomus (C. anthracinus. C. athalassicus. and C. tentans). Research to date suggests that s a l i n i t y and  coexistence  problems are the major factors influencing the d i s t r i b u t i o n of the chironomid  fauna of fresh and saline lakes i n the  Cariboo and C h i l c o t i n areas of central B r i t i s h Columbia. The difference i n the d i s t r i b u t i o n of these three Chironomus species i s p a r t i c u l a r l y interesting.  The investigation of  the importance of s a l i n i t y to t h e i r d i s t r i b u t i o n consisted of a study of ( i ) the s a l i n i t y tolerance of each species, ( i i ) t h e regulation of the haemolymph, and ( i i i ) the influx and efflux of sodium and chloride ions. Several conclusions were drawn from the investigation. A difference i n the s a l i n i t y tolerance of each species  was  found i n the laboratory: C. anthracinus and C. tentans did not survive at lake water conductivities above 9000 microo mhos/cm at 25 C, C. athalassicus survived i n lake water conductivities at least as high as 15»000 micromhos/cm at o 25 C.  Temperature affected the survival of each species i n a  similar way;  at high temperatures survival time decreased.  Sodium, potassium, and chloride as well as the concentration of the haemolymph were regulated by the three species at low s a l i n i t i e s .  C. athalassicus was the only species able to  conform at higher external concentrations.  C. athalassicus  iii  had a low sodium a f f i n i t y and a powerful sodium uptake system compared to the other species.  Chloride a f f i n i t y and the  power of the uptake system exceeded that of C. anthracinus and C. tentans. The general conclusion reached was that s a l i n i t y does affect the d i s t r i b u t i o n of the three Chlronomus species. More research i s c a l l e d for i n t h i s and related areas i n order to more f u l l y understand the d i s t r i b u t i o n of the chironomid  fauna.  iv  TABLE OF CONTENTS A. P r e l i m i n a r y Pagess  Page  T i t l e Page Abstract Table of Contents L i s t of Tables L i s t of F i g u r e s Acknowledgement  i i i iv v vi viii  B. T e x t : I. Introduction  1.  I I . M a t e r i a l s and Methods  h.  a) b) c) d)  C o l l e c t i o n and I d e n t i f i c a t i o n S a l i n i t y Tolerance of Larvae R e g u l a t i o n of Haemolymph Sodium and C h l o r i d e F l u x  h. 5. 7. 8. m-.  I I I . Results a) S a l i n i t y Tolerance of Larvae b) R e g u l a t i o n of Haemolymph c) Sodium and C h l o r i d e F l u x  1^f. 19. 29.  IV. D i s c u s s i o n a) S a l i n i t y Tolerance of Larvae b) R e g u l a t i o n of Haemolymph c) Sodium and C h l o r i d e F l u x  51. 62.  V. C o n c l u s i o n Literature  h1. k-5.  Cited  6h.  V  LIST OF TABLES TABLE 1 The Occurrence of Chironomidae i n the Lakes at the One Meter Depth.  PAGE 3»  TABLE 2 The Composition of Rock Lake Water.  6.  TABLE 3 The Composition of A r t i f i c i a l Lake Water Solutions.  11  TABLE h The Equations ^escribing the Results of Flux Measurements.  36  TABLE 5 The Sodium Flux Rates of Several Invertebrate Species.  57 58  vi LIST OF FIGURES PAGE FIGURE 1; The Survival of Fourth Instar C. anthracinus. 1 5. and C. tentans Larvae i n Rock L. Water. FIGURE 2 The Survival of Fourth Instar C. athalassicus 1 7 . Larvae i n Rock L. Water. FIGURE 3 The Survival of Fourth Instar C. athalassicus Larvae i n Rock L. Water Conductivities Above 10,000 micromhos/cm (at 25°C).  18.  FIGURE h The Effect of Transfer to a Medium of D i f f e r ent Sodium Concentration on the Sodium Concentration of the Haemolymph of Chironomus Larvae.  20.  FIGURE 5 The Effect of Transfer to a Medium of D i f f erent Concentration on the Freezing Point Depression of the Haemolymph of Chironomus Larvae.  21.  FIGURE 6 The Freezing Point depression of the Haemolymph of Fourth Instar C. anthracinus. C. athalassicus. and C. tentans.Larvae Over a Range of External Medium Concentrations.  23.  FIGURE 7 The Sodium Concentration of the Haemolymph 25. of Fourth Instar C. anthracinus C. athalassicus, and C. tentans Larvae Over a Range of External Sodium Concentrations t  FIGURE "'8 The Potassium Concentration of the Haemolymph of Fourth Instar C. anthracinus. C. athalassicus. and C. tentans Larvae Over a Range of External Potassium Concentrations.  27.  FIGURE 9 The Chloride Concentration of the Haemolymph of Fourth Instar C. anthracinus. C. a t h a l a s s i c u s and C. tentans Larvae Over a Range of External Chloride Concentrations.  28.  FIGURE 10^The Uptake of Sodium-22 by C. anthracinus, £• athalassicus. and £. tentans Over a 72 Hour Period.  30.  f  vii LIST OF FIGURES CONTINUED PAGE FIGURE 11 The Uptake of Chloride-36 by C. anthracinus. 3 1 . C. athalassicas and C. tentans Over a 120 Hour Period. T  FIGURE 12 The Effect of the External Sodium Concentrat i o n on the Sodium Flux Rate of Fourth Instar C. anthracinus Larvae.  33.  FIGURE 13 The Effect of the External Sodium Concentrat i o n on the Sodium Flux Rate of Fourth Instar C. athalassicus Larvae.  3*+.  FIGURE llf The Effect of the External Sodium Concentrat i o n on the Sodium Flux Rate of Fourth Instar C. tentans Larvae.  35.  FIGURE 15 The Effect of the External Chloride Concentration on the Chloride Flux Rate of Fourth Instar C. anthracinus Larvae.  38.  FIGURE 16 The Effect of the External Chloride Concentration of the Chloride Flux Rate of Fourth Instar C. athalassicus Larvae.  39.  FIGURE 17 The E f f e c t of the External Chloride Concentration on the Chloride Flux &ate of Fourth Instar C. tentans Larvae.  1+0.  FIGURE 18 The Daily Temperature Range i n Three of the Lakes Where Chironomus Species are Abundant.  k2.  FIGURE 19 The Survival Limits of C. anthracinus. C. athalassicus. and C. tentans as Determined by Laboratory Experiments.  ^6.  viii  ACKNOWLEDGEMENT I wish to thank Drs. Scudder, Acton, and P h i l l i p s for their advice and interest i n this thesis.  As a supervisor Dr. Scudder  has been both i n s p i r a t i o n a l and understanding.  This has made  my l i f e as a graduate student more enjoyable. A research topic such as t h i s depends upon certain technical skills.  Joan Martin has generously shared her knowledge  of these s k i l l s , and her time.  Rob Cannings and the graduate  students under Dr. Scudder's supervision have also helped me and I am grateful.  1.  I. INTRODUCTION Saline water, depending on i t s composition and d i l u t i o n , o f f e r s a variety of problems to the survival of i t s inhabitants.  In high s a l i n i t i e s there are problems with water  economy and the balance of the various ions within the body. In low s a l i n i t i e s the problems are with ion retention and the excretion of excess water. Topping and Scudder (1977) have investigated the  chemical  and physical features of many of the fresh and saline lakes in the Cariboo and C h i l c o t i n regions of B r i t i s h Columbia. Topping (1972) and Cannings and Scudder (1978) have documented the d i s t r i b u t i o n of the chironomid fauna i n r e l a t i o n to various c h a r a c t e r i s t i c s of the lakes.  The research to date  suggests  that s a l i n i t y and coexistence problems are the major factors influencing the d i s t r i b u t i o n of the chironomid fauna. d i s t r i b u t i o n of three chironomid  The  species of the genus Chironomus  (C. anthracinus Zetterstedt, C. athalassicus Cannings, and C. tentans Fabricius) i s p a r t i c u l a r l y interesting (Table 1). The difference i n the d i s t r i b u t i o n of the three species i s the focus of t h i s thesis, particular attention i s paid to the importance of s a l i n i t y .  The investigation consisted of  ( i ) the s a l i n i t y tolerance of each species, ( i i ) the regulat i o n of ion levels i n the haemolymph, ( i i i ) the influx of sodium and chloride ions. Knowing what s a l i n i t i e s each species can tolerate i s important  for i t may explain why C. anthracinus and C. tentans  2.  are absent from certain lakes where C. athalassicus i s present. It i s also important to know not only i f s a l i n i t y affects chironomid d i s t r i b u t i o n i n the study area, but how s a l i n i t y affects the individual larvae.  The best way to determine  how s a l i n i t y affects the larvae i s to compare certain aspects of the physiology of the three species i n different  salinities.  Thus the effect of external environment on the haemolymph of the insects was studied by monitoring the osmotic  concentra-  t i o n , and the concentration of sodium, potassium, and chloride of the body f l u i d while each of the parameters was varied i n the external environment.  Regulation of the haemolymph i s  c r i t i c a l because i t i s the haemolymph which bathes the tissues themselves and determines the well-being of the organism as a whole. A further area of chironomid  physiology examined was  the influx and efflux of sodium and chloride ions.  These  ions may be c r i t i c a l to the d i s t r i b u t i o n of C. anthracinus« C. athalassicus. and C. tentans.  Shaw ( 1 9 6 D has suggested  that certain flux values (Km and Vmax) characterize species from fresh, saline, or brackish water habitats. While t h i s study cannot completely explain the effects of s a l i n i t y on chironomid d i s t r i b u t i o n , i t can determine i f s a l i n i t y does i n fact affect chironomid d i s t r i b u t i o n and i n doing so contribute to the understanding physiology.  of chironomid  3a.  TABLE 1  The Occurrence of Chironomidae i n the Lakes at the One Meter Depth. After Topping (1969) and Cannings ( 1 9 7 3 ) .  3.  CONDUCTIVITY SODIUM (micromhos/cm) (mM) @ 25°C  SPECIES  LAKE  W  e o a o  a •H  O CO  • H -p  a  X5 O cd  CO  a o  a o  o  w  CO  a o  •H cn  CO  £  m cO •H £5  o cO -P •H cj  o  o  si  +3  CO  CHLORIDE (mM)  CD  Barnes L.  X  12,000  130.26  36.97  Round-Up L.  X  7,000  71.35  23.69  L. Lye  X  6,600  70.25  21.90  Boitano L.  X  X  X  *+,200  '3^.03  *f.20  L. Jackson  X  X  A  2,600  15.^2  3.28  L. Greer  X  X  X  1 ,600  13.25  1.25  Rock L.  X  X  X  1 ,500  1^.13  1.85  Nr. Phalarope  X  X  X  1,325  7.25  0A9  Westwick L.  X  X  X  1,280  it.61  0.11  Sorenson L.  X  X  X  1,500  hM  0.3+  Nr. Op. Crescent  X  X  X  800  3.^1  0.25  Box 17  X  X  X  7^0  3.31  0.1V  Barkley L.  X  X  X  600  •1.85  o.T5'  East L.  X  X  X  3.08  0.26  0.15  0.07  Box 27  If.  I I . MATERIALS AND METHODS a. C o l l e c t i o n and I d e n t i f i c a t i o n Larvae of Chironomus anthracinus. C. athalassicus. and C. tentans were collected from lakes i n the central i n t e r i o r of B r i t i s h Columbia.  The characteristics of the l o c a l i t i e s  have been described by Topping and Scudder (1977).  C. anthracinus  and C. athalassicus were collected from Jackson L. on the C h i l c o t i n plateau, while C. tentans was collected from Westwick L. i n the Cariboo region. Eckraan dredge samples of bottom mud were collected and s i f t e d i n mesh-bottom tubs.  Fourth instar larvae, recognized  by the enlarged prothoracic segments, were picked but by hand. They were then transported to the laboratory i n one gallon Thermos jugs containing lake water.  