The signal transducer and activator of transcription-3 (STAT3) induces transcription of genes that control differentiation, inflammation, proliferation, and tumor cell invasion. Cytokines such as interleukin-6 and interferon stimulate the specific tyrosine phosphorylation of STAT3, which confers its ability to bind consensus DNA targets. In addition, unphosphorylated STAT3 has been demonstrated to induce specific gene expression. STAT3 must gain entrance to the nucleus to impact transcription, however access to the nucleus is a tightly regulated process. Because nuclear trafficking is critical to the function of STAT3, we investigated the molecular mechanisms by which STAT3 is imported to the nucleus. Live cell imaging techniques were used with STAT3 tagged with green fluorescence protein (GFP) or photoactivatable GFP to follow the cellular dynamics of both unphosphorylated and tyrosine phosphorylated forms. Cytokine activation did not alter the rate of STAT3 nuclear import or nuclear export. In addition, Förster resonance energy transfer experiments revealed homomeric interaction of unphosphorylated STAT3 dependent on its amino terminus, but this dimerization is not necessary for its nuclear import. Previous work demonstrated the adapter importin-α3 binds to STAT3 and is required for nuclear import. To determine whether STAT3 nuclear import is mediated by the importin-α/importin-β1 heterodimer, the effects of siRNA to importin-β1 were evaluated. Results indicate STAT3 nuclear import is dependent on the function of importin-β1. Since the Ran GTPase is necessary to bind importin-β1 in the nucleus for release of importin-α-cargo, the effect of a GTPase deficient mutant of Ran was tested. Expression of the Ran interfering mutant inhibited STAT3 nuclear import. This study defines importin-α/importin-β1/Ran as the molecular mechanism by which STAT3 traffics to the nucleus. The biological functions of the signal transducer and activator of transcription 3 (STAT3) are diverse. Deletion of the STAT3 gene in mice results in embryonic lethality, and studies with conditional gene targeting have revealed impaired differentiation, proliferation, migration, or apoptosis in a wide variety of tissues Access to the cellular genome is limited by passage through nuclear pore complexes (NPC) within the nuclear membrane The direction of protein transport into or out of the nucleus is controlled by a gradient of Ran-GTP Regulation of transcription factor access to the nucleus provides a means to turn on or to turn off specific gene expression. Nuclear trafficking of STAT3 is therefore pivotal to its function as a transcription factor. We have demonstrated previously that nuclear import of STAT3 is independent of its tyrosine phosphorylation, and provided evidence for the requirement of importin-α3 Genetically encoded fluorescent proteins allow the study of molecular dynamics and protein-protein interactions in real time without artifacts of fixation necessary for immunofluorescence. As a signaling molecule and transcription factor, STAT3 transmits signals to the nucleus to change gene transcription. In order to evaluate STAT3 nuclear trafficking in a live cell, we tagged STAT3 with the enhanced green fluorescent protein (GFP). We first made certain that the function of STAT3-GFP was comparable to untagged STAT3. Two critical parameters of STAT3-GFP function were evaluated; tyrosine phosphorylation and transcriptional induction. Cells expressing STAT3-GFP or STAT3 were treated with interferon-α (IFNα), and specific tyrosine 705 phosphorylation was evaluated. Results show robust tyrosine phosphorylation of STAT3-GFP similar to the untagged STAT3  as has bSeveral studies have reported STAT3 accumulates in the nucleus only following tyrosine phosphorylation To study STAT3 movement in living cells, a variant of the GFP molecule was used with very low endogenous fluorescence that increases more than 100-fold following photoactivation The nuclear import rate of STAT3-GFP was evaluated prior to and following tyrosine phosphorylation using nuclear fluorescence recovery after photobleaching (FRAP) STAT3 cellular localization is dynamic, as it has been shown to enter and exit the nucleus To assess more accurately the nuclear export rate of STAT3-GFP in untreated or IFNα-treated cells, we used the technique of cytoplasmic fluorescence loss in photobleaching (FLIP) The crystal structure of tyrosine phosphorylated STAT3 bound to DNA has been solved, and it revealed a dimer in which monomer subunits interact via reciprocal SH2 domains and phosphotyrosine residues To evaluate unphosphorylated and tyrosine phosphorylated STAT3 interaction and its impact on nuclear import, we used the technique of Förster resonance energy transfer (FRET) after acceptor photobleaching We determined the contribution of the N-terminus of STAT3 (1–134a.a.) to promote interactions of unphosphorylated molecules by measuring FRET between STAT3-YFP and STAT3-CFP molecules lacking the N-terminus (a.a.135–770). The results showed little FRET between unphosphorylated STAT3 deletion mutants, indicating the N-terminus to be critical for interaction between unphosphorylated STAT3 monomers . The finStudies using cell fractionation techniques have reported that a portion of STAT3 is localized and functional within mitochondria Active transport is required for large molecules such as STAT3 to enter the nucleus, however the mechanism by which STAT3 gains entrance to the nucleus remained unresolved. Our studies clearly demonstrated the requirement of specific interaction with the adapter importin-α3 for STAT3 nuclear import To determine if a classical role of Ran GTPase is involved in the nuclear import of STAT3 by the importin-α/importinβ1 heterodimer, we evaluated the influence of a GTPase mutant of Ran, RanQ69L The involvement of Ran-GTP in the regulation of STAT3 nuclear import along with our previous finding of the role of importin-α3 suggested that importin-β1 would play a critical role. To directly evaluate the requirement of importin-β1 for STAT3 nuclear import we tested the effect of RNA interference. siRNA duplexes corresponding to a control gene (vimentin) or to importin-β1 were transfected into cells to decrease mRNA and protein levels. Subsequently STAT3-GFP was transfected into cells and evaluated for cellular localization . There wSTAT3 is activated by tyrosine phosphorylation in response to a variety of cytokines