The microbiome of an invasive Antarctic insect, Eretmoptera murphyi (Diptera: Chironomidae), and its potential role in nutrient cycling

The microbiome of an invasive Antarctic insect, Eretmoptera murphyi (Diptera: Chironomidae), and its potential role in nutrient cycling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The microbiome of an invasive Antarctic insect, Eretmoptera murphyi (Diptera: Chironomidae), and its potential role in nutrient cycling Octavia D. M. Brayley, Kirsty McCready, Shengwei Liu, Peter Convey, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7744438/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Microbial Ecology → Version 1 posted 14 You are reading this latest preprint version Abstract Eretmoptera murphyi is a flightless chironomid midge endemic to South Georgia in the sub-Antarctic. In the 1960s it was accidentally introduced to Signy Island (in the maritime Antarctic), where it is now considered an invasive species. This study set out to assess the archaeal and bacterial microbiome of E. murphyi to enhance understanding of the ecological impacts of this non-native species. Previous work has revealed that the detritivorous larvae of E. murphyi can increase soil nitrogen levels by up to five times compared with similar uncolonized substrates, although the mechanisms involved are not known. As nutrient availability in Antarctic terrestrial habitats is an important limitation to biodiversity and the development of communities, any significant change could impact native microarthropod and plant communities as well as open new pathways for future non-native species establishment. Dominant archaea and bacteria were: Crenarchaeota, Actinobacteriota, Chloroflexi, Proteobacteria and Planctomycetota, many of which have known roles in nutrient cycling. The microbiome of E. murphyi appears to be more diverse than those of the few Antarctic invertebrates studied to date. Further work is required to confirm which microorganisms are true endosymbionts and their specific roles in nutrient cycling in polar terrestrial ecosystems. Archaea bacteria invertebrate nitrogen cycling nutrient release polar Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The diversity and ecological success of insects is partly due to their associated microbiomes [ 1 , 2 , 3 ]. These communities of bacteria, archaea, fungi, protozoa and viruses influence many aspects of host biology [ 4 ], facilitated by long co-evolutionary relationships [ 5 , 6 ]. Bacteria are the most abundant and diverse microorganisms present in the microbiome [ 4 ]. Mutualistic species can boost energy metabolism and play essential roles in nutrient acquisition [ 7 ], as well as in the host immune system [ 8 ], developmental processes [ 9 ] and fecundity [ 2 ]. Microbiome composition changes seasonally in some insects [ 10 ] and can contribute to winter/cold adaptation [ 11 ]. Studies of the relationships of archaea with their hosts are currently under-represented in the literature, primarily because this domain represents a smaller proportion of the microbiome compared to other groups [ 12 ]. Nonetheless, archaea can also play an important role in insect metabolism, such as the fruit fly, Anastrepha obliqua , with communities changing across developmental stages and depending on diet [ 13 ]. Methanogenic archaea are important to digestion in detritus-feeding insects including beetles and termites [ 5 ]. Furthermore, as with gut bacteria, archaea can provide their insect hosts with additional nitrogen [ 5 , 14 ], contributing to nitrogen cycling processes such as nitrogen fixation [ 15 , 16 , 17 ] and nitrogenous waste recycling [ 18 ]. Investigating the microbiome of insects that inhabit typically nutrient-poor polar terrestrial ecosystems may provide unique insights into how these species establish and persist in these environments. Maistrenko et al. (2023) characterised the gut microbiome of the Antarctic continent’s only endemic insect, Belgica antarctica Jacobs (Diptera: Chironomidae), noting a surprisingly limited overall diversity compared to temperate species [ 20 ]. However, this study did not consider the nutrient cycling roles of any of the microorganisms identified. A closely related chironomid, Eretmoptera murphyi Schaeffer 1914 [ 19 ] (Diptera: Chironomidae; Fig. 1 ), is endemic to sub-Antarctic South Georgia and molecular phylogenetic analyses suggest that it is a sister species of B. antarctica , albeit with a multimillion-year evolutionary separation [ 21 ]. Brayley et al. (2025) noted the absence of the bacterium Wolbachia in E. murphyi [ 22 ], as is the case in all Antarctic invertebrate microbiomes studied to date [ 23 ] but did not examine this species’ microbiome further. Eretmoptera murphyi is now an established and invasive non-native species on Signy Island (South Orkney Islands, maritime Antarctic), where it was accidentally introduced most likely in the 1960s [ 24 ]. The fly’s larvae (Fig. 1 ) are detritivorous [ 25 , 26 ] and their presence is associated with an increase in available soil nitrate of up to five times compared with similar uncolonized substrate on the island [ 27 ]. The hypothesis that the microbiome of E, murphyi facilitates this increase remains unexamined, but is important to investigate, because such a significant change in nutrient availability could impact native microarthropod and plant communities and may also facilitate establishment of further non-native species [ 28 ]. The aims of this metagenomic study were to (1) conduct the first microbiome characterisation of E. murphyi , (2) suggest which groups of microorganisms may contribute to the elevated nutrient availability associated with this species on Signy Island, and (3) compare the E. murphyi microbiome with information available for other Antarctic invertebrates. In addition, given how little is known about Antarctic insect microbiomes, this study enhances understanding of the ecological roles of Antarctic invertebrates and contributes data to the region's under-represented microbial reference databases. Methods Sample collection Larvae of E. murphyi were collected on Signy Island (South Orkney Islands, maritime Antarctic) during the austral summer in February 2023. They were obtained from the ‘Backslope’ (unofficial name) adjacent to the British Antarctic Survey’s Signy Station on Berntsen Point, where the species is now patchily abundant [ 28 ]. The flora of the island, and specifically the study location, is dominated by cryptogams and extensive moss banks [ 27 , 29 ] and the soil is highly organic and ‘peaty’ [ 30 , 31 ]. Larvae were kept in a container under field conditions on Signy before being rinsed and then stored in 96% ethanol at -80ºC during their return to the UK by ship. Samples were maintained at -80ºC at the University of Birmingham until analysis at the University of Warwick in April 2024. Seven larvae of the same size and developmental stage (L3 instar) were selected for metagenomic analysis to avoid introducing any differences associated with developmental stage [ 32 , 33 ]. All larvae were checked to ensure absence of visible substrate on their cuticles before DNA extraction. Microbial DNA extraction and sequencing Total DNA was extracted from each individual larva separately using the DNeasy PowerSoil Pro Kit (QIAGEN). Larvae were first manually fragmented with a sterilised pipette tip in solution C1 before the other extraction steps were carried out following the manufacturer’s protocol. Extracted DNA was quantified using a NanoDrop ND-2000 (Wilmington) and then used as a template for PCR amplification. Universal primers were used to target and amplify the bacterial V4 region of the 16S rRNA, 515F (5ʹ - GTGCCAGCMGCCGCGGTAA − 3ʹ) and 806R (5ʹ - GGACTACHVGGGTWTCTAAT − 3ʹ) [ 34 ]. Customised primers were also used to target and amplify the archaeal V4 region, 519F (5ʹ - CAGYMGCCRCGGKAAHACC − 3ʹ) and 806R (5ʹ - GGACTACNSGGGTMTCTAAT − 3ʹ) [ 35 ]. Other PCR components were prepared using the NEBiolabs Q5 PCR kit following the manufacturer's protocol. Two separate PCR reactions were carried out for each extraction, with 28 cycles for bacterial DNA and 32 cycles for archaeal (95ºC for 30 s, 53ºC for 30 s), with a final extension step at 72ºC for 30 s. The target region of ~ 300 bp was obtained by gel electrophoresis, followed by extraction and purification using the QIAquick Gel Extraction Kit (QIAGEN) following the manufacturer’s protocol. The concentration of recovered DNA was again quantified using a NanoDrop ND-2000 and the five samples with the highest concentrations of archaeal and bacterial DNA were selected for sequencing. Library preparation and sequencing were carried out commercially on an Illumina PE250 platform by Novogene (UK). Sequencing analyses The DADA2 pipeline (V1.32.0) [ 36 ] was used for quality trimming, error rate estimation, merging, chimera removal and amplicon sequence variant (ASV) feature table construction. A total of 333,809 bacterial and 355,215 archaeal reads were obtained. Primer sequences were removed from the 5’ region of forward and reverse reads (19 bp and 20 bp, respectively) and reads were truncated at the first instance of a quality score ≤ 10. Following dereplication, merging and chimera removal, 69.7–73.1% bacterial reads and 61.9–78.4% archaeal reads were retained for further analysis. Taxonomy was assigned to genus level using the SILVA 138.1 prokaryotic SSU taxonomic training data, formatted for DADA2 and Zenodo [ 37 ]. ASVs classified as chloroplast, mitochondria or archaea were removed from the bacterial sequence dataset, and those classified as chloroplast, mitochondria or bacteria were removed from the archaeal