Nitrogen partitioning and blood meal utilization by Aedes aegypti (Diptera Culicidae)

.I. Insect Physiol., Vol. 25, pp. 841 to 846. Pergamon Press Ltd. 1919. Printed in Great’ Britain.

NITROGEN PARTITIONING AND BLOOD MEAL UTILIZATION BY AEDES AEG YPTI (DIPTERA CULICIDAE) K. R. FRANCE* and C. L. JUDSON Department of Entomology, University of California, Davis, CA, U.S.A. (Received

5 December

1978; revised 22 May

1979)

Abstract-The

utilization of the blood meal by mosquitoes was investigated by first feeding females quantities of blood ranging from 1 to 5 mg, and then analyzing the faeces for the various by-products 01 protein catabolism that were subsequently eliminated. The nitrogeneous waste products in order of importance were uric acid, histidine, ammonia and arginine. Only traces of the other amino acids were excreted. The total amount ofeach faecal substance varied linearly with thequantity ofblood ingested, however their relative proportions did not change. Regardless of blood meal size the quantity of uric acid and ammonia produced indicates that about 80% of the non-histidine and arginine amino acids are deaminated and utilized for metabolic purposes other than egg protein synthesis. Most of the histidine and about one half of the arginine content of the blood were excreted as free amino acids, but the other amino acids were lost in trace amounts. Nineteen per cent of the total ingested amino acids was incorporated into soluble yolk proteins and this proportion was constant even for small blood meals that result in a reduction in the numbers of eggs produced. The comparative aspects of nitrogen partitioning and blood meal utilization by haematophagous insects. as well as the factors that affect blood meal utilization and fecundity in A. aegypti are discussed. Key Word Index:

Aedes aegypti, nitrogen

utilization-excretion

INTRODUCTION THE NUTRITIONAL dependency of Aedes aegypti on blood for energy and reproduction carries with it the problem of eliminating a large quantity of waste nitrogen without excessive water loss or energy expenditure. Except for the excellent work of BURSELL (1960, 1965) on Glossina morsitans, little has been done to evaluate the physiological problems associated with blood feeding or the compensatory mechanisms that insects have evolved to meet them. Various aspects of nitrogen excretion by sugar-fed (IRREVERRE and TERZIAN, 1959) and blood-fed (TERZIAN et al., 19.57; THAYER et al., 1971) female A. aegypti have been investigated. Although these studies described the kinds and relative amounts of nitrogeneous waste products contained in the faeces, the relationship of these substances to ingested protein has never been studied. The goals of this work were to investigate some of the physiological mechanisms utilized by A. aegypti for eliminating waste nitrogen and to provide insight into the utilization of the ingested blood proteins. This species is ideally suited for a study of blood protein metabolism and utilization because blood ingestion is rapid, can be manipulated experimentally and is easily measured. Human blood is well defined chemically, and almost 99% of its nitrogen is contained in the protein component (ALBRITTON, 1951). The amino acid composition of human blood (BURSELL, 1965) *Present address: The Medical College of Wisconsin, Allen-Bradley Medical Science Laboratory, Dept. of Environmental Medicine, 8700 West Wisconsin Avenue, Milwaukee, Wisconsin 53226, U.S.A. I.P.2511 I--n

841

and mosquito known.

egg

proteins

MATERIALS

(CHANG, 1976) is also

AND METHODS

Biological material Larvae of the Liverpool strain of A. aegypti were reared on liver powder at 27°C. Adults were maintained on sucrose for at least 4 days after emergence and prior to blood feeding to allow for the excretion of nitrogenous wastes accumulated during the immature stages. After this period the loss of nitrogen in the urine of unfed females was found to be quantitatively unimportant. At 4-6 days, post emergence, females were weighted, .fed on a human hand, and reweighed within 45 set of feeding. They were then placed in separate vials for 72 hr at 25°C and 75% r.h. by which time ovarian development and the elimination of blood meal wastes was usually completed. For certain experiments females were depleted of carbohydrate and triglyceride reserves by maintaining them on water only, for 5 days after emergence (NAYAR and SAUERMAN, 1975a). Uric acid Uric acid was dissolved from the faecal residue that accumulated in the vial with 0.01 M Li,CO,. Since the quantity of uric acid produced per female varied with blood meal size, the volume of Li,CO, was adjusted to give uric acid solutions of approximately equal concentrations. Volumes ranged from 1 ml for 1 mg blood meals to 10 ml of Li,CO, for blood meals of 5 mg. Anew method for the quantitative determination of

