Locus-specific protocol for nine different innate immune genes (antimicrobial peptides: β-defensins) across passerine bird species reveals within-species coding variation and a case of trans-species polymorphisms

We present a locus-specific protocol suitable for the investigation, from extracted DNA, into natural inter- and intra-specific genetic variation in a group of nine innate immune genes, all belonging to the β-defensin gene family. The products of these genes encode peptides with antimicrobial properties and form part of the innate immune system. The protocol amplifies the exon coding for the peptide that interacts with invading pathogens and is applicable across a wide range of passerine bird species, although with varying success depending on species. In several species tested, we found individuals to be heterozygous at several of the genes, highlighting the existence of coding genetic variation in this part of the immune system. Furthermore, for several of the genes, alleles have been conserved at the amino acid level across taxonomically distant bird species. In one case, we observed the existence of trans-species polymorphisms, often taken as evidence of balancing selection. The method will make it possible to investigate a part of the immune system for which there exists very little information of the genetic variation in wild vertebrate populations, thus making it possible to start investigating the selective forces under which the genes are evolving and the extent to which the found genetic variation is associated with pathogen susceptibility in wild populations.     

“Balancing” balancing selection? Assortative mating at the major histocompatibility complex despite molecular signatures of balancing selection

In vertebrate animals, genes of the major histocompatibility complex (MHC) determine the set of pathogens to which an individual's adaptive immune system can respond. MHC genes are extraordinarily polymorphic, often showing elevated nonsynonymous relative to synonymous sequence variation and sharing presumably ancient polymorphisms between lineages. These patterns likely reflect pathogen‐mediated balancing selection, for example, rare‐allele or heterozygote advantage. Such selection is often reinforced by disassortative mating at MHC. We characterized exon 2 of MHC class II, corresponding to the hypervariable peptide‐binding region, in song sparrows (Melospiza melodia). We compared nonsynonymous to synonymous sequence variation in order to identify positively selected sites; assessed evidence for trans‐species polymorphisms indicating ancient balancing selection; and compared MHC similarity of socially mated pairs to expectations under random mating. Six codons showed elevated ratios of nonsynonymous to synonymous variation, consistent with balancing selection, and we characterized several alleles similar to those occurring in at least four other avian families. Despite this evidence for historical balancing selection, mated pairs were significantly more similar at MHC than were randomly generated pairings. Nonrandom mating at MHC thus appears to partially counteract, not reinforce, pathogen‐mediated balancing selection in this system. We suggest that in systems where individual fitness does not increase monotonically with MHC diversity, assortative mating may help to avoid excessive offspring heterozygosity that could otherwise arise from long‐standing balancing selection.     

Fitness in invasive social wasps: the role of variation in viral load,immune response and paternity in predicting nest size and reproductive output

Within any one habitat, the relative fitness of organisms in a population can vary substantially. Social insects like the common wasp are among the most successful invasive animals, but show enormous variation in nest size and other fitness‐related traits. Some of this variation may be caused by pathogens such as viruses that can have serious consequences in social insects, which range from reduced productivity to colony death. Both individual immune responses and colony‐level traits such as genetic diversity are likely to influence effects of pathogen infections on colony fitness. Here we investigate how infections with Kashmir Bee Virus (KBV), immune response and intracolony genetic diversity (due to queen polyandry) affect nest size in the invasive common wasp Vespula vulgaris. We show that KBV is highly prevalent in wasps and expression of antiviral immune genes is significantly increased with higher viral loads across individuals. Patriline membership within a nest did not influence KBV susceptibility or immune response. A permutational MANCOVA revealed that polyandry, viral load and expression of the immune gene Dicer were significant predictors of variation in nest size. High intracolony genetic diversity due to polyandry has previously been hypothesized to improve colony‐level resistance to parasites and pathogens. Consistent with this hypothesis, we observed genetically diverse colonies to be significantly larger and to produce more queens, although this effect was not driven by the pathogen we investigated. Invasive wasps clearly suffer from pathogens and expend resources, as indicated here by elevated immune gene expression, toward reducing pathogen‐impact on colony fitness.     

