Parentage assignment in hybrid abalones (Haliotis rufescens × Haliotis discus hannai) based on microsatellite DNA markers

Parentage analysis in aquaculture determines genealogical relationships between broodstock and progeny when the parents are unknown. Thus, parentage analysis is a useful tool to establish pedigree reports in molecular‐assisted selection programs. Here, we evaluated 10 heterologous microsatellite markers for parentage assignment in abalone hybrids produced from 43 abalone broodstocks of red abalone (Haliotis rufescens) and Japanese abalone (H. discus hannai). The allele frequencies, exclusion probabilities and broodstock contributions were calculated using CERVUS, PAPA and GERUD software. The polymorphic information content (PIC) values showed that most of the microsatellite loci were highly informative (>0.7) and more than 90% of parentage assignment was possible with a minimum of 5–6 microsatellite markers. Parentage assignment for hybrid and pure‐red progeny showed a better performance than pure‐Japanese progeny. This result could be due to the high level of allele loss in the parental genotypes. In addition, results indicated that only two sires contributed over 80% and 90% of red and hybrid progenies, respectively. This study gives a new molecular tool to support marker‐assisted selection in abalone hybrids produced in Chile.     

Substitution effect of Undaria pinnatifida with citrus (Citrus unshiu,Marcovitch) peel by‐product in feed on the growth,body composition and air exposure stressor of juvenile abalone (Haliotis discus,Reeve 1846)

Substitution effect of Undaria pinnatifida with citrus peel by‐product (CPB) on growth, body composition and air exposure stressor of abalone was determined. A total of 1,080 abalone were distributed into 18 net cages. Five formulated diets were prepared in triplicate. The CPB0 diet contained 200 g/kg U. pinnatifida. The 250, 500, 750 and 1,000 g/kg U. pinnatifida were substituted with the equal amount of CPB, referred to as the CPB250, CPB500, CPB750 and CPB1000 diets, respectively. Finally, dry U. pinnatifida was prepared. Abalone were fed for 16 weeks and then subjected to air exposure stressor for 24 hr. The cumulative mortality of abalone was monitored for the following 4 days after 24‐hr air exposure. Survival, weight gain and specific growth rate (SGR) of abalone fed all formulated diets were greater than those of abalone fed the U. pinnatifida. The greatest weight gain and SGR were achieved in abalone fed the CPB500 diet. The chemical composition of the soft body of abalone was not affected by the experimental diets. Higher cumulative mortality was observed in abalone fed the CPB0 and dry U. pinnatifida at 16 hr after 24‐hr air exposure compared to abalone fed all other diets. In conclusion, U. pinnatifida could be completely substituted with CPB in abalone feed.     

Embryonic and larval development of a hybrid between kelp grouper Epinephelus moara ♀ × giant grouper E. lanceolatus ♂ using cryopreserved sperm

Hybridization was used to take advantage of desirable traits in offspring. In the present study, we applied the cryopreserved Epinephelus lanceolatus sperm into interspecific hybridization with E. moara. Successful hybridization between these two species was achieved and cultured in 23–24°C seawater (34‰). There was no difference in survival rate between hybrid (E. moara ♀ × E. lanceolatus ♂) and non‐hybrid (E. moara ♀ × E. moara ♂) at 24 hrs post hatch (HPH), but less hybrid (14.35 ± 8.02%) hatched than non‐hybrid (93.60 ± 1.65%), which might be due to irregularity cell cleavage and skeletal deformities from the formation of embryonic body to the later embryonic development. Similar phenomenon was found in hybrid embryos from fertilization with fresh sperm, indicating that species variations between parents, rather than cryopreserved sperm, resulted in deformities in embryos. Mean hatch time of the hybrid was 1 hr faster than that of E. moara. The hybrid was 1.95 ± 0.06 mm in total length when newly hatched and reached 38.00 mm at 58 days post hatch (DPH), which showed faster growth than E. moara (recorded in the previous study). Considering its faster growth, E. moara × E. lanceolatus hybrid was a potential breeding production in aquaculture. Over 212,000,000 larvae have been produced and launched in the market since 2015. The results of this study also shed some lights on further comparative studies in grouper hybrid performance.     

