Flow cytometric analysis of hepatopancreatic cells from Armadillidium vulgare highlights terrestrial isopods as efficient environmental bioindicators in ex vivo settings.

Several studies have reported the high bioindication capacity of Isopoda (Crustacea, Oniscidea), which is related to their important ability to accumulate contaminants, usefulness in soil ecotoxicology and bioindication activities. Any change in the isopod population, diversity and life cycle can indicate relevant pollution levels. The analysis of target tissues, such as the hepatopancreas, is another emerging approach (from a cytologic/histological level) to detect contaminant accumulation from different sources. In this study, tissue disaggregation procedures were optimised in the hepatopancreas, and flow cytometry (FC) was applied to detect cell viability and several cell functions. After disaggregation, two hepatopancreatic cell types, small (S) and big (B), were still recognisable: they differed in morphology and behaviour. The analyses were conducted for the first time on isopods from sites under different conditions of ecological disturbance through cytometric re-interpretation of ecological-environmental parameters. Significant differences in cell functional parameters were found, highlighting that isopod hepatopancreatic cells can be efficiently analysed by FC and represent standardisable, early biological indicators, tracing environmental-induced stress through cytologic/histologic analyses.

Intra-island patterns of cryptic diversity within an oceanic island: Insights from the differentiation of Schizidium Verhoeff, 1901 (Oniscidea, Armadillidiidae) within Cyprus, with descriptions of two new species.

Cyprus is a Mediterranean island of volcanic origin isolated for at least 5.3 Myr from surrounding continental areas. The present study focuses on the diversification of the isopod genus Schizidium within the island, including also specimens from surrounding continental areas. The genus Schizidium sensu lato is probably non monophyletic, comprising 26 species distributed from Greece to Iran. Up to date the only representative of the genus reported from Cyprus was Schizidium fissum. Aiming to investigate the patterns of genetic diversity within the focal island, to evaluate the morphology-based taxonomy of the species in the genus, and at the same time to explore phylogenetic relationships with mainland populations, we applied genome-wide ddRADseq as well as Sanger sequencing targeting three mitochondrial (16S, COI and 12S) and the nuclear NaK loci. Results of phylogenetic analyses support the existence of two distinct epigean Schizidium clades with well-defined geographic boundaries that conform to the known paleogeography of Cyprus, plus one endogean clade with restricted distribution within the island. Genetic data and morphology corroborate the assignment of this latter endogean clade to a new species, Schizidium myrrae n. sp. The two epigean clades are also considered as distinct species, one corresponding to the known S. fissum at the eastern part of the island (Pentadaktylos massif) and the other to the newly described S. christosi n. sp. distributed along the western part of the island (Troodos massif). Even though detailed examination of many specimens could not retrieve any morphological differences among representatives of these two clades, clado-chronological analysis indicates a long isolation between them, estimated at âˆ¼ 9 Mya, as well as the sharing of a common ancestor with S. tiberianum from Israel at âˆ¼ 15 Mya. Hence, we can consider these epigean Schizidium species as one more case of cryptic diversity on Cyprus, exhibiting similar patterns with the recently described case in the genus Armadillo.

Reactivity to pseudorandom environmental patterns in woodlice: Effects of hydric deprivation and light exposure.

Exploratory activity is an essential component of animal behavior, including among invertebrate species. This study examined the effects of hydric deprivation and their possible modulation by light exposure on locomotion and rearing-up behavior in two woodlice species, Porcellio scaber (Latreille 1804) and Armadillidium vulgare (Latreille 1804). It was also an attempt to replicate previous findings on the stimulation of these behaviors in P. scaber, exposed to (pseudo)random vs. regular visual and tactile patterns in a small enclosure. In Experiment 1, two groups of P. scaber and two groups of A. vulgare were exposed to randomly vs. regularly distributed visual (black and white) and tactile (grained and smooth) patterns for approximately 20 min. No rewards were present in the environment and the woodlice were tested without preliminary hydric deprivation. In Experiment 2, the same procedure was used but the woodlice were tested following a 20-min hydric deprivation under a bottle cap (darkness). Experiment 3 replicated this procedure with the 20-min hydric deprivation spent in a plastic cup (light exposure). The results of Experiments 1 and 3 provide partial replication with A. vulgare, but not P. scaber, of the previous findings: Random patterns stimulate rearing-up behavior on the apparatus' vertical walls more than regular patterns. Also, a more aversive stimulation in Experiment 3 compared to Experiment 2, increased locomotion, especially in the random environment. The role of hydric deprivation and light exposure in the process of escaping from a hostile environment is discussed, as well as the effects of the treatments used in these experiments.

