Australian Abalone Research Articles

Optimisation of marker assisted selection for abalone breeding programs

A computer simulation model was used to optimise strategies for marker assisted selection with best linear unbiased prediction (MBLUP) for quantitative traits in a selective breeding program for the southern Australian abalone industry. The simulation model assumed five marker loci associated with genes affecting growth rate explained 50% of the genetic variance for this trait. Use of markers associated with improved disease resistance was also modelled. Parameters and simulation scheme used are described. The simulations accounted for the possibility of early induction of reproductive condition to reduce generation intervals and for selective genotyping. Affects on profitability were calculated using estimates from the industry of income and expenses for a typical farm and accounting for the costs associated with marker testing. While the extra genetic gains from MAS were in the order of 15%, if the cost of genotyping each marker per animal was $10 AUS, then MBLUP for growth was only profitable when 75 progeny per family were genotyped. Early selection (halving the generation interval) was advantageous for MBLUP and BLUP provided the number of progeny per family was greater 5, as the advantage of two generations of selection more than compensated for the decreased accuracy of selection for early growth. However accurate estimates of parameters, such as the genetic correlation between early growth rate and growth and age at first spawning, would need to be obtained before considering implementing this strategy. The advantage of MBLUP was greater for disease resistance than for growth rate. Selective genotyping, which would greatly reduced genotyping costs of implementing MBLUP to improve disease resistance, did not greatly reduce the additional response achieved with marker assisted selection.

Differentiation of Australian and New Zealand abalone species based on partialmitochondrial gene sequences of 12S rRNA, 16S rRNA and ND2 (NADHdehydrogenase subunit 2)

Partial sequences of mitochondrial ND2 (NADH dehydrogenase subunit 2), 12S rRNA and 16S rRNA genes were obtained from four Australian abalone species and two New Zealand abalone species. Phylogenetic analyses were conducted using the parsimony method. The resulting phylogenetic trees revealed two clades, one consisting of four Australian abalone species and the other containing two New Zealand abalone species. The possible application of ND2, 12S and 16S sequence for the investigation of abalone poaching is discussed.

Effects of temperature on growth of juvenile blackfoot abalone, Haliotis iris Gmelin

The effects of temperature on growth and survival of juvenile blackfoot abalone, Haliotis iris, were investigated. Animals of 10, 30 or 60 mm initial shell length were exposed to ambient (6–10°C), 14, 18, 22 and 26°C for 112 days in a flow-through culture system. Maximum growth occurred at 22°C for the 10 and 30 mm size classes and at 18°C for the 60 mm size class. Regression analysis identified the optimal temperature for growth (ToptG) at around 21°C for the 10 and 30 mm size classes and at 17–18°C for the largest size class. In a second experiment, the critical thermal maximum of H. iris was determined as a measure of thermal tolerance. Abalone were subjected to increasing water temperatures at a rate of 2°C h−1 until they detached from the substrate. Abalone of 10 mm displayed greater thermal tolerance than abalone of 30 and 60 mm in length. CT50 temperatures were 28.8, 27.7 and 27.8°C, yielding deduced ToptG values of 19.7, 18.3 and 18.4°C for the 10, 30 and 60 mm size classes respectively. The size-dependent nature of the relationship between growth and temperature could be capitalized upon in recirculating aquaculture systems.

