Molecular identification of four species of trematode larvae in freshwater snails from Qiqihar area

doi:10.12140/j.issn.1000-7423.2024.03.012

Abstract

Abstract:

Objective Molecular identification of four species of trematode larvae isolated from freshwater snails in the Nenjiang River Basin in Qiqihar City. Methods Freshwater snails were collected from the Liuyuan section of the Nenjiang River in Qiqihar City from March to July 2023. After classification and identification, the shell was crushed, the visceral mass was observed under a microscope, and the trematode larvae in the snails were isolated. The total DNA of different trematode larvae was extracted, and the internal transcribed spacer region 2 (ITS2) of trematode larvae was amplified by PCR, and the amplified products were sequenced. After splicing using Contig Express software, the sequence consistency was compared on the NCBI website. The phylogenetic tree was using the neighbor-joining method, and the genetic distance was calculated using MEGA 11.0 software. Results A total of 7 species of freshwater snails (2 771 snails) were collected, of which 3 species were positive, including Koreoleptoxis amurensis (282 snails), Cipangopaludina chinensis (709 snails) and Bellamya limnophila (142 snails). A total of 4 species of trematode larvae were detected, and each freshwater snail only parasitized 1 species of trematode larvae, which were larvae a (positive rate 25.23%, 107/424) parasitized in K. amurensis and B. limnophila, larvae b (positive rate 2.82%, 20/709) parasitized in C. chinensis, larvae c (positive rate 0.70%, 2/282) parasitized in K. amurensis and larvae d (positive rate 0.56%, 4/709) parasitized in C. chinensis. The amplified lengths of ITS2 target sequences of larvae a-d were about 523 bp, 701 bp, 960 bp and 554 bp, respectively. The results of gene sequencing and sequence alignment showed that the larvae a ITS2 sequence had the highest identity with Notocotylus ephemera (GenBank: OP720890.1) sequence, which was 98.49%. It had the closest genetic distance with N. ephemera and Notocotylidae sp., both of which were 0.014. It was speculated that larva a was a trematode of the Notocotylidae. The larvae b ITS2 sequence had the highest consistency with the sequence of Echinostoma revolutum (GenBank: GQ463130.1), which was 94.56%. The genetic distance with E. revolutum is the closest, which is 0.085. It is speculated that larva b is a trematode of the genus Echinostoma in the family Echinostomatidae. The larvae c ITS2 sequence had the highest consistency with Echinochasmus suifunensis (GenBank: MT447049.1) sequence (99.82%). The genetic distance with E. milvi was the closest, less than 0.001, suggesting that larva c was a trematode of the genus Echinochasmus of the family Echinostomatidae. The larvae d ITS2 sequence had the highest consistency with the sequence of Asymphylodora markewitschi (GenBank: OP106430.1), which was 92.36%. The genetic distance with A. parasquamosa is the closest, which is 0.090, suggesting that the larva d was a trematode of the genus Asymphylodora of the family Monorchiidae. Conclusion Freshwater snails in the Nenjiang River Basin of Qiqihar may harbour trematodes of the Notocotylidae, Echinostoma and Echinochasmus of the Echinostomatidae, and Asymphylodora of the Monorchiidae, potentially endangering the health of fish, poultry and mammals.

Key words: Freshwater snail, Trematodes, Cercaria, ITS2 sequence, Species identification

CLC Number:

R383.2

LI Jianke, ZHANG Jing, LIU Liu, LIU Qianhao, ZHANG Hao. Molecular identification of four species of trematode larvae in freshwater snails from Qiqihar area[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2024, 42(3): 360-366.

