Heat shock proteins (HSPs) are highly conserved cellular proteins that contribute to adaptive responses of organisms to a variety of stressors. In response to stressors, cellular levels of HSPs are increased and play critical roles in protein stability, folding and molecular trafficking. The mRNA expression pattern of two well-known heat shock protein transcripts, HSP70 and HSP90 were studied in two tissues of nerve ganglia, cerebral ganglion and pleuropedal ganglion of Pacific abalone (Haliotis discus hannai). It was observed that both HSP70 and HSP90 transcripts were upregulated under heat stress in both ganglion tissues. Expression level of HSP70 was found higher than HSP90 in both ganglia whereas cerebral ganglion showed higher expression than pleuropedal ganglion. The HSP70 and HSP90 showed higher expression at Day-1 after exposed to heat stress, later decreased at Day-3 and Day-7 onwards. The present result suggested that HSP70 and HSP90 synthesize in nerve ganglion tissues and may provide efficient protection from stress.

Karyotype analysis is a major work in the process of triploid abalone production for the purpose of productivity and quality improvement. However, the metaphase spreads for karyotype analysis have been prepared just from the larvae at trochophore stage, which has restricted the spectrum of sample correction inhibiting more efficient analysis. Here, we investigated the feasibility of preparing metaphase spreads from the larvae at veliger stage that is the next developmental stage of trochophore. For this, diploid and triploid larvae at trochophore and veliger stages from Pacific abalone (Haliotis discus hannai ) were subjected to metaphase spread preparation and its efficiencies were measured and compared each other. As the results, although the efficiencies of metaphase spread preparation were significantly lower in the larvae at veliger stage compared to the ones at trochophore stage regardless of ploidy status, we found that the preparation of metaphase spreads, which showed the clear chromosomal images containing the normal number of chromosomes, was possible from the veliger stage larvae. On the other hands, all larvae used in this study regardless of developmental stage and ploidy did not show colchicine sensitivity. Moreover, no significant difference was observed in cell cycle distribution of the cells comprising larvae between two developmental stages regardless of ploidy status. These suggested that the details of protocol to prepare metaphase spreads from abalone larvae should be optimized depending on its developmental stages. Taken together, we demonstrated the feasibility of preparing metaphase spreads from H. discus hannai veliger stage larvae for karyotype analysis.

The level at which analyses of DNA content might contribute more significantly to the genetic mechanisms of evolution lies in the events of speciation. The object of this study was to investigate the DNA content of abalone (Haliotis discus hannai) and determine the optimal tissue samples for measuring the DNA content of abalone by flowcytometry without fixation. The DNA content (pg/nucleus) of gill tissue (2.5±0.08), which was contaminated with protozoa, was significantly lower than that of muscle tissue (3.2±0.02), mantle tissue (3.2±0.02) (p<0.05), and a standard reference standard, while the DNA contents of muscle tissue and mantle tissue were higher than that of the standard reference. Considering the results of this study, DNA content analysis with flowcytometry is an acute and rapid method by which muscle tissue and mantle tissue are the most appropriate sample for measuring the DNA content of abalone without fixation.

Potential utility of 14 candidate housekeeping genes as normalization reference for RT-qPCR analysis with developmental samples (fertilized eggs to late veliger larvae) in Pacific abalone Haliotis discus hannai was evaluated using four different statistical algorithms (geNorm, NormFinder, BestKeeper and comparative ΔCT method). Different algorithms identified different genes as the best candidates, and geometric mean-based final ranking from the most to the least stable expression was as follow: RPL5, RPL4, RPS18, RPL8, RPL7, UBE2, RPL7A, GAPDH, RPL36, PPIB, EF1A, ACTB and B-TU. The findings were further validated via relative quantification of metallothionein (MT) transcripts using the stable and unstable reference genes, and expression levels of MT were greatly influenced according to the choice of reference genes. In overall, our data suggest that RPL5 and RPS18, either singly or in combination, are appropriate for normalizing gene expression in developmental samples of this abalone species, whereas ACTB, B-TU and EF1A are less stable and not recommended. In addition, our findings propose that standard deviations in geometric ranking as well as geometric mean itself should also be taken into account for the final selection of reference gene(s). This study could be a useful basis to facilitate the generation of accurate and reliable RT-qPCR data with developmental samples in this abalone species.

Recently, mass mortality of the young abalone Haliotis discus hannai has occurred in commercial seed production farms in Korea. The mortality rate was above 50% of the total cultured organisms in the farm, and the shell length of the moribund organisms was about 3cm. The mortal phenomenon was that the young abalones were weakly scattered on the bottom of the pond from the attachment matrix, or that they could not be moved back to their normal positions. The diseased farmed Pacific abalone had abdominal edema. From the edema in the moribund individuals, three bacterial strains were isolated and all the strains were identified as Vibrio harveyi. These strains were compared with thirty six strains isolated from the fish. The results was that the Vibrio harveyi from the fish were sorted into genogroup A or B; however, the three strains of the diseased farmed Pacific abalone were sorted into genogroup A and the new genogroup C. The identical mortality and pathological symptoms of the naturally infected organisms were reproduced by artificial infection with WA AG-1 and WA CS-5 strains. The LD50 of WA AG-1 and WA CS-5 were each 1.0×103 cfu animal-1 and 1.7×104 cfu animal-1.

