Anopheles (Anopheles) forattinii: a new species in Series Arribalzagia (Diptera: Culicidae).

Anopheles (Anopheles) forattinii new species, a member of the Series Arribalzagia, is described with illustrations of the larval and pupal stages, and male and female genitalia. It is contrasted with 2 similar species, An. (Anopheles) costai Fonseca & Ramos and An. (Anopheles) mediopunctatus (Lutz). This species, and An. costai, occur over much of South America where both have been misidentified as An. mediopunctatus, a species presently only known from southeastern Brazil.     

Species composition and distribution of adult Anopheles (Diptera: Culicidae) in Panama

Anopheles (Diptera: Culicidae) species composition and distribution were studied using human landing catch data over a 35-yr period in Panama. Mosquitoes were collected from 77 sites during 228 field trips carried out by members of the National Malaria Eradication Service. Fourteen Anopheles species were identified. The highest average human biting rates were recorded from Anopheles (Nyssorhynchus) albimanus (Wiedemann) (9.8 bites/person/night) and Anopheles (Anopheles) punctimacula (Dyar and Knab) (6.2 bites/person/night). These two species were also the most common, present in 99.1 and 74.9%, respectively, of the sites. Anopheles (Nyssorhynchus) aquasalis (Curry) was encountered mostly in the indigenous Kuna Yala Comarca along the eastern Atlantic coast, where malaria case history and average human biting rate (9.3 bites/person/night) suggest a local role in malaria transmission. An. albimanus, An. punctimacula, and Anopheles (Anopheles) vestitipennis (Dyar and Knab) were more abundant during the rainy season (May-December), whereas An. aquasalis was more abundant in the dry season (January-April). Other vector species collected in this study were Anopheles (Kerteszia) neivai (Howard, Dyar, and Knab) and Anopheles (Anopheles) pseudopunctipennis s.l. (Theobald). High diversity of Anopheles species and six confirmed malaria vectors in endemic areas of Panama emphasize the need for more detailed studies to better understand malaria transmission dynamics.     

Analysis of reproductive isolation between sibling species anopheles Albitarsis sensu stricto and Anopheles deaneorum, two malaria vectors belonging to the Albitarsis complex (Diptera: Culicidae)

Complexes of sibling species are common among mosquitoes, and their existence within vector species can have important epidemiological consequences. Anopheles albitarsis sensu stricto and Anopheles deaneorum Rosa-Freitas are two putative vectors of malaria parasites belonging to the Albitarsis species complex (Diptera: Culicidae). Using an induced mating technique, we studied the reproductive isolation between these two closely related species and their reciprocal hybrids. Evidence for hybrid male sterility consistent with Haldane's rule was found. The results indicate that male hybrids show very low insemination rates, probably due to abnormalities in their reproductive organs. In addition, the data show that hybrid males carrying an X chromosome derived from An. deaneorum perform significantly worse than hybrid males carrying an An. albitarsis s.s. X chromosome.     

Natural Plasmodium infections in Anopheles darlingi and Anopheles benarrochi (Diptera: Culicidae) from eastern Peru

Malaria, both Plasmodium falciparum (Welch) and Plasmodium vivax (Grassi & Feletti), has reemerged as a significant public health disease issue in Peru, especially in forested areas in the eastern part of the country. The spread of Anopheles darlingi Root, the principal South American malaria vector, into new areas of Peru is thought to be a factor in this resurgence. However, epidemiological evidence suggests that in malaria endemic areas of eastern Peru where An. darlingi does not occur, other species are involved in malaria transmission. The objective of this study was to analyze Anopheles species collected from 11 provinces within four departments in eastern Peru during 2001 and 2002 for infections with P. falciparum and P. vivax. More than 84,000 Anopheles mosquitoes representing 13 species were tested by enzyme-linked immunosorbent assay for the presence of Plasmodium circumsporozoite (CS) proteins. Of these, only An. darlingi and Anopheles benarrochi Gabaldón, Cova García & López were found positive. In total, 14 (0.98%) of 1,432 pools of An. darlingi were positive for Plasmodium species; specifically 10 (0.70%) pools were positive for P. falciparum, two (0.14%) were positive for P. vivax VK210, and two (0.14%) were positive for P. vivax VK247 proteins. Nine (0.14%) of 6,323 pools of An. benarrochi were positive for Plasmodium; five (0.08%) of 6,323 pools were positive for P. falciparum, two (0.03%) were positive for P. vivax VK247, one (0.02%) was positive for mixed P. vivax VK210/VK247 infections, and one (0.02%) was positive for mixed P. falciparum and P. vivax VK210 CS-proteins. Although infection rates in An. benarrochi were significantly lower (0.14%) than rates found for An. darlingi (0.98%), our data suggest that An. benarrochi may play a role in transmitting and maintaining Plasmodium species in various malaria endemic areas of eastern Peru.     

