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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 British 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 presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I further agree that permission for extensive copying of th i s thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion o f th i s thes is f o r f i nanc ia l gain sha l l not be allowed without my writ ten permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i i ABSTRACT This thesis deals with the importance of salinity to the distribution of three chironomid species of the genus Chironomus (C. anthracinus. C. athalassicus. and C. tentans). Research to date suggests that salinity and coexistence problems are the major factors influencing the distribution of the chironomid fauna of fresh and saline lakes in the Cariboo and Chilcotin areas of central British Columbia. The difference in the distribution of these three Chironomus species is particularly interesting. The investigation of the importance of salinity to their distribution consisted of a study of (i) the salinity tolerance of each species, (ii)the 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 in the salinity tolerance of each species was found in the laboratory: C. anthracinus and C. tentans did not survive at lake water conductivities above 9000 micro-o mhos/cm at 25 C, C. athalassicus survived in lake water conductivities at least as high as 15»000 micromhos/cm at o 25 C. Temperature affected the survival of each species in 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 i i i had a low sodium aff i n i t y and a powerful sodium uptake system compared to the other species. Chloride affinity and the power of the uptake system exceeded that of C. anthracinus and C. tentans. The general conclusion reached was that salinity does affect the distribution of the three Chlronomus species. More research is called for in this and related areas in order to more f u l l y understand the distribution of the chironomid fauna. iv TABLE OF CONTENTS A. Preliminary Pagess Page T i t l e Page i Abstract i i Table of Contents iv L i s t of Tables v L i s t of Figures v i Acknowledgement v i i i B. Text: I. Introduction 1. I I . Materials and Methods h. 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 h. b) S a l i n i t y Tolerance of Larvae 5. c) Regulation of Haemolymph 7. d) Sodium and Chloride Flux 8. I I I . Results m-. a) S a l i n i t y Tolerance of Larvae 1^ f. b) Regulation of Haemolymph 19. c) Sodium and Chloride Flux 29. IV. Discussion a) S a l i n i t y Tolerance of Larvae h1. b) Regulation of Haemolymph k-5. c) Sodium and Chloride Flux 51. V. Conclusion 62. Literature Cited 6h. V LIST OF TABLES PAGE TABLE 1 The Occurrence of Chironomidae in the Lakes 3» at the One Meter Depth. 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 11 Solutions. TABLE h The Equations ^escribing the Results of Flux 36 Measurements. TABLE 5 The Sodium Flux Rates of Several Invertebrate 57 Species. 58 vi LIST OF FIGURES PAGE FIGURE 1; The Survival of Fourth Instar C. anthracinus. 1 5. and C. tentans Larvae in Rock L. Water. FIGURE 2 The Survival of Fourth Instar C. athalassicus 17. Larvae in Rock L. Water. FIGURE 3 The Survival of Fourth Instar C. athalassicus 18. Larvae in Rock L. Water Conductivities Above 10,000 micromhos/cm (at 25°C). FIGURE h The Effect of Transfer to a Medium of Differ- 20. ent Sodium Concentration on the Sodium Con-centration of the Haemolymph of Chironomus Larvae. FIGURE 5 The Effect of Transfer to a Medium of Diff-erent Concentration on the Freezing Point Depression of the Haemolymph of Chironomus Larvae. FIGURE 6 The Freezing Point depression of the Haemo-lymph of Fourth Instar C. anthracinus. C. athalassicus. and C. tentans.Larvae Over a Range of External Medium Concentrations. FIGURE 7 The Sodium Concentration of the Haemolymph of Fourth Instar C. anthracinus t C. athalassicus, and C. tentans Larvae Over a Range of External Sodium Concentrations 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. FIGURE 9 The Chloride Concentration of the Haemolymph of Fourth Instar C. anthracinus. C. athalassicus f and C. tentans Larvae Over a Range of External Chloride Concentrations. FIGURE 10^The Uptake of Sodium-22 by C. anthracinus, £• athalassicus. and £. tentans Over a 72 Hour Period. 21. 23. 25. 27. 28. 30. v i i LIST OF FIGURES CONTINUED PAGE FIGURE 11 The Uptake of Chloride-36 by C. anthracinus. 3 1 . C. athalassicas T and C. tentans Over a 120 Hour Period. FIGURE 12 The Effect of the External Sodium Concentra- 33 . tion on the Sodium Flux Rate of Fourth Instar C. anthracinus Larvae. FIGURE 13 The Effect of the External Sodium Concentra- 3*+. tion on the Sodium Flux Rate of Fourth Instar C. athalassicus Larvae. FIGURE llf The Effect of the External Sodium Concentra- 35 . tion on the Sodium Flux Rate of Fourth Instar C. tentans Larvae. FIGURE 15 The Effect of the External Chloride Concent- 38. ration on the Chloride Flux Rate of Fourth Instar C. anthracinus Larvae. FIGURE 16 The Effect of the External Chloride Concent- 39. ration of the Chloride Flux Rate of Fourth Instar C. athalassicus Larvae. FIGURE 17 The Effect of the External Chloride Concent- 1+0. ration on the Chloride Flux &ate of Fourth Instar C. tentans Larvae. FIGURE 18 The Daily Temperature Range in Three of the k 2 . Lakes Where Chironomus Species are Abundant. FIGURE 19 The Survival Limits of C. anthracinus. ^6. C. athalassicus. and C. tentans as Determined by Laboratory Experiments. v i i i ACKNOWLEDGEMENT I wish to thank Drs. Scudder, Acton, and Phillips for their advice and interest in this thesis. As a supervisor Dr. Scudder has been both inspirational and understanding. This has made my l i f e as a graduate student more enjoyable. A research topic such as this depends upon certain tech-nical s k i l l s . 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 its composition and dilution, offers a variety of problems to the survival of its inhabit-ants. In high salinities there are problems with water economy and the balance of the various ions within the body. In low salin 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 Chilcotin regions of British Columbia. Topping (1972) and Cannings and Scudder (1978) have documented the distribution of the chironomid fauna in relation to various characteristics of the lakes. The research to date suggests that salinity and coexistence problems are the major factors influencing the distribution of the chironomid fauna. The distribution of three chironomid species of the genus Chironomus (C. anthracinus Zetterstedt, C. athalassicus Cannings, and C. tentans Fabricius) is particularly interesting (Table 1). The difference in the distribution of the three species is the focus of this thesis, particular attention is paid to the importance of salinity. The investigation consisted of (i) the salinity tolerance of each species, ( i i ) the regula-tion of ion levels in the haemolymph, ( i i i ) the influx of sodium and chloride ions. Knowing what salinities each species can tolerate is important for i t may explain why C. anthracinus and C. tentans 2. are absent from certain lakes where C. athalassicus is present. It is also important to know not only i f salinity affects chironomid distribution in the study area, but how salinity affects the individual larvae. The best way to determine how salinity affects the larvae is to compare certain aspects of the physiology of the three species in different s a l i n i t i e s . Thus the effect of external environment on the haemolymph of the insects was studied by monitoring the osmotic concentra-tion, and the concentration of sodium, potassium, and chloride of the body f l u i d while each of the parameters was varied in the external environment. Regulation of the haemolymph is c r i t i c a l because i t is 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 distribution 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 this study cannot completely explain the effects of salinity on chironomid distribution, i t can determine i f salinity does in fact affect chironomid distribution and in doing so contribute to the understanding of chironomid physiology. 