Terrestrial Isopods Research Articles

Molecular approach challenges the monophyly of the genus Benthana Budde-Lund, 1908 (Oniscidea, Philosciidae)

ABSTRACT Although new data and methodologies are gaining prominence in the field of taxonomy, the vast majority of terrestrial isopod species are still described solely based on morphology. The genus Benthana was recently revised and currently comprises 30 species found in South America. However, the broad geographic distribution of some species, coupled with the presence of few diagnostic characters, raises doubts about the validity of the described species. This study aimed to test the applicability of DNA Barcoding to assess the congruence between morphological and molecular identification of Benthana species. We analyzed 72 individuals belonging to 13 species from the scientific collection of the UFRGS Crustacean Laboratory. We obtained 24 sequences from ten Benthana species. The molecular phylogenies demonstrated that Benthana forms a paraphyletic group. Even at the species level, paraphyletic taxa were observed. Species delimitation methods revealed the presence of eight molecular entities within Benthana. Regarding genetic distances, we observed low intra-specific distances and significantly high inter-specific distances. Our results show that molecular analysis does not support the current taxonomy of Benthana. Therefore, a reassessment of the genus systematics based on integrative taxonomy is warranted.

Redescription of the terrestrial isopod Armadillo mayeti Simon, 1885 and its comparison with Armadillo officinalis Duméril, 1816 from Tunisian populations

ABSTRACT The genus Armadillo is represented by 26 species, three of which are recorded from Tunisia: the Mediterranean A. officinalis Duméril, 1816, and the endemic A. mayeti Simon, 1885 and A. pseudomayeti Mattern, 1999. In this paper, the endemic species A. mayeti is redescribed, its morphological diagnostic characters are illustrated, and its stridulation apparatus structure and biochemical composition are highlighted for the first time using optical and scanning electronic microscopy. The stridulation apparatus, visible dorsally on propodus of pereopods 4 and 5, is structured in a row of chain scales. Regarding its chemical composition, the scale contains numerous elements with a high carbon content (C = 58.71%) followed by calcium (Ca = 19.63%), oxygen (O = 13.98), aluminium (Al = 4.81), copper (Cu = 1.02), iron (Fe = 0.63), potassium (K = 0.43), and silicon (Si = 0.20). Its distribution is extended and its status on the International Union for Conservation of Nature (IUCN) Red List is reported. Armadillo mayeti is then compared with the widespread Mediterranean A. officinalis for diagnostic purposes on the bases of morphological features and the stridulation apparatus structure focusing on the chemical composition. The taxonomic status of A. pseudomayeti is discussed.

Dual roles of terrestrial isopods in seed predation and seed dispersal in Phacellanthus tubiflorus (Orobancheaceae)

AbstractTerrestrial isopods are known to play significant roles in litter decomposition and seed predation. The present study examines the function of terrestrial isopods, particularly Armadillidium vulgare and Armadillidium nasatum, in the seed dispersal of a nonphotosynthetic plant, Phacellanthus tubiflorus. These isopods were observed ingesting seeds in the natural habitat of P. tubiflorus in Hyogo Prefecture, Japan. Contrary to the prevailing belief that invertebrates do not serve as effective internal seed dispersers, our analysis demonstrated that some seeds ingested by A. vulgare and A. nasatum were excreted whole, with viability rates comparable to those of seeds directly sourced from fruits. This indicates that these isopods could act not only as seed predators but also as seed dispersers. The research highlights the importance of further exploration into the ecological contributions of isopods and other invertebrates as seed dispersers.

