Identification and characterisation of the Mpeg1 homologue in the South African abalone, Haliotis midae

Although Haliotis midae is the most economically important cultured abalone species in South Africa, infectious diseases have the potential to severely limit the production of this shellfish. Consequently, it is becoming increasingly important to characterise the abalone immune system in order to better understand their ability to combat infection. This study reports the identification and characterisation of a perforin-like protein, designated hmMpeg1, which is believed to be involved in the H. midae immune system. hmMpeg1 encodes for a 78 kDa protein that has significant sequence similarity to Mpeg proteins from other abalone species and includes the conserved cytolytic membrane attack complex/perforin (MACPF) domain of perforin. Real-time quantitative PCR (qPCR) analysis demonstrated expression of hmMpeg1 mRNA in haemocytes and epipodia samples from H. midae exposed to a heat-killed, Gram-negative bacterial pathogen, Vibrio anguillarum 5676. hmMpeg1 mRNA in haemocytes increased significantly 48 h post-infection (h.p.i) (8.2 fold; P < 0.05), coinciding with a decrease in the total number of circulating haemocytes, and reached a maximum at 96 h.p.i (17.2 fold; P < 0.05). Similarly, a significant increase in the level of hmMpeg1 mRNA occurred at 24 h.p.i in epipodia samples (3.8 fold; P < 0.05), reaching a maximum at 48 h.p.i (4.5 fold; P < 0.05). In addition, western blot analysis detected a significant increase in hmMpeg1 between 24 h.p.i (4.2 fold; P < 0.05) and 48 h.p.i (3.1 fold; P < 0.05) in the epipodia, and between 48 h.p.i (1.7 fold; P < 0.05) and 96 h.p.i (1.9 fold; P < 0.05) in haemocytes, sampled from abalone exposed to the abalone pathogen V. anguillarum 5676. The importance of hmMpeg1, in terms of its function and importance in the H. midae immune response, is discussed.     

Isolation and characterization of microsatellite markers in the South African abalone (Haliotis midae)

We report the isolation and characterization of 11 polymorphic microsatellite loci in the South African abalone Haliotis midae. These loci showed a range of five to 21 alleles per locus and observed heterozygosities ranging from 0.14 to 0.93 in a wild population of 32 individuals. All loci except four conformed to Hardy–Weinberg expectations and did not show linkage disequilibrium. The polymorphism exhibited at these loci indicate that they would be useful in determining levels of genetic variability in natural and commercial Haliotis midae populations as well as in parentage and Quantitative Trait Loci (QTL) analysis in hatchery reared abalone.     

Effects of wild and farm-grown macroalgae on the growth of juvenile South African abalone Haliotis midae Linnaeus

The effect of various macroalgal diets on the growth of grow-out (>20 mm shell length) South African abalone Haliotis midae was investigated on a commercial abalone farm. The experiment consisted of four treatments: fresh kelp blades (Ecklonia maxima (Osbeck) Papenfuss) (c. 10% protein); farmed, protein-enriched Ulva lactuca Linnaeus (c. 26% protein) grown in aquaculture effluent; wild U. lactuca (c. 20% protein); and a combination diet of kelp blades + farmed U. lactuca. Abalone grew best on the combination diet (0.423 ± 0.02% weight d^?1 SGR [specific growth rate]; 59.593 ± 0.02 ?m d^?1 DISL [daily increment in shell length]; 1.093 final CF [condition factor]) followed by the kelp only diet (0.367 ± 0.02% weight d^?1 SGR; 53.148 ± 0.02 ?m d^?1 DISL; 1.047 final CF), then the farmed, protein-enriched U. lactuca only diet (0.290 ± 0.02% weight d^?1 SGR; 42.988 ± 0.03 um d”¹ DISL; 1.013 final CF) that in turn outperformed the wild U. lactuca only diet (-0.079 ± 0.01% weight d^?1 SGR; 3.745 ± 0.02 ?m d”^?1 DISL; 0.812 final CF). The results suggest that protein alone could not have accounted for the differences produced by the varieties of U. lactuca and that the gross energy content is probably important.     

