Study on the population evolution of Ascaris lumbricoides and Ascaris suum based on whole genome resequencing

Journal Pre-proof Study on the Population Evolution of Ascaris lumbricoides and Ascaris suum based on Whole Genome Resequencing Chunhua Zhou (Conceptualization) (Writing - original draft) (Writing - review and editing) (Project administration), Jinping Chen (Software) (Formal analysis)Data Curation), Hongyan Niu (Methodology) (Validation) (Investigation) (Writing original draft), Shan Ouyang (Visualization) (Supervision), Xiaoping Wu (Resources) (Funding acquisition)

PII:

S0304-4017(20)30042-X

DOI:

https://doi.org/10.1016/j.vetpar.2020.109062

Reference:

VETPAR 109062

To appear in:

Veterinary Parasitology

Received Date:

22 October 2019

Revised Date:

21 February 2020

Accepted Date:

24 February 2020

Please cite this article as: Zhou C, Chen J, Niu H, Ouyang S, Wu X, Study on the Population Evolution of Ascaris lumbricoides and Ascaris suum based on Whole Genome Resequencing, Veterinary Parasitology (2020), doi: https://doi.org/10.1016/j.vetpar.2020.109062

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Study on the Population Evolution of Ascaris lumbricoides and Ascaris suum based on Whole Genome Resequencing Chunhua Zhou* [email protected], Jinping Chen, Hongyan Niu, Shan Ouyang, Xiaoping Wu* [email protected]

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School of Life Science, Nanchang University, Nanchang 330031, People’s Republic of China *Corresponding author.

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Highlights

The whole genomes of two Ascaris populations were resequenced.

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The A. lumbricoides population was more primitive than the A. suum population.

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Two KEGG pathways but no GO terms were enriched in the A. lumbricoides population.

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Five GO entries and one KEGG pathway were enriched in the A. suum population.

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Ascaris population sizes peaked about 1 million years ago and then declined.

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Abstract

Ascaris lumbricoides and Ascaris suum are parasitic nematodes that mainly parasitize the small intestines of people and pigs, respectively. Ascariasis seriously endangers human health and causes huge economic losses in the pig industry. A. lumbricoides and A. suum have similar morphologies and genetic structures, and occasionally these organisms cross-infect the alternate host. Therefore, their taxonomies are controversial. In this study, the whole genomes of A. lumbricoides (n = 6) and A. suum (n = 6) were 1

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resequenced using a HiSeq X Ten sequencing platform. Phylogenetic, principal component, and population structure analyses showed clear genetic differentiation between the two Ascaris populations. Linkage disequilibrium analysis indicated that the A. lumbricoides population was more primitive than the A. suum population. In the selective elimination analysis, 160 and 139 candidate regions were screened in A. lumbricoides and A. suum, respectively, and the selected regions were analyzed by Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The A. lumbricoides population had no significant enrichment in GO terms, but two KEGG pathways, the RNA degradation and tyrosine metabolism pathways, were significantly enriched. Five GO entries and one KEGG pathway, the alanine, aspartate, and glutamate metabolism signaling pathway, were significantly enriched in the A. suum population. An analysis of the demographic histories of Ascaris populations revealed that A. lumbricoides and A. suum had similar trends in effective population size in different historical periods. Ascaris populations peaked about 1 million years ago and then began to decline. In the last glacial period, they dropped to a historical low and continued at this level until the last glacial maximum. This phenomenon may be associated with the cold climate at that time. This study provides new information on the genetic differentiation, evolutionary relationships, gene functional enrichment, and population dynamics of Ascaris populations, with implications for host differences, evolution, and classification of A. lumbricoides and A. suum.

