Pacific Abalone Research Articles

Experimental hybridization and genetic identification of Pacific abalone Haliotis discus hannai and green abalone H. fulgens

Abstract The temperate-water Pacific abalone, Haliotis discus hannai , has replaced the local subtropical species Haliotis diversicolor , as the main cultured abalone species in southern China in recent years. Although the ability to tolerate high temperature has increased for Pacific abalone, summer mortality still has a serious effect on cultivation success, as the temperature in southern China is significantly higher than the temperature in natural habitats. Hybridization has been demonstrated as an effective way to improve adaptability to particular environmental conditions. In this study, we introduced green abalone, Haliotis fulgens , from North America into China and successfully conducted hybridization trials with Pacific abalone. Fertilization rate for H. discus hannai ♀ × H. fulgens ♂ was at an acceptable level for commercial production. The hybrids exhibited hybrid vigor for growth and survival, as well as a higher temperature tolerance, showing great potential for application in the abalone aquaculture industry in southern China. Moreover, two-step PCR methods using three primers were developed, which can be used for quick genetic identification of green abalone, Pacific abalone and their reciprocal hybrids. Statement of relevance Summer mortality still has a serious effect on cultivation success of abalone farming in China. In this study, green abalone, H. fulgens were introduced and successfully conducted hybridization trials with Pacific abalone. The hybrids exhibited exciting hybrid vigor and adaptation to the higher temperature tolerance, which showed great potentials on application to the abalone aquaculture industry in southern China.

Effects of waterborne nickel on the physiological and immunological parameters of the Pacific abalone Haliotis discus hannai during thermal stress.

In this study, the 96-h LC50 at 22 and 26 °C values was 28.591 and 11.761 mg/L, respectively, for NiCl2 exposure in the abalone. The alteration of physiological and immune–toxicological parameters such as the total hemocyte count (THC), lysozyme, phenoloxidase (PO), and phagocytosis activity was measured in the abalone exposed to nickel (200 and 400 μg/L) under thermal stress for 96 h. In this study, Mg and THC decreased, while Ca, lysozyme, PO, and phagocytosis activity increased in the hemolymph of Pacific abalone exposed to NiCl2 when compared to a control at both 22 and 26 °C. However, these parameters were not affected by a rise in temperature from 22 to 26 °C in non-exposed groups. Our results showed that NiCl2 below 400 μg/L was able to stimulate immune responses in abalone. However, complex stressors, thermal changes, or NiCl2 can modify the immunological response and lead to changes in the physiology of host–pollutant interactions in the abalone.

Development of microparticulate diets with special reference to Pacific bluefin tuna, abalone, and Japanese spiny lobster: a review

Despite considerable progress in understanding the nutritional requirements of marine fish larvae and processing technology of dietary ingredients, microparticulate diets, which can completely replace live food such as rotifers and Artemia, remain to be developed. Recent reviews have generally covered a wide range of aspects of fish larvae nutrition and the formulation of microparticulate diets as well as technical limitations. However, most of them focused on widely cultured finfish species such as seabream and seabass, but not invertebrate species and novel finfish species. This review describes the recent development of microparticulate diets for the larvae and juveniles of three species of aquatic seawater animals: Pacific bluefin tuna, abalone, and Japanese spiny lobster phyllosoma.

Hepatoprotective Activity of H aliotis discus hannai Ino Extract on Lipopolysaccharide-Induced Liver Damage in Rats

The hepatoprotective activity of Haliotis discus hannai Ino extract (HDE) against lipopolysaccharide (LPS)-induced liver damage was investigated in rats. LPS (5 mg/kg body weight) produced hepatic damage that was manifested by significant increases in the activities of marker enzymes (aspartate transaminase, alanine transaminase) and reduced antioxidant (superoxide dismutase, catalase, glutathione peroxidase, malondialdehyde) activities. These antioxidant activities were elevated by HDE administration to LPS-treated rats. These results suggested that HDE extracts had a potential hepatoprotective effect in LPS-induced liver damage without any acute toxicity. In addition, reducing power of the extracts and their ability to scavenge free radicals were evaluated by applying DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt]. This study demonstrates that these extracts have potential hepatoprotective activity which is mainly attributed to the antioxidant potential. Practical Applications Pacific abalone is an important marine food resource in many Asian countries. A number of experimental studies have demonstrated numerous health benefits of Pacific abalone. In South Korea, abalone has important economic significance as a food and as a functional food. In addition, numerous recent research studies have focused on the nutritional and pharmaceutical values of abalone.