3a. TABLE 1 The Occurrence of Chironomidae in the Lakes at the One Meter Depth. After Topping (1969) and Cannings (1973). 3 . LAKE SPECIES CONDUCTIVITY (micromhos/cm) @ 25°C SODIUM (mM) CHLORIDE (mM) W CO o w a •H e •H a cn a o O o CO o a CO a CO £ o o o cO m cO -P • H -p •H £5 •H cj X5 a si +3 CD O cd o CO o Barnes L. Round-Up L. L. Lye Boitano L. L. Jackson L. Greer Rock L. Nr. Phalarope Westwick L. Sorenson L. Nr. Op. Crescent Box 17 Barkley L. East L. Box 27 X X X X X X X X X X X X X X X X X X X X X X X X X X A X X X X X X X X X 12,000 7,000 6,600 *+,200 2,600 1 ,600 1 ,500 1,325 1,280 1,500 800 7^0 600 130.26 71 .35 70 .25 '3^.03 15.^2 13.25 1^.13 7 .25 it.61 hM 3.^1 3.31 •1.85 3.08 0 .15 36.97 23.69 21.90 * f . 2 0 3.28 1.25 1.85 0 A 9 0.11 0.3+ 0 .25 0 . 1 V o.T5' 0.26 0 .07 If. II. MATERIALS AND METHODS a. Collection and Identification Larvae of Chironomus anthracinus. C. athalassicus. and C. tentans were collected from lakes in the central interior of British Columbia. The characteristics of the loc 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 Chilcotin plateau, while C. tentans was collected from Westwick L. in the Cariboo region. Eckraan dredge samples of bottom mud were collected and sifted in mesh-bottom tubs. Fourth instar larvae, recognized by the enlarged prothoracic segments, were picked but by hand. They were then transported to the laboratory in one gallon Thermos jugs containing lake water. In the laboratory, the larvae were either used immediately, or were held in constant o temperature cabinets at 5 C and under long photoperiod (16 hr light: 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 intervals. The larvae were identified by use of the key prepared by Bassett (1967)• Bassett's species IV, V, and VII refer to C. anthracinus t C. athalassicus T and C. tentans. respectively. A l l three species are of the C. dorsalis type, as outlined by Miall and Hammond (1900), in that they have four long, paired blood g i l l s on the posterior ventral surface. Identification 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 identified by reference to the description of Cannings (1975) . Fourth instar larvae of the three species were collected together where possible so that they were acclimated to similar physical and chemical conditions. However, since only a limited number of larvae were thus obtained, those collected from different 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 in the spring, and hence were overwintered forms. Attempts were made to rear the larvae and obtain egg masses for experimental use. Unfortunately, while adults could be obtained in some cases, the resulting numbers of egg1 masses were too small for experimentation. b. Salinity Tolerance of Larvae o Larvae were held at 5 C until required. Experiments were conducted in millipore f i l t e r e d (HA 0 A 5 JU) Rock L. water which was diluted (with d i s t i l l e d water) or air evaporated to produce the desired s a l i n i t i e s . Rock L. water was used because of i t s chemical similarity to Jackson L. and Westwick L. water, these being the lakes from which the larvae were collected. Larvae were acclimated by sequential increases of 1000 micromho/cm every 12 hours until the desired salinity was reached, following the method used by Beadle (1939) with Aedes detritus. The range of salinity tested depended upon the upper limit of salinity to which the species could acclimate. TABLE 2 The Composition of Rock Lake Water. After Topping and Scudder, 1969. 6 . THE COMPOSITION OF ROCK L. ION M-EQUIV./L. Na + 1^.91 K + O .98 C a + + 0 . 6 2 Mg + + 1.71+ WATER PER CENT TOTAL CATIONS 8 1 . 7 5 A . 9 . 5 PER CENT TOTAL ANIONS CO" M-.32 2 3 . 5 HCO3 1 2 . 3 5 6 7 . 0 CI* 1 .57 8 . 5 SO"" 0 . 1 8 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 their respective conductivities from which they were collected. For each species, duplicate samples of ten fourth instar larvae were placed in 100 ml of the test salinity at 5°C ( + 1 ° ) , 15°C ( + 1 ° ) , and 23°C ( + 3 ° ) . Acclimation to the higher temp-eratures was at a rate of 10°C per 12 hour period. Light conditions were 16 hours light: 8 hours dark. Air was gently bubbled through a l l test salinities 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 for each experimental combination of temperature and salinity; these were compared to control groups of the same species. c. Regulation of Haemolymph Fourth instar larvae were acclimated to the experimental sal i n i t i e s in steps of 1000 micromhos/cm per 12 hour period. Larvae were acclimated to the Rock L. experimental salinity for 96 hours at 5°C Haemolymph was analyzed from C. athalassicus acclimated to salinities 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 salinities 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 salinity, samples of haemolymph and of the external medium were taken. Haemolymph samples were obtained by removing a larva from the test medium, rinsing with d i s t i l l e d water, drying briefly on a Kimwipe, placing on Parafilm M, and puncturing the cuticle. Samples were taken up in one microliter Microcap pipettes (Drummond Sci. Co., Brommall, Pa.). Freezing point depression was determined from samples taken up in a sandwich of paraffin o i l contained in non-alkaline glass pipettes. The osmotic pressure (Ag) and the concentration of sodium, potassium, and chloride of the samples were determined. A nanoliter osmometer (Clifton Technical Physics, Hartford, N.Y.) was used to read the freezing point depression of the samples. Chloride concentration determined with a microtitration buret (Misco Microchemical Specialties micro-burette coupled to a Radiometer 25 voltmeter, Copenhagen, Denmark). One microlitre samples were titrated with a 0 . 0 2 $ AgNO^ solution to an electrical endpoint (Ramsay, Brown, and Croghan, 1955). The concentrations of sodium and potassium were determined with an atomic absorption spectrophotometer (Varian Techtron Pty, Ltd., Melbourne, Australia), 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, 1 9 6 0 ) . In the experiments described below, sodium-22 as NaCl and 9 . chloride - 3 6 as Na-^Cl Were used to estimate the sodium flux and the chloride flux in fourth instar larvae of C. anthracinus« C. athalassicus, and C. tentans. The sodium and chloride flux were investigated in separate experiments, but the methods used were similar. A l l experiments were conducted at a room temperature of 23°C ( + 3 ° )* the temperature was monitored by continous recording equipment. Wet weights of individual larvae were obtained by surface drying with Kimwipes (Scott Paper Co.) then weighing on an August Sauter 50 mg balance (Ebingen, Wurtbg., Germany) to an accuracy of 0.01i mg. The radioactivity of samples was determined with a Nuclear-Chicago endwindow G-M counter (Model *+70) using stainless steel planchets. Larvae were macerated in the planchets using a standardized technique. Solution samples were measured with an automatic pipette (Oxford Sampler Micro-pipetting System), or a Drummond Microcap pipette of appro-priate volume. Samples were air dried previous to counting. A correction factor for sample self-absorption was established by f i r s t finding the radioactivity of a known amount of isotope, then recounting with a sample in place on top of the isotope. Larvae removed from the radioactive loading medium were rinsed for at least a half minute in a current of non-radioactive Rock L. water. An estimate of the radioactivity that remained on the outside of the larval body was found by placing a non-radioactive larva in radio-active 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 radioactivity. To establish 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). The freezing point depression of each solution was determined and where necessary sucrose was added to raise the osmotic concentration. Sodium and chloride concentrations were ascertained according to the methods described above. To establish that sodium and chloride enter the body at sites separate from the mouth, larvae of the three species were ligated just posterior to the head capsule with silk thread. Ligated and non-ligated larvae were placed in sol-utions containing sodium-22 Qrd6hloride-36. At the end of the experiment the larvae were rinsed, weighed, and carcass radioactivity was determined. The radioactivity of ligated larvae was compared to that of non-ligated larvae. It was fQtjn&f;tRat the radioactivity was similar in 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. Fifty-five fourth instar larvae of each species were acclimated in Rock L. water at 23°C for 2h hours. They were placed in plastic vials containing a known quantity of isotope (2.28 microCurie 2 2Na or 5.50 microGurie ^ C l ) 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^ in a 1;3 ratio. 