Studies on techniques for reliable production of seed of barfin flounder Verasper moseri
Barfin flounder, Verasper moseri, are large flat fish inhabiting cold sea water basins in the northern Pacific off Japan. This species is suitable for aquaculture and resource enhancement in Hokkaido due to its high commercial value and growth rate at low temperatures. Recently, a developmental program for brood stock management of barfin flounder was proposed by the Hokkaido government; one of the program objectives was to establish a reliable method for seed production of this species. At the Hokkaido Institute of Mariculture, technique for seed production of barfin flounder have been developed since 1990. However, the annual output of seedlings has been highly variable, and a consistant supply of seedlings could not be realized. This was primarily due to the very limited knowledge about reproduction, growth and habits of this species. Therefore, in the present study, the author firstly examined the mechanism of sexual maturation, spawning and larval development in cultured barfin flounder. This knowledge was subsequently applied to determine the most suitable system by which a large amount of seed can be consistently produced.
When barfin flounder were reared in culture from the larval stage, both male and female fish matured normally. However, there was an obvious difference in the timing during which eggs or sperm could be obtained, because testicular development progress faster than ovarian development. Therefore, the effects of water temperature on gonadal development, and the potential to synchronize maturation among sexes by water temperature manipulation were subsequently examined. In experiments conducted in 1997 and 1998, 50 cultured barfin flounder aged 3 years were reared from October to April under two water temperature regimes. As reflented by the gonadosomatic index (GSI) and by histological observation, males reared at ambient seawater temperature reached functional maturation of testes in February. For females in this group, both GSI and oocyte diameter linearly increased from October and first ovulation was observed in April. These results indicated that there was a 2-month difference in the timing of spawning between male and female flounders reared at ambient seawater temperature. However, in males reared at 18oC from October to December, functional maturation did not occur until April, because spermatogenesis was inhibited at 18 oC. While in females vitellogenesis proceeded normally at 18 oC and ovulation commenced in April, which is the same period as females reared at ambient seawater temperature. By maintaining the rearing temperature of barfin flounder at 18 oC from October to December, and then gradually shifting to ambient seawater temperature over one month, it is thus possible to delay the maturation of males and thereby synchronize the spawning periods among sexes.
Obtaining eggs with good quality is very important for seed production of fish, because a deterioration of egg quality results in a decrease in fertilization rate or low vitality of larvae. Therefore, we examined the cause of a deterioration of egg quality in barfin flounder. Female barfin flounder has a regular ovulation rhythm that is 3.5 days on average. However, under conditions of cultivation, spontaneous egg release is limited and deterioration of egg quality due to the retention of the eggs in the ovarian cavity for a prolonged period (over-ripening) is likely to occur. Firstly, in order to clarify the relationship between over-ripening of eggs and a decrease in fertilization rate, three morphos of artificially stripped eggs (transparent eggs, opaque eggs, over-ripening eggs) were incubated for 72 hours at 4 oC. When only incubating transparent eggs that seemed to just have undergone ovulation, the fertilization rates remained high throughout the experimental period. In contrast, when mixing transparent eggs and over-ripening eggs and incubating them together, the fertilization rate sharply plunged and became zero even after 24 hours of incubation. These results indicated that over- ripening caused a rapid decrease in fertilization rate, but it also atributed to the deterioration of newly ovulated eggs by some means or another. Accordingly, to obtain good quality eggs, it is necessary to prevent the ovulated eggs from over-ripening either by periodic egg stripping or by the development of a method inducing spontaneous spawning.
In order to examine influencing larval vitality, the survival activity index (SAI) was calcurated for 33 batches of larvae. Relating SAI with the morphological characters of brood stocks, eggs and larvae, a positive correlation with total length of larvae just after hatching was shown. Accordingly, the hatching larvae which are bigger size are thought to have superior vitality and the size of larvae was suggested to be a suitable indicator for identification of a good batch, one with the potential to survive at a high rate during seed production.
In this thesis, the effect of rearing environment of brood stocks such as water temperature or diets, on larval vitality was also examined, and yielded the following results; 1) rearing of brood stocks at an elevated temperature in winter could accelerate their growth as well as improving the larval vitality. 2) both the content and composition of lipids in the eggs are strongly influenced by those in the diets which the brood stock were fed. 3) it seemed possible to obtain larvae with superior vitality by feeding brood stocks diets containing higher concentrations of highly unsaturated fatty acids.
