Plasma cortisol and faecal cortisol metabolites concentrations in stereotypic and non-stereotypic horses: do stereotypic horses cope better with poor environmental conditions?

Background

Stereotypic behaviours, i.e. repetitive behaviours induced by frustration, repeated attempts to cope and/or brain dysfunction, are intriguing as they occur in a variety of domestic and captive species without any clear adaptive function. Among the different hypotheses, the coping hypothesis predicts that stereotypic behaviours provide a way for animals in unfavourable environmental conditions to adjust. As such, they are expected to have a lower physiological stress level (glucocorticoids) than non-stereotypic animals. Attempts to link stereotypic behaviours with glucocorticoids however have yielded contradictory results. Here we investigated correlates of oral and motor stereotypic behaviours and glucocorticoid levels in two large samples of domestic horses (N _Study1 = 55, N _Study2 = 58), kept in sub-optimal conditions (e.g. confinement, social isolation), and already known to experience poor welfare states. Each horse was observed in its box using focal sampling (study 1) and instantaneous scan sampling (study 2). Plasma samples (collected in study 1) but also non-invasive faecal samples (collected in both studies) were retrieved in order to assess cortisol levels.

Results

Results showed that 1) plasma cortisol and faecal cortisol metabolites concentrations did not differ between horses displaying stereotypic behaviours and non-stereotypic horses and 2) both oral and motor stereotypic behaviour levels did not predict plasma cortisol or faecal cortisol metabolites concentrations.

Conclusions

Cortisol measures, collected in two large samples of horses using both plasma sampling as well as faecal sampling (the latter method minimizing bias due to a non-invasive sampling procedure), therefore do not indicate that stereotypic horses cope better, at least in terms of adrenocortical activity.     

Whole genome sequencing reveals a novel deletion variant in the KIT gene in horses with white spotted coat colour phenotypes

White spotting phenotypes in horses can range in severity from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for such phenotypes in horses. For the present study, we re‐investigated a large horse family segregating a variable white spotting phenotype, for which conventional Sanger sequencing of the candidate genes’ individual exons had failed to reveal the causative variant. We obtained whole genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous ~1.9‐kb deletion spanning exons 10–13 of the KIT gene (chr3:77,740,239_77,742,136del1898insTATAT). In continuity with previously named equine KIT variants we propose to designate the newly identified deletion variant W22. We had access to 21 horses carrying the W22 allele. Four of them were compound heterozygous W20/W22 and had a completely white phenotype. Our data suggest that W22 represents a true null allele of the KIT gene, whereas the previously identified W20 leads to a partial loss of function. These findings will enable more precise genetic testing for depigmentation phenotypes in horses.     

The short-term effects of increasing meal frequency on stereotypic behaviour of stabled horses

In this study, we investigated the effect of increasing the number of meals of concentrate (whilst maintaining the same daily intake) on the behaviour of stabled horses with particular reference to stereotypic activities. The study was carried out on a working equestrian yard with stables for up to 50 horses. A pilot study was used to record incidence of stereotypic behaviour and to select subjects for the main study. In this, the behaviour of 30 warm-blooded horses was recorded during their morning (08:30 h) and afternoon (16:30 h) concentrate feeds. Whilst there was a low incidence of stereotypic behaviour (5.6% of scans) in the population, they were more commonly observed in the afternoon (7.1%) than the morning observations (4.2%; P < 0.05). The higher incidence in the afternoon observation appeared to be related to the lower availability of high fibre forage during the afternoon meal. In the main study nine horses were fed their normal ration of concentrate divided between two, four or six equally sized meals. Their behaviour was compared with seven control horses, which received two meals per day throughout the trial. As the number of meals increased, the treatment horses showed a decrease in oral stereotypies (P < 0.01), but an increase in weaving (P < 0.05) and nodding (P < 0.01) prior to feeding. The control group increased weaving, nodding and oral stereotypies (all P < 0.05) as their yard-mates received more meals. Consequently there was an overall increase in incidence of stereotypy in both treatment and control horses with the increase in meal frequency. The study, therefore, suggests that dividing the stabled horses’ concentrate ration into a number of smaller meals may be an effective means of reducing oral stereotypies, but that pre-feeding stereotypies may persist and that the practise may increase the frequency of stereotypic behaviour on unfed horses in visual contact.     

