Anopheles (Kerteszia) homunculus Komp

Sallum, Maria Anice Mureb, Dos, Cecilia L. S. & Wilkerson, Richard C., 2009, Studies on Anopheles (Kerteszia) homunculus Komp (Diptera: Culicidae), Zootaxa 2299, pp. 1-18 : 4-9

Anopheles (Kerteszia) homunculus Komp

Anopheles (Kerteszia) homunculus Komp, 1937: 509 . Lectotype male (no. 3) with associated larval skin and male genitalia on separate microscope slides, deposited in the National Museum of Natural History (NMNH), USA. Typelocality: Restrepo, Meta, Colombia.

Anopheles (Kerteszia) anoplus Komp, 1937: 514 . Holotype male with associated larval exuviae and male genitalia on separate microscope slides, deposited in the National Museum of Natural History (NMNH), USA. Type-locality: Restrepo, Meta, Colombia. Lane 1953: 287 (syn. An. homunculus ).

Anopheles (Kerteszia) homunculu s of Coutinho, 1946: 149 (first record in Brazil, systematics); Coutinho 1947: 13 (distribution); Rachou 1958: 149 (malaria vector status); Lima 1952: 401 (systematics, distribution); Lane 1953: 287 (systematics, distribution); Martins 1958: 429 (adult female identification); Forattini 1962: 441 (M*) (systematics, distribution); Aragão 1964: 73 (distribution, bionomics); Ferreira 1964: 329 (distribution, bionomics); Zavortink 1973: 20 (in part, specimens from Brasil, systematics); Calado and Navarro-Silva 2005: 1128 (PCR-RAPD, PCR- RFLP identification); Sallum et al. 2008: 671 (first record, Espírito Santo state).

Morphological characterization. Anopheles homunculus can be recognized as described below. In the male genitalia the ventral claspette is densely spiculose mesally ( Fig. 1 A,B), possessing a broad, large, lateral expansion that is somewhat sinuous at the lateral edges, curved posteriorly in ventral direction and possessing a long, sharp retrorse point anteriorly ( Fig. 1 C,D); the lobes of the ventral claspette are separated by a deep funnel-shaped emargination, and are connected at base ( Fig. 1 E); the dorsal claspette has two groups of long setae, a dorsal group composed of five long, twisted, flattened, swollen at mid-length, then tapering, becoming slender at apical 0.3 and ending in a narrow, pointed apex, and the ventral group is composed of three setae that are narrow, contorted and flattened ( Fig. 1 C,D); the aedeagus is long and slender with a pair of distinctive subapical leaflets ( Fig. 1 E) and the apical opening is bordered by two minute, incomplete lateral sclerites ( Fig. 1 F). Adult females can be recognized by a lack of scales on abdominal terga II-VII, the mesepimeron has both upper and middle patches of scales, the hindtarsomeres 2-5 possess broad apical bands of white scales, the maxillary palpomere 3 has scales slightly to moderately outstanding, scales on the palpomere 4 are decumbent or moderately outstanding, and the abdominal terga are dark, mauve when whole mounted in Canada balsam on a microscope slide. Larvae are distinctly purple with a dorsal pattern of dark pigment on the thorax and abdomen ( Fig. 2 A). The thorax is entirely bright purple, with dark pigment forming a complex pattern ( Fig. 2 A). On the abdomen the purple color is more evident on segments III, VI, VII and VIII, and laterally on the remaining segments. The abdominal dark pigment pattern on segments I and II is concentrated dorsomedially, the dark pattern of segment I is connected to that on segment II by a narrow dark bridge, in the abdominal segment III, the dark pattern extends laterally at the level of seta 6, with two large unpigmented areas bordering a central unpigmented area, the dark pigment pattern reaches the posterior border of the segment with a median dark line extending anteriorly into the unpigmented medial portion ( Fig. 2 B), the dark pattern on segments IV and V are distinct, restricted to the longitudinal anteroposterior portion, the dark pattern on segment IV is connected to that on segment V by a narrow dark bridge, on segments VI, VII and VIII, the dark pattern is distinct, extending to lateral sides ( Fig. 2 C). The fourth-instar larva can be recognized as follow: seta 4-A usually single; seta 3-C stronger than 2-C ( Fig. 2 D); 4-C stronger than 2-C, single or forked at apex; 5,7-C long; 6-C moderately long or long, reaching or extending beyond insertion of 4-C; 1-I-VII palmate, small, with pointed or blunt leaflets; 5-II-V branched near base; 6-VI long, plumose; 4a- X usually weakly developed, shorter or slightly longer than anal saddle; pecten alternating long and short spines medially, spicules restricted to basal portion of external edge; and saddle darkly pigmented dorsally. The pupa can be recognized by: possessing a trumpet darkly pigmented at mid-length, somewhat yellow at base and apex ( Fig. 2 E); seta 9-V moderately long, pointed, dissimilar to 9-III; paddle obovate, unpigmented or very weakly pigmented, lighter than posterior abdominal segments ( Fig. 2 F); outer edge of paddle distal to external buttress without spicules, marginal spicules along distal portion of external buttress closely spaced, moderately long, numerous; and, by its distinct purple color.