THE ROLES OF BACTERIA AND MICRO AND MACRO ALGAE IN ABALONE AQUACULTURE: A REVIEW

Abalone aquaculture is dependent on cultured algae to induce larval settlement and as a food source for the early life stages of abalone until formulated feed or macroalgae such as Macrocystis sp., Porphyra sp. and Ulva sp. are introduced into the growout system. In the natural environment, abalone larvae settle on coralline red algae, which provide one of the strongest and most consistent settlement cues available for abalone larvae. However, propagation of coralline red algae is not practical commercially. Abalone farms in Japan successfully settle abalone larvae (Haliotis discus hannai) on the green alga Ulvella lens. U. lens also proved to be suitable to enhance settlement of cultured southern Australian abalone species (Haliotis laevigata, H. rubra). Most abalone farms in Australia are now growing U. lens for that purpose. U. lens is easy to culture, no specific facilities are needed and the alga can be grown on PVC settlement plates in commercial nursery tanks. However, U. lens has limited value as a feed for young postlarvae. Instead, cultured diatoms can be added after larvae successfully settle and start feeding. Juvenile abalone (>3 mm in shell length) can consume U. lens and grow rapidly on this alga. Diatom cultures and biofilms developing on settlement plates are not axenic and the role of bacteria in early postlarvae feeding is poorly understood. It has been suggested that bacteria may perform metabolic activities in the undeveloped gut of young postlarvae. At later stages of the nursery phase it becomes increasingly difficult to maintain adequate feed on the plates and this is still regarded as a significant bottleneck for the abalone aquaculture industry. Recent investigations have indicated that sporelings of macroalgae like Ulva sp. or diatoms that can provide more biomass may provide a suitable additional food source for juveniles (>3 mm in shell length).

The effects of tactile stimulants on feeding, growth, behaviour, and meat quality of cultured Blackfoot abalone, Haliotis iris

The New Zealand Blackfoot abalone or paua, Haliotis iris uses both tactile and chemosensory cues to detect and feed on drifting seaweed in its natural habitat. In aquaculture situations, abalone are usually provided with static artificial food pellets, effectively removing the tactile stimulus. This study investigated the effects of tactile stimuli from suspended particles on pellet ingestion, growth, behaviour and meat quality of juvenile paua (length 40–45 mm). Over eleven months, individuals were offered a commercial pellet diet (AbFeed™) and small quantities of one of four particulate materials: macerated seaweed (Gracilaria spp. and Macrocystis pyrifera) and 2 sizes of synthetic PVC fragments. There was seasonal variation in the ingestion rate of abalone from all treatments and the control (no stimulants) with lowest rates during winter (June to September) and highest rates in summer (December to February).The specific growth rate (% body weight gain per day) and shell length increase varied seasonally. Of the four treatments, the only effective stimulant was Gracilaria spp. which resulted in summer shell growth of 110.6±3.2 μm d−1 compared with 86.9±4.0 μm d−1 for control abalone. Behavioural observations showed that when Gracilaria particles were present, abalone spent >80% of their time engaged in feeding-related activity. Control abalone, without the algal stimulant, spent most of their time in a sedentary position. None of the particulate stimulants tested had any significant effect on food conversion ratio, water content, protein, lipid and glycogen levels or meat tenderness. These parameters were, however, significantly affected by season. A preliminary trial of the Gracilaria particle treatment in a commercial system over 90 days enhanced the growth of cultured H. iris (45±5 mm shell length) by 15.3%. The main commercial implication of this research is that the addition of algal stimulants is suggested as a cost-effective means of improving abalone performance using pre-existing culture systems and food types.

Oxygen uptake, diffusion limitation, and diffusing capacity of the bipectinate gills of the abalone, Haliotis iris (Mollusca: Prosobranchia)

Extant abalone retain an ancestral system of gas exchange consisting of paired bipectinate gills. This paper examines the hypothesis that fundamental inefficiencies of this arrangement led to the extensive radiation observed in prosobranch gas exchange organs. Oxygen uptake at 15 °C was examined in the right gill of resting adult blackfoot abalone, Haliotis iris Martyn 1784. Pre- and post-branchial haemolymph and water were sampled and oxygen content, partial pressure ( Po 2), pH, and haemocyanin content measured; in vivo haemolymph flow rate was determined by an acoustic pulsed-Doppler flowmeter. During a single pass across the gills, mean seawater Po 2 fell from 138.7 Torr to 83.4 Torr, while haemolymph Po 2 rose from 37.2 Torr to 77.0 Torr raising total O 2 content from 0.226 to 0.346 mmol L − 1 . Haemolymph flowed through the right gill at a mean rate of 9.6 mL min − 1 and carried 0.151 to 0.355 mmol L − 1 of haemocyanin (mean body mass 421 g). Only 34.7% of the oxygen carried in the arterial haemolymph was taken up by the tissues and less than half of this was contributed by haemocyanin. A diffusion limitation index ( L diff) of 0.47–0.52, a well-matched ventilation–perfusion ratio (1.2–1.4) and a diffusing capacity ( D) of 0.174 μmol O 2 kg − 1 Torr − 1 indicate that the gills operate efficiently and are able to meet the oxygen requirements of the resting abalone.

