SEPT12 is reported as a critical gene for spermatogenesis. This gene encodes a testis specific member of Septin proteins, a family of polymerizing GTP-binding proteins. SEPT12 in association with other Septins is an essential annulus component in mature sperm. So, it is hypothesized that genetic alterations of SEPT12 may be concerned in male infertility.Male infertility is a multifactorial disorder, which affects approximately 10% of couples at childbearing age with substantial clinical and social impact. Genetic factors are associated with the susceptibility to spermatogenic impairment in humans. Recently, SEPT12 gene association with idiopathic male infertility in Iranian men.The objective of this research is exploration of new single nucleotide polymorphism G5508A in the SEPT12, using polymerase chain reaction sequencing technique. Fisher exact test was used for statistical analysis and p<0.05 was considered as statistically significant.In this case control study, 67 infertile men and 100 normal controls were analyzed for genetic alterations in the active site coding region of SEPT12 alleles were distributed in three peaks of frequency in both control and diseases groups. Categorization of the alleles into (GG), (GA), (AA) types revealed a significant difference between infertile patients (azoospermic and asthenospermic) and normal controls (p=0.005).Genotype analysis indicated that G5508A polymorphic SEPT12 gene can affect spermatogenesis in men, the opinion needs more investigation in different populations.According to our finding we suggest that G5508A polymorphism in  Subfertility is one of the major clinical, social and economic concern. In up to 55% of couples seeking medical attention, the male partner is diagnosed with spermatogenic failure, defined as one or more semen parameters falling below the world health organization (WHO) cut-off for normozoospermia . In the Spermatogenesis is governed by the parallel and serial actions of thousands of genes, alterations in any of them or their expression may cause male infertility -10. In rRecently, a number of reports have linked altered expression of Septin genes to a range of diseases, including male infertility , 13. SinSEPT12 is dominantly expressed in testis tissue of adults, with an established essential role in annulus structure of mature sperm , while this mRNA was not detectable in men with sterility resulting from inability to produce mature spermatozoa. So, it has been considered that SEPT12 is crucial for the process of spermatogenesis in mammals  who were referred to Royan Instiute, Tehran, Iran, during 2012-2013. Also, 100 normozospermic men with female factor subfertility were analyzed as control . Using pGenomic DNA was extracted from peripheral blood specimens using a QIAamp DNA minikit (Qiagen Germany), according to the instructions provided by the manufacturer.Polymerase Chain Reaction (PCR)-Sequencing analysisSEPT12 including exones 2-3 and their interval intron, and also exon 6 and its exon–intron boundaries were screened by polymerase chain reaction (PCR)-directed sequencing, using the specific primers designed by Primer3 software (Primer3.ut.ee)  .°C for 5 min, followed by 30 cycles of denaturation at 95°C for 45 sec, annealing at 60°C for 45 sec and extension at 72°C for 45 sec. The PCR products were confirmed by running on 1.5% agarose gel, and were applied for sequencing by an ABI 3730XL automated DNA sequencer .To analyze the aforementioned DNA sequences, PCR-Sequencing technique was performed with an initial denaturation at 95Statistical analysisFisher exact test was used to investigate the relationship between three sample groups  and allele frequency. All statistical analysis were performed using SPSS  software. A p<0.05 was considered as statistically significant.SEPT12 gene of the patients with respect to normal controls . Also, the frequency of the normal G allele was significantly higher in the control group compared with the patients groups (p=0.001). However, there was no significant change between the two groups of azoospermic and asthenospermic for this allele (p=0.05). On the other side, the frequency of the A allele in normal group was significantly lower compared with the both patients groups (p=0.001). Again, no significant change was observed between the two patients groups (p=0.05).Sequence analysis data revealed the nucleotide transition G5508A in controls . The patSEPT12 is one of these genes which its lower expression in the testicular biopsies of infertile men is significantly related with azoospermia. In this study, we hypothesized genetic variations of SEPT12 gene may be associated with male infertility caused by spermatogenesis failure.There are several reports introducing novel genes involved in spermatogenesis, in the way that their down-regulation in the testes tissues of infertile men has been identified by high throughput expression analyses . SEPT12 SEPT12 gene, among patients with azoospermia and asthenospermia with respect to control men with normal spermograms. Although the number of analyzed patients is not enough to have a final decision, our preliminary finding observed in Iranian patients suggests that this G>A alteration may play a causative role in disruption of spermatogenesis. According to the recent reports showing that some SEPT12 SNPs may predispose men to spermatogemic failure (The present association study revealed significantly different allele frequency of G5508A in  failure -18, the Today, in vitro fertilization has been established as an efficient technique to resolve infertility in couples with female factors, but it cannot be useful for severe male factors with lacks in spermatogenesis. Although testicular sperm extraction-intracytoplasmic sperm injection is now successfully used for these cases, however, it cannot benefit patients with complete failure in spermatogenesis. So, genetic diagnosis of severe male infertility is a critical subject for assisted reproductive technology.SEPT12 gene may be associated with idiopathic male infertility in Iranian men. However, as this genetic alteration is in the intronic region, the molecular mechanism of its effectiveness is really unknown. Further study is needed to test this synonymous coding polymorphism for potential alteration of splicing between exonic elements.In conclusion, we suggest that the novel G5508A polymorphism in


Tscherskia triton) from Raoyang, Guan and Shunyi counties of the North China Plain. The results showed that (i) high genetic diversity and differentiation exist in three geographic populations with patchy distributions; (ii) gene flow occurs among these three populations with physical barriers of Beijing city and Hutuo River, which potentially restricted the dispersal of the animal; (iii) the gene flow is negatively correlated with the geographic distance, while the genetic distance shows the positive correlation. Our results suggest that the effect of the physical barriers is conditional-dependent, including barrier capacity or individual potentially dispersal ability. Geographic distance also acts as an important factor affecting dispersal for the patchy distributed geographic populations. So, gene flow is effective, even at relatively long distances, in balancing the effect of geographic barrier in this study.Dispersal is a fundamental process in ecology influencing the genetic structure and the viability of populations. Understanding how variable factors influence the dispersal of the population is becoming an important question in animal ecology. To date, geographic distance and geographic barriers are often considered as main factors impacting dispersal, but their effects are variable depending on different conditions. In general, geographic barriers affect more significantly than geographic distance on dispersal. In rapidly expanding populations, however, geographic barriers have less effect on dispersal than geographic distance. The effects of both geographic distance and geographic barriers in low-density populations with patchy distributions are poorly understood. By using a panel of 10 microsatellite loci we investigated the genetic structure of three patchy-distributed populations of the Greater long-tailed hamster ( Population persistence strongly depends on its own evolutionary capacity which in turn relies on the genetic variability. The capacity of genetic variability within and among populations results from many processes involving mutation, dispersal, genetic drift and selection. Dispersal refers to the movement of an organism from one place to another, which plays a fundamental role in population biology and conservation because it influences the genetic structure as well as the persistence of populations It is well known that dispersal is conditional-dependent. There are a number of potential driving forces identified for dispersal including kin competition, inbreeding, resource competition and environmental stochasticity Some previous studies have focused on the effects of several main factors on dispersal, such as population density Maniola jurtina uses a non-random, systematic dispersal strategy and can detect and orient towards habitat from distances of 100–150 m