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Differences between the real 3D-shape of the male genitalia and traditional 2D pictures obtained during examination of balsam slides are revealed. Original ...
ISSN 0013-8738, Entomological Review, 2010, Vol. 90, No. 4, pp. 511–532. © Pleiades Publishing, Inc., 2010. Original Russian Text © A.V. Khalin, 2009, published in Parazitologiya, 2009, Vol. 43, No. 5, pp. 389–410.

Three-Dimensionality of the Male Genitalia Shape and Species Identification in the Mosquito Genus Aedes Meigen, 1818 (Diptera, Culicidae) A. V. Khalin Zoological Institute, Russian Academy of Sciences, St. Petersburg, 199034 Russia e-mail: [email protected] Received May 7, 2009

Abstract—The significance of the 3D-shape of the mosquito male genitalia is demonstrated in the species of the genus Aedes1 (Diptera, Culicidae) as an example. Differences between the real 3D-shape of the male genitalia and traditional 2D pictures obtained during examination of balsam slides are revealed. Original descriptions of 3Dshaped coxites, claspettes, lobes of tergite IX, and proctiger in 30 species of the subgenus Ochlerotatus are given. This study allowed finding new diagnostic characters, including the basic 3D-shape of the basal lobe of the coxite, of the claspette filament, and of some other structures. DOI: 10.1134/S0013873810040111

Mosquitoes of the genus Aedes form a significant fraction of a complex of bloodsucking dipterans in Russia, because the females of the majority of species of this genus are active bloodsuckers attacking humans and causing them considerable inconvenience. The most dangerous are, however, pathogens transmitted by mosquitoes, such as mosquito fevers and encephalitis. In northwestern European Russia, Okelbo viruses, pathogens of the Karelian fever, are transmitted by mosquito species Aedes (Ochlerotatus) communis (DeGeer, 1776), А. (О.) punctor (Kirby, 1837), A. (O.) hexodontus Dyar, 1916, A. (O.) pullatus (Coquillett, 1904), A. (O.) diantaeus Howard, Dyar et Knab, 1913, and A. (O.) impiger (Walker, 1848) (Lvov et al., 1989). The study of relations between different groups of mosquitoes of the family Culicidae, transmitters of pathogens, and different groups of these pathogens needs accurate identification of vector species; this identification is frequently hampered by the use of commonly accepted taxonomic characters. Some of these characters are unreliable (e.g., coloration of tho_____________ 1

In the present publication, the author uses Edwards’s (1932) classification, according to which, the taxon Ochlerotatus is considered as a subgenus of the genus Aedes. Other points of view on the composition of the genus Aedes (e.g., Reinert, 2000a, 2000b; Reinert et al., 2004, 2006, 2008) were presented earlier (Khalin, 2005b, 2007; Khalin and Gornostaeva, 2008)].

racic scales); some other characters are treated differently by different authors; besides, many diagnostic structures (especially setae and scales) are usually lost during collecting. These circumstances ask for the use of new approaches to revealing new diagnostic characters. The body of a bloodsucking mosquito and all the appendages of this body, including wings, are volumetric structures. This fact should be taken into consideration when we use body structures as diagnostic characters. Any analysis of body fragments pictured in the literature needs understanding that these figures show us only the projection plane of a structure, but not its real 3D model. Projection of the same organ at another angle can look quite different, misinforming entomologists. The study of slides with mosquitoes also results in obtaining information on the plane projection of contours of certain structures, but not on their real 3D shape. For example, plane projection of a sphere will look as a circle at any angle (Fig. 1, 1). The majority of mosquito body structures are, however, not spheres. Even simple formations, such as conical prominences of the cuticle (Fig. 1, 2), can possess different plane projections, namely a circle or a triangle. Besides, a pyramid can also look like a triangle in plane projection (Fig. 1, 3); some modified cuticular outgrowths resemble pyramids. Thus, different plane projections of the same structure can differ, whereas some projec-

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Fig. 1. Projections of 3D-figures onto a 2D plane: (1) sphere; (2) cone; (3) pyramid.

tions of structurally different objects can be similar (Fig. 1, 1–3). This fact is confirmed by the results of our studies (Khalin, 2005a, 2005b, 2006, 2006a, 2006b, 2007, 2009) and by literary data (Stackelberg, 1937; Rubtsov, 1951, 1953; Maslov, 1967; Gutzevich et al., 1970; Carpenter and La-Casse, 1955; Snodgrass, 1957; Barr, 1958; Belkin, 1962; Mohrig, 1969; Knight and Laffoon, 1971; Becker et al., 2003). The male genitalia are one of the most complicated morphological structures in mosquitoes of the family Culicidae Meigen, 1818. Traditionally, however, it was examined only as a single plane projection (Stackelberg, 1937; Carpenter and LaCasse, 1955; Gutzevich et al., 1970; Becker et al., 2003) that evidently gave no complete data on its real 3D shape (Fig. 2, 1). Barr (1958) pointed to the expediency of the study of the male genitalia in different views, offering preliminary examination of the male genitalia in temporary creosote slides before placing them into balsam. He noted that an object can be turned, e.g., sidewise, in order to examine such structures as the shape of the claspette wing. In his monograph, Barr (1958) gives pictures of the male genitalia in mosquitoes of the family Culicidae in ventral view, and also lateral views of the claspettes and the coxite in some representatives of the subgenus Ochlerotatus. The study of some structures of bloodsucking mosquitoes (larvae, the head of the imago, the mail genitalia, etc.) in a single projection is associated with preparation of permanent balsam slides. Although permanent slides are convenient for study and preservation, they also possess very significant disadvantages. An object can be examined only in a single view, it is impossible to turn or shift it. As a result, the most part of information on its real 3D shape is lost. Unfortunately, it is rather hard to solve this problem. The most complete information on the 3D shape

of a structure can be obtained using a scanning electron microscope (SEM). The methods of optical microscopy (OM) are usually less informative in relation to the 3D structure of mosquito bodies. Some data on the basic 3D shape of, e.g., mosquito male genitalia can be obtained during examination of temporary slides under a binocular microscope. The use of the latter is, however, limited by its low resolution. An optical microscope allows reaching higher magnification, but information on the 3D shape of the object is, as a rule, lost. This information can be obtained mainly by examination of a series of fine cuts. Thus, a combined use of binocular and optical microscopes allows describing the 3D shape of a structure, even if partly. Some practical recommendations for specialists determining bloodsucking mosquitoes and preparing slides are given below. Permanent slides (e.g., of the male genitalia) are traditionally prepared for the diagnostics of species of the genus Aedes; we, however, do not recommend fixing the material in balsam till its identification. Temporary preparations are more preferable for examination, because several projections of an object can be obtained. In our opinion, examination of the male genitalia in a 60–70% glycerin aquatic solution is the most convenient. The general view of the genitalia can be examined in a drop of glycerin solution on a piece of glass. In such a way it is possible to reveal relative arrangement and basic 3D shape of large structures (valves, claspettes, proctiger, etc.). Small details (the structure of the basal wart, of wings and stems of claspettes, of lobes of the abdominal tergite IX, of the aedeagus, etc) are better revealed under an optical microscope. Manipulations with a fine adjustment knob allow obtaining some information on the 3D shape of these structures. ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

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Fig. 2. Aedes cantans (Meigen, 1818), male genitalia, ventral view: (1) according to Stackelberg (1937), (2) scheme (tergite IX removed); cl, claspette; wcl, wing of claspette; scl, stem of claspette; cox, coxite; par, paraproct; sta, stylus appendage; st, stylus; aed, aedeagus; st IX, abdominal sternite IX; t IX, abdominal tergite IX.