In general, egg acquisition by induced spontaneous spawning is extremely useful to obtain a sufficient quantity of good quality eggs in an efficient and consistent manner. Although female barfin flounder that started ovulation during spawning season, occasionally released eggs even in the aquarium, spawning behavior between male and female rarely occurred spontaneously. Especially from cultured fish, few fertilized eggs have ever been obtained from spawning in an aquarium. Therefore, the present study examined the potential to induce spontaneous spawning in cultured barfin flounder using increased temperature as a stimulus; that is, increasing the rearing temperature from 6 to 8 to 9°C over 6 h in the morning and reducing it to 6°C the following morning. In 1997 and 1998, cultured barfin flounder, aged 3 years, were reared under two water temperature regimens. Among brood stock groups reared at fixed temperatures (6 or 8-9°C) during the spawning season, all females ovulated normally and released eggs into the aquarium; however, almost all eggs were unfertilized. In contrast, from groups reared with periodic stimulation with increased temperature, the number of eggs collected per day was greater than that from groups without stimulation. Furthermore, fertilization was achieved frequently throughout the experimental term and the number of fertilized eggs obtained was much greater. Estimation of fertilization time indicated that fertilization occurred largely within 24 h after the stimulation was conducted. Moreover, in 2000, the fertilization rate of fish reared with periodic stimulation was very much higher than that in fish without stimulation. These results demonstrate that performing periodic increased temperature stimulation during the spawning season is effective in accelerating egg release in females and also in inducing fertilization of cultured barfin flounder in rearing conditions. Furthermore, the environmental factors inhibiting spawning were experimentally deduced to be either the rearing of brood stock at excessively high densities or a low turnover rate of the rearing water. It thus became possible to obtain sufficient numbers of fertilized eggs from cultured barfin flounder by induced spontaneous spawning as well as by artificial fertilization techniques.
The occurrence of mass mortality in the larval phase was the most serious problem for seed production of barfin flounder. Therefore, a study was conducted to elucidate the cause of mass mortality, and to develop a new method to rear the larvae at high survival rates. Survival statistics and morphological observations of dead larvae during ordinal larval rearing indicated that mass mortality tended to occur when larvae were either 18-20 days-old (preflexion larva, Stage D) or 25- 28 days-old (flexion larva, Stage E). To clarify the cause of the former mortality, the effect of environmental factors on initial feeding of larvae was examined, because morphological observation revealed that almost all larvae that died at age 18-20 days had empty digestive tracts. The feeding activity of larvae during early development was closely related to 1) the density of the unicellular alga Nannnochloropsis oculata in rearing water, 2) light intensity, 3) the density (amount) of diet in the rearing water, 4) water temperature. The density of Nannnochloropsis oculata in the rearing water turned out to be the most important factor affecting feeding. Besides, larval rearing in water supplemented with Nannnochloropsis oculata at higher densities is an effective means not only to accelerate initial feeding, but also to drastically increase survival.Observations on swimming behavior of flexion larvae further showed that most of the larvae suddenly sank from the upper layer to the bottom of the tank at 25 days of age, and that mass mortality occurred just after sinking (flexion-stage mortality). Coinciding with the sinking of larvae was an obvious increase in body density, especially in larvae from 24 to 28 days-old. In order to investigate the effects of turbulence on sudden sinking and mortality, 10 day-old larvae were reared at various aeration levels, i.e., no aeration, gentle aeration and strong aeration. Larvae maintained under no or gentle aeration sank suddenly when 24-25 days-old and high mortality occurred. On the contrary, in the strong aeration groups, almost all larvae swam in the upper or middle layer throughout the experimental period. The survival rates by day 36 were 74.3-89.0%, clearly superior to those in other experimental groups. These results suggest that sudden sinking of flexion larvae is caused by a rapid increase in body density during a period of rapid ontogenetic change. It appears that manipulating aeration to establish vertical mixing is very successful to prevent flexion larvae from sinking, and hence, to avoid mass mortality. The larval rearing system was redesigned on the basis of this knowledge, resulting the prevention of early mass mortality and the production of large numbers of barfin flounder seed with high survival rates.
Among artificially reared barfin flounder, morphologically abnormal fish such as pseudoalbino fish and ambicolored fish (metamorphosis-related morphological abnormality), were frequently observed. Moreover, artificially reared barfin flounder frequently had a male-biased sex ratio. This could possibly cause a landing shortage or price fluctuations on markets, or serious fluctuations of genetic characteristics among wild fish, if these abnormal fish released into the wild. Therefore, the mechanisms responsible for metamorphosis-related abnormalities in morphology and sex determination were examined in a rearing experiment. It was found that 1) the rearing temperature during larval development affect the incidence of morphological abnormality, i.e. larval rearing at suitable temperature, 14 oC, could produce morphologically normal fish at high rates. 2) Normal larval development up to Stage E (just before metamorphosis) is crucial to ensure that larvae can metamorphose into normal morphology. 3) it is necessary to keep the larvae under 14 oC during the temperature-sensitive period (ranging from 10 mm to 35mm in total length) to ensure that gonadal tissue will undergo sex differentiation according to genetic sex.

