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Transovarial transmission is the most reliable way of passing on essential nutrient-providing endosymbionts from mothers to offspring. However, not all endosymbiotic microbes follow the complex path through the female host tissues to oocytes on their own. Here, we demonstrate an unusual transmission strategy adopted by one of the endosymbionts of the planthopper Trypetimorpha occidentalis (Hemiptera: Tropiduchidae) from Bulgaria. In this species, an Acetobacteraceae endosymbiont is transmitted transovarially within deep invaginations of cellular membranes of an ancient endosymbiont Sulcia-strikingly resembling recently described plant virus transmission. However, in males, Acetobacteraceae colonizes the same bacteriocytes as Sulcia but remains unenveloped. Then, the unusual endobacterial localization of Acetobacteraceae observed in females appears to be a unique adaptation to maternal transmission. Further, the symbiont's genomic features, including encoding essential amino acid biosynthetic pathways and its similarity to a recently described psyllid symbiont, suggest a unique combination of the ability to horizontally transmit among species and confer nutritional benefits. The close association with Acetobacteraceae symbiont correlates with the so-far-unreported level of genomic erosion of ancient nutritional symbionts of this planthopper. In Sulcia, this is reflected in substantial changes in genomic organization, reported for the first time in the symbiont renowned for its genomic stability. In Vidania, substantial gene loss resulted in one of the smallest genomes known, at 108.6 kb. Thus, the symbionts of T. occidentalis display a combination of unusual adaptations and genomic features that expand our understanding of how insect-microbe symbioses may transmit and evolve.IMPORTANCEReliable transmission across host generations is a major challenge for bacteria that associate with insects, and independently established symbionts have addressed this challenge in different ways. The facultatively endobacterial localization of Acetobacteraceae symbiont, enveloped by cells of ancient nutritional endosymbiont Sulcia in females but not males of the planthopper Trypetimorpha occidentalis, appears to be a unique adaptation to maternal transmission. Acetobacteraceae's genomic features indicate its unusual evolutionary history, and the genomic erosion experienced by ancient nutritional symbionts demonstrates the apparent consequences of such close association. Combined, this multi-partite symbiosis expands our understanding of the diversity of strategies that insect symbioses form and some of their evolutionary consequences.
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Hemípteros , Simbiosis , Animales , Hemípteros/microbiología , Hemípteros/fisiología , Femenino , Masculino , Acetobacteraceae/genética , Acetobacteraceae/fisiología , Genoma Bacteriano/genética , Filogenia , Adaptación FisiológicaRESUMEN
The more insects there are, the more food there is for insectivores and the higher the likelihood for insect-associated ecosystem services. Yet, we lack insights into the drivers of insect biomass over space and seasons, for both tropical and temperate zones. We used 245 Malaise traps, managed by 191 volunteers and park guards, to characterize year-round flying insect biomass in a temperate (Sweden) and a tropical (Madagascar) country. Surprisingly, we found that local insect biomass was similar across zones. In Sweden, local insect biomass increased with accumulated heat and varied across habitats, while biomass in Madagascar was unrelated to the environmental predictors measured. Drivers behind seasonality partly converged: In both countries, the seasonality of insect biomass differed between warmer and colder sites, and wetter and drier sites. In Sweden, short-term deviations from expected season-specific biomass were explained by week-to-week fluctuations in accumulated heat, rainfall and soil moisture, whereas in Madagascar, weeks with higher soil moisture had higher insect biomass. Overall, our study identifies key drivers of the seasonal distribution of flying insect biomass in a temperate and a tropical climate. This knowledge is key to understanding the spatial and seasonal availability of insects-as well as predicting future scenarios of insect biomass change.
