Genetic certification of presumed hybrids of blue × red abalone (Haliotis fulgens Philippi and H. rufescens Swainson)

Presumed hybrids between Haliotis rufescens and H. fulgens abalones, produced in a commercial abalone laboratory, were evaluated for progeny viability, gonad characteristics and for their ‘true’ status as hybrids by means of allozyme and microsatellite analyses. Four out of 10 evaluated allozyme loci proved to be useful for the genetic certification of hybrids, Gpi, Mdh‐1, Mdh‐2 and Sod, as they had exclusive alleles for each of the abalone reference species from which the presumed hybrids were produced. Three microsatellite loci were also evaluated for their usefulness in abalone hybrid certification. Two were not useful for hybrid certification as the reference abalone species shared some of the same alleles. The third loci (Hka 56) also had shared alleles between the two species, but at very different frequencies. Therefore, this permitted a presumptive certification of hybrids, which, however, was not error‐proof as two allozyme certified hybrids were wrongly classified as H. rufescens rather than as hybrids. Progeny viability of the only female certified to be a hybrid was significantly lower compared with one viability of the other females spawned, which were certified as H. rufescens. However, the number of eggs spawned by the only certified hybrid female was as large as those spawned by females certified as H. rufescens. Gonads of abalones sampled for the macroscopic appearance of sterility indicated that in the absence of any gonad development, no distinctive difference allows for hybrid identification, as there were undifferentiated individuals that were both hybrid and non‐hybrid. On the other hand, two certified hybrid males were characterized by spermatocytes being the most abundant gamete type, although one had some spermatozoa present, indicating only partial sterility. Among the two female hybrids found, vitellogenesis was observed to proceed up to vitellogenic oocytes.     

Cryopreservation of strip spawned sperm using non‐programmable freezing technique in the blue mussel Mytilus galloprovincialis

In this study, a non‐programmable freezing technique has been developed with the strip spawned blue mussel (Mytilus galloprovincialis) sperm. The key parameters optimized including (1) cryoprotectant agents (CPAs); (2) cooling temperature; (3) thawing temperature; (4) sugar and amino acid supplementation and (5) sperm to egg ratio. The fertilization rate and/or integrity of plasma membrane and acrosome were used as sperm quality assessment indicators. The highest post‐thaw sperm fertilization rate of 95% was achieved, when sperm were cryopreservated in 8% dimethyl sulfoxide at 7.8 cm above the liquid nitrogen surface and thawed in a 60°C seawater bath. The addition of glucose, sucrose or trehalose in dimethyl sulfoxide did not, whereas 0.8% glycine did significantly improve the post‐thaw sperm fertilization rates. The fluorescent evaluation has demonstrated that the addition of glycine significantly improved the post‐thaw sperm acrosome integrity, revealing a positive role of glycine in the improvement of post‐thaw sperm quality in blue mussels.     

Thermal preference and tolerance of green abalone Haliotis fulgens (Philippi, 1845) and pink abalone Haliotis corrugata (Gray, 1828)

The thermoregulatory behaviour of green abalone Haliotis fulgens and pink abalone H. corrugata was investigated. Haliotis fulgens juveniles ranging in wet weight from 3.0 to 3.3 g and from 28.7 to 30.5 mm shell length and of H. corrugata 2.0 g and 25.7 mm in shell length were exposed to 19°C for 30 days in a flow‐through water system. Temperature preference was determined in a horizontal thermal gradient and was found to be 25.4°C for green abalone and 25.0°C for pink abalone. Displacement velocity was 4.3 cm h⁻¹ for H. fulgens and 12.8 cm h⁻¹ for H. corrugata. The optimum temperature for growth calculated for both abalone species was 24.6 and 24.5°C respectively. The critical thermal maxima (CTMax) of H. fulgens and H. corrugata were determined as a measure of thermal tolerance. Abalones were subjected to increasing water temperatures at a rate of 1°C on 30 min until they detached from the substrate. The CTMax at 50% were 33.6 and 32.0°C for green and pink abalone respectively. The results are discussed in relation to site selection and commercial rearing.     