Factors Affecting Primary Cultures of Abalone Haliotis discus hannai Ovary-dissociated Cells and General Culture Aspects

We investigated factors affecting primary cultures of Pacific abalone Haliotis discus hannai ovary-dissociated cells to identify general aspects of their early-phase culture. Ninety-seven cell populations derived from 30 individuals were cultured in different media with varying compositions of medium supplements, and initial attachment, subculture, and survival for <TEX>${\geq}10$</TEX> weeks were assessed according to medium composition and individual. We also examined the time required for subculture and the rate of cell death according to both culturing period and passage number within 10 weeks. A lack of fetal bovine serum (FBS) and hemolymph significantly inhibited the growth of cultured cells, while we detected no significant effect of medium composition on initial cell attachment. Through data reallocation, with the omission of data from cell populations cultured in FBS-free and hemolymph-free media, we showed that growth inhibition was also affected by individual differences among the abalones used. During the culture, we observed four different types of cell morphology. Moreover, considerable time was required for subculture-18.4 and 19.5 days for first and second subcultures, respectively-and cell death did not occur within 30 days or for passage 0. Our results will provide valuable information for developing universal cell culturing guidelines in abalone species and suggest the feasibility of culturing abalone ovary-dissociated cells.

Anti-Inflammatory Effects of H aliotis discus hannai Ino on Dextran Sulfate Sodium-Induced Colitis in Mice

In this study, an ethanol extract (HDE1) and a fermented extract (HDE2) of Haliotis discus hannai Ino (Pacific abalone) were administered to mice with dextran sulfate sodium (DSS)-induced colitis. Both extracts were administered at 50 mg/kg and evaluated for their anti-inflammatory effects. Histological evaluations indicated that HDE1 effectively suppressed colonic tissue damage in mice with DSS-induced colitis. In addition, the expression levels of immune-related cytokines and transcription factors such as interferon-γ (IFN-γ), phosphorylated signal transducer and activator of transcription-1 (pSTAT1), interleukin-4 (IL-4), phosphorylated signal transducer and activator of transcription-6 (pSTAT6) and Gata3 were also downregulated by both extracts. These results indicated that both HDE1 and HDE2 suppressed inflammatory cytokines and mediators. However, histological examinations clearly suggested that HDE1 had greater efficacy in attenuating colonic tissue damage by DSS than HDE2. Therefore, HDE1 may be a potential anti-inflammatory agent for the treatment of ulcerative colitis. Practical Applications Pacific abalone is an important marine food source in many Asian countries. The health benefits of Pacific abalone have been demonstrated in a number of studies. This study investigated the anti-inflammatory effects of Pacific abalone extracts in a mouse model of ulcerative colitis. An ethanol extract (HDE1) of Pacific abalone potently suppressed the mucosal tissue damage and crypt loss in the colons of mice exposed to DSS. In addition, inflammatory cytokines and mediators were effectively suppressed by HDE1. Therefore, HDE1 may be a potential therapy for ulcerative colitis.

Anti-Inflammatory Effect of By-Products fromHaliotis discus hannaiin RAW 264.7 Cells

Several reports promoted the potential of shellfish due to their ability to act as antioxidant, anti-inflammatory, and antimicrobial agents. Pacific abalone,Haliotis discus hannaiviscera is, reported to possess bioactivities such as antioxidative stress and anti-inflammatory. In this study, anti-inflammatory potential of mucus-secreting glands from shell-shucking waste ofH. discus hannaiwas evaluated using RAW 264.7 mouse macrophage cell model. Results indicated that presence ofH. discus hannaimucosubstance by-products (AM) significantly lowered the nitric oxide (NO) production along the expressional suppression of inflammatory mediators such as cytokines TNF-α, IL-1β, and IL-6 and enzymes iNOS and COX-2. Also, AM was shown to increase expression of anti-inflammatory response mediator HO-1. Presence of AM also scavenged the free radicalsin vitro. In conclusion, by-products ofH. discus hannaiare suggested to possess notable anti-inflammatory potential which promotes the possibility of utilization as functional food ingredient.

