Hans Briegel

Metabolic relationship between female body size, reserves, and fecundity of Aedes aegypti

Abstract The body size of individual male and female Aedes aegypti , recorded as the cubic value of wing length, was correlated with total protein, lipid, and carbohydrate reserves at eclosion. Protein and carbohydrate had a linear relationship, whereas lipids were positively allometric and sex-specific; per unit size, males had substantially higher lipid levels than females. A wide spectrum of imaginal body size resulted by varying larval rearing conditions (crowding, starvation, and optimal food supply), but regardless of treatment, teneral reserves conformed to regression equations related to body size. Therefore, larval food supply was of primary importance in determining imaginal body size and reserves. The minimal irreducible amounts of female reserve components were 69–86% of the teneral protein, and 16–46% of teneral lipids; carbohydrates were mobilized nearly completely. The extent of reserve mobilization was dependent on the availability of drinking water. With free access to sucrose, maximal reserves were synthesized, dominated by tremendous lipogenesis and leading to a linear correlation of body size with total caloric reserves at the outset of the gonotrophic cycle. Blood consumption by large females was more than twice that of small females, but fecundity increased about 4-fold. The efficiency of blood meal utilization for yolk synthesis was proportional to body size. Large females revealed a threshold level in fecundity, below which maternal reserves were required for completion of oogenesis. The total caloric content per mature oocyte however, was constant under all experimental conditions, with variations in the relative distribution of its protein and lipid, depending on maternal body size. In large females the gonotrophic cycle was completed in a significantly shorter period than in smaller ones that ingested blood of equal volumes.

Aedes albopictus (Diptera: Culicidae): Physiological Aspects of Development and Reproduction

Abstract Basic ecophysiological data are presented on the development and reproduction of Aedes albopictus (Skuse) that were reared and maintained at four temperatures between 12 and 32°C. Median larval developmental time from hatching to pupation was correlated inversely with temperature, lasting 7 d at 32°C and up to 28 d at 12°C. Duration of the pupal period also varied from 2–3 d at 32°C to 7–12 d at 12°C. This extension of larval development elongated the phagoperiod and gave rise to larger imagoes. Based on wing length measurements, body sizes varied from 10 to 57 mm3 for females and from 10 to 30 mm3 for males. The caloric protein content at emergence showed a linear and significant regression with body size, independent of sex, treatment, or temperature. Teneral lipid content also followed a linear relationship with body size at warmer temperatures, whereas at low temperatures it increased exponentially with body size. Glycogen was always below 10% of the protein or lipid levels. Reserves at emergence determined median adult survival times, which ranged from 16 d at 32°C to 100 d at 17°C. Access to sucrose solution allowed females to increase their teneral glycogen up to fourfold within 1 wk, and their lipids up to 10-fold within 2 wk. Despite a broad variation, the number of mature oocytes (15–110 eggs per female) was correlated positively with body size, but inversely with the rearing and maintenance temperature. Utilization of the blood meal protein for oogenesis ranged between 35 and 50%, again inversely correlated with temperature; absolute compositions per oocyte were 6.3–6.5 mcal of protein but ranged from 5 to 7 mcal of lipid.

Mosquito reproduction: Incomplete utilization of the blood meal protein for oögenesis

Fecundity, measured by utilization of dietary protein for the production ofyolk protein was examined in several mosquito species (A. aegypti, A. simpsoni, A. atropalpus, A. triseriatus) using blood from eight vertebrate hosts over a whole range of biological blood mean volumes. All experimental blood meals were injected by enema in measured amounts. The total ovarian and excretory nitrogen produced during each gonotrophic cycle was quantified. Blood proteins are utilized for oogenesis only to a limited extent: two thirds, at the most, are detectable in the mature ovaries. This efficiency decreases with increasing blood meal size to about one fifth. The nitrogen content of each mature egg is constant and species-specific, irrespective of blood meal size or source. There are no significant differences in the efficiency of conversion of protein from different types of vertebrate blood into oocyte protein except for human blood which led to a drastically reduced efficiency. This is shown to be due to a lack of isoleucine which is essential for reproduction. Hominid primates lack this amino acid in their haemoglobin molecule and it exists at a low titre in human plasma. Apparently, in other vertebrates the amino acids are better balanced in the haemoglobin or, can be mobilized in sufficient amounts from various non-haemoglobin protein fractions. All blood protein is catabolized and the excess nitrogen excreted quantitatively; excretory nitrogen therefore reflects the quantitative demands of a gonotrophic cycle. These processes are not dependent upon receiving a mating stimulus.

