Effects of laminarin on growth performance and resistance against Pseudomonas plecoglossicida of large yellow croaker （Larimichthys crocea）.

Laminarin (LAM) is widely used as an immunopotentiator in aquaculture, but its protective mechanism is still unclear. In this study, the effects of LAM on the growth performance and resistance against Pseudomonas plecoglossicida of large yellow croaker were studied in vitro and in vivo. The 42 d-feeding trial in large yellow croaker showed that dietary LAM could obviously promote the fish growth by improving the weight gain rate (WGR), specific growth rate (SGR), and feed conversion rate (FCR). Dietary LAM could also improve the survival rate of large yellow croakers subjected to P. plecoglossicida infection, and 500 mg/kg LAM produced the highest relative percent survival (RPS) of 35.00 %. LAM improved fish antioxidant level by enhancing serum total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity, and reducing malondialdehyde (MDA) content. In addition, LAM also improved fish innate immunity by increasing serum acid phosphatase (ACP) and alkaline phosphatase (AKP) activities and complement 3 (C3) content under P. plecoglossicida infection. What is more, on 9 d post P. plecoglossicida challenge, LAM could significantly decrease the bacteria load in head kidneys, spleens and livers of fish, and the lowest bacterial load was found in 500 mg/kg LAM group. In vitro, LAM exerted a protective role against inactivated P. plecoglossicida-triggered inflammatory injury in primary head kidney macrophages (PKM) of large yellow croaker by recovering cell viability, suppressing NO production, and reversing pro-inflammatory cytokine expression (IL-1ß, IL-6, and IL-8). All these findings therefore will provide insights into the protection mechanism of LAM in fish, facilitating its application in prevention and control of fish bacteriosis.

Effects of Dietary Fish Meal Replaced by Fish Steak Meal on Growth Performance, Antioxidant Capacity, Intestinal Health and Microflora, Inflammatory Response, and Protein Metabolism of Large Yellow Croaker Larimichthys crocea.

Although fish steak meal (FSM) is a potentially available protein source, its efficiency as a fish meal (FM) substitute remains unclear to date. To this end, this study was carried out to determine the effects of dietary FM replaced by FSM on growth performance, antioxidant capacity, intestinal health and microflora, inflammatory response, and protein metabolism of large yellow croaker. Five isolipidic and isonitrogenous diets were formulated by substituting FM with FSM at levels of 0% (FSM0, control diet), 25% (FSM25), 50% (FSM50), 75% (FSM75), and 100% (FSM100), and were fed to juvenile large yellow croaker for 8 weeks. Compared with the control diet, the replacement of 25% dietary FM with FSM did not markedly alter the weight gain (WG) and specific growth rate (SGR). When the FM substitution level was over 25%, WG and SGR markedly reduced. The intestinal structure observation found that the FSM75 and FSM100 diets markedly decreased villus height, villus width, and muscle thickness of the anterior intestine. The FSM75 and FSM100 diets significantly decreased enzyme activities of amylase (AMS), lipase (LPS), trypsin, catalase (CAT), and total superoxide dismutase (T-SOD) and the total antioxidant capacity (T-AOC), and increased the malondialdehyde (MDA) content in the liver of large yellow croaker. The mRNA expression levels of intestinal barrier and inflammatory response-related genes suggested that the FSM50, FSM75, and FSM100 diets significantly decreased the mRNA abundances of intestinal barrier-related genes and anti-inflammatory response-related genes, and increased the mRNA abundances of proinflammatory gene il-6 in the anterior intestine. The compositions of intestinal microflora displayed that the FSM50, FSM75, and FSM100 diets decreased relative abundances of Firmicutes phylum and increased relative abundances of Proteobacteria phylum. In addition, the results of protein expression levels showed that the phosphorylation level of mammalian target of rapamycin (mTOR) and 4E-binding protein 1 (4E-BP1) in FSM75 and FSM100 groups were markedly reduced. In conclusion, FSM can replace up to 25% dietary FM without compromising the growth performance, intestinal health, and protein metabolism of the large yellow croaker.

