Manuela Oliveira Ramalho

Mansonia humeralis is a neotropical mosquito common in floodplain ecosystems and a potential host of Mayaro virus in the Amazon region. Studies show that bacterial communities associated with the midgut of mosquito vectors can mediate insecticide detoxification and modulate vector competence. Understanding the characteristics of the intestinal microbiota, and the factors that shape them, is essential for effective mosquito control. This study investigated the influence of bacterial communities in water from the larval environment on the composition and bacterial diversity in Ma. humeralis larval intestines and characterized the midgut microbiota of adult females with and without apparent blood. Samples were obtained from the Jirau Hydroelectric Plant, Madeira River, AM, Brazil, and their bacteria characterized using the 16S rRNA molecular marker. A total of 402 bacterial Amplicon Sequencing Variants (ASVs) were identified to genus level. The sample groups (water, larvae, blood + and blood-) had distinct bacterial communities, especially the larvae, probably due to the intense feeding activity which occurs at this stage. The phylum Proteobacteria was dominant in water, blood + and blood- (52.5; 99 and 98%), respectively, while Firmicutes dominated in larvae (45%). The most abundant taxa in water were: ASV Comamonadaceae (11.7%) and hgcI_clade (9.6%), in larvae: ASV Synergistaceae (11.1%) and ASV Rhodocyclaceae (7.8%), in blood-: Serratia (46.9%) and Asaia (12.6%) and, in blood+: ASV Enterobacteriaceae (54.5%) and Serratia (21.1%). Some 19% of the taxonomic groups from water were also recorded in larvae, while 46.7% of blood- microbiota were present in blood+. Wolbachia, Acinetobacter and Enterobacter bacteria were recorded at low frequency in all samples. This result provides a powerful tool for understanding the ecology of the intestinal microbiota of Ma. humeralis and the relationships with the ambient microbiota derived from larval filter, and adult blood, feeding. The results will also be useful for future studies which focusing the development of potential tools for the biological control of this vector.

Wolbachia is a widespread and well-known bacterium that can induce a wide range of changes within its host. Ants specifically harbor a great deal of Wolbachia diversity and are useful systems to study endosymbiosis. The turtle ants (Cephalotes) are a widespread group of tropical ants that rely on gut microbes to support their herbivorous diet for their survival, yet little is known of the extent of this diversity. Therefore, studying their endosymbionts and categorizing the diversity of bacteria within Cephalotes hosts could help to delimit species and identify new strains and can help lead to a further understanding of how the microbiome leads to survival and speciation in the wild. In our study, 116 individual samples were initially tested for positive infection with the wsp gene. Of the initial 116 samples, 9 samples were infected with only one strain of Wolbachia, and 7 were able to be used successfully for multilocus sequence typing (MLST). We used the new MLST data to infer a phylogeny with other Formicidae samples from the MLST online database to identify new Wolbachia strains and related genes, of which only one came back as an exact match. The 18 Wolbachia-positive samples ranged across 15 different species and 7 different countries, which we further test for species identity and geographic correlation. This study is the first comprehensive look into the diversity of Wolbachia in the turtle ants, providing insight into how endosymbionts are oriented in widespread species and providing a strong foundation for further research in host-microbe interactions.

Ants of the genus Solenopsis are globally distributed, presenting high diversity and many generalist species. In South America, the dominant species is Solenopsis saevissima (Smith, 1855), commonly found nesting in grassy fields surrounding humanized areas. In spite of being so common, there has been no research evaluating the effect of human disturbances on the mitochondrial DNA (mtDNA) haplotype diversity in this species. In this context, we here characterized the mtDNA haplotype diversity in S. saevissima nests by highway roadsides, dust roads, and forest borders of Atlantic Forest, based on partial sequences of cytochrome c oxidase subunit I (COI). Based on the facts that the species is a rapid colonizer of disturbed habitats, we specifically probed how the genetic diversity of native S. saevissima is impacted by highways and roads infrastructure expanding around the rainforest. Species diagnosis was established both by morphological characters and obtained mtDNA COI sequences. Overall, the species exhibited high haplotypes and nucleotide diversity, particularly around forest borders; though all haplotypes seemed closely related across the different habitats. We identified seven mitochondrial haplotypes (H1 to H7), where haplotype H1 was exclusively found in highway roadside nests, and H7 on dust roads; the remaining haplotypes were recorded from all habitats. Haplotype H1 was geographically isolated to the south of the Atlantic Forest, supporting previous suggestions that it acts as a biogeographical barrier. The pattern is suggestive of a recent species expansion, probably resulting from extensive habitat fragmentation. Taken together, our data demonstrates fire ant haplotypes prevailing in some anthropized habitats, characterizing how a native species lining the remnants of the Brazilian Atlantic Forest might be a concern for environmental conservation.