Determining the most appropriate feeding regime for the South African abalone Haliotis midae Linnaeus grown on kelp

Beach-cast kelp (the most widely used feed for commercially grown South African abalone) is plentiful during winter months when periodic storms cause kelp to wash ashore. During summer, however, this resource is not always readily available and farmed abalone are often starved for short periods. The aim of this research was to assess how periodic kelp starvation influences growth of the commercially grown abalone, Haliotis midae Linnaeus. Growth of grow-out abalone was monitored on a commercial abalone farm over a period of six months and consisted of 3 treatments with 2 replicates (n = ±250 abalone per replicate). The treatments were: Control (abalone always given more kelp than what they typically needed); Treatment 1 (abalone fed their weekly ration only once a week); Treatment 2 (abalone fed half their weekly ration every 3 and then 4 days respectively). While the data at first suggest that the control animals outperform the treatment animals, after undergoing an initial adjustment period to the new feeding regime, the treatment animals perform better. Weight gain and feed conversion efficiencies show that the treatment animals perform better overall. The control animals generally required much more feed to produce comparable increases in both length and weight compared to the treatment animals. This study has shown that periodic bouts of starvation is beneficial to Haliotis midae, allowing variable growth spurts when returned to full feed rations. This paper has not been submitted elsewhere in identical or similar form.     

Development of an experimental laboratory fertilization protocol for the South African abalone,Haliotis midae (Linnaeus 1758)

In this study, effective gamete concentrations, egg viability, and fertilization volumes were evaluated for Haliotis midae (L.). Sperm concentrations between 5?×?10³ and 5?×?10⁴?mL^?1 (p?>?0.05) consistently resulted in high hatch-out rates (96?±?1%). At concentrations higher than 5?×?10⁵?mL^?1, hatch-out rates decreased to 69?±?7% (p??1 resulted in high fertilization rates, with 50?eggs?mL^?1 being the ideal concentration for fertilization in H. midae. Egg viability was consistently high up to 100?min post-spawning, with a decrease in hatch-out success, when eggs were fertilized 120?min post-spawning. Fertilization volumes did not affect successful hatch-out. The results from this study can be implemented by South African abalone farms to increase hatch-out rates and subsequent culture. It can also be used as basis for the development of fertilization protocols in other marine invertebrate species.     

Settlement pattern and survival of a shell-infesting sabellid polychaete,Terebrasabella heterouncinata,on South African abalone,Haliotis midae,fed two diets

This study tested whether abalone diet influences larval settlement success of the shell-dwelling sabellid polychaete Terebrasabella heterouncinata and their survival over the first 96h after settlement on host abalone. Shell area preferences of the sabellid were also investigated. Abalone diet (fresh kelp, Ecklonia maxima or Abfeed?) did not affect sabellid settlement success (F_1,30 = 1.05; p <= 0.31). There were significant differences in settlement between shell regions, with the highest number of sabellids settling near the respiratory pores (F_2,30 = 61.0; p < 0.001). Percentage occupation rate of tubes 96h after settlement did not differ between dietary treatments. There were no significant interactions between diet and shell region for sabellid settlement (F_2,30 = 0.77, p = 0.69) and tube formation (F_2,30 = 1.02, p = 0.67) or for the percentage occupation rate of the tubes (F_9,180 = 0.11, p = 0.73). Thus, infestation intensity in relation to shell region can be estimated independent of abalone diet. For treatment purposes, total settlement after 36 days of exposure can be used to identify the shell areas on which most sabellids will be found.     

