Primary Malaria Research Articles

Identification of Anopheles species in Sud Kivu, Democratic Republic of Congo, using molecular tools.

The mosquito fauna of the Democratic Republic of Congo remains understudied, including that of the province of Sud Kivu. To improve understanding of species presenting Sud Kivu, adult mosquitoes were collected from houses and larvae were collected from standing water at altitudes between 1627 and 1875 m above sea level. Morphological and molecular methods were used to identify the species of Anopheles collected. Six species were found, including several primary and potential secondary malaria vectors. Further work is needed to characterize mosquito populations in Sud Kivu, as well as to improve methods for identifying Anopheles in general.

ICATE: infection control awareness through education

Background: Education should be understood and used as a tool for social transformation, and as a resource to achieve social, cultural and economic equality. Therefore, ICATE is a project addressed to lower-income countries and/or rural communities that brings together universities, hospitals, schools and the local communities creating collaborative learning communities (CLC) to improve infection control. Methods & Materials: Infection Control Awareness Through Education (ICATE) is an educational project for children, teachers and health professionals that directly tackles both: three of the eight Millennium Development Goals: (i) to help achieve universal primary education, (ii) combating HIV/AIDS, malaria, and other diseases, and (iii) to create a global partnership for development; and two of the seventeen Sustainable Development Goals: (i) ensure healthy lives and promote well-being for all at all ages, and (ii) ensure inclusive and equitable quality education and promote lifelong learning opportunities for all. ICATE tackled these issues through a multidisciplinary team of clinicians, biologists, microbiologists and anthropologists that provided the scientific knowledge to elaborate resources, namely books, eBooks, informative flyers, and to organize courses, workshops for children, educators and general population. Results: ICATE was already implemented in Brazil impacting on teachers and children. After its implementation a partnership for development was created to exponentiate the impact of this project. Conclusion: Across African and South American countries, three major issues raise the need for awareness: HIV/AIDS, Malaria and Tuberculosis. ICATE takes advantage of the CLC to facilitate the transfer of knowledge and educational expertise from the academic media to schools, hospitals and general community, empowering the population with a set of tools, which will allow them to deal and/or participate in the infection control more pro-actively.

Optimal Discriminating Concentrations of Six Synthetic Pyrethroids for Monitoring Insecticide Susceptibility in Anopheles minimus (Diptera: Culicidae), a Primary Malaria Vector in Thailand.

Malaria is a serious mosquito-borne disease transmitted to humans by Anopheles mosquitoes. Seven species of Anopheles are important malaria vectors in Thailand and all remain susceptible to pyrethroid insecticides, despite decades of use in public health mosquito control programs. However, for a more refined and accurate approach to temporal monitoring of susceptibility patterns to commonly used pyrethroids to control adult Anopheles mosquitoes, it is preferred to have specific concentrations for more discriminating testing. A laboratory strain of Anopheles minimus Theobald was used to establish baseline concentrations for susceptibility to six different synthetic pyrethroids-deltamethrin, permethrin, bifenthrin, cypermethrin, α-cypermethrin, and λ-cyhalothrin using the World Health Organization (WHO) adult bioassay system. Final discriminating concentrations of each active ingredient were determined based on doubling LC99 for each chemical to arrive at the following percent concentrations: deltamethrin (0.009%), permethrin (0.551%), bifenthrin (0.12%), cypermethrin (0.104%), α-cypermethrin (0.034%), and λ-cyhalothrin (0.019%). For permethrin, deltamethrin, α-cypermethrin, and λ-cyhalothrin, all revised percentages were lower than currently recommended discriminating concentrations by WHO. Using the newly derived concentrations, insecticide susceptibility tests were followed in a field population of An. minimus s.l. from Kanchanaburi Province. Deltamethrin, bifenthrin, and λ-cyhalothrin produced 100% kill, whereas permethrin, cypermethrin, and α-cypermethrin gave between 92.9 and 97.7% mortality, indicating lower sensitivity (tolerance). Routine monitoring of insecticide susceptibility in An. minimus wild populations throughout Thailand should continue to ensure that insecticides in use are effective to control this vector species.

