piggyBac transposon remobilization and enhancer detection in Anopheles mosquitoes

Technical advances in mosquito biology are enabling the development of new approaches to vector control. Absent are powerful forward-genetics technologies, such as enhancer and gene traps, that permit determination of gene functions from the phenotypes arising from transposon insertion mutations. We show that the piggyBac transposon is highly active in the germline of the human malaria vector Anopheles stephensi. Up to 6% of the progeny from transgenic A. stephensi containing a single 6-kb piggyBac element with a marker gene expressing EGFP had the vector in new genomic locations when piggyBac transposase was provided in trans from a second integrated transgene. The active transposition of piggyBac resulted in the efficient detection of enhancers, with ~10% of the progeny with piggyBac in new locations with novel patterns of EGFP expression in third and fourth instar larvae and in adults. The availability of advanced transgenic capabilities such as efficient transposon-based forward-genetics technologies for Anopheles mosquitoes not only will accelerate our understanding of mosquito functional genomics and the development of novel vector and disease transmission control strategies, but also will enable studies by evolutionary developmental biologists, virologists, and parasitologists.     

First detection of Anopheles species mosquitoes breeding in water-filled cellars

Studies of the breeding of blood-sucking mosquitos in water-filled cellars of Astrakhan were carried out in 1980-1986 using routine entomological techniques of assessment, collection and determination of blood-sucking mosquitos. In July, 1986, Anopheles sp. breeding in the cellars of 2 multistory buildings was registered along with commonly observed breeding of C. pipiens. The rate of preimago development stages of Anopheles sp. was 2 to 10 sp. per 1 cubic meter of water surface. Water-filled cellars are full of ground water mixed with sewage. It increases epidemiological value of the fact of mosquitos' breeding in populated areas, which should be taken into account during mosquito control.     

Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes

Over evolutionary time, Wolbachia has been repeatedly transferred between host species contributing to the widespread distribution of the symbiont in arthropods. For novel infections to be maintained, Wolbachia must infect the female germ line after being acquired by horizontal transfer. Although mechanistic examples of horizontal transfer exist, there is a poor understanding of factors that lead to successful vertical maintenance of the acquired infection. Using Anopheles mosquitoes (which are naturally uninfected by Wolbachia) we demonstrate that the native mosquito microbiota is a major barrier to vertical transmission of a horizontally acquired Wolbachia infection. After injection into adult Anopheles gambiae, some strains of Wolbachia invade the germ line, but are poorly transmitted to the next generation. In Anopheles stephensi, Wolbachia infection elicited massive blood meal-induced mortality, preventing development of progeny. Manipulation of the mosquito microbiota by antibiotic treatment resulted in perfect maternal transmission at significantly elevated titers of the wAlbB Wolbachia strain in A. gambiae, and alleviated blood meal-induced mortality in A. stephensi enabling production of Wolbachia-infected offspring. Microbiome analysis using high-throughput sequencing identified that the bacterium Asaia was significantly reduced by antibiotic treatment in both mosquito species. Supplementation of an antibiotic-resistant mutant of Asaia to antibiotic-treated mosquitoes completely inhibited Wolbachia transmission and partly contributed to blood meal-induced mortality. These data suggest that the components of the native mosquito microbiota can impede Wolbachia transmission in Anopheles. Incompatibility between the microbiota and Wolbachia may in part explain why some hosts are uninfected by this endosymbiont in nature.Bacteria in the genus Wolbachia are maternally transmitted Rickettsia-like endosymbionts that infect an estimated 40–69% of arthropod species (1, 2). In many cases, Wolbachia manipulate host reproduction to spread throughout arthropod populations (3). Incongruence between Wolbachia and host phylogenies indicate that horizontal transfer of the symbiont has been commonplace over evolutionary time (4, 5), enabling Wolbachia to invade new species. However, there is a poor understanding of barriers to horizontal transmission and why some species remain uninfected. An understanding of these factors is important from an evolutionary perspective given that Wolbachia influences speciation (6, 7), and from an applied perspective as Wolbachia is being transinfected into vector species for the control of arthropod-borne disease (8–10).The ability to invade the host germ line is an important feature of Wolbachia biology that facilitates horizontal transmission, leading to the pervasive nature of this bacterium across invertebrate taxa. In order for Wolbachia to become established in a naïve host species, it must be acquired horizontally and successfully transmitted vertically (i.e., to offspring) to maintain the infection in the population. Multiple mechanisms of Wolbachia horizontal transmission have been proposed, including cohabitation, hemolymph transfer, predation, and parasitoid infection (11–15). After microinjection into Drosophila, Wolbachia infects the stem cell niches in the germ line (16, 17), and both Wolbachia-derived and host factors appear to influence tropism and bacterial density during oogenesis (17–20). Alternatively, somatic tissue may act as a reservoir for Wolbachia infection of the developing oocyte (20–23). Although pathways of horizontal transmission have been characterized in some species, identification of barriers to vertical transmission of the acquired Wolbachia infection remains elusive.Microbial conflict or incompatibility within arthropods is a potential barrier to transmission of heritable symbionts. Studies in the tick Dermacentor variabilis demonstrate competitive exclusion between maternally inherited bacteria. Transovarial transmission of Rickettsia montanensis (formerly Rickettsia montana) and Rickettsia rhipicephali is inhibited by infection with the reciprocal species (24). Similarly, infection exclusion has been observed in D. variabilis between conspecific strains of Anaplasma marginale where one strain inhibits the infection of the other (25). Competitive inter- and intraspecific microbial interactions have also been observed with Wolbachia (26, 27).Anopheles mosquitoes provide a unique system to examine microbial barriers to Wolbachia transmission. With few exceptions, Anophelines (which transmit the Plasmodium parasites that cause human malaria) are naturally uninfected with Wolbachia (28–31), suggesting the potential presence of innate barriers to infection in this genus. However, in vitro and in vivo studies indicate that Wolbachia are capable of infecting cultured Anopheles cells (32, 33), ex vivo cultured tissues (34), in vivo somatic tissue (35–37), and can stably infect the mosquito germ line (38). We investigated the ability of the native microbial community to influence vertical transmission of Wolbachia in Anopheles mosquitoes. We found that bacteria in the genus Asaia were responsible for inhibiting Wolbachia maternal transmission in this important mosquito genus.     