Isolation, Characterization, and Expression Analysis of Three Actin Genes in the New Zealand Black-Footed Abalone, Haliotis iris

Three actin genes -- H. irisA1, H. irisA2, and H. irisA3 -- from the mollusc Haliotis iris (New Zealand black-footed abalone) were isolated by polymerase chain reaction (PCR). The genes were similar to molluscan (84.1% to 94.9%) and vertebrate (84.5% to 86.6%) actins. The sequence similarity between the genes ranged from 88.5% to 93.2%. The greatest disparity, 32.3%, was found over a 99-nt region located at nt 808-906 of H. irisA1, corresponding to amino acids 212-244 of the three actins. The H. iris actin gene family contains at least eight members. Reverse transcription (RT)-PCR analysis of the three genes showed H. irisA1 and H. irisA2 were expressed at low levels in fertilized eggs and blastula stages and at high levels in trochophore and veliger larvae. H. irisA3 was detected in fertilized eggs; it was not detected in the blastula stages and at high levels in the trochophore and veliger larvae. The structure and expression of the three actin genes are discussed.

Extracellular fluid volume, urine filtration rate and haemolymph mixing time in the abalone, Haliotis iris Martyn: a comparison of 51Cr-EDTA and 14C-inulin as extracellular markers

Measurement of extracellular fluid volume (ECFV, haemolymph or blood volume) of abalone is important for understanding respiratory and circulatory functions and for investigation of factors affecting meat recovery in the fishery. The compounds 14C-inulin and 51Cr-ethylenediaminetetraacetic acid (EDTA) were evaluated as markers for the determination of ECFV and primary urine formation (filtration) in New Zealand blackfoot abalone or paua, Haliotis iris. The volume of distribution of either marker reliably estimated ECFV after correction for clearance (56.6 ± 3.5 mL 100 g–1 soft tissue). 51Cr-EDTA was cleared from the haemolymph three to five times faster than 14C-inulin and was sequestered in the kidneys, digestive gland and mucous gland. 51Cr-EDTA was therefore unsuitable as a filtration marker. Urine filtration rate was best estimated from the appearance of inulin in the seawater (16.2 ± 2.1 mL 100 g–1 soft tissue day–1). The ECFV (inulin space) of isolated tissues ranged from 12–17 mL 100 g–1 tissue in the digestive gland, foot and adductor muscle to 60–70 mL 100 g–1 tissue in the kidneys, right ctenidium and epipodium. Equalisation of both markers between the efferent ctenidial vein and haemolymph lacunae of the shell adductor muscle occurred extremely slowly (t1/2 ~2 h, t0.95 ~8 h), confirming the poor perfusion of this anaerobic muscle.

Comparison of n-3 polyunsaturated fatty acid contents of wild and cultured Australian abalone

The fatty acid contents of wild and cultured Australian adult blacklip abalone, Haliotis rubra, were analysed by gas liquid chromatography. Wild abalone contained significantly higher levels of total n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3) and α-linolenic acid (18:3n-3) than cultured abalone (P<0.05). The predominant n-3 PUFA was docosapentaenoic acid in wild abalone, while in cultured abalone a high level of eicosapentaenoic acid was found. The concentration of docosahexaenoic acid (22:6n-3) was low in both wild and cultured abalone, and cultured abalone had a significantly higher percentage composition of this fatty acid than wild abalone (P<0.01). Significantly higher levels of arachidonic acid (20:4n-6), 22:2n-6, 22:4n-6 and total n-6 PUFA were also found in wild abalone than in cultured animals (P<0.05). The ratio of n-3 PUFA to n-6 PUFA was the same in wild and cultured abalone. Manipulation of nutrient sources of cultured abalone may influence their lipid composition. Consumption of either wild or cultured abalone will contribute to dietary n-3 PUFA intake, with benefits to human health.

