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Non-MHC immunity genes do not affect parasite load in European invasive populations of common raccoon

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Authors:
  • Institute of Nature Conservation, Polish Academy of Sciences

Abstract and Figures

Understanding the evolutionary mechanisms behind invasion success enables predicting which alien species and populations are the most predisposed to become invasive. Parasites may mediate the success of biological invasions through their effect on host fitness. The evolution of increased competitive ability (EICA) hypothesis assumes that escape from parasites during the invasion process allows introduced species to decrease investment in immunity and allocate resources to dispersal and reproduction. Consequently, the selective pressure of parasites on host species in the invasive range should be relaxed. We used the case of the raccoon Procyon lotor invasion in Europe to investigate the effect of gastrointestinal pathogen pressure on non-MHC immune genetic diversity of newly established invasive populations. Despite distinct differences in parasite prevalence between analysed populations, we detected only marginal associations between two analysed SNPs and infection intensity. We argue that the differences in parasite prevalence are better explained by detected earlier associations with specific MHC-DRB alleles. While the escape from native parasites seems to allow decreased investment in overall immunity, which relaxes selective pressure imposed on immune genes, a wide range of MHC variants maintained in the invasive range may protect from newly encountered parasites.
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Non‑MHC immunity genes
do not aect parasite load
in European invasive populations
of common raccoon
Aleksandra Biedrzycka
1*, Maciej K. Konopiński
1, Marcin Popiołek
2, Marlena Zawiślak
2,
Magdalena Bartoszewicz
3 & Agnieszka Kloch
4
Understanding the evolutionary mechanisms behind invasion success enables predicting which
alien species and populations are the most predisposed to become invasive. Parasites may mediate
the success of biological invasions through their eect on host tness. The evolution of increased
competitive ability (EICA) hypothesis assumes that escape from parasites during the invasion process
allows introduced species to decrease investment in immunity and allocate resources to dispersal and
reproduction. Consequently, the selective pressure of parasites on host species in the invasive range
should be relaxed. We used the case of the raccoon Procyon lotor invasion in Europe to investigate
the eect of gastrointestinal pathogen pressure on non‑MHC immune genetic diversity of newly
established invasive populations. Despite distinct dierences in parasite prevalence between analysed
populations, we detected only marginal associations between two analysed SNPs and infection
intensity. We argue that the dierences in parasite prevalence are better explained by detected earlier
associations with specic MHC‑DRB alleles. While the escape from native parasites seems to allow
decreased investment in overall immunity, which relaxes selective pressure imposed on immune
genes, a wide range of MHC variants maintained in the invasive range may protect from newly
encountered parasites.
Invasive species disrupt ecological communities, drive population declines and species extinctions. Parasites
may aect the invasion success of their hosts through their eect on tness and thus on host population growth
and stability14. According to the enemy release hypothesis, newly established populations of non-native species
harbour fewer enemies (pathogens and parasites) in the introduced range compared to the native range. As a
result population regulation is reduced and spatial expansion accelerates5. According to the EICA hypothesis, i.e.
evolution of increased competitive ability such an escape from parasites should favour introduced species that
can decrease investment in immunity and allocate resources to dispersal and reproduction, thereby enhancing
their invasive potential6. Along with leaving pathogens behind, the selective pressure on host species should be
lower in invasive than in native ranges. However, non-native populations may come into contact with native
species and accumulate novel pathogens, gaining high infection intensities over a relatively short time (e.g.7). If
this is the case, ability to ght those infections is crucial to a successful invasion.
A recent meta-analysis indicated that parasite success is limited by the hosts genetic diversity8. Since patho-
gens exert strong selection pressure on their hosts, immunity-related genes are presumed to be under selection
due to host–pathogen coevolution9, 10. However, such relationships have been rarely studied in invasive popula-
tions. e importance of immune gene diversity is supported by signicant associations between specic alleles
and susceptibility to infections. is has been reported in many species; however, most of these examples are
based on the components of the adaptive immunity with a wide range of studies focussing on MHC genes1115.
