Tailed bacteriophages (Caudoviricetes) dominate the microbiome of a diseased stingless bee.

Bacteriophages, viruses that infect bacterial hosts, are known to rule the dynamics and diversity of bacterial populations in a number of ecosystems. Bacterial communities residing in the gut of animals, known as the gut microbiome, have revolutionized our understanding of many diseases. However, the gut phageome, while of apparent importance in this context, remains an underexplored area of research. Here we identify for the first time genomic sequences from tailed viruses (Caudoviricetes) that are associated with the microbiome of stingless bees (Melipona quadrifasciata). Both DNA and RNA were extracted from virus particles isolated from healthy and diseased forager bees, the latter showing symptoms from an annual syndrome that only affects M. quadrifasciata. Viral contigs from previously sequenced metagenomes of healthy and diseased forager bees were used for the analyses. Using conserved proteins deduced from their genomes, we found that Caudoviricetes were only present in the worker bee gut microbiome from diseased stingless bees. The most abundant phages are phylogenetically related to phages that infect Gram-positive bacteria from the order Lactobacillales and Gram-negative bacteria from the genus Gilliamella and Bartonella, that are common honey bee symbionts. The potential implication of these viruses in the M. quadrifasciata syndrome is discussed.

Temporal Changes in Gut Microbiota Composition and Pollen Diet Associated with Colony Weakness of a Stingless Bee.

Compared to honeybees and bumblebees, the effect of diet on the gut microbiome of Neotropical corbiculate bees such as Melipona spp. is largely unknown. These bees have been managed for centuries, but recently an annual disease is affecting M. quadrifasciata, an endangered species kept exclusively by management in Southern Brazil. Here we report the results of a longitudinal metabarcoding study involving the period of M. quadrifasciata colony weakness, designed to monitor the gut microbiota and diet changes preceding an outbreak. We found increasing amounts of bacteria associated to the gut of forager bees 2 months before the first symptoms have been recorded. Simultaneously, forager bees showed decreasing body weight. The accelerated growth of gut-associated bacteria was uneven among taxa, with Bifidobacteriaceae dominating, and Lactobacillaceae decreasing in relative abundance within the bacterial community. Dominant fungi such as Candida and Starmerella also decreased in numbers, and the stingless bee obligate symbiont Zygosaccharomyces showed the lowest relative abundance during the outbreak period. Such changes were associated with pronounced diet shifts, i.e., the rise of Eucalyptus spp. pollen amount in forager bees' guts. Furthermore, there was a negative correlation between the amount of Eucalyptus pollen in diets and the abundance of some bacterial taxa in the gut-associated microbiota. We conclude that diet and subsequent interactions with the gut microbiome are key environmental components of the annual disease and propose the use of diet supplementation as means to sustain the activity of stingless bee keeping as well as native bee pollination services.

Transposable element abundance correlates with mode of transmission in microsporidian parasites.

The extreme genome reduction and physiological simplicity of some microsporidia has been attributed to their intracellular, obligate parasitic lifestyle. Although not all microsporidian genomes are small (size range from about 2 to 50 MB), it is suggested that the size of their genomes has been streamlined by natural selection. We explore the hypothesis that vertical transmission in microsporidia produces population bottlenecks, and thus reduces the effectiveness of natural selection. Here we compare the transposable element (TE) content of 47 microsporidian genomes, and show that genome size is positively correlated with the amount of TEs, and that species that experience vertical transmission have larger genomes with higher proportion of TEs. Our findings are consistent with earlier studies inferring that nonadaptive processes play an important role in microsporidian evolution.

The virome of an endangered stingless bee suffering from annual mortality in southern Brazil.

Meliponiculture - the management of stingless bee colonies - is an expanding activity in Brazil with economic, social and environmental potential. However, unlike in apiculture, the pathogens that impact on meliponiculture remain largely unknown. In southern Brazil, every year at the end of the summer, managed colonies of the stingless bee Melipona quadrifasciata manifest a syndrome that eventually leads to collapse. Here we characterize the M. quadrifasciata virome using high-throughput sequencing, with the aim of identifying potentially pathogenic viruses, and test whether they are related to the syndrome outbreaks. Two paired viromes are explored, one from healthy bees and another from unhealthy ones. Each virome is built from metagenomes assembled from sequencing reads derived either from RNA or DNA. A total of 40 621 reads map to viral contigs of the unhealthy bees' metagenomes, whereas only 11 reads map to contigs identified as viruses of healthy bees. The viruses showing the largest copy numbers in the virome of unhealthy bees belong to the family Dicistroviridae - common pathogenic honeybee viruses - as well as Parvoviridae and Circoviridae, which have never been reported as being pathogenic in insects. Our analyses indicate that they represent seven novel viruses associated with stingless bees. PCR-based detection of these viruses in individual bees (healthy or unhealthy) from three different localities revealed a statistically significant association between viral infection and symptom manifestation in one meliponary. We conclude that although viral infections may contribute to colony collapses in the annual syndrome in some meliponaries, viruses spread opportunistically during the outbreak, perhaps due to colony weakness.