Blood-induced differential gene expression in Anopheles dirus evaluated using RNA sequencing.

Malaria parasites are transmitted through blood feeding by female Anopheline mosquitoes. Unveiling the blood-feeding process will improve understanding of vector biology. Anopheles dirus (Diptera: Culicidae) is one of the primary malaria vectors in the Greater Mekong Subregion, the epicentre of malaria drug resistance. In this study, differential gene expression between sugar- and blood-fed An.dirus was investigated by RNA sequencing (RNA-seq). A total of 589 transcripts were found to be upregulated and 703 transcripts downregulated as a result of blood feeding. Transcriptional differences were found in genes involved in blood digestion, peritrophic matrix formation, oogenesis and vitellogenesis. The expression levels of several genes were validated by quantitative reverse transcription polymerase chain reaction. The present results provide better understanding of An.dirus biology in relation to its blood feeding.

Decreasing proportion of Anopheles darlingi biting outdoors between long-lasting insecticidal net distributions in peri-Iquitos, Amazonian Peru

BackgroundIn Loreto Department, Peru, a successful 2005–2010 malaria control programme (known as PAMAFRO) included massive distribution of long-lasting insecticidal nets (LLINs). Additional local distribution of LLINs occurred in individual villages, but not between 2012 and 2015. A 2011–2012 study of the primary regional malaria vector Anopheles darlingi detected a trend of increased exophagy compared with pre-PAMAFRO behaviour. For the present study, An. darlingi were collected in three villages in Loreto in 2013–2015 to test two hypotheses: (1) that between LLIN distributions, An. darlingi reverted to pre-intervention biting behaviour; and, (2) that there are separate sub-populations of An. darlingi in Loreto with distinct biting behaviour.ResultsIn 2013–2015 An. darlingi were collected by human landing catch during the rainy and dry seasons in the villages of Lupuna and Cahuide. The abundance of An. darlingi varied substantially across years, villages and time periods, and there was a twofold decrease in the ratio of exophagic:endophagic An. darlingi over the study period. Unexpectedly, there was evidence of a rainy season population decline in An. darlingi. Plasmodium-infected An. darlingi were detected indoors and outdoors throughout the night, and the monthly An. darlingi human biting rate was correlated with the number of malaria cases. Using nextRAD genotyping-by-sequencing, 162 exophagic and endophagic An. darlingi collected at different times during the night were genotyped at 1021 loci. Based on model-based and non-model-based analyses, all genotyped An. darlingi belonged to a homogeneous population, with no evidence for genetic differentiation by biting location or time.ConclusionsThis study identified a decreasing proportion of exophagic An. darlingi in two villages in the years between LLIN distributions. As there was no evidence for genetic differentiation between endophagic and exophagic An. darlingi, this shift in biting behaviour may be the result of behavioural plasticity in An. darlingi, which shifted towards increased exophagy due to repellence by insecticides used to impregnate LLINs and subsequently reverted to increased endophagy as the nets aged. This study highlights the need to target vector control interventions to the biting behaviour of local vectors, which, like malaria risk, shows high temporal and spatial heterogeneity.

Molecular Identification of Four Members of the Anopheles dirus Complex Using the Mitochondrial Cytochrome C Oxidase Subunit I Gene.

Precise mosquito species identification is an essential step for proper management and control of malaria vectors. Misidentification of members in the Anopheles dirus complex, some which are primary malaria vectors in Thailand and mainland Southeast Asia, remains problematic because of indistinguishable or overlapping morphological characters between sibling species. Moreover, there is a need for alternative methods, since the existing molecular techniques in the literature are not entirely satisfactory in differentiating all members in the An. dirus complex. The nucleotide polymorphisms in the mitochondrial cytochrome c oxidase subunit I (COI) sequences were developed to identify the 4 species within the An. dirus complex using an allele-specific (AS) multiplex polymerase chain reaction (PCR). The identified primers amplified and clearly differentiated the 4 members of the complex found in Thailand, Anopheles dirus, An. cracens, An. scanloni, and An. baimaii with PCR products 428/104, 236, 625, and 428 bp, respectively. These results demonstrate that an AS-PCR based on the COI region can accurately identify 4 members of An. dirus complex and would be useful as an alternative PCR-based method for accurate species identification.