dataset. To remove sequences potentially arising from the host insect species, a local BLAST search was performed against the assembled genome of the closely related Antarctic chironomid Belgica antarctica [ 38 ] using rBLAST, as genomic sequence data are not available for E. murphyi . ASVs with > 90% sequence similarity across 90% of the query length were removed [ 39 ]. The bacterial and archaeal datasets were rarefied to depths of 42,723 and 31,182, respectively, using the rrarefy function from the R package vegan v 2.6–6.1, with default settings [ 40 ]. ASVs that represented ≥ 1% of the total abundance in either of the sequence datasets were classed as ‘dominant’ (for both bacteria and archaea). The most abundant bacterial ASVs at different taxonomic levels were defined as those with abundance in at least one individual (sample) of ≥ 10% for phylum, ≥ 6% for class, ≥ 25% for order, ≥ 10% for family and ≥ 5% for genus. The most abundant archaea were defined as those with abundance of ≥ 84% across all ranks. These thresholds were chosen based on the distribution of relative abundances across the individuals and to reflect the taxa that contributed considerably to the composition in at least one individual, avoiding including groups that were consistently found in low abundances and/or with sporadic representation. Statistical analyses Statistical analyses were carried out using RStudio, version 2024.09.1 + 394. Beta-diversity measures were calculated using the phyloseq package [ 41 ] with a Bray-Curtis distance metric [Supplementary Information (S1, S2)]. Associated statistics were calculated using PERMANOVA [ 42 ] with 999 permutations to obtain p -values, using the vegan function. The R² value was used to estimate the proportion of variation between larvae. Results Beta diversity Across the five individual larvae analysed, there were no significant differences between the beta diversity of the bacterial (R 2 = 0.37, p = 0.125) or archaeal samples (R 2 = 0.18, p = 0.525). Bacterial ASV diversity A total of 2013 bacterial ASVs were assigned. Of these, 1971 were classified to at least phylum level. A mean of 677 (range: 480–752) unique ASVs were detected in each individual larva. Bacteria therefore represented 97.4% of the total assigned microbiome. Representatives of 24 bacterial phyla were assigned. The most abundant phyla were Actinobacteriota, Chloroflexi, Planctomycetota and Proteobacteria (Fig. 2 a). Of the 46 classes detected, the most abundant were Acidimicrobia, Actinobacteria, AD3, Alphaproteobacteria, Planctomycetes, Ktedonobacteria and Thermoleophilia (Fig. 2 b). The most abundant of the 113 orders detected were Frankiales, Micrococcales, Rickettsiales and Solirubrobacterales (Fig. 2 c). The most abundant families (148 detected) were Rickettsiaceae and Solirubrobacteraceae (Fig. 2 d). Of the 200 genera assigned, the most abundant were Acidothermus , Conexibacter , Humibacillus , Jatrophihabitans , Nakamurella and Rickettsia (Fig. 2 e). Only five ASVs were assigned to species, Tomitella biformata , M ethylocella palustris , Nakamurella panacisegeti , Faecalibacterium prausnitzii and Clostridium putrefaciens . Archaeal ASV diversity A total of 47 ASVs were assigned using archaeal primers. Of these, 29 were assigned to archaea and classified to at least phylum level. Four ASVs were assigned to Eukaryota and the remaining 14 ASVs were not classified; these were not included in the subsequent analyses. A mean of 18 (range: 13–29) unique archaeal ASVs were obtained from the five individual larvae. Archaea comprised 2.6% of the total ASVs recovered. The phylum Crenarchaeota dominated the assigned sequences [Supplementary Information (S3)]. Representatives of the single class Nitrososphaeria were detected and all but one ASVs represented the order Nitrososphaerales (the exception was assigned to the Group 1.1c order), family Nitrososphaeraceae and genus Candidatus Nitrocosmicus . No ASVs were classified to species level. Discussion Neither the overall microbial community composition (beta diversity) nor the relative abundance of archaeal and bacterial phyla differed significantly between the five individual larvae. This could be attributed to all samples being the same developmental life stage (L3 larvae) [ 32 ], as well as being sourced from the same habitat and soil type on Signy Island. It is not possible in a study of this type to confirm which of the archaea and bacteria assigned are true symbionts or commensal microorganisms of E. murphyi , as the DNA extraction method may have also detected the presence of microbial DNA on the external surface of larvae. Nonetheless, we suggest that the very high abundance of ASVs representing the phylum Crenarchaeota indicates that there are some species within this group that are endosymbiotic. Further, representatives of the bacterial order Rickettsiales (phylum Proteobacteria, class Alphaproteobacteria) are also likely to be symbionts as they are known to be obligate intracellular organisms within eukaryotic cells [ 43 ]. Finally, one of the few bacterial ASVs assigned to species level, Faecalibacterium prausnitzii , is primarily associated in the literature with the human gut microbiome [ 44 ]. Bartlett et al. (2023) concluded that E. murphyi larvae are associated with an increase in soil inorganic nitrogen, particularly nitrate, and elevated soil organic carbon [ 27 ]. To account for the increase in total inorganic nitrogen, we hypothesise that groups of microorganisms that were introduced to Signy Island in association with E. murphyi are capable of nitrogen fixation, whereby atmospheric nitrogen is reduced to ammonia or ammonium ions (Fig. 3 ). In particular, the elevated nitrates may indicate the presence of some groups associated with E. murphyi that are capable of ammonia oxidation, which is a limiting step during nitrification in the nitrogen cycle where ammonia is oxidised to nitrite [ 45 ]. Subsequent oxidation of nitrite to nitrate by other microorganisms is then required to release bioavailable nitrogen to plants and microarthropods [ 46 ]. Bacteria The most abundant bacterial phyla, Actinobacteriota, Chloroflexi, Proteobacteria and Planctomycetota, have all previously been reported from Signy soils [ 47 – 49 ]. Considering these abundant phyla, representatives of Actinobacteriota are known to be highly tolerant of desiccation and other stresses [ 50 ]. Chloroflexi are functionally diverse, including chemoautotrophs, photoautotrophs and thermophiles [ 51 ] and Proteobacteria are Gram-negative bacteria that are commonly reported in soils globally [ 52 ]. These three phyla play similar roles in the nitrogen cycle, including nitrogen fixation [ 53 ] (Fig. 3 ) and denitrification, where nitrate is converted back to nitrogen gas and released from the soil [ 54 ]. Further, Actinobacteriota play an important role in the carbon cycle, assisting with the decomposition of organic material and facilitating carbon sequestration in plants [ 55 ]. Representatives of Planctomycetota are also widely distributed, primarily in marine environments, and are known to be important in the nitrogen and carbon cycles [ 56 ], including in ammonia oxidation [ 57 ]. Within the abundant bacterial classes, Acidimicrobia (phylum Actinobacteria), are generally associated with low soil pH conditions (3.0–6.5) [ 58 ], consistent with the low pH of Signy soil. They play an important role in the carbon cycle, possessing genes associated with the breakdown of organic molecules such as carbohydrates (e.g., cellulose) and nitrogen-containing compounds (forming bacterial cellulose), and are significant decomposers within the soil community [ 59 ]. They may provide nutrition to E. murphyi by facilitating decomposition, as well as potentially conferring desiccation resistance through the formation of intestinal biofilms [ 60 ]. Studies of the genes associated with Ktendonobacteria (phylum Chloroflexi) have suggested that members of this class can carry out autotrophic carbon fixation [ 61 , 62 ]. Such activity could reduce E. murphyi ’s dependence on organic material as its primary carbon source [ 63 ]. Members of the family Solirubrobacteraceae (phylum Actinobacteriota, class Thermoleophilia) have been detected in desert environments exposed to high UV radiation where they increase nitrogen availability and absorption [ 64 ], again potentially contributing to E. murphyi nutrient uptake. Archaea The most abundant archaea, Crenarchaeota (also known as the Thaumarchaeota) [ 65 , 66 ], are associated with sulphur-dependent thermophiles [ 67 ] and are generally found in high- or low-temperature environments [ 68 ]. Members of Crenarchaeota are known to perform ammonia oxidation [ 53 , 68 , 69 ] (Fig. 3 ). This process may be facilitated by the most abundant archaeal class identified in the microbiome here, Nitrosophaeria, whose members are typically found in nitrogen-limited and low pH soils, such as those occurring on Signy, and also in the McMurdo Dry Valleys [ 70 ]. This process is also undertaken by the bacterial class Planctomycetes [ 71 , 72 ]. Crenarchaeota have been reported in the guts of detritivorous insects, including termites ( Cubitermes orthognathus ) [ 73 ] and beetles ( Oryctes nasicornis and Amphimallon solstitiale ) [ 12 ]. This, combined with the particularly high abundance of Crenarchaeota found here, suggests that ammonia oxidation may contribute to the survival of E. murphyi larvae by allowing them to utilise the released nutrients for their growth. This may be a particularly important adaptation supporting the presence of the fly on Signy where, like much of Antarctica, nutrients (particularly nitrogen-containing