Metagenomics, paratransgenesis and the Anopheles microbiome: a portrait of the geographical distribution of the anopheline microbiota based on a meta-analysis of reported taxa

Anophelines harbour a diverse microbial consortium that may represent an extended gene pool for the host. The proposed effects of the insect microbiota span physiological, metabolic and immune processes. Here we synthesise how current metagenomic tools combined with classical culture-dependent techniques provide new insights in the elucidation of the role of the Anopheles-associated microbiota. Many proposed malaria control strategies have been based upon the immunomodulating effects that the bacterial components of the microbiota appear to exert and their ability to express anti-Plasmodium peptides. The number of identified bacterial taxa has increased in the current “omics” era and the available data are mostly scattered or in “tables” that are difficult to exploit. Published microbiota reports for multiple anopheline species were compiled in an Excel® spreadsheet. We then filtered the microbiota data using a continent-oriented criterion and generated a visual correlation showing the exclusive and shared bacterial genera among four continents. The data suggested the existence of a core group of bacteria associated in a stable manner with their anopheline hosts. However, the lack of data from Neotropical vectors may reduce the possibility of defining the core microbiota and understanding the mosquito-bacteria interactive consortium.

Is there an efficient trap or collection method for sampling Anopheles darlingi and other malaria vectors that can describe the essential parameters affecting transmission dynamics as effectively as human landing catches? - A Review

Distribution, abundance, feeding behaviour, host preference, parity status and human-biting and infection rates are among the medical entomological parameters evaluated when determining the vector capacity of mosquito species. To evaluate these parameters, mosquitoes must be collected using an appropriate method. Malaria is primarily transmitted by anthropophilic and synanthropic anophelines. Thus, collection methods must result in the identification of the anthropophilic species and efficiently evaluate the parameters involved in malaria transmission dynamics. Consequently, human landing catches would be the most appropriate method if not for their inherent risk. The choice of alternative anopheline collection methods, such as traps, must consider their effectiveness in reproducing the efficiency of human attraction. Collection methods lure mosquitoes by using a mixture of olfactory, visual and thermal cues. Here, we reviewed, classified and compared the efficiency of anopheline collection methods, with an emphasis on Neotropical anthropophilic species, especially Anopheles darlingi, in distinct malaria epidemiological conditions in Brazil.

Anopheles species composition explains differences in Plasmodium transmission in La Guajira, northern Colombia

Malaria in La Guajira, the most northern state of Colombia, shows two different epidemiological patterns. Malaria is endemic in the municipality of Dibulla whereas in Riohacha it is characterised by sporadic outbreaks. This study aimed to establish whether differences in transmission patterns could be attributed to different vector species. The most abundant adult female species were Anopheles aquasalis, exclusive to Riohacha, and Anopheles darlingi, restricted to Dibulla. Anopheles mosquitoes were identified using morphology and the molecular markers internal transcribed spacer 2 and cytochrome c oxidase I. All specimens (n = 1,393) were tested by ELISA to determine natural infection rates with Plasmodium falciparum and Plasmodium vivax. An. darlingi was positive for P. vivax 210, with an infection rate of 0.355% and an entomological inoculation rate of 15.87 infective bites/person/year. Anopheles albimanus larvae were the most common species in Riohacha, found in temporary swamps; in contrast, in Dibulla An. darlingi were detected mainly in permanent streams. Distinctive species composition and larval habitats in each municipality may explain the differences in Plasmodium transmission and suggest different local strategies should be used for vector control.

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.

Anopheles darlingi polytene chromosomes: revised maps including newly described inversions and evidence for population structure in Manaus

Salivary gland polytene chromosomes of 4th instar Anopheles darlingi Root were examined from multiple locations in the Brazilian Amazon. Minor modifications were made to existing polytene photomaps. These included changes to the breakpoint positions of several previously described paracentric inversions and descriptions of four new paracentric inversions, two on the right arm of chromosome 3 and two on the left arm of chromosome 3 that were found in multiple locations. A total of 18 inversions on the X (n = 1) chromosome, chromosome 2 (n = 7) and 3 (n = 11) were scored for 83 individuals from Manaus, Macapá and Porto Velho municipalities. The frequency of 2Ra inversion karyotypes in Manaus shows significant deficiency of heterozygotes (p < 0.0009). No significant linkage disequilibrium was found between inversions on chromosome 2 and 3. We hypothesize that at least two sympatric subpopulations exist within the An. darlingi population at Manaus based on inversion frequencies.