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 mM, 3.0 mM, 9.0 mM, and 27.0 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 KH2POL 1.0 mM NaHCO^ lf .0 mM NaCO. 2 .0 mM 12. 55ml of millipore f i l t e r e d Rock L. water. The activity of the solution was determined at time T Q. The water was aerated throughout the experiment. At intervals of 1 hr., 2 hr., 3 hr., 5 hr., 10 hr., 2k hr., *+8 hr., 96 hr., and 120 hr. three to five larvae were removed from each v i a l . The larvae were rinsed, weighed, and carcass radioactivity was determined. Corrections were made for rinse-off residue and for self-absorp-tion. Stobbart ( i 9 6 0 ) describes the sequence of ion flux across a body wall. For a short time specific ions move in one direction only: from the outside medium into the body. After a period of time there is flux both into and out of the body, eventually leading to a point where there is no net flux in either direction. During the i n i t i a l influx period the departure from linearity of the time versus rate of influx relationship would be slight, and i t can be assumed that backflux would be neglegible. The length of that short period of unidirectional flux was estimated from the results of these experiments, ^ach of the three species was dealt with separately. The estimate of the unidirectional flux period was taken as a suitable time for short term flux experiments. To establish the individual 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 for the predetermined time period. Five larvae were placed in a 12 dram plastic vial 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 2 2Na, or 2 . 6 9 microCurie -^Cl). The radioactivity 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 radioactivity were corrected for rinse-off residue and self-absorption. Influx rates were expressed as mmolesAg wet weight/hour. The individual efflux rates of sodium and chloride from C. anthracinus. C. athalassicus. and C. tentans larvae were estimated by allowing them to equilibrate in radioactive sodium-22 or chloride - 3 6 solutions over a 96 hour period at 5°C, then moving the larvae into non-radioactive water. This was sufficient time for the sodium-22 specific activity in the larvae to equilibrate with the external environment. Once equilibrated the larvae were rinsed and placed individually in 12 dram plastic vials containing five 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. After three hours the larvae were removed and weighed. Two 0 . 5 ml samples of the solution were taken from each v i a l , air dried, and the radioactivity 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. III. RESULTS a. Salinity Tolerance of Larvae The mean survival of fourth instar C. anthracinus and C. tentans larvae is shown in Figure 1 . Both species survived upon acclimation to a range of 0 to 9000 micromhos/cm at 25°C$ over twice that found in the f i e l d (Cannings and Scudder, 1978). Acclimation to the higher conductivities is possible only i f the conductivity is 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 survival 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 tests. The results showed that survival throughout the range of cond-uctivities tested was similar to or better than survival at the control conductivity in each temperature regime. This suggests that C. anthracinus 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 is 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 in the control cond-uctivity (1500 micromhos/cm at 25°C) by chi-square tests, limits to the survival of C. tentans could be designated 1 5 a . FIGURE 1 The Survival of Fourth Instar C. anthracinus and tentans larvae i n Rock L. water. Experiments at three temperatures are depicted: 5°C (—•—), 15°C (—*—), and 23°C (—o—). Each conductivity was determined at 25°C. One standard error is indicated for each survival value. (n= 20 larvae for each point) C O N D U C T I V I T Y X 1 0 0 0 ( M I C R O M H O S / C M A T 2 5 ° C ) 16. for 5°C and 15°C. No such limits 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 significantly 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 significantly reduced at 7000 and 9000 micromhos/cm (at 25°C). The similarity 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 is 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 in 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. This was borne out by chi-square tests which compared survival at each conductivity to survival at a designated conductivity ( 1 7 , 0 0 0 micromhos/cm at 25°C). No limits to survival could be set on the basis of the information contained in Figure 2 . Figure 3 shows the results of a further experiment to find the highest conductivity at which C. athalassicus larvae can survive. An attempt was made to acclimate larvae to conductivities as high as 3 5 , 0 0 0 micromhos/cm (at 25°C). The highest conductivity to which a significant 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. Standard errors are omitted for cl a r i t y . (n= 20 larvae for each point) 17. 18a. FIGURE 3 The Survival of Fourth Instar C. athalassicus Larvae in 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 is indicated for each survival value. (n= 20 larvae for each point) 19. conductivities above 2 0 , 0 0 0 micromhos/cm (at 25°C), however, the larvae appeared to be in poor health with a lack of activity and a shrivelled appearance. This observation is supported by the results shown in Figure 3 . At 2 0 , 0 0 0 micromhos/cm (at 25°C) in the 5 ° c and 15°C temperature regimes there was a marked reduction in 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 survival of C. anthracinus. C. athalassicus. and G. tentans. The results displayed in Figures 1 , 2 , and 3 show that in 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 in Rock L. water of various s a l i n i t i e s . At time intervals up to 96 hours after introduction, larvae were removed and the sodium concentration and freezing point depression of their haemolymph were determined. During the f i r s t 2k hours major shifts in each parameter were observed (Figures *f and 5 ) . No s t a t i s t i c a l l y significant changes were, however, observed after ^8, 72 , and 96 hours. Based on the results shown in Figures h and 5 the larvae were acclimated for 96 hours before subsequent experiments were carried out. No problem was encountered in 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 is shown as C — ) . Transfer from a medium of 1 9 2 . 5 mM Na to 1 0 9 . 5 mM Na is shown as (—o—). One standard error is indicated for each value. 20. HAEMOLYMPH SODIUM (mM SODIUM) I 2 1 a . 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 is followed over a 96 hour period. Transfer from a medium of A E 0.^7°C to a medium of A E 0.65°C is shown as ( — 6 — ) . Transfer from a medium of Ag 0.>+70C to a medium of A g 0.32°C is shown as (—o—). One standard error is indicated for each value. 