Vector Biology and Integrated Management of Malaria Vectors in China.

Malaria is an infectious disease caused by Plasmodium parasites, transmitted by Anopheles sinensis, Anopheles lesteri, Anopheles minimus, and Anopheles dirus in China. In 2021, the disease was eliminated in China after more than 70 years of efforts implementing an integrated mosquito management strategy. This strategy comprised indoor residual spray, insecticide-treated bed nets, irrigation management, and rice-fish coculture based on an understanding of taxonomic status and ecological behaviors of vector species, in conjunction with mass drug administration and promotion of public education. However, China still faces postelimination challenges, including the importation of approximately 2,000-4,000 cases of malaria into the country each year, as well as widespread resistance to pyrethroid insecticides in An. sinensis; these challenges require long-term vector surveillance to understand the distribution, population density, and development of resistance in vector mosquitoes to prevent local epidemics caused by imported malaria cases.

Knockdown of Anopheles dirus far upstream element-binding protein gene lower oocyst numbers of Plasmodium vivax.

The modulation of gene expression levels of Anopheles dirus on Plasmodium vivax infection at the ookinete and oocyst stages was previously reported. In the present study, several upregulated An. dirus genes were selected based on their high expression levels and subcellular locations to examine their roles in P. vivax infection. Five An. dirus genes-carboxylesterase, cuticular protein RR-2 family, far upstream element-binding protein, kraken, and peptidase212-were knocked down by dsRNA feeding using dsRNA-lacZ as a control. The dsRNA-fed mosquitoes were later challenged by P. vivax-infected blood, and the oocyst numbers were determined. The expression of these five genes was examined in many organs of both male and female mosquitoes. The results showed that the decreased expression level of the far upstream element-binding protein gene could lower the oocyst numbers, whereas the others showed no effect on P. vivax infection. The expression levels of these genes in ovaries were found, and in many organs, they were similar between male and female mosquitoes. The reduction of these five gene expressions did not affect the lifespan of the mosquitoes. In addition, the malaria box compound, MMV000634, demonstrated the lowest binding energy to the far upstream element-binding protein using virtual screening. This protein might be a target to block malaria transmission.

Effectiveness of Herbal Essential Oils as Single and Combined Repellents against Aedes aegypti, Anopheles dirus and Culex quinquefasciatus (Diptera: Culicidae).

Mosquito repellents reduce human-vector contact of vector-borne diseases. We compared the repellent activity of 10 undiluted essential oils (anise, basil, bergamot, coriander, patchouli, peppermint, petitgrain, rosemary, sage and vetiver) against A. aegypti, A. dirus and C. quinquefasciatus using the arm-in-cage method. Petitgrain oil was the most effective against A. aegypti (270 min). Peppermint oil was the most effective against A. dirus (180 min). Interestingly, all single oils had attributes of repellency against C. quinquefasciatus (ranged, 120−360 min). Moreover, we integrated their binary combinations of highly effective essential oils against A. aegypti and A. dirus to potentially increase the protection time. A 1:1 combination of petitgrain/basil, petitgrain/coriander, basil/coriander and basil/sage reduced the median complete-protection time of 150 min for A. aegypti; a combination of sage and patchouli oils prolonged the median complete-protection time of 270 min for A. dirus. Combining essential oils effect protection time from these two mosquito species.

Time of Test Periods Influence the Behavioral Responses of Anopheles minimus and Anopheles dirus (Diptera: Culicidae) to DEET.

Information on factors influencing the behavioral responses of mosquitoes to repellents is lacking and poorly understood, especially in the Anopheles species, night-biting mosquitoes. Our goal was to investigate the impact of different time periods on circadian activity and behavioral responses of two malaria vectors, Anopheles minimus and An. dirus, to 5% DEET using an excito-repellency test system. Each mosquito species was exposed to the repellent during the daytime (06.00-18.00) and nighttime (18.00-06.00), and time of observation was further divided into four 3-h intervals. Significant escape responses were observed between daytime and nighttime for An. minimus in both noncontact and contact tests. An. dirus showed statistical differences in contact irritancy escape response, whereas no significant difference was found in noncontact repellency tests. Both mosquito species showed more significantly higher escape responses when exposed to DEET during the afternoon and late in the night. This finding indicates that the time of testing may affect the behavioral responses of mosquitoes to repellents, especially in An. minimus and An. dirus. A better understanding of nocturnally active mosquito behavioral responses spanning from dusk to dawn would assist in optimizing product development, screening, and effective evaluation.

