Autogenous Mosquito Research Articles

Insulin-like peptide 3 stimulates hemocytes to proliferate in anautogenous and facultatively autogenous mosquitoes.

Most mosquito species are anautogenous, which means they must blood feed on a vertebrate host to produce eggs, while a few are autogenous and can produce eggs without blood feeding. Egg formation is best understood in the anautogenous mosquito Aedes aegypti, where insulin-like peptides (ILPs), ovary ecdysteroidogenic hormone (OEH) and 20-hydroxyecdysone (20E) interact to regulate gonadotrophic cycles. Circulating hemocytes also approximately double in abundance in conjunction with a gonadotrophic cycle, but the factors responsible for stimulating this increase remain unclear. Focusing on Ae. aegypti, we determined that hemocyte abundance similarly increased in intact blood-fed females and decapitated blood-fed females that were injected with ILP3, whereas OEH, 20E or heat-killed bacteria had no stimulatory activity. ILP3 upregulated insulin-insulin growth factor signaling in hemocytes, but few genes - including almost no transcripts for immune factors - were differentially expressed. ILP3 also stimulated circulating hemocytes to increase in two other anautogenous (Anopheles gambiae and Culex quinquefasciatus) and two facultatively autogenous mosquitoes (Aedes atropalpus and Culex pipiens molestus), but had no stimulatory activity in the obligately autogenous mosquito Toxorhynchites amboinensis. Altogether, our results identify ILPs as the primary regulators of hemocyte proliferation in association with egg formation, but also suggest this response has been lost in the evolution of obligate autogeny.

Genetic Diversity Based on Autogeny and ISSR Markers in Culex pipiens Mosquitoes from Northern Coast of Egypt

The heredity of autogeny in Culex pipiens from northern coast, Alexandria, Egypt, was investigated. Parental females laid more autogenous egg rafts than F1, F3, and F4 females. No significant difference was found in the autogeny rate when F3 female mosquitoes from an autogenous colony mated with males either from an autogenous or anautogenous colony. Similarly, when F3 males from an autogenous colony mated with females either from an autogenous or anautogenous colony, statistically comparable autogeny rates were recorded. However, when females from an anautogenous colony were allowed to mate with males from an autogenous colony, the autogeny rate was statistically higher than that when females from an autogenous colony mated with males from an anautogenous colony. Molecular analysis using ISSR was suitable for detecting polymorphic banding patterns between autogenous and anautogenous specimens of mosquitoes. The ISSR technique succeeded in showing 13 different molecular markers which can be relied upon in distinguishing between populations. Molecular distances among populations were low, ranging between 0.114 and 0.284. On the other hand, cluster and principal coordinate analyses showed that anautogenous specimens were contiguous in one group and separate from autogenous specimens. The ISSR technique is therefore able to detect genetic differentiation among the studied populations, and to distinguish between autogenous and anautogenous mosquitoes from the northern coast of Egypt.

Analysis of ovary-specific genes in relation to egg maturation and female nutritional condition in the mosquitoes Georgecraigius atropalpus and Aedes aegypti (Diptera: Culicidae)

Analysis of the reproductive physiology of anautogenous mosquitoes at the molecular level is complicated by the simultaneity of ovarian maturation and the digestion of a blood meal. In contrast to anautogenous mosquitoes, autogenous female mosquitoes can acquire greater nutrient stores as larvae and exhibit higher ovarian production of ecdysteroids at adult eclosion. These features essentially replace the role of a blood meal in provisioning the first batch of eggs and initiating egg development. To gain insight into the process of ovary maturation we first performed a transcript analysis of the obligatory autogenous mosquito Georgecraigius atropalpus (formerly Ochlerotatus atropalpus). We identified ESTs using suppressive subtractive hybridization (SSH) of transcripts from ovaries at critical times during oogenesis in the absence of blood digestion. Preliminary expression studies of genes such as apolipophorin III (APO) and oxysterol binding protein (OSBP) suggested these genes might be cued to female nutritional status. We then applied our findings to the medically important anautogenous mosquito Aedes aegypti. RNAi-based analyses of these genes in Ae. aegypti revealed a reduction in APO transcripts leads to reduced lipid levels in carcass and ovaries and that OSBP may play a role in overall lipid and sterol homeostasis. In addition to expanding our understanding of mosquito ovarian development, the continued use of a comparative approach between autogenous and anautogenous species may provide novel intervention points for the regulation of mosquito egg production.

