Heritable beneficial bacterial endosymbionts have been crucial for the evolutionary success of numerous insects by enabling the exploitation of nutritionally limited food sources. Herein, we describe a previously unknown dual endosymbiosis in the psyllid genus Cacopsylla, consisting of the primary endosymbiont "Candidatus Carsonella ruddii" and a co-occurring Enterobacteriaceae bacterium for which we propose the name "Candidatus Psyllophila symbiotica." Its localization within the bacteriome and its small genome size confirm that Psyllophila is a co-primary endosymbiont widespread within the genus Cacopsylla. Despite its highly eroded genome, Psyllophila perfectly complements the tryptophan biosynthesis pathway that is incomplete in the co-occurring Carsonella. Moreover, the genome of Psyllophila is almost as small as Carsonella's, suggesting an ancient dual endosymbiosis that has now reached a precarious stage where any additional gene loss would make the system collapse. Hence, our results shed light on the dynamic interactions of psyllids and their endosymbionts over evolutionary time.

• Keywords
• comparative genomics, endosymbionts, psyllids, symbiosis,
• MeSH
• Bacteria MeSH
• Enterobacteriaceae genetics   MeSH
• Phylogeny MeSH
• Hemiptera * genetics   MeSH
• Symbiosis genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

Insects that live exclusively on vertebrate blood utilize symbiotic bacteria as a source of essential compounds, e.g., B vitamins. In louse flies, the most frequent symbiont originated in genus Arsenophonus, known from a wide range of insects. Here, we analyze genomic traits, phylogenetic origins, and metabolic capacities of 11 Arsenophonus strains associated with louse flies. We show that in louse flies, Arsenophonus established symbiosis in at least four independent events, reaching different stages of symbiogenesis. This allowed for comparative genomic analysis, including convergence of metabolic capacities. The significance of the results is twofold. First, based on a comparison of independently originated Arsenophonus symbioses, it determines the importance of individual B vitamins for the insect host. This expands our theoretical insight into insect-bacteria symbiosis. The second outcome is of methodological significance. We show that the comparative approach reveals artifacts that would be difficult to identify based on a single-genome analysis.

• Keywords
• bacterial symbiosis, coevolution, genome evolution, hematophagy,
• MeSH
• Anoplura * MeSH
• Bacteria MeSH
• Diptera * microbiology   MeSH
• Enterobacteriaceae MeSH
• Phylogeny MeSH
• Gammaproteobacteria * genetics   MeSH
• Insecta MeSH
• Symbiosis MeSH
• Vitamin B Complex * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Vitamin B Complex * MeSH

BACKGROUND: The contribution of the microbiome to pesticide breakdown in agricultural pests remains unclear. We analyzed the effect of pirimiphos-methyl (PM) on four geographically different cultures of the stored product pest mite Acarus siro (6 L, 6Tu, 6Tk and 6Z) under laboratory experiments. The effect of PM on mite mortality in the impregnated filter paper test was compared. RESULTS: The mite sensitivity to PM decreased in the order of 6 L, 6Tu, 6Tk, and 6Z. Then, the mites were cultured on PM residues (0.0125 and 1.25 µg·g-1), and population growth was compared to the control after 21 days of exposure. The comparison showed two situations: (i) increasing population growth for the most sensitive cultures (6 L and 6Tu), and (ii) no effect on mite population growth for tolerant cultures (6Z and 6Tk). The microbiome of mites was analyzed by quantification of 16S DNA copies based on quantitative polymerase chain reaction (qPCR) and by barcode sequencing of the V4 fragment of 16S DNA on samples of 30 individuals from the control and PM residues. The microbiome comprised primarily Solitalea-like organisms in all cultures, except for 6Z, followed by Bacillus, Staphylococcus, and Lactobacillus. The microbiomes of mite cultures did not change with increasing population density. The microbiome of cultures without any differences in population density showed differences in the microbiome composition. A Sodalis-like symbiont replaced Solitalea in the 1.25 µg·g-1 PM in the 6Tk culture. Sodalis and Bacillus prevailed in the microbiomes of PM-treated mites of 6Z culture, while Solitalea was almost absent. CONCLUSION: The results showed that the microbiome of A. siro differs in composition and in response to PM residues in the diet. The results indicate that Sodalis-like symbionts can help recover mites from pesticide-induced stress.

• Keywords
• Pesticide; Storage; Interaction; Tolerance; Symbionts,
• MeSH
• Acaridae * MeSH
• Bacteroidetes MeSH
• Humans MeSH
• Microbiota * MeSH
• Pesticide Residues * MeSH
• Mites * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• pirimiphos methyl MeSH Browser
• Pesticide Residues * MeSH

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.