Large Neurological Component to Genetic Differences Underlying Biased Sperm Use in Drosophila

Sperm competition arises as a result of complex interactions among male and female factors. While the roles of some male factors are known, little is known of the molecules or mechanisms that underlie the female contribution to sperm competition. The genetic tools available for Drosophila allow us to identify, in an unbiased manner, candidate female genes that are critical for mediating sperm competition outcomes. We first screened for differences in female sperm storage and use patterns by characterizing the natural variation in sperm competition in a set of 39 lines from the sequenced Drosophila Genetic Reference Panel (DGRP) of wild-derived inbred lines. We found extensive female variation in sperm competition outcomes. To generate a list of candidate female genes for functional studies, we performed a genome-wide association mapping, utilizing the common single-nucleotide polymorphisms (SNPs) segregating in the DGRP lines. Surprisingly, SNPs within ion channel genes and other genes with roles in the nervous system were among the top associated SNPs. Knockdown studies of three candidate genes (para, Rab2, and Rim) in sensory neurons innervating the female reproductive tract indicate that some of these candidate female genes may affect sperm competition by modulating the neural input of these sensory neurons to the female reproductive tract. More extensive functional studies are needed to elucidate the exact role of all these candidate female genes in sperm competition. Nevertheless, the female nervous system appears to have a previously unappreciated role in sperm competition. Our results indicate that the study of female control of sperm competition should not be limited to female reproductive tract-specific genes, but should focus also on diverse biological pathways.     

Exploration into natural variation for genes associated with fruit shape and size among Capsicum chinense collections

Phenotype diversity within cultivated Capsicum chinense is particularly evident for fruit shape and size. We used this diversity in C. chinense to further unravel the genetic mechanisms underlying fruit shape variation in pepper and related Solanaceous species. We identified candidate genes for C. chinense fruit shape, explored their contribution to population structure, and characterized their potential function in pepper fruit shape. Using genotyping by sequencing, we identified 43,081 single nucleotide polymorphisms (SNPs) from diverse collections of C. chinense. Principal component, neighbor-joining tree, and population structure analyses resolved 3 phylogenetically robust clusters associated with fruit shapes. Genome-wide association study (GWAS) was used to identify associated genomic regions with various fruit shape traits obtained from image analysis with Tomato Analyzer software. In our GWAS, we selected 12 SNPs associated with locule number trait and 8 SNP markers associated with other fruit shape traits such as perimeter, area, obovoid, ellipsoid and morphometrics (5y, 6y and 7y). The SNPs in CLAVATA1, WD-40, Auxin receptor, AAA type ATPase family protein, and RNA polymerase III genes were the major markers identified for fruit locule number from our GWAS results. Furthermore, we found SNPs in tetratricopeptide-repeat thioredoxin-like 3, enhancer of ABA co-receptor 1, subunit of exocyst complex 8 and pleiotropic drug resistance proteins associated with various fruit shape traits. CLAVATA1, WD-40 and Auxin receptor genes are known genes that affect tomato fruit shape. In this study, we used Arabidopsis thaliana T-DNA insertion knockout mutants and expression profiles for functional characterization of newly identified genes and to understand their role in fruit shape.     

Whole‐genome sequencing reveals a large deletion in the MITF gene in horses with white spotted coat colour and increased risk of deafness

White spotting phenotypes in horses are highly valued in some breeds. They are quite variable and may range from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for white spotting phenotypes in horses. For the present study, we investigated an American Paint Horse family segregating a phenotype involving white spotting and blue eyes. Six of eight horses with the white‐spotting phenotype were deaf. We obtained whole‐genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous ~63‐kb deletion spanning exons 6–9 of the MITF gene (chr16:21 503 211–21 566 617). We confirmed the breakpoints of the deletion by PCR and Sanger sequencing. PCR‐based genotyping revealed that all eight available affected horses from the family carried the deletion. The finding of an MITF variant fits well with the syndromic phenotype involving both depigmentation and an increased risk for deafness and corresponds to human Waardenburg syndrome type 2A. Our findings will enable more precise genetic testing for depigmentation phenotypes in horses.     

