Study on genetic differentiation of Schistosome japonicum intermediate hosts Oncomelania hupensis robertsoni in hilly regions of China: using the complete mitochondrial genome

Objective Oncomelania hupensis robertsoni is the only intermediate host of Schistosoma japonicum in western China, its genetic differentiation directly impacts the susceptibility of Schistosoma japonicum. This study aimed to sequence the complete mitochondrial genome of Oncomelania hupensis robertsoni Yunnan strain and analyze the genetic differentiation of Oncomelania hupensis robertsoni in hilly regions of China. Methods Samples were from 14 administrative villages in Yunnan Province of China, with 30 Oncomelania hupensis per village, and the complete mitochondrial genome was sequenced. Additional, we retrieved 14 other region Oncomelania hupensis of complete mitochondrial sequences from GenBank, and a comprehensive analysis of the genetic differentiation of Oncomelania hupensis robertsoni was conducted by constructing phylogenetic trees, calculating genetic distances, and analyzing homogeneity. Results A total of 26 complete mitochondrial sequences were determined. The length of genome ranged from 15,181 to 15,187 bp, and the base composition of the genome was A+T (67.5%) and G+C content (32.5%). This genome encoded 37 genes, including 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding region rich in A+T. Using the Philippines genotypes as outgroup, the phylogenetic trees and homology analysis confirmed the existence of two distinct phylogroups, Oncomelania hupensis robertsoni and the remaining 9 provincial genotypes. Oncomelania hupensis robertsoni is subdivided into Oncomelania hupensis robertsoni Yunnan strain and Sichuan strain, with a genetic distance of 0.0834. Oncomelania hupensis robertsoni Yunnan strain is subdivided into two subbranches, “Yunnan North” and “Yunnan South”, with a genetic distance of 0.0216, and the samples exhibited over 97% homology. Conclusion Oncomelania hupensis robertsoni Yunnan strain exhibits a higher level of genetic homology and clear north-south differentiation, the distribution characteristics were closely associated with watershed distribution. This work reported the first mitochondrial genome of Oncomelania hupensis robertsoni Yunnan strain, which could be used as an important reference genome for Oncomelania hupensis, and also provide a theoretical basis for explaining the distribution pattern of Oncomelania hupensis robertsoni and control of schistosomiasis. Author Summary Oncomelania hupensis (O. hupensis) is the only intermediate host of Schistosoma japonicum (S. japonicum), O. hupensis residing in different geographical regions display morphological differences and genetic variations, along with varying susceptibility to S. japonicum. In this study, we sequenced 26 complete mitochondrial genome of O. hupensis robertsoni Yunnan strain (O. h. r. Yunnan strain), the length of genome ranged from 15,181 to 15,187 bp, and the base composition of the genome was A+T (67.5%) and G+C content (32.5%). This genome encoded 37 genes, including 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding region rich in A+T. Additional, we retrieved 14 other region O. hupensis of complete mitochondrial sequences from GenBank. The phylogenetic trees and homology analysis confirmed that O. hupensis robertsoni is subdivided into Yunnan strain and Sichuan strain, and O. h. r. Yunnan strain is subdivided into two subbranches, “Yunnan North” and “Yunnan South”, the samples exhibited over 97% homology. This work reported the first mitochondrial genome of O. h. r. Yunnan strain, which could be used as an important reference genome for O. hupensis, and also provide a molecular biology-based theoretical foundation for understanding the genetic differentiation of O. hupensis.

hupensis primarily inhabits the 12 southern provinces in the middle and lower reaches of the Yangtze River [4], its geographical distribution range is extensive, with notable climate variations and complex array of environmental types [5].O. hupensis residing in different geographical regions display morphological differences and genetic variations, along with varying susceptibility to S. japonicum [4,6,7].Considering the close genetic interaction between S. japonicum and its intermediate host, the O.
hupensis, in terms of coevolution [8,9], and the fact that O. hupensis with higher genetic diversity are more susceptible to S. japonicum [10], conducting research on the genetic differentiation and classification of O. hupensis is of great significance for understanding the transmission of schistosomiasis and guiding disease control measures.
Based on the morphological characteristics, biological traits, and molecular Yunnan strain (O.h.r.Yunnan strain).However, only one study focused on the complete mitochondrial genome of O. hupensis [17], but lacking the Yunnan Province, this merits more research of mitochondrial genome of O. h.r.Yunnan strain attention.
In addition, endemic areas for schistosomiasis in Yunnan Province exhibit a distinct "island-like" pattern [22], prompting many researchers to speculate about the potential existence of on unique genetic characteristics within this regional O. hupensis.

