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     

Midgut and salivary gland barriers to La Crosse virus dissemination in mosquitoes of the Aedes triseriatus group

Vector competence for La Crosse virus (LACV) was compared for four species in the Aedes (Protomacleaya) triseriatus group: Ae. triseriatus (Say), Ae. hendersoni Cockerell, Ae. zoosophus Dyar and Knab and Ae. brelandi Zavortink (Diptera: Culicidae). Rates of replication and dissemination of virus in the mosquito hosts were compared and rates of oral transmission of virus to suckling mice were determined. Barriers to virus dissemination which limited the ability of each species to transmit virus were identified. Ae. zoosophus displayed the highest vector competence for LACV. Both infection and transmission rates were high: 99% and 85% respectively; no significant barriers to LACV were found. Disseminated infection of Ae. triseriatus with LACV was controlled primarily be a midgut escape barrier. When virus was introduced directly into the haemocoel, transmission rates were significantly increased (37% v. 79%). Ae. hendersoni showed high susceptibility to LACV infection but a very low rate of oral transmission (7%). Ae. brelandi was also highly susceptible to infection by LACV and transmitted virus at an intermediate rate (27%). Modulation of vector competence in both Ae. hendersoni and Ae. brelandi resulted from a salivary gland escape barrier. As these four species of mosquitoes comprise a closely related monophyletic series, their differences of vector competence for LACV provide an excellent model for studying the genetic basis of the barriers involved.     

A comparison of Aedes vigilax larval population densities and associated vegetation categories in a coastal wetland,Northern Territory,Australia

Darwin's northern suburbs border an extensive coastal reed and upper mangrove wetland recognized as an important larval habitat for Aedes vigilax (Skuse), the northern salt marsh mosquito, an established vector for Ross River and Barmah Forest viruses and an appreciable pest species. We sought to identify the most important vegetation categories associated with Ae. vigilax breeding to maximize the efficiency of mosquito control efforts. Using a generalized linear model with negative binominal distribution and log link, this study compares larval densities, determined by focused dipping, between 13 discernable vegetation categories. The incidence rate ratios (RR) generated can be used to compare the magnitude of larval densities for each vegetation category, compared with the reference category. Aedes vigilax larval densities were almost ten times greater in artificial drainage areas (RR=9.82), followed by tide‐affected reticulate (Sporobolus/Xerochloa) areas (RR=8.15), then Schoenoplectus/mangroves (RR=2.29), compared with the reference vegetation category “lower mangroves.” Furthermore, larval densities were highest in May, due to tidal inundation, for drainage areas and tide‐affected reticulates (RR=12.2, 11.7, respectively) compared with March, the reference month. Thus, to maximize the efficiency of aerial salt marsh mosquito control operations in this wetland, larval control is best accomplished by concentrating on drains, Schoenoplectus/mangroves, and tide‐affected reticulate areas, commencing early after the wet season. These results should apply to other areas of salt marsh mosquito breeding across northern Australia.     

Anti-mosquito antibodies reduce the susceptibility of Aedes aegypti to arbovirus infection

Aedes aegypti (L.) mosquitoes showed a significant reduction in susceptibility to infection with Ross River virus and Murray Valley encephalitis virus when they were fed on a blood-virus mixture containing rabbit antibodies to mosquito midgut components. Presence of the antibodies did not demonstrably affect virus titres in infected mosquitoes, nor the transmission of virus from infected mosquitoes to vertebrates.     

Vector competence of Coquillettidia linealis (Skuse) (Diptera: Culicidae) for Ross River and Barmah Forest viruses

Abstract Coquillettidia linealis is a severe pest on some of the Moreton Bay islands in Queensland, Australia, but little is known of its breeding habitats and biology. Because of its high abundance and its association with Ross River (RR) and Barmah Forest (BF) viruses by field isolation, its vector competence was evaluated in the laboratory by feeding dilutions of both viruses in blood. For RR, Cq. linealis was of comparable efficiency to Ochlerotatus vigilax (Skuse), recognised as being a major vector. Results were as follows for Cq. linealis and Oc. vigilax , respectively: dose to infect 50%, 10^2.2 and <10^1.7 CCID₅₀/mosquito; 88% and 90% disseminated infection at 4 days postinfection; transmission at 4 days with rates of 68−92% and 25−60%. For BF dose to infect 50%, 10^2.7 and 10^2.0; disseminated infection rates on first transmission day (day 6), 40% and 70%; transmission rates of 8−16% and 0−10%. As a capillary-tube method was used rather than suckling mice to demonstrate transmission, transmission rates may be underestimates. This, the first study of the vector competence of Cq. linealis in Australia, demonstrates that this species deserves control on the southern Moreton Bay islands.     