Complete mitochondrial genome of the terrestrial isopod Cubaris murina Brandt, 1833: new family gene order and novel tRNA secondary structures

ABSTRACT Among isopods (order Isopoda), species belonging to the suborder Oniscidea are the most successful colonisers of terrestrial habitats and the largest family in this order is Armadillidae. In this study, the complete mitochondrial genome of an armadillid isopod, Cubaris murina, was assembled and characterised in detail for the first time. The gene order and secondary structure of transfer RNA (tRNA) genes were compared among isopods. The 14,212-bp-long mitogenome consists of 13 protein-coding genes, 20 tRNAs, two ribosomal RNAs, and four non-coding regions (NCRs), each longer than 100 bp. Two tRNA genes (trnG and trnI) were missing and several other tRNA genes exhibited secondary structures that departed from the typical cloverleaf pattern. Deletions and nucleotide insertions in the T- and D-arms and mismatches in the acceptor stem were commonly observed in these tRNAs. Notably, the mitogenome of C. murina exhibited a new gene order containing four tRNA rearrangements, and differences in the location of the NCRs were detected when compared to those reported for other isopods, including oniscid species. Interestingly, the second NCR had a similar number of secondary structures and repetitive elements to those reported in the control region of other crustaceans. The novel gene order, tRNA secondary structures, and NCR organisation reported herein for the mitogenome of C. murina confirms the hyper-variability of mitochondrial genomes in isopods. This study is the first to characterise in detail the mitogenome of the invasive isopod C. murina, belonging to the species-rich family Armadillidae within the Isopoda, and advances our understanding of mitochondrial genome architecture in isopods.

Redescription of Terrestrial Isopod Porcellio laevis Collected From Sohag Governorate, Egypt with an Overview of Epicuticular Structures Using Scanning Electron Microscopy

Redescription of Terrestrial Isopod Porcellio laevis Collected From Sohag Governorate, Egypt with an Overview of Epicuticular Structures Using Scanning Electron Microscopy

Supralittoral isopod (Oniscidea) diversity at three ecoregions along the Colombian Caribbean: useful data for environmental management

Introduction: Terrestrial isopods (Oniscidea) play important roles in the ecological processes of the soil in tropical ecosystems and are employed as indicators of environmental impact. Despite their importance, studies on the ecology of this suborder in the Neotropics are scarce. Objective: To assess spatial changes in alpha and beta diversity of Oniscidea in the supralittoral zone across Archipiélagos Coralinos (ARCO), Magdalena (MAG), and Morrosquillo (MOR) ecoregions in the Colombian Caribbean. Methods: We conducted Direct Intuitive Searches of specimens and measured soil temperature and moisture in 19 transects with logs, leaf litter, coral remains, and decomposing plant material. Results: A total of 1 970 individuals representing 17 species were collected, with Tylidae, Halophilosciidae, and Ligiidae being the most abundant families. Alpha diversity orders were higher in ARCO than MAG and MOR ecoregions. MAG and MOR differed in observed richness. The structure of the assemblage varied in dominant species and abundances. In ARCO, the indicator species were Tylos niveus, Littorophiloscia denticulata, Halophiloscia trichoniscoides, and Ligia baudiniana; in MOR, it was Tylos marcuzzii, and in MAG, it was Littorophiloscia tropicalis. High beta diversity (> 60 %), with significant differences in the assemblage structure among ecoregions, was confirmed by the NMDS, which distinctly separated each group. CCA analysis revealed a negative relationship between most species with soil temperature and moisture, with a positive relationship observed with T. marcuzzii. Conclusions: This is the first effort to describe spatial changes in the diversity of oniscideans in the supralittoral zone of the Neotropical region, providing a baseline for future studies. This information could be instrumental in identifying priority areas for conservation efforts.

Temporal stability of sex ratio distorter prevalence in natural populations of the isopod Armadillidium vulgare.

