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Magnesium regulation in Aedes campestris larvae Kiceniuk, Joe Willie 1971

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MAGNESIUM REGULATION IN AEDES CAMPESTRIS LARVAE  by  JOE WILLIE KIC2NIUK B.Sc,  University of Alberta, 1969  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Zoology We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October, 1971  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r  an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y  a v a i l a b l e f o r r e f e r e n c e and study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e  copying of t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s .  I t i s understood that copying or p u b l i c a t i o n  of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my written permission.  Department o f  Zoology  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  Date  December 2, 1971  i ABSTRACT Regulation of hemolymph and whole-body Kg"*"" concentration was 1  studied i n the larvae of Aedes campestris Dyar and Knab from a s a l t lake containing 190 m Eq Mg**/litre.  Kemolymph Mg** concentration of  the larvae responded quickly to a change in external Mg+t concentration and reached a new level after one day.  Over a wide range (0.02 to  ,|.|  200 m Bq/litre) of external Mg  concentrations the- blood Mg++  concentration changed only from 4 to 8 in B q / l i t r e . of Mg  The rate of entry  into the larvae by drinking was 19 to 57 n Eq/rag x hr.  Drinking  rate was found to be independent of temperature (10C- 22C), Kg** concentration (100- 300 m E q / l i t r e ) , and presence of particles in the medium.  More than 95$ of the ingested Mg*"*" was absorbed from the  midgut.  Whole-body Mg  content of larvae remained low> indicating  that excess Mg** was not stored i n tissue.  Measurement of urine Mg**  concentrations of animals i n different media showed that excretion via urine could account for a l l of the ingested Mg"*". 1-  Anal papillae need  not therefore be implicated i n Mg"***" excretion i n Aedes campestris larvae.  ii TABLE OF CONTENTS PAGE 1  INTRODUCTION  4  MATERIALS AND METHODS Animals  4  Media  4  Handling of larvae  5  Washing and drying  5  Sampling o f hemolymph  5  Kg*"*" determination  5  Wet  6  ashing  Evaluation of the use of P V P I ^ f o r drinking rate determination  6  Drinking rate  7  Mg  8  12  absorption i n the gut  Weighing  8  Urine sampling  9  Ashing of parts of the excretory system  10  Ligations  11  M o r t a l i t y curves  11  RESULTS  12  Blood Mg"*"  12  Mortality  12  Drinking rate  16  Relative r a d i o a c t i v i t y of various components of the larvae exposed t o PVPI - f o r 40 hrs  16  Mg** uptake  22  Body Mg"*"  22  1  25  1  -j |„  Urine Mg  concentration  24  iii PAGE Mg  26  content of parts of larvae  ++ Permeability of body wall to Mg  26  DISCUSSION  31  LITERATURE CITED  36  APPENDIX  38  iv LIST OF TABLES TABLE I II III IV  PAGE Distribution of PVPI 5 i n larvae  17  Whole body Mg  23  Mg""* concentration of excretory products  25  Mg"*"*" content of parts of larvae  27  12  1  LIST OF FIGURES FIGURE 1  PAGE Changes i n blood [Mg ] with time f o l l o w i n g t r a n s f e r of animals from normal Ctenocladus pond water (190 m E q / l i t r e ) t o media of d i f f e r e n t [Mg++]. The steady-state r e l a t i o n s h i p between hemolymph [Mg ] and medium [Mg" "].  13 14  |  4-1  3  M o r t a l i t y among f o u r t h i n s t a r larvae during hemolymph [Mg ] experiment ( F i g . l ) .  15  4  E f f e c t o f temperature on d r i n k i n g r a t e i n normal Ctenocladus pond water u s i n g PVPI125 as an i n d i c a t o r .  19  5  E f f e c t o f presence o f p a r t i c l e s on d r i n k i n g r a t e as measured .using PVPT.125 as an i n d i c a t o r .  20  6  E f f e c t o f [Mg* "] on d r i n k i n g r a t e a t 10 C.  21  7  T o t a l Mg l o s s from animals i n d i v i d u a l l y confined i n 1 u l of d i s t i l l e d water under o i l .  28  8  Mean Mg++ l o s s /animal from 20 animals i n 1 m l o f d i s t i l l e d water.  29  9  Percent m o r t a l i t y o f f i r s t i n s t a r larvae i n : A. Ctenocladus pond water [Mg**] = 180 m E q / l i t r e B. Pure s o l u t i o n of MgSO^ (200 mM/litre)  39  10  4  Per cent m o r t a l i t y o f f i r s t i n s t a r larvae i n : A. Pure s o l u t i o n of MgSO; (250 mM/litre) and NaCl (50 mM/litre) B. Pure s o l u t i o n o f MgSO (250 mM/litre), KCO3 (1 inM/litre) a n d C a C l (0.25 m M / l i t r e ) .  40  4  2  11  Per cent m o r t a l i t y o f : A. T h i r d i n s t a r larvae i n pure s o l u t i o n of MgSO; (200 mM/litre) B. F i r s t i n s t a r larvae i n pure s o l u t i o n o f MgSO^ (250 mM/litre) and NaHC03 (5 mM/litre)  41  ACKNOWLEDGEMENTS I wish to thank my supervisor, Dr. John P h i l l i p s , for his guidance during this study.  I thank Drs. G.G.E. Scudder, A.B. Acton,  and T.H. Carefoot for their criticism of the manuscript.  I also  gratefully acknowledge the assistance of Miss Joan Meredith with the tracer experiments and my wife with the typing of the manuscript.  INTRODUCTION Maintenance of salt and water balance i n insects has been a much investigated problem i n view of the variety of different habitats these animals occupy.  The larvae of many species of insects are aquatic;  most of them inhabit fresh water, but several are adapted to l i f e i n hypertonic media (Shaw and Stobbart, 1963).  Probably the most studied  aquatic larvae with regards to ionic and osmotic regulation are those of mosquitoes, particularly of the genus Aedes. Most mosquito larvae inhabit fresh water and as i n a l l fresh water organisms they gain water by osmosis and lose ions by diffusion across permeable parts of the body wall.  However several species live  i n a l k a l i lakes and salt marshes where the external medium may vary widely in chemical composition and t o t a l solute concentrations (Topping, 1969j Scudder, 1969; Beadle 1939).  The external osmotic pressure in  fact may exceed that of the hemolymph by two or three fold.  