Thus, the goal of the present publication is to bring the importance of study of the 3D shape of insect body structures to the notice of entomologists dealing with dipteran diagnostics. In this work, structures of the male genitalia in the genus Aedes are analyzed as an example; however it would be worthwhile to study 3D shape of other organs of bloodsucking mosquitoes. The study of the spatial configuration of the male genitalia and also of other body structures would clarify species diagnostics not only in the family Culicidae, but in other dipteran families as well. MATERIALS AND METHODS The work was performed in the Laboratory of Parasitology, the Zoological Institute, Russian Academy of Sciences (ZIN RAS). The author examined the material deposited at ZIN RAS (mosquitoes on entomological needles and slides with the genitalia), and also self-collected material. A total of more than 300 specimens belonging to 30 species of the subgenus Ochlerotatus were examined, including about 50 specimens of 19 species examined under a scanning electron microscope and more than 250 specimens, belonging to 30 species of the subgenus studied were examined under an optical microscope. The structure of the genitalia was examined in the following species: A. annulipes (Meigen, 1830); ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

A. behningi Martini, 1926; A. cantans (Meigen, 1818); A. cyprius Ludlow, 1920; A. euedes Howard, Dyar et Knab, 1913; A. excrucians (Walker, 1856); A. flavescens (Muller, 1764); A. kasachstanicus Gutzevich, 1962; A. mercurator Dyar, 1920; A. riparius Dyar et Knab, 1907; A. caspius (Pallas, 1771); A. dorsalis (Meigen, 1830); A. pulchritarsis (Rondani, 1872); A. stramineus Dubitzkij, 1970; A. cataphylla Dyar, 1916; A. communis (De Geer, 1776); A. detritus (Haliday, 1833); A. diantaeus Howard, Dyar et Knab, 1913; A. hexodontus Dyar, 1916; A. impiger (Walker, 1848); A. intrudens Dyar, 1919; A. leucomelas (Meigen, 1804); A. montchadskyi Dubitsky, 1968; A. nigrinus (Eckstein, 1918); A. nigripes (Zetterstedt, 1838); A. pionips Dyar, 1919; A. pullatus (Coquillett, 1904); A. punctor (Kirby, 1837); A. simanini Gutzevich, 1966; A. sticticus (Meigen, 1838). In our morphological studies, we used methods of scanning electron microscopy (SEM) and computerized optical microscopy. Electron microscopy allows characterizing surface microrelief, three-dimensional configuration of microstructures, and their arrangement in relation to each other. Optical microscopy is the most fruitful in the study of flat semitransparent objects that cannot be examined in SEM. Light microscopic images (Laboval 4, Leica 9, and Leica DM5000B) were captured with a Panasonic WV-

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PB330/GE, Panasonic WV-CP460, Nikon Coolpix 4500, or Leica DFC320 camera and transferred into a Pentium IV or Coretm2 PC. The digital images were processed using Corel Photo-Paint 9 and Corel Draw 9 software. Preparation of objects for SEM studies was performed as follows. The objects observed under binocular optical microscopes (MBI-3, MBS-10, and Leica MZ95) were dissected with wolfram needles pointed by electrolysis; the dissected objects were fixed on special stubs with glue or double-sided sticky film and coated with platinum in a vacuum coater. Special drying was used for examination of specimens stored in 70% alcohol in SEM. The ultimate abdominal segments were cut off and treated with 10% alkaline solution, then washed in distilled water, dehydrated in a series of alcohols and in acetone. Alkaline treatment was used for cleaning of surface structures; no visible deformation of the integument was observed during this procedure. The objects were dried in CO2, using the method of critical point drying. The objects were glued on special stubs with double-sided sticky film and coated with platinum. Balsam slides were prepared for the study of mosquito morphology under an optical microscope. The objects were dehydrated in alcohols, and after staining in lavender oil and xylene were placed in balsam on glass slides. In order to avoid deformations of the genitalia under the cover glass, small stalks made of cover glass pieces were used. Terminology of Male Genital Structures in the Family Culicidae2 In bloodsucking mosquitoes, the male genitalia are represented by the strongly modified abdominal segment IX (Fig. 2, 1, 2). The copulative organ proper (aedeagus) is situated on this segment together with specialized sexual appendages (valves and claspettes) used for the capture of female abdomen during copulation. Traditionally, the proctiger (a highly integrated complex enveloping the anal aperture) is also included into the male genitalia. It is formed by abdomen segment X and probably also by the structures of the _____________ 2

In this section, the structure of the genitalia is described according to the literary data (Edwards, 1932; Stackelberg, 1937; Marshall, 1938; Rubtsov, 1951, 1953; Carpenter and LaCasse, 1955; Snodgrass, 1957; Belkin, 1962; Maslov, 1967; Mohrig, 1969; Gutzevich et al., 1970; Knight and Laffoon, 1971; Wood, 1991; Sinclair, 2000; Becker et al., 2003).

strongly reduced segment XI, and mainly fulfils the excretory function. Rotation of abdominal segment VIII (and all the subsequent segments) by 180º in relation to its longitudinal axis is the characteristic feature of males of the family Culicidae. This rotation occurs during the first day after emergence of an adult from a pupa; as a result, tergites occupy a ventral position and sternites, a dorsal one. Together with the term “male genitalia,” many authors use such terms as “hypopygium,” e.g., Stackelberg (1937), Gutzevich et al. (1970), Becker et al. (2003) or “male terminalia” (Carpenter and LaCasse, 1955). In my opinion, it is not necessary to use some special term for the designation of this structure; therefore, in the present work I use the term “male genitalia” the most commonly accepted for all the insects. Many authors (Snodgrass, 1957; Knight and Laffoon, 1971; Becker et al., 2003) suggest using the terms “dorsal” and “ventral” for the parts of the male genitalia independently of their definitive position (i.e., dorsal for the lower part and ventral for the upper part). For example, the dorsal surface of the coxite will correspond to the lower surface, etc. Belkin (1962), a well-known American entomologist, used the terms “tergal” and “sternal” for the description of these structures. However, Knight and Laffoon (1971) did not recommend using such names, because they point to the origin of a structure (from tergite or sternite, respectively), which is not always true. In our work we use the terms “dorsal” and “ventral” in order to avoid erroneous interpretation. The terms “tergal” and “sternal” are used only as applied to the segments themselves, when the origin of the structure from the tergite or the sternite is underlined. The main part of the male genitalia, abdominal segment IX, or the sexual segment (Maslov, 1967), bears highly specialized appendages serving for the capture of the female abdomen, and also the copulative organ itself. According to some authors (Rubtsov, 1951, 1953; Maslov, 1967), the tergal, sternal, and pleural parts in abdominal segment IX are distinguished. The tergal part is represented by tergite IX (“epandrium” after Sinclair, 2000), divided frequently into paired prominences (or lobes), connected by a narrower bridge. The sternal part of abdominal segment IX consists of the sternite (“hypandrium” after Sinclair, 2000), which is fused with the tergite in a common ring in the majority of species of the family Culicidae. ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

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Some authors (Rubtsov, 1951, 1953; Maslov, 1967) also include the gonofurca, a special formation serving for the holding of the female postgenital plate during copulation, into the sternal part. The gonofurca is a sclerite with paired lobes, connected by a bridge. In some genera of the family Culicidae, the lobes of the gonofurca are also named claspettes, or basal warts [“basal lobe,” “basal mesal lobe” after Stackelberg (1937), Gutzevich et al. (1970), Carpenter and LaCasse (1955), Snodgrass (1957), Knight and Laffoon (1971), Becker et al. (2003)]. In this case, such a plurality of terms is superfluous and brings confusion, because the term “basal wart” is used for the designation of coxite’s outgrowth (see below). Within the family Culicidae, the structure of these lobes is very diverse, which makes their homologation difficult. The large movable lateral outgrowths (valves or gonopods, see Figs. 3, 4) are treated by some authors (Rubtsov, 1951, 1953; Maslov, 1967) as outgrowths of pleural part of segment IX, i.e., as homologues of thoracic legs. Each valve includes the coxite (homologue of the coxa) and the stylus (homologue of the telopodite of lower arthropods). According to Snodgrass (1957), however, the valves cannot be homologized directly with thoracic legs, but are “parameres” originating from primary phallic lobes of abdominal segment IX. The phallic complex as a whole and also claspettes appeared as a result of fusion and following differentiation of “mesomeres,” i.e. secondary lobes developed at the base of parameres. The coxite (Gutzevich et al., 1970), “the 1st segment of valves” (Stackelberg, 1937), “gonocoxite,” “coxite” (Edwards, 1932; Carpenter and LaCasse, 1955; Knight and Laffoon, 1971; Becker et al., 2003), “sidepiece” (Belkin, 1962) is the basal and the largest valval segment, characterized by a complicated structure, very diverse within the family Culicidae. The inner, dorsal, and ventral surfaces can possess prominences (lobes, warts) bearing different cuticular formations. In the subgenus Ochlerotatus, the coxite is elongated, weakly bent, and bean-shaped in the transverse cross-section; its dorsal and ventral surfaces are, as a rule, sclerotized, whereas the inner surface is membranous. The dorsal part of the coxite bears outgrowths on its inner margin (they are also named warts, lobes, or blades), including the apical/basal lobe and the basal mesal lobe (Edwards, 1932; Carpenter and LaCasse, 1955; Knight and Laffoon 1971; Becker ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

