Biology subjects

Lukasik, P.

Publications and source records attributed to Lukasik, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Whole forest in a pouch? Methods converge in uncovering wood ants fungal and bacterial microbiota

Despite their importance for individual fitness and population processes, the microbiota of many ecologically significant insects remains poorly explored. Even less is known about the interactions between microbial communities inhabiting insects and their surrounding environment. Ant infrabuccal pockets (IBPs), representing the interface between the digestive tract and the external environment, provide an opportunity to study these interactions. Here, we aimed to characterize ant-microbial interaction networks in the forest floor by profiling fungal and bacterial communities associated with the IBP of Formica polyctena ants, known as ecosystem engineers in temperate forests. We used direct microscopy, culturing, and sequencing amplicons of ITS1, ITS2, 18S rRNA marker regions to describe fungal communities, and 16S rRNA metabarcoding to characterize bacterial communities. Classical methods combined with a multi-marker amplicon sequencing allowed for a comprehensive description of the IBP microbiota. Fungal communities consistently contained representatives of 15 ecologically diverse genera, including insect-associated yeasts and primarily saprotrophic or endophytic fungi. Bacterial communities were dominated by genera previously reported from ant guts, mainly Bacilli and Alphaproteobacteria, and showed greater stability among ant colonies than fungal communities. Further studies on red wood ants IBP microbiota would enhance our understanding of their role in shaping ecological networks in forest ecosystems.

ecology↗

bioRxiv2026

Accuracy of occurrence and abundance estimates from insect metabarcoding

1. DNA metabarcoding--high-throughput sequencing of barcode regions from bulk samples--has become a key tool for insect biodiversity assessment. Yet, how methodological choices affect the accuracy of metabarcoding data remains insufficiently explored. In this paper, we ask: (1) How does the lysis method (non-destructive lysis vs. destructive homogenization) affect community recovery? (2) How comprehensively does metabarcoding capture species richness? (3) To what extent can spike-ins improve abundance estimates? (4) How accurately can species abundances be estimated? 2. We evaluated the accuracy of insect metabarcoding using 4,749 bulk samples from a large-scale biodiversity survey subjected to mild lysis. Of these samples, 856 were also homogenized, allowing a systematic comparison of the effect of alternative treatments. To potentially improve abundance estimates, we added six biological spike-ins (i.e., foreign insects) to all samples, and two synthetic spike-ins (artificial DNA fragments) to the homogenization treatment. In addition, we established the contents of 15 samples by individually barcoding all specimens, enabling direct assessment of occurrence and abundance estimates. 3. Our results revealed consistent differences between destructive and non-destructive treatments. While both methods reliably detected the majority of species, small and soft-bodied taxa were more often recovered after mild lysis than after homogenization, while the reverse was true for heavily sclerotized, hairy, and large taxa. Using biological spike-ins for calibration reduced the variance in read numbers per specimen considerably, especially in homogenized samples, while synthetic spike-ins were less effective. In a Bayesian analysis, where species data were matched to the best-fitting spike-in calibration curve, accurate abundance estimates (+/-1 individual) were obtained for 72.9% of species occurrences. 4. Our results show that it is possible to obtain reasonably accurate abundance estimates from metabarcoding data, and that mild lysis and homogenization result in different taxon-specific biases in terms of occurrence data, with neither method outperforming the other. Accuracy is improved by homogenization rather than mild lysis of samples, and by the use of biological rather than synthetic spike-ins. Together, these findings provide a major step towards robust, quantitative biodiversity monitoring using DNA-metabarcoding.

zoology↗

bioRxiv2025

The tiniest genomes shrink much further: extreme reductive evolution in planthopper symbionts

