Host-parasite interactions in non-native invasive species are dependent on the levels of standing genetic variation at the immune locus.

BACKGROUND: Parasites may mediate the success of biological invasions through their effect on host fitness and thus, on host population growth and stability. However, a release from the pressure of parasites is strongly related to the genetic differentiation of the host. In invasive host populations, the number of available genetic variants, allowing them to 'fight' the infection, are likely to be influenced by founder events and genetic drift. The level standing genetic variation of invasive populations may be crucial in successfully adapting to new environments and resisting diseases. We studied invasive populations of raccoon that experienced a random reduction in genetic diversity during the establishment and evaluated the relationship between host immune genetic diversity and intestinal parasites infection. RESULTS: We distinguished two different genetic clusters that are characterized by different sets of functionally relevant MHC-DRB alleles. Both clusters were characterized by considerably different allele-parasite associations and different levels of parasite infection. The specific resistance MHC-DRB alleles explained the lower prevalence of Digenea parasites. An increased infection intensity was related to the presence of two MHC-DRB alleles. One of these alleles significantly decreased in frequency over time, causing a decrease of Digenea abundance in raccoons in consecutive years. CONCLUSIONS: Our findings suggest that intestinal parasites can exert selective pressure on an invasive host with lowered levels of immune genetic diversity and contribute to promoting local adaptation over time. The random genetic drift that created the two different genetic clusters in the invasive raccoon range imposed completely different MHC-parasite associations, strongly associated with the infection status of populations. Our findings underline the role of standing genetic variation in shaping host-parasite relationships and provide empirical support that functional genetic variation may be, at least partly, responsible for differences in the success of invasive populations.

Blood parasites shape extreme major histocompatibility complex diversity in a migratory passerine.

Pathogens are one of the main forces driving the evolution and maintenance of the highly polymorphic genes of the vertebrate major histocompatibility complex (MHC). Although MHC proteins are crucial in pathogen recognition, it is still poorly understood how pathogen-mediated selection promotes and maintains MHC diversity, and especially so in host species with highly duplicated MHC genes. Sedge warblers (Acrocephalus schoenobaenus) have highly duplicated MHC genes, and using data from high-throughput MHC genotyping, we were able to investigate to what extent avian malaria parasites explain temporal MHC class I supertype fluctuations in a long-term study population. We investigated infection status and infection intensities of two different strains of Haemoproteus, that is avian malaria parasites that are known to have significant fitness consequences in sedge warblers. We found that prevalence of avian malaria in carriers of specific MHC class I supertypes was a significant predictor of their frequency changes between years. This finding suggests that avian malaria infections partly drive the temporal fluctuations of the MHC class I supertypes. Furthermore, we found that individuals with a large number of different supertypes had higher resistance to avian malaria, but there was no evidence for an optimal MHC class I diversity. Thus, the two studied malaria parasite strains appear to select for a high MHC class I supertype diversity. Such selection may explain the maintenance of the extremely high number of MHC class I gene copies in sedge warblers and possibly also in other passerines where avian malaria is a common disease.

Extreme MHC class I diversity in the sedge warbler (Acrocephalus schoenobaenus); selection patterns and allelic divergence suggest that different genes have different functions.

