Potential Spatial Distribution of the Newly Introduced Long-horned Tick, Haemaphysalis longicornis in North America.

The North American distributional potential of the recently invaded tick, Haemaphysalis longicornis, was estimated using occurrence data from its geographic range in other parts of the world and relevant climatic data sets. Several hundred candidate models were built using a correlative maximum entropy approach, and best-fitting models were selected based on statistical significance, predictive ability, and complexity. The median of the best-fitting models indicates a broad potential distribution for this species, but restricted to three sectors-the southeastern United States, the Pacific Northwest, and central and southern Mexico.

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.

Molecular systematics of the Philippine malaria vector Anopheles flavirostris.

Allozyme and molecular sequence data from the malaria vector Anopheles flavirostris (Ludlow) (Diptera: Culicidae) were analysed from 34 sites throughout the Philippines, including the type locality, to test the hypothesis that this taxon is a single panmictic species. A finer-scaled allozyme study, of mainly Luzon samples, revealed no fixed genetic differences in sympatric sites and only low levels of variation. We obtained data from partial sequences for the internal transcribed spacer 2 (ITS2) (483 bp), the third domain (D3) (330 bp) of the 28S ribosomal DNA subunit and cytochrome c oxidase subunit I (COI) of mitochondrial DNA (261 bp). No sequence variation was observed for ITS2, only a one base pair difference was observed between Philippine and Indonesian D3 sequences and An. flavirostris sequences were unique, confirming their diagnostic value for this taxon. Sixteen COI haplotypes were identified, giving 25 parsimony informative sites. Neighbour-Joining, Maximum Parsimony, Maximum Likelihood and Bayesian phylogenetic analysis of COI sequences for An. flavirostris and outgroup taxa revealed strong branch support for the monophyly of An. flavirostris, thus confirming that Philippine populations of this taxon comprise a single separate species within the Minimus Subgroup of the Funestus Group. Variation in the behaviour of An. flavirostris is likely to be intraspecific rather than interspecific in origin.

Population structure of an island malaria vector.

The impact of islands on the population structure of Anopheles flavirostris (Ludlow) (Diptera: Culicidae), the primary malaria vector in the Philippines, was assessed. A phylogenetic analysis of 16 cytochrome oxidase subunit 1 (CO1) haplotypes revealed three clades: one basal clade containing genetically disparate haplotypes from Mindanao, and two derived clades, one of which was largely confined to the largest island, Luzon, and one that was widespread except for Luzon. For the Luzon clade, nested clade analysis revealed an isolation-by-distance effect, and a mismatch distribution analysis diagnosed a recent demographic expansion (sum of squared deviation, SDD = 0.0093, P= 0.075), which mirrors demographic attributes found in mainland primary malaria vectors and could inflate estimates of gene flow from F(ST). For the widespread clade, evidence of range expansion and past fragmentation and/or long distance colonization from the Visayas or Mindanao to Palawan is suggested. A south-to-north range expansion of An. flavirostris is suggested; estimates of coalescence for the Luzon clade was 214 000 years ago (ya) (95% confidence interval 35 600-298 000 ya), i.e. late Pleistocene. Present day rather than Pleistocene island association and some, but not all, sea barriers appeared to be important for An. flavirostris population structure. Our results suggest that endemic island malaria vector species need to be considered before any generalizations are made about the population structure of primary and secondary vectors.

Population structure of the peridomestic mosquito Ochlerotatus notoscriptus in Australia.