근친교배에 의한 북방전복 (Haliotis discus hannai)의 근교약세 현상

북방전복의 근친교배로 인한 근교약세현상의 유무를 명확히 하기 위하여 근친교배 2가계 (AA 및 BB)와 비근친교배 (ww1 및 ww2) 2가계를 같은날 동시에 생산하여 유생단계부터 동일한 수조에 혼합 사육관리한 후 친자확인 기술을 통하여 4개월째 및 10개월째 각각의 가계별 생존율 및 성장을 비교분석하였다. 성장의 경우, 4개월째 근친교배구 AA가계만이 비근친교배구 ww1 및 ww2가계보다 유의적으로 낮은 값으로 나타났지만, 10개월째는 근친교배구 AA가계 및 BB가계 모두 비근친교배구 ww1 및 ww2가계보다 유의적으로 낮은 값을 나타냈다. 또한 생존율에 있어서도 근친교배구가 비근친교배구에 비하여 낮은 값을 나타내었다. 이것은 한세대의 근친교배(근교계수 : 0.25) 에 있어서도 근교약세현상이 나타난다는 것을 보고하고 있다. Inbreeding depression may be an avoidable phenomenon for abalone culture. However, only a few studies were carried out on inbreeding depression. In the present study, to demonstrate inbreeding depression in growth trait of Pacific abalone, H. discus hannai, inbreeding and outbreeding families were produced in 2010. Inbred and outbred families from each experiment were reared in same tank until 10 month for the same breed environment. The individual of inbred and outbred were distinguished by paternity test using microsatellite DNA. The shell length between inbred and outbred families was compared. At the results, significantly higher shell length was observed in the outbred families at 10 mon (P < 0.05). These results indicate that inbreeding depression is obviously observed in growth traits in the first generation of full-sib family of the H. discus hannai.

HdhCTL1 is a novel C-type lectin of abalone Haliotis discus hannai that agglutinates Gram-negative bacterial pathogens

C-type lectins (CTLs) are Ca2+-dependent carbohydrate recognition proteins, which play important roles in the innate immunity of both vertebrates and invertebrates. In this study, we identified and characterized a C-type lectin (named HdhCTL1) from Pacific abalone, Haliotis discus hannai. HdhCTL1 is composed of 176 amino acid residues and shares low (23.9%) identity with the known CTL of abalone. HdhCTL1 possesses a putative signal peptide and a carbohydrate-recognition domain (CRD) typical of CTLs. The CRD of HdhCTL1 contains four disulfide bond-forming cysteine residues that are highly conserved in CTLs. HdhCTL1 mRNA was detected in a wide range of tissues and expressed abundantly in the digestive gland. Experimental infection with the bacterial pathogen Vibrio anguillarum significantly upregulated HdhCTL1 expression in a time-dependent manner. Recombinant HdhCTL1 (rHdhCTL1) purified from Escherichia coli was able to agglutinate Gram-negative bacterial pathogens. The agglutinating ability of rHdhCTL1 was abolished in the presence of mannose. These results suggest that HdhCTL1 is a novel CTL which is likely to be involved in host defense against bacterial infection.

Dietary ascorbic acid modulates the expression profile of stress protein genes in hepatopancreas of adult Pacific abalone Haliotis discus hannai Ino

