Heritability estimates for growth in the tropical abalone Haliotis asinina using microsatellites to assign parentage

The tropical abalone Haliotis asinina is a wild-caught and cultured species throughout the Indo-Pacific as well as being an emerging model species for the study of haliotids. H. asinina has the fastest recorded natural growth rate of any abalone and reaches sexual maturity within one year. As such, it is a suitable abalone species for selective breeding for commercially important traits such as rapid growth. Estimating the amount of variation in size that is attributable to heritable genetic differences can assist the development of such a selective breeding program. Here we estimated heritability for growth-related traits at 12 months of age by creating a single cohort of 84 families in a full-factorial mating design consisting of 14 sires and 6 dams. Of 500 progeny sampled, 465 were successfully assigned to their parents based on shared alleles at 5 polymorphic microsatellite loci. Using an animal model, heritability estimates were 0.48 ± 0.15 for shell length, 0.38 ± 0.13 for shell width and 0.36 ± 0.13 for weight. Genetic correlations were > 0.98 between shell parameters and weight, indicating that breeding for weight gains could be successfully achieved by selecting for shell length.

Egg-laying-hormone immunoreactivity in the neural ganglia and ovary of Haliotis asinina Linnaeus

Immunoreactivity against the abalone egg-laying hormone (aELH) was detected in the fine granules of type 1 and 2 neurosecretory (NS) cells, neurites in the neuropil, and blood sinuses in the connective tissue sheath of the cerebral, pleuropedal, and visceral ganglia of the tropical abalone, Haliotis asinina Linnaeus. The number of positive NS cells, and the intensity of staining in the ganglia, varied and might be related to the stage of ovarian cycle. At any stage, positive cells were most numerous in the pleuropedal, and least numerous in the visceral ganglion. In addition, several cells of the statocyst and associated nerves also exhibited the immunoreactivity. In the ovary, the most intense reactivity was detected in the follicular and granular cells adjacent to mature oocytes, in the trabeculae and the ovarian capsule. The cytoplasm of mature oocytes was also moderately stained. The results indicate that the cerebral, pleuropedal, and visceral ganglia are the main sites of aELH-producing cells. The ovary may also produce aELH locally.

Larval settlement of the tropical abalone, Haliotis asinina Linnaeus, using natural and artificial chemical inducers

Many kinds of chemical and biological materials have been used as inducers of settlement of abalone larvae, as well as other species of marine gastropods, with responses being highly variable, even to the same chemical cue. The present study tested chemical inducers, γ-aminobutyric acid (GABA), δ-aminovaleric acid (5-AVA) and l-glutamic acid (GA) and the effects they have on larval settlement of Haliotis asinina. Additionally, a relatively inexpensive commercial substance, monosodium glutamate (MSG), was trialed. The datum provided shows all chemicals to be active inducers of settlement in this study, in order of effectiveness of 5-AVA, GABA, MSG to GA. Induction as adjudged from larval numbers settled was best at 6 h 62%, with 10⁻¹ mM 5-AVA. At 24 h, induction was the highest at 78% when exposed to 10⁻² mM 5-AVA. Larvae that were allowed to settle up to 72 h showed the highest numbers of settled larvae, and declined back to 60% when exposed to 10⁻² 5-AVA and 10⁻¹ mM GABA respectively. Monosodium glutamate, although third in settlement standings would bypass the other chemicals, with regard to cost versus yield. The assessment of settlement surface, rough or smooth proved to be irrelevant, which had no significant impact on larval settlement.

The existence of gonadotropin-releasing hormone-like peptides in the neural ganglia and ovary of the abalone, Haliotis asinina L.

