Inimicus Japonicus Research Articles

Molecular cloning and characterization of the two putative toxins expressed in the venom of the devil stinger Inimicus japonicus

Various proteins are involved in fish venom toxicity, but limited information is available regarding their structure and mode of action. Here, we analyzed RNA transcripts in the dorsal spine of the devil stinger Inimicus japonicus using next-generation sequencing (NGS), and identified two putative protein toxins, a natterin-like protein (Ij-natterin) and a phospholipase A2 (Ij-PLA2), as well as a previously reported stonustoxin-like protein. The deduced amino acid sequence of Ij-natterin suggested that it acts as a pore-forming toxin through the cooperation of the N-terminal lectin-like domain and the C-terminal pore-forming domain. Ij-PLA2 showed significant homology with secreted Ca2+-dependent PLA2s from snake venom and mammals (sPLA2-I/II). The recombinant Ij-PLA2 protein exhibited PLA2 activity in the absence of Ca2+, in contrast to canonical sPLA2-I/II. Comparison of the amino acid sequences of Ij-PLA2 with the other sPLA2-I/II suggests that the C-terminal extended peptide region of Ij-PLA2 is involved in its Ca2+-independent activity.

The complete mitochondrial genome sequence and phylogenetic analysis for Inimicus japonicus (Scorpaeniformes: Synanceiidae)

The complete mitochondrial genome (mitogenome) of the Inimicus japonicus was analyzed by next-generation sequencing in this study. The mitogenome is 16,978 base pairs (bp) long and codes for 22 transfer RNA (tRNA) genes, 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNA), and 1 non-coding control region. The overall nucleotide composition of the mitogenome is: 29.61% for A, 29.16% for T, 25.26% for C, and 15.97% for G. Twenty-two tRNAs range from 67 to 74 bp in length, and 2 rRNA (12S and 16S) were 953 and 1,687 bp long, respectively. Phylogenetic analysis by neighbor-joining (NJ) method indicated that I. japonicus showed considerable genetic similarity (82%), and had a closer relationship in the phylogenetic tree to Synanceia verrucosa.

The complete mitochondrial genome of Inimicus japonicus and its phylogenetic analysis

The complete mitochondrial genome was sequenced from the marine teleost fish Inimicus japonicus. The genome sequence is 16,830 bp in size and has a base composition of A (29.25%), T (29.01%), C (20.7%), and G (21.03%). Moreover, 13 protein-coding genes (PCGs) encoded 3210 amino acids in total. The phylogenetic analysis showed that I. japonicus belongs to Synancejidae family. The complete mitochondrial genome sequences provided here would be useful for further understanding the evolution and conservation genetics of I. japonicus.

Effect of exposure to low salinity water on plasma ion regulation and survival rates in artificially wounded devil stinger <i>Inimicus japonicus</i>

We investigated the effect of exposure to low salinity water on plasma ion regulation and survival rates in artificially wounded devil stinger Inimicus japonicus. All fishes survived in 33% seawater (SW), while survival rate in 100% SW was 5.1% at 24 h. In 100% SW, plasma Na+, K+, Mg2+, and Ca2+ concentrations significantly increased to 238 ± 49.9, 9.6 ± 2.4, 15.1 ± 3.5 and 5.0 ± 0.7 mmol/l at 6 h, respectively; the gill Na+/K+–ATPase (NKA) activity was almost stable, although only one fish survived to 24 h. In 33% SW, plasma Na+ and K+ concentrations remained at the same level, and plasma Mg2+ and Ca2+ concentrations gradually increased to 16.2 ± 0.7 and 4.5 ± 0.2 mmol/l until 24 h, respectively. The NKA activity significantly increased to 5.1 ± 1.1 µmol ADP/mg protein per h at 6 h. A positive correlation was observed between the wound surface area against body weight and the plasma ion concentrations, although no difference was observed in the restoration rate of the wounded area between 100 and 33% SW. These results indicate that exposure of wounded fish to low salinity water improves survivability by favoring plasma ion regulation without influencing the restoration rate.

A new species of bomolochid Orbitacolax brevispinus n. sp. and redescription of taeniacanthid Cirracanthus inimici (Yamaguti et Yamasu, 1959) (Crustacea: Copepoda: Cyclopoida) parasitic on marine fishes of Korea.

