Introduction
Many hosts are exposed to parasites, especially helminths, when they encounter infective stages that are clumped together within discrete packets. Examples include the ingestion by definitive hosts of clumps of larvae located within intermediate hosts and the penetration of hosts by aggregates of free-living larvae or eggs. Compared to the mode of ‘trickle’ transmission involving repeated exposure of hosts to low doses of infective larvae (e.g., Walker et al. Reference Walker, Hall and Basanez2010), there has been little attention devoted to characterizing patterns of infection in samples of hosts, especially intermediate hosts, exposed to clumped infective stages. This is a shortcoming because clumped transmission is known to influence patterns of parasite aggregation within exposed hosts (Walker et al. Reference Walker, Hall and Basanez2010), the rate of development of host immunity (Glover et al. Reference Glover, Columbo, Thornton and Grencis2019), the distribution of clonal populations of worms in intermediate hosts (Criscione et al. Reference Criscione, van Paridon, Gilleard and Goater2020, Reference Criscione, Hulke and Goater2022), and the rate of evolution of drug resistance (Cornell et al. Reference Cornell, Isham, Smith and Grenfell2003).
The lancet liver fluke, Dicrocoelium dendriticum, has a three-host life cycle that includes an obligate clumped transmission stage. Following reproduction of hermaphroditic worms located within the bile ducts of grazing mammals, embryonated eggs are passed into the external environment via host faeces. Terrestrial snails encounter fluke eggs while foraging on the soil surface or on vegetation. Following ingestion, miracidia contained within an egg hatch, penetrate the gut, and then undergo cycles of asexual reproduction with two sporocyst generations, ultimately leading to the production of motile, tailed cercariae. These infective larvae are packaged into discrete, fluid-filled, mucous balls (= slimeballs) within the snail’s lung that are secreted onto substrate (Krull and Mapes, Reference Krull and Mapes1952, Reference Krull and Mapes1953). To continue the life cycle, slimeballs must be encountered on the substrate, presumably by individual foraging worker ants, then broken apart to release individual cercariae prior to ingestion.
The lancet liver fluke has been introduced into a parkland region in southern Alberta, Canada (van Paridon et al. Reference van Paridon, Colwell, Goater and Gilleard2017a). Within the confines of Cypress Hills Interprovincial Park (CHIP), the life cycle has become established within various grazing herbivores (Beck et al. Reference Beck, Goater and Colwell2015), three species of Oreohelid land snails (Dempsey et al. Reference Dempsey, Burg and Goater2019), and two species of Formicid ants (van Paridon et al. Reference van Paridon, Gilleard, Colwell and Goater2017b). Although spatiotemporal patterns of D. dendriticum infection within this region have been described in snail intermediate hosts (Dempsey et al. Reference Dempsey, Burg and Goater2019), there is little information available on how factors such as season, location, year, host species, or host size contribute to variation in mean metacercariae intensity in samples of ant intermediate hosts.
In this study, we describe spatial and temporal variation in prevalence and intensity of D. dendriticum metacercariae within samples of two species of ants, Formica aserva and F. podzolica, collected from nests in CHIP. The first aim was to assess how variation in metacercariae counts in ant samples was associated with variation in site, year, and month. A second aim was to characterize the timing of larval fluke recruitment in ants during the annual cycle of ant activity. Lastly, given the absence of information regarding parasites of ants in this region, we report the results of a host survey involving other endoparasites found in samples of F. aserva and F. podzolica collected from this parkland region.
Materials and methods
Site descriptions
van Paridon et al. (Reference van Paridon, Gilleard, Colwell and Goater2017b) and Criscione et al. (Reference Criscione, van Paridon, Gilleard and Goater2020) provide general descriptions of the elevated (maximum 1,465 m above sea level) Cypress Hills sky island located at the northern edge of the North American Great Plains in southeastern Alberta. Two of the three sites (Staff Camp – SC and Ski Hill – SH) contained known infected ant nests that have provided a source of infected and uninfected ants for our previous surveys of infected ants (van Paridon et al. Reference van Paridon, Gilleard, Colwell and Goater2017b) and land snails (Dempsey et al. Reference Dempsey, Burg and Goater2019) and for our behaviour (Li et al. Reference Li, Goater and Wasmuth2025) and population genetics (Criscione et al. Reference Criscione, van Paridon, Gilleard and Goater2020) studies. Sites SC and SH are approximately 2 km apart. Each of these sites is located on gently sloped, south-facing forest stands under canopies dominated by mature trembling aspen, lodgepole pine, and white spruce. Formica aserva nests are commonly observed in this habitat type, typically located within woody downfall and/or at the base of tree stumps.