2 1 . " " ~" 6 6 d HAEMOLYMPH FREEZING POINT DEPRESSION (°C) 22 water on the freezing point depression of the haemolymph of C. anthracinus. C. athalassicus. and C. tentans is shown in Figure 6 . A l l three species hyperregulated throughout the range of external concentrations provided in the experiment. The haemolymph of each species was relatively constant in freezing point depression over the range of Ag 0 . 0 0 to 0.32°C, with no significant difference among the species. At external concentrations above A g 0.32°C the freezing point depression of a l l three species rose sharply with C. anthracinus and C. tentans unable to survive in media significantly 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 until the external concentration reached Ag 0.67°C where the haemolymph concent-ration increased sharply and thereafter varied considerably. The results shown in Figure 6 suggest that the three species regulated their haemolymph osmotic pressure in 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 limit of their survival range (Ag 0.39°C is 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 line. 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 is shown as (—*—). C. athalassicus is shown as (- _ A _ _). C. tentans is shown as (-<>-—). One standard error is indicated for each value. (n= h to 6 larvae for each point) 23. 2k. ( A g 0.67°C) which is near the upper survival limit for C. athalassicus larvae. Such variation in the freezing point depression of C. athalassicus may be linked to the survival problems encountered by C. athalassicus larvae above 15,000 micromhos/cm (at 25°C) in Rock L. water. The effects of external sodium concentration on each of the three Chironomus species are presented in Figure 7. A l l three species hyperregulated haemolymph sodium over the range 0 to 110 mM sodium; no significant 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 and C. tentans did not survive well in the media that contained sodium levels above 110 mM sodium (equivalent to Rock L. water conductivity of 9000 micromhos/cm at 25°C) so i t is 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, and the haemo-lymph sodium concentrations 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. athalassicus T and C. tentans Larvae Over a Range 6f External Sodium Concentrations. C. anthracinus i s shown as (—•—). C. athalassicus is shown as (—*--). C_. tentans is shown as (—o~). One standard error is indicated for each value. (n= *f to 6 larvae for each point) 25 26. and C. tentans did not hyporegulate, while C. athalassicus survived at considerably higher sodium concentrations and appeared to hyporegulate the haemolymph sodium concentration. The results of the potassium concentration determinations are shown i n Figure 8 . Below external potassium concentrations of 8 . 0 mM potassium the haemolymph potassium concentration of the three species was only s l i g h t l y above or equal to that of the external medium. At potassium concentrations above 8 . 0 mM potassium (equivalent to a Rock L. water conductivity above 10,000 micromhos/cm at 25°C) C. athalassicus haemolymph potassium concentration rose sharply and varied considerably. At no time did the haemolymph potassium concentration of any of the three species f a l l below that of the external medium. These findings suggest that the potassium concentration i n the haemolymph of the three species conforms to the potassium concentration of the external environment, and that they do not hyporegulate potassium. The results presented i n Figure 9 indicate considerable v a r i a t i o n i n the haemolymph chloride concentration of each species over the range of external concentrations that were tested. The response of the three species was probably si m i l a r , but the reason for the i r r e g u l a r i t i e s i n Figure 9 i s unknown. The chloride concentration was hyperregulated by the three species at a l l external chloride concentrations, with the exception of the reaction of C. athalassicus at 3 0 . 3 mM chloride (equivalent to a Rock L. water conductivity of 3 0 , 0 0 0 micromhos/cm at 2 5°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 is shown as (—*—). C. tentans is shown as ( - -o- ) . One standard error is indicated for each value. (n= h to 6 larvae for each point) 27. CM r -POTASSIUM CONCENTRATION 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 is shown as (—•—). C. athalassicus is shown as (---*—). C. tentans is shown as (—o—). One standard error is indicated for each value. (n= h to 6 larvae for 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 five 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 directly 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 in 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 is shown by Figures 10 and 11 to be sufficient for the three species to reach a steady-state. No significant difference in carcass radioactivity per unit wet weight was found after hQ hours acclimation. The influx and efflux of sodium was investigated in steady-state larvae only; no depletion tests were carried out since the objective was to compare C. anthracinus. C. athalassicus. and C. tentans.larvae in terms of the conditions encountered in 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 is shown as (—•—). C. athalassicus is shown as (--a---), c. tentans is shown as (—o—). One standard error is indicated for each value. 12 0' 10CH 0 10 20 30 40 50 60 70 80 TIME (HOURS) 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 is displayed as disintegrations (counts) per mg wet weight of larva per minute. C. anthracinus is shown as (—•—). C. athalassicus is shown as ( — ) . C. tentans is shown as (-0-). One standard error is indicated for each value. 3 1 . 3 2 . at which half the maximum flux rate is obtained. The sodium influx and efflux rates of £. anthracinus. C. athalassicus. and C. tentans larvae are shown in Figures 12 ,13,and 1*f. The equations that describe the curves in these figures are presented in Table k. The sodium influx curves of the three species differed markedly: C. tentans had the lowest Vmax (maximum influx rate) of 0 . 9 6 mmole Na/Kg/hr and the lowest Km value ( 1 . 0 5 mM Na), while C. anthracinus was intermediate with a maximum influx rate of 2 . 1 9 mmole Na/Kg/hr, and a Km value of 2 . 1 5 mM Na. The maximum rate of C. athalassicus was 3.1*+ 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 differed only slightly amongst the three species as shown in Figures 1 2 , 1 3 ,and 1*f. There was very l i t t l e increase in the sodium efflux rate of any of the three species as external sodium concentration was increased. Also the efflux rates of the three species differed 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 0 r 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, for C. tentans i t was 3*30 mM sodium Chloride flux 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 is shown as (—•—). Sodium efflux is shown as (--*--). The influx Vmax is 2.19 mmole Na/Kg wet weight/hr; the Km is 2 .15 mM Na. The equation which describes the efflux line is (Y - 9.0 • 10" 1 + X + Q.h7). One standard error is indicated for each value. 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 is shown as (—•—). Sodium efflux is shown as (--^--). The influx Vmax is 3 . 1 ^ mmole Na/Kg wet weight/hr; the Km is 5.65 mM Na. The equation which describes the efflux line is (Y = 3 3 . ^ • 1 0 ' ^ + 1.02) . One standard error is indicated for each value. 35a. FIGURE llf The Effect of the External Sodium Concentration on the Sodium Flux Rate of Fourth Instar C. tentans Larvae. Sodium influx is shown as (—•—). Sodium efflux is shown as ( - - A - - ) . The influx Vmax is 0.96 mmole Na/Kg wet weight/hr; the Km is 1.05 mM Na. The equation which describes the efflux line is (Y = 9.1 • 10 - l + + 0.69). One standard error i s indicated for each value. 