The Importance of Males: Larval Diet and Adult Sugar Feeding Influences Reproduction in Culex molestus

Culex molestus is an obligatory autogenous mosquito that is closely associated with subterranean habitats in urban areas. The objective of our study was to investigate the influence of larval and adult nutrition on the role of males in determining the expression of autogeny in Cx. molestus. Mosquitoes raised at low and high larval diets had sex ratio, wing length, mating rates, autogenous egg raft size, and hatching rates recorded. There was a higher ratio of males to females when raised at a low larval diet. Mean wing lengths of both males and females were significantly greater when raised at the high larval diet regime. Regardless of larval or adult diet, males mated with only a single female. Mosquitoes raised at the higher larval diet regimes developed significantly more autogenous eggs. However, the egg raft size was reduced when adult females were denied access to sugar. The results of this study indicate that the performance of males in the reproductive process is influenced by both larval diet and adult sugar feeding.

Vitellogenin gene expression in autogenous Culex tarsalis

Autogeny, the ability of a mosquito to mature an initial batch of eggs without blood feeding, is an alternative reproductive strategy with important implications for vector-borne disease transmission. Regulation of the major yolk protein (vitellogenin; Vg) genes during bloodmeal-induced oogenesis is well studied, but little is known about regulation of vitellogenesis in autogenous mosquitoes. We characterized the expression of four vitellogenin genes (Vg1a, Vg1b, Vg2a and Vg2b) in an autogenous strain of the West Nile Virus vector, Culex tarsalis. All vitellogenin genes were expressed during autogenous reproduction and following a bloodmeal, although the intensity and duration of expression varied amongst genes. Quantitative PCR analysis of vitellogenin transcription during autogeny revealed a similar temporal pattern to known vitellogenin expression profiles in anautogenous Aedes aegypti. Vitellogenin transcript, primarily produced from the Vg1b gene, was also detected in the larval and pupal stages of development, but no detectable vitellogenin protein was produced during this time period.

The Hypopharynx of Male and Female Mosquitoes

In blood-feeder female mosquitoes, the hypopharynx stylet is one part of the fascicle, the structure that pierces into the host skin during blood feeding. As other parts, the hypopharynx is a free stylet. However, since male mosquitoes do not feed blood, their mouthparts are less developed. The hypopharynx fuses with the inner wall of the labium, while maxillae and mandibles are much shorter than the labium. Only the labrum and the labium are well developed and func- tion as food canal and its sheath, respectively. Light microscopy (LM) and scanning electron microscopy (SEM) were done to compare the hypopharynx of males of several mosquito genera and, in addition, females of autogenous mosqui- toes. The hypopharynxs of males of both autogenous and anautogenous mosquitoes fuse with the labium inner wall as long as the labium length, but are distinctly different structures from the labium. Dissociation occurs on the hypopharynx of fe- male autogenous mosquitoes: Toxorhynchites spp. have a free hypopharynx as in anautogeny mosquitoes, whereas it fuses with the labium wall in Malaya genurostris Leicester, as in male mosquitoes.

Reproductive Costs of the Immune Response of the Autogenous Mosquito Culex pipiens molestus Against Inoculated Dirofilaria immitis

Culex pipiens molestus is an autogenous mosquito, a vector of Dirofilaria immitis in Madeira Island, and it mounts a melanotic encapsulation response when inoculated intrathoracically with D. immitis microfilariae (mf). Because Cx. p. molestus is autogenous, this mosquito is a good model from which to gain a better understand the relationship between oviposition and melanization, independent of the signaling pathways related to blood feeding. The present work assessed the impact follicle growth might have on melanization of intrathoracically inoculated mf. The ovaries from mosquitoes undergoing melanotic encapsulation developed more eggs than those that could not melanize the mf. Possible explanations are discussed in this article.