Ochlerotatus notoscriptus (Skuse) (Diptera: Culicidae) is the predominant peridomestic mosquito in Australia where it is the primary vector of dog heartworm, Dirofilaria immitis (Leidy), and a potentially important vector of arboviruses (Barmah Forest, Ross River) with geographical variation of vector competence. Although widespread, Oc. notoscriptus has low dispersal ability, so it may have isolated subpopulations. The identification of gene flow barriers may assist in understanding arbovirus epidemiology and disease risk, and for developing control strategies for this species. We investigated the population structure of Oc. notoscriptus from 17 sites around Australia, using up to 31 putative allozyme loci, 11 of which were polymorphic. We investigated the effect of larval environment and adult morphology on genetic variation. At least five subpopulations were found, four in New South Wales (NSW) and one unique to Darwin. Perth samples appear to be a product of recent colonization from the Australian east coast. For NSW sites, a Mantel test revealed an isolation by distance effect and spatial autocorrelation analysis revealed an area of effective gene flow of 67 km, which is high given the limited dispersal ability of this species. No consistent difference was observed between 'urban' and 'sylvan' habitats, which suggests frequent movement between these sites. However, a finer-scaled habitat study at Darwin revealed small but significant allele frequency differences, including for Gpi. No fixed allozyme differences were detected for sex, size, integument colour or the colour of species-diagnostic pale scales on the scutum. The domestic habit of Oc. notoscriptus and assisted dispersal have helped to homogenize this species geographically but population structure is still detectable on several levels associated with geographical variation of vector competence.

Host-dependent Anopheles flavirostris larval distribution reinforces the risk of malaria near water.

Malaria control strategies are more likely to be successful if groups at high risk can be accurately predicted. Given that mosquitoes have an obligate aquatic phase we were interested in determining how vector larval abundance relates to the spatial distribution of human malaria infection. We examined the relationship between malaria parasite prevalence and distance from vector larval habitat, and vector larval abundance and distance from human habitation, in separate studies in rural, low-endemic areas of the Philippines. Parasite prevalence among symptomatic patients was significantly higher among those living in proximity (< or = 50 m) to potential larval habitats of the major vector, Anopheles flavirostris (adjusted odds ratio [AOR] = 2.64, P = 0.02 and AOR = 3.43, P = 0.04). A larval survey of A. flavirostris revealed a higher density of early and late instars near human habitation (adjusted P < 0.05). The results suggest that larvae are associated with human habitation, thereby reinforcing malaria risk in people living close to larval habitats. This has implications for understanding the interaction between vectors, hosts, and parasites, and the potential for success of localized malaria control measures.

A morphological study of the Anopheles punctulatus group (Diptera: Culicidae) in the Solomon Islands, with a description of Anopheles (Cellia) irenicus Schmidt, sp.n.

A description of Anopheles (Cellia) irenicus Schmidt, sp.n. (formerly A. farauti No. 7) is provided. This species is one of six recorded from the Solomon Islands within the A. punctulatus group, which contains the major vectors of the causative agents of malaria and lymphatic filariasis in the southwest Pacific. Morphological markers are described for adult females, fourth-instar larvae and pupae that identify most specimens of A. irenicus. Keys are presented to distinguish members of the A. punctulatus group in the Solomon Islands.

Stream-bank shade and larval distribution of the Philippine malaria vector Anopheles flavirostris.

The principal malaria vector in the Philippines, Anopheles flavirostris (Ludlow) (Diptera: Culicidae), is regarded as 'shade-loving' for its breeding sites, i.e. larval habitats. This long-standing belief, based on circumstantial observations rather than ecological analysis, has guided larval control methods such as 'stream-clearing' or the removal of riparian vegetation, to reduce the local abundance of An. flavirostris. We measured the distribution and abundance of An. flavirostris larvae in relation to canopy vegetation cover along a stream in Quezon Province, the Philippines. Estimates of canopy openness and light measurements were obtained by an approximation method that used simplified assumptions about the sun, and by hemispherical photographs analysed using the program HEMIPHOT. The location of larvae, shade and other landscape features was incorporated into a geographical information system (GIS) analysis. Early larval instars of An. flavirostris were found to be clustered and more often present in shadier sites, whereas abundance was higher in sunnier sites. For later instars, distribution was more evenly dispersed and only weakly related to shade. The best predictor of late-instar larvae was the density of early instars. Distribution and abundance of larvae were related over time (24 days). This pattern indicates favoured areas for oviposition and adult emergence, and may be predictable. Canopy measurements by the approximation method correlated better with larval abundance than hemispherical photography, being economical and practical for field use. Whereas shade or shade-related factors apparently have effects on larval distribution of An. flavirostris, they do not explain it completely. Until more is known about the bionomics of this vector and the efficacy and environmental effects of stream-clearing, we recommend caution in the use of this larval control method.

