Phellinus noxius (Corner) Cunningham has become a major tree disease in Taiwan. This fungal pathogen can infect more than 200 hardwood and softwood tree species, causing gradual to fast decline of the trees. For effective control, we must determine how the pathogen is disseminated and how the new infection center of brown root rot is established. We performed Illumina sequencing and de novo assembly of a single basidiospore isolate Daxi42 and obtained a draft genome of ~40 Mb. By comparing the 12,217 simple sequence repeat (SSR) regions in Daxi42 with the low-coverage Illumina sequencing data for four additional P. noxius isolates, we identified 154 SSR regions with potential polymorphisms. A set of 13 polymorphic SSR markers were then developed and used to analyze 329 P. noxius isolates collected from 73 tree species from urban/agricultural areas in 14 cities/counties all around Taiwan from 1989 to 2012. The results revealed a high proportion (~98%) of distinct multilocus genotypes (MLGs) and that none of the 329 isolates were genome-wide homozygous, which supports a possible predominant outcrossing reproductive mode in P. noxius. The diverse MLGs exist as discrete patches, so brown root rot was most likely caused by multiple clones rather than a single predominant strain. The isolates collected from diseased trees near each other tend to have similar genotype(s), which indicates that P. noxius may spread to adjacent trees via root-to-root contact. Analyses based on Bayesian clustering, FST statistics, analysis of molecular variance, and isolation by distance all suggest a low degree of population differentiation and little to no barrier to gene flow throughout the P. noxius population in Taiwan. We discuss the involvement of basidiospore dispersal in disease dissemination.Since the 1990s, brown root rot caused by  Phellinus noxius (Corner) Cunningham, a white rot fungus, occurs in tropical and subtropical areas worldwide. P. noxius is a member of the family Hymenochaetaceae, order Hymenochaetales, phylum Basidiomycota of the kingdom Fungi. When grown in potato dextrose agar (PDA), it forms colonies that are initially white to yellowish brown and with age are decorated with irregularly shaped lines or patches of dark brown tissues. Moreover, trichocysts and arthospores are formed in culture, although no clamp connection is observed   , 2 mM MgCl2, 40 nM locus-specific SSR primers, 80 nM fluorescence dye-labeled tagF primer (5’- VIC/FAM/NED/PET-ACGACGTTGTAAAA), 80 nM unlabeled tagR primer (5’- GTTTCTTTTCCCATTA), and 0.25 U Immolase DNA polymerase (Bioline). The thermal cycling parameters were 1 cycle of 95°C for 10 min, 20 cycles of 92°C for 30 s, 63°C for 90 s, and 72°C for 60 s, followed by 40 cycles of 92°C for 15 s, 54°C for 30 s, and 72°C for 60 s, and a final extension step of 72°C for 30 min. Amplicons labeled with different fluorescent dyes were multiplexed (VIC:FAM:NED:PET = 2:4:4:6), mixed with formamide and GeneScan-500 LIZ size standard , and analyzed on the Applied BioSystems 3730xl DNA Analyzer at Genomics BioSci & Tech. . The sizes of amplicons were scored by using GeneMapper v3.0 (Applied Biosystems).Genotypic analysis followed a modified fluorescence-based SSR genotyping method . To minie primer . Each PCA total of 329 isolates were genotyped for the 14 newly-developed SSR markers. For each of the 14 SSR loci, number of alleles, major allele frequency, number of genotypes, genetic diversity, heterozygosity, and polymorphism information content (PIC) were analyzed by using PowerMarker v3.25 . Before K). Individual genotypic data were used to evaluate models assuming different numbers of genetic clusters  based on the posterior probabilities. Under the assumptions of admixture and correlated allele frequencies, the program was run with each K-value replicated 10 times, with the burn-in period and Markov Chain Monte Carlo (MCMC) lengths both set to 500,000. Appropriate number of K was estimated following the ΔK method by Evanno et al. [K inferred from the STRUCTURE analysis.The Bayesian genetic clustering analyses were implemented in STRUCTURE 2.3.4  to detero et al. . For theo et al.  , we arbitrarily divided the whole population into six geographical subpopulations: Taipei (TP), Hsinchu-Miaoli Hills (HM), Central West (CW), Southern West (SW), Yilan (YL), and East Rift Valley (EV) (NA), allelic richness averaged over loci (AR) (rarefaction-based estimation), observed heterozygosity (HO), and unbiased expected heterozygosity (HS) [http://www.ccmar.ualg.pt/maree/software.php?soft=genclon) [Since the STRUCTURE analysis did not detect significant clustering structure for the 329 ley (EV) . To assety (HS)  were calgenclon) .F statistics [FST values across all the subpopulations and pairwise FST values between pairs of subpopulations were calculated with FSTAT v2.9.3.2 [FIS) were calculated with FSTAT v2.9.3.2 [P values estimated by a Markov chain method . To evaluate the proportions of genetic variance explained by the effects of geographical subpopulations, families of host plants, and collection years, we used AMOVA [The degree of genetic differentiation was evaluated by Wright’s atistics . Global v2.9.3.2 ,37, withv2.9.3.2  and the ed AMOVA  performeed AMOVA . The tesAnalysis of isolation by distance between pairs of individuals was performed as described Rousset  with GenSSR genotypes of the 329 isolates were used to construct a phylogenetic dendrogram based on simple matching coefficient by the neighbor-joining (NJ) method with DARwin 6.0.5 . BootstrP. noxius, we performed Illumina sequencing and de novo assembly with DNA from a single-basidiospore isolate, Daxi42. We also generated low-coverage sequencing data for four other P. noxius isolates from Taiwan, Japan, and Indonesia . After quality trimming, we obtained approximately 25 Gb paired-end and 164 Mb mate-paired sequences for Daxi42 and approximately 2 Gb paired-end sequences for each of the other four isolates. The assembly resulted in 15,966 contigs with a N50 scaffold length of 47,281 bp, and the size of the assembled genome was 39,988,869 bp. A total of 12,217 SSR regions were identified in the Daxi42 genome, including repeat regions of 1,653 dinucleotide, 4,012 trinucleotide, 3,126 tetranucleotide, and 3,426 pentanucleotide. By comparing Daxi42 SSRs with nucleotide sequences of the other four isolates, 154 SSR regions with potential polymorphisms were identified, from which 76 SSR primer sets were designed. To validate the applicability of these markers, we analyzed the corresponding SSR loci for eight P. noxius isolates collected from different areas of Taiwan. To verify that these markers were locus-specific and the observed differences in lengths were allelic variations, we sequenced the amplicons of two dominant and two rare alleles for each marker by the dideoxy termination method. Based on the PCR amplification efficiency, reproducibility, and discriminating capacity , a set of 14 SSR markers were chosen and used for subsequent analyses  in some individual isolates . In tota. noxius , we specs remain . TherefoP. noxius isolates sampled across all of Taiwan revealed the presence of 131 alleles detected by the 14 SSR markers, ranging from 4 alleles at the SSR05 locus to 16 alleles at the SSR19 locus (P = 0.00055) was observed between SSR05 and SSR16, as well as between SSR27 and five other loci . The SSR05 locus was excluded from subsequent analyses because it was less informative than SSR16 and the rest of the markers  and average allelic richness (AR) values were 9.77 and 5.285 in the overall population, and 4.69–7.62 and 4.619–5.379 in the six geographical subpopulations. Within all the sampled 329 P. noxius isolates and isolates in the subpopulations, the mean unbiased heterozygosity (HS) ranged from 0.524 to 0.558 and the observed heterozygosity (HO) from 0.275 to 0.372. Heterozygosity deficit and significant deviations from Hardy-Weinberg expectations (P < 0.001) were observed . Tests for Hardy-Weinberg expectation were also conducted for each SSR locus for the overall population, and a significant departure (P = 0) was detected for all the 13 SSR loci. Among the 265 P. noxius isolates successfully genotyped for the 13 SSR loci (~20% of the isolates had a few missing data), 259 MLGs were identified, so most individuals (~98%) showed unique genotypes. In summary, for each of the abovementioned statistics, similar results were obtained for the overall population and the six subpopulations, which suggested comparable level of genetic diversity among P. noxius isolates collected from different areas of Taiwan.The computed values of K plot, peaks were observed at K = 2, 3, 5, and 7. With STRUCTURE constrained to estimate 2, 3, 5, or 7 clusters, most individuals appeared highly admixed, which suggested no significant clustering structure, at least for K from 1 to 20. The whole population was then arbitrarily divided into six geographical subpopulations for subsequent analyses. The global FST value across the six geographical subpopulations was low (FST = 0.015) but significantly differed from zero (P < 0.001)  . All pai effects .P. noxius isolates in this subpopulation were sampled from an area of 7 x 4 km2. Among them, 85 isolates were sampled from a small urban area of 3 x 1.2 km2, where three campuses are located , whereas clusters C, D, and E were highly differentiated . Moderate pairwise FST values were obtained between clusters A and C/D/E (FST = 0.130–0.163) and between clusters B and C/D/E (FST = 0.191–0.265). The samples assigned to a specific cluster did not necessarily originate from neighboring areas  for all Taiwan, 0.013 (P = 0.153) for TP, 0.018 (P = 0.047) for HM, 0.016 (P = 0.103) for CW, 0.040 (P = 0) for SW, 0.037 (P = 0) for YL, and -0.002 (P = 0.157) for EV. Similar non-significant patterns were observed when considering only one isolate representative of the genetically highly identical isolates collected from diseased trees/stumps located near each other.We performed analysis of isolation by distance to investigate the relationship between spatial distance and genetic similarity for the entire Taiwan population and for the six geographical subpopulations, and none of the datasets were found significant. Slopes of the regression lines were 0.005 , isolation and sequencing of SSR containing clones, and primer design and optimization. Alternatively, SSR markers can be derived from cloning and sequencing of the amplicons of PCR-based molecular markers [Phytophthora ramorum [Anisogramma anomala [P. noxius, we performed Illumina sequencing and de novo assembly of the genome of a single basidiospore isolate. Similar to the microsatellite patterns found in many other fungi (including Basidiomycetes), microsatellites identified in P. noxius genome are enriched in the short repeat motifs, and the numbers of repeat units are greater for the short than long repeat motifs [P. noxius.The  markers . With th ramorum  and Anis anomala . In thist motifs –49. The P. noxius isolates sampled across sufficiently large geographic areas and diverse host trees were used to infer the occurrence and dissemination pattern of brown root rot in Taiwan. High levels of genetic diversity were detected in the overall population and the six geographical subpopulations of P. noxius in Taiwan. There seems little to no barrier to gene flow throughout the P. noxius population in Taiwan, which may be attributed to a combined effect of basidiospore dispersal and the migration of P. noxius via infected seedlings, trees, and debris. The widespread distribution of diverse MLGs indicated that the epidemic is caused by diverse clones rather than a single predominant highly virulent strain of P. noxius. In closely related Phellinus species such as P. linteus, P. weirii, P. gilvus, and P. tremulae, sexual reproduction is governed by bipolar or tetrapolar heterothallic systems [P. noxius. However, with the lack of clamp connections for diagnosing compatibility, the mating system of P. noxius remains to be clarified.Abundant  systems –53. The P. noxius isolates collected within a small area of 3 x 1.2 km2 showed a high level of genetic diversity. Nonetheless, isolates from neighboring infected trees showed identical or nearly identical genotypes, so P. noxius might have spread from diseased to adjacent healthy trees through root-to-root contact. In support of our result, Hattori et al. (1996) identified up to 25 different P. noxius clones, mostly broadly distributed in five small sampling plots (each 15–25 × 10–20 m2) in a windbreak [P. noxius between neighboring trees. The reason that genotypes of some isolates from neighboring trees are not perfectly identical may attribute to the inclusion of different sets of nuclei during the process of single-hyphal-tip or single-arthospore isolation.Our intensive sampling at three nearby campuses in Taipei further revealed that even indbreak . Becauseindbreak , the mycFST statistics, and AMOVA all suggested low population differentiation for P. noxius in Taiwan. For each maker locus, most alleles (except for the