Samples of F. podzolica were collected from a third site (Trans Canada Trail – TCT) on the same day as the F. aserva samples. This site is located approximately 12 km southeast of the SC and SH sites. In contrast to the forested F. aserva sites, TCT is open prairie dominated by grasses and shrubs that are characteristic of the rough fescue/parry oat grass plant community type. The site is south facing, with a gently sloping aspect and sandy substrate.
Ant collection
Samples of F. aserva and F. podzolica were collected from the three sites during the last weeks of May, June, July, August, and September in summers of 2021 and 2022. Ants were collected between 8:00 and 10:00 a.m. when cooler temperatures favoured attachment of ants on plants (Gasque and Fredensborg Reference Gasque and Fredensborg2023). At SC and SH, approximately 10 large (20–100 cm diameter) F. aserva nests were conspicuous within the approximately 100 m × 100 m site. At TCT, there were 15 conspicuous F. podzolica nests (10–30 cm diameter) located within the 20 m × 10 m site. Individual nests at the three sites contained hundreds to thousands of active workers. For monthly F. aserva collections at SC and SH, ants that were associated with a single nest opening were sampled. These were the largest nests at the two sites and were located approximately 10 m from the next visible nest. These were the same nests sampled in previous studies (Criscione et al. Reference Criscione, van Paridon, Gilleard and Goater2020; Li et al. Reference Li, Goater and Wasmuth2025; van Paridon et al. Reference van Paridon, Gilleard, Colwell and Goater2017b). In contrast, F. podzolica collections each month involved pooling samples from multiple nest entrances within the 20 × 10 m site.
We collected a maximum of 200 ants from each site each month. A portion of these were sampled from plants located within 2 m of a nest entrance. These ants were easily observed with their mandibles firmly attached to the petals of flowers. Attached ants were gently removed from their flower and immediately fixed in ethanol. Concurrently, a sample of ants was collected directly from the nest. For collection, gentle pressure was applied to the surface of a nest with an open-topped plastic, 1 L container. After approximately 100–200 workers had entered the container, ethanol was added for fixation. We assumed that ants collected at the nest entrance and those collected attached to plants originated from the same nest. The purpose of collecting ants directly from a nest was to provide an estimate of total D. dendriticum prevalence within an individual nest.
Enumeration of metacercariae in ants
Dissection methods used to assess metacercariae intensity (= number of parasites/host) in individual ants followed van Paridon et al. (Reference van Paridon, Gilleard, Colwell and Goater2017b). Ants were placed in a Petri dish with ~5 mL water and then the haemocoel was isolated and teased apart with forceps at 10× magnification with a dissecting microscope. Following counts, metacercariae from individual ants were pooled, removed from solution with a pipette, mounted on a microscope slide, and then observed under a compound microscope. Metacercariae were classified as mature if they were enveloped by a thick, double-layered cyst wall (Faltynkova et al. Reference Faltynkova, Karvonen and Valtonen2011); immature if they were not (Fig. 1). In rare cases, ants collected during their attachment to plants did not contain metacercariae in the haemocoel. In these cases, brain tissue was carefully teased apart using the methods described above to scan for an unencysted ‘brainworm’.
Figure 1. Light microscope images of immature (A) and mature (B) Dicrocoelium dendriticum metacercariae dissected from the haemocoel of ants, Formica aserva (10× magnification).
Concurrent with the collection of metacercariae count data in ants, the presence and number of other endoparasites were recorded. The size of individual ants was approximated by measuring the length of the right hind tibia with a micrometre at 4× under a compound microscope.
