The Journl of Experimentl Biology 207, 2313-2321 Published by The Compny of Biologists 4 doi:10.1242/jeb.01024 2313 Osmotic regultion in dult Drosophil melnogster during dehydrtion nd rehydrtion Meliss A. Albers* nd Timothy J. Brdley Deprtment of Ecology nd Evolutionry Biology, University of Cliforni, Irvine, CA 92612, USA *Author for correspondence (e-mil: mlbers@uci.edu) Accepted 13 April 4 Summry We hve exmined the osmoregultory cpcities of lbortory popultions of the insect Drosophil melnogster by mesuring hemolymph osmotic concentrtion during desicction nd upon recovery from bout of desicction. Recovery tretments entiled llowing the flies ccess to distilled wter, sline solution or sline+sucrose solution fter desicction bout shown to reduce hemolymph volume by ~60%. Prior to desicction, the hemolymph osmotic concentrtion ws 353±11 mosm. We found tht Drosophil disply strict osmotic regultion under prolonged conditions of dehydrtion. Osmotic regultion continued during recovery from desicction, regrdless of the fluid provided. This result is evidence tht this insect does not require n externl source of osmolytes or energy to regulte its hemolymph osmotic concentrtion or to restore hemolymph volume, which is reduced during desicction. We lso exmined popultions tht hve been selected for over 250 genertions for enhnced desicction resistnce to identify physiologicl chrcters tht hve evolved in response to the selection regime. The selected lines displyed reduced pre-desicction hemolymph osmotic concentrtion (315±7 mosm) nd mrginlly improved cpcity for osmoregultion. Key words: osmoregultion, hemolymph, desicction, rehydrtion, Drosophil melnogster. Introduction Terrestril insects re prticulrly susceptible to dehydrtion due to their reltively smll size nd lrge surfce-to-volume rtio in reltion to other clsses of terrestril nimls. Insect popultions exposed to dehydrting conditions on regulr bsis will either perish or become dpted to the given environment (Brdley et l., 1999; Wtnbe et l., 2). The dnger of desicction is mplified in insects tht rely on flight for trnsporttion to food sources (Mrkow nd Cstrezn, 0), for mting (Eiko et l., 2; Norio, 2) or for migrtion (Coyne et l., 1987; Drke nd Gtehouse, 1995). Wter loss through the respirtory system is enhnced during flight (Lehmnn, 1). In ddition to respirtory wter loss, insects lso lose wter through the cuticle (Rmsy, 1935) nd vi excretion (Brdley, 1985). On the other hnd, insects cn gin wter through food nd drink, metbolic wter (Showler nd Morn, 3) nd (rrely) wter vpor bsorption (Rmsy, 1964; Grimstone et l., 1968; Mchin 1980, 1983). Given the potentil for desicction in insects, they re remrkble t withstnding even the most rid environments. Severl osmotic strtegies hve been observed in insects to del with the stress of desicction; these include tolernce to osmotic vribility (Nidu nd Httingh, 1986; Grrett nd Brdley, 1994; Ptrick nd Brdley, 0), osmotic regultion by sequestrtion (Hytt nd Mrshll, 1977, 1985) nd osmotic regultion by excretion (Brdley, 1985; Hdley, 1994). Although numerous studies hve been conducted exmining osmoregultion in insects, studies on smll terrestril insects re notbly bsent. An exception to this sttement is found in investigtions regrding wter vpor bsorption in smll insects (Holmstrup et l., 1). We chose to study osmoregultion in smll insect tht is of gret importnce in the fields of genetics, evolution nd moleculr biology, nmely Drosophil melnogster. The pucity of informtion regrding the ptterns nd mechnisms of osmotic regultion in this species is regrettble, given the numerous studies investigting the evolution nd popultion genetics of enhnced desicction resistnce in both wild nd lbortory-selected Drosophil popultions (Dobzhnsky, 1952; Gibbs et l., 1997, 3; Brdley et l., 1999; Hoffmnn nd Hrshmn, 1999; Nghiem et l., 0; Pfeiler nd Mrkow, 1; Mrron et l., 3). In the current study, we hve undertken n nlysis of ptterns of osmoregultion in dult Drosophil during desicction. Since some degree of desicction is inevitble in these smll insects s they fly bout seeking mtes, food sources nd oviposition sites, the pttern of osmoregultion during wter loss nd subsequent rehydrtion re of considerble interest. We chose to study these phenomen in