compounds) are generally limited [ 74 , 75 ]. Crenarchaeota have previously been reported from Signy (although not from soil), contributing 77% of the total sequences obtained from cryoconite holes [ 76 ]. Elsewhere in Antarctica, this phylum represented 80% of all archaeal sequences in soil from the McMurdo Dry Valleys [ 53 , 77 ]. Such observations suggest that there may be a strong link between the archaeal microbiome of E. murphyi and that of the local environment. Comparison of E. murphyi microbiome with other Antarctic invertebrates Maistrenko et al. (2023) provide the only available data on the bacterial community associated with an insect endemic to the Antarctic continent, B. antarctica [ 20 ]. This study combined data from both adults and larvae but only reported the presence of five bacterial phyla. In contrast, our E. murphyi (larvae only) dataset recovered 24 bacterial phyla (Fig. 2 A), which is comparable to temperate chironomids such as Chironomus ramosus in India (22 phyla) [ 79 ] and C. transvaalensis in Israel (20 phyla) [ 32 ]. Four of the bacterial phyla identified in B. antarctica were shared with E. murphyi - Actinobacteria, Bacterioidetes, Firmicutes and Proteobacteria. The fifth, Fusobacteria, was not detected in the current study. Both insect species share representatives of two assigned genera, Humibacillus and Pseudomonas , although only two ASVs were assigned to these taxa here. Other genera assigned in B. antarctica but not detected in E. murphyi were Arthrobacter, Cutibacterium, Porphyromonas, Lactococcus, Pelmonas, Janthinobacterium, Neisseria, Escherichia, Serratia and Yersinia . This comparison suggests that the bacterial communities associated with these two sister but biogeographically isolated insect species from the Antarctic region [ 21 ] are very different and unique. Maistrenko et al. (2023) utilised existing and archival whole-genome sequencing data to identify bacterial species associated with B. antarctica , which likely only gave a very limited characterisation as this is not a targeted approach. Further studies are also required to disentangle potential changes in each species microbiome across different developmental stages. As both species are detritivores [ 24 , 80 ], it is likely that their gut microbial communities will reflect the substrate upon which they feed. Thus, some of the differences in microbial diversity found may be due to differences in the soil properties and associated microbial communities between the distinct geographical locations from which they originate. Belgica antarctica is endemic to the South Shetland Islands and western coastal regions of the Antarctic Peninsula, while E. murphyi is endemic to sub-Antarctic South Georgia with an introduced population on maritime Antarctic Signy Island [ 81 , 82 ]. The highest densities of B. antarctica larvae are found in moss [ 83 ]; by contrast, E. murphyi on Signy favours dead organic matter and soil/peat substrata [ 84 ]. There may also be differences in the morphology of the gut [ 5 ] between the two species, although detailed gut description is currently only available for B. antarctica [ 80 ]. Leo et al. (2021) employed metagenomic sequencing to investigate the bacterial microbiomes of four Antarctic springtail species, Cryptopygus antarcticus (Collembola: Isotomidae) from the South Shetland Islands (maritime Antarctic), Friesea antarctica (Collembola: Neanuridae) from the South Shetland Islands and Lagoon Island (Marguerite Bay) and C. terranovus (Collembola: Isotomidae) and F. propria (Collembola: Neanuridae) from continental Antarctica (North Victoria Land) [ 85 ]. Of the six most abundant phyla identified, all but Fusobacteria were shared with E. murphyi , and of the most abundant bacterial orders, only Betaproteobacteriales, Flavobacteriales and Fusobacteriales were not shared with E. murphyi . (Fig. 4 ). As with B. antarctica , these springtail species had lower diversity bacterial microbiomes than E. murphy i. Holmes et al. (2019) characterised the microbiome of the Antarctic oribatid mite, Alaskozetes antarcticus (Oribatida: Trhypochthoniidae), from Cormorant Island, south of Anvers Island off the western Antarctic Peninsula [ 86 ]. The dominant bacterial classes detected in mite tritonymphs were Actinobacteriota, Flavobacteriia, Sphingobacteriia, Gammaproteobacteria and Betaproteobacteria. These fall within the phyla Actinobacteriota, Bacteroidetes and Proteobacteria, which are all shared with E. murphyi (Fig. 4 ). Representatives of 31 families were detected, 117 less than in E. murphyi , again suggesting systematic differences in diversity of invertebrate-associated microbial communities, although in this case a higher diversity than present in mites from lower latitudes. Finally, Vecchi et al. (2018) assessed the bacterial microbiome of the Antarctic tardigrade, Acutuncus antarcticus (Parachela: Hypsibiidae), from Victoria Land (continental Antarctica) [ 87 ]. This, again, suggested many similarities with E. murphyi in terms of shared phyla, with only Gemmatimonadetes not found in our study (Fig. 4 ). Overall, the relatively diverse microbiome of E. murphyi differs from other Antarctic invertebrates examined to date, although the possible effects of methodological differences cannot be discounted. Finally, not all previous studies have characterised microorganisms to genus level, and those that have all utilise different threshold levels and pipelines, so in-depth comparisons remain difficult. To test hypotheses on whether E. murphyi ’s microbiome has facilitated its success as an invader on Signy, future research must address which archaea and bacteria are true symbionts of E. murphyi . This could be achieved by using bleach to remove external microorganisms [ 88 ] or dissecting the digestive tract and isolating the associated microorganisms [ 89 ]. Analysis of microbial community differences between larval stages and the non-feeding adults would also contribute. Access to fresh material from the species’ native South Georgia, where the fly is increasingly hard to find within areas recently colonised by non-native and aggressive predatory beetles [ 90 , 91 ], would also allow assessment of the extent to which the fly’s microbiome has altered in the c. 60 years since its introduction to Signy Island. Although amplicon sequencing is a powerful tool, it should be noted that this methodology does not confirm the activity of microorganisms, nor does it differentiate between expressed and non-expressed genes [ 92 ]. To resolve this, a combination of ‘omics’ technologies could be applied, including metatranscriptomics, to assess the activity of the microbiome community [ 64 ], their associated functions in insect nutrition [ 93 ] and, potentially, the wider environment. Conclusions We provide the first assessment of the archaeal and bacterial microbiomes associated with E. murphyi on Signy Island, maritime Antarctic. The most abundant archaeal and bacterial phyla were Crenarchaeota, Actinobacteriota, Chloroflexi, Proteobacteria and Planctomycetota, all of which have known roles in nitrogen cycling, with some possibly also involved in carbon cycling. Association with representatives of these phyla may allow E. murphyi to access additional energy sources in the nutrient-depleted soils of Signy Island which, in turn, could release key nutrients into the peaty soil. This may provide a mechanism for the elevated inorganic nitrogen and organic carbon levels previously reported in soil associated with the insect. Eretmoptera murphyi appears to have a more diverse microbiome (at the phylum level) than several native Antarctic invertebrates studied to date, but differences in methodologies across studies currently limit robust comparisons. Further research is also required to confirm which organisms are true endosymbionts/commensals. Finally, comparison of the Signy E. murphyi microbiome with that of the original source population from South Georgia would enable the first confirmation of the combined transfer of microbiota in an introduction event to Antarctica. Declarations Acknowledgements The authors are very grateful to Monica Aquilino for sample collection. Funding O. Brayley and S. Liu are funded by NERC CENTA2 grant NE/S007350/1. O. Brayley’s PhD is also supported by the British Antarctic Survey. S. Hayward is supported by a Leverhulme Research Fellowship RF-2024-396/2. P. Convey is supported by NERC core funding to the British Antarctic Survey ‘Biodiversity, Evolution and Adaptation’ Team. S. Hayward, P. Convey and K. McCready were also supported by NSFGEO-NERC grant NE/T009446/1. N. Teets is supported by the National Science Foundation Grant OPP-1850988 and USDA National Institute of Food and Agriculture Hatch Project 700545. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author contributions ODMB performed the DNA extractions, analysed the data and wrote the initial draft. SL assisted with DNA extractions. KM assisted with the bioinformatic analysis and edited the pipeline. PC, YC, SU, NT and SALH contributed to manuscript writing and editing. All authors gave final approval for manuscript submission. Data Availability The ASVs generated during the current study and their taxonomic assignment are available in the Supplementary Information. References Feldhaar, H., 2011. Bacterial symbionts as mediators of ecologically important traits of insect hosts. Ecological Entomology 36, 533–543. https://doi.org/10.1111/j.1365-2311.2011.01318.x Schmidt, K., Engel, P., 2021. 