31P NMR spectra of mosquito larvae parasitized with Romanomermis culicivorax

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JOURNAL

OF INVERTEBRATE

PATHOLOGY

48, 52-59 ( 1986)

31P NMR Spectra of Mosquito Larvae Parasitized Romanomermis culicivorax ROBIN

with

M. GIBLIN~

W. K. LEE

ROBERT

AND EDWARD

G. PLATZER

Received September 30. 1985: accepted December 19. 1985 The energy status of Rorncrnorrzrr-/nis c,rt/ic,i,,o,.a.r-parasitized and control CltlcJ.t-pipietls larvae was studied in vivo with jlP nuclear magnetic resonance (NMR) at 4-6°C. Peak assignments were made for inorganic phosphorus, phosphoarginine. AMP, ADP. and ATP. The mean ATPiADP ratios were 4.45 for control and 4.02 and 2.94 for parasitized mosquito larvae with I .3 and 2.3 parasites per host, respectively. The ATPiADP ratios for control mosquito larvae reared with a pH of 7 and 4.5 were not significantly different. R. ctrlici\wtrx parasitism had no significant effect on the ATP level in whole mosquito larvae. The mean ATP concentrations were 9.86 and I I .64 nmolimg dry weight mosquito for control and parasitized larvae. respectively. R. c~rrlici~wrcrx postparasites t IO days post-infection) were shown to have a very weak 31PNMR signal and it was concluded that the parasites contribute little to the NMR spectra of infected mosquito larvae. Enzymatic analysis of perchloric acid-extracted control mosquito larvae yielded 9.3 nmol ATPimg dry wt and 2.6 nmol ATPimg dry weight for R. ctrlicirwux postparasites at IO days post-infection. I 19x6 Academ>c PX\\. KEY

Inc. WORDS:

Culex

nematode: Romcrnomennis

pipiens: in vivo ctrlic~i~wrtrs.

31P NMR spectra: ATPiADP

INTRODUCTION

The nematode Romanomeumis culiciv0~a.x grows significantly in its mosquito host starting at 4 days post-infection (PI) (Castillo et al., 1982). The host’s fat body reserves, hemolymph protein, and carbohydrate levels were severely reduced as the parasitism proceeded (Schmidt and Platzer, 1980). The respiration rate of parasitized mosquito larvae indicated that the host was not significantly stressed by parasitism until 4 days PI (Powers and Platzer, 1984). The host continued to osmoregulate and maintain the pH of the hemolymph r Present address: University of Florida. IFAS. 3205 College Avenue. Fort Lauderdale, Florida 33314

0022-2011186 $ I .50 Copyright i 1986 hy Academic Pre\\. Inc All rtght, of reproduction in any term rewrvcd.

ratio; ATP quantification:

throughout parasitism (Giblin and Platzer, 1984; Powers et al., 1984). Thus, the parasite appears to act as a “nutritional sink” and by day 4 PI absorbs increasing amounts of osmotic effecters, i.e., amino acids and monosaccharides, for growth and development. The host catabolizes and eventually depletes its fat body reserves to maintain osmotic balance (Powers et al.. 1984). Since adenosine triphosphate (ATP) is hydrolyzed to ADP and inorganic phosphorus by organisms when energy is needed for chemical, osmotic, and mechanical work, these are the key phosphorus metabolites to measure in a comparison of energy status. 31P NMR is a noninvasive technique that

31P NMR

SPECTRA

OF PARASITIZED

has been applied recently in whole living organisms for analyzing phosphorus metabolites (Gadian et al., 1979). Although 31P NMR is now used routinely to study the diseased state in mammalian tissues (Barany and Glonek, 1984), very little research has been done to examine the potential of 31P NMR for studying the diseased state in invertebrates (Thompson and Lee, 1986). We used 31P NMR to examine the effects of R. culici\~ouz.u parasitism on the adenylate levels and energy status of parasitized and control mosquito larvae. MATERIALS