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.     

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.     

Surveillance of the mosquito Aedes aegypti and its biocontrol with the copepod Mesocyclops aspericornis in Australian wells and gold mines

Abstract. A survey of the dengue vector mosquito Aedes aegypti was undertaken using runnel traps to detect immature stages (larvae and pupae) in flooded disused mine shafts and wells in Charters Towers, Queensland, Northern Australia. The town has a history of dengue fever since 1885 when goldminers were the first recorded victims. During the latest dengue epidemic in 1993, 2% of the population had laboratory-confirmed dengue virus Type 2, despite source reduction of Ae.aegypti breeding-sites at ground level or above. This led to suspicions that dengue vector Ae.aegypti breeding-sites might be below ground level. When surveyed in March 1994, Ae.aegypti immatures were found in 9/10 wells and 1/6 mine shafts. The water in wells and mines had similar characteristics -except that turbidity was higher in the mines, which more often contained predators of mosquito immatures.
The copepod Mesocyclops aspericornis was collected from water in 1/10 wells and 2/6 mine shafts. Laboratory predation trials resulted in 95.5–100% predation by 25 copepods/1 on Ae.aegypti first-instar larvae up to 200 larvae/1. Five wells containing Ae.aegypti in the survey were inoculated with fifty indigenous M.aspericornis , and five wells (one positive and four negative in the survey) were left untreated as controls. Nine months later, in December 1994, Ae.aegypti had been eliminated from all five treated wells but all untreated control wells contained Ae.aegypti , except for one well that contained a natural population of M.aspericornis. The role of wells and mines as winter/ dry season refuges of Ae.aegypti in northern Australia is reviewed, and we recommend the use of M.aspericornis as a cost-effective, environmentally acceptable and persistent agent for the sustainable control of Ae.aegypti , especially in inaccessible breeding sites.     

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.     

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.     

Cross-induction of detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes aegypti: impact on larval tolerance to chemical insecticides

The effect of exposure of Aedes aegypti larvae to sub-lethal doses of the pyrethroid insecticide permethrin, the organophosphate temephos, the herbicide atrazine, the polycyclic aromatic hydrocarbon fluoranthene and the heavy metal copper on their subsequent tolerance to insecticides, detoxification enzyme activities and expression of detoxification genes was investigated. Bioassays revealed a moderate increase in larval tolerance to permethrin following exposure to fluoranthene and copper while larval tolerance to temephos increased moderately after exposure to atrazine, copper and permethrin. Cytochrome P450 monooxygenases activities were induced in larvae exposed to permethrin, fluoranthene and copper while glutathione S-transferase activities were induced after exposure to fluoranthene and repressed after exposure to copper. Microarray screening of the expression patterns of all detoxification genes following exposure to each xenobiotic with the Aedes Detox Chip identified multiple genes induced by xenobiotics and insecticides. Further expression studies using real-time quantitative PCR confirmed the induction of multiple CYP genes and one carboxylesterase gene by insecticides and xenobiotics. Overall, this study reveals the potential of xenobiotics found in polluted mosquito breeding sites to affect their tolerance to insecticides, possibly through the cross-induction of particular detoxification genes. Molecular mechanisms involved and impact on mosquito control strategies are discussed.     