1. Introduction

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Keywords: genome resequencing; Ascaris lumbricoides; Ascaris suum; population history

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Ascaris lumbricoides is one of the most prevalent parasites in the world. Ascaris suum is the most common parasitic infection in pigs, leading to huge economic losses in the swine industry. The taxonomic statuses of the two species have long been controversial because of the difficulty in distinguishing between their morphologies and frequent reports of cross-infections from one host to the other. There have been four views in academia. The first is that these are two different species with a possible common ancestor (perhaps before pig domestication) (Leles et al., 2012). The second view holds that pig Ascaris was the ancestor of human Ascaris, which emerged in humans due to host transfers in different regions (Peng et al., 1998a). The third view, which contrasts with the second, suggests that human Ascaris was the ancestor of pig Ascaris (Loreille et al., 2003; Zhou et al., 2011). The fourth idea is that A. lumbricoides and A. suum are the same species because of morphological similarities, low genetic distance between them, and microRNA profiles, but their host specificity and the genetic evidence do not support this view (Peng et al., 2003, 2005, 2006; Liu et al., 2012; Shao et al., 2014). It 2

will take time to resolve this uncertainty, with contributions from molecular ecology, population genetics, parasite archaeology of parasite, and other fields. However, the classification status of the two is directly related to control of Ascaris infection. One practical question is whether Ascaris infection in local pigs must be controlled at the same time as Ascaris infections in humans, and vice versa, in the endemic areas where both coexist. Further study of these organisms is necessary, because ascariasis is recognized as a neglected parasitic disease (Holland et al., 2013).

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In the past 20 years, with the development of modern molecular biology and genetics, especially the identification of various molecular markers of DNA polymorphisms combined with modern data analysis methods, there was new thinking on the relationship between A. lumbricoides and A. suum (Peng et al., 2012). A. lumbricoides and A. suum are generally believed to be two genetically independent populations. According to different genetic markers such as ITS-1, the G1 type represented humanderived Ascaris, and the G3 type represented pig-derived Ascaris (Peng et al., 2003). In terms of mitochondrial DNA (mtDNA) markers, human Ascaris is mainly H9 type, whereas pig-derived Ascaris is mainly P1 and P5 types (Peng et al., 2005). In the case of microsatellite markers, pure human-type Ascaris accounted for most human-derived Ascaris, and pure pig Ascaris accounted for the overwhelming majority of pig-derived Ascaris (Criscione et al., 2007; Zhou et al., 2012). Human-derived and pig-derived Ascaris are believed to have relatively independent transmission cycles due to genetic separation (Anderson et al., 1997; Peng et al., 1998b, 2007). However, the same genotypes were found among human-derived and pig-derived Ascaris, based on nucleic acid electrophoresis band patterns (Peng et al., 1998b, 2007; Anderson et al., 1993), type G2 prevalences (about 20%) (Peng et al., 2003), and identical H9 and P9 sequences (Peng et al., 2005). This suggested the possibility of cross-infection or even hybridization. Indeed, human infections of A. suum were reported in North America (Anderson 1995), Denmark (Nejsum et al., 2005), and Japan (Arizono et al., 2010), confirming the existence of cross-infections between A. lumbricoides and A. suum and their hosts. In non-epidemic areas of A. lumbricoides infection, such as North America, Denmark, and Japan, Ascaris infections in humans are mainly due to pig-derived Ascaris. Recently, in epidemic areas where both human and pig Ascaris infections occur, cross-infection has been noted. Peng et al. (2006, 2007) infected animals with known genotypes of Ascaris. The host specificity of different genotypes of Ascaris (mainly G1 of human Ascaris and G3 of pig Ascaris) and its mechanism were examined, and the results suggested that A. lumbricoides did not easily parasitize and develop into adults in pigs, and that the characteristics of Ascaris from the two sources were different in early-stage infection and host immune response (Peng et al., 2006, 2007). Danish scientists also infected pigs with pig-derived Ascaris of different mtDNA genotypes and discovered that different genotypes of Ascaris had different infection characteristics 3

