FACTORS AFFECTING THE FERTILIZATION SUCCESS IN LABORATORY HYBRIDIZATION BETWEEN HALIOTIS DISCUS HANNAI AND HALIOTIS GIGANTEA

In this study, effects of sperm concentration and gamete age on fertilization success of Haliotis discus hannai (D) X H. gigantea (G) were investigated. Results showed that the fertilization rates of heterologous crosses H. discus hannai female x H. gigantea male (DG) and H. gigantec female X H. discus hannai male (GD) were consistently lower than those of homospecific groups H. discus hannai female x H. discus hannai male (DD) and H. gigame female X H. gigantea male (GG). In sperm concentration experiments, the sperm concentrations that yielded maximum fertilization rates with the least abnormality in subsequent development were 4.66 X 10(7) sperm/mL for the DG cross and 2.6 x 10(7) sperm/mL for the reciprocal cross GD. In gamete age experiments, the optimal fertilization rates were achieved in heterologous crosses when freshly spawned ova were fertilized with sperm that were released within 0.5 h. Furthermore, in heterospecific crosses, when ova were fertilized 10 min after being spawned, fertilization rates declined significantly with increasing ova age. It is suggested to use fresh gametes and higher sperm concentrations for hybridization between H. discus hannai and H. gigantea

ALLOGYOGENETIC PROGENY ARE PRODUCED FROM A HYBRID ABALONE CROSS OF FEMALE HALIOTIS DIVERSICOLOR AND MALE HALIOTIS DISCUS DISCUS

Interspecific hybrid families of female Haliotis diversicolor X male H. discus discus were produced and analyzed using amplified fragment length polymorphism (AFLP) technology to reveal the genetic makeup of F1 progenies. The survival rates of the hybrid F1 were very low, ranging from 0-0.13%. Twenty hybrid F1 from 3 families along with 3 different female parents and their common male parent were analyzed with 3 AFLP primer combinations. In total, 266 markers were detected. Genetic relationships among the progenies and the parents were evaluated by generating a similarity and genetic distance matrix. The genetic divergence between Haliotis diversicolor and Haliotis discus was at a high level, with genetic distance ranging from 1.471-1.492. The AFLP band patterns of hybrid F1 progeny were similar to those of the female parents, but were quite different from that of the male parent. The mean genetic distance between hybrid F1 and their female parents were 0.024-0.039, slightly less than that among the female parents, which indicates that the hybrid F1 shared high genetic similarity with their female parents, Haliotis diversicolor. However, 0-0.8% of total AFLP bands of each individual were found to be parental bands, and 0-3.3% were found to be nonparental bands. The possible reason for the presence of paternal-specific and nonparental bands is discussed

GENETIC CORRELATIONS TO MORPHOLOGICAL TRAITS OF SMALL ABALONE HALIOTIS DIVERSICOLOR

A total of 241 samples of small abalone (Nation's diversicolor) from 8 full-sib families of market size were used in this experiment. Shell length (X-1), shell width (X-2), shell height (X-3), apex height (X-4), body weight (Y-1), muscle weight (Y-2), and shell weight (Y-3) 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 (Y-1) had the highest correlation coefficient with shell length (X-1), and muscle weight (Y-2) had the highest correlation coefficient with shell width (X-2). The results of high correlation index would be useful for selecting important growth-related traits in genetic breeding program of small abalone

KARYOLOGICAL STUDIES OF THE HYBRID LARVAE OF HALIOTIS DISVERSICOLOR SUPERTEXTA FEMALE AND HALIOTIS DISCUS DISCUS MALE

To determine the genomic composition of the interspecific hybrid between Haliotis diversicolor supertexta female and H. discus discus male at an early developmental stage, veliger larvae produced from hybrid (SJ-5 and SJ-50) and pure species crosses (SS and JJ) were sampled and analyzed using standard karyological methods and genomic in situ hybridization. In hybrid metaphase spreads, chromosomes from both parents were detected, except one metaphase, which showed the H. diversicolor supertexta haploid karyotype. The genomic composition of the hybrid was also confirmed through preliminary genomic in situ hybridization results. Many more aneuploids and chromosome fragments were found in the hybrids than those in the control pure species crosses, indicating genome instability and chromosome loss in the hybrids. In the hybrid hypodiploid metaphase spreads, two intact sets of H. diversicolor supertexta chromosomes and several H. discus discus chromosomes were detected by pairing. Spontaneous diploidization of the maternal chromosome set was shown to occur in hybrid larvae, as 2.2% heterogeneous triploid and 17.9% hypodiploids with two intact H. diversicolor supertexta chromosome sets for SJ-5. The current findings suggest that uniparental chromosome elimination along with spontaneous diploidization of maternal chromosome sets may be the reason for allogynogenesis production in H. diversicolor supertexta X H. discus discus hybridization