2314 M. A. Albers nd T. J. Brdley five replicte popultions of Drosophil tht hve been mintined in the lbortory for over 250 genertions (C popultions). We lso exmined five replicte popultions tht hve undergone selection for enhnced desicction resistnce (D popultions) to determine if osmotic regultion or ptterns of rehydrtion hve evolved during this selection process. Previous studies involving the popultions of flies used in this experiment hve estblished tht, reltive to the C popultions, the D popultions hve reduced rte of wter loss both prior to nd during bout of desicction (Gibbs et l., 1997; Willims et l., 1998) nd greter body wter content (Gibbs et l., 1997). The mjority of this dditionl wter is found extrcellulrly s hemolymph (Folk et l., 1). It hs lso been demonstrted tht hemolymph volume decreses substntilly in the C nd D popultions during desicction nd tht some ions re removed from this fluid comprtment nd excreted (Folk nd Brdley, 3). Wht is not known is whether the flies llow their hemolymph osmollity (number of solutes per kg of wter) to increse substntilly or whether they re strictly regulting their internl fluids. The bsence of regultion could result in intolerble concentrtions of osmolytes tht led to cellulr nd metbolic mlfunctioning. In this study, we mesure the hemolymph osmollity of flies of five D popultions nd five C popultions before desicction, during desicction nd upon recovery from desicction to determine the osmoregultory cpbilities of the C nd D popultions under these circumstnces. In ddition, we exmine the bility of the C nd D popultions to replenish hemolymph volume during recovery from desicction. Mterils nd methods Fly stock The popultions of Drosophil melnogster Meigen used in this experiment hve been involved in long-term, on-going selection study. They were derived from five lrge, outbred popultions selected for postponed reproduction (O popultions; Rose, 1984). From ech of the five O popultions, two dditionl lines were creted, D popultion nd C popultion. There exist, therefore, five pired D nd C popultions. The D popultions undergo selection for enhnced desicction resistnce every genertion, nd the C popultions serve s pired controls. Popultion sizes t ech genertion were >1000 flies to prevent inbreeding. All flies were mintined t 25 C with 24 h light. Stndrd Rose lb bnn food ws used for the rering of ll popultions. This food consisted of 1.1 liter wter, 16.7 g gr, 150 g peeled bnns, 18.3 ml light corn syrup, 18.3 ml drk corn syrup, 27.5 ml brley mlt nd 40 g ctive dry yest dissolved in 95% ethnol (n nti-fungl solution). The yest ws inctivted during cooking by vigorous boiling. Flies were provided with diet rich in yest for source of protein prior to egg collection. Eggs were collected from flies ~6 9 dys post-eclosion, nd lrve were grown t moderte densities of 60 80 per 40 ml vil contining pproximtely 10 ml of fly food. Adult flies were trnsferred to Plexigls cges (24 cm 19 cm 13 cm) 14 dys fter egg collection. All flies on which physiologicl mesurements were to be mde were rered seprtely from the ongoing colony without selection for two genertions to eliminte prentl nd grndprentl phenotypic effects. Only femles were used for experimentl purposes. Selection regime The D popultions were selected for enhnced desicction resistnce t every genertion. Fourteen dys fter egg collection (t pproximtely 4 dys post-eclosion), ech C nd D popultion ws trnsferred from food vils to seprte lrge Plexigls cges, one for ech popultion. At this point, selection ws initited. The D popultions were plced in cges long with cheesecloth bg of desiccnt (Drierite; W. A. Hmmond, Drierite Compny, Ltd, Xeni, OH, USA) nd no food or wter. Cge entrnces were seled with plstic wrp to retrd the entrnce of wter vpor from the mbient environment. The C popultions were plced in identicl cges but with wter source ( non-nutritive gr plte), no food nd no desiccnt. When ech D popultion reched 80% mortlity, selection ws removed nd food ws presented to both the D popultion nd its pired control popultion. Therefore, the difference in the tretment of the C nd the D popultions took plce in the dult stge nd consisted only of the presence or bsence of wter. Mesuring hemolymph osmollity Hemolymph osmollity ws mesured in individul flies of ll 10 popultions (N=10). Hemolymph smples were collected by piercing the lterl thorcic segment of individul