LARVAL GROWTH AND BIOSYNTHESIS OF RESERVES IN MOSQUITOES

Developmental instars of four species of mosquitoes have been analyzed for growth and synthesis of biomass with respect to their caloric content of protein, lipids, and carbohydrates for each instar of Aedes aegypti and Culex pipiens of the subfamily Culicinae, and Anopheles albimanus, and An. gambiae of the subfamily Anophelinae. The diameter of the thorax grows during the intermolt, reflecting continuous increase in biomass because it correlates significantly with the larval synthesis of total protein, lipids, and carbohydrates. For Ae. aegypti the fourth instar was sexed to disclose the sex-specific synthetic potential. In Ae. aegypti the protein increased in linear proportion with larval body size, whereas lipid synthesis followed a significant, exponential regression, which was clearly steeper in male larvae and most pronounced in the last instar. When normalized for size, the size-specific protein and lipid contents showed minimal levels of 0.25 and 0.1, respectively, regardless of standard or crowded rearing conditions. The rate of lipid synthesis in Ae. aegypti was determined by incubating fourth instar larvae with (14)C-acetate and estimating the lipids. The rate was highest in the early larvae and decreased towards the end shortly before pupation; in male larvae incorporation was twice the rate of female larvae. Cx. pipiens reached the largest body sizes of all species tested, with protein and lipids increasing linearly with size. Their minimal levels of size-specific caloric contents were around 0.35 for protein and 0.25 for lipids. Anopheles also showed a linear relationship between larval size and caloric protein and lipid contents. Their minimal threshold levels in size-specific contents were 0.35 for protein and 0.2 for total lipids, similar to Culex, but slightly higher than in Aedes. Starvation of Ae. aegypti larvae and subsequent feeding partially improved their lipid contents, but never to the levels of non-starving, optimal controls. Conversely, well-fed final instars exposed to complete starvation showed a tremendous reduction of the protein and lipids contents in the surviving imagines, accompanied by 73% mortality. These results demonstrate the biosynthetic plasticity and the significance of the phagoperiod in Ae. aegypti during the final fourth instar for growth. The characteristic differences between these two subfamilies in their larval physiology are discussed in relation to ecological factors as encountered in the field under natural conditions, and in relation to our earlier findings on the reproductive physiology.

Mosquito trypsin: immunocytochemical localization in the midgut of blood-fed Aedes aegypti (L.)

SummaryA polyclonal antibody was raised against trypsin purified from the midgut of blood-fed Aedes aegypti. Using this antibody and our modification of the peroxidase-antiperoxidase immunocytochemical reaction, strong activity was found in the lumen of the midgut at the light-microscopical level. The activity was localized mainly in the posterior part of the distensible, abdominal midgut, along the periphery of the blood bolus and within the peritrophic membrane. Immunoreactivity appeared 8 h after the blood meal and was most prominent around 24 h, coinciding with our previous spectrophotometric determinations of trypsin.At the electron-microscopical level, secretory granules, immunocytochemically labelled with anti-trypsin antibody and protein A-colloidal gold, were first detected about 12 h after the blood meal. At 18 h, the secretory pathway could be followed immunocytochemically from the formation of granules in the Golgi complex until their release by exocytosis in the midgut lumen. By 24 h, there was a reduction in secretory granules, and large lysosomes appeared.The process of secretion described for this mosquito is comparable to similar events in vertebrate secretory systems and the presence of an intracellular trypsinogen is suggested.

Arrest, resorption, or maturation of oöcytes in Aedes aegypti: dependence on the quantity of blood and the interval between blood meals

ABSTRACT. After emergence, the follicles of A. aegypti double in length and the oöcytes may deposit a small amount of yolk, but within 2 days growth is arrested. Renewed growth and vitellogenesis, as well as the number of eggs finally produced, depends on the quantity of blood ingested. All females, given either a small (1 μl) or large (4 μl) meal of rat blood by enema, began yolk deposition in a nearly equal number of oöcytes, and each oöcyte had about the same amount of yolk 8 h later. Within 48 h, females fed 4 μl had each produced more than 100 eggs, whereas females fed 1 μl either had continued yolk deposition in some oöcytes, while most degenerated, or had all re‐entered oögenic arrest. Consequently, 48 h after the 1 μl meal, a female had either c. 50 or 0 eggs. Even by 14 h after a 1‐μl meal, females were either committed to re‐enter oögenic arrest or to complete maturation of some oöcytes and resorb the yolk of others. This was shown by removing and examining one ovary 14 h after a blood meal and then giving a second blood meal. The second meal stimulated meal maturation in the remaining ovary, but only in those females whose oöcytes had been in oögenic arrest when the first ovary was examined; the second meal had no effect on females whose first ovary had contained both vitellogenic and degenerating oöcytes. Oösorption was not reversed by a second blood meal. Our results do not support the hypothesis that the female ‘evaluates’ the ingested meal and begins vitellogenesis in an ‘appropriate’ number of oöcytes. The results demonstrate that the ovary is an unreliable indicator of the frequency of blood‐feeding, when females take a small meal.

