Identification of Heterochaerus australis as a new enemy species in the cultured ponds of Apostichopus japonicus and its harm
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摘要: 为了对导致辽宁大连、山东东营的2家养殖场池塘养殖刺参大量化皮死亡的新的敌害生物进行鉴定并确定其对养殖刺参的危害。本实验通过形态学观察、分子鉴定及系统发育分析确定了涡虫的分类地位，通过生态学方法确定了其生态适应条件，通过切割后培养的方法观测了其再生能力，通过与刺参苗种的共培养实验测试了该物种对刺参的危害及其危害方式。形态学观察结果显示，该涡虫体长0.96~3.26 mm，体宽0.49~1.93 mm，外观黄色或黄褐色，头部钝圆，具一对暗红色棒状眼点，尾部具两条并列的尾垂；显微镜镜检发现其表皮下分布密集的虫黄藻，体表周生纤毛，雌雄同体，口后具有两个生殖孔；对该物种 COⅠ及18S rDNA基因片段扩增测序结果进行分析，并构建基于18S rDNA基因的系统发育树，结果显示该生物与澳洲异尾涡虫序列同源性达99.64%，根据其形态学特征，并结合18S rDNA分子鉴定结果，将该生物鉴定为澳洲异尾涡虫；进一步对其生活习性进行了研究，结果显示，该生物具有避光性，其适宜温度为18~24 °C，适宜pH为5.5~8.0，适宜盐度为20~40；再生实验表明，该物种具有很强的前后轴极性再生能力；该生物与刺参的共培养实验表明，澳洲异尾涡虫对刺参体表表现出很强的趋向性，可以吸附在刺参体表导致刺参苗种溃疡、化皮甚至死亡，但刺参的体腔、肠道、呼吸树内均未发现虫体寄生。研究表明，澳洲异尾涡虫是营自由生活的池塘养殖刺参的一种新的敌害生物，在养殖过程中需要密切关注并防范该敌害生物。Abstract: There was an outbreak of Bacterial Ulceration Syndrome (BUS) of Apostichopus japonicus in two culture ponds located in Dalian, Liaoning Province and Dongying, Shandong Province, in October 2019. Epidemiological investigation detected a kind of free-living turbellarians in the culture system including the body surface of A. japonicus, the water and the sediment. Morphological observation of this species revealed that the body length ranged from 0.96 to 3.26 mm and the body width range from 0.49 to 1.93 mm. The body color was yellow or yellowish-brown. Its head was blunt round shape, with a pair of dark red rodlike ocellus. Two caudal lappets lay coordinately on both sides of its tail.Microscopic examination revealed that symbiotic Zooxanthella within the epidermis and its body was covered with cilia. It was hermaphrodite, with two genital openings behind the mouth. The 18S rDNA gene sequence showed 99.64% identity with Heterochaerus australis. Based on the morphological and genetic analysis, it was identified as H. australis. The ecological study showed that this species was photophobic, its suitable temperature was 18-24 °C, its suitable pH was 5.5-8.0 and its suitable salinity was 20-40. This species also showed strong ability of polar regeneration. The co-cultivation experiment of this species and A. japonicus showed that H. australis can adsorb on the surface of sea cucumber, causing ulceration on the body wall and eventually the death of sea cucumber. However, there was no parasitic phenomenon in the body cavity, intestine and respiratory tree of the sea cucumber. All the results indicate that H. australis is a new enemy species for A. japonicus in pond culture.
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Key words:
- Apostichopus japonicus /
- Heterochaerus australis /
- enemy species /
- morphology /
- regeneration /
- histopathology /
- harm
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图 2 再生实验的切割方式示意图
Figure 2. Schematic diagram of cutting mode in regeneration experiment
图 图版II 澳洲异尾涡虫的石蜡切片标本显微观察
Figure 图版II. Microscopical observation of paraffinic section of H. australis
图 3 基于18S rDNA基因序列构建的系统发育树
Figure 3. Phylogenetic tree based on 18S rDNA gene sequence
图 图版III 再生能力测试中各组分割块的形态变化
Figure 图版III. Morphological changes of each group (tissue section) in the regeneration experiment
图 4 涡虫与刺参共培养实验中涡虫大量聚集于刺参体表
Figure 4. A large number of turbellarian gathered on the body surface of A. japonicus in the co-culture experiment
图 图版IV 刺参体壁组织的病理观察(横切)
Figure 图版IV. Pathological observation of the body wall of A. japonicus (horizontal section)
表 1 用于扩增18S rDNA、COI基因的PCR引物
Table 1. PCR primers used to amplify 18S rDNA genes and COI genes
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表 2 本研究分离涡虫的形态学特征统计学结果
Table 2. Statistical results of morphological characteristics of turbellarian worm isolated in this study
项目
items最小值
minimum最大值
maximum平均值
mean标准差
standard deviation标准误
standard error变异系数/%
coefficient of variation体长/μm body length 960.00 3 260.00 1 838.67 620.44 20.68 33.74 体宽/μm body width 490.00 1 930.00 1 054.33 343.58 11.45 32.59 眼点长/μm ocellus length 71.00 89.00 80.13 4.60 0.15 5.74 纤毛长/μm cilia length 6.00 8.00 6.90 0.76 0.03 11.00 虫黄藻直径/μm diameter of Zooxanthellae 23.00 240.00 27.67 4.00 2.13 14.47
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表 3 共培养实验中涡虫对刺参危害的统计结果
Table 3. Statistical results of the harm of turbellarian worm to A. japonicus in the co-cultivation experiment
涡虫与刺参
数量之比
the ratio of turbellarian worm to A. japonicus涡虫的数量/只
the number of turbellarian worm刺参的数量/头
the number of
A. japonicus附着于刺参体表的
涡虫总数/只
the number of turbellarian worm attached to the body surface of A. japonicus刺参个体体表最多
附着涡虫数/只
the maximum number of turbellarian worm on the surface of A. japonicus刺参化皮
起始时间/h
starting time of the skin ulceration of A. japonicus刺参化皮率/%
the ratio of A. japonicus with skin ulceration0∶10 0 10 0 0 0.00±0.00 1∶10 1 10 1 1 0.00±0.00 1∶1 10 10 9 3 0.00±0.00 2.5∶1 25 10 21 6 76 16.67±0.06 5∶1 50 10 43 11 61 33.33±0.06 10∶1 100 10 84 21 43 56.67±0.06 15∶1 150 10 127 39 20 73.33±0.06
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