Pathology of mosquito iridescent virus of Aedes taeniorhynchus in cell cultures of Aedes aegypti.

An electron microscopical study was conducted on the pathology of the mosquito iridescent virus (MIV) of Aedes taeniorhynchus in monolayer cultures of Aedes aegypti cells. The sequence of events in the pathology, from the initiation of attachment through maturation and release, is presented.MIV attaches to cells and is taken up by the process of viropexis (phagocytosis) within 15 min after inoculation. Intact virions are released into the cytoplasm at 30–60 min by disruption of the phagocytic vesicles. Discrete foci of replication (viroplasm) develop in the cytoplasm within 1 day after infection. Progeny virus is assembled in the viroplasm within 2 days after infection and later appears at the cell surface, where it acquires an envelope from the plasma membrane upon budding from the cell. Virus does not accumulate to form aggregates in the cytoplasm; instead, it buds from the cell after assembly.     

Susceptibility of mosquito and lepidopteran cell lines to the mosquito iridescent virus (IIV-3) from Aedes taeniorhynchus

Mosquito iridescent viruses (MIV) are members of the genus Chloriridovirus that currently contains only the type IIV-3 from Aedestaeniorhynchus. The complete genome of invertebrate iridescent virus -3 (IIV-3) has been sequenced and the availability of a tissue culture system would facilitate functional genomic studies. This investigation, using quantitative PCR and electron microscopy, has determined that the mosquito cell lines Aedes aegypti (Aag2), Aedes albopictus (C6/36) and Anopheles gambiae (4a3A) as well as the lepidopteran cell line from Spodoptera frugiperda (SF9) are permissive to IIV-3 infection. However, IIV-3 infection remained longer in Aag2 and C6/36 cells. Virus produced in C6/36 cell line was infectious to larvae of A. taeniorhynchus by injection and per os. Ultrastructural examination of 4a3A and SF9 cells infected with IIV-3 revealed an unusual feature, where virions were localized to mitochondria. It is speculated that containment with mitochondria may play a role in the lack of persistence in these cell lines.     

The replication of a cytoplasmic polyhedrosis virus from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) in Spodoptera frugiperda cells

A cytoplasmic polyhedrosis virus (CPV) from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) replicated in Spodoptera frugiperda cells. Low rates of infection were achieved, even at high multiplicities of infection and TCID₅₀ assays showed that there was negligible release of virus particles from infected cells. In an infected focus assay, based on formation of PIB, the dose-response data demonstrated that a single particle could initiate infection. No loss of infectivity occurred in virus preparations stored at 4°, ?20°, or ?90°C, but infectivity of virus stored at 20°C declined sharply. A small isometric virus contaminant was present in some CPV preparations and its interaction with the CPV is discussed. Limited CPV infection was achieved in Trichoplusia ni cells, but attempts to infect Aedes aegypti cells were unsuccessful.     

Physiological effects of carbohydrates on survival, metabolism, and flight potential of female Aedes taeniorphynchus

Starved Aedes taeniorhynchus and Aedes aegypti females 2 days after emergence were fed ad lib. a mixture of 10% test carbohydrate solution and 5% sorbose solution (as a phagostimulant) and maintained for survival. Also, starved A. taeniorhynchus females 5 days after emergence were fed, with a micro-pipette, 2 μl/female of a mixture of 20% test carbohydrate and 5% sorbose. These females were analysed for glycogen accumulation 18 hr after feeding, and they were flown tethered on a flight mill immediately as well as 24 hr after feeding. Twenty-eight carbohydrates including sugar alcohols were tested.     

Establishment and characterization of a new <Emphasis Type="Italic">Aedes aegypti</Emphasis> (L.) (Diptera: Culicidae) cell line with special emphasis on virus susceptibility

