Pillbug: A Toolbox for Feminisation.

Wolbachia-induced feminisation is an extended phenotype that has been observed in only three models: isopods, butterflies and leafhoppers. Even among these, the process of feminisation differs markedly, especially since sexual differentiation is coordinated body-wide by a hormone in isopods, whereas in insects it is a chromosomally determined process and enforced cell by cell. For isopods, Armadillidium vulgare has contributed substantially to an understanding of Wolbachia-mediated feminisation, because a wide array of know-how and methods has been tailored to this model. Here, we describe establishment and maintenance of Wolbachia-infected laboratory lineages from animals collected in the field, the grafting of an androgenic gland to demonstrate the mode of action of Wolbachia (i.e. the disruption of the Androgenic Hormone pathway), and transinfection of naïve adults with Wolbachia. Finally, we describe selection of a ZZ genetic background in lineages created from transinfected WZ females, which is necessary to benefit from a property intrinsic to the naturally infected lineages: In a ZZ background, the vertical transmission rate of Wolbachia directly equates to the female ratio in the progeny. These protocols provide the essential framework for investigating molecular aspects of Wolbachia-induced feminisation.

Prudent burrow-site selection in a landscape of fear.

Prey should select safer breeding sites over riskier sites of otherwise similar habitats. This preference, however, may differ between conspecifics of different competitive abilities if the costs of intraspecific competition overpower the benefits of breeding in a safer site. Our goal was to test this hypothesis by exploring the burrow-site selection of different-sized desert isopods (Hemilepistus reaumuri) near and away from a scorpion burrow. We found that larger females are more likely to occupy burrows than smaller females, regardless of whether these burrows were close or away from scorpion burrows. We also found that larger females stayed longer in safer burrows and that smaller females tended to stay longer in riskier sites even in the absence of direct competition, implying a prudent burrow-site selection. We found no association between male size and the tendency to occupy or to spend time in a burrow, regardless of whether these burrows were close or away from scorpion burrows. Our work highlights the need to consider intraspecific competition when exploring how predators regulate prey behaviour.

The circadian clock gene bmal1 is necessary for co-ordinated circatidal rhythms in the marine isopod Eurydice pulchra (Leach).

Circadian clocks in terrestrial animals are encoded by molecular feedback loops involving the negative regulators PERIOD, TIMELESS or CRYPTOCHROME2 and positive transcription factors CLOCK and BMAL1/CYCLE. The molecular basis of circatidal (~12.4 hour) or other lunar-mediated cycles (~15 day, ~29 day), widely expressed in coastal organisms, is unknown. Disrupting circadian clockworks does not appear to affect lunar-based rhythms in several organisms that inhabit the shoreline suggesting a molecular independence of the two cycles. Nevertheless, pharmacological inhibition of casein kinase 1 (CK1) that targets PERIOD stability in mammals and flies, affects both circadian and circatidal phenotypes in Eurydice pulchra (Ep), the speckled sea-louse. Here we show that these drug inhibitors of CK1 also affect the phosphorylation of EpCLK and EpBMAL1 and disrupt EpCLK-BMAL1-mediated transcription in Drosophila S2 cells, revealing a potential link between these two positive circadian regulators and circatidal behaviour. We therefore performed dsRNAi knockdown of Epbmal1 as well as the major negative regulator in Eurydice, Epcry2 in animals taken from the wild. Epcry2 and Epbmal1 knockdown disrupted Eurydice's circadian phenotypes of chromatophore dispersion, tim mRNA cycling and the circadian modulation of circatidal swimming, as expected. However, circatidal behaviour was particularly sensitive to Epbmal1 knockdown with consistent effects on the power, amplitude and rhythmicity of the circatidal swimming cycle. Thus, three Eurydice negative circadian regulators, EpCRY2, in addition to EpPER and EpTIM (from a previous study), do not appear to be required for the expression of robust circatidal behaviour, in contrast to the positive regulator EpBMAL1. We suggest a neurogenetic model whereby the positive circadian regulators EpBMAL1-CLK are shared between circadian and circatidal mechanisms in Eurydice but circatidal rhythms require a novel, as yet unknown negative regulator.

