Observation of Agonistic Behavior in Pacific White Shrimp (Litopenaeus vannamei) and Transcriptome Analysis.

Agonistic behavior has been identified as a limiting factor in the development of intensive L. vannamei aquaculture. However, the characteristics and molecular mechanisms underlying agonistic behavior in L. vannamei remain unclear. In this study, we quantified agonistic behavior through a behavioral observation system and generated a comprehensive database of eyestalk and brain ganglion tissues obtained from both aggressive and nonaggressive L. vannamei employing transcriptome analysis. The results showed that there were nine behavior patterns in L. vannamei which were correlated, and the fighting followed a specific process. Transcriptome analysis revealed 5083 differentially expressed genes (DEGs) in eyestalk and 1239 DEGs in brain ganglion between aggressive and nonaggressive L. vannamei. Moreover, these DEGs were primarily enriched in the pathways related to the energy metabolism process and signal transduction. Specifically, the phototransduction (dme04745) signaling pathway emerges as a potential key pathway for the adjustment of the L. vannamei agonistic behavior. The G protein-coupled receptor kinase 1-like (LOC113809193) was screened out as a significant candidate gene within the phototransduction pathway. Therefore, these findings contribute to an enhanced comprehension of crustacean agonistic behavior and provide a theoretical basis for the selection and breeding of L. vannamei varieties suitable for high-density aquaculture environments.

Targeted cultivation of diatoms in mariculture wastewater by nutrient regulation and UV-C irradiation.

Mariculture wastewater poses environmental challenges due to pollution and eutrophication. Targeted cultivation of diatoms in wastewater can help alleviate these issues while generating beneficial algae biomass, however reliable operating methods are lacking. We proposed a novel method for treating mariculture wastewater that employed UV-C irradiation and nutrient regulation to achieve targeted diatom cultivation. This study first examined growth of four diatom species (Nitzschia closterium, Chaetoceros muelleri, Cyclotella atomus, and Conticribra weissflogii) in mariculture wastewater. C. muelleri and C. weissflogii demonstrated better adaptability compared to N. closterium and C. atomus. Additionally, the growth and nutrient utilization of C. muelleri were studied under varying concentrations of silicate, phosphate, ammonium, and trace elements in wastewater. Optimal growth was observed at 500 µmol/L silicate, 0.6 mg/L phosphate, and 4 mg/L ammonium. Ammonium proved to be a more effective nitrogen source than urea and nitrate in promoting growth at this low level. Surprisingly, trace element supplementation did not significantly impact growth. Finally, this study utilized UV-C irradiation as a pre-treatment method for wastewater prior to nutrient adjustment, significantly enhancing the growth of C. muelleri. Overall, this study provides guidance on regulating key nutrients and pre-treatment method to optimize diatom biomass production from mariculture wastewater. This approach not only addresses environmental challenges associated with mariculture but also contributes to sustainable aquaculture practices through the recovery of valuable aquatic resources.

The transcriptomic and biochemical responses of blood clams (Tegillarca granosa) to prolonged intermittent hypoxia.

The blood clam (Tegillarca granosa), a marine bivalve of ecological and economic significance, often encounters intermittent hypoxia in mudflats and aquatic environments. To study the response of blood clam foot to prolonged intermittent hypoxia, the clams were exposed to intermittent hypoxia conditions (0.5 mg/L dissolved oxygen, with a 12-h interval) for 31 days. Initially, transcriptomic analysis was performed, uncovering a total of 698 differentially expressed genes (DEGs), with 236 upregulated and 462 downregulated. These genes show enrichments in signaling pathways related to glucose metabolism, sugar synthesis and responses to oxidative stress. Furthermore, the activity of the enzyme glutathione peroxidase (GPx) and the levels of gpx1 mRNA showed gradual increases, reaching their peak on the 13th day of intermittent hypoxia exposure. This observation suggests an indirect protective role of GPx against oxidative stress. The results of this study make a significantly contribute to our broader comprehensive of the physiological, biochemical responses, and molecular reactions governing the organization of foot muscle tissue in marine bivalves exposed to prolonged intermittent hypoxic conditions.

