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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 . S c , 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 presenting 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 at the U n i v e r s i t y of B r i t i s h Columbia, I agree that 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 reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or 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 gain s h a l l not be allowed without my w r i t t e n permission. Department of Zoology The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date December 2, 1971 i ABSTRACT Regulation of hemolymph and whole-body Kg"*"1" concentration was studied in the larvae of Aedes campestris Dyar and Knab from a salt-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 Bq/litre. The rate of entry of Mg 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 Eq/l itre) , 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 in tissue. Measurement of urine Mg** concentrations of animals in 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 in Mg"***" excretion in Aedes campestris larvae. i i TABLE OF CONTENTS PAGE INTRODUCTION 1 MATERIALS AND METHODS 4 Animals 4 Media 4 Handling of larvae 5 Washing and drying 5 Sampling of hemolymph 5 Kg*"*" determination 5 Wet ashing 6 Evaluation of the use of PVPI 1 2^ for drinking rate 6 determination Drinking rate 7 Mg absorption i n the gut 8 Weighing 8 Urine sampling 9 Ashing of parts of the excretory system 10 Ligations 11 Mortality curves 11 RESULTS 12 Blood Mg"*" 12 Mortality 12 Drinking rate 16 Relative radioactivity of various components of the larvae 16 exposed to PVPI 1- 2 5 for 40 hrs Mg** uptake 22 Body Mg"*-1" 22 -j |„ Urine Mg concentration 24 i i i PAGE Mg content of parts of larvae 26 ++ Permeability of body wall to Mg 26 DISCUSSION 31 LITERATURE CITED 36 APPENDIX 38 i v LIST OF TABLES TABLE PAGE I Distribution of PVPI125 in larvae 17 II Whole body Mg 23 III Mg"1"* concentration of excretory products 25 IV Mg"*"*" content of parts of larvae 27 LIST OF FIGURES FIGURE PAGE 1 Changes i n blood [Mg ] with time following transfer 13 of animals from normal Ctenocladus pond water (190 m E q / l i t r e ) to 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"4-1"]. 14 3 M o r t a l i t y among fourth i n s t a r larvae during hemolymph 15 [Mg ] experiment (F i g . l ) . 4 E f f e c t of temperature on drinking rate i n normal 19 Ctenocladus pond water using PVPI125 as an i n d i c a t o r . 5 Effect of presence of p a r t i c l e s on drinking rate as 20 measured .using PVPT.125 as an i n d i c a t o r . 6 Eff e c t of [Mg*4"] on drinking rate at 10 C. 21 7 Total Mg loss from animals i n d i v i d u a l l y confined 28 i n 1 u l of d i s t i l l e d water under o i l . 8 Mean Mg++ loss /animal from 20 animals i n 1 ml of 29 d i s t i l l e d water. 9 Percent mo r t a l i t y of f i r s t i n s t a r larvae i n : 39 A. Ctenocladus pond water [Mg**] = 180 m E q / l i t r e B. Pure solu t i o n of MgSO^ (200 mM/litre) 10 Per cent mo r t a l i t y of f i r s t i n s t a r larvae i n : 40 A. Pure solu t i o n of MgSO; (250 mM/litre) and NaCl (50 mM/litre) B. Pure solu t i o n of MgSO (250 mM/litre), KCO3 (1 inM/litre) and 4CaCl 2 (0.25 mM/litre). 11 Per cent m o r t a l i t y of: 41 A. Third i n s t a r larvae i n pure solution of MgSO; (200 mM/litre) B. F i r s t i n s t a r larvae i n pure solu t i o n of MgSO^ (250 mM/litre) and NaHC03 (5 mM/litre) ACKNOWLEDGEMENTS I wish to thank my supervisor, Dr. John Phill ips, 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 in insects has been a much investigated problem in 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 fe in 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 in 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 in alkali lakes and salt marshes where the external medium may vary widely in chemical composition and total 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 in their tissues at suitable concentrations, and at proportions different from those encountered in their environment. The organs responsible for this regulation include the excretory system (Malpighian tubules, hindgut, and rectal system) and extra-renal mechanisms located in 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 in 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 in a wide range of concentrations of media after a suitable period of adaptation. Ion-depleted larvae took up Na+ and Cl~ 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 in regulation in 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 measure-ment of low concentrations of divalent inorganic ions, l i t t l e work has ++ ++ been done on the regulation of Ca and Mg in any group of .insects, either larval or adult. Clark