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.

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.

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.

Detection of bancroftian filariasis in human blood samples from Sorsogon province, the Philippines by polymerase chain reaction.

The polymerase chain reaction (PCR) was used for diagnosing Wuchereria bancrofti infection in a small village in the province of Sorsogon, the Philippines. Of 54 night-time blood samples collected, 4 (7.4%) were found to be microfilaremic as determined by combined direct blood film examination and membrane filtration of blood followed by blood film examination. However, utilization of the SspI PCR assay to detect repeated W. bancrofti DNA sequences in human blood doubled the number of microfilaremic individuals to 8 (13.0%). The results of this survey suggest that utilization of diagnostic tools based on microscopy could underestimate the true prevalence of W. bancrofti in the Philippines.

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.

Components of the vectorial capacity of Aedes poicilius for Wuchereria bancrofti in Sorsogon province, Philippines.

Components of vectorial capacity (biting density, survival, and host preferences) of a population of Aedes (Finlaya) poicilius, the principal vector of Wuchereria bancrofti in the Philippines, were studied in 1994-1995 in Sorsogon province. Aedes poicilius comprised 20.7% of 3243 mosquitoes of 24 species taken in 42 nights of human-biting collections, and 1.4% of 7586 mosquitoes of 27 species taken in 18 concurrent nights of carabao-trap collections. There were, on average, 16 bites by Ae. poicilius per person per night. As there was no relationship between body size of the female Ae. poicilius and parity status, body size was not a variable affecting survivorship of Ae. poicilius. The probability of daily survival was estimated to be 0.85, and the human blood index was 0.67. None of the 862 Ae. poicilius examined contained W. bancrofti larvae, probably because of distribution of diethylcarbamazine to microfilaraemic people prior to the study. The vectorial capacity of the Ae. poicilius population under study was estimated to be 2.4 new cases per primary case per day. However, the basic reproductive rate (i.e. the number of new cases of W. bancrofti infection generated from a single infective case) was estimated to be very low because of inefficiency in parasite transmission.

Vector abundance and behaviour in an area of low malaria endemicity in Bataan, the Philippines.

The vectorial importance of known and potential vectors in Morong, Bataan, Philippines was assessed based on human and animal baited collections of adult mosquitoes and on larval collections. Anopheles flavirostris, the principal vector in the Philippines, was the most abundant among human landing catches, followed by An. maculatus sensu lato (s.l.). Both showed similar seasonal abundance with a peak during the early drier part of the year, which coincided with the peak in malaria cases. Both An. flavirostris and An. maculatus s.l. fed throughout the night with the broad peak of capture from 00:00 to 04:00 and from 22:00 to 00:00, respectively. The two species had similar parous rates (0.76 and 0.72, respectively) giving an average life span equivalent to four feeding cycles. Neither vector was abundant with average human landing rates on collectors of 0.6 and 0.4 mosquitoes per person per night, respectively over the study period. An. maculatus s.l. showed a stronger preference for outdoor feeding compared to An. flavirostris. An. maculatus s.l. was markedly zoophilic with a biting rate on water buffalo 50 times the human landing rate. An. flavirostris was less zoophilic with a corresponding ratio of 7.5. It was concluded that in this area, An. flavirostris is the principal vector. The combination of localised transmission, late night biting pattern and localised breeding sites of An. flavirostris suggest that the use of bed nets and environmental management are relevant control measures that can be implemented through community participation.