Introduction
The host-parasite relationship is one of nature’s most diverse and complex ecological interdependencies. In mammals, these interactions span from intracellular Trypanosoma to ectoparasitic fleas (Goater et al. Reference Goater, Goater and Esch2014). The complexity is further compounded by a suite of ecological, biological, and anthropogenic factors, including host population size and density, host behaviour, sex and age, and the introduction of alien species (Kołodziej-Sobocińska Reference Kołodziej-Sobocińska2019). Parasites also engage in complex interactions with the host immune system and can impose selective pressure on host phenotypes, meaning that the host’s immune and genetic profile also play important roles in shaping the host-parasite relationship (Goater et al. Reference Goater, Goater and Esch2014). These drivers may exert independent and direct effects, or act synergistically. Studying the influences and interactions of the wide range of factors shaping the host-parasite relationship is challenging, particularly given the complex statistical analysis and interpretation of results (Behnke et al. Reference Behnke, Lewis, Zain and Gilbert1999). Consequently, there is a general lack of consensus regarding the strength and direction of effect for many predictors of parasite infection.
Intestinal nematodes and their rodent hosts provide a well-studied system for investigating these factors. Specifically, host sex has been debated extensively as a determinant of intestinal nematode prevalence and abundance, with two mutually non-exclusive hypotheses proposed to explain sex-biased parasitism. On the one hand, the immunocompetence handicap hypothesis (ICHH) emphasises the immunosuppressive effect of testosterone, which makes males more susceptible to infection (Bordes et al. Reference Bordes, Ponlet, de Bellocq, Ribas, Krasnov and Morand2012; Folstad and Karter Reference Folstad and Karter1992; Klein Reference Klein2004; Krasnov et al. Reference Krasnov, Bordes, Khokhlova and Morand2012; Schalk and Forbes Reference Schalk and Forbes1997; Wesołowska Reference Wesołowska2022; Zuk Reference Zuk2009; Zuk and McKean Reference Zuk and McKean1996). Conversely, life-history theory suggests a trade-off, where males compromise immune function to maximise competitive success and mating opportunities (Nunn et al. Reference Nunn, Lindenfors, Pursall and Rolff2009; Rolff Reference Rolff2002; Scantlebury et al. Reference Scantlebury, Maher McWilliams, Marks, Dick, Edgar and Lutermann2010; Stoehr and Kokko Reference Stoehr and Kokko2006; Zuk Reference Zuk2009; Zuk and McKean Reference Zuk and McKean1996). Both perspectives converge on the idea that evolution has placed males in a sort of ‘cruel bind’, where they are forced to choose between allocating resources either into reproductive success at the cost of weakened immune response, or into immunocompetence, which would reduce the probability of infection but diminish their chances of fathering offspring (Zuk Reference Zuk2009). While theory predicts a broad trend of male-biased infection, empirical studies are inconsistent, reporting heavier intestinal nematode infections in either male (Abu-Madi et al. Reference Abu-Madi, Behnke, Lewis and Gilbert1998, Reference Abu-Madi, Behnke, Lewis and Gilbert2000; Behnke et al., Reference Behnke, Lewis, Zain and Gilbert1999) or female rodents (Sanchez et al. Reference Sanchez, Devevey and Bize2011), or finding no discernible host sex effect (Čabrilo et al. Reference Čabrilo, Jovanović, Čabrilo, Budinski, Blagojević and Vujošević2018; Fuentes et al. Reference Fuentes, Sáez, Trelis, Galán-Puchades and Esteban2004, Reference Fuentes, Sainz-Elipe and Galán-Puchades2007, Reference Fuentes, Sainz-Elipe, Sáez-Durán and Galán-Puchades2010; Goüy de Bellocq et al. Reference Goüy de Bellocq, Sarà, Casanova, Feliu and Morand2003; Gregory et al. Reference Gregory, Montgomery and Montgomery1992; Milazzo et al. Reference Milazzo, Di Bella, Casanova, Ribas and Cagnin2010). This discrepancy suggests that sex-biased parasitism is highly context-dependent, affected by factors such as parasite and host species, site, seasonality, and local environmental conditions (Krasnov et al. Reference Krasnov, Morand, Hawlena, Khokhlova and Shenbrot2005, Reference Krasnov, Bordes, Khokhlova and Morand2012).
