Morphology of the larvae, male genitalia and DNA sequences of Anopheles (Kerteszia) pholidotus (Diptera: Culicidae) from Colombia.

Since 1984, Anopheles (Kerteszia) lepidotus has been considered a mosquito species that is involved in the transmission of malaria in Colombia, after having been incriminated as such with epidemiological evidence from a malaria outbreak in Cunday-Villarrica, Tolima. Subsequent morphological analyses of females captured in the same place and at the time of the outbreak showed that the species responsible for the transmission was not An. lepidotus, but rather Anopheles pholidotus. However, the associated morphological stages and DNA sequences of An. pholidotus from the foci of Cunday-Villarrica had not been analysed. Using samples that were caught recently from the outbreak region, the purpose of this study was to provide updated and additional information by analysing the morphology of female mosquitoes, the genitalia of male mosquitoes and fourth instar larvae of An. pholidotus, which was confirmed with DNA sequences of cytochrome oxidase I and rDNA internal transcribed spacer. A total of 1,596 adult females were collected in addition to 37 larval collections in bromeliads. Furthermore, 141 adult females, which were captured from the same area in the years 1981-1982, were analysed morphologically. Ninety-five DNA sequences were analysed for this study. Morphological and molecular analyses showed that the species present in this region corresponds to An. pholidotus. Given the absence of An. lepidotus, even in recent years, we consider that the species of mosquitoes that was previously incriminated as the malaria vector during the outbreak was indeed An. pholidotus, thus ending the controversy.

Mosquitoes of eastern Amazonian Ecuador: biodiversity, bionomics and barcodes.

Two snapshot surveys to establish the diversity and ecological preferences of mosquitoes (Diptera: Culicidae) in the terra firme primary rain forest surrounding the Tiputini Biodiversity Station in the UNESCO Yasuní Biosphere Reserve of eastern Amazonian Ecuador were carried out in November 1998 and May 1999. The mosquito fauna of this region is poorly known; the focus of this study was to obtain high quality link-reared specimens that could be used to unequivocally confirm species level diversity through integrated systematic study of all life stages and DNA sequences. A total of 2,284 specimens were preserved; 1,671 specimens were link-reared with associated immature exuviae, all but 108 of which are slide mounted. This study identified 68 unique taxa belonging to 17 genera and 27 subgenera. Of these, 12 are new to science and 37 comprise new country records. DNA barcodes [658-bp of the mtDNA cytochrome c oxidase (COI) I gene] are presented for 58 individuals representing 20 species and nine genera. DNA barcoding proved useful in uncovering and confirming new species and we advocate an integrated systematics approach to biodiversity studies in future. Associated bionomics of all species collected are discussed. An updated systematic checklist of the mosquitoes of Ecuador (n=179) is presented for the first time in 60 years.

The insupportable validity of mosquito subspecies (Diptera: Culicidae) and their exclusion from culicid classification.

Beginning about 80 years ago, the recognition of morphological varieties of mosquitoes was gradually replaced by the recognition of subspecies. As an examination of revisionary and detailed taxonomic studies of mosquitoes clearly shows, subspecies are untenable concepts which have been synonymized with nominotypical forms or recognized as distinct species. Thus, from our perspective, subspecies is not a functional or practical taxonomic rank. Consequently, in this study we critically assessed the taxonomic status of the 120 nominal taxa distinguished as subspecies before now to determine whether they should be recognized as separate species or synonymous names. As a result, 96 subspecies are formally elevated to specific rank, 22 are relegated to synonymy with nominotypical forms, one is considered a nomen dubium, one a species inquirenda and the names of four nominal species regarded as synonyms are revalidated. The subspecies and their new status are listed in a conspectus. The revalidated species include Anopheles argentinus (Brèthes, 1912), from synonymy with An. pseudopunctipennis Theobald, 1901c; An. peruvianus Tamayo, 1907, from synonymy with An. pseudopunctipennis as nomen dubium; Culex major Edwards, 1935, from synonymy with Cx. annulioris consimilis Newstead, 1907; and Trichoprosopon trichorryes (Dyar & Knab, 1907), from synonymy with Tr. compressum Lutz, 1905. Additionally, the type locality of Anopheles sergentii Theobald, 1907 is restricted to El Outaya, Biskra Province, Algeria. A complete list of species to be retained, added to or removed from the Encyclopedia of Life, with a few corrections, is provided.