Geographic distance is also an important factor affecting dispersal. As the costs of movement increase with distance, a successful dispersal is often considered to occur when the distance between patches decreases In addition to distance, geographic barriers are also thought to be the important factors affecting dispersal Tscherskia triton) is widely distributed in croplands of North China The Greater long-tailed hamster . All researchers had received appropriate training and affirmed before conducting animal studies.2 asphyxiation immediately. These operations were approved by the Institute of Zoology, Chinese Academy of Sciences. Captured hamsters were numbered, sexed, weighed, dissected, and measured to provide estimates of body size, age, and reproductive condition The Greater long-tailed hamsters were captured using wooden-iron traps in Raoyang, Guan and Shunyi Counties of the North China Plain in autumn, 2000. The wooden-iron traps are box-shaped, with a wooden-side at the bottom and iron sheets with holes at other sides. One small side of the traps could be open or closed, and the other sides are fixed. The wooden-iron trap works by the leverage principle. The trap is 20 cm in length, 12 cm in width and 14 cm in height. The wooden side as bottom is baited with peanuts and equipped with spring device. When the Greater long-tailed hamsters step on the wood plate to eat and the spring set up release, the hamsters will be closed in the trap and then euthanized by COTissues were fixed in 90% ethanol, preserved in formalin solution and kept in the animal ecological Laboratory, Institute of Zoology, Chinese Academy of Sciences for more than 10 yrs. The genomic DNA was extracted from liver tissues using an improved phenol-chloroform extraction method 2, 0.2 mM each dNTP, 1 U of Taq DNA polymerase (Promega), 10 pM forward and reverse primers, and approximately 2 ng of template DNA in 25 µL. Amplifications began with a 5-min denaturing step at 94°C, followed by 30–35 cycles of the following thermal reaction: denaturing at 94°C for 45 s, annealing at 47∼55°C for 45 s, and extending at 72°C for 1 min, with a final extension for 5 min at 72°C. All products were analyzed in an ABI 377 instrument  and the gel analysis was performed by using GENESCAN3.1 (Perkin-Elmer Applied Biosystem).Ten microsatellite loci of the Greater long-tailed hamster  were useR, number of alleles independent of sample size), Shannon's Information index (I), observed (HO) and expected (HE) heterozygosity. FSTAT version 2.9.2 FST value Genetic variation within populations were assessed by using the measures including allelic richness  with Arlequin version 2.000 Geographic distances between sampling sites were calculated based on the approximate center of the sampling areas. Correlations between geographic distances and Nei's standard genetic distance Among 10 microsatellite loci, there are 3 to 11 alleles with a mean of 6.1 alleles per locus . Within Some alleles were more restricted, while others showed wide range of geographic distribution as shown in RA), Shannon's Information index (I), observed (HO) and expected (HE) heterozygosity for the microsatellites in each population are shown in RA, I, Ho, and He were 3.57, 1.93, 0.56 and 0.60 for Raoyang population, 3.31, 1.86, 0.63 and 0.62 for Guan population and 3.47, 1.97, 0.68 and 0.65 for Shunyi population, respectively. The parameters of RA, I, Ho, and He show that genetic diversity exists in Raoyang, Guan and Shunyi populations. No significant difference on the genetic diversity level was detected from the used parameters among the three populations.Estimates of allelic richness  was observed between the populations of Raoyang and Shunyi. The lowest STF value  was observed between the populations of Guan and Shunyi. The STF value  between the populations of Raoyang and Guan was moderate. The STF values of populations separated by the Hutuo River were higher than the ones separated by Beijing city,which indicates that the Hutuo River has higher barrier capacity than Beijing city for the Greater long-tailed hamsters. This leads to the higher genetic difference between the two sides populations of the Hutuo River than those of Beijing city.Three pairwise estimates of F-statistics revealed the significance for ISF = 0.0281 (P = 0.0032), ITF = 0.0437 (P = 0.0053) and STF = 0.0382 (P = 0.0039). Wright (1978) identified the problem of interpreting STF values as an absolute value based on highly polymorphic loci and proposed that a STF<0.05 could indicate a considerable population differentiation STF value suggests that the genetic differentiation exists among three tested Greater long-tailed hamster populations.The AMOVA showed that 71.6% of the variance is explained by within-individual variation, 8.9% by variation between individuals within population, and 19.5% by variation between populations. Although the total genetic variation is mainly from the variation of within-individuals, there was more than twice as much variation between populations as the one between individuals within populations. The overall r = 0.98, P = 0.008), as shown in Genetic and geographic distances among the three examined patchy distributed geographic populations were summarized in r = −0.99, P = 0.017) as shown in Gene flows between the different examined geographic populations were also summarized in Generally, the populations that are isolated from one another by geographic barrier can evolve variable characters in order to adapt to local environments. Genetic variation of a population may lead to the character differentiation resulting from natural selection. To some extent analyzing the genetic structure of populations is very important to understand the population dynamics From our study, we examined the genetic structure of three populations of Greater long-tailed hamster and found the significant genetic differentiation exist among these three patchy-distributed populations, suggesting the geographic distance plays an critical role in the genetic differentiation of these three patchy populations despite geographic barriers act on them to a certain degree. We have identified the correlation among genetic distance, gene flow and geographic distance and found that geographic distance is positively correlated with the genetic distance and is negatively correlated with the gene flow.RA) are 3.566, 3.314, and 3.474, and expected heterozygosities (He) are 0.601, 0.615, and 0.647, respectively for Raoyang, Guan and Shunyi populations. The values of the expected heterozygosity (He) for individual geese ranged from 0.38 to 0.51He) detected in three populations are higher than those of the geese By using 10 microsatellites, we were able to detect comparable values of expected heterozygosity and allelic richness in three patchy distributed geographic populations. Mean allelic richnesses  exist among the three geographic populations. Population differentiation can result from two genetic processes. 1) Populations may be isolated from one another by geographic barriers and therefore gene flow will be reduced among local populations. 2) Population differentiation level may increase with the increased geographic distance because the magnitude of gene flow declines with extended length of the geographic distance.In this study, significant genetic differences were found among three geographic populations. High values of differentiation  indicating no gene flow exists between the populations from two sides of the barriers Ursus arctos) was found to be reduced or sometimes absent between four insular populations when separated by stretches of sea water of a few kilometres, but continuous gene flow between populations was detected when the stretches of sea water were much narrower at approximately 600 m Populations separated by the Hutuo river showed high genetic differentiation than populations on opposite sides of the Beijing city, suggesting that Hutuo river has higher barrier capacity than Beijing city for the Greater long-tailed hamster populations. In research of small mammal species, such as voles and ground beetles, barrier effect of roads to gene flow has clearly been shown Genetic diversity and genetic differentiation exist in the three examined hamster populations. The barriers in this study acting on populations are less restricted than expected. Furthermore, the genetic differentiation is positively correlated with the geographic distance, while the gene flow shows a negative correlation. Geographic distance may act as one of main causes for the genetic differentiation of the patchy distributed hamster populations.Table S1Tscherskia triton).Characterization of the microsatellite loci in Greater long-tailed hamster ((DOC)Click here for additional data file.Table S2Tscherskia triton populations.Alleles and their frequencies for the ten microsatellite markers in the three (DOC)Click here for additional data file.Table S3Tscherskia triton populations at ten microsatellite markers.Genetic diversity indices within three (DOC)Click here for additional data file.Table S4Genotypes for the Greater long-tailed hamsters examined in this study at 10 microsatellite loci.(DOC)Click here for additional data file.


