Protein and lactate dehydrogenase levels in Aedes aegypti

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Insect Biochem., 1975, Vol. 5, pp. 331 to 335. Pergamon Press. Printed in Great Britain

PROTEIN AND LACTATE DEHYDROGENASE LEVELS IN AEDES A E G Y P T I C H A R L E S S. BARNES, P A U L M. T O O M and E D D I E CUPP* Department of Chemistry and Department of Biology, University of Southern Mississippi, Hattiesburg, Mississippi 39401, U.S.A. (Received 10 August 1974)

Abstract--Lactate dehydrogenase (LDH) and total protein concentrations of adult female Aedes aegypti were determined following emergence from the pupal cuticle through a period of 20 days, during which time 2 blood meals were taken. Preceding the blood meal, LDH exhibited distinct peaks 5 and 10 days following emergence. LDH activity remained constant for 2 hr following a blood meal, then rose rapidly to a value 10 times pre-blood meal values 7 hr after feeding. This activity then steadily declined to reach pre-blood meal values within 72 hr. This pattern was repeated in mosquitoes given a second blood meal as well as in virgin mosquitoes taking blood, suggesting that the blood meal stimulates either the synthesis or activation of LDH. INTRODUCTION THE PHYSIOLOGICALand endocrinological mechanisms involved in ovarian maturation and vitellogenesis in insects have received considerable attention (ENGELMANN, 1970; HAGEDORN and JUDSON, 1972; ROTH and PORTER, 1964). Of particular interest are reports establishing that in Aedes aegypti, the yellow fever mosquito, yolk protein is synthesized in the fat body (HAoEDORN and JUBSON, 1972) rather than in the midgut as suggested by ROTH and PORTER(1964). Further investigations by HAaEDORN et al. (1973) have shown production of vitellogenin (yolk protein) to be stimulated by the blood meal which ultimately initiates the synthesis of both messenger and ribosomal RNA. This synthetic process appears to be controlled by a hormone produced by the medial neurosecretory cells (LEA, 1967). Thus, while the general physiological processes describing the activation of vitellogenin can be described, little is known about the biochemical processes underlying these events. The purpose of this paper is to report on the increase in protein concentration and lactate dehydrogenase activity at different times in a laboratory population of adult mosquitoes, including the apparent stimulation of lactate dehydrogenase by the blood meal. MATERIALS AND METHODS Adult Aedes aegypti (Tampa strain) which had been raised by the standard laboratory techniques described by ABOUALYand HORSFALL(1968) were used for all experimentation. Larvae were fed live yeast whereas adults were maintained on 5 % sucrose and held at 27°. Adults 10 to 30 days after emergence were fed on a human hand for 5 min. Individuals not engorged at the end of 5 min time were discarded. Replete mosquitoes were *Present address: Department of Entomology, Cornell University, Ithaca, New York, 14850, U.S.A. 331

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CHARLESS. B~-ES, PAULM. TOOM, AND EDDIE CuPP

collected, frozen and weighed. For each time period to be analyzed, five individuals were homogenized in 200 gl distilled water and centrifuged at 2000 g for 15 min. Total protein was determined by a modified method of Lowry as described by H~Tm~ (1972). Lactate dehydrogenase activity was assayed by incubating 100 gl of mosquito homogenate and 2"5 ml of 0"3 mM NADH in 0"1 M phosphate buffer (pH 7.4) at 37° for 30 rain. The lactate dehydrogenase specific reaction was then initiated by the addition of 0"2 ml of 10 mM sodium pyruvate with the decrease in absorbance at 340 nm measured continuously on a Beckman model DBG spectrophotometer with the cuvets thermostated at 37°. All activities were expressed in international units per mg protein at 25 ° (Tim'z, 1970),