Relapse of a Plasmodium vivax Infection in an Iranian Patient: A Case Report

Introduction: Plasmodium vivax is the most prevalent in the malaria endemic areas of Iran. The number of Plasmodium vivax sporozoites that get to the liver remain latent, and after several months or years of inappropriate treatment period, they can reactivate and leads to the clinical manifestation of malaria. Case Presentation: A 49-year-old male was referred to a private clinical center in the capital city of Iran, Tehran, in October 2016 due to fever, chills, nausea, appetite, headache, and weakness. The patient had a history of clinical vivax malaria 1-year prior in Chabahar, in the province of Sistan and Baluchistan. The light microscope and nested PCR were applied for detecting the vivax malaria infection. In this patient, the relapse of P. vivax occurred after about 12 months, following the primary malaria infection. Conclusions: The report underlines that relapses should be considered in febrile individuals returning from malaria endemic areas of the country to malaria free areas. In addition, it highlights the occurrence of relapse in the patient with history using primaquine for a radical cure of liver latent parasites.

Ivermectin susceptibility, sporontocidal effect, and inhibition of time to re-feed in the Amazonian malaria vector Anopheles darlingi

BackgroundOutdoor malaria transmission hinders malaria elimination efforts in the Amazon region and novel vector control tools are needed. Ivermectin mass drug administration (MDA) to humans kills wild Anopheles, targets outdoor-feeding vectors, and can suppress malaria parasite transmission. Laboratory investigations were performed to determine ivermectin susceptibility, sporontocidal effect and inhibition of time to re-feed for the primary Amazonian malaria vector, Anopheles darlingi.MethodsTo assess ivermectin susceptibility, various concentrations of ivermectin were mixed in human blood and fed to An. darlingi. Mosquito survival was monitored daily for 7 days and a non-linear mixed effects model with Probit analysis was used to calculate lethal concentrations of ivermectin that killed 50% (LC50), 25% (LC25) and 5% (LC5) of mosquitoes. To examine ivermectin sporonticidal effect, Plasmodium vivax blood samples were collected from malaria patients and offered to mosquitoes without or with ivermectin at the LC50, LC25 or LC5. To assess ivermectin inhibition of mosquito time to re-feed, concentrations of ivermectin predicted to occur after a single oral dose of 200 μg/kg ivermectin were fed to An. darlingi. Every day for 12 days thereafter, individual mosquitoes were given the opportunity to re-feed on a volunteer. Any mosquitoes that re-blood fed or died were removed from the study.ResultsIvermectin significantly reduced An. darlingi survivorship: 7-day-LC50 = 43.2 ng/ml [37.5, 48.6], -LC25 = 27.8 ng/ml [20.4, 32.9] and -LC5 = 14.8 ng/ml [7.9, 20.2]. Ivermectin compound was sporontocidal to P. vivax in An. darlingi at the LC50 and LC25 concentrations reducing prevalence by 22.6 and 17.1%, respectively, but not at the LC5. Oocyst intensity was not altered at any concentration. Ivermectin significantly delayed time to re-feed at the 4-h (48.7 ng/ml) and 12-h (26.9 ng/ml) concentrations but not 36-h (10.6 ng/ml) or 60-h (6.3 ng/ml).ConclusionsIvermectin is lethal to An. darlingi, modestly inhibits sporogony of P. vivax, and delays time to re-feed at concentrations found in humans up to 12 h post drug ingestion. The LC50 value suggests that a higher than standard dose (400-μg/kg) is necessary to target An. darlingi. These results suggest that ivermectin MDA has potential in the Amazon region to aid malaria elimination efforts.

