Differential analysis of the haemolymph proteome of Carcinus maenas parasitized by Sacculina carcini (Cirripeda, Rhizocephala) reveals potential mechanisms of parasite control.

Sacculina carcini is an endoparasite of the green crab, Carcinus maenas. This parasite induces behavioural changes in its host and affects its metabolism by inhibiting moulting and reproduction. Using a proteomic approach in mass spectrometry, we studied the haemolymph proteomes of healthy and parasitized wild green crabs from Brittany, France to identify proteins that are differentially expressed as a consequence of parasitization. We also investigated specific proteins involved in reproduction, moulting, and immunity. We detected 77 proteins for females and 53 proteins for males that were differentially present between the healthy and parasitized crabs, some of which were sex-specific. Detection of these differentially expressed proteins suggests that the parasite can inhibit and promote different aspects of the immune response of the host. Sacculina appears to inhibit host melanisation for self-protection, while promoting the presence of immune factors, such as antimicrobial peptides to cope with possible bacterial superinfections. Moreover, one protein, juvenile hormone esterase-like carboxylesterase, was 17-times more abundant in parasitized crabs than in healthy crabs and may be responsible for inhibiting moulting and reproduction in parasitized crabs, thus ensuring the success of Sacculina reproduction.

Discovery of a Digenean (Cryptogonimidae) Living in a Cleft-Lipped Goby, Sicyopterus cynocephalus (Teleostei: Gobiidae) from Ranongga Island, Solomon Islands: Analysis of Multiple Ribosomal DNA Regions.

This study results from a continued investigation of the occurrence and diversity of parasites of freshwater fish in the Solomon Islands. Thus, we revealed a new host as well as a new site of infection and a new geographical area for the cryptogonimid parasite, Stemmatostoma cribbi (Digenea: Cryptogonimidae). The cryptogonimid species was identified based on general morphology and on molecular data of metacercariae found in the gills of the cleft-lipped goby, Sicyopterus cynocephalus, from Ranongga Island, Western Province of the Solomon Islands. This is the first report of a Stemmatostoma sp. digenean parasitizing fish of the genus Sicyopterus in the Indo-Pacific region and the first report of S. cribbi infection in a fish from the Solomon Islands. Phylogenetic analysis performed by Bayesian inference and maximum likelihood confirmed the presence of the cryptogonimid in a well-supported subclade of Stemmatostoma spp.

New leptestherid clam shrimps (Pancrustacea: Branchiopoda: Spinicaudata: Leptestheriidae) from peninsular India.

Spinicaudatan clam shrimps are an evolutionarily ancient lineage restricted to temporary freshwater pools. Use of classical morpho-taxonomic approaches alone have led to some issues in the taxonomy of this group, which are now being resolved through integrative taxonomy. Here, we describe two new leptestherid spiny clam shrimps Leptestheria chalukyae sp. nov. and Leptestheria gomantaki sp. nov. from peninsular India based on their unique morphological characters and distinct phylogenetic position. We also re-describe Leptestheria nobilis and present an overview of the morphological characters of all the Indian leptestherids. Most of the conventional taxonomic characters appear to overlap among all the Indian species, although the combination of occipital condyle shape and the cercopod marginal spines arrangement in combination, appear to be useful in separating leptestherid species.

Assigning the Australian clam shrimp Eoleptestheria (Branchiopoda: Spinicaudata: Leptestheriidae) to a new species status.

We assign the only Australian leptestherid clam shrimp, Eoleptestheria ticinensis (Balsamo-Crivelli, 1859) to a new species status, Leptestheria timmsi sp. nov., based on the latest molecular phylogeny of spinicaudatan clam shrimps from a previous study. We provide a redescription of the species, highlighting morphological details not explored previously for this species. We also present a comparative account of all Eoleptestheria species and populations throughout the world. The head morphology of Leptestheria timmsi sp. nov. overlaps with other populations of Eoleptestheria and a few Leptestheria species. The telsonic and cercopod marginal spines seem to increase in size posteriorly in L. timmsi sp. nov., unlike in many other Eoleptestheria populations.

Phylogenetic analysis and characterization of a new parasitic cnidarian (Myxosporea: Myxobolidae) parasitizing skin of the giant mottled eel from the Solomon Islands.

