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In rare cases, giant cell tumor of bone (GCTB) can undergo primary or secondary malignant transformation to malignant giant cell tumor of bone (MGCTB), but the details of the molecular alterations are still unclear. The present study aimed to elucidate the clinicopathologic and molecular features of MGCTBs based on immunohistochemistry, fluorescence in situ hybridization (FISH) and next generation sequencing (NGS) of nine MGCTBs (five primary and four secondary). Seven (78%) of 9 MGCTBs were immunohistochemically positive for H3.3 G34W. In two (22%) patients, although GCTB components were focally or diffusely positive for H3.3 G34W, their malignant components were entirely negative for H3.3 G34W, which was associated with heterozygous loss of H3F3A by FISH. NGS on four MGCTBs revealed pathogenic mutations in TP53 (n = 3), EZH2 (n = 1) and several other genes. Immunohistochemical analysis of the nine MGCTBs confirmed the p53 nuclear accumulation (n = 5) and loss of H3K27me3 expression (n = 3) and showed that they were mutually exclusive. In addition, four (80%) of five cases of pleomorphic or epithelioid cell-predominant MGCTBs were positive for p53, while three (75%) of four cases of spindle cell-predominant MGCTBs were negative for trimethylation at lysine 27 of histone 3 (H3K27me3). The results suggested that p53 alteration and dysfunction of histone methylation as evidenced by H3K27me3 loss may play an important role in the malignant progression of GCTB, and might contribute to the phenotype-genotype correlation in MGCTB. The combined histologic, immunohistochemical and molecular information may be helpful in part for the diagnosis of challenging cases.

AimsDedifferentiation is a histological phenomenon characterised by abrupt transition of histology to a sarcomatous component with high-grade malignant potential in solitary fibrous tumour (SFT). The authors histologically reviewed SFT cases to reveal the histological background of dedifferentiated SFTs.MethodsClinicopathological and histopathological findings of 145 SFT cases were reviewed. Immunohistochemical staining and genetic analysis were also performed.ResultsThe non-dedifferentiated components showed a cellular component in 45 of 145 (31%), high mitotic rate (≥4/10 high-powered field) in 12 of 145 (8.2%) tumours, necrosis in 7 of 145 (4.8%) tumours, multinodular growth pattern in 39 of 132 (29.5%) available tumours and intratumoural fibrous septa in 37 of 131 (28.2%). Immunohistochemically, the non-dedifferentiated components were positive for CD34 in 128 of 141 (90.7%), bcl-2 in 101 of 133 (75.9%), nuclear pattern of β-catenin in 64 of 127 (50.3%) and p16 in 22 of 140 (15.7%). Loss of Rb protein expression was detected in 17 of 110 (15.4%) cases. Statistically, cellular component, multinodular structure, p16 overexpression and Rb protein loss were significantly associated with dedifferentiation. Moreover, cellular component and multinodular structure were significantly associated with p16 overexpression and Rb protein loss. All the non-deddifferentiated components showed wild type of p53 expression. The dedifferentiated components of all 10 dedifferentiated tumours presented positivity for p16 in 9 of 10 (90%) and mutational type of p53 in 5 of 10 (50%). Loss of Rb protein expression was detected in 6 of 10 (60%).ConclusionsThe authors propose that cellular or multinodular transformation may be associated with dedifferentiation. They also suggest that cellular and multinodular transformation may be associated with p16 overexpression and Rb downregulation.

The scientific name Cottus kazika has been used for over a century as valid for the catadromous fourspine sculpin endemic to Japan. However, recent DNA sequence analyses have indicated a sister relationship between two catadromous species, so-called C. kazika and Trachidermus fasciatus with a great phylogenetic divergence, separated from a monophyletic freshwater spawning clade including the other Cottus species and Baikalian sculpins. Morphological, life-historical, ecological, and karyological characteristics have also suggested that the fourspine sculpin so-called C. kazika is neither a member of Cottus nor Trachidermus. Therefore, the fourspine sculpin is transferred to the genus Rheopresbe. The genus and R. kazika are re-described. The genus Rheopresbe is discriminated from related genus Trachidermus by not having bony ridges on both nape and cheeks and also by presence of entopterygoid, and from the genus Cottus by having teeth on palatines and four spines on preopercle ridges.

Acanthopagruslatus and A.schlegelii (Sparidae) are closely related species distributed in northern tropical to temperate coastal waters of eastern Asia. Early life histories of the two species were investigated by seine net sampling in the Tien Yen estuary, northern Vietnam, which is probably the southernmost co-occurrence locality for these species. In northern Vietnam, early juveniles of both the Acanthopagrus species occurred in the Tien Yen estuary from December to March (A.schlegelii) or December to April (A.latus) when temperatures ranged from ca. 16 to 25 °C. The given temperature range is observed twice (spring and autumn) a year in Japan, and A.latus and A.schlegelii early juveniles are abundant respectively in each season. Thus, a thermoperiodicity of early juveniles of the two Acanthopagrus is common Asia-wide. The two species occurred as the same developmental stage, being markedly more abundant and having larger sizes in A.latus. The Acanthopagrus early juveniles inhabited bank waters of the estuary. Although waters continued to be mixed horizontally by temperatures but to be stratified by salinities during the study period, there was little variation in developmental stage and size among stations for both species. The two Acanthopagrus species from Vietnam spawn for the same season and their early juveniles simultaneously use the same nursery ground.