and Craig (1953) measured the concentration . . j j of Ca and Mg in the hemolymph of a number of insects and found for Mg"1-1" a range of 0.012 /ig/mg in Vespula pensylvanica (adult) to 0.673 pg/mg in Laphygma exigua larvae. No studies have been done to determine how hemolymph Mg+ +levels respond to changes in external Mg"1"4" concentration. Wigglesworth (1931) found Mg"*"*" in the excreta of Rhodnius in 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 saline lakes of B.C. by Scudder (1969) and the chemical analysis of these lakes by Topping (1969) and Blinn (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 to 395 m Bq/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 either: 1) have some unusual regulatory mechanism which permits them to maintain their Mg content at constant and low physiological levels 2) exhibit an unusual tolerance of high blood Mg^+levels 3) prevent entry of Mg*4" into the hemolymph by reducing drinking rates 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 under-taken f i r s t to determine the degree of regulation of hemolymph Mg i n larvae after rapid transfer into media of different Mg++ concentrations . ++ (to test hypothesis 2 ) . After finding that the hemolymph Mg remained ++ independent of external Mg concentrations, experiments were conducted to determine whether Mg-H- was excluded from the larvae by reduction of drinking rate and/or whether the larvae were impermeable to Mg (hypothesis 3). In these l a t t e r experiments i t was found that the gut was permeable to Mg and that a large amount of Mg was taken into the hemolymph i n larvae subjected to high Mg concentrations. Further experiments were therefore undertaken to determine the steady state regulatory mechanisms responsible for maintenance of constant -H- ++ hemolymph Mg concentrations i n animals l i v i n g i n media with high Mg concentration. In other words what i s the fate of absorbed Mg i n the larvae? MATERIALS AND METHODS 4 Animals Aedes campestris larvae were collected in 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 in one gallon insulated picnic jugs for transportation to the laboratory at U.B.C. Water from the pond was filtered through glass wool and transported in five gallon polyethylene carboys. In the laboratory the animals were sorted into plastic trays containing normal pond water, where they were stored in a constant temperature cabinet at 10 C £ 0.5 C. Natural particulate material in the water provided food for the larvae in a l l cases except for larvae treated in a r t i f i c i a l media, in which case tropical fish food was used. The density of animals in storage containers was approximately 2 0/litre, which is less than that in the pond (about 4 0/litre). 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 in 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 in a nylon tea strainer in 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 in the range of 0 to 80 p. Eq/l itre Mg . A l l samples were diluted in swamp solution ( l ml for hemolymph samples) contained in polyethylene vials and run on a Techtron model AA 120 absorption spectrophotometer (Varian) as directed in the instrument manual. 6 Wet ashing. To measure total body or tissue Mg , weighed material was fi r s t dried overnight in an oven at 60 C in scintillation vials made of borosilicate glass. The vials and contents were then placed in a sandbath on a hotplate and 0.1 ml of modified Pirie's reagent (Pirie, 1932), consisting of one volume of 60% perchloric acid in three volumes of concentrated nitric acid was added to the vials. The latter were heated until white fumes began to form, at which point more concentrated nitr i c acid was added to prevent explosion due to production of perchlorates. The vials were further heated to 260-280 C until a dry white residue was formed. The residue was then dissolved in swamp solution for determination of Mg as previously described. Evaluation of the use of PVPI 1 2 5 for drinking rate determination Fifty 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 C a C l 2 A i t r e 0.0625 mC PVPI 1 2 5(Polyvinyl pyrrolidone 1125/50 ml After 5 min animals were removed, washed in running tap water for 5 min and the following samples were taken, placed in planchets, dried and counted: background (blank) external medium 1 jul hemolymph whole larvae After one day in 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 in no loss of contents). The gut, along with the attached Malpighian tubules and anal segment, was washed in twenty separate drops of ar t i f i c ia l medium., Cuticle (remainder of animal) left after this operation was washed in 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 ty 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 in several drops of water in planchets. The samples were