Mineralogical Variation in Shells of the Blackfoot Abalone, Haliotis iris (Mollusca: Gastropoda: Haliotidae), in Southern New Zealand

The New Zealand blackfoot abalone, Haliotis iris Gmelin, is among the few gastropods that precipitate both calcite and aragonite in their shells. The location, composition, and thickness of these mineral layers may affect color, luster, and strength of the shell, which is locally important in jewelry manufacture. Skeletal mineralogy and shell structure of H. iris from three southern New Zealand locations were determined using X-ray diffractometry, scanning electron micrography, and mineral staining. In H. iris an outer calcitic layer is separated from an inner aragonitic surface by both calcified and noncalcified organic layers running longitudinally through the shell. Skeletal mineralogy within individual shells varies from 29 to 98% aragonite, with older shell having significantly higher aragonite content than young sections. Variation within populations ranges from 40 to 98% aragonite, and among three populations from 34 to 98% aragonite. Shell thickness, too, varies within individual shells from 0.2 to 4.2 mm, with a significant positive relationship with age. Withinpopulation variation in shell thickness ranges from 2.1 to 5.4 mm, with no significant difference in shell thickness variation among populations. The high degree of variability within and among individual shells suggests that it is essential to test replicate samples from individual mollusk shells, especially when they have complex bimineral structure.

A Growth Assessment of Juvenile Abalone Haliotis laevigata Fed Enriched Macroalgae Ulva rigida

The experiment was conducted to assess the growth of juvenile greenlip abalone Haliotis laevigata fed a range of Ulva rigida diets with different nitrogen contents and an artificial pelleted diet. The minimal nutrient concentration resulting in significant enrichment of U. rigida was 2.25 mg N l−1. The maximum nitrogen content obtained by U. rigida in this study was 6.099%, which resulted from an inorganic nutrient concentration of 7 mg N l−1. However, no further significant increase in enrichment occurred from nutrient concentrations of 10 and 15 mg N l−1. Abalone specific growth rate in shell length (SGRL) and wet weight (SGRW) and foot muscle yield were significantly higher for abalone fed the artificial diet compared to those fed any U. rigida diet. Abalone fed U. rigida enriched in less than 15 mg N l−1 had similar growth rates compared to abalone fed unenriched U. rigida. Abalone fed the artificial diet contributed a higher percentage of weight gain into the valuable foot muscle. The foot muscle moisture content of abalone fed the artificial diet was significantly lower than that of abalone fed any U. rigida diet. The conclusions reached by this study should assist Australian abalone producers in assessing the potential benefits of using U. rigida as an effluent biofilter and subsequent food source for the commercially important juvenile greenlip abalone.

Molecular analysis of a haplosporidian parasite from cultured New Zealand abalone Haliotis iris.

In the Austral summer and autumn of 2000 and 2001, mortalities of black-footed abalone Haliotis iris (Martyn, 1784) occurred in a commercial facility in New Zealand. Histological analyses suggested that infection by a haplosporidian parasite was responsible. To confirm identification as a haplosporidian and to help determine if this parasite represented a new, undescribed species, DNA was extracted from infected host tissues scored as positive for infection by histological examination. Small-subunit rRNA (SSU rRNA) gene sequences from both the host abalone and a parasitic organism were amplified by PCR and characterized. Although the sequence for this parasite was novel, not matching any known SSU rRNA gene sequences, phylogenetic analyses strongly supported grouping this parasite with the haplosporidians. Parsimony analyses placed the parasite at the base of the phylum Haplosporidia, ancestral to Urosporidium crescens and the Haplosporidium, Bonamia, and Minchinia species. Sequencing of multiple parasite DNA clones revealed a single polymorphic site in the haplosporidian SSU rRNA gene sequence.

Characterization of microsatellite loci in the Australian Blacklip abalone (Haliotis rubra, Leach).

Characterization of microsatellite loci in the Australian Blacklip abalone (Haliotis rubra, Leach).

Corrigendum to: "PauA: a novel plasminogen activator from streptococcus uberis".

