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Viral edema of carp is caused by Carp edema virus and is a major carp disease listed on the WOAH list of emerging infectious diseases. However, an effective experimental infection method that can control virus copy number has not been established. In this study, we compared three experimental infection methods：immersion infection using gill suspensions from diseased fish (immersion method), infection by rearing water from diseased fish (rearing water method) and co-infection method. Cumulative mortality in the immersion groups ranged from 40 to 60%, while that in the rearing water infected and co-infected groups ranged from 80 to 100%. The rearing water method can be used to control the viral DNA copy number and to perform experimental infections when frozen storage is possible. Thus, we performed the same experimental infection using frozen rearing water and found no disease or mortality, indicating that stable experimental infection using the rearing water method is difficult. The immersion method has a lower mortality rate than the other two methods, and it requires a large amount of gill tissue because of the low number of virus DNA copies that can be obtained from the gill tissue. However, it is possible to control the number of virus DNA copies and to preserve the gill tissue suspension by freezing. Therefore, the immersion method was considered the most suitable for stable experimental infection under the same conditions,

On board research were carried out for investigate the quality of fishing boat under the circumstances, which are observed experimentally in ordinary use on the seas. The present research was prepared in the ordinary operation of maintenances in dock. As the results, it was revealed that fuel consumption was reduced remarkably by cleaning bottom of ship hull, propeller and rudder. And in the condition of foul of ship bottom before cleaning, reducing the rotation speed of the main engine capable of largely improving a fuel efficiency, in comparison to the rate of decrease in ship speed.

To induce breeding of the freshwater pufferfish Colomesus asellus, artificial maturation by hormone injection (hCG, 10 IU per gram) and insemination were performed, and subsequent development observed. The rearing environment for the parental fish was freshwater, maintained at 26.0 ± 1.5 ℃ under L12：D12 lighting cycle. Two hormone treatments were applied, one month apart, to two females and four males, ovulation being confirmed 47 hours after the second treatment. Three hundred and sixty-three and 548 eggs with a mean diameter of 1.11 mm (n = 20) were collected from the two females, 334 (92.0 %) and 395 (72.1 %) being fertilized, respectively. The larvae and juveniles were fed with S-type Brachionus sp. fortified initially with freshwater chlorella, followed by Artemia larvae and Chironomid sp. larvae. The pufferfish were reared in a salinity of 7 ‰ for 24 days after hatching, the water then being gradually changed to freshwater over the following 10 days. Two individuals survived for 225 days after hatching, growing to average standard and total lengths of 29.24 mm and 38.72 mm, respectively. The Gonperz growth formulae were as follows：SL：Lt = 29.4075×exp (-exp (-0.02301(t-39.1213))) TL：Lt = 38.51177×exp (-exp (-0.023253(t-41.25841))) Further investigations of rearing conditions, such as salinity, are required to improve the breeding techniques for this species.

We investigated the effect of temperature on disk abalone Haliotis (Nordotis) discus discus hemolymph CO_2 solubility coefficient (αco_2) and the apparent dissociation constant of carbonic acid (pKapp). The disk abalone hemolymph was equilibrated with a standard CO_2 gas mixture between 10℃ and 20℃, to obtain expressions for αco_2 and pKapp as a function of temperature. The relationship between αco_2 and temperature (T) is expressed as follows： αco_2 = 74.005 - 1.2936 • T - 0.00944 • T^2. And the relationship between pKapp and temperature expressed as follows： pKapp = 6.4675 - 0.08682 • T + 0.003996 • T^2. In these equations, the parameter units are ℃ for T and µM/L/torr for αco_2. The non-bicarbonate buffer values (ꞵ_NB), obtained as a regression coefficient relating pH and [HCO_3^–], were 2.5 Slykes at 10℃, 2.2 Slykes at 15℃, and 3.4 Slykes at 20℃. These equations enable estimation of hemolymph αco_2 and pKapp between temperatures of 10℃ and 20℃.

