The Allium Derivate Propyl Propane Thiosulfinate Exerts Anti-Obesogenic Effects in a Murine Model of Diet-Induced Obesity.

Allium species and their organosulfur-derived compounds could prevent obesity and metabolic dysfunction, as they exhibit immunomodulatory and antimicrobial properties. Here, we report the anti-obesogenic potential and dose-dependent effects (0.1 or 1 mg/kg/day) of propyl propane thiosulfinate (PTS) in a murine model of diet-induced obesity. The obesogenic diet increased body weight gain and adipocyte size, and boosted inflammatory marker (Cd11c) expression in the adipose tissue. Conversely, PTS prevented these effects in a dose-dependent manner. Moreover, the higher dose of PTS improved glucose and hepatic homeostasis, modulated lipid metabolism, and raised markers of the thermogenic capacity of brown adipose tissue. In the colon, the obesogenic diet reduced IL-22 levels and increased gut barrier function markers (Cldn3, Muc2, Reg3g, DefaA); however, the highest PTS dose normalized all of these markers to the levels of mice fed a standard diet. Gut microbiota analyses revealed no differences in diversity indexes and only minor taxonomic changes, such as an increase in butyrate producers, Intestimonas and Alistipes, and a decrease in Bifidobacterium in mice receiving the highest PTS dose. In summary, our study provides preclinical evidence for the protective effects of PTS against obesity, which if confirmed in humans, might provide a novel plant-based dietary product to counteract this condition.

Ontogeny, species identity, and environment dominate microbiome dynamics in wild populations of kissing bugs (Triatominae).

BACKGROUND: Kissing bugs (Triatominae) are blood-feeding insects best known as the vectors of Trypanosoma cruzi, the causative agent of Chagas' disease. Considering the high epidemiological relevance of these vectors, their biology and bacterial symbiosis remains surprisingly understudied. While previous investigations revealed generally low individual complexity but high among-individual variability of the triatomine microbiomes, any consistent microbiome determinants have not yet been identified across multiple Triatominae species. METHODS: To obtain a more comprehensive view of triatomine microbiomes, we investigated the host-microbiome relationship of five Triatoma species sampled from white-throated woodrat (Neotoma albigula) nests in multiple locations across the USA. We applied optimised 16S rRNA gene metabarcoding with a novel 18S rRNA gene blocking primer to a set of 170 T. cruzi-negative individuals across all six instars. RESULTS: Triatomine gut microbiome composition is strongly influenced by three principal factors: ontogeny, species identity, and the environment. The microbiomes are characterised by significant loss in bacterial diversity throughout ontogenetic development. First instars possess the highest bacterial diversity while adult microbiomes are routinely dominated by a single taxon. Primarily, the bacterial genus Dietzia dominates late-stage nymphs and adults of T. rubida, T. protracta, and T. lecticularia but is not present in the phylogenetically more distant T. gerstaeckeri and T. sanguisuga. Species-specific microbiome composition, particularly pronounced in early instars, is further modulated by locality-specific effects. In addition, pathogenic bacteria of the genus Bartonella, acquired from the vertebrate hosts, are an abundant component of Triatoma microbiomes. CONCLUSION: Our study is the first to demonstrate deterministic patterns in microbiome composition among all life stages and multiple Triatoma species. We hypothesise that triatomine microbiome assemblages are produced by species- and life stage-dependent uptake of environmental bacteria and multiple indirect transmission strategies that promote bacterial transfer between individuals. Altogether, our study highlights the complexity of Triatominae symbiosis with bacteria and warrant further investigation to understand microbiome function in these important vectors. Video abstract.

Methodological Insight Into Mosquito Microbiome Studies.