Limiting factor for growtn of Alexandrium tamarense in the coastal water, northeastern part of Hokkaido, Japan in summer.

Light condition, temperature and nutrients (nitrate and phosphate) in the coastal water, northeastern part of Hokkaido, Japan were investigated to clarify that these conditions were fit for grwoth of Alexandrium tamarense or not from June to August 2003. Although light condition and temperature would be fit for the growth, nitrate and phosphate were relatively low levels during survey. These results suggest that the growth of A. tamarense might be limited by nutrients concentration in this water.

Predictive model for the harvest failure of maricultured scallop, Mizuhopecten yessoensis, in the coastal area, northeastern part of Hokkaido, Japan.

The predictive model for harvest failure of maricultured scallop, Mizuhopecten yessoensis, in the coastal area, northeastern part of Hokkaido, Japan, was constructed by scallop, temperature and chlorophyll a data observed from 1992 to 2004. The appropriate logistic regression model was selected by AIC method, so that the model using spring chlorophyll a data was adapted.

Cryopreservation of flatfish sperm.
II. Artificial hybridization among flatfish using cryopreserved sperm

Scanning electron microscope observations on eggs of Japanese flounder (Paralichthys olivaceus), and cryopreserved spermatozoa in seven species of flatfish, Japanese flounder, Pacific halibut (Hippoglossus stenolepis), barfin flounder (Verasper moseri), brown sole (Pleuronectes herzensteini), crest head flounder (Pleuronectes schrenki), marbled flounder (Pleuronectes yokohamae) and slime flounder (Microstomus achne). One micropyle which average diameter was 5 to 7μm on the surface of egg was opened and spiral structure was observed in micropyle tunnel and many pits were present on the egg surface. The external shape of spermatozoa of seven species of flounder was closely resembled one another. The spermatozoa of those species had almost round-shaped head, a middle piece, and a long tail. Measurements of head length, head width, and tail width of each spermatozoon were done, but there were no significant difference (p>0.01). The present result indicated at least these six species of flounder sperm could run through the micropyle of Japanese flounder egg. Hybridizations among flatfish were attempted by artificial insemination and chromosome manipulation technique using Japanese flounder eggs and cryopreserved flatfish sperm, but embryo forming and hatching rates were extremely low and no hybrid fish was produced.

