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.     

Molecular identification of interspecific hybrids between Haliotis discus hannai Ino and Haliotis gigantea Gmelin using amplified fragment‐length polymorphism and microsatellite markers

Interspecific hybrids between Haliotis discus hannai Ino and Haliotis gigantea Gmelin were produced in this study. The hybridity of the interspecific hybrids was confirmed by using the methods of amplified fragment‐length polymorphism (AFLP) and microsatellite [simple sequence repeats (SSR)] markers. Five AFLP primer combinations were used to develop the AFLP profiles of H. discus hannai, H. gigantea and their reciprocal hybrids. AFLP analysis revealed that genetic variations of H. discus hannai and H. gigantea were relatively diverse and each species holds species‐specific bands. The AFLP profiles of reciprocal hybrids showed that all of the hybrids inherited bands specific to H. discus hannai and H. gigantea. Of a total of 20 microsatellite loci, which were selected from H. discus hannai microsatellite markers evaluated, eight loci were polymorphic in H. gigantea samples, with an average of 3.375 alleles per locus. Preliminary screening showed that, two of these eight microsatellite loci (Awb002 and Awb022) could be used as species‐specific markers to distinguish the hybrids and their parental species. Simple sequence repeats analysis showed that the reciprocal hybrids inherited one allele from each parent for both of the two SSR loci investigated. These data strongly suggest that the induced interspecific hybrid is a true hybrid between H. discus hannai and H. gigantea.     

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.     

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.     

Parentage assignment in hybrid abalones (Haliotis rufescens × Haliotis discus hannai) based on microsatellite DNA markers

Parentage analysis in aquaculture determines genealogical relationships between broodstock and progeny when the parents are unknown. Thus, parentage analysis is a useful tool to establish pedigree reports in molecular‐assisted selection programs. Here, we evaluated 10 heterologous microsatellite markers for parentage assignment in abalone hybrids produced from 43 abalone broodstocks of red abalone (Haliotis rufescens) and Japanese abalone (H. discus hannai). The allele frequencies, exclusion probabilities and broodstock contributions were calculated using CERVUS, PAPA and GERUD software. The polymorphic information content (PIC) values showed that most of the microsatellite loci were highly informative (>0.7) and more than 90% of parentage assignment was possible with a minimum of 5–6 microsatellite markers. Parentage assignment for hybrid and pure‐red progeny showed a better performance than pure‐Japanese progeny. This result could be due to the high level of allele loss in the parental genotypes. In addition, results indicated that only two sires contributed over 80% and 90% of red and hybrid progenies, respectively. This study gives a new molecular tool to support marker‐assisted selection in abalone hybrids produced in Chile.     

Comparative analysis of the predominant carotenoids and chemical components in the common and orange‐muscle mutant of Haliotis gigantea

The Xishi abalone (Haliotis gigantea) is an economically significant aquaculture species in southern China. We identified a novel orange‐muscle mutation present in less than 2% of the cultured population. High‐performance liquid chromatography and electrospray ionization mass spectrometry identified zeaxanthin and β‐carotene as the two main pigments present in the orange‐muscle abalone. The concentrations of zeaxanthin and β‐carotene detected in the orange‐muscle abalones were significantly higher than those detected in the common abalones (p < 0.01). Notably, the concentration of zeaxanthin was approximately 16.5 times higher in orange‐muscle abalones than in common abalones, which was also the species with highest carotenoid content reported in shellfish to date. Furthermore, the contents of total amino acids and essential amino acids in the orange‐muscle abalone were higher than that in the common abalone, though not significantly, while the content of non‐essential amino acids was significantly higher in the orange‐muscle abalone than that in the common abalone (p < 0.05). The saturated fatty acid content in the orange‐muscle abalones was slightly lower than that in the common abalones, and the unsaturated fatty acid content was higher in the orange‐muscle abalones than that in the common abalones. These results could provide a basis for characterizing the mechanism by which carotenoids accumulate in abalones and further guide the breeding of abalone with orange‐muscle mutants in the future.     