Symbiotic bacteria affect competence for pathogen transmission in insect vectors, including mosquitoes. However, knowledge on mosquito-microbiome-pathogen interactions remains limited, largely due to methodological reasons. The current, cost-effective practice of sample pooling used in mosquito surveillance and epidemiology prevents correlation of individual traits (i.e., microbiome profile) and infection status. Moreover, many mosquito studies employ laboratory-reared colonies that do not necessarily reflect the natural microbiome composition and variation in wild populations. As a consequence, epidemiological and microbiome studies in mosquitoes are to some extent uncoupled, and the interactions among pathogens, microbiomes, and natural mosquito populations remain poorly understood. This study focuses on the effect the pooling practice poses on mosquito microbiome profiles, and tests different approaches to find an optimized low-cost methodology for extensive sampling while allowing for accurate, individual-level microbiome studies. We tested the effect of pooling by comparing wild-caught, individually processed mosquitoes with pooled samples. With individual mosquitoes, we also tested two methodological aspects that directly affect the cost and feasibility of broad-scale molecular studies: sample preservation and tissue dissection. Pooling affected both alpha- and beta-diversity measures of the microbiome, highlighting the importance of using individual samples when possible. Both RNA and DNA yields were higher when using inexpensive reagents such as NAP (nucleic acid preservation) buffer or absolute ethanol, without freezing for short-term storage. Microbiome alpha- and beta-diversity did not show overall significant differences between the tested treatments compared to the controls (freshly extracted samples or dissected guts). However, the use of standardized protocols is highly recommended to avoid methodological bias in the data.

Seasonal and Sexual Differences in the Microbiota of the Hoopoe Uropygial Secretion.

The uropygial gland of hoopoe nestlings and nesting females hosts bacterial symbionts that cause changes in the characteristics of its secretion, including an increase of its antimicrobial activity. These changes occur only in nesting individuals during the breeding season, possibly associated with the high infection risk experienced during the stay in the hole-nests. However, the knowledge on hoopoes uropygial gland microbial community dynamics is quite limited and based so far on culture-dependent and molecular fingerprinting studies. In this work, we sampled wild and captive hoopoes of different sex, age, and reproductive status, and studied their microbiota using quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH) and pyrosequencing. Surprisingly, we found a complex bacterial community in all individuals (including non-nesting ones) during the breeding season. Nevertheless, dark secretions from nesting hoopoes harbored significantly higher bacterial density than white secretions from breeding males and both sexes in winter. We hypothesize that bacterial proliferation may be host-regulated in phases of high infection risk (i.e., nesting). We also highlight the importance of specific antimicrobial-producing bacteria present only in dark secretions that may be key in this defensive symbiosis. Finally, we discuss the possible role of environmental conditions in shaping the uropygial microbiota, based on differences found between wild and captive hoopoes.

Microbiomes of North American Triatominae: The Grounds for Chagas Disease Epidemiology.

Insect microbiomes influence many fundamental host traits, including functions of practical significance such as their capacity as vectors to transmit parasites and pathogens. The knowledge on the diversity and development of the gut microbiomes in various blood feeding insects is thus crucial not only for theoretical purposes, but also for the development of better disease control strategies. In Triatominae (Heteroptera: Reduviidae), the blood feeding vectors of Chagas disease in South America and parts of North America, the investigation of the microbiomes is in its infancy. The few studies done on microbiomes of South American Triatominae species indicate a relatively low taxonomic diversity and a high host specificity. We designed a comparative survey to serve several purposes: (I) to obtain a better insight into the overall microbiome diversity in different species, (II) to check the long term stability of the interspecific differences, (III) to describe the ontogenetic changes of the microbiome, and (IV) to determine the potential correlation between microbiome composition and presence of Trypanosoma cruzi, the causative agent of Chagas disease. Using 16S amplicons of two abundant species from the southern US, and four laboratory reared colonies, we showed that the microbiome composition is determined by host species, rather than locality or environment. The OTUs (Operational Taxonomic Units) determination confirms a low microbiome diversity, with 12-17 main OTUs detected in wild populations of T. sanguisuga and T. protracta. Among the dominant bacterial taxa are Acinetobacter and Proteiniphilum but also the symbiotic bacterium Arsenophonus triatominarum, previously believed to only live intracellularly. The possibility of ontogenetic microbiome changes was evaluated in all six developmental stages and feces of the laboratory reared model Rhodnius prolixus. We detected considerable changes along the host's ontogeny, including clear trends in the abundance variation of the three dominant bacteria, namely Enterococcus, Acinetobacter, and Arsenophonus. Finally, we screened the samples for the presence of Trypanosoma cruzi. Comparing the parasite presence with the microbiome composition, we assessed the possible significance of the latter in the epidemiology of the disease. Particularly, we found a trend toward more diverse microbiomes in Trypanosoma cruzi positive T. protracta specimens.