Abundance and distribution of Anopheles mosquitoes in a malaria endemic area along the Thai-Lao border.

Malaria is an important public health problem in Thailand, especially along international borders. In this study, we conducted a longitudinal entomological survey in six villages and rubber plantation sites to address the spatio-temporal abundance and behavior of malaria vectorsin Ubon Ratchathani Provincealong the Thailand-Laosborder. Adult female mosquitoes were collected by human landing collections (indoor and outdoor) and by cattle bait collections twice per year, during rainy and dry seasons. Mosquitoes were morphologically identified and sibling species were determined byallele-specific PCR. Of the 10,024Anopheles, 9,328 (93.1%) and 696 (6.9%) were collected duringtherainy and dry seasons, respectively. A total of 9,769 (97.5%) and 255 (2.5%) was collected on cattle and human baits, respectively. Very few primary and secondary malaria vectors were collected, consisting of 12 specimens of An. dirus, eight An. minimus, and seven An. aconitus. Of the 152 specimens of the Maculatus Group, only three were identified to An sawadwongporni by molecular methods. The others were 112 An. rampae, a non-vector, that were not amplified or were misidentified as other non-vectors. Theverylow density of primary malaria vectors found in the study villages suggeststhat entomological risk and malaria transmission is higher in neighboring forest areas. Further studies on malaria vector distribution, as well as human behaviors, are needed to understand malaria transmission dynamics intheprovince and to develop suitable vector control methods.

Plasmodium falciparum variant erythrocyte surface antigens: a pilot study of antibody acquisition in recurrent natural infections

BackgroundDuring intra-erythrocytic replication Plasmodium falciparum escapes the human host immune system by switching expression of variant surface antigens (VSA). Piecemeal acquisition of variant specific antibody responses to these antigens as a result of exposure to multiple re-infections has been proposed to play a role in acquisition of naturally acquired immunity.MethodsImmunofluorescence was used to explore the dynamics of anti-VSA IgG responses generated by children to (i) primary malaria episodes and (ii) recurrent P. falciparum infections.ResultsConsistent with previous studies on anti-VSA responses, sera from each child taken at the time of recovery from their respective primary infection tended to recognize their own secondary parasites poorly. Additionally, compared to patients with reinfections by parasites of new merozoite surface protein 2 (MSP2) genotypes, baseline sera sampled from patients with persistent infections (recrudescence) tended to have higher recognition of heterologous parasites. This is consistent with the prediction that anti-VSA IgG responses may play a role in promoting chronic asymptomatic infections.ConclusionsThis pilot study validates the utility of recurrent natural malaria infections as a functional readout for examining the incremental acquisition of immunity to malaria.

Phylogenetic analysis of simian Plasmodium spp. infecting Anopheles balabacensis Baisas in Sabah, Malaysia.

BackgroundAnopheles balabacensis of the Leucospyrus group has been confirmed as the primary knowlesi malaria vector in Sabah, Malaysian Borneo for some time now. Presently, knowlesi malaria is the only zoonotic simian malaria in Malaysia with a high prevalence recorded in the states of Sabah and Sarawak.Methodology/Principal findingsAnopheles spp. were sampled using human landing catch (HLC) method at Paradason village in Kudat district of Sabah. The collected Anopheles were identified morphologically and then subjected to total DNA extraction and polymerase chain reaction (PCR) to detect Plasmodium parasites in the mosquitoes. Identification of Plasmodium spp. was confirmed by sequencing the SSU rRNA gene with species specific primers. MEGA4 software was then used to analyse the SSU rRNA sequences and bulid the phylogenetic tree for inferring the relationship between simian malaria parasites in Sabah.PCR results showed that only 1.61% (23/1,425) of the screened An. balabacensis were infected with one or two of the five simian Plasmodium spp. found in Sabah, viz. Plasmodium coatneyi, P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Sequence analysis of SSU rRNA of Plasmodium isolates showed high percentage of identity within the same Plasmodium sp. group. The phylogenetic tree based on the consensus sequences of P. knowlesi showed 99.7%–100.0% nucleotide identity among the isolates from An. balabacensis, human patients and a long-tailed macaque from the same locality.Conclusions/SignificanceThis is the first study showing high molecular identity between the P. knowlesi isolates from An. balabacensis, human patients and a long-tailed macaque in Sabah. The other common simian Plasmodium spp. found in long-tailed macaques and also detected in An. balabacensis were P. coatneyi, P. inui, P. fieldi and P. cynomolgi. The high percentage identity of nucleotide sequences between the P. knowlesi isolates from the long-tailed macaque, An. balabacensis and human patients suggests a close genetic relationship between the parasites from these hosts.