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Supplementary Files SupplementaryInformationOctaviaBrayley.docx SupplementaryInformationAssignmentofASVs.txt SupplementaryInformationASVsgenerated.txt Cite Share Download PDF Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Microbial Ecology → Version 1 posted Editorial decision: Revision requested 03 Nov, 2025 Reviews received at journal 03 Nov, 2025 Reviews received at journal 28 Oct, 2025 Reviews received at journal 28 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviews received at journal 03 Oct, 2025 Reviewers agreed at journal 02 Oct, 2025 Reviewers agreed at journal 01 Oct, 2025 Reviewers agreed at journal 01 Oct, 2025 Reviewers agreed at journal 01 Oct, 2025 Reviewers invited by journal 01 Oct, 2025 Editor assigned by journal 01 Oct, 2025 Submission checks completed at journal 30 Sep, 2025 First submitted to journal 29 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7744438","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":525850259,"identity":"105b6e9c-e17a-48d3-84be-b7393a4d98d9","order_by":0,"name":"Octavia D. M. 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M.","lastName":"Brayley","suffix":""},{"id":525850260,"identity":"e809f061-7856-4563-8451-1fb62545d30e","order_by":1,"name":"Kirsty McCready","email":"","orcid":"","institution":"University of Liverpool","correspondingAuthor":false,"prefix":"","firstName":"Kirsty","middleName":"","lastName":"McCready","suffix":""},{"id":525850261,"identity":"25f289af-5c0c-44f1-820e-c9bc543fd406","order_by":2,"name":"Shengwei Liu","email":"","orcid":"","institution":"University of Warwick","correspondingAuthor":false,"prefix":"","firstName":"Shengwei","middleName":"","lastName":"Liu","suffix":""},{"id":525850262,"identity":"616f9b94-9178-40a7-8bc3-8c398d56041b","order_by":3,"name":"Peter Convey","email":"","orcid":"","institution":"British Antarctic Survey","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Convey","suffix":""},{"id":525850263,"identity":"b58ca499-d4ab-4dc7-a26e-a00fe3c0b80d","order_by":4,"name":"Yin Chen","email":"","orcid":"","institution":"University of Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Yin","middleName":"","lastName":"Chen","suffix":""},{"id":525850264,"identity":"95ececf3-354b-41a8-8546-f901dfc262b9","order_by":5,"name":"Sami Ullah","email":"","orcid":"","institution":"University of Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Sami","middleName":"","lastName":"Ullah","suffix":""},{"id":525850265,"identity":"434657b3-6892-4009-9d6c-1f874fe4ec2a","order_by":6,"name":"Nicholas Teets","email":"","orcid":"","institution":"University of Kentucky","correspondingAuthor":false,"prefix":"","firstName":"Nicholas","middleName":"","lastName":"Teets","suffix":""},{"id":525850266,"identity":"44e23c62-4e1a-48bc-b197-bc477f758829","order_by":7,"name":"Scott A.L. Hayward","email":"","orcid":"","institution":"University of Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Scott","middleName":"A.L.","lastName":"Hayward","suffix":""}],"badges":[],"createdAt":"2025-09-29 17:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7744438/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7744438/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00248-026-02706-5","type":"published","date":"2026-02-28T15:59:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":93533410,"identity":"c7704600-610d-464f-890d-eff683264cba","added_by":"auto","created_at":"2025-10-14 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23:24:02","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":209739,"visible":true,"origin":"","legend":"","description":"","filename":"d3e484b75d8d43f2a59ede3b949b488f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7744438/v1/ee4734b04216f913a629921d.xml"},{"id":93533530,"identity":"ce97b832-0308-4a3a-a2fe-ae4ea55839c1","added_by":"auto","created_at":"2025-10-14 23:32:01","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":227339,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7744438/v1/71905d21280ee52cc09b5298.html"},{"id":93533525,"identity":"e6f23d8d-ec42-40d2-bca5-653a39c67dfd","added_by":"auto","created_at":"2025-10-14 23:32:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":522687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEretmoptera murphyi \u003c/em\u003eSchaeffer 1914 [19] (Diptera: Chironomidae) adult (left) and larvae (right). Images: British Antarctic Survey\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7744438/v1/7f90e464ec44ea57e223c16f.png"},{"id":93533406,"identity":"05ca1900-e48e-425c-b8c3-682316bb4bd0","added_by":"auto","created_at":"2025-10-14 23:24:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":504134,"visible":true,"origin":"","legend":"\u003cp\u003eStacked bar plots showing relative abundances (as percentage of sequences obtained) of the dominant (≥ 1%) bacterial phyla (A), classes (B), orders (C), families (D) and genera (E) detected in each individual \u003cem\u003eE. murphyi \u003c/em\u003elarva (sample).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7744438/v1/6e52740ea090cb93a5b88836.png"},{"id":93533413,"identity":"2de3b5e7-fc1f-4ad9-8201-7d3401c13b0b","added_by":"auto","created_at":"2025-10-14 23:24:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":321089,"visible":true,"origin":"","legend":"\u003cp\u003eA simplified illustration of the soil nitrogen cycle, indicating candidate archaeal and bacterial phyla present in the microbiome of \u003cem\u003eE. murphyi\u003c/em\u003e that may facilitate certain steps. A large proportion of microorganisms involved with nitrogen fixation and nitrification may contribute to the enhancement of nitrate concentrations in Signy soil. Re-drawn from Makhalanyane et al.\u003cem\u003e \u003c/em\u003e(2016) [78].\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7744438/v1/9aac84318e10504948259c9a.png"},{"id":93533528,"identity":"bbb96d3b-ebd6-429e-947a-5c34429d9823","added_by":"auto","created_at":"2025-10-14 23:32:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":189173,"visible":true,"origin":"","legend":"\u003cp\u003ePresence/absence heat map comparing the most abundant bacterial phyla associated with \u003cem\u003eE. murphyi\u003c/em\u003e and other studied Antarctic invertebrates.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7744438/v1/50c1bb0ef38e5db10ed45f67.png"},{"id":103765676,"identity":"3156bc24-27f0-41ff-ad85-c4574df06858","added_by":"auto","created_at":"2026-03-02 16:07:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2171592,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7744438/v1/c982455c-2219-4324-bbe5-ff220bf7dcbf.pdf"},{"id":93533407,"identity":"0aa913b1-99cb-4928-9273-88dd8eb53316","added_by":"auto","created_at":"2025-10-14 23:24:01","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":284373,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationOctaviaBrayley.docx","url":"https://assets-eu.researchsquare.com/files/rs-7744438/v1/3c3daad2e660b4a39b4574a2.docx"},{"id":93533408,"identity":"83c57841-d2b2-41fc-951a-ebbd626e9c26","added_by":"auto","created_at":"2025-10-14 23:24:01","extension":"txt","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":168781,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationAssignmentofASVs.txt","url":"https://assets-eu.researchsquare.com/files/rs-7744438/v1/7d60e7aa584915cac8b7d1fc.txt"},{"id":93533420,"identity":"f80750e6-75b1-4048-8dd5-32bfaa69ac0b","added_by":"auto","created_at":"2025-10-14 23:24:01","extension":"txt","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":562923,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationASVsgenerated.txt","url":"https://assets-eu.researchsquare.com/files/rs-7744438/v1/84e3d460c7be5380f80374d4.txt"}],"financialInterests":"No competing interests reported.","formattedTitle":"The microbiome of an invasive Antarctic insect, Eretmoptera murphyi (Diptera: Chironomidae), and its potential role in nutrient cycling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe diversity and ecological success of insects is partly due to their associated microbiomes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These communities of bacteria, archaea, fungi, protozoa and viruses influence many aspects of host biology [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], facilitated by long co-evolutionary relationships [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Bacteria are the most abundant and diverse microorganisms present in the microbiome [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Mutualistic species can boost energy metabolism and play essential roles in nutrient acquisition [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], as well as in the host immune system [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], developmental processes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and fecundity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Microbiome composition changes seasonally in some insects [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and can contribute to winter/cold adaptation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Studies of the relationships of archaea with their hosts are currently under-represented in the literature, primarily because this domain represents a smaller proportion of the microbiome compared to other groups [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Nonetheless, archaea can also play an important role in insect metabolism, such as the fruit fly, \u003cem\u003eAnastrepha obliqua\u003c/em\u003e, with communities changing across developmental stages and depending on diet [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Methanogenic archaea are important to digestion in detritus-feeding insects including beetles and termites [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, as with gut bacteria, archaea can