Strictly heritable endosymbiotic bacteria that provide limiting nutrients to sap-sucking hemipteran insects are known for their highly reduced genomes conserved in organization and function. Here, we show how in ancestral endosymbionts of planthoppers, Sulcia and Vidania, gradually losing genes during [~]263 my of co-diversification with hosts, co-infections by additional microbes and host ecological switches coincided with more dramatic genomic changes. At its extremes, this has resulted in the smallest non-organellar bacterial genomes known, at barely 50-52 kb. Such minuscule Vidania genomes evolved convergently in two planthopper superfamilies, and are strikingly similar in gene contents, including the ability to produce a single amino acid (phenylalanine) for the host. Losing many additional cell-function genes places them among mitochondria in the level of host dependence, further blurring the bacteria-organelle boundary. One-sentence summaryWe present the smallest bacterial genomes known and evaluate the patterns and processes of their reductive evolution

evolutionary biology↗

bioRxiv2025

The conserved nature of nitrogen recycling within the bacterial pouch of Tetraponera ants

Microbial symbioses are fundamental to the nutrition of many animal groups, yet the mechanisms of nutrient provisioning remain poorly understood -- even in social insects, a diverse clade whose global ecological dominance and sophisticated caste systems make them disproportionately well-studied models for symbiosis research. Here, we investigate the functional significance of a specialized and unusual symbiotic organ-- the bacterial pouch--in four ant species within the Tetraponera nigra-group, focusing on the symbionts roles in nitrogen recycling and colony fitness. Bacterial pouch houses a microbial community consistently dominated by the core bacterial symbiont Tokpelaia. Metagenomic sequencing and targeted {superscript 1}N-labeled urea feeding experiments demonstrate that these symbionts assimilate nitrogen from urea into amino acids, transferring them to adult workers and developing larvae that incorporate recycled nitrogen into their tissues. Disruption of this nitrogen-recycling symbiosis severely impairs larval growth and overall colony fitness. Our study highlights the critical role of the bacterial pouch in sustaining colony fitness in nitrogen-limited environments, providing new insights into the pivotal role of microbial symbioses in the ecological success of social insects.

ecology↗

bioRxiv2024

HAPP: High-Accuracy Pipeline for Processing deep metabarcoding data

We introduce HAPP, a high-accuracy pipeline for processing deep metabarcoding data, leveraging data richness to enhance the signal-to-noise-ratio. Starting with denoised amplicon sequence variants, the pipeline consists of four steps: (1) additional chimera removal, using UCHIME and a strict sample-based approach; (2) taxonomic annotation, combining k-mer matching (SINTAX) to a reference library with phylogenetic placement (EPA-NG) on a reference tree; (3) OTU clustering using SWARM, an open-source algorithm with precision and recall comparable to RESL used in circumscribing BOLD BINs; and (4) noise filtering (NUMTs and sequencing errors), using a new algorithm introduced here, NEEAT, which combines "echo" signals across samples with detection of unusual evolutionary signatures among clusters with similar DNA sequences. HAPP computations are parallelized across taxa, making analyses tractable on very large datasets. The performance of HAPP was validated through extensive benchmarks, involving CO1 data from BOLD and Malaise trap data, demonstrating significant improvements over the state of the art.

bioinformatics↗

bioRxiv2024

Cold and lonely: low-abundance microbiota and no core microbiome across a broad geographic sampling of a mosquito species in Greenland

Microbial symbionts are key players in insect ecology and evolution, but microbiota vary widely across and within host species. The microbiota of insect clades such as high-latitude mosquitoes remain understudied, despite their abundance and potential economic and medical relevance. Here, we resolve temporal and spatial variation in the abundance and diversity of bacteria and fungi associated with Ochlerotatus mosquitoes in Greenland. To this aim, we apply a comprehensive workflow combining the simultaneous characterization of host, bacterial, and fungal marker regions, contamination filtering, as well as bacterial quantification and joint species distribution modelling. We find that 573 mosquitoes collected in five regions of Greenland between 2009 and 2024 represented two species (O. nigripes and O. impiger). Their microbial communities were low-abundance, highly diverse, and dominated by environmentally versatile taxa, showing high individual-to-individual-rather than spatio-temporal variation, although taxa such as Pseudomonas and Serratia exhibited sex-specific associations. DNA from local vertebrates and potential vertebrate pathogens (e.g., Bartonella) was detected, likely reflecting blood meals. These findings suggest that Greenland Ochlerotatus mosquitoes do not rely on core microbiota, but engage in flexible associations with diverse, low-abundance microbial communities. They also point to mosquitoes as likely vectors of bacterial and fungal pathogens in the Arctic.

