A comparative study of magnesium sulphate tolerance in saline-water mosquito larvae

J. Insrcf Ph~siol. Vol. 28. No. 7, pp. 641-646, Printed in Great Britain.

0022-19101821070641-06803.0010 0 1982 Pergamon Press Lrd

1982

A COMPARATIVE STUDY OF MAGNESIUM SULPHATE TOLERANCE IN SALINE-WATER MOSQUITO LARVAE ANTHONY W. SHEPLAY and Department of Developmental and Cell Biology, (Rewired

TIMOTHY J. BRADLEY

University

15 December

of California.

Irvine. CA 92717. U.S.A.

1981)

Abstract-The waters in which saline-water mosquito larvae are found can differ substantially in the types and ratios of ions present. Inland saline waters are more likely to contain high levels of MgS04 than are coastal ones. We undertook a study to determine if Aedes dorsalis. an inland species, is more tolerant of MgSO.+ than Aedes taeniorhynchus. a coastal one. The effects of MgSO, concentration were tested on all of the aquatic life stages of both species. Egg hatching was adversely affected at higher concentrations yet eggs would hatch in waters in which larvae could not survive. Younger larvae were more sensitive to MgS04 toxicity than the third and fourth instars. No differences in larval tolerance to MgSO, were observed between the two species indicating that neither species is ‘preadapted’ for MgSO, tolerance. Pupae of both species were completely insensitive to the levels of MgSO, tested. Larvae of A. taeniorhynchus reared in MgSO,-rich media showed substantial increases in tolerance to subsequent systems. Increased tolerance MgS04 stress. possibly through an induction of Mg*+ and SOi- transport to MgSO, required the presence of both Mg2+ and SOi- in the rearing medium. When larvae of both species were subjected to steadily increasing concentrations of seawater. A. taekx%ynchus survived much higher concentrations than did A. dorsalis. Therefore, while the process of induction is very important in extending the ionic range over which larvae can survive and develop. the possible ionic range for each species is finite. The differences in ionic range for each species probably accounts for the diversity of ionic tolerance and species distribution observed in the field.

INTRODUCTION THE LARVAE of several species of Aedes mosquitoes are able to survive in saline-water environments. RAMSAY (1950) found that the larvae of Aedes detritus survive in hypersaline environments by producing a concentrated urine in the rectum. Using Aedes taeniorhynchus and Aedes campestris. BRADLEYand PHILLIPS (1975. 1977a) showed that this concentrated urine was produced by the secretion of a hyperosmotic fluid in the posterior rectum, a specialized rectal region not found in freshwater species (RAMSAY, 1950; MEREDITH and PHILLIPS, 1974). The rectum is capable of actively transporting K+. Na+, Mg”‘. Cl-, and HCO;, i.e. most of the ions commonly found in high concentrations in saline-water environments (BRADLEY and PHILLIPS, 1977b). Of the above ions, Mg*+ was found to be the most toxic to A. campestris (KICENIUK and PHILLIPS, 1975) with the upper limit being about 95-125 mM Mg 2+ in Ctenocladus pond water, an unusual saline pond high in Na+. Mg*+, and SOibut very low in K+ and Cl-. The one ion which the rectum seems unable to transport actively is sulphate. which must be cleared from the blood by the Malpighian tubules (MADDRELL and PHILLIPS, 1975; BRADLEY and PHILLIPS, 1977a). MADDRELL and PHILLIPS (1978) showed that sulphate transport in the Malpighian tubules of A. taeniorhynchus could be increased through induction by rearing the animals in a sulphate-rich medium. However, since the Malpighian tubules cannot produce a hyperosmotic fluid. the location of the sulphate-excreting mechanism in this tissue puts a relatively low upper limit (compared e.g. to Cl-) on