A new cell line from the neonate larvae of Aedes aegypti (L) mosquito was established and characterized. The cell line at the 50th passage (P) level consisted of three prominent cell types, i.e., epithelial-like cells (92%), fibroblast-like cells (7%), and giant cells (∼1%). Karyological analysis showed diploid (2n = 6) number of chromosomes in >75% cells at P-50. The growth kinetics studied at 52nd passage level showed approximately tenfold increase in cell number over a 10-d study period. The species specificity studies using DNA amplification fingerprinting profile analysis using RAPD primers demonstrated 100% homology with the host profile showing the integrity of the cell line. Electron microscopy revealed the absence of mycoplasma or other adventitious agents. The cell line supported the multiplication of seven arboviruses, i.e., Chikungunya (CHIK), Japanese encephalitis, West Nile, dengue 2 (DEN-2), Chandipura, vesicular stomatitis, and Chittoor viruses. The cell line did not replicate Ganjam and Kaisodi viruses. CHIK virus yield in the new cell line was approximately 3log and 0.5log 50% tissue culture infective dose (TCID₅₀)/mL higher than Vero E6 and C6/36 cell lines, respectively. In the case of DEN-2 virus, it yielded 1log TCID₅₀/mL higher than Vero E6, but lesser than C6/36 cell line. Due to its high susceptibility to a broad spectrum of viruses, the new cell line may find application in virus isolation during epidemics and in antigen production.     

Oral Susceptibility of Singapore Aedes (Stegomyia) aegypti (Linnaeus) to Zika Virus

Background

Zika virus (ZIKV) is a little known flavivirus that caused a major outbreak in 2007, in the South-western Pacific Island of Yap. It causes dengue-like syndromes but with milder symptoms. In Africa, where it was first isolated, ZIKV is mainly transmitted by sylvatic Aedes mosquitoes. The virus has also been isolated from Ae. aegypti and it is considered to be the vector involved in the urban transmission of the virus. Transmission of the virus by an African strain of Ae. aegypti has also been demonstrated under laboratory conditions. The aim of the present study is to describe the oral susceptibility of a Singapore strain of Ae. aegypti to ZIKV, under conditions that simulate local climate.

Methodology/Principal Findings

To assess the receptivity of Singapore''s Ae. aegypti to the virus, we orally exposed a local mosquito strain to a Ugandan strain of ZIKV. Upon exposure, fully engorged mosquitoes were maintained in an environmental chamber set at 29°C and 70–75% RH. Eight mosquitoes were then sampled daily from day 1 to day 7, and subsequently on days 10 and 14 post exposure (pe). The virus titer of the midgut and salivary glands of each mosquito were determined using a tissue culture infectious dose₅₀ (TCID₅₀) assay. High midgut infection and salivary gland dissemination rates were observed. By day 5 after the infectious blood meal, ZIKV was found in the salivary glands of more than half of the mosquitoes tested (62%); and by day 10, all mosquitoes were potentially infective.

Conclusions/Significance

This study showed that Singapore''s urban Ae. aegypti are susceptible and are potentially capable of transmitting ZIKV. The virus could be established in Singapore should it be introduced. Nevertheless, Singapore''s current dengue control strategy is applicable to control ZIKV.     

Serial passage of Heliothis zea singly embedded nuclear polyhedrosis virus in a homologous cell line

This paper describes the replication and serial passage of Heliothis zea nuclear polyhedrosis virus (NPV) in a H. zea cell line. It was demonstrated that long-term serial passages of the H. zea NPV in homologous host cell culture decreased both the total number of polyhedral inclusion bodies (PIBs) produced and the infectivity of the supernatant as measured by TCID₅₀. The growth curve indicated that infectious material was released from cells 24 hr postinfection (p.i.) and approached a maximal titer 3 days p.i. The kinetics of H. zea NPV decay at 4°, 27°, and 37°C were determined. Infectivity was not detected after 3 weeks at 37°C, but approximately 10^3.5 TCID₅₀/ml activity was still present after 3 and 8 weeks storage at 27° and 4°C, respectively. Electron microscopy confirmed the presence of single embedded virions in the inoculated cells.     

Dengue infection increases the locomotor activity of Aedes aegypti females

Background

Aedes aegypti is the main vector of the virus causing Dengue fever, a disease that has increased dramatically in importance in recent decades, affecting many tropical and sub-tropical areas of the globe. It is known that viruses and other parasites can potentially alter vector behavior. We investigated whether infection with Dengue virus modifies the behavior of Aedes aegypti females with respect to their activity level.

Methods/Principal Findings

We carried out intrathoracic Dengue 2 virus (DENV-2) infections in Aedes aegypti females and recorded their locomotor activity behavior. We observed an increase of up to ∼50% in the activity of infected mosquitoes compared to the uninfected controls.

Conclusions

Dengue infection alters mosquito locomotor activity behavior. We speculate that the higher levels of activity observed in infected Aedes aegypti females might involve the circadian clock. Further studies are needed to assess whether this behavioral change could have implications for the dynamics of Dengue virus transmission.     

