Mathematical Modelling of Parasite Dynamics: A Stochastic Simulation-Based Approach and Parameter Estimation via Modified Sequential-Type Approximate Bayesian Computation.

The development of mathematical models for studying newly emerging and re-emerging infectious diseases has gained momentum due to global events. The gyrodactylid-fish system, like many host-parasite systems, serves as a valuable resource for ecological, evolutionary, and epidemiological investigations owing to its ease of experimental manipulation and long-term monitoring. Although this system has an existing individual-based model, it falls short in capturing information about species-specific microhabitat preferences and other biological details for different Gyrodactylus strains across diverse fish populations. This current study introduces a new individual-based stochastic simulation model that uses a hybrid τ -leaping algorithm to incorporate this essential data, enhancing our understanding of the complexity of the gyrodactylid-fish system. We compare the infection dynamics of three gyrodactylid strains across three host populations. A modified sequential-type approximate Bayesian computation (ABC) method, based on sequential Monte Carlo and sequential importance sampling, is developed. Additionally, we establish two penalised local-linear regression methods (based on L1 and L2 regularisations) for ABC post-processing analysis to fit our model using existing empirical data. With the support of experimental data and the fitted mathematical model, we address open biological questions for the first time and propose directions for future studies on the gyrodactylid-fish system. The adaptability of the mathematical model extends beyond the gyrodactylid-fish system to other host-parasite systems. Furthermore, the modified ABC methodologies provide efficient calibration for other multi-parameter models characterised by a large set of correlated or independent summary statistics.

Depletion of MHC supertype during domestication can compromise immunocompetence.

The major histocompatibility complex (MHC) plays an important role in infectious disease resistance. The presence of certain MHC alleles and functionally similar groups of MHC alleles (i.e., supertypes) has been associated with resistance to particular parasite species. Farmed and domesticated fish stocks are often depleted in their MHC alleles and supertype diversity, possibly as a consequence of artificial selection for desirable traits, inbreeding (loss of heterozygosity), genetic drift (loss of allelic diversity) and/or reduced parasite biodiversity. Here we quantify the effects of depletion of MHC class II genotype and supertype variation on resistance to the parasite Gyrodactylus turnbulli in guppies (Poecilia reticulata). Compared to the descendants of wild-caught guppies, ornamental fish had a significantly reduced MHC variation (i.e., the numbers of MHC alleles and supertypes per individual, and per population). In addition, ornamental fish were significantly more susceptible to G. turnbulli infections, accumulating peak intensity 10 times higher than that of their wildtype counterparts. Four out of 13 supertypes were associated with a significantly reduced parasite load, and the presence of some supertypes had a dramatic effect on the intensity of infection. Remarkably, the ornamental and wildtype fish differed in the supertypes that were associated with parasite resistance. Analysis with a genetic algorithm showed that resistance-conferring supertypes of the wildtype and ornamental fish shared two unique amino acids in the peptide-binding region of the MHC that were not found in any other alleles. These data show that the supertype demarcation captures some, but not all, of the variation in the immune function of the alleles. This study highlights the importance of managing functional MHC diversity in livestock, and suggests there might be some immunological redundancy among MHC supertypes.

Functional immunogenetic variation, rather than local adaptation, predicts ectoparasite infection intensity in a model fish species.

Natural host populations differ in their susceptibility to infection by parasites, and these intrapopulation differences are still an incompletely understood component of host-parasite dynamics. In this study, we used controlled infection experiments with wild-caught guppies (Poecilia reticulata) and their ectoparasite Gyrodactylus turnbulli to investigate the roles of local adaptation and host genetic composition (immunogenetic and neutral) in explaining differences in susceptibility to infection. We found differences between our four study host populations that were consistent between two parasite source populations, with no indication of local adaptation by either host or parasite at two tested spatial scales. Greater values of host population genetic variability metrics broadly aligned with lower population mean infection intensity, with the best alignments associated with major histocompatibility complex (MHC) "supertypes". Controlling for intrapopulation differences and potential inbreeding variance, we found a significant negative relationship between individual-level functional MHC variability and infection: fish carrying more MHC supertypes experienced infections of lower severity, with limited evidence for supertype-specific effects. We conclude that population-level differences in host infection susceptibility probably reflect variation in parasite selective pressure and/or host evolutionary potential, underpinned by functional immunogenetic variation.