High Levels of Diversity in Anopheles Subgenus Kerteszia Revealed by Species Delimitation Analyses.

The Anopheles subgenus Kerteszia is a poorly understood group of mosquitoes that includes several species of medical importance. Although there are currently twelve recognized species in the subgenus, previous studies have shown that this is likely to be an underestimate of species diversity. Here, we undertake a baseline study of species delimitation using the barcode region of the mtDNA COI gene to explore species diversity among a geographically and taxonomically diverse range of Kerteszia specimens. Beginning with 10 of 12 morphologically identified Kerteszia species spanning eight countries, species delimitation analyses indicated a high degree of cryptic diversity. Overall, our analyses found support for at least 28 species clusters within the subgenus Kerteszia. The most diverse taxon was Anopheles neivai, a known malaria vector, with eight species clusters. Five other species taxa showed strong signatures of species complex structure, among them Anopheles bellator, which is also considered a malaria vector. There was some evidence for species structure within An. homunculus, although the results were equivocal across delimitation analyses. The current study, therefore, suggests that species diversity within the subgenus Kerteszia has been grossly underestimated. Further work will be required to build on this molecular characterization of species diversity and will rely on genomic level approaches and additional morphological data to test these species hypotheses.

Phylogenomics reveals the history of host use in mosquitoes.

Mosquitoes have profoundly affected human history and continue to threaten human health through the transmission of a diverse array of pathogens. The phylogeny of mosquitoes has remained poorly characterized due to difficulty in taxonomic sampling and limited availability of genomic data beyond the most important vector species. Here, we used phylogenomic analysis of 709 single copy ortholog groups from 256 mosquito species to produce a strongly supported phylogeny that resolves the position of the major disease vector species and the major mosquito lineages. Our analyses support an origin of mosquitoes in the early Triassic (217 MYA [highest posterior density region: 188-250 MYA]), considerably older than previous estimates. Moreover, we utilize an extensive database of host associations for mosquitoes to show that mosquitoes have shifted to feeding upon the blood of mammals numerous times, and that mosquito diversification and host-use patterns within major lineages appear to coincide in earth history both with major continental drift events and with the diversification of vertebrate classes.

Mosquito bisection as a variable in estimates of PCR-derived malaria sporozoite rates.

BACKGROUND: Highly sensitive polymerase chain reaction (PCR) methods offer an alternative to the light microscopy examination of mosquito salivary glands for the determination of malaria sporozoite rates in wild caught female Anopheles. Removal of mosquito abdomens is assumed to eliminate false positives caused by malaria oocyst DNA in the midgut. This assumption has not been tested with current gold standard PCR assays, and for the variety of conditions that specimens could encounter in the laboratory and field. METHODS: Laboratory Anopheles stephensi were used that had been infected with Plasmodium falciparum 6-7 days and 14 days post infection (p.i.), when oocysts only and oocysts + sporozoites, respectively, are developed. Mosquitoes were killed and immediately frozen, air dried before being frozen, or stored under humid conditions overnight before being frozen, to simulate a range of conditions in the field. Additionally, abdomens were removed anterior to, at, or posterior to the junction of the abdomen and thorax, and both portions were processed using a standard nested PCR of the small sub-unit nuclear ribosomal genes (ssrDNA) with products visualized on agarose gels. RESULTS: Overall, 4.1 % (4/97) of head + thorax samples that were 6-7 days p.i. gave apparent false positives for sporozoites, compared to 9.3 % (9/97) that were positive for abdomens. No positives (0/52) were obtained when similar specimens were bisected anterior to the junction of the thorax and abdomen, compared to 21.2 % (11/52) that were positive for posterior portions. Multiple bands were noted for positives from the 'Frozen' treatment and the rate of false negatives due to DNA degradation appears higher under the 'Humid' treatment. Reproducibility of results for the 'Frozen' treatment was 90 %. CONCLUSIONS: Despite the importance of specimen condition and the bisection step in determining sporozoite rates, little attention has been paid to them in the literature. Recommendations from this study are that: 1) care needs to be taken to reduce DNA degradation in the field; 2) mosquito abdomens be separated anterior to the junction of the thorax and abdomen; and 3) DNA sequencing of a subsample of positive results should be undertaken if possible.