and growth factor receptor tyrosine kinases. For this reason it is not surprising that STAT3 serves critical functions in diverse physiological responses. However, persistent activity of STAT3 in response to unregulated hormone signaling or oncogenic tyrosine kinases leads to the induction of genes that positively control proliferation in cancer, tumor cell invasion, or chronic inflammation that can progress into life-long autoimmune diseases To transmit extracellular signals to the nucleus, STAT3 needs the ability to shuttle between cytoplasmic and nuclear compartments. We have provided evidence with live cell imaging that the nuclear import of STAT3 and nuclear export of STAT3 occurs continually independent of its state of tyrosine phosphorylation. In addition, the rate of import and export does not change following phosphorylation. Our studies are not dependent on a fixation process for immunofluorescence that can perturb cellular architecture or can be influenced by cross-reactive antibodies. The results are consistent with previous evidence identifying a nuclear import signal residing within the coiled coil domain of STAT3 We have provided evidence that unphosphorylated STAT3 forms dimers or oligomers by homomeric interaction between their N-termini (1–135a.a.). The FRET technique with photobleaching revealed STAT3 interactions in both tyrosine phosphorylated and unphosphorylated forms. Although the crystal structure of unphosphorylated STAT3 core fragment was solved as a monomer, this core fragment lacked the N-terminus. Our results support the concept that the structures of unphosphorylated and tyrosine phosphorylated STAT3 may be similar to that of STAT1. The tyrosine phosphorylated dimers associate via the SH2 domain and phosphotyrosine in a ‘parallel’ conformation, whereas the unphosphorylated dimers associate via N-termini in an ‘anti-parallel’ conformation The spatial and temporal localization of a protein within the cell can dictate its biological effects. Two studies have used cell fractionation to report STAT3 localization within mitochondria to regulate oxidative phosphorylation The mechanism by which STAT3 is imported to the nucleus has been proposed to occur independent of active transport and importins, and alternatively to require association with a Rac-GAP In conclusion, our live cell imaging studies clearly show that STAT3 is continuously imported to the nucleus and exported independent of tyrosine phosphorylation, and nuclear import is mediated by the importin-α/importin-β1-Ran system. This knowledge of the molecular interactions that mediate STAT3 nuclear trafficking is critical to provide a basis to develop strategies to block its action in cancer and autoimmunity.HeLa, Hep3B, and 293FT cells (ATCC) were cultured in DMEM with 10% fetal bovine serum, 1 mM L-glutamine, and 1% v/v penicillin/streptomycin. For microscopy, cells were seeded in glass bottom tissue culture dishes (Mattek Corp.) or on glass coverslips. Transfections were performed with TransIT-LT1 reagent (Mirus Bio LLC). Cells were cultured in serum-free media for 12 hours prior to experiments. Cells were treated with 20 ng/ml IL-6 , IFNα 1000 units/ml , or TNF (Invitrogen PHC3015).STAT3  was cloned into the vectors pEGFP-N1 (STAT3-GFP), pEYFP-N1 (STAT3-YFP), and pECFP-N1 (STAT3-CPF) (Clontech). STAT3 DNA-binding mutant (VVV) was cloned into pEGFP-N1 Cells were treated with small interfering RNA (siRNA) corresponding to importin-β1 or vimentin control (Qiagen) as described Western blots were performed as described previously using the following antibodies: anti-phospho-tyrosine (p705) STAT3 , anti-STAT3 (Santa Cruz sc-482), anti-importin-β1 (Santa Cruz H-7 sc-137016), anti-tubulin (Sigma B5-1-2), anti-rabbit (Alexa-labeled Invitrogen A21109), and anti-mouse HRP  2 control  Live cell imaging was performed using a Zeiss LSM 510 META NLO Two-Photon Laser Scanning Confocal Microscope System and cell chamber system with 37°C temperature control  and COFigure S1Accurate tyrosine phosphorylation of endogenous STAT3 and STAT3-GFP in HeLa and Hep3B cells in response to cytokines. Cells were transiently transfected with STAT3-GFP and serum-starved overnight. HeLa cells were treated with 1000 U/ml IFNα for one hour and Hep3B cells were treated with 20 ng/ml IL-6 for one hour. Endogenous STAT3 (ENDO) and STAT3-GFP were detected by Western blot with antibodies to STAT3 (bottom panel) or to tyrosine phosphorylated STAT3 (pY-STAT3) (top panel).(TIF)Click here for additional data file.Figure S2STAT3 intra-nuclear mobility reduced with DNA binding. Nuclear FRAP experiments were performed in HeLa cells expressing STAT3-GFP or a mutant of STAT3 that cannot bind DNA (STAT3-VVV-GFP) with or without IFNα stimulation. A limited ROI strip area (31 µm2) was selected for photobleaching, and kinetics of fluorescence recovery into this area was quantified. A) Time-lapse imaging of STAT3-GFP to evaluate intranuclear mobility. B) Graphic quantitation of multiple experimental results with STAT3-GFP in serum-starved cells untreated  or IFNα-treated (dashed line). C) The half-time of fluorescence recovery in seconds was calculated from multiple experiments in serum starved cells untreated  or IFNα-treated (black bars) for wild type STAT3-GFP  or the DNA binding mutant STAT3-VVV-GFP .(TIF)Click here for additional data file.Figure S3STAT3-GFP exclusion from mitochondria in Hep3B cells. Hep3B cells expressing STAT3-GFP were treated with MitoTracker Orange, a cell permeable dye for mitochondria. Live cell imaging was used to detect the localization of STAT3-GFP and mitochondria. Images captured with Zeiss LSM 5 using maximal vertical resolution (<1 µm) with 40× oil objective or the 63× C-Apochromat (water) objective.(TIF)Click here for additional data file.Figure S4STAT3 nuclear import independent of Rac1.A) HeLa cells co-expressing STAT3-GFP and T7-Rac-N17 were serum-starved and untreated (control) or stimulated with IFNα Nuclear prominence of STAT3-GFP in cells expressing RacN17 is shown in left panels. Immunofluorescence of T7-RacN17 is shown in center panels with anti-T7 antibody. Merged images clearly indicate nuclear localization of STAT3-GFP in cells expressing Rac1-N17. B) The dominant negative action of Rac1-N17 was demonstrated by its ability to block Rac1-mediated activation of NF-κB in a dose-reponsive manner (TIF)Click here for additional data file.


