Caenorhabditis brenneri as a complementary model organism to C. elegans: Insights from ecological, molecular and comparative biology

Caenorhabditis brenneri as a complementary model organism to C. elegans: Insights from ecological, molecular and comparative biology | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Caenorhabditis brenneri as a complementary model organism to C. elegans: Insights from ecological, molecular and comparative biology MD TALUKDER, Md. Rajiur Rabbi, Md. Sajib, Nurnabi Ahmed, Md Makshuder Zim, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6856196/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The Caenorhabditis elegans is a well-established model in the advancement of cell and molecular biology, lead-compound discovery and multidisciplinary research. However, its sibling species C. brenneri, remains relatively underexplored despite its hyperdiverse genome and ecological significance. This study sought to discover sister species of C. elegans through ecological surveys, genetic characterization, and biological comparisons with C. elegans (N2), with the aim of positioning of wild isolates as complementary model organisms. Sampling was conducted across 11 distinct bio-ecological zones, followed by cultivation and independent propagation of bacterivorous nematodes. Distinct morphological traits with the amplification of ITS2 and 18s rRNA regions confirmed isolates of C. brenneri, Oscheus sp., and surprisingly, Tarantobrlus jeffdanielsi and Rhabditella axei. The genetic diversity analysis based on ITS2 sequences of C. brenneri isolates revealed that diversities and polymorphisms are consistent with neutral evolution rather than selection. A comparative analysis of C. elegans and wild C. brenneri isolates including lifespan, fecundity, thermo-tolerance, anthelmintic sensitivity and bacterial killing assay revealed that, compared to C. elegans N2 strain, C. brenneri isolates live longer, produce fewer eggs than C. elegans, exhibited higher survival under prolonged heat stress and significantly greater survival against bacteria and transient lethality in anthelmintics. This pioneering investigation confirms the availability of C. brenneri and other previously undiscovered nematodes in different regions of Bangladesh, outlines its evolutionary context, resilience to stressors and warns altered host adaptability. These findings highlight the potential of C. brenneri as a robust complementary model for research into species-specific responses to environmental or pathogenic stressors. Biological sciences/Genetics Scientific community and society/Developing world C. brenneri Bangladesh Ecology Evolution Stressors Resistance C. elegans Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights This study investigates the lesser-known nematode C. brenneri to understand its ecology and genetic diversity compared to C. elegans . Eleven distinct bio-ecological areas in Bangladesh were selected for sampling from pre-made baits were used to collect cultivate and analyze nematodes in a controlled laboratory setting. The findings indicate that C. brenneri has a longer lifespan and lower fecundity than C. elegans , with distinct morphological traits aiding in identification. Molecular analysis confirmed the species identity through amplification of the rDNA ITS2 and 18S rRNA regions, establishing a phylogenetic relationship within the Caenorhabditis nematodes This research highlights the genetic diversity of C. brenneri as a valuable resource for future studies on environmental stress responses, drug discovery, and comparative genomics with C. elegans . Introduction Caenorhabditis elegans , a cornerstone model organism, has made important contributions to biological fields namely developmental biology, neuroscience, and cell biology with current scientific interest also directed towards its natural ecology and evolutionary lineage (Barrière and Félix, 2005; Schulenburg and Félix, 2017). Its morphological simplicity and maintenance-ease allows more precise investigations into the neural circuitry. Furthermore, ithas been extensively studied genetically and molecularly (Rahmani et al., 2021), providing a wealth of data regarding chromosome mutations and facilitating genetic manipulation (Singh, 2021) and loci mapping (Andersen et al., 2022). The C. elegans model has been successfully utilized in the field of apoptosis (Lettre and Hengartner, 2006), muscle differentiation (Fox et al., 2007) and high-throughput screening(HTS) for drug discovery (Zwirchmayr et al., 2020), mode of action of anthelmintic (Driscoll et al., 1989), anthelmintic resistance (Collins et al., 2024), host-microbe interactions (Backes et al., 2021; Kumar et al., 2020), heat-stress response (Nehammer et al., 2015) and toxicology (Hunt et al., 2020; Tejeda-Benitez and Olivero-Verbel, 2016) serving as an impetus model to solve important research questions. As a free-living nematode, C. elegans can help address key questions such as identifying the minimum number of genes necessary for a nematode to become parasitic and understanding the essential genetic requirements for successful parasitism (Geary et al., 2001). The morphological traits of Caenorhabditis species often display convergence, posing challenges in species identification (Sudhaus et al., 2007). Therefore, a combination of cross-breeding for species identification and molecular sequence comparisons, alongside traditional morphological analysis, proves instrumental in accurate species diagnosis. C. elegans and its close kin, including C. briggsae, C. remanei, and C. brenneri are widely distributed in organic-rich microenvironments like decomposing plant material and compost heaps (Félix et al., 2013), distinct from typical soil-dwelling nematodes. The geographic distribution of the different species is also quite different, especially for C. brenneri , which unlike the others has not yet been found in Europe or North America (Kiontke et al., 2005). In terms of reproduction, C. elegans and C. briggsae are hermaphroditic, whereas C. remanei and C. brenneri are gonochoristic (Sudhaus and Kiontke, 2007). Global sampling efforts have led to the discovery of over 50 putative species from the genus Caenorhabditis (Stevens et al., 2019), many of which await formal species description. C. brenneri , a nematode species, is known for its molecular diversity, exhibiting 14.1% polymorphic-synonymous sites, making it one of the most diverse eukaryotes globally (Stevens et al., 2019). C. brenneri has been found in pan-tropical geography with extensive genetic diversity, allowing for viable genetic crosses between continents and showing weak geographic genetic structure (Dey et al., 2013). It was first isolated from the terrestrial slug Philippinella moellendorffi in the Philippines (Dalan et al., 2022). This cryptic nematode has also been documented in Eastern India (Dey et al., 2013), though no records exist for its presence in Bangladesh. Understanding the ecological distribution and genetic diversity of Caenorhabditis nematodes worldwide is crucial for studying their adaptation to different environments and their potential implications for human health and agriculture. The introduction of C. brenneri has expanded the understanding of nematode diversity and evolutionary complexity, providing a valuable model for studying genetic evolution and functional elements within genomes (Thomas, 2008). South Asian countries, particularly Bangladesh, are rich in medicinal flora with potential for discovering natural anthelmintic products; however, research in this area is significantly hindered by limited funding, inadequate model organism utilization, weak industry-academia partnerships and a lack of technological innovation (Kamal et al., 2023). A locally adapted, free-living nematode closely related to C. elegans could help bridge the research gap in Bangladesh and other regions facing similar obstacles to nematode research. The survey aimed to position C. brenneri as a complementary model with biological studies in different environment along with C. elegans , while also examining the genetic diversity of Caenorhabditis nematodes in different ecological niches and geographical regions of Bangladesh. The aim of this study is to gain insights into the population genetics and ecology of Caenorhabditis nematodes in Bangladesh, which can further contribute to our understanding of the evolutionary ecology, adaptation and responses to environmental stressors of these nematodes. These observations will highlight the versatility and widespread distribution of Caenorhabditis and other bacterivore nematodes across various ecosystems. Additionally, the comparative biology will reflect the differences and relativeness between two sibling species, ultimately providing a valuable insight into the utility of acclimatized nematodes such as C. brenneri as complementary model organisms. Methods Study regions The present study covered 11 regions in Bangladesh, including Thakurgaon, Rangpur, Bogura, Mymensingh, Dhaka, Gazipur, Sylhet, Chattogram, Bhola, Barishal, and Jhenaidah, located in distinct bio-ecological zones such as the Himalayan piedmont plain, Barind tract, Teesta and Ganges floodplains, offshore islands, Chittagong hills, Madhupur sal forest, and the Brahmaputra-Jamuna and Surma-Kushiyara floodplains (Supplementary Figure 1). A total of 225 samples were collected in triplicate, with each replicate buried one foot apart (Supplementary Figure 2) and later pooled into a single sample. Sample collection took place between October 2023 and November 2024. Optimized protocol for cultivation, collection and freezing of nematodes Samples were procured from diverse geographical locations. For sampling, on Day 0, small tomato-cuts of 1-1.5 inch were excised and buried at a depth of 1-2 inches beneath the soil surface or just covered with a thin layer of loose soil in shaded areas to avoid direct sunlight exposure and feeding by birds, in triplicate and with each site being demarcated. Subsequently, on Day 1, Nematode Growth Media (NGM) was prepared, and a pure culture of E. coli OP50 bacteria was cultivated (Stiernagle, 2006) and spread onto NGM plates. On Day 3, the decomposed tomatoes were retrieved and the decaying segments were carefully arranged along the periphery of NGM plates, instigating the emergence of worms. Following this, the samples were dispatched to C. elegans model Sci. and Tech lab, Bangladesh Agricultural University using expedited shipping methods, ensuring temperature regulation (11°C to 25°C). On Day 4, surveillance of the Petri dishes began. Worms exhibited distinct morphological characteristics were isolated onto seeded NGM plates. To maintain homogeneity, 4 to 5 gravid individual worms were transferred using worm picker and allowed to reproduce naturally at 20-25ºC cooling incubator. This sub-culturing process was repeated at least three times for each isolate (Crombie et al., 2022). From the homogenous population of each isolate, freshly starved age synchronized cohorts (L1-L2) were produced that were then stored indefinitely in liquid nitrogen or -80ºC freezer. In brief, soft agar freezing solution was melted using an autoclave and equilibrated in a 50°C water bath for at least 15 minutes. Freshly starved L1–L2 worms were harvested by washing and subsequently an equal volume of soft agar freezing solution was added, mixed thoroughly and 1 ml of the mixture was aliquoted into labeled 1.8 ml cryovials. The vials were packed in a styrofoam box and frozen at −80°C overnight. The following day, the vials were transferred to permanent storage and worm viability was assessed from one vial (Brenner, 1974). Morphological analysis Based on previous studies (Sudhaus and Kiontke, 2007; Dalan et al. 2022), worms having double bulbed pharynx and morphology similar to that of C. elegans or Caenorhabditis spp, wereadvanced for micro-morphology. For microphotography, worms were placed in a drop of M9 buffer containing 5% Levamisole on 2% agar pads and studied at different magnification with Olympus (Olympys Corporation, Tokyo, Japan) light microscope BX53 equipped with Olympus DP27 microscope digital camera and cellSens imaging software (Olympus Corporation, Tokyo, Japan). Nucleic acid preparation and polymerase chain reaction (PCR) Worms having similar morphology to C. elegans or Caenorhabditis sp. and also bacterivore nematodes were subjected to DNA extraction. The genomic DNA was extracted from (n = 15-20) adult worms including C. elegans adults using the TIANamp Genomic DNA Kit (TIANGEN Biotech (Beijing Co., Ltd.) according to the manufacturer's instructions. Subsequently, the extracted genomic DNAs were subjected to PCR amplification using two pairs of primers. This study performed two separate PCRs for each sample. The first primer set amplifies a 2,000-bp fragment of the internal transcribed spacer region between the 5.8S and 28S rDNA genes (ITS2); oECA1687F CTGCGTTACTTACCACGAATTGCARAC, oECA202R GCGGTATTTGCTACTACCAYYAMGATCTGC. The second set of primers, CE1209F (5′-TACTGTCAGTTTCGACTGACTC-3′) and CE2250R (5′-ATACGAACCCGAAGATTCGCC-3′), which amplify about 1,000 bp of the 18S rRNA gene of Caenorhabditis nematodes. PCRs were performed in 25 μl reaction volume containing 12.5 μl Premix Taq™ (TaKaRa Bio, Japan), 1.5 μl forward primer, 1.5 μl reverse primer, 4.5 μl molecular grade water, 5 μl genomic DNA. The cycling profile for CE1209F + CE2250R: 2 min at 95°C, followed by 35 cycles of 20 s at 95°C, 1 min 55 °C, 1 min 30 s at 72°C and a final extension of 10 min at 72°C (Haber et al., 2005). Another cycling profile for oECA1687 + oECA202R: 3 min at 95 °C, followed by 35 cycles of 15 s at 95°C, 15 s at 55°C, 2 min 72°C Got to step 2, a final extension of 5 min at 72°C and hold at 12°C (Kiontke et al., 2011). The PCR products were analyzed by gel electrophoresis (1.5%) stained with Midori Green Advance (NIPPON Genetics EUROPE). Gene sequencing PCR products were run on an agarose gel and then purified. The single-stranded products were generated from purified products using cycle sequencing PCR with forward or reverse primers. The amplified products were run on a Sanger machine using the di-deoxy chain termination method at Genecreate Biotech, China (Sanger et al., 1977). Sequence alignment, phylogenetic relationship and genetic diversity analysis Generated sequences were BLASTn-searched to compare with all sequences deposited in the National Centre for Biotechnology (NCBI) (Altschul et al., 1990) for closely related species. All obtained sequences were trimmed and annotated. Phylogenetic analysis was done with other identified closely related species based on the BLASTn output. Multiple sequence alignment was made using MUSCLE in MEGA v.11 (Tamura et al., 2021) followed by manual trimming to remove poorly aligned regions. Phylogenetic modeling and tree visualization were achieved by applying MEGA v.11 with the Maximum Likelihood(ML) method (Tamura et al., 2021). The phylogenetic tree was validated by running the analysis on 1000 bootstrapped input datasets and cross-referencing it against the Tamura-Nei substitution model. Population nucleotide diversity indices such as nucleotide diversity per site(π), average number of nucleotide differences(k), mean genetic diversity(Hd), number of segregating sites(S), average number of nucleotide substitutions per site(K), pairwise divergence(D XY ) and neutrality tests including Tajima’s D and Fu Li’s F test were calculated using DnaSP ver. 6.12.03 (Rozas et al., 2017). Comparative biology of C. brenneri isolates and C. elegans (N2) Fecundity assay Fecundity assay was performed in according to the previous study (Woodruff et al., 2019). In brief, larvae production was monitored daily following overnight mating and under continuous male exposure. For all observations, L4 of four C. brenneri isolatesand C. elegans (N2) strains, maintained at 25°C, were isolated and reared to adulthood. During overnight mating, single adult females were transferred to fresh plates and mated with at least six males overnight. The following day, males were removed and embryos and larvae were counted daily. Egg-laying females were transferred to new plates and new progeny were recorded until females ceased laying. In the continuous mating condition, females were always paired with six males. Males that crawled up the plate or died before the female stopped laying were replaced with young adult males. These continuous mating observations were conducted at 25°C. Intra and inter species outcrossing For all mating experiments, L4-stage females were paired with males on seeded NGMs and left overnight at 25°C for mating. The following day, males were removed and embryos and larvae were counted daily. A 1:2 female-male ratio was used for all crosses, except for interspecies crosses with C. elegans males, where a 1:3 ratio was employed due to the lower mating efficiency of C. elegans males compared to dioecious males. Egg-laying females were transferred to new plates and new progeny were recorded until females ceased laying (Jessica et al., 2024). Mating tests were counted as positive if viable progeny were produced and a fertile second or third generation was observed (Sudhaus and Kiontke, 2007). Lifespan assay Synchronized population (L4) of C. brenneri isolates and C. elegans (N2) were collected using a standard bleaching assay. A total of 12–15 healthy L4 larvae were transferred to freshly seeded plates, creating a stack of 6 plates per isolates or strains, which were labeled and returned to the cooling incubator in order to reach their adulthood. The following day, parents were transferred to fresh plates, ensuring no larvae or eggs were carried over and maintaining the consistency of the plates. After animals stopped producing progeny, lifespans were scored every other day until all animals had been scored as dead or censored. The worm was counted as alive if it moves in response to the tapping. All dead or censored worms were removed from the plate to avoid confusion and recounting the same animal. Worms that flee were typically removed from analysis. This assay was done by maintaining the temperature of 25 °C (Amrit et al., 2014). Assessment of thermal tolerance Thermal