K. R. FRANCEAND C. L. JUDSON

842

uric acid was developed, based on the loss of peak absorbancy at 295 nm when uric acid is oxidized after incubation with sodium hydroxide and cupric sulphate (GRIFFITHS, 1952). This method was compared with the uricase procedure of LIDDLE et al. (1959) on 20 samples of mosquito faecal extract. No significant difference between the two methods was noted at the 95% significance level using a paired comparison t test. The two stock solutions used in the procedure were a 7.5 N NaOH and a 0.21% CuSO;SH,O in 7.5 N NaOH. The second was made by adding a 1% aqueous cupric sulphate solution to 7.5 N NaOH. Water free of metallic ions was used throughout and both solutions were stored in Pyrex bottles. Prior to the assay the 7.5 N NaOH was diluted to a 0.0925 N working solution. Two millilitres of the working solution were added to 1 ml of Li,CO, sample solution, mixed, and the A,, nm recorded immediately. Twenty-five microlitres or about one drop from a Pasteur pipette of the cupric sulphate stock solution was added to each tube, mixed and incubated at 60°C for 15 min. After incubation, tubes were cooled to room temperature with tap water and the Azg5nmrecorded. Uric acid was calculated by the formula

A295nm initia1-A295 nm final = ~guric

acid/ml

0.0803 Amino acids and ammonia The faecal residue was dissolved by adding 5 ml of 0.01 M Li,CO,. Uric acid and haematin were then precipitated by acidification with HCl. After centrifugation the supernatant solution was removed and reduced to dryness under nitrogen. Amino acids and NH,Cl were then taken up in pH 2.2 sodium citrate buffer (0.2 N, N $) and assayed in a Beckman model 121 amino acid analyzer. Protein Ovarian protein was extracted from mature ovaries with Hayes’s mosquito saline and colourimetrically assayed (LOWRY et al., 1951). Results agreed well with those obtained using a micro-biuret method (ITZHAKI and GILL, 1964). The weight of protein was converted to the equivalent weight of amino acids by adding 14% to correct for water of hydrolysis. The nitrogen content of egg proteins was calculated from their amino acid composition (CHANG, 1976). RESULTS Nitrogen partitioning The data in Table 1 lists the occurrence of nitrogenous substances in the faeces. These values are expressed as a percentage of the total ingested protein nitrogen. The proportionality between ingested nitrogen and the nitrogen excreted in each category of faecal substances was uniform for blood meals which ranged from 1 to 5 mg. About 60% of the ingested nitrogen was excreted as uric acid and ammonia. It originates primarily from the deamination of amino acids other than histidine and arginine. The high urate and ammonia levels indicate the utilization of a large proportion of these amino acids for metabolic purposes; while the very

Table 1. The ratio between ingested amino nitrogen* and the nitrogen content of faecal components

Faecal component Uric acid Ammonia Histidine Arginine Other amino acids?