생존율은 염분과 관계없이 8℃, 10℃일 때는 98.7~100%였으나, 4℃일 때는 생존율이 25~55%로 나타났다. 혈림프액 내 SOD와 glutathione 농도와 같은 스트레스 지표는 모든 염분구에서 4℃, 6℃일 때 높게 나타났다. THC, hemocyte population, apoptotic cell및 necrotic cell의 비율은 염 분 별로는 26 psu구에서 가장 높게 나타났고, 4~6℃일 때 다른 실험구에 비해 유의하게 높았 다. 이러한 결과들은 26 psu의 염분과 6℃ 이하의 수온에서는 참전복의 스트레스 반응이 증가 하였으나, 30, 34 psu의 염분과 8, 10℃의 수온에서는 대조구와 차이가 없어, 참전복의 수송을 위한 적정 수온 및 염분 조건은 8~10℃, 30~34 psu인 것으로 나타난다.

To characterize the female or male transcriptome of the Pacific abalone and further increase genomic resources, we sequenced the mRNA of full-length complementary DNA (cDNA) libraries derived from pooled tissues of female and male Haliotis discus hannai by employing the Iso-Seq protocol of the PacBio RSII platform. We successfully assembled whole full-length cDNA sequences and constructed a transcriptome database that included isoform information. After clustering, a total of 15,110 and 12,145 genes that coded for proteins were identified in female and male abalones, respectively. A total of 13,057 putative orthologs were retained from each transcriptome in abalones. Overall Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyzed in each database showed a similar composition between sexes. In addition, a total of 519 and 391 isoforms were genome-widely identified with at least two isoforms from female and male transcriptome databases. We found that the number of isoforms and their alternatively spliced patterns are variable and sex-dependent. This information represents the first significant contribution to sex-preferential genomic resources of the Pacific abalone. The availability of whole female and male transcriptome database and their isoform information will be useful to improve our understanding of molecular responses and also for the analysis of population dynamics in the Pacific abalone.

Gonadotropin-releasing hormone (GnRH) is a key neuropeptide regulating reproduction in humans and other vertebrates. Here, we evaluated the reproductive control system mediated by GnRH in the Pacific abalone Haliotis discus hannai. We cloned a prepro-GnRH cDNA (Hdh-GnRH) from the pleural-pedal ganglion (PPG) in H. discus hannai, and analyzed its spatiotemporal gene expression pattern. The open reading frame of Hdh-GnRH encodes a protein of 101 amino acids, consisting of a signal peptide, a GnRH dodecapeptide, a cleavage site, and a GnRH-associated peptide. This structure and sequence are highly similar to GnRH-like peptides reported for mollusks and other invertebrates. Quantitative polymerase chain reaction demonstrated that Hdh-GnRH mRNA was more strongly expressed in the ganglions (PPG and cerebral ganglion [CG]) than in other tissues (gonads, gills, intestine, hemocytes, muscle, and mantle) in both sexes. In females, the expression levels of Hdh-GnRH mRNA in the PPG and branchial ganglion (BG) were significantly higher at the ripe and partial spent stages than at the early and late active stages. In males, Hdh-GnRH mRNA levels in the BG showed a significant increase in the partial spent stage. Unexpectedly, Hdh-GnRH levels in the CG were not significantly different among the examined stages in both sexes. These results suggest that Hdh-GnRH mRNA expression profiles in the BG and possibly the PPG are tightly correlated with abalone reproductive activities.

For the study of population genetic structure with mtDNA, it is essential to measure genetic diversity at each mtDNA regions. Also, to evaluate the variation according to the each region should follow as well as to see if there are differences. In this study, we delved into the variations and dendrogram among samples of seven mtDNA regions (NDⅡ, NDⅤ, NDⅣ, NDⅣL, NDⅥ, NDⅠ, 12SrRNA) from wild Pacific abalone, Haliotis discus hannai collected in Yeosu, Korea. The region with the highest genetic variation was NDⅣ region (Haplotype diversity = 1.0000, Nucleotide diversity = 0.010823) with two to five times higher variation than the others. Furthermore, the study to see if there is a difference between the regions of samples showed that similar aspects of dendrogram in NDⅡ and NDⅠ(divergence of 90% and 87%), which forms a group with hd4, 7, 8 and 10 at bootstrap support, based on 1000 replications. Also, pair-wise FST between clusters within the regions showed high values; 0.4061 (P=0.0000), 0.4805 (P=0.0000) respectively. Therefore we can infer that it is the most efficient and accurate way to analyze the region of NDⅣ with the highest variation in addition to the regions of NDⅡ and NDⅠ, which formed clusters with high bootstrap value, for study of population genetic structure in this species.