Identification keys to the Anopheles mosquitoes of South America (Diptera: Culicidae). IV. Adult females

Background Morphological identification of adult females of described species of the genus Anopheles Meigen, 1818 in South America is problematic, but necessary due to their differing roles in the transmission of human malaria. The increase in the number of species complexes uncovered by molecular taxonomy challenges accurate identification using morphology. In addition, the majority of newly discovered species have not been formally described and in some cases the identities of the nominotypical species of species complexes have not been resolved. Here, we provide an up-to-date key to identify Neotropical Anopheles species using female external morphology and employing traditionally used and new characters. Methods Morphological characters of the females of South American species of the genus Anopheles were examined and employed to construct a species/group identification key. Photographs of key characters were obtained using a digital Canon Eos T3i, attached to a microscope. The program Helicon Focus was used to build single in-focus images by stacking multiple images of the same structure. Results A morphological identification key to the adult females of species of the genus Anopheles described in South America is presented. Definitions and illustrations of the key characters are provided to facilitate use of key. Conclusions Identification of species of the genus Anopheles based on female morphology is challenging because some key characters can be variable and overlapping among species. In addition, the majority of key characters are linked to color and shape of scales, their distribution on the head, scutum, abdomen, maxillary palpi, labium and legs, and pattern of pale and dark scales on dorsal and ventral surfaces of the wing veins. Thus, it is understandable that a specimen needs to be in good condition to be accurately identified. Morphologically similar species, such as those of the Konderi, Oswaldoi, Nuneztovari, Benarrochi and Albitarsis Complexes, and the Triannulatus and Strodei Groups, among others, cannot be accurately identified using characters included in the key. Further investigation will be required to exploit morphological characteristics for identification of members of those complexes, with formal description of new species.


Background
General introductory comments, distributions and species authors and publication dates are given in Part I [1] of this series of four articles. Keys to fourth-instar larvae and male genitalia are in Parts II [2] and III [3], respectively. Despite many recent studies have focused on the importance of DNA sequences for uncovering species complexes [4][5][6][7][8][9][10][11][12][13], the identification of Anopheles species is primarily based on morphological characters of female, male, and fourth-instar larvae [1]. This paper provides an illustrated dichotomous morphological key for the identification of females of Anopheles species of South America.

Methods
The primary types (holotypes and paratypes) and other field-collected specimens deposited in the Coleção Entomológica de Referência, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil (FSP-USP), Museo de Entomología, Universidad del Valle, Santiago de Cali, Colombia (MUSENUV) and the US National Mosquito Collection, Smithsonian Institution, Washington, DC, USA (USNMC) were selected and morphologically studied to discover additional characters to be used in the female key [1]. In addition, original descriptions, keys, summaries, and revisions from the published literature were examined. Photomicrographs of relevant characters for the female key were taken using a digital Canon Eos T3i (Canon, USA), attached to a stereomicroscope, using the program Helicon Focus software (https ://www.helic onsof t.com/helic onsof t-produ cts/helic onfocus /), which was used to build single in-focus images by stacking multiple images of the same structure. Photomicrographs were further processed in Adobe Photoshop (https ://www.photo shop.com/en) to embed names and labels. Table 1 in Sallum et al. [1] shows the traditional classification of the genus Anopheles. The female key was modified from Forattini [14], Wilkerson & Strickman [15], and Harrison et al. [16] with further characters proposed herein.

Results and discussion
Identification of species of the genus Anopheles based on female morphology can, for various reasons, be inaccurate. Morphological similarities and overlapping characters are common in species of the genus Anopheles and will increase with further taxonomic studies using molecular tools to address identification, phylogeny and establish species complexes. In addition, increased sampling in remote and poorly sampled regions of South America will propitiate discovery of new species and improvement in the taxonomic knowledge and nomenclature of the group as well. The newly proposed identification key compiled morphological information for identification of females, however, ideally characters of the male genitalia, fourth-instar larvae, and scanning electron microscope of the eggs should be examined to increase accuracy. Employment of this key to identify both unknown species and those already defined by molecular approach should be considered with caution. Likely, a specimen that may belong to a species that was not formally named will be identified to a morphologically similar species. Thus, when facing morphological variations, further investigations will be necessary to verify if those observed differences can indicate an unknown species. It is highly recommended to examine all life stages to reach an accurate species identification using morphology.

Morphological features
The terminology of Harbach & Knight [17,18] is followed in the key below. Valid species of the genus Anopheles of the subgenera Anopheles, Kerteszia, Lophopodomyia, and Stethomyia found in South America are provided in Table 1 in Sallum et al. [1]. In addition to the morphological traits that identify members of the Culicidae Meigen, 1818, most females of the subfamily Anophelinae Grassi, 1900 differ from those of the subfamily Culicinae Meigen, 1818 by having the maxillary palpi as long as the proboscis. In the Anophelinae, the majority of the species of the genera Anopheles Meigen, 1818 and Bironella Theobald, 1905 have the posterior margin of the scutellum rounded, not developed with median and lateral lobes. Consequently, the scutellar setae are uniformly distributed along the posterior border (Fig. 1). However, it is noteworthy that some species of the subgenera Anopheles and Cellia Theobald, 1902 exhibit a shallow subdivision into three lobes, but the distinction between the median and lateral lobes is not as evident as in species of the genus Chagasia Cruz, 1906 (Fig. 2).