Sample size

2; of total ingested amino N excreted

rt

100 23 23 22 4

52.0 7.80 11.9 2.30 <0.50

0.884 0.939 0.986 0.861 0.970

*Blood meal sizes ranged from 1 to 5 mg: proportional values did not differ within this range. t This group of amino acids is related on the basis of total weight ingested versus total weight excreted. Histidine, arginine and ammonia were assayed simultaneously in the same samples. small quantity of amino acids lost in the faeces suggests a highly efficient conservation mechanism. In another experiment, both uric acid and ammonia were measured in the same eight individuals and it was found that the two substances occurred in a molar ratio near unity. Individual urate to ammonia ratios ranged from 1.02 to 1.39 (X = 1.19, SD. = 0.131). This phenomenon has been reported by VAN HANDEL (1975) for ‘several species of mosquitoes,’ but the species were not named and no data was included. Histidine and arginine constitute 10.4% of the ingested amino acids but are important to nitrogen balance because they contain about 25% of the nitrogen content of the blood proteins. In contrast to urate and ammonia, they are produced directly from the digestion of the blood proteins. Haematin was also identified in the faeces by its absorption spectrum, and was quantitatively estimated by its peak absorbancy at 387 nm. It appeared to be excreted in amounts approximating those ingested. Amino acid partitioning All of the amino acids are utilized in egg production. Estimates of the proportion of amino acids incorporated into egg protein (Table 2) were based on a total of 42 females, subdivided into three blood meal size categories: 23 had ingested between 1.85 and 2.92 mg of blood, 7 between 3.09 and 3.91 mg and 12 between 4.02 and 4.86 mg. These three groups converted 19.7, 17.3 and 18.7%, respectively, of ingested protein into egg protein, giving an overall average of 19%. Sixteen starved females, which ingested between 2.1 and 4.86 mg of blood, converted an average of 16.5% of ingested protein into egg protein. Incontrast to the other amino acids ingested, most of the histidine and much of the arginine that were not utilized for egg protein production, were excreted. It can be estimated from the quantity of nitrogen eliminated as urate and ~ammonia that 80% of the amino acids excluding arginine and histidine were deaminated and became available for metabolic purposes. Our data (Table 1) indicates that this pattern of amino acid partitioning remains unchanged even for small blood meals which result in a reduction in the numbers of eggs produced (WOKE et al., 1956).

Blood meal utilization by Aedes aegypti

843

Table 2. The partitioning of amino acids derived from ingested blood Total Amino acid

Egg* proteins

Faecest

CatabolizedS

Accounted for

Arginine Histidine Others

24.0 12.0 15.3

25.0 78.3 <0.50

? 80.0

49.0 90.3 95.8

Unaccounted for 51.0 9.1 4.2

Values are percentages of total quantity of each ingested. *Calculated from protein content of mature ovaries and amino acid composition of egg protein (CHANG,1976). t Derived from data in Table 1. $ Estimated from the quantity of nitrogen excreted as uric acid and NH ‘, (Table 1) and the nitrogen content of the amino acids excluding arginine and histidine, in the ratio that they occur in the blood proteins. THAYERet Q/.(1971) showed that there is a slight rise in tissue levels of most amino acids by the time blood meal assimilation, oiigenesis, and excretion are completed, but that it is quantitatively insignificant relative to the quantities ingested. Since they found that there is no retention of arginine, we suggest that the proportion of ingested arginine we were unable to account for may be catabolized.

DISCUSSION

Nitrogen excretion Histidine and arginine were first recognized as true nitrogenous waste products by BURSELL (1965), who recovered 82% and 95% respectively of the ingested histidine and arginine from the faeces of blood-fed G. morsitans. Since histidine and arginine have a nitrogen content rivalling that of uric acid, (27, 32 and 33% respectively), BURSELL (1965) suggested that their catabolism would be metabolically wasteful to the insect because the resulting waste nitrogen must be eliminated as uric acid. A. aegypti is very similar to G. morsitans in the excretion of histidine, and we believe that this aspect of nitrogen excretion may be widespread among other haematophagous species. Calculations from the data of BRIEGEL (1969) show that 74% of the ingested histidine can be accounted for in the eggs and faeces of Culex pipiens fatigans. In addition, analysis of the faeces of the blood-sucking bug, Rhodnius pvolixus (HARINGTON, 1956) showed that large quantities of histidine, and its decarboxylation product histamine, were excreted relative to the other amino acids. No such generality is apparent in the excretion of arginine by the few haematophagous insects studied. In contrast to G. morsitans, R. prolixus excretes arginine only occasionally and then in trace amounts (HARINGTON, 1961). Our findings indicate that A. aegypti may occupy an intermediate position between these two extremes. An insect could economically gain energy from arginine despite its high nitrogen content if it possessed the enzyme arginase which converts arginine to urea and ornithine. The initial arginase step does not require energy and reduces the nitrogen content or arginine by 50% with the loss of a carbon atom. The excretion of amino acids tends to work against