provide their insect hosts with additional nitrogen [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], contributing to nitrogen cycling processes such as nitrogen fixation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and nitrogenous waste recycling [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eInvestigating the microbiome of insects that inhabit typically nutrient-poor polar terrestrial ecosystems may provide unique insights into how these species establish and persist in these environments. Maistrenko et al. (2023) characterised the gut microbiome of the Antarctic continent\u0026rsquo;s only endemic insect, \u003cem\u003eBelgica antarctica\u003c/em\u003e Jacobs (Diptera: Chironomidae), noting a surprisingly limited overall diversity compared to temperate species [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, this study did not consider the nutrient cycling roles of any of the microorganisms identified. A closely related chironomid, \u003cem\u003eEretmoptera murphyi\u003c/em\u003e Schaeffer 1914 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] (Diptera: Chironomidae; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), is endemic to sub-Antarctic South Georgia and molecular phylogenetic analyses suggest that it is a sister species of \u003cem\u003eB. antarctica\u003c/em\u003e, albeit with a multimillion-year evolutionary separation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Brayley et al. (2025) noted the absence of the bacterium \u003cem\u003eWolbachia\u003c/em\u003e in \u003cem\u003eE. murphyi\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], as is the case in all Antarctic invertebrate microbiomes studied to date [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] but did not examine this species\u0026rsquo; microbiome further. \u003cem\u003eEretmoptera murphyi\u003c/em\u003e is now an established and invasive non-native species on Signy Island (South Orkney Islands, maritime Antarctic), where it was accidentally introduced most likely in the 1960s [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The fly\u0026rsquo;s larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) are detritivorous [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and their presence is associated with an increase in available soil nitrate of up to five times compared with similar uncolonized substrate on the island [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The hypothesis that the microbiome of \u003cem\u003eE, murphyi\u003c/em\u003e facilitates this increase remains unexamined, but is important to investigate, because such a significant change in nutrient availability could impact native microarthropod and plant communities and may also facilitate establishment of further non-native species [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe aims of this metagenomic study were to (1) conduct the first microbiome characterisation of \u003cem\u003eE. murphyi\u003c/em\u003e, (2) suggest which groups of microorganisms may contribute to the elevated nutrient availability associated with this species on Signy Island, and (3) compare the \u003cem\u003eE. murphyi\u003c/em\u003e microbiome with information available for other Antarctic invertebrates. In addition, given how little is known about Antarctic insect microbiomes, this study enhances understanding of the ecological roles of Antarctic invertebrates and contributes data to the region's under-represented microbial reference databases.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSample collection\u003c/h2\u003e\u003cp\u003eLarvae of \u003cem\u003eE. murphyi\u003c/em\u003e were collected on Signy Island (South Orkney Islands, maritime Antarctic) during the austral summer in February 2023. They were obtained from the \u0026lsquo;Backslope\u0026rsquo; (unofficial name) adjacent to the British Antarctic Survey\u0026rsquo;s Signy Station on Berntsen Point, where the species is now patchily abundant [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The flora of the island, and specifically the study location, is dominated by cryptogams and extensive moss banks [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and the soil is highly organic and \u0026lsquo;peaty\u0026rsquo; [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Larvae were kept in a container under field conditions on Signy before being rinsed and then stored in 96% ethanol at -80\u0026ordm;C during their return to the UK by ship. Samples were maintained at -80\u0026ordm;C at the University of Birmingham until analysis at the University of Warwick in April 2024. Seven larvae of the same size and developmental stage (L3 instar) were selected for metagenomic analysis to avoid introducing any differences associated with developmental stage [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. All larvae were checked to ensure absence of visible substrate on their cuticles before DNA extraction.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMicrobial DNA extraction and sequencing\u003c/h3\u003e\n\u003cp\u003eTotal DNA was extracted from each individual larva separately using the DNeasy PowerSoil Pro Kit (QIAGEN). Larvae were first manually fragmented with a sterilised pipette tip in solution C1 before the other extraction steps were carried out following the manufacturer\u0026rsquo;s protocol. Extracted DNA was quantified using a NanoDrop ND-2000 (Wilmington) and then used as a template for PCR amplification. Universal primers were used to target and amplify the bacterial V4 region of the 16S rRNA, 515F (5ʹ - GTGCCAGCMGCCGCGGTAA \u0026minus;\u0026thinsp;3ʹ) and 806R (5ʹ - GGACTACHVGGGTWTCTAAT \u0026minus;\u0026thinsp;3ʹ) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Customised primers were also used to target and amplify the archaeal V4 region, 519F (5ʹ - CAGYMGCCRCGGKAAHACC \u0026minus;\u0026thinsp;3ʹ) and 806R (5ʹ - GGACTACNSGGGTMTCTAAT \u0026minus;\u0026thinsp;3ʹ) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Other PCR components were prepared using the NEBiolabs Q5 PCR kit following the manufacturer's protocol. Two separate PCR reactions were carried out for each extraction, with 28 cycles for bacterial DNA and 32 cycles for archaeal (95\u0026ordm;C for 30 s, 53\u0026ordm;C for 30 s), with a final extension step at 72\u0026ordm;C for 30 s. The target region of ~\u0026thinsp;300 bp was obtained by gel electrophoresis, followed by extraction and purification using the QIAquick Gel Extraction Kit (QIAGEN) following the manufacturer\u0026rsquo;s protocol. The concentration of recovered DNA was again quantified using a NanoDrop ND-2000 and the five samples with the highest concentrations of archaeal and bacterial DNA were selected for sequencing. Library preparation and sequencing were carried out commercially on an Illumina PE250 platform by Novogene (UK).\u003c/p\u003e\n\u003ch3\u003eSequencing analyses\u003c/h3\u003e\n\u003cp\u003eThe DADA2 pipeline (V1.32.0) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] was used for quality trimming, error rate estimation, merging, chimera removal and amplicon sequence variant (ASV) feature table construction. A total of 333,809 bacterial and 355,215 archaeal reads were obtained. Primer sequences were removed from the 5\u0026rsquo; region of forward and reverse reads (19 bp and 20 bp, respectively) and reads were truncated at the first instance of a quality score\u0026thinsp;\u0026le;\u0026thinsp;10. Following dereplication, merging and chimera removal, 69.7\u0026ndash;73.1% bacterial reads and 61.9\u0026ndash;78.4% archaeal reads were retained for further analysis. Taxonomy was assigned to genus level using the SILVA 138.1 prokaryotic SSU taxonomic training data, formatted for DADA2 and Zenodo [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. ASVs classified as chloroplast, mitochondria or archaea were removed from the bacterial sequence dataset, and those classified as chloroplast, mitochondria or bacteria were removed from the archaeal dataset. To remove sequences potentially arising from the host insect species, a local BLAST search was performed against the assembled genome of the closely related Antarctic chironomid \u003cem\u003eBelgica antarctica\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] using rBLAST, as genomic sequence data are not available for \u003cem\u003eE. murphyi\u003c/em\u003e. ASVs with \u0026gt;\u0026thinsp;90% sequence similarity across 90% of the query length were removed [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The bacterial and archaeal datasets were rarefied to depths of 42,723 and 31,182, respectively, using the rrarefy function from the R package vegan v 2.6\u0026ndash;6.1, with default settings [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. ASVs that represented\u0026thinsp;\u0026ge;\u0026thinsp;1% of the total abundance in either of the sequence datasets were classed as \u0026lsquo;dominant\u0026rsquo; (for both bacteria and archaea). The most abundant bacterial ASVs at different taxonomic levels were defined as those with abundance in at least one individual (sample) of \u0026ge;\u0026thinsp;10% for phylum, \u0026ge; 6% for class, \u0026ge; 25% for order, \u0026ge; 10% for family and \u0026ge;\u0026thinsp;5% for genus. The most abundant archaea were defined as those with abundance of \u0026ge;\u0026thinsp;84% across all ranks. These thresholds were chosen based on the distribution of relative abundances across the individuals and to reflect the taxa that contributed considerably to the composition in at least one individual, avoiding including groups that were consistently found in low abundances and/or with sporadic representation.