Cell-free Hemolymph Research Articles

The venom of Habrobracon hebetor induces alterations in host metabolism.

The ability of parasitic wasps to manipulate a host's metabolism is under active investigation. Components of venom play a major role in this process. In the present work, we studied the effect of the venom of the ectoparasitic wasp Habrobracon hebetor on the metabolism of the greater wax moth host (Galleria mellonella). We identified and quantified 45 metabolites in the lymph (cell-free hemolymph) of wax moth larvae on the second day after H. hebetor venom injection, using NMR spectroscopy and liquid chromatography coupled with mass spectrometry. These metabolites included 22 amino acids, nine products of lipid metabolism (sugars, amines and alcohols) and four metabolic intermediates related to nitrogenous bases, nucleotides and nucleosides. An analysis of the larvae metabolome suggested that the venom causes suppression of the tricarboxylic acid cycle, an increase in the number of free amino acids in the lymph, an increase in the concentration of trehalose in the lymph simultaneously with a decrease in the amount of glucose, and destructive processes in the fat body tissue. Thus, this parasitoid venom not only immobilizes the prey but also modulates its metabolism, thereby providing optimal conditions for the development of larvae.

Insects as a Prospective Source of Biologically Active Molecules and Pharmaceuticals-Biochemical Properties and Cell Toxicity of Tenebrio molitor and Zophobas morio Cell-Free Larval Hemolymph.

Insects are of great interest as novel sources of alternative proteins and biologically active compounds, primarily anticancer agents. Protein-rich insect larval hemolymph is a prospective candidate for pharmaceutical and food industry-related research. In this study, selected biochemical properties and cell toxicity of larval hemolymph from two mealworm species, Tenebrio molitor and Zophobas morio, were analyzed. Total proteins and carbohydrates, antioxidant capacity, and the level of lipid peroxidation were determined. Human cancer (U-87) and normometabolic (MRC-5) cells were treated with different concentrations of larval hemolymph proteins, and the effects on cell viability were assayed 24, 48, and 72 h after treatments. Z. morio hemolymph was shown to be richer in total proteins, showing a higher antioxidant capacity and lipid peroxidation level than T. molitor hemolymph, which was richer in total carbohydrates. Cytotoxicity assays showed that T. molitor and Z. morio hemolymphs differently affect the viability of U-87 and MRC-5 cells in cell type-, dose-, and time-dependent manners. Hemolymph from both species was more cytotoxic to U-87 cells than to MRC-5 cells, which was particularly prominent after 48 h. Additionally, a more potent cytotoxic effect of Z. morio hemolymph was observed on both cell lines, likely due to its higher antioxidant capacity, compared to T. molitor hemolymph.

Dietary Clostridium autoethanogenum protein has dose-dependent influence on the gut microbiota, immunity, inflammation and disease resistance of abalone Haliotis discus hannai

Clostridium autoethanogenum protein (CAP) is an eco-friendly protein source and has great application potential in aquafeeds. The present study aimed to investigate the effects of dietary CAP inclusion on the anti-oxidation, immunity, inflammation, disease resistance and gut microbiota of abalone Haliotis discus hannai after a 110-day feeding trial. Three isonitrogenous and isolipidic diets were formulated by adding 0 % (control), 4.10 % (CAP4.10) and 16.25 % (CAP16.25) of CAP, respectively. A total of 540 abalones with an initial mean body weight of 22.05 ± 0.19 g were randomly distributed in three groups with three replicates per group and 60 abalones per replicate. Results showed that the activities of superoxide dismutase and glutathione peroxidase in the cell-free hemolymph (CFH) were significantly decreased and the content of malondialdehyde in CFH was significantly increased in the CAP16.25 group. The diet with 4.1 % of CAP significantly increased the activities of lysozyme and acid phosphatase in CFH. The expressions of pro-inflammatory genes such as tumor necrosis factor-α (tnf-α), nuclear factor-κb (nf-κb) and toll-like receptor 4 (tlr4) in digestive gland were downregulated, and the expressions of anti-inflammatory genes such as β-defensin and mytimacin 6 in digestive gland were upregulated in the CAP4.10 group. Dietary CAP inclusion significantly decreased the cumulative mortality of abalone after the challenge test with Vibrio parahaemolyticus for 7 days. Dietary CAP inclusion changed the composition of gut microbiota of abalone. Besides, the balance of the ecological interaction network of bacterial genera in the intestine of abalone was enhanced by dietary CAP. The association analysis showed that two bacterial genera Ruegeria and Bacteroides were closely correlated with the inflammatory genes. In conclusion, the 4.10 % of dietary CAP enhanced the immunity and disease resistance as well as inhibited the inflammation of abalone. The 16.25 % of dietary CAP decreased the anti-oxidative capacity of abalone. The structure of the gut microbiota of abalone changed with dietary CAP levels.

