In recent years, abalone fisheries around the world have shown similar trends, with landings rapidly increasing and then falling as abalone stocks were overfished. A rapid increase in the illegal exploitation of abalone has contributed to population crashes and, sometimes, to the complete decommercialization of abalone fishing industries. Concurrently, however, there has been a rapid increase in abalone farming and, to some extent, farm production has helped to ensure that the overall relationship between supply and demand has not changed too much from what existed during the 1970s. What has changed, however, is the overall availability of abalone on the world market, which has almost doubled from about 20,000 mt during the 1970s to almost 40,000 mt in 2008. Also changed is the species mix of the overall demand, with production and demand in China being a dominant factor but, unfortunately, not necessarily impacting as much as many people expected on overall world demand. The reasons for this will be explored and suggestions will be made on what might be appropriate responses from the abalone farming industry.

During the 2008 season, 318 mt (meat weight) of abalone (Haliotis spp.) were commercially fished in México. The species composition of the catch was H. fulgens (70.8%), H. corrugata (28.9%), and H. cracherodii (0.3%). During the past 5 y, total catches increased 29% as a result of the recovery of natural populations of the 2 main species. In particular, H. corrugata catches increased from 50.4 t in 2004 to 91.7 t in 2008 (up by 82%). Currently, 3 private farms culture abalone (mostly H. rufescens) to market size (approximately >7 cm), and 29.6 t of this product were sold in 2008, mostly to markets in Asia and the United States. One of these companies is also producing abalone pearls and jewelry. A new private farm with restocking and growout objectives has just started operations. In addition, 6 hatcheries of fishermen cooperatives produce larvae and seed for restocking purposes and at least 2 of these have started grow-out trials. During the past 2 y, more than 130 million larvae and 350 thousand seeds (approximately > 1.5 cm) of H. fulgens and H. corrugata have been released in the wild stocks by these cooperative hatcheries.

The black abalone (Haliotis cracherodii Leach, 1814) is a relatively large prosobranch gastropod mollusc ranging from approximately Point Arena in northern California to Bahia Tortugas and Isla Guadalupe, Mexico. In the United States, populations of black abalone on offshore islands, especially those of southern California, were particularly large prior to the mid 1980s. Analysis of long-term fishery-dependent and -independent data revealed that fishing pressure in combination with a lethal disease, withering syndrome, has resulted in mass mortalities of 95% or greater in black abalone populations south of Monterey County, CA. Reduction in local densities below the threshold necessary for successful fertilization (0.34/m²) has been a widespread and pervasive consequence of population reductions by withering syndrome and other factors. The most significant current and future threat that the black abalone faces is that imposed by the spread of withering syndrome, known to be enhanced by periods of ocean warming. Other factors, such as illegal take, ocean pollution, and natural predation, also pose risks to remaining populations and those that may be restored via active management in the future. Without identification, development, and implementation of effective measures to counter the population-level effects of withering syndrome, remaining black abalone populations may experience further declines.

Settlement and metamorphosis of Haliotis tuberculata coccínea larvae were examined in the presence of different settlement cues reported effective in larval settlement of other abalone species (crustose coralline algae, Ulvella lens, conspecific mucus, γ-aminobutyric acid, and 4 benthic diatom species). In addition, larval density effect was tested on crustose coralline algae substrate. Larval settlement was highest on crustose coralline algae followed by U. lens. Settlement was very low on conspecific mucus and γ-aminobutyric acid, varying between 1% and 2%. U. lens inoculated with the diatom Navicula incerta induced a reduced settlement of 9% compared with the 22% obtained on films of U. lens alone. The settlement induction efficiency of all the benthic diatoms tested (Amphora sp., N. incerta, Proschkinia sp. and Nitzschia sp.) was very low and not significantly different among diatom species. Larval settlement at 48 h after addition and survival after 1 mo were higher for the lower larval density, whereas postlarval growth rates were not influenced by larval density.