Toxicokinetics of Ag from Ag2S NP exposure in Tenebrio molitor and Porcellio scaber: Comparing single-species tests to indoor mesocosm experiments.

Determining the potential for accumulation of Ag from Ag2S NPs as an environmentally relevant form of AgNPs in different terrestrial organisms is an essential component of a realistic risk assessment of AgNP emissions to soils. The objectives of this study were first to determine the uptake kinetics of Ag in mealworms (Tenebrio molitor) and woodlice (Porcellio scaber) exposed to Ag2S NPs in a mesocosm test, and second, to check if the obtained toxicokinetics could be predicted by single-species bioaccumulation tests. In the mesocosms, mealworms and woodlice were exposed together with plants and earthworms in soil columns spiked with 10 µg Ag g-1 dry soil as Ag2S NPs or AgNO3. The total Ag concentrations in the biota were measured after 7, 14, and 28 days of exposure. A one-compartment model was used to calculate the Ag uptake and elimination rate constants. Ag from Ag2S NPs appeared to be taken up by the mealworms with significantly different uptake rate constants in the mesocosm compared to single-species tests (K1 = 0.056 and 1.66 g dry soil g-1 dry body weight day-1, respectively), and a significant difference was found for the Ag bioaccumulation factor (BAFk = 0.79 and 0.15 g dry soil g-1 dry body weight, respectively). Woodlice did not accumulate Ag from Ag2S NPs in both tests, but uptake from AgNO3 was significantly slower in mesocosm than in single-species tests (K1 = 0.037 and 0.26 g dry soil g-1 dry body weight day-1, respectively). Our results are of high significance because they show that single-species tests may not be a good predictor for the Ag uptake in mealworms and woodlice in exposure systems having greater levels of biological complexity. Nevertheless, single-species tests could be used as a fast screening approach to assess the potential of a substance to accumulate in biota before more complex tests are conducted.

Everything is similar, everything is different! Trichorhina (Oniscidea, Platyarthridae) from Brazilian caves, with descriptions of 11 new species / Tudo é parecido, tudo é diferente! Trichorhina (Oniscidea, Platyarthridae) de cavernas brasileiras, com descrições de 11 novas espécies

Abstract The present work describes 11 new species of the genus Trichorhina from Brazilian caves in the states of Bahia, Mato Grosso do Sul, Minas Gerais, Pará, and São Paulo. Moreover, the distribution of T. tomentosa has been expanded to include the states of Mato Grosso and Minas Gerais. Additionally, a distribution map and a key for all Brazilian species of Trichorhina, including epigean ones, are provided.

Resumo O presente trabalho descreve 11 novas espécies do gênero Trichorhina de cavernas brasileiras nos estados da Bahia, Mato Grosso do Sul, Minas Gerais, Pará e São Paulo. Além disso, a distribuição de T. tomentosa foi ampliada para incluir os estados de Mato Grosso e Minas Gerais. Adicionalmente, são fornecidos um mapa de distribuição e uma chave para todas as espécies brasileiras de Trichorhina, incluindo as epígeas.

Comparison of exploratory behavior of male and female woodlice (Armadillidium vulgare)

There are several studies in the literature showing that male and female rats explore novel environments and exhibit different exploration patterns when submitted to different apparatuses. In general, female rats spend more time moving and exploring the apparatuses than males do. A previous study showed that male woodlice (Armadillidium vulgare) explore novel environments in a very similar way to male rats (Rattus norvegicus) when tested in apparatuses analogous to the open-field test and light/dark box. Since that study was conducted only with male rats and woodlice, and since they exhibited very similar patterns of behavior, the present experiment aimed at investigating whether male and female woodlice explore novel environments with different behavioral patterns. Female and male woodlice were tested in the open-field and in the dry/moist box. Results obtained in the open-field test showed that both males and females remained longer in the corners than along the walls and avoided staying in the center. However, females remained longer along the walls and less in the corners. In the dry/moist box, there were no significant differences between the sexes: both females and males remained significantly longer in the moist compartment.