Chemosensory gene expression in olfactory organs of the anthropophilic Anopheles coluzzii and zoophilic Anopheles quadriannulatus

BackgroundAnopheles (An.) coluzzii, one of Africa’s primary malaria vectors, is highly anthropophilic. This human host preference contributes greatly to its ability to transmit malaria. In contrast, the closely related An. quadriannulatus prefers to feed on bovids and is not thought to contribute to malaria transmission. The diverged preference for host odor profiles between these sibling species is likely reflected in chemosensory gene expression levels in the olfactory organs. Therefore, we compared the transcriptomes of the antennae and maxillary palps between An. coluzzii and An. quadriannulatus, focusing on the major chemosensory gene families.ResultsWhile chemosensory gene expression is strongly correlated between the two species, various chemosensory genes show significantly enhanced expression in one of the species. In the antennae of An. coluzzii the expression of six olfactory receptors (Ors) and seven ionotropic receptors (Irs) is considerably enhanced, whereas 11 Ors and 3 Irs are upregulated in An. quadriannulatus. In the maxillary palps, leaving aside Irs with very low level of expression, one Ir is strongly enhanced in each species. In addition, we find divergence in odorant binding protein (Obp) gene expression, with several highly expressed Obps being enhanced in the antennae and palps of An. coluzzii. Finally, the expression of several gustatory receptors (Grs) in the palps appears to be species-specific, including a homolog of a sugar-sensing Drosophila Gr.ConclusionsA considerable number of Ors and Irs are differentially expressed between these two closely related species with diverging host preference. These chemosensory genes could play a role in the human host preference of the malaria vector An. coluzzii. Additionally, divergence in Obp expression between the two species suggests a possible role of these odor carrier proteins in determining host preference. Finally, divergence in chemosensory expression in the palps may point towards a possible role for the maxillary palps in host differentiation.

Composition of Anopheles mosquitoes, their blood-meal hosts, and Plasmodium falciparum infection rates in three islands with disparate bed net coverage in Lake Victoria, Kenya

BackgroundSmall islands serve as potential malaria reservoirs through which new infections might come to the mainland and may be important targets in malaria elimination efforts. This study investigated malaria vector species diversity, blood-meal hosts, Plasmodium infection rates, and long-lasting insecticidal net (LLIN) coverage on Mageta, Magare and Ngodhe Islands of Lake Victoria in western Kenya, a region where extensive vector control is implemented on the mainland.ResultsFrom trapping for six consecutive nights per month (November 2012 to March 2015) using CDC light traps, pyrethrum spray catches and backpack aspiration, 1868 Anopheles mosquitoes were collected. Based on their cytochrome oxidase I (COI) and intergenic spacer region PCR and sequencing, Anopheles gambiae s.l. (68.52%), Anopheles coustani (19.81%) and Anopheles funestus s.l. (11.67%) mosquitoes were differentiated. The mean abundance of Anopheles mosquitoes per building per trap was significantly higher (p < 0.001) in Mageta than in Magare and Ngodhe. Mageta was also the most populated island (n = 6487) with low LLIN coverage of 62.35% compared to Ngodhe (n = 484; 88.31%) and Magare (n = 250; 98.59%). Overall, 416 (22.27%) engorged Anopheles mosquitoes were analysed, of which 41 tested positive for Plasmodium falciparum infection by high-resolution melting (HRM) analysis of 18S rRNA and cytochrome b PCR products. Plasmodium falciparum infection rates were 10.00, 11.76, 0, and 18.75% among blood-fed An. gambiae s.s. (n = 320), Anopheles arabiensis (n = 51), An. funestus s.s. (n = 29), and An. coustani (n = 16), respectively. Based on HRM analysis of vertebrate cytochrome b, 16S rRNA and COI PCR products, humans (72.36%) were the prominent blood-meal hosts of malaria vectors, but 20.91% of blood-meals were from non-human vertebrate hosts.ConclusionsThese findings demonstrate high Plasmodium infection rates among the primary malaria vectors An. gambiae s.s. and An. arabiensis, as well as in An. coustani for the first time in the region, and that non-human blood-meal sources play an important role in their ecology. Further, the higher Anopheles mosquito abundances on the only low LLIN coverage island of Mageta suggests that high LLIN coverage has been effective in reducing malaria vector populations on Magare and Ngodhe Islands.