The effect of temperature and conditioning interval on the spawning success of wild-caught blacklip (Haliotis rubra, Leach 1814) and greenlip (H. laevigata, Donovan 1808) abalone

Wild‐caught blacklip (Haliotis rubra, Leach 1814) and greenlip (H. laevigata, Donovan 1808) abalone fed a formulated feed were held at 16 or 18°C for different conditioning intervals ranging from 114 to 235 days and induced to spawn using ultraviolet‐irradiated seawater. They were conditioned again for a second identical period before another induction. For H. rubra, mean spawning rate of both sexes was higher in groups held at 18°C than at 16°C, as was the repeat spawning rate. Conversely, animals held at 16°C produced significantly more gametes than those at 18°C. Egg production peaked in groups held at 16°C for ≥165 days. While both mean and total sperm production of H. rubra varied significantly, both figures were always high. Unlike H. rubra, the spawning rate, repeat spawning rate and gamete production of both sexes of H. laevigata were higher when cultured at 16°C than at 18°C. Egg production peaked in groups conditioned at 16°C for ≥212 days. Both mean and total sperm production by H. laevigata were much lower than for H. rubra. This study demonstrates that year‐round hatchery production of seedstock of both species is possible providing broodstock are held under favourable environmental conditions, preferably 16°C.     

Greenlip abalone (Haliotis laevigata Donovan, 1808) sperm cryopreservation using a programmable freezing technique and testing the addition of amino acid and vitamin

This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate; (3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post‐thaw sperm motility was achieved with the sperm being frozen at a cooling rate of ?5°C min^?1 to ?30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post‐thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L‐ascorbic acid, on the other hand, significantly improved the post‐thaw sperm motility. However, only the addition of 0.6% glycine improved the post‐thaw sperm fertilization rate, which was further confirmed by the improvement of the post‐thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.     

Genetic characterization and gonad development of artificially produced interspecific hybrids of the abalones, Haliotis discus discus Reeve, Haliotis gigantea Gmelin and Haliotis madaka Habe

Hybridization among abalone species has been suggested as a possible means to increase their growth rates for aquaculture. As a first step to test the usefulness of the hybrids of Japanese abalone species (Haliotis discus discus, Haliotis gigantea and Haliotis madaka) for aquaculture, we characterized the genetic background and gonad development of hybrids that were produced by artificial insemination. The hybrid status of the resulting offspring was confirmed by assaying 14 allozymes and by RFLP analysis of the 16s rRNA and cytochrome oxidase I (COI) regions of mtDNA using 13 restriction enzymes. Histological examination of the gonads of the hybrids was conducted in comparison with those of the parental species. Cross‐breeding among the three species was conducted successfully in all combinations although with lower fertilization rates (means of 1.3–60.8%) than the parental species (34.3–90%). Crosses between H. discus discus and H. madaka had higher fertilization rates (22.4–60.8%) than those involving H. gigantea (1.3–19.9%). The hybrids were ascertained by the presence of both parental genotypes at the LDH‐A, ME‐A, MDH‐A and GPI loci. The maternal origin of the hybrid mtDNA was confirmed by digestion with DdeI, TaqI, HpaII of the COI region. No polymorphism was observed in the 16S rRNA region. The hybrids had gonadal development and maturity stages similar to the parental species up to fully mature oocytes and sperm. They spawned upon stimulation and produced viable offspring with high fertilization rates and successful development to the juvenile stage in back‐ and homologous hybrid crosses.     