Two species of cyclopoid copepods are recorded in this study. (1) A new species of bomolochid, Orbitacolax brevispinus n. sp. (Crustacea) is described based on adult females collected from the gill filaments and inner surface of the opercula of red barracuda, Sphyraena pinguis Günther (Perciformes: Sphyraenidae), captured in Korean waters. The new species differs from its congeners by the possession of two pairs of spines on the dorsal surface of the cephalothorax located just posterior to the rostrum and a different setal formula on the distal exopodal segments of legs 2-4. (2) A taeniacanthid Cirracanthus inimici (Yamaguti et Yamasu, 1959) (Crustacea) is redescribed based on the specimens collected from the gill filaments and inner surface of the opercula of devil stinger, Inimicus japonicus (Cuvier) (Scorpaeniformes: Synancellidae). This finding is the first record in Korean waters and the first description of male. A checklist of parasitic copepods of the families Bomolochidae Sumpf, 1871 and Taeniacanthidae Wilson, 1911of Korea is also provided.

Effect of exposure to low salinity water on plasma ion regulation and survival rates in artificially wounded devil stinger Inimicus japonicus

We investigated the effect of exposure to low salinity water on plasma ion regulation and survival rates in artificially wounded devil stinger Inimicus japonicus. All fishes survived in 33% seawater (SW), while survival rate in 100% SW was 5.1% at 24 h. In 100% SW, plasma Na+, K+, Mg2+, and Ca2+ concentrations significantly increased to 238 ± 49.9, 9.6 ± 2.4, 15.1 ± 3.5 and 5.0 ± 0.7 mmol/l at 6 h, respectively; the gill Na+/K+–ATPase (NKA) activity was almost stable, although only one fish survived to 24 h. In 33% SW, plasma Na+ and K+ concentrations remained at the same level, and plasma Mg2+ and Ca2+ concentrations gradually increased to 16.2 ± 0.7 and 4.5 ± 0.2 mmol/l until 24 h, respectively. The NKA activity significantly increased to 5.1 ± 1.1 µmol ADP/mg protein per h at 6 h. A positive correlation was observed between the wound surface area against body weight and the plasma ion concentrations, although no difference was observed in the restoration rate of the wounded area between 100 and 33% SW. These results indicate that exposure of wounded fish to low salinity water improves survivability by favoring plasma ion regulation without influencing the restoration rate.

Activities of digestive enzymes and histology of digestive system during larval development of devil stinger (Inimicus japonicus)

Devil stinger is a valuable demersal scorpaenid fish while the rearing of stinger larvae still relies on live prey. This study was conducted to illustrate the development of the main digestive enzymes and digestive system during larval development of this species to provide evidence for the application of artificial feeds. Enzymatic and histological assays were conducted from 1 day post hatching (dph) to 36 dph in larvae. The result showed that the selected digestive enzyme activities increased significantly after 15 dph. Specifically, the total trypsin activities increased significantly from 18 dph to 33 dph. The total pepsin and amylase activities increased significantly first and thereafter decreased significantly. The lipase activities followed the similar pattern with trypsin. With regard to the histological study, the stinger larvae open their mouth to first feeding at 3 dph and turned into totally exogenous nutritional stage at 6 dph. In addition, mucous membrane, rich in goblet cells, was widely distributed in oesophagus epithelium at 18 dph. The height and amounts of gastric gland in cardia and main body of the stomach increased gradually with the development of stinger larvae after 15 dph. The intestine length of stinger larvae was short, and goblet cell was abundant in anterior intestine after 12 dph, not the posterior intestine. The ontogeny of liver and pancreas started from newly hatched stage, and the differentiation of liver was prior to pancreas. The above findings would provide evidence for the use of artificial feeds from the larval stage of stinger larvae (at least from 21 dph).

Influence of salinity on the early development and biochemical dynamics of a marine fish, Inimicus japonicus

Fertilised eggs of the devil stringer (Inimicus japonicus) were incubated at different salinity levels (21, 25, 29, 33, and 37), and then the hatching performances, morphological parameters, and biochemical composition (protein, lipid and carbohydrate) of the larvae were assayed to determine the influence of salinity on the early development of I. japonicus. The tested salinity levels did not affect the times of hatching or mouth opening for yolk-sac larvae. However, the salinity significantly influenced the hatching and survival rates of open-mouthed larvae, as well as the morphology of yolk-sac larvae. The data indicated that 30.5 to 37.3 and 24.4 to 29.8 were suitable salinity ranges for the survival of embryos and larvae of I. japonicus, respectively. Larvae incubated at a salinity level of 29 had the greatest full lengths, and decreasing yolk volume was positively correlated with the environmental salinity. With increasing salinity, the individual dry weights of newly hatched larvae or open-mouthed larvae decreased significantly. Newly hatched larvae incubated at a salinity level of 29 had the greatest metabolic substrate contents and gross energy levels, while the openmouthed larvae’s greatest values occurred at a salinity level of 25. Larvae incubated in the salinity range of 33 to 37 had the lowest nutritional reserves and energy values. Thus, the I. japonicus yolk-sac larvae acclimated more readily to the lower salinity level than the embryos, and higher salinity levels negatively influenced larval growth and development. In conclusion, the environmental salinity level should be maintained at 29–33 during embryogenesis and at 25–29 during early larval development for this species. Our results can be used to provide optimum aquaculture conditions for the early larval development of I. japonicus.

