Sex‐peptides bind to two molecularly different targets in Drosophila melanogaster females

Sex‐Peptide (SP) and the peptide DUP99B elicit two postmating responses in Drosophila melanogaster females: receptivity is reduced and oviposition is increased. Both are synthesized in the male genital tract and transferred into the female during copulation. To elucidate their function, we characterized the binding properties of SP and DUP99B in females. Cryostat sections of adult females were incubated with alkaline phosphatase (AP)‐tagged peptides. In virgin females, both peptides have specific target sites in the nervous system and in the genital tract. The binding pattern is almost identical for both peptides. Incubation of sections of mated females confirm that some of these target sites correspond to the in vivo targets of the two peptides. Neuronal binding is dependent on an intact C‐terminal sequence of SP, binding in the genital tract is less demanding in terms of amino acid sequence requirement. On affinity blots the AP–SP probe binds to membrane proteins extracted from abdomen and head plus thorax, respectively. The binding proteins in the nervous system and the genital tract differ in their molecular properties. Calculation of dissociation constants (K_d), and also determination of the minimal peptide concentrations necessary for binding, indicate that SP is the more important peptide inducing the postmating responses. Our results suggest that binding of SP in the nervous system is responsible for eliciting the postmating responses, whereas binding in the genital tract reflects the presence of a peptide transporter. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 372–384, 2003     

Spatiotemporal dynamics and genome‐wide association analysis of desiccation tolerance in Drosophila melanogaster

Water availability is a major environmental challenge to a variety of terrestrial organisms. In insects, desiccation tolerance varies predictably over spatial and temporal scales and is an important physiological determinant of fitness in natural populations. Here, we examine the dynamics of desiccation tolerance in North American populations of Drosophila melanogaster using: (a) natural populations sampled across latitudes and seasons; (b) experimental evolution in field mesocosms over seasonal time; (c) genome‐wide associations to identify SNPs/genes associated with variation for desiccation tolerance; and (d) subsequent analysis of patterns of clinal/seasonal enrichment in existing pooled sequencing data of populations sampled in both North America and Australia. A cline in desiccation tolerance was observed, for which tolerance exhibited a positive association with latitude; tolerance also varied predictably with culture temperature, demonstrating a significant degree of thermal plasticity. Desiccation tolerance evolved rapidly in field mesocosms, although only males showed differences in desiccation tolerance between spring and autumn collections from natural populations. Water loss rates did not vary significantly among latitudinal or seasonal populations; however, changes in metabolic rates during prolonged exposure to dry conditions are consistent with increased tolerance in higher latitude populations. Genome‐wide associations in a panel of inbred lines identified twenty‐five SNPs in twenty‐one loci associated with sex‐averaged desiccation tolerance, but there is no robust signal of spatially varying selection on genes associated with desiccation tolerance. Together, our results suggest that desiccation tolerance is a complex and important fitness component that evolves rapidly and predictably in natural populations.     

Genome‐wide patterns of latitudinal differentiation among populations of Drosophila melanogaster from North America

Understanding the genetic underpinnings of adaptive change is a fundamental but largely unresolved problem in evolutionary biology. Drosophila melanogaster, an ancestrally tropical insect that has spread to temperate regions and become cosmopolitan, offers a powerful opportunity for identifying the molecular polymorphisms underlying clinal adaptation. Here, we use genome‐wide next‐generation sequencing of DNA pools (‘pool‐seq’) from three populations collected along the North American east coast to examine patterns of latitudinal differentiation. Comparing the genomes of these populations is particularly interesting since they exhibit clinal variation in a number of important life history traits. We find extensive latitudinal differentiation, with many of the most strongly differentiated genes involved in major functional pathways such as the insulin/TOR, ecdysone, torso, EGFR, TGFβ/BMP, JAK/STAT, immunity and circadian rhythm pathways. We observe particularly strong differentiation on chromosome 3R, especially within the cosmopolitan inversion In(3R)Payne, which contains a large number of clinally varying genes. While much of the differentiation might be driven by clinal differences in the frequency of In(3R)P, we also identify genes that are likely independent of this inversion. Our results provide genome‐wide evidence consistent with pervasive spatially variable selection acting on numerous loci and pathways along the well‐known North American cline, with many candidates implicated in life history regulation and exhibiting parallel differentiation along the previously investigated Australian cline.     

