Malaria vectors on Buka and Bougainville islands, Papua New Guinea

Anophelines were sampled from 82 locations oil 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 faraunti 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 Lis larvae from only I site on Bougainville. Anopheles farauti s.s. led 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 farauti 2 is unlikely to be involved in malaria transmission on Buka or Bougainville islands.

Malaria vectors in the Brazilian Amazon: Anopheles of the subgenus Nyssorhynchus

Various species of Anopheles (Nyssorhynchus) were studied in the Amazon with the objective of determining their importance as malaria vectors. Of the 33 known Anopheles species occurring in the Amazon, only 9 were found to be infected with Plasmodium. The different species of this subgenus varied both in diversity and density in the collection areas. The populations showed a tendency towards lower density and diversity in virgin forest than in areas modified by human intervention. The principal vector, An. darlingi, is anthropophilic with a continuous activity cycle lasting the entire night but peaking at sunset and sunrise. These species (Nyssorhynchus) are peridomiciliary, entering houses to feed on blood and immediately leaving to settle on nearby vegetation. Anopheles nuneztovari proved to be zoophilic, crepuscular and peridomiciliary. These habits may change depending on a series of external factors, especially those related to human activity. There is a possibility that sibling species exist in the study area and they are being studied with reference to An. darlingi, An. albitarsis and An. nuneztovari. The present results do not suggest the existence of subpopulations of An. darlingi in the Brazilian Amazon.

SEASONAL DISTRIBUTION OF MALARIA VECTORS (DIPTERA: CULICIDAE) IN RURAL LOCALITIES OF PORTO VELHO, RONDÔNIA, BRAZILIAN AMAZON

We conducted a survey of the malaria vectors in an area where a power line had been constructed, between the municipalities of Porto Velho and Rio Branco, in the states of Rondônia and Acre, respectively. The present paper relates to the results of the survey of Anopheles fauna conducted in the state of Rondônia. Mosquito field collections were performed in six villages along the federal highway BR 364 in the municipality of Porto Velho, namely Porto Velho, Jaci Paraná, Mutum Paraná, Vila Abunã, Vista Alegre do Abunã, and Extrema. Mosquito captures were performed at three distinct sites in each locality during the months of February, July, and October 2011 using a protected human-landing catch method; outdoor and indoor captures were conducted simultaneously at each site for six hours. In the six sampled areas, we captured 2,185 mosquitoes belonging to seven Anopheles species. Of these specimens, 95.1% consisted of Anopheles darlingi, 1.8% An. triannulatus l.s., 1.7% An. deaneorum, 0.8% An. konderi l.s., 0.4 An. braziliensis, 0.1% An. albitarsis l.s., and 0.1% An. benarrochi. An. darlingi was the only species found in all localities; the remaining species occurred in sites with specific characteristics.

Fecundity, parity, and adult feeding relationships among Nyssorhynchus malaria vectors from Venezuela

Relative to their pre-engorgement weights, nulliparous Anopheles nuneztovari consumed significantly smaller blood meals than A. marajoara, A. triannulatus or A. aquasalis. When females were deprived of sugar before blood feeding, only one-quarter of A. nuneztovari, but more than two-thirds of A. marajoara, A. triannulatus and A. aquasalis matured eggs. Sugar feeding before blood, or two sucessive blood meals by sugar-deprived females, increased the proportion of nulliparous a. nuneztovari which developed eggs, but not significantly so. Nearly all individuals of nulliparous, sugar-fed A. marajoara, A. triannulatus and A. aquasalis matured eggs after one blood feeding. Among A. nuneztovari, A. marajoara and A. aquasalis that matured some eggs in the laboratory, there were no positive correlations between the number of eggs developed and relative vlood mealsize. However, blood meals larger than the mean size significantly increased the chance that A. nuneztovari would develop some eggs. Mean fecundities of gravid A. nuneztovari and A. marajoara reared in the laboratory were significantly lower than those of the same species captured at human bait in nature. Post-engorgement access to sugar by A. nuneztovari (captured at human bait) did not influence fecundity, but significantly enhanced survivorship and the proporticon of individuals which retained eggs. Release-recapture experiments revealed that relatively small blood meals are typical of A. nuneztovari only during the first gonotrophic cycle. We suggest that multiple blood feeding, seemingly necessary for most A. nuneztovari to develop a first clutch of eggs, may increase the probability of infection with Plasmodium vivax where this mosquito species is a primary vector.

