Identification And Seasonal Dyanamic Of A Novel Species Of Limnotrachelobdella On Bighead Carp And Screening Of Anthelmintic Drugs

Fish leeches（Piscicolids）are common ectoparasites of fish and are widely distributed in fresh and marine ecosystems.They feed on the blood of their hosts and cause mechanical damage.Infection of fish with leeches can lead to serious health problems.In recent years,one species of fish leech was found on the bighead carp（Hypophthalmichthys nobilis）and silver carp（H.molitrix）,in reservoirs and lakes in Hubei,Zhejiang and Jiangxi province.To clarify the pathogenesis and damage of this disease,this study conducted the species identification,histopathological analysis,epidemiology and host specificity,as well as the infestation dynamics.In order to effectively prevent and control the disease,in vitro drug screening against leeches and acute toxicity experiments were performed.The main results are as follows:1.Based on the morphology,molecular analyses and host specificity,a novel species Limnotrachelobdella hypophthalmichthysa n.sp.was identified from bighead carp and silver carp.L.hypophthalmichthysa n.sp.is consistent with the identification of the genus Limnotrachelobdella,but the morphological characters are not consistent with the known species:with 10 pairs of pulsatile vesicles,no eye or 1-2 pairs of symmetrical or asymmetrical eyes,5 pairs of testisacs and 7 pairs of crop caeca.The molecular analyses showed that the cox1 and ITS sequence similarity within the three specimens（from Zhejiang,Hubei and Jiangxi,respectively）is as high as 98.6%-99.5%,suggesting they were of the same species.Also,L.hypophthalmichthysa n.sp.showed 87.8%ITS sequence identity with L.sinensis and 85.0%and 86.1%cox1 sequence identity with L.sinensis and L.okae,respectively.The uncorrected p distance based on the cox1 sequence was 15.1%and 14.0%for L.sinensis and L.okae,respectively,showing interspecific variation.Phylogenetic analyses based on the combination of 18S and cox1 sequences showed that the newly discovered leeches were related to Limnotrachelobdella species.Among the 17 species of fish surveyed in the same reservoir,only bighead and silver carp were infected with this leech.Histopathology showed that attachment of the leech to the gill rakers and gill arches caused loss of connective tissue,hemorrhage,and ulceration.2.The mean abundance of infestation had obvious seasonal dynamics.The parasitic cycle lasts 8 months and the infection period is from December to July of the following year.There is a peak in May,when the prevalence,mean infection intensity and mean abundance could be as high as 96.7%,13.0±11.2 and 12.9±10.1,respectively.During the parasitic period,there were obvious seasonal variations in the growth indicators of L.hypophthalmichthysa n.sp.,with each indicator being smallest at the beginning of infection and gradually increasing within the parasitic period.Based on the above results,the life cycle of L.hypophthalmichthysa n.sp.was suggested to be 1 year.3.Preliminary studies on the in vitro experiments of four drugs against L.hypophthalmichthysa n.sp.showed that dinotefuran,fenobucarb and formalin,except for avermectin,had a certain insecticidal effect(24 h LC₅₀was 390.1 mg/L,2.6 mg/L and144.4μL/L,respectively).But the safe concentration（SC）of these three drugs on juvenile bighead carp was 346.6 mg/L,0.3 mg/L and 6.5μL/L,respectively.The 24 h LC₅₀of dinotefuran against leech was slightly higher than SC.Neurological paralysis occurred in leech after exposure to dinotefuran,suggesting that dinotefuran is an effective and safe insect repellent which can prevent and control the leech probably.