The term “neutrophilic” and “neutrophilic granule” was first introduced by Paul Ehrlich to identify specific granules of major polymorphonuclear leukocytes (neutrophils). He used an original staining method, triacid stain, for this purpose. The staining solution, called “neutral mixture”, which was mixture of acidic dye solution and basic dye solution, with soluble form in water (not neutral dye). In this paper, we speculate the structure and characteristics of the dye complex in the neutral mixture. The dye complex has free acidic groups and is expected to behave as an acidic dye. The dye complex bound to the neutrophilic granules stains the granules with the color tone of the dye complex (purple) because the acid and basic dyes do not dissociate. On the other hand, in the vicinity of the eosinophilic granules and nucleus, the dye complex dissociates into acid and basic dyes, and the former bind to the acid dye and the latter to the basic dye. It was inferred that this dye complex is not formed during staining with stains containing neutral dyes, such as May-Grünwald (methylene blue eosinate), Wright (polychromtic methylene blue eosinate), and Giamsa (containing azure II eosinate).

Neutrophil granules (NG) of West African lungfish Protopterus annectens were classified into two types of stratified [two-layer; inner layer (L0) and outer layer (L1)] granules (type A, NG-A; type B, NG-B). The L1 of NG-A and both layers of NG-B were chromophobic, and L0 of NG-A showed polychromatophilic [eosinophilic or basophilic (orthomethylenophilic or metaazurophilic)]. The L0 of NG-A showed metachromatic (reddish purple) with toluidine blue. On the other hand, L0 of NG-B were positive for acid phosphatase, α-naphthyl acetate esterase, α-naphthyl butyrate esterase and naphthol AS-D chloroacetate esterase. Both types of granules were negative in periodic acid Schiff reaction, alcian blue, Sudan black B, Sudan III, and oil red O. Alkaline phosphatase, β-glucuronidase, and peroxidase were not detected in either granule.

Structure and development of eosinophilic granules (EG1) in eosinophils from larva (ammocoetes) of far eastern brook lamprey Lethenteron reissneri were speculated. EG1 is stratified granules (two-layer) and consists of inner eosinophilic layer (L0) and chromophobic outer layer (L1). Three subtypes of EG1 are identified based on the optical artificial image (OAI) of inclusion structure (IS) in L0: EG1a, EG1b, and EG1c. The EG1a had no OAI (probably no IS). The EG1b and EG1c contain IS in L0. The OAI of both EG1b and EG1c were larger than IS. The former was round or oval chromophobic area (OAI-1), and the latter was expanded and rugged (three-dimensional) image (OAI-2) surrounded with OAI-1. EG1a was thought to be a prototype of EG1, which would develop into EG1b and then EG1c.

Monocytes were observed in the blood of inshore hagfish Eptatretus burgei. The monocytes were round or oval, high nucleus/cytoplasm ratio, and have several round or oval eosinophilic granules, which show dark red or blackish red color when stained with May-Grünwald (MG), Giemsa, and MG-Giemsa stain. The granules were positive for alkaline phosphatase, acid phosphatase, and α-naphthyl butyrate esterase. Also, the granules show metachromatic (reddish purple) with toluidine blue. However, ꞵ-glucuronidase, α-naphthyl acetate esterase, naphthol AS-D chloroacetate esterase and peroxidase were not detected in the monocytes, and negative for periodic acid Schiff reaction, alcian blue, Sudan black B, Sudan III, and oil red O. The monocytes engulfed many yeast particles (zymosan).

Since the construction of class timetables in the university requires a great deal of effort and time, many studies have been conducted in Japan and abroad on the automatic construction of class timetables. Similarly, at the National Fisheries University, labor and time are devoted to the creation of class timetables. Therefore, author aims to conduct basic research on automatic timetable construction with the simulated annealing method in this study. This paper describes the proposed method and algorithm for generating timetables using the simulated annealing method. The generated timetables by the proposed method are also reported.