The hypothesis that most cancers are of monoclonal origin is often accepted as a fact in the scientific community. This dogma arose decades ago, primarily from the study of hematopoietic malignancies and sarcomas, which originate as monoclonal tumors. The possible clonal origin of malignant mesothelioma (MM) has not been investigated. Asbestos inhalation induces a chronic inflammatory response at sites of fiber deposition that may lead to malignant transformation after 30-50 years latency. As many mesothelial cells are simultaneously exposed to asbestos fibers and to asbestos-induced inflammation, it may be possible that more than one cell undergoes malignant transformation during the process that gives rise to MM, and result in a polyclonal malignancy.To investigate the clonality patterns of MM, we used the HUMARA (Human Androgen Receptor) assay to examine 16 biopsies from 14 women MM patients. Out of 16 samples, one was non-informative due to skewed Lyonization in its normal adjacent tissue. Fourteen out of the 15 informative samples revealed two electrophoretically distinct methylated HUMARA alleles, the Corrected Allele Ratio (CR) calculated on the allele peak areas indicating polyclonal origin MM.Our results show that MM originate as polyclonal tumors and suggest that the carcinogenic “field effect” of mineral fibers leads to several premalignant clones that give rise to these polyclonal malignancies.The online version of this article (doi:10.1186/s12967-014-0301-3) contains supplementary material, which is available to authorized users. BAP1 gene cause a novel cancer syndrome characterized by a very high incidence of MM, and other malignancies [Malignant mesothelioma (MM) is an aggressive cancer arising from the transformation of the mesothelial lining of the pleura, peritoneum and pericardium. It is a lethal cancer affecting approximately 3,200 individuals each year in the US, most of them die within 1 year from diagnosis . Asbestognancies .MMs show different histology. There are 3 main subtypes: about 50% of MM show an epithelioid morphology and they look like carcinomas, 10% have a spindle cell morphology similar to sarcomas, and about 35% are biphasic, composed of both epithelioid and spindle cells in different proportions. In addition there are less common histological variants . DiagnosHUMARA) locus, is considered the most accurate method to assess clonality [Multistep carcinogenesis is the currently accepted hypothesis to explain genetic diversity in tumors -10. Thislonality . During lonality . Therefolonality ,14.To the best of our knowledge, the clonal origin of MM has never been investigated. However MMs are “assumed” to be monoclonal as most tumors are usually assumed to be. Here we tested the hypothesis that MMs are monoclonal using the HUMARA assay on 16 MM biopsies from 14 female patients. We found that human MMs are polyclonal in origin.Human MM biopsies were collected at the following institutions: Department of Cardiothoracic Surgery, New York University, New York, NY; MedStar Washington Hospital Center, Washington, DC; University of Wisconsin School of Medicine and Public Health Department of Surgery, Madison, WI, and at the Department of Surgery, Penn Presbyterian Medical Center, Philadelphia, PA.All tissue collections were approved by the Institutional Review Boards. Written and informed consent was obtained from all patients included in the study according to the guidelines set forth by the Institutional Review Board and in agreement with the Helsinki Declaration of 1975, as revised in 1983. Specimens were de-identified prior to analysis. Tissues collected during surgical tumor resection were immediately frozen and processed for laser microdissection, DNA extraction and immunohistochemistry. Clinical features are included in Table HUMARA assay . DNAs were then digested with HpaII enzyme as previously described [escribed : brieflyU = A1U/A2U) of the non-HpaII digested sample by the ratio (RD = A1D/A2D). The AR calculation (AR = RU/RD) corrects for any preferential amplification of one allele that might occur if the alleles are different in length. The clonality ratio (CR) is then calculated by dividing the AR of the tumor DNA by the AR calculated for the normal tissue (CR = ART÷ARN). This final calculation corrects for a potential skewed Lyonization [For each sample, the allele intensities were measured as the peak areas of both alleles, which are proportional to the molar amount of DNA. The allele ratios (AR) were first calculated by dividing the ratio , with the less frequent allele detectable when present at a fraction greater than or equal to 1/8 (12.5% of the input copies)  Figure C.Figure HUMARA clonality assessment was conducted on 16, stage I to III, frozen biopsies from 14 cases of female MMs (Table BAP1 mutations were found to be polyclonal. All these MM samples were from women, as the HUMARA assay used here to assess clonality, cannot be used on specimen from men. MMs in women are rare and have a slightly better prognosis than MM in man [M in man , a findiM in man . HoweverM in man . Thus, cFOXP3 [HUMARA assay is unique in its capability of detecting the actual origin of a cell population. Our data indicate that MMs may arise as polyclonal tumors due to concurrent transformation of multiple mesothelial cells (Figure X-chromosome inactivation based assays may detect a seemingly monoclonal tumor when transformation occurs in multiple cells having the same inactivated X chromosome. Although possible, this is a rare event, mainly dependent on the X-inactivation patch size during early developmental Lyonization. This event leads few of the progeny of a single embryonic stem cell to be grouped together in the adult, forming patches. This phenomenon has been described in breast tissues, which display a rather large patch size . AlternaFOXP3 ,23. The FOXP3 . Despites Figure . Recent Our data indicate that MM arise as polyclonal tumors, a finding that has both pathogenesis and clinical implications. For example, MM patients whose tumors are removed at Stage Ia, most often experience recurrence after surgery in spite of apparent successful MM eradication .BAP1 mutations. Therefore, MM may arise from a large pool of independent and mostly covert cancers, as observed in some other malignancies [Our data suggest that, in contrast to current dogma, recurrence may represent novel malignancies, occurring because of the carcinogenic “field effect” of asbestos, its related chronic inflammation, and/or because of ubiquitous genetic predisposition in patients carrying germline gnancies . Accordi


PRDM9 and CPLX1 to have significant effects on genome-wide recombination rate in both sexes. Two novel loci, NEK9 and REC114, were associated with recombination rate in both sexes, whereas three loci, MSH4, SMC3 and CEP55, affected recombination rate in females only. Among the multiple PRDM9 paralogues on the bovine genome, our GWAS of recombination hotspot usage together with linkage analysis identified the PRDM9 paralogue on chromosome 1 to be associated in the U.S. Holstein data. Given the largest sample size ever reported for such studies, our results reveal new insights into the understanding of cattle and mammalian recombination.Meiotic recombination is an essential biological process that generates genetic diversity and ensures proper segregation of chromosomes during meiosis. From a large USDA dairy cattle pedigree with over half a million genotyped animals, we extracted 186,927 three-generation families, identified over 8.5 million maternal and paternal recombination events, and constructed sex-specific recombination maps for 59,309 autosomal SNPs. The recombination map spans for 25.5 Morgans in males and 23.2 Morgans in females, for a total studied region of 2,516 Mb . The male map is 10% longer than the female map and the sex difference is most pronounced in the subtelomeric regions. We identified 1,792 male and 1,885 female putative recombination hotspots, with 720 hotspots shared between sexes. These hotspots encompass 3% of the genome but account for 25% of the genome-wide recombination events in both sexes. During the past forty years, males showed a decreasing trend in recombination rate that coincided with the artificial selection for milk production. Sex-specific GWAS analyses identified  PRDM9 gene to be associated with recombination hotspot usage. These results provide new insights for understanding cattle and mammalian genome recombination.Previous studies on cattle recombination largely focused on males. Using a large Holstein sample from the USDA national database, we studied both male and female recombination by assembling paternal and maternal recombination events in at least three generations. This unique data set provides unprecedented statistical power to study cattle genome recombination in the two sexes: (1) We report for the first time that bulls have more recombination than cows, contrary to the common perception that females have more recombination than males as observed in many mammalian species including humans and mice, and that the sex difference in recombination primarily occurs near the subtelomeric regions of all bovine autosomes; (2) We identify several genes associated with cattle recombination in both females and males, and genes affecting female recombination only; (3) We define putative recombination hotspots and find the cattle  In eukaryotes, meiotic recombination through reciprocal crossovers is an essential biological process that ensures the proper segregation of homologous chromosomes during meiosis –4. Any mCPLX1, RNF212 and PRDM9 [PRDM9 has been found to be associated with the percentage of crossovers in hotspots that is termed as ‘hotspot usage’ [PRDM9 alleles in humans [PRDM9 homologue such as cattle.Considerable variation in recombination rate among individuals has been documented from pedigree-based studies in humans and mice ,10. In hnd PRDM9 –13. Addind PRDM9 –13. Recond PRDM9 –16, and n humans . While aPRDM9 in bulls, but localized the gene to chromosome X. However, PRDM9 has four paralogues in the bovine genome and previous studies have found signals of positive selection associated with the copy on chromosome 1 [PRDM9 paralogue on chromosome 1 is associated with recombination hotspot usage in the U.S. Holstein population.Recombination rate varies considerably between the two sexes in many species, particularly in mammals ,18,19. Imosome 1 . This stCattle are a uniparous species where the population structure generally lacks female recombination information that requires at least three generations. The United States Department of Agriculture (USDA) has received genotypes for over half a million Holstein cattle spanning several generations for genomic selection. This large multi-generational structure overcomes the problem of lacking female recombination information in cattle and provides a unique opportunity to study genome recombination in both females and males with unprecedented statistical power. Utilizing this large sample, the present study seeks to comprehensively