This study was conducted to investigate the effects of dietary ascorbic acid (AA) on transcriptional expression patterns of antioxidant proteins, heat shock proteins (HSP) and nuclear factor kappa B (NF-κB) in the hepatopancreas of Pacific abalone Haliotis discus hannai Ino (initial average length: 84.36 ± 0.24 mm) using real-time quantitative PCR assays. L-ascorbyl-2-molyphosphate (LAMP) was added to the basal diet to formulate four experimental diets containing 0.0, 70.3, 829.8 and 4967.5 mg AA equivalent kg−1 diets, respectively. Each diet was fed to triplicate groups of adult abalone in acrylic tanks (200 L) in a flow-through seawater system. Each tank was stocked with 15 abalone. Animals were fed once daily (17:00) to apparent satiation for 24 weeks. The results showed that the dietary AA (70.3 mg kg−1) could significantly up-regulate the expression levels of Cu/Zn superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), feritin (FT) and heat shock protein 26 (HSP26) in the hepatopancreas of abalone in this treatment compared to the controls. However, the expression levels of Mn-SOD, glutathione peroxidase (GPX), thioredoxin peroxidase (TPx), selenium-binding protein (SEBP), HSP70 and HSP90 were significantly down-regulated. Compared with those in the group with 70.3 mg kg−1 dietary AA, the expression levels of CAT, GST and HSP26 were decreased in abalone fed with very high dietary AA (4967.5 mg kg−1). In addition, significant up-regulations of expression levels of Mn-SOD, GPX, TPx, SEBP, FT, HSP70, HSP90 and NF-κB were observed in abalone fed with apparently excessive dietary AA (829.8 and 4967.5 mg kg−1) as compared to those fed 70.3 mg kg−1 dietary AA. These findings showed that dietary AA influenced the expression levels of antioxidant proteins, heat shock proteins and NF-κB in the hepatopancreas of abalone at transcriptional level. Levels of dietary AA that appeared adequate (70.3 mg kg−1) reduced the oxidative stress by influencing gene expression of antioxidant proteins, but excessive dietary AA (829.8 and 4967.5 mg kg−1) induced oxidative stress in Pacific abalone H. discus hannai.

Molecular characterization and gene expression analysis of a metalloprotease from Pacific abalone Haliotis discus hannai

We isolated a metalloprotease (MP) homologue from an abalone muscle cDNA library. A 3284-kb full-length cDNA encoding a predicted polypeptide of 667 amino acids was sequenced. The abalone MP Haliotis discus hannai (HdMP) exhibited a domain structure typical of the peptidase M4 family, a 21-amino acid N-terminal hydrophobic signal sequence followed by a long propeptide sequence of 347 amino acids and the mature protease domain comprising 299 amino acids. The mature region contains features characteristic of a zinc protease, including a zinc-binding motif (HEXXH) and an active site. The protein showed 32–38% amino acid sequence identity with other known MP sequences and with a hypothetical protein from owl limpet. The mRNA transcript is expressed in almost all tissues, with high expression in the mantle and adductor muscle of healthy abalones, and is expressed constitutively during the early developmental stages after fertilization. Lipopolysaccharide or poly I:C stimulation induced the expression of the HdMP transcript in the digestive track and gills of abalones. Collectively, these results suggest that HdMP could play multiple roles in defense, the immune response, growth, and regulation of reproduction.

Amplified fragment length polymorphism analysis to assess crossover interference and homozygosity in gynogenetic diploid Pacific abalone (Haliotis discus hannai)

Recombination analysis in gynogenetic diploids is a powerful tool for assessing the degree of inbreeding, investigating crossover events and understanding chiasma interference during meiosis. To estimate the marker-centromere recombination rate, the inheritance pattern of 654 amplified fragment length polymorphism (AFLP) markers was examined in the 72-h veliger larvae of two meiogynogenetic diploid families in the Pacific abalone (Haliotis discus hannai). The second-division segregation frequency (y) of the AFLP loci ranged from 0.00 to 0.96, with 23.9% of loci showing y-values higher than 0.67, evidencing the existence of interference. The average recombination frequency across the 654 AFLP loci was 0.45, allowing estimation of the fixation index of 0.55, indicating that meiotic gynogenesis could provide an effective means of rapid inbreeding in the Pacific abalone. The AFLP loci have a small proportion (4.4%) of y-values greater than 0.90, suggesting that a relatively low or intermediate degree of chiasma interference occurred in the abalone chromosomes. The information obtained in this study will enhance our understanding of the abalone genome and will be useful for genetic studies in the species.

Complete mitochondrial genome of Pacific abalone (Haliotis discus hannai) from Korea

We have sequenced and characterized the complete mitochondrial DNA of an economically and ecologically important Pacific abalone, Haliotis discus hannai (Haliotidae, Gastropoda). The mitogenome of the Pacific abalone is 16,886 nt total length with a 39.6% G + C composition. Thirty-seven genes were identified including 13 protein-coding, 2 rRNA and 22 tRNA genes. We compared the mitogenome of the Pacific abalone to a putative relative species, H. rubra.