Dichotomous miR expression and immune responses following primary blood-stage malaria.

Clinical responses to infection or vaccination and the development of effective immunity are characterized in humans by a marked interindividual variability. To gain an insight into the factors affecting this variability, we used a controlled human infection system to study early immune events following primary infection of healthy human volunteers with blood-stage Plasmodium falciparum malaria. By day 4 of infection, a dichotomous pattern of high or low expression of a defined set of microRNAs (miRs) emerged in volunteers that correlated with variation in parasite growth rate. Moreover, high-miR responders had higher numbers of activated CD4+ T cells, and developed significantly enhanced antimalarial antibody responses. Notably, a set of 17 miRs was identified in the whole blood of low-miR responders prior to infection that differentiated them from high-miR responders. These data implicate preexisting host factors as major determinants in the ability to effectively respond to primary malaria infection.

Host attraction and biting behaviour of Anopheles mosquitoes in South Halmahera, Indonesia

BackgroundIndonesia is home to a variety of malaria vectors whose specific bionomic traits remain largely uncharacterized. Species-specific behaviours, such as host feeding preferences, impact the dynamics of malaria transmission and the effectiveness of vector control interventions.MethodsTo examine species-specific host attraction and feeding behaviours, a Latin square design was used to compare Anopheles mosquitoes attracted to human, cow, and goat-baited tents. Anopheles mosquitoes were collected hourly from the inside walls of each baited tent. Species were morphologically and then molecularly identified using rDNA ITS2 sequences. The head and thorax of individual specimens were analysed for Plasmodium DNA using PCR. Bloodmeals were identified using a multiplex PCR.ResultsA total of 1024, 137, and 74 Anopheles were collected over 12 nights in cow, goat, and human-baited tents, respectively. The species were identified as Anopheles kochi, Anopheles farauti s.s., Anopheles hackeri, Anopheles hinesorum, Anopheles indefinitus, Anopheles punctulatus, Anopheles tessellatus, Anopheles vagus, and Anopheles vanus, many of which are known to transmit human malaria. Molecular analysis of blood meals revealed a high level of feeding on multiple host species in a single night. Anopheles kochi, An. indefinitus, and An. vanus were infected with Plasmodium vivax at rates comparable to primary malaria vectors.ConclusionsThe species distributions of Anopheles mosquitoes attracted to human, goat, and cow hosts were similar. Eight of nine sporozoite positive samples were captured with animal-baited traps, indicating that even predominantly zoophilic mosquitoes may be contributing to malaria transmission. Multiple host feeding and flexibility in blood feeding behaviour have important implications for malaria transmission, malaria control, and the effectiveness of intervention and monitoring methods, particularly those that target human-feeding vectors.

Potential distribution of mosquito vector species in a primary malaria endemic region of Colombia.

