Avaliação da atratividade de hamster dourado (Mesocricetus auratus) infectado com Leishmania (Leishmania) amazonensis para Lutzomyia longipalpis (Diptera:Psychodidae)
Data
2022-03-30
Autores
da Rocha Silva, Flávia Benini [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
As leishmanioses são zoonoses causadas por protozoários flagelados do gênero Leishmania e transmitidas por insetos hematófagos denominados flebotomíneos. Clinicamente se apresentam como visceral e tegumentar. A leishmaniose visceral tem como agente etiológico a Leishmania (Leishmania) infantum chagasi e principal vetor nas Américas os flebotomíneos da espécie Lutzomyia longipalpis. Além disso, Lu. longipalpis é considerado como vetor permissivo para L. (L.) amazonensis, um dos agentes etiológicos da leishmaniose tegumentar americana. Para localização dos hospedeiros e realização do repasto sanguíneo, os insetos hematófagos utilizam compostos orgânicos voláteis (COV) liberados pelos mesmos, denominados cairomônios. Alguns estudos demonstram que hospedeiros infectados por parasitas podem liberar compostos diferentes dos não infectados e influenciar na atratividade de tais hospedeiros para os insetos hematófagos vetores. O presente estudo buscou investigar se hamsters não infectados e infectados com L. (L.) amazonensis apresentavam diferença na atratividade para Lu. longipalpis; além da diferença de atratividade, buscou avaliar o volume de sangue ingerido pelos flebotomíneos e os COV emitidos pelos diferentes grupos de hamsters. Complementar a isso, também foram avaliados os COV liberados in vitro em cultura de macrófagos infectados ou não com L. (L.) amazonensis e de amastigotas livres dessa espécie. A atratividade dos animais foi avaliada por meio de testes realizados em gaiolas, onde se contabiliza a taxa de alimentação das fêmeas dos flebotomíneos. As fêmeas alimentadas foram então individualizadas em microtubos, maceradas e homogeneizadas para leitura da absorbância a 540nm. Tal leitura permite estimar o volume de sangue ingerido por cada fêmea, a partir de curva padrão de absorbância versus volume de sangue obtida previamente. Os COV liberados pelos diferentes grupos de animais foram obtidos por meio de uma fibra de microextração em fase sólida (SPME) e analisados por meio de cromatografia gasosa acoplada à espectrometria de massas. Após algumas etapas de desenvolvimento da metodologia, ficou estabelecida extração dos voláteis com uso de fibra de SPME revestida por polidimetilsiloxano/divinilbenzeno e com a amostra aquecida em banho-maria a 90°C por um período de 50 minutos. Os testes de atratividade foram realizados com um total de 10 hamsters – antes e após a infecção – onde foi possível observar uma taxa média de alimentação das fêmeas de 64,25% antes da infecção e de 64,75% após a infecção. Na avaliação do volume de sangue ingerido por fêmea, foram obtidas médias de 0,98μL e 1,10μL antes e após a infecção, respectivamente. Os voláteis extraídos a partir de amostras de pelos dos animais utilizados nos testes de atratividade e das culturas in vitro foram identificados com auxílio de biblioteca de espectros e cálculo dos índices de retenção. Os COV identificados foram submetidos à análise de componentes principais para verificar possíveis correlações com os animais e o status da infecção. A partir dos resultados obtidos para os testes in vivo, não foi possível observar uma influência da infecção por L. (L.) amazonensis na taxa de alimentação das fêmeas de Lu. longipalpis em hamsters e no perfil de COV liberados por esses animais. Para os COV identificados a partir dos testes in vitro com culturas infectadas com L. (L.) amazonensis, foi possível observar a presença de undecan-2-ona exclusivamente nas formas amastigotas-like livres; octan-1-ol e 2-feniletanol nas formas amastigotas (independente da maneira de obtenção); e de 4-fenilciclohexeno em todas as amostras, porém em maior teor nas amastigotas livres de lesão. Tais compostos podem ser estudados futuramente quanto à sua atratividade para flebotomíneos.
