Intraespecific variation on the aedeagus of Anopheles oswaldoi (Peryassú) (Diptera: Culicidae)

Variação intraespecífica no edeago de Anopheles oswaldoi (Peryassú) (Diptera: Culicidae)

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Intraespecific variation on the aedeagus of Anopheles oswaldoi (Peryassú) (Diptera: Culicidae)

Variação intraespecífica no edeago de Anopheles oswaldoi (Peryassú) (Diptera: Culicidae)

Maysa T Motoki; Cecília L S dos Santos; Maria A M Sallum

Depto. de Epidemiologia, Faculdade de Saúde Pública, Univ. de São Paulo, Avenida Dr. Arnaldo 715, 01246-904, São Paulo, SP; [e-mail], [e-mail], [e-mail]

ABSTRACT

Anopheles (Nyssorhynchus) oswaldoi (Peryassú) comprises a species complex in South America. Specimens from two localities in east Mata Atlântica were characterized both at the morphological and molecular level as An. oswaldoi s.s. Intraspecific variation of the shape of the apex of the aedeagus of the male genitalia of specimens of Anopheles (Nyssorhynchus) oswaldoi s.s. from Vale do Ribeira, Mata Atlântica, São Paulo State, Brazil, was observed. Distinctive aedeagus of the specimens from Vale do Ribeira, Mata Atlântica, were evaluated, illustrated and compared to that of An. oswaldoi s.s.

Key words: ITS2, Nyssorhynchus, ITS2, male genitalia, identification

RESUMO

Anopheles (Nyssorhynchus) oswaldoi (Peryassú) compreende complexo de espécies crípticas na América do Sul. Espécimes de duas localidades situadas no leste da Mata Atlântica foram empregados para caracterizar morfologica e molecularmente An. oswaldoi s.s. Foram observadas e avaliadas variações na forma do ápice do edeago da genitália masculina de espécimes de Anopheles (Nyssorhynchus) oswaldoi s.s. do Vale do Ribeira, Mata Atlântica, estado de São Paulo, e nas sequências do segundo espaçador interno transcrito (ITS2). Os espécimes com edeagos distintos apresentaram seqüências idênticas de ITS2. Os tipos distintos de edeago encontrados nos exemplares do Vale do Ribeira, Mata Atlântica, foram ilustrados.

Palavras-chave: ITS2, Nyssorhynchus, ITS2, genitália masculina, identificação

Anopheles (Nyssorhynchus) oswaldoi (Peryassú) comprises a species complex in South America (Marrelli et al 1999, Ruiz et al 2005, Scarpassa & Conn 2006). It is considered an efficient malaria vector in some regions of Brazil (Branquinho et al 1996), while a secondary or unimportant vector in others.

Anopheles oswaldoi s.s. is morphologically similar and thus was considered conspecific with Anopheles konderi Galvão and Damasceno by Lane (1953). Recently, Flores-Mendoza et al (2004) validated and resurrected An. konderi from synonymy with An. oswaldoi. Motoki et al. (2007), using morphological characters of all life stages except the eggs, redescribed An. oswaldoi s.s. and designated the lectotype. Results of these studies showed that males of An. oswaldoi s.s. and An. konderi can be distinguished by the shape of the apical part of the aedeagus, which is usually longer than broad and somewhat ovate in An. oswaldoi s.s., whereas it is broader than long and somewhat conical in An. konderi (Flores-Mendoza et al 2004, Motoki et al 2007).

Male genitalia characteristics are important and largely used to distinguish among species of Anopheles (Nyssorhynchus) (Faran 1980, Linthicum 1988, Bergo et al 2007, Motoki et al 2007). However, Hribar (1994) observed geographic variation in the male genitalia characteristics of Anopheles nuneztovari Gabaldón from several regions. Similarly, morphological variation on the male genitalia was reported in other groups of insects. Schulmeister (2003a, b) observed morphological variations in the terminal abdominal segments of Hymenoptera species, whereas Pires et al (1998) studied distinct male genitalia characteristics in three Triatoma infestans Klug populations.

While examining the male genitalia of specimens of An. oswaldoi s.l. collected in Pariquera Mirim district, Pariquera-Açú, São Paulo State, Brazil, we observed differences in the shape of apex of the aedeagus. In considering only the shape of the apex of the aedeagus, those specimens could be misidentified as An. galvaoi Causey, Deane and Deane, when using Faran's (1980) identification key, because the apex of the aedeagus either was broader than long or as broad as long. However, based on the characteristics of the immature stages and adult male and female, those specimens were identified as An. oswaldoi s.l. To confirm the identity of these morphologically variant specimens, the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA was sequenced for those specimens obtained in Pariquera Mirim district, Pariquera-Açú and compared with those generated by Motoki et al. (2007) for An. oswaldoi s.s.