Host–parasite coevolution: genetic variation in a virus population and the interaction with a host gene

Host–parasite coevolution is considered to be an important factor in maintaining genetic variation in resistance to pathogens. Drosophila melanogaster is naturally infected by the sigma virus, a vertically transmitted and host‐specific pathogen. In fly populations, there is a large amount of genetic variation in the transmission rate from parent to offspring, much of which is caused by major‐effect resistance polymorphisms. We have found that there are similarly high levels of genetic variation in the rate of paternal transmission among 95 different isolates of the virus as in the host. However, when we examined a transmission‐blocking gene in the host, we found that it was effective across virus isolates. Therefore, the high levels of genetic variation observed in this system do not appear to be maintained because of coevolution resulting from interactions between this host gene and parasite genes.     

Not all non‐natives are equally unequal: reductions in herbivore β‐diversity depend on phylogenetic similarity to native plant community

Effects of host plant α‐ and β‐diversity often confound studies of herbivore β‐diversity, hindering our ability to predict the full impact of non‐native plants on herbivores. Here, while controlling host plant diversity, we examined variation in herbivore communities between native and non‐native plants, focusing on how plant relatedness and spatial scale alter the result. We found lower absolute magnitudes of β‐diversity among tree species and among sites on non‐natives in all comparisons. However, lower relative β‐diversity only occurred for immature herbivores on phylogenetically distinct non‐natives vs. natives. Locally in that comparison, non‐native gardens had lower host specificity; while among sites, the herbivores supported were a redundant subset of species on natives. Therefore, when phylogenetically distinct non‐natives replace native plants, the community of immature herbivores is likely to be homogenised across landscapes. Differences in communities on closely related non‐natives were subtler, but displayed community shifts and increased generalisation on non‐natives within certain feeding guilds.     

Virulence phenotypes result from interactions between pathogen ploidy and genetic background

Studying fungal virulence is often challenging and frequently depends on many contexts, including host immune status and pathogen genetic background. However, the role of ploidy has often been overlooked when studying virulence in eukaryotic pathogens. Since fungal pathogens, including the human opportunistic pathogen Candida albicans, can display extensive ploidy variation, assessing how ploidy impacts virulence has important clinical relevance. As an opportunistic pathogen, C. albicans causes nonlethal, superficial infections in healthy individuals, but life‐threatening bloodstream infections in individuals with compromised immune function. Here, we determined how both ploidy and genetic background of C. albicans impacts virulence phenotypes in healthy and immunocompromised nematode hosts by characterizing virulence phenotypes in four near‐isogenic diploid and tetraploid pairs of strains, which included both laboratory and clinical genetic backgrounds. We found that C. albicans infections decreased host survival and negatively impacted host reproduction, and we leveraged these two measures to survey both lethal and nonlethal virulence phenotypes across the multiple C. albicans strains. In this study, we found that regardless of pathogen ploidy or genetic background, immunocompromised hosts were susceptible to fungal infection compared to healthy hosts. Furthermore, for each host context, we found a significant interaction between C. albicans genetic background and ploidy on virulence phenotypes, but no global differences between diploid and tetraploid pathogens were observed.     

Isolation,purification and de novo sequencing of TBD‐1, the first beta‐defensin from leukocytes of reptiles

A novel peptide with antimicrobial activity was isolated from leukocytes of the European pond turtle Emys orbicularis and purified to homogeneity by preparative gel electrophoresis followed by reversed phase chromatography. It was highly active in vitro against Escherichia coli, Listeria monocytogenes, methicillin‐resistant Staphylococcus aureus, and Candida albicans. The isolated peptide was sequenced de novo by tandem mass spectrometry using both collision‐induced and electron‐transfer dissociation in combination with different chemical derivatization techniques. The 40‐residue peptide, called TBD‐1 (turtle β‐defensin 1), represents the first defensin isolated from reptilian leukocytes. It contains three disulfide bonds and shows high structural similarities to β‐defensins isolated from birds and mammals.     