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Biomasa , Estaciones del Año , Temperatura , Clima Tropical , Animales , Suecia , Madagascar , Insectos/fisiología , Agua , EcosistemaRESUMEN
Microbial symbionts play crucial roles in insect biology, yet their diversity, distribution, and temporal dynamics across host populations remain poorly understood. In this study, we investigated the spatio-temporal distribution of bacterial symbionts within the widely distributed and economically significant leafhopper genus Macrosteles, with a focus on Macrosteles laevis. Using host and symbiont marker gene amplicon sequencing, we explored the intricate relationships between these insects and their microbial partners. Our analysis of the cytochrome oxidase subunit I (COI) gene data revealed several intriguing findings. First, there was no strong genetic differentiation across M. laevis populations, suggesting gene flow among them. Second, we observed significant levels of heteroplasmy, indicating the presence of multiple mitochondrial haplotypes within individuals. Third, parasitoid infections were prevalent, highlighting the complex ecological interactions involving leafhoppers. The 16S rRNA data confirmed the universal presence of ancient nutritional endosymbionts-Sulcia and Nasuia-in M. laevis. Additionally, we found a high prevalence of Arsenophonus, another common symbiont. Interestingly, unlike most previously studied species, M. laevis exhibited only occasional cases of infection with known facultative endosymbionts and other bacteria. Notably, there was no significant variation in symbiont prevalence across different populations or among sampling years within the same population. Comparatively, facultative endosymbionts such as Rickettsia, Wolbachia, Cardinium and Lariskella were more common in other Macrosteles species. These findings underscore the importance of considering both host and symbiont dynamics when studying microbial associations. By simultaneously characterizing host and symbiont marker gene amplicons in large insect collections, we gain valuable insights into the intricate interplay between insects and their microbial partners. Understanding these dynamics contributes to our broader comprehension of host-microbe interactions in natural ecosystems.
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Bacterias , Hemípteros , Microbiota , ARN Ribosómico 16S , Simbiosis , Animales , Hemípteros/microbiología , ARN Ribosómico 16S/genética , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Filogenia , Complejo IV de Transporte de Electrones/genéticaRESUMEN
Microbiota are considered significant in the biology of tardigrades, yet their diversity and distribution remain largely unexplored. This is partly due to the methodological challenges associated with studying the microbiota of small organisms that inhabit microbe-rich environments. In our study, we characterized the microbiota of 31 species of cultured tardigrades using 16S rRNA amplicon sequencing. We employed various sample preparation strategies and multiple types of controls and estimated the number of microbes in samples using synthetic DNA spike-ins. We also reanalysed data from previous tardigrade microbiome studies. Our findings suggest that the microbial communities of cultured tardigrades are predominantly composed of bacterial genotypes originating from food, medium, or reagents. Despite numerous experiments, we found it challenging to identify strains that were enriched in certain tardigrades, which would have indicated likely symbiotic associations. Putative tardigrade-associated microbes rarely constituted more than 20% of the datasets, although some matched symbionts identified in other studies. We also uncovered serious contamination issues in previous tardigrade microbiome studies, casting doubt on some of their conclusions. We concluded that tardigrades are not universally dependent on specialized microbes. Our work underscores the need for rigorous safeguards in studies of the microbiota of microscopic organisms and serves as a cautionary tale for studies involving samples with low microbiome abundance.
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Bacterias , Microbiota , ARN Ribosómico 16S , Simbiosis , Tardigrada , Microbiota/genética , Animales , ARN Ribosómico 16S/genética , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Filogenia , ADN Bacteriano/genética , Análisis de Secuencia de ADN/métodosRESUMEN
BACKGROUND: Various animal taxa have specialized to living with social hosts. Depending on their level of specialization, these symbiotic animals are characterized by distinct behavioural, chemical, and morphological traits that enable close heterospecific interactions. Despite its functional importance, our understanding of the feeding ecology of animals living with social hosts remains limited. We examined how host specialization of silverfish co-habiting with ants affects several components of their feeding ecology. We combined stable isotope profiling, feeding assays, phylogenetic reconstruction, and microbial community characterization of the Neoasterolepisma silverfish genus and a wider nicoletiid and lepismatid silverfish panel where divergent myrmecophilous lifestyles are observed. RESULTS: Stable isotope profiling (δ13C and δ15N) showed that the isotopic niches of granivorous Messor ants and Messor-specialized Neoasterolepisma exhibit a remarkable overlap within an ant nest. Trophic experiments and gut dissections further supported that these specialized Neoasterolepisma silverfish transitioned to a diet that includes plant seeds. In contrast, the isotopic niches of generalist Neoasterolepisma silverfish and generalist nicoletiid silverfish were clearly different from their ant hosts within the shared nest environment. The impact of the myrmecophilous lifestyle on feeding ecology was also evident in the internal silverfish microbiome. Compared to generalists, Messor-specialists exhibited a higher bacterial density and a higher proportion of heterofermentative lactic acid bacteria. Moreover, the nest environment explained the infection profile (or the 16S rRNA genotypes) of Weissella bacteria in Messor-specialized silverfish and the ant hosts. CONCLUSIONS: Together, we show that social hosts are important determinants for the feeding ecology of symbiotic animals and can induce diet convergence.