Analyses
Due to heterogeneity in sample sizes between F. aserva and F. podzolica each month and because the factors ‘site’ and ‘host species’ are confounded, worm count data for the two host species were separately analyzed. For F. aserva counts, a generalized linear mixed model (GLMM) fit to a negative binomial distribution using a log-link function was used to assess differences in mean metacercariae counts between samples. Month, year, and host size were fixed effects; site was a random effect. For F. podzolica counts, a generalized linear model (GLM) fit to a negative binomial distribution using a log-link function with month, year, and host size as fixed effects was used. Non-significant two-way interactions were removed from each model after a likelihood ratio test concluded that they do not significantly increase the power of the models. Statistical analyses were completed in R Statistical Software (v4.1; R Core Team Reference Team2024).
The low number of metacercariae observed with incomplete cyst walls (5.3% of 17,909 total observed metacercariae) limited our ability to evaluate temporal variation in metacercariae recruitment into ants. Fisher’s exact tests were used to evaluate differences in the proportion of ants within a sample that contained immature metacercariae. The proportion of non-encysted metacercariae within those ants that contained at least one immature larva was also reported.
Results
Host survey of parasites in ants
Dicrocoelium dendriticum metacercariae were the most commonly encountered endoparasite within the total sample of F. aserva and F. podzolica (Table 1). Three other parasites were encountered, but only in 0.07–1.2% of the total sample. In the case of the unidentified larval mermithid nematode and an unidentified larval cestode, only two hosts within the total sample were infected (Table 1). Single larvae of the parasitoid wasp, Elasmosoma sp., were found in 27 of the total sample of 2,766 ants, representing 0.2–1.2% within F. aserva samples and 0–1.2% within F. podzolica samples.
Table 1. Summary host survey data for macroparasites recovered from ants (Total n = 2766), Formica aserva and F. podzolica, collected from Cypress Hills Interprovincial Park, Alberta
Variation in metacercariae intensity
The range in metacercariae intensity in the total sample of infected F. aserva was 1–168 (n = 490, 25.5 ± 25.4). The results of the GLMM analysis indicated that none of this variation could be explained by month or year. Overall, there was a significant positive correlation between ant size and metacercaria intensity (Supplementary Fig. 1a; Table 2). The range in metacercariae intensity in the total sample of attached F. podzolica was 1–106 (n = 142; mean = 29.4 ± 23.3). The results of the GLM analysis indicated that none of this variation could be explained by month or year (Table 2). The correlation between F. podzolica hind tibia length and metacercariae intensity was not significant (Supplementary Fig. 1b; Table 2).
Table 2. Results of a generalized linear mixed model (Formica aserva) and of a generalized linear model (Formica podzolica) investigating the effects of month, year, and host size on the mean numbers of Dicrocoelium dendriticum metacercariae in samples of two species of ants collected from Cypress Hills Interprovincial Park, Alberta
Twenty-six (5.3%) of the total of 490 F. aserva that were collected from plants did not contain metacercariae in the haemocoel. Of these, 19 contained a single unencysted metacercariae within the suboesophageal ganglia. Similarly, there were 9 (6.3% of 142) F. podzolica that did not have encysted metacercariae in the haemocoel, 7 of which contained a single unencysted brainworm.
Patterns of metacercariae recruitment
95% of the 17,909 metacercariae assessed in this study were enveloped by thick cyst walls. The remaining 5% lacked the characteristic bilayered cyst wall (Fig. 1a) and were considered new recruits. Counts of thin-walled metacercariae were highly variable and inconsistent between months and years for both species (Table 3). With a single exception in June 2022, observations of new F. aserva recruits were restricted to ant samples collected in mid- to late-summer. For samples of F. aserva collected in 2021, only 2 of 223 infected ants contained populations with non-encysted metacercariae (Table 3). In contrast, there was a significant increase in metacercariae recruitment between May and July samples in 2022 (p = 0.007). There was a significant increase in metacercariae recruitment in samples of F. podzolica collected in 2021 (p = 0.020), with a single peak in July samples (Table 3). The observed peak in July 2022 was not significant (p = 0.720).
Table 3. Seasonal changes in the percentage of immature Dicrocoelium dendriticum metacercariae in two species of ants, Formica aserva and F. podzolica, collected from three sites in Cypress Hills Interprovincial Park, Alberta
* Numbers in brackets indicate sample size.
Mean immature metacercariae intensity in ants was 32.6 ± 32.8 (Table 3). Of 26 ants that contained immature metacercariae, 23 (89%) contained only immature metacercariae and were collected from nests and not attached to plants. The remaining 11% of ants that contained immature metacercariae were collected while they were attached to plants. In each of the latter cases, infected ants contained a mixture of mature and immature metacercariae. In ants with mixed infections, an average of 33 ± 43% of the total numbers of metacercariae in an ant were immature.