Analysis of the evolutionary forces shaping mitochondrial genomes of a Neotropical malaria vector complex.

Many vectors of human malaria belong to complexes of morphologically indistinguishable cryptic species. Here we report the analysis of the newly sequenced complete mitochondrial DNA molecules from six recognized or putative species of one such group, the Neotropical Anopheles albitarsis complex. The molecular evolution of these genomes had been driven by purifying selection, particularly strongly acting on the RNA genes. Directional mutation pressure associated with the strand-asynchronous asymmetric mtDNA replication mechanism may have shaped a pronounced DNA strand asymmetry in the nucleotide composition in these and other Anopheles species. The distribution of sequence polymorphism, coupled with the conflicting phylogenetic trees inferred from the mitochondrial DNA and from the published white gene fragment sequences, indicates that the evolution of the complex may have involved ancient mtDNA introgression. Six protein coding genes (nad5, nad4, cox3, atp6, cox1 and nad2) have high levels of sequence divergence and are likely informative for population genetics studies. Finally, the extent of the mitochondrial DNA variation within the complex supports the notion that the complex consists of a larger number of species than until recently believed.

The AFHSC-Division of GEIS Operations Predictive Surveillance Program: a multidisciplinary approach for the early detection and response to disease outbreaks.

The Armed Forces Health Surveillance Center, Division of Global Emerging Infections Surveillance and Response System Operations (AFHSC-GEIS) initiated a coordinated, multidisciplinary program to link data sets and information derived from eco-climatic remote sensing activities, ecologic niche modeling, arthropod vector, animal disease-host/reservoir, and human disease surveillance for febrile illnesses, into a predictive surveillance program that generates advisories and alerts on emerging infectious disease outbreaks. The program's ultimate goal is pro-active public health practice through pre-event preparedness, prevention and control, and response decision-making and prioritization. This multidisciplinary program is rooted in over 10 years experience in predictive surveillance for Rift Valley fever outbreaks in Eastern Africa. The AFHSC-GEIS Rift Valley fever project is based on the identification and use of disease-emergence critical detection points as reliable signals for increased outbreak risk. The AFHSC-GEIS predictive surveillance program has formalized the Rift Valley fever project into a structured template for extending predictive surveillance capability to other Department of Defense (DoD)-priority vector- and water-borne, and zoonotic diseases and geographic areas. These include leishmaniasis, malaria, and Crimea-Congo and other viral hemorrhagic fevers in Central Asia and Africa, dengue fever in Asia and the Americas, Japanese encephalitis (JE) and chikungunya fever in Asia, and rickettsial and other tick-borne infections in the U.S., Africa and Asia.

Malaria vector species in Colombia: a review.

Here we present a comprehensive review of the literature on the vectorial importance of the major Anopheles malaria vectors in Colombia. We provide basic information on the geographical distribution, altitudinal range, immature habitats, adult behaviour, feeding preferences and anthropophily, endophily and infectivity rates. We additionally review information on the life cycle, longevity and population fluctuation of Colombian Anopheles species. Emphasis was placed on the primary vectors that have been epidemiologically incriminated in malaria transmission: Anopheles darlingi, Anopheles albimanus and Anopheles nuneztovari. The role of a selection of local, regional or secondary vectors (e.g., Anopheles pseudopunctipennis and Anopheles neivai) is also discussed. We highlight the importance of combining biological, morphological and molecular data for the correct taxonomical determination of a given species, particularly for members of the species complexes. We likewise emphasise the importance of studying the bionomics of primary and secondary vectors along with an examination of the local conditions affecting the transmission of malaria. The presence and spread of the major vectors and the emergence of secondary species capable of transmitting human Plasmodia are of great interest. When selecting control measures, the anopheline diversity in the region must be considered. Variation in macroclimate conditions over a species' geographical range must be well understood and targeted to plan effective control measures based on the population dynamics of the local Anopheles species.

Mosquito species composition and Plasmodium vivax infection rates on Baengnyeong-do (island), Republic of Korea.

