Populations of the Intertidal Crab Macrophthalmus banzai are Not Grouped by Latitude: Taiwan Population is Genetically Closer to the Populations in the Japanese Mainland Than the Ryukyu Islands.

Macrophthalmus banzai is an intertidal crab species of Macrophthalmidae inhabiting muddy tidal flats in the northwestern Pacific. A previous study on the population genetic structure of Japanese M. banzai based on the mitochondrial COI gene sequences revealed the presence of two genetically distinguished groups, i.e., the northern group (Honshu, Shikoku, and Kyushu Islands) and the southern group (the Ryukyu Islands). In the present study, we newly determined the COI gene sequences of M. banzai collected from Taiwan and conducted population genetic analyses of these sequences together with Japanese sequences obtained from GenBank to reveal the genetic relationship of this species between Japan and Taiwan. The SAMOVA and pairwise ΦST analysis showed that the Taiwan population is more closely related to the northern group than the southern group. This indicates that the populations of M. banzai are not genetically differentiated by latitude but probably by the pathway of the Kuroshio Current, resulting in the isolation of the population in the Ryukyu Islands. Such a pattern is consistent with the population genetic structure of the fiddler crab Tubuca arcuata shown by a previous study, whereas the pattern differs from those of other intertidal invertebrates. The difference in the larval durations may have influenced the difference in population genetic structures among species. The present study provides a further case of the genetic structure of intertidal species that are not simply regulated by geographic distances.

A novel symbiotic relationship between ascidians and a new tunic-boring polychaete (Annelida: Spionidae: Polydora).

Polydora tunicola Abe, Hoshino Yamada, sp. nov., a new spionid species currently considered an obligate symbiont of styelid ascidians, is described based on materials collected from Polycarpa cf. cryptocarpa kroboja (Oka, 1906) and Cnemidocarpa sp. in Izu-Oshima Island and Polycarpa sp. in Wakayama Prefecture, Japan. Polychaeteascidian symbiotic relationships are known only in two syllid species: Myrianida pinnigera (Montagu, 1808) and Proceraea exoryxae Martin, Nygren Cruz-Rivera, 2017. The latter has been the only polychaete known to bore into the tunic of an ascidian. Polydora tunicola sp. nov. is the second known example of a tunic-boring polychaete, which constructs U-shaped burrows in the tunic of the host ascidians. Worms were often concentrated near the host siphons and assumed to use water currents created by the filter-feeding host for suspension feeding. Although the boring mechanism into ascidian tunica is unknown, the plate assay and zymography results consistently detected cellulase activities, suggesting that cellulose digestion may enable the worms to bore into the cellulose-rich ascidian tunics. Polydora tunicola sp. nov. is morphologically similar to P. aura Sato-Okoshi, 1998, P. cornuta Bosc, 1802, P. fusca Radashevsky Hsieh, 2000, P. glycymerica Radashevsky, 1993, P. latispinosa Blake Kudenov, 1978, P. lingulicola Abe Sato-Okoshi, 2020, P. nanomon Orensky Williams, 2009, P. robi Williams, 2000, and P. vulgaris Mohammad, 1972 in having a single median antenna on the caruncle and chaetiger 5 without dorsal superior capillaries but with ventral capillaries. The new species is unique in having a black-rimmed pygidium, distinguishing it from these species. The phylogenetic analyses of the concatenated 18S, 28S, and 16S sequences recovered P. tunicola sp. nov. as the sister species to P. aura within a well-supported clade also including P. lingulicola and P. cf. glycymerica. The bright yellow body color of P. tunicola sp. nov. in life is similar to that of P. aura, however, these two species are distinguished by the former not having modified posterior notochaetae. The symbiotic nature of the association between P. tunicola sp. nov. and styelid ascidians is discussed.

A novel parasite strain of Amoebophrya sp. infecting the toxic dinoflagellate Alexandrium catenella (Group I) and its effect on the host bloom in Osaka Bay, Japan.

