Characterization of the fragmented mitochondrial genome of domestic pig louse Haematopinus suis (Insecta: Haematopinidae) from China

doi:10.21203/rs.3.rs-2446385/v1

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Characterization of the fragmented mitochondrial genome of domestic pig louse Haematopinus suis (Insecta: Haematopinidae) from China

https://doi.org/10.21203/rs.3.rs-2446385/v1

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The domestic pig louse Haematopinus suis is found worldwide wherever pig is raised. This ectoparasite causes direct damage to their hosts and also acts as a vector of various infectious disease agents. Despite its significance, molecular genetics, biology and systematics of H. suis has not been studied in detail. Herein, the entire mitochondrial (mt) genome of H. suis from China was sequenced, characterized and compared with that of H. suis from Australia. We identified 37 mt genes located on nine circular mt minichromosomes in H. suis from China, with 2.9 kb-4.2 kb in size and contains 2–8 genes and one non-coding region (1,957 bp-2,226 bp). The number of minichromosomes, gene content, and gene order in the mt genomes of H. suis from China and Australia is identical. The identity of both mt genomes (except for non-coding regions) was 96.3% between H. suis from China and Australia. For the 13 protein-coding genes, sequence differences in each gene at both the nucleotide and amino acid were 2.8–6.5% and 0.5–5.3%, respectively. The present study determined the entire mt genome of H. suis from China that provides novel genetic markers for studying the molecular genetics, biology and systematics of sucking louse.

Sucking louse

Mitochondrial genome

Minichromosome

Mitochondrial DNA

Sucking louse is one of the common ectoparasites of mammals (Durden and Musser 1994). There are about 540 species, which can be categorized into 15 families and 50 genera (Kim and Ludwig 1978; Jiang et al. 2013). The 21 species that comprise the genus Haemophilus are persistent, mammalian-specific body parasites (Meleney and Kim 1974; Barker 1994; Light et al. 2010; Scofield et al. 2012). The domestic pig louse Haematopinus suis is an ubiquitous ectoparasite of domestic pigs that causes itching, hair loss, skin irritation, or even anemia (Tsai et al. 2011). Numerous louse-borne diseases, including African swine fever virus, swine fever virus, Mycoplasma suis, and anaplasma (Thibault et al. 1998; Zikmundová et al. 2014; Acosta et al. 2019; Saegerman et al. 2021), have been found from pig louse, raising considerable threat to the swine health.

Metazoan mitochondrial (mt) genomes are usually circular DNA molecules (13–20 kb) with 36–37 genes that contain 12–13 protein-coding genes, two rRNA genes and 22 tRNA genes (Wolstenholme 1992; Lavrov 2007). Sucking lice have an unusual, fragmented mt genome organization. Fragmentation of the mt genome was first found in the human body louse, Pediculus humanus corporis (suborder Anoplura) (Shao et al. 2009). To date, the mt genomes of 21 sucking lice species (12 complete mt genomes and 9 incomplete mt genomes) have been sequenced, all are extensively fragmented with different numbers of minichromosomes (Shao et al. 2012; Jiang et al. 2013; Dong et al. 2014; Fu 2022). In the genus Haematopinus, the complete fragmented mt genome was available for only four species (Song et al. 2014; Nie et al. 2022; Fu 2022). The fragmented mt genome of H. suis from Australia has been sequenced, but H. suis isolated from different geographic origins may have some differences. A previous study indicated that a pseudo-trnV gene has been found in H. suis and H. apri from Australia (Jiang et al. 2013). However, this pseudo-trnV gene has not been reported in H. apri from China (Nie et al. 2022).

The objectives of the present study were: (i) to characterise the fragmented mt genome of H. suis from China, (ii) to compare this fragmented mt genome with that of H. suis from Australia.

Sample collection and DNA extraction

Adult specimens were collected from a domestic pig in Sichuan province, China. Those lice were recognized as H. suis based on morphological features and host information (Fig.S1) (Kim and Ludwig 1978). Those lice were washed five times in physiological saline solution and stored in 70% (v/v) ethanol at − 40°C until DNA extraction. The total genomic DNA of those lice were extracted using a commercially available kit (Promega, Madison, USA) per manufacturer's introduction. Each louse was further confirmed by PCR with primers reported previously (Fu 2020). Partial mt cox1 and rrnS sequences of lice were then blast and showed 97.3% and 97.4% similarity with H. suis isolated from Australia (GenBank accession no. HM241908 and KC814610), respectively.