Occurrence of Anopheles hermsi (Diptera: Culicidae) in Arizona and Colorado

Historically, malaria was a significant cause of morbidity and mortality throughout the western United States, and Anopheles freeborni Aitken was thought to be the vector west of the Continental Divide. In 1989, Anopheles hermsi Barr & Guptavanij was described and subsequently found to be an effective laboratory vector of Plasmodium. The adults of these two species are morphologically indistinguishable, and therefore polymerase chain reaction was used to analyze the DNA from 48 mosquitoes collected in Arizona and Colorado (identified morphologically as An. freeborni). All specimens were identified as An. hermsi. This was the first report of An. hermsi in Arizona and Colorado and indicated that this Anopheles species historically may have been a malaria vector in these two western states.     

Unassisted isolated-pair mating of Anopheles gambiae (Diptera: Culicidae) mosquitoes

Female Anopheles mosquitoes usually mate only once, but mating is seldom seen in small containers containing only one female and male. Therefore, matings are often performed among many adults in large cages or by forced copulation. Isolated-pair mating of Anopheles gambiae G3 strain-derived mosquitoes without forced copulation in small vials is described. We observed that the experimental variables eye color and male number were significant factors in the mating frequency. Females mated more frequently when three males were present over only one male. White-eyed females were more likely to be mated than wild-eyed females, but wild males mated more frequently than did white-eyed males. Experiments were also conducted to determine when mating was occurring by using wild-eye-color mosquitoes in isolated pairs. Almost no matings were observed before day 6 rather than the frequencies typically observed after 1-2 d in standard large-cage matings among large numbers of adults.     

Seasonal abundance and blood feeding activity of Anopheles minimus Theobald (Diptera: Culicidae) in Thailand

Anopheline mosquito larvae and adults were sampled at Ban Pu Teuy, Tri-Yok District, Kanchanaburi Province, western Thailand, from January 2000 to December 2001. Over the period of 2 yr, Anopheles minimus sensu lato was the most commonly collected species, followed by Anopheles swadiwongporni and Anopheles dirus sensu lato; all three species are important vectors of malaria in Thailand. Attempted blood feeding by An. minimus occurred throughout the night, with two distinct feeding peaks: strong activity immediately after sunset (1800-2100 hours), followed by a second, less pronounced, rise before sunrise (0300-0600 hours). Anopheles minimus were more abundant during the wet season compared with the dry and hot seasons, although nocturnal adult feeding patterns were similar. Anopheles minimus fed readily on humans inside and outside of houses, showing a slight preference for exophagy. The human-biting peak of An. minimus in our study area differed from other localities sampled in Thailand, indicating the possible existence of site-specific populations of An. minimus exhibiting different host-seeking behavior. These results underscore the importance of conducting site-specific studies to accurately determine vector larval habitats and adult activity patterns and linking their importance in malaria transmission in a given area.     

Attraction of Anopheles (Diptera: culicidae) to volatile chemicals in Western Kenya

Anopheles gambiae s.l. and Anopheles funestus Giles are the primary vectors of malaria in East Africa. Identification of host-location olfactory cues may increase trap sensitivity for vector control and surveillance programs. Solid-state army miniature light traps were operated near sleeping humans in huts at night without lights and augmented with the potential attractants: L-lactic acid, Limburger cheese volatiles, hexanoic acid, and carbon dioxide. Mosquito response varied between species and gender. Female An. funestus exhibited a greater response to traps baited with L-lactic acid in combination with carbon dioxide than carbon dioxide alone in two different experiments.     

