Introduction
Ascaris lumbricoides is a parasitic worm that infects the human intestine, leading to a condition known as ascariasis. It is recognized as one of the Neglected Tropical Diseases and is currently targeted in global eradication initiatives. This parasite is estimated to infect around 1.2 billion people across the globe, with children in Asia, sub-Saharan Africa, and parts of Latin America being the most affected populations (Brooker et al. Reference Brooker, Nikolay, Balabanova and Pullan2015; Mitra et al. Reference Mitra and Mawson2017; WHO 2023). In humans, A. lumbricoides infections can become severe and even life-threatening when the number of parasites is high. Such heavy infections may lead to complications like intestinal blockages, ruptures, and the movement of adult worms into the bile ducts or other organs (Umetsu et al. Reference Umetsu, Sogo, Iwasawa, Kondo, Tsunoda, Oikawa-Kawamoto, Komatsu, Inui and Fujisawa2014). Long-term infections can also contribute to stunted physical growth and impaired cognitive development in children (Blouin et al. Reference Blouin, Casapia, Joseph and Gyorkos2018; Moncayo et al. Reference Moncayo, Lovato and Cooper2018; Wang et al. Reference Wang and Davis2020; Holland et al. Reference Holland, Sepidarkish, Deslyper, Abdollahi, Valizadeh, Mollalo, Mahjour, Ghodsian, Ardekani, Behniafar, Gasser and Rostami2022). High worm burdens typically result from repeated exposure and continual reinfection in environments contaminated with human faeces (Carvalho-Costa et al. Reference Carvalho-Costa, Gonçalves, Lassance, Silva Neto, Salmazo and Bóia2007). While ascariasis has been effectively managed in developed nations through improved sanitation and proper waste disposal, it remains a major public health concern in many developing regions, where both the prevalence and severity of infections remain high.
Strategies to control ascariasis primarily rely on mass drug administration (MDA) programs, particularly across Africa, Asia, and Latin America. These programs typically involve administering a single 400 mg dose of albendazole annually or biannually, depending upon the prevalence (Belew et al. Reference Belew, Getachew, Suleman, Mohammed, Deti, D’Hondt, Wynendaele, Mekonnen, Vercruysse, Duchateau, De Spiegeleer and Levecke2015; Betson et al. Reference Betson, Evangelista, Jones and Woolfe2025; Bronzan et al. Reference Bronzan, Dorkenoo, Agbo, Halatoko, Layibo, Adjeloh, Teko, Sossou, Yakpa, Tchalim, Datagni, Seim and Sognikin2018; Farrell et al. Reference Farrell, Coffeng, Truscott, Werkman, Toor, de Vlas and Anderson2018). The primary goals of this approach are to lower infection rates, decrease the number of parasites within individuals, and ultimately reduce disease transmission. In 2015, ‘India’s Ministry of Health and Family Welfare (MoHFW)’ initiated the National Deworming Day (NDD), one of the world’s largest mass drug administration (MDA) efforts. This program targets children between the ages of 1 and 19, providing them with albendazole to combat parasitic worm infections (Kaliappan et al. Reference Kaliappan, Ramanujam, Manuel, Farzana, Janagaraj, Laxmanan, Muliyil, Sarkar, Kang, Walson and Ajjampur2022; Sachdeva Reference Sachdeva2017). Molecular techniques help in detection of light infection intensity and play important role in epidemiology and phylogenetics studies (Khurana et al. Reference Khurana and Sethi2017)
Traditionally considered a disease confined to humans, ascariasis has been shown to potentially cross species boundaries, affecting both humans and pigs. This suggests active transmission at the human-animal interface (Bendall et al. Reference Bendall, Barlow, Betson, Stothard and Nejsum2011; Boeira et al. Reference Boeira, Bezagio, Carvalho, Gandra, Abreu, Massara, Colli and Toledo2025; Dutto et al. Reference Dutto and Petrosillo2013; Easton et al. Reference Easton, Gao, Lawton, Bennuru, Khan, Dahlstrom, Oliveira, Kepha, Porcella, Webster, Anderson, Grigg, Davis, Wang and Nutman2020; Miller et al. Reference Miller, Colby, Manning, Hoenig, McEvoy, Montgomery, Mathison, de Almeida, Bishop, Dasilva and Sears2015). However, the role of such zoonotic transmission and interspecies infections in sustaining the disease and contributing to its persistence within certain populations remains inadequately understood and requires further investigation.