Microarray and RNAi Analysis of P450s in Anopheles gambiae Male and Female Steroidogenic Tissues: CYP307A1 Is Required for Ecdysteroid Synthesis.

In insects, the steroid hormone 20-hydroxyecdysone (20E) coordinates major developmental transitions. While the first and the final steps of 20E biosynthesis are characterized, the pathway from 7-dehydrocholesterol to 5β-ketodiol, commonly referred as the "black box", remains hypothetical and whether there are still unidentified enzymes is unknown. The black box would include some oxidative steps, which are believed to be mediated by P450 enzymes. To identify new enzyme(s) involved in steroid synthesis, we analyzed by small-scale microarray the expression of all the genes encoding P450 enzymes of the malaria mosquito Anopheles gambiae in active steroidogenic organs of adults, ovaries from blood-fed females and male reproductive tracts, compared to inactive steroidogenic organs, ovaries from non-blood-fed females. Some genes encoding P450 enzymes were specifically overexpressed in female ovaries after a blood-meal or in male reproductive tracts but only three genes were found to be overexpressed in active steroidogenic organs of both females and males: cyp307a1, cyp4g16 and cyp6n1. Among these genes, only cyp307a1 has an expression pattern similar to other mosquito steroidogenic genes. Moreover, loss-of-function by transient RNAi targeting cyp307a1 disrupted ecdysteroid production demonstrating that this gene is required for ecdysteroid biosynthesis in Anopheles gambiae.

Ultrastructure of the eggs of two species of Anopheles (Anopheles) Meigen (Diptera, Culicidae)

Egg ultrastructure of two morphologicaly similar species of the Arribalzagia Series, Anopheles (A.) costai Fonseca & Ramos, 1939 and A. (A.) mediopunctatus (Lutz, 1903) are described and illustrated using scanning electron micrographs. Although similar in the adult stage, male genitalia structures and larval and pupal stages, the eggs of these species are distinct. The eggs of A. costai and A. mediopunctatus are compared with that of A. forattinii Wilkerson & Sallum, 1999. Incomplete illustration of egg of A. forattinii is provided for comparison.

Revision of the Leucosphyrus group of Anopheles (Cellia) (Diptera, Culicidae)

Revision of the Leucosphyrus Group of Anopheles (Cellia) (Diptera, Culicidae). This is a comprehensive revision of 20 species of the Leucosphyrus Group of the Neomyzomyia Series of Anopheles (Cellia). Morphological description of the adults, male and female, male genitalia, pupa and fourth-instar larva are provided for each taxon in addition to bionomics, distribution data and systematic discussion for each species, including diagnostic characters. Identification keys for females and fourth-instar larvae are provided. When possible medical importance of each species is included. Illustrations of of the adults, fourth-instar larvae and pupae are provided. Distribution maps for each species are mainly based on the material examined; however, when possible published data were also used. Tables on adult character variations, fourth-instar larval and pupal setal branching are included as appendices. A neotype for An. takasagoensis Morishita and An. sulawesi Koesoemawinangoen, and a lectotype for An. balabacensis Baisas, are designated. The authorship of An. sulawesi previously cited as Waktoedi is corrected to Koesoemawinangoen.

Papel da Coagulação na Resposta do Mosquito Anopheles Gambiae ao Parasita da malária Plasmodium Berghei

