实验海洋生物学重点实验室知识库

本研究以凡纳滨对虾（Litopenaeus vannamei）这一重要经济养殖种类为研究对象，分别以生物絮团、青贮鱼粉和发酵豆粕部分替代凡纳滨对虾饲料中的鱼粉，进行养殖实验，从mTOR信号通路、肠道菌群、代谢组和蛋白质组等方面探究凡纳滨对虾对不同鱼粉替代物的响应机制。具体研究结果如下：

1、生物絮团替代凡纳滨对虾饲料中鱼粉的生产应用评估

以初始体重为7.76 ± 0.61 g的凡纳滨对虾为研究对象，在实际生产的尺度上探究生物絮团对凡纳滨对虾饲料中鱼粉的替代效果。实验设计和制作了两种等氮等脂的饲料：分别为全鱼粉组（FM）和以生物絮团替代15%的鱼粉组（BM），并选用一种商品化饲料作为对照（CD），在温棚内进行为期6周的生产尺度养殖。结果发现：BM组相对于FM组，在生长表现上并无显著性差异（P > 0.05），而BM组和FM组在生长表现上显著优于CD组（P < 0.05）；在体成分方面，BM组和FM组的粗蛋白含量显著高于CD组（P < 0.05）；在消化酶指标方面，BM组和FM组的胰蛋白酶酶活和基因表达水平也显著高于CD组（P < 0.05）；在mTOR信号通路关键基因表达量上，BM组和FM组的tor基因和s6k基因的表达量并无显著性差异（P > 0.05），且二者的表达量较CD组均显著上调（P < 0.05）。以上结果说明以生物絮团替代15%的鱼粉在生产上是可行的，mTOR信号通路与生长之间具有密切关系。

2、凡纳滨对虾mTOR信号通路和肠道菌群对饲料中青贮鱼粉替代鱼粉的响应

以初始体重为0.26 ± 0.03 g的凡纳滨对虾为研究对象，以青贮鱼粉替代凡纳滨对虾饲料中的鱼粉，通过养殖实验确定青贮鱼粉替代鱼粉的合理替代水平，并从mTOR信号通路和肠道菌群两方面研究凡纳滨对虾对青贮鱼粉这一鱼粉替代物的响应机制。实验设计了五种等氮等脂的饲料：分别为全鱼粉组（FM）以及分别以青贮鱼粉替代25%（FS25%）、50%（FS50%）、75%（FS75%）和100%（FS100%）的鱼粉，在温棚内进行为期8周的养殖实验。结果发现：鱼粉组（FM）和25%鱼粉替代组（FS25%）在生长表现上要显著优于其他高替代水平处理组（P < 0.05）；在mTOR信号通路关键基因表达量上，FM和FS25%的tor基因和s6k基因的表达量也显著上调（P < 0.05）；在肠道菌群方面，摄食不同替代水平青贮鱼粉对肠道菌群的组成和Beta多样性造成一定影响，而对肠道菌群丰度、Alpha多样性和功能影响并不显著（P > 0.05）。以上结果说明以青贮鱼粉替代凡纳滨对虾饲料中25%的鱼粉是较为合理的替代方案，摄食不同替代水平的青贮鱼粉很可能通过影响mTOR信号通路，进一步影响生长，且肠道菌群也是凡纳滨对虾对青贮鱼粉替代鱼粉响应机制中的重要因素。

3、凡纳滨对虾mTOR信号通路和肠道菌群对饲料中发酵豆粕替代鱼粉的响应

以初始体重为0.27 ± 0.04 g的凡纳滨对虾为研究对象，以发酵豆粕替代凡纳滨对虾饲料中的鱼粉。实验设计了五种等氮等脂的饲料：分别为全鱼粉组（FM）以及分别以发酵豆粕替代10%（FSM10%）、20%（FSM20%）、30%（FSM30%）和40%（FSM40%）的鱼粉，在温棚内进行为期8周的养殖实验。结果发现：以发酵豆粕替代20%鱼粉处理组（FSM20%）在生长表现上要显著优于其他处理组（P < 0.05）；在mTOR信号通路关键基因表达量上，FSM20%的tor基因和s6k基因的表达量也显著上调（P < 0.05）；在肠道菌群方面，摄食不同替代水平发酵豆粕对肠道菌群的组成、多样性和功能均不造成显著影响（P > 0.05）。以上结果说明以发酵豆粕替代凡纳滨对虾饲料中20%的鱼粉是较为合理的替代水平，mTOR信号通路与生长之间具有密切关系，是凡纳滨对虾对发酵豆粕替代鱼粉响应机制中的重要一环。

4、基于代谢组学和蛋白质组学筛选凡纳滨对虾体内对发酵豆粕替代鱼粉响应显著的信号通路

本实验选取发酵豆粕替代鱼粉实验中生长表现差异较大的FM、FSM20%和FSM40%为研究对象，分别使用LC-MS和TMT技术进行代谢组学和蛋白质组学分析。结果发现：在FM与FSM40%、FM与FSM20%和FSM20%与FSM40%的比较中，分别鉴定到差异代谢产物29种、97种和61种，主要差异代谢产物集中在蛋白质消化和吸收、ABC转运蛋白和癌症中的中心碳代谢通路；在三个处理组中，共鉴定蛋白总数3877个，在FM与FSM40%、FM与FSM20%和FSM20%与FSM40%的比较中，分别发现差异表达蛋白202个、184个和179个，主要差异表达蛋白集中在胰高血糖素、溶酶体和蛋白质消化和吸收通路；代谢组学和蛋白质组学联合分析显示，蛋白质消化和吸收信号通路在三个处理组两两比较中均发生显著变化，说明凡纳滨对虾的这一信号通路对饲料中发酵豆粕替代鱼粉后做出显著响应。

