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Specific and seasonal variation in survival and sodium balance at low pH in five species of waterboatmen… Needham, Karen Merrie 1990

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SPECIFIC AND SEASONAL VARIATION IN SURVIVAL AND SODIUM BALANCE AT LOW pH IN FIVE SPECIES OF WATERBOATMEN (HEMIPTERA: CORIX1DAE) By KAREN MERRIE NEEDHAM B.Sc, The University of British Columbia, 1986 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 1990 © Karen Merrie Needham, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada TH DE-6 (2/88) ABSTRACT Sodium balance and mortality rates were examined ' i n five species of adult waterboatmen (Hemiptera: Corixidae) exposed to neutral and low pH waters. The f i v e species were chosen to r e f l e c t a wide range of pH conditions i n waters where they naturally occur. Cenocorixa bifida and C. expleta normally inhabit high pH waters, whereas C. blaisdelli and Hesperocorixa atopodonta can be found most often at neutral pH. Sigara omani occur i n a c i d i c waters. Haemolymph [Na], whole-body [Na], and sodium i n f l u x rates were recorded during exposures of 6-9 days at pH 7.0, 4.5, and 3.0. C. blaisdelli and H. atopodonta were studied throughout the year (spring, summer, and f a l l ) . C. bifida and C. expleta were examined i n the f a l l , while S. omani were tested i n the spring. Overall, these corixids appeared to be tolerant of short-term exposure to low pH. Mortality for a l l species remained below 50% in both neutral and a c i d i c pH le v e l s throughout the year. Add i t i o n a l l y , differences i n haemolymph [Na], whole-body [Na], and sodium i n f l u x rates from pH 7.0 to either pH 4.5 or 3.0 were ra r e l y s i g n i f i c a n t . However, both i n t e r - and i n t r a s p e c i f i c v a r i a t i o n i n sodium balance over the range of pH level s tested were apparent. Most notably, C. blaisdelli and H. atopodonta exhibited t h e i r highest concentrations of haemolymph and whole-body Na i n the summer, under both natural conditions and i n the laboratory at a l l pH l e v e l s . For C. blaisdelli, summer was also the time of highest mortality, with mortality increasing as pH was lowered. The observed c o r r e l a t i o n between high haemolymph/whole-body [Na] and high mortality i n the summer appears to r e s u l t from a r e l a t i v e l y large decrease i n haemolymph and whole-body [Na] when bugs were exposed to pH 3.0, at a time when these values were i n i t i a l l y high. F a l l was the season of lowest haemolymph/whole-body [Na], and also of lowest mortality, for both C. blaisdelli and H. atopodonta. I n t e r s p e c i f i c v a r i a t i o n i n the a b i l i t y to tole r a t e low pH did not appear to r e f l e c t v a r i a t i o n i n the pH of water these bugs normally inhabit. Of the f i v e species tested, H. atopodonta appeared best able to maintain i n t e r n a l homeostasis under a c i d i c conditions, despite being common to neutral waters. Haemolymph [Na], whole-body [Na], and sodium i n f l u x rates d i d not change s i g n i f i c a n t l y from pH 7.0 to pH 3.0 i n any season. Furthermore, C. blaisdelli, which was c o l l e c t e d from the same pond as H. atopodonta, seemed to be least able to regulate i n t e r n a l milieu i n low pH waters. In C. blaisdelli, exposure to pH 3.0 usually resulted i n decreased haemolymph and whole-body [Na], r e l a t i v e to values recorded i n those individuals exposed to pH 7.0. The difference i n the size of these two species (H. atopodonta i s approximately twice that of C. blaisdelli) may account for the observed variations i n t h e i r respective sodium balance at low pH. i v T A B L E OF CONTENTS Page Abstract i i Table of Contents i v L i s t of Tables v L i s t of Figures v i L i s t of Appendices v i i Acknowledgements v i i i I n troduction 1 M a t e r i a l s and Methods 6 Results 15 Discussion 48 Summary 62 References 64 Appendices 70 V LIST OF TABLES Page Table 1 Major ion composition and pH of waters from the c o l l e c t i o n s i t e s i n B.C 9 Table 2 Haemolymph [Na], whole-body [Na], and sodium i n f l u x rates of f i v e species of c o r i x i d s i n t h e i r n a t u r a l waters 16 Table 3 Q u a l i t a t i v e summary of changes i n haemolymph [Na], whole-body [Na], and sodium i n f l u x rates of f i v e species of c o r i x i d s upon exposure to low pH 53 v i LIST OF FIGURES Page F i g u r e 1 Map of v a r i o u s c o l l e c t i o n s i t e s i n B.C 7 F i g u r e 2 R e l a t i o n s h i p between haemolymph [Na] and sodium c o n c e n t r a t i o n or pH of the n a t u r a l waters f o r f i v e s p e c i e s of c o r i x i d s 17 F i g u r e 3 R e l a t i o n s h i p between whole-body [Na] and sodium c o n c e n t r a t i o n or pH of the n a t u r a l waters f o r f i v e s p e c i e s of c o r i x i d s 19 F i g u r e 4 R e l a t i o n s h i p between sodium i n f l u x r a t e s and sodium c o n c e n t r a t i o n or pH of the n a t u r a l waters f o r f o u r s p e c i e s of c o r i x i d s 21 F i g u r e 5 Seasonal v a r i a t i o n i n cumulative percent m o r t a l i t y f o r C . b l a i s d e l l i and H. atopodonta 25 F i g u r e 6 Cumulative percent m o r t a l i t y d u r i n g the haemolymph/whole-body experiments f o r f i v e s p e c i e s of c o r i x i d s 28 F i g u r e 7 Cumulative percent m o r t a l i t y d u r i n g the i n f l u x experiments f o r f i v e s p e c i e s of c o r i x i d s 30 F i g u r e 8 Seasonal v a r i a t i o n i n haemolymph [Na], whole-body [Na], and sodium i n f l u x r a t e s f o r C . b l a i s d e l l i 33 F i g u r e 9 Seasonal v a r i a t i o n i n haemolymph [Na], whole-body [Na], and sodium i n f l u x r a t e s f o r H.atopodonta 36 F i g u r e 10 Haemolymph [Na] of f i v e s p e c i e s of c o r i x i d s a t t h r e e pH l e v e l s i n t h r e e seasons 39 F i g u r e 11 Whole-body [Na] of f i v e s p e c i e s of c o r i x i d s a t t h r e e pH l e v e l s i n t h r e e seasons 41 F i g u r e 12 Sodium i n f l u x r a t e s of f o u r s p e c i e s of c o r i x i d s a t two pH l e v e l s i n two seasons.... 44 v i i LIST OF APPENDICES Page Appendix A Raw data from the haemolymph [Na] experiments 70 Appendix B Raw data from the whole-body [Na] experiments 7 6 Appendix C Raw data from the sodium i n f l u x experiments 81 v i i i ACKNOWLEDGEMENTS I w o u l d l i k e t o t h a n k my s u p e r v i s o r , D r . G . G . E . S c u d d e r , f o r h i s w e a l t h o f k n o w l e d g e a n d h i s i n s p i r a t i o n . I w o u l d a l s o l i k e t o t h a n k my c o m m i t t e e m e m b e r s , D r . M . I s m a n a n d D r . T . N o r t h c o t e , f o r t h e i r v a l u a b l e c o m m e n t s , w h i c h g r e a t l y i m p r o v e d t h i s m a n u s c r i p t . I n a d d i t i o n , a v e r y s p e c i a l t h a n k -y o u g o e s o u t t o my o l ' p a l L o c k e f o r h i s u n r e l e n t i n g i n t e r e s t i n my p r o j e c t , h i s e x p e r t k n o w l e d g e o f t h e s u b j e c t a r e a , a n d h i s i n s i s t e n c e u p o n q u a l i t y , c o n c i s e n e s s , a n d a c c u r a c y . A r t i s t i c a s s i s t a n c e a n d , m o r e i m p o r t a n t l y , c a m a r a d e r i e w e r e g r a c i o u s l y p r o v i d e d b y E d i e , S h o n a , a n d L o i s . R i c h c o n t r i b u t e d t e c h n i c a l a s s i s t a n c e a n d h u m o u r t h r o u g h o u t t h e e x p e r i m e n t a l s t a g e o f t h i s p r o j e c t . A n d f i n a l l y , u n e n d i n g g r a t i t u d e t o t h e " f o u r t h - f l o o r g a n g " , f o r t u r n i n g U . B . C . f r o m a m e r e i n s t i t u t i o n i n t o a s e c o n d home a n d a p l e a s a n t p l a c e i n w h i c h t o b e . 1 INTRODUCTION Anthropogenic a c i d i f i c a t i o n of natura l freshwaters i s a serious problem, in f luenc ing both the d i s t r i b u t i o n and abundance of many aquatic l i f e forms (Haines, 1981; D i l l o n et al., 1984; Baker et al., 1990). A c i d i f i c a t i o n of surface waters can affect aquatic b io ta e i ther d i r e c t l y , as a re su l t of H + t o x i c i t y (Packer and Dunson, 1970), or i n d i r e c t l y , v i a recruitment f a i l u r e (Fraser and Harvey, 1984) or a l t e r a t i o n of a food chain (Eriksson et al., 1980) . Within taxa, both i n t e r - and i n t r a s p e c i f i c di f ferences i n a c i d to lerance can be seen (Pierce, 1985). I n t e r s p e c i f i c d i f ferences may be re la ted to H + permeabi l i ty of g i l l s and other exchange surfaces (Potts and Fryer , 1979) or eco log i ca l s trategies (Servos et al., 1985), while i n t r a s p e c i f i c d i f ferences can often be corre la ted with habitat h i s t o r y ; more to l erant populations are usua l ly found l i v i n g i n the more a c i d i c natura l waters (Pierce, 1985). While reports of species losses from a c i d i f i e d natura l waters are quite common i n the l i t e r a t u r e , few studies have attempted to corre la te p h y s i o l o g i c a l constra ints at low pH with observed species d i s t r i b u t i o n s (for exceptions, see Havas and Hutchinson, 1983; Rowe et al., 1989). Within any one taxon of animals, studies must f i r s t be conducted on species that are adapted for s u r v i v a l i n these waters. Then, other species of the same group that are not current ly found i n t h i s type of habitat can be subjected to acc l imat ion and experimentation i n low pH water, and 2 these two sets of results may be compared (Scudder, 1987). Acid freshwaters present some i n t e r e s t i n g osmoregulatory and ionoregulatory problems. In such d i l u t e conditions, water may enter the body passively through the integument or through the gut v i a ingestion of the medium. Excess water must be excreted, while at the same time necessary ions must be retained. In addition, i f animals are to inhabit these environs they must be able to deal with the presence of a high concentration of H+ i n the external medium. H+ can compete with Na+ for exchange s i t e s on the external surfaces of aquatic animals, or these ions may a l t e r the conformation of sodium uptake s i t e s so that they no longer function properly (Havas, 1980; Wood, 1989; Vangenechten et a l . , 1989). Whatever