AND

METHODS

At~irnd tnairztenance. Cu1e.u pipiem was reared at 27°C according to the procedures of Platzer and Stirling (1978) and rearing waler was acidified to pH 4.5 with acetic acid for fungus control on day 4 PI only. Some experiments were performed on mosquitoes reared without acid treatment. The pH in this case was ca 7.0. R. rrrliciborax infections were done as described by Giblin and Platzer (1985). The dose of the preparasites was altered depending upon the desired level of parasitism. Mosquito eggs were collected 3-4 days prior to infection and held at 15°C. At day 0, control and parasitized larvae were set up at 27°C and infections were started. At day 6 PI mosquito larvae were separated from any pupae present with ice water and rinsed three times on a 35-mesh screen. The larvae were scraped from the screen into a Petri dish containing 6- 10 ml of deuterium oxide (D,O) and acclimated for ca. 30 min at 4-6°C. A 12-mm x 25-cm NMR tube (Wilmad Glass Co., Buena, N.J.) was precalibrated to 4 ml and ca. 500 mosquito larvae were transferred in D,O to the tube. The tube was cooled in an ice water bath and a 13-mm serum filter/separator (Accufilter TM, Acculab, Norwood, N.J.) was used to pack the mosquitoes into the 4-ml volume. The temperature of the tube was controlled between 4” and 6°C (3.8”C = freezing point of D,O). It had been previously determined that 93% of the third-

MOSQUITOES

53

and fourth-instar larvae of C. pipiens survived under such conditions. NMR analysis was complete within 3 hr from starting time. After the analysis, the larvae were returned to room temperature, diluted in distilled water, counted, and staged as described by Giblin and Platzer (1985). Each larva was examined with a dissecting scope and its viability was assessed by the presence or absence of a heartbeat. In addition, at least 100 parasitized mosquitoes per infected cohort were examined for the presence and number of parasites. R. czrficivoraa postparasites were collected from infected mosquitoes according to the procedures of Platzer and Stirling (1978) on day 8 PI and held in tap water until day 10 PI when they were transferred to ca. 10 ml of D,O and incubated and processed for NMR observations as above. The number of postparasites was quantified, and sex and viability were checked and recorded. A subsample of mosquito larvae or nematodes was taken for wet and dry weight determinations. Organisms were lyophilized for dry weight measurements as described by Giblin and Platzer (1985). An experiment was conducted to determine if the contents of the mosquito gut represented a significant contribution to the 31P NMR spectra of control and infected mosquito larvae. Approximately 5000 fourth-instar control C. pipiens at day 6 PI were placed in a Chinese ink suspension in D20 (Giblin and Platzer, 1985) for I hr. The resulting fecal material was suspended in 50% Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden) in an NMR tube and analyzed. Carbon-glutted C. pipiens larvae were prepared for analysis as above. After analysis the larvae were suspended in water and fed for 1 hr prior to a subsequent 31P NMR spectral examination. NMR analysis. 31P NMR spectroscopy of intact C. pipiens and postparasitic R. c’w licivorax was conducted at 4-6°C on a wide-bore Nicolet 300 MHz spectrometer at 121,469 MHz as described by Thompson

54

GIBLIN,

LEE, AND PLATZER

and Lee (1986). Data were acquired using a Nicolet 1280 computer in pulsed Fourier transform mode. A D,O field frequency lock was used at the beginning of each experiment. A single-pulse sequence was conducted (30 ksec. 45” pulse followed by a 1 set delay before collection of FID data) to maximize the signal-to-noise ratio of all resonances and shorten the data acquisition time. Larger pulse angles resulted in longer relaxation times for the nuclei of interest and were not used. Gated proton decoupling without nuclear Overhauser effect did not increase spectral resolution and was not used. Although the sample tube was spun in the observation coil, no increase in signal resolution was obtained. The tube was spun to keep the sample suspended homogeneously. Spectral transformations were done with 5-10 Hz line broadening and chemical shifts were measured relative to an external standard of 85% phosphoric acid. Two data acquisition runs of 1200-2400 scans each were obtained for most samples. The first acquisition run established the ATP/ADP ratio (Fig. 1A). Computation of this ratio was done with computer-integrated peak areas. The ATP integral was arbitrarily fixed. The relative level of ADP was estimated by subtraction of @ATP from the a-ATP + P-ADP integral. The second data acquisition run was for ATP quantification. This was done with a coaxial inner cell (i.d. = 4.0 mm) (Wilmad Glass Co.) containing a calibrated internal standard solution of 8 mM ATP (non-magnesium-chelated), 8 mM K,HPO,, 100 mM 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (Hepes), and 20% D,O, pH 7 (Fig. IA’). This inner cell was gently placed in the NMR tube prior to the second run. The chemical shift between magnesium-bound p-ATP in the sample and non-magnesium-bound l3-ATP in the inner cell allowed integral comparisons and quantification of sample ATP (Fig. IA). The amount of ATP was expressed on a per milligram dry weight tissue basis. Enzyme analysis. ATP was also mea-

d

!