BACKGROUND: Recent work suggests that gene duplications may play an important role in the evolution of immunity genes. Passerine birds, and in particular Sylvioidea warblers, have highly duplicated major histocompatibility complex (MHC) genes, which are key in immunity, compared to other vertebrates. However, reasons for this high MHC gene copy number are yet unclear. High-throughput sequencing (HTS) allows MHC genotyping even in individuals with extremely duplicated genes. This HTS data can reveal evidence of selection, which may help to unravel the putative functions of different gene copies, i.e. neofunctionalization. We performed exhaustive genotyping of MHC class I in a Sylvioidea warbler, the sedge warbler, Acrocephalus schoenobaenus, using the Illumina MiSeq technique on individuals from a wild study population. RESULTS: The MHC diversity in 863 genotyped individuals by far exceeds that of any other bird species described to date. A single individual could carry up to 65 different alleles, a large proportion of which are expressed (transcribed). The MHC alleles were of three different lengths differing in evidence of selection, diversity and divergence within our study population. Alleles without any deletions and alleles containing a 6 bp deletion showed characteristics of classical MHC genes, with evidence of multiple sites subject to positive selection and high sequence divergence. In contrast, alleles containing a 3 bp deletion had no sites subject to positive selection and had low divergence. CONCLUSIONS: Our results suggest that sedge warbler MHC alleles that either have no deletion, or contain a 6 bp deletion, encode classical antigen presenting MHC molecules. In contrast, MHC alleles containing a 3 bp deletion may encode molecules with a different function. This study demonstrates that highly duplicated MHC genes can be characterised with HTS and that selection patterns can be useful for revealing neofunctionalization. Importantly, our results highlight the need to consider the putative function of different MHC genes in future studies of MHC in relation to disease resistance and fitness.

Monitoring of species' genetic diversity in Europe varies greatly and overlooks potential climate change impacts.

Genetic monitoring of populations currently attracts interest in the context of the Convention on Biological Diversity but needs long-term planning and investments. However, genetic diversity has been largely neglected in biodiversity monitoring, and when addressed, it is treated separately, detached from other conservation issues, such as habitat alteration due to climate change. We report an accounting of efforts to monitor population genetic diversity in Europe (genetic monitoring effort, GME), the evaluation of which can help guide future capacity building and collaboration towards areas most in need of expanded monitoring. Overlaying GME with areas where the ranges of selected species of conservation interest approach current and future climate niche limits helps identify whether GME coincides with anticipated climate change effects on biodiversity. Our analysis suggests that country area, financial resources and conservation policy influence GME, high values of which only partially match species' joint patterns of limits to suitable climatic conditions. Populations at trailing climatic niche margins probably hold genetic diversity that is important for adaptation to changing climate. Our results illuminate the need in Europe for expanded investment in genetic monitoring across climate gradients occupied by focal species, a need arguably greatest in southeastern European countries. This need could be met in part by expanding the European Union's Birds and Habitats Directives to fully address the conservation and monitoring of genetic diversity.

Molecular characterization of putative Hepatozoon sp. from the sedge warbler (Acrocephalus schoenobaenus).

We characterized partial sequences of 18S rDNA from sedge warblers infected with a parasite described previously as Hepatozoon kabeeni. Prevalence was 47% in sampled birds.We detected 3 parasite haplotypes in 62 sequenced samples from infected animals. In phylogenetic analyses, 2 of the putative Hepatozoon haplotypes closely resembled Lankesterella minima and L. valsainensis. The third haplotype grouped in a wider clade composed of Caryospora and Eimeria. None of the haplotypes showed resemblance to sequences of Hepatozoon from reptiles and mammals. Molecular detection results were consistent with those from microscopy of stained blood smears, confirming that the primers indeed amplified the parasite sequences. Here we provide evidence that the avian Hepatozoon-like parasites are most likely Lankesterella, supporting the suggestion that the systematic position of avian Hepatozoon-like species needs to be revised.

Complex patterns shape immune genes diversity during invasion of common raccoon in Europe - Selection in action despite genetic drift.