Hybridization between Haliotis rufescens and Haliotis discus hannai: evaluation of fertilization,larval development,growth and thermal tolerance

Two introduced abalone species are currently produced in Chile, red abalone Haliotis rufescens and Japanese abalone Haliotis discus hannai. However, red abalone accounts for 99% of total production, while the Japanese abalone has not adapted well to Chilean coastal waters. This study reports the hatching, growth and thermal tolerance performance in interspecific hybrids produced between red (R) and Japanese (J) abalone. Our results show that egg age and sperm concentration were critical factors to produce hybrids. The cross R♀ × J♂ showed a fertilization rate of 55.3 ± 3.5% using 20‐min‐old eggs and sperm concentrations of 14 × 10⁶ cells mL^?1, while the reciprocal cross (J♀ × R♂) was not successful. Further, larval development stages were similar in RR, JJ and RJ hybrid abalones. Among the experimental trials, settlement rate varied from 12.3% to 18.6% and final survival from 20.1% to 31.7%, being the RJ hybrid rates intermediate between parental species. The final shell lengths were similar between RR and RJ hybrids, but significantly higher in JJ abalones. In addition, thermal tolerance was ascertained due its pivotal role for the abalone physiology. Thus, RJ hybrids showed the highest HSP70 gene expression and offers new possibilities to expand Chilean abalone production in warm waters zones.     

三角帆蚌kinase X基因的克隆及功能初探

三角帆蚌是我国特有的淡水育珠蚌,在养殖业中占有重要地位,在实际养殖过程中,雄性个体有着明显的产珠优势,因此对性别决定的相关研究至关重要。研究发现Sox9基因在许多物种中起到性别决定的作用,kinase X基因是蛋白激酶(PKA)合成中至关重要的基因,而Sox9基因很可能受到PKA激酶的调控。实验通过RACE法克隆kinase X基因的全长cDNA序列,使用荧光定量PCR的方法检测该基因在2龄雌雄三角帆蚌各组织中的相对表达量,利用RNA干扰技术研究干扰链对kinase X基因及下游基因Sox9基因表达的影响。结果显示,kinase X基因全长1 652 bp,编码430个氨基酸;荧光定量PCR结果显示,kinase X基因在雄性性腺中表达量最高,雌雄间差异极显著;RNA干扰结果显示,合成的干扰链均对kinase X基因有着一定的干扰效率,其中干扰链1的干扰率最高,在雌性中的干扰率为83.1%,雄性中为81.9%,同时干扰链1干扰后Sox9基因的表达量在雌性中下降了90.3%,雄性中下降了56.6%,推测这两个基因可能参与性别决定过程。本实验为三角帆蚌性别决定和雄性单性化育种研究提供理论基...     

Use of macroalgae supplemented with probiotics in the Haliotis rufescens (Swainson, 1822) culture in Northern Chile

In this study, we evaluate the use of macroalgae as vectors of probiotics bacteria into the digestive tract of abalone to improve their survival and growth. It is shown that when abalone Haliotis rufescens of different sizes were fed with a natural diet composed of fronds of the macroalga Macrocystis integrifolia supplemented with a mixture of Vibrio sp. C21‐UMA, Agarivorans albus F1‐UMA and Vibrio sp. F15‐UMA bacteria, there was a significant increase (P<0.05) in the average monthly growth rate and survival (%)in a period of 210 days, compared with the control without a probiotic supplement. The permanence of the probiotics in the digestive tract of the animals was monitored, and it was found that the number of culturable C21‐UMA and F1‐UMA bacteria decreased significantly (P<0.05) in recently weaned and adult abalone, and they almost disappeared completely on day 19 of the bioassay. However, the culturable Vibrio sp. F15‐UMA disappeared completely from the digestive tract on day 22 of the bioassay, and these were the bacteria that remained at the highest concentration compared with the other two bacterial strains over the experimental period. It is therefore shown that it is feasible to use a probiotic mixture to improve the profitability of the H. rufescens culture.     

