饲粮α-亚麻酸水平对意大利蜜蜂工蜂幼虫生理机能的影响

doi:10.3864/j.issn.0578-1752.2019.13.015

摘要/Abstract

摘要：

【目的】探究饲粮中α-亚麻酸的添加水平对意大利蜜蜂（Apis mellifera ligustica）工蜂幼虫抗氧化活性和免疫能力的影响。【方法】移取1日龄意大利蜜蜂工蜂幼虫1 200只,随机分为5组,每组5个重复,每个重复48只;其中1组为对照组,饲喂不添加α-亚麻酸的基础饲粮,4组为处理组,分别饲喂α-亚麻酸添加水平为0.02%、0.04%、0.06%和0.08%的饲粮。按照室内蜜蜂幼虫饲养方法,将1日龄幼虫用移虫针移至温度适宜的加入200 μL饲粮的24孔细胞培养板内,培养板置于恒温培养箱中（温度33℃,相对湿度55%）,试验期间每天更换饲粮。饲养至第6天末或第7天初,幼虫开始有直立或排便现象时,将幼虫转移至提前铺好灭菌纸的24孔细胞培养板内准备化蛹。从饲养第1天开始,每天检查并记录幼虫和蛹的死亡数量,并将死亡个体及时移除,直至未死亡的蛹全部羽化新蜂,记录成功化蛹和羽化新蜂个体数量,统计幼虫化蛹率和羽化率。各组分别取5、6和7日龄幼虫测定抗氧化、免疫、脂质代谢指标及相关基因表达量。【结果】饲粮中α-亚麻酸的添加水平为0.02%和0.04%时,化蛹率和羽化率显著高于与其他处理组（P＜0.05）,而工蜂幼虫血淋巴中甘油三酯（TG）、总胆固醇（TC）和低密度脂蛋白（LDL）含量显著低于对照组,高密度脂蛋白（HDL）的含量却显著高于对照组（P＜0.05）。饲粮中α-亚麻酸的添加水平为0.04%时,工蜂幼虫超氧化物歧化酶（T-SOD）的活性较对照组显著增加,丙二醛（MDA）的含量显著降低（P＜0.05）。饲粮中α-亚麻酸的添加水平为0.02%、0.04%和0.06%时,6日龄工蜂幼虫的溶菌酶（lysozyme）和酚氧化酶（PO）活性显著高于对照组（P＜0.05）。饲粮中α-亚麻酸添加水平为0.04%时,6日龄工蜂幼虫脂肪酸合成酶（FAS）和乙酰辅酶A羧化酶（ACC）活性显著低于对照组（P＜0.05）。饲粮中α-亚麻酸添加水平为0.04%时,5和7日龄工蜂幼虫lysozyme和PO相对表达量显著高于对照组,但饲粮中α-亚麻酸添加水平为0.08%时,lysozyme相对表达量会显著降低（P＜0.05）。【结论】α-亚麻酸对意大利蜜蜂工蜂幼虫抗氧化活性和免疫能力有一定影响,幼虫饲粮中α-亚麻酸适宜添加水平为0.02%—0.04%。

关键词: 意大利蜜蜂, 工蜂, α-亚麻酸, 添加水平, 抗氧化活性, 免疫

Abstract:

【Objective】 The objective of this study is to explore the effect of dietary α-linolenic acid levels on the antioxidant activity and immunity of Apis mellifera ligustica worker bee larvae. 【Method】 Twelve hundreds 1-day-old worker bee larvae were randomly divided into 5 group, with 5 replicates in each group and 48 worker bee larvae in each replicate. The control group was fed with basic diets without α-linolenic acid, and the 4 treatment groups were fed experimental diets containing different amounts of α-linolenic acid (0.02%, 0.04%, 0.06%, and 0.08%). According to the indoor feeding in vitro method, the 1-day-old worker bee larvae were moved to a 24-well cell culture plate with 200 μL diet at suitable temperature. The larvae were moved by worm-moving needle. The culture plate was placed in a constant temperature incubator (temperature 33℃, relative humidity 55%), the diet was changed daily during the test. Feeding until the end of the 6th day or the beginning of the 7th day, when the larvae started to stand upright or defecate, the larvae were moved to the 24-well cell culture plate covered with sterilized paper in advance to prepare for pupation. From the 1st day of feeding, the death number of larvae and pupae was checked and recorded every day, and the dead individuals were removed in time until all adult emerged. The number of undead pupae and emergence new bees was recorded, and pupation rate and emergence rate were calculated. The 5-, 6- and 7-day-old worker bee larvae were used to measure antioxidant, immune, lipid metabolism indexes and related gene expression. 【Result】 When the addition level of α-linolenic acid in the diet was 0.02% and 0.04%, the pupation rate and emergence rate were significantly higher than those in other treatment groups (P＜0.05), while the contents of triglyceride (TG), total cholesterol (TC) and low density lipoprotein (LDL) in the hemolymph of worker bee larvae were significantly lower than those of the control group, the content of high density lipoprotein (HDL) was significantly higher than that of the control group (P＜0.05). Compared with the control group, the activity of superoxide dismutase (SOD) was significantly increased, while the content of malondialdehyde (MDA) was significantly decreased when the addition level of α-linolenic acid was 0.04% (P＜0.05). When the addition level of α-linolenic acid was 0.02%, 0.04% and 0.06%, the activities of lysozyme and phenoloxidase (PO) of 6-day-old worker bee larvae were significantly higher than those in the control group (P＜0.05). When the addition level of α-linolenic acid was 0.04%, the activities of fatty acid synthase (FAS) and acetyl CoA carboxylase (ACC) of 6-day-old worker bee larvae were significantly lower than those in the control group (P＜0.05). When the addition level of α-linolenic acid was 0.04%, the relative expression levels of lysozyme and PO of 5- and 7-day-old worker bee larvae were significantly higher than those in the control group, but when the addition level of α-linolenic acid was 0.08%, the relative expression level of lysozyme was significantly decreased (P＜0.05). 【Conclusion】 Different dietary supplementation levels of α-linolenic acid have significant effects on antioxidant activity and immunity of A. m. ligustica worker bee larvae, and the appropriate addition level of α-linolenic acid in larval diet is 0.02%-0.04%.