Bioassays for comparative infectivity of mermithid nematodes (Romanomermis iyengari,Romanomermis culicivorax and Strelkovimermis spiculatus) for culicine mosquito larvae

Two improved bioassays were developed to establish infectivity baselines for selection experiments using mermithid nematode variants. Comparative infectivity of Romanomermis iyengari, Romanomermis culicivorax and Strelkovimermis spiculatus using larvae of three mosquito spp. Aedes sierrensis, Aedes aegypti and Culex pipiens were evaluated with “plate” and “tray” bioassays at selected intensity of infections. Using the “plate” bioassay, single mosquito larvae were immersed in 2 ml of water within individual depressions of 12-well, polystyrene tissue culture plates. One, three, or five preparasitic juveniles (J2) were added to each well. In the “tray” bioassay, polyethylene trays containing 500 ml water and 100 mosquito larvae were exposed to 500 (5:1, nematode:insect host) or 1000 (10:1) J2s. Percentage infection (PINF, infectivity) and intensity of infection (IINF, #nematodes per infected larvae) number were determined only after emergence of post-parasitic J3 juveniles. Under the bioassay conditions, all three species of nematodes resulted in infections in all mosquito hosts, but R. iyengari exhibited better effectiveness in the parasitism of mosquito larvae. The three species of mosquitoes presented high levels of susceptibility to each of the three species of nematodes, but in general Cx. pipiens and Ae. sierrensis were slightly more susceptible than Ae. aegypti. The “plate” bioassay was more efficient in measurement of infectivity of the mermithid species and in establishing baseline characteristics for these mosquito-parasitic nematodes. The “tray” bioassay was an effective bioassay for large cohorts of both infective juveniles and host larvae and, potential for field interactions.     

Dehiscence of Coelomomyces psorophorae sporangia from Aedes taeniorhynchus: Induction by amines and amino acids

The effect of various chemicals on induction of dehiscence of nonsterile Coelomomyces psorophorae sporangia from Aedes taeniorhynchus larvae was examined. Tests were made with various salts, reducing agents, chelating agents, buffers, alcohols, carbohydrates, fatty acids and derivatives, amino acids and derivatives, peptides, amines, purines and pyrimidines, antibiotics, and plant hormones. The most active compound was Tris [tris(hydroxymethyl)aminomethane]. Tris was most effective at pH 8–9, and a concentration of 1–20 mM. Structure-activity studies indicated that active compounds had a basic requirement for NH₂ and either COOH or CH₂OH attached to the alpha carbon, but only certain amino acids and amines were highly active. Preparations virtually free of host debris were not responsive to Tris, but addition of bacteria-free homogenates of A. taeniorhynchus larvae restored responsiveness. Dehiscence of C. psorophorae sporangia from Psorophora howardii was also enhanced by exposure to Tris.     

Isolation and characterization of ovarian ecdysteroidogenic hormones from the mosquito,Aedes aegypti

Five molecular species of ecdysteroidogenic peptides were isolated from female heads of the mosquito, Aedes aegypti. Three groups of fractions, separated by conventional liquid chromatography, had gonadotropic activity in an in vivo bioassay using autogenous Aedes atropalpus. The active peptides from one of the three groups were purified to homogeneity with ion-exchange and reversed phase HPLC. Aedes atropalpus decapitated at eclosion do not deposit yolk, whereas injection of 12–15 pg of the purified peptides elicited yolk deposition. In an in vitro assay, the same peptides also stimulated ovaries of A. aegypti to secrete ecdysteroids, as measured in a RIA.The purified peptides have a molecular weight between 6500 and 13,000. Amino acid composition analysis of one species revealed 92 amino acid residues, and the number of basic residues substantiated the basic nature of the peptide observed during chromatography. Since the peptides were purified to homogeneity and are functional in both bioassays, we consider the peptides to be “ovarian ecdysteroidogenic hormones”.     

Chromosomal differentiation in two species of Aedes and their hybrids revealed by giemsa C-banding

Giemsa C-banding patterns in two species of mosquitoes, Aedes aegypti and Aedes mascarensis, their hybrids and backcross progeny revealed differences in the sex chromosome pair. In A. aegypti, the female determining or the m chromosome in both males and females shows a conspicuous band in the centromere region and another band in one arm. The male determining or the M chromosome is devoid of any bands. Progeny of crosses involving A. aegypti females and A. mascarensis males showed interesting albeit unexpected results. The intercalary band was suppressed in both sons and daughters. When such F₁ sons were backcrossed to A. aegypti females, a proportion of males developed into intersexes. These intersex progeny differed from the normal males in terms of their banding pattern. In the reciprocal cross (A. mascarensis female × A. aegypti male), the F₁ and the backcross progeny yielded the expected C-banding patterns. The implications of the reversible expression of the intercalary band on the A. aegypti m chromosome and its relevance to genetic regulation are discussed.     