The endoparasitic dinoflagellates belonging to the genus Amoebophrya can infect a broad range of free-living marine dinoflagellates, including harmful/toxic species. The parasite kills its host; the high prevalence of the parasite has been suggested to be a significant factor for the termination of dinoflagellate blooms in marine systems. The issues involved in culturing host-parasite systems have greatly restricted further research on Amoebophrya biology. Here, we established the culture of a novel strain of Amoebophrya sp. ex Alexandrium catenella (Group I) from Osaka Bay, Japan, and studied its genetic diversity, host specificity, and prevalence in the field. Genetic analysis established that the strain we isolated was a novel culture strain infecting A. catenella. Among the host species tested, the Amoebophrya sp. could infect the genera Alexandrium and Prorocentrum in culture, and the infection was also confirmed in the genus Tripos in a field sample. A maximum prevalence of 73% was recorded during the Alexandrium bloom period in Osaka Bay, after which the host cell density rapidly declined. Our results indicated that the existence of the parasite had a significant effect on the dynamics of A. catenella, especially on the termination of the blooms.

Molecular identification and larval morphology of spionid polychaetes (Annelida, Spionidae) from northeastern Japan.

Planktonic larvae of spionid polychaetes are among the most common and abundant group in coastal meroplankton worldwide. The present study reports the morphology of spionid larvae collected mainly from coastal waters of northeastern Japan that were identified by the comparison of adult and larval 18S and 16S rRNA gene sequences. The molecular analysis effectively discriminated the species. Adult sequences of 48 species from 14 genera (Aonides Claparède, 1864; Boccardia Carazzi, 1893; Boccardiella Blake & Kudenov, 1978; Dipolydora Verrill, 1881; Laonice Malmgren, 1867; Malacoceros Quatrefages, 1843; Paraprionospio Caullery, 1914; Polydora Bosc, 1802; Prionospio Malmgren, 1867; Pseudopolydora Czerniavsky, 1881; Rhynchospio Hartman, 1936; Scolelepis Blainville, 1828; Spio Fabricius, 1785; Spiophanes Grube, 1860) and larval sequences of 41 species from 14 genera (Aonides; Boccardia; Boccardiella; Dipolydora; Laonice; Paraprionospio; Poecilochaetus Claparède in Ehlers, 1875; Polydora; Prionospio; Pseudopolydora; Rhynchospio; Scolelepis; Spio; Spiophanes) of spionid polychaetes were obtained; sequences of 27 of these species matched between adults and larvae. Morphology of the larvae was generally species-specific, and larvae from the same genus mostly shared morphological features, with some exceptions. Color and number of eyes, overall body shape, and type and arrangement of pigmentation are the most obvious differences between genera or species. The morphological information on spionid larvae provided in this study contributes to species or genus level larval identification of this taxon in the studied area. Identification keys to genera and species of planktonic spionid larvae in northeastern Japan are provided. The preliminary results of the molecular phylogeny of the family Spionidae using 18S and 16S rRNA gene regions are also provided.

Molecular approach for analysis of in situ feeding by the dinoflagellate Noctiluca scintillans.

The red tide forming heterotrophic dinoflagellate Noctiluca scintillans is common in temperate to tropical waters around the world. Understanding the in situ prey of N. scintillans is essential for elucidating its role in marine microbial food webs. In this study, we applied two polymerase chain reaction (PCR)-based cloning techniques, a predator-specific restriction enzyme, and a blocking primer. The PCR of nuclear 18S rDNA was performed on single N. scintillans cells that were collected from Ishinomaki Bay, Japan, in May 2018. The maximum detection rates of non-Noctiluca sequences were 56% using the restriction enzyme method and 87% with the blocking primer method, representing a broad taxonomic range of organisms, including diatom, dinoflagellate, bolidophyte, haptophyte, euglenophyte, green algae, golden algae, ciliate, heliozoa, copepod, brown seaweed, sponge, bivalve, and polychaete. The diverse DNA was probably ingested by N. scintillans directly or indirectly through secondary predation or ingestion of marine snow or detritus containing many organisms. The application of molecular approaches to various species may reveal undiscovered interactions within the phytoplankton community, including prey-predator, or symbiotic relationships.