In the laboratory, the  larvae were either used immediately, or were held i n constant o temperature cabinets at 5 C and under long photoperiod  (16 hr  l i g h t : 8 hr dark): water was continuously aerated and food, in the form of a mixture of powdered nettle leaves and milk powder, was provided at i n t e r v a l s . The larvae were i d e n t i f i e d by use of the key prepared by Bassett (1967)• Bassett's species IV, V, and VII refer to C. anthracinus  t  C. athalassicus and C. tentans. respectively. T  A l l three species are of the C. dorsalis type, as outlined by M i a l l and Hammond (1900), i n that they have four long, paired blood g i l l s on the posterior ventral surface. I d e n t i f i c a t i o n of species was confirmed by rearing individual larvae to the adult stage.  Males were named by  5. reference to the key of Townes (19*+5)J C. athalassicus  was  i d e n t i f i e d by reference to the description of Cannings ( 1 9 7 5 ) . Fourth instar larvae of the three species were collected together where possible so that they were acclimated similar physical and chemical conditions.  to  However, since  only a limited number of larvae were thus obtained,  those  collected from d i f f e r e n t lakes were acclimated to a single lake water, namely that of Rock L.  (See Table 2 Rock Lake  Composition) A l l larvae were collected i n the spring, and hence were overwintered forms.  Attempts were made to rear the larvae  and obtain egg masses for experimental use. while adults could be obtained  Unfortunately,  i n some cases, the r e s u l t i n g  numbers of egg masses were too small for experimentation. 1  b. S a l i n i t y Tolerance  of Larvae o  Larvae were held at 5 C u n t i l required.  Experiments  were conducted i n m i l l i p o r e f i l t e r e d (HA 0 A 5 JU) Rock L. water which was  d i l u t e d (with d i s t i l l e d water) or a i r  evaporated to produce the desired s a l i n i t i e s . was  Rock L. water  used because of i t s chemical s i m i l a r i t y to Jackson L.  and Westwick L. water, these being the lakes from which the larvae were c o l l e c t e d .  Larvae were acclimated by  sequential  increases of 1000 micromho/cm every 12 hours u n t i l the desired s a l i n i t y was  reached, following the method used by  Beadle (1939) with Aedes d e t r i t u s . The range of s a l i n i t y tested depended upon the upper l i m i t of s a l i n i t y to which the species could  acclimate.  TABLE 2  The Composition of Rock Lake Water. and Scudder, 1 9 6 9 .  After Topping  6.  THE COMPOSITION OF ROCK L. WATER ION Na K  +  M-EQUIV./L. 1^.91  PER CENT TOTAL CATIONS 81.7  O.98  +  Ca  + +  0.62  Mg  ++  1.71+  5 A .  9.5 PER CENT TOTAL ANIONS CO"  M-.32  23.5  HCO3  12.35  67.0  CI*  1.57  8.5  SO""  0.18  1.0  7.  C. anthracinus was tested from 0 to 9000 micromhos/cm ( 0 . 0 0 0 to 0 . 0 9 ^ g-mole/i NaCI), C. tentans from 0 to 9000 micromhos/cm ( 0 . 0 0 0 to 0 . 0 9 ^ g-mole/1 NaCI), and C. athalassicus from 0 to 3 0 , 0 0 0 micromhos/cm ( 0 . 0 0 0 to 0.3W-3 g-mole/1 NaCI).  Control  experiments consisted of placing larvae at t h e i r respective conductivities from which they were collected. For  each species, duplicate samples of ten fourth instar  larvae were placed i n 100 ml of the test s a l i n i t y at 5°C 15°C ( + 1 ° ) ,  and 23°C ( + 3 ° ) .  (+1°),  Acclimation to the higher temp-  eratures was at a rate of 10°C per 12 hour period. conditions were 16 hours l i g h t : 8 hours dark.  Light  A i r was gently  bubbled through a l l test s a l i n i t i e s during the experiments. The length of time that each individual survived was recorded to the nearest half day.  The average time of survival  was calculated f o r each experimental combination of temperature and s a l i n i t y ; these were compared to control groups of the same species. c. Regulation of Haemolymph Fourth instar larvae were acclimated to the experimental s a l i n i t i e s i n steps of 1000 micromhos/cm per 12 hour period. Larvae were acclimated to the Rock L. experimental s a l i n i t y for 96 hours at 5 ° C  Haemolymph was analyzed from  C. athalassicus acclimated to s a l i n i t i e s within the range 0 to 3 0 , 0 0 0 micromhos/cm at 25°C ( 0 . 0 0 0 to 0.3^-3 g-mole/1 NaCI).  The haemolymph of C. anthracinus and C. tentans  was analyzed from larvae acclimated to s a l i n i t i e s within^ the  range 0 to 9000 micromhos/cm at 25°C ( 0 . 0 0 0 to 0.09h g-mole/1  8. NaCl). After 96 hours at the designated s a l i n i t y , samples of haemolymph and of the external medium were taken.  Haemolymph  samples were obtained by removing a larva from the test medium, r i n s i n g with d i s t i l l e d water, drying b r i e f l y on a Kimwipe, placing on Parafilm M, and puncturing the c u t i c l e .  Samples  were taken up i n one microliter Microcap pipettes (Drummond S c i . Co., Brommall, Pa.). determined  Freezing point depression was  from samples taken up i n a sandwich of p a r a f f i n  o i l contained i n non-alkaline glass pipettes.  The osmotic  pressure (Ag) and the concentration of sodium, potassium, and chloride of the samples were  determined.  A nanoliter osmometer ( C l i f t o n Technical Physics, Hartford, N.Y.) was used to read the freezing point depression of the samples.  Chloride concentration determined  with a  m i c r o t i t r a t i o n buret (Misco Microchemical Specialties microburette coupled to a Radiometer 25 voltmeter, Copenhagen, Denmark).  One m i c r o l i t r e samples were t i t r a t e d with a  0 . 0 2 $ AgNO^ solution to an e l e c t r i c a l endpoint (Ramsay, Brown, and Croghan, 1955).  The concentrations of sodium  and potassium were determined spectrophotometer  with an atomic absorption  (Varian Techtron Pty, Ltd., Melbourne,  A u s t r a l i a ) , according to the method of Wright (1975^). d. Sodium and Chloride Flux The terms influx, efflux, net uptake, and net loss are used according to the scheme of Stobbart (1959,  1960).  the experiments described below, sodium-22 as  NaCl and  In  9.  c h l o r i d e - 3 6 as Na-^Cl e r e used to estimate the sodium flux W  and the chloride flux i n fourth instar larvae of C. anthracinus« C. athalassicus, and C. tentans.  The sodium and chloride  f l u x were investigated i n separate experiments, but the methods used were similar. A l l experiments  were conducted at a room temperature  of 23°C ( + 3 ° ) * the temperature recording equipment.  was monitored by continous  Wet weights of individual larvae were  obtained by surface drying with Kimwipes (Scott Paper Co.) then weighing on an August Sauter 50 mg balance Wurtbg., Germany) to an accuracy of 0.01i  (Ebingen,  mg.  The r a d i o a c t i v i t y of samples was determined with a Nuclear-Chicago endwindow G-M stainless steel planchets.  counter (Model *+70) using  Larvae were macerated i n the  planchets using a standardized technique.  Solution samples were  measured with an automatic pipette (Oxford Sampler Micropipetting System), or a Drummond Microcap pipette of appropriate volume.  Samples were a i r dried previous to counting.  A correction factor f o r sample self-absorption was established by f i r s t finding the r a d i o a c t i v i t y of a known amount of isotope, then recounting with a sample i n place on top of the isotope.  Larvae removed from the radioactive  loading medium were rinsed f o r at least a half minute i n a current of non-radioactive Rock L. water.  An estimate of  the r a d i o a c t i v i t y that remained on the outside of the l a r v a l body was found by placing a non-radioactive larva i n radioactive medium for three minutes.  The larva was then subjected  to the standard rinse-off procedure, weighed, and placed on  10. the G-M  counter.  A rinse-off residue correction was  thus  obtained and applied to the estimates of carcass r a d i o a c t i v i t y . To e s t a b l i s h the effect of the external concentration of sodium or chloride ions on the rate of the ion's flux a series of a r t i f i c i a l lake water solutions were prepared. These solutions were composed of fixed concentrations of various salts to which was added a known concentration of sodium or chloride (Table 3). of each solution was was  The freezing point depression  determined and where necessary  added to raise the osmotic concentration.  sucrose  Sodium and  chloride concentrations were ascertained according to the methods described above. To e s t a b l i s h that sodium and chloride enter the body at s i t e s separate from the mouth, larvae of the three species were ligated just posterior to the head capsule with s i l k thread.  Ligated and non-ligated larvae were placed i n s o l -  utions containing sodium-22 Qrd6hloride-36.  At the end of  the experiment the larvae were rinsed, weighed, and r a d i o a c t i v i t y was determined. larvae was  The r a d i o a c t i v i t y of ligated  compared to that of non-ligated larvae.  fQtjn& ;tRat the r a d i o a c t i v i t y was f  carcass  It was  similar i n the two groups,  therefore a site of ion entry other than the mouth must exist. Experiments were conducted to monitor the uptake of sodium-22 and chloride-36 by C. anthracinus. C. athalassicus, and C. tentans.  F i f t y - f i v e fourth instar larvae of each  species were acclimated i n Rock L. water at 23°C for 2h hours. They were placed i n p l a s t i c vials containing a known quantity of isotope (2.28 microCurie  22  N a or 5.50 microGurie  ^Cl)  a n (  i  11a.  TABLE 3 The Composition of A r t i f i c i a l Lake Water Solutions. Where sodium concentration was varied, sodium was added as NaCl and NaHCO^ i n a 1;3 r a t i o .  The sodium  concentrations prepared were: 0.0 mM, 0 . 5 mM, 1.0 mM,  2.0 mM,  k.O mM,  8.0 mM, 16.0 mM, and 2^.0 mM.  Where chloride concentration was varied, chloride was added as NaCl.  The chloride concentrations  prepared were: 0.0 mM, 0.2 mM, 0 . 5 mM, 1.0 mM, 2.0  3.0 mM, 9.0 mM, and 27.0 mM.  mM,  11.  THE COMPOSITION OF ARTIFICIAL LAKE WATER SOLUTIONS Sodium Concentration Varied: COMPOUND  MOLES ADDED  KHCO^  3 . 0 mM  MgSO^.  1.0 mM  MgCl  1.0 mM  CaCO^  1.0 mM  KHgPO^.  1.0 mM  Chloride Concentration Varied: COMPOUND  MOLES ADDED  MgSO^.  1.0 mM  CaCO^  1.0 mM  KH POL  1.0 mM  NaHCO^  l f . 0 mM  NaCO.  2 . 0 mM  2  12. 55ml of m i l l i p o r e f i l t e r e d Rock L. water. the solution was determined at time T . Q  throughout the experiment.  The a c t i v i t y of The water was aerated  At intervals of 1 hr., 2 hr.,  3 hr., 5 hr., 10 hr., 2k hr., *+8 hr., 96 hr., and 120 hr. three to f i v e larvae were removed from each v i a l .  The larvae  were rinsed, weighed, and carcass r a d i o a c t i v i t y was determined. Corrections were made f o r rinse-off residue and f o r self-absorption. Stobbart a body wall.  ( i 9 6 0 ) describes the sequence of ion f l u x across For a short time s p e c i f i c ions move i n one  d i r e c t i o n only: from the outside medium into the body.  After  a period of time there i s f l u x both into and out of the body, eventually leading to a point where there i s no net flux i n either d i r e c t i o n .  During the i n i t i a l influx period the  departure from l i n e a r i t y of the time versus rate of influx relationship would be s l i g h t , and i t can be assumed that backflux would be neglegible.  The length of that short  period of u n i d i r e c t i o n a l flux was estimated from the results of these experiments, with separately.  ^ach of the three species was dealt  The estimate of the u n i d i r e c t i o n a l flux  period was taken as a suitable time f o r short term flux experiments. To establish the i n d i v i d u a l influx rates of sodium and chloride into C. anthracinus. C. athalassicus. and C. tentans larvae, fourth instar larvae of each species were exposed to loading medium f o r the predetermined time period.  Five  larvae were placed i n a 12 dram p l a s t i c v i a l containing  13. 15 ml of a r t i f i c i a l lake water solution and a quantity of radioisotope (*+.90 microCurie  22  N a , or 2 . 6 9 microCurie  -^Cl).  The r a d i o a c t i v i t y of each solution was determined at time T Q . After the experiment, larvae were rinsed, weighed, and prepared for the G-M counter.  Estimates of carcass r a d i o a c t i v i t y  were corrected f o r rinse-off residue and self-absorption. Influx rates were expressed as mmolesAg wet weight/hour. The i n d i v i d u a l efflux rates of sodium and chloride from C. anthracinus. C. athalassicus. and C. tentans larvae were estimated by allowing them to equilibrate i n radioactive sodium-22 or c h l o r i d e - 3 6 solutions over a 96 hour period at 5°C, then moving the larvae into non-radioactive water. This was s u f f i c i e n t time f o r the sodium-22 s p e c i f i c a c t i v i t y i n the larvae to equilibrate with the external environment. Once equilibrated the larvae were rinsed and placed i n d i v i d u a l l y i n 12 dram p l a s t i c v i a l s containing f i v e m i l l i l i t e r s of a r t i f i c i a l lake water solution. were removed and weighed.  After three hours the larvae  Two 0 . 5 ml samples of the solution  were taken from each v i a l , a i r dried, and the r a d i o a c t i v i t y was determined.  Corrections were made for self-absorption  and the efflux rates were calculated as mmole/Kg wet weight/hour. In each of the short term experiments at least three repetitions of each combination of species and medium were carried out.  I I I . RESULTS a. S a l i n i t y Tolerance  of Larvae  The mean s u r v i v a l of fourth instar C. anthracinus and C. tentans larvae i s shown i n Figure 1.  Both species  survived  upon acclimation to a range of 0 to 9000 micromhos/cm at 25°C$ over twice that found i n the f i e l d (Cannings and Scudder, 1978). Acclimation to the higher conductivities i s possible only i f the conductivity i s raised slowly over a long period of time. Such a demonstration of acclimation a b i l i t y does not, however, imply that the larvae could mature and produce a second generation of likewise adapted larvae. The  s u r v i v a l of C. anthracinus  larvae at each conductivity  was compared to the survival at a control conductivity (3500 micromhos/cm at 25°C) using chi-square  t e s t s . The  results showed that survival throughout the range of condu c t i v i t i e s tested was similar to or better than survival at the control conductivity i n each temperature regime. suggests that C. anthracinus  This  larvae can survive at conduct-  i v i t i e s up to 9000 micromhos/cm (at 25°C).  Based on the number  of larvae from a group that were able to acclimate to a conductivity of 9000 micromhos/cm (at 25°C) i t i s doubtful that C. anthracinus  larvae could acclimate and survive at  higher conductivities. When the mean survival of C. tentans larvae at each conductivity was compared to survival i n the control condu c t i v i t y (1500 micromhos/cm at 25°C) by chi-square t e s t s , l i m i t s to the s u r v i v a l of C. tentans could be designated  15a.  FIGURE 1 The Survival of Fourth Instar C. anthracinus and tentans larvae i n Rock L. water. at three temperatures 15°C  are depicted: 5°C  ( — * — ) , and 23°C ( — o — ) .  was determined  Experiments  at 25°C.  One  Each conductivity standard error i s  indicated f o r each survival value. (n= 20 larvae for each point)  (—•—),  CONDUCTIVITY  X  1000  (MICROMHOS/CM  AT  25°C)  16. for  5°C and 15°C. No such l i m i t s could be set at 2 3 ° C  At  5°C C. tentang survived equally well at conductivities from 0 micromhos/cm (at 25°C) ( d i s t i l l e d water) to between 7000 and 9000 micromhos/cm (at 25°C).  Survival at 9000 micromhos/cm  was s i g n i f i c a n t l y less than at the control conductivity. At 15°C  C. tentans survived equally well at conductivities from  0 to 5000 micromhos/cm (at 25°0\ but survival was s i g n i f i c a n t l y reduced at 7000 and 9000 micromhos/cm (at 25°C).  The s i m i l a r i t y  between the laboratory and the f i e l d results suggest that C. tentans does not survive well above 5000 micromhos/cm (at 25°C)when the temperature of the water i s 15°C or more. Figure 2 shows the survival of C. athalassicus larvae over a range of conductivities from 0 to 2 5 , 0 0 0 micromhos/cm (at 25°C).  From Figure 2 i t appears that the larvae survived  markedly better i n the 15,000 to 17,000 micromhos/cm (at 25°C) range with no obvious trend toward reduced survival time at either extreme of conductivity. chi-square  tests which compared s u r v i v a l at each conductivity  to s u r v i v a l at a designated at 25°C).  This was borne out by  conductivity ( 1 7 , 0 0 0 micromhos/cm  No l i m i t s to survival could be set on the basis  of the information contained  i n Figure 2 .  Figure 3 shows the results of a further experiment to f i n d the highest conductivity at which C. athalassicus larvae can survive.  An attempt was made to acclimate larvae to  conductivities as high as 35,000 micromhos/cm (at 25°C). The highest conductivity to which a s i g n i f i c a n t number of larvae acclimated was 3 0 , 0 0 0 micromhos/cm (at 25°C). At  17a.  FIGURE 2 The Survival of Fourth Instar C. athalassicus larvae in Rock L. Water.  Experiments at three temperatures  are depicted: 5°C ( — • — ) ,  15°C  and 23°C ( — o — ) .  Each conductivity was determined at 25°C. errors are omitted f o r c l a r i t y . (n= 20 larvae f o r each point)  Standard  17.  18a.  FIGURE 3 The Survival of Fourth Instar C. athalassicus Larvae i n Rock L. Water Conductivities Above 10,000 micromhos/cm (at 25°C).  Experiments at three temperatures are  depicted: 5°C ( — — ) , 15°C ( — * — ) ,  and 23°C ( — o — ) .  Each conductivity was determined at 25°C.  One  standard error i s indicated f o r each s u r v i v a l value. (n= 20 larvae f o r each point)  19. conductivities above 2 0 , 0 0 0 micromhos/cm (at 25°C), however, the larvae appeared to be i n poor health with a lack of a c t i v i t y and a s h r i v e l l e d appearance.  This observation i s supported  by the results shown i n Figure 3 .  At 2 0 , 0 0 0 micromhos/cm  (at 25°C) i n the 5 ° c and 15°C temperature regimes there was a marked reduction i n the period of survival.  At 23°C  survival was low at conductivities above 15,000 micromhos/cm (at 25°C). Temperature had an important effect on the s u r v i v a l of C. anthracinus. C. athalassicus. and G. tentans. displayed i n Figures 1 , 2 ,  The results  and 3 show that i n every case the  larvae survived longest at 5°C at a l l conductivities, and that survival dropped as temperature increased. b. Regulation of the Haemolymph An acclimation period was established by placing larvae i n Rock L. water of various s a l i n i t i e s .  At time i n t e r v a l s  up to 96 hours after introduction, larvae were removed and the sodium concentration and freezing point depression of t h e i r haemolymph were determined.  During the f i r s t 2k hours  major s h i f t s i n each parameter were observed (Figures *f and 5 ) . No s t a t i s t i c a l l y s i g n i f i c a n t changes were, however, observed after ^8, 72,  and 96 hours.  Based on the results shown i n  Figures h and 5 the larvae were acclimated f o r 96 hours before subsequent experiments were carried out. No problem was encountered i n any of the experiments concerning  the survival  of larvae during the acclimation period. The effects of various external concentrations of Rock L.  Z3<su  FIGURE k The Effect of Transfer to a Medium of Different Sodium Concentration on the Sodium Concentration of the Haemolymph of Chironomus larvae. The acclimation process was followed over a 96 hour period.  Transfer from a medium of 1 9 2 . 5 mM Na  to 2 8 ^ . 5 mM Na i s shown as C — ) . Transfer from a medium of 1 9 2 . 5 mM Na to 1 0 9 . 5 mM Na i s shown as ( — o — ) . each value.  One standard error i s indicated f o r  20.  HAEMOLYMPH  SODIUM  ( m M SODIUM)  I  21a.  FIGURE 5 The Effect of Transfer to a Medium of Different Concentration on the Freezing Point Depression of the Haemolymph of Chironomus larvae.  The acclimation  process i s followed over a 96 hour period. from a medium of A  E  i s shown as ( — — ) . 6  0.^7°C to a medium of A  Transfer E  0.65°C  Transfer from a medium of  Ag 0.>+7C to a medium of A 0  g  0.32°C i s shown as (—o—).  One standard error i s indicated f o r each value.  21.  "  HAEMOLYMPH  "  ~"  FREEZING POINT  DEPRESSION  6  (°C)  6  d  22 water on the freezing point depression of the haemolymph of C. anthracinus. C. athalassicus. and C. tentans i s shown i n Figure 6 .  A l l three species hyperregulated  throughout the  range of external concentrations provided i n the experiment. The haemolymph of each species was  r e l a t i v e l y constant i n  freezing point depression over the range of Ag 0 . 0 0 with no s i g n i f i c a n t difference among the species. concentrations above A  g  to 0.32°C, At external  0.32°C the freezing point depression  of a l l three species rose sharply with C. anthracinus  and  C. tentans unable to survive i n media s i g n i f i c a n t l y above a freezing point depression of A  2  0.39°C.  Above Ag 0.32°C  the freezing point depression of C. athalassicus haemolymph rose sharply but continued to be hyperosmotic to the medium, following the slope of the isosmotic line u n t i l the external concentration reached Ag 0.67°C where the haemolymph concentration increased sharply and thereafter varied considerably. The results shown i n Figure 6 suggest that the three species regulated their haemolymph osmotic pressure i n media up to about Ag 0.32°C, then began to conform to the osmotic pressure of the external medium.  C. tentans and C.  anthracinus  reached the upper l i m i t of their survival range (Ag 0.39°C i s equivalent to a conductivity of 9000 micromhos/cm at 25°C Rock L. water) before the freezing point depression of the haemolymph rose very high, or crossed the isosmotic l i n e . The freezing point depression of the haemolymph of C. athalassicus larvae increased smoothly to a point that corresponds to a Rock L. water conductivity of 15,000 micromhos/cm (at 25°C)  23a.  FIGURE 6 The Freezing Point Depression of the Haemolymph of Fourth Instar C. anthracinus  T  C. athalassicus, and  C. tentans larvae over a range of external medium concentrations.  C. anthracinus i s shown as ( — * — ) .  C. athalassicus i s shown as ( -  _A__  ).  C. tentans i s  shown as (-<>-—). One standard error i s indicated for each value.  (n= h to 6 larvae f o r each point)  23.  2k.  ( A g 0.67°C) which i s near the upper survival l i m i t f o r C. athalassicus larvae. point depression  Such variation i n the freezing  of C. athalassicus may be linked to the  survival problems encountered by C. athalassicus larvae above 15,000 micromhos/cm (at 25°C) i n Rock L. water. The effects of external sodium concentration on each of the three Chironomus species are presented i n Figure A l l three species hyperregulated  7.  haemolymph sodium over the  range 0 to 110 mM sodium; no s i g n i f i c a n t difference was ^ observed amongst them.  Above 110 mM sodium the haemolymph  sodium concentration of each species was below the sodium concentration of the external medium.  C. anthracinus  C. tentans did not survive well i n the media that  and  contained  sodium levels above 110 mM sodium (equivalent to Rock L. water conductivity of 9000 micromhos/cm at 25°C) so i t i s doubtful that the two species are able to hyporegulate sodium. C. athalassicus survived at external media  concentrations  between 110 mM and 180 mM sodium and appeared to hyporegulate sodium.  Above 180 mM sodium (equivalent to Rock L. water  conductivity above 17,000 micromhos/cm at 25°C) the larvae did not survive well and may have been unable to excrete excess sodium. In summary the responses of the three species were similar at low external sodium concentrations, lymph sodium concentrations  and the haemo-  became isotonic with the external  sodium concentration at approximately the same external sodium concentration.  Above that sodium concentration C.  anthracinus  25a.  FIGURE 7 The Sodium Concentration of the Haemolymph of Fourth Instar C. anthracinus« C. a t h a l a s s i c u s and C. tentans T  Larvae Over a Range 6f External Sodium Concentrations. C. anthracinus i s shown as ( — • — ) . shown as (—*--).  C. athalassicus i s  C_. tentans i s shown as ( — o ~ ) .  One standard error i s indicated f o r each value. (n= *f to 6 larvae f o r each point)  25  26. and C. tentans d i d not hyporegulate,  while C. a t h a l a s s i c u s  s u r v i v e d at c o n s i d e r a b l y higher sodium c o n c e n t r a t i o n s appeared to hyporegulate The  of 8 . 0  the haemolymph sodium c o n c e n t r a t i o n .  r e s u l t s of the potassium c o n c e n t r a t i o n  are shown i n F i g u r e 8. mM  and  determinations  Below e x t e r n a l potassium  concentrations  potassium the haemolymph potassium c o n c e n t r a t i o n of  the three s p e c i e s was  only s l i g h t l y above or equal t o t h a t of  the e x t e r n a l medium.  At potassium c o n c e n t r a t i o n s above  8.0  mM  potassium ( e q u i v a l e n t to a Rock L. water c o n d u c t i v i t y  above 10,000 micromhos/cm at 25°C) C. a t h a l a s s i c u s haemolymph potassium c o n c e n t r a t i o n rose s h a r p l y and v a r i e d c o n s i d e r a b l y . At no time d i d the haemolymph potassium c o n c e n t r a t i o n of  any  of the three species f a l l below t h a t of the e x t e r n a l medium. These f i n d i n g s suggest  t h a t the potassium c o n c e n t r a t i o n i n  the haemolymph of the three species conforms to the  potassium  c o n c e n t r a t i o n of the e x t e r n a l environment, and t h a t they not hyporegulate The  do  potassium.  r e s u l t s presented  i n Figure 9 indicate considerable  v a r i a t i o n i n the haemolymph c h l o r i d e c o n c e n t r a t i o n of each s p e c i e s over the range of e x t e r n a l c o n c e n t r a t i o n s t h a t were tested.  The  response of the three s p e c i e s was  probably  s i m i l a r , but the reason f o r the i r r e g u l a r i t i e s i n F i g u r e 9 i s unknown.  The  c h l o r i d e c o n c e n t r a t i o n was  hyperregulated  by the three s p e c i e s at a l l e x t e r n a l c h l o r i d e c o n c e n t r a t i o n s , w i t h the e x c e p t i o n of the r e a c t i o n of C. a t h a l a s s i c u s at 30.3  mM  c h l o r i d e ( e q u i v a l e n t to a Rock L. water c o n d u c t i v i t y  of 3 0 , 0 0 0 micromhos/cm at 25°C).  27a.  FIGURE 8 The Potassium Concentration of the Haemolymph of Fourth Instar C. anthracinus, C. athalassicus, and £• tentans Larvae Over a Range of External Potassium Concentrations.  C. anthracinus i s shown as ( — • — ) .  C. athalassicus i s shown as ( — * — ) .  C. tentans i s  shown as ( - - o - ) .  One standard error i s indicated  for each value.  (n= h to 6 larvae f o r each point)  27.  CM  POTASSIUM  CONCENTRATION  r-  OF  THE  HAEMOLYMPH frnM/l )  28a.  FIGURE 9 The Chloride Concentration of the Haemolymph of Fourth Instar C. anthracinus, C. athalassicus, and C. tentans Larvae Over a Range of External Chloride Concentrations.  C. anthracinus i s shown as ( — • — ) .  C. athalassicus i s shown as (---*—). shown as (—o—). for each value.  C. tentans i s  One standard error i s indicated (n= h to 6 larvae f o r each point)  28  CHLORIDE  CONCENTRATION  OF  THE  HAEMOLYMPH  (rr^M/l)  29.  c. Sodium and Chloride Flux The appropriate period for a short term loading experiment involving sodium-22 v/as found to be two to f i v e hours for C. anthracinus. C. athalassicus. and C. tentans larvae. Figure 10 shows the f i r s t segment of the curves where inflow of sodium-22 was d i r e c t l y proportional to time.  For a short  term loading experiment involving chloride-36 the appropriate period was found to be about three hours as shown by Figure 11. The  loading period that was employed i n the influx and efflux  experiments described below was two and a half hours. A period of 96 hours was allowed for these species to acclimate to the test solutions.  Such an acclimation period  i s shown by Figures 10 and 11 to be s u f f i c i e n t for the three species to reach a steady-state.  No s i g n i f i c a n t difference  i n carcass r a d i o a c t i v i t y per unit wet weight was found after hQ hours acclimation. The  influx and efflux of sodium was investigated i n  steady-state  larvae only; no depletion tests were carried  out since the objective was to compare C. anthracinus. C. athalassicus. and C. tentans.larvae  i n terms of the  conditions encountered i n saline lakes. The relationship between sodium influx and external sodium concentration can be described approximately by the Michaelis-Menton equation, Influx = Vmax x C Km + C Where Vmax = the maximum rate of sodium transport, C = external sodium concentration, and Km = the external sodium concentration  30a.  FIGURE 10 The Uptake of Sodium-22 by C. anthracinus. C. athalassicus, and C. tentans over a 72 hour period.  Radioactivity of the haemolymph i s displayed  as disintegrations (counts) per mg wet weight of larva per minute.  C. anthracinus i s shown as ( — • — ) .  C. athalassicus i s shown as (--a---), shown as ( — o — ) . for each value.  c. tentans i s  One standard error i s indicated  12 0'  10CH  0  10  20  30  TIME  40  (HOURS)  50  60  70  80  31a.  FIGURE 11 The Uptake of Chloride-36 by C. anthracinus. C. athalassicus, and C. tentans Over a 120 Hour Period.  Radioactivity of the haemolymph i s  displayed as disintegrations (counts) per mg wet weight of larva per minute. shown as ( — • — ) .  C. anthracinus i s  C. athalassicus i s shown as ( — ) .  C. tentans i s shown as (-0-). i s indicated f o r each value.  One standard error  31.  32. at which half the maximum f l u x rate i s obtained. The sodium influx and efflux rates of £. anthracinus. C. athalassicus. and C. tentans larvae are shown i n Figures 12,13,and 1*f.  The equations that describe the  curves i n these figures are presented i n Table k. The sodium influx curves of the three species d i f f e r e d markedly: C. tentans had the lowest Vmax (maximum influx rate) of 0 . 9 6 mmole Na/Kg/hr and the lowest Km (1.05  mM Na), while C. anthracinus was intermediate  a maximum influx rate of 2.19 of 2 . 1 5 3.1*+  value  mM Na.  with  mmole Na/Kg/hr, and a Km value  The maximum rate of C. athalassicus was  mmole Na/Kg/hr which was well above that of the other  two species.  The Km value was 5 . 6 5 mM Na which was also  the highest of the three species. The sodium efflux curves d i f f e r e d only s l i g h t l y amongst the three species as shown i n Figures 12,13,and 1*f.  There  was very l i t t l e increase i n the sodium efflux rate of any of the three species as external sodium concentration was increased.  Also the efflux rates of the three species d i f f e r e d  only marginally at a single external sodium concentration. For each species there was an external sodium concentration at which influx and efflux of sodium were equal.  F r 0  C. anthracinus the concentration at which net flux was zero was 0 . 7 5 mM sodium.  For C. athalassicus the concentration  was 2.3*4- mM sodium, f o r C. tentans i t was 3*30 mM sodium Chloride f l u x measurements were carried out on steady-state fourth instar larvae onlyj no flux measurements were made on  33a.  FIGURE 12 The Effect of the External Sodium Concentration on the Sodium Flux Rate of Fourth Instar C. anthracinus Larvae.  Sodium influx i s shown as ( — • — ) .  efflux i s shown as (--*--).  Sodium  The influx Vmax i s  2.19 mmole Na/Kg wet weight/hr; the Km i s 2 . 1 5 mM Na. The equation which describes the efflux l i n e i s (Y - 9 . 0 • 1 0 " X 1+  +  Q.h7).  i s indicated f o r each value.  One standard error  33.  CM  34a.  FIGURE 13 The Effect of the External Sodium Concentration on the Sodium Flux Rate of Fourth Instar C. athalassicus Larvae.  Sodium influx i s shown as ( — • — ) .  efflux i s shown as (--^--).  Sodium  The influx Vmax i s  3 . 1 ^ mmole Na/Kg wet weight/hr; the Km i s 5 . 6 5 mM Na. The equation which describes the efflux l i n e i s (Y = 3 3 . ^ • 1 0 ' ^ +  1.02).  i s indicated f o r each value.  One standard error  35a.  FIGURE llf The Effect of the External Sodium Concentration on the Sodium Flux Rate of Fourth Instar C. tentans Larvae.  Sodium influx i s shown as ( — • — ) .  efflux i s shown as ( - - A - - ) .  Sodium  The influx Vmax i s  0.96 mmole Na/Kg wet weight/hr; the Km i s 1.05 mM Na. The equation which describes the efflux l i n e i s (Y = 9.1 • 1 0 indicated  - l +  + 0.69).  f o r each value.  One standard error i s  35  -03  +4  O  o  I  o SODIUM  Q FLUX  r  (mMOLES  O Na/Kg  WET WEIGHT/ H O U R )  36a.  TABLE h The Equations Describing the Results of Flux Measurements.  Sodium and chloride influx, and  sodium and chloride e f f l u x equations are l i s t e d . The symbols are explained i n the text.  36  THE EQUATIONS DESCRIBING THE RESULTS OF FLUX MEASUREMENTS SPECIES C. anthracinus  MEASUREMENT  EQUATION  Sodium influx  2.19 x C 2.15  Sodium efflux Chloride influx Chloride efflux C. athalassicus  (Y=(9.0 • 10-^)X + 0 A 7 ) 5.61 x C 7.77 + C (Y=(12.0 • 10~3)x + 0 . 5 0 )  l.1*f  Sodium influx  xC 5.65 + C  Sodium efflux  (Y=(33A '  Chloride influx Chloride efflux C. tentans  +c  Sodium influx Sodium e f f l u x Chloride influx Chloride efflux  io~Sx +  1.02)  5t70 x c 2.90 + C (Y=(2.0- 10~ )X + 0.9^) 3  +c  0.96 x C 1.05  (Y=(9.1 • 10  )X + 0 . 6 2 )  2.36 x C if. 99 + C (Y=(2.0- 10" )X + ) . 6 8 ) 3  37. chloride depleted larvae.  The relationship between chloride  influx and the external chloride concentration can, as i n the case of sodium i n f l u x , be described approximately by the Michaelis-Menton equation (see above). The influx and e f f l u x measurements of C. anthracinus, C. athalassicus, and C. tentans are shown i n Figures 15, and 17 respectively.  16,  The equations that describe the flux  r e s u l t s are given i n Table h (page 3 6 ) .  