Sequencing, assembling and annotation

The concentration of total DNA was tested using the Qubit system (Thermo Fisher Scientific, Waltham, MA, USA) to construct genomic library (with 350 bp inserts). The raw data (paired-end reads) was sequenced and obtained based on Illumina HiSeq 4000 Platform. Reads with adapter, repetitive “N-” bases and low quality (Phred quality < 5) were filtered to obtain clean data based on FastQC 0.11.9 and Skewer v0.2.2 (Jiang et al. 2014). Using partial cox1 and rrnS sequences mentioned before as references, clean reads were used to assembled mitogenome of H. suis by Geneious Prime v2022.1.11 (Kearse et al. 2012). Minichromosomes of H. suis were individually assembled as previous description (Fu 2020). Locations of protein-coding genes (PCGs) and rRNAs (rrnL and rrnS) and gene boundaries of each minichromosome were predicted and confirmed by aligning with Haematopinus species. All 22 tRNAs were found based on ARWEN and tRNAscan-SE (Laslett and Canbäck 2008; Lowe and Chan 2016).

Verification of mitochondrial minichromosomes

The size and organization of each mt minichromosome of H. suis from China were amplified and verified by PCR (Fig. S2) using designed primers which were listed in (Table S1). Forward and reverse primers of each pair were next to each other with a small gap (10–90 bp). To obtain full-length sequences of each minichromosome, the amplicons of nine full-length mt minichromosome for H. suis were sequenced in a separate batch with high throughput sequencing as described above. The clean data of each minichromosomes was obtained and assembled as described above, respectively.

General features of minichromosomes organization

A total of 19,867,822×2 clean reads was obtained after quality filtration. Complete mitogenome of H. suis isolated from China was assembled with 37 genes scattering into nine minichromosomes (Fig. 1; Table 1), consistent with previous studies (Jiang et al. 2013; Song et al. 2014). The nucleotide sequences of each circle across H. suis were uploaded into the GenBank database under the accession numbers ON585586- ON585594. Each minicircle was from 3.0–4.8 kb in length containing 2–8 genes and a wide NCR (Table 1). The size of coding regions varied the size from 789 to 2,668 bp, similar to previous reports of pig lice H. suis and H. apri with 786–2,669 bp and 788–2,669 bp coding regions (Jiang et al. 2013). The longest and shortest NCRs were 2,226 bp and 1,957 bp, which located in trnL₁-rrnL minichromosome and trnQ-nad1-trnT-trnG-nad3-trnW minichromosome (Table 1), respectively. However, the length of the longest NCR isolated from China was 144 bp shorter than that of Australia (Jiang et al. 2013). Except for trnQ-nad1-trnT gene block, all other genes were transcribed in a clockwise direction, consistent with H. suis isolated from Australia, H. apri, and H. asini.

Table 1

Mitochondrial minichromosomes of the louse *Haematopinus suis* from China, identified by Illumina sequencing.
Minichromosomes	Size (bp)	Size of coding region (bp)	Size of non-coding region (bp)	Intergenic region (bp)
nad2-trnI-cox1-trnL₂	4800	2668	2132	0
trnD-trnY-cox2-trnS₁-trnS₂-trnP-cox3-trnA	4031	1830	2179	22
trnE-cytb-trnV	3280	1216	2062	2
trnQ-nad1-trnT-trnG-nad3-trnW	3473	1509	1957	7
trnK-nad4-atp8-atp6-trnN	4461	2296	2157	8
trnR-nad4L-nad6-trnM	3022	867	2091	64
trnH-nad5-trnF	3930	1769	2152	9
rrnS-trnC	2882	789	2093	0
trnL₁-rrnL	3453	1227	2226	0
Total	33332	14171	19049	112

Annotation

The use of initiation/termination codons were similar in H. suis from Australia and China except for cox1 gene. ATN (ATA, ATT, ATG) was the most frequent initiation codon used in 13 PCGs of pig louse H. suis from China, followed by TTG and GTG. ATA was used in cox3, nad5, nad6 and atp6 genes, and ATG was used in nad1, nad2 and nad4L genes. ATT was only applied for cox2 and cytb genes. Though TTG and GTG were rare initiation codons, they were used in cox1, nad3, atp8, and nad4. This mitogenome had four codons (TAA, TAG, TA, and T) as termination. Complete termination codon TAA was the most common codon used to stop translation of six protein genes. TAG was used only in atp8 and nad4L. Incomplete codons TA (cox1 and cox2) and T (cox3, nad2 and nad3) were high frequency of being used as stop codons, which also found in other mitogenomes of lice, including blood-sucking lice, lice genera Haematopinus (Jiang et al. 2013; Song et al. 2014), Polyplax (Dong et al. 2014), Pediculus (Herd et al. 2015), Microthoracius (Shao et al. 2017), Hoplopleura (Fu 2020), and Pthirus speices (Shao et al. 2012), and chewing lice, such as vulture lice Falcolipeurus quadripustulatus (Song et al. 2019), elephant louse Haematomyzus elephantis (R et al. 2015), and screamer louse Bothriometopus macrocnemis (Cameron et al. 2007). In the mitogenome of domestic pig louse H. suis, the lengths of rRNAs, rrnS and rrnL genes, were 721 bp and 1,161 bp, respectively. The 22 tRNA genes of H. suis were from 63 to 73 bp in length. The secondary structure predictions in H. suis from China (not shown) were similar to those of H. asini, H. apri and H. suis from Australia (Jiang et al. 2013; Song et al. 2014).