First Report of the Morphology and rDNA Sequences of Two Pseudopolydora Species (Annelida: Spionidae) from Japan.

The morphology of two Pseudopolydora species, P. cf. reticulata Radashevsky and Hsieh, 2000 and P. achaeta Radashevsky and Hsieh, 2000 are reported from Japan for the first time. Pseudopolydora cf. reticulata was collected from Japanese tidal flats, and individuals possess the characteristic netlike pigmentation on the dorsum of anterior chaetigers and the longitudinal black band along midline of caruncle. Pseudopolydora achaeta was collected from subtidal bottom mud of Onagawa Bay, and individuals have distinctive characteristics, such as intensive black pigmentation on dorsal and ventral sides of the anterior body and nearly straight vertical rows of major spines on the fifth chaetiger. The morphology of P. cf. reticulata is very similar to that of P. cf. kempi, with which it had been confused in Japan. We analyzed the 18S and 28S rRNA gene sequences of all five Pseudopolydora species recorded from Japan and found strong evidence that they are genetically distinct. Our analysis also suggests that boring polydorids have evolved among non-boring ones; the genus Pseudopolydora, which mostly shows the non-boring form, appears to have remained in a more ancestral condition.

Cosmopolitan or Cryptic Species? A Case Study of Capitella teleta (Annelida: Capitellidae).

Capitella teleta Blake et al., 2009 is an opportunistic capitellid originally described from Massachusetts (USA), but also reported from the Mediterranean, NW Atlantic, and North Pacific, including Japan. This putatively wide distribution had not been tested with DNA sequence data; intraspecific variation in morphological characters diagnostic for the species had not been assessed with specimens from non-type localities, and the species status of the Japanese population(s) was uncertain. We examined the morphology and mitochondrial COI (cytochrome c oxidase subunit I) gene sequences of Capitella specimens from two localities (Ainan and Gamo) in Japan. Specimens from Ainan and Gamo differed from C. teleta from Massachusetts in methyl-green staining pattern, shape of the genital spines, and shape of the capillary chaetae; we concluded that these characters vary intraspecifically. Species delimitation analyses of COI sequences suggested that worms from Ainan and Massachusetts represent C. teleta; these populations share a COI haplotype. The specimens from Gamo may represent a distinct species and comprise a sister group to C. teleta s. str.; we refer to the Gamo population as Capitella aff. teleta. The average Kimura 2-parameter (K2P) distance between C. teleta s. str. and C. aff. teleta was 3.7%. The COI data indicate that C. teleta actually occurs in both the NW Atlantic and NW Pacific. Given the short planktonic larval duration of C. teleta, this broad distribution may have resulted from anthropogenic dispersal.

A new species of Protodrilus (Annelida, Protodrilidae), covering bone surfaces bright red, in whale-fall ecosystems in the northwest Pacific.

There are unique ecosystems in the ocean that are driven by chemosynthesis. Whale-fall communities are one of these reducing habitats, and many unknowns are left to be resolved to understand their uniqueness. A new species of the genus Protodrilus was discovered on the exposed bones of sperm whale carcasses found in the waters off Cape Nomamisaki in the northwest Pacific. Protodrilus puniceus sp. nov. was the most abundant annelid to be found on the 2.5-y-old carcasses; the exposed bone surfaces appeared bright red due to the coloration of the innumerable worms covering them. Closer inspection revealed that this species was found in the small pores of both the internal and external surfaces of the bones. P. puniceus shows simple morphology; it has paired palps and pygidial lobes, but no eyespots, nuchal organs, statocysts, or ciliary rings dorso-anteriorly-an exceptional finding in this group. A ventral ciliary band was conspicuous, extending over each segment of the animal. The male of the species possessed paired, separated lateral organs on segments 6-11; salivary glands were inconspicuous. From morphological, biological, and ecological characteristics, P. puniceus showed differences from the already known Protodrilus group of shallow interstitial inhabitants. P. puniceus is thought to be a unique deposit feeder, acquiring nutrients by adhering to organic substances from whale carcasses. This is the first description of this group to be found in the organically enriched whale-fall environments of the deep sea below 200 m and from Japanese waters. Information on a nuclear, 18S ribosomal RNA gene sequence is included.