Mosquito Vitellogenin Genes: Comparative Sequence Analysis, Gene Duplication, and the Role of Rare Synonymous Codon Usage in Regulating Expression

Comparative sequence analysis of mosquito vitellogenin (Vg) genes was carried out to gain a better understanding of their evolution. The genomic clones of vitellogenin genes were isolated and sequenced from all three subfamilies of the family Culicidae including Culicinae (Aedes aegypti, Ochlerotatus atropalpus, Ae. polynesiensis, Ae. albopictus, Ochlerotatus triseriatus and Culex quinquefasciatus), Toxorhynchitinae (Toxorhynchites amboinensis), and Anophelinae (Anopheles albimanus). Genomic clones of vitellogenin genes Vg-B and Vg-C were isolated from Ae. aegypti and sequenced. A comparison of Vg-B and Vg-C, with the previously characterized vitellogenin gene, Vg-A1, suggests that Vg-A1 and Vg-B probably arose by a recent gene duplication, and Vg-C apparently diverged from the two other members of the gene family in an earlier gene duplication event. Two vitellogenin genes orthologous to Vg-C were cloned from a Cx. quinquefasciatus DNA library, one of which is truncated at the N-terminal end. Single vitellogenin genes, orthologous to Vg-C, were cloned from the An. albimanus and Tx. amboinensis libraries. Incomplete sequences orthologous to Vg-B and Vg-C were isolated from the Oc. atropalpus library. Only partial sequences were isolated from Ae. polynesiensis, Ae. albopictus and Oc. triseriatus. Inferred phylogenetic relationships based on analysis of these sequences suggest that Vg-C was the ancestral gene and that a recent gene duplication gave rise to Vg-A1 and Vg-B after the separation of the genus Aedes.The deduced amino acid composition of mosquito vitellogenin proteins exhibits higher tyrosine and phenylalanine composition than other mosquito proteins except for the hexamerin storage proteins. Analysis of vitellogenin coding sequences showed that a majority of amino acid substitutions were due to conserved and moderately conserved changes suggesting that the vitellogenins are under moderately selective constrains to maintain tertiary structure. The vitellogenin genes of the three anautogenous mosquitoes, that require a blood meal to develop eggs, had very high synonymous codon usage biases similar to highly expressed genes of other organisms. On the other hand, the vitellogenin genes of autogenous mosquitoes, that develop at least one batch of eggs without a blood meal, exhibited low synonymous codon usage bias. An unusual pattern of synonymous codon usage was observed in the first 15 amino acid residues encoding the signal peptide in the vitellogenin genes, where a high number of rarely used synonymous codons are present. It is hypothesized that rare synonymous codons have selectively accumulated in the signal peptide region to down-regulate the rate of translation initiation in the absence of a blood meal. Real-time PCR gene expression experiments showed that all three Ae. aegypti vitellogenin genes were highly expressed after a blood meal, and expressed in non-blood-fed females, males, larvae and pupae at trace levels. Sequences were deposited in GenBank (accession numbers: Ae. aegypti Vg-B, AY380797, Vg-C, AY373377; Oc. atropalpus Vg-B, AY691321, Vg-C, AY691322; Ae. polynesiensis Vg-A1, AY691318, Vg-B, AY691319, Vg-C, AY691320; Ae. albopictus Vg-A1, AY691316, Vg-C, AY691317; Oc. triseriatus Vg-C, AY691323; Cx. quinquefasciatus Vg-C1, AY691324, Vg-C2, AY691325; Tx. amboinensis Vg-C, AY691326; An. albimanus Vg-C, AY691327).