Acipenser naccarii. A critically endangered endemic species of the Adriatic Sea tributaries, its persistence is strictly linked to the ex situ conservation of a single captive broodstock currently decimated to about 25 individuals, which represents the last remaining population of Adriatic sturgeon of certain wild origin. The genetic variability of three F1 broodstocks available as future breeders was estimated based on mitochondrial and microsatellite information and compared with the variability of the parental generation. Genetic data showed that the F1 stocks have only retained part of the genetic variation present in the original stock due to the few parent pairs used as founders. This prompts for the urgent improvement of the current F1 stocks by incorporating new founders that better represent the genetic diversity available. Following parental allocation based on band sharing values, we set up a user-friendly tool for selection of candidate breeders according to relatedness between all possible parent-pairs that secures the use of non-related individuals. The approach developed here could also be applied to other endangered tetraploid sturgeon species overexploited for caviar production, particularly in regions lacking proper infrastructure and/or expertise.While the current expansion of conservation genetics enables to address more efficiently the management of threatened species, alternative methods for genetic relatedness data analysis in polyploid species are necessary. Within this framework, we present a standardized and simple protocol specifically designed for polyploid species that can facilitate management of genetic diversity, as exemplified by the ex situ conservation program for the tetraploid Adriatic sturgeon  Management of captive breeding programs require routinely use of parentage analysis and, to a lesser extent, estimators of relatedness in order to evaluate the relationship among founders in the absence of parentage data. Pedigree information on all aquaculture broodstock is necessary for the estimation and management of inbreeding. When aiming at either livestock breeding or wildlife conservation, it is essential to control and minimize the rate of inbreeding, and therefore avoid mating among close relatives. Parentage assignment using molecular markers implies that parents and offspring are all genotyped at a given number of unlinked genetic markers, and that the information on genotypes is used to assign progeny to the correct parental pair http://www.iucnredlist.org). Since 1998, international trade in all species of sturgeons has been regulated under CITES (http://www.cites.org) owing to concerns over the impact of unsustainable harvesting of and illegal trade in sturgeon populations in the wild. Despite protective fishing regulations, sturgeon populations have been decimated due to human activities including historic exploitation for caviar (unfertilized sturgeon eggs), poaching, damming of rivers and habitat degradation ex situ conservation is strongly encouraged through the reproduction of the few wild breeders available and the subsequent rearing of the progenies This holds particularly true for sturgeons (order Acipenseriformes), of which about 50% of the species are polyploid. Sturgeons were once widely distributed and abundant in the Holarctic region but today exist in the wild only as fragmented and isolated populations with a limited geographic distribution Acipenser naccarii) a tetraploid species endemic for the North Adriatic region http://www.cites.org) and its status was updated in 2010 from “Vulnerable” to “Critically Endangered” by the IUCN (http://www.iucnredlist.org). In common with most Eurasian sturgeons, catch data show a dramatic population decline from over 2,000 kg per year in the early 1970s to about 200 kg per year in the early 1990s to only 19 individuals caught in 1993 Our species of interest is the Adriatic sturgeon , funded by local administrations, and are composed by about 100–200 animals each. Unfortunately, until now the breeding program has been conducted without any genetic input and breeders used for artificial reproduction were randomly paired. No information is available on how many or which F0 parent pairs were used to produce these F1 animals and, consequently, relatedness between individuals is unknown.A breeding program for the Adriatic sturgeon was initiated in 1977 with the transfer of immature wild specimens from the Po River  to the fish plant Azienda Agricola VIP . Following the successful reproduction in captivity of the Adriatic sturgeon for the first time in 1988, restocking practices were conducted in the period 1988–2009 in tributaries of the Po river et al. A. gueldenstaedtii haplotypes, pointing to a possible ancient introgression episode. However, nuclear variation using microsatellite and AFLP (Amplified Fragment Length Polymorphism) analysis did not support the Po group subdivision while confirming the differentiation between Po and Buna rivers.In the only available study on the evolutionary history of the Adriatic sturgeon, Ludwig The present study characterized the distribution of relatedness values within all remaining Adriatic sturgeons of certain wild origin. This included most of the animals that were employed by Azienda Agricola VIP as F0 parents back in 1977, even if presently dead, and whose progeny was either released in the restocking practices conducted in the period 1988–2009 or reared to be part of the F1 stocks currently available as future source of breeders. Ultimately, we propose a standardized method for the analysis of genetic relatedness data in polyploid species to be used as a simple approach to support practical breeding activities.d =  0.153). A two-fold drop in nucleotide diversity was also observed in all F1 samples  and across individuals. Since the homology between repeats could only be unequivocally established for the flanking region and the last repeat, the rest of repeats were discarded. Analysis of the 42 parental stock specimens revealed 14 variable sites , which defined 6 haplotypes . No chan samples .ST =  0.266–0.321) than among F1 samples (FST =  0.003–0.087) . Accordingly, pairwise genetic differences were higher between F0-F1 samples (F3–0.087) .A. naccarii control region sequence. A Blast search revealed the three haplotype sequences as belonging to the white sturgeon Acipenser transmontanus, a North American species frequently employed in Italy for aquaculture production