Both  fresh-water and salt-water larvae are faced with the problems of maintaining the osmotic pressure of their hemolymph at the required level and of maintaining the ions i n their tissues at suitable concentrations, and at proportions different from those encountered i n their environment.  The organs responsible for this regulation include  the excretory system (Malpighian tubules, hindgut, and rectal system) and extra-renal mechanisms located i n the anal papillae (Shaw and Stobbart, 1963). Wigglesworth (1933)> working with Aedes aegyptiL. found that the anal papillae were very permeable to water, whereas the rest of the animal was not.  He also showed that these larvae drink negligible amounts of  water with their food.  Anal papillae of A. aegypti larvae have since  2 been shown to be the site of active uptake of Na (Stobbart, 1959* +  1960$ Treherne, 1954) and K+ (Ramsay, 1953) from fresh water.  The  uptake of Na has been shown to take place independent of CI- uptake, while K+ uptake is associated with CI" (Stobbart, 1967). On the other hand, A. detritus Edw., a brackish water mosquito, was found to take up water via the gut and, unlike A. aegypti, is able to osmoregulate i n hyperosmotic media by the production of strongly hyperosmotic urine (Beadle, 1939). Aedes campestris Dyar and Knab larvae have been found to be capable of survival i n a wide range of concentrations of media after a suitable period of adaptation. Ion-depleted larvae took up Na and C l ~ from dilute media via the anal +  papillae, since blockage of the mouth had no effect,but ligation of the anal papillae abolished their uptake (Phillips and Meredith, 1969). The implication of anal papillae i n regulation i n hyperosmotic media has been suggested by the latter authors, but this role is not yet clearly defined. Due to the absence of good ultramicro techniques for the measurement of low concentrations of divalent inorganic ions, l i t t l e work has  ++ been done on the regulation of Ca either larval or adult. .. j j of Ca  and Mg  ++ and Mg  i n any group of .insects,  Clark and Craig (1953) measured the concentration  i n the hemolymph of a number of insects and found for Mg" " 1-1  a range of 0.012 /ig/mg i n Vespula pensylvanica (adult) to 0.673 pg/mg i n Laphygma exigua larvae.  No studies have been done to determine how  hemolymph Mg levels respond to changes i n external Mg""" concentration. ++  1 4  Wigglesworth (1931) found Mg"*"*" i n the excreta of Rhodnius i n the late stages of digestion of a blood meal.  Ramsay (1956) found that Mg** was excreted  by the Malpighian tubules of the stick insect Dixippus morosus (1956).  3 Investigation of the fauna of s a l i n e lakes of B.C. by Scudder (1969) and the chemical analysis of these lakes by Topping (1969) and B l i n n (l97l) have shown that Aedes campestris larvae inhabit some a l k a l i lakes i n B.C. which have high Mg  content (up t o 395 m B q / l ) .  This  information, along with the findings of P h i l l i p s (unpublished) that these animals have a high drinking rate (15-100$ of body weight per day), suggested that these animals must e i t h e r : 1) have some unusual regulatory mechanism which permits them to maintain t h e i r Mg 2)  content at constant and low p h y s i o l o g i c a l l e v e l s  e x h i b i t an unusual tolerance of high blood Mg^+levels  3) prevent entry of Mg* " i n t o the hemolymph by reducing drinking rates 4  j j  or by impermeability of the gut to ingested Mg  or both.  With these three p o s s i b i l i t i e s i n mind, experiments were undertaken f i r s t t o determine the degree of r e g u l a t i o n of hemolymph Mg i n larvae a f t e r r a p i d t r a n s f e r i n t o media of d i f f e r e n t Mg++ concentrations  ++  . (to t e s t hypothesis 2 ) .  A f t e r f i n d i n g that the hemolymph Mg  remained  ++ independent of external Mg t o determine whether Mg-H- was  concentrations, experiments were conducted excluded from the larvae by reduction of  drinking rate and/or whether the larvae were impermeable to Mg (hypothesis 3). was  In these l a t t e r experiments i t was  permeable t o Mg  and that a large amount of Mg  the hemolymph i n larvae subjected to high Mg  found that the gut was taken i n t o  concentrations.  Further experiments were therefore undertaken t o determine the steady state regulatory mechanisms responsible f o r maintenance of constant -H-  hemolymph Mg concentration. larvae?  ++  concentrations i n animals l i v i n g i n media with high Mg In other words what i s the fate of absorbed Mg  i n the  4 MATERIALS AND METHODS Animals Aedes campestris larvae were collected i n late April and early May from Ctenocladus pond (located 12 miles west of Kamloops on Highway l ) , using a dip net.  Large numbers of larvae were placed i n  one gallon insulated picnic jugs for transportation to the laboratory at U.B.C. Water from the pond was f i l t e r e d through glass wool and transported i n five gallon polyethylene carboys.  In the laboratory  the animals were sorted into plastic trays containing normal pond water, where they were stored i n a constant temperature cabinet at 10 C £ 0 . 5 C. Natural particulate material i n the water provided food for the larvae i n a l l cases except for larvae treated i n a r t i f i c i a l media, i n which case tropical f i s h food was used. The density of animals i n storage containers was approximately 2 0 / l i t r e , which i s less than that i n the pond (about 4 0 / l i t r e ) .  Media Ctenocladus water on May 7» 1970 had a conductivity of 28 m mho (22 C) with a Mg** content of 190 m Eq/litre and a Ma content of 350 +  m Eq/litre.  The water temperature at the time of collection of fourth  instar larvae ranged from 10 C at night to 19 C at midday. The pH of the water was 8.8.  On the date of collection i n 1971 (May 4) the  conductivity was 25 m mho (at 22 C), the water temperature was 18 C at noon, and the pH was 8 . 