In the terrestrial isopod Armadillidium vulgare, many females produce progenies with female-biased sex ratios due to two feminizing sex ratio distorters (SRD): Wolbachia endosymbionts and a nuclear non-mendelian locus called the f element. To investigate the potential impact of these SRD on the evolution of host sex determination, we analyzed their temporal distribution in six A. vulgare populations sampled between 2003 and 2017, for a total of 29 time points. SRD distribution was heterogeneous among populations despite their close geographic locations, so that when one SRD was frequent in a population, the other SRD was rare. In contrast with spatial heterogeneity, our results overall did not reveal substantial temporal variability in SRD prevalence within populations, suggesting equilibria in SRD evolutionary dynamics may have been reached or nearly so. Temporal stability was also generally reflected in mitochondrial and nuclear variation. Nevertheless, in a population, a Wolbachia strain replacement coincided with changes in mitochondrial composition but no change in nuclear composition, thus constituting a typical example of mitochondrial sweep caused by endosymbiont rise in frequency. Rare incongruence between Wolbachia strains and mitochondrial haplotypes suggested the occurrence of intraspecific horizontal transmission, making it a biologically relevant parameter for Wolbachia evolutionary dynamics in A. vulgare. Overall, our results provide an empirical basis for future studies on SRD evolutionary dynamics in the context of multiple sex determination factors co-existing within a single species, to ultimately evaluate the impact of SRD on the evolution of host sex determination mechanisms and sex chromosomes.

Patterns of molecular evolution in a parthenogenic terrestrial isopod (Trichoniscus pusillus).

The "paradox of sex" refers to the question of why sexual reproduction is maintained in the wild, despite how costly it is compared to asexual reproduction. Because of these costs, one might expect nature to select for asexual reproduction, yet sex seems to be continually selected for. Multiple hypotheses have been proposed to explain this incongruence, including the niche differentiation hypothesis, the Red Queen hypothesis, and accumulation of harmful mutations in asexual species due to inefficient purifying selection. This study focuses on the accumulation of mutations in two terrestrial isopods, Trichoniscus pusillus, which has sexual diploid and parthenogenic triploid forms, and Hyloniscus riparius, an obligately sexual relative. We surveyed sex ratios of both species in an upstate New York population and obtained RNA-seq data from wild-caught individuals of both species to examine within- and between-species patterns of molecular evolution in protein-coding genes. The sex ratio and RNA-seq data together provide strong evidence that this T. pusillus population is entirely asexual and triploid, while the H. riparius population is sexual and diploid. Although all the wild T. pusillus individuals used for sequencing shared identical genotypes at nearly all SNPs, supporting a clonal origin, heterozygosity and SNP density were much higher in T. pusillus than in the sexually reproducing H. riparius. This observation suggests this parthenogenic lineage may have arisen via mating between two divergent diploid lineages. Between-species sequence comparisons showed no evidence of ineffective purifying selection in the asexual T. pusillus lineage, as measured by the ratio of nonsynonymous to synonymous substitutions (dN/dS ratios). Likewise, there was no difference between T. pusillus and H. riparius in the ratios of nonsynonymous to synonymous SNPs overall (pN/pS). However, pN/pS ratios in T. pusillus were significantly higher when considering only SNPs that may have arisen via recent mutation after the transition to parthenogenesis. Thus, these recent SNPs are consistent with the hypothesis that purifying selection is less effective against new mutations in asexual lineages, but only over long time scales. This system provides a useful model for future studies on the evolutionary tradeoffs between sexual and asexual reproduction in nature.

Comparisons of developmental processes of air-breathing organs among terrestrial isopods (Crustacea, Oniscidea): implications for their evolutionary origins

BackgroundThe acquisition of air-breathing organs is one of the key innovations for terrestrialization in animals. Terrestrial isopods, a crustacean lineage, can be interesting models to study the evolution of respiratory organs, as they exhibit varieties of air-breathing structures according to their habitats. However, the evolutionary processes and origins of these structures are unclear, due to the lack of information about their developmental processes. To understand the developmental mechanisms, we compared the developmental processes forming different respiratory structures in three isopod species, i.e., ‘uncovered lungs’ in Nagurus okinawaensis (Trachelipodidae), ‘dorsal respiratory fields’ in Alloniscus balssi (Alloniscidae), and pleopods without respiratory structures in Armadilloniscus cf. ellipticus (Detonidae).ResultsIn N. okinawaensis with uncovered lungs, epithelium and cuticle around the proximal hemolymph sinus developed into respiratory structures at post-manca juvenile stages. On the other hand, in Al. balssi with dorsal respiratory fields, the region for the future respiratory structure was already present at manca 1 stage, immediately after hatching, where the lateral protrusion of ventral epithelium occurred, forming the respiratory structure. Furthermore, on pleopods in Ar. cf. ellipticus, only thickened dorsal cuticle and the proximal hemolymph sinus developed during postembryonic development without special morphogenesis.ConclusionsThis study shows that the respiratory structures in terrestrial isopods develop primarily by postembryonic epithelial modifications, but the epithelial positions developing into respiratory structures differ between uncovered lungs and dorsal respiratory fields. This suggests that these two types of respiratory structures do not result from simple differences in the degree of development. Future analysis of molecular developmental mechanisms will help determine whether these are the result of heterotopic changes or have different evolutionary origins. Overall, this study provides fundamental information for evolutionary developmental studies of isopod respiratory organs.

