| In recent years,with the development of the cultivation of Apostichopus japonicus in China,there are more and more diseases and new enemy species of A.japonicus,which has become an important factor limiting the development of the cultivation of A.japonicus breeding industry.It can provide data support for disease prevention and control of cultured A.japonicus to identify the enemy species and carry out drug screening.In this study,species identification,hazard analysis and prevention and control drug screening were carried out on the new species found in pond culture A.japonicus in main producing areas of Shandong and Liaoning in 2019,so as to provide scientific support for the healthy development of A.japonicus breeding industry.The main results are as follows:1.Identification of Heterochaerus australis as a new enemy species in the cultured ponds of Apostichopus japonicus and its harmThere was an outbreak of Bacterial Ulceration Syndrome of Apostichopus japonicus in two culture ponds located in Dalian,Liaoning Province and Dongying,Shandong Province,in October 2019.Investigation on the characteristics of the disease 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 reveals that the body length range from 0.96 to 3.26 mm and the body width range from 0.49 to 1.93 mm.The body color is yellow or yellowish-brown.Its head was blunt round shape,with a pair of dark red rodlike ocellus.Two caudal lappets laid coordinately on both sides of its tail.Microscopic examination revealed that symbiotic Zooxanthella within the epidermis and its body is covered with cilia.It was hermaphrodite,with two genital openings behind the mouth.The 18 S r DNA 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 ?,its suitable p H was 5.5-8 and its suitable salinity was 20-40.This species also shows strong ability of polar regeneration.The cocultivation 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 cause 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 of A.japonicus in pond culture,which needs to be paid close attention to and prevented in the process of culture.2.Screening of killing drugs against Heterochaerus australis and its safety to Apostichopus japonicusIn this study,7 chemicals including copper sulfate,ferrous sulfate,5:2 mixture of copper sulfate and ferrous sulfate,hydrogen peroxide,copper acetate,metronidazole and eucalyptus oil,and 3 Chinese herbal medicines including Artemisia annua,Compound Chinese herbal medicine for killing ciliates and fructic acid were used to the acute toxicity of Heterochaerus australis.The results showed that ferrous sulfate and metronidazole had no lethal effect.While copper sulfate,copper sulfate and ferrous sulfate 5:2 mixture,hydrogen peroxide,copper acetate,eucalyptus oil,Artemisia annua water extract,Compound Chinese herbal medicine for killing ciliates and fruit acid extract had 100% killing effect on H.australis.The minimum lethal concentrations are 0.50?0.25?3.00?0.25?10?5000?2500?500 mg/L.According to the experimental results of acute toxicity of 10 kinds of drugs to H.australis and its feasibility,5 drugs were selected to conduct safety experiments on 3 varieties of A.japonicus.The results showed that only copper acetate had no significant toxic effect on A.japonicus.Therefore,copper acetate can be selected as the candidate exterminating drug for H.australis,with the drug concentration of 0.25 mg/L.3.Analysis of comptete mitochondrial genome sequences of Heterochaerus australisThe next-generation sequencing technology was used to obtain the whole mitochondrial genome sequences of H.australis.The mitochondrial gene was annotated and its structure was analyzed.And its phylogenetic analysis was carried out.The results showed that the sequence length was 13885 bp,and the base composition was A(39.8%),G(31.0%),C(14.9%),and T(14.3%).The A+T content of the complete mitochondrial genome sequence was 70.8%.The base composition showed a tendency of high AT and AT negative skewness.The mitochondrial genome encodes 12 proteins,20 t RNAs and 2 r RNAs.16 of them were encoded on the heavy strand(H strand)and 18 on the light strand(L strand).1 of 20 t RNA genes failed to predict the secondary structure,13 showed the shortage of T ? C-loops,the other 6 could be folded in clover shape.The results of mitochondrial genome analysis of Acoela showed that there was no similarity among the five species and complex gene rearrangement.Phylogenetic analysis showed that most of the species of Acoela had a distant relationship with other species of Turbellaria.4.Identification of Melanochlamys aquilina sp.nov.as a new enemy species in the cultured ponds of Apostichopus japonicus and its harmThere was an outbreak of Bacterial Ulceration Syndrome of Apostichopus japonicus in one culture ponds located in Dongying,Shandong Province,in October 2019.Investigation on the characteristics of the disease detected a kind of free-living species of Opisthobranchia in the sediment of culture system.This species is cylindrical in shape,with a body length of 9-15 mm and a body width of 3-6mm.Cephalic shield occupying about half of the length of the body,almost oblong-quadrate but anterior part slightly wider.Posterior shield elongate,ending in two short and wide caudal lobes of similar size.Parapodia extending for most of the length of the animal,developed and folded up to the sides of the body.A large muzzle,can turn out of the body,whose size about 1/3 of the body.Animal with velvety black in colour.The internal anatomical structure showed that the shell was brown,well-calcified but thin,fragile.Spire small,with a rounded apex.Shell surface sculptured by weak irregular growth lines.Prostate elongate,simple,of equal width along its entire length,and attached to a tissue mass,where the penis,the spermatic bulb and the excurrent sperm groove connected.The results of whole paraffin section showed that the organism had mucous glands and complicated digestive and reproductive systems.The internal organs of this species are arranged in a spiral shape,and the diaphragm extends vertically from the top to the bottom of the body.After careful taxonomic studies,the species was identified as a new species and named Melanochlamys aquilina sp.nov.The results of phylogenetic analysis show that M.aquilina sp.nov.has formed a well evolutionary branch.The co-cultivation experiment of this species,its egg mass,mucus and A.japonicus showed that it had no harmful effect on A.japonicus.However,the disease rates of the A.japonicus that coexist with their egg mass and mucus is 60% and 30% respectively,the mortality rates is 30% and 10% respectively.It is preliminarily concluded that the cause of the disease is that a large number of ciliates in the egg mass invade the respiratory tree.However,its pathogenesis is not clear.All the results indicate that M.Aquilina sp.nov.is a new enemy species of A.japonicus in pond culture.5.Analysis of comptete mitochondrial genome sequences of Melanochlamys aquilina sp.nov.The next-generation sequencing technology was used to obtain the whole mitochondrial genome sequences of M.aquilina sp.nov..The mitochondrial gene was annotated and its structure was analyzed.And its phylogenetic analysis was carried out.The results showed that the sequence length was 13795 bp,and the base composition was A(37.9%),G(30.3%),C(17.1%),and T(14.7%).The A+T content of the complete mitochondrial genome sequence was 68.2%.The base composition showed a tendency of high AT and AT positive skewness.The mitochondrial genome encodes 12 proteins,20 t RNAs and 2 r RNAs.10 of them were encoded on the heavy strand(H strand)and 24 on the light strand(L strand).5 of 20 t RNA genes showed the shortage of T ? C-loops,the other 15 could be folded in clover shape.The results of mitochondrial genome analysis of Opisthobranchia showed thatthe mitochondrial genes of M.aquilina sp.nov.have multiple rearrangements.The ML-tree based on the complete mitochondrial DNA genomes of 9 species of Opisthobranchia.The results showed that M.aquilina sp.nov.had distant relationship with the other 8 species.In this study,we found two new types of harmful species in the pond culture of A.japonicus,and carried out species identification and preliminary control research,providing technical support and reference for disease control and healthy culture of A.japonicus. |
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