Integrative taxonomy on the rare sky-island Ligidium species from southwest China (Isopoda, Oniscidea, Ligiidae).

BACKGROUND: The sky-island Ligidium species fauna in southwest China is poorly known. Before this study, six of the seven sky-island species of the genus were known to be endemic to southwest China. In morphology, Ligidium species are often difficult to identify, and an appraisal of integrative taxonomy is needed. RESULTS: We integrated morphology and molecular analyses to delimit Ligidium species. Molecular species delimitation based on distance- and evolutionary models recovered seven-candidate lineages from five gene markers (COI, 12S rRNA, 18S rRNA, 28S rRNA and NAK). We also estimated that the species divergences of sky-island Ligidium in southwest China started in late Eocene (40.97 Mya) to middle Miocene (15.19 Mya). Four new species (L. duospinatum Li, sp. nov., L. acuminatum Li, sp. nov., L. rotundum Li, sp. nov. and L. tridentatum Li, sp. nov.) are described. Morphological confusion among L. denticulatum Shen, 1949, L. inerme Nunomura & Xie, 2000 and L. sichuanense Nunomura, 2002 is clarified by integrative taxonomy. CONCLUSION: This work confirms that an integrative approach to Ligidium taxonomy is fundamental for objective classification, and deduced the uplift of Qinghai-Tibetan Plateau in the late Eocene and middle Miocene as one of the principal reasons for the species divergences of sky-island Ligidium in southwest China. We also inferred that sky-island mountains have a huge reserve of higher Ligidium species diversity.

New records of Trachelipusvespertilio (Budde-Lund, 1896) (Isopoda, Oniscidea) with a description of the male.

Background: The existing descriptions of the woodlouse Trachelipusvespertilio are based on a single female collected in Croatia in the nineteenth century. No further information on the occurrence of this species has been reported in published literature and the morphology of the male, which may offer additional reliable diagnostic characters, has remained unknown. New information: On the basis of new material collected in Slovenia, a description of the male morphology of T.vespertilio is provided along with a species diagnosis. The rediscovery of this woodlouse after more than a century extends its distribution range to Slovenia.

Characterization of a Sex-Determining Region and Its Genomic Context via Statistical Estimates of Haplotype Frequencies in Daughters and Sons Sequenced in Pools.

Sex chromosomes are generally derived from a pair of autosomes that have acquired a locus controlling sex. Sex chromosomes may evolve reduced recombination around this locus and undergo a long process of molecular divergence. At that point, the original loci controlling sex may be difficult to pinpoint. This difficulty has affected many model species from mammals to birds to flies, which present highly diverged sex chromosomes. Identifying sex-controlling loci is easier in species with molecularly similar sex chromosomes. Here we aimed at pinpointing the sex-determining region (SDR) of Armadillidium vulgare, a terrestrial isopod with female heterogamety (ZW females and ZZ males) and whose sex chromosomes appear to show low genetic divergence. To locate the SDR, we assessed single-nucleotide polymorphism (SNP) allele frequencies in F1 daughters and sons sequenced in pools (pool-seq) in several families. We developed a Bayesian method that uses the SNP genotypes of individually sequenced parents and pool-seq data from F1 siblings to estimate the genetic distance between a given genomic region (contig) and the SDR. This allowed us to assign more than 43 Mb of contigs to sex chromosomes, and to demonstrate extensive recombination and very low divergence between these chromosomes. By taking advantage of multiple F1 families, we delineated a very short genomic region (∼65 kb) that presented no evidence of recombination with the SDR. In this short genomic region, the comparison of sequencing depths between sexes highlighted female-specific genes that have undergone recent duplication, and which may be involved in sex determination in A. vulgare.

Impact of transposable elements on genome size variation between two closely related crustacean species.

