Susceptibility of Four Abalone Species, Haliotis gigantea, Haliotis discus discus, Haliotis discus hannai and Haliotis diversicolor, to Abalone asfa-like Virus

Abalone amyotrophia is a viral disease that causes mass mortality of juvenile Haliotis discus and H. madaka. Although the cause of this disease has yet to be identified, we had previously postulated a novel virus with partial genome sequence similarity to that of African swine fever virus is the causative agent and proposed abalone asfa-like virus (AbALV) as a provisional name. In this study, three species of juvenile abalone (H. gigantea, H. discus discus, and H. diversicolor) and four species of adult abalone (the above three species plus H. discus hannai) were experimentally infected, and their susceptibility to AbALV was investigated by recording mortality, quantitatively determining viral load by PCR, and conducting immunohistological studies. In the infection test using 7-month-old animals, H. gigantea, which was previously reported to be insusceptible to the disease, showed multiplication of the virus to the same extent as in H. discus discus, resulting in mass mortality. H. discus discus at 7 months old showed abnormal cell masses, notches in the edge of the shell and brown pigmentation inside of the shell, which are histopathological and external features of this disease, while H. gigantea did not show any of these characteristics despite suffering high mortality. Adult abalones had low mortality and viral replication in all species; however, all three species, except H. diversicolor, became carriers of the virus. In immunohistological observations, cells positive for viral antigens were detected predominantly in the gills of juvenile H. discus discus and H. gigantea, and mass mortality was observed in these species. In H. diversicolor, neither juvenile nor adult mortality from infection occurred, and the AbALV genome was not increased by experimental infection through cohabitation or injection. Our results suggest that H. gigantea, H. discus discus and H. discus hannai are susceptible to AbALV, while H. diversicolor is not. These results confirmed that AbALV is the etiological agent of abalone amyotrophia.

The role of diversity in mediating microbiota structural and functional responses in two sympatric species of abalone under stressed withering syndrome conditions

Background: The withering syndrome (WS) is an infectious disease initially affecting the gastro-intestinal tract (GI) of wild abalone populations of the coasts of Baja California. In spite of its high incidence, structural and functional changes in abalone GI microbiotas under WS-stressed conditions remain poorly investigated. Moreover, it is equally uncertain if interspecific microbiota features, such as the presence or absence of certain bacterial species, their abundances, and their functional capabilities, may prevent or at least lead to different microbiota responses. Healthy Haliotis fulgens and Haliotis corrugata from Baja California Sur (Mexico) harbor species-specific structural and functional microbiota profiles; hence, we hypothesize a distinctive microbiota response under WS-stressed conditions. Here, we compared both the structural arrangements and functional capabilities of healthy and dysbiotic microbiotas using 454 pyrosequencing high throughput sequencing technologies and PICRUSt v.2 outputs, respectively.Results: Our findings suggest that the extent to which WS may involve structural and functional changes in GI microbiotas is contingent on the microbiota diversity itself. Indeed, we report significant structual alterations in the less complex microbiotas of H. fulgens, which in turn led to a significant downregulation of several metabolic activities conducted by GI bacteria. Conversely, the effects of WS were marginal in more complex bacterial communities, as in H. corrugata, in which no significant structural and functional changes were detected. Conclusions: Our results provide new insights concerning the role of microbiome diversity in abalone health and the etiology of WS. Notably, complex bacterial communities appear to be less affected by WS than less complex microbiotas. Moreover, our insights suggest that structural changes observed under WS-stressed conditions may be considered stochastic, as predicted by the Anna Karenina principle, and result in the downregulation of several ecological functions conducted by GI bacteria. Overall, our results support the hypothesis that the occurrence of WS may be associated with shifts in GI microbiotas. Moreover, we propose that the susceptibility to WS that has been reported among abalone species may reflect the natural degree of complexity of the GI microbiomes harbored by each species.

