Effects of AeDNV infection on Aedes aegypti lifespan and reproduction

The effects of Aedes Densovirus (AeDNV) infections on survival, fertility, fecundity and vertical transmission in Aedes aegypti (Diptera: Culicidae) were measured in laboratories in Kiev, Ukraine and Colorado, USA and incorporated into a predictive model of the effects of AeDNV on vector capacity. Adult lifespan and daily survival were reduced in AeDNV infected mosquitoes. This effect was dependent on the dose of the virus. Infected females had decreased fecundity. The oviposition rate was less in infected females and the hatch rate declined in eggs laid by infected females. The amounts of AeDNV in infected females and the infection rate of their offspring were measured with real-time PCR. The average filial transmission rate was 70% and larval infection rates from infected females varied between 42 and 62%. Vertically infected larvae, and individual eggs contained 1 × 10⁵ AeDNV genome equivalents (geq). Modeling the effects of AeDNV infection on Ae. aegypti populations suggested a large decrease in the numbers of eggs, larvae, pupae, and adults arising from infected mothers and suggested that AeDNV treatment of larvae could cause up to a 76% reduction of infectious mosquito days.     

Identification of novel LTR retrotransposons in the genome of Aedes aegypti

We have detected seventy-six novel LTR retrotransposons in the genome of the mosquito Aedes aegypti by a genome wide analysis using the LTR_STRUC program. We have performed a phylogenetic classification of these novel elements and a distribution analysis in the genome of A. aegypti. These mobile elements belong either to the Ty3/gypsy or to the Bel family of retrotransposons and were not annotated in the mosquito LTR retrotransposon database (TEfam). We have found that  1.8% of the genome is occupied by these newly detected retrotransposons that are distributed predominantly in intergenic genomic sequences and introns. The potential role of retrotransposon insertions linked to host genes is described and discussed. We show that a retrotransposon family belonging to the Osvaldo lineage has peculiar structural features, and its presence is likely to be restricted to the A. aegypti and to the Culex pipiens quinquefasciatus genomes. Furthermore we show that the ninja-like group of elements lacks the Primer Binding Site (PBS) sequence necessary for the replication of retrotransposons. These results integrate the knowledge on the complicate genomic structure of an important disease vector.     

Iron loaded ferritin secretion and inhibition by CI-976 in Aedes aegypti larval cells

Ferritin is a multimer of 24 subunits of heavy and light chains. In mammals, iron taken into cells is stored in ferritin or incorporated into iron-containing proteins. Very little ferritin is found circulating in mammalian serum; most is retained in the cytoplasm. Female mosquitoes, such as Aedes aegypti (yellow fever mosquito, Diptera), require a blood meal for oogenesis. Mosquitoes receive a potentially toxic level of iron in the blood meal which must be processed and stored. We demonstrate by ⁵⁹Fe pulse-chase experiments that cultured A. aegypti larval CCL-125 cells take up iron from culture media and store it in ferritin found mainly in the membrane fraction and secrete iron-loaded ferritin. We observe that in these larval cells ferritin co-localizes with ceramide-containing membranes in the absence of iron. With iron treatment, ferritin is found associated with ceramide-containing membranes as well as in cytoplasmic non-ceramide vesicles. Treatment of CCL-125 cells with iron and CI-976, an inhibitor of lysophospholipid acyl transferases, disrupts ferritin secretion with a concomitant decrease in cell viability. Interfering with ferritin secretion may limit the ability of mosquitoes to adjust to the high iron load of the blood meal and decrease iron delivery to the ovaries reducing egg numbers.     

Chemical composition and larvicidal effects of essential oil of Dendropanax morbifera against Aedes aegypti L.

Essential oils obtained from the flowers of Dendropanax morbifera were extracted and the chemical composition and larvicidal effects were studied. The analyses were conducted by gas chromatography and mass spectroscopy (GC–MS) revealed that the essential oil of D. morbifera contained 27 compounds. The major chemical components identified were γ-elemene (18.59%), tetramethyltricyclohydrocarbon (10.82%), β-selinene (10.41%), α-zingibirene (10.52%), 2-isopropyl-5-methylbicylodecen (4.2%), β-cubebene (4.19), and 2,6-bis(1,1-Dimethylethyl)-4-phenol (4.01%). The essential oil had a significant toxic effect against early fourth-stage larvae of Aedes aegypti L. with an LC₅₀ value of 62.32 ppm and an LC₉₀ value of 131.21 ppm. The results could be useful in search for newer, safer, and more effective natural larvicidal agents against A. aegypti.     