Feeding behaviour of female Aedes aegypti: effects of diet temperature, bicarbonate and feeding technique on the response to ATP

Abstract. The effects of variation in diet temperature, mouthpart deployment and the addition of bicarbonate to a saline-ATP diet were investigated for their effects on amount ingested and diet destination in female Aedes aegypti (L.). Mouthpart deployment was achieved by having the insects feed through a membrane, or from a free-liquid surface with mouthparts intact, or with the fascicle separated from the labial groove.
Under the 'feeding through membrane' protocol, a 6-fold increase in the percentage of Aedes feeding on ATP-diets was recorded at 37C compared with 21C. Bicarbonate (9.5 or 19 mM) induced a 3-fold increase in numbers feeding at both 21C and 37C. Neither diet temperature nor bicarbonate content appeared to affect meal size. The pH of all diets was adjusted to 7.0. Diet was directed primarily to the midgut for all diets tested under this regimen, with the exception of the saline-ATP-19 mM NaHC0₃ at 37C which caused the diet to be directed primarily to the crop.
Under the 'feeding without membrane through feeding tube' protocol, females showed little control over diet destination. Female Aedes aegypti exhibited no strong response to ATP when the mouthparts were immersed in 21C diets in feeding tubes and few of these insects ingested large meals. The addition of bicarbonate to the ATP diets did not enhance feeding under these test conditions.     

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.     

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 effects of allatectomy and juvenile hormone replacement on the development of host-seeking behaviour and lactic acid receptor sensitivity in the mosquito Aedes aegypti

Host-seeking behaviour in newly emerged Aedes aegypti (L.) females is not expressed immediately after adult eclosion but develops gradually over a period of approximately 3-4 days. This development is accompanied by an apparent maturation of the antennal chemosensory afferent neurons involved in the detection of the airborne host attractant lactic acid. Since these events coincide in time with juvenile hormone-dependent previtellogenic ovarian growth and since the expression of other reproduction-associated behaviour has previously been shown to be dependent on juvenile hormone, the effects of juvenile hormone removal and replacement on the development of host-seeking behaviour and the response characteristics of the lactic acid-sensitive receptors were investigated. No effect of juvenile hormone removal by allatectomy or juvenile hormone replacement or augmentation by topical application of the juvenile hormone mimic methoprene was found. It was concluded that this hormone is not involved in the appearance of host-seeking behaviour or the apparent maturation of the lactic acid receptors that occurs during early imaginal life.     

Inhibitory and enhancing effects of insertion of central polypurine tract sequence on gene expression with vectors derived from human immunodeficiency virus type 1

Reportedly, in human immunodeficiency virus type 1 (HIV) vectors, insertion of central polypurine tract (cPPT) increased expression of transgenes for a short period. To test this for a stable condition, we constructed a series of vectors carrying a Neo(r) gene as a stable marker driven by a synthetic thymidine kinase (hTK) promoter. Transduction efficiency was increased in about 2-fold and decreased in about 8-fold by insertion of the reported 178bp and our 282bp cPPTs, respectively. PCR analyses revealed that insertion of 282bp cPPT, but not 178bp cPPT, impaired integration, although it did not deteriorate nuclear transport much. Furthermore, we found that insertion of 282bp cPPT between hTK promoter and an upstream LTR sequence reduced reporter gene activity in about 5-fold. This inhibitory effect of 282bp cPPT may partly account for the observed decrease in transduction efficiency. We suggest that actual effect of cPPT insertion should be examined in each HIV vector.     

Wound-induced changes in tomato leaves and their effects on the feeding patterns of larval lepidoptera

Several studies have shown changes in the patterns of damage from feeding insects associated with changes in palatability and overall consumption as a result of wound-induced chemical changes in plants. This paper describes how the pattern of feeding damage made by the larvae of Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) on tomato is affected by changes in palatability of the leaves. Two sorts of responses to leaves from plants that had received prior damage were observed. Larvae offered a choice of leaves tended to take fewer meals on leaves from previously-wounded plants than on control leaves, frequently rejecting the former after sampling them. On wounded plants this rejection behaviour was associated with a shift in feeding site towards the base of the plant. However, starved larvae offered only a single excised leaf readily ate leaves from wounded plants but took shorter meals on these leaves than on controls. Although it was not directly tested it is possible that this difference in response reflected changes in food selectivity with a differing level of satiation. The results are considered in relation to the adaptive significance of the plant of changes in within-plant distributions of herbivore damage.