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Hormigas , Conducta Alimentaria , Simbiosis , Animales , Hormigas/fisiología , Hormigas/microbiología , Conducta Alimentaria/fisiología , Filogenia , Isótopos de Nitrógeno/análisis , Isótopos de Carbono/análisis , Perciformes/fisiología , Perciformes/microbiologíaRESUMEN
Background: Symbiotic relationships with diverse microorganisms are crucial for many aspects of insect biology. However, while our understanding of insect taxonomic diversity and the distribution of insect species in natural communities is limited, we know much less about their microbiota. In the era of rapid biodiversity declines, as researchers increasingly turn towards DNA-based monitoring, developing and broadly implementing approaches for high-throughput and cost-effective characterization of both insect and insect-associated microbial diversity is essential. We need to verify whether approaches such as high-throughput barcoding, a powerful tool for identifying wild insects, would permit subsequent microbiota reconstruction in these specimens. Methods: High-throughput barcoding ("megabarcoding") methods often rely on non-destructive approaches for obtaining template DNA for PCR amplification by leaching DNA out of insect specimens using alkaline buffers such as HotSHOT. This study investigated the impact of HotSHOT on microbial abundance estimates and the reconstructed bacterial community profiles. We addressed this question by comparing quantitative 16S rRNA amplicon sequencing data for HotSHOT-treated or untreated specimens of 16 insect species representing six orders and selected based on the expectation of limited variation among individuals. Results: We find that in 13 species, the treatment significantly reduced microbial abundance estimates, corresponding to an estimated 15-fold decrease in amplifiable 16S rRNA template on average. On the other hand, HotSHOT pre-treatment had a limited effect on microbial community composition. The reconstructed presence of abundant bacteria with known significant effects was not affected. On the other hand, we observed changes in the presence of low-abundance microbes, those close to the reliable detection threshold. Alpha and beta diversity analyses showed compositional differences in only a few species. Conclusion: Our results indicate that HotSHOT pre-treated specimens remain suitable for microbial community composition reconstruction, even if abundance may be hard to estimate. These results indicate that we can cost-effectively combine barcoding with the study of microbiota across wild insect communities. Thus, the voucher specimens obtained using megabarcoding studies targeted at characterizing insect communities can be used for microbiome characterizations. This can substantially aid in speeding up the accumulation of knowledge on the microbiomes of abundant and hyperdiverse insect species.
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To understand insect abundance, distribution and dynamics, we need to understand the relevant drivers of their populations and communities. While microbial symbionts are known to strongly affect many aspects of insect biology, we lack data on their effects on populations or community processes, or on insects' evolutionary responses at different timescales. How these effects change as the anthropogenic effects on ecosystems intensify is an area of intense research. Recent developments in sequencing and bioinformatics permit cost-effective microbial diversity surveys, tracking symbiont transmission, and identification of functions across insect populations and multi-species communities. In this review, we explore how different functional categories of symbionts can influence insect life-history traits, how these effects could affect insect populations and their interactions with other species, and how they may affect processes and patterns at the level of entire communities. We argue that insect-associated microbes should be considered important drivers of insect response and adaptation to environmental challenges and opportunities. We also outline the emerging approaches for surveying and characterizing insect-associated microbiota at population and community scales. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.