선발 3세대 북방전복의 성장형질에 대한 가계변이 및 유전모수 추정

본 연구에서는 전복의 성장형질에 대한 가계변이를 비교분석하기 위한 목적으로 2011년에 생산된 선발 3세대 북방전복 5,334마리에 대한 18개월령 성장형질인 각장, 각폭 및 중량의 전체 계측자료를 이용하여 유전모수와 육종가를 추정하였으며, 그 중 개체수가 많은 상위 10가계의 865마리 대한 가계변이를 조사하였다. 가계효과를 추정하기 위해 개체모형에 근거한 선형모형을 이용하였고, 유전모수 및 육종가는 생산시기를 고정 효과로 처리하고, EM-REML algorithm을 전산 프로그램화한 REMLF90을 이용하여 최적선형불편예측법에 의해 추정하였다. 본 연구에서 조사된 18개월령 북방전복의 표현형에 있어 각장, 각폭 및 중량의 전체평균은 각각 54.5 mm, 36.8 mm 및 21.3 g로 나타났고, 중량의 변이계수가 51.0%로 나타나 각장의 21.1% 및 각폭의 20.7% 보다 자료의 변동성이 크게 나타났다. 개체수가 많은 상위 10가계를 대상으로 각장과 중량의 관계를 산점도로 표시한 결과 상관관계식이 TW = <TEX>$0.0002SL^{2.8796}$</TEX> (<TEX>$R^2$</TEX> = 0.9864) 과 같이 지수곡선식으로 추정되었다. 가계효과에 있어서는 각 가계별로 유의적인 차이를 보였으며 (p < 0.05), 각장, 각폭 및 중량의 유전력은 각각 0.370, 0.382 및 0.367로 중도의 유전력을 보여 개체선발 보다는 가계선발이 유리할 것으로 사료된다. 또한 각 형질 간 상관계수는 매우 높은 정의 상관관계를 보여 한 형질만의 개량으로 다른 형질의 개량효과를 기대할 수 있을 것으로 판단된다. 추정된 각장 및 중량의 육종가를 토대로 개체수가 많은 상위 10가계에 대한 각 개체별 분포와 순위를 조사하기 위해 표준화육종가로 변환하여 이용하였으며, 각장을 기준으로 상위 5.4%의 개체 수는 152마리, 하위 5.4%의 개체 수는 8마리로 조사되었고, 중량의 경우 상위 5.4%의 개체 수는 164마리, 하위 5.4%의 개체 수는 1마리로 조사되었다. 이와 같이 가계간의 표현형 및 유전적인 다양한 변이를 확인 할 수 있었고, 다음 세대 생산을 위한 모집단의 유전모수와 육종가를 추정하여 선발과 교배에 적절히 이용한다면 보다 나은 육종효과를 기대할 수 있을 것으로 사료된다. The purpose of this paper is to compare and analyze family variations for growth-related traits of Pacific abalone, Haliotis discus hannai. Genetic parameters and breeding values were estimated using all measurement data like shell length, shell width, and total weight as 18-month-old growth traits of 5,334 individuals of selected third generation's Pacific abalone produced in 2011. Family variations of 865 individuals of the upper 10 families with the largest number were inspected. Overall mean in phenotypic traits of 18-month-old Pacific abalone which was investigated in this study showed 54.5 mm of shell length, 36.8 mm of shell width and 21.3 g of total weight respectively. And, variation coefficient of total weight was 51.0%, so variability of data was shown to be higher than 21.1% of shell length and 20.7% of shell width. The family effects showed significant difference by each family (p < 0.05), and heritability of shell length, shell width, and total weight was medium with 0.370, 0.382, and 0.367 respectively. So it is considered that family selection is more advantageous than individual selection. On the basis of breeding values of estimated shell length and total weight, to investigate distribution and ranking by each individual about the upper 10 families with the largest number of individuals, the values were used by being changed into standardized breeding values. Based on shell length, it was investigated that the individual number of the upper 5.4% is 152 and the number of the lower 5.4% is 8. In case of total weight, it was inspected that the individual number of the upper 5.4% is 164 and the number of the lower 5.4% is 1. Like these, phenotypic and genetic diverse variations between families could be checked. By estimating genetic parameters and breeding values of a population for production of the next generation, if they are used properly in selection and mating, it is considered that more breeding effects can be expected.

