Dissemination barriers to Ross River virus in Aedes vigilax and the effects of larval nutrition on their expression

Effects of larval nutrition on vector competence of the mosquito Aedes vigilax (Skuse) (Diptera: Culicidae) from Townsville, north Queensland, for Ross River virus (RR) were examined. Larvae were reared on three different diets to create three significantly different size classes of adult mosquito. These were fed on serial dilutions of RR and then sampled on alternate days so the progression of the virus through the mosquito could be examined. No differences of vector competence could be attributed to larval nutrition. Barriers to infection and the correlation between infection rate, viral titre and transmission of RR by Ae. vigilax were also examined. The mesenteronal barrier was the only infection barrier expressed. No correlation between viral titre and transmission was detected, but a strong correlation was found between salivary gland infection and transmission rate. From this it was possible to estimate that 98.7+/-1.3% of Ae. vigilax with infected salivary glands transmit RR.     

Competition and resistance to starvation in larvae of container-inhabiting Aedes mosquitoes

Abstract. 1. Hypotheses about declining populations of container-inhabiting Aedes mosquitoes following the invasion by additional species were tested.
2. The larval competition hypothesis was studied experimentally in pure and mixed cultures of Aedes aegypti (L.), A.albopictus (Skuse) and A.triseriatus (Say). The experiments used decomposing leaf litter in the laboratory, as opposed to most previous research which used non-natural food.
3. Resistance to starvation is introduced as a new measure of larval performance and competitiveness. The hypothesis is that more successful larvae store larger energy reserves and resist the lack of food longer.
4. Contrary to previous research showing better performance of A.aegypti in mixed cultures, A.albopictus developed faster and had greater survival when natural food was used.
5. Resistance to starvation was greater in the better performing species (i.e. A.aegypti with non-natural food and A.albopictus with leaf litter). Oxygen consumption by starved larvae was similar in the three container species, and in the ground-water mosquito, A.taeniorhynchus (Wied.), whose resistance to starvation was comparatively very low.     

Horizontal transfer of the insect growth regulator pyriproxyfen to larval microcosms by gravid Aedes albopictus and Ochlerotatus triseriatus mosquitoes in the laboratory

The insect growth regulator (IGR) pyriproxyfen is highly active against mosquitoes (Diptera: Culicidae). Through continuous emersion of large larvae (instars 3-4) the concentration causing 50% inhibition of adult emergence (EI50) was determined as 0.200 p.p.b. for Aedes albopictus (Skuse) and 3.5 to 7 times less for Ochlerotatus triseriatus (Say): IE50 0.0288 p.p.b. As a possible method of application to larval microcosms of these species that oviposit in water containers and phytotelmata, the horizontal transfer of pyriproxyfen to larval microcosms by adult mosquitoes was evaluated under laboratory conditions. Gravid females were forced to walk on surfaces treated with pyriproxyfen (tarsal contact exposure) and then allowed to oviposit in larval microcosms. Using replicate bioassay cages, each with an oviposition container, and a factorial experimental design, we assessed Ae. albopictus for the effects of (i) pyriproxyfen concentration (0.2, 0.3 and 0.4 mg/cm2) contacted by gravid females, and (ii) the number of treated gravid females added to bioassay cages (one, three or five females/cage), on the mortality of larvae in oviposition containers. Only 0.2 mg/cm2 treatment rate was tested on Oc. triseriatus. A significant (P < 0.05) curvilinear response in inhibition of emergence (IE) was achieved on both species. Densities of one or three treated Oc. triseriatus females/cage yielded IE rates of only 21-27%, whereas five treated females/cage resulted in 70% inhibition. With Ae. albopictus, densities of three or five treated females/cage yielded 48-67% and 59-73% IE, respectively, whereas one treated female/cage gave only 4-30% inhibition. Use of IGR-treated oviposition containers to achieve horizontal transfer of pyriproxyfen to mosquito oviposition sites could be a field management technique based on mosquito biology and behaviour. In binary choice tests with Ae. albopictus, horizontal transfer of pyriproxyfen from a container with a treated ovistrip (0.3 or 0.4 mg/cm2) to an untreated microcosm resulted in 14-38% inhibition. In larval bioassays, pyriproxyfen activity declined markedly within 10 days. Forcibly exposing gravid female mosquitoes to pyriproxyfen-treated paper surface did not affect their fecundity. However, from the 1st to 2nd gonotrophic cycles the egg hatch rate declined by 30% (P < 0.05). Some variation of results could be due to interactions between females at the oviposition site, possibly causing disproportionate transfer of pyriproxyfen to larval microcosms. Comparative studies of the oviposition behaviour of each mosquito are warranted and would potentially provide information needed to improve the technique.     