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Insectos , Microbiota , Simbiosis , Animales , Insectos/microbiología , Insectos/fisiología , Microbiota/fisiología , BiodiversidadRESUMEN
The transmission of microbial symbionts across animal species could strongly affect their biology and evolution, but our understanding of transmission patterns and dynamics is limited. Army ants (Formicidae: Dorylinae) and their hundreds of closely associated insect guest species (myrmecophiles) can provide unique insights into interspecific microbial symbiont sharing. Here, we compared the microbiota of workers and larvae of the army ant Eciton burchellii with those of 13 myrmecophile beetle species using 16S rRNA amplicon sequencing. We found that the previously characterized specialized bacterial symbionts of army ant workers were largely absent from ant larvae and myrmecophiles, whose microbial communities were usually dominated by Rickettsia, Wolbachia, Rickettsiella and/or Weissella. Strikingly, different species of myrmecophiles and ant larvae often shared identical 16S rRNA genotypes of these common bacteria. Protein-coding gene sequences confirmed the close relationship of Weissella strains colonizing army ant larvae, some workers and several myrmecophile species. Unexpectedly, these strains were also similar to strains infecting dissimilar animals inhabiting very different habitats: trout and whales. Together, our data show that closely interacting species can share much of their microbiota, and some versatile microbial species can inhabit and possibly transmit across a diverse range of hosts and environments.
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Hormigas , Escarabajos , Microbiota , Animales , Hormigas/genética , Hormigas/microbiología , Filogenia , ARN Ribosómico 16S/genética , Larva , Bacterias/genética , SimbiosisRESUMEN
1: Metabarcoding (high-throughput sequencing of marker gene amplicons) has emerged as a promising and cost-effective method for characterizing insect community samples. Yet, the methodology varies greatly among studies and its performance has not been systematically evaluated to date. In particular, it is unclear how accurately metabarcoding can resolve species communities in terms of presence-absence, abundances, and biomass. 2: Here we use mock community experiments and a simple probabilistic model to evaluate the effect of different DNA extraction protocols on metabarcoding performance. Specifically, we ask four questions: (Q1) How consistent are the recovered community profiles across replicate mock communities?; (Q2) How does the choice of lysis buffer affect the recovery of the original community?; (Q3) How are community estimates affected by differing lysis times and homogenization?; and (Q4) Is it possible to obtain adequate species abundance estimates through the use of biological spike-ins? 3: We show that estimates are quite variable across community replicates. In general, a mild lysis protocol is better at reconstructing species lists and approximate counts, while homogenization is better at retrieving biomass composition. Small insects are more likely to be detected in lysates, while some tough species require homogenization to be detected. Results are less consistent across biological replicates for lysates than for homogenates. Some species are associated with strong PCR amplification bias, which complicates the reconstruction of species counts. Yet, with adequate spike-in data, species abundance can be determined with roughly 40% standard error for homogenates, and with roughly 50% standard error for lysates, under ideal conditions. In the latter case, however, this often requires species-specific reference data, while spike-in data generalizes better across species for homogenates. 4: We conclude that a non-destructive, mild lysis approach shows the highest promise for presence/absence description of the community, while also allowing future morphological or molecular work on the material. However, homogenization protocols perform better for characterizing community composition, in particular in terms of biomass.
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Symbiotic bacteria have played crucial roles in the evolution of sap-feeding insects and can strongly affect host function. However, their diversity and distribution within species are not well understood; we do not know to what extent environmental factors or associations with other species may affect microbial community profiles. We addressed this question in Philaenus spittlebugs by surveying both insect and bacterial marker gene amplicons across multiple host populations. Host mitochondrial sequence data confirmed morphology-based identification of six species and revealed two divergent clades of Philaenus spumarius. All of them hosted the primary symbiont Sulcia that was almost always accompanied by Sodalis. Interestingly, populations and individuals often differed in the presence of Sodalis sequence variants, suggestive of intra-genome 16S rRNA variant polymorphism combined with rapid genome evolution and/or recent additional infections or replacements of the co-primary symbiont. The prevalence of facultative endosymbionts, including Wolbachia, Rickettsia, and Spiroplasma, varied among populations. Notably, cytochrome I oxidase (COI) amplicon data also showed that nearly a quarter of P. spumarius were infected by parasitoid flies (Verralia aucta). One of the Wolbachia operational taxonomic units (OTUs) was exclusively present in Verralia-parasitized specimens, suggestive of parasitoids as their source and highlighting the utility of host gene amplicon sequencing in microbiome studies.