e role of other immunity genes in coping with parasite infection is oen studied using a small number of Toll-
like receptor (TLR) or cytokine genes1622 although several hundred genes are involved in innate and acquired
immune response. Genes evolving under balancing selection maintain high levels of diversity. MHC genes, which
are usually characterised by dozens to hundreds of allelic variants segregating in natural populations15 or TLRs23,
OPEN
1Institute of Nature Conservation, Polish Academy of Sciences, Al. Mickiewicza 33, 31-120, Kraków,
Poland. 2Department of Parasitology, Faculty of Biological Sciences, University of Wrocław, Przybyszewskiego
63/67, 51-148 Wrocław, Poland. 3Ekspertyzy Przyrodnicze, Szpitalna 2, 66-436 Słońsk, Poland. 4Faculty of Biology,
University of Warsaw, Miecznikowa 1, 02-089 Warszawa, Poland. *email: biedrzycka@iop.krakow.pl
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may play an important role in the immune defence of invasive species24. is is because the high level of the
invasive populations’ immune standing genetic variation, is crucial for their pathogen resistance (reviewed by25).
e innate immune response is responsible for recognizing the pathogen and then initiating the acquired immune
response26, 27. e PRRs (pattern recognition receptors) are the rst line of pathogen detection28). Cytokines are
signalling molecules capable of triggering and modulating the immune response, are the crucial link between
innate and adaptive immunity. ey mediate in removing the larval stages of gastrointestinal parasites and indi-
rectly regulate the functioning of the mechanisms of the acquired immune response and enhancing the response
from 2 lymphocytes27, 28. Nevertheless, the type of selection shaping the diversity of innate immune genes
usually leads to maintaining limited number of genetic variants. In case of positive or purifying selection that is
found to shape diversity of many classes of innate immunity genes29 low frequency variants may be lost due to
bottleneck occurring when the invasive population is established16, 24. erefore, we may expect dierent roles of
dierent groups of immune related genes in the response to the pathogen pressure in an invasive environment.
In the present study, we used the case of the raccoon Procyon lotor invasion in Europe to investigate the
associations between a large set of immune genes and gastrointestinal parasites in newly-established invasive
populations. e raccoon is a medium-sized carnivore whose native distribution in North America extends
from southern Canada to Panama30. e rst successful introduction in Europe occurred in Germany in the
1930s with a limited number of individuals31. Recently, approximately 1,000,000 raccoons were estimated to be
living in Germany, and the range of the species in Europe has extended to the west, east and south of the inva-
sion core32, 33. Native populations of raccoon are a reservoir of numerous viral (rabies virus, canine distemper
virus), bacterial (Leptospira spp., Francisella tularensis) and parasitic (Baylisascaris procyonis, Toxoplasma gondii)
pathogens3436, but invasive populations are relatively mildly infected with parasites3739 in comparison with native
raccoon populations and other carnivore species in the invasive range4044. Multiple genetic clusters detected
throughout the European range of the raccoon provide the evidence for multiple independent introductions45, 46.
Previously reported associations between specic MHC-DRB resistance and susceptibility alleles and digenean
parasite Isthmiophora melis infection suggest an important role of this gene region in providing local adaptation
to intestine parasites37. Specic infection-associated alleles were partly xed in populations established from
dierent introductions, causing extreme infection-level dierences. is nding underlines the role of standing
genetic variation, at least in MHC loci, in shaping host-parasite relationships in invasive populations and provides
empirical support that functional genetic variation may be responsible for dierences in invasion success37. In
the present work, we analysed four previously studied invasive raccoon populations, varying considerably in the
prevalence of intestinal parasites. e rst population was located at the invasion core in central Germany and
possessed relatively high levels of genetic variation, possibly due to mixing with divergent raccoon populations46.
Two others, one from eastern Germany and another from western Poland, were located towards the invasion front
and form the eastern edge of the raccoon invasion. e fourth population, located in northern Czech Republic,
was established putatively from individuals which escaped from captivity was separated from the remaining ones
and was characterised by a signicantly lower level of genetic diversity16, 47.
e objective of this study was to test if a wide range of single nucleotide polymorphisms (SNPs) located in
non-MHC immune related genes, in addition to MHC-DRB locus diversity, shape the levels of gastrointestinal
infections in invasive raccoon populations. We aimed to reveal the main genetic determinants of infection in
invasive raccoon populations. We expected that the associations between infection levels and genetic diversity
may dier for the MHC gene, where high levels of genetic variation were retained in invasive populations and
non-MHC immunity genes were characterised by relatively lower numbers of genetic variants. Our second objec-
tive was to discuss our ndings in light of the EICA hypothesis. Invasive raccoon populations have lower parasitic
infection levels in comparison to both the native raccoon populations and other carnivores that share a habitat
with the raccoons in the invasive range. e putative release from enemies could allow invaders to outcompete
native species and invest less into immune responses. e lowered parasite pressure might have resulted in relaxed
selection pressure. en visible as lack of associations between immune genetic variants and intestinal parasites
is expected. e relaxation in selection pressure should be visible mainly in terms of costly innate immunity.