• Keywords
• Bacteria, Blood, Hematophagous, Insect, Microbiome, Ontogeny, Pathogen, Triatominae, Vector,
• MeSH
• Chagas Disease parasitology   MeSH
• Animals, Wild classification   microbiology   MeSH
• Microbiota genetics   physiology   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Triatominae classification   microbiology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Video-Audio Media MeSH
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Ribosomal, 16S MeSH

Animals interact with a diverse array of both beneficial and detrimental microorganisms. In insects, these symbioses in many cases allow feeding on nutritionally unbalanced diets. It is, however, still not clear how are obligate symbioses maintained at the cellular level for up to several hundred million years. Exact mechanisms driving host-symbiont interactions are only understood for a handful of model species and data on blood-feeding hosts with intracellular bacteria are particularly scarce. Here, we analyzed interactions between an obligately blood-sucking parasite of sheep, the louse fly Melophagus ovinus, and its obligate endosymbiont, Arsenophonus melophagi. We assembled a reference transcriptome for the insect host and used dual RNA-Seq with five biological replicates to compare expression in the midgut cells specialized for housing symbiotic bacteria (bacteriocytes) to the rest of the gut (foregut-hindgut). We found strong evidence for the importance of zinc in the system likely caused by symbionts using zinc-dependent proteases when acquiring amino acids, and for different immunity mechanisms controlling the symbionts than in closely related tsetse flies. Our results show that cellular and nutritional interactions between this blood-sucking insect and its symbionts are less intimate than what was previously found in most plant-sap sucking insects. This finding is likely interconnected to several features observed in symbionts in blood-sucking arthropods, particularly their midgut intracellular localization, intracytoplasmic presence, less severe genome reduction, and relatively recent associations caused by frequent evolutionary losses and replacements.

• Keywords
• B-vitamins, RNA-Seq, immunity, interactions, parasites, symbiotic bacteria, zinc,
• MeSH
• Bacteria classification   genetics   isolation & purification   MeSH
• Biological Evolution MeSH
• Diptera genetics   microbiology   MeSH
• DNA, Bacterial analysis   genetics   MeSH
• Phylogeny MeSH
• Genes, Insect * MeSH
• Disease Vectors MeSH
• Host-Pathogen Interactions MeSH
• Sheep parasitology   MeSH
• Gastrointestinal Microbiome * MeSH
• Symbiosis MeSH
• Transcriptome MeSH
• Digestive System microbiology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Bacterial MeSH

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.

• Keywords
• Rhodnius prolixus, Triatominae, Trypanosoma cruzi, microbiome, ontogeny,
• Publication type
• Journal Article MeSH

Symbiotic interactions between insects and bacteria are ubiquitous and form a continuum from loose facultative symbiosis to greatly intimate and stable obligate symbiosis. In blood-sucking insects living exclusively on vertebrate blood, obligate endosymbionts are essential for hosts and hypothesized to supplement B-vitamins and cofactors missing from their blood diet. The role and distribution of facultative endosymbionts and their evolutionary significance as seeds of obligate symbioses are much less understood. Here, using phylogenetic approaches, we focus on the Hippoboscidae phylogeny as well as the stability and dynamics of obligate symbioses within this bloodsucking group. In particular, we demonstrate a new potentially obligate lineage of Sodalis co-evolving with the Olfersini subclade of Hippoboscidae. We also show several likely facultative Sodalis lineages closely related to Sodalis praecaptivus (HS strain) and suggest repeated acquisition of novel symbionts from the environment. Similar to Sodalis, Arsenophonus endosymbionts also form both obligate endosymbiotic lineages co-evolving with their hosts (Ornithomyini and Ornithoica groups) as well as possibly facultative infections incongruent with the Hippoboscidae phylogeny. Finally, we reveal substantial diversity of Wolbachia strains detected in Hippoboscidae samples falling into three supergroups: A, B, and the most common F. Altogether, our results prove the associations between Hippoboscoidea and their symbiotic bacteria to undergo surprisingly dynamic, yet selective, evolutionary processes strongly shaped by repeated endosymbiont replacements. Interestingly, obligate symbionts only originate from two endosymbiont genera, Arsenophonus and Sodalis, suggesting that the host is either highly selective about its future obligate symbionts or that these two lineages are the most competitive when establishing symbioses in louse flies.

• Keywords
• Arsenophonus, Louse flies, Phylogeny, Replacements, Sodalis, Wolbachia,
• Publication type
• Journal Article MeSH

Candidatus Arsenophonus lipopteni (Enterobacteriaceae, Gammaproteobacteria) is an obligate intracellular symbiont of the blood feeding deer ked, Lipoptena cervi (Diptera: Hippoboscidae). The bacteria reside in specialized cells derived from host gut epithelia (bacteriocytes) forming a compact symbiotic organ (bacteriome). Compared to the closely related complex symbiotic system in the sheep ked, involving four bacterial species, Lipoptena cervi appears to maintain its symbiosis exclusively with Ca. Arsenophonus lipopteni. The genome of 836,724 bp and 24.8 % GC content codes for 667 predicted functional genes and bears the common characteristics of sequence economization coupled with obligate host-dependent lifestyle, e.g. reduced number of RNA genes along with the rRNA operon split, and strongly reduced metabolic capacity. Particularly, biosynthetic capacity for B vitamins possibly supplementing the host diet is highly compromised in Ca. Arsenophonus lipopteni. The gene sets are complete only for riboflavin (B2), pyridoxine (B6) and biotin (B7) implying the content of some B vitamins, e.g. thiamin, in the deer blood might be sufficient for the insect metabolic needs. The phylogenetic position within the spectrum of known Arsenophonus genomes and fundamental genomic features of Ca. Arsenophonus lipopteni indicate the obligate character of this symbiosis and its independent origin within Hippoboscidae.

• Keywords
• Arsenophonus, Hippoboscidae, Symbiosis, Tsetse,
• Publication type
• Journal Article MeSH
• Case Reports MeSH