Holobionts and their hologenomes: Evolution with mixed modes of inheritance.

Symbioses are ubiquitous and have played an influential role in the evolution of life on Earth. Genomic studies are now revealing a huge diversity of associations among hosts and their microbiotas, allowing us to characterize their complex ecological and evolutionary dynamics. The different transmission modes and the asynchronous cell proliferation of the numerous symbionts associated with one host generate a genomic conflict ought to be solved. Two disputing views have been used to model and predict the outcome of such conflicts. The traditional view is based on community ecology, and considers that selection at the level of individuals is sufficient to explain longstanding associations among species. A new perspective considers that the host and its associated microbiota constitute a biological entity called holobiont, and that regarding it as a higher-level unit of selection is unavoidable to understand phenotypic evolution. Novel extended phenotypes are often built through symbiotic interactions, allowing the holobiont to explore and survive in distinct environmental conditions, and may evolve in a Lamarckian fashion.

Holobionts and their hologenomes: Evolution with mixed modes of inheritance

Abstract Symbioses are ubiquitous and have played an influential role in the evolution of life on Earth. Genomic studies are now revealing a huge diversity of associations among hosts and their microbiotas, allowing us to characterize their complex ecological and evolutionary dynamics. The different transmission modes and the asynchronous cell proliferation of the numerous symbionts associated with one host generate a genomic conflict ought to be solved. Two disputing views have been used to model and predict the outcome of such conflicts. The traditional view is based on community ecology, and considers that selection at the level of individuals is sufficient to explain longstanding associations among species. A new perspective considers that the host and its associated microbiota constitute a biological entity called holobiont, and that regarding it as a higher-level unit of selection is unavoidable to understand phenotypic evolution. Novel extended phenotypes are often built through symbiotic interactions, allowing the holobiont to explore and survive in distinct environmental conditions, and may evolve in a Lamarckian fashion.

Report on the microbiota of Melipona quadrifasciata affected by a recurrent disease.

Melipona quadrifasciata is an eusocial stingless bee traditionally used for honey production in Brazil. In the last decades, the species disappeared from the wild in Southern Brazil, being kept exclusively in managed colonies for commercial and recreational purposes. Stingless beekeepers from this region report annual losses of their colonies due to a syndrome of yet unknown causes. We investigate whether it is associated to pathogenic microorganisms already known to cause disease in bees. These results provide a starting point for future studies aimed at clarifying the relationship between the microbial community of stingless bees and their colony collapses.

Genetic diversity and phylogeography of highly zoonotic Echinococcus granulosus genotype G1 in the Americas (Argentina, Brazil, Chile and Mexico) based on 8279bp of mtDNA.

Echinococcus granulosus is a taeniid cestode and the etiological agent of an infectious zoonotic disease known as cystic echinococcosis (CE) or hydatid disease. CE is a serious public health concern in many parts of the world, including the Americas, where it is highly endemic in many regions. Echinococcus granulosus displays high intraspecific genetic variability and is divided into multiple genotypes (G1-G8, G10) with differences in their biology and etiology. Of these, genotype G1 is responsible for the majority of human and livestock infections and has the broadest host spectrum. However, despite the high significance to the public and livestock health, the data on genetic variability and regional genetic differences of genotype G1 in America are scarce. The aim of this study was to evaluate the genetic variability and phylogeography of G1 in several countries in America by sequencing a large portion of the mitochondrial genome. We analysed 8279bp of mtDNA for 52 E. granulosus G1 samples from sheep, cattle and pigs collected in Argentina, Brazil, Chile and Mexico, covering majority of countries in the Americas where G1 has been reported. The phylogenetic network revealed 29 haplotypes and a high haplotype diversity (Hd=0.903). The absence of phylogeographic segregation between different regions in America suggests the importance of animal transportation in shaping the genetic structure of E. granulosus G1. In addition, our study revealed many highly divergent haplotypes, indicating a long and complex evolutionary history of E. granulosus G1 in the Americas.