Vivax malaria is a significant military and civilian health threat in the north of the Republic of Korea (ROK). The island of Baengnyeong-do is the westernmost point of the ROK and is located close to the southwestern coast of the Democratic People's Republic of Korea (DPRK). Mosquitoes were collected using a black light trap on Baengnyeong-do, and Anopheles spp. were assayed by PCR, to identify the species, and screened for sporozoites of Plasmodium vivax. Of a subsample of 257 mosquitoes, Anopheles lesteri was the most frequently collected (49.8%), followed by Anopheles sinensis (22.6%), Anopheles pullus (18.7%), Anopheles kleini (7.8%), and Anopheles belenrae (1.2%). The overall sporozoite rate was 3.1%, with the highest rates observed in An. kleini (15.0%), An. sinensis (5.2%), and An. lesteri (1.6%). No sporozoite positive An. pullus or An. belenrae were observed. The results extend our knowledge of the distribution and potential role in malaria transmission of An. kleini, An. lesteri, and An. sinensis, for an area previously considered to be at a low risk for contracting vivax malaria.

Molecular species delimitation reveals high diversity in the mosquito Anopheles tessellatus Theobald, 1901 (Diptera, Culicidae) across its range.

Anopheles tessellatus is a potentially important vector found across South, East and Southeast Asia. While it was formerly considered a formidable vector of human Plasmodium and filarial parasites in the Maldives, and of lesser importance as a vector of human Plasmodium in Sri Lanka and parts of Indonesia, it is currently of little or unknown health importance in many other parts of its range. This study describes the genetic diversity and evolutionary relationships among An. tessellatus populations in nine Asian countries at the COI gene using maximum-likelihood and Bayesian phylogenetic inference tree and cluster-based species delimitation approaches. These analyses reveal exceptional levels of genetic diversity in An. tessellatus populations across its known range, and identify up to six putative species in the newly determined Tessellatus Complex. The existence of such cryptic diversity has potentially important consequences for vector management and disease control. Differences in the ecologies and life histories among these species may have considerable impact on vectorial capacity and may go some way towards explaining why An. tessellatus s.l. has such varying degrees of public health importance across its range.

Phylogenetic Network of Mitochondrial COI Gene Sequences Distinguishes 10 Taxa Within the Neotropical Albitarsis Group (Diptera: Culicidae), Confirming the Separate Species Status of Anopheles albitarsis H (Diptera: Culicidae) and Revealing a Novel Lineage, Anopheles albitarsis J.

The Neotropical Albitarsis Group is a complex assemblage of essentially isomorphic species which currently comprises eight recognized species-five formally described (Anopheles albitarsis Lynch-Arribalzaga, An. deaneorum Rosa-Freitas, An. janconnae Wilkerson and Sallum, An. marajoara Galvao and Damasceno, An. oryzalimnetes Wilkerson and Motoki) and three molecularly assigned (An. albitarsis F, G & I)-and one mitochondrial lineage (An. albitarsis H). To further explore species recognition within this important group, 658 base pairs of the mitochondrial DNA cytochrome oxidase subunit I (COI) were analyzed from 988 specimens from South America. We conducted statistical parsimony network analysis, generated estimates of haplotype, nucleotide, genetic differentiation, divergence time, and tested the effect of isolation by distance (IBD). Ten clusters were identified, which confirmed the validity of the eight previously determined species, and confirmed the specific status of the previous mitochondrial lineage An. albitarsis H. High levels of diversity were highlighted in two samples from Pará (= An. albitarsis J), which needs further exploration through additional sampling, but which may indicate another cryptic species. The highest intra-specific nucleotide diversity was observed in An. deaneorum, and the lowest in An. marajoara. Significant correlation between genetic and geographical distance was observed only in An. oryzalimnetes and An. albitarsis F. Divergence time within the Albitarsis Group was estimated at 0.58-2.25 Mya, during the Pleistocene. The COI barcode region was considered an effective marker for species recognition within the Albitarsis Group and a network approach was an analytical method to discriminate among species of this group.

Phylogenetic analysis of the Neotropical Albitarsis Complex based on mitogenome data.

