Improving taxonomic classification of marine zooplankton by molecular approach: registration of taxonomically verified 18S and 28S rRNA gene sequences.

Background: Zooplankton plays an important role in the marine ecosystem. A high level of taxonomic expertise is necessary for accurate species identification based on morphological characteristics. As an alternative method to morphological classification, we focused on a molecular approach using 18S and 28S ribosomal RNA (rRNA) gene sequences. This study investigates how the accuracy of species identification by metabarcoding improves when taxonomically verified sequences of dominant zooplankton species are added to the public database. The improvement was tested by using natural zooplankton samples. Methods: rRNA gene sequences were obtained from dominant zooplankton species from six sea areas around Japan and registered in the public database for improving the accuracy of taxonomic classifications. Two reference databases with and without newly registered sequences were created. Comparison of detected OTUs associated with single species between the two references was done using field-collected zooplankton samples from the Sea of Okhotsk for metabarcoding analysis to verify whether or not the newly registered sequences improved the accuracy of taxonomic classifications. Results: A total of 166 sequences in 96 species based on the 18S marker and 165 sequences in 95 species based on the 28S marker belonging to Arthropoda (mostly Copepoda) and Chaetognatha were registered in the public database. The newly registered sequences were mainly composed of small non-calanoid copepods, such as species belonging to Oithona and Oncaea. Based on the metabarcoding analysis of field samples, a total of 18 out of 92 OTUs were identified at the species level based on newly registered sequences in the data obtained by the 18S marker. Based on the 28S marker, 42 out of 89 OTUs were classified at the species level based on taxonomically verified sequences. Thanks to the newly registered sequences, the number of OTUs associated with a single species based on the 18S marker increased by 16% in total and by 10% per sample. Based on the 28S marker, the number of OTUs associated with a single species increased by 39% in total and by 15% per sample. The improved accuracy of species identification was confirmed by comparing different sequences obtained from the same species. The newly registered sequences had higher similarity values (mean >0.003) than the pre-existing sequences based on both rRNA genes. These OTUs were identified at the species level based on sequences not only present in the Sea of Okhotsk but also in other areas. Discussion: The results of the registration of new taxonomically verified sequences and the subsequent comparison of databases based on metabarcoding data of natural zooplankton samples clearly showed an increase in accuracy in species identification. Continuous registration of sequence data covering various environmental conditions is necessary for further improvement of metabarcoding analysis of zooplankton for monitoring marine ecosystems.

Expression of GnRH genes is elevated in discrete brain loci of chum salmon before initiation of homing behavior and during spawning migration.

Our previous studies suggested the importance of gonadotropin-releasing hormones (GnRHs) for initiation of spawning migration of chum salmon, although supporting evidence had been not available from oceanic fish. In farmed masu salmon, the amounts of salmon GnRH (sGnRH) mRNAs in the forebrain increased in the pre-pubertal stage from winter through spring, followed by a decrease toward summer. We thus hypothesized that gene expression for GnRHs in oceanic chum salmon changes similarly, and examined this hypothesis using brain samples from winter chum salmon in the Gulf of Alaska and summer fish in the Bering Sea. They were classified into sexually immature and maturing adults, which had maturing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GnRH mRNAs were determined by real-time PCRs. The amounts of sGnRH mRNA in the maturing winter adults were significantly larger than those in the maturing summer adults. The amounts of sGnRH and chicken GnRH mRNAs then peaked during upstream migration from the coast to the natal hatchery. Such changes were observed in various brain loci including the olfactory bulb, terminal nerve, ventral telencephalon, nucleus preopticus parvocellularis anterioris, nucleus preopticus magnocellularis and midbrain tegmentum. These results suggest that sGnRH neurons change their activity for gonadal maturation prior to initiation of homing behavior from the Bering Sea. The present study provides the first evidence to support a possible involvement of neuropeptides in the onset of spawning migration.

Changes in gene expression for GH/PRL/SL family hormones in the pituitaries of homing chum salmon during ocean migration through upstream migration.

Gene expression for growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family hormones in the pituitaries of homing chum salmon were examined, because gene expression for these hormones during ocean-migrating phases remains unclear. Fish were collected in the winter Gulf of Alaska, the summer Bering Sea and along homing pathway in the Ishikari River-Ishikari Bay water system in Hokkaido, Japan in autumn. The oceanic fish included maturing adults, which had developing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GH, PRL and SL mRNAs in the pituitaries of the maturing adults in the summer Bering Sea were 5- to 20-fold those in the winter Gulf of Alaska. The amount of GH mRNA in the homing adults at the coastal seawater (SW) areas was smaller than that in the Bering fish, while the amount of PRL mRNA remained at the higher level until fish arrived at the Ishikari River. The gill Na(+),K(+)-ATPase activity in the coastal SW fish and the plasma Na(+) levels in the brackish water fish at the estuary were lowered to the levels that were comparable to those in the fresh water (FW) fish. In conclusion, gene expression for GH, PRL and SL was elevated in the pituitaries of chum salmon before initiation of homing behavior from the summer Bering Sea. Gene expression for GH is thereafter lowered coincidently with malfunction of SW adaptability in the breeding season, while gene expression for PRL is maintained high until forthcoming FW adaptation.

