Identification keys to the Anopheles mosquitoes of South America (Diptera: Culicidae). II. Fourth-instar larvae

Background Accurate species identification of South American anophelines using morphological characters of the fourth-instar larva is problematic, because of the lack of up-to-date identification keys. In addition, taxonomic studies, employing scanning electron microscopy of the eggs and DNA sequence data, have uncovered multiple complexes of morphologically similar species, and resulted in the resurrection of other species from synonymy, mainly in the subgenus Nyssorhynchus. Consequently, the identification keys urgently need to be updated to provide accurate morphological tools to identify fourth-instar larvae of all valid species and species complexes. Methods Morphological characters of the fourth-instar larvae of South American species of the genus Anopheles were examined and employed to elaborate a fully illustrated identification key. For species for which no specimens were available, illustrations were based on published literature records. Results A fully illustrated key to the fourth-instar larvae of South American species of the genus Anopheles (Diptera: Culicidae) is presented. Definitions of the morphological terms used in the key are provided and illustrated. Conclusions Morphological identification of South American Anopheles species based on the fourth-instar larvae has been updated. Characters of the spiracular apparatus were determined useful for the identification of morphologically similar species, in the Strodei Group and some taxa in the Myzorhynchella Section. The single versus branched abdominal seta 6-IV used to differentiate Myzorhynchella species from other Nyssorhynchus species was shown to be variable in Myzorhynchella species. Also, the abdominal setae 1-IV,V of Anopheles atacamensis and Anopheles pictipennis were shown to be slightly serrate at the edges. Recognition of this character is important to avoid inaccurate identification of these species as members of the subgenus Anopheles.


Background
General introductory comments, distributions and species authors and publication dates are given in Part I [1] of this series of four articles. Despite the continuous interventions to control and eliminate malaria, this disease remains an important cause of morbidity and mortality in endemic tropical and subtropical countries throughout the world. Malaria occurrence is affected by several factors including those of the parasites, human host, anopheline mosquitoes and environment [2]. Because of its intrinsic complexity, in order to reach an effective and sustainable control of malaria it is necessary to adopt an integrated approach delineated on the evidence of local characteristics of the disease and transmission dynamics [3]. Thus, the public health policy for malaria control demands interventions focused on the local anopheline vector species and empirical field-knowledge on the characteristics of the local populations behavior and ecology [4]. Different mosquito species exhibit distinct preferences for bloodfeeding on animals or humans, place where females take their blood-feeding, peak biting time, resting places and habitats. These biological characteristics of vector species need to be considered for the choice of an effective vector control intervention. It is important to note that vector control interventions need to be delineated for the local vector species, thus an accurate species identification is of vital importance for control, surveillance, and field malariology studies. Misidentification will cause error for a decision-making control programme that is based on metrics of transmission and local vector species [3]. Actions for vector control encompasses activities focused on the larval habitat to prevent the production of adult mosquitoes. This requires specific measures that are defined taken into consideration field-collected data of the vector population, and accurate identification of species and their habitats. The fastest, easiest, and cheapest way to identify field-collected mosquitoes for surveillance and control programmes is based on morphological characteristics. This activity is usually conducted by field-entomologists conducting mosquito collections, dissections of females in malaria endemic areas. A dichotomous morphological key is proposed with the purpose of providing a tool for identification of Anopheles species in South America based on the morphology of the fourth-instar larvae. The key includes major vectors, local vectors and species that have not been involved in the malaria transmission but can emerge and become dominant in human-dominated environments.

Methods
Morphological characters of the fourth-instar larvae of South American species of the genus Anopheles Meigen were examined and employed to construct a fully illustrated identification key. 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 examined to discover characters to be used in the key based on larval morphology. For species that we could not access, drawings were based on published illustrations. Photomicrographs of relevant characters for the key were taken from the fourth-instar larval exuviae mounted on microscope slides and covered with a coverslip. Specimens were obtained either as field-collected larvae or progenies of field-collected females linked to either adults. The identification was based on the morphology of the male genitalia and females. Photomicrographs were taken with a digital Canon Eos T3i (Canon, USA) camera, attached to a Leitz Diaplan microscope, using the Helicon Focus software (https ://www.helic onsof t.com/helic onsof t-produ cts/helic on-focus /) that was used to build single in-focus images by stacking multiple images of the same structure. These in-focus images were, then, employed to draw the line illustrations of the characters in CorelDRAW software (https ://www.corel draw.com/en/produ ct/corel draw/ essen tials /?topNa v=en). Except for one figure, illustrations are not to scale, but the proportions of the characters in the drawings are maintained. The morphological terminology employed in the key are defined and illustrated in accordance with Harbach & Knight [5,6]. The key is modified after Faran & Linthicum [7] and Forattini [8], with additional characters proposed herein. The species included in this Part II are listed in Table 1

