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The mechanisms of oceanic animal migration remain enigmatic. Adult Japanese eels start their long-distance oceanic migration from coastal areas to breed near the West Mariana Ridge. We tracked acoustically tagged eels released in the Kuroshio Current (KC) area near Japan (five silver-phase eels, three of which had impaired swim bladders) and a tropical/subtropical (TS) area near/in the spawning area (two yellow-phase and three silver-phase eels). We analyzed their active swimming and transport by water currents. The strong flow of the KC dominated the eels' movements in the north, and TS area; their swimming influenced their movements. In the KC area, greater distances were covered at night than during the day, because eels swam in shallower layers with strong currents at night. Three and one eel in the TS and KC area in the upper 400 m showed counterclockwise and clockwise movements around the time of solar culmination, respectively. The meta-analysis showed that eels released at middle latitudes (20°-34° N) generally swam southward through currents, whereas those released at low latitudes (12°-13° N) generally swam northward through currents. Our study suggests the influence of the surrounding current and a potential effect of solar cues on the movements of Japanese eels., (© 2022. The Author(s).)

Morphological descriptions of phyllosoma larvae are essential for correct species identification and investigating the spatiotemporal distribution and recruitment process of spiny and slipper lobsters. Species identification of the phyllosoma larvae in the Scyllarinae subfamily is particularly difficult because of the morphological similarities among species and the scarcity of morphological information describing correct species identity. We extracted mid-to final-stage (V to VIII) phyllosoma larvae ( n = 12) belonging to the subfamily Scyllarinae from several plankton samples collected in the Pacific and then performed molecular species identification using mitochondrial DNA COI and 16S rDNA sequence analyses. Three larvae collected around the Ryukyu Archipelago were identified as Chelarctus aureus (stage VI to VIII), and four collected around the Ryukyu Archipelago and Ogasawara Islands were identified as C. virgosus (V to VIII). One larva (V) collected in the central South Pacific was determined to be a subspecies of C. crosnieri . DNA barcodes could not be made for the remaining four larvae (V to VIII) collected around the Ryukyu Archipelago (designated by ? Chelarctus sp-1). Based on the morphological characteristics of the C. virgosus phyllosoma described in this study and the adult distributions reported to date, C. cultrifer phyllosomas previously reported in Japanese and Taiwanese waters are likely to be C. virgosus . This paper also presents a set of diagnostic morphological characteristics that can be used to discriminate among these four species of Chelarctus and from other genera in the subfamily Scyllarinae.

Partial mtDNA cytochrome oxidase subunit I (COI) fragments and near entire stretch of 12S rDNA (12S) and control region (Dloop) of the Japanese spiny lobster (Panulirus japonicus) (n = 3) were amplified by PCR and used for direct nucleotide sequencing and for clone library-based nucleotide sequence analysis. Nucleotide sequences of a total of 75 clones in COI, 77 in 12S and 92 in Dloop were determined. Haplotypes of the clones matched with those obtained by direct sequencing were determined to be genuine mtDNA sequence of the individual. Phylogenetic analysis revealed several distinct groups of haplotypes in all three regions. Genuine mtDNA sequences were observed to form a group with their closely related variables, and most of these variables may be due to amplification error but a few to be heteroplasmy. Haplotypes determined as nuclear mitochondrial pseudogenes (NUMTs) formed distinct groups. Nucleotide sequence divergence (K2P distance) between genuine haplotypes and NUMTs were substantial (7.169-23.880% for COI, 1.336-23.434% for 12S, and 7.897-71.862% for Dloop). These values were comparable to or smaller than those between species of the genus Panulirus, indicating that integration of mtDNA into the nuclear genome is a continuous and dynamic process throughout pre- and post-speciation events. Double peaks in electropherograms obtained by direct nucleotide sequencing were attributed to common nucleotides shared by multiple NUMTs. Information on the heteroplasmy and NUMTs would be very important for addressing their impact on direct nucleotide sequencing and for quality control of nucleotide sequences obtained., (© 2021. The Author(s).)