New addition to the mosquito fauna of United States,Anopheles grabhamii (Diptera: Culicidae)

An anopheline mosquito new to the United States was collected in Monroe County, FL, USA. It is Anopheles (Anopheles) grabhamii Theobald, a species common throughout the Greater Antilles area and often found in association with Anopheles albimanus Wiedemann.     

Environmental factors associated with spatial and temporal distribution of Anopheles (Diptera: Culicidae) larvae in Sukabumi, West Java, Indonesia

A 12-mo ecological study of the spatial-temporal distribution of immature stages of Anopheles species was conducted in Sukabumi District, West Java, Indonesia. The study characterized 1,600 sites from a contiguous coastal and hill zone (0-800-m elevation) of which 64% contained Anopheles larvae. Principal component and multiple logistic regression analyses identified ecological parameters associated with presence of nine [Anopheles aconitus Doenitz, Anopheles annularis Van de Wulp, Anopheles barbirostris Van der Wulp, Anopheles flavirostris (Ludlow), Anopheles insulaeflorum (Swellengrebel and Swellengrebel de Graaf), Anopheles kochi Doenitz, Anopheles maculatus Theobald, Anopheles sundaicus (Rodenwaldt), and Anopheles vagus Doenitz] of 15 Anopheles species collected. Combined data for all nine species showed increased Anopheles presence associated with wet season periods and higher elevation habitats exhibiting reduced tree canopy coverage, higher water temperatures, and shallower water depths. Habitat variables measured included topography (elevation), water conditions (temperature, pH, salinity depth, and velocity), habitat characteristics (substrate and canopy cover), density and type of aquatic vegetation coverage (riparian, floating, and emergent), and distance from nearest human habitation. Significant relationships were found for nine species when using all habitats in the analysis. Habitat characteristics for three species were refined. An. aconitus and An. barbirostris were associated with higher elevation rice, Oryza savita L., paddies with relatively shallow water depths, higher water temperatures, higher acidity and salinity concentrations, and a greater average distance from human habitation. An. vagus presence in rice paddies was associated with lower elevation fields, deeper and cooler water, less acidic and saline conditions, and habitats closer to human dwellings. Overall, the distribution of Anopheles species in Sukabumi was found to be nonrandom and predictable on the basis of habitat characteristics.     

Potential distribution of two species in the medically important Anopheles minimus complex (Diptera: Culicidae)

Anopheles minimus Theobald (=An. minimus A) and possibly Anopheles harrisoni Harbach & Manguin (=An. minimus C) are important malaria vector species in the Minimus Complex in Southeast Asia. The distributions of these species are poorly known, although detailed information could benefit malaria vector incrimination and control. We used published collection records of these species and environmental geospatial data to construct consensus ecological niche models (ENM) of each species' potential geographic distribution. The status of the Indian taxon An. fluviatilis S as a species distinct from An. harrisoni has been debated in the literature, so we tested for differentiation in ecological niche characteristics. The predicted potential distribution of An. minimus is more southerly than that of An. harrisoni: Southeast Asia is predicted to be more suitable for An. minimus, and China and India are predicted more suitable for An. harrisoni, so An. harrisoni seems to dominate under cooler conditions. The distribution of An. minimus is more continuous than that of An. harrisoni: disjunction in the potential distribution of the latter is suggested between India and Southeast Asia Anopheles fluviatilis S occurrences are predicted within the An. harrisoni ecological potential, so we do not document ecological differentiation that might reject conspecificity. Overall, model predictions offer a synthetic view of the distribution of this species complex across the landscapes of southern and eastern Asia.     

Redescriptions of Anopheles punctimacula and An. malefactor (Diptera: Culicidae)

Anopheles malefactor Dyar and Knab is elevated from synonymy with An. punctimacula Dyar and Knab, and both species are described and illustrated. Primary morphological differences in the adult, pupal, and larval stages are provided for the separation and recognition of these species. An. punctimacula occurs from Mexico to northern Colombia and Venezuela, whereas An. malefactor is restricted to Panama and northwestern Colombia. Literature records indicate that An. punctimacula is naturally infected with malaria parasites in Panama and Colombia.     

