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

A bioassay of mosquito iridescent virus (MIV) of Aedes taeniorhynchus was developed using cell cultures of Aedes aegypti. The dilution end point technique was based on the occurrence of cytopathic effects which were optimum at 31°C. Peleg's A. aegypti cell line was more sensitive and reliable than Singh's A. aegypti cell line for infectivity titration of the “R” and “T” strains of MIV. The highest tissue culture infectivity dose 50s (TCID₅₀) were elicited by virion:cell ratios of approximately 10. TCID₅₀ titers were significantly reduced by virus neutralization with either homologous or heterologous antiserum to either RMIV or TMIV. The virus propagated in either cell line was not infectious to A. taeniorhynchus larvae, or to the respective cells from which the virus was produced. All plaque assay attempts were unsuccessful.     

Pathology of a mosquito iridescent virus (MIV) infecting Aedes taeniorhynchus

An investigation was initiated to study the pathology and biology of the regular mosquito iridescent virus (RMIV) in the black salt marsh mosquito, Aedes taeniorhynchus. RMIV was capable of infecting a variety of tissues within its host. Cells of the fat body, tracheal epithelium, imaginal discs, and epidermis were the primary sites of viral replication. Extensive destruction of the fat body by this virus resulted in the death of most infected mosquitoes before they reached the adult stage. Other tissues which were involved to a lesser extent were hemocytes, esophagus, nerve, muscle, and both larval and adult ovaries.     

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.     

A new microsporidian parasite of the mosquito Aedes taeniorhynchus

A new species of microsporidium is described from the mosquito Aedes taeniorhynchus. This microsporidium is vertically transmitted and exhibits dimorphic development with one sequence leading to formation of short pyriform, uninucleate spores in male fourth instar larvae, pupae, and adults, and the other sequence leading to formation of diplocaryotic stages and cylindrical spores in adult females. Vertical transmission is apparently limited to a single generation, and the uninucleate spores are not transmissible per os to larvae.     

Osmoregulation in larvae of the salt-marsh mosquito, Aedes taeniorhynchus

Larvae of Aedes taeniorhynchus (Wiedemann) were reared in media with salinities from that of distilled water up to and including 300% of that of sea water to investigate certain aspects of their potential physiological range. Regulation of hemolymph osmotic pressure and chloride ions was also studied.Larvae showed normative growth rate in all concentrations from distilled water to 150% sea water (SW), but in salinities between 150% to 300% growth was retarded. Hemolymph osmotic pressure and hemolymph chloride were both hyper-and hypoosmotically regulated. Anal papillae size was inversely related with increased concentration of the sea water medium, e.g., from 443×142 in distilled water to 116×62 in 100% SW. The average hemolymph osmotic pressure was higher in fed larvae than in starved larvae. Hemolymph osmotic pressure increased for 7 hr before equilibrating with the medium when larvae reared to the 4th instar in 10% SW were transferred to 100% SW, whereas larvae reared in 100% SW and transferred to 10% SW showed a decrease in hemolymph osmotic pressure before equilibrating. Regulation of hemolymph chloride was found to be a function of the anal papillae, as chloride levels dropped significantly in larvae with chemically cauterized anal papillae when they were maintained in lower concentrations. It is suggested that the limitations of A. taeniorhynchus larvae primarily to salt-marshes are not due to an inability to survive and grow successfully in fresh water, but due to other ecological interactions.

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Zusammenfassung Larven von Aedes taeniorhynchus wurden in Medien mit einer Salinität von a.ddest. bis zu der von 300% Meerwasser (MW) gehalten, um die folgenden Aspekte einer möglichen physiologischen Wirkung zu untersuchen: a) Überleben und Wachstum der Larven, b) Osmotischer Druck der Hämolymphe (HL), sowie Grösse der Analpapillen in Abhängigkeit vom Zuchtmedium, c) Wirkung von Fütterung und Hungern auf den osmotischen Druck der HL, d) Wirkung der Übertragung von niedrigerer zu höherer Salinität und umgekehrt auf den osmotischen Druck und die Analpapillengrösse und e) Regulation des Chloridions in der HL.Osmotischer Druck der HL wurde bestimmt mit Hilfe des Mikrocryoskops, die Chloridio-nenkonzentration der HL durch Ultramikro-Volhard-Titration.Die Larven zeigten normales Wachstum und normale Überlebensrate bei allen Konzentrationen von a. dest. bis 150% MW, zwischen 150% und 300% MW war das Wachstum verzögert. Osmotischer Druck der HL und Chlorid der HL waren hyperosmotisch reguliert bis 10% MW und hypotonisch zwischen 25% und 300% MW. Die Grösse der Analpapillen nahm mit zunehmender Konzentration des MW-Mediums ab, z. B. von 443×142 m in a. dest. auf 116×62 m in 100% MW. Der durchschnittliche osmotische Druck der HL war bei gefütterten Larven höher als bei gehungerten. Wenn Larven, die bis zum 4. Stadium in 10% MW gehalten wurden, in 100% MW übertragen wurden, stieg der osmotische Druck der HL weit über die für 100% MW festgestellte Gleichgewichtslage hinaus an und näherte sich dieser (also durch Abnahme) erst nach 7 Stunden; bei Übertragung von 100% auf 10% MW erfolgte entsprechend zunächst eine übernormale Abnahme des osmotischen Drucks der HL vor Erreichen des Gleichgewichts. Die Regulation der HL-Chloride erwies sich als eine Funktion der Analpapillen, da der Chloridspiegel signifikant abfiel bei Larven, die mit chemisch kautorisierten Analpapillen in niedrigen Konzentrationen gehalten wurden. Es ist anzunehmen, dass die Beschränkung des Vorkommens von Aedes taeniorhynchus-Larven auf Aussenmarschen nicht verursacht wird durch die Unfähigkeit, in Süsswasser zu überleben und sich zu entwickeln, sondern durch andere ökologische Einflüsse.