flies, under oil, with pulled micropipette (micropipette puller; Nrishig Scientific Instruments Lb, Setgy-Ku, Tokyo, Jpn). Through cpillry ction, hemolymph ws drwn into the micropipette. Oil ws collected in the micropipette before nd fter hemolymph collection to void evportive wter loss from the hemolymph smple. The smples were immeditely expelled vi mouth pipetting into oil wells of clibrted nnoliter osmometer (Clifton Technicl Physics, Hrtford, NY, USA) under dissecting microscope (500 ), nd osmollity (mosm) ws determined by melting point depression (Brdley nd Phillips, 1975). Mesured hemolymph smples rnged from volumes of ~0.05 to 1.4 nl. No melniztion of the hemolymph ws observed subsequent to collection. Hemolymph osmollity during desicction Hemolymph osmollity ws determined in 10 individul femle flies in ech popultion t vrious time intervls during bout of desicction stress. The D flies were desiccted for 8, 16, 24 nd 48 h nd the C flies for 8 nd 16 h. Five flies were plced in 40 ml glss vil contining pproximtely 5 g of indictor Drierite. Flies were llowed to occupy the lower three-qurters of the chmber nd were isolted from the Drierite by thin fom plug. Entrnces to the desiccting chmbers were seled with Prfilm (Americn Cn Compny, Greenwich, CT, USA). After the llotted desicction period, live flies were removed from desiccting chmbers nd directly
Osmotic regultion in Drosophil 2315 submerged in oil. Hemolymph smples were drwn nd osmollity ws mesured s described bove. Recovery We lso exmined hemolymph osmollity nd hemolymph volumes following recovery from bout of desicction. After 8 h of desicction in the C popultions nd 24 h desicction in the D popultions, live flies were removed from desiccting chmbers nd plced in recovery chmbers. Recovery chmbers were 40 ml vils contining Kimwipe (Kimberly- Clrk, Roswell, GA, USA) sturted with 1.5 ml of one of three recovery fluids: (1) distilled wter, (2) sline solution (25 mmol l 1 KCl, 135 mmol l 1 NCl) or (3) sline+sucrose solution (5% sucrose, 35 mmol l 1 KCl, 135 mmol l 1 NCl). The flies were llowed to recover in these chmbers for 24 h (five flies per vil). Hemolymph osmollities were then obtined s described bove nd the hemolymph volumes were estimted grvimetriclly using the blotting technique described by Folk et l. (1). Dt nlysis Initil hemolymph osmotic concentrtions nd those of dehydrted flies were plotted ginst proportionl dehydrtion of the hemolymph. Proportionl dehydrtion of the hemolymph is defined by the expression V i /V e (Hdley, 1994), where V i is the initil volume of the hemolymph (i.e. the hemolymph volume prior to desicction) nd V e is the volume of hemolymph remining fter given mount of time in the desiccting chmber. Volumes were obtined in previous study exmining rtes of wter loss nd ion regultion in the C nd D popultions (Folk nd Brdley, 3). Using grvimetric blotting technique, these uthors determined the verge extrctble hemolymph volume in individul flies of ech popultion before desicction nd fter 8, 16, 24 nd 48 h of desicction (8 nd 16 h in the C flies). To determine the degree to which flies of ech popultion osmoregulte, theoreticl regression line ws constructed. This theoreticl line ws the osmollity for given hemolymph volume if no ion regultion were to occur. The theoreticl osmollity (osmollity e ) ws defined by: Osmollity e = (osmollity i V i )/V e, where V is the volume of hemolymph nd subscripts i nd e re initil (prior to desicction) nd experimentl (fter given increment of desicction period), respectively. If the observed hemolymph osmollity nd corresponding volume fll on the theoreticl line, then the flies filed to osmoregulte. If they fll below the theoreticl line, the flies osmoregulted to some extent. To quntify the extent of osmoregultion, modifiction of method proposed by Riddle (1985) ws used. The rtio of observed osmollity slope/theoreticl osmollity slope served s n index of the extent of osmoregultion. If this rtio is equl to 1, there is no evidence of osmoregultion. The closer this vlue is to 0, the greter the extent of osmoregultion. This index llows comprison between the C nd D popultions becuse it ignores differences in initil hemolymph osmollity or volume (Hdley, 1994). Sttistics We tested the difference in the mens of hemolymph osmollity for ech popultion (N=5) prior