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that is conserved in both vertebrate and invertebrate species. In this study, we have demonstrated the presence and distribution of two isoforms of GnRH-like peptides in neural ganglia and ovary of reproductively mature female abalone, Haliotis asinina, using immunohistochemistry. We found significant immunoreactivities (ir) of anti-lamprey(I) GnRH-III and anti-tunicate(t) GnRH, but with variation of labeling intensity by each anti-GnRH type. IGnRH-III-ir was detected in numerous type1 neurosecretory cells (NS1) throughout the cerebral and pleuropedal ganglia, whereas tGnRH-I-ir was detected in only a few NS1 cells in the dorsal region of cerebral and pleuropedal ganglia. In addition, a small number of type2 neurosecretory cells (NS2) in cerebral ganglion showed lGnRH-III-ir. Long nerve fibers in the neuropil of ventral regions of the cerebral and pleuropedal ganglia showed strong tGnRH-I-ir. In the ovary, lGnRH-III-ir was found primarily in oogonia and stage I oocytes, whereas tGnRH-ir was observed in stage I oocytes and some stage II oocytes. These results indicate that GnRH produced in neural ganglia may act in neural signaling. Alternatively, GnRH may also be synthesized locally in the ovary where it could induce oocytes development.

The tetrapeptide APGW-amide induces somatic growth in Haliotis asinina Linnaeus

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.

Identification of an attractin-like pheromone in the mucus-secreting hypobranchial gland of the abalone Haliotis asinina linnaeus.

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.

Stimulatory effects of egg-laying hormone and gonadotropin-releasing hormone on reproduction of the tropical abalone, Haliotis asinina linnaeus

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 (1GnRH-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.

Distribution of Gaba in the nerve ganglia of haliotis asinina linnaeus

Gamma-aminobutyric acid (GABA) is a major neurotransmitter and effective settlement inducer in abalone aquaculture. This study aimed to explore the distribution of GABA within neural tissues of Haliotis asinina. Gamma-aminobutyric acid was found in neuronal cell type 1 of 3 major ganglia (i.e., cerebral, pleuropedal, and visceral ganglia) of both sexes. The distribution of GABA-immunoreactive (-ir) cells in the cerebral ganglion was concentrated mostly in the cortex region of the dorsal horn, whereas it was scattered throughout the pleuropedal ganglion, with more in the upper half. Gamma-aminobutyric acid-ir nerve fibers were found throughout the neuropils of the ganglia. The visceral ganglion had the least numbers of GABA-ir neurons compared with the other 2 ganglia. The cells were distributed mainly in the dorsal horn. We also observed GABA to be colocalized with 2 other neurotransmitters: serotonin (5-HT) and dopamine (DA). In the cerebral ganglion, fluorescence double staining of GABA and 5-HT, and GABA and DA showed immunoreactivity in separate cells and was also colocalized in the same cells. In the pleuropedal ganglion, the staining pattern was similar to the cerebral ganglion, but positive-staining cells were less numerous. In the visceral ganglion, GABA and DA, and GABA and 5-HT were colocalized in the same cell types. Overall, we found that GABAergic cells were most numerous in the cerebral ganglion of H. asinina. Further studies are required to determine the functions of these neurotransmitters in relation to their distribution.

Correlating gene expression with larval competence and the effect of age and parentage on metamorphosis in the tropical abalone Haliotis asinina

The non-geniculate crustose coralline alga (CCA) Mastophora pacifica can induce the metamorphosis of competent Haliotis asinina (Vetigastropoda) larvae. The ability to respond to this natural cue varies considerably with larval age, with a higher proportion of older larvae (e.g. 90 h) able to metamorphose in response to M. pacifica than younger larvae (e.g. 66 h). Here we document the variation in time to acquisition of competence within a larval age class. For example, after 18 h of exposure to M. pacifica, approximately 15 and 36% of 84 and 90-h-old H. asinina larvae had initiated metamorphosis, respectively. This age-dependent response to M. pacifica is also observed when different aged larvae are exposed to CCA for varying periods. A higher proportion of older larvae require shorter periods of exposure to CCA than younger larvae in order to initiate metamorphosis. In this experiment, as in the previous, a small proportion of young larvae were able to respond to brief periods of CCA exposure, suggesting that they had developed the same state of competency as the majority of their older counterparts. Comparisons of the proportions of larvae undergoing metamorphosis between families reveals that parentage also has a significant (P < 0.05) affect on whether an individual will initiate metamorphosis at a given age. These familial differences are more pronounced when younger, largely pre-competent larvae (i.e. 66 h old) are exposed to M. pacifica, with proportions of larvae undergoing metamorphosis differing by as much as 10 fold between families. As these data suggest that variation in the rate of development of the competent state has a genetic basis, and as a first step towards identifying the molecular basis to this variation, we have identified numerous genes that are differentially expressed later in larval development using a differential display approach. Spatial expression analysis of these genes suggests that they may be directly involved in the acquisition of competence, or may play a functional role in the postlarva following metamorphosis.