Illustrated keys to the mosquitoes of Thailand. IV. Anopheles

Illustrated keys for the identification of the larvae and adult female Anopheles mosquitoes of Thailand are presented along with distribution maps, tabulated bionomics information, and a checklist. A total of 73 species are treated, including 71 previously and newly described species (An. cracens = dirus B, An. scanloni = dirus C, An. baimaii = dirus D, An. latens = leucosphyrus A, and An. epiroticus = sundaicus A). Also, two undescribed species are included, i.e., An. minimus C and a new species near An. gigas. Thirty-four chromosomal forms of 14 species are discussed, with suggestions provided for resolving their taxonomic status.     

按蚊拟除虫菊酯类杀虫剂抗性相关分子机理的研究进展

在杀虫剂的选择压力下蚊虫形成抗药性,不同类型的杀虫剂,由于其作用机制不同,其抗性分子机制也会有所不同。本文对按蚊拟除虫菊酯类杀虫剂抗药性相关因素酯酶、细胞色素P450单加氧酶、谷胱甘肽-S-转移酶及神经轴突钠离子通道等的研究进展进行了综述。     

按蚊免疫相关基因及其多态性研究进展

病原体感染媒介按蚊后,会引起一系列免疫反应,其过程涉及数量众多的免疫相关基因,包括病原体识别、信号调节、信号转导及效应反应等.初步研究表明按蚊免疫相关基因多态性与其易感性和进化相关.该文就近期按蚊免疫相关基因及其多态性研究进展进行综述.     

Malaria mosquitoes of the Anopheles maculipennis (Diptera, Culicidae) complex in Georgia

Malaria mosquito larvae and imagoes underwent morphological, cytogenetic, and molecular genetic analyses in West and East Georgia. In the areas under study, the authors identified three related types of malaria mosquitoes of the maculipennis complex: An. maculipennis Meigen, 1818; An. melanoon Hacket, 1934; An. sacharovi Favre, 1903. The authors revealed the types An. maculipennis and An. melanoon in the Kolkhida cavity (West Georgia) and An. maculipennis and An. sacharovi in the Iveria cavity (East Georgia). The morphology of eggs was defined in the study types of mosquitoes. An. melanoon ovipositions similar in the exochorion pattern with An. messeae eggs were found in West Georgia.     

Effects of anticoagulants on Plasmodium vivax oocyst development in Anopheles albimanus mosquitoes

Artificial membrane feeding (AMF) assays are used to determine malaria transmission-blocking activity in Anopheles. The purpose of this study was to determine the effect of the most widely used anticoagulants, EDTA and heparin, on development of the Plasmodium vivax sporogonic cycle. Blood samples collected from 60 patients carrying P. vivax infections were used to feed An. albimanus using AMF. Seven days after feeding, mosquitoes were dissected to assess mosquito infection. Mosquitoes fed with blood containing EDTA showed a lower mean oocyst number as compared with those fed blood with heparin. However, this effect was minimized upon reduction of EDTA concentrations in the serum. This result may be explained by the fact that microgametocytes require Ca(2+), Mn(2+), and Mg(+2) to activate enzymes important for exflagellation process and for motility of ookinetes. We therefore recommend that heparin be used as the anticoagulant of choice for blood used in AMF assays.     