to desicction between the C popultions nd the D popultions using pired Student s t-test to determine whether the popultions were t the sme hemolymph osmollity t the onset of desicction. We determined whether the hemolymph osmotic concentrtion of the C nd D popultions incresed s result of desicction by performing liner regression test, which estblished if the observed slopes differed from zero. To evlute osmoregultory bilities of flies within given selection tretment, pired Student s t-test ws performed compring the five C n (or D n ) slopes of the observed regression lines to the five slopes of the theoreticl regression lines. To determine whether the C popultions were osmoregulting differently from the D popultions, we compred the rtios of the slopes of the observed regression line with the slopes of the theoreticl regression lines. This rtio, clculted for ech popultion, ws used to evlute the extent of osmoregultion. The rtio did not stisfy the ssumptions of prmetric test; therefore, onetiled Wilcoxon signed rnk test ws used to determine whether the D popultions exhibit greter extent of osmoregultion thn their pired control popultions. We performed two nlysis of vrince (ANOVA) tests with Bonferroni/Dunn post-hoc tests on hemolymph volume nd nother two for hemolymph osmollity to determine differences between initil vlues, vlues fter the prescribed desicction nd vlues fter recovery on wter, sline or sline+sucrose. One ANOVA detected differences within the C popultions nd the other within the D popultions. Hemolymph volume dt obtined from Folk nd Brdley (3) were used for the pre-desicction volume nd volume fter desicction. Results Hemolymph osmollity prior to desicction The hemolymph osmotic concentrtion vries considerbly between individuls, even within single popultion (Tble 1). Tble 1. Men hemolymph osmollities of Drosophil melnogster Osmollity Osmollity Popultion (mosm) Popultion (mosm) C 1 338±11 D 1 317±8 C 2 352±15 D 2 314±6 C 3 387±9 D 3 324±12 C 4 325±3 D 4 289±6 C 5 365±15 D 5 331±7 C men 353±11 D men 315±7 C popultions (C 1 5) re control flies nd D popultions (D 1 5) re those selected for enhnced desicction resistnce. Vlues re given in mosm ± S.E.M.
2316 M. A. Albers nd T. J. Brdley Hemolymph osmollity (mosm) 0 1800 1600 1400 1 1000 800 600 400 1 1.5 2 2.5 3 3.5 4 4.5 Proportionl dehydrtion of the hemolymph Fig. 1. Observed hemolymph osmollity ( ) nd theoreticl hemolymph osmollity ( ) in C popultions s function of proportionl dehydrtion of the hemolymph. Proportionl dehydrtion of the hemolymph represents decline of hemolymph s numbers increse. For exmple, t vlue of 2, the flies lost 50% of their hemolymph, t 4 they lost 75%, etc. The solid nd broken lines re the mens of the slopes for the five observed nd theoreticl slopes, respectively. Hemolymph osmollity (mosm) 5 4700 4 3700 3 2700 2 1700 1 700 1 3 5 7 9 11 13 Proportionl dehydrtion of the hemolymph Fig. 2. Observed hemolymph osmollity ( ) nd theoreticl hemolymph osmollity ( ) in D popultions s function of proportionl dehydrtion of the hemolymph. The solid nd broken lines re the mens of the slopes for the five observed nd five theoreticl slopes, respectively. Tble 2. The rtio of observed to theoreticl slopes for five C popultions (C 1 5 ) nd five D popultions (D 1 5 ) Popultion Obs./theor. Popultion Obs./theor. C 1 0.231 D 1 0.066 C 2 0.483 D 2 0.006 C 3 0.005 D 3 0.003 C 4 0. D 4 0.073 C 5 0.148 D 5 0.115 The rtio of observed to theoreticl slopes serves s n index of the extent of osmoregultion. The closer this vlue is to 0, the greter the osmoregultory bility in given popultion. 15 5 Prior to desicction, when ll flies were presumbly in fully hydrted stte, the D popultions hd lower men hemolymph osmollity (315±7 mosm) thn the C popultions (353±11 mosm) (P<0.05). Hemolymph osmollity during desicction Throughout desicction, the hemolymph osmollity in flies from both selection tretments incresed grdully s hemolymph volume decresed, tht is to sy s proportionl dehydrtion of the hemolymph incresed (Figs 1, 2). The liner reltionship for ech selection tretment is the regression line of men vlues of ech popultion (C 1 5 or D 1 5 ). The positive slopes of these men lines were found to be sttisticlly significntly different from zero (P<0.05). The point tht represents the lrgest vlue on the x-xis is