Material and Methods

Immature collections were conducted in Pariquera Mirim (24º42'54"S, 44º52'52"W), Pariquera-Açú municipality, São Paulo State, Brazil. The ITS2 nucleotide sequences were derived from six individually reared adult male specimens, with associated fourth-instar larval and/or pupal exuviae, and the adult male genitalia were slide mounted and kept as vouchers. Species identification was based on both characters of both the male genitalia and fourth-instar larvae. Specimens examined are as follow: SP22-9 (Le, Pe, male), SP22-13 (Le, Pe, female), SP22-17 (Le, Pe, female), SP22-21 (Le, Pe, male, DNA), SP22-33 (Le, Pe, male, DNA), SP22-48 (Le, Pe, female), SP22-54 (Le, Pe, female), SP22-55 (Le, Pe, female), SP22-63 (Le, Pe, female), SP22-70 (Le, Pe, male, DNA), SP22-80 (Le, Pe, female), SP22-103 (Pe, male, DNA) and SP22-104 (Pe, male, DNA).

DNA was extracted from the field collected specimens, following the DNA extraction protocol provided by the QIAgen DNeasy^® Blood and Tissue Kit (QIAgen Ltd., Crawley, UK). All buffers were supplied in the kit. The elution step was repeated and stored in a separate tube, as DNA often remains bound to the membrane following the first elution.

One µl of the first elution was used as DNA template in the PCR reactions. Amplification of the ITS2 region was carried out using the 5.8SF (- ATC ACT CGG CTC GTG GAT CG -) and 28SR (- ATG CTT AAA TTT AGG GGG TAG TC -) primers recommended by Ruiz et al. (2005). PCR products were amplified in 25 µl reaction mix containing: 1 µl DNA; 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 2.5 µl DMSO; 5 pM of each primer; 200 µM each dNTPs; and 2.5 U New England Biolabs^®Taq polymerase. PCR amplification protocol consisted of a 2-min denaturation at 94ºC, 34 cycles at 94ºC, 57ºC and 72ºC for 30 sec each, followed by a 10 min extension at 72ºC. PCR products were electrophoresed in 1% TAE agarose gels stained with ethidium bromide. ITS2 PCR amplicons were purified directly from bands excised from agarose gel using the QIAQuick Gel Extraction kit, and directly sequenced.

Sequencing reactions were carried out in both directions using the PCR primers and the Big Dye Terminator Kit v.3.1 (PE Applied Biosystems, Warrington, England). Sequences were analyzed on a 377 - ABI Sequencer (Applied Biosystems, Foster City, CA, USA).

Template DNA from this study is retained at -70ºC in the Faculdade de Saúde Pública (FSP-USP) for future reference (DNA reference numbers SP22-9, SP22-21, SP22-33, SP22-70, SP22-103 and SP22-104). Linked immature exuviae and male genitalia slides of specimens used for DNA extraction are deposited in the FSP-USP collection. Sequences were edited using Sequencer Navigator (version 1.0.1, PE Applied Biosystems^®), aligned in CLUSTAL X (Thompson et al. 1997) and optimized manually in MacClade version 4.3 (Maddison and Maddison 2000). Sequence similarity of the ITS2 sequences generated in this study (GenBank accession number FJ 425910-FJ425915), with those previously available in GenBank (EF457237 & EF457239, Motoki et al 2007) were assessed using FASTA search (http:/www.ncbi.nlm.nih.gov/BLAST), and aligned using MEGA3 software (Kumaret al 2004).

Results

The ITS2 sequences obtained from adults of An. oswaldoi s.s. collected in Pariquera Mirim, Pariquera-Açú, SP, were compared with the sequences of An. oswaldoi s.s. deposited in GenBank (Espírito Santo, EF457228-37; São Paulo EF457238-9). ITS2 sequences of all specimens were identical (Fig 1), and thus the identity of all specimens used in this study were determined as An. oswaldoi s.s.