Effects of Broodstock Age and Sperm Collection Time over a Natural Spawning Period on Sperm Cryopreservation in Farmed Greenlip Abalone,Haliotis laevigata

This study investigated the effects of broodstock age (2‐ and 3‐yr‐old), and sperm collection time at the beginning, middle, and end of a natural spawning period, on the ability of sperm to tolerate cryopreservation in farmed greenlip abalone, Haliotis laevigata. The quality of sperm was assessed by motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential (MMP), and acrosome integrity (AI). The sperm collected at the middle of a natural spawning period had significantly higher quality than those collected at the beginning and the end of the spawning period in terms of plasma membrane integrity, MMP, and AI of fresh and post‐thaw sperm, post‐thaw sperm motility, and fertilization rate. No significant difference was found between 2‐ and 3‐yr‐old animals in most quality parameters evaluated. The results suggested that sperm collected during the middle of the spawning season should be used for cryopreservation. The efficiency of genetic improvement programs can be further enhanced by using sperm collected from 2‐yr‐old abalone which has a similar ability to tolerate cryopreservation as 3‐yr‐old counterparts.     

Evaluation of cryodamage in small abalone,Haliotis diversicolor,spermatozoa by using a flow cytometer

We used flow cytometry to determine the quality of small abalone, Haliotis diversicolor, sperm before freezing and after thawing. We investigated the effects of cryopreservation on the characteristics of small abalone semen and determined the motility and fertilization capacity of the pre-freezing and post-thawed sperm. The percentages of motility and fertility were 61 ± 2% and 67 ± 1%, respectively, for the post-thawed sperm and 90 ± 4% and 92 ± 0%, respectively, for the pre-freezing sperm. Sperm cells were stained with specific fluorescent dyes to measure plasma and acrosomal membrane integrity, mitochondrial status, oxidation level, DNA compaction, and viability through flow cytometry. The frozen–thawed sperm exhibited significantly higher mitopotential activity (p < 0.05, damaged mitochondria; 25.01 ± 1.18%) and oxidation value (p < 0.01, free radicals; 63.79 ± 3.93%) compared with the pre-freezing sperm. The oxidation level was the most sensitive signal of the cryopreservation-induced small abalone sperm damage. Flow cytometry is valuable for the objective, accurate, and rapid assessment of pre-freezing and post-thaw small abalone sperm quality.     

Maturation and spawning performance of pond-reared Penaeus merguiensis in different combinations of temperature, light intensity and photoperiod

The combined effects of temperature (23 and 27 °C), light intensity (1100 and 2 lux or ≈ 21.5 and 0.04 μEinst m^?2 s respectively) and photoperiod (10 h light/14 h dark and 14 h light/10 h dark) on ovarian maturation and spawning performance of ablated pond‐reared Penaeus merguiensis were investigated in a 51‐day experiment. The results showed that temperature was the most influential factor, followed by light intensity, whereas the effect of photoperiod was minor. As the maturation process progressed, the effects of temperature and light intensity became stronger while that of photoperiod became less significant. Temperature significantly affected all the reproductive parameters assessed. Prawns in the 27 °C treatments outperformed those in the 23 °C treatments (P < 0.05). The effect of light intensity was found to have a significant effect (P < 0.05) only on the proportion of spawners (prawns that spawned) and spawning rate. More prawns spawned at a higher rate under dim light (2 lux) than under strong light (1100 lux). Photoperiod affected only the proportion of prawns reaching stage III of ovarian development (P < 0.05). There were interactions between temperature and light intensity affecting the proportion of prawns reaching stage III and, although not significantly, the proportion of spawners (P = 0.177), fecundity (P = 0.134) and survival (P = 0.061). Overall, it is recommended that a combination of 27 °C, 2 lux and 10 h light/14 h dark is suitable for the maturation of pond‐reared P. merguiensis. There were indications that temperature can be used to control the rate of ovarian maturation. Also, it is possible to increase light intensity up to 1100 lux in P. merguiensis hatcheries if prawns are ablated.     