Homogeneous environmental selection mainly determines the denitrifying bacterial community in intensive aquaculture water.

Nitrate reduction by napA (encodes periplasmic nitrate reductase) bacteria and nitrous oxide reduction by nosZ (encodes nitrous oxide reductase) bacteria play important roles in nitrogen cycling and removal in intensive aquaculture systems. This study investigated the diversity, dynamics, drivers, and assembly mechanisms of total bacteria as well as napA and nosZ denitrifiers in intensive shrimp aquaculture ponds over a 100-day period. Alpha diversity of the total bacterial community increased significantly over time. In contrast, the alpha diversity of napA and nosZ bacteria remained relatively stable throughout the aquaculture process. The community structure changed markedly across all groups over the culture period. Total nitrogen, phosphate, total phosphorus, and silicate were identified as significant drivers of the denitrifying bacterial communities. Network analysis revealed complex co-occurrence patterns between total, napA, and nosZ bacteria which fluctuated over time. A null model approach showed that, unlike the total community dominated by stochastic factors, napA and nosZ bacteria were primarily governed by deterministic processes. The level of determinism increased with nutrient loading, suggesting the denitrifying community can be manipulated by bioaugmentation. The dominant genus Ruegeria may be a promising candidate for introducing targeted denitrifiers into aquaculture systems to improve nitrogen removal. Overall, this study provides important ecological insights into aerobic and nitrous oxide-reducing denitrifiers in intensive aquaculture, supporting strategies to optimize microbial community structure and function.

The Effects of Shaking Duration on the Abundance and the Community of Aerobic Denitrifying Bacteria in Shrimp Pond Water and Sediment Samples.

With the rapid development of intensive aquaculture, the considerable release of nitrogenous organic compounds has become a serious threat to aquatic organisms. Currently, isolating autochthonous aerobic denitrifying bacteria (ADB) from aquaculture environments is essential for the biological elimination of nitrogenous pollutants. In this study, the enrichment of ADB from shrimp pond water and sediment samples was conducted under different shaking durations. The absolute abundance of total bacteria, nosZ-type, and the napA-type ADB was measured using qPCR. High-throughput sequencing of 16S rRNA, nosZ, and napA genes was performed to reveal the community structure of bacteria and ADB, respectively. Our data revealed that absolute abundance and the community structure of the total bacteria, nosZ-type and napA-type ADB, were significantly altered under different shaking durations. Specifically, the order Pseudomonadales, possessing both nosZ and napA genes, was significantly enriched in water and sediment samples under both 12/12 and 24/0 shaking/static cycles. However, in water samples, compared to the 24/0 shaking/static cycles, the 12/12 shaking/static cycles could lead to a higher enrichment rate of aerobic denitrification bacteria indicated by the higher absolute abundance of bacteria and the higher accounting percentage of orders Oceanospirillales and Vibrionales. Moreover, although the order Pseudomonadales notably increased under the 12/12 of shake/static cycle compared to the 24/0 shaking/static cycle, considering the relative higher abundance of ADB in 24/0 shaking/static cycle, the enrichment of ADB in sediment may be efficient with the 24/0 shaking/static cycle.

Changes in Bacterial Communities of Kumamoto Oyster Larvae During Their Early Development and Following Vibrio Infection Resulting in a Mass Mortality Event.

Vibrio and Ostreid herpesvirus 1 are responsible for mass mortalities of oyster larvae in hatcheries. Relevant works have focused on their relationships with the disease when larval mortality occurs. On the contrary, little is known about how the resident microbiota in oyster larvae responds to Vibrio-infected disease causing mortality as the disease progressed, whereas this knowledge is fundamental to unveil the etiology of the disease. Here, we analyzed the temporal succession of the microbiome of Kumamoto oyster (Crassostrea sikamea) larvae during their early development, accompanied by a Vibrio-caused mortality event that occurred at the post D-stage of larval development in a shellfish hatchery in Ningbo, China, on June 2020. The main causative agent of larval mortality was attributable to Vibrio infection, which was confirmed by linearly increased Vibrio abundance over disease progression. Larval bacterial communities dramatically changed over host development and disease progression, as highlighted by reduced α-diversity and less diverse core taxa when the disease occurred. Null model and phylogenetic-based mean nearest taxon distance analyses showed that the relative importance of deterministic processes governing larval bacterial assembly initially increased over host development, whereas this dominance was depleted over disease progression. Furthermore, we screened the disease-discriminatory taxa with a significant change in their relative abundances, which could be indicative of disease progression. In addition, network analysis revealed that disease occurrence remodeled the co-occurrence patterns and niche characteristics of larval microbiota. Our findings demonstrate that the dysbiosis of resident bacterial communities and the shift of microecological mechanisms in the larval microbiome may contribute to mortality during oyster early development.

