The effect of putative diuretic factors on in vivo urine production in the mosquito, Aedes aegypti.

Changes in total urine production were measured in the mosquito, Aedes aegypti, following injection of 5-hydroxytryptamine (5-HT), Culex salinarius diuresin, culekinin depolarizing peptides (CDP-I, II and III) or the A. aegypti leucokinin peptides (ALP-I, II and III). All stimulated total urine production in a dose dependent manner. 5-HT was the least potent in urine production experiments with ED(50) values nearly 100-fold higher than other diuretic agonists. Doses greater than 2x10(-4) &mgr;moles inhibited urine production, suggesting either the occurrence of receptor down regulation, more than one type of 5-HT receptor, or increases in hindgut resorption of urine. The ALPs had relatively low ED(50) values compared to the CDPs suggesting that the endogenous peptides may have higher receptor binding affinities. Injection of mosquitoes with polyclonal antisera raised against either ALP-I or C. salinarius diuresin significantly reduced the response to injections of the respective peptides. The evidence presented above suggests that mosquito leucokinins and the C. salinarius diuresin function in the neuroendocrine regulation of urine production in the mosquito.

Effects of putative diuretic factors on intracellular second messenger levels in the Malpighian tubules of Aedes aegypti.

Intracellular levels of the second messengers, 3',5'-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP(3)) were measured in the Malpighian tubules of Aedes aegypti following the in vitro application of 5-hydroxytryptamine (5-HT) and the putative mosquito diuretic peptides, Culex salinarius diuresin and mosquito leucokinins (culekinin depolarizing peptides (CDPs) I, II, III, A. aegypti leucokinin peptides (ALPs) I, II, III). The C. salinarius diuresin significantly (p<0.05) increased tubule intracellular cAMP concentrations. Treatment of tubules with either 5-HT or CDP-II resulted in significant increases in both intracellular cAMP and IP(3) concentrations. All of the mosquito leucokinins, with the exception of CDP-I, significantly stimulated intracellular IP(3) in isolated tubules. These data suggest that the mosquito leucokinins may function on the Malpighian tubules of A. aegypti by increasing the intracellular Ca(2+) levels through the release of IP(3) sensitive Ca(2+) stores. The physiological relevance of these data to the regulation of mosquito Malpighian tubule function is discussed.

Vitelline envelope genes of the yellow fever mosquito, Aedes aegypti.

Vitelline envelope genes from the mosquito Aedes aegypti were analyzed with respect to their DNA sequences, genomic representation, temporal and spatial expression profiles and response to 20-hydroxyecdysone. Genomic clones of three vitelline envelope genes, 15a-1, 15a-2 and 15a-3 were isolated. Southern analysis indicates that all three genes are represented by a single copy in the genome. The deduced amino acid sequences of all three vitelline envelope genes contain a conserved region of 46 residues that overlaps with a region that is conserved in four Drosophila melanogaster vitelline envelope genes. DNA was sequenced flanking the 15a-1, 15a-2 and 15a-3 coding regions. A 360 bp sequence 5' of the 15a-2 coding region was identified with 72% identity to a sequence upstream of the Ae. aegypti VgA1 vitellogenin gene. The temporal patterns of 15a-1, 15a-2 and 15a-3 expression, as determined by Northern analysis, were similar. The spatial patterns of expression, as determined by whole-mount in situ hybridization, differed between the three genes. 15a-1 and 15a-3 were only expressed in the middle and posterior regions of the follicle, while 15a-2 was also expressed at the anterior region. Vitelline envelope gene expression was higher in ovaries that were dissected at 0, 2 and 10 h following a blood meal and then incubated in vitro for 10 h in medium containing 10(-5) M 20-hydroxyecdysone, compared to ovaries that were incubated without hormone.

Biochemical, molecular, and phylogenetic analysis of pyruvate carboxylase in the yellow fever mosquito, Aedes aegypti.