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

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 female x G male (DG) and G female x D male (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 X 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

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

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

Types and distribution of mucous cells of the abalone Haliotis diversicolor

The types and distribution of mucous cells of Haliotis diversicolorwere observed and analyzed using the alcian blue and periodic acid schiffs (AB-PAS) reaction and histological procedures. According to the color of the mucous cells, they were divided into four types: Type I, pure red; type II, pure blue; type III, purple reddish; type IV, blue purple. Most of the mucous cells in the mantle were type II, with cup- or stick-like shape. Gill axis and gill filament epithelia were rich in mucous cells and most of them were type II and III, with circle-, cup- and stick-like shapes. There were a few mucous cells in the pedal epithelia, mainly type II, whereas, the pedal gland had a great density of mucous cells, which were large or small and mainly type II and IV. There were many mucous cells in the epithelia around the mouth, most of which were medium-sized cup-shaped type II. In the esophagus, the number of mucous cells decreased gradually from the anterior to posterior, whereas in the distal intestine mucous cells, there were more than in the proximal one and they were more cup–shaped and circle–shaped, mainly type II.Keywords: Haliotis diversicolor; mucous cells, types, distributio

Microbiota characterization of the green mussel Perna viridis at the tissue scale and its relationship with the environment

Research on the microbiota associated with marine invertebrates is important for understanding host physiology and the relationship between the host and the environment. In this study, the microbiota of the green mussel Perna viridis was characterized at the tissue scale using 16S rRNA gene high-throughput sequencing and compared with the microbiota of the surrounding environment. Different mussel tissues were sampled, along with two environmental samples (the mussel's attachment substratum and seawater). The results showed that the phyla Proteobacteria, Bacteroidetes, and Spirochaetae were dominant in mussel tissues. The bacterial community composition at the family level varied among the tissues of P. viridis. Although the microbiota of P. viridis clearly differed from that of the surrounding seawater, the composition and diversity of the microbial community of the foot and outer shell surface were similar to those of the substratum, indicating their close relationship with the substratum. KEGG prediction analysis indicated that the bacteria harbored by P. viridis were enriched in the degradation of aromatic compounds, osmoregulation, and carbohydrate oxidation and fermentation, processes that may be important in P. viridis physiology. Our study provides new insights into the tissue-scale characteristics of mussel microbiomes and the intricate connection between mussels and their environment

Different Transcriptomic Responses to Thermal Stress in Heat-Tolerant and Heat-Sensitive Pacific Abalones Indicated by Cardiac Performance

The Pacific abalone Haliotis discus hannai is one of the most economically important mollusks in China. Even though it has been farmed in southern China for almost 20 years, summer mortality remains the most challengeable problem for Pacific abalone aquaculture recently. Here, we determined the different heat tolerance ability for five selective lines of H. discus hannai by measuring the cardiac performance and Arrhenius breakpoint temperature (ABT). The Red line (RL) and Yangxia line (YL) were determined as the most heat-sensitive and most heat-tolerant line, respectively. Heart rates for RL were significantly lower than those of the YL at the same temperature (p &lt; 0.05). The differentially expressed genes (DEGs), which were enriched in several pathways including cardiac muscle contraction, glutathione metabolism and oxidative phosphorylation, were identified between RL and YL at control temperature (20°C) and heat stress temperature (28.5°C, the ABT of the RL) by RNA-seq method. In the RL, 3370 DEGs were identified between the control and the heat-stress temperature, while only 1351 DEGs were identified in YL between these two temperature tests. Most of these DEGs were enriched in the pathways such as protein processing in endoplasmic reticulum, nucleotide binding and oligomerization domain (NOD) like receptor signaling, and ubiquitin mediated proteolysis. Notably, the most heat-tolerant line YL used an effective heat-protection strategy based on moderate transcriptional changes and regulation on the expression of key genes