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FIG. 1. Protein concentrations as a function of age before blood meal (a) and after blood meal (b). 9~--O--Q---Q ~tg protein/rag body weight; 0 - - 0 - - 0 - - 0 Ilg protein/mosquito. Protein was assayed by a modified Lowry technique. Each point represents the average of 30 individuals which were 4-6 hr in age of one another. RESULTS Both total protein per mosquito and protein per mg body weight decreased for 2 days following emergence from the pupal cuticle (Fig. la). These concentrations then remained constant at about 50% of the immediate, post emergence levels until day 8 when protein levels (both total and per mg mosquito body weight) rose sharply, reaching peak values on day 10 well in excess of levels seen immediately after emergence. This high protein concentration was only temporary, for by day 11, values were near or below pre-emergence levels and by day 14 were once again about 50% of the immediate post emergence levels. Following a blood meal (Fig. lb), protein values rapidly decreased for 3 days, after which time they remained constant at a value slightly higher than values immediately preceding the blood meal. Total lactate dehydrogenase activity was found to rapidly decrease the first day following emergence (Fig. 2). Between emergence and the taking of the first blood meal, 2 peaks of lactate dehydrogenase activity were found--one occurring on day 5, the second occurring on day 10. Following the blood meal, no rise in lactate dehydrogenase activity was noted for 2 hr (Fig. 3). However, following

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FIG. 2. Lactate dehydrogenase activity as a function of age before blood meal. Activity is expressed as International Units/rag protein at 25 °. Each point represents the average lactate dehydrogenase activity of 15 individuals.

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FIG. 3. Lactate dehydrogenase activity as a function of age following a blood meal. Activity is expressed as International Units/rag protein at 25 °. Each point represents the average lactate dehydrogenase activity of 15 individuals. Blood meal was taken (0 time) 15 days following emergence from the pupal cuticle with a second blood meal taken 72 hr after the first. 4 ~ - - Q - - q ~ - - O mosquitoes mated prior to blood meal; O m O - - O m O mosquitoes not mated prior to blood meal.

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CHARLESS. BARNES,PAULM. TOOM, AND EDDIE CUPP

this 2 hr lag period, lactate dehydrogenase activity rose rapidly, reaching a value approximately 10 times higher than pre-blood meal values within 7 hr of ingestion of blood. Lactate dehydrogenase activity then decreased until pre-blood meal values were reached approximately 72 hr after the blood meal. A second blood meal produced a similar response, although lactate dehydrogenase activity did not peak at quite so high a value at 7 hr. A similar response was observed in both inseminated and virgin mosquitoes (Fig. 3). DISCUSSION AND CONCLUSIONS The decrease in both total protein and in protein per mg body weight for the first 2 days following emergence from the pupal stage can probably be attributed to the partial utilization of pupal and even larval protein as an energy source. It has been shown that larval abdominal muscles in Culex pipiens are histolysed following emergence of the adult (ROUBAUD, 1932) and adult Aedes aegypti excrete large amounts of uric acid for several days after emergence (TE~IAN et al., 1957). Our finding of a decrease in total protein concentration for this same period time would strongly suggest that much of the uric acid nitrogen is indeed derived from protein and thus the mosquito is using protein as an energy source for the first hours following adult emergence. The two peaks of protein concentration at days 5 and 10 are difficult to explain. Since this strain of Aedes aegypti is anautogenous, the likelihood that protein is being distinctly sequestered for egg production on those days can be discounted. Examination of developing ovaries revealed that following extended sucrose feedings, these organs were in the usual resting stage and would require a blood meal for maturation of follicles. Therefore, the endogenous rhythm which acts to concentrate protein at days 5 and 10 is most likely associated with fat body development as a result of sucrose feeding as well as enzyme production for basic catabolic processes. The steady decrease in total protein and in protein per mg body weight following the blood meal but preceding the deposition of eggs shows that only a portion of the protein derived from the blood meal is ultimately incorporated into yolk protein. It is known that during and for several hours after engorgement, this species excretes a clear urine containing fluid (CHRISTOPHERS, 1960) which could in turn contain small amounts of undigested and partially digested protein. However, whether the insect is utilizing some of the protein from the blood meal as an energy source or whether all of the digested protein is being incorporated into egg protein, must await further investigation. It is generally recognized that in mammalian tissue, lactate dehydrogenase serves as a means of rapidly generating ATP under anaerobic conditions. The increased level of this enzyme 5 and 10 days after emergence of the adult mosquito would suggest that the enzyme serves the same function in the mosquito and that the metabolic activity in the insect (and thus energy requirements) reach relatively high values during these times. These results are similar in principle to the report of VED BRAT and WHirr (1974) who observed an increased lactate dehydrogenase

