IDENTIFICATION OF SANDFLIES (Diptera: Psychodidae: Phlebotominae) BLOOD MEALS IN AN ENDEMIC LEISHMANIASIS AREA IN BRAZIL

Identificação do repasto sanguíneo de flebotomíneos (Diptera: Psychodidae: Phlebotominae) provenientes de área endêmica de leishmaniose no Brasil

SUMMARY

The aim of this study was to identify blood meals of female sandflies captured in the municipality of Governador Valadares, an endemic area of visceral and cutaneous leishmaniasis, in the State of Minas Gerais, Brazil. From May 2011 to January 2012, captures were performed using HP light traps in four districts. There were 2,614 specimens (2,090 males and 524 females) captured; 97 engorged females were identified belonging to the species Lutzomyia longipalpis (82.1%) and Lutzomyia cortelezzii (17.9%). Considering simple and mixed feeding, the enzyme-linked immunosorbent assay revealed a predominance of chicken blood (43.6%) in Lutzomyia longipalpis, showing the important role that chickens exert around the residential areas of Governador Valadares. This finding increases the chances of sandflies contact with other vertebrates and consequently the risk of leishmaniasis transmission.

KEYWORDS:
Sandflies; Lu. cortelezzii; Lu. longipalpis; Blood meal identification; ELISA.

RESUMO

O objetivo deste estudo foi identificar o repasto sanguíneo de fêmeas de flebotomíneos capturadas no município de Governador Valadares, área endêmica de leishmaniose visceral e tegumentar no Estado de Minas Gerais, Brasil. Entre maio de 2011 e janeiro 2012 foram realizadas capturas com armadilhas luminosas HP em quatro bairros. Foram capturados 2.614 exemplares (2.090 machos e 524 fêmeas). Noventa e sete fêmeas ingurgitadas foram identificadas como pertencentes às espécies Lutzomyia longipalpis(82,1%) e Lutzomyia cortelezzii(17,9%). Considerando a alimentação simples e a mista, o ensaio imunoenzimático revelou em Lutzomyia longipalpisuma predominância de sangue de galinhas (43,6%), mostrando o importante papel que galinhas podem exercer no peridomicílio, aumentando a chance de contato dos flebotomíneos com outros vertebrados e, consequentemente, o risco de transmissão da leishmaniose.

INTRODUCTION

In the Americas, the transmission of Leishmania (Kinetoplastida, Trypanosomatidae) occurs mainly through the bite of female sandflies species of the genus Lutzomyia (Diptera: Psychodidae: Phlebotominae). These females need blood for the maturation of their ovarioles; they suck many vertebrates, including amphibians, reptiles, birds and mammals, the latter retaining reservoirs of Leishmania^10,16.

Sandfly species have the ability to adapt to different habitats. The diversity of species found next to human dwellings, with a plasticity in the face of changes brought about by humans, have contributed to the urbanization of leishmaniasis^3,23. The identification of sandfly blood meal has been shown as an alternative to know how domestic and synanthropic animals can be effectively used as blood sources^6,18. The identification of these blood sources can provide data on possible reservoirs of Leishmania, as well as the role that some animals might play in the transmission of parasites⁷. Many techniques have been used to identify blood sources for bloodsucking insects (i.e., precipitin test, gel electrophoresis, PCR and ELISA), highlighting the most commonly

used, the immunoenzymatic techniques^12,14,20,24.

In Brazil, Lutzomyia longipalpis (Lutz & Neiva 1912) is a frequent subject of studies on blood meal^1,15, probably because of its importance as a main vector of Leishmania infantum in different geographical regions¹³. In contrast, the literature on the feeding behavior of other nonvector species is quite rare. ROSA et al. (2012)²² raised the possibility of Lutzomyiacortelezzii complex be participating in the transmission of Leishmania in areas of cutaneous leishmaniasis because they have been found naturally infected and in high abundance in these areas. In Brazil, CARVALHO et al. (2008)⁸ found this species naturally infected by Le. infantum (syn Leishmania chagasi) in the Santa Luzia municipality, in the State of Minas Gerais.

The aim of this study was to identify the blood meals of females from the species Lu. longipalpis and Lu. cortelezzii in an area of simultaneous occurrence of visceral and cutaneous leishmaniasis, to contribute to the knowledge of the biology and behavior of these species and their roles in the epidemiology of leishmaniasis.

MATERIAL AND METHODS

STUDY AREA: In the municipality of Governador Valadares (18°51'12'' S - 41°56'42'' W), Minas Gerais State, Brazil (Fig. 1), from May 2011 to January 2012, phlebotomine captures were performed using 16 HP light traps¹⁹ exposed inside and outside the homes of eight fixed residences in four neighborhoods in the urban area with reported human cases of visceral (VL) and cutaneous leishmaniasis (ACL) (Altinopolis, Mae de Deus, Nossa Senhora das Graças and Santa Helena). In general, the residences had courtyards, with the continuous presence of domestic animals such as dogs, chickens, and cats. The presence of rodents was mentioned by residents. Sandflies were captured with traps assembled at 4:00 P.M. and removed the following day at 8:00 A.M., for three consecutive nights each month.

Fig. 1
- Geographical localization of the Municipality of Governador Valadares, Minas Gerais State, Brazil.

