Authors: Maria Anice Mureb Sallum, Ranulfo González Obando, Nancy Carrejo, Richard C. Wilkerson
Categories: Research, Illustrated key, Male genitalia, Morphology, South America
Source: Parasites & Vectors
Accurate identification of the species of Anopheles Meigen, 1818 requires careful examination of all life stages. However, morphological characters, especially those of the females and fourth-instar larvae, show some degree of polymorphism and overlap among members of species complexes, and sometimes even within progenies. Characters of the male genitalia are structural and allow accurate identification of the majority of species, excluding only those in the Albitarsis Complex. In this key, based on the morphology of the male genitalia, traditionally used important characters are exploited together with additional characters that allow robust identification of male Anopheles mosquitoes in South America.
Morphological characters of the male genitalia of South American species of the genus Anopheles were examined and employed to construct a comprehensive, illustrated identification key. For those species for which specimens were not available, illustrations were based on published illustrations. Photographs of key characters of the genitalia were obtained using a digital Canon Eos T3i attached to a light Diaplan Leitz microscope. The program Helicon Focus was used to build single in-focus images by stacking multiple images of the same structure.
An illustrated key to South American species of Anopheles based on the morphology of the male genitalia is presented, together with a glossary of morphological terms. The male genitalia of type-specimens of previously poorly documented species were also examined and included in the key, e.g. Anopheles (Anopheles) tibiamaculatus (Neiva, 1906) which has a unique quadrangular-shaped aedeagus with an apical opening.
Male genitalia of South American species of Anopheles possess robust characters that can be exploited for accurate species identification. Distortion that can occur during the dissection and mounting process can obstruct accurate identification; this is most evident with inadvertent damage or destruction of unique features and interferes with correctly assigning shapes of the features of the ventral claspette. In some species, the shape, and anatomical details of the aedeagus also need to be examined for species identification. For members of the Myzorhynchella Series, both ventral and dorsal claspettes possess multiple characteristics that are herein used as reliable characters for species identification.
Keywords: Anopheles, Illustrated key, Male genitalia, Morphology, South America
General introductory comments, distributions and species authors and publication dates are given in Part I [1] of this series of four articles. Keys to the fourth-instar larvae and adult females are provided in Parts II [2] and IV [3], respectively. A list of species treated here is included in Part I [1].
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 male genitalia key. For species that we could not access, illustrations were based on published illustrations. Photomicrographs of relevant characters for the male genitalia were taken using a digital Canon Eos T3i (Canon, USA), attached to a Diaplan Leitz microscope, using the program Helicon Focus software (https://www.heliconsoft.com/heliconsoft-products/helicon-focus/), 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.photoshop.com/en) to embed names and labels. The institutional sources of specimens are recorded on each photograph. The nomenclature adopted is that of Harbach & Knight [4, 5]. The water marks embedded in photomicrographs of the male genitalia show the institution where the vouchers are deposited, Universidade de Sao Paulo (USP) and Universidad del Valle.
The key includes most of the species registered in South America, except for 15 species that are poorly known. They are the Anopheles (Ano.) annulipalpis Lynch Arribálzaga; An. (Ano.) bustamantei Galvão; An. (Ano.) evandroi da Costa Lima; An. (Ano.) pseudomaculipes (Chagas); An. (Ano.) pseudopunctipennis levicastilloi Leví-Castillo; An. (Ano.) pseudopunctipennis neghmei Mann; An. (Ano.) pseudopunctipennis noei Mann; An. (Ano.) pseudopunctipennis patersoni Alvarado & Heredia; An. (Ano.) pseudopunctipennis rivadeneirai Leví-Castillo; An. (Ano.) rachoui Galvão; An. (Ker.) auyantepuiensis Harbach & Navarro; An. (Ker.) boliviensis (Theobald); An. (Ker.) rollai Cova García, Pulido F. & Escalante de Ugueto; An. (Nys.) nigritarsis (Chagas); and An. (Nys.) sanctielii Senevet & Abonnenc. For these species, it will be necessary to conduct field collections in the type-localities and further taxonomic investigations.