Partial Substitution of Fish Meal with Soy Protein Concentrate on Growth, Liver Health, Intestinal Morphology, and Microbiota in Juvenile Large Yellow Croaker (Larimichthys crocea).

The present study investigated the growth performance, feed utilization, intestinal morphology, and microbiota communities of juvenile large yellow croaker (Larimichthys crocea) fed diets containing different proportions of soy protein concentrate (SPC) (0, 15%, 30%, and 45%, namely FM, SPC15, SPC30, and SPC45) as a substitute for fish meal (FM) for 8 weeks. The weight gain (WG) and specific growth rate (SGR) in fish fed SPC45 were significantly lower than those fed FM and SPC15 but not differ with these fed SPC30. The feed efficiency (FE) and protein efficiency ratio (PER) decreased sharply when the dietary SPC inclusion level was higher than 15%. The activity of alanine aminotransferase (ALT) and expression of alt and aspartate aminotransferase (ast) were significantly higher in fish fed SPC45 than those fed FM. The activity and mRNA expression of acid phosphatase were opposite. The villi height (VH) in distal intestine (DI) showed a significant quadratic response to increasing dietary SPC inclusion levels and was highest in SPC15. The VH in proximal intestine, middle intestine decreased significantly with increasing dietary SPC levels. The 16S rRNA sequences in intestine revealed that fish fed SPC15 had higher bacterial diversity and abundance of Phylum Firmicutes such as order Lactobacillales and order Rhizobiaceae than those fed other diets. Genus vibrio, family Vibrionaceae and order Vibrionales within phylum Proteobacteria were enriched in fish fed FM and SPC30 diets. Tyzzerella and Shewanella that belongs to phylum Firmicutes and Proteobacteria, respectively, were enriched in fish fed SPC45 diet. Our results indicated that SPC replacing more than 30% FM could lead to lower quality diet, retard growth performance, ill health, disordered intestine structure, and microbiota communities. Tyzzerella could be the bacteria indicator of intestinal in large yellow croaker fed low quality diet due to high SPC content. Based on the quadratic regression analysis of WG, the best growth performance could be observed when the replacement of FM with SPC was 9.75%.

Adhesion and Colonization of the Probiotic Lactobacillus plantarum HC-2 in the Intestine of Litopenaeus Vannamei Are Associated With Bacterial Surface Proteins.

Surface proteins are a type of proteins expressed on the surface of bacteria that play an important role in cell wall synthesis, maintenance of cell morphology, and signaling with the host. Our previous study showed that the probiotic Lactobacillus plantarum HC-2 improved the growth performance and immune response of Litopenaeus vannamei. To further investigate the probiotic mechanism, we determined the automatic aggregation ability of the bacteria and surface hydrophobicity of HC-2 after being treated with 5 M of lithium chloride (LiCl) and observed the morphology and adhesion of the bacteria to HCT116 cells. The results showed that with the removal of the HC-2 surface protein, the auto-aggregation ability and surface hydrophobicity of HC-2 decreased, and the crude mucus layer coated on the bacterial surface gradually dissociated. The adhesion rate of HC-2 to HCT116 cells decreased from 98.1 to 20.9%. Moreover, a total of 201 unique proteins were identified from the mixture of the surface proteins by mass spectrometry (MS). Several proteins are involved in transcription and translation, biosynthetic or metabolic process, cell cycle or division, cell wall synthesis, and emergency response. Meanwhile, a quantitative real-time PCR qPCR_ showed that HC-2 was mainly colonized in the midgut of shrimp, and the colonization numbers were 15 times higher than that in the foregut, while the colonization rate in the hindgut was lower. The adhesion activity measurement showed that the adhesion level of HC-2 to crude intestinal mucus of L. vannamei was higher than that of bovine serum albumin (BSA) and collagen, and the adhesion capacity of the bacterial cells decreased with the extension of LiCl-treatment time. Finally, we identified the elongation factor Tu, Type I glyceraldehyde-3-phosphate dehydrogenase, small heat shock protein, and 30S ribosomal protein from the surface proteins, which may be the adhesion proteins of HC-2 colonization in the shrimp intestine. The above results indicate that surface proteins play an important role in maintaining the cell structure stability and cell adhesion. Surface proteomics analysis contributes to describing potential protein-mediated probiotic-host interactions. The identification of some interacting proteins in this work may be beneficial to further understand the adhesion/colonization mechanism and probiotic properties of L. plantarum HC-2 in the shrimp intestine.