Certain differences  amongst the three species are obvious i n the figures. C. anthracinus  and C. athalassicus had similar maximum influx  rates (Vmax) of 5 . 6 3 and 5.70 mmole Cl/Kg/hr, respectively, while C. tentans was less able to take up chloride, having a Vmax of 2 . 3 6 mmole Cl/Kg wet weight/hr.  The a f f i n i t y of  the chloride uptake systems also varied markedly; C. athalassicus had a Km value of 2 . 9 0 mM CI, C. tentans had a Km value of *+.99 mM CI, and C. anthracinus  had a Km value of 7.77  mM CI.  C. athalassicus was the best able to take up chloride, having the greatest a f f i n i t y f o r chloride and the most powerful i n f l u x system. The relationship between chloride e f f l u x and the external chloride concentration was similar amongst the three  species,  with the rate of chloride efflux r i s i n g only s l i g h t l y as the external chloride concentration  increased.  At a certain  external chloride concentration the rates of influx and efflux were equal and net f l u x f e l l to zero.  For C. anthracinus  concentration was O.76 mM CI, f o r 0 . athalassicus i t was O.83 mM CI, and f o r C. tentans i t was 2 . 2 9 mM CI.  that  38a.  FIGURE 15 The Effect of the External Chloride Concentration on the Chloride Flux Rate of Fourth Instar C. anthracinus Larvae. as ( — • — ) .  Chloride influx i s shown  Chloride efflux i s shown as  (--A--).  The i n f l u x Vmax i s 5.63 mmole Cl/Kg wet weight/hr; the Km i s 7.77 mM C l .  The equation which describes  the efflux l i n e i s (Y = 1 1 . 9 x 10" X + 0 . 5 0 ) . 3  One standard error i s indicated f o r each value.  33  39a.  FIGURE 16 The E f f e c t of the E x t e r n a l C h l o r i d e C o n c e n t r a t i o n on the C h l o r i d e F l u x Rate of F o u r t h I n s t a r C. a t h a l a s s i c u s Larvae. as ( — • — ) . The  C h l o r i d e i n f l u x i s shown  Chloride efflux  i s shown as ( - - a - - ) .  i n f l u x Vmax i s 5.70 mmole Cl/Kg wet weight/hr$  the Km i s 2.90 mM C l .  The equation which d e s c r i b e s  the e f f l u x l i n e i s (Y = 2.0 x 10" X + 0.9*+). 3  standard e r r o r i s i n d i c a t e d f o r each v a l u e .  One  39  40a.  FIGURE 17 The E f f e c t of the E x t e r n a l C h l o r i d e  Concentration  on the C h l o r i d e ^lux. Rate of F o u r t h C. tentans Larvae. (—•—). The  Instar  C h l o r i d e i n f l u x i s shown as  Chloride efflux  i s shown as  (--A--).  i n f l u x Vmax i s 2.36 mmole Cl/Kg wet weight/hr;  the Km i s ^ . 9 9 mM CI. the e f f l u x l i n e  The equation  which d e s c r i b e s  i s (Y = 19.8 x 10"^X + 0.68).  1+1.  IV DISCUSSION a. S a l i n i t y Tolerance of Larvae Kinne (1963, f$6k)  has discussed the effects of temper-  ature and s a l i n i t y f a c t o r s , and has demonstrated that temperature and s a l i n i t y are i n t e r r e l a t e d i n t h e i r effects on ^. a c t i v i t y and metabolism.  They therefore cannot be e a s i l y  considered separately. C. anthracinus. C. athalassicus. and C. tentans have been subjected i n t h i s study to combinations of temperatures and s a l i n i t i e s .  The findings support the argument put  forward by Kinne (1963) which states that the effect of ~ s a l i n i t y on survival i s dependent upon the temperature of the environment.  An example of t h i s relationship i s shown  in Figure 1 (page 1 5 ) .  C. tentans survives a mean of 16  days at 5°C i n a 7000 micromhos/cm (at 25°C) solution, but survives a mean of only four days i n the same solution at 23°C.  As Figure 18 shows the lake temperature can then have  an important effect on survival since water temperatures are  low i n the spring (5°C) and high i n the summer (25°C). One of the objectives of the present study i s to compare  the  range of s a l i n i t i e s that C. anthracinus  and C. tentans can t o l e r a t e .  t  C. athalassicus,  While they are affected by  temperature i n a similar way, the range of s a l i n i t y that the species survive d i f f e r s s i g n i f i c a n t l y .  C. athalassicus can ..,  survive at: s a l i n i t i e s at least 6000 micromhos/cm above the highest tolerated by C. anthracinus or C. tentans. £» anthracinus and C. tentans survive over a similar range  42a.  FIGURE 18 The Daily Temperature Range i n Three of the Lakes Where Chironomus Species are Abundant. (After Cannings, 1973)  (°  n e  meter depth)  2 5-  L. J A C K S O N  h2.  2CH  ml |'  I,  i'  I.I.'-I  .I'  15-H |II,I-| „...||.'' ,  10-  I  il  5H  MAY  JUNE  JULY  AUGUST  E A S T L.  3 0 n  O  25H  2  0-  l'l"„  cr. H  15.I'l  or  I''  •"  10I"  LU  -  I  "-'•'•I'.ll  "II..,.".  UJ  Q.  I" 1  <  ''Il  5H  cc  M  LU t<  W E S T W I C K L.  £ 3on 2 5H 2 OH  15H  ioH 5H  M  ^3. of  salinities. Scudder (1969) carried out temperature and s a l i n i t y  experiments to compare the survival of Cenocorixa b i f i d a and C. expleta.  C. b i f i d a survives to an upper conductivity  l i m i t of between 2 0 , 0 0 0 and 2 ^ , 0 0 0 micromhos/cm (at 25°C), while C. expleta survives to an upper l i m i t of between 3 3 , 0 0 0 and 3 5 , 0 0 0 micromhos/cm (at 25°C).  Scudder compared  these findings to the results of f i e l d collections and found that the f i e l d and laboratory information agreed well. C. b i f i d a was collected i n waters up to 2 0 , 0 0 0 micromhos/cm (at  25°C), and C. expleta was collected i n waters up to  3 0 , 0 0 0 micromhos/cm (at 25°C). S i m i l a r l y the results of the laboratory experiments can be compared to data obtained from f i e l d c o l l e c t i o n s of C. anthracinus, C. athalassicus, and £. tentans. Such collections were carried out by Cannings ( 1 9 7 3 ) ,  working  i n the one meter depth zone of lakes i n the Cariboo and C h i l c o t i n areas (see annings and Scudder, c  1978).  C. anthracinus was collected from lakes of conductivities between  hQ$  (East L.) and  klOQ  micromhos/cm at 25°C (Boitano L.)  C. athalassicus was collected from kQ5 (East L.) to 15,000 micromhos/cm at 25°C (Barnes L . ) .  C. tentans was collected  from lakes ranging from *+85 (East L.) to ^108 micromhos/cm at 25°C (Boitano L . ) . The laboratory and f i e l d results compare very well as f a r as C. athalassicus i s concerned.  C. anthracinus and  C. tentans, however, survived i n the laboratory at conductivities  well above those that they were collected from i n the f i e l d . Such discrepancies between laboratory and f i e l d results have been found i n other studies (Doudoroff, marine f i s h ) .  1938  working with  A p a r t i a l explanation for the discrepancy  l i e s i n the effects of s a l i n i t y on the organism and i t s ecology.  As Kinne (196^) points out, s a l i n i t y may  affect  functional and structural properties of the organism through changes i n osmo-concentration, solute concentration, c o e f f i c ients of absorption, density, and v i s c o s i t y . a f f e c t the species composition  Salinity will  of the eco-system, the a v a i l -  a b i l i t y of food, and the effects and l i k e l i h o o d of disease. Another factor which must be considered i s that survival i s limited by the tolerances of the most vulnerable l i f e  stage.  In the laboratory only the survival of fourth instar larvae was  studied, i n the natural habitat fourth instar larvae w i l l  be found only i f the preceding stages have at least an equal a b i l i t y to survive. Though the means by which s a l i n i t y i s acting upon C. anthracinus, C. athalassicus, and C. tentans i s not apparent, i t i s obvious that C. athalassicus can survive i n the lakes occupied by £. anthracinus and C. tentans. and that i t can survive i n lakes much more saline than those occupied by the other two species.  It i s important  to relate  t h i s d i s t r i b u t i o n difference amongst the species to the effects of s a l i n i t y on haemolymph composition,  and to the e f f e c t s of  the concentration of c e r t a i n ions i n the lake water. Although the species are affected d i f f e r e n t l y by the  h5. s a l i n i t y of the external medium, the length of time that the species survive decreases i n a l l cases as temperature r i s e s . At a fixed temperature, survival decreases increased past a certain l e v e l .  i f salinity i s  Figure 19 depicts t h i s  relationship between temperature and s a l i n i t y  (expressed  as conductivity), b. Regulation of the Haemolymph The osmotic and ionic regulation of several f r e s h and saline water insect species have been well documented (Wigglesworth, 1938; Beadle, 1939; Ramsay, 1953; Shaw, 1955; S u t c l i f f e , 1961a, 1961b; Scudder et a l . 1972; and Nayer et a l . 197 +). 1  Animals such as insect species that are r e s t r i c t e d  to low s a l i n i t i e s are i n general unable to hyporegulate the haemolymph i n waters of high s a l i n i t y (Beadle, 1969). C. anthracinus, C. athalassicus. and C. tentans are examples of  such animals.  They regulate the osmotic pressure of the  haemolymph i n lake water concentrations up to A  0.32°C,  but at higher levels the freezing point depression of the haemolymph r i s e s with the concentration of the medium. Lauer (1969) describes the osmotic regulation of Chironomus plumosus (L.) and Procladius nubifer.  The osmotic response  curves of these species are similar to those of C. anthracinus. C. athalassicus. and G. tentans, although Lauer (1969) thought C. plumosus and P. hubifer showed some hyporegulation at high s a l i n i t i e s .  Beadle (1959) suggests that species may be  c l a s s i f i e d according to the type of inland saline water they occupy.  C. anthracinus and C. tentans would be c l a s s i f i e d  46a.  FIGURE 19 The Survival Limits of C. anthracinus. C. athalassicus. and C. tentans as Determined by Laboratory Experiments.  These are expressed  as a combination of temperature and lake water conductivities.  ^7 as normal inhabitants of freshwater which can tolerate external concentrations approximately equal to the concentrat i o n of the haemolymph.  C. athalassicus, because i t i s known  to l i v e i n concentrations well above those tolerated by C. anthracinus and C. tentans, may  belong to another group  of species which show a preference for more saline water, but may  also occur i n lower s a l i n i t i e s and fresh water.  Some species i n t h i s group, though C. athalassicus i s not an example, may have developed a mechanism of hypoosmotic regulation. Some of the fresh water species that have osmotic response curves similar to those of Chironomus are: S i a l l s l u t a r i a (Beadle and Shaw, 1959; Aedes aegypti L. (Wigglesworth,  1938;  Shaw, 1 9 5 5 ) , Ramsay, 1953),  Limnephilus stigma (Curtis) and Anabolia nervosa (Leach) ( S u t c l i f f e , 1961b), and Cenocbrixa b i f i d a (Hung.) and C. expleta (Uhler) (Scudder et a l . 1972).  These species  do not hyporegulate the haemolymph i n the higher s a l i n i t i e s . C. anthracinus. C. athalassicus. and C. tentans have similar patterns of sodium, potassium, and chloride regulation. Since C. athalassicus survives at higher s a l i n i t i e s , i t s response  covers a greater range of external ion concentrations  than do the responses of C. anthracinus and C. tentans.  