Population structure, mitochondrial polyphyly and the repeated loss of human biting ability in anopheline mosquitoes from the southwest Pacific.

Australia and New Guinea contain high levels of endemism and biodiversity, yet there have been few evaluations of population-level genetic diversity in fauna occurring throughout the Australo-Papuan region. Using extensive geographical sampling, we examined and compared the phylogenetic relationships, phylogeography and population structure of Anopheles farauti, An. hinesorum and An. irenicus throughout their ranges in the southwest Pacific using mitochondrial (mtDNA COI) and nuclear (ribosomal protein S9 and ribosomal DNA ITS2) loci. Phylogenetic analyses suggest that the ability to utilize humans as hosts has been lost repeatedly, coincident with independent colonizations of the Solomon Islands. As some of the species under investigation transmit malaria in the region, this is a medically important finding. Maximum likelihood and Bayesian phylogenetic analyses of nuclear loci also showed that the three species are monophyletic. However, putative introgression of An. hinesorum mtDNA onto a nuclear background of An. farauti was evident in populations from Queensland, Torres Strait and southern New Guinea. Haplotype networks and pairwise F(ST) values show that there is significant genetic structure within New Guinea and Australia in both An. farauti and An. hinesorum, consistent with a long-term history of low gene flow among populations.

Field evaluation of deet against Anopheles farauti at Ndendo (Santa Cruz) Island, Solomon Islands.

Field efficacy studies comparing two formulations of deet (N,N-diethyl-3-methylbenzamide) against mosquitoes were conducted on Ndendo Island, Solomon Islands. The repellent study was conducted at Pala village in November 2008, and the only mosquito species collected was Anopheles farauti Laveran. A formulation containing 35% deet in a gel provided >95% protection for 2 h, whereas a formulation containing 40% deet in ethanol in a spray applicator provided >95% for only 1 h. This field study demonstrated again that repellents containing deet provide a relatively short period of complete protection against Anopheles spp.

Malaria vectors of Papua New Guinea.

Understanding malaria transmission in Papua New Guinea (PNG) requires exact knowledge of which Anopheles species are transmitting malaria and is complicated by the cryptic species status of many of these mosquitoes. To identify the malaria vectors in PNG we studied Anopheles specimens from 232 collection localities around human habitation throughout PNG (using CO(2) baited light traps and human bait collections). A total of 22,970mosquitoes were individually assessed using a Plasmodium sporozoite enzyme-linked immunosorbent assay to identify Plasmodiumfalciparum, Plasmodiumvivax and Plasmodiummalariae circumsporozoite proteins. All mosquitoes were identified to species by morphology and/or PCR. Based on distribution, abundance and their ability to develop sporozoites, we identified five species as major vectors of malaria in PNG. These included: Anophelesfarauti, Anopheleshinesorum (incriminated here, to our knowledge, for the first time), Anophelesfarauti 4, Anopheleskoliensis and Anophelespunctulatus. Anopheleslongirostris and Anophelesbancroftii were also incriminated in this study. Surprisingly, An. longirostris showed a high incidence of infections in some areas. A newly identified taxon within the Punctulatus Group, tentatively called An. farauti 8, was also found positive for circumsporozoite protein. These latter three species, together with Anopheleskarwari and Anophelessubpictus, incriminated in other studies, appear to be only minor vectors, while Anophelesfarauti 6 appears to be the major vector in the highland river valleys (>1500m above sea level). The nine remaining Anopheles species found in PNG have been little studied and their bionomics are unknown; most appear to be uncommon with limited distribution and their possible role in malaria transmission has yet to be determined.

Rapid identification of Aedes albopictus, Aedes scutellaris, and Aedes aegypti life stages using real-time polymerase chain reaction assays.

In 2005, a widespread infestation of Aedes albopictus was discovered in the Torres Strait, the region between northern Australia and New Guinea. To contain this species, an eradication program was implemented in 2006. However, the progress of this program is impeded by the difficulty of morphologically separating Ae. albopictus larvae from the endemic species Aedes scutellaris. In this study, three real-time TaqMan polymerase chain reaction assays that target the ribosomal internal transcribed spacer 1 region were developed to rapidly identify Aedes aegypti, Ae. albopictus, and Ae. scutellaris from northern Australia. Individual eggs, larvae, pupae, and adults, as well as the species composition of mixed pools were accurately identified. The assay method was validated using 703 field-collected specimens from the Torres Strait.

Identification of Anopheles minimus complex and related species in Vietnam.