Immunogenetic novelty confers a selective advantage in host-pathogen coevolution.

The major histocompatibility complex (MHC) is crucial to the adaptive immune response of vertebrates and is among the most polymorphic gene families known. Its high diversity is usually attributed to selection imposed by fast-evolving pathogens. Pathogens are thought to evolve to escape recognition by common immune alleles, and, hence, novel MHC alleles, introduced through mutation, recombination, or gene flow, are predicted to give hosts superior resistance. Although this theoretical prediction underpins host-pathogen "Red Queen" coevolution, it has not been demonstrated in the context of natural MHC diversity. Here, we experimentally tested whether novel MHC variants (both alleles and functional "supertypes") increased resistance of guppies (Poecilia reticulata) to a common ectoparasite (Gyrodactylus turnbulli). We used exposure-controlled infection trials with wild-sourced parasites, and Gyrodactylus-naïve host fish that were F2 descendants of crossed wild populations. Hosts carrying MHC variants (alleles or supertypes) that were new to a given parasite population experienced a 35-37% reduction in infection intensity, but the number of MHC variants carried by an individual, analogous to heterozygosity in single-locus systems, was not a significant predictor. Our results provide direct evidence of novel MHC variant advantage, confirming a fundamental mechanism underpinning the exceptional polymorphism of this gene family and highlighting the role of immunogenetic novelty in host-pathogen coevolution.

Gastrodermis ultrastructure of the different life stages of the polyopisthocotylean monogenean gill parasite Discocotyle sagittata.

The polyopisthocotylean Discocotyle sagittata is a blood-feeding monogenean that infects the gill lamellae of rainbow trout, Oncorhynchus mykiss, and brown trout, Salmo trutta. The ultrastructure of their alimentary tract, at different stages of the life cycle, was previously unknown. Here, we show that the gastrodermis of the oncomiracidium, subadult, and adult D. sagittata follows the same structural organization as that of other blood-feeding polyopisthocotyleans, being composed of digestive cells alternating with a connecting syncytium. Digestive cells of the oncomiracidium are found in three developmental forms: undifferentiated, developing differentiated, and differentiated (presumably functioning) cells whereas those of adult and subadult are present in a single functioning state with variable size and content. The apical cytoplasm of adult digestive cells forms conical outgrowths, a feature which is absent in the oncomiracidium. The connecting syncytium of the oncomiracidium has no evidence of metabolic activity, while that of adult and subadult is metabolically active. The lamellae of the connecting syncytium of adults and subadults are more numerous and larger, and their terminal portions are expanded, compared with those of the oncomiracidium. Parallel, tubular, membranous structures are characteristic of the apical cytoplasm of the connecting syncytium of the oncomiracidium. Luminal lamella in the oncomiracidium, subadult, and adult form balloon-like structures enclosing some luminal contents, but those of the oncomiracidium are larger, bounded by nucleated cytoplasmic layer, and enclose more luminal contents. The possible functions of these structures and mechanism of digestion in both oncomiracidium and adult are discussed.

RNA-Seq analysis of the guppy immune response against Gyrodactylus bullatarudis infection.

