Weiwei You

The role of hybridization in improving the immune response and thermal tolerance of abalone

Recently, frequent death of cultured abalone drew our attention to the stress tolerance of abalone. Hybridization is an effective way of genetic improvement in aquaculture, which can introduce improved traits to the hybrids. In this study, we challenged the hybrids between Haliotis discus hannai and Haliotis gigantea, and their parents with bacteria (vibrio harveyi, vibrio alginolyticus and vibrio parahemolyticus), then held them at 20 °C and 28 °C, survival rates of the parental populations and hybrid populations were recorded. Then we tested the immune responses and thermal-induced responses of the four populations at different temperatures. Total hemocyte count (THC), respiratory burst, superoxide dismutase activity (SOD), acid phosphatase activity (ACP), alkaline phosphatase activity (AKP), myeloperoxidase activity (MPO), and HSP70 expression were determined on day 1 and day 7 of the temperature exposure. Results showed higher survival rates of the hybrids than their parents against bacteria challenge. For immune parameters, THCs were evaluated at 28 °C, while increased THC was also observed in H. discus hannai ♀ × H. gigantea ♂ (DG) and H. discus hannai ♀ × H. discus hannai ♂ (DD) at 12 °C (day 7); at 28 °C, respiratory burst was activated (day 1 and 7), while SOD activity first rose then fell over 7-days exposure; AKP activity was elevated at 12 °C and 28 °C (day 1), most notably in DG, and an increased level of ACP was observed in DG at 28 °C (day 7); MPO activity was suppressed at 12 °C and 28 °C on day 1, but recovered on day 7. For HSP70, increased HSP70 levels were observed in all populations at 28 °C (day 1), and DD got the lowest HSP70 level after 7-days exposure at 28 °C. Overall, the results suggest that temperature changes could significantly affect the physiological status of abalone, and hybrids may be more resistant to disease and thermal stresses than their parents.

GENETIC CORRELATIONS TO MORPHOLOGICAL TRAITS OF SMALL ABALONE HALIOTIS DIVERSICOLOR

ABSTRACT A total of 241 samples of small abalone (Haliotis diversicolor) from 8 full-sib families of market size were used in this experiment. Shell length (X1), shell width (X2), shell height (X3), apex height (X4), body weight (Y1), muscle weight (Y2), and shell weight (Y3) were measured, and the correlation coefficient matrix was calculated. The shell shape traits were used as independent variables, then body weight and muscle weight were used as dependent variable for path analysis. Path coefficients, determination coefficients, and correlation index were calculated. The results showed that correlation coefficients between each shell shape trait and body weight, muscle weight, and shell weight were all significant (P < 0.01). For the 4 shell morphological traits, body weight (Y1) had the highest correlation coefficient with shell length (X1), and muscle weight (Y2) had the highest correlation coefficient with shell width (X2). The results of high correlation index would be useful for selecting important growth-related traits in genetic breeding program of small abalone.

HERITABILITY OF GROWTH TRAITS FOR SMALL ABALONE HALIOTIS DIVERSICOLOR ESTIMATED FROM SIB MATINGS

ABSTRACT Twelve half-sib groups and 36 full-sib groups of small abalone Haliotis diversicolor were obtained by the unbalanced nest design using artificial fertilization of 3 females by each male. Heritabilities of growth-related traits were estimated from postlarva to market size at days 10, 40, 120, 220, 320, and 420. The estimated heritabilities based on sire components for shell length and shell width were 0.15–0.37 and 0.18–0.42, respectively. Heritability estimate for shell length and shell width based on dam component were larger than those based on sire component. The results in the current study indicate that genetic improvement through selective breeding conducted on small abalone might be available and would be an efficient method to obtain a positive response to selection.

Genetic changes in muscle protein following hybridization between Haliotis diversicolor reeve Japan and Taiwan populations revealed using a proteomic approach.

Protein expression patterns were compared in a Japan and Taiwan population of Haliotis diversicolor and in a hybrid between them using 2DE and MALDI‐TOF‐TOF analyses. Using the software PDQuest, 924 ± 7 protein spots were detected in the Japan population (RR), 861 ± 11 in the Taiwan population (TT), and 882 ± 9 in the F1 hybrid (TR). RR and TR were clustered together, but the distance between RR and TT was the maximum using hierarchical cluster analysis. A total of 46 gel spots were identified and a total of 15 spots matched with abalone proteins (a 33.6% identification rate). Hybrid exhibiting additivity or overdominance accounted for 73.9% of these 46 identified proteins. The 46 differentially expressed proteins were shown to be involved in major biological processes, including muscle contraction and regulation, energy metabolism, and stress response. The proteins involved in energy metabolism included adenosine triphosphate (ATP) synthase β subunit, fructose 1, 6‐bisphosphate aldolase, triosephosphate isomerase, enolase, arginine kinase, and tauropine dehydrogenase. These proteins exhibited additivity in their offspring. The proteins involved in stress responses included HSP Hsp70 (exhibiting overdominance in the offspring) and Cu/Zn‐superoxide dismutase (exhibiting additivity). These results suggested that proteomic approach is suitable for analysis of heterosis and functional prediction of abalone hybridization.