35 -03 +4 O o o SODIUM I r Q FLUX (mMOLES O Na/Kg WET WEIGHT/ HOUR) 36a. TABLE h The Equations Describing the Results of Flux Measurements. Sodium and chloride influx, and sodium and chloride efflux equations are liste d . The symbols are explained in the text. 36 THE EQUATIONS DESCRIBING THE RESULTS OF FLUX MEASUREMENTS SPECIES C. anthracinus C. athalassicus C. tentans MEASUREMENT EQUATION Sodium influx 2.19 x C 2.15 + c Sodium efflux (Y=(9.0 • 10-^)X + 0 A 7 ) Chloride influx 5.61 x C 7.77 + C Chloride efflux (Y=(12.0 • 10~3)x + 0.50) Sodium influx l.1*f x C 5.65 + C io~Sx + Sodium efflux (Y=(33A ' 1.02) Chloride influx 5t70 x c 2.90 + C Chloride efflux (Y=(2.0- 10~ 3)X + 0.9^) Sodium influx 0.96 x C 1.05 + c 10 )X + Sodium efflux (Y=(9.1 • 0.62) Chloride influx 2.36 x C if. 99 + C Chloride efflux (Y=(2.0- 10" 3)X + ) .68) 37. chloride depleted larvae. The relationship between chloride influx and the external chloride concentration can, as in the case of sodium influx, be described approximately by the Michaelis-Menton equation (see above). The influx and efflux measurements of C. anthracinus, C. athalassicus, and C. tentans are shown in Figures 15, 16, and 17 respectively. The equations that describe the flux results are given in Table h (page 3 6 ) . Certain differences amongst the three species are obvious in 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 aff 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 af f i n i t y for chloride and the most powerful influx system. The relationship between chloride efflux and the external chloride concentration was similar amongst the three species, with the rate of chloride efflux rising only slightly as the external chloride concentration increased. At a certain external chloride concentration the rates of influx and efflux were equal and net flux f e l l to zero. For C. anthracinus that concentration was O.76 mM CI, for 0 . athalassicus i t was O .83 mM CI, and for C. tentans i t was 2 . 2 9 mM CI. 38a. FIGURE 15 The Effect of the External Chloride Concentration on the Chloride Flux Rate of Fourth Instar C. anthracinus Larvae. Chloride influx is shown as (—•—). Chloride efflux is shown as ( - - A - - ) . The influx Vmax is 5.63 mmole Cl/Kg wet weight/hr; the Km is 7.77 mM Cl. The equation which describes the efflux line i s (Y = 11.9 x 10"3X + 0 . 5 0 ) . One standard error is indicated for each value. 33 39a. FIGURE 16 The Ef f e c t of the External Chloride Concentration on the Chloride Flux Rate of Fourth Instar C. athalassicus Larvae. Chloride influx i s shown as ( — • — ) . Chloride efflux i s shown as (--a--). The in 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 describes the efflux l i n e i s (Y = 2.0 x 10"3X + 0.9*+). One standard error i s indicated for each value. 39 40a. FIGURE 17 The E f f e c t of the External Chloride Concentration on the Chloride ^lux. Rate of Fourth Instar C. tentans Larvae. Chloride influx i s shown as ( — • — ) . Chloride efflux i s shown as ( - - A - - ) . The i n f l u x Vmax i s 2.36 mmole Cl/Kg wet weight/hr; the Km i s ^.99 mM CI. The equation which describes the e f f l u x l i n e i s (Y = 19.8 x 10"^X + 0.68). 1+1. IV DISCUSSION a. Sal inity Tolerance of Larvae Kinne (1963, f$6k) has discussed the effects of temper-ature and sal inity factors, and has demonstrated that temper-ature and sal in ity are interrelated in their effects on .^ act iv i ty and metabolism. They therefore cannot be easily considered separately. C. anthracinus. C. athalassicus. and C. tentans have been subjected inthis study to combinations of temperatures and sa l in i t ies . The findings support the argument put forward by Kinne (1963) which states that the effect of ~ sa l in i ty on survival is dependent upon the temperature of the environment. An example of this relationship is shown in Figure 1 (page 15) . C. tentans survives a mean of 16 days at 5°C in a 7000 micromhos/cm (at 25°C) solution, but survives a mean of only four days in 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 in the spring (5°C) and high in the summer (25°C). One of the objectives of the present study is to compare the range of sa l in i t ies that C. anthracinus t C. athalassicus, and C. tentans can tolerate. While they are affected by temperature in a similar way, the range of sal inity that the species survive dif fers s ignif icantly. C. athalassicus can .., survive at: sa l in i t ies 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 in Three of the Lakes Where Chironomus Species are Abundant. (After Cannings, 1973) ( ° n e meter depth) 2 5-2CH 15-H 1 0 -5H i l |II,I,-|I„...||.'' M A Y ml | ' I.I.'-I h2. I, .I' i' L. J A C K SON J U N E J U L Y A U G U S T 3 0 n O 2 5 H 2 0 -cr. H 15-< or UJ Q. 1 0 -LU - 5 H l ' l "„ .I'l I 1" •" "II..,.". I" cc LU t -< £ 3on 2 5H 2 OH 1 5 H ioH 5H M M EAST L. ''Il I I'' "-'•'•I'.ll WESTWICK L. ^ 3 . of s a l i n i t i e s . Scudder (1969) carried out temperature and salinity experiments to compare the survival of Cenocorixa bifida and C. expleta. C. bifida survives to an upper conductivity limit of between 2 0 , 0 0 0 and 2 ^ , 0 0 0 micromhos/cm (at 25°C), while C. expleta survives to an upper limit 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. bifida was collected in waters up to 2 0 , 0 0 0 micromhos/cm (at 25°C), and C. expleta was collected in waters up to 3 0 , 0 0 0 micromhos/cm (at 25°C). Similarly the results of the laboratory experiments can be compared to data obtained from f i e l d collections of C. anthracinus, C. athalassicus, and £. tentans. Such collections were carried out by Cannings ( 1 9 7 3 ) , working in the one meter depth zone of lakes in the Cariboo and Chilcotin areas (see cannings and Scudder, 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 far as C. athalassicus is concerned. C. anthracinus and C. tentans, however, survived in the laboratory at conductivities well above those that they were collected from in the f i e l d . Such discrepancies between laboratory and f i e l d results have been found in other studies (Doudoroff, 1938 working with marine f i s h ) . A partial explanation for the discrepancy l i e s in the effects of salinity on the organism and i t s ecology. As Kinne (196^) points out, salinity may affect functional and structural properties of the organism through changes in osmo-concentration, solute concentration, coeffic-ients of absorption, density, and viscosity. Salinity w i l l affect the species composition of the eco-system, the avail-a b i l i t y of food, and the effects and likelihood of disease. Another factor which must be considered is that survival is 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, in 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 salinity is acting upon C. anthracinus, C. athalassicus, and C. tentans is not apparent, i t is obvious that C. athalassicus can survive in the lakes occupied by £. anthracinus and C. tentans. and that i t can survive in lakes much more saline than those occupied by the other two species. It is important to relate this distribution difference amongst the species to the effects of salinity on haemolymph composition, and to the effects of the concentration of certain ions in the lake water. Although the species are affected differently by the h5. salinity of the external medium, the length of time that the species survive decreases in a l l cases as temperature rises. At a fixed temperature, survival decreases i f salinity is increased past a certain level. Figure 19 depicts this relationship between temperature and salinity (expressed as conductivity), b. Regulation of the Haemolymph The osmotic and ionic regulation of several fresh and saline water insect species have been well documented (Wigglesworth, 1938; Beadle, 1939; Ramsay, 1953; Shaw, 1955; Sutcliffe, 1961a, 1961b; Scudder et a l . 1972; and Nayer et a l . 