四种鲍45S rDNA在染色体上的比较定位

为了研究皱纹盘鲍、西氏鲍、绿鲍和杂色鲍等4种鲍的核型特征,实验利用荧光原位杂交(fluorescence in situ hybridization, FISH)技术比较定位了上述4种鲍的45S rDNA位点。皱纹盘鲍中约83%的中期细胞均检出2对45S rDNA位点,分别位于13号和16号染色体的长臂端部。西氏鲍中约75%的中期细胞均检出3对45S rDNA位点,分别位于6号染色体短臂端部、14号和17号染色体长臂端部。绿鲍中约85%的中期细胞均检出3对45S rDNA位点,分别位于4号、6号和8号染色体长臂的端部。杂色鲍中约65%的中期细胞均检出3对45S r DNA,位点,分别位于3号、4号和12号染色体短臂的端部。此外,4种鲍均有少数中期相的45S rDNA位点数高于众数,这提示,除了明确的45S rDNA位点外,4种鲍可能均有若干个不稳定的45S rDNA位点。实验结果丰富了鲍细胞遗传学研究资料,同时为鲍的遗传育种研究提供了基础数据。     

皱纹盘鲍4个养殖群体杂交子代生长与存活状况比较

为利用杂种优势培育皱纹盘鲍(Haliotis discus hannai)优良新品种,本研究以4个不同养殖群体[黄岛(HD)、荣成(RC)、日本(JP)、大连(DL)]的皱纹盘鲍为亲本,设计4×4完全双列杂交,建立了4个自交家系和12个正反杂交家系,在利用微卫星标记进行亲子鉴定的基础上,对各家系的F1在1、5、13、17月龄时的生长性状、杂种优势率、生长速度和存活情况进行比较,分析杂交效应。结果表明,在各个生长阶段均有部分杂交家系与自交家系相比表现出显著的生长优势;HDRC、HDDL和JPDL家系的生长速度较高;HDDL、HDJP、RCDL、JPRC及RCHD家系有着较高的存活率;在杂种优势方面,HDRC、HDDL与DLHD家系在各生长参数与生长速度上有明显的杂种优势,HDDL、RCDL、DLHD家系在存活率上表现出明显的杂种优势。本研究筛选出的具有优势的交配组合,可作为皱纹盘鲍生产上种苗来源的参考,也为利用杂种优势培育皱纹盘鲍新品种提供了基础资料。     

Trophic structure in natural habitats of the abalone Haliotis discus hannai with distinct algal vegetation of kelp and crustose coralline algae: implication of ontogenetic niche shifts

The abundance, species composition, and stable isotope ratios of benthic organisms were investigated to determine the trophic structures in abalone (Haliotis discus hannai) habitats, which are characterized by contrasting vegetation of crustose coralline algae (CCA) and kelp beds. A size–frequency analysis revealed that juvenile abalones with shell lengths (SLs) smaller than ~30 mm primarily inhabited CCA beds, whereas adults were abundant in kelp beds. Stable isotope analyses indicated that CCA beds were composed of a single food chain, whereas kelp beds supported multiple food chains. The abalone were divided into three size groups to estimate potential species interactions during their ontogeny. A small gastropod, Homalopoma sangarense, was the most abundant species, but is suspected to be less competitive with abalone, especially in CCA beds. An abundant starfish Asterina pectinifera appeared to function as a potential predator of juvenile abalones in both CCA and kelp beds. We concluded that CCA beds are essential for immediate post-settlement processes of abalones, whereas kelp beds are more important for providing refuge and food sources for adult abalones. The present study highlights that ontogenetic niche shifting can be a successful life-history strategy to sustain the abalone population in a subtidal rocky shore ecosystem.     