BACKGROUND: Some of the most important malaria vectors in South America belong to the Albitarsis Complex (Culicidae; Anophelinae; Anopheles). Understanding the origin, nature, and geographical distribution of species diversity in this important complex has important implications for vector incrimination, control, and management, and for modelling future responses to climate change, deforestation, and human population expansion. This study attempts to further explore species diversity and evolutionary history in the Albitarsis Complex by undertaking a characterization and phylogenetic analysis of the mitogenome of all 10 putative taxa in the Albitarsis Complex. METHODS: Mitogenome assembly and annotation allowed for feature comparison among Albitarsis Complex and Anopheles species. Selection analysis was conducted across all 13 protein-coding genes. Maximum likelihood and Bayesian inference methods were used to construct gene and species trees, respectively. Bayesian methods were also used to jointly estimate species delimitation and species trees. RESULTS: Gene composition and order were conserved across species within the complex. Unique signatures of positive selection were detected in two species-Anopheles janconnae and An. albitarsis G-which may have played a role in the recent and rapid diversification of the complex. The COI gene phylogeny does not fully recover the mitogenome phylogeny, and a multispecies coalescent-based phylogeny shows that considerable uncertainty exists through much of the mitogenome species tree. The origin of divergence in the complex dates to the Pliocene/Pleistocene boundary, and divergence within the distinct northern South American clade is estimated at approximately 1 million years ago. Neither the phylogenetic trees nor the delimitation approach rejected the 10-species hypothesis, although the analyses could not exclude the possibility that four putative species with scant a priori support (An. albitarsis G, An. albitarsis H, An. albitarsis I, and An. albitarsis J), represent population-level, rather than species-level, splits. CONCLUSION: The lack of resolution in much of the species tree and the limitations of the delimitation analysis warrant future studies on the complex using genome-wide data and the inclusion of additional specimens, particularly from two putative species, An. albitarsis I and An. albitarsis J.

Lineage divergence detected in the malaria vector Anopheles marajoara (Diptera: Culicidae) in Amazonian Brazil.

BACKGROUND: Cryptic species complexes are common among anophelines. Previous phylogenetic analysis based on the complete mtDNA COI gene sequences detected paraphyly in the Neotropical malaria vector Anopheles marajoara. The "Folmer region" detects a single taxon using a 3% divergence threshold. METHODS: To test the paraphyletic hypothesis and examine the utility of the Folmer region, genealogical trees based on a concatenated (white + 3' COI sequences) dataset and pairwise differentiation of COI fragments were examined. The population structure and demographic history were based on partial COI sequences for 294 individuals from 14 localities in Amazonian Brazil. 109 individuals from 12 localities were sequenced for the nDNA white gene, and 57 individuals from 11 localities were sequenced for the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2). RESULTS: Distinct A. marajoara lineages were detected by combined genealogical analysis and were also supported among COI haplotypes using a median joining network and AMOVA, with time since divergence during the Pleistocene (<100,000 ya). COI sequences at the 3' end were more variable, demonstrating significant pairwise differentiation (3.82%) compared to the more moderate 2.92% detected by the Folmer region. Lineage 1 was present in all localities, whereas lineage 2 was restricted mainly to the west. Mismatch distributions for both lineages were bimodal, likely due to multiple colonization events and spatial expansion (~798-81,045 ya). There appears to be gene flow within, not between lineages, and a partial barrier was detected near Rio Jari in Amapá state, separating western and eastern populations. In contrast, both nDNA data sets (white gene sequences with or without the retention of the 4th intron, and ITS2 sequences and length) detected a single A. marajoara lineage. CONCLUSIONS: Strong support for combined data with significant differentiation detected in the COI and absent in the nDNA suggest that the divergence is recent, and detectable only by the faster evolving mtDNA. A within subgenus threshold of >2% may be more appropriate among sister taxa in cryptic anopheline complexes than the standard 3%. Differences in demographic history and climatic changes may have contributed to mtDNA lineage divergence in A. marajoara.

Species composition, larval habitats, seasonal occurrence and distribution of potential malaria vectors and associated species of Anopheles (Diptera: Culicidae) from the Republic of Korea.