Gyrodactylids are ubiquitous ectoparasites of teleost fish, but our understanding of the host immune response against them is fragmentary. Here, we used RNA-Seq to investigate genes involved in the primary response to infection with Gyrodactylus bullatarudis on the skin of guppies, Poecilia reticulata, an important evolutionary model, but also one of the most common fish in the global ornamental trade. Analysis of differentially expressed genes identified several immune-related categories, including IL-17 signalling pathway and Th17 cell differentiation, cytokine-cytokine receptor interaction, chemokine signalling pathway, NOD-like receptor signalling pathway, natural killer cell-mediated cytotoxicity and pathways involved in antigen recognition, processing and presentation. Components of both the innate and the adaptive immune responses play a role in response to gyrodactylid infection. Genes involved in IL-17/Th17 response were particularly enriched among differentially expressed genes, suggesting a significant role for this pathway in fish responses to ectoparasites. Our results revealed a sizable list of genes potentially involved in the teleost-gyrodactylid immune response.

Smell of Infection: A Novel, Noninvasive Method for Detection of Fish Excretory-Secretory Proteins.

Chemical signals are produced by aquatic organisms following predatory attacks or perturbations such as parasitic infection. Ectoparasites feeding on fish hosts are likely to cause release of similar alarm cues into the environment due to the stress, wounding, and immune response stimulated upon infection. Alarm cues are often released in the form of proteins, antimicrobial peptides, and immunoglobulins that provide important insights into bodily function and infection status. Here we outline a noninvasive method to identify potential chemical cues associated with infection in fish by extracting, purifying, and characterizing proteins from water samples from cultured fish. Gel free proteomic methods were deemed the most suitable for protein detection in saline water samples. It was confirmed that teleost proteins can be characterized from water and that variation in protein profiles could be detected between infected and uninfected individuals and fish and parasite only water samples. Our novel assay provides a noninvasive method for assessing the health condition of both wild and farmed aquatic organisms. Similar to environmental DNA monitoring methods, these proteomic techniques could provide an important tool in applied ecology and aquatic biology.

Half the story: Thermal effects on within-host infectious disease progression in a warming climate.

Immune defense is temperature dependent in cold-blooded vertebrates (CBVs) and thus directly impacted by global warming. We examined whether immunity and within-host infectious disease progression are altered in CBVs under realistic climate warming in a seasonal mid-latitude setting. Going further, we also examined how large thermal effects are in relation to the effects of other environmental variation in such a setting (critical to our ability to project infectious disease dynamics from thermal relationships alone). We employed the three-spined stickleback and three ecologically relevant parasite infections as a "wild" model. To generate a realistic climatic warming scenario we used naturalistic outdoors mesocosms with precise temperature control. We also conducted laboratory experiments to estimate thermal effects on immunity and within-host infectious disease progression under controlled conditions. As experimental readouts we measured disease progression for the parasites and expression in 14 immune-associated genes (providing insight into immunophenotypic responses). Our mesocosm experiment demonstrated significant perturbation due to modest warming (+2°C), altering the magnitude and phenology of disease. Our laboratory experiments demonstrated substantial thermal effects. Prevailing thermal effects were more important than lagged thermal effects and disease progression increased or decreased in severity with increasing temperature in an infection-specific way. Combining laboratory-determined thermal effects with our mesocosm data, we used inverse modeling to partition seasonal variation in Saprolegnia disease progression into a thermal effect and a latent immunocompetence effect (driven by nonthermal environmental variation and correlating with immune gene expression). The immunocompetence effect was large, accounting for at least as much variation in Saprolegnia disease as the thermal effect. This suggests that managers of CBV populations in variable environments may not be able to reliably project infectious disease risk from thermal data alone. Nevertheless, such projections would be improved by primarily considering prevailing thermal effects in the case of within-host disease and by incorporating validated measures of immunocompetence.

Transmission risk predicts avoidance of infected conspecifics in Trinidadian guppies.

