Sequencing and analysis of the mitochondrial genome of Hoplopleura edentula

doi:10.12140/j.issn.1000-7423.2022.02.010

Abstract

Abstract:

Objective To conduct sequencing and analysis the mitochondrial (mt) genome of Hoplopleura edentula to understand the structure and variation of the mt genome of Hoplopleura. Methods We used stratified random sampling to capture the Eothenomys miletus in Mount Cangshan Geopark Dali, and collected all sucking lice on the surface of E. miletus by complete catching method and identified the species of the sucking lice. The DNA of each H. edentula was extracted using the Dneasy Tissue Kit. Universal primers were used to amplify the short fragment of small ribosomal submit RNA (rrnS) and large ribosomal submit RNA (rrnL) genes of H. edentula. Subsequently, the specific primers in the conserved regions of the short fragment were designed to amplify long fragment sequences of rrnS and rrnL genes, and the specific primers in the minichromosomes conserved region were designed to amplify all the coding regions of the minichromosomes. The successfully amplified PCR products were purified and sequenced by Illumina HiSeq X Ten platform high-throughput sequencing method. The structure and variation of the mt genome were analyzed by bioinformatics tools such as Geneious, tRNAscan, CodonW, BLAST, etc. Results A tatol of 6 812 606 bp sequence reads were obtained from the H. edentula mt genome and 24 mt genes (7 protein-coding genes, 15 tRNA genes and 2 rRNA genes) of common genes in arthropod mt genomes were identified after assembly. The mt genome of H. edentula fragmented into 8 minichromosomes (GenBank accession number: MW835203-MW835210). These genes were unevenly distributed on minichromosomes. The coding region of each minichromosome contains 1-4 genes, at least one protein-coding gene or rRNA gene. AT content of the coding region is 61.0%. All start codon of protein-coding genes were ATN, except for cox2, which start codon was TTG. All protein-coding genes use TAA and TAG as stop codons. The codon AUU is the most frequently used (RSCU: 1.53). The secondary structure of 15 tRNA genes is a typical clover like structure. There are 31 mismatches in tRNA genes, mainly G-U mismatch. The rrnS and M-L₁(tag)-rrnL-V minichromosomes have all the non-coding regions, and there are 2 types of tandem repetitive sequences, and they were 88.0%-90.0% identical. An AT-rich motif (50 bp, 64.0% A&T) is present in the non-coding region upstream of the 5′-end of the coding region, whereas a GC-rich motif (42 bp, 85.7% G&C) is present downstream of the 3′-end of the coding region. We also sequenced parts of the non-coding regions upstream and downstream of the coding regions of the other 6 H. edentula minichromosomes, and the identity reached to 86.5%-88.5%. Comparing the mt genomes of H. edentula, H. akanezumiand H. kitti showed that the mt genomes of all three species were fragmented. There were four minichromosomes that have the same gene composition and sequence: E-cob-S₁(tct)-S₂(tga), I-cox1, K-nad4 and rrnS. The H-nad5-F-T minichromosome of H. edentula was not found in the other two Hoplopleura species. trnT gene translocated frequently and distributed in the different minichromosomes of three Hoplopleura species. trnS₁(tct) of H. edentula has a typical clover-leaf structure, while trnS₁(tct) of the other two Hoplopleura species lacks D-arm. Conclusion H. edentula has 24 genes distributed unevenly on eight minichromosomes, each of them contains a coding region and a non-coding region, and is of AT rich. The number of base mismatches of tRNA is considerably high. The trnS₁(tct) of H. edentulais shows a typical clover like structure. The structure of Hoplopleura mt genome varies, and its particularity may be related to the genome cracking.

Key words: Hoplopleura edentula, Mitochondrial minichromosomes, RNA genes, Protein-coding genes, Non-coding regions

CLC Number:

R384.3

SUN Jia-ning, CHEN Ting, DONG Wen-ge. Sequencing and analysis of the mitochondrial genome of Hoplopleura edentula[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(2): 194-203.