I

1

I

I

30 20 IO 0 -10 -20 - 30 FIG. 1. Typical jLP NMR spectra of Ro~~unomen~is clr/i~i~,o~ax-parasitized and control Ctclex pipiens larvae at 4-6°C. (A’) Diagram of NMR tube with inner cell containing internal standard inserted for ATP quantification (see text for description). (BY Diagram of NMR tube used for ATPiADP ratio determinations (see text for description). (A) 3iP NMR spectra (2400 scans) from an ATPiADP ratio run of R. dici~wu.r infected (2.56 k 1.42 parasites per host) C. pipirrn larvae (484 mosquitoesM-ml vol in NMR tube) with inner cell containing internal standard inserted. (B) 3’P NMR spectra (1200 scans) of the same R. c~r/ic~i~wasinfected C. pipiensin (A) without inner cell. (Cl 3’P NMR spectra (1200 scans) from an ATPiADP ratio run of control mosquito larvae (550 mosquitoesi4-ml vol in NMR tube) without inner cell.

sured in perchloric acid (PCA) extracts of whole mosquito larvae and R. culicivorax postparasites. More than 300 control mosquito larvae at day 6 PI or 1150 R. culicivorax postparasites at day 10 PI were weighed and their volume measured by fluid displacement. R. culicivorax postpar-

31P NMR SPECTRA OF PARASITIZED

asites were acclimated at 4-6°C for 1 hr prior to extraction, One volume of tissue was extracted in 3-6 vol of 7% PCA in a tissue homogenizer over ice. The homogenate was centrifuged and the tissue-free supernatant was removed for titration to pH 6-7 with a saturated solution of KOH. The precipitated potassium perchlorate was removed by centrifugation and the sample was frozen at -70°C until used. Each sample was thawed and analyzed for ATP content using the coupled enzymatic assay [phosphoglycerate phosphokinase (PGK) and glyceraldehyde phosphate dehydrogenase (GAPD)] described by Adams ( 1963). The disappearance of NADH which correlates directly with the quantity of ATP in the sample was monitored by observing the decrease in absorbance at 340 nm on a Gilford 240 Spectrophotometer. Statistical comparisons were done with an ANOVA and Duncan’s multiple range test.

55

MOSQUITOES

suggests that the intracellular pH of parasitized and control C. pipiens fourth instars was probably close to 7. The 31P NMR spectra of control C. pipiens larvae that were killed by asphyxiation in CO, at 25°C for 15 min is depicted in Figure 2A. The inorganic phosphorus peak has shifted to 1.1 ppm, indicating acidification of the tissue. All other phosphorus metabolites are severely reduced or absent, except an unidentified peak (possibly a sugar phosphate) which has shifted from 4.6 ppm in regular control and parasitized mosquito spectra (Fig. 1) to 3.4 ppm (Fig. 2A).

A

RESULTS

Typical 31P NMR spectra of runs for parasitized and control C. pipiens larvae are shown in Figure 1. The spectra for parasitized and control mosquito larvae are qualitatively very similar. The chemical shifts of ca. - 18.9, - 10.2, and -5.2 ppm were similar to reports for magnesium-bound B-ATP, a-ATP + wADP, and CX-ATP + B-ADP, respectively, at pH 6-8 (Fig. 1) (Thompson and Lee, 1986; Gadian et al., 1979). Phosphoarginine (invertebrate phosphagen), which was present in both control and parasitized mosquitoes (Fig. l), had a chemical shift of - 3.2 ppm and was identified by comparison of the chemical shift with reports in the literature (Barrow et al., 1980; Thompson and Lee, 1986). Inorganic phosphorus (Pi) was identified using a Hepes-buffered internal standard with H,PO, (PH 7.0). The chemical shift of the internal standard Pi (ca. 2.2 ppm) agrees with the value reported by Gadian et al. (1979) and coincides with the inorganic phosphorus peak present in both control and infected C. pipiens larvae (Fig. 1). This

B

1

I

I

I

30

20

IO

0

-10

,

I

-20

-30

PPM

FIG. 2. jlP NMR spectra at 4-6°C of (A) control C. pipiens larvae (371 mosquitoes/2.2-ml vol in NMR tube with spacer) that had been killed with 15-min exposure to CO, at room temperature (608 scans). (B) Fecal material from 5000 control C. pipiens larvae that were carbon-glutted for 1 hr (4800 scans). tC) Rornanomermis cdicivomu postparasites at 10 day PI (65001 4-ml vol in NMR tube) (2400 scans).