Unlike host sex and major histocompatibility alleles, certain intrinsic genetic factors, such as B chromosomes (Bs), have received limited attention in parasitology, despite their potential to shape parasite infrapopulations and infection dynamics. B chromosomes are supernumerary, dispensable chromosomes found across eukaryotes (D’Ambrosio et al. Reference D’Ambrosio, Alonso-Lifante, Barros, Kovařík, Mas De Xaxars and Garcia2017); unlike A chromosomes, they are not essential for normal growth and development (Ferree et al. Reference Ferree, Blagojević, Houben, Martins, Trifonov and Vujošević2024). Despite this, they are remarkably prevalent in rodents, with the genus Apodemus alone comprising six B-chromosome-bearing species (Vujošević et al. Reference Vujošević, Rajičić and Blagojević2018). Two of the most intensively studied rodent models in this context are the Korean field mouse (A. peninsulae) and the yellow-necked mouse (A. flavicollis), both of which exhibit stable B chromosome polymorphisms (Borisov and Zhigarev Reference Borisov and Zhigarev2018; Vujošević Reference Vujošević1992; Vujošević and Blagojević Reference Vujošević and Blagojević2004; Wójcik et al. Reference Wójcik, Wójcik, Macholán, Piálek and Zima2004). Although direct investigations of B chromosome effects on intestinal nematodes remain rare, existing studies suggest biologically meaningful links between Bs, host immune regulation, and parasite life-history traits. A study on A. flavicollis has shown that Bs may modify the internal host environment experienced by parasites: Bs significantly altered nematode infrapopulation sex ratios, shifting them toward a higher proportion of males (Jovanović et al. Reference Jovanović, Čabrilo, Budinski, Bjelić-Čabrilo, Adnađević, Blagojević and Vujošević2019). Additionally, reduced expression of the immunomodulatory TGF-β gene was detected in A. flavicollis individuals carrying Bs, potentially facilitating parasite establishment and survival (Adnađević et al. Reference Adnađević, Jovanović, Blagojević, Budinski, Čabrilo, Bijelić-Čabrilo and Vujošević2014). Recent advances in B chromosome research have fundamentally changed their conceptual status, demonstrating that Bs can harbour actively transcribed genes (Chen et al. Reference Chen, Bartoš, Boudichevskaia, Voigt, Rabanus-Wallace, Dreissig, Tulpová, Šimková, Macas, Kim, Buhl, Bürstenbinder, Blattner, Fuchs, Schmutzer, Himmelbach, Schubert and Houben2024; Huang et al. Reference Huang, Du, Zhao and Jin2016; Rajpal et al. Reference Rajpal, Sharma, Sehgal, Sharma, Wadhwa, Dhakate, Chandra, Thakur, Deb, Rama Rao, Mir and Raina2023), influence A chromosome gene expression, and participate in epigenetic regulation (Ahmad and Martins Reference Ahmad and Martins2019; Houben et al. Reference Houben, Banaei-Moghaddam, Klemme and Timmis2014, Reference Houben, Jones, Martins and Trifonov2019). Long-read and low-pass sequencing further reveal the mosaic origin of Bs from multiple A chromosomes and, in some cases, organellar DNA, underscoring their dynamic and context-dependent functional potential (Banaei-Moghaddam et al. Reference Banaei-Moghaddam, Martis, Macas, Gundlach, Himmelbach, Altschmied, Mayer and Houben2015; Makunin et al. Reference Makunin, Rajičić, Karamysheva, Romanenko, Druzhkova, Blagojević, Vujošević, Rubtsov, Graphodatsky and Trifonov2018; Rajpal et al. Reference Rajpal, Sharma, Sehgal, Sharma, Wadhwa, Dhakate, Chandra, Thakur, Deb, Rama Rao, Mir and Raina2023). Rajičić et al. (Reference Rajičić, Romanenko, Karamysheva, Blagojević, Adnađević, Budinski, Bogdanov, Trifonov, Rubtsov and Vujošević2017) showed that Bs in A. flavicollis have a common origin, most likely derived from the sex chromosomes (Makunin et al. Reference Makunin, Rajičić, Karamysheva, Romanenko, Druzhkova, Blagojević, Vujošević, Rubtsov, Graphodatsky and Trifonov2018), with additional shared sequences from small autosomes. Despite wide geographic separation and number variation, all Bs show the same structure, indicating a single origin and a shared evolutionary pathway (Rajičić et al. Reference Rajičić, Romanenko, Karamysheva, Blagojević, Adnađević, Budinski, Bogdanov, Trifonov, Rubtsov and Vujošević2017).
Recent research has revealed interesting and potentially ecologically significant relationships between Bs, host sex and gene expression. Rajičić et al. (Reference Rajičić, Makunin, Adnađević, Trifonov, Vujošević and Blagojević2021) used RT-PCR to show that transcription of B-chromosome–associated sequences varies with B chromosome number and age, and exhibits sex-dependent differences, with higher activity in the splenic tissue of females. B-linked genes and/or standard genome genes in the presence of Bs show higher transcription levels in young females. For some genes, transcription correlates with the number of Bs, peaking in individuals with a single B chromosome and decreasing as the number increases. This female-biased transcriptional activity is consistent with observations from other taxa, where the expression of B-linked sequences is often associated with the female sex. For example, a correlation between B chromosome sequence expression and females has been reported in some cichlids (Clark et al. Reference Clark, Conte and Kocher2018; Yoshida et al. Reference Yoshida, Terai, Mizoiri, Aibara, Nishihara, Watanabe, Kuroiwa, Hirai, Hirai, Matsuda and Okada2011). This association between B chromosomes and females may suggest increased adaptive value in female Bs carriers (Clark et al. Reference Clark, Conte, Ferreira-Bravo, Poletto, Martins and Kocher2017). Altogether, existing evidence supports the assumption that the presence of B chromosomes in the host genome may directly or indirectly affect parasite population structure through complex genetic and immunological mechanisms.
This study addresses existing knowledge gaps on the effects of host sex and B chromosome presence on parasite infection by investigating three key questions: (i) Are males truly ‘the sicker sex’ as immunological and evolutionary theories suggest? (ii) Do Bs facilitate parasite establishment and survival? (iii) Do host sex and Bs interact synergistically? We hypothesize that if both males and B carriers are immunologically disadvantaged, males with Bs will harbour disproportionately greater parasite loads. We addressed these questions by analysing intestinal nematode abundance in a large sample of A. flavicollis from Serbia. To account for the highly aggregated, negative binomial distribution typical of parasite populations (Wilson et al. Reference Wilson, Grenfell and Shaw1996), we employed a Monte Carlo randomisation approach, allowing for robust statistical testing without the restrictive assumptions of standard parametric analyses (Gotelli and Ellison Reference Gotelli and Ellison2013).