Terrestrial isopods (Isopoda: Oniscidea) in the Upper Mežica valley

The distribution of terrestrial isopods in many parts of Slovenia is still poorly documented. This article is a contribution to the knowledge of the terrestrial isopod fauna in the Upper Mežica Valley, which is known for its long mining tradition. Isopods were collected from April to October 2022 by hand from 49 localities in the wider area of two towns – Žerjav and Črna na Koroškem in the area of Podpeca, the area above the Bistra Valley, and in the areas Spodnje Javorje and Javorje. Twelve different taxa of terrestrial isopods were determined, eleven of them to the species level. The most frequently found isopod was Trachelipus ratzeburgii as the researched area is mainly forest covered. However, the highest diversity of species was recorded in urban and semiurban areas. Oniscus asellus was found in the town Črna na Koroškem, which is probably the second documented location of O. asellus in Slovenia.

Morphological, Molecular Identification and Genetic Diversity Assessment of Three Terrestrial Isopods from the High-Altitude Region

Terrestrial specimens were collected from Ashshafa, a south-western highland area in Saudi Arabia. Three species, i.e., Porcellio laevis, Porcellionides pruinosus (Porcellionidae), and Armadillidium vulgare (Armadillidiidae), were identified in this study based on their morphological characteristics. Partial mitochondrial cytochrome C oxidase subunit I (COI) gene sequences were used for DNA barcoding and biodiversity assessments. A phylogenetic tree of 22 haplotypes from 35 specimens of the three isopod species was drawn from the most similar sequences obtained from BLAST with the associated accession numbers. The tree included two clades. The first clade included samples of P. laevis and P. pruinosus, whereas the second clade included samples of A. vulgare. Each identified species formed a distinct subclade within the main clade, along with similar sequences obtained from the NCBI database. The heat map of genetic distance among haplotypes shows the haplotype diversity (Hd) ranged from 0.590 to 0.933 (mean = 0.767) and total nucleotide diversity (πT) ranged from 0.001 to 0.089 (mean=0.049), with a similar trend observed for nucleotide diversity per site (θw) ranged from 0.001 to 0.80 (mean = 0.049). In contrast, synonymous nucleotide diversity (πs), mean=0.009, was low compared to nonsynonymous nucleotide diversity (πs), mean=0.060, across all species. In conclusion, the morphological identification of terrestrial isopods was confirmed using COI gene sequencing of mitochondrial DNA. These results will be helpful in developing a deeper isopod identification method.

Phylogenomics resolves long-standing questions about the affinities of an endangered Corsican endemic fly.

Recent studies on oestroidean Diptera (Brachycera) are providing a comprehensive and nuanced understanding of the evolutionary history of this remarkably diverse clade of holometabolous insects. The Oestroidea, which includes formidable pests such as various blowflies, botflies, and flesh flies that infest livestock, pets and humans, are mostly composed of beneficial species that act as scavengers or parasitoids on various pest insects. In our research, we used genomic methods to elucidate the phylogenetic position of Nesodexia corsicanaVilleneuve, 1911(Diptera: Calliphoridae), a mysterious oestroid species endemic to Corsica and characterized by distinctive morphological features that have puzzled taxonomists for years. Contrary to initial hypotheses, our results place Nesodexia Villeneuve, 1911within the Calliphoridae subfamily Rhinophorinae, a small lineage of terrestrial isopod parasitoids. Through detailed morphological analysis of adults of both sexes and eggs, we uncovered significant insights consistent with our phylogenomic reconstruction. The unique morphological features of the species, coupled with its restricted and fragmented habitat, highlight its potential conservation importance. We delineated the area of occupancy for N. corsicana and assessed its "threatened" category using specific IUCN Red List criteria. In addition, we mapped the available habitat within its range and determined potential key biodiversity areas (KBA) triggered by N. corsicana. New potential KBAs are only partially covered by the Corsican Regional Park. Finally, we mapped the distribution of habitats on the island to assess the potential distribution of the species beyond its currently known geographic range.