The  sodium response curves of these Chironomus species are unlike those of S i a l i s l u t a r i a (Shaw, 1955), Aedes aegypti (Ramsay, 1953), and Limnephilus  stigma ( S u t c l i f f e , 1961b) which  maintain the sodium concentration i n the haemolymph above  »f8.  that of the external medium, even at high external concentrations of sodium.  The sodium response curves of C. anthracinus,  C. athalassicus. and C. tentans can be compared to those of Cenocorixa expleta and C. b i f i d a (Scudder et a l . 1972), and the caddis larva Limnephilus a f f i n i s ( S u t c l i f f e , 1961a), which maintain a constant haemolymph sodium l e v e l over a range of external sodium concentrations.  These species are known to  tolerate f r e s h and r e l a t i v e l y saline waters. The chloride regulation curves of C. anthracinus, C. athalassicus. and C. tentans are comparable at low chloride concentrations; haemolymph chloride levels are above the external concentration.  Only C. athalassicus survived at  the higher s a l i n i t i e s and i t maintained haemolymph chloride hypertonic to the external chloride l e v e l s .  The range of  chloride concentrations studied i n t h i s investigation compared to the work carried out on other f r e s h water species, i s small. Both S i a l i s l u t a r i a (Shaw, 1955) and Aedes aegypti (Wigglesworth, 1938)  regulate the haemolymph chloride l e v e l hypertonic at  low external chloride concentrations, and hypotonic at high external chloride concentrations (for example S i a l i s above 120 mmole C l / 1 ) .  Wright (1975c) measured the haemolymph  chloride concentration of C. tentans larvae over a range of external chloride concentrations from 0 . 5 to 100 mM C l . Throughout that chloride range the haemolymph chloride concentr a t i o n of C. tentans was 30 mM C l .  The haemolymph chloride  concentration of C. anthracinus. C. athalassicus. and C. tentans varied from 10 to 29 mM C l over an external chloride range  h9. of 0 to 5 mM CI. The chloride concentration i n the haemolymph of C. tentans as determined by Wright (1975c) and the three Chironomus species of the present study are low compared to other species such as S i a l i s l u t a r i a (Shaw, 1955) and Aedes aegypti (Wigglesworth, 1938), which both have a chloride concentration of 30 mM CI or greater. Scudder et al.(1972) studied Cenocorixa b i f i d a and C. expleta over a range of chloride concentrations similar to that used i n t h i s study.  Both the species regulate the  haemolymph chloride concentration within narrow l i m i t s and hypertonic to the external medium at low chloride concentrations. Both the Chironomus and the Cenocorixa species were collected from inland saline lakes i n which chloride concentrations were low (the major anions are: carbonate, bicarbonate, and sulphate).  The chloride concentrations range from 0 . 0 7 mM CI  to 3 6 . 9 6 mM CI, approximately  the range of chloride i n v e s t i -  gated i n t h i s study. At external potassium  concentrations below 10 mmole/1  C. anthracinus, C. athalassicus. and £. tentans maintain haemolymph potassium hypertonic to the external potassium concentration.  At s a l i n i t i e s where only C. athalassicus  survives, the potassium  concentration of the haemolymph  becomes more hypertonic as the external potassium tion rises.  concentra-  These Chironomus species regulate potassium i n  a way similar to Cenocorixa b i f i d a and C. expleta (Scudder et al.1972).  S i a l i s l u t a r i a maintains the haemolymph  concentration at approximately  potassium  5 mmole K / l over an external  50. potassium range of 0 to 3 * mmoie/1 (Shaw, 1955). 1  Over the  same range of potassium concentration, C. athalassicus has a haemolymph potassium concentration s l i g h t l y hypertonic to the external concentration.  The difference between the  species i s that S. l u t a r i a regulates while C. athalassicus i n effect maintains  the potassium concentration of the  haemolymph close to that of the external environment. P h i l l i p s (1970) regards the a b i l i t y to produce a urine that i s hyperosmotic to the haemolymph as a prerequisite f o r the survival of hyporegulating salinity.  species i n waters of high  No analyses of the urine of these Chironomus  species were carried out, but i n the l i g h t of the osmotic and ionic responses described above i t i s doubtful that a hyperosmotic urine could be elaborated even by C. athalassicus. The osmotic and ionic balance i n C. anthracinus. C. athalassicus. and £. tentans are similar except that C. athalassicus can control i t s i n t e r i o r milieu over a wider range of external concentrations.  These responses are similar to those of  f r e s h water insects i n general (Wigglesworth, 1938; Shaw, 1955; Beadle, 1959; S u t c l i f f e , 1961b; Beadle, 19&9; and Scudder  et a l . 1972). The question remains as to why C. athalassicus can survive and thrive i n s a l i n i t i e s twice as high as those endured by C. anthracinus and C. tentans.  S u t c l i f f e (1961a) suggests  that external concentrations greater than 30 to ^O per cent seawater are rapidly f a t a l to fresh water insects.  As the  external salt concentration increases to a l e v e l roughly  51. equivalent to the normal t o t a l concentration of the haemolymph, the regulation of the haemolymph salt concentration begins to break:down.  If the concentration of the haemolymph (Ag)  c o l l e c t e d larvae i s considered  of  to be representative, then the  external concentration at which the regulation of the haemolymph of £. athalassicus begins to break down i s 50 per cent higher than that of C. anthracinus  and C. tentans.  (195*+) has suggested that t h i s break-down may  Ramsay  be due to an  i n a b i l i t y to increase the amino acid content of the muscle c e l l s , ultimately a f f e c t i n g body metabolism i n general.  It  i s perhaps the fact that break-down does not begin at such a low external concentration that enables C. athalassicus to survive at higher external concentrations and C.  than C.  anthracinus  tentans.  While i t i s not possible on the basis of t h i s study to state precisely why  C. athalassicus survives at higher  salinities  than C. anthracinus  and C. tentans* t h i s study does show that  the three species are fresh water insects and that they possess equal a b i l i t i e s to regulate at the lower external  concentrations.  c. Sodium and Chloride Flux The control of internal salt and water balance has been studied i n a wide variety of animals. particular i s now  Sodium regulation i n  understood i n some d e t a i l , especially as  i t relates to fresh water adaptation and Shaw, 1967a; Greenaway, 1970;  (Shaw, 19615  Maetz, 19715  Sutcliffe  Stobbart,  1971;  S u t c l i f f e , 197*+ S Wright, 1975a, 1975b). The sodium regulation of some saline water species  has  52. also been characterized and i n some cases they have been compared to fresh water species (for example S u t c l i f f e , 1967, The  1968,  1971)-  parameters of such a characterization are; the maximum  f l u x rate (Vmax), the external concentration at half maximal f l u x (Km),  the sodium loss rate, and the minimum concentration  at which the animal balances sodium uptake and  loss.  C. anthracinus. C. athalassicus. and C. tentans were studied at concentrations  that ranged from fresh to saline  according to the Venice Symposium ( 1 9 5 9 ) . between fresh and mixohaline water was 0 . 5 P?M  The  distinction  a r b i t r a r i l y placed at  s a l i n i t y , or approximately 7 mmole/.l.sodium ( S u t c l i f f e ,  1967b). Comparing the sodium regulatory a b i l i t i e s of the three species studied i n t h i s thesis, C. tentans shows the a f f i n i t y f o r sodium (Km of 1.05  mM Na), but has the least  powerful uptake system (Vmax of O.96 C. anthracinus  greatest  mmole Na/Kg/hr).  has a lower a f f i n i t y for sodium (Km of 2.15  Na) but i t s uptake system i s more powerful (Vmax of 2*19 per Kg per h r ) .  mM  mM  Na  C. athalassicus has a Km value over f i v e times  as large (Km of 5.65 mM Na) as C. tentans. suggesting lower sodium a f f i n i t y than have C. anthracinus The Vmax of C. athalassicus O . l ^  or C.  a much tentans.  mmole Na/Kg/hr) i s over  three times as great as that of C. tentans.  C. athalassicus  has, therefore, the least a f f i n i t y f o r sodium of the  three  species, but has the most powerful uptake system. Wright (1975a, 1975b, 1975c) characterizes the sodium f l u x of Chironomus dorsalis and C. tentans under low sodium  53. conditions (0.0002 to 6.0 mmole/1 sodium).  Wright (1975b)  established a Km value of 0.75 mmole/1 sodium f o r both C. d o r s a l i s , and £. tentans, including the depleted individuals. This low Km value indicates a high a f f i n i t y for sodium; as one might expect i n populations acclimatized to low external sodium concentrations.  The Vmax of the species studied by  Wright are: C. dorsalis 3.0 mmole Na/Kg/hr, and C. tentans 1.9 mmole Na/Kg/hr.  If these values are compared to the  Vmax values of C. anthracinus, C. athalassicus, and C. tentans. the differences are found to be small.  Comparing Km values,  however, C. dorsalis and C. tentans (Wright, 1975b) appear much better adapted to fresh water habitats of very low conductivity. In the present study the relationship between efflux and external sodium concentration i s best described by a straightl i n e equation, with a slight increase i n efflux with increased external sodium concentration.  Maetz (1971) found a similar  relationship between sodium efflux and external sodium concentration working with the fresh water flounder. (1975b) found that sodium  Wright  efflux displays t y p i c a l saturation  k i n e t i c s i n C. dorsalis and £. tentans.  The difference between  the study by Wright and the present study may be the difference i n the ranges of sodium u t i l i z e d i n each study.  The low  concentrations of the Wright study may have emphasized the •"first order" k i n e t i c s , while the r e l a t i v e l y higher  concent-  rations of sodium i n the present study may obscure  such  kinetics.  5V. The  maximum sodium e f f l u x r a t e s of C .  anthracinus  (0.*+9 mmole Na/Kg/hr), C . a t h a l a s s i c u s (1.10 and C . tentans (0.71 those p u b l i s h e d (1.6 the  and  1.5,  mmole Na/Kg/hr),  mmole Na/Kg/hr) were s i m i l a r , but  by Wright (1975b) f o r C . d o r s a l i s and  respectively).  The  below C . tentans  lower r a t e s of e f f l u x of  s a l i n e water adapted i n s e c t s are d i f f i c u l t  to e x p l a i n  s i n c e s a l i n e water organisms would be expected t o l o s e much more sodium than f r e s h water adapted organisms, although sodium l o s s may  be handled adequately by the e x c r e t o r y  Speaking i n general  (1968) found t h a t  terms S u t c l i f f e  sodium l o s s r a t e s are s i m i l a r i n f r e s h and crustaceans,  while the  f o u r to ten times No C. and  balancing  anthracinus.  b r a c k i s h water  l o s s r a t e of marine crustaceans  experiments were c a r r i e d out  was  involving  C . a t h a l a s s i c u s , and C . tentans.  e f f l u x r a t e s of these s p e c i e s were equal at  The  influx  external (1975a).  