Sallum et al. Parasites Vectors (2020) 13:583 https://doi.org/10.1186/s13071-020-04298-6 Parasites & Vectors RESEARCH Open Access Identifcation keys to the Anopheles mosquitoes of South America (Diptera: Culicidae). I. Introduction Maria Anice Mureb Sallum1*, Ranulfo González Obando2, Nancy Carrejo2 and Richard C. Wilkerson3,4,5 Abstract Background: The worldwide genus Anopheles Meigen, 1918 is the only genus containing species evolved as vectors of human and simian malaria. Morbidity and mortality caused by Plasmodium Marchiafava & Celli, 1885 is tremendous, which has made these parasites and their vectors the objects of intense research aimed at mosquito identifcation, malaria control and elimination. DNA tools make the identifcation of Anopheles species both easier and more difcult. Easier in that putative species can nearly always be separated based on DNA data; more difcult in that attaching a scientifc name to a species is often problematic because morphological characters are often difcult to interpret or even see; and DNA technology might not be available and afordable. Added to this are the many species that are either not yet recognized or are similar to, or identical with, named species. The frst step in solving Anopheles identi- fcation problem is to attach a morphology-based formal or informal name to a specimen. These names are hypoth- eses to be tested with further morphological observations and/or DNA evidence. The overarching objective is to be able to communicate about a given species under study. In South America, morphological identifcation which is the frst step in the above process is often difcult because of lack of taxonomic expertise and/or inadequate identifca- tion keys, written for local fauna, containing the most consequential species, or obviously, do not include species described subsequent to key publication. Methods: Holotypes and paratypes and other specimens deposited in the Coleção Entomológica de Referência, Faculdade de Saúde Pública (FSP-USP), Museo de Entomología, Universidad del Valle (MUSENUV) and the US National Mosquito Collection, Smithsonian Institution (USNMC) were examined and employed to illustrate the identifcation keys for female, male and fourth-instar larvae of Anopheles. Results: We presented, in four concurrent parts, introduction and three keys to aid the identifcation of South Ameri- can Anopheles based on the morphology of the larvae, male genitalia and adult females, with the former two keys fully illustrated. Conclusions: Taxonomic information and identifcation keys for species of the genus Anopheles are updated. The need for further morphology-based studies and description of new species are reinforced. Keywords: South America, Anopheles, Identifcation keys, Introduction Background Malaria continues to be a serious public health prob- *Correspondence: [email protected] lem. In 2016, there were an estimated 216 million cases 1 Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, Avenida Doutor Arnaldo 715, São Paulo, São of malaria in 91 countries, an increase of 5 million cases Paulo CEP01246-904, Brazil over 2015. Malaria deaths reached 445,000 in 2016, a Full list of author information is available at the end of the article similar number (446,000) to 2015 (http://www.who. © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sallum et al. Parasites Vectors (2020) 13:583 Page 2 of 12 int/news-room/fact-sheet s/detai l/malar ia) [1]. In 2017, can also be employed to study live specimens for eco- there were an estimated 219 million cases of malaria logical studies, such as capture-mark-release-recapture. worldwide, mostly in developing countries, resulting in Even though there are many keys addressing morpho- an estimated 435,000 deaths of which 266,000 deaths logical identifcation of Neotropical Anopheles, they are were children under 5 years of age [2]. limited in geographical or taxonomic scope and/or are In Central and South America, malaria transmission outdated. For this reason, we present comprehensive keys occurs primarily at altitudes of less than 1000 meters (based on the morphology of male genitalia, females, and above sea level. It is not surprising therefore that the fourth-instar larvae) to all Anopheles species recorded in incidence of malaria is highest in lowland countries, South America. such as those situated in the Amazon basin: Brazil, Mosquitoes (family Culicidae Meigen, 1818), belong to Colombia, Venezuela, Ecuador, Peru, and the Guia- order Diptera (true fies, i.e. insects with two wings), and nas. Brazil, Colombia, Peru, and Venezuela contrib- like many other insect orders (Coleoptera, Hymenoptera, uted approximately 82% of the 875,000 malaria cases etc.) they are holometabolous, meaning they have dis- reported in the region in 2016: Brazil (18.0%); Colom- tinctly diferent egg, larval, pupal and adult stages. Te bia (15.3%); Peru (14.3%) and Venezuela (34.4%) [1]. In culicids are recognized as a monophyletic group (with 2017, an increase in malaria cases was reported in Bra- a single common ancestor) [10–12], that diverged from zil, Ecuador, Mexico, Nicaragua, and Venezuela [2]. its nearest relative, family Chaoboridae Edwards, 1912, Variability in the epidemiological components of about 255 million years ago (mya) [13]. Te two culicid malaria transmission (host(s), parasites, environment) subfamilies, Anophelinae Grassi, 1900 and Culicinae varies widely by locality, with diferences in physiogra- Meigen, 1818, diverged 229–192 mya [14]. phy, regional and local ecological characteristics, vector Adult mosquitoes (Fig. 1) can be distinguished [15] by: competence and vectorial capacity of individual species scales on wing veins and usually also on the head, legs, of Anopheles Meigen, 1818. Mosquito vectors can dif- thorax and abdomen; proboscis long, extending well fer in physiology, behavior, and ecology, as well as in beyond the clypeus; characteristic wing venation (also morphological characters used for their identifcation. found, however, in families Dixidae Schifner, 1868 and Tese diferences taken together can facilitate the def- Chaoboridae) [16] (i.e. subcostal (Sc) vein ending near or nition of species and are the source of characteristics beyond midpoint of the costal vein (C), Sc and vein R1 for species identifcation. Te most accessible method both reach C in the apical half of the wing in front of R 2 of identifcation is by morphological characteristics. and R3, and vein M three-branched); antennal pedicel However, if basic (alpha) morphological taxonomy is prominent and nearly always larger than the scape. Mos- incomplete, it is possible to make incorrect identifca- quito (and chaoborid) larvae [16, 17] have the three tho- tions of morphologically similar species of very dif- racic segments fused into a rounded composite structure ferent public health importance. Tis is especially the that is wider than the head or abdomen; abdominal seg- case for species complexes that include both vector ment X with a fan-like ventral brush; lateral tufts of long and non-vector species. Te defnition and delinea- setae on most thoracic and abdominal segments; labrum tion of morphologically similar species often requires with a distinctive brush of long setae on either side (Culi- auxiliary tools, for example molecular markers, which cidae only); and antenna moderately long, usually with a include DNA sequences. DNA not only has given us number of apical setae (Culicidae only). evolutionary insights but also many new characters for While the above characters serve for identifcation, the recognition and identifcation of species. However, another overlapping set of characters further character- molecular methods are not available in many malaria- ize the family and can be used for phylogenetic analyses endemic areas in South America. Recently, multiple [18]. According to [10], the synapomorphies of Culicidae studies (e.g. [3–9]) have reported satisfactory resolu- are internal premandibles in the larvae, apparently with- tion of taxonomic problems using a combination of out residual external sclerites, and a long proboscis, with molecular markers, and/or morphological characters stilettes corresponding to the maxillae, mandibles and from all developmental stages. Tese studies have led the labrum-hypopharynx, all encased in the labium [16]. to description or re-description of multiple species, and In a study using characters of the adults and fourth-instar the recognition of complexes of morphologically simi-