survey the sex-specific patterns of meiotic recombination and to understand the genetic basis of individual differences in recombination in males and females. We also aim to generate the first SNP-based recombination maps in the two sexes and to evaluate the trend of meiotic recombination features that might be associated with the long-term artificial selection for dairy production.We extracted a total of 185,917 three-generation families that included one offspring, both parents, and two grandsires per family genotyped by various SNP chips ranging from 3K, 7K, to 770K SNPs from the large Holstein cattle pedigree with over half million genotyped cattle . In eachR = 0.636), which is similar to the results in humans [The recombination map was calculated as the recombination rates between adjacent SNPs based on the AGIL SNP map. Using an EM algorithm , we consn humans . To evalThe sex-specific recombination maps comprised of 59,309 SNP markers for all 29 bovine autosomes, with an average physical distance of 42.4 kb between adjacent SNPs and an average genetic distance of 0.043 cM in males and 0.039 cM in females, respectively. The 59,309 autosomal SNPs covered a total physical length of 2,516 Mb with 986 kb/cM in males and 1,085 kb/cM in females. The physical and genetic lengths of bovine autosomes had strong positive correlations of 0.960 in males and 0.985 in females across the 29 autosomes . Our estCattle male and female recombination rates are unevenly distributed along the genome , consistAlthough our approach has a measure to minimize the effect of genotyping and genome assembly errors by requiring at least three informative markers for a crossover call, we caution that big chunks of genome assembly errors may still lead to spurious recombination hotspots . To filtWe assessed the relationship between recombination rate and chromosomal locations, as recombination rates are known to differ considerably across chromosomal locations, including telomeres and centromeres. After removing the 2-Mb regions at the two ends of each chromosome where the power for identifying crossovers was reduced due to low SNP coverage , we fittAll cattle autosomes are acrocentric with the centromere located at the beginning and the telomere at the end of each chromosome . Males hTo evaluate whether crossover interference contributed to the bi-modal distribution of recombination events along the chromosome , we sepa−7 from the Bonferroni correction.We conducted GWAS analysis of genome-wide recombination rates for 3,224 sires and 53,125 dams separately. We corrected for the effect of SNP number so that the average number of recombination events was the same regardless of the genotyping assay used  Tables. Pfemale = 2.95×10−51; Pmale = 7.34×10−30), was located in the intron of the CPLX1 gene, which was associated with genome-wide recombination rate in humans [Pfemale = 2.62×10−51; Pmale = 1.07×10−16), which was located downstream of PABPN1. A conditional analysis identified four independently associated SNPs at this locus, spanning a 9-Mb window that consisted of several meiosis-related genes, including REC8, REC114, and FMN1 . This SNP was located 10 kb downstream of NEK9 that was related to spindle organization and cell cycle progression during mouse oocyte formation [A total of thirteen loci were identified to have significant effects on recombination rate, four loci on male recombination rate and nine loci on female recombination rate, among which three loci were shared between the two sexes . The thrn humans . Using tand FMN1 . The RECn cattle . The topormation . A condiP-values in females in general . This SNP was also nominally associated in males , exhibiting the same direction of effect in both sexes. Taking into account the difference in power, this association is more likely to be shared between sexes than a female-specific effect. Moreover, this association was 35 kb downstream of the PRDM9 gene, which has been associated with both recombination rate and hotspot usage in the two sexes [Pfemale = 1.53×10−13; Pmale = 6.68×10−5) in the first locus on chromosome 3, showing the same effect direction in both sexes, are less likely to be female-specific.Although the top three associated loci were shared between sexes, differences between sexes were observed among the less significant associations. We observed a trend of smaller  general , indicat general . The femwo sexes ,12. SimiPmale = 1.27×10−7). Among the four female-specific associations, two were inside or near genes closely related to the meiotic pathway. Rs136642773  was located in the intron of MSH4, which is a meiosis-specific MutS homologue that affects crossing over [Pfemale = 1.25×10−7) was upstream of SMC3 that encodes a protein related to meiotic chromosomes and synaptonemal complexes [CEP55  and rs42382307 on the X chromosome .Potential sex-specific associations with recombination rate were found at five loci on chromosomes 3, 26 and X, with one male-specific and four female-specific . SNP rs1ing over ,42. SNP omplexes . The othP-values become smaller from the joint analysis than in the single-marker analysis, for those independent associations in both males and females because of the reduced residual errors [P-values of the single-marker and joint analyses was 1014 for the association at rs137264867 in females.To comprehensively evaluate the associations and estimate their effects, we conducted a joint analysis by including all the significantly associated SNPs in one model as fixed effects . As expel errors . The larPRDM9 and hotspot usage.Based on the 1,792 male and 1,885 female recombination hotspots, we calculated the proportion of recombination events occurring in the hotspots genome-wide, i.e., hotspot usage, for the sire and dam in a three-generation family. To increase accuracy, we only included the high-quality meioses, where the offspring, the parent and the grandsire were genotyped by the 50K SNP chip. We also used the average of multiple measurements of hotspot usage as the phenotype, resulting in a sample size of 1,772 and 12,756 for males and females respectively. We then tested the association between hotspot usage and each of the 310,442 imputed SNPs. To evaluate the effect of different definitions of hotspot, we tested a range of cutoff values, 2, 2.5, 3, 5, and 10 standard deviations, and found that the cutoff value of 2.5 standard deviations had the clearest signal for the association between PRDM9 . This SNP was also associated with genome-wide recombination rate . In females, this SNP was only nominally associated with hotspot usage from the joint analysis, but with an opposite effect , and some of them were potentially sex-specific . We also confirmed the putative role of PRDM9 in the genome-wide recombination rate in both sexes. From GWAS of hotspot usage, we confirmed the cattle PRDM9 gene in both sexes to be the paralogue on chromosome 1 in our population, although the cattle genome encompasses multiple paralogues of PRDM9 and a previous study localized the associated PRDM9 to chromosome X [Our GWAS analyses identified several loci influencing genome-wide recombination rate. Some of these loci had significant influence in both sexes  than those from the original GWAS  at USDA, with one offspring, both parents and two grandsires with SNP genotypes in each family . In eachThe animals in the selected families were genotyped by various genotyping assays , rangingTo study recombination, we included up to 59,309 genome-wide SNPs after quality control filtering and used the USDA-AGIL SNP coordinates that showed a higher quality than the UMD3.1 assembly . PreviouTo compare sex differences on an equal footing, we phased the raw genotypes for the paternal and maternal meioses within a three-generation family without using the possible additional information from multiple offspring , becauseAfter phasing the genotypes of the offspring and parents, we inferred recombination events in the paternal and maternal haplotypes of the offspring by comparing the offspring’s paternal haplotype to the two haplotypes of the sire as well as by comparing the offspring’s maternal haplotype to the two haplotypes of the dam. In the offspring haplotype, a recombination event was defined as a transition from the parent's paternal to maternal haplotype or vice versa. Note that the recombination events defined here were observed crossovers so that the number of observed crossovers could differ from the number of true crossovers when multiple crossover events occurred at the same site. This potential inconsistency between observed and true crossovers typically is addressed by a map function that translates a recombination frequency into a map distance in terms of crossovers. However, the physical distances between two adjacent SNPs were small so that the use of a map function virtually would not make a numerical difference. Therefore, our estimates of crossovers based on recombinants should be close to the true number of underlying crossovers. To further reduce false positives, we required a crossover call to be supported by at least three consecutive informative heterozygous SNPs . In totam to each of the m adjacent SNP intervals in a crossover region, the EM algorithm proceeded in the following iteration steps: 1) M-step: considering a total of n meioses, the overall expected count attributed to a SNP interval divided by n were the maximum likelihood estimate of the probability, and 2) E-step: for a crossover region, the expected count assigned to a SNP interval was estimated as proportional to the current estimate of the probability of crossover for that SNP interval. The M and E steps were iterated until convergence.A recombination event was assigned to a region spanned by two informative SNPs that may not be adjacent to each other. To construct a recombination map, we used an EM-algorithm to calculate the probability of crossing over per meiosis or recombination rate between each pair of consecutive SNPs based on the observed crossover regions . After aWe constructed the male and female recombination maps for 59,309 autosomal SNPs based on >1.8 million paternal and >1.4 million maternal recombination crossovers, which were identified from 70,715 male and 61,616 female meioses that are most informative where the offspring, the parent, and the grandsire were genotyped by 50K SNP chips. Note that some granddams were genotyped by a low density chip or even not genotyped. As described earlier, a correction for the number of SNPs of the granddam was employed in the two sexes separately such that the total number of crossovers after correction was the same regardless of the SNP numbers for granddams. We used the expected number of crossovers per meiosis or recombination rate between adjacent SNPs as the genetic distance in our recombination map, since one crossover event on average corresponds to a genetic distance of 1 Morgan and recombination rate is almost the same as genetic distance for small intervals . AlternaPRDM9 and recombination hotspot usage. For validation purposes, we also defined