Evidence for immune priming specificity and cross-protection against sympatric and allopatric Vibrio splendidus strains in the oyster Magalana (Crassostrea) gigas

Infections with pathogenic Vibrio strains are associated with high summer mortalities of Pacific oysters Magalana (Crassostrea) gigas, affecting production worldwide. This raises the question of how M. gigas cultures can be protected against deadly Vibro infection. There is increasing experimental evidence of immune priming in invertebrates, where previous exposure to a low pathogen load boosts the immune response upon secondary exposure. Priming responses, however, appear to vary in their specificity across host and parasite taxa. To test priming specificity in the Vibrio – M. gigas system, we used two closely related Vibrio splendidus strains with differing degrees of virulence towards M. gigas. These V. splendidus strains were either isolated in the same location as the oysters (sympatric, opening up the potential for co-evolution) or in a different location (allopatric). We extracted cell-free haemolymph plasma from infected and control oysters to test the influence of humoral immune effectors on bacterial growth in vitro. While addition of haemolypmph plasma in general promoted growth of both strains, priming by an exposure to a sublethal dose of bacterial cells lead to inhibitory effects against a subsequent challenge with a potentially lethal dose in vitro. Inhibitory effects and immune priming was strongest when oysters had been primed with the sympatric Vibrio strain, but inhibitory effects were seen both when challenged with the sympatric as well as against allopatric V. splendidus, suggesting some degree of cross protection. The stronger immune priming against the sympatric strain suggests that priming could be more efficient against matching local strains potentially adding a component of local adaptation or co-evolution to immune priming in oysters. These in vitro results, however, were not reflected in the in vivo infection data, where we saw increased bacterial loads following an initial challenge. This discrepancy might suggests that that it is the humoral part of the oyster immune system that produces the priming effects seen in our in vitro experiments.

Bioaccumulation and sub-lethal physiological effects of metal mixtures on mussel, Mytilus edulis: Continuous exposure to a binary mixture of mercury and cadmium

In the natural ecosystem, aquatic organisms are exposed to a cocktail of chemicals that may result in toxicological responses differing from those of individual chemicals. In the present study, mussels were exposed using a semi-static and triplicated design to either control (no added metal), 50 µg l−1 (Hg alone), 50 µg l−1 (Cd alone), or 50 µg l−1 Hg plus 50 µg l−1 Cd (Hg + Cd) mixture for 14 days. Tissues were collected on days 0, 2, 4, 8, and 14 for metal analysis and sub-lethal responses using a suite of assays. Tissue metal concentrations were not significantly different in the single metal (Hg or Cd) compared to the Hg plus Cd mixture treatment for all tissues, apart from the gill of the Cd alone treatment. At the end of the experiment, the gill Cd concentration was significantly increased in the Hg plus Cd mixture compared to the Cd alone treatment, suggesting the influence of Hg on Cd uptake. The percentage increases of the Hg plus Cd mixture compared to the arithmetic sum of the individual metals were ( %): 20.2, 9.3, 25.1, 23.8, 10.7, and 12.4 for adductor muscle, digestive gland, gill, gonad, remaining soft tissue, and haemolymph, respectively. There were no observed treatment effects on total haemocyte count, haemolymph protein, or glucose concentration in the cell-free haemolymph. Neither was there any treatment effect on osmotic pressure, ions in the tissues, or in the cell-free haemolymph. At the end of the experiment, Hg-mediated oxidative damage, as an increase of thiobarbituric reactive substances (TBARS) and apparent depletion of total glutathione. This was observed in the gill and digestive gland of the Hg alone and Hg plus Cd mixture. Histopathology examination showed similar pathology in the Hg alone and the Hg plus Cd treatment. In conclusion, despite some oxidative stress and pathology during metal exposure, the accumulation of metals and effects on mussel health were similar between single exposures and a mixture of Hg plus Cd. In terms of risk assessment, regulations for the individual metals should suffice to protect against the mixture of Hg plus Cd, at least for adult M. edulis in full-strength seawater.

Characterization of Aspergillus fumigatus secretome during sublethal infection of Galleria mellonella larvae.

Introduction. The fungal pathogen Aspergillus fumigatus can induce prolonged colonization of the lungs of susceptible patients, resulting in conditions such as allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis.Hypothesis. Analysis of the A. fumigatus secretome released during sub-lethal infection of G. mellonella larvae may give an insight into products released during prolonged human colonisation.Methodology. Galleria mellonella larvae were infected with A. fumigatus, and the metabolism of host carbohydrate and proteins and production of fungal virulence factors were analysed. Label-free qualitative proteomic analysis was performed to identify fungal proteins in larvae at 96 hours post-infection and also to identify changes in the Galleria proteome as a result of infection.Results. Infected larvae demonstrated increasing concentrations of gliotoxin and siderophore and displayed reduced amounts of haemolymph carbohydrate and protein. Fungal proteins (399) were detected by qualitative proteomic analysis in cell-free haemolymph at 96 hours and could be categorized into seven groups, including virulence (n = 25), stress response (n = 34), DNA repair and replication (n = 39), translation (n = 22), metabolism (n = 42), released intracellular (n = 28) and cellular development and cell cycle (n = 53). Analysis of the Gallerial proteome at 96 hours post-infection revealed changes in the abundance of proteins associated with immune function, metabolism, cellular structure, insect development, transcription/translation and detoxification.Conclusion. Characterizing the impact of the fungal secretome on the host may provide an insight into how A. fumigatus damages tissue and suppresses the immune response during long-term pulmonary colonization.