Primer design and PCR amplification
The primers and PCR amplification reaction conditions were based on the literature [24] (Table 1).The entire genome was divided into 16 overlapping fragments according to the sequence length, and the primers were designed separately, except for the reverse primer of primer pair 10 and the primer pair 14, which were not consistent with the literature.The primer synthesis, PCR amplification, PCR product purification and sequence sequencing were performed by Shanghai Xianghong Biotechnology Co.

Sequence assembly and editing
All sequence fragments were filtered to remove those that were not normally reported, such as PCR "amplification failure" and sequencing "bimodal mutation".The SeqMan module of the DNAstar software package was used to splice the 16 sequences of each sample, remove the sequences that could not be assembled into closed circular forms.The overlapping regions of adjacent sequences that could be assembled into closed circular forms were trimmed, and the entire sequence was manually checked for base identification based on peak shape.The assembled and edited sample sequences were submitted to GenBank for sequence alignment and direction confirmation.If the sequence orientations were inconsistent, the reverse complementary sequences of the sample sequences were obtained using the free online tool "SMS2 Nanjing Detai Biological Mirror", and then submitted to GenBank for sequence alignment.Based on the gene order of the nearest mitochondrial sequence (the closest mitochondrial gene sequence to all sample sequences is the SCXC strain of O. hupensis mitochondrion, GenBank ID: JF284691), the sample sequences were trimmed and ordered to have the same starting gene sequence as the COX1 gene in the SCXC strain of O. hupensis mitochondrion.

Sequence alignment
The edited self-test sequences and all complete mitochondrial sequences of O.

Sequence composition analysis
The MEGA-formatted alignment results were imported into MEGA11.0.9 software, and the sequence component analysis was performed using the menu "statistics-Nucleotide Composition".The start and end points of each gene were determined by combining MEGA11.0.9 software with NCBI's BLAST to perform homology-based gene annotation analysis.

Construction of phylogenetic tree
Phylogenetic trees were constructed using MEGA11.0.

Sequence genetic distance and homology analysis
Genetic distances within and between groups were calculated using the "distance" menu in MEGA11.0.9 software based on alignment results.Sequence homology was calculated using the "View-Sequence distance" option in MegAlign of the DNAstar package.The average nucleotide similarity among 40 snail sequences was calculated using Fastani software, homology analysis was performed by comparing genome sequence similarities combined with Blast, and the Average nucleotide identity (ANI) heat map was generated by Tbtools.

Sequence base composition analysis
The length of the complete sequence of O. h.r.

Genetic distance and homology analysis
The average genetic distance within O. h.r.Yunnan strain was 0.0129, while the genetic distance between the northern and southern branches was 0.