1971+). Animals such as insect species that are restricted to low salinities are in general unable to hyporegulate the haemolymph in waters of high salinity (Beadle, 1969). C. anthracinus, C. athalassicus. and C. tentans are examples of such animals. They regulate the osmotic pressure of the haemolymph in lake water concentrations up to A 0.32°C, but at higher levels the freezing point depression of the haemolymph rises 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 classified according to the type of inland saline water they occupy. C. anthracinus and C. tentans would be classified 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 concentra-tion of the haemolymph. C. athalassicus, because i t is known to live in 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 in lower salinities and fresh water. Some species in this group, though C. athalassicus is 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 lutaria (Beadle and Shaw, 1959; Shaw, 1 9 5 5 ) , Aedes aegypti L. (Wigglesworth, 1938; Ramsay, 1 9 5 3 ) , Limnephilus stigma (Curtis) and Anabolia nervosa (Leach) (Sutcliffe, 1961b), and Cenocbrixa bifida (Hung.) and C. expleta (Uhler) (Scudder et a l . 1972). These species do not hyporegulate the haemolymph in 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 lutaria (Shaw, 1 9 5 5 ) , Aedes aegypti (Ramsay, 1 9 5 3 ) , and Limnephilus stigma (Sutcliffe, 1961b) which maintain the sodium concentration in the haemolymph above » f 8 . that of the external medium, even at high external concent-rations of sodium. The sodium response curves of C. anthracinus, C. athalassicus. and C. tentans can be compared to those of Cenocorixa expleta and C. bifida (Scudder et a l . 1972), and the caddis larva Limnephilus aff i n i s (Sutcliffe, 1961a), which maintain a constant haemolymph sodium level over a range of external sodium concentrations. These species are known to tolerate fresh and relatively 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 salinities and i t maintained haemolymph chloride hypertonic to the external chloride levels. The range of chloride concentrations studied in this investigation compared to the work carried out on other fresh water species, is small. Both S i a l i s lutaria (Shaw, 1955) and Aedes aegypti (Wigglesworth, 1938) regulate the haemolymph chloride level hypertonic at low external chloride concentrations, and hypotonic at high external chloride concentrations (for example Si a l i s above 120 mmole Cl / 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 Cl. Throughout that chloride range the haemolymph chloride concent-ration of C. tentans was 30 mM Cl. The haemolymph chloride concentration of C. anthracinus. C. athalassicus. and C. tentans varied from 10 to 29 mM Cl over an external chloride range h9. of 0 to 5 mM CI. The chloride concentration in 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 Si a l i s lutaria (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 bifida and C. expleta over a range of chloride concentrations similar to that used in this study. Both the species regulate the haemolymph chloride concentration within narrow limits and hypertonic to the external medium at low chloride concentrations. Both the Chironomus and the Cenocorixa species were collected from inland saline lakes in which chloride concentrations were low (the major anions are: carbonate, bicarbonate, and sulphate). The chloride concentrations range from 0 .07 mM CI to 36.96 mM CI, approximately the range of chloride investi-gated in this 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 salinities where only C. athalassicus survives, the potassium concentration of the haemolymph becomes more hypertonic as the external potassium concentra-tion rises. These Chironomus species regulate potassium in a way similar to Cenocorixa bifida and C. expleta (Scudder et al.1972). S i a l i s lutaria maintains the haemolymph potassium concentration at approximately 5 mmole K/l over an external 50. potassium range of 0 to 31* mmoie/1 (Shaw, 1955). Over the same range of potassium concentration, C. athalassicus has a haemolymph potassium concentration slightly hypertonic to the external concentration. The difference between the species is that S. lutaria regulates while C. athalassicus in effect maintains the potassium concentration of the haemolymph close to that of the external environment. Phillips (1970) regards the a b i l i t y to produce a urine that is hyperosmotic to the haemolymph as a prerequisite for the survival of hyporegulating species in waters of high salinity. No analyses of the urine of these Chironomus species were carried out, but in the light of the osmotic and ionic responses described above i t is doubtful that a hyperosmotic urine could be elaborated even by C. athalassicus. The osmotic and ionic balance in C. anthracinus. C. athalassicus. and £. tentans are similar except that C. athalassicus can control i t s interior milieu over a wider range of external concentrations. These responses are similar to those of fresh water insects in general (Wigglesworth, 1938; Shaw, 1955; Beadle, 1959; Sutcliffe, 1961b; Beadle, 19&9; and Scudder et a l . 1972). The question remains as to why C. athalassicus can survive and thrive in salinities twice as high as those endured by C. anthracinus and C. tentans. Sutcliffe (1961a) suggests that external concentrations greater than 30 to ^ O per cent seawater are rapidly fatal to fresh water insects. As the external salt concentration increases to a level roughly 51. equivalent to the normal total concentration of the haemolymph, the regulation of the haemolymph salt concentration begins to break:down. If the concentration of the haemolymph (Ag) of collected larvae is considered to be representative, then the external concentration at which the regulation of the haemo-lymph of £. athalassicus begins to break down is 50 per cent higher than that of C. anthracinus and C. tentans. Ramsay (195*+) has suggested that this break-down may be due to an inability to increase the amino acid content of the muscle cel l s , ultimately affecting body metabolism in general. It is 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 than C. anthracinus and C. tentans. While i t is not possible on the basis of this study to state precisely why C. athalassicus survives at higher salinities than C. anthracinus and C. tentans* this 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 in a wide variety of animals. Sodium regulation in particular is now understood in some detail, especially as i t relates to fresh water adaptation (Shaw, 19615 Sutcliffe and Shaw, 1967a; Greenaway, 1970; Maetz, 19715 Stobbart, 1971; Sutcliffe, 197*+ S Wright, 1975a, 1975b). The sodium regulation of some saline water species has 52. also been characterized and in some cases they have been compared to fresh water species (for example Sutcliffe, 1967, 1968, 1971)-The parameters of such a characterization are; the maximum flux rate (Vmax), the external concentration at half maximal flux (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 (1959). The distinction between fresh and mixohaline water was arbitrarily placed at 0 . 5 P?M salinity, or approximately 7 mmole/.l.sodium (Sutcliffe, 1967b). Comparing the sodium regulatory a b i l i t i e s of the three species studied in this thesis, C. tentans shows the greatest a f f i n i t y for sodium (Km of 1.05 mM Na), but has the least powerful uptake system (Vmax of O.96 mmole Na/Kg/hr). C. anthracinus has a lower af f i n i t y for sodium (Km of 2.15 mM Na) but i t s uptake system is more powerful (Vmax of 2*19 mM Na per Kg per hr). C. athalassicus has a Km value over five times as large (Km of 5.65 mM Na) as C. tentans. suggesting a much lower sodium aff i n i t y than have C. anthracinus or C. tentans. The Vmax of C. athalassicus O . l ^ mmole Na/Kg/hr) is over three times as great as that of C. tentans. C. athalassicus has, therefore, the least affinity for sodium of the three species, but has the most powerful uptake system. Wright (1975a, 1975b, 1975c) characterizes the sodium flux 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 for both C. dorsalis, 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 in 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 is best described by a straight-line equation, with a slight increase in efflux with increased external sodium concentration. Maetz (1971) found a similar relationship between sodium efflux and external sodium con-centration working with the fresh water flounder. Wright (1975b) found that sodium efflux displays typical saturation kinetics in C. dorsalis and £. tentans. The difference between the study by Wright and the present study may be the difference in the ranges of sodium utilized in each study. The low concentrations of the Wright study may have emphasized the •"first order" kinetics, while the relatively higher concent-rations of sodium in the present study may obscure such kinetics. 