tolerance of the C. elegans (N2) and a local isolate of C. brenneri (Syt11) was evaluated at 36ºC. For each trial, 20 to 30 L4 larval stages of each strain were transferred to fresh NGM plates seeded with OP50 E. coli . In this assay, we have modified the protocol described previously (Lithgow et al., 1994; Wolf et al., 2008 and Golden et al., 2020). In brief, synchronized worms were incubated at 36ºC for 2h, followed by a rehabilitation phase at 20ºC in a cold incubator for 15-20 minutes. This cycle was repeated until all worms were dead. Worms were gently prodded after each rehabilitation phase with a platinum worm pick and those that failed to respond were recorded as dead. This assay was repeated twice and standard male female ratio was maintained. Nematicidal sensitivity assay of anthelmintics Age synchronized cohorts of L4 larvae from C. elegans (N2) and C. brenneri isolate (Syt11) were utilized for this assay. The assay was performed using 24 well microtitre plates, each well containing 250 µL of serially diluted anthelmintics with three replicates per concentration. The test compounds - ivermectin, levamisole and albendazole were prepared with 0.1% DMSO at concentrations of 0.05 mg/mL, 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL. Worm mortality rate was determined after 1hr, 3 h, 6 h, 12 h, 24 h and 48 h at 20 °C. A total of (20 to 30) L4 C. elegans (N2) and local isolate of C. brenneri were pipetted per well, thereafter the plates were sealed and incubated at 25ºC. Nematodes are considered dead when the body is straight, there is no head thrusting, body bending, tail movement or pharyngeal pumping for a continuous 10s of observation period (Skantar et al., 2005). Standard male:female ratio was maintained and repeated counting was ensured. Bacterial killing assay All assays were conducted at 25°C and with standard sex ratio. Worms were scored as alive or dead by gentle prodding with a platinum wire and sometimes with clear or transparent body. The counting was done in every hour for the first five hours and then at hour 24. Experiments were performed at least twice. S. aureus killing assays Assays were performed as described (Sifri et al., 2003). Briefly, NCTC8325 (or mutant derivatives, as noted) was grown overnight in tryptic soy broth (TSB, BD, Sparks, MD) with 10 µg/ml nalidixic acid (Sigma). Almost 5–10 µl of overnight cultures were seeded on 35 mm tryptic soy agar (TSA) plates with 10 µg/ml nalidixic acid. A total of 25–35 L4 stage of both worms were transferred to each of three replicate plates per species. Animals that died as a consequence of a bursting vulva or crawled off the agar were removed. P. aeruginosa killing assays Briefly, PA14 was cultured in Luria broth (LB), seeded on plates with 5–10 µl culture and incubated first for 15h at 37°C and then for 24 h at 25°C. A total of 25–35 L4 stage both C. elegans (N2) and C. brenneri isolate was transferred to each of three replicate plates per strain. Worms that don’t response while prodding and appears clear counted as dead and then removed from the plate. Statistical analysis All data and statistical analyses were done with Microsoft Excel, SPSS and R depending on suitability. Data was compared using ANOVA (Analysis of Variance), T-tests, Tukey HSD test for post-hock analysis, at 95% confidence intervals (CI) and presented as mean ± standard error(SE) using SPSS version 29. Correlation coefficients were assessed using Pearson correlation in R to evaluate relationships between variables. To make Kaplan-Meier survival curves survival package (Rstudio) and then pairwise comparisons between survival curves was obtained using the log rank test or Cox proportional hazards regression analyses (Rstudio) to test for differences between species and interactions with treatments. Results Detection of worms across the bio-ecological regions of Bangladesh Out of 225 samples collected from 11 regions, only those from Sylhet (n=29), Thakurgaon (n=25), Rangpur (n=27), Gazipur (n=10), Dhaka (n=6), Bogura (n=21), Mymensingh (n=30), and Bhola (n=29) tested positive for bacterivorous worms, while no bacterivore worms were found in samples from Chattogram (n=15), Barishal (n=16) and Jhenaidah (n=18) (Table 1). With the positive samples, worms were sorted from heterogeneous population on the basis of morphology and cultured separately to prepare homogenous cohorts in a cooling incubator at 25ºC. Nematodes with a double-bulbed pharynx and a brown to transparent color were identified as potential Caenorhabditis nematodes and were further examined under microscopy for detailed morphology. Furthermore, all bacterivore worms culture was maintained for nucleic acid extraction to perform molecular characterization. Phenotypic profiling Nematodes similar with the micromorphology of Caenorhabditis nematodes were taken into account for detailed phenotypic characterization. Most phenotypic traits found - Fine transverse ridge in the cuticle; Buccal tube was longer than lip; pharynx exhibited a strong, oval median bulb and a rounded terminal bulb; In male: Testis was single. Tail was covered with a fan shaped closed bursa, comprised with pairs of rod like genital papillae or rays; Spicules are long and slender with a pointed tip; Gubernaculum is also slender; In female: Tail is conical with filiform tip. Anus and rectum are clearly visible. Uterus carried embryos (oviparous to ovoviviparous) at different stages of development (Figure 1). Phylogenetic analysis and taxonomic positioning of C. brenneri strains and other nematodes All 64 bacterivorous worm samples tested positive for ITS2 amplification, while only 4 samples were positive for 18S rRNA amplification. Of the ITS2 -positive samples, 10 were selected for sequencing, and all 18S rRNA -positive samples were also sequenced. The ITS2 amplified query sequences IT11, IT8, IT20, and IT21 showed 93.24%, 95.01%, 96.10%, and 96.95% identity, respectively, to Caenorhabditis brenneri strain SB129 (JN636110.1) from Sumatra (Bohorok). Similarly, IT3, IT4, IT14, IT16, and IT17 exhibited 99.38%, 98.77%, 99.26%, 99.38%, and 98.75% identity, respectively, to Oscheius sp. (MT482317.1) from South Africa. Furthermore, IT13 shared 98.80% identity with Rhabditella axei isolate (PP135624.1) from Riccio, Italy, while IT15 showed 98.58% identity to Tarantobelus jeffdanielsi isolate (OQ703326.1) from the USA. The 18S rRNA amplified query sequences CE11, CE8, CE20, and CE21 was found to be 99.03%, 99.00%, 99.36% and 99.25%, respectively, similar to the subject sequence of Caenorhabditis brenneri strain CB5161 (U13930.1) from Trinidad. The phylogenetic relationship of the ITS2 query sequences designated as IT11, IT8, IT20 and IT21 formed a distinct monophyletic clade within the C. brenneri lineage, clustering closely with the reference C. brenneri SB129 strain (JN636110.1) from Sumatra. Within the clade, IT20 and IT21 appeared more closely related to each other than to IT11 and IT8, forming a well-supported subclade (Figure 2). The query sequences from Oscheius sp., Rhabditella axei , and Tarantobelus jeffdanielsi formed sister clades with their respective top BLAST hits, indicating close phylogenetic relationships. Similarly, ML analysis of query sequences of 18S rRNA gene revealed that C. wallacei as sister to the group (Figure 3) consisting of the sister taxa CE8, CE11, CE20, CE21 + Caenorhabditis sp. ( C. brenneri ) CB5161 and the Elegans group. Within this clade, the query taxa CE8, CE11, CE20, and CE21 clustered within the Elegans group, showing closest relatedness to C. brenneri CB5161. Notably, CE11 and CE20 formed one subclade, while CE8 and CE21 formed another, indicating close phylogenetic relationships. To add more, C. macrosperma , C. nouraguensis , C. panamensis , C. japonica , C. waitukubuli are placed in Japonica group which is sister to C. sulstoni and the Elegans group (Kiontke et al., 2011). Genetic Diversity Genetic diversity analysis of ITS2 sequences revealed that Group 1, comprising IT8, IT11, IT20 and IT21 exhibited higher nucleotide diversity (π = 0.03762 ± 0.008) and a greater number of segregating sites (S = 42) compared to Group 2, which included sequences JN636110.1, JN636107.1, JN636108.1, and JN636109.1, with a lower nucleotide diversity (π = 0.00470 ± 0.001) and fewer segregating sites (S = 6). Both groups showed haplotype diversity (Hd) of 1. Neutrality tests indicated no significant deviation from neutrality in either group, with Tajima’s D and Fu and Li’s F values being non-significant (P > 0.10). The overall dataset displayed a moderate nucleotide diversity (π = 0.03420 ± 0.001) with a total of 48 segregating sites, further supporting the presence of genetic variation across the studied taxa (Table 2). Comparison of reproductive output and lifespan between C. brenneri and C. elegans The brood size for the four C. brenneri isolates (Syt8, Syt11, Syt20 and Syt21) were 128 ± 6.65, 117.6 ± 4.72, 119.2 ± 5.92 and 121.8 ± 5.82 which exhibit significantly lower reproducibility (p<0.01) compared to the C. elegans N2 wild-type: 237.41 ± 13.2. But there was no significant variation among C. brenneri isolates (Figure 4). The outcrossing experiment within four C. brenneri isolates show higher progeny numbers, indicating strong outcrossing success among these introgressed strains (Table 3). In contrast, all crosses involving C. elegans N2 produce very few progeny (6–13), suggesting significant reproductive isolation or hybrid incompatibility between the C. brenneri lineages and the N2 wild type. For the lifespan experiment, numbers of worms that were alive or dead were recorded each day for each strain tested. The count of number of worms alive on each day was not used directly in lifespan analysis. The number of worms that died on each day was inverted to calculate the proportion of worms alive on each day which is plotted graphically as a survival curve (Figure 5). The day of the timed egg laying is considered day 0. There was no significant difference in longevity among the four C. brenneri isolates and sex did not have a significant effect on lifespan either (Figure 5B). Kaplan-Meier survival analysis was performed to compare C. elegans (N2) with the C. brenneri isolate (Syt11). This C. brenneri isolate was chosen for lifespan and all subsequent analyses as a representative and having biological similarity with other isolates. Hermaphroditic wild type C. elegans worms show 100% lethality after 30 days and survival is significantly higher(p<0.01) in gonochoristic C. brenneri isolate (Syt11). Comparative assessment of thermal tolerance in C. elegans and C. brenneri under prolonged heat stress The thermal tolerance assay was performed to compare the thermal tolerance profile of C. elegans (N2) and C. brenneri isolate (Syt11). By 2 hours of exposure at 36°C, C. elegans (N2) shows a slight decrease to 91.06%, while the C. brenneri isolate maintains a higher survival rate at 97.65% (Figure 6). Thereafter, survival percentages declined gradually for both species. Notably, by 6 hours, a significant reduction ( p<0.01 ) in survival was observed, with C. brenneri isolate (Syt11) showing higher survival percentages (49.64%) compared to C. elegans (22.23%). By the 8-hour mark, all individuals of both species were dead. These results suggest that while both species differ in thermo-tolerance profiles, C. brenneri demonstrate a higher resistance to prolonged thermal stress. Nematicidal activity of P. aeruginosa and S. aureus In this P. aeruginosa (PA14 seeded plates) killing assay, C. brenneri isolate (Syt11) demonstrated markedly stronger resistance compared to C. elegans N2. By 5 hours, C. elegans N2 survival plummeted to 32–41%, while C. brenneri isolate (Syt11) maintained 72–79%, and by 24 hours, C. elegans N2 exhibited 100% lethality, whereas C. brenneri isolate (Syt11) retained 38–45% of its population which indicate significant variability (p<0.0001) after the pairwise comparisons between survival curves (Figure 7B). A similar trend was observed on S. aureus (NCTC8325 seeded plates) infected plates (Figure 7A), where C. brenneri isolate (Syt11) shows significantly (p<0.0001) higher survival rate than C. elegans N2. Anthelmintic sensitivity In both species, Ivermectin and Levamisole demonstrated rapid and potent effects, with significantly faster mortality at higher concentrations, especially notable in C. elegans , where survival dropped sharply within the first 6 hours for doses of 0.25 mg/ml and above (Figure 8A). In contrast, C. brenneri displayed a slightly more gradual response to these drugs, though the overall trend of faster decline with increased doses. Similar trend of sensitivity was observed in response to levamisole (Figure 8B). Albendazole showed a slower reduction in survival in both species, with C. elegans exhibited more pronounced sensitivity over time compared to C. brenneri isolate (Syt11), which retained a significant (p<0.001) higher survival percentage across all concentrations up to 48 hours (Figure 8C). This indicated that both speciesshowed almost similar response to all three drugs, particularly at higher doses and earlier time points, whereas C. brenneri tends to exhibit a more gradual response in lower doses, suggesting C. brenneri isolate shows a greater and robust resistance to albendazole compared to C. elegans . The correlation co-efficients between drug category and dose concentration with the survival percentage ranged from strong to moderate (r = -0.27 to 0.68) in C. brenneri (Supplementary Table 2). In contrast, the correlation coefficients for drug category and dose concentration with the survival index were moderate to weak (r = 0.15 to -0.49) in C. elegans (Supplementary Table 1).Additionally, the time period exhibited a negative correlation ( r= -0.41) in both species with the survival index as time increased. The analysis of variances (ANOVA) of two species indicated that all factors and their interactions have a highly significant impact ( p< 0.001 ) on the survival rate (Supplementary Table 3). Post-hoc analysis of drug category, dose concentrations, and observation time revealed that, within the first hour, Albendazole exhibited a slower lethality at a concentration of 0.05 mg/mL compared to the other drugs in both species. Furthermore, significant intra-group differences ( p<0.01 ) were observed across all variables in terms of their effect on the survival. Discussion Most of the free-living Caenorhabditis nematodes habitat in ephemeral patches rich in microbial blooms and sometimes in sympatry (Crombie et al., 2022). This study discovered new wild isolates of C. brenneri from baits in Mymensingh and Sylhet, Oscheius sp. fromThakurgoan, Rangpur, Mymensingh, Bhola and Sylhet, Rhabditella axei from Gazipur and Tarantobelus jeffdanielsi from Dhaka, Bangladesh. For the baiting procedure, this study tried two different approach as baits on the surface mostly attracted by predators and rodents, which lead to chance of sample missing in Bangladeshi context. This study tried also an innovative approach to identify Caenorhabditis and other closely related nematodes. The place where the baits were buried was wet and shaded. The temperature during collection was 18-20ºC. Sylhet is characterized by its cooler climate throughout the year compared to other parts of the country, may provide favorable conditions for the colonization of C. brenneri . Despite samples being collected in pre-winter and winter seasons, the overall climate of Sylhet supports a variety of species that thrive in cooler temperatures. Similarly, the riverside of old Brahmaputra river in Mymensingh, known for its fertile soil and microbial blooms may support greater ecological diversity of bacterivore nematodes. Mymensingh and Sylhet are also located in the north of the country not far from the Himalayan plains where temperatures are milder compared to the southern parts such as Chattogram. The milder conditions may support growth of bacterivore species in the Caenorhabditis genus, although as this study has shown, there can be significant differences between the sibling species with respect to resistance to thermal stress, drug and microbial insults. Altogether, these environmental and geographical factors contribute to the successful colonization of this species in the region. Endemic gonochoristic elegans group species, which are quite numerous in eastern Asia (Dey et al., 2013) and South America (Sloat et al., 2022), appear to be absent from the neo-tropics. Recently, another nematode, Oscheius tipulae (Family- Rhabditidae) was proposed as a model organism for NP-based anthelmintic screen (Kamal et al., 2023) in tropics. This species is phylogenetically more closely related to the insect parasite Heterohabditis and the vertebrate parasites Strongylida (Kiontke et al., 2005). However, C. brenneri possesses many advantages over O. tipulae , such as being a sister species to the more well-established C. elegans , and the fact that molecular biology reagents and techniques are standardized for C. brenneri as they are for C. elegans. It can be maintained in the laboratory and cryopreserved using the same set-ups used for C. elegans. Other nematode Rhabditella axei , which can be both parasitic or free living (Sciandra et al., 2024), feeds mainly on bacteria present in decaying organic matter, soil and other substrates. However, in its parasitic form, it can colonize some species of snails. This nematode was found in the Gazipur region may be due to abundant decaying organic matter and bacteria for its free-living form, warm humid conditions favoring proliferation and potential snail hosts enabling parasitic colonization, supported by agricultural and waste management practices. Additionally, tarantula-parasitic nematode, Tarantobelus jeffdanielsi was first reported in Los Angeles, California, USA (Baniya et al., 2023) and it was also reported by this study for first time in Dhaka, Bangladesh. That may be for the climate adaptability of the nematode, or previously undetected native presence now identified. Previous study reported that T. jeffdanielsi successfully infected several different species of tarantulas as well as wax worms ( Galleria mellonella ), indicating that this nematode may have a broad host range (Schurkman et al., 2022) . Most interestingly, R. axei and T. jeffdanielsi can be cultured with OP50 seeded NGM petri plates. This warrants further study in their biology and host adaptability. A total of 11 isolates of different species namely C. brenneri (4), Oscheius sp . (5), T. jeffdanielsi (1) and R. axei (1) were successfully preserved in -80ºC. Additionally, all of the nematode strains used in this study, including the four new C. brenneri isolates are being maintained in the C. elegans model Science and Technology Lab, Department of Parasitology, Bangladesh Agricultural University and are available for research use upon request. The description of fine transverse ridges in the cuticle and the proximal and terminal bulbs, distinct features of the buccal tube, pharynx and spicules, gubernaculum, genital papillae covered with bursa in male and filiform tail with anal pore, gravid uteri in female, enable important information on the anatomical nature of the species. This study corroborates previous observations of fine transverse ridges in the cuticle of C. brenneri (Sudhaus and Kiontke, 2007). Additionally, the elongated buccal tube and distinct pharyngeal structure observed in the specimens align with descriptions of C. brenneri morphology provided by Stevens et al., 2019. Furthermore, the observations provide insight for the taxonomic identification of the nematode of Caenorhabditis genera. The molecular and phylogenetic analyses revealed distinct genetic identities and evolutionary relationships among the studied nematode isolates. The higher sequence similarity of query sequences to C. brenneri isolate from Sumatra and their monophyletic clustering suggest either recent common ancestry or ongoing gene flow between populations, potentially facilitated by natural dispersal mechanisms. The closer relationship between query IT20 and IT21 sequence reflect localized genetic differentiation due to isolation or selective pressures in their specific microhabitats. The strong identity of other five isolates to Oscheius sp. from South Africa could indicate shared environmental conditions favoring these bacterial-feeding nematodes in similar ecological niches. The IT13's identity with Italian R. axei may result from its cosmopolitan distribution in organic-rich habitats. The relatively low similarity of IT15 to T. jeffdanielsi from the USA suggests a divergent local strain, potentially arising from rapid evolution in parasitic nematodes adapting to new hosts. The 18S rRNA results showing remarkable identity to Trinidad's C. brenneri CB5161 and their Elegans group placement likely reflect conserved ribosomal sequences in this widely distributed species, with subclade formation possibly indicating incipient speciation or microevolutionary adaptations to different ecological conditions. These patterns align with known nematode biogeography (Kiontke et al., 2011) where both natural dispersal and human activities contribute to the global distribution of Caenorhabditis nematodes, while local environmental factors drive genetic differentiation at finer scales. To analyze the population genetic diversity analysis based on ITS2 sequences highlighted notable differentiation between the two identified groups, reflecting underlying evolutionary dynamics within the studied taxa. The elevated diversity in Group1 may suggest either a larger effective population size, historical admixture events, or exposure to heterogeneous environmental conditions that promoted mutation accumulation and lineage diversification (Booy et al., 2000). Interestingly, neutrality tests, including Tajima’s D and Fu and Li’s F, yielded non-significant results for both groups, suggesting that the observed polymorphisms are consistent with neutral evolution rather than being driven by strong directional selection or demographic expansion/contraction (Nei, 2005). The moderate nucleotide diversity observed across the entire dataset along with 48 segregating sites supports the conclusion that there is substantial genetic variation present, which could have taxonomic, epidemiological, or ecological implications depending on the identity of the organisms studied. Overall, this pattern of genetic variation reflects both intra- and inter-group diversity, possibly shaped by a combination of evolutionary forces such as mutation, drift, and historical demography. Future studies involving broader genomic regions and additional populations could further elucidate the evolutionary relationships and demographic history of these groups. The study presented a comparative analysis of reproductive output and lifespan among the isolates of C. brenneri and C. elegans (N2), revealing key differences that may reflect evolutionary trade-offs between reproductive strategies and longevity (Reznick, 1983). The result reveled that C. elegans (N2) produced significantly (p<0.01) more compared to the isolates of C. brenneri . This finding is consistent with previous studies suggesting that self-fertilizing species like C. elegans tend to have higher reproductive rates compared to species that require mating, such as C. brenneri (Cutter, 2008). The higher progeny yield from outcrosses among the four C. brenneri isolates suggests robust reproductive compatibility, likely facilitated by limited genetic divergence and possible local adaptation or recent gene flow among these strains. This result reinforces the notion that C. brenneri , while genetically diverse, retains high levels of fertility across its lineages (Cutter, 2008; Dey et al., 2013). In contrast, the markedly low progeny output in all crosses involving C. elegans N2 indicates strong pre- or post-zygotic reproductive isolation. This finding is consistent with prior studies that have shown C. elegans to be largely reproductively isolated from other Caenorhabditis species, particularly from C. brenneri , due to a combination of genetic divergence, karyotypic incompatibility, and rapid evolution of reproductive proteins (Dey et al., 2013; Nigon and Félix, 2018). Hybrid incompatibility in Caenorhabditis species has often been attributed to Dobzhansky-Muller incompatibilities, where interacting loci from divergent lineages result in reduced hybrid viability or fertility (Bundus et al., 2018). Lifespan analysis highlighted significant differences between the two species. C. elegans exhibited 100% lethality by day 31, whereas more than 50% of C. brenneri (Syt11) individuals survived beyond 20 days, with the survival difference being highly significant. These findings align with the broader theory that species with lower reproductive rates often exhibit extended lifespans, as seen in other model organisms (Partridge & Gems, 2002). The significant longevity observed in C. brenneri may be driven by distinct physiological mechanisms, potentially linked to differences in genetic pathways regulating aging, such as the insulin/IGF-1 signaling pathway (Murphy & Hu, 2013). Conversely, no notable differences were observed among the C. brenneri isolates, even when analyzed based on sex-specific comparisons. Having biological similarity in terms of longevity and brood size, the study utilized the Syt11 strain ( C. brenneri ) as a representative reference for other comparative analyses in response to the stressors. The thermos-tolerance assay revealed C. brenneri isolate (Syt11) likely to have higher survival% than C. elegans (N2). These findings align with the research of Lithgow et al., (1994) and Golden et al., (2020), which reported similar susceptibility to prolonged heat stress. The marginally higher thermal tolerance of C. brenneri may reflect species-specific adaptations, potentially linked to enhanced expression of heat shock proteins or stress response mechanisms, as suggested in previous studies (Jhaveri et al., 2023). This highlights the need for further molecular investigations to understand the genetic basis of interspecies differences in thermal resilience. On PA14-seeded plates, C. brenneri showed consistently higher survival than C. elegans (N2). A similar trend was observed on S. aureus plates, where C. brenneri isolate (Syt11) again outperformed C. elegans . These findings align with studies like Tan et al. (1999), which documented C. elegans susceptibility to P. aeruginosa , and suggest that C. brenneri possesses enhanced stress resilience. This striking difference suggests C. brenneri possesses superior innate immunity possibly through enhanced antimicrobial peptide production, more effective pathogen avoidance or improved detoxification mechanisms making it a compelling model for studying bacterial resistance evolution. Further genetic and molecular comparisons between these strains could reveal key defensive pathways against bacterial pathogenesis. The survival curves for C. elegans and C. brenneri in response to Ivermectin, Levamisole, and Albendazole demonstrate distinct species-specific drug sensitivities but variable effects on the survivability shows similar trend, influenced by dose and time. The genetic differences among C . elegans strains mediate differential susceptibilities across the majority of (Macrocyclic lactones) MLs, (Benzimidazoles) BZs and nAChR agonists (Shaver et al., 2023). Ivermectin and Levamisole induced rapid, dose-dependent mortality in both species, with C. elegans showing a sharper decline within the first 6 hours, particularly at higher concentrations, while C. brenneri responded more gradually. Unlike antimicrobial resistance gene transfer between pathogenic and non-pathogenic bacteria (Acar & Rostel, 2001), no analogous pathway has been established for anthelmintic resistance. In contrast, Albendazole displayed a transient effect, maintaining a higher survival% in C. brenneri which is indicative of resilience. As variation in response to BZs is driven by genetic difference among naturally diverse strain, it may be indicative that closely related siblings follow the same trajectories as the intra-species lineages do. Pearson correlation analysis revealed stronger associations between drug category and dose with survival in C. brenneri , compared to moderate-to-weak correlations in C. elegans , while time showed a negative correlation with survival for both species. ANOVA results underscored the significant effects of drug category, dose, and time, along with their interactions, on survival ( p < 0.001 ). These findings reinforce results from previous studies demonstrating genetic variability within C. elegans and its impact on drug resistance, emphasizing the role of genetic factors and interspecies differences in anthelmintic efficacy (Shaver et al., 2023). C. elegans remains a robust model for assessing the impact of genetic differences on phenotypic variation (Widmayer et al., 2022) and high-throughput assay (HTA) to screen natural compounds (Nyaanga et al., 2021). Similarly, its close relative C. brenneri can be used as a model for these purposes, especially in its ecological environment. Parasitic nematodes are known to be more genetically diverse than C. elegans and are capable of infecting nearly all animal species, making it crucial to understand how genetic diversity affects AR (Hahnel et al., 2020). Additionally, accurate AR testing in these genetically diverse parasites is challenging due to several factors: limited access to relevant life cycle stages, the absence of comprehensive global sample collections, host-dependent and costly laboratory life cycles, the complexity or lack of in vitro culture systems, and a restricted set of molecular tools (Hahnel et al., 2020). But, but the hyperdiversive C. brenneri, with its maintenance ease, can be complimentary to C. elegans model to identify and validate the responsibility of gene variants in AR. A notable factor contributing to the differences in Albendazole dose-response curves is the presence of circulating anthelmintic residues in soil and the vegetation growing on it (Navrátilová et al., 2023). Albendazole and its metabolites can cycle through ecosystems, from sheep dung to plants and back to livestock, impacting the expression of drug-metabolizing enzymes in parasitic nematodes like Haemonchus contortus (Navrátilová, 2024). These residues may also influence soil invertebrates, exerting selective pressure on free-living nematodes and promoting the development of resistance (Vokřál et al., 2023). Altogether, the continued characterization of C. brenneri presented additional avenues for investigating its morphology, genetics and phylogeny. Moreover, future work may elaborate on the functional relevance of genetic differences among C. brenneri as well as Caenorhabditis populations, with possible implications for adaptation, reproductive strategies and matters related to their interactions within the ecosystem. Conclusion This is a very first approach in Bangladesh to perform an ecological survey to browse Caenorhabditis nematodes, evaluating and comparing its biology with C. elegans . Additionally, the study was able to optimize an innovative sampling technique that led to the discovery of four wild isolates of C. brenneri as well as two nematodes, T. jeffdanielsi and R. axei, all for the first time in Bangladesh. This study needs more sampling efforts to find other species. Survival assays in different environment on C. brenneri and C. elegans can play key roles in identifying novel genetic factors that regulate many aspects of animal physiology, such as biology of aging and lifespan, stress response, and immunity against pathogens. Comparative biology of two nematodes can shed light on evolutionary dynamics: how reproductive modes evolve and adapt to different environments? how animals are keeping balance between lifespan and reproductive output? The hyperdiversive C. brenneri can be used as a genetic resource and useful to investigate species-specific responses to environmental stressors, disease pathogenesis, drug discovery and many other discipline. With the ease of maintenance, genome homology with C. elegans , hyperdiversive C. brenneri strains can be deployed to identify genes involvement in AR and can create a new dimension on species-specific host-microbe interaction. The report of getting two parasitic nematode indicate the host adaptability in different geography. Declarations Conflict of interest 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. Acknowledgments The authors gratefully acknowledge Prof. Dr. Md. Tanvir Rahman, Department of Microbiology & Hygiene, Bangladesh Agricultural University (BAU) and Dr. Muntasir Kamal, Department of Molecular Genetics, University of Toronto, Canada for generously providing the bacterial strains and N2 (wild type) C. elegans respectively. The abstracts were presented in the Hydra Conference on Parasitic Helminths - New Perspectives in Biology and Infection at the Hotel Bratsera on the island of Hydra in Greece from 1-6 September 2024 in a poster presentation and a short talk entitled “Ecological survey and molecular profiling of C. brenneri for the first time in Bangladesh” and also in the 10th Asia Pacific Worm Meeting (APWM 2024) during 13-16 June, 2024 in Bangalore, India hosted by the Indian Institute of Science (IISc). MHT had been awarded a travel and registration bursary by the organizers of Hydra 2024. Author contributions MRRR: Review of literature, investigation, resources, methodology, formal analysis, original draft preparation, writing – review and editing. MMRS, MMRZ, MA and NA: Methodology, investigation, formal analysis, writing – review and editing. BCR and MK: Co-supervision, conceptualization, resources, methodology, formal analysis, writing – review and editing. MHT: Conceptualization, methodology, project administration, supervision, validation, visualization, investigation, resources, writing – review and editing. Research ethics statement Not applicable Funding The authors gratefully acknowledge the funding (to Prof. MH Talukder; LS20222078), from the Grants for the Advanced Research and Education (GARE) by the Ministry of Education, Govt. of the Peoples Republic of Bangladesh on the 'Screening anthelmintic potency of plant extracts and compounds for biodiscovery using model nematode Caenorhabditis elegans and Trichostrongylid parasites (Grant award No. LS20222078). 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Toxicology . 