then dried under a heat lamp. The total dry weight of material per planchet did not exceed 3 mg, which is 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 in 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 in a l l parts of this experiment. A l l 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 in ten changes of dis t i l led water, blot-dried in f i l ter paper, weighed and placed in 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 ia l , dried and counted for r ^ 5 activity; the remainder of each sample was used for determination of Mg** concentration. The Mg^/PVP ratios were then calculated for the samples. Mg^/PVP ratios for Groups I and II were not different, so the results were pooled. The Mg /PVP ratio in the external medium was similarly determined. If no absorption of -H- + + Mg occurred in 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 in 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 Mg4 "^ was regulated via the Malpighian tubules or the rectum, urine samples were taken in the following manner: Several hundred animals were acclimated for 1 week in each of five media ranging in Mg concentration from 2 to 170 m Eq/ l i t r e . After one week of acclimation, larvae were removed as required, dabbed dry on tissue paper and placed under paraffin o i l in 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 in 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 in 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 irst partially f i l l ed 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, fitted inside a disposable hematocrit tube, and centrifuged for 20 minutes in a Clay-Adams hematocrit centrifuge. The 30 ul pipet was then removed, cut off above the urine-oil interface, and mounted in a micromanipulator in a horizontal position. A siliconized micropipet, made by drawing out a Drummond 1 u l disposable pipet in a bunsen flame to yield a pipet of about 0.25 ul 10 volume, was mounted in 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 in 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 irst 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 in 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 in 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 in 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 in 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 in 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 total accumulated. Per cent mortality was then calculated (number dead X 100/25 ). 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"*""1" and 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 dist i l led 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. The results are shown in Fig. 1. The blood Mg"*""*" levels respond quickly to the change in medium Mg (within one day) and stabilize thereafter. When the steady-state level of blood Mg (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"*4" (0.02 to 200 m Eq/litre) the blood Mg"*~*" concentration does not increase significantly from 6 m Eq/ l i tre . Mortality During this experiment mortality of remaining (unsampled) animals was recorded (% dead of remaining animals). Fig. 3 shows that mortality was high, but remained more or less constant for a l l media in the range of 23 p. Eq/l itre to 190 m Eq/l i tre . It was observed that larvae could not survive a molt in 23 p. Eq medium. In the other media some larvae survived indefinitely. FIGURE 1 Changes in blood [Mg*4"] with time following transfer of animals from normal Ctenocladus pond water (190 m Eq/litre) to media of different [Mg ] O animals in 190 m Eq/litre (normal Ctenocladus water) • animals in 100 m Eq/litre (Ctenocladus water diluted 2X) • animals in 300 m Eq/l itre (Ctenocladus water + 100 m Eq MgSO/j/litre) • animals in 7 . 5 m Eq/litre (Ctenocladus water diluted 100X) Vertical bars indicate standard error of the mean of ten animals FIGURE 2 The steady state relationship between hemolymph [Mg ] and medium [Mg**]. 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*4"] experiment (Fig. 1) T 23 p. Eq M g+Vlitre (Ctenocladus water diluted 1000X) • 100 m Eq Mg^/l i tre (Ctenocladus water diluted 2X) • 190 m Eq Mg^/ l i tre (normal Ctenocladus water) O 300 m Eq Mg^/ l i tre (Ctenocladus water + 200 m Eq MgSO/yiitre) % MORTALITY/DAY Drinking rate Having obtained evidence that the Mg*4" level of the hemolymph -H-was constant, work was done to determine whether Mg is excluded 131 from the body by depressed drinking rate. PVPI has been employed by other authors (Smith, 1969) to determine drinking rate in an aquatic species (Artemia salina), but i t has not been used to measure drinking rate in a mosquito larva. It was therefore f irst 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~^^ is contained in the gut and almost none passes into the hemolymph,(A .small fraction of unbound l l 2 5 would account for the latter activity.) The fact that almost a l l of this j_125 Was confined to the gut contents means that PVPI125 is a good indicator of drinking rate in 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. The larvae might then concentrate the microorganisms. This would tend to produce high estimates of drinking rate. In view of these poss-ib i l i t i e s i t was decided to make drinking rate measurements under the following conditions: 1. normal Ctenocladus pond water at 10 C (Experimental temp.) 2. normal Ctenocladus pond water at 22 C (Room temp) 3 . Millipore-filtered Ctenocladus pond water at 10 C 4. 