Associating with conspecifics afflicted with infectious diseases increases the risk of becoming infected, but engaging in avoidance behaviour incurs the cost of lost social benefits. Across systems, infected individuals vary in the transmission risk they pose, so natural selection should favour risk-sensitive avoidance behaviour that optimally balances the costs and benefits of sociality. Here, we use the guppy Poecilia reticulata-Gyrodactylus turnbulli host-parasite system to test the prediction that individuals avoid infected conspecifics in proportion to the transmission risk they pose. In dichotomous choice tests, uninfected fish avoided both the chemical and visual cues, presented separately, of infected conspecifics only in the later stages of infection. A transmission experiment indicated that this avoidance behaviour accurately tracked transmission risk (quantified as both the speed at which transmission occurs and the number of parasites transmitting) through the course of infection. Together, these findings reveal that uninfected hosts can use redundant cues across sensory systems to inform dynamic risk-sensitive avoidance behaviour. This correlation between the transmission risk posed by infected individuals and the avoidance response they elicit has implications for the evolutionary ecology of infectious disease, and its explicit inclusion may improve the ability of epidemic models to predict disease spread.

East-West Divide: temperature and land cover drive spatial variation of Toxoplasma gondii infection in Eurasian otters (Lutra lutra) from England and Wales.

Toxoplasma gondii, a zoonotic parasite of global importance, infects all endothermic vertebrates, with extensive health implications. The prevalence of this parasite is seldom monitored in wildlife. Here, a semi-aquatic species, the Eurasian otter (Lutra lutra) was used as a model to assess the potential effect of climate, land cover and biotic factors on T. gondii seroprevalence in British wildlife. The Sabin-Feldman cytoplasm-modifying dye test identified T. gondii antibodies in 25·5% of blood samples from otters found dead, mainly as road kill, in England and Wales, between 2004 and 2010. Otters in the east of England were more likely to be infected with T. gondii than those in western regions. Land cover and temperature are key determinants of T. gondii infection risk, with more infection in arable areas and lower infection where temperatures are higher. The probability of T. gondii infection increased with host age, reflecting cumulative exposure with time, but there was no association between T. gondii seroprevalence and cause of host death.

Scanning and transmission electron microscopy of the histopathological impact of Macrogyrodactylus clarii (Monogenea: Gyrodactylidae) on the gills of catfish, Clarias gariepinus.

Scanning and transmission electron microscopy (TEM) were used to study the histopathological effects of the monogenean Macrogyrodactylus clarii Gussev, 1961 on the gills of the catfish Clarias gariepinus (Burchell). Suction generated during attachment created 'footprints' on host surfaces in which the host tissues were elevated above the general gill surface. 'Footprints' were bordered by four clefts caused by the muscular flaps on the anterior, lateral and posterior margins of the haptor. The hamuli points penetrate the gill tissue but no evidence was found for the insertion of the marginal hooklets. At the site of attachment, host cells adjacent to the lateral flaps often appeared compressed and widely spaced with large intercellular spaces. Desquamation of these surface epithelia was also apparent and some of the widely spaced epithelial cells had pseudopodium-like processes. Cells within the upper surface epithelial layer of the host were vacuolated and necrotic. Ruptured blood capillaries (blood spaces) in the secondary gill lamellae contained atypical compressed erythrocytes, agranular and granular leucocytes and evidence of haemorrhaging. Cells with fibrotic cytoplasm, putative phagocytes and host mucous cells were evidence of a host response at the site of parasite attachment. The possible role of these cells is discussed in relation to host resistance against infection.

Parasites of Trinidadian guppies: evidence for sex- and age-specific trait-mediated indirect effects of predators.

Predation pressure can alter the morphology, physiology, life history, and behavior of prey; each of these in turn can change how surviving prey interact with parasites. These trait-mediated indirect effects may change in direction or intensity during growth or, in sexually dimorphic species, between the sexes. The Trinidadian guppy, Poecilia reticulata presents a unique opportunity to examine these interactions; its behavioral ecology has been intensively studied in wild populations with well-characterized predator faunas. Predation pressure is known to have driven the evolution of many guppy traits; for example, in high-predation sites, females (but not males) tend to shoal, and this anti-predator behavior facilitates parasite transmission. To test for evidence of predator-driven differences in infection in natural populations, we collected 4715 guppies from 62 sites across Trinidad between 2003 and 2009 and screened them for ectosymbionts, including Gyrodactylus. A novel model-averaging analysis revealed that females were more likely to be infected with Gyrodactylus parasites than males, but only in populations with both high predation pressure and high infection prevalence. We propose that the difference in shoaling tendency between the sexes could explain the observed difference in infection prevalence between males and females in high-predation sites. The infection rate of juveniles did not vary with predation regime, probably because juveniles face constant predation pressure from conspecific adults and therefore tend to shoal in both high- and low-predation sites. This represents the first evidence for age- and sex-specific trait-mediated indirect effects of predators on the probability of infection in their prey.