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et al., 2003). Some authors, e.g., Belkin (1962), underline their belonging to the dorsal part, naming them “a basal/apical tergomesal lobe.” In Russian literature (Stackelberg, 1937; Gutzevich et al., 1970; Gutzevich and Dubitsky, 1981), these formations are called “warts;” we use this term in the present work. The shape and chaetotaxy of the basal wart is very important taxonomically. This structure can bear microtrichiae, scales, and setae. The spine-like setae are the most important [= “strong setae” (Stackelberg, 1937); “spines” (Gutzevich et al., 1970; Gutzevich and Dubitsky, 1981) “spine-like (or strong) setae of the basal lobe” (Becker et al., 2003); “spines of the basal lobe” (Edwards, 1932; Knight and Laffoon, 1971)]. The second segment of valves is named the stylus (Gutzevich et al., 1970); the 2nd valval segment (Stackelberg, 1937); gonotelopodite (Maslov, 1967); “gonostylus,” “style” (Edwards, 1932; Knight and Laffoon, 1971; Becker et al., 2003); “dististyle” (Carpenter and LaCasse, 1955); “clasper” (Belkin, 1962). By contrast to the coxite, the stylus is finer; in the majority of mosquitoes, it is cone-shaped and weakly bent. A digitiform or the spine-like appendage is articulated to the stylus at the apex. The male copulatory organ in the family Culicidae is named the aedeagus (Stackelberg, 1937; Gutzevich et al., 1970; Maslov (1967) or the phallosoma (Edwards, 1932; Belkin, 1962; Knight and Laffoon, 1971). The aedeagus consists of the phallosoma and a system of associated levers: the parameres and the basal plate (apodeme of the parameres). In this case, there is a confusion of terms, because the terms phallosoma and aedeagus are used by different authors either for the designation of the whole copulatory complex, including levers, or only for the copulatory organ itself. The phallosoma (Gutzevich et al., 1970), designated by some authors as mesosoma (Stackelberg, 1937), phallus (Maslov, 1967), aedeagus (Edwards, 1932; Belkin, 1962; Knight and Laffoon, 1971; Becker et al., 2003), or phallosome (Carpenter and LaCasse, 1955) is a more or less sclerotized formation bearing the sperm duct. The shape of the phallosoma can vary even within a single genus. For example, some subgenera of the genus Aedes are characterized by a solid bean-shaped or cylindrical phallosoma; in other subgenera, it is subdivided into a pair of lateral plates bearing apical teeth. The parameres of the aedeagus (Stackelberg, 1937; Gutzevich et al., 1970), also

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known as gonepisternites (Maslov, 1967) or parameres (Edwards, 1932; Carpenter and LaCasse, 1955; Knight and Laffoon, 1971; Becker et al., 2003) look like elongated plates situated at sides of the phallosoma and articulating with the latter at the base. The basal plate (Stackelberg, 1937; Gutzevich et al., 1970) also known as the gonepimerite (Maslov, 1967), the basal piece (Edwards, 1932; Belkin, 1962), the basal plate (Carpenter and LaCasse, 1955), the parameral apodeme (Knight and Laffoon, 1971) is articulated with the apodeme of the coxite and with parameres (occasionally, also with the proctiger). The structure of the postgenital abdominal segments, or the proctiger (Edwards, 1932; Belkin, 1962; Knight and Laffoon, 1971; Becker et al., 2003), i.e., abdominal segments X and reduced XI, in the family Culicidae is characterized by a comparatively narrower diversity in comparison with segment IX. The homology of proctiger sclerites is not clear yet; therefore, many authors use only descriptive names for their designation. Paired, frequently strongly sclerotized sclerites forming the paraproct (Edwards, 1932; Belkin 1962; Knight and Laffoon, 1971; Becker et al., 2003), frequently homologized with the sternite of abdominal segment X (Gutzevich et al., 1970), occupy a lateral position on the proctiger. It is also homologized with abdominal sternite X (Gutzevich et al., 1970). The basolateral parts of the proctiger (proximal continuations of the paraproct), also known as basolateral sclerotizations (Belkin, 1962), are occasionally homologized with the abdominal tergite X (Knight and Laffoon, 1971). Some authors homologize the dorsal part of the proctiger with cerci (Knight and Laffoon, 1971; Becker et al., 2003) and, hence, treat the sclerites that are found there as cercal ones. Other authors (Maslov, 1967; Gutzevich et al., 1970) treat the dorsal part of the proctiger as tergite X. RESULTS AND DISCUSSION The combined SEM and OM methods were used for the detailed study of the male genitalia in 30 species of the subgenus Ochlerotatus of the genus Aedes. As a result, we significantly improved our knowledge of the structure of appendages of abdominal segment IX (valves and claspettes) as the basic structures for species identification. Our study revealed the fact that the real 3D configuration of the male genitalia in the genus Aedes strongly differs from pictures made on the basis of examination of balsam slides. The use of scanning electron microscopy for the study of the male

genitalia has demonstrated that some structures are more complicated than it was believed after examination of these structures under an optical microscope. The main advantages of SEM included the ability to see the non-warped 3D shape of objects rather than high magnifications (we used mainly magnifications varying from 500 to 1000). This result can hardly be achieved when studying balsam slides with an optical microscope. In many species of the genus Aedes, such complicated structures as, e.g., coxites, claspettes, and proctiger are deformed during preparation of balsam slides; differing periods of alkaline treatment and also cover-glass pressure play their roles. Therefore, it is very hard to reveal the real shape of the male genital structures under an optical microscope. In this connection, descriptions of these structures in the literature are incomplete even in such cases, when they are very important for diagnostics. Descriptions of the coxite, basal part of valves, claspettes, tergite IX, and proctiger in the examined species made on the basis of original studies of the author are given below. The coxite. The results of our studies (Khalin, 2005b, 2006a, b, 2007) demonstrated the complicated character of the 3D shape of coxites (the basal part of valves in the male genitalia). A single projection (Fig. 2) is insufficient for characterizing the coxite shape in the genus Aedes. Additional projections (Figs. 3, 4) are more informative for the description of the 3D shape of the coxite. Our SEM and OM study of 25 species of the subgenus Ochlerotatus allowed revealing the natural shape of the coxite, including all its prominences, lobes, and emarginations (Figs. 3, 4). In A. communis, A. punctor, A. caspius, A. cantans, and A. diantaeus, this structure was examined in more detail; we used the critical point method for drying of specimens. Using this method it was possible to minimize the damage done to the fine cuticle of the coxite when it is air-dried. The general shape of the coxite was already characterized in the last century (Marshall, 1916). This task was solved by methods of optical microscopy: examination of the male genitalia under the binocular microscope gives us some information on the coxite shape. Finer structural details (shape of its prominences and chaetotaxy), however, usually stay beyond the optical possibilities of a binocular microscope; at the same time, it is hardly possible to reveal their 3D shape under a transmitting optical microscope. ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

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Fig. 3. Aedes Meigen, 1818, male genitalia, scheme: (1–3) coxite; (4–5) basal wart of coxite [(1, 4) A. punctor (Kirby, 1837); (2, 3, 5) A. diantaeus Howard, Dyar et Knab, 1913; (1, 4) ventral-inner view; (2) inner view; (3) caudal view; (5) dorsal-inner view]; bw and aw, basal and apical warts of the coxite; cob and coa, base and apex of the coxite; vens, ins, dors, ventral, inner, and dorsal surfaces of the coxite; dp. bw, pp. bw, distal and proximal parts of basal wart of the coxite; dset and prset, dorsal and proximal spine-shaped setae of the basal wart; pos. bw and ans. bw, posterior and anterior surface of basal wart; lars, large setae; m, membrane on inner surface of coxite; mitr, microtrichiae; s. set, small setae; s. tf, tuft of setae on coxite; art, place of articulation between coxite and stylus; sp. set, spineshaped seta of basal wart; p. m, proximal margin of basal wart; v. m, ventral margin of basal wart.