Effects of Time-Dependent Protein Restriction on Growth Performance, Digestibility, and mTOR Signaling Pathways in Juvenile White Shrimp Litopenaeus vannamei.

A 6-week feeding strategy experiment was conducted to investigate the effects of time-dependent protein restriction and subsequent recovery on shrimp. Diets with protein levels of 43 and 36% were used as adequate and restricted diets, respectively. Shrimp with an initial body weight of 6.52 ± 0.46 g were given four feeding strategies: feeding on an adequate diet for six weeks (T1, the control), having protein-restricted diet in weeks 1 and 4 (T2), being given a protein-restricted diet in weeks 1, 3, and 5 (T3), and having protein-restricted diet in weeks 1, 2, 4, and 5 (T4). WG, SGR, FE, and PER of shrimp in T1-T3 showed no significant difference (P > 0.05), these indicators of T4 were significantly reduced (P < 0.05). No significant differences were found in digestive enzyme activities of shrimp among all treatments (P > 0.05). Crude protein content of shrimp muscle in T4 was lower than that of T1-T3. The expression level of tor in T4 was lower than that in other treatments, while 4e-bp was higher than that of other treatments. To balance saving on feeding cost and growth performance, giving the shrimp a protein-restricted diet for 1 week with subsequent refeeding (T2 and T3) is suitable for shrimp under high-density conditions.

Dietary Different Replacement Levels of Fishmeal by Fish Silage Could Influence Growth of Litopenaeus vannamei by Regulating mTOR at Transcriptional Level.

Fish silage (FS) has been confirmed as a high-quality feed ingredient because of its balanced nutrition, low cost, and environmental friendliness. In the present study, we evaluated the performance of replacing fishmeal by FS in the diet of white shrimp, Litopenaeus vannamei. Five isonitrogenous (410 g kg-1) and isolipidic (75 g kg-1) diets were formulated with replacement of fishmeal by 0% (FM), 25% (FS25%), 50% (FS50%), 75% (FS75%), and 100% (FS100%) FS. After an 8-week trial, shrimps fed low FS diets (FM and FS25%) had significantly higher final weight (FW), weight gain (WG), and specific growth ratio (SGR) (P < 0.05). No significant differences were found in body composition and most antioxidant enzyme activities of all groups (P > 0.05). Compared to high FS groups (FS75% and FS100%), low FS replacement levels (0 and 25%) had enhanced trypsin activity. And trypsin transcriptional level presented a similar trend with trypsin activity. In terms of intestinal histopathology, no obvious histological damage was observed in the intestine of all groups. tor and s6k of low replacement level groups (FM and FS25%) were significantly upregulated (P < 0.05), which indicated activation of mammalian target of rapamycin (mTOR) signaling pathway in low FS groups at transcriptional level. The enhanced performances of growth and mTOR signaling pathway in low FS groups (FM and FS25%) provided us some insights into the regulation mechanism of nutrient signal on growth. Based on the above, dietary FS could influence the growth of shrimp by regulating mTOR at the transcriptional level, and FS is a potential substitute of fishmeal in shrimp feed.

The effect of rearing density on immune responses of hepatopancreas and intestine in Litopenaeus vananmei against Vibrio paraheamolyticus E1 challenge.