Lunar-linked biological rhythms in the immune system of freshwater three-spined stickleback.

Immune responses are widely accepted to be under circadian regulation via a molecular clock, with many practical consequences, but much less is known of how other biological rhythms could affect the immune system. In this study, we search for lunar rhythms (circalunar, circasemilunar, and circatidal cycles) in the immune expression of the recently marine-derived freshwater fish, the low-plate morph of the three-spined stickleback. We employed time series of immune expression (mRNA) measurements for 14 immune-associated genes, representing a variety of immunological pathways. Times series measurements were taken on fish populations in the wild, in seminatural outdoor mesocosms, and in the laboratory, according to sampling regimens originally designed to study circannual variation but with the additional potential to provide information about lunar variation. Our evidence best supported the existence of a very small endogenous tidal rhythm. This is consistent with previous suggestions of the existence of a primordial tidal endogenous clock, some elements of which may be conserved in animals evolving outside the marine environment.

Can parasites use predators to spread between primary hosts?

Parasites typically have low reproductive fitness on paratenic hosts. Such hosts offer other significant inclusive fitness benefits to parasites, however, such as increased mobility and migration potential. The parasite fauna of the guppy (Poecilia reticulata) is dominated by the directly transmitted ectoparasites Gyrodactylus bullatarudis and Gyrodactylus turnbulli. In the wild, close predatory and competitive interactions occur between the guppy and the killifish Rivulus hartii. Previous observations suggest that these fish can share gyrodactylids, so we tested experimentally whether these parasites can use R. hartii as an alternative host. In aquaria, G. bullatarudis was the only species able to transmit from prey to predator. Both parasite species transferred equally well to prey when the predator was experimentally infected. However, in semi-natural conditions, G. bullatarudis transmitted more successfully to the prey fish. Importantly, G. bullatarudis also survived significantly longer on R. hartii out of water. As R. hartii can migrate overland between isolated guppy populations, G. bullatarudis may have an enhanced ability to disperse and colonize new host populations, consistent with its wider distribution in the wild. To our knowledge, this is the first empirical study demonstrating a predator acting as a paratenic host for the parasites of its prey.

Mitochondria-derived organelles in the diplomonad fish parasite Spironucleus vortens.

In some eukaryotes, mitochondria have become modified during evolution to yield derived organelles (MDOs) of a similar size (hydrogenosomes), or extremely reduced to produce tiny cellular vesicles (mitosomes). The current study provides evidence for the presence of MDOs in the highly infectious fish pathogen Spironucleus vortens, an organism that produces H2 and is shown here to have no detectable cytochromes. Transmission electron microscopy (TEM) reveals that S. vortens trophozoites contain electron-dense, membranous structures sometimes with an electron-dense core (200 nm-1 µm), resembling the hydrogenosomes previously described in other protists from habitats deficient in O2. Confocal microscopy establishes that these organelles exhibit autofluorescence emission spectra similar to flavoprotein constituents previously described for mitochondria and also present in hydrogenosomes. These organelles possess a membrane potential and are labelled by a fluorescently labeled antibody against Fe-hydrogenase from Blastocystis hominis. Heterologous antibodies raised to mitochondrial proteins frataxin and Isu1, also exhibit a discrete punctate pattern of localization in S. vortens; however these labelled structures are distinctly smaller (90-150 nm) than hydrogenosomes as observed previously in other organisms. TEM confirms the presence of double-membrane bounded organelles of this smaller size. In addition, strong background immunostaining occurs in the cytosol for frataxin and Isu1, and labelling by anti-ferredoxin antibody is generally distributed and not specifically localized except for at the anterior polar region. This suggests that some of the functions traditionally attributed to such MDOs may also occur elsewhere. The specialized parasitic life-style of S. vortens may necessitate more complex intracellular compartmentation of redox reactions than previously recognized. Control of infection requires biochemical characterization of redox-related organelles.

