Effect of cooling rate and cryoprotectant concentration on intracellular ice formation of small abalone (Haliotis diversicolor) eggs

The intracellular ice formation (IIF) behavior of Haliotis diversicolor (small abalone) eggs is investigated in this study, in relation to controlling the cooling rate and the concentration of dimethyl sulfoxide (DMSO). The IIF phenomena are monitored under a self-developed thermoelectric cooling (TEC) cryomicroscope system which can achieve accurate temperature control without the use of liquid nitrogen. The accuracy of the isothermal and ramp control is within ±0.5 °C. The IIF results indicate that the IIF of small abalone eggs is well suppressed at cooling rates of 1.5, 3, 7 and 12 °C/min with 2.0, 2.5, 3.0 and 4.0 M DMSO in sea water. As 2.0 M DMSO in sea water is the minimum concentration that has sufficient IIF suppression, it is selected as the suspension solution for the cryopreservation of small abalone eggs in order to consider the solution’s toxicity effect. Moreover, IIF characteristics of the cumulative probability of IIF temperature distribution are shown to be well fitted by the Weibull probabilistic distribution. According to our IIF results and the Weibull distribution parameters, we conclude that cooling at 1.5 °C/min from 20 to −50 °C with 2.0 M DMSO in sea water is more feasible than other combinations of cooling rates and DMSO concentrations in our experiments. Applying this protocol and observing the subsequent osmotic activity, 48.8% of small abalone eggs are osmotically active after thawing. In addition, the higher the cooling rate, the less chance of osmotically active eggs. A separate fertility test experiment, with a cryopreservation protocol of 1.5 °C/min cooling rate and 2.0 M DMSO in sea water, achieves a hatching rate of 23.7%. This study is the first to characterize the IIF behavior of small abalone eggs in regard to the cooling rate and the DMSO concentration. The Weibull probabilistic model fitting in this study is an approach that can be applied by other researchers for effective cryopreservation variability estimation and analysis.     

Isolation and characterization of microsatellite DNA markers in the Pacific abalone,Haliotis discus hannai

We developed 17 new microsatellite markers in Haliotis discus hannai. All loci were found to be polymorphic with an average of 13.1 alleles per locus (range 3–28). The mean observed and expected heterozygosities were 0.77 (range 0.17–1.00) and 0.79 (range 0.42–0.96), respectively. Six loci deviated significantly from Hardy–Weinberg proportions, and thus should be used with caution. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for studies of trait mapping, kinship and population genetics.     

Influence of temperature and spawning effort on Haliotis tuberculata mortalities caused by Vibrio harveyi: an example of emerging vibriosis linked to global warming

Since 1998, Haliotis tuberculata mass mortalities have been occurring regularly in wild abalone populations in France during their reproductive period and in conjunction with seawater summer temperature maxima and Vibrio harveyi presence. To confirm the importance of bacterial exposure, temperature and reproductive status on abalone susceptibility, experimental infections via bath exposure were performed using abalone ranging from immature to reproductively mature. Ripe abalone were more susceptible to the bacterium than immature specimens ( P <0.001), and a difference of only 1 °C in temperature had a highly significant impact on the mortalities ( P <0.001). The natural mortalities that were surveyed during summer 2007 confirmed that recent epidemic losses of European abalone appeared in conjunction with host reproductive stress, elevated temperatures and presence of the pathogen V. harveyi . In view of the elevation of the mean summer temperatures observed in Brittany and Normandy over the last 25 years, this temperature-dependent vibriosis represents a new case of emerging disease associated with global warming.     

幼龄皱纹盘鲍唾液腺和消化腺的超微结构与组织化学

以透射电镜观察和组织化学方法研究了45日龄皱纹盘鲍的唾液腺和消化腺。唾液腺由粘液细胞和纤毛细胞组成,粘液细胞含发达的粗面内质网和大量的粘原颗粒,分泌中性和酸性混合粘多糖。消化腺由消化细胞和嗜碱性细胞组成,消化细胞呈现活跃的内吞和细胞内消化,并具蛋白酶和非特异性酯酶活性。嗜碱性细胞含发达的粗面内质网和大量含铁的折光小体,折光小体的电子密度较低。     

皱纹盘鲍消化腺的超微结构

在电镜下研究了大连地区皱纹盘鲍消化腺的超微结构。结果表明,皱纹盘鲍同其它腹足纲软体动物一样,消化腺由消化细胞、排泄细胞和钙细胞三种细胞类型组成。消化细胞呈柱状或锥体形,数量最多,细胞表面具微绒毛,顶端胞质内具顶颗粒,细胞中央区域具有许多“具电子致密核心池”结构,细胞基部胞质内含成簇的脂滴,线粒体球形或不规则,游离核糖体丰富。胞核椭圆形或不规则,位于细胞的基部,染色质多呈块状散布于核内,亦有少量贴核膜分布,核仁一个,十分明显。排泄细胞形状和结构与消化细胞相似,其主要特征是在细胞的在部区域内具有许多含高电子致密物的大液泡。钙细胞呈锥体形,大多分布在消化细胞和排泄细胞的基部,其主要特征是细胞内具有许多呈同心环状的钙粒子。     

The implication of ambient temperature with the outbreak of vibriosis in cultured small abalone Haliotis diversicolor supertexta Lischke

(1) Outbreaks of mass mortality among cultured small abalone Haliotis diversicolor supertexta Lischke with vibriosis occurred in May and September of 1998 in Kao-Hsiung, Taiwan. (2) The effect of different temperature treatments on the susceptibility of small abalone to vibrio and its extracellular products (ECP) was investigated. (3) Two bacterial strains, Vibrio alginolyticus H11 and V. parahaemolyticus B4 originally isolated from the haemolymph of the moribund small abalone at 28 and 32°C, respectively, were used in this study for susceptibility tests. (4) The results reveal that at higher temperatures, the small abalone were more susceptible to vibrio and ECP challenge indicating that the outbreak of vibriosis is associated with warm water conditions.     