Anopheles minimus A and C and several closely related species of mosquitoes have been identified in Vietnam, where some have been implicated in malaria transmission. Morphological variation within and between Anopheles minimus A and C makes identification using alpha taxonomy difficult and several molecular techniques have been developed to separate them. However the difficulties of applying these techniques and the benefits of morphological identification in the field have seen morphological characteristics, such as the humeral pale spot on the costa, being used to separate these two species. In this study, the morphological and molecular examinations of 2,206 specimens collected in Vietnam indicate that pale scaling on the proboscis reliably separates An. aconitus from An. minimus s.l., but hind tarsal banding cannot separate An. jeyporiensis from An. minimus s.l., and the presence or absence of the humeral pale spot is not a reliable characteristic for differentiating An. minimus A from C due to variation of this characteristic in An. minimus C.

Evaluation of Bistar 80SC (bifenthrin) as a tent treatment for protection against mosquitoes in Northern Territory, Australia.

A field trial to assess the efficacy of Bistar 80 SC as a barrier treatment of Australian military tents was conducted over 10 d at Mount Bundey Military Training Area, Northern Territory, Australia, in March 2003. Four pairs of standard eight-person tents were erected, with a single tent in each pair treated with 0.1% Bistar 80 SC as a course spray, and the remainder left as untreated control tents. Carbon dioxide-baited traps were operated in each tent nightly, and biting collections conducted over 8 nights. There was a mean increase in protection of 81% for mosquitoes entering treated tents and 90.4% increase in protection against biting of predominantly Culex annulirostris Skuse. In addition, bifenthrin applied to the military tents enhances the protection of occupants against bites from this important arbovirus vector.

Evaluation of the inhibition of egg laying, larvicidal effects, and bloodfeeding success of Aedes aegypti exposed to permethrin- and bifenthrin-treated military tent fabric.

Laboratory studies were conducted to evaluate the effects of treating military canvas tent fabric with bifenthrin and permethrin on the survival of the eggs and larvae of Aedes aegypti. Gravid female Ae. aegypti were able to oviposit on tent canvas treated with either bifenthrin or permethrin. However, none of the eggs laid on treated canvas hatched, and no larvae added to water in treated trays survived. Low residual concentrations of bifenthrin and permethrin on treated canvas prevented the development of eggs and larvae of Ae. aegypti. Inhibition of bloodfeeding was shown when Ae. aegypti adults were exposed to lower concentrations (10-50% of operational concentrations) of bifenthrin- and permethrin-treated canvas tent fabric. These experiments have shown that military tent canvas treated with either bifenthrin or permethrin can reduce the development of Ae. aegypti eggs and larvae and reduce bloodfeeding success of adults.

A molecular phylogeny of Anopheles annulipes (Diptera: Culicidae) sensu lato: the most species-rich anopheline complex.

The Australasian Annulipes Complex is the most species-rich among Anopheles mosquitoes, with at least 15 sibling species suspected. Members of this complex are the most likely vectors of malaria in the past in southern Australia and are involved in the spread of myxomatosis among rabbits. In this, the first comprehensive molecular study of the Annulipes Complex, 23 ITS2 rDNA variants were detected from collections throughout Australia and Papua New Guinea, including diagnostic variants for the previously identified An. annulipes species A-G. Specimens of each ITS2 variant were sequenced for portions of the mitochondrial COI, COII and nuclear EF-1alpha genes. Partitioned Bayesian and Maximum Parsimony analyses confirmed the monophyly of the Annulipes Complex and revealed at least 17 clades that we designate species A-Q. These species belong to two major clades, one in the north and one mainly in the south, suggesting that climate was a driver of species radiation. We found that 65% (11) of the 17 sibling species recorded here had unique COI sequences, suggesting that DNA barcoding will be useful for diagnosing species within the Annulipes Complex. A comparison of the taxa revealed morphological characters that may be diagnostic for some species. Our results substantially increase the size of the subgenus Cellia in Australasia, and will assist species-level studies of the Annulipes Complex.

The anopheline fauna of Papua New Guinea.

Surveys for anopheline mosquitoes were conducted throughout the mainland of Papua New Guinea from 1992 to 1998 with the aim of mapping the distribution of the anopheline fauna. Larval collections, adult trap, and human landing collections indicated the presence of seven species (other than those belonging to the Anopheles punctulatus group); these were An. bancroftii, An. annulipes, An, karwari, An. longirostris, An. meraukensis, An. novaguinensis, and An. subpictus. The distribution and ecology of these species is discussed.