In mosquitoes of the subgenus Ochlerotatus, the coxite (Figs. 3, 4) resembles a strongly elongated ellipsoid, more (Fig. 3, 2) or less (Fig. 3, 1) curved and bearing a longitudinal emargination. In order to make the description more convenient, we conditionally subdivided the entire surface of the coxite into 4 parts: dorsal (lower), ventral (upper), external, and internal surfaces (Fig. 3, 4). The dorsal, ventral, and external surfaces are more or less plain, lacking any promiENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

nences and lobes. The surface of the coxite is uniformly covered with microtrichiae, it also bears setae of different length and scales on the external surface. The inner surface is divided into dorsal and ventral parts by the longitudinal emargination. This emargination is represented by a membranous part of different width. For example, in A. communis it is comparatively wide and in A. caspius, more narrow. In proximal and medial parts of the coxite, the emargination

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Fig. 4. Aedes Meigen, 1818, coxite under scanning electron microscope (SEM): (1, 2) A. punctor (Kirby, 1837); (3, 4) A. diantaeus Howard, Dyar et Knab, 1913 [(1, 3) ventral view; (2) ventral-inner view; (4) inner view]; dp. co and pp. co, distal and proximal parts of coxite; th. set, thickened setae. For other designations, see Fig. 3.

reaches closer to its ventral part and in its distal part, it is smoothly bent dorsally. Thus, the distal margin of the dorsal part of the internal surface of the coxite forms a lobe named the apical wart. This apical wart may look as a noticeable prominence (e.g., in A. diantaeus, A. cantans, A. communis, and A. punctor) or like only a fold (in A. caspius). The apical wart looks differently at different angles (Figs. 4, 5); it is necessary to take this fact into consideration using this character as a diagnostic one.

In A. diantaeus, the coxite (Figs. 3, 4, 5) is rather strongly modified in comparison with coxites of other representatives of the subgenus Ochlerotatus (e.g., A. communis, A. punctor, A. cantans, and A. caspius). In this coxite, together with dorsal, ventral, external, and internal surfaces, we can conditionally distinguish its proximal and distal parts (Fig. 4, 3). The distal part is somewhat dilated, because it bears the apical wart. This structure is represented by a strongly projecting lobe situated on internal and partly on dorsal surfaces ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

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Fig. 5. Aedes Meigen, 1818, male genitalia in SEM: (1) coxite; (2) apical wart of coxite; (3–6) basal wart of coxite; [(1, 2) A. diantaeus Howard, Dyar et Knab, 1913; (3–6) A. caspius (Pallas, 1771); (1, 4, 5) caudal view; (2) dorsal view; (3) ventral view; (6) anterior view]. For other designations, see Fig. 3.

of the coxite (Figs. 3, 4, 5). The study of the shape of the apical wart is hampered by its position: it forms an acute angle with the longitudinal axis of the coxite. In ventral view, the apical wart looks like a digitiform outgrowth with a weak band in the middle (Fig. 4, 3); the wart is more than twice as long as its width at the band. In apical-caudal view, the wart looks like an asymmetrical outgrowth with rounded apex and diENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

lated base; at the same time, its width at the base exceeds the length of the wart (Fig. 5, 2). The dorsal surface of the wart is moderately sclerotized and covered with microtrichiae and short setae of different width. Among these setae, 2 thickest ones are situated at the base of the wart (Fig. 4, 4). The ventral surface of the wart is membranous (Fig. 5, 2) and covered only with microtrichiae. A vast membranous area is situated

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on the internal surface of the coxite above the apical wart. Proximally, it reaches the posterior part of the basal wart and then continues above its ventral margin Fig. 4, 4). A hardly visible carina is situated on the border between the membrane and the moderately sclerotized ventral surface of the coxite (Figs. 3, 3; 5, 1). A wide tuft of long setae pointed inwards is situated not on the internal, but on dorsal surface of the coxite (Figs. 3, 3; 4, 4; 5, 1). The bases of tuft setae are arranged approximately in a line order; this line is situated at a small angle to the longitudinal axis of the coxite. At some distance from the base, the setae are strongly bent; as a result, in ventral view (Fig. 4, 3), the setae seem to be situated just on the internal surface of the coxite, as it was mentioned by several authors (Stackelberg, 1937; Barr, 1958; Mohrig, 1969; Gutzevich et al., 1970; Becker et al., 2003). In representatives of the genus Ochlerotatus, a more or less complicated outgrowth (the basal wart) is situated at the base of the coxite on its internal surface (Fig. 3, 4–8). The shape of the basal wart significantly differs in species examined by us; the basic structure, however, is more or less similar in some species examined. For example, in A. caspius and closely related A. dorsalis, the basal wart possesses rather simple structure (Fig. 5, 3–6). Its shape somewhat resembles the shape of a polypore growing on a tree trunk. Let us conditionally, for the description of the basal wart, subdivide the latter into anterior and posterior surfaces, oriented more or less perpendicularly to the longitudinal axis of the coxite, and also dorsal and ventral margins. We treat the length of the basal wart as the shortest distance between its apex and the internal surface of the coxite (Fig. 5, 3). The anterior surface of the basal wart (Fig. 5, 6) is not smooth; it is covered with folds and lacks any cuticular outgrowths. The posterior surface of the wart (Fig. 5, 4) is weakly concave, forming an angle of slightly more than 90° between the wart and the inner surface of the coxite; at the same time, no distinct border between the surfaces of the wart and the coxite is visible. The entire surface of the basal wart is uniformly covered with microtrichiae and small setae. In order to describe the chaetotaxy of the basal wart, we distinguished conditionally several types of setae. The small setae include setae as long as the basal wart or shorter; the large setae include setae significantly longer than the basal wart. The basal diameter of the large setae at the base is more than twice as long as that of the small setae. The basal wart of A. caspius bears about 10 small

setae; each seta is situated on a separate tubercle (Fig. 5, 4, 5); similar setae are also situated on the inner surface of the coxite posterior to the basal wart. More than 10 large setae are situated on its dorsal and ventral margins. The dorsal margin also bears structures, in the majority of published papers designated as “spines of the basal wart” (e.g., Mohrig, 1969; Gutzevich et al., 1970). This name is rather insufficient, because this structure is not a spine (the socket is well visible, Fig. 5, 5). In my opinion, the term “spine-shaped seta” (Stackelberg, 1937; Becker et al., 2003) is more convenient. In A. caspius, these structures look as thickened setae (Fig. 5, 3–6); two setae are present in this area, including a small spine-shaped seta (this seta is shorter than the basal wart and is situated at the base of the wart) and the large spine-shaped seta, which is 1.5–2 times as long as the wart and is situated distally to the small seta. In A. dorsalis, spineshaped setae are usually situated at longer distances from each other (this distance is approximately 3.5–4.0 times as long as the diameter of the main spine-shaped seta) than in A. caspius, where this distance is approximately 2.5 times as long as the diameter of the main spine-shaped seta. Our examination of A. caspius specimens from the collection, however, revealed the presence of the third additional seta situated dorsally to the large spine-shaped seta; the distance between these setae can vary. Some aspects of the structure of the setae situated on the basal wart of the coxite should be especially noted. For example, the basal diameter of the large spine-shaped seta of the basal wart is approximately twice as long as that of the small one. The diameter of the small spine-shaped seta, in its turn, is approximately equal to that of setae situated ventrally to the large seta (Fig. 5, 4–6) and is 4–5 times as long as that of the short setae on the posterior surface of the basal wart. The surface of the small spine-shaped seta bears longitudinal ribs, similarly to the short setae situated on the posterior surface of the basal wart. The large spine-shaped seta and setae situated ventrally, all bear shallow longitudinal grooves with smoothed margins, significantly differing from ribs of other setae (Fig. 5, 5). The length of the setae situated ventrally to the large spine-shaped seta decreases with the distance from the latter (Fig. 5, 4–6). The shape of the basal wart of A. communis (Fig. 6, 1) is similar to that of A. caspius; its posterior surface is, however, more concave. Thus, the shape of ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