The terminology of the male genitalia used in this key follows that of Harbach & Knight [4, 5]. Also known as the male terminalia, Harbach & Knight [4] recommended instead to use “the genitalia” to avoid confusion with other terminal structures. The composite male genitalia are structures formed from elements of the posterior segments IX and X of the abdomen. These modified structures are involved in mating, copulation, and insemination. After emergence of the adult male, the posterior part of the abdomen beyond segment VII makes a 180° rotation. Thus, the ventral segmental surfaces become dorsal in relation to the rest of the abdomen, and vice versa. In Culicidae Meigen, 1818, the male genitalia are therefore inverted in relation to the female genitalia. This means that when coupling occurs, both individuals have the same upright orientation, instead of the male ending up vulnerable, and upside down in relation to the female, as is the case in the family Tabanidae Latreille, 1802 (horse flies), which do not have male genital rotation [6]. This phenomenon must be considered when describing the position of the various elements of the genitalia.
Tergum IX, which usually varies little, can exhibit useful morphological variation in certain species. Species of the Arribalzagia Series of the subgenus Anopheles Meigen, 1818 possess ninth tergal lobes (IX-Te lobes) of variable size and development, features which can be useful for species recognition. The internal margin of tergum IX is attached to the proctiger that is formed by tergum X, the cerci, the cercal sclerites and the paraprocts. Dorsally there are two sclerotized plates called the cercal sclerites. The two structures attached laterally on the most posterior part of the abdomen are called the gonocoxopodites. They are adapted to facilitate insemination by grasping the female during copulation. The gonocoxopodites are composed of a proximal gonocoxite and a distal gonostylus.
The gonocoxite (Fig. 1) is a large, relatively long, and somewhat conical structure, much wider than the gonostylus. The external ventral surface is convex, while the internal surface is slightly concave, especially basally at the attachment of the claspettes [7]. The gonocoxites bear a large number of scales and setae, the larger of which are sometimes called spines; for purposes of this key, the term seta will be used. One or two parabasal setae are inserted on the dorsobasal portion of the gonocoxite. In species of the subgenera Nyssorhynchus Blanchard, 1902 and Kerteszia Theobald, 1905, the setae are inserted on prominent parabasal lobes that are situated dorsobasally (in a prerotational sense). The parabasal lobes are absent in Neotropical species belonging to the subgenera Anopheles, Lophopodomyia Antunes, 1937 and Stethomyia Theobald, 1902. The parabasal setae are instead inserted directly on the surface or on relatively small projections on the surface of the gonocoxite. In species of the subgenus Stethomyia, the parabasal setae are absent.
Fig. 1 Schematic drawing of the male genitalia of An. pseudopunctipennis Theobald, 1901. The proctiger (anal lobe) was removed to permit an unobstructed view of the ventral structures (redrawn after Komp [7])
Species of the subgenera Nyssorhynchus (Fig. 2) and Kerteszia have a single dorsally directed seta that is inserted on the parabasal lobe. The Neotropical species of the subgenera Anopheles and Lophopodomyia have two parabasal setae (Fig. 3b, c). The internal seta is inserted on the ventral surface of the gonocoxite, on the distal half or near mid-length. Species of the subgenera Nyssorhynchus and Kerteszia have a pair of accessory setae inserted on the dorsal surface of the gonocoxite (Figs. 2, 3a).
Fig. 2 Dorsal view of the male genitalia of An. albimanus Wiedemann. Segment IX and the proctiger (anal lobe) were removed to facilitate the observation of structures that occupy the ventral position
Fig. 3 Gonocoxites showing the accessory setae, internal seta and parabasal setae of species of the subgenera Nyssorhynchus (a), Anopheles (b), Stethomyia (c). b and c redrawn after Komp [7]
The gonostylus corresponds to the stylus of the gonocoxopodite. It is a well-sclerotized structure, moveable and articulated, on or near the apex of the gonocoxite. It is somewhat thickened and curved. At its apex is a small spiniform structure called the gonostylar claw [8] (Fig. 3).