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(Nejsum et al., 2009). Hybrid A. lumbricoides-A. suum individuals were reported for the first time by Criscione et al. (Criscione et al., 2007). These individuals were produced by Ascaris cross-infections, with the first generation of immigrants entering non-permanent hosts and mating with Ascaris in permanent hosts. Subsequently, Zhou et al. (2012) used microsatellite markers and nuclear gene sequences to explore the frequency of cross-infection and hybrid individuals in China and their distribution in human and pig hosts and in northern and southern regions, and different genotypes (G1–G3) (Zhou et al., 2012). The results indicated that pig Ascaris had become an important source of human ascariasis in a developing country (Zhou et al., 2012). This was true also in developed countries (Beston et al., 2014). This progress in resolving the taxonomic uncertainty of these species is largely due to the application of multiple molecular genetic markers and the use of new molecular markers in epidemiological and animal studies.

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The rapid development of high-throughput sequencing technologies has revolutionized nematode genome research. The ability to predict proteins encoded in the nematode genome has been conducive to the rapid identification of biomolecules related to pathology. Abad et al. (2008) screened specific secretory proteins by comparing Ascaris genomes to that of Meloidogyne incognita, and most secretory proteins were found to be related to the parasitic life of nematodes (Abad et al., 2008). Fosu-Nyarko et al. (2016) screened 500 factors related to beet cyst nematode parasitism by highthroughput sequencing (Fosu-Nyarko et al., 2016). In addition, also based on highthroughput sequencing, some parasitic helminths have been found to cause host lesions. For example, an exudate of Schistosoma haematobium can cause tumors in its host (Young et al., 2012), and granular protein growth factor in Clonorchis sinensis can induce cholangiocarcinoma in human hosts under specific circumstances (Wang et al., 2011). Moreover, many sites related to metabolism, growth, reproduction, and immune evasion were identified by whole genome sequencing (Zheng et al., 2013; Zhou et al., 2009). The premise of genome resequencing is that genomes of the species are known, but that variation in different individuals or populations of species are analyzed to identify a large number of variable sites (e.g., SNP, InDel, SV, and CNV). Resequencing lays a foundation for subsequent genetic diversity analyses, selective elimination, and functional gene mining (Stratton, 2008). Resequencing technology has great potential to be applied to helminth evolution, transmission, invasion, drug resistance, and immune escape (Wit et al., 2017).

In this study, differences in A. lumbricoides and A. suum populations were evaluated at the whole-genome level for the first time. Genomic differences between the two Ascaris populations were elucidated, selected regions of A. lumbricoides and A. suum were screened, and candidate parasitism-related regions were determined. This approach 4

provides new evidence to clarify the taxonomies of A. lumbricoides and A. suum.

2. Material and methods 2.1. Sample collection and genomic DNA extraction

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Ascaris lumbricoides (n = 6) and Ascaris suum (n = 6) were collected from people and pigs in Xinjian County, Jiangxi Province, China. The samples were stored at –80℃. Genomic DNA was extracted using a Wizard SV Genomic DNA Purification System (Promega). Femal Ascaris might contain fertilized eggs, which can cause spurious genotypes (Anderson et al., 2003). The uteruses of female Ascaris were removed to prevent contamination by male sperm during DNA extraction. Identification of A. lumbricoides and A. suum was carried out using microsatellite and ITS markers combined with Hae III enzyme digestion (Zhou et al., 2012; Zhu et al., 1999). 2.2. Genome resequencing and variant detection

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The purified DNA samples were segmented into approximately 350 bp. Then, the segmented sequences were repaired and spliced, and the genomic library was constructed by PCR amplification. The whole genomes of A. lumbricoides and A. suum were resequenced on an Illumina HiSeq X Ten sequencing platform. The sequencing depth of each sample was 30×. High-quality sequencing data were compared with the Ascaris suum reference genome using BWA software (http://parasite.wormbase.org/Ascaris_suum_prjna62057/Info/Index/), and the sequencing depth and coverage of each sample was determined. Duplicates in the alignment results were removed by SAMTOOLS. Clean data were identified and screened for single nucleotide polymorphisms (SNPs). SNPs below Q20 and less than 5 bp with an interval between adjacent sites were removed. The coverage depth of SNPs was between one-third and 10× the average depth. Then, the filtered SNPs were functionally annotated using ANNOVAR software.