Nestedness and β‐diversity in ectoparasite assemblages of small mammalian hosts: effects of parasite affinity,host biology and scale

We asked whether (a) variation in species composition of parasite assemblages on the same host species follows a non‐random pattern and (b) if so, manifestation of this non‐randomness across space and time differs among parasites, hosts and scales. We assessed nestedness and its contribution to β‐diversity of fleas and gamasid mite assemblages exploiting small mammals across three scales: (a) within the same region across different locations; (b) within the same location across different times and (c) across distinct geographic regions. We estimated (a) the degree of nestedness (N_COL) and (b) the proportional contribution of nestedness to the total amount of β‐diversity across locations, times and regions (β_NESP). In the majority of host species, parasite assemblages were nested significantly across all three scales. In mites, but not fleas, N_COL correlated with the contribution of nestedness to the total amount of β‐diversity. In fleas, N_COL did not differ among assemblages at the two local scales, but was significantly lower at regional scale. In mites, N_COL was the highest in assemblages at local spatial scale. β_NESP was significantly higher (a) in flea than in mite assemblages at both local scales and (b) in mite than in flea assemblages at regional scale. In fleas, β_NESP was higher at both local scales, whereas in mites it was higher at both local temporal and regional scales. Sheltering habits and geographic range of a host species did not affect either N_COL or β_NESP in flea assemblages, but both metrics significantly decreased with an increase of geographic range of a host species in mite assemblages. We conclude that flea and mite assemblages across host populations at smaller and larger spatial scales and at temporal scale were characterized by nestedness which, in turn, contributed to an important degree to the total amount of β‐diversity of these assemblages.     

Comparative genomic study of arachnid immune systems indicates loss of beta‐1,3‐glucanase‐related proteins and the immune deficiency pathway

Analyses of arthropod genomes have shown that the genes in the different innate humoral immune responses are conserved. These genes encode proteins that are involved in immune signalling pathways that recognize pathogens and activate immune responses. These immune responses include phagocytosis, encapsulation of the pathogen and production of effector molecules for pathogen elimination. So far, most studies have focused on insects leaving other major arthropod groups largely unexplored. Here, we annotate the immune‐related genes of six arachnid genomes and present evidence for a conserved pattern of some immune genes, but also evolutionary changes in the arachnid immune system. Specifically, our results suggest that the family of recognition molecules of beta‐1,3‐glucanase‐related proteins (βGRPs) and the genes from the immune deficiency (IMD) signalling pathway have been lost in a common ancestor of arachnids. These findings are consistent with previous work suggesting that the humoral immune effector proteins are constitutively produced in arachnids in contrast to insects, where these have to be induced. Further functional studies are needed to verify this. We further show that the full haemolymph clotting cascade found in the horseshoe crab is retrieved in most arachnid genomes. Tetranychus lacks at least one major component, although it is possible that this cascade could still function through recruitment of a different protein. The gel‐forming protein in horseshoe crabs, coagulogen, was not recovered in any of the arachnid genomes; however, it is possible that the arachnid clot consists of a related protein, spätzle, that is present in all of the genomes.     

Patterns of divergence of a large family of nodule cysteine‐rich peptides in accessions of Medicago truncatula

The nodule cysteine‐rich (NCR) groups of defensin‐like (DEFL) genes are one of the largest gene families expressed in the nodules of some legume plants. They have only been observed in the inverted repeat loss clade (IRLC) of legumes, which includes the model legume Medicago truncatula. NCRs are reported to play an important role in plant–microbe interactions. To understand their diversity we analyzed their expression and sequence polymorphisms among four accessions of M. truncatula. A significant expression and nucleotide variation was observed among the genes. We then used 26 accessions to estimate the selection pressures shaping evolution among the accessions by calculating the nucleotide diversity at non‐synonymous and synonymous sites in the coding region. The mature peptides of the orthologous NCRs had signatures of both purifying and diversifying selection pressures, unlike the seed DEFLs, which predominantly exhibited purifying selection. The expression, sequence variation and apparent diversifying selection in NCRs within the Medicago species indicates rapid and recent evolution, and suggests that this family of genes is actively evolving to adapt to different environments and is acquiring new functions.     

KRE genes are required for β‐1,6‐glucan synthesis,maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans

The polysaccharide β‐1,6‐glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in β‐1,6‐glucan synthesis. The H99 deletion mutants kre5Δ and kre6Δskn1Δ contained less cell wall β‐1,6‐glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI‐anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5Δ and kre6Δskn1Δ. Our results indicate that KRE5, KRE6 and SKN1 are involved in β‐1,6‐glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer.     

