Speciation and distribution of the members of the Anopheles punctulatus (Diptera: Culicidae) group in Papua New Guinea

Mosquito collections were made throughout the mainland of Papua New Guinea to identify the members of the Anopheles punctulatus group present and to determine their distribution. Identification was made using morphology, DNA hybridization, and polymerase chain reaction (PCR)-RFLP analysis. Nine members of the group were identified: An. farauti s.s. Laveran, An. farauti 2, An. koliensis Owen, and An. punctulatus D?nitz, were common and widespread; An. farauti 4 was restricted to the north of the central ranges where it was common; An. farauti 6 was found only in the highlands above 1,000 m; and An. farauti 3, An. sp. near punctulatus and An. clowi Rozeboom & Knight were uncommon and had restricted distributions. Identification of An. koliensis and An. punctulatus using proboscis morphology was found to be unreliable wherever An. farauti 4 occurred. The distribution and dispersal of the members of the An. punctulatus group is discussed in regard to climate, larval habitats, distance from the coast, elevation, and proximity to human habitation.     

Lymphatic filariasis in Vanuatu

Lymphatic filariasis, in the Archipelago of Vanuatu, is due to Wuchereria bancrofti. The vector is Anopheles farauti, endophilic mosquito, which transmits also malaria. The level of endemicity was very high in the beginning of this century (microfilariae in blood: 60.9%, elephantiasis: 21%), and has dramatically decline now by detection and treatment of sick people (70 cases in 1986), mass chimioprophylaxis and protection against the vectors. But these actions must be continued to obtain the eradication of filariasis in the Vanuatu.     

Seasonal abundance of Anopheles farauti (Diptera: Culicidae) sibling species in far north Queensland, Australia

In the Cairns area of far north Queensland, Australia, the seasonal abundance of Anopheles farauti Laveran sibling species was studied at 6 locations, representing 3 habitat types, between August 1995 and September 1997. A total of 45,401 An. farauti s.l. was collected using CO2 + octenol baited CDC light traps, and consisted of 29,565 An. farauti No. 2, 14,214 An. farauti No. 3, and 1,622 An. farauti s.s. The relative abundance of all 3 species differed significantly by season and location. An. farauti No.2 was the dominant species except in Cairns, where An. farauti s.s. was most abundant, and at Ninds Creek, where An. farauti No. 3 predominated. The dominant species at each location was present year round, although peaks in seasonal abundance were observed. An. farauti s.s. populations were highest during the wet season (January-April). In lowland freshwater swamp habitats and 1 brackish location, An. farauti No. 2 was more abundant during the wet season. However, at the highland freshwater swamp habitat, populations of An. farauti No. 2 were highest during the late dry season and early wet season (October-December). There was a significant positive correlation of both temperature and rainfall with An. farauti s.s. and An. farauti No. 2 trap collections. There was a negative correlation between An. farauti No. 3 and temperature, indicating that this species may be more abundant during cool weather. Although there were significant relationships among weather variables and An. farauti s.l. collections, correlation values were generally low, indicating that other factors may contribute to variability among An. farauti sibling species trap collections.     

Effects of local geographic barriers and latitude on population structure in Anopheles punctipennis (Diptera: Culicidae)

We sampled Anopheles punctipennis (Say) from 11 localities throughout Vermont to examine the effects of latitude and two local geographical boundaries, Lake Champlain and the Green Mountains, on the population genetic structure of this species. Thirty-five mitochondrial haplotypes were detected in 104 individuals using a variable region of the COI gene. When latitude was examined, we detected significant structure within localities and among localities within latitudinal regions. For geographic analysis, significant genetic structure was detected only within localities. Estimates of gene flow across geographic regions indicate that the Green Mountains, but not Lake Champlain, is a barrier to dispersal for this species. We found no correlation between genetic and geographic distances for An. punctipennis.     

Rediscovery of Anopheles (Cellia) clowi (Diptera: Culicidae), a rarely recorded member of the Anopheles punctulatus group

Anopheline specimens collected in Papua New Guinea were morphologically identified as the rarely recorded Anopheles clowi Rozeboom & Knight. Amplification of the rDNA ITS2 region of this material revealed a fragment of 750 bp confirming its placement in the Anopheles punctulatus group. This group contains 12 species and includes the major malaria vectors in the islands of the southwest Pacific. Digestion of the ITS2 with the restriction enzyme MspI produced restriction fragment-length polymorphism with bands at 380, 300, and 150 bp, a pattern shared by no other members of this group. Phylogenetic analysis involving the sequencing of a 2 kb region of the rDNA 18S gene indicated that An. clowi was monophyletic and basal to the rest of the group and showed considerable independent evolution from the other members. This is the first record of An. clowi in Papua New Guinea and only the third collection of this species since its discovery in 1945.     