Background Undifferentiated pleomorphic sarcoma (UPS) is a sarcoma with a poor prognosis. A clinical trial, SARC028, revealed that treatment with anti-PD-1 drugs was effective against UPS. Studies have reported that UPS expresses PD-L1, sometime strongly (≥ 50%). However, the mechanism of PD-L1 expression in UPS has remained still unclear. CKLF-like MARVEL transmembrane domain containing 6 (CMTM6) was identified as a novel regulator of PD-L1 expression. The positive relationship between PD-L1 and CMTM6 has been reported in several studies. The aim of this study was to examine CMTM6 expression in UPS and evaluate the relationship between PD-L1 and CMTM6. Materials and methods Fifty-one primary UPS samples were subjected to CMTM6 and PD-L1 immunostaining. CMTM6 expression was assessed using proportion and intensity scores. CMTM6 gene copy number was also evaluated using a real-time PCR-based copy number assay. We also analyzed the mRNA expression and copy number variation of PD-L1 and CMTM6 in The Cancer Genome Atlas (TCGA) data. Results TCGA data indicated that the mRNAs encoded by genes located around 3p22 were coexpressed with CMTM6 mRNA in UPS. Both proportion and intensity scores of CMTM6 positively correlated with strong PD-L1 expression (≥ 50%) (both p = 0.023). CMTM6 copy number gain increased CMTM6 expression. Patients with UPS with a high CMTM6 intensity score had worse prognosis for overall survival. Conclusions CMTM6 expression was significantly correlated with PD-L1 expression. CMTM6 expression induced strong PD-L1 expression (≥ 50%). CMTM6 copy number gain promoted CMTM6 expression and increased PD-L1 expression in UPS.

Summary Loss of SMARCB1 protein expression has recently been identified in a variety of tumor types such as poorly differentiated chordoma (PCh) and malignant rhabdoid tumor (MRT) including atypical teratoid/rhabdoid tumor (AT/RT). PCh is characterized by poorly differentiated epithelioid tumor cells, sheet arrangement, and coexpression of nonepithelial and epithelial markers. Rhabdoid cells are sometimes present. Therefore, the differentiation of these tumors is often difficult. Brachyury is a transcription factor within the T-box family typically expressed in notochord tissue and chordomas. Some studies have reported high specificity and sensitivity of brachyury expression in chordomas. In the present study, we analyzed immunohistochemical brachyury expression in SMARCB1-deficient tumors and discuss important clinicopathological and diagnostic points, especially in cases of intracranial SMARCB1-deficient tumors with brachyury expression. Brachyury and cytokeratin immunoexpression status was examined in 42 formalin-fixed paraffin-embedded SMARCB1-deficient tumor specimens (PCh, 6 cases; extra-central nervous system [CNS] MRT, 26 cases; AT/RT, 10 cases) and 25 cases of conventional chordoma (CCh). All cases of PCh and CCh showed diffuse immunopositivities for cytokeratin 8, pan-cytokeratin, and brachyury. Brachyury immunoexpression was present in 2 extra-CNS MRT (8%) and 5 AT/RT (50%) cases, but immunopositivity was focal not diffuse. Indeed, in almost all cases of AT/RT (cytokeratin 8, 7/10 cases; pan-cytokeratin, 7/10 cases) and extra-CNS MRT (cytokeratin 8, 23/26 cases; pan-cytokeratin, 25/26 cases), fewer than 50% of cells showed immunoreactivity. Although the histological and clinical features of PCh resemble those of AT/RT, semiquantitative evaluations of the degree of brachyury and cytokeratin immunoexpressivity may help to distinguish PCh from AT/RT.

Undifferentiated pleomorphic sarcoma (UPS) is a sarcoma with a poor prognosis. A clinical trial, SARC028, revealed that treatment with anti-PD-1 drugs was effective against UPS. Studies have reported that UPS expresses PD-L1, sometimes strongly (≥ 50%). However, the mechanism of PD-L1 expression in UPS has remained unclear. CKLF-like MARVEL transmembrane domain containing 6 (CMTM6) was identified as a novel regulator of PD-L1 expression. The positive relationship between PD-L1 and CMTM6 has been reported in several studies. The aim of this study was thus to examine CMTM6 expression in UPS and evaluate the relationship between PD-L1 and CMTM6 in this disease. Fifty-one primary UPS samples were subjected to CMTM6 and PD-L1 immunostaining. CMTM6 expression was assessed using proportion and intensity scores. CMTM6 gene copy number was also evaluated using a real-time PCR-based copy number assay. We also analyzed the mRNA expression and copy number variation of PD-L1 and CMTM6 in The Cancer Genome Atlas (TCGA) data. TCGA data indicated that the mRNAs encoded by genes located around 3p22 were coexpressed with CMTM6 mRNA in UPS. Both proportion and intensity scores of CMTM6 positively correlated with strong PD-L1 expression (≥ 50%) (both p = 0.023). CMTM6 copy number gain increased CMTM6 expression. Patients with UPS with a high CMTM6 intensity score had a worse prognosis for overall survival. UPS showed variation in CMTM6 copy number and CMTM6 expression. CMTM6 expression was significantly correlated with PD-L1 expression, especially with strong PD-L1 expression.