Descriptions of the Anopheles (Cellia) farauti complex of sibling species (Diptera: Culicidae) in Australia.

Descriptions of the three sibling species of the Anopheles farauti complex in Australia, A. farauti Laveran (formerly A. farauti No. 1), A. hinesorum Schmidt sp.n. (formerly A. farauti No. 2) and A. torresiensis Schmidt sp.n. (formerly A. farauti No. 3) are provided. These species form a part of the punctulatus group, which contains the major malaria vectors in the southwest Pacific. Morphological markers are described for adult females, fourth instar larvae and pupae which identify most specimens, and are presented in keys.

Ecological distribution of mosquito larvae of the Anopheles punctulatus group on Niolam (Lihir) Island, Papua New Guinea.

We surveyed the larval habitats of members of the Anopheles punctulatus group of mosquitoes on Niolam (Lihir) Island, Papua New Guinea. Identification of this group was undertaken by polymerase chain reaction-restriction fragment length polymorphism analysis of the amplified internal transcribed spacer unit 2 of rDNA, because morphologic separation of member species is unreliable. The most widespread malaria vector species and their most common larval habitats were identified to aid source-reduction programs for malaria control. The most ubiquitous species was An. punctulatus, followed by An. farauti no. 2. then An. farauti s.s. Anopheles punctulatus has increased relative to An. farauti s.l. since the start of development projects on Lihir Island. The most common larval habitats were shallow temporary pools with clay substrate and with plants or floatage. These habitats, mostly encountered alongside poorly drained roads, may be increased by development projects.

Ribosomal DNA sequence markers differentiate two species of the Anopheles maculatus (Diptera: Culicidae) complex in the Philippines.

The Anopheles maculatus Theobald complex includes important vectors of malaria. Based on chromosomal and morphological evidence, two species in this complex occur in the Philippines. Because separation of these species, An. dispar Rattanarithikul & Harbach and An. greeni Rattanarithikul & Harbach, is problematic due to the difficulty or unreliability of the identification methods currently available, we sought a molecular technique for identifying these two species. We sequenced two regions of nuclear ribosomal DNA; the second internal transcribed spacer (ITS2) and the third domain (D3) of the 28S gene, from An. maculatus sensu lato (s.l.) collected throughout the Philippines. Two sequence groups were identified that corresponded morphologically to An. dispar and An. greeni. Four percent of the 318-320 bp ITS2 and 2.5% of the 367 bp D3 differed between the two species. No evidence of intraspecific variation in sequences was found. From the sequence data, we developed a more reliable and easier method for identifying An. dispar and An. greeni, based on a HaeII restriction fragment-length polymorphism in a polymerase chain reaction amplified fragment of ITS2. This method will facilitate future vector studies, which will be necessary, as previous data collected on An. maculatus s.l. in the Philippines is unreliable given the multispecies nature of this taxon.

Shared salinity tolerance invalidates a test for the malaria vector Anopheles farauti s.s. on Guadalcanal, Solomon Islands [corrected].

Among the Punctulatus Group of Anopheles mosquitoes (Diptera: Culicidae), first-instar larvae of the medically unimportant freshwater Anopheles farauti species No. 7 survives a seawater tolerance test (STT) that was previously thought to be diagnostic for the saltwater-tolerant malaria vector species, An. farauti Laveran s.s. Salt tolerance in these two closely related isomorphic species appears to be a shared derived character within the Farauti Complex. Failure to differentiate An. farauti s.s. from An. farauti No.7 will overestimate potential malaria vector numbers and waste limited larval control resources. Use of the STT should therefore be discontinued on Guadalcanal and other techniques such as allozyme electrophoresis used instead [corrected].