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2.3. Population structure analysis

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Based on SNPs detected in a whole gene analysis, a distance matrix was calculated in TreeBeST 1.9.2. The evolutionary tree was constructed using the adjacent method. Principal component analysis (PCA) analysis was carried out using EIGENSOFT software, and the population clustering analysis was conducted in PLINK. 2.4. Linkage disequilibrium analysis Linkage disequilibrium (LD) analysis was performed using the linkage disequilibrium coefficient (R2) and SNP intervals to construct a coordinate diagram. 2.5. Selective elimination analysis The selective elimination analysis was executed by constructing a frequency 5

distribution map based on Fst and π of high-quality SNP loci. Fst and π was an effective method to detect selective regions. When excavating functional area closely related to the living environment, it can often detect strong selection signals. Fst and π were analyzed using vcftools software with a window size of 40 kb and step size of 20 kb. 2.6. Gene functional enrichment analysis

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Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted for the selected areas and filtered on the basis of p-values. The selected regions and GO entries in the GO database were compared during the GO enrichment analysis. The number of genes enriched for different entries was counted, and then hypergeometric distribution was tested to determine GO entries significantly enriched in candidate regions. Similarly, the KEGG analysis was conducted using the pathway as the test unit, and significantly enriched pathways were obtained. In all categories, enrichment items with p values less than 0.05 after correction were selected for further analysis. 2.7. Historical dynamic analysis of populations

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The size of effective populations was estimated by determining the linkage disequilibrium between SNPs. The size of effective populations in different historical periods was calculated using the pairwise sequentially Markovian coalescent (PSMC) method (Li and Durbin, 2011).

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3. Results 3.1. Sequencing data statistics

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Raw data (137.745 Gb) were obtained by resequencing the whole genomes of A. lumbricoides and A. suum. After strict filtration, a total of 134.085 Gb of high-quality (Q20 ≥ 97%, Q30 ≥ 91%) data were obtained. The GC distribution, as well as other indexes, were normal. Database construction was successful. The sequencing data for each sample is shown in Supplementary Table 1. High-quality sequences were compared to the A. suum reference genome (273M) using BWA software. After removing repetitions, the alignment rate was more than 80% (Supplementary Table 2). The average sequencing depth of the genome was 24.78 bases (only those with a comparison mass greater than 0). Coverage of at least one base was more than 88%, and coverage of at least 4 bases was more than 86%. The sequencing sequence alignment rate was high for each Ascaris population. All sequences had very good depth and coverage. Comparison information is shown in Supplementary Table 2.

3.2. Mutation detection A total of 10.79 million high-quality SNPs were obtained after quality control of highquality data from Ascaris populations using SAMTOOLS software. There were 4.26 million SNPs in the A. lumbricoides population and 6.53 million SNPs in the A. suum 6

population. The average number of SNPs detected per sample was 0.9 million. The distribution of SNPs on chromosomes of Ascaris in the two populations is shown in Table 1. Most of the mutation sites were located in non-coding regions such as intronic, intergenic, and regulatory regions. A. suum and A. lumbricoides had 2,223,890 (22.9%) and 3,393,616 (52.2%) intronic SNPs and 975,525 (22.3%) and 148,1945 (52.0%) intergenic SNPs, respectively. In the coding region, there were 93,646 (2.2%) and 14,8410 (2.3%) SNPs, respectively. The number of nonsynonymous mutations was significantly higher than that of synonymous mutations. In addition, the ts/tv ratios of SNP mutation sites in A. suum and A. lumbricoides population were 2.379 (ts: 3000064, tv: 1260803) and 2.378 (ts: 4593765, tv: 1931313), respectively.