4 無吸血産卵性イナトミシオカの継代飼育について(一般講演,第55回日本衛生動物学会南日本支部大会講演要旨)

4 無吸血産卵性イナトミシオカの継代飼育について(一般講演,第55回日本衛生動物学会南日本支部大会講演要旨)

The effect of larval and adult nutrition on successful autogenous egg production by a mosquito

Females of most mosquito species require a blood meal to provision eggs and can be medical problems because of this dependency. Autogenous mosquitoes do not require blood to mature an initial egg batch and, instead, acquire nutrients for egg provisioning as larvae. We studied the importance of larval and adult nourishment for Ochlerotatus atropalpus which is obligatory autogenous for its first egg cycle but may ingest blood for subsequent cycles. Larval nourishment strongly influenced autogenous egg production: female larvae that were nutritionally stressed emerged as smaller adults, produced fewer eggs and emerged with less protein, lipid and glycogen stores. Female Oc. atropalpus are 100% autogenous, regardless of larval diet quality or whether females are fed sugar or water at emergence. Upon completion of the first egg batch, only females emerging from a poor larval diet ingested blood and produced a second egg batch.

Maxillae and mandibles of male mosquitoes and female autogenous mosquitoes (Diptera: Culicidae).

Maxillae and mandibles of males of 44 species of 12 mosquito genera and females of three autogenous genera and two partially autogenous species were examined under light microscopy and scanning electron microscopy. The maxillae and mandibles of male mosquitoes are delicate, tape-like structures with lengths characterizing genera or higher level classification units. Five patterns are recognized: (A) long maxillae and mandibles with mandibles longer than maxillae in Anopheles; (B) long maxillae and mandibles with maxillae longer than mandibles in Toxorhynchites; (C) short or intermediate lengths of maxillae with short mandibles in Aedes, Armigeres, Culex, Ochlerotatus, Orthopodomyia, and Uranotaenia; (D) short or intermediate length of maxillae with no mandibles in Mimomyia and Tripteroides; and (E) no maxillae and mandibles in Malaya and Topomyia. Maxillary and mandibular lengths of male mosquitoes show a positive correlation. Length of maxillae and mandibles of autogenous females are reduced to the same level as conspecific males. In contrast, females of partially autogenous species have complete maxillae and mandibles as in females of anautogenous species.

Female-specific expression of a hexamerin gene in larvae of an autogenous mosquito.

Fourth-instar larvae of the autogenous mosquito, Aedes atropalpus, synthesize three hexamerins or hexameric storage proteins which are distinguished by different methionine and aromatic amino-acid contents. One protein, Hexamerin-1.2 (AatHex-1.2) is only found in female larvae and pupae. In order to investigate the molecular basis for this sex-specific accumulation, we have cloned and sequenced the cDNA encoding AatHex-1.2 and isolated and sequenced over 1 kb of the 5' flanking region of the AatHex-1.2 gene. The AatHex-1.2 transcript encodes a 81.6-kDa hexamerin subunit which contains 19.8% phenylalanine, tyrosine and tryptophan and 8.6% methionine residues. The single-copy AatHex-1.2 gene consists of three exons and two small introns located at its 5' end. A 2.3-kb AatHex-1.2 mRNA accumulates only in female larvae and pupae and is expressed at very low levels in adult female mosquitoes. The temporal expression profile of this transcript is typical of other mosquito hexamerin genes, with rapid disappearance of the mRNA shortly after pupation. Hence this is the first observation of exclusively female-specific gene activity during preadult development of an insect. In the 5' flanking region of the AatHex-1.2 gene, we identified putative binding sites for transcription factors, such as GATA, C/EBP and Doublesex, typically involved in fat body- and female-specific gene activity in Diptera. These findings suggest that mechanisms for sex-specific transcription in the fat body may be well conserved between flies and mosquitoes.

A role for storage proteins in autogenous reproduction in Aedes atropalpus

In the autogenous mosquito, Aedes atropalpus, storage proteins accumulated during the larval stage may serve as an amino acid reserve for oogenesis, in addition to metamorphosis. Hexameric storage proteins accumulate during larval development and include subunits of three different masses: 62.5, 66, 72.5 kDa. All three types of subunits are found in the female but only the larger two are in males. In females, storage proteins are only partially depleted by the time of eclosion. The remaining protein amounts to about 40% of the original store. Males, in contrast, exhaust their supply of stored protein during metamorphosis. In the female, the storage proteins disappear over the first days after eclosion, and are depleted before vitellogenin/vitellin levels reach their maximum. This suggests that the amino acids held in storage proteins are transferred to vitellogenesis, enabling autogenous egg development. The fact that these amino acids are not available for egg development until after eclosion, later than in many other insects, probably reflects a relatively recent evolution from blood-feeding ancestors.

