¹ Programa de Pós-Graduação em Diversidade Biológica – ICB, Universidade Federal do Amazonas, Manaus, AM, Brazil.

¹ Instituto de Biologia – ICB, Universidade Federal do Amazonas, Manaus, AM, Brazil.

² Laboratório de Ecologia de Doenças Transmissíveis na Amazônia – Centro de Pesquisas Leônidas e Maria Deane, Fundação Oswaldo Cruz, Manaus, AM, Brazil.

*Autor de correspondencia: afariajosedeoliveira@gmail.com

SUMMARY

Phlebotomine sand flies are responsible for the transmission of the etiological agents of several diseases, including protozoan parasites that cause leishmaniases. In the Lutzomyia França genus, the females of some species are morphologically similar, and the morphological characters normally used to identify them are unreliable. The aim of this study was to distinguish between females of cryptic Lutzomyia França species from the subgenus Psathyromyia Barretto by analyzing the armature in the genital atrium (AGA). The species studied were Lutzomyia dendrophyla Mangabeira, L. scaffi Damasceno & Arouck and L. shannoni sensulato Dyar. Lab-reared females were dissected to isolate the genital atrium and spermathecae. The AGA of each species was then photographed, and the arrangement of spines described. We conclude that the AGA can be a useful tool for distinguishing between female cryptic species of the subgenus Psathyromyia.

Key words: morphology; taxonomy; phlebotomine

Descripción del atrio genital de tres especies de Lutzomyia del subgénero Psathyromyia (Diptera, Psychodidae, Phlebominae) de la Amazonía Central Brasileña

Los flebotomíneos (Diptera, Psychodidae, Phlebotominae) son responsables por la transmisión de agentes etiológicos de varias enfermedades, incluyendo protozoarios parásitos que causan las leishmaniosis. En el género Lutzomyia França, las hembras de algunas especies tienen morfologías semejantes y los carácteres morfológicos utilizados para identificarlas no son confiables. El objetivo de este estudio fué distinguir las hembras de especies crípticas del subgénero Psathyromyia Barretto analizando la armadura del área genital (AGA). Las especies estudiadas fueron Lutzomyia dendrophyla Mangabeira-Filho, Lutzomyia scaffi Damansceno & Arouck y Lutzomyia shannoni sensulato Dyar. Las hembras criadas en laboratorio fueron diseccionadas para aislar la genitalia y la espermateca, las armaduras fueron fotografiadas y la distribución de las espinas fue descrita. Concluímos que la AGA puede ser una herramienta útil para distinguir las hembras crípticas del subgénero Psathyromyia.

Palabras clave: morfología, taxonomía, flebótomo

Recibido el 28/11/2015 Aceptado el 08/04/2016

Phlebotomine sand flies are insects of medical and veterinary importance, because they can transmit etiological agents that cause leishmaniasis, bartonellosis, and arboviruses (e.g. Guerra et al., 2006. Cad.SaúdePública 22: 2319-2327; Cohnstaedt et al., 2011. Am. J. Trop. Med. Hyg. 84: 913-922; Comer et al., 1994. J. Med. Entomol. 31: 850-854). In the Amazon region, these insects exhibit high species richness and high indices of local diversity (Alves et al., 2012. Rev. Bras. Entomol. 56: 220-227; Oliveira et al., 2013. Uakari. 9: 55-59). Several species of sandflies, are considered cryptic (e.g. Arrivillaga & Feliciangeli 2001. J. Med. Entomol. 38: 783-790; De La Riva et al., 2001. Mem Inst. Oswaldo Cruz. 96: 1089-1094; Dujardin et al., 2005. Infect. Genet. Evol. 5: 362-365; Pinto et al., 2010. J. Med. Entomol. 47: 16-21; Freitas et al., 2015. Paras. Vectors. 8: 448).

In the subgenus Psathyromyia, of the genus Lutzomyia, females of some species have morphologically similar external structures, and similar spermathecal characters e.g., Lutzomyia dendrophyla, L. scaffi and L. shannoni sensulato. Among these species, similar females are distinguished only by discrete chromatic characters of the pleura of the thorax, and these characters usually disappear when specimens are clarified for slide mounting (Young & Duncan 1994. Mem. Am. Entomol. Inst. 54: 1-881). In the Amazon, these groups of species are also difficult to identify because they share the same resting place: tree trunks (Pessoa et al., 2007. Mem. Inst. Oswaldo Cruz. 102: 593-599).

According Valenta et al., 1999 (Ann. Trop. Med. Parasitol. 93: 389-399), the genital atrium is a structure with a thinchitinous membrane bearing an armature of small spines. The armature in the genital atrium (AGA) is located between the arms of the furca (Fig. 1). Armature morphology is consistent for individuals of the same species,but different species exhibit variations in the size, shape, number, and arrangement of spines andspine comb-sets. The size and width of ewach AGA is showed in the Table I.