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activity before emergence of Anopheles albimanus. This morphogenetic period is also one of great metabolic activity since it is during this time that the adult is formed. The fact that lactate dehydrogenase activities do not rise for the first 2 hr following the blood meal shows that increased lactate dehydrogenase values are not due to ingested enzyme. Thus, the sharp rise in lactate dehydrogenase activity following this 2 hr lag period both after the first and second blood meal and in both inseminated and virgin mosquitoes strongly suggests that this increase in activity is stimulated by the blood meal. This sharp rise in lactate dehydrogenase activity parallels the release of ' b r a i n ' hormones determined from previous reported ligature experiments (LEA, 1967), as well as vitellogenin synthesis within the fat body of Aedes aegypti (HAOEDORNet al., 1973). In the latter report it was shown that RNA concentrations began to rise two hours after the blood meal, and reached their highest concentrations 12 hr following the blood meal. Additionally, the synthesis of yolk proteins by the fat body began after the blood meal and reached its maximum rate in 24 hr. It would thus appear that one of the enzymes coded for in the newly synthesized RNA is lactate dehydrogenase. If this is indeed true, it is the first reported energy yielding enzyme whose synthesis can be stimulated by the taking of a blood meal by Aedes aegypti. REFERENCES ABOUALYA. and HORSFALLW. R. (1968) Bionomics of Paorophora varipes, a model laboratory mosquito. J. econ. Ent. 61, 1657-1660. CHRISTOPrmRSS. R. (1960) Aedes aegypti (L.) the Yellow Fever Mosquito. Life History Bionomics and Structure, p. 493. Cambridge University Press, Cambridge. ENCELMANNF. (1970) The Physiology of Insect Reproduction, pp. 45-56. Pergamon Press, New York. HACEDORN,H. H., FALLONA. M., and LAUFERH. (1973) Vitellogenin synthesis by the fat body of the mosquito Aedes aegypti. Develop. Biol. 31, 285-294. HAGEDORNH. H. and JUDSONC. L. (1972) Purification and site of synthesis of Aedes aegypti yolk proteins. J. exp. Zool. 182, 367-378. HARTREEE. F. (1972) Determination of protein: A modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48~ 422--427. LEA A. O. (1967) The medial neurosecretory cells and egg maturation in mosquitoes. ft. Insect Physiol. 13, 419-429. ROTH T. F. and PORTERK. R. (1964) Yolk protein uptake in the oocyte of the mosquito Aedes aegypti. ~. Cell Biol. 20, 313-332. ROUBAUDE. (1932) Des phenomenes d'histolyse larvaire post-nymphale et d'alimentation imaginal autotrophe chez le moustique commun (Culex pipiens). C. 17. Acad. Sci., Paris 194, 389-391. TIETZ N. W. (Ed.) (1970) Fundamentals of Clinical Chemistry, pp. 438-441. W. B. Saunders, Philadelphia. TERZAXNL. A., IRR~eEaREF., and STAHLERN. (1957) A study of nitrogen and uric acid patterns in the excreta and body tissues of adult Aedes aegypti. ~. Insect Physiol. 1, 221-228. VED BRATS. S. and WHXTTG. S. (1974) Lactate dehydrogenase and glycerol-3-phosphate dehydrogenase gene expression during ontogeny of the mosquito (Anopheles albimanus). ft. exp. Zool. 187, 135-140.