the concentration of SOiwhich the animals can withstand in the external medium (BRADLEY and PHILLIPS, 1977a). BRADLEYand PHILLIPS (1977a) found that A. campestris (an inland species) was better able to withstand waters high in Mg’+ and SOi- than A. tarniorhvnthus (a coastal species). The salts in coastal saline swamps generally derive from influxes of seawater during storm tides and are therefore high in NaCl. The ions in inland ponds derive from surrounding sediments which are often rich in Na2HC0, or Na + MgSOh. BRADLEY and PHILLIPS (1977a) showed that a single strain of A. campestris could survive in water of any one of the above ionic types, indicating an osmoregulatory versatility that is perhaps associated with the ionic diversity they encounter in their inland habitats. The studies reviewed above elucidate in substantial detail the mechanisms and cellular locations of ion transport in saline-water mosquito larvae. What is presently lacking is an understanding of how these transport processes and the ions in the environment mutually determine the distribution and survival of various saline-water mosquito species. While many factors in the field undoubtedly contribute to oviposition and survival (NAYAR and SAUERMAN. 1975). the above studies suggest that sulphate and perhaps magnesium ion concentrations are critical parameters which limit the distribution of saline-water mosquito larvae in saline ponds. We undertook this study to examine the tolerance of the eggs, larvae and pupae of saline-water mosquitoes to various levels of MgSOL. in order to determine what these limits might be and in which portion

641

642

ANTHONY W. SHEPLAYand

of the life cycle they might operate. In addition. we wished to determine if increased MgSO.+ tolerance was a general characteristic of inland saline-water species. We therefore compared a coastal species, A. tneniorhynchus, to A. dorsalis, an inland species common in the Great Basin of western North America. Finally, we examined the effects of rearing animals in solutions high in Mg2* and SO:-, on later survival of the larvae upon challenge with MgSO+ Previous workers investigating the ionic tolerance of saline-water mosquito larvae have frequently used pure ionic solutions. PARKFR (1979), for instance, studied the survival of A. dorsalis in pure NaCl solutions. Natural waters are, of course, ionically much more diverse than this and the presence of a variety of ions generally promotes survival due to ionic antagonism (BRADLEY and PERKINS, 1975). We therefore tested the effects of MgSO, on A. taeniorhynchus and A. dorsaiis by adding it to 50% seawater. This assured a hyperosmotic solution and supplied the larvae with a more natural medium containing ions (e.g. Ca’+, Cl-) necessary for their survival and development, while still allowing us to substantially vary their exposure to MgS04.

MATERIALS

AND

METHODS

Experimental animals Colonies of Aedes taeniorhynchus and Aedes dorsalis were maintained as described previously (BRADLEY and PHILLIPS, 1975). The larvae were reared at 27°C using a 12:12 hght:dark cycle with 200 larvae in 500 ml of solution. They were fed dried yeast (Fleischmanns) and dried liver (Bacto). The Aedes taeniorhynthus eggs used to establish the colony were a gift of Dr. J. K. Nayar, Florida Medical Entomology Lab., Vero Beach, Florida. The Aedes dorsalis eggs were kindly provided by Dr. Laura Kramer, Dept. of Biomedical and Environmental Health Sciences, School of Public Health, Berkeley, CA. The effects of MgSO, hatching

on larval survival and egg

The ability of each species to survive transfer to waters of higher MgS04 content was tested. For this purpose larvae were reared in 50% seawater to the proper larval instar and transferred to solutions containing 50% seawater plus one of the following combinations of salts: (a) 0 mM MgS04 + 500 mM NaCl (abbreviated as O/500), (b) 100/400, (c) 200/300, (d) 300/200, (e) 100/400, (f) 500/O. All of these solutions contained 50% seawater plus 5OOmM of additional salt and differed only in the ratio of MgSO, to NaCl which had been added. Since NaCl shows a higher ionization ratio than MgS04, the osmotic concentration, as measured on a Wescor vapour-pressure osmometer. differed in the final solutions (50% seawater + 500mM MgSO, = 1030mOSM; 50% seawater + 500mM NaCl = 1530mOSM). As shown in the results section, the toxicity of each solution was directly related to the MgSO, concentration and not the osmotic strength. The toxicity of these solutions to each of the larval instars and the pupal instar was tested by transferring the animals from 507; seawater to the test solution.