9 . The Mg concentration was 170 m Eq/litre and the Na concentration was 330 m Eq/litre. +  5 Handling of larvae Individual larvae were usually transferred with a medicine dropper with a glass tube large enough to accommodate large larvae. Transfer of large numbers of larvae was accomplished by using a nylon tea strainer.  When dry larvae were to be handled (individual animals)  they were picked up by the anal siphon using needle-pointed forceps with minimal pressure.  Washing and drying Larvae were floated i n a nylon tea strainer i n an overflow bucket through which tap water was circulated.  After rinsing for  3 min, the larvae were pipetted onto clean paper towelling and were dabbed dry with clean "Scotties".  Sampling of hemolymph After washing and drying, animals were individually transferred to clean Parafilm, broken open with needle-pointed forceps (being careful not to rupture the gut), and hemolymph was taken up into a 1 Ail Drummond disposable pipet.  I|  Mg  determination A commercial standard solution (Harleco) containing 80 m Eq/litre  Mg  was diluted with 3% EDTA (hereafter referred to as swamp) to  produce five standards i n the range of 0 to 80 p. E q / l i t r e Mg .  All  samples were diluted i n swamp solution ( l ml for hemolymph samples) contained i n polyethylene vials and run on a Techtron model AA 120 absorption spectrophotometer (Varian) as directed i n the instrument manual.  6 Wet ashing. To measure t o t a l body or tissue Mg  , weighed material was  first  dried overnight i n an oven at 60 C i n s c i n t i l l a t i o n vials made of borosilicate glass. The vials and contents were then placed i n a sandbath on a hotplate and 0.1 ml of modified Pirie's reagent (Pirie, 1932), consisting of one volume of 60% perchloric acid i n three volumes of concentrated n i t r i c acid was added to the v i a l s .  The latter were  heated u n t i l white fumes began to form, at which point more concentrated n i t r i c acid was added to prevent explosion due to production of perchlorates.  The vials were further heated to 260-280 C u n t i l a dry  white residue was formed.  The residue was then dissolved i n swamp  solution for determination of Mg  Evaluation of the use of P V P I  125  as previously described.  for drinking rate determination  F i f t y fourth instar larvae were placed in 50 ml of the following a r t i f i c i a l medium: 200 mM NaHC0„/litre 50 mM MgSOi/iLtre 5 mM KCl/litre 2 mM  CaCl Ai  t r e  2  0.0625 mC PVPI (Polyvinyl pyrrolidone 1125/50 ml 125  After 5 min animals were removed, washed i n running tap water for 5 min and the following samples were taken, placed i n planchets, dried and counted: background (blank) external medium 1 jul hemolymph whole larvae After one day i n the medium the same sampling pattern was repeated.  Forty  hours after i n i t i a l exposure to PVP some animals were removed, washed and  7  dissected in non-radioactive medium as follows: The head was removed, body s l i t dorsally and the gut separated from the body.  The gut was cut anterior to the valve in front of the crop  (resulting i n no loss of contents).  The gut, along with the attached  Malpighian tubules and anal segment, was washed in twenty separate drops of a r t i f i c i a l medium., Cuticle (remainder of animal) left after this operation was washed i n the same way and counted.  After the  gut was washed using the above procedure i t was placed in a planchet, broken open and the peritrophic membrane and contents removed to be counted separately.  Drinking: rate Groxips of f i f t y animals were placed in 1 ml of various experimental media to which 0.0625 mC of polyvinyl pyrrolidone (mol wt 30,000) labelled with 1125 (PVPI125) obtained from New England Nuclear had been added.  The animals were rinsed before introduction into the media.  Immediately after introduction into the medium, ten animals were removed, rinsed, weighed and broken up individually i n several drops of water i n planchets.  The samples were then dried under a heat lamp.  The t o t a l dry weight of material per planchet did not exceed 3 mg, which i s too small to cause appreciable self absorption of I  1 2 5  .  This  i n i t i a l determination gave a maximum estimation of error due to surface contamination with T.125. The same sampling procedure was followed at various times after adding animals to radioactive media. The drinking rate could therefore be calculated from the increase i n radioactivity per larva per unit time and from the activity of the external medium per unit volume.  Temperature and salinity-adapted  8 animals were used i n a l l parts of this experiment.  All  samples  were counted on a Nuclear Chicago Model 1042 planchet counter.  MR absorption in the gut Animals were put into normal Ctenocladus water containing pvpjl25  f  o  r  30 hours (Group I) and 6 days (Group II).  At the end of  exposure the animals were removed by pipet, rinsed in running tap water, the whole midgut dissected out, rinsed i n ten changes of d i s t i l l e d water, blot-dried i n f i l t e r paper, weighed and placed i n 4 ml of swamp to measure the external activity (PVPI-^5) and Mg"*""'" concentration.  After 8 days at room temperature to permit breakdown  of the wall and release of PVP, the midguts were broken up with forceps.  One ml was removed from each sample v i a l , dried and  counted for r ^ 5 activity; the remainder of each sample was used for determination of Mg** concentration. calculated for the samples.  The Mg^/PVP ratios were then  Mg^/PVP ratios for Groups I and II were  not different, so the results were pooled.  The Mg /PVP ratio i n  the external medium was similarly determined.  If no absorption of  -HMg occurred i n the gut the external and internal Mg /PVP ratios +  +  should be the same.  Weighing A l l weighings of animals and parts of animals were done on a 50 mg torsion balance (Sauter).  Utmost care was taken to ensure  that the balance was properly zeroed and standardized at a l l times to insure accuracy.  Animal parts were kept i n humidity chambers or  under o i l to prevent loss of weight due to evaporation prior to or  9 during the weighing operation.  