Molecular cloning,expression and functional analysis of a predicted HdiQM gene in small abalone,Haliotis diversicolor

The small abalone (Haliotis diversicolor) is a mollusc and was cultured in south of china. Problems such as the decreasing pathogen‐resistance and their mass mortality during the summer. The increased immunity of small abalone populations is a key factor in resolving these problems. Thus, the study of immunity‐related genes in small abalone has become important. In this study, three bacterial species were initially isolated from small abalone carcasses. The regression of infection was analysed, which revealed that the bacteria species could cause rapid morbidity in small abalone. A QM‐like gene (HdiQM) was found and bacterial challenge tests showed that HdiQM gene expression was induced by the bacterial isolates from small abalone carcasses. Therefore, our results implied that HdiQM was found to be an inflammatory stress‐inducible gene associated with pathogen infection, with important functions in small abalone immunity.     

Cryopreservation of sperm in farmed blacklip abalone (Haliotis rubra Leach, 1814)

This study developed a technique of sperm cryopreservation using liquid nitrogen (LN) vapour in farmed blacklip abalone Haliotis rubra through evaluating the following five key factors: (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature; (3) thawing temperature; (4) sperm to egg ratio and (5) sugar addition, using sperm motility or fertilization rate as quality assessment indicators. The results demonstrated that 6% dimethyl sulfoxide (DMSO) was the best single CPA for sperm cryopreservation in this species. The highest post‐thaw sperm motility was achieved when sperm were exposed to LN vapour for 10 min at 5.2 cm above the LN surface and thawed at 60°C and recovered at 16°C in seawater baths. Post‐thaw sperm motility was found to be significantly higher when 6% DMSO was used in combination with 1% or 2% glucose than 6% DMSO alone. Further evaluation of fertilization rate between these CPAs showed that 6% DMSO+2% glucose achieved the highest fertilization rate of 70% at a sperm to egg ratio of 10 000:1.     

Isolation of alginate lyase‐producing bacteria and screening for their potential characteristics as abalone probionts.

This study is aimed at the isolation and screening of alginate lyase‐producing bacteria from the gastrointestinal tracts of hybrid abalone, Haliotis rubra x H. laevigata, as probiotic candidates. Six bacterial isolates were detected to produce alginate lyase. Of these, the isolate with the highest alginate‐lyase activity was identified as Enterobacter ludwigii strain EN‐119, displaying 99% similarity of 16S rDNA sequence. Further assays indicated that E. ludwigii showed good viability and stability when it was incorporated into manufactured pellets and stored at 4°C for 7 days. The isolate also had high tolerance of high salinity (35 mg/L), low pH in simulated stomach juice (5) and to simulated intestinal juice containing surfactants such as bile salts and gastric enzymes (pepsin and trypsin). Additionally, a short, preliminary study indicated that supplementation of E. ludwigii via manufactured pellets improved the total weight gain and specific growth rate of hybrid abalone. These results suggest that E. ludwigii is a potential probiont for shortening the culture period of hybrid abalone.     

Effects of dietary α‐lipoic acid on the growth and antioxidative responses of juvenile abalone Haliotis discus hannai Ino

A feeding experiment was conducted to determine the effects of α‐lipoic acid (LA) on the growth and antioxidative responses of juvenile abalone Haliotis discus hannai Ino. Six purified diets supplemented with 0, 200, 400, 800, 1600 and 3200 mg kg^?1 of LA, respectively, were fed to abalone for 16 weeks in a flow‐through water system. The results showed that the weight gain ratio (WGR) increased with the dietary LA levels, and reached the highest value in the group with 800 mg kg^?1 dietary LA supplement. Glutathione peroxidase activity in the 800 mg kg^?1 dietary LA group was significantly higher than that in the dietary LA‐deficient (0 mg kg^?1) group. Superoxide dismutase activities in the 200, 400 and 800 mg kg^?1 groups were significantly increased. Supplements of 200, 400 and 800 mg kg^?1 dietary LA elevated the total antioxidative capacity significantly. The glutathione level in the hepatopancreas increased significantly with the dietary LA supplements in a dose‐dependent manner (except for the 200 mg kg^?1 group). Catalase and malondialdehyde in the hepatopancreas were not significantly affected by dietary LA. In conclusion, dietary LA promoted the growth and stimulated the antioxidative defence capacity of abalone. Based on the data of WGR, the optimal dietary LA supplement for juvenile abalone was found to be 709 mg kg^?1 using piece‐wise linear analysis.     