The effect of stocking density on growth performance of Thai abalone, Haliotis asinina, Linnaeus 1758, was investigated over an 8-mo period (July 2008 to March 2009). A semiclosed recirculating land-based system was used with stocking densities of 368, 735, and 1,100 individuals/m². Each stocking density had 3 replicates. An artificial diet, modified from a shrimp commercial diet, was used throughout this experiment. In July 2008, initial sizes of abalone (mean shell length (SL) ± SE) were not statistically different, with values of 1.28 ± 0.0062, 1.28 ± 0.0131, and 1.30 ± 0.0339 cm for 368, 735, and 1,100 individuals/m², respectively (P > 0.05). After 8 mo, SLs were significantly different, with values of 3.63 ± 0.092, 3.25 ± 0.1173, and 3.05 ± 0.0985 cm for the 3 stocking densities. As the stocking density increased, mean specific growth rates, measured by the percent increase in SL during the 8-mo period, decreased, with values (mean ± SD) of 1.97 ± 0.0495, 1.66 ± 0.1595, and 1.53 ± 0.0544. The mean specific growth rate of the density 368 individuals/m² was significantly different from 735 individuals/m² and 1,100 individuals/m². The variations of specific growth rate, calculated in term of the coefficient of variation (mean ± SD) were increased with increasing stocking densities: 20.16 ± 0.251, 22.17 ± 3.146, and 23.66 ± 0.886. The difference was significant only between the 368 individuals/ m² and the 1,100 individuals/m². The best-fit linear equations between the natural logarithm of specific growth rate and initial size of abalone for 3 stocking densities were estimated. These equations demonstrated that specific growth rates of the larger size abalone decreased at higher rates than that of the smaller size as the stocking density increased. This demonstrated a nonrandom effect of stocking density on abalone size. This also implied that grading of large abalone should be done regularly. Allometric equations between shell width (SW) and SL as well as between total weight (TW) and SL were calculated: SW = 0.6531 SL^0.777 (r² = 0.721) and TW = 0.3276 SL^2.720 (r² = 0.823), respectively. Throughout this 8-mo experiment, survival rates of abalone were not significantly different, with values of 95.1 ± 5.05, 92.9 ± 6.19, and 95.7 ± 0.513 at stocking densities of 368, 735, and 1,100 individuals/m², respectively.

Changes in ocean temperature can have direct and indirect effects on the population dynamics of marine invertebrates. We examined the impacts of warm water, starvation, and disease on reproduction in red abalone (Haliotis rufescens). We found that sperm production was highly sensitive to warm water and starvation, suggesting there may be a dramatic temperature threshold above which sperm production fails. Wild males from northern (72%) and southern (81%) California had sperm. In contrast, only 30% of the males exposed to warm water (18°C) for 6 mo or starvation for 13 mo had sperm, with spermatogenesis dropping dramatically from 300,000 presperm cells/mm³ (wild) to 46,000 presperm cells/mm³ (warm water) and 84,000 presperm cells/mm³ (starvation). In a longer warm-water experiment (12 mo), males had total reproductive failure in temperatures greater than 16°C, irrespective of food treatment. Egg production was less sensitive to warm water, but was impacted more by starvation, especially food quantity relative to quality. Wild females from northern (97%) and southern (100%) California had mature oocytes averaging 3 million eggs and 21 million eggs, respectively. Females exposed to 18°C water for 6 mo had diminished fecundity, averaging only 400,000 mature eggs whereas females in the starvation experiment did not produce any mature eggs. Normal sperm and egg production was found in abalone testing positive for Rickettsiales-like-prokaryote (RLP), the agent of Withering Syndrome in cool water. However, abalone with RLP also exposed to warm water developed the disease withering syndrome and did not produce any mature gametes. The temperature-mediated lethal and sublethal effects on red abalone reproduction described here, combined with temperature's known impacts on abalone growth, kelp abundance, and disease status, clearly demonstrate population-level consequences. We suggest that temperature needs to be explicitly incorporated into red abalone recovery and management planning, because California's ocean has warmed and is predicted to warm in the future.

A major bottleneck in the aquaculture of abalone is feeding during the nursery stage of production, from larval settlement to approximately 10 mm in shell length (SL). Most commercial abalone nurseries settle larvae onto vertical plastic plates coated in a film of algae that acts as a settlement cue and an important postsettlement food source. As abalone grow, their food consumption increases exponentially, and it becomes increasingly difficult to maintain an adequate supply of algal food. Even grazing-resistant algae, like Ulvella lens, are often depleted when abalone reach 3–5 mm SL, requiring nurseries to provide additional algae or to wean the juveniles prematurely onto manufactured diets. Early weaning typically results in slower growth and higher mortality relative to live algal feeds. The current study tested various algal species to aid early weaning of the abalone Haliotis iris. Two experiments were conducted pairing nursery tanks of similar environmental conditions to test a total of three algal treatments: (1) the benthic diatom Nitzschia longissima, (2) a film of N. longissima and U. lens, and (3) a film of N. longissima and a naturally occurring mixture of green algal species that remained on weaning tank surfaces after a previous cohort of abalone had been harvested from the tank. Algae were established prior to the introduction of juvenile (SL, 2–7 mm) H. iris at a density of 15,000–30,000 abalone per tank (2.5 m³ water volume, 13 m² surface area). The abalone were raised to approximately 15 mm SL with twice-weekly supplementary feeding of suspended N. longissima during the first 4 wk, and the addition of manufactured feed 3 times a day for the duration of the experiment. The N. longissima films were completely removed by grazing within 4–5 days whereas the green algal films persisted for 60–120 days. Despite the difference in resilience of algal films, there was no significant difference (P > 0.05) in abalone performance between treatments 1 (98.7% survival, growth of 85 µm/day) and 2 (98.3% survival, growth of 92 µm/day). In a second experiment, treatment 2 provided slightly greater growth than the mixed algal film of treatment 3(101 µm/day vs. 96 µm/day, P = 0.034), but survival rates were similar (98.2% vs. 98.3%, P = 0.86). All 3 algal treatments offer practical and effective means of weaning H. iris onto manufactured feeds at an SL of more than 3 mm. However, both treatments 1 and 3 can be established more rapidly than U. lens and are therefore likely to be more efficient for commercial weaning of H. iris and potentially for other species of abalone.