The post ovulatory follicle (POF) is an important and reliable tissue structure used to investigate the spawning history in teleost fish. Fresh POFs shortly after spawning are comprised of cellular (follicular cells) and acellular (basement membrane and fibrils such as elastic fibers) components. The cellular components are quickly disintegrated by means of apoptosis, while the acellular components persist for a longer period. Since cellular components are well visualized by conventional hematoxylin-eosin (HE) staining but acellular components are not stained well, old POFs that have lost cellular components are difficult to identify. In this study, periodic acid-Schiff and Victoria blue staining, which can distinctly visualize acellular POF components, were applied to the ovarian tissues of Japanese eel ( Anguilla japonica ) ( n = 9) captured from June to August of 2008, 2009, and 2013 at the southern West Mariana Ridge, a spawning area for Japanese eels. Only new POFs were observed in seven females caught in June, and these females had ovaries with early-to mid-vitellogenic stage oocytes. Both fresh and old POFs were observed in a female caught in July, and only mid-vitellogenic stage oocytes were observed. Only old POFs and no vitellogenic stage oocyte were observed in a female caught in August. A progressive decrease in muscle lipid content, gonad somatic index, and condition factors was observed from June to August. Thus, the female Japanese eel can spawn at least twice or three times at most during spawning season, depending on energy reserve.

Eel larvae apparently feed on marine snow, but many aspects of their feeding ecology remain unknown. The eukaryotic 18S rRNA gene sequence compositions in the gut contents of four taxa of anguilliform eel larvae were compared with the sequence compositions of vertically sampled seawater particulate organic matter (POM) in the oligotrophic western North Pacific Ocean. Both gut contents and POM were mainly composed of dinoflagellates as well as other phytoplankton (cryptophytes and diatoms) and zooplankton (ciliophoran and copepod) sequences. Gut contents also contained cryptophyte and ciliophoran genera and a few other taxa. Dinoflagellates (family Gymnodiniaceae) may be an important food source and these phytoplankton were predominant in gut contents and POM as evidenced by DNA analysis and phytoplankton cell counting. The compositions of the gut contents were not specific to the species of eel larvae or the different sampling areas, and they were most similar to POM at the chlorophyll maximum in the upper part of the thermocline (mean depth: 112 m). Our results are consistent with eel larvae feeding on marine snow at a low trophic level, and feeding may frequently occur in the chlorophyll maximum in the western North Pacific.

Natural diets of leptocephalus larvae have been enigmatic. In this study, we collected DNA samples from the gut contents and body surface of leptocephali belonging to the five Anguilliform families (Anguillidae, Chlopsidae, Congridae, Muraenidae, and Serrivomeridae) from the northwest Pacific and performed next-generation 18S rDNA sequencing. Wide variety of eukaryotes was detected in both samples, from which eight eukaryotic groups (jellyfish, conoid parasite, tunicate, copepod, krill, segmented worm, fungi, and dinoflagellate) were selected on the basis of abundance. All groups except conoid parasites were common in both the samples. Cnidarian 18S rDNA reads were the most abundant in both the samples; however, the number of samples having cnidarian reads and the read counts were significantly higher in the body surface scraping samples than in the gut content samples, regardless of careful rinsing of the body surface. These results indicate that the cnidarian DNAs are most likely found because of cross contamination from the body surface and/or environment. 18S rDNA read counts of copepod and tunicate in the gut contents were greater than or comparable with those in the body surface scraping samples, which may correspond to the previous observations of fecal pellets and larvacean houses in the leptocephali gut. Thus, the present study supports previous implications that leptocephali utilize detritus materials, so called marine snow., Competing Interests: The authors have declared that no competing interests exist.