The male genitalia characteristics of specimens of An. oswaldoi s. s. from Espírito Santo state and São Paulo state are very similar (Fig 2A, E), except for six specimens collected in Pariquera Mirim, Pariquera-Açú municipality, that showed a dissimilar aedeagus apex when compared with those of specimens of An. oswaldoi s. s. described by Motoki et al (2007). By examining these specimens, we observed three distinct aedeagus apex shapes, i.e., the width and length of the apex of aedeagus is the same (Fig 2B,C), the apex is broader than long (not shown), and the apex is slightly longer than broad (Fig 2D), but less than in the specimens of An. oswaldoi s.s. (Fig 2F).

Discussion

Molecular and morphological data are convenient to test relative rates of evolution (Huber 1993). In insects, the male genitalia characters are complex and the differences in genitals may have evolved as barriers to insemination. Additionally, the genital structures may have diverged through the sexual selection that occurs after insemination for any device that increases fertilization success (Gwinne 1998). Schulmeister (2003a, b) simultaneously analyzed morphological and molecular characters of the terminal abdominal segments to characterize Hymenoptera. The characters of male genitalia provided support for some nodes in the phylogenetic tree. Pires et al (1998) employed both the presence and absence of denticles on endosoma process of the external male genitalia of three populations of T. infestans, two from Brazil (Minas Gerais and Bahia) and one from Bolivia (Cochabamba Valley) to evaluate intraspecific variation. These differences on the external male genitalia were used to characterize three populations of T. infestans.

Having said that, it is noteworthy that in the Culicidae, it is usually necessary to examine the male genitalia characteristics to ensure accurate species identification. This is the case in most species of Anopheles (Nyssorhynchus). In the revision of the Albimanus Section, Faran (1980) distinguished An. evansae (Brethes) (as An. noroestensis Galvão and Lane) from An. oswaldoi s.l.,An. galvaoi and An. aquasalis Curry by the shape of the apex of the aedeagus, which is truncate in An. evansae, whereas it is rounded in the other species. Additionally, Peyton (1993) validated and resurrected An. dunhami Causey from the synonymy of An. nuneztovari based on characteristics of the male genitalia, including aspects of the aedeagus, and Florez-Mendoza et al. (2004) considered An. konderi to be a valid name based on the shape of the apex of the aedeagus.

In considering characteristics of the aedeagus, Hribar (1994) analyzed morphological variation of male genitalia structures of several specimens of An. nuneztovari cytotype A (Amazonian), B (western Venezuela, southeast of the Andes) and C (from Colombia and western Venezuela northwest of the Andes), and concluded that, despite of geographical variations, there are four characters that distinguish cytotype B from both cytotypes A and C. Interestingly, Sierra et al (2004) examined the variability of four populations of An. nuneztovari from Colombia using the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA sequences and demonstrated the cytotypes B and C to be conspecific. Consequently, the male genitalia characteristics observed by Hribar (1994) which distinguished the cytotype B from both A and C may represent morphological polymorphisms.

According to Faran (1980), male genitalia characteristics of An. oswaldoi s.s. are somewhat similar to those of An. galvaoi. These taxa can be distinguished by aspects of the ventral claspette, i.e., the length of the setae along basal margin of basal lobules and shape and size of the preapical plate. Additionally, Faran (1980) reported that in An. galvaoi, the apex of the aedeagus was rounded, about as wide as it is long, distinguishing it from that of An. oswaldoi which is longer. Consequently, in considering only the apex of the aedeagus to distinguish between An. oswaldoi s .s. and An. galvaoi, it is plausible to consider that a specimen with an anomalous aedeagus could be misidentified as An. galvaoi. Although the variation on the shape of the apex of the aedeagus reported for the specimens obtained in Pariquera Mirim, Pariquera-Açú, the fourth-instar larvae and the adult female were identified as An. oswaldoi s.l. Because An. oswaldoi s.l. is a species complex, it is important to evaluate any morphological difference observed specially in the male genitalia to ascertain the species identification. Furthermore, other species of the complex can be distinguished by characteristics of the apex of the aedeagus, for example An. konderi (Flores-Mendoza et al 2004) and other unnamed species of the complex that occur in Brazil (Sallum et al 2008).

Acknowledgments

This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grant 05/53973-0) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant 472485/2006-7).

Received 03/IV/08. Accepted 02/I/09.