Attached to the internal surface of the gonocoxite is the claspette. This is a membranous structure, usually divided into ventral and dorsal lobes, both exhibiting great variability according to subgenus and species within the subgenera (Fig. 4). For purposes of this key, the ventral and dorsal lobes of the claspette [4] are referred to as the ventral claspette and dorsal claspette, respectively. These are terms also used by Faran [9].
Fig. 4 Ventral and dorsal lobe claspettes of the male of An. neomaculipalpus Curry, 1931(redrawn after Komp [7])
In species of the subgenera Anopheles, Lophopodomyia and Kerteszia, the dorsal claspette is divided into ventral and dorsal lobes. These lobes exhibit interspecific variability in the form of the setae, as well as the size and distribution of spicules, which makes them useful in taxonomy. In species of the subgenera Anopheles (Fig. 4), Lophopodomyia and Kerteszia, the ventral lobe is divided apically, with the distance between them being more pronounced in species of the subgenera Kerteszia. In species of the subgenus Nyssorhynchus, the ventral claspette is not subdivided, instead the two ventral claspettes are fused and the composite structure occupies a median position between the gonocoxites. The structure is rich in morphological variation and is therefore useful for species identification. In some taxa, the ventral claspette is smooth (i.e. without spicules) while in others the spicules can be short or long and variously distributed on the claspette.
The apex of the ventral claspette can be rounded, truncated or angular, and, in some species can bear apicolateral expansions that resemble lobes. The presence of these apicolateral lobes in An. (Nys.) triannulatus (Neiva & Pinto, 1922) allows separation of this species from the otherwise morphologically similar An. (Nys.) halophylus Silva-do- Nascimento & Lourenço-de-Oliveira, 2002. Other structures that make up parts of the ventral claspette and are employed in taxonomy treatments include the preapical plate and basoventral lobes (Fig. 5). In species of the subgenera Nyssorhynchus, Kerteszia, Anopheles and Lophopodomyia, there is a short basal portion on the dorsal claspette upon which variable numbers of setae are attached dorsoventrally. These setae are variable in form, point of insertion, development, and quantity (Fig. 6). In species of the subgenus Stethomyia, the dorsal claspette is absent and the ventral claspette is columnar, with two subdivisions that support apical setae that are variously developed [7].
Fig. 5 Ventral claspette of An. strodei Root, 1926 (redrawn after Faran & Linthicum [10])
Fig. 6 Basal portion of dorsal claspette of species of Anopheles (Nyssorhynchus). a An. braziliensis (Chagas, 1907). b An. argyritarsis Robineau-Desvoidy, 1827 (redrawn after Faran & Linthicum [10])
The aedeagus is part of the phallosome, which includes, in addition to the aedeagus, the parameres and the basal pieces. The aedeagus is articulated basally to the parameres, which are connected to the basal pieces by an acetabulum that is on the median lateral area. The basal pieces, responsible for movement of the aedeagus during copulation, are connected to the gonocoxal apodemes. The aedeagus is the central organ of the phallosome and serves as the intromittent organ [4]. In species of Anopheles, the aedeagus is a tubular structure, dorsally curved, with the walls unequally sclerotized and with a circular opening near or at the apex. The apical part of the aedeagus is variable in form and development. In species of the subgenus Nyssorhynchus, the apical part of the aedeagus is variable, and often used in species identification. The presence of leaflets subapically on the aedeagus, as well as the number of these structures, their form, development, and presence of marginal serrations, permit identification of many species of the genus (Fig. 7).
Fig. 7 Aedeagus of the male genitalia. a Subgenus Anopheles. b Subgenus Nyssorhynchus (redrawn after Komp [7])
In species of the subgenus Anopheles, the apical leaflets, when present, can vary in number, position, form, and development. Some species possess a single pair of leaflets that can have smooth or serrate margins, be uniformly or unevenly sclerotized, and be short or long. In species of the subgenera Kerteszia and Nyssorhynchus, the leaflets may be present or absent. When present, they occur as a single subapical pair.