Occurrence of heat-dissociable ribosomal RNA in insects: the presence of three polynucleotide chains in 26 S RNA from cultured Aedes aegypti cells

When RNA extracted from a mixture of cultured mosquito (Aedes aegypti) and hamster (BHK) cells is heated at 60 °C for five minutes the 26 S mosquito RNA but not the 28 S BHK RNA is converted to 18 S products. These products are not separable from each other or from pre-existent 18 S RNA on 2.4% acrylamide gels and have molecular weights near 0.7 × 10⁶. The large ribosomal RNA from insects belonging to ten different orders shows a similar conversion, although this property is absent in two species of aphid.A. aegypti 26 S RNA dissociates over a narrow temperature range. The reaction equilibrium favours dissociation and is dependent on ionic strength, showing a 6 deg. C change in T_m′ (the temperature of 50% dissociation) with tenfold change in salt concentration. Although the T_m of 26 S RNA from Drosophila melanogaster and A. aegypti is markedly different, reflecting the difference in base composition, the T_m′ of the two RNA species was virtually the same.High molecular weight ribosomal RNA from Escherichia coli, BHK cells and A. aegypti cells was terminally labelled with [³H]isonicotinic acid hydrazide. The specific activities of the large RNA species show the presence of one, two and three polynucleotide chains in 23 S, 28 S and 26 S RNA, respectively. A. aegypti 26 S RNA contains a small, heat-dissociable “IRNA” similar in relative amount and mobility to that found in BHK cells.     

Incrimination of the mosquito,Aedes taeniorhynchus,as the primary vector of heartworm,Dirofilaria immitis,in coastal Yucatan,Mexico

Mosquito collections were carried out on microfilaraemic dogs, positive for Dirofilaria sp., for 18 consecutive nights in the coastal town of Celestún, Yucatan, southeast Mexico, during the rainy season (August) of 2007. A total of 292 female mosquitoes representing 12 species of dipteran Culicidae were collected: Anopheles albimanus (Wiedemann); Anopheles crucians (Wiedemann); Anopheles pseudopunctipennis (Theobald); Culex coronator (Dyar & Knab); Culex interrogator (Dyar & Knab); Culex nigripalpus (Theobald); Culex quinquefasciatus (Say); Culex salinarius (Coquillett); Aedes aegypti (Linnaeus); Aedes scapularis (Rondani); Aedes sollicitans (Walker), and Aedes taeniorhynchus (Wiedemann). Aedes taeniorhynchus and Cx. quinquefasciatus were the species found most commonly feeding on the dogs. Filarial nematodes were observed by microscopy in nine of the mosquito species collected; however, third‐instar larvae were only observed in Ae. taeniorhynchus and An. crucians. Of 76 Ae. taeniorhynchus specimens found positive for Dirofilaria sp. by dissection, 14 were confirmed to be positive for Dirofilaria immitis (Leidy) by polymerase chain reaction (PCR). The resulting infection rate for D. immitis confirmed by PCR (6.2%) is higher than any infection rate for Ae. taeniorhynchus previously reported from the Americas.     

Larvicidal activity of Bacillus thuringiensis subsp. israelensis, serovar H14 in Aedes aegypti: Histopathological studies

Bacillus thuringiensis subsp. israelensis, serovar H14, when applied as a primary commercial powder, caused the rapid death of Aedes aegypti larvae. Mortality started 6 min after application of 4 μg/ml of the pathogen and reached a maximum 27 min later. When the LC₅₀ (10 ng/ml) was applied, mortality began after 37 min and reached a maximum 120 min later. Histopathological changes in B. thuringiensis israelensis-treated larvae could be observed only in the midgut and caeca. In B. thuringiensis israelensis-treated “dead larvae”, the epithelial layer is disorganized, most of the cells have disappeared and the peritrophic membrane is broken. The epithelium in the B. thuringiensis israelensis-treated “living larvae” still maintains its monolayer structure, but with marked cellular hypertrophy and vacuolized cytoplasm. Also, the “brush border” is thinner and disrupted. Based on the fact that mortality of A. aegypti is a quick process, and because the histopathological changes caused by B. thuringiensis israelensis are similar to those found in lepidopterous larvae treated with pure δ-endotoxin of other B. thuringiensis variants, it is suggested that larvicidal activity of B. thuringiensis israelensis in A. aegypti is due to its δ-endotoxin.     