Isolation of a novel lectin from the dorsal spines of the devil stinger, Inimicus japonicus

A novel lectin was purified from the dorsal spines of the devil stinger, Inimicus japonicus using a combination of affinity chromatography techniques. A single band was detected on a native PAGE gel with a relative molecular mass of 97 kDa. The N-terminal partial amino acid of the intact 75 kDa subunit of the 97 kDa lectin was found to be DHEDS. The agglutination of rabbit erythrocytes by the 97 kDa lectin was inhibited most effectively by methyl α-D-mannoside. The 97 kDa lectin stimulated mitogenesis in murine splenocytes. This is the first study to examine the dorsal lectin of I. japonicus and one of the very few studies on venom lectins from venomous scorpaeniform fish. These results suggest that the devil stinger, I. japonicus, may be a novel resource for biologically active substances.

Effects of Aeration Rate and Salinity Gradient on the Survival and Growth in the Early Life Stages of the Devil Stinger Inimicus japonicus

We examined the effects of a flow field in the rearing tank exhibited by different aeration rate and salinity gradient on the larviculture of the devil stinger Inimicus japonicus. Two different rearing experiments using 1 kl rearing tanks were conducted from hatching (day 0) to settlement. In the Experiment 1, fish were reared until day 21 in 5 different aeration rates (0 -1200 ml/min). There was a significant and positive relationship between survival and aeration rate, and fish survival became stable at an aeration rate of more than 300 ml/min. In Experiment 2, the salinity gradient in a rearing tank was formed by pumping brackish water (22 ppt) from the sur- face and seawater (34 ppt) from the bottom at the same time. Control rearing tanks (34 ppt) were aerated at 300 ml/min and fish were reared until day 23. Growth and development was synchro- nized in the salinity gradient tanks with little standard deviation, although average survival at settle- ment in the salinity gradient tank (47.3%) was lower than the aeration tank (68.2%). The optimal aeration rate for devil stinger larviculture is stronger than 300 ml/min and introducing a salinity gradient can be a novel approach for enhancing performance of the fish.

OBSERVATION OF EMBRYONIC AND LAVAL DEVELOPMENT OF DEVIL STINGER, INIMICUS JAPONICUS

Artificial rearing technique for Inimicus japonicus was conducted at Xishan experimental ground of Zhejiang Marine Fisheries Research Institute from May to June 2011.After obtaining fertilized eggs by artificial insemination,we observed the embryonic and larval development of devil stinger using microscope and described the development time and morphological characteristics of each development stage,which provide basic information for the artificial breeding and early development.The results showed that fertilized eggs had perfectly spherical shape and were transparent,and average egg diameter was(1.42±0.04) mm and without oil globule.Furthermore,the eggs showed buoyancy in seawater at salinity 31.Embryos were incubated in seawater(salinity 31) at(22±0.5)℃.The time-table of major embryonic development stages of devil stinger were as follows: blastodisc forming stage at 15min post fertilization(pf);2-cell stage at 25min pf;multicellular stage at 4h and 10min pf;bastula stage at 7h and 20min pf;gastrula stage at 13h and 20min pf;neurula stage at 25h 40min pf;organogenesis stage at 26h 10min pf;tail bud stage at 38h 10min pf;heart beat stage at 40h 30min pf;muscular contraction stage at 46h 20min pf;pro-hatching stage at 52h 10min pf;hatching stage at 52h 10min pf.Newly hatched larvae usually lied on the water surface and had 3.0 mm in average in total length(TH) with yolk sac length of 2.2 mm and height 1.7 mm.The larvae relied on their yolk for nourishment before the 3rd day post hatching at(21±0.5) ℃.4 days later,the yolk sac was consumed completely and started ingesting rotifers.About 13 days after hatching,the whole body length was(7.1±0.35) mm and fin ray appeared on the dorsal fin,indicating the larvae began turned into juvenile.On day 15 post hatching,the whole body length was(7.92±0.61) mm,and golden yellow maculation occurred on each fin.On day 25,whole body length was(13.66±0.55) mm,metamorphosis was completed with yellow brown of total body and the fish turned into young fish,while accompanied by a change from planktonic habits to demersal habits.The morphological development and changes of pigment pattern from embryonic to juvenile stage were observed.Pectoral fin bud was developed early after the later stage of embryo together with the fan-shaped membrane.Three days later,3—5 melanin pigments appeared in the neighbor of pectoral fin border of larvae,and yellow pigments were clearly seen in the border with arc distribution.Six days later,a small amount of melanin pigment was developed in the distal end of pectoral fin and yellow pigments showed more intensity.Twenty days later,vivid golden yellow maculation occurred on the outsized? fan-shaped pectoral fin.Two fin rays at the below of pectoral fin of adult fish were connected without membrane,presenting the dissociation state.In the juvenile stage,these fin rays,like the other fin rays,was connected by membrane,subsequently became the free fin ray.From the morphological observation,it is thought that two dissociated fin rays on the pectoral fin were differentiated from the pectoral fin rays.