Urine sampling; I  To determine whether Mg^" was regulated via the Malpighian 4  tubules or the rectum, urine samples were taken i n the following manner:  Several hundred animals were acclimated for 1 week i n  each of five media ranging i n Mg litre.  concentration from 2 to 170 m Eq/  After one week of acclimation, larvae were removed as required,  dabbed dry on tissue paper and placed under paraffin o i l i n a siliconized petri dish ( a l l glassware used was siliconized).  At  f i r s t , attempts were made to collect urine directly as i t was released, but i t was found that the urine clung so tenaciously to the fecal pellets that i t was impossible i n the available time to get samples large enough to centrifuge (aboxit 3/4>il)«  Centrifugation was found  to be necessary because particles of fecal material i n the urine produced unreplicable results.  A technique was developed using a 30 jul  disposable pipet broken off at a 45° angle and a small glass rod to collect the fecal pellets. The pipet was f i r s t p a r t i a l l y f i l l e d with paraffin o i l , then fecal pellets were transferred under o i l into the pipet.  When  enough fecal material was collected, the 30 ,ul pipet was sealed with beeswax, f i t t e d inside a disposable hematocrit tube, and centrifuged for 20 minutes i n a Clay-Adams hematocrit centrifuge.  The 30 u l  pipet was then removed, cut off above the u r i n e - o i l interface, and mounted i n a micromanipulator i n a horizontal position. A siliconized micropipet, made by drawing out a Drummond 1 u l disposable pipet i n a bunsen flame to y i e l d a pipet of about 0.25 u l  10 volume, was mounted i n a glass tube with beeswax, and held horizontally in another micromanipulator.  The micropipet was then fitted with a  rubber tube and micrometer syringe.  Under a microscope the pipet was  manoeuvered into the sample pipet, through the o i l into the urine sample. With adequate care, samples thus taken contained no o i l or particulate fecal matter.  The sample of urine was then delivered from  the micropipet into 1 ml of swamp solution along with several rinsings from the pipet.  After washing the micropipet, a sample of external  medium was taken (using the same pipet) and diluted i n 1 ml of swamp.  Ashing of parts of the excretory system Normal animals were removed from Ctenocladus water, dabbed dry and dissected as follows:  The head was f i r s t removed; then, holding  the body with one pair of forceps, the anal segment and gut were pulled from the animal.  In this manner the gut and attached Malpighian  tubules were removed and divided into parts i n the following categories: The midgut usually shrank so that the contents within the peritrophic sheath protruded from i t .  The contents could be picked up  easily and were pooled from many animals on a piece of Parafilm set on a wad of cotton i n a covered petri dish. The Malpighian tubules were pulled off and, after removal of excess hemolymph, were pooled on Parafilm, as were the midgut contents above. The rectum was removed from the remainder of the gut and from the anal segment and pooled with the other recta i n a petri dish as described above.  A l l of these dissections were done using the hemolymph of the animal as a dissecting medium.  To check that serious overestimation  of Mg"*"*" did not result from dehydration of tissues during collection and pooling of the latter, some determinations were repeated on organs which had been transferred directly to, and stored under, o i l .  Ligations Animals were ligated where necessary using washed human hair tied on the animal with a double knot.  Mortality Curves Three repetitions each consisting of 25 animals were done for each of the different media and instars used.  The larvae i n each repetition  were transferred gradually from normal Ctenocladus pond water to the test solution by increasing the proportion of test solution on each of four days i n the following proportions- 1/4,1/2, 3/4 and f u l l strength test medium.  The number of dead was counted each day and  the t o t a l accumulated. (number dead X 100/25 ).  Per cent mortality was then calculated The average (of three repetitions) mortality  for each day was then graphed (See Appendix).  12 RESULTS Blood Mg"*~*" To determine the degree of regulation of hemolymph Mg"*""" and 1  the range of external Mg  concentrations over which fourth instar  larvae are able to regulate hemolymph Mg"*"*", the following experiments were carried out:  Animals were rapidly transferred from Ctenocladus  water to various media prepared by diluting the latter with d i s t i l l e d water or by addition of MgSO^.  Twice as many animals were used as  required for sampling to allow for mortality.  At various times after  transfer, ten animals were removed from each medium for estimation of hemolymph Mg"*~*" concentration, and sampled. F i g . 1. Mg  The results are shown in  The blood Mg"*""*" levels respond quickly to the change in medium  (within one day) and stabilize thereafter.  level of blood Mg  When the steady-state  (level after 4 days) is plotted against external  concentration (Fig. 2) i t is clearly evident that these larvae exhibit a remarkable degree of regulation.  Over a 10,000-fold range of external  concentrations of Mg"*" (0.02 to 200 m E q / l i t r e ) the blood Mg"*~*" 4  concentration does not increase significantly from 6 m E q / l i t r e .  Mortality During this experiment mortality of remaining (unsampled) animals was recorded (% dead of remaining animals).  F i g . 3 shows that mortality  was high, but remained more or less constant for a l l media in the range of 23 p. E q / l i t r e to 190 m E q / l i t r e . not survive a molt i n 23 p. Eq medium. survived indefinitely.  It was observed that larvae could In the other media some larvae  FIGURE 1 Changes i n blood [Mg*"] with time following transfer of animals from normal Ctenocladus pond water (190 m Eq/litre) to media of different [Mg ] 4  O animals i n 190 m E q / l i t r e (normal Ctenocladus water) •  animals i n 100 m E q / l i t r e (Ctenocladus water diluted 2X)  •  animals i n 300 m E q / l i t r e (Ctenocladus water + 100 m Eq MgSO/j/litre)  •  animals i n 7 . 