the mechanism, the re s u l t i s usually the same - sodium uptake i s p a r t i a l l y i n h i b i t e d or arrested completely i n many animals exposed to low pH (Havas and Hutchinson, 1983; McDonald, 1983a; Vangenechten et al., 1989). Disruption of ion regulation leads to decreases i n both haemolymph and whole-body [Na], and f i n a l l y to death i f exposure i s prolonged (McDonald, 1983b; Vangenechten et al., 1989). Despite these problems, some aquatic animals are able to survive and breed i n acid waters. Examples of acid tolerance are taxonomically diverse, including species of f i s h (Dunson et al., 1977; McWilliams, 1982), amphibians (Pierce, 1985), crustaceans ( B e r r i l l et al., 1985), and insects (Henrikson and Oscarson, 1981, 1985). Since the ionoregulatory processes of these groups are very si m i l a r (Motais and Garcia-Romeu, 1972; Komnick, 1977), there may be common mechanisms by which these animals adapt to 3 l i f e at low pH. Therefore, explorat ions of these mechanisms within any one taxa might provide information appl icable to studies of other taxa. By understanding the extent of these s i m i l a r i t i e s , the reasons for the di f ferences between taxa i n the a b i l i t y to survive at low pH might become more apparent. Insects appear to be more to l erant of low pH than e i ther crustaceans, amphibians, or f i s h (Bel l and Nebeker, 1969; Lech le i tner et al., 1985; Rowe et al., 1989). While some insect species are l o s t from a c i d i f i e d waters, s e n s i t i v i t y var ies between and wi th in orders (Okland and Okland, 1986). Diptera (Mierle et al., 1986; Havas, 1980), Tr ichoptera (Be l l , 1971), and Hemiptera (Vangenechten and Vanderborght, 1980) appear to be the most a c i d - t o l e r a n t . Within Hemiptera, some species of the family Corixidae have, i n p a r t i c u l a r , been extremely successful i n t h e i r co lon iza t ion of ac id freshwaters (Henrikson and Oscarson, 1981, 1985; Scudder, 1987). Often, they become the top predators i n a c i d i f i e d , f i s h l e s s lakes (Henrikson and Oscarson, 1981). However, because some c o r i x i d species are most common i n ac id waters while others p r i m a r i l y inhabi t a l k a l i n e environments, we might expect the ac id tolerances of d i f f erent species of c o r i x i d s to r e f l e c t these d i s junct d i s t r i b u t i o n s . Vangenechten et al. (1979b) and Scudder (1987) report species di f ferences i n c o r i x i d ion regulat ion a b i l i t i e s at low pH, which may have been r e l a t e d to di f ferences i n habitat c h a r a c t e r i s t i c s . Thus, one can hypothesize that d i f f erent species of c o r i x i d s w i l l show dif ferences i n s u r v i v a l and sodium balance at low pH, depending on t h e i r natura l hab i ta t . S p e c i f i c a l l y , i t i s predic ted 4 that species taken from high pH waters should f i n d i t d i f f i c u l t to survive under low pH condit ions for any length of time, while species which occur n a t u r a l l y i n a c i d i c environments should be bet ter able to regulate t h e i r i n t e r n a l mi l i eu under a range of a c i d i c condit ions i n the laboratory . Species which are found most often i n neutra l waters w i l l be expected to have a regulat ion a b i l i t y that i s intermediate i n i t s e f fect iveness . To tes t t h i s hypothesis, f ive species from three d i f ferent waters varying i n pH were studied i n the laboratory at neutral and low pH. Cenocorixa bifida and C. expleta were c o l l e c t e d from waters with a pH of 9.0-10.0, while Sigara omani was taken from n a t u r a l l y a c i d i c waters of pH 4.5. C. blaisdelli and Hesperocorixa atopodonta came from neutral waters (pH 7.0) . P h y s i o l o g i c a l parameters measured are l i s t e d below. Seasonal v a r i a t i o n i n the a b i l i t y of animals to survive and regulate ions have been demonstrated i n several studies (Vangenechten et al., 1979b; Stuart and Morr i s , 1985; Rowe et al., 1988a; F r i s b i e and Dunson, 1988c). Cooper et al. (1988) reported seasonal d i f ferences i n Malpighian tubule secret ion rates i n C. blaisdelli. The lowest rates occurred i n tubules treated with head extract derived from cor ix ids c o l l e c t e d i n the spr ing . Therefore, the second hypothesis of t h i s study i s that c o r i x i d s w i l l show a seasonal v a r i a t i o n i n t h e i r a b i l i t y to maintain i n t e r n a l homeostasis under a c i d i c condi t ions . The c o r i x i d species studied to date overwinter as adul ts , not feeding or feeding very l i t t l e during t h i s time (Jansson and Scudder, 1974). Consequently, sodium leve l s are considerably depleted by 5 the spring (P. Cooper, pers. comm.). So, i t i s probable that sodium balance i n acid waters could be more d i f f i c u l t i n the spring than at other times of the year. In contrast, summer i s a time of high a c t i v i t y , including feeding (pers. obs.). Presumably, then, i n t e r n a l sodium concentrations are high and exposure to low pH water should be tolerated more e a s i l y . In the f a l l , many in t e r n a l processes are slowed down i n preparation for overwintering (G. Scudder, pers. comm.). This results i n the modification of metabolic a c t i v i t i e s such that corixids may not be readi l y able to deal with unexpected changes i n t h e i r external environment. F a l l , then, may again be a season of poor sodium balance c a p a b i l i t i e s i n low pH water. To te s t these hypotheses, four parameters were measured i n the fi v e species examined. Mortality, haemolymph [Na], whole-body [Na], and sodium i n f l u x rates were quantified i n the spring for S. omani, i n the f a l l for C. bifida and C. expleta, and i n the spring, summer, and f a l l for both C. blaisdelli and H. atopodonta. A l l species were not available for te s t i n g i n a l l seasons. Exposures were 6-9 days i n duration, and pH level s used i n a l l three seasons were 7.0, 4.5, and 3.0. As i n previous studies (Vangenechten et a l . , 197 9ab; Lechleitner et al., 1985; Rowe et a l . , 1988a, 1989), changes i n haemolymph [Na], whole-body [Na], or sodium i n f l u x upon exposure to low pH were used as indicators of ion regulation disruption. 6 MATERIALS AND METHODS Adult corixids were obtained from various l o c a l i t i e s around B r i t i s h Columbia (Figure 1). Cenocorixa blaisdelli (Hungerford) and Hesperocorixa atopodonta (Hungerford) were collected from Jericho Pond, Vancouver (49°16'N, 123°12'W; Figure 1A) . Cenocorixa bifida hungerford! (Lansbury) were taken from Lake Lye on Becher's P r a i r i e , C h i l c o l t i n Plateau (52°00'N, 122°30'W; Figure IB), while Cenocorixa expleta (Uhler) were obtained from the lake LB2, Kamloops (50°45'N, 120°25'W; Figure IC) (Topping & Scudder 1977). Sigara omani (Hungerford) were c o l l e c t e d from naturally a c i d i c bog pools located along the eastern coast of Graham Island, Queen Charlotte Islands. Most of the Queen Charlotte Island specimens came from a shallow, extensive pool 2 km south of Port Clements (53°41'N, 132°11'W; Figure ID) . The major ion composition and pH of the waters from the c o l l e c t i o n s i t e s are l i s t e d i n Table 1. Insects were transported to the laboratory i n 4 l i t r e vacuum flasks containing 2-3 l i t r e s of lake water and some weeds from the c o l l e c t i o n s i t e (Juncus spp.), to which the insects could c l i n g . Measurements of pH were made i n the f i e l d and again i n the laboratory, and did not change more than one-tenth of a unit during transport. In the laboratory, insects were transferred to shallow plexiglass containers (30 x 24 x 10 cm) containing water of o r i g i n and some weeds from the c o l l e c t i o n s i t e (Juncus spp.). Corixids were maintained at 5° C i n the dark and without food. 7 F i g u r e 1 Map of various c o l l e c t i o n s i t e s i n B.C. A) Jericho Pond, Vancouver, B) Lake Lye, Becher's P r a i r i e , C h i l c o t i n Plateau, C) lake LB2, Kamloops and D) Port Clements, Queen Charlotte Islands. Insect: Cenocorixa expleta. 9 Table 1 Major ion composition and pH of waters from the collection sites in B.C. Water Location Species Na+ K+ cr (mM) (mM) (mM) Total CO2 (mM) PH Jericho Pond Vancouver Cenocorixa blaisdelli Hesperocorixa atopodonta 6 1 7 3 7.0 Lake Lye Riske Creek (Becher's Prairie) Cenocorixa bifida 50 4 15 29 9.0 LB2 Kamloops Cenocorixa expleta 230 11 18 105 9.5 Bog Pools Port Clements (Q.C.I.) Sigara omani 6 2 4 — 4.5 10 Indiv iduals c o l l e c t e d i n the f a l l have an adequate reserve of fat body and therefore need not be fed (Scudder et al., 1972). Withholding food i n such insects assures that a l l animals are i n a s i m i l a r p h y s i o l o g i c a l state before experimentation and are not rece iv ing any sodium v i a t h e i r food. At 5° C, which mimics the overwintering condit ion of adults i n the f i e l d , adult cor ix ids can l i v e for long periods of time (2-3 months) i n an apparently healthy state (pers. o b s . ) . However, experimental insects were always used within 1-2 weeks of capture, to minimize the p o s s i b i l i t y of p h y s i o l o g i c a l s tress due to c a p t i v i t y . Experiments were designed to measure haemolymph and whole-body sodium concentrations, and sodium in f lux for each species of c o r i x i d . These measurements were- repeated several times a year (spring, summer and f a l l ) , to examine seasonal v a r i a t i o n . Prel iminary experiments showed no s i g n i f i c a n t di f ferences between males and females; therefore sexes were pooled. A l l experiments were conducted at 5° C. De ta i l s of these experiments are given below. Haemolymph and Whole-body Sodium Measurements Af ter a 3-day acc l imat ion per iod at 5° C (Scudder et al., 1972), insects were t rans ferred to 1 L beakers containing a 5 x 7 cm piece of p l a s t i c screen for the insects to c l i n g to and 800 ml of f i l t e r e d Jer icho Pond water (0.45 yum M i l l i p o r e HA f i l t e r s ) . Treatments were pH l eve l s 7.0, 4.5, and 3.0. In most experiments two rep l i ca te s of each pH were used. pH was es tabl i shed using 0.5 M H2S04. Haemolymph and whole-body sodium values were determined 11 on the day of transfer (Day 