Distribution. Anopheles homunculus s.l. has a large, discontinuous distribution in South America. It was reported from localities in Colombia, Venezuela, Bolivia, the Guianas, Peru and Brazil ( Zavortink 1973). In Brazil, An. homunculus is known from areas in the Mata Atlântica in Santa Catarina, Paraná, São Paulo and Espírito Santo states ( Forattini 1962, 2002, Marrelli et al. 2007, Sallum et al. 2008).

Considering the possibility that An. homunculus may be misidentified as An. cruzii , it would be plausible to suppose that the geographical distribution of the taxon may be more extensive than is reported in the published literature. The species may occur along the Atlantic coast, and also in areas in west Brazil.

Bionomics. Habitats for the immature stages of both forms of An. homunculus are the leaf axils of epiphytic and terrestrial bromeliads in areas of dense humid primary forest.

Medical importance. Anopheles homunculus was observed to be a highly anthropophilic and endophylic species in Blumenau, Santa Catarina ( Coutinho 1947), and a secondary or local vector of human malaria in localities in eastern Mata Atlântica, southern Brazil ( Rachou 1958; Forattini 1962, 2002). Females were found naturally infected with both oocysts and sporozoits in localities in Santa Catarina state by Coutinho (1946), Rachou (1946), and Rachou and Ferreira (1947).

The role of An. homunculus in Plasmodium transmission in Trinidad has been recorded in the literature as either a vector or suspected vector ( Pittendrigh 1948; Forattini 1962; Chadee and Kitron 1999; Chadee et al. 1999).

ITS2 molecular characterization. The second internal transcribed spacer (ITS2) of the nuclear rDNA cistron was sequenced for six individuals of An. homunculus from Cananéia, Mata Atlântica, Brazil (GenBank accession numbers FJ176945 - FJ176950 ), one direct sequence and eight clones from a single individual from Colombia (GenBank accession numbers FJ176951 - FJ176959 ), and three specimens of An. homunculus l. s. from Trinidad (GenBank accession numbers FJ176960 - FJ176962 ). The ITS2 sequences consist of the following base composition: 0.17544% T, 0.20468% A, 0.31871% C and 0.30117% G for Cananéia, Brazil; 0.17289% T, 0.20465% A, 0.32079% C and 0.30168% G for Colombia; and 0.17544% T, 0.19883% A, 0.31823% C and 0.30750% G for An. homunculus l. s. for Trinidad.

A single ITS2 sequence of this species was available in GenBank ( DQ364655 , Brazil, Malafronte et al. 2006, unpublished). The ITS2 sequences of An. homunculus from Mata Atlântica, Cananéia, São Paulo (n = 5), and Santa Teresa, Espírito Santo (n = 1) share 100% similarity. However, one individual from Parque Estadual Turístico do Alto Ribeira (Petar Reserve), southern São Paulo state ( DQ364655 ), exhibited only 99% similarity with these specimens. Along a 344 bp alignment, three bases varied, one singleton polymorphic site (position 341), and two 1-bp indels (positions 339 and 344, Fig. 3 ). Mean uncorrected “P” distance among ITS2 sequences of An. homunculus from Cananéia / Santa Teresa and DQ364655 is 0.00292.

Comparing the ITS2 cloned sequences from a single Colombian specimen with those from Cananéia / Santa Teresa, excluding DQ364655 , the mean uncorrected P distance ranged from 0.00 to 0.00887. One cloned ITS2 sequence shares 100% similarity with sequences from samples collected in Cananéia / Santa Teresa. Sequences from the Colombian specimen showed superimposed chromatogram peaks apparently due to intragenomic indel variation in a CG repeat at positions 312 and 313, and a polymorphic C or T indel at position 337. Single base pair polymorphism in highly conserved regions may, however, represent sequencing/cloning artifacts. Apparently, there is no fixed difference between the Colombian and Cananéia / Santa Teresa specimens. Two clones showed either a T or C insertion at position 337, whereas the majority showed a deletion in this position. In contrast, sequences from the Cananéia / Santa Teresa specimens showed no polymorphism, and all five individuals were directly sequenced from PCR products, with a T at position 337 in all individuals.