Effects of dietary supplementation of Bacillus, β-glucooligosaccharide and their synbiotic on the growth, digestion, immunity, and gut microbiota profile of abalone, Haliotis discus hannai

Probiotics, prebiotics, and synbiotics are known to improve aquatic animals' growth and health conditions. In this study, we investigated the effect of Bacillus sp. KRF-7 (KRF-7), Bacillus sp. PM8313 (PM8313), β-glucooligosaccharide (BGO), and their synbiotics on the growth, digestive physiology, immunity, and gut microbiota of abalone, Haliotis discus hannai. The control diet (CON) was sprayed with 108 CFU/g KRF-7 (PRO1) or PM8313 (PRO2) as probiotics, 0.10% BGO (PRE) as prebiotic, and their combinations (SYN1: PRO1 + BGO; SYN2: PRO2+ BGO) as synbiotic treatments. These diets (triplicates) were given once daily to the abalone (initial body weight: 1.32 ± 0.05 g, initial shell length: 21. 69 ± 0.19 mm) for 12 weeks. The results showed that the PRE diet induced a significant 15% shell length and body weight increase against the CON (SLGR: 45.77% vs. 53.04%; WGR: 182.77% vs. 210.22%), the highest among dietary supplements (P < 0.05). The level of IGF and IGFBP in the cell-free hemolymph, protein expression of IGF signaling, and gene expression of myosin heavy chain in the adductor muscle increased significantly (P < 0.05) following dietary supplementation. Correspondingly, PRE-, PRO1-, and PRO2-fed abalone had the highest trypsin, lipase, and amylase secretion in the hepatopancreas (P < 0.05) and increased histomorphometry (stomach epithelial thickness and intestinal villi height) (P < 0.05). Regarding antioxidative capacity and immunity, the hepatopancreatic expression of SOD, CAT, IRAK-4, NF-Kb, TNF-α, and β-defensin were significantly upregulated in abalone fed with dietary additives compared to the CON (P < 0.05). In terms of gut microbiota profile, PRE-fed abalone had a significant reduction in microbial richness (P < 0.05). The phylum level was similarly occupied, while a diet-specific core microbiota at the genus level was apparent. The findings illustrated that BGO, KRF-7, and PM8313 can be used in abalone aquaculture as growth-promoting and immune-stimulating supplements.

Immune priming modulates Galleria mellonella and Pseudomonas entomophila interaction. Antimicrobial properties of Kazal peptide Pr13a.

Galleria mellonella larvae repeatedly infected with Pseudomonas entomophila bacteria re-induced their immune response. Its parameters, i.e. the defence activities of cell-free hemolymph, the presence and activity of antimicrobial peptides, and the expression of immune-relevant genes were modulated after the re-challenge in comparison to non-primed infected larvae, resulting in better protection. No enhanced resistance was observed when the larvae were initially infected with other microorganisms, and larvae pre-infected with P. entomophila were not more resistant to further infection with other pathogens. Then, the peptide profiles of hemolymph from primed- and non-primed larvae infected with P. entomophila were compared by quantitative RP-HPLC (Reverse Phase - High Performance Liquid Chromatography). The level of carbonic anhydrase, anionic peptide-1, proline peptide-2, and finally, unknown so far, putative Kazal peptide Pr13a was higher in the primed infected animals than in the larvae infected with P. entomophila for the first time. The expression of the Pr13a gene increased two-fold after the infection, but only in the primed animals. To check whether the enhanced level of Pr13a could have physiological significance, the peptide was purified to homogeneity and checked for its defence properties. In fact, it had antibacterial activity: at the concentration of 15 µM and 7.5 µM it reduced the number of P. entomophila and Bacillus thuringiensis CFU, respectively, to about 40%. The antibacterial activity of Pr13a was correlated with changes observed on the surface of the peptide-treated bacteria, e.g. surface roughness and adhesion force. The presented results bring us closer to finding hemolymph constituents responsible for the effect of priming on the immune response in re-infected insects.

Effects of Alpinia officinarum stems and leaves extract on growth performance, non-specific immunity, and intestinal microflora of Litopenaeus vannamei