Materials and methods
Sample collection
A total of 294 A. flavicollis were captured from 18 sites in Serbia (Figure 1) between October 2011 and October 2015. A part of this sample was previously analysed in Jovanović et al. (Reference Jovanović, Čabrilo, Budinski, Bjelić-Čabrilo, Adnađević, Blagojević and Vujošević2019). Sampling was conducted in old deciduous forests, which represent the preferred habitat of the species, using Longworth traps baited with canned fish. Hay bedding and wheat were provided to improve survival. For each captured individual, sex and biometric data were recorded.
A map of Serbia showing the 18 trapping sites. 1 – Avala, 2 – Bukulja, 3 – Cer, 4 – Gučevo, 5 – Jabukovac, 6 – Košutnjak, 7 – Lisine, 8 – Liškovac, 9 – Milošev Do, 10 – Misača, 11 – Mlekominci, 12 – Petnica, 13 – Rudnik, 14 – Temska, 15 – Treska, 16 – Vlasina, 17 – Zlot, 18 – Župa.
Figure 1. Long description
A map of Serbia plotted on a coordinate grid with Latitude on the Y axis (43 degrees North to 46 degrees North) and Longitude on the X axis (19 degrees East to 23 degrees East). The map shows internal district boundaries in white. 18 black dots representing trapping sites are distributed as follows:
* Central and Northern Cluster: Site 6 (Košutnjak) and Site 1 (Avala) are located near the center-north. Site 10 (Misača), Site 2 (Bukulja), and Site 13 (Rudnik) form a vertical cluster in the central region.
* Western Region: Site 3 (Cer) and Site 4 (Gučevo) are on the western border. Site 12 (Petnica) is located further inland to the east of Site 3.
* Eastern Region: Site 8 (Liškovac) and Site 5 (Jabukovac) are in the northeast. Site 7 (Lisine) and Site 17 (Zlot) are in the east-central area.
* Southern and Southeastern Region: Site 9 (Milošev Do) is in the southwest. Sites 18 (Župa) and 15 (Treska) are in the south-central area. Site 14 (Temska), Site 16 (Vlasina), and Site 11 (Mlekominci) are clustered in the southeast corner.
Host cytogenetic analysis
Chromosome preparation was carried out directly from bone marrow cells, following the standard procedure (Hsu and Patton Reference Hsu, Patton and Benirschke1969). B chromosome presence was determined by analysing 30 metaphase figures per individual. Animals with chromosome counts above the standard complement for A. flavicollis (2n = 48) were classified as B-carriers (B+). Following cytogenetic analysis, gastrointestinal tracts were stored at −20°C for parasitological analysis.
Parasitological analysis
Thawed gastrointestinal tracts were separated into four sections (stomach, small intestine, caecum and large intestine), placed in water and opened longitudinally to release their contents. Water containing the intestinal sections and their contents was decanted from the Petri dishes into 250 ml conical glasses, which were filled up to full volume to allow settling of particles. After 10–15 minutes, the intestinal sections and their contents, including endoparasites, formed a layer at the bottom of the cone, allowing for the excess water containing larger undigested food particles to be poured out, after which the glass was once again filled up with tap water. This process was repeated until the water in the glass was clear. At this point, excess water was poured out one last time, leaving a small amount of fluid containing intestinal sections, small particles, and endoparasites at the bottom of the glass. This fluid was examined under a stereomicroscope. Nematodes were collected, preserved in 70% ethanol, and cleared in a drop of lactic acid for identification. Species were identified based on morphological and morphometric criteria using an optical microscope (40× and 100× magnification). Nine species were identified: Aonchotheca annulosa (Dujardin 1845), Aspiculuris tetraptera Nitzsch, 1821, Eucoleus bacillatus (Eberth 1863), Heligmosomoides polygyrus Dujardin, 1845, Mastophorus muris (Gmelin 1790), Rictularia proni Seurat, 1915, Syphacia frederici Roman, 1945, Syphacia stroma Linstow, 1884, and Trichuris muris Schrank, 1788.
Statistical analysis
Hosts were grouped by sex (male or female) and B chromosome presence (present – B+ and absent – B0). Differences in group proportions were assessed using two-tailed one-sample proportion tests, whereas Pearson’s Chi-squared test with Yates’ continuity correction was used to test sex-dependent differences in Bs occurrence. Mean abundance (MA), defined as the average number of parasites per host including uninfected hosts, and prevalence (P%), defined as the percentage of hosts infected by a parasite (Bush et al. Reference Bush, Lafferty, Lotz and Shostak1997), were calculated for total intestinal nematodes and individual species in the total host sample, as well as separated by sex, Bs status and sex + Bs status. 95% confidence intervals for prevalence were calculated using the exact (Clopper-Pearson) method. Differences in MA between host groups were tested using the Mann-Whitney U-test.
To assess the effect of host sex, B chromosomes, and their interaction on the nematode abundance, a Monte Carlo analysis was performed to test three specific hypotheses. The first hypothesis (H 1) was that host sex has a significant effect on intestinal nematode abundance. Specifically, in line with data on behavioural and physiological differences between the sexes, male A. flavicollis should be more heavily infected. The second hypothesis (H 2) was that hosts with and without Bs should significantly differ in intestinal nematode abundance. Since the effects of B chromosomes on their hosts remain an open question, this hypothesis claims that Bs somehow affect mean nematode abundance without making any assumptions on the direction of this effect. The third hypothesis (H 3) proposed a synergistic interaction between host sex and B chromosome status. While male A. flavicollis are expected to be more infected, once again, there is no reason to presume a directional effect of Bs on nematode abundance, so testing this hypothesis would simply uncover a significant interaction between the two factors if there was one.