An Evolution-Based Screen for Genetic Differentiation between Anopheles Sister Taxa Enriches for Detection of Functional Immune Factors

Nucleotide variation patterns across species are shaped by the processes of natural selection, including exposure to environmental pathogens. We examined patterns of genetic variation in two sister species, Anopheles gambiae and Anopheles coluzzii, both efficient natural vectors of human malaria in West Africa. We used the differentiation signature displayed by a known coordinate selective sweep of immune genes APL1 and TEP1 in A. coluzzii to design a population genetic screen trained on the sweep, classified a panel of 26 potential immune genes for concordance with the signature, and functionally tested their immune phenotypes. The screen results were strongly predictive for genes with protective immune phenotypes: genes meeting the screen criteria were significantly more likely to display a functional phenotype against malaria infection than genes not meeting the criteria (p = 0.0005). Thus, an evolution-based screen can efficiently prioritize candidate genes for labor-intensive downstream functional testing, and safely allow the elimination of genes not meeting the screen criteria. The suite of immune genes with characteristics similar to the APL1-TEP1 selective sweep appears to be more widespread in the A. coluzzii genome than previously recognized. The immune gene differentiation may be a consequence of adaptation of A. coluzzii to new pathogens encountered in its niche expansion during the separation from A. gambiae, although the role, if any of natural selection by Plasmodium is unknown. Application of the screen allowed identification of new functional immune factors, and assignment of new functions to known factors. We describe biochemical binding interactions between immune proteins that underlie functional activity for malaria infection, which highlights the interplay between pathogen specificity and the structure of immune complexes. We also find that most malaria-protective immune factors display phenotypes for either human or rodent malaria, with broad specificity a rarity.     

Prevalence of and risk factors for colic in horses that display crib-biting behaviour

Background

Crib-biting and windsucking (CBWS) behaviour in horses has been associated with increased risk of colic in general, recurrence of colic and specific forms of colic. The aims of the present study were to determine the prevalence of colic within a population of horses that display CBWS behaviour and to identify risk factors for colic.

Methods

Owners/carers of horses in the general UK equine population that display CBWS behaviour were invited to participate in a questionnaire-based survey about the management and health of these horses. Data were obtained for a number of variables considered to be possible risk factors for colic. The prevalence of colic was calculated and multivariable logistic regression was used to identify associations between horse- and management-level variables for two outcomes of interest: a history of colic ever and a history of colic in the previous 12 months.

Results

Data were obtained for 367 horses. One or more episodes of colic had been observed in 130 horses (35.4%). A total of 672 colic episodes were reported and 13 colic episodes required surgical intervention in 12 horses. Where the horse/pony had been in that persons care over the previous 12 months (n=331), colic had been observed in 67 horses (20.2%) during that time. A total of 126 colic episodes were reported in the preceding 12 months of which veterinary attendance was required in 69 (54.8%) episodes. Increased duration of ownership, increased duration of stabling in the Autumn months (September-November), crib-biting/windsucking behaviour associated with eating forage and horses that were fed haylage were associated with increased risk of colic (ever). Increasing severity (frequency) of CBWS behaviour and increased duration of stabling in the Autumn were associated with increased risk of colic in the previous 12 months.

Conclusions

The prevalence of colic in a population of horses that display CBWS appeared to be relatively high. The results of this study can be used to identify horses that display CBWS who are at increased risk of colic and identifies areas for further research to determine if there are ways in which this risk might be reduced.