of caviar. Consequently, these three individuals were excluded from further analyses.At the Piacenza F1 stock, 3 out of the 50 individuals analyzed presented an allochthonous haplotype, not present in the parental stock, and highly divergent from any et al. Phylogenetic network analyses confirmed the presence of two clearly distinct haplogroups, designated as Po1 and Po2 in the previous study of Ludwig The 24 microsatellite loci analyzed using a band sharing approach were moderately polymorphic, revealing 2 to 16 alleles per locus among the 42 individuals from the original parental stock . Each inST =  0.207–0.262; p<0.001) and among F1 stocks   was lower in comparison with the F0 stock (11.13) . Accordip<0.001) .We conducted a Multidimensional Scaling analysis using genetic distances at 8 microsatellite loci among all F1 stocks . When plParental allocation data indicated the F1 stocks to result from few founders, only five males and six females, in a total of nine pairings. As shown in A high correspondence was observed between observed and simulated distributions of pairwise genetic distance . Using tThe parental wild stock showed a moderately high level of genetic variation as indicated by high mtDNA haplotype diversities and the observation of a distinct microsatellite profile for each F0 individual. Since the bottleneck is recent, it is possible that the loss of genetic diversity is not yet detectable. This observation corroborates other recent empirical studies suggesting that long generation times may act as a buffering effect contributing to a reduction in the pace of genetic diversity erosion in theatened species Comparison of the 2005 and the present parental stock showed that a considerable part of genetic variability has been retained, despite the recent drop in population numbers . No loss of haplotypes was observed at the mtDNA level, with similar haplotype and nucleotide diversity values found between samples, while microsatellite data only showed a slight reduction in number of alleles in the present sample.By contrast, a drastic decline in genetic variability based on the mtDNA was found between F0 and F1 groups, which was less apparent at the microsatellite level. Both haplotype and nucleotide diversity diminished by 50%, from 6 haplotypes observed in the parental stock to 3 haplotypes in each F1 stock. The loss of genetic variation is probably a consequence of relatedness, with F1 individuals being more related than the parental wild stock. Parentage allocation assigned all F1 progeny to only five males and six females (nine pairings), and about 95% of the allocated individuals were assigned to four single families, with two of the crosses producing 55 and 52 of the F1 individuals, respectively.A. transmontanus, frequently used in Italy for aquaculture caviar production. This exotic species is reared at the Azienda Agricola VIP plant where A. transmontanus x A. naccarii hybrids are produced. We hypothesize that three of those hybrid individuals have been accidentally introduced in the rearing tanks with the F1 A. naccarii stocks. Those hybrids have been pointed out to the operators in the facilities and appropriately removed from the stock.Mitochondrial DNA analysis also detected the presence of three individuals with a highly divergent haplotype, identified as belonging to the North American species et al.et al.In agreement with the study of Ludwig The combination of eight highly polymorphic nuclear loci with additional mtDNA information resulted to be highly efficient in order to assign the F1 progeny to the correct F0 parental pair. All positive allocations were unequivocal, with no ambiguities due to multiple allocations. Only 13 out of 147 F1 individuals presented a profile that was incompatible with all possible parent-pair combinations, which suggests that those parents died between 1988 and 2005 and were not included in our sampling. It should be noted that the high allocation power in our analysis might be due to the limited number of parent pairs, and that the assignment of a progeny to a larger parental stock might require a higher number of marker loci.Besides its applicability in the selection of candidate breeders for artificial reproduction, parental assignment can be useful for the identification of individuals recaptured in the wild. A positive match would indicate an F1 produced at the Orzinuovi plant and released into the river Po as part of the frequent restocking activities conducted in the region since the late 1980s. A negative match would suggest either an individual of wild origin not derived from the Orzinuovi stock or an F1 produced at Orzinuovi whose parents died in the period 1988–2005 and were not included in our sampling.A first mandatory step toward an adequate restoration program for the Adriatic sturgeon is to conduct a complete and thorough genetic tagging of all the individuals currently reared in captivity. This includes (i) the three F1 broodstocks of Treviso, Piacenza and Orzinuovi, of which a sub-set of 150 individuals has been genetically analyzed in the present study; (ii) additional F1 individuals produced by the Azienda Agricola VIP through the years that have been maintained in the facilities.As our study pointed out, the F1 stocks have only retained a part of the genetic variation present in the original F0 stock due to the few founders used for reproduction, which underlines how little attention was paid to genetic diversity at the time of the establishment of those F1 stocks in the late 1980s/early 1990s. In that respect, a second action urgently needed is the improvement of the current F1 stocks by incorporating new founders with the aim of maximizing the amount of genetic diversity transmitted to coming generations.A. oxyrinchus) in the Baltic rivers Because of its higher genetic variability, the F0 parental stock should have priority over the F1s in any breeding scheme. A very low effective population size (<20) was recently estimated for the founder population of the Atlantic sturgeon , the relationship among all possible parent-pairs and their adequacy as future breeders is colour-coded according to inbreeding risk: white for unrelated individuals with low risk of inbreeding, light grey for half-sibs with medium risk, and, dark grey for full-sibs with high inbreeding risk. The implementation of this intuitive and easy-to-use method can facilitate the choice of candidate breeders