Environmental factors associated with distribution and range limits of malaria vector Anopheles farauti in Australia.

Ecological factors associated with the narrow coastal distribution of Anopheles farauti Laveran s.s. were investigated using decision tree software and a recently developed software tool that permits analysis of environmental gradients across distributional boundaries. Significant variables identified by these procedures were then used to develop ecological niche models that permitted detailed--and improved--predictions of the species' overall distribution. These methods identified seven climatic factors (four of temperature factors and three atmospheric moisture factors) from among 40 environmental variables related to the range of this species. In addition, the gradient-analysis tool identified elevation as being particularly important. The distributional hypothesis predicted using ecological niche modeling of these factors included all of the record sites from which An. farauti s.s. was collected in northern Australia and successfully reconstructed its narrow limitation to coastal areas. Omission of elevation from analyses resulted in unrealistic predictions of potential distributional areas > 100 km inland, where the species has not been found.

R-253 disrupts microtubule networks in multiple tumor cell lines.

PURPOSE: The design and development of synthetic small molecules to disrupt microtubule dynamics is an attractive therapeutic strategy for anticancer drug discovery research. Loss of clinical efficacy of many useful drugs due to drug resistance in tumor cells seems to be a major hurdle in this endeavor. Thus, a search for new chemical entities that bind tubulin, but neither are a substrate of efflux pump, P-glycoprotein 170/MDR1, nor cause undesired side effects, would potentially increase the therapeutic index in certain cancer treatments. EXPERIMENTAL DESIGN: A high-content cell-based screen of a compound library led to the identification of a new class of compounds belonging to a thienopyrimidine series, which exhibited significant antitumor activities. On structure-activity relationship analysis, R-253 [N-cyclopropyl-2-(6-(3,5-dimethylphenyl)thieno[3,2-d]pyrimidin-4-yl)hydrazine carbothioamide] emerged as a potent antiproliferative agent (average EC(50), 20 nmol/L) when examined in a spectrum of tumor cell lines. RESULTS: R-253 is structurally unique and destabilizes microtubules both in vivo and in vitro. Standard fluorescence-activated cell sorting and Western analyses revealed that the effect of R-253 on cell growth was associated with cell cycle arrest in mitosis, increased select G(2)-M checkpoint proteins, and apoptosis. On-target activity of R-253 on microtubules was further substantiated by immunofluorescence studies and selected counter assays. R-253 competed with fluorescent-labeled colchicine for binding to tubulin, indicating that its binding site on tubulin could be similar to that of colchicine. R-253 neither is a substrate of P-glycoprotein 170/MDR1 nor is cytotoxic to nondividing human hepatocytes. CONCLUSION: Both biochemical and cellular mechanistic studies indicate that R-253 could become a promising new tubulin-binding drug candidate for treating various malignancies.

Does 1-octen-3-ol enhance trap collections of Japanese encephalitis virus mosquito vectors in northern Australia?

The responses of Japanese encephalitis virus (JEV) mosquito vectors to 1-octen-3-ol (octenol) and CO2 were evaluated using Centers for Disease Control (CDC) light traps at 3 sites in northern Australia. There was no significant difference between the number of Culex sitiens subgroup mosquitoes or Cx. gelidus collected in CDC light traps baited with either CO2 alone or CO2 + octenol on Badu Island. At both mainland locations, using octenol in combination with CO2 significantly increased collections of Cx. sitiens subgroup mosquitoes. Collections of nontarget species, such as Ochlerotatus spp., Anopheles spp., and Verrallina spp. were also significantly increased with the addition of octenol. At all 3 locations, reducing collections of nontarget mosquitoes by not using octenol increased the proportion of Culex spp. collected, thus potentially reducing the time and resources required to sort and process collections for JEV detection. Our results also indicate that trials into the efficacy of using octenol as an attractant should be carried out in each area prior to the implementation of a mosquito-based arbovirus surveillance system.

Field evaluation of commercial repellent formulations against mosquitoes (Diptera: Culicidae) in Northern Territory, Australia.