Leishmaniasis are zoonosis caused by flagellated protozoa of Leishmania genus, transmitted by hematophagous insects called sand flies, and present two main clinical manifestations: visceral and cutaneous. Visceral leishmaniasis has as Leishmania (Leishmania) infantum chagasi as aetiological agent and Lutzomyia longipalpis sand flies as main vector in the America. Furthermore, Lu. longipalpis is considered as a permissive vector to L. (L.) amazonensis, one of aetiological agents of American cutaneous leishmaniasis. To ensure a successful Leishmania transmission, the female sand fly needs to detect a vertebrate host for blood meal. For this, sand flies females and other haematophagous insects use some chemical cues called kairomones, which are volatiles organic compounds (VOCs) released by the hosts. Some studies have demonstrated that parasites-infected hosts can release different compounds when compared to uninfected, presenting a higher attractiveness to haematophagous vectors. In the present study, we evaluated possible differences on the attractiveness of golden hamsters uninfected versus L. (L.) amazonensis-infected to Lu. longipalpis females and the blood volume ingested by the sand flies females fed on those animals; also, we investigated the VOCs released by the animals before and after infection with L. (L.) amazonensis. Additionally, we evaluate possible differences in the VOCs released by host macrophages, uninfected or infected by L. amazonensis, and by free amastigotes in culture medium. Hamsters’ attractiveness trials were performed in Barraud cages, by counting of the number of blood fed sand flies females, which after each trial were individualized in microtubes, macerated and homogenized for absorbance reading at 540nm. Such reading allows estimating the blood volume ingested by each sand fly female, by using a standard curve of absorbance versus blood volume previously obtained. VOCs realeased by the different groups of animals were obtained by a solid phase microextraction fiber and analysed by gas chromatography-linked mass spectrometry. After some development steps, we established the VOCs extraction conditions: SPME fiber coated with polydimethylsiloxane/divinylbenzene, with the sample heated at 90°C for 50 minutes. Attractiveness trials were performed with a total of 10 hamsters – before and after infection – and we could observer means of blood fed females of 64.25% before the infection and 64.75% after the infection. For the blood volume ingested by sand fly female, we obtain means of 0.98μL and 1.10μL before and after the infection, respectively. VOCs extracted from animals samples and from in vitro cultures were identified by comparing mass spectra to the library of the machine and by comparing the retention indexes to those of a mixture of C8-C20 n-alkanes. Identified VOCs were subjected to principal components analysis to verify possible correlations with animals and status of infection. The results obtained from the in vivo trials did not showed a possible influence of L. (L.) amazonensis infection on the blood meal rates of Lu. longipalpis in hamsters and on the VOCs profiles released by these animals. For the VOCs identified from in vitro L. (L.) amazonensis-infected cell cultures, it was possible to observe the presence of undecan-2-one exclusively in amastigote-like cells; octan-1-ol and 2-phenylethanol in amastigote forms (independently of the obtained method); and 4-phenycyclohexene in all of the samples, but with large amount in amastigotes from lesion site. Such compounds can be future evaluated for their attractiveness for sand flies.
Leishmaniasis are zoonosis caused by flagellated protozoa of Leishmania genus, transmitted by hematophagous insects called sand flies, and present two main clinical manifestations: visceral and cutaneous. Visceral leishmaniasis has as Leishmania (Leishmania) infantum chagasi as aetiological agent and Lutzomyia longipalpis sand flies as main vector in the America. Furthermore, Lu. longipalpis is considered as a permissive vector to L. (L.) amazonensis, one of aetiological agents of American cutaneous leishmaniasis. To ensure a successful Leishmania transmission, the female sand fly needs to detect a vertebrate host for blood meal. For this, sand flies females and other haematophagous insects use some chemical cues called kairomones, which are volatiles organic compounds (VOCs) released by the hosts. Some studies have demonstrated that parasites-infected hosts can release different compounds when compared to uninfected, presenting a higher attractiveness to haematophagous vectors. In the present study, we evaluated possible differences on the attractiveness of golden hamsters uninfected versus L. (L.) amazonensis-infected to Lu. longipalpis females and the blood volume ingested by the sand flies females fed on those animals; also, we investigated the VOCs released by the animals before and after infection with L. (L.) amazonensis. Additionally, we evaluate possible differences in the VOCs released by host macrophages, uninfected or infected by L. amazonensis, and by free amastigotes in culture medium. Hamsters’ attractiveness trials were performed in Barraud cages, by counting of the number of blood fed sand flies females, which after each trial were individualized in microtubes, macerated and homogenized for absorbance reading at 540nm. Such reading allows estimating the blood volume ingested by each sand fly female, by using a standard curve of absorbance versus blood volume previously obtained. VOCs realeased by the different groups of animals were obtained by a solid phase microextraction fiber and analysed by gas chromatography-linked mass spectrometry. After some development steps, we established the VOCs extraction conditions: SPME fiber coated with polydimethylsiloxane/divinylbenzene, with the sample heated at 90°C for 50 minutes. Attractiveness trials were performed with a total of 10 hamsters – before and after infection – and we could observer means of blood fed females of 64.25% before the infection and 64.75% after the infection. For the blood volume ingested by sand fly female, we obtain means of 0.98μL and 1.10μL before and after the infection, respectively. VOCs extracted from animals samples and from in vitro cultures were identified by comparing mass spectra to the library of the machine and by comparing the retention indexes to those of a mixture of C8-C20 n-alkanes. Identified VOCs were subjected to principal components analysis to verify possible correlations with animals and status of infection. The results obtained from the in vivo trials did not showed a possible influence of L. (L.) amazonensis infection on the blood meal rates of Lu. longipalpis in hamsters and on the VOCs profiles released by these animals. For the VOCs identified from in vitro L. (L.) amazonensis-infected cell cultures, it was possible to observe the presence of undecan-2-one exclusively in amastigote-like cells; octan-1-ol and 2-phenylethanol in amastigote forms (independently of the obtained method); and 4-phenycyclohexene in all of the samples, but with large amount in amastigotes from lesion site. Such compounds can be future evaluated for their attractiveness for sand flies.
Descrição
Palavras-chave
leishmanioses, flebotomíneos, ecologia química, compostos orgânicos voláteis, leishmaniasis, sand flies, chemical ecology, volatile organic compounds