ecology↗

bioRxiv2024

A new tool in a toolbox: Addressing challenges in high-throughput microbiota surveys across diverse wild insects

With their significant effects on the biology of higher organisms, host-associated microbiota has attracted the research communitys attention. The rapid progress in sequencing techniques has greatly facilitated microbial community characterization. However, the most popular surveying technique, marker gene amplicon sequencing, has multiple caveats that are not often addressed satisfactorily, including the uncertainty about the identity of the surveyed wild-caught specimens, variable and sometimes very low abundance of microbes in some samples, or reagent- and cross-contamination. As a result, researchers often obtain incomplete, biased, and sometimes totally incorrect microbial community profiles. Here, we present a versatile, cost-effective, and high-throughput quantitative multi-target amplicon sequencing workflow for the characterization of host-associated microbial communities, combining laboratory and bioinformatic steps and addressing most of the known methodological issues. Optimized for the study of the microbiota of wild insects, it can be easily adapted for other sample types. Outputs include contamination-controlled data on the absolute abundance and identity of microbes present in insect samples, both at genotype- and OTU-level, as well as host barcodes alongside information on parasite infections. Using 1384 samples from Zackenberg Valley, NE Greenland, we demonstrate the potential of the workflow to study insect and symbiont diversity patterns across a large portion of a diverse natural community.

ecology↗

bioRxiv2024

Phylogenomics resolves the relationship and the evolutionary history of planthoppers (Insecta: Hemiptera: Fulgoromorpha)

Planthoppers (Hemiptera: Fulgoromorpha) are a species-rich and globally distributed insect clade with high economic, ecological, and evolutionary importance. However, the relationships among planthopper lineages and families remain unclear. Previous efforts based on inconsistent morphological traits, a few genes, or limited sampling often resulted in conflicting tree topologies. Here, we used genome-level data to assemble 1164 nuclear single-copy genes and 13 mitochondrial protein-coding genes for 149 planthopper species representing 19 out of 21 extant families. Additional markers were added from published mitogenomes, expanding our sampling to 285 species. These markers were used for Maximum Likelihood-based tree inference and dating analyses. The newly inferred phylogenies validated well-accepted relationships and recovered novel placements. Taxonomic conclusions include the establishment of a new family Borysthenidae stat. rev. within Delphacoidea and a new superfamily Meenoploidea superfam. nov. including redefined Kinnaridae stat. rev. and Meenoplidae stat. rev., the confirmation of the monophyletic family Achilixiidae outside the Achilidae-Derbidae clade, and the transfer of tribes Lyncidini and Amyclini to Dictyopharidae and the genus Madagascaritia to Fulgoridae. The time analyses based on 57 nuclear markers and 30 fossils dated the origin of crown Fulgoromorpha back to Guadalupian, Permian ([~]263 Ma), close to the maximum constraint at 267.3 Ma, while applying an older root constraint resulted in an origin in Mississippian, Carboniferous ([~]332 Ma). While future sampling of unstudied fauna in unexplored regions or habitats may change the topology, the current phylogenomic analysis will serve as a solid foundation for research into planthopper ecology, evolution, and significance.

evolutionary biology↗

bioRxiv2024

Implementing high-throughput insect barcoding in microbiome studies: impact of non-destructive DNA extraction on microbiome reconstruction

