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Submitted by Sheila MONTEIRO (sheila.monteiro@ufma.br) on 2021-05-18T14:09:21Z No. of bitstreams: 1 ROSA-SILVA.pdf: 4540270 bytes, checksum: 76da788e7af61d8d5e31361fb06975a2 (MD5) Made available in DSpace on 2021-05-18T14:09:21Z (GMT). No. of bitstreams: 1 ROSA-SILVA.pdf: 4540270 bytes, checksum: 76da788e7af61d8d5e31361fb06975a2 (MD5) Previous issue date: 2021-04-13 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão - FAPEMA Studies about vectorial capacity and competence for the transmission of certain parasites require laboratory colonization of their biological vectors. The sand fly Lutzomyia longipalpis is incriminated as the main vector of Leishmania (Leishmania) infantum, but it has been shown to be permissive for several Leishmania species that cause american cutaneous leishmaniasis (ACL). Therefore, we demonstrate the competence of Lu. longipalpis in transmitting Leishmania (Leishmania) amazonensis in mice, causing clinical manifestation of ACL in these animals. Initially, we stablished a colony of Lu. longipalpis from females sampled in the municipality of Raposa, MA, using CDC light traps. The colony produced ten generations (F10) from the parental generation (P). The first four generations showed the highest productivity. The whole life cycle lasted on average 28 + 0.5 days at 27°C and 80% relative humidity. Nonviable eggs represented more than 50% of the total number of eggs produced by the engorged females, while pupae had a mortality rate of only 2%. In the tests for vectorial capacity and competence 291 uninfected females of Lu. longipalpis of generation F1, F2 and F3 performed a first blood feeding on mice infected with Le. amazonensis (strain IFLA / BR / 1968 / PH8). After blood-feeding, 52.6% of the females were positive for Leishmania DNA. Of these, 17 (of which 58,8% were positive for Leishmania by dissection of the intestine) performed a second feeding on uninfected mice. At 27 days post-infection, one mouse presented leishmaniotic lesions. The limiting dilution test and immunohistopathological analyses confirmed the occurrence of Leishmania promastigote and amastigote forms, respectively, in the skin of the animal. Parasite DNA was also detected in lesions on paws and inguinal lymph node. DNA sequencing and analysis confirmed the Leishmania species infecting both insects and mice. Our results confirm that Lu. longipalpis can sustain infection and experimentally transmit Le. amazonensis to rodents, inducing leishmaniastic lesions. From the epidemiological perspective of the urban ACL, the successful transmission demonstrated supports the role of Lu. longipalpis as a vector of Le. amazonensis in the studied region and possibly in others throughout Brazil and the New World, where both sand fly and parasite are widespread. Estudos de capacidade e competência vetorial requerem a disponibilidade de colônias de insetos vetores de doenças em condições laboratoriais. O táxon Lutzomyia longipalpis é incriminado como vetor natural de Leishmania (Leishmania) infantum, porém tem se demonstrado permissivo para várias espécies de Leishmania que causam leishmaniose tegumentar americana (LTA). Por esta razão, o trabalho teve como objetivo demonstrar a competência de Lu. longipalpis em transmitir Leishmania (Leishmania) amazonensis em camundongo causando manifestação clínica de LTA no animal. Inicialmente, foi estabelecida uma colônia de Lu. longipalpis a partir de fêmeas capturadas no município da Raposa, MA, utilizando-se armadilha luminosa do tipo CDC. A colônia produziu dez gerações (F10) a partir da geração parental (P). A maior produtividade da colônia foi nas quatro primeiras gerações. O ciclo de vida completo durou, em média, 28 + 0,5 dias, a 27°C e umidade relativa de 80%. Os ovos inviáveis representaram mais de 50% do total de ovos produzidos pelas fêmeas ingurgitadas, enquanto as pupas apresentaram índice de mortalidade de apenas 2%. Nos ensaios de capacidade e competência vetorial, 291 fêmeas de Lu. longipalpis de geração F1, F2 e F3 (livre de infecção de Leishmania) realizaram um primeiro repasto sanguíneo em camundongos infectados com Le. amazonensis (linhagem IFLA/BR/1968/PH8). Após a alimentação, 52,6% positivaram DNA de Leishmania. Destes, 17 (dos quais 58,8% foram positivos para Leishmania por dissecação do intestino) efetuaram um segundo repasto sanguíneo em camundongos não infectados. Após 27 dias de infecção, um dos camundongos apresentou um edema no membro anterior direito característico de lesão leishmaniótica. O teste de diluição limitante e as análises imunohistopatológicas confirmaram a ocorrência de formas promastigotas e amastigostas de Leishmania, respectivamente, na lesão do membro anterior do animal. Também foi detectado DNA do parasita em lesões do membro anterior esquerdo e linfonodo poplíteo. O sequenciamento do DNA confirmou a espécie Leishmania tanto em insetos como no camundongo. Os resultados confirmam a capacidade de Lu. longipalpis sustentar a infecção e transmitir experimentalmente Le. amazonensis a roedores, induzindo lesões leishmanióticas. Do ponto de vista epidemiológico da LTA urbana, onde tanto Lu. longipalpis como Le. amozonensis coabitam extensivamente pelo Novo Mundo, inclusive em várias regiões do Brasil, a suscetibilidade do teste de transmissão confirma a possibilidade do papel vetorial deste flebotomíneo para transmitir Le. amazonensis na região estudada.

Laboratory colonies of phlebotomine sand flies are necessary for experimental study of their biology, behaviour and mutual relations with disease agents and for testing new methods of vector control. They are indispensable in genetic studies and controlled observations on the physiology and behaviour of sand flies, neglected subjects of high priority. Colonies are of particular value for screening insecticides. Colonized sand flies are used as live vector models in a diverse array of research projects, including xenodiagnosis, that are directed toward control of leishmaniasis and other sand fly-associated diseases. Historically, labour-intensive maintenance and low productivity have limited their usefulness for research, especially for species that do not adapt well to laboratory conditions. However, with growing interest in leishmaniasis research, rearing techniques have been developed and refined, and sand fly colonies have become more common, enabling many significant breakthroughs. Today, there are at least 90 colonies representing 21 distinct phlebotomine sand fly species in 35 laboratories in 18 countries worldwide. The materials and methods used by various sand fly workers differ, dictated by the availability of resources, cost or manpower constraints rather than choice. This paper is not intended as a comprehensive review but rather a discussion of methods and techniques most commonly used by researchers to initiate, establish and maintain sand fly colonies, with emphasis on the methods proven to be most effective for the species the authors have colonized. Topics discussed include collecting sand flies for colony stock, colony initiation, maintenance and mass-rearing procedures, and control of sand fly pathogens in colonies.