Exploring the value of the double source CT angiography in diagnosing in-stent restenosis in lower limb artery.

OBJECTIVES: This paper is aimed to explore the value of double source CT angiography (DS-CTA) for diagnosing in-stent restenosis in lower limb artery. METHODS: From January 2016 to October 2018, all patients with stent in lower limb artery in our hospital were investigated by both DS-CTA and digital subtraction angiography. We measured the minimum lumen diameter and the diameter of the proximal normal vessels under each stent placement. The in-stent restenosis is defined as restenosis when the lumen area decreased by more than 50%. Digital subtraction angiography was performed within 1 week after DS-CT scan. Relationship between DS-CTA and digital subtraction angiography for diagnosing in-stent restenosis in lower limb artery was analyzed. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of DS-CTA for diagnosis of in-stent restenosis were analyzed with digital subtraction angiography as the reference standard. A total of 68 stents were placed in 51 patients. Among these patients, 27 cases were diagnosed as in-stent restenosis, presenting as endovascular contrast agent bias or crescent filling defect with the lumen area reducing over 50%, 6 cases of which had no significant in-stent restenosis by digital subtraction angiography analysis. Furthermore, 12 cases were occlusion, in which there was no high density contrast agent in stents; the remaining 41 stents were unobstructed and the contrast agent was filled well, 8 cases of which had significant in-stent restenosis by digital subtraction angiography analysis. In addition, four stents were deformed or distorted. Statistical analysis demonstrated the concentrations of DS-CTA and digital subtraction angiography in diagnosing in-stent restenosis for lower limb artery were closely related, and the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of DS-CTA were 72.4%, 84.6%, 77.8%, 80.5%, and 79.4%, respectively. CONCLUSION: DS-CTA has a potential reliability for diagnosis of in-stent restenosis in lower limb artery, which may be further improved to be used for clinical interventional treatment of vascular diseases.

Transcriptomic Analysis of the Cold-Pretreated Larimichthys crocea Showing Enhanced Growth Fitness in Cold Water.

The large yellow croaker Larimichthys crocea is an economically important marine fish species endemic to China and East Asia. Ningde area of Fujian Province is a major L. crocea aquaculture and spawning center in China. L. crocea cultivated at the Zhoushan area appears to be popular but suffered high mortality in cold water during winter seasons. To reduce the mortality rate, we pretreated fish with cold shocks prior to shift to cold water. In this study, we show that cold-pretreated L. crocea 12 days after shift to cold water increase the viability by 5.77-fold compared to the unpretreated (live fish 75 versus 13, p value = 1.775e-06, n = 100). The highest loss of 31 out of 100 fish in the unpretreated group occurred in day 3 after temperature shift. To identify the pretreatment-induced transcriptional changes that may be attributed to cold-resistance and survival, we performed RNA-seq analysis of a total of 48 fish that were prior to and 48 h, 54 h, and 72 h after temperature shift in pretreated and unpretreated groups in sextuplicate. Transcriptomic profiling analysis indicates that pretreatment-induced transcriptional alterations of enzymes involved in FASI, ß-oxidation, PUFA synthesis, oxidative phosphorylation, and molecular chaperones persisted after temperature shift, suggesting that these metabolic pathways may play a role in L. crocea cold-resistance and survival. Our study provides insights on how the pretreatment enhances the L. crocea growth fitness in cold water.

Immunity-associated long non-coding RNA and expression in response to bacterial infection in large yellow croaker (Larimichthys crocea).