Impacts of the 2011 tsunami on the subtidal polychaete assemblage and the following recolonization in Onagawa Bay, northeastern Japan.

The ecological impacts of the 2011 Great East Japan Earthquake and tsunami and the following recolonization of the subtidal benthic polychaete community were examined by monthly pre- and post-quake field surveys that were conducted in Onagawa Bay from 2007 to 2013. Before the tsunami, the species composition in this benthic community was constant and was dominated by cirratulid and magelonid polychaetes. The density and biomass of benthic polychaetes drastically decreased after the tsunami, and the polychaete community fluctuated during the 2 years after the natural disaster. Spionid and capitellid polychaetes were dominant at this period. In June 2013, the community entered a new constant stage dominated by maldanids, which is different from the pre-quake community. Ecological impacts due to chemical pollution were suggested in addition to the tsunami disturbance. These overlapping effects and physical, chemical and biological factors affected the recovery and recolonization of the polychaete community after the natural disaster.

Swimming behavior of the spoon worm Urechis unicinctus (Annelida, Echiura).

Large numbers of swimming and stranding Urechis unicinctus were observed at night during low tide in Sasuhama, Miyagi Prefecture, northeastern Japan, during the periods from January to February in 2012 and 2013. Worms did not drift passively but swam actively, therefore hinting at a certain purpose for such behavior. As trochophore larvae of U. unicinctus were observed to occur simultaneously in the plankton, we infer the possibility that this is an event of reproductive swarming. Anatomical observations of both swimming and stranding U. unicinctus showed that none of the specimens had gametes, which may suggest that these were completely spent after spawning. Urechis unicinctus seemed to begin swimming after dusk and the observed swimming behavior occurred during the evening ebb tide throughout the night low tide during winter time. Stranding U. unicinctus have long been known in Japan and have been attributed to sea storms. The present study shows for the first time the possibility that U. unicinctus swims in order to reproduce at night and that this swimming behavior is closely linked to the stranding of U. unicinctus individuals.

Accumulation of organotins in wharf roach (Ligia exotica Roux) and its ability to serve as a biomonitoring species for coastal pollution.

In this study, we measured the accumulation of tributyltin (TBT) in wharf roach (Ligia exotica Roux) and examined the species' ability to be used for TBT biomonitoring in coastal environments. In an exposure test, wharf roach were exposed to TBT via diet for 2d. TBT was accumulated in wharf roach, and its metabolite dibutyltin was detected. The concentrations of these compounds gradually decreased during the depuration period, but they were still detected 12d after exposure ceased (TBT 290±140ng/g; dibutyltin 1280±430ng/g). The biological half-life of TBT in wharf roach was estimated to be about 4d. In a field study conducted in 2011-2012, wharf roach were collected from 15 coastal sites in Japan and 3 sites in Manado, Indonesia. TBT was detected in both Japanese and Indonesian samples. The highest concentration of TBT was found in wharf roach collected at Bitung ferry port, Manado (57.9±16.5ng/g), which is close to a shipyard, and the highest concentration at a Japanese site was 12.3±6.2ng/g. Thus, we were able to detect organotins in the coastal environments by testing wharf roach, suggesting that L. exotica might serve as a good bioindicator for monitoring organotin pollution.