Methods
Ethics statement. Our study involved the raccoon as a study species but no animal was captured or
killed specically for the purpose of the project. No experiments involving live animals were performed for
the purpose of this particular study. Most of the carcasses used in the study were obtained courtesy of hunt-
ers controlling this species in accordance to national hunting laws in all three countries (Poland: e Hunting
Act of 13th October 1995 https:// isap. sejm. gov. pl/ isap. nsf/ downl oad. xsp/ WDU19 95147 0713/U/ D1995 0713Lj.
pdf, Germany: https:// ljv- sachs en- anhalt. de/? wpdmp ro= jagdz eiten- in- sachs en- anhalt, https:// www. ljv- meckl
enburg- vorpo mmern. de/ Jagd- in-M- V/ Jagd- und- Schon zeiten/ and the Czech Republic: e Hunting Act of
27th November 2001 https:// www. zakon yprol idi. cz/ cs/ 2001- 449). Additional carcasses were obtained from road
accidents. For these reasons, there were no experimental protocols to be approved by a named institutional and/
or licensing committee. All methods were carried out in accordance with relevant guidelines and regulations and
are reported in accordance with ARRIVE guidelines.
Sample collection. We collected 240 raccoon carcasses from the European part of the introduced range of
the species: 116 from Poland, 18 from D1 population and 34 from D3 in Germany, and 72 from CZ population in
Czech Republic. e sampling took place between 2012 and 2017. e localities of the sampling sites are shown
in Fig.1. e carcasses were obtained from hunters culling raccoons as part of game management activities to
reduce the number of invasive species populations in all three countries and collected as road-killed individuals.
No animal was killed or used in order to conduct this study. We collected only animals that were proven to be
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Figure1. Geographical locations of invasive raccoon populations and the DAPC scatterplot of genetic
dierentiation across individuals resulting from DAPC analysis performed on 197 SNPs located in exon
fragments of immune genes. e number of seven retained PCs chosen using a-score procedure. e map was
generated using ArcGIS soware by Esri. ArcGIS and ArcMap are the intellectual property of Esri and are used
herein under license. Copyright Esri. All rights reserved. For more information about Esri soware, please visit
http:// www. esri. com.
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killed not longer than several hours earlier, with no signs of decomposition. e tissue samples for DNA analyses
were collected as ear fragments or other tissues preserved in ethanol. e carcasses were kept frozen at − 20°C
prior to dissection.
Parasite screening. e detailed procedure describing parasite screening is described in37. Briey, each
animal was sexed, weighed and measured for body length during dissection. Based on body length, weight
and month of death, animals were classied as juvenile or adult. e whole alimentary tract was examined and
macroscopically screened for the presence of helminths. All the isolated helminths were rinsed, counted and
preserved in 70 or 90% ethyl alcohol.
DNA extraction and immune diversity screening. DNA was extracted using the NucleoSpin Tissue
Kit (Macherey and Nagel, Dueren, Germany). We selected a set of 246 non-MHC genes, proved to be associated
with helminth infection, most of which are involved in innate immunity48. To perform targeted resequencing of
the exon fragments for selected genes, we developed Molecular Inversion Probes (MIPs49). A detailed protocol
describing MIP design and subsequent resequencing in studied individuals is described in47. e list of genes
where analysed SNPs are located is given in Supplementary TableS1, online.
Population structure. To visualise the population structure displayed at the SNP loci and identify discrete
population clusters for inter-population description of parasite infections and dening the cofactor for models
used in associations analysis, we used discriminant analysis of principal components (DAPC) implemented in
the R package adegenet50. Multivariate analyses are the method of choice when the assumptions of Hardy–Wein-
berg and linkage equilibrium within populations are violated, which might be the case for genes under selection.