Effect of Different Wall Surface Coverage With Deltamethrin-Treated Netting on the Reduction of Indoor-Biting Anopheles Mosquitoes (Diptera: Culicidae).

Indoor residual spray with deltamethrin remains the most common tool for reducing malaria transmission in Thailand. Deltamethrin is commonly used to spray the entire inner surfaces of the walls to prevent mosquitoes from resting. This study compared the mosquito landing responses on humans inside three experimental huts treated with deltamethrin at three different extents of wall coverage (25%, 50%, and full coverage), with one clean/untreated hut serving as a control. There were no significant differences between the numbers of Anopheles mosquitoes landing in the 50% and full coverage huts, whereas, in comparison to both of these, there was a significantly greater number landing in the 25% coverage hut. This study demonstrates that varying the percent coverage of indoor surfaces with deltamethrin-treated netting influences the blood-feeding success of wild Anopheles, and our findings suggest that it may be possible to reduce the extent of insecticide surface treatment while maintaining equivalent mosquito avoidance action to that seen in fully treated structures.

[Identification, molecular structure and expression characteristics of Torso like gene in Anopheles dirus].

OBJECTIVE: To characterize Torso-like (tsl) gene and investigate its expression characteristics in Anopheles dirus, so as to provide a theoretical basis for subsequent functional studies of the tsl gene. METHODS: According to the coding sequences of Drosophila melanogaster and An. gambiae tsl genes, the complete genome of An. dirus was retrieved and the An. dirus tsl gene was characterized. Specific primers were designed and the target gene was amplified using PCR and reverse-transcription PCR assays. The physicochemical properties, signal peptide, transmembrane structure, secondary structure and tertiary structure of the encoded protein TSL were analyzed using bioinformatics tools, and a phylogenetic analysis was performed. In addition, the specific expression of the tls gene was detected in various tissues of An. dirus using a quantitative real-time PCR assay. RESULTS: The An. dirus tsl gene was 16 751 bp in length with a CDS region of 1 134 bp, encoding 377 amino acids, and the encoded TSL protein was a stably hydrophilic protein. The TSL protein was predicted to be a secretory protein that was located in extra-membrane regions containing signal peptides. The secondary structure of the TSL protein contained α-helix (51.72%), extended strand (12.20%), ß-bridge (4.78%) and random coil (31.30%) in the secondary structure, and a 3D homology model was generated using 5cj9.1.A as a template. Phylogenetic analysis revealed a close genetic relationship in the TSL protein between An. dirus and An. farauti. In addition, quantitative real-time PCR assay detected the tsl gene expression in the head, chest, abdomen and foot of An. dirus, with the highest expression in the head and low expression in the foot. CONCLUSIONS: The tsl gene is characterized in An. dirus at a genomic level, and the prediction of the TSL protein structure and the elucidation of the tissue-specific tsl gene expression in An. dirus provide a basis for the further studies on the gene functions.

New insights into malaria vector bionomics in Lao PDR: a nationwide entomology survey.