rare ones) could be detected in different geographical subpopulations. The phylogenetic tree also showed that the isolates sampled from diverse areas of Taiwan were randomly clustered. Moreover, analysis of the isolation by distance revealed no spatial pattern of genetic diversity in all of Taiwan or within the six geographical subpopulations. Heterozygosity deficit within the population, significant deviation from Hardy-Weinberg expectation across all loci, and the high FIS values detected in the overall population and subpopulations may reflect the fact of non-random mating, which, in our populations, may result from sibling mating, vegetative hyphal fusion, and/or sampling bias. Because we observed low population differentiation across Taiwan, and the P. noxius individuals in different areas were somewhat genetically related, the effect of inbreeding between closely related individuals (siblings) is not surprising. Vegetative hyphal fusion may be a common phenomenon and a possible source of new genetic variability in P. noxius because we could detect multinucleate and heterokaryotic individuals by SSR genotyping and DAPI staining. Small sample sizes from distinct genetic clones within the population also may have contributed to high FIS values [P. noxius can be asymptomatic, but the isolates we collected were solely from symptomatic trees or decaying stumps.Analyses based on Bayesian clustering, S values , as sampArmillaria cepistipes [Armillaria mellea [Cylindrobasidium argenteum [Fomitopsis pinicola [Serpula lacrymans [P. noxius. Basidiocarps and basidiospores of P. noxius are rarely seen in the field [P. noxius were able to infect fresh stumps of hoop pine, although the rate of successful inoculation was low [Fomitopsis rosea, Cystostereum murraii and Phlebia centrifuga at sites 3 km away from the nearest fruit bodies [Heterobasidion annosum trapped 50–500 km apart from the inoculum source [Peniophora aurantiaca captured ~1000 km from a known natural occurrence [P. noxius basidiospores by using available spore trapping techniques [Previous studies based on (indirect) population genetic analyses or (direct) trapping of airborne spores support the involvement of basidiospores in the dissemination of many wood-decay Basidiomycetes fungi, including pistipes , Armillaa mellea , Cylindrrgenteum , Fomitoppinicola , and Seracrymans . Our finhe field . However was low . The dis was low  and envi was low ; althoug was low ,62,63, lt bodies , the spom source , and thecurrence . Furtherchniques  would beA. mellea, Ganoderma australe, and H. annosum, was proposed for P. noxius, but the role of basidiospores has long been equivocal [P. noxius may spread over short distances by hyphal extension via root-to-root contact of the hosts, and the genetically variable basidiospores are likely responsible for long-distance dispersal and the establishment of unique clones in new infection sites. Basidiospores may infect plants directly through fresh wounds [P. weirii, the causal agent of laminated root rot of certain conifers, could germinate and colonize previously frozen and scalded wood disks [P. noxius basidiospores can survive for 3 to 4.5 months in soils of varying moisture levels, and P. noxius can survive on woody debris for more than 10 years [P. noxius basidiospores on woody materials would help explain how the basidiospores can still be a key player when the occurrence of P. noxius basidiocarps is relatively low. For long-distance spread of brown root rot disease, the movement of diseased plants or debris by humans cannot be ruled out. However, the effect of human activities is difficult to evaluate because the records for cultivation and transplanting are poorly maintained in most cases. Nevertheless, the inoculum of P. noxius, originating from distant infection sites, may be more common in the environment than we imagined. Moreover, whether a tree will become diseased seems to depend largely on its health status. In urban areas in Taiwan, brown root rot disease has often been observed on weakened trees surrounded by cement or grown in small tree wells. Damage caused by transplantation and the increasing frequency of extreme weather events may also threaten the health of trees and make trees more susceptible to P. noxius infection. Overall, molecular evidence from this study provides important insight into the population genetic structure of P. noxius as well as local, regional, and distant spread of brown root rot disease caused by this fungus.A disease cycle similar to other root-rotting basidiomycetes, such as quivocal . In agrequivocal , our resh wounds  or indirod disks . Chang  mycelium, (b) arthospores, (c) basidiospores, and (d) a germinating basidiospore examined in the light- (left) and fluorescent-field (right) under microscope. .(PPTX)Click here for additional data file.S2 FigP. noxius samples isolated from neighboring trees/stumps at 23 sites.The neighbor-joining (NJ) tree was constructed on the basis of the genotypes of 13 SSR markers. Numbers at branch points refer to bootstrap values (2000 iterations). Isolate IDs in different colors indicate that they were from different geographical areas. The black asterisks indicate the 60 (PPTX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableThe allelic fragments for SSR markers are shown as the sizes (bp) of PCR products. For each SSR locus, only the alleles with the first and second greatest fluorescence intensity (data in the first two columns) were used for analyses in the study. The symbol "?" denotes missing data.(XLSX)Click here for additional data file.S3 TableTP: Taipei; HM: Hsinchu-Miaoli Hills, CW: Central West, SW: Southern West, YL: Yilan, and WV: East Rift Valley.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Galaxea fascicularis, from four regions in the subtropical Nansei Islands in the northwestern Pacific Ocean. Two types (soft and hard types) of nematocyst morphology are known in G. fascicularis and are significantly correlated with the length of a mitochondrial DNA noncoding sequence . Using microsatellites, significant genetic differentiation was detected between the mitochondrial DNA sequence types in all regions. We also found a third genetic cluster (mt‐L+), and this unexpected type may be a cryptic species of Galaxea. High clonal diversity was detected in both mt‐L and mt‐S types. Significant genetic differentiation, which was found among regions within a given type , may result from the shorter larval development than in other broadcast‐spawning corals, such as the genus Acropora. Nevertheless, intraspecific genetic diversity and connectivity have been maintained, and with both sexual and asexual reproduction, this species appears to have a potential for the recovery of populations after disturbance.Population connectivity resulting from larval dispersal is essential for the maintenance or recovery of populations in marine ecosystems, including coral reefs. Studies of species diversity and genetic connectivity within species are essential for the conservation of corals and coral reef ecosystems. We analyzed mitochondrial DNA sequence types and microsatellite genotypes of the broadcast‐spawning coral,  This is the first report of a population genetic study of the genus Galaxea focusing on a large geographic range in an island reef system.Scleractinian reef‐building corals constitute the framework of reef ecosystems and are especially endangered due to a multitude of rapid, oceanic environmental changes caused by anthropogenic disturbances, including extreme thermal stress from increasing sea surface temperatures   . Also, local genetic differentiation  was detected among sites in C. aspera, which employs planula brooding as well as broadcast spawning  were analyzed using an automated capillary‐based DNA sequencer  and GeneMapper ver. 3.7 (Thermo Fisher Scientific).Genomic DNA was extracted using a DNeasy Blood & Tissue kit . To analyze polymorphism and amplification of designed primer sets and to identify the mitochondrial DNA sequence type , we used the tailed primer method to perform PCR. We developed 11 microsatellite markers from cyt b and nad 2 to the one reported previously, PCR products amplified using 188‐1 and 188‐R3 (5′‐CATCATTATCCTCTTCAAGG‐3′) for ten colonies  were employed to classify clonal replicates  using GenClone ver. 2.0  occurring R = (MLL−1)/(N−1) . We removed clonal replicates from the dataset, and MLLs were retained for the following population genetic analyses.Clonal diversity was estimated with the following index proposed by Dorken and Eckert : R = , and a deviation index (FIS) from Hardy–Weinberg equilibrium (HWE) for each type were evaluated with GenAlEx for each region. INEST ver. 2.0 (Chybicki and Burczyk AR) at each region using FSTAT ver. 1.2  and excluding null alleles (ENA) effects using FreeNA . A burn‐in of 100,000 iterations followed by 1,000,000 Markov chain Monte Carlo (MCMC) replications was used for population clustering without prior information under the admixture model and assuming correlated allele frequencies  that best fit the data, was determined using the method of Evanno et al.  and type. Each chain was iterated 1,000,000 times including burn‐in with 100,000 iterations. Determination of the number of K clusters that best fit the data was conducted using the method of Evanno et al. . Fourteen of these 21 are indeterminate colonies, and distinct and clear fragment peaks for both mitochondrial types were detected, although only one peak is usually derived from the mitochondrial noncoding region between cyt b and nad 2. Both peaks were derived from both types of mitochondrial noncoding region by sequencing. We excluded these 21 colonies and used genotypes from the remaining 510 colonies for all other analyses. Detailed site data are provided in Table Of 531 colonies of PSEX value. We added this doubled MLL as a different lineage for subsequent analyses. In these MLLs, there is a large variation in clonality at each site. Values ranged from 0 to 1 for both mt‐L and mt‐S types, although MLLs were few at some sites because of high clonal diversity within types. Three MLLs  in mt‐L and two MLLs  in mt‐S showed the opposite genetic cluster in STRUCTURE , and this result was supported by a relatively high frequency of null alleles (Null freq. = 0.061 to 0.140 among regions) and inbreeding influences (Avg Fi = 0.021 to 0.133 among regions) from the results of INEST . These tendencies are identical with the results of pairwise FST excluding the effect of null alleles, and no large influence of null alleles for the calculation of these genetic differentiation indexes (Table S2). The results from STRUCTURE, based on Bayesian statistical model‐based clustering, also indicated that these three types are genetically isolated . This application of the method of Evanno et al. . STRUCTURE results .Allelic richness and the expected heterozygosity of mt‐L and mt‐S at northern regions were comparable to those of southern regions Table . Alleliclts Fig. B suggestG. fascicularis, in the Nansei Islands of Japan, this study analyzed mitochondrial types and compared these types with genotypes of microsatellites to estimate the species diversity of Galaxea. Mitochondrial type is related to microsatellite genotype in all regions. Values of clonal diversity are variable among sites for each type. Genetic differentiation is significant, but low among regions, and genetic diversity has been maintained for each type.For the broadcast‐spawning coral, Galaxea has an extensive range in the Indo‐western Pacific, and it is not clear what sort of divergence has occurred in the Coral Triangle itself. In Okinawa, mt‐L and mt‐S types can be distinguished not only by morphology, but also by the timing of spawning   markers, or amino acid sequences derived from all coding genes on mitochondrial genome) are needed for the exact estimation of species diversity. With nematocyst types, fine‐scale investigations of the corallite and subcorallite features observed with scanning electron microscopy (SEM) and thin sections may reveal defining characters each type.The mitochondrial type, mt‐L+, found at O‐Kum and M‐Uen, was unexpected, indicating greater genetic differentiation; however, the sequence of the mitochondrial noncoding region is interestingly similar to the E lineage of mt‐L reported by Watanabe et al.  Fig. 2)2). AlthoAcropora species, A. digitifera and a cryptic species Acropora sp. 1 aff. digitifera . Mean Ln P(D) values (K = 1 to 11) across 10 iterations per K, and ΔK values (K = 2 to 10) using the method of Evanno et al.  and mt‐S (b) by estimation of the optimal number of genetic clusters of InStruct. Mean Ln P(D) values (K = 1 to 5) across 10 iterations per K, and ΔK values (K = 2 to 4) using the method of Evanno et al.  and a nuclear gene (28S rRNA) from GenBank split nominal species, suggesting that some Galaxea species may either be misidentified or may contain cryptic species. These networks were constructed by TCS ver. 1.21  model and lower diagonal are by INA  model.Click here for additional data file.