300 m Eq/l itre Mg** medium at 10 C 5. 100 m Eq/litre Mg*4" medium at 10 C TABLE I Distribution of PVPI125 in larvae Sample PVPI 1 2 5 Activity (cpm± SE) 40 hrs 3 min Background 12± 0.58 9.7 - 0.88 External medium ( l pl) 173 - 1.7 240 ± 2.65 Hemolymph ( l pl) 72 - 14-7 16.7 ± 2.19 Whole body 2950 i 350 54 * 13.0 Whole gut including contents 2300 ± 230 Whole gut without contents 333 ± 33 .3 Whole gut contents 3533 ± 8 1 9 . 2 Animal excluding gut 423 i 78.8 18 Temperature and salinity-adapted animals (one week at experimental conditions) were used in a l l cases. Drinking rates of animals in ordinary Ctenocladus pond water at 10 C and 22 C are compared in Fig. 4« There was a large individual variation in uptake of the medium, but no difference seems evident in 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 total body weight (4-6 mg) per day (assuming that the rate does not undergo any diurnal rbythmn. Fig. 5 shows the comparison of drinking rate determination in Millipore-filtered and ordinary Ctenocladus pond water. No difference is evident between the two experimental conditions; ie the drinking rates seem in agreement with the previous estimate. Fig. 6 shows the results of drinking rate determinations in media of different concentrations. From the latter results i t seems reasonable to assume that drinking rate does not change in response to concentration. In conclusion, the drinking rate by fourth instar larvae does not seem -H-to be affected by temperature in the range of 10 C to 22 C, or by Mg concentration over a range of 100 to 300 m Eq/ l i tre . Moreover, the high estimates of drinking rates obtained by the PVP method are not due to experimental error associated with concentration of PVPI^^ in a food source of the larvae (eg bacteria). These results show that the larvae are ingesting large amounts of Mg**4", therefore negating the possibility that low blood Mg"*4" concentration is due to reduced entry of Mg into the hemocoel as a consequence of lower drinking rate. FIGURE 4 Effect of temperature on drinking rate in 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 in Millipore-filtered Ctenocladus pond water A animals at 10 C in nonfiltered Ctenocladus pond water Vertical bars indicate standard error of the mean of ten animals FIGURE 6 Effect of frig*"1"] on drinking rate at 10 C T animals in 190 m Eq Mg^/l i tre (Ctenocladus pond water) • animals in 300 m Eq Mg^/l i tre (Ctenocladus pond water + 100 mM MgSO^/litre) • animals in 100 m Eq M g+Vlitre (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 is not absorbed. The Mg^/PVP ratio for midgut contents was 0.0606 ± 0.0086 (n = 19) and for the external medium 7.6-0.26 (n = 3 ) . This indicates that 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 in 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"1"*" in these experimental larvae are low compared 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 Whole body Mg -4-4- | | Acclimation Medium [Mg"""] Whole body Mg content (m Eq/litre) 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 abil it ies indicated by studies of blood Mg"t~t" levels. 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 in 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"*4" concentrations were analysed. The concentration of Mg++ in urine (urine/medium- U/M ratios- Table III) was higher than that in the medium in 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"*4" was increased from 2 to 170 m Eq/ l i tre . Furthermore, the Mg44" concentration in fecal material is high under a l l conditions, possibly due to binding to fixed charges in 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 fragi l i ty of anal papillae. However, simple calculations indicate that elimination of Mg"*4" by the excretory system alone balances the rate of entry by drinking (see discussion). 