PCR identification and distribution of Anopheles daciae (Diptera,Culicidae) in Germany

Based primarily on nucleotide polymorphisms in the internal transcribed spacer 2 (ITS2) of the ribosomal DNA, Anopheles daciae was recently described as an additional member of the Maculipennis Group of species, separate from Anopheles messeae with which it had previously been confused due to morphological and genetic similarity. Species differentiation between A. messeae and A. daciae was possible only by ITS2 polymerase chain reaction (PCR) amplification followed by DNA sequencing or RFLP analysis. In addition to its siblings, Anopheles maculipennis, Anopheles atroparvus and A. messeae, A. daciae has been shown to occur in Germany, although with limited distribution. We here describe additional collection sites for this species in Germany, showing concentrations in East Germany and the northern Upper Rhine Valley in Southwest Germany. A species-specific multiplex PCR assay is presented that is able to differentiate the four Maculipennis Group sibling species occurring in Germany plus Anopheles sacharovi, Anopheles melanoon and Anopheles labranchiae. The correct identification and detailed knowledge of the biology of A. daciae are of relevance since it might be a vector of disease agents, as suggested by the vector potential of its siblings and the recent finding of an A. daciae female infected with Dirofilaria repens in southern Germany.     

Revisited ITS2 phylogeny of Anopheles (Anopheles) Hyrcanus group mosquitoes: reexamination of unidentified and misidentified ITS2 sequences

The Anopheles (Anopheles) Hyrcanus group of mosquitoes is very important for human health because some are malarial vector mosquitoes. Despite their pathological importance, some unidentified internal transcribed spacer 2 (ITS2) sequences have been reported from members of this group, and their phylogenetic relationships have been rarely understood. In the present study, 84 ITS2 sequences for the Hyrcanus group members were retrieved from GenBank. The detailed sequence comparison unambiguously revealed that the unknown1 sequences of Li et al. (Zootaxa 939:1–8, 2005), YM-2004 (or sp.2) of Ma and Xu [J Med Entomol 42(4):610–619, 2005], Anopheles sp. of Ree et al. (2005), and Anopheles lesteri reported by Gao et al. [J Med Entomol 41(1):5–11, 2004] are identical to Anopheles belenrae [Rueda (Zootaxa 941:1–26, 2005)] and that YM-2003 (or sp.1) of Ma and Xu [J Med Entomol 42(4):610–619, 2005] is closely related to A. lesteri. In addition, some candidate species that may be synonymized are suggested in addition to the possibility that A. lesteri may be divided into at least three types: A, B, and C. The neighbor joining, maximum parsimony, and maximum likelihood trees show that the 23 examined Hyrcanus group members could be divided into four subgroups. The phylogenetic relationships among them are generally well resolved with high bootstrapping values (60–100%), and they are consistently supported by all three trees without any conflicts. These results may strongly suggest that the morphology-based groupings of the Hyrcanus group members should be seriously reconsidered. Further study must be conducted to address the following issues: the three or more multitypified A. lesteri; the unidentified YM-2003 closely related to A. lesteri; and the synonymies of Anopheles peditaeniatus/Anopheles nigerrimus, Anopheles kunmingensis/Anopheles liangshanensis, Anopheles pullus/Anopheles junlianensis, and Anopheles engarensis/Anopheles kleini. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Ministry of Science and Technology (MOST) of Korea (R01-2004-000-10930-0) awarded to UWH.     

Anopheles kleini, Anopheles pullus, and Anopheles sinensis: potential vectors of Plasmodium vivax in the Republic of Korea