Ontogenesis from embryo to juvenile and salinity tolerance of Japanese devil stinger Inimicus japonicus during early life stage.

Embryonic development and morphological characteristics of Japanese devil stinger Inimicus japonicus during early life stage were investigated. Larvae were hatched out 50 h after fertilization at temperature 21°C. Total length of the newly hatched larva was 4.03 mm, the mouth of the larva opened at 3 days after hatching (DAH), and the yolk sac of the larva disappeared at 5 DAH. After hatching, the pectoral fin first developed, then the tail fin, dorsal fin, anal fin and pelvic fin continuously developed, and all fins formed completely at 15 DAH. The metamorphosis was complete at 25 DAH, and the body color and habit of the metamorphosed individuals were different from the larvae. At 30 DAH, the morphology and habit of the juveniles were the same to adults. In order to determine the suitable salinity for larviculture of I. japonicus, salinity tolerance at different early developmental stages was compared in terms of the survival activity index (SAI) and mean survival time (MST). The results indicated that salinity tolerance varied with development stages. The optimum salinity range for newly hatched larvae was 10–25‰. Larvae showed low tolerance to low salinity (5‰) before the mouth opened, and the suitable salinities for the larvae with open mouth, yolk-sac larvae, post yolk-sac larvae were 10–15‰. The flexion larvae showed a wider salinity tolerance with range of 5–20‰. After metamorphosis, the juveniles showed a preferable adaptability of salinities of 15–20‰. The SAI and MST of individuals at various stages under different salinity conditions were positively correlated.

Proteinaceous toxins from three species of scorpaeniform fish (lionfish Pterois lunulata, devil stinger Inimicus japonicus and waspfish Hypodytes rubripinnis): Close similarity in properties and primary structures to stonefish toxins

The crude toxins from three species of venomous fish (lionfish Pterois lunulata, devil stinger Inimicus japonicus and waspfish Hypodytes rubripinnis) belonging to the order Scorpaeniformes exhibited mouse-lethal, hemolytic, edema-forming and nociceptive activities. In view of the antigenic cross-reactivity with the stonefish toxins, the primary structures of the stonefish toxin-like toxins from the three scorpaeniform fish were determined by cDNA cloning using primers designed from the highly conserved sequences of the stonefish toxins. Based on the data obtained in gel filtration, immunoblotting and cDNA cloning, each toxin was judged to be a 160 kDa heterodimer composed of 80 kDa α- and β-subunits. The three scorpaeniform fish toxins contain a B30.2/SPRY domain (∼200 amino acid residues) in the C-terminal region of each subunit, as reported for the toxins from two species of lionfish and two species of stonefish. With respect to the amino acid sequence similarity, the scorpaeniform fish toxins are divided into the following two groups: toxins from three species of lionfish and those from devil stinger, two species of stonefish and waspfish. The phylogenetic tree generated also clearly supports the classification of the toxins.

Temperature effects on early development and biochemical dynamics of a marine fish, Inimicus japonicus