The vector competence of Culex annulirostris, Aedes sagax and Aedes alboannulatus for Murray Valley encephalitis virus at different temperatures

Culex annulirostris Skuse, colonized from Brisbane, Queensland, and Mildura, Victoria, Australia, were effective vectors of Murray Valley encephalitis virus at 20, 27 and 32-35 degrees C with full extrinsic incubation periods of 15, 10 and 4 days respectively. At 20 degrees C, 7-11 days post-infection, transmission by the Mildura colony (0-20%) was less efficient than the Brisbane colony (30-70%) but both were capable of 75-100% transmission after longer extrinsic incubation periods. Discriminant analysis of body and salivary gland titres showed that these were not satisfactory indicators of transmission. Wild-caught Aedes sagax (Skuse) and Cx annulirostris from the Murray Valley showed equal competence, but Aedes alboannulatus (Macquart) was a poor vector. The results provide data on rural amplification of Murray Valley encephalitis virus during spring and suggest that further work on the potential of Ae. sagax as a natural vector is warranted.     

Oral susceptibility of Aedes albopictus to dengue type 2 virus: a study of infection kinetics, using the polymerase chain reaction for viral detection

Female Aedes albopictus mosquitoes, aged 1 week, were infected with DEN-2 dengue virus. The kinetics of infection in mosquito brain and mesenteron were monitored using DNA probes with polymerase chain reaction (PCR) amplification of target DNA sequences coding for DEN-2 virus envelope protein, compared with the standard immunofluorescence assay technique (IFA). Rates of virus detection in the mesenteron of orally infected mosquitoes rose to 38% by day 4 post-inoculation, then declined until day 8, followed by irregular peaks around days 11-14 and subsequently. In mosquito head squashes, virus was detected from day 4 onwards, reaching 38% positive by day 18. Salivary glands of all the same females were found to be positive for virus by day 8 onwards. Parenterally infected Ae.albopictus females were all positive for DEN-2 in the brain and salivary glands 8 days post-inoculation. In every case, results obtained with the PCR matched those from the IFA. Our DNA probe with PCR procedure can therefore be utilized as a sensitive and reliable method for studies of DEN-2 vectors.     

Panveld oviposition sites of floodwater Aedes mosquitoes and attempts to detect transovarial transmission of Rift Valley fever virus in South Africa

Floodwater aedine mosquito eggs were recovered from soil samples taken from grassland depressions, called pans, in the Orange Free State Province of South Africa. A sedge, Mariscus congestus (Vahl) C.B.Cl., was a useful indicator of Aedes (Ochlerotatus) juppi McIntosh oviposition areas. No transovarial transmission of virus was demonstrated by Ae.juppi females reared from the eggs and allowed to feed shortly after eclosion on hamsters. No virus was recovered from 557 pools of 5425 adult Ae.juppi that were collected as eggs and reared to the adult stage in the laboratory. Rift Valley fever virus replicated to high titres in experimentally infected Ae.juppi females, but horizontal transmission experiments proved inconclusive.     