Identifying and quantifying genome size variation among species and understanding the underlying causes is a long-standing objective in evolutionary biology. Here, we investigated the basis of genome size variation between two closely related species of terrestrial isopods: Armadillidium vulgare and Armadillidium nasatum. The two species diverged 25 million years ago and the A. vulgare genome is ~500 megabases larger than the A. nasatum genome (1.7 vs. 1.2 gigabases, respectively). Our analyses indicated that genome size difference is essentially attributed to transposable elements (TEs). We found that the deletion rate may be slightly higher in A. nasatum than in A. vulgare, but it is unlikely to explain the observed genome size difference. As the two genomes largely share the same TE families, differential transpositional activity also contributes to the observed variation. Analyses of TE expression suggested that the cumulative expression level of all expressed TEs was higher in A. nasatum than in A. vulgare. Assuming TE expression level is a good proxy for TE transpositional activity, our results suggest that the two species may have recently been experiencing different TE transposition dynamics. Overall, our results illustrate the important impact TEs can have on genome structure and evolution between closely related species.

Sphaerillo boninensis Nunomura, 1990 (Crustacea, Isopoda, Oniscidea) is a junior synonym of a pantropical species, Venezillo parvus (Budde-Lund, 1885).

Re-examination of the holotype and paratype of Sphaerillo boninensis Nunomura, 1990 from Chichijima Island of the Ogasawara archipelago, which is registered as a UNESCO World Heritage Site, indicates that this species is a junior synonym of a pantropical species, Venezillo parvus (Budde-Lund, 1885).

Shedding Light on the Antimicrobial Peptide Arsenal of Terrestrial Isopods: Focus on Armadillidins, a New Crustacean AMP Family.

In crustaceans, antimicrobial peptides (AMPs) are clustered into four major groups according to their amino acid composition and structure: (1) single-domain peptides containing cysteine residues such as anti-lipopolysaccharide-factor (ALF), (2) multi-domain or chimeric AMPs such as crustins, (3) non-conventional AMPs, and (4) linear single-domain AMPs. The majority of AMPs has been described in commercially exploited crustaceans, particularly decapods living in aquatic environments (crab, shrimp, lobster, and crayfish). Here, we aimed at establishing the AMPs repertoire of terrestrial isopods (Oniscidea), an original suborder of crustaceans adapted to life outside of the aquatic environment. Using transcriptomic data from 21 species, we identified 110 ALF and 73 crustin sequences. We also characterized the full-length sequence of armadillidins from 17 species, similar to the AMP previously described in the terrestrial isopod Armadillidium vulgare. Furthermore, we tested the antimicrobial activity of three armadillidin peptides characterized from three distantly related species. This analysis revealed similar activity spectra against pathogens, despite extensive structural variation among the tested peptides. In addition to conventional crustacean AMPs, our work highlights armadillidins as a new and independent family of AMPs specific to the Oniscidea, thus opening new perspectives concerning the study of the immune system of terrestrial isopods.

Two new species and new records of terrestrial isopods (Crustacea, Isopoda, Oniscidea) from Brazilian caves.

To date, approximately 190 species of terrestrial isopods are known from Brazil and only 14 are considered troglobiotic. After the examination of a large collection from caves in Bambuí and Una geomorphological areas, along the states of Bahia, Minas Gerais and Goiás, two new troglobiotic species were recognized. Pectenoniscus liliae Campos-Filho, Bichuette Taiti sp. n. (Styloniscidae) is described from Serra do Ramalho karst area, and Benthana xiquinhoi Campos-Filho, Bichuette Taiti sp. n. (Philosciidae) from sandstone caves of Chapada Diamantina region. The latter constitutes the second troglomorphic species of the genus. Xangoniscus aganju (Styloniscidae) is also recorded from two caves in the Serra do Ramalho karst area. The systematic position of Iuiuniscus iuiuensis is briefly discussed and Venezillo congener (Armadillidae) is redescribed from São Domingos karst area.

Taxonomy of Alpioniscus (Illyrionethes): A. magnus and three new species from the Dinaric Karst (Isopoda: Oniscidea: Trichoniscidae).

Alpioniscus (Illyrionethes) is a widespread subgenus of Trichoniscidae in the Dinaric Karst with 15 nominal troglobiotic species. Here we redescribe Alpioniscus (Illyrionethes) magnus and describe three new species (A. lossinii n. sp., A. drazinai n. sp., and A. mandalinae n. sp.) restricted to coastal areas. As a result, 18 nominal Illyrionethes species are known from the Dinaric Karst.