A study was undertaken to evaluate the development and metabolic substrates (carbohydrate, protein, lipid and fatty acids) dynamics during the embryonic and yolk-sac larval stages of the devil stinger, Inimicus japonicus, at different water temperatures (18, 21, 24, 27 and 30°C). The hatching time was significantly shortened when the temperature increased (P<0.05). Only 14.5% of embryos hatched at 30°C, while the hatching rate was 92.5% at 24°C. Larvae incubated at 21°C displayed the greatest survival at mouth-opened larva stage (96.5%), but the rate dropped sharply when the temperature surpassed 24°C (P<0.05). The full length and yolk-sac volume of newly hatched larva at 18°C and 30°C were significantly smaller than those at 21, 24 and 27°C. Larva at 21°C always had the largest full length during early development. The advent of mouth-opened larvae was closely related to temperature, so were the full length and yolk volume of the mouth-opened larva. As the incubation temperature declined, the mouth-opened larva obtained longer full length and less yolk remain. Lipid was, to a large extent (47.0% of the initial amount), a fuel for development when the embryo was incubated at 18°C. The depletion of protein in the embryo was significantly higher at 30°C (67.6%) than that at 21°C (31.3%). Consumption of carbohydrate was observed only in embryo at 18°C. During the development of yolk-sac larva, a lower incubation temperature induced more protein anabolism and less lipid consumption. In I. japonicus, polar lipids (PL) constituted the principal class of lipid in eggs (74.0% of the total lipid); and it was the most consumed portion during embryogenesis in all treatments. The lipid composition in mouth-opened larva was affected by the incubation temperature. The storage triglycerides (TG) tended to comprise a higher proportion in larva incubated at 30°C than in those at lower temperatures. The PL proportions in larva at 18°C and 30°C were lower than those in larvae at 21°C, 24°C and 27°C. Low concentrations of polyunsaturated fatty acid (PUFA) in embryos and larvae were correlated with increased temperatures; this indicated that the rapid development at higher temperatures was at the cost of more PUFA loss. It is concluded that 21–24°C is an appropriate range for incubation of I. japonicus eggs and larvae and that 24°C is optimal in terms of shortening the endogenous nutritional stage and improving metabolic efficiency.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 August 2012 – 30 September 2012

This article documents the addition of 83 microsatellite marker loci and 96 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Bembidion lampros, Inimicus japonicus, Lymnaea stagnalis, Panopea abbreviata, Pentadesma butyracea, Sycoscapter hirticola and Thanatephorus cucumeris (anamorph: Rhizoctonia solani). These loci were cross-tested on the following species: Pentadesma grandifolia and Pentadesma reyndersii. This article also documents the addition of 96 sequencing primer pairs and 88 allele-specific primers or probes for Plutella xylostella.

Effectiveness of Marking by Extracting Dorsal Fin Spines of Devil Stinger Inimicus japonicus

Effectiveness of Marking by Extracting Dorsal Fin Spines of Devil Stinger Inimicus japonicus

Validity of an External Marking on Young Devil Stinger Inimicus japonicus by Clipping the Branchial Mantle

Validity of an External Marking on Young Devil Stinger Inimicus japonicus by Clipping the Branchial Mantle

Transitional Reactive Oxygen Species (ROS) Production in Fertilized Egg Embryos of Devil Stinger (Inimicus japonicus), a Marine Fish Species

A time-course analysis of reactive oxygen species (ROS) generation in fertilized eggs of the devil stinger (Inimicus japonicus) from 0 h post-fertilization (hpf) to the early larval stage indicated that the ROS level was highest in the 22 hpf embryo, and declined thereafter. Phorbol myristate acetate (PMA) had no effect on ROS generation by the 22 hpf embryo, whereas PMA significantly increased larval ROS generation, suggesting that the ROS generation mechanisms of the 22 hpf embryo and larva are different at least in terms of PMA-responsiveness. Our results suggest the presence of a specific ROS generation system in devil stinger embryo which can be transitionally activated during embryogenesis.

Isolation and characterization of 19 highly polymorphic microsatellite markers in the devil stinger, Inimicus japonicus (Synanceiidae)

Inimicus japonicus, the devil stinger, has an extensive distribution along the coast of China, Japan and the Korean Peninsula. Nineteen highly polymorphic microsatellite markers were isolated and characterized in I. japonicus. Twenty-eight individuals from a wild population were tested for polymorphism using this set of polymorphic microsatellite markers. The number of alleles per locus ranged from 4 to 14. The ranges of observed and expected heterozygosity were 0.500-0.892 and 0.521-0.910, respectively. Significant deviations from Hardy-Weinberg equilibrium were detected at two loci. To the best of our knowledge, these were the first microsatellite loci characterized from the Synanceiidae; they can be used for estimating genetic diversity, population structure studies, parentage analysis, genetic linkage map construction, germplasm classification and identification, gene identification, quantitative trait loci mapping, and marker-assisted selection in breeding of I. japonicus and other species of this family.

Optimum Salinity for Hatching and Salinity Tolerance of Larvae and Juveniles in the Devil Stinger, Inimicus japonicus

Optimum Salinity for Hatching and Salinity Tolerance of Larvae and Juveniles in the Devil Stinger, Inimicus japonicus