TIMOTHYJ. BRADLEY

After 24 hr the number of dead and surviving animals was determined by pushing the larvae gently below the surface. Only those larvae which swam to the top to re-establish tracheal contact with the air were counted as living. This assay is simple, unambiguous and highly repeatable (BRADLEYand PERKINS. 1975). The effects of the above spectrum of solutions on egg hatching were tested as well. Eggs from each species were taken from storage, washed with distilled water, mixed to randomize them, and separated into groups of 100 eggs. Each group was placed in a separate porcelain crucible with 2ml of one of the above solutions of 507; SW + MgSO, + NaCl. All of the crucibles in one experimental run were placed together in a vacuum desiccator and subjected to reduced atmospheric pressure (150 mm Hg) for 45 min, after which time the number of hatched larvae was determined. The eggs hatch in response to reduced partial pressure of oxygen. The reduced atmospheric pressure does not adversely affect hatching or larval survival, and is indeed the routine hatching method used by Dr. Kramer with Aedes dorsalis (personal communication). In addition to the above test solutions, a parallel control was run in distilled water. our usual hatching medium. Increased tolerance to MgS04 following exposure We wished to determine if larvae reared in the presence of high levels of MgSO, showed increased tolerance to MgSO, stress. Larvae were hatched and reared for one day in 507: SW. On each successive day the MgS04 concentration was raised by 50 mM/l., by adding MgSO, as a dry salt. On day six the 4th-instar larvae were removed from the rearing pan, which now contained 50% SW + 200mM MgSO,, and placed in 50% SW + 300mM MgSU, + 200 mM NaCl. The number of surviving larvae was determined after 24 hr. Parallel groups of larvae were treated identically with the exception that the medium was progressively concentrated by the daily addition of 33 mM Na,SO,, 33 mM MgCl, or 50 mM NaCl. In each case, 6-day-old, 4th-instar larvae were placed in 50% SW + 300mM MgSO, + 200mM NaCI. Comparisons could therefore be made of the MgSO, tolerance of larvae reared in high MgS04, high SO:-, high Mg2+ or a similar high osmotic concentration produced with NaCI. Salinity tolerance In order to determine the comparative salinity tolerance of Aedes taeniorhynchus and Aedes dorsalis, larvae were reared in progressively more concentrated seawater solutions under the following schedule: days 1 and 2,50x SW; days 3 and 4,100x SW; days 4 and 5, 150% SW, etc. Concentrations above 100% SW were produced by dissolving appropriate quantities of artificial sea salt (LYMAN and FLEMING, 1940) in fullstrength seawater. The number of live larvae or pupae was determined each day as described above. RESULTS AS described in the Materials and Methods section, the solutions used for testing MgS04 tolerance in this study were composed of 50% seawater + 500 mM/l. of

MgSO,

Aedes

1234P

1234P

0 mM

100

643

tolerance in mosquitoes taeniorhynchus

1234P

1234P 300

mM

mhl

I2 400

3 4P mM

I2

3

4P

M9SO4

dorsalis

1234P 0 mM

I23

4P 100 mM

I2 200

3

4P

mM

300

MS04

mM

hSO4

400

mM

500

mM

MS04

Fig. 1. The relationship of larval and pupae survival in both species to MgSO, concentration In external medium. Percentage survival over 24 hr for each of the four larval (1. 2, 3. 4) stages and pupal stage (P) are shown at each MgSO, concentration from 0 to 5OOmM. See the Materials Methods section for the complete composition of each medium. Sample size for each determinatmn 20 animals.

salt. The MgSO, concentration to which the animals were exposed was varied by changing the ratio of MgSO, to NaCI in the salt added. The amount of MgSO, added proved to be the only parameter which varied directly with the toxicity of the test solutions (see Results below). Therefore, for the sake of simplicity and economy of space, the solutions are referred to by the amount of MgSO, added. It is to be understood, however, that for example the solution referred to as 300mM MgSO, contains SO”;, seawater + 300 mM MgSO, + 200 mM NaCI.