Lactic acid sensitive receptors in the autogenous mosquito Aedes atropalpus

Host seeking behavior is not observed during the first gonotrophic cycle in Aedes atropalpus. The objectives of this study were to examine the possible role of the peripheral sensory system in this phenomenon. The results show that the rate of maturation of the lactic acid sensitive neurons is relatively rapid in this species so the absence of host-seeking during the first gonotrophic cycle cannot be attributed to delayed development of the peripheral sensory system. Furthermore, lactic acid receptors of high sensitivity are present on the antennae of females that are gravid and in the early stages of egg development. These results support the conclusion that a humoral mechanism of host-seeking inhibition is not operative in this species, at least during autogenous egg development, and show that receptors for at least one host attractant are fully mature early in the first autogenous gonotrophic cycle.

Host-seeking behavior in the autogenous mosquito Aedes atropalpus

The absence of host-seeking behavior during the first gonotrophic cycle in Aedes atropalpus is not due to hemolymph-borne factors. The removal of all developing eggs from nulliparous, gravid individuals by ovariectomy did not result in host-seeking. Furthermore, hemolymph transfers from non-host-seeking, nulliparous, gravid females into host-seeking, parous, non-gravid females did not inhibit host-seeking in the recipients. Circumstantial evidence supports the hypothesis that distention inhibits host-seeking during the first gonotrophic cycle in this species. Host-seeking can only be elicited after oviposition of the first egg clutch. Although the frequency of sugar feeding is high, particularly in the parous, non-gravid population, host-seeking is not correlated with the presence of sugar in the crop. Host-seeking and non-host-seeking individuals were equally likely to have recently fed on sugar. The control of host-seeking behavior during the first gonotrophic cycle in Ae. atropalpus is thus very different from that in the anautogenous mosquito Aedes aegypti, in which host-seeking is inhibited by hemolymph-borne factors associated with egg development.

Autogenous and Anautogenous Mosquitoes: a Mathematical Analysis of Reproductive Strategies

A mathematical model is presented to compare the relative advantage of an anautogenous mosquito population (in which females blood feed throughout life) with an obligate autogenous population (in which females do not feed on blood for the first oviposition). The advantage is measured by the intrinsic rate of natural increase. Autogeny was more advantageous than anautogeny when host searching time (ts), the ratio of the fecundity of the first autogenous to anautogenous oviposition (p), the fecundity in one anautogenous oviposition (n), or the instantaneous death rate of the adult population (D) was large, or when the preimaginal period (xo), the instantaneous death rate during the preimaginal period (D'), or survival during blood feeding (s) was low. The parameters most sensitive to the advantage of autogeny were ts, p, and s. The value of n was insensitive, and xo, D, and D' were intermediately sensitive to autogeny. Conditions when autogeny was advantageous were equivalent to conditions conducive to high autogeny rates in facultatively autogenous species, which alter the expression of autogeny depending upon environmental stimuli. Data on several facultatively autogenous species are discussed qualitatively and quantitatively to demonstrate the utility of our model in considering the evolution of autogeny and the autogeny rate.

Viability of Culex pipiens pipiens eggs affected by nutrition and aposymbiosis

Raisins were a better source of carbohydrate than sucrose for reproduction by autogenous Culex pipiens. A blood meal increased the number of eggs per raft from 49 autogenously to 114. Eggs of aposymbiotic females produced autogenously did not hatch, but 34% of the eggs were viable if the mosquitoes fed on chickens. With repeated blood meals the number of eggs per raft and the rate of embryonation and hatching declined in each successive gonotrophic cycle. In about 1 4 of the unhatched eggs of normal females there were no fully developed embryos, while many more of the unhatched eggs of aposymbiotic females contained no evidence of embryonic development. After the fifth blood meal, neither normal nor aposymbiotic insects oviposited. The ovaries of the nulliparous females contained approximately 10% of the potential number of mature terminal oocytes. Proximally in the ovarioles there were dilatations and coiled tracheoles indicating egg resorption. There were fewer parous follicles in aposymbiotic than in normal females. Larval rearing water, i.e., infusion in which larvae had been reared, was more attractive than fresh infusion for oviposition by normal, autogenous mosquitoes. The degree of embryonation of the eggs was lower and the hatching success rate was poorer in fresh infusion than in larval rearing water.