The hypothesis that ascariasis may be a zoonotic disease involving cross-species infections stems from the high degree of similarity between Ascaris lumbricoides (infecting humans) and Ascaris suum (infecting pigs). Molecular evolutionary genetics studies reveal a high degree of similarity in both nucleotide and amino acid sequences between Ascaris lumbricoides and Ascaris suum, indicating they may be genetically indistinguishable (Landeryou et al. Reference Landeryou, Maddren, Hearn, Belachew, Gomez, Liyew, Forbes, Mengistu, Lawton, Eze, Tasew, Angulo and Anderson2025). The fact that both humans and pigs can harbour infections from either species further reinforces the idea that these nematodes may not be separate entities but rather a single zoonotic species capable of cross-infecting both hosts (Iñiguez et al. Reference Iñiguez, Leles, Jaeger, Carvalho-Costa and Araújo2012; Leles et al. Reference Leles, Araújo, Vicente and Iñiguez2009, Reference Leles, Araújo, Vicente and Iñiguez2010, Reference Leles, Gardner, Reinhard, Iñiguez and Araujo2012; Nejsum et al. Reference Nejsum, Betson, Bendall, Thamsborg and Stothard2012; da Silva Alves et al. Reference da Silva Alves, Conceição and Leles2016).
The aim of this study was to investigate the mitochondrial genetic diversity of Ascaris lumbricoides from human populations in northern India and to contextualize these sequences within a global phylogenetic framework to assess evolutionary relationships and potential historical zoonotic connectivity with Ascaris suum. While previous studies have examined mitochondrial diversity of Ascaris in other endemic regions, molecular phylogenetic data from India remain extremely limited. The present work represents the first comprehensive mitochondrial phylogenetic and haplotype network analysis of Ascaris from India, expands the available dataset through the inclusion of newly generated sequences, and provides novel insights into the genetic structure, evolutionary relationships, and possible zoonotic linkages of Indian Ascaris populations.
Material and methods
Study design, population, and detection of A. lumbricoides in faecal samples
The scientific and ethics committee of the ‘Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, approved this study (IEC-INT/2022/PhD-849)’. This study is a continuation of our previous work (Thakur et al. Reference Thakur, Mewara, Lakshmi, Guleria and Khurana2025), in which A. lumbricoides was identified in human faecal samples. Here, we used these samples for further phylogenetic analysis.
Isolation of genomic DNA, conventional PCR, and sanger sequencing
DNA was extracted from stool sample using the ‘QIAamp Fast DNA Stool Mini Kit (Cat. No./ID: 51604, QIAGEN, Hilden, Germany)’. The concentration and purity of the extracted DNA were assessed by ‘NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Thermo Scientific Instruments, USA)’. The cytochrome oxidase 1 (450 bp of the cox1 gene) was amplified by PCR with primers Forward (5′-TTTTTTGGTCATCCT GAGGTTTAT-3′) and Reverse (5′-ACATAATGAAAATGA CTAACAAC-3′), and the NADH dehydrogenase subunit 1 (400 bp of the nad1 gene) with primers Forward (5′-TATGAGCGTCATTTATTGGG-3′) and Reverse (5′-GCATCACAATAGCCAACAAATAC-3′) (Peng et al. Reference Peng, Yuan, Hu, Zhou and Gasser2005). Primers were used at a final concentration of 10 nM. PCR amplification was carried out with an initial denaturation at 95°C for 1 minute, followed by 34 cycles of denaturation at 94°C, annealing at 54°C for 1 minute, and extension at 72°C for 30 seconds for cox1 gene and annealing at 56 C for 1 minute for nad1 gene. This was followed by a final extension step at 72°C for 5 minutes. The PCR product size of 450 bp (cox1) and 400 bp (nad1) was verified using 2% agarose gel electrophoresis (Figure S1 and S2), then amplicons were purified using ‘QIAquick Gel Extraction Kit (Cat no. ID. 28704 QIAGEN, Hilden, Germany)’ and were sequenced with the Sanger method.