Gut bacteria of phytophagous and omnivorous marine invertebrates often possess alginate lyases (ALGs), which are key enzymes for utilizing macroalgae as carbon neutral biomass. We hypothesized that the exclusive feeding of a target alga to marine invertebrates would shift the gut bacterial diversity suitable for degrading the algal components. To test this hypothesis, we reared sea hare (Dolabella auricularia) and sea snail (Batillus cornutus) for two to four weeks with exclusive feeding of a brown alga (Ecklonia cava). Pyrosequencing analysis of the gut bacterial 16S rRNA genes revealed shifts in the gut microbiota after rearing, mainly due to a decrease in the variety of bacterial members. Significant increases in six and four 16S rRNA gene phylotypes were observed in the reared sea hares and sea snails, respectively, and some of them were phylogenetically close to known alginate-degrading bacteria. Clone library analysis of PL7 family ALG genes using newly designed degenerate primer sets detected a total of 50 ALG gene phylotypes based on 90% amino acid identity. The number of ALG gene phylotypes increased in the reared sea hare but decreased in reared sea snail samples, and no phylotype was shared between them. Out of the 50 phylotypes, 15 were detected only after the feeding procedure. Thus, controlled feeding strategy may be valid and useful for the efficient screening of genes suitable for target alga fermentation.

Background: Endotracheal intubation (ETI) is the gold standard for the out-of-hospital emergency airway management in severely injured patients. Due to time-critical circumstances, poor patient presentation and hostile environments, it may be prone for mechanical complications and failure., Methods: In a retrospective study (January 2011 to December 2013), all patients who underwent out-of-hospital ETI before admittance to a level 1 trauma center were analyzed consecutively. Patients with supraglottic airways, being under cardiopulmonary resuscitation and interfacility transports were excluded. The main study endpoint was the incidence of unrecognized tube malposition; secondary endpoints were Glasgow Outcome Scale (GOS) and in-hospital mortality adjusted to on-scene Glasgow Coma Scale (GCS), Injury Severity Score (ISS), Abbreviated Injury Scale head (AIS head), and on-scene time., Results: Out of 1176 patients, 151 underwent out-of-hospital ETI. At hospital admission, tube malpositions were recognized in nine patients (5.9%). Accidental and unrecognized esophageal intubation was detected in five patients (3.3%) and bronchial intubation in four patients (2.7%). Although ISS (p=0.053), AIS head (p=0.469), on-scene GCS (p=0.151), on-scene time (p=0.530), GOS (p=0.748) and in-hospital mortality (p=0.431) were similar compared with correctly positioned ETI tubes, three esophageal intubation patients died due to hypoxemic complications., Discussion: In our study sample, out-of-hospital emergency ETI in severely injured patients was associated with a considerable tube misplacement rate. For safety, increased compliance to consequently use available technologies (eg, capnography, video laryngoscopy) for emergency ETI should be warranted., Level of Evidence: Level of Evidence IIA., Competing Interests: Competing interests: None declared.

The Japanese eel ( Anguilla japonica ), European eel ( Anguilla anguilla ), and American eel ( Anguilla rostrata ) are migratory, catadromous, temperate zone fish sharing several common life cycle features. The population genetics of panmixia in these eel species has already been investigated. Our extensive population genetics analysis was based on 1400 Gb of whole-genome sequence (WGS) data from 84 eels. It demonstrated that a Japanese eel group from the Kuma River differed from other populations of the same species. Even after removing the potential adapted/selected single nucleotide polymorphism (SNP) data, and with very small differences (fixation index [Fst] = 0.01), we obtained results consistently indicating that panmixia does not occur in Japanese eels. The life cycle of the Japanese eel is well-established and the Kuma River is in the center of its habitat. Nevertheless, simple reproductive isolation is not the probable cause of non-panmixia in this species. We propose that the combination of spawning area subdivision, philopatry, and habitat preference/avoidance accounts for the non-panmixia in the Japanese eel population. We named this hypothesis the "reproductive isolation like subset mapping" (RISM) model. This finding may be indicative of the initial stages of sympatric speciation in these eels.