1 1 TABLE III Mg concentration of excretory products Media [Mg**] Urine [Mg*4"] Feces [Mg**4"] in Eq/l itre m Eq/litre n Eq/mg wet wt 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"H^emolymph Mg Where unspecified n = 3 26 Mg"*""*" content of parts of larvae In an attempt to determine what organ of the body was responsible for the Mg"*4" excretion, animals were dissected and various parts pooled, • i i ashed, and Mg content determined. Although the number of separate determinations i s small (one or two) each determination involved 20 animals. While the number of observations i s small (Table IV) , i t seems quite clear that the Malpighian tubules have a very high content of Mg per unit weight. The recta were quite often empty due to defecation during handling, possibly explaining their observed low Mg content. These results implicate the Malpighian tubules as secretory organs for Mg . Permeability of body wall to Mg"14-To determine the extent of movement of Mg"*4" across the body wall of the larvae, and to determine the rate of excretion by the whole larva, the following experiment was performed: Mg"*4" loss from animals ligated at the anal segment, from animals ligated at the penultimate abdominal segment and from normal (unligated) animals was measured by monitoring the increase i n Mg concentration of a small volume of d i s t i l l e d water i n which individual animals were confined under paraffin o i l (to prevent volume reduction by desiccation). The Mg concentration was then plotted against time (Figs. 7 and 8). The i n i t i a l small loss (Fig. 7) by animals ligated at the anal segment i s probabjy due largely to surface contamination, since after five minutes there i s no net loss of Kg"*4" with time. This indicates that loss of Mg from the larvae can only occur by excretion ( or by way of anal papillae) but there i s no Mg efflux 27 Mg concentration n Eq/mg wet wt collected in humid collected under o i l n = 1 TABLE IV Mg-"" content of parts of larvae Organ or fluid  chamber 111.0 (95, 127)* 566.7 (443, 690)* 62.4 (65, 60)* 18 (n = 3) Midgut contents Malpighian tubules Rectum Hemolymph after dissection n = number of repetitions each using 20 animals * = average, with individual values in parentheses 123 720 FIGURE 7 Total MR4-*" loss from animals individually confined in 1 ul of distilled water under oil 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 in 1 ml of dist i l led 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 in 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 in previous experiment), 20 normal animals were dabbed dry and placed in 1 ml of dist i l led water in a v i a l . Five ul 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+ in 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 in which there are few other species to compete with them (Scudder, 1969 ) . 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 in conjunction with blood Mg"*-*" experiments show that there is a lot of variation in individual animals and that the rate of mortality over a range of 0 .02 m Eq to 300 m Eq remains relatively constant at about 10$ per day. It is diff icult , i f not impossible, to determine whether the mortality observed in these animals is a result of a developmental abnormality or whether i t is the result of selection for regulatory abil ity (in this case for Mg**). Preliminary mortality studies on f irst 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 inal concentration, a few survive indefinitely. The above indicates that individuals within the Ctenocladus pond population vary greatly in their abil i ty to survive in 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 in the lake. The bottom of the lake was covered with dead larvae in mid-June 1970. At this time some of the animals had pupated and emerged, while others were s t i l l larvae. These remaining larvae seemed grossly shrunken and unhealthy ( i .e . slow moving) compared to those of earlier in 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"14" concentration of the 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 in experiments are a random sample of the animals in 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**4" concentrations. However, since i t was also observed that Ctenocladus animals in late instars survived as well i f not better in GR2 lake water (high in 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 in A. campestris l iving in Ctenocladus pond, the absorption of Mg44" from the gut, and the rapid response of the blood Mg44" concentration to changes in Mg44" concentration of the I I medium indicate the necessity for Mg regulation. The measurement of Mg44" concentration of urine reveals high U/M ratios (Urine [Mg44"]/ medium [Mg 3 ) (Table III) which indicate that the larvae are ridding themselves of excess Mg44" via the urine. In media o.f lower Mg44" 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 retention alone since the urine 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 with the high Mg content of the feces are i n d i c a t i v e that the animals are perhaps feeding on