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Hemípteros , Wolbachia , Humanos , Animales , ARN Ribosómico 16S/genética , Filogenia , Enterobacteriaceae/genética , Bacterias/genética , Hemípteros/microbiología , Simbiosis/genética , Wolbachia/genéticaRESUMEN
The evolutionary success of sap-feeding hemipteran insects in the suborder Auchenorrhyncha was enabled by nutritional contributions from their heritable endosymbiotic bacteria. However, the symbiont diversity, functions, and evolutionary origins in this large insect group have not been broadly characterized using genomic tools. In particular, the origins and relationships among ancient betaproteobacterial symbionts Vidania (in Fulgoromorpha) and Nasuia/Zinderia (in Cicadomorpha) are uncertain. Here, we characterized the genomes of Vidania and Sulcia from three Pyrops planthoppers (family Fulgoridae) to understand their metabolic functions and evolutionary histories. We find that, like in previously characterized planthoppers, these symbionts share nutritional responsibilities, with Vidania providing seven out of ten essential amino acids. Sulcia lineages across the Auchenorrhyncha have a highly conserved genome but with multiple independent rearrangements occurring in an early ancestor of Cicadomorpha or Fulgoromorpha and in a few succeeding lineages. Genomic synteny was also observed within each of the betaproteobacterial symbiont genera Nasuia, Zinderia, and Vidania, but not across them, which challenges the expectation of a shared ancestry for these symbionts. The further comparison of other biological traits strongly suggests an independent origin of Vidania early in the planthopper evolution and possibly of Nasuia and Zinderia in their respective host lineages. This hypothesis further links the potential acquisition of novel nutritional endosymbiont lineages with the emergence of auchenorrhynchan superfamilies.
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Betaproteobacteria , Hemípteros , Animales , Hemípteros/microbiología , Filogenia , Simbiosis/genética , Bacterias/genética , Insectos , Betaproteobacteria/genéticaRESUMEN
Insects are diverse and sustain essential ecosystem functions, yet remain understudied. Recent reports about declines in insect abundance and diversity have highlighted a pressing need for comprehensive large-scale monitoring. Metabarcoding (high-throughput bulk sequencing of marker gene amplicons) offers a cost-effective and relatively fast method for characterizing insect community samples. However, the methodology applied varies greatly among studies, thus complicating the design of large-scale and repeatable monitoring schemes. Here we describe a non-destructive metabarcoding protocol that is optimized for high-throughput processing of Malaise trap samples and other bulk insect samples. The protocol details the process from obtaining bulk samples up to submitting libraries for sequencing. It is divided into four sections: 1) Laboratory workspace preparation; 2) Sample processing-decanting ethanol, measuring the wet-weight biomass and the concentration of the preservative ethanol, performing non-destructive lysis and preserving the insect material for future work; 3) DNA extraction and purification; and 4) Library preparation and sequencing. The protocol relies on readily available reagents and materials. For steps that require expensive infrastructure, such as the DNA purification robots, we suggest alternative low-cost solutions. The use of this protocol yields a comprehensive assessment of the number of species present in a given sample, their relative read abundances and the overall insect biomass. To date, we have successfully applied the protocol to more than 7000 Malaise trap samples obtained from Sweden and Madagascar. We demonstrate the data yield from the protocol using a small subset of these samples.
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Biodiversidad , Ecosistema , Animales , Código de Barras del ADN Taxonómico/métodos , Insectos/genética , Etanol , ADN/genéticaRESUMEN
Bacteria that form long-term intracellular associations with host cells lose many genes, a process that often results in tiny, gene-dense, and stable genomes. Paradoxically, the some of the same evolutionary processes that drive genome reduction and simplification may also cause genome expansion and complexification. A bacterial endosymbiont of cicadas, Hodgkinia cicadicola, exemplifies this paradox. In many cicada species, a single Hodgkinia lineage with a tiny, gene-dense genome has split into several interdependent cell and genome lineages. Each new Hodgkinia lineage encodes a unique subset of the ancestral unsplit genome in a complementary way, such that the collective gene contents of all lineages match the total found in the ancestral single genome. This splitting creates genetically distinct Hodgkinia cells that must function together to carry out basic cellular processes. It also creates a gene dosage problem where some genes are encoded by only a small fraction of cells while others are much more abundant. Here, by sequencing DNA and RNA of Hodgkinia from different cicada species with different amounts of splitting-along with its structurally stable, unsplit partner endosymbiont Sulcia muelleri-we show that Hodgkinia does not transcriptionally compensate to rescue the wildly unbalanced gene and genome ratios that result from lineage splitting. We also find that Hodgkinia has a reduced capacity for basic transcriptional control independent of the splitting process. Our findings reveal another layer of degeneration further pushing the limits of canonical molecular and cell biology in Hodgkinia and may partially explain its propensity to go extinct through symbiont replacement.