Each of the three hypotheses was associated with its own test statistic that represented the difference in mean intestinal nematode abundance between different sample categories. The test statistic used to test H 1 was:
where TS 1 is the observed difference of mean abundances, and MAM and MAF are the mean abundances of intestinal nematodes in males and females, respectively. The test statistic used to test H 2 was:
where MAB and MAB0 are the mean abundances of intestinal nematodes in mice carrying and not carrying B chromosomes, respectively. Since H 2 assumes that there is a difference between B+ and B0 animals without specifying its direction, we were interested in both unusually small and unusually large differences between MAB0 and MAB. In order to detect all of these differences in the lower tail of the null distribution of the simulated test statistics under the Monte Carlo approach, the expression was squared. Finally, H 3 was tested using the test statistic:
where the indices of each MA value indicate a specific combination of host sex and B chromosome status. Once again, the expression was squared in order to detect both types of differences between B+ and B0 individuals in the lower tails of null distributions of the simulated test statistics.
Quantitative infection parameters indicated that three species (A. tetraptera, E. bacillatus, and M. muris) had very low prevalences (less than 3%) (Table 1). These three species were excluded from Monte Carlo simulations due to low sample size. Observed values of each test statistic were calculated from the full dataset, for intestinal nematodes in general and each nematode species. Following this, simulated values of the test statistic were generated using randomisation. This was achieved by breaking the association between predictor variables (sex or/and Bs) and response variables (abundance of intestinal nematodes). Random sampling without replacement was performed on the predictor column(s) to generate a new column (or new columns) of equal length containing randomised values of the original column. The test statistic was then calculated for the randomised dataset, using the randomized column(s) as predictor(s) and intestinal nematode abundance as the response. To generate a null distribution of test statistics, this process was repeated 10,000 times. Since we were interested in unusually high values of the test statistic for all three hypotheses, a right-tailed test was used, and observed values were tested against the generated null distributions to find the proportion of simulated values equal to or greater than the relevant observed value. This proportion corresponds to the p-value from the Monte Carlo analysis.
Selected quantitative parameters of infection for individual intestinal nematode species, as well as total intestinal nematodes, in the sample of 294 A. flavicollis from Serbia
Table 1. Long description
The table consists of five columns: Species, min-max (range of nematodes per infected mouse), N I (number of infected mice), M A (mean abundance), and P percent (prevalence with 95 percent confidence intervals).
* Aonchotheca annulosa: min-max 1-74, N I 31, M A 0.918, P percent 10.5 (7.3-14.6).
* Aspiculuris tetraptera: min-max 1-163, N I 8, M A 0.687, P percent 2.7 (1.2-5.3).
* Eucoleus bacillatus: min-max 1-10, N I 5, M A 0.078, P percent 1.7 (0.6-3.9).
* Heligmosomoides polygyrus: min-max 1-738, N I 197, M A 15.150, P percent 67.0 (61.3-72.4).
* Mastophorus muris: min-max 1-1, N I 3, M A 0.010, P percent 1.0 (0.2-3.0).
* Rictularia proni: min-max 1-24, N I 33, M A 0.466, P percent 11.2 (7.8-15.4).
* Syphacia frederici: min-max 1-144, N I 35, M A 3.932, P percent 11.9 (8.4-16.2).
* Syphacia stroma: min-max 1-978, N I 67, M A 15.078, P percent 22.8 (18.1-28.0).
* Trichuris muris: min-max 1-289, N I 63, M A 3.527, P percent 21.4 (16.9-26.6).
* All intestinal nematodes: min-max 1-1063, N I 238, M A 39.847, P percent 81.0 (76.0-85.3).
min-max – minimum to maximum range of the number of nematodes per infected mouse.
NI – number of infected mice.
MA – mean abundance.
P% – prevalence. Exact (Clopper-Pearson) 95% confidence intervals are given in parentheses.
Statistical analysis was performed in R version 4.6.0 (R Core Team 2026). In addition to base packages, the tidyverse metapackage (Wickham et al. Reference Wickham, Averick, Bryan, Chang, McGowan, François, Grolemund, Hayes, Henry, Hester, Kuhn, Pedersen, Miller, Bache, Müller, Ooms, Robinson, Seidel, Spinu, Takahashi, Vaughan, Wilke, Woo and Yutani2019), as well as the broom (Robinson et al. Reference Robinson, Hayes and Couch2026), knitr (Xie, Reference Xie2025), patchwork (Pedersen Reference Pedersen2025), prevalence (Devleesschauwer et al. Reference Devleesschauwer, Torgerson, Charlier, Levecke, Praet, Roelandt, Smit, Dorny, Berkvens and Speybroeck2022), and ggtext (Wilke and Wiernik Reference Wilke and Wiernik2022) packages were also used.
Results
Host sample structure
The host sample consisted of 294 individuals: 148 males (50.3%) and 146 females (49.7%). The sample sex ratio did not significantly deviate from unity (z = 0.003, p = 0.953). Regarding B chromosome presence, 198 mice (105 males and 93 females) had no Bs, whereas 96 (43 males and 53 females) carried at least one B chromosome. The proportion of B+ individuals (32.7%) was significantly lower than that of B0 individuals (67.3%; z = 34.697, p <0.0001), whereas no significant sex-related differences in B chromosome occurrence were detected (χ2 = 1.4413, df = 1, p = 0.230).
Intestinal nematode abundance and prevalence
On average, each mouse in the sample carried around 40 intestinal nematodes (Table 1). The mean abundance (MA) of individual nematode species varied from 0.01 (M. muris) to 15.15 (H. polygyrus). Four out of the nine recorded species had a mean abundance greater than 1. Regarding prevalence, 81% of mice harboured at least one species of intestinal nematode, with prevalences of individual species ranging from 1% (M. muris) to 67% (H. polygyrus).
