Analysis of ribosomal and mitochondrial genes of Baylisascaris procyonis

doi:10.12140/j.issn.1000-7423.2022.05.005

Abstract

Abstract:

Objective To characterize the ribosomal and mitochondrial genes of Baylisascaris procyonis, and their phylogenetic relationship with other Baylisascaris spp. Methods Adult worms of B. procyonis were collected from the intestine of a deceased raccoon at the Kunming Zoo in Kunming, China. After morphological identification, 20 adult worms comprising 10 males and 10 females were selected for morphological measurement and related molecularbiological study. The DNA was extracted from the tissue sample of the parasite using a DNA extraction kit. For amplification of ribosome internal transcribed spacer (ITS), cytochrome c oxidase 1 (cox1), cox2, cox3, cytochrome b (cytb), nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1), nad5 and tRNA-nad2-tRNA by PCR, which were sequenced and aligned with the database deposited in GenBank using BLAST. Single nucleotide polymorphisms (SNP) were analyzed using MEGA X software, and phylogenetic trees based on ITS, cox1 and cox2 sequences were constructed respectively, using the maximum likelihood (ML) method. Results The adult worms were in ivory color, at a size of (9.15-14.50) cm × (0.20-0.32) cm for females and (6.45-8.70) cm × (0.15-0.20) cm for males. Adult male has copulatory spicule at the caudal ends. The eight genetic markers of B. procyonis were successfully sequenced, and all sequences were deposited in GenBank under the accession numbers MZ092850-MZ092855, MZ164969, MZ172987, MZ172988, MZ172982-MZ172986, MZ172989, MZ172981, and MZ172980 for ITS, cox1, cox2, cox3, cytb, nad1, nad5, and tRNA-nad2-tRNA, respectively. Of the eight loci, the newly obtained sequence shared identities of 98.6%-100%, 98.5%-99.5%, 99.4%-99.7%, 98.3%, 98.8%-99.0%, 98.2%, 97.8% and 99.2%, respectively, with those of B. procyonis previously deposited in GenBank. Additionally, the newly obtained ITS, cox1, cox2 and tRNA-nad2-tRNA sequences had identities of 98.6%-100%, 99.2%-100%, 99.4%-100% and 99.6%-99.9%, respectively, with those of B. columnaris previously deposited in GenBank. SNP analysis showed that the newly obtained ITS, cox1, cox2 and tRNA-nad2-tRNA sequences had 28, 7 and 12 SNP sites, respectively, compared to those of B. procyonis and B. columnaris previously deposited in GenBank. However, none of the SNP sites was suitable to distinguish B. procyonis from B. columnaris. Meanwhile, the newly obtained ITS sequences contained 7 to 9 GA tandem repeats, and the 7 and 8 repeats constituted the first records discovered in B. procyonis. Phylogenetic analysis inferred from ITS, cox1 or cox2 sequences indicated that B. procyonis and B. columnaris obtained from different geographical areas completely aggregated, and formed an individual clade, that clustered with B. devosi. Conclusion The markers sequences and SNP characteristics of B. raccoonus were highly similar to those of B. cylindrica, whereas phylogenetic analysis could not effectively differentiate B. procyonis from B. columnaris.

Key words: Raccoon, Baylisascaris procyonis, Nuclear and mitochondrial genes, Single nucleotide polymorphisms, Phylogeny

CLC Number:

S582.731

WEI Kai-wen, ZENG Hong-xia, HE Mu, HU Jun-jie. Analysis of ribosomal and mitochondrial genes of Baylisascaris procyonis[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(5): 594-602.

Figures/Tables 10

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DNA 区域 DNA region	引物名字 Primer name	引物序列（5′→3′） Primer sequence (5′→3′)	参考文献 Reference
ITS	18sF	ACTGCTGTTTCGAGACCTTT-CGAG	[7]
	28sR	TAGCACCTTCTTTGGACTAT-AGCC
tRNA-nad2-tRNA	Amplicon 1F	CCGTTGGCCTTAACTGTTCG	[9]
	Amplicon 1R	GGCAACCCAACAACCATAG
cox1	Amplicon 2F	CGGGTTTTCTGTTCCTGTGG	[9]
	Amplicon 2R	ACCTGATTGGAAGTCAGGTG
cox2	Amplicon 3F	GTGGATAAGGGGCCTTGTG	[9]
	Amplicon 3R	ATAAACCCCCGCCAGTTCTC
cox3	BPCO3F	GGCTCTTTAGGTTTGACCA-GTT	a
	BPCO3R	GCAAACTCCAGACCAAGATGAT
cytb	BPCytbF	GGGTTCCAGATTTTAACA-GGT	a
	BPCytbR	ACCAAACACTGCCCAAGC
nad1	BPNAD1F	TTTTGAAGAAGGAGCAGATGAC	a
	BPNAD1R	CGAGGATAAGCCCTACGA-AC
nad5	BPNAD5F	TTGGGATAGTTGTAGAGGGG	a
	BPNAD5R	CAAAACGACCACAACAGA-AT