Mosquito Microbiome Dynamics, a Background for Prevalence and Seasonality of West Nile Virus.

Symbiotic microbial communities augment host phenotype, including defense against pathogen carriage and infection. We sampled the microbial communities in 11 adult mosquito host species from six regions in southern Ontario, Canada over 3 years. Of the factors examined, we found that mosquito species was the largest driver of the microbiota, with remarkable phylosymbiosis between host and microbiota. Seasonal shifts of the microbiome were consistently repeated over the 3-year period, while region had little impact. Both host species and seasonal shifts in microbiota were associated with patterns of West Nile virus (WNV) in these mosquitoes. The highest prevalence of WNV, with a seasonal spike each year in August, was in the Culex pipiens/restuans complex, and high WNV prevalence followed a decrease in relative abundance of Wolbachia in this species. Indeed, mean temperature, but not precipitation, was significantly correlated with Wolbachia abundance. This suggests that at higher temperatures Wolbachia abundance is reduced leading to greater susceptibility to WNV in the subsequent generation of C. pipiens/restuans hosts. Different mosquito genera harbored significantly different bacterial communities, and presence or abundance of Wolbachia was primarily associated with these differences. We identified several operational taxonomic units (OTUs) of Wolbachia that drive overall microbial community differentiation among mosquito taxa, locations and timepoints. Distinct Wolbachia OTUs were consistently found to dominate microbiomes of Cx. pipiens/restuans, and of Coquilletidia perturbans. Seasonal fluctuations of several other microbial taxa included Bacillus cereus, Enterococcus, Methylobacterium, Asaia, Pantoea, Acinetobacter johnsonii, Pseudomonas, and Mycoplasma. This suggests that microbiota may explain some of the variation in vector competence previously attributed to local environmental processes, especially because Wolbachia is known to affect carriage of viral pathogens.

Nest Bacterial Environment Affects Microbiome of Hoopoe Eggshells, but Not That of the Uropygial Secretion.

The study of associations between symbiotic bacterial communities of hosts and those of surrounding environments would help to understand how bacterial assemblages are acquired, and how they are transmitted from one to another location (i.e. symbiotic bacteria acquisition by hosts). Hoopoes (Upupa epops) smear their eggshells with uropygial secretion (oily secretion produced in their uropygial gland) that harbors antibiotic producing bacteria. Trying to elucidate a possible role of nest material and cloaca microbiota in determining the bacterial community of the uropygial gland and the eggshells of hoopoes, we characterized bacterial communities of nest material, cloaca, uropygial gland and eggshells by the ARISA fingerprinting. Further, by adding material with scarce bacteria and antimicrobial properties, we manipulated the bacterial community of nest material and thus tested experimentally its effects on the microbiomes of the uropygial secretion and of the eggshells. The experiment did not influence the microbiome of the uropygial secretion of females, but affected the community established on eggshells. This is the first experimental evidence indicating that nest material influences the bacterial community of the eggshells and, therefore, probability of embryo infection. Some of the bacterial strains detected in the secretion were also in the bacterial communities of the nest material and of cloaca, but their occurrence within nests was not associated, which suggests that bacterial environments of nest material and cloaca are not sources of symbiotic bacteria for the gland. These results do not support a role of nest environments of hoopoes as reservoirs of symbiotic bacteria. We discuss possible scenarios explaining bacterial acquisition by hoopoes that should be further explored.

