Phylogenomics revealed migration routes and adaptive radiation timing of Holarctic malaria mosquito species of the Maculipennis Group.

BACKGROUND: Phylogenetic analyses of closely related species of mosquitoes are important for better understanding the evolution of traits contributing to transmission of vector-borne diseases. Six out of 41 dominant malaria vectors of the genus Anopheles in the world belong to the Maculipennis Group, which is subdivided into two Nearctic subgroups (Freeborni and Quadrimaculatus) and one Palearctic (Maculipennis) subgroup. Although previous studies considered the Nearctic subgroups as ancestral, details about their relationship with the Palearctic subgroup, and their migration times and routes from North America to Eurasia remain controversial. The Palearctic species An. beklemishevi is currently included in the Nearctic Quadrimaculatus subgroup adding to the uncertainties in mosquito systematics. RESULTS: To reconstruct historic relationships in the Maculipennis Group, we conducted a phylogenomic analysis of 11 Palearctic and 2 Nearctic species based on sequences of 1271 orthologous genes. The analysis indicated that the Palearctic species An. beklemishevi clusters together with other Eurasian species and represents a basal lineage among them. Also, An. beklemishevi is related more closely to An. freeborni, which inhabits the Western United States, rather than to An. quadrimaculatus, a species from the Eastern United States. The time-calibrated tree suggests a migration of mosquitoes in the Maculipennis Group from North America to Eurasia about 20-25 million years ago through the Bering Land Bridge. A Hybridcheck analysis demonstrated highly significant signatures of introgression events between allopatric species An. labranchiae and An. beklemishevi. The analysis also identified ancestral introgression events between An. sacharovi and its Nearctic relative An. freeborni despite their current geographic isolation. The reconstructed phylogeny suggests that vector competence and the ability to enter complete diapause during winter evolved independently in different lineages of the Maculipennis Group. CONCLUSIONS: Our phylogenomic analyses reveal migration routes and adaptive radiation timing of Holarctic malaria vectors and strongly support the inclusion of An. beklemishevi into the Maculipennis Subgroup. Detailed knowledge of the evolutionary history of the Maculipennis Subgroup provides a framework for examining the genomic changes related to ecological adaptation and susceptibility to human pathogens. These genomic variations may inform researchers about similar changes in the future providing insights into the patterns of disease transmission in Eurasia.

First report on natural infection of the Phlebotomus tobbi by Leishmania infantum in northwestern Iran.

Visceral leishmaniasis (VL) is an important health problem in Ardebil, where it borders Azerbaijan in the northwestern Iran. In spite of the presence of both cutaneous and visceral leishmaniasis (CL and VL) in northwestern Iran, previous researches have consistently revealed the etiologic agent of VL in the region to be Leishmania infantum. This is the first report of natural infection of Phlebotomus tobbi with L. infantum in Bilesavar district in the northern part of Ardebil province bordering Azerbaijan. Polymerase chain reaction (PCR) of kDNA, ITS1-rDNA, and CPB genes of the parasite followed by restriction fragment length polymorphism (RFLP) and gene sequencing analyses revealed presence of L. infantum in six out of 433 tested female sand fly specimens. Although sand flies of P. tobbi were infrequent, two out of 32 (6.25%) females captured in the area were found infected with the parasite. Phlebotomus perfiliewi transcaucasicus, the known vector of VL in the area, were the most dominant species but only four out of 273 (1.47%) tested were infected with L. infantum. This study showed that P. tobbi similar to P. perfiliewi transcaucasicus could play a significant role in the transmission of the L. infantum. However more investigations are needed to demonstrate that L. infantum is the only species circulating in the focus.

Lectin-carbohydrate recognition mechanism of Plasmodium berghei in the midgut of malaria vector Anopheles stephensi using quantum dot as a new approach.