5V. The maximum sodium e f f l u x rates of C . anthracinus (0.*+9 mmole Na/Kg/hr), C . athalassicus (1 . 10 mmole Na/Kg/hr), and C . tentans (0.71 mmole Na/Kg/hr) were si m i l a r , but below those published by Wright (1975b) for C . dorsalis and C . tentans (1 . 6 and 1.5, r e s p e c t i v e l y ) . The lower rates of efflux of the saline water adapted insects are d i f f i c u l t to explain since saline water organisms would be expected to lose much more sodium than fresh water adapted organisms, although sodium loss may be handled adequately by the excretory organs. Speaking i n general terms S u t c l i f f e (1968) found that the sodium loss rates are similar i n fresh and brackish water crustaceans, while the loss rate of marine crustaceans was four to ten times higher. No balancing experiments were carried out involving C . anthracinus. C . athalassicus, and C . tentans. The i n f l u x and efflux rates of these species were equal at external sodium concentrations well above those c i t e d by Wright (1975a). C . dorsalis 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 10 micromoles (per l i t e r ) external sodium concentration, respectively. Influx and efflux rates are equal i n C . anthracinus at 0 .75 mM Na, C . athalassicus at 2.3V mM Na, and C . tentans at 3 .30 mM Na. Direct comparisons between the f l u x of fresh and saline water acclimatized chironomids have not been carried out previously. However, e a r l i e r studies have characterized the sodium regulatory a b i l i t i e s of other fresh and saline water invertebrates. S u t c l i f f e (1967a, 1967b, 1968, 1971, 197^) 55. compared populations of the amphipod Gammarus duebeni Lilljeborg 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 correlation between the half-saturation level (Km) of sodium influx and the external sodium concentration of the particular habitat of that pop-ulation. Populations from similar habitats shared the same Km values, but differed from populations in other habitats. Sutcliffe (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 fu l l y saturated at about 10 mmole/1 sodium. The marine species had values six to ten times higher for the respective parameters. From these findings Sutcliffe suggested that the evblutionary scheme has been the colonization of fresh waters by brackish water species, with the marine environment being the original habitat of the species. It also i s suggested that brackish species could probably inhabit most fresh waters if sodium levels were sufficient. The mechanism of evolution in that case appears to be one of phenotypic selection where the f i t individuals are those able to regulate sodium in accordance with the requirements of the habitat. For instance, those able to regulate sodium in 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 origin. Insects have probably evolved from fresh water habitats to also inhabit saline and brackish water habitats to gain ecological advantages such as in 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, 1975b). Sutcliffe (1968) cites a similar difference between fresh and brackish populations of amphipods but adds that a much greater difference exists between these and marine populations, the latter having a very low aff i n i t y for sodium. Shaw ( 1 9 6 D has shown that the Km value is progressively lower in species which have increasely strong powers of osmoregulation in fresh water. This suggests that the Km value is one way to compare species from different habitats since i t is indicative of the species' sodium af f i n i t y . Table 5 is a collation 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 in research techniques), there is a trend toward lower sodium af 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. SPECIES Aedes aegypti Asellus  communis A. aquaticus A. meridianus Chironomus  anthracinus C. dorsalis C. athalassicus C. tentans C. tentans Gammarus  duebeni G. lacustris STUDY Stobbart 1965 Sutcliffe 197^ Sutcliffe 197^ Sutcliffe 197>+ present study Wright 1975 present study Wright 1975 present study Sutcliffe 1967b Sutcliffe Shaw 1967b ACCLIMATION ORIGIN (mM/1 Na) 2 . 0 FW 0 . 0 7 5 - 2 . 0 0 . 1 - 2 . 0 FW FW 0 . 1 - 0 . 7 FW 1 5 . 0 sw 2 . 0 FW 1 5 . 0 SW 2 . 0 FW 1 5 . 0 SW 0 . 2 5 - 0 . 3 5 BW 0 . 1 0 FW TEMP. Km Vmax T^CT (mMNa)(mmoleNa/Kg/hr) 28 0 . 5 5 20 0 . 1 2 1.7 20 0 . 6 2 6 . 5 20 0.91 1 8 . 2 23 2 . 1 5 2 . 1 9 21 0 . 7 5 3 . 0 23 5 . 6 5 3 . 1 ^ 21 0 . 7 5 1 . 9 23 1 . 0 5 0 . 9 6 10 1 . 5 - 2 . 0 2 0 . 0 10 0 . 1 0 - 0 . 1 5 2 . 6 EFFLUX (mmoleNa/Kg/hr) 0 . 8 3 0 . 3 - 0 . 7 0 . 9 - 2 . 1 1 . 7 - 2 . 7 0 . 5 0 1 . 6 1.11 1 . 5 7 .77 3 . 8 1 A 58a. TABLE 5 continued The Sodium Flux Rates of Several Invertebrate Species. SPECIES STUDY ACCLIMATION (mM/1 Na) Gammarus Sutcliffe 0.10 pulex 1967a G. Sutcliffe 20% sea H?0 tierinus 1968 Limnaea Greenaway 0 .35 staenalis 1970 Marinogammarus Sutcliffe 0 . 5 f inmarchus 1968 Mesidotea Croghan 0 .7 entomon 1968 Sphaeroma Harris 10.0 rugicauda 1972 S. Harris 8 5 . 0 serrtum 1972 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 . 1 5 MW 10 6 . 0 2 0 . 0 20.0 - 1 0 . 0 FW 10 0 . 2 5 1 .5-2.0 0.08 BW 10 1.0 20.0 7.0 - 1 . 5 FW 5-10 2.6 15.0 1.6 BW 16-20 2.3 15.0 *+.1 BW 16-20 15.0 30.0 16.5 59. species from brackish and marine habitats. The findings of the present study of Chironomus species from saline habitats conform to that trend, with sodium af 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 is best adapted for 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 kinetics prevailed in the sodium and chloride uptake of the crayfish Astacus pallipes. The maximum chloride influx rates in the present study varied from 2.36 mmole Cl/Kg/hr (C. tentans) to 5.70 mmole Cl/Kg/hr (C. athalassicus). The chloride influx systems were half saturated at 2 .90 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 affinity for chloride, as well as the most powerful chloride uptake system. The chloride uptake abi l i t y of C. athalassicus may give i t an advantage in its natural habitat since chloride is low in a l l of the saline lakes inhabitated by these species (Table 1 ) . Chloride flux values are few in 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 study (chloride flux around 2 mmole Cl/Kg/hr). Kerstetter and Kirschner (1972), 60. and Payan et al.(1978) report on active chloride transport by the g i l l s of rainbow trout (Salmo gairdneri). The i n vivo chloride flux was lower ( 2 5 . 0 microEq/hr/100g) than the values c i t e d i n t h i s study. The chloride e f f l u x rates did not show saturation k i n e t i c s over the range of chloride concentrations studied. The maximum eff l u x rates of the three species were under 1 mM Cl/Kg/hr. If compared to the euryhaline brine shrimp Artemia salina (Croghan, 1958; Smith, 1969), which has a maximum eff l u x rate of 57 ml1! Cl/1. haemolymph/hr, the rates of the Chironomus species are much lower. According to the transport model of Maetz (1971) chloride and sodium flux are related i n d i r e c t l y . The chloride and sodium e f f l u x rates of Artemia sal i n a were compared by Thuet et al.