Rapid adaptation is common in invasive populations and is crucial to their long-term success. The primary target of selection in the invasive species' new range is standing genetic variation. Therefore, genetic drift and natural selection acting on existing variation are key evolutionary processes through which invaders will evolve over a short timescale. In this study, we used the case of the raccoon Procyon lotor invasion in Europe to identify the forces shaping the diversity of immune genes during invasion. The genes involved in the defence against infection should be under intense selection pressure in the invasive range where novel pathogens are expected to occur. To disentangle the selective and demographic processes shaping the adaptive immune diversity of its invasive and expanding populations, we have developed species-specific single-nucleotide polymorphism markers located in the coding regions of targeted immune-related genes. We characterised the genetic diversity of 110 functionally important immune genes in two invasive and one native raccoon genetic clusters, each presenting a different demographic history. Despite the strong effect of demographic processes in the invasive clusters, we detected a subset of genes exhibiting the diversity pattern suggestive of selection. The most likely process shaping the variation in those genes was balancing selection. The selected genes belong to toll-like receptors and cytokine-related genes. Our results suggest that the prevalence of selection depends on the level of diversity, that is - less genetically diverse invasive population from the Czech Republic displayed fewer signs of selection. Our results highlight the role of standing genetic variation in adapting to new environment. Understanding the evolutionary mechanisms behind invasion success would enable predicting how populations may respond to environmental change.

Cytokine gene polymorphism and parasite susceptibility in free-living rodents: Importance of non-coding variants.

Associations between genetic variants and susceptibility to infections have long been studied in free-living hosts so as to infer the contemporary evolutionary forces that shape the genetic polymorphisms of immunity genes. Despite extensive studies of proteins interacting with pathogen-derived ligands, such as MHC (major histocompatilbility complex) or TLR (Toll-like receptors), little is known about the efferent arm of the immune system. Cytokines are signalling molecules that trigger and modulate the immune response, acting as a crucial link between innate and adaptive immunity. In the present study we investigated how genetic variation in cytokines in bank voles Myodes glareolus affects their susceptibility to infection by parasites (nematodes: Aspiculuris tianjensis, Heligmosomum mixtum, Heligmosomoides glareoli) and microparasites (Cryptosporidium sp, Babesia microti, Bartonella sp.). We focused on three cytokines: tumour necrosis factor (TNF), lymphotoxin alpha (LTα), and interferon beta (IFNß1). Overall, we identified four single nucleotide polymorphisms (SNPs) associated with susceptibility to nematodes: two located in LTα and two in IFNß1. One of those variants was synonymous, another located in an intron. Each SNP associated with parasite load was located in or next to a codon under selection, three codons displayed signatures of positive selection, and one of purifying selection. Our results indicate that cytokines are prone to parasite-driven selection and that non-coding variants, although commonly disregarded in studies of the genetic background of host-parasite co-evolution, may play a role in susceptibility to infections in wild systems.

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

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.

Does reduced MHC diversity decrease viability of vertebrate populations?

Loss of genetic variation may render populations more vulnerable to pathogens due to inbreeding depression and depletion of variation in genes responsible for immunity against parasites. Here we review the evidence for the significance of variation in genes of the Major Histocompatibility Complex (MHC) for conservation efforts. MHC molecules present pathogen-derived antigens to the effector cells of the immune system and thus trigger the adaptive immune response. Some MHC genes are the most variable functional genes in the vertebrate genome. Their variation is clearly of adaptive significance and there is considerable evidence that its maintenance is mainly due to balancing selection imposed by pathogens. However, while the evidence for selection shaping MHC variation on the historical timescale is compelling, a correlation between levels of MHC variation and variation at neutral loci is often observed, indicating that on a shorter timescale drift also substantially affects MHC, leading to depletion of MHC diversity. The evidence that the loss of MHC variation negatively affects population survival is so far equivocal and difficult to separate from effects of general inbreeding. Some species with depleted MHC variation seem to be particularly susceptible to infection, but other species thrive and expand following severe bottlenecks that have drastically limited their MHC variation. However, while the latter demonstrate that MHC variation is not always critical for population survival, these species may in fact represent rare examples of survival despite of the loss of MHC variation. There is clearly a compelling need for data that would disclose the possible consequences of MHC diversity for population viability. In particular, we need more data on the impact of MHC allelic richness on the abundance of parasites or prevalence of disease in populations, while controlling for the role of general inbreeding. Before such evidence accumulates, captive breeding programs and other conservation measures aimed at inbreeding avoidance should be favoured over those protecting only MHC variation, especially since inbreeding avoidance programs would usually conserve both types of genetic diversity simultaneously.