伊蚊C型凝集素mosGCTL-2是登革病毒感染相关的重要蛋白质

C型凝集素是一类含有糖结合结构域的蛋白质,从节肢动物到哺乳动物的C型凝集素都具有共同的基序,它在进化上相当保守,在免疫反应中发挥重要作用. 埃及伊蚊表达30多种C型凝集素蛋白,它是登革病毒的关键传播媒介,这些蛋白质对病毒和细菌感染均有至关重要的作用. 最近研究表明,C型凝集素mosGCTL-3与二型登革热病毒包膜蛋白具有相互作用,能够增强登革病毒对埃及伊蚊的感染. 在本文中,我们发现了C型凝集素蛋白mosGCTL-2具有与mosGCTL-3类似的功能. 两种C型凝集素mosGCTL-2和mosGCTL-3的氨基酸残基序列一致性高达43.56％. 为研究mosGCTL-2在登革病毒蚊媒传播中的作用,我们通过果蝇S2细胞表达系统表达纯化了mosGCTL-2蛋白. 结果表明,mosGCTL-2与二型登革热病毒包膜蛋白的结合具有钙离子依赖性. 进一步的研究表明,埃及伊蚊感染登革病毒能够诱导mosGCTL2表达上调,是二型登革热病毒感染埃及伊蚊所必需的蛋白质. 以上研究说明,mosGCTL-2蛋白可能是在登革热病毒感染埃及伊蚊中起重要作用的一种模式识别受体.     

Transient expression of the Drosophila melanogaster cinnabar gene rescues eye color in the white eye (WE) strain of Aedes aegypti

The lack of eye pigment in the Aedes aegypti WE (white eye) colony was confirmed to be due to a mutation in the kynurenine hydroxylase gene, which catalyzes one of the steps in the metabolic synthesis of ommochrome eye pigments. Partial restoration of eye color (orange to red phenotype) in pupae and adults occurred in both sexes when first or second instar larvae were reared in water containing 3-hydroxykynurenine, the metabolic product of the enzyme kynurenine hydroxylase. No eye color restoration was observed when larvae were reared in water containing kynurenine sulfate, the precursor of 3-hydroxykynurenine in the ommochrome synthesis pathway. In addition, a plasmid clone containing the wild type Drosophila melanogaster gene encoding kynurenine hydroxylase, cinnabar (cn), was also able to complement the kynurenine hydroxylase mutation when it was injected into embryos of the A. aegypti WE strain. The ability to complement this A. aegypti mutant with the transiently expressed D. melanogaster cinnabar gene supports the value of this gene as a transformation reporter for use with A. aegypti WE and possibly other Diptera with null mutations in the kynurenine hydroxylase gene.     

Effects of antennectomy on blood-feeding behavior of Aedes aegypti mosquitoes

Completely flagellectomized females of Aedes aegypti usually do not locate a person of moderate attractiveness at very close range (0 to 4.5 cm from hand), but all may feed on an individual of great attractiveness under similar conditions. Approximately seven flagellar segments on one or two antennae are normally required for a high degree of blood avidity towards a person of average attractiveness in a cubic cage with 30.5-cm sides.

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Résumé Le test consiste à offrir la main et (ou) l'avant-bras d'un individu préalablement reconnu peu ou très attractif, à des moustiques enfermés dans de petites cages cylindriques plus ou moins longues; cette longueur conditionne la distance à laquelle les moustiques peuvent déceler l'appât.L'ablation bilatérale des flagelles antennaires supprime toute alimentation des moustiques opérés, sur un individu reconnu comme modérément attractif, mais ces mêmes insectes peuvent encore déceler un individu hautement attractif et s'alimenter. Les diverses amputations pratiquées montrent qu'il faut que soient conservés approximativement sept segments flagellaires (répartis sur une ou deux antennes) pour maintenir un comportement hématophage à l'égard d'une personne médiocrement attractive, les insectes étant enfermés dans une cage de 30,5×30,5×30,5 cm.