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Fig. 6. Aedes Meigen, 1818, basal wart of the coxite (SEM): (1) A. communis (DeGeer, 1776); (2) A. punctor (Kirby, 1837); (3) A. cantans (Meigen, 1818); (4) A. diantaeus Howard, Dyar et Knab, 1913 [(1–3) ventral view; (4) inner view]; hs, hooked setae of basal wart; d. m, dorsal margin of basal wart, . For other designations, see Figs. 3, 4.

this structure resembles a basket, as it was noted by Mohrig (1969). The chaetotaxy of the basal wart of the coxite includes only a single spine-shaped seta and more than 30 setae of lesser diameter (half as thick as the spine-shaped seta or thinner). The spine-shaped seta occupies the most dorsal position on the wart; in this region, the border between the wart and the dorsal surface of the coxite is indistinct. Straight or weakly bent setae, some of which are more than twice as long as the wart, are situated ventrally to it along the margin of the wart and also on its posterior surface. The largest of them occupy the most dorsal (close to the spine-shaped seta) and ventral positions. The ventral setae differ from all the others, being strongly bent in the middle or distally to the middle. As in A. caspius, the entire posterior surface of the wart is covered with microtrichiae. ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

According to our preliminary data, the similar shape is characteristic of the basal wart in A. cataphylla and A. leucomelas, and also, probably, in A. cyprius and A. mercurator. These species were examined in SEM and OM as dry collection material; hence, the shape of the wart could be deformed during air-drying. The main difference noted by us includes the fact that the margin of the wart is bent backwards in the ventral part, smoothly passing into the ventral part of the coxite. These species also possess a single spineshaped seta situated in the most dorsal part of the wart and numerous thinner setae situated ventrally to it along the wart’s margin. The structure of the basal wart in A. punctor (Figs. 3, 4; 6, 2), a species closely related to A. communis, differs from that in A. caspius by the presence

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of a vast distal part, situated on the internal surface of the coxite. The shape of the basal wart in this species is rather complicated for the description: it possesses a proximal part (on the whole, similar to the basal wart of A. caspius) and a distal part, a flat prominence situated directly beyond the proximal part. The border between these two parts is indistinct; the border between the distal part of the wart and the inner surface of the coxite is also rather conditional. Similarly to A. caspius, the proximal part of the basal wart resembles a polypore. The posterior surface of the proximal part is, however, oriented not perpendicularly to the longitudinal axis of the coxite, but at an angle of 45–60° to it (Fig. 6, 2). The posterior surface looks like a triangle with rounded sides and its dorsal margin is 2–3 times as long as the ventral one. Nearly all the posterior surface is uniformly covered with small setae similar to those found in A. caspius. More than 10 large setae, however, are accumulated in the area of the dorsal angle and the spine-shaped seta of the basal wart occupies the most dorsal position (Fig. 6, 2). This seta is distinctly distinguished among others not only by its thickness, but also by its structure: deep longitudinal grooves are revealed on its surface at higher magnifications (1,000–5,000). These grooves are significantly deeper than ordinary ribs distinguished in the neighboring setae and grooves on the spine-shaped seta of A. caspius. By contrast to A. caspius and A. communis, in A. punctor, microtrichiae occupy only a small area of the wart along its dorsal margin (Fig. 3, 4). The distal part of the basal wart, most distinctly expressed in A. punctor, is a continuation of the posterior surface of the proximal part of the wart; it looks like a low irregular prominence with rounded margins (Fig. 3, 4) covered with short setae situated on prominences similar to the proximal part of the wart; no microtrichiae are found in this region. In A. cantans, the basal wart possesses a structure different from that found in A. caspius, A. communis, and A. punctor. In the basal wart region, the coxite forms a cone-shaped prominence with a rounded apex, slightly flattened distally-proximally (Fig. 6, 3). An elongated lobe directed ventrally-distally-medially at some angle runs from the prominence ventrally. Thus, in caudal view, the basal wart looks like a digitiform outgrowth. The posterior surface of the wart is weakly concave and covered with setae of different sizes and with microtrichiae in the dorsal part. It should be noted that a group of large setae, including the spine-shaped seta, is situated in the most dorsal

part of the wart; this group is similar to that found in A. communis. The rest of the posterior surface of the wart is irregularly covered with setae: rather thickened but short setae (up to half length of the wart) are grouped in the ventral part of the wart (Fig. 6, 3). The spine-shaped seta of the wart is distinctly distinguished among the surrounding setae (similarly to A. communis), the diameter of this seta is 3–7 times as long as that of the surrounding setae. The length of the spine-shaped seta and nearby setae is approximately equal to that of the wart. In A. diantaeus, the structure of the basal wart (Figs. 3, 5; 6, 4) also differs from that found in the abovementioned species (A. caspius, A. communis, A. punctor, and A. cantans). Its main part is situated on the interior surface of the coxite and only a small proximal part occupies the area adjoining the dorsal surface. On the whole, in the inside view, the basal wart of the coxite looks like a flat triangular prominence with strongly rounded apices (Fig. 6, 4). The height of this structure is different: the wart is the highest at the proximal margin and then it gradually decreases in height distally along the ventral margin (Fig. 7, 1). The dorsal margin of the basal wart is indistinct: in the proximal part, the border between the basal wart and the interior surface of the coxite is slightly visible and in the distal part, virtually absent. We treat the distance between proximal and distal margins of the wart as its length; the latter is 0.4 times as long as the coxite. In A. diantaeus, the basal wart of the coxite bears cuticular outgrowths of a different type; the largest of the latter include three spineshaped setae. Among these setae, the proximal spineshaped seta is the longest (Fig. 7, 1); it is as long as the basal wart and is situated in the proximal part of this wart on the border between the interior and the dorsal surfaces of the coxite. The distal spine-shaped setae are slightly shorter, their length constituting about two thirds of the length of the basal wart and their thickness, slightly more than three fourths of the thickness of the proximal seta. They are situated in the distal part of the basal wart, closer to the ventral surface of the coxite. A detailed study of the surface of spineshaped setae revealed longitudinal grooves similar to those found on the surface of other setae. No characters demonstrating the fusion of several setae, as it was found, e.g., in the spine-shaped seta of A. cantans, were revealed. Together with spine-shaped setae, about 20 smaller setae are also found on the basal wart; they are situated mainly in its distal half. The ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

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Fig. 7. Aedes Meigen, 1818, basal wart of the coxite (SEM): (1) A. diantaeus Howard, Dyar et Knab, 1913; (2) A. pullatus (Coquillett, 1904); (3) A. sticticus (Meigen, 1838); (4) A. excrucians (Walker, 1856); (5) A. behningi Martini, 1926 [(1) ventral-inner view; (2, 4, 5) inner view; (3) ventral view]; bbw, band of basal wart; fl. s, flattened setae of basal wart. For other designations, see Figs. 3, 6.