The Microbiome of the Uropygial Secretion in Hoopoes Is Shaped Along the Nesting Phase.

Microbial symbiont acquisition by hosts may determine the effectiveness of the mutualistic relationships. A mix of vertical and horizontal transmission may be advantageous for hosts by allowing plastic changes of microbial communities depending on environmental conditions. Plasticity is well known for gut microbiota but is poorly understood for other symbionts of wild animals. We here explore the importance of environmental conditions experienced by nestling hoopoes (Upupa epops) during the late nesting phase determining microbiota in their uropygial gland. In cross-fostering experiments of 8 days old nestlings, "sibling-sibling" and "mother-offspring" comparisons were used to explore whether the bacterial community naturally established in the uropygial gland of nestlings could change depending on experimental environmental conditions (i.e., new nest environment). We found that the final microbiome of nestlings was mainly explained by nest of origin. Moreover, cross-fostered nestlings were more similar to their siblings and mothers than to their stepsiblings and stepmothers. We also detected a significant effect of nest of rearing, suggesting that nestling hoopoes acquire most bacterial symbionts during the first days of life but that the microbiome is dynamic and can be modified along the nestling period depending on environmental conditions. Estimated effects of nest of rearing, but also most of those of nest of origin are associated to environmental characteristics of nests, which are extended phenotypes of parents. Thus, natural selection may favor the acquisition of appropriated microbial symbionts for particular environmental conditions found in nests.

The Hoopoe's Uropygial Gland Hosts a Bacterial Community Influenced by the Living Conditions of the Bird.

Molecular methods have revealed that symbiotic systems involving bacteria are mostly based on whole bacterial communities. Bacterial diversity in hoopoe uropygial gland secretion is known to be mainly composed of certain strains of enterococci, but this conclusion is based solely on culture-dependent techniques. This study, by using culture-independent techniques (based on the 16S rDNA and the ribosomal intergenic spacer region) shows that the bacterial community in the uropygial gland secretion is more complex than previously thought and its composition is affected by the living conditions of the bird. Besides the known enterococci, the uropygial gland hosts other facultative anaerobic species and several obligated anaerobic species (mostly clostridia). The bacterial assemblage of this community was largely invariable among study individuals, although differences were detected between captive and wild female hoopoes, with some strains showing significantly higher prevalence in wild birds. These results alter previous views on the hoopoe-bacteria symbiosis and open a new window to further explore this system, delving into the possible sources of symbiotic bacteria (e.g. nest environments, digestive tract, winter quarters) or the possible functions of different bacterial groups in different contexts of parasitism or predation of their hoopoe host.

Preening as a Vehicle for Key Bacteria in Hoopoes.

Oily secretions produced in the uropygial gland of incubating female hoopoes contain antimicrobial-producing bacteria that prevent feathers from degradation and eggs from pathogenic infection. Using the beak, females collect the uropygial gland secretion and smear it directly on the eggshells and brood patch. Thus, some bacterial strains detected in the secretion should also be present on the eggshell, beak, and brood patch. To characterize these bacterial communities, we used Automatic Ribosomal Intergenic Spacer Analysis (ARISA), which distinguishes between taxonomically different bacterial strains (i.e. different operational taxonomic units [OTUs]) by the size of the sequence amplified. We identified a total of 146 different OTUs with sizes between 139 and 999 bp. Of these OTUs, 124 were detected in the uropygial oil, 106 on the beak surface, 97 on the brood patch, and 98 on the eggshell. The highest richness of OTUs appeared in the uropygial oil samples. Moreover, the detection of some OTUs on the beak, brood patch, and eggshells of particular nests depended on these OTUs being present in the uropygial oil of the female. These results agree with the hypothesis that symbiotic bacteria are transmitted from the uropygial gland to beak, brood patch, and eggshell surfaces, opening the possibility that the bacterial community of the secretion plays a central role in determining the communities of special hoopoe eggshell structures (i.e., crypts) that, soon after hatching, are filled with uropygial oil, thereby protecting embryos from pathogens.