56

GIBLIN,

LEE,

AND

The ATP/ADP ratios for R. culicivovnxparasitized and control C. pipiens are reported in Table 1. There was a significant decline in the ratio (P d 0.05) in C. pipiens larvae with parasitemias of 2.3 + 0.32 parasites per host when compared with control mosquitoes. The addition of acetic acid for fungal control in the mosquito rearing water had no significant effect (P d 0.05) on the ATPi ADP ratios for control C. pipiens larvae. The ratios were 4.30 ? 1.37 (N = 4 runs) and 4.51 + 1.04 (N = 5) for mosquitoes reared at pH 7 and 4.5, respectively. Carbon glutting of control mosquito larvae for 2 hr depressed the ATPiADP ratio to 3.85 (2400 scans). The ratio of the same cohort of control larvae increased to 4.55 (2400 scans) when they were fed for 1 hr and returned to the NMR for spectral analysis. There were no discernible 31P NMR spectra for the voided gut contents of 5000 C. pipiens fourth instars (Fig. 2B). This indicates that the gut microflora does not contribute to the 31P NMR spectra of control or infected C. pipierzs larvae. NMR quantifications of ATP for R. crrlicirorax-parasitized and control C. pipiens at day 6 PI are reported in Table 2. There TABLE ATP/ADP

RATIOS*

FOR Rornat7ornemi.s

was no significant difference (P s 0.05) in concentrations of ATP in parasitized and control mosquitoes. Enzymatic determinations of ATP in PCA extracts from control C. pipierzs yielded values of 9.3 nmol ATPi mg dry wt. 1.69 nmol ATP/mg wet wt, or 5.1 nmol ATP per mosquito. These values agree with 31P NMR-derived values for C. pipiens control larvae and are close to the value determined enzymatically for ATP in the larval stage of the gall fly, Eurosta solidcrgirzis (Storey et al., 1981). A typical 31P NMR spectrum for IO-day PI R. culici~wax postparasites (6500 parasites/4 ml NMR tube volume) is shown in Figure 2C. There are discernible peaks at 4.6, 2.5, 0, -5.2, and - 10 ppm which probably represent an unidentified sugar phosphate, Pi, an unidentified compound, Mg-bound a-ATP + B-ADP, and Mg-bound CI-ATP + a-ADP, respectively. Because a peak at - 18.9 was not resolvable, no attempts were made to determine at ATPi ADP ratio or to quantify ATP with 31P NMR. Enzyme analysis of ATP in PCA-extracted R. crrlicivomx postparasites yielded 2.6 nmol ATPimg dry wt or 0.52 nmol ATP per nematode (two replicates). We contend that the contribution of I

c,lr/ic,i~,o~tr.r-P~~~sl~l~~~

AND

LarvaeiNMR tubeb

CONTROL

Cku

Q Survivalc

pipiens 57 4th instar+

ratio

Range

N

4.45

? I. I PAf

(3.10-6.24)

9

502 k

121A

93 k 5A

99 ? IwA

4.02

2 0.53AB

(3.55-4.70)

4

590 t

127A

97 k 3A

83 ? 20B

2.94

2 0.61B

(2.39-3.74)

4

709 t 266A

97 k 2A

71 t 24B

ATPiADP Cdex pipiens (control) C. pipiens (1.28 k 0.33 parasites/host)h C. pipiens (2.30 k 0.32 parasites/host)

PLATZER

n Data acquired with ,tP NMR spectroscopy (1200-2400 scans each run). b Mean number of C. pipiens larvae in a 4-ml volume of a NMR tube. c Mean percentage of C. pipirns larvae per NMR tube that survived 3 hr of ,‘P NMR analysis d Mean percentage of fourth-instar C. pipiens per NMR tube (the remaining larvae were third e Values presented as mean t SD. f Means followed by different capital letters in a column are significantly different (P < 0.05). g Data arc sine-transformed for analysis of variance. h Parasitemias represent the number of parasites per infected and uninfected hosts.

at 4-6°C. instars).