Pyruvate carboxylase (PC, pyruvate: carbon dioxide ligase [ADP-forming], EC 6.4.1.1) was purified from the yellow fever mosquito, Aedes aegypti. The purified PC showed two polypeptides of similar M(r) (133 and 128 k). The N-terminal sequences of both polypeptides were shown to be very similar, if not identical. A polyclonal antiserum against the 133 kDa polypeptide cross-reacted strongly with the 128 kDa polypeptide. PC was found in all tissues examined. Using a semi-quantitative Western blot assay, PC was shown to be concentrated in the indirect flight muscles and fat body preparations. The ratios of the 133 to 128 kDa polypeptides were shown to differ in various tissues and an Aedes albopictus cell line. The indirect flight muscle was the only tissue in which the 128 kDa polypeptide was more abundant, while both the midgut and the cell line showed almost exclusively the 133 kDa polypeptide. Both peptides were present in varying amounts in brain, malpighian tubule, ovary and fat body preparation. The two isoforms of PC could play different roles in the flight muscle and other tissues. Clones covering a complete cDNA of PC of A. aegypti were obtained using a directional approach. The 3952 bp nucleotide sequence, including a 3585 bp coding region, was determined from these cDNA clones. The deduced 1195 amino acid sequence has a calculated M(r) of 132,200. A putative mitochondrial targeting sequence was determined by comparing the deduced amino acid sequence to the N-terminal sequences of the mature protein. The presence of a mitochondrial targeting sequence indicates that the mosquito PC encoded by the cloned cDNA may be localized in the mitochondria. After the targeting sequence, three functional domains were identified in the following order; biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP). The mosquito PC showed very high similarity to PCs from other sources (55.1-75.2% identity). Genomic Southern analysis indicated that there could be two similar PC genes or a single PC gene with allelic polymorphism in the A. aegypti genome. The evolutionary relationship of PCs among different organisms was consistent with the accepted evolutionary relationship of their host organisms. The evolution of the domain structures of the biotin-dependent carboxylases including PC was also investigated. This analysis indicates that biotin-dependent carboxylases evolved from a common origin. The analysis also provides evidence for early gene duplication events that shaped the family of biotin-dependent carboxylases. Clear evidence for the coevolution of BC and BCCP domains is presented, although they are associated with very different CT domains and the relative position of the three functional domains varies between members of the biotin-dependent carboxylases.

Vitellin of the sweet potato whitefly, Bemisia tabaci: biochemical characterization and titer changes in the adult.

SDS-PAGE of the sweet potato whitefly (Bemisia tabaci) egg extract showed one major band (approximately 190 kDa) and two minor bands (approximately 75 kDa and 67 kDa). A distinct 190 kDa band was also present in male extract. On SDS gels the vitellin band of the greenhouse whitefly (Trialeurodes vaporarium) was larger, about 220 kDa. The native molecular mass of sweet potato whitefly vitellin was estimated to be 375 kDa using 4-20% native porelimiting gel electrophoresis. Its isoelectric point was estimated to be 7.3 using isoelectric focusing. Two-dimensional gel electrophoresis and densitometry were used to estimate vitellin subunit composition; the data suggest that the sweet potato whitefly vitellin is likely to be a 380 kDa native molecule formed by two 190 kDa subunits. The two minor bands (75 kDa and 67 kDa) may be breakdown products of the native vitellin. This conclusion was supported by a Western blot of an SDS-PAGE gel of partially degraded female and egg extracts, which showed that polyclonal antiserum raised against the 190 kDa polypeptide recognized the 75 kDa and 67 kDa bands. Seven hybridoma cell lines secreting monoclonal antibodies against the 190 kDa band were screened, and one of them (S1A2G9H2) was mass produced. The antibody recognized the 190 kDa band in a Western blot. All the screened monoclonal antibodies were female and egg-specific by ELISA and/or Western blot, suggesting that the 190 kDa band in male extract was not a vitellin. A sensitive ELISA was established that could detect as little as 1/40 of an egg equivalent of vitellin using the monoclonal antibody from S1A2G9H2. Profiles of female sweet potato whitefly reproductive activities (egg laying, amount of vitellin in the female, and total vitellin produced by a female) within 2 days after eclosion were determined. Arch. Insect Biochem.

Analysis of a vitellogenin gene of the mosquito, Aedes aegypti and comparisons to vitellogenins from other organisms.

A genomic clone of the Aedes aegypti vitellogenin A1 gene was sequenced including 2015 bp of 5' untranscribed sequence, 6369 bp of open reading frame interrupted by two introns, and a short 3' untranslated region. Primer extension was used to identify the transcription initiation site. The amino termini of the large and small subunits were located by N-terminal sequencing of vitellin purified from eggs. The length of the signal sequence and the position of the cleavage site between the two subunits were also determined. Three sequential imperfect repeats were found near the beginning of the small subunit. The sequence of the coding region appears to be polymorphic. Comparison of the signal sequences of seven insect vitellogenin genes revealed several conserved leucines, and a conserved position of an intron. However, the signal sequences are not conserved between these genes and the yolk protein genes of Cyclorraphid Dipteran insects. The cleavage sites between the small and large subunits in the vitellogenins of the mosquito, A. aegypti, sawfly, Athalia rosae, boll weevil, Anthonomus grandis, and silkworm, Bombyx mori are flanked by sequences rich in serine. Pairwise dot matrix analysis at the protein level showed that the mosquito, boll weevil and silkworm vitellogenins are significantly related with approx. 50% similarity. One region of the three insect vitellogenin genes, near the N-terminal of the large subunit, showed the highest levels of similarity, from 57.5 to 64.4%. The position of cysteines in insect vitellogenins is conserved, particularly in the C-terminus of the large subunit. Dot matrix comparison of the mosquito vitellogenin with that of Xenopus laevis and Caenorhabditis elegans showed much lower, but still significant degrees of relationship. Pairwise comparisons of the mosquito vitellogenin and the Drosophila melanogaster yolk proteins did not show significant similarities. Potential regulatory regions in the mosquito VgA1 gene were identified by comparison to regulatory elements known from other organisms, especially D. melanogaster, which could provide useful information for further functional analysis.