Key words: Apis mellifera ligustica, worker bee, α-linolenic acid, addition level, antioxidant activity, immunity

于静,张卫星,马兰婷,胥保华. 饲粮α-亚麻酸水平对意大利蜜蜂工蜂幼虫生理机能的影响[J]. 中国农业科学, 2019, 52(13): 2368-2378.

YU Jing,ZHANG WeiXing,MA LanTing,XU BaoHua. Effect of Dietary α-Linolenic Acid Levels on Physiological Function of Apis mellifera ligustica Worker Bee Larvae[J]. Scientia Agricultura Sinica, 2019, 52(13): 2368-2378.

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原料 Ingredient	α-亚麻酸添加水平 Level of α-linolenic acid (%)
原料 Ingredient	0	0.02	0.04	0.06	0.08
蜂王浆 Royal jelly 葡萄糖 Glucose 果糖 Fructose 酵母提取物 Yeast extract 无菌水 Sterile water α-亚麻酸α-linolenic acid 合计Total	50.00 6.00 6.00 1.00 37.00 0 100.00	50.00 6.00 6.00 1.00 37.00 0.02 100.00	50.00 6.00 6.00 1.00 37.00 0.04 100.00	50.00 6.00 6.00 1.00 37.00 0.06 100.00	50.00 6.00 6.00 1.00 37.00 0.08 100.00

目的基因 Target gene	引物序列 Primer sequence (5′-3′)	产物长度 Product length (bp)	GenBank登录号 GenBank accession number
酚氧化酶基因 PO	F: ATGAGGACTTCCTGTCTGTTGCAG R: CTTCGGTAAGCGGTGGTACTGG	132	XM_026444741.1
溶菌酶基因 lysozyme	F: TCGCACTACTCGCCACGATA R: TTTGATGCAGAGCGAGAGCG	114	NM_001011614.1

项目 Item	α-亚麻酸添加水平Level of α-linolenic acid (%)					P值 P-value
项目 Item	0	0.02	0.04	0.06	0.08	P值 P-value
化蛹率 Pupation rate (%)	82.86±0.80a	86.31±1.07a	84.65±0.26a	73.70±0.71b	51.14±0.56c	<0.0001
羽化率 Emergence rate (%)	60.00±0.39a	62.45±0.86a	52.50±0.35b	23.33±1.33c	16.67±0.66d	<0.0001

项目 Item	α-亚麻酸添加水平Level of α-linolenic acid (%)					P值 P-value
项目 Item	0	0.02	0.04	0.06	0.08	P值 P-value
总蛋白TP (g·L^-1)	40.80±0.37ab	39.38±1.31b	36.08±1.27c	42.42±0.74a	40.70±1.36ab	0.0013
甘油三酯TG (mmol·L^-1)	3.36±0.39a	1.59±0.04b	1.58±0.01b	2.01±0.21b	1.56±0.13b	0.0002
总胆固醇TC (mmol·L^-1)	0.94±0.01a	0.59±0.03c	0.63±0.02c	0.77±0.01b	0.78±0.03b	<0.0001
高密度脂蛋白HDL (mmol·L^-1)	0.38±0.01c	0.60±0.01b	0.79±0.03a	0.66±0.07b	0.40±0.03c	<0.0001
低密度脂蛋白LDL (mmol·L^-1)	0.17±0.01a	0.13±0.01b	0.09±0.01c	0.14±0.01b	0.18±0.00a	<0.0001

项目 Item	α-亚麻酸添加水平 Level of α-linolenic acid (%)					P值 P-value
项目 Item	0	0.02	0.04	0.06	0.08	P值 P-value
5日龄幼虫 5-day-old larva	7.89±0.54c	13.80±0.47b	9.50±0.40c	16.30±0.23a	13.62±1.47b	0.0002
7日龄幼虫 7-day-old larva	12.05±0.82c	13.49±0.11abc	12.35±0.07bc	14.61±0.87a	15.76±1.01a	0.0190