Molecular cloning of two molluscan caspases and gene functional analysis during Crassostrea angulata (Fujian oyster) larval metamorphosis

Caspases have been demonstrated to possess important functions in apoptosis and immune system in vertebrate. But there is less information reported on the oyster larval development. In the present work, two full-length molluscan caspase genes, named Cacaspase-2 and Cacaspase-3, were characterized for the first time from Fujian oyster, Crassostrea angulata. Which respectively encode two predicted proteins both containing two caspase domains of p20 and p10 including the cysteine active site pentapeptide “QACRG” and the histidine active site signature. Otherwise Cacaspase-2 also contains a caspase recruitment domain. Homology and phylogenetic analysis showed that Cacaspase-2 shared high similarity with initiator caspase-2 groups, but Cacaspase-3 clustered together with executioner caspase-3 groups. Cacaspase-2 and Cacaspase-3 mRNA were both highly expressed in gills and labial palp and were significantly expressed highly in larvae during settlement and metamorphosis. Through the whole mount in situ hybridization, the location of Cacaspase-2 is in the foot of the oyster larvae and the location of Cacaspase-3 is in both the foot and velum tissues. These results implied that Cacaspase-2 and Cacaspase-3 genes play a key role in the loss of foot and Cacaspase-3 gene has an important function in the loss of velum during larvae metamorphosis in C. angulata.

ESTIMATES OF CORRELATIONS FOR SHELL MORPHOLOGICAL TRAITS ON BODY WEIGHT OF INTERSPECIFIC HYBRID ABALONE (HALIOTIS DISCUS HANNAI AND HALIOTIS GIGANTEA)

ABSTRACT In this study, shell length, shell width, shell height, apex height, and body weight of reciprocal hybrid abalone (Haliotis discus hannai ♀ × H. gigantea ♂ (DG) and H. gigantea ♀ × H. discus hannai ♂ (GD)) were measured, and the correlation coefficients were estimated. Path coefficients, determination coefficients, and correlation index were calculated. The results showed that phenotypic correlations between morphological traits and body weight in reciprocal hybrids were positive. The association of the morphological traits and body weight were found to be different in these 2 hybrid abalone. For DG, shell height was highly correlated with body weight among the 4 shell morphological traits, whereas shell width was highly correlated with body weight in GD. Multiple regression equations were obtained to estimate shell length, &KHgr;1 (in millimeters); shell width, &KHgr;2 (in millimeters); apex height, &KHgr;3 (in millimeters); and shell height, &KHgr;4 (in millimeters); against body weight, Y (in grams) of reciprocal hybrids (DG and GD). For DG: Y = -181.723 + 2.4327&KHgr;1 + 1.2548&KHgr;2 + 1.2128&KHgr;3 + 2.2128&KHgr;4; for GD: Y = -241.131 + 3.1122&KHgr;1 + 2.0166&KHgr;2 + 1.7543&KHgr;3 + 2.7312&KHgr;4. These results will help abalone breeding practices and be useful for selecting growthrelated traits in genetic breeding program of hybrid abalone.

AFLP ANALYSIS OF POPULATIONS OF HALIOTIS DISCUS HANNAI, HALIOTIS GIGANTEA, AND THEIR HYBRIDS

ABSTRACT Amplified fragment length polymorphism (AFLP) analysis of the population genetic structure and genetic diversity of Haliotis discus hannai (D), Haliotis gigantea (G), and their reciprocal hybrids D ♀ × G ♂ (DG) and G ♀ × D ♂ (GD) was carried out in this study. A total of 479 unambiguous and highly repeatable AFLP markers, 311 of which (64.93%) were polymorphic, were obtained using 7 primer combinations. The reciprocal hybrids inherited bands from both parents, indicating that the hybrids were truly heterogeneous. The Shannon diversity index for D, G, and their reciprocal hybrid populations DG and GD was 0.169 ± 0.188, 0.211 ± 0.227, 0.236 ± 0.267, and 0.231 ± 0.242, respectively. Analysis of molecular variance revealed that 29.58% of the variance was among populations, whereas 71.42% of variance was within populations. Genetic distance was maximum (0.681) between D and G, and was minimum (0.482) between GD and G. The 4 populations were clustered into 2 major clades using the unweighted pair group method with arithmetic mean. All genetic parameters indicated that there was plentiful genetic diversity in the reciprocal hybrids of D × G. Results of this study suggest that these AFLP markers can be used in the future to enhance current breeding practices in abalone culture because of the large numbers of polymorphic markers.