Head
Anopheles, like all other mosquitoes, have the antenna made up of 13 elongate flagellomeres. Each flagellomere possesses short setae dispersed around it and a number of longer, stronger setae arising apically ( Fig. 3). In the males, the antenna possesses a higher concentration of longer and stronger setae disposed apically that form the flagellar whorl. The maxillary palpus of the females and males is made up of five palpomeres (Fig. 3). Palpomere 1 (MPlp 1 ) is the shortest, arising laterally to the clypeus. Palpomere 5 (MPlp 5 ) is longer than palpomere 1 but shorter than palpomeres 2, 3 and 4 (MPlp 2-4 ), which are elongate. Scales covering the maxillary palpus vary in color from silvery white to cream to yellowish to dark brown and black. The pattern of distribution of pale and dark scales on the maxillary palpus can help identify some species of the genus Anopheles.

Thorax
The thorax of the majority of the species of the genus Anopheles is elongate and as in all mosquitoes is represented mostly by the mesonotum (Fig. 4). The color of the scutal integument varies from blackish to brownish to grayish and exhibits patterns of color and scale distributions that can be employed for identification of species, species groups and subgenera. Scales can  Wilkerson, 1991 be absent or present. When present, scales are usually sparse and dispersed on some areas of the thoracic pleura (Fig. 5). The patterns of distribution of the scales on the mesokatepisternum and mesepimeron are frequently used to identify species of the subgenus Kerteszia Theobald, 1905 (Fig. 6).

Legs
The legs of anophelines are predominantly dark but can have pale and dark scales in defined patterns or distributed without a characteristic pattern in the form of speckling. Some species have a defined pattern of scales, but there is also intraspecific and intra-individual variability. In other species, the legs are mostly dark-scaled, with pale scales forming rings and bands of variable size and distribution. On the hindlegs, the majority of species of the Arribalzagia Series of the subgenus Anopheles, as well as Nyssorhynchus Blanchard, 1902 and Kerteszia, have well-defined patterns of pale and dark scales that are often used for species identification. In species of the subgenus Nyssorhynchus, hindtarsomeres 2-5 are dark-scaled but show distinct patterns of pale scales that are employed for species identification (Fig. 7).

Wings
Independent of the shading or dark patterns that are sometimes seen on the wing membrane, the coloration of the scales that cover most of the wing veins is what defines the color of the wings. The scales vary from dark to pale, making the wings appear completely dark or with pale and dark areas that form patterns that are species-specific or group specific (Figs. 8,9,10,11). This is usually evident on the longitudinal veins. The nomenclature adopted in the identification key is that proposed by Wilkerson & Peyton [19]. The wing spots are named with reference to the pale and dark spots observed in An.

Abdomen
Females of the genus Anopheles possess a variable pattern of scales, ranging from a dense covering (Fig. 12), i.e. Anopheles pharoensis Theobald, 1901 (an African species), to scales grouped in patches that are more evident on the dorsal portions of the segments, to almost entirely bare. The absence of scales on the abdominal segments is variable and is observed in species of diverse subgenera of the genus Anopheles. However, the abdomen is always covered with setae of variable development. The majority of the species of the subgenus Nyssorhynchus and some species of the subgenus Anopheles possess patches of scales grouped laterally at the posterior end of segments II-VII or III-VII or IV-VII. These patches of scales are called posterolateral scale-tufts (Fig. 11). In other species, scales are either absent or present only on segments VII and VIII and the cerci (Fig. 13). Abdominal sternum I is small and closely associated with the metathorax. Consequently, it is usually not easy to examine characteristics of sternum I when the specimen is dry-pinned, and the abdomen droops. Traits of sternum I are more easily seen if the individual is examined from a posterior view. In some species of the subgenus Nyssorhynchus, sternum I possesses sparse scales, or the scales are arranged in a longitudinal line (Fig. 14).

Conclusions
Our identification key, based on morphological characters of adult females, can be used to separate South American subgenera and species of the genus Anopheles. This key will serve a wide range of users. It will be: (i) reliable to a large degree in that many species can be identified definitively using morphological characters, especially if characters from additional life stages can be included; (ii) cost-effective for many. Morphological identification is still much less expensive and less technology-dependent than molecular methods; (iii) a unique research resource for the identification of specimens to morphospecies, which is needed as a basis for molecular studies. Molecular tools are increasingly effective for enhancing Anopheles taxonomy by uncovering similar species, species complexes and sibling species. Identification to morphospecies allows for focus on a subset of individuals rather than having to broadly sample throughout a wide geographical distribution; (iv) a resource for control. Control actions can be justified based on morphological identifications that narrow down to a vector group. Even with the potential of misidentification it is better to assume one is dealing with an effective vector, and that control action is required, rather than to not act at all. This identification key, however, does not allow separation of individual species in a number of informally named groups: i.e. Konderi, Oswaldoi, Nuneztovari, Benarrochi and Albitarsis Complexes, and the Triannulatus and Strodei Groups. In the key these are given species names and designated as "sensu lato". To include component species in future keys, taxonomic studies are needed to name and describe them and to uncover differential characters.