Molecular analysis of the intestinal bacterial flora in cage‐cultured adult small abalone,Haliotis diversicolor

To characterize the composition of the intestinal bacterial flora in cage‐cultured adult small abalone, Haliotis diversicolor, we used both culture‐dependent and culture‐independent approaches. In addition, in order to investigate the presence of probable pathogens, the intestinal bacterial community structures of healthy and diseased adult abalones were compared using the denaturing gradient gel electrophoresis (DGGE) fingerprinting technique. Bacteria isolated from the healthy abalone intestine belonged to the genera Vibrio, Flammeovirga, Shewanella, Persicobacter and Paraferrimonas. From the 16S rRNA gene clone library analysis, the intestinal bacterial community comprised two groups: the Proteobacteria and Tenericutes. Betaproteobacteria (18.48%), Deltaproteobacteria (30.43%) and Mollicutes (35.87%) were the most dominant components in the clones and the Mollicutes were mainly Mycoplasma‐affiliated clones. The DGGE band pattern demonstrated that more bands appeared in the healthy abalones (H group) than in the diseased abalones (D group). Although few potential pathogens were detected from the D group, a clustering dendrogram indicated that individuals of the H and D groups (except for D6) formed two separate clusters. An unidentified Deltaproteobacteria species and Mycoplasma sp. were the dominant components in the intestine of both the H and the D group.     

N and P budgets of Haliotis discus hanai,Apostichopus japonicas,and Sebastes schlegeli in a polyculture system

The viability of placing abalones (Haliotis discus hannai), sea cucumbers (Apostichopus japonicas) and rockfish (Sebastes schlegeli) in a polyculture system, the effect of this mixed species group on the system's nitrogen (N) and phosphorous (P) budgets, and the growth and food intake of the organisms in the system were examined using a recirculating aquaculture system. Four replicates were set up for each of three treatment groups (abalone only (C), abalone‐sea cucumber (AS) and abalone‐sea cucumber‐rockfish (ASF)) with an experimental period of 60 d. Compared with the C group, in the AS group the abalone survival rate and specific growth rate (SGR) of body weight increased and the harvested abalones from the polyculture system became the main source of N and P output of the polyculture system. However, the N and P output in the water layer did not differ significantly from that in the C group (p > 0.05), and the N utilization rate was significantly higher than that in the C and ASF groups (p < 0.05). Compared with the AS group, in the ASF treatment the SGR of body weight as well as the protease and amylase activities of sea cucumbers were significantly higher (p < 0.05), the water layer and faeces became the main sources of N and P output in the system. These results showed that the AS polyculture mode significantly improved the N and P utilization rates in the system and led to increased aquaculture production.     

Effect of Hybrid Abalone,Haliotis discus hannai × Haliotis discus discus,Cultivation on the Carbon Cycle: Carbon Source/Sink

Respiration, calcification, and bio‐deposition of hybrid abalone, Haliotis discus hannai × Haliotis discus discus, fed on different foodstuffs have been measured to evaluate the effect of hybrid abalone culture on carbon source/sink in coastal areas. Fed with Laminaria japonica, Undaria pinnatifida, Gracilaria lemaneiformis, U. pinnatifida, and Ulva pertusa, alternated mutually, the carbon bio‐deposition rate of hybrid abalone was 24.29 ± 6.39, 65.40 ± 10.55, 21.48 ± 5.99, and 29.28 ± 6.47 µg/g/h, respectively. Hybrid abalone fed on U. pinnatifida had a higher carbon bio‐deposition rate compared to that fed on other foodstuff (P < 5%). Rate of CO₂ released by respiration of hybrid abalone fed on the experimental foodstuff was 24.53 ± 8.57, 32.73 ± 7.99, 29.31 ± 6.39, and 33.67 ± 12.37 µg/g/h, respectively. Results indicated that calcification presented less relationship with body weight type of the foodstuff. The rate of CO₂ released by calcification into seawater and atmosphere was 2.77 ± 1.89 and 6.53 ± 3.36 µg/g/h, respectively. The total rate of CO₂ released because of bio‐deposition, respiration, and calcification processes was 16.19 ± 4.67 µg/g/h, while the total rate of carbon sequestered in shells and tissues was 8.94 ± 2.07 µg/g/h. The study revealed that hybrid abalone culture is a source of CO₂.     