Populations of the south-west Pacific malaria vector Anopheles farauti s.s. revealed by ribosomal DNA transcribed spacer polymorphisms.

Malaria in the south-west Pacific is transmitted by members of the Anopheles punctulatus group which comprises 12 cryptic species with overlapping morphology. The most widely distributed species of the group is Anopheles farauti s.s. (An. farauti 1) found throughout northern Australia, Papua New Guinea, eastern Indonesia, the Solomon Islands and Vanuatu. A study of the population structure of this species using PCR-RFLP analysis on the ribosomal DNA internal transcribed spacer 1 reveals five genotypes which had distinct geographical distributions. Where these distributions overlap, genotype hybrids can be identified. Heteroduplex analysis of the ITS2 region reveals combinations of nonhomogenized ITS2 sequences and subsequently seven identifiable genotypes, reflecting the ITS1 distribution. Sequence analysis of these ITS2 polymorphisms reveals a minimum of 13 ITS2 sequence types present in heterogeneous combinations in individual mosquitoes. It appears that there are different levels of evolution occurring within the ITS1 and ITS2 regions. These data suggest that An. farauti s.s. may contain multiple loci for the rDNA gene family or that the homogenization of these regions is relatively slow and can be used in genetic studies of population distribution and structure.

Shared salinity tolerance invalidates a test for the malaria vector Anopheles farauti s.s. on Guadalcanal, Solomon Islands.

Among the Punctulatus Group of Anopheles mosquitoes (Diptera: Culicidae), first-instar larvae of the medically unimportant freshwater Anopheles farauti species No. 7 survives a seawater tolerance test (SST) that was previously thought to be diagnostic for the saltwater-tolerant malaria vector species, An. farauti Laveran s.s. Salt tolerance in these two closely related isomorphic species appears to be a shared derived character within the Farauti Complex. Failure to differentiate An. farauti s.s. from An. farauti No. 7 will overestimate potential malaria vector numbers and waste limited larval control resources. Use of the SST should therefore be discontinued on Guadalcanal and other techniques such as allozyme electrophoresis used instead.

The potential of ivermectin to control the malaria vector Anopheles farauti.

We investigated mortality in Anopheles farauti mosquitoes, a major coastal malaria vector in the south-west Pacific, fed on a volunteer who had taken a 250 micrograms/kg dose of ivermectin. High mortality was recorded in mosquitoes feeding during the first week after treatment of the volunteer, for instance 100-80% failed to survive 3 days. A long-term residual effect of ivermectin in the blood was indicated by a small but significantly higher mortality in mosquitoes fed 6 weeks after ivermectin was taken. These effects were included in malaria transmission models that incorporated host choice and host-induced mortality parameters. For the zoophilic An. farauti, ivermectin treatment of animals resulted in a greater reduction in malaria than ivermectin treatment of humans alone, whereas for an anthropophilic vector, treatment of humans was more important. This suggests that ivermectin treatment of animals could have an important role in malaria control where An. farauti is the vector. Improvement in the health of humans and domestic animals through control of parasitic worms and mites might encourage community participation in strategies involving ivermectin.

Oviposition preference for freshwater in the coastal malaria vector, Anopheles farauti.

Oviposition preference of the Australasian coastal malaria vector Anopheles farauti s.s. for water of varying salinity was determined in the laboratory to help understand the distribution and control of this species in the field. Numbers of eggs laid showed an inverse relationship with salinity; of 5 NaCl concentrations most eggs were laid in distilled water but some were laid in 3.17% NaCl (the salinity of seawater). The association of An. farauti with coastal areas occurs in spite of an aversion to salt water by ovipositing females. Factors other than salinity must be the primary determinants of distribution. Increasing the salinity of larval habitats will not totally prevent An. farauti from laying eggs. Elimination of this species may not occur unless salinity is kept high enough to prevent complete larval development.