Signal transducer and activators of transcription-3 (STAT3) regulates diverse biological functions including cell growth, differentiation, and apoptosis. In addition, STAT3 plays a key role in regulating host immune and inflammatory responses and in the pathogenesis of many cancers. Several studies reported differential regulation of STAT3 in a range of viral infections. Interestingly, STAT3 appears to direct seemingly contradictory responses and both pro- and antiviral roles of STAT3 have been described. This review summarized the currently known functions of STAT3 in the regulation of viral replication and pathogenesis of viral infections. Some of the key unanswered questions and the gap in our current understanding of the role of STAT3 in viral pathogenesis are discussed. Signal transducers and activators of transcription (STATs) are a family of transcription factors that play crucial roles in regulating a number of diverse biological functions including cell proliferation, differentiation, apoptosis, inflammatory response, immunity, and angiogenesis . There aSTATs were first discovered through their capacity to mediate signalling from interferon (IFN) and interleukin-6 (IL-6) receptors following binding of cytokines , 4, 6–8.STAT proteins are activated by phosphorylation of specific tyrosine residues, following which they form stable homodimers or heterodimers with other STAT proteins through reciprocal phosphotyrosine-SRC homology 2 (SH2) domain interactions . STAT diIn addition to the canonical model of JAK/STAT signalling, STATs also form dimers in the absence of the activating tyrosine phosphorylation . In the Of all the STAT family proteins, STAT3 is unique as it is known to direct seemingly contradictory responses  and is eThe structure of STAT3 is similar to the other STAT family members comprising six structural regions, namely, N-terminal domain (ND), coiled-coil domain (CCD), DNA-binding domain (DBD), linker domain, SH2 domain, and a C-terminal transcriptional activation domain (TAD) . The corγ, TNF-α, LIGHT, a member of the TNF superfamily, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), CCL-5/RANTES, stem cell factor (SCF), and oncostatin M (OSM) [STAT3 is activated by phosphorylation at tyrosine 705 in the C-terminal domain  and more M (OSM) –38.2+/calmodulin-dependent protein kinase IIγ (CaMKIIγ), bile acids, leptin and low pH, black soy peptides, diazoxide, isoliquiritigenin, and olanzapine have been found to activate STAT3 , hepatocyte growth factor receptors (HGFRs), fibroblast growth factor receptors (FGFRs), platelet-derived growth factor receptors (PDGFRs), insulin-like growth factor receptors (IGFRs), and vascular endothelial growth factor receptors (VEGFRs) . In addil., 2014 ).Growth factor or cytokine receptor-ligand interaction results in dimerization of gp130, a signal transducer protein in cytoplasm . This isα5/NPI-1 [The phosphorylation of STAT3 promotes homodimerization, wherein the SH2 domain of each STAT3 monomer interacts with the Y705 residue on another monomeric STAT3 [α5/NPI-1  and bindα5/NPI-1 –47 AA-3 . FurtherεC, TC45, and SHP-1 have been implicated in the termination of STAT3 signaling. Because only STAT3 dimers bind to DNA, the nuclear PTP TC45 may be important in the termination of STAT3-mediated transcriptional activation [Activation of JAK/STAT pathway begins with the binding of extracellular signalling proteins (ESPs) to specific receptors associated with JAKs. Activated JAKs then activate STAT proteins by phosphorylation which otherwise remain latent in the cytoplasmic , 50. A rtivation , 53.Protein inhibitors of activated STAT (PIAS) family members are involved in blocking the DNA-binding ability of STAT proteins thereby inhibiting their function as transcription factors. PIAS family of proteins shares a highly conserved domain structure comprising the N terminus SAP domain that can bind to AT-rich DNA sequences and a Pro-Ile-Asn-Ile-The (PINIT) motif, which is involved in the nuclear retention of PIAS proteins. PIAS3 has been shown to specifically bind to STAT3 and block its DNA-binding activity and transcriptional activation , 52, 54.The family of suppressors of cytokine signalling (SOCS) acts as classical feedback inhibitors of the JAK/STAT pathway. STAT3 activates the transcription of SOCS3 which can block STAT3 signalling either by direct binding and inhibition of JAKs, by competing with STAT3 for pY-binding sites on activated receptor chains, or by binding signalling proteins and targeting them for proteasomal degradation , 56 Fig.STAT3 is a signaling mediator of IL-6 and IL-10 family members and other cytokines such as leptin and G-CSF , 57. N-t Toxoplasma gondii exploits host STAT3 to prevent LPS-triggered proinflammatory cytokine production in infected mouse macrophages [ in vivo and in vitro [STAT3 activation results in either activation or suppression of inflammatory response depending on the physiological status of the cells. STAT3 is found to be constitutively activated in cancer cells and the persistent activation of STAT3 in cancer cells mediates tumour-promoting inflammation . STAT3 arophages . IL-10 rrophages  and STATin vitro –64. WhilA number of viruses are known to regulate STAT family members including STAT1, STAT2, and STAT3. Epstein-Barr virus infection results in transcriptional activation of STAT1 through latent membrane protein 1 (LMP1) . Dengue Several studies described the involvement of STAT3 in the replication and pathogenesis of viruses in humans and animals. Notably, both pro- and antiviral functions of STAT3 have been documented and its precise role in the pathogenesis of viral infections is not yet fully established. A number of DNA and RNA viruses are known to regulate STAT3, which is summarized in  Herpesvirus saimiri subgroup A strain 11 (STP-A11) [STAT3 is either positively or negatively regulated in a range of viral infections depending on the type of virus involved. STP-A11) , and VarSTP-A11)  promote In contrast, influenza A virus (IAV) nonstructural protein 1 (NS-1)  and huma+ [+ T cells [+ T cell-mediated responses to viruses. For example, STAT3 plays an important role in the activation of CD8+ T cells effective response during herpes simplex virus (HSV-1) infection [STAT3 plays an important role in adaptive immune response, in particular in the regulation of T lymphocyte function. STAT3 mediates IL-6-dependent T cell proliferation by preventing apoptosis . Further+  and CD8+ T cells . Furthernfection .It is evident that viruses either promote or disrupt STAT3-mediated gene transcription and host immune responses against viruses. Further, activation or inhibition of STAT3-mediated signalling appears to be dependent on virus and host cell type involved. Inhibition of STAT3 activation could be a strategy of viruses to subvert host immune responses. On the other hand STAT3-mediated gene transcription of proviral factors could be essential for certain other viruses. It is possible that the timing of STAT3 regulation during virus infection may be critical and hence require further in depth studies to profile STAT3 regulation during different stages of viral infection.The role of STAT3 in virus replication appears to be complex, as it appears to function as a proviral factor in some viral infections and antiviral factor in others. STAT3 cooperatively interacts with hepatocyte nuclear factor 3 (HNF-3) and activates hepatitis B virus (HBV) gene expression . There iHCV constitutively activates STAT3 in liver cells which plays an important role in HCV RNA replication . HCV repHCMV primarily utilizes unphosphorylated STAT3 to promote, either directly or indirectly, the initiation of HCMV DNA replication . HCMV in in vitro and also in human skin xenografts in SCID mice in vivo. STAT3 activation induces the antiapoptotic protein survivin and both STAT3 and surviving are essential for VZV replication as inhibition of STAT3 phosphorylation and survivin results in restricted VZV replication [STAT3 activation was shown to be critical for replication of VZV that causes varicella (chickenpox) during primary infection and zoster (shingles) upon reactivation . VZV trilication .Mumps virus V protein functions as a ubiquitin ligase that targets STAT3 for degradation and STAT3 evasion has been proposed to be beneficial to the replication of Paramyxoviruses . FurtherInfluenza A virus (IAV) NS1 protein interferes with IFN production  which co in vivo.Viruses such as HBV, HCV, and HCMV appear to exploit host genes transcriptionally regulated by STAT3 for their gene replication which could be independent of the regulatory effects of STAT3 on type 1 IFN response. Whereas STAT3 inhibition appears to be a strategy of viruses such as Paramyxo- and Orthomyxoviruses to evade host innate and adaptive immune responses, it is plausible that inhibition of STAT3 signalling could provide a much broader spectrum of cytokine and growth factor suppression to allow replication and spread of these virusesγ-herpesviruses, KSHV, EBV, and Herpesvirus saimiri [STAT3 role in viral pathogenesis appears to be complex with reports suggesting both promotion of innate antiviral response and contribution to the detrimental effects of viral infection. STAT3 is constitutively phosphorylated in neoplastic cells  and many saimiri , 73, 89.STAT3 role has been well characterized in the pathogenesis of viral infections resulting in liver disease in humans. HCV infection in liver cells causes constitutive activation of STAT3, which plays a central role in chronic hepatitis and often results in liver cirrhosis and hepatocellular carcinoma . HCV corγ-herpesviruses, such as KSHV [STAT3 activation and upregulation of antiapoptotic protein survivin play an important role in the pathogenesis of lytic as well as tumorigenic herpesviruses . STAT3 a as KSHV . STAT3 a as KSHV .STAT3 is also known to promote host defense against virus infections and play a protective role in regulating virus mediated proinflammation. Gp130-STAT3 signalling plays an important role in the innate immune response in cardiac myocytes against coxsackievirus B3 infection . Dysregu In vitro studies using cell culture systems are a valuable tool to dissect the molecular basis of STAT3 role in innate response to viruses. However, in vivo studies are essential to elucidate STAT3 role in adaptive immune response to viruses. Complementary in vitro and in vivo experiments are therefore essential to better understand the seemingly contradictory role of STAT3 in viral infections.In summary, STAT3 plays a significant role in the complex interplay between viruses and their hosts and functions as either a pro- or antiviral factor depending on the virus and host cell type involved. While STAT3 role has been reasonably well characterized in the pathogenesis of oncogenic viruses and viruses causing liver pathology, its role in many other viral infections is less well understood. STAT3 regulates antiviral and proinflammatory responses, either through transcriptional regulation of other cellular factors or through pathways independent of its role as a transcription factor. The seemingly contradictory roles of STAT3 in viral infections raise a number of interesting questions: “what factors determine the switch between pro- and anti-inflammatory functions of STAT3?,” “how are viruses able to exploit STAT3 signalling for their gene replication?,” and “does STAT3 either negatively or positively regulate type 1 IFN response depending on the virus type involved?” Further in depth studies to dissect the role of STAT3 in viral infections could provide valuable insights into viral pathogenesis and development of novel antiviral therapies.