3.3. Population structure analyses

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Population structure analyses based on SNPs were conducted in TreeBeST 1.9.2, EIGENSOFT and PLINK. A neighbor-joining phylogenetic tree showed that Ascaris populations divided into two clades (Figure 1a). The A. suum population clustered in one branch, and the A. lumbricoides population belonged to the other branch. Principal component analysis can separate individuals with different genetic backgrounds and bring together individuals with similar genetic backgrounds. In this study, the three most important feature vectors PC1, PC2, and PC3, were plotted. The results indicated that A. suum and A. lumbricoides population were clearly distinguishable. However, individuals in the A. suum population clustered together, and the A. lumbricoides population was relatively dispersed (Figure 1b). The results of the cluster analysis showed that the two Ascaris populations could be separated when K = 2. When K = 3, two colors appeared in the A. suum population, but it was still distinguishable from the A. lumbricoides population. Although there were two different colors in the A. suum and A. lumbricoides populations, the two populations did not exist as a mixture when K = 4.

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3.4. Linkage disequilibrium analysis

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Linkage disequilibrium (LD) values are usually shown as R2 (a linkage disequilibrium coefficient). A larger R2 represents a stronger linkage relationship and smaller corresponding SNP spacing. The value of linkage disequilibrium decay (LD-decay) is represented by corresponding SNP spacing when R2 decays to half. The slower the linkage disequilibrium decays, the longer the corresponding LD-decay, which indicates a stronger linkage relationship between SNPs in the two species and lower genetic diversity. Figure 2 shows that the attenuation distances in the two populations were very different. LD-decay in the A. lumbricoides population decreased faster, and the LDdecay in the A. suum population was longer. Compared with the A. lumbricoides population, the LD-decay in the A. suum population was slower and the probability of linkage between SNPs was higher. 3.5. Selective elimination analysis Regions with significantly higher Fst values (Fst > 0.441) and higher log2 (PiP/PiH) 7

values (π ratio > 0.590) were selected and screened for strong selection signals. A total of 160 selected regions were obtained from the A. lumbricoides population. For the A. suum population, regions with Fst > 0.441 and log2 (PiH/PiP) > 1.510 were screened, and 139 regions were selected. Compared with the whole genome sites, the candidate regions had higher Fst and π values. The results of the Fst and π selective elimination analysis of A. lumbricoides and A. suum are shown in Figure 3. 3.6. Gene functional enrichment analysis

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The results of the GO analysis of the A. suum population showed that 2171 GO entries were enriched in the candidate region, and a total of 283 candidate genes were annotated (Figure 4a). The candidate genes were mainly enriched in molecular functions and biological processes. Anatomical structure morphogenesis (19 genes) and anatomical structure formation involved in morphogenesis (10 genes) were enriched in candidate genes. Both of these were biological processes. Five GO entries were significantly enriched, namely three biological processes (inositol biosynthetic process [GO:0006021, 4 genes], polyol biosynthetic process [GO:0046173, 4 genes], and transcription initiation from RNA polymerase III promoter [GO:0006384, 4 genes] and two molecular functions (intramolecular lyase activity [GO:0016872, 6 genes] and inositol-3-phosphate synthase activity [GO:0004512, 4 genes].

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The population of A. lumbricoides was enriched in 2001 GO entries, but none of these entries were significantly enriched. The 278 candidate genes were annotated as enriched in molecular functions, biological processes, and cell components. Cell components, including membrane parts (128 genes), integral to membranes (109 genes), and intrinsic to membranes (109 genes), accounted for most enriched candidate genes (Figure 4b).