BackgroundSymbiotic 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. MethodsHigh-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. ResultsWe 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. ConclusionOur 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.

microbiology↗

bioRxiv2024

Limited variation in microbial communities across populations of Macrosteles leafhoppers (Hemiptera: Cicadellidae)

Microbial symbionts play important roles in insect biology, but their diversity, distribution, and dynamics over time across host populations are poorly understood. We surveyed the spatio-temporal distribution of bacterial symbionts in the broadly distributed and economically significant leafhopper genus Macrosteles, with emphasis on Macrosteles laevis, using host and symbiont marker gene amplicon sequencing. The cytochrome oxidase I (COI) gene data revealed no strong genetic differentiation across M. laevis populations, significant levels of heteroplasmy, and multiple cases of parasitoid infections. 16S rRNA data confirmed the universal presence of the ancient nutritional endosymbionts Sulcia and Nasuia and a high prevalence of Arsenophonus. Interestingly, in contrast to most previously surveyed species, in M. laevis we found only occasional cases of infection with facultative endosymbionts and other bacteria. There was no significant variation in symbiont prevalence across populations, or among sampling years for the same population. Facultative endosymbionts including Rickettsia, Wolbachia, Cardinium, and Lariskella, were more common in other Macrosteles species. Combined, our data demonstrate that not all species show clear spatial and temporal variation in genetic structure and microbial prevalence. However, simultaneous characterization of host and symbiont marker gene amplicons in large insect collections can help understand the dynamics of host-microbe interactions.

evolutionary biology↗

bioRxiv2023

Co-habiting ants and silverfish display a converging feeding ecology

O_LISpecies from a great number of 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 specialized animals living with social hosts remains limited. C_LIO_LIIn this study, 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. C_LIO_LIStable isotope profiling ({delta}13C and {delta}15N) showed that the isotopic niches (proxy for trophic 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. C_LIO_LIThe 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. C_LIO_LITogether, we show that social hosts are important determinants for the feeding ecology of symbiotic animals, and can induce diet convergence. C_LI

ecology↗

bioRxiv2023

Facultatively intra-bacterial localization of a planthopper endosymbiont as an adaptation to its vertical transmission

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 adapted 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, symbionts genomic features, including encoding essential amino acid biosynthetic pathways and very similar to a recently described psyllid symbiont, suggest a unique combination of 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 renown for its genomic stability. In Vidania, substantial gene loss resulted in one of the smallest genomes known, at 109 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. Significance StatementReliable 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 association 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. Acetobacteraceaes genomic features indicate its unusual evolutionary history, and the genomic erosion experienced by ancient nutritional symbionts demonstrates 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.

cell biology↗

bioRxiv2022

Variable organization of symbiont-containing tissue across planthoppers hosting different heritable endosymbionts

Sap-feeding hemipteran insects live in associations with diverse heritable endosymbiotic bacteria and fungi that provide essential nutrients deficient in their diets. These symbionts typically reside in bacteriomes, dedicated organs made up of specialized cells termed bacteriocytes. The bacteriome organization 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, 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 tissues and cell types, including bacteriocytes 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.

evolutionary biology↗

bioRxiv2022

Genome comparison reveals inversions and alternative evolutionary history of nutritional endosymbionts in planthoppers (Hemiptera: Fulgoromorpha)

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. 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. Originality-Significance StatementWe sequenced and characterized the genomes of two ancient nutritional symbionts, Sulcia and Vidania, in three species from the genus Pyrops in the species- and symbiont-rich but understudied insect clade, Fulgoromorpha (planthoppers). We describe--for the first time--several independent genome rearrangements in Sulcia, which is often cited as a premier example of extreme genome stability spanning hundreds of millions of years. We also show a global lack of synteny across the genomes of the Auchenorrhynchan betaproteobacterial symbionts (Vidania, Nasuia, and Zinderia). This result is unexpected given previous hypotheses of a common origin for these symbionts >250 million years ago alongside Sulcia. Taken together, we suggest an independent origin of Vidania and possibly of Nasuia and Zinderia symbiont lineages as well. This hypothesis further links the potential acquisition of novel nutritional endosymbiont lineages with the emergence of auchenorrhyncham superfamilies.