Echinococcus granulosus sensu lato and Taenia hydatigena in pig in southern Brazil.

This study aimed to identify the parasitical etiologic agents of visceral cysts in pigs from the central/northern region of Rio Grande do Sul State, Brazil. Fifty-eight cysts were found in livers during veterinary inspection of swine slaughtered from January 2008 to 2012. Collected samples were submitted to macroscopic and molecular analyzes. Polymerase chain reaction (PCR), DNA sequencing and BLAST alignment of sequences was used to molecular characterization of the samples. By PCR 10.3% (6/58) of tested samples were positive for Echinococcus granulosus sensu lato and 56.9% (33/58) for Cysticercus tenuicollis. In this study, it was verified the occurrence of larval forms of E. granulosus sensu lato and Taenia hydatigena in pig herds from the central/northern region of Rio Grande do Sul State. The presence of both parasites is relevant due to the economic losses for the meat industry. Additionally, E. granulosus sensu lato has zoonotic importance and may be infecting pig herds in southern Brazil.

Echinococcus granulosus sensu lato and Taenia hydatigena in pig in southern Brazil / Echinococcus granulosus sensu lato e Taenia hydatigena em suínos no sul do Brasil

This study aimed to identify the parasitical etiologic agents of visceral cysts in pigs from the central/northern region of Rio Grande do Sul State, Brazil. Fifty-eight cysts were found in livers during veterinary inspection of swine slaughtered from January 2008 to 2012. Collected samples were submitted to macroscopic and molecular analyzes. Polymerase chain reaction (PCR), DNA sequencing and BLAST alignment of sequences was used to molecular characterization of the samples. By PCR 10.3% (6/58) of tested samples were positive for Echinococcus granulosus sensu lato and 56.9% (33/58) for Cysticercus tenuicollis. In this study, it was verified the occurrence of larval forms of E. granulosus sensu lato and Taenia hydatigena in pig herds from the central/northern region of Rio Grande do Sul State. The presence of both parasites is relevant due to the economic losses for the meat industry. Additionally, E. granulosus sensu lato has zoonotic importance and may be infecting pig herds in southern Brazil.

Neste estudo, buscou-se identificar os agentes etiológicos de origem parasitária em cistos nas vísceras de suínos oriundos sa região centro/norte do sEstado do Rio Grande do Sul, Brasil. Cinquenta e oito cistos foram encontrados durante a inspeção veterinária em fígados de suínos abatidos entre janeiro de 2008 a 2012. As amostras foram submetidas às análises macroscópicas e moleculares. Reação em cadeia da polimerase (PCR), sequenciamento de DNA e alinhamento das sequências no BLAST (bases de dados do GenBank) foram utilizados na caracterização molecular das amostras. Das amostras analisadas, 10,3% (6/58) foram positivas para Echinococcus granulosus sensu lato e 56,9% (33/58) para Cysticercus tenuicollis. Evidenciou-se a ocorrência de larvas de E. granulosus sensu lato e Taenia hydatigena em rebanhos suínos na região centro/norte do RS. A presença de ambos os agentes é relevante devido às perdas econômicas para a indústria de carnes. Além disso, destaca-se que E. granulosus sensu lato possui importância zoonótica e pode contaminar os rebanhos suínos no sul do Brasil.

The Ordospora colligata genome: Evolution of extreme reduction in microsporidia and host-to-parasite horizontal gene transfer.