The potential for utilizing fouling macroalgae as feed for abalone Haliotis discus hannai

In order to assess the potential of utilizing fouling macroalgae as feed for abalone Haliotis discus hannai, the species, biomass and total stock of fouling macroalgae attached on the aquaculture facility‐lines were investigated and assessed at Sungo Bay, China, in August 2007. The nutritional value of four fouling macroalgae (Codium fragile, Sargassum muticum, Ulva pertusa and Laminaria japonica) and one cultured macroalgae (Gracilaria lemaneiformis) as the diet of abalone was assessed by analysis of the chemical composition of the macroalgae and by measuring bioavailability and digestibility to abalone H. discus hannai under laboratory conditions in August 2007. The results showed that the biomass of fouling macroalgae at the two sites was 243.61 ± 26.23 and 1078.15 ± 50.28 g m^?1 respectively. The contents of protein of G. lemaneiformis and C. fragile were 18.30% and 17.60%, respectively, which were significantly higher than the others. The five macroalgae were accepted by the abalone, and growth energies (P) and growth efficiency (%) were positive. However, significant differences were found among the different macroalgae. The growth efficiencies of abalone show a negative relationship with the levels of dietary lipid and a positive relationship with the protein contents. These results suggest that fouling macroalgae have a great potential to be used as diets of abalone.     

杂色鲍与九孔鲍种质资源研究进展

杂色鲍和九孔鲍是我国南方鲍养殖业的主要对象,九孔鲍与杂色鲍的分类关系,学术界一直存在着不同看法。文章综述了我国杂色鲍和九孔鲍在形态学、染色体核型、生化遗传学及分子遗传学方面种质研究的主要进展,得出二者间差异属于种群差异,台湾的九孔鲍和大陆的杂色鲍的种质差异达不到亚种水平的结论。提出了自己的观点,并提出了合理引种及保护原种的建议。     

Heat-shock protein70 gene expression in four hatchery Pacific Abalone Haliotis discus hannai Ino populations using for marker-assisted selection

Heat shock proteins (Hsps) are molecular chaperones that help organisms cope with stressful conditions. Here, we report on the growth rates and Hsp70 expressions in inbred and hybrid populations of abalone Haliotis discus hannai Ino. In abalone, inbred populations expressed more Hsp70 than hybrid populations at all temperatures, except at very high temperatures close to the physiological limit. At benign temperatures, there was a clear trend towards higher Hsp70 expression in inbred than hybrid populations, whereas at higher temperatures, a trend in the opposite direction was observed. The temperature of maximal Hsp70 expression (T_peak) varied with the population type. The T_peak of inbred populations (26°C) was lower than that of the hybrid populations (28°C). The maximal inducible Hsp70 of inbred populations was higher than that of hybrid populations. The results showed a trend towards higher expression in inbred population at a lower temperature. These results provide direct experimental evidence that hybrids can cope with the intrinsic stress even at non‐stressful temperatures. The constitutive Hsp70 may therefore be used for marker‐assisted selection in a breeding programme.     

Time to recover the upright posture in juvenile abalones (Haliotis discus discus Reeve, H. gigantea Gmelin and H. madaka Habe)

The adoption of an upright posture in abalones is essential to enable them to use their foot, and hence to be able to move and seek shelter, as well as to avoid exposure of soft parts and possible predation. In mass restocking programs for abalone, juveniles are released by divers near the seabed, but without control over their posture when they reach the bottom. Thus, the time to recover the upright posture is an important consideration in abalone restocking programs as the quicker they assume this posture the higher the likelihood of survival. This study reports significant differences in the speed of recovering the upright posture between juveniles of the abalones Haliotis discus discus, H. gigantea and H. madaka and between tests conducted under stagnant and flowing water conditions. Longer times were required for recovery in all species in stagnant than flowing water. On average, juveniles of H. discus discus (17.16 and 10.43 s) and H. gigantea (22.54 and 11.89 s) recovered faster than those of H. madaka (161.13 and 49.02 s) under stagnant and flowing water conditions respectively. These results suggest that different species require different levels of care and that water flow or current at the time of release may affect post‐release survival.     