Gastrointestinal symbionts of chimpanzees in Cantanhez National Park, Guinea-Bissau with respect to habitat fragmentation.

One of the major factors threatening chimpanzees (Pan troglodytes verus) in Guinea-Bissau is habitat fragmentation. Such fragmentation may cause changes in symbiont dynamics resulting in increased susceptibility to infection, changes in host specificity and virulence. We monitored gastrointestinal symbiotic fauna of three chimpanzee subpopulations living within Cantanhez National Park (CNP) in Guinea Bissau in the areas with different levels of anthropogenic fragmentation. Using standard coproscopical methods (merthiolate-iodine formalin concentration and Sheather's flotation) we examined 102 fecal samples and identified at least 13 different symbiotic genera (Troglodytella abrassarti, Troglocorys cava, Blastocystis spp., Entamoeba spp., Iodamoeba butschlii, Giardia intestinalis, Chilomastix mesnili, Bertiella sp., Probstmayria gombensis, unidentified strongylids, Strongyloides stercoralis, Strongyloides fuelleborni, and Trichuris sp.). The symbiotic fauna of the CNP chimpanzees is comparable to that reported for other wild chimpanzee populations, although CNP chimpanzees have a higher prevalence of Trichuris sp. Symbiont richness was higher in chimpanzee subpopulations living in fragmented forests compared to the community inhabiting continuous forest area. We reported significantly higher prevalence of G. intestinalis in chimpanzees from fragmented areas, which could be attributed to increased contact with humans and livestock.

Fish response to the presence of hydrokinetic turbines as a sustainable energy solution.

Hydrokinetic turbines such as vertical axis turbines (VATs) may provide decentralised, clean, sustainable energy for remote communities that lack access to the main energy grid or renewable resources. As traditional hydropower adversely alters aquatic ecosystems, it is essential to evaluate the environmental consequences of deploying VATs in riverine ecosystems to meet current and future energy needs. This study explores the implications of VATs on fish movement by observing fish swimming behaviour under two discharges, turbine operation states, and cross-sections confinements using scaled laboratory experiments. Our findings reveal that for cross-sectional confined conditions neither discharge, turbine presence, nor device operation, prevented fish from passing around and through the turbine both in the up- and downstream directions. However, fish spent the least time near the turbine vicinity and within the turbine's turbulent, low-velocity wake, indicating avoidance behaviour. Swimming in a less confined test section further reduced the time spent within the turbine's vicinity and wake, increasing the distance fish kept away from the device. Our results contribute to an understanding of VATs as low-risk hazards for fish swimming behaviour, advancing the potential of deploying VATs in rivers, estuaries or sea as a renewable energy solution for remote communities.

Parasites pitched against nature: Pitch Lake water protects guppies (Poecilia reticulata) from microbial and gyrodactylid infections.