Mean abundance and prevalence values of nematodes varied between males and females, as well as B+ and B0 mice (Table 2). When mean abundances of intestinal nematodes were compared between male and female mice, the Mann-Whitney U-test produced a statistically significant result for only two species, S. stroma and T. muris. In both species, mean abundance was greater in male mice. Comparison of mean abundance between B+ and B0 hosts also produced a significant result in two species, A. tetraptera and M. muris. Mean abundances of both nematodes were greater in B+ mice (Table 3).
Quantitative parameters of infection of A. flavicollis separated by sexes and Bs presence. Sample sizes are given in parentheses.
Table 2. Long description
The image consists of two data tables. Both tables list nine nematode species and a total for all intestinal nematodes in the first column. The columns for data are M A (mean abundance) and P % (prevalence with 95% confidence intervals).
Top Table: Categorized by Sex.
- Males (n = 148): M A ranges from 0.007 (Mastophorus muris) to 17.047 (Syphacia stroma). P % is highest for Heligmosomoides polygyrus at 67.6%.
- Females (n = 146): M A ranges from 0.014 (Mastophorus muris) to 15.918 (Heligmosomoides polygyrus). P % is highest for Heligmosomoides polygyrus at 66.4%.
- All intestinal nematodes: Males M A 43.108, P % 81.1; Females M A 36.541, P % 80.8.
Bottom Table: Categorized by B chromosomes.
- B plus (n = 96): M A ranges from 0.031 (Mastophorus muris) to 22.812 (Heligmosomoides polygyrus). P % is highest for Heligmosomoides polygyrus at 72.9%.
- B 0 (n = 198): M A ranges from 0 (Mastophorus muris) to 14.167 (Syphacia stroma). P % is highest for Heligmosomoides polygyrus at 64.1%.
- All intestinal nematodes: B plus M A 53.156, P % 82.3; B 0 M A 33.394, P % 80.3.
Species listed in both tables: Aonchotheca annulosa, Aspiculuris tetraptera, Eucoleus bacillatus, Heligmosomoides polygyrus, Mastophorus muris, Rictularia proni, Syphacia frederici, Syphacia stroma, and Trichuris muris.
MA – mean abundance.
P% – prevalence. Exact (Clopper-Pearson) 95% confidence intervals are given in parentheses.
Results of the Mann-Whitney U-test for comparison of mean abundance of intestinal nematodes in A. flavicollis with regard to host sex and Bs presence
Table 3. Long description
The table consists of three columns: Nematode species, Host sex, and B chromosomes. Each cell contains a test statistic followed by a p-value in parentheses. Significant p-values (p < 0.05) are bolded.
* Aonchotheca annulosa: Host sex 11316.5 (0.188); B chromosomes 9658.0 (0.674).
* Aspiculuris tetraptera: Host sex 10796.0 (0.971); B chromosomes 10002.0 (0.010).
* Eucoleus bacillatus: Host sex 10580.0 (0.171); B chromosomes 9412.0 (0.550).
* Heligmosomoides polygyrus: Host sex 10875.5 (0.921); B chromosomes 10400.5 (0.181).
* Mastophorus muris: Host sex 10729.0 (0.557); B chromosomes 9801.0 (0.013).
* Rictularia proni: Host sex 10116.5 (0.085); B chromosomes 9712.0 (0.580).
* Syphacia frederici: Host sex 10580.5 (0.586); B chromosomes 9791.5 (0.455).
* Syphacia stroma: Host sex 12129.0 (0.013); B chromosomes 8915.0 (0.241).
* Trichuris muris: Host sex 9642.5 (0.026); B chromosomes 9688.0 (0.708).
* All intestinal nematodes: Host sex 11757.0 (0.190); B chromosomes 10220.5 (0.293).
Each result is given as a test statistic, with the associated p-value in parentheses.
Statistically significant results are marked in bold.
Hypothesis 1: Effect of host sex
The observed test statistic used to test H 1 (TS 1) varied across nematode species. Positive values were recorded for total intestinal nematodes and four individual species, indicating a higher mean abundance of parasites in males. Conversely, two species showed negative values, indicating higher MA in female hosts (Figure 2). Despite these trends, a statistically significant effect of host sex was confirmed for only one species, A. annulosa (Figure 2B).
Effect of host sex on intestinal nematode abundance: the distributions of simulated values of TS 1 for total intestinal nematodes (A), Aonchotheca annulosa (B), Heligmosomoides polygyrus (C), Rictularia proni (D), Syphacia frederici (E), S. stroma (F) and Trichuris muris (G). In all plots, the full blue line indicates the observed value of TS 1, and the dashed red line indicates the critical value of the test statistic calculated as the 0.95 quantile of a null distribution of test statistics generated from 10,000 randomisations of the observed data. p values are given in the upper right of each plot.
Figure 2. Long description
A multi-panel set of seven histograms labeled A through G. Each graph plots frequency on the y-axis against the T S 1 test statistic on the x-axis. A solid blue vertical line represents the observed value, and a dashed red vertical line represents the 0.95 quantile critical value.
* Panel A: Large top histogram for total nematodes. The distribution is bell-shaped, centered near 0. The blue line is at approximately 7 and the red line is at approximately 18. p equals 0.298.
* Panel B: Aonchotheca annulosa. A bimodal distribution. The blue line and red line are nearly overlapping on the far right tail near 1.1. p equals 0.048.
* Panel C: Heligmosomoides polygyrus. A wide bimodal distribution. The blue line is near -2 and the red line is near 9. p equals 0.567.