Edited by Roberto Antonio Zucchi - ESALQ/USP

References

• Bergo E S, Souto R N P, Galardo A K R, Nagaki S S, Calado D C, Sallum M A M (2007) Systematic notes on Anopheles Meigen (Diptera: Culicidae) species in the State of Amapá, Brazil. Mem Inst Oswaldo Cruz 102: 373-376.
• Branquinho M S, Araújo M S, Natal D, Marrelli M T, Rocha M R, Taveira F A L, Kloetzel J K (1996) Anopheles oswaldoi a potential malaria vector in Acre, Brazil. Trans R Soc Trop Med Hyg 90: 233.
• Faran M E (1980) Mosquito studies (Diptera: Culicidae) XXXIV. A revision of the Albimanus section of the subgenus Nyssorhynchus of Anopheles Cont Amer Entomol Inst 15: 1-215.
• Flores-Mendoza C, Peyton E L, Wilkerson R C, Lourenço-de-Oliveira R (2004) Anopheles (Nyssorhynchus) konderi Galvão and Damasceno: neotype designation and resurrection from synonymy with Anopheles (Nyssorhynchus) oswaldoi (Peryassú) (Diptera: Culicidae). Proc Entomol Soc Washington 106: 118-132.
• Gwinne D T (1998) Genitally does it. Nature 393: 734-735.
• Hribar L J (1994) Geographic variation of male genitalia of Anopheles nuneztovari (Diptera: Culicidae). Mosq Syst 26: 132-144.
• Lane J (1953) Neotropical Culicidae, v I, Universidade de São Paulo, São Paulo, 1112p.
• Linthicum K J (1988) A revision of Argyritarsis section of the subgenus Nyssorhynchus of Anopheles (Diptera: Culicidae). Mosq Syst 20: 98-271.
• Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings Bioinform 5:150-163.
• Maddison D R, Maddison W P (2000) MacClade 4: analysis of phylogeny and characters evolution, version 4.0, Sunderland. MA, Sinauer Associates, Inc.
• Marrelli M T, Malafronte R S, Flores-Mendoza C, Lourenço-de-Oliveira R, Kloetzel J K, Marinotti O (1999) Sequence analysis of the second internal transcribed spacer of ribosomal DNA in Anopheles oswaldoi (Diptera: Culicidae). J Med Entomol 36: 679-684.
• Motoki M T, Linton Y M, Ruiz F, Flores-Mendoza C, Sallum M A M (2007) Redescription of Anopheles oswaldoi (Peryassú, 1922), with formal lectotype designation. Zootaxa 1588: 31-51.
• Peryassú A (1922) Duas novas espécies de mosquitos do Brasil. A Folha Médica 3: 179.
• Peyton E L (1993) Anopheles (Nyssorhynchus) dunhami, ressurected from synonymy with Anopheles nuneztovari and validated as a senior synonym of Anopheles trinkae (Diptera: Culicidae). Mosq Syst 25: 151-156.
• Pires H H R, Barbosa S E, Margonari C, Jurberg J, Diotaiuti L (1998) Variations of the external male genitalia in three population of Triatoma infestans Klug 1834. Mem Inst Oswaldo Cruz 93: 479-483.
• Ruiz F, Quiñones M L, Erazo H F, Calle D A, Alzate J F, Linton Y M (2005) Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and Anopheles (Nys) oswaldoi from southern Colombia. Mem Inst Oswaldo Cruz 100: 155-160.
• Sallum M A M, Marrelli M T, Nagaki S S, Laporta G Z, Santos C L S (2008) Insights into Anopheles (Nyssorhynchus) (Diptera: Culicidae) species from Brazil. J Med Entomol 45: 970-981.
• Scarpassa V M, Conn J E (2006) Molecular differentiation in natural populations of Anopheles oswaldoi sensu lato (Diptera: Culicidae) from the Brazilian Amazon, using sequences of the COI gene from mitochondrial DNA. Gen Mol Res 5: 493-502.
• Schulmeister S (2003a) Genitalia and terminal abdominal segments of male basal Hymenoptera (Insecta): morphology and evolution. Org Divers Evol 3: 253-279.
• Schulmeister S (2003b) Simultaneous analysis of basal Hymenoptera (Insecta): introducing robust-choice sensitivity analysis. Biol J Linn Soc 79: 245-275.
• Sierra D M, Velez I D, Linton Y M (2004) Malaria vector Anopheles (Nyssorhynchus) nuneztovari comprises one genetic species based on homogeneity of nuclear ITS2 rDNA. J Med Entomol 41: 302-307.
• Thompson J D, Gibson T J, Plewniak F, Jeanmougin F, Higgins D G (1997) The ClustalX windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24: 4876-4882.

Publication Dates

• Publication in this collection
  27 Mar 2009
• Date of issue
  Feb 2009

History

• Received
  03 Apr 2008
• Accepted
  02 Jan 2009