Despite the existence of two recent natural hybrid lineages, a pervasive view is that recombination has been restrained at an evolutionary scale and is of little epidemiological relevance to contemporary parasite populations. This article reviews the growing number of field studies which indicate that natural hybridization in T. cruzi may be frequent, non-obligatory and idiosyncratic; potentially involving independent exchange of kinetoplast and nuclear genetic material as well as canonical meiotic mechanisms. Together these observations now challenge the traditional paradigm of preponderate clonal evolution in T. cruzi and highlight the need for additional, intensive and appropriately sampled field surveys, complemented by high resolution, combined nuclear and mitochondrial population genetics analyses.Many eukaryotic pathogenic microorganisms that were previously assumed to propagate clonally have retained cryptic sexual cycles. The principal reproductive mode of  At the Trypanosoma cruzi, the aetiological agent of Chagas disease, often fulfils some key assumptions of PCE, which have been cited as compelling evidence that it is essentially a clonal organism, namely strong linkage disequilibrium, deviations from Hardy–Weinberg allele frequencies and structuring of populations into stable, distinct genetic clades, or discrete typing units (DTUs)  . T. cruzase foci . Currentase foci . Genotypase foci . Moleculase foci , possiblase foci . These hT. cruzi strains is crucial to establish the epidemiological risk associated with recombinant genotypes and to reconcile the implications parasite hybridization has at both the generational and evolutionary scales. However, detecting genetic exchange among natural populations is inherently complicated by choice of samples and marker resolution, given that strains most likely to be recombining may be closely related and potentially indistinguishable.A clear understanding of the impact of genetic exchange on the ecological and geographical distributions and clinical characteristics of 2T. cruzi may be frequent, non-obligatory and idiosyncratic; potentially involving independent exchange of kinetoplast and nuclear genetic material as well as canonical meiotic mechanisms  . The oriAt the intra-lineage level, genetic exchange is increasingly reported, particularly among TcI populations. It is unclear whether this is due to the examination of representatives from intensely sampled populations that are minimally-subdivided spatially and temporally, and therefore more likely to undergo hybridization, or if it truly reflects the analysis of strains that are more permissive to recombination . The undIn general, genetic exchange at the nuclear level has been demonstrated by Hardy–Weinberg allele frequencies, linkage equilibrium between loci, a lack of repeated multilocus genotypes (MLGs)  and morevs. <0.1% of the nuclear genome); without whole nuclear genome sequences for introgression hybrids and parental isolates, it is impossible to distinguish between these two hypotheses. However, by analogy to other medically important trypanosome species, the presence of alternate, covert sexual mechanisms within the same species is not entirely unexpected , is thus far low  and in vivo (mice and triatomine bugs) cycles  cycles . Isolatigenotype .Candida albicans  can inflate genetic linkage statistics and mask recombination events occurring between more closely related individuals  is a well-documented phenomenon among parasitic protozoa (Leishmania hybrids indicate that genetic exchange can impact vector permissibility (Importantly, the effects of genetic exchange on protozoa . Observasibility , increassibility  or altersibility  virulencsibility , as wellsibility ; a scenasibility . The pat6T. cruzi is both contemporary and historical, responsible for shaping current parasite population structures, as well as the evolution of distinct T. cruzi DTUs. Together these observations challenge the traditional paradigm of PCE in T. cruzi and highlight the need for additional, intensive and appropriately sampled field surveys, complemented by high resolution, combined nuclear and mitochondrial population genetics analyses.The majority of field studies now indicate that natural genetic exchange in T. cruzi population genetics until imminently superseded by comparative population genomics.The precedent in experimental design established by such studies may represent the most promising intermediary in


Over the last two decades, the zebrafish has joined the ranks of premier model organisms for biomedical research, with a full suite of tools and genomic resources. Yet we still know comparatively little about its natural history. Here I review what is known about the natural history of the zebrafish, where significant gaps in our knowledge remain, and how a fuller appreciation of this organism's ecology and behavior, population genetics, and phylogeny can inform a variety of research endeavors.DOI:http://dx.doi.org/10.7554/eLife.05635.001 Like so many model organisms, the zebrafish was chosen for its tractability and, especially, its potential for genetic analysis and cellular observation. The founder of ‘modern’ zebrafish research, George Streisinger, had a passion for fish but it seems unlikely that he chose this particular minnow because of any prior insights into its natural history or its phylogenetic position within the teleosts (bony fishes). Rather, the zebrafish was readily available, it was easy to breed, and its lovely, transparent embryo was quick to develop. Streisinger's efforts at the University of Oregon, and the hard work of many early adopters of the species, most notably other labs in Oregon, Tübingen and Boston, propelled zebrafish into the top tier of NIH-funded biomedical models . The zebGiven all of these resources, one might wonder how much is known about zebrafish as an organism (rather than as a system), and whether it matters. Here, I review briefly what we do and don't know about wild zebrafish, and reflect upon the ways in which a deeper appreciation of zebrafish in their natural habitat can inform a range of biological enquiries.Danio rerio . Formally described in the state of Bihar in northeastern India, zebrafish have also been collected in the south and west of peninsular India, past the city of Bangalore, and beyond India, as far north as Pakistan and Nepal, as well as east into Bangladesh and possibly Myanmar , knifefish (Notopterus) and catfish, birds such as kingfishers and herons, and even dragonfly larvae, are all likely predators of adults, and a great many species probably eat zebrafish eggs and larvae  that likely derive from intentional selection on some traits and relaxed selection on others . IncludiGenomics is another arena in which wild zebrafish are providing valuable insights. Most lab zebrafish represent any of several commonly used strains initiated with founders obtained from the pet trade or, in some cases, from collections in the wild. Some strains have been maintained to preserve allelic variation, others to intentionally minimize genetic diversity, to facilitate genetic mapping and genome editing, and to control genetic background effects on mutant and other phenotypes . One theWhen samples from wild populations are analyzed, it becomes clear that even the extensive variation in lab strains represents but a tiny fraction of total zebrafish genetic diversity . MoreoveThe considerable genetic diversity of zebrafish is put into perspective by comparisons with the human genome. For instance, complete genomic sequences of just two gynogenetic . Genetic analyses of lab strains have identified chromosomal regions associated with sex determination, yet, surprisingly, these differed between studies . AnalyseDanio dangila   . InteresDanio itself, 20 species are considered valid taxonomically  provided only weak support for the sister relationship between D. rerio and D. aesculapii. It will be exciting to further unravel how speciation is proceeding in this group as new Danio are discovered and new sequences gathered for analysis, and as roles for hybridization, as well as for geography and other potential isolating mechanisms, are defined.Phylogenetic reconstructions of  unclear . A recen unclear  to examisculapii  as a cansculapii . InteresDanio and for more distant cyprinids. These additional genomes will greatly facilitate the identification of gene regulatory domains and how they evolve  that impinge upon zebrafish survival and reproduction in the wild?Are there subtle—or not so subtle—differences in morphology, physiology or behavior among natural populations, and are such differences adaptive?Do female zebrafish choose their mates in nature, and if so, what criteria do they use?What are the specific genetic changes underlying the evolution of divergent sex determination mechanisms between wild fish and lab strains?Danio species are there, what are their evolutionary relationships and what factors have influenced speciation and morphological diversification in the genus?How many more DOI:http://dx.doi.org/10.7554/eLife.05635.005The differences between wild and lab zebrafish cited above also serve as a cautionary tale about generalizing from particular populations to the species as whole. For example, given their range and genetic variability, a single set of optimal conditions for the growth and development of wild zebrafish is unlikely, let alone for lab strains that have experienced very different selective regimes. Likewise, it remains unclear whether environmental enrichment, to mimic the habitat complexity that wild fish can experience, also benefits lab strains, particularly when such interventions are balanced against costs, such as the increased difficulty of observing fish and the increased accumulation of detritus, which themselves can lead to morbidity and mortality. What is clear is that a deeper understanding of zebrafish natural history will benefit the health and well being of fish in the lab, as well as research productivity, when combined with a holistic view of variation in behavior, genetics, and the broader goals of lab research. The promise of integrating a deeper understanding of zebrafish ‘the organism’ with zebrafish ‘the system’ suggests exciting times to come for the devotees of Hamilton's beautiful fish.