Corrigendum to: ``PauA: a novel plasminogen activator from Streptococcus uberis'' [FEMS Microbiol. Lett. 178 (1999) 27^33]1 E.L. Rosey b, R.A. Lincoln a, P.N. Ward a, R.J. Yancey Jr. b, J.A. Leigh a;* a Institute for Animal Health Compton Laboratory, Compton, Newbury, Berks RG20 7NN, UK b Central Research Division, P¢zer, Animal Health Biological Discovery, Eastern Point Road, Groton, CT 06340, USA

Spermatozoa of the Australian ‘greenlip’ abalone Haliotis laevigata Donovan: ultrastructure and comparison with other gastropods, especially other Haliotidae (Vetigastropoda, Molluscs)

Summary Spermatozoa of a commercial Australian abalone, Haliotis laevigata Donovan, 1808 (the ‘greenlip’ abalone) are examined for the first time using transmission electron microscopy. As is characteristic of other externally fertilizing molluscs, the spermatozoa of H. laevigata are of the structurally simple type (classic ect-aquasperm). In anterior- posterior sequence, each spermatozoon consists of a conical acrosomal vesicle (length 1.35–1.38 μm, deeply invaginated posteriorly and associated with a subacrosomal axial rod and flocculent deposits), a barrel-shaped nucleus (length 2.1–2.2 μm, with prominent lacunae), a short midpiece (consisting of a pair of orthogonally arranged, triplet substructure centrioles surrounded by a five spherical mitochondria), nine satellite fibres (connecting the distal centriole to the plasma membrane) and a 40–44 μm long flagellum (featuring a 9 + 2 microtubular pattern axoneme). Total length of spermatozoa ranges from 44–48 μm. The contents of the acrosomal vesicle can be divided into anterior and posterior portions based on differing electron densities and the occurrence anteriorly of a thin, ‘truncated cone’ structure at the periphery of the vesicle. Preliminary examination of spermiogenic stages indicates that like other vetigastropods the acrosomal vesicle of H. laevigata develops through coalescence of several moderately large, electron-dense, pro-acrosomal vesicles. Comparison with other species of Haliotis which have been studied reveals that spermalozoa of H. laevigata (type of subgenus Schismotis) most closely resemble those of the Asian species H. japonica aquatilis and H. diversicolor supertexta (members of subgenus Sulculus). Such spermatozoa differ from those of other investigated haliotids (H. discus, H. mfescens of subgenus Nordotis and H. midae of subgenus Euhaliotis) all of which have appreciably longer acrosomal and nuclear regions. The use of sperm ultrastructure for probing species relationships within the Haliotidae appears to be a most promising area for future research, particularly when directed at type species of nominal subgeneric units.

Virus-like Particles Detected from Juvenile Abalones (Nordotis discus discus) Reared with an Epizootic Fatal Wasting Disease

Haemocytes play an important role in innate immune responses within invertebrate organisms. However, identification and quantification of different types of haemocytes can be extremely challenging, and has led to numerous inconsistencies and misinterpretations within the literature. As a step to rectify this issue, we present a comprehensive and detailed approach to characterize haemocytes using a combination of classical (cytochemical and phagocytosis assays with optical microscopy) and novel (flow cytometry with Sysmex XN-1000 and Muse® Cell analyser) techniques. The Sysmex XN-1000 is an innovative fluorescent flow cytometric analyser that can effectively detect, identify and count haemocytes, while the Muse® Cell analyser provides accurate and rapid haemocyte cell counts and viability. To illustrate this approach, we present the first report on morphological and functional features of New Zealand black-footed abalone (Haliotis iris) haemocyte cells. Two types of haemocytes were identified in this study, including type I (monocyte-like) and type II (lymphocyte-like) cells. Granular cells, which have been reported in other molluscan species, were not detected in H. iris. Cell types were categorized based on shape, size, internal structures and function. The lymphocyte-like haemocytes were the most abundant hemocytes in the haemolymph samples, and they had large nuclei and basic cytoplasms. Monocyte-like cells generally were larger cells compared to lymphocyte-like cells, and had low nucleus-cytoplasm ratios. Monocyte-like cells showed higher phagocytic activity when encountering Zymosan A particles compared to lymphocyte-like cells. The present study provides a comprehensive and accurate new approach to identify and quantify haemocyte cells for future comparative studies on the immune system of abalone and other molluscan species.