Phylogenetic analysis, genetic diversity, and haplotype structure
The Basic Local Alignment Search Tool (BLAST; NCBI, https://blast.ncbi.nlm.nih.gov/Blast.cgi) was employed to confirm sequence similarity with existing Ascaris sequences. Representative orthologous sequences capturing the genetic diversity of Ascaris species were retrieved from GenBank (NCBI, https://www.ncbi.nlm.nih.gov/nucleotide/) for comparative analysis. Multiple sequence alignment was performed using ClustalW integrated in MEGA11 software (Saitou et al. Reference Saitou and Nei1987; Tamura et al. Reference Tamura, Stecher and Kumar2021). Phylogenetic relationships were reconstructed using the Neighbor-Joining (NJ) method (Saitou and Nei Reference Saitou and Nei1987), with branch support assessed through 1000 bootstrap replicates (Felsenstein Reference Felsenstein1985). Evolutionary distances were estimated using the Maximum Composite Likelihood method (Tamura et al. Reference Tamura, Nei and Kumar2004) and expressed as the number of base substitutions per site. Positions with less than 95% site coverage (i.e., containing >5% gaps, ambiguous bases, or missing data) were excluded via the partial deletion option. All evolutionary analyses were conducted in MEGA11 (Tamura et al. Reference Tamura, Stecher and Kumar2021). DNA Sequence Polymorphism analysis was carried out using DnaSP version 6.12 (Rozas et al. Reference Rozas, Ferrer-Mata, Sánchez-DelBarrio, Guirao-Rico, Librado, Ramos-Onsins and Sánchez-Gracia2017). Prior to analysis, sequences were aligned and trimmed to equal lengths (cox1: ~450 bp; nad1: ~400 bp) to exclude terminal gaps and ensure comparability across datasets. Sites containing ambiguous bases or gaps were excluded from haplotype analyses. Haplotypes were defined based on nucleotide sequence variation or nucleotide diversity across the final aligned regions. and a median-joining haplotype network was constructed using POPART (Population Analysis with Reticulate Trees) software (Leigh et al. Reference Leigh and Bryant2015). Default settings were applied, with no epsilon value specified, and networks were visualized without pruning. Host origin (human or pig) and geographic origin were annotated for each sequence to facilitate interpretation of shared and host-specific haplotypes.
Results
Sequences
Seven cox1 Ascaris sequences and 18 nad1 Ascaris sequences (Table 1) were obtained and submitted to GenBank. Alignments were performed with our new sequences and Ascaris spp. reference sequences (32 cox1 and 31 nad1 sequences) from GenBank (S1 Table).
Number of sequences for cox1 and nad1 genes from the present study and their accession numbers
Phylogenetic analysis
Phylogenetic tree based on mitochondrial cytochrome oxidase 1 gene (cox1 gene)
The tree includes (Figure 1) reference sequences of A. lumbricoides and A. suum from various geographical regions (GenBank accession numbers indicated), along with sequences generated in the present study (labelled as SeqSK1–SeqSK6 & SeqSK8). Ancylostoma ceylanicum (KC247732.1) was used as an outgroup to root the tree. Bootstrap values (>25) based on 1,000 replicates are shown at the nodes. The tree illustrates the genetic relationships among Ascaris sequences from humans and pigs across different countries, indicating possible host and geographical clustering. Notably, A. suum sequences from Tanzania, Uganda, Denmark, and the United Kingdom formed a distinct clade, indicating a close evolutionary relationship among pig-derived sequences across these regions. These clustered separately from the A. lumbricoides clade, although with some overlap, suggesting possible historical gene flow or hybridization events.
Maximum Likelihood phylogenetic tree based on mitochondrial cox1 gene sequences of Ascaris species.
A. lumbricoides sequences from present study (SeqSK1–SeqSK6, SeqSK8) formed a clearly defined and separate clade, closely related to other A. lumbricoides sequences from Nepal, Kenya, and Uganda. These Indian sequences were all based on cox1 markers and grouped distinctly with strong bootstrap support (100%), reinforcing their genetic uniqueness and potential regional adaptation.
Several clades included sequences from both humans and pigs (e.g., Brazil, China, and the United Kingdom), further supporting the hypothesis of zoonotic transmission and host-sharing between A. suum and A. lumbricoides. For example, the Czech Republic pig-derived sequences clustered within the A. lumbricoides group, showing a close relationship with European human-derived sequences.