Collecting Marine Gnathiid Isopod Fish Parasites with Light Traps.

A method to collect marine gnathiid isopod fish parasites with the use of light traps is presented. Gnathiid isopods are a major group of marine fish parasites that feed on blood and fluid from host fishes, mostly at night. Like ticks and mosquitos on land, they associate only temporarily with their host and spend most of their life free-living in the benthos. Given their high mobility and transient and predominantly nocturnal association with hosts, they cannot easily be collected by capturing free-living hosts. However, they are readily attracted to underwater light sources, creating the opportunity to collect them in light traps. Here the design and individual steps involved in the deployment and processing of specially adapted light traps for collecting free-living stages of gnathiid isopods are outlined. Sample results and possible modifications of the basic protocol for a variety of different sampling needs are presented and discussed.

Evaluation of newly designed flushing techniques for on-site remediation of arsenic-contaminated excavated debris.

Excavated debris (soil and rock) contaminated with geogenic arsenic (As) is an increasing concern for regulatory organizations and construction stakeholders. Chelator-assisted soil flushing is a promising method for practical on-site remediation of As-contaminated soil, offering technical, economic, and environmental benefits. Ethylenediaminetetraacetic acid (EDTA) is the most prevalent chelator used for remediating As-contaminated soil. However, the extensive environmental persistence and potential toxicity of EDTA necessitate the exploration of eco-compliant alternatives. In this study, the feasibility of the conventional flushing method pump-and-treat and two newly designed immersion and sprinkling techniques were evaluated at the laboratory scale (small-scale laboratory experiments) for the on-site treatment of As-contaminated excavated debris. Two biodegradable chelators, L-glutamic acid-N,N'-diacetic acid (GLDA) and 3-hydroxy-2,2'-iminodisuccinic acid (HIDS), were examined as eco-friendly substitutes for EDTA. Additionally, this study highlights a useful post-treatment measure to ensure minimal mobility of residual As in the chelator-treated debris residues. The pump-and-treat method displayed rapid As-remediation (t, 3 h), but it required a substantial volume of washing solution (100 mL g-1). Conversely, the immersion technique demonstrated an excellent As-extraction rate using a relatively smaller washing solution (0.33 mL g-1) and shorter immersion time (t, 3 h). In contrast, the sprinkling technique showed an increased As-extraction rate over an extended period (t, 48 h). Among the chelators employed, the biodegradable chelator HIDS (10 mmol L-1; pH, 3) exhibited the highest As-extraction efficiency. Furthermore, the post-treatment of chelator-treated debris with FeCl3 and CaO successfully reduced the leachable As content below the permissible limit.

Unraveling the host-parasite interaction: immune response in Oreochromis mossambicus to Cymothoa eremita (Isopoda, Cymothoidae) infection.

The host immune response in Oreochromis mossambicus during natural infection with the isopod Cymothoa eremita was investigated. The naturally infected fishes were examined for enzyme profile, viz., respiratory burst activity (RBA), myeloperoxidase activity (MPO), and expression of immune-related genes, viz., toll-like receptor 22 (TLR 22), interleukin-1ß (IL-1ß), tumor necrosis factor α (TNF-α), complement component (C3), chemokine (CXCa), and ß-actin in tissues of various organs (buccal cavity, gills and anterior kidney). Significant reduction (P < 0.05) in RBA and MPO was observed in the parasite-infected fishes when compared to the uninfected control fishes. In the buccal cavity, the expression of the immune-related genes was significantly (P < 0.05) upregulated, whereas all the genes except IL-1ß were significantly (P < 0.05) upregulated in the anterior kidney. In the case of gill tissue, the expressed genes showed a varied type of regulation. The immunological responses in O. mossambicus during isopod infection have not been investigated in detail so far, and this is the first study unveiling such insights. Hence, this study will help to improve our molecular understanding of the host-immune response to parasitic isopod infection.