Myxosporeans are microscopic cnidarians associated with severe diseases in aquaculture and wild fish populations. This group of parasitic cnidarians thus warrants close attention concerning its potential impact on susceptible fish stocks. At present, little is known about this group of parasites infecting anguillid eels. From myxospore specimens collected from a freshwater eel (Anguilla marmorata) in the Solomon Islands, we describe a new species belonging to the genus Myxobolus based on an integrative taxonomic analysis of morphological, biological traits and molecular data. Furthermore, we determined the phylogenetic position and relationships of this species among other platysporine myxosporeans. Molecular phylogenetic assessment of small subunit ribosomal DNA showed that the species clusters together with Myxobolus portucalensis and Echinactinomyxon type 5 Özer, Wootten and Shinn, 2002, in a well-supported subclade. This is the first report of a myxosporean parasite infecting fish from the Solomon Islands.

Phylogeny and Biogeography of Spinicaudata (Crustacea: Branchiopoda).

Spinicaudata (spiny clam shrimp) is a taxon of Branchiopoda occurring since the Devonian and today it occurs nearly globally in temporary water bodies. We present the most species-rich phylogenetic analyses of this taxon based on four molecular loci: COI, 16S rRNA, EF1α and 28S rRNA. Our results support previous findings that Cyzicidae sensu lato is paraphyletic. To render Cyzicidae monophyletic we establish a fourth extant spinicaudatan family to accommodate Eocyzicus. Within Cyzicidae, none of the genera Cyzicus, Caenestheria or Caenestheriella are monophyletic, and the morphological characters used to define these genera (condyle length and rostrum shape) are not associated with well-delimited clades within Cyzicidae. There is insufficient resolution to elucidate the relationships within Leptestheriidae. However, there is sufficient evidence to show that the leptestheriid genera Eoleptestheria and Leptestheria are non-monophyletic, and there is no support for the genus Leptestheriella. Molecular clock analyses suggest that the wide geographic distribution of many spinicaudatan taxa across multiple continents is largely based on vicariance associated with the break-up of Pangea and Gondwana. Trans-oceanic dispersal has occurred in some taxa (e.g., Eulimnadia and within Leptestheriidae) but has been relatively rare. Our results highlight the need to revise the taxonomy of Cyzicidae and Leptestheriidae and provide evidence that the global spinicaudatan diversity may be underestimated due to the presence of numerous cryptic species. We establish Eocyzicidae fam. nov. to accommodate the genus Eocyzicus. Consequently, Cyzicidae comprises only two genera -Cyzicus and Ozestheria. Ozestheria occurs also in Africa and Asia and Ozestheria pilosa new comb. is assigned to this genus.

Description of the First Leptestheria Species (Branchipoda, Spinicaudata) from Brazil.

Leptestheria (Spinicaudata: Leptestheriidae) was collected for the first time from Brazil. This species was found during a study on temporary pools in the municipality of Palmas de Monte Alto in Bahia state, which is in the Caatinga climatic zone. A morphological description of this new species is presented and compared with other known South American species. Our new species differs from the other described species by the form of the head and the telson. The associated fauna is comparable to what was observed by Gurney in 1931 in Paraguay or by Roessler in 1995 in Colombia, indicating possible connections between the dry regions of South America.

A new species of genus Eulimnadia Packard, 1874 (Branchiopoda: Spinicaudata: Limnadiidae) from India with an updated key for some Indian species.

We describe a new species of Eulimnadia, Eulimnadia bondi sp. nov., from the state of Goa, India. This is the 8th species to be described from the country and first from this state. This species differs from all other Indian species in having unique egg morphology-cylindrical egg with dilated borders at both ends and a specific internal eggshell structure. Specimens showed intra-population morphological variation as observed in other species from the genus. Eulimnadia bondi sp. nov. egg resembles the South American E. colombiensis and the Australian E. australiensis. These species are however distinguished on egg morphological details and by some morphological characteristic of adults. Based on the available information, we present an overview of the distribution of the different Indian Eulimnadia species and an updated identification key based on egg morphology.

Chirocephalus sanhadjaensis sp. nov., a new chirocephalid species (Branchiopoda: Anostraca) from Numidia (Algeria).