5 m E q / l i t r e (Ctenocladus water diluted 100X)  Vertical bars indicate standard error of the mean of ten animals  FIGURE 2 The steady state relationship between hemolymph [Mg medium [Mg**].  ] and  Dotted line is the isotonic line Vertical bars indicate standard error of the mean of ten animals  FIGURE 3 Mortality among fourth instar larvae during hemolymph [Mg*"] experiment (Fig. 1) 4  T 23 p. Eq M g + V l i t r e (Ctenocladus water diluted 1000X) • 100 m Eq M g ^ / l i t r e (Ctenocladus water diluted 2X) • 190 m Eq M g ^ / l i t r e (normal Ctenocladus water) O 300 m Eq M g ^ / l i t r e (Ctenocladus water + 200 m Eq MgSO/yiitre)  % MORTALITY/DAY  Drinking rate Having obtained evidence that the Mg* " level of the hemolymph 4  -Hwas constant, work was done to determine whether Mg i s excluded 131 from the body by depressed drinking rate.  PVPI  has been employed  by other authors (Smith, 1969) to determine drinking rate i n an aquatic species (Artemia salina), but i t has not been used to measure drinking rate i n a mosquito larva.  It was therefore f i r s t necessary  to evaluate the application of this method to mosquitos. Relative radioactivity of various components of the larvae exposed to PVPI125 for 40 hrs Table I shows the breakdown of the results.  It is evident  that about 78$ of the J~^^ i s contained i n the gut and almost none passes into the hemolymph,(A .small fraction of unbound l l 2 5 would account for the latter a c t i v i t y . )  The fact that almost a l l of this  j_125 as confined to the gut contents means that PVPI125 is a good W  indicator of drinking rate i n these animals. In addition to concentration of the medium, temperature and presence of microorganisms may affect the determination of drinking rate.  Temperature could possibly affect the animals' activity.  Microorganisms could possibly ingest PVP and concentrate 1^25. larvae might then concentrate the microorganisms. to produce high estimates of drinking rate.  The  This would tend  In view of these poss-  i b i l i t i e s i t was decided to make drinking rate measurements under the following conditions: 1. 2. 3. 4. 5.  normal Ctenocladus pond water at 10 C (Experimental temp.) normal Ctenocladus pond water at 22 C (Room temp) Millipore-filtered Ctenocladus pond water at 10 C 300 m E q / l i t r e Mg** medium at 10 C 100 m E q / l i t r e Mg*" medium at 10 C 4  TABLE I Distribution of PVPI125 i n larvae Sample  PVPI Activity (cpm± SE) 40 hrs 3 min 125  Background  12± 0.58  9.7 -  External medium ( l pl)  173 - 1.7  240 ± 2.65  Hemolymph ( l pl)  72 - 14-7  1 6 . 7 ± 2.19  Whole body  2950 i 350  54 * 13.0  Whole gut including contents  2300 ± 230  Whole gut without contents  333 ± 3 3 . 3  Whole gut contents  3533 ± 8 1 9 . 2  Animal excluding gut  423 i  78.8  0.88  18 Temperature and salinity-adapted animals (one week at experimental conditions) were used i n a l l cases.  Drinking rates of animals i n  ordinary Ctenocladus pond water at 10 C and 22 C are compared i n F i g . 4« There was a large individual variation i n uptake of the medium, but no difference seems evident i n the drinking rate of animals at the two different temperatures.  The slope of the line for the pooled data  gives an average drinking rate of 0.1 to 0.3 ul/mg X hr, or approximately 500$ of t o t a l body weight (4-6 mg) per day (assuming that the rate does not undergo any diurnal rbythmn. F i g . 5 shows the comparison of drinking rate determination i n Millipore-filtered and ordinary Ctenocladus pond water.  No difference  i s evident between the two experimental conditions; ie the drinking rates seem i n agreement with the previous estimate. F i g . 6 shows the results of drinking rate determinations i n media of different concentrations.  From the latter results i t seems reasonable  to assume that drinking rate does not change i n response to concentration. In conclusion, the drinking rate by fourth instar larvae does not seem -H-  to be affected by temperature i n the range of 10 C to 22 C, or by Mg concentration over a range of 100 to 300 m E q / l i t r e .  Moreover, the  high estimates of drinking rates obtained by the PVP method are not due to experimental error associated with concentration of PVPI^^ i n a food source of the larvae (eg bacteria).  These results show that  the larvae are ingesting large amounts of Mg**", therefore negating 4  the possibility that low blood Mg"*" concentration is due to reduced 4  entry of Mg  into the hemocoel as a consequence of lower drinking rate.  FIGURE 4 Effect of temperature on drinking rate i n normal Ctenocladus pond water using PVPI125 as an indicator. •  10 C  A  22 C  Vertical bars indicate standard error of the mean of ten animals  HOURS AFTER TRANSFER INTO PVP SOLUTION  FIGURE 5 Effect of presence of particles on drinking rate as measured using PVPI125 as an indicator. •  animals at 10 C i n Millipore-filtered Ctenocladus pond water  A  animals at 10 C i n nonfiltered Ctenocladus pond water  Vertical bars indicate standard error of the mean of ten animals  FIGURE 6 Effect offrig*" "]on drinking rate at 10 C 1  T animals i n 190 m Eq M g ^ / l i t r e (Ctenocladus pond water) •  animals in 300 m Eq M g ^ / l i t r e (Ctenocladus pond water + 100 mM MgSO^/litre)  •  animals i n 100 m Eq M g + V l i t r e (Ctenocladus pond water diluted 2X)  Vertical bars indicate standard error of the mean of 10 animals  21  HOURS  MR*"*" uptake Since the drinking rate and consequent fluid absorption in the gut (observed lack of fluid)were found to be high (Fig. 4-6), the question remained:  Is the gut wall permeable to Mg , or is Mg" " 1-1  accumulated within the gut so that i t is excreted without ever entering the hemocoel?  To answer this question, gut absorption of Mg"**" was -  estimated by comparing the PVPll25/Mg++ ratio in the medium with that of the midgut contents.  