The morphological key using the external characters of the male genitalia can aid in identifying species of the genus Anopheles of the South America. Unnamed species of the known complexes can be identified as morphologically similar valid species. In the key, species complex is labelled as (s.l.). For these groups, further investigations will be necessary to define characters of the male genitalia for accurate identification. The key was modified from [10, 11, 13, 14], with additional characters provided herein.
Fig. 8 aAn. thomasi Shannon, 1933. b An. antunesi Galvao & Franco do Amaral, 1940
Fig. 9 aAn. kompi Edwards, 1930. b An. thomasi
Fig. 10 An. kompi
Fig. 11 aAn. thomasi. bAn. nimbus (Theobald, 1902)
Fig. 12 aAn. acanthotorynus Komp, 1937 (redrawn after Komp [12]). b, c An. nimbus (c drawn from b)
Fig. 13 aAn. braziliensis. bAn. costai da Fonseca & da Silva Ramos, 1940
Fig. 14 aAn. darlingi Root, 1926. b An. peryassui Dyar & Knab, 1908
Fig. 15 a, bAn. homunculus Komp, 1937. c An. darlingi Root, 1926
Fig. 16 Aedeagus of An. neivai (Lane & Coutinho, 1940) (a) and An. cruzii Dyar & Knab, 1908 (b)
Fig. 17 a Ventral claspette of An. bambusicolus Komp, 1937. b, c An. homunculus (b redrawn after Zavortink [13])
Fig. 18 An. homunculus
Fig. 19 aAn. neivai. bAn. homunculus
Fig. 20 aAn. pholidotus Zavortink, 1973. b An. lepidotus Zavortink, 1973 (redrawn after Zavortink [13])
Fig. 21 a, bAn. homunculus (a redrawn after Zavortink [13]). c An. laneanus Correa & Cerqueira, 1944
Fig. 22 aAn. cruzii. bAn. bellator Dyar & Knab, 1906
Fig. 23 aAn. bellator. bAn. laneanus
Fig. 24 aAn. triannulatus. bAn. strodei
Fig. 25 aAn. triannulatus. bAn. guarani Shannon, 1928
Fig. 26 aAn. triannulatus. bAn. halophylus
Fig. 27 aAn. guarani. bAn. albitarsis Lynch Arribálzaga, 1938
Fig. 28 aAn. parvus (Chagas, 1907). b An. antunesi Galvão & Franco do Amaral, 1940
Fig. 29 aAn. atacamensis González & Sallum, 2010. b An. darlingi
Fig. 30 aAn. antunesi. bAn. atacamensis
Fig. 31 aAn. antunesi. bAn. pristinus Nagaki & Sallum, 2010
Fig. 32 aAn. pictipennis (Philippi, 1865). b An. lutzii Cruz, 1901
Fig. 33 aAn. atacamensis. bAn. pictipennis
Fig. 34 a, bAn. lutzii. c, dAn. guarani Shannon, 1928
Fig. 35 a, bAn. darlingi. c, d, eAn. lanei Galvão & Franco do Amaral, 1938
Fig. 36 aAn. lanei. b, cAn. argyritarsis
Fig. 37 aAn. sawyeri Causey, Deane, Deane & Sampaio, 1943. b An. argyritarsis
Fig. 38 aAn. albimanus (redrawn after Faran [9]). b An. marajoara Galvão & Damasceno, 1942
Fig. 39 aAn. albitarsis Lynch Arribálzaga, 1878. b An. marajoara
Fig. 40 a, bAn. braziliensis. c, dAn. marajoara
Fig. 41 aAn. strodei. bAn. nuneztovari Gabaldon, 1940
Fig. 42 a, bAn. benarrochi Gabaldon, Cova-Garcia & Lopez, 1941. c An. rondoni (Neiva & Pinto, 1922). d An. strodei
Fig. 43 a, bAn. rondoni. c, dAn. strodei
Fig. 44 aAn. arthuri Unti, 1941. b An. albertoi Unti, 1941
Fig. 45 aAn. strodei. bAn. albertoi
Fig. 46 aAn. ininii (redrawn after Faran [9]). b An. goeldii Rozeboom & Gabaldon, 1941
Fig. 47 aAn. ininii Senevet & Abonnenc, 1938 (redrawn after Faran [9]). b, c An. nuneztovari