     

Biochemical analysis of a blood meal-induced Aedes aegypti glutamine synthetase gene

Glutamine synthetase (GS) in the mosquito, Aedes aegypti, is induced in the midgut following a blood meal. Mosquito GS message is detected as soon as 1 h post-blood feeding and remains stable for 18 h. Using a PCR product encoding mosquito GS, a λgt10 adult female mosquito cDNA library was screened. A cDNA clone, pCl5A2, encoding the full translation product of mosquito GS was isolated and sequence analyses performed. Mosquito GS cDNA is 2.5 kb in length and its putative translation product shares all the conserved regions characteristic of the GS gene family, including the presumed ATP biding site. Glutamine synthetase activity in the mosquito midgut is highest at 18 h post-blood feeding. Activity can be detected over a broad pH range, from 6.0 to 7.5. Unlike other cellular GS enzymes, mosquito GS is not active in the presence of ATP. Very low dosages (0.05 mM) of L-methionine S-sulfoximine are sufficient to partially inhibit mosquito GS activity. Inhibition of GS disrupts the normal formation of the midgut peritrophic matrix, suggesting that GS enzyme might be involved in the initial pathway of chitin synthesis. The unique expression pattern and inducible nature of the mosquito GS gene make it an interesting candidate for studying promoter function. Additionally, the blood meal activation of the GS gene makes this a potentially valuable tool in mosquito transformation studies.     

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.     

Lecithin in synthetic larval diet for Aedes aegypti improves larval and adult performance

A chemically defined synthetic rearing medium was used to compare larval growth of Aedes aegypti with or without crude animal lecithin or synthetic dipalmitoyl lecithin. Pupal weights, adult female life spans and oviposition histories of pupae and adults derived from synthetic diet rearings and from crude culture (liver powder) rearings were also compared. Both lecithins improved larval growth rate; optimal concentrations reduced time to pupation by 2 and 1 days for animal and synthetic lecithins respectively, and animal lecithin was better tolerated by the mosquito larvae at the higher concentrations tested. Addition of crude animal lecithin to the basal synthetic rearing medium had little effect on the weights of male and female pupae, but it increased adult female life spans and improved weekly egg production. In no case did mosquitoes from axenic rearings in synthetic media deviate greatly from limits specified by earlier workers for field-derived mosquitoes.

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Résumé Les développements larvaires d'Aedes aegypti ont été comparés sur des substrats alimentaires avec ou sans lecithine animale brute ou DL-a-dipalmitoyle lecithine synthétique.Les comparaisons ont porté aussi sur les poids nymphaux, la longé-vité et la fécondité de femelles élevées sur régime synthétique ou à partir de poudre de foie.Les deux types de lecithines améliorent le développement larvaire; les concentrations optimales des deux régimes (lecithine animale et lecithine synthétique) avancent respectivement la pupaison de 2 et 1 jour; les lecithines animale et synthétique sont tolérées par les larves respectivement jusqu'aux concentrations de 0,008 et 0,006%.L'addition de lecithine animale brute au régime contenant de la lecithine synthétique a eu peu d'effets sur les poids des nymphes mâles et femelles mais a augmenté la longévité et la fécondité hebdomadaire des femelles.Les performances des moustiques élevés sur substrat aseptique synthétique n'ont jamais été très éloignées des limites indiquées antérieurement par les travaux sur moustiques provenant de la nature.

     

Alteration of feeding behavior and fertility in Aedes aegypti by the chemosterilant apholate

Studies have demonstrated that the inhibition of ovarian development, as contrasted to inhibition of oogenesis, by the chemosterilant Apholate, causes a significant increase in the biting rate of female Aedes aegypti. The material also reduces the fertility of males of this species.The possibility exists that materials with similar modes of action might increase the vector efficiency of this species, at least temporarily, while the population is being reduced at a slower rate. Assessment of this possibility should be made in other vectors, of both plant and animal diseases, as well as with other chemosterilants.