In this study, effects of sperm concentration and gamete age on fertilization success of Haliotis discus hannai (D) × H. gigantea (G) were investigated. Results showed that the fertilization rates of heterologous crosses H. discus hannai♀ × H. gigantea♂ (DG) and H. gigantea♀ × H. discus hannai♂ (GD) were consistently lower than those of homospecific groups H. discus hannai♀ × H. discus hannai♂ (DD) and H. gigantea♀ × H. gigantea♂ (GG). In sperm concentration experiments, the sperm concentrations that yielded maximum fertilization rates with the least abnormality in subsequent development were 4.66 × 10⁷ sperm/mL for the DG cross and 2.6 × 10⁷ sperm/mL for the reciprocal cross GD. In gamete age experiments, the optimal fertilization rates were achieved in heterologous crosses when freshly spawned ova were fertilized with sperm that were released within 0.5 h. Furthermore, in heterospecific crosses, when ova were fertilized 10 min after being spawned, fertilization rates declined significantly with increasing ova age. It is suggested to use fresh gametes and higher sperm concentrations for hybridization between H. discus hannai and H. gigantea.

Egg-laying hormone (ELH) is a neuropeptide hormone that stimulates ovulation of gastropods, including Aplysia californica and Lymnaea stagnalis. Other neuropeptides, gonadotropin releasing hormones (GnRHs), also play important roles in controlling reproduction in both vertebrates and invertebrates. In the current study, the effects of abalone ELH (aELH) and several GnRHs on somatic growth, sex differentiation, gonad maturation, and spawning of Haliotis asinina were investigated in 3 experiments. In experiment 1, groups of 4-mo-old juveniles (11.8 ± 0.03 mm shell length (SL) and 0.33 ± 0.04 g body weight (BW)) were injected with aELH and GnRHs, including buserelin (mammalian GnRH analogue), octopus GnRH (octGnRH), and tunicate GnRH-I (tGnRH-I), at doses of 20 ng/g BW and 200 ng/g BW. The aELH induced early sex differentiation with a bias toward females, but with normal somatic growth, whereas the different isoforms of GnRH had no effect on sexual differentiation or somatic growth. In experiment 2, groups of 1-y-old-abalone (SL, 4.04 ± 0.02 cm; BW, 20.15 ± 0.25 g) were injected with aELH and the 3 isoforms of GnRH including buserelin, octGnRH, and lamprey GnRH (IGnRH-I) at doses of 500 ng/g BW and 1,000 ng/g BW, and all produced stimulatory effects. For each peptide treatment, the gonads reached full maturation within 5–6 wk and spawning occurred, whereas control groups took 8 wk to reach maturity. In experiment 3, injections of ripe abalone with aELH stimulated spawning of both sexes in a dose-dependent manner. Buserelin had a lesser effect on inducing spawning, and octGnRH had no apparent effect. The gametes released from induced spawnings by aELH and GnRH showed normal fertilization and development of larvae. Altogether, these findings provide further knowledge on manipulating abalone reproduction, which is important in improving abalone aquaculture.

Energy budget parameters consisting of feed intake, food digestibility, absorption efficiency, oxygen consumption, and ammonia excretion were investigated in 3 size classes of Thai abalone Haliotis asinina (2.22 ± 0.13, 3.27 ± 0.02, and 4.16 ± 0.05 cm in shell length). These abalone were fed with artificial diets (approximately 28% crude protein) and held at 28.0 ± 1.0°C and 31.0 ± 1.0 psu salinity. The daily feeding intake of H. asinina decreased with size class, ranging from 0.75% to 0.48% of their dry weight tissue. However, high absorption efficiencies were observed in all 3 size classes, with an average of 81.04%. Further calculations of the obtained energy from the artificial diet for the 3 size classes were 273.6, 666.9, and 1,744.2 J/day, for the 2-, 3-, and 4-cm size classes, respectively. More than 30% of the obtained energy was used for metabolism (ranging from 33.2–42.5%), whereas the energy used for ammonia excretion was low (ranging from 0.8–1.8%). Scope for growth of the abalone varied between size classes (ranging from 37.4–45.8% of the obtained energy). Results from this study suggest the Thai abalone H. asinina fed with this artificial diet and reared in a semiclosed recirculating water system were in optimal culture conditions, because the feed intake resulted in high-energy absorption that allowed a positive energy balance.