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Alphaproteobacteria , Flavobacteriaceae , Hemípteros , Animales , Filogenia , Hemípteros/microbiología , Simbiosis/genética , Flavobacteriaceae/genética , Alphaproteobacteria/genética , Genoma Bacteriano , Dosificación de Gen , Evolución MolecularRESUMEN
The safety and efficacy of Hyruan ONE (test product), an intra-articular cross-linked sodium hyaluronate injection, to treat mild-to-moderate knee osteoarthritis was compared with that of Durolane (comparator) in a prospective, active-controlled, parallel-group, double-blind (masked-observed), multicenter non-inferiority study. European patients (n = 284) were randomized 1:1 (test product:comparator) and received one injection of cross-linked hyaluronic acid (60 mg/3 mL). In total, 280 patients completed the study. The primary endpoint of mean change in Western Ontario and McMaster University (WOMAC)-Likert Pain sub-scores from baseline at week 13 revealed changes of -5.59 and -5.54 for the test and comparator groups, respectively, demonstrating non-inferiority of the test product (difference, -0.05 [95% confidence interval, -0.838 to 0.729]). Secondary endpoint results, which included changes in WOMAC-Likert Pain sub-score from baseline to 26 weeks post-injection and changes in WOMAC-Likert Total score and Physical Function and Stiffness sub-scores, changes in patients' and investigators' global assessments, use of rescue medication, and responder rates at 13 and 26 weeks post-injection were similar between the groups. Incidence of adverse events was also similar. In both groups, most treatment-emergent adverse events were mild/moderate. Hyruan ONE was non-inferior to the comparator at 13 weeks post-injection in European patients with mild-to-moderate knee osteoarthritis.
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Sap-feeding hemipteran insects live in associations with diverse heritable symbiotic microorganisms (bacteria and fungi) that provide essential nutrients deficient in their hosts' diets. These symbionts typically reside in highly specialized organs called bacteriomes (with bacterial symbionts) or mycetomes (with fungal symbionts). The organization of these organs varies between insect clades that are ancestrally associated with different microbes. As these symbioses evolve and additional microorganisms complement or replace the ancient associates, the organization of the symbiont-containing tissue becomes even more variable. Planthoppers (Hemiptera: Fulgoromorpha) are ancestrally associated with bacterial symbionts Sulcia and Vidania, but in many of the planthopper lineages, these symbionts are now accompanied or have been replaced by other heritable bacteria (e.g., Sodalis, Arsenophonus, Purcelliella) or fungi. We know the identity of many of these microbes, but the symbiont distribution within the host tissues and the bacteriome organization have not been systematically studied using modern microscopy techniques. Here, we combine light, fluorescence, and transmission electron microscopy with phylogenomic data to compare symbiont tissue distributions and the bacteriome organization across planthoppers representing 15 families. We identify and describe seven primary types of symbiont localization and seven types of the organization of the bacteriome. We show that Sulcia and Vidania, when present, usually occupy distinct bacteriomes distributed within the body cavity. The more recently acquired gammaproteobacterial and fungal symbionts generally occupy separate groups of cells organized into distinct bacteriomes or mycetomes, distinct from those with Sulcia and Vidania. They can also be localized in the cytoplasm of fat body cells. Alphaproteobacterial symbionts colonize a wider range of host body habitats: Asaia-like symbionts often colonize the host gut lumen, whereas Wolbachia and Rickettsia are usually scattered across insect tissues and cell types, including cells containing other symbionts, bacteriome sheath, fat body cells, gut epithelium, as well as hemolymph. However, there are exceptions, including Gammaproteobacteria that share bacteriome with Vidania, or Alphaproteobacteria that colonize Sulcia cells. We discuss how planthopper symbiont localization correlates with their acquisition and replacement patterns and the symbionts' likely functions. We also discuss the evolutionary consequences, constraints, and significance of these findings.