Enzyme-linked immunosorbent assay: After the capture, the specimens were killed by freezing them at -20 °C for complete paralysis of the digestive process. Then, the specimens' head and last two segments of the abdomen were removed to identify the species of sandfly according to the classification proposed by YOUNG & DUNCAN (1994)²⁵. The identification of blood meal was performed according to the enzymelinked immunosorbent assay proposed by BURKOT et al. (1981)⁷ modified by DUARTE (1997)¹¹, using four antisera: chicken, dog, rodent and human, for each specimen (see, MARASSÁ et al. 2004)¹⁴.

RESULTS

In the municipality of Governador Valadares, 2,614 specimens were captured (2,090 males and 524 females) (see, BARATA et al. 2013) of which 97 were engorged. The distribution of phlebotomine females per district and environment is shown in Table 1. Engorged females were identified as belonging to the species Lu. longipalpis (82.1%) and Lu. cortelezzii (17.9%). The identification of blood-feeding females according to the ELISA test can be viewed in Table 2. The record of these species occurred predominantly in the peridomicile (69.4%), but also inside homes (30.6%). Two other species were captured in this study: Lutzomyia intermedia (Lutz & Neiva 1912) and Lutzomyia whitmani (Antunes & Coutinho, 1939) (data not shown)⁵, but they were not engorged. Simple feeding was observed in 79.6% of females analyzed while 20.4% fed on more than one source of blood (Table 2).

Table 1
Distribution of phlebotomine females collected per district and environment, in the municipality of Governador Valadares, state of Minas Gerais, between May 2011 and January 2012

Species	District								Total
	Altinópolis		Mae de Deus		N. Sra das Graças		Santa Helena
	Inside	Outside	Inside	Outside	Inside	Outside	Inside	Outside
Lu. cortelezzii	1	2	2	-	-	3	-	3	11
Lu. longipalpis	11	3	6	35	4	24	1	2	86
Total	12	5	8	35	4	27	1	5	97

Table 2
Percentage of engorged female sandflies females analyzed by ELISA, collected in the municipality of Governador Valadares, state of Minas Gerais, between May 2011 and January 2012

Feeding	Antisera	Lu. cortelezzii		Lu. longipalpis		N	Total (%)
		Inside (%)	Outside (%)	Inside (%)	Outside (%)
Simple	chicken	5.2	-	5.2	28.2	37	38.6
	dog	-	7.7	12.7	5.2	25	25.6
	human	-	-	-	5.2	5	5.2
	rodent	-	2.5	2.5	5.2	10	10.2
	Subtotal	5.2	10.2	20.4	43.8	77	79.6
Mixed	chicken + dog	-	-	-	2.5	2	2.5
	chicken + human	-	-	2.5	-	2	2.5
	chicken + rodent	-	2.5	-	5.2	8	7.7
	dog + rodent	-	-	2.5	5.2	8	7.7
	Subtotal	0	2.5	5.0	12.9	20	20.4
Total		17.9		82.1		97	100

• N = absolute number of sand flies engorded.

DISCUSSION

Considering simple and mixed feeding, we observed more positive results to chicken antiserum in Lu. longipalpis, but also found sandflies engorged with the blood of dogs, rodents and humans. Lutzomyia cortelezzii seems to follow the same eclectic feeding behavior and preference of Lu. longipalpis. The presence of more than one source of blood in some females reinforces the food eclecticism of these species, demonstrating that sandflies usually adjust their feeding pattern according to the availability of hosts¹⁷.

Attraction of Lu. longipalpis to chickens has already been reported in the literature^2,4. It is evident that this association contributes to the domiciliation of Lu. longipalpis. Chickens attract insects in the peridomicile area increasing the chances of sandflies contact with other vertebrates and thus increasing the risk of leishmaniasis transmission. This finding can play an important epidemiological role, contributing to the maintenance of breeding places for sandflies.

Dogs play an important role in the maintenance of visceral leishmaniasis in the human environment, serving as reservoirs for this intracellular parasite²¹. Analyzing Table 2, dogs also appeared as alternative sources of blood meal. Thus, the presence of sandflies inside.and outside homes, as mentioned earlier, can increase the contact of sandflies with humans, maximizing the probability of infection by Leishmania.

Human blood was detected only in Lu. longipalpis. The observation that Lu. longipalpis females fed on humans demonstrates that this species exhibits an anthropophilic behavior. In addition, the ability to feed on a variety of host species and also mixed feeding profiles (chicken+dog/ chicken+human/ chicken+rodent/ dog+rodent) reinforces the role of sandflies as the main vectors of VL in Brazil.

Finally, rodents were also in the group of animals used in sandflies blood meals. This finding reinforces the need to study rodents as possible reservoirs of Leishmania sp., as suggested by CORREDOR et al. (1989)⁹, because they constitute an important link between the forest and domiciliary environments.

ACKNOWLEDGMENTS

To the Fundação de Amparo à Pesquisa do Estado de Minas Gerais(APQ-0098/10) for the financial support and to Miss Debra Kelci ReyesBrannon (FullBright English Teaching Assistant, Brazil, 2014) who kindly revised the English manuscript.

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Publication Dates

• Publication in this collection
  Jul-Aug 2015

History

• Received
  15 May 2014
• Accepted
  03 Nov 2014