Molecular Analysis of Atypical Family 18 Chitinase from Fujian Oyster Crassostrea angulata and Its Physiological Role in the Digestive System.

Chitinolytic enzymes have an important physiological significance in immune and digestive systems in plants and animals, but chitinase has not been identified as having a role in the digestive system in molluscan. In our study, a novel chitinase homologue, named Ca-Chit, has been cloned and characterized as the oyster Crassostrea angulate. The 3998bp full-length cDNA of Ca-Chit consisted of 23bp 5-UTR, 3288 ORF and 688bp 3-UTR. The deduced amino acids sequence shares homologue with the chitinase of family 18. The molecular weight of the protein was predicted to be 119.389 kDa, with a pI of 6.74. The Ca-Chit protein was a modular enzyme composed of a glycosyl hydrolase family 18 domain, threonine-rich region profile and a putative membrane anchor domain. Gene expression profiles monitored by quantitative RT-PCR in different adult tissues showed that the mRNA of Ca-Chit expressed markedly higher visceral mass than any other tissues. The results of the whole mount in-situ hybridization displayed that Ca-Chit starts to express the visceral mass of D-veliger larvae and then the digestive gland forms a crystalline structure during larval development. Furthermore, the adult oysters challenged by starvation indicated that the Ca-Chit expression would be regulated by feed. All the observations made suggest that Ca-Chit plays an important role in the digestive system of the oyster, Crassostrea angulate.

Cytogenetic Analysis and Chromosomal Characteristics of the Polymorphic 18S rDNA of Haliotis discus hannai from Fujian, China

We report on novel chromosomal characteristics of Haliotis discus hannai from a breeding population at Fujian, China. The karyotypes of H. discus hannai we obtained from an abalone farm include a common type 2n = 36 = 10M + 8SM (82%) and two rare types 2n = 36 = 11M + 7SM (14%) and 2n = 36 = 10M + 7SM + 1ST (4%). The results of silver staining showed that the NORs of H. discus hannai were usually located terminally on the long arms of chromosome pairs 14 and 17, NORs were also sometimes located terminally on the short arms of other chromosomes, either metacentric or submetacentric pairs. The number of Ag-nucleoli ranged from 2 to 8, and the mean number was 3.61 ± 0.93. Among the scored interphase cells, 41% had 3 detectable nucleoli and 37% had 4 nucleoli. The 18S rDNA FISH result is the first report of the location of 18S rDNA genes in H. discus hannai. The 18S rDNA locations were highly polymorphic in this species. Copies of the gene were observed in the terminal of long or/and short arms of submetacentric or/and metacentric chromosomes. Using FISH with probe for vertebrate-like telomeric sequences (CCCTAA)3 displayed positive green FITC signals at telomere regions of all analyzed chromosome types. We found about 7% of chromosomes had breaks in prophase. A special form of nucleolus not previously described from H. discus hannai was observed in some interphase cells. It consists of many small silver-stained nucleoli gathered together to form a larger nucleolus and may correspond to prenucleolar bodies.

Genetic Variation Analysis in Wild and Cultured Subpopulations of Small Abalone Haliotis diversicolor Estimated by Microsatellite Markers

Abstract The small abalone Haliotis diversicolor is an economically important shellfish species in southern China. Wild (Japanese, Guangdong, and Hainan) and cultured (Taiwanese and Vietnamese) subpopulations of small abalone were examined using seven microsatellite loci to assess the levels of genetic diversity and the degree of genetic differentiation among them. These seven microsatellite markers showed a high level of polymorphism. A total of 209 alleles were detected, with an average of 29.86 alleles per locus among the five subpopulations. The average observed heterozygosities per locus ranged from 0.733 in the Japanese subpopulation to 0.804 in the Hainan subpopulation, while the average expected heterozygosities per locus ranged from 0.909 in the Vietnamese subpopulation to 0.940 in the Guangdong subpopulation. Values for the differentiation index D est varied from 0.1071 to 0.4020 among different subpopulations. The values for genetic distance (D A) ranged from 0.244 to 0.409, with an average of ...