Malaria in Vanuatu. Recent epidemiological data

The Republic of Vanuatu, ex-Condominium of New-Hebrides, is an archipelago in the South Pacific, between Solomon Islands and New-Caledonia. Although the health system is quite satisfactory, malaria has become the major health problem in the whole country. Its epidemiological characteristics are reviewed, and particularly the biology of the only known vector, the Anopheles farauti complex. A sudden and severe aggravation of the malaria morbidity since 1980 is then evidenced. This increase of the yearly incidence (from 21% to 183% between 1980 and 1984) has privileged the rising of Plasmodium falciparum which reached 75% of the national paraside index. Moreover, it has clearly followed a geographical progression from north to south within the group during the four years. Various possible causes to this alarming situation are examined, among which the emergence of chloroquin-resistant strains of P. falciparum, and the possibility of modifications in the vector's population, particularly hold the attention.     

Molecular evidence of speciation between island and continental populations of Anopheles (Cellia) sundaicus (Diptera: Culicidae), a principal malaria vector taxon in Southeast Asia

Anopheles sundaicus s.l. is a principal malaria vector taxon on islands and along the coastal areas of Southeast Asia. It has a wide geographical distribution and exhibits a high level of ecological and behavioral variability. Study of this taxon is crucial for understanding its biology and implementing effectise vector control measures. We compared populations of An. sundaicus from Vietnam, Thailand, and Malaysian Borneo by using two mitochondrial DNA markers: cytochrome oxidase I and cytochrome b. Genetic divergence, geographic separation, and cladistic analysis of relationships revealed the presence of two cryptic species: Anopheles sundaicus s.s. on Malaysian Borneo and An. sundaicus species A in coastal areas of Thailand and Vietnam. A polymerase chain reaction (PCR) assay was developed to easily identify these two species throughout their geographic distributions. The assay was based on sequence characterized amplified region derived from random amplified polymorphic DNA. This PCR identification method needs to be validated and adapted for the recognition of other possible species in the Sundaicus Complex.     

An analysis of correspondence between unique rabies virus variants and divergent big brown bat (Eptesicus fuscus) mitochondrial DNA lineages

The literature supports that unique rabies virus (RABV) variants are often compartmentalized in different species of bats. In Colorado, two divergent mtDNA lineages of big brown bats (Eptesicus fuscus) co-occur. RABV associated with this species also segregates into two clades. We hypothesized that unique RABV variants might be associated with mtDNA lineages of Colorado big brown bats. DNA was extracted from brain tissue of rabid big brown bats, the ND2 gene was amplified to determine mtDNA lineage, and the lineage was compared to a previously derived phylogenetic analysis of the RABV N gene. No correspondence was found between host bat lineage and RABV variant.     

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.     

Insecticide susceptibility status of Anopheles gambiae s.l. (Diptera: Culicidae) in the Republic of Cameroon

A large-scale survey of Anopheles gambiae Giles, 1902 susceptibility to DDT, dieldrin, permethrin, and deltamethrin was conducted in the Republic of Cameroon. 15 field populations from various geographical areas were tested using World Health Organization test kits for adult mosquitoes. The laboratory Kisumu susceptible reference strain was tested as a control. Results showed that dieldrin and DDT resistance was still present in some populations, and indicated permethrin or deltamethrin resistance. Within the Anopheles gambiae complex, resistant individuals belonged to An. gambiae s.s. and An. arabiensis species. Both M and S molecular forms of An. gambiae s.s. were found resistant. In most of resistant populations, the knockdown times were 2-5-folds increased. However, none of the surviving mosquitoes was positive to the kdr "Leu-Phe" mutation using polymerase chain reaction (PCR) diagnostic test. These results likely suggested involvement of other resistance mechanism(s), such as enzyme detoxification or kdr "Leu-Ser" mutation. Researches on An. gambiae s.l. resistance should be promoted in Cameroon, to improve malaria vector control programs and to implement resistance management strategies.     