     

Independent of Calorie Intake,Short-term Alternate-day Fasting Alleviates NASH,With Modulation of Markers of Lipogenesis,Autophagy, Apoptosis,and Inflammation in Rats

Non-alcoholic steatohepatitis (NASH) is a worldwide health problem. Alternate-day fasting (ADF), although thought to be aggressive, has proven safety and efficacy. We aimed to evaluate the effect of short-term ADF against already established high-fat-fructose (HFF)-induced NASH, independent of the amount of calorie intake, and to study the effect of ADF on lipogenesis, apoptosis, and hepatic inflammation. Male Sprague Dawley rats were divided into two groups: (1) negative control and (2) NASH group fed on HFF for 9 weeks, and then randomized into two subgroups of either HFF alone or with ADF protocol for 3 weeks. The ADF could improve HFF-related elevation in serum lactate dehydrogenase and could decrease the mRNA expression of lipogenesis genes; acetyl CoA carboxylase, peroxisome proliferator-activated receptor γ, and peroxisome proliferator-activated receptor α; apoptotic genes caspase-3, p53, and inflammatory cyclo-oxygenase 2; and immunohistochemical staining for their proteins in liver with upregulation of LC3 and downregulation of P62 immunoexpression. Moreover, ADF ameliorated HFF-induced steatosis, inflammation, ballooning, and fibrosis through hematoxylin and eosin, Oil Red O, and Sirius Red staining, confirmed by morphometric analysis, without significant weight loss. Significant correlation of morphometric parameters with levels of gene expression was found. These findings suggest ADF to be a safe effective therapeutic agent in the management of NASH     

TRAIL/TRAIL receptor system and susceptibility to multiple sclerosis

The TNF-related apoptosis inducing ligand (TRAIL)/TRAIL receptor system participates in crucial steps in immune cell activation or differentiation. It is able to inhibit proliferation and activation of T cells and to induce apoptosis of neurons and oligodendrocytes, and seems to be implicated in autoimmune diseases. Thus, TRAIL and TRAIL receptor genes are potential candidates for involvement in susceptibility to multiple sclerosis (MS). To test whether single-nucleotide polymorphisms (SNPs) in the human genes encoding TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 are associated with MS susceptibility, we performed a candidate gene case-control study in the Spanish population. 59 SNPs in the TRAIL and TRAIL receptor genes were analysed in 628 MS patients and 660 controls, and validated in an additional cohort of 295 MS patients and 233 controls. Despite none of the SNPs withstood the highly conservative Bonferroni correction, three SNPs showing uncorrected p values<0.05 were successfully replicated: rs4894559 in TRAIL gene, p = 9.8×10⁻⁴, OR = 1.34; rs4872077, in TRAILR-1 gene, p = 0.005, OR = 1.72; and rs1001793 in TRAILR-2 gene, p = 0.012, OR = 0.84. The combination of the alleles G/T/A in these SNPs appears to be associated with a reduced risk of developing MS (p = 2.12×10⁻⁵, OR = 0.59). These results suggest that genes of the TRAIL/TRAIL receptor system exerts a genetic influence on MS.     

The Landscape of Candidate Driver Genes Differs between Male and Female Breast Cancer

The rapidly growing collection of diverse genome-scale data from multiple tumor types sheds light on various aspects of the underlying tumor biology. With the objective to identify genes of importance for breast tumorigenesis in men and to enable comparisons with genes important for breast cancer development in women, we applied the computational framework COpy Number and EXpression In Cancer (CONEXIC) to detect candidate driver genes among all altered passenger genes. Unique to this approach is that each driver gene is associated with several gene modules that are believed to be altered by the driver. Thirty candidate drivers were found in the male breast cancers and 67 in the female breast cancers. We identified many known drivers of breast cancer and other types of cancer, in the female dataset (e.g. GATA3, CCNE1, GRB7, CDK4). In contrast, only three known cancer genes were found among male breast cancers; MAP2K4, LHP, and ZNF217. Many of the candidate drivers identified are known to be involved in processes associated with tumorigenesis, including proliferation, invasion and differentiation. One of the modules identified in male breast cancer was regulated by THY1, a gene involved in invasion and related to epithelial-mesenchymal transition. Furthermore, men with THY1 positive breast cancers had significantly inferior survival. THY1 may thus be a promising novel prognostic marker for male breast cancer. Another module identified among male breast cancers, regulated by SPAG5, was closely associated with proliferation. Our data indicate that male and female breast cancers display highly different landscapes of candidate driver genes, as only a few genes were found in common between the two. Consequently, the pathobiology of male breast cancer may differ from that of female breast cancer and can be associated with differences in prognosis; men diagnosed with breast cancer may consequently require different management and treatment strategies than women.     