Discussions
In recent years, genetic sequence analysis has gained widespread utilization in the fields of phylogenetics and population genetics [25][26][27][28].MtDNA has many advantages over nuclear genes for phylogenetic inference and classification due to its easy amplification with a large number of available conserved primers, lack of recombination, introns, and non-coding sequences, maternal inheritance, absence of recombination, which simplify the complexity of phylogenetic studies [25,[29][30][31][32].
Furthermore, mtDNA evolves rapidly, exhibits high variability within populations, and has high sensitivity for resolving closely related species [33].Currently, mitochondrial gene fragments or mitochondrial genomes are the common markers for analyzing genetic polymorphism and genetic variation of O. hupensis.However, individual gene fragments may no longer provide the necessary level of identification and comprehensive phylogenetic analysis [20,21].While previous studies have reported the mitochondrial genome sequence of O. hupensis and its phylogenetic analysis, they have not conducted a systematic analysis of O. h.r.Yunnan strain, and the number of samples involved in the previous research on the mitochondrial genome was also limited [17].The infection of O. hupensis by S. japonicum cercariae is an important link in the prevalence of schistosomiasis, and its susceptibility determines to some extent the extent of the disease's spread [4].O. hupensis shows different levels of genetic differentiation, leading to varying degrees of susceptibility to S. japonicum [34][35][36].The study herein obtained 26 complete mitochondrial sequences of O. h.r.Yunnan strain, these sequences represent a diverse range of breeding environments characterized by different altitudes, distribution water systems, and environmental types.In this study, the full-length sequences of the 26 mtDNA genomes ranged from 15,181 bp to 15,187 bp, with an average length of 15,185bp, in which the A+T content (67.5%) was significantly higher than the G+C content (32.5%).The mitochondrial genome encoded 37 genes, including 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding region rich in A+T.The gene composition and distribution closely mirrored that of the mitochondrial genome of O. hupensis Gredler [17].
The phylogenetic trees constructed by the MP, ML, ME and NJ methods exhibited consistent topologies, and most of the branch bootstrap values of the four methods were greater than 90, indicating a high degree of reliability [37].Major strengths of this study lie in obtaining the complete mitochondrial genome data of O. h.r.Yunnan strain, and we conducted a comprehensive analysis of the genetic differentiation using constructing phylogenetic trees, calculating genetic distances, and analyzing homogeneity, which ensured a high level of reliability in our results [37].However, there is limitation to this study.Due to extensive schistosomiasis control efforts, the snails in Gejiu city, located in the southern part of Yunnan Province, have been eliminated [41].Consequently, during O. hupensis sampling for this study, we were unable to collect O. hupensis from this region.Gejiu city was relatively isolated within the previous endemic regions of schistosomiasis in Yunnan Province, the genetic differentiation analysis of O. hupensis in this area was not included, which may introduce some limitations to our findings.

Conclusion
In summary, our study successfully obtained the complete mitochondrial genome of O. h.r.Yunnan strain and conducted a comprehensive analysis of its mitochondrial genome phylogeny.Our results indicate that O. h.r.Yunnan strain exhibits a higher level of genetic homology and clear north-south differentiation.Notably, O. h.r.
Yunnan strain does not exhibit distinct "island" characteristics but instead shows a close association with watershed distribution.Taken together, the findings provide a molecular biological basis for the study of genetic evolution and resistance genes of O.
hupensis.Additionally, it may provide useful clues for the development of snail-killing drugs, and serve as a theoretical basis and scientific evidence for the control of schistosomiasis, particularly with regard to snail control.

1
Introduction Oncomelania hupensis (O.hupensis) is the only intermediate host of Schistosoma japonicum (S. japonicum), and its distribution area directly determines the epidemic range of schistosomiasis[1].Despite differences in morphology among O. hupensis from different countries and regions, they can still be classified as the same species based on their genetic characteristics of reproductive isolation and chromosomal homology[2, 3].While it's currently recognized that there are eight subspecies of O. hupensis, the classification of these subspecies has been controversial due to factors such as identification methods and sample sources.Nevertheless, the classification of O. hupensis robertsoni (O.h.robertsoni) remains relatively well-defined.In China, O.
0216.The average genetic distance between the O. hupensis of Yunnan strain and Sichuan strain was 0.0834.The average genetic distance between the O. hupensis Gredler (Zhejiang, Guangdong, Jiangsu, Anhui, Hunan, Hubei, Jiangxi Provinces) was 0.0216.The average genetic distance between the O.h. robertsoni and O.hupensis Gredler was 0.113.The average genetic distance among provinces of the O.hupensis Gredler, Fujian, Guangxi was 0.0291.Some of the clustering information in the phylogenetic tree can be visualized in the ANI heat map (Figure 7 ANI heat map of mitochondrial sequence of O.hupensis), as well as the degree of homology among the samples.As shown in figure6, the homology among the samples of O. h.r.Yunnan strain was above 97%, forming an orange rectangle, which contained two small red rectangles, namely "North Yunnan branch" and "South Yunnan branch".The other subspecies of O. hupensis, except for the O. h.robertsoni and Philippine O. hupensis, formed a rectangle in the lower right corner.