w e l l above those c i t e d by Wright  d o r s a l i s and p_. tentans are able to achieve a s a t i s f a c t o r y  sodium balance i n 25 micromoles and e x t e r n a l sodium c o n c e n t r a t i o n ,  10 micromoles (per  respectively.  e f f l u x r a t e s are equal i n C . anthracinus C.  the  higher.  sodium c o n c e n t r a t i o n s C.  organs.  a t h a l a s s i c u s at 2.3V  mM  Na,  and  Influx  at 0 . 7 5  mM  and Na,  C . tentans at 3.30  D i r e c t comparisons between the f l u x of f r e s h and water a c c l i m a t i z e d chironomids have n o t previously.  liter)  mM  saline  been c a r r i e d out  However, e a r l i e r s t u d i e s have c h a r a c t e r i z e d  sodium r e g u l a t o r y a b i l i t i e s of other f r e s h and invertebrates.  Sutcliffe  (1967a,  1967b,  Na.  1968,  the  s a l i n e water 1971,  197^)  55. compared populations of the amphipod Gammarus duebeni L i l l j e b o r g from fresh and brackish locations throughout the United Kingdom.  L i t t l e difference was found between the pop-  ulations concerning  the regulation of sodium uptake and loss  at external concentrations below 10 mmole Na/1.  Evidence  was found, however, of a direct c o r r e l a t i o n between the h a l f saturation l e v e l (Km) of sodium influx and the external sodium concentration of the p a r t i c u l a r habitat of that population.  Populations from similar habitats shared the same  Km values, but d i f f e r e d from populations i n other habitats. S u t c l i f f e (1968) compared the sodium regulatory a b i l i t i e s of amphipod species from marine, fresh, and brackish habitats. The  species from fresh and brackish waters had Km values of  about 1 mmole/1 sodium and were f u l l y saturated at about 10 mmole/1 sodium.  The marine species had values six to ten  times higher for the respective parameters.  F  rom these findings  S u t c l i f f e suggested that the evblutionary scheme has been the colonization of fresh waters by brackish water species, with the marine environment being the o r i g i n a l habitat of the species. probably  I t also i s suggested that brackish species could inhabit most fresh waters i f sodium levels were  sufficient.  The mechanism of evolution i n that case appears  to be one of phenotypic selection where the f i t individuals are those able to regulate sodium i n accordance with the requirements of the habitat.  For instance, those able to  regulate sodium i n fresh water with low sodium levels w i l l survive.  56. Beadle (19^3) has pointed out that insect larvae tend to maintain their haemolymph near the levels of the fresh water insects, despite the external sodium concentration, revealing their fresh water o r i g i n .  Insects have probably evolved from  fresh water habitats to also inhabit saline and brackish water habitats to gain ecological advantages such as i n food  sources  or reduced competition. The chironomid  populations from saline lakes (C. anthracinus,  C. athalassicus. and C. tentans) have a lower sodium a f f i n i t y than populations from very fresh lakes (C. dorsalis and C. tentans. Wright, 1 9 7 5 b ) .  S u t c l i f f e (1968) c i t e s a similar  difference between fresh and brackish populations of amphipods but adds that a much greater difference exists between these and marine populations, the l a t t e r having a very low a f f i n i t y f o r sodium. Shaw ( 1 9 6 D has shown that the Km value i s progressively lower i n species which have increasely strong powers of osmoregulation  i n fresh water.  This suggests that the Km  value i s one way to compare species from different habitats since i t i s indicative of the species' sodium a f f i n i t y . Table 5 i s a c o l l a t i o n of the findings of a wide variety of studies.  The table compares the Km, Vmax, and sodium  loss rates of species from fresh, brackish, and marine habitats.  With the variation amongst the studies considered  (temperature, habitat variations, differences i n research techniques), there i s a trend toward lower sodium a f f i n i t y , and higher maximum sodium influx and efflux rates amongst  57a.  TABLE 5 The Sodium Flux Rates of Several Invertebrate Species.  FW = freshwater, SW = saline water,  BW = brackish water, MW = marine water.  ACCLIMATION (mM/1 Na) 2.0  ORIGIN  EFFLUX TEMP. Km Vmax T^CT (mMNa)(mmoleNa/Kg/hr) (mmoleNa/Kg/hr) 0.83 28 0.55  SPECIES  STUDY  Aedes aegypti  Stobbart 1965  Asellus communis  Sutcliffe 197^  0.075-2.0  FW  20  0.12  1.7  0.3-0.7  A.  Sutcliffe 197^  0.1-2.0  FW  20  0.62  6.5  0.9-2.1  Sutcliffe  0.1-0.7  FW  20  0.91  18.2  1.7-2.7  15.0  sw  23  2.15  2.19  0.50  Wright  2.0  FW  21  0.75  3.0  1.6  present study  15.0  SW  23  5.65  3.1^  1.11  2.0  FW  21  0.75  1.9  1.5  15.0  SW  23  1.05  0.96  7.77  0.25-0.35  BW  10  1.5-2.0  20.0  3.8  0.10  FW  10  0.10-0.15  2.6  1A  aquaticus A.  meridianus Chironomus anthracinus C. dorsalis C. athalassicus C. tentans C. tentans Gammarus duebeni G. lacustris  197>+  present study  1975  Wright 1975 present study Sutcliffe 1967b Sutcliffe Shaw 1967b  FW  58a.  TABLE 5 continued  The Sodium Flux Rates of Several  Invertebrate Species.  SPECIES  STUDY  ACCLIMATION (mM/1 Na) 0.10  Gammarus pulex  Sutcliffe 1967a  G. tierinus  S u t c l i f f e 20% sea H?0 1968  Limnaea staenalis  Greenaway 1970  ORGIN TEMP. Km Vmax EFFLUX P^cT (mMNa)(mmole Na/Kg/hr)(mmole Na/Kg/hr) FW 10 0.10 3.1 1.7 -0.15 MW  10  6.0 -10.0  20.0  20.0  0.35  FW  10  0.25  1.5-2.0  0.08  Marinogammarus S u t c l i f f e f inmarchus 1968  0.5  BW  10  1.0 -1.5  20.0  7.0  Mesidotea entomon  Croghan 1968  0.7  FW  5-10  2.6  15.0  1.6  Sphaeroma rugicauda  Harris 1972  10.0  BW  16-20 2 . 3  15.0  *+.1  S. serrtum  Harris 1972  85.0  BW  16-20 15.0  30.0  16.5  59. species from brackish and marine habitats. the present  The findings of  study of Chironomus species from saline habitats  conform to that trend, with sodium a f f i n i t y below that of chironomids from fresh water habitats (Wright, 1975a,  1975b).  Of the three chironomid species studied here C. athalassicus i s best adapted f o r high s a l i n i t i e s , with a high Km value and a high Vmax value that characterize other saline adapted species. Shaw (1960) found that similar saturation k i n e t i c s prevailed i n the sodium and chloride uptake of the c r a y f i s h Astacus p a l l i p e s . present  The maximum chloride influx rates i n the  study varied from 2.36 mmole Cl/Kg/hr (C. tentans)  to 5.70 mmole Cl/Kg/hr (C. athalassicus).  The chloride  i n f l u x systems were half saturated at 2 . 9 0 mM CI (C. athalassicus)« >+.99 mM CI (C. tentans). and 7.77 mM CI (C. anthracinus). This information suggests that C. athalassicus has the greatest a f f i n i t y for chloride, as well as the most powerful chloride uptake system. The chloride uptake a b i l i t y of C. athalassicus may give i t an advantage i n i t s natural habitat since chloride i s low i n a l l of the saline lakes inhabitated by these species (Table  1).  Chloride flux values are few i n the literature, of insects,but uptake studies have been carried out.  Stobbart (1967)  has investigated the chloride uptake of Aedes aegypti and has reported results similar to those of the present flux around 2 mmole Cl/Kg/hr).  study (chloride  Kerstetter and Kirschner  (1972),  60. and Payan et al.(1978) r e p o r t on a c t i v e c h l o r i d e t r a n s p o r t by the g i l l s of rainbow t r o u t (Salmo g a i r d n e r i ) . The i n vivo c h l o r i d e f l u x was lower ( 2 5 . 0 microEq/hr/100g) the values The  c i t e d i n t h i s study.  c h l o r i d e e f f l u x r a t e s d i d not show s a t u r a t i o n  k i n e t i c s over the range of c h l o r i d e c o n c e n t r a t i o n s The  than  maximum e f f l u x r a t e s of the three  1 mM Cl/Kg/hr.  studied.  species were under  I f compared to the e u r y h a l i n e  brine  shrimp  Artemia s a l i n a (Croghan, 1958; Smith, 1969), which has a maximum e f f l u x r a t e of 57 ml! C l / 1 . haemolymph/hr, the r a t e s 1  of the Chironomus species are much lower. According  to the t r a n s p o r t model of Maetz (1971)  c h l o r i d e and sodium f l u x are r e l a t e d i n d i r e c t l y . and  sodium e f f l u x r a t e s of Artemia s a l i n a were compared by  Thuet e t a l . ( 1 9 6 8 ) ; sodium e f f l u x r a t e . and  The c h l o r i d e  the c h l o r i d e e f f l u x rate was twice the No p a t t e r n i s obvious when the c h l o r i d e  sodium e f f l u x r a t e s of Chironomus are compared, they  vary from s i m i l a r t o twelve times l a r g e r (Table The  c h l o r i d e i n f l u x r a t e s can be compared t o sodium  r a t e s t h a t have been p u b l i s h e d . c h l o r i d e are about twice present  study (Table h).  page 36). influx  The maximum i n f l u x r a t e s of  those of sodium as o u t l i n e d i n the Compared t o the maximum sodium  i n f l u x r a t e s of s e v e r a l other f r e s h water s p e c i e s , the c h l o r i d e r a t e s of Chironomus are a l s o about twice as l a r g e . Sodium and c h l o r i d e f l u x were s e l e c t e d f o r study because each can be compared t o s i m i l a r f l u x s t u d i e s on other  species.  T h i s i s e s p e c i a l l y t r u e i n the case of sodium f l u x , but c h l o r i d e  61. f l u x i s receiving further study.  Such comparisons between  species are important not only from a physiological point of view, but also from an ecological point of view since the flux c h a r a c t e r i s t i c s have been related to environmental c h a r a c t e r i s t i c s by researchers such as Shaw ( 1 9 6 1 ) , Sutcliffe  and  (197^).  Sodium and chloride are of course not the only important ions i n the saline lake habitat of C. anthracinus. C. and C. tentans.  athalassicus,  If a complete picture of the adaptation to  saline lakes i s to be obtained, the importance of other ions as well as other factors such as t o t a l dissolved s o l i d s , organic and  inorganic carbon, and the ecological effects of  s a l i n i t y must be evaluated.  62. V. CONCLUSIONS This thesis has investigated the importance of as a factor i n the d i s t r i b u t i o n of C. C. athalassicus. and C. tentans.  salinity  anthracinus.  I t has been shown that  there i s a difference i n the s a l i n i t y tolerance of the species. C. anthracinus and C. tentans  larvae can survive i n cond-  u c t i v i t i e s only as high as 9000 micromhos/cm (at 25°C) while C. athalassicus can survive to at least 15,000 micromhos/cm (at 25°C).  A l l three species are, however, affected by the  temperature of the water which may  a f f e c t s u r v i v a l at certain  times of the year. C. anthracinus. C. athalassicus, and C. tentans  regulate  the haemolymph In similar ways at low external concentrations, but only C. athalassicus can continue to function at high concentrations.  There i s a c o r r e l a t i o n between the a b i l i t y  to regulate and the s u r v i v a l of C. athalassicus; the regulatory a b i l i t i e s appear to break down at s a l i n i t i e s above 15,000 micromhos/cm (at 25°C). of C. anthracinus  Evidence of break-down i n the regulation and C. tentans i s not as obvious, but neither  survives at haemolymph concentrations greater than that of the external environment.  