Unravelling the molecular mechanisms of nickel in woodlice.

During the last few years, there has been an alarming increase in the amount of nickel (Ni) being released into the environment, primarily due to its use in the production of stainless steel but also from other sources such as batteries manufacturing and consequent disposal. The established biotic ligand models provide precise estimates for Ni bioavailability, in contrast, studies describing the mechanisms underpinning toxicological effect of Ni are scarce. This study exploits RNA-seq to determine the transcriptomic responses of isopods using Porcellionides pruinosus as an example of a terrestrial metal-resistant woodlouse. Furthermore, the recently proposed model for Ni adverse outcome pathways (Ni-AOP) presents an unprecedented opportunity to fit isopod responses to Ni toxicity and define Porcellionides pruinosus as a metalomic model. Prior to this study, P. pruinosus represented an important environmental sentinel, though lacking genetic/omic data. The reference transcriptome generated here thus represents a major advance and a novel resource. A detailed annotation of the transcripts obtained is presented together with the homology to genes/gene products from Metazoan and Arthropoda phylum, Gene Ontology (GO) classification, clusters of orthologous groups (COG) and assignment to KEGG metabolic pathways. The differential gene expression comparison was determined in response to nickel (Ni) exposure and used to derive the enriched pathways and processes. It revealed a significant impact on ion trafficking and storage, oxidative stress, neurotoxicity, reproduction impairment, genetics and epigenetics. Many of the processes observed support the current Ni-AOP although the data highlights that the current model can be improved by including epigenetic endpoints, which represents key chronic risks under a scenario of Ni toxicity.

Woodlice of Belgium: an annotated checklist and bibliography (Isopoda, Oniscidea).

Woodlice are key organisms for nutrient cycling in many terrestrial ecosystems; however, knowledge on this invertebrate group is limited as for other soil fauna taxa. Here, we present an annotated checklist of the woodlice of Belgium, a small but densely populated country in Western Europe. We reviewed all 142 publications on Belgian woodlice, the oldest dating back to 1831 and re-identified all doubtful specimens from the Royal Belgian Institute of Natural Sciences (RBINS) collection. These data is complemented with observations from extensive field surveys dating from March 2014 until December 2017. We report 36 species of woodlice with free-living populations for Belgium. Nine species can be added compared to the latest checklist published in 2000 being Hyloniscusriparius (C. Koch, 1838), Miktoniscuspatiencei Vandel, 1946, Trichoniscoidessarsi Patience, 1908, Haplophthalmusmontivagus Verhoeff, 1941, Porcelliomonticola Lereboullet, 1853, Metatrichoniscoidesleydigii (Weber, 1880), Trichoniscusalemannicus Verhoeff, 1917, Elumacaelata (Miers, 1877) and Philosciaaffinis Verhoeff, 1908. Two species are deleted from the checklist (Ligidiumgermanicum Verhoeff, 1901 and Armadillidiumdepressum Brandt, 1833) because records are doubtful and no material has been preserved. Additionally the data of the field surveys is used to determine a species status of occurrence in Belgium. For each species, a short overview of their first records is provided and their confirmation as part of the Belgian fauna, their current status, as well as a complete bibliography of the species in Belgium.

Terrestrial isopods (Oniscidea) of the White Carpathians (Czech Republic and Slovakia).

This paper summarizes data regarding the terrestrial isopods of the White Carpathians range in the Western Outer Carpathians based on field research undertaken during the past several decades in natural meadow pasture and forest localities. Using a combination of four collection methods 19 species belonging to nine families were recorded. The most common representatives were Protracheoniscuspolitus Trachelipusrathkii and Ligidiumhypnorum. In general the biodiversity of isopod communities in the studied area was considerable with half of the localities explored inhabited by six to ten species. The composition of the isopod assemblages was determined by the character of the biotope and its geographical location. Forest habitats were considerably richer in species than the meadow and pasture ecosystems. Some xerotermic localities in the Slovak part of the area were inhabited by Trachelipusnodulosus and Orthometoponplanum thermophilic species typically associated with warmer parts of Europe. Two relic species (Hyloniscusmariae and Ligidiumgermanicum) were confirmed for this area. Except for only one finding of Porcellioscaber no other evidently introduced or synanthropic species were recorded. Based on the data analyzed the high nature conservancy value of the given area is emphasised.