A homologue of dermatopontin from Haliotis diversicolor and its response to pathogenic infection

Dermatopontin (DPT), a component of the extracellular matrix, plays important roles in cell‐matrix interactions and matrix assembly. Some studies have revealed that it has more general functions in biological activities. However, its function in molluscs is poorly understood. In this study, a molluscan DPT gene, saDPT2, was cloned from small abalone Haliotis diversicolor. The full‐length cDNA of saDPT2 sequence is 620 bp, with a 531 bp open reading frame encoding a protein of 177 amino acids (aa). Amino acid sequence analysis revealed that saDPT2 shares conserved signature motifs with other DPT proteins, including three repeats of 10‐residue motif (S‐X‐H‐X‐N‐X‐Y‐E‐D‐R), which is similar to mammalian 6‐residue repeating sequence of D‐R‐E/Q‐W‐X‐F/Y. Quantitative real‐time PCR was employed to investigate the tissue distribution of saDPT2 mRNA, its expression at different developmental stages, and in abalone under bacteria challenge. The saDPT2 mRNA could be detected in all examined tissues and developmental stages. Moreover, the saDPT2 mRNA was up‐regulated in haemocytes and gills after bacteria injection. The results indicate that the saDPT2 could respond to pathogenic infection and may play a role in adult abalone immune system.     

Parentage assignment in Haliotis midae L.: a precursor to future genetic enhancement programmes for South African abalone

The establishment and evaluation of family lines using pedigree information provides an advanced understanding of the variability that exists for complex, economically valuable traits and is a necessary step in the execution of an effective breeding programme. The aim of this study was to assign parentage to mass‐spawned Haliotis midae juveniles using species‐specific microsatellite markers. Screening of wild abalone individuals revealed that the nine loci selected complied with the minimum requirements for parentage analyses: a null allele frequency <5% as well as a high number and frequency of alleles per locus. A total of 598 individuals were genotyped (198 breeding individuals and 400 F1 progeny) from two farms, with parentage results yielding 91% and 90% successful assignment for Farms A and B respectively. This study, therefore, provided the necessary pedigree information required for controlled breeding of individual adult abalone and indicated the usefulness of the panel of microsatellite markers selected for parentage assignment.     

Temperature and size range for the transport of juvenile donkey's ear abalone Haliotis asinina Linne

Live transport of hatchery‐produced juvenile donkey's ear abalone Haliotis asinina Linne was examined to evaluate the effect of transportation on the survival of juvenile abalone. Simulated transport experiments were conducted to determine the appropriate temperature using 5, 10 and 20 g L^?1 of ice to air volume for 8 h and the appropriate size using two size groups (Size A, 15–20 mm, 0.5–1.3 g, and Size B, 30–35 mm, 5.3–8.5 g) up to 24‐h out‐of‐water live transport. Survival was significantly higher (P<0.001) when 10 g L^?1 of ice was used to decrease the temperature to the range of 17–23 °C. At this temperature, both size groups subjected to simulated transport for 8 and 10 h had 100% survival after 48 h, while mortality occurred in abalones subjected to 16 and 24 h of simulated transport. The Size B abalone subjected to 24 h of transport had significantly higher survival (64.4 ± 2.9%) (P<0.001) than the Size A abalone (5.5 ± 1.6%) after 48 h. Live juvenile abalone were successfully transported to the field applying the protocols developed in the lab experiment. This study serves as a guide for handling and shipping live juvenile abalone.