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eStatistical analyses were carried out using RStudio, version 2024.09.1\u0026thinsp;+\u0026thinsp;394. Beta-diversity measures were calculated using the phyloseq package [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] with a Bray-Curtis distance metric [Supplementary Information (S1, S2)]. Associated statistics were calculated using PERMANOVA [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] with 999 permutations to obtain \u003cem\u003ep\u003c/em\u003e-values, using the vegan function. The R\u0026sup2; value was used to estimate the proportion of variation between larvae.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eBeta diversity\u003c/h2\u003e\u003cp\u003eAcross the five individual larvae analysed, there were no significant differences between the beta diversity of the bacterial (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.37, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.125) or archaeal samples (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.18, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.525).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBacterial ASV diversity\u003c/h3\u003e\n\u003cp\u003eA total of 2013 bacterial ASVs were assigned. Of these, 1971 were classified to at least phylum level. A mean of 677 (range: 480\u0026ndash;752) unique ASVs were detected in each individual larva. Bacteria therefore represented 97.4% of the total assigned microbiome. Representatives of 24 bacterial phyla were assigned. The most abundant phyla were Actinobacteriota, Chloroflexi, Planctomycetota and Proteobacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Of the 46 classes detected, the most abundant were Acidimicrobia, Actinobacteria, AD3, Alphaproteobacteria, Planctomycetes, Ktedonobacteria and Thermoleophilia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The most abundant of the 113 orders detected were Frankiales, Micrococcales, Rickettsiales and Solirubrobacterales (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The most abundant families (148 detected) were Rickettsiaceae and Solirubrobacteraceae (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Of the 200 genera assigned, the most abundant were \u003cem\u003eAcidothermus\u003c/em\u003e, \u003cem\u003eConexibacter\u003c/em\u003e, \u003cem\u003eHumibacillus\u003c/em\u003e, \u003cem\u003eJatrophihabitans\u003c/em\u003e, \u003cem\u003eNakamurella\u003c/em\u003e and \u003cem\u003eRickettsia\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Only five ASVs were assigned to species, \u003cem\u003eTomitella biformata\u003c/em\u003e, M\u003cem\u003eethylocella palustris\u003c/em\u003e, \u003cem\u003eNakamurella panacisegeti\u003c/em\u003e, \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e and \u003cem\u003eClostridium putrefaciens\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003eArchaeal ASV diversity\u003c/h3\u003e\n\u003cp\u003eA total of 47 ASVs were assigned using archaeal primers. Of these, 29 were assigned to archaea and classified to at least phylum level. Four ASVs were assigned to Eukaryota and the remaining 14 ASVs were not classified; these were not included in the subsequent analyses. A mean of 18 (range: 13\u0026ndash;29) unique archaeal ASVs were obtained from the five individual larvae. Archaea comprised 2.6% of the total ASVs recovered. The phylum Crenarchaeota dominated the assigned sequences [Supplementary Information (S3)]. Representatives of the single class Nitrososphaeria were detected and all but one ASVs represented the order Nitrososphaerales (the exception was assigned to the Group 1.1c order), family Nitrososphaeraceae and genus \u003cem\u003eCandidatus Nitrocosmicus\u003c/em\u003e. No ASVs were classified to species level.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNeither the overall microbial community composition (beta diversity) nor the relative abundance of archaeal and bacterial phyla differed significantly between the five individual larvae. This could be attributed to all samples being the same developmental life stage (L3 larvae) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], as well as being sourced from the same habitat and soil type on Signy Island. It is not possible in a study of this type to confirm which of the archaea and bacteria assigned are true symbionts or commensal microorganisms of \u003cem\u003eE. murphyi\u003c/em\u003e, as the DNA extraction method may have also detected the presence of microbial DNA on the external surface of larvae. Nonetheless, we suggest that the very high abundance of ASVs representing the phylum Crenarchaeota indicates that there are some species within this group that are endosymbiotic. Further, representatives of the bacterial order Rickettsiales (phylum Proteobacteria, class Alphaproteobacteria) are also likely to be symbionts as they are known to be obligate intracellular organisms within eukaryotic cells [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Finally, one of the few bacterial ASVs assigned to species level, \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e, is primarily associated in the literature with the human gut microbiome [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBartlett et al. (2023) concluded that \u003cem\u003eE. murphyi\u003c/em\u003e larvae are associated with an increase in soil inorganic nitrogen, particularly nitrate, and elevated soil organic carbon [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To account for the increase in total inorganic nitrogen, we hypothesise that groups of microorganisms that were introduced to Signy Island in association with \u003cem\u003eE. murphyi\u003c/em\u003e are capable of nitrogen fixation, whereby atmospheric nitrogen is reduced to ammonia or ammonium ions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In particular, the elevated nitrates may indicate the presence of some groups associated with \u003cem\u003eE. murphyi\u003c/em\u003e that are capable of ammonia oxidation, which is a limiting step during nitrification in the nitrogen cycle where ammonia is oxidised to nitrite [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Subsequent oxidation of nitrite to nitrate by other microorganisms is then required to release bioavailable nitrogen to plants and microarthropods [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eBacteria\u003c/h2\u003e\u003cp\u003eThe most abundant bacterial phyla, Actinobacteriota, Chloroflexi, Proteobacteria and Planctomycetota, have all previously been reported from Signy soils [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Considering these abundant phyla, representatives of Actinobacteriota are known to be highly tolerant of desiccation and other stresses [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Chloroflexi are functionally diverse, including chemoautotrophs, photoautotrophs and thermophiles [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and Proteobacteria are Gram-negative bacteria that are commonly reported in soils globally [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. These three phyla play similar roles in the nitrogen cycle, including nitrogen fixation [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and denitrification, where nitrate is converted back to nitrogen gas and released from the soil [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Further, Actinobacteriota play an important role in the carbon cycle, assisting with the decomposition of organic material and facilitating carbon sequestration in plants [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Representatives of Planctomycetota are also widely distributed, primarily in marine environments, and are known to be important in the nitrogen and carbon cycles [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], including in ammonia oxidation [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWithin the abundant bacterial classes, Acidimicrobia (phylum Actinobacteria), are generally associated with low soil pH conditions (3.0\u0026ndash;6.5) [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], consistent with the low pH of Signy soil. They play an important role in the carbon cycle, possessing genes associated with the breakdown of organic molecules such as carbohydrates (e.g., cellulose) and nitrogen-containing compounds (forming bacterial cellulose), and are significant decomposers within the soil community [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. They may provide nutrition to \u003cem\u003eE. murphyi\u003c/em\u003e by facilitating decomposition, as well as potentially conferring desiccation resistance through the formation of intestinal biofilms [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Studies of the genes associated with Ktendonobacteria (phylum Chloroflexi) have suggested that members of this class can carry out autotrophic carbon fixation [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Such activity could reduce \u003cem\u003eE. murphyi\u003c/em\u003e\u0026rsquo;s dependence on organic material as its primary carbon source [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Members of the family Solirubrobacteraceae (phylum Actinobacteriota, class Thermoleophilia) have been detected in desert environments exposed to high UV radiation where they increase nitrogen availability and absorption [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], again potentially contributing to \u003cem\u003eE. murphyi\u003c/em\u003e nutrient uptake.