Helicoverpa armigera is one of the primary agricultural pests in the Old World, whereas H. zea is predominant in the New World. However, H. armigera was first documented in Brazil in 2013. Therefore, the geographical distribution, range of hosts, invasion source, and dispersal routes for H. armigera are poorly understood or unknown in Brazil. In this study, we used a phylogeographic analysis of natural H. armigera and H. zea populations to (1) assess the occurrence of both species on different hosts; (2) infer the demographic parameters and genetic structure; (3) determine the potential invasion and dispersal routes for H. armigera within the Brazilian territory; and (4) infer the geographical origin of H. armigera. We analyzed partial sequence data from the cytochrome c oxidase subunit I (COI) gene. We determined that H. armigera individuals were most prevalent on dicotyledonous hosts and that H. zea were most prevalent on maize crops, based on the samples collected between May 2012 and April 2013. The populations of both species showed signs of demographic expansion, and no genetic structure. The high genetic diversity and wide distribution of H. armigera in mid-2012 are consistent with an invasion period prior to the first reports of this species in the literature and/or multiple invasion events within the Brazilian territory. It was not possible to infer the invasion and dispersal routes of H. armigera with this dataset. However, joint analyses using sequences from the Old World indicated the presence of Chinese, Indian, and European lineages within the Brazilian populations of H. armigera. These results suggest that sustainable management plans for the control of H. armigera will be challenging considering the high genetic diversity, polyphagous feeding habits, and great potential mobility of this pest on numerous hosts, which favor the adaptation of this insect to diverse environments and control strategies. Helicoverpa Hardwick and Heliothis Ochsenheimer genera Helicoverpa genus contains two of the primary Heliothinae pest species: Helicoverpa armigera (Hübner) (Old World bollworm) and Helicoverpa zea (Boddie) (New World bollworm). Although the exact evolutionary relationship between H. armigera and H. zea remains uncertain, these insects are considered to be ‘twin’ or ‘sibling’ species, and they are able to copulate and produce fertile offspring under laboratory conditions H. zea evolved from a small portion of the larger H. armigera population  that reached the American continent approximately 1.5 million years ago, which is consistent with previous phylogeographic analyses of H. armigera and H. zea individuals The Heliothinae (Lepidoptera: Noctuidae) subfamily has 381 described species, many of which are important agricultural pests from the H. armigera is considered to be one of the most important agricultural pests in the world. This insect is widely distributed throughout Asia, Africa, Europe, and Australia, and it has been shown to attack more than 100 host species from 45 different plant families H. zea is restricted to the American continent and is of lesser economic importance; it is a secondary pest of cotton, tomato, and, most significantly, maize crops H. armigera individuals, which are considered to be A1 quarantine pests, were officially reported within the Brazilian territory H. armigera lineages showing tolerance/resistance to insecticides and genetically modified plants H. armigera will cause a loss of more than US$2 billion to the 2013/14 Brazilian agriculture crop because of direct productivity losses and resources spent on phytosanitary products for soybean, cotton, and maize, which are the main crops of Brazilian agribusinesses. Therefore, H. armigera is now one of the most important pest species with respect to agriculture in Brazil Helicoverpa spp. and the resulting economic damages to cultivated plants have been reported in different regions of Brazil, in particular in the Western state of Bahia H. armigera in Brazil. This atypical and confusing scenario was likely caused by the significant morphological similarities between H. zea and H. armigeraBacillus thuringiensis (Bt) genes in Brazil.High population densities of H. armigera individuals within the Brazilian territory, many basic pieces of information concerning this species, including its geographical distribution, the types of hosts it attacks, its invasion source, and its dispersal routes, remain poorly understood or completely unknown. Therefore, we attempted to address some of these outstanding questions using a phylogeographic approach by analyzing genetic sequence data from a portion of the cytochrome c oxidase subunit I (COI) gene of Helicoverpa spp. specimens isolated from different hosts and regions of Brazil. This study was performed with the following goals in mind: (1) to confirm and evaluate the occurrence of H. armigera and H. zea individuals from different hosts and regions of Brazil; (2) to assess the demographic parameters and genetic structure of H. armigera and H. zea populations within the Brazilian territory, with a focus on the region, season, and host; (3) to assess the potential invasion (single or multiple) and dispersal routes for H. armigera within the Brazilian territory; and (4) to determine the geographical origin of the H. armigera populations present in Brazil. This information will be essential for understanding the genetic diversity and population dynamics of these pests as well as for guiding both immediate control strategies  and subsequent long-term integrated management programs for the Helicoverpa spp. complex in Brazil.Aside from the identification of Helicoverpa spp. specimens initially sampled were identified as H. armigera (98–100% homology) and 134 individuals were identified as H. zea (98–100% homology)  . H. armigera was not found on maize crops in the Midwest, Southeast, or South of Brazil. H. zea was primarily found on maize crops and was present in all sampled regions during both the winter and summer growing seasons. Of the winter crops, millet and cotton were exceptional in that they could simultaneously support H. zea and H. armigera  and specific hosts  . H. armiarmigera . We founic hosts .H. armigera individuals, and four non-synonymous substitutions were observed in eight H. zea individuals. However, considering the relatively high mutation rate reported for the COI gene in the Helicoverpa genus Following alignment and editing, we were unable to identify indels or stop codons in the sequences from either species. However, using the most common haplotype for each species as a reference, eight non-synonymous substitutions were observed in 17 H. armigera individuals sampled, which yielded 31 haplotypes with a haplotype diversity (Hd) of 0.821 and a nucleotide diversity (Pi) of 0.0028. Sequence analysis of the 134 sampled H. zea individuals identified 19 polymorphic sites, which yielded 20 haplotypes with an Hd of 0.420 and a Pi of 0.0011  sampled on summer crops; however, Fu's Fs test was significant (p<0.01). The Tajima's D and Fu's Fs test results for both H. armigera and H. zea were negative and significant when the individuals were tested as a single group and when the individuals were split into groups based on the crop on which they were sampled . These results indicate an excess of low frequency polymorphisms and are consistent with either population expansion or purifying selection  or H. zea .Twenty-six polymorphic sites were found among the 139 f 0.0011 . No signf 0.0011 . The reselection . In addiH. armigera (ΦST = 0.071) and 94.22% for H. zea (ΦST = 0.058) , host group , and each host type  (H. armigera was not significant for any of the three tested groups (p>0.10). The AMOVA with three hierarchical levels divided the H. zea individuals into groups by time (winter and summer crops), which showed a larger variation within populations ; the group division was not significant (p>0.10)  with two hierarchical levels showed that the greatest amount of total variation was accounted for by differences among individuals within populations: 92.89% for = 0.058) . For the= 0.068) . The gro(p>0.10) .Helicoverpa spp. represented in the haplotype network revealed a close genetic relation between H. armigera and H. zea, which were separated by only 13 mutational steps (H. armigera: H1 (31.65%) and H3 (23.02%), which were located at the center of the haplotype network. The other haplotypes of H. armigera, with the exception of haplotype H2 (15.83%), all had frequencies below 5%. Haplotypes H19, H18, H16, H12, H21, and H25 formed an outer cluster within the haplotype network of H. armigera  surrounded by low-frequency haplotypes (<5%)  .Helicoverpa spp. specimens sampled in Brazil into two monophyletic clades (H. armigera and H. zea) with an associated probability of 99%  was separated from the other individuals with an associated probability of 98%. Finally, Helicoverpa gelotopoeon showed a closer phylogenetic relationship to H. armigera and H. zea compared with H. assulta . The estimated model parameters were based on empirical base frequencies , with the proportion of invariable sites (I) set to 0.7393 and the gamma distribution shape parameter set to 0.5778. The consensus tree generated by the Bayesian analysis divided the y of 99% . The pro assulta .The haplotype network constructed using the edited sequences collected in Brazil, along with numerous Old World sequences, identified 38 distinct haplotypes . H1 28%.H. armigera throughout the Midwest and Northeast of Brazil on a variety of crops, particularly dicotyledons, beans, soybeans, and cotton as well as, to a lesser extent, millet, sorghum, and maize. This pest was not found on maize crops in the Midwest, Southeast, or South of Brazil, despite the fact that these crops were initially identified as sources of H. armigera in this system. H. armigera individuals associated with maize crops were only found at a single sampling site in the Northeast (state of Bahia) during February 2013. In contrast, H. zea individuals were essentially found only on maize crops, with the exception of a few individuals collected from millet and cotton crops, where H. zea individuals were found alongside H. armigera individuals. Before the documentation of H. armigera in Brazil in 2013, we had hypothesized that major