Fig. 48 An. goeldii
Fig. 49 aAn. rangeli Gabaldon, Cova-Garcia & Lopez, 1940. b An. dunhami Causey, 1945
Fig. 50 aAn. dunhami. bAn. galvaoi Causey, Deane & Deane, 1943
Fig. 51 aAn. evansae (Brèthes, 1926). b An. galvaoi
Fig. 52 aAn. aquasalis Curry, 1932 (redrawn after Faran [9]). b An. konderi Galvão & Damasceno, 1942
Fig. 53 a, bAn. galvaoi. c, eAn. oswaldoi (Peryassú, 1922). d, f An. konderi
Fig. 54 aAn. squamifemur Antunes, 1937. b An. pseudotibiamaculatus Galvão & Barretto, 1941
Fig. 55 aAn. pseudotibiamaculatus. bAn. gilesi (Neiva, 1908)
Fig. 56 aAn. pseudotibiamaculatus. bAn. gomezdelatorrei Levi-Castillo, 1955
Fig. 57 aAn. vargasi Gabaldon, Cova-Garcia & Lopez, 1941 (redrawn after Levi-Castillo [15]). b An. gilesi (Neiva, 1908)
Fig. 58 a, bAn. gilesi. c, dAn. oiketorakras Osorno-Mesa, 1947 redrawn after Lane [16]
Fig. 59 aAn. tibiamaculatus Neiva, 1906. b An. costai
Fig. 60 aAn. mediopunctatus (Lutz, 1903). b An. eiseni Coquillett, 1902
Fig. 61 a, bAn. mediopunctatus. cAn. costai. dAn. forattinii Wilkerson & Sallum, 1999
Fig. 62 aAn. costai. bAn. forattinii redrawn after Wilkerson & Sallum [17]
Fig. 63 aAn. mattogrossensis Lutz & Neiva, 191163. b An. peryassui
Fig. 64 a, bAn. vestitipennis Dyar & Knab, 1906 (redrawn after Komp [7]). c An. mattogrossensis Lutz & Neiva, 1911
Fig. 65 aAn. mattogrossensis. b, cAn. eiseni geometricus Correa, 1944
Fig. 66 aAn. peryassui. bAn. calderoni
Fig. 67 a, bAn. peryassui. c, dAn. minor da Costa Lima, 1929
Fig. 68 aAn. anchietai Corrêa & Ramalho, 1968. b An. fluminensis Root, 1927
Fig. 69 aAn. maculipes (Theobald, 1903). b An. anchietai
Fig. 70 aAn. apicimacula Dyar & Knab, 1906. b An. pseudopunctipennis (both redrawn after Komp [7])
Fig. 71 aAn. apicimacula. b, cAn. punctimacula Dyar & Knab, 1906 (both redrawn after Komp [7])
Fig. 72 a, bAn. calderoni Wilkerson, 1991. c, d An. shannoni Davis, 1931 (redrawn after Wilkerson et al. [18])
Fig. 73 aAn. medialis Harbach, 2018 (redrawn after Wilkerson et al. [18]). b An. fluminensis
Fig. 74 aAn. medialis. bAn. fluminensis
Fig. 75 a, bAn. neomaculipalpus (b redrawn from [7], c An. pseudopunctipennis (redrawn after Komp [7])
Male genitalia possess characters that can be employed for accurate species identification. However, caution is necessary during the entire multi-step preparation procedure, including staining in a solution of acid fuchsine, separation of some parts with extra-fine needles, and repositioning each part on the microscope slide before covering with a coverslip. Any distortion during the dissection and mounting process presents an obstruction for accurate identification. In particular, the ventral claspette must not be distorted, because this will obstruct recognition of its unique features and shape that are essential for identification. For some species, the shape, and anatomical details of the aedeagus must be examined for species identification. For members of the Myzorhynchella Series, the ventral and dorsal claspettes possess multiple characteristics that are here employed for species identification.