MgSO, tolerance Figure 1 shows the percentage of larvae and pupae surviving for 24 hr upon transfer from 50°, seawater to solutions containing various concentrations of MgSO+ Two general trends, common to both species. are evident in Fig. 1, (I) that increasing the concentration of MgSO,. substantially reduces the percentage survival of all larval instars and (2) that earlier instars are less able to survive transfer to solutions high in MgSO, than are the older larvae. This latter trend is most pronounced at 200mM MgS04 where survival is near 0”, for first instars and near IOO”,, for fourths. It is interesting that no major differences in the response to MgSO, were observed between the two species. It is important to note that since NaCl has a higher dissociation constant than MgSO,, the solutions from left to right in Fig. 1 are decreasing slightly in osmolarity and increasing greatly in MgSO, concentration. The results indicate therefore that. in these solutions.

the the and was

toxicity observed is directly proportional to MgSO, concentration. not the osmolarity. Pupae of both species are essentially unaffected by changes in the MgSO, concentration of the medium. Even in 500 mM MgSO.+. where all the larvae of each instar die within 24 hr, 1009, of the pupae of both species remained viable. Eficts

on hatching

Having demonstrated a substantial increase in larval mortality with increasing MgSO, concentration, we were interested in determining whether MgSO, levels might also influence egg hatching in these two species of saline-water mosquito. Figure 2 shows the percentage of eggs which hatched in each solution under identical hatching stimulus. The number hatching is compared to that in OmM MgSO, which is normalized to IOO”,. In Aedes rueniorhynchus. no significant differences were found in the hatching rate of eggs in 0. 100 and 200 mM MgSO, and in distilled water (indicated by arrows on Fig. 2). At higher magnesium concentrations, the eggs show a trend of decreasing hatching success. In 500 mM MgSO,, the hatching rate is statistically significantly lower than in IOOmM MgSO, (P < 0.05). Ardes dorsalis shows a greater reduction in hatching rate at high MgSOS concentrations than does Ardcs taeniorhynchus, with 300mM MgSO,. and SOOmM MgS04 all being 400 mM MgSO,. lower than the rate at mM MgSOS (all P values < 0.001). Interestingly. a larger percentage of eggs hatched in OmM MgSO,. which is a rather concentrated sol-

ANTHONY

644 Aedes

taenlorhynchus

Aedes --

mM

W.

SHEPLAY

dorsalls

MgSO,

Fig. 2. A comparison of percentage egg hatching success for both species in media containing MgSO, concentrations ranging from 0 to 500 mM. See the Materials and Methods section for the complete composition of each medium. One hundred eggs were used for each determination.

and

TIMOTHYJ. BRADLEY

in 50% seawater (Fig. 1). we raised larvae in waters high in Mgzi and SO:- to investigate the possible adaptive significance of the induction of Mg’+ and SO:transport (MADDRELL and PHILLIPS, 1978). Table 1 shows the percentage survival of Aedes taeniorhynchus larvae reared in various solutions and then stressed with 50% seawater + 300mM MgSO, + 200 mM NaCI. Fourth-instar larvae reared in daily increasing levels of MgS04 showed a high level of survival (83%) upon transfer to the MgSO, stress solution. This contrasts with a survival of 187; for 4th-instar larvae similarly stressed but reared in daily increasing levels of NaCI. It is clear that it is the MgSO, concentration which is toxic and not the osmotic increment or handling stress. since animals reared in daily increasing levels of NaCl and then stressed with NaCl (SOY/,seawater + 500mM NaCI) show a survival of 94%.

ution (50% seawater + 500mM NaCl). than in distilled water (P < 0.05) the solution in which the eggs of both species are routinely hatched in the lab. Possible induction mechanisms

Having established that MgSO, is highly toxic to saline-water mosquito larvae which had been reared