The Tiger Pufferfish (Takifugu rubripes) is a staple in Japanese cuisine, with over ten species of the Takifugu genus found in the surrounding seas. Given that certain parts of the pufferfish are toxic, they are predominantly prepared by trained professionals. Furthermore, species within the Takifugu genus are susceptible to hybridization, leading to an increase in hybrid numbers. However, identifying these hybrids is a challenging and time-consuming task, even for experts. To address this, we developed a transfer learning model using the pretrained VGG16 model to differentiate between pufferfish species. The VGG16 model, commonly used in image recognition, is built on convolutional neural networks. We also implemented Gradient-weighted Class Activation Mapping (Grad-CAM) for visual interpretation of the model. Grad-CAM generates a heat map that highlights the areas focused on by the AI model in the image, allowing us to identify factors contributing to misjudgment and make further improvements. We used seven species from the Takifugu genus (excluding hybrids), and approximately 15 colored images of each species were prepared for machine learning. The results showed that our model was able to distinguish between pufferfish species with relatively high accuracy, although some misclassification occurred among species with similar body patterns. The Grad-CAM results revealed that the model was able to distinguish body patterns, but some misclassifications occurred due to gravel and background objects being recognized as patterns.

In aquaculture, leaving dead fish that have sunk to the bottom of the net for a long time causes harmful components from the dead fish body. This risk making other fish in the aquaculture net sick. The early collection of dead fishes is required, but when divers work in the sea, high water pressure and low water temperatures make it difficult for them to work for a long time, and the work efficiency of the entire aquaculture facility is reduced due to the need for manpower. To protect aquaculture fishes from disease, it is useful to install devices and robots to detect and collect the dead fish at an early stage. If the target is small fishes, there is a pumping method. But if the target is medium to large fishes, it is not reasonable. Therefore, we devised a system for collecting dead fishes using AUV(autonomous underwater vehicle) and wire. This report describes the configuration and operation of this system, and conducts simple experiments to confirm its feasibility.

Effects of water temperature on feeding and growth of the 0-year-old catadromous fourspine sculpin Rheopresbe kazika were studied using 8 different temperatures from 12 to 26℃ for 60 days. At each of the 8 temperatures, 20 fish were reared in aquaria. In the ranges of 16 to 24℃ and 22 to 26℃ the daily growth rate and daily feeding rate, respectively, were higher than the others. Also, the optimal water temperatures were calculated 19.6℃ for daily growth rate and 25.5℃ for daily feeding rate. The optimal water temperature for daily growth rate was lower than that for daily feeding rate. While, feeding efficiency were showed high values in the range from 12 to 22℃. These results indicate that the optimal water temperature range is from 16 to 22℃, and that a water temperature of 12℃ is too low and above 24℃ is too high for sustainable growth during culture of R.kazika.

We investigated the hemolymph oxygen and acid–base status of Akoya pearl oysters, Pinctada fucata martensii, exposed to hypoxic seawater to elucidate the acid–base balance. Akoya pearl oysters cannulated to the anterior aorta for hemolymph collection from the submerged animals showed oxygen and acid–base disturbance of the hemolymph during environmental hypoxia for 24 h (O_2 partial pressure in seawater, Pwo_2 8 torr). The hemolymph O_2 partial pressure (Po_2) decreased from 72.2 torr to 13.6 torr, pH decreased from 7.581 to 7.129, and CO_2 partial pressure (Pco_2) increased from 0.86 torr to 3.31 torr during hypoxia. The hemolymph total CO_2 concentration (Tco_2) and bicarbonate ion concentration ([HCO_3^–]) were 1.93–1.95 mM/L and 1.80–1.91 mM/L, respectively, and there was no statistically significant change between pre-hypoxia and hypoxia for 24 h. When normoxic seawater was resumed after the hypoxia, the hemolymph Po_2, pH, and Pco_2 returned to their initial levels for about 3 h, and hemolymph Tco_2 and [HCO_3^–] gradually increased. These results showed that Akoya pearl oysters undergo hypoxemia and respiratory acidosis in the hypoxic environments for 24 h (Pwo_2 8 torr). In post-hypoxia, most of the disturbances disappeared within 3–24 h, and the increase in hemolymph [HCO_3^–] which was a secondary change compensated for respiratory disturbance.