153292 Wolf M, Nunes F, Henkel A, Heinick A, Paul RJ (2008) The MAP kinase JNK-1 of Caenorhabditis elegans : Location, activation, and influences over temperature-dependent insulin-like signaling, stress responses, and fitness. J Cell Physiol 214:721–729 Woodruff GC, Johnson E, Phillips PC (2019) A large close relative of C. elegans is slow-developing but not long-lived. BMC Evol Biol 19:1–4 Zwirchmayr J, Kirchweger B, Lehner T, Tahir A, Pretsch D, Rollinger JM (2020) A robust and miniaturized screening platform to study natural products affecting metabolism and survival in Caenorhabditis elegans . Sci Rep 10:12323 Tables Table 1. Distribution of bacterivorous worms across bio-geographical zones in selected regions. Locations Bio-geographical zones No of Samples No. of bacterivore worm containing samples ITS2 positive samples 18s rRNA positive samples Identified organism No. of strain (sequenced) Thakurgaon Himalayan piedmont plain 27 13 13 - Oscheus sp. 1 Rangpur Teesta floodplains 25 11 11 - Oscheus sp. 1 Bogura Barind tract 21 4 - - - - Mymensingh Brahmaputra-Jamuna floodplains 30 15 15 1 C. brenneri 1 Oscheus sp. 1 Bhola Offshore islands 28 2 2 - Oscheus sp. 1 Sylhet Surma-Kushiyara floodplains 29 19 19 3 C. brenneri 3 Oscheus sp. 1 Gazipur Madhupur sal forest 10 3 3 - Rhabditella axei 1 Dhaka 6 1 1 - Tarantobelus jeffdanielsi 1 Chattogram Chittagong hills 15 - - - - - Jhenaidah Ganges floodplains 18 - - - - - Barishal 16 - - - - - Total 225 68 64 4 11 Table 2. Genetic diversity and neutrality indices of ITS2 gene sequences across two distinct groups of C. brenneri isolates Gene Group Seq ID π±SD Hd S K D XY Tajima D Fu and Li's F test ITS 2 Group1 IT8 0.03762±0.008 1 42 24 0.04399 0.24617 (P > 0.10) 0.29927 (P > 0.10) IT20 IT21 PV324956.1 Group 2 JN636110.1 0.00470±0.001 1 6 3 -0.80861 (P > 0.10) -0.80861 (P > 0.10) JN636107.1 JN636108.1 JN636109.1 Total 0.03420±0.001 1 48 0 0 Hn: Number of Haplotypes, Hd: Haplotype (gene) diversity, π: Nucleotide diversity (per site), K: Average number of nucleotide differences between two randomly chosen sequences from within in the population, S: Number of variable/segregating sites. Dxy: The average number of nucleotide substitutions per site between populations Table 3. Intra and inter-species outcrossing experiment between C. brenneri isolates and C. elegans (N2). C. brenneri isolates C. elegans N2 XX Syt IT11 XX Syt IT8 XX Syt IT20 XX Syt IT21 XX Syt IT 11 XY X 142 105 133 7 Syt IT8 XY 118 X 131 172 6 Syt IT20 XY 123 107 X 158 11 Syt IT21 XY 165 204 215 X 13 Data within the table indicate the number of progeny in F2 generation resulting from selective outcrossing. Here, XX=male, XY=female Additional Declarations There is no duality of interest Supplementary Files Supplementaryfigure1.tif Supplementary figure 1. Supplementaryfigure2.tif Supplementary figure 2. SupplementaryTables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6856196","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":476995589,"identity":"30071790-5079-4094-96ec-2e4c1da1312d","order_by":0,"name":"MD TALUKDER","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYFAC5gbGBjYgLQHm2SSAKR4GhgSGA9g18DAwomhJI13LYcJa7NkPtknOKLPJ153d/PjDzx3n8/hnJDA+eNvGkMeHyxaexDbJDefSLLfdOWYm2XvmdrHEjQRmw7ltDMWSOB0G1PKw7bCB2Y0EMwbettuJDTcS2KR52xgSN+DSwv8QpOU/UEv6549/284lzr+RwP4brxYJoC0b2w4AteQYAA0/kLgBaAszXi03HjZbzjiXDNJSJi3bllxseOZhs+SccxI4/cLen3zwZk+ZHchhmz++bbPLkzuefPDDmzIbnCGGDTA2MMCiaRSMglEwCkYBeQAA7b9mkSA1feUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9538-2520","institution":"Bangladesh Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"MD","middleName":"","lastName":"TALUKDER","suffix":""},{"id":476995590,"identity":"6517a3ad-ff56-4c27-aba8-2ad6c9fb47e3","order_by":1,"name":"Md. Rajiur Rabbi","email":"","orcid":"https://orcid.org/0009-0004-2852-7362","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Md.","middleName":"Rajiur","lastName":"Rabbi","suffix":""},{"id":476995591,"identity":"2489cfb1-1795-43d6-b07e-6d12896bb6ab","order_by":2,"name":"Md. Sajib","email":"","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Md.","middleName":"","lastName":"Sajib","suffix":""},{"id":476995592,"identity":"6f5f4265-a723-4a90-a4fa-a1a2961b8918","order_by":3,"name":"Nurnabi Ahmed","email":"","orcid":"","institution":"International Centre for Diarrhoeal Diseases Research","correspondingAuthor":false,"prefix":"","firstName":"Nurnabi","middleName":"","lastName":"Ahmed","suffix":""},{"id":476995593,"identity":"567078ca-7058-4d69-8ee1-670445d68128","order_by":4,"name":"Md Makshuder Zim","email":"","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"Makshuder","lastName":"Zim","suffix":""},{"id":476995594,"identity":"f3b8497f-08f5-4b80-ad85-d3e26484c054","order_by":5,"name":"Mostak Ahmed","email":"","orcid":"https://orcid.org/0009-0009-5878-2184","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Mostak","middleName":"","lastName":"Ahmed","suffix":""},{"id":476995595,"identity":"10efdd76-e8ee-4b7f-a7d9-2832ff216286","order_by":6,"name":"Babul Roy","email":"","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Babul","middleName":"","lastName":"Roy","suffix":""},{"id":476995596,"identity":"76325b35-6734-4b9e-8515-62630b592800","order_by":7,"name":"Muntasir Kamal","email":"","orcid":"","institution":"Krembil Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Muntasir","middleName":"","lastName":"Kamal","suffix":""}],"badges":[],"createdAt":"2025-06-09 16:20:56","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6856196/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6856196/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85761916,"identity":"397ac20f-ab78-448f-b0f2-2447f7ea5453","added_by":"auto","created_at":"2025-07-01 11:47:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1475302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicromorphology of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. brenneri\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (adult).\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(A)\u003c/strong\u003e A female with a uterus containing eggs; \u003cstrong\u003e(B)\u003c/strong\u003eStoma of an adult with wide lips and long buccal cavity; \u003cstrong\u003e(C)\u003c/strong\u003e Anterior part with procorpus, bulbed metacorpus, isthmus and pear-shaped terminal bulb; \u003cstrong\u003e(D)\u003c/strong\u003ePosterior end ventral showing bursa, spicules, gubernaculum and genital papillae; (E) Mid posterior part of the body with numerous embryonated eggs and the vulva; \u003cstrong\u003e(F)\u003c/strong\u003e The striped pattern of the cuticle, anal pore and the filiform tail.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6856196/v1/70160aec7eb4d4abb70709f3.png"},{"id":85761426,"identity":"5fa68e86-dbed-47e1-b0e8-88ea43a1f59d","added_by":"auto","created_at":"2025-07-01 11:39:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":377249,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of ITS2 query sequences using maximum-likelihood(ML) algorithm and the number on each node correspond to the bootstrap analysis of 1000 replicates. The query sequences are highlighted in boldface type letters and the arrows in the node indicate bootstrap values.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6856196/v1/ce21ae24840e55ebbd83c874.png"},{"id":85762771,"identity":"d584b6b5-1d99-4a4e-96d3-aeb089a31c68","added_by":"auto","created_at":"2025-07-01 11:55:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":329650,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of query sequences of \u003cem\u003e18s rRNA\u003c/em\u003egene using maximum-likelihood(ML) algorithm and the number on each node correspond to the bootstrap analysis of 1000 replicates. The query sequences are highlighted in boldface type letters and the arrows in the node indicate bootstrap values.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6856196/v1/03acd74b1b41250f3141f650.png"},{"id":85761435,"identity":"782faddf-eb37-4310-89e9-77cd6f973947","added_by":"auto","created_at":"2025-07-01 11:39:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":55801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFecundity assay of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. elegans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. brenneri.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eData is presented in the bars as mean ± se.\u003cstrong\u003e \u003c/strong\u003eHere\u003cstrong\u003e, \u003c/strong\u003e*** = p\u0026lt; 0.0001 at 95% confidence intervals; ns= non-significant.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6856196/v1/fce68da91c07798d95ac097c.png"},{"id":85761427,"identity":"6c4b93e4-e00d-452d-9589-292e12ac5521","added_by":"auto","created_at":"2025-07-01 11:39:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":243812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLifespan assay of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. elegans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (N2) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. brenneri \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eisolates.\u003c/strong\u003e\u003cem\u003e \u003c/em\u003eData is presented in the bars as mean ± se.\u003cstrong\u003e \u003c/strong\u003eHere\u003cstrong\u003e, \u003c/strong\u003ens= non-significant and p\u0026lt;0.0001 indicate significant variation at 95% confidence intervals. (A) Sex linked longevity of four isolates of \u003cem\u003eC. brenneri\u003c/em\u003e. (B) Life span comparison of wild type strain N2 of \u003cem\u003eC. elegans \u003c/em\u003eand Cb Syt11 isolate of \u003cem\u003eC. brenneri.\u003c/em\u003e ns= non-significant.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6856196/v1/c48e345730889b0f34edef04.png"},{"id":85761918,"identity":"7befba91-68b1-4244-b078-dfeacec6883d","added_by":"auto","created_at":"2025-07-01 11:47:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":42079,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaplan-Meier survival analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. elegans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. brenneri\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eunder thermal stress at 36°C. \u003c/strong\u003eL4 larvae of N2 (wild type) \u003cem\u003eC. elegans\u003c/em\u003e and a local isolate (Cb Syt11) of \u003cem\u003eC. brenneri\u003c/em\u003e were subjected to repeated cycles of 2-hour exposure at 36°C followed by a rehabilitation phase at 20°C for 15–20 minutes. Data points represent the mean survival percentage of three independent trials (n = 20–30 worms per trial) with the quartiles.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6856196/v1/1b9e4f179272cd62622232ff.png"},{"id":85761915,"identity":"b9194c14-bec6-48ed-8473-c5bac3155c63","added_by":"auto","created_at":"2025-07-01 11:47:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":63026,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe survival percentage of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. elegans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (N2 strain) and a \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. brenneri\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e isolate over 24 hours following their transfer to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plates. \u003c/strong\u003eThe data, presented with Kaplan-Meier survival analysis suggest a time-dependent reduction in survival under pathogenic stress. \u003cstrong\u003e(A) \u003c/strong\u003eSurvival curves of two species on \u003cem\u003eS. aureus\u003c/em\u003e plate over a 24-hour period. \u003cstrong\u003e(B) \u003c/strong\u003eSurvival curves of two species in \u003cem\u003eP. aeruginosa\u003c/em\u003e plate over 24-hour period.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6856196/v1/0ecbadac40b4f33c919e4b02.png"},{"id":85761434,"identity":"bb6e21c6-0f99-4867-9821-22e392cb72f5","added_by":"auto","created_at":"2025-07-01 11:39:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":522453,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative survivability of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. elegans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (N2) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. brenneri\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e isolate (Cb Syt11) under three anthelmintic treatments at different time frame.\u003c/strong\u003e The survival percentages of \u003cem\u003eC. elegans\u003c/em\u003e(N2) and \u003cem\u003eC. brenneri\u003c/em\u003e isolate were assessed under three drug treatments— Ivermectin, Levamisole, and Albendazole— at varying concentrations. Data are presented in the survival curves as mean survival percentage.\u003cem\u003e C. brenneri \u003c/em\u003eisolate and \u003cem\u003eC. elegans \u003c/em\u003e(N2) exhibited similar trend in survival percentage at different concentration of \u003cstrong\u003e(A)\u003c/strong\u003e Ivermectin and\u003cstrong\u003e(B)\u003c/strong\u003e Levamisole\u003cstrong\u003e. (C)\u003c/strong\u003e\u003cem\u003e \u003c/em\u003eSignificant variation in the survival % of\u003cem\u003eC. elegans \u003c/em\u003e(N2) and\u003cem\u003e C. brenneri \u003c/em\u003eisolate in response to albendazole.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6856196/v1/efb8ef563b0a2145c33b894c.png"},{"id":91317419,"identity":"2cf7ff70-e44b-4dd2-bde4-64937273cef7","added_by":"auto","created_at":"2025-09-15 08:33:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4603555,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6856196/v1/a9f1c460-1985-4d6e-84f3-7a5a3ca3258f.pdf"},{"id":85761429,"identity":"9fb833d8-9bd8-4dfe-a2e2-2dce4ea1b524","added_by":"auto","created_at":"2025-07-01 11:39:43","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":818622,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figure 1.\u003c/p\u003e","description":"","filename":"Supplementaryfigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6856196/v1/537cf15d3ccfb33216322d66.tif"},{"id":85761433,"identity":"98933f7b-0a22-442a-b9a5-db4e8718f821","added_by":"auto","created_at":"2025-07-01 11:39:43","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2082822,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figure 2.\u003c/p\u003e","description":"","filename":"Supplementaryfigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6856196/v1/c3301a44c311b1b9ed88ab68.tif"},{"id":85761425,"identity":"80bdfca8-ddf7-494a-8033-7f59d2f6c157","added_by":"auto","created_at":"2025-07-01 11:39:43","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20168,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6856196/v1/83e8e90baf3a54b8c012112d.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Caenorhabditis brenneri as a complementary model organism to C. elegans: Insights from ecological, molecular and comparative biology","fulltext":[{"header":"Highlights","content":"\u003col start=\"1\" type=\"1\"\u003e\n \u003cli\u003eThis study investigates the lesser-known nematode \u003cem\u003eC. brenneri\u003c/em\u003e to understand its ecology and genetic diversity compared to \u003cem\u003eC. elegans\u003c/em\u003e.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eEleven distinct bio-ecological areas in Bangladesh were selected for sampling from pre-made baits were used to collect cultivate and analyze nematodes in a controlled laboratory setting.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eThe findings indicate that \u003cem\u003eC. brenneri\u003c/em\u003e has a longer lifespan and lower fecundity than \u003cem\u003eC. elegans\u003c/em\u003e, with distinct morphological traits aiding in identification.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMolecular analysis confirmed the species identity through amplification of the rDNA ITS2 and 18S rRNA regions, establishing a phylogenetic relationship within the\u003cem\u003e\u0026nbsp;Caenorhabditis\u003c/em\u003e nematodes\u003c/li\u003e\n \u003cli\u003eThis research highlights the genetic diversity of \u003cem\u003eC. brenneri\u003c/em\u003e as a valuable resource for future studies on environmental stress responses, drug discovery, and comparative genomics with \u003cem\u003eC. elegans\u003c/em\u003e.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eCaenorhabditis elegans\u003c/em\u003e, a cornerstone model organism, has made important contributions to biological fields namely developmental biology, neuroscience, and cell biology with current scientific interest also directed towards its natural ecology and evolutionary lineage (Barrière and Félix, 2005; Schulenburg and Félix, 2017). Its morphological simplicity and maintenance-ease allows more precise investigations into the neural circuitry. Furthermore, ithas been extensively studied genetically and molecularly (Rahmani et al., 2021), providing a wealth of data regarding chromosome mutations and facilitating genetic manipulation (Singh, 2021) and loci mapping (Andersen et al., 2022). The \u003cem\u003eC. elegans\u003c/em\u003e model has been successfully utilized in the field of apoptosis (Lettre and Hengartner, 2006), muscle differentiation (Fox et al., 2007) and high-throughput screening(HTS) for drug discovery (Zwirchmayr et al., 2020), mode of action of anthelmintic (Driscoll et al., 1989), anthelmintic resistance (Collins et al., 2024), host-microbe interactions (Backes et al., 2021; Kumar et al., 2020), heat-stress response (Nehammer et al., 2015) and toxicology (Hunt et al., 2020; Tejeda-Benitez and Olivero-Verbel, 2016) \u0026nbsp; serving as an impetus model to solve important research questions. As a free-living nematode, \u003cem\u003eC. elegans\u003c/em\u003e can help address key questions such as identifying the minimum number of genes necessary for a nematode to become parasitic and understanding the essential genetic requirements for successful parasitism (Geary et al., 2001). The morphological traits of \u003cem\u003eCaenorhabditis\u003c/em\u003e species often display convergence, posing challenges in species identification (Sudhaus et al., 2007). Therefore, a combination of cross-breeding for species identification and molecular sequence comparisons, alongside traditional morphological analysis, proves instrumental in accurate species diagnosis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. elegans\u003c/em\u003e and its close kin, including C. \u003cem\u003ebriggsae, C. remanei,\u003c/em\u003e and \u003cem\u003eC. brenneri\u003c/em\u003e are widely distributed in organic-rich microenvironments like decomposing plant material and compost heaps (Félix et al., 2013), distinct from typical soil-dwelling nematodes. The geographic distribution of the different species is also quite different, especially for \u003cem\u003eC. brenneri\u003c/em\u003e, which unlike the others has not yet been found in Europe or North America (Kiontke et al., 2005). In terms of reproduction, \u003cem\u003eC. elegans\u003c/em\u003e and \u003cem\u003eC. briggsae\u003c/em\u003e are hermaphroditic, whereas \u003cem\u003eC. remanei\u003c/em\u003e and \u003cem\u003eC. brenneri\u003c/em\u003e are gonochoristic (Sudhaus and Kiontke, 2007). Global sampling efforts have led to the discovery of over 50 putative species from the genus \u003cem\u003eCaenorhabditis\u003c/em\u003e (Stevens et al., 2019), many of which await formal species description. \u003cem\u003eC. brenneri\u003c/em\u003e, a nematode species, is known for its molecular diversity, exhibiting 14.1% polymorphic-synonymous sites, making it one of the most diverse eukaryotes globally (Stevens et al., 2019). \u003cem\u003eC. brenneri\u003c/em\u003e has been found in pan-tropical geography with extensive genetic diversity, allowing for viable genetic crosses between continents and showing weak geographic genetic structure (Dey et al., 2013). It was first isolated from the terrestrial slug \u003cem\u003ePhilippinella moellendorffi\u003c/em\u003e in the Philippines (Dalan et al., 2022). This cryptic nematode has also been documented in Eastern India (Dey et al., 2013), though no records exist for its presence in Bangladesh. Understanding the ecological distribution and genetic diversity of \u003cem\u003eCaenorhabditis\u003c/em\u003e nematodes worldwide is crucial for studying their adaptation to different environments and their potential implications for human health and agriculture. The introduction of \u003cem\u003eC. brenneri\u0026nbsp;\u003c/em\u003ehas expanded the understanding of nematode diversity and evolutionary complexity, providing a valuable model for studying genetic evolution and functional elements within genomes (Thomas, 2008). South Asian countries, particularly Bangladesh, are rich in medicinal flora with potential for discovering natural anthelmintic products; however, research in this area is significantly hindered by limited funding, inadequate model organism utilization, weak industry-academia partnerships and a lack of technological innovation (Kamal et al., 2023). A locally adapted, free-living nematode closely related to \u003cem\u003eC. elegans\u003c/em\u003e could help bridge the research gap in Bangladesh and other regions facing similar obstacles to nematode research. The survey aimed to position \u003cem\u003eC. brenneri\u003c/em\u003e as a complementary model with biological studies in different environment along with \u003cem\u003eC. elegans\u003c/em\u003e, while also examining the genetic diversity of \u003cem\u003eCaenorhabditis\u003c/em\u003e nematodes in different ecological niches and geographical regions of Bangladesh. The aim of this study is to gain insights into the population genetics and ecology of \u003cem\u003eCaenorhabditis\u003c/em\u003e nematodes in Bangladesh, which can further contribute to our understanding of the evolutionary ecology, adaptation and responses to environmental stressors of these nematodes. These observations will highlight the versatility and widespread distribution of \u003cem\u003eCaenorhabditis\u003c/em\u003e and other bacterivore nematodes across various ecosystems. Additionally, the comparative biology will reflect the differences and relativeness between two sibling species, ultimately providing a valuable insight into the utility of acclimatized nematodes such as \u003cem\u003eC. brenneri\u003c/em\u003e as complementary model organisms.