Saline tolerance in larvae We observed no difference in MgSO, tolerance in Aedes taeniorhynchus and Aedes dorsalis (Fig. 1). Since Aedes taeniorhynchus is a coastal species and Aedes dorsalis an inland one, we compared the effects of increasing the osmolality of the medium using the ionic ratios found in seawater. Table 2 shows the percentage survival for both species in increasing concentrations of seawater. Age specific survival is included for each 24-hr period. For both species. increasing salinity is associated with ad-

Table 1. Percentage survival for larvae reared in daily increasing levels of various salts (Column I) and stressed as fourth instars with 50:~ seawater + 500mM MgSO, or 509; seawater + 500 mM NaCl (column 2) Reared in

Transferred to

MgSO, MgClz Na2S0, NaCl NaCl

(y,;) survival

n

83 30* 13* 18* 94NS

MgS04 MgSO, MgSO, MgS04 NaCl

30 40 15 33 33

‘n’ indicates sample size for each determination. *Indicates those values significantly different (P < 0.001) from that of animals reared in MgS04 and stressed with MgSO+ No significant difference is indicated by NS.

Table 2. Percentage survival and age specific survival for larvae increasing

concentrations

of both

Aedes tarniorhpnchus

Day

Concentration of seawater (“/b)

0 1 2 3 4 5 6

50 75 100 150 2M) 250

species

in daily

of seawater Aedes dorsalis

(“/,) Survival

Age-specific survival (%)

(%) Survival

Age-specific survival (%)

100 100 100 95 83 12 52

100 100 95 87 87 72

100 97 94 83 54 32 0

97 91 88 65 59 0

Percentage survival indicates the percentage of original larvae alive at the end of each day. Age-specific survival shows the percentage of larvae surviving each 24 hr period relative to those alive at the start of that period. Sample size equals 100 larvae for each species,

MgSO, tolerance in mosquitoes ditional mortality. Not only are fewer larvae found alive at the higher concentrations but the age specific mortality increases as well. In contrast to these similarities between species, the data shows markedly better survival under these conditions for Aedes raeniorhynchus than for Aedes dorsa/is. When the experiment was terminated on day six, 52 Ardes taeniorhynchus had completed their larval development and had pupated, while the Aedes dorw/is raised under identical conditions had all died as larvae. Aedes raeniorhynchus, a coastal species, therefore shows better survival in concentrated waters containing sea salt than does the inland species A. dorw/is.

DISCUSSION Saline-water mosquito larvae are among the best ionic- and osmoregulators in the animal kingdom. Aedes raeniorhynchus, for example, can survive and develop in solutions ranging from distilled water to 2%300”,, seawater (NAYAR and SAUERMAN, 1975; this study). Aedes campestris can tolerate waters containing 100 mM Mg’ ‘, 250 mM SO: and extremely low concentrations of Cl- (2 14 mM) (KICENIUK and PHILLIPS, 1974; BRADLEYand PHILLIPS, 1977a). Aedes dorsalis can regulate haemolymph pH in concentrated saline waters with a pH of 10.5 (STRANGE and PHILLIPS,1980). In spite of the above phenomenal regulatory abilities, there are limitations to the ionic concentrations and ratios which saline-water mosquitoes can tolerate. More importantly, the limitations are frequently reached and exceeded in natural waters (KICENIUK and PHILLIPS. 1974; BRADLEY and PHILLIPS, 1977a). As we pointed out in the introduction, previous studies have suggested that, of the commonly occurring ions in natural bodies of water, Mg*+ and SOiappeared to be the most toxic to saline-water mosquito larvae. The results of this study provide experimental evidence supporting this suggestion and pinpoint the portions of the life cycle most sensitive to these ions. The efSects of MgS04 on eggs, larvae and pupae This study is the first systematic investigation of the effects of an environmentally common salt on the entire life cycle of a saline-water mosquito larva. The data in Fig. 1 show the profound influence that MgS04 concentration can have on larval survival. Early instars are particularly sensitive and development is completely halted for first instars at 200 mM MgSO+ The tolerance of the larvae rapidly increase as they enter the later instars. Tbis result has been observed in other transfer experiments (BRADLEYand PHILLIPS, 1977a) and probably reflects the greater surface-to-volume ratio in the younger larvae. The differences observed between first and fourth-instar larvae are extreme in the present study with some fourthinstar larvae surviving in media containing twice the MgSO, concentration needed to kill first instars. Pupae appear to be almost totally unaffected by the external MgSO, concentration. This is probably a consequence of the fact that pupae do not drink (CLEMENTS,1963).