Glossary of morphological terms
All mosquitoes pass through four larval instars (stadia, stages). As in all insects, mosquitoes have three body regions. These are well differentiated in mosquito larvae: the head (C), thorax (T) and abdomen (A) (Fig. 1). Mosquito larvae are metapneustic, meaning there is a single pair of respiratory openings caudally. Species of the subfamily Anophelinae Grassi, 1900, which includes the genus Anopheles Meigen, 1818, differ from the other culicid subfamily, Culicinae Meigen, 1818, in that the larval stages do not possess a respiratory siphon, but instead have paired spiracular openings on a clearly differentiated plate.
The larval cuticle (exoskeleton) bears a number of features of taxonomic utility. The most distinctive of these are the numbered, serially homologous (comparable between segments) setae that can almost always be directly compared to all other mosquito species. The setae can vary in number, form, and position (also referred to as chaetotaxy). Non-segmental structures, such as antennae and mouthparts, also have homologous numbered setae. Since setae are added with each of the four instars, the numbering system is based on the last (4th) instar. Not all setae have taxonomic value. Therefore, we only discuss those that appear in our key, or may appear in other commonly used keys. Larvae usually possess 222 pairs of setae [5], 54 on the head (including antennae and mouthparts), 42 on the thorax and 126 on the abdomen. The primary types of setae, i.e. palmate, dendritic, branched, simple, aciculate, bifurcate, and plumose are illustrated (Fig. 2). The setae are designated with a number and a structure/ segment abbreviation, e.g. 2-C is seta 2 on the head, and 1-A is seta 1 on the antenna. Any given seta on a segment is expressed in the singular even though there are two with the same number per segment, e.g. seta 2-C in the above example is expressed with a singular verb, i.e. "seta 2-C is…. " not "setae 2-C are…. ". Singular is also used for the same numbered seta on different segments, e.g. seta 1 on abdominal segments II through VII would be "seta 1-II-VII is…. ". For different numbered setae on the same or multiple segment(s) the plural is used, e.g. setae 3 and 4 on abdominal segments II and III would be "setae 3,4-II, III are…. ". On any given structure/segment the setae are numbered in ascending order beginning anteriorly or from the dorsomesal line. Species of the genus Anopheles can be differentiated from species of the genera Chagasia Cruz, 1906 and Bironella Theobald, 1905, also the Anophelinae, by characters of the spiracular apparatus. In Chagasia, the posterolateral spiracular lobes possess a fringe of setae laterally, and the anterior spiracular lobe is produced into an elongate process. Species of Anopheles and Bironella lack the fringe of setae and the anterior spiracular lobe is knob-like (see [9]). Note however that Bironella spp. occur in the Australasian Region, including Papua New Guinea, Solomon Islands, Bismarck and Queensland, Australia.