Studies on southern Australian abalone (genus Haliotis) XVII. A chronology of H. laevigata

The abalone Haliotis laevigata deposits 2 rings yr-1 in the spire during the first 4–6 years of life at 11 sites in southern Australia. At two sites it deposits 3 rings yr-1 , and at another, 3 rings yr-1 for the first 3 years and 2 yr-1 in the next 3 years. These rates of ring deposition appear to be dependent on the growth rate; they are not exact for every individual due to individual variation in growth but appear to be a statistical property of the population. The tendency for an integer number of rings to be deposited annually may be related to exogenous cues. Hence at one site where 3 rings yr-1 are laid down they are deposited in about June, September and December each year, corresponding to winter, the spawning season and summer. Long term tag recaptures suggest that these rates of deposition persist in older shells but better validation is required. Erosion of the shell causes the loss of rings, and the attack of muricid and polydorid borers causes the deposition of adventitious rings as well as pigmented rings, called brown rings. These complications need to be considered in estimating the age of a shell from ring counts.

Studies on southern Australian abalone (genus Haliotis) XVIII. Ring formation in H. scalaris

The abalone Haliotis scalaris deposits two fine rings a year in the spire, one in about December and the other from May to July, corresponding roughly to summer and winter sea temperature extremes. Additional fine rings, and in particular brown rings, are deposited in response to boring annelids and drilling muricid gastropods. Multiple regression analysis revealed a relationship between the number of rings in the spire and the variables: age of the shell, extent of infestation by annelids, and the number of brown rings. The analysis showed that additional fine rings’ and brown rings are deposited according to the degree of parasitic attack.

Effect of diet on the lipid composition of wild and cultured abalone

Foot-muscle tissue from wild-caught adults of the two main commercial species of Australian abalone, Haliotis laevigata and H. rubra, were analysed for fatty acid and sterol composition. Foot muscle tissue from juvenile H. laevigata and hybrid H. laevigata X H. rubra fed an artificial diet was compared with that of juvenile H. laevigata fed the green alga Ulva australis. The major fatty acids in all samples of muscle were 16:0, 18:0, 18:1(n-9), 18:1(n-7), 20:4(n-6), 20:5(n-3) and 22:5(n-3). The abundance of 22:5(n-3) is unusual as most marine animals have much more 22:6(n-3). The muscle of juvenile abalone fed an artificial diet high in total lipids and 18:2(n-6) contained elevated total lipid levels and elevated levels of 18:2(n-6) and 20:2(n-6). The muscle of abalone fed U. australis, which was high in C 16 and C 18(n-3) polyunsaturated fatty acids (PUFA), contained elevated proportions of their chain-elongation products C 20 and C 22(n-3) PUFA, which is similar to wild-caught adult abalone. The major sterol in all samples of abalone muscle was cholesterol, irrespective of diet. The muscle of juvenile H. laevigata fed an artificial diet high in 24-methylcholest-5-enol and 24-ethylcholest-5-enol contained elevated proportions of these sterols compared with the muscle of juveniles fed U. australis and wild-caught adult abalone. Observed differences in fatty acid and sterol compositions were attributed to the accumulation of lipid in the foot muscle of abalone fed an artificial diet.

Digestive efficiency of the Australian abalone Haliotis rubra in relation to growth and feed preference

The digestibilities of dry and organic matter, energy and nitrogen were determined for Haliotis rubra feeding on a range of monospecific algal diets. These digestibilities, in conjunction with total intakes, were used to estimate the daily intakes of nitrogen and energy of abalone feeding on those algal species used by Fleming (1995). These were then used to explain the growth rates, intakes and chemosensory preferences of abalone in that study. The chemosensory preferences of abalone were strongly related to the digestible nitrogen content of the algae and to a slightly lesser extent the nitrogen content of the algae, indicating the important role nitrogen plays in setting preferences in this abalone. The intakes of digestible nitrogen were significantly related to the growth rates of abalone, suggesting that nitrogen is a limiting nutrient to abalone growth. Thus, H. rubra selects those foods which maximise the intake of nitrogen and result in maximum growth rates. Energy does not appear to play a major role in setting abalone preference as the digestible energy contents of algae in this study (and in general) are similar and did not correlate with preference. The digestible nitrogen content was lowest in the 3 brown algae tested and digestibility was related to the polyphenolic content of the brown algae. This suggests that the low growth of H. rubra on these species results from the action of polyphenolic compounds, reducing nitrogen digestibility.