UNLABELLED: Microsporidia are a group of obligate intracellular parasites that are best known for their unique infection mechanism and their unparalleled levels of genomic reduction and compaction. We sequenced the genome of Ordospora colligata, a gut parasite of the microcrustacean Daphnia sp. and the closest known relative to the microsporidia characterized by the most extreme genomic reduction, the model genus Encephalitozoon. We found that the O. colligata genome is as compact as those of Encephalitozoon spp., featuring few introns and a similar complement of about 2,000 genes, altogether showing that the extreme reduction took place before the origin of Encephalitozoon spp. and their adaptation to vertebrate hosts. We also found that the O. colligata genome has acquired by horizontal transfer from its animal host a septin that is structurally analogous to septin 7, a protein that plays a major role in the endocytosis-based invasion mechanism of the fungal pathogen Candida albicans. Microsporidian invasion is most often characterized by injection through a projectile tube, but microsporidia are also known to invade cells by inducing endocytosis. Given the function of septins in other systems, we hypothesize that the acquired septin could help O. colligata induce its uptake by mimicking host receptors. IMPORTANCE: The smallest known eukaryotic genomes are found in members of the Encephalitozoon genus of microsporidian parasites. Their extreme compaction, however, is not characteristic of the group, whose genomes can differ by an order of magnitude. The processes and evolutionary forces that led the Encephalitozoon genomes to shed so much of their ancestral baggage are unclear. We sequenced the genome of Ordospora colligata, a parasite of the water flea Daphnia sp. and the closest known relative of Encephalitozoon species, and show that this extreme reduction predated the split between the two lineages. We also found that O. colligata has acquired a septin gene by host-to-parasite horizontal transfer and predicted that the encoded protein folds like a septin 7, which plays a major role in endocytosis. We hypothesize that this acquisition could help O. colligata parasitize its hosts by facilitating endocytic infection, a mechanism that occurs in microsporidia but that is not yet well understood.

Cytological and molecular description of Hamiltosporidium tvaerminnensis gen. et sp. nov., a microsporidian parasite of Daphnia magna, and establishment of Hamiltosporidium magnivora comb. nov.

We describe the new microsporidium Hamiltosporidium tvaerminnensis gen. et sp. nov. with an emphasis on its ultrastructural characteristics and phylogenetic position as inferred from the sequence data of SSU rDNA, alpha- and beta-tubulin. This parasite was previously identified as Octosporea bayeri Jírovec, 1936 and has become a model system to study the ecology, epidemiology, evolution and genomics of microsporidia - host interactions. Here, we present evidence that shows its differences from O. bayeri. Hamiltosporidium tvaerminnensis exclusively infects the adipose tissue, the ovaries and the hypodermis of Daphnia magna and is found only in host populations located in coastal rock pool populations in Finland and Sweden. Merogonial stages of H. tvaerminnensis have isolated nuclei; merozoites are formed by binary fission or by the cleaving of a plasmodium with a small number of nuclei. A sporogonial plasmodium with isolated nuclei yields 8 sporoblasts. Elongated spores are generated by the most finger-like plasmodia. The mature spores are polymorphic in shape and size. Most spores are pyriform (4·9-5·6×2·2-2·3 µm) and have their polar filament arranged in 12-13 coils. A second, elongated spore type (6·8-12·0×1·6-2·1 µm) is rod-shaped with blunt ends and measures 6·8-12·0×1·6-2·1 µm. The envelope of the sporophorous vesicle is thin and fragile, formed at the beginning of the sporogony. Cytological and molecular comparisons with Flabelliforma magnivora, a parasite infecting the same tissues in the same host species, reveal that these two species are very closely related, yet distinct. Moreover, both cytological and molecular data indicate that these species are quite distant from F. montana, the type species of the genus Flabelliforma. We therefore propose that F. magnivora also be placed in Hamiltosporidium gen. nov.

The EG95 antigen of Echinococcus spp. contains positively selected amino acids, which may influence host specificity and vaccine efficacy.

Echinococcosis is a worldwide zoonotic parasitic disease of humans and various herbivorous domestic animals (intermediate hosts) transmitted by the contact with wild and domestic carnivores (definitive hosts), mainly foxes and dogs. Recently, a vaccine was developed showing high levels of protection against one parasite haplotype (G1) of Echinococcus granulosus, and its potential efficacy against distinct parasite variants or species is still unclear. Interestingly, the EG95 vaccine antigen is a secreted glycosylphosphatydilinositol (GPI)-anchored protein containing a fibronectin type III domain, which is ubiquitous in modular proteins involved in cell adhesion. EG95 is highly expressed in oncospheres, the parasite life cycle stage which actively invades the intermediate hosts. After amplifying and sequencing the complete CDS of 57 Echinococcus isolates belonging to 7 distinct species, we uncovered a large amount of genetic variability, which may influence protein folding. Two positively selected sites are outside the vaccine epitopes, but are predicted to alter protein conformation. Moreover, phylogenetic analyses indicate that EG95 isoform evolution is convergent with regard to the number of beta-sheets and alpha-helices. We conclude that having a variety of EG95 isoforms is adaptive for Echinococcus parasites, in terms of their ability to invade different hosts, and we propose that a mixture of isoforms could possibly maximize vaccine efficacy.