Malária é uma doença parasitária infecciosa aguda ou crónica, causada pelo protozoário do género Plasmodium que é transmitido ao homem através de picada de mosquitos fêmeas do género Anopheles. Causa a morte a milhões de pessoas por ano, na sua maioria crianças até aos 5 anos de idade. A inexistência de estratégias eficazes, contra a transmissão da malária, deve-se sobretudo à falta de conhecimento de moléculas cruciais ao desenvolvimento do parasita no vector. Mecanismos de reconhecimento do parasita e a resposta imune do mosquito à infecção são claramente alvos de novas estratégias de controlo da malária. O ciclo natural de transmissão de Plasmodium requer a conclusão com sucesso, do ciclo esporogónico no intestino médio e nas glândulas salivares do mosquito Anopheles, um processo que demora cerca de duas semanas. Este processo de desenvolvimento pode ser bloqueado pelo sistema imune inato do mosquito, resultando assim na eliminação do parasita do vector. A resposta imunológica envolve vários mecanismos como a fagocitose, encapsulação, nodulação, síntese de péptidos antimicrobianos e coagulação, que são acompanhadas pela activação proteolítica da pró-fenoloxidase presente na hemolinfa.O sistema imune é um factor determinante da capacidade vectorial do mosquito, pelo que vários estudos têm sido feitos para melhor compreender as respostas do mosquito Anopheles, principal vector da malária, ao parasita Plasmodium. Uma das principais respostas desencadeadas pelo sistema imune do mosquito é a coagulação. Estudos recentes demonstram que a coagulação da hemolinfa de Anopheles requer actividade da fenoloxidase e difere de Drosophila s.p na formação, estrutura e composição. O objectivo deste trabalho é estudar o papel da coagulação na resposta do mosquito Anopheles gambiae ao parasita da malária Plasmodium berghei, através do estudo da transglutaminase. Para tal foi feita a caracterização dos genes que codificam para a transglutaminases em A. gambiae, através da sequenciação destes genes e dos seus transcritos, verificando-se alguns polimorfismos quando comparada com a sequência do genoma disponível na base de dados. Para caracterizar do papel desta enzima durante a infecção com P. berghei, foi feita a inibição da actividade enzimática dos genes AGAP009097 e AGAP009098 que codificam para a transglutaminases. Foi feita a descrição da dinâmica de transcrição durante a infecção e o silenciamento destes mesmos genes usando dsRNA. Observou-se um aumento da taxa de infecção e do número médio de oocistos por intestino médio nos mosquitos em que as transglutaminases foram inibidas quimicamente bem como naqueles que possuíam os genes silenciados, quando comparados com os grupos controlo. Com este trabalho espera-se ter contribuído para uma melhor compreensão do funcionamento da capacidade imunológica dos mosquitos na resposta ao parasita da malária e a possibilidade de manipular o sistema imune dos mesmos de modo a eliminar o parasita e contribuir para a diminuição/irradicação da malária, uma das principais doença e causa de morte a nível mundial.

PCR-RAPD and PCR-RFLP polymorphisms detected in Anopheles cruzii (Diptera, Culicidae)

The aim of the present study was to examine genetic variability in populations of An. cruzii by employing PCR-RAPD and PCR-RFLP markers. All analyses were carried out using individuals of the F1 generation of wild caught females obtained in Santa Catarina State (Florianópolis and São Francisco do Sul), Paraná State (Morretes, Paranaguá and Guaratuba) and São Paulo State (Cananéia). In the PCR-RAPD experiments, seven primers were used for comparisons within and among populations. The restriction profile of the ITS2 including a fragment of both 5.8S and 28S regions of the rDNA was obtained with the enzymes BstUI, HaeIII, TaqI, HhaI, Sau96I, HinfI, HincII and NruI. The PCR-RAPD method detected a large number of polymorphic bands. Genetic distance among populations of An. cruzii varied from 0,0214 to 0,0673, suggesting that all individuals used in the analyses belong to a single species. The number of migrants per generation (Nm) was 4.3, showing the existence of gene flow among populations. The restriction profile of the ITS2, 5.8S and 28S gene regions was similar in all An. cruzii samples, whereas the results obtained by using HhaI and NruI are indicative that the individuals analyzed have nucleotide sequences distinct from those of An. cruzii samples from Peruíbe and Juquiazinho deposited in GenBank.

Records of Anopheles (Nyssorhynchus) (Diptera, Culicidae) in artificial containers in Ribeirão Preto City, State of São Paulo, Brazil

Thirty immatures of Anopheles darlingi Root, Anopheles argyritarsis Robineau-Desvoidy and Anopheles rondoni (Neiva & Pinto) (were found in an artificial lagoon situated near Pardo River, in Ribeirão Preto City, State of São Paulo, Brazil. The presence of immatures of Anopheles darlingi breeding in a lagoon is suggestive of the potential capacity of that species to adapt and occupy new habitats and to invade urban areas along the species distribution range and also disperse to new localities.