Contrasting patterns of selection and drift between two categories of immune genes in prairie‐chickens

Immune‐receptor genes of the adaptive immune system, such as the major histocompatibility complex (MHC), are involved in recognizing specific pathogens and are known to have high rates of adaptive evolution, presumably as a consequence of rapid co‐evolution between hosts and pathogens. In contrast, many ‘mediating’ genes of the immune system do not interact directly with specific pathogens and are involved in signalling (e.g. cytokines) or controlling immune cell growth. As a consequence, we might expect stronger selection at immune‐receptor than mediating genes, but these two types of genes have not been compared directly in wild populations. Here, we tested the hypothesis that selection differs between MHC (class I and II) and mediating genes by comparing levels of population differentiation across the range of greater prairie‐chickens (Tympanuchus cupido). As predicted, there was stronger population differentiation and isolation by distance at immune receptor (MHC) than at either mediating genes or neutral microsatellites, suggesting a stronger role of local adaptation at the MHC. In contrast, mediating genes displayed weaker differentiation between populations than neutral microsatellites, consistent with selection favouring similar alleles across populations for mediating genes. In addition to selection, drift also had a stronger effect on immune receptor (MHC) than mediating genes as indicated by the stronger decline of MHC variation in relation to population size. This is the first study in the wild to show that the effects of selection and drift on immune genes vary across populations depending on their functional role.     

Detecting evolutionary rate heterogeneity among mangroves and their close terrestrial relatives

Mangroves form the dominant intertidal ecosystems and differ morphologically and physiologically from their close terrestrial relatives. We investigate the molecular evolutionary pattern of the typical mangrove family, i.e. Rhizophoraceae, and rate heterogeneity for the plastid matK and rbcL genes in different species of the family, as revealed by phylogenetic analyses and relative‐rate tests. Our study documents evolutionary rate heterogeneity in the Rhizophoraceae for the two genes: the mangrove genus Bruguiera has relatively slow substitution rates compared to the terrestrial genus Carallia at both synonymous and non‐synonymous sites in the matK sequences, and the synonymous and non‐synonymous substitution matrices are correlated. However, the rbcL non‐synonymous sites exhibit a high degree of rate heterogeneity among mangroves and related terrestrial groups, and uncoupling of rates with the synonymous sites. Selection is probably an important influence on the rate variation, suggesting further investigation for better understanding of various forces contributing to the rate heterogeneity and molecular adaptation in mangroves.     

Drift,not selection,shapes toll‐like receptor variation among oceanic island populations

Understanding the relative role of different evolutionary forces in shaping the level and distribution of functional genetic diversity among natural populations is a key issue in evolutionary and conservation biology. To do so accurately genetic data must be analysed in conjunction with an unambiguous understanding of the historical processes that have acted upon the populations. Here, we focused on diversity at toll‐like receptor (TLR) loci, which play a key role in the vertebrate innate immune system and, therefore, are expected to be under pathogen‐mediated selection. We assessed TLR variation within and among 13 island populations (grouped into three archipelagos) of Berthelot's pipit, Anthus berthelotii, for which detailed population history has previously been ascertained. We also compared the variation observed with that found in its widespread sister species, the tawny pipit, Anthus campestris. We found strong evidence for positive selection at specific codons in TLR1LA, TLR3 and TLR4. Despite this, we found that at the allele frequency level, demographic history has played the major role in shaping patterns of TLR variation in Berthelot's pipit. Levels of diversity and differentiation within and across archipelagos at all TLR loci corresponded very closely with neutral microsatellite variation and with the severity of the bottlenecks that occurred during colonization. Our study shows that despite the importance of TLRs in combating pathogens, demography can be the main driver of immune gene variation within and across populations, resulting in patterns of functional variation that can persist over evolutionary timescales.     