Comparing raccoon major histocompatibility complex diversity in native and introduced ranges: Evidence for the importance of functional immune diversity for adaptation and survival in novel environments.

The adaptive potential of invasive species is related to the genetic diversity of the invader, which is influenced by genetic drift and natural selection. Typically, the genetic diversity of invaders is studied with neutral genetic markers; however, the expectation of reduced diversity has not been consistently supported by empirical studies. Here, we describe and interpret genetic diversity at both neutral microsatellite loci and the immune-related MHC-DRB locus of native and invasive populations of raccoon to better understand of how drift and selection impact patterns of genetic diversity during the invasion process. We found that despite the loss of many MHC (major histocompatibility complex) alleles in comparison with native populations, functional MHC supertypes are preserved in the invasive region. In the native raccoon population, the number of supertypes within individuals was higher than expected under a neutral model. The high level of individual functional divergence may facilitate the adaptation to local conditions in the invasive range. In the invasive populations, we also detected increased population structure at microsatellites compared to the MHC locus, further suggesting that balancing selection is acting on adaptively important regions of the raccoon genome. Finally, we found that alleles known to exhibit resistance to rabies in the native range, Prlo-DRB*4, Prlo-DRB*16 and Prlo-DRB*102, were the most common alleles in the European populations, suggesting directional selection is acting on this locus. Our research shows empirical support for the importance of functional immune diversity for adaptation and survival in novel environments.

Population fragmentation and major histocompatibility complex variation in the spotted suslik, Spermophilus suslicus.

The fragmentation of populations typically enhances depletion of genetic variation, but highly polymorphic major histocompatibility complex (MHC) genes are thought to be under balancing selection and therefore retain polymorphism despite population bottlenecks. In this study, we investigate MHC DRB (class II) exon 2 variation in 14 spotted suslik populations from two regions differing in their degree of habitat fragmentation and gene flow. We found 16 alleles that segregated in a sample of 248 individuals. The alleles were highly divergent and revealed the hallmark signs of positive selection acting on them in the past, showing a significant excess of nonsynonymous substitutions. This excess was concentrated in putative antigen-binding sites, which suggests that past selection was driven by pathogens. MHC diversity was significantly lower in fragmented western populations than in the eastern populations, characterized by significant gene flow. In contrast to neutral variation, amova did not reveal genetic differentiation between the two regions. This may indicate similar selective pressures shaping MHC variation in both regions until the recent past. However, MHC allelic richness within a population was correlated with that for microsatellites. F(ST )outlier analyses have shown that population differentiation at DRB was neither higher nor lower than expected under neutrality. The results suggest that selection on MHC is not strong enough to counteract drift that results from recent fragmentation of spotted suslik populations.

AmpliSAS and AmpliHLA: Web Server Tools for MHC Typing of Non-Model Species and Human Using NGS Data.

AmpliSAS and AmpliHLA are web server tools for automatic genotyping of MHC genes from high-throughput sequencing data. AmpliSAS is designed specifically to analyze amplicon sequencing data from non-model species and it is able to perform de-novo genotyping without any previous knowledge of the reference alleles. AmpliHLA is a human-specific version, it performs HLA typing by comparing sequenced variants against human reference alleles from the IMGT/HLA database. Here we describe four genotyping protocols: the first two use amplicon sequencing data to genotype the MHC genes of a passerine bird and human respectively; the third and fourth present the HLA typing of a human cell line starting from RNA and exome sequencing data respectively.

Testing genotyping strategies for ultra-deep sequencing of a co-amplifying gene family: MHC class I in a passerine bird.