Changes in the plasma levels of insulin-like growth factor-I from the onset of spawning migration through upstream migration in chum salmon.

An increase in activity of the pituitary-gonadal axis (PG-axis) and gonadal development are essential for the onset of spawning migration of chum salmon from the Bering Sea. In the Bering Sea, fish with larger body sizes initiated gonadal development and commenced spawning migration to the natal river by the end of summer. We thus hypothesized that insulin-like growth factor-I (IGF-I), a somatotropic signal that interacts with the PG-axis, can be one of such factors responsible for the onset of migration, and examined changes in plasma levels and hepatic expression of IGF-I gene in oceanic and homing chum salmon in 2001-2003. The plasma IGF-I levels and corresponding body sizes in maturing adults, which had developing gonads, were significantly higher than those in immature fish in all years examined. Such increase in the plasma IGF-I levels in maturing fish was observed even in the Gulf of Alaska during February 2006, while coincident increase was not observed in the hepatic amounts of IGF-I mRNA. In autumn, the plasma IGF-I levels in homing adults decreased during upstream migration in the Ishikari River-Ishikari bay water system in Hokkaido, Japan. In conclusion, the plasma IGF-I levels increased with gonadal development when chum salmon migrated from the winter Gulf of Alaska to the summer Bering Sea. Circulating IGF-I may interact with the PG-axis and promote gonadal development that is inseparable from the onset of spawning migration. Circulating IGF-I levels were thereafter lowered in accordance with final maturation during upstream migration in the breeding season.

Elevation of the plasma level of insulin-like growth factor-I with reproductive maturation prior to initiation of spawning migration of chum salmon.

When, where, and how oceanic chum salmon initiate spawning migration is unknown although gonadal development and elevation of the activity of the pituitary-gonadal axis (PG-axis) are essential. Insulin-like growth factor-I (IGF-I) is a somatotropic signal that interacts with the PG-axis for gametogenesis. We thus examined the plasma level of IGF-I in immature and maturing chum salmon in the Bering Sea and the Gulf of Alaska. The maturing adults which had maturing gonads left the Bering Sea for the natal river by the end of summer, because almost all fish were immature in September. The plasma level of IGF-I and corresponding body size in the maturing adults were two- to threefold that of immature fish. The plasma IGF-I level correlated positively with the pituitary contents of follicle-stimulating hormone and the plasma levels of 11-ketotestosterone and estradiol-17beta. Therefore, the plasma level of IGF-I increased with elevation of the PG-axis activity prior to the initiation of spawning migration from the Bering Sea. Circulatory IGF-I from visceral organs may inform the status of body growth to the PG-axis for gonadal development that is inseparable from decision of chum salmon whether to initiate homing behavior from the Bering Sea or not to initiate spawning migration by the coming spawning season.

Activity of the pituitary-gonadal axis is increased prior to the onset of spawning migration of chum salmon.

The activity of the pituitary-gonadal axis (PG axis) in pre-migratory and homing chum salmon was examined because endocrine mechanisms underlying the onset of spawning migration remain unknown. Pre-migratory fish were caught in the central Bering Sea in June, July and September 2001, 2002 and 2003, and in the Gulf of Alaska in February 2006. They were classified into immature and maturing adults on the basis of gonadal development. The maturing adults commenced spawning migration to coastal areas by the end of summer, because almost all fish in the Bering Sea were immature in September. In the pituitaries of maturing adults, the copy numbers of FSHbeta mRNA and the FSH content were 2.5- to 100-fold those of the immature fish. Similarly, the amounts of LHbeta mRNA and LH content in the maturing adults were 100- to 1000-fold those of immature fish. The plasma levels of testosterone, 11-ketotestosterone and estradiol were higher than 10 nmol l(-1) in maturing adults, but lower than 1.0 nmol l(-1) in immature fish. The increase in the activity of the PG-axis components had already initiated in the maturing adults while they were still in the Gulf of Alaska in winter. In the homing adults, the pituitary contents and the plasma levels of gonadotropins and plasma sex steroid hormones peaked during upstream migration from the coast to the natal hatchery. The present results thus indicate that the seasonal increase in the activity of the PG axis is an important endocrine event that is inseparable from initiation of spawning migration of chum salmon.

Genetic stock identification of chum salmon in the Bering Sea and North Pacific Ocean using mitochondrial DNA microarray.

A newly developed DNA microarray was applied to identify mitochondrial (mt) DNA haplotypes of more than 2200 chum salmon in the Bering Sea and North Pacific Ocean in September 2002 and also 2003, when the majority of maturing fish were migrating toward their natal river. The distribution of haplotypes occurring in Asian and North American fish in the surveyed area was similar in the 2 years. A conditional maximum likelihood method for estimation of stock compositions indicated that the Japanese stocks were distributed mainly in the north central Bering Sea, whereas the Russian stocks were mainly in the western Bering Sea. The North American stocks were abundant in the North Pacific Ocean around the Aleutian Islands. These results indicate that the Asian and North American stocks of chum salmon are nonrandomly distributed in the Bering Sea and the North Pacific Ocean, and further the oligonuleotide DNA microarray developed by us has a high potential for identification of stocks among mixed ocean aggregates of high-seas chum salmon.