Flatworm cocoons in the abyss: same plan under pressure.

While knowledge of early ontogeny in abyssal animals is highly limited in general, it was completely lacking for abyssal, free-living platyhelminths. We discovered flatworm egg capsules (or 'cocoons') on rocks collected at depths of 6176-6200 m on the abyssal slope of the Kuril-Kamchatka Trench, northwestern Pacific. The egg capsules were black and spherical, around 3 mm in diameter, and contained three to seven individuals (n = 4) at the same developmental stage, either the spherical (putative early embryo) or vermiform (putative late embryo) stages. A molecular phylogenetic analysis based on 18S and 28S rRNA sequences revealed that the flatworms belong in suborder Maricola in Tricladida and suggested that they may have colonized from shallow to deep waters. This study provides the deepest record for free-living flatworms and the first information on their early life stages in the abyssal zone, which were very similar to those in shallow-water forms. This similarity in development between the relatively benign shallow-water and the extreme abyssal environments suggests that triclads adapting to the latter faced primarily physiological and/or ecological adaptive challenges rather than developmental ones.

Ontogenetic Changes in Pigmentation Pattern in Mesanthura miyakoensis (Crustacea: Isopoda: Anthuridae).

Species in the anthurid isopod genus Mesanthura have specific, dorsal dark pigmentation patterning on the body. Though Mesanthura species have traditionally been distinguished mainly by differences in the dorsal pigmentation pattern in females, the stability of the pigmentation pattern within species had not been investigated, and information was lacking on ontogenetic variation in the pattern. Our study showed the following for M. miyakoensis. (1) Mancae begin to show dorsal pigmentation in the marsupium roughly 9 days before their release. (2) The pigmentation pattern in the first-instar mancae (first free-living stage) differs from that in later instars. (3) The pigmentation pattern in females is discrete and stable from putative second-instar mancae through females lacking oostegites, and distorted but recognizable in ovigerious females. (4) The pattern in males is different from and less discrete than that in females; it remains similar through the molt from subadult to adult male but changes markedly with age, leading to heavy pigmentation of the body. (5) The pigmentation pattern in mancae and females remains stable and observable after storage in ethanol for at least 13.7 months. Our results suggest that comparisons of pigmentation pattern across species in Mesanthura taxonomy should be restricted to females in the post-manca or later stages.

Nectonema horsehair worms (Nematomorpha) parasitic in the Tanner crab Chionoecetes bairdi, with a note on the relationship between host and parasite phylogeny.

Nectonema nematomorphs utilize marine crustacean hosts in their life cycle; 16 decapod and 1 isopod genera have been reported to date as host genera. This study reports the first case of Nectonema parasitic in the Tanner crab Chionoecetes bairdi, adding another known host genus. A single nematomorph juvenile was recovered from the body cavity of each of 2 ovigerous female crabs. A nucleotide sequence for the 18S rRNA gene (1854 bp) was determined from 1 Nectonema individual. The 18S sequence showed Kimura 2-parameter (K2P) distances of 10.0, 2.0, and 1.7% from 18S sequences from Nectonema sp. from an isopod host, N. agile, and N. munidae, respectively. In an 18S-based tree, the unknown species was the sister taxon to a clade comprising N. agile and N. munidae, both of which also utilize decapod hosts. The phylogenetic relationships among the 3 Nectonema species parasitic in decapods were not congruent with the phylogeny of the hosts, not supporting a hypothesis of nematomorph-host co-evolution.

Phototaxis in Two Shallow-Water Zeuxo Species (Crustacea: Tanaidacea).

Different crustacean species can differ in their response to light. In Tanaidacea, a small group of aquatic, benthic crustaceans, previous studies suggested that several species may be positively phototactic based on their attraction to nocturnal light traps, but no experimental investigations of phototaxis had been conducted on this group. Here we show experimentally that two species in the genus Zeuxo are phototactic but exhibit opposite reactions to light; Zeuxo ezoensis, which inhabits the blades and stipes of seaweeds, was positively phototactic, whereas Zeuxo molybi, which inhabits muddy sediments overlying bedrock, was negatively phototactic. This differential response may reflect differences in photoenvironment between these species' microhabitats.

A new Sphaeronella species (Copepoda: Siphonostomatoida: Nicothoidae) parasitic on Euphilomedes sp. (Ostracoda: Myodocopa: Philomedidae) from Hokkaido, Japan, with an 18S molecular phylogeny.