Development of microsatellite loci in pinto abalone (Haliotis kamtschatkana)

Twelve novel di‐, tri‐ and tetranucleotide microsatellite loci to the pinto abalone (Haliotis kamtschatkana) are described. Over 400 individuals were analysed at each microsatellite locus. Observed heterozygosities ranged from 0.44 to 0.93, and numbers of alleles from 20 to 63. Six of the loci contained excesses in homozygosity indicative of inbreeding, nonrandom mating, population admixture, or null alleles.     

Cryopreservation of sperm in farmed Australian greenlip abalone Haliotis laevigata

This study investigated factors important to the development of the liquid nitrogen (LN) vapor sperm cryopreservation technique in farmed greenlip abalone Haliotis laevigata, including (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature (height above LN surface); (3) thawing temperature; (4) sperm to egg ratio; and (5) sugar supplementation, using sperm motility, fertilization rate or integrity/potential of sperm components and organelles as quality assessment indicators. Results suggested that among the single CPAs evaluated 6% dimethyl sulfoxide (Me2SO) would be the most suitable for sperm cryopreservation in this species. The highest post-thaw sperm motility was achieved with the sperm that had been exposed to LN vapor for 10 min at 5.2 cm above the LN surface, thawed and recovered in 60 and 18 °C seawater bathes, respectively after at least 2 h storage in LN. The highest fertilization rates were achieved at a sperm to egg ratio of 10,000:1 or 15,000:1. Addition of 1% glucose or 2% sucrose produced significantly higher post-thaw sperm motility than 6% Me2SO alone. Among the three cryoprotectant solutions further trialled, 6% Me2SO + 1% glucose produced the highest fertilization rate of 83.6 ± 3.7%. Evaluation of sperm has shown that the addition of glucose could significantly improve the sperm plasma membrane integrity and mitochondrial membrane potential. These results demonstrated a positive role of glucose in the improvement of sperm cryopreservation in farmed greenlip abalone.     

A review of the world marine gastropod fishery: evolution of catches, management and the Chilean experience

Marine mollusks are among the most importantinvertebrate fisheries in the world. The mainclasses of mollusk fished are Cephalopoda,Bivalvia and Gastropoda. Marine gastropodsrepresent approximately 2% of the mollusksfished in the world. Several species ofgastropods, such as Haliotis spp., Strombus spp., Busycon spp. and Concholepas concholepas, have high economicvalue in international markets and playimportant social roles in small-scale artisanalfisheries. In the past 25 years wild-stockcatches of marine gastropods have increasedfrom 75,000 mt in 1979 to 103,000 mt in 1996.During the same period the countries involvedin gastropod landings rose from 23 to 47.Gastropods are fished mainly in: (1) theAmerican continent, dominated by the extractionof the muricid ``loco', C. concholepas, inChile and Peru; strombid conchs, Strombusspp., in the Caribbean, and abalone, Haliotis spp., in California and the west coastof the Baja California peninsula; (2) Asia andOceania, with the dominate abalone fishery,mainly in Australia and New Zealand, and thehorned turban snail, Turbo truncatus, inJapan and Korea; (3) in Africa and Europe, thedominate species extracted are Haliotismidae, heavily fished in South Africa, and thecommon periwinkle, Littorina littorea,and the whelk Buccinum undatum, which areextracted in Europe. This review summarizes the fishery of abalonespecies in California and the west coast of theBaja California peninsula. We highlightoverfishing situations and the utilization ofadaptive management tools, such as those inoperation in Baja California, where small-scalefisher associations (cooperatives) havereceived exclusive access rights to abaloneextraction within specific fishing zones, since1936. We also review the abalone fishery inAustralia, and the use of IndividualTransferable Quotas (ITQs) and Total AllowableCommercial Catches (TACC), which have been inuse since the mid-1980's. We describe thegastropod fisheries in Chile, mainly C. concholepas, highlighting their economic andsocial importance. We provide information onthe evolution of catches and exports anddiscuss the development of novel managementadaptive tools, such as the implementation ofthe Benthic Regime for Extraction andProcessing (BREP), the introduction ofNon-Transferable Individual Quotas (NTIQs) andterritorial users rights for benthic fisheries,such as the Management and Exploitation Areas(MEAS). Finally, we present and discuss thenecessary steps for the sustainable managementof marine gastropods and other benthic resources.     

Purification and characterization of α-D-galactosidase produced by ADG cell line established from abalon digestive gland

ADG cell line was established from an abalonedigestive gland and previously characterized. ADGcells have the potential to grow in protein-freeculture and secrete l3 types of glycosidases. Inthis article, we determined the origin of ADG cell line,using electron microscopy, and purified a glycosidasesecreted by these cells. The electron microscopicanalysis showed that ADG cell line contains severalnuclei, which suggests that they may be derived fromprotist cells. Moreover, -D-galactosidasethat hydrolyzes p-nitorophenyl galactopyranosidewas purified 130-fold from the spent culture medium ofADG cells. The molecular weight of the enzyme,determined by sodium dodecyl sulfate polyacrylamidegel electrophoresis and gel filtration analysis, wasshown to be 43 and 42 kDa, respectively, and itappeared to consist of a single polypeptide chain. The purified enzyme preparation was practically freefrom other glycosidases secreted from the cells. Catalytic activity was optimal at pH 5.5 and at atemperature of 37 °C. The enzyme was also the most stable at pH 5.5.