Mean uncorrected P distance among three ITS2 sequences generated from An. homunculus l. s. collected in Trinidad is 0.0000. There is a single ambiguity, which may be due to a sequencing artifact ( Fig. 3 ). The mean uncorrected P distance among the ITS2 sequences of An. homunculus l. s. from Trinidad and An. homunculus from Colombia and Brazil ranged from 0.00298 to 0.01190. ITS2 sequences from Trinidad and from Colombia and Mata Atlântica varied at five sites, including two 2-bp indels (bases 211, 212 and 282, 283) and one singleton polymorphic site at position 190 ( Fig. 3 ). The mean uncorrected P distance among clones from Colombia ranged from 0.00 to 0.01173. The most divergent clone is E10225clone15. The mean uncorrected P distance between this clone and the remaining clones varied from 0.00887 to 0.01173.

Secondary structures were predicted in Sfold by centroids in Botzmann-weighted ensemble, which generated a set of clusters, and from them, the best cluster centroid was determined that reflected the highfrequency base-pair in the structure sample. Sfold also compares the centroids with suboptimal minimum free energy (MFE) structures. Based on sequence of one clone from Colombia ( FJ176955 ), the Srna module of Sfold generated the ensemble centroid shown in Fig. 4A, whereas for FJ176953 the ensemble centroid is shown in Fig. 4B, and for the clone ( FJ176945 ) from Cananéia, Mata Atlântica, Brazil, the ensemble centroid is shown in Fig. 5. For An. homunculus B ( FJ176961 ) from Trinidad, the ensemble centroid is shown in Fig. 6. In comparing the ensemble centroids of these three populations of An. homunculus , we identified helix I, II, and III. Helix II can be recognized by the characteristic pyrimidine-pyrimidine mismatch (C-U) (Fig. 6). Helix III is the longest helix and can be recognized by having its most conserved region on the 5’ side, near the tip (in brackets). Also, a highly conserved pairing (GGU) is in helix III on positions 131-133, and a UGGU motif is at positions 200-203 on the 3’side of the most conserved region of helix III. The indels observed in specimens from Trinidad are in loop regions, i.e., deletion of a UA at position 211-212 is located in a loop between two side arms, and the UG insertion, positions 280-281, is on a loop at the apex of a sidearm of helix III (Fig. 6). This sidearm is absent in sequences from Colombia and Mata Atlântica, Brazil (Fig. 4B, 5), except in one clone from Colombia ( FJ176955 , Fig. 4A). No compensatory base changes (CBC) were observed among sequences from the three populations.

FIGURE 4. A, ITS2 ensemble centroid structure diagram for clone FJ176955 from a specimen of Anopheles homunculus from Colombia, generated in Sfold software (http://sfold.wadsworth.org/srna.pl), using Botzmann weighted ensemble features of RNA secondary structures. Folding temperature 37o C, ionic conditions 1M NaCl. Δ˚37 = -160.50; B, clone FJ176953 , Δ˚37 = -159.90.

Figures

FIGURE 1. Male genitalia of Anopheles homunculus Komp (lectotype) from Restrepo, Colombia, and Anopheles homunculus from Mata Atlântica, Brazil. A, B, detail of the ventral claspette showing mesal spicules; C, D, detail of the ventral claspette showing the anterior retrorse process; E, aedeagus showing the lateral leaflets; F, detail of the apex of the aedeagus showing the minute apicolateral sclerite. B and D represent the lectotype of Anopheles homunculus.

FIGURE 2. Fourth-instar larva and pupa structures of Anopheles homunculus from Mata Atlântica, Brazil. A, fourthinstar larva showing the bright purple color in the thorax and abdominal segments I-VIII; B, fourth-instar larva showing the darkly pigmented pattern in the abdominal segments I-VIII; C, cephalothorax of the pupa, showing a dark ring in the middle of the trumpet; D, metanotum and abdomen of the pupa.

FIGURE 3. A 498 bp 5.8 S, ITS 2 and 28 S sequence alignment of Anopheles homunculus from Brazil, Colombia and Trinidad. (-) indicates either an indel or missing data at the 5 ’ and 3 ’ ends.