Rapid transformation of natural ecosystems changes ecological conditions for important human disease vector species; therefore, an essential task is to identify and understand the variables that shape distributions of these species to optimize efforts toward control and mitigation. Ecological niche modeling was used to estimate the potential distribution and to assess hypotheses of niche similarity among the three main malaria vector species in northern Colombia: Anopheles nuneztovari, An. albimanus, and An. darlingi. Georeferenced point collection data and remotely sensed, fine-resolution satellite imagery were integrated across the Urabá –Bajo Cauca–Alto Sinú malaria endemic area using a maximum entropy algorithm. Results showed that An. nuneztovari has the widest geographic distribution, occupying almost the entire study region; this niche breadth is probably related to the ability of this species to colonize both, natural and disturbed environments. The model for An. darlingi showed that most suitable localities for this species in Bajo Cauca were along the Cauca and Nechí river. The riparian ecosystems in this region and the potential for rapid adaptation by this species to novel environments, may favor the establishment of populations of this species. Apparently, the three main Colombian Anopheles vector species in this endemic area do not occupy environments either with high seasonality, or with low seasonality and high NDVI values. Estimated overlap in geographic space between An. nuneztovari and An. albimanus indicated broad spatial and environmental similarity between these species. An. nuneztovari has a broader niche and potential distribution. Dispersal ability of these species and their ability to occupy diverse environmental situations may facilitate sympatry across many environmental and geographic contexts. These model results may be useful for the design and implementation of malaria species-specific vector control interventions optimized for this important malaria region.

Phylogenetic Relationships Among Malaria Vectors and Closely Related Species in Thailand Using Multilocus DNA Sequences.

The evolutionary and taxonomic status is important for understanding speciation events and phylogenetic relationships between closely related vector and nonvector species. This information is useful for targeting important disease vector species groups for the development of novel genetic-based vector and pathogen control methods. In this study, different phylogenetic analyses were performed to reconstruct phylogenetic trees for the primary malaria vectors in Thailand based on sequence information of 4 DNA fragments from the nuclear and mitochondrial regions. The primary Anopheles species in the subgenus Cellia involved in malaria transmission in Thailand separate clearly into 3 distinct clades: the Leucosphyrus group, Minimus subgroup, and Maculatus group. The phylogenetic trees based on different reconstructed algorithms and different gene regions provided congruent phylogenetic status of the mosquito species studied. The phylogenetic relationships of malaria vector species examined followed similar patterns based on morphological characters. An estimate of the divergence time among the Anopheles species infers that they were present during the Eocene and Miocene periods (>41 million years ago). Congruent phylogenetic analysis of malaria vectors is presented with different algorithms and gene regions. The nuclear TOLL6 fragment appears useful for molecular phylogenetic, species DNA barcode, and Anopheles population genetic analyses.

Vector bionomics and malaria transmission along the Thailand-Myanmar border: a baseline entomological survey.

Baseline entomological surveys were conducted in four sentinel sites along the Thailand-Myanmar border to address vector bionomics and malaria transmission in the context of a study on malaria elimination. Adult Anopheles mosquitoes were collected using human-landing catch and cow-bait collection in four villages during the rainy season from May-June, 2013. Mosquitoes were identified to species level by morphological characters and by AS-PCR. Sporozoite indexes were determined on head/thoraces of primary and secondary malaria vectors using real-time PCR. A total of 4,301 anopheles belonging to 12 anopheline taxa were identified. Anopheles minimus represented >98% of the Minimus Complex members (n=1,683), whereas the An. maculatus group was composed of two dominant species, An. sawadwongporni and An. maculatus. Overall, 25 Plasmodium-positive mosquitoes (of 2,323) were found, representing a sporozoite index of 1.1% [95%CI 0.66-1.50]. The transmission intensity as measured by the EIR strongly varied according to the village (ANOVA, F=17.67, df=3, P<0.0001). Our findings highlight the diversity and complexity of the biting pattern of malaria vectors along the Thailand-Myanmar border that represent a formidable challenge for malaria control and elimination.