Exploring Valid Reference Genes for Quantitative Real-time PCR Analysis in Plutella xylostella (Lepidoptera: Plutellidae)

Abstract: Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental conditions. RefFinder, a web-based analysis tool, integrates four major computational programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCt method to comprehensively rank the tested candidate genes. Elongation factor 1 (EF1) was the most suited reference gene for the biotic factors (development stage, tissue, and strain). In contrast, although appropriate reference gene(s) do exist for several abiotic factors (temperature, photoperiod, insecticide, and mechanical injury), we were not able to identify a single universal reference gene. Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. Our finding is the first step toward establishing a standardized qRT-PCR analysis of this agriculturally important insect pest.     

Comparative Genome of GK and Wistar Rats Reveals Genetic Basis of Type 2 Diabetes

The Goto-Kakizaki (GK) rat, which has been developed by repeated inbreeding of glucose-intolerant Wistar rats, is the most widely studied rat model for Type 2 diabetes (T2D). However, the detailed genetic background of T2D phenotype in GK rats is still largely unknown. We report a survey of T2D susceptible variations based on high-quality whole genome sequencing of GK and Wistar rats, which have generated a list of GK-specific variations (228 structural variations, 2660 CNV amplification and 2834 CNV deletion, 1796 protein affecting SNVs or indels) by comparative genome analysis and identified 192 potential T2D-associated genes. The genes with variants are further refined with prior knowledge and public resource including variant polymorphism of rat strains, protein-protein interactions and differential gene expression. Finally we have identified 15 genetic mutant genes which include seven known T2D related genes (Tnfrsf1b, Scg5, Fgb, Sell, Dpp4, Icam1, and Pkd2l1) and eight high-confidence new candidate genes (Ldlr, Ccl2, Erbb3, Akr1b1, Pik3c2a, Cd5, Eef2k, and Cpd). Our result reveals that the T2D phenotype may be caused by the accumulation of multiple variations in GK rat, and that the mutated genes may affect biological functions including adipocytokine signaling, glycerolipid metabolism, PPAR signaling, T cell receptor signaling and insulin signaling pathways. We present the genomic difference between two closely related rat strains (GK and Wistar) and narrow down the scope of susceptible loci. It also requires further experimental study to understand and validate the relationship between our candidate variants and T2D phenotype. Our findings highlight the importance of sequenced-based comparative genomics for investigating disease susceptibility loci in inbreeding animal models.     

No Association between Personality and Candidate Gene Polymorphisms in a Wild Bird Population

Consistency of between-individual differences in behaviour or personality is a phenomenon in populations that can have ecological consequences and evolutionary potential. One way that behaviour can evolve is to have a genetic basis. Identifying the molecular genetic basis of personality could therefore provide insight into how and why such variation is maintained, particularly in natural populations. Previously identified candidate genes for personality in birds include the dopamine receptor D4 (DRD4), and serotonin transporter (SERT). Studies of wild bird populations have shown that exploratory and bold behaviours are associated with polymorphisms in both DRD4 and SERT. Here we tested for polymorphisms in DRD4 and SERT in the Seychelles warbler (Acrocephalus sechellensis) population on Cousin Island, Seychelles, and then investigated correlations between personality and polymorphisms in these genes. We found no genetic variation in DRD4, but identified four polymorphisms in SERT that clustered into five haplotypes. There was no correlation between bold or exploratory behaviours and SERT polymorphisms/haplotypes. The null result was not due to lack of power, and indicates that there was no association between these behaviours and variation in the candidate genes tested in this population. These null findings provide important data to facilitate representative future meta-analyses on candidate personality genes.     

Evaluation and Validation of Reference Genes for Normalization of Quantitative Real-Time PCR Based Gene Expression Studies in Peanut

The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut.