Pathogenesis of vibriosis in cultured juvenile red abalone, Haliotis rufescens Swainson

Abstract. Morbidity of intensively cultured red abalone, Haliotis rufescens , as well as experimentally stressed (elevated temperature and hyper-oxygenation) abalone, was studied using clinical, histological, immunofluorescent and bacteriological techniques. Histological study showed a typical pattern of bacterial infection from all groups studied, characterized by epithelial exfoliation or rupture and systemic growth of the bacteria along vascular sinuses and along neural sheaths. Peripheral neurons degenerated rapidly and a responsive host cellular infiltrate did not appear to effectively retard the advancement of the infection. Nine bacterial isolates from the culture system water or sick animals were characterized biochemically. All were Gram-negative, facultatively anaerobic, non-aerogenic, oxidase-positive rods with single polar flagella and thus appeared related to the Vibrio group. Further characterization showed that most isolates did not correspond to specifically characterized vibrios, Antiserum prepared to the isolates contained antibody both to common group antigens (from all nine strains) and to strain-specific antigens. Selection of antiserum and subsequent absorption permitted the use of the antiserum for specific recognition of each isolate. Immunofluorescent studies clearly demonstrated that antiserum to an isolate corresponding to Vibrio alginolyticus was the predominant antiserum producing positive staining of infecting bacteria in the typical lesions in abalone tissues. The pattern of positive staining corresponded to histopathological observations of the disease. The disease can be managed in husbandry systems by both limiting the number of potentially pathogenic bacteria and by limiting the exposure of the animals to physico-chemical stresses.     

Comparative performance of juvenile red abalone, Haliotis rufescens, reared in laboratory with fresh kelp and balanced diets

Juvenile Haliotis rufescens were reared in the laboratory in order to investigate the extent to which fresh kelp and formulated feeds with 250 g kg⁻¹ (25P) and 380 g kg⁻¹ protein content (38P) affected their growth rate, gut residence time (GRT), food consumption ( C ), food conversion ratio (FCR) and digestibility. Abalone from 38P attained the highest growth rate (70.5 ± 4.2 μm day⁻¹; 98.3 ± 6.95 μg day⁻¹), followed by 25P (47.9 ± 2.79 μm day⁻¹; 67.4 ± 2.82 μg day⁻¹) and kelp (23.6 ± 3.36 μm day⁻¹; 28.2 ± 4.11 μg day⁻¹). No significant differences were observed in consumption rate among treatments (0.61–0.68% body weight per day), yet kelp-fed abalone exhibited higher FCR (2.44), protein efficiency ratio (4.42), and apparent digestibility of dry matter (69.5%), protein (69.8%) and gross energy (79.2%) than 38P organisms (59.8, 62.4 and 62.2%, respectively). They also showed longer GRT (23.1 ± 0.93 h). This study demonstrated that formulated diets with 250 g kg⁻¹ and 380 g kg⁻¹ protein inclusion can sustain higher growth rates of juvenile H. rufescens than fresh algae. These differences seem to be due to the amount of dietary protein. Kelp meal appears to improve the consumption and digestibility of balanced diets, and its inclusion in formulated diets is recommended.     

Genetic certification of presumed hybrids of blue × red abalone (Haliotis fulgens Philippi and H. rufescens Swainson)

Presumed hybrids between Haliotis rufescens and H. fulgens abalones, produced in a commercial abalone laboratory, were evaluated for progeny viability, gonad characteristics and for their ‘true’ status as hybrids by means of allozyme and microsatellite analyses. Four out of 10 evaluated allozyme loci proved to be useful for the genetic certification of hybrids, Gpi, Mdh‐1, Mdh‐2 and Sod, as they had exclusive alleles for each of the abalone reference species from which the presumed hybrids were produced. Three microsatellite loci were also evaluated for their usefulness in abalone hybrid certification. Two were not useful for hybrid certification as the reference abalone species shared some of the same alleles. The third loci (Hka 56) also had shared alleles between the two species, but at very different frequencies. Therefore, this permitted a presumptive certification of hybrids, which, however, was not error‐proof as two allozyme certified hybrids were wrongly classified as H. rufescens rather than as hybrids. Progeny viability of the only female certified to be a hybrid was significantly lower compared with one viability of the other females spawned, which were certified as H. rufescens. However, the number of eggs spawned by the only certified hybrid female was as large as those spawned by females certified as H. rufescens. Gonads of abalones sampled for the macroscopic appearance of sterility indicated that in the absence of any gonad development, no distinctive difference allows for hybrid identification, as there were undifferentiated individuals that were both hybrid and non‐hybrid. On the other hand, two certified hybrid males were characterized by spermatocytes being the most abundant gamete type, although one had some spermatozoa present, indicating only partial sterility. Among the two female hybrids found, vitellogenesis was observed to proceed up to vitellogenic oocytes.     

Low‐density culture of red abalone juveniles,Haliotis rufescens Swainson 1822, recirculating aquaculture system and flow‐through system

Commercial abalone culture is carried out using flow‐through systems with a high water volume exchange in Baja California, Mexico. The objective of this work was to compare the growth rate and survival of red abalone cultured in two systems. Flow through (daily water exchange rate of 800%) and recirculating systems consisted of a 250 L fibreglass tank and constant aeration, but biofiltration in the recirculating system was provided with a 28 L (1 ft³) bubble‐washed bead filter. Water variables were measured either daily (dissolved oxygen, temperature, pH and salinity) or three times a week (total ammonia nitrogen, nitrate‐nitrogen, nitrite‐nitrogen and alkalinity). Shell length was measured every 2 weeks for 18 weeks. Only the alkalinity and pH were significantly different due to the addition of sodium bicarbonate to the recirculating system. Abalone growth rate was 26.1 ± 15.96 μm day^?1 in the recirculating systems and 22.21 ± 18.69 μm day^–1 in the flow‐through systems. The final survival was 78.74% in the recirculating systems and 71.82% in the flow‐through systems. Significant differences in the final size and survival of the abalones were found between systems (P<0.05). Therefore, recirculating aquaculture systems is a feasible alternative for juvenile red abalone culture.     

Histological evidence of accumulation of iron in postlarvae of red abalone,Haliotis rufescens

The effect of iron on abalone postlarvae (Haliotis rufescens) was investigated in a controlled‐culturing system. Three iron concentrations (0.15, 1.5 and 15 mg L^?1 of Fe) and a control (no iron added) were used to culture H. rufescens postlarvae while being fed the diatom Navicula inserta over 10 days. Results indicate that H. rufescens postlarvae accumulate iron granules in the stomach, digestive gland and mantle, but not in the gills or other tissues. The number and diameter of iron granules in tissues increased with increasing iron concentration in the culturing environment. The iron accumulation is assumed to have been acquired in the digestive system through the iron‐enriched diatom feed and in the mantle through subcutaneous iron transfer. The lack of iron granules in the gills suggests that iron is not absorbed through the respiratory system, as is the case for many filter feeding bivalves. Exposure to the highest iron concentration (15 mg L^?1) resulted in tissue abnormalities where granules accumulated, and may have significantly affected the health of H. rufescens postlarvae. These findings provide valuable information for the regulation of appropriate iron levels within aquaculture settings and highlights the importance of monitoring iron levels within abalone larval culturing environments.     

Bioeconomic effect from the size selection in red abalone intensive culture Haliotis rufescens as a production strategy

The variability in growth is a common characteristic in mollusks breeding. Effects rising from the variability in the individual growth rate and the consequent dispersion of sizes in cultivation are important in financial terms. To manage this heterogeneity many firms use size selection, which can happen in two stages: toward the end of the stage of growing, or in the phase of growing of seeds. A bioeconomic model simulating the operation of a firm producing red abalone was implemented in spreadsheets. The firm produces 70 tons yearly. The model was structured in three sub-models. A biological sub-model detailed a batch’s dynamics, in terms of survival and growth, considering individual variation of size around a central value for each age. A technological sub-model described raw materials, the quantity of food and the energy required. Finally, the simulation model is completed with an economic integrated sub-model, where net present value is calculated considering income and costs over the time. Results of the alternative production strategies (with or without selection) are assessed according to: quantity of larvae and necessary spawners to reach the desired level of production; net present value (NPV) and necessary time to recover the investment. The number of larvae was approximately 17 millions larger for the case of the strategy of production with sizes selection and 73% more of available spawners is required for this larger amount of larvae. In the short term, the size-selection strategy increases the production costs at the initial time, compared with the strategy without selection. However, in the long term, this strategy generates greater NPV. The span for investment recovery was shorter for the case of the strategy with size selection and living product (nearly 2,140 days) than frozen (nearly 2,232 days); while without sizes selection a 15-year simulation showed the investment is not recovered. Finally, could be verified that size selection can be an interesting strategy to explore, since it improves the financial result, the same way other more expensive technological changes would.     

Factors influencing half‐pearl (mabe) production and quality in the red abalone Haliotis rufescens

The factors influencing half‐pearl (mabe) production in the red abalone Haliotis rufescens were investigated, as a strategy to optimize the technology for the development of a pearling industry in Chile. The effects of abalone size (small, large), implant position (dorsal, central, ventral and their combinations), and number of nuclei (one, two, three) were analysed on the quantity, thickness of the nacre layer and quality (shape, colour, lustre, defects) of the mabe produced. The size of abalone did not exert a clear influence on the quantity and quality of mabe, but the position on the shell (firstly) and number of nuclei (secondly) did. More mabe, with thicker nacre, brighter lustre and fewer defects occurred in small or large abalone implanted with only one nucleus on dorsal position. In contrast, small and large abalone implanted in the ventral position formed less mabe, with thinner nacre, poor lustre and more defects. This likely occurs because mantle tissue folds at the ventral position and does not always cover the nucleus, leaving it partially or totally exposed. Most of the pearls were categories AA in small abalone and A in large abalone; only three AAA gems were produced in large abalone with one nucleus on dorsal position or with three nuclei (DCV). Based on these results, we recommend implanting abalone at 60–100 mm shell length, with only one nucleus on the dorsal position.     

Use of macroalgae supplemented with probiotics in the Haliotis rufescens (Swainson, 1822) culture in Northern Chile

In this study, we evaluate the use of macroalgae as vectors of probiotics bacteria into the digestive tract of abalone to improve their survival and growth. It is shown that when abalone Haliotis rufescens of different sizes were fed with a natural diet composed of fronds of the macroalga Macrocystis integrifolia supplemented with a mixture of Vibrio sp. C21‐UMA, Agarivorans albus F1‐UMA and Vibrio sp. F15‐UMA bacteria, there was a significant increase (P<0.05) in the average monthly growth rate and survival (%)in a period of 210 days, compared with the control without a probiotic supplement. The permanence of the probiotics in the digestive tract of the animals was monitored, and it was found that the number of culturable C21‐UMA and F1‐UMA bacteria decreased significantly (P<0.05) in recently weaned and adult abalone, and they almost disappeared completely on day 19 of the bioassay. However, the culturable Vibrio sp. F15‐UMA disappeared completely from the digestive tract on day 22 of the bioassay, and these were the bacteria that remained at the highest concentration compared with the other two bacterial strains over the experimental period. It is therefore shown that it is feasible to use a probiotic mixture to improve the profitability of the H. rufescens culture.     

Effects of oxygen supersaturation and temperature on juvenile greenlip, Haliotis laevigata Donovan, and blacklip, Haliotis rubra Leach, abalone

Growth and survival of juvenile greenlip (39.03 (SD 3.80) mm (n=524)) and blacklip (31.92 (SD 4.19) mm (n=531) abalone were investigated at high dissolved oxygen levels (95–120% saturation) between 17 and 19°C. Abalone were fed the same artificial diet and each species was contained in groups of approximately 30 individuals within triplicate tanks for each of six treatments and were exposed to flow through water adjusted to give experimental conditions for up to 75 days. Blacklip abalone held at 16.9°C and 97% oxygen saturation grew in shell length significantly faster than all other treatments of blacklip abalone held at 19°C, and significantly faster than blacklip abalone maintained at 111% oxygen saturation and 17.5°C. Both temperature and oxygen saturation significantly affected the survival of this species. Blacklip abalone held at 19°C had significantly lower survival for both 96% oxygen saturation and 120% oxygen saturation, compared with blacklip abalone maintained at either 110% oxygen saturation and 19°C, or for any 17°C treatment. No significant differences were noted for greenlip abalone within the range tested in terms of growth rate, food consumption rate or survival, indicating that greenlip abalone tolerated these conditions better than did blacklip abalone.     

Effect of three bacterial isolates from a commercial hatchery on early red abalone (Haliotis rufescens) postlarvae

Three bacterial strains, GHrC11, GHrC13 and GHrC15, were isolated from an abalone postlarval culture system in a commercial farm at Baja California, México. The strains were phenotypically characterized and sequenced (16S rDNA). Strain GHrC11 was a Gram-positive coccobacillum, while strains GHrC13 and GHrC15 were Gram-negative bacilli. Strain GHrC11 was identified as Exiguobacterium sp. The strains GHrC13 and GHrC15 were identified as Vibrio splendidus. The effects of these strains for the development of early abalone postlarvae (2 days old) were evaluated following a completely randomized design with three replicates using 5-mL-volume Petri dishes as experimental units. The experiment considered two different bacterial concentrations of each strain (10³ and 10⁵?cells?ml^?1) and two controls (with and without the benthic diatom Navicula incerta). After 10?days of experimentation, the highest mortality (90?±?5.8?%) and the lowest growth rate (4.1?±?0.1?μm?day^?1) were recorded for the strain GHrC11. In contrast, the lower mortality (16.7?±?3.3?%) and the highest growth rate (11.2?±?0.9?μm?day^?1) corresponded to the control fed N. incerta. Our results suggest that pathogenic effects of these bacterial strains were stronger than any potential benefits derived from the ingestion of bacteria by early abalone postlarvae. In conclusion, the most pathogenic strain was GHrC11, and the intensity of pathogenicity could be ordered as Exiguobacterium sp.?>?V. splendidus (C13)?>?V. splendidus (C15).     