Interestingly, some sequences from Bangladesh (e.g., KF719106.1 to KF719104.1) grouped together in a tightly packed cluster with low divergence, indicating localized transmission and limited genetic variability. Conversely, sequences from China, Korea, and Brazil exhibited broader distribution across different clades, highlighting higher intra-regional diversity.
Phylogenetic tree based on mitochondrial NADH dehydrogenase subunit 1 (nad1 gene)
The phylogenetic tree (Figure 2) illustrates the evolutionary relationships between Ascaris suum and Ascaris lumbricoides using mitochondrial nad1 gene sequences. The tree is rooted with Enterobius vermicularis (NC_056632.1) as the outgroup. The tree demonstrates clear separation between A. suum and A. lumbricoides clades, with multiple well-supported subclades. Chinese A. suum sequences (e.g., AJ968358.1, AJ968360.1, AJ968355.1) clustered tightly together with strong bootstrap support (values between 36 and 41), suggesting a high degree of sequence similarity and possible localized evolution. These formed a distinct clade, separate from most A. lumbricoides sequences.
Maximum Likelihood phylogenetic tree based on mitochondrial nad1 gene sequences of Ascaris species.
Chinese A. lumbricoides sequences (AJ968351.1 to AJ968350.1) and Korean (KY576136.1) and Brazilian sequences (GU326960.1, HM602028.1) clustered closely with Chinese A. suum sequences, indicating potential shared ancestry or interspecies gene flow. Interestingly, several Chinese and Brazilian sequences clustered together in mixed clades, reflecting the complexity of the evolutionary relationship between A. suum and A. lumbricoides.
The Indian sequences generated in this study (SeqSK1–SeqSK18) displayed a broad distribution across multiple clades. Most Indian A. lumbricoides sequences grouped closely (e.g., SeqSK5, SK11, SK7, SK13–SK16) with a major cluster including sequences from China and Brazil, supported by high bootstrap values (e.g., 80, 51, 59, 83). This indicates a close genetic affinity of Indian sequences with international sequences, particularly those from Asia and South America, and highlights possible historical or zoonotic transmission routes.
Other Indian sequences (e.g., SeqSK2, SK4, SK3, SK1) formed a separate basal clade near the root of the tree, with moderate to high bootstrap support, suggesting the presence of genetically divergent variants. One clade including SeqSK6, SK14, SK15, and SKA3–SKA5 was well supported and distantly located from the main clusters, indicating localized diversification within Indian populations.
Sequences from Brazil (GU326951.1, GU326959.1, KX022399.1) and China (A. lumbricoides and A. suum) were widely dispersed throughout the tree, forming both exclusive and mixed clusters. This reflects high intra-regional diversity and possibly overlapping host usage.
Genetic diversity and haplotype network
Haplotype network for cox1 gene
A total of 25 haplotypes (Hap_1 to Hap_25) were identified from the mitochondrial cox1 gene sequences (details provided in Supplementary File S1). The haplotype network (Figure 3) revealed distinct Clusters. Two major clusters are observed within Cluster B: one cantered around Hap_1 and another around Hap_7, each radiating out to several closely related haplotypes, indicating possible recent population expansions. A deep branch separates Hap_5 and Hap_6 from the main network, suggesting high divergence. The network reflects high haplotype diversity and evidence of genetic structuring within the population. One major central haplotype (Hap_7) formed a star-like structure, connecting with 11 other haplotypes (Hap_4, Hap_12–14, Hap_16–21, and Hap_25), suggesting recent population expansion or a potential ancestral lineage. Similarly, Hap_1 exhibited high connectivity, linking with eight haplotypes (Hap_3, Hap_8–10, Hap_23–24, etc.), representing another prominent node. A deep mutational branch was observed between Hap_5 and the rest of the haplotypes, indicating high genetic divergence. Statistical analysis showed high haplotype diversity (Hd = 0.950 ± 0.021; variance = 0.00046), reflecting a genetically diverse population. Nucleotide diversity (π) was estimated at 0.1969, with 224 segregating sites, of which 218 were parsimony informative. Tajima’s D value was –1.20095 (p = 0.879), indicating a negative but statistically non-significant deviation from neutrality, suggesting either population expansion or purifying selection.