Differential responses of juvenile and adult South African abalone (Haliotis midae Linnaeus) to low and high oxygen levels

Marine invertebrates have evolved multiple responses to naturally variable environmental oxygen, all aimed at either maintaining cellular oxygen homeostasis or limiting cellular damage during or after hypoxic or hyperoxic events. We assessed organismal (rates of oxygen consumption and ammonia excretion) and cellular (heat shock protein expression, anti-oxidant enzymes) responses of juvenile and adult abalone exposed to low (~ 83% of saturation), intermediate (~ 95% of saturation) and high (~ 115% of saturation) oxygen levels for one month. Using the Comet assay, we measured DNA damage to determine whether the observed trends in the protective responses were sufficient to prevent oxidative damage to cells. Juveniles were unaffected by moderately hypoxic and hyperoxic conditions. Elevated basal rates of superoxide dismutase, glutathione peroxidase and catalase were sufficient to prevent DNA fragmentation and protein damage. Adults, with their lower basal rate of anti-oxidant enzymes, had increased DNA damage under hypoxic and hyperoxic conditions, indicating that the antioxidant enzymes were unable to prevent oxidative damage under hypoxic and hyperoxic conditions. The apparent insensitivity of juvenile abalone to decreased and increased oxygen might be related to their life history and development in algal and diatom biofilms where they are exposed to extreme diurnal fluctuations in dissolved oxygen levels.     

Preliminary investigation to determine the cytotoxicity of various cryoprotectants on southern African abalone (Haliotis midae) embryos

Cryopreservation could provide stock quantities of embryos for transgenic research. This study aimed to determine the least toxic cryoprotective agent for Haliotis midae embryos. They were exposed for 30 min to concentrations varying from 5% to 20% of the following cryopreservatives: methanol (MET), polyethylene glycol (PEG), dimethyl sulfoxide (ME₂SO) and glycerol (GLY). In contrast to cryopreservation studies done in other molluscs, PEG showed the least toxicity to H. midae embryos in concentrations ranging from 5% to 15%. MET was also less toxic than ME₂SO and GLY at correlating concentrations. GLY showed the most toxic effects with most embryos dead or abnormal at concentrations above 15%.     

Historical isolation and hydrodynamically constrained gene flow in declining populations of the South-African abalone, Haliotis midae

Over the past two decades, the South African abalone (Haliotis midae), has been under serious threat mainly due to overexploitation. To assure successful management and conservation of wild stocks, the consideration of species-specific evolutionary and population dynamic aspects is critical. In this study, eight microsatellites and 12 single nucleotide polymorphic loci (SNPs) were applied to determine genetic structure in nine populations sampled throughout the species?? natural distribution range. It spans along three biogeographical regions of the South African coastline: temperate in the West coast, warm temperate in the South coast and subtropical in the East coast. Data analysis applying frequentist and Bayesian-based clustering methods indicated weak genetic differentiation between populations of the West, South and East coast. Spatial Bayesian inference further revealed clinal variation along a longitudinal gradient and a transitional zone in the South coast. Coalescent analysis of long-term migration showed restricted interchange among the sampling locations of the South coast while estimates of effective population size were comparable between coastal regions. Furthermore demographic analysis of microsatellite data suggested population expansion, probably reflecting range expansion that occurred following glacial retreat during the Pleistocene. Overall, population structure analysis suggested contemporary (hydrographical conditions) as well as historical (Pleistocene contraction of habitat) restrictions to gene flow. This study provides the foundation for the establishment of an integrated management policy for preserving the natural diversity and adaptive potential of H. midae.     