Changes of heritability and genetic correlations in production traits over time in red abalone (Haliotis rufescens) under culture

Red abalone Haliotis rufescens is one of the most valuable mollusks in the international market, but it has a low growth rate. A breeding program is being developed to increase its growth rate in Chile. We estimated the changes in direct heritability (h²), maternal/common environments heritability (m²) and genetic correlations (r_G) of growth traits (shell length and width, total mass, flesh mass and foot protein as an indicator of meat quality) measured during 2 years (every 4 months) from the juvenile stage (27 months) to the adult harvesting age (51 months), in 60 full‐sib red abalone families. Heritabilities for growth traits measured in juveniles and young adults (27–35 months of age), were low (0.07–0.17) and not significant. Initial low h² were associated with significant amounts of maternal/common environmental effects (m² = 0.4). In young adults and abalone near the harvest age (39–51 months of age) h² were much higher (0.32–0.75). These results emphasize the importance of multiple estimations of h² over time. Among meat quality traits, only the h² for the flesh mass for adults at harvesting age was significant (0.15). We observed strong positive r_G (>0.9) between shell sizes (easy to measure) and total and flesh masses (trait more related to market value than shell sizes but harder to measure) for adults at harvesting age. Thus, if the 5% largest 51 month old abalone were selected from the population as broodstock we expect a positively correlated response on flesh mass of 23.4%.     

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 (T_optG) 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 T_optG 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.     

Growth performance of weaning red abalone (Haliotis rufescens) fed with Macrocystis pyrifera plantlets and Porphyra columbina compared with a formulated diet

A feeding experiment was carried out to evaluate two natural diets versus a formulated feed on the performance of weaning red abalone Haliotis rufescens . Four treatment diets were then investigated: a formulated diet; plantlets from culture Macrocystis pyrifera, Porphyra columbina from natural beds; and a mixed diet consisting of a blend of fresh P. columbina together with the formulated diet. This study was performed in a shallow aquaculture system with a horizontal water flow. After 90 days, significant differences were observed between treatments. The highest growth was obtained with Porphyra (3.3 mm month⁻¹), followed by the mixed diet (2.6 mm month⁻¹), then Macrocystis (2.1 mm month⁻¹) and lastly the formulated diet (1.4 mm month⁻¹). Moreover, after the diets were tested for stability and remnant nutrients after a 12-h water immersion, a positive trend appeared to be related to the protein/energy (P:E). It is concluded that fronds of P. columbina resulted in the best diet for weaning H. rufescens under horizontal water flow systems, even if apparently the water stability of the formulated diet had a negative impact on abalone performance, particularly due to a poor nutrient retention capacity. Therefore, formulated diets should be improved before being recommended for weaning red abalone.     

Effects of dietary guaiacol on shell biomineralization of juvenile abalone Haliotis discus hannai, Ino

A study was conducted to investigate the effects of guaiacol on shell biomineralization of abalone Haliotis discus hannai Ino. Seven semi-purified diets were formulated with graded levels of guaiacol (0, 5, 10, 50, 100, 500 and 1000 mg kg⁻¹). Abalone (initial weight: 0.29±0.01 g; initial shell length: 8.55±0.27 mm) were fed with these diets in a re-circulated water system for 152 days. The results showed that the contents of calcite in the shell increased significantly with dietary guaiacol levels, but the aragonite content decreased significantly. Dietary guaiacol significantly decreased the contents of calcium and strontium, but significantly increased the magnesium content in the shell. The effects of dietary guaiacol on the contents of phosphorus and zinc in shell were not significant. It was obvious that the ratio of potential acidic/basic amino acids [(Asx+Glx)/(Lys+Arg+His)] in the shell was elevated by dietary guaiacol. In conclusion, dietary guaiacol has effects on both the mineralogy and the amino acid composition in the shell. Furthermore, changes in mineralogy are related to the ratio of (Asx+Glx)/(Lys+Arg+His) in the shell.     