recombination hotspots via using a standardized recombination rate that was calculated by dividing the original recombination rate between two SNPs by the physical length. After this adjustment of physical lengths, the standard recombination rates vary even less so we used a cutoff value of 0.6 standard deviations to define recombination hotspots.We defined recombination hotspots as the SNP intervals with a recombination rate >2.5 standard deviations from the genome-wide average in males and females separately, because 2.5 standard deviations are highly significant departures from the average recombination in cattle given our large sample sizes in both sexes, consistent with the observations from a recent recombination study in cattle . We testTo ensure the quality of the hotspots identified, we calculated pairwise linkage disequilibrium (LD) statistics between each of the SNPs in or near a recombination hotspot and all other SNPs on the same chromosome using Locusmap . The paiAs expected, the power for identifying recombination events was affected by the number of genotyped SNPs. For male meiosis, the average number of crossovers identified varied from 25.9 to 17.7 depending on the number of measured SNPs of the animals in a three-generation family . OverallTo evaluate the power for identifying crossovers using 50K SNP genotypes, we simulated 50K SNP genotypes for all animals in the real pedigree and used the same phasing and crossover identification procedures as described in previous sections. By defining a positive result as an identified crossover interval overlapping with the true location of a crossover, we calculated the power of crossover identification as the proportion of positive results across all simulated crossover events. As a result, our approach had a power of 97.6% for the three-generation families genotyped by 50K SNP chip, which means that on average only 2.4% crossovers were missed in our recombination map.β = −0.28 and P-value = 0.006; Females: β = −0.11 and P-value = 3.2 × 10−12). To investigate the common pattern across the 29 autosomes, we pooled all the autosomes together using the relative physical position and fitted a smooth spline model for all the data combined , or small minor allele frequency (MAF < 0.001), we retained over 310K genome-wide SNPs in the association studies [Imputed genotypes of 777,962 SNPs on the Illumina BovineHD Genotyping BeadChip (HD) were obtained by running Findhap on measured genotypes with 3K to 50K SNP chips using a reference population of 2,433 animals directly measured with HD SNP chips . After fy = a vector of the phenotype, X = a design matrix of the fixed effects g, including a population mean and the additive effect of the candidate SNP, Z = a design matrix for a random animal effect a, and e = a vector of random residuals. We assume that A is the genomic relationship matrix and R is a diagonal matrix with the ith diagonal element equal to 1/w, where w is the number of phenotypic measurements for the ith animal.We tested for association between each SNP and a phenotype using a linear mixed model with variable residual variances that are inversely proportional to the number of repeated measures of the phenotype, i.e., residual variance is smaller for individuals with more measurements of the phenotype. The model equation in matrix notation isP-values for both recombination rate and hotspot usage in this study Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S7 Fig(DOCX)Click here for additional data file.S8 Fig(DOCX)Click here for additional data file.S9 Fig(DOCX)Click here for additional data file.S10 Fig(DOCX)Click here for additional data file.S11 Fig(DOCX)Click here for additional data file.S12 Fig(DOCX)Click here for additional data file.S13 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


The successful application of Augmentative Biological Control (ABC) to control pest fruit flies (Diptera: Tephritidae) confronts two fundamental requirements: (1) the establishment of efficient mass rearing procedures for the species to be released, and (2) the development of methodologies for the packing and release of parasitoids that permit a uniform distribution and their optimal field performance under an area-wide approach. Parasitoid distributions have been performed by ground and by air with moderate results; both options face challenges that remain to be addressed. Different devices and strategies have been used for these purposes, including paper bags and the chilled adult technique, both of which are commonly used when releasing sterile flies. However, insect parasitoids have morphological and behavioral characteristics that render the application of such methodologies suboptimal. In this paper, we discuss an alternate strategy for the augmentative release of parasitoids and describe packing conditions that favor the rearing and emergence of adult parasitoids for increased field performance. We conclude that the use of ABC, including the packaging of parasitoids, requires ongoing development to ensure that this technology remains a viable and effective control technique for pest fruit flies.  Augmentative Biological Control (ABC) is defined as the strategy by which "a very large number of natural enemies are reared and released in critical periods for the suppression of pest populations in the short term" . Some auMany integrated pest management (IPM) programs are focused on a sustainable approach to control pests, in order to mitigate the adverse effects commonly associated with the indiscriminate use of pesticides . Severali.e., a higher intrinsic rate of growth) and dispersal abilities than their parasitoids ) control . In Meximergence . It was Fopius arisanus (Sonan) (Hymenoptera: Braconidae), an egg parasitoid that successfully attacks Bactrocera spp. and C. capitata in Hawaii [For parasitoid species that require long pre-oviposition periods, the design of an appropriate packing container could be more elusive. For instance, n Hawaii , requiren Hawaii ,23. The n Hawaii ,25. i.e., remains of bags and pupae) [The chilled adult release method for sterile fruit flies is the most common release system because it favors a more homogenous distribution in the field and avoids the dissemination of undesirable residue (d pupae) ,27. Insed pupae) ) for thed pupae) ,27. The d pupae) . This syC. capitata on coffee plantations. Paper bags containing chilled adults were dropped from a single engine airplane at an height of ~ 100 km and a rate of 25 bags/min. The second application utilized the Auger Sterile Release Machine to release the chilled adults from an aircraft. In this study the authors collected the falling adults in specific release zones on the ground where eight technicians spaced 25m apart aspirated released parasitoids for subsequent quality control bioassays, but they failed to find evidence of damage or mortality on the released insects [et al. [D. longicaudata, D. tryoni and Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae).However, the utility of the chilled adult technique for the release of fruit fly parasitoids is undetermined because little is known about the efficiency of these methods and their effects on the performance of released parasitoids. The first report evaluating the effect of the chilling system on parasitoids was in 2000 under two alternative scenarios . The fir insects . In a co [et al. , the chiD. longicaudata are highly sensitive to packing conditions and manipulation during the chilling process. The authors evaluated three packing densities  in two different types of PARC: 1) A standard PARC , and 2) a modified PARC with larger lateral windows (40cm × 25cm) to increase ventilation. They also evaluated three different packing densities  per sieve of 66cm × 72cm × 4 cm arranged in “Guatemala” eclosion towers  and the chilling process exerted a significant and negative influence on longevity (with water and food) and flight percentage but not on fecundity. This work also found that the packing condition and especially the chilling process [A more recent study  has showers  the stress experienced by the insects under high packing densities, as has been noted for fruit flies , and (2)In Mexico, D. longicaudata is mass reared on third-instar (8-day-old) A. ludens larvae, previously irradiated at 45 Gy to prevent the emergence of adult flies from any unparasitized pupae . Two day2, which can hold approximately 6,000–6,500 adult parasitoids [A modified circular plastic container  with two windows, one opposite the other, each measuring 35 cm x 11 cm, to maintain ventilation inside the container was developed to release parasitoids terrestrially a. The wiasitoids . Dietary requirements of eclosed adult parasitoids consist of a mixture of honey and tissue paper (17g of honey: 1g shredded paper); which has proven to be an excellent food . Both in2 [Parasitoids are packaged as pupae inside of the plastic container. A packing density of 10,000 pupae per bucket produces approximately 6,500 emergent adults, with a density of approximately 1 adult/2cm2 . The honAdults are reared in a growth room at 25 ± 1 °C and 70 ± 5% RH and a light-dark photoperiod of 12:12 h for optimal emergence. Adult emergence takes on average three days; males emerge first, and two days later, females begin to emerge. To homogenize the sexual maturity of insects, the release containers are moved to a second room at 22 ± 1 °C (same photoperiod) to facilitate copulation as it is favorable to release copulated adults . The fieTo assess the quality of eclosed adults four parameters are evaluated: adult emergence, sex ratio, longevity  without water and food and flight see ,41,42). ,42. 41,4Releases are based on preliminary technical plans in which the periods and densities of release are defined according to specific conditions, including fruit phenology, the size of the release area and the host trees present. Geographical Positional Satellite (GPS) services are used to precisely locate the release areas. Containers holding eclosed parasitoids are placed in air-conditioned vehicles to move the parasitoids to the target area. This is performed in the early morning (6:00–8:00 h) to provide the best conditions under which to release the parasitoids. Once in the release area, central points are selected to open the containers and permit the escape of parasitoids.As indicated previously, releases are focused near commercial orchards, in marginal areas  with high densities of alternate host fruits, which are considered “hot points” for fly populations ,16. Relei.e., adequate roads to reach the target zones), size and spatial distribution of releasing areas. In the Northwest of Mexico, terrestrial releases have been successful [The release of parasitoids using the road transportation release strategy detailed above could be a viable option, depending on several local circumstances such as topography, infrastructure (ccessful  because ccessful . HoweverAugmentative biological control is considered to be a sound strategy for fruit fly control under an area-wide basis ,7,16,47.