645

As shown in Fig. 2. increasing MgSO, concentration leads to a substantial decrease in hatching rate for both species at higher MgSOL concentrations. This decrease is statistically significant but for the following reasons is of little biological importance. All larvae hatching out of eggs in the wild must thereafter attempt to survive, as first-instar larvae. in the water in which they hatch. We can see from Fig. 1 that first-instar larvae from both species die within 24 hr in any medium containing 200 mM MgSO, or more. In Fig. 2. however, we see that larvae will hatch in large numbers into waters in which they cannot survive. The slight, but statistically significant reductions in hatching observed in the higher MgSO, concentrations become biologically unimportant when one considers that these larvae cannot survive in these waters. It would appear therefore that it is the tolerance of the earliest-larval instars that defines the critical ionic regulatory niche for each species. The increased tolerance to MgSO, observed in the later instars is also of considerable interest when one considers the ionic characteristics of the natural waters in which these larvae are found. Saline-water Aedes are floodwater mosquitoes. meaning that they generally hatch into a dilute pond recently flooded by rain or runoff. An exception to this is found in coastal pools where high storm tides may initiate hatching in full-strength seawater. In either circumstance further larval development proceeds during a period when the medium is increasing in concentration by evaporation. The ability of the older larvae to withstand higher concentrations therefore closely parallels the likely increase in ionic concentration which the larvae will encounter during development. Since the pupae are essentially insensitive to external MgSO, concentration, the mosquitoes are in a race with time to complete their larval development and become pupae before the external ionic concentration reaches toxic levels. Possible induction mrchanisms MADDRELL and PHILLIPS (1978) demonstrated that the Malpighian tubules of Aedes raeniorh~nchus larvae. reared in the presence of high external levels of SO:-. showed higher SOi- transport rates in ritro than those from larvae reared in sulphate-free seawater. They further showed that this increased transport capacity was due to an inducible pumping mechanism which required about 8 hr for activation, We reared Aedes taeniorhynchus in daily increasing levels of MgSO, in order to maximize the induction of transporting mechanisms. Our Mg’+ and SOiresults (Table 1) indicate a marked increase in MgSO, tolerance in animals reared in this way. suggesting that the induction of transport in these animals has considerable bearing on survival in the field. The ability of the animals to survive in high MgSO, media is strongly dependent on previous exposure to MgSO,. For example, exposure to increasing levels of NaCl does not benefit the animals in withstanding later MgSO, stress. Additionally, animals reared in daily increasing levels of MgCI, or Na2S0, showed no better survival in subsequent MgSO, stress than did animals exposed to increased NaCl. This indicates that larvae must be exposed to both Mg2+ and SOiin order for the animals to best survive subsequent

646

ANTHONY W. SHEPLAYand TIMOTHY J. BRADLEY

MgS04 stress. While no one has yet demonstrated induction of Mg2+ at the cellular level in saline-water mosquito transport tissues, the results of this study indicate that the search for such a mechanism is warranted. A comparison of ionic and osmoregulatory saline-water mosquito larvae