Cataphryxus zapoteca sp. nov. (Isopoda, Bopyridae) and new hosts and records of bopyrid isopods from the Mexican Eastern Pacific.

Based on the examination of diverse crustacean taxa collected along the Mexican Pacific and deposited in the Colección Nacional de Crustáceos of the Instituto de Biología, UNAM, six species of bopyrid isopods were detected. New hosts and localities are reported for Munidion pleuroncodis Markham, 1975, Probopyrus pacificensis Román-Contreras, 1993, Probopyrus markhami Román-Contreras, 1996, Progebiophilus bruscai Salazar-Vallejo & Leija-Tristán, 1990 and Schizobopyrina striata (Nierstrasz & Brender à Brandis, 1929). Cataphryxus zapoteca sp. nov., is described as abdominal parasite of the shrimp Lysmata galapagensis Schmitt; this bopyrid is the second species described in the genus Cataphryxus Shiino, 1936 and the first registered on the American continent. Taxonomic characters, distribution and some reproductive data for five of the six species examined are provided in order to update the knowledge of this parasite group in this Eastern Pacific region.

Chemosensory proteins as putative semiochemical carriers in the desert isopod Hemilepistus reaumurii.

The order Isopoda contains both aquatic and terrestrial species, among which Hemilepistus reaumurii, which lives in arid environments and is the most adapted to terrestrial life. Olfaction has been deeply investigated in insects while it has received very limited attention in other arthropods, particularly in terrestrial crustaceans. In insects, soluble proteins belonging to two main families, Odorant Binding Proteins (OBPs) and Chemosensory Proteins (CSPs), are contained in the olfactory sensillar lymph and are suggested to act as carriers of hydrophobic semiochemicals to or from membrane-bound olfactory receptors. Other protein families, namely Nieman-Pick type 2 (NPC2) and Lipocalins (LCNs) have been also reported as putative odorant carriers in insects and other arthropod clades. In this study, we have sequenced and analysed the transcriptomes of antennae and of the first pair of legs of H. reaumurii focusing on soluble olfactory proteins. Interestingly, we have found 13 genes encoding CSPs, whose sequences differ from those of the other arthropod clades, including non-isopod crustaceans, for the presence of two additional cysteine residues, besides the four conserved ones. Binding assays on two of these proteins showed strong affinities for fatty acids and long-chain unsaturated esters and aldehydes, putative semiochemicals for this species.

110-million-years-old fossil suggests early parasitism in shrimps.

Direct evidence of paleo-parasitism in crustaceans is very scarce. Epicaridean isopods are obligatory parasites of crustaceans, including decapods such as crabs, shrimps, and lobsters. Their interaction with hosts is known from fossils as far back as the Jurassic through deformations of the branchial cuticle on the hosts. Their small size and low fossilization potential, outside of those larvae that have been found in amber, makes understanding the group's evolution challenging. Here, we report the oldest evidence of paleo-parasitism in marine shrimps and an imprint of a putative adult parasite that appears to be an epicaridean isopod. Our results suggest that the parasite-host interaction between epicaridean isopods and marine shrimps started at least 110 million years ago, and the Tethys Sea was a possible dispersal pathway for this lineage of parasites during the Jurassic and Cretaceous, as known for other marine organisms through most of the Mesozoic and Cenozoic. The oldest fossil records of bopyrid swellings associated with a large number of decapods from the Jurassic in Europe suggest that the Tethys region was a center of epicaridean distribution as a whole. Recent parasitic isopods found on dendrobranchiate shrimps are restricted to the Indo-Pacific and may represent a relict group of a lineage of parasites more widely distributed in the Mesozoic.

Progress in the discovery of isopods (Crustacea: Peracarida)-is the description rate slowing down?