Non-coding regions

We obtained complete NCR sequences of all nine mt minichromosomes in domestic pig louse H. suis, which range from 1,957 bp (trnQ-nad1-trnT-trnG-nad3-trnW minichromosome) to 2,226 bp (trnL₁-rrnL minichromosome) (Table 1; Fig. 1), with 82.2–95.1% pairwise identity to each other. Those NCR sequences showed some consensus sequences in each minichromosome based on nucleotide polymorphisms in the NCR (Fig. 2). There was partial sequence (about 62 bp in size) repeated 3 times in the non-coding region of each mt minicircle except for trnQ-nad1-trnT-trnG-nad3-trnW minichromosome for which partial sequence was 32 bp and only repeated twice. Similarly, those mt minicircles also had a highly conserve sequence (149 bp, AT-rich, 70.5% A + T) in NCR in which they were usually found at upstream of the 5′-end of the coding region (Fig. 2). Highly conserved regions also presented in other blood-sucking lice, such as H. suis (Jiang et al. 2013), H. apri (Jiang et al. 2013), P. obtusus (Fu et al. 2020), P. badii (Fu et al. 2020)d praelongiceps (Shao et al. 2017). Previous studies found there was a GC-rich region downstream of the 3′-end of the coding region in mt minnichromosomes (Shao et al. 2012; Jiang et al. 2013), interestingly, in the present study, this region was replaced by AT-rich that was 190 bp in length with 63.7% A + T.

Comparative mt genomic analyses of H. suis from China and Australia

The H. suis mt genome sequence from China was 7 bp shorter than that of H. suis from Australia. Comparisons of nucleotide (NT) and amino acid (AA) sequences of 13 PCGs of H. suis from China (HsC) and Australia (HsA) were given in Table 2. The NT homology of complete coding-regions between HsC and HsA was 96.3%, and the homology of NT and AA of 13 PCGs was 96% and 97.9%, respectively. Pairwise comparisons of NT and AA across HsC and HsA revealed differences of 2.8–5.6% and 0.5–5.3% (Table 2), respectively, and the gene atp6 and atp8 were the most conserved and the least. The divergence of rrnL and rrnS between HsC and HsA was 5.5% and 6.5% and that of 22 tRNAs was only 3.0%. The difference of complete coding-regions between HsC and HsA was 2.8–6.5%, indicating HsC was the same species as HsA.

Table 2

Nucleotide (nt) and/or predicted amino acid (aa) sequence differences in mitochondrial genes between the pig louse *H. suis* from China (HsC) and *H. suis* from Australia (HsA) upon pairwise comparison.
Gene/region	Nt sequence length		Nt difference (%)	Number of aa		aa difference (%)
Gene/region	HsC	HsA	HsC/HsA	HsC	HsA	HsC/HsA
atp6	666	666	5.6	221	221	0.5
atp8	174	174	4.6	57	57	5.3
nad1	900	900	2.9	299	299	2.0
nad2	997	997	4.8	332	332	1.0
nad3	346	346	3.8	115	115	3.5
nad4	1317	1317	4.0	438	438	2.5
nad4L	282	282	2.8	93	93	1.1
nad5	1638	1638	4.0	545	545	2.8
nad6	450	450	4.0	149	149	2.7
cox1	1529	1529	3.9	509	509	2.1
cox2	665	665	3.0	221	221	0.8
cox3	767	767	5.5	255	255	2.7
cytb	1083	1083	4.5	360	360	2.5
rrnS	721	718	5.5
rrnL	1161	1161	6.5
tRNAs	1475	1485	3.0

In conclusion, the present study determined and characterized the complete fragmented mt genome sequence of H. suis from China, which is slightly shorter (7 bp) than that of H. suis from Australia and both mt genomes differed by 3.7% only. These mt genome sequences should provide novel genetic data for addressing further questions in systematics and population genetics of sucking louse.