Growth of immature Lates japonicus in eelgrass (Zostera japonica) beds of the Tomouchi River located at around 4.3 km upstream from the river mouth of the Gokase River, Kyushu Island, Japan was studied by the mark and recapture method and scale reading from September 1998 to March 2004. Newly occurring 0-year-old L. japonicus specimens of 13.9–78.0 mm SL (standard length) in Z. japonica beds of the Tomouchi River in August or September reached sizes ranging 58.8–125.7 mm SL by April and 203.4–232.8 mm SL between October and November of the following year. Monthly changes in specific growth rates of individuals demonstrated that rapid growth months of immature fish were limited to the period of high water temperature (22–28℃) from June to October. Also, it was supposed that the large-sized fish of the 0-year-old fish migrated away from Z. japonica beds after December, however a few fish remained in Z. japonica beds until the following November.

In order to research the actual operation status of fishing vessels by AIS, an effective extraction method is needed even when vessel name, MMSI, and type of ship information are insufficient. In this study, we analysis on the illegal use of AIS for fishing pole to indicate the location of operations and proposes a method for estimating fishing grounds using AIS buoys. Extracted by Vendor ID and vessel name notation and visualized using the kernel density estimation to show the major fishing grounds. The use of AIS buoys is illegal, but if the reception rate of satellite AIS improves, it may be easier to determine the location of operations and prevent damage to fishing gear.

Based on the findings of past literature, we speculated the existence of two types of stratified [two-layer; inner layer (L0) and outer layer (L1)] granules in the neutrophils (granulocytes; blood cells) of amphioxus (Cephalocordata), especially Branchiostoma japonicum. Type 1 neutrophil granules (NG1a) consist of chromophobic L0 and chromatophilic L1. Other type (type 2; NG2) have chromatophilic L0 and chromophobic L1. The L1 of NG1a and L0 of NG2 show metaazurophilic (purple to blackish purple) in Giemsa staining preparation. Ultrastructurally, these two granule types will not be separately identified. These granules contain intragranular particles (granulons, g) in the L0 of granules. At least, three types of granulons (g-1, g-2, and g-3) are recognized in a granule and localized in L0. Two of them form an agglomerate with short rod shape and two layers (inner layer consisted by g-2, and outer layer by g-3). The agglomerate have been considered as tubular structure or microtubule (misinterpretation). Neutrophils of cyclostome (hagfish and lamprey) also have NG1 [NG1a or NG1b (chromophobic L0 and L1)] and NG2. Further, neutrophil granules of cyclostome also contain granulons (without forming of agglomerate).

Neutrophil granules (NG) of adult (mature) far eastern brook lamprey Lethenteron reissneri [L. sp. S (souther form)] were classified into two types of stratified [two-layer; inner layer (L0) and outer layer (L1)] granules (type 1, NG1a; type 2, NG2), like as larva (ammocoetes) of this species. The L0 of NG1a and L0 of NG2 in adult neutrophils were chromophobic, and L1 of NG1a and L0 of NG2 showed chromatophilic. The stainability of those chromatophilic layers with May-Grünwald (MG), Giemsa, or MG-Giemsa were varied [chromophobic, eosinophilic or basophilic (orthomethylenophilic or metaazurophilic)] with a slight difference to larva. As seen in larval neutrophils, these layers were positive for alkaline phosphatase,α-naphtyl acetate esterase and Sudan black B, and some enzymes (acid phosphatase, ꞵ-glucuronidase, and naphthol AS-D chloroacetate esterase) were detected in the L0 of NG2. Alpha-naphtyl acetate esterase was positive in L0 of NG2 of adult neutrophils. Both larval and adult neutrophils lacked peroxidase.