Colonization of the Gastrointestinal Tract of the Farmed South African Abalone Haliotis midae by the Probionts Vibrio midae SY9, Cryptococcus sp. SS1, and Debaryomyces hansenii AY1

Viable cell counts and/or in situ hybridization were used to determine whether the probionts Vibrio midae SY9, Cryptococcus sp. SS1, and Debaryomyces hansenii AY1 can colonize the gastrointestinal tract of the South African abalone Haliotis midae. The number of culturable probiotic cells reisolated from H. midae fed probiotic-supplemented feed for 3 weeks ranged from 10⁶ to 10⁷ cfu/g gut material. A significant decrease (P < 0.05) in probiont numbers 2 days after feeding the probiotic-supplemented feed had been halted correlated with a significant decrease (P < 0.05) in intestinal protease and amylase activity. There was a positive correlation between Cryptococcus sp. SS1 and amylase activity (r² = 0.681) and V. midae SY9.8 and protease activity (r² = 0.711) in the H. midae intestine. Although culturable probionts were isolated from abalone that had not been fed probiotic-supplemented feed for a 2-week period, the drop in the number of probiotic cells colonizing the abalone digestive tract 2 days after feeding with the probiotic-supplemented feed had been halted indicates that farmed abalone should be fed probiotic-supplemented feed at least every second day for maximum benefit.     

Macrocystis (Laminariales,Phaeophyceae) in South Africa: potential for cultivation through holdfast fragmentation and use as feed for the aquacultured abalone,Haliotis midae

Journal of Applied Phycology - Macrocystis pyrifera (Linnaeus) C. Agardh, known in some regions as ‘giant kelp’, occurs along temperate coastal rocky reefs throughout the southern...     

Comparison of population genetic estimates amongst wild,F1 and F2 cultured abalone (Haliotis midae)

Haliotis midae is South Africa's most important aquaculture species. The reproduction cycle is currently not closed as many farms rely on wild‐caught broodstock for seed production. However, there is an increasing interest in genetic improvement in commercial stocks, with a growing number of producers implementing selective breeding strategies. High throughput commercial production and mass spawning make it difficult to maintain breeding records; therefore, mostly mass selection is practised. The high fecundity and unequal parental contributions also often lead to increased levels of inbreeding. This study therefore aimed to assess the genetic effects of such breeding practices on commercial populations of H. midae. Using microsatellite loci, the genetic properties of a wild, an F1 and an F2 population were estimated and compared. Although there was no significant loss of genetic diversity amongst the cultured populations in comparison with the wild progenitor population, there was low‐to‐moderate genetic differentiation between populations. Relatedness amongst the F2 population was significant, and the rate of inbreeding was high. The effective population size for the F2 (±50) was also comparatively small with respect to the wild (∞) and F1 (±470) populations. These results suggest that farms need to give caution to breeding practices beyond the first (F1) generation and aim to increase effective population sizes and minimise inbreeding to ensure long‐term genetic gain and productivity. This study also confirms the usefulness of population genetic analyses for commercial breeding and stock management in the absence of extensive pedigree records.     

Influence of elevated temperatures on the immune response of abalone, Haliotis rubra

Elevated water temperature can act as a stressor impacting the immune responses of molluscs, potentially increasing their susceptibility to microbial infections. Abalone are commercially important marine molluscs that have recently experienced disease outbreaks caused by a herpesvirus and Vibrio bacteria. Sampling of wild-caught Haliotis rubra showed a significant correlation between water temperature and both antiviral and antibacterial activity, with higher activity in summer than in winter months. However, antibacterial activity was compromised in favour of antiviral activity as the water temperatures peaked in summer. A controlled laboratory experiment was then used to investigate several immune responses of H. rubra, including total haemocyte count (THC), stimulated superoxide anion production (SO), antiviral activity against a model herpesvirus, herpes simplex virus type 1 and antibacterial activity against a representative pathogenic bacterium, Vibrio anguillarum, over one week after raising water temperature from 18 to 21 or 24 °C. THC and SO increased at day 1 and then dropped back to control levels by days 3 and 7. By comparison, the humoural immune parameters showed a delayed response with antibacterial and antiviral activity significantly increasing on days 3 and 7, respectively. Consistent with the field study, antibacterial activity became significantly depressed after prolonged exposure to elevated temperatures. A principal components analysis on the combined immune parameters showed a negative correlation between antiviral and antibacterial activity. SO was positively correlated to THC and neither of these cellular parameters were correlated to the humoural antimicrobial activity. Overall, this study indicates that abalone may have more resilience to viruses than bacterial pathogens under conditions of elevated temperature, such as those predicted under future climate change scenarios.     