mesured vlue during non-lethl prescribed bout of desicction nd does not represent the hemolymph osmotic concentrtion t deth. The observed hemolymph osmotic concentrtion of the C nd D popultions during dehydrtion (Figs 1, 2) is plotted djcent to slopes representing the theoreticl osmollities tht would rise if no osmotic regultion occurred. The observed increse in hemolymph during dehydrtion is substntilly lower (P<0.001) thn the theoreticl increse for both C nd D popultions. This discrepncy between observed nd theoreticl slopes is cler indiction tht ll popultions were osmoregulting. The rtio generted by dividing the observed chnge in hemolymph osmotic concentrtion by the theoreticl concentrtions cn be used s mesure of the extent of osmoregultion tht occurred during dehydrtion (Tble 2). There is some vrition in this rtio mong popultions within selection tretments; however, when we compre the pired popultions (i.e. compre C n with D n ) ech D popultion hs lower rtio thn its pired control popultion. Therefore, t lest with regrd to this prmeter, selection for enhnced desicction resistnce hs led to greter cpcity for osmoregultion (P<0.05). Recovery (hemolymph volume) Simple visul observtions of the flies under dissecting scope following 24 h of recovery suggested tht the responses during recovery were not entirely uniform. A few flies ppered to hve drunk nothing t ll nd were still in rther dehydrted stte while other individuls hd very little hemolymph despite lrgely distended gut. Nonetheless, most flies were ble to increse their hemolymph volume during recovery. Pre- nd post-desicction vlues re included in Figs 3 6 to show wht is hppening to hemolymph volume nd hemolymph osmollity during desicction. Following drop in hemolymph volume during n 8 h desicction bout, the C popultions incresed hemolymph
Osmotic regultion in Drosophil 2317 Hemolymph volume (nl) 120 100 80 60 40 20 0,b Initil 8 h W S S+S Fig. 3. Men hemolymph volume of the C popultions prior to desicction, fter 8 h of desicction nd fter recovery from desicction on distilled wter (W), sline (S) or sline+sucrose (S+S). Broken-lined brs represent dt published in previous study (Folk nd Brdley, 3). Error brs represent S.E.M. b,c c c Hemolymph osmollity (mosm) 500 450 400 350 300 250 150 100 50 0,b Initil b 8 h S+S Fig. 5. Men hemolymph osmollity of the C popultions prior to desicction, fter 8 h desicction nd fter recovery from desicction on distilled wter (W), sline (S) or sline+sucrose (S+S). Error brs represent S.E.M. W S volume fter rehydrtion on the three recovery solutions (Fig. 3). Hemolymph volumes verged 84±9 nl on wter, 99±11 nl on sline nd 101±7 nl on sline+sucrose. The type of recovery fluid hd no significnt effect on the men hemolymph volume following the 24 h rehydrtion period (P<0.05). Flies tht recovered on sline or sline+sucrose hd significntly higher hemolymph volume thn when they were in their pre-desiccted stte (P<0.05). The D popultions showed different response following the recovery tretment. Hemolymph volumes were 185±44 nl, 177±41 nl nd 273±47 nl for wter, sline nd sline+sucrose, respectively (Fig. 4). There were no sttisticlly significnt differences in hemolymph volume between the flies hydrted on wter, sline or sline+sucrose (P>0.05). These volumes lso did not differ from either preor post-desicction hemolymph volumes (P>0.05), lthough when compring hemolymph volumes of pre- nd postdesicction, there ws sttisticlly significnt difference (P<0.05). Recovery (hemolymph osmollity) Hemolymph osmollity following recovery ws not dependent on the recovery tretment. The hemolymph osmollities in flies of the C popultions were 298±19 mosm, 334±18 mosm nd 329±4 mosm fter recovery on wter, sline nd sline+sucrose, respectively (Fig. 5). These vlues were not sttisticlly different from pre-desiccted hemolymph osmollities (P>0.05). Following recovery, the osmollity of the hemolymph in the D popultions ws mesured s 296±22 mosm, 315±14 mosm nd 305±24 mosm fter rehydrtion on wter, sline or sline+sucrose, respectively (Fig. 6). These vlues re not Hemolymph volume (nl) 400 350 300 250 150 100 50 0 Initil b 24 h S+S Fig. 4. Men hemolymph volume of the D popultions prior to desicction, fter 24 h of desicction nd fter recovery from desicction on distilled wter (W), sline (S) or sline+sucrose (S+S). Broken-lined brs represent dt published in previous study (Folk nd Brdley, 3). Error brs represent S.E.M.,b W,b S,b Hemolymph osmollity (mosm) 500 450 400 350 300 250 150 100 50 0 Initil b 24 h S+S Fig. 6. Men hemolymph osmollity of the D popultions prior to desicction, fter 24 h desicction nd fter recovery from desicction on distilled wter (W), sline (S) or sline+sucrose (S+S). Error brs represent S.E.M. W S