largest of them are almost half as long as the basal wart; their thickness constituting less than 1/4 of that of the proximal spine-shaped seta. The entire surface of the wart is covered with microtrichiae; near the dorsal margin of the wart, they are arranged not so densely as on the rest of wart surface. According to the preliminary data, the basal wart shape in several species closely related to A. diantaeus (A. intrudens and A. pullatus, Fig. 7, 2) is similar (we examined only the dry collection material in SEM and OM: in these specimens, the shape of the wart could be deformed). In A. pullatus, only a single spineshaped seta is present, and 2 distal setae are flattened (Fig. 7, 2). ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

The basal wart of A. sticticus (Fig. 7, 3) differs from that examined in other species (only air-dried collection material was examined by us in SEM and OM; the data on the shape of basal wart are preliminary). This structure is represented by a distinctly isolated cupshaped outgrowth with a characteristic band at the base. The interior surface of the wart is concave and covered with short setae (their length constituting less than half the distance between the distal and the proximal margins of the wart). The longer setae and a single spine-shaped seta are situated at the base of the wart on its dorsal side. The basal wart of A. excrucians (Fig. 7, 4), as well as warts of closely related species A. behningi

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Fig. 8. Aedes Meigen, 1818, male genitalia: (1) basal wart of coxite; (2) claspette; (3–6) claspette’s wing [(1, 5) A. flavescens (Muller, 1764); (2) A. diantaeus Howard, Dyar et Knab, 1913; (3, 4, 6) A. caspius (Pallas, 1771) [(1, 2, 5, 6) under SEM; (3, 4) under an optical microscope; (1, 4, 5) inner view; (2) dorsal view; (3) ventral view; (6) caudal view]; dil. clp, hyaline dilatation of claspette; st. clp, stem of claspette; hl. clp, handle of claspette. For other designations, see Figs. 2, 3.

(Fig. 7, 5) and A. flavescens (Fig. 8, 1), looks like a more or less isolated triangular lobe situated on the internal surface of the coxite. The lobe of the wart is projected more or less inwards and is pointed mainly ventrally. In A. excrucians, the basal wart passes into the inner surface of the coxite without any borders. It almost does not project inside and in the dorsal direction. The wart is 2–3 as long as wide, constituting approximately half of the coxite length. The surface of the wart is evenly covered with small setae (their length constituting about one third of the wart length), pointed mainly ventrally; microtrichiae are also pre-

sent in the proximal part of the wart. In A. behningi, by contrast, the basal wart looks like a well-separated lobe on the inner side of the coxite, distinctly projecting inwards. It is approximately as wide as long, constituting no more than a quarter the length of the coxite. The proximal and dorsal parts of the wart slightly project dorsally. Setae of different length (never longer than the length of the wart) are situated on the dorsal surface of the wart. In A. flavescens, the length of the basal wart constitutes approximately one third of the coxite length. The wart is slightly raised in the proximal part; its length gradually decreases disENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

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tally. A single thickened spine-shaped seta (slightly shorter than the length of the wart) is situated on the dorsal surface of the coxite at the base of the wart closer to its proximal margin. The dorsal surface of the wart bears shorter (constituting about half the wart length) and finer (constituting 1/10 of the width of the spine-shaped seta) weakly bent setae. Thus, in species of the subgenus Ochlerotatus, the basal wart of the coxite possesses a complicated structure; its characters are important for species diagnostics. In this connection, it is necessary to take into consideration the fact that the study of the basal wart from a single point of view (in ventral view) results in obtaining incomplete and erroneous results. For example, the basal warts of A. caspius and A. behningi look similar on balsam microslides, although their real structure significantly differs. The basal wart of A. caspius looks like a distinctly isolated prominence shaped as a polypore; its posterior surface bears numerous setae and is oriented virtually perpendicular to the longitudinal axis of the coxite (Fig. 5, 3–6). In A. behningi, this structure looks somewhat different and is represented by a lobe slightly projecting dorsally (Fig. 7, 5). The dorsal surface bears numerous setae and is oriented along the longitudinal axis of the coxite. Thus, in the structure of the basal wart, A. behningi appears to be more closely related to A. excrucians and A. flavescens (Fig. 7, 4; 8, 1), than to A. caspius. Claspettes. The study of structures not so complicated as coxites also needs their examination in several projections. Such formation includes, e.g., claspettes. In all the mosquitoes of the subgenus Ochlerotatus of the genus Aedes are articulated to abdominal sternite IX and consist of two parts: the proximal part is represented by the claspette’s stem, and the dorsal part, by its wing (Figs. 2; 8, 2). The shape of these structures also significantly differs in different species of the subgenus; therefore, they are important for species diagnostics. Differences in descriptions and figures of the claspette wing are found in the literature devoted to some species of the subgenus Ochlerotatus (Stackelberg, 1937; Barr, 1958; Mohrig, 1969; Gutzevich et al., 1970; Becker et al., 2003). SEM studies demonstrated that the claspette wing looked quite different when turned at different angles (Figs. 9, 6–7; 10, 1). This is associated not only with the fact that the wing is oriented at different angles in relation to the claspette ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

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stem, but also with the irregularity of the wing plate (Figs. 8, 6; 10, 1). In many representatives of the subgenus Ochlerotatus examined by us (e.g., A. caspius, A. dorsalis, A. cataphylla, A. leucomelas, A. punctor, A. cantans, A. excrucians, A. flavescens, A. behningi, A. cyprius, A. euedes, A. mercurator, A. pullatus, A. simanini, A. sticticus, and A. stramineus), claspettes possess rather a simple structure. The stem of the claspette is oblong-cylindrical, usually slightly narrowing toward apex and more or less bent in such a way that its distal part is pointed dorsally. In these species the wing of the claspette is represented by a more or less elongated blade-shaped (Fig. 8, 4, 5), spear-shaped (Fig. 9, 2), triangular (Fig. 9, 7), or differently shaped appendage (Fig. 10, 2), flattened laterally. In A. punctor, the claspette wing is uniformly sclerotized (Fig. 9, 1), whereas in other species of the subgenus Ochlerotatus, it possesses both strongly and weakly sclerotized areas. As a rule, they are represented by a single median distinctly sclerotized area (“the stem of the claspette wing,” Fig. 8, 5) and one (or, less frequently, two, e.g., in A. sticticus; Fig. 9, 4) weakly sclerotized area (“the hyaline dilation of the claspette wing”) (terminology according to Gutzevich et al., 1970). Such at first sight comparatively simple structure as the claspette wing appears to be rather complicated for the description under an optical microscope. During examination of the male genitalia in species of the subgenus Ochlerotatus, it is very hard to characterize the shape of the claspette wing in ventral view because in this position it can be seen only as its edge (Fig. 8, 3). In lateral view, the shape of the claspette wing is more accessible to description (Fig. 8, 4); the caudal view of the genitalia, however, demonstrates that the wing is sinuously bent (Fig. 8, 6); a noticeable curve is also present at the base. It should be noted that in some species (e.g., in A. leucomelas, A. pullatus, A. simanini, A. mercurator, A. flavescens, and A. euedes; Fig. 9, 2) the claspette wing is oriented at a greater or lesser angle to the longitudinal body axis of the insect. This fact hampers studies of the male genitalia under an optical microscope, especially in permanent balsam microslides. The claspette wing of A. communis is characterized by the presence of several (usually 3–5) longitudinal grooves on its external surface; at least one of these grooves is distinct, strongly projecting in lateralcaudal direction, frequently nearly reaching the poste-

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Fig. 9. Aedes Meigen, 1818, claspettes under SEM: (1–4, 6, 7) claspette wing; (5) claspette stem [(1) A. punctor (Kirby, 1837); (2) A. mercurator Dyar, 1920; (3) A. intrudens Dyar, 1919; (4) A. sticticus (Meigen, 1838); (5–7) A. diantaeus Howard, Dyar et Knab, 1913; (1, 4) inner view; (2) inner-caudal view; (3, 7) caudal view; (5) dorsal view; (6) ventral view]; w. wcl, window of claspette wing; prom. st, prominence of stem; st. st, seta of stem. For other designations, see Figs. 2, 3, 8.

rior margin of the claspette wing (Fig. 10, 3). The inner surface of the claspette wing possesses corresponding depressions; thus, the wing plate is corrugated; this corrugation is visible even under an optical microscope. According to our studies under an optical microscope, distinct longitudinal grooves are also present in the claspette wing of A. annulipes; in this species, however, they project to a lesser extent than in A. communis.