BACKGROUND: In Laos, the malaria burden remains high despite a significant reduction of cases during the last decade. In the context of the disease elimination by 2030, a nationwide entomological survey was conducted to better understand the distribution, abundance and behaviour of major malaria vectors (Anopheles spp.) in the country. METHODS: Mosquito collections were implemented in ten villages from ten provinces during the rainy and dry seasons of 2014 and 2015 by using human landing catch (HLC) and cow bait collection (CBC) methods. After morphological identification in the field, molecular identification of the sibling species of Anopheles mosquitoes from the Funestus, Leucosphyrus, and Maculatus groups were determined using PCR specific alleles. A screening of Plasmodium falciparum and Plasmodium vivax infections in the vectors was carried out by quantitative PCR assays. RESULTS: A total of 14,146 adult mosquitoes representing 25 different Anopheles species were collected and morphologically identified. Molecular identification revealed the presence of 12 sibling species within the main primary vector groups, including Anopheles maculatus, Anopheles rampae, Anopheles sawadwongporni, Anopheles pseudowillmori, Anopheles dravidicus, Anopheles minimus, Anopheles aconitus, Anopheles pampanai, Anopheles harrisoni, Anopheles dirus, Anopheles baimaii, Anopheles nemophilous. Anopheles maculatus and An. minimus were predominant during both the dry and rainy seasons, but showed highly zoophilic preferences (Zoophilic index of 98% and 95%, respectively). Overall, 22% of the total malaria vectors were collected between 10:00 PM and 5:00 AM indoors when people are sleeping. Twenty-seven percent of primary and secondary vectors were collected outdoors before 10:00 PM or after 5:00 AM, times when people are usually awake and outdoors. Only two specimens were positive for P. falciparum, one An. aconitus from Phongsaly and one An. minimus from Vientiane Province CONCLUSIONS: The results indicate that people living in rural areas in Laos are constantly exposed to malaria vectors throughout the year and specifically outdoors. The use of LLINs/IRS remains important but innovative tools and new strategies are needed to address locally, the early and outdoor malaria transmission. Lack of expertise in general entomological methods may further exacerbate the situation.

Bacillus sphaericus exposure reduced vector competence of Anopheles dirus to Plasmodium yoelii by upregulating the Imd signaling pathway.

BACKGROUND: Vector control with Bacillus sphaericus (Bs) is an effective way to block the transmission of malaria. However, in practical application of Bs agents, a sublethal dose effect was often caused by insufficient dosing, and it is little known whether the Bs exposure would affect the surviving mosquitoes' vector capacity to malaria. METHODS: A sublethal dose of the Bs 2362 strain was administrated to the early fourth-instar larvae of Anopheles dirus to simulate shortage use of Bs in field circumstance. To determine vector competence, mosquitoes were dissected and the oocysts in the midguts were examined on day 9-11 post-infection with Plasmodium yoelii. Meanwhile, a SYBR quantitative PCR assay was conducted to examine the transcriptional level of the key immune molecules of mosquitoes, and RNA interference was utilized to validate the role of key immune effector molecule TEP1. RESULTS: The sublethal dose of Bs treatment significantly reduced susceptibility of An. dirus to P. yoelii, with the decrease of P. yoelii infection intensity and rate. Although there existed a melanization response of adult An. dirus following challenge with P. yoelii, it was not involved in the decrease of vector competence as no significant difference of melanization rates and densities between the control and Bs groups was found. Further studies showed that Bs treatment significantly increased TEP1 expression in the fourth-instar larvae (L4), pupae (Pu), 48 h post-infection (hpi) and 72 hpi (P < 0.001). Further, gene-silencing of TEP1 resulted in disappearance of the Bs impact on vector competence of An. dirus to P. yoelii. Moreover, the transcriptional level of PGRP-LC and Rel2 were significantly elevated by Bs treatment with decreased expression of the negative regulator Caspar at 48 hpi, which implied that the Imd signaling pathway was upregulated by Bs exposure. CONCLUSIONS: Bs exposure can reduce the vector competence of An. dirus to malaria parasites through upregulating Imd signaling pathway and enhancing the expression of TEP1. The data could not only help us to understand the impact and mechanism of Bs exposure on Anopheles' vector competence to malaria but also provide us with novel clues for wiping out malaria using vector control.

The impact of transfluthrin on the spatial repellency of the primary malaria mosquito vectors in Vietnam: Anopheles dirus and Anopheles minimus.