genomics↗

bioRxiv2022

Till evolution do us part: The diversity of symbiotic associations across populations of Philaenus spittlebugs

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 dont know to what extent environmental factors or associations with other species may affect microbial community profiles. Here, we sequenced host and bacterial marker gene amplicons to survey the bacterial community composition across multiple populations of Philaenus spittlebugs. Host mitochondrial sequence data confirmed morphology-based identification of 6 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, COI amplicon data also showed that nearly a quarter of P. spumarius were infected by parasitoid flies (Verralia aucta). One of the Wolbachia 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.

ecology↗

bioRxiv2022

Microbial symbionts are shared between ants and their associated beetles

The transmission of microbial symbionts across individuals and generations can be critical for animal development and survival. Likewise, the transmission of microbes across closely interacting species could also affect host biology. Army ants (Formicidae: Dorylinae) and their hundreds of closely associated insect species (myrmecophiles) can provide a unique insight into interspecific 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 symbionts of army ant workers (Unclassified Firmicutes and Unclassified Entomoplasmatales) were largely absent from ant larvae and from 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 common bacteria. In particular, army ant larvae, some workers, and several myrmecophile species often hosted identical Weissella (Lactobacillales), based on 16S rRNA and also protein-coding gene sequences. Also, we found high relatedness between some newly characterized Weissella and animal-associated strains from aquatic and marine habitats. Looking more broadly, we found Weissella OTUs in 11.6% of samples from nearly all habitats and environments characterized by the Earth Microbiome Project. Together, our data show that unrelated but closely interacting species can share much of their microbiota. The high relatedness of strains found across such disparate hosts as ants, beetles, trout, and whales suggests that some versatile microbes move between hosts and habitats despite few opportunities for direct interaction.

ecology↗

bioRxiv2022

Optimizing insect metabarcoding using replicated mock communities

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. Here we use mock community experiments and a simple probabilistic model to evaluate the performance of different metabarcoding protocols. 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? 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. Tiny 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. 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.

ecology↗

bioRxiv2021

Alternative transmission patterns in independently acquired nutritional co-symbionts of Dictyopharidae planthoppers

Sap-sucking hemipterans host specialized, heritable microorganisms that supplement their unbalanced diet with essential nutrients. These microbes show unusual features that provide a unique perspective on the evolution of life but have not been systematically studied. 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 all species examined, the ancestral nutritional endosymbionts Sulcia and Vidania are complemented by co-primary symbionts, either Arsenophonus or Sodalis, acquired several times independently by different host lineages. Like in other obligate sap-feeders, the ancestral symbionts produce essential amino acids, whereas co-primary symbionts contribute 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 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 (vitellogenic) oocytes and next gather in their perivitelline space. In contrast, in other species, Sodalis colonizes the cytoplasm of the anterior pole of young (previtellogenic) oocytes forming a cluster separate from the "symbiont ball" formed by late-invading Sulcia and Vidania. Our data add to evidence on frequent replacements of gammaproteobacterial symbionts combined with the relative functional stability of the nutritional functions during the evolution of sap-feeding insects, and show how newly-arriving microbes may utilize different strategies to establish long-term heritable symbiosis. Significance statementSup-sucking hemipterans host ancient heritable microorganisms that supplement their unbalanced diet with essential nutrients, and which have repeatedly been complemented or replaced by other microorganisms. They need to be reliably transmitted to subsequent generations through the reproductive system, and often they end up using the same route as the ancient symbionts. 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 temporarily. These data show how newly-arriving microbes may utilize different strategies to establish long-term heritable symbiosis.

evolutionary biology↗