that secure the use of non-related parents in captive breeding programs. For example, assuming that all breeders are tagged with a microchip, given an ovulating female, operators simply need to cross-check in the distance matrix which of the candidate males are adequate or not according to the colour-code, and a matching male can easily be selected for milt collection.A. gueldenstaedtii have been conducted through artificial reproduction and restocking practices in Russia and Iran, while other countries in the region  are only at the starting point in the development of conservation plans. Those countries do not currently have the capacity to conduct genetic analysis but plan to set up genetic laboratories shortly, and would greatly benefit from a simple and effective protocol for the genetic characterization of polyploid sturgeons like the one presented in this paper.The method is specifically designed for polyploid species and the band sharing approach is used due to the impossibility to infer the true genotype, thus it can be applied to all polyploid sturgeon species. The set up of a simple standardized protocol might be particularly relevant in regions with scarcity of facilities or lack of adequate expertise for the management of genetic diversity in sturgeon hatcheries. This is the case of the Caspian Sea region, where many sturgeon species with high commercial value for caviar production occur. The technical capacity within Caspian Sea caviar exporting countries to perform genetic research is variable. For instance, conservation activities in the Russian sturgeon ex situ restoration program utilizing genetic information and aiming at maximizing the original genetic diversity is extremely urgent. There is no evidence of natural reproduction, which makes the Adriatic sturgeon effectively dependent on captive breeding programs. In parallel with all genetic work, a successful reintroduction of Adriatic sturgeons obtained and raised in captivity into the wild can only be achieved if factors affecting the natural habitat are monitored, including restoration of spawning sites an establishment of fish passages at dams. Detailed guidelines and recommendations are listed in the Ramsar Declaration on Global Sturgeon Conservation Due to the high rate of mortality observed in the parental stock, decimated from 42 individuals in 2005 to 24 individuals at present, the establishment of an adequate The University of Padova ethic board C.E.A.S.A.  exempted this study from review because it was an extra moenia activity. Nevertheless we tried to minimize the impact of sample collection on the animals. All samples were collected in aquaculture facilities by the owners. Fish were kept into the pond water to minimize stress and were released immediately after sample collection, which consisted of painless clippings from the caudal fin. No mortality, pain or stress was observed.A. naccarii) were obtained at Azienda Agricola VIP  from the parental stock constituted by individuals caught in the river Po and transferred to the fish plant in 1977, which is the last remaining Italian population of wild origin.Samples of Adriatic sturgeon  were sampled for genetic analysis and tagged for the first time with a microchip. The sample set was completed with additional individuals from a previous sampling conducted in 1999/2000, in which a sub-set of the parental stock was sampled following individual identification ‘by eye’. Nine of the individuals sampled in 1999/2000 were not present in the 2005 sampling as they died sometime in the period 2000–2005. Those animals were added to the 2005 set as they might have been used as founders in producing the F1 stocks. Following a period of high mortalities in recent years, the current stock has been decimated to 24 individuals . All analyses have been conducted separately for the full data set  and considering only those individuals presently alive .Additionally, genetic analysis included a total of 150 individuals corresponding to the three F1 stocks obtained from unrecorded artificial reproduction using the Azienda Agricola VIP parental stock of wild origin, currently reared as future breeders at three aquaculture plants in Treviso, Piacenza and Orzinuovi (N =  50 at each site).Finally, two complete families each composed by both parents plus 10 fingerlings were also sampled following artificial reproduction, and genotyped at genetic markers in order to test the reliability of the parental allocation procedure.5′-CACCCTTAACTCCCAAAGC-3′) and phenylalanine tRNA (Phe1R: 5′- CCCATCTTAACATCTTCAGT-3′), respectively. PCR reactions consisted of about 10 ng template DNA, 2.5 µl 10x buffer, 200 µM of each dNTP, 1.5 mM MgCl2, 10 pmol of each primer, 1 U Taq DNA polymerase , and water up to 25 µl. PCR conditions were as follows: 3 min at 94°C, 35 cycles of 30 sec at 94°C, 45 sec at 53°C and 1 min at 72°C, and final elongation for 5 min at 72°C. Following enzymatic purification with ExoSAP-IT™ (Usb), sequence reactions were performed using primer Phe1R, which allowed to obtain unambiguous sequences of the entire control region. By contrast, the use of primer Pro1F yielded sequencing failures due to the co-sequencing of different heteroplasmic variants linked to a 82 bp tandem repeat region located at the 5′ region Genomic DNA was extracted from fin clips (10–100 mg) using the DNAeasy Blood and Tissue Extraction kit (Qiagen). The entire mitochondrial control region including partial sequences of the flanking tRNA genes was amplified and sequenced for all F0 and F1 individuals. Primers used matched the proline . In order to achieve maximum scoring accuracy, analysis was conducted in parallel in two separate laboratories, including independent tissue extractions for each animal. Microsatellite loci were amplified following the conditions reported in the original references .ST values were calculated in ARLEQUIN et al. Mitochondrial DNA sequences were aligned using CLUSTAL W A. naccarii, e.g. the four peaks for a complete heterozygous genotype did not always show the same height even if they correspond to a single copy of each allele, thus it was not possible to estimate gene dosages on the basis of peak heights. Using the classic bandsharing approach, microsatellite data were considered as presence/absence of bands, disregarding the number of alleles present in each individuals. Data from all microsatellite loci were combined by creating individual profiles