Taxonomic species are the best standardised metric of biodiversity. Therefore, there is broad scientific and public interest in how many species have already been named and how many more may exist. Crustaceans comprise about 6% of all named animal species and isopods about 15% of all crustaceans. Here, we review progress in the naming of isopods in relation to the number of people describing new species and estimate how many more species may yet be named by 2050 and 2100, respectively. In over two and a half centuries of discovery, 10,687 isopod species in 1,557 genera and 141 families have been described by 755 first authors. The number of authors has increased over time, especially since the 1950s, indicating increasing effort in the description of new species. Despite that the average number of species described per first author has declined since the 1910s, and the description rate has slowed down over the recent decades. Authors' publication lifetimes did not change considerably over time, and there was a distinct shift towards multi-authored publications in recent decades. Estimates from a non-homogeneous renewal process model predict that an additional 660 isopod species will be described by 2100, assuming that the rate of description continues at its current pace.

Homeotic transformation in a terrestrial isopod: insights into the appendage identity in crustaceans.

In many crustacean species, an individual possesses both uniramous and biramous appendages that enable us to compare the two types on the same genetic background. Therefore, among the diverse morphologies of arthropod appendages, crustacean biramous appendages provide interesting subjects for studying the developmental mechanisms underlying appendage modifications. In this study, we report a malformed specimen of the terrestrial isopod Porcellio scaber, in which one of the pleopods was transformed into a different structure. Morphological observations of exoskeletons and musculatures by confocal scanning laser microscopy revealed that the transformed appendage was three-segmented, with at least the apical two segments having pereopod-like musculoskeletal structures. The apical segment of the transformed appendage lacked muscles, and the following segment had a pair of muscle bundles. These findings together with those of some previous studies of gene expression patterns in this species suggest that this anomaly could be caused by homeotic transformation of a flap-like pleopod into a three-segmented pereopod tip, which may be a homologous structure of the pleopod.

History of the terrestrial isopod genus Ligidium in Japan based on phylogeographic analysis.

BACKGROUND: Phylogeographical approaches explain the genetic diversity of local organisms in the context of their geological and geographic environments. Thus, genetic diversity can be a proxy for geological history. Here we propose a genus of woodland isopod, Ligidium, as a marker of geological history in relation to orogeny and the Quaternary glacial cycle. RESULTS: Mitochondrial analysis of 721 individuals from 97 sites across Japan revealed phylogenetic divergence between the northeastern and southwestern Japan arcs. It also showed repeated population expansions in northeastern Japan in response to Quaternary glacial and interglacial cycles. Genome-wide analysis of 83 selected individuals revealed multiple genetic nuclear clusters. The genomic groupings were consistent with the local geographic distribution, indicating that the Ligidium phylogeny reflects its regional history. CONCLUSION: Ligidium DNA sequence analysis can provide insight into the geological, geographical, and paleoenvironmental history of the studied region.

Integration of transcriptomic and metabolomic analyses reveal the molecular responses of the mud crab Scylla paramamosain to infection by an undescribed endoparasite Portunion sp.

Portunion is a rare endoparasitic isopod genus, recently observed inhabiting the hemocoel of the commercially important mud crab, Scylla paramamosain. For better understanding of the host-parasite interaction between S. paramamosain and Portunion sp., the metabolomic and transcriptomic changes in the hemolymph of the S. paramamosain were analyzed. We detected a total of 143 and 126 differentially accumulated metabolites in the positive and negative modes, respectively. Pathways related to amino acids and vitamin synthesis, such as Aminoacyl-tRNA biosynthesis, Tyrosine metabolism, Cysteine and methionine metabolism, Vitamin B6 metabolism, and Biotin metabolism were significantly enriched. Based on the transcriptomic data, a total of 942 differentially expressed genes were identified, of which 25 and 36 were significantly related to the immune system and metabolic pathways, respectively. Based on the metabolomic and transcriptomic data, 90 correlated metabolite-gene pairs were selected to build a regulatory network. Common significantly enriched pathways, including Starch and sucrose metabolism, Metabolism of xenobiotics by cytochrome P450, Aminoacyl-tRNA biosynthesis, Nitrogen metabolism, and Galactose metabolism were detected. On the basis of our analysis, the endoparasite Portunion sp. places a heavy metabolic burden on the host, particularly with respect to fundamental resources, such as amino acids, vitamins, carbohydrates, and lipids. In summary, these data provide an overview of the global metabolic and transcriptomic changes of the S. paramamosain resulting from Portunion sp. infection.