BACKGROUND: The complexity of mosquito-borne diseases poses a major challenge to global health efforts to mitigate their impact on people residing in sub-tropical and tropical regions, to travellers and deployed military personnel. To supplement drug- and vaccine-based disease control programmes, other strategies are urgently needed, including the direct control of disease vectors. Modern vector control research generally focuses on identifying novel active ingredients and/or innovative methods to reduce human-mosquito interactions. These efforts include the evaluation of spatial repellents, which are compounds capable of altering mosquito feeding behaviour without direct contact with the chemical source. METHODS: This project examined the impact of airborne transfluthrin from impregnated textile materials on two important malaria vectors, Anopheles dirus and Anopheles minimus. Repellency was measured by movement within taxis cages within a semi-field environment at the National Institute of Hygiene and Epidemiology in Hanoi, Vietnam. Knockdown and mortality were measured in adult mosquito bioassay cages. Metered-volume air samples were collected at a sub-set of points in the mosquito exposure trial. RESULTS: Significant differences in knockdown/mortality were observed along a gradient from the exposure source with higher rates of knockdown/mortality at 2 m and 4 m when compared with the furthest distance (16 m). Knockdown/mortality was also greater at floor level and 1.5 m when compared to 3 m above the floor. Repellency was not significantly different except when comparing 2 m and 16 m taxis cages. Importantly, the two species reacted differently to transfluthrin, with An. minimus being more susceptible to knockdown and mortality. The measured concentrations of airborne transfluthrin ranged from below the limit of detection to 1.32 ng/L, however there were a limited number of evaluable samples complicating interpretation of these results. CONCLUSIONS: This study, measuring repellency, knockdown and mortality in two malaria vectors in Vietnam demonstrates that both species are sensitive to airborne transfluthrin. The differences in magnitude of response between the two species requires further study before use in large-scale vector control programmes to delineate how spatial repellency would impact the development of insecticide resistance and the disruption of biting behaviour.

Efficient Transmission of Mixed Plasmodium falciparum/vivax Infections From Humans to Mosquitoes.

BACKGROUND: In Southeast Asia, people are often coinfected with different species of malaria (Plasmodium falciparum [Pf] and Plasmodium vivax [Pv]) as well as with multiple clones of the same species. Whether particular species or clones within mixed infections are more readily transmitted to mosquitoes remains unknown. METHODS: Laboratory-reared Anopheles dirus were fed on blood from 119 Pf-infected Cambodian adults, with 5950 dissected to evaluate for transmitted infection. Among 12 persons who infected mosquitoes, polymerase chain reaction and amplicon deep sequencing were used to track species and clone-specific transmission to mosquitoes. RESULTS: Seven of 12 persons that infected mosquitoes harbored mixed Pf/Pv infection. Among these 7 persons, all transmitted Pv with 2 transmitting both Pf and Pv, leading to Pf/Pv coinfection in 21% of infected mosquitoes. Up to 4 clones of each species were detected within persons. Shifts in clone frequency were detected during transmission. However, in general, all parasite clones in humans were transmitted to mosquitoes, with individual mosquitoes frequently carrying multiple transmitted clones. CONCLUSIONS: Malaria diversity in human hosts was maintained in the parasite populations recovered from mosquitoes fed on their blood. However, in persons with mixed Pf/Pv malaria, Pv appears to be transmitted more readily, in association with more prevalent patent gametocytemia.

Discriminating Lethal Concentrations for Transfluthrin, a Volatile Pyrethroid Compound for Mosquito Control in Thailand.

Pyrethroids are commonly used to control malaria and dengue vectors in Thailand. The lack of specific lethal discriminating concentrations (DCs) for specific mosquito species has possibly compromised more accurate assessments of physiological susceptibility to various chemicals over time. Previous studies have established DCs of various residual pyrethroids against specific mosquitoes in Thailand. However, DCs for transfluthrin (TFT), a highly volatile pyrethroid compound, against mosquito vectors in Thailand has been lacking. The aim of this study was to determine the DCs and susceptibility baselines of TFT against pyrethroid-susceptible laboratory strains of Aedes aegypti, Anopheles minimus, and An. dirus using the World Health Organization adult susceptibility tube method. Final DCs of TFT of each species were determined based on doubling the 99% lethal concentration at the following percentages: Ae. aegypti (0.06824%), An. minimus (0.06382%), and An. dirus (0.01508%). Subsequently, the respective TFT DCs were used to test field-collected populations of Ae. aegypti, An. harrisoni (Minimus Complex species), and An. dirus. Anopheles harrisoni and An. dirus were found completely susceptible (100% mortality), whereas Ae. aegypti from Nonthaburi Province was resistant to TFT. The suitability of the testing system and procedures is discussed. Routine assessment of insecticide susceptibility should include pyrethroids with high-vapor-pressure characteristics for informing control programs and consumers of product and chemical effectiveness.