The HLA‐B landscape of Africa: Signatures of pathogen‐driven selection and molecular identification of candidate alleles to malaria protection

Human leukocyte antigen (HLA) genes play a key role in the immune response to infectious diseases, some of which are highly prevalent in specific environments, like malaria in sub‐Saharan Africa. Former case–control studies showed that one particular HLA‐B allele, B*53, was associated with malaria protection in Gambia, but this hypothesis was not tested so far within a population genetics framework. In this study, our objective was to assess whether pathogen‐driven selection associated with malaria contributed to shape the HLA‐B genetic landscape of Africa. To that aim, we first typed the HLA‐A and ‐B loci in 484 individuals from 11 populations living in different environments across the Sahel, and we analysed these data together with those available for 29 other populations using several approaches including linear modelling on various genetic, geographic and environmental parameters. In addition to relevant signatures of populations’ demography and migrations history in the genetic differentiation patterns of both HLA‐A and ‐B loci, we found that the frequencies of three HLA alleles, B*53, B*78 and A*74, were significantly associated with Plasmodium falciparum malaria prevalence, suggesting their increase through pathogen‐driven selection in malaria‐endemic environments. The two HLA‐B alleles were further identified, by high‐throughput sequencing, as B*53:01:01 (in putative linkage disequilibrium with one HLA‐C allele, C*04:01:01:01) and B*78:01 in all but one individuals tested, making them appropriate candidates to malaria protection. These results highlight the role of environmental factors in the evolution of the HLA polymorphism and open key perspectives for functional studies focusing on HLA peptide‐binding properties.     

Alanine substitutions of noncysteine residues in the cysteine‐stabilized αβ motif

The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine‐stabilized αβ motif (CSαβ) consists of an α‐helix and an antiparallel triple‐stranded β‐sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSαβ motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty‐two of 46 residue positions of VrD1 are altered by site‐directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSαβ motif. The information is valuable to the scaffold with the CSαβ motif and protein engineering.     

Bos indicus introgression into (peri‐)alpine cattle breeds – evidence from the analysis of bovine whey protein variants

The major bovine whey proteins, α‐lactalbumin (α‐LA) and β‐lactoglobulin (β‐LG), exhibit breed‐specific genetic variation. The aim of this study was to identify possible new protein variants and determine the distribution of variants across a variety of 18 taurine and indicine cattle breeds applying a DNA‐based sequencing approach. To this end, the open reading frames of the respective genes (LALBA and LGB) were sequenced in 476 animals. Within the LALBA gene, a previously unknown synonymous and a previously undesignated non‐synonymous nucleotide exchange were identified. Furthermore, two known α‐LA variants (A and B) and four known β‐LG variants (A, B, C and W) were determined. The occurrence of typical indicine variants in some taurine cattle breeds, such as Suisse Eringer, German Hinterwälder and Hungarian Grey Steppe, further supports the hypothesis of ancient Bos indicus introgression into (peri‐)alpine cattle breeds.     

Lactobacillus helveticus SBT2171 upregulates the expression of β‐defensin and ameliorates periodontal disease caused by Porphyromonas gingivalis

Antimicrobial peptides play important roles in the innate immune system of various organisms, and they may also be considered to prevent the organisms from infections. In particular, β‐defensins, mainly produced in epithelial cells, are recognized as one of the major antimicrobial peptides in mammals, including humans. In this study, we showed that Lactobacillus helveticus SBT2171 (LH2171), one of the several species of lactic acid bacteria, upregulates the production of β‐defensins in oral epithelial cells in vitro. Moreover, LH2171 reduced the increase of proinflammatory cytokine expression, induced by Porphyromonas gingivalis stimulation, in gingival epithelial cells. These data suggested that LH2171 suppresses P. gingivalis‐induced inflammation by upregulating the expression of β‐defensins in gingival epithelial cells. We subsequently investigated the effects of LH2171 in vivo and revealed that β‐defensin expression was increased in the oral cavities of LH2171‐fed mice. Furthermore, LH2171 decreased alveolar bone loss, gingival inflammation, and amounts of P. gingivalis‐specific 16S ribosomal RNA in the gingiva of P. gingivalis‐inoculated mice. Taken together, our results showed that LH2171 upregulates the expression of β‐defensins in oral cavity, thereby decreasing the number of P. gingivalis consequently ameliorating the experimental periodontal disease.