in which bands were coded as a string of 0s (absent) and 1s (present). Band sharing (BS) between individuals was used as a measure of similarity. Genetic distances were calculated as 1-BS, and graphically represented by Multi-Dimensional Scaling (MDS) analysis using STATISTICA version 7.1 (StatSoft). Pairwise FST values were calculated in ARLEQUIN Microsatellite data were analyzed using a phenetic approach, with microsatellite alleles scored as dominant markers. Interpretation of microsatellite patterns in tetraploid species is not straightforward due to the presence of multiple alleles at each locus that can be present in more than one copy, thus the true genotype cannot be accurately resolved. Alternatively, some authors propose to infer allele frequencies from gene dosages, assuming a correspondence between number of allele copies and peak heights in the chromatogram Assignment of F1 individuals to the correct F0 parental pair was conducted in two steps. First, the microsatellite profiles of all putative parent-pairs were combined and compared with the profile of each F1 individual to be allocated. Parent-pairs not showing all bands observed in the F1 were discarded as possible parents. Among all compatible pairs, a second selection step consisted in comparing the mitochondrial DNA haplotype between mother and progeny. Prior to the application of the allocation procedure, accuracy was tested on 20 F1 individuals (two groups of full-sibs) of known pedigree. Using the 42 individuals of the F0 group as putative founders, all 20 F1 individuals were allocated to the correct parent pair with a 100% accuracy.Genetic distance (1-BS) was calculated between all pairs of full sibs, half sibs and unrelated individuals. Using the observed distribution of genetic distances within groups, a threshold value was established above which the probability to exclude that two individuals are related  is >99%.Simulations were used to verify if the observed distributions of pairwise genetic distances in the different groups of relatives  correspond to the expected ones. Diploid gametes drawn from all F0 animals were combined to produce a simulated tetraploid genotype for 8 independent microsatellite loci for each F1 individual. In order to take into account the tetraploid nature of the species, two alleles per parent were randomly drawn. When less than four alleles were observed at a given locus, the possibility to segregate the same allele twice was accepted. With this approach we assumed a tetrasomic pattern of inheritance, in which the different homolog chromosomes can be transmitted to gametes in all possible combinations. The obtained microsatellite profiles were used to reconstruct the distribution of genetic distances (1-BS) in simulated full sibs, simulated half sibs and simulated unrelated individuals. For each group, the threshold value that excluded related individuals with a >99% chance was calculated using the simulated data, and compared with the threshold values previously obtained with the observed data. The program used for virtual offspring simulation and estimation of pairwise distances within different classes of relatedness is available upon request, as well as the one used for parental allocation.


Erophylla sezekorni/E. bombifrons and Macrotus waterhousii (Chiroptera: Phyllostomidae). Using a landscape genetics approach, we asked if geographic distance, island size, or climatological winds mediate historical gene flow in this system. Samples from 13 islands spanning Erophylla's range clustered into five genetically distinct populations. Samples of M. waterhousii from eight islands represented eight genetically distinct populations. While we found evidence that a majority of historical gene flow between genetic populations was asymmetric for both lineages, we were not able to entirely rule out incomplete lineage sorting in generating this pattern. We found no evidence of contemporary gene flow except between two genetic populations of Erophylla. Both lineages exhibited significant isolation by geographic distance. Patterns of genetic structure and gene flow, however, were not explained by differences in relative effective population sizes, island area, sex-biased dispersal (tested only for Erophylla), or surface-level climatological winds. Gene flow among islands appears to be highly restricted, particularly for M. waterhousii, and we suggest that this species deserves increased taxonomic attention and conservation concern.Observed patterns of genetic structure result from the interactions of demographic, physical, and historical influences on gene flow. The particular strength of various factors in governing gene flow, however, may differ between species in biologically relevant ways. We investigated the role of demographic factors  and physical features  on patterns of genetic structure and gene flow for two lineages of Greater Antillean bats. We used microsatellite genetic data to estimate demographic characteristics, infer population genetic structure, and estimate gene flow among island populations of  The factors promoting or restricting gene flow among populations have important consequences for a broad range of ecological and evolutionary processes A wide variety of demographic and physical processes can lead to asymmetric migration between populations To date, landscape genetics research has focused on systems where genetic connectivity is influenced by the spatial arrangement of different quality habitat and of physical barriers. In these systems, genetic connectivity among locations is predicted to be symmetric. In contrast, many natural systems are subject to strong directional forces that may have important effects on gene flow among populations. A valuable and logically appealing next step in landscape genetics is to extend the concept of ‘effective distance’ to situations with anisotropic forces. For example, wind and water currents have been demonstrated to influence gene flow of some passively dispersing organisms Erophylla sezekorni/bombifrons and Macrotus waterhousii, Chiroptera: Phyllostomidae) and to explore the potential role of a variety of mechanisms that may drive these patterns. Specifically, we addressed the following questions: (1) What are the patterns of genetic structure throughout the Greater Antilles for these lineages of bats? (2) How much gene flow occurs among genetic populations in each of these taxa and is it