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To better understand the signal pathway of candidate genes in the two Ascaris populations, the screened candidate genes were subjected to KEGG pathway analysis. A total of 14 pathways were enriched in the A. suum population (Figure 5a). Only one pathway (alanine, aspartate, and glutamate metabolism, 3 genes) was significantly enriched. The signaling pathway with the most enriched genes was the metabolic pathway (6 genes). A total of 12 pathways were enriched in the A. lumbricoides population (Figure 5b). Two pathways were significantly enriched, namely the RNA degradation (2 genes) and tyrosine metabolism (1 gene) pathways. The RNA degradation showed high enrichment for many genes.

3.7. Population history Variations in the effective population sizes of A. lumbricoides and A. suum populations during different historical periods were estimated using PSMC software (Figure 6). According to the simulation trajectory of the effective populations, trends in changes of the effective population sizes of the two Ascaris populations were the same through the passage of time. However, the effective population changed greatly from 100 8

thousand to 2 million years ago. As a whole, the effective A. suum population was lower than that of A. lumbricoides. Ascaris ancestors began to expand 2 million years ago and reached a peak 1 million years ago. Then, the population declined with a bottleneck period and again 100 thousand years ago. The population dropped to an all-time low at around 60,000 years ago and continued to do so until 10,000 years ago.

4. Discussion

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During evolution of a long period, many species experience strong positive selection. These selected areas exhibit selective elimination signals and lead to the decline of SNPs and increase in LD. The LD analysis in this study showed slower attenuation of LD in the A. suum population and a relatively large linkage probability between SNPs. Domesticated populations usually show longer LDs than wild populations because of positive selection. Thus, species with faster LD attenuation are more primitive (Guo et al., 2011; Xia et al., 2015). Compared with the LD value of the A. suum population, the LD value of the A. lumbricoides population was lower, suggesting a lower probability of SNP linkages and the A. lumbricoides population is more primitive. Here, we verified the hypothesis that the ancestors of A. suum could have been human A. lumbricoides (Zhou et al., 2011).

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In the phylogenetic tree based on the genetic distance between SNPs, A. lumbricoides and A. suum populations were on separate branches, showing obvious genetic differentiation. The results of clustering based on three eigenvectors in the principal component analysis indicated that the Ascaris populations were still divided into two independent groups, although one A. lumbricoides individual was quite different from the other individuals in that population. All individuals in the A. suum population clustered together. These results were consistent with those of the phylogenetic analysis. The population structure analysis showed that A. lumbricoides and A. suum populations are distinguishable at K = 2. The population of A. suum showed internal differentiation at K = 3 and 4, and individual H 24 crossed with other individuals in A. lumbricoides population at K = 4. This phenomenon may be the result of gene exchange. The results of the population structure analysis were consistent with those of the phylogenetic and principal component analyses. All these analyses showed that there was significant genetic differentiation between the A. lumbricoides and A. suum populations. These findings agree with those from studies based on nuclear genes and mitochondrial DNA fragments from sympatric A. lumbricoides and A. suum populations (Anderson et al., 1993, 1998; Peng et al., 1998b, 2005). Molecular epidemiological studies on Ascaris from humans and pigs from all over the world using microsatellite and mitochondrial markers also showed significant genetic differentiation between the two Ascaris populations (Beston et al., 2014).

This genome-level study of two Ascaris populations was helpful to identifying proteins 9

that play important roles in the physiological function of Ascaris. In this study, there were five GO entries significantly enriched in the A. suum population, among which intramolecular lytic enzyme (GO:0016872) and inositol-3-phosphate synthase (GO:0004512) activities were enriched biological processes related to biological macromolecules in Ascaris. They might be involved in parasite immune maintenance in A. suum. The A. lumbricoides population was not significantly enriched in GO entries, but the G protein-coupled receptor signal pathway (GO:0007186) with a large number of genes enriched in biological processes might be a drug target site in A. lumbricoides. The G protein-coupled receptor signal pathway is associated with many voltage-gated channels, which are known targets of deworming drugs such as high-efficiency macrolide and levamisole nematicides combined with ligand-gated ion channels (Kaminsky et al., 2008).

