THE OCCURRENCE OF AHPND IN POND CULTURED LITOPENAEUS VANNAMEI AND ITS INFLUENCE ON ENVIRONMENT, PATHOGEN AND IMMUNE FACTORS
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摘要: 为研究虾急性肝胰腺坏死病(Acute Hepatopancreas Necrosis Disease, AHPND)的发生与环境、病原和虾体免疫间的相互关系, 文章对池塘养殖凡纳滨对虾(Litopenaeus vannamei)AHPND发生及其环境、病原、虾体免疫因子进行持续性跟踪监测。结果表明, 试验点的气温、水温、溶解氧(DO)、pH、盐度、氨氮(NH4-N)和亚硝态氮(NO2-N)波动范围为21—29℃、24.8—31℃、1.4—8.32 mg/L、8—8.91、34—50、0.01—0.26 mg/L和0.005—0.212 mg/L; 水体可培养细菌和弧菌数量变化范围为3×103—2.4×105和2×102—1.8×104 CFU/mL, 虾体肝胰腺内可培养细菌和弧菌数量变化范围为9.8×104—8.8×106和3.9×103—3.61×106 CFU/g; 16S rDNA鉴定结果显示, 在可培养优势菌株中, 弧菌检出数量达到135株, 占全部鉴定菌株的61.6%, 其中主要弧菌种类有欧文氏弧菌(Vibrio owensii)、坎贝氏弧菌(V. campbellii)、副溶血弧菌(V. parahaemolyticus)、溶藻弧菌(V. alginolyticus)和哈维氏弧菌(V. harveyi); 虾体中ACP、AKP、SOD、LZM和PO等免疫酶活的变化范围分别为7.5—75、1—8.5、2.4—11.07、1.3—43和6.23—28 U/mg。结合AHPND发生前后各理化因子变化的相关性分析表明, 水温、虾体肝胰腺内可培养细菌和弧菌数量、DO、LZM和PO可作为池塘养殖模式下对虾警示AHPND的指示因子。相关研究结果为指导池塘养殖凡纳滨对虾健康养殖及AHPND临床防控提供数据支撑和科学依据。Abstract: Acute Hepatopancreatic Necrosis Disease (AHPND) is an important limiting factor affecting the healthy cultivation of shrimp in recent years, which has brought huge economic losses to the global shrimp industry. In order to explore the relationship between the occurrence of AHPND and environment, pathogen and shrimp immunity factors, the pond cultured Litopenaeus vannamei systems were continuously monitored. Based on the 35 days continuously monitoring showed that, the air temperature, water temperature, dissolved oxygen (DO), pH, salinity, ammonia nitrogen (NH4-N) and nitrite (NO2-N) were fluctuated in the range of 21—29℃, 24.8—31℃, 1.4—8.32 mg/L, 8—8.91, 34—50, 0.01—0.26 mg/L and 0.005—0.212 mg/L, respectively; the culturable bacteria and Vibrio sp. in water and shrimp hepatopancreas were fluctuated in the range of 3×103—2.4×105 CFU/mL, 2×102—1.8×104 CFU/mL, 9.8×104—8.8×106 and 3.9×103—3.61×106 CFU/g, respectively; 135 isolates of Vibrio sp. were detected based on 16S rDNA, and the main Vibrio species was V. owensii, V. campbellii, V. parahaemolyticus, V. alginolyticus and V. harveyi; the activities of ACP, AKP, SOD, LZM and PO were 7.5—75, 1.0—8.5, 2.4—11.07, 1.3—43 and 23—28 U/mg, respectively. Combined the physical and chemical factors before and after the occurrence of AHPND, the results showed that, water temperature, culturable bacteria and Vibrio sp. in hepatopancreas, DO, LZM and PO could be used as the early warning candidate factors for the occurrence of AHPND in pond culture. The results provide a scientific basis for the healthy culture of L. vannamei and the prevention and control of AHPND disease.
