Microbiota intestinal de Melipona spp.: caracterização e impactos da paisagem e do uso de agroquímicos

Abstract

A microbiota intestinal desempenha um papel fundamental na preservação da saúde de abelhas. As abelhas eusociais corbiculadas (Apini, Bombini e Meliponini) apresentam uma microbiota densa e relativamente simples, entretanto pouco se conhece sobre a composição e papel da microbiota de Melipona. Além disso, são escassos e necessários estudos que analisem o impacto de diferentes paisagens e do uso de agroquímicos na microbiota intestinal das abelhas sem ferrão, visando o desenvolvimento de novas estratégias de preservação. Nesse contexto, o presente trabalho teve como objetivos analisar a composição microbiana intestinal de abelhas do gênero Melipona coletadas em diferentes regiões do Brasil, a fim de caracterizar a microbiota core dessas abelhas e elucidá-la ao longo do trato digestório de Melipona quadrifasciata. Adicionalmente, analisar os efeitos da paisagem na microbiota de M. capixaba, e os efeitos de doses subletais de dimetoato na microbiota intestinal de M. quadrifasciata e M. mondury. O trabalho foi dividido em três capítulos. No primeiro, a diversidade microbiana intestinal em Melipona spp. coletadas em diversos estados brasileiros, tiveram o DNA intestinal extraído e sequenciado para o gene 16S rRNA, e a caracterização de novos simbiontes em diferentes partes do intestino de M. quadrifasciata foram abordadas. A microbiota core de Melipona spp. incluiu Bifidobacterium, Lactobacillus, Apilactobacillus, Floricoccus e Bombella. Dentre eles, Apilactobacillus e Bombella dominaram no papo, enquanto o ventrículo foi dominado por Apilactobacillus e Lactobacillus. Foi confirmada a ausência de Snodgrassella e Gilliamella no íleo, no qual verificou-se um novo simbionte filogeneticamente próximo a Floricoccus, bem como a presença de Bifidobacterium, Lactobacillaceae e Bombella. O reto foi dominado por Bifidobacterium e Lactobacillus. No segundo capítulo, abordou-se a influência da paisagem e da sazonalidade na microbiota intestinal de M. capixaba. As abelhas foram coletadas em áreas urbanas, naturais, agrícolas e de agricultura orgânica, ao final do verão e do inverno. O DNA intestinal foi extraído e o gene 16S rRNA sequenciado. A microbiota das abelhas de áreas urbanas diferiu significativamente da microbiota das abelhas coletadas em outras áreas. Abelhas coletadas no verão/2023 também apresentaram uma composição microbiana diferente daquelas coletadas no verão/2022. Entretanto, mais estudos são necessários com um maior número de amostras para elucidar os efeitos da sazonalidade na microbiota de M. capixaba. No terceiro capítulo, foram discutidos os efeitos de doses subletais de dimetoato na microbiota intestinal de M. quadrifasciata e M. mondury. Os grupos de abelhas que apresentaram uma taxa de sobrevivência acima de 65% tiveram seus intestinos extraídos e sequenciados para a região 16S rRNA. Observou-se que as doses subletais de dimetoato não impactaram significativamente a microbiota intestinal de ambas as abelhas testadas. Porém, as abelhas participantes do experimento tiveram uma composição microbiana dissimilar das abelhas do grupo controle de campo, indicando um possível efeito das condições a que foram expostas em laboratório.Palavras-chave: Meliponini; Microbiota; Diversidade microbiana; Dimetoato; Simbiontes; Intestino.The symbiotic relationship between microorganisms and hosts is essential for the maintenance of species and ecosystems. In the case of animal hosts, specifically insects, the gut microbiota plays a fundamental role in preserving the health of these invertebrates, from their development to protection against infections and detoxification of the organism. Eusocial corbiculate bees (Apini, Bombini, and Meliponini) have a dense and relatively simple microbiota that performs various functions in maintaining the health of these insects, such as pollen digestion and detoxification, nectar fermentation, protection against pathogens, and detoxification of these animals from chemicals commonly used in agriculture. Among corbiculate bees, stingless bees stand out as the main pollinators of native species in tropical and subtropical ecosystems, constituting the most diverse group among eusocial bees. In recent decades, these bees have faced numerous challenges contributing to the decline of their populations, such as climate change, indiscriminate use of agrochemicals, and anthropogenic alterations in their natural habitat. Little is known about the composition, relevance, and role of the microbiota of Melipona in the face of challenges faced by these bees. Currently, it is known that Melipona lacks two main symbionts present in other bees, Gilliamella and Snodgrassella, but still lacks information on the acquisition of new symbionts and their role for the host. Additionally, studies analyzing the impact of different environments and the use of agrochemicals on the gut microbiota of stingless bees are scarce and extremely necessary to develop new preservation strategies for these insects. In this context, this study aimed to analyze the gut microbial composition of Melipona collected in different regions of Brazil to characterize the core microbiota of these bees and investigate the different parts of the gut of Melipona quadrifasciata anthidioides to elucidate the distribution of microorganisms along the digestive tract of these insects. Additionally, the effects of landscape on the microbiota of M. capixaba and the effects of sublethal doses of dimethoate on the gut microbiota of M. quadrifasciata and M. mondury were analyzed. To achieve these objectives, the study was divided into three chapters. In the first chapter, the gut microbial diversity in Melipona spp. and the characterization of new symbionts in different gut parts of Melipona quadrifasciata anthidioides were addressed. Different bee species were collected in various Brazilian states, with gut DNA being extracted, sequenced for the 16S rRNA gene, and analyzed together with previously published data. Subsequently, the gut of M. quadrifasciata anthidioides was sectioned, and each part was sequenced for the 16S rRNA gene. The core microbiota of Melipona spp. included Bifidobacterium, Lactobacillus, Apilactobacillus, Floricoccus, and Bombella. Among them, Apilactobacillus and Bombella dominated the crop, while the venticulus was dominated by Apilactobacillus and Lactobacillus. The absence of Snodgrassella and Gilliamella in the ileum of these bees was confirmed, which contained a new symbiont phylogenetically close to Floricoccus, as well as the presence of Bifidobacterium, Lactobacillaceae, and Bombella in this gut region. The rectum was dominated by Bifidobacterium and Lactobacillus. In the second chapter, the influence of landscape and seasonality on the gut microbiota of M. capixaba was addressed. Bees were collected in urban, natural, agricultural, and organic farming areas, at the end of the hot and rainy season (summer) and the cold and dry season (winter). Bee gut DNA was extracted and sequenced for the 16S rRNA region. The microbiota of bees collected in urban areas differed significantly from the microbiota of bees collected in other areas. Bees collected at the end of the summer of 2023 also had a different microbial composition from those collected in the summer of 2022 and winter of 2022. Further studies with a larger number of samples are needed to elucidate the effects of seasonality on the microbiota of these bees. In the third chapter, the effects of sublethal doses of dimethoate on the gut microbiota of M. quadrifasciata and M. mondury were analyzed. Bee groups with a survival rate above 65% had their gut extracted and sequenced for the 16S rRNA region. It was observed that sublethal doses of dimethoate did not significantly impact the microbiota of both tested bees. However, bees participating in the experiment had a dissimilar microbial composition from the field control bees, which were bees sequenced shortly after being removed from the hives, indicating a possible effect of the conditions they were exposed to in the laboratory. Keywords: Meliponini; Microbiota; Microbial diversity; Dimethoate; Symbiont; Gut.