Diversity and biting patterns of Anopheles species in a malaria endemic area, Umphang Valley, Tak Province, western Thailand.

Malaria is highly endemic in Umphang Valley, a district in the western edge of Tak Province, along the boundary with Kayin State of Myanmar. Although there are high indigenous malaria cases in this area every year, nothing about malaria vectors and their transmission role have been investigated before this study. The objective of this work is to characterize the Anopheles species diversity and trophic behavior of malaria vectors in the transmission area of Umphang Valley. Females of Anopheles mosquitoes were collected every two months during a two-year period. Mosquito collections were using standard collection technique, indoor and outdoor human landing collections and outdoor cattle bait collection. Anopheles mosquitoes were identified using morphological characters and multiplex AS-PCR assay for the identification of sibling species within groups and complexes present. From a total of 16,468 Anopheles females, 2723 specimens (16.54%) were collected from humans and 13,745 specimens (83.46%) were captured from cattle. From human landing collections, 2447 specimens (89.86%) of Anopheles minimus were obtained, followed by 119 Anopheles peditaeniatus (4.37%), 62 Anopheles maculatus (2.28%), 17 Anopheles dirus (0.6%), 15 Anopheles aconitus (0.5%) and 6 Anopheles sawadwongporni (0.2%) respectively. Seven putative malaria vectors, including An. minimus, An. dirus, An. baimaii, An. sawadwongporni, An. maculatus, An. pseudowillmori and An. aconitus were documented from this study and trophic behavior of each respective species were observed. Such information is definitely crucial for defining the vector capacity of each single species and for designing appropriate vector prevention and control strategies against target 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.

Therapeutic and Transmission-Blocking Efficacy of Dihydroartemisinin/Piperaquine and Chloroquine against Plasmodium vivax Malaria, Cambodia.

We assessed the efficacy of standard 3-day courses of chloroquine and dihydroartemisinin/piperaquine against Plasmodium vivax malaria. Compared with chloroquine, dihydroartemisinin/piperaquine was faster in clearing asexual P. vivax parasites and blocking human-to-mosquito transmission. This drug combination was also more effective in preventing potential recurrences for >2 months.

Natural Plasmodium vivax infections in Anopheles mosquitoes in a malaria endemic area of northeastern Thailand.

There was recently an outbreak of malaria in Ubon Ratchathani Province, northeastern Thailand. In the absence of information on malaria vector transmission dynamics, this study aimed to identify the anopheline vectors and their role in malaria transmission. Adult female Anopheles mosquitoes were collected monthly by human-landing catch in Na Chaluai District of Ubon Ratchathani Province during January 2014-December 2015. Field-captured mosquitoes were identified to species using morphology-based keys and molecular assays (allele-specific polymerase chain reaction, AS-PCR), and analysed for the presence of Plasmodium falciparum and Plasmodium vivax using an enzyme-linked immunosorbent assay (ELISA) for the detection of circumsporozoite proteins (CSP). A total of 1,229 Anopheles females belonging to 13 species were collected. Four anopheline taxa were most abundant: Members of the Anopheles barbirostris complex, comprising 38% of the specimens, species of the Anopheles hyrcanus group (18%), Anopheles nivipes (17%) and Anopheles philippinensis (12%). The other nine species comprised 15% of the collections. Plasmodium infections were detected in two of 668 pooled samples of heads/thoraces, Anopheles dirus (1/29) and An. philippinensis (1/97). The An. dirus pool had a mixed infection of P. vivax-210 and P. vivax-247, whereas the An. philippinensis pool was positive only for the latter protein variant. Both positive ELISA samples were confirmed by nested PCR. This study is the first to incriminate An. dirus and An. philippinensis as natural malaria vectors in the area where the outbreak occurred. This information can assist in designing and implementing a more effective malaria control programme in the province.

Molecular epidemiology of mosquitoes for the transmission of forest malaria in south-central Vietnam.

