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Memórias do Instituto Oswaldo Cruz

Print version ISSN 0074-0276

Mem. Inst. Oswaldo Cruz vol.105 no.7 Rio de Janeiro Nov. 2010 



Sex pheromone and period gene characterization of Lutzomyia longipalpis sensu lato (Lutz & Neiva) (Diptera: Psychodidae) from Posadas, Argentina



Oscar D SalomónI, +; Alejandra S ArakiII; James GC HamiltonIII; Soraya A AcardiIV; Alexandre A PeixotoII

ICentro Nacional de Diagnóstico e Investigación en Endemo-epidemias, Av. Paseo Colon 568, 1063 CA Buenos Aires, Argentina
IILaboratório de Biologia Molecular de Insetos, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, RJ, Brasil
IIICentre for Applied Entomology and Parasitology, Keele University, Staffordshire, United Kingdom
IVLaboratorio de Biologia Molecular Aplicada, Universidad Nacional de Misiones, Posadas, Argentina




Lutzomyia longipalpis s.l. is the primary vector of Leishmania (L.) infantum in the New World. In this study, male Lutzomyia longipalpis specimens from Posadas, Argentina were characterized for two polymorphic markers: the male sex pheromone and the period (per) gene. The male sex pheromone was identified as (S)-9-methylgermacrene-B, the same compound produced by Lu. longipalpis from Paraguay and many populations from Brazil. The analysis of per gene sequences revealed that the population from Argentina is significantly differentiated from previously studied Brazilian populations. Marker studies could contribute to the understanding of the distribution and spread of urban American visceral leishmaniasis, thus aiding in the design of regional surveillance and control strategies.

Key words: Lutzomyia longipalpis - sex pheromone - period gene



Fourteen autochthonous human cases of leishmaniasis with visceral involvement were reported at various times and locations in Argentina from 1925-1989. The primary vector of this disease, Lutzomyia longipalpis, was captured only twice in Argentina during this time period; in both cases, the vectors were captured in localities near the city of Posadas, province of Misiones, where there had been no previous reports of visceral leishmaniasis cases (Salomón et al. 2001).

This sparse transmission scenario changed after the southern spread of urban American visceral leishmaniasis (AVL) in Brazil during the last decades of the previous century (Gontijo & Melo 2004, Lainson & Rangel 2005). The incidence of AVL changed from sporadic cases to clustered cases in regions such as the state of Mato Grosso do Sul (MS), Brazil (Oliveira et al. 2006, Botelho & Natal 2009) and, subsequently, Asunción, Paraguay (Canese 2000, Cousiño 2006). In 2004, Lu. longipalpis was captured in the city of Clorinda, Argentina, on the border with Paraguay (Salomón & Orellano 2005). In 2006, the first human case of AVL associated with Leishmania infantum infected dogs and Lu. longipalpis was reported in the city of Posadas, Argentina (Salomón et al. 2008). Furthermore, from May 2006-November 2009, 39 clustered cases of human AVL, five of them fatal, were reported in Posadas, where thousands of dogs were found to be infected (Fernández et al. 2010). Lu. longipalpis and canine AVL were also identified 350 km south of Posadas, in Corrientes, Argentina (Salomón et al. 2009b) and in the state of Rio Grande do Sul, Brazil (Souza et al. 2009).

If these southern outbreaks are thought of as a unique emergent urban pattern, then epidemiological studies can be extrapolated among foci (Correa Antonialli et al. 2007, Oliveira et al. 2008, Pangrazio et al. 2009, Salomón et al. 2009a, Fernández et al. 2010) and a common regional control strategy can be proposed. Lu. longipalpis is a species complex with polymorphic markers that show both a heterogeneous geographic distribution and divergent trends; in addition, there are putative differences in vectorial competence among populations (Lainson & Rangel 2005, Bauzer et al. 2007, Maignon et al. 2008). Therefore, to contribute to the characterization of Lu. longipalpis vectors in the southern AVL foci, two polymorphic markers of individuals from Posadas were studied: the male sex pheromone and the period (per) gene. Both markers have been analyzed in a number of Lu. longipalpis populations from Brazil (Hamilton et al. 1999, 2004, 2005, Bauzer et al. 2002a, b, Araki et al. 2009) and from Paraguay (Brazil et al. 2009).

Homosesquiterpene (C16) and diterpene (C20) are sex pheromones released by male Lu. longipalpis prior to and during courtship, with molecular weights of 218 and 272, respectively. At present, four different sex pheromone-producing populations (chemotypes) are recognized (Hamilton et al. 2004). The expression patterns of the per gene, which controls circadian rhythms and affects interpulse interval periodicity of the male courtship songs in Drosophila melanogaster and close relatives, suggest the existence of two main groups of Lu. longipalpis in Brazil. One group is represented by a single species with males producing burst-type copulation songs and cembrene-1 pheromones. The other group is more heterogeneous and probably represents a number of incipient species producing different combinations of pulse-type songs and pheromones (Araki et al. 2009).