0), and on every second day after t h i s , up to and including Day 8. In every experiment, pH was adjusted d a i l y and mortality noted. Following the methodology of Scudder et al. (1972), haemolymph samples were co l l e c t e d by removing the right forewing of each c o r i x i d with a pa i r of forceps after the insect had been blotted dry on f i l t e r paper. Upon removal of the forewing, haemolymph would well up on the pronotum of the insect, and could then be col l e c t e d by c a p i l l a r y action i n a 1 /al disposable glass micropipette (Microcap). Usually i t was not possible to c o l l e c t more than 1 /al of haemolymph per insect. However, samples were never pooled. Haemolymph was d i l u t e d i n 1 ml of d i s t i l l e d (deionized) water and analyzed for sodium content on a Techtron Atomic Absorption Spectrophotometer (Model 120). To determine whole-body sodium values of corixids, insects that had not been used for haemolymph sodium determinations were removed from each te s t solution and blotted dry on f i l t e r paper. After recording a wet weight of each insect (Mettler Gram-atic balance; + 0.1 mg accuracy), they were placed i n a drying oven and dried to a constant weight (80° C; 24 hours) . The dry weights were recorded, and the corixids were transferred to a muffle furnace (540° C) and ashed. Ash was dissolved f i r s t with 1 ml of concentrated HN03, and then made up to 25 ml with d i l u t e (0.2 M) HN03. This assured that the samples were within a molar range suitable for analysis. Sodium content was determined on the atomic absorption spectrophotometer. 12 Sodium Influx Insects held i n t h e i r natural pond water i n vacuum flasks were ' acclimated to 5° C for 3 days. They were then transferred to a 1 L beaker containing approximately 800 ml of saline solution (ions (mM) : Na+ 6, K+ 1, CI- 8, t o t a l C02 3) set at pH 7.0 with 0.5 M H2S04. This . solution was intended to mimic the chemical composition of Jericho Pond water (Table 1), while at the same time eliminating the number of possible unknown substances i n the l a t t e r . Adjustments to pH and mortality counts were made da i l y throughout the acclimation periods and during the experiments. After a further 3-day acclimation at 5° C to t h i s a r t i f i c i a l s aline solution, insects were placed i n t h e i r respective test solutions: 800 ml of pH 7.0, pH 4.5 or pH 3.0 saline (chemical composition as above), with a 5 x 7 piece of p l a s t i c screening i n each beaker. On the day of transfer (Day 0), and on each subsequent day up to and including Day 6, insects were removed from the beakers and placed i n 5 ml of r a d i o l a b e l l e d (22Na -purchased from Dupont as 2 2NaCl i n water) saline (0.68 /iCi/ml) i d e n t i c a l i n chemical composition and pH to the saline from which they had been removed. After 3 hours at 5° C, corixids were removed from the radioactive solution, rinsed f i v e times with cold (5° C) saline, weighed, and assayed for r a d i o a c t i v i t y i n a Packard Minaxi (Auto-Gamma RIA 5000 Series) gamma counter. Again, pH adjustments and mortality counts were made d a i l y for each experiment. By knowing the s p e c i f i c a c t i v i t y of the external medium and the amount of r a d i o a c t i v i t y i n the whole-body of each insect, 13 i n f l u x could be calculated using the equation: Mx = l / s 0 x dQ/dt where Mx i s the i n f l u x of Na (nmoles/mg wet weight»hr); S0 i s the s p e c i f i c a c t i v i t y of the external solution (counts per minute^umole Na); Q i s the amount of r a d i o a c t i v i t y i n each c o r i x i d (counts per minute/mg wet weight); and t i s the time (hr) (Havas & Likens, 1985). Preliminary experiments determined that the departure from l i n e a r i t y (time vs. influx) i n the f i r s t three hours of uptake was minimal. O r i g i n a l l y , e f f l u x measurements were attempted on insects that had been used i n the i n f l u x experiments described above. After i n f l u x values had been determined, insects were transferred to 5 ml of cold (unlabelled) saline and held at 5° C for a week. Then, hourly measurements of the amount of r a d i o a c t i v i t y i n the medium were recorded. However, mortality was extremely high throughout the e f f l u x experiments and results could therefore not be considered v a l i d . The e f f l u x experiments were subsequently discontinued. S t a t i s t i c a l Analyses Preliminary s t a t i s t i c a l tests showed few s i g n i f i c a n t differences between data c o l l e c t e d on the various days. Therefore, data from a l l days were pooled for subsequent s t a t i s t i c a l analyses. When three or more categories of any one variable (pH, species, or season) were compared, a one-way analysis of variance was performed. This was followed by a Tukey's Test to determine between which pH le v e l s , species, or 14 seasons s i g n i f i c a n t di f ferences i n haemolymph and whole-body sodium concentrations and sodium inf luxes had occurred (Zar, 1984). When only two categories of any one v a r i a b l e were compared (usually due to high morta l i ty i n pH 3.0) , an independent Student T-test was used. In a l l cases, means were considered s i g n i f i c a n t l y d i f f erent i f p<.05. Symbols on a l l graphs represent mean values, while bars on these symbols ind icate + one standard error of the mean. Characters beside these symbols s i g n i f y one of the fo l lowing: * s i g n i f i c a n t l y d i f f erent from pH 7.0 + s i g n i f i c a n t l y d i f f eren t from C. blaisdelli # s i g n i f i c a n t l y d i f f erent from s p r i n g . Sample s izes v a r i e d from 2 to 10 insects per mean value; usua l ly e i ther 5 or 10 insects were used. Exact sample s izes can be found i n the f igure legends. Means, standard dev ia t ions , standard e r r o r s , and numbers sampled for each experiment can also be found i n Appendices A (haemolymph), B (whole-body), and C (influx) . 15 RESULTS Sodium Balance: Natural Waters A comparison of c o r i x i d species taken from the same natural waters and of species from d i f f e r e n t water bodies reveals v a r i a t i o n i n haemolymph [Na], whole-body [Na], and sodium i n f l u x rates both between species and between seasons. In general, haemolymph [Na], whole-body [Na], and sodium i n f l u x increased as sodium concentration i n the natural waters increased (Figures 2-4a) . C. expleta, which came from the water with the highest sodium concentration (230 mM; Table 1), exhibited the highest haemolymph [Na] (188.4 mM) and the highest whole-body [Na] (0.42/umoles/mg dry weight) of any species tested (Table 2). These values were 27% (p<.005) and 320% (p<.05) higher, respectively, than values found for C. blaisdelli within the same season ( f a l l ) . Figures 2-4 (a) also show that values recorded at the lowest water sodium concentration (6 mM) cover an extensive range, and display marked seasonal v a r i a t i o n . For example, C. blaisdelli exhibited t h e i r highest haemolymph and whole-body [Na] i n the summer. On the other hand, H. atopodonta, which came from the same pond as C. blaisdelli, reached t h e i r peak i n the f a l l . fi. atopodonta also had lower values of haemolymph [Na], whole-body [Na], and sodium i n f l u x than C. blaisdelli o v e r a l l . These were s i g n i f i c a n t l y lower i n the spring (WB[Na]: 60% lower, p<.05; INFLUX: 48% lower, p<.005) and i n the summer (WB[Na]: 53% lower, Table 2 Haemolymph [Na], whole-body [Na], and sodium influx rates of five species of corixids in their natural waters. Season Species pHof Haemolymph Whole-body Sodium natural [Na] [Na] Influx water (mM) (umoles/mg dry (nmoles/mg wet weight) weight • hr.) Spring Sigaraomani 4.5 171.1 ±10.4 0.39±0.04 0.55±0.02* (5) (5) (5) Hesperocorixa atopodonta 7.0 126.1 ±19.4 0.10 ±0.02* 0.22 ±0 .01 * (2) (2) (5) Cenocorixa blaisdelli 7.0 123.4 ±16.0 0.25 ±0.03 0.42 ±0.05 (5) (5) (5) Summer Hespercorixa atopodonta 7.0 157.6 ±7.0 0.19 ±0.02* 0.10 ±0.02 (5) (5) (5) Cenocorixa blaisdelli 7.0 173.2 ±3.4 0.40 ±0.04 (5) (5) Fall Hesperocorixa atopodonta 7.0 162.3 ±4.0 0.22 ±0.00 0.24 ±0.02 (2) (2) (5) Cenocorixa blaisdelli 7.0 148.0 ±11.3 0.10 ± 0.03 0.51 ± 0.08 (3) (2) (5) Cenocorixa bifida 9.0 147.3 ±4.5 0.34 ±0.07 (5) (5) Cenocorixa expleta 9.5 188.4 ±1 .7* 0.42 ±0.02* 0.49 ±0.10 (5) (5) (5) * = significantly different from C. blaisdelli within the same season (p< .05) Numbers in parentheses indicate sample size 17 Figure 2 Haemolymph [Na] of f i v e species of corixids i n three seasons plo t t e d against (a) sodium concentration of t h e i r natural waters and (b) pH of t h e i r natural waters. + = s i g n i f i c a n t l y d i f f e r e n t from C . b l a i s d e l l i within the same season (p<.05). n = 5, except where noted i n Table 2. | = C . b l a i s d e l l i - spring ± = H.atopodonta - spring 9 = S.omani - spring 0 = C . b l a i s d e l l i - summer £ = H.atopodonta - summer • = C . b l a i s d e l l i - f a l l A = H.atopodonta - f a l l O = C.b i f i d a - f a l l V = C.expleta - f a l l 18 a 190-r 180 + 140 + 130-H 120+1 b 190 + 180 + 170+ 160+ 140 + 130+ 120 + 50 100 150 200 250 [Na] of Natural Waters (mM) i 4 H h 2 4 6 6 10 pH of Natural Waters 19 Figure 3 Whole-body [Na] of f i v e species of corixids i n three seasons plotted against (a) sodium concentration of t h e i r natural waters and (b) pH of t h e i r natural waters. + = s i g n i f i c a n t l y d i f f e r e n t from C . b l a i s d e l l i within the same season (p<.05). n = 5, except where noted i n Table 2. • = C . b l a i s d e l l i - spring • = H.atopodonta - spring # = S.omani - spring 0 = C . b l a i s d e l l i - summer A. = H.atopodonta - summer • = C . b l a i s d e l l i - f a l l A = H.atopodonta - f a l l 0 = C.bifida - f a l l V = C.expleta - f a l l 20 0.40 ft 0.35 + 0.30+ § 0.254* 3 0.20+, 0.15-0.10-0.05 + 0.40 + ? 