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Zusammenfassung Untersuchungen mit dem Chemosterilans Apholat weisen eine zweifache Wirkung auf Aedes aegypti nach. Werden frisch geschlüpfte Larven 10–20 ppm des Stoffes ausgesetzt, so werden die Prozesse der Oogenese und Spermatogenese in so starkem Maße beeinflußt, daß ein hoher Anteil unfruchtbarer Eier abgelegt wird. Bei einer Konzentration von 30 ppm wird jedoch die Entwicklung der Ovarien selbst gehemmt, so daß den Erwachsenen diese Organe fehlen. Solche Weibchen nahmen signifikant mehr Blut auf als unbehandelte Weibchen, obwohl gar keine Eier gebildet werden konnten.Diese Änderung im Nahrungsaufnahme-Verhalten könnte wichtige Folgen für die Übertragung von Pathogenen durch behandelte Vektoren von Tier- und Pflanzenkrankheiten haben. Einige Aspekte dieses Problems werden erörtert.

     

Lipophorin as a yolk protein precursor in the mosquito, Aedes aegypti

We examined expression of the lipophorin (Lp) gene, lipophorin (Lp) synthesis and secretion in the mosquito fat body, as well as dynamic changes in levels of this lipoprotein in the hemolymph and ovaries, during the first vitellogenic cycle of females of the yellow fever mosquito, Aedes aegypti. Lipophorin was purified by potassium bromide (KBr) density gradient ultracentrifugation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Polyclonal antibodies were produced against individual Lp apoproteins, apolipoprotein-I (apoLp-I) and apolipoprotein-II (apoLp-II), with molecular weights of 240 and 75 kDa, respectively. We report here that in the mosquito A. aegypti, Lp was synthesized by the fat body, with a low level of the Lp gene expression and protein synthesis being maintained in pre- and postvitellogenic females. Following a blood meal, the Lp gene expression and protein synthesis were significantly upregulated. Our findings showed that the fat body levels of Lp mRNA and the rate of Lp secretion by this tissue reached their maximum at 18 h post-blood meal (PMB). 20-Hydroxyecdysone was responsible for an increase in the Lp gene expression and Lp protein synthesis in the mosquito fat body. Finally, the immunocytochemical localization of Lp showed that in vitellogenic female mosquitoes, this protein was accumulated by developing oocytes where it was deposited in yolk granules.     

Sensitivity of Aedes aegypti adults (Diptera: Culicidae) to the vapors of Eucalyptus essential oils

Vapors of essential oils extracted from various species of Eucalyptus (E. gunnii, E. tereticornis, E. grandis, E. camaldulensis, E. dunnii, E. cinerea, E. saligna, E. sideroxylon, E. globulus ssp. globulus, E. globulus ssp. maidenii, E. viminalis and the hybrids E. grandis × E. tereticornis and E. grandis × E. camaldulensis) and their major components were found to be toxic to Aedes aegypti adults, the yellow fever mosquito.An aliquot of each oil was placed in a cylindrical test chamber and the number of knocked-down mosquitoes was recorded as function of time. Knockdown time 50% was then calculated. Results showed that E. viminalis had the fastest knockdown time at of 4.2 min, on the same order as dichlorvos, a standard knockdown agent. A correlation was observed between the content of 1,8-cineole in the Eucalyptus essential oils and the corresponding toxic effect.The correlation between KT₅₀ values and calculated vapor pressures of the essential oil components showed that the fumigant activity of simple organic compounds in insects is correlated with their volatility.     

The effects of various oxygen tensions on embryogeny and larval responses of Aedes aegypti

Eggs of Aedes aegypti (L.) were submerged in water containing dissolved oxygen at levels ranging from less than 1 to 14 parts per million, at 1, 24, 48, 72 and 96 hours after being laid. After a 4.5 day exposure period, which encompasses the normal period of embryogeny, the eggs were subjected to the hatching stimulus as a measure of maturity.The whole of embryogeny occurred at a normal rate under levels of 3.8 to 14 ppm dissolved oxygen. An oxygen level of 0.95 ppm was lethal to all eggs except those exposed only in the advanced stages of development. A level of 1.9 ppm dissolved oxygen caused a retardation of developmental rate, with 6.5 days being required to achieve maturation.Immature, but advanced, embryos could be hatched artificially, with completion of development to normal adults.