Ecology of malaria vectors in the Americas and future direction

The resurgence of malaria in the Americas has renewed interest in Anopheles biology. Anopheles darlingi, An. albimanus, An. nuneztovai and An aquasalis are reconfirmed as major malaria vectors and other species are playing important roles in regional malaria transmission. Adultbiting activity and larval ecology are discussed in detail. Seasonal abundance and daily biting activity of Anopheles vary considerably among species and geographically for the same species. Anopheles albimanus has the least amount of variation in biting activity over its range and An. darlingi has the greatest. All species studied are more exophilic and exophagic than endophilic and endophagic. Anopheles darling is more antropophilic, endophilic and endophagic than other Anophelines. Larval studies remain more descriptive than comprehensive. Research on Anophelines is becoming more integrated and biologists are using new biochemical techniques and ecological principles to answer critical questions. This "pluralization" will help us understand species complexes, population dynamics and malaria transmission. integrated control programs will require more regional, in-depth ecological studies.

Malaria vectors in the municipality of Serra do Navio, State of Amapá, Amazon Region, Brazil

We conducted a survey to determine the vectors of malaria in six localities of Serra do Navio municipality, State of Amapá, from 1990 to 1991. Malaria infection rates of 29.3%, 6.2% and 20.4% were detected by human blood smears in Colônia Água Branca, Porto Terezinha and Arrependido, respectively. There was no malaria infection detected in Serra do Navio. Fifteen species were identified among 3,053 anopheline mosquitoes collected by human bait and 64.4% were identified as Anopheles albitarsis s.l., 16.7% An. braziliensis, 9.5% An. nuneztovari and 5.8% An. triannulatus. An. darlingi, the main vector of malaria in the Amazon region of Brazil, was scare. Using enzyme-linked immunosorbent assay (ELISA), a total positive rate of 0.8% (23/2876) was found for six species: fifteen An. albitarsis s.l., four An. nuneztovari, and one of each: An. braziliensis, An. triannulatus, An. oswaldoi and An. rangeli. Nine of 23 positive mosquitoes were infected with Plasmodium malariae, eight with P. vivax VK210, three with P. vivax VK247 and three with P. falciparum. Since An. albitarsis s.l. was collected feeding on humans, was present in the highest density and was positive by ELISA for malaria sporozoites, it probably plays an important role in malaria transmission in this area.

The distribution of two major malaria vectors, Anopheles gambiae and Anopheles arabiensis, in Nigeria

The distribution of Anopheles gambiae and An. arabiensis across the ecological zones of Nigeria (arid savanna in the north gradually turns into humid forest in the south) was investigated. Results of the present study were compared to the distributions determined from samples of indoor-resting females reported by an earlier study over 20 years ago. Larvae were sampled in the rainy seasons of 1997 and 1999 from 24 localities, 10 of which were sampled in both years. Specimens were identified by the polymerase chain reaction method. Results showed that species composition changed significantly among the 10 localities in both years (chi2=13.62, P = 0.0002), but this change was significant in only four of the 10 localities. The identity of the prevalent (more abundant) species changed between 1997 and 1999 in only three of 10 localities. An. arabiensis was prevalent in several localities in the southern Guinea savanna, an area where it was virtually absent over 20 years ago. The data suggest that An. arabiensis has extend its range, although differences in sampling technique (larval sampling versus adult collection) can not be ruled out as a possible explanation.