Salivary gland apyrase determines probing time in anopheline mosquitoes

Salivary gland homogenates of adult female anopheline mosquitoes, of three different species, hydrolysed ATP and ADP, thereby demonstrating an apyrase activity. Total enzyme activity was greatest in the vector species A. freeborni (20.7 ± 2.4 mU/pair of glands) and least in the autogenous mosquito A. sp. nr. salbaii (3.0 ± 0.4 mU/pair of glands); another vector species, A. stephensi, produced intermediate levels of the enzyme (7.8 ± 0.7 mU/pair of glands). In all cases, the reaction was activated by divalent cations and maximal at pH 9.0 and in the presence of 2-mercaptoethanol. Apyrase activity in each salivary gland correlated with the degree of inhibition of ADP-induced platelet aggregation in vitro. Duration of probing correlated inversely with salivary apyrase content. We conclude that salivary apyrase largely determines a mosquito's ability to locate blood. Differential selective pressures for facility of blood location would have influenced the level of salivary apyrase in these mosquitoes.

Hormonal regulation of ovarian ecdysteroid production in the autogenous mosquito, Aedes atropalpus

The effects of juvenile hormone (JH) and egg development neurosecretory hormone (EDNH) on ovarian ecdysteroid production during vitellogenesis in the autogenous mosquito, Aedes atropalpus, were investigated using in vitro techniques coupled with radioimmunoassay (RIA) and high-pressure liquid chromatography (HPLC). Normal females were characterized by quantitative, qualitative, and temporal patterns of in vitro ovarian ecdysteroid production. Females decapitated at emergence showed little ovarian ecdysteroid production and did not undergo vitellogenesis. A 500-ng dose of JH-I applied topically to decapitated females restored normal patterns of ecdysteroid production. In both normal and experimental females, ecdysone constituted the major portion of the ecdysteroids secreted by the ovaries in vitro. However, significant amounts of other RIA-active materials were detected, one of which was probably 20-OH-ecdysone. Fat body incubations indicated that these tissues produce little RIA-active material during the peak of vitellogenesis. During that period, the ovaries were the major source of ecdysteroid. Various doses of JH-I, applied to adbomens isolated at emergence, enhanced ovarian responsiveness to subsequent applications of head extracts containing EDNH both in vivo and in vitro. A 500-ng dose of JH-I, applied topically to blood-fed, decapitated Aedes aegypti, stimulated a significant increase in in vitro ovarian ecdysteroid production. Similarities between these data and those demonstrating prothoracicotropic effects of JH in the Lepidoptera are discussed.

INDUCTION OF OVARIAN DEVELOPMENT IN AUTOGENOUSAEDES ATROPALPUSBY JUVENILE HORMONE AND 20-HYDROXYECDYSONE

Aedes atropalpus is an autogenous mosquito characterized by a first gonadotropic cycle which results in approximately 200 mature oocytes without a bloodmeal. Ovarian development is completely inhibited if these animals are decapitated or ligated between the thorax and abdomen shortly after adult emergence. Injection of 4.8 ng of 20-hydro- xyecdysone into decapitated females 12 h after eclosion restores ovarian development in all females so treated. However, the same amount of 20-hydroxyecdysone injected into isolated abdomens obtained shortly after adult emergence had no discernible effect on vitellogenesis. In contrast, all abdomens which received 0.5 ng of topically applied JH I followed by the injection of 4.8 ng 20-hydroxyecdysone produced mature oocytes. Isolated abdomens were also capable of oocyte maturation when treated with excess amounts of JH alone; JH I was the most effective followed by JH II and then JH III. Polyacrylamide gel electrophoretic analysis of vitellin extracted from the ovaries o...