symmetric? (3) Can instances of asymmetric gene flow be explained by differences in population size, island area, sex-biased dispersal, or climatological winds?Island systems, including those of the Caribbean basin, provide excellent opportunities to explore the mechanisms governing gene flow among populations. Despite little direct information about the ability of organisms to travel among islands, population genetic tools can provide insight into the patterns of colonization history and population dynamics. In particular, comparative studies provide a powerful means to reveal shared and disparate mechanisms mediating gene flow and to expose species differences that may have important conservation implications Erophylla and Macrotus waterhousii. The genus Erophylla contains two currently recognized species: E. sezekorni (the buffy flower bat), distributed in the western Greater Antilles  and Bahamas, and E. bombifrons (the brown flower bat), which occurs in the eastern Greater Antilles (Hispaniola and Puerto Rico) The two lineages of phyllostomid bats examined in this study are Macrotus occur throughout the Bahamas and Greater Antilles (except Puerto Rico), as well as on the mainland from the southwestern United States south to Guatemala Macrotus is considered the basal genus of Phyllostomidae M. waterhousii occurs in tropical dry forests of western Mexico and the Greater Antilles.Bats of the genus unpublished data). Erophylla bats are omnivores, feeding on nectar and fruits as well as insects Macrotus are considered to be insectivorous gleaners Adults of both genera weigh 12–20 g and have head/body lengths of 50–75 mm Erophylla (N = 293) and 8 islands for M. waterhousii (N = 190) throughout the Bahamas and Greater Antilles  and pairwise FST values Erophylla for evidence of sex-biased dispersal (sex data were insufficient to test M. waterhousii) through seven independent tests of differential genetic divergence between the sexes fstat version 2.9.3 micro-checkerWe genotyped individuals of structure v.2 Erophylla) and K = 1–8 (M. waterhousii) for 20×105 MCMC generations with a 20×104 burn-in period. We selected the value of K (hereafter referred to as groups) to use in subsequent gene flow analyses based on the average maximum estimated log-likelihood of P(X|K) across trials. We assigned islands to each of the K groups based on the maximum estimated membership coefficient  averaged for samples within islands. To provide an independent assessment of genetic structure, we used the group level analysis in baps v.5.1 Erophylla samples, we also ran these analyses separately for E. sezekorni and E. bombifrons (results not shown); results did not differ from the combined analyses.We used two Bayesian clustering analyses to identify the number of genetically distinct populations (K) within the study area. In ftp://ftp.cdc.noaa.gov/pub/Datasets), which includes monthly mean magnitude and direction from 1948 to 2005 at 2.5° lat/long (≈278 km2) resolution  and azimuth of the shortest lines connecting all island pairs Wij, was calculated as the product of the ‘friction’ due to wind and the Euclidean distance between islands. We evaluated the magnitude of asymmetry of effective distance using a normalized index of DW asymmetry for all island pairs :Dw ranges from 0 (complete symmetry) to 1 (complete asymmetry).First, we determined the Euclidean distance (Dm/μ) using maximum likelihood implemented in migrate v.2.1.3 Erophylla are from 20 short chain searches  followed by 3 long chain searches  after a 104 burn-in period. M. waterhousii results are from 15 short chain searches  followed by 3 long chain searches  after a 104 burn-in period. The results of the final long chain searches were averaged over three independent runs. We identified asymmetric gene flow between genetic populations by examining 95% confidence intervals of M and calculated a normalized index of gene flow asymmetry (RM) for island or group pairs:M ranges from 0 (complete symmetry) to 1 (complete asymmetry) and pairs with overlapping 95% confidence intervals of migration rate were assigned RM = 0. To determine if historic gene flow was biased from larger populations into smaller populations, we used linear regression to examine the relationship between θ and emigration. Similarly, we determined if historic gene flow was mediated by island size by examining the relationship between island area and migration rate.We estimated relative effective population size (θ) and levels of historical gene flow between genetic populations  with a sampling frequency of 2×103. To assess the reliability of estimated parameters, we ran four additional short MCMC runs for each lineage. Short runs were 5×106 generations (1×106 burn in) with the same sampling frequency. All other options were left at their default settings. In contrast to migrate, bayesass estimates all pairwise migration rates rather than a user-defined migration matrix.We used NA), the effective sizes of the two daughter populations (N1 and N2), directional migration rates between the two daughter populations (M1 and M2), the divergence time for the two daughter populations (t), and the proportion of the ancestral population that founded daughter population 1 (S). All parameters, except for S, were estimated as mutation-scaled rates: θX = 4NXμ; τ = tμ; and mX = MX/μ. We assumed a mutation rate of 10−5 mutations per locus per generation for these calculations. Populations were analyzed in pairwise combinations using the MCMC composite Bayesian/likelihood framework implemented in im v.3_5_2007 S, t, 1M and 2M. Uniform prior distributions were explored for S from 0–1, for t from 0–20, and for m from 0–20. These bounds were expanded when initial runs indicated that the full marginal posterior density was not being included. We emphasize that im produces marginal, not joint posteriors, so that failure of one parameter to converge is no reflection of uncertain inference for other parameters. Parameters were estimated from at least 3 jobs, each consisting of 10 Metropolis-Hastings coupled chains that were run for at least 5×105 steps. Analyses were run on either a dispersed computing grid at the University of Arizona or at Cornell University's Computational Biology Service Unit (http://cbsuapps.tc.cornell.edu/index.aspx). Convergence on the underlying stationary distributions of the model parameters was assured by the use of multiple independent runs, many coupled