First, using the function nd.clusters, we identied the number of clusters that best reects the genetic structur-
ing in the data without a prior assignment of samples to given populations, using BIC scores (Bayesian Informa-
tion Criterion50). A two-step procedure was used to establish an optimal number of principal components used
in the DAPC. First, maximum number of principal components was established with a cross-validation function
Xval.Dapc. e cross validation was performed as follows: 14,000 replicates were used to establish the optimal
value from group mean, the maximum number of tested PCs was 80 and the training set comprised of 80% of
observations, e resulting number of PCs was used to perform the DAPC. e number of PCs was further
rened using α-score (a-score function). e resulting number of the optimal PCs was used in a second round
of the DAPC. e results of the DAPC were plotted using the scatter function, with a population as a grouping
factor.
Association analysis. Of the initial le containing 254 individuals genotyped in 1772 SNPs, we removed
individuals with over 20% of ungenotyped loci. Next, we removed variants with minor allele frequency
MAF < 0.05, variants not in Hardy–Weinberg equilibrium, and those in lineage disequilibrium (threshold value
of r2 > 0.65). Selection can aect HWE, but there can also be also other reasons for disequilibrium. We decided
to remove loci not in HWE, following recommendations for association analysis (PLINK manual for example).
is step is advised as such variants may result from genotyping errors. Furthermore, using R we checked for
correlations between SNPs situated in dierent chromosomes, as it was not possible to test for associations
between them using LD. From each group of loci correlated with r > 0.7 we randomly selected one locus for fur-
ther analyses. ese rather conservative ltering criteria resulted in 197 SNPs located in exon fragments of 115
immune related genes (Supplementary TableS1, online) that were used in the association analysis in R.
Due to the relatively low number of infected individuals, we could not test for the associations between
parasite load and genetic variants for all parasite species detected in the analysed samples. We focused on the
presence/absence of parasites rather than the intensity of the infection, and we considered higher parasite taxa
(class/phylum) rather than species. e exception was the most frequent uke I. melis, which infected 38% of
racoons, and we tested for associations between genotypes and infection intensity only for this parasite.
e eects of SNPs on the presence/absence of parasites (I. melis, Digenea, Cestoda and Nematoda) were
tested in R package SNPassoc51 under ve genetic models (codominant, dominant, recessive, overdominant and
log-additive) with genetic cluster membership, identied by the DAPC, included as a cofactor. Eect sizes were
estimated using odds ratio implemented in the package SNPassoc.
e SNPs’ eects on infection intensity with I. melis were tested in R using zero inated models implemented
in R package glmmTMB52 with population (genetic cluster) as random eect. We used negative binomial rather
than Poisson distribution to control for the overdispersion. Eect size was estimated using the coecient of
determination R2 calculated separately for xed eects and the whole model using r.squared GLMM function
from the MuMIn package53. In several models we encountered a problem of complete separation what resulted
in glmmTMB function failing to estimate model parameters. e separation occurred when the rarest genotype
(present in fewer than 5 individuals) was observed in just one of four studied populations. us, were removed
such genotypes before tting the models what solved the separation problem. In the case of six SNPs we could
not apply this procedure, as these SNPs had only two genotypes so aer removing the one causing separation,
only one genotype would have remained. In 10 models, the glmmTMB function returned convergence problem
due to non-positive-denite Hessian matrix. Following the package tutorial we removed the zero-inated part
resulting in at mixed-eect models with negative binomial distributions. In all models, we applied the most
conservative correction for multiple comparisons which is the Bonferroni correction.
e prevalence diered between populations (Table1). Parasite prevalence in PL and D3 populations was
considerably higher than in the two remaining ones, so we tted models two types of models: (1) using all data
and (2) using only data from the two clusters with the highest prevalence (PL and D3).
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A common problem in association analysis is the diculty to detect variants of low eects (low odds ratio) or
in low frequencies in a population54. Such variants usually require large sample sizes which is hard to achieve in
a study of free-living species. us, to check a detectability threshold of our study, we performed power analysis
using epi.sscc function from the package epiR55 following the example 8.18 from Woodward56.