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eStudy regions\u003c/p\u003e\n\u003cp\u003eThe present study covered 11 regions in Bangladesh, including Thakurgaon, Rangpur, Bogura, Mymensingh, Dhaka, Gazipur, Sylhet, Chattogram, Bhola, Barishal, and Jhenaidah, located in distinct bio-ecological zones such as the Himalayan piedmont plain, Barind tract, Teesta and Ganges floodplains, offshore islands, Chittagong hills, Madhupur sal forest, and the Brahmaputra-Jamuna and Surma-Kushiyara floodplains (Supplementary Figure 1). A total of 225 samples were collected in triplicate, with each replicate buried one foot apart (Supplementary Figure 2) and later pooled into a single sample. Sample collection took place between October 2023 and November 2024.\u003c/p\u003e\n\u003cp\u003eOptimized protocol for cultivation, collection and freezing of nematodes\u003c/p\u003e\n\u003cp\u003eSamples were procured from diverse geographical locations. For sampling, on Day 0, small tomato-cuts of 1-1.5 inch were excised and buried at a depth of 1-2 inches beneath the soil surface or just covered with a thin layer of loose soil in shaded areas to avoid direct sunlight exposure and feeding by birds, in triplicate and with each site being demarcated. Subsequently, on Day 1, Nematode Growth Media (NGM) was prepared, and a pure culture of \u003cem\u003eE. coli\u003c/em\u003e OP50 bacteria was cultivated (Stiernagle, 2006) and spread onto NGM plates. On Day 3, the decomposed tomatoes were retrieved and the decaying segments were carefully arranged along the periphery of NGM plates, instigating the emergence of worms. Following this, the samples were dispatched to \u003cem\u003eC. elegans\u003c/em\u003e model Sci. and Tech lab, Bangladesh Agricultural University using expedited shipping methods, ensuring temperature regulation (11\u0026deg;C to 25\u0026deg;C). On Day 4, surveillance of the Petri dishes began. Worms exhibited distinct morphological characteristics were isolated onto seeded NGM plates. To maintain homogeneity, 4 to 5 gravid individual worms were transferred using worm picker and allowed to reproduce naturally at 20-25\u0026ordm;C cooling incubator. This sub-culturing process was repeated at least three times for each isolate (Crombie et al., 2022). From the homogenous population of each isolate, freshly starved age synchronized cohorts (L1-L2) were produced that were then stored indefinitely in liquid nitrogen or -80\u0026ordm;C freezer. \u0026nbsp;In brief, soft agar freezing solution was melted using an autoclave and equilibrated in a 50\u0026deg;C water bath for at least 15 minutes. Freshly starved L1\u0026ndash;L2 worms were harvested by washing and subsequently an equal volume of soft agar freezing solution was added, mixed thoroughly and 1 ml of the mixture was aliquoted into labeled 1.8 ml cryovials. The vials were packed in a styrofoam box and frozen at \u0026minus;80\u0026deg;C overnight. The following day, the vials were transferred to permanent storage and worm viability was assessed from one vial (Brenner, 1974).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMorphological analysis\u003c/p\u003e\n\u003cp\u003eBased on previous studies (Sudhaus and Kiontke, 2007; Dalan et al. 2022), worms having double bulbed pharynx and morphology similar to that of \u003cem\u003eC. elegans or Caenorhabditis spp,\u0026nbsp;\u003c/em\u003ewereadvanced for micro-morphology. For microphotography, worms were placed in a drop of M9 buffer containing 5% Levamisole on 2% agar pads and studied at different magnification with Olympus (Olympys Corporation, Tokyo, Japan) light microscope BX53 equipped with Olympus DP27 microscope digital camera and cellSens imaging software (Olympus Corporation, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNucleic acid preparation and polymerase chain reaction (PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWorms having similar morphology to \u003cem\u003eC. elegans\u003c/em\u003e or \u003cem\u003eCaenorhabditis sp.\u0026nbsp;\u003c/em\u003eand also bacterivore nematodes were subjected to DNA extraction. The genomic DNA was extracted from (n = 15-20) adult worms including \u003cem\u003eC. elegans\u003c/em\u003e adults using the TIANamp Genomic DNA Kit (TIANGEN Biotech (Beijing Co., Ltd.) according to the manufacturer\u0026apos;s instructions. Subsequently, the extracted genomic DNAs were subjected to PCR amplification using two pairs of primers. This study performed two separate PCRs for each sample. The first primer set amplifies a 2,000-bp fragment of the internal transcribed spacer region between the 5.8S and 28S rDNA genes (ITS2); oECA1687F CTGCGTTACTTACCACGAATTGCARAC, oECA202R GCGGTATTTGCTACTACCAYYAMGATCTGC. The second set of primers, CE1209F (5\u0026prime;-TACTGTCAGTTTCGACTGACTC-3\u0026prime;) and CE2250R (5\u0026prime;-ATACGAACCCGAAGATTCGCC-3\u0026prime;), which amplify about 1,000 bp of the 18S rRNA gene of \u003cem\u003eCaenorhabditis\u003c/em\u003e nematodes. PCRs were performed in 25 \u0026mu;l reaction volume containing 12.5 \u0026mu;l Premix Taq\u0026trade; (TaKaRa Bio, Japan), 1.5 \u0026mu;l forward primer, 1.5 \u0026mu;l reverse primer, 4.5 \u0026mu;l molecular grade water, 5 \u0026mu;l genomic DNA. The cycling profile for CE1209F\u003cstrong\u003e+\u003c/strong\u003eCE2250R: 2 min at 95\u0026deg;C, followed by 35 cycles of 20 s at 95\u0026deg;C, 1 min 55 \u0026deg;C, 1 min 30 s at 72\u0026deg;C and a final extension of 10 min at 72\u0026deg;C (Haber et al., 2005). Another cycling profile for oECA1687\u003cstrong\u003e+\u003c/strong\u003eoECA202R: 3 min at 95 \u0026deg;C, followed by 35 cycles of 15 s at 95\u0026deg;C, 15 s at 55\u0026deg;C, 2 min 72\u0026deg;C Got to step 2, a final extension of 5 min at 72\u0026deg;C and hold at 12\u0026deg;C (Kiontke et al., 2011). The PCR products were analyzed by gel electrophoresis (1.5%) stained with Midori Green Advance (NIPPON Genetics EUROPE).\u003c/p\u003e\n\u003cp\u003eGene sequencing\u003c/p\u003e\n\u003cp\u003ePCR products were run on an agarose gel and then purified. The single-stranded products were generated from purified products using cycle sequencing PCR with forward or reverse primers. The amplified products were run on a Sanger machine using the di-deoxy chain termination method at Genecreate Biotech, China (Sanger et al., 1977).\u003c/p\u003e\n\u003cp\u003eSequence alignment, phylogenetic relationship and genetic diversity analysis\u003c/p\u003e\n\u003cp\u003eGenerated sequences were BLASTn-searched to compare with all sequences deposited in the National Centre for Biotechnology (NCBI) (Altschul et al., 1990) for closely related species. All obtained sequences were trimmed and annotated. Phylogenetic analysis was done with other identified closely related species based on the BLASTn output. Multiple sequence alignment was made using MUSCLE in MEGA v.11 (Tamura et al., 2021) followed by manual trimming to remove poorly aligned regions. Phylogenetic modeling and tree visualization were achieved by applying MEGA v.11 with the Maximum Likelihood(ML) method (Tamura et al., 2021). The phylogenetic tree was validated by running the analysis on 1000 bootstrapped input datasets and cross-referencing it against the Tamura-Nei substitution model. Population nucleotide diversity indices such as nucleotide diversity per site(\u0026pi;), average number of nucleotide differences(k), mean genetic diversity(Hd), number of segregating sites(S), average number of nucleotide substitutions per site(K), pairwise divergence(D\u003csub\u003eXY\u003c/sub\u003e) and neutrality tests including Tajima\u0026rsquo;s D and Fu Li\u0026rsquo;s F test were calculated using DnaSP ver. 6.12.03 (Rozas et al., 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eComparative biology of \u003cem\u003eC. brenneri\u003c/em\u003e isolates and \u003cem\u003eC. elegans\u003c/em\u003e (N2)\u003c/p\u003e\n\u003cp\u003eFecundity assay\u003c/p\u003e\n\u003cp\u003eFecundity assay was performed in according to the previous study (Woodruff et al., 2019). In brief, larvae production was monitored daily following overnight mating and under continuous male exposure. For all observations, L4 of four \u003cem\u003eC. brenneri\u0026nbsp;\u003c/em\u003eisolatesand \u003cem\u003eC. elegans\u003c/em\u003e (N2) strains, maintained at 25\u0026deg;C, were isolated and reared to adulthood. During overnight mating, single adult females were transferred to fresh plates and mated with at least six males overnight. The following day, males were removed and embryos and larvae were counted daily. Egg-laying females were transferred to new plates and new progeny were recorded until females ceased laying. In the continuous mating condition, females were always paired with six males. Males that crawled up the plate or died before the female stopped laying were replaced with young adult males. These continuous mating observations were conducted at 25\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eIntra and inter species outcrossing\u003c/p\u003e\n\u003cp\u003eFor all mating experiments, L4-stage females were paired with males on seeded NGMs and left overnight at 25\u0026deg;C for mating. The following day, males were removed and embryos and larvae were counted daily. A 1:2 female-male ratio was used for all crosses, except for interspecies crosses with \u003cem\u003eC. elegans\u003c/em\u003e males, where a 1:3 ratio was employed due to the lower mating efficiency of \u003cem\u003eC. elegans\u003c/em\u003e males compared to dioecious males. \u0026nbsp;Egg-laying females were transferred to new plates and new progeny were recorded until females ceased laying (Jessica et al., 2024). Mating tests were counted as positive if viable progeny were produced and a fertile second or third generation was observed (Sudhaus and Kiontke, 2007).\u003c/p\u003e\n\u003cp\u003eLifespan assay\u003c/p\u003e\n\u003cp\u003eSynchronized population (L4) of \u003cem\u003eC. brenneri\u003c/em\u003e isolates and \u003cem\u003eC. elegans\u003c/em\u003e (N2) were collected using a standard bleaching assay. \u0026nbsp;A total of 12\u0026ndash;15 healthy L4 larvae were transferred to freshly seeded plates, creating a stack of 6 plates per isolates or strains, which were labeled and returned to the cooling incubator in order to reach their adulthood. The following day, parents were transferred to fresh plates, ensuring no larvae or eggs were carried over and maintaining the consistency of the plates. After animals stopped producing progeny, lifespans were scored every other day until all animals had been scored as dead or censored. The worm was counted as alive if it moves in response to the tapping. All dead or censored worms were removed from the plate to avoid confusion and recounting the same animal. Worms that flee were typically removed from analysis. This assay was done by maintaining the temperature of 25 \u0026deg;C (Amrit et al., 2014).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of thermal tolerance\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThermal tolerance of the \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003e\u003cem\u003e(N2)\u0026nbsp;\u003c/em\u003eand a local isolate of \u003cem\u003eC. brenneri\u003c/em\u003e (Syt11) was evaluated at 36\u0026ordm;C. For each trial, 20 to 30 L4 larval stages of each strain were transferred to fresh NGM plates seeded with OP50 \u003cem\u003eE. coli\u003c/em\u003e. In this assay, we have modified the protocol described previously (Lithgow et al., 1994; Wolf et al., 2008 and Golden et al., 2020). In brief, synchronized worms were incubated at 36\u0026ordm;C for 2h, followed by a rehabilitation phase at 20\u0026ordm;C in a cold incubator for 15-20 minutes. This cycle was repeated until all worms were dead. Worms were gently prodded after each rehabilitation phase with a platinum worm pick and those that failed to respond were recorded as dead. This assay was repeated twice and standard male female ratio was maintained.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNematicidal sensitivity assay of anthelmintics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAge synchronized cohorts of L4 larvae from \u003cem\u003eC. elegans\u003c/em\u003e (N2) and \u003cem\u003eC. brenneri\u003c/em\u003e isolate (Syt11) were utilized for this assay. The assay was performed using 24 well microtitre plates, each well containing 250 \u0026micro;L of serially diluted anthelmintics with three replicates per concentration. The test compounds - ivermectin, levamisole and albendazole were prepared with 0.1% DMSO at concentrations of 0.05 mg/mL, 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL. Worm mortality rate was determined after 1hr, 3 h, 6 h, 12 h, 24 h and 48 h at 20 \u0026deg;C. A total of (20 to 30) L4 \u003cem\u003eC. elegans\u003c/em\u003e (N2) and local isolate of \u003cem\u003eC. brenneri\u003c/em\u003e were pipetted per well, thereafter the plates were sealed and incubated at 25\u0026ordm;C. Nematodes are considered dead when the body is straight, there is no head thrusting, body bending, tail movement or pharyngeal pumping for a continuous 10s of observation period (Skantar et al., 2005). \u0026nbsp;Standard male:female ratio was maintained and repeated counting was ensured.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial killing assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll assays were conducted at 25\u0026deg;C and with standard sex ratio. Worms were scored as alive or dead by gentle prodding with a platinum wire and sometimes with clear or transparent body. The counting was done in every hour for the first five hours and then at hour 24. Experiments were performed at least twice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;killing assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAssays were performed as described (Sifri et al., 2003). Briefly, NCTC8325 (or mutant derivatives, as noted) was grown overnight in tryptic soy broth (TSB, BD, Sparks, MD) with 10 \u0026micro;g/ml nalidixic acid (Sigma). Almost 5\u0026ndash;10 \u0026micro;l of overnight cultures were seeded on 35 mm tryptic soy agar (TSA) plates with 10 \u0026micro;g/ml nalidixic acid. A total of 25\u0026ndash;35 L4 stage of both worms were transferred to each of three replicate plates per species. Animals that died as a consequence of a bursting vulva or crawled off the agar were removed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;killing assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBriefly, PA14 was cultured in Luria broth (LB), seeded on plates with 5\u0026ndash;10 \u0026micro;l culture and incubated first for 15h at 37\u0026deg;C and then for 24 h at 25\u0026deg;C. A total of 25\u0026ndash;35 L4 stage both \u003cem\u003eC. elegans\u003c/em\u003e (N2) and \u003cem\u003eC. brenneri\u003c/em\u003e isolate was transferred to each of three replicate plates per strain. Worms that don\u0026rsquo;t response while prodding and appears clear counted as dead and then removed from the plate.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data and statistical analyses were done with Microsoft Excel, SPSS and R depending on suitability. Data was compared using ANOVA (Analysis of Variance), T-tests, Tukey HSD test for post-hock analysis, at 95% confidence intervals (CI) and presented as mean \u0026plusmn; standard error(SE) using SPSS version 29. Correlation coefficients were assessed using Pearson correlation in R to evaluate relationships between variables. \u0026nbsp;To make Kaplan-Meier survival curves survival package (Rstudio) and then pairwise comparisons between survival curves was obtained using the log rank test or Cox proportional hazards regression analyses (Rstudio) to test for differences between species and interactions with treatments.