* Panel D: Rictularia proni. A jagged bell-shaped distribution. The blue line is at -0.2 and the red line is at 0.4. p equals 0.718.
* Panel E: Syphacia frederici. A smooth bell-shaped distribution. The blue line is at 1 and the red line is at 3. p equals 0.358.
* Panel F: S. stroma. A broad bell-shaped distribution. The blue line is at 4 and the red line is at 14. p equals 0.350.
* Panel G: Trichuris muris. A bell-shaped distribution. The blue line is at 2 and the red line is at 4. p equals 0.294.
Hypothesis 2: Effect of B chromosomes presence
Differences in MA (prior to squaring) indicated a general trend of higher intestinal nematode abundance in B+ mice for all species except T. muris. A significant effect of B chromosome presence was found for S. frederici, with a marginally significant effect observed in H. polygyrus (Figure 3C and E).
Effect of Bs’ presence on intestinal nematode abundance: the distributions of simulated values of TS 2 for total intestinal nematodes (A), Aonchotheca annulosa (B), Heligmosomoides polygyrus (C), Rictularia proni (D), Syphacia frederici (E), S. stroma (F), and Trichuris muris (G). In all plots, the full blue line indicates the observed value of TS 2, and the dashed red line indicates the critical value of the test statistic calculated as the 0.95 quantile of a null distribution of test statistics generated from 10,000 randomisations of the observed data. p values are given in the upper right of each plot.
Figure 3. Long description
A multi-panel figure containing seven histograms labeled A through G. Each histogram represents a null distribution of test statistics with frequency on the y-axis and T S 2 values on the x-axis.
* Panel A: Total intestinal nematodes. The distribution peaks near 0 and tails off toward 1500. A solid blue line is at approximately 400 and a dashed red line is at approximately 550. p equals 0.101.
* Panel B: Aonchotheca annulosa. The distribution tails off toward 5. A solid blue line is near 0 and a dashed red line is at 1.5. p equals 0.898.
* Panel C: Heligmosomoides polygyrus. The distribution tails off toward 400. A solid blue line and dashed red line are nearly overlapping around 130. p equals 0.055.
* Panel D: Rictularia proni. The distribution tails off toward 0.9. A solid blue line is at 0.1 and a dashed red line is at 0.25. p equals 0.391.
* Panel E: Syphacia frederici. The distribution tails off toward 60. A dashed red line is at 15 and a solid blue line is at 22. p equals 0.017.
* Panel F: S. stroma. The distribution tails off toward 1000. A solid blue line is near 0 and a dashed red line is at 280. p equals 0.804.
* Panel G: Trichuris muris. The distribution tails off toward 80. A solid blue line is at 2 and a dashed red line is at 25. p equals 0.690.
Hypothesis 3: Interaction between host sex and B chromosomes presence
The differences in MA among the four host categories (sex × Bs status) varied considerably, ranging from −2.39 (S. frederici) to 57.71 (total intestinal nematodes), indicating the absence of a uniform trend across all recorded parasite species (Table 4). Depending on species, parasite abundance was highest either in males with Bs (A. annulosa, R. proni, S. frederici), males without Bs (T. muris), or females with Bs (total intestinal nematodes, H. polygyrus, S. stroma). However, a significant interactive effect was found for total intestinal nematodes and S. stroma (Figure 4A and F). In both statistically significant cases, mean nematode abundance was highest in females carrying B chromosomes.
Quantitative parameters of infection for intestinal nematodes in general and individual species included in the Monte Carlo analysis of A. flavicollis separated by both sexes and B presence. Sample sizes are given in parentheses.
Table 4. Long description
The table is divided into two main columns for Males (n = 148) and Females (n = 146). Each sex is further subdivided into B plus (presence of B-chromosomes) and B 0 (absence of B-chromosomes). For each group, two metrics are reported: M A (mean abundance) and P % (prevalence with 95% confidence intervals).
Data rows for species:
* A a (Aonchotheca annulosa): Highest M A in B plus males (1.535) and lowest in B 0 females (0.236).
* H p (Heligmosomoides polygyrus): Shows high prevalence across all groups, peaking at 75.5% in B plus females with an M A of 29.604.
* R p (Rictularia proni): M A ranges from 0.190 in B 0 males to 0.860 in B plus males.
* S f (Syphacia frederici): M A is highest in B plus males (8.604) compared to other groups.
* S s (S. stroma): Notable high M A in B plus females (27.113) and B 0 males (22.210).
* T m (Trichuris muris): Prevalence is highest in B plus females (30.2%).
* Nem (Total intestinal nematodes): Summarizes overall infection. B plus females show the highest M A (67.000) and prevalence (83.0%), while B 0 females show the lowest M A (19.183).
Aa – Aonchotheca annulosa, Hp – Heligmosomoides polygyrus, Rp – Rictularia proni, Sf – Syphacia frederici, Ss – S. stroma, Tm – Trichuris muris, Nem – total intestinal nematodes.
MA – mean abundance.
P% – prevalence. Exact (Clopper-Pearson) 95% confidence intervals are given in parentheses.
Effect of the interaction between host sex and Bs’ presence: the distributions of simulated values of TS 3 for total intestinal nematodes (A), Aonchotheca annulosa (B), Heligmosomoides polygyrus (C), Rictularia proni (D), Syphacia frederici (E), S. stroma (F), and Trichuris muris (G). In all plots, the full blue line indicates the observed value of TS 3, and the dashed red line indicates the critical value of the test statistic calculated as the 0.95 quantile of a null distribution of test statistics generated from 10,000 randomisations of the observed data. p values are given in the upper right of each plot.