BACKGROUND: Larval mosquito habitats of potential malaria vectors and related species of Anopheles from three provinces (Gyeonggi, Gyeongsangbuk, Chungcheongbuk Provinces) of the Republic of Korea were surveyed in 2007. This study aimed to determine the species composition, seasonal occurrence and distributions of Anopheles mosquitoes. Satellite derived normalized difference vegetation index data (NDVI) was also used to study the seasonal abundance patterns of Anopheles mosquitoes. METHODS: Mosquito larvae from various habitats were collected using a standard larval dipper or a white plastic larval tray, placed in plastic bags, and were preserved in 100% ethyl alcohol for species identification by PCR and DNA sequencing. The habitats in the monthly larval surveys included artificial containers, ground depressions, irrigation ditches, drainage ditches, ground pools, ponds, rice paddies, stream margins, inlets and pools, swamps, and uncultivated fields. All field-collected specimens were identified to species, and relationships among habitats and locations based on species composition were determined using cluster statistical analysis. RESULTS: In about 10,000 specimens collected, eight species of Anopheles belonging to three groups were identified: Hyrcanus Group - Anopheles sinensis, Anopheles kleini, Anopheles belenrae, Anopheles pullus, Anopheles lesteri, Anopheles sineroides; Barbirostris Group - Anopheles koreicus; and Lindesayi Group - Anopheles lindesayi japonicus. Only An. sinensis was collected from all habitats groups, while An. kleini, An. pullus and An. sineroides were sampled from all, except artificial containers. The highest number of Anopheles larvae was found in the rice paddies (34.8%), followed by irrigation ditches (23.4%), ponds (17.0%), and stream margins, inlets and pools (12.0%). Anopheles sinensis was the dominant species, followed by An. kleini, An. pullus and An. sineroides. The monthly abundance data of the Anopheles species from three locations (Munsan, Jinbo and Hayang) were compared against NDVI and NDVI anomalies. CONCLUSION: The species composition of Anopheles larvae varied in different habitats at various locations. Anopheles populations fluctuated with the seasonal dynamics of vegetation for 2007. Multi-year data of mosquito collections are required to provide a better characterization of the abundance of these insects from year to year, which can potentially provide predictive capability of their population density based on remotely sensed ecological measurements.

Molecular phylogenetics of Aedes japonicus, a disease vector that recently invaded Western Europe, North America, and the Hawaiian islands.

We used two mitochondrial loci (nicotinamide adenine dinucleotide dehydrogenase subunit 4 and cytochrome oxidase II) and a nuclear locus (28S-D2 spacer) for a total of 1337 bp to evaluate the relationships among the four subspecies of Aedes (Finlaya) japonicus Theobald. Ae. j. japonicus was recently introduced into the United States and has been expanding rapidly. We also included in our analysis a morphologically very closely related species, Aedes (Finlaya) koreicus Edwards, as well as three more distantly related species: Aedes (Finlaya) togoi Theobald, Aedes (Finlaya) hatorii Yamada, and Aedes (Aedimorphus) vexans Meigen. We found that the four subspecies in the Ae. japonicus complex are genetically quite distinct but seem to form a monophyletic group that surprisingly also includes Ae. koreicus, suggesting the need for a taxonomic reconsideration of the group. We also found that the two southern subspecies are more closely related to each other than to any of the remaining subspecies or to Ae. koreicus and may indicate an ancient north-south split of the lineage. Considering the overlap between Ae. j. japonicus and Ae. koreicus, but the stronger association between Ae. koreicus and humans, we are surprised it also has not expanded from its original range. As a proactive reaction to this possibility, we designed and tested a DNA-based rapid assay to differentiate Ae. koreicus from some of the species with which it may be confused in the United States. These Aedes are putative vectors of several important viral encephalitides.

MosquitoMap and the Mal-area calculator: new web tools to relate mosquito species distribution with vector borne disease.