Figures/Tables 7

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

References 23

[1]	Pyrka E, Kanarek G, Gabrysiak J, et al. Life history strategies of Cotylurus spp. Szidat, 1928 (Trematoda, Strigeidae) in the molecular era-evolutionary consequences and implications for taxonomy[J]. Int J Parasitol Parasites Wildl, 2022, 18: 201-211.
[2]	Li CP, Sun ET, Zhu YX, et al. Zoonotic trematode species identified in domestic animals from Huainan Area[J]. Chin J Schisto Control, 2014, 26(1): 38-41. (in Chinese)
	(李朝品, 孙恩涛, 朱玉霞, 等. 淮南地区禽畜体内寄生人兽共患吸虫的种类[J]. 中国血吸虫病防治杂志, 2014, 26(1): 38-41.)
[3]	Mahulu A, Clewing C, Stelbrink B, et al. Cryptic intermediate snail host of the liver fluke Fasciola hepatica in Africa[J]. Parasit Vectors, 2019, 12(1): 573.
[4]	Hu YY, Sun X, Wu ZD. Freshwater snails and snail-borne infectious diseases in China[J]. China Trop Med, 2022, 22(4): 374-381. (in Chinese)
	(胡云逸, 孙希, 吴忠道. 我国淡水螺及螺传性传染病[J]. 中国热带医学, 2022, 22(4): 374-381.) doi: 10.13604/j.cnki.46-1064/r.2022.04.17
[5]	Du CH, Lü S, Zhang Y, et al. Molecular identification of Tricula spp. and the parasitized trematode cercariae in schistosomiasis-endemic areas of Yunnan Province[J]. Chin J Schisto Control, 2020, 32(2): 159-167. (in Chinese)
	(杜春红, 吕山, 张云, 等. 云南省血吸虫病流行区一种拟钉螺及其寄生吸虫尾蚴的分子鉴定[J]. 中国血吸虫病防治杂志, 2020, 32(2): 159-167.)
[6]	Nong WY, Yu YF, Aase-Remedios ME, et al. Genome of the ramshorn snail Biomphalaria straminea: an obligate intermediate host of schistosomiasis[J]. Gigascience, 2022, 11: giac012.
[7]	Luo J, Chen JQ, Jiang DW, et al. Intermediate host population and infection status of Paragonimus in southeastern Youxi County, Fujian Province[J]. Chin J Parasitol Parasit Dis, 2021, 39(5): 646-651. (in Chinese)
	(罗鋆, 陈及清, 江典伟, 等. 福建省尤溪县东南部并殖吸虫中间宿主种群及其感染情况[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(5): 646-651.) doi: 10.12140/j.issn.1000-7423.2021.05.022
[8]	He T, Zhou Y, Cheng N, et al. Molecular identification and genetic polymorphism analysis of Fasciola flukes in Nanning, Guangxi[J]. J Pathog Biol, 2021, 16(5): 557-563. (in Chinese)
	(何婷, 周岩, 程娜, 等. 广西南宁地区片形吸虫的分子鉴定及遗传多态性分析[J]. 中国病原生物学杂志, 2021, 16(5): 557-563.)
[9]	Curran SS, Gonzales RD, Bullard SA. Molecular character ization of sporocystsand cercariae (Digenea ∶ Bucephalidae) infecting the eastern oyster Crassostrea virginica from Virginia[J]. J Parasitol, 2023, 109(3): 259-263.
[10]	Zhang FY, Liu L, Zhang J, et al. Prevalence of trematode metacercariae in Pseudorasbora parva and species identification in Qiqihaer Area[J]. Chin J Parasitol Parasit Dis, 2023, 41(1): 112-116. (in Chinese)
	(张凤玉, 刘柳, 张静, 等. 齐齐哈尔地区麦穗鱼吸虫囊蚴感染情况及其种类鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(1): 112-116.) doi: 10.12140/j.issn.1000-7423.2023.01.018
[11]	Du FX. Species Identification and initial study of nervous system on the trematode of subclass Aspidogastrea in anodonta from the basin of Nenjiang River[D]. Huainan: Anhui University of Science & Technology, 2008: 14-27. (in Chinese)
	(杜凤霞. 嫩江流域河蚌体内寄生盾盘吸虫的种类鉴定及神经系统初步研究[D]. 淮南: 安徽理工大学, 2008: 14-27.)
[12]	Liu YY, Zhang WZ, Wang YX. Medical malacology[M]. Beijing: Ocean Press, 1993: 27-77. (in Chinese)
	(刘月英, 张文珍, 王耀先. 医学贝类学[M]. 北京: 海洋出版社, 1993: 27-77.)