Esterase-6 and the pheromonal effects of cis-vaccenyl acetate in Drosophila melanogaster

In Drosophila melanogaster the polymorphic enzyme Est-6 and a male specific lipid, cis-vaccenyl acetate (cVA), are components of the seminal fluid. It has been suggested that cVA could be a physiological substrate for Est-6, constituting, together with the hypothetical hydrolytic product, cis-vaccenyl alcohol (cVOH), a pheromonal system active in the regulation of the sexual attractiveness of mated females. However, some doubt exists concerning the physiological conversion of cVA to cVOH in females. Est-6 is also known to affect sperm motility, sperm use and female fecundity. The results of a study on qualitative and quantitative effects of topical treatment of females with cis-vaccenyl acetate and cis-vaccenyl alcohol on courtship behaviour are presented. The inhibition of courtship produced by cVOH was more persistent; this was possibly related to its lower volatility. The hypothesis was tested that different Est-6 allozymes could produce different amounts of cis-vaccenyl alcohol in vivo by differential hydrolysis of cis-vaccenyl acetate, and thus affect the timing of remating. For this purpose highly homogeneous Est-6^S and Est-6^F homozygous lines, with almost certainly identical levels of Est-6 and cVA, were used. They were obtained after 100 generations of inbreeding and selection of the heterozygotes at each generation. The Est-6^S and Est-6^F products of the Est-6 structural gene did not differentially affect the timing of remating. This could imply that the two allozymes have similar catalytic properties. However, such results could be obtained even if cVA is not converted to cVOH in vivo by Est-6. The effects of Est-6 allozymes on sperm use after remating were also investigated. The general effect of second males was a very high elimination of first male fertilizations. No evidence for a different contribution of the two allozymes to the fitness through sperm predominance was obtained. Remating and sperm predominance experiments were performed at three temperatures: 18, 25 and 30° C. A more general—not species specific—role of cis-vaccenyl esters is suggested by preliminary results on their presence in the ejaculate of other Drosophila species.     

Secondary contact and local adaptation contribute to genome‐wide patterns of clinal variation in Drosophila melanogaster

Populations arrayed along broad latitudinal gradients often show patterns of clinal variation in phenotype and genotype. Such population differentiation can be generated and maintained by both historical demographic events and local adaptation. These evolutionary forces are not mutually exclusive and can in some cases produce nearly identical patterns of genetic differentiation among populations. Here, we investigate the evolutionary forces that generated and maintain clinal variation genome‐wide among populations of Drosophila melanogaster sampled in North America and Australia. We contrast patterns of clinal variation in these continents with patterns of differentiation among ancestral European and African populations. Using established and novel methods we derive here, we show that recently derived North America and Australia populations were likely founded by both European and African lineages and that this hybridization event likely contributed to genome‐wide patterns of parallel clinal variation between continents. The pervasive effects of admixture mean that differentiation at only several hundred loci can be attributed to the operation of spatially varying selection using an F_ST outlier approach. Our results provide novel insight into the well‐studied system of clinal differentiation in D. melanogaster and provide a context for future studies seeking to identify loci contributing to local adaptation in a wide variety of organisms, including other invasive species as well as temperate endemics.     