Influences of dietary antimicrobial peptide APSH‐07 on the growth performance,immune response and vibriosis resistance of abalone Haliotis discus hannai Ino

A 115‐day feeding trial and subsequently a 10‐day challenge test with Vibrio parahaemolyticus were conducted to investigate the effects of dietary antimicrobial peptide APSH‐07 on growth performance, immune response, antioxidative status and vibriosis resistance of abalone Haliotis discus hannai (initial body weight: 2.06 ± 0.01 g; initial shell length: 25.42 ± 0.18 mm). Four artificial diets were designed with 0 (artificial diet control), 7.5, 15.0 and 22.5 mg/kg of APSH‐07, respectively. The brown alga Laminaria japonica was used as the live food control. Results showed that the specific growth rates of abalone in the groups with 7.5 and 15.0 mg/kg dietary APSH‐07 were significantly higher than those in the controls (p < .05). The total haemocyte counts and respiratory burst activity in haemolymph of abalone in the group with 7.5 mg/kg of dietary APSH‐07 were significantly higher than those in the groups with 0 and 22.5 mg/kg of dietary APSH‐07 (p < .05). The gene expression levels of Mn‐superoxide dismutase and thioredoxin peroxidase 2, and the activities of glutathione peroxidase in the group with 7.5 mg/kg of dietary APSH‐07 were significantly higher than those in the other groups (p < .05). Cumulative mortality of abalone after the challenge test was significantly decreased in the group with 7.5 mg/kg of dietary APSH‐07 supplementation. Supplementation of 22.5 mg/kg dietary APSH‐07 significantly increased the cumulative mortality. In conclusion, 7.5 mg/kg of dietary APSH‐07 supplementation had the better growth performance, higher antioxidation, immune and disease resistance capacity of abalone. Excessive supplementation of dietary antimicrobial peptide APSH‐07 (22.5 mg/kg) had significantly negative effects. Further studies are needed to determine the optimal level of dietary APSH‐07 supplementation for abalone.     

N and P budgets of Haliotis discus hanai,Apostichopus japonicas,and Sebastes schlegeli in a polyculture system

The viability of placing abalones (Haliotis discus hannai), sea cucumbers (Apostichopus japonicas) and rockfish (Sebastes schlegeli) in a polyculture system, the effect of this mixed species group on the system's nitrogen (N) and phosphorous (P) budgets, and the growth and food intake of the organisms in the system were examined using a recirculating aquaculture system. Four replicates were set up for each of three treatment groups (abalone only (C), abalone‐sea cucumber (AS) and abalone‐sea cucumber‐rockfish (ASF)) with an experimental period of 60 d. Compared with the C group, in the AS group the abalone survival rate and specific growth rate (SGR) of body weight increased and the harvested abalones from the polyculture system became the main source of N and P output of the polyculture system. However, the N and P output in the water layer did not differ significantly from that in the C group (p > 0.05), and the N utilization rate was significantly higher than that in the C and ASF groups (p < 0.05). Compared with the AS group, in the ASF treatment the SGR of body weight as well as the protease and amylase activities of sea cucumbers were significantly higher (p < 0.05), the water layer and faeces became the main sources of N and P output in the system. These results showed that the AS polyculture mode significantly improved the N and P utilization rates in the system and led to increased aquaculture production.     