0.30 + D 0.15 + 0.05 + 50 100 150 200 [Na] of Natural Waters (mM) 250 0.35 + | 0.25 + 0.20+ 0.10 + 10 pH of Natural Waters 21 Figure 4 Sodium i n f l u x rates of four species of corixids i n three seasons plotted against (a) sodium concentration of t h e i r natural waters and (b) pH of t h e i r natural waters. + = s i g n i f i c a n t l y d i f f e r e n t from C . b l a i s d e l l i within the same season (p<.05). n = 5. Note: data not available for C . b l a i s d e l l i (summer) or for C.bifida ( f a l l ) . c . b l a i s d e l l i - spring H .atopodonta - spring S .omani - spring H .atopodonta - summer C . b l a i s d e l l i - f a l l H .atopodonta - f a l l V = C.expleta - f a l l 22 a O.6O-1-0.50 0.40-0.304 I-0.204 0.10-k b o.60-r 0.504 0.404 0.304 0.204 0.104 50 100 1 50 200 250 [Na] of Natural Waters (mM) 4 6 8 10 p H of Natural Waters 23 p<.005). In contrast to t h i s , S. omani, which came from a di f f e r e n t water body than C. blaisdelli and H. atopodonta but one i n which the sodium concentration was the same (6 mM) , had extremely high haemolymph [Na], whole-body [Na], and sodium i n f l u x rates. The i n f l u x rates were s i g n i f i c a n t l y higher than those of C. blaisdelli i n the same season (SPRING: 31% higher, p<.05). These high values may be related to the fact that 5. omani was c o l l e c t e d from waters with the lowest pH (4.5). When pH of the water i s considered, the highest values of haemolymph [Na], whole-body [Na], and sodium i n f l u x occurred at the extremes - pH 4.5 and pH 9.5 (Figures 2-4b). Values recorded for these three parameters i n C. blaisdelli and H. atopodonta at pH 7.0 varied greatly, but again seasonal differences are evident. S. omani at pH 4.5 i n the spring and C. expleta at pH 9.5 i n the f a l l , exhibited extremely high values of haemolymph [Na], whole-body [Na], and sodium i n f l u x . Summer C. blaisdelli measurements were also high, and these three high sodium records show a l i n e a r r e l a t i o n s h i p of increasing haemolymph and whole-body [Na] with increasing pH. Considering only the three highest data points for sodium i n f l u x produces the opposite trend; S. omani had the highest i n f l u x rate at pH 4.5, while C. expleta had the lowest at pH 9.5. F a l l C. blaisdelli rates l i e i n between. Survivorship: Season Mortality varied greatly between seasons i n C. blaisdelli, while H. atopodonta exhibited only small seasonal v a r i a t i o n i n survivorship. However, for both species mortality was always 24 lowest i n the f a l l . During the haemolymph/whole-body experiments, C. blaisdelli showed the highest mortality i n the summer i n both test pH levels (34%, pH 4.5; 100%, pH 3.0; Figure 5a). In the control or natural water pH (7.0), mortality was highest i n the spring, but reached only 30%. At a l l pH le v e l s , the best survivorship occurred in the f a l l , with 30% mortality i n pH 3.0, and no mortality i n either pH 4.5 or pH 7.0. While conducting the i n f l u x experiments, mortality for C. blaisdelli was highest i n the spring i n both the control pH (7.0, 54%) and i n the test pH (4.5, 49%; Figure 5c). Again, mortality was lowest i n the f a l l ; no animals died i n either pH. H. atopodonta exhibited almost no mortality during the haemolymph/whole-body experiments i n any season. Only i n the spring, and only i n pH 3.0, did a 5% mortality occur (Figure 5b). Results i n the in f l u x experiments were more variable, but mortality s t i l l remained under 35% i n every season. The highest mortality occurred i n the summer i n both pH le v e l s (34%, pH 7.0; 23%, pH 4.5), while no mortality occurred i n the f a l l (Figure 5d) . Mortality data for 5. omani, C. bifida, and C. expleta are discussed i n the following section, since each of these species was only tested i n one season (SPRING: S. omani; FALL: C. bifida and C. expleta). Survivorship: Species Survivorship varied between species i n a l l seasons. 25 Figure 5 Seasonal v a r i a t i o n i n cumulative percent mortality during the haemolymph/whole-body experiments for (a) C . b l a i s d e l l i and (b) H.atopodonta, and during the i n f l u x experiments for (c) C . b l a i s d e l l i and (d) H.atopodonta. • = C . b l a i s d e l l i - PH 7 .0 • = c . b l a i s d e l l i - pH 4 .5 A = c . b l a i s d e l l i " PH 3 .0 • = H .atopodonta " PH 7 .0 O = H .atopodonta " PH 4 .5 A = H .atopodonta - pH 3 .0 Day 8 Cumulative % Mortality (H/WB) Day 8 Cumulative % Mortality (H/WB) Ch 27 C. blaisdelli usua l ly exhib i ted the highest morta l i ty within any one season, and fl. atopodonta frequently exhibi ted the lowest. Of the three species tested i n the spr ing , C. blaisdelli had a cons i s tent ly higher morta l i ty at a l l pH l eve l s i n both the haemolymph/whole-body and the in f lux experiments. While 5. omani had morta l i ty values ranging from 5-23%, and fl. atopodonta exhib i ted m o r t a l i t i e s from 0-18%, C. blaisdelli displayed morta l i ty values ranging from 10-54% (Figures 6a and 7a) . In the summer, C. blaisdelli had a much higher morta l i ty than fl. atopodonta i n the haemolymph/whole-body experiments. Although fl. atopodonta exhib i ted no morta l i ty at any pH, morta l i ty i n C. blaisdelli reached 100% i n pH 3.0 by day 5 of the 8-day experiment (Figure 6b) . During the summer i n f l u x experiments, fl. atopodonta had a s l i g h t l y higher morta l i ty than C. blaisdelli (23-34% versus 14-15%; Figure 7b) . This was the only time a reversa l i n the morta l i ty trend of these two species occurred, though. Four species were tes ted i n the f a l l . C. expleta exh ib i ted the highest morta l i ty during the haemolymph/whole-body experiments at a l l pH l eve l s (20-32%; Figure 6c) , but C. bifida had a 100% morta l i ty by day 2 of the 6-day sodium i n f l u x experiment (Figure 7c) . Values for C. blaisdelli and fl. atopodonta were low for both sets of experiments i n the f a l l ; fl. atopodonta showed no morta l i t y , while C. blaisdelli only died at pH 3.0, and only i n the haemolymph/whole-body experiment (27% m o r t a l i t y ) . Although morta l i ty was general ly highest i n the lowest pH (3.0) during the haemolymph/whole-body experiments, morta l i ty for 28 Cumulative percent mortality during the haemolymph/whole-body experiments for f i v e species of corixids at three pH leve l s i n (a) spring, (b) summer and (c) f a l l . • = C . b l a i s d e l l i - spring A = H.atopodonta - spring • = S.omani - spring E3 = C . b l a i s d e l l i - summer A = H.atopodonta - summer • = C . b l a i s d e l l i - f a l l A = H.atopodonta - f a l l O = C.bifida - f a l l V = C.expleta - f a l l Day 8 Cumulative % Mortality (H/WB) Day 8 Cumulative % Mortality (H/WB) Day 8 Cumulative % Mortality (H/WB) VO 30 Figure 7 Cumulative percent mortality during the i n f l u x experiments for f i v e species of corixids at two pH levels i n (a) spring, (b) summer and (c) f a l l . • = C . b l a i s d e l l i - spring • = H.atopodonta - spring • = S.omani - spring E3 = C . b l a i s d e l l i - summer A = H.atopodonta - summer • = C . b l a i s d e l l i - f a l l A = H.atopodonta - f a l l O = C.bifida - f a l l V = C.expleta - f a l l 32 four out of the f ive species tested (C. bifida being the exception) was always lower i n pH 4.5 than i n pH 7.0 during the i n f l u x experiments. H+ T o x i c i t y : Season Haemolymph [Na], whole-body [Na], and sodium inf lux showed v a r i a t i o n with season i n both C. blaisdelli and H. atopodonta. In general , the highest values of haemolymph and whole-body [Na] occurred i n the summer, while the lowest values occurred i n the f a l l for C. blaisdelli and i n the spring for H. atopodonta. Sodium i n f l u x showed a less d e f i n i t e trend. In C. blaisdelli, summer values of haemolymph and whole-body [Na] were s i g n i f i c a n t l y higher than spring values i n a l l pH l eve l s (H[Na]: 22% higher, p<.001; WB[Na]: 49% higher, p<.01), with the exception of the haemolymph [Na] at pH 3.0 (p>.05; Figure 8a) . A d d i t i o n a l l y , f a l l values of haemolymph [Na] were s i g n i f i c a n t l y lower than spring values i n both pH 4.5 (21% lower, p<.001) and pH 3.0 (38% lower, p<.001). Whole-body [Na] was also lowest i n the f a l l at a l l pH l eve l s , but only s i g n i f i c a n t l y so at pH 7.0 (18% lower than spr ing , p<.05; Figure 8b). This may re la te to the fact that morta l i ty i n C. blaisdelli during the haemolymph/whole-body experiments was the highest i n the summer (corresponding to the highest haemolymph/whole-body [Na]) and was the lowest i n the f a l l (when haemolymph/whole-body values were the lowest) . Sodium i n f l u x i n C. blaisdelli was highest i n the spr ing and lowest i n the f a l l i n both pH 7.0 and pH 4.5 ( r e l i a b l e summer 33 Figure 8 Seasonal v a r i a t i o n i n (a) haemolymph [Na], (b) whole-body [Na] and (c) sodium i n f l u x rates for C . b l a i s d e l l i at three pH l e v e l s . * = s i g n i f i c a n t l y d i f f e r e n t from pH 7.0 (p<.05); # = s i g n i f i c a n t l y d i f f e r e n t from spring (p<.05). n = 5 or 10 for (a) and (b) ; n = 5 for (c) , except where noted i n Appendices A, B, or C. • = pH 7.0 # = pH 4.5 A = PH 3.0 Spring Summer Fall 35 values are not a v a i l a b l e ) . However, t h i s decrease was only s i g n i f i c a n t i n pH 4.5 (31% decrease, p=.005; Figure 8c) . Again, the season with the highest morta l i ty ( spr ing- inf lux experiment) corresponded to the time of highest sodium i n f l u x , while the lowest sodium in f lux values occurred i n the season with no morta l i ty ( f a l l - i n f l u x experiment). In H. atopodonta (as i n C. blaisdelli), summer haemolymph and whole-body [Na] were general ly the highest . Haemolymph [Na] i n both tes t pH leve l s (4.5 and 3.0) were s i g n i f i c a n t l y higher i n the summer than i n the spr ing , by 10% (p<.05) and 13% (p<.01), re spec t ive ly (Figure 9a) . Whole-body [Na] i n the summer were higher than i n the spr ing i n pH 7.0 (29%, p<.05) and i n pH 4.5 (28%, p<.05), but not i n pH 3.0 (p>.05; Figure 9b). The lowest values of haemolymph and whole-body [Na] i n H. atopodonta were found i n the spr ing i n a l l cases, unl ike with C. blaisdelli, whose lowest concentrations occurred i n the f a l l . R e c a l l that for H. atopodonta, almost no morta l i ty occurred i n any season during these experiments. Sodium i n f l u x i n H. atopodonta was lowest i n the summer i n pH 7.0, but lowest i n the spr ing i n pH 4.5 (Figure 9c) . Values were highest i n both pH leve l s i n the f a l l . The highest morta l i ty occurred i n the summer (which may correspond to the low sodium i n f l u x values seen i n t h i s season) and l i t t l e or no morta l i ty occurred i n the spring and i n the f a l l . 