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eArchaea\u003c/h2\u003e\u003cp\u003eThe most abundant archaea, Crenarchaeota (also known as the Thaumarchaeota) [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], are associated with sulphur-dependent thermophiles [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] and are generally found in high- or low-temperature environments [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Members of Crenarchaeota are known to perform ammonia oxidation [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This process may be facilitated by the most abundant archaeal class identified in the microbiome here, Nitrosophaeria, whose members are typically found in nitrogen-limited and low pH soils, such as those occurring on Signy, and also in the McMurdo Dry Valleys [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. This process is also undertaken by the bacterial class Planctomycetes [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Crenarchaeota have been reported in the guts of detritivorous insects, including termites (\u003cem\u003eCubitermes orthognathus\u003c/em\u003e) [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e] and beetles (\u003cem\u003eOryctes nasicornis\u003c/em\u003e and \u003cem\u003eAmphimallon solstitiale\u003c/em\u003e) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This, combined with the particularly high abundance of Crenarchaeota found here, suggests that ammonia oxidation may contribute to the survival of \u003cem\u003eE. murphyi\u003c/em\u003e larvae by allowing them to utilise the released nutrients for their growth. This may be a particularly important adaptation supporting the presence of the fly on Signy where, like much of Antarctica, nutrients (particularly nitrogen-containing compounds) are generally limited [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Crenarchaeota have previously been reported from Signy (although not from soil), contributing 77% of the total sequences obtained from cryoconite holes [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Elsewhere in Antarctica, this phylum represented 80% of all archaeal sequences in soil from the McMurdo Dry Valleys [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Such observations suggest that there may be a strong link between the archaeal microbiome of \u003cem\u003eE. murphyi\u003c/em\u003e and that of the local environment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eComparison of\u003c/em\u003e E. murphyi \u003cem\u003emicrobiome with other Antarctic invertebrates\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMaistrenko et al. (2023) provide the only available data on the bacterial community associated with an insect endemic to the Antarctic continent, \u003cem\u003eB. antarctica\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This study combined data from both adults and larvae but only reported the presence of five bacterial phyla. In contrast, our \u003cem\u003eE. murphyi\u003c/em\u003e (larvae only) dataset recovered 24 bacterial phyla (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), which is comparable to temperate chironomids such as \u003cem\u003eChironomus ramosus\u003c/em\u003e in India (22 phyla) [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e] and \u003cem\u003eC. transvaalensis\u003c/em\u003e in Israel (20 phyla) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Four of the bacterial phyla identified in \u003cem\u003eB. antarctica\u003c/em\u003e were shared with \u003cem\u003eE. murphyi\u003c/em\u003e - Actinobacteria, Bacterioidetes, Firmicutes and Proteobacteria. The fifth, Fusobacteria, was not detected in the current study. Both insect species share representatives of two assigned genera, \u003cem\u003eHumibacillus\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e, although only two ASVs were assigned to these taxa here. Other genera assigned in \u003cem\u003eB. antarctica\u003c/em\u003e but not detected in \u003cem\u003eE. murphyi\u003c/em\u003e were \u003cem\u003eArthrobacter, Cutibacterium, Porphyromonas, Lactococcus, Pelmonas, Janthinobacterium, Neisseria, Escherichia, Serratia\u003c/em\u003e and \u003cem\u003eYersinia\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThis comparison suggests that the bacterial communities associated with these two sister but biogeographically isolated insect species from the Antarctic region [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] are very different and unique. Maistrenko et al. (2023) utilised existing and archival whole-genome sequencing data to identify bacterial species associated with \u003cem\u003eB. antarctica\u003c/em\u003e, which likely only gave a very limited characterisation as this is not a targeted approach. Further studies are also required to disentangle potential changes in each species microbiome across different developmental stages. As both species are detritivores [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e], it is likely that their gut microbial communities will reflect the substrate upon which they feed. Thus, some of the differences in microbial diversity found may be due to differences in the soil properties and associated microbial communities between the distinct geographical locations from which they originate. \u003cem\u003eBelgica antarctica\u003c/em\u003e is endemic to the South Shetland Islands and western coastal regions of the Antarctic Peninsula, while \u003cem\u003eE. murphyi\u003c/em\u003e is endemic to sub-Antarctic South Georgia with an introduced population on maritime Antarctic Signy Island [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. The highest densities of \u003cem\u003eB. antarctica\u003c/em\u003e larvae are found in moss [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]; by contrast, \u003cem\u003eE. murphyi\u003c/em\u003e on Signy favours dead organic matter and soil/peat substrata [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. There may also be differences in the morphology of the gut [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] between the two species, although detailed gut description is currently only available for \u003cem\u003eB. antarctica\u003c/em\u003e [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLeo et al. (2021) employed metagenomic sequencing to investigate the bacterial microbiomes of four Antarctic springtail species, \u003cem\u003eCryptopygus antarcticus\u003c/em\u003e (Collembola: Isotomidae) from the South Shetland Islands (maritime Antarctic), \u003cem\u003eFriesea antarctica\u003c/em\u003e (Collembola: Neanuridae) from the South Shetland Islands and Lagoon Island (Marguerite Bay) and \u003cem\u003eC. terranovus\u003c/em\u003e (Collembola: Isotomidae) and \u003cem\u003eF. propria\u003c/em\u003e (Collembola: Neanuridae) from continental Antarctica (North Victoria Land) [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Of the six most abundant phyla identified, all but Fusobacteria were shared with \u003cem\u003eE. murphyi\u003c/em\u003e, and of the most abundant bacterial orders, only Betaproteobacteriales, Flavobacteriales and Fusobacteriales were not shared with \u003cem\u003eE. murphyi\u003c/em\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). As with \u003cem\u003eB. antarctica\u003c/em\u003e, these springtail species had lower diversity bacterial microbiomes than \u003cem\u003eE. murphy\u003c/em\u003ei.\u003c/p\u003e\u003cp\u003eHolmes et al. (2019) characterised the microbiome of the Antarctic oribatid mite, \u003cem\u003eAlaskozetes antarcticus\u003c/em\u003e (Oribatida: Trhypochthoniidae), from Cormorant Island, south of Anvers Island off the western Antarctic Peninsula [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. The dominant bacterial classes detected in mite tritonymphs were Actinobacteriota, Flavobacteriia, Sphingobacteriia, Gammaproteobacteria and Betaproteobacteria. These fall within the phyla Actinobacteriota, Bacteroidetes and Proteobacteria, which are all shared with \u003cem\u003eE. murphyi\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Representatives of 31 families were detected, 117 less than in \u003cem\u003eE. murphyi\u003c/em\u003e, again suggesting systematic differences in diversity of invertebrate-associated microbial communities, although in this case a higher diversity than present in mites from lower latitudes. Finally, Vecchi et al. (2018) assessed the bacterial microbiome of the Antarctic tardigrade, \u003cem\u003eAcutuncus antarcticus\u003c/em\u003e (Parachela: Hypsibiidae), from Victoria Land (continental Antarctica) [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. This, again, suggested many similarities with \u003cem\u003eE. murphyi\u003c/em\u003e in terms of shared phyla, with only Gemmatimonadetes not found in our study (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Overall, the relatively diverse microbiome of \u003cem\u003eE. murphyi\u003c/em\u003e differs from other Antarctic invertebrates examined to date, although the possible effects of methodological differences cannot be discounted. Finally, not all previous studies have characterised microorganisms to genus level, and those that have all utilise different threshold levels and pipelines, so in-depth comparisons remain difficult.