Madulo-Leblond et al., 1991 (Parasitol. 33: 387-391) noted that the AGA of the female Phlebotomus papatasi Scopoli exhibited spines of a different length than those found in the female P. dubosqi Neveu-Lemaire. Spine length was used as a diagnosis character to distinguish between the two species. Sandfly AGA was used subsequently as a taxonomic tool to assess African and Asian species of the genus Phlebotomus Rondani, and was applied successfully to the identification of subgenera Phlebotomus Rondani & Brete, Larroussious Nitzulescu, and Paraphlebotomus Theodor (Killick-Kendrick et al., 1994. Ann. Trop. Med. Parasitol. 88: 433-437; Killick-Kendrick et al., 1997. Ann. Trop. Med. Parasitol. 91: 417-428; Pessonet al., 1994. Ann. Trop. Med. Parasitol. 88: 539-542; Depaquitet al., 1998. Bull. Soc. Path. Exot. 91: 346-352; Kakarsulemankhel 2003. Parassitol. 45: 103-118).

The aim of this study was to describe the AGA of the species L. dendrophyla, L. scaffi, and L. shannoni s.l. These species have very similar females and share the same ecotypes in the Central Brazilian Amazon.

Sand fly collections carried out in solid ground forest located at the Experimental Farm of the Federal University of Amazonas, on BR - 174 high way, Km 38 (2º 38‘59.46 "S, 60º 3’16:47" W). Live sand flies were taken to the entomological facility at the Leônidas & Maria Deane Institute. The samples taken, the females who were with eggs or engorged were separated into individual tubes oviposition. For all three species, the first generation of laboratory reared females was obtained according to the methodology described by Killick-Kendrick & Killick-Kendrick 1991 (Parasitol. 33: 315-320) and Pessoa et al., 2008 (Zootaxa. 1740: 1-14). The identification of cryptic species females was confirmed by identification of the reared males morphological characters. The identification key and nomenclature used were Young & Duncan 1994 (Amer. Entomol. Inst. 54: 1-881) proposals.

To obtain the furca of each species studied, we separated the last three abdominal segments and placed them overnight in 10% KOH. The samples were then washed in 10% acetic acid and water, and placed in glycerin for dissection under a stereoscopic microscope. The segments were separated using fine needles to open them ventrally and expose the furca within the atrium (Killick-Kendrick et al., 1994. Ann. Trop. Med. Parasitol. 88: 433-437). The atria were then mounted in Berlese’s fluid. The images were obtained using a Leica DM 1000optical microscope with a 100x objective. Images were recorded with a JVC - 3 CCD digital cameraand a computer, using Auto Montage 4.0 (Syncroscopy).

In this study, we slide mounted nine furcae from each of the species studied. We did not found any intraspecific differences in the AGA of all individuals examined. In L. scaffi, there were three to six spines in each comb, the combs formed horizontal strips, and spine-sets were abundant across the membrane (Fig. 2A, 3A). In L. shannoni s.l., spine clusters in the middle of the membrane formed combs of three to six short spines, while spines on the lateral edges of the membrane were large and ungrouped (Fig. 2B, 3B). In L. dendrophyla, spines were large and clustered in sets of one to three; grouped and ungrouped spines were present across the membrane (Fig. 2C, 3C).

In this study, the species examined showed specific differences in AGA distinguished at specific levels. Valenta et al., 1999 (Ann. Trop. Med. Parasitol. 93: 389-399) distinguished between important species of the verrucarum group that are involved in the transmission of leishmaniasis. Farias et al., 2014 (Acta Amaz. 45: 81-88) distinguished L. lenti (Mangabeira) from L. carmelinoi Ryan, Fraiha, Lainson & Shaw, and also distinguished L. longipalpis (Lutz & Neiva) from L. cruzi (Mangabeira) species that are vectors of Leishmania infantum, and have very similar females. This study has shown that there are morphological differences in the atria of L. dendrophyla, L. shannoni s.l., and L. scaffi. Two of these species are important in vector competence studies: L. dendrophyla may be infected by Leishmania amazonensis (Ryan et al., 1986. Trans. R. Soc. Trop. Med. Hyg. 80: 164-165); L. shannoni s.l. has been incriminated as a vector of vesicular stomatitis virus (Comer et al., 1990. Am. J. Trop. Med. Hyg. 42: 483-490; 1991. Parassitol. 33: 151-158; 1994. J. Med. Entomol. 31: 850-854; Corn et al., 1990. Am. J. Trop. Med. Hyg. 42: 476-482).This study concludes that morphological differences in the AGA can be adopted as important taxonomic characters for differentiating between cryptic species of the subgenus Psathyromyia in the Central Brazilian Amazon.