Comparative analysis of the predominant carotenoids and chemical components in the common and orange‐muscle mutant of Haliotis gigantea

The Xishi abalone (Haliotis gigantea) is an economically significant aquaculture species in southern China. We identified a novel orange‐muscle mutation present in less than 2% of the cultured population. High‐performance liquid chromatography and electrospray ionization mass spectrometry identified zeaxanthin and β‐carotene as the two main pigments present in the orange‐muscle abalone. The concentrations of zeaxanthin and β‐carotene detected in the orange‐muscle abalones were significantly higher than those detected in the common abalones (p < 0.01). Notably, the concentration of zeaxanthin was approximately 16.5 times higher in orange‐muscle abalones than in common abalones, which was also the species with highest carotenoid content reported in shellfish to date. Furthermore, the contents of total amino acids and essential amino acids in the orange‐muscle abalone were higher than that in the common abalone, though not significantly, while the content of non‐essential amino acids was significantly higher in the orange‐muscle abalone than that in the common abalone (p < 0.05). The saturated fatty acid content in the orange‐muscle abalones was slightly lower than that in the common abalones, and the unsaturated fatty acid content was higher in the orange‐muscle abalones than that in the common abalones. These results could provide a basis for characterizing the mechanism by which carotenoids accumulate in abalones and further guide the breeding of abalone with orange‐muscle mutants in the future.     

Growth performance and the soft body composition of juvenile abalone,Haliotis discus,Reeve 1846, fed the extruded pellets substituting fish meal and macroalgae with tunic meal of sea squirt,Halocynthia roretzi

Growth performance and the soft body composition of juvenile abalone fed the extruded pellets (EPs) substituting fish meal (FM) and macroalgae (MA) with tunic meal of sea squirt (SS) was investigated. A total of 1,260 abalone were distributed into 18 containers. Six experimental diets were prepared in triplicate. Five diets were pelletized by an extruder pelleter. The 140 g/kg FM and 250 g/kg mixture of MA were included into the control (Con) diet. Five hundred and 1,000 g/kg of each FM and MA were substituted with an equal amount of tunic meal of SS, referred to as the FM50, FM0, MA50 and MA0 diets, respectively. Finally, dry Undaria pinnatifida was prepared. Weight gain and specific growth rate of abalone fed all EPs were greater than those fed U. pinnatifida. Weight gain of abalone fed MA50 and FM50 diets was greater than Con and FM0 diets, but not different from MA0 diet. Higher crude protein and lipid contents were observed in soft body of abalone fed all EPs compared to U. pinnatifida. In conclusion, FM and MA up to 500 and 1,000 g/kg, respectively, could be replaced with tunic meal of SS in EPs without retardation in growth of abalone.     

Isolation,identification and pathogenicity of Vibrio harveyi,the causal agent of skin ulcer disease in juvenile hybrid groupers Epinephelus fuscoguttatus × Epinephelus lanceolatus

The hybrid grouper, Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂), is a newly bred cultivated marine fish species of high economic value. However, a skin ulcer disease with high mortality has occurred, and the responsible pathogen remains unknown. In this study, we summarized the epidemic status and external signs of this disease. We screened potential pathogens and finally isolated one bacterial strain ML01 from affected fish. We subjected healthy juvenile hybrid groupers to bacterial challenge tests with the isolate by immersion, immersion after dermal abrasion and intraperitoneal injection, respectively. Within 14 days post‐infection, the isolate ML01 caused mass mortality of juveniles infected via immersion after dermal abrasion or intraperitoneal injection. Diseased juveniles displayed obvious signs of skin ulcers. The median lethal dose of ML01 by intraperitoneal injection was 1.10 × 10⁵ colony‐forming units. ML01 was identified as Vibrio harveyi by bacterial morphology, analytical profile index identification, 16S rDNA sequencing and multilocus sequence analysis. Antibiotic susceptibility tests showed that ML01 was sensitive to ceftriaxone, doxycycline and minocycline. The results of this study suggest that V. harveyi is the causal agent of skin ulcer disease in juvenile hybrid groupers, thus providing a basis for effective control and prevention of this disease.     