Evolution and systematics of Anopheles: insights from a molecular phylogeny of Australasian mosquitoes.

Relationships among the genus Anopheles and its many sibling species-groups are obscure despite the importance of anophelines as the vectors of human malaria. For the first time, the interrelationships and the origin of Australasian members of the subgenus Cellia are investigated by a cladistic analysis of sequence variation within the mitochondrial cytochrome oxidase subunit II gene. Estimated divergence times between many Australasian and Oriental taxa predate the mid Miocene collision of Australasia and Southeast Asia. Phylogenetic analysis suggests that two-way exchanges with Oriental mosquitoes rather than only immigration may have been a characteristic of anopheline paleobiogeography in Australasia. The Australasian fauna is mostly included in a large clade. The medically important Punctulatus Group is monophyletic and appears derived from Oriental stock. Populations within this group from as far apart as Australia and Vanuatu were in contact in the recent past (i.e., 0.35-2.44 mya), supporting dispersal rather than vicariance explanations. Some support for the monophyly of the Myzomyia, Neomyzomyia, and Pyretophorus Series was found. However, the subgenera Anopheles and Cellia and the Neocellia Series are paraphyletic, but branch support at these taxonomic levels was poor. The COII gene shows promise for questions concerning alpha taxonomy but appears to be of limited use for resolving deeper relationships within the Anopheles.

Anopheline mosquitoes of the western province of Papua New Guinea.

A survey of the Anopheles species of Western Province, Papua New Guinea, was made in April-May 1992. A total of 6,427 specimens was collected from 74 sites within the province using carbon dioxide-baited light traps and larval sampling. Eleven species were identified using morphological characteristics, allozyme analysis, and species-specific DNA probes. These were, in order of prevalence: Anopheles farauti 2 (51 sites), An. bancroftii (17 sites), An. farauti s. s. (16 sites), An. longirostris (9 sites), An. farauti 3 (7 sites), An. punctulatus (4 sites), An. koliensis (4 sites), Anopheles sp. near punctulatus (4 sites), An. meraukensis (4 sites), An. farauti 4 (3 sites), and An. novaguinensis (2 sites). Members of the An. farauti complex made up 93.3% of the specimens collected with An. farauti 2 being the most abundant and widespread species inland and An. farauti s. s. the dominant species on the coast. The abundance and distribution of the species are discussed.

DNA probes for the Anopheles punctulatus complex.

Genomic DNA probes were made for two recently identified members of the Anopheles punctulatus complex; Anopheles sp. near punctulatus from Papua New Guinea and Anopheles farauti No. 7 from the Solomon Islands. The probes are species-specific and with the use of 32P labeling sensitive enough so that a squash blot of only a small segment of the mosquito is required for identification. The 119-basepair (bp) probe for An. sp. near punctulatus and the 1,106-bp probe for An. farauti No. 7 have been sequenced in full and the probes have been tested on field collected specimens. These probes now make it possible to distinguish An. sp. near punctulatus and An. farauti No. 7 from the other eight members of the An. punctulatus complex. A pan-species probe was also made from the 18S ribosomal DNA that binds to DNA from all members of the complex. These three probes complete the set required for distinguishing all known members of the An. punctulatus complex by DNA hybridization.

Malaria transmission and climate change in Australia.

Although endemic malaria was eradicated from Australia by 1981, the vectors remain and transmission from imported cases still occurs. Climate modelling shows that global warming will enlarge the potential range of the main vector, Anopheles farauti sensu stricto; by the year 2030 it could extend along the Queensland coast to Gladstone, 800 km south of its present limit. Vigilance and a dispassionate assessment of risk are needed to meet this challenge.