Influence of body size on the dual role of isopod Porcellio scaber in seed dispersal and predation of the mycoheterotrophic plant Monotropastrum humile

Abstract Endozoochorous seed dispersal by invertebrates, although infrequently documented, is gaining attention for its feasibility in plants with small seeds. Recent studies have highlighted terrestrial isopods as potential dual agents, acting both as seed dispersers and predators, especially for herbaceous plants with fleshy fruits and tiny seeds. However, the determinants of these contrasting roles are not well understood. The present study explores intraspecific variation in Porcellio scaber, examining its dual role as a seed predator and disperser for Monotropastrum humile (Ericaceae), a plant with fleshy fruits and minute seeds (0.30–0.35 mm in length). We particularly focus on the impact of the sex and body size (5.44–11.99 mm) of the isopod P. scaber on seed dispersal or predation. Our results indicate a significant correlation between seed dispersal effectiveness and isopod body size, with some variation attributed to sex differences. Smaller P. scaber individuals predominantly prey on seeds, whereas larger individuals are more likely to facilitate seed dispersal, maintaining seed viability. Overall, our findings reveal that body size is a crucial factor in defining the ecological role of P. scaber. This challenges the conventional approach of using species‐level average data in seed dispersal studies, emphasising the need to consider intraspecific variations for a comprehensive understanding of seed dispersal.

Hidden diversity in eastern North America: The genus Ligidium (Oniscidea, Ligiidae) in the southern Appalachian Mountains

AbstractThe terrestrial isopod genus Ligidium includes 58 species from Europe, Asia, and North America. In Eastern North America four species are recognized: L. floridanum and L. mucronatum, known just from their type localities in Florida and Louisiana respectively, L. blueridgensis, endemic to the southern Appalachians, and L. elrodii, widespread from Georgia to Ontario. The genus shows a marked morphological conservatism, and species are differentiated mostly by small morphological differences; it is not always easy to determine if such variability represents inter‐ or intraspecific variation. Here, we explore the diversity of Ligidium from the southern Appalachian Mountains, exploring the congruence of morphologically defined groups with multilocus phylogenetic reconstructions and molecular species delimitation methods. We have studied a total of 130 specimens from 37 localities, mostly from the southern Appalachians, and analysed mtDNA (Cox1) and nuclear (28S, NaK) sequences. Morphologically, we recognized eight morphotypes, most of them assignable to current concepts of L. elrodii and L. blueridgensis. Phylogenetic analyses supported the evolutionary independence of all morphotypes, and suggest the existence of 8–9 species, including limited cryptic diversity. Single‐locus delimitation analyses based on mtDNA data suggest the existence of a much higher number of species than the multilocus analyses. The estimated age of the ancestors of sampled lineages indicates a long presence of the genus in eastern North America and old speciation events through the Miocene. Our results indicate a higher diversity than previously thought among the Ligidium populations present in the southern Appalachian Mountains, with several species to be described.