According to the  classification  of Beadle ( 1 9 5 9 ) , a l l three species are fresh water types, but C. athalassicus alone may  belong to a group which prefer'  more saline water. The chloride and sodium flux studies are important for two reasons.  They allow the chironomid species to be compared  to other species, and the flux c h a r a c t e r i s t i c s themselves  63  are new  information to science since no saline adapted  chironomids have been examined to date. Throughout this thesis a trend of information C. athalassicus has become apparent.  concerning  C. athalassicus i s  c l e a r l y able to tolerate and thrive at high s a l i n i t i e s . It can regulate the haemolymph at s a l i n i t i e s equal to those occupied  i n the f i e l d .  The sodium c h a r a c t e r i s t i c s suggest  that i t i s similar to species adapted to highly saline or brackish waters.  It has a low sodium a f f i n i t y , but a powerful  uptake system. The conclusion to be drawn from t h i s study l i s t h a t r  s a l i n i t y does indeed affect the d i s t r i b u t i o n of C.  1  anthracinus,  C. athalassicus. and C. tentans i n the saline lakes of the Cariboo-Chilcotin region of central B r i t i s h Columbia.  This  study does not, however, exhaust the possible ways i n which s a l i n i t y i s important  (see Kinne, 196V), and i t cannot be  assumed that because s a l i n i t y a f f e c t s d i s t r i b u t i o n that i t i s the only factor of importance to the d i s t r i b u t i o n of C. anthracinus. C. athalassicus. and C.  tentans.  6h. LITERATURE CITED Bassett, M.C. 1967. A cytotaxonomic study of the most common l a r v a l chironomidae i n a series of saline waters i n the southern i n t e r i o r of B r i t i s h Columbia. M.Sc. Diss. U.B.C. Vancouver, B.C. Beadle, L. 1939. Regulation of the haemolymph i n the saline water mosquito larva Aedes detritus Edw. J . Exp. B i o l . 16: 3 ^ 6 - 3 6 2 . Beadle, L. 19*+3. Osmotic regulation and the faunas of inland waters. B i o l . Rev. 18: 172-183. Beadle, L. 1959. Osmotic regulation i n r e l a t i o n to the c l a s s i f i c a t i o n of brackish and inland saline waters. Archivo Di Oceanografia E Limnologia Vol. XI: 1V3-151. Beadle, L. 1969Osmotic regulation and the adaptation of freshwater animals to inland saline waters. Verh. Internat. Verein. Limnol. 17: **21-4-29. Beadle, L., & J . Shaw. 1950. The retention of s a l t and the regulation of the non-protein nitrogen f r a c t i o n i n the blood of the aquatic larva, S i a l i s l u t a r i a . J. Exp. B i o l . 27: 9 6 - 1 0 9 . Cannings, R.A. 1973. An ecological study of some of the Chironomidae inhabiting a series of saline lakes i n central B r i t i s h Columbia with special reference to Chironomus tentans Fabricius. M.Sc. Diss. U.B.C. Vancouver, B.C. Cannings, R.A. 1975. A new species of Chironomus (Diptera: Chironomidae) from saline lakes i n B r i t i s h Columbia. Can. Ent. 107: ^ 7 - ^ 5 0 . Cannings, R.A., & G.G.E. Scudder. 1978. The l i t t o r a l Chironomidae (Diptera) of saline lakes i n central B r i t i s h Columbia. Can. J . Zool. 56: 1 1 ^ - 1 1 5 5 . Croghan, P..C. 1958. J . Exp. B i o l . 3 5 :  Ionic fluxes i n Artemia salina (L.)«' ^25-^36.  Doudoroff, P. 1938. Reactions of marine fishes to temperature gradients. B i o l . B u l l . 7 5 : *+9l+-509. Fry, F.E.J. 91^7. Effects of the environment on animal a c t i v i t y . Publ. Ontario F i s h . Res. Lab. 6 8 : 1-62.  65. Greenaway, P. 1970. Sodium regulation i n the fresh water mollusc Limnaea stagnalis (L.) (Gastropoda:Pulmonata). J . Exp. B i o l . 53« W - 1 6 3 . Harris, R.R. 1972. Aspects of sodium regulation i n a brackish water and a marine species of the isopod genus Sphaeroma. Mar. B i o l . 12: 18-27. Kerstetter, T.H., & L.B. Kirschner. 1972. Active chloride transport by the g i l l s of rainbow trout (Salmo gairdneri). J . Exp. B i o l . 5 6 : 263-272. K i h n e , 0 . 1963. The effects of temperature and s a l i n i t y on marine and brackish water animals. I.Temperature. Oceanogr. Mar. B i o l . Ann. Rev. 1 : 301-3H-0. Kinne, 0 . 196V. The effects of temperature and s a l i n i t y on marine and brackish water animals. I I . S a l i n i t y and temperature s a l i n i t y combinations. Oceanogr. Mar. B i o l . Ann. Rev. 2 : 2 8 1 - 3 3 9 . Koch, H.J. 1938. The absorption of chloride ions by the anal papillae of dipteran larvae. J . Exp. B i o l . 15* 152-160. Krogh, A. 1939. Osmotic Regulation In Aquatic Animals. Cambridge Univ. Press. 2*+2Pp. Lauer, G.J. 1969. Osmotic regulation of Tanypus nubifer, Chironomus plumosus, and Enallagma clausum i n various concentrations of saline lake water. Physiological Zoology H - 2 : 381-387. Maetz, J . 1971. Fish g i l l s : mechanisms of salt transfer i n fresh water and sea water. P h i l . Trans. Roy. Soc. Lond. B 262: 2 0 9 - 2 ^ 9 . M i a l l , L.C., & A.R. Hammond. 1900. The Structure and L i f e History of the Harlequin F l y (Chironomus). Oxford, Clarendon Press. Nayar, J.K., & D.M. Sauerman, J r . 197*+. Osmo-regulation i n larvae of the salt-marsh mosquito Aedes taeniorhynchus. Ent. Exp. & Appl. 17: 3 6 7 - 3 8 0 . Payan, P., P. P i c , & G. De Renzis. 1978. Comparaison des echanges branchiaux de CI" en eau douce chez Salmo gairdneri i n vivo ou i n v i t r o sur l a tete isolee perfusee. J . Fish Res. Board Can. 3 5 * ^77-^79• P h i l l i p s , J.E. 1970. excretory systems.  Apparent transport of water by insect Am. Zool. 10: H-1 3 - ^ 3 6 .  66 Ramsay, J.A. 1953. Active transport of potassium by the Malpighian tubules of insects. J . Exp. B i o l . 30: 358-369. Ramsay, J.A. 1954-. Movement of water and electrolytes i n invertebrates. Symp:. Soc. Exp. B i o l . 8: 1-15. Ramsay, J.A., R.H.J. Brown, & P.C. Croghan. 1955. Electrometric t i t r a t i o n of chloride i n small volumes. J . Exp. B i o l . 32: 822-829. Remane, A., & C. Schlieper. 1971. Biology of Brackish Water. Wiley Interscience Division. John Wiley & Sons, Inc. " Toronto. 372Pp (English Ed.) Sadler, W.O. 1935. Biology of the midge C. tentans Fabricius and methods for i t s propogation. Cornell Univ. Agric. Exp. Stn. Memoir 173 Scudder, G.G.E. 1969a. The d i s t r i b u t i o n of two species of Cenocorixa i n inland saline lakes of B r i t i s h Columbia. J . Entomol. Soc. B r i t . Columbia 66: 32-4-1. Scudder, G.G.E. 1969b. The fauna of saline lakes on the Fraser Plateau of B r i t i s h Columbia. Verh. Internat. Verein. Limnol. 17: ^30-4-39. icudder, G.G.E., M.S. J a r i a l , & S.K. Choy. 1972. Osmotic and Ionic balance i n two species of Cenocorixa (Hemiptera). J . Insect Physiol. 18: 883-895. Shaw,J; 1955. Ionic regulation and water balance i n the aquatic larva of S i a l i s l u t a r i a . J . Exp. B i o l . 32: 353-382. Shaw,J. i960. The absorption of C l ions by the c r a y f i s h Astacus pallipes Lereboullet. J . Exp. B i o l . 37: 557-572. Shaw, J . 1961. Sodium balance i n Eriocheir sinensis (M. Edw.). The adaptation of the Crustacea to fresh water. J . Exp. B i o l . 38: 153-162. Smith, P.G. 1969• The ionic relations of Artemia salina (L.). I I . *luxes of sodium, chloride and water. J . Exp. B i o l . 51: 739-757. Stobbart, R.H. 1959. Studies on the exchange and regulation of sodium i n the larva of Aedes aegypti ( L . ) . I. The steady state exchange. J . Exp. B i o l . 36: 64-1-653. Stobbart, R.H. i960. Studies on the exchange and regulation of sodium i n the larva of Aedes aegypti (L.). I I . The net transport and the fluxes associated with i t . J . Exp. B i o l . 37: 594--608.  67. Stobbart, R.H. 1965. The effect of some anions and cations upon the fluxes and net uptake of sodium i n the larva of Aedes aegypti (L.). J . Exp. B i o l . 4-2: 29-4-3. Stobbart, R.H. 1967. The effectof some anions and cations upon the fluxes and net uptake of chloride i n the larva of Aedes aegypti (L.), and the nature of the uptake mechanisms for Nat and C l " . J . Exp. B i o l , 4-7: 35-57. Stobbart, R.H. 1971. ^actors a f f e c t i n g the control of body volume i n the larvae of the mosquitoes Aedes aegypti (L.) and Aedes detritus Edw. J . Exp. B i o l . 54-: 67-82. S u t c l i f f e , D.W. 19618- Studies on salt and water balance i n caddis larvae (Trichoptera): I. Osmotic and ionic regu l a t i o n of body f l u i d s i n Limnephilus a f f i n i s C u r t i s . J. Exp. B i o l . 3 8 : 501-519. S u t c l i f f e , D.W. 1961b- Studies on s a l t and water balance i n caddis larvae (Trichoptera): I I . Osmotic and ionic regulat i o n of body f l u i d s i n Limnephilus stigma Curtis and Anabolia nervosa Leach. J . Exp. B i o l . 3 8 : 521-530. S u t c l i f f e , D.W. 1967a. Sodium regulation i n the fresh water amphipod, Gammarus pulex (L.). J . Exp. B i o l . 4-6:- 4-99-518. S u t c l i f f e , D.W. 1967b. Sodium regulation i n the amphipod Gammarus duebeni from brackish-water and fresh-water localities in Britain. J . Exp. B i o l . 4-6: 529-550. S u t c l i f f e , D.W. 1968. Sodium regulation and adaptation to fresh water i n Gammarid crustaceans. J . Exp. B i o l . 4-8: 359-380. S u t c l i f f e , D.W. 1971. Sodium influx and loss i n freshwater and brackish-water populations of the amphipod Gammarus duebeni L i l l j e b o r g . J . x p . B i o l . 54-: 2 5 5-26 8. £  S u t c l i f f e , D.W. 1974-. Sodium regulation and adaptation t o fresh water i n the isopod genus Asellus. J . Exp. B i o l . 6 1 : 719-736. S u t c l i f f e , D.W., & J . Shaw. 1967. The sodium balance mechanism i n the fresh water amphipod, Gammarus l a c u s t r i s Sars. J . Exp. B i o l . 4-6: 519-528. Thuet, P., R. Motais, & J . Maetz. 1968. Les mecanismes de 1'euryhalinite chez l e c r u s t a c 6 de salines, Artemia salina L. Comp. Biochem. Physiol. 2 6 : 793-818.  68. Topping, M.S. 1969. Giant chromosomes, ecology, and adaptat i o n i n Chironomus tentans. Ph.D. Diss. U.B.C., Vancouver, Topping, M.S. 1971. Ecology of the larvae of C. tentans in saline lakes i n central B r i t i s h Columbia. Can. Entom. 103* 328-338. Topping, M.S. 1972. D i s t r i b u t i o n of C. tentans ie some lakes i n central B r i t i s h Columbia i n r e l a t i o n to some physical and chemical factors. Proc. XIII Int. Congr. Ent. 3* 4-77-4-73. Topping, M.S., & G.G.E. Scudder. 1977. ome physical and chemical features of saline lakes i n central B r i t i s h Columbia. Syesis 10: 14-5-166. s  Townes, H.K. 194-5. The Nearctic species of Tendipedini. Amer. Midi. Nat. 34-: 1-206. Wigglesworth, V.B. 1938. The regulation of osmotic pressure and chloride concentration i n the haemolymph of mosquito larvae. J . Exp. B i o l . 15: 235-24-7. Wright, D.A. 1975a. Sodium regulation i n the larvae of Chironomus dorsalis (Meig.) and Camptochironomus tentans, (Fabr.): the effect of salt depletion and some observations on temperature changes. J . Exp. B i o l . 6 2 : 121-139. Wright, D.A. 1975b. The effect of external sodium concentrat i o n upon sodium fluxes i n Chironomus dorsalis (Meig.) and Camptochironomus tentans (Fabr.), and the effect of other ions on sodium influx i n C. tentans. J . Exp. B i o l . 6 2 : 1M-155. Wright, D.A. 1975c The relationship between t r a n s e p i t h e l i a l sodium movement and potential difference i n the larva of Camptochironomus tentans (Fabr.) and some observations on the accumulation of other ions. J . Exp. B i o l . 6 2 : 157-174-.  B.C.  

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