Cryopreservation of flatfish sperm
III. Long-term preservation of sperm from sex reversed gynogenetically induced clone Japanese flounder (Paralichthys olivaceus)

Hatching rates of Japanese flounder (Paralichthys olivaceus) eggs inseminated by the cryopreserved or fresh sperm from sex reversed clonal Japanese flounder were compared. Hatching rates increased with the number of straw or pellet when using cryopreserved sperm frozen by straw or pellet method. And they were over the values of using fresh semen when four straws or four pellets were applied to the insemination. Abnormal fry rates using cryopreserved semen were not so different compared with them using fresh ones. Hatching rates of eggs inseminated by the cryopreserved sperm which were stored under the three different cryopreserved temperatures, -80℃(deep freezer), -150℃(deep freezer) and -196℃ (LN2) for about one year were examined for comparison. In case of -80℃, hatching rates of eggs inseminated by cryopreserved semen were zero, but when preserved at -150℃, they were almost as same as when stored in LN2.

Feeding effects of enriched rotifers on the survival , growth and starvation resistance of Brown Sole　(Pleuronectes herzensteini ) larvae

Feeding experiments of rotifers enriched with different levels of n-3HUFA (highly unsaturated fatty acid) by two kinds of commercial algae (MARINE GLOS and MARINE ALPHA ) to larval brown sole (Pleuronectes herzensteini ) for the purpose of improving the survival, growth and starvation resistance were conducted using 200L and 5L rearing tanks for 27days.
Suitable concentration of n-3HUFA were estimated more than 433.1～590.1mg/100g on a dry matter basis for the survival ,growth and starved resistance of larvae. Especially feeding of rotifers enriched both DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) was more effective to the survival of larvae than that only enriched with DHA.
Ability of starvation resistance of larvae was apparently raised with feeding rotifers enriched by DHA rather than EPA.

Change of triglyceride concentration in juveniles of Japanease scallop (Mizuhopecten yessoensis) under a short term starvation culture.

Change of triglyceride (TG) concentration in juvenile of Japanease scallops (Mizuhopecten yessoensis) were examined under a short term starvation culture experiment (7 days). The fifth trials were carried out in different periods from August to November, 2004, using the juveniles collected from the cage culture off Yakumo or Oshamanbe in Funka Bay. Shell length and initial TG concentration of the juveniles used for the fifth trials were 3.6 times and 6.7 times differ in maximum among the experiments, respectively. The TG concentrations on one day after the experiments were 70 to 80 % of the values at start points. The TG concentrations on seven days after the experiments were 10 to 20 % of the values at start points. A positive linear regression is admitted between the TG concentrations and daily TG consumption under the starvation cultures. A quickly respons of TG concentration in the scallop juveniles to a short term starvation was confirmed. The regression coleration between TG concentrations and daily TG consumptions means that TG concentration of the scallop juveniles can indicate the metaboric activity. From these results, TG concentration has a future as in situ index of physiological condition of the scallop juveniles.

Growth and maturation of biennial kelp (Laminaria sp.) on the coast of Iwanai, Hokkaido, Japan

This study used SCUBA survey data of 1995-1998 to investigate growth and maturation of biennial kelp (Laminaria sp.) populations off Iwanai. Blade width, blade wet weight, stipe and holdfast weight, and blade substantiality value of second-year sporophytes were greater than those of the first-year sporophytes. In June 1995 mean standing crop of first-year sporophytes had nearly 20 kg wet weight/㎡. Substantiality values of both plants were higher at the shallower stations after July. The sorus formation rates of first-year plant blades were higher on lower surfaces of blades and at shallower stations, whereas regeneration rates of the plants were higher at deeper stations. Blade substantiality values and maturity values of first-year plants showed negative correlation with frond regeneration rates. “Small type”first-year sporophytes were found in 1998. They had lower maturity values and higher regeneration rates than the normal growth type (large type). Biennial kelp populations are distributed widely around Iwanai waters of 3-10 m depth. It is necessary to preserve and nurse these populations and to use the growing varieties.

Outbreak of silicoflagellates in Lake Notoro, in fall 2004（Short Paper）