A new species of Chirocephalus collected in temporary freshwater ponds in Northeastern Algeria is described. Chirocephalus sanhadjaensis sp. nov. seems to be restricted to Ain-Magroun and Belkroun pools (Skikda Province). The most similar species is C. marchesonii Ruffo Vesentini, 1957, which is endemic to Italy. The new taxon, belonging to the "diaphanus" species group of the genus Chirocephalus, is identifiable from all congeners primarily by the shape of the antennal appendages and that of the apex of the distal antennomere of the antennae. The resting eggs of C. sanhadjaensis sp. nov. are similar to those of C. diaphanus or C. salinus but are larger (448.24 ± 30.93 µm). The restricted distribution of this species confirms the high biological diversity of the area of the Guerbes-Sanhadja eco-complex.

Comparative Study of Chemosensory Organs of Shrimp From Hydrothermal Vent and Coastal Environments.

The detection of chemical signals is involved in a variety of crustacean behaviors, such as social interactions, search and evaluation of food and navigation in the environment. At hydrothermal vents, endemic shrimp may use the chemical signature of vent fluids to locate active edifices, however little is known on their sensory perception in these remote deep-sea habitats. Here, we present the first comparative description of the sensilla on the antennules and antennae of 4 hydrothermal vent shrimp (Rimicaris exoculata, Mirocaris fortunata, Chorocaris chacei, and Alvinocaris markensis) and of a closely related coastal shrimp (Palaemon elegans). These observations revealed no specific adaptation regarding the size or number of aesthetascs (specialized unimodal olfactory sensilla) between hydrothermal and coastal species. We also identified partial sequences of the ionotropic receptor IR25a, a co-receptor putatively involved in olfaction, in 3 coastal and 4 hydrothermal shrimp species, and showed that it is mainly expressed in the lateral flagella of the antennules that bear the unimodal chemosensilla aesthetascs.

Review of the Eulimnadia (Branchiopoda: Spinicaudata: Limnadiidae) from Argentina with the description of a new species.

A new spinicaudatan species, Eulimnadia pampa sp. nov., is described from Argentina mostly based on the differential egg morphology. This species was collected from rainwater pools from Buenos Aires City and the provinces of Buenos Aires and Entre Ríos, and compared to other Neotropical species. Previous Argentine record of E. brasiliensis is shown to belong to our new species, which represents the southernmost Neotropical Eulimnadia. We also present new records of the Paraguayan Eulimnadia ovisimilis Martin and Belk, 1989 from northeastern Argentina. Eulimnadia santiaguensis is treated as a species inquirenda, thus we recognize only three Eulimnadia species in Argentina: E. pampa sp. nov., E. ovilunata and E. ovisimilis, and we provide a brief indentification key. We present the first description and SEM images of the putative Eulimnadia spermatophore, which we found in the new species.

A 365-Million-Year-Old Freshwater Community Reveals Morphological and Ecological Stasis in Branchiopod Crustaceans.

Branchiopod crustaceans are represented by fairy, tadpole, and clam shrimps (Anostraca, Notostraca, Laevicaudata, Spinicaudata), which typically inhabit temporary freshwater bodies, and water fleas (Cladoceromorpha), which live in all kinds of freshwater and occasionally marine environments [1, 2]. The earliest branchiopods occur in the Cambrian, where they are represented by complete body fossils from Sweden such as Rehbachiella kinnekullensis [3] and isolated mandibles preserved as small carbonaceous fossils [4-6] from Canada. The earliest known continental branchiopods are associated with hot spring environments [7] represented by the Early Devonian Rhynie Chert of Scotland (410 million years ago) and include possible stem-group or crown-group Anostraca, Notostraca, and clam shrimps or Cladoceromorpha [8-10], which differ morphologically from their modern counterparts [1, 2, 11]. Here we report the discovery of an ephemeral pool branchiopod community from the 365-million-year-old Strud locality of Belgium. It is characterized by new anostracans and spinicaudatans, closely resembling extant species, and the earliest notostracan, Strudops goldenbergi [12]. These branchiopods released resting eggs into the sediment in a manner similar to their modern representatives [1, 2]. We infer that this reproductive strategy was critical to overcoming environmental constraints such as seasonal desiccation imposed by living on land. The pioneer colonization of ephemeral freshwater pools by branchiopods in the Devonian was followed by remarkable ecological and morphological stasis that persists to the present day.

