Biology subjects

Nowak, K. H.

Publications and source records attributed to Nowak, K. H..

7 recordsLinked to original sources

bioRxiv2026

Insect COI barcoding data as an untapped resource for surveying Wolbachia symbioses

O_LIDNA barcoding of the mitochondrial cytochrome c oxidase I (COI) gene is widely used to characterise insect diversity and distributions; however, its potential to reveal information on species interactions, including host-symbiont associations, remains largely unexplored. Here, we assess whether COI amplicon data can be used to identify Wolbachia - one of the most widely distributed bacterial symbionts known to profoundly affect their hosts biology. C_LIO_LIWe demonstrate that several commonly used invertebrate COI primer sets perfectly match many reference Wolbachia genomes, leading to frequent co-amplification. C_LIO_LIBy screening 7,901 individual-insect COI amplicon libraries obtained with the popular BF3-BR2 primer set, we detected Wolbachia sequences in over 35% of samples, revealing that co-amplification is indeed widespread. After removing low-abundance reads, Wolbachia detection based on COI amplicons showed over 90% agreement with simultaneously generated 16S-V4 rRNA amplicon data from the same specimens. The degree of agreement, however, varied depending on the thresholds used, among datasets and insect clades. C_LIO_LIFurther, we show that Wolbachia abundance inferred from COI amplicons correlated with their abundance in metagenomic datasets for 152 specimens, supporting the quantitative relevance of the signal. C_LIO_LIFinally, we find that Wolbachia COI sequences provide greater phylogenetic resolution than 16S-V4 rRNA data (mean pairwise genetic distance of COI sequences - 9.6%, 16S-V4 rRNA - 2.8%), and the reconstructed Wolbachia COI-based genotype network largely agrees with genome-based phylogenies. C_LIO_LICollectively, our results demonstrate that off-target Wolbachia sequences recovered from standard insect COI barcoding data may reliably detect symbiont presence, provide phylogenetic insight, and guide sample selection for metagenomics. Given the rapid expansion of global insect barcoding initiatives, these findings highlight an opportunity for cost-effective monitoring of their most prevalent bacterial symbionts, offering new perspectives on how host-microbe interactions may shape insect communities. C_LI

ecology↗

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

Dissecting multitrophic interactions: the relationships among Entomophthora, their dipteran hosts, and associated bacteria

Interactions with microorganisms from across a range of mutualism-to-pathogenicity spectrums shape the biology of insects at all levels - from individual traits to populations and communities. However, the understanding of pathogens attacking non-model insect species in natural ecosystems, or their interactions with other insect-associated microorganisms, is fragmentary. Here, we tested a conceptually novel approach - the simultaneous sequencing of insect, fungal, and bacterial marker gene amplicons - as a means of dissecting interactions among entomopathogenic fungi in the genus Entomophthora and their dipteran hosts in South Greenland. We aimed to describe the taxonomic diversity of Entomophthora-killed flies, their pathogens, and the bacterial diversity within a set of field-collected dead insects exhibiting signs of Entomophthora infection. Across nine collected dipteran species, we identified multiple Entomophthora genotypes, with strong but not perfect patterns of host-specificity across the five targeted marker regions. Additionally, we found consistent differences in bacterial community composition among fungus-killed fly species and sampling sites. Our results substantially expand the knowledge of Entomopthora diversity and host associations while providing the very first insights into associated bacteria and their potential roles. We also conclude that multi-target amplicon sequencing can be a powerful tool for addressing broad questions about biological interactions in diverse natural communities.

ecology↗

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

What lurks beneath the humped back? - The patterns of bacterial abundance, diversity and distribution across communities of a dipteran family

Symbiotic microorganisms can profoundly impact insects, their life history traits, population dynamics, and evolutionary trajectories. However, microbiota remain poorly understood in natural insect communities, especially in dark taxa - i.e., hyperdiverse, yet understudied clades. Here, we implemented a novel multi-target amplicon sequencing approach to study microbiota in complex, species-rich communities. It combines four methodological innovations: (1) To establish a host taxonomic framework, we sequenced amplicons of the host marker gene (COI) and reconstructed barcodes alongside microbiota characterisation. (2) To assess microbiota abundance, we incorporated spike-in-based quantification. (3) To improve the phylogenetic resolution for the dominant endosymbiont, Wolbachia, we analysed bycatch data from the COI amplicon sequencing. (4) To investigate the primary drivers of host-microbe associations in massive multi-dimensional datasets, we performed HMSC modelling. Applying this approach to 1,842 wild-caught scuttle flies (Diptera: Phoridae) from northern Sweden, we organised them into 480 genotypes and 186 species and gained unprecedented insights into their microbiota. We found orders-of-magnitude differences in bacterial abundance and massive within-population variation in microbiota composition. Patterns and drivers differed among microbial functional categories: the distribution and abundance of facultative endosymbionts (Wolbachia, Rickettsia, Spiroplasma) were shaped by host species, genotype and sex. In contrast, many other bacterial taxa were broadly distributed across species and sites. This study highlights facultative endosymbionts as key players in insect microbiota and reveals striking variations in distributional patterns of microbial clades. It also demonstrates the power of integrative sequencing approaches in uncovering the ecological complexity and significance of symbiotic microorganisms in multi-species natural communities.

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↗