Characterization of highly duplicated genes, such as genes of the major histocompatibility complex (MHC), where multiple loci often co-amplify, has until recently been hindered by insufficient read depths per amplicon. Here, we used ultra-deep Illumina sequencing to resolve genotypes at exon 3 of MHC class I genes in the sedge warbler (Acrocephalus schoenobaenus). We sequenced 24 individuals in two replicates and used this data, as well as a simulated data set, to test the effect of amplicon coverage (range: 500-20 000 reads per amplicon) on the repeatability of genotyping using four different genotyping approaches. A third replicate employed unique barcoding to assess the extent of tag jumping, that is swapping of individual tag identifiers, which may confound genotyping. The reliability of MHC genotyping increased with coverage and approached or exceeded 90% within-method repeatability of allele calling at coverages of >5000 reads per amplicon. We found generally high agreement between genotyping methods, especially at high coverages. High reliability of the tested genotyping approaches was further supported by our analysis of the simulated data set, although the genotyping approach relying primarily on replication of variants in independent amplicons proved sensitive to repeatable errors. According to the most repeatable genotyping method, the number of co-amplifying variants per individual ranged from 19 to 42. Tag jumping was detectable, but at such low frequencies that it did not affect the reliability of genotyping. We thus demonstrate that gene families with many co-amplifying genes can be reliably genotyped using HTS, provided that there is sufficient per amplicon coverage.

Development of novel associations between MHC alleles and susceptibility to parasitic infections in an isolated population of an endangered mammal.

The role of pathogens in dynamics of endangered species is not fully understood, and the effect of infection often interacts with other processes affecting those species, such as fragmentation and isolation or loss of genetic variation. Small, isolated populations are prone to losing functional alleles due to demographic processes and genetic drift, which may diminish their ability to resist infection if immune genes are affected. Demographic processes may also alter the selective pressure exerted by a parasite, as they influence the rate of parasite transmission between individuals. In the present paper we studied changes in parasite infection levels and genetic variability in an isolated population of spotted suslik (Spermophillus suslicus). Over a three-year period (approx. three generations), when the population size remained relatively stable, we observed a considerable increase in parasite prevalence and infection intensity, followed by the development of novel associations between MHC DRB alleles and parasite burden. Contrary to expectations, the change in MHC allele frequency over time was not consistent with the effect of the allele - for instance, Spsu-DRB*07, associated with higher intensity of infection with a nematode Capillaria sp., increased in frequency from 11.8 to 20.2%. Yet, we found no signatures of selection in the studied loci. Our results show that an isolated, stable population may experience a sudden increase in parasitic infections, resulting in a development of novel associations between MHC alleles and parasite susceptibility/resistance, even though no signatures of selection can be found.

Tissue oximetry in anaesthesia and intensive care.

Conventional monitoring during surgery and intensive care is not sufficiently sensitive to detect acute changes in vital organs perfusion, while its good quality is critical for maintaining their function. Disturbed vital organ perfusion may lead to the development of postoperative complications, including neurological sequel and renal failure. Near-infra-red spectroscopy (NIRS) represents one of up-to-date techniques of patient monitoring which is commonly used for the assessment of brain oximetry in thoracic aorta surgery, and - increasingly more often -in open-heart surgery. Algorithms for maintaining adequate brain saturation may result in a decrease of neurological complications and cognitive dysfunction following cardiac surgery. The assessment of kidney and visceral perfusion with tissue oximetry is gaining increasing interest during pediatric cardiac surgery. Attempts at decreasing complications by the use of brain oximetry during carotid endarterectomy, as well as thoracic and abdominal surgery demonstrated conflicting results. In recent years NIRS technique was proposed as a tool for muscle perfusion assessment under short term ischemia and reperfusion, referred to as vascular occlusion test (VOT). This monitoring extension allows for the identification of early disturbances in tissue perfusion. Results of recent studies utilizing VOT suggest that the muscle saturation decrease rate is reduced in septic shock patients, while decreased speed of saturation recovery on reperfusion is related to disturbed microcirculation. Being non-invasive and feasible technique, NIRS offers an improvement of preoperative risk assessment in cardiac surgery and promises more comprehensive intraoperative and ICU patient monitoring allowing for better outcome.

Aortic cross-clamping phase of cardiopulmonary bypass is related to decreased microvascular reactivity after short-term ischaemia of the thenar muscle both under intravenous and volatile anaesthesia: a randomized trial.