Figure 4. Long description
A multi-panel set of seven histograms labeled A through G. Each graph plots frequency on the y-axis and T S 3 values on the x-axis. The data is represented by light blue vertical bars forming a right-skewed distribution, with the highest frequency near zero. In every plot, a solid blue vertical line represents the observed value and a dashed red vertical line represents the critical value at the 0.95 quantile.
* Panel A: Large top panel. The solid blue line is far to the right of the dashed red line. p equals 0.011.
* Panel B: The solid blue line is at the far left, near zero, well before the dashed red line. p equals 0.885.
* Panel C: The solid blue line is to the left of the dashed red line. p equals 0.076.
* Panel D: The solid blue line is to the left of the dashed red line. p equals 0.143.
* Panel E: The solid blue line is at the far left, near zero. p equals 0.572.
* Panel F: The solid blue line is to the right of the dashed red line. p equals 0.015.
* Panel G: The solid blue line is at the far left, near zero. p equals 0.605.
Discussion
This study evaluated the effect of two intrinsic factors, host sex and B chromosome presence, and their interaction, on intestinal nematode abundance in a sample of 294 yellow-necked mice from Serbia. Our findings revealed a complex landscape where sex and genetic background exert differential effects depending on the parasite species involved. While host sex significantly predicted the abundance of A. annulosa, whose abundance was significantly higher in male hosts, B chromosome presence emerged as a significant predictor for S. frederici, which had significantly higher mean abundance in B+ mice. Notably, a significant interaction was detected for total intestinal nematodes and S. stroma abundance, with B-carrying females exhibiting the highest infection loads.
Host sex as a determinant of infection
The higher mean abundance of A. annulosa in male hosts is consistent with the immunocompetence handicap hypothesis (ICHH) and life-history/sexual selection theories, suggesting that testosterone-mediated immunosuppression or behavioural trade-offs render males more susceptible to infection (Zuk Reference Zuk2009). However, the underlying mechanism remains open to interpretation. Differences in dietary preferences between male and female yellow-necked mice could drive exposure, for example, if males consume more intermediate hosts, but the life cycle of A. annulosa remains ambiguously described in the literature: as direct (Goüy de Bellocq et al. Reference Goüy de Bellocq, Sarà, Casanova, Feliu and Morand2003), indirect (Fuentes et al. Reference Fuentes, Sainz-Elipe, Sáez-Durán and Galán-Puchades2010), or unknown (Fuentes et al. Reference Fuentes, Cerezuella and Galán-Puchades2000, Reference Fuentes, Sáez, Trelis, Galán-Puchades and Esteban2004; Pisanu et al. Reference Pisanu, Lebailleux and Chapuis2009). An alternative explanation could be the immunosuppressive effect of testosterone. This physiological mechanism could be tested in the future by correlating the immune phenotypes of collected mice with A. annulosa abundance in male A. flavicollis, highlighting the necessity for collaboration between parasite ecologists and immunologists. Crucially, however, we found no significant host sex effect for the remaining five species, or for total intestinal nematode abundance. This aligns with a substantial body of research that failed to uncover a sex-bias in intestinal nematode parasitism of rodents (e.g. Fuentes et al. Reference Fuentes, Sáez, Trelis, Galán-Puchades and Esteban2004, Reference Fuentes, Sainz-Elipe and Galán-Puchades2007, Reference Fuentes, Sainz-Elipe, Sáez-Durán and Galán-Puchades2010; Goüy de Bellocq et al. Reference Goüy de Bellocq, Sarà, Casanova, Feliu and Morand2003). However, while our data suggest that males of A. flavicollis are not universally ‘the sicker sex’ for each parasite species, sex-dependent dynamics may still exist outside of simple abundance metrics, with males and females differentially contributing to parasite dynamics even in the absence of a sex-bias in infection (Ferrari et al. Reference Ferrari, Cattadori, Nespereira, Rizzoli and Hudson2004, Reference Ferrari, Rosà, Pugliese and Hudson2007).
The effect of B chromosomes
The significant association between the presence of B chromosomes and increased abundance and aggregation of S. frederici requires attention. Unlike species with free-living stages, mature Syphacia females lay their eggs in the perianal region of the host’s body, allowing for autoinfection via grooming (Anderson Reference Anderson2006; Klimpel et al. Reference Klimpel, Förster and Schmahl2007), leading to high aggregation in a few host individuals (Behnke et al. Reference Behnke, Bajer, Harris, Newington, Pidgeon, Rowlands, Sheriff, Kuliś-Malkowska, Siński, Gilbert and Barnard2008; Müller-Graf et al. Reference Müller-Graf, Durand, Feliu, Hugot, O’callaghan, Renaud, Santalla and Morand1999). While our correlational data cannot confirm causality, these results suggest that Bs may modify the host environment in a way that facilitates nematode parasite infrapopulation establishment and/or survival. If we assume that the higher abundance of S. frederici in certain mice was caused by the presence of Bs, this would indicate support for the view that supernumerary chromosomes negatively affect their carriers, in this case by leading to increased parasite load. However, it is important to tread very carefully when making such assumptions: the results of Adnađević et al. (Reference Adnađević, Jovanović, Blagojević, Budinski, Čabrilo, Bijelić-Čabrilo and Vujošević2014) suggest that potentially beneficial effects of Bs are secondary byproducts of their selection for transmission and accumulation in germinative cells. This suggests highly complex mechanisms of Bs maintenance that are determined by conflict or cooperation with the A genome. In A. flavicollis, Bs harbour transcriptionally active sequences (Rajičić et al. Reference Rajičić, Makunin, Adnađević, Trifonov, Vujošević and Blagojević2021) whose products may affect multiple biological pathways, potentially modulating the host’s responses to external stressors and thereby contributing to variation in parasite susceptibility.