Figures/Tables 8

Fig. 1

Table 1

Table 2

Table 3

Table 4

Fig. 2

Fig. 3

Fig. 4

References 46

[1]	Durden LA,, Musser GG. The sucking lice (Insecta, Anoplura) of the world: a taxonomic checklist with records of mammalian hosts and geographical distributions[J]. Bull Am Mus Nat Hist, 1994, 218: 1-90.
[2]	Chin TH. Taxonomy and fauna of sucking lice (Anoplura) in China[M]. Beijing: Science Press, 1999: 44-45. (in Chinese)
	(金大雄. 中国吸虱的分类与检索[M]. 北京: 科学出版社, 1999: 44-45.)
[3]	Luo ZX,, Chen W,, Gao W. Fauna sinica[M]. Beijing: Science Press, 2000: 449-451. (in Chinese)
	(罗泽珣,, 陈卫,, 高武. 中国动物志[M]. 北京: 科学出版社, 2000: 449-451.)
[4]	Zhu WL,, Jia T,, Lian X, et al. Seasonal variations of maximum metabolic rate in Eothenomys miletusin Hengduan mountains region[J]. Acta Ecol Sin, 2010, 30(5): 1133-1139. (in Chinese)
	(朱万龙,, 贾婷,, 练硝, 等. 横断山脉大绒鼠最大代谢率的季节性差异[J]. 生态学报, 2010, 30(5): 1133-1139.)
[5]	Guo M,, Dong XQ. Development situation of the plague foci Apodemus chevrieri and Eothenomys miletus in northwest Yunnan Province[J]. Chin J Control of Endem Dis, 2008, 23(1): 27-31. (in Chinese)
	(郭牧,, 董兴齐. 滇西北齐氏姬鼠、大绒鼠鼠疫疫源地的发展概况[J]. 中国地方病防治杂志, 2008, 23(1): 27-31.)
[6]	Dong WG,, Guo XG,, Men XY, et al. Ectoparasites of Eothenomys miletus in the focus of plague in northwest Yunnan[J]. Sichuan J Zool, 2009, 28(5): 683-690.
[7]	Peng PY,, Guo XG,, Song WY, et al. Investigation of ectoparasites on body surface of Eothenomys miletus in some places of Guizhou[J]. Guizhou Agric Sci, 2015, 43(2): 75-79. (in Chinese)
	(彭培英,, 郭宪国,, 宋文宇, 等. 贵州省部分地区大绒鼠体表寄生虫调查[J]. 贵州农业科学, 2015, 43(2): 75-79.)
[8]	Zhang YZ,, Zhang HL,, Mi ZQ, et al. Monitoring of hemorrhagic fever with renal syndrome in Yunnan Province, China, 2005[J]. Chin J Vector Biol Control, 2008, 19(2): 148-150. (in Chinese)
	(张云智,, 张海林,, 米竹青, 等. 2005年云南省肾综合征出血热监测研究[J]. 中国媒介生物学及控制杂志, 2008, 19(2): 148-150.)
[9]	Boore JL. Animal mitochondrial genomes[J]. Nucleic Acids Res, 1999, 27(8): 1767-1780. pmid: 10101183
[10]	Cameron SL. Insect mitochondrial genomics: implications for evolution and phylogeny[J]. Annu Rev Entomol, 2014, 59(1): 95-117. doi: 10.1146/annurev-ento-011613-162007
[11]	Jiang H,, Barker SC,, Shao R. Substantial variation in the extent of mitochondrial genome fragmentation among blood-sucking lice of mammals[J]. Genome Biol Evol, 2013, 5(7): 1298-1308. doi: 10.1093/gbe/evt094
[12]	Song SD,, Barker SC,, Shao R. Variation in mitochondrial minichromosome composition between blood-sucking lice of the genus Haematopinus that infest horses and pigs[J]. Parasit Vectors, 2014, 7: 144. doi: 10.1186/1756-3305-7-144
[13]	Shao R,, Kirkness EF,, Barker SC. The single mitochondrial chromosome typical of animals has evolved into 18 minichromosomes in the human body louse, Pediculus humanus[J]. Genome Res, 2009, 19(5): 904-912. doi: 10.1101/gr.083188.108
[14]	Shao R,, Zhu XQ,, Barker SC, et al. Evolution of extensively fragmented mitochondrial genomes in the lice of humans[J]. Genome Biol Evol, 2012, 4(11): 1088-1101. doi: 10.1093/gbe/evs088
[15]	Dong WG,, Song S,, Guo XG, et al. Fragmented mitochondrial genomes are present in both major clades of the blood-sucking lice(Suborder Anoplura): evidence from two Hoplopleura rodent lice (family Hoplopleuridae)[J]. BMC Genom, 2014, 15(1): 1-13. doi: 10.1186/1471-2164-15-1