Since PVP is not significantly absorbed in j j  the gut, this ratio should not change i f Mg  i s not absorbed. The  Mg^/PVP ratio for midgut contents was 0.0606 ± 0.0086 (n = 19) and (n = 3 ) . This indicates that  for the external medium 7.6-0.26  virtually a l l of the ingested Mg"**' was absorbed -  from the midgut,  and thus entered the hemolymph. Body MR"*"*"  Since Mg  was shown to be ingested rapidly i n concentrated  media and was found to be almost completely absorbed from the midgut, while the hemolymph Mg"**" concentration remains the same, excretion or -  storage in other tissues or both must be taking place. To determine ++ whether absorbed Mg was stored in the larvae, whole animals ++ acclimated to different Mg  concentrations for a minimum of 7 days  were ashed and their Mg"*"*" content measured (Table.II). The levels of Mg""*" i n these experimental larvae are low compared 1  to the Mg  levels in normal Ctenocladus water (about 50 m Eq/litre  body water, compared to 200 m Eq/litre for Ctenocladus water).  For  a forty-fold change in external concentration the total body content j j  of Mg  changed less than two-fold; this reflects the regulatory  TABLE II -4-4-  Whole body Mg  Acclimation Medium [Mg"""] (m E q / l i t r e )  |  |  Whole body Mg content n Eq Mg*'*' SE ~ mg body vrt  6.5  29.5 *  2.65  10  100  38.6 ±  4.88  5  200  51.4 -  4-03  14  250  43.1 -  2.9  10  a b i l i t i e s indicated by studies of blood Mg"~" levels. t t  For example,  j ,|  in Ctenocladus water (170 m Eq Mg / l i t r e ) , using the conservative value for drinking rate of 0.1 u l Mg"*""*"/hr, the entire body Mg"*" 4  content appears to turn over i n just under 3 hours. Urine Mg  concentration  Having established that Mg  is absorbed but not accumulated  in any tissue, i t follows that this ion must be excreted at the same rate as i t is absorbed.  The possible sites of excretion are:  1. anal papillae 2. Malpighian tubules 3. rectum To determine whether Mg"*"*" was regulated via the Malpighian tubules or the rectum, urine samples from animals adapted to different Mg"*" concentrations were analysed. 4  urine  The concentration of Mg++ i n  (urine/medium- U/M ratios- Table III) was higher than that i n  the medium i n a l l cases, as expected i f the excretory system was playing a regulatory role.  The U/H (urine to hemolymph) ratios (Table III)  increased drastically from 0.7 to 23.0 as the external concentration of Mg"*" was increased from 2 to 170 m E q / l i t r e . 4  Furthermore, the Mg " 44  concentration i n fecal material is high under a l l conditions, possibly due to binding to fixed charges i n the latter. Excretion of Mg+'t" by anal papillae could not be studied directly, as no techniques have been developed to date, owing to the small size and f r a g i l i t y of anal papillae.  However, simple calculations indicate  that elimination of Mg"*" by the excretory system alone balances the 4  rate of entry by drinking (see  discussion).  1  TABLE III Mg Media [Mg**] in E q / l i t r e  1  concentration of excretory products  Urine [Mg*"] m Eq/litre 4  Feces [Mg**"] n Eq/mg wet wt 4  U/M  U/H  2  5.6 i 1.34  250 (n = 1)  2.8  0.7  10  22 ± 4.3  353 ± 24.40  2.2  2.3  33  46 ± 3.83  376 i 8.05  1.4  5.2  70  84 ± 5.72  413 ± 68.25  1.2  9.3  170  178 ± 8.01  441 *- 13.36  1.2  23.0  Note:  U/M = urine Mg /medium Mg *" -1-  U/H = urine Mg" ^emolymph Mg H  Where unspecified n = 3  26 Mg"*""*" content of parts of larvae In an attempt t o determine what organ of the body was responsible f o r the Mg"*" excretion, animals were dissected and various parts pooled, 4  •  ii  ashed, and Mg determinations animals.  content determined.  Although the number o f separate  i s small (one or two) each determination involved 20  While the number of observations i s small (Table I V ) , i t  seems quite c l e a r that the Malpighian tubules have a very high of Mg  per u n i t weight.  content  The r e c t a were quite often empty due t o  defecation during handling, p o s s i b l y explaining t h e i r observed low Mg  content.  These r e s u l t s implicate the Malpighian  secretory organs f o r Mg  tubules as  .  Permeability of body w a l l t o Mg"  14-  To determine the extent of movement of Mg"*" across the body w a l l 4  of the larvae, and t o determine the rate of excretion by the whole l a r v a , the f o l l o w i n g experiment was performed:  Mg"*" l o s s from animals 4  l i g a t e d at the anal segment, from animals l i g a t e d at the  penultimate  abdominal segment and from normal (unligated) animals was measured by monitoring the increase i n Mg  concentration of a small volume o f  d i s t i l l e d water i n which i n d i v i d u a l animals were confined under p a r a f f i n o i l (to prevent volume reduction by d e s i c c a t i o n ) .  The Mg  concentration was then p l o t t e d against time (Figs. 7 and 8). The i n i t i a l small loss ( F i g . 7) by animals l i g a t e d a t the anal segment i s probabjy due l a r g e l y t o surface contamination, a f t e r f i v e minutes there i s no net loss of Kg"*" with time. 4  i n d i c a t e s that l o s s of Mg  since This  from the larvae can only occur by  excretion ( or by way of anal p a p i l l a e ) but there i s no Mg  efflux  27  TABLE IV Mg"" content of parts of larvae -  Organ or f l u i d  Midgut contents Malpighian tubules Rectum Hemolymph after dissection  Mg concentration n Eq/mg wet wt collected i n humid collected under colle oil n = 1 chamber  111.0 (95, 127)* 566.7 (443, 690)* 62.4 (65, 60)*  18 (n = 3) n = number of repetitions each using 20 animals * = average, with individual values in parentheses  123 720  FIGURE 7  Total MR *" loss from animals individually confined in 1 u l of distilled water under o i l 4-  v unligated animals •  larvae ligated at the penultimate abdominal segment  O larvae ligated at the anal segment Standard errors were smaller than points (n = 10)  MINUTES  FIGURE 8 Mean Mg** loss/animal from 20 animals i n 1 ml of d i s t i l l e d water  ON  160 f  MINUTES  across the body wall.  This also indicates that probably Mg++ cannot  move into larvae by diffusion across the body wall from media of high Mg  concentration.  The fact that the loss from animals ligated at  the penultimate abdominal segment stops after five minutes i n both non-ligated animals and animals ligated at the penultimate abdominal segment (identical rate) is due to the fact that animals tend to defecate when handled (see discussion).  To investigate the validity  of results for animals confined to small volumes of water under o i l (10 u l i n previous experiment), 20 normal animals were dabbed dry and placed i n 1 ml of d i s t i l l e d water i n a v i a l .  Five u l samples  were taken at various times for determination of Mg** concentrations. The results of this experiment (Fig. 8) were almost identical to those for unligated animals in the previous experiment.  DISCUSSION ++ The investigation of blood Mg  concentrations showed that Aedes  campestris larvae are able to regulate over a wide range of Mg** concentrations as was previously shown for K* and Na i n the same +  larvae (Phillips and Meredith, 1 9 6 9 ) .  This in no doubt a valuable  adaptation for these animals since they are thus able to inhabit very productive (in terms of organic matter) alkaline lakes i n which there are few other species to compete with them (Scudder, 1 9 6 9 ) .  They are  thus able to maintain a dense population in an area where most of the ponds suitable for mosquito breeding have high salt contents. Mortality studies done i n conjunction with blood Mg"*-*" experiments show that there is a lot of variation i n individual animals and that the rate of mortality over a range of 0 . 0 2 m Eq to 300 m Eq remains relatively constant at about 10$ per day.  It is d i f f i c u l t , i f not  impossible, to determine whether the mortality observed i n these animals is a result of a developmental abnormality or whether i t is the result of selection for regulatory a b i l i t y (in this case for Mg**). Preliminary mortality studies on f i r s t instar and third instar larvae (see Appendix) indicate that regardless of how high the i n i t i a l mortality was for animals transferred through different Mg  concentra-  tions to a f i n a l concentration, a few survive indefinitely.  The above  indicates that individuals within the Ctenocladus pond population vary greatly in their a b i l i t y to survive i n media having high Mg concentrations. I feel that I was working in the laboratory with a sample of animals which are representative of the Ctenocladus pond population for  the following reasons:  A great mortality was observed repeatedly  32 amongst the natural population of larvae i n the lake.  The bottom of  the lake was covered with dead larvae i n mid-June 1970.  At this time  some of the animals had pupated and emerged, while others were s t i l l larvae. (i.e.  These remaining larvae seemed grossly shrunken and unhealthy  slow moving) compared to those of earlier i n the summer and to  those of less concentrated lakes.  These last-of-the-season larvae  when taken to the laboratory showed extremely high mortality rates and none pupated.  By this time of year the Mg" " concentration of the 14  lake had increased to 250 m Eq/litre and the water smelled of nitrogenous wastes.  It seems reasonable, therefore, to assume that the animals  used i n experiments are a random sample of the animals i n the lake, and not a selected group.  This does not mean that both of these groups of  animals have not been selected during one generation for those best able to survive under the existing Mg**" concentrations. 4  However, since i t  was also observed that Ctenocladus animals i n late instars survived as well i f not better i n GR2 lake water (high i n NaC03), i t seems that i f there is selection of any sort i t would be for general ionic regulation, not for Mg  specifically.  The high drinking rate i n A. campestris l i v i n g i n Ctenocladus pond, the absorption of Mg " from the gut, and the rapid response of 44  the blood Mg " concentration to changes i n Mg " concentration of the 44  44  I I  medium indicate the necessity for Mg  regulation.  The measurement  of Mg " concentration of urine reveals high U/M ratios (Urine [Mg "]/ 44  44  medium [Mg 3 ) (Table III) which indicate that the larvae are ridding themselves of excess Mg " via the urine. 44  In media o.f lower Mg " 44  concentration than that of blood (the 2 m Eq/litre medium), even though the urine Mg  concentration is lower than the blood Mg  concentration,  (U/H = 0.7), equilbrium connot be achieved by r e t e n t i o n alone since the u r i n e Mg  concentration i s s t i l l higher than the medium Mg""" concen-  t r a t i o n (U/M = 2.8).  The high U/H r a t i o s f o r animals i n hypotonic  medium, along w i t h the high Mg  content o f the feces are i n d i c a t i v e  t h a t t h e animals are perhaps feeding on m a t e r i a l having a higher l e v e l o f Mg  than the water.  This i s a p o s s i b i l i t y since these  animals are bottom feeders and could be i n g e s t i n g m a t e r i a l which has bound Mg  . Another explanation i s t h a t they have a way o f t a k i n g up  Mg"*"- independently o f feeding. 1  Larvae i n d i l u t e media may be capable  of t a k i n g up Mg""" v i a the a n a l p a p i l l a e ( i . e . t u r n i n g on a c t i v e transport).  The t u r n i n g on o f a c t i v e t r a n s p o r t has been observed  f o r N a and C l " ( P h i l l i p s and Meredith, 1969). +  The high Mg " content of feces i s p o s s i b l y due t o binding of -  -  ++ Mg  t o charged groups on p r o t e i n s i n the feces ( f i x e d charges), or  t o incomplete separation of u r i n e and feces (the f e c a l samples may have had a s u b s t a n t i a l amount o f u r i n e i n them).  Another p o s s i b i l i t y  i s t h a t the Malpighian tubules are s e c r e t i n g Kg"*""*" i n some p r e c i p i t a t e d or granular form as i s suggested by t h e i r very high Mg""" content.  On  observation, Malpighian tubule contents appear granular o r c r y s t a l l i n e | j  i n nature.  