Diet and trophic role of octopus on an abalone sea ranch

Gastric tract (crop and stomach) and stable isotope analyses (SIA) were used to evaluate the dietary composition of resident, commercially important Octopus aff. O. tetricus Gould, and its importance as a predator of Haliotis laevigata Donovan, on artificial abalone habitats. Taxonomic diversity and resolution of diet were higher in crops (13 taxa) than stomachs (10 taxa), with the latter dominated by unidentifiable material (%Volume = 74%). Crops contained mainly molluscs (~54%, including 31% abalone) and crustaceans (33%), suggesting octopus are important predators of abalone on the sea ranch. Dietary composition of crops did not differ with increasing octopus weight (<300 g, 300–999 g, ≥1,000 g wet weight), paralleling the findings of SIA and reflecting the high stocking densities of abalone that are able to be consumed by octopus of all sizes. The SIA also revealed that octopus occupy a mid‐trophic level, slightly below teleosts and loliginid squids.     

Comparison of Edwardsiella ictaluri isolates from different hosts and geographic origins

The intraspecific variability of E. ictaluri isolates from different origins was investigated. Isolates were recovered from farm‐raised catfish (Ictalurus punctatus) in Mississippi, USA, tilapia (Oreochromis niloticus) cultured in the Western Hemisphere and zebrafish (Danio rerio) propagated in Florida, USA. These isolates were phenotypically homologous and antimicrobial profiles were largely similar. Genetically, isolates possessed differences that could be exploited by repetitive‐sequence‐mediated PCR and gyrB sequence, which identified three distinct E. ictaluri genotypes: one associated with catfish, one from tilapia and a third from zebrafish. Plasmid profiles were also group specific and correlated with rep‐PCR and gyrB sequences. The catfish isolates possessed profiles typical of those described for E. ictaluri isolates; however, plasmids from the zebrafish and tilapia isolates differed in both composition and arrangement. Furthermore, some zebrafish and tilapia isolates were PCR negative for several E. ictaluri virulence factors. Isolates were serologically heterogenous, as serum from a channel catfish exposed to a catfish isolate had reduced antibody activity to tilapia and zebrafish isolates. This work identifies three genetically distinct strains of E. ictaluri from different origins using rep‐PCR, 16S, gyrB and plasmid sequencing, in addition to antimicrobial and serological profiling.     

Hybridization between Haliotis rufescens and Haliotis discus hannai: evaluation of fertilization,larval development,growth and thermal tolerance

Two introduced abalone species are currently produced in Chile, red abalone Haliotis rufescens and Japanese abalone Haliotis discus hannai. However, red abalone accounts for 99% of total production, while the Japanese abalone has not adapted well to Chilean coastal waters. This study reports the hatching, growth and thermal tolerance performance in interspecific hybrids produced between red (R) and Japanese (J) abalone. Our results show that egg age and sperm concentration were critical factors to produce hybrids. The cross R♀ × J♂ showed a fertilization rate of 55.3 ± 3.5% using 20‐min‐old eggs and sperm concentrations of 14 × 10⁶ cells mL^?1, while the reciprocal cross (J♀ × R♂) was not successful. Further, larval development stages were similar in RR, JJ and RJ hybrid abalones. Among the experimental trials, settlement rate varied from 12.3% to 18.6% and final survival from 20.1% to 31.7%, being the RJ hybrid rates intermediate between parental species. The final shell lengths were similar between RR and RJ hybrids, but significantly higher in JJ abalones. In addition, thermal tolerance was ascertained due its pivotal role for the abalone physiology. Thus, RJ hybrids showed the highest HSP70 gene expression and offers new possibilities to expand Chilean abalone production in warm waters zones.     