(1968); the chloride e f f l u x rate was twice the sodium e f f l u x rate. No pattern i s obvious when the chloride and sodium e f f l u x rates of Chironomus are compared, they vary from similar to twelve times larger (Table page 36). The chloride i n f l u x rates can be compared to sodium i n f l u x rates that have been published. The maximum inf l u x rates of chloride are about twice those of sodium as outlined i n the present study (Table h). Compared to the maximum sodium in f l u x rates of several other fresh water species, the chloride rates of Chironomus are also about twice as large. Sodium and chloride flux were selected for study because each can be compared to similar f l u x studies on other species. This i s especially true i n the case of sodium f l u x , but chloride 61 . flux is 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 characteristics have been related to environmental characteristics by researchers such as Shaw (1961) , and Sutcliffe (197^) . Sodium and chloride are of course not the only important ions in the saline lake habitat of C. anthracinus. C. athalassicus, and C. tentans. If a complete picture of the adaptation to saline lakes is to be obtained, the importance of other ions as well as other factors such as total dissolved solids, organic and inorganic carbon, and the ecological effects of salinity must be evaluated. 6 2 . V. CONCLUSIONS This thesis has investigated the importance of salinity as a factor in the distribution of C. anthracinus. C. athalassicus. and C. tentans. It has been shown that there is a difference in the salinity tolerance of the species. C. anthracinus and C. tentans larvae can survive in cond-uctivities only as high as 9000 micromhos/cm (at 25°C) while C. athalassicus can survive to at least 1 5 , 0 0 0 micromhos/cm (at 25°C). A l l three species are, however, affected by the temperature of the water which may affect survival 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 is a correlation between the ab i l i t y to regulate and the survival of C. athalassicus; the regulatory a b i l i t i e s appear to break down at salinities above 15,000 micro-mhos/cm (at 25°C). Evidence of break-down in the regulation of C. anthracinus 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 characteristics themselves 63 are new information to science since no saline adapted chironomids have been examined to date. Throughout this thesis a trend of information concerning C. athalassicus has become apparent. C. athalassicus is clearly able to tolerate and thrive at high s a l i n i t i e s . It can regulate the haemolymph at salinities equal to those occupied in the f i e l d . The sodium characteristics suggest that i t is similar to species adapted to highly saline or brackish waters. It has a low sodium af f i n i t y , but a powerful uptake system. The conclusion to be drawn from this study lis rthat 1 salinity does indeed affect the distribution of C. anthracinus, C. athalassicus. and C. tentans in the saline lakes of the Cariboo-Chilcotin region of central Br i t i s h Columbia. This study does not, however, exhaust the possible ways in which salinity is important (see Kinne, 196V), and i t cannot be assumed that because salinity affects distribution that i t is the only factor of importance to the distribution of C. anthracinus. C. athalassicus. and C. tentans. 6h. LITERATURE CITED Bassett, M.C. 1967. A cytotaxonomic study of the most common larval chironomidae in a series of saline waters in the southern interior of British Columbia. M.Sc. Diss. U.B.C. Vancouver, B.C. Beadle, L. 1939. Regulation of the haemolymph in the saline water mosquito larva Aedes detritus Edw. J. Exp. Bi o l . 16: 3 ^ 6 - 3 6 2 . Beadle, L. 19*+3. Osmotic regulation and the faunas of inland waters. Bi o l . Rev. 18: 172-183. Beadle, L. 1959. Osmotic regulation in relation to the classification of brackish and inland saline waters. Archivo Di Oceanografia E Limnologia Vol. XI: 1V3-151. Beadle, L. 1969- Osmotic 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 salt and the regulation of the non-protein nitrogen fraction in the blood of the aquatic larva, S i a l i s lutaria. J. Exp. Biol. 27 : 9 6 - 1 0 9 . Cannings, R.A. 1973. An ecological study of some of the Chironomidae inhabiting a series of saline lakes in central British 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 in British 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 in central B r i t i s h Columbia. Can. J. 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Studies on the exchange and regulation of sodium in the larva of Aedes aegypti (L.). I. The steady state exchange. J. Exp. Biol. 36: 64-1-653. Stobbart, R.H. i960. Studies on the exchange and regulation of sodium in the larva of Aedes aegypti (L.). II. The net transport and the fluxes associated with i t . J. Exp. Biol. 37: 594--608. 67. Stobbart, R.H. 1965. The effect of some anions and cations upon the fluxes and net uptake of sodium in the larva of Aedes aegypti (L.). J. Exp. Biol. 4-2: 29-4-3. Stobbart, R.H. 1967. The effectof some anions and cations upon the fluxes and net uptake of chloride in the larva of Aedes aegypti (L.), and the nature of the uptake mechanisms for Nat and Cl " . J. Exp. Biol, 4-7: 35-57. Stobbart, R.H. 1971. ^actors affecting the control of body volume in the larvae of the mosquitoes Aedes aegypti (L.) and Aedes detritus Edw. J. Exp. Biol. 54-: 67-82. S u t c l i f f e , D.W. 19618- Studies on salt and water balance in caddis larvae (Trichoptera): I. Osmotic and ionic reg-ulation of body fluids in Limnephilus af f i n i s Curtis. J. Exp. Biol. 3 8 : 501-519. Sutcliffe, D.W. 1961b- Studies on salt and water balance in caddis larvae (Trichoptera): II. Osmotic and ionic regula-tion of body fluids in Limnephilus stigma Curtis and Anabolia nervosa Leach. J. Exp. Biol. 38: 521-530. Sutcliffe, D.W. 1967a. Sodium regulation in the fresh water amphipod, Gammarus pulex (L.). J. Exp. Biol. 4-6:- 4-99-518. Sutcliffe, D.W. 1967b. Sodium regulation in the amphipod Gammarus duebeni from brackish-water and fresh-water loc a l i t i e s in Britain. J. Exp. Bio l . 4-6: 529-550. Sutcliffe, D.W. 1968. Sodium regulation and adaptation to fresh water in Gammarid crustaceans. J. Exp. Biol. 4-8: 359-380. Sutcliffe, D.W. 1971. Sodium influx and loss in freshwater and brackish-water populations of the amphipod Gammarus  duebeni Lilljeborg. J. £xp. Biol. 54-: 2 5 5-26 8. Sutcliffe, D.W. 1974-. Sodium regulation and adaptation to fresh water in the isopod genus Asellus. J. Exp. Biol. 6 1 : 719-736. Sutcliffe, D.W., & J. Shaw. 1967. The sodium balance mechanism in the fresh water amphipod, Gammarus lacustris Sars. J. Exp. Biol. 4-6: 519-528. Thuet, P., R. Motais, & J. Maetz. 1968. Les mecanismes de 1'euryhalinite chez le c r u s t a c 6 de salines, Artemia  salina L. Comp. Biochem. Physiol. 26: 793-818. 68. Topping, M.S. 1969. Giant chromosomes, ecology, and adapta-tion in Chironomus tentans. Ph.D. Diss. U.B.C., Vancouver, B.C. Topping, M.S. 1971. Ecology of the larvae of C. tentans in saline lakes in central British Columbia. Can. Entom. 103* 328-338. Topping, M.S. 1972. Distribution of C. tentans ie some lakes in central British Columbia in relation to some physical and chemical factors. Proc. XIII Int. Congr. Ent. 3* 4-77-4-73. Topping, M.S., & G.G.E. Scudder. 1977. some physical and chemical features of saline lakes in central B r i t i s h Columbia. Syesis 10: 14-5-166. 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 in the haemolymph of mosquito larvae. J. Exp. Biol. 15: 235-24-7. Wright, D.A. 1975a. Sodium regulation in the larvae of Chironomus dorsalis (Meig.) and Camptochironomus tentans, (Fabr.): the effect of salt depletion and some observations on temperature changes. J. Exp. Biol. 6 2 : 121-139. Wright, D.A. 1975b. The effect of external sodium concentra-tion upon sodium fluxes in Chironomus dorsalis (Meig.) and Camptochironomus tentans (Fabr.), and the effect of other ions on sodium influx in C. tentans. J. Exp. B i o l . 6 2 : 1 M - 1 5 5 . Wright, D.A. 1975c The relationship between transepithelial sodium movement and potential difference in the larva of Camptochironomus tentans (Fabr.) and some observations on the accumulation of other ions. J. Exp. Biol. 6 2 : 157-174-. 

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