This work would not have been possible without the examination and permission for photographs of specimens from one of the major collections of mosquitoes in South America, repository of valuable reference specimens of multiple species of Anopheles of the Neotropical Region, including primary and secondary type specimens, at the Coleção Entomológica de Referência da Faculdade de Saúde Pública, Universidade de São Paulo, Brazil (FSP-USP). We also thank the Museum of Entomology at the Universidad del Valle, Colombia, for allowing the use of photographic resources and preparing digital images of various parts of its entomological collection. MAMS extends her thanks to the Saúde Pública, Universidade de São Paulo, for their continued support for research projects and the logistics facility for the preparation and maintenance of thousands of specimens of the mosquito collection; and the Fundação de Amparo à Pesquisa do Estado São Paulo for continuous financial support that allowed the execution of hundreds of field sampling efforts for research in the systematics and ecology of mosquitoes (FAPESP Grants #2014/26229-7; #2011/20397-7; #2005/53973-0; CNPq # 301877/2016 to MAMS). RGO and NSC give special thanks to the Facultad de Ciencias Naturales y Exactas de la Universidad del Valle, Colombia, for continuous support and the logistics facility. Project Amazon Malaria Initiative (AMI) - Amazon Network for the Surveillance of Antimalarial Drug Resistance (RAVREDA) provided partial financial support with assistance from USAID and coordination with PAHO/WHO. We are in debt to Yvonne-Marie Linton (Walter Reed Army Institute of Research) and Bruce Harrison (in memoriam) for their thoughtful review of the first version of the identification keys for females, males, and larvae, Caio Cesar Moreira, Faculdade de Saúde Pública, Universidade de São Paulo, for final editing of all illustrations, Ralph E. Harbach (Natural History Museum, London, UK) for his thoughtful revision and valuable contribution for the manuscript, and Aneta Kostadinova for her suggestions, corrections, editorial edits that greatly improved the Part III. The activities undertaken at WRBU were performed in part under a Memorandum of Understanding between the Walter Reed Army Institute of Research (WRAIR) and the Smithsonian Institution, with institutional support provided by both organizations. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the U.S. Army, the Department of Defense, or the U.S. Government.
MAMS and RCW conceived the study. MAMS, RGO and RCW constructed the identification key. MAMS, RGO and NC prepared all illustrations. MAMS, RCW and RGO wrote the manuscript. All authors revised successive drafts of the key. All authors read and approved the final manuscript.
This study was funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grant no. 2014/26229-7, CNPq grant no. 301877/2016-5 to MAMS; the Armed Forces Health Surveillance Board – Global Emerging Infectious Disease Surveillance (AFHSB-GEIS) [P0116_19_WR_05 and P0140_20_WR_05].
Specimens used in the current study are deposited and available in the Coleção Entomológica de Referência, Faculdade de Saúde Pública, Universidade de São Paulo (FSP-USP), São Paulo State, Brazil, the US National Mosquito Collection, Smithsonian Institution, Washington, DC, USA (USNMC), and the Facultad de Ciencias Naturales y Exactas de la Universidad del Valle, Colombia.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Maria Anice Mureb Sallum, Email: masallum@usp.br.
Ranulfo González Obando, Email: ranulfo.gonzalez@correounivalle.edu.co.
Nancy Carrejo, Email: nancy.carrejo@correounivalle.edu.co.
Richard C. Wilkerson, Email: wilkersonr@si.edu
Specimens used in the current study are deposited and available in the Coleção Entomológica de Referência, Faculdade de Saúde Pública, Universidade de São Paulo (FSP-USP), São Paulo State, Brazil, the US National Mosquito Collection, Smithsonian Institution, Washington, DC, USA (USNMC), and the Facultad de Ciencias Naturales y Exactas de la Universidad del Valle, Colombia.