Human infection caused by non-human primate malarial parasites, such as Plasmodium knowlesi and Plasmodium cynomolgi, occurs naturally in Southeast Asian countries, including Vietnam. Members of the Anopheles dirus species complex are known to be important vectors of human malarial parasites in the forested areas of southern and central Vietnam, including those in Khanh Phu commune and Khanh Hoa Province. Recent molecular epidemiological studies in Vietnam have reported cases of co-infection with Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and P. knowlesi in An. dirus. The commonly found macaques in the forest in the forested areas are suspected to be bitten by the same An. dirus population that bites humans. A recent epidemiological study identified six species of malarial parasites in sporozoite-infected An. dirus using polymerase chain reaction, of which P. vivax was the most common, followed by P. knowlesi, Plasmodium inui, P. cynomolgi, Plasmodium coatneyi, and P. falciparum. Based on a gametocyte analysis, the same allelic gametocyte types were observed in both humans and mosquitoes at similar frequencies. These observations suggest that people who stay overnight in the forests are frequently infected with both human and non-human primate malarial parasites, leading to the emergence of novel zoonotic malaria. Moreover, it is suggested that mosquito vector populations should be controlled and monitored closely.

Quantitative real-time PCR analysis of Anopheles dirus TEP1 and NOS during Plasmodium berghei infection, using three reference genes.

Quantitative reverse transcription PCR (qRT-PCR) has been an integral part of characterizing the immunity of Anopheles mosquitoes towards Plasmodium invasion. Two anti-Plasmodium factors of Anopheles, thioester-containing protein 1 (TEP1) and nitric oxide synthase (NOS), play a role in the refractoriness of Anopheles towards Plasmodium infection and are generally expressed during infection. However, these are less studied in Anopheles dirus, a dominant malaria vector in Southeast Asia. Furthermore, most studies used a single reference gene for normalization during gene expression analysis without proper validation. This may lead to erroneous quantification of expression levels. Therefore, the present study characterized and investigated the expression profiles of TEP1 and NOS of Anopheles dirus during P. berghei infection. Prior to that, the elongation factor 1-alpha (EF1), actin 1 (Act) and ribosomal protein S7 (S7) genes were validated for their suitability as a set of reference genes. TEP1 and NOS expressions in An. dirus were found to be significantly induced after P. berghei infection.

Detection of the Plasmodium falciparum Kelch-13 gene P553L mutation in sporozoites isolated from mosquito salivary glands in South-Central Vietnam.

BACKGROUND: Plasmodium falciparum has developed resistance against artemisinin in Southeast Asia. Mutations in the P. falciparum Kelch-13 (Pfk13) gene are associated with artemisinin resistance in vitro and in vivo. We investigated the prevalence of mutations in PfK13 from sporozoite-stage parasites isolated from the salivary glands of Anopheles dirus mosquitoes. METHODS: Mosquitoes were caught by human-landing catches at two locations within the Khanh Phu commune, South-Central Vietnam. Identification of Anopheles species was performed based on morphological features and nucleotide sequence analysis. Sporozoite-infected salivary glands were stored on filter paper and at 4-6 °C. A nested-PCR targeting the small subunit ribosomal RNA gene was used for Plasmodium species identification. Pfk13 was amplified by nested PCR, and subjected to nucleotide sequencing. RESULTS: Five of 33 P. falciparum sporozoite samples carried the P553L mutation at the PfK13 locus. This mutation has been recorded previously in Vietnam, but not in Khanh Hoa province, were surveys of K13 polymorphism have not previously been carried out. CONCLUSION: These results demonstrate the utility of mosquito-stage malaria parasite samples for studies on the molecular epidemiology of drug resistance.

Infectivity of symptomatic and asymptomatic Plasmodium vivax infections to a Southeast Asian vector, Anopheles dirus.

Plasmodium vivax is now the predominant species causing malarial infection and disease in most non-African areas, but little is known about its transmission efficiency from human to mosquitoes. Because the majority of Plasmodium infections in endemic areas are low density and asymptomatic, it is important to evaluate how well these infections transmit. Using membrane feeding apparatus, Anopheles dirus were fed with blood samples from 94 individuals who had natural P. vivax infections with parasitemias spanning four orders of magnitude. We found that the mosquito infection rate was positively correlated with blood parasitemia and that infection began to rise when parasitemia was >10parasites/µl. Below this threshold, mosquito infection is rare and associated with very few oocysts. These findings provide useful information for assessing the human reservoir of transmission and for establishing diagnostic sensitivity required to identify individuals who are most infective to mosquitoes.

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.