Ecological and eco‐social models for the introduction of the abalone Haliotis discus hannai into benthic systems of north‐central Chile: sustainability assessment

• 1. Loop models of ecological and socio‐economic systems were developed to analyse and predict the impact of a possible accidental introduction (escapes) of the abalone Haliotis discus hannai into a benthic community of north‐central Chile.
• 2. Although the ‘new’ ecological system resulting from a successful invasion of abalone would be locally stable, the establishment of a self‐enhanced dynamic of recruits would transform this into an unstable system.
• 3. The harvest of the kelp, Lessonia trabeculata and other macroalgae is not recommended because this destabilizes the system. The harvest of abalone adults is only sustainable if they do not exert a negative effect upon other native invertebrates.
• 4. The eco‐social model showed three important results: (1) if the variables ‘Price’ and ‘Farming’ are in expansion and stationary dynamics, then the models were found unstable; (2) a self‐enhanced dynamic of abalone recruits tends toward instability; and (3) the harvest of the kelp L. trabeculata and other macroalgae would be non‐sustainable.
• 5. Based on our results, the sustainable development of extensive farming of H. discus hannai in the sea would be not reached. If it is done, an intensive monitoring of the community after introduction into the system is strongly recommended. Likewise, the farming of macroalgal species (source of food for abalone) should be promoted in order to avoid harvesting of macroalgae from natural systems.

Copyright © 2006 John Wiley & Sons, Ltd.     

Determination of genome size of Haliotis discus hannai and H.?diversicolor aquatilis (Haliotidae) and phylogenetic examination of this family

Genome size (C value) is an important index for phylogenetic studies. Haliotidae (abalones) includes many species widely distributed throughout the world??s oceans, which makes this family interesting for phylogenetic studies. To examine Haliotidae phylogeny, we determined the C value and adenine and thymine base pair content (AT?%) of Haliotis discus hannai and H.?diversicolor aquatilis by using flow cytometry. The C values of H.?discus hannai and H.?diversicolor aquatilis were 1.84 and 1.45?pg, with AT?% of 62.3 and 66.3?%, respectively. These data represent the first report of abalones classified as Pacific Northwest (H.?discus hannai) and Indo-Pacific (H.?diversicolor aquatilis) groups, and provides new validation for previous theories related to Haliotidae phylogeny.     

Suitability of cage culture for Pacific abalone Haliotis discus hannai Ino production in China

Pacific abalone (Haliotis discus hannai Ino) aquaculture is a thriving industry in China. This study describes a novel submerged cage culture system for abalone rearing in Fujian, South China. The cage consisted of five vertical slots that were oriented perpendicular to the flow of water. The slots were separated by six vertically connected plastic plates for abalone attachment and shelter at the bottom of the cage. Experiment 1 was designed to determine the appropriate stocking density at the start of the abalone sea‐based production cycle. Eight‐month‐old hatchery reared and size‐graded juveniles were transferred to the sea‐based culture system. For different stocking densities, shell length of juveniles obtained in this novel culture system on 2, 3.5 and 5 months, respectively, was compared with shell lengths obtained in a traditionally multi‐tier basket culture system. In Experiment 2, daily growth rates (DGRs) in shell length and biomass in terms of wet weight of 2‐year‐old abalones reared in cage and tiered basket culture systems were compared over a 6‐month period. Results of Experiment 1 showed that growth of abalone in the cage culture system is density‐dependent; the mean final shell length of juveniles obtained was 6.7–15.9% higher than in tiered baskets system even at the same initial stocking density. In Experiment 2, DGRs in shell length of 53.83–78.38 μm day^?1 obtained in cage system were significantly higher than that in tiered baskets (P < 0.01). And in terms of wet weight biomass, it was 1.48–3.01 times higher in the cage system compared with the traditional system. Abalone survival was more than 87.5% in both culture systems in both experiments. Advantages of the newly established cage culture system included better growth performance of the animals reared and potential improvement of rearing conditions, such as improved water flow velocity and dissolved oxygen.