Results
Population structure. e DAPC analysis performed on 197 SNPs with seven PCs retained (Fig.1)
revealed clear dierentiation between raccoons from four locations. Individuals from the introduction core in
Central Germany and Czech Republic formed two separate clusters. Two other more closely related clusters were
created by individuals from Eastern Germany and Western Poland, the relatively smaller distance between them
is in accordance with the raccoon expansion from Germany towards the east (Fig.1).
Parasite prevalence and intensity. A total of 240 raccoons (132 males and 108 females) were screened
for parasite infection. e analyses revealed a total of 15 parasite species taxa representing four main groups:
Digenea, Cestoda, Nematoda and Acanthocephala (Table1). e most prevalent parasite was trematode I. melis,
which infected nearly 40% of the hosts. Two species of tapeworms were present in approximately 8% of the hosts.
Interestingly, the racoon-specic Baylisascaris procyonis was found in only 9 hosts. Other nematode species,
similar to Acantocephala, were found in less than 1% of hosts (Supplementary TableS2a, online). We detected
pronounced dierences in the basic parasitological indices between analysed populations. e prevalence of
infection was very dierent for raccoons from populations CZ and D1 than from PL and D3 both for Digenea
(7.58% in CZ; 11.11% in D1; 42.86% in D3; 63.64% in PL, Table1) and Cestoda infection (6.06% in CZ; 0% in
D1; 65.71% in D3; 9.09% in PL, Table1).
SNPs–parasite associations. When we considered all four populations, we found a signicant eect of
SNP 26438_387 on infection intensity with the uke I. melis (chi2 = 26.295, padj = 0.004, (Fig. S1 online). is
SNP is located in GMNN gene that codes for geminin, DNA replication inhibitor. Individuals with genotype
T/T had signicantly higher parasite burden (301.55 ukes) compared with heterozygotes and homozygotes
C/C (49.77 and 23.11 respectively). Among heterozygotes T/T we found two individuals of the highest parasite
load (858 and 1042). Notably, those two individuals were clearly outliers, as the parasite load in other infected
individuals did not exceed 480, and the mean number of ukes per infected host was 109. When the model was
tted without the outliers, the eect of SNP was no longer signicant (chi2 = 9.3404, df = 2, p = 0.009; below the
adjusted p = value threshold of 0.00025). We also found a signicant eect of the SNP 194809_624, located in
IL6ST; interleukin 6 signal transduction gene (chi2 = 22.874, df = 2, padj = 0.0021, Fig.S1 online) but again, the
strong eect of the genotype GG can be explained by the fact that it was present in a single individual with sec-
ond-highest parasite load (890 ukes). When this individual was removed, the association of SNP 194809_624
with I. melis infection intensity was no longer signicant (chi2 = 2.8609, df = 2, p = 0.091). ese same eect was
observed for both SNPs when two populations with the lowest parasite load were removed from the models.
We found no signicant associations between risk of infection (presence/absence of a given parasite) and
genotype for any parasite group tested (I. melis, Digenea, Cestoda, Nematoda) under ve analysed genetic mod-
els. e exact p-values for each SNP and each model are given in (Supplementary TableS2a online). e tables
presenting odds-ratio tests for each SNP and each parasite are given in Supplementary Information 1–11 online.
When we rejected two populations with the lowest parasite load (D1 and CZ), we detected one signicant
association aer correcting for multiple analyses. e SNP located in NRCAM gene was associated with the
prevalence of Nematoda infection under recessive model: in the infected group, individuals with the genotype
TT constituted 57% compared to the other genotypes combined (CC and CT). In the non-infected group, they
represented just 11% of individuals. Still, taking into account that the number of infected individuals was only
4 and 3 in D3 and PL, respectively, we consider this result as an artefact. e exact p-values are given in Sup-
plementary TableS2b online. e tables presenting odds-ratio tests for each SNP and each parasite are given in
Supplementary Information 1–11 online.
e power analysis (Fig.S2 online) showed generally for a given odds-ratio, higher power was achieved when
infection rate was low. However, the generally satisfactory power of 80% for infection levels > 70% was achieved
only when the odds-ratio was higher than 2.25. is suggest that in our sample size and infection levels, we were
only able to detect relatively strong eects, and weaker eects might have been unnoticed.