36 Figure 9 Seasonal v a r i a t i o n i n (a) haemolymph [Na], (b) whole-body [Na] and (c) sodium i n f l u x rates for H.atopodonta at three pH l e v e l s . * = s i g n i f i c a n t l y d i f f e r e n t from pH 7.0 (p<.05); # = s i g n i f i c a n t l y d i f f e r e n t from spring (p<.05). n = 5 or 10 for (a) and (b) ; n = 5 for (c) , except where noted i n Appendices A, B, or C. • = pH 7.0 O = PH 4.5 A = pH 3.0 38 H+ T o x i c i t y : Species Of the f ive species tested across three seasons, a l l except C. blaisdelli were able to maintain a remarkably constant haemolymph [Na] with decreasing pH. Whole-body [Na] was also constant for most species; however, C. bifida and C. expleta, the two species that l i v e normally i n a l k a l i n e , sa l ine lakes, exhibi ted a decrease i n whole-body [Na] with lowered pH i n the f a l l . Sodium in f lux was constant for a l l species tes ted i n a l l seasons. In the spr ing , species were able to maintain a constant haemolymph [Na] as pH decreased, with only S. omani having a s l i g h t increase i n haemolymph [Na] (9%, p<.005) at pH 3.0 (Figure 10a) . This general re su l t corresponds wel l with the r e l a t i v e l y low and stable morta l i ty rates seen i n the spr ing for H. atopodonta (0-18%) and S. omani (5-23%). However, the r e l a t i v e l y higher morta l i ty rate for C. blaisdelli i n the spring (10-54%), while not c o r r e l a t i n g with reduced haemolymph [Na] at low pH, might help to explain the o v e r a l l lowered haemolymph [Na] measured i n t h i s species . C. blaisdelli had a lower haemolymph [Na] than S. omani i n a l l three pH leve l s (22%-28% lower, p<.001), and a s i g n i f i c a n t l y lower haemolymph [Na] than H. atopodonta at pH 7.0 (12% lower, p=.01). Spring whole-body [Na] and sodium in f lux rates were constant i n a l l pH l eve l s for a l l species t es ted . In add i t i on , whole-body [Na] were approximately the same for H. atopodonta, C. blaisdelli, and S. omani (Figure 11a). However, H. atopodonta exhibi ted a much lower sodium in f lux i n the spring than the other species examined; for example, sodium in f lux values were 7 9% 39 Figure 10 Haemolymph [Na] of f i v e species of corixids at three pH levels i n (a) spring, (b) summer and (c) f a l l . * = s i g n i f i c a n t l y d i f f e r e n t from pH 7.0 (p<.05); + = s i g n i f i c a n t l y d i f f e r e n t from C . b l a i s d e l l i (p<.05). n = 5 or 10 except where noted i n Appendix A. • = C . b l a i s d e l l i - spring • = H . atopodonta - spring • = S .omani - spring 0 = C . b l a i s d e l l i - summer £ = H .atopodonta - summer • = C . b l a i s d e l l i - f a l l A = H .atopodonta - f a l l 0 = C.bifida - f a l l V = C.expleta - f a l l 'Haemolymph [Na] (mM) Haemolymph |Na) (mM) "Haemolymph [Nal (mM) 41 Figure 11 Whole-body [Na] of f i v e species of cor i x i d s at three pH le v e l s i n (a) spring, (b) summer and (c) f a l l . * = s i g n i f i c a n t l y d i f f e r e n t from pH 7.0 (p<.05); + = s i g n i f i c a n t l y d i f f e r e n t from C . b l a i s d e l l i (p<.05). n = 5 or 10 except where noted i n Appendix B. • = C . b l a i s d e l l i - spring A = H.atopodonta - spring 9 = S.omani - spring E3 = C . b l a i s d e l l i - summer A = H.atopodonta - summer • = C . b l a i s d e l l i - f a l l A = H.atopodonta - f a l l O = C.bifida - f a l l V = C.expleta - f a l l Whole-Body [Nal (umoles/mgDW) Whole-Body [Na] (umoles/mgDW) Whole-Body [Na] (umotes/rngDW) X u i 43 lower i n H. atopodonta than i n C. blaisdelli (p<.001; Figure 12a) . While H. atopodonta was able to maintain a constant haemolymph [Na] i n the summer, C. blaisdelli exhib i ted a s i g n i f i c a n t decrease i n haemolymph [Na] with decreasing pH. Haemolymph [Na] at pH 4.5 were 9% lower than values at pH 7.0 (p<.005); haemolymph [Na] at pH 3.0 were 16% lower than the contro l (p<.001; Figure 10b). Whole-body [Na] remained constant with a lowered pH for both species . However, values measured for H. atopodonta were s i g n i f i c a n t l y lower than those measured for C. blaisdelli at a l l pH l eve l s (36-46% lower, p<.01; Figure l i b ) . It should be noted that morta l i ty i n the summer was high for C. blaisdelli, reaching 100% i n pH 3.0, while H. atopodonta exhibi ted no morta l i ty i n the summer. Re l iab le sodium in f lux values are ava i lab le only for H. atopodonta i n the summer, so a species comparison cannot be made i n t h i s season. In the f a l l , of a l l the species tested, only C. blaisdelli showed a response to decreased pH by e x h i b i t i n g a lower haemolymph [Na] i n pH 4.5 (19% lower, p<.001) and i n pH 3.0 (38% lower, p<.001) than i n pH 7.0 (Figure 10c). C. bifida d i d have a s l i g h t l y elevated haemolymph [Na] i n pH 3.0, but t h i s was only marginal ly s i g n i f i c a n t (9% increase, p<.05). C. blaisdelli also had an o v e r a l l lower haemolymph [Na] than a l l other species i n the f a l l ; values measured at pH 4.5 were 30% lower (p, .001), and those at pH 3.0 were 45% lower (p<.005). C. bifida and C. expleta both experienced a decrease i n whole-body [Na] as pH was lowered. For C. bifida, t h i s decrease was 44 Figure 12 Sodium i n f l u x rates of four species of corixids at two pH l e v e l s i n (a) spring and (b) f a l l . + = s i g n i f i c a n t l y d i f f e r e n t from C . b l a i s d e l l i (p<.05). n = 5 except where noted i n Appendix C. Note: data not available for summer or for C.bifida ( f a l l ) . • = C . b l a i s d e l l i - spring • = H.atopodonta - spring # = S.omani - spring • = C . b l a i s d e l l i - f a l l A = H.atopodonta - f a l l V = C.expleta - f a l l 46 s i g n i f i c a n t only at pH 3.0 (32% lower, p=.001). For C. expleta, whole-body . [Na] was s i g n i f i c a n t l y lower than the contro l value at both pH 4.5 (24% lower, p=.01) and at pH 3.0 (28% lower, p<.005; Figure 11c). C. blaisdelli also exhibi ted a decrease i n whole-body [Na] with decreasing pH, s i g n i f i c a n t only at pH 3.0 (31% decrease, p<.001). C. blaisdelli cons i s tent ly had the lowest values measured o v e r a l l . Whole-body [Na] were 37% lower than those found i n C. bifida and C. expleta (p<.05), but were not s i g n i f i c a n t l y lower than concentrations obtained for H. atopodonta (p>.05). M o r t a l i t y i n the f a l l haemolymph/whole-body experiments was low and f a i r l y stable for a l l species tes ted . C. expleta had the highest morta l i ty o v e r a l l , which might be a r e s u l t of the observed decrease i n whole-body [Na], but the mor ta l i t y rate remained below 32%. C. blaisdelli was the only species to show a d r a s t i c increase i n morta l i ty rate with decreasing pH; morta l i ty went from 0% i n pH 7.0 and pH 4.5 to 27% i n pH 3.0. This corresponds wel l with the drop i n both haemolymph and whole-body [Na] experienced by C. blaisdelli i n the f a l l at both test pH l e v e l s , and may also explain the o v e r a l l lower values found i n C. blaisdelli compared with other species tested i n t h i s season. Sodium i n f l u x values were not s i g n i f i c a n t l y d i f f erent at pH 4.5 than at pH 7.0 for any of the species measured i n the f a l l , but, as i n the spr ing , H. atopodonta had a decreased sodium in f lux value (62% lower, p<.001) when compared with the other two species examined (Figure 12b). S u r p r i s i n g l y , the high morta l i ty rate experienced by C. expleta i n these experiments i s not r e f l e c t e d i n the in f lux rates themselves. Influx rates for C. bifida i n the f a l l are not ava i lab le owing to the complete morta l i ty (100% i n pH 3.0) during t h i s set of experiments. 48 DISCUSSION Sodium Balance: Natural Waters Although many studies have compared the chemical composition of a habi tat with eco log i ca l parameters such as density/abundance (Hall et al., 1980; Bendel l , 1986) or species d i v e r s i t y ( S u t c l i f f e and C a r r i c k , 1973), few have attempted to explain species d i s t r i b u t i o n s by c o r r e l a t i n g chemical composition of the aquatic environment with p h y s i o l o g i c a l measurements of the animals that l i v e there . In the present study, pH and sodium concentrations of the various c o r i x i d habi tats i n B . C . were compared with c e r t a i n p h y s i o l o g i c a l c h a r a c t e r i s t i c s of the f ive species examined. Some patterns could be discerned. Most notably, the species which came from the water with the highest sodium concentration and pH (C. expleta) had the highest concentrations of haemolymph and whole-body sodium. Freda and Dunson (1984) found that , i n amphibians, a normally high whole-body [Na] i s corre la ted with a low tolerance to a c i d i c condi t ions . This may explain the high concentrations seen i n C. expleta, a species which does not usual ly inhabit waters below pH 9.0 (Scudder, 1969ab) . However, H a l l et al. (1988) report that a species of mayfly, Leptophlebia cupida, c o l l e c t e d from a stream with a high sodium concentration and pH ac tua l ly had a lower whole-body [Na] than the same species taken from a stream with a much lower [Na] and pH. The authors a t t r i b u t e t h i s to a permanent or long-term environmental 49 inf luence on cat ion accumulation i n the eggs or nymphs. But, s ince adult c o r i x i d s often disperse before overwintering or breeding (Jansson and Scudder, 1974), adaptations i n the egg or l a r v a l stages are not as l i k e l y to play a r o l e i n adult to lerances to high or low pH waters. Also noted i n the present study was that under natura l condit ions haemolymph [Na] and whole-body [Na] were higher i n the summer for C. blaisdelli than i n e i ther the spr ing or f a l l . Rowe et al. (1988a) also found that the highest concentrations of whole-body Na occurred i n the summer for the mayfly, Stenonema femoratum, under contro l condi t ions . For c o r i x i d s , summer i s a time of high a c t i v i t y and, consequently, extensive feeding (Jansson and Scudder, 1974). Feeding most c e r t a i n l y serves to elevate [Na] i n the blood and body above those values recorded i n other seasons. Survivorship: Species/Season A l l c o r i x i d s tes ted to date have