Background: Gene duplication is considered important to increasing the genetic diversity in animals. In fish, visual pigment genes are often independently duplicated, and the evolutionary significance of such duplications has long been of interest. Eels have two rhodopsin genes ( rho ), one of which (freshwater type, fw-rho ) functions in freshwater and the other (deep-sea type, ds-rho ) in marine environments. Hence, switching of rho expression in retinal cells is tightly linked with eels' unique life cycle, in which they migrate from rivers or lakes to the sea. These rho genes are apparently paralogous, but the timing of their duplication is unclear due to the deep-branching phylogeny. The aim of the present study is to elucidate the evolutionary origin of the two rho copies in eels using comparative genomics methods., Results: In the present study, we sequenced the genome of Japanese eel Anguilla japonica and reconstructed two regions containing rho by de novo assembly. We found a single corresponding region in a non-teleostean primitive ray-finned fish (spotted gar) and two regions in a primitive teleost (Asian arowana). The order of ds-rho and the neighboring genes was highly conserved among the three species. With respect to fw-rho , which was lost in Asian arowana, the neighboring genes were also syntenic between Japanese eel and Asian arowana. In particular, the pattern of gene losses in ds-rho and fw-rho regions was the same as that in Asian arowana, and no discrepancy was found in any of the teleost genomes examined. Phylogenetic analysis supports mutual monophyly of these two teleostean synteny groups, which correspond to the ds-rho and fw-rho regions., Conclusions: Syntenic and phylogenetic analyses suggest that the duplication of rhodopsin gene in Japanese eel predated the divergence of eel (Elopomorpha) and arowana (Osteoglossomorpha). Thus, based on the principle of parsimony, it is most likely that the rhodopsin paralogs were generated through a whole genome duplication in the ancestor of teleosts, and have remained till the present in eels with distinct functional roles. Our result indicates, for the first time, that teleost-specific genome duplication may have contributed to a gene innovation involved in eel-specific migratory life cycle.

Alginate-degrading bacteria play an important role in alginate degradation by harboring highly efficient and unique alginolytic genes. Although the general mechanism for alginate degradation by these bacteria is fairly understood, much is still required to fully exploit them. Here, we report the isolation of a novel strain, Falsirhodobacter sp. alg1, the first report for an alginate-degrading bacterium from the family Rhodobacteraceae. Genome sequencing reveals that strain alg1 harbors a primary alginate degradation pathway with only single homologs of an endo- and exo-type alginate lyase, AlyFRA and AlyFRB, which is uncommon among such bacteria. Subsequent functional analysis showed that both enzymes were extremely efficient to depolymerize alginate suggesting evolutionary interests in the acquirement of these enzymes. The exo-type alginate lyase, AlyFRB in particular could depolymerize alginate without producing intermediate products making it a highly efficient enzyme for the production of 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Based on our findings, we believe that the discovery of Falsirhodobacter sp. alg1 and its alginolytic genes hints at the potentiality of a more diverse and unique population of alginate-degrading bacteria.