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KEGG pathway analysis of the screened candidate regions revealed that the alanine, aspartate, and glutamate metabolism signal pathway enriched in the A. suum population might be related to immune evasion and parasite maintenance. The enzyme content in the pathway increases after exposure to deworming drugs (Wang et al., 2008). As an important component of protein metabolism, glutamate is involved in many chemical reactions in organisms. It is one of the classical neurotransmitters in nematodes (Yang et al., 2017a), an excitatory neurotransmitter in animals (Cleland, 1996), and an inhibitory neurotransmitter in invertebrates (Gration et al., 1979; Shoop et al., 1995). When glutamate binds to postsynaptic receptors in invertebrates, glutamate chloride channels open (Cully et al., 1996). These channels are part of an inhibitory ion transmembrane protein complex that includes subunits α and β. The α subunit is mainly involved in depolarization of nerve muscle cells. The β subunit is primarily related to the gating. Moreover, the chloride channel has only been found in invertebrates such as Caenorhabditis elegans and Haemonchus contortus. It is a target for anthelmintic drugs, including macrolides such as ivermectin (Greenberg et al., 2014). Therefore, candidate genes enriched in this pathway may encode target sites for deworming drugs. The signal pathways significantly enriched by KEGG analysis in the A. lumbricoides population were RNA degradation and tyrosine metabolism, which is associated with amino acid metabolism in parasitic worms. The amino acid metabolism pathway was also enriched in the A. suum population. Studies have shown that amino acid metabolism is related to the growth and development of nematode parasites (Yang et al., 2017b). The cortex of the parasite plays an important protective role in their specific living environment. These selected areas probably contributed to immune avoidance and parasite maintenance. A signal pathway analysis of the two Ascaris populations indicated that they had common parasite-related amino acid metabolism pathways, but that there were differences in other specific pathways. It also showed that Ascaris from pigs and humans were host specific (Peng et al., 2006). Therefore, we speculated that alanine, aspartate, and glutamate metabolism in A. suum and tyrosine metabolism in A. lumbricoides are associated with host specificity. The historical dynamic analysis revealed that there was a large amplification of the 10

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effective population of the two Ascaris species during the interglacial period. Compared with the glacial epoch, the global temperature of the interglacial period was rebounding. During the Triassic period to interglacial period (about 200 million to 2 million years ago), the Ascaris population expanded. Because of melting glaciers, the movement of biological communities and climate zones toward high latitudes was easy, leading to the rapid growth and propagation of Ascaris populations. After peaking at 1 million years ago, the Ascaris populations began to decline; this coincided with the Pleistocene glacial period, including the Naynayxungla glaciation (780,000 to 500,000 years ago) (Ehlers et al., 2007) and second-to-last glaciation (300,000 to 130,000 years ago) (Zhao et al., 2013). These two periods were the two longest ice ages in the Pleistocene, and there was a significant contraction of the population in both periods. With the advent of the last glacial period (110,000 years to 12,000 years ago), the Ascaris population size decreased to its lowest level (Nadachdownska-Brzyska et al., 2015). In the last glacial maximum (Zhao et al., 2013) (3.2 million to 1.5 million years ago), when the temperature dropped to its lowest, the population size was at a lower level than that in the interglacial period. The Ascaris population began to decay rapidly in the harsh cold environment.

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Conclusions

Credit Author Statement:

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Genome resequencing of A. lumbricoides and A. suum populations was first carried out by high-throughput sequencing technology in the present study. New information that had not been shown or fully verified before was mined, including evolutionary relationships, gene function enrichment, and population history dynamics. Results of this study have implications for the evolution and classification of A. lumbricoides and A. suum and are of great significance to the prevention and control of ascariasis.

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Chunhua Zhou: Conceptualization, Writing-Original Draft, Writing-Reviewing and Editing, Project administration.

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Jinping Chen: Software, Formal analysis, Data Curation. Hongyan Niu: Methodology, Validation, Investigation, Writing-Original Draft.