Response of the terrestrial isopod Porcellio scaber to lipopolysaccharide challenge after microplastic and insecticide exposure

The exposure of organisms to microplastics could compromise their ability to cope with other environmental stressors, such as infections. In this context, we investigated the effects of a 14-day exposure of the terrestrial isopod Porcellio scaber to tire particles in soil (1.5 % w w−1 dry weight) on the organisms' response to a secondary exposure, i.e., injection of the bacterial endotoxin lipopolysaccharide. In addition, the insecticide chlorpyrifos (2 mg kg−1 dry weight) was tested as a positive control. The survival and immune response of P. scaber was assessed at the end of the 7- and 14-day primary exposure and two days after the secondary exposure, by analyzing selected haemolymph immune parameters (total haemocyte count, differential haemocyte count, and haemocyte viability). No change in survival was observed after primary exposure of P. scaber to tire particles or chlorpyrifos. However, primary exposure to chlorpyrifos triggered a strong activation of the immune response, which was not the case following exposure to the tire particles. Further injection of lipopolysaccharide into the body did not affect the survival of animals exposed to tire particles or chlorpyrifos, while a strong immunomodulatory change was observed, particularly with chlorpyrifos, and to some extent, tire particles. Based on these results, we conclude that exposure of P. scaber to tire particles or chlorpyrifos has no significant effect on the susceptibility of the organism to lipopolysaccharide in terms of their mortality, but primary exposure to an insecticide significantly modulates the immune response of the organisms to a second stressor. We discuss the “stress on stress” approach for testing low-toxic substances, such as microplastics, where an environmentally realistic exposure is followed by a secondary exposure.

Una nueva especie troglobia del Alto Atlas de Marruecos: Castellanethes ubachi n. sp. (Crustacea, Isopoda, Olibrinidae)

In this work, a new troglobitic species of terrestrial isopod present in the High Atlas of Morocco belonging to the family Olibrinidae and the Castellanethes genus is described. The new species is differentiated from the rest of the genus by its strong dorsal tubercles on the cephalon and pereion, the antennula with three petaliform aesthetascs, the antennal flagellum with seven or eight articles, the molar penicillium of the mandibles with two branches and the endopod of the first pleopod of the male with fifteen large scales at the end. To facilitate the identification of the species of the genus, a key is included.

Effects of leaf litter traits on terrestrial isopod and millipede consumption, assimilation and growth

Abstract Nutrient cycling through leaf litter consumption is an essential ecological function performed by macrodetritivorous invertebrates such as isopods and millipedes. Leaf litter consumption rates can vary greatly depending on the environment, consumer identity and litter traits, but generalizations about the effects of plant traits on macrodetritivore leaf litter consumption, assimilation and growth are not well established and are mostly indirectly inferred. We conducted a systematic search of the global literature and obtained 456 standardized measures from laboratory experiments of relative consumption (RCR), assimilation (RAR) and growth (RGR) rates of terrestrial isopods and millipedes, extracted from 56 different articles. We investigated if commonly measured leaf traits, plant functional groups, prior microbial conditioning of leaves and climatic conditions affected these rates. We obtained data on commonly measured leaf traits from the TRY global plant trait database, inferred plant functional groups from taxonomic groupings and obtained climatic data from information reported within articles. RCR, RAR and RGR varied greatly among macrodetritivore and plant species, but overall, there were no differences between isopods and millipedes. Microbial conditioning of litter greatly increased RCR. Plant functional group was an important predictor of RCR, with eudicot trees and forbs being consumed in greater quantities than magnoliid trees and grasses. Fresh leaf N:P ratio had a positive effect on RAR, and leaf N and C:N ratio had positive and negative effects on RGR, respectively, while climatic variables had weak effects on the three rates. Our work shows that plant traits (both those associated with plant functional groups and commonly measured leaf traits) exert strong effects on resource processing rates by terrestrial macrodetritivores. Further, prior microbial conditioning of leaf litter has a large and globally consistent positive effect on macrodetritivore litter consumption, suggesting that they may consume little, if any, freshly senesced leaf material when microbially conditioned litter is available. Our results suggest that, where extremes of temperature or precipitation do not occur, variables reflective of food quality (leaf traits and microbe conditioning) are more important drivers of macrodetritivore leaf litter consumption than are extrinsic climatic variables. Read the free Plain Language Summary for this article on the Journal blog.