SUMMARY The enemy release hypothesis proposes that in parasite depleted habitats, populations will experience relaxed selection and become more susceptible (or less tolerant) to pathogenic infections. Here, we focus on a population of guppies (Poecilia reticulata) that are found in an extreme environment (the Pitch Lake, Trinidad) and examine whether this habitat represents a refuge from parasites. We investigated the efficacy of pitch in preventing microbial infections in Pitch Lake guppies, by exposing them to dechlorinated water, and reducing gyrodactylid infections on non-Pitch Lake guppies by transferring them to Pitch Lake water. We show that (i) natural prevalence of ectoparasites in the Pitch Lake is low compared to reference populations, (ii) Pitch Lake guppies transferred into aquarium water develop microbial infections, and (iii) experimentally infected guppies are cured of their gyrodactylid infections both by natural Pitch Lake water and by dechlorinated water containing solid pitch. These results indicate a role for Pitch Lake water in the defence of guppies from their parasites and suggest that Pitch Lake guppies might have undergone enemy release in this extreme environment. The Pitch Lake provides an ideal ecosystem for studies on immune gene evolution in the absence of parasites and long-term evolutionary implications of hydrocarbon pollution for vertebrates.

Moving towards improved surveillance and earlier diagnosis of aquatic pathogens: From traditional methods to emerging technologies.

Early and accurate diagnosis is key to mitigating the impact of infectious diseases, along with efficient surveillance. This however is particularly challenging in aquatic environments due to hidden biodiversity and physical constraints. Traditional diagnostics, such as visual diagnosis and histopathology, are still widely used, but increasingly technological advances such as portable next generation sequencing (NGS) and artificial intelligence (AI) are being tested for early diagnosis. The most straightforward methodologies, based on visual diagnosis, rely on specialist knowledge and experience but provide a foundation for surveillance. Future computational remote sensing methods, such as AI image diagnosis and drone surveillance, will ultimately reduce labour costs whilst not compromising on sensitivity, but they require capital and infrastructural investment. Molecular techniques have advanced rapidly in the last 30 years, from standard PCR through loop-mediated isothermal amplification (LAMP) to NGS approaches, providing a range of technologies that support the currently popular eDNA diagnosis. There is now vast potential for transformative change driven by developments in human diagnostics. Here we compare current surveillance and diagnostic technologies with those that could be used or developed for use in the aquatic environment, against three gold standard ideals of high sensitivity, specificity, rapid diagnosis, and cost-effectiveness.

Spatial and temporal parasite dynamics: microhabitat preferences and infection progression of two co-infecting gyrodactylids.

BACKGROUND: Mathematical modelling of host-parasite systems has seen tremendous developments and broad applications in theoretical and applied ecology. The current study focuses on the infection dynamics of a gyrodactylid-fish system. Previous experimental studies have explored the infrapopulation dynamics of co-infecting ectoparasites, Gyrodactylus turnbulli and G. bullatarudis, on their fish host, Poecilia reticulata, but questions remain about parasite microhabitat preferences, host survival and parasite virulence over time. Here, we use more advanced statistics and a sophisticated mathematical model to investigate these questions based on empirical data to add to our understanding of this gyrodactylid-fish system. METHODS: A rank-based multivariate Kruskal-Wallis test coupled with its post-hoc tests and graphical summaries were used to investigate the spatial and temporal parasite distribution of different gyrodactylid strains across different host populations. By adapting a multi-state Markov model that extends the standard survival models, we improved previous estimates of survival probabilities. Finally, we quantified parasite virulence of three different strains as a function of host mortality and recovery across different fish stocks and sexes. RESULTS: We confirmed that the captive-bred G. turnbulli and wild G. bullatarudis strains preferred the caudal and rostral regions respectively across different fish stocks; however, the wild G. turnbulli strain changed microhabitat preference over time, indicating microhabitat preference of gyrodactylids is host and time dependent. The average time of host infection before recovery or death was between 6 and 14 days. For this gyrodactylid-fish system, a longer period of host infection led to a higher chance of host recovery. Parasite-related mortalities are host, sex and time dependent, whereas fish size is confirmed to be the key determinant of host recovery. CONCLUSION: From existing empirical data, we provided new insights into the gyrodactylid-fish system. This study could inform the modelling of other host-parasite interactions where the entire infection history of the host is of interest by adapting multi-state Markov models. Such models are under-utilised in parasitological studies and could be expanded to estimate relevant epidemiological traits concerning parasite virulence and host survival.