This study was to investigate the effects of Alpinia officinarum Hance stems and leaves extract (AOE) on the growth, non-specific immunity, intestinal microflora of shrimp (Litopenaeus vannamei). A total of 540 juvenile L. vannamei (2.69 ± 0.03 g) were randomly divided into six groups, and feed experiment with diet supplement of 0 g/kg (CG), 0.1 g/kg (AO-1), 0.2 g/kg (AO-2), 0.3 g/kg (AO-3), 0.5 g/kg (AO-4) and 0.7 g/kg(AO-5) AOE was carried out for 8 weeks, respectively. Compared with the control group (CG), the WGR and SGR of the AO-1, AO-3, and AO-4 groups were significantly increased. Crude protein content in the muscle of L. vannamei in AO-2 was significantly increased, while the water content was significantly decreased. The crude lipid content of each extract group was significantly decreased. Compared with the control group, activities of GSH-Px, SOD, and T-AOC in the cell-free hemolymph of AO-3 group were significantly enhanced, and the MDA content of each extract group was significantly decreased. Activities of CAT, SOD, and T-AOC in the hepatopancreas of AO-2 group were significantly increased. Gene expression levels of lvcat, lvgsh-px, and lvmn-sod in AO-3 group were obviously higher than those in the control group, while lvpropo expression was dramatically higher in AO-2 and AO-3 groups. Moreover, AOE addition in feed changed the composition and species abundance of intestinal microflora of L. vannamei at phylum and genus levels. Through α diversity analysis, the biodiversity of the flora was improved, while β diversity analysis changed the similarity of the flora between the control and addition groups. Therefore, addition of 0.2–0.3 g/kg AOE could improve the growth, immunity and optimize the composition of intestinal microorganisms of L. vannamei.

Effects of dietary bile acids levels on growth performance, anti-oxidative capacity, immunity and intestinal microbiota of abalone Haliotis discus hannai

Abalone Haliotis discus hannai (initial weight: 38.79 ± 0.70 g) was used as the experimental animal in a 105-day feeding trial to investigate the influence of dietary bile acids levels on the growth, anti-oxidation, immune response and intestinal microbiota. Six isonitrogenous and isolipidic diets were prepared by adding 0 (control group), 15, 30, 60, 120 and 240 mg/kg of bile acids, respectively (named BA0, BA15, BA30, BA60, BA120 and BA240, respectively). It was found that survival of abalone between groups had no significant difference (P > 0.05). Compared to the control, significant improvements in weight gain rate (WGR) were observed in the groups of BA30 and BA60 (P < 0.05). Based on WGR, the broken line regression model analysis showed that the optimum demand for dietary bile acids for abalone was 35.47 mg/kg. Dietary bile acids increased the total anti-oxidative capacity and activities of catalase, superoxide dismutase, lysozyme and alkaline phosphatase, meanwhile decreased the content of malondialdehyde, alanine aminotransferase and aspartate aminotransferase activities in the cell-free hemolymph (P < 0.05). When bile acids were added to the diets, mRNA levels of genes related to pro-inflammatory factors and apoptosis in the digestive gland were down-regulated (P < 0.05). In contrast, the expression of genes related to anti-oxidation was significantly up-regulated (P < 0.05). The Firmicutes, Actinobacteriota and Proteobacteria were the most abundant phyla in intestine. And dietary bile acids significantly decreased the abundance of Actinobacteria and increased the abundance of Firmicutes (P < 0.05). In conclusion, supplementation of dietary bile acids within 120 mg/kg significantly increased the growth of abalone. The 34.62 mg/kg of dietary bile acids significantly increased the anti-oxidative capacity of abalone. Appropriate levels of dietary bile acids (34.62–61.75 mg/kg) promote the immunity of abalone. Application of appropriate levels of bile acids in diets (34.62 mg/kg) changed the intestinal microbiota and promoted the intestinal health of abalone.

Impacts of replacing dietary fish meal with poultry by-product meal on growth, digestive enzymes and gut microbiota, biomarkers of metabolic and immune response, and resistance to Vibrio challenge in abalone (Haliotis discus hannai)

A 110-day feeding trial was carried out to investigate the impacts of replacing dietary fish meal (FM) with poultry by-product meal (PBM) on growth performance, digestion, gut microbiota, immunity, and disease resistance of abalone Haliotis discus hannai (initial weight: 22.53 ± 0.21 g). The control diet (PBM0) without PBM contained 20% of FM. Based on PBM0, the other four isonitrogenous (about 30% of crude protein) and isolipidic (about 4% of crude lipid) diets were formulated replacing 25, 50, 75 and 100% of FM by PBM (designated as PBM25, PBM50, PBM75 and PBM100), respectively. Every diet was fed to triplicate cages (40 × 35 × 70 cm) with 60 abalones per cage. At the end of the feeding trial, abalones were starved for 3 days and anesthetized with 5% ethanol before sampling. Results indicated that biomass gain of the abalone fed PBM75 (916.71 g) and PBM100 (659.93 g) was significantly lower than that of the abalone fed PBM0 (1222.51 g). Feed conversion ratio of the abalone fed PBM50 (2.93), PBM75 (2.89) and PBM100 (4.04) were significantly higher than that of the abalone fed PBM0 (2.37). The muscle crude protein content of the abalone fed PBM100 (16.01% wet weight) was significantly lower than that of the abalone fed PBM0 (16.67% wet weight). Activities of trypsin and α-amylase in the digestive gland in the PBM100 group (309.21 and 0.064 U/mg prot., respectively) were significantly lower than the PBM0 group (463.57 and 0.078 U/mg prot., respectively). When the substitution levels rose over 50%, expressions of phosphatidylinositol 3 kinase, threonine kinase/protein B, mammalian target of rapamycin and eukaryotic translation initiation factor 4E in muscle were significantly reduced, while 4E binding protein was significantly increased. The most abundant phyla across the abalones' gut were Proteobacteria and Firmicutes. Catalase (from 2.90 to 1.69 U/mL), superoxide dismutase (from 98.93 to 74.39 U/mL), lysozyme (from 345.86 to 247.81 U/mL) and acid phosphatase (from 21.68 to 15.71 U/100 mL) activities in cell-free hemolymph were markedly decreased with substitution level increasing. When the replacement levels rose over 50%, myeloid differentiation primary response gene 88 and tumor necrosis factor α expressions in the digestive gland were markedly upregulated, while β-defensin and mytimacin 6 expressions were markedly downregulated. The cumulative mortality of abalone challenged with Vibrio parahaemolyticus (1.0 × 107 CFU/mL) for 7 days was remarkably increased in the PBM50 (83.49%), PBM75 (89.56%) and PBM100 (100%) groups compared with the PBM0 group (58.82%). In conclusion, 50% of dietary FM could be replaced by PBM without influencing abalone growth. When the replacement level was over 50%, the growth and immunity of abalone were decreased, and the mTOR pathway was negatively affected.