source of Helicoverpa spp. attacking different host plant was maize crops. However, our findings showed that targeting the control of H. armigera on maize crops may not be effective because H. zea was the predominant species in this host plant. The possibility of the formation of hybrid individuals between these two species, which has been reported under laboratory conditions Our results indicate a widespread distribution for H. armigera and H. zea populations within the Brazilian territory. Population expansions were also consistent with the Haplotype network structure, which was characteristic of species undergoing processes of demographic expansion H. armigera individuals showed two primary maternal lineages, whereas H. zea showed a single primary lineage, all of which were surrounded by numerous lower-frequency haplotypes. Therefore, these central high-frequency haplotypes represent the ancestral haplotypes, with the low-frequency haplotypes more recently derived H. armigera population expansion are likely because of the recent introduction of this pest into Brazil. Following the founder event, during which a portion of the overall genetic diversity of the species was introduced to Brazil, the H. armigera population further propagated. According to Nibouche et al. H. armigera can migrate as far as 2,000 km, which likely facilitated the colonization of a variety of crops. The migration and colonization of crop areas by a small group of individuals can cause bottleneck effects, which, combined with plague population-suppression strategies , can lead to the types of demographic expansions observed for H. zea and H. armigera in Brazil H. zea. Additionally, assuming that not all COI variation is neutral, Helicoverpa spp. populations could be suffering selection, especially considering that populations have colonized new environments recently. However, further studies using a larger number of molecular markers from nuclear and mitochondrial genome regions would answer these questions. The H. armigera and H. zea population genetics were not structured according to space, time (winter and summer crops), or host (crops). Unstructured genetic networks have been reported for other populations of these two pest species in other parts of the world, which were based on several molecular markers, including mtDNA, allozymes, and microsatellites Demographic analyses using neutrality tests and a Mismatch Distribution Analysis indicated an expansion of the H. armigera and the wide distribution of the two ancestral maternal lineages within the Brazilian territory did not allow us to infer any hypothetical invasion or dispersal routes for this species within the region. However, we noted that the haplotype and nucleotide diversities found for H. armigera in Brazil are similar to or greater than those reported for natural H. armigera populations in the Old World H. armigera haplotype network, formed by haplotypes H19, H18, H16, H12, H21, and H25, is noteworthy for having the greatest genetic distance from the central haplotypes (H1 and H3), and these haplotypes have yet to be identified in Old World populations H. armigera during the first half of 2012, support the hypothesis of an invasion period prior to the first reports of this species in Brazil. Alternatively, these findings are also consistent with a more recent invasion that involved a large gene pool, multiple invasion events, or some combination of these events.The unstructured population of H. armigera and H. zea in the haplotype network analysis and the Bayesian phylogeny confirms the close genetic relatedness of these two species. Therefore, the reported co-occurrence of these species in time and space, as well as on the same hosts (as described here), could allow for the formation of hybrid individuals, which has been reported under laboratory conditions H. armigera, which is reportedly resistant to control methods, and H. zea, which has adapted to the environmental conditions of the American continent, may enable gene transfer and fixation in some individuals. Therefore, hybridization may enable the selection of breeds with enhanced hybrid vigor and the ability to rapidly adapt to current management and suppression methods.The low genetic divergence observed between H. armigera and H. zea in Brazil. Therefore, the sustainable management of H. armigera will likely become a significant challenge for Brazilian entomology in the coming years, especially considering the polyphagous feeding habit, the great dispersal ability, and the numerous reports of resistance to insecticides and Bt crops for this insect H. armigera expanding throughout the American territory and perhaps reaching agricultural areas in Central and North America. However, it was not possible to trace the invasion and dispersal routes of H. armigera in the Brazilian territory. Nevertheless, the hypotheses of an invasion period prior to the first reports in the literature and/or an invasion that involved a diverse gene pool are both consistent with the observed high incidence and rapid adaptation of H. armigera in the Brazilian territory. Our confirmation that the predominant maternal lineages in the Brazilian territory are the same compared with those in Europe and Asia may represent a starting point to guide H. armigera management programs. Indeed, control strategies have a greater chance of success when reliable information is gathered in the regions where the pests, their hosts, and their natural enemies have co-evolved over a significant period of time.The population studies described in this study indicate a recent demographic expansion and a high mitochondrial genetic diversity for Permit access to collect material used in our research at various crop sites was granted by respective growers. GPS coordinates of each location are listed in H. zea population dynamics and primarily involved maize fields. However, attacks on soybean, cotton, bean, sorghum, and millet crops were also reported between May 2012 and April 2013 . Therefore, we directed our sampling efforts towards a variety of crops and regions throughout Brazil. We also focused on the Western region of Bahia State, Brazil, which was the site of numerous Helicoverpa spp. attacks, to determine whether maize crops were the main source of H. zea in the Brazilian agricultural system. A total of 274 Helicoverpa caterpillars were collected at 19 sampling sites from six different crops  and HCO(R) (5′ - TAA ACT TCA GGG TGA CCA AAA AAT CA - 3′) 2, 0.003 mg.mL−1 BSA, 6.25 mM dNTPs, 10 pmol each primer, 1 U Taq DNA Polymerase , and 10% 10× Taq Buffer in a final volume of 25 µL. The PCR program consisted of an initial denaturation step at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 45°C for 30 s, and polymerization at 72°C for 1.5 min, with a final extension step at 72°C for 10 min. Following amplification, the aliquots were visually inspected using agarose gel (1.5% w/v) electrophoresis. The amplicons were purified by ethanol precipitation, and a second round of amplification was performed using the Big Dye Terminator v3.1 Cycle Sequencing system , which was followed by further purification. DNA sequencing was performed using the ABI3500xl automated genetic analyzer  at the State University of Campinas .Genomic DNA was isolated from the thorax of each adult using an Invisorb Spin Tissue Kit , according to the manufacturer's protocol. A fragment of the COI mitochondrial gene was amplified by polymerase chain reaction (PCR) with the primers LCO(F) ; (ii) stop codons leading to premature protein termination; and (iii) increased rates of non-synonymous mutations. The presence of signatures (i) and (ii) was considered sufficient to regard a sequence as a COI numt. In the presence of signatures (i) or (ii), signature (iii) was used to confirm the sequence as a numt. The presence of signature (iii) alone was not considered sufficient to define a sequence as a numt.The MEGA version 4 P-value was below 0.02. The diversity estimates and neutrality tests were performed using all sampled individuals from each species, which were divided into winter-crop and summer-crop groups. A Mismatch Distribution Analysis using a spatial expansion model P-value support).Haplotype and nucleotide diversity parameters for each species were estimated using the DnaSP version 5 Using Arlequin 3.1, we also performed an AMOVA at the two- and three-hierarchy levels Helicoverpa spp. haplotypes were determined by constructing a maximum parsimony network Genetic differences and connections among Helicoverpa assulta (Guenée) (GenBank Accession number: EU768937), H. gelotopoeon Dyar (EU768938), and H. virescens (IN799050) sequences were included as outgroups for the Bayesian analysis. We obtained a 50%-majority-rule consensus tree with posterior probabilities that were equal to the bipartition frequencies.We used the distance matrix option in the PAUP *4.0 software program to calculate the inter- and intra-species genetic distances, which were inferred using the nucleotide substitution model and the Akaike Information Criteria H. armigera sequences we collected in Brazil. In particular, 73 sequences were obtained from specimens collected in China (N = 35) [GenBank Accession numbers GQ892840 - GQ892855, GQ995232 - GQ995244 H. armigera populations in Brazil.Seventy-three sequences from a variety of Old World sites that were present in GenBank were included with the 139 Figure S1Bayesian phylogenetic tree of H. armigera and H. zea individuals sampled in Brazil. This phylogenetic tree is based on partial COI haplotype sequences and includes H. assulta and H. gelotopoeon sequences. Numbers near the interior branches indicate the posterior probability  values. The outgroup used was Heliothis virescens. H. armigera COI haplotypes and Genbank Accession numbers can be found in (TIF)Click here for additional data file.Table S1Hierarchical analysis of molecular variance (AMOVA), for population genetics structure of Helicoverpa armigera and H. zea with a mithocondrial (COI) region marker.(DOCX)Click here for additional data file.Table S2Global Helicoverpa armigera including the Brazilian H. armigera haplotypes, and relevant GenBank Accession numbers. Numbers of individuals sequenced from each locality are indicated in parentheses.(DOCX)Click here for additional data file.