OBJECTIVES: The purpose of the present study was to assess, by near-infrared spectroscopy with an INVOS oximeter during the vascular occlusion test (VOT), the influence of cardiopulmonary bypass (CPB) on tissue saturation in the thenar muscle. The secondary aim was to compare the effects of propofol and sevoflurane anaesthesia on tissue saturation. METHODS: This was a prospective, randomized, open-label study. Sixty cardiac surgery patients received either propofol or sevoflurane anaesthesia. Three-minute VOT was performed at the following time points: 30 min after anaesthesia induction, directly after sternotomy, 20 and 40 min after aortic cross-clamping, 20 min after aortic cross-clamp removal and 45 min after weaning of cardiopulmonary bypass. Group and time effects on tissue saturation were analysed with RM-ANOVA and the post hoc Tukey test. RESULTS: In both groups at baseline, the lowest and the highest tissue saturation and the rate of saturation recovery during the reperfusion phase of the vascular occlusion test were lower during aortic cross-clamping in comparison to the values before CPB. Lower nadir tissue saturation during ischaemia was observed under propofol in comparison to sevoflurane anaesthesia (P = 0.018). CONCLUSIONS: This study demonstrated that the aortic cross-clamping phase of CPB cardiac surgery is associated with lower values of tissue saturation and a decreased rate of saturation recovery under both propofol and sevoflurane anaesthesia. Aortic cross-clamp release is followed by accelerated tissue desaturation during VOT. Propofol anaesthesia for CPB cardiac surgery results in greater reduction of nadir tissue saturation during the ischaemic phase of VOT in comparison to that of sevoflurane. TRIAL REGISTRATION NUMBER: NCT02593448.

Comparison of haemodynamics and myocardial injury markers under desflurane vs. propofol anaesthesia for off-pump coronary surgery. A prospective randomised trial.

BACKGROUND: Several studies have highlighted that volatile anaesthetics improve myocardial protection in cardiopulmonary bypass coronary surgery. However, the haemodynamic effect of desflurane in off-pump coronary surgery has not been clarified yet. Our study hypothesis was that desflurane-fentanyl anaesthesia could decrease myocardial injury markers and improve haemodynamics compared to propofol-fentanyl in patients undergoing off-pump coronary surgery. DESIGN: Prospective, randomised open-lable study. Sixty elective patients with left ventricular ejection fraction above 30% received either desflurane (group D, n = 32) or propofol (group P, n = 28), in addition to fentanyl and vecuronium bromide anaesthesia for off-pump coronary surgery. Assessment of haemodynamic function included thermodilution continuous cardiac output and right ventricular end diastolic volume. RESULTS: No significant differences in cardiac output, stroke volume and mean arterial pressure were noted between groups. The only observed difference in haemodynamic profile was that group D demonstrated improved stability, expressed as left ventricular stroke work index (LVSWI). Decrease in LVSWI after performing distal anastomoses was smaller in D compared to P (median value: -14.3 and -19.8 [g m m⁻² beat⁻¹]), respectively (P = 0.029). Oxygen uptake index (VO2I) and oxygen extraction ratio (OER) after skin incision were lower in D, while blood lactate concentration was slightly higher after surgery in D compared to P. The groups did not differ with respect to CK-MB and troponin I concentration. CONCLUSIONS: This study demonstrated no difference between desflurane and propofol anaesthesia for off-pump coronary surgery in major haemodynamic parameters, as well as in myocardial injury markers and the long-term outcome. However, the study indicated that desflurane might accelerate recovery of myocardial contractility, as assessed by LVSWI. Lower oxygen uptake and elevated lactate under desflurane anaesthesia indicated a discrete shift towards anaerobic metabolism. CLINICAL TRIAL REGISTRATION INFORMATION: NCT00528515 (http://www.clinicaltrials.gov/ ct2/show/NCT00528515?term = NCT00528515&rank = 1).