Isolation of two AKH-related peptides from cicadas.

Two peptides related to locust adipokinetic hormone and crustacean red pigment concentrating hormone were isolated by high performance liquid chromatography from the cicadas Cacama valavata and Diceroprocta semicincta. Both species have the same peptides. The structure of one of the peptides is pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Asn-amide. The mass spectrum, amino acid composition, and amino acid sequence of the other peptide suggest that it is almost identical to the first peptide. However, the exact nature of the difference between the two peptides could not be determined.

A comparative study of leucokinin-immunoreactive neurons in insects.

Antisera were raised against leucokinin IV, a member of the leucokinin peptide family. Immunohistochemical localization of leucokinin immunoreactivity in the brain of the cockroach Nauphoeta cinerea revealed neurosecretory cells in the pars intercerebralis and pars lateralis, several bilateral pairs of interneurons in the protocerebrum, and a group of interneurons in the optic lobe. Several immunoreactive interneurons were found in the thoracic ganglia, while the abdominal ganglia contained prominent immunoreactive neurosecretory cells, which projected to the lateral cardiac nerve. The presence of leucokinins in the abdominal nerve cord was confirmed by HPLC combined with ELISA. Leucokinin-immunoreactive neurosecretory cells were also found in the pars intercerebralis of the cricket Acheta domesticus and the mosquito Aedes aegypti, but not in the locust Schistocerca americana or the honey bee Apis mellifera. However, all these species have leucokinin-immunoreactive neurosecretory cells in the abdominal ganglia. The neurohemal organs innervated by abdominal leucokinin-immunoreactive cells were different in each species.

Structure, expression, and hormonal control of genes from the mosquito, Aedes aegypti, which encode proteins similar to the vitelline membrane proteins of Drosophila melanogaster.

Genomic and cDNA clones of a gene expressed after a blood meal in the mosquito, Aedes aegypti, were identified as having significant similarity to the vitelline membrane protein genes of Drosophila melanogaster. The predicted protein had unusually high contents of alanine, histidine, and proline and contained a region of hydrophobic amino acids that was highly conserved in the predicted protein of the D. melanogaster vitelline membrane protein genes. The 15a gene was expressed from 5 to 40 hr after a blood meal. It was expressed only in the follicle cells of the ovary, particularly in the cells surrounding the oocyte. The 15a gene was expressed in ovaries of the blood-fed, decapitated female in response to an injection of 20-hydroxyecdysone, and in ovaries from non-blood-fed females incubated with the hormone, even in the presence of cycloheximide. A second gene, with weaker homology to 15a, is presumably another member of a family of related genes, as is the case with D. melanogaster vitelline membrane protein genes. This second gene contained a coding sequence similar to a decapeptide recently isolated from mosquito ovaries as an "oostatic factor" (Borovsky et al., FASEB J. 4, 3015-3020, 1990).

Rapid isolation of a neurohormone from mosquito heads by high-performance liquid chromatography.

Methods were developed for the isolation of the egg development neurosecretory hormone, EDNH, from heads of the mosquito Aedes aegypti. This hormone stimulates ecdysone production by ovaries. Methods used for the successful isolation of insulin-like peptides from vertebrate tissues were modified to develop a four-step procedure involving extraction in acidified ethanol, precipitation by neutralization, followed by sequential separation on size-exclusion, ion-exchange and reversed-phase high-performance liquid chromatography columns.

Defensive role of tropical tree resins: antitermitic sesquiterpenes from Southeast Asian Dipterocarpaceae.

Chemical components demonstrating antiinsectan properties were isolated and identified from crude resins of fourDipterocarpus species. The most active compounds against Southeast Asian termites (Neotermes spp.) proved to be alloaromadendrene, humulene, and caryophyllene. These results suggest that these sesquiterpenes, which occur in many dipterocarps, play an important role in defense against insects.

Defensive sesquiterpenoids from a dipterocarp (Dipterocarpus kerrii).

Four sesquiterpenoids (2, 4, 7, and9) have been isolated and characterized from the termiticidal fraction ofDipterocarpus kerrii resin. The major constituent of this resin is α-gurjunene (1).

The vitellogenin gene family of Aedes aegypti.