The use of fish parasitic isopods as element accumulation indicators in marine pollution monitoring.

Marine ecosystems are continuously under threat due to pollutants, which endanger marine biodiversity. The present study determines the potential use of the parasitic isopod, Cinusa tetrodontis Schjödte et Meinert, 1884, together with its fish host, Amblyrhynchotes honckenii (Bloch) for marine bioaccumulation monitoring. The concentrations of As, Cd, Cu, Mn, Ni, Pb, Se, and Zn were determined in muscle and liver tissues of infested and uninfested fish, and male and female parasites on the South African temperate south coast. The concentrations of Cu and Ni in C. tetrodontis differed significantly between two sampling sites, a near-pristine (Breede River Estuary, Witsand) and a more polluted site (harbour area in Mossel Bay). Mossel Bay isopods had higher concentrations of Ni, while Witsand isopods had higher concentrations of Cu. In contrast to fish hosts, parasitic isopods accumulated significantly higher levels of all elements except Cd. Most significant relationships between elements accumulated by C. tetrodontis and an increase of elements in fish tissues were seen in liver, rather than muscle tissue samples. Specimens of C. tetrodontis can be defined as good bioindicators for elements such as As, Cu, Mn, Ni, Pb, Se, and Zn, as they possess high bioaccumulation capabilities. This study addresses one of several future directions needed within environmental parasitology and highlights the importance of studying and utilising this host-ectoparasite model system.

Redescription and molecular characterization of Mothocya parvostis Bruce, 1986 (Crustacea: Isopoda: Cymothoidae) parasitic on Japanese halfbeak, Hyporhamphus sajori (Temminck & Schlegel, 1846) (Hemiramphidae) with Mothocya sajori Bruce, 1986 placed into synonymy.

Two species of Mothocya have previously been recorded from Hyporhamphus sajori: M. parvostis Bruce, 1986 and M. sajori Bruce, 1986. Mothocya parvostis is re-described based on the ovigerous female type and additional materials collected from the host from in and around the type locality. Morphological re-examination of fresh specimens and the type materials together with genetic data show that the M. sajori and M. parvostis are the same species, differing primarily in size, therefore we have placed Mothocya sajori Bruce, 1986 into a junior synonym of Mothocya parvostis Bruce, 1986. Mothocya parvostis is characterized by the following combinations of characters: 1) body slightly to moderately twisted to one side; 2) pereonite 7 posterior margin moderately to deeply recessed; 3) uropodal rami extending to pleotelson posterior margin; and 4) uropod rami bluntly rounded, exopod 1.5 times as long as peduncle. The differences of four morphological features for M. parvostis and M. sajori was quantified. Furthermore, a total of 635 isopods infesting H. sajori were collected from all over Japan to conduct quantitative morphological and molecular sequence analyses (mitochondrial cytochrome c oxidase subunit I and 16S rRNA). Although the four quantitative features did not overlap between the two species in type specimens, all quantitative morphological values of newly collected specimens in this study did not display a bimodal distribution. In addition, our molecular analyses found only a single clade for our newly collected specimens in neighbor-joining tree.

The genus Armadillo Latreille, 1802 (Oniscidea, Armadillidae) from Cyprus, with descriptions of two new species.

The present work aims to morphologically characterize the species of Armadillo from Cyprus. Moreover, two new species of the genus are described, A. konstantinoui sp. nov. from several places along the island, and A. karametae sp. nov. from Pafos region. The circum-Mediterranean species A. officinalis is also redescribed.

Three new species of the family Cirolanidae Dana, 1852 (Isopoda, Cymothoida) from the shallow Brazilian bathyal.