First faunal inventory of large branchiopods (Crustacea: Branchiopoda) of Western Maharashtra, India with taxonomical and distributional comments.

An updated inventory of large branchiopods of the Western Maharashtra state, India is presented for the first time. Employing current identification keys, we have detailed the egg morphology of Eulimnadia indocylindrova, which shares some similarities with E. geayi from South America and E. alluaudi from Madagascar, based on these egg characters. A thorough re-description of few morphological traits of the same species is also presented. We also have described the female of a Cyzicus species seen rarely from the studied region. Distributional comments on few species are also reported.

Molecular phylogeny of the Notostraca.

We used a combined analysis of one nuclear (28S rDNA) and three mitochondrial markers (COI, 12S rDNA, 16S rDNA) to infer the molecular phylogeny of the Notostraca, represented by samples from the six continents that are inhabited by this group of branchiopod crustaceans. Our results confirm the monophyly of both extant notostracan genera Triops and Lepidurus with good support in model based and maximum parsimony analyses. We used branchiopod fossils as a calibration to infer divergence times among notostracan lineages and accounted for rate heterogeneity among lineages by applying relaxed-clock models. Our divergence date estimates indicate an initial diversification into the genera Triops and Lepidurus in the Mesozoic, most likely at a minimum age of 152.3-233.5 Ma, i.e., in the Triassic or Jurassic. Implications for the interpretation of fossils and the evolution of notostracan morphology are discussed. We further use the divergence date estimates to formulate a biogeographic hypothesis that explains distributions of extant lineages predominantly by overland dispersal routes. We identified an additional hitherto unrecognised highly diverged lineage within Lepidurus apus lubbocki and three additional previously unknown major lineages within Triops. Within T. granarius we found deep differentiation, with representatives distributed among three major phylogenetic lineages. One of these major lineages comprises T. cancriformis, the T. mauritanicus species group and two hitherto unrecognised T. granarius lineages. Samples that were morphologically identified as T. granarius diverged from the most basal nodes within this major lineage, and divergence dates suggested an approximate age of 23.7-49.6 Ma for T. cancriformis, indicating the need for a taxonomic revision of Triassic and Permian fossils that are currently attributed to the extant T. cancriformis. We thus elevate T. cancriformis minor to full species status as Triops minorTrusheim, 1938 and include in this species the additional Upper Triassic samples that were attributed to T. cancriformis. We further elevate T. cancriformis permiensis to full species status as Triops permiensisGand et al., 1997.

Adaptation to thermally variable environments: capacity for acclimation of thermal limit and heat shock response in the shrimp Palaemonetes varians.

In the context of climate change, there is a sustained interest in understanding better the functional mechanisms by which marine ectotherms maintain their physiological scope and define their ability to cope with thermal changes in their environment. Here, we present evidence that the variable shrimp Palaemonetes varians shows genuine acclimation capacities of both the thermal limit (CT(max)) and the heat shock response (hsp70 induction temperature). During cold acclimation to 10 °C, the time lag to adjust the stress gene expression to the current environmental temperature proved to exceed 1 week, thereby highlighting the importance of long-term experiments in evaluating the species' acclimation capacities. Cold and warm-acclimated specimens of P. varians can mobilise the heat shock response (HSR) at temperatures above those experienced in nature, which suggests that the species is potentially capable of expanding its upper thermal range. The shrimp also survived acute heat shock well above its thermal limit without subsequent induction of the HSR, which is discussed with regard to thermal adaptations required for life in highly variable environments.

Acetylcholinesterase activity in Clytia hemisphaerica (Cnidaria).

Cholinesterase activity is known in representatives of all living organisms phyla but the origin of the cholinergic system as known in bilaterian animals is still undeciphered. In particular the implication of cholinesterases in the nervous system of non-bilaterian Metazoa is not well known. We thus chose to investigate this activity in the Clytia hemisphaerica (Cnidaria) medusa. In toto histochemical staining revealed an acetylcholinesterase activity in the tentacle bulbs but not in the nervous system. Sequences homologous to acetylcholinesterase were searched within Clytia ESTs and compared to other sequences found in public databases.