Plants  were checked three times per season for ant presence. Results showed that 21 species (29%) and 266 individuals (35%) possessed EFNs. These plants attracted 38 ant species . In the rainy season, plants with EFNs had higher ant abundance/richness than plants without EFNs, but in the dry season, EFN presence did not influence ant visitation. Plant phenology affected ant richness and abundance in different ways: plants with young leaves possessed higher ant richness in the rainy season, but in the dry season ant abundance was higher on plants possessing old leaves or flowers. The species composition of plant-associated ant communities, however, did not differ between plants with and without EFNs in either season. These findings suggest that the effect of EFN presence on a community of plant-visiting ants is context dependent, being conditioned to seasonal variation.Plants bearing extrafloral nectaries (EFNs) are common in the Brazilian cerrado savanna, where climatic conditions having marked seasonality influence arboreal ant fauna organization. These ant-plant interactions have rarely been studied at community level. Here, we tested whether: 1) EFN-bearing plants are more visited by ants than EFN-lacking plants; 2) ant visitation is higher in the rainy season than in dry season; 3) plants producing young leaves are more visited than those lacking young leaves in the rainy season; 4) during the dry season, plants with old leaves and flowers are more visited than plants with young leaves and bare of leaves or flowers; 5) the composition of visiting ant fauna differs between plants with and without EFNs. Field work was done in a cerrado reserve near Uberlândia, MG State, Brazil, along ten transects (total area 3,000 m Interspecific interactions are considered important processes that can influence species variation and adaptation  as well The presence of renewable and predictable food sources on the vegetation favors its use as substrate for foraging by ants and explains the high frequency of interactions observed in these systems , 9, 13. The availability of highly energetic liquid resources on the vegetation, such as extrafloral nectar, can influence ant survival  and commStudies in Neotropical regions have suggested that changes in associations between ants and plants with EFNs can be related to the existence of climatic conditions having striking seasonality , 22. ThuTo understand the role and adaptive significance of EFN-mediated ant-plant associations in a community, it is necessary to investigate the distribution of plants with EFNs in the flora and its visiting ant fauna . In the In the cerrado vegetation, new leaves are very abundant and possess more active EFNs during the rainy season, while in the dry season leaves with active EFNs are not common, as well as the volume of extrafloral nectar produced is reduced –33. A reIn the present study, we evaluated whether EFN-plants are important to structure the visiting ant community on the local flora and how these interactions vary over time. The main objectives were to verify whether there are seasonal differences in terms of richness and abundance of visiting ants, whether phenological factors affect the presence of these visitors and to compare the composition of ant fauna between plants with and without EFNs. The following hypotheses were tested: 1) EFN-bearing plants are more visited than EFN-lacking plants, because sugar sources are vital to ant nutrition and colony survivorship; 2) Ant visitation on plants with EFNs is higher during the rainy season, because there are more plants offering extrafloral nectar during this period than in the dry season; 3) EFN-bearing plants with young leaves are more visited than EFN-lacking plants with young leaves during the rainy season, because extrafloral nectar is a valuable resource to ants, and plants could be more vulnerable to herbivores during this period; 4) in the dry season, plants with remaining leaves or flowers are more visited than plants with young leaves or bare of leaves and flowers, as a shortage of extrafloral nectar increases the importance of other resources to ants; 5) the composition of visiting ant fauna differs between plants with and without EFNs, because ant species that depend on extrafloral nectar would tend to visit plants containing this resource more frequently.strictu sensu, consisting of a woody layer of trees and large shrubs 2–8 m tall, and a ground layer composed of grasses, herbs and small shrubs. Köppen [Field work was conducted between August 2010 and July 2011 (90 days and 600 hours of sampling efforts) in a private natural savanna reserve  in Uberlândia, Minas Gerais State, southeastern Brazil. The Biology Institute of Universidade Federal de Uberlândia has a memorandum of understanding with CCPIU, an agreement between Mr. Nilson Dias, head of CCPIU, and Dr. Kleber Del-Claro, director of Biology Institute, that enables ecological studies in the area. The vegetation is dominated by cerrado . Köppen  classifistrictu sensu area, shrubs and trees 1–5 m tall were selected randomly along five transects of 50 x 4 m and five transects of 100 x 4 m, apart from each other by at least 50 m. For this selection, transects were divided into plots of 1 m2, resulting in 200 or 400 plots, depending on transect size. The plots on each transect were numbered, and 50 or 100 plots per transect, totaling 750, were chosen by drawing pieces of numbered paper. In the field, one plant was marked in each plot, but there were cases in which two plants were marked because stems were very close to each other. This procedure added 12 plants, totaling 762 studied plants.In a cerrado To record abundance and richness of visiting ants, each plant was sampled once in the morning 7h30–10h30), once in the afternoon (13h30–16h30) and once at night (traps were kept over the course of 14 hours from 17h00 until 7h00), to ensure a better representation of ant fauna. Sampling was carried out once per season for each plant. Samplings in the morning, afternoon and night for plants of the same transect were carried out within a maximum of three days. This procedure lasted 45 days and 300 hours per season and was designed for detecting differentiation in ant visitation between the seasons. Samplings were made avoiding cloudy days or days with precipitation both in rainy  and dry seasons . Air temperature across days varied between 25–35°C in rainy season and between 20–30°C in dry season. Additionally, phenological state of the plants was registered in both seasons by recording whether plants had old leaves, young leaves, flowers  and/or fruits [–10h30, o®), that were fixed with plated pins (size #29) around the stem at 30 cm above the ground—the adhesive traps stayed on the plants for 14 hours. Plants possessing girth up to 10 cm, measured at 30 cm the ground, received traps of 10 x 7 cm, while plants possessing girth between 10 and 20 cm received traps of 20 x 7 cm. For plants possessing girth greater than 20 cm, the two kinds of trap were used together.Ant sampling during the morning and afternoon periods was conducted by direct observation, and each plant was observed for five minutes. All ants observed on the plants, in any position 30 cm above ground, were registered through instantaneous counting. To avoid double counting of co-specific individuals, plant’s parts were observed separately and ants were counted as they were seen (‘snapshot’ ). Ant saAt least one individual from each ant species observed by plant was collected and placed in 70% ethanol. Identification of ant species was carried out by comparing the collected individuals with those specimens identified at the Museu de Zoologia da Universidade de São Paulo. Voucher specimens were deposited at the Museu da Biodiversidade do Cerrado at the Universidade Federal de Uberlândia. Identification of plant species was mostly carried out in the field as most plants were in a vegetative state. Voucher specimens were deposited at the Herbarium Uberlandense at Universidade Federal de Uberlândia.phia package [To determine whether ant visitation levels varied between plants with or without EFNs, between seasons and according to plant phenological factors, we constructed generalized linear mixed models (GLMMs). Sampling transect and plant species were factors grouping and reducing independence of our sampling units , and hence were included as random variables. Because we aimed to estimate total ant activity on the plant (and not to focus on any particular period), ant visitation levels at the scale of individual plants were measured as the total number of individuals and ant species observed per plant over a full day. Although differences in sampling methods between day and night may affect absolute values of each period, this effect is shared for all categories of our predictive variables. Separated models were constructed for ant abundance and richness per plant, which were treated as response variables. In these models, the effects of sampling season and presence of EFNs were used as fixed variables. In both models we also included the phenological factors  as fixed variables modeling either ant abundance or richness. Given that the response variable was a count, and to avoid overdispersion, we fitted models with a negative binomial distribution correcting the standard errors with the dispersion parameter. We used a Laplace approximation to estimate model parameters. The effects of the fixed variables and their interactions were evaluated by comparing concurrent models  using likelihood ratio tests. This process was repeated until the minimal adequate model was obtained , 38. Con package . All ana package .To verify whether composition of the visiting ant fauna differs between EFN-bearing plants and EFN-lacking plants in both seasons, we used non-metric multidimensional scaling (NMDS), followed by an analysis of similarities (ANOSIM) . Ant comCamponotus crassus, Cephalotes pusillus and Pseudomyrmex gracilis, respectively  were bare of leaves . In rainX2 = 28.34; P < 0.001; X2 = 5.35; P = 0.021; X2 < 0.8; P > 0.4; X2 = 10.49; P = 0.001; X2 = 2.57; P = 0.11). A similar pattern was found for plants with flowers, which were more visited during the dry season . Composition of visiting ant fauna did not differ between plants with and without EFNs (P = 0.36) or dry season .Ant community was significantly affected by the interaction between seasonality and presence of EFNs: EFN-plants had higher ant richness (out EFNs  in the rDiospyros burchelli (Ebenaceae), a species without EFNs, was considerably visited by ants due to aggregations of an unidentified Aleyrodidae (Sternorrhyncha) species found on all D. burchelli plants (= 