Figure 1 Distribution of sampling points of O. hupensis in Yunnan Province of China). 14
The study herein aimed to sequence the complete mitochondrial genome of the O. h.r.Yunnan strain, and to explore intraspecific developmental relationships of the O. h.robertsoni and the differentiation characteristics of O. h.r.Yunnan strain.Our results

Figure 2 Representative genome map representing the mitochondrial genome circular molecule of O. h. r. Yunnan strain). 3.3 Phylogenetic tree construction
located in the Jinsha River Watershed in northern Yunnan Province.The South branch included samples from ten administrative villages in seven counties of Dali, Weishan, region rich in A+T.Detailed information about the organization of O. h.r.Yunnan strain mitochondrial genome in 13 sampling sites are presented eTable1~eTable9 in the supplement.The topology of the phylogenetic tree constructed by the four methods of MP, ML, ME, and NJ (Figures 3, 4, 5 and 6 Phylogenetic trees constructed by MP, ML, ME, and NJ method, respectively) were all largely consistent.The genotype from the Philippines was used as the outgroup, and the other sequences clustered into 2 major branches: one composed of genotypes from Yunnan and Sichuan provinces, and the other including genotypes from nine other provinces.The branch containing Sichuanand Yunnan genotypes was further divided into two sub-branches, named "Sichuan branch" and "Yunnan branch", respectively.The remaining nine provincial branches were divided into three sub-branches originating from Fujian, Guangxi, and the other seven provinces, respectively.The bootstrap values of all these branches were greater than 70, with most exceeding 90.According to the results of phylogenetic tree, the O. h.r.Yunnan strain were divided into North branch and South branch.The North branch included samples from four administrative villages in three counties of Yongsheng, Gucheng, and Heqing, [39,40]]tering of Yunnan and Sichuan O. hupensis into one large branch, supporting the previous classification of the O. h.robertsoni[2,4,38].In addition, the Yunnan and Sichuan populations formed two distinct branches, showing clear differentiation.It's worth noting that the genetic distance between Yunnan and Sichuan populations was significantly greater than that observed between other subspecies of O. hupensis, but the genetic distance between other subspecies of O. hupensis was narrower, reflecting higher homology and less pronounced differentiation.These results align with those of previous studies by Han et al. and Bai, which examined the COX1 gene of O. hupensis, while the bootstrap values of some branches in the phylogenetic trees constructed by these two researchers were relatively small[39,40].Phylogenetic tree analysis showed that O. h.r.Yunnan strain could be subdivided into two subgroups, namely the "North Yunnan Branch" and "South Yunnan Branch".However, when considering the ANI heat map and genetic distance, the average genetic distance within the O. h.r.Yunnan strain was 0.0129, indicating a high degree of homogeneity and limited differentiation.Although the O. h.r.Yunnan strain is divided into two small subgroups, genetic differentiation between these subgroups is not significant, as evidenced by a genetic distance of 0.0216, which is similar to the average genetic distance observed between provinces of O. hupensis Gredler in this study.O. of the river watershed.Notably, the O. hupensis in the "North Yunnan" subgroup exclusively occupies the Jinsha River watershed, while the remaining sampling points are distributed across the other three river watershed, with the exception of Wuxing Village in Dali City, located on the southern edge of the Jinsha River Watershed.Although three river watersheds are separated by obvious geographical barriers, samples from each watershed cluster together in the same branch of the phylogenetic tree, indicating a high degree of homogeneity and no apparent differentiation based on the distribution of river watershed.The divergence between the northern and southern lineages of Yunnan snails may result from either unique natural factor in the Jinsha River Watershed or geographic isolation due to the significant distance separating the two lineages.Further research is required to determine the exact cause of this divergence.In addition, Wuxing Village in Dali City, situated within the Jinsha River Watershed, grouped with the "South Yunnan" lineage on the phylogenetic tree.This deviation may be due to its position on the edge of the Jinsha River Watershed, making it more susceptible to gene flow with other snail populations in Dali that are province were included in this study, and further research with larger sample sizes is needed to verify the actual differentiation status of O. hupensis in Sichuan Province.
[22]acteristics in O. h.r.Yunnan strain[22], but according to the data in this study, there isn't a clear "island" feature.Instead, these seems to be a correlation with the distribution pattern the Philippine O. hupensis, this suggested a lower degree of homogeneity and more pronounced differentiation.However, only two geographical strains of O. hupensis from Sichuan