With about 80 species, Sphaeronella is the most species-rich genus in the copepod family Nicothoidae. To date, 20 named Sphaeronella species have been reported as ectoparasites on ostracod crustaceans. Here we describe Sphaeronella uyenoi sp. nov. parasitic on the philomedid ostracod Euphilomedes sp. collected from Akkeshi Bay, Hokkaido, Japan, northwestern Pacific. Sphaeronella uyenoi most closely resembles S. monothrix (Bowman & Kornicker, 1967), parasitic on the cylindroleberidid Parasterope pollex Kornicker in Bowman & Kornicker in the northwestern Atlantic, but differs from the latter in having (1) the submedian skeleton containing paired, strongly chitinized, Λ-shaped areas and paired wide oblong holes bearing a strongly chitinized fringe posteriorly, and (2) maxillipedal segment 3 with antero-subdistal serration. We determined partial sequences for the cytochrome c oxidase subunit I (COI) and 18S rRNA (18S) genes for S. uyenoi and constructed an 18S-based phylogenetic tree of copepods. In our tree, Nicothoidae was not monophyletic, and S. uyenoi was the sister taxon to Cancerilla sp. in Cancerillidae (ectoparasites on brittle stars).

Digenean Metacercariae Parasitic in a Staurozoan Cnidarian.

I report digenean metacercariae from Staurozoa, which were not previously known as digenean hosts. The host species, Haliclystus tenuis Kishinouye, 1910, was collected from algae in Oshoro Bay, Hokkaido, Japan, and contained metacercariae in the mesoglea. The metacercariae were encysted; cysts were oval, 93 µm long by 64 µm wide in one live individual. For the digenean, I generated partial sequences for the 18S rRNA (1585 bp) and 28S rRNA (1672 bp) genes, and the region spanning the 3' end of the cytochrome c oxidase subunit gene and the 5' end of the 16S rRNA gene, including the threonine tRNA gene (868 bp in total). Phylogenetic reconstructions based on combined 18S + 28S datasets showed the digenean to belong in Opecoelidae, members of which utilize marine or freshwater teleost fishes as definitive hosts, and placed it in Plagioporinae (sensu lato) clade C within Opecoelidae.

Observations on Predation in Paranthura japonica Richardson, 1909 (Isopoda: Cymothoida: Paranthuridae).

Although isopods in Anthuroidea are predators, there is little information on their predatory behavior. In this study, we investigated predation by the paranthurid Paranthura japonica, which was originally described from northern Japan but has recently been reported as an invasive alien species in western America and Europe. Six crustacean species (two isopods, two amphipods, and two tanaidaceans, one of which does not co-occur with P. japonica in the wild) and one pycnogonid species were used as prey candidates in our experiments. Paranthura japonica preyed on all candidate species except the pycnogonid, grasping them with its falciform pereopods, inserting its piercing-type mouthparts, and sucking out the internal contents of the prey. Cannibalism or scavenging was observed when several P. japonica individuals were put in a single aquarium. This study showed that P. japonica is an aggressive predator; it consumed various crustaceans, including one it never encounters in the wild. Our results suggest that P. japonica will have a high impact on alien ecosystems it invades as a predator on native crustaceans. Cytochrome c oxidase subunit (COI) nucleotide sequences for putative P. japonica from Oshoro, Japan and a topotypic individual from Muroran confirmed that the population we dealt with was P. japonica.

Three Free-Living Marine Nematodes from Sagami Bay, Japan, with a Description of Wiesoncholaimus jambio sp. nov. (Nematoda: Oncholaimidae).

We describe three free-living marine nematode species from Sagami Bay, Japan. Wiesoncholaimus jambiosp. nov. is the second species of this genus. It differs from the type species, Wiesoncholaimus mawsonae Inglis, 1966, by its dorsal tooth, which is as long as the other two teeth; its conico-cylindrical tail, which is as long as 4.6-4.8 cloacal body diameters; and the absence of the gubernaculum. Thalassironus cf. britannicus de Man, 1889 agrees well with a redescription of Thalassironus britannicus de Man, 1889 based on specimens collected near the type locality. However, as it is possible that T. britannicus sensu lato includes two or more species, our species may not be conspecific to T. britannicus sensu stricto. Vasostoma cf. longispicula Huang and Wu, 2010 closely resembles Vasostoma longispicula Huang and Wu, 2010 originally described from China, but minor differentiations are observed in body diameter, body cuticle thickness, and type of precloacal supplements.

Description of a New Hamatipeda Species, with an 18S Molecular Phylogeny (Crustacea: Tanaidacea: Typhlotanaidae).