Haplotype network of Ascaris spp. based on mitochondrial cox1 gene sequences using a median-joining network. Each circle represents a distinct haplotype, and the size of the circle is proportional to the number of sequences sharing that haplotype. The lines connecting haplotypes represent mutational steps, with hash marks indicating the number of nucleotide differences.
Haplotype network for nad1 gene
Analysis of the mitochondrial nad1 gene revealed a total of 27 distinct haplotypes (details provided in Supplementary File S1), as visualized in the median-joining haplotype network (Figure 4). The network showed a complex and reticulate structure, with no single dominant haplotype, suggesting a high degree of genetic variation. Several haplotypes, such as Hap_2, Hap_5, and Hap_8, were centrally located and connected to multiple others, indicating their potential role as intermediate or ancestral nodes. Multiple long branches with numerous mutational steps (e.g., between Hap_21 and Hap_12, and between Hap_17 and Hap_19) reflect significant sequence divergence within the population. Statistical analysis supported these observations, revealing a high haplotype diversity (Hd = 0.94048), with an average number of nucleotide differences (Kt) of 6.8852. Nucleotide diversity (π) was 0.1279, indicating moderate polymorphism. A total of 71 segregating sites were identified, of which 13 were parsimony informative. Tajima’s D value was –1.95129 (p = 0.985572), suggesting an excess of low-frequency polymorphisms, possibly due to recent population expansion or purifying selection.
Haplotype network of Ascaris spp. based on mitochondrial nad 1 gene sequences using a median-joining network. Each circle represents a distinct haplotype, and the size of the circle is proportional to the number of sequences sharing that haplotype. The lines connecting haplotypes represent mutational steps, with hash marks indicating the number of nucleotide differences.
Discussion
In the present study, we investigated the genetic diversity of Ascaris spp. in mitochondrial markers that are cox1 and nad1, from human stool samples collected from north India. Both the Maximum Likelihood (ML) phylogenetic tree and median-joining (MJ) network analyses revealed three major clusters (A–C) using the cox1 gene. This clustering pattern is in agreement with previous studies on mitochondrial genes in Ascaris, which typically identify two major lineages: one comprising clusters A and C, and the other consisting of cluster B (Monteiro et al. Reference Monteiro, Calegar, Santos, Bacelar, Coronato-Nunes, Reis, Boia, Carvalho-Costa and Jaeger2019; Nejsum et al. Reference Nejsum, Hawash, Betson, Stothard, Gasser and Andersen2017; Sadaow et al. Reference Sadaow, Sanpool, Phosuk, Rodpai, Thanchomnang, Wijit, Anamnart, Laymanivong, Aung, Janwan, Maleewong and Intapan2018).
The present study A. lumbricoides cox1 sequences that are SeqSK1-6 and SeqSK8 form a distinct cluster in phylogenetic analysis that suggests genetic similarity, which is likely from the same region or community. A. suum and A. lumbricoides are intermixed, indicating a close evolutionary relationship or host overlap. Many countries show no strict geographical clustering, especially Bangladesh and Brazil, which suggests shared genetic diversity (Betson et al. Reference Betson, Nejsum, Bendall, Deb and Stothard2014; Iñiguez et al. Reference Iñiguez, Leles, Jaeger, Carvalho-Costa and Araújo2012). Chinese and Korean samples (Peng et al. Reference Peng, Yuan, Hu, Zhou and Gasser2005, Hong et al. Reference Hong, Oh, Seo, Chai and Shin2017) stand apart, indicating regional evolution, emphasizing that present sequences are genetically distinct from reference sequences, which supports the novelty of present data, which further discusses cross-host phylogenetic mixing (pig and human Ascaris). The haplotype network analysis of mitochondrial cox1 sequences revealed significant genetic diversity among A. lumbricoides populations across different geographical regions. A total of 25 distinct haplotypes were identified, with the present study sequences clustering into three haplotypes that are Hap_5, 6, and 7. In our study two haplotypes are unique (Hap_5 and 6), suggesting