To demonstrate inbreeding depression in various traits of Pacific abalone Haliotis discus hannai, 12 inbreeding full-sib families (brother-sister mating) and 13 outbreeding full-sib families were produced by factorial mating using the parents from two full-sib families. All 25 full-sib families were reared for 158 days after settlement, and 8 of the inbred and outbred full-sib families were reared until 3 y 4 mo of age. Inbreeding depression traits between inbred and outbred crosses were compared. No significant differences in fertilization rate, hatchability, and veliger survival rate were observed between the inbreeding and outbreeding crosses. Deformity rate in veliger larvae from the inbred crosses was significantly higher than that among the outbred crosses (t-test, P < 0.01). Significantly lower survival rates were observed in the inbreeding crosses at 109 days after settlement (t-test, P < 0.01) and between 1 y 4 mo-old and 3 y 4 mo-old crosses (t-test, P < 0.05). Mean increases in shell length were not different between the inbred and outbred crosses. Gonad indexes in both crosses were investigated in 3 y 4 mo-old families. The gonad index among inbreeding crosses was significantly lower than that of the outbreeding crosses (analysis of covariance: df = 1, SS = 13.141, F = 25.834, P < 0.01). Significant inbreeding depression in larval deformity rate, juvenile and adult survival rates, and gonad index of Pacific abalone was detected. Although only 2 full-sib families were used as parental broodstock, this is the first long-term rearing examination, from fertilization to adult, of inbreeding depression in Pacific abalone.

Gill and heart tissues from the abalone Haliotis asinina were cultured in vitro using Leibovitz L15 medium supplemented with insulinlike growth factor and basic fibroblast growth factor. Culture conditions at a pH of 7.8–8.0, a temperature of 27–28°C, and 31–32% salinity yielded cell growth expansion from the explanted tissue. Dissociated, floating spherical cells were found along with spindle-shaped fibroblastlike cells, forming a cell monolayer. This study provides basic knowledge in the development of in vitro culture technology of abalone tissues and is the first report on H. asinina cell culture.

Studies on the feeding habits of postlarval abalone in their natural habitats have never been carried out primarily because of the lack of an investigating tool. In this study, carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N) were tested to detect nutritional sources during the early life stages of the abalone Haliotis discus hannai in laboratory experiments to evaluate stable isotope analyses as an appropriate investigative tool for field studies. Fertilized eggs, larvae, and the parent spawner muscle were analyzed to detect a nutritional source during the nonfeeding period. The δ¹³C of fertilized eggs showed slight depletion, about 2‰, compared with that of the parent muscle, and was not detectable after lipid extraction of fertilized eggs. As for the larvae, the isotopic values in postlarvae after settlement and metamorphosis were 2‰ more enriched, become similar to that of the spawner muscle. These results imply that the lipid portion of the eggs is the primary nutritional source during the nonfeeding period of abalone. Laboratory feeding experiments were conducted during which early postlarval abalone (approximately 1 mm in shell length (SL)) were reared for 7 days and supplied with 4 benthic diatoms respectively. The benthic diatom Cylindrotheca closterium produced high growth and survival rates for early postlarval abalone, and there was a significant isotopic shift in their δ¹³C, approaching that of C. closterium. Larger postlarval abalone (SL, >1.3 mm) were fed 1 of 5 benthic diatoms and a gametophyte of a macroalga, Undaria pinnatifida, for 21 days. Isotopic changes approaching the δ¹³C of the provided benthic diatoms were detected from postlarvae fed the benthic diatoms Achnanthes longipes, Cocconeis scutellum, C. closterium, and the gametophyte of U. pinnatifida, which provided high survival rates and growth rates for the postlarvae. From these results, stable isotope ratios of postlarval abalone are suggested to be useful to infer the origin of the assimilated diets. The current study highlights that stable isotope ratios are a promising tool to investigate the diet composition of postlarval abalone in their natural habitat as well as in abalone hatcheries.

Haliotis australis is a medium-size New Zealand abalone with aquaculture potential. H. australis can be readily ripened and spawned, but larval settlement has proved difficult. The current study addressed aspects of larval settlement (i.e., attachment and metamorphosis) in laboratory experiments and small-scale hatchery trials. Competence to metamorphose was first seen weakly in larvae at 8 days old at 14.5°C, but larval metamorphosis rates increased progressively up to at least 12 days of age. In laboratory experiments, larvae showed a dose-dependent response to dissolved γ-aminobutyric acid (GABA), with settlement peaking at 1 µM (57% metamorphosis 4 days after settlement induction) and declining at higher and lower doses. Across a range of cues, metamorphosis was highest on crustose coralline algae (97 ± 4% metamorphosis after 4 days), intermediate on 1 µM GABA (55 ± 14%), and low on diatom films (<20%). GABA combined additively with diatom film to give strong metamorphosis (81 ± 6%). Metamorphosis induction by a diatom film was doubled if the film was pregrazed by juvenile H. australis (76% vs. 35%, P < 0.0001), but the grazing effect appeared to be species specific, because pregrazing by Haliotis iris had no effect (41% vs. 35%, P = 0.720). The species-specific response to trail mucus appears to preclude GABA as being the main inducer of metamorphosis in this case. On hatchery plates, settlement on pregrazed biofilms was approximately 4-fold higher when the plates were horizontal rather than vertical (41 % vs. 10% metamorphosis, P = 0.003). Settlement on ungrazed horizontal plates remained low even if those plates had been held in water with adult conspecifics, but without contacting the abalone. We conclude that tanks offering sloped or horizontal surfaces will be more effective than vertical plates for settlement of H. australis, and that pregrazed biofilms and/or GABA are promising settlement cues.