[13]	Sugiyama H, Morishima Y, Kameoka Y, et al. Polymerase chain reaction (PCR)-based molecular discrimination between Paragonimus westermani and P. miyazakii at the metacercarialstage[J]. Mol Cell Probes, 2002, 16(3): 231-236.
[14]	Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11[J]. Mol Biol Evol, 2021, 38(7): 3022-3027. doi: 10.1093/molbev/msab120 pmid: 33892491
[15]	Sun L, Li M. Diagnosis and treatment of leptospirosis in Wulong Goose[J]. Heilongjiang Anim Sci Vet Med, 2016(18): 148. (in Chinese)
	(孙莉, 李明. 五龙鹅细背孔吸虫病的诊治[J]. 黑龙江畜牧兽医, 2016(18): 148.)
[16]	Huang RJ, Dai JW, Li WP, et al. Morphological and molecular biological identification of Echinostoma from red-crowned crane[J]. Contemp Anim Husb, 2020(10): 60-63. (in Chinese)
	(黄润基, 代军威, 李婉萍, 等. 丹顶鹤源棘口吸虫的形态及分子生物学鉴定[J]. 当代畜牧, 2020(10): 60-63.)
[17]	Liu XQ, Shi HH. Two cases infected by Echinostoma hortense in Guangxi[J]. Chin J Parasitol Parasit Dis, 2011, 29(1): 77. (in Chinese)
	(刘晓泉, 石焕焕. 广西圆圃棘口吸虫感染2例报告[J]. 中国寄生虫学与寄生虫病杂志, 2011, 29(1): 77.)
[18]	Tang CT, Tang ZZ. Trematologyin China[M]. 2nd ed. Beijing: Science Press, 2015: 773-774. (in Chinese)
	(唐崇惕, 唐仲璋. 中国吸虫学[M]. 2版. 北京: 科学出版社, 2015: 773-774.)
[19]	Tang ZZ. Progenetic development of Asymphylodora stenothyraen sp.[J]. Acta Hydrobiol Sin, 1980, 4(2): 231-244. (in Chinese)
	(唐仲璋. 窄口螺侧殖吸虫的发育史及早熟现象[J]. 水生生物学集刊, 1980, 4(2): 231-244.)
[20]	Prastowo J, Priyowidodo D, Sahara A, et al. Molecular identification of cercaria Fasciola gigantica in lymnaeid snails in Kulon Progo, Yogyakarta[J]. Vet Parasitol Reg Stud Reports, 2022, 30: 100707.
[21]	Faltýnková A, Kudlai O, Pantoja C, et al. Prey-mimetism in cercariae of Apatemon (Digenea, Strigeidae) in freshwater in northern latitudes[J]. Parasitol Res, 2023, 122(3): 815-831.
[22]	Cai ZH, Zhang ZP, Zhuo MY, et al. Trematode egg internal transcribed space-2 sequence based analysis for diagnosis of Opisthorchis viverrin and Opisthorchis sinensis infections[J]. Chin J Parasitol Parasit Dis, 2023, 41(4): 427-433. (in Chinese)
	(蔡长煌, 张芝平, 卓鸣莺, 等. 基于虫卵内转录间隔区2序列分析诊断麝猫后睾吸虫和华支睾吸虫感染[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 427-433.) doi: 10.12140/j.issn.1000-7423.2023.04.005
[23]	Chen WQ, Jiang TT, Deng Y, et al. Sequence analysis of Paragonimus internal transcribed spacer 2 and cyclooxygenase 1 genes in freshwater crabs in Henan Province[J]. Chin J Schisto Control, 2023, 35(5): 501-507. (in Chinese)
	(陈伟奇, 蒋甜甜, 邓艳, 等. 河南省溪蟹体内并殖吸虫内转录间隔区2与环氧化酶1基因序列分析[J]. 中国血吸虫病防治杂志, 2023, 35(5): 501-507.)

	幼虫a（n = 5） Larva a (n = 5)	幼虫b（n = 5） Larva b (n = 5)	幼虫c（n = 5） Larva c (n = 5)	幼虫d（n = 5） Larva d (n = 5)
体部大小/mm Body size/mm	0.607 × 0.072	1.118 × 0.576	0.544 × 0.374	1.387 × 0.368
尾部大小/mm Tail size/mm	0.789 × 0.104	0.876 × 0.153	0.990 × 0.216	-
口吸盘大小/mm Oral sucker size/mm	0.062 × 0.063	0.158 × 0.106	0.191 × 0.089	0.212 × 0.179
腹吸盘大小/mm Ventral sucker size/mm	-	0.155 × 0.152	0.083 × 0.089	0.245 × 0.229
口腹吸盘距离/mm Distence between oral sucker and ventral sucker/mm	-	0.612	0.228	0.453
特征性结构 Characteristic structure	眼点2个 2 ocellis	头冠处可见一排小棘 A row of small spines is visible at the crown of the head	尾部宽大，末端有透明囊状物 Tail broad, terminating in a transparent vesicle	睾丸、卵巢各一 One testicle, one oarium