Head
The head is the most heavily sclerotized part of the body. It is usually longer than wide and, as in other culicids, is made up of various named sclerites (sclerotized plates). The head articulates with the thorax in such a way that is possible for larvae to rotate their heads up to 180°, resulting in great mobility while feeding. The head exhibits well-developed mandibles, maxillae, maxillary palpi and associated structures (for details see [5,6]). These are not utilized here but can be seen elsewhere in the literature. Anterolaterally there is a pair of antennae (A), which are made up of a very short scape and a long tubular structure formed by fusion of the antennal pedicel and flagellum. All insects have some variation of these three parts of the antenna (scape, pedicel and flagellum). On the antenna there are six pairs of setae, some of which are often called by informal names: antennal seta (1-A), a terminal antennal seta (4-A) and two setae in the form of a sabre (2-A and 3-A), one dorsal and the other ventral (Fig. 3). Seta 1-A is often used in identification since its branching and position can vary. Seta 4-A can be two-or threebranched, with symmetrical or asymmetrical branching.
The head has 15 pairs of setae, a number of which have taxonomic utility. These setae are often referred to by informal names. Head setae 2-C, 3-C and 4-C are the clypeal setae. Setae 2-C and 3-C are located toward the most anterior part of the head and are called the anterior clypeal setae, with the lateral seta (3-C) called external  These setae can be simple, aciculate, barbed, plumose, branched with simple branches, or dendritic, and can vary in length. The two seta 2-C can be variably separated and are considered close when the distance between them is less than the distance between setae 2-C and 3-C on either side. If the distance between the two setae 2-C is equal or more than the distance between 2-C and 3-C they are considered well separated. The ratio between the distances between 2-C and 3-C on one of the sides with relation to the distance between the bases of the pair of 2-C, constitutes the clypeal index (distance between 2-C and 3-C on one side / distance separating setae 2-C [7]). Posterior to the anterior clypeal setae is the posterior clypeal seta (4-C), which can be single, bifurcate, forked, or multi-branched, short or long. Length is judged by how far forward the seta extends toward the bases of setae 2,3-C, and development compared to development of setae 2,3-C. Medially on the head are three pairs of setae often called the frontal setae: 5-C, 6-C and 7-C (5-7-C) (Fig. 3). They are generally long and extend at least past seta 4-C. They can be simple or branched, and sometimes appear simple but are slightly branched only apically. They usually occur in a line, but 7-C can be more anterior. Seta 8-C is posterolateral to 5-7-C. Setae 9-14-C are on an area called the lateralia (lat) (lateral and ventral areas of the head lateral to the ecdysial lines (Fig. 3). Of these, 9-C is located immediately lateral of the frontal ecdysial line and near 8-C, while 10-C is lateral of 9-C. Seta 11-C can also have taxonomic utility for the diagnosis of some species, and is found dorsolaterally near the base of the antenna.

Thorax
The thorax is longer than wide and, as in all insects, is composed of three segments. In mosquito larvae the segments are not clearly differentiated. Using known setal groups as landmarks, one can recognize them even without distinct demarcations. They are the prothorax (P) (nearest the head), the mesothorax (M) and the metathorax (T) (Fig. 4). There are 42 pairs of setae on the thorax, many with informal names. Near the midline on the prothorax are setae 1-3-P, the submedian prothoracic group. These are often referred to their relative positions: internal (1-P), median (2-P) and external (3-P). Seta 1-P originates closest to the dorsomesal line and is generally branched or palmate. It is close to seta 2-P. Seta 3-P is usually simple and originates close to 1,2-P, which can share the same basal support plate or occur individually. These three setae are of great taxonomic utility. On the mesothorax and metathorax setae 1 and 2 (1-M,T and 2-M,T) can be closer to each other and well separated from seta 3. The variable form of seta 3-T makes it useful in the identification of some species. Setae 9-12-P,M,T, the pro-, meso-and metathoracic pleural groups, originate on a common tubercle in a lateroventral position on all three segments. Some of the setae vary in form, size, and number of branches, which often makes them taxonomically useful.

Abdomen
As in all Culicidae, the abdomen is composed of 10 segments, nine of them visible. Segments are identified by Roman numerals (I-X). The larva usually has the following pairs of setae: up to 12 on segment I, 15 on II-VII, seven on VIII, 13 on the spiracular apparatus and pecten, and four on X [5]. In the case of the spiracular apparatus and pecten, they are referenced by the letter S, to indicate that they are equivalent to the setae of the siphon in species of the subfamily Culicinae. The setal nomenclature starts with the number 0 (zero). Setae 8 and 14 are absent from segment I (Fig. 1). The following principal diagnostic setae are all dorsal or lateral. Seta 0-II-VII is the most anterior seta. It is usually poorly developed or inconspicuous, but its variability makes it useful for the identification of some species. Seta 1-I-VII is posterolateral, nearly equidistant between the midline and lateral margins (Fig. 5). In Anopheles, this seta, at least on segments III-VI, is usually palmate (form suggesting a palm frond or fan) or with filiform branches. In species of Chagasia, seta 1 is also palmate, but the leaflets are distinctive paddle-like structures with a long slender hair-like extension. Some species of Anopheles (found in tree holes) do not have palmate setae, or the palmate setae are not present on all segments. Palmate setae are variously developed, but the usual form is for the individual leaflets to have a short stem with many expanded leaflets. However, diagnostic variation includes having the margins smooth or strongly to moderately toothed, apices narrowed, filamentous, diamond-shaped, or truncate. Seta 6-I-VII is lateral, generally on a tubercle. These are informally called lateral setae and are often the longest setae. Seta 6-IV-VII is usually simple or with a few branches but on segments I-III as they can be plumose. Segments VIII and X bear the pecten plate and the spiracular apparatus, both of which present various structures of taxonomic value. These include the anterior spiracular lobe, a median plate, two posterolateral spiracular lobes, and two anterolateral spiracular lobes (Fig. 6). The two spiracles are found behind the anterior spiracular lobe and on the sides of the anterior margin of the median plate. In species of the subgenus Stethomyia Theobald, 1902, the spiracular openings are well separated and located at the base of each anterolateral lobe. Usually, the median plate has lateral wings or arms of variable development and length that are utilized in the identification of some species or groups, especially in the subgenus Nyssorhynchus Blanchard, 1902 (Fig. 6). The posterolateral spiracular lobes can be rounded or include posterior projections, somewhat similar to spines, as observed in An. pseudopunctipennis pseudopunctipennis Theobald, 1901. On either side of the spiracular apparatus is the pecten plate bearing pecten spines (Fig. 7), which is homologous in part with the siphon in the species of the Culicinae. It is a triangular plate with spines that resemble a comb. Frequently, variations in this structure are used for characterization of species of the subgenus Kerteszia Theobald, 1905; in some species the spines have an irregular arrangement, in others they have a regular arrangement of alternating long and short spines, whereas others exhibit equally-sized spines. There are 13 pairs of spiracular setae (1-13-S). Seta 1-S is generally the most developed and branched (with various simple branches). It is  inserted posterior to the pecten plate below the posterolateral spiracular lobe. Seta 2-S is inserted at the base of the pecten plate. Setae 3-5-S, are generally small and indistinct, and 3-S is often only represented by an alveolus. These three setae are borne laterally on the anterior spiracular lobe. Setae 6-S and 7-S are on the proximal and distal anterolateral spiracular lobes, respectively. Setae 8-S and 9-S are on the proximal and distal lateral margins of the posterolateral spiracular lobe. Seta 10-S is borne on the internal posterior margin, while 11-S and 12-S are found on the posterior border of the lobe (Fig. 7). Seta 13-S can be of taxonomic importance, it is borne medially on the anterior margin of the internal surface of the lobe. It is generally small and somewhat stout. In the case of An. darlingi Root, 1926, it is usually much longer than the dorsal length of the saddle.
The dichotomous identification key for the genus Anopheles, using morphological characters of the fourthinstar larva, includes species of the subgenera Anopheles pseudopunctipennis neghmei, An. pseudopunctipennis patersoni, An. pseudopunctipennis rivadeneirai, An. rachoui, An. sanctielii and An. striatus cannot be identified using this key. They are poorly known, and the original descriptions were based on the morphology of the eggs, or females, or males with no further association of all life stages. In addition, An. pseudopuntipennis encompasses six subspecies that are morphologically similar in all life stages, including the male genitalia. Thus, specimens of these subspecies will be identified as An. pseudopunctipennis. However, it is highly recommended to use geographical localities as additional information for identification. Anopheles striatus belongs to the Strodei Subgroup of the Oswaldoi Group. This subgroup includes Anopheles albertoi, Anopheles arthuri, Anopheles rondoni (Neiva & Pinto, 1922), Anopheles striatus and Anopheles strodei. These species can be identified using DNA sequences of mitochondrial and nuclear genes [10][11][12], and morphological characters of the eggs, larvae, pupae, male genitalia, and females [13][14][15]. However, a detailed comparative morphological investigation will be necessary for description of all life stages and accurate species identification. In this key, specimens of species of the Strodei Subgroup will be identified as An. strodei/An. rondoni.