Discussion
Mean metacercariae intensity within the total sample of ants (n = 677; mean = 27 ± 25) was comparable with the results of our previous studies involving single samples of F. aserva collected from other sites in CHIP (Criscione et al. Reference Criscione, van Paridon, Gilleard and Goater2020; Li et al. Reference Li, Goater and Wasmuth2025; van Paridon et al. Reference van Paridon, Gilleard, Colwell and Goater2017b). Similar mean intensities were reported in a field survey involving 4 species of D. dendriticum-infected Formicid ants collected over 2 years from sheep pastures in northwest Spain (Manga-Gonzalez et al. Reference Manga-Gonzalez, Gonzalez-Lanza, Cabanas and Campo2001; range in mean intensities/host ± SE = 26 ± 16; 28 ± 9; 42 ± 20; 97 ± 21). Although metacercariae intensities in individual Formica spp. can be in the hundreds (review in Manga-Gonzalez et al. Reference Manga-Gonzalez, Gonzalez-Lanza, Cabanas and Campo2001), mean intensities appear to be consistent at approximately 25–30 metacercariae per host. Consistent patterns of infection across continent-wide spatial scales and between species of ant that vary in size, social structure, and behaviour imply common processes of snail-to-ant transmission.
Seasonal peaks in mean metacercariae intensity in samples of ants were not observed in our study. Significant differences in mean intensity between ant nests were also absent. A lack of spatiotemporal patterns of infection also occurred in four species of Formicid ants collected from pastures in Spain (Manga-Gonzalez et al. Reference Manga-Gonzalez, Gonzalez-Lanza, Cabanas and Campo2001). These results involving counts of D. dendriticum metacercariae are in sharp contrast to studies involving numerous metacercariae/host interactions where seasonal infection dynamics, high year-to-year variation, and high spatial variation are frequently reported (reviews in Poulin Reference Poulin2020; Paterson et al. Reference Paterson, Poulin and Selbach2024). This common pattern typically results from heterogeneities in the rates that cercariae penetrate second intermediate hosts, often leading to high seasonal and spatial variation in mean metacercariae intensities. It is unlikely that the absence of spatiotemporal patterns in the Dicrocoelium/ant interaction arises from a lack of heterogeneity in factors such as temperature, precipitation, and snail densities. Indeed, temperature and precipitation extremes are characteristic of these elevated ‘sky islands’. Furthermore, land snail densities in these habitats, including for species of Oreohelix spp. that are known intermediate hosts in CHIP (Dempsey et al. Reference Dempsey, Burg and Goater2019), are seasonally and spatially variable (Anderson et al. Reference Anderson, Weaver and Guralnick2007). In short, such heterogeneities acting upon snail intermediate hosts typically lead to easily detected spatiotemporal patterns of infection, yet such patterns are absent in samples of ants infected with D. dendriticum metacercariae.
The absence of spatiotemporal patterns of mean metacercariae intensity contrasts with the observed seasonal pattern of metacercariae recruitment in ants. Although the total number of infected ants that we observed with immature metacercariae was low, instances in which this occurred in 2021 were restricted to a single sample collected in July for both species of ant. Similarly, a single July peak in recruitment occurred in F. podzolica samples the following year. These results indicate that the exposure of ants to D. dendriticum cercariae is restricted to a narrow seasonal window each summer, but seasonal exposure to slimeballs did not lead to seasonal patterns in metacercariae intensity. Perhaps higher monthly sample sizes would increase our ability to detect seasonal patterns, if present. Furthermore, our sampling scheme could explain these counter-intuitive results. Multiple generations of workers and queens are common within the same nests of wood ants (Stockan et al. Reference Stockan, Robinson, Trager, Yao, Seifert, Stockan and Robinson2016). Formica aserva nests can comprise one to several founding queens, together with several generations of workers (Scarparo et al. Reference Scarparo, West, Brelsford and Purcell2024). Our samples likely included workers that ranged in age from a few weeks to several years. Heterogeneity in host age and history of exposure within our ant samples likely masked seasonal patterns that would otherwise be detectable if we could follow infection rates within cohorts of infected ants.