Abalone Viral Ganglioneuritis

Abalone viral ganglioneuritis (AVG), caused by Haliotid herpesvirus-1 (HaHV-1; previously called abalone herpesvirus), is a disease that has been responsible for extensive mortalities in wild and farmed abalone and has caused significant economic losses in Asia and Australia since outbreaks occurred in the early 2000s. Researchers from Taiwan, China, and Australia have conducted numerous studies encompassing HaHV-1 genome sequencing, development of molecular diagnostic tests, and evaluation of the susceptibility of various abalone species to AVG as well as studies of gene expression in abalone upon virus infection. This review presents a timeline of the most significant research findings on AVG and HaHV-1 as well as potential future research avenues to further understand this disease in order to develop better management strategies.

Microbial community analysis in the gills of abalones suggested possible dominance of epsilonproteobacterium in Haliotis gigantea

Gills are important organs for aquatic invertebrates because they harbor chemosynthetic bacteria, which fix inorganic carbon and/or nitrogen and provide their hosts with organic compounds. Nevertheless, in contrast to the intensive researches related to the gut microbiota, much is still needed to further understand the microbiota within the gills of invertebrates. Using abalones as a model, we investigated the community structure of microbes associated with the gills of these invertebrates using next-generation sequencing. Molecular identification of representative bacterial sequences was performed using cloning, nested PCR and fluorescence in situ hybridization (FISH) analysis with specific primers or probes. We examined three abalone species, namely Haliotis gigantea, H. discus and H. diversicolor using seawater and stones as controls. Microbiome analysis suggested that the gills of all three abalones had the unclassified Spirochaetaceae (one OTU, 15.7 ± 0.04%) and Mycoplasma sp. (one OTU, 9.1 ± 0.03%) as the core microbes. In most libraries from the gills of H. gigantea, however, a previously unknown epsilonproteobacterium species (one OTU) was considered as the dominant bacterium, which accounted for 62.2% of the relative abundance. The epsilonproteobacterium was only detected in the gills of H. diversicolor at 0.2% and not in H. discus suggesting that it may be unique to H. gigantea. Phylogenetic analysis performed using a near full-length 16S rRNA gene placed the uncultured epsilonproteobacterium species at the root of the family Helicobacteraceae. Interestingly, the uncultured epsilonproteobacterium was commonly detected from gill tissue rather than from the gut and foot tissues using a nested PCR assay with uncultured epsilonproteobacterium-specific primers. FISH analysis with the uncultured epsilonproteobacterium-specific probe revealed that probe-reactive cells in H. gigantea had a coccus-like morphology and formed microcolonies on gill tissue. This is the first report to show that epsilonproteobacterium has the potential to be a dominant species in the gills of the coastal gastropod, H. gigantea.

Molecular Identification, Characterization, and Expression Analysis of a Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Pacific Abalone, Haliotis discus hannai

A full-length cDNA sequence encoding a GnRH receptor was cloned from the pleuropedal ganglion of the Pacific abalone, Haliotis discus hannai. The cloned sequence is 1499-bp in length encoding a protein of 460 amino acid residues, with a molecular mass of 52.22 kDa and an isoelectric point (pI) of 9.57. The architecture of HdhGnRH-R gene exhibited key features of G protein-coupled receptors (GPCRs), including seven membrane spanning domains, putative N-linked glycosylation motifs, and phosphorylation sites of serine and threonine residues. It shared 63%, 52%, and 30% sequence identities with Octopus vulgaris, Limulus polyphemus, and Mizuhopecten yessoensis GnRH-R II sequences, respectively. Phylogenetic analysis indicated that HdhGnRH-R gene was clustered with GnRH-R II of O. vulgaris and O. bimaculoides. qPCR assay demonstrated that the mRNA expression level of this receptor was significantly higher in the pleuropedal ganglion than that in any other examined tissue. Transcriptional activities of this gene in gonadal tissues were significantly higher in the ripening stage. The mRNA expression of this gene was significantly higher in pleuropedal ganglion, testis, and ovary at higher effective accumulative temperature (1000 °C). In situ hybridization revealed that HdhGnRH-R mRNA was expressed in neurosecretory cells of pleuropedal ganglion. Our results suggest that HdhGnRH-R gene synthesized in the neural ganglia might be involved in the control of gonadal maturation and gametogenesis of H. discus hannai. This is the first report of GnRH-R in H. discus hannai and the results may contribute to further studies of GPCRs evolution or may useful for the development of aquaculture method of this abalone species.