Notes on the holotype of Anopheles marajoara Galvão & Damasceno (Diptera, Culicidae)

During studies on the dynamics of malaria transmission in Marajó Island, State of Pará, Brazil, Galvão & Damasceno (1942) collected a single specimen of a new species that they named Anopheles (Nyssorhynchus) marajoara Galvão & Damasceno, 1942. Now, examining genitalia slide associated to the holotype, we observed that the ventral claspette of the male genitalia is distinct from those of all other species of the Argyritarsis Section and consequently from members of the complex Anopheles albitarsis Lynch Arribalzaga, 1878. The male genitalia of the slide belong to a specimen of Anopheles aquasalis Curry, 1932, nevertheless, it was originally labeled as Anopheles marajoara. To solve this problem, we are setting aside the male genitalia slide associated with the holotype of Anopheles marajoara and excluding it from the type material. Illustrations of the male genitalia and adult male are included.

Aspectos ecológicos de Anopheles cruzii e Culex ribeirensis (Diptera, Culicidae) da Mata Atlântica de Morretes, Paraná, Brasil

Um estudo foi realizado sobre mosquitos adultos de abril 1995 a março 1996 na Mata Atlântica do município de Morretes, Paraná, Brasil. A investigação foi procedida com auxílio de duas armadilhas luminosas CDC-M instaladas em estratos verticais diferentes. Os mosquitos foram capturados mensalmente durante um ano, com início às 18 horas e término às 6 horas do dia seguinte, totalizando 144 horas de trabalho de campo. Obteve-se 1.408 exemplares de mosquitos (409 na copa e 999 próximo ao solo), pertencentes a 10 gêneros e 31 espécies. Anopheles cruzii e Culex ribeirensis foram predominantes e são objetos do presente estudo. Não foi observada diferença entre as armadilhas para Anopheles cruzii. Mas Culex ribeirensis foi coletado em maior número pela CDC-M/solo. Anopheles cruzii, quanto à freqüência horária, apresentou picos nas primeiras horas da noite, depois a sua atividade decresceu progressivamente até o crepúsculo matutino, sem apresentar um pico secundário. Em referência a distribuição mensal, Anopheles cruzii foi mais freqüente nos meses de abril e maio de 1995 e março de 1996. Não houve correlação do número de exemplares com a temperatura ou precipitações pluviométricas. Culex ribeirensis apresentou maior atividade de vôo de 22h às 4h, mas não houve picos significativos. Nas coletas obtidas por mês, Culex ribeirensis teve picos em dezembro e janeiro. Houve correlação do número de espécimes deste culicíneo com a temperatura e precipitações pluviométricas. Constituem os primeiros registros para o Estado do Paraná: Ochlerotatus rhyacophilus, Culex misionensis, Culex pedroi, Culex ribeirensis e Culex zeteki.

Primeiro registro de Anopheles (Kerteszia) homunculus Komp (Diptera, Culicidae) no Estado do Espírito Santo, Brasil

Em pesquisa de campo realizada no interior do Estado do Espírito Santo, Brasil, em janeiro de 2006, como parte de projeto sobre a transmissão de Plasmodium, foram coletadas larvas de anofelinos em bromélias. Os imaturos foram mantidos no laboratório até a obtenção dos adultos machos e fêmeas associados com as exúvias das larvas e das pupas, para serem identificados. Conseqüentemente, verificou-se que dois espécimes pertenciam a Anopheles (Kerteszia) homunculus Komp, 1937. Este é o primeiro registro dessa espécie de Kerteszia no Espírito Santo. O encontro evidencia a importância de estudos adicionais de modo a estabelecer a distribuição geográfica do An. homunculus, bem como o status taxonômico e a importância epidemiológica da espécie na dinâmica da transmissão da malária em áreas de Mata Atlântica.

Anopheles goeldii Rozeboom & Gabaldón (Diptera, Culicidae): a species of the Nuneztovari Complex of Anopheles Meigen

Anopheles (Nyssorhynchus) goeldiiRozeboom & Gabaldón, 1941, a species of the Nuneztovari Complex, was described based on morphological characteristics of the male, female, larva, pupa, and eggs. The type locality is Boa Vista (= Fordlândia), a district in the vicinity of Rio Tapajós, in the municipality of Aveiro, in the state of Pará, Brazil. Anopheles goeldii is redescribed based on morphological traits of the fourth instar larva, pupa, egg, and male and female. DNA sequences from the cytochrome oxidase subunit I (COI barcode region) of the mitochondrial genome were utilized for species characterization. Specimens of An. goeldii from the Pará, Amapá, and Amazonas states were employed to redescribe the species and to compare with morphologically similar taxa.