Iron-isopods: new records and new species of terrestrial isopods (Isopoda, Oniscidea) from Brazilian Amazon iron ore caves

Abstract. Two new species of terrestrial isopods are described from iron ore caves in Brazil, within the Amazon biome, Circoniscus mendesi López-Orozco, Campos-Filho & Bichuette sp. nov. and C. xikrin López-Orozco, Campos-Filho & Carpio-Díaz sp. nov. (Scleropactidae). In addition, the knowledge of the distribution of Ctenorillo ferrarai Campos-Filho, Araujo & Taiti, 2014 (Armadillidae) is extended to Parauapebas, and Benthanoides tarzan Cardoso & Ferreira, 2023 to south area of the Campos Ferruginosos National Park, both in the State of Pará. Moreover, a distribution map and photographs of the species are given.

A TERRESTRIAL ISOPOD ( ARMADILLIDIUM ARCANGELII STROUHAL, 1929) AS A POTENTIAL AGRICULTURAL PEST: A CASE STUDY ON MELON CROP (CUCUMIS MELO L.) IN ITALY

Terrestrial isopods are generally considered as detritivore invertebrates, but in recent years and in different parts of the world they seem to become potential agricultural pests for different crops. The crops affected so far have been mainly arable crops, but damages to few horticultural crops were also documented. Until now, no referenced problems with isopods as crop pests had ever occurred in Italy. In March/April 2023 on two organic farms in northern Italy newly transplanted seedlings of smooth melon in greenhouses were damaged by isopods. Among farms, the loss was estimated between 40% and 50% of the seedlings. The isopod that caused the damage was identified as Armadillidium arcangelii, and, until now, the species had never been reported as a crop pest. Key Words: New potential pest, Armadillidium arcangelii, terrestrial isopod, melon crop

Metagenome-assembled genomes of three Hepatoplasmataceae provide insights into isopod-mollicute symbiosis.

The digestive organs of terrestrial isopods harbour bacteria of the recently proposed mollicute family Hepatoplasmataceae. The only complete genome available so far for Hepatoplasmataceae is that of 'Candidatus Hepatoplasma crinochetorum'. The scarcity of genome sequences has hampered our understanding of the symbiotic relationship between isopods and mollicutes. Here, we present four complete metagenome-assembled genomes (MAGs) of uncultured Hepatoplasmataceae members identified from shotgun sequencing data of isopods. We propose genomospecies names for three MAGs that show substantial sequence divergence from any previously known Hepatoplamsataceae members: 'Candidatus Tyloplasma litorale' identified from the semiterrestrial isopod Tylos granuliferus, 'Candidatus Hepatoplasma vulgare' identified from the common pill bug Armadillidium vulgare, and 'Candidatus Hepatoplasma scabrum' identified from the common rough woodlouse Porcellio scaber. Phylogenomic analysis of 155 mollicutes confirmed that Hepatoplasmataceae is a sister clade of Metamycoplasmataceae in the order Mycoplasmoidales. The 16S ribosomal RNA gene sequences and phylogenomic analysis showed that 'Candidatus Tyloplasma litorale' and other semiterrestrial isopod-associated mollicutes represent the placeholder genus 'g_Bg2' in the r214 release of the Genome Taxonomy Database, warranting their assignment to a novel genus. Our analysis also revealed that Hepatoplasmataceae lack major metabolic pathways but has a likely intact type IIA CRISPR-Cas9 machinery. Although the localization of the Hepatoplasmatacae members have not been verified microscopically in this study, these genomic characteristics are compatible with the idea that these mollicutes have an ectosymbiotic lifestyle with high nutritional dependence on their host, as has been demonstrated for other members of the family. We could not find evidence that Hepatoplasmataceae encode polysaccharide-degrading enzymes that aid host digestion. If they are to provide nutritional benefits, it may be through extra-copy nucleases, peptidases, and a patatin-like lipase. Exploration of potential host-symbiont interaction-associated genes revealed large, repetitive open reading frames harbouring beta-sandwich domains, possibly involved with host cell adhesion. Overall, genomic analyses suggest that isopod-mollicute symbiosis is not characterized by carbohydrate degradation, and we speculate on their potential role as defensive symbionts through spatial competition with pathogens to prevent infection.