DNA barcode (COI‐5P) surveys of the macroalgal flora along the northwest Atlantic coast, we discovered a population of Ceramium secundatum in Narragansett Bay, Rhode Island, USA. This species is regarded as common and widespread in the northeast Atlantic, ranging from Norway to Morocco, but until now has not been reported from the western Atlantic. Several lines of evidence suggest that C. secundatum may be introduced to Narragansett Bay: (1) despite extensive collecting, specimens have only been obtained from a limited geographic range in the northwest Atlantic; (2) three other nonindigenous seaweed species are reportedly introduced in this region, which is thought to be a consequence of shipping; and (3) this species is introduced to South Africa and New Zealand. To investigate this suspected introduction, we applied population genetic analyses (using the cox2‐3 spacer) to compare the Narragansett Bay C. secundatum population to native populations in the Republic of Ireland and the United Kingdom. Collectively, analyses of biogeographical and molecular data indicate that C. secundatum is likely introduced to Narragansett Bay. The implications of this discovery are discussed.During ongoing  Monitoring of marine algal diversity is critical not only for understanding species richness across biogeographical ranges, but for detecting biological changes in the marine environment such as the introduction of nonindigenous species to novel regions. In recent studies, molecular tools have improved environmental monitoring, as well as facilitated the rapid detection of nonindigenous and potentially invasive species  has unquestionably made a significant contribution toward establishing accurate accounts of species diversity and distributions  have permitted the assignment of one of these genetic groups to Ceramium secundatum Lyngbye, a new record for the Northwest Atlantic flora. In this study, we assess the presence of C. secundatum in the northwest Atlantic using population genetic tools to ascertain whether it is native or introduced to this region. To accomplish this, we employed the noncoding intergenic region between the cytochrome oxidase subunit 2 and 3 genes (the cox2‐3 spacer), a quickly evolving haploid  genetic marker that has proven useful at the population level in red algae  lower genetic diversity within the Narragansett Bay population than comparable populations within this species' native range; and (2) haplotypes within the Narragansett Bay population represent a subset of those present within the native population . Subsequently, additional specimens of C. secundatum were collected from Narragansett Bay and four sites within the reported native range (within the Republic of Ireland and the United Kingdom) for a total of 91 individuals to be included in population genetic analyses (Table S2. Supporting information). In the field, specimens were collected from subtidal environments by SCUBA, snorkeling, wading at low tide or from pools in the lower intertidal. All specimens were dried and preserved as vouchers, either as a herbarium press or in a vial of silica, and a subsample of material was taken for DNA sequencing (Saunders and McDevit As part of an ongoing floristic survey (COI‐5P), 188 specimens assignable to the “morphospecies” rbcL sequence data for representatives of each resulting COI‐5P genetic group  . To identify haplotypes, quantify their abundance within each population, and generate a 95% parsimony network, we used TCS  (GWS018041 (GenBank accession = KT250273) is a 100% match to GenBank accession #AF439287; Fig. C. secundatum was morphologically consistent with what is reported as C. virgatum for the northwest Atlantic  and were almost exclusively subtidal (the exception being one tidepool collection), C. virgatum specimens were collected from numerous localities along the northwest Atlantic coast from both subtidal and intertidal habitats .While conducting a routine COI‐5P survey of the genus atum n = 6  for 16 of the 8 bp for Eleven haplotypes (HAP A‐K) were identified by our genetic analyses Fig. ; Table 2C. secundatum was lower than populations within the native range and that the haplotypes (with the exception of HAP C) were subsets of those within the native range, all consistent with the expected characteristics of a species introduction.Collectively, our results indicated that the genetic variation within the Narragansett Bay population of Introduced species have the potential to compromise native biodiversity, potentially endangering or further threatening rare species through the disruption of the ecological community balance , C. secundatum would most likely have remained cryptic in the northwest Atlantic flora due to morphological attributes shared with C. virgatum , and lower nucleotide diversity (π) than populations within the native range . This may be an indication that C. secundatum was recently established at Dorn Lagoon, but was more likely a consequence of the limited area that specimens were found growing at this site, as a small population is more subject to genetic drift in the absence of gene flow , and rarefaction analyses do not make predictions for haplotypes beyond the sampled range. Therefore, it is likely that there are even more than 51 haplotypes for this species within its native range, which makes it more likely that HAP C was overlooked in the native range. The fact that each population had at least one unique haplotype, with the exception of Dingle, further emphasizes the probability that there are unsampled haplotypes within this species' native range.Interestingly, there was one individual in the Narragansett Bay population that had a unique haplotype, HAP C, which was contrary to expectations for a population arising from an introduction. It is possible that HAP C is the result of a mutation within the Narragansett Bay population; however, rarefaction analyses  estimated upwards of 51 haplotypes for the sampled region; and acknowledging that we collected specimens representing 11 of these haplotypes there is a considerable possibility that HAP C in our Narragansett Bay population represents an unsampled haplotype within the native range. Notably, we only sampled a small portion , we feel these circumstances favor the hypothesis that C. secundatum was introduced to this region.From our comparisons of genetic variation and haplotypes within the C. secundatum in the northwest Atlantic, we review what is known about the biology of this species relative to the new habitats. One weakness of this approach is that what is known about the biology of this species is based on observations within its native range and species may behave unpredictably when introduced to novel ecosystems  Fensholt, M. Bruce, pers. observation), epilithically, or on artificial materials in marinas  from all other northwest Atlantic Ceramium species identified during our ongoing DNA barcoding surveys , we do not regard C. secundatum as having the potential to hybridize with the native species and thus “genetic pollution” is not a concern.In considering reproduction, we did not find any reports of s et al.  eluded tC. secundatum is not an aggressive competitor with the native flora and has had a longer presence in the region. While it's reasonable to postulate the mode of introduction given the extensive shipping history of Narragansett Bay and the putative mode of other macroalgal introductions  may reflect a recent introduction that has yet to spread, but could also be because Grateloupia turuturu Yamada in the northwest Atlantic and Sargassum muticum in the northeast Pacific ; NCBI SRA:COI5‐P sequences: GenBank accessions (Table S1); NSBI SRA:additional specimen information available in BOLDTable S1. Collection and GenBank information for specimens included in our DNA barcoding (COI‐5P) survey of northwest Atlantic Ceramium spp.Table S2. Ceramium secundatum collections included in population genetic (cox2‐3 spacer) analyses and their respective GenBank accessions.Table S3. Output from EstimateS analyses conducted in order to investigate the possibility of unsampled haplotypes within each of the four sites sampled within the native range of C. secundatum.Click here for additional data file.


Objectives. The present study aimed to investigate the effects of guided imagery training on heart rate variability in individuals while performing spaceflight emergency tasks. Materials and Methods. Twenty-one student subjects were recruited for the experiment and randomly divided into two groups: imagery group (n = 11) and control group (n = 10). The imagery group received instructor-guided imagery (session 1) and self-guided imagery training (session 2) consecutively, while the control group only received conventional training. Electrocardiograms of the subjects were recorded during their performance of nine spaceflight emergency tasks after imagery training. Results. In both of the sessions, the root mean square of successive differences (RMSSD), the standard deviation of all normal NN (SDNN), the proportion of NN50 divided by the total number of NNs (PNN50), the very low frequency (VLF), the low frequency (LF), the high frequency (HF), and the total power (TP) in the imagery group were significantly higher than those in the control group. Moreover, LF/HF of the subjects after instructor-guided imagery training was lower than that after self-guided imagery training. Conclusions. Guided imagery was an effective regulator for HRV indices and could be a potential stress countermeasure in performing spaceflight tasks. Numerous studies and anecdotes showed that astronauts experienced many negative emotions in space, such as anxiety, stress, and depression, which would deteriorate even further under emergency situations and ultimately lead to unreliable or incorrect operations . Some reAutonomic cardiac control is crucial in health and social behavior. As a marker of cardiac autonomic nervous system function, heart rate variability (HRV) has been reported to indicate cardiac health, cardiac mortality and morbidity, and overall mortality risk . CardiacAs a mind-body relaxation technique, guided imagery has been widely used for decreasing patient's stress and anxiety and increasing an athlete's sport performance , 5. PrevHRV is the change in the time interval between heartbeats, from beat to beat. It is controlled by the autonomic nervous system including the sympathetic nervous system (SNS) and the parasympathetic nervous system (PNS). Generally, the SNS activity increases heart rate, and the PNS activity decreases heart rate. HRV is believed to be an indicator of the dynamic interaction and balance between the SNS and the PNS , 14. ThuGiven the high demand of operation pressure in manned spaceflight, the present study aimed to explore the impact of guided imagery training and self-guided imagery training on HRV of Chinese operators when performing spaceflight emergency tasks. This study revealed the enhancing effects of guided imagery on the indices of HRV and identified how instructor-guided imagery plays a more effective role in intervention than self-guided imagery.The study was approved by the ethics commission of China Astronaut Research and Training Center. All subjects signed consent forms prior to participating in the experiment.A between-subjects experiment was designed to explore the effects of guided imagery training on physiological variables. The presence of training is a between-subject factor which has two groups: one is imagery group, and the other is control group. For the imagery group, subjects received two kinds of imagery training: instructor-guided and self-guided imagery. On the contrary, subjects in the control group just used the corresponding time to sit still and review the operations. In addition to investigating whether training is effective, another purpose of this study is to investigate if self-guided imagery has the same effect as instructor-guided imagery does. Therefore, both instructor-guided imagery and self-guided imagery belong to the imagery group, but the reference within such group was instructor-guided imagery condition.n = 11) and control group (n = 10). Their ages ranged from 21 to 26 years .The best subjects for this study may be current Chinese astronauts. Unfortunately, it was impossible to have Chinese astronauts participating in this study for the limited number and the free time of them. In the future, some new astronauts will be selected from among scientists and engineers. Besides, most of Chinese astronauts are male. Thus, we recruited twenty-one male students from the astronautics department of Tsinghua University to meet the astronaut selection criterion from the aspect of education background and gender. They were randomly divided into two groups: imagery group  and the noise was avoided. Second, the subjects were asked to sit in a chair fixed in the ground and to avoid abrupt and severe body movement. Only forearms and fingers were asked to finish the nine spaceflight operations. These controls generally reduce, if not eliminate, possible artefacts that could influence the HRV analyses.There are various ways to practice mental imagery. Generally, mental imagery was guided by some instructions given by an experienced psychology expert , 11, whiBefore the formal experiment, the two groups learned how to perform the nine spaceflight emergency tasks for 90 minutes to make sure the subjects established the correct mental operational model and reach a skill plateau. The nine spaceflight emergency tasks were chosen from the Chinese spaceflight operation handbook. One of the emergency tasks is a procedural activity to handle one malfunction of a spacecraft subsystem. Those activities only induced some minor body movements such as brief reporting, mouse clicking, or putting/loosening the button. The nine tasks appeared in a random sequence for each subject of one group. One subject took about 10 minutes to finish all the nine tasks and called one trail.The formal experiment included two sessions. In session 1, the imagery group received instructor-guided imagery training, which was conducted by an experienced psychologist. Before guided imagery, the subjects were asked to review the operations. Then, the subjects were led to a quiet room where they lay down on a comfortable chair and closed their eyes. Firstly, the imagery group received intervention instructions and was guided to an image while staying in a welcoming, secure, and comfortable place, to experience some fine things and sceneries through visual, auditory, and body sensation in imagery. The guided words were developed by a Swedish psychological therapist and several clinical psychological experts in Astronaut Center of China. Then