In the present study, we replaced fish meal with biofloc meal, fish silage and fermented soybean meal in the diet of white shrimp (Litopenaeus vannamei). And we further investigated the response mechanism of shrimp to fish meal substitutes in terms of mTOR signaling pathway, intestinal microbiota, metabonomics and proteomics. All main results are as follows:

1. Evaluation of biofloc meal as an ingredient in diets for Litopenaeus vannamei under practical conditions

This study evaluated the potential for the replacement of fish meal with biofloc meal in the diet of Litopenaeus vannamei under practical conditions. Two isonitrogenous and isolipidic diets were formulated, in which fish meal was replaced with biofloc meal at 0% (FM) and 15% (BM). A commercial diet (CD) was used as reference. A 6-week feeding trail was conducted in concrete tanks in green house with 8000 shrimps (initial weight 7.76 ± 0.61 g) per tank. Compared to FM, BM did not make negative effect on growth performance, digestive enzymes and mTOR signaling pathway (P > 0.05). The parameters for growth performance of both FM and BM were significantly higher than that of CD (P < 0.05). Through real-time quantitative PCR analysis, relative expression levels of trypsin, tor and s6k of CD were significantly down-regulated (P < 0.05). Based on the above data, 15% replacement of fishmeal with biofloc meal did not make any difference on shrimps compared with fish meal group. And mTOR signaling pathway is closed to growth of shrimp.

2. Response to replacement of fish meal with fish silage of mTOR signaling pathway and intestinal microbiota in Litopenaeus vannamei

This study evaluated the potential for the replacement of fish meal with fish silage in the diet of Litopenaeus vannamei. And an Illumina-based highthroughput sequencing method was further used to analyse the intestinal microbiota of shrimp. Five isonitrogenous and isolipidic diets were formulated: a control diet (FM) containing and four experimental diets in which the fishmeal in control diet was replaced by fish silage at 25 (FS25%), 50 (FS50%), 75 (FS75%) and 100% (FS100%). An 8-week feeding trail was conducted in fifteen fiberglass tanks with 50 shrimps (initial weight 0.26 ± 0.03 g) per tank. After 8 weeks trial, shrimps fed with diets FM and FS25% had significantly enhanced growth performance in terms of FW, PWG and SGR and PER (P < 0.05). No significant difference was found in body composition of all groups (P > 0.05). Through real-time quantitative PCR analysis, relative expression levels of trypsin, tor and s6k of low replacement level groups (FM and FS25%) were significantly uo-regulated (P < 0.05). Based on the Illumina sequencing of 15 samples, family Vibrionaceae and genus Photobacterium were more abundant in high replacement level groups (50%, 75% and 100%) than those in low replacement level groups (0% and 25%), which may be related to the worse growth performance of high replacement level groups. However, dietary different levels of fish silage did not make significant difference on intestinal bacterial richness, diversity and metabolism functions (P > 0.05), which indicated the limited impact of dietary fish silage on intestinal microbiota.

3. Response to replacement of fish meal with fermented soybean meal of mTOR signaling pathway and intestinal microbiota in Litopenaeus vannamei

This study evaluated the potential for the replacement of fish meal with fermented soybean meal in the diet of Litopenaeus vannamei. And an Illumina-based highthroughput method was further used to analyse the intestinal microbiota of shrimp. Five isonitrogenous and isolipidicdiets were formulated: a control diet and four experimental diets in which FM in control diet was replaced by FSM at 10 (FSM10%), 20 (FSM20%), 30 (FSM30%) and 40% (FSM40%). An 8-week feeding trial was conducted in fifteen fibreglass tanks with 100 shrimps (initial weight 0.27 ± 0.04 g) per tank. After 8 weeks trial, FSM20% had significantly enhanced growth performance (P < 0.05). No significant difference was found in body composition and digestive enzyme activities of all groups (P > 0.05). Through real-time quantitative PCR analysis, tor and s6k expression levels of FSM20% were significantly up-regulated (P < 0.05). Based on the Illumina sequencing of 15 samples, dietary different levels of fermented soybean meal did not make any difference on intestinal bacterial composition, richness, diversity and metabolism functions (P > 0.05).

4. Screening of signaling pathway which is sensitive to replacement of fish meal with fermented soybean meal in Litopenaeus vannamei using metabolomics and proteomics method

In the present study, we investigated the metabolomics and proteomics of FM, FSM20% and FSM40% by LC-MS and TMT technology. Based on results of metabolomics, there were 29, 97 and 61 differential metabolites in FM and FSM40%, FM and FSM20%, FSM 20% and FSM40%, respectively. These differential metabolites enriched in protein digestion and absorption, ABC transporters and central carbon metabolism in cancer signaling pathway. Based on results of proteomics, there were 202, 184 and 179 differential proteins in FM and FSM40%, FM and FSM20%, FSM 20% and FSM40%, respectively. These differential proteins enriched in glucagon, lysosome and protein digestion and absorption signaling pathway. Based on results of conjoint analysis of metabolomics and proteomics, protein digestion and absorption signaling pathway changed significantly, which indicated its signicant response to replacement of fish meal with fermented soybean meal in the diet of Litopenaeus vannamei.