Induction of salivation in biting midges and mosquitoes, and demonstration of virus in the saliva of infected insects

Culicoides biting midges and Aedes aegypti (Linnaeus) mosquitoes were induced to salivate by the topical application of pilocarpine, neostigmine, malathion and dimethoate; of these, malathion was the most effective. Drops of saliva produced by virus-infected midges and mosquitoes were shown to contain virus. The method could be used to demonstrate transmission in insects infected with a variety of pathogens.     

Blood-feeding in mosquitoes: probing time and salivary gland anti-haemostatic activities in representatives of three genera (Aedes, Anopheles, Culex)

Mosquitoes (Diptera: Culicidae) face their hosts' haemostatic mechanisms when attempting to feed on blood. Accordingly, they antagonize haemostasis by salivary agents that include anti-clotting, anti-platelet and vasodilatory compounds. Because haemostasis is a complex and redundant physiological response that varies between vertebrates, it is to be expected that haematophagous animals have a salivary armoury that most efficiently counteracts their preferred hosts. The mosquito Culex quinquefasciatus Say, which has a strong tendency to ornithophagy, appears to have only recently adapted to mammals and may not have evolved efficient mechanisms to counteract mammalian platelet responses, while birds only have relatively inefficient thrombocytes. Accordingly, we compared the probing behaviour of Cx. quinquefasciatus with two other mosquito species from different backgrounds: Aedes aegypti (L.) and Anopheles albimanus Weidemann, that have apparently had a longer evolutionary association with mammals. Culex takes much more time to find blood on a mammalian host (human or mouse) when compared to the two other mosquito species, but does not differ in probing behaviour when feeding on a chicken. Salivary anti-haemostatic components were also measured in those three species of mosquito and results are discussed in context with the probing behaviour.     

Differential responses of Aedes and Culex mosquitoes to octenol or light in combination with carbon dioxide in Queensland, Australia

Abstract. Field studies were conducted with EVS (encephalitis vector surveillance) traps in south-eastern Queensland, Australia, to determine the relative response rates of mosquitoes to three levels (0.1, 4.5 and 30mg/h) of 1-octen-3-ol (octenol) in combination with a standard bait of 2200 g carbon dioxide (CO₂), compared with CO₂ alone or CO₂ with light from a 6V incandescent bulb. Compared to CO₂ alone, Aedes vigilax collections increased significantly when CO₂ was supplemented by all three octenol emission levels, but not by the addition of light. Furthermore, the 4.5 and 30 mg/h release rate of octenol gave a significant increase in numbers of Ae.vigilax relative to that from CO₂+ light. In contrast, collections of Culex annulirostris and Culex sitiens were not enhanced significantly by either the addition of light or octenol at all three levels. Fewer Cx sitiens were collected with octenol released at 4.5 mg/h in comparison to CO₂ alone. These differential sampling rates should be taken into account when using EVS traps.     

First record in America of Aedes albopictus naturally infected with dengue virus during the 1995 outbreak at Reynosa, Mexico

Abstract Mosquito collections were conducted during a dengue outbreak in Reynosa, Tamaulipas, Mexico, July-December 1995. A total of 6694 adult mosquitoes (four genera and nine species) were captured, of which 2986 (78.3% females and 21.7% males) were Aedes albopictus and 2339 (39.7% females and 60.3% males) were Ae.aegypti. These two species comprised 84.2% of the total collection. Specimens were grouped into pools, nearly 50% of them processed for detection of virus by cythopathic effect in C6-36 and VERO cell cultures and by haemagglutination test. Five pools gave positive haemagglutin-ation reactions and were examined by immunofluorescence using monoclonal antibodies to flavivirus and to dengue virus. One pool of ten Ae.albopictus males was positive for dengue virus: serotypes 2 and 3 were identified by serotype-specific monoclonal antibodies arid confirmed by RT-PCR. This is the first report of Ae.albopictus naturally infected with dengue virus in America. Also, it is the very first time Ae.albopictus males have been found infected with dengue virus in the wild.     