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eDetection of worms across the bio-ecological regions of Bangladesh\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOut of 225 samples collected from 11 regions, only those from Sylhet (n=29), Thakurgaon (n=25), Rangpur (n=27), Gazipur (n=10), Dhaka (n=6), Bogura (n=21), Mymensingh (n=30), and Bhola (n=29) tested positive for bacterivorous worms, while no bacterivore worms were found in samples from Chattogram (n=15), Barishal (n=16) and Jhenaidah (n=18) (Table 1). With the positive samples, worms were sorted from heterogeneous population on the basis of morphology and cultured separately to prepare homogenous cohorts in a cooling incubator at 25\u0026ordm;C. Nematodes with a double-bulbed pharynx and a brown to transparent color were identified as potential \u003cem\u003eCaenorhabditis\u003c/em\u003e nematodes and were further examined under microscopy for detailed morphology. Furthermore, all bacterivore worms culture was maintained for nucleic acid extraction to perform molecular characterization.\u003c/p\u003e\n\u003cp\u003ePhenotypic profiling\u003c/p\u003e\n\u003cp\u003eNematodes similar with the micromorphology of \u003cem\u003eCaenorhabditis\u003c/em\u003e nematodes were taken into account for detailed phenotypic characterization. Most phenotypic traits found - Fine transverse ridge in the cuticle; Buccal tube was longer than lip; pharynx exhibited a strong, oval median bulb and a rounded terminal bulb; In male: Testis was single. Tail was covered with a fan shaped closed bursa, comprised with pairs of rod like genital papillae or rays; Spicules are long and slender with a pointed tip; Gubernaculum is also slender; In female: Tail is conical with filiform tip. Anus and rectum are clearly visible. Uterus carried embryos (oviparous to ovoviviparous) at different stages of development (Figure 1).\u003c/p\u003e\n\u003cp\u003ePhylogenetic analysis and taxonomic positioning of \u003cem\u003eC. brenneri\u003c/em\u003e strains and other nematodes\u003c/p\u003e\n\u003cp\u003eAll 64 bacterivorous worm samples tested positive for \u003cem\u003eITS2\u003c/em\u003e amplification, while only 4 samples were positive for \u003cem\u003e18S rRNA\u003c/em\u003e amplification. Of the \u003cem\u003eITS2\u003c/em\u003e-positive samples, 10 were selected for sequencing, and all 18S \u003cem\u003erRNA\u003c/em\u003e-positive samples were also sequenced. The \u003cem\u003eITS2\u003c/em\u003e amplified query sequences IT11, IT8, IT20, and IT21 showed 93.24%, 95.01%, 96.10%, and 96.95% identity, respectively, to \u003cem\u003eCaenorhabditis brenneri\u003c/em\u003e strain SB129 (JN636110.1) from Sumatra (Bohorok). Similarly, IT3, IT4, IT14, IT16, and IT17 exhibited 99.38%, 98.77%, 99.26%, 99.38%, and 98.75% identity, respectively, to \u003cem\u003eOscheius\u003c/em\u003e sp. (MT482317.1) from South Africa. Furthermore, IT13 shared 98.80% identity with \u003cem\u003eRhabditella axei\u003c/em\u003e isolate (PP135624.1) from Riccio, Italy, while IT15 showed 98.58% identity to \u003cem\u003eTarantobelus jeffdanielsi\u003c/em\u003e isolate (OQ703326.1) from the USA. The \u003cem\u003e18S rRNA\u003c/em\u003e amplified query sequences CE11, CE8, CE20, and CE21 was found to be 99.03%, 99.00%, 99.36% and 99.25%, respectively, similar to the subject sequence of \u003cem\u003eCaenorhabditis brenneri\u003c/em\u003e strain CB5161 (U13930.1) from Trinidad.\u003c/p\u003e\n\u003cp\u003eThe phylogenetic relationship of the \u003cem\u003eITS2\u003c/em\u003e query sequences designated as IT11, IT8, IT20 and IT21 formed a distinct monophyletic clade within the \u003cem\u003eC. brenneri\u003c/em\u003e lineage, clustering closely with the reference \u003cem\u003eC. brenneri\u003c/em\u003e SB129 strain (JN636110.1) from Sumatra. Within the clade, IT20 and IT21 appeared more closely related to each other than to IT11 and IT8, forming a well-supported subclade (Figure 2). \u0026nbsp;The query sequences from \u003cem\u003eOscheius\u003c/em\u003e sp., \u003cem\u003eRhabditella axei\u003c/em\u003e, and \u003cem\u003eTarantobelus jeffdanielsi\u003c/em\u003e formed sister clades with their respective top BLAST hits, indicating close phylogenetic relationships. Similarly, ML analysis of query sequences of \u003cem\u003e18S rRNA\u003c/em\u003e gene revealed that \u003cem\u003eC. wallacei\u003c/em\u003e as sister to the group (Figure 3) consisting of the sister taxa CE8, CE11, CE20, CE21 + \u003cem\u003eCaenorhabditis sp.\u0026nbsp;\u003c/em\u003e(\u003cem\u003eC. brenneri\u003c/em\u003e)\u0026nbsp;CB5161\u0026nbsp;and the Elegans group. Within this clade, the query taxa CE8, CE11, CE20, and CE21 clustered within the Elegans group, showing closest relatedness to \u003cem\u003eC. brenneri\u003c/em\u003e CB5161. Notably, CE11 and CE20 formed one subclade, while CE8 and CE21 formed another, indicating close phylogenetic relationships. To add more, \u003cem\u003eC. macrosperma\u003c/em\u003e, \u003cem\u003eC. nouraguensis\u003c/em\u003e, \u003cem\u003eC. panamensis\u003c/em\u003e, \u003cem\u003eC. japonica\u003c/em\u003e, \u003cem\u003eC. waitukubuli\u003c/em\u003e are placed in Japonica group which is sister to \u003cem\u003eC. sulstoni\u003c/em\u003e and the Elegans group (Kiontke et al., 2011).\u003c/p\u003e\n\u003cp\u003eGenetic Diversity\u003c/p\u003e\n\u003cp\u003eGenetic diversity analysis of ITS2 sequences revealed that Group 1, comprising IT8, IT11, IT20 and IT21 exhibited higher nucleotide diversity (\u0026pi; = 0.03762 \u0026plusmn; 0.008) and a greater number of segregating sites (S = 42) compared to Group 2, which included sequences JN636110.1, JN636107.1, JN636108.1, and JN636109.1, with a lower nucleotide diversity (\u0026pi; = 0.00470 \u0026plusmn; 0.001) and fewer segregating sites (S = 6). Both groups showed haplotype diversity (Hd) of 1. Neutrality tests indicated no significant deviation from neutrality in either group, with Tajima\u0026rsquo;s D and Fu and Li\u0026rsquo;s F values being non-significant (P \u0026gt; 0.10). The overall dataset displayed a moderate nucleotide diversity (\u0026pi; = 0.03420 \u0026plusmn; 0.001) with a total of 48 segregating sites, further supporting the presence of genetic variation across the studied taxa (Table 2).\u003c/p\u003e\n\u003cp\u003eComparison of reproductive output and lifespan between \u003cem\u003eC. brenneri\u003c/em\u003e and \u003cem\u003eC. elegans\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe brood size for the four \u003cem\u003eC. brenneri\u003c/em\u003e isolates (Syt8, Syt11, Syt20 and Syt21) were 128 \u0026plusmn; 6.65, 117.6 \u0026plusmn; 4.72, 119.2 \u0026plusmn; 5.92 and 121.8 \u0026plusmn; 5.82 which exhibit significantly lower reproducibility (p\u0026lt;0.01) compared to the \u003cem\u003eC. elegans\u003c/em\u003e N2 wild-type: 237.41 \u0026plusmn; 13.2. But there was no significant variation among \u003cem\u003eC. brenneri\u003c/em\u003e isolates (Figure 4). The outcrossing experiment within four \u003cem\u003eC. brenneri\u003c/em\u003e isolates show higher progeny numbers, indicating strong outcrossing success among these introgressed strains (Table 3). In contrast, all crosses involving \u003cem\u003eC. elegans\u003c/em\u003e N2 produce very few progeny (6\u0026ndash;13), suggesting significant reproductive isolation or hybrid incompatibility between the \u003cem\u003eC. brenneri\u003c/em\u003e lineages and the N2 wild type.\u003c/p\u003e\n\u003cp\u003eFor the lifespan experiment, numbers of worms that were alive or dead were recorded each day for each strain tested. The count of number of worms alive on each day was not used directly in lifespan analysis. The number of worms that died on each day was inverted to calculate the proportion of worms alive on each day which is plotted graphically as a survival curve (Figure 5). The day of the timed egg laying is considered day 0. There was no significant difference in longevity among the four \u003cem\u003eC. brenneri\u003c/em\u003e isolates and sex did not have a significant effect on lifespan either (Figure 5B). Kaplan-Meier survival analysis was performed to compare \u003cem\u003eC. elegans\u003c/em\u003e (N2) with the \u003cem\u003eC. brenneri\u003c/em\u003e isolate (Syt11). This \u003cem\u003eC. brenneri\u003c/em\u003e isolate was chosen for lifespan and all subsequent analyses as a representative and having biological similarity with other isolates. Hermaphroditic wild type \u003cem\u003eC. elegans\u003c/em\u003e worms show 100% lethality after 30 days and survival is significantly higher(p\u0026lt;0.01) in gonochoristic \u003cem\u003eC. brenneri\u003c/em\u003e isolate (Syt11).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative assessment of thermal tolerance in \u003cem\u003eC. elegans\u003c/em\u003e and \u003cem\u003eC. brenneri\u003c/em\u003e under prolonged heat stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe thermal tolerance assay was performed to compare the thermal tolerance profile of \u003cem\u003eC. elegans\u003c/em\u003e (N2) and \u003cem\u003eC. brenneri\u0026nbsp;\u003c/em\u003eisolate (Syt11). By 2 hours of exposure at 36\u0026deg;C, \u003cem\u003eC. elegans\u003c/em\u003e (N2) shows a slight decrease to 91.06%, while the \u003cem\u003eC. brenneri\u003c/em\u003e isolate maintains a higher survival rate at 97.65% (Figure 6). Thereafter, survival percentages declined gradually for both species. Notably, by 6 hours, a significant reduction (\u003cem\u003ep\u0026lt;0.01\u003c/em\u003e) in survival was observed, with \u003cem\u003eC. brenneri\u003c/em\u003e isolate (Syt11) showing higher survival percentages (49.64%) compared to \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003e(22.23%). By the 8-hour mark, all individuals of both species were dead. These results suggest that while both species differ in thermo-tolerance profiles, \u003cem\u003eC. brenneri\u003c/em\u003e demonstrate a higher resistance to prolonged thermal stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNematicidal activity of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this \u003cem\u003eP. aeruginosa\u003c/em\u003e (PA14 seeded plates)\u0026nbsp;killing assay, \u003cem\u003eC. brenneri\u003c/em\u003e isolate (Syt11) demonstrated markedly stronger resistance compared to \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003eN2. By 5 hours, \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003eN2 survival plummeted to 32\u0026ndash;41%, while \u003cem\u003eC. brenneri\u003c/em\u003e isolate (Syt11) maintained 72\u0026ndash;79%, and by 24 hours, \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003eN2 exhibited 100% lethality, whereas \u003cem\u003eC. brenneri\u003c/em\u003e isolate (Syt11) retained 38\u0026ndash;45% of its population which indicate significant variability (p\u0026lt;0.0001) after the pairwise comparisons between survival curves (Figure 7B). A similar trend was observed on \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003e(NCTC8325 seeded plates) infected plates (Figure 7A), where \u003cem\u003eC. brenneri\u003c/em\u003e isolate (Syt11) shows significantly (p\u0026lt;0.0001) higher survival rate than \u003cem\u003eC. elegans\u003c/em\u003e N2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnthelmintic sensitivity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn both species, Ivermectin and Levamisole demonstrated rapid and potent effects, with significantly faster mortality at higher concentrations, especially notable in \u003cem\u003eC. elegans\u003c/em\u003e, where survival dropped sharply within the first 6 hours for doses of 0.25 mg/ml and above (Figure 8A). In contrast, \u003cem\u003eC. brenneri\u003c/em\u003e displayed a slightly more gradual response to these drugs, though the overall trend of faster decline with increased doses. Similar trend of sensitivity was observed in response to levamisole (Figure 8B). Albendazole showed a slower reduction in survival in both species, with \u003cem\u003eC. elegans\u003c/em\u003e exhibited more pronounced sensitivity over time compared to \u003cem\u003eC. brenneri\u003c/em\u003e isolate (Syt11), which retained a significant (p\u0026lt;0.001) higher survival percentage across all concentrations up to 48 hours (Figure 8C). This indicated that both speciesshowed almost similar response to all three drugs, particularly at higher doses and earlier time points, whereas \u003cem\u003eC. brenneri\u003c/em\u003e tends to exhibit a more gradual response in lower doses, suggesting \u003cem\u003eC. brenneri\u003c/em\u003e isolate shows a greater and robust resistance to albendazole compared to \u003cem\u003eC. elegans\u003c/em\u003e. The correlation co-efficients between drug category and dose concentration with the survival percentage ranged from strong to moderate \u003cem\u003e(r = -0.27 to 0.68)\u003c/em\u003e in \u003cem\u003eC. brenneri\u0026nbsp;\u003c/em\u003e(Supplementary Table 2). In contrast, the correlation coefficients for drug category and dose concentration with the survival index were moderate to weak \u003cem\u003e(r = 0.15 to -0.49)\u0026nbsp;\u003c/em\u003ein \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003e(Supplementary Table 1).Additionally, the time period exhibited a negative correlation (\u003cem\u003er= -0.41)\u003c/em\u003e in both species with the survival index as time increased. The analysis of variances (ANOVA) of two species indicated that all factors and their interactions have a highly significant impact (\u003cem\u003ep\u0026lt; 0.001\u003c/em\u003e) on the survival rate\u0026nbsp;(Supplementary Table 3). Post-hoc analysis of drug category, dose concentrations, and observation time revealed that, within the first hour, Albendazole exhibited a slower lethality at a concentration of 0.05 mg/mL compared to the other drugs in both species. Furthermore, significant intra-group differences (\u003cem\u003ep\u0026lt;0.01\u003c/em\u003e) were observed across all variables in terms of their effect on the survival. \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMost of the free-living \u003cem\u003eCaenorhabditis\u003c/em\u003e nematodes habitat in ephemeral patches rich in microbial blooms and sometimes in sympatry (Crombie et al., 2022). This study discovered new wild isolates of \u003cem\u003eC. brenneri\u003c/em\u003e from baits in Mymensingh and Sylhet, \u003cem\u003eOscheius sp.\u0026nbsp;\u003c/em\u003efromThakurgoan, Rangpur, Mymensingh, Bhola and Sylhet, \u003cem\u003eRhabditella\u003c/em\u003e \u003cem\u003eaxei\u003c/em\u003e from Gazipur and \u003cem\u003eTarantobelus\u003c/em\u003e \u003cem\u003ejeffdanielsi\u003c/em\u003e from Dhaka, Bangladesh. For the baiting procedure, this study tried two different approach as baits on the surface mostly attracted by predators and rodents, which lead to chance of sample missing in Bangladeshi context. This study tried also an innovative approach to identify \u003cem\u003eCaenorhabditis\u003c/em\u003e and other closely related nematodes. The place where the baits were buried was wet and shaded. The temperature during collection was 18-20ºC. Sylhet is characterized by its cooler climate throughout the year compared to other parts of the country, may provide favorable conditions for the colonization of \u003cem\u003eC. brenneri\u003c/em\u003e. Despite samples being collected in pre-winter and winter seasons, the overall climate of Sylhet supports a variety of species that thrive in cooler temperatures. Similarly, the riverside of old Brahmaputra river in Mymensingh, known for its fertile soil and microbial blooms may support greater ecological diversity of bacterivore nematodes. Mymensingh and Sylhet are also located in the north of the country not far from the Himalayan plains where temperatures are milder compared to the southern parts such as Chattogram. The milder conditions may support growth of bacterivore species in the \u003cem\u003eCaenorhabditis\u003c/em\u003e genus, although as this study has shown, there can be significant differences between the sibling species with respect to resistance to thermal stress, drug and microbial insults. Altogether, these environmental and geographical factors contribute to the successful colonization of this species in the region. Endemic gonochoristic elegans group species, which are quite numerous in eastern Asia (Dey et al., 2013) and South America (Sloat et al., 2022), appear to be absent from the neo-tropics. Recently, another nematode, \u003cem\u003eOscheius tipulae\u0026nbsp;\u003c/em\u003e(Family- Rhabditidae) was proposed as a model organism for NP-based anthelmintic screen (Kamal et al., 2023) in tropics. This species is phylogenetically more closely related to the insect parasite \u003cem\u003eHeterohabditis\u003c/em\u003e and the vertebrate parasites \u003cem\u003eStrongylida\u0026nbsp;\u003c/em\u003e(Kiontke et al., 2005). However, \u003cem\u003eC. brenneri\u003c/em\u003e possesses many advantages over \u003cem\u003eO. tipulae\u003c/em\u003e, such as being a sister species to the more well-established \u003cem\u003eC. elegans\u003c/em\u003e, and the fact that molecular biology reagents and techniques are standardized for \u003cem\u003eC. brenneri\u003c/em\u003e as they are for \u003cem\u003eC. elegans.\u003c/em\u003e It can be maintained in the laboratory and cryopreserved using the same set-ups used for \u003cem\u003eC. elegans.