material having a higher l e v e l of 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 ingesting material which has bound Mg . Another explanation i s that they have a way of taking up Mg"*"1- independently of feeding. Larvae i n d i l u t e media may be capable of taking up Mg""" v i a the anal papillae ( i . e . turning on active transport). The turning on of active transport has been observed f o r Na + and C l " ( P h i l l i p s and Meredith, 1969). The high Mg-"- content of feces i s possibly due to binding of ++ Mg to charged groups on proteins i n the feces ( f i x e d charges), or to incomplete separation of urine and feces (the f e c a l samples may have had a sub s t a n t i a l amount of urine i n them). Another p o s s i b i l i t y i s that the Malpighian tubules are secreting Kg"*""*" i n some precipitated or granular form as i s suggested by t h e i r very high Mg""" content. On observation, Malpighian tubule contents appear granular or c r y s t a l l i n e | j i n nature. The Mg could possibly be secreted i n conjunction with urates as has been reported i n the case of the u t r i c l e s of B l a t e l l a  germanica (Ballan-Dufrancais, 1970). The r e s u l t s of a l i g a t i o n experiment (Figs 7,8) cannot be used to calculate instantaneous loss of Mg""" from animals on the basis of the loss i n the f i r s t f i v e minutes, since i t i a a common observation that these animals tend to defecate when handled so that rate of loss of the ion i s extremely large during handling. The small loss of Mg from 34 animals ligated at the anal segment is most likely due to surface contamination of the animal. The very rapid i n i t i a l loss (first five minutes) in the case of both the animals ligated at the penultimate abdominal segment and of normal animals is most likely due to defecation brought on by handling. The fact that a l l the loss in the case of animals ligated at the penultimate abdominal segment occurs within five minutes is indicative that the animals are emptying the posterior part of the gut a l l at once (defecating). If this is the case, then the next part of the curve for normal animals should represent the rate of loss in undisturbed animals. When the latter argument is 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 is in the range of drinking rate determined for these animals. Since the amount of water lost osmotically in these animals via the cuticle is small (0.3 ul/animal X day or 0.13$ of the amount drunk) (Phillips, unpublished) the Mg concentration in urine should be slightly higher than the Mg"1-1" concentration of the medium in the case of hyperionic media to achieve regulation. In actual fact the urine/ media Mg concentration ratio is in the order of 1.2. It is therefore possible to account for the excretion of a l l of the ingested Mg"*4" via the normal excretory process. In conclusion, i t has been found that the large amounts of Mg"*4" ingested by Aedes campestris larvae living in 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 in 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 in the saline water mosquito larva Aedes detritus Edw. J. Exp. Biol. 16:346-362. Blinn, D.W. 1971. Dynamics of major ions in some permanent and semipermanent saline systems. Hydrobiologia 38:225-238. Clark, E.W. and R. Craig. 1 9 5 3 . The calcium and magnesium content in the hemolymph of certain insects. Physiol. Zool. 26:101-107. Phillips, 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. Pirie, N.W. 1932. The metabolism of methionine and related sulphides. Biochem. J. 26:2041-2045. Ramsay, J.A. 1953. Exchange of sodium and potassium in mosquito larvae. 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 in British Columbia. Verh. Internat. Verein. Limnol. 17:430-439. Shaw, J. and R.H. Stobbart. 1963. Osmotic and ionic regulation in 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 in 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. Biol . 31:386-401. Wigglesworth, V.B. 1931. The physiology of excretion in a blood-sucking insect, Rhodnius prolixus (Hemiptera, Reduviidae). III. The mechanism of uric acid excretion. J . Exp. Biol . 8:411-451. Wigglesworth, V.B. 1933. The function of the anal g i l l s of the mosquito larva. J . Exp. Biol . 10:16-26. 38 APPENDIX FIGURE 9 Per cent mortality of f irst instar larvae in: A . Ctenocladus pond water [Mg4-1"] = 180 m Eq/litre B. Pure solution of MgSO^  (200 mM/litre) Standard error smaller than points. 39 FIGURE 10 Per cent mortality of f irst instar larvae in: 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 in pure solution of MgSO^  (200 mM/litre) B. First instar larvae in pure solution of MgSO^  (250 mM/litre) and NaHCO^  (5 mM/litre) Standard errors smaller than points. % MORTALITY 

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