31P NMR

SPECTRA

OF PARASITIZED TABLE

QUANTIFICATIONS

OF ATP IN Romanomermis nmol

C44lex pipiens (control) C. pipiens (1.68 L 0.58 parasites/hostY

ATPimg dry wt mosquito

9.86

? 1.39’Af

11.64 12.91’

2 2.53A t 2.90A

2

cu/icivoru.~-PARASITIZED

N

57

MOSQUITOES

Mosquito IarvaeiNMR

AND

tubeb

CONTROL

Cftlex

% Survival’

pipiens % 4th instar+

3

626 2 112A

97 -c ?A

99 k IZA

5

696 k 230A -

98 k

75 2 25A

-

u-h See Table 1. i Dry weight contribution of the parasite burden was calculated subtracted from the mosquito dry weight and the parasite burden

phosphorus-containing metabolites by the parasite to the parasitized C. pipiens 31P NMR spectra was negligible. Comparisons of the dry weight contribution of the parasite burden to the total weight of the parasitized host (ca. 11%) and the low concentration of ATP in the parasite (one-fifth that of the host) suggest that the parasite ATP contributes very little (ca. 2.4%) to the parasitized host’s 31P NMR spectrum. Also, there was no significant difference (P 2 0.05) between the quantity of ATP in infected mosquitoes with or without adjustment for the parasite-contributed ATP (Table 2). DISCUSSION

Barany and Glonek (1984) have emphasized the importance of 31P NMR analysis in the qualitative and quantitative detection, characterization, and study of diseased states in mammalian tissues. It is only recently that the diseased status of invertebrates has been examined with the use of 31P NMR (Thompson and Lee, 1986). These authors noted that although the ATP/ADP ratio of the vector snail Biornphalaria glabrata was not affected by parasitism with the trematode Schistosoma mansoni, there were large differences in the relative abundance of other phosphorus metabolites, such as a phosphonate compound (at +22 ppm). Results from our work show that the 31P NMR spectra ob-

IA

(using 33 pg dry wt/6-day PI parasite) contribution for ATP was also subtracted.

and

tained from parasitized and control C. pipiens larvae (Fig. 1) were qualitatively the same, and very similar to values reported for another dipteran larva, E. solidaginis (Storey et al., 1984) and to another invertebrate, Tapes watlingi (see Barrow et al., 1980). Also, heavy parasitism by R. culicivorax caused a significant drop in the ATPI ADP ratio in the C. pipiens host (Table I). This drop coincided with a significant delay in mosquito development (higher proportion of third instars in infected C. pipiens, see Table 1). This is consistent with Giblin and Platzer’s (1985) report that development was significantly delayed by parasitism with R. culicivorax by day 6 PI. This should not affect the ATP/ADP ratio unless there are large stage-specific differences in ATP/ADP ratios. Giblin and Platzer (1985) noticed significant dry weight differences between R. clrlicivorax-parasitized and control C. pipiens, but only when the mosquitoes were heavily burdened. Bailey and Gordon (1973) reported that high R. culicivorax parasitemias in Aedes aegypti caused more gross pathology than occurred in lightly infected hosts. Thus, data on the ATP/ADP ratio from this paper support the idea that the parasitized host compromises itself in the long term by mobilizing fat body reserves and postponing imaginal disc formation in order to maintain the integrity of its energetic, osmotic, and hydrogen ion balance. It is also apparent that burdens with larger numbers of parasites stress the

58

GIBLIN,

LEE,

host more than lighter burdens both in the short and long terms. Giblin and Platzer (1984) reported a pH of ca. 7.4 for the hemolymph of C. pipiens third- and fourth-instar larvae using pH microelectrodes. Inorganic phosphorus from the spectra of control and parasitized mosquitoes was at ca. 2.2 ppm (Fig. I). Inorganic phosphorus in the inner cell was also at ca. 2.2 ppm, suggesting an intracellular pH of 7. This comparison can be used only as a rough approximation of pH since ionic strength and other factors in the environment can alter the chemical shift of Pi (Barrow et al., 1980) and the internal standard solution we used was not similar to the intracellular or hemolymph composition. ATP/ADP ratios and ATP levels in live larvae of diseased and healthy C. pipierzs were effectively compared by 31P NMR. The major problem involved temperature. C. pipiens larvae could not survive for 2 hr in an oxygenated and perfused NMR tube at 25-27°C. When large numbers of larvae were packed into the 4-ml volume, access to the small breathing space was insufficient and the larvae died. This difficulty was overcome by running the experiments at 4-6°C. However, this temperature is too low for timed course experiments for metabolic inhibitors or anoxia. Thus, the experimental design described herein may be realistic only for interval comparisons in the development of the mosquito and/or the progress of a disease. Energy status comparisons with 31P NMR would be best for nonmetabolic pathogens such as viruses since the energetic contribution of the parasite or pathogen would not need consideration. ACKNOWLEDGMENTS We thank Dr. Ken Yamada, Biology Department, University of California, Riverside, for his assistance, ideas, and suggestions during this project; Dr. S. Nelson Thompson. Department of Entomology, University of California. Riverside. for his support. sug-

AND

PLATZER

gestions, and critical review of the manuscript: and Ms. Donna Viverette for general assistance. This project was supported in part by Research Grant Al-15717 from NIAID, U.S. Public Health Service, National Institutes of Health.