\u0026nbsp;\u003c/em\u003eOther nematode \u003cem\u003eRhabditella\u003c/em\u003e \u003cem\u003eaxei\u003c/em\u003e, which can be both parasitic or free living (Sciandra et al., 2024), feeds mainly on bacteria present in decaying organic matter, soil and other substrates. However, in its parasitic form, it can colonize some species of snails. This nematode was found in the Gazipur region may be due to abundant decaying organic matter and bacteria for its free-living form, warm humid conditions favoring proliferation and potential snail hosts enabling parasitic colonization, supported by agricultural and waste management practices. Additionally, tarantula-parasitic nematode, \u003cem\u003eTarantobelus\u003c/em\u003e \u003cem\u003ejeffdanielsi\u0026nbsp;\u003c/em\u003ewas first reported in Los Angeles, California, USA (Baniya et al., 2023) and it was also reported by this study for first time in Dhaka, Bangladesh. That may be for the climate adaptability of the nematode, or previously undetected native presence now identified. Previous study reported that \u003cem\u003eT. jeffdanielsi\u003c/em\u003e successfully infected several different species of tarantulas as well as wax worms ( \u003cem\u003eGalleria mellonella\u003c/em\u003e ), indicating that this nematode may have a broad host range\u0026nbsp;\u003ca href=\"https://pmc.ncbi.nlm.nih.gov/articles/PMC10176109/#R10\"\u003e(Schurkman et al., 2022)\u003c/a\u003e . Most interestingly, \u003cem\u003eR. axei\u003c/em\u003e and \u003cem\u003eT. jeffdanielsi\u003c/em\u003e can be cultured with OP50 seeded NGM petri plates. This warrants further study in their biology and host adaptability. A total of 11 isolates of different species namely \u003cem\u003eC. brenneri\u003c/em\u003e (4), \u003cem\u003eOscheius sp\u003c/em\u003e. (5), \u003cem\u003eT. jeffdanielsi\u003c/em\u003e (1) and \u003cem\u003eR. axei\u003c/em\u003e (1) were successfully preserved in -80ºC. Additionally, all of the nematode strains used in this study, including the four new \u003cem\u003eC. brenneri\u003c/em\u003e isolates are being maintained in the \u003cem\u003eC. elegans\u003c/em\u003e model Science and Technology Lab, Department of Parasitology, Bangladesh Agricultural University and are available for research use upon request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe description of fine transverse ridges in the cuticle and the proximal and terminal bulbs, distinct features of the buccal tube, pharynx and spicules, gubernaculum, genital papillae covered with bursa in male and filiform tail with anal pore, gravid uteri in female, enable important information on the anatomical nature of the species. This study corroborates previous observations of fine transverse ridges in the cuticle of \u003cem\u003eC. brenneri\u0026nbsp;\u003c/em\u003e(Sudhaus and Kiontke, 2007). Additionally, the elongated buccal tube and distinct pharyngeal structure observed in the specimens align with descriptions of \u003cem\u003eC. brenneri\u003c/em\u003e morphology provided by Stevens et al., 2019. Furthermore, the observations provide insight for the taxonomic identification of the nematode of \u003cem\u003eCaenorhabditis\u003c/em\u003e genera.\u003c/p\u003e\n\u003cp\u003eThe molecular and phylogenetic analyses revealed distinct genetic identities and evolutionary relationships among the studied nematode isolates. The higher sequence similarity of query sequences to \u003cem\u003eC. brenneri\u003c/em\u003e isolate from Sumatra and their monophyletic clustering suggest either recent common ancestry or ongoing gene flow between populations, potentially facilitated by natural dispersal mechanisms. The closer relationship between query IT20 and IT21 sequence reflect localized genetic differentiation due to isolation or selective pressures in their specific microhabitats. The strong identity of other five isolates to \u003cem\u003eOscheius sp.\u003c/em\u003e from South Africa could indicate shared environmental conditions favoring these bacterial-feeding nematodes in similar ecological niches. The IT13's identity with Italian \u003cem\u003eR. axei\u003c/em\u003e may result from its cosmopolitan distribution in organic-rich habitats. The relatively low similarity of IT15 to \u003cem\u003eT. jeffdanielsi\u003c/em\u003e from the USA suggests a divergent local strain, potentially arising from rapid evolution in parasitic nematodes adapting to new hosts. The \u003cem\u003e18S rRNA\u003c/em\u003e results showing remarkable identity to Trinidad's \u003cem\u003eC. brenneri\u003c/em\u003e CB5161 and their Elegans group placement likely reflect conserved ribosomal sequences in this widely distributed species, with subclade formation possibly indicating incipient speciation or microevolutionary adaptations to different ecological conditions. These patterns align with known nematode biogeography (Kiontke et al., 2011) where both natural dispersal and human activities contribute to the global distribution of \u003cem\u003eCaenorhabditis\u003c/em\u003e nematodes, while local environmental factors drive genetic differentiation at finer scales.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo analyze the population genetic diversity analysis based on \u003cem\u003eITS2\u003c/em\u003e sequences highlighted notable differentiation between the two identified groups, reflecting underlying evolutionary dynamics within the studied taxa. The elevated diversity in Group1 may suggest either a larger effective population size, historical admixture events, or exposure to heterogeneous environmental conditions that promoted mutation accumulation and lineage diversification (Booy et al., 2000). Interestingly, neutrality tests, including Tajima’s D and Fu and Li’s F, yielded non-significant results for both groups, suggesting that the observed polymorphisms are consistent with neutral evolution rather than being driven by strong directional selection or demographic expansion/contraction (Nei, 2005). The moderate nucleotide diversity observed across the entire dataset along with 48 segregating sites supports the conclusion that there is substantial genetic variation present, which could have taxonomic, epidemiological, or ecological implications depending on the identity of the organisms studied. Overall, this pattern of genetic variation reflects both intra- and inter-group diversity, possibly shaped by a combination of evolutionary forces such as mutation, drift, and historical demography. Future studies involving broader genomic regions and additional populations could further elucidate the evolutionary relationships and demographic history of these groups.\u003c/p\u003e\n\u003cp\u003eThe study presented a comparative analysis of reproductive output and lifespan among the isolates of \u003cem\u003eC. brenneri\u003c/em\u003e and \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003e(N2), revealing key differences that may reflect evolutionary trade-offs between reproductive strategies and longevity (Reznick, 1983). The result reveled that \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003e(N2) produced significantly (p\u0026lt;0.01) more compared to the isolates of \u003cem\u003eC. brenneri\u003c/em\u003e. This finding is consistent with previous studies suggesting that self-fertilizing species like \u003cem\u003eC. elegans\u003c/em\u003e tend to have higher reproductive rates compared to species that require mating, such as \u003cem\u003eC. brenneri\u003c/em\u003e (Cutter, 2008). The higher progeny yield from outcrosses among the four \u003cem\u003eC. brenneri\u003c/em\u003e isolates suggests robust reproductive compatibility, likely facilitated by limited genetic divergence and possible local adaptation or recent gene flow among these strains. This result reinforces the notion that \u003cem\u003eC. brenneri\u003c/em\u003e, while genetically diverse, retains high levels of fertility across its lineages (Cutter, 2008; Dey et al., 2013). In contrast, the markedly low progeny output in all crosses involving \u003cem\u003eC. elegans\u003c/em\u003e N2 indicates strong pre- or post-zygotic reproductive isolation. This finding is consistent with prior studies that have shown \u003cem\u003eC. elegans\u003c/em\u003e to be largely reproductively isolated from other \u003cem\u003eCaenorhabditis\u003c/em\u003e species, particularly from \u003cem\u003eC. brenneri\u003c/em\u003e, due to a combination of genetic divergence, karyotypic incompatibility, and rapid evolution of reproductive proteins (Dey et al., 2013; Nigon and Félix, 2018). Hybrid incompatibility in \u003cem\u003eCaenorhabditis\u003c/em\u003e species has often been attributed to Dobzhansky-Muller incompatibilities, where interacting loci from divergent lineages result in reduced hybrid viability or fertility (Bundus et al., 2018).\u003c/p\u003e\n\u003cp\u003eLifespan analysis highlighted significant differences between the two species. \u003cem\u003eC. elegans\u003c/em\u003e exhibited 100% lethality by day 31, whereas more than 50% of \u003cem\u003eC. brenneri\u003c/em\u003e (Syt11) individuals survived beyond 20 days, with the survival difference being highly significant. These findings align with the broader theory that species with lower reproductive rates often exhibit extended lifespans, as seen in other model organisms (Partridge \u0026amp; Gems, 2002). The significant longevity observed in \u003cem\u003eC. brenneri\u003c/em\u003e may be driven by distinct physiological mechanisms, potentially linked to differences in genetic pathways regulating aging, such as the insulin/IGF-1 signaling pathway (Murphy \u0026amp; Hu, 2013). Conversely, no notable differences were observed among the \u003cem\u003eC. brenneri\u003c/em\u003e isolates, even when analyzed based on sex-specific comparisons. Having biological similarity in terms of longevity and brood size, the study utilized the Syt11 strain (\u003cem\u003eC. brenneri\u003c/em\u003e) as a representative reference for other comparative analyses in response to the stressors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe thermos-tolerance assay revealed \u003cem\u003eC. brenneri\u0026nbsp;\u003c/em\u003eisolate (Syt11) likely to have higher survival% than \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003e(N2). These findings align with the research of Lithgow et al., (1994) and Golden et al., (2020), which reported similar susceptibility to prolonged heat stress. The marginally higher thermal tolerance of \u003cem\u003eC. brenneri\u003c/em\u003e may reflect species-specific adaptations, potentially linked to enhanced expression of heat shock proteins or stress response mechanisms, as suggested in previous studies (Jhaveri et al., 2023). This highlights the need for further molecular investigations to understand the genetic basis of interspecies differences in thermal resilience.\u003c/p\u003e\n\u003cp\u003eOn PA14-seeded plates, \u003cem\u003eC. brenneri\u003c/em\u003e showed consistently higher survival than \u003cem\u003eC. elegans\u003c/em\u003e (N2). A similar trend was observed on \u003cem\u003eS. aureus\u003c/em\u003e plates, where \u003cem\u003eC. brenneri\u0026nbsp;\u003c/em\u003eisolate (Syt11) again outperformed \u003cem\u003eC. elegans\u003c/em\u003e. These findings align with studies like Tan et al. (1999), which documented \u003cem\u003eC. elegans\u003c/em\u003e susceptibility to \u003cem\u003eP. aeruginosa\u003c/em\u003e, and suggest that \u003cem\u003eC. brenneri\u003c/em\u003e possesses enhanced stress resilience. This striking difference suggests \u003cem\u003eC. brenneri\u003c/em\u003e possesses superior innate immunity possibly through enhanced antimicrobial peptide production, more effective pathogen avoidance or improved detoxification mechanisms making it a compelling model for studying bacterial resistance evolution. Further genetic and molecular comparisons between these strains could reveal key defensive pathways against bacterial pathogenesis.\u003c/p\u003e\n\u003cp\u003eThe survival curves for \u003cem\u003eC. elegans\u003c/em\u003e and \u003cem\u003eC. brenneri\u003c/em\u003e in response to Ivermectin, Levamisole, and Albendazole demonstrate distinct species-specific drug sensitivities but variable effects on the survivability shows similar trend, influenced by dose and time. The genetic differences among \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eelegans\u003c/em\u003e strains mediate differential susceptibilities across the majority of (Macrocyclic lactones) MLs, (Benzimidazoles) BZs and nAChR agonists (Shaver et al., 2023). Ivermectin and Levamisole induced rapid, dose-dependent mortality in both species, with \u003cem\u003eC. elegans\u003c/em\u003e showing a sharper decline within the first 6 hours, particularly at higher concentrations, while \u003cem\u003eC. brenneri\u003c/em\u003e responded more gradually. Unlike antimicrobial resistance gene transfer between pathogenic and non-pathogenic bacteria (Acar \u0026amp; Rostel, 2001), no analogous pathway has been established for anthelmintic resistance. In contrast, Albendazole displayed a transient effect, maintaining a higher survival% in \u003cem\u003eC. brenneri\u0026nbsp;\u003c/em\u003ewhich is indicative of resilience. As variation in response to BZs is driven by genetic difference among naturally diverse strain, it may be indicative that closely related siblings follow the same trajectories as the intra-species lineages do. Pearson correlation analysis revealed stronger associations between drug category and dose with survival in \u003cem\u003eC. brenneri\u003c/em\u003e, compared to moderate-to-weak correlations in \u003cem\u003eC. elegans\u003c/em\u003e, while time showed a negative correlation with survival for both species. ANOVA results underscored the significant effects of drug category, dose, and time, along with their interactions, on survival (\u003cem\u003ep \u0026lt; 0.001\u003c/em\u003e). These findings reinforce results from previous studies demonstrating genetic variability within \u003cem\u003eC. elegans\u003c/em\u003e and its impact on drug resistance, emphasizing the role of genetic factors and interspecies differences in anthelmintic efficacy (Shaver et al., 2023). \u003cem\u003eC. elegans\u003c/em\u003e remains a robust model for assessing the impact of genetic differences on phenotypic variation (Widmayer et al., 2022) and high-throughput assay (HTA) to screen natural compounds (Nyaanga et al., 2021). Similarly, its close relative \u003cem\u003eC. brenneri\u003c/em\u003e can be used as a model for these purposes, especially in its ecological environment. Parasitic nematodes are known to be more genetically diverse than \u003cem\u003eC. elegans\u003c/em\u003e and are capable of infecting nearly all animal species, making it crucial to understand how genetic diversity affects AR (Hahnel et al., 2020). Additionally, accurate AR testing in these genetically diverse parasites is challenging due to several factors: limited access to relevant life cycle stages, the absence of comprehensive global sample collections, host-dependent and costly laboratory life cycles, the complexity or lack of in vitro culture systems, and a restricted set of molecular tools (Hahnel et al., 2020). But, but the hyperdiversive \u003cem\u003eC. brenneri,\u003c/em\u003e with its maintenance ease, can be complimentary to \u003cem\u003eC. elegans\u003c/em\u003e model to identify and validate the responsibility of gene variants in AR. A notable factor contributing to the differences in Albendazole dose-response curves is the presence of circulating anthelmintic residues in soil and the vegetation growing on it (Navrátilová et al., 2023). Albendazole and its metabolites can cycle through ecosystems, from sheep dung to plants and back to livestock, impacting the expression of drug-metabolizing enzymes in parasitic nematodes like \u003cem\u003eHaemonchus contortus\u003c/em\u003e (Navrátilová, 2024). These residues may also influence soil invertebrates, exerting selective pressure on free-living nematodes and promoting the development of resistance (Vokřál et al., 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAltogether, the continued characterization of \u003cem\u003eC. brenneri\u003c/em\u003e presented additional avenues for investigating its morphology, genetics and phylogeny. Moreover, future work may elaborate on the functional relevance of genetic differences among \u003cem\u003eC. brenneri\u003c/em\u003e as well as \u003cem\u003eCaenorhabditis\u003c/em\u003e populations, with possible implications for adaptation, reproductive strategies and matters related to their interactions within the ecosystem.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis is a very first approach in Bangladesh to perform an ecological survey to browse \u003cem\u003eCaenorhabditis\u003c/em\u003e nematodes, evaluating and comparing its biology with \u003cem\u003eC. elegans\u003c/em\u003e. Additionally, the study was able to optimize an innovative sampling technique that led to the discovery of four wild isolates of \u003cem\u003eC. brenneri\u003c/em\u003e as well as two nematodes, \u003cem\u003eT. jeffdanielsi\u003c/em\u003e and \u003cem\u003eR. axei,\u0026nbsp;\u003c/em\u003eall for the first time in Bangladesh. This study needs more sampling efforts to find other species. Survival assays in different environment on \u003cem\u003eC. brenneri\u003c/em\u003e and \u003cem\u003eC. elegans\u003c/em\u003e can play key roles in identifying novel genetic factors that regulate many aspects of animal physiology, such as biology of aging and lifespan, stress response, and immunity against pathogens. Comparative biology of two nematodes can shed light on evolutionary dynamics: how reproductive modes evolve and adapt to different environments? how animals are keeping balance between lifespan and reproductive output? The hyperdiversive \u003cem\u003eC. brenneri\u0026nbsp;\u003c/em\u003ecan be used as a genetic resource and useful to investigate species-specific responses to environmental stressors, disease pathogenesis, drug discovery and many other discipline. With the ease of maintenance, genome homology with \u003cem\u003eC. elegans\u003c/em\u003e, hyperdiversive \u003cem\u003eC. brenneri\u003c/em\u003e strains can be deployed to identify genes involvement in AR and can create a new dimension on species-specific host-microbe interaction. The report of getting two parasitic nematode indicate the host adaptability in different geography.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge Prof. Dr. Md. Tanvir Rahman, Department of Microbiology \u0026amp; Hygiene, Bangladesh Agricultural University (BAU) and Dr. Muntasir Kamal, Department of Molecular Genetics, University of Toronto, Canada for generously providing the bacterial strains and N2 (wild type) \u003cem\u003eC. elegans\u003c/em\u003e respectively. The abstracts were presented in the Hydra Conference on Parasitic Helminths - New Perspectives in Biology and Infection at the Hotel Bratsera on the island of Hydra in Greece from 1-6 September 2024 in a poster presentation and a short talk entitled \u0026ldquo;Ecological survey and molecular profiling of \u003cem\u003eC. brenneri\u003c/em\u003e for the first time in Bangladesh\u0026rdquo; and also in the 10th Asia Pacific Worm Meeting (APWM 2024) during 13-16 June, 2024 in Bangalore, India hosted by the Indian Institute of Science (IISc). MHT had been awarded a travel and registration bursary by the organizers of Hydra 2024.