A long-spined sea urchin Diadema-sp reported from Japanese waters was genetically distinct from all known Diadema species, but it remained undescribed. Extensive field surveys in Japan with molecular identification performed in the present study determined five phenotypes (I to V) in Diadema-sp according to the presence and/or shape of a white streak and blue iridophore lines in the naked space of the interambulacral area. All phenotypes were distinct from Diadema setosum (Leske, 1778) and Diadema savignyi (Audouin, 1829), of which a major type (I) corresponded to Diadema clarki Ikeda, 1939 that was questioned and synonymized with Diadema setosum by Mortensen (1940). The holotype of Diadema clarki has not been found, but three unlabeled dried tests of Diadema were found among Ikeda's original collection held in the Kitakyushu Museum of Natural History and Human History, Fukuoka, Japan. A short mtDNA COI fragment (ca. 350bp) was amplified from one of the tests, and the nucleotide sequence determined (275bp) was nearly identical with that of Diadema-sp. Arrangements of the primary tubercles on the coronal plates in Diadema-sp and the museum specimen also conformed with Diadema clarki, indicating that Diadema-sp is identical to Diadema clarki and a valid species. Narrow latitudinal distribution (31°N to 35°N) of Diadema clarki in Japan was observed, where it co-existed with abundant Diadema setosum and rare Diadema savignyi. No Diadema clarki was found in the southern islands in Japan, such as Satsunan Islands to Ryukyu Islands and Ogasawara Island, where Diadema setosum and Diadema savignyi were commonly observed.

The purpose of this study was to develop a novel electrical retrieval method (ER method) for living sponge-associated microorganisms from marine sponges frozen at -80 °C. A -0.3-V vs. Ag/AgCl constant potential applied for 2 h at 9 °C induced the attachment of the sponge-associated microorganisms to an indium tin oxide/glass (ITO) or a gallium-doped zinc oxide/glass (GZO) working electrode. The electrically attached microorganisms from homogenized Spirastrella insignis tissues had intact cell membranes and showed intracellular dehydrogenase activity. Dead microorganisms were not attracted to the electrode when the homogenized tissues were autoclaved for 15 min at 121 °C before use. The electrically attached microorganisms included cultivable microorganisms retrieved after detachment from the electrode by application of a 9-MHz sine-wave potential. Using the ER method, we obtained 32 phyla and 72 classes of bacteria and 3 archaea of Crenarchaeota thermoprotei, Marine Group I, and Thaumarchaeota incertae sedis from marine sponges S. insignis and Callyspongia confoederata. Employment of the ER method for extraction and purification of the living microorganisms holds potential of single-cell cultivation for genome, transcriptome, proteome, and metabolome analyses of bioactive compounds producing sponge-associated microorganisms.

Short-time tracking (one to eight days) of the Japanese eel (Anguilla japonica) using ultrasonic transmitter was performed in the tropical-subtropical area adjacent to the spawning area and temperate area off the Japanese Archipelago. Of 16 eels (11 wild and five farmed) used, 10 wild eels displayed clear diel vertical migration (DVM) from the beginning, while the other five farmed eels tracked for 19 to 66 hours did not. During daytime, a significantly positive correlation between migration depth and light intensity recorded on the vessel was observed in the 10 wild eels, indicating that the eels were sensitive to sunlight even at the middle to lower mesopelagic zone (500 to 800 m). During nighttime, the eel migration depth was observed to be associated with the phase, rising and setting of the moon, indicating that the eels were sensitive to moonlight at the upper mesopelagic zone (<300 m). Two of 10 wild eels were in the yellow stage but shared similar DVM with the silver stage eels. Swimbladders of three silver stage eels were punctured before releasing, but very little effect on DVM was observed. The eels very punctually initiated descent upon nautical dawn and ascent upon sunset, enabling us to determine local times for sunrise and sunset, and hence this behavior may be used for geolocating eels. In fact, estimated positions of eels based on the depth trajectory data were comparable or even better than those obtained by light-based archival tag in other fish species.