Three new species of isopod crustaceans of the family Cirolanidae-Dolicholana ivanyae spec. nov., Natatolana hadassae spec. nov. and Politolana cleoniceae spec. nov.-are described from the shallow waters of the Brazilian bathyal. The specimens were collected from the shallow bathyal zone of the Potiguar Basin, off the Brazilian state of Rio Grande do Norte, from Pernambuco and Bahia, and the Campos Basin, off Rio de Janeiro and São Paulo states. Dolicholana ivanyae spec. nov. can be distinguished from its congeners by the subrectangular shape of the head, its very long frontal lamina, which surpasses article 2 of the peduncle of antenna 1, and its densely setose pereopod 7. Natatolana hadassae spec. nov. is unique due to the subacute posteroventral angle of epimeron 4, ischial pereopod 3 as produced as those of pereopods 1 and 2, and the distal margin of the pleotelson with 10 robust distal setae. Politolana cleoniceae spec. nov. differs from its congeners based on the combination of the following traits: the pereon-pleon articulation is tight, with pleonite 1 clearly visible, and not covered completely by pereonite 7, while the pleon (in the dorsal-ventral view) is arched in a uniform sequence with the pereon, and the uropodal endopod is shorter than the exopod. In addition to these findings, the known geographical distribution of Dolicholana brucei, which was described recently from Brazil, is extended southward to include Bahia, Brazil. This study increases the number of cirolanid species recorded from Brazilian waters to 23.

New troglobitic species of Benthana Budde-Lund, 1908 and Benthanoides Lemos de Castro, 1958 from iron-ore caves and their important record in the Amazon biome (Crustacea: Isopoda: Philosciidae).

A new cave-restricted species of Benthana and two cave-restricted species of Benthanoides are described from iron-ore caves located in the Serra dos Carajás complex, Pará state, in the Amazon biome. Benthana alba n. sp. occurs only in one cave in the south of Serra Norte, in the plateau N5 (cave N5SM2_0077); Benthanoides amazonicus n. sp. occurs in several caves in the north of Serra Norte, in the plateau N1; and Benthanoides tarzan n. sp. occurs in several caves in the Serra do Tarzan. This record represents an important distribution data for Benthana that typically occurs in the Brazilian Atlantic Forest, indicating that such species may represent a relict lineage of this genus. Additionally, efforts must be directed towards the conservation of these species, all of which are associated with caves occurring in areas of intense mining activity.

Combining morphological and mitochondrial DNA data to describe a new species of Austroniscus Vanhöffen, 1914 (Isopoda, Janiroidea, Nannoniscidae) linking abyssal and hadal depths of the Puerto Rico Trench.

Hadal trenches are perceived as a unique deep-sea ecosystem with fundamentally different communities compared to the nearby abyss. So far, however, scarce information exists about how populations are genetically linked within a trench and about mechanisms for species divergence. The present study presents the morphological and molecular-genetic characterization and description of a new nannoniscid species within the genus Austroniscus Vanhöffen, 1914 obtained from abyssal and hadal depths of the Puerto Rico Trench, NW Atlantic. Samples were collected as part of the Vema-TRANSIT expedition onboard RV Sonne in January 2015. Because of the large depth differences between sampling locations (4,552-8,338 m), we expected to find different species within the genus inhabiting abyssal and hadal sites. Initial morphological examination using traditional light microscopy and Confocal Laser Scanning Microscopy was paired with subsequent molecular analysis based on mtDNA (COI and 16S). Contrary to our assumptions, combined morphological and molecular species delimitation analyses (sGMYC, mPTP, ABGD) revealed the presence of only one species spanning the abyssal and hadal seafloor of the Puerto Rico Trench. In addition, comparison with type material could show that this species belongs to a new species, Austroniscus brandtae n. sp., which is described herein. Incongruence between some species delimitation methods suggesting the presence of multiple species is interpreted as strong genetic population structuring within the trench, which is also supported by the analysis of the haplotype networks. The geographic and bathymetric distribution of Austroniscus species is discussed. The species described herein represents the first in the genus Austroniscus from the Atlantic Ocean and the deepest record of the genus to date, and hence significantly expanding previously known limits of its geographic and bathymetric range.