Survival of Salmonella Typhimurium in the hemolymph of the German cockroach vector is limited by both humoral immune factors and hemocytes but not by trehalose metabolism.

The German cockroach (Blattella germanica) has been linked to transmission of Salmonella enterica serovar Typhimurium (S. Typhimurium), but infection dynamics within this vector are poorly characterized. Our recent work has focused on S. Typhimurium infection in the cockroach gut. However, microbial dissemination to the hemolymph is an essential aspect of many vector-borne pathogen transmission cycles and could potentially contribute to S. Typhimurium colonization of cockroaches. Therefore, the goal of this study was to examine the ability of S. Typhimurium to disseminate, survive, and proliferate in the hemolymph of cockroaches after oral infection. We detected only low numbers of bacteria in the hemolymph of a minority of insects (~26%) after oral infection. Further, S. Typhimurium was unable to survive overnight in cell-free hemolymph. Several hypotheses to explain the inability of S. Typhimurium to colonize hemolymph were tested. First, we investigated the ability of S. Typhimurium to metabolize trehalose, the primary sugar in hemolymph. S. Typhimurium grew efficiently in vitro using trehalose as a sole carbon source and mutant strains lacking trehalose metabolism genes exhibited no growth deficiencies in media mimicking the composition of hemolymph, suggesting that trehalose metabolism ability is not a factor involved in restricting survival in hemolymph. On the other hand, heat-inactivated cell-free hemolymph was permissive of S. Typhimurium growth, demonstrating that survival in hemolymph is limited specifically by heat-labile humoral factors. The involvement of cellular immune responses was also investigated and cockroach hemocytes in culture were observed to internalize S. Typhimurium within 1 h of exposure. Most hemocytes harbored few to no bacteria after 24 h, indicating that hemocyte responses are additionally involved in clearing infection from the hemolymph. However, dense intracellular clusters of S. Typhimurium were observed sporadically, suggesting a small subset of hemocytes may serve as reservoirs for bacterial replication. Together, our results reveal that a minute proportion of ingested S. Typhimurium is able to escape the cockroach gut and enter the hemolymph, but this systemic population is limited by both humoral effectors and hemocytes. Thus, we conclude that invasion of the hemolymph appears minimally important for colonization of the cockroach vector and that colonization of the gut is the main driver of vector-borne transmission. Our insight into the antimicrobial mechanisms of cockroach hemolymph also highlights the strong ability of these prevalent pests/vectors to cope with frequent infectious challenges in septic habitats.

Dietary arachidonic acid affects the growth performance, fatty acid profile, immunity and resistance to heat stress in juvenile abalone Haliotis discus hannai Ino

A 93-day feeding trial was performed to evaluate the effects of dietary arachidonic acid (ARA) on the growth performance, antioxidative capacity, immunity and anti-stress capacity of juvenile abalone Haliotis discus hannai Ino with an initial mean body weight of 4.26 ± 0.11 g. Six practical diets with 0.04% (the control), 0.31%, 0.44%, 0.68%, 0.94% and 1.34% of ARA were designed. After the feeding trial, abalones were subjected to a heat stress test. The findings showed that the survival of abalone (91.7–96.7%) after the feeding trial was not significantly influenced by dietary ARA. The 0.31–0.44% of dietary ARA significantly increased the weight gain rate (WGR). The feed conversion ratio (FCR) was decreased by 0.31–0.68% of dietary ARA. The 0.44% of dietary ARA significantly enhanced the activity of trypsin, lipase and α-amylase in the intestine. Besides, dietary ARA supplementation significantly elevated the total antioxidative capacity and activity of superoxide dismutase, catalase and lysozyme in the cell-free hemolymph. Higher levels of dietary ARA (≥0.94%) significantly up-regulated the mRNA levels of nuclear factor κB, inhibitor of κBα and transcription factor AP-1 in the digestive gland. The 0.68% of dietary ARA significantly decreased the falling rate of abalone after heat stress. In conclusion, appropriate dietary ARA levels improved the growth performance and feed utilization of abalone. The optimal dietary ARA content for juvenile abalone was 0.52% based on the quadratic regression analysis of WGR. The 0.44–0.68% of dietary ARA improved the antioxidative capacity, immunity and anti-heat stress of abalone.