We have been interested in identifying genes that play a role in reproduction of the mosquito Aedes aegypti. Our interests are currently focused on the vitellogenin genes which in the mosquito are expressed only in the fat body in response to the insect steroid hormone, 20-hydroxyecdysone. Four of the five vitellogenin genes in the genome have been cloned. We have examined the relationships between these genes and find that they form a small gene family exhibiting different levels of relationship.

Peptide nature of two mosquito natriuretic factors.

High-pressure liquid chromatography (HPLC) of saline extracts of Aedes aegypti heads yields three fractions (from a total of 108) that affect transepithelial voltage and/or fluid secretion in isolated Aedes Malpighian tubules. In this study we investigated the physical and chemical nature of the active materials in these fractions. Gel-filtration chromatography revealed that the molecular weights of the three fractions were between 1,900 and 2,700. To test their thermostability the fractions were repeatedly frozen and thawed over a period of 110 days without loss of biological activity. Boiling at 100 degrees C for 5 min failed to significantly reduce their biological effects in isolated Malpighian tubules. In contrast, treatment with the proteolytic enzyme mixture, pronase, destroyed activity in all three. Fraction I no longer depolarized the transepithelial voltage of in vitro perfused Malpighian tubules, and fractions II and III completely lost their ability to stimulate fluid secretion and to affect transepithelial voltage. We conclude that our HPLC isolation yields a heterogeneous group of three polar low-molecular weight peptides. Expression of their biological activities in Malpighian tubules depends on intact peptide bonds.

Egg maturation and ecdysiotropic activity in extracts of mosquito (Aedes aegypti) heads.

Blood-fed, decapitated female Aedes aegypti mosquitoes matured eggs when injected with an extract of heads, but non-blood-fed females did not. The response to head extract was optimal when the head was allowed to remain for 2 hr after feeding. A dose response to injected egg development neurosecretory hormone (EDNH) was observed in vivo that was similar to in vitro dose responses previously reported. Blood-fed decapitated females responded equally well to boiled or unboiled head extract. When blood-fed decapitated females were injected with head extract, ecdysteroid levels increased. Partial purification of head extract using high-pressure liquid chromatography yielded a fraction at 34% acetonitrile that showed egg maturation activity in vivo when injected into blood-fed decapitated females, and ecdysiotropic activity when incubated in vitro with ovaries. In addition, a fraction at 30% acetonitrile was found that showed activity in vivo but not in vitro and may be a precursor. Occasionally, the fraction at 37% acetonitrile showed activity in the in vitro assay but had little activity in vivo and may be a metabolite. These results suggest that the same hormone was being assayed in vivo and in vitro and is EDNH.

Preliminary isolation of mosquito natriuretic factor.

A natriuretic factor that triggers diuresis in isolated Malpighian tubules of the mosquito was isolated from the head of the yellow-fever mosquito Aedes aegypti by passing a saline extract of mosquito heads through low-pressure and then high-pressure liquid chromatography (HPLC) columns. Three fractions with biologic activity eluted during a reverse-phase HPLC linear acetonitrile gradient run. Fraction I depolarized the transepithelial voltage (Vt) of isolated perfused Malpighian tubules but did not not stimulate fluid secretion in the Ramsay assay (J. A. Ramsay, J. Exp. Biol. 31: 104-113, 1954). Fraction II depolarized and fraction III hyperpolarized Vt, and both stimulated fluid secretion three- to fourfold. Even though the effects of fractions II and III on Vt differed, both stimulated fluid secretion by increasing the rate of NaCl secretion without affecting K secretion. The selective stimulation of active secretory Na transport by fraction III is mimicked by cyclic AMP (cAMP), suggesting the second messenger role of cAMP in the effects of fraction III. Because fraction III stimulates a NaCl-rich, as opposed to KCl-rich, fluid, the term mosquito natriuretic factor is proposed for this active fraction.

Partial purification of egg development neurosecretory hormone with reverse-phase liquid chromatographic techniques.

We report the use of reverse-phase liquid chromatographic techniques for the isolation of a steroidogenic neuropeptide (EDNH) from mosquito heads. Activity of fractions was assayed by measuring the ability of ovaries to produce ecdysteroid in vitro. Dose response profiles using crude head extracts or partially purified EDNH were nearly identical, indicating that the methods of preparation did not alter biological activity. EDNH activity eluted from a reverse-phase HPLC (RP-HPLC) column primarily near 35 percent acetonitrile using a linear gradient. Methods developed with an analytical RP-HPLC column were successfully adapted for preparative work. Active fractions from the preparative RP-HPLC were further purified on a second analytical column under isocratic conditions at 30% acetonitrile. Two adjacent UV absorbing peaks were found, each with EDNH activity. Activity was sensitive to proteolysis.