We describe a new typhlotanaid species, Hamatipeda kohtsukai sp. nov., collected from between 167 and 488 m depth in the Sagami Sea, Japan. This is the first record of Hamatipeda from the northern hemisphere. Hamatipeda kohtsukai resembles Hamatipeda trapezoida from the Subantarctic region in having pereonites 1-3 widest anteriorly (not rectangular), but differs from it in the length ratio of antennal articles 4/5; the number of setae on the dactyli of pereopods 1-3, ischia of pereopods 4-6, and carpi of pereopods 4-6; the shape of the unguis of pereopods 4-6; and the shape of the uropodal endopod. We determined partial sequences for the cytochrome c oxidase subunit I (COI; cox1) and 18S rRNA (18S) genes in H. kohtsukai. A phylogenetic reconstruction based on the 18S sequences recovered a highly supported Typhlotanaidae clade containing H. kohtsukai and Typhlotanais mixtus, with Paranarthrura sp. (Agathotanaidae) as the sister taxon. A key to species of Hamatipeda is presented.

First Complete Mitochondrial Genome of a Tanaidacean Crustacean (Arctotanais alascensis).

We present a complete mitochondrial genomic sequence for the tanaidacean Arctotanais alascensis (Richardson, 1899); this is the first complete mitogenome reported from the order Tanaidacea. The mitogenome is 13,988 bp long and contains 13 protein coding and two ribosomal RNA genes (as is typical for animal mitogenomes), and 21 of 22 transfer RNAs; we did not detect an isoleucine transfer RNA (trnI) gene. The gene order differed markedly from the hypothetical ground pattern for Pancrustacea; only four clusters (trnM + nad2; trnC + trnY + cox1 + trnL2 + cox2; trnD + atp8 + atp6 + cox3; trnH + nad4 + nad4l) ancestrally present were retained. In a malacostracan phylogenetic tree reconstructed from mitogenome data, basal relationships were marginally supported or incongruent with the traditional morphology-based classification and the latest phylogenetic reconstructions from large transcriptomic datasets. Relationships involving more recent divergences were better supported in our tree, suggesting that complete mitogenome sequences are more suitable for phylogenetic analyses within malacostracan orders, presumably including Tanaidacea.

Heterocypris spadix sp. nov. (Crustacea: Ostracoda: Cypridoidea) from Japan, with Information on Its Reproductive Mode.

We describe the cypridoidean ostracod Heterocypris spadix sp. nov. from brackish water on Okinawa Island, Japan. The species closely resembles Heterocypris salina (Brady, 1868) but differs in that (1) the marginal infolds on valves are less developed, (2) the tubercles on the anterior margin of the right valve are completely covered by the selvage and invisible in inner view, and (3) the calcified inner lamella on the ventral margin of the left and right valves is scarcely evident in inner view, as the ventral margins of the valves bend inwardly. We determined partial sequences for the cytochrome c oxidase subunit I (COI; cox1) and 18S rRNA genes in H. spadix for future DNA barcoding and phylogenetic analyses. Our sample contained only females. A breeding experiment revealed that H. spadix females reproduce parthenogenetically. Another experiment showed that H. spadix has low tolerance to desiccation, with all individuals at 25°C dying between 1-2 hours after removal from water. We amplified and sequenced a partial 16S rRNA sequence for the endosymbiotic bacterium Cardinium from H. spadix. Infection by Cardinium may be related to the parthenogenetic reproductive mode we observed in H. spadix.

First report of marine horsehair worms (Nematomorpha: Nectonema) parasitic in isopod crustaceans.

Nectonema, the only horsehair worm (Nematomorpha) genus found in marine environments, was previously known to be parasitic only in decapod crustaceans. We report Nectonema sp. as the first record of a marine nematomorph parasitic in isopod crustaceans. This is also the third record of marine nematomorphs from the North Pacific. Six infected isopods (Natatolana japonensis) collected from 1425 m of depth in the Sea of Japan each contained one to seven (mean 2.33) nematomorphs in the body cavity in the pereon. There was no correlation between the host body length and number of parasites. For Nectonema sp., we describe and illustrate morphological features of the parasitic juvenile stage and present nucleotide sequences for the cytochrome c oxidase subunit I gene (COI or cox1; 451 nt), 18S rRNA gene (1777 nt), and region spanning the internal transcribed spacer 1 (ITS1) and the 28S rRNA gene including the 5.8S rRNA gene and ITS2 (1218 nt in total). In an 18S maximum-likelihood tree that included 24 nematomorph species, Nectonema sp. grouped with N. agile from the northwestern Atlantic; the 18S gene from these two taxa was divergent by 11.8% K2P distance, suggesting that they are different species. Nectonema species may have a broader range of host groups than previously suspected, but may have been previously misidentified as nematode parasites.