Comparative evaluation on the effects of dietary docosahexaenoic acid on growth performance, fatty acid profile and lipid metabolism in two sizes of abalone Haliotis discus hannai Ino

A 93-day feeding trial was conducted to comparatively evaluate the effects of graded levels of dietary docosahexaenoic acid (DHA) (0.06%, 0.46%, 0.99%, 1.57%, 1.95% and 2.25%) on growth performance, fatty acid profile and lipid metabolism in two different sizes of abalone Haliotis discus hannai (initial body weight: 2.83 ± 0.03 g and 26.81 ± 0.17 g, respectively). Results showed that 0.99%–1.95% of dietary DHA contents significantly increased the weight gain rate (WGR) in large size of abalone (P < 0.05), while the WGR in small size of abalone was significantly increased by the 0.46%–0.99% of dietary DHA contents (P < 0.05). There was no significant difference in the survival of abalone among all the six groups (P > 0.05). Based on the WGR, the optimal dietary DHA content for large and small size abalone were estimated to be 1.32% and 0.88%, respectively. Dietary DHA supplementation significantly increased the activities of trypsin and lipase in the intestine, the content of ∑n-3 polyunsaturated fatty acids (PUFA) and ∑n-3/n-6 PUFA ratio in digestive gland in both sizes of abalone (P < 0.05). The total glyceride concentration in the cell-free hemolymph in two sizes of abalone was decreased. In large size of abalone, 1.95%–2.25% of dietary DHA reduced the lipid content in digestive gland. Lipogenic genes in the digestive gland, including sterol regulatory element binding protein-1 (srebp-1) and diacylglycerol acyltransferase, were up-regulated in the 0.46%–1.57% DHA group (P > 0.05). The mRNA expression levels of hormone sensitive lipase (hsl) and carnitine palmitoyl transferase-1 (cpt-1) of lipolytic genes in the digestive gland were significantly increased in the 0.46% and 0.99% DHA groups, respectively (P < 0.05). In the small size of abalone, the lipid content in digestive gland and soft body decreased significantly in the 1.95% and 2.25% DHA groups, respectively (P < 0.05). Lipogenic genes in the digestive gland, including srebp-1 and acetyl-CoA carboxylase, were significantly down-regulated in the 0.99%–1.95% DHA group (P < 0.05). Lipolytic genes in the digestive gland, including hsl and cpt-1, were not significantly different from the control group (P > 0.05). In conclusion, based on the WGR, the dietary DHA requirements for large and small size of abalone were estimated to be 1.32% and 0.88%, respectively. Dietary DHA reduced lipid deposition in both sizes of abalone by promoting lipid decomposition and inhibiting lipid synthesis.

Effects of dietary methionine on growth and body composition, indicators of digestion, protein metabolism and immunity, and resistance to heat stress of abalone Haliotis discus hannai

The present study was conducted to investigate the effects of dietary methionine on growth performance, protein metabolism, immunity and resistance to heat stress of abalone Haliotis discus hannai. Seven isonitrogenous (about 32% of crude protein) and isolipidic (about 3% of crude lipid) diets were designed with graded levels of methionine (0.46%, 0.77%, 0.97%, 1.19%, 1.41%, 1.71% and 2.07%, respectively). The experimental diets were named as M1 (control), M2, M3, M4, M5, M6 and M7, respectively. An 84-day feeding trial was conducted in sea cages (40 cm × 35 cm × 70 cm) for abalones (initial body weight: 4.10 ± 0.01 g), which were fed once every two days. The results showed that the biomass gain (BG) of abalone significantly increased (from 560.20 g to 643.83 g) with the increasing dietary methionine levels from 0.46% to 1.19%, and then decreased (from 643.83 g to 484.29 g) (P < 0.05). Based on the broken-line regression analysis of BG, the requirement of dietary methionine for abalone was determined to be 0.99% (dry matter of diet). The highest trypsin activity (862.10 U/mg prot.) and α-Amylase activity (0.28 U/mg prot.) in the intestine of abalone were observed in the M4 and M5 group, respectively. In the digestive gland, the gene expression of mtor, s6k and eif4e showed a significant quadratic trend with the increasing dietary methionine levels, and all of them reached their highest values in the M4 group. The highest mRNA level of gcn2 was observed in the M1 group. The glutathione content and total anti-oxidative capacity in cell-free hemolymph (CFH) were significantly increased as dietary methionine levels increasing from 0.46% to 1.19%, and then decreased (P < 0.05). The lowest gene expression level of keap1, nf-κb and tnf-α, and the highest gene expression level of nrf2 and iκbα in the digestive gland were found in the M4 group. After the heat stress test at the end of feeding trial, the minimum falling rate (19.44%) and the highest gene expression level of hsp70 and hsp90 in the digestive gland of abalone were found in the group with 1.19% of dietary methionine. In conclusion, appropriate dietary methionine (0.97%–1.19%) could improve the growth, protein synthesis, immunity and resistance to heat stress of abalone.

