Ammonia nitrogen, a major oxygen-consuming pollutant in the environment, can adversely affect aquatic organisms such as fish, bivalves, and crustaceans. We investigated the toxic effects of ammonia nitrogen on the Hong Kong oyster, Crassostrea hongkongensis, using flow cytometry and ¹H nuclear magnetic resonance metabolomics. Exposure to ammonia nitrogen caused time- and concentration-dependent alterations in various immune parameters in hemocytes and impaired the metabolic profiles of the gills. We observed changes in the rate of apoptosis, esterase activity, lysosomal mass, hemocyte counts, phagocytic activity, and mitochondrial mass. Exposure affected metabolic pathways involved in energy metabolism, osmotic balance, and oxidative stress. We concluded that ammonia nitrogen induces metabolic and hematological dysfunction in C. hongkongensis, and our findings provide insights into the biochemical defense strategies of bivalves exposed to acute high-concentration ammonia nitrogen.

In recent years, more and more studies have shown that early pathogenic bacterial infection in invertebrates can enhance immunity and significantly reduce mortality when reinfected with the same pathogen. There are mechanisms to explain this phenomenon, but they are relatively few. In addition, dose-dependent primary infection is also associated with increased immunity. In the present study, the initial infection dose and mortality of abalone Haliotis diversicolor after reinfection with Vibrio harveyi were recorded, and the mechanism of immune enhancement was investigated by the transcriptomic response of abalone after two successive stimuli with V. harveyi. Priming with different concentrations of pathogen can enhance immunity; however, higher concentration is not always better. Compared with the first exposure, more genes were up-regulated after the second exposure. Among the commonly expressed genes, the immune related genes were significantly or persistently highly expressed after two infections and included pattern recognition receptors as well as immune effectors, such as toll-like receptors, perlucin 4, scavenger receptor class B-like protein, cytochrome P450 1B1-like, glutathione S-transferase 6, lysozyme and so on; in addition, these immune-related genes were mainly distributed in the pathways related to phagocytosis and calcium signaling. Among the specifically expressed genes, compared with the first infection, more genes were involved in the immune, metabolic and digestive pathways after the second infection, which would be more conducive to preventing the invasion of pathogens. This study outlined the mechanism of immune enhancement in abalone after secondary infection at the global molecular level, which is helpful for a comprehensive understanding of the mechanism of immune priming in invertebrates.

Perkinsosis, a disease caused by the protist Perkinsus, is responsible for mass mortalities of many molluscan species worldwide. The rapid, early and accurate detection of Perkinsus infection is necessary to react to outbreaks, and manage disease transmission. Current methods for diagnosis of Perkinsus spp. are time-consuming or require professional equipment and experienced personnel, rendering them unsuitable for field application. Recombinase polymerase amplification (RPA) assay is a highly sensitive and selective isothermal amplification technique that operates at temperatures of 37–42 °C, requires minimal sample preparation, and is capable of amplifying as low as 1–10 target DNA copies in less than 20 minutes. We report a novel RPA assay that amplifies the internal transcriber spacer (ITS) region of P. beihaiensis, which, followed by rapid detection of amplicons using a lateral flow (LF) strip, enables easy visualization of results by the naked eye. The LF-RPA assay successfully amplified P. beihaiensis DNA using a set of primers of 20–25 bp in length. After incubation at 37 °C for 25 min, results were read within 5 min by the naked eye on a lateral flow strip. Our LF-RPA assay was comparably sensitive to qPCR assay, and capable of detecting as few as 26 copies of P. beihaiensis DNA. Cross-amplification occurred with other two Perkinsus species, P. olseni and P. chesapeaki, but not with other potential pathogen taxa in culture environments. We compared the performance of LF-RPA, conventional PCR and qPCR assays on 60 oyster samples. While LF-RPA assay results were 86.2% as sensitive, 77.4% as specific, and generally in agreement with those of conventional PCR results, they were more (93.3%) sensitive, (86.7%) specific, and agreed better with qPCR assay results. Future research should focus on developing simple DNA extraction methods that do not require professional laboratories and complicated extraction procedures, to facilitate application of this LF-RPA assay in the field. Our LF-RPA assay provides a rapid and efficient method for detecting species of Perkinsus. This novel assay has potential to be used in field applications.

Gender differences in the hemocyte immune response of Hong Kong oyster Crassostrea hongkongensis to Vibrio harveyi and lipopolysaccharide (LPS) infection exist. To determine if a gender difference also exists, we use a ¹H NMR-based metabolomics method to investigate responses in C. hongkongensis hepatopancreas tissues to V. harveyi and LPS infection. Both infections induced pronounced gender- and immune-specific metabolic responses in hepatopancreas tissues. Responses are mainly presented in changes in substances involved in energy metabolism (decreased glucose, ATP, and AMP in males and increased ATP and AMP in LPS-infected females), oxidative stress (decreased glutathione in males and decreased tryptophan and phenylalanine and increased choline and proline in LPS-infected females), tricarboxylic acid (TCA) cycle (decreased α-ketoglutarate acid and increased fumarate in LPS-infected males, and decreased fumarate in LPS-infected females), and osmotic regulation (decreased trigonelline and increased taurine in V. harveyi-infected males and decreased betaine in V. harveyi-infected females). Results suggest that post-spawning-phase male oysters have a more significant energy metabolic response and greater ability to cope with oxidative stress than female oysters. We propose that the impact of oyster gender should be taken into consideration in the aftermath of oyster farming or oyster disease in natural seas.