Rearing density and disease management are considered as pivotal factors determining shrimp farm productivity and profitability. To systematically investigate the potential mechanisms for density-related differences between disease susceptibility and rearing densities, we conducted comparative transcriptome analysis of the molecular differences between hepatopancreas and intestine of Litopenaeus vannamei under two different rearing densities (800- and 400- shrimp/m3) for 15 d and further analyzed the differences in immune response to Vibrio parahaemolyticus E1 (VPE1) raised under two density conditions. Totally 45 different expression genes (DEGs) were identified in the hepatopancreas under two different rearing densities, the DEGs were grouped into four processes or pathways related to animal immune system. Then, exposure to the VPE1 resulted in 639 DEGs, involved into fourteen immune related processes or pathways. In the intestine, seventeen processes or pathways related to the immune system were identified among the 5470 DEGs under two different rearing densities. 279 DEGs were identified post VPE1 challenge, classified into five processes or pathways associated with the immune system. Meanwhile, the results of growth performance, histopathology and the activities of antioxidant enzymes in the hepatopancreas and intestines of shrimp showed that high density decreased weight gain rate (63.20 ±â€¯1.67% and 18.73 ±â€¯3.35% in the high and low rearing density groups, respectively), severely destroyed the histopathology and inhibited the antioxidant enzymes activities. This study demonstrated that rearing density in L. vannamei significantly impacts susceptibility to the VPE1, via altered transcriptional challenge responses, and thus higher mortality due to disease.

Growth Performance, Digestive Enzymes, and TOR Signaling Pathway of Litopenaeus vannamei Are Not Significantly Affected by Dietary Protein Hydrolysates in Practical Conditions.

Protein hydrolysates have been widely reported as the protein source of aquatic feed. However, previous studies on protein hydrolysates focused on fish under experimental conditions. In this study, a 6-week feeding trial in a greenhouse was conducted to investigate the effects of partially replacing fishmeal by protein hydrolysates on growth performance, digestive enzymes, and TOR signaling pathway of Litopenaeus vannamei under practical conditions. This involved randomly selecting 72,000 shrimps (initial body weight 2.26 ± 0.02 g) and placing them in groups inside nine concrete tanks (L 5 m × W 5 m × H 1 m) (3 treatments × 3 replicates × 8000 individuals per concrete tank). Two isonitrogenous (430 g kg-1) and isolipidic (80 g kg-1) diets were prepared: fishmeal diet (FM) containing 400g kg-1 fishmeal, and protein hydrolysates diet (PH) in which 15% of the fishmeal was replaced by protein hydrolysates. A commercial diet (CD) was used as reference. The final weight (FW), percent weight gain (PWG), specific growth ratio (SGR), and total weight for each tank (TW) of L. vannamei fed with FM and PH diets were not significantly different (P > 0.05). However, shrimp fed with PH diet had significantly higher FW, PWG, SGR, and TW values than those fed with CD diet (P < 0.05). Trypsin activity was significantly lower in shrimp fed with CD diet, than in shrimp fed with FM and PH diets (P < 0.05). However, trypsin activity of L. vannamei fed with FM and PH diets were not significantly different (P > 0.05). The mRNA expression of tor, s6k, and 4e-bp genes were not significantly affected between FM and PH diets, while tor and s6k expression levels of CD diet were significantly down-regulated. Based on the above data, 15% replacement of fishmeal with protein hydrolysates did not make any difference on shrimps compared with FM group. Therefore, protein hydrolysates can partially replace fishmeal as the protein source of shrimp formula feed in practical conditions.

Effects of high ammonia concentrations on three cyprinid fish: Acute and whole-ecosystem chronic tests.