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo test hypotheses on whether \u003cem\u003eE. murphyi\u003c/em\u003e\u0026rsquo;s microbiome has facilitated its success as an invader on Signy, future research must address which archaea and bacteria are true symbionts of \u003cem\u003eE. murphyi\u003c/em\u003e. This could be achieved by using bleach to remove external microorganisms [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e] or dissecting the digestive tract and isolating the associated microorganisms [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. Analysis of microbial community differences between larval stages and the non-feeding adults would also contribute. Access to fresh material from the species\u0026rsquo; native South Georgia, where the fly is increasingly hard to find within areas recently colonised by non-native and aggressive predatory beetles [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e], would also allow assessment of the extent to which the fly\u0026rsquo;s microbiome has altered in the c. 60 years since its introduction to Signy Island. Although amplicon sequencing is a powerful tool, it should be noted that this methodology does not confirm the activity of microorganisms, nor does it differentiate between expressed and non-expressed genes [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. To resolve this, a combination of \u0026lsquo;omics\u0026rsquo; technologies could be applied, including metatranscriptomics, to assess the activity of the microbiome community [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], their associated functions in insect nutrition [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e] and, potentially, the wider environment.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe provide the first assessment of the archaeal and bacterial microbiomes associated with \u003cem\u003eE. murphyi\u003c/em\u003e on Signy Island, maritime Antarctic. The most abundant archaeal and bacterial phyla were Crenarchaeota, Actinobacteriota, Chloroflexi, Proteobacteria and Planctomycetota, all of which have known roles in nitrogen cycling, with some possibly also involved in carbon cycling. Association with representatives of these phyla may allow \u003cem\u003eE. murphyi\u003c/em\u003e to access additional energy sources in the nutrient-depleted soils of Signy Island which, in turn, could release key nutrients into the peaty soil. This may provide a mechanism for the elevated inorganic nitrogen and organic carbon levels previously reported in soil associated with the insect. \u003cem\u003eEretmoptera murphyi\u003c/em\u003e appears to have a more diverse microbiome (at the phylum level) than several native Antarctic invertebrates studied to date, but differences in methodologies across studies currently limit robust comparisons. Further research is also required to confirm which organisms are true endosymbionts/commensals. Finally, comparison of the Signy \u003cem\u003eE. murphyi\u003c/em\u003e microbiome with that of the original source population from South Georgia would enable the first confirmation of the combined transfer of microbiota in an introduction event to Antarctica.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cu\u003eAcknowledgements\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are very grateful to Monica Aquilino for sample collection.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eFunding\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eO. Brayley and S. Liu are funded by NERC CENTA2 grant NE/S007350/1. O. Brayley\u0026rsquo;s PhD is also supported by the British Antarctic Survey. S. Hayward is supported by a Leverhulme Research Fellowship RF-2024-396/2. P. Convey is supported by NERC core funding to the British Antarctic Survey \u0026lsquo;Biodiversity, Evolution and Adaptation\u0026rsquo; Team. S. Hayward, P. Convey and K. McCready were also supported by NSFGEO-NERC grant NE/T009446/1. N. Teets is supported by the National Science Foundation Grant OPP-1850988 and USDA National Institute of Food and Agriculture Hatch Project\u0026nbsp;700545.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cu\u003eCompeting Interests\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cu\u003eAuthor contributions\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eODMB performed the DNA extractions, analysed the data and wrote the initial draft. SL assisted with DNA extractions. KM assisted with the bioinformatic analysis and edited the pipeline. PC, YC, SU, NT and SALH contributed to manuscript writing and editing. All authors gave final approval for manuscript submission.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cu\u003eData Availability\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe ASVs generated during the current study and their taxonomic assignment are available in the Supplementary Information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFeldhaar, H., 2011. Bacterial symbionts as mediators of ecologically important traits of insect hosts. Ecological Entomology 36, 533\u0026ndash;543. https://doi.org/10.1111/j.1365-2311.2011.01318.x\u003c/li\u003e\n\u003cli\u003eSchmidt, K., Engel, P., 2021. Mechanisms underlying gut microbiota\u0026ndash;host interactions in insects. Journal of Experimental Biology 224, jeb207696. https://doi.org/10.1242/jeb.207696\u003c/li\u003e\n\u003cli\u003eRupawate, P.S., Roylawar, P., Khandagale, K., Gawande, S., Ade, A.B., Jaiswal, D.K., Borgave, S., 2023. 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Host-plant adaptation in xylophagous insect-microbiome systems: Contributions of longicorns and gut symbionts revealed by parallel metatranscriptome. iScience 26, 106680. https://doi.org/10.1016/j.isci.2023.106680\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meco","sideBox":"Learn more about [Microbial Ecology](https://www.springer.com/journal/248)","snPcode":"248","submissionUrl":"https://submission.nature.com/new-submission/248/3","title":"Microbial Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Archaea, bacteria, invertebrate, nitrogen cycling, nutrient release, polar","lastPublishedDoi":"10.21203/rs.3.rs-7744438/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7744438/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eEretmoptera murphyi\u003c/em\u003e is a flightless chironomid midge endemic to South Georgia in the sub-Antarctic. In the 1960s it was accidentally introduced to Signy Island (in the maritime Antarctic), where it is now considered an invasive species. This study set out to assess the archaeal and bacterial microbiome of \u003cem\u003eE. murphyi\u003c/em\u003e to enhance understanding of the ecological impacts of this non-native species. Previous work has revealed that the detritivorous larvae of \u003cem\u003eE. murphyi\u003c/em\u003e can increase soil nitrogen levels by up to five times compared with similar uncolonized substrates, although the mechanisms involved are not known. As nutrient availability in Antarctic terrestrial habitats is an important limitation to biodiversity and the development of communities, any significant change could impact native microarthropod and plant communities as well as open new pathways for future non-native species establishment. Dominant archaea and bacteria were: Crenarchaeota, Actinobacteriota, Chloroflexi, Proteobacteria and Planctomycetota, many of which have known roles in nutrient cycling. The microbiome of \u003cem\u003eE. murphyi\u003c/em\u003e appears to be more diverse than those of the few Antarctic invertebrates studied to date. Further work is required to confirm which microorganisms are true endosymbionts and their specific roles in nutrient cycling in polar terrestrial ecosystems.\u003c/p\u003e","manuscriptTitle":"The microbiome of an invasive Antarctic insect, Eretmoptera murphyi (Diptera: Chironomidae), and its potential role in nutrient cycling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-14 23:23:56","doi":"10.21203/rs.3.rs-7744438/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-03T17:18:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-03T16:12:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T22:26:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T17:19:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"307387920403482010773523694286823551782","date":"2025-10-07T08:27:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-03T15:13:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334251604869361604517648643185517832955","date":"2025-10-02T10:23:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"276116304279302922154485237810736994428","date":"2025-10-01T21:29:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40051728258560223315007855576384459842","date":"2025-10-01T15:28:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114433619309844853463423072606636637493","date":"2025-10-01T15:11:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-01T15:05:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-01T15:03:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-30T11:25:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Ecology","date":"2025-09-29T17:31:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meco","sideBox":"Learn more about [Microbial Ecology](https://www.springer.com/journal/248)","snPcode":"248","submissionUrl":"https://submission.nature.com/new-submission/248/3","title":"Microbial Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"56080669-640c-4ea6-b71b-049c5a15fd3f","owner":[],"postedDate":"October 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:04:19+00:00","versionOfRecord":{"articleIdentity":"rs-7744438","link":"https://doi.org/10.1007/s00248-026-02706-5","journal":{"identity":"microbial-ecology","isVorOnly":false,"title":"Microbial Ecology"},"publishedOn":"2026-02-28 15:59:02","publishedOnDateReadable":"February 28th, 2026"},"versionCreatedAt":"2025-10-14 23:23:56","video":"","vorDoi":"10.1007/s00248-026-02706-5","vorDoiUrl":"https://doi.org/10.1007/s00248-026-02706-5","workflowStages":[]},"version":"v1","identity":"rs-7744438","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7744438","identity":"rs-7744438","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}