Two types of stratified (two-layered) granules (type 1, NG1a; type 2, NG2) in the neutrophils of inshore hagfish Eptatretus burgei showed eosin-positive (positive site: outer layer (L1) of NG1a and inner layer (L0) of NG2). In contrast, both eosin-positive sites have been reported to be basophilic. From present result, it is clarified that both sites exhibit as follows: L1 of NG1a, eosinophilic and mataazurophilic; L0 of NG2, eosinophilic, orthomethylenophilic and mataazurophilic. The inshore hagfish neutrophils phagocytosed zymosan particles, in vitro. All blood cells showing phagocytosis were identified as neutrophil.

Embryonic development in giant cuttlefish Sepia latimanus was studied using eggs laid from broodstocks reared in captive condition. 26 embryonic stages were examined in this study and the detail process of organ development was clarified under microscope observation. Blastdisc was visible at the animal pole on the yolk at 2.5 hours after fertilizaton (HAF). The first cleavage occurred at 8.6 HAF. Then embryo developed to the morula stage at 22 HAF. Almost surface of the egg was covered with blastoderm at 10 days after fertilization (DAF). Primordia of main organs such as arms, mouth, mantle, funnel, optic vesicle, gills, statocyst, lens and optic ganglion were formed till 14 DAF. At 15 DAF calcification began in the first striation on the shell. Primordium of the shell became visible at 18 DAF. The number of striations on the shell reached to 5, 9 and 14 at 26, 32 and 40 DAF, respectively. Embryo needed 45 days for hatching. Newly hatched larva was 14 mm in mantle length and had 18 striations on the shell. The process of embryonic development in S. latimanus was very similar as S. lycidas, S. esucurenta and Sepiella japonia. S. latimanus is characterized with large newly hatched larva in Japanese sepiids. It was revealed that the large yolk of S. latimanus induces the growth of the body over a long period in the embryonic stage.

Viral edema of carp (VEC) caused by the carp edema virus (CEV) causes economic losses for Japanese koi farms. In this study, we investigated the infectivity and pathogenicity of a domestic CEV isolate (genogroup IIa) in koi carp, common carp and goldfish. The challenge test consisted of 9 groups (n = 15):3 groups each of koi carp, common carp, and goldfish, at 15, 20 and 25℃. These groups were challenged with CEV (3.0×10^3 copies/µL) in duplicate. All koi carp died in the 15 and 20℃ groups, but all survived in the 25℃ group. The surviving koi carp in 25℃ groups showed high PCR positive rates of 66.7 and 73.3%, with VEC histopathological changes observed. For the common carp, 1 and 2 fish died in the 20℃ groups, but no deaths or VEC symptoms were observed in the 15 and 25℃ groups. In all common carp groups, PCR-positive fish were observed along with histopathological changes. For all goldfish groups, no deaths or VEC symptoms were observed. As with the common carp, PCR-positive fish were found in all goldfish groups, yet no VEC histopathological changes were detected. These results demonstrate infectivity of this CEV strain in koi carp, common carp, and goldfish, but low pathogenicity in common carp and goldfish.

In aquaculture and fixed-net fishing, fishing nets need to be fixed in the water for a long period of time. Therefore, attached organisms such as shellfish, algae, and parasite eggs easily stick to the nets, making periodic cleaning indispensable. A common method is for divers to dive into the water and clean the nets directly with their hands. However, this method is dangerous because it can be fatal to divers due to high water pressure and low water temperature. Therefore, this study considered the possibility of using underwater robot to clean fishery nets in place of divers to avoid these dangers. A mobile mechanism that can move freely along the sides of fixed fishery net in the water has already been developed in this laboratory. In this study, we propose the cleaning system using autonomous robots that automatically cleans aquaculture nets underwater and the charging station using coils for wireless power supply. This robot and a charging station will be installed on the aquaculture nets, and the robot will be driven periodically to clean the nets underwater with less labor and in a safe manner.