2318 M. A. Albers nd T. J. Brdley sttisticlly distinguishble from the pre-desicction vlues (P>0.05). Discussion In the present study, we demonstrte tht Drosophil melnogster re very strong osmoregultors. They cn mintin their hemolymph osmotic concentrtion in nrrow rnge even when subjected to bout of desicction tht reduces their hemolymph volume to less thn 25% of its initil vlue. Hemolymph osmollity prior to desicction Insects exhibit much lrger vrition in the concentrtion of the extrcellulr fluid, both individully nd in response to environmentl vrition, thn do vertebrtes (Buck, 1953; Jeuniux, 1971; Bosquet, 1977). It hs been rgued tht the selectively permeble sheth tht protects the insect centrl nervous system permits greter tolernce of both osmotic nd ionic vribility in the hemolymph (Treherne nd Pichon, 1973; Jones, 1977; Ashhurst, 1985). In the present study, the control popultions hve men osmotic concentrtion prior to desicction of 353 mosm. This vlue is considerbly higher thn the single other mesurement of 251±9 mosmol l 1 from individul D. melnogster (Singleton nd Woodruff, 1994). Other vlues from dult dipterns include 400 mosm for blowflies (Phillips, 1969) nd 354 mosm for the mosquito Aedes egypti (Willims et l., 1983). Interestingly, the osmotic concentrtion of the hemolymph of the control popultions is higher thn tht of the D flies under the sme conditions. The hemolymph osmollity in the O popultions, the ncestors of both the C nd D popultions, is unknown. It is therefore uncler whether the C popultions hve experienced n increse in hemolymph concentrtion reltive to their ncestor following reproductive isoltion from the D popultions or whether the D popultions hve evolved lower hemolymph osmollity in response to selection for enhnced desicction resistnce. Hemolymph osmollity during desicction As wter is lost during desicction, the hemolymph osmollity increses in ll popultions tested. This increse in hemolymph osmollity is still well below the theoreticl osmollity vlues, which re expected in the bsence of osmoregultion, demonstrting strong cpcity for osmoregultion in D. melnogster. Other insects hve similr pttern of osmotic regultion, including, for exmple, the orthoptern Crusius morosus (Nicolson et l., 1974) nd the coleopterns Stips stli (Nidu nd Httingh, 1986) nd Onymcris pln (Nicolson, 1980). Phillips (1969) found tht the diptern Clliphor erythrocephl incresed its hemolymph osmotic concentrtion during dehydrtion by 25% fter two dys of wter deprivtion, concomitntly incresing its urine concentrtion 15-fold. Wll (1970) reported similr trend in the cockroch Periplnet mericn during dehydrtion. In exmining popultions tht hve undergone 250 genertions of selection for enhnced desicction resistnce, we found tht their cpcities for osmotic regultion hve been mrginlly improved in response to this selection regime. This difference in osmotic regultory cpcity is not the physiologicl trit tht is considered the most importnt for enhnced desicction resistnce, however. More importnt evolved physiologicl differences between the C nd D popultions re reduced rte of wter loss (Gibbs et l., 1997; Willims et l., 1998) nd n increse in wter content (Gibbs et l., 1997; Folk nd Brdley, 3). The mechnistic detils of osmoregultion in Drosophil re yet to be worked out. Folk nd Brdley (3) found tht s the flies lose wter, both C nd D popultions excrete sodium, potssium nd chloride. The qulity of these excreted solutes, s reported in their study, does not ccount for ll of the osmolytes removed from the hemolymph to mintin hemolymph osmollities s were mesured during desicction. Further studies re required to determine wht dditionl solutes re removed nd to where they re trnsferred. Of the orgns engged in osmoregultion in Drosophil, only the Mlpighin tubules hve been exmined in mechnistic detil (Mddrell nd O Donnell, 1992; Dow et l., 1994; O Donnell nd Mddrell, 1995; O Donnell et