Mosquitoes catalogued and ready for study

Thematic Project conducted by the USP Public Health School organizes cataloguing of Anopheles genus mosquitoes and increases the number of known species by 76%. The material will be used in research for years to come.

By Fábio Reynol

Agência FAPESP – The vectors of several diseases that affect humans, mosquitoes of the Anopheles genus, are still not completely understood by science. Spread around Brazil, many species of these insects have been catalogued within a gene pool that has clear morphological distinctions, with veritable gaps between the groups.

 

With the objective of improving the taxonomical organization of these mosquitoes, over a period of six years researchers from Universidade de São Paulo’s School of Public Health (FSP-USP), in collaboration with five other institutions, developed the Thematic Project “Systematics of the Anopheles (Nyssorhynchus),” funded by FAPESP.

 

The study targeted the Nyssorhynchus and Anopheles darlingi subgenuses and resulted in the collection of insects from 19 Brazilian states split among four regions: the Cerrado (savanna areas), the Eastern Atlantic Rainforest, the Western Atlantic Rainforest and Amazonia.

 

The scientists collected 659 samples of 289 species, totaling approximately 30,000 mosquitoes. Of the total, 20,000 samples were frozen for molecular research and 10,000 were dried and deposited at FSP-USP’s reference collection for morphological studies.

 

“It is an immense quantity that will serve as research material for at least 10 years,” notes Maria Anice Mureb Sallum, professor at FSP-USP and coordinator of the Thematic Project, which began in 2006 and ended in 2012. During this period, the project resulted in a 76% increase in the number of known species or phylogenetic lines of the Nyssorhynchus subgenus.

 

The project formally describes four new species, and three others are currently in the description phases. In addition, four species are no longer synonymous, and 11 phylogenetic lines were discovered or corroborated. In total, 22 species or phylogenetic lines were added to the subgenus, which in 2006 had 29 valid species.

 

An analysis of molecular markers confirmed the hypothesis among researchers that populations of the mosquito vector Anopheles darlingi, found in regions of north and southeast Brazil, belong to distinct groups. The morphological studies included observations of wing configurations, which allowed for the detection of possible new divisions of populations of the insect. 

 

To analyze the origin of each insect, the samples were georeferenced and the coordinates were incorporated into the data of each sample.

 

The study was also careful to analyze the morphological variations within each species. To this end, scientists carried out reproduction in the laboratory allowing for larvae, pupae, and eggs to be compared to distinguish normal variations among siblings and those that indicate divisions among species.

 

The Thematic Project also boasted genetic sequencing technology applied to parts of the genome considered important for differentiation of the species. “It is an initial step that will allow sequencing of the genomes of several Anopheles species in the future,” explains Sallum.

 

In the current project, researchers are conducting sequencing of the mitochondrial genome of all species and of the phylogenetic lines detected to date.

 

International cooperation 

 

In addition to the USP team, participants in the study included researchers Cecília Luiza Simões Santos of the Adolfo Lutz Institute (IAL), Eduardo Sterlino Bergo of the Endemic Disease Control Superintendency (Sucen), and Mário Antonio Navarro da Silva of Universidade Federal do Paraná (UFPR). The Thematic Project also had two foreign contributors: Peter Foster of the Natural History Museum (United Kingdom) and Richard Wilkerson of the Smithsonian Institute (United States).

 

The study also serves as the basis for three post-doctoral studies, five doctorates, and 11 master’s dissertations. In total, eight students of these three modalities received FAPESP fellowships.

 

Irish national Brian Patrick Bourke, one of the project’s post-doctoral fellows, is currently studying the Anopheles cruzii mosquito, a malaria vector, in the Atlantic Rainforest within the context of genetic landscape, which associates environmental characteristics with genetic variations.

 

Bourke is also a contributor to the project “Morphological and molecular taxonomy and phylogeny of the Nyssorhynchus subgenus of the Anopheles (Diptera: Culicidae)”. Initiated in 2012, this project is also coordinated by Sallum with FAPESP funding, and it continues the advances made in the Thematic Project.

 

The new study focuses on Anopheles darlingi in the Atlantic Rainforest. The objective is to go beyond studies related to public health.

 

Sallum calls attention to the fact that the majority of the studies on mosquitoes are directed at species that act as vectors for infectious agents. “This results in a common misperception that the anophelines are a taxonomically well-known group,” she said.

 

The researcher considers knowledge of these insects fundamental for several other fields, and for this reason, the taxonomical study will likely be broad. “Mosquitoes have a lot to offer basic science, for example, as indicators of Neotropical biodiversity, of the degree of degradation of the natural environment, of the varied forms of soil use for evolutionary studies and of speciation,” she said.

 

The results obtained also indicate that some species that were considered vectors of Plasmodium in previous studies could represent complexes.

 

“In this manner, there will be a need to verify which species are real vectors and to understand aspects of biology, ecology, behavior, and how they participate in malaria transmission,” says Sallum.

 

According to the researcher, only a minority of the 3,523 known species of mosquitoes are a significant plague for human beings.

 

The results of the study will soon be published by PLoS magazine in an article entitled “Phylogenetic analysis and DNA-based species confirmation in Anopheles.” In another article, “A multi-locus approach to barcoding in the Anopheles strodei Subgroup (Diptera: Culicidae)”, which will soon be published in Parasites and Vectors, the researchers show that a given species could represent a complex of four species.