Man biting rate seasonal variation of malaria vectors in Roraima, Brazil

Malaria control has been directed towards regional actions where more detailed knowledge of local determinants of transmission is of primary importance. This is a short report on range distribution and biting indices for Anopheles darlingi and An. albitarsis during the dry and rainy season that follows river level variation in a savanna/alluvial forest malaria system area in the Northern Amazon Basin. Distribution range and adult biting indices were at their highest during the rainy season for both An. darlingi and An. albitarsis. During the rainy season the neighboring alluvial forest was extensively flooded. This coincided with highest rates in malaria transmission with case clustering near the river. As the river receded, anopheline distribution range and density decreased. This decrease in distribution and density corresponded to a malaria decrease in the near area. An exponential regression function was derived to permit estimations of An. darlingi distribution over specified distances. Anopheline spatio-temporal variations lead to uneven malaria case distribution and are of important implications for control strategies.

Characterization of the 3-HKT gene in important malaria vectors in India, viz: Anopheles culicifacies and Anopheles stephensi (Diptera: Culicidae)

The 3-hydroxykynurenine transaminase (3-HKT) gene plays a vital role in the development of malaria parasites by participating in the synthesis of xanthurenic acid, which is involved in the exflagellation of microgametocytes in the midgut of malaria vector species. The 3-HKT enzyme is involved in the tryptophan metabolism of Anophelines. The gene had been studied in the important global malaria vector, Anopheles gambiae. In this report, we have conducted a preliminary investigation to characterize this gene in the two important vector species of malaria in India, Anopheles culicifacies and Anopheles stephensi. The analysis of the genetic structure of this gene in these species revealed high homology with the An. gambiae gene. However, four non-synonymous mutations in An. stephensi and seven in An. culicifacies sequences were noted in the exons 1 and 2 of the gene; the implication of these mutations on enzyme structure remains to be explored.

Is there an efficient trap or collection method for sampling Anopheles darlingi and other malaria vectors that can describe the essential parameters affecting transmission dynamics as effectively as human landing catches? - A Review

Distribution, abundance, feeding behaviour, host preference, parity status and human-biting and infection rates are among the medical entomological parameters evaluated when determining the vector capacity of mosquito species. To evaluate these parameters, mosquitoes must be collected using an appropriate method. Malaria is primarily transmitted by anthropophilic and synanthropic anophelines. Thus, collection methods must result in the identification of the anthropophilic species and efficiently evaluate the parameters involved in malaria transmission dynamics. Consequently, human landing catches would be the most appropriate method if not for their inherent risk. The choice of alternative anopheline collection methods, such as traps, must consider their effectiveness in reproducing the efficiency of human attraction. Collection methods lure mosquitoes by using a mixture of olfactory, visual and thermal cues. Here, we reviewed, classified and compared the efficiency of anopheline collection methods, with an emphasis on Neotropical anthropophilic species, especially Anopheles darlingi, in distinct malaria epidemiological conditions in Brazil.

Aspects of spatial and habitat ecology of multiple Anopheles species (Diptera: Culicidae): malaria vectors in the highlands and foothills of Ecuador