been extremely to l erant of low pH (Vangenechten et al., 197 9ab; Vangenechten and Vanderborght, 1980; Scudder, 1987). It i s not s u r p r i s i n g , then, that the f ive c o r i x i d species examined i n t h i s study were also remarkably to l erant of increased a c i d i t y . Although morta l i ty i n the present study increased from pH 7.0 to pH 3.0 (at l east i n the haemolymph/whole-body experiments), i t remained below 50% i n both neutra l and a c i d i c pH leve l s throughout the year. However, s p e c i f i c and seasonal di f ferences i n morta l i ty were apparent. In p a r t i c u l a r , C. blaisdelli 50 experienced greater morta l i ty than any other species tested in e i ther the spring or summer. As we l l , t h i s same species suffered much greater morta l i ty i n the summer (100% i n pH 3.0) than i n the spring or f a l l during the haemolymph/whole-body experiments. Cooper et al. (1987) also found increased morta l i ty i n C. blaisdelli i n the summer - 50% (at pH 7.0) versus only 5% i n the f a l l . Rowe et al. (1988a) report summer as the time of highest morta l i ty for the mayfly, Stenonema femoratum, i n both pH 6.5 and 3.5. High summer morta l i ty , seen i n C. blaisdelli but not in H. atopodonta, supports the hypothesis that surv ivorship d i f f e r s i n d i f f eren t species of c o r i x i d s and i n d i f f erent seasons. However, the expected resu l t would have been for morta l i ty to reach i t s peak i n e i ther the spr ing or the f a l l , when c o r i x i d s are less r e a d i l y able to deal with environmental change (see Introduct ion) . Thus, increased morta l i ty i n the summer, i n both the present study and an e a r l i e r one (Cooper et al., 1987), may instead have been due to the fact that insects were c o l l e c t e d from pond water that was 20°C-25°C, and then acclimated to the 5°C experimental condi t ion . In the spr ing and, i n p a r t i c u l a r , the f a l l , pond water i s much c loser i n temperature to the experimental . temperature used. Poss ib ly r e l a t e d , then, i s the observation that f a l l was the time of lowest morta l i ty for both C. blaisdelli and H. atopodonta. Cooper et al. (1987) and Rowe et al. (1988a) also found f a l l to be the season with the least morta l i ty for t h e i r tes t species . 51 One other p o s s i b i l i t y to explain high summer mortality, as noted by Rowe et al. (1988a), i s that t h i s i s the time of highest whole-body [Na]. Indeed, C. blaisdelli and H. atopodonta did exhibit t h e i r highest l e v e l s of haemolymph [Na] and whole-body [Na] i n the summer. Because these l e v e l s are so high i n the summer, the whole-body [Na] (haemolymph [Na]) losses which occur when animals are exposed to low pH are greater, r e l a t i v e l y , than at other times of the year. It i s t h i s greater r e l a t i v e loss that i s associated with mortality i n S. femoratum (Rowe et al., 1988a), and which may also explain the higher summer mortality seen i n the c o r i x i d , C. blaisdelli, studied here. H+ T o x i c i t y : Season/Species A further i n d i c a t i o n that a l l f i v e c o r i x i d species examined in the present study were extremely tolerant of exposure to low pH was t h e i r a b i l i t y to maintain haemolymph [Na], whole-body [Na], and sodium i n f l u x rates within t i g h t boundaries as pH decreased. Differences between values recorded at pH 7.0 and those recorded at either pH 4.5 or pH 3.0 were rarely s i g n i f i c a n t . However, season did af f e c t l e v e l s of a l l three ph y s i o l o g i c a l parameters measured, and there were s i g n i f i c a n t differences i n o v e r a l l values between species within any one season. Both C. blaisdelli and H. atopodonta exhibited t h e i r highest lev e l s of haemolymph [Na] and whole-body [Na] i n the summer at a l l three pH l e v e l s tested. As mentioned previously, summer i s a time of profuse feeding for these species, and diet i s known to 52 be an extremely important source of sodium for many aquatic insects (Frisbie and Dunson, 1988abc; S u t c l i f f e and Hildrew, 1989). Unlike haemolymph [Na] and whole-body [Na], sodium i n f l u x did not appear to be affected by season. No other seasonal study of sodium i n f l u x i n insects has been reported to date. Although, i n general, a l l corixids tested were tolerant of exposure to low pH, species differences did occur. It i s apparent that the corixids examined d i f f e r i n io n i c balance under acidic conditions. Table 3 summarizes the changes, or lack thereof, when data for the three parameters measured are compared at pH 7.0 versus pH 3.0 (haemolymph/whole-body experiments) or at pH 7.0 versus pH 4.5 (influx experiments). E f f l u x trends are inf e r r e d when possible from the patterns seen i n the measured variab l e s . Scudder (1987) and Vangenechten et al. (1979ab) also report species differences i n the sodium regulating a b i l i t i e s of co r i x i d s . Overall, H. atopodonta seem best able to balance t h e i r i n t e r n a l milieu (Table 3) . In every season, haemolymph [Na] and whole-body [Na] remained constant as pH decreased, i n d i c a t i n g a strong a b i l i t y to maintain homeostasis under a l l conditions tested. In contrast, C. blaisdelli, obtained from the same pond as H. atopodonta, exhibited s i g n i f i c a n t decreases i n both haemolymph [Na] and whole-body [Na] from pH 7.0 to pH 3.0 i n the summer and f a l l . A d ditionally, concentrations recorded for C. blaisdelli were o v e r a l l much lower than those recorded for H. atopodonta. This v a r i a t i o n i n tolerance to a c i d i c conditions may be due to the size difference i n these two species. H. atopodonta 53 Table 3 Qualitative summary of changes in haemolymph [Na], whole-body [Na], and sodium influx rates of five species of corixids upon exposure to low pH. Season Species Haemolymph Whole-body [Na] [Na] (pH 7.0 vs (pH 7.0 vs. pH 3.0) pH 3.0) Sodium Influx (pH 7.0 vs. pH 4.5) Sodium Efflux (Inferred) Spring Sigara omani Increased No change No change Decreased Hesperocorixa atopodonta No change No change No change No change Cenocorixa blaisdelli No change No change No change No change Summer Hespercorixa atopodonta No change No change Increased Increased Cenocorixa blaisdelli Decreased No change — — Fall Hesperocorixa atopodonta No change No change No change No change Cenocorixa blaisdelli Decreased Decreased No change Increased Cenocorixa bifida Increased Decreased — — Cenocorixa expleta No change Decreased No change No change or Increased For actual values, see graphs in Results section or Appendices A.B, and C. 54 i s approximately twice the s ize of C. blaisdelli (average wet weights: 45 mg and 20 mg, r e s p e c t i v e l y ) . Since smaller animals have a greater surface to volume r a t i o than larger animals, passive losses of sodium at low pH should be greater in smaller animals than i n larger ones (Rowe, 1986). The dif ference i n s u s c e p t i b i l i t y to H+ t o x i c i t y i s r e f l e c t e d by these species' d i s t r i b u t i o n patterns; while H. atopodonta can be found natura l ly occurring i n waters of e i ther neutra l or a c i d i c pH, C. blaisdelli most often occur i n ponds with a circumneutral pH (Scudder, 1987). C. bifida and C. expleta, which are found almost exc lus ive ly i n high pH waters (Scudder, 1969ab), also showed a decreased whole-body [Na] i n pH 4.5 and pH 3.0 r e l a t i v e to pH 7.0. Rowe et al. (1989) found that whole-body [Na] decreased s i g n i f i c a n t l y i n the mayflies Stenonema femoratum and Leptophlebia cupida a f ter 8 days of exposure to pH 3.5 r e l a t i v e to pH 6.5. In add i t i on , Lech le i tner et al. (1985) found a decreased whole-body [Na] i n pH 3.0 versus pH 8.0 for the stonef ly Pteronarcys proteus. In teres t ing ly , these l a t t e r authors a t t r i b u t e the whole-body sodium loss to damage of the osmoregulatory c e l l s (chloride c e l l s ) of the stonef ly g i l l . Cor ix ids are known to possess s i m i l a r c e l l s i n t h e i r integument (Komnick, 1977; Komnick and Schmitz, 1977), p a r t i c u l a r l y on t h e i r l a b i a ( J a r i a l et al. (1969), and Komnick (1977) has suggested that waterbugs, as wel l as other aquatic insects , use these ch lor ide c e l l s for Na and CI absorpt ion. 55 S. omani, the only species tested which normally inhabits ac id waters of pH 4.5, were ac tua l ly able to increase t h e i r haemolymph [Na] when exposed to pH 3.0 experimental condi t ions . Presumably t h i s was accomplished v i a a decreased sodium e f f lux , as ne i ther whole-body [Na] nor sodium in f lux changed with varying pH. Since increased e f f lux i s often an i n d i c a t i o n of animals suf fer ing from i o n i c s tress i n low pH water (McDonald et al., 1983; Havas and Likens, 1985), perhaps a decreased e f f lux suggests an a b i l i t y to deal with these s t r e s s f u l condit ions success fu l ly . This i s supported by the observation that o v e r a l l S. omani had the highest l eve l s of haemolymph [Na] of any species tested i n a l l pH l e v e l s , even though they came from water with an extremely low sodium concentration (6 mM) . Unfortunately, e f f lux could not be measured i n t h i s study. The a b i l i t y to maintain haemolymph i o n i c concentrations against a steep concentration gradient with respect to the external medium i s c h a r a c t e r i s t i c of many of the Corix idae (Frick et al., 1972; Vangenechten et al., 197 9b; Scudder, 1987). Vangenechten and Vanderborght (1980) studied the waterboatman Corixa punctata, a species which also occurs n a t u r a l l y i n low pH waters (pH 3 .5) . These authors found no s i g n i f i c a n t loss of Na and CI from the haemolymph at pH 3.0 as compared to pH 6.0. Af ter 7 days of s tarvat ion , though, Vangenechten et al. (197 9b) d id f i n d that haemolymph [Na] was elevated, but external pH appeared to have no af fect on t h i s increase . In the f lux studies that have been conducted to date on c o r i x i d s , the general conclusion has been that these insects are 56 capable of u t i l i z i n g sodium from the external environment for i n t e r n a l sodium regulat ion (Vangenechten et al., 197 9a; Vangenechten and Vanderborght, 1980; Witters