Optimal protein level in a semipurified diet for juvenile greenlip abalone Haliotis laevigata

To optimize dietary protein level in relation to growth, semipurified diets with an essential amino acid profile similar to that of the soft body profile were fed for 59 days to young greenlip abalone, Haliotis laevigata (initial shell length, 15–25 mm). Animals were housed in 10-L acrylic tanks, with flow-through seawater supplied at 1 L min^–1 (20 °C, salinity= 36 g L^–1). Protein level of feeds ranged from 122 g kg^–1 to 461 g kg^–1 crude protein (CP) on an 'as is' basis. Second-order polynomial regression analysis of specific growth rate indicated that maximal growth occurs at 270 g kg^–1 CP. The protein and energy components of the feed were estimated to have a digestibility of 71.7% and 55.6%, respectively.     

Growth and biochemical composition of juvenile green abalone Haliotis fulgens fed rehydrated macroalgae

Under controlled laboratory conditions, juvenile green abalone Haliotis fulgens were fed rehydrated natural feeds to determine the effects on growth, survival, feed efficiency and biochemical composition of the digestive gland and muscle. Five macroalgae, Ulva sp. (Chlorophyta), Eisenia arborea, Macrocystis pyrifera, Egregia menziesii (Phaeophyta) and Porphyra perforata (Rhodophyta) were tested. The macroalgae promoted growth, although, depending on the species, there were considerable differences in growth, feed efficiency and biochemical composition of the digestive gland and muscle. Mean growth rate in length and weight and survival rate varied within the ranges 1.6–15.1 μm day^?1, 1.4–8.1 mg day^?1 and 44–69%, respectively. Significantly, higher growth rates were obtained from Egregia menziesii, M. pyrifera and P. perforata. Feed conversion ratio ranged from 6.5 to 42.4 for P. perforata and Ulva sp. Protein, carbohydrates and lipid contents in the digestive gland ranged from 113 to 180, 98.3 to 448 and 17.2 to 23.3 mg g^?1_, respectively. In muscle, the ranges were 66.9–123, 9.5–23.2 and 2.8–3.9 mg g^?1, respectively. This study shows that rehydrated Egregia menziesii, M. pyrifera and, particularly, P. perforata are more efficient in promoting growth than Ulva sp. and E. arborea which match results reported by other authors when using the same fresh macroalgae.     

Temperature and size range for the transport of juvenile donkey's ear abalone Haliotis asinina Linne

Live transport of hatchery‐produced juvenile donkey's ear abalone Haliotis asinina Linne was examined to evaluate the effect of transportation on the survival of juvenile abalone. Simulated transport experiments were conducted to determine the appropriate temperature using 5, 10 and 20 g L^?1 of ice to air volume for 8 h and the appropriate size using two size groups (Size A, 15–20 mm, 0.5–1.3 g, and Size B, 30–35 mm, 5.3–8.5 g) up to 24‐h out‐of‐water live transport. Survival was significantly higher (P<0.001) when 10 g L^?1 of ice was used to decrease the temperature to the range of 17–23 °C. At this temperature, both size groups subjected to simulated transport for 8 and 10 h had 100% survival after 48 h, while mortality occurred in abalones subjected to 16 and 24 h of simulated transport. The Size B abalone subjected to 24 h of transport had significantly higher survival (64.4 ± 2.9%) (P<0.001) than the Size A abalone (5.5 ± 1.6%) after 48 h. Live juvenile abalone were successfully transported to the field applying the protocols developed in the lab experiment. This study serves as a guide for handling and shipping live juvenile abalone.     

Karyotype analysis of interspecific hybrids between Haliotis rufescens and Haliotis discus HANNAI

Evidence of hybridization in Haliotis has been mainly supported by hatchery experiences and collection of wild hybrid abalones among several species from natural populations worldwide. However, despite the importance to understand the role of the hybridization process through Haliotidae evolution, and also its impact on the abalone aquaculture, genetic studies in hybrid abalones have been poorly developed. Herein, cytogenetic approach allows studying the genetic conformation in hybrid organisms at the chromosome level. This paper reports a quantitative karyotype analysis in Haliotis rufescens, Haliotis discus hannai and their interspecific hybrid. Thus, to characterize chromosome pairs and establish cytogenetic comparisons, chromosome banding with distamycin‐A/4,6‐diamidino‐2‐phenylindole fluorochromes and morphologic measurements were performed. The results showed that the hybrids are successfully viable and their karyotypes evidenced a conservative chromosome number of 2n=36. The karyo‐idiogram showed a high correspondence in chromosome pair morphology among the hybrids and their parental species, except for a single heteromorphic pair that corresponds to the chromosome 16 from H. rufescens andH. d. hannai respectively. The implications of the abalone hybrid viability derived from its chromosome composition are discussed.