Interactive effects of host sex and Bs presence
A pronounced pattern emerged from the host sex × B chromosome status interaction, wherein females bearing Bs displayed significantly elevated abundances of S. stroma and total intestinal nematodes. We posit that Bs impair host fitness, indirectly altering parasite burdens through physiological stress. Bs typically diminish fitness in animals via depressed fertility, growth rates, and body size, with effects that intensify with increasing number of Bs (Beukeboom et al. Reference Beukeboom, Seif, Plowman, de Ridder and Michiels1998). For instance, in the planarian flatworm Polycelis nigra, Bs curtailed cocoon production in one population and juvenile growth in another (Beukeboom et al. Reference Beukeboom, Seif, Plowman, de Ridder and Michiels1998). Sex-differential impacts are documented in the grasshopper Dichroplus elongatus, where Bs were associated with reduced body size and male mating success, yet elevated embryo production per clutch in certain females (Rosetti et al. Reference Rosetti, Vilardi and Remis2007). Similarly, in another grasshopper species, Myrmeleotettix maculatus, embryonic development decelerated in males but not females (Hewitt and East Reference Hewitt and East1978). Zima et al. (Reference Zima, Piálek and Macholán2003) further reported elevated body mass in Bs-carrying males of A. flavicollis. Given Bs’ recurrent derivation from sex chromosomes, as exemplified in A. flavicollis (Rajičić et al. Reference Rajičić, Romanenko, Karamysheva, Blagojević, Adnađević, Budinski, Bogdanov, Trifonov, Rubtsov and Vujošević2017), sex-specific effects are anticipated, plausibly interfacing with stress responses or parasite dynamics.
Methodological implications and limitations
To the best of our knowledge, this is the first study to utilise the Monte Carlo statistical framework to test hypotheses regarding the effects of intrinsic factors on intestinal nematode abundance in rodents. While generalized linear models (GLMs) and generalized linear mixed models (GLMMs) are the standard for analysing highly aggregated parasite abundance datasets, where errors tend to have a negative binomial distribution (Wilson et al. Reference Wilson, Grenfell and Shaw1996, Reference Wilson, Bjørnstad, Dobson, Merler, Poglayen, Randolph, Read, Skorping, Hudson, Rizzoli, Grenfell, Heesterbeek and Dobson2002), the Monte Carlo approach allows researchers to design an analysis from the ground up, customizing the test statistic and randomisation method for each dataset without making any assumptions on error distribution (Gotelli and Ellison Reference Gotelli and Ellison2013). Given the highly aggregated nature of parasite abundance/infrapopulations, we encourage parasitologists studying intestinal nematodes of rodents to include Monte Carlo analyses in their statistical toolkit, as they can be a valuable asset in addition to parametric models. An important limitation of the current study is the binary coding of B chromosome presence (present/absent). While most B+ yellow-necked mice carry a single B chromosome, with two or more Bs being a much rarer occurrence (e.g. Vujošević et al. Reference Vujošević, Jojić, Bugarski-Stanojević and Blagojević2007, Reference Vujošević, Blagojević, Jojić-Šipetić, Bugarski-Stanojević, Adnađević and Stamenković2009; Jojić et al. Reference Jojić, Blagojević and Vujošević2011), the exact number of Bs would be a valuable additional source of information that allows for explicit tests of dosage effects.
Conclusion
In conclusion, a Monte Carlo analysis of the effects of host sex and Bs presence on the intestinal nematode parasites of A. flavicollis in Serbia revealed interesting patterns that merit further attention. The abundance of A. annulosa was significantly higher in male mice, in line with expectations of immunocompetence-handicap and sexual selection hypotheses. At the same time, the absence of sex-bias in the remaining species, as well as intestinal nematodes in general, when taken with existing evidence of differential effects of males and females on parasite dynamics that are independent of parasite abundance, provides the impetus to reassess the relevance of sex-biases in parasite load in parasitological studies. A significantly higher abundance of S. frederici in mice with B chromosomes and of S. stroma in females with Bs points the way to a potentially fruitful line of study. However, disentangling the potential effects of Bs from other closely interwoven factors will require experimental studies. Considering this, perhaps the most important conclusion of the current study is that there is still much work to be done if we wish to understand the way biological, ecological, and anthropogenic factors act in concert to shape the abundance of parasites in their hosts. Monte Carlo analyses offer a powerful statistical tool that can aid parasitologists in navigating the demands of such complex studies, with the end goal of gaining a better understanding of the host-parasite relationship.
Acknowledgements
The authors wish to thank Srđan Milićević for his kind assistance with the statistical analysis of the data. The results presented in this manuscript are in line with Sustainable Development Goal 13 (Climate action) of the United Nations 2030 Agenda.
Financial support
This research was supported by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia, Contract Nos. 173003 and 451-03-136/2025-03/200007. The authors gratefully acknowledge the additional financial support of the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Grants No. 451-03-33/2026-03/200125 & 451-03-34/2026-03/200125).
Competing interests
The authors declare no conflict of interests.
Ethical standard
All procedures involving animals were carried out in compliance with Directive 2010/63/EU of the European Parliament and of the Council (22 September 2010) on the protection of animals used for scientific purposes. The experimental protocol was approved by the Ethical Committee for the Use of Laboratory Animals of the Institute for Biological Research ‘Siniša Stanković’, University of Belgrade. Animals were euthanized using ether anaesthesia in accordance with the Committee’s recommendations.
Data availability
The raw data, metadata and Quarto report containing R code for statistical analysis have been uploaded to a data repository (DOI: 10.5281/zenodo.20660767).
Open access