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Key words:
- AHPND /
- Environmental factors /
- Culturable bacteria /
- Immune factors /
- Early warning factors /
- Litopenaeus vannamei
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图 1 实验周期内池塘水质因子变化趋势
Figure 1. Trends of environmental factors in the ponds water during the experiment period
图 2 实验周期内池塘水体和对虾肝胰腺内细菌和弧菌数量变化趋势
Figure 2. Trend of the number of culturable bacteria in water and shrimp hepatopancreas during the experiment period
图 3 四个实验点监测周期内对虾机体门免疫酶活性的变化趋势
Figure 3. Four experimental points monitor the trend of changes in shrimp phyla immune enzyme activity during the period
表 1 试验池塘基本信息
Table 1. Basic information of the four test ponds
编号
No.面积
Aera (hm2)水深
Depth (m)苗种投放密度
Density of initial
shrimp postlarva
(ind./m2)养殖时间
Breeding time (d)投喂时间
Feeding timePond 1 2.40 1.50 22.5 68 5:00; 16:30 Pond 2 3.33 1.30 35.0 61 6:30; 16:30 Pond 3 3.33 1.30 26.0 70 5:00; 16:30 Pond 4 3.20 1.30 21.5 60 5:30; 16:40 
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表 2 各实验点对虾出现典型AHPND发病和死亡时的信息统计
Table 2. AHPND occurrence and distribution information in each test sites
实验点
Groups对虾AHPND检测阳性且具有典型肝胰腺坏死症状
发生时间Time points of AHPND positive with typical hepatopancreas necrosis symptom7.7 7.10 7.12 7.15 7.16 7.26 Pond 1 Pond 2 + + + Pond 3 + + Pond 4 + + + 注: “+”代表发病, 空白代表未发病Note: The “+” means outbeak of AHPND and the blank means absence points
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表 3 基于16S rDNA的对虾肝胰腺可培养优势菌株鉴定信息
Table 3. The composition of dominant bacteria in the L. vannamei pond culture system
菌株
Strain数量
Number菌株
Strain数量
NumberVobrio弧菌属 135 Bacillus芽孢杆菌属 38 欧文氏弧菌V. owensii 41 枯草芽孢杆菌B. subtilis 17 坎贝氏弧菌V. campbellii 23 暹罗芽孢杆菌B. siamensis 10 溶藻弧菌V. alginolyticus 21 特基拉芽孢杆菌B. tequilensis 4 副溶血弧菌V. parahaemolyticus 10 弯曲芽孢杆菌B. flexus 2 肝弧菌V. hepatarius 8 短小芽孢杆菌B. pumilus 2 需钠弧菌V. natriegens 6 黄海芽胞杆菌B.marisflavi 2 V. hyugaensis 5 B. cabrialesii 1 哈维氏弧菌V. harveyi 4 Photobacterium发光杆菌属 26 轮虫弧菌V. rotiferianus 3 美人鱼发光杆菌P. damselae 25 蓝弧菌V. azureus 2 海洋发光杆菌P. marinum 1 日本弧菌V. japonicus 2 Exiguobacterium微小杆菌属 6 沙蚕弧菌V. nereis 2 墨西哥微小杆菌E. mexicanum 3 变异弧菌V. variabilis 2 印度微小杆菌E. indicum 2 锡那罗州弧菌V. sinaloensis 1 深海微小杆菌E. profundum 1 V. ruber 1 Acinetobacter不动杆菌属 5 萨迦米弧菌V. sagamiensis 1 约氏不动杆菌A. johnsonii 2 拟态弧菌V. mimicus 1 琼氏不动杆菌A. junii 2 非典型弧菌V. atypicus 1 皮特不动杆菌A. pittii 1 巴西弧菌V. brasiliensis 1 Pseudomonas假单胞菌属 2 Shewanella希瓦氏菌属 3 P. oryzihabitans 1 海滨希瓦氏菌S. litorisediminis 2 恶臭假单胞菌P. alloputida 1 瓦氏希瓦氏菌S. waksmanii 1 维氏气单胞菌Aeromonas veronii 2 假交替单胞菌 Pseudoalteromonas shioyasakiensis 2
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表 4 各理化因子的描述统计量
Table 4. Descriptive statistics of each physical and chemical factor
统计量
Statistic全距
Full range极大值
Maximum极小值
Minimum均值
Average value标准差
Standard deviation方差
Variance水温Water temperature (℃) 35 4.9 30.3 25.4 27.4 1.132 1.28 DO (mg/L) 35 4.79 5.93 4.46 5.5 1.15 1.32 盐度Salinity (‰) 35 8 48 40 43 2.21 4.9 pH 35 0.64 8.99 8.35 8.59 0.14 0.02 氨氮NH4-N (mg/L) 35 0.15 0.16 0.01 0.07 0.042 0.0018 亚硝态氮NO2-N (mg/L) 35 0.199 0.212 0.013 0.12 0.044 0.002 SOD 35 5 8.23 3.22 5.94 1.53 2.35 PO 35 5.27 28.12 22.85 25.14 1.33 1.76 AKP 35 10.35 12.53 2.15 4.23 1.89 1.83 ACP 35 41.3 74.55 33.32 5.81 8.86 76 LZM 35 37.25 42.84 5.59 27 9.69 72 水体细菌
Culturable bacteria in water35 1.79×105 1.86×105 7000 6.02×107 4.72×104 2.23×109 水体弧菌
Culturable Vibrios in water35 1.37×105 1.38×105 800 3.08×107 3.38×104 1.14×109 虾体细菌
Culturable bacteria in shrimp hepatopancreas35 7.88×107 7.90×107 1.76×105 4.54×1012 1.30×107 1.68×1014 虾体弧菌
Culturable Vibrios in shrimp hepatopancreas35 4.47×106 4.52×106 5.50×104 2.88×1010 1.03×106 1.07×1012
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