The Mg  could p o s s i b l y be secreted i n conjunction with  urates as has been reported i n the case o f the u t r i c l e s o f B l a t e l l a germanica ( B a l l a n - D u f r a n c a i s , 1970). The r e s u l t s o f a l i g a t i o n experiment (Figs 7,8) cannot be used t o c a l c u l a t e instantaneous l o s s o f Mg""" from animals on the basis o f the l o s s i n t h e f i r s t f i v e minutes, since i t i a a common observation t h a t these animals tend t o defecate when handled so t h a t r a t e of l o s s of the i o n i s extremely l a r g e during handling.  The s m a l l l o s s o f Mg  from  34 animals ligated at the anal segment i s most l i k e l y due to surface contamination of the animal.  The very rapid i n i t i a l loss ( f i r s t  five minutes) i n the case of both the animals ligated at the penultimate abdominal segment and of normal animals i s most l i k e l y due to defecation brought on by handling. The fact that a l l the loss i n the case of animals ligated at the penultimate abdominal segment occurs within five minutes i s indicative that the animals are emptying the posterior part of the gut a l l at once (defecating).  If this i s the case, then the  next part of the curve for normal animals should represent the rate of loss i n undisturbed animals.  When the latter argument i s used, | j  the calculated rate of loss (240 m Eq Mg /animal X hr) would require a drinking rate of 1.4 ul/animal X hr or 0.28 ul/mg X hr, which i s i n the range of drinking rate determined for these animals.  Since the  amount of water lost osmotically i n these animals via the cuticle i s small (0.3 ul/animal X day or 0.13$ of the amount drunk) (Phillips, unpublished) the Mg  concentration i n urine should be slightly  higher than the Mg" " concentration of the medium i n the case of 1 1  hyperionic media to achieve regulation. media Mg  In actual fact the urine/  concentration ratio i s i n the order of 1.2.  It i s therefore  possible to account for the excretion of a l l of the ingested Mg"*" via 4  the normal excretory process.  In conclusion, i t has been found that  the large amounts of Mg"*" ingested by Aedes campestris larvae living i n 4  Ctenocladus pond water are excreted by the Malpighian tubules. A number of interesting problems have come to mind as a result of this work.  Can the  Malpighian tubules of A. campestris i n fact  secrete ions as crystalline or granular complexes?  Electron microscopy  studies of these Malpighian tubules, as well as isolated preparations  would be most illuminating.  The problem of possible uptake of  ++ and indeed Ca  by anal papillae is also an interesting one.  LITERATURE CITED  36  Ballan-Dufrancois, Cristiane. 1970. Donnees cytophysiologiques sur un organe excreteur particulier d'un insecte, Blatella germanica L. (Dictyoptere). Z. Zellforsch Mikroskop Anat. 109:336-355. Beadle, L.C. 1939. Regulation of the haemolymph i n the saline water mosquito larva Aedes detritus Edw. J . Exp. Biol. 16:346-362. Blinn, D.W. 1971. Dynamics of major ions i n some permanent and semipermanent saline systems. Hydrobiologia 38:225-238. Clark, E.W. and R. Craig. 1953. The calcium and magnesium content i n the hemolymph of certain insects. Physiol. Zool. 26:101-107. P h i l l i p s , J.E. and J . Meredith. 1969. Active sodium and chloride transport by anal papillae of a salt water mosquito larva (Aedes campestris). Nature 222(5819):l68-l69. P i r i e , N.W. 1932. The metabolism of methionine and related sulphides. Biochem. J . 26:2041-2045. Ramsay, J.A. larvae.  1953. Exchange of sodium and potassium i n mosquito J . Exp. Biol. 30:79-89.  Ramsay, J.A. 1956. Excretion by the Malpighian tubules of the stick insect Dixippus morosus (Othoptera, Phasmidae): Calcium, magnesium, chloride, phosphate and hydrogen ions. J . Exp. Biol. 33:697-709. Scudder, G.G.E. 1969. The fauna of saline lakes of the Fraser Plateau i n British Columbia. Verh. Internat. Verein. Limnol. 17:430-439. Shaw, J . and R.H. Stobbart. 1963. Osmotic and ionic regulation i n insects. Adv. Ins. Physiol. 1:315-399. Smith, P.G. 1969b. The ionic relations of Artemia salina L. II. Fluxes of sodium, chloride and water. J . Exp. Biol. 51:739-757. Stobbart, R.H. 1959. Studies on the exchange and regulation of sodium in the larva of Aede3 aegypti L. I. The steady-state exchange. J. Exp. Biol. 36:641-653. Stobbart, R.H. I960. Studies on the exchange and regulation of sodium in the larva of Aedes aegypti L. II. The net transport and fluxes associated with i t . J . Exp. Biol. 37:594-608. Stobbart, R.H. 1967• The effect of some anions and cations upon the fluxes and net uptake of chloride i n the larva of Aedes aegypti and the nature of the uptake mechanisms for sodium and chloride. J. Exp. Biol. 47:35-57.  Topping, M.S. 1969. Giant chromosomes, ecology, and adaptation in Chironomus tentans. Ph.D. Thesis. Univ. of British Columbia. Treherne, J . E . 195k- The exchange of labelled sodium in the larva of Aedes aegypti L . J . Exp. B i o l . 31:386-401. Wigglesworth, V.B. 1931. The physiology of excretion in a bloodsucking insect, Rhodnius prolixus (Hemiptera, Reduviidae). III. The mechanism of uric acid excretion. J . Exp. B i o l . 8:411-451. Wigglesworth, V.B. 1933. The function of the anal g i l l s of the mosquito larva. J . Exp. B i o l . 10:16-26.  38  APPENDIX  FIGURE 9 Per cent mortality of f i r s t instar larvae i n : A.  Ctenocladus pond water [Mg "] = 180 m Eq/litre  B.  Pure solution of MgSO^ (200 mM/litre)  4-1  Standard error smaller than points.  39  FIGURE 10 Per cent mortality of f i r s t instar larvae i n : A.  Pure solution of MgSO^ (250 mM/litre) and NaCl (50 mM/litre)  B.  Pure solution of MgSO^ (250 mM/litre), KCO^ ( l mM/litre) and CaCl  2  (0.25 mM/litre)  Standard errors smaller than points  % MORTALITY  FIGURE 11 Per cent mortality of: A.  Third instar larvae i n pure solution of MgSO^ (200 mM/litre)  B.  First instar larvae i n pure solution of MgSO^ (250 mM/litre) and NaHCO^ (5 mM/litre)  Standard errors smaller than points.  % MORTALITY  


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