Giant cell tumor of bone (GCTB) is an intermediate malignant bone tumor that is locally aggressive and rarely metastasizes. Denosumab, which is a receptor activator of nuclear factor kappa B ligand (RANKL) inhibitor, can be used to treat GCTB. We focused on potential immunotherapy for GCTB and investigated the tumor microenvironment of GCTB. Programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1) expression and signal-regulatory protein alpha (SIRPα), forkhead box P3 (FOXP3), and cluster of differentiation 8 (CD8) infiltration were assessed by immunohistochemical studies of 137 tumor tissues from 96 patients. Of the naive primary specimens, 28% exhibited PD-L1 expression and 39% exhibited IDO1 expression. There was significantly more SIRPα+, FOXP3+, and CD8+ cell infiltration in PD-L1- and IDO1-positive tumors than in PD-L1- and IDO1-negative tumors. The frequency of PD-L1 expression and SIRPα+ cell infiltration in recurrent lesions treated with denosumab was significantly higher than in primary lesions and recurrent lesions not treated with denosumab. PD-L1 expression and higher SIRPα+ cell infiltration were significantly correlated with shorter recurrence-free survival. PD-L1 and SIRPα immune checkpoint inhibitors may provide clinical benefit in GCTB patients with recurrent lesions after denosumab therapy.

Malignant rhabdoid tumor (MRT) is a rare, highly aggressive sarcoma with an uncertain cell of origin. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is very poor. Novel antitumor agents are needed for MRT patients. Forkhead box transcription factor 1 (FOXM1) is overexpressed and is correlated with the pathogenesis in several human malignancies. In this study, we identified the clinicopathological and prognostic values of the expression of FOXM1 and its roles in the progression of MRT. We investigated the FOXM1 expression levels and their clinical significance in 23 MRT specimens using immunohistochemistry and performed clinicopathologic and prognostic analyses. We also demonstrated correlations between the downregulation of FOXM1 and oncological characteristics using small interfering RNA (siRNA) and FOXM1 inhibitor in MRT cell lines. Histopathological analyses revealed that primary renal MRTs showed significantly low FOXM1 protein expression levels (p = 0.032); however, there were no significant differences in other clinicopathological characteristics or the survival rate. FOXM1 siRNA and FOXM1 inhibitor (thiostrepton) successfully downregulated the mRNA and protein expression of FOXM1 in vitro and the downregulation of FOXM1 inhibited cell proliferation, drug resistance to chemotherapeutic agents, migration, invasion, and caused the cell cycle arrest and apoptosis of MRT cell lines. A cDNA microarray analysis showed that FOXM1 regulated FANCD2 and NBS1, which are key genes for DNA damage repair. This study demonstrates that FOXM1 may serve as a promising therapeutic target for MRT.

Ischemic fasciitis is a benign myofibroblastic lesion, occurring in the sacral region or proximal thigh of elderly or bedridden individuals. The pathogenesis of ischemic fasciitis is thought to be based on ischemic condition; however, it has never been demonstrated. In this study, we examined the expression of ischemia-associated proteins in ischemic fasciitis by immunohistochemical and genetic methods. Specifically, this study aimed to reveal the expression of HIF-1α, MDM2, CDK4, p16, and gene amplification of MDM2 gene. Seven cases of ischemic fasciitis from among the soft-tissue tumors registered at our institution were retrieved. Histopathological findings were as follows: poorly demarcated nodular masses, a proliferation of spindle-shaped fibroblastic or myofibroblastic cells with oval nuclei and eosinophilic or pale cytoplasm, zonal fibrinous deposition, pseudocystic degeneration, granulation-like proliferation of capillary vessels, ganglion-like cells, myxoid or hyalinized stroma, and chronic inflammatory infiltration. Immunohistochemically, the spindle cells were positive for HIF-1α (7/7 cases), MDM2 (4/7 cases), CDK4 (4/7 cases), p16 (7/7 cases), p53 (2/7 case), cyclin D1 (7/7 cases), and alpha-smooth muscle actin (6/7 cases). Neither MDM2 gene amplification nor USP6 gene split signal was detected in any case. Overexpression of the above proteins may be associated with the pathogenic mechanism of ischemic fasciitis. It is noted that the immunohistochemical positivity of MDM2, CDK4, and p16 do not necessarily indicate malignant neoplasm such as dedifferentiated liposarcoma.

The original version of this article unfortunately contained mistakes introduced during the production process. Tables 1 and 2 were presented incorrectly. The original article has been corrected.

The RNA-binding protein HuC/D displays a neuron-specific expression and is involved in neuronal differentiation and the maintenance of the nervous system. Here we investigated the diagnostic value of HuC/D in neuroblastomas. We evaluated 85 neuroblastic tumors: 81 neuroblastomas; 3 ganglioneuroblastomas, intermixed; 1 ganglioneuroma, maturing; and 101 other tumors consisting of 34 Ewing sarcomas, 14 nephroblastomas, 11 rhabdomyosarcomas, 15 pulmonary small cell carcinomas, 18 pancreatic neuroendocrine tumors, and 9 pheochromocytomas. Immunohistochemistry for HuC/D, PHOX2B, and tyrosine hydroxylase was performed. The immunoreactivity for HuC/D was semiquantified using the total score (TS; range, 0-8). HuC/D positivity was defined as a TS ≥6. The TS of the neuroblastic tumors (mean TS, 7.94) was significantly higher than those of the other small round cell tumors and neuroendocrine tumors (P < .001) except for the pheochromocytomas (mean TS, 6.89; P = .074). HuC/D was positive in all 85 neuroblastic tumors, 1 (2.9%) Ewing sarcoma, 1 (6.7%) pulmonary small cell carcinoma, and 8 (89%) pheochromocytomas. PHOX2B was positive in all of the neuroblastic tumors and pheochromocytomas. Tyrosine hydroxylase was positive in 80 (94%) neuroblastic tumors, 1 (9.1%) rhabdomyosarcoma, and all of the pheochromocytomas. Therefore, HuC/D serves as a highly sensitive diagnostic marker to distinguish neuroblastomas from other small round cell tumors. The combination of HuC/D and PHOX2B staining may be valuable for the diagnosis of neuroblastic tumors, especially in the assessment of small sections. HuC/D expression in tumors may be related to catecholamine production or a neural crest-derived cell origin.