069 Advances in shellfish germplasm cryopreservation: Current status and future directions

Cryopreservation can be a powerful tool in shellfish aquaculture for selective breeding programmes and hatchery production. Most species that are commercially farmed are broadcast spawners, releasing sperm and eggs into the environment where they fertilize and develop. However, some species are brooders (e.g. flat oysters (Ostrea chilensis) ), which retain and rear embryos until they reach later developmental stages. Sperm cryopreservation has been successfully achieved for several farmed species including the Pacific oyster ( Crassostrea gigas ), greenshell™ mussel ( Perna canaliculus ) and abalone ( Haliotis iris ). In general, dimethyl sulphoxide and trehalose are the preferred cryoprotectants (CPAs) and simple methods to control cooling rate (e.g. floating rack on liquid nitrogen; methanol/dry ice bath) have worked well for our main species of interest (Pacific oyster, greenshell™ mussel and abalone). Interestingly, sperm perform better in CPAs that are prepared in Milli-Q water as opposed to seawater, indicating that a lower salt concentration during freezing is beneficial. Recent studies with flat oysters and geoducks ( Panopea zelandica ) have also resulted in successful sperm cryopreservation. Shellfish oocyte and larvae cryopreservation has proven more challenging, however, successful cryopreservation of Pacific oyster oocytes has been achieved. Pacific oyster oocytes are able to be fertilized and develop to normal spat post-thawing. However, for other species (e.g. greenshell™ mussel) and for larvae, cryo-injuries are not always immediately apparent post-thawing but manifest during development and are often lethal. greenshell™ mussel oocytes are irretrievably damaged when they reach -6 to -8°C and attempts to overcome this sub-zero chilling injury by vitrification have been unsuccessful. Towards understanding these injuries and their effects, we investigated the effect of cryopreservation on the meiotic spindle of greenshell™ mussel oocytes. Results show that the meiotic spindle undergoes major changes during both CPA addition and cooling. However, the spindle is able to re-form normally in most instances given sufficient time post thawing. For a proportion of oocytes, cryopreservation appears to trigger oocyte activation. The effect of cryopreservation on oxidative stress in greenshell™ mussel oocytes has also been investigated. Although, there are changes in oxidative stress parameters as a result of cryopreservation and strong correlations between fertilization, development and oxidative stress parameters (lipid hydroperoxides, superoxide dismutase, protein carbonyls, total glutathione and glutathione state), preliminary attempts to mitigate oxidative stress using antioxidants have so far failed to enable normal development following cooling, suggesting that oxidative stress may be a secondary rather than primary stress event. Research on shellfish larval cryopreservation utilizing SEM and confocal microscopy has also been carried out for greenshell™ mussel. Later larval stages (D-larvae) appear more resilient to cryopreservation than trochophore larvae, but abnormalities are still apparent (e.g.shell abnormalities and delayed organogenesis). Future studies are aimed toward elucidating mechanisms of injury using new tools such as proteomics and then targeting susceptible cellular processes for protection during cryopreservation. Source of funding: This research was funded by the New Zealand Ministry of Business Innovation and Employment (CAWX0802). Conflict of interest: None declared. serean.adams@cawthron.org.nz

Analysis of heat, cold or salinity stress-inducible genes in the Pacific abalone, Haliotis discus hannai, by suppression subtractive hybridization

In order to investigate environmental stress inducible genes in abalone, we analyzed differentially expressed transcripts from a Pacific abalone, Haliotis discus hannai, after exposure to heat-, cold- or hyposalinity-shock by suppression subtractive hybridization (SSH) method. 1,074 unique sequences from SSH libraries were composed to 115 clusters and 986 singletons, the overall redundancy of the library was 16.3%. From the BLAST search, of the 1,316 ESTs, 998 ESTs (75.8%) were identified as known genes, but 318 clones (24.2%) did not match to any previously described genes. From the comparison results of ESTs pattern of three SSH cDNA libraries, the most abundant EST was different in each SSH library: small heat shock protein p26 (sHSP26) in heat-shock, trypsinogen 2 in cold-shock, and actin in hyposalinity SSH cDNA library. Based on sequence similarities, several response-to-stress genes such as heat shock proteins (HSPs) were identified commonly from the abalone SSH libraries. HSP70 gene was induced by environmental stress regardless of temperature-shock or salinity-stress, while the increase of sHSP26 mRNA expression was not detected in cold-shock but in heat-shock condition. These results suggest that the suppression subtractive hybridization method is an efficient way to isolate differentially expressed gene from the invertebrate environmental stress-response transcriptome.