Population structure and dispersal of the saltmarsh mosquito Aedes vigilax in Queensland, Australia

Population genetics of the mosquito Aedes vigilax (Skuse) (Diptera: Culicidae), a major vector of arboviruses (e.g. Barmah Forest, Ross River), were investigated to obtain an indirect estimate of mosquito dispersal characteristics in typical habitats of Aedes vigilax in south-east Queensland: on the off-shore islands of Moreton Bay and on the mainland where disjunct breeding populations of Ae. vigilax are distributed along intertidal marsh. Six allozyme loci were assessed for genetic differentiation between samples from 11 localities. Significant larval variation between some breeding sites was attributed to site-specific selection. Nonsignificant genetic differentiation was found among collections of adult mosquitoes caught in light traps throughout the study area (exceeding 60x27 km), indicating widespread dispersal. As distances of < or = 9 km over water did not appear to act as effective barriers to Ae. vigilax dispersal, localized control activities applied to Ae. vigilax breeding sites are unlikely to be effective against the vagile adult population. Therefore, the contiguous shires programme of broad acre control is endorsed to prevent the spread of arboviruses carried by Ae. vigilax     

Vector competence of South African Culicoides species for bluetongue virus serotype 1 (BTV-1) with special reference to the effect of temperature on the rate of virus replication in C. imicola and C. bolitinos

Abstract. The oral susceptibility of 22 South African livestock associated Culicoides species to infection with bluetongue virus serotype 1 (BTV‐1) and its replication rate in C. imicola Kieffer and C. bolitinos Meiswinkel (Diptera: Ceratopogonidae) over a range of different incubation periods and temperatures are reported. Field‐collected Culicoides were fed on sheep blood containing 7.5 log₁₀TCID₅₀/mL of BTV‐1, and then held at constant different temperatures. Virus replication was measured over time by assaying individual flies in BHK‐21 cells using a microtitration procedure. Regardless of the incubation temperatures (10, 15, 18, 23.5 and 30°C) the mean virus titre/midge, infection rates (IR) and the proportion of infected females with transmission potential (TP = virus titre/midge ≥ 3 log₁₀ TCID₅₀) were found to be significantly higher in C. bolitinos than in C. imicola. Results from days 4–10 post‐infection (dpi), at 15–30°C, shows that the mean IR and TP values in C. bolitinos ranged from 36.7 to 87.8%, and from 8.4 to 87.7%, respectively; in C. imicola the respective values were 11.0–13.7% and 0–46.8%. In both species the highest IR was recorded at 25°C and the highest TP at 30°C. The time required for the development of TP in C. bolitinos ranged from 2 dpi at 25°C to 8 dpi at 15°C. In C. imicola it ranged from 4 dpi at 30°C to 10 dpi at 23.5°C; no individuals with TP were detected at 15°C. There was no evidence of virus replication in flies held at 10°C. When, at various points of incubation, individual flies were transferred from 10°C to 23.5°C and then assayed 4–10 days later, virus was recovered from both species. The mean virus titres/midge, and proportion of individuals with TP and IR, were again significantly higher in C. bolitinos than in C. imicola. Also the infection prevalence in C. magnus Colaço was higher than in C. imicola. Low infection prevalences were found in C. bedfordi Ingram & Macfie, C. leucostictus Kieffer, C. pycnostictus Ingram & Macfie, C. gulbenkiani Caeiro and C. milnei Austen. BTV‐1 was not detected in 14 other Culicoides species tested; however, some of these were tested in limited numbers. The present study indicates a multivector potential for BTV transmission in South Africa. In C. imicola and C. bolitinos the replication rates are distinct and are significantly influenced by temperature. These findings are discussed in relation to the epidemiology of bluetongue in South Africa.     

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.     