the reabsorption of water since they increase the osmotic strength of the urine. However, the substitution of equimolar quantities of urea for arginine would leave the osmotic strength of the urine unchanged. If an insect catabolizes argiiiine via this pathway, the quantity of urea excreted should equal the quantity of arginine ingested minus that which is utilized for protein synthesis, retained in tissue pools, or otherwise stored. Such a quantitative relationship has been demonstrated in the phytophagous bug Nezara viridula (POWLES et al., 1972). Urea is a significant component in the faeces of several species of mosquitoes including A. aegypti (IRPLEVERREan’d TERZIAN, 1959), and large quantities are excreted by R. prolixus (HARINGTON, 1961), but only trace amounts occur in the faeces of G. morsitans which excretes almost all of the ingested arginine (BURSELL, 1965). Since mammalian and chicken bloods contain considerable quantities of pre-formed urea, the metabolic production of urea from arginine in haematophagous insects remains to be demonstrated. Uric acid and ammonia are unique among the waste products because they are produced as a result of the breakdown of amino acids rather than from the digestion of protein. Most (86%) of nitrogen from amino acid catabolism is excreted as uric acid but a significant proportion (14%) is ultimately eliminated as ammonia. The high levels of uric acid are consistent with the findings for other haematophagous insects, and ammonia has also been reported as a faecal constituent in many. However, our results are the first to demonstrate that the quantity of ammonia is directly related to both the quantity of protein ingested, and to the amount of uric acid produced. Ammonia production may result from the breakdown of uric acid in the lumen of the hind-gut or directly from amino acid deamination. If faecal ammonia is originally produced from amino acid deamination, the metabolic cost of uric acid synthesis would be reduced. Furthermore, deamination itself would produce additional energy and thus might constitute another ivay for gaining energy from ingested amino acids. The presence of both uricotelic and ammonia-excreting mechanisms would be adaptive because the two systems functioning together would permit the insect to reach an optimum between water balance and the conservation of metabolities. An ammonia-excreting mechanism involving amino acid deamination by hind-gut tissue and the direct secretion of ammonia

844

K. R. FRANCEANDC. L. JUDSON

ions against a concentration gradient into the hind-gut lumen has been elucidated by PRUSCH (1972) in Sarcophaga bullata, The precise molar ratio between uric acid and ammonia in A. aegypti, and in other uricotelic animals such as some birds and reptiles (VAN HANDEL, 1975), may favour the hypothesis that ammonia excretion is an integral part of the nitrogen metabolism of uricotelic organisms rather than an artifact of the decomposition of uric acid or related purines.

The utilization of amTno acid metabolities Deamination converts amino acids to their ketoacid analogues, some of which are TCA intermediates, which can be utilized for energy or for the synthesis of other substances. Considerable data exists concerning the major processes that draw on amino acid metabolites during blood meal assimilation and oiigenesis, and it is possible to estimate how A. aegypti partitions the metabolites derived from the blood meal (Table 3). Based on our uric acid and ammonia measurements we calculate that 69 pg of carbon in the form of amino acid metabolites becomes available for each mg of blood ingested. From this total, 14 pg of carbon, in the form of formate and glycine, are utilized for the synthesis of uric acid. This represents most of the metabolic cost of uricoteliim but does not include the energy of synthesis. Triglyceride production is another process that uses available metabolites both as a source of substrate and for the energy of synthesis. Triglyceride production was measured by NAYAR and SAUERMAN (1975a), who found that female A. aegypti that had been depleted of triglyceride and glycogen reserves, acquire new triglyceride stores by 72 hr after blood ingestion. If no further nourishment is provided these new reserves undergo a steady decline; presumably due to their utilization for maintenance energy. Maximal triglyceride levels at 72 hr are equal to 0.43 cal, or 47.4 pg triglyceride, per female. Assuming triglyceride to be 80% carbon and dividing by the total mg of blood ingested, it can be estimated that 10 pg C are incorporated into triglycerides for each mg of blood ingested. A large proportion of the remaining metabolites are utilized for energy (HUESNER and LAVOIPIERRE, 1973a,b). Their studies show that oxygen consumption during the 72 hr period of blood meal assimilation and oGgenesis consist of two components. The first is the same in unfed and blood fed individuals, and represents the energy required for Table 3. The utilization of deaminated amino acids Carbon /%

1. 2. 3. 4.