Field trials comparing commercially available repellent formulations containing picaridin (1-piperidinecarboxylate acid, 2-(2-hydroxyethyl)-1-methylpropylester) and deet (N,N-diethyl-3-methylbenzamide) against mosquitoes in Northern Territory, Australia, were conducted. Three repellents were compared: Autan Repel containing 9.3% picaridin, RID containing 10% deet, and Bushman Ultra containing 80% deet in a gel. The predominant mosquito species collected were Culex annulirostris Skuse (63.2%), Ochlerotatus normanensis (Taylor) (19.6%), and Anopheles meraukensis Venhuis (8.6%). Autan Repel provided >95% protection against all mosquitoes for 2 h, RID for 7 h, and Bushman for >8 h. Against Cx. annulirostris, Autan Repel provided >95% protection for 5 h, RID for 7 h, and Bushman for >8 h. The study showed that both deet formulations provided significantly better protection against mosquitoes than picaridin (Autan Repel). All 3 repellents provided good protection against Cx. annulirostris, an important vector of arboviruses in Australia.

The effectiveness of light, 1-octen-3-ol, and carbon dioxide as attractants for anopheline mosquitoes in Madang Province, Papua New Guinea.

The effectiveness of light, 1-octen-3-ol (octenol), carbon dioxide (CO2) and a combination of CO2 and octenol were compared as mosquito attractants using encephalitis vector surveillance traps in 2 villages in Madang Province, Papua New Guinea (PNG). Five species were collected, Anopheles koliensis, Anopheles farauti 2, Anopheles farauti 4, Anopheles longirostris, and Anopheles bancroftii. Light alone was not attractive to any of these species, and the attractiveness of octenol alone, though greater than light, was less than that of CO2 or the CO2 + octenol combination. With An. longirostris, the addition of octenol to CO2 resulted in a statistically significant increase in trap numbers; however, for the other species, any increase was not significant, and with An. koliensis and An. bancroftii, trap numbers were actually reduced when the CO2 + octenol bait was used. In PNG, the use of octenol alone would be effective in attracting more anophelines than if light alone was used; however, octenol by itself was not as effective as CO2.

Field evaluation of repellent formulations containing deet and picaridin against mosquitoes in Northern Territory, Australia.

Field efficacy of repellent formulations containing picaridin (1-methyl-propyl 2-(2-hydroxyethyl)-1-piperidinecarboxylate) or deet (N,N,-diethyl-3-methylbenzamide) against mosquitoes in Northern Territory, Australia, was evaluated. The following repellent treatments were evaluated: 19.2% picaridin (Autan Repel Army 20), a solution of 20% deet in ethanol, and 35% deet in a gel (Australian Defense Force [ADF]). The predominant mosquito species were Culex annulirostris Skuse (57.8%), Anopheles merankensis Venhuis (15.4%), and Anopheles bancroftii Giles (13.2%). The protection provided by repellents against Anopheles spp. was relatively poor, with 19.2% picaridin and ADF deet providing >95% protection for only 1 h, whereas 20% deet provided <95% protection at 1 h after repellent application. In contrast, the repellents provided good protection against Cx. annulirostris, with 19.2% picaridin providing >95% protection for 5 h and both deet formulations providing >95% protection for 7 h when collections ceased. This study provides additional field data showing tolerance of Anopheles spp. for repellents. The response of field populations of Cx. annulirostris, an important vector of arboviruses in Australia, to repellents containing deet and picaridin is reported for the first time.

Molecular identification of the malaria vectors Anopheles anthropophagus and Anopheles sinensis (Diptera: Culicidae) in central China using polymerase chain reaction and appraisal of their position within the Hyrcanus group.

In central China, Anopheles anthropophagus is considered the primary malaria vector and Anopheles sinensis is a secondary vector. Identification of these two cryptic species would facilitate studies on malaria transmission and the application of control measures. At present, the only reliable morphological markers occur in the egg stage, making this approach impractical for any large scale field studies. In this study, we report on the development of a polymerase chain reaction (PCR)-restriction fragment length polymorphism procedure involving the ribosomal DNA ITS2 region for discrimination of these species. The PCR-amplified product size of the ITS2 was 574 bp for An. anthropophagus and 594 bp for An. sinensis. Diagnostic restriction fragment length polymorphisms appeared with the restriction enzymes RsaI or HinfI. This diagnostic PCR was tested on mosquitoes collected from different locations throughout China. Specimens identified morphologically as An. anthropophagus in the adult and egg stage from one location in Quangdong Province were found to be An. sinensis, while specimens from Liaoning Province, which were variable in their egg morphology, were found to be An. anthropophagus. The presence of An. anthropophagus in Liaoning Province extends the range of this species north to 42 degrees N. The ITS2 spacer sequence was used in a maximum parsimony phylogenetic reconstruction of six members of the Hyrcanus group, two members of the Lesteri subgroup, and one member of the Nigerrimus subgroup, with the resulting molecular groupings at odds with the current morphological groupings.