Octopus as predators of abalone on a sea ranch

This study examined the occurrence of octopus across an abalone, Haliotis laevigata, Donovan, sea ranch in south‐western Australia, to understand how octopus may be impacting abalone production. Commercial divers removed 654 octopus and 17,666 empty abalone shells during regular, 2 to 4‐weekly surveys over 27 months. A negative binomial generalised linear model estimated a 78% increase in empty shells per artificial abalone habitat per day, when octopuses were present, after adjusting for location and season. Of the 408 shells examined for evidence of predation, 19% had a small, slightly ovoid hole consistent with those made by octopus. The mean (± 1 SE) length of shells with boreholes (70.3 ± 2.2 mm) was significantly longer than those without (59.8 ± 0.5 mm), and boreholes were concentrated over the adductor, respiratory organs and heart. This study provides important insights into the adaptable feeding regimes of octopus and their potential to impose strong top‐down controls on sea ranching operations.     

Multilocus sequence typing detects new Piscirickettsia salmonis hybrid genogroup in Chilean fish farms: Evidence for genetic diversity and population structure

Piscirickettsia salmonisis the causative bacterial pathogen of piscirickettsiosis, a salmonid disease that causes notable mortalities in the worldwide aquaculture industry. Published research describes the phenotypic traits, virulence factors, pathogenicity and antibiotic‐resistance potential for various P. salmonisstrains. However, evolutionary and genetic information is scarce for P. salmonis. The present study used multilocus sequence typing (MLST) to gain insight into the population structure and evolution of P. salmonis. Forty‐two Chilean P. salmonisisolates, as well as the type strain LF‐89^T, were recovered from diseased Salmo salar, Oncorhynchus kisutchand Oncorhynchus mykissfrom two Chilean Regions. MLST assessed the loci sequences of dnaK, efp, fumC, glyA, murG, rpoD and trpB. Bioinformatics analyses established the genetic diversity among P. salmonis isolates (H = 0.5810). A total of 23 sequence types (ST) were identified, 53.48% of which were represented by ST1, ST5 and ST2. Population structure analysis through polymorphism patterns showed few polymorphic sites (218 nucleotides from 4,010 bp), while dN/dS ratio analysis indicated purifying selection for dnaK, epf, fumC, murG, and rpoD but neutral selection for the trpB loci. The standardized index of association indicated strong linkage disequilibrium, suggesting clonal population structure. However, recombination events were detected in a group of seven isolates. Findings included genogroups homologous to the LF‐89^T and EM‐90 strains, as well as a seven‐isolate hybrid genogroup recovered from both assessed regions (three O. mykiss and four S. salar isolates). The presented MLST scheme has comparative potential, with promising applications in studying distinct P. salmonis isolates (e.g., from different hosts, farms, geographical areas) and in understanding the epidemiology of this pathogen.     

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%.     

Aeromonas shuberti as a cause of multi‐organ necrosis in internal organs of Nile tilapia,Oreochromis niloticus

A disease with white spots in internal organs of Nile tilapia occurred in Zhanjiang, southern China. Multiple, white nodules, 0.8–2.2 mm in diameter, were scattered throughout the liver, spleen and kidney of diseased fish. Signs of nodules reproduced after artificial infection with the isolated strain. Isolated bacteria were Gram‐negative, facultative anaerobic, motile, short rod‐shaped, with a length of 1.2–2.2 μm. Morphological and biochemical tests, as well as phylogenetic analysis, all strongly indicated that the isolate from tilapia is identical to Aeromonas schubertii (A. schubertii) which temporary named LF1708 strain. Antibiotic sensitivity assays showed the LF1708 is sensitive to 24 of 27 tested antibiotics. Pathogenicity test revealed that the isolate at the dose of 3.75 × 10⁶ CFU/g killed 100% of experimental tilapia within 2 days and the dose of 1 × 10⁷ CFU/g killed 100% of experimental zebrafish within 1 day. Histopathology of diseased tilapia infected with A. schubertii showed numerous necrotic lesions widely distributed in spleen, liver and kidney, and infiltration with a large number of bacteria. To our knowledge, this was the first report that associated A. schubertii with mortality in tilapia.     