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Die wirkungen unterschiedlicher sauerstoffspannungen auf die embryogenese und larvalreaktionen von Aedes aegypti

Zusammenfassung Eier von Aedes aegypti wurden 1, 24, 48, 72 und 96 Stunden nach der Ablage in Wasser getaucht, das gelösten Sauerstoff in Mengen von weniger als 1 bis 14 Teilen pro Million enthielt. Nach einer Behandlungszeit von 4,5 Tagen, die dem normalen Zeitraum der Embryonalentwicklung entspricht, wurden die Eier als Maß ihrer Reife dem Schlüpfreiz unterworfen.Die gesamte Embryonalentwicklung verlief bei Sauerstoffspannungen von 3,8 bis 14 ppm in normalem Ausmaß. Eine Sauerstoffspannung von 0,95 ppm war für alle Eier lethal mit Ausnahme derjenigen, die ihr erst in fortgeschrittenen Entwicklungsstadien ausgesetzt wurden. Eine Menge von 1,9 ppm gelösten Sauerstoffs verursachte eine Verzögerung der Entwicklungsgeschwindigkeit, bei der 6,5 Tage zur Erreichung der Schlüpfreife benötigt wurden.Unreife, aber fortgeschrittene Embryonen konnten künstlich zum Schlüpfen gebracht werden, bei vollständiger Weiterentwicklung zu normalen Imagines.

     

Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes

Background  

Arthropod-borne viruses (arboviruses) can persistently infect and cause limited damage to mosquito vectors. RNA interference (RNAi) is a mosquito antiviral response important in restricting RNA virus replication and has been shown to be active against some arboviruses. The goal of this study was to use a recombinant Sindbis virus (SINV; family Togaviridae; genus Alphavirus) that expresses B2 protein of Flock House virus (FHV; family Nodaviridae; genus Alphanodavirus), a protein that inhibits RNAi, to determine the effects of linking arbovirus infection with RNAi inhibition.     

Release of egg development neurosecretory hormone in Aedes aegypti and Aedes taeniorhynchus induced by an ovarian factor

Ovariectomized Aedes aegypti do not synthesize vitellogenin after a blood meal, unless an ovary from a blood-fed donor is implanted. Decapitation, however, prior to implantation inhibits vitellogenin synthesis. A female ovariectomized and decapitated 6 hr after a blood meal, synthesizes vitellogenin if an ovary from a blood-fed donor is implanted. On the other hand, females that are fed on blood and immediately decapitated can not be stimulated to synthesize vitellogenin with implanted ovaries removed from blood-fed donors. These experiments led to the hypothesis that the blood meal stimulates the ovary to secrete a corpus cardiacum stimulating factor, that in turn promotes release of egg development neurosecretory hormone stored in the corpus cardiacum.Injection of 20-hydroxy-ecdysone or ovarian extract prepared from ovaries removed from unfed females does not release egg development neurosecretory hormone. Thus corpus cardiacum stimulating factor is not 20-hydroxy-ecdysone, and ovaries removed from unfed females do not store it.The rate of inactivation of egg development neurosecretory hormone released from the corpus cardiacum after a blood meal was investigated by implanting an ovary into females that were blood fed for various intervals than decapitated and ovariectomized. Seventy per cent of implants grow when the operation is done 18 hr after feeding, and 30% when the operation is done between 18 and 24 hr after feeding, indicating that egg development neurosecretory hormone is stable for the first 18 hr after a blood meal.Aedes taeniorhynchus females ovariectomized 24 hr after adult emergence do not synthesize vitellogenin. When such a female is implanted with an ovary removed from a sugar-fed or blood-fed Aedes aegypti donor vitellogenin synthesis is initiated, and the implant grows. Decapitation prior to implantation inhibit vitellogenin synthesis and implants do not grow. These results indicate that corpus cardiacum stimulating factor is not species specific.     

Larvicidal activity against Aedes aegypti of some plants native to the West-Central region of Brazil

A total of 42 ethanolic extracts from 30 different plant species, native to the Pantanal and Cerrado of the West-Central region of Brazil, have been evaluated for their larvicidal activity against Aedes aegypti larvae, the vector of dengue and dengue hemorrhagic fevers. Among the extracts tested, that obtained from the trunk bark of Ocotea velloziana was the most active. Using a bioassay-directed fractionation of this extract, the active constituent was isolated and characterized as the aporphine alkaloid (+)-dicentrine. Its structure was established on the basis of ¹H and ¹³C NMR spectra, optical rotation and by comparison with an authentic sample. This is the first report on the larvicidal activity against A. aegypti of this alkaloid. Our results suggest that (+)-dicentrine may be considered as a promising natural mosquito larvicidal agent.