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Gut bacterial symbionts can support animal nutrition by facilitating digestion and providing valuable metabolites. However, changes in symbiotic roles between immature and adult stages are not well documented, especially in ants. Here, we explored the metabolic capabilities of microbiomes sampled from herbivorous turtle ant (Cephalotes sp.) larvae and adult workers through (meta)genomic screening and in vitro metabolic assays. We reveal that larval guts harbor bacterial symbionts with impressive metabolic capabilities, including catabolism of plant and fungal recalcitrant dietary fibers and energy-generating fermentation. Additionally, several members of the specialized adult gut microbiome, sampled downstream of an anatomical barrier that dams large food particles, show a conserved potential to depolymerize many dietary fibers. Symbionts from both life stages have the genomic capacity to recycle nitrogen and synthesize amino acids and B-vitamins. With help of their gut symbionts, including several bacteria likely acquired from the environment, turtle ant larvae may aid colony digestion and contribute to colony-wide nitrogen, B-vitamin and energy budgets. In addition, the conserved nature of the digestive capacities among adult-associated symbionts suggests that nutritional ecology of turtle ant colonies has long been shaped by specialized, behaviorally-transferred gut bacteria with over 45 million years of residency.
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Hormigas , Microbioma Gastrointestinal , Animales , Bacterias/genética , Fibras de la Dieta , Nitrógeno , Filogenia , SimbiosisRESUMEN
Sap-sucking hemipterans host specialized, heritable microorganisms that supplement their diet with essential nutrients. These microbes show unusual features that provide a unique perspective on the coevolution of host-symbiont systems but are still poorly understood. Here, we combine microscopy with high-throughput sequencing to revisit 80-year-old reports on the diversity of symbiont transmission modes in a broadly distributed planthopper family, Dictyopharidae. We show that in seven species examined, the ancestral nutritional symbionts Sulcia and Vidania producing essential amino acids are complemented by co-primary symbionts, either Arsenophonus or Sodalis, acquired several times independently by different host lineages and contributing to the biosynthesis of B vitamins. These symbionts reside within separate bacteriomes within the abdominal cavity, although in females Vidania also occupies bacteriocytes in the rectal organ. Notably, the symbionts are transovarially transmitted from mothers to offspring in two alternative ways. In most examined species, all nutritional symbionts simultaneously infect the posterior end of the full-grown oocytes and next gather in their perivitelline space. In contrast, in other species, Sodalis colonizes the cytoplasm of the anterior pole of young oocytes, forming a cluster separate from the "symbiont ball" formed by late-invading Sulcia and Vidania. Our results show how newly arriving microbes may utilize different strategies to establish long-term heritable symbiosis. IMPORTANCE Sup-sucking hemipterans host ancient heritable microorganisms that supplement their unbalanced diet with essential nutrients and have repeatedly been complemented or replaced by other microorganisms. These symbionts need to be reliably transmitted to subsequent generations through the reproductive system, and often they end up using the same route as the most ancient ones. We show for the first time that in a single family of planthoppers, the complementing symbionts that have established infections independently utilize different transmission strategies, one of them novel, with the transmission of different microbes separated spatially and temporally. These data show how newly arriving microbes may utilize different strategies to establish long-term heritable symbioses.