Dietary consumption of monosodium L‐glutamate induces adaptive response and reduction in the life span of Drosophila melanogaster

Adaptive response is the ability of an organism to better counterattack stress‐induced damage in response to a number of different cytotoxic agents. Monosodium L‐glutamate (MSG), the sodium salt of amino acid glutamate, is commonly used as a food additive. We investigated the effects of MSG on the life span and antioxidant response in Drosophila melanogaster (D. melanogaster). Both genders (1 to 3 days old) of flies were fed with diet containing MSG (0.1, 0.5, and 2.5‐g/kg diet) for 5 days to assess selected antioxidant and oxidative stress markers, while flies for longevity were fed for lifetime. Thereafter, the longevity assay, hydrogen peroxide (H₂O₂), and reactive oxygen and nitrogen species levels were determined. Also, catalase, glutathione S‐transferase and acetylcholinesterase activities, and total thiol content were evaluated in the flies. We found that MSG reduced the life span of the flies by up to 23% after continuous exposure. Also, MSG increased reactive oxygen and nitrogen species and H₂O₂ generations and total thiol content as well as the activities of catalase and glutathione S‐transferase in D. melanogaster (P < .05). In conclusion, consumption of MSG for 5 days by D. melanogaster induced adaptive response, but long‐term exposure reduced life span of flies. This study may therefore have public health significance in humans, and thus, moderate consumption of MSG is advocated by the authors.     

A genome‐wide SNP scan reveals two loci associated with the chicken resistance to Marek's disease

Marek's disease (MD) is a neoplastic disease in chickens, caused by the Marek's disease virus (MDV). To investigate host genetic resistance to MD, we conducted a genome‐wide association study (GWAS) on 67 MDV‐infected chickens based on a case and control design, including 57 susceptible chickens in the case group and 10 resistant chickens as controls. After searching 38 655 valid genomic markers, two SNPs were found to be associated with host resistance to MD. One SNP, rs14527240, reaching chromosome‐wide significance level (P < 0.01) was located in the SPARC‐related modular calcium‐binding 1 (SMOC1) gene on GGA5. The other one, GGaluGA156129, reaching genome‐wide significance (P < 0.05), was located in the protein tyrosine phosphatase, non‐receptor type 3 (PTPN3) gene on GGA2. In addition, expression patterns of these two genes in spleens were detected by qPCR. The expression of SMOC1 was significantly up‐regulated (P < 0.05), whereas the expression of PTNP3 did not show significance when the case group was compared with the control group. Up‐regulation of SMOC1 in susceptible spleens suggests its important roles in MD tumorigenesis. This is the first study to investigate MD‐resistant loci, and it demonstrates the power of GWASs for mapping genes associated with MD resistance.     

Metabolite‐based genome‐wide association study enables dissection of the flavonoid decoration pathway of wheat kernels

The marriage of metabolomic approaches with genetic design has proven a powerful tool in dissecting diversity in the metabolome and has additionally enhanced our understanding of complex traits. That said, such studies have rarely been carried out in wheat. In this study, we detected 805 metabolites from wheat kernels and profiled their relative contents among 182 wheat accessions, conducting a metabolite‐based genome‐wide association study (mGWAS) utilizing 14 646 previously described polymorphic SNP markers. A total of 1098 mGWAS associations were detected with large effects, within which 26 candidate genes were tentatively designated for 42 loci. Enzymatic assay of two candidates indicated they could catalyse glucosylation and subsequent malonylation of various flavonoids and thereby the major flavonoid decoration pathway of wheat kernel was dissected. Moreover, numerous high‐confidence genes associated with metabolite contents have been provided, as well as more subdivided metabolite networks which are yet to be explored within our data. These combined efforts presented the first step towards realizing metabolomics‐associated breeding of wheat.     