In A. diantaeus, and also in the closely related A. intrudens, claspettes strongly differ from those described above. They look like complicated elongated outgrowths, flattened and dilated at the base and pointed inside in the apical part (Fig. 8, 2). In A. diantaeus, the stem of the claspette is an irregular formation (Fig. 9, 5) bearing a small median outgrowth and slightly bent at apex. The stem is more than 6.5 times as long as its median width. The base of the claspette stem is strongly dilated in the place of articulation ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

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Fig. 10. Aedes Meigen, 1818, male genitalia: (1, 3) claspette wing; (4–7) lobes of tergite IX [(1) A. diantaeus Howard, Dyar et Knab, 1913; (2) A. pullatus (Coquillett, 1904); (3) A. communis (DeGeer, 1776); (4) A. cataphylla Dyar, 1916; (5) A. simanini Gutzevich, 1966; (6) A. leucomelas (Meigen, 1804); (7) A. kasachstanicus Gutzevich, 1962; (1–3, 5, 7) under SEM; (4, 6) under an optical microscope; (1, 3) inner view; (2) caudal view; (4–7) ventral view]. For other designations, see Fig. 2.

with abdominal sternite IX. The entire surface of the stem, except for its apical third, is covered with microtrichiae pointed inwards (Fig. 9, 5). The microtrichiae situated on the stem are significantly larger than those situated on the coxite; their length is nearly 3 times as great as the length of the microtrichiae of the basal wart and constitutes more than 0.75 times of the width of the claspette stem at its middle. Several setae are ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

also present on the inner surface of the stem, including a single longest seta on the apex of claspette stem outgrowth and 4 shorter setae on its inner surface. Claspette wing of A. diantaeus (Fig. 9, 6, 7) is represented by an irregular triangular plate oriented nearly perpendicularly to the longitudinal axis of the claspette wing. In ventral view, the wing looks like a hooked outgrowth (Fig. 9, 6); in caudal view, it looks quite dif-

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ferent (Fig. 9, 7). Irregularity of the wing plate complicates the study of the claspette wing shape: in the inside view (Fig. 10, 1), a wavy character of the wing is evident. During preparation of balsam slides of the male genitalia, the wing is usually turned at some angle because of the pressure of the cover glass; thus, the claspette wing can occupy a different position intermediate between those shown in Figs. 9, 6 and 9, 7. Significant differences in pictures published by different authors (Stackelberg, 1937; Barr, 1958; Mohrig, 1969; Gutzevich et al., 1970; Becker et al., 2003) may be explained by this fact; analysis of these pictures makes an impression that these authors described quite different structures. Different authors (Barr, 1958; Mohrig, 1969) pointed to the so-called “fenestrated” (“der Fensterflecken” in German) structure of the claspette wing in A. diantaeus. Our studies under an optical microscope demonstrated that the claspette wing was not uniform, possessing strongly and weakly sclerotized areas; the latter look like orifices. In SEM, it is evident that the claspette wing is solid; its windows (fenestra) are shallow depressions (Fig. 9, 7). In contrast to A. diantaeus, A. intrudens possesses a more elongated claspette wing, looking like a bird’s head with a bent apex (Fig. 9, 3). This structure is oriented perpendicularly to the stem; its apex is pointed dorsally and somewhat anteriorly. Weakly sclerotized “windows” on the wing are indistinct. Abdominal tergite IX. Several plain projections are needed for examination of such seemingly flat formation as lobes of tergite IX. In species of the subgenus Ochlerotatus, tergite IX consists of broad lateral parts connected by a narrow central bridge (Figs. 11, 1, 2, 4). A noticeable emargination is visible on the anterior margin of the tergite in its middle, whereas its posterior margin bears paired lobes; thus, in ventral view, the tergite resembles the letter M. The lobes of tergite IX are, as a rule, slightly asymmetrical; they can possess different shapes in different species, projecting forward in relation to the rest of the tergite (Fig. 11, 4). In A. diantaeus, the posterior margin of each lobe is rounded and weakly oblique towards the middle (Fig. 11, 1); 8–15 strongly thickened setae of similar size are situated on this margin and nearby on the dorsal surface. The length of these setae exceeds that of the lobe; their width constitutes about half of the width of the proximal spine-shaped seta of the basal wart of the coxite. Due to the setae on lobes of tergite IX being arranged in several rows, it is fre-

quently impossible to calculate their number under an optical microscope (Figs. 11, 1, 2, 4). Studies of the intraspecific variability of this character in some other species (e.g., A. communis, A. cantans, A. cataphylla, and A. leucomelas) have demonstrated that the number and arrangement of setae on the lobes of tergite IX strongly varies. For example, the number of these setae in A. communis varies from 6 to 11, in A. cantans and A. cataphylla, from 6 to 13, and in A. leucomelas, from 8 to 14. The shape of lobes themselves also varies; they project dorsally above the rest of the tergite surface; therefore, their shape also looks different from different points. The ratio between the length of the seta and the length of the lobe appeared to be more constant. For example, we noted that in A. cantans, setae were significantly longer than in A. diantaeus and A. communis. Among all the species examined by us, the longest setae on the lobes of tergite IX were revealed in A. kasachstanicus (Fig. 10, 7), their length more than twice exceeds the width of the lobe and is approximately equal to that of claspettes; the shortest setae were found in A. stramineus (Fig. 10, 5); their length is approximately equal to the lobe width, constituting slightly more than 0.25 of the claspette length. The position of lobes of tergite IX (the distance between them in relation to the lobe width) also seems to be a relatively stable character. Probably, this character may be used as an additional feature distinguishing closely related species A. cataphylla and A. leucomelas (Fig. 10, 4, 6): in A. cataphylla, the distance between the lobes is approximately as long as the lobe width, whereas in A. leucomelas, this distance constitutes about half of the lobe width. Proctiger. In mosquitoes of the family Culicidae, the structures situated posteriorly to abdominal segment IX form a more or less compact structure, or proctiger, formed mainly by elements of abdominal segment X (Fig. 11, 1–4). The structures of the proctiger are virtually not used for the species diagnostics in the genus Aedes of the fauna of Russia. Nevertheless, this complex is rather complicated and its characters may be valuable for species diagnostics of bloodsucking mosquitoes. In the majority of the species of the subgenus Ochlerotatus examined by us (A. caspius, A. dorsalis, A. communis, A. cataphylla, A. leucomelas, A. punctor, A. cantans, A. excrucians, A. flavescens, A. behningi, A. cyprius, A. euedes, A. mercurator, A. pullatus, A. simanini, A. sticticus, and A. stramineus), the proctiger is represented by paired hooked ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

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Fig. 11. Aedes Meigen, 1818, male abdominal segment IX and proctiger (SEM): (1, 2, 4) A. diantaeus Howard, Dyar et Knab, 1913; (3) A. communis (DeGeer, 1776) [(1) ventrolateral view; (2) caudolateral view; (3) lateral view; (4) caudal view]. l IX, lobes of tergite IX; t X, abdominal tergite X; cer. s, cercal setae. For other designations, see Figs. 2, 3.

strongly sclerotized formations; their hooked apices are pointed dorsally (Fig. 11, 3). In the ventral view of the genitalia of the above species, the proctiger possesses a conical shape, whereas in lateral view, it is hooked (Fig. 11, 3). Small membranous structures are present on dorsal and partly on external surfaces of each hooked structure of the proctiger; a large central membranous area is also present caudally to the lobes of tergite IX. Small cercal setae are situated at the sides of the proctiger not far from its apex (Fig. 11, 3). The proctiger of A. diantaeus possesses a vast membranous area on its dorsal surface; this area forms small lateral lobes in the distal part (Fig. 11, 1). Thus, in the ventral view, the proctiger of A. diantaeus looks like a fishtail, differing from the conic shape of the ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

proctiger typical of the majority of other species. The almost entire dorsal surface of the proctiger bears numerous microtrichiae with cercal setae situated closer to the lateral areas. Paraprocts (sclerotized lateral formations of the proctiger) are hooked not only anteriorly, but also inwards (Fig. 11, 1, 2, 4). This feature also determines the characteristic shape of the proctiger in A. diantaeus, because in many species of the subgenus Ochlerotatus, the apices of the paraproct are hooked forward. It should be noted that the proctiger is usually strongly deformed in balsam slides owing to the cover glass pressure. During preparation of such slides, paraprocts usually turn their apices inwards and move away from each other; therefore, it is hard to distinguish the shape of the complex as a whole.