Among insects, Wolbachia is an exceedingly common bacterial endosymbiont with a range of consequences of infection. Despite the frequency of Wolbachia infection, very little is known about this bacteria’s diversity and role within hosts, especially within ant hosts. In this study, we analyze the occurrence and diversity of Wolbachia across the spiny ants (Polyrhachis), a large and geographically diverse genus. Polyrhachis samples from throughout the host genus’ phylogenetic and biogeographical range were first screened for single infections of Wolbachia using the wsp gene and Sanger sequencing. The multilocus sequence typing (MLST) scheme was then used on these singly infected samples to identify the Wolbachia strains. A Wolbachia phylogeny was inferred from the Polyrhachis samples analyzed in this study as well as other Formicidae MLST profiles from the MLST online database. We hypothesized that three key host factors were impacting Wolbachia diversity within the Polyrhachis genus: biogeography, phylogeny, and species level. The results suggest that the phylogeny and biogeography of Polyrhachis hosts have no impact on Wolbachia diversity; however, species level may have some limited influence. Additionally, Wolbachia strains appear to group according to being either Old World or New World strains. Among the taxa able to form complete MLST allelic profiles, all twenty are seemingly new strains.

The present investigation deals with some aspects of the diversity of fire ants (Hymenoptera: Formicidae) in their native range. The Red Imported Fire Ant Solenopsis invicta is native to the tropical and subtropical inland territories of South America. In Brazil, it mainly occurs around the Pantanal region and across the Paraguay river, a region composed of grasslands which are seasonally flooded. Recent studies have evidenced this fire ant species is gradually spreading to other regions of Brazil. In the present investigation, we surveyed the molecular diversity of S. invicta populations across fragments of Atlantic Forest in São Paulo, Brazil, using mtDNA COI haplotypes. Fire ant nests were sampled along the highways lining the northern and southern slope sides of the mountain range Serra do Mar, SP, Brazil. Four haplotypes were identified (H1–H4), which were assessed for similarity to deposited records by other authors, revealing that the haplotypes H1 and H2 are likely of foreign origin through recent reintroduction via a marine port to the south of the Serra do Mar mountain range. On the other hand, the haplotypes H3 and H4, predominating among the inland samples from the northern side of the mountain range, were most similar to previous records from more central regions of Brazil. Haplotypes clustered into distinct supergroups, further pointing to the occurrence of two separate expansion waves of S. invicta in the region. We suggest the obtained pattern indicates the mountain range may function as a geographical barrier deferring gene flow.

Background: To understand the patterns of biodiversity it is important to consider symbiotic interactions as they can shape animal evolution. In several ant genera symbiotic interactions with microbial communities have been shown to have profound impacts for the host. For example, we know that for Camponotini the gut community can upgrade the host’s diet and is shaped by development and colony interactions. However, what is true for one ant group may not be true for another. For the microbial communities that have been examined across ants we see variation in the diversity, host factors that structure these communities, and the function these microbes provide for the host. In the herbivorous turtle ants (Cephalotes) their stable symbiotic interactions with gut bacteria have persisted for 50 million years with the gut bacteria synthesizing essential amino acids that are used by the host. Although we know the function for some of these turtle ant-associated bacteria there are still many open questions.Results: In the present study we examined microbial community diversity (16S rRNA and 18S rRNA amplicons) of more than 75 species of turtle ants across different geographic locations and in the context of the host’s phylogenetic history. Our results show (1) that belonging to a certain species and biogeographic regions are relevant to structuring the microbial community of turtle ants; (2) both bacterial and eukaryotic communities demonstrated correlations and cooccurrence within the ant host; (3) within the core bacterial community, Burkholderiaceae bacterial lineage were the only group that showed strong patterns of codiversification with the host, which is remarkable since the core bacterial community is stable and persistent.Conclusions: We concluded that for the turtle ants there is a diverse and evolutionarily stable core bacterial community, which leads to interesting questions about what microbial or host factors influence when these partner histories become evolutionarily intertwined.