Greenlip Abalone (Haliotis laevigata) Genome and Protein Analysis Provides Insights into Maturation and Spawning

Wild abalone (Family Haliotidae) populations have been severely affected by commercial fishing, poaching, anthropogenic pollution, environment and climate changes. These issues have stimulated an increase in aquaculture production; however production growth has been slow due to a lack of genetic knowledge and resources. We have sequenced a draft genome for the commercially important temperate Australian ‘greenlip’ abalone (Haliotis laevigata, Donovan 1808) and generated 11 tissue transcriptomes from a female adult abalone. Phylogenetic analysis of the greenlip abalone with reference to the Pacific abalone (Haliotis discus hannai) indicates that these abalone species diverged approximately 71 million years ago. This study presents an in-depth analysis into the features of reproductive dysfunction, where we provide the putative biochemical messenger components (neuropeptides) that may regulate reproduction including gonad maturation and spawning. Indeed, we isolate the egg-laying hormone neuropeptide and under trial conditions induce spawning at 80% efficiency. Altogether, we provide a solid platform for further studies aimed at stimulating advances in abalone aquaculture production. The H. laevigata genome and resources are made available to the public on the abalone ‘omics website, http://abalonedb.org.

Dual Transcriptomic Analysis Reveals a Delayed Antiviral Response of Haliotis diversicolor supertexta against Haliotid Herpesvirus-1

Haliotid herpesvirus-1 (HaHV-1) is the first identified gastropod herpesvirus, causing a highly lethal neurologic disease of abalone species. The genome of HaHV-1 has been sequenced, but the functions of the putative genes and their roles during infection are still poorly understood. In the present study, transcriptomic profiles of Haliotis diversicolor supertexta at 0, 24 and 60 h post injection (hpi) with HaHV-1 were characterized through high-throughput RNA sequencing. A total of 448 M raw reads were obtained and assembled into 2.08 × 105 unigenes with a mean length of 1486 bp and an N50 of 2455 bp. Although we detected increased HaHV-1 DNA loads and active viral expression at 24 hpi, this evidence was not linked to significant changes of host transcriptomic profiles between 0 and 24 hpi, whereas a rich immune-related gene set was over-expressed at 60 hpi. These results indicate that, at least at the beginning of HaHV-1 infection, the virus can replicate with no activation of the host immune response. We propose that HaHV-1 may evolve more effective strategies to modulate the host immune response and hide during replication, so that it could evade the immune surveillance at the early stage of infection.

Haliotis papulata Reeve 1846 (Vetigastropoda: Haliotidae), the pimpled abalone, is the correct name for the small Indo-West Pacific abalone referred to as Haliotis thailandis Dekker & Patamakanthin 2001 and not as a synonym of Haliotis varia Linnaeus 1758

The syntypes of Haliotis papulata Reeve 1846 have been re-examined and are found to not represent specimens of H. varia Linnaeus 1758. Haliotis papulata appears to be the correct name for specimens that are currently considered H. thailandis Dekker & Patamakanthin 2001. The synonymy of Haliotis thailandis within H. papulata provides a more detailed understanding of the distribution of the species and its probable close phylogenetic relationships with the Indo-Pacific abalone species, Haliotis unilateralis Lamarck 1822 and H. clathrata Reeve 1846