First in Situ Observations of Behavior in Deep-Sea Tanaidacean Crustaceans.

We report on the behavior of a deep-sea tanaidacean, Gigantapseudes sp. (Apseudomorpha: Gigantapseudidae), recorded at the depths of 6446-6447 m by the manned submersible Shinkai 6500. From recordings of at least three individuals walking on the sea floor, we confirm that Gigantapseudes sp. is epibenthic, as previously inferred from leg shape. One individual was recorded entering a hole. All individuals in the videos kept pereopods 4 raised from the seafloor while walking, implying that those legs have a function other than for walking, such as mechano- or chemoreception, or posture control. Our in situ observations of behavior are the first for any deep-sea tanaidacean and illustrate the importance of recording high-resolution videos in the deep sea and archiving them for future use. Our identification of Gigantapseudes sp. from video footage provides the first record of this genus from Japanese waters and extends the northern limit of the known generic distribution.

Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex.

The microcrustacean Daphnia pulex is an important model for environmental, ecological, evolutionary and developmental genomics because its adaptive life history displays plasticity in response to environmental changes. Even though the whole-genome sequence is available and omics data have actively accumulated for this species, the available tools for analyzing gene function have thus far been limited to RNAi (RNA interference) and TALEN (the transcription activator-like effector nuclease) systems. The development of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is thus expected to further increase the genetic tractability of D. pulex and to advance the understanding of this species. In this study, we developed a genome editing system for D. pulex using CRISPR/Cas9 ribonucleoprotein complexes (Cas9 RNPs). We first assembled a CRISPR single-guide RNA (sgRNA) specific to the Distal-less gene (Dll), which encodes a homeodomain transcription factor essential for distal limb development in invertebrates and vertebrates. Then, we injected Cas9 RNPs into eggs and evaluated its activity in vivo by a T7 endonuclease I assay. Injected embryos showed defective formation of the second antenna and disordered development of appendages, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene.

Shell-Exchange Behavior in a Hermit-Crab-Like Tanaidacean (Crustacea: Malacostraca).

This study describes shell-exchange behavior in the hermit-crab-like tanaidacean Macrolabrum sp. (Pagurapseudidae: Pagurapseudinae) under captive conditions. I observed one shell exchange by Macrolabrum sp., the behavioral sequence of which was as follows: a shell-carrying tanaidacean 1) grasped the edge of the aperture of an empty gastropod shell with its right cheliped; 2) inspected the condition inside the shell four times by inserting the anterior portion of its body into the shell; and 3) moved into the shell, posterior end (pleotelson) first. The elapsed time from the initial grasping of the empty shell to completing the move into it was 2 min 20 sec. In contrast to a Pagurapseudes tanaidacean and hermit crabs, the individual of Macrolabrum sp. did not examine the external surface of the shell during the single shell exchange observed.

Correction to: A new species of Xenoturbella from the western Pacific Ocean and the evolution of Xenoturbella.

After publication of Nakano et al. (2017) [1], the authors became aware of the fact that the new species-group name erected for the two specimens of a Japanese xenoturbellid species in the article is not available because Nakano et al. (2017) [1] does not meet the requirement of the amendment of Article 8.5.3 of the International Code of Zoological Nomenclature (the Code) [2]. The authors therefore describe the two xenoturbellids as a new species again in this correction article. Methods for morphological observation, DNA extraction and sequencing were as described in Nakano et al. (2017) [1]. The holotype and paratype specimens are deposited in the National Museum of Nature and Science, Tsukuba (NSMT), Japan. The DNA sequences obtained were deposited in the International Nucleotide Sequence Database (INSD).

Molecular Phylogeny of the Family Capitellidae (Annelida).