The SWI/SNF chromatin-remodeling complex, which is composed of evolutionarily conserved core subunits such as SMARCB1/INI1 (INI1), SMARCA4/BRG1 (BRG1), SMARCC1/BAF155 (BAF155), and SMARCC2/BAF170 (BAF170), can be viewed as the prototype of an epigenetic regulator of gene expression that is involved in tumor suppression. Epithelioid sarcoma, which classified as a tumor of uncertain differentiation, shows an almost complete loss of INI1. However, some cases of epithelioid sarcoma have preserved INI1, and the clinicopathologic features of these cases are uncertain. To date, there has been no investigation focused on the SWI/SNF chromatin-remodeling complex in INI1-preserved epithelioid sarcoma cases. First, an investigation of INI1 immunoexpression statuses in 60 formalin-fixed paraffin-embedded epithelioid sarcoma specimens (proximal type, 29 cases; conventional type, 31 cases) was performed. In the available INI1-preserved epithelioid sarcoma cases, we analyzed the BRG1, BAF155, and BAF170 protein expressions. INI1 preservation was observed in 6 of 29 (21%) proximal-type and 2 of 31 (6%) conventional-type epithelioid sarcoma cases. Six cases of INI1-preserved epithelioid sarcomas of proximal type were available for further immunohistochemical study. One proximal type showed loss of BAF170, and 2 proximal-type cases revealed loss of BRG1 with preservation of the other remaining core subunit proteins. One proximal-type case showed a mosaic pattern of BRG1 and loss of BAF155. However, in the remaining 2 proximal-type cases, all core subunit proteins were preserved. Overall, these results suggest that loss of expression of SWI/SNF chromatin-remodeling complex proteins has an important role in tumorigenesis. The remaining 2 INI1-preserved epithelioid sarcoma cases may have had other abnormalities causing dysfunction of SWI/SNF chromatin remodeling.

Marine fish generally show higher genetic diversity and shallower population structure than freshwater fish, but data for rare species are lacking. A latid fish, akame (Lates japonicus), is a rare, large predatory fish endemic to Japan, occurring mainly in the estuaries and coastal waters of Kochi and Miyazaki prefectures. Among species of Lates, akame is the only one adapted to temperate waters, while the others are distributed in tropical waters. Despite conservation concerns for this species, almost nothing is known about its population genetic characteristics. We used amplified fragment length polymorphism (AFLP) and mitochondrial DNA (mtDNA) markers to describe the genetic variation and its partitioning among juvenile samples collected from multiple localities and over different years. Twenty-four primer combinations detected a total of 522 AFLP loci, only 40 (7.7 %) of which were polymorphic. Sequence analysis of the entire mtDNA control region identified only five haplotypes defined by four variable sites. The level of genetic diversity revealed by both genetic markers was unusually low compared to other marine fish and comparable to levels observed for some endangered freshwater fish exhibiting some of the lowest levels of genetic diversity among vertebrate populations. Analysis of molecular variance for the AFLP data indicated a weak but significant population structure (F CT = 0.07, P

To examine reproduction of Coilia nasus, an engraulid endemic fish to Ariake Bay, in the freshwaters upstream of the reclamation dike of Isahaya inlet, ichthyoplankton was collected at the surface with a larva net. Eggs and larvae of this species were captured in abundance, especially in the retention basin. All eggs had reached middle or late embryonic stages, and larvae from the preflexion with yolk to the juvenile stage (4.1–39.3 mm) were found. This suggests that the freshwaters of retention basin supply spawning and nursery grounds for C. nasus.

Phosphaturic mesenchymal tumour, mixed connective tissue variant (PMT-MCT), is a tumour of uncertain differentiation, characterised by 'smudgy/grungy' calcification and vitamin D-resistant phosphaturic osteomalacia. Fibroblast growth factor (FGF)23 is recognised as a reliable marker of PMT-MCT, but quantitative evaluation has never been performed. We reviewed cases of tumour-associated osteomalacia or histologically definitive PMT-MCT without osteomalacia using histological, immunohistochemical and genetic methods and evaluated the diagnostic significance of these findings.A total of 19 tumours from 14 cases diagnosed previously as PMT-MCT were retrieved, on which immunohistochemical staining, reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence in-situ hybridisation (FISH) analysis were performed. Histologically, fibrous capsule, calcification and giant cell reaction tended to be observed in soft-tissue PMT-MCT, while PMT-MCT of bone and multiple PMT-MCT showed an infiltrative growth pattern. The immunohistochemical results were as follows: the tumour cells were positive for FGF23 (nine of 12, 75%), FGFR1 (11 of 11, 100%), CD56 (12 of 14, 85.7%) and E26 oncogene homologue (ERG) (5 of 13, 38.4%). The sole malignant tumour was positive for p53. FGF23 mRNA was detected in seven of 14 formalin-fixed paraffin-embedded (FFPE) specimens and all five frozen specimens by RT-PCR. The level of FGF23 mRNA, which was determined by real-time PCR, varied among the phosphaturic cases. Two of 17 tumours were positive for FGFR1 gene rearrangement.It was considered that PMT-MCT is a histopathological entity with or without phosphaturia, with varying levels of FGF23 mRNA, and with or without fibronectin 1 (FN1)-FGFR1 fusion gene. The authors propose that the histology of PMT-MCT differs depending on its location, such as bone or soft tissue, which could complicate the differential diagnosis.