17). In the dry season, fewer plants possessed the aggregations. Due to the scarcity of trophobionts, comparisons of ant-trophobiont interactions between plants with and without EFNs were not possible.There were few plants (< 30) possessing sap-sucking hemipterans (trophobiont insects) during the rainy season, however Presence and activity of EFNs, among other factors, would be decisive in the capacity of myrmecophilic plants to attract ants , 43, 44.Camarota and co-workers  also obsAlso, Schoereder and co-workers  studied Q. multiflora, Ouratea hexasperma, O. spectabilis and Caryocar brasiliense were the plant species with the highest occurrences, contrasting to Q. grandiflora, Q. parviflora, Stryphnodendron adstringens and Lafoensia pacari, which stood out in the registers of that study.The divergence between our results and those of Schoereder and co-workers  may haveCamponotus, Cephalotes, Pseudomyrmex and Azteca were represented among the most common ants on plants, especially on those possessing EFNs, as seen in other tropical regions and cerrado areas [The identity of interacting species is relevant to explain the conditional outcomes in ant-plant systems , 54–56. do areas , 57–59. do areas ). Therefsensu [There are evidences that the outcomes of ant-plant-herbivore interactions are strongly influenced by plant phenology , 32. In sensu ) to explsensu , 63.We observed that EFN presence only influences ant visitation on plants during the rainy season, but not during the dry season. Based on this, we can assume that, in the rainy season, when extrafloral nectar is abundant on vegetation, the consumption of this resource would be greater due to its greater availability over this period. On the contrary, when extrafloral nectar is produced in low quantity or quality (e.g. during the dry season), ants are obligated to search for other alternatives sources of food and water on vegetation , 26, 29.Records of plants with young leaves being visited often by ants during the rainy season are common for many seasonal tropical ecosystems , 29, 32,Considering that some plants become bare of leaves and few plants have young leaves during the dry period, it can be inferred that plants remaining with old leaves and possessing flowers or floral buds may increase in importance, because they represent sources of additional food items, such as prey herbivorous insects , floral Although we did not investigate flower visitation by ants in this study, our results suggest that it is possible for floral tissue or nectar act as an attractive resource, especially in contexts of reduction/absence of extrafloral nectar. This suggestion is based on our finding that during the dry season ants tended to visit plants with flowers instead of those without flowers. Besides this, ants were seen several times feeding on flowers during the sampling.Studies about the mechanisms of nectar production and regulation of its secretion are scarce  and neceCaryocar brasiliense, a common cerrado species, as a host plant showed that the leaf phenology registered in the first sampling was similar to the second one carried out approximately ten years later [Although the present study was performed in only one year, it is reasonable to expect that the observed patterns may be similar across years, considering that the periodicity of leaf emergence and fall is well identified for cerrado plant species , anrs later , 33.It is known that ants prefer nectar rich in amino acids , showingSeveral possibilities could explain the resemblance in the composition of ant fauna between plants with and without EFNs at cerrado vegetation. Firstly, connections between plants (bearing and lacking EFNs) favored by overlaps of crowns that bring together trunks and branches of different plants (which is very common at cerrado), would promote a shared visiting fauna. This explanation is supported by the study of Powell and co-workers , carriedWe observed very few aggregations of trophobiont insects on plants during the rainy season (and even less in the dry season), thus, comparisons of ant-trophobiont interactions between plants with and without EFNs were not possible. This may suggest that trophobionts have low influence on the studied community. However, previous studies showed that the honeydew produced by trophobionts has a role in structuring the visiting ant community of cerrado plants , 81. It According to Díaz-Castelazo and co-workers , the facWe conclude that ant presence varies on cerrado vegetation through time as a consequence of changes in the availability of resources across seasons. However, these changes are not restricted to differential production of extrafloral nectar but also to variations in the phenological conditions of plants. Chamberlain and Holland  argued tS1 Table1 Total abund = Total abundance of ants per season; 2 (% on EFN) = how much of the total ant abundance was observed on EFN-bearing plants; 3 Mean ± sd (n obs) = mean number ± standard-deviation of ants per plant when ant species was observed (number of observations); 4 Period: (M) morning, (A) afternoon, (N) night.(DOCX)Click here for additional data file.S2 Table1 According to [2 N indiv = number of plant individuals; 3 Ant abundance per plant = mean ± standard-deviation of the total number observed once in all periods  of each season.rding to , 16; 2 N(DOCX)Click here for additional data file.


Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), which has a strong dispersal capacity and has had a significant impact on several cropping systems, including peach (Prunus persica (L.)). Management of H. halys has relied on intensive insecticide use, and thus a better understanding of its dispersal behavior may assist in developing improved management strategies. In order to investigate H. halys movement and distribution patterns within a peach orchard we applied ecologically safe, food protein markers to the trees along the orchard border  and to the trees within the orchard interior (bovine casein in the form of cow’s milk). We used enzyme-linked immunosorbent assays (ELISA) to assess whether collected H. halys were “marked” with either of the two protein markers, revealing where in the orchard the bugs had visited. From the density data we determined that H. halys is a perimeter-driven pest in peaches, with a significantly higher density of bugs collected along the orchard border. Interestingly, this trend is primarily driven by the distribution of male bugs. The protein marking data revealed that a small proportion of male H. halys move equally between the orchard border and interior, while a small proportion of females move predominately to the border after visiting the interior. The verification of a strong edge-effect, although potentially sex-specific, implies that H. halys displays a dispersal behavior that may also be exploited for management, which may help growers more efficiently and more effectively manage H. halys.In this study we focus on the invasive brown marmorated stink bug,  Dispersal, as defined by an individual’s movement in space to promote gene flow , is a bein situ is an approach that minimally impacts natural movement and dispersal over space and time. As a method to investigate insect dispersal and movement patterns, previous studies have relied on immunomarking  (Diptera: Tephritidae), as adults disperse into apple orchards from the surrounding natural habitat (Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae)  (Hemiptera: Pentatomidae)   and browtomidae) . Feeding borders , suggestHalyomorpha halys has more than a hundred reported host plants, including many economically important crops species, such as peach (Prunus persica (L.)), apple , and soybean Glycine max L. Merrill  from mid-late May through harvest  at the Rutgers Agriculture Research and Extension Center in Bridgeton, NJ. A forested edge on the North, soybeans on the East, an apple orchard on the South, and a mixed varietal peach orchard on the West bordered the research orchard.®, Michael Foods, Inc., Minnetonka, MN) and a 20% milk solution , both diluted in tap water  at a rate of 935 L/ha. The egg white solution was applied to only the edge trees (“border”) along the orchard perimeter (76 trees), whereas the milk solution was applied to the orchard “interior” (216 trees), leaving a two tree row buffer zone between the “border” and “interior” application areas .H. halys within the orchard was conducted by sampling for stink bug adults at 16 sites  within the orchard , sex was recorded and then the individual was placed in a 1.5 ml microcentrifuge tube and frozen for later analyses by the ELISAs described below. Stink bugs were sampled 1, 3, 5, and 7 days after the proteins were applied to the orchard. Additionally, stink bugs were collected from other orchards prior to protein application in order to be used as negative controls.Identification of the distribution and location of  orchard . The eigIn order to assess the uniformity of protein application to the trees, we collected leaves from the 16 insect sampling sites sample within the orchard. After the protein solution was dry and again 4–6 days later, three leaves from each of three trees were randomly collected and combined in individual plastic bags for each sampling sites. In the laboratory, a 7-mm-diameter leaf disc was removed with a cork borer (cleaned with 70% ethanol after each use) from one of the randomly chosen leaves from each of the 16 sampling sites. Leaf discs were placed in individual 1.5 ml microcentrifuge tubes and frozen for later analyses by the ELISAs described below. Negative controls for leaves were collected following the same protocols prior to protein application to the orchard.Separate immunoassays were performed as indirect enzyme-linked immunosorbent assays (ELISA), following slightly modified methods from One ml of the extraction buffer solution, tris-buffered saline  plus 0.3 g/liter EDTA, was added to each sample tube, and stink bugs and leaf discs, including negative control samples, were soaked for 3 min and then discarded. An 80 µl aliquot of each sample was transferred via pipette into individual wells of a 96-well microplate . Each 96 well microplate (8 × 12 wells) was laid out as follows: first column (eight wells) was extraction buffer only, second column was negative control samples, columns three through ten were the samples to be tested, column eleven was deionized water, and column twelve was the positive control samples (5% egg white or 20% milk solution). Samples were incubated at 37 °C for 2 h on an orbital plate shaker  and the contents of each well were then discarded.2SO4  was added to stop the reaction. The optical density (OD) for each sample