Solenopsis invicta and Solenopsis saevissima are the most common fire ants in Brazil. Environmental disturbances favour the colony dispersal of both species, particularly those with an anthropogenic origin. However, the frequencies of the species can vary according to the amount of anthropogenic disturbances. Crops near native vegetation fragments is a common landscape in the Brazilian Atlantic Forest. Here, we analyse if there is influence of native vegetation cover on the occurrence of these fire ants and their molecular diversity in crops. Our hypothesis is that these species are antagonistic in terms of occurrence and molecular diversity when compared in the same habitat. We selected crops near fragments with around 50% of native vegetation cover, a percentage informative enough to detect biological responses from insects belonging to a same order. Nests were collected and the species were identified using external morphology, and mtDNA COI sequences. We reject our hypothesis, which suggest that the species colonize the vegetation patches around crops in a similar way. Solenopsis invicta is not limited by vegetation cover, and has a high haplotype diversity when compared to S. saevissima , particularly when the vegetation cover is greater than 50%. Additionally, the vegetation cover seems to not have influence in the total number of haplotypes, but the species have haplotypes that are exclusive to each landscape. Our findings suggest that S. invicta is expanding to native vegetation areas that it has not occupied before, as S. saevissima was dominant there.

Bacterial communities in animals are often necessary for hosts to survive, particularly for hosts with nutrient-limited diets. The composition, abundance, and richness of these bacterial communities may be shaped by host identity and external ecological factors. The turtle ants (genus Cephalotes) are predominantly herbivorous and known to rely on bacterial communities to enrich their diet. Cephalotes have a broad Neotropical distribution, with high diversity in the South American Cerrado, a geologically and biologically diverse savanna. Using 16S rRNA amplicon sequencing, we examined the bacterial communities of forty-one Cephalotes samples of sixteen different species collected from multiple locations across two sites in the Cerrado (MG, Brazil) and compared the bacterial communities according to elevation, locality, species, and species group, defined by host phylogeny. Beta diversity of bacterial communities differed with respect to all categories but particularly strongly when compared by geographic location, species, and species group. Differences seen in species and species groups can be partially explained by the high abundance of Mesorhizobium in Cephalotes pusillus and Cephalotes depressus species groups, when compared to other clades via the Analysis of Composition of Microbiome (ANCOM). Though the Cephalotes bacterial community is highly conserved, results from this study indicate that multiple external factors can affect and change bacterial community composition and abundance.

In South America, Solenopsis saevissima and S. invicta are the most common fire ants. Nests are founded in areas under anthropic interference like urban or rural areas, but S. invicta is found preferentially in those with the greatest anthropic interference. However, we do not know the rates at which they exist in anthropized areas next to high density of native vegetation. Areas with 60 to 90% of native Atlantic Forest were selected to verify the occurrence of both species in rural and urban areas. We investigated the molecular diversity and applied the reconstruction of the ancestral state analysis for each species. A total of 186 nests were analyzed and we found that the two species had the same proportion in the urban area. However, S. saevissima had a higher rate of prevalence in the rural area, in addition to having a greater number of haplotypes and ancestry associated with this type of habitat for the region. S. invicta had the same number of haplotypes in both rural and urban regions, and less haplotypic diversity. We conclude that S. saevissima is a species typically associated with rural areas and S. invicta, although present, is not dominant in urban areas.

Bacteria of the genus Wolbachia are widely distributed in arthropods, particularly in ants; nevertheless, it is still little explored with the Multilocus Sequence Typing (MLST) methodology, especially in the genus Solenopsis, which includes species native to South America. Ants from this genus have species distributed in a cosmopolitan way with some of them being native to South America. In Brazil, they are widely spread and preferentially associated with areas of human activity. This study aimed to investigate the diversity of Wolbachia in ants of the genus Solenopsis through the MLST approach and their phylogenetic relationship, including the relationship between mtDNA from the host and the related Wolbachia strain. We also tested the geographic correlation between the strains to infer transmission and distributional patterns. Fifteen new strains and eleven previously unknown alleles were obtained by sequencing and analyzing the five genes that make up the MLST. The phylogenetic relationship between the strains showed a polyphyletic pattern, indicative of the complexity of the evolutionary history of these bacteria in the analyzed species. We detected the correlation of host’s mitochondrial DNA with Wolbachia diversity which imply that related strains exist in related hosts, strongly suggesting the occurrence of vertical transfer. We found no specificity of the Wolbachia strain for a given geographic region that could indicate either that there is no horizontal transfer of the strain from the environment for the host or that the human action could be shuffling the distribution of the Solenopsis ants and the endosymbiont Wolbachia, as well. Our study highlights the complexity and novelty of Wolbachia diversity with this specific group of ants and the need for further studies that focus on understanding of this intricate relationship.