To examine regional specific diversity in development and growth of Ayu (Plecoglossus altivelis) larvae, we collected and compared collections from the Kalong estuary in Vietnam, and the Shimanto and Muko estuary, and the Niigata coast in Japan. Among the four areas, most of the morphometrics through ontogeny were similar except that the snout tended to be shorter and the anus hardly migrated in Kalong larvae. The snout length increased gradually with growth in the Vietnamese larvae, while this value increased significantly until ca. 10 mm BL, subsequently being constant up to 30 mm body length (BL) in the Japanese larvae. The water temperature when the larvae were collected was higher in the Kalong than in most of the Japan sites. Growth-rates estimated from otolith increments were from highest to lowest, Niigata (mean = 0.54 mm/day), Kalong (0.47), Shimanto (0.38) and Muko (0.34). The higher growth-rates were obtained not in Niigata of highest latitudinal region, but in Kalong of lowest latitudinal region. This indicates that Ayu could experience their early developmental stages from the cool temperate to tropical regions, implying the potential biodiversity of this fish species in the world.

The early life history of Ayu (Plecoglossus altivelis) was investigated in the Kalong and Tien Yen River systems, northern Vietnam, which is probably the most southern distribution locality for this species, during the period of November 2010 to February 2011. A total of 248 larvae were captured in the Kalong, and none were collected in the Tien Yen. There was little difference in development between the Kalong larvae and those of P. a. altivelis and P. a. ryukyuensis. Temperatures and salinities when the larvae were collected ranged from ca. 12 to 21°C and from ca. 3.5 to 30 psu. The preflexion to flexion larvae (primarily preflexion with yolk, 5.2–12.9 mm BL) occurred in the central current from December to February, with a peak abundance in early January. The flexion to postflexion (primarily postflexion, 14.1–23.8 mm BL) larvae occurred in the bank waters from early January to late February. The larval occurrence in the Kalong was 1–2 months later than for P. a. altivelis in Japan and P. a. ryukyuensis in the Ryukyu Islands, probably because of the delay until a reasonable photoperiod for the start of spawning in the lower latitudinal region. The larvae were never collected from the sea, where the temperatures were lower than in the river and estuary in January and February, unlike in Japan.

To examine the importance of the upper estuarine areas of Ariake Bay as a nursery ground for fish, assemblages of larvae and juveniles were compared among various aquatic habitats. The upper estuaries of the bay (the Rokkaku and Hayatsue estuaries) are brackish, highly turbid waters with high tidal velocities, and differ substantially from the Isahaya area, which has been separated from the bay by a man-made dike, to the middle estuary (the Kikuchi estuary). Abundances of larvae and juveniles were higher in the estuaries than in the open bay and Isahaya areas. Abundant species in the upper estuaries were similar to each other, but differed from those of the middle estuary. This was primarily due to larvae and juveniles of fishes that occurred almost entirely in the upper estuaries, such as Acanthogobius hasta, Boleophthalmus pectinirostris, Coilia nasus, Cynoglossus abbreviatus, Nibea albiflora, Odontamblyopus lacepedii, Trachidermus fasciatus and Tridentiger barbatus. These results suggest that the upper estuaries play an important role as nursery grounds for fishes.

Little is known about where anguilliform fishes complete metamorphosis from the leptocephalus to juvenile stage. Conger myriaster leptocephali in the final stages of metamorphosing were collected from the estuarine portions of the lower Rokkaku River and in the northwest region of Ariake Bay. Three metamorphosing leptocephali with PAM/TM ratios of 0.27–0.34 were collected in the lower river, and five (PAM/TM, 0.30–0.46) were collected at sea stations in the bay. The collection of metamorphosing larvae of this species in a river estuary with lower salinity and high turbidity raises the question of how much these habitats are used by the metamorphosing larvae of this marine eel species.

Preflexion to postflexion larvae (2.1–8.0 mm) of Diagramma pictum (Haemulidae) occur in coastal Tosa Bay, Japan from May to September. They can be distinguished from the other haemulid larvae by head spination, location of anus, and melanophore patterns. Compared with larvae identified as the same species from Australia, there are significantly morphological differentiations between the two areas. Thus, these significant differences are likely a reflection of subspecific differences.

In order to study the physiological characteristics of neoteny in teleosts, the development of the thyroid axis and digestive tract of the ice fish, Salangichthys microdon (Salmonoidei), were examined using wild caught samples in the Yura River and its estuary. In all developmental stages examined in this study, the digestive tract was straight, and no gastric gland was observed, even in adults. Since these features are only observed in larval stages of a related species, ayu, Plecoglossus altivelis (Salmonoidei), it is suggested that the ice fish retains larval features, not only in appearance but also in internal organs. Additionally, the thyroid axis exhibited a unique characteristic. Until the end of the juvenile stage, thyroid glands remained inactive, and no thyroid stimulating hormone (TSH) cells were observed. Activation of the thyroid gland and the first appearance of TSH cells occurred in adults. Since activation of the thyroid axis was generally known to occur by the time larval have transformed to juveniles in several teleosts, a delayed functioning of the thyroid axis was suggested as a specific characteristic, and probably a major factor contributing to neotenic development of the ice fish.