Anopheles sinensis Wiedemann (63.3%) was the most abundant Anopheles mosquito captured at cowshed resting collections in malaria high-risk areas (northern Gyeonggi Province) near the demilitarized zone (DMZ) in Korea during 2005, followed by Anopheles kleini Rueda (24.7%) and Anopheles pullus M. Yamada (8.7%). At cowshed resting collections in malaria low-risk areas (Jeonnam and Gyeongnam provinces), An. sinensis accounted for 96.8% of all Anopheles spp. collected, followed by An. kleini Rueda (2.7%), whereas no An. pullus were collected. Three species, An. kleini (50.9%), An. pullus (29.0%), and An. sinensis (13.8%), accounted for nearly all of the 224 Anopheles spp. captured by New Jersey light trap near the DMZ. In addition, An. pullus and An. kleini captured by New Jersey light trap near the DMZ and assayed by enzyme linked immunosorbent assay for Plasmodium vivax circumsporozoite antigen concentrations were higher than An. sinensis sensu stricto (s.s.), indicating higher levels of sporozoites. In laboratory studies of four concurrent artificial membrane feedings on malaria-infected blood from patients, F1 progeny of An. kleini and An. pullus had higher infection rates (8.8 and 7.5%, respectively) than An. sinensis s.s. (4.2%). These data suggest that An. kleini and An. pullus and An. sinensis are vectors of malaria in Korea. Further studies are required to determine the role of these species in the transmission of P. vivax in the Republic of Korea.     

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.     

Sequence analysis of the rDNA internal transcribed spacer 2 and polymerase chain reaction identification of Anopheles fluminensis (Diptera: Culicidae: Anopheles) in Bolivia

Anopheles fluminensis Root is a member of the Arribalzagia Series in the subgenus Anopheles. We report the first record of this species in the department of Cochabamba, Bolivia. This species was sampled from two locations in the foothills of the eastern Andes Mountains within the Chapare Valley. Larvae were collected in fast-flowing, shaded streams at the edges of rocky pools. We provide the first sequence data for the rDNA of An. fluminensis, a partial sequence of the 5.8S and the internal transcribed spacer 2 (ITS2). The ITS2 of An. fluminensis, sequenced from two individuals at one site, was at least 596 bp, had 56.5% GC, and included three large repeats (approximately equal to 125 bp each). We describe a polymerase chain reaction protocol and species-specific primers for identifying this species in the Chapare Valley, Bolivia.     

Analysis of female salivary gland proteins of the Anopheles barbirostris complex (Diptera: Culicidae) in Thailand

Electrophoretic protein profiles of female salivary glands of five sibling species within the Anopheles barbirostris complex, namely A. barbirostris species A1 (Forms A, B, and D), A2, A3, and A4 and Anopheles campestris-like (Forms B and E), were analyzed. At least eight major and several minor protein bands were detected in the glands of each species, of which each morphological region contained different major proteins. The protein profiles distinguished the five sibling species. The variability in major proteins among species was observed in the 40–48, 32–37, and 10–18 kDa ranges. No difference in protein profiles was found in different cytogenetic forms. Polymorphism of the protein profiles within species was only noted in species A4. The lowest major protein (marker) band of each species showed remarkably different relative mobility on SDS–polyacrylamide gels. NanoLC-MS analysis revealed that the marker protein of some species matched with a protein involving in blood feeding, gSG6, of Anopheles gambiae and Anopheles freeborni. These results might be useful for construction of an additional tool to distinguish the five sibling species and lead to further study on the evolution of blood feeding and pathogen transmission.     

Bloodmeal hosts of Anopheles species (Diptera: Culicidae) in a malaria-endemic area of the Brazilian Amazon

Hosts of blood-fed anophelines (Diptera: Culicidae) were determined in three riverine villages, 1.5-7.0 km apart, along the Matapí River, Amapá state, Brazil, by enzyme-linked immunosorbent assay midgut analysis for IgG of common vertebrates. Anopheles marajoara Galv?o & Damsceno and Anopheles darlingi Root had higher human blood indices (HBI) than Anopheles nuneztovari Gabaldón, Anopheles triannulatus (Neiva and Pinto), and Anopheles intermedius (Chagas), which were relatively zoophilic. HBIs of An. darlingi varied significantly among villages, attributable to a low proportion of human-fed mosquitoes in Santo Ant?nio. Significantly higher incidence of An. marajoara and An. nuneztovari fed on pig blood at two villages, associated with a low number of pigs in Santo Ant?nio. The incidences of bovine blood varied significantly among villages for all three of the most common anopheline species. The incidence of mixed meals ranged from 7.1 to 27.6% among common species, and, for An. marajoara, varied significantly among villages. This study demonstrates differences in host selection patterns among villages only a few kilometers apart, which may be influenced by host availability and have important epidemiological consequences.