Table 1. Gastrointestinal parasite prevalence by population. All species except I. melis had a prevalence < 10%
and were summarised as higher taxa. n number of individuals sampled per population, prevalence given in %
CZ D1 D3 PL
n 72 18 34 116
I. melis 6.06 0 42.86 60.33
Digenea 7.58 11.11 42.86 63.64
Cestoda 6.06 0 65.71 9.09
Nematoda 6.06 55.56 11.43 3.31
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Discussion
In this study, we used a targeted sequencing approach to investigate how the associations between the non-MHC
immunity gene diversity and gastrointestinal parasite infections are shaped in invasive populations of the rac-
coon. We studied four populations of invasive raccoons with dierent levels of parasite infection. In our previous
study, we identied MHC-DRB alleles altering the probability of Digenean (I. melis). e presence/absence of
MHC-DRB alleles in dierent populations reected demographic process related to population establishment
during invasion but, in turn, allowed for creating strong dierences in population susceptibility to infection.
Here we studied the importance of a wide range of non-MHC immunity genes in controlling gastrointestinal
infections in invasive raccoon populations.
Studied populations varied considerably in the prevalence of Digenea and Cestoda, with D1 and CZ popula-
tions having an order of magnitude lower prevalence than D3 and PL populations. Nevertheless, using association
analyses, we did not identify SNPs that would explain such sharp dierences in pathogen prevalence. Notably, the
only signicant association with the infection intensity could be contributed to the presence of three individu-
als with extra high parasite load. Although statistically valid, this result is hard to interpret biologically. Aer
removing the outliers, the eect of SNPs was no longer visible. us, it is dicult to conclude whether those
SNPs really aect susceptibility or rather the individuals with enormous parasite load had other traits making
them prone to such an abundant infection.
e most common intestinal parasite group was Digeneans (with I. melis being the most frequent parasite). Its
prevalence in D3 and PL was 0.412 and 0.69, respectively (Table1) and was much lower in CZ and D1 populations
(0.042 and 0.059, respectively; Table1). According to the available data I. melis is widespread in Europe and uses
a wide range of denitive vertebrate hosts typical for environment inhabited by raccoon genetic clusters studied
here57. e trematodes have a complex life cycle, and the raccoon gets infected by ingesting infected snails, sh
or amphibians58. Since the intermediate hosts may disperse on their own, it is not likely that the observed dif-
ferences in infection levels between clusters are due to the dierential occurrence of I. melis in the environment.
e second most frequent parasite group infecting racoons was Cestodes. Atriotaenia incisa is a parasite of
badgers, and Mesocestoides are common in medium and large carnivores; both have also been described in inva-
sive raccoon populations59. Similarly to trematodes, an infection occurs aer consuming infected intermediate
land-dwelling hosts, such as rodents. To the contrary, nematodes detected in the current study have a simple
lifecycle, where infection occurs directly via contact with faeces shredded by an infected carnivore. us, the
infection risk with trematodes and cestodes is associated with feeding habits, while infection with nematodes
occurs during direct or indirect contact with other carnivores or other vertebrate species. Interactions of rac-
coons with other species in the invasive range may dier from the native range, resulting in a lower infection
prevalence. Moreover, generally low parasite load in invasive raccoons was previously described60, which sup-
ports the enemy release hypothesis.
Several factors may contribute to the dierences in parasite load between populations. Each of them pre-
sents a dierent demographic history and forms well separated geographical units. Although potentially con-
nected, German populations (D1 and D3) located in the continuous range of invasive raccoon populations are
characterised by dierent frequencies of mitochondrial haplotypes and were probably established by separate
introductions46. e Polish population was formed by the expansion of raccoons towards the east and is geneti-
cally and geographically close to German population D3. A case such as this, we can expect that, along with
genetic variants transferred during population spread, the transmission of I. melis also occurred, although there
is a prerequisite of the presence of intermediate hosts in the expansion range. e CZ population was established
from separate introduction, putatively from individuals from a dierent source population where dierent host-
parasite genetic associations might have evolved. It is possible considering the intraspecic variability of this
trematode species57. erefore individuals that established CZ population might have evolved specic immune
protection to the I. melis present in the native range, while such associations did not arise in native populations
that acted as a source for German and Polish populations. ere are also habitat dierences between the studied
genetic clusters. Both D3 and PL populations inhabit marshes, wetlands and lake regions with a high abundance
of intermediate hosts58, while D1 occupies dry woodland habitat. ese dierences may contribute to the dif-
ferential prevalence of Digenea between D1 vs. D3 and PL populations but not between CZ vs. D3 and PL as the
Czech population also inhabits the lake region and wetlands (Table1).