Early-stage morphologies of the mandarinfish Siniperca chuatsi are described on the basis of an ontogenetic series of reared specimens in an aquarium. Spherical eggs (diameter 1.70–1.82 mm) with a single oil globule (0.40–0.48 mm) were free-demersal and easily floated when agitated. Hatching occurred about 3 days after spawning at about 24°C. Newly hatched larvae (3.8–4.2 mm in notochord length: NL) had many melanophores on the yolk sac. After reaching ca. 5.5 mm NL (8–9 + 19–20 = 27–28 myomeres), larvae had almost completely absorbed the yolk, possessed a large mouth and sharp teeth, and were starting to prey on other fish larvae. Three large preopercle spines appeared at ca. 5.5 mm NL, five spines by ca. 8.5 mm NL, and eight by ca. 21 mm in standard length (SL). The interopercle bore a single spine at ca. 8.5 mm NL and two spines at ca. 13.5 mm SL. A single spine appeared at the supracleithrum and another at the opercle at ca. 10 mm SL. Dorsal fin spines and pelvic, anal, and caudal fins were completed at ca. 10 mm SL. Dorsal fin rays and pectoral fins were completed at ca. 13.5 mm SL. Four ontogenetic characters (free-demersal eggs, large jaws with large teeth, conspicuous head spination, and precocious completion of dorsal fin spines) are rare among freshwater percoids.

The escape behavior of wild and hatchery-reared juvenile Japanese flounder Paralichthys olivaceus from a dummy predator, gray goblinfish Minous monodactylus, was investigated under artificial light in an experimental tank. When the dummy predator approached, both the juveniles quickly emerged from the sand, escaping primarily by swimming close to the bottom (benthic swimming) rather than off the bottom (off-bottom swimming). It is suggested that juvenile flounders might escape in this manner from a diurnal visual feeder in the daytime, owing to the flounder being virtually undetectable visually when close to the bottom. The average escape swimming speed of an individual of 9.3 total length (TL)/s in wild fish and 9.8 TL/s in hatchery-reared fish was similar to the maximum speed of many fish species. Juvenile flounder escaped more quickly and had a higher swimming speed (v cm/s), when their distance (d cm) to the dummy predator decreased (v = −2.7d + 82.1, r = −0.33; anova, P

In order to clarify the mechanisms of neoteny in the ice goby (Leucopsarion petersii), we examined effects of thyroid hormone and thiourea (TU) treatments on their neotenic characteristics and the pituitary-thyroid axis. Adult ice goby were exposed to 3, 5, 3'-triiodothyronine (T3, 0.1 ppm), TU (inhibitor of thyroid hormone synthesis, 30 ppm), or the combination of the two for 2 weeks. Observations of whole body T3 levels, thyroid follicles and TSH immunoreactive cells in the pituitary suggests the presence of a functioning thyroidal system. However, all of the neotenic features did not disappear in T3-treated fish, suggesting the absence of T3 responsiveness in peripheral tissues. These results indicate the similarity between neoteny of the ice goby and obligatory-type neoteny of urodeles.

Larval and juvenile ayu from seven year-groups (1986, 1987, 1992, 1995, 1996, 1999 and 2000) were collected in the Shimanto Estuary and adjacent coastal waters. The present study focuses on the variation among year-groups in the hatching period estimated by age determination using the otolith. The hatching period of the dominant cohorts in the 1986, 1987 and 1992 year-groups occurred from late October to mid-November. A delay of the hatching period of the dominant cohort was observed from the 1995 year-group, which occurred in late November to early December, and the dominant periods in the 1996, 1999 and 2000 year-groups were observed from early to late December. It appears that the delay of the hatching period of dominant cohorts was not due to a delay of spawning, but a high mortality of early hatched larvae. The water temperature in autumn in the coastal waters adjacent to the Shimanto River has tended to rise over the past 20 years and this trend was especially notable in the late 1990s. The delay in the hatching period of the dominant cohort observed from the 1995 year-group was likely to be related to the rise in seawater temperatures in autumn.

To elucidate the recruitment and movement process of larval ayuin the surf zone of a sandy beach facing Tosa Bay, 31 collectionswere made with a small seine net at intervals of approximately 5days from November 1990 to April 1991. On the basis of seasonalvariations in larval abundance, size, age, duration of residence,and the occurrence patterns of ayu in the surf zone, larval ayuwere classified into two groups: short-term resident (

To clarify the intra- and interspecific relationships of four Gymnogobius species, G. urotaenia, G. isaza, Gymnogobius sp. 1 (sumiukigori), and Gymnogobius sp. 2 (shimaukigori), partial mitochondrial cytochrome b gene sequences of 639 bp were obtained for a total of 31 specimens from Japan and Korea, plus 2 outgroup specimens. Twenty-nine haplotypes were identified in the ingroup, with a total of 122 variable sites (19.1%). The individuals regarded as the same species morphologically were monophyletic genetically. Sequence differences between amphidromous individuals of three species distributed in both Japan and Korea were relatively small (0.16–1.25%). The largest intraspecific sequence difference was observed between individuals of G. urotaenia from Lake Biwa and those from other localities (1.25–2.19%). Interspecific sequence differences ranged from 4.07% to 13.46%; neighbor-joining, maximum-parsimony, and maximum-likelihood methods indicated that Gymnogobius sp. 2 diverged first, followed by G. isaza, with Gymnogobius sp. 1 and G. urotaenia being monophyletic. The estimated divergence time of each species, based on estimated divergence rates for mitochondrial protein-coding genes already reported (0.8–2.8%/my), suggested that speciation occurred mainly in the Pliocene (possibly Miocene), with G. isaza (a Lake Biwa endemic) diverging significantly earlier (probably Lake Kouga stage) than estimated in previous studies. In contrast, according to the previous hypothesis, the substitution rates were highly overestimated to about 12–20%/my.