The resurgence of malaria in highland regions of Africa, Oceania and recently in South America underlines the importance of the study of the ecology of highland mosquito vectors of malaria. Since the incidence of malaria is limited by the distribution of its vectors, the purpose of this PhD thesis was to examine aspects of the ecology of Anopheles mosquitoes in the Andes of Ecuador, South America. A historical literature and archival data review (Chapter 2) indicated that Anopheles pseudopunctipennis transmitted malaria in highland valleys of Ecuador prior to 1950, although it was eliminated through habitat removal and the use of chemical insecticides. Other anopheline species were previously limited to low-altitude regions, except in a few unconfirmed cases. A thorough larval collection effort (n=438 attempted collection sites) in all road-accessible parts of Ecuador except for the lowland Amazon basin was undertaken between 2008 - 2010 (Chapter 3). Larvae were identified morphologically and using molecular techniques (mitochondrial COl gene), and distribution maps indicated that all five species collected (Anopheles albimanus, An. pseudopunctipennis, Anopheles punctimacula, Anopheles oswaldoi s.l. and Anopheles eiseni) were more widespread throughout highland regions than previously recorded during the 1940s, with higher maximum altitudes for all except An. pseudopunctipennis (1541 m, 1930 m, 1906 m, 1233 m and 1873 m, respectively). During larval collections, to characterize species-specific larval habitat, a variety of abiotic and biotic habitat parameters were measured and compared between species-present and species-absent sites using chi-square tests and stepwise binary logistic regression analyses (Chapter 4). An. albimanus was significantly associated with permanent pools with sand substrates and An. pseudopunctipennis with gravel and boulder substrates. Both species were significantly associated with floating cyanobacterial mats and warmer temperatures, which may limit their presence in cooler highland regions. Anopheles punctimacula was collected more often than expected from algae-free, shaded pools with higher-than-average calculated dissolved oxygen. Anopheles oswaldoi s.l., the species occurring on the Amazonian side of the Andes, was associated with permanent, anthropogenic habitats such as roadside ditches and ponds. To address the hypothesis that human land use change is responsible for the emergence of multiple highland Anopheles species by creating larval habitat, common land uses in the western Andes were surveyed for standing water and potential larval habitat suitability (Chapter 5). Rivers and road edges provided large amounts of potentially suitable anopheline habitat in the western Andes, while cattle pasture also created potentially suitable habitat in irrigation canals and watering ponds. Other common land uses surveyed (banana farms, sugarcane plantations, mixed tree plantations, and empty lots) were usually established on steep slopes and had very little standing water present. Using distribution and larval habitat data, a GIS-based larval habitat distribution model for the common western species was constructed in ArcGIS v.l 0 (ESRI 2010) using derived data layers from field measurements and other sources (Chapter 6). The additive model predicted 76.4 - 97.9% of the field-observed collection localities of An. albimanus, An. pseudopunctipennis and An. punctimacula, although it could not accurately distinguish between species-absent and speciespresent sites due to its coarse scale. The model predicted distributional expansion and/or shift of one or more anopheline species into the following highland valleys with climate warming: Mira/Chota, Imbabura province, Tumbaco, Pichincha province, Pallatanga and Sibambe, Chimborazo province, and Yungilla, Azuay province. These valleys may serve as targeted sites of future monitoring to prevent highland epidemics of malaria. The human perceptions of malaria and mosquitoes in relation to land management practices were assessed through an interview-based survey (n=262) in both highlands and lowlands, of male and female land owners and managers of five property types (Chapter 7). Although respondents had a strong understanding of where the disease occurs in their own country and of the basic relationship among standing water, mosquitoes and malaria, about half of respondents in potential risk areas denied the current possibility of malaria infection on their own property. As well, about half of respondents with potential anopheline larval habitat did not report its presence, likely due to a highly specific definition of suitable mosquito habitat. Most respondents who are considered at risk of malaria currently use at least one type of mosquito bite prevention, most commonly bed nets. In conclusion, this interdisciplinary thesis examines the occurrence of Anopheles species in the lowland transition area and highlands in Ecuador, from a historic, geographic, ecological and sociological perspective.

Concordant Phylogeographies of 2 Malaria Vectors Attest to Common Spatial and Demographic Histories