Assessment of the functionality of genome‐wide canine SNP arrays and implications for canine disease association studies

Domestic dogs share a wide range of important disease conditions with humans, including cancers, diabetes and epilepsy. Many of these conditions have similar or identical underlying pathologies to their human counterparts and thus dogs represent physiologically relevant natural models of human disorders. Comparative genomic approaches whereby disease genes can be identified in dog diseases and then mapped onto the human genome are now recognized as a valid method and are increasing in popularity. The majority of dog breeds have been created over the past few hundred years and, as a consequence, the dog genome is characterized by extensive linkage disequilibrium (LD), extending usually from hundreds of kilobases to several megabases within a breed, rather than tens of kilobases observed in the human genome. Genome‐wide canine SNP arrays have been developed, and increasing success of using these arrays to map disease loci in dogs is emerging. No equivalent of the human HapMap currently exists for different canine breeds, and the LD structure for such breeds is far less understood than for humans. This study is a dedicated large‐scale assessment of the functionalities (LD and SNP tagging performance) of canine genome‐wide SNP arrays in multiple domestic dog breeds. We have used genotype data from 18 breeds as well as wolves and coyotes genotyped by the Illumina 22K canine SNP array and Affymetrix 50K canine SNP array. As expected, high tagging performance was observed with most of the breeds using both Illumina and Affymetrix arrays when multi‐marker tagging was applied. In contrast, however, large differences in population structure, LD coverage and pairwise tagging performance were found between breeds, suggesting that study designs should be carefully assessed for individual breeds before undertaking genome‐wide association studies (GWAS).     

Longevity and resistance to cold stress in cold‐stress selected lines and their controls in Drosophila melanogaster

Thermal environments can influence many fitness‐related traits including life span. Here, we assess whether longevity in Drosophila melanogaster can experimentally evolve as a correlated response to cold‐stress selection, and whether genotype‐by‐temperature and sex‐by‐temperature interactions are significant components of variation in life span. Three replicated S lines were cold‐stress selected and compared with their respective unselected controls (Clines) in the 16^th generation of thermal selection. Cold‐stress resistance exhibited a substantial direct response to selection, and also showed a significant interaction between sex and type of line. Mean longevity exhibited a significant interaction between adult test temperature (14 and 25 °C) and line (with suggestive evidence for increased longevity of S lines when tested at 14 °C), but there was no evidence for increased longevity in S lines at normal temperatures (i.e. 25 °C). Another temperature‐dependent effect was sex‐specific, with males being the longer lived sex at 25 °C but the less long‐lived sex at 14 °C. Additionally, we tested in an exploratory way the relationship between longevity and cold‐stress resistance by also measuring resistance to a prefreezing temperature before and after one generation of longevity selection at 14 °C (selection intensity, i = 1.47 for S lines, and 1.42 for C lines). In this longevity selection, we found that cold‐stress resistance increased by about 6% in S lines and 18% in C lines. However, taken together, the results indicate no simple relationship between longevity and cold‐stress resistance, with genotype‐by‐sex interactions in both traits. Temperature dependent interaction in longevity is apparent between S and C lines, and sex‐specific variation in mean longevity also depends on temperature.     

Three novel quantitative trait loci for skin thickness in swine identified by linkage and genome‐wide association studies

Skin is the largest organ in the pig body and plays a key role in protecting the body against pathogens and excessive water loss. Deciphering the genetic basis of swine skin thickness would enrich our knowledge about the skin. To identify the loci for porcine skin thickness, we first performed a genome scan with 194 microsatellite markers in a White Duroc × Erhualian F₂ intercross. We identified three genome‐wide significant QTL on pig chromosomes (SSC) 4, 7 and 15 using linkage analysis. The most significant QTL was found on SSC7 with a small confidence interval of ~5 cM, explaining 23.9 percent of phenotypic variance. Further, we conducted a genome‐wide association study (GWAS) using Illumina PorcineSNP60 Beadchips for the F₂ pedigree and a population of Chinese Sutai pigs. We confirmed significant QTL in the F₂ pedigree and replicated QTL on SSC15 in Chinese Sutai pigs. A meta‐analysis of GWASs on both populations detected a genomic region associated with skin thickness on SSC4. GWAS results were generally consistent with QTL mapping. Identical‐by‐descent analysis defined QTL on SSC7 in a 683‐kb region harboring an interesting candidate gene: HMGA1. On SSC15, the linkage disequilibrium analysis showed a haplotype block of 2.20 Mb that likely harbors the gene responsible for skin thickness. Our findings provide novel insights into the genetic basis of swine skin thickness, which would benefit further understanding of porcine skin function.     