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Aedeagus. The structures of the aedeagus, and, mainly, that of the phallosoma are very important for diagnostics. In different subgenera of the genus Aedeagus, this structure is different. In representatives of the nominotypical subgenus Aedes, as well as in the subgenera Aedimorphus Theobald, 1903 and Stegomyia Theobald, 1901, it is subdivided into two symmetrical lateral parts. In other subgenera (e.g., in Ochlerotatus, Finlaya Theobald, 1903, and Rusticoidus Shevchenko et Prudkina, 1973), the phallosoma is solid (Reinert, 2000). It is according to this character that Reinert (2000) divided the genus Aedes into two subgenera, Aedes and Ochlerotatus. We have noted that in some representatives of the subgenus Ochlerotatus, the phallosoma is not divided into lateral areas completely and that a more or less deep emargination is present in its distal margin. The deepest emargination, constituting two thirds of the length of the phallosoma, was observed in Aedes diantaeus. In addition, the study of the genitalia in representatives of the subgenus Rusticoidus [e.g., A. rusticus (Rossi, 1790), A. subdiversus Martini, 1926, etc] demonstrated that their phallosoma is rather complicated. It possesses a sclerotized elongated median part and two lateral areas. These characters were not mentioned as diagnostic ones, although the phallosoma of species in the subgenus Rusticoidus significantly differs from that of the subgenus Ochlerotatus. CONCLUSION A detailed study of the male genitalia in 30 species of the subgenus Ochlerotatus of the genus Aedes allowed us to reveal new diagnostic characters. These characters include the general 3D shape of the coxite and, in particular, of its basal wart. The shape of the basal wart was frequently used for species diagnostics in the subgenus Aedes (e.g., for separation of such closely related species as A. caspius and A. dorsalis, A. nigrinus and A. sticticus, and A. cantans and A. riparius). However, as it was mentioned above, only the 2D shape of this structure in the ventral view (i.e., the view of the wart on a balsam microslide) was taken into account. Because the coxite on the whole, as wel as its basal wart, possess a complicated spatial structure, it is important to observe them in different views. The results of our study allowed clarifying the characters of the basal wart separating the following species: A. diantaeus, A. intrudens, A. pullatus, A. caspius, A. dorsalis, A. nigripes, A. impiger, A. communis, A. pionips, A. punctor, A. hexodontus, A. cata-

phylla, A. leucomelas, A. sticticus, A. nigrinus, A. cantans, A. riparius, A. flavescens, A. mercurator, A. cyprius, A. behningi, A. annulipes, A. excrucians, and A. euedes. In some cases, characters of the claspettes were also incorrectly used in the literature for the species diagnostics; these structures were described as flat figures. The 3D structure of claspettes allows separating such species as A. diantaeus, A. intrudens, and A. pullatus; A. communis, A. punctor, and A. hexodontus; A. annulipes, A. excrucians, and A. euedes, and also some other species. In particular, we have found that the claspette’s wing in A. communis significantly differs from that observed in closely related species (A. punctor and A. hexodontus) in the presence of distinct longitudinal carinae (Fig. 10, 3). Some authors (Carpenter and LaCasse, 1955) mentioned that A. communis and A. pionips differed by this character. According to these authors, the claspette’s wing in A. pionips lacks any distinct grooves. We had in our disposition the genitalia of only a single specimen of A. pionips; in this specimen, the wing of the claspette is similar to that in A. communis. Some structures that were proposed in the literature as diagnostic ones but, in reality, cannot serve as the latter, should also be mentioned. They include a number of thickened setae in the lobes of tergite IX; this number frequently widely varies within one species. For example, this character was mentioned as a significant diagnostic character for separation of A. cataphylla and A. leucomelas (Gutzevich et al., 1970). In our opinion, some elements of the chaetotaxy of the basal wart of coxite are also insufficient for species diagnostics. They include the shape of spine-shaped setae in A. caspius and A. dorsalis, mentioned by Becker et al. (2003), and also in A. cataphylla and A. leucomelas (Mohrig, 1969). We did not find any difference in the structure of hooked setae which, according to Becker et al. (2003) are more strongly bent in A. communis than in A. pionips. In order to estimate the diagnostic value of these characters, it is necessary to examine the male genitalia in a larger number of accurately identified specimens of A. communis, A. pionips, A. caspius, A. dorsalis, A. cataphylla, and A. leucomelas. These species are very closely related; reliable diagnostic characters allowing determination of many life stages in these species are absent. Therefore, it is rather hard to obtain a large number of reliably identified specimens of these species. No reliable diagnostic characters allowing separation of such speENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

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cies as A. punctor and A. hexodontus, A. excrucians and A. euedes, and of some other species exist. Diagnostic problems hamper obtaining a sufficient number of specimens reliably belonging to certain species. Different characters of the male genitalia are very important for species diagnostics in the genus Aedes. Since some structures of the male genitalia are rather complicated they should be studied in more detail. Otherwise, the results obtained will be incorrect, hampering the following species identification. In our opinion, difficulties concerning examination of the male genitalia are to a greater extent associated with the study of permanent balsam microslides. Any reliable information on complicated 3D structure of certain objects can be obtained only after its examination in different views; the data obtained only after examination in a single (ventral) view is incomplete. It may be assumed that the study of the 3D shape of the male genitalia is important not only for the species of the genus Aedes. The study of the real spatial configuration of the male genitalia would add new data to the species diagnostics of other mosquito genera, not only in the family Culicidae, but also in many dipteran families where species diagnostics is rather difficult. ACKNOWLEDGMENTS The author is grateful to Dr. N.A. Filippova (Zoological Institute, Russian Academy of Sciences, St. Petersburg [ZIN]) for valuable consultations and critical comments to the manuscript. The work was performed on the basis of the collection of ZIN (UFK ZIN reg. no. 2-2.20) and of the contract with Rosnauka 02.452.11.7031 (2006-RI-26.0/001/070). The work was financially supported by the Russian Foundation for Basic Research, project no. 08-04-00216-a and the grant of the President of Russian Federation for the support of leading scientific schools NS-1664.2003.4. REFERENCES 1. Barr, A.R., The Mosquitoes of Minnesota (Diptera: Culicidae: Culicinae) (Techn. Bull., 228, 1958). 2. Becker, N., Petrić, D., Zgomba, M., Boase, C., Dahl, C., Lane, J., and Kaiser, A., Mosquitoes and Their Control (Plenum Publ., New York, Boston, Dordrecht, London, Moscow, 2003). 3. Belkin, J.N., The Mosquitoes of the South Pacific (Diptera, Culicidae) (Univ. California Press, Berkeley and Los Angeles, 1962). Vol. 2. 4. Carpenter, S.J. and LaCasse, W.J., Mosquitoes of North America (North of Mexico) (Univ. California Press, 1955). ENTOMOLOGICAL REVIEW Vol. 90 No. 4 2010

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