Molecular evolution and phylogeny of dengue type 4 virus in the Caribbean

We sequenced the E gene and adjacent prM/M and NS1 junctions (1940 bp) of 48 Dengue-4 (DEN-4) isolates collected between 1981 and 1999 from 8 Caribbean islands and from 7 South and Central American countries. Phylogenetic analysis confirms a single introduction in the early 1980s and a high degree of gene flow resulting in a pattern of evolution defined more by time period than geographic origin, especially within the Caribbean basin. A modern Caribbean clade consisting of four distinct lineages has arisen, comprised of isolates from Caribbean islands and nearby regions of South America. This clade is defined by three amino acid substitutions in the E (aa 163 and 351) and NS1 (aa 52) proteins. These findings highlight the importance of migration and gene flow in dengue viral change and suggest that efforts to understand disease dynamics in the Caribbean basin need to focus at regional, rather than local scales.     

Polymerase chain reaction identification of three members of the Anopheles sundaicus (Diptera: Culicidae) complex, malaria vectors in Southeast Asia

Anopheles sundaicus s.l., a major malaria vector taxon, occurs primarily along coastal areas and on islands in Southeast Asia. Our previous studies using cytochrome oxidase I, cytochrome-b, and internal transcribed spacer 2 markers discriminated three allopatric species: An. sundaicus s.s. in northern Borneo, An. epiroticus in Southeast Asia, and An. sundaicus E on Sumatra and Java, Indonesia. Morphological comparisons of three developmental stages did not reveal unique diagnostic characters that could reliably distinguish the three species. Therefore, we developed a multiplex polymerase chain reaction (PCR) assay based on two mitochondrial DNA markers to unambiguously identify them. This PCR was tested on 374 specimens from 24 different geographical populations, expanding our knowledge of the distribution of these species.     

Estimation of the sporozoite rate of malaria vectors using the polymerase chain reaction and a mathematical model

We developed a sensitive polymerase chain reaction (PCR) method for the detection of Plasmodium falciparum DNA from mosquitoes collected in the field. Plasmodium falciparum was detected from 15.2% of 1-parous mosquitoes, Anopheles farauti, in the Solomon Islands through use of the PCR method. A novel mathematical model was developed to estimate the sporozoite rate based on the malaria-positive rate of 1-parous mosquitoes. Using this model, the sporozoite rate of Anopheles farauti in the Solomon Islands was calculated to be 0.09%. This method enables estimation of the sporozoite rate based on a relatively small number (100-200) of mosquitoes compared with the number needed for the ELISA method.     

Dispersal ghosts in Oceania.

Anthropological genetics helps expand our understanding of human phenotypes in the Pacific, in part because of its focus on gene genealogies to infer past episodes of dispersal and to differentiate these events from adaptations due to long-duration directional selection. Sewall Wright's 1949 seminal paper on population structure emphasized that there were two strong forces that exerted systematic and therefore determinant pressure on the gene pool: recurrent immigration and gene flow. These are important topics to all discussions of human dispersal in any region of the world. Furthermore, Wright listed five unique kinds of events that produced indeterminate or unpredictable changes that could lead to phenotypic and genotypic effects. In this category, he placed unique selective incidents, unique hybridization events, unique reductions in number, swamping by mass immigration, and mutational drive due to an allele always being favored since its origin or introduction. This discussion of human dispersal in the Pacific will touch on these topics, since they provide a second level of complexity in knowing who moved about a region of the world found already settled when rediscovered by colonial explorers during the 16-18th centuries.     

Effect of Plasmodium falciparum on the survival of naturally infected afrotropical Anopheles (Diptera: Culicidae)

The effect of the malarial parasite, Plasmodium falciparum Welch, on the daily survival rates and longevity of Anopheles gambiae Giles sensu lato and Anopheles funestus Giles was determined for wild-caught, naturally infected females from western Kenya. Mosquitoes were collected inside houses and held in cages until death, after which they were assayed for P. falciparum circumsporozoite protein by an enzyme-linked immunosorbent assay (ELISA). Survival rates of field populations determined by parity rates were significantly higher than survival rates estimated by regression for Anopheles dying in cages. Overall, An. gambiae s.l. had a significantly higher daily mortality rate (means = 17.5%) than An. funestrus (means = 13.2%). P. falciparum ELISA infection rates, which were higher for An. gambiae s.l. (means = 19.8%; n = 1,221) than for An. funestus (means = 11.9%; n = 1,128), did not increase as a function of time for caged Anopheles. For An. gambiae s.l., there was a significant negative correlation between holding time and P. falciparum ELISA absorbance, suggesting that detectable circumsporozoite protein and perhaps the number of sporozoites may decrease with time in the vector. In western Kenya, an area where Anopheles populations often have extremely high malaria infection rates. Plasmodium infections did not reduce vector survivorship.     