Acknowledgements

Not applicable.

Ethics approval and consent to participate

All procedures involving animals in the present study were approved and this study was approved by the Animal Ethics Committee of Hunan Agricultural University (No. 43321503).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported in part, by the National Natural Science Foundation of China (Grant No. 32172884).

Authors’ contributions

GHL and RL conceived and designed the study, and critically revised the manuscript. RL and YN performed the experiments. RL YTF and YPD analyzed the data. RL YTF and YPD drafted the manuscript. WW and PPM helped in study design, study implementation, and manuscript preparation. All authors read and approved the final manuscript.

Data availability

The complete mitochondrial genome of Haematopinus suis from China have been deposited in the GenBank under the accession numbers ON585586- ON585594.

Acosta DB, Ruiz M, Sanchez JP (2019) First molecular detection of Mycoplasma suis in the pig louse Haematopinus suis (Phthiraptera: Anoplura) from Argentina. Acta Trop 194:165–168. https://doi.org/10.1016/j.actatropica.2019.04.007
Barker SC (1994) Phylogeny and classification, origins, and evolution of host associations of lice. Int J Parasitol 24:1285–1291. https://doi.org/10.1016/0020-7519(94)90195-3
Cameron SL, Johnson KP, Whiting MF (2007) The mitochondrial genome of the screamer louse Bothriometopus (phthiraptera: ischnocera): effects of extensive gene rearrangements on the evolution of the genome. J Mol Evol 65:589–604. https://doi.org/10.1007/s00239-007-9042-8
Dong W-G, Song S, Jin D-C, et al (2014) 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. BMC Genomics 15:44. https://doi.org/10.1186/1471-2164-15-44
Durden LA, Musser GG (1994) The mammalian hosts of the sucking lice (Anoplura) of the world: A host-parasite list. bulletin of the society for vector ecology
Fu Y-T (2022) Human pediculosis, a global public health problem. Infect Dis Poverty 11:58. https://doi.org/10.1186/s40249-022-00986-w
Fu Y-T (2020) Variation of mitochondrial minichromosome composition in Hoplopleura lice (Phthiraptera: Hoplopleuridae) from rats. Parasites Vectors 13:506. https://doi.org/10.1186/s13071-020-04381-y
Fu Y-T, Dong Y, Wang W, et al (2020) Fragmented mitochondrial genomes evolved in opposite directions between closely related macaque louse Pedicinus obtusus and colobus louse Pedicinus badii. Genomics 112:4924–4933. https://doi.org/10.1016/j.ygeno.2020.09.005
Herd KE, Barker SC, Shao R (2015) 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. BMC Genomics 16:661. https://doi.org/10.1186/s12864-015-1843-3
Jiang H, Barker SC, Shao R (2013) Substantial Variation in the Extent of Mitochondrial Genome Fragmentation among Blood-Sucking Lice of Mammals. Genome Biology and Evolution 5:1298–1308. https://doi.org/10.1093/gbe/evt094
Jiang H, Lei R, Ding S-W, Zhu S (2014) Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15:182. https://doi.org/10.1186/1471-2105-15-182
Kearse M, Moir R, Wilson A, et al (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649. https://doi.org/10.1093/bioinformatics/bts199
Kim KC, Ludwig HW (1978) The family classification of the Anoplura. System Entomol 3:249–284. https://doi.org/10.1111/j.1365-3113.1978.tb00120.x
Laslett D, Canbäck B (2008) ARWEN: a program to detect tRNA genes in metazoan mitochondrial nucleotide sequences. Bioinformatics 24:172–175. https://doi.org/10.1093/bioinformatics/btm573
Lavrov DV (2007) Key transitions in animal evolution: a mitochondrial DNA perspective. Integr Comp Biol 47:734–743. https://doi.org/10.1093/icb/icm045
Light JE, Smith VS, Allen JM, et al (2010) Evolutionary history of mammalian sucking lice (Phthiraptera: Anoplura). BMC Evol Biol 10:292. https://doi.org/10.1186/1471-2148-10-292
Lowe TM, Chan PP (2016) tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Res 44:W54-57. https://doi.org/10.1093/nar/gkw413
Meleney WP, Kim KC (1974) A comparative study of cattle-infesting Haematopinus, with redescription of H. quadripertusus Fahrenholz, 1916 (Anoplura: Haematopinidae). J Parasitol 60:507–522