BACKGROUND: Mosquitoes are important vectors of diseases but, in spite of various mosquito faunistic surveys globally, there is a need for a spatial online database of mosquito collection data and distribution summaries. Such a resource could provide entomologists with the results of previous mosquito surveys, and vector disease control workers, preventative medicine practitioners, and health planners with information relating mosquito distribution to vector-borne disease risk. RESULTS: A web application called MosquitoMap was constructed comprising mosquito collection point data stored in an ArcGIS 9.3 Server/SQL geodatabase that includes administrative area and vector species x country lookup tables. In addition to the layer containing mosquito collection points, other map layers were made available including environmental, and vector and pathogen/disease distribution layers. An application within MosquitoMap called the Mal-area calculator (MAC) was constructed to quantify the area of overlap, for any area of interest, of vector, human, and disease distribution models. Data standards for mosquito records were developed for MosquitoMap. CONCLUSION: MosquitoMap is a public domain web resource that maps and compares georeferenced mosquito collection points to other spatial information, in a geographical information system setting. The MAC quantifies the Mal-area, i.e. the area where it is theoretically possible for vector-borne disease transmission to occur, thus providing a useful decision tool where other disease information is limited. The Mal-area approach emphasizes the independent but cumulative contribution to disease risk of the vector species predicted present. MosquitoMap adds value to, and makes accessible, the results of past collecting efforts, as well as providing a template for other arthropod spatial databases.

Confirmation of Anopheles (Anopheles) calderoni Wilkerson, 1991 (Diptera: Culicidae) in Colombia and Ecuador through molecular and morphological correlation with topotypic material.

The morphologically similar taxa Anopheles calderoni, Anopheles punctimacula, Anopheles malefactor and Anopheles guarao are commonly misidentified. Isofamilies collected in Valle de Cauca, Colombia, showed morphological characters most similar to An. calderoni, a species which has never previously been reported in Colombia. Although discontinuity of the postsubcostal pale spots on the costa (C) and first radial (R1) wing veins is purportedly diagnostic for An. calderoni, the degree of overlap of the distal postsubcostal spot on C and R1 were variable in Colombian specimens (0.003-0.024). In addition, in 98.2% of larvae, seta 1-X was located off the saddle and seta 3-C had 4-7 branches in 86.7% of specimens examined. Correlation of DNA sequences of the second internal transcribed spacer and mtDNA cytochrome c oxidase subunit I gene (COI) barcodes (658 bp of the COI gene) generated from Colombian progeny material and wild-caught mosquitoes from Ecuador with those from the Peruvian type series of An. calderoni confirmed new country records. DNA barcodes generated for the closely related taxa, An. malefactor and An. punctimacula are also presented for the first time. Examination of museum specimens at the University of the Valle, Colombia, revealed the presence of An. calderoni in inland localities across Colombia and at elevations up to 1113 m.

Molecular comparison of topotypic specimens confirms Anopheles (Nyssorhynchus) dunhami Causey (Diptera: Culicidae) in the Colombian Amazon.

The presence of Anopheles (Nyssorhynchus) dunhami Causey in Colombia (Department of Amazonas) is confirmed for the first time through direct comparison of mtDNA cytochrome c oxidase I (COI) barcodes and nuclear rDNA second internal transcribed spacer (ITS2) sequences with topotypic specimens of An. dunhami from Tefé, Brazil. An. dunhami was identified through retrospective correlation of DNA sequences following misidentification as Anopheles nuneztovari s.l. using available morphological keys for Colombian mosquitoes. That An. dunhami occurs in Colombia and also possibly throughout the Amazon Basin, is of importance to vector control programs, as this non-vector species is morphologically similar to known malaria vectors including An. nuneztovari, Anopheles oswaldoi and Anopheles trinkae. Species identification of An. dunhami and differentiation from these closely related species are highly robust using either DNA ITS2 sequences or COI DNA barcode. DNA methods are advocated for future differentiation of these often sympatric taxa in South America.

Validation of ecological niche models for potential malaria vectors in the Republic of Korea.

Data on molecularly identified adult and larval mosquitoes collected from 104 sites from the Republic of Korea (ROK) in 2007 were used to test the predictive ability of recently reported ecological niche models (ENMs) for 8 potential malaria vectors. The ENMs, based on the program Maxent and the least presence threshold criterion, predicted 100% of new collection locations for Anopheles sinensis, An. belenrae, An. pullus, and An. sineroides; 96% of locations for An. kleini; and 83% for An. lesteri, but were relatively unsuccessful for the infrequently collected non-Hyrcanus group species An. koreicus and An. lindesayi japonicas. The ENMs produced with the use of Maxent had fewer omission errors than those using the Genetic Algorithm for Rule-Set Prediction program. The results emphasize the importance of independent test data for validation and improvement of ENMs, and lend support for the further development of ENMs for predicting the distribution of malaria vectors in the ROK.