Immunological changes in response to herpesvirus infection in abalone Haliotis laevigata and Haliotis rubra hybrids

Australian abalone production has been affected by outbreaks of abalone viral ganglioneuritis (AVG) caused by a herpesvirus (AbHV). In this study, we undertook experimental transmission trials by immersion to study the abalone immune response to infection with AbHV. Representative cellular and humoural immune parameters of abalone, including total haemocyte count (THC), superoxide anion (SO) and antiviral activity against herpes simplex virus type 1 (HSV-1), were examined in apparently healthy (sub-clinical) and moribund abalone after challenge. In the early infection, sub-clinical stage (days 1–3), THC was found to increase significantly in infected abalone. TaqMan qPCR confirmed 20.5% higher viral load in moribund abalone compared to apparently healthy abalone, indicating that the abundance of AbHV within abalone is linked to their clinical signs. At the clinical stage of infection, THC was significantly lower in moribund abalone, but increased in AbHV-exposed but apparently healthy abalone, in comparison to non-infected controls. SO was reduced in all abalone that were PCR-positive for AbHV. THC and SO level were found to be negatively correlated with the presence of AbHV in abalone, but no effect of AbVH exposure was observed on the haemolymph antiviral activity. These results suggest that abalone mount an initial cellular immune response to AbHV infection, but this response cannot be sustained under high viral loads, leading to mortality.     

Observations on the composition and larval developmental modes of polydorid pests of farmed oysters (Crassostrea gigas) and abalone (Haliotis midae) in South Africa

This study provides preliminary insights into how the culture method of molluscs and the larval developmental mode of pest polydorids may determine which species become pests. A total of 10 species were identified on oysters and abalone in offshore and oysters in onshore culture on the west coast of South Africa. One-factor PERMANOVA showed that abalone and oysters cultured offshore housed similar communities of worms, but that they differed significantly from the communities housed by oysters cultured onshore. Oysters from onshore were infested mainly by Polydora hoplura, but also by Polydora cf. websteri and Boccardia pseudonatrix. All these species produce adelphophagic larvae or a mixture of these and planktotrophic larvae. Adelphophagic larvae usually only emerge soon before they settle, which would probably enhance their ability to recruit locally and establish local populations. By contrast, worms from molluscs grown offshore were infested by high abundances of P. hoplura and lower abundances of up to five more species per individual host. All hatched broods observed with these female P. hoplura contained planktotrophic larvae which have a long planktonic phase before settling. This would presumably enhance their ability to reach hosts in the offshore.     

Vibrio carchariae,a pathogen of the abalone Haliotis tuberculata

Since 1997, mass mortality of the abalone Haliotis tuberculata L. has occurred in the natural environment along the French coast. The outbreak of disease started on the south coast of Brittany near Concarneau in 1997, then spread to the north of Brittany (in 1998) and the west coast of Normandy (Golfe de St. Malo in 1999). Between 60 and 80% of the abalone died. In 1999, mortality also affected a land-based abalone farm in Normandy during the summer. At this farm, a Vibrio sp. was isolated in abundance from abalone that had just died. The disease was experimentally reproduced by inoculation or by introducing the pathogen into the surrounding water. This vibrio, identified by genotypic and phenotypic characters, is related to V carchariae. It is similar to the V carchariae, responsible for mortality in the Japanese abalone Sulculus diversicolor supratexta, but some phenotypic characters differentiate both strains. In 2000, healthy abalone placed in 2 sites on the north and south coasts of Brittany died, and the pathogen V carchariae could be isolated from dead individuals, demonstrating that the pathogen was probably the cause of the abalone disease that has been occurring since 1997 in Brittany.