Nie Y, Fu Y-T, Wang W, et al (2022) Comparative analyses of the fragmented mitochondrial genomes of wild pig louse Haematopinus apri from China and Japan. International Journal for Parasitology: Parasites and Wildlife 18:25–29. https://doi.org/10.1016/j.ijppaw.2022.03.013
R S, Sc B, H L, et al (2015) Fragmented mitochondrial genomes in two suborders of parasitic lice of eutherian mammals (Anoplura and Rhynchophthirina, Insecta). Scientific reports 5:. https://doi.org/10.1038/srep17389
Saegerman C, Bonnet S, Bouhsira E, et al (2021) An expert opinion assessment of blood-feeding arthropods based on their capacity to transmit African swine fever virus in Metropolitan France. Transbound Emerg Dis 68:1190–1204. https://doi.org/10.1111/tbed.13769
Scofield A, Campos KF, da Silva AMM, et al (2012) Infestation by Haematopinus quadripertusus on cattle in São Domingos do Capim, state of Pará, Brazil. Rev Bras Parasitol Vet 21:315–318. https://doi.org/10.1590/s1984-29612012000300027
Shao R, Kirkness EF, Barker SC (2009) The single mitochondrial chromosome typical of animals has evolved into 18 minichromosomes in the human body louse, Pediculus humanus. Genome Res 19:904–912. https://doi.org/10.1101/gr.083188.108
Shao R, Li H, Barker SC, Song S (2017) The Mitochondrial Genome of the Guanaco Louse, Microthoracius praelongiceps: Insights into the Ancestral Mitochondrial Karyotype of Sucking Lice (Anoplura, Insecta). Genome Biology and Evolution 9:431–445. https://doi.org/10.1093/gbe/evx007
Shao R, Zhu X-Q, Barker SC, Herd K (2012) Evolution of Extensively Fragmented Mitochondrial Genomes in the Lice of Humans. Genome Biol Evol 4:1088–1101. https://doi.org/10.1093/gbe/evs088
Song F, Li H, Liu G-H, et al (2019) Mitochondrial Genome Fragmentation Unites the Parasitic Lice of Eutherian Mammals. Systematic Biology 68:430–440. https://doi.org/10.1093/sysbio/syy062
Song SD, Barker SC, Shao R (2014) Variation in mitochondrial minichromosome composition between blood-sucking lice of the genus Haematopinus that infest horses and pigs. Parasit Vectors 7:144. https://doi.org/10.1186/1756-3305-7-144
Thibault S, Drolet R, Alain R, Dea S (1998) Congenital swine pox: A sporadic skin disorder in nursing piglets. 6:
Tsai Y-L, Chang C-C, Chuang S-T, Chomel BB (2011) Bartonella species and their ectoparasites: Selective host adaptation or strain selection between the vector and the mammalian host? Comparative Immunology, Microbiology and Infectious Diseases 34:299–314. https://doi.org/10.1016/j.cimid.2011.04.005
Wolstenholme DR (1992) Genetic novelties in mitochondrial genomes of multicellular animals. Curr Opin Genet Dev 2:918–925. https://doi.org/10.1016/s0959-437x(05)80116-9
Zikmundová J, Georgieva S, Faltýnková A, et al (2014) Species diversity of Plagiorchis Lühe, 1899 (Digenea: Plagiorchiidae) in lymnaeid snails from freshwater ecosystems in central Europe revealed by molecules and morphology. Syst Parasitol 88:37–54. https://doi.org/10.1007/s11230-014-9481-8

No competing interests reported.

Fig.S1.tif
Fig. S1 Dorsal and ventral Haematopinus suis from China.
Fig.S2.tif
Fig. S2 PCR verification of the nine mt minichromosomes of the domestic pig louse Haematopinus suis from China. Lane M: DL5000 DNA marker; Lane M0: Negative control, Lane M1–M9: trnD-trnY-cox2-trnS₁-trnS₂-trnP-cox3-trnA, trnE-cytb-trnV, trnH-nad5-trnF, trnK-nad4-atp8-atp6-trnN, trnL₁-rrnL, nad2-trnI-cox1-trnL₂, trnQ-nad1-trnT-trnG-nad3-trnW, trnR-nad4L-nad6-trnM, rrnS-trnC
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Characterization of the fragmented mitochondrial genome of domestic pig louse Haematopinus suis (Insecta: Haematopinidae) from China

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Abstract

Figures

Introduction

Methods And Materials

Sample collection and DNA extraction

Sequencing, assembling and annotation

Verification of mitochondrial minichromosomes

Results And Discussion

General features of minichromosomes organization

Annotation

Non-coding regions

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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