l., 1996; Linton nd O Donnell, 1999; O Donnell nd Spring, 0; Rheult nd O Donnell, 1). It would be vluble to determine the reltive roles of the osmoregultory orgns in Drosophil, prticulrly the rectum, which Phillips (1969) demonstrted is the site of urine concentrtion in dult dipterns. The specific osmolytes tht re importnt in the vrious fluid comprtments (intrcellulr fluid, hemolymph, urine) re lso unknown. Recovery The full selection regime of these fly popultions involves not only resistnce to desicction but lso the cpcity for recovery. We were therefore interested in the cpcity of the flies to resist desicction nd their cpcity for osmotic recovery. We therefore exmined recovery on vrious fluids. Recovery in the control popultions Recovery on distilled wter. Following n 8 h bout of desicction, the C popultions lost lmost 60% of their hemolymph volume yet did not increse their hemolymph osmotic concentrtion significntly. During 24 h recovery period on distilled wter, the C popultions were ble to increse hemolymph volume to pre-desicction vlues. Hemolymph osmollity fter rehydrtion on this fluid ws lso returned to pre-desicction vlues. It follows tht the flies must hve obtined osmolytes from the body comprtment in order to replce hemolymph volume t the pproprite osmotic concentrtion. Folk nd Brdley (3) exmined the chnges in ion content of the C popultions under identicl conditions of desicction. They found tht the flies excrete some sodium during desicction but retin pproximtely 85% of whole body sodium content, 83% of potssium nd 60% of chloride. A detiled study of the loction of these ions following desicction nd the degree to which they re mobilized upon
Osmotic regultion in Drosophil 2319 rehydrtion hs yet to be crried out. Dipter normlly hve firly sodium-rich hemolymph compred with other insects (Sutcliffe, 1963). It might be expected tht ion mobiliztion, prticulrly tht of sodium, would be mjor spect of hemolymph reconstitution during recovery in Drosophil. The ft body in Periplnet mericn cts s sink for sodium nd potssium ions from the hemolymph during dehydrtion (Hytt nd Mrshll, 1977). Upon rehydrtion on deionized wter, these ions re removed from the ft body nd replced in the hemolymph (Hytt nd Mrshll, 1985). In Drosophil, however, role for other osmolytes, including mino cids, orgnic cids nd peptides, in the rehydrtion process cnnot t this time be ruled out. Dipterns nd other species of insects hve been shown to brek down proteins into osmoticlly ctive mino cids in response to perturbtions in hemolymph osmotic concentrtion (Collett, 1976,b; Woodring nd Blkeney, 1980). Further studies will be required to determine hemolymph composition before nd fter recovery in D. melnogster s well s the source of hemolymph osmolytes. Recovery on sline or sline+sucrose solution. In the C flies, rehydrtion on sline solution isosmotic to the hemolymph resulted in n increse in hemolymph volume following decline during the 8 h desicction period. The restored hemolymph volume ctully surpssed predesicction volume nd ws sttisticlly indistinguishble from tht of flies rehydrted on distilled wter. As in the flies tht recovered on distilled wter, the hemolymph osmollity ws returned to the originl pre-desiccted vlues fter rehydrtion on the sline solution. It is cler tht Drosophil cn fully rehydrte nd mintin osmotic concentrtion using only sline without supplementl energy source. Hemolymph volume subsequent to bout of dehydrtion nd recovery hs been shown to increse beyond levels of pre-stressed vlues in the orthoptern Chortoicetes terminifer (Djjkusumh nd Miles, 1966). When the control popultions were rehydrted on sline+sucrose solution, hemolymph volume ws higher thn pre-desicction vlues. Clerly, Drosophil cn restore wter lost from the hemolymph by the consumption of fluids of vrible composition. Hemolymph osmollity ws lso restored fter recovery on the sline+sucrose solution. Recovery in the popultions selected for enhnced desicction resistnce Following 24 h bout of desicction, the D popultions hd lost on verge 66% of their hemolymph volume nd incresed their hemolymph osmollity by ~100 mosm. When provided with ny of the three recovery fluids, the D flies were ble to return to hemolymph volume sttisticlly indistinguishble from the initil hemolymph volume. The finl hemolymph volumes