Water management for controlling the breeding of Anopheles mosquitoes in rice irrigation schemes in Kenya

An experiment to assess the impact of intermittent irrigation on Anopheles larval populations, rice yields and water use was conducted in the Mwea rice irrigation scheme in Kenya. Four water regimes including intermittent irrigation were tested in a complete randomized block experimental design. Intermittent irrigation was carried out on a weekly schedule, with flooded conditions from Saturday through Tuesday morning. Larval sampling at each plot was conducted every Monday and prior to draining of intermittently irrigated subplots on Tuesday. All the adult anopheline mosquitoes emerging from larvae collected in the experimental plots were identified as being An. arabiensis. By far the highest numbers of An. arabiensis 1st instar larvae were found in the intermittently irrigated subplots, indicating that the water regime provided the most attractive environment for egg laying. However, the ratio between the 4th and 1st instar larvae in the subplots was only 0.08, indicating very low survival rates. In contrast, the 4th/1st instar ratio for subplots with other water management regimes ranged between 0.27 and 0.68, suggesting a correspondingly higher survival than observed with intermittent irrigation. The total number of 4th instars was almost the same in the intermittently irrigated subplots and the irrigation system normally practised by the farmers. The failure to eliminate larval development up to the 4th instar in the former method was attributed to residual pools of water. Larval abundance fluctuated throughout the 12-week sampling period. The highest larval densities were recorded in the 3 weeks after transplanting the rice seedlings. Afterwards, larval numbers dropped dramatically as the height of rice plants increased. Rice yields at harvest did not show statistically significant differences among subplots with different water regimes. The average yield per hectare ranged from 4.8-5.3 metric tonnes. The average daily water percolation/seepage rate was 3.6 mm and did not significantly differ among different water management regimes. Further research is necessary to, among other things, determine whether rice yields could be increased by having flooded and drained intervals that were different from those used in this study. It would likewise be important to assess on a wider scale the feasibility of implementing intermittent irrigation with respect to farmer acceptance and required changes in irrigation system design and management.     

Taxonomical observations on Anopheles philippinensis/nivipes group mosquitoes in north-east India

To examine the distribution pattern of Anopheles philippinensis and An. nivipes, mosquitoes belonging to An. philippinensis/nivipes group were collected from five sites in north-east Indian state of Assam and its adjoining areas and identified. Presence of both An. philippinensis and An. nivipes, with predominance of the latter taxon, was recorded with conformity on the basis of pupal paddle characteristics. Based on adult wing characters, 91.4% specimens were identified as An. philippinensis and 8.6% as An. nivipes with gradually increasing proportion of An. nivipes in westwardly direction. An. philippinensis/ nivipes was noted to be mid night biter, mainly exophagic and exclusively exophilic. Of the two species, An. philippinensis appears to be mostly anthropophilic and available in broken forest areas whereas An. nivipes seems to be mostly zoophilic and generally found in ecotone zones in plains areas.     

Prevention of sporogony of Plasmodium vivax in Anopheles dirus mosquitoes by transmission-blocking antimalarials

The sporontocidal activity of four dihydroacridine-diones (WR-233602, WR-243251, WR-250547, and WR-250548) and three fluoroquinolones (WR-279135, WR-279298, and WR-279288) was determined against naturally circulating isolates of Plasmodium vivax. Laboratory-reared Anopheles dirus mosquitoes were infected with P. vivax by feeding them on gametocytemic volunteers reporting to local malaria clinics in Kanchanaburi and Tak provinces, Thailand. Four days after the infectious feed, mosquitoes were re-fed on uninfected mice treated 90 minutes previously with a given drug at a dose of 100 mg base drug/kg mouse body weight. Sporontocidal activity was determined by assessing both oocyst and sporozoite development. None of the fluoroquinolones exhibited sporontocidal activity against P. vivax, whereas all 4 dihydroacridine-diones affected sporogonic development to some degree. WR-233602 affected oocyst development, but had no impact on sporozoite production, WR-250548 affected oocyst development and had a limited effect on sporozoite production, and WR-243251 and WR-250547 had a marked impact on all phases of sporogony. These data demonstrate that experimental dihydroacridine-diones are capable of interrupting the sporogonic development of P. vivax. These compounds may be useful in preventing malaria transmission.