Comparative study of immunological biomarkers in the carpet shell clams (Ruditapes decussatus) from metal-contaminated sites in the South Lagoon of Tunis (Tunisia)

The South Lagoon of Tunis (Tunisia) is a Mediterranean lagoon adversely affected by industrial contaminants, harbour activity and untreated urban sewage. In this lagoon, the clam Ruditapes decussatus has been widely used as a biomonitor of seawater pollution through measurements of parameters related to oxidative stress and neurotoxicity. However, few studies have considered parameters of the immune system of this species in the South Lagoon of Tunis. Therefore, the aim of the present work was to evaluate several immune-related parameters in the cell-free haemolymph of carpet shell clams sampled during August and February from three polluted sites in the South Lagoon of Tunis (S1, S2 and S3) and one less polluted site as a reference site (RS) in order to identify suitable biomarkers for environmental quality assessments of this ecosystem. Concerning the immune-related parameters, seasonal factors modulated phenoloxidase, lysozyme, protease and esterase activity, with lower values measured for samples collected in August than for samples collected in February. In fact, bactericidal activity against two of the pathogenic bacteria tested and the activity of most immune-related enzymes were reduced in the cell-free haemolymph of clams collected from the most sampling sites in August compared to February one. In addition, values of abiotic parameters (temperature, salinity and pH) and metal (cadmium, copper, iron, lead and zinc) concentrations in the clams’ soft tissues, previously obtained and published by the authors, as well as the values of immune-related parameters were integrated using principal component analyses. Results indicated that the values of all measured immune-related parameters were negatively correlated with the temperature values and the variations most of these parameters highlighted that the chemical industrial area (S3) was the most impacted location within the South Lagoon of Tunis. The present study illustrates that the immune-related parameters measured in carpet shell clam cell-free haemolymph represent suitable biomarkers for environmental quality assessments because they provide effective seasonal and spatial discrimination.

Effects of replacing dietary fish meal with enzyme-treated soybean meal on growth performance, intestinal microbiota, immunity and mTOR pathway in abalone Haliotis discus hannai

In addition to eliminating most of the anti-nutritional factors in soybean meal, enzyme-treated soybean meal (ESBM) can also increase the proportion of small peptides. It was found that ESBM can replace fish meal (FM) either partially or completely in diets for some fish and shrimp species. In the present study, the effects of replacing dietary FM with ESBM on growth performance, intestinal microbiota, immunity and mTOR pathway in abalone Haliotis discus hannai (initial weight: 16.75 ± 0.09 g) were investigated after a 100-day feeding trial. Five experimental diets were designed to replace 0%, 25%, 50%, 75% and 100% of dietary FM by ESBM, which were named as ESBM0 (control), ESBM25, ESBM50, ESBM75 and ESBM100, respectively. Results showed that ESBM could replace up to 75% of FM in the diet without significant effect on the weight gain rate (WGR, 118.05%–124.16%) of abalone. The increasing dietary ESBM levels significantly decreased the trypsin activity from 418.52 to 286.52 U/mg protein in the digestive gland. No significant differences in the contents of total cholesterol (T-CHO), ammonia (BLA) and malondialdehyde (MDA) in cell-free hemolymph were observed among the groups with replacement levels of dietary FM by ESBM from 0% to 75%. Excessive replacement level of FM with ESBM (ESBM100) significantly increased the MDA content (2.33 nmol/mg prot.) and pro-inflammatory-related gene expression in digestive gland. Compared with the control group, the mTOR pathway in muscle was significantly upregulated in the ESBM75 group. The digestive gland in the ESBM100 group contained more golden refractile spherules than those in the other groups. The abundance of intestinal microbes such as Halomonas, Zobellella and Bacillus was decreased in the ESBM100 group. In conclusion, up to 75% of replacement of dietary FM by ESBM had no negative effects on the growth performance, intestinal microbiota, immunity and mTOR pathway of abalone.

Preferential binding of DAP-PGs by major peptidoglycan recognition proteins found in cell-free hemolymph of Manduca sexta