Specific alleles at immune genes,rather than genome‐wide heterozygosity,are related to immunity and survival in the critically endangered Attwater's prairie‐chicken

The negative effects of inbreeding on fitness are serious concerns for populations of endangered species. Reduced fitness has been associated with lower genome‐wide heterozygosity and immune gene diversity in the wild; however, it is rare that both types of genetic measures are included in the same study. Thus, it is often unclear whether the variation in fitness is due to the general effects of inbreeding, immunity‐related genes or both. Here, we tested whether genome‐wide heterozygosity (20 990 SNPs) and diversity at nine immune genes were better predictors of two measures of fitness (immune response and survival) in the endangered Attwater's prairie‐chicken (Tympanuchus cupido attwateri). We found that postrelease survival of captive‐bred birds was related to alleles of the innate (Toll‐like receptors, TLRs) and adaptive (major histocompatibility complex, MHC) immune systems, but not to genome‐wide heterozygosity. Likewise, we found that the immune response at the time of release was related to TLR and MHC alleles, and not to genome‐wide heterozygosity. Overall, this study demonstrates that immune genes may serve as important genetic markers when monitoring fitness in inbred populations and that in some populations specific functional genes may be better predictors of fitness than genome‐wide heterozygosity.     

Validation of reference genes for quantitative real‐time polymerase chain reaction in Drosophila melanogaster exposed to two chemicals

Drosophila melanogaster is attracted to chemicals produced by fermentation and it is abundantly found in rotten fruits. Considering its habitat, the fruit fly is reported to be tolerant to environmental chemicals. Quantitative real‐time polymerase chain reaction was employed to investigate the expression pattern and physiological function of genes putatively involved in chemical detoxification. In quantitative real‐time polymerase chain reaction assays, normalization of target gene expression with internal reference genes is required. These reference genes should be stably expressed during chemical exposure and in chemical‐free conditions. In this study, therefore, we used two programs (geNorm and BestKeeper) to evaluate the expression stability of five reference genes (nd, rpL18, ef1β, hsp22 and tbp) in female adult flies exposed to various concentrations of methanol and ethyl acetate. Four genes (nd, rpL18, ef1β and tbp) were found to be suitable for use as reference genes in methanol‐treated flies and three genes (ef1β, nd, tbp) were found to be suitable for use as reference genes in ethyl acetate‐treated flies. These results suggested that a combination of two genes among these stably expressed genes can be used for accurate normalization of target gene expression in quantitative real‐time polymerase chain reaction‐based determination of gene expression profiles in D. melanogaster treated with both chemicals.     

A genome‐wide search for local adaptation in a terrestrial‐breeding frog reveals vulnerability to climate change

Terrestrial‐breeding amphibians are likely to be vulnerable to warming and drying climates, as their embryos require consistent moisture for successful development. Adaptation to environmental change will depend on sufficient genetic variation existing within or between connected populations. Here, we use Single Nucleotide Polymorphism (SNP) data to investigate genome‐wide patterns in genetic diversity, gene flow and local adaptation in a terrestrial‐breeding frog (Pseudophryne guentheri) subject to a rapidly drying climate and recent habitat fragmentation. The species was sampled across 12 central and range‐edge populations (192 samples), and strong genetic structure was apparent, as were high inbreeding coefficients. Populations showed differences in genetic diversity, and one population lost significant genetic diversity in a decade. More than 500 SNP loci were putatively under directional selection, and 413 of these loci were correlated with environmental variables such as temperature, rainfall, evaporation and soil moisture. One locus showed homology to a gene involved in the activation of maturation in Xenopus oocytes, which may facilitate rapid development of embryos in drier climates. The low genetic diversity, strong population structuring and presence of local adaptation revealed in this study shows why management strategies such as targeted gene flow may be necessary to assist isolated populations to adapt to future climates.