cDNA cloning and characterization of two gelatinases from Japanese flounder

SUMMARY: Toughness is one of the most important elements that define the commercial value of the raw meat of fish. Degradation of the extracellular matrix is thought to be a cause of postmortem tenderization of fish meat. A previous study has suggested that this tenderization is caused mainly by metalloproteinases. The present study seeks to identify the proteinase(s) involved in tenderization; hence, cloned cDNA of two gelatinases from Japanese flounder, which showed high homology with mammalian matrix metalloproteinase (MMP)-2 and MMP-9, were designated as jfMMP-2 and jfMMP-9 , respectively. Northern blot analysis revealed that jfMMP-2 mRNA was expressed almost ubiquitously in adult tissues including the brain, muscle, gill, heart, gut, kidney, spleen, testis, and ovary. In contrast, the expression of jfMMP-9 mRNA was observed in those tissues which were abundant in blood cells, such as kidney, spleen, heart, and gill. Both recombinant proteins (jfMMP-2 and jfMMP-9) produced with the COS-7 cell system exhibited gelatin-degrading activity that was sensitive to 1,10-phenanthroline, a typical metalloproteinase inhibitor.     

The effect of trigger depth on self-feeder utilisation, growth and the vertical distribution of tank-held rainbow trout Oncorhynchus mykiss (Walbaum)

This study investigated the effect of placing a self-feeding trigger at either a) 1 cm below the water surface or b) mid-water (35 cm below the surface), on self-feeder usage, growth, feed wastage and fin damage of juvenile rainbow trout, Oncorhynchus mykiss (Walbaum). The vertical distribution of trout was also measured for the first 31 days of self-feeding. Group-held trout (n = 10 fish group^− 1, 5 groups treatment^− 1), weighing 51.06 g ± 8.09 g (mean ± SD) were held in 200 l cylindroconical tanks (depth = 75 cm) for 62 days. Trigger placement had no significant effect upon self-feeder conditioning, and upper trigger group (UTG) and lower trigger group (LTG) fish took an average of 16 ± 7.4 days and 14 ± 9.8 days to reach a stable level of self-feeding, respectively. Specific growth rate was significantly higher in UTG fish (1.83 ± 0.20 vs. 0.95 ± 0.21 for UTG vs. LTG fish, respectively) whilst condition factor and self-feeder utilisation (ration size, 1.21 ± 0.18 vs. 0.52 ± 0.15 kg tank^− 1 for UTG vs. LTG fish) were also significantly higher in UTG fish. Trigger depth had no significant effect on size heterogeneity, mortality, feed wastage, feed conversion ratio (FCR) or the incidence of caudal and dorsal fin splitting and fin erosion. Dorsal fin splitting decreased with time in UTG but not in LTG fish. Caudal fin erosion increased with time in UTG (upper lobe only) and LTG (both lobes). The vertical distribution of fish also differed between treatments and more LTG fish preferred the lower part of the tank. Placing a self-feeding trigger at a mid-water depth can significantly limit growth, condition factor and self-feeder utilisation in juvenile rainbow trout, in addition to hindering recovery from dorsal fin erosion.     

Isolation of microsatellite markers by in silico screening implicated for genetic linkage mapping in Japanese pufferfish Takifugu rubripes

ABSTRACT:   The search for dinucleotide repeat microsatellites within scaffolds 1–25 of genome database JGI Fugu v3.0 for the pufferfish Takifugu rubripes revealed that 80% of microsatellite loci consisted of five to 13-fold repeats with locus-specific differences in density. Eleven out of 15 microsatellite loci isolated from the database with which genotyping using wild pufferfish was successfully performed showed polymorphism; that is, the means of the number of alleles and expected and observed heterozygosities at these 11 loci were 21.8, 0.915 and 0.829, respectively. It was confirmed that eight out of the 11 polymorphic loci were inherited through the Mendelian law and one pair of microsatellite loci derived from the same scaffold was linked. These results demonstrated that these loci are useful for constructing a linkage map in the pufferfish as DNA markers.     

Osteoinductivity of gelatin/β-tricalcium phosphate sponges loaded with different concentrations of mesenchymal stem cells and bone morphogenetic protein-2 in an equine bone defect model

Fracture is one of the most life-threatening injuries in horses. Fracture repair is often associated with unsatisfactory outcomes and is associated with a high incidence of complications. This study aimed to evaluate the osteogenic effects of gelatin/β-tricalcium phosphate (GT) sponges loaded with different concentrations/ratios of mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) in an equine bone defect model. Seven thoroughbred horses were used in this study. Eight bone defects were created in the third metatarsal bones of each horse. Then, eight treatments, namely control, GT, GT/M-5, GT/M-6, GT/M-5/B-1, GT/M-5/B-3, GT/M-6/B-1, and GT/M-6/B-3 were applied to the eight different sites in a randomized manner (M-5: 2?×?10⁵ MSCs; M-6: 2?×?10⁶ MSCs; B-1: 1 μg of BMP-2; B-3: 3 μg of BMP-2). Repair of bone defects was assessed by radiography, quantitative computed tomography (QCT), and histopathological evaluation. Radiographic scores and CT values were significantly lower in the control group than in the other groups, while they were significantly higher in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The amount of mature compact bone filling the defects was greater in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The present study demonstrated that the GT sponge loaded with MSCs and BMP-2 promoted bone regeneration in an equine bone defect model. The GT/MSC/BMP-2 described here may be useful for treating horses with bone injuries.     