Peptidoglycan recognition proteins (PGRPs) detect invading bacteria to trigger or modulate immune responses in insects. While these roles are established in Drosophila, functional studies are not yet achieved at the PGRP family level in other insects. To attain this goal, we selected Manduca sexta PGRP12 and five of the nine secreted PGRPs for recombinant expression and biochemical characterization. We cloned PGRP2−5, 12 and 13 cDNAs, produced the proteins in full (PGRP2–5, 13) or in part (PGRP3s, 12e, 13N, 13C) in Sf9 cells, and tested their bindings of two muramyl pentapeptides by surface plasmon resonance, two soluble peptidoglycans by competitive ELISA, and four insoluble peptidoglycans and eight whole bacteria by a pull-down assay. Preferential binding of meso-diaminopimelic acid-peptidoglycans (DAP-PGs) was observed in all the proteins containing a peptidoglycan binding domain and, since PGRP6, 7 and 9 proteins were hardly detected in cell-free hemolymph, the reportoire of PGRPs (including PGRP1 published previously) in M. sexta hemolymph is likely adapted to mainly detect Gram-negative bacteria and certain Gram-positive bacteria with DAP-PGs located on their surface. After incubation with plasma from naïve larvae, PGRP2, 3f, 4, 5, 13f and 13N considerably stimulated prophenoloxidase activation in the absence of a bacterial elicitor. PGRP3s and 12e had much smaller effects. Inclusion of the full-length PGRPs and their regions in the plasma also led to proHP8 activation, supporting their connections to the Toll pathway, since HP8 is a Spӓtzle-1 processing enzyme in M. sexta. Together, these findings raised concerns on the common belief that the Toll-pathway is specific for Gram-positive bacteria in insects.

CgHMGB1 functions as a broad-spectrum recognition molecule to induce the expressions of CgIL17-5 and Cgdefh2 via MAPK or NF-κB signaling pathway in Crassostrea gigas

High-mobility group box 1 (HMGB1), a highly conserved nucleoprotein, functions in immune recognition, inflammation and antibacterial immunization in vertebrates. In the present study, the mediation mechanism of CgHMGB1 in activating MAPK and NF-κB/Rel signaling pathways to induce the expressions of immune effectors was investigated. CgHMGB1 mRNA was detected in all tested developmental stages from fertilized egg to D-larvae, with the higher expressions in 4 cells and 8 cells stages. CgHMGB1 proteins were mainly distributed in haemocyte granulocytes. The expressions of CgHMGB1 mRNA in haemocytes increased significantly after Vibrio splendidus stimulation, and CgHMGB1 protein translocated into the haemocyte cytoplasm and release into cell-free haemolymph. The phosphorylation of CgERK and CgP38 were induced, the nuclear translocation of CgRel were promoted, and the mRNA expressions of CgIL17-5 and Cgdefh2 increased significantly after rCgHMGB1 treatment. Obvious branchial swelling and cilium shedding were observed after rCgHMGB1 treatment. rCgHMGB1 exhibited binding activity to different polysaccharides, bacteria, and fungi. rCgHMGB1 also displayed obvious antibacterial activity to V. splendidus and E. coli. These results indicated that CgHMGB1 functioned as an immune recognition molecule to recognize various PAMPs and bacteria to induce the mRNA expressions of CgIL17-5 and Cgdefh2 via the activation of MAPK and NF-κB signaling pathways in oysters.

Effects of dietary chromium yeast levels on growth performance, anti-oxidative capacity, immune response and flesh quality of abalone Haliotis discus hannai

A 110-day feeding trial was conducted to investigate the effects of dietary chromium (Cr) on the growth performance, immune response, anti-oxidative capacity, stress resistance, flesh quality of abalone Haliotis discus hannai. Seven isonitrogenous and isolipidic experimental diets were formulated with graded levels of Cr (0.04, 0.67, 1.21, 1.58, 2.21, 2.72 and 3.33 mg/kg) from Cr yeast. The results showed that weight gain rate (WGR) was highest in the group with 1.21 mg/kg of dietary Cr. Based on the broken-line regression analysis for WGR, the dietary Cr requirement of abalone was estimated to be 1.32 mg/kg. With the increase of dietary Cr level from 0.04 to 1.21 mg/kg, the total anti-oxidative capacity and lysozyme activity in the cell-free hemolymph were increased and then decreased when the dietary Cr level exceeded 1.21 mg/kg. After the challenge of Vibrio parahaemolyticus, the cumulative mortality of abalone was significantly decreased in the group with 1.21 mg/kg of dietary Cr compared with that in the control group. Meanwhile, after the heat stress test, the cumulative mortality of abalone was significantly reduced in the groups with 1.58, 2.21 and 2.72 mg/kg of dietary Cr compared with that in the control group. The liquid and water loss of foot muscle were significantly increased in the groups with 2.72 and 3.33 mg/kg of dietary Cr. With the increase of dietary Cr levels, the hardness of foot muscle was significantly elevated. The chewiness of foot muscle was significantly increased with the increase of dietary Cr level from 0.04 to 1.21 mg/kg, and thereafter showed no significant difference when dietary Cr exceeded 1.21 mg/kg. In conclusion, based on the WGR, dietary Cr requirement of abalone (initial weight: 22.31 ± 0.31 g) was estimated to be 1.32 mg/kg. Appropriate dietary Cr levels (1.21–1.58 mg/kg) had positive effects on growth performance, immune response, anti-oxidation, anti-stress and flesh quality of abalone. Overhigh dietary Cr level (2.72–3.33 mg/kg) decreased the flesh quality. Excessive dietary Cr level (3.33 mg/kg) had a negative effect on the health of abalone.