et al., 1984). Chlor ide c e l l s on the head and legs are responsible for t h i s act ive uptake i n both Corixa punctata and Corixa dentipes (Komnick and Schmitz, 1977; Vangenechten, 1983). Influx occurs v i a an uptake mechanism, poss ib ly a Na + /H + exchanger, which displays Michaelis-Menten saturat ion k i n e t i c s and which i s inf luenced by the pH of the external so lu t ion (Vangenechten et al., 1979a). The labium may also be a s i t e of ch lor ide c e l l concentrations i n some c o r i x i d species ( J a r i a l et al., 1969). In teres t ing ly , while Vangenechten et al. (1979a) found that sodium in f lux i n both C. punctata and C. dentipes was depressed at ac id pH and a low ambient [Na] (<1.4 mM), in f lux was ac tua l ly higher at pH 3.0 versus pH 6.0 i n water with a [Na] greater than t h i s . As water used i n the present study had a [Na] of 6 mM, perhaps i t i s not so s u r p r i s i n g , then, that i n f l u x at pH 4.5 was seldom lower than i n f l u x at pH 7.0 for any species tested i n any season. Indeed, H. atopodonta, l i k e C. punctata and C. dentipes, exhib i ted an increase i n sodium i n f l u x from pH 7.0 to pH 4.5 i n a l l seasons, although only s i g n i f i c a n t l y so i n the summer. One dilemma throughout the present study has been the low i n f l u x rates recorded for a l l B . C . species tested, i n comparison with those rates reported by Vangenechten and h i s coworkers for c o r i x i d species i n Belgium (nmoles/hour versus /amoles/hour - see references above). However, Cooper et al. (1987) d i d report values i n the nmole range for ca l cu la ted sodium i n f l u x rates v i a 57 dr ink ing i n C. blaisdelli. In addi t ion , these authors found that dr ink ing rates were approximately twice as high i n freshwater as i n sa l t water (Cooper et al., 1987). F r i c k and Sauer (1974) suggest that dr ink ing may be important for the uptake of s p e c i f i c , e s sent ia l solutes from a d i l u t e media i n the c o r i x i d , Corisella edulis. The above r e s u l t s , i n conjunction with those of the present study, seem to ind icate that the major route of sodium entry into c o r i x i d s i n freshwater (in the absence of food) may be v i a dr ink ing , rather than v i a an external uptake mechanism such as a ch lor ide c e l l . In support of t h i s , Cannings (1981) found that c u t i c u l a r permeabi l i ty was low i n C. bifida c o l l e c t e d from low s a l i n i t y waters. This emphasizes the importance of sodium entry v i a the mouth rather than through the c u t i c l e under freshwater condi t ions . Scudder (1965) found that dr ink ing was s i g n i f i c a n t and v a r i e d with environmental condit ions i n both C. bifida and C. expleta. Survival at Low pH While a c i d i f i c a t i o n of surface waters general ly re su l t s i n a loss of species, almost every group of animals has some members which have managed to make the t r a n s i t i o n into a c i d waters. The success of these species i s due i n part to p h y s i o l o g i c a l or behav iour ia l compensatory mechanisms which allow for s u r v i v a l i n a low pH environment. Molluscs are one of the most ac id - sens i t i ve groups of aquatic organisms (Okland and Okland, 1986). In soft water, decreased 58 growth, decreasing dens i t i e s , and a reduction i n the number of species often occur with increas ing a c i d i t y (Rooke and Mackie, 1984; Servos et al., 1985; Okland and Okland, 1986). However, a few species of molluscs appear to be l a r g e l y unaffected by low pH. Pisldium equilaterale d i d not exhib i t reduced growth in ac id lakes and P. ferrugineum exhibi ted only s l i g h t l y reduced fecundity i n a c i d waters (Servos et al., 1985). This tolerance may be accounted for by the d i f f e r e n t eco log i ca l habits of these species . P i s i d i i d clams usual ly burrow i n the sediment, which i s less a c i d i c than the surrounding waters (Servos et al., 1985). As we l l , larvae of these clams are protected from low pH, since embryonic development occurs wi th in the mother's s h e l l (Servos et al., 1985; Okland and Okland, 1986). There i s a wide var i e ty of to lerance responses to low pH amongst crustaceans. M o r t a l i t y of the more sens i t ive species appears to re su l t from blood ac idos i s and/or ionoregulatory f a i l u r e (Wood and Rogano, 1986). In c r a y f i s h , where tolerances vary widely between species ( B e r r i l l et al., 1987), the sens i t ive Orconectes. rusticus, 0. propinquus, and Procambarus corkii exhibi ted elevated l eve l s of haemolymph [Ca] and decreased haemolymph [Na] fo l lowing exposure to low pH, while i n the more to l erant Cambarus robustus no change i n ion l eve l s were detected (Morgan and McMahon, 1982; H o l l e t t et al., 1986; Wood and Rogano, 1986) . This increase i n blood [Ca] appears to be the re su l t of d i s s o l u t i o n of the carapace to obtain CaC0 3, which can act as a buffer against blood ac idos i s (Wood and Rogano, 1986). 59 Delayed moulting as a r e s u l t of decreased calcium uptake i n ac id waters has also been noted i n crustaceans (Havas, 1980) . In Orconectes virilis, calcium uptake was reduced at pH 5.75 and i n h i b i t e d below pH 4.0 (Malley, 1980). Post-moult ind iv idua l s are more sens i t ive to low pH than e i ther pre-moult or inter-moult i n d i v i d u a l s (Malley, 1980) . As prev ious ly mentioned, insects are one of the most a c i d -to lerant groups of aquatic organisms. When morta l i ty at low pH does occur, i t i s usua l ly the r e s u l t of ionoregulatory d i srupt ion (Havas, 1980). Komnick (1977) has suggested that several aquatic insect species use ch lor ide c e l l s for N a / C l uptake. Rowe (1986) found that moulting increased i n Leptophlebia cupida at pH leve l s of 3.5 and 4.5. Fol lowing moulting, whole-body [Na] and [CI] were s t a b i l i z e d . Therefore, increased moulting i n ac id waters could be a mechanism for increas ing the number of ch lor ide c e l l s ava i lab le for ion absorption (Rowe, 1986). Rowe et al. (1988b) also studied the egg stage of L . cupida, along with that of Habrophlebia vibrans, Stenonema femoratum, and Baetis spp. They found that hatching and development rates d id not vary with pH, but they observed morta l i ty i n the more sens i t ive species (S. femoratum and Baetis spp.) before hatching was complete at pH < 6.5. The authors have suggested that the mayfly egg s h e l l i n some species may provide protec t ion for the embryo against ambient H+ (Rowe et al., 1988b). Like insec t s , most species of amphibians appear to be r e l a t i v e l y to l erant of low pH (Pierce, 1985). Since many adult amphibians are t e r r e s t r i a l , the e f fects of ac id water are 60 general ly r e s t r i c t e d to the egg and l a r v a l stages (Mierle et al., 1986). These ef fects include morta l i ty (Pough, 1976; Dale et al., 1985; Freda, 1986), reduced hatching (Clark and LaZerte, 1985), the "cur l ing defect", character ized by the embryo c u r l i n g within the v i t e l l i n e membrane (Freda, 1986), and d i srupt ion of Na /Cl balance (Freda and Dunson, 1984, 1985; Freda, 1986). A c i d to lerance i n amphibians can often be corre lated with habitat pH (Pierce, 1985). For example, several species of frogs from a c i d i c bogs i n New Jersey were found to r e s i s t a c i d i t y bet ter than these same species breeding i n nearby habitats of a higher pH (Pierce, 1985) . S i m i l a r l y , i n the larvae of the wood frog , Rana sylvatica, varying ac id to lerances of several populations were corre la ted with t h e i r d i f f e r e n t habitat pH l eve l s (Pierce, 1985) . As w e l l , i t has been reported that a c i d -to lerant species have lower i n i t i a l whole-body [Na] and lose less sodium when exposed to low pH than a c i d - s e n s i t i v e species (Freda and Dunson, 1984). Thus, i t seems that the v u l n e r a b i l i t y to a c i d i f i c a t i o n of amphibian breeding habitats ( t y p i c a l l y smal l , temporary forest ponds) has over time conferred t h i s group with a to lerance of ac id condi t ions , poss ib ly v i a se l ec t ion for the maintenance of low whole-body sodium l e v e l s . F i s h are the most thoroughly s tudied taxa with respect to p h y s i o l o g i c a l d i s tress i n low pH water. Populations have decreased or are ser ious ly threatened i n many a c i d i f i e d lakes (Schofie ld, 1976). In f i s h , a c i d water inf luences ionoregulat ion, acid/base balance, secret ion of mucous, and g i l l s tructure (McDonald, 1983a). Opinions vary as to the r e l a t i v e importance of 61 each of these changes (Wood, 1989) . However, even with these serious problems, a few species of f i s h can s t i l l be found in ac id (pH 4.0-5.0) waters (Dunson et al., 1977; McWilliams, 1982). Upon exposure to low pH, many f i s h w i l l increase the release of mucous from c e l l s i n the g i l l s and skin (McDonald, 1983a). The th i cker coating on the g i l l s may be a mechanism for correct ing i o n i c disturbances; t h i s would be p a r t i c u l a r l y useful i f increased mucous resu l ted i n a higher [Ca] at the g i l l surface. H + normally d i sp lace Ca + + from the g i l l i n ac id waters. Therefore, increased {Ca] at the g i l l surface could have a large mi t igat ing ef fect on H+ t o x i c i t y (McDonald, 1983a). The a b i l i t y of the g i l l ionoregulatory mechanism to r e s i s t high [H] has been demonstrated i n a few a c i d - t o l e r a n t f i s h species . In card ina l t e tras (Chelrodon axelrodi), a pH of 3.0 was required to produce net sodium losses comparable to those which occurred i n an a c i d - s e n s i t i v e species at pH 4.0 (Dunson et al., 1977). In the perch, Perca fluviatilis, sodium in f lux and ef f lux d i d not change from contro l values even i n pH 4.0 (McWilliams, 1982). Both the p h y s i o l o g i c a l bas is for a c i d tolerance and the behav iour ia l modi f icat ion u t i l i z e d to allow for s u r v i v a l i n such condit ions require further study i n a l l taxonomic groups. Only through t h i s type of experimentation w i l l the s i m i l a r i t i e s among compensatory mechanisms i n d i f f eren t taxa be e luc idated and the species losses i n a c i d i f i e d surface waters f u l l y understood. 