Total amino carbon available for metabolic purposes per mg of ingested blood Utilized for uric acid synthesis Utilized for triglyceride synthesis Utilized for maintenance energy Utilized associated

for the energy with oiigenesis

69 14 10* 9.57

expenditure 16.5

*Calculation from NAYAUand SAUERMAN (1975a). t HEUSNERand LAVOIPIERRE (1973a).

maintenance. (1.02 ~1 O,/ml live weight/hr). The secdnd component appears as a sharp increase in 0, uptake occurring within hours of blood feeding. It equals a constant value of 71 ~1 O,/mg blood regardless of the total amount of blood ingested. We believe that this sharp rise in oxygen consumption is due, in part, to the energy demands of digestion, uriC acid, triglyceride, and protein synthesis, and to the specific dynamic action of protein. These authors point out that the total oxygen consumption during this period represents the catabolism for energy of about 30% of the total ingested protein regardless of blood meal size. Thus uric acid synthesis, triglyceride synthesis, and energy expenditure for maintenance and to support the metabolic processes associated with oiigenesis and blood meal assimilation account for the utilization of 72% of the total deaminated amino acids. (Table 3). Significant quantities of chitin, for egg chorion formation, are also synthesized during oiigenesis. However, since little is known of this process in the mosquito, an estimate of its quantitative importance is currently impossible.

Utilization of amino acids for reproduction A dietary source ,of amino acids is essential for reproduction in A. aegypti, and the quantity of yolk protein produced is correlated with the number of eggs matured. Since deamination and protein synthesis occur simultaneously, factors that alter the partitioning of amino acids between the two processes are potential determinants of fecundity. A. aegypti produces more eggs from the blood of some host species than from others, and this has been attributed to a better amino acid balance in the blood proteins of the former (LEA et al., 1958). We believe that a better amino acid balance increases the protein synthetic rate and results in a greater proportion of the ingested amino acids captured for egg production. Except for this shift in the proportionality between deamination and protein synthesis, the kind of blood ingested should not otherwise greatly affect the manner in which the insect metabolizes and utilizes the blood meal. NAYAR and SAUERMAN (1975b) have shown that females depleted of triglyceride stores prior to blood ingestion produce fewer eggs per mg of ingested blood. Our results show that these reserve-depleted females also convert a smaller proportion of ingested amino acids into egg proteins (16.5% vs 19%). Although a correlation exists between the absence of energy reserves and reduced protein synthesis, or egg production, the validity of the assumption that this is a causal relationship, as claimed by these authors, is uncertain. Starvation produces a highly stressed and physiologically weakened individual in which atrophy of the fat body (the site of yolk protein synthesis) iS evident. Thus the reduction in egg protein could be explained on the basis of a reduced rate of protein synthesis. In addition, the conclusions of these authors that “blood feeding after sugar feeding contributes to egg production only” and that only starved females utilize blood for energy, are untenable in light of the evidence presented here. Egg numbers are influenced by hormonal, as well as by nutritional factors, and for this reason using the

Blood meal utilization by Aedes aegypti

0.6

I

EaAmmonia q Protein

0

U-ic odd

0.6

0.6

C t p,

z f P f ;

0.4

04

B *

ITI t

:.

‘1

0.2

r 0.2 -

~ A

8

Fig. 1. A comparison of the proportion of the ingested protein channelled into protein synthesis and the proportjon of ingested amino acids catabolized for metabolic purposes (as estimated by the proportion of ingested protein nitrogen excreted as uric acid and ammonia) by three haematophagous insects. A. A. aegypti; B. G. morsitans, constructed from the data of BURSELL(1965); C. C. pipiens fatigans, constructed from the data of BRIEGEL(1969), and IRREVERRE and TERZIAN(1959).

number of eggs produced as an end point for evaluating nutritional effects should be approached with caution. For example, WOKE et al. (1956) reported that maximal numbers of eggs were reached by A. aegypti at a blood meal size of about 2.0mg and that no further increase occurred up to approx. 5.0 mg

of ingested blood. Our results, over the same meal size range, show that a constant 19% of the blood meal is converted to ovarian proteins. These results suggest that the protein level per egg might vary, as well as the total number of eggs, with differing amounts of ingested protein. Figure 1compares the utilization of ingested amino acids by A. aegypti, G. morsitans and Culex pipiens fatigans. Protein comparisons were based on the soluble yolk proteins of A. aegypti, ovarian protein hydrolysates of C. pipiens fatigans, and the thoracic muscle proteins synthesized during the first feeding cycle by male G. morsitans. Only the nitrogenous