New perspective to control of tenacibaculosis in sea bass Dicentrarchus labrax L

This work was carried out to investigate the effects of injection of Tenacibaculum maritimum formalin‐killed cells (FKC), extracellular products (ECPs) and crude lipopolysaccharide (LPS) as well as 1% feed supplements of oil extracts of Echinacea purpurea and Origanum vulgare on sea bass immunity improvement to the favour of T. maritimum experimental infection control after 4 weeks of the experiment. Tenacibaculum maritimum isolated from naturally infected sea bass showed brown to yellowish‐brown lesions (sores) on gills, skin and/or fins and identified by different biochemical methods and polymerase chain reaction technique. Immune parameters namely, total protein, globulin and lysozyme activity, as well as the relative level of protection were improved by T. maritimum (FKC), (LPS), (ECPs), O. vulgare and E. purpurea, respectively compared with control. Histopathological examination of T. maritimum naturally infected sea bass indicated many pathological changes in gill, skin and musculatures. Present study could be concluded that application of T. maritimum (FKC), (LPS), (ECPs), O. vulgare or E. purpurea improved sea bass immunity to the favour of disease resistance against T. maritimum. Further investigations on the combination between the previous control methods and the vaccine application methods will be needed.     

Protective effects of dietary selenium on abalone Haliotis discus hannai against the toxicity of waterborne cadmium

This study was conducted to investigate protective effects of dietary selenium (Se) on abalone Haliotis discus hannai Ino against the toxicity of cadmium (Cd). A 60‐day feeding trial was conducted with abalone (initial weight: 3.17 ± 0.01 g), which were exposed to 0.34 mg/L of waterborne Cd. During a feeding trial, abalone were fed graded levels of Se at 0.10 (controls), 1.31, and 4.20 mg/kg diet respectively. Results showed that there was no significant difference in specific growth rate and survival rate of abalone among the three treatments. Compared with the controls, dietary Se significantly decreased Cd concentrations in serum, muscle, mantle, gill, mantle, and hepatopancreas of abalone. Besides, compared with the controls, dietary Se significantly increased metallothionein concentration in the hepatopancreas of abalone. Additionally, compared with the controls, dietary Se significantly decreased concentrations of malondiadehyde and protein carbonyl in hepatopancreas of abalone. Meanwhile, compared with the controls, dietary Se significantly increased activities of glutathione peroxidase, thioredoxin reductase, and thioredoxin peroxidase, and concentration of glutathione in the hepatopancreas of abalone. Based on the data above, in abalone, dietary Se showed protective effects against waterborne Cd.     

The effects of elevated pCO2 on growth,shell production and metabolism of cultured juvenile abalone,Haliotis iris

Reduced seawater pH and elevated pCO₂ are important considerations in tank‐based abalone aquaculture, while sea‐based farms may be at risk to ocean acidification reductions in pH. Juvenile Haliotis iris (5–13 and 30–40 mm shell length) were reared in two, 100‐day experiments at ambient pH_nbs (~ 8.1, 450 μatm CO₂), pH 7.8 (~1000 μatm CO₂) and pH 7.6 (~1600 μatm CO₂). Seawater pH was measured and adjusted automatically by bubbling CO₂ into water in replicated flow through tanks. Two separate trials were run, in winter (8.8°C) and summer (16.5°C). Survival and growth were monitored every 30 days, and post experiment measurements of morphometrics and respiration rate undertaken. Growth of shell length and wet weight were negatively affected by reduced pH, with a 2 to 3‐fold reduction in growth of both size classes between ambient and pH 7.6 treatments in the summer experiment. For small juveniles, growth reductions were in conjunction with decreases to shell weight, while large juveniles showed greater resilience in shell production. No changes to respiration rate occurred, suggesting that juveniles may maintain physiological functioning while tolerating dissolution pressure or that they are unable to upregulate metabolism to compensate for pH effects. These data show that CO₂ driven reductions in pH can impact growth, metabolism and biomineralization of abalone, and indicate that water quality and ocean acidification are of importance in aquaculture of the species.