Detrimental effects of reduced seawater pH on the early development of the Pacific abalone

The hatching process of the Pacific abalone Haliotis discus hannai was prolonged at a pH of 7.6 and pH 7.3, and the embryonic developmental success was reduced. The hatching rate at pH 7.3 was significantly (10.8%) lower than that of the control (pH 8.2). The malformation rates at pH 7.9 and pH 8.2 were less than 20% but were 53.8% and 77.3% at pH 7.6 and pH 7.3, respectively. When newly hatched larvae were incubated for 48h at pH 7.3, only 2.7% of the larvae settled, while more than 70% of the larvae completed settlement in the other three pH treatments. However, most 24h old larvae could complete metamorphosis in all four pH treatments. Overall, a 0.3-unit reduction in water pH will produce no negative effect on the early development of the Pacific abalone, but further reduction in pH to the values predicted for seawater by the end of this century will have strong detrimental effects.

북방전복, Haliotis discus hannai 3배체의 생식능력

참전복의 배수체별 생식소 조직 관찰에서 2배체 난소의 경우에는 성숙기를 거쳐 방출기로 들어가고 있는 상태이며, 2배체 정소의 경우에는 방출기의 상태였다, 이에 비해 3배체 전복의 생식소는 발달이 억제되어 난소는 성장기 혹은 성숙기의 상태이고, 정소는 성숙기의 단계에 머물고 있다. 따라서 전복은 완전한 불임이 되는 것은 아니고 어느 정도 성숙이 억제된다고 생각된다. 3배체 참전복의 산란 유발 실험에서 산란 유발에 반응한 개체는 2배체는 암수 전부 반응해서 100%의 산란 유발율을 나타냈고, 3배체는 암컷 50%, 수컷은 25%의 산란 유발율을 나타냈다. 산란된 알과 정자를 배수체별로 조사한 결과, 2배체의 정자는 장경 17.47 <TEX>${\mu}m$</TEX>, 단경 11.40 <TEX>${\mu}m$</TEX> 및 정자의 길이 103.94 <TEX>${\mu}m$</TEX>이었던 데 비해 3배체 정자는 장경 10.86 <TEX>${\mu}m$</TEX>, 단경 8.47 <TEX>${\mu}m$</TEX> 및 정자의 길이 85.06 <TEX>${\mu}m$</TEX>로서 2배체 정자가 3배체 정자보다 장경, 단경, 정자의 길이 모두에서 유의적으로 길었다 (P < 0.05). 알의 직경은 2배체가 199.50 <TEX>${\mu}m$</TEX>, 3배체가 192.50 <TEX>${\mu}m$</TEX>로서 2배체가 컸으나 유의한 차는 없었다 (P > 0.05). 배수체별 교배 실험에서 2배체 <TEX>${\times}$</TEX> 2배체는 정상적으로 수정되어서 발생을 했으나 2배체 알 <TEX>${\times}$</TEX> 3배체 정자와 3배체 알 <TEX>${\times}$</TEX> 2배체 정자의 경우에는 수정이 되지 않았다. The gonadal development of triploid and diploid Pacific abalones, Haliotis discus hannai was histologically investigated in spawning season. Diploid abalones had matured oocytes and spermatozoa, but most triploid had spermatocytes or developing oocytes that was slightly retarded in gonadal development compared to diploid abalones. In spawning experiment of triploid and diploid abalones, spawning rates of diploid male and female were 100%, but those of triploid female was 50% and male was 25% respectively. Investigation of spawned abalone eggs and spermatozoa revealed that length of diploid sperms head were 17.47 <TEX>${\mu}m$</TEX>, breadth of head were 10.31 <TEX>${\mu}m$</TEX>, length of spermatozoa were 130.72 <TEX>${\mu}m$</TEX>, but those of triploid spermatozoa were 11.83 <TEX>${\mu}m$</TEX>, 7.89 <TEX>${\mu}m$</TEX> and 103.36 <TEX>${\mu}m$</TEX> respectively. Triploid spermatozoa were significantly small to diploid spermatozoa (p < 0.05). The eggs of diploid and triploid were not different in size. The cross experiment between oocytes produced by triploids and spermatozoa by diploids (<TEX>$3n{\times}2n$</TEX> cross) revealed that no fertilization were occurred, and <TEX>$2n{\times}3n$</TEX> cross also revealed same result.