Although it is hard to dene ecological factors explaining why two genetic clusters (D1 and CZ) present
very low infection levels, genetic factors may play a major role in shaping these dierences. As we previously
detected, the MHC-DRB allele found in CZ cluster, but not in others, was associated with resistance to I. melis.
It could suggest that adaptive immune response, linked mainly to MHC loci variation, could play a primary role
in ghting I. melis infection. MHC genes that exhibit extremely high polymorphism maintained by balancing
selection may be more important in coping with infections in recently established invasive populations than other
immune genes. A wide range of genetic variants maintained by balancing selection creates relatively high standing
genetic variation that enables invasive populations to respond quickly to novel environmental conditions37, 61. e
release from enemies believed to occur during invasion helps invaders outcompete native species. According to
the increased competitive ability (EICA) hypothesis, invaders are predicted to invest less in immune responses5,
but this lowered response is expected mainly for costly innate immunity. Innate defences are mobilised quickly
and are eective against novel pathogens but impose higher metabolic and inammatory costs62 and may be
downregulated in highly active and dispersing organisms63. On the other hand, the adaptive immune response
is specic to various pathogens but acts eectively only aer primary contact with the pathogen. In the case of
invading species that can get rid of pathogens during the invasion and do not encounter many novel enemies
in the new range, adaptive immune response, that is associated with lower cost, may act more eectively. is
process was observed in invasive edge populations of cane toads64 or in expansive populations of neotropical
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thrushes that exhibited lower infection levels and lowered inammation compared to native populations65. e
level of gastrointestinal infections in invasive raccoon populations is relatively low compared to native species.
It suggests the "escape" from higher pathogenic load and potential decrease in the innate immune response. e
studied 197 SNPs are located in non-MHC, mostly innate immunity genes. In the case of the suppressed innate
immune response, the associations between studied SNPs and parasite load would rather not be established,
especially in relatively recently formed populations. e lack of associations suggests that analysed SNPs of non-
MHC genes do not show signs of pathogen-induced selection. At least they are not under a divergent selection in
invasive raccoon populations, as we did not nd associations explaining between-populations dierentiation in
infection level. Although it is established that specic groups of innate immunity genes follow divergent evolu-
tion pattern66, 67, a short time since the establishment of the invasive population may be not enough to observe
any clear selection pattern. On the contrary, a wide range of specic functional MHC variants transferred to the
new range16 may constitute sucient basis of genetic variation on which pathogen mediated selection could act.
Conclusions
Our study shows that non-MHC innate immunity gene diversity does not play a crucial role in ghting gastro-
intestinal infections in invasive raccoon populations. is observation is in line with the increased competitive
ability hypothesis that relates the downregulation of innate immunity with higher dispersal and survival in
novel environmental conditions. At the same time, we suggest that invasive populations may acquire immune
response via adaptive immunity as specic MHC-DRB variants regulate gastrointestinal infection levels in studied
invasive populations.
Data availability
e VCF le containing SNP genotypes used in this work have been deposited on GitHub (https:// github. com/
konop inski/ racco on). All the data necessary to reproduce the analysis presented in the manuscript are provided
as supplementary material.
Received: 8 February 2023; Accepted: 30 August 2023
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Acknowledgements
We thank all game species managers and hunters for their help in the sample collection. Grants from the National
Science Centre, Poland, awarded to Aleksandra Biedrzycka, project nos.: 2014/15/B/NZ8/00261 and 2020/37/B/
NZ8/03801 supported this work. e text was professionally proof-edited by PRS company. We would like to
thank Olek Michalski and Patryk Czortek for their helpful tips on the statistical analysis.
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Author contributions
A.B. designed the study, coordinated sample collection and wrote the manuscript, A.K. performed statistical
analysis of SNP-infection associations and contributed to manuscript writing, M.K.K. performed raw data pro-
cessing, SNP ltering and population structure analysis, statistical analyses, M.P. and M.Z. performed parasito-
logical analysis and M.B. collected a subset of samples and contributed to manuscript writing. All authors have
read and approved the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 023- 41721-1.
Correspondence and requests for materials should be addressed to A.B.
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