Lu. longipalpis were collected overnight in February 2007 using CDC light traps in the backyard of an urban house in the AVL endemic area of Posadas (S27º22'18.2"W 55º54'52.3") (Salomón et al. 2008, Fernández et al. 2010). One trap was placed in a chicken coop containing 24 chickens and another was placed 25 m from the chicken coop in a kennel containing two dogs. Taxonomic identifications were made according to the method described by Young and Duncan (1994); Lu. longipalpis was the only species found.

The male specimens used for the pheromone analysis were processed as previously reported for the Paraguayan samples (Brazil et al. 2009). Briefly, 58 males (29 from each site) were placed individually in glass ampoules made from Pasteur pipettes with a small volume of n-hexane (20 ¼L) that were then flame sealed. Prior to analysis, extracts were removed from the vials and filtered through glass wool to remove the flies and fly hairs before the volume was reduced to 1 ¼L under N2. Mass spectra and gas chromatography retention times were compared with authentic (S)-9-methylgermacrene-B (Hamilton et al. 1999). Peak enhancement studies were performed by co-injecting extracts of Lu. longipalpis from Lapinha (Minas Gerais, Brazil) and Lu. longipalpis from Posadas. Gas chromatography coupled mass spectrometry (GC/MS) analysis was carried out on a Hewlett Packard 5890 II GC with a HP-5MS capillary column, 30 m x 0.25 mm i.d. and 0.25 mm film thickness that was directly coupled to a Hewlett Packard 5972A bench top MS (EI, 70eV, 165°C). Samples were introduced via an on-column injector (40°C). The GC was temperature programmed for an initial 2 min at 40°C, followed by an increase of 10°C min-1 to a final isothermal period at 250ºC (10 min).

The pheromone of the male Lu. longipalpis complex member from Argentina was identified as (S)-9-methylgermacrene-B, by comparison of mass spectral [218 (M+, 22), 165(49), 135(76), 121(100), 119(40), 107(62), 93(71), 91(44), 79(40), 67(78), 41(66)] and retention time data. Peak enhancement studies confirmed that Argentinian Lu. longipalpis males produce the same compound as Lu. longipalpis from Paraguay (Brazil et al. 2009) and Brazil (Hamilton et al. 1999, 2005, Araki et al. 2009).

The per gene sequences were generated as reported previously (Bauzer et al. 2002a, b, Araki et al. 2009). Briefly, sand fly DNA was extracted according to Jowett (1998) and a 266-bp fragment of the Lu. longipalpis per gene was amplified as described in Bauzer et al. (2002b). PCR fragments were purified using a Wizard PCR prep kit (Promega) and cloned using a pMOS Blunt Ended cloning kit (GE Healthcare). Plasmid DNA was prepared using the alkaline lysis method in 96-well micro-plates and was filtered in Multiscreen-filter plates. Sequencing of cloned fragments was performed using an ABI 3730 DNA Analyzer and an ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction V3.1 kit (Applied Biosystems). Between five and eight sequences per individual were edited and aligned using BioEdit software version (Hall 1999). Fst analysis was performed using both DnaSP 5.1 and Proseq software (Filatov 2002, Librado & Rozas 2009).

Sequences were obtained from 29 Lu. longipalpis males collected from the two sites in Posadas. There was no evidence of microgeographic differentiation between sites (Fst = -0.03; p > 0.05); therefore, the two samples were pooled and compared to the previously published sequences of Brazilian populations of Lu. longipalpis (Bauzer et al. 2002a, b, Araki et al. 2009). The results indicated that the population from Argentina is different from previously analyzed Brazilian populations, with significant (p < 0.01) Fst values ranging from 0.17-0.43. These values are quite high and suggest that the Posadas population of Lu. longipalpis might be a different sibling species from those found in Northeast and Southeast Brazil (Araki et al. 2009).

These findings illustrate the importance of studies further evaluating the relationship between the Lu. longipalpis populations that are producing the emergent phenomena of urban AVL outbreaks in MS, Paraguay and Argentina over the last decade. These studies, together with research on the ecological and social variables that modulate the outbreaks and dispersion of both the parasites and vectors, could aid in the design of improved regional surveillance and control strategies based on vector behavior and distribution (Salomón et al. 2009a, Bray et al. 2010, Fernández et al. 2010).



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Received 14 April 2010
Accepted 26 July 2010
Financial support: HHMI, FIOCRUZ, FAPERJ, CNPq (to AAP) ODS and SAA are a researcher and a fellow, respectively, of the CONICET, Argentina.



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