Characterization and description of a virus causing salivary gland hyperplasia in the housefly,Musca domestica

Abstract. A double-stranded DNA virus was isolated from hyperplasic salivary glands of male and female houseflies, Musca domestica L. (Diptera: Muscidae), collected from a dairy in Alachua County, Florida, U.S.A. Sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) of this housefly salivary gland hyperplasia (SGH) virus revealed the presence of two major and eight minor structural polypeptides. Restriction endonuclease analysis indicated that the c. 137 kilobase pair DNA was double-stranded. Weekly sweep-net sampling of the fly population throughout the season (May-October, 1991) showed that 1.5-18.5% of the dissected flies possessed hyperplasic salivary glands. The virus replicated within the nuclei of the salivary gland cells and was transmitted per os to newly-emerged healthy adult flies.     

La, PTB, and PAB proteins bind to the 3(') untranslated region of Norwalk virus genomic RNA

Noroviruses are human enteric caliciviruses for which no cell culture is available. Consequently, the mechanisms and factors involved in their replication have been difficult to study. In an attempt to analyze the cis- and trans-acting factors that could have a role in NV replication, the 3(')-untranslated region of the genome was studied. Use of Zuker's mfold-2 software predicted that NV 3(')UTR contains a stem-loop structure of 47 nts. Proteins from HeLa cell extracts, such as La and PTB, form stable complexes with this region. The addition of a poly(A) tail (24 nts) to the 3(')UTR permits the specific binding of the poly(A) binding protein (PABP) present in HeLa cell extracts, as well as the recombinant PABP. Since La, PTB, and PABP are important trans-acting factors required for viral translation and replication, these RNA-protein interactions may play a role in NV replication or translation.     

Sex ratio distortion and reduced lifespan of Glossina pallidipes infected with the virus causing salivary gland hyperplasia

A virus infection, associated with enlargement of the salivary glands (ESG) and gonadal pathology in Glossina pallidipes Austen (Diptera: Glossinidae), was studied in field-caught and laboratory-reared flies. The lifespan of both sexes of infected (ESG-) flies was significantly shorter than that of flies with normal salivary glands (NSG). NSG-females, mated to infected males only or to both infected and normal males, produced predominantly male progeny. In general, NSG-parents produced only NSG-progeny, and ESG-females only ESG-progeny. ESG-males were usually sterile, but a few NSG-females inseminated by ESG-males produced NSG-progeny. One NSG-female, mated first to an ESG-male and then to an NSG-male, produced 3 ESG-sons. As will be discussed, this virus may have important effects (reduced insemination rates, fecundity and lifespan, and sex ratio distortion) on laboratory colonies of G. pallidipes as well as on the regulation of its natural populations.

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Résumé C'est à partir de mouches capturées et élevées au laboratoire à Kibwezi au Kenya, qu'a été étudiée l'infection virale, accompagnée d'hyperplasie des glandes salivaires (ESG) et la pathologie des gonades de G. pallidipes Aust. La durée de vie des mouches contaminées (ESG)_des 2 sexes était significativement plus brève que celle des mouches aux glandes salivaires normales (NSG). Des femelles NSG accouplées uniquement à des mâles contaminés ou à des mâles sains et contaminés, ont donné une descendance majoritairement mâle.En général, des parents NSG ont donné uniquement des enfants NSG, et des femelles ESG des enfants ESG. Les mâles ESG étaient généralement stériles, mais quelques femelles NSG inséminées par des mâles ESG ont donné des enfants NSG. Une femelle NSG accouplée en premier avec un mâle ESG et ensuite à un mâle NSG, a donné 3 fils ESG. Bien que les transmissions orales et sexuelles puissent être naturellement des modes de contamination, la transmission verticale de la mère à la descendance semble être le mécanisme le plus significatif du maintien de ce virus. L'apparition d'enfants ESG de parents NSG est dans quelques cas vraisemblablement due à la présence du virus chez quelques mouches NSG sans qu'il y ait apparemment infection, sans symptômes visibles. Le virus ESG peut avoir des effets importants (diminution des taux d'insémination, de la fécondité, de la durée de vie, et modification de la fréquence des sexes) dans les souches de laboratoire et aussi sur la régulation des populations naturelles de ce vecteur des trypanosomiases africaines.