Occurrence of Ross River virus and Barmah Forest virus in mosquitoes at Shoalwater Bay military training area, Queensland, Australia.

Shoalwater Bay military training area (SWBTA), 2,713 km2 of land located 50-80 km north of Rockhampton, Queensland, Australia, is used by Australian and allied forces for training purposes. Between March 1998 and February 2000, monthly collections of mosquitoes at 15 sites were conducted using carbon dioxide-baited traps to study the seasonal occurrence of mosquitoes and Ross River virus (RRV) and Barmah Forest virus (BFV) in mosquitoes. A total of 72,616 mosquitoes, comprising 3,897 pools were collected, and 2,428 pools were tested using a reverse transcriptase-polymerase chain reaction. A total of 15 pools of mosquitoes were positive for virus, 10 RRV and five BFV. Blood meals from an additional 763 mosquitoes were tested by a gel diffusion assay, and the majority (96%) of those identified were from kangaroo, which was the most common mammal in the study area. The results indicate that Culex annulirostris Skuse and Ochlerotatus vigilax (Skuse) are the main vectors of RRV at SWBTA.

Efficacy of a new self-supporting low-profile bednet for personal protection against Anopheles farauti (Diptera: Culicidae) in a village in Papua New Guinea.

A new United States (U.S.) self-supporting low-profile bednet was designed by Walter Reed Army Institute of Research in collaboration with Breakthrough Technologies. The bednet incorporated permethrin-impregnated screening into a frame that erected automatically when removed from its bag. The new U.S. bednet was compared with the current Australian Defense Force (ADF) mosquito bednet at Buka Island, North Solomons Province, Papua New Guinea, in March 1999. At the time of the test, Anopheles farauti Laveran was the most abundant biting mosquito. Both bednet types provided >97.8% protection compared with an unprotected collector. The untreated U.S. Army prototype bednet provided better protection than the untreated ADF bednet against mosquitoes entering the bednet during the night.

Extensive multiple test centre evaluation of the VecTest malaria antigen panel assay.

To determine which species and populations of Anopheles transmit malaria in any given situation, immunological assays for malaria sporozoite antigen can replace traditional microscopical examination of freshly dissected Anopheles. We developed a wicking assay for use with mosquitoes that identifies the presence or absence of specific peptide epitopes of circumsporozoite (CS) protein of Plasmodium falciparum and two strains of Plasmodium vivax (variants 210 and 247). The resulting assay (VecTest Malaria) is a rapid, one-step procedure using a 'dipstick' test strip capable of detecting and distinguishing between P. falciparum and P. vivax infections in mosquitoes. The objective of the present study was to test the efficacy, sensitivity, stability and field-user acceptability of this wicking dipstick assay. In collaboration with 16 test centres world-wide, we evaluated more than 40 000 units of this assay, comparing it to the standard CS ELISA. The 'VecTest Malaria' was found to show 92% sensitivity and 98.1% specificity, with 97.8% accuracy overall. In accelerated storage tests, the dipsticks remained stable for > 15 weeks in dry conditions up to 45 degrees C and in humid conditions up to 37 degrees C. Evidently, this quick and easy dipstick test performs at an acceptable level of reliability and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.

Malaria vectors on Buka and Bougainville Islands, Papua New Guinea.

Anophelines were sampled from 82 locations on Buka and Bougainville islands in Papua New Guinea by larval collections, carbon dioxide-baited mosquito traps, and human biting catches. Anopheles farauti s.s. was collected in larval surveys but infrequently in mosquito traps on both islands; on Buka Island this species was readily collected in human biting catches. Anopheles farauti 2 was commonly collected in larval surveys on both islands; however, it was not collected in either mosquito traps or human biting catches. Anopheles punctulatus was found only on Buka Island, where it was commonly collected as larvae, but rarely in human biting catches and mosquito traps. Anopheles lungae was collected as larvae from only 1 site on Bougainville. Anopheles farauti s.s. fed consistently throughout the night (1900-0600 h); small peaks at midnight and dawn were not statistically significant. Of 1,156 An. farauti s.s. specimens examined by enzyme-linked immunosorbent assay for malaria sporozoites, 20 were found to be positive; 12 were positive for Plasmodium falciparum and 8 were positive for P. vivax (247 variant = 5; 210 variant = 3). Anopheles farauti s.s. seems to be the major malaria vector on these islands, whereas An. punctulatus may play a minor role on Buka Island. Anophele jarauti 2 is unlikely to be involved in malaria transmission on Buka or Bougainville islands.