The phylogeography of South American lineages is a topic of heated debate. Although a single process is unlikely to describe entire ecosystems, related species, which incur similar habitat limitations, can inform the history for a subsection of assemblages. We compared the phylogeographic patterns of the cytochrome oxidase I marker from Anopheles triannulatus (N = 72) and previous results for A. darlingi (N = 126) in a broad portion of their South American distributions. Both species share similar population subdivisions, with aggregations northeast of the Amazon River, in southern coastal Brazil and 2 regions in central Brazil. The average (ST) between these groups was 0.39 for A. triannulatus. Populations northeast of the Amazon and in southeastern Brazil are generally reciprocally monophyletic to the remaining groups. Based on these initial analyses, we constructed the a priori hypothesis that the Amazon and regions of high declivity pose geographic barriers to dispersal in these taxa. Mantel tests confirmed that these areas block gene flow for more than 1000 km for both species. The efficacy of these impediments was tested using landscape genetics, which could not reject our a priori hypothesis but did reject simpler scenarios. Results form summary statistics and phylogenetics suggest that both lineages originated in central Amazonia (south of the Amazon River) during the late Pleistocene (579 000 years ago) and that they followed the same paths of expansion into their contemporary distributions. These results may have implications for other species sharing similar ecological limitations but probably are not applicable as a general paradigm of Neotropical biogeography.

Morphometrical diagnosis of the malaria vectors Anopheles cruzii, An. homunculus and An. bellator

Background: Anopheles (Kerteszia) cruzii is a primary vector of Plasmodium parasites in Brazil's Atlantic Forest. Adult females of An. cruzii and An. homunculus, which is a secondary malaria vector, are morphologically similar and difficult to distinguish when using external morphological characteristics only. These two species may occur syntopically with An. bellator, which is also a potential vector of Plasmodium species and is morphologically similar to An. cruzii and An. homunculus. Identification of these species based on female specimens is often jeopardised by polymorphisms, overlapping morphological characteristics and damage caused to specimens during collection. Wing geometric morphometrics has been used to distinguish several insect species; however, this economical and powerful tool has not been applied to Kerteszia species. Our objective was to assess wing geometry to distinguish An. cruzii, An. homunculus and An. bellator. Methods: Specimens were collected in an area in the Serra do Mar hotspot biodiversity corridor of the Atlantic Forest biome (Cananeia municipality, State of Sao Paulo, Brazil). The right wings of females of An. cruzii (n= 40), An. homunculus (n= 50) and An. bellator (n= 27) were photographed. For each individual, 18 wing landmarks were subjected to standard geometric morphometrics. Discriminant analysis of Procrustean coordinates was performed to quantify wing shape variation. Results: Individuals clustered into three distinct groups according to species with a slight overlap between representatives of An. cruzii and An. homunculus. The Mahalanobis distance between An. cruzii and An. homunculus was consistently lower (3.50) than that between An. cruzii and An. bellator (4.58) or An. homunculus and An. bellator (4.32). Pairwise cross-validated reclassification showed that geometric morphometrics is an effective analytical method to distinguish between An. bellator, An. cruzii and An. homunculus with a reliability rate varying between 78-88%. Shape analysis revealed that the wings of An. homunculus are narrower than those of An. cruzii and that An. bellator is different from both of the congeneric species. Conclusion: It is possible to distinguish among the vectors An. cruzii, An. homunculus and An. bellator based on female wing characteristics.

Molecular biological approaches to the study of vectors in relation to malaria control

To a large extent, control of malaria vectors relies on the elimination of breeding sites and the application of chemical agents. There are increasing problems associated with the use of synthetic insecticides for vector control, including the evolution of resistance, the high cost of developing and registering new insecticides and an awareness of pollution from insecticide residues. These factors have stimulated interest in the application of molecular biology to the study of mosquito vectors of malaria; focussing primarily on two aspects. First, the improvement of existing control measures through the development of simplified DNA probe systems suitable for identification of vectors of malaria. The development of synthetic, non-radioactive DNA probes suitable for identification of species in the Anopheles gambiae complex is described with the aim of defining a simplified methodology wich is suitable for entomologist in the field. The second aspect to be considered is the development of completely novel strategies through the development of completely novel strategies through the genetic manipulation of insect vectors of malaria in order to alter their ability to transmit the disease. The major requirements for producing transgenic mosquitoes are outlined together with the progress wich has been made to date and discussed in relation to the prospects which this type of approach has for the future control of malaria.