To clarify the habitats of the ayu in the early stages of development in the Shimanto Estuary, the size and hatching date of the larval and juvenile ayu were examined. Sizes of larvae and juveniles were larger in the center of the flow area than in the waters adjacent to the estuary’s banks, which suggests that larvae shift habitat to the center of flow from the waters along the estuary’s banks. This shift began at approximately 20 mm body length. However, the resident term in the waters adjacent to the estuary’s banks changed with the hatching dates; that is, the early and late-hatched larvae became short and long-term residents, respectively. A similar phenomenon was found also in the juveniles collected in the freshwater section that were migrating upstream. The growth rates of larval ayu in the waters adjacent to the banks tended to decrease with hatching dates. The fluctuations in growth rate with hatching date appear to be a factor leading to the variation of resident term.

To clarify the characteristics of the larval and juvenile fish community in a Japanese estuary, monthly collections were conducted in the shallow waters of the Shimanto Estuary, Japan from July 1985 to June 1987. A total of 49 101 fishes of more than 100 species from 44 families were sampled over the 2-year period. Numerically, Gerres equulus was most common, accounting for 20.02% of the total abundance. Rhyncopelates oxyrhynchus was ranked second, followed by Acanthopagrus latus, Acanthogobius flavimanus, Mugil cephalus cephalus, Parioglossus dotui, and Acanthopagrus schlegeli. Thus, fishes observed in the estuary were mostly marine species that spawn in the sea. The majority of these fish first appeared within the estuary at late postlarval and/or early juvenile stages. Based on their utilization of the shallow waters in the estuary, the majority of species could be categorized as residents. In the Shimanto Estuary, shallow waters are studded with shelters, especially eelgrass beds, which serve as important nursery grounds for coastal and euryhaline fishes.

Meristic counts of early and late wild settled juveniles of Japanese flounder, Paralichthys olivaceus (Paralichthyidae), in Wakasa Bay, Japan, were compared, with specimens collected from 26 sites spread along ca. 1700 km of coastline facing the Japan Sea. Mean dorsal and anal ray counts of the early settled group at Kyoto were significantly larger than those of the late settled group. A discontinuous geographic boundary in mean dorsal and anal ray counts was found around Wakasa Bay and the Noto Peninsula with both means significantly larger in the southern group than in the northern group. Mean ray counts of the early and late settlers at Kyoto were similar to those of the southern and northern groups, respectively. The effect of water temperature on meristic characters was tested on two groups of laboratory-reared juveniles. Mean dorsal and anal ray counts were larger in juveniles reared at higher water temperatures. The early and late settler groups were estimated to hatch in late February and late April, respectively, representing negative response of meristic characters to water temperatures if these two groups hatched out in Wakasa Bay. Differences in the dorsal and anal ray counts between the early and late settled groups at Kyoto can, therefore be attributed to genotypic, not phenotypic variation. This suggests the possibility of transport of larvae of the early group from areas further south to Wakasa Bay by the warm Tsushima Current, a branch of the Kuroshio, which flows northeastward along the coast of the Japan Sea, particularly when the temperature gradation between the south and north areas during these seasons is considered.

To clarify the early life history of Cottus kazika (Cottidae), the ontogeny, osteology, distribution, migration and food habit were examined based on larvae and juveniles collected from coasts and estuaries in western Japan. The sequence of fin completion was pectoral (P1)-dorsal soft-ray part (D2)-anal (A)-dorsal spine part (D1)-pelvic (P2). However, that of calcification was P1-P2-D1-D2-A. Larvae and juveniles of C. kazika are distinguished from those of coastal sculpins of Japan by large preopercle spines, conspicuous parietal spines, the large and densely pigmented pectoral fin and the relatively deep body. C. kazika and Myoxocephalus polyacanthocephalus plus Enophrys bison share these four ontogenetic characters. From the relationship between the growth ring count on the sagittae and total length, the size of embryos at hatching is estimated at 5.2 mm. Larvae are estimated to reach 7 mm in 10 days, 9 mm in 20 days and 11 mm in 30 days after hatching. Hatching dates for larvae collected in the Shimanto estuary in 1987 peaked during the period around the new moon in January. Many free embryos ranging from 4.3 to 5.9 mm were collected using an aqualamp at a rocky shore outside the Yura River mouth, at high salinity (> 33 ppt), but none were collected in the estuary. From beach samples, seasonal changes in body size varied from year to year, and no consistent growth pattern was found, although juveniles were generally larger in late March and April than in January and February. Very few larvae were collected along shorelines where there were no adjacent rivers. Food habit of larvae and juveniles changed with growth. Polychaete larvae, cladocerans and copepods were found in preflexion larvae less than 6 mm. Gammarids were one of the important food items for larvae over 6 mm. Juveniles over 12 mm almost exclusively fed on gammarids. In the Shimanto estuary, larvae and juveniles stayed near the river mouth in January and February, within a stagnant layer where temperature and salinity remained about 12 °C and 20 ppt, respectively. Juveniles tended to move in March when the stagnant layer disappeared in the estuary.

The near-shore migration and settlement phases of Japanese and American flounders of the genus Paralichthys are compared and discussed relative to differences in coastal environments. Field sampling was conducted in Wakasa Bay, Kyoto Prefecture, Japan, where the shelf is narrow, estuarine habitat limited and tidal range slight, and in Onslow Bay, North Carolina, USA, where the shelf is broad, estuarine habitat extensive and tide relatively strong. Distribution of larvae and juveniles suggests Japanese flounder spawn in close proximity to nursery grounds relative to flounder in Onslow Bay. Sampling of planktonic and benthic flounder just seaward of nursery grounds resulted in capture of a wide range of developmental stages in Wakasa Bay (early planktonic to juvenile), but was limited to metamorphosing larvae in Onslow Bay. Vertical distribution of larvae during the night also differed between the Bays. At night most larvae in Wakasa Bay remained near the bottom regardless of tidal stage. In contrast, larvae in Onslow Bay exhibited selective tidal stream transport. Laboratory experiments conducted to examine the behaviour of recently captured wild and laboratory-reared larvae indicated that wild flounder from Onslow Bay had an endogenous rhythm of activity that corresponded to the tide at the time of capture. In contrast, wild flounder from Wakasa Bay and laboratory-reared larvae showed no distinct activity pattern. These results suggest that physical characteristics of the environment influence the early life history patterns of flounders by modifying behaviour of migrating larvae.