The lipid and fatty acid composition of the muscle of the wild Japanese freshwater eel, Anguilla japonica, was analyzed between the initial and terminal stages of spawning migration to clarify the relationship between lipid physiology and maturation. Triacylglycerols were the only major component in the initial-phase eels, which contained high levels of lipids, while comparatively low triacylglycerol levels were observed in terminal-phase eels (Mariana silvers) at spawning area. Significant levels of plasmalogens were found in its phosphatidylethanolamine, different from other common fish species, which have their little levels. The major fatty acids in A. japonica depot triacylglycerols were 14:0, 16:0, 18:0, 16:1n-7, 18:1n-7, 18:1n-9, and 18:2n-6. Noticeable levels of 20:4n-6, EPA, 22:5n-3, and DHA were also found in initial-phase sample TAG at the yellow and initial silver stages. High 18:2n-6 levels in all A. japonica lipids were similar to those in other common freshwater fishes. In all A. japonica tissue phospholipids, high levels of n-6 and n-3 PUFA, such as 20:4n-6, EPA, 22:5n-3, and DHA, were observed except for the matured terminal female sample. High n-6 PUFA levels in terminal-phase samples caught at the spawning area suggest that A. japonica maintains and uses initial fatty acids from inland waters without feeding during long spawning migrations. The post-spawning sample, containing low levels of 20:4n-6 and DHA with unusually high levels of its degradation products (18:3n-6, 20:2n-6, and 18:4n-3), indicates that A. japonica may finally use its most important PUFA as energy for spawning before ending its life.

Four long-spined sea urchin species in the genus Diadema are known to occur around the Japanese Archipelago. Three species (D. savignyi, D. setosum, and D. paucispinum) are widely distributed in the Indo-Pacific Ocean. The fourth species was detected by DNA analysis among samples originally collected as D. savignyi or D. setosum in Japan and the Marshall Islands and tentatively designated as Diadema-sp, remaining an undescribed species. We analyzed nucleotide sequences of the cytochrome oxidase I (COI) gene in the "D. savignyi-like" samples, and found all 17 individuals collected in the mainland of Japan (Sagami Bay and Kyushu) to be Diadema-sp, but all nine in the Ryukyu Archipelago (Okinawa and Ishigaki Islands) to be D. savignyi, with large nucleotide sequence difference between them (11.0%±1.7 SE). Diadema-sp and D. savignyi shared Y-shaped blue lines of iridophores along the interambulacrals, but individuals of Diadema-sp typically exhibited a conspicuous white streak at the fork of the Y-shaped blue iridophore lines, while this feature was absent in D. savignyi. Also, the central axis of the Y-shaped blue lines of iridophores was approximately twice as long as the V-component in D. savignyi whereas it was of similar length in Diadema-sp. Two parallel lines were observed to constitute the central axis of the Y-shaped blue lines in both species, but these were considerably narrower in Diadema-sp. Despite marked morphological and genetic differences, it appears that Diadema-sp has been mis-identified as D. savignyi for more than half a century.

The Western Australian rock lobster fishery has been both a highly productive and sustainable fishery. However, a recent dramatic and unexplained decline in post-larval recruitment threatens this sustainability. Our lack of knowledge of key processes in lobster larval ecology, such as their position in the food web, limits our ability to determine what underpins this decline. The present study uses a high-throughput amplicon sequencing approach on DNA obtained from the hepatopancreas of larvae to discover significant prey items. Two short regions of the 18S rRNA gene were amplified under the presence of lobster specific PNA to prevent lobster amplification and to improve prey amplification. In the resulting sequences either little prey was recovered, indicating that the larval gut was empty, or there was a high number of reads originating from multiple zooplankton taxa. The most abundant reads included colonial Radiolaria, Thaliacea, Actinopterygii, Hydrozoa and Sagittoidea, which supports the hypothesis that the larvae feed on multiple groups of mostly transparent gelatinous zooplankton. This hypothesis has prevailed as it has been tentatively inferred from the physiology of larvae, captive feeding trials and co-occurrence in situ. However, these prey have not been observed in the larval gut as traditional microscopic techniques cannot discern between transparent and gelatinous prey items in the gut. High-throughput amplicon sequencing of gut DNA has enabled us to classify these otherwise undetectable prey. The dominance of the colonial radiolarians among the gut contents is intriguing in that this group has been historically difficult to quantify in the water column, which may explain why they have not been connected to larval diet previously. Our results indicate that a PCR based technique is a very successful approach to identify the most abundant taxa in the natural diet of lobster larvae.