locally evolved or region-specific variants. In contrast, Hap_7 was shared with sequences from Brazil, Angola, and Tanzania (Betson et al. Reference Betson, Halstead, Nejsum, Imison, Khamis, Sousa-Figueiredo, Rollinson and Stothard2011; Iñiguez et al. Reference Iñiguez, Leles, Jaeger, Carvalho-Costa and Araújo2012), suggesting ancestral linkage or historical gene flow across continents. The central position of Hap_1 (Denmark) (Søe et al. Reference Søe, Nejsum, Fredensborg and Kapel2015) in the network and its multiple connections to other haplotypes suggest it may represent an ancestral or common lineage. The Bangladesh sequences (Betson et al. Reference Betson, Nejsum, Bendall, Deb and Stothard2014) were distributed across several haplotypes, reflecting high endemic diversity. Meanwhile, sequences derived from Ascaris suum (Hap_22–24) formed a genetically distinct cluster, separated by multiple mutational steps, supporting host-associated divergence within the Ascaris complex. Overall, the network highlights both localized variation and global connectivity among Ascaris populations. The pig-derived A. suum sequences (Hap_22–24) (Betson et al. Reference Betson, Nejsum, Bendall, Deb and Stothard2014; Iñiguez et al. Reference Iñiguez, Leles, Jaeger, Carvalho-Costa and Araújo2012), apart from human haplotypes, suggest limited cross-transmission between pig and human haplotypes. But the overall genetic relatedness and shared ancestry within the Ascaris complex still support the hypothesis of zoonotic transmission. The geographically diverse human sequences within closely related haplotypes (Hap_7) also indicate the potential role of human migration or environmental exposure in the global dissemination of Ascaris lineages.
The phylogenetic tree for nad1 gene, notably the Chinese sequences of A. suum, form a distinct group which is monophyletic with moderate bootstrap support (Chang et al. Reference Chang, Gao, Sheng, Lou, Zheng and Wang2015; Peng et al. Reference Peng, Yuan, Hu, Zhou and Gasser2005), indicating limited genetic divergence between them. Brazilian sequences (Iñiguez et al. Reference Iñiguez, Leles, Jaeger, Carvalho-Costa and Araújo2012; da Silva Alves et al. Reference da Silva Alves, Conceição and Leles2016) form separate clades, some showing closer relation to present study sequences, suggesting a historical or zoonotic link. The Indian sequences (SeqSK1,5,8) are distributed across multiple branches, clustering with Chinese A. suum and Brazilian A. lumbricoides sequences. This pattern indicates the existence of genetic variability within Indian Ascaris populations and supports previous findings consistent with hybridization or gene flow between human and pig Ascaris species. The median-joining haplotype network constructed using mitochondrial nad1 sequences reveals a complex pattern of genetic relationships among Ascaris isolates from diverse geographical origins, including Brazil, China, Korea, and India (new sequences labelled as SeqSK). A total of 27 distinct haplotypes (Hap_1 to Hap_27) were identified in the dataset. Hap_1 is the most frequent haplotype, comprising 8 sequences, predominantly from Brazil (Chang et al. Reference Chang, Gao, Sheng, Lou, Zheng and Wang2015; Peng et al. Reference Peng, Yuan, Hu, Zhou and Gasser2005; Søe et al. Reference Søe, Nejsum, Fredensborg and Kapel2015). This central haplotype likely represents an ancestral haplotype, showing its role in connecting different regional variants. Indian A. lumbricoides sequences (SeqSK1–SeqSK18) are dispersed across multiple haplotypes, indicating high genetic diversity. For example: SeqSK6 and SeqSK10 cluster into Hap_2, shared with Brazilian sequences, suggesting possible historical gene flow or shared ancestry and SeqSK5, SeqSK11, SeqSK13, and SeqSK17 cluster together in Hap_15, indicating a closely related group within Indian sequences, unique haplotypes such as Hap_18 to Hap_22 are exclusively occupied by single Indian sequences (e.g., SeqSK9, SeqSK12, SeqSK14, etc.), suggesting local differentiation or recent mutations. Hap_3, another major haplotype with 8 members, includes sequences from Korea and China, supporting a geographically distinct cluster, possibly linked to A. suum.
