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.

The complete mitochondrial genome of major malaria vector Anopheles anthropophagus (Diptera: Culicidae) in China.

Anopheles anthropophagus (Xu and Feng 1975) is the major vector of malaria in Eastern and Southern China. The species An. anthropophagus is considered a synonym of An. lesteri (Baisas & Hu, 1936), although they differ in several key biological characteristics. Here, we report the complete mitochondrial genome of An. anthropophagus for the first time. The mitogenome of An. anthropophagus is a typical circular, double-stranded molecule with a total length of 15,413 base pairs, and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and an AT-rich control region. A phylogenetic analysis of the complete mitogenomes of 16 species of Anopheles (Culicidae) revealed that An. anthropophagus is closely related to An. sinensis (Wiedemann 1828), in the family Culicidae. The An. anthropophagus mitogenome provides new data for further taxonomic and phylogenetic studies of the genus Anopheles.

Immune responses of Aedes togoi, Anopheles paraliae and Anopheles lesteri against nocturnally subperiodic Brugia malayi microfilariae during migration from the midgut to the site of development.

BACKGROUND: Lymphatic filariasis is a mosquito-borne disease caused by filarioid nematodes. A comparative understanding of parasite biology and host-parasite interactions can provide information necessary for developing intervention programmes for vector control. Here, to understand such interactions, we choose highly susceptible filariasis vectors (Aedes togoi and Anopheles lesteri) as well as Anopheles paraliae, which has lower susceptibility, infected them with nocturnally subperiodic (NSP) Brugia malayi microfilariae (mf) and studied the exsheathment, migration and innate immune responses among them. METHODS: Mosquito-parasite relationships were systematically investigated from the time mf entered the midgut until they reached their development site in the thoracic musculature (12 time points). RESULTS: Results showed that exsheathment of B. malayi mf occurred in the midgut of all mosquito species and was completed within 24 h post-blood meal. The migration of B. malayi mf from the midgut to thoracic muscles of the highly susceptible mosquitoes Ae. togoi and An. lesteri was more rapid than in the low susceptibility mosquito, An. paraliae. Melanisation and degeneration, two distinct refractory phenotypes, of mf were found in the midgut, haemocoel and thoracic musculature of all mosquito species. Melanisation is a complex biochemical cascade that results in deposition of melanin pigment on a capsule around the worms. Also, some biological environments in the body are inhospitable to parasite development and cause direct toxicity that results in vacuolated or degenerated worms. Even though Ae. togoi is highly susceptible to B. malayi, melanisation responses against B. malayi mf were first noted in the haemocoel of Ae. togoi, followed by a degeneration process. In contrast, in An. lesteri and An. paraliae, the degeneration process occurred in the haemocoel and thoracic musculature prior to melanisation responses. CONCLUSION: This study provides a thorough description of the comparative pathobiology of responses of mosquitoes against the filarial worm B. malayi.

Effects of cross-mating on susceptibility of synonymous mosquitoes, Anopheles paraliae and Anopheles lesteri to infection with nocturnally subperiodic Brugia malayi.

In Southeast Asia, Anopheles lesteri (recently synonymized with An. paraliae) is a competent vector for Plasmodium parasites, but its ability to transmit parasites that cause lymphatic filariasis has yet to be determined. In this study, the susceptibility of An. lesteri and An. paraliae to Brugia malayi parasites was determined by comparing with the control mosquito, Aedes togoi. We found that the infection prevalence per infected mosquito in An. paraliae was significantly lower than that in Ae. togoi in all experiments (p < 0.05). Reciprocal crosses (female An. paraliae x male An. lesteri) produced highly susceptible F1-hybrid progeny, with increased infection prevalence when compared to parental stocks (p < 0.05). Subsequently, the possibilities of introgression between high and low/moderate parasite susceptibility genes were investigated by cross-mating experiments (parental, reciprocal crosses, back crosses and repeated backcrosses). The results showed the possibility of introgression of B. malayi-susceptible genes between An. paraliae (low/moderate susceptibility) and An. lesteri (high susceptibility) based on increasing or decreasing susceptibility and normal larval development in the thoracic muscles of F3-hybrids. Additionally, melanization, an innate immune response with proven involvement in the susceptibility or refractoriness of mosquitoes to B. malayi parasites, was examined. Parasite degeneration and cell aggregation, and melanization were observed for first-stage larvae in the thoracic muscle fibers of hybrid mosquitoes.

Structural divergence of chromosomes between malaria vectors Anopheles lesteri and Anopheles sinensis.

BACKGROUND: Anopheles lesteri and Anopheles sinensis are two major malaria vectors in China and Southeast Asia. They are dramatically different in terms of geographical distribution, host preference, resting habitats, and other traits associated with ecological adaptation and malaria transmission. Both species belong to the Anopheles hyrcanus group, but the extent of genetic differences between them is not well understood. To provide an effective way to differentiate between species and to find useful markers for population genetics studies, we performed a comparative cytogenetic analysis of these two malaria vectors. RESULTS: Presented here is a standard cytogenetic map for An. lesteri, and a comparative analysis of chromosome structure and gene order between An. lesteri and An. sinensis. Our results demonstrate that much of the gene order on chromosomes X and 2 was reshuffled between the two species. However, the banding pattern and the gene order on chromosome 3 appeared to be conserved. We also found two new polymorphic inversions, 2Lc and 3Rb, in An. lesteri, and we mapped the breakpoints of these two inversions on polytene chromosomes. CONCLUSIONS: Our results demonstrate the extent of structural divergence of chromosomes between An. lesteri and An. sinensis, and provide a new taxonomic cytogenetic tool to distinguish between these two species. Polymorphic inversions of An. lesteri could serve as markers for studies of the population structure and ecological adaptations of this major malaria vector.