Presence of a stridulatory apparatus in the manca stages of isopods (Crustacea, Isopoda, Oniscidea).

Armadilloofficinalis Duméril, 1816 (Armadillidae) is a widespread terrestrial isopod species in the Mediterranean basin and on the western coasts of the Black Sea. The species is adapted to live in xeric environments and has mainly nocturnal habits. This species is capable of producing stridulations, which is nowadays recognized as a synapomorphy of the genus. In both sexes, these vibrations are produced by a line of scales on the propodus of pereopod 4 and 5. The main goals of this study are: to describe the manca stages of Armadilloofficinalis; to detect the presence of the stridulatory apparatus in the manca stages; to evaluate the differences of such apparatus in the various manca stages. The manca stages (I, II, III) of Armadilloofficinalis are described for the first time showing: i, the shortest duration (known in literature) of the manca stage I (approximately 30 minutes); ii, the presence of a rudimental stridulatory organ that may be of great importance in terms of evolutionary aspects and adaptation to terrestrial life. Notes on the reproductive biology are also reported. Furthermore, some considerations on future perspectives for A.officinalis as a model species in biotremology are also discussed.

Investigating the Molecular Genetic Basis of Cytoplasmic Sex Determination Caused by Wolbachia Endosymbionts in Terrestrial Isopods.

In animals, sexual differences between males and females are usually determined by sex chromosomes. Alternatively, sex may also be determined by vertically transmitted intracellular microbial endosymbionts. The best known cytoplasmic sex manipulative endosymbiont is Wolbachia which can, for instance, feminize genetic males into phenotypic females in the terrestrial isopod Armadillidium vulgare. However, the molecular genetic basis of cytoplasmic sex determination is unknown. To identify candidate genes of feminization induced by Wolbachia strain wVulC from A. vulgare, we sequenced the genome of Wolbachia strain wCon from Cylisticus convexus, the most closely related known Wolbachia strain to wVulC that does not induce feminization, and compared it to the wVulC genome. Then, we performed gene expression profiling of the 216 resulting wVulC candidate genes throughout host developmental stages in A. vulgare and the heterologous host C. convexus. We identified a set of 35 feminization candidate genes showing differential expression during host sexual development. Interestingly, 27 of the 35 genes are present in the f element, which is a piece of a feminizing Wolbachia genome horizontally transferred into the nuclear genome of A. vulgare and involved in female sex determination. Assuming that the molecular genetic basis of feminization by Wolbachia and the f element is the same, the 27 genes are candidates for acting as master sex determination genes in A. vulgare females carrying the f element.

Joint effects of chlorpyrifos and mancozeb on the terrestrial isopod Porcellionides pruinosus: A multiple biomarker approach.

The exposure to pesticides by nontarget soil biota has long been regarded as a serious downside of modern agricultural regimes and the subject of heated debate. Of utmost relevance is the exposure to pesticide mixtures because their effects have been shown to not necessarily reflect the individual toxicity of their components, and even the simple addition of effects may lead to consequences not clearly anticipated. In the present study, a multiple biomarker approach was employed to identify the mechanistic and time effects underlying several single and mixture treatments of chlorpyrifos (CPF) and mancozeb (MCZ) in juveniles and adults of the terrestrial isopod Porcellionides pruinosus. The effects of the individual pesticides and the mixtures at recommended doses were mostly transitory under these controlled conditions and one-pulse exposure. Whereas imbalances were identified on detoxification and oxidative stress-related enzymes, the isopods generally showed the ability to recover through the end of the experiment. However, juveniles displayed greater vulnerability than adults. Most of the differences between life stages occurred in energy-related parameters where distinct performances and stress-handling behaviors were observed, suggesting higher metabolic costs to juveniles. Our results stress that understanding the time dependence of the underlying mechanisms governing the joint effects of the pesticides can help in assessing and anticipating the effects of the pesticide mixtures. Moreover, we emphasize the importance of taking life stage-related differences into consideration when evaluating the environmental risks of pesticides and pesticide mixtures. Environ Toxicol Chem 2018;37:1446-1457. © 2018 SETAC.