[16]	Dong WG,, Song S,, Jin DC, et al. Fragmented mitochondrial genomes of the rat lice, Polyplax asiatica and Polyplax spinulosa: intra-genus variation in fragmentation pattern and a possible link between the extent of fragmentation and the length of life cycle[J]. BMC Genom, 2014, 15: 44. doi: 10.1186/1471-2164-15-44
[17]	Shao R,, Li H,, Barker SC, et al. The mitochondrial genome of the guanaco louse, Microthoracius praelongiceps: insights into the ancestral mitochondrial karyotype of sucking lice (Anoplura, Insecta)[J]. Genome Biol Evol, 2017, 9(2): 431-445. doi: 10.1093/gbe/evx007
[18]	Fu YT,, Dong Y,, Wang W, et al. Fragmented mitochondrial genomes evolved in opposite directions between closely related macaque louse Pedicinus obtusus and colobus louse Pedicinus badii[J]. Genomics, 2020, 112(6): 4924-4933. doi: 10.1016/j.ygeno.2020.09.005
[19]	Herd KE,, Barker SC,, Shao R. The mitochondrial genome of the chimpanzee louse, Pediculus schaeffi: insights into the process of mitochondrial genome fragmentation in the blood-sucking lice of great apes[J]. BMC Genom, 2015, 16: 661. doi: 10.1186/s12864-015-1843-3
[20]	Kearse M,, Moir R,, Wilson A, et al. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data[J]. Bioinformatics, 2012, 28(12): 1647-1649. doi: 10.1093/bioinformatics/bts199
[21]	Laslett D,, Canbäck B. ARWEN: a program to detect tRNA genes in metazoan mitochondrial nucleotide sequences[J]. Bioinformatics, 2008, 24(2): 172-175. pmid: 18033792
[22]	Lowe TM,, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence[J]. Nucleic Acids Res, 1997, 25(5): 955-964. pmid: 9023104
[23]	Altschul SF,, Madden TL,, Schäffer AA, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[J]. Nucleic Acids Res, 1997, 25(17): 3389-3402. doi: 10.1093/nar/25.17.3389 pmid: 9254694
[24]	Gish W,, States DJ. Identification of protein coding regions by database similarity search[J]. Nat Genet, 1993, 3(3): 266-272. pmid: 8485583
[25]	Puigbò P,, Bravo IG,, Garcia-Vallve S. CAIcal: a combined set of tools to assess codon usage adaptation[J]. Biol Direct, 2008, 3: 38. doi: 10.1186/1745-6150-3-38 pmid: 18796141
[26]	Perna NT,, Kocher TD. Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes[J]. J Mol Evol, 1995, 41(3): 353-358. pmid: 7563121
[27]	Shao R,, Barker SC,, Li H, et al. Fragmented mitochondrial genomes in two suborders of parasitic lice of eutherian mammals (Anoplura and Rhynchophthirina, Insecta)[J]. Sci Rep, 2015, 5: 17389. doi: 10.1038/srep17389
[28]	Song F,, Li H,, Liu GH, et al. Mitochondrial genome fragmentation unites the parasitic lice of eutherian mammals[J]. Syst Biol, 2019, 68(3): 430-440. doi: 10.1093/sysbio/syy062 pmid: 30239978
[29]	Shi GH,, Cui ZX,, Hui M, et al. The complete mitochondrial genomes of Umalia orientalis and Lyreidus brevifrons: the phylogenetic position of the family Raninidae within Brachyuran crabs[J]. Mar Genom, 2015, 21: 53-61. doi: 10.1016/j.margen.2015.02.002
[30]	Chen XX,, Yuan ZW,, Yuan XW, et al. Advances in mitochondrial genome complete sequence structure of leafhopper[J]. Genom Appl Biol, 2020, 39(6): 2565-2577. (in Chinese)
	(陈晓晓,, 袁周伟,, 苑晓伟, 等. 叶蝉线粒体基因组全序列结构研究进展[J]. 基因组学与应用生物学, 2020, 39(6): 2565-2577.)