Characterization of an α-glucosidase, HdAgl, from the digestive fluid of Haliotis discus hannai

Previously, we isolated two α-amylase isozymes, HdAmy58 and HdAmy82, from the digestive fluid of the Pacific abalone Haliotis discus hannai (Kumagai et al., 2013, Comp. Biochem. Physiol., B 164, 80-88). These enzymes degraded starch producing maltooligosaccharides but not glucose. However, the digestive fluid itself could produce glucose from starch, indicating that the digestive fluid contains α-glucosidase-like enzymes together with the α-amylases. Thus, in the present study, we isolated this α-glucosidase-like enzyme from the digestive fluid and characterized it to some extent. Isolation of this enzyme was carried out by ammonium sulfate fractionation followed by conventional column chromatographies and FPLC. The purified enzyme showed an apparent molecular mass of 97kDa on SDS-PAGE, and optimal temperature and pH of 45°C and 3.8-5.5, respectively. This enzyme could degrade various sizes of maltooligosaccharides into glucose and released glucose from starch producing no appreciable intermediate oligosaccharides. We concluded that this enzyme is an α-glucosidase (EC 3.2.1.20) exolitically acting on polymer substrate and named HdAgl. HdAgl efficiently degraded maltose but hardly degraded p-nitrophenyl α-D-glucopyranoside (α-pNPG) and isomaltose. This enzyme was regarded as a maltase-like α-glucosidase that preferably degrades maltose but scarcely aryl glucosides. When starch was used as a substrate, HdAgl converted approximately 40% (w/w) of the starch to glucose. If an abalone α-amlylase HdAmy58 was added to the reaction mixture, the glucose yield increased to 60% (w/w). These results suggested that both HdAgl and HdAmy58 play important roles for the production of glucose from dietary starch in the digestive fluid. The amino-acid sequence of 887 residues for HdAgl was deduced by the cDNA method. This sequence showed 41-46% amino-acid identities to those of mammalian and avian α-glucosidases belonging to glycoside-hydrolase-family31.

Development of Pacific Abalone (Haliotis Discus HannaiIno) Broodstock: Initial Characterization of Early Growth Traits of F1Progeny

Families of Pacific abalone (Haliotis discus hannai Ino) were initiated from fertilized gametes from broodstock taken from Japan and China. Three wild stocks from Hokkaido (Hkd), Iwate (Iwt), and Miyagi (Myg), Japan, and a local farmed stock (Zzd) at Zhangzidao Island, Dalian, China, were collected and conditioned at Zhangzidao Island during the winter in 2010. In 2011, 95 full-sib families generated by a single-mating method within each stock were tested for growth performance under a hatchery and sea-based culture system for a 1-y period. Progeny from the 4 stocks were reared in separated tanks under identical conditions until 5 mo posthatch, then 4,925 juveniles were tagged and evaluated in a growth trial that consisted of 2 replicate large pools. Growth data of juvenile abalone at the age of 1 y posthatch were collected and used to investigate differences among the stock in daily growth rate. Stock effect and full-sib effect on abalone daily growth rate from tagging to the end of the 1-y culture period were both significant. Variation among individuals within the full-sib families is comparable with the variation among stock. Top-ranked full-sib families based on 1-y growth performance were from varying Pacific abalone stock. The implications of the results is that, when comparing the performance of different H. discus hannai stock or initiating a selective breeding program for H. discus hannai, it is important to test many breeders from each stock. In terms of growth performance, top-ranked candidate F-1 families from varying stock may be crossbred to generate F-2 families if potential positive growth heterosis occurs.