遗传标记 Genetic marker	本研究序列的登录号 Accession number of the sequence in this study	与GenBank序列的比对结果 Comparison result with sequences in GenBank
遗传标记 Genetic marker	本研究序列的登录号 Accession number of the sequence in this study	种名 Species	GenBank登录号 Accession number in GenBank	相似性/% （均值/%） Similarity/% (Average/%)
ITS	MZ092850~MZ092855	柱状贝氏蛔虫	KC543484、KC543485、MH030594、MH030595、LN600405、KX774493	98.6~100 (99.4)
		浣熊贝氏蛔虫	OU974763、MH030596、MH030597、MZ092854、JQ403615、KJ698593~KJ698602、KC543488	98.6~100 (99.2)
		德氏贝氏蛔虫	MN960312、KY465505、MH030598	96.1~97.3 (96.7)
cox1	MZ164969	柱状贝氏蛔虫	KY580736~KY580739	99.2~100 (99.6)
		浣熊贝氏蛔虫	MW385416~MW385530、MH795149、MH795150、KJ698533~KJ698567	98.5~99.5 (99.0)
		德氏贝氏蛔虫	MH795151	95.4
cox2	MZ172987	柱状贝氏蛔虫	FJ357429、KC543478、KC543480、KY580740~KY580742、MH469662、MH469663	99.4~100 (99.8)
		浣熊贝氏蛔虫	MH469664、MH469665、KP843602、KP843603、KC543482、AF179908、JF951366	99.4~99.7 (99.6)
		德氏贝氏蛔虫	MH469666	96.6
cox3	MZ172988	浣熊贝氏蛔虫	JF951366	98.3
		猪蛔虫	HQ912104	90.6
cytb	MZ172982~MZ172986	浣熊贝氏蛔虫	JF951366、JQ340492	98.8~99.0 (98.9)
		转移贝氏蛔虫	HQ671079、JQ340493、JQ340494	87.3~88.0 (87.5)
nad1	MZ172989	浣熊贝氏蛔虫	JF951366	98.2
		转移贝氏蛔虫	HQ671079	91.9
tRNA-nad2-tRNA	MZ172980	柱状贝氏蛔虫	KY580744~KY580747	99.6~99.9 (99.7)
		浣熊贝氏蛔虫	JF951366	99.2
		小熊猫贝氏蛔虫	HQ671080	89.7
nad5	MZ172981	浣熊贝氏蛔虫	JF951366	97.8
		转移贝氏蛔虫	HQ671079	88.1

种类（登录号） Species (Accession number)	ITS中的SNP位点 SNP site in ITS								ITS中的GA串联重复 GA tandem repeat in ITS
种类（登录号） Species (Accession number)	位置号 Position number								位置号（重复数） Position number (No. repeat)
	143^a	480^a (158)^b	523^a (201)^b	865^a	866^a	867^a	868^a	1 129^a	849~870
Bc (MH030594)	C	-	T	-	-	-	-	-	GAGAGAGAGAGAGAAA (6)
Bc (MH030595)	-	-	T	-	-	-	-	-	GAGAGAGAGAGAGAAA (6)
Bc (KC543484)	-	-	T	-	-	-	-	-	GAGAGAGAGAGAGAAA (6)
Bc (KC543485)	-	A	T	-	-	-	-	-	GAGAGAGAGAGAGAGAAA (7)
Bc (KX774493)	-	-	T	-	-	-	-	-	GAGAGAGAGAGAGAAA (6)
Bp (JQ403615)	-	-	C	G	A	G	A	-	GAGAGAGAGAGAGAGAGAGAAA (9)
Bp (MH030596)	-	-	T	G	A	G	A	-	GAGAGAGAGAGAGAGAGAGAAA (9)
Bp (MH030597)	-	-	C	G	A	G	A	-	GAGAGAGAGAGAGAGAGAGAAA (9)
Bp (KC543488)	-	-	C	G	A	G	A	-	GAGAGAGAGAGAGAGAGAGAAA (9)
Bp#1 (MZ092850)	-	A	T	-	-	-	-	C	GAGAGAGAGAGAGAGAAA (7)
Bp#5 (MZ092851)	-	A	T	G	-	-	A	C	GAGAGAGAGAGAGAGAGAAA (8)
Bp#7 (MZ092852)	-	-	C	G	A	G	A	T	GAGAGAGAGAGAGAGAGAGAAA (9)
Bp#11 (MZ092853)	C	A	T	-	-	-	-	C	GAGAGAGAGAGAGAGAAA (7)
Bp#12 (MZ092854)	C	-	C	G	A	G	A	T	GAGAGAGAGAGAGAGAGAGAAA (9)
Bp#19 (MZ092855)	-	-	C	G	-	-	A	T	GAGAGAGAGAGAGAGAGAAA (8)