chieved were intermedite to the pre-desicction nd post-desicction levels. Although the flies did not fully recover lost hemolymph, they did mnge to regin their originl hemolymph osmollity fter recovery on distilled wter. Like the C popultions, the D popultions cn replce substntil volumes of hemolymph t the pproprite osmollity while imbibing only distilled wter, s well s by drinking isosmotic sline or sline+sucrose solutions. Clerly, their cpcities for osmotic regultion of the hemolymph re substntil nd the flies re cpble of deling with vriety of environmentl conditions. Osmoregultion in Drosophil Drosophil fce intermittent desicction in their norml environment. Competition for mtes nd serching for food nd oviposition sites inevitbly led the insects wy from dietry sources of wter. Their exceptionlly smll surfce re to volume rtio excerbtes wter loss, with rtes of wter loss of 20 30 µl g 1 h 1 being reported for control popultions in the lbortory (Gibbs et l., 1997; Willims et l., 1998) under non-flying conditions. Lehmnn (1) mesured the rte of wter loss in D. melnogster during flight nd found tht, s metbolic demnd incresed, spircles must be open more frequently nd the rte of wter loss increses ccordingly (60 140 µl g 1 h 1 ). The present study ws designed to determine the degree of osmotic regultion tht occurs in Drosophil during the periods of wter loss, s well s during the rehydrtion events tht must occur when the insects gin encounter source of wter. We found tht Drosophil disply surprisingly strict osmotic regultion under conditions of dehydrtion, being ble to regulte osmotic concentrtion when over two-thirds of the hemolymph volume hs been lost. Similrly, recovery of hemolymph volume cn be chieved with vriety of recovery fluids, including distilled wter. Neither externl sources of sodium nor energy in the form of sugr re required. This implies tht Drosophil could rehydrte in the wild using vriety of sources of wter such s rinwter or dew, nectr nd the fruit juices ssocited with their oviposition sites. Hoffmnn (1990) reported n cclimtion response in D. melnogster when subjected to non-lethl dry environment. Subsequent to this temporry bout of desicction, the flies becme more resistnt to further desicction stress. We now know tht this species osmoregultes; therefore, it would be interesting to determine the pttern of osmoregultion during the second bout of desicction. Potentilly, this increse in desicction resistnce could result from higher tolernce to the elevtion of hemolymph osmotic concentrtion (due perhps to the presence of het shock proteins; Lindquist, 1986), decrese in cuticulr wter loss, n increse in body wter during the recovery phse or nother physiologicl mechnism. When Hoffmnn (1991) crried out these experiments on vrious field-collected Drosophil species of differing hbitts, he found tht the cclimtion response ws well estblished in Drosophil with the exception of the species D. birchii, which is found exclusively in tropicl rinforests. It would be interesting to mesure physiologicl mechnisms behind this result nd to determine other osmoregultory differences in this tropicl species. In the course of exmining osmotic regultion in Drosophil, very interesting new questions hve risen. Of the popultions of Drosophil exmined in this study (both control
2320 M. A. Albers nd T. J. Brdley nd selected), ll re ble to replce hemolymph lost during desicction using only distilled wter while osmoregulting, suggesting tht the flies cn restore this hemolymph using internlly stored or produced osmolytes. Clerly, this result deserves further study. The processes by which these two osmotic strtegies, osmotic regultion nd volume homeostsis, re mintined in Drosophil re of considerble interest, given the extensive physiologicl, moleculr, genetic nd now genomic techniques vilble for their investigtion. This work ws supported by Ntionl Science Foundtion grnt IBN 0079501 wrded to T.J.B. References Ashhurst, D. E. (1985). Connective Tissues. In Comprehensive Insect Physiology, Biochemistry nd Phrmcology, vol. 3 Integument, Respirtion nd Circultion (ed. G. A. Kerkut nd L. I. Gilbert), pp. 249-287. Oxford: Pergmon Press. Bosquet, G. (1977). Hemolymph modifictions during strvtion in Philosmi cynthi wlkeri (Ferber) I. 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