Aside from their critical role in reproduction, abalone gonads serve as an indicator of sexual maturity and energy balance, two key considerations for effective abalone culture. Temperate abalone farmers face issues with tank restocking with highly marketable abalone owing to inefficient spawning induction methods. The identification of key proteins in sexually mature abalone will serve as the foundation for a greater understanding of reproductive biology. Addressing this knowledge gap is the first step towards improving abalone aquaculture methods. Proteomic profiling of female and male gonads of greenlip abalone, Haliotis laevigata, was undertaken using liquid chromatography-mass spectrometry. Owing to the incomplete nature of abalone protein databases, in addition to searching against two publicly available databases, a custom database comprising genomic data was used. Overall, 162 and 110 proteins were identified in females and males respectively with 40 proteins common to both sexes. For proteins involved in sexual maturation, sperm and egg structure, motility, acrosomal reaction and fertilization, 23 were identified only in females, 18 only in males and 6 were common. Gene ontology analysis revealed clear differences between the female and male protein profiles reflecting a higher rate of protein synthesis in the ovary and higher metabolic activity in the testis.
This study investigated the use of a recombinant protein of Neoparamoeba perurans, the causative agent of Amoebic gill disease (AGD), as an immunogen to generate systemic and mucosal antibody responses against the parasite. Genes encoding N. perurans homologs of mannose-binding protein (MBP) from Acanthamoeba spp. have been identified. From these, a Neoparamoeba MBP - like EST has been identified and produced as a recombinant fusion protein. Attachment of N. perurans to the gill might be reduced by antibody-mediated interference of this protein, but this is dependent on the presence and level of functional antibodies in the mucus. Fish were immunized with the protein via i.p. injection with Freund's complete adjuvant (FCA); and serum and skin mucus samples were collected before and after immunization. Antibodies (IgM) present in samples were characterized via Western blot and their levels measured with an ELISA. The immunization was able to induce a systemic IgM response 8 weeks after primary exposure and a mucosal response 4 weeks post initial immunization, which were specific to the recombinant protein but not to antigens obtained from crude amoebic preparations. However, adherence of the antibodies to the parasite was observed using immunocytochemistry, and both, serum and skin mucus IgM, were able to bind the surface of formalin-fixed N. perurans. This finding may contribute to further research into the development of a vaccine for AGD.
RNA interference (RNAi) has been extensively used to study gene function in non-model organisms and has the potential to identify parasite target molecules in order to develop alternative treatment strategies. This technology could assist in further development of preventive methods against amoebic gill disease (AGD), the main health problem affecting the Atlantic salmon aquaculture industry in Tasmania (Australia) and now a significant emerging issue in Europe. Using ?-actin and EF1-? as candidate genes, we investigated the feasibility of gene knockdown by double-stranded RNA (dsRNA) in Neoparamoeba pemaquidensis, the non-infective strain closely related to the causative agent of AGD, Neoparamoeba perurans. Bacterially expressed dsRNA targeting the selected target genes was administered by soaking (2, 20 and 50 ?g/mL) and a time course sampling regime performed. Quantitative real-time PCR analysis showed that candidate genes were successfully downregulated with silencing efficiency and duration both target and dose-dependent. Additionally, ?-actin deficient trophozoites unexpectedly transformed into a cyst-like stage, which has not been previously reported in this species. An effective RNAi model system for N. pemaquidensis was validated in the current study. Such findings will greatly facilitate further application of RNAi in the aetiological agent of AGD. To our knowledge, this is the first time that RNAi-mediated technology has been successfully employed in a member of the Neoparamoeba genus.
Aquatic animal diseases are one of the most significant constraints to the development and management of aquaculture worldwide. As a result, measures to combat diseases of fish and shellfish have assumed a high priority in many aquaculture-producing countries. RNA interference (RNAi), a natural mechanism for post-transcriptional silencing of homologous genes by double-stranded RNA (dsRNA), has emerged as a powerful tool not only to investigate the function of specific genes, but also to suppress infection or replication of many pathogens that cause severe economic losses in aquaculture. However, despite the enormous potential as a novel therapeutical approach, many obstacles must still be overcome before RNAi therapy finds practical application in aquaculture, largely due to the potential for off-target effects and the difficulties in providing safe and effective delivery of RNAi molecules in vivo. In the present review, we discuss the current knowledge of RNAi as an experimental tool, as well as the concerns and challenges ahead for the application of such technology to combat infectious disease of farmed aquatic animals.
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