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Betaproteobacteria/metabolismo , Gammaproteobacteria/metabolismo , Hemípteros/microbiología , Nutrientes/metabolismo , Simbiosis , Animales , Betaproteobacteria/genética , Femenino , Gammaproteobacteria/genética , Hemípteros/anatomía & histología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Microscopía/métodos , FilogeniaRESUMEN
Traditionally, insects collected for scientific purposes have been dried and pinned, or preserved in 70% ethanol. Both methods preserve taxonomically informative exoskeletal structures well but are suboptimal for preserving DNA for molecular biology. Highly concentrated ethanol (95-100%), preferred as a DNA preservative, has generally been assumed to make specimens brittle and prone to breaking. However, systematic studies on the correlation between ethanol concentration and specimen preservation are lacking. Here, we tested how preservative ethanol concentration in combination with different sample handling regimes affect the integrity of seven insect species representing four orders, and differing substantially in the level of sclerotization. After preservation and treatments (various levels of disturbance), we counted the number of appendages (legs, wings, antennae, or heads) that each specimen had lost. Additionally, we assessed the preservation of DNA after long-term storage by comparing the ratio of PCR amplicon copy numbers to an added artificial standard. We found that high ethanol concentrations indeed induce brittleness in insects. However, the magnitude and nature of the effect varied strikingly among species. In general, ethanol concentrations at or above 90% made the insects more brittle, but for species with robust, thicker exoskeletons, this did not translate to an increased loss of appendages. Neither freezing the samples nor drying the insects after immersion in ethanol had a negative effect on the retention of appendages. However, the morphology of the insects was severely damaged if they were allowed to dry. We also found that DNA preserves less well at lower ethanol concentrations when stored at room temperature for an extended period. However, the magnitude of the effect varies among species; the concentrations at which the number of COI amplicon copies relative to the standard was significantly decreased compared to 95% ethanol ranged from 90% to as low as 50%. While higher ethanol concentrations positively affect long-term DNA preservation, there is a clear trade-off between preserving insects for morphological examination and genetic analysis. The optimal ethanol concentration for the latter is detrimental for the former, and vice versa. These trade-offs need to be considered in large insect biodiversity surveys and other projects aiming to combine molecular work with traditional morphology-based characterization of collected specimens.
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The aim of the study was to analyze the prevalence of ocular demodicosis and ocular surface conditions in patients selected for cataract surgery. Eyelashes from 73 patients selected for cataract surgery were evaluated at × 40 and × 100 magnification using light microscopy. The anterior segment was assessed with the slit lamp. Additionally, Schirmer I and break up time (BUT) tests were carried out before surgery and 1 and 3 months postoperatively. A specially designed questionnaire containing e.g. information about chronic skin and eye diseases, previous ophthalmic surgeries, and patient's hygiene habits was used to assess the demographic variables. A majority of patients were at the age of 70-79 years, and there were more females (83%) in the study group. Demodex folliculorum was found in 48% of the patients. There was a correlation between the number of parasites and the presence of blepharitis, discharge at eyelid margins, and conjunctival hyperemia. Schirmer I and BUT test results were lower in patients with Demodex infestation before and after cataract surgery. The higher number of mites was correlated with lower Schirmer I test results postoperatively. The presence of Demodex mites influences the conjunctiva and lid margins leading to inflammation. The higher number of Demodex mites disturbs the tear film over time after cataract surgery.
RESUMEN
The fungal genus Massospora (Zoopagomycota: Entomophthorales) includes more than a dozen obligate, sexually transmissible pathogenic species that infect cicadas (Hemiptera) worldwide. At least two species are known to produce psychoactive compounds during infection, which has garnered considerable interest for this enigmatic genus. As with many Entomophthorales, the evolutionary relationships and host associations of Massospora spp. are not well understood. The acquisition of M. diceroproctae from Arizona, M. tettigatis from Chile, and M. platypediae from California and Colorado provided an opportunity to conduct molecular phylogenetic analyses and morphological studies to investigate whether these fungi represent a monophyletic group and delimit species boundaries. In a three-locus phylogenetic analysis including the D1-D2 domains of the nuclear 28S rRNA gene (28S), elongation factor 1 alpha-like (EFL), and beta-tubulin (BTUB), Massospora was resolved in a strongly supported monophyletic group containing four well-supported genealogically exclusive lineages, based on two of three methods of phylogenetic inference. There was incongruence among the single-gene trees: two methods of phylogenetic inference recovered trees with either the same topology as the three-gene concatenated tree (EFL) or a basal polytomy (28S, BTUB). Massospora levispora and M. platypediae isolates formed a single lineage in all analyses and are synonymized here as M. levispora. Massospora diceroproctae was sister to M. cicadina in all three single-gene trees and on an extremely long branch relative to the other Massospora, and even the outgroup taxa, which may reflect an accelerated rate of molecular evolution and/or incomplete taxon sampling. The results of the morphological study presented here indicate that spore measurements may not be phylogenetically or diagnostically informative. Despite recent advances in understanding the ecology of Massospora, much about its host range and diversity remains unexplored. The emerging phylogenetic framework can provide a foundation for exploring coevolutionary relationships with cicada hosts and the evolution of behavior-altering compounds.