Capitellids have emerged as monophyletic in most but not all recent molecular phylogenies, indicating that more extensive taxon sampling is necessary. In addition, monophyly of most or all capitellid genera was questionable, as some diagnostic characters vary ontogenetically within individuals. We tested the monophyly of Capitellidae and eight capitellid genera using phylogenetic analyses of combined 18S, 28S, H3, and COI gene sequences from 36 putative capitellid species. In our trees, Capitellidae formed a monophyletic sister group to Echiura, and Capitella was also monophyletic, separated by a long branch from other capitellids. Well-supported clades each containing representatives of different genera, or containing a subset of species within a genus, indicated that Barantolla, Heteromastus, and Notomastus are likely not monophyletic. We mapped three morphological characters traditionally used to define capitellid genera (head width relative to width of first segment, number of thoracic segments, and number of segments with capillary chaetae) onto our tree. While Capitella showed unique character states, states in the other genera were decidedly not phylogenetically informative. Morphology-based capitellid taxonomy will require a fine-scale reevaluation of character states and detection of new characters.

A new species of Xenoturbella from the western Pacific Ocean and the evolution of Xenoturbella.

BACKGROUND: Xenoturbella is a group of marine benthic animals lacking an anus and a centralized nervous system. Molecular phylogenetic analyses group the animal together with the Acoelomorpha, forming the Xenacoelomorpha. This group has been suggested to be either a sister group to the Nephrozoa or a deuterostome, and therefore it may provide important insights into origins of bilaterian traits such as an anus, the nephron, feeding larvae and centralized nervous systems. However, only five Xenoturbella species have been reported and the evolutionary history of xenoturbellids and Xenacoelomorpha remains obscure. RESULTS: Here we describe a new Xenoturbella species from the western Pacific Ocean, and report a new xenoturbellid structure - the frontal pore. Non-destructive microCT was used to investigate the internal morphology of this soft-bodied animal. This revealed the presence of a frontal pore that is continuous with the ventral glandular network and which exhibits similarities with the frontal organ in acoelomorphs. CONCLUSIONS: Our results suggest that large size, oval mouth, frontal pore and ventral glandular network may be ancestral features for Xenoturbella. Further studies will clarify the evolutionary relationship of the frontal pore and ventral glandular network of xenoturbellids and the acoelomorph frontal organ. One of the habitats of the newly identified species is easily accessible from a marine station and so this species promises to be valuable for research on bilaterian and deuterostome evolution.

Difference in Size at Maturity in Annual and Overwintering Generations in the Tanaidacean Zeuxo sp. in Oshoro Bay, Hokkaido, Japan.

We investigated the life cycle of the tanaidid Zeuxo sp. 1 (Crustacea: Peracarida: Tanaidacea), which lives epiphytically in dwelling tubes on the algae Sargassum spp. and Neorhodomela aculeata in Oshoro Bay, Hokkaido, Japan. We obtained data on its population size distribution, age structure, and reproductive phenology through monthly sampling from April 2011 to June 2012. From these data, we detected an overwintering group (F0) that arises from mancae that are released in autumn, overwinters, reproduces the following spring, and then disappears. The overwintering group produces mancae (F1) that reach maturity and themselves reproduce within six weeks after release; the F1, mancae give rise to an F2 and possibly an F3 generation within a single summer. We refer to individuals that reproduce in the same summer that they were released as the 'annual group'. Reproduction overall was restricted to the period from May to October, when the seasurface temperature exceeded 10°C. During both years, overwintering females first became reproductive in May. Reproduction in the annual group began in June and continued through mid-October. Mancae were observed in samples from June through October. The two groups differed significantly in size at reproduction; compared to the annual group, females in the overwintering group reproduced at larger minimum and average body sizes, and males began to express enlarged chelae, a secondary sexual character, at a larger size. The difference in size at maturity may be related to the differences in water temperature during the main period of growth and maturation.

Multiple processes generate productivity-diversity relationships in experimental wood-fall communities.

Energy availability has long been recognized as a predictor of community structure, and changes in both terrestrial and marine productivity under climate change necessitate a deeper understanding of this relationship. The productivity-diversity relationship (PDR) is well explored in both empirical and theoretical work in ecology, but numerous questions remain. Here, we test four different theories for PDRs (More-Individuals Hypothesis, Resource-Ratio Theory, More Specialization Theory, and the Connectivity-Diversity Hypothesis) with experimental deep-sea wood falls. We manipulated productivity by altering wood-fall sizes and measured responses after 5 and 7 years. In November 2006, 32 Acacia sp. logs were deployed at 3203 m in the Northeast Pacific Ocean (Station Deadwood: 36.154098 degrees N, 122.40852 degrees W). Overall, we found a significant increase in diversity with increased wood-fall size for these communities. Increases in diversity with wood-fall size occurred because of the addition of rare species and increases of overall abundance, although individual species responses varied. We also found that limited dispersal helped maintain the positive PDR relationship. Our experiment suggests that multiple interacting mechanisms influence PDRs.