[31]	Kolesnikov AA,, Gerasimov ES. Diversity of mitochondrial genome organization[J]. Biochemistry(Mosc), 2012, 77(13): 1424-1435.
[32]	Li JJ. Research on phylogeny and acoustic signal evolution of Ensifera based on complete mitochondrial genome[D]. Chang-chun:Northeast Normal University, 2018: 13-28. (in Chinese)
	(李君健. 基于线粒体基因组的螽亚目昆虫系统发育与鸣声进化研究[D]. 长春:东北师范大学, 2018: 13-28.)
[33]	Ou J. Complete mitochondrial genomes sequences of six stored grain beetles[D]. Xi’an:Shanxi Normal University, 2015: 15-57. (in Chinese)
	(欧静. 六种储粮甲虫线粒体基因组测定及分析[D]. 西安:陕西师范大学, 2015: 15-57.)
[34]	Zhou F. Complete mitochondrial genomes sequences of four locust and analyzed the phylogeny of Orthoptera[D]. Xi’an:Shanxi Normal University, 2015: 27-50. (in Chinese)
	(周飞. 四种蝗虫线粒体基因组测定及直翅目系统发生分析[D]. 西安:陕西师范大学, 2015: 27-50.)
[35]	Wu YM. Genome sequencing and transcriptome analysis of chemosensory genes of two meloids[D]. Guiyang:Guizhou University, 2018: 36-41. (in Chinese)
	(吴渊明. 两种芫菁的全基因组测序和化学感受器转录组基因的鉴定与分析[D]. 贵阳:贵州大学, 2018: 36-41.)
[36]	Xia LY,, Sun WW,, Cui M, et al. Sequencing and analysis of mitochondrial genome of Lasioderma serricorne[J]. Tob Sci Technol, 2020, 53(12): 1-8. (in Chinese)
	(夏丽媛,, 孙为伟,, 崔淼, 等. 烟草甲线粒体基因组序列测定与分析[J]. 烟草科技, 2020, 53(12): 1-8.)
[37]	Liu J,, Bian X. Characteristics of the Orthoptera mitogenome and its application[J]. J Guangxi Norm Univ Nat Sci Ed, 2021, 39(1): 17-28. (in Chinese)
	刘静,, 边迅. 直翅目昆虫线粒体基因组的特征及应用[J]. 广西师范大学学报(自然科学版), 2021, 39(1): 17-28.)
[38]	Yan D,, Tang Y,, Hu M, et al. The mitochondrial genome of Frankliniella intonsa: insights into the evolutionof mitochondrial genomes at lower taxonomic levels in Thysanoptera[J]. Genomics, 2014, 104(4): 306-312. doi: 10.1016/j.ygeno.2014.08.003
[39]	Chen S. Complete mitochondrial genomes sequences of six species of Spilomelinae[D]. Xi’an: Shanxi Normal University, 2017: 34-37. (in Chinese)
	(陈汕. 六种斑野螟亚科昆虫全线粒体基因组的序列测定与分析[D]. 西安:陕西师范大学, 2017: 34-37.)
[40]	Varani G,, Mcclain WH. The G x U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems[J]. Embo Rep, 2000, 1(1): 18-23. pmid: 11256617
[41]	Wang XM. Structural characteristics of mitochondrial genome and preliminary phylogenetic analysis for Lepus hainanus[D]. Jinan: Shandong University, 2012: 3-4. (in Chinese)
	(王晓明. 海南兔的线粒体基因组结构特征与系统演化初探[D]. 济南:山东大学, 2012: 3-4.)
[42]	Broughton RE,, Dowling TE. Length variation in mitochondrial DNA of the minnow Cyprinella spiloptera[J]. Genetics, 1994, 138(1): 179-190. doi: 10.1093/genetics/138.1.179 pmid: 8001785
[43]	Ladoukakis ED,, Zouros E. Direct evidence for homologous recombination in mussel (Mytilus galloprovincialis) mitochondrial DNA[J]. Mol Biol Evol, 2001, 18(7): 1168-1175. pmid: 11420358
[44]	Okada K,, Yamazaki Y,, Yokobori S, et al. Repetitive sequences in the lamprey mitochondrial DNA control region and speciation of Lethenteron[J]. Gene, 2010, 465(1/2): 45-52. doi: 10.1016/j.gene.2010.06.009
[45]	Ray DA,, Densmore LD. Repetitive sequences in the crocodilian mitochondrial control region: Poly-A sequences and heteroplasmic tandem repeats[J]. Mol Biol Evol, 2003, 20(6): 1006-1013. doi: 10.1093/molbev/msg117
[46]	Shi W,, Kong XY,, Wang ZM, et al. Pause-melting misalignment: a novel model for the birth and motif indel of tandem repeats in the mitochondrial genome[J]. BMC Genom, 2013, 14: 103. doi: 10.1186/1471-2164-14-103