种类（登录号） Species (Accession number)	位置号 Position number
种类（登录号） Species (Accession number)	37^a	168^a		201^a		231^a,c		378^a		429^a		484^a		549^a		696^a		786^a (43)^b		802^a (59)^b		804^a,d (61)^b		822^a (79)^b		832^a (89)^b
Bc (KY580736)	A	A		C		G		G		A		G		T		C		C		T		G		T		T
Bc (KY580738)	G	G		T		G		A		G		A		C		C		C		C		G		T		T
Bc (KY580739)	A	A		C		G		G		G		G		T		G		C		T		A		T		T
Bc (MH795147)	-	-		-		-		A		G		A		C		C		T		T		G		T		C
Bc (MH795148)	-	-		-		-		A		G		A		C		C		C		C		G		T		T
Bc (KC543475)	-	-		-		-		-		-		-		-		-		C		T		G		C		T
Bp (MH795149)	-	-		-		-		G		G		G		T		T		C		T		A		T		T
Bp (MH795150)	-	-		-		-		G		G		G		T		C		C		T		G		T		T
Bp (JF951366)	A	A		T		A		G		G		G		T		T		C		T		A		T		T
Bp#1 (MZ164969)	G	G		T		G		A		G		A		C		C		C		C		G		T		T
种类（登录号） Species (Accession number)	位置号 Position number
种类（登录号） Species (Accession number)	834^a,d (91)^b		957^a (214)^b		972^a (229)^b		1 002^a,c,d (259)^b		1 046^a (303)^b		1 266^a,c		1 305^a		1 311^a		1 315^a,c		1 350^a		1 378^a		1 379^a		1 491^a,c		1 506^a,c
Bc (KY580736)	A		T		A		G		T		T		A		T		G		T		A		T		T		T
Bc (KY580738)	A		T		A		G		T		T		A		C		G		T		G		T		T		T
Bc (KY580739)	G		T		A		G		T		T		A		T		G		C		A		T		T		T
Bc (MH795147)	A		C		A		G		C		T		A		T		G		T		G		T		T		-
Bc (MH795148)	A		T		A		G		T		T		A		C		G		T		G		T		-		-
Bc (KC543475)	A		T		G		G		T		-		-		-		-		-		-		-		-		-
Bp (MH795149)	G		T		A		A		T		C		G		T		G		C		A		C		C		-
Bp (MH795150)	A		T		A		G		T		T		A		T		G		T		A		T		-		-
Bp (JF951366)	G		T		A		A		T		C		G		T		A		C		A		C		C		C
Bp#1 (MZ164969)	A		T		A		G		T		T		A		C		G		T		G		T		T		T

种类（登录号） Species (Accession number)	位置号 Position number
种类（登录号） Species (Accession number)	9^a	168^a (66)^b	219^a (117)^b	306^a (204)^b	348^a (246)^b	382^a (280)^b	582^a (480)^b
Bc (FJ357429)	-	G	A	A	T	C	G
Bc (MH469662)	-	G	A	A	C	C	G
Bc (MH469663)	-	G	A	A	T	C	G
Bc (KC543478)	-	G	A	A	T	C	G
Bc (KC543480)	-	G	G	A	T	T	T
Bc (KY580740)	T	G	A	A	C	T	G
Bc (KY580741)	T	A	A	A	C	T	G
Bc (KY580742)	G	G	A	A	T	C	G
Bp (MH469664)	-	A	A	A	T	T	G
Bp (MH469665)	-	G	A	A	C	T	G
Bp (KP843602)	-	G	A	A	C	T	G
Bp (KP843603)	-	A	A	G	T	T	G
Bp (KC543482)	-	A	A	A	T	T	G
Bp (AF179908)	-	A	A	A	T	T	G
Bp (JF951366)	T	A	A	A	T	T	G
Bp#1 (MZ172987)	G	G	A	A	T	C	G