wastes produced by the deamination of amino acids were considered since they indicate the proportion of amino acids catabolized for metabolic purposes. Data for nitrogen excretion by C. pipiens fatigans was derived from two sources. BRIEGEL (1969) did not measure ammonia, yet this substance had been shown to comprise 10% of thk total excreted nitrogen of this species (IRREVERREand TERZIAN, 1959). A. aegypti and G. morsitans are remarkably similar in their

partitioning of amino acids, differing mainly in the excretion of ammonia. In contrast, amino acid partitioning by C. pipiens fatigaRs differs markedly from that of the other two species, which deaminate twice the proportion of ingested amino acids and incorporate only about a third as large a proportion into protein. A part of the increased efficiency of C. pipiens fatigans for incorporating amino acids into eggs can be attributed to the better amino acid balance of the chicken blood used in BRIEGEL’S(1968) study atid to the fact that his measurements included the structural proteins of the eggs. However, since these factors can only account for a relatively small proportion of the observed difference and in view of

845

the simultaneous reduction in the proportion of amino acids deaminated for metabolic purposes by C. pipiens fatigans, we believe that an important difference in this species’ strategy of blood meal utilization is indicated. Although both the amino acid balance of the ingested blood and prior starvation can affect the partitioning of amino acids between deamination and protein synthesis, A. aegypti does not appear to be able to increase the proportion of amino acids channelled into egg production by mobilizing previously acquired body stores to meet its energy requirements during the reproductive period. If it had this capability it could attain maximal fecundity at smaller blood meal sizes. However, our results show that females with ample triglyceride reservesdeaminate the same proportion of the ingested amino acids regardless of the quantity of blood ingested. C. pipiens fatigans deaminates only one half the proportion of amino acids deaminated by A. aegypti, and the difference is approximately equal to the proportion of amino acids utilized by A. aegypti for energy. This suggests that the difference between the two species may be that C. pipiens fatigans can make more efficient use of ingested amino acids for reproduction by mobilizing body reserves to meet its energy requirements during the period of blood meal assimilation and o&genesis. We point out that the closely related autogenous Culex pipc’ens pipiens, which produces the first batch of eggs without blood feeding, furnishes both amino acids and energy substrates from previously acquired body stores. Thus C. pipiensfatigans may be a physiological intermediate between the truly autogenous mosquito and the fully anautogenous A. aegypti, which appears to deaminate a proportion of the ingested amino acids sufficient to meet all of its energy and non-protein synthetic requirements during the critical reproductive period.

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GRIFFITHSM. (1952) Oxidation of uric acid catalyzed by copper and the cytochrome
HARINCTON J. S. (1956) Histamine and histidine in excreta of the blood-sucking bug Rhodnius prolixus. Nature Lond. 268.

HARINGTONj. S. (1961) Studies of the amino acids of Rhodnius prolixus II. Analysis Parasitology 51, 319-326.

of the excretory

material.

HEUSNERA. A. and LAVOIPIERRE M. (1973a) finergktique de repas sanguin chez Aedes aegypti. C. r. hebd. Seam. Acad. Sci., Paris 276, 1725-l 728.

HEUSNER A. A. and LAVOIPIERRE M. (1973b) Etude cinetique

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de l’effet metabolique d’un repas sanguin chez Aedes aegypti C. r. hebd. Si~nc. Acad Sci. Paris 277,2017-2020. IRREVERRE F. and TER~IANL. A. (1959) Nitrogen partition in excreta of three species of mosquitoes. Science 129, 1358-1359. ITZHAKIR. F. and GILL D. M. (1964) A micro-biuret method for estimating protein. Analyt. Biochem. 9, 401-410. LEAA. O., DIMONDJ. B. and DELONGD. M. (1958) Some nutritional factors in egg production by Aedes aexypti. Proc. Tenth Int. Congr.~in;. (1956) Vol. 3, 793-?96..^ LIDDLEL.. SEEGMILLER J. E. and LASTERL. (1959) The \ enzymatic spectrophotometric method for determination of uric acid. J. Lab cfin. Med. 54, 903-913. LOWRY0. H., ROSEBROUCH N. J., FARRA. L. and RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-215. NAYARJ. K. and SAUERMAN D. M. (1975a) The effects of nutrition on survival and fecundity in Florida mosquitoes. Part 2. Utilization of a blood meal for survival. J. med. Ent. 12,99-103. NAYARJ. K. and SAUERMAN D. M. (1975b) The effects of I

C. L. JUDSON

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