In the amphidromous form of ayu Plecoglossus altivelis Temminck et Schlegel, generally the prelarvae hatched in rivers in autumn are drifted down into the sea, where they live throughout the larval and early juvenile stages until spring. In the Shimanto estuary, however, an appreciable number of postlarval ayu occurred not only during March to May, but also during October to February. Furthermore, there was little difference in the size and age between the larvae occurring in the estuary and sea. In the estuary, they reside in the bank waters, during 10-75 day ages and 10-30mm SL. Thereafter, they seem to ascend the river without going out to the sea. The growth rate estimated from the otolith daily rings was higher in the estuary than in the sea beach.

In Shijiki Bay of Hirado Island, diel discrete-depth horizontal tows with a larva net in May 1983 resulted in a total of 2, 350 larval and juvenile red sea bream (2.1-10.7mm SL), and 347 larval black sea bream (2.4-6.2mm SL) being collected. During the daytime, black sea bream larvae were concentrated near the surface, but red sea bream larvae near the bottom, with these tendencies becoming more evident with their growth. During the nighttime, the larvae of both species were dispersed throughout the water column. In the surf zone of the sandy-beaches located in the innermost part of the bay, collections with a small seine net in June and July 1983 gave a total of 241 late-larval and early-juvenile black sea bream (6.2-20.1mm SL), but no red sea bream. The separation of juvenile habitats between these related species seemed to be a consequence of the species-specific vertical distribution pattern differentiated during the pelagic larval period. A possible mechanism for inshore migration in the black sea bream was discussed in relation to their surface occurrence.

In order to study the characteristics of neoteny in teleosts, development of the thyroid system and digestive tract of a neotenic goby (ice goby, Leucopsarion petersii) and a non-neotenic goby (ukigori, Gymnogobius urotaenia) were compared. In juvenile ukigori, the intestine was found to be convoluted once in the antero-midpart, and gastric glands were present. In the ice goby, the alimentary canal was straight, and no gastric gland was observed even in adult, suggesting that the ice goby retains larval features, not only in appearance but also in internal organs. A marked difference was also found in the thyroid system. In ukigori, activity of the thyroid gland and thyroid stimulating hormone (TSH) cells increased between flexion and postflexion larval phases. However, in the ice goby, thyroid glands remained inactive, and no TSH cells were observed. A delayed development of the thyroid system was suggested as a major factor contributing to neoteny in the ice goby.

The larval distribution and feeding habits of Cynoglossus abbreviatus and Cynoglossus lighti were investigated around the Rokkaku estuary in Ariake Bay during October 2004 and March and April 2005. Cynoglossus abbreviatus started and completed metamorphosis at larger sizes than C. lighti. Developmental phases consisted primarily of individuals at metamorphosis and postmetamorphosis, which were mainly distributed inside and just outside the Rokkaku River in March–April for C. abbreviatus and October for C. lighti. Tidal changes in vertical distribution just outside the river mouth differed between the closely related species; C. abbreviatus was distributed in the surface and middle layers at flood tide, and aggregated near the bottom (where the current speed was lowest) at ebb tide. Cynoglossus lighti stayed mainly near the bottom during all tidal phases. Larvae of both Cynoglossus species selectively fed on copepods (Pseudobradya sp.), which were scarce in the waters. These facts suggest that the nursery grounds of the two Cynoglossus species were different in terms of their tidal vertical distribution patterns.

A total of 211 larval and juvenileNibea mitsukurii (4.0–19.0 mm SL) was collected with a small seine in surf zones of Tosa Bay during the period of May 1981 to June 1984. They had morphological characteristics common to the larvae and juveniles of Sciaenidae, but were distinguished from the others by the distribution pattern of melanophores on body and spines at the anterior tip of maxillary. They occurred only in surf zone of Tei out of three locations facing Tosa Bay from middle May to middle August. Temperatures and salinities at the place when they were collected ranged from 21.7 to 29.5°C and from 24.5 to 31.3‰, respectively. Good catches were observed when minute dusts floated abundantly in the surf zone. In past studies using traditional larval nets or minnow-nets in coastal or shallow waters of Tosa Bay, larval and juvenileN. mitsukurii were not reported. It seems that they occur in association with minute dusts in extremely shallow waters such as surf zones.

A total of 343 larval and juvenile Lateolabrax latus (9.2–27.0 mm TL) was collected with a small seine in surf zones of Tosa Bay. They are very similar in general morphology to those of the closely related L. japonicus, but can be distinguished from them by the distribution pattern of melanophores on the tail, head spination, proportion of head length to standard length and the 15 or 16 dorsal fin soft-rays. They occurred in surf zones of Tosa Bay from early January to middle May, being most abundant in middle April. Temperatures and salinities of waters where any number of them were collected ranged from 12.2 to 24.0°C and from 23.7 to 34.5%, respectively. Larval and juvenile L. latus have not been reported so far in coastal and shallow waters, and eelgrass beds of southern Japan. It may be possible that they have been confounded with those of L. japonicus. Their occurrence seems to be limited in extremely shallow waters such as surf zones.