Dempsey et al. (Reference Dempsey, Burg and Goater2019) showed that D. dendriticum-infected Oreohelix spp. are common (prevalence = 10–25%) in microhabitats immediately surrounding infected ant nests in CHIP and that sporocysts and cercariae appeared to be fully developed within the snails throughout the summer months. Their survey results indicate that infected snails are common throughout the summer, but slimeballs are either rarely produced, rarely released onto pasture, or both. These results are consistent with the laboratory studies reported by Krull and Mapes (Reference Krull and Mapes1952). It appears that whereas infected Oreohelix spp. likely contain life-long infections, they only release slimeballs under specific environmental conditions. Like many land snails, Oreohelids frequently enter periods of metabolic quiescence (aestivation) in summer to reduce the risk of desiccation (Rees and Hand, Reference Rees and Hand1993). This phenomenon has been observed in each of the three sympatric Oreohelix spp. located at the study sites in CHIP (Dempsey et al. Reference Dempsey, Burg and Goater2019). High densities of Oreohelids have also been observed at the study sites during periodic rains in mid-summer, particularly when the rains are followed by consecutive days of high humidity. In the absence of experimental data, it is expected that specific environmental conditions (e.g., soil moisture and relative humidity) provide triggers that lead to the release of cercariae from snails within slimeballs. These conditions, in turn, may lead to restricted and highly variable rates of encounter between clumps of cercariae and foraging wood ants.
A pattern of restricted seasonal recruitment is consistent with the results of population genetics data obtained from metacercariae in F. aserva collected at the same nests. Criscione et al. (Reference Criscione, van Paridon, Gilleard and Goater2020) showed that clonal diversity of metacercariae in samples of infected ants was lower than for any other species of trematode. The authors suggested that the exceptionally low diversity of clonemates in ants resulted from infrequent exposure, perhaps just once in a lifetime, of genetically identical cercariae located within slimeballs. Unfortunately, our ability to detect bouts of cercariae recruitment based on the structure of the cyst wall does not have the resolution of these genetic data. However, the contrasting approaches lead to a broadly similar conclusion of clumped and restricted cercariae recruitment into ants. Intriguingly, the genetic data presented by Criscione et al. (Reference Criscione, van Paridon, Gilleard and Goater2020) further showed that ants in the same nest share clones of cercariae. From a transmission perspective, this pattern could only arise if nestmates forage on the same slimeball within their nest, or forage on multiple slimeballs that originate from the same infected snail.
The temporal patterns of metacercariae recruitment that we observed can also be used to inform aspects of the iconic behavioural manipulation in ants. As expected, only D. dendriticum-infected ants were found attached to plants. Even in rare cases in which metacercariae were not recovered in the haemocoel, these ants contained a metacercariae in the brain. Thus, the results confirm the assumption (Manga-Gonzalez et al. Reference Manga-Gonzalez, Gonzalez-Lanza, Cabanas and Campo2001) that at least one metacercariae is required to orchestrate the complex attach/detach/repeat sequence of manipulation in ants. A further striking observation is that in those few ants that contained a single cohort of immature metacercariae, none were found attached to plants. The implication is that the onset of ant manipulation requires a period of development for the metacercariae in the hemocoel to reach infectivity or for the brainworm to complete development within the suboesophageal ganglion. Other manipulative parasites require an obligate period of development prior to the onset of host manipulation (Hughes et al. Reference Hughes, Brodeur and Thomas2012; Moore Reference Moore2002). Taken together, the results support the idea that classical demographic surveys of parasite hosts can lead to a more complete understanding of complex host manipulations.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X25101028.
Acknowledgements
We thank personnel in Cypress Hills Provincial Park, especially Peter Swain and Chris Dodds, for logistical support. We thank Liam Burns for assistance with fieldwork and ant dissections, and we thank Shelley Hoover and Rob Laird for sharing their expertise.
Author contribution
LE contributed to field collections, dissections, analyses, and writing. CG conceived the project and contributed supervisorship, project management, writing, and editing.
Financial support
This research was supported by the Natural Sciences and Engineering Research Council of Canada to CG and the Alberta Conservation Association Grants in Biodiversity Program to LE.
Competing interests
The authors declare there are no conflicts of interest.
Ethical standard
Not applicable
Open access
