Anal fin morphology and gonadal histopathology in mosquitofish (Gambusia holbrooki) exposed to treated municipal sewage effluent.

Feral mosquitofish living in undiluted treated municipal sewage in Queensland, Australia, were examined for morphologic abnormalities indicative of reproductive dysfunction. Male and female mosquitofish were captured at two sites receiving undiluted treated sewage and compared with those captured at a reference site. Several morphologic end points were examined, including length of the fourth and sixth anal fin rays and gonadal histology. Both male and female mosquitofish at one site exhibited minor elongation of the fourth anal fin ray consistent with exposure to androgenic stimulation, although the spatial extent of the effect was limited and not significant at other sites further downstream. No incidences of intersex were found. These findings suggest that the level of treatment of domestic sewage at the two sewage treatment plants assessed is adequate to prevent reproductive abnormalities in exposed mosquitofish populations.

Quantification of vitellogenin mRNA induction in mosquitofish (Gambusia affinis) by reverse transcription real-time polymerase chain reaction (RT-PCR).

A method to quantify induction of vitellogenin (Vtg) mRNA in adult male mosquitofish was developed. Male mosquitofish were exposed to 0, 1, 20 and 250 ng l(-1) 17beta-oestradiol (E(2)) for 4 and 8 days in static exposures, and liver Vtg mRNA and 18S rRNA expression were quantified in duplex RT-PCR. Liver 18S rRNA expression was very consistent among individuals, and there was a highly significant increase in Vtg mRNA expression after exposure of mosquitofish for just 4 days at 250 ng l(-1) E(2). Lower doses did not induce Vtg mRNA expression even at 4 or 8 days. This method could be used as a rapid test to detect exposure of mosquitofish to oestrogenic chemicals. Further work is needed to determine if increased Vtg mRNA levels in male mosquitofish induce Vtg synthesis, and to determine the usefulness of the method in field sampling.

Population structure and dispersal of the freshwater mosquitoes Culex annulirostris and Culex palpalis (Diptera: Culicidae) in Papua New Guinea and northern Australia.

In 1995 and 1998, outbreaks of Japanese encephalitis (JE) virus occurred for the first time in Australia. Virus isolation from pools of mosquitoes indicated Culex annulirostris Skuse was the most likely vector. It was hypothesized that wind-blown mosquitoes introduced JE from Papua New Guinea to the Torres Strait and Cape York, northern Australia. This study used levels of genetic differentiation, as indirect evidence of dispersal of mosquitoes between the two continents. The results have demonstrated that in the region incorporating Western Province in PNG, the Torres Strait and Cape York Peninsula, Cx. annulirostris is represented by a panmictic population, indicating frequent widespread dispersal throughout the region of investigation. The closely related Cx. Palpalis Taylor was also present at some locations but the population structure of this species is uncertain. This supports the hypothesis that wind-blown Cx. annulirostris and possibly Cx. Palpalis could be a possible mechanism for introduction of JE virus into Australia.

Development and evaluation of a species diagnostic polymerase chain reaction-restriction fragment-length polymorphism procedure for cryptic members of the Culex sitiens (Diptera: Culicidae) subgroup in Australia and the southwest Pacific.

Members of the Culex sitiens subgroup are important vectors of arboviruses, including Japanese encephalitis virus, Murray Valley encephalitis virus and Ross River virus. Of the eight described species, Cx. annulirostris Skuse, Cx. sitiens Wiedemann, and Cx. palpalis Taylor appear to be the most abundant and widespread throughout northern Australia and Papua New Guinea (PNG). Recent investigations using allozymes have shown this subgroup to contain cryptic species that possess overlapping adult morphology. We report the development of a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) procedure that reliably separates these three species. This procedure utilizes the sequence variation in the ribosomal DNA ITS1 and demonstrates species-specific PCR-RFLP profiles from both colony and field collected material. Assessment of the consistency of this procedure was undertaken on mosquitoes sampled from a wide geographic area including Australia, PNG, and the Solomon Islands. Overlapping adult morphology was observed for Cx. annulirostris and Cx. palpalis in both northern Queensland and PNG and for all three species at one site in northwest Queensland.

Definition of species in the Culex sitiens subgroup (Diptera: Culicidae) from Papua New Guinea and Australia.

Japanese encephalitis is a disease of major importance in Asia, which from 1995 has extended its range into the north of Australia. Culex annulirostris Skuse of the Culex sitiens subgroup of Culex was incriminated as the most likely mosquito vector. Because misidentifications previously have occurred within this subgroup, our paper defines electrophoretic methods to identify Culex specimens from Queensland and the Northern Territory in Australia and from Western Province in Papua New Guinea. In collections from 1996 to 1998 Culex sitiens Wiedemann, Cx. palpalis Taylor, and Cx. annulirostris comprised 93.1% of the total banded proboscis Culex mosquitoes collected; 6.9% remained unidentified to species by morphological or electrophoretic examination. The prevalence of Cx. palpalis was variable and appeared to be associated with below average rainfall during 1997. In Papua New Guinea, this species comprised up to 97.4% of banded proboscis Culex, with smaller numbers collected from the Torres Strait Islands and Cape York; none was collected from other Queensland localities. Cx. annulirostris was more prevalent during years of average or above average rainfall and was predominant in most localities, especially in the Torres Strait and Queensland. These data confirmed the problems in separating Cx. sitiens from Cx. palpalis and Cx. annulirostris using morphological characters.

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

A method for assessing the effects of runneling on salt marsh grapsid crab populations.

Runneling, a type of habitat modification using shallow channels, is an effective method for controlling mosquitoes that breed in intertidal salt marshes. Grapsid crab populations were studied to assess the environmental effects of runneling on nontarget species. Pitfall traps provided a means of monitoring crab distribution and relative abundance at Coomera Island in southeastern Queensland. The study indicated that although runneling does not have a significant effect on the total number of crabs, it may have a significant impact on species distribution. Significantly greater numbers of Parasesarma erythrodactyla were found at the runneled site. In contrast, Helograpsus haswellianus was more abundant at the unrunneled control site. Associations were also found between species abundance, distance from the tidal inlet, and vegetation type. Helograpsus haswellianus was associated with mixed vegetation, whereas Parasesarma erythrodactyla was associated with tall, dense salt marsh couch. No correlation was found between the number of crab burrows present at the study site and the number of crabs caught in the traps. However, the runneled site had approximately twice the number of crab burrows of the unrunneled control site. These results were consistent for 3 consecutive monthly sampling periods. The method has proven suitable for further studies on the long-term environmental impact of runneling.