Potential targets of Plasmodium ookinetes at the mosquito midgut walls were investigated in relation to interfering malarial transmission. In this study, the essential application of Quantum Dots (QDs) was used to examine the interaction between Plasmodium berghei ookinetes and the Anopheles stephensi midgut, based on lectin-carbohydrate recognition. Two significant lectins were utilized to determine this interaction. Two QDs, cadmium telluride (CdTe)/CdS and cadmium selenide (CdSe)/CdS, were employed in staining Plasmodium ookinete to study its interaction in the midgut of the mosquito vector in vivo. Concurrently, two lectins, wheat germ agglutinin (WGA) and concanavalin A (Con A), were inadvertently exploited to mask lectin binding sites between ookinetes and mosquito midgut cells. The numbers of ookinetes in both lumen and epithelial cells were eventually counted, following adequate preparation of wax sections extracted from whole midgut, and subsequent examination using a differential interference contrast a fluorescence microscopic technique. Interestingly, we detected that neither of the QDs mutated ookinete invasion into the midgut cells of the investigated mosquitoes. QD staining of ookinetes remained permanent despite the effective embedding procedure. The massive binding potency of ookinetes to midgut cells of the cross-examined mosquitoes undoubtedly revealed that Con A did not interrupt ookinete penetration into the midgut wall. In contrast, WGA inhibited ookinete invasion into the midgut cells. The results proved that QD nanoparticles are biocompatible, non-toxic to P. berghei and stable to photobleaching. The QDs staining, which was successfully implemented for ookinete labelling, is a simple and effective tool which plays a crucial role in bioimaging including the study of parasite-vector interactions.

Mosquito genomics. Highly evolvable malaria vectors: the genomes of 16 Anopheles mosquitoes.

Variation in vectorial capacity for human malaria among Anopheles mosquito species is determined by many factors, including behavior, immunity, and life history. To investigate the genomic basis of vectorial capacity and explore new avenues for vector control, we sequenced the genomes of 16 anopheline mosquito species from diverse locations spanning ~100 million years of evolution. Comparative analyses show faster rates of gene gain and loss, elevated gene shuffling on the X chromosome, and more intron losses, relative to Drosophila. Some determinants of vectorial capacity, such as chemosensory genes, do not show elevated turnover but instead diversify through protein-sequence changes. This dynamism of anopheline genes and genomes may contribute to their flexible capacity to take advantage of new ecological niches, including adapting to humans as primary hosts.

Preferential feeding success of laboratory reared Anopheles stephensi mosquitoes according to ABO blood group status.

Recent epidemiological evidences revealed a higher rate of O blood group in the residents of malaria-endemic areas suggesting that groups A, B, and AB associated with a higher disease severity and fatality. Also recent data showed the low prevalence of AB group within the malaria-endemic residents in south of Iran and India. The aim of this study was to determine the ABO blood groups preference of Anopheles stephensi which is the main malaria vector in Iran, southwest Asia, and India. An. stephensi mosquitoes were fed either artificially on A/B/O/AB membrane blood feeders or directly on human volunteer hands and forearms of A/B/O/AB groups in a cage under lab conditions. Phenotype and genotype analyzes of 450-blood-fed mosquito specimens using agglutination and multiplex-allele-specific PCR revealed a significant blood preference of An. stephensi to AB group (40%) than other groups of A (24%), B (21%), and O (15%) in combination of both experiments. High preference of An. stephensi to AB group might increase malaria infection and fatality in this blood group and resulted in low frequency of AB group in the residents of malaria endemic areas. The data suggested that malaria vectors, like parasites may have selection pressure on human genotypes.

Community sleeping pattern and anopheline biting in southeastern Iran: a country earmarked for malaria elimination.

An important variable in determining the vectorial capacity of malaria mosquito species is the degree of mosquito-human contact. This parameter can be affected by community sleeping behavior and the host-feeding habits of vectors. A cross-sectional study of 775 randomly selected inhabitants, including 385 Baluchi residents and 390 Afghani refugees, was conducted in a malarious area in Sabaz District, Sistan-Baluchestan Province, southeastern Iran. In addition, monitoring of human landing periodicity of main malaria vectors was carried out during an entire transmission season. Afghanis and Baluchis showed diversity in sleeping behavior. Most (79.6%) respondents were familiar with symptoms of malaria and also aware of an association between mosquitoes and malaria. Despite this familiarity, 94.6% of Afghan refugees, 74.8% of Baluch residents, and 87.2% of study participants did not use self-protection preventive measures. Overall, only 8.8% of participants reported using bed nets regularly. Surveyed persons used bed nets mainly during second quarter of night. Three major species of malaria vectors (Anopheles culicifacies, An. fluviatilis, and An. stephensi) started biting by sunset and continued throughout the night. The results of present study indicated that synchronization of encounters between inhabitants and mosquito vectors was caused by poor self-protection and sleeping behavior of inhabitants. In addition, diversity in culture and behavior of the two communities may cause the prevalence of malaria to be different between them. Therefore, promoting awareness of self-protection against mosquito bites could promote community participation in malaria elimination program in this malaria-endemic region.