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Shan Ouyang: Visualization, Supervision. Xiaoping Wu: Resources, Funding acquisition. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Conflict of interest: All authors declare no conflict of interest. 11

Acknowledgements This work was funded by the National Natural Science Foundation of China (81460318). Thanks to Professor Weidong Peng at Nanchang University, China, for his help in data analysis. Thanks also to Dr. Tao Wang at the University of Melbourne, Australia, for kindly providing reference data.

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Zhou, Y., Zheng, H., Chen, Y., et al., 2009. The Schistosoma japonicum genome reveals features of host-parasite interplay. Nature 460, 345-351. Zhou, C., Li, M., Yuan, K., Hu, N., Peng, W., 2011. Phylogeography of Ascaris lumbricoides and A. suum from China. Parasitol. Res. 109, 329-338. Zhou, C., Li, M., Yuan, K., Deng, S., Peng, W., 2012. Pig Ascaris: An important source of human ascariasis in China. Infect. Genet. Evol. 12, 1172-1177. Zhu, X., Chilton, N.B., Jacobs, D.E., Boes, J., Gasser, R.B., 1999. Characterisation of Ascaris from human and pig hosts by nuclear ribosomal DNA sequences. Int. J. Parasit. 29, 469-478.

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Figure 1: Population structure. (a) NJ tree of sequences from all samples. (b) PCA analysis of A. lumbricoides and A. suum populations. (c) Genetic structure of the two Ascaris populations.

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Figure 2: Linkage disequilibrium (LD) analysis of A. lumbricoides and A. suum populations. The transverse coordinates represent SNP spacing, and the longitudinal coordinates represent the linkage disequilibrium coefficient.

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Figure 3: Selective sweep analysis of A. lumbricoides and A. suum populations. The transverse coordinates were π ratios and the longitudinal coordinates were Fst values, which corresponded to the left and right frequency distribution maps, respectively. The dot plot in the middle represents the corresponding Fst values and π ratios in different windows. The red region indicates the top 5% of regions selected by π and Fst. The area below the black curve indicates the enriched area.

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Figure 4: The most enriched gene ontology (GO) terms in the (a) A. suum and (b) A. lumbricoides populations.

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Note: * indicates significant enrichment of GO terms.

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Figure 5: Statistical analysis of KEGG pathway enrichment in (a) A. suum population and (b) A. lumbricoides populations. Smaller p-values indicate more significant enrichment and red circles indicate significantly enriched pathways. The size of the circle indicates the number of genes. 18

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Figure 6: Demographic history of Ascaris populations.

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Note: The horizontal axis represents different historical periods, and the vertical axis represents the effective Ascaris population size. In the figure, the generation period is 90 days, and the mutation rate of each generation is 1.6 × 10–9.

A. suum population

A. lumbricoides population

155472

233768

2069

3398

546

891

Synonymous

33101

51596

Non-synonymous

57930

92525

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Table 1. SNP distribution in A. suum and A. lumbricoides chromosomes.

Intronic

2223890

3393616

Splicing

1961

3427

Downstream

125662

190270

Upstream/Downstream

27156

40976

Intergenic

975525

1481945

ts

3000064

4593765

tv

1260803

1931313

ts/tv

2.379

2.378

Total

4260867

6525078

Upstream Stop gain

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Stop loss

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Upstream: Variants are located in the 1-Kb region upstream of the gene; Stop gain: Variants introduce a stop codon; Stop loss: Variants cause the loss of a stop codon; Synonymous: Synonymous variants; Nonsynonymous: Non-synonymous variants; Intronic: Variants are located in an intronic region; Splicing: Variants are within 2 bp of a splice junction; Downstream: Variants are located in the 1-Kb region downstream of the gene; Upstream/Downstream: Variants are located in the 1-Kb regions upstream and downstream of two different genes; Intergenic: Variants are located in an intergenic region; ts: Conversion; tv: Transversion; Total: Total number of SNPs.

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