abilities in

Saline-water mosquito larvae from various environments seem to be extremely well adapted to their ionic surroundings. Each species demonstrates an impressive ability to regulate the ions found in the waters it inhabits. What is unclear from these observations is the degree to which these abilities reflect inherent species differences. This study was designed to look for differences between coastal and inland species with regard to their tolerance to MgSOc. One could hypothesize that the inland species, Aedes dorsalis, should show better MgSO, regulation since MgSOl is more prevalent in inland waters. We find, however, no difference in MgSO, tolerance in Aedes dorsalis and Aedes taeniorhynchus reared under identical conditions. Neither species seems ‘preadapted’ therefore for waters rich in MgS04. Interestingly, the tolerance of Aedes taeniorhynchus can be greatly increased, however, by rearing the animals in MgS04. It would seem therefore that induction plays a very important role in determining the abilities of the larvae to withstand later salt stress. With regard to MgS04 tolerance in this study, the previous ionic environment in which the larva was reared was more important than whether the species was inland or coastal. We can conclude that the physiological phenomenon of induction is of considerable ecological significance in extending the ‘ionic niches’ of saline-water mosquitoes. Some differences which we observe between species cannot, however, be explained on the basis of differences in induction. For example, in this study. A. taeniorhgnchus and A. dorsalis were reared identically in daily increasing concentrations of sea salt. The opportunity for induction of transport systems was identical for both groups, yet A. taeniorhynchus showed substantially better survival than A. dorsak It would seem therefore that despite the ability of the process of ion transport induction to increase survival in media of high ionic concentration, each species of saline-water mosquito has a finite range of ionic and osmotic concentration which it can tolerate. These differences in total range are probably the basis for

the differences in ionic tolerance and species distribution that are observed in the field. Acknowledyrments-This study was supported by a grant from the California Special State Fund for Mosquito Research and by NIH grant GM 27919.

REFERENCES BRADLEY T. J. and PERKINS D. L. (1975) Ionic antagonism in mosquito larvae, Culrx pipiens. Comp. Biochem. Pkysiol. 52A, 403407. BRADLEY T. J. and PHILLIPS J. E. (1975) The secretion of hyperosmotic fluid by the rectum of a saline-water mosquito larva, Ardes taeniorhynchus. J. r.~p. Biol. 63. 331-342. BRADLEY T. J. and PHILLIPS J. E. (1977a) Regulation of rectal secretion in saline-water mosquito larvae living in waters of diverse ionic composition. J. rup. Biol. 66, 83-96. BRADLEY T. J. and PHILLIPS J. E. (1977b) The location and mechanism of hyperosmotic fluid secretion in the rectum of the saline-water mosquito larvae. Ardcs taeniorhynthus. J. rxp. Biol. 66, 11 l-126. CLEMENTSA. N. (1963) The Physiology ofMosquitoes. Mac-

Millan, New York. KICENIUK J. W. and PHILLIPS J. E. (1974) Magnesium regulation in mosquito larvae, Aedes campestris, living in waters of high MgS04 content. J. exp. Biol. 61, 749-760. LYMAN J. and FLEMING R. H. (1940) Composition of sea water. J. mar. Res. 3. 134-146. MADDRELL S. H. P. and PHILLIPS J. E. (1975) Active transport of magnesium by the Malpighian tubules of the larva of the mosquito, Aedes compestris. J. exp. Biol. 61, 761-771. MADDRELL S. H. P. and PHILLIPS J. E. (1978) Induction of sulphate transport and hormonal control of fluid secretion by Malpighian tubules of larvae of the mosquito Aerces taeniorhynchus. J. exp. Biol. 72, 181-202. MEREDITH J. and PHILLIPS J. E. (1973) Rectal ultrastructure in salt- and freshwater mosquito larvae in relation to physiological state. Z. Zellforsch. 138, l-22. NAYAR J. K. and SAUERMAN D. M. (1975) Osmoregulation in larvae of the salt-marsh mosquito. Aedes tueniorhynthus Wiedeman. Ent. exp. appl. 17, 367-380. PARKER B. M. (1979) Development of the mosquito Ardes dorsalis (Diptera:Culicidae) in relation to temperature and salinity. Ann. ent. Sot. Am. 72, 105-108. RAMSAY J. A. {1950) Osmotic regulation in mosquito larvae. J. exp. Biol. 27, 145-1.57. STRANGE K. and PHILLIPS J. (1980) pH and ionic regulation in saline water mosquito larvae inhabiting athalassohaline Na2C03-HC03 lakes. Am. Zoo/. 20(4). 939.