微环染色体 Minichromosome	编码区大小/bp Size of coding region/bp	序列读数/bp No. sequence read/bp
E-cob-S₁(tct)-S₂(tga)	1 303	456 433
I-cox1	1 599	206 917
D-Y-cox2	791	822 133
Q-nad1-G-nad3	1 446	712 883
K-nad4	1 322	960 072
H-nad5-F-T	1 889	441 440
M-L₁_(tag)-rrnL-V	1 311	1 681 291
rrnS	874	1 290 852
合计Total	10 535	6 572 021

特征 Feature	缺齿甲胁虱 H. edentula	克氏甲胁虱 H. kitti	红姬甲胁虱 H. akanezumi
微环染色体组成Composition of minichromosome	-	atp8-atp6-N	atp8-atp6-N
	E-cob-S₁(tct)-S₂(tga)	E-cob-S₁(tct)-S₂(tga)	E-cob-S₁(tct)-S₂(tga)
	I-cox1	I-cox1	I-cox1
	D-Y-cox2	D-Y-cox2-	D-Y-cox2
	-	R-nad4L-P-cox3-A	R-nad4L-P-cox3-A-
	Q-nad1-G-nad3	Q-nad1-G-nad3	-
	-	nad2	nad2
	K-nad4	K-nad4	K-nad4
	H-nad5-F-	-	-
	-	C-nad6-W-L₂(taa)	C-nad6-W-L₂(taa)
	M-L₁(tag)-rrnL-V	M-L₁(tag)-rrnL-V	-rrnL-V
	rrnS	rrnS	rrnS
微环染色体数No. minichromosome	8	11	11
基因数No. gene	24	34	28

基因序列 Gene sequence	长度/bp Length/bp	占比/% Proportion/%					AT偏倚值 AT-skew	GC偏倚值 GC-skew
基因序列 Gene sequence	长度/bp Length/bp	A	T	G	C	A + T	AT偏倚值 AT-skew	GC偏倚值 GC-skew
编码区Coding region	10 545	26.9	34.1	20.7	18.3	61.0	-0.118	0.062
蛋白质编码基因 Protein-coding gene	7 563	25.0	34.8	19.1	21.1	59.8	-0.164	-0.050
密码子第1位点 The 1st codon position	2 521	28.2	28.4	25.6	17.8	56.6	-0.004	0.180
密码子第2位点 The 2nd codon position	2 521	16.9	43.5	18.0	21.6	60.4	-0.440	-0.091
密码子第3位点 The 3rd codon position	2 521	26.8	35.7	19.5	18.0	62.5	-0.426	0.040
tRNAs	995	31.5	32.8	19.6	16.1	64.3	-0.020	0.098
rrnL	1 117	31.5	32.2	21.1	15.2	63.7	-0.011	0.163
rrnS	874	31.2	31.5	19.2	18.1	62.7	-0.005	0.029

氨基酸 Amino acid	密码子 Codon	使用次数 No. use	相对同义密码子使用频率 Relative synonymous codon usage	氨基酸 Amino acid	密码子 Codon	使用次数 No. use	相对同义密码子使用频率 Relative synonymous codon usage
丙氨酸 Ala	GCU	39	1.65	脯氨酸 Pro	CCU	63	2.23
	GCC	64	1.01		CCC	23	0.81
	GCA	32	0.83		CCA	16	0.57
	GCG	20	0.52		CCG	11	0.39
半胱氨酸 Cys	UGU	21	1.08	谷氨酰胺 Gln	CAA	15	0.77
	UGC	18	0.92		CAG	24	1.23
天冬氨酸 Asp	GAU	31	1.24	精氨酸 Arg	CGU	6	0.27
	GAC	19	0.76		CGC	10	0.45
谷氨酸 Glu	GAA	41	1.32		CGA	10	0.45
	GAG	21	0.68		CGG	12	0.54
苯丙氨酸 Phe	UUU	129	1.50		AGA	43	1.93
	UUC	43	0.50		AGG	53	2.37
甘氨酸 Gly	GGU	24	0.57	丝氨酸 Ser	UCU	77	2.23
	GGC	25	0.60		UCC	40	1.16
	GGA	50	1.20		UCA	40	1.16
	GGG	68	1.63		UCG	12	0.35
组氨酸 His	CAU	24	0.92		AGU	21	0.61
	CAC	28	1.08		AGC	17	0.49
异亮氨酸 Ile	AUU	137	1.53	苏氨酸 Thr	ACU	46	1.72
	AUC	38	0.43		ACC	26	0.97
	AUA	93	1.04		ACA	30	1.12
赖氨酸 Lys	AAA	25	0.82		ACG	5	0.19
	AAG	36	1.18	缬氨酸 Val	GUU	75	1.42
亮氨酸 Leu	UUA	124	1.97		GUC	32	0.60
	UUG	46	0.73		GUA	65	1.23
	CUU	91	1.45		GUG	40	0.75
	CUC	32	0.51	色氨酸 Trp	UGG	32	1.00
	CUA	41	0.65		UGA	45	2.60
	CUG	43	0.68	酪氨酸 Tyr	UAU	46	1.12
蛋氨酸 Met	AUG	68	1.00		UAC	36	0.88
天冬酰胺 Asn	AAU	45	1.25	终止密码子* Stop codon*	UAA	6	0.35
	AAC	27	0.75	终止密码子* Stop codon*	UAG	1	0.06