Mosquito (Diptera: Culicidae) dispersal: implications for the epidemiology of Japanese and Murray Valley encephalitis viruses in Australia

One hypothesis to explain the southern extension of Japanese encephalitis (JE) virus from Papua New Guinea into the Torres Strait islands in 1995 and to mainland Australia in 1998 is the dispersal of infected mosquitoes, particularly Culex annulirostris Skuse from which JE virus has been isolated repeatedly. To investigate whether this species disperses in this manner, mosquitoes were identified from 368 aerial kite trap collections operated at 50-310 m (altitude) at inland New South Wales between November 1979 to December 1984. Forty samples (9 during daylight and 31 at night) contained mosquitoes, of which 221 could be identified as Culex australicus Dobrotworsky & Drummond (58.8%), Culex annulirostris (21.3%), Anopheles annulipes Walker s.l. (10.4%), Aedes theobaldi (Taylor) (7.2%), Aedes rubrithorax (Macquart) (1.4%), and Aedes sagax (Skuse) (< 0.9%). During the night, mosquitoes were found in 22.6% of the collections at a mean density (+/- SD) of 91.3 +/- 151.7/10(6) m3 of air sampled. During the day, only 3.8% were positive at a mean density 125.3 +/- 152.1. When examined in relation to possible flying time and wind speed, mean +/- SD dispersal distances by day and night were 23.9 +/- 15.3 km and 152.4 +/- 116.3 km, respectively. These data provide circumstantial evidence that aerial carriage southward approximately 200 km from Papua New Guinea to Cape York peninsula is feasible, but that southern dispersal of Murray Valley encephalitis virus infected mosquitoes from tropical to temperate Australia is unlikely.     

Structure of Anisakis simplex s.l. populations in a region sympatric for A. pegreffii and A. simplex s.s. Absence of reproductive isolation between both species

The aim of this study was to determine the genetic structure of A. simplex s.l. populations. This was done by applying PCR-RFLP and RAPD-PCR to 42 specimens morphologically identified as third stage larvae of A. simplex s.l. Of these larvae, 59.26% of those of Mediterranean origin are identified by PCR-RFLP as A. pegreffii. In Atlantic waters, this percentage dropped to 20.00% while A. simplex s.s. represents 66.67%. However, findings seem to suggest that the taxonomic status of both species should be reconsidered owing to: (i) the high gene flow value that we detected between A. simplex s.s. and A. pegreffii; (ii) the short genetic distance between both members of the A. simplex complex; (iii) the fact that hybrid genotypes represent 16.67% of the parasites analyzed and are represented in all the populations studied, except for the Cantabrian one. When the genetic variation is estimated between the 42 individual A. simplex s.l. specimens studied these can be classified into four groups. The genetic distances and gene flow between three of them are compatible with the existence of three different genetic populations. The fourth is comprised of a single specimen of L3, identified both by PCR-RFLP and by RAPD-PCR as a new genotype.     

A survey of the Anopheles funestus (Diptera: Culicidae) group of mosquitoes from 10 sites in Kenya with special emphasis on population genetic structure based on chromosomal inversion karyotypes

We sampled Anopheles funestus Giles mosquitoes from 10 sites in Kenya to determine (1) the different members of this group present at these sites and (2) the population genetic structure of these species based on chromosomal inversion karyotypes with particular reference to the role of the Eastern arm of the Great Rift Valley and geographical distance as barriers to gene flow. Four members of this species group, namely An. funestus s.s., An. parensis, An. leesoni, and An. rivulorum, were found. An. funestus was found almost exclusively inside human dwellings while An. rivulorum was found almost exclusively outdoors. An. parensis was found both indoors and outdoors in comparable proportions. Earlier attempts to collect this group of mosquitoes from two of the sites yielded no mosquitoes, suggesting that numbers of mosquitoes change during the year, sometime reaching zero or very near zero. Significant levels of differentiation were observed among some An. funestus s.s. populations studied. Results suggest that the Rift Valley acts as a barrier to gene flow for An. funestus, but that levels of differentiation are not associated with the geographical distance between populations. The results are discussed in the light of the fact that inversions may have some adaptive significance to differences in environmental conditions.