REFERENCES ADAMS, H. 1963. Adenosine 5’-triphosphate, determination with phosphoglycerate kinase. In “Methods of Enzymatic Analysis.” (H. U. Bergmeyer. ed.). pp. 539-543. Academic Press, New York. BAILEY, C. H.. AND GORDON. R. 1973. Histopathology of Aedes uegypti (Diptera: Culicidae) larvae parasitized by Reesimermis nielseni (Nematoda: Mermithidae). J. Inrlerfehr. P~thol., 22, 435-441. BARANY, M.. AND GLONEK, T. 1984. Identification of diseased states by phosphorus-31 NMR. 112 “Phosphorus-3 1 NMR. Principles and Applications.” (D. G. Gorenstein. ed.). pp. 511-548. Academic Press, Orlando. Fla. BARROW, K. D.. JAMIESON. D. D.. AND NORTON, R. S. 1980. 3iP nuclear-magnetic-resonance studies of energy metabolism in tissue from the marine invertebrate Tupes hvatlingi. Eur. J. Biochem.. 103, 289-297. CASTILLO. J. M., CHIN, P.. AND ROBERTS, D. W. 1982. Growth and development of Romanomermis culiciIWUX in ritr-o. J. Nemtrtol.. 14, 476-485. GADIAN. D. G., RADDA. G. K., RICHARDS, R. E.. AND SEELEY, P. J. 1979. ,iP NMR in living tissue: The road from a promising to an important tool in biology. In “Biological Applications of Magnetic Resonance.” (R. G. Shulman. ed.). pp. 463-535. Academic Press, New York. GIBLIN. R. M.. AND PLATZER, E. G. 1984. Hemolymph pH of the larvae of three species of mosquitoes. and the effect of Rornunornerrnis culicivovax parasitism on the blood pH of Culex pipiens. J. Invertehr-. Pathol.. 44, 63-66. GIBLIN, R. M.. AND PLATZER. E. G. 1985. Rornrrnomerrnis cu/ici\~ora.u parasitism and the development. growth. and feeding rates of two mosquito species. J. Invertehr. Pathol., 46, I I- 19. PLATZER. E. G.. AND STIRLING, A. M. 1978. Improved rearing procedures for Romanomerrnis cu1ici~wtr.u. Proc. Calif. Mosq. Vector Control Assw., 46, 87-88. POWERS, K. S., AND PLATZER. E. G. 1984. Oxygen consumption in mosquito larvae parasitized by Romanomermis culicivorax (Nematoda). Comp. Biothem. Physiol. A, 78, 119- 122. POWERS, K. S., PLATZER. E. G., AND BRADLEY, T. J. 1984. The effect of nematode parasitism on the osmolality and major cation concentration in the haemolymph of three larval mosquito species. J. Insect Physiol., 30, 547-550.

31P NMR

SPECTRA

OF PARASITIZED

SCHMIDT. S. P.. AND PLATZER, E. G. 1980. Changes in body tissues and hemolymph composition of Culex pipiens in response to infection by Rornanomennis

culicivorax. J. Invertebr. Pathol, 36, 240-254. STOREY. K. B., BAUST. J. G.. AND STOREY, J. M. 1981. Intermediary metabolism during low temperature acclimation in the overwintering gall fly larva, Elcrosta solidoginis. J. Cotnp. Physiol. B.. 144, l83- 190.

MOSQUITOES

59

STOREY, K. B., MICELI, M., BUTLER, K. W., SMITH, I. C. P., AND DESLAURIERS. R. 1984. “P-NMR studies of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis. Eur. J. Biochern.. 142, 591-595. THOMPSON. S. N., AND LEE, R. W. K. 1986. 31P NMR studies on adenylates and other phosphorus metabolites in the schistosome vector Biomphalaria g/ubrata. J. Parmitol.. in press.

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