the subjects were guided to imagine the operations of the nine tasks. It generally took about half an hour for instructor-guided imagery training. After guided imagery, the subject practiced the nine tasks for two trials. And the physiological data was recorded when subjects were performing the nine spaceflight emergency tasks.In session 2, self-guided imagery training was conducted by the subjects themselves in accordance with the experience of instructor-guided imagery. After self-guided imagery, the subject practiced the nine tasks for two trials and the physiology data was recorded.Subjects in the control group also attend two session's experiment. In each session, they practiced the nine tasks for two trials as the imagery group without imagery training. And the physiology data was recorded at the corresponding trials.Self-reports were given by the subjects in imagery group after mental imagery training. The reports were about the effect imagery training had on the subjects.Electrocardiogram (ECG) was recorded for all subjects by one dynamic multiparametric physiological detector called KF2 . KF2 recorded ECG with 3 leads at a rate of 250 Hz. KF2 was worn around the chest of each subject when performing the nine spaceflight emergency tasks. Disposable silver/silver chloride ECG electrodes were placed at the standard location for ECG recording. And the ECG data were stored on a Secure Digital Card for offline analysis by HRV processing software.The HRV processing software provides HRV indices in time and frequency domains through the following steps. Firstly, ECG data was automatically corrected firstly using an artefact detection algorithm . Then FaHeart rate variability was analyzed in time and frequency domains . Time doFrequency domain  measurement of HRV was obtained by Fast Fourier Transform (FFT) which is one of spectral analysis methods to determine the power at different frequencies and helps to distinguish the contribution of the PNS and SNS to the variability in heart rate. Frequency domain measurements included very low frequency , low frequency , and high frequency  power, as well as normalized values , the total power , and LF/HF ratio. The LF and HF components are considered as an index of sympathetic and parasympathetic modulation, respectively, and the LF to HF ratio reflects the global sympathovagal balance . Total pStationarity is an important prerequisite in analyzing time series data such as heart rate. Therefore, we checked whether our data is stationary before carrying out the formal analyses. We used the widely used Augmented Dickey-Fuller test which seems to be more suitable as our time series data is quite long ; therefore the parameters recommended by Magagnin et al.  might noHRV indices of each subject were calculated over a short duration of 5 minutes by the HRV processing software. Before the statistical analysis, a homogeneity test of HRV between the imagery and control groups was conducted. The data were acquired from the last two trails of the 90 min practicing period before the formal experiment.P < 0.05 were considered significant. The power of the ANOVA was calculated as reported in this study.The Shapiro-Wilk test and Levene's test were used to test whether the given HRV data presented with a normal distribution. Two-way repeated measures ANOVA was used to assess the group effect (control group versus imagery group), the effect of training techniques, and interaction between them. All analyses were carried out in SPSS software Release 20 and all statistical tests with a Homogeneity testing revealed that heart rate variability consisting of SDNN, RMSDD, PNN50, CV, VLF, LF, HF, LF/HF, and TP was equally distributed between the imagery group and control group.P = 0.002, 0.033, and 0.001, resp.; power = 0.900, 0.579, and 0.941, resp.), CV of imagery group was marginally significantly higher than that of control group , and HR of two groups has no statistical significance . The interaction effect of imagery training and training technique was not significant. Moreover, analysis of HR, RMSDD, SDNN, CV, and PNN50 showed that there was no statistical significance between the two techniques. Two-way repeated measures ANOVA was conducted on the HRV indices in the time domain including HR, RMSDD, SDNN, CV, and PNN50. The results showed that RMSDD, SDNN, and PNN50 of imagery group were significantly higher than those of control group , and VLF, LF, HF, and TP of imagery group were higher than those of control group. The effect of the training technique on LF/HF was marginally significant ; LF/HF of the subjects after instructor-guided imagery training was lower than that after self-guided imagery training. Two-way repeated measures ANOVA was conducted on the HRV indices in frequency including VLF, LF, HF, LF/HF, TP, LFNU, and HFNU. The results showed that the effect of imagery training on the VLF, LF, HF, and TP was significant  is a marker of cardiac autonomic nervous system function and has been presented as a good tool to study physiological effects of work-related stress , 54 and Guided imagery is a simple, easily taught, and acquired method, but it has rarely been implemented as a training method for spaceflight operations. It was typically used as a psychological intervention tool before a manned spaceflight mission or applied to astronauts when some negative emotional symptoms appear. Plessinger recommended that athlete training incorporates mental imagery along with physical practice . Not onlIt should be noted that this study has some limitations. First of all, small population size is a continuous worrisome issue for researchers like us whose job is to train a special group of operators. As performing the tasks in our research requires a complex simulation system which is quite costly to use, it is very difficult to use a large size of subjects. Specifically, subjects in this study were all students from the same university excluding the Chinese astronauts. Therefore, some of the results will be tested further in astronaut training and other experiments. Moreover, the effect of mental imagery is limited by the engagement of subjects, which was reported by subjective reports from subjects. Further investigation requires the combination of electroencephalogram (EEG) and other physiological indicators to evaluate the engagement of subjects and thus to objectively analyze the role of mental imagery and the mechanism itself. In addition, virtual reality technology can be used to design mental imagery training programs to improve the effect of imagery training.The results of this study showed that guided imagery training significantly increased HRV indices in Chinese subjects while performing spaceflight emergency tasks. It implied that guided imagery training is one effective way to decrease stress and therefore improve operation performance. From the current data, we can conclude that the training is at least effective during the 10-minute operations before which the astronauts were asked to do such imagery work either instructor-guided or self-guided. As concluded from this study, guided imagery training seems promising and beneficial to ensure astronauts well-being and stable performance in orbit. Hence, a program with operation practice combined with imagery training was recommended for astronaut operation training. In practice, self-guided imagery was applied to the astronauts in Shenzhou Nine and Shenzhou Ten before they conducted Manually Rendezvous and Docking. The effect of imagery was committed by the astronauts.


The diversity and genetic relationships among two accessions of tall coconut palms collected in Brazil and seven accessions introduced from different geographic regions of the world were analyzed using 25 microsatellite primers, 19 of which were polymorphic and detected between 4 and 10 alleles per locus, with an average of 6.57. The observed and expected heterozygosity ranged from 0.25 and 0.40 in the Rennell Islands Tall (RIT) accession to 0.54 and 0.62 in the Polynesian Tall (PYT) accession. The analysis of genetic structure resulted in the formation of five distinct groups. The first group was formed by the accessions Brazilian Tall—Praia do Forte (BRTPF), Brazilian Tall—Merepe (BRTMe) and West African Tall (WAT); the second group consisted of Malaysian Tall (MLT); the third group of RIT; the fourth group of Vanuatu Tall (VTT); and the fifth group of Rotuman Tall (RTMT), Tonga Tall (TONT) and PYT. The dendrogram based on the nearest-neighbor method detected the formation of two main groups and five subgroups, indicating that the genetic relationships of the accessions are based on their geographic regions of origin. The analyses revealed genetic relationships between the accessions collected in Brazil and the accession from Africa, and among palms from South East Asia and the South Pacific, confirming the common origin of these accessions. The information obtained in this study can guide decisions on germplasm conservation activities and the efficient selection of genetically divergent parents for use in coconut breeding programs in Brazil, which are attempting to select for disease resistance, mainly to lethal yellowing, among other characteristics. Cocos nucifera L.) has played an important role in the mobility of humans as source of food and water. Its evolution, dissemination and classification can be considered as a logical sequence of spread by floating, selection by cultivation, hybridization and introgression of the two contrasting types—representing two important gene sets (Niu Kafa and Niu Vai)—which resulted in its wide range of varieties and a pantropical distribution [The coconut palm . Different characterization and evaluation activities have been conducted at the genebank, and the Brazilian Tall—Praia do Forte and Brazilian Tall—Merepe populations have shown to be superior in terms of their fruit components, primarily regarding fruit weight, solid albumen and coconut water volume [In 2006, the Germplasm Bank of Brazil was associated with the International Coconut Genetic Resources Network (COGENT) and was renamed the International Coconut Genebank for Latin America and the Caribbean (ICG-LAC). One of the many objectives of ICG-LAC is to be the host location for conserving of the genetic variability of the species in this region. There are 29 accessions of dwarf and tall coconut palms conserved at ICG-LAC, and despite the small number of accessions, it is the second most important genebank in the Americas, after that of Jamaica . The SSR marker analyses were performed as previously described [™ FS96 system , with DNF900 gel kit, for 120 minutes, at a current of 7.50 Kw to separate the DNA fragments, with a 5 pg/μL sensitivity and a 5 bp resolution. For this analysis, 1 μL of each amplification product was diluted in 79 μl of ultrapure water and placed in 96-well microplates. A 50-bp molecular weight marker (Invitrogen USA) was included in the last well of the microplate to determine the size of the amplified fragments.DNA was extracted from 500 mg of leaflets of the first leaf using the CTAB Protocol  with somescribed . The PCRE) and observed heterozygosity (HO) and Shannon indexThe program GENALEX 6.3  was emplE−HO)/HE = 1−(HO /HE)) and polymorphism information content (PIC) were calculated with PowerMarker version 3.25 [The coefficient of inbreeding  , which iThe presence of a secondary ΔK  suggestsThe third group was strongly mixed and formed by MLT and VTT, while the fourth clear group was formed by the RIT accession. The fifth group was formed by RTMT and PYT, while the sixth with the TONT accession, both of them with high levels of allele sharing. These results follow patterns similar to those reported in Southeast Asia and Oceania by other authors , who alsCluster analysis detected the formation of two main groups . The firThe genetic proximity between the accessions collected in Brazil and the WAT accession confirms the common origin of these accessions, as do the historical records that report that the tall coconut was introduced from the Cape Verde Islands. The strategic geographical location of the archipelago of Cape Verde in the Portuguese sea routes between Brazil and Africa contributed to these islands being used as a supply and trading outpost. The origin of these coconut palms would have been East Africa, India and Sri Lanka , when VaOther studies have confirmed genetic relationships between the Dominican Republic-Tall and the Mexican Atlantic Tall classified in the Indo-Atlantic Group, and populations of Brazilian Tall coconuts . These fIn Brazil, populations of tall coconut were initially formed by a few closely related individuals, representing only a small fraction of the genetic variation in the source population. From the State of Bahia in the northeast region of Brazil, tall coconut expanded all along the northeast coast, which is a region that provides optimal soil and climate conditions for the development of this species, allowing this region to become the largest coconut producer in the country  and the Within this scenario it is possible to suggest that some genetic variation exists between the materials introduced in Brazil five centuries ago and the WAT. In fact, this study detected significant differentiation between WAT and BRT accessions, both by the genetic structure analysis  as well The populations collected in Brazil belong to the Indo-Atlantic group and this group proved to be susceptible to lethal yellowing (LY) , hence iThe use of SSR markers increased the available information on the genetic relationships among the accessions collected in Brazil and those introduced from different geographic regions of the world that are preserved in the ICG-LAC. The accessions were assigned to the two main genetically distinct groups. The genetic relationships between accessions collected in Brazil and the African accession, as well as between the accessions of Southeast Asia and the South Pacific, agree with what is known about coconut in those regions. The information gathered in this study may guide decisions on germplasm conservation activities and the efficient selection of genetically divergent parents for use in coconut breeding programs in Brazil, that are attempting to select for resistance to lethal yellowing, among other characteristics.S1 Fig(TIF)Click here for additional data file.S2 FigPopulation assignments for each accession are shown at K = 2 to K = 9 groups. BRTPF = Brazilian Tall Praia do Forte, BRTMe = Brazilian Tall Merepe, WAT = West African Tall, MLT = Malayan Tall, RIT = Rennell Islands Tall, VTT = Vanuatu Tall, RTMT = Rotuman Tall, TONT = Tonga Tall and PYT = Polynesian Tall.(BMP)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.