The stomach contents of the larvae of marine animals are usually very small in quantity and amorphous, especially in invertebrates, making morphological methods of identification very difficult. Nucleotide sequence analysis using polymerase chain reaction (PCR) is a likely approach, but the large quantity of larval (host) DNA present may mask subtle signals from the prey genome. We have adopted peptide nucleic acid (PNA)-directed PCR clamping to selectively inhibit amplification of host DNA for this purpose. The Japanese spiny lobster (Panulirus japonicus) and eel (Anguilla japonica) were used as model host and prey organisms, respectively. A lobster-specific PNA oligomer (20 bases) was designed to anneal to the sequence at the junction of the 18 S rDNA gene and the internal transcribed spacer 1 (ITS1) of the lobster. PCR using eukaryote universal primers for amplifying the ITS1 region used in conjunction with the lobster-specific PNA on a mixed DNA template of lobster and eel demonstrated successful inhibition of lobster ITS1 amplification while allowing efficient amplification of eel ITS1. This method was then applied to wild-caught lobster larvae of P. japonicus and P. longipes bispinosus collected around Ryukyu Archipelago, Japan. ITS1 sequences of a wide variety of animals (Ctenophora, Cnidaria, Crustacea, Teleostei, Mollusca, and Chaetognatha) were detected.

The natural reproductive ecology of freshwater eels remained a mystery even after some of their offshore spawning areas were discovered approximately 100 years ago. In this study, we investigate the spawning ecology of freshwater eels for the first time using collections of eggs, larvae and spawning-condition adults of two species in their shared spawning area in the Pacific. Ovaries of female Japanese eel and giant mottled eel adults were polycyclic, suggesting that freshwater eels can spawn more than once during a spawning season. The first collection of Japanese eel eggs near the West Mariana Ridge where adults and newly hatched larvae were also caught shows that spawning occurs during new moon periods throughout the spawning season. The depths where adults and newly hatched larvae were captured indicate that spawning occurs in shallower layers of 150-200 m and not at great depths. This type of spawning may reduce predation and facilitate reproductive success.

The pronghorn spiny lobster, Panulirus penicillatus, is a circumtropical species which has the widest global distribution among all the species of spiny lobster, ranging throughout the entire Indo-Pacific region. Partial nucleotide sequences of mitochondrial DNA COI (1,142-1,207 bp) and 16S rDNA (535-546 bp) regions were determined for adult and phyllosoma larval samples collected from the Eastern Pacific (EP)(Galápagos Islands and its adjacent water), Central Pacific (CP)(Hawaii and Tuamotu) and the Western Pacific (WP)(Japan, Indonesia, Fiji, New Caledonia and Australia). Phylogenetic analyses revealed two distinct large clades corresponding to the geographic origin of samples (EP and CP+WP). No haplotype was shared between the two regional samples, and average nucleotide sequence divergence (Kimura's two parameter distance) between EP and CP+WP samples was 3.8±0.5% for COI and 1.0±0.4% for 16S rDNA, both of which were much larger than those within samples. The present results indicate that the Pacific population of the pronghorn spiny lobster is subdivided into two distinct populations (Eastern Pacific and Central to Western Pacific), with no gene flow between them. Although the pronghorn spiny lobster have long-lived teleplanic larvae, the vast expanse of Pacific Ocean with no islands and no shallow substrate which is known as the East Pacific Barrier appears to have isolated these two populations for a long time (c.a. 1MY).