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMRRR: Review of literature, investigation, resources, methodology, formal analysis, original draft preparation, writing \u0026ndash; review and editing. MMRS, MMRZ, MA and NA: Methodology, investigation, formal analysis, writing \u0026ndash; review and editing. BCR and MK: Co-supervision, conceptualization, resources, methodology, formal analysis, writing \u0026ndash; review and editing. MHT: Conceptualization, methodology, project administration, supervision, validation, visualization, investigation, resources, writing \u0026ndash; review and editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch ethics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the funding (to Prof. MH Talukder; LS20222078), from the Grants for the Advanced Research and Education (GARE) by the \u0026nbsp; Ministry of Education, Govt. of the Peoples Republic of Bangladesh on the \u0026apos;Screening anthelmintic potency of plant extracts and compounds for biodiscovery using model nematode \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e and Trichostrongylid parasites (Grant award No. LS20222078).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData archiving\u003c/p\u003e\n\u003cp\u003eAll kind of data is available within the manuscript\u003c/p\u003e\n\u003cp\u003eSupplementary information\u003c/p\u003e\n\u003cp\u003eSupporting information has been provided. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAcar J, Rostel B (2001) Antimicrobial resistance: an overview. \u003cem\u003eRev Sci Tech Off Int Epiz\u003c/em\u003e 20:797\u0026ndash;810\u003c/li\u003e\n \u003cli\u003eAltschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. \u003cem\u003eJ Mol Biol\u003c/em\u003e 215:403\u0026ndash;410\u003c/li\u003e\n \u003cli\u003eAmrit FRG, Ratnappan R, Keith SA, Ghazi A (2014) The \u003cem\u003eC. elegans\u003c/em\u003e lifespan assay toolkit. \u003cem\u003eMethods\u003c/em\u003e 68:465\u0026ndash;475\u003c/li\u003e\n \u003cli\u003eAmrit FR, Boehnisch CM, May RC (2010) Phenotypic covariance of longevity, immunity and stress resistance in the \u003cem\u003eCaenorhabditis\u003c/em\u003e nematodes. \u003cem\u003ePLoS One\u003c/em\u003e 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(Panagrolaimomorpha; Panagrolaimidae), a nematode parasite of tarantulas. \u003cem\u003eJ Parasitol\u003c/em\u003e 108:30\u0026ndash;43\u003c/li\u003e\n \u003cli\u003eSchulenburg H, F\u0026eacute;lix MA (2017) The natural biotic environment of \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e. \u003cem\u003eGenetics\u003c/em\u003e 206:55\u0026ndash;86\u003c/li\u003e\n \u003cli\u003eShaver AO, Wit J, Dilks CM, Crombie TA, Li H, Aroian RV, Andersen EC (2023) Variation in anthelmintic responses are driven by genetic differences among diverse \u003cem\u003eC. elegans\u003c/em\u003e wild strains. \u003cem\u003ePLoS Pathog\u003c/em\u003e 19:e1011285\u003c/li\u003e\n \u003cli\u003eSifri CD, Begun J, Ausubel FM, Calderwood SB (2003) \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e as a model host for \u003cem\u003eStaphylococcus aureus\u003c/em\u003e pathogenesis. \u003cem\u003eInfect Immun\u003c/em\u003e 71:2208\u0026ndash;2217\u003c/li\u003e\n \u003cli\u003eSingh J (2021) Harnessing the power of genetics: fast forward genetics in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e. \u003cem\u003eMol Genet Genomics\u003c/em\u003e 296:1\u0026ndash;20\u003c/li\u003e\n \u003cli\u003eSkantar AM, Agama K, Meyer SL, Carta LK, Vinyard BT (2005) Effects of geldanamycin on hatching and juvenile motility in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e and \u003cem\u003eHeterodera glycines\u003c/em\u003e. \u003cem\u003eJ Chem Ecol\u003c/em\u003e 31:2481\u0026ndash;2491\u003c/li\u003e\n \u003cli\u003eSloat SA, Noble LM, Paaby AB, Bernstein M, Chang A, Kaur T, Yuen J, Tintori SC, Jackson JL, Martel A, Salome Correa JA (2022) \u003cem\u003eCaenorhabditis\u003c/em\u003e nematodes colonize ephemeral resource patches in neotropical forests. \u003cem\u003eEcol Evol\u003c/em\u003e 12:e9124\u003c/li\u003e\n \u003cli\u003eStevens L, F\u0026eacute;lix MA, Beltran T, Braendle C, Caurcel C, Fausett S, Fitch D, Fr\u0026eacute;zal L, Gosse C, Kaur T, Kiontke K (2019) Comparative genomics of 10 new \u003cem\u003eCaenorhabditis\u003c/em\u003e species. \u003cem\u003eEvol Lett\u003c/em\u003e 3:217\u0026ndash;236\u003c/li\u003e\n \u003cli\u003eStiernagle T (2021) Maintenance of \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e. \u003cem\u003eWormBook\u003c/em\u003e 3\u0026ndash;4\u003c/li\u003e\n \u003cli\u003eSudhaus W, Kiontke K (2007) Comparison of the cryptic nematode species \u003cem\u003eCaenorhabditis brenneri\u003c/em\u003e sp. n. and \u003cem\u003eC. remanei\u003c/em\u003e (Nematoda: Rhabditidae) with the stem species pattern of the \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e group. \u003cem\u003eZootaxa\u003c/em\u003e 1456:45\u0026ndash;62\u003c/li\u003e\n \u003cli\u003eTamura K, Stecher G, Kumar S (2021) MEGA11: molecular evolutionary genetics analysis version 11. \u003cem\u003eMol Biol Evol\u003c/em\u003e 38:3022\u0026ndash;3027\u003c/li\u003e\n \u003cli\u003eTejeda-Benitez L, Olivero-Verbel J (2016) \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e, a biological model for research in toxicology. \u003cem\u003eRev Environ Contam Toxicol\u003c/em\u003e 237:1\u0026ndash;35\u003c/li\u003e\n \u003cli\u003eThomas JH (2008) Genome evolution in \u003cem\u003eCaenorhabditis\u003c/em\u003e. \u003cem\u003eBrief Funct Genomics Proteomics\u003c/em\u003e 7:211\u0026ndash;216\u003c/li\u003e\n \u003cli\u003eVokř\u0026aacute;l I, Podlipn\u0026aacute; R, Matou\u0026scaron;kov\u0026aacute; P, Sk\u0026aacute;lov\u0026aacute; L (2023) Anthelmintics in the environment: Their occurrence, fate, and toxicity to non-target organisms. \u003cem\u003eChemosphere\u003c/em\u003e. 140446\u003c/li\u003e\n \u003cli\u003eWidmayer SJ, Crombie TA, Nyaanga JN, Evans KS, Andersen EC (2022) \u003cem\u003eC. elegans\u003c/em\u003e toxicant responses vary among genetically diverse individuals. \u003cem\u003eToxicology\u003c/em\u003e. 153292\u003c/li\u003e\n \u003cli\u003eWolf M, Nunes F, Henkel A, Heinick A, Paul RJ (2008) The MAP kinase JNK-1 of \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e: Location, activation, and influences over temperature-dependent insulin-like signaling, stress responses, and fitness. \u003cem\u003eJ Cell Physiol\u003c/em\u003e 214:721\u0026ndash;729\u003c/li\u003e\n \u003cli\u003eWoodruff GC, Johnson E, Phillips PC (2019) A large close relative of \u003cem\u003eC. elegans\u003c/em\u003e is slow-developing but not long-lived. \u003cem\u003eBMC Evol Biol\u003c/em\u003e 19:1\u0026ndash;4\u003c/li\u003e\n \u003cli\u003eZwirchmayr J, Kirchweger B, Lehner T, Tahir A, Pretsch D, Rollinger JM (2020) A robust and miniaturized screening platform to study natural products affecting metabolism and survival in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e. \u003cem\u003eSci Rep\u003c/em\u003e 10:12323\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Distribution of bacterivorous worms across bio-geographical zones in selected regions.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"670\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocations\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBio-geographical zones\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo of Samples\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of bacterivore worm containing samples\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eITS2 positive samples\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e18s rRNA positive samples\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIdentified organism\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of strain (sequenced)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eThakurgaon\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003eHimalayan piedmont plain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cem\u003eOscheus sp.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRangpur\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003eTeesta floodplains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cem\u003eOscheus sp.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBogura\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003eBarind tract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMymensingh\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 97px;\"\u003e\n \u003cp\u003eBrahmaputra-Jamuna floodplains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 69px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 86px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 68px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 68px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. brenneri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cem\u003eOscheus sp.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBhola\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003eOffshore islands\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cem\u003eOscheus sp.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSylhet\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 97px;\"\u003e\n \u003cp\u003eSurma-Kushiyara floodplains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 69px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 86px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 68px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 68px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cem\u003eC. brenneri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cem\u003eOscheus sp.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGazipur\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 97px;\"\u003e\n \u003cp\u003eMadhupur sal forest\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cem\u003eRhabditella \u0026nbsp;axei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDhaka\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cem\u003eTarantobelus jeffdanielsi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eChattogram\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003eChittagong hills\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eJhenaidah\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 97px;\"\u003e\n \u003cp\u003eGanges floodplains\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBarishal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e225\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable 2. Genetic diversity and neutrality indices of \u003cem\u003eITS2\u003c/em\u003e gene sequences across two distinct groups of \u003cem\u003eC. brenneri\u003c/em\u003e isolates\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"690\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSeq ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026pi;\u0026plusmn;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHd\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD\u003csub\u003eXY\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTajima D\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFu and Li\u0026apos;s F test\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"8\" style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cem\u003eITS 2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 63px;\"\u003e\n \u003cp\u003eGroup1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003eIT8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.03762\u0026plusmn;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 37px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 32px;\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 30px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"8\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0.04399\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 89px;\"\u003e\n \u003cp\u003e0.24617\u003c/p\u003e\n \u003cp\u003e(P \u0026gt; 0.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 138px;\"\u003e\n \u003cp\u003e0.29927\u003c/p\u003e\n \u003cp\u003e(P \u0026gt; 0.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003eIT20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003eIT21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003ePV324956.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 63px;\"\u003e\n \u003cp\u003eGroup 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003eJN636110.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.00470\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 37px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 32px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 30px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 89px;\"\u003e\n \u003cp\u003e-0.80861\u003c/p\u003e\n \u003cp\u003e(P \u0026gt; 0.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 138px;\"\u003e\n \u003cp\u003e-0.80861\u003c/p\u003e\n \u003cp\u003e(P \u0026gt; 0.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003eJN636107.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003eJN636108.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003eJN636109.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e0.03420\u0026plusmn;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eHn: Number of Haplotypes, Hd: Haplotype (gene) diversity, \u0026pi;: Nucleotide diversity (per site), K: Average number of nucleotide differences between two randomly chosen sequences from within in the population, S: Number of variable/segregating sites.\u0026nbsp;Dxy: The average number of nucleotide substitutions per site between populations\u003c/p\u003e\n\u003cp\u003eTable 3. Intra and inter-species outcrossing experiment between \u003cem\u003eC. brenneri\u003c/em\u003e isolates and \u003cem\u003eC. elegans\u003c/em\u003e (N2).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"642\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 409px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. brenneri\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;isolates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. elegans\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eN2\u003csub\u003eXX\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSyt IT11\u003csub\u003eXX\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSyt IT8\u003csub\u003eXX\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSyt IT20\u003csub\u003eXX\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSyt IT21\u003csub\u003eXX\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSyt IT 11\u003csub\u003eXY\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e133\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSyt IT8\u003csub\u003eXY\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e131\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSyt IT20\u003csub\u003eXY\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSyt IT21\u003csub\u003eXY\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e204\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e215\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;Data within the table indicate the number of progeny in F2 generation resulting from selective outcrossing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere, XX=male, XY=female\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"C. brenneri, Bangladesh, Ecology, Evolution, Stressors, Resistance, C. elegans","lastPublishedDoi":"10.21203/rs.3.rs-6856196/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6856196/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The Caenorhabditis elegans is a well-established model in the advancement of cell and molecular biology, lead-compound discovery and multidisciplinary research. However, its sibling species C. brenneri, remains relatively underexplored despite its hyperdiverse genome and ecological significance. This study sought to discover sister species of C. elegans through ecological surveys, genetic characterization, and biological comparisons with C. elegans (N2), with the aim of positioning of wild isolates as complementary model organisms. Sampling was conducted across 11 distinct bio-ecological zones, followed by cultivation and independent propagation of bacterivorous nematodes. Distinct morphological traits with the amplification of ITS2 and 18s rRNA regions confirmed isolates of C. brenneri, Oscheus sp., and surprisingly, Tarantobrlus jeffdanielsi and Rhabditella axei. The genetic diversity analysis based on ITS2 sequences of C. brenneri isolates revealed that diversities and polymorphisms are consistent with neutral evolution rather than selection. A comparative analysis of C. elegans and wild C. brenneri isolates including lifespan, fecundity, thermo-tolerance, anthelmintic sensitivity and bacterial killing assay revealed that, compared to C. elegans N2 strain, C. brenneri isolates live longer, produce fewer eggs than C. elegans, exhibited higher survival under prolonged heat stress and significantly greater survival against bacteria and transient lethality in anthelmintics. This pioneering investigation confirms the availability of C. brenneri and other previously undiscovered nematodes in different regions of Bangladesh, outlines its evolutionary context, resilience to stressors and warns altered host adaptability. These findings highlight the potential of C. brenneri as a robust complementary model for research into species-specific responses to environmental or pathogenic stressors.","manuscriptTitle":"Caenorhabditis brenneri as a complementary model organism to C. elegans: Insights from ecological, molecular and comparative biology","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-01 11:39:38","doi":"10.21203/rs.3.rs-6856196/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ea01cff5-83be-4f9b-9297-726e5295fa89","owner":[],"postedDate":"July 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50653055,"name":"Biological sciences/Genetics"},{"id":50653056,"name":"Scientific community and society/Developing world"}],"tags":[],"updatedAt":"2025-09-15T08:25:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-01 11:39:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6856196","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6856196","identity":"rs-6856196","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}