The haplotype network analyses highlight the complex population structure of Ascaris spp. across different geographic regions. Shared haplotypes observed among sequences from multiple countries may reflect historical gene flow and widespread dispersal of Ascaris lineages. In several regions, haplotypes are shared between A. lumbricoides and A. suum, a pattern that is consistent with previous reports suggesting either zoonotic transmission or the persistence of a shared ancestral population, particularly in settings where close contact between humans and pigs is common (Monteiro et al. Reference Monteiro, Calegar, Santos, Bacelar, Coronato-Nunes, Reis, Boia, Carvalho-Costa and Jaeger2019; Peng et al. Reference Peng, Yuan, Hu, Zhou and Gasser2005). In contrast, the presence of unique haplotypes restricted to specific geographic regions, including several haplotypes identified exclusively among Indian human isolates in the present study, suggests local diversification and region-specific transmission dynamics. Such patterns may be influenced by differences in sanitation, human population movement, and the long-term implementation of mass drug administration programmes. Altogether, the haplotype network analyses support the coexistence of global connectivity and localized evolutionary processes within Ascaris populations. These findings highlight the importance of a One Health approach that integrates human and animal surveillance to better understand transmission dynamics and inform more effective control strategies for Ascaris in endemic settings.
A key limitation of the present study is the limited availability of comparable sequences from Indian sequences, which may have constrained the resolution of genetic and phylogenetic analyses. The limitation of the study is that, although we initially compiled all available nad1 and cox1 sequences, including those derived from complete mitochondrial genomes and wild sequences, several datasets contained incomplete or inconsistent gene regions that caused repeated errors during alignment and haplotype network construction. To ensure analytical reliability, only high-quality sequences with comparable lengths were retained. Future work incorporating standardized, fully annotated mitochondrial gene regions from wild sequences would further enhance the comprehensiveness of Ascaris population analyses. Nevertheless, the findings highlight the complex genetic structure of Ascaris populations and underscore the importance of continued molecular surveillance in both human and animal hosts to better understand transmission dynamics and evolutionary patterns. Although the observed clustering and shared haplotypes are consistent with historical gene flow and possible cross-host transmission between A. lumbricoides and A. suum, these inferences are based solely on mitochondrial markers. As mitochondrial DNA represents a single maternally inherited locus, it cannot clearly distinguish among incomplete lineage sorting, introgression, or true hybridization. Therefore, our findings should be interpreted as suggestive rather than conclusive. Future studies using nuclear and multilocus or genome-wide data will be necessary to more definitively resolve patterns of hybridization and zoonotic transmission within the Ascaris species complex.
Conclusion
The phylogenetic and haplotype network analyses revealed substantial genetic diversity and complex evolutionary relationships among Ascaris sequences from humans and pigs across multiple regions. Shared haplotypes between Indian, Brazilian, and Chinese sequences suggest historical gene flow or common ancestry, while the presence of unique Indian haplotypes indicates localized diversification (or variants). The genetic distinction between pig-derived A. suum (reference sequences) and human A. lumbricoides sequences from the present study supports host-associated divergence, although in previous studies, some overlap highlights possible historical zoonotic transmission. Overall, the data underscore both regional diversification and global connectivity in Ascaris populations, emphasizing the need for integrated, cross-host surveillance to understand transmission dynamics.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X26101242.
Data availability statement
All newly generated sequences in this study have been submitted to GenBank. The corresponding accession numbers are provided in the manuscript.
Acknowledgements
We thank the participants and the Department of Medical Parasitology at the Post Graduate Institute of Medical Education and Research for providing the platform necessary for conducting the project.
Author contribution
Mamta Thakur: Conception of study, sample collection and processing, investigation, experimentation, acquisition of data, analysis and interpretation of data, draft writing, final editing. Abhishek Mewara: Supervision and review of the study, revised the manuscript, final editing. PVM Lakshmi: Supervision and review of the study, conception of study, sample collection, revised the manuscript, final editing. Sucheta Guleria: Writing manuscript, analysis, review. Sumeeta Khurana: Supervision and review of the study, contributed to conception of study, investigation, acquisition of data, validation of results, revised manuscript, final editing.
Financial support
This work is funded by ICMR, New Delhi, via file no. Zon/69/2022/ECD-II dated 01-03-2023. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests
The authors declare no competing interests.