Developmental expression of two Haliotis asinina hemocyanin isoforms

Hemocyanins are large copper-containing respiratory proteins that play a role in oxygen transport in many molluscs. In some species only one hemocyanin isoform is present while in others two are expressed. The physiological relevance of these isoforms is unclear and the developmental and tissue-specific expression of hemocyanin genes is largely unknown. Here we show that two hemocyanin genes in the gastropod Haliotis asinina, which encode H. asinina hemocyanin (HaH1) and HaH2 isoforms, are developmentally expressed. These genes initially are expressed in a small number of mesenchyme cells at trochophore and pre-torsional veliger stages, with HaH1 expression slightly preceding HaH2. These cells largely are localized to the visceral mass, although a small number of cells are present in head and foot regions. Following metamorphosis the isoforms show overlapping as well as isoform-specific expression profiles, suggesting some degree of isoform-specific function.

Digenean trematodes infecting the tropical abalone Haliotis asinina have species-specific cercarial emergence patterns that follow daily or semilunar spawning cycles

Approximately 1-2% of the tropical abalone Haliotis asinina inhabiting Heron Island Reef are infected with opecoelid digeneans. These largely inhabit the haemocoel surrounding the cerebral ganglia and digestive gland-gonad complex, and infected abalone typically have significantly reduced or ablated gonads. Observations of infected abalone reveal two distinct cercarial emergence patterns, one which correlates tightly with the abalone's highly regular and synchronous fortnightly spawning cycle, and the other which occurs in a circadian pattern. The former appears to be a novel emergence strategy not previously observed in digeneans. While the cercariae in all abalone are morphologically indistinguishable, comparison of sequences from the internal transcribed spacer 2 (ITS 2) region of the ribosomal DNA reveals a 5.7% difference between cercariae displaying different emergence patterns, indicating these are two distinct species that probably belong to the same genus. The ITS 2 sequences of the species with the daily emergence pattern are identical to that of an undescribed adult opecoelid from the gut of the barramundi cod, Cromileptes altivelis. Combined molecular, morphological and emergence data suggest that while these opecoelid cercariae use the same first intermediate host and are closely related species-members of the genus Allopodocotyle-they fill different ecological niches that are likely to include different definitive hosts.

Heritability estimates for growth in the tropical abalone Haliotis asinina using microsatellites to assign parentage

The tropical abalone Haliotis asinina is a wild-caught and cultured species throughout the Indo-Pacific as well as being an emerging model species for the study of haliotids. H. asinina has the fastest recorded natural growth rate of any abalone and reaches sexual maturity within one year. As such, it is a suitable abalone species for selective breeding for commercially important traits such as rapid growth. Estimating the amount of variation in size that is attributable to heritable genetic differences can assist the development of such a selective breeding program. Here we estimated heritability for growth-related traits at 12 months of age by creating a single cohort of 84 families in a full-factorial mating design consisting of 14 sires and 6 dams. Of 500 progeny sampled, 465 were successfully assigned to their parents based on shared alleles at 5 polymorphic microsatellite loci. Using an animal model, heritability estimates were 0.48 +/- 0.15 for shell length, 0.38 +/- 0.13 for shell width and 0.36 +/- 0.13 for weight. Genetic correlations were > 0.98 between shell parameters and weight, indicating that breeding for weight gains could be successfully achieved by selecting for shell length. (c) 2006 Elsevier B.V. All rights reserved.