Ordered progression of nematogenesis from stem cells through differentiation stages in the tentacle bulb of Clytia hemisphaerica (Hydrozoa, Cnidaria).

Nematogenesis, the production of stinging cells (nematocytes) in Cnidaria, can be considered as a model neurogenic process. Most molecular data concern the freshwater polyp Hydra, in which nematocyte production is scattered throughout the body column ectoderm, the mature cells then migrating to the tentacles. We have characterized tentacular nematogenesis in the Clytia hemisphaerica hydromedusa and found it to be confined to the ectoderm of the tentacle bulb, a specialized swelling at the tentacle base. Analysis by a variety of light and electron microscope techniques revealed that while cellular aspects of nematogenesis are similar to Hydra, the spatio-temporal characteristics are markedly more ordered. The tentacle bulb nematogenic ectoderm (TBE) was found to be polarized, with a clear progression of successive nematoblast stages from a proximal zone (comprising a majority of undifferentiated cells) to the distal end where the tentacle starts. Pulse-chase labelling experiments demonstrated a continuous displacement of differentiating nematoblasts towards the tentacle tip, and that nematogenesis proceeds more rapidly in Clytia than in Hydra. Compact expression domains of orthologues of known nematogenesis-associated genes (Piwi, dickkopf-3, minicollagens and NOWA) were correspondingly staggered along the TBE. These distinct characteristics make the Clytia TBE a promising experimental system for understanding the mechanisms regulating nematogenesis.

Knockdown of spalt function by RNAi causes de-repression of Hox genes and homeotic transformations in the crustacean Artemia franciscana.

Hox genes play a central role in the specification of distinct segmental identities in the body of arthropods. The specificity of Hox genes depends on their restricted expression domains, their interaction with specific cofactors and selectivity for particular target genes. spalt genes are associated with the function of Hox genes in diverse species, but the nature of this association varies: in some cases, spalt collaborates with Hox genes to specify segmental identities, in others, it regulates Hox gene expression or acts as their target. Here we study the role of spalt in the branchiopod crustacean Artemia franciscana. We find that Artemia spalt is expressed in the pre-segmental 'growth zone' and in stripes in each of the trunk (thoracic, genital and post-genital) segments that emerge from this zone. Using RNA interference (RNAi), we show that knocking down the expression of spalt has pleiotropic effects, which include thoracic to genital (T-->G), genital to thoracic (G-->T) and post-genital to thoracic (PG-->T) homeotic transformations. These transformations are associated with a stochastic de-repression of Hox genes in the corresponding segments of RNAi-treated animals (AbdB for T-->G and Ubx/AbdA for G-->T and PG-->T transformations). We discuss a possible role of spalt in the maintenance of Hox gene repression in Artemia and in other animals.

Posterior patterning genes and the identification of a unique body region in the brine shrimp Artemia franciscana.

All arthropods share the same basic set of Hox genes, although the expression of these genes differs among divergent groups. In the brine shrimp Artemia franciscana, their expression is limited to the head, thoracic/trunk and genital segments, but is excluded from more posterior parts of the body which consist of six post-genital segments and the telson (bearing the anus). Nothing is currently known about the genes that specify the identity of these posterior structures. We examine the expression patterns of four candidate genes, Abdominal-B, caudal/Cdx, even-skipped/Evx and spalt, the homologues of which are known to play an important role in the specification of posterior structures in other animals. Abdominal-B is expressed in the genital segments of Artemia, but not in the post-genital segments at any developmental stage. The expression of caudal, even-skipped and spalt in the larval growth-zone suggests they may play a role in the generation of body segments (perhaps comparable with the role of gap and segmentation genes in insects), but not a direct role in defining the identity of post-genital segments. The expression of caudal at later stages suggests a role in the specification of anal structures. A PCR screen designed to isolate Hox genes expressed specifically in the posterior part of the body failed to identify any new Hox genes. We conclude that the post-genital segments of Artemia are not defined by any of the genes known to play a role in the specification of posterior segments in other arthropods. We argue that these segments constitute a unique body region that bears no obvious homology to previously characterised domains of Hox gene activity.