Abalone larvae settle (attach and metamorphose) in response to crustose coralline algae (CCA), but it is not known whether the settlement cues arise from the CCA or from microbes on their surface. The CCA Phymatolithon repandum induced close to 100% metamorphosis of Haliotis iris larvae. Pebbles and shells with a biofilm but without visible macroalgae induced, on average, 58% metamorphosis on H. iris larvae within 2 days, and biofilms are commonly used to cue settlement in H. iris hatcheries. This suggests that settlement on P. repandum might be triggered by the surface biofilm rather than the CCA itself. Treatment of P. repandum with antibiotics, germanium dioxide, and scrubbing did not significantly reduce settlement of H. iris larvae. Diatom strains isolated from P. repandum induced little attachment or metamorphosis at the low diatom densities (10³–10⁴ cells/cm²) found on P. repandum, but induced moderate attachment (30–85% after 4 days) and metamorphosis (2–40%) at high densities (5 × 10⁴-1 × 10⁶ cells/cm²). Nine bacterial strains isolated from P. repandum induced low-percent attachment (0–45% after 7 days) and metamorphosis (0–20%) if assayed in the presence of antibiotics. Parallel assays without antibiotics had higher attachment (40–80% after 7 days) and metamorphosis (5–50%), but this activity may have arisen from bacteria introduced with the larvae. Although bacteria and diatoms did induce some attachment and metamorphosis of H. iris larvae, they were less effective than P. repandum (95% within 2 days). Treatments that greatly reduced the biofilm on P. repandum had no effect on its settlement-inducing activity. This evidence supports the view of CCA as an especially potent source of inducers for temperate abalone larval settlement.

The efficacy of clove oil as an anesthetic was evaluated in adult Haliotis tuberculata coccinea (shell length, 42–80 mm) and was compared with the commonly used 2-phemoxyethanol. The clove oil was previously mixed at 50% with absolute ethanol, and 4 different concentrations were tested: 0.1, 0.3, 0.5, and 0.7 mL/L. The trial was replicated 3 times. Relaxation and recovery time, muscle condition, and mortality after 1 wk were considered to evaluate the efficiency of anesthetics. The results showed that clove oil at 0.5 mL/L was the lowest concentration that removed all abalone from the rearing shelters in 8.9 ± 3.5 min, showing 2 ± 1.5 min of recovery time. Although clove oil is appropriate for handling animals, it was not suitable for pearl culture because it left the abalone muscle hard, making access to the pearl nuclei insertion site difficult. In comparison with 2-phenoxyethanol, clove oil was effective in 10-fold lower doses.

A total of 241 samples of small abalone (Haliotis diversicolor) from 8 full-sib families of market size were used in this experiment. Shell length (X₁), shell width (X₂), shell height (X₃), apex height (X₄), body weight (Y₁), muscle weight (Y₂), and shell weight (Y₃) were measured, and the correlation coefficient matrix was calculated. The shell shape traits were used as independent variables, then body weight and muscle weight were used as dependent variable for path analysis. Path coefficients, determination coefficients, and correlation index were calculated. The results showed that correlation coefficients between each shell shape trait and body weight, muscle weight, and shell weight were all significant (P < 0.01). For the 4 shell morphological traits, body weight (Y₁) had the highest correlation coefficient with shell length (X₁), and muscle weight (Y₂) had the highest correlation coefficient with shell width (X₂). The results of high correlation index would be useful for selecting important growth-related traits in genetic breeding program of small abalone.