An effective method for the identification and separation of Anopheles minimus, the primary malaria vector in Thailand, and its sister species Anopheles harrisoni, with a comparison of their mating behaviors

BackgroundSpecies of the Anopheles minimus complex are considered to be the primary vectors of malaria in South and Southeast Asia. Two species of the complex, Anopheles minimus and Anopheles harrisoni, occur in Thailand. They are sympatric and difficult to accurately distinguish based on morphological characters. The aim of this study was to investigate the potential of antennal sensory organs to distinguish these two species. Additionally, we investigated their ability to mate in cages of different sizes, as well as the possible mechanism(s) that evokes stenogamous behavior.MethodsLarge sensilla coeloconica present on the antennae of females of An. minimus and An. harrisoni were counted under a conventional light microscope and various types of antennal sensilla were examined under a scanning electron microscope (SEM). Determinations of mating ability were carried out in 20 and 30 cm3 cages with a density resting surface (DRS) of 7.2. The insemination rate, frequency of clasper (gonocoxopodite) movement of the male genitalia during induced copulation and duration of mating of the two species were compared.ResultsThe mean numbers of large sensilla coeloconica on antennal flagellomeres 1–8 and the mean number of large sensilla coeloconica on each flagellum in An. minimus (26.25) and An. harrisoni (31.98) were significantly different. Females of both species bear five types of antennal sensilla: chaetica, trichodea, basiconica, coeloconica and ampullacea. Marked differences in the structure of the large sensilla coeloconica were observed between the two species. Furthermore, only An. minimus could copulate naturally in the small cages. The frequency of clasper movement in the stenogamous An. minimus was significantly higher than in An. harrisoni, but there was no difference in the duration of mating.ConclusionsTo our knowledge, this study is the first to examine and discover the usefulness of large sensilla coeloconica on the antennae of females and the frequency of clasper movement in males for distinguishing the sibling species An. minimus and An. harrisoni. The discovery provides an effective and relatively inexpensive method for their identification. Additionally, the greater frequency of clasper movement of An. minimus might influence its ability to mate in small spaces.

Genetic diversity and population structure of the primary malaria vector Anopheles sinensis (Diptera: Culicidae) in China inferred by cox1 gene

BackgroundAnopheles sinensis is a primary vector for Plasmodium vivax malaria in most regions of China. A comprehensive understanding of genetic variation and structure of the mosquito would be of benefit to the vector control and in a further attempt to contribute to malaria elimination in China. However, there is only inadequate population genetic data pertaining to An. sinensis currently.MethodsGenetic variations and structure among populations of An. sinensis was examined and analyzed based on the nucleotide sequences of a 662 nt variable region of the mitochondrial cox1 gene among 15 populations from 20 collection sites in China.ResultsA total of 453 individuals in 15 populations were analyzed. The cox1 gene sequences were aligned, and 247 haplotypes were detected, 41 of these shared between populations. The range of haplotype diversity was from 0.709 (Yunnan) to 0.998 (Anhui). The genealogic network showed that the haplotypes were divided into two clusters, cluster I was at a high level of homoplasy, while cluster II included almost all individuals from the Yunnan population. The Yunnan population displayed a significantly high level of genetic differentiation (0.452−0.622) and a restricted gene flow with other populations. The pairwise FST values among other populations were lower. The AMOVA result showed that the percentage of variation within populations (83.83%) was higher than that among populations (16.17%). Mantel test suggested that geographical distance did not significantly contribute to the genetic differentiation (R2 = 0.0125, P = 0.59). Neutral test and mismatch analysis results showed that the An. sinensis population has undergone demographic expansions.ConclusionsAnopheles sinensis populations showed high genetic polymorphism by cox1 gene. The weak genetic structure may be a consequence of low genetic differentiation and high gene flow among populations, except the Yunnan samples. The Yunnan population was isolated from the other populations, gene flow limited by geographical distance and barriers. These findings will provide a theoretical basis for vector surveillance and vector control in China.