Polymerase chain reaction (PCR)-clamping using blocking primer and DNA-analogs, such as peptide nucleotide acid (PNA), may be used to selectively amplify target DNA for molecular diet analysis. We investigated PCR-clamping efficiency by studying PNA position and mismatch with complementary DNA by designing PNAs at five different positions on the nuclear rDNA internal transcribed spacer 1 of the Japanese eel Anguilla japonica in association with intra-specific nucleotide substitutions. All five PNAs were observed to efficiently inhibit amplification of a fully complementary DNA template. One mismatch between PNA and template DNA inhibited amplification of the template DNA, while two or more mismatches did not. DNA samples extracted from dorsal muscle and intestine of eight wild-caught leptochephalus larvae were subjected to this analysis, followed by cloning, nucleotide sequence analysis, and database homology search. Among 12 sequence types obtained from the intestine sample, six were identified as fungi. No sequence similarities were found in the database for the remaining six types, which were not related to one another. These results, in conjunction with our laboratory observations on larval feeding, suggest that eel leptocephali may not be dependent upon living plankton for their food source.

To clarify the natural diet of phyllosoma larvae of the Japanese spiny lobster Panulirus japonicus, the sources of 18S rDNA clones obtained from the hepatopancreas were investigated. Of a total of 1537 clones examined, 160 had different restriction profiles from the host larvae, in which 21 restriction types were observed. Nucleotide sequences of 16 of 21 restriction types were successfully determined and their assignments were investigated by homology search and phylogenetic analysis. From seven late-stage larvae collected in spring to early summer, eukaryote DNA molecules of Teleostei, Oomycetes, Mycetozoa, and Fungi were identified. Exogenous DNA from four younger phyllosoma larvae collected in late autumn could not be recovered. A previous study identified DNAs of cnidarians and urochordates in late-stage phyllosoma larvae of a closely related species collected in winter. This indicates that the phyllosoma larvae are opportunistic carnivores, whose diets correlate with the relative abundance of prey organisms in the ambient water.

To identify lobster phyllosoma larvae of the genus Panulirus occurring in waters adjacent to Japan, genetic variation within and between 10 Indo-Pacific lobster species was investigated using restriction fragment length polymorphism (RFLP) analysis for the 1300-base pair mitochondrial cytochrome oxidase I (COI) gene. RFLP analysis using two endonucleases (AluI and TaqI) enabled discrimination of all species, including the P. longipes complex. The diagnostic DNA markers, supplemented with nucleotide sequence analysis, were applied to 44 mid- to late-stage phyllosoma larvae (7.4 to 27.7 mm in body length) collected in the northwestern Pacific. These samples were unexpectedly variable in species composition, comprising P. japonicus (n = 16), P. longipes bispinosus (21), P. longipes longipes (1), P. "aka" (1), and P. penicillatus (5). Comparison of larval size at similar stages revealed that P. l. bispinosus larvae were significantly larger than P. japonicus.

Twenty local populations of the Japanese freshwater shrimp Palaemon paucidens were electrophoretically and morphologically surveyed. Based on the diagnostic distributions of some alleles at Gpi, Mpi, Mdh-1, and Mdh-2, these populations were largely classified into two types (A and B). The A type occurred in lakes, ponds, and rivers, while the B type was observed only in rivers. Average Nei's genetic distance (D) between the two types fell into the subspecies range (D¯=0.1186). The coefficient of gene differentiation, G ST , varied considerably between the two types. In 12 populations of the A type, with a G ST value of 0.281, nine pond and lake populations showed a higher G ST (0.246) than the three river populations (0.151). On the other hand, G ST was 0.036 for the eight local populations of the B type. The lower rostrum tooth number had a mode of two in type A and three in type B. Type-A populations largely varied in the upper rostrum tooth number and egg size but type B did not. Under laboratory conditions, mating frequently occurred within each type, but not between types. Furthermore, no embryonic development was observed in the few cases of intertype mating. These results indicate that the A and B types had experienced cladogenic separation with pre- or postmating isolation, whereafter the A type, under geographic isolation, underwent genetic and phenotypic differentiation, while the B type, under extensive gene flow, did not undergo differentiation., (© 1988 The Society for the Study of Evolution.)