Toxicity of Bacillus thuringiensis toward Aedes aegypti larvae.

Among six strains of Bacillus thuringiensis and five other species of Bacillus, only two strains of B. thuringiensis, strains HD-1 and BA-068, were toxic to Aedes aegypti larvae within 24 hr. The LC₅₀s were 5.6 × 10⁴ and 2.4 × 10⁵ spores/ml for strains HD-1 and BA-068, respectively. The toxic factor(s) was heat sensitive and γ ray resistant and preliminary evidences indicated that it was associated with the crystalline body of B. thuringiensis.     

Selective inhibitors of digestive enzymes from Aedes aegypti larvae identified by phage display

Dengue is a serious disease transmitted by the mosquito Aedes aegypti during blood meal feeding. It is estimated that the dengue virus is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies have been based on controlling the vector, Ae. aegypti, using insecticide, but the emergence of resistance poses new challenges. The aim of this study was the identification of specific protease inhibitors of the digestive enzymes from Ae. aegypti larvae, which may serve as a prospective alternative biocontrol method. High affinity protein inhibitors were selected by all of the digestive serine proteases of the 4th instar larval midgut, and the specificity of these inhibitors was characterized. These inhibitors were obtained from a phage library displaying variants of HiTI, a trypsin inhibitor from Haematobia irritans, that are mutated in the reactive loop (P1–P4′). Based on the selected amino acid sequence pattern, seven HiTI inhibitor variants were cloned, expressed and purified. The results indicate that the HiTI variants named T6 (RGGAV) and T128 (WNEGL) were selected by larval trypsin-like (IC₅₀ of 1.1 nM) and chymotrypsin-like enzymes (IC₅₀ of 11.6 nM), respectively. The variants T23 (LLGGL) and T149 (GGVWR) inhibited both larval chymotrypsin-like (IC₅₀ of 4.2 nM and 29.0 nM, respectively) and elastase-like enzymes (IC₅₀ of 1.2 nM for both). Specific inhibitors were successfully obtained for the digestive enzymes of Ae. aegypti larvae by phage display. Our data also strongly suggest the presence of elastase-like enzymes in Ae. aegypti larvae. The HiTI variants T6 and T23 are good candidates for the development as a larvicide to control the vector.     

Brugia pahangi: Development in Aedes aegypti reared axenically on a defined synthetic diet

Adult Aedes aegypti axenically reared on a chemically defined dietary medium had low infectivity to Brugia pahangi, and harbored few third-stage parasitic juveniles. Some of these parasites appeared not to have completed the second molt at the end of 10 days of incubation. Mortality was high in mosquitoes from axenic larval rearings, especially during filarial infection. It appeared probable that adverse effects on the worm resulted from suboptimal host nutrition. Thus, manipulations of the chemically defined dietary medium used to rear A. aegypti may be used to study the influence of specific nutrients on filarial development.     

Post-Integration Silencing of piggyBac Transposable Elements in Aedes aegypti

The piggyBac transposon, originating in the genome of the Lepidoptera Trichoplusia ni, has a broad host range, making it useful for the development of a number of transposon-based functional genomic technologies including gene vectors, enhancer-, gene- and protein-traps. While capable of being used as a vector for the creation of transgenic insects and insect cell lines, piggyBac has very limited mobility once integrated into the genome of the yellow fever mosquito, Aedes aegypti. A transgenic Aedes aegypti cell line (AagPB8) was created containing three integrated piggyBac elements and the remobilization potential of the elements was tested. The integrated piggyBac elements in AagPB8 were transpositionally silent in the presence of functional transposase, which was shown to be capable of catalyzing the movement of plasmid-borne piggyBac elements in the same cells. The structural integrity of one of the integrated elements along with the quality of element-flanking DNA, which is known to influence transposition rates, were tested in D. melanogaster. The element was found to be structurally intact, capable of transposition and excision in the soma and germ-line of Drosophila melanogaster, and in a DNA sequence context highly conducive to element movement in Drosophila melanogaster. These data show that transpositional silencing of integrated piggyBac elements in the genome of Aedes aegypti appears to be a function of higher scale genome organization or perhaps epigenetic factors, and not due to structural defects or suboptimal integration sites.