62 SUMMARY The f i v e c o r i x i d species tested i n t h i s study (C. b l a i s d e l l i , H. atopodonta, C. b i f i d a , C. expleta, and S. omani) were, in general, tolerant of exposure to low pH. However, both seasonal and s p e c i f i c differences were detected i n each of the parameters measured (haemolymph [Na], whole-body [Na], and sodium i n f l u x ) . The most important seasonal trend observed was that the time of greatest mortality (summer) corresponded to the time of highest haemolymph and whole-body [Na] i n C. b l a i s d e l l i . F a l l was a time of low mortality and low i n t e r n a l sodium concentrations. A l l f i v e species varied i n t h e i r strategies of sodium balance at low pH. H. atopodonta appeared to be the best adapted for l i f e i n low pH waters, maintaining a constant haemolymph [Na] and whole-body [Na] as pH decreased. C. b l a i s d e l l i seemed to regulate t h e i r i n t e r n a l milieu poorly under a c i d i c conditions, often exh i b i t i n g decreased l e v e l s of haemolymph [Na] and whole-body [Na] i n low pH waters. As species from an alkaline environment, C. b i f i d a and C. expleta did better than expected i n acid conditions, while S. omani performed as had been predicted for a species n a t u r a l l y occurring i n acid waters. F i n a l l y , drinking may be an important route of entry for sodium i n the f i v e species of corixids examined here. Sodium i n f l u x rates were extremely low i n the d i l u t e , freshwater medium used, and are therefore probably not i n d i c a t i v e of balance v i a the s p e c i a l i z e d external structures known as chloride c e l l s . 63 T h e s e c e l l s h a v e b e e n r e p o r t e d as t h e m a i n i o n o r e g u l a t o r y s t r u c t u r e s i n c o r i x i d s p e c i e s t e s t e d t o d a t e . C h l o r i d e c e l l s , a s w e l l a s o t h e r c o m p e n s a t o r y m e c h a n i s m s f o r s u r v i v a l a t l o w p H , w e r e d i s c u s s e d f o r s e v e r a l g r o u p s o f a q u a t i c o r g a n i s m s . 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New Jersey. 70 APPENDIX A Raw data from the haemolympyh [Na] experiments 71 HAEMOLYMPH [Na] (mM) Season Species PH Day Mean Standard Deviation Standard Error Number Sampled Spring Sigara omani 7.0 2 172.5 17.4 5.5 10 - 4 163.8 15.1 4.8 10 6 169.1 14.4 4.6 10 8 168.0 9.3 2.9 10 4.5 2 172.2 13.6 4.3 10 4 166.1 50.8 16.1 10 6 176.6 19.0 6.0 10 8 175.8 10.8 3.4 10 3.0 2 186.9 12.4 3.9 10 4 193.6 15.6 4.9 10 6 174.2 33.9 10.7 10 8 185.6 16.2 5.1 10 Hesperocorixa atopodonta 7.0 4 158.3 17.0 7.6 5 8 146.7 4.5 2.0 5 4.5 4 155.3 19.6 8.8 5 8 151.4 5.3 2.4 5 3.0 4 151.4 6.7 3.0 5 8 135.0 3.2 1.4 5 Cenocorixa blaisdelli 7.0 4 132.1 15.1 6.8 5 8 137.5 14.9 6.7 5 cont... 72 HAEMOLYMPH [Na] (mM) cont.. Season Species pH Day Mean Standard Deviation Standard Error Number Sampled Spring Cenocorixa blaisdelli 4.5 4 141.6 14.2 6.3 5 8 134.7 6.4 2.9 5 3.0 4 136.8 18.6 8.3 5 8 136.0 15.6 7.8 4 Summer Hesperocorixa atopodonta 7.0 2 158.9 19.8 8.9 5 4 161.3 26.1 11.7 5 6 152.4 24.1 10.8 5 8 156.1 12.2 5.5 5 4.5 2 164.5 7.4 3.3 5 4 169.0 7.2 3.2 5 6 164.9 7.5 3.3 5 8 160.9 17.3 7.8 5 3.0 2 153.6 9.9 4.4 5 4 168.5 3.2 1.4 5 6 162.5 4.9 2.2 5 8 152.8 29.8 13.3 5 Cenocorixa blaisdelli 7.0 2 175.9 13.7 4.3 10 4 177.3 12.5 4.0 10 6 180.4 19.9 6.3 10 8 174.4 26.5 8.4 10 cont.., 73 HAEMOLYMPH [Na] (mM) cont... Season Species pH Day Mean Standard Deviation Standard Error Number Sampled Summer Cenocorixa blaisdelli 4.5 2 157.0 12.6 4.0 10 4 157.1 17.2 5.4 10 6 161.9 11.8 3.7 10 B 155.1 7.1 2.9 6 3.0 2 134.7 20.9 6.6 10 4 147.0 — — 1 Fall Hesperocorixa atopodonta 7.0 4 159.8 13.5 6.8 4 8 146.9 19.4 9.7 4 4.5 4 157.9 8.9 4.5 4 8 153.9 8.3 4.1 4 3.0 4 152.4 6.3 3.1 4 8 153.9 8.5 4.2 4 Cenocorixa blaisdelli 7.0 2 143.1 6.2 2.0 10 4 131.2 12.4 3.9 10 6 123.5 7.1 2.2 10 8 119.5 8.7 2.8 10 4.5 2 115.6 5.0 1.6 10 4 112.3 10.2 3.2 10 6 98.3 11.4 3.6 10 8 81.7 14.9 4.7 10 cont... 74 HAEMOLYMPH [Na] (mM) cont.. Season Species pH Day Mean Standard Standard Number Deviation Error Sampled Fall Cenocorixa blaisdelli 3.0 2 53.8 23.2 8.2 8 4 91.2 35.2 11.1 10 6 73.4 26.1 8.3 10 6 83.1 28.8 14.4 4 Cenocorixa bifida 7.0 2 135.9 36.1 11.4 10 4 155.1 20.5 6.5 10 6 158.5 10.8 3.4 10 8 151.8 30.1 9.5 10 4.5 2 149.7 57.4 19.1 9 4 158.9 29.0 9.2 10 6 145.0 26.9 8.5 10 8 163.9 5.3 1.7 10 3.0 2 170.1 18.2 5.7 10 4 158.7 29.0 9.2 10 6 173.4 9.4 3.0 10 8 161.0 31.0 9.8 10 Cenocorixa expleta 7.0 2 121.4 11.9 3.8 10 4 125.5 8.7 2.7 10 6 133.8 18.7 5.9 10 8 126.0 14.4 5.5 7 cont... 75 HAEMOLYMPH [Na] (mM) cont... Season Species pH Day Mean Standard Deviation Standard Error Number Sampled Fall Cenocorixa expleta 4.5 2 131.6 21.8 6.9 10 4 134.3 15.6 4.9 10 6 128.2 21.5 6.8 10 8 136.9 14.7 6.6 5 3.0 2 114.9 34.0 10.8 10 4 107.1 40.2 12.7 10 6 116.6 53.2 16.8 10 8 119.5 31.9 16.0 4 end 76 A P P E N D I X B Raw data from the whole-body [Na] experiments 77 WHOLE-BODY [Na] (ujnoles/mg dry weight) Season Species pH Day Mean Standard Deviation Standard Error Number Sampled Spring Sigara omani 7.0 2 0.19 0.06 0.02 10 4 0.19 0.05 0.02 10 6 0.12 0.04 0.01 9 8 0.20 0.08 0.03 6 4.5 2 0.19 0.06 0.02 10 4 0.18 0.04 0.01 10 6 0.14 0.07 0.02 10 8 0.13 0.06 0.03 5 3.0 2 0.22 0.05 0.02 10 4 0.22 0.06 0.02 10 6 0.17 0.07 0.02 10 8 0.15 0.05 0.02 5 Hesperocorixa atopodonta 7.0 4 0.16 0.02 0.01 5 8 0.15 0.03 0.01 5 4.5 4 0.16 0.02 0.01 5 8 0.17 0.04 0.02 5 3.0 4 0.18 0.08 0.03 5 8 0.15 0.02 0.01 4 Cenocorixa blaisdelli 7.0 4 0.24 0.11 0.05 5 8 0.35 — — 1 cont, 78 WHOLE-BODY [Na] (umoles/mg dry weight) cont... Season Species pH Day Mean Standard Deviation Standard Error Number Sampled Spring Cenocorixa blaisdelli 4.5 4 0.14 0.08 0.04 5 8 0.13 0.02 0.01 3 3.0 4 0.15 0.05 0.02 5 Summer Hesperocorixa atopodonta 7.0 2 0.17 0.03 0.01 5 4 0.21 0.05 0.02 5 6 0.19 0.02 0.01 5 4.5 2 0.21 0.01 0.01 5 4 0.19 0.04 0.02 5 6 0.18 0.02 0.01 5 3.0 2 0.20 0.04 0.02 5 4 0.19 0.02 0.01 5 6 0.15 0.01 0.00 5 Cenocorixa blaisdelli 7.0 2 0.36 0.08 0.03 10 4 0.39 0.09 0.03 10 6 0.29 0.06 0.02 10 8 0.33 0.07 0.03 4 4.5 2 0.28 0.11 0.03 10 4 0.26 0.09 0.03 10 6 0.32 0.08 0.02 10 8 0.36 0.06 0.05 2 cont... 79 WHOLE-BODY [Na] (umoles/mg dry weight) cont... Season Species pH Day Mean Standard Deviation Standard Error Number Sampled Summer Cenocorixa blaisdelli 3.0 2 0.23 0.11 0.04 8 Fall Hesperocorixa atopodonta 7.0 4 0.16 0.02 0.01 4 8 0.20 0.06 0.03 3 4.5 4 0.16 0.03 0.01 4 8 0.21 0.01 0.01 3 3.0 4 0.17 0.02 0.01 4 8 0.21 0.01 0.00 3 Cenocorixa blaisdelli 7.0 2 0.26 0.05 0.02 10 4 0.22 0.06 0.02 10 6 0.15 0.06 0.02 9 8 0.23 0.06 0.02 10 4.5 2 0.19 0.05 0.02 10 4 0.18 0.05 0.02 10 6 0.18 0.04 0.01 10 8 0.21 0.05 0.02 10 3.0 2 0.13 0.07 0.02 10 4 0.15 0.06 0.02 10 6 0.16 0.05 0.02 10 Cenocorixa bifida 7.0 2 0.32 0.16 0.05 10 4 0.38 0.19 0.06 10 cont.. 80 WHOLE-BODY [Na] (umoles/mg dry weight) cont... Season Species PH Day Mean Standard Deviation Standard Error Number Sampled Fall Cenocorixa bifida 7.0 6 0.32 0.14 0.04 10 8 0.44 0.25 0.09 7 4.5 2 0.31 0.18 0.06 10 4 0.28 0.18 0.06 10 6 0.18 0.09 0.03 9 8 0.41 0.13 0.05 7 3.0 2 0.25 0.14 0.05 10 4 0.20 0.05 0.02 10 6 0.19 0.07 0.02 10 8 0.29 0.08 0.03 9 Cenocorixa expleta 7.0 2 0.30 0.09 0.03 10 4 0.28 0.09 0.03 10 6 0.34 0.13 0.04 10 4.5 2 0.22 0.07 0.02 10 4 0.24 0.12 0.04 10 6 0.16 0.04 0.02 7 3.0 2 0.23 0.08 0.03 10 4 0.20 0.07 0.02 10 6 0.18 0.06 0.02 10 end APPENDIX C Raw data from the sodium i n f l u x experiments 3 82 SODIUM INFLUX (nmoles/mg wet welghMiour) Season Species pH Day Mean Standard Deviation Standard Error Number Sampled Spring Sigara omani 7.0 3 0.73 0.19 0.09 5 7 0.73 0.09 0.04 5 9 0.35 0.02 0.01 5 4.5 3 0.48 0.15 0.07 5 7 0.74 0.16 0.07 5 9 0.43 0.08 0.04 4 Hesperocorixa atopodonta 7.0 1 0.20 0.07 0.03 5 2 0.15 0.04 0.02 5 3 0.09 0.03 0.01 5 4 0.12 0.05 0.02 5 5 0.06 0.03 0.01 5 6 0.09 0.03 0.02 5 4.5 1 0.18 6.06 0.04 5 2 0.21 0.08 0.03 5 3 0.13 0.07 0.03 5 4 0.12 0.05 0.02 5 5 0.08 0.04 0.02 5 6 0.06 0.03 0.01 5 Cenocorixa blaisdelli 7.0 1 0.52 0.20 0.09 5 2 0.85 0.41 0.18 5 cont... 83 SODIUM INFLUX (nmoles/mg wet weight-hour) cont... Season Species pH Day Mean Standard Deviation Standard Error Number Sampled Spring Cenocorixa blaisdelli 7.0 3 0.56 0.22 0.11 4 5 0.58 0.32 0.19 3 4.5 1 0.56 0.05 0.02 5 2 0.86 0.26 0.12 5 3 0.88 0.39 0.17 5 5 0.70 0.41 0.19 5 Summer Hesperocorixa atopodonta 7.0 1 0.13 0.03 0.02 5 2 0.07 0.02 0.01 5 3 0.07 0.01 0.01 5 4 0.08 0.01 0.00 5 5 0.14 0.04 0.02 4 6 0.10 0.02 0.01 4 4.5 1 0.24 0.10 0.04 5 2 0.22 0.09 0.04 5 3 0.15 0.06 0.02 5 4 0.13 0.05 0.02 5 5 0.21 0.05 0.02 5 - 6 0.13 0.04 0.02 5 Cenocorixa blaisdelli (not used) 7.0 2 12.30 7.41 3.31 5 3 14.04 3.84 1.72 5 cont.. 84 SODIUM INFLUX (nmoles/mg wet weight>hour) cont.. Season Species pH Day Mean Standard Deviation Standard Error Number Sampled Summer Cenocorixa blaisdelli (not used) 7.0 4 9.55 3.18 1.42 5 5 10.56 2.50 1.12 5 6 13.87 1.64 0.74 5 4.5 2 2.01 1.14 0.51 5 3 0.05 0.07 0.03 5 4 10.78 2.61 1.17 5 5 9.94 3.52 1.57 5 6 14.59 4.05 1.81 5 Fall Hesperocorixa atopodonta 7.0 1 0.10 0.03 0.02 5 2 0.10 0.02 0.01 5 3 0.10 0.01 0.01 5 4 0.21 0.03 0.01 5 6 0.17 0.02 0.01 5 4.5 1 0.22 0.01 0.00 5 2 0.17 0.05 0.02 5 3 0.19 0.03 0.01 5 4 0.13 0.02 0.01 5 6 0.09 0.02 0.01 5 Cenocorixa blaisdelli 7.0 1 (1988-not used) 10.75 3.64 1.63 5 2 7.00 2.06 0.92 5 cont... 85 SODIUM INFLUX (nmoles/mg wet weight-hour) cont.. Season Species pH Day Mean Standard Deviation Standard Error Number Sampled Fall Cenocorixa blaisdelli 7.0 3 -(1988-not used) 1.43 0.50 0.22 5 4 16.40 7.08 3.17 5 5 15.58 5.79 2.59 5 6 1.87 0.90 0.40 5 4.5 1 11.52 3.12 1.39 5 2 11.78 2.51 1.12 5 3 10.24 5.77 2.58 5 4 17.12 9.78 4.37 5 5 4.65 1.03 0.46 5 6 2.66 0.96 0.43 5 Cenocorixa blaisdelli(1989) 7.0 1 0.66 0.10 0.04 5 2 0.54 0.08 0.04 5 3 0.45 • 0.03 0.01 5 4 0.50 0.12 0.05 5 6 0.46 0.23 0.10 5 4.5 1 0.39 0.12 0.05 5 2 0.49 0.08 0.04 5 3 0.41 0.15 0.07 5 4 0.55 0.08 0.03 5 6 0.40 0.06 0.03 5 cont.., 86 SODIUM INFLUX (nmoles/mg wet weight-hour) cont... Season Species pH Day Mean Standard Deviation Standard Error Number Sampled Fall Cenocorixa bifida (not used) 7.0 3 36.65 6.49 2.90 5 3.0 3 27.43 17.78 8.89 4 Cenocorixa expleta 7.0 3 0.39 0.12 0.06 4 6 0.44 0.10 0.06 3 4.5 3 0.27 0.05 0.03 4 6 0.47 0.09 0.04 4 end 

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