The synthetic pathway of trypsin in the mosquito Aedes aegypti L. (Diptera: Culicidae) and in vitro stimulation in isolated midguts

Abstract Mosquito trypsin is synthesized in vivo and in vitro in two groups of forms with differing molecular sizes: one group of 32–36 KD forms is noted immediately after the blood meal, followed by the principal forms with Mr around 30 KD about 10 h later; synthesis is terminated at about 24 h after blood meal. Similar results were obtained after in vitro translation of mRNA. Trypsin precursor and trypsin mRNA were not detected by our assay of midguts of unfed females. Trypsin synthesis is induced in a dose-dependent manner by injection of either blood or sugar solutions into isolated midguts. It is concluded that the stimulus for initial trypsin synthesis is mechanical and/or osmotic stress acting independently of the nervous system. Processing of trypsin in the midgut cells involves cleavage of putative signal peptides: in vitro translation in the presence of microsomes led to a constant shift in molecular weights of 1–2 KD prior to secretion. Exposure of washed midgut epithelia from different stages to native blood in vitro inhibited final processing of the intracellular trypsin to the extracellular forms while stimulation of protein synthesis was observed. Consequently the role of the peritrophic membrane in compartmentalization of the digestive process is further emphasized.

Reproductive physiology of Aedes (Aedimorphus) vexans (Diptera: Culicidae) in relation to flight potential.

Abstract Total protein, lipid, and glycogen of Aedes vexans (Meigen) were related linearly to body size at eclosion. Starvation after emergence led to the determination of minimal irreducible amounts of protein, lipid, and glycogen and the availability of the teneral reserves, whereas access to sucrose revealed the potential for reserve synthesis. Glycogenesis and lipogenesis increased reserves ≈10-fold the teneral value within 1 and 2 wk after emergence, respectively. Carbohydrate feeding was an essential behavior before blood feeding and oogenesis commenced. Female flight was tested on a flight mill. Maximal flights of 10–17 km in a single night occurred at 2 wk posteclosion and paralleled maximal reserve syntheses. Comparisons of our laboratory data to host-seeking mosquitoes in the field confirmed our data. The vast majority of maternal lipid was transferred to the yolk when a blood meal was taken, but only a quarter of the blood protein was recovered from mature ovaries. Maternal glycogen was used mainly for flight. Fecundity varied between 20 and 120 eggs per female and was determined largely by body size and blood meal volume. At 27°C, maximal egg numbers were produced, but at 22 and 17°C the caloric yolk content was greater. Females from the southern United States were smaller than females from northern areas. However, southern females had similar fecundity as northern females, and their flight performances were similar. Differences in the reproductive physiology between this species and Ae. aegypti were discussed.

Lipid metabolism during sequential gonotrophic cycles in large and small female Aedes aegypti

The lipid metabolism was investigated during six gonotrophic cycles of Aedes aegypti. Females of constant body size were analyzed for their total lipid content: large females with a body size of 41.06 (wing length cubed) and small females with 15.63. Their lipid contents at eclosion were compared to lipid values after two days of sugar-feeding, shortly before a blood meal, after oviposition, of their total egg batches, and again before the next blood meal, with intermittent access to sugar for two days for six gonotrophic cycles.Large females transferred most of their pre-blood meal lipid into the ovaries. Their low lipid content after oviposition was restored by synthesis from intermittent sugar meals. After the third gonotrophic cycle, they withheld more and more of the resynthesized lipid in their fat body, thus gradually reducing their fecundity. Since blood consumption was not altered significantly during these six cycles, age-related reduction of fecundity was clearly caused by limitations of yolk lipid.Small females transferred a considerably smaller, but constant segment of sugar-derived lipids to the ovaries. In both size classes, lipid content per oocyte was constant throughout all cycles with 9 mcal/oocyte in large and 7 mcal/oocyte in small females. Total fecundity reached 450 eggs in large and 280 eggs in small females. Large females that were maintained on water without sucrose took large blood meals from which part of the yolk lipid was synthesized. Extrapolations suggest that only one or two additional gonotrophic cycles would be possible without additional carbohydrate sources, despite lipogenesis from blood protein.

Isolation of trypsin isozymes from the mosquito Aedes aegypti (L.)

Trypsin has been isolated from midgut homogenates of blood-fed females of Aedes aegypti by a simple two-step purification procedure: ion-exchange chromatography followed by affinity chromatography. The resulting mosquito trypsin contains a number of isozymes, among which 5 major SDS-PAGE bands are recognized with molecular weights of 26.7, 28.5, 29.7, 31.0 and 32.0 kdaltons, as are some minor bands above and below this range. The isozymic pattern is comparable to that in crude homogenates. Isoelectric focussing of purified trypsin however, revealed over 20 tryptic isozymes, demonstrating that several isozymes segregate into subforms. A high correlation between TAME-active fractions and their DFP equivalent was demonstrated by using 3H-labelled DFP as a marker for trypsin on native acrylamide gels. The purification factor and the specific activities are discussed with respect to the unusual amounts of protein of dietary origin present in the midgut homogenates. Interference of blood-borne coagulation factors of a tryptic nature is unlikely.