Spawned eggs of a tropical abalone Haliotis asinina have 2 protective barriers: the egg jelly coat and the vitelline envelope. At the electron microscopic level, the egg jelly is composed of a network of large fibers (40–50 nm thick) cross-linked by smaller fibers (15–20 nm thick), whereas the vitelline envelope is a thin, tough sheet containing pores that might be channels for sperm contact and entry. Electrophoretically, the egg jelly contains 2 major glycoproteins at 107 kDa and 178 kDa, whereas the vitelline envelope contains a broad spectrum of protein bands ranging from 15–200 kDa, which also includes the corresponding egg jelly protein bands. Glycoproteins of egg jelly and vitelline envelope exhibit strong cross-reactivities, and they appear in late oocytes (Oc4, Oc5). Glucose is the major sugar composition of both egg jelly and vitelline envelope glycoproteins, whereas minor proportions of arabinose, fructose, galactose, and fucose are present in both the egg jelly and vitelline envelope. Our findings suggest that a sperm acrosome reaction could be induced by isolated vitelline envelope glycoproteins, whereas acceleration of sperm motility could be stimulated by egg jelly glycoproteins.

Pheromones are chemicals used to communicate between animals of the same species, and are thought to be used by most marine animals. With limited vision, abalone primarily sense their world chemically, and pheromones may play an important role in settlement, attraction, recognition, alarm, and reproduction. Despite this, there has been no detailed investigation into pheromone substances, both in their precise biochemical nature or pheromonal function. In this study, we investigated the presence of pheromonelike substances from the hypobranchial gland of the abalone Haliotis asinina using bioassays, immunohistochemistry, Western blotting, and reverse-phase high-performance liquid chromatography (RP-HPLC). The hypobranchial gland of many prosobranchial marine molluscs has been classified as a sex auxiliary gland releasing unknown substances during spawning. In our study, cephalic tentacle assays demonstrated that the cell extracts of the hypobranchial gland contain chemical cues that are sensed by conspecifics. An antibody against the sea slug “attractin” pheromone was used as a probe to localize a similar protein in the mucin-secreting cells of the epithelial lining the hypobranchial gland of both male and female abalone. The approximate molecular weight of this abalone attractin-like protein is 30 kDa in both males and females. Fractionation of hypobranchial gland extracts by C5 RP-HPLC could not selectively purify this protein, and no sex-specific differences were observed. We predict that the attractin-like protein could be one of a number of important proteins involved in maturation, aggregation, and/or spawning behavior of abalone. In future research, additional hypobranchial gland components will be tested further for these types of behavior.

Twelve half-sib groups and 36 full-sib groups of small abalone Haliotis diversicolor were obtained by the unbalanced nest design using artificial fertilization of 3 females by each male. Heritabilities of growth-related traits were estimated from postlarva to market size at days 10, 40, 120, 220, 320, and 420. The estimated heritabilities based on sire components for shell length and shell width were 0.15–0.37 and 0.18–0.42, respectively. Heritability estimate for shell length and shell width based on dam component were larger than those based on sire component. The results in the current study indicate that genetic improvement through selective breeding conducted on small abalone might be available and would be an efficient method to obtain a positive response to selection.

To evaluate the current genetic status of Chilean abalone hatchery populations, we tested 25 heterologous microsatellite loci in Haliotis rufescens Swainson 1822. Successful cross-amplification was obtained with 13 microsatellite loci, and 7 were used to estimate genetic variability in 4 separate hatchery populations: 2 located in Caldera, 1 in Coquimbo (northern Chile), and one more on Chiloe Island (southern Chile). The number of alleles per locus (N_A) ranged from 2–16, with an average of 8.86. Among populations, mean H_o and H_e values were 0.510 and 0.746, respectively, with significant deviations from Hardy-Weinberg equilibrium at 23–28 of the population loci analyzed (82.1%). The F_IS values calculated showed no significant differences among the different hatcheries analyzed. However, F_IS values from the Caldera populations were close to panmixia values, whereas Coquimbo and Chiloe populations showed a slight tendency to endogamy. According to the F_ST and R_ST values, there are genetic differences among the populations, with the 3 northern hatcheries from Caldera and Coquimbo more related than the abalone population of southern Chile. In addition, comparison between California wild and Chilean hatchery abalone populations showed a loss of genetic diversity evidenced by microsatellite markers. The current study contributes to the genetic knowledge of the Chilean red abalone aquaculture resource and reveals the necessity to perform genetic monitoring in hatchery populations.

The Chilean abalone aquaculture industry is only supported with two species introduced during the late 1970s: red abalone Haliotis rufescens and Japanese abalone H. discus hannai. At the moment, red abalone accounts for 97% of total production due to its adaptability for full-cycle culture and faster growing than the Japanese species. However, Japanese abalone has a better acceptance and higher prices in Asian markets. These dualities have been merged by the successful hybridization between red and Japanese abalone. The goal of this study was to carry out a genetic analysis on red and Japanese abalone populations and their interspecific hybrids. Microsatellite markers were applied in three hatchery populations (HRed, HJap, and HHyb) to assess the genetic diversity and to certificate hybrid status. Allelic diversity was similar between HRed and HHyb populations (12 alleles), whereas for HJap it was significantly lower (7.8 alleles). Mean observed and expected heterozygosity (H_o, H_e) were 0.533 (0.045) and 0.786 (0.031), with no significant differences among populations (P ≤ 0.05). In most cases, Hovalues were lower than H_e, indicating significant deviations from Hardy-Weinberg equilibrium. Comparison among populations showed that the hybrids are comparatively more similar to H. rufescens than H. discus hannai. Furthermore, hybrid status was confirmed by the presence of species-specific bands for each parental species of microsatellite locus Hco97. This work is the first approach to characterize genetically hybrids of H. rufescens × H. discus hannai produced in Chile.