Genetic compatibility between Anopheles lesteri from Korea and Anopheles paraliae from Thailand

To assess differentiation and relationships between Anopheles lesteri and Anopheles paraliae we established three and five iso-female lines of An. lesteri from Korea and An. paraliae from Thailand, respectively. These isolines were used to investigate the genetic relationships between the two taxa by crossing experiments and by comparing DNA sequences of ribosomal DNA second internal transcribed spacer (ITS2) and mitochondrial DNA cytochrome c oxidase subunit I (COI) and subunit II (COII). Results of reciprocal and F1-hybrid crosses between An. lesteri and An. paraliae indicated that they were compatible genetically producing viable progenies and complete synaptic salivary gland polytene chromosomes without inversion loops in all chromosome arms. The pairwise genetic distances of ITS2, COI and COII between these morphological species were 0.040, 0.007-0.017 and 0.008-0.011, respectively. The specific species status of An. paraliae in Thailand and/or other parts of the continent are discussed.

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

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

Mosquito Species Composition and Plasmodium vivax Infection Rates on Baengnyeong-do (Island), Republic of Korea

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

Population dynamics of five anopheles species of the Hyrcanus group in northern Gyeonggi-do, Korea.

To investigate the population densities of potential malaria vectors, Anopheles species were collected by light traps in malaria endemic areas, Paju and Gimpo, Gyeonggi-do of Korea. Five Anopheles Hyrcanus sibling species (An. sinensis, An. pullus, An. lesteri, An. kleini, and An. belenrae) were identified by PCR. The predominant species, An. pullus was collected during the late spring and mid-summer, while higher population consists of An. sinensis were collected from late summer to early autumn. These 2 species accounted for 92.1% of all Anopheles mosquitoes collected, while the other 3 species accounted for 7.9%. Taking into account of these population densities, late seasonal prevalence, and long-term incubation period (9-13 months) of the Korean Plasmodium vivax strain, An. sinensis s.s is thought to play an important role in the transmission of vivax malaria in the study areas.

Population Dynamics of Five Anopheles Species of the Hyrcanus Group in Northern Gyeonggi-do, Korea

To investigate the population densities of potential malaria vectors, Anopheles species were collected by light traps in malaria endemic areas, Paju and Gimpo, Gyeonggi-do of Korea. Five Anopheles Hyrcanus sibling species (An. sinensis, An. pullus, An. lesteri, An. kleini, and An. belenrae) were identified by PCR. The predominant species, An. pullus was collected during the late spring and mid-summer, while higher population consists of An. sinensis were collected from late summer to early autumn. These 2 species accounted for 92.1% of all Anopheles mosquitoes collected, while the other 3 species accounted for 7.9%. Taking into account of these population densities, late seasonal prevalence, and long-term incubation period (9-13 months) of the Korean Plasmodium vivax strain, An. sinensis s.s is thought to play an important role in the transmission of vivax malaria in the study areas.

Vector competence of Anopheles lesteri Baisas & Hu (Diptera: Cullicidae) to Plasmodium vivax in Korea

Three anopheline mosquitoes in Korea were studied for their abilities as vectors for Plasmodium vivax. The female mosquitoes of Anopheles lesteri, An. pullus and An. sinensis were allowed to suck malaria patient blood until fully fed, and they were then bred for 2 weeks to develop from malaria parasites to sporozoites. The result from the above confirmed the sporozoites in one An. lesteri of one individual and five An. sinensis of six individuals. We also reconfirmed that An. sinensis was the main vector to transmit malaria and An. lesteri as well as An. sinensis were able to carry Korean malaria parasites. Therefore, we propose that diversified study is needed to manage malaria projects.

ON MOLECULAR IDENTIFICATION AND TAXONOMIC STATUS OF ANOPHELES LESTERI AND ANOPHELES ANTHROPOPHAGUS IN CHINA (DIPTERA:CULICIDAE) / 中国寄生虫学与寄生虫病杂志

Objective] To clarify the taxonomic status of Anopheles lesteri and An.anthropophagus in China. [Methods] Using molecular identification (PCR assay and rDNA\|ITS2 sequencing) to examine the field anopheline mosquito specimens from Liaoning and Shandong. According to the ITS2 sequences, molecular phylogenetic tree was made. [Results] According to the molecular identification, An.lesteri and An.anthropophagus were distributed both in Liaoning Province and Shandong Province. The length and GC content of rDNA\|ITS2 sequence were 451 bp, 46 2% in An.lesteri (n=6), and 448 bp, 46 0% in An.anthropophagus (n=10), respectively. The ITS2 sequences from presentation sites were same in An.lesteri, while the intraspecies difference in An.anthropophagus was 0 88%. The specific difference between An.lesteri and An.anthropophagus was 25 7%. By analyzing molecular phylogenetic tree, the relationship between An.lesteri and An.sinensis, An.anthropophagus and An.liangshanensis was found to be closer. [Conclusion] According to the molecular identification, it was defined that An.lesteri and An.anthropophagus were sympatric independent species in China.

Historical Review on the Classification and Rectification of Anopheles anthropophagus to An. lesteri in China / 中国寄生虫学与寄生虫病杂志

This paper deals with the taxonomic status and specific names of Anopheles anthropophagus and An. lesteri, the important malaria transmitting vector in China. Based on a historical review of the literature recorded from the country, substantial evidence from morphological and molecular biological studies gives reason to convince that An. anthropophagus is a synonym of An. lesteri. A resurrection of the specific name of An. lesteri Baisas et Hu, 1936 brooks no delay.