Long non-coding RNA refers to an RNA transcript of a non-coding protein with a sequence length greater than 200 bp. More and more reports indicated that lncRNA was involved in the regulation of gene expression as a signalling molecule, an inducing molecule, a leader molecule and a scaffold molecule. Previous studies have sequenced the draft genome and several transcriptome data sets for protein-coding genes of the large yellow croaker (Larimichthys crocea), but little is known about the expression and function of lncRNAs in this species. In order to obtain a catalogue of lncRNAs for this croaker, Vibrio parahaemolyticus infection challenge experiment was conducted and long non-coding RNA sequences were obtained. Using high-throughput sequencing of lncRNA, a total of 73,233 high-confidence transcripts were reconstructed in 32,726 loci, recovering most of the expressed reference transcripts, and 6473 novel expressed loci were identified. The tissue expression profile revealed that most lacunas were specifically enriched in distinct tissues. A set of 163 lncRNAs were identified as being specifically expressed in the spleen and may be involved in the immune response. It is the first time to identify specific lncRNAs in the L. crocea systematically in this croaker, aiming to benefit the future genomic study of this species.

Molecular cloning and expression analysis of a cytosolic heat shock protein 70 gene from mud crab Scylla serrata.

Heat shock protein 70s (Hsp70s) play important roles in resisting environmental stresses and stimulating innate immune system. To understand the immune defense mechanisms of Scylla serrata, a full-length cytosolic Hsp70 cDNA of S. serrata (designated as SSHsp70) was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends (RACE). The full-length of SSHsp70 cDNA was 2235 bp, with a 5' untranslated region of 105 bp, a 3' untranslated region of 174 bp, and an open reading frame of 1956 bp encoding a polypeptide of 651 amino acids with an estimated molecular mass of 71.3 kDa and an estimated isoelectric point of 5.55. The cloned SSHsp70 belonged to a cytosolic Hsp70 family. Three typical Hsp70 signature motifs were detected in SSHsp70 by InterPro analysis. Quantitative PCR (qPCR) was used to detect tissue distribution and mRNA expression levels of SSHsp70 under different stress conditions. The obviously high levels of SSHsp70 transcript were in hemocyte, heart, hepatopancreas and gill, whereas low levels were detected in muscle, eyestalk, stomach, and gut. In different temperature treatments, the expression levels of SSHsp70 in low or high temperatures were higher than those in temperate temperature. In pathogen challenge treatments, the mRNA expression level of SSHsp70 reached a maximum level after 18 h and then dropped progressively. In different salt concentration treatments, the mRNA expression level of SSHsp70 had a minimum level at 25‰ salt concentration and high expression levels at high or low salt concentration. In different nitrite concentration treatments, the mRNA expression level of SSHsp70 increased progressively with the increase of nitrite concentration. The results confirmed Hsp70 could be used as a tool for evolution and phylogenetic analysis, a kind of potential biomarker, and a disease resistance factor used in application.

[Isolation and identification of the pathogenic strain of Vibrio harveyi from Miichthys miiuy].

OBJECTIVE: An outbreak of disease on the cultured Miichthys miiuy occurred in Zhoushan of Zhejiang province. The symptom displayed as skin ulceration and the inside apparatus turned white. METHODS: We isolated a dominant bacterial strain from the diseased Miichthys and assigned it as strain 090212. The artificial infection test proved that the isolate 090212 was the pathogenic bacterium that caused the disease. We applied physiological and biochemical characterization and API system in the bacterial classification. In order to confirm the result, we amplified a 1458bp sequence of 090212' s 16S rDNA and compared with other Vibrio in GenBank. RESULTS: The results turned out that 090212 was Gram negative and short rod with single polar flagellum. Homology analysis and phylogenetic study showed that strain 090212 had the highest similarity to Vibrio harveyi, with 99% identity. The sensitivity test of strain 090212 to 28 kinds of antibiotics revealed that the pathogen was sensitive to drugs such as Florfenicol and Tetracycline. CONCLUSION: This paper revealed for the first time that the causative pathogen, Vibrio harveyi, lead to the mass mortality of Miichthys, which will be helpful in the disease control and health management during Miichthys cultivation.