Pancreatic Ductal Adenocarcinoma in a Wistar Hannover GALAS Rat

There are no reported spontaneous cases of pancreatic ductal adenocarcinoma (PDAC), and there are few reports about chemically-induced PDAC in rats. We encountered a PDAC in a Wistar Hannover GALAS rat that had been subjected to a medium-term multiorgan carcinogenicity bioassay. This article describes the histological and histochemical findings of the tumor. The tumor was located in the pancreatic tissue and had not invaded the liver parenchyma or the mucosal layer of the alimentary tract. The tumor cells were atypical and were mainly arranged in small tubules. In addition, abundant stroma and mucus production were observed in the tumor. In an immunohistochemical examination, the tumor cells were positive for cytokeratin, Sox9 and pancreas duodenum homeobox 1 and negative for amylase 2A and insulin. Therefore, the tumor was diagnosed as a PDAC based on its histological and histochemical findings. We considered that the tumor was caused by the carcinogens administered during the abovementioned bioassay.     

Interaction of rumen bacteria as assumed by colonization patterns on untreated and alkali‐treated rice straw

Colonization patterns of representative rumen bacteria were compared between untreated rice straw (UTS) and sodium hydroxide‐treated rice straw (SHTS). UTS and SHTS were incubated in the rumen of sheep for 10 min, 1, 2, 6, 12, 24, 48 and 96 h using the nylon bag method. The population sizes of 13 representative bacterial species or groups were quantified by real‐time PCR. The total bacterial population size (abundance) was similar in both UTS and SHTS. Fibrobacter succinogenes showed a higher population size compared to other fibrolytic species and was detected at a higher level in SHTS (3.7%) than in UTS (2.6%). Ruminococcus albus and Ruminococcus flavefaciens were also detected at higher levels in SHTS (0.15% and 0.29%) than in UTS (0.03% and 0.18%). Population sizes of non‐fibrolytic species, such as Selenomonas ruminantium, Anaerovibrio lipolytica and Succinivibrio dextrinosolvens were higher in UTS than in SHTS. Coefficient of determination (r²) on population changes between bacterial species or groups were higher in UTS than in SHTS, suggesting the necessity of stronger bacterial interactions for UTS digestion. Therefore, not only colonization of fibrolytic species, but also synergistic interactions between different bacterial species may be key to the ruminal digestion of rice straw.     

A low body-color regulating ability of spotted halibut Verasper variegatus: Evaluation of the roles of melanin-concentrating hormone and proopiomelanocortin systems

In teleosts, melanin-concentrating hormone (MCH) induces body brightening. On the other hand, α-melanophore-stimulating hormone (α-MSH), derived from the precursor proopiomelanocortin (POMC), leads to body darkening. In many teleosts, the expression level of pmch is high in light backgrounds, while that of pomc is low in dark backgrounds. In the present study, we investigated the ability of spotted halibut Verasper variegatus to regulate body coloration. Intraperitoneal administration of MCH resulted in a dose-dependent brightening of body color. The body color and brain contents of pmch1 and pmch2 in spotted halibut acclimated to the black or white background were not almost affected by background color. In addition, pituitary mRNA levels of pomc-a, pomc-b, and pomc-c genes in fish acclimated to the white background often exceed those levels of fish kept under the black background. These results suggest that the weak response of expression levels of pmch genes in response to background color is a factor in the blunt ability of spotted halibut to regulate body color. The different profiles of pomc gene expression to background color compared to that reported in other teleosts may be another factor for the weak ability of body color change in the spotted halibut.

     

Linking environmental drivers,juvenile growth,and recruitment for Japanese jack mackerel Trachurus japonicus in the Sea of Japan

Growth traits of juvenile Japanese jack mackerel (Trachurus japonicus) were estimated based on otolith daily increments in spring during 2011–2016 in the southwestern part of the Sea of Japan (SWSJ). Spatial variability in growth traits was examined between the western and eastern areas of the study area. Back-calculated body length at 50 days post hatch varied among areas and years between 26 and 40 mm. Body lengths at age in the eastern area were significantly smaller than those in the western area. Slower growth rate in the eastern area was associated with the lower ambient temperature in the eastern area than in the western area. In the eastern area, interannual variations in the instantaneous growth rate positively correlated with ambient temperature. Furthermore, interannual variations in back-calculated body length during the late larval and early juvenile stages positively correlated with the juvenile abundance. These results suggest that the thermal conditions in the eastern area are indicative of the growth rate and abundance of juvenile T. japonicus in SWSJ. We further examined relationships between juvenile abundance and thermal conditions during 2003–2019 using archived data of this survey and found that the ambient temperature in the eastern area positively correlated with the juvenile abundance and also the strength of the Tsushima Warm Current. Thus, higher temperatures derived from the Tsushima Warm Current result in a faster growth rate during the late larval and early juvenile stages and subsequently higher abundance of T. japonicus juveniles in SWSJ.