was measured with a BioTek Synergy™ 4 microplate reader  at 450 nm, using 490 nm as the reference standard. All samples were scored positive for the presence of the protein marker if the ELISA OD reading was three standard deviations greater than the mean negative control result  plus 0.09% Triton-X100  (PBST). Then 300 µl/well of blocker solution was added to the microplates, which was composed of PBS plus 1,300 ppm Silwet L-77  plus 20% bovine serum  for the egg white assays or 10% ethanolamine  for the milk assays. After blocking for 1 h, the microplates were washed twice with 300 µl/well of the PBST solution, and 80 µl/well of the diluted primary antibodies were added. The primary antibodies were diluted at a ratio of 1:6,000 for the egg white assay and 1:1,000 for the milk assay in a solution of PBS plus 1,300 ppm Silwet L-77 and 20% bovine serum. The secondary antibodies were diluted in the same solution at a ratio of 1:28,000 for the egg white assay and 1:20,000 for the milk assay . The prl result .t-test.The number of marked leaves for each protein (egg white and milk) were pooled separately for each location (border and interior) and replicated by sample period. The percentages of leaf samples per location that were marked with only egg white, only milk, or both proteins were compared separately against zero using a one sample H. halys during the seven days after protein application, the total densities of collected H. halys were pooled separately for each orchard location, averaged across sampling periods, and compared between the two locations for each of the four sampling days  using the nonparametric Kruskal–Wallis test with chi-square approximation. Furthermore, in order to review the longevity of the egg and milk proteins, we plotted the mean percentages of all bugs marked with egg only, milk only, or both proteins, averaged across sampling periods for each of the four sampling days.Adult densities were compared between orchard border and interior locations using a generalized linear mixed model (GLMM). The densities of bugs were pooled for each of the 16 sample sites for each of the sampling periods . The single fixed factor was sample location blocked by sampling period as the random factor, with a Poisson distribution and log link function. This was repeated to compare density of bugs collected between each location for each sex. The total number of females and males collected were pooled separately for the entire season for each of the 16 sampling sites and the means were compared using the nonparametric Kruskal–Wallis test blocking by sampling period with chi-square approximation, after data did not meet assumptions of normality. Additionally, to assess the distribution and movement of all collected The densities of positively marked stink bugs collected per sampling site were compared between crop border and interior locations using a GLMM. Bugs marked with either egg white only, milk only, or both proteins were pooled separately for each sampling site and sampling period. The single fixed factor was sample location (border or interior) blocked by sampling period as a random factor, with a Poisson distribution and log link function. The densities of positively marked stink bugs were summed for each location/sample period to calculate the percentage of marked bugs along the border and within the interior of the orchard. We compared the percentages (arcsine transformed) of positively marked specimens between crop border and interior locations using a one-way analysis of variance (ANOVA), blocked by sampling period as the random factor. This was repeated to compare percentage of marked bugs collected between each location, separately for each sex.H. halys adults were assumed to be positively marked with the appropriate protein if collected within the respective orchard location regardless of ELISA results. For example, if a specimen was collected along the crop border, but tested only positive for milk protein, that stink bug was corrected to be marked as “both” because the border was treated with egg white protein. The corrected percentages of positively marked stink bugs between crop border and interior locations were then compared using an ANOVA as described above. This was repeated separately for males and females. Statistical analyses were performed with SPSS v.20.0 .As all the collected bugs did not test positive for proteins through the ELISA analysis, additional statistical analyses were performed on “corrected” percentages. To better represent movement between border and interior, The ELISA analysis of the 64 leaf samples collected from each orchard location (border and interior) revealed that the majority of the leaves along the orchard border and within the orchard interior were successfully marked with their respective protein marker . MarkingH. halys were collected across all sampling sites in the peach orchard, with 140 collected along the crop border and 81 from the interior. Over the entire season there were significantly more stink bugs collected along the orchard border than the orchard interior . Of the H. halys adults collected, there was no difference in the average abundance (±SEM) of females (1.51 ± 0.27) compared to males (1.94 ± 0.37) . However, the distribution of H. halys within a peach orchard may be influenced by the location of male bugs. Female H. halys were evenly distributed between the border and interior, with no significant difference in the density of female bugs collected between locations , whereas significantly more males were collected along the orchard border compared to the interior .A total of 221 adult H. halys revealed that after the first day of sampling there were significantly more bugs collected along the orchard border than within the interior . Even though an average of 12 bugs were removed from each location during the first day of sampling, we were able to continue collecting bugs the following days, suggesting that H. halys continually colonizes peach orchards. The abundance of bugs collected by the third day dropped for both locations with no significant difference between the two . By the fifth and seventh days, there were significantly higher numbers of bugs collected along the crop border . Additionally, even though percentages of positively marked H. halys fluctuated over the seven day period, the egg and milk proteins were persistent enough in the orchard that we were able to consistently collect bugs marked with either egg only, milk only, or both proteins across the seven day period , 32 with milk only (interior), and 21 with both protein markers. There was a significant difference in the mean density (±SEM) of stink bugs marked with egg white only per sampling site along the border (2.5 ± 0.7) compared with those collected within the interior . Although overall fewer bugs were marked with only milk, significantly more were marked with milk per sampling site that were collected from the interior (0.72 ± 0.22) than from along the edge . There were more stink bugs marked with both proteins collected per sampling site along the border (0.5 ± 0.16) compared with those collected within the interior (0.16 ± 0.08), but this difference was not statistically significant .From the 221 adult H. halys, we found that a total of 63.3% were successfully marked. As with the density of all stink bugs collected, a significantly higher percentage of H. halys collected along the orchard border were marked with only egg white proteins compared to those collected within the interior . We studied within crop dispersal to investigate how an insect species may utilize different aspects of a monoculture. An understanding of this behavior has implications for density-dependent interactions, but can also be exploited for management. Through this approach we were able to determine that ld crops , ornamenld crops , and vinld crops , and is ld crops . The per borders .H. halys would be more likely to disperse into the interior of an resource-filled orchard. One explanation may be the preference-performance hypothesis, where females will maximize their fitness by ovipositing eggs on hosts in which their offspring will perform the best  with both proteins, implying that there is significant movement within the orchard. Identifying the bugs marked with both proteins revealed that female H. halys appear to move more readily from the orchard interior to the orchard border with over double the percentage (corrected) of females marked with both proteins collected along the border. Conversely, the density of male stink bugs were nearly double along the crop border, but the percentages (corrected) of males marked with both were nearly identical in both locations. This supports previous demonstrations that H. halys has a strong dispersal capacity (Although capacity .H. halys in emerging as a key pest of many annual and perennial crops in the mid-Atlantic region of the United States. Currently, many peach growers rely on either repeated 10–14-day whole block or 7-day alternate-row-middle application of insecticides to manage H. halys, which has disrupted previous pest management programs (H. halys management in soybean (C. nenuphar (H. halys management while reducing insecticide inputs (The strong capacity for dispersal and its polyphagous behavior has aided programs . The neeprograms . Similar soybean  and to onenuphar , exploite inputs .H. halys adults in an agricultural landscape will help growers more efficiently and more effectively manage H. halys through techniques that exploit insect behavior, such as border spray applications that will reduce overall insecticide use and help preserve beneficial insects in the agro-ecosystem. These results will be used to support further research on the effectiveness of the systems-level approach exploiting the perimeter-driven behavior of H. halys and to better understand the mechanisms behind the border spray approach and how it could be further optimized. Further testing, utilizing a more appropriate sampling method, a higher concentration of milk protein solution, and investigating female reproductive status is needed.Understanding the movement and distribution patterns of 10.7717/peerj.1997/supp-1Data S1Halyomorpha halys and peach leaves collected to assess the movement and distribution of the bugs within the peach orchard.The raw data for the protein marking of adult Click here for additional data file.10.7717/peerj.1997/supp-2Figure S1Orchard layout and sampling map, illustrating locations of protein marker application and bug collection sites. Satellite image source: 39°30′57.64″N and 75°12′1.77″W; Google Earth, 21/06/2015. Accessed 22/03/2016.Click here for additional data file.