Interspecific hybrid families of female Haliotis diversicolor × male H. discus discus were produced and analyzed using amplified fragment length polymorphism (AFLP) technology to reveal the genetic makeup of F1 progenies. The survival rates of the hybrid F1 were very low, ranging from 0–0.13%. Twenty hybrid F1 from 3 families along with 3 different female parents and their common male parent were analyzed with 3 AFLP primer combinations. In total, 266 markers were detected. Genetic relationships among the progenies and the parents were evaluated by generating a similarity and genetic distance matrix. The genetic divergence between Haliotis diversicolor and Haliotis discus was at a high level, with genetic distance ranging from 1.471–1.492. The AFLP band patterns of hybrid F1 progeny were similar to those of the female parents, but were quite different from that of the male parent. The mean genetic distance between hybrid F1 and their female parents were 0.024–0.039, slightly less than that among the female parents, which indicates that the hybrid F1 shared high genetic similarity with their female parents, Haliotis diversicolor. However, 0–0.8% of total AFLP bands of each individual were found to be parental bands, and 0–3.3% were found to be nonparental bands. The possible reason for the presence of paternal-specific and nonparental bands is discussed.

Amplified fragment length polymorphism (AFLP) analysis of the population genetic structure and genetic diversity of Haliotis discus hannai (D), Haliotis gigantea (G), and their reciprocal hybrids D ♀ × G ♂ (DG) and G ♀ × D ♂ (GD) was carried out in this study. A total of 479 unambiguous and highly repeatable AFLP markers, 311 of which (64.93%) were polymorphic, were obtained using 7 primer combinations. The reciprocal hybrids inherited bands from both parents, indicating that the hybrids were truly heterogeneous. The Shannon diversity index for D, G, and their reciprocal hybrid populations DG and GD was 0.169 ± 0.188, 0.211 ± 0.227, 0.236 ± 0.267, and 0.231 ± 0.242, respectively. Analysis of molecular variance revealed that 29.58% of the variance was among populations, whereas 71.42% of variance was within populations. Genetic distance was maximum (0.681) between D and G, and was minimum (0.482) between GD and G. The 4 populations were clustered into 2 major clades using the unweighted pair group method with arithmetic mean. All genetic parameters indicated that there was plentiful genetic diversity in the reciprocal hybrids of D × G. Results of this study suggest that these AFLP markers can be used in the future to enhance current breeding practices in abalone culture because of the large numbers of polymorphic markers.

To determine the genomic composition of the interspecific hybrid between Haliotis diversicolor supertexta ♀ and H. discus discus ♂ at an early developmental stage, veliger larvae produced from hybrid (SJ-5 and SJ-50) and pure species crosses (SS and JJ) were sampled and analyzed using standard karyological methods and genomic in situ hybridization. In hybrid metaphase spreads, chromosomes from both parents were detected, except one metaphase, which showed the H. diversicolor supertexta haploid karyotype. The genomic composition of the hybrid was also confirmed through preliminary genomic in situ hybridization results. Many more aneuploids and chromosome fragments were found in the hybrids than those in the control pure species crosses, indicating genome instability and chromosome loss in the hybrids. In the hybrid hypodiploid metaphase spreads, two intact sets of H. diversicolor supertexta chromosomes and several H. discus discus chromosomes were detected by pairing. Spontaneous diploidization of the maternal chromosome set was shown to occur in hybrid larvae, as 2.2% heterogeneous triploid and 17.9% hypodiploids with two intact H. diversicolor supertexta chromosome sets for SJ-5. The current findings suggest that uniparental chromosome elimination along with spontaneous diploidization of maternal chromosome sets may be the reason for allogynogenesis production in H. diversicolor supertexta × H. discus discus hybridization.

There is currently a paucity of specific genetic data regarding growth and reproduction-related processes in abalone, marine vetigastropods of commercial value. However, strong inferences about these processes can be drawn from other molluscs. For example, ganglia from the gastropods Aplysia and Lymnaea are known to produce neuropeptides related to growth, feeding behavior, and reproduction. Here, we use suppression subtractive hybridization to identify expressed genes that may be linked to the control of growth and feeding in the tropical abalone Haliotis asinina. Two pools of suppression subtractive hybridization clones were obtained from messenger RNA derived from H. asinina cerebral and pleuropedal ganglia, corresponding to genes differentially expressed in (1) well-nourished animals relative to food-deprived ones and (2) well-nourished animals relative to reproductively active ones. From these subtractions, respectively, 204 and 214 unigenes were identified in 222 and 231 sequenced clones, with 18 of these unigenes common to both subtractions. A subset of the putative differentially expressed genes was confirmed by quantitative polymerase chain reaction, validating this approach. The transcripts that are differentially expressed in the ganglia of growing H. asinina fall into a wide range of functional categories, including biological regulation, cell proliferation, and metabolic process, and include genes encoding Phe-Met-Arg-Phe-NH₂ (or, FMRF-amide), myomodulin, CDC123, RAB37, and dermatopontin.

APGW-amide is a well-known neurohormone modulator in several molluscs, and is involved in motor activities, feeding, and sexual behavior. In this report we show that injections of APGW-amide into 4-mo-old juvenile Haliotis asinina stimulate growth of body weight and, to a lesser degree, shell length. The injections were given at 0 (control), 20, and 200 ng/g body weight (BW), at 1-wk intervals for 14 wk. BW and shell length (SL) were measured every week, and growth rates were calculated. When compared with control animals, there was an approximate 2-fold increase in body growth rates of animals given 20 ng/g BW and 200 ng/g BW APGW-amide (P ≤ 0.05), whereas only 20 ng/g BW APGW-amide produced significantly greater SL than controls (P ≤ 0.05), with an approximate 1.2-fold increase. Using an immunoperoxidase technique, we showed the presence of APGW-amide in neuronal cells of the cerebral ganglia and nerve fibers. Overall, these data indicate that APGW-amide is an important neurohormone/neuromodulator in the nervous system of H. asinina and plays a role in controlling the body growth of H. asinina.

To obtain the highest yield during in vitro fertilization of tropical abalone Haliotis asinina, optimal proportion of the gametes, the timing of sperm-egg interaction, and subsequent development were investigated. The highest yield of fertilization (75%) with fewest abnormal eggs was obtained when incubating eggs and sperm at the ratio of 1:100 in seawater with a salinity of 27.5 ppt, a pH of 7.8, and a temperature range from 27–29°C. After incubation, sperm swim through the eggjelly coat and become bound to the vitelline envelope within 30 sec, followed by an acrosomal reaction at 1 min. The fertilized egg extrudes the first and second polar bodies at 8–10 min, and then the zygote begins cleavage at 15–20 min. This is followed by the second cleavage, and development through the stages of blastula, gastrula, trochophore, veliger, and early creeping larvae, which were completed within 3 days. Noticeably, occurrence of egg jelly condensation after penetration of the first sperm would not allow other sperm bind to the egg jelly and to penetrate through its vitelline envelope. This event is thought to be a weak blocking against polyspermy, because the classic cortical reaction initiated by cortical granule exocytosis could not be observed in this species.

Expressed sequence tag analysis of normal and subtractive complementary DNA (cDNA) libraries of Thai abalone (Haliotis asinina Linneaus) was carried out. Several reproduction-related transcripts—for example, vitelline envelope zona pellucida domain 2 (Ha-VEZPD2), vitellogenin (Ha-VTG1), sperm lysin (Ha-SL), fertilization protein (Ha-FP), tektin A1 (Ha-TekA1), axonemal protein 66.0 (Ha-Axp66.0), and double-sex male abnormal-3-related transcription factor 1—(Ha-DMRT1)—were identified. The full-length cDNA of Ha-SL (665 bp containing an open reading frame (ORF) of 453 bp corresponding to a polypeptide of 150 amino acids) was obtained. In addition, that of Ha-VEZPD2.1 (1,094 bp with an ORF of 942 bp, 313 aa) and Ha-VEZPD2.7 (1,130 bp with an ORF of 1,002 bp, 333 aa) were also deduced after cluster analyses of ESTs encoding Ha-VEZPD2. Tissue distribution analysis indicated that Ha-Axp66.0 and Ha-DMRT1 were detected in testes but not in other tissues. Conversely, Ha-VEZPD2.1, Ha-VEZPD2.3, Ha-VEZPD2.17, Ha-VEZPD2.18, and Ha-VTG1 were expressed in ovaries but not in other tissues of male and female broodstock. Expression of these sex-related transcripts during gonad development was estimated by semiquantitative reverse transcription polymerase chain reaction. Relative expression levels of Ha-Axp66.0, Ha-TekA1, and Ha-DMRT1 in testes were significantly higher in stages II, III, and IV of testicular development (P < 0.05). In contrast, Ha-SL and Ha-FP levels were not significantly different during testicular development of H. asinina (P > 0.05). The expression levels of Ha-VEZPD2.1, Ha-VEZPD2.2, Ha-VEZPD2.3, Ha-VEZPD2.7, Ha-VEZPD2.17, Ha-VEZPD2.18, and Ha-VTG1 during stage III were significantly lower than those in stage I ovaries of H. asinina (P < 0.05).