A number of studies have revealed ammonia to be toxic to aquatic organisms; however, little is known about its effects under natural conditions. To elucidate the role of ammonia, we conducted 96-h acute toxicity tests as well as a whole-ecosystem chronic toxicity test for one year in ten 600-m2 ponds. Three common cyprinids, silver carp Hypophthalmichthys molitrix Val. (H.m.), bighead carp Aristichthys nobilis Richardson (A.n.), and gibel carp Carassius auratus gibelio Bloch (C.g.), were used as test organisms. The 96-h LC50 values of un-ionized ammonia (NH3) for H.m., A.n., and C.g. were 0.35, 0.33, and 0.73mgL-1, respectively. In the ponds, annual mean NH3 ranged between 0.01 and 0.54mgL-1, with 4 ponds having a NH3 higher than the LC50 of A.n. (lowest LC50 in this study). No fish were found dead in the high-nitrogen ponds, but marked histological changes were found in livers and gills. Despite these changes, the specific growth rate of H.m. and A.n. increased significantly with NH3. Our pond results suggest that fish might be more tolerant to high ammonia concentrations in natural aquatic ecosystems than under laboratory conditions. Our finding from field experiments thus suggests that the existing regulatory limits for reactive nitrogen (NH3) established from lab toxicity tests might be somewhat too high at the ecosystem conditions. Field-scale chronic toxicity tests covering full life histories of fish and other aquatic organisms are therefore encouraged in order to optimize determination of the effects of ammonia in natural environments.

Effects of high nitrogen concentrations on the growth of submersed macrophytes at moderate phosphorus concentrations.

Eutrophication of lakes leading to loss of submersed macrophytes and higher turbidity is a worldwide phenomenon, attributed to excessive loading of phosphorus (P). However, recently, the role of nitrogen (N) for macrophyte recession has received increasing attention. Due to the close relationship between N and P loading, disentanglement of the specific effects of these two nutrients is often difficult, and some controversy still exists as to the effects of N. We studied the effects of N on submersed macrophytes represented by Vallisneria natans (Lour.) Hara in pots positioned at three depths (0.4 m, 0.8 m, and 1.2 m to form a gradient of underwater light conditions) in 10 large ponds having moderate concentrations of P (TP 0.03 ± 0.04 mg L(-1)) and five targeted concentrations of total nitrogen (TN) (0.5, 2, 10, 20, and 100 mg L(-1)), there were two ponds for each treatment. To study the potential shading effects of other primary producers, we also measured the biomass of phytoplankton (ChlaPhyt) and periphyton (ChlaPeri) expressed as chlorophyll a. We found that leaf length, leaf mass, and root length of macrophytes declined with increasing concentrations of TN and ammonium, while shoot number and root mass did not. All the measured growth indices of macrophytes declined significantly with ChlaPhyt, while none were significantly related to ChlaPeri. Neither ChlaPhyt nor ChlaPeri were, however, significantly negatively related to the various N concentrations. Our results indicate that shading by phytoplankton unrelated to the variation in N loading and perhaps toxic stress exerted by high nitrogen were responsible for the decline in macrophyte growth.

[Alleles of HLA class II DRB1 of patients with essential hypertension in Yunnan Hans].

OBJECTIVE: To investigate the association between the alleles of human leukocyte antigens (HLA)-DRB1 and essential hypertension (EH) in the Hans living in Yunnan of China. METHODS: The alleles of HLA-DRB1 were typed by polymerase chain reaction-sequence specific primers (PCR-SSP) technique in samples from 83 patients with essential hypertension and 91 healthy controls. All of the subjects, their grandparents and grand-grandparents are Hans living in Yunnan of China. RESULTS: The frequency of DRB1*1501/2 in the EH patients (0.219) was significantly higher than that in the controls(0.060), chi quare=18.331, P<0.01. Relative risk (RR) was 4.46, and etiologic factor (EF) was 0.34. The frequency of DRB1*0901 significantly decreased in the group of patients with EH in comparison to the controls (0.081 versus 0.192) chi-square=8.704 P<0.05. RR was 0.41 and prevention factor(PF) was 0.19. CONCLUSION: HLA-DRB1*1501/2 is associated with the susceptibility to essential hypertension in the Yunnan Hans of China. DRB1*0901 may protect one from the disease.