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Contribution no. 315, Florida State Division of Health, Florida Medical Entomology Laboratory, P.O. Box 520, Vero Beach, Florida 32960.     

Egg chorion tanning in Aedes aegypti mosquito

The biochemical pathway of egg chorion tanning in the mosquito, Aedes aegypti, is described and compared with chorion protein crosslinking in Drosophila and silkmoths and the biochemical pathways of cuticular tanning in insects. Phenol oxidase, dopa decarboxylase and tyrosine are critical components involved in egg chorion tanning in A. aegypti. Tanning of the mosquito egg chorion is initiated following activation of phenol oxidase, which then catalyzes the hydroxylation of tyrosine to dopa and further oxidizes dopa and dopamine to their respective o-quinones. Because intramolecular cyclization is much slower in dopaminequinone than dopaquinone, the chance to react with external nucleophiles to participate in protein crosslinking reactions also is much greater in dopaminequinone than dopaquinone. This might partly explain the necessity for the involvement of dopa decarboxylase in mosquito chorion tanning. Intramolecular cyclization of dopaquinone and dopaminequinone to form dopachrome and dopaminechrome, respectively, the structural rearrangement of these aminochromes to produce 5,6-dihydroxyindole, and the subsequent oxidation of 5,6-dihydroxyindole by phenol oxidase also lead to melanin formation during egg chorion tanning.     

FLP-mediated recombination in the vector mosquito, Aedes aegypti.

The activity of a yeast recombinase, FLP, on specific target DNA sequences, FRT, has been demonstrated in embryos of the vector mosquito, Aedes aegypti. In a series of experiments, plasmids containing the FLP recombinase under control of a heterologous heat-shock gene promoter were co-injected with target plasmids containing FRT sites into preblastoderm stage mosquito embryos. FLP-mediated recombination was detected between (i) tandem repeats of FRT sites leading to the excision of specific DNA sequences and (ii) FRT sites located on separate plasmids resulting in the formation of heterodimeric or higher order multimeric plasmids. In addition to FRT sites originally isolated from the yeast 2 microns plasmid, a number of synthetic FRT sites were also used. The synthetic sites were fully functional as target sites for recombination and gave results similar to those derived from the yeast 2 microns plasmid. This successful demonstration of yeast FLP recombinase activity in the mosquito embryo suggests a possible future application of this system in establishing transformed lines of mosquitoes for use in vector control strategies and basic studies.     

Genome of invertebrate iridescent virus type 3 (mosquito iridescent virus)

Iridoviruses (IVs) are classified into five genera: Iridovirus and Chloriridovirus, whose members infect invertebrates, and Ranavirus, Lymphocystivirus, and Megalocytivirus, whose members infect vertebrates. Until now, Chloriridovirus was the only IV genus for which a representative and complete genomic sequence was not available. Here, we report the genome sequence and comparative analysis of a field isolate of Invertebrate iridescent virus type 3 (IIV-3), also known as mosquito iridescent virus, currently the sole member of the genus Chloriridovirus. Approximately 20% of the 190-kbp IIV-3 genome was repetitive DNA, with DNA repeats localized in 15 apparently noncoding regions. Of the 126 predicted IIV-3 genes, 27 had homologues in all currently sequenced IVs, suggesting a genetic core for the family Iridoviridae. Fifty-two IIV-3 genes, including those encoding DNA topoisomerase II, NAD-dependent DNA ligase, SF1 helicase, IAP, and BRO protein, are present in IIV-6 (Chilo iridescent virus, prototype species of the genus Iridovirus) but not in vertebrate IVs, likely reflecting distinct evolutionary histories for vertebrate and invertebrate IVs and potentially indicative of genes that function in aspects of virus-invertebrate host interactions. Thirty-three IIV-3 genes lack homologues in other IVs. Most of these encode proteins of unknown function but also encode IIV3-053L, a protein with similarity to DNA-dependent RNA polymerase subunit 7; IIV3-044L, a putative serine/threonine protein kinase; and IIV3-080R, a protein with similarity to poxvirus MutT-like proteins. The absence of genes present in other IVs, including IIV-6; the lack of obvious colinearity with any sequenced IV; the low levels of amino acid identity of predicted proteins to IV homologues; and phylogenetic analyses of conserved proteins indicate that IIV-3 is distantly related to other IV genera.     

Relationship of hemolymph phenol oxidase and mosquito age in Aedes aegypti.

Monophenol oxidase (MPO) and diphenol oxidase (DPO) activity in hemocytes and cell-free plasma perfused from 7-, 14-, 21-, and 28-day-old Aedes aegypti mosquitoes were compared. A progressive decrease of enzyme activity was detected as mosquito age increased, and this decrease was significant in both hemocytes and cell-free plasma when mosquitoes were 28 days old as compared with that found in 7-day-old mosquitoes. There was no significant difference in total hemolymph protein as mosquito age increased. Although this decreased MPO and DPO activity might be partially responsible for the reduced immune response against filarial worms previously reported for older mosquitoes, other factors undoubtedly play a significant role.