We aimed to clarify the microhabitat preferences of Atyidae shrimps in the middle to upper reaches of the Hiwasa R. , Kitagawatidani R. , and Mugi R. , Tokushima Prefecture, southern central Japan. Relationships between the shrimp abundance and physical environmental variables were surveyed at six sites in 2011 fall and 2012 spring by setting 5-m line transects delineated with ten consecutive 50-cm quadrats along the riverbank. The dominant shrimp species collected from the riverbanks were Caridina multidentata, Paratya compressa and Neocaridina denticulata. C. multidentata was shown to prefer loose boulder or cobble beds with interstices. When interstices were scarce, the shrimps were found inhabiting among the vegetation. P. compressa was shown to prefer vegetation and boulder or cobble bed in pools. N. denticulata tended to prefer vegetation in darker, gently-flowing areas. Our results insist that the three shrimp species segregate microhabitat under base-flow conditions, but flow reduction may cause microhabitat overlap between C. multidentata and P, compressa.

We investigated the hemolymph oxygen and acid–base status of akoya pearl oysters, Pinctada fucata martensii, exposed to air for a short time (4 h) to elucidate the acid–base balance and CO_2 dynamics. The hemolymph O_2 partial pressure (Po_2) in air-exposed akoya pearl oysters decreased from 88.7 torr (mean value) to 29.4 torr at 1 h, and the low Po_2 continued for the next 3 h during air exposure. The hemolymph pH decreased from 7.586 to 7.082 during air exposure for 1 h and reached 6.851 at 4 h. The hemolymph CO_2 partial pressure increased from 0.9 torr to 4.4 torr at 1 h and reached 7.3 torr after 4 h of air exposure. The hemolymph bicarbonate concentration and calcium ion concentration at 0 h (control) were 1.9 mM/L and 9.0 mM/L, respectively, and these properties did not significantly change during air exposure. From these results, it was determined that the akoya pearl oysters had hypoxemia caused by hypoventilation at an early phase of the short-term air exposure. The akoya pearl oysters inhibited the discharge of CO_2 by hypoventilation, and respiratory acidosis was caused due to the excessive accumulation of CO_2. Bicarbonate was not mobilized from the shell valve into the hemolymph during the short-term air exposure.

An unusual instance of biting behavior by the pufferfish Arothron meleagris, possibly related to spawning behavior was observed during low tide of new moon spring tide while scuba-diving off the Maldives Islands. Multiple males were following, biting and clinging persistently to a female. Subsequent internet searches revealed that similar biting behavior was common among several congeners, possibly being a characteristic spawning-related behavior of the genus.

To improve breeding techniques for aquarium freshwater pufferfishes, hormone injection (hCG, 10 IU per g)-induced maturity and insemination of Auriglobus modestus from Thailand were undertaken, and morphological development of eggs and early stage larvae observed. Artificial insemination was performed two to five days after hormone injection. Although no fertilized eggs resulted from artificial insemination utilizing a wet (freshwater) method, an isotonic (sodium lactate ringer solution) method produced 458 fertilized eggs out of 787 eggs (fertilization rate 61.7%) spawned by one female, although only 8 larvae (1.7%) eventually hatched. Eggs were oval [long axis 2.05 ± 0.06 mm, short axis 1.68 ± 0.06 mm (n = 5)], translucent, demersal, and adhesive, and contained a number of small yellow oil globules. Oval eggs are unusual among pufferfishes, although common in the genus Chonerinos, an indication of the genetic closeness of the latter and Auriglobus, but also suggesting ecological similarity. The eggs hatched after 2 days, larvae one day after hatching [4.54 ± 0.34 mm NL (n = 9)] having a large yolk sac. However, the mouth remained unopened, the optic vesicle uncolored, and the pectoral fin membrane undeveloped, except for small knob-like rudiments. Seven days after hatching, larvae [5.22 ± 0.10 mm NL (n = 3)] had lost the yolk sac and acquired rudimentary soft rays in the pectoral, dorsal and anal fins. All larvae died after eight days. DNA barcoding comparisons (COI gene, 652 bp) of several pufferfish genera indicated a close genetic relationship of Auriglobus and Chonerhinos, being nested in the same clade.