chains, and long run times. Where convergence could not be obtained for specific parameters despite these efforts, we do not provide those parameter estimates.Recognizing that populations may share alleles through either shared history or ongoing gene flow, we used coalescent analyses based on the non-equilibrium isolation-with-migration model to distinguish the influence of these two processes on population pairs. This model focuses on seven demographic parameters: the effective size of the ancestral population (GEO), between island groups. If mean surface winds mediate gene flow, we expected genetic differentiation to be more strongly correlated with a measure of effective distance than with Euclidean distance. We examined the correlation between genetic differentiation and the natural log of the minimum DW for each pair of independent genetic populations. As an alternative approach, we used linear regression to evaluate the relationship between the index of migration asymmetry (RM) and the index of distance asymmetry (RDw) for each group pair. Statistical significance of all IBD relationships was assessed using Mantel tests 4 randomizations in fstat v.2.9.3 We tested our data for evidence of isolation by Euclidean distance   . IslandsD 98.50)  and the structure analysis , and all islands had a maximum average Q-value >0.76. Estimated log likelihood values from island samples in the baps analysis also reached a maximum for K = 8 (mean −5576.2 +/− SD 132.78)  in the analysis . The mea 132.78) . These r 132.78) .Macrotus) and groups (Erophylla) are shown in st are provided with Euclidean distances between genetic populations in Erophylla dataset, 142 total alleles were detected at 12 loci from all genotyped individuals (n = 293). Average observed heterozygosity over all loci was 0.66, and the estimated total FIS over all loci was 0.039 (95% CI −0.007–0.072). Hardy-Weinberg proportions occurred in all groups (p>0.05), there was no evidence of linkage disequilibrium, and all pairwise tests of differentiation were significant (p<0.05) after Bonferroni correction. We found no statistical support for sex-biased gene flow . Average observed heterozygosity over all loci was 0.67, Hardy-Weinberg proportions occurred at all sites, and we found no evidence of linkage disequilibrium. The estimated total FIS over all loci was 0.034 (95% CI -0.009–0.059), and all pairwise tests of differentiation were significant (p<0.05) after Bonferroni correction. Allelic richness per locus was similar between the two lineages .Microsatellite marker characteristics for islands (ene flow . In the m/μ) and θ estimates between genetic populations of both lineages are shown in migrate for Erophylla ranged from 0.08 to 15.31 (mean 5.04 +/− SD 5.23). Four of the five pairwise comparisons exhibited significantly asymmetric rates of gene flow based on non-overlapping 95% confidence intervals. The bayesass analysis suggested no detectable contemporary gene flow among genetic populations with one exception . When the program was run using only data for E. bombifrons, however, gene flow from PUE to HIS was 0.019 (95% CI 0.00–0.07) and from HIS to PUE was 0.089 (95% CI 0.00–0.26). The percentage of changes accepted for these runs was 53.5 (+/− SD 0.055), falling within the range indicating acceptable algorithm performance Historical migration rate  approximately 30–40 thousand years ago (kya). An exception to this pattern is the founding of Hispaniolan E. bombifrons from the Puerto Rican population approximately 10 kya. Current levels of gene flow among islands was extremely low, averaging 11.24×10−6 (95% CI = 0.55–41.5×10−6), with only the GBB-LBB pair showing significantly asymmetric rates based on non-overlapping 95% confidence intervals.The  islands . HistoriMacrotus ranged from 0.00 to 13.20 . Two approaches were used to address this potential problem: (1) we randomly resampled 11 individuals from each other island and reran the analysis to investigate sample size bias, and (2) we reran the program including only samples from islands for which gene flow with Cuba was estimated  using the same settings as described above. These runs yielded qualitatively similar results in that migration was still biased toward CUB, suggesting adequate sample size to achieve consistent parameter estimates. The bayesass analysis estimated zero contemporary gene flow among all island pairs for M. waterhousii . While we found no statistical support for asymmetric gene flow between any pair of islands with the im analysis, the 95% confidence intervals were quite large in most cases, reducing the power of this assessment. Instead, these analyses attributed most genetic similarity between islands to common ancestry and historical founding events. In general, the oldest of these founding events  connect islands of the Bahamas with near islands of the Greater Antilles (Hispaniola and Cuba). Except for Jamaica, founding events between islands of the Greater Antilles appear to be significantly more recent . Founding events among islands of the Bahamas are, in most cases, even more recent  with Exuma indicated as a significant source population.Migration rate scaled for mutation rate for ward CUB . The relerhousii . A low perhousii  were alsmigrate were not correlated with historical emigration rate for either lineage . Additionally, neither θ nor M were correlated with the island (M. waterhousii) or total group area (Erophylla) . These results suggest that asymmetric gene flow is not mediated by differences in estimated population size or island area.Theta values estimated by effective distance metrics are shown in GEO for Erophylla when considering all islands separately  as well as among groups , and for M. waterhousii among islands . Minimum pairwise effective distance between groups was not significantly correlated with genetic differentiation for Erophylla  but was for M. waterhousii . However, genetic differentiation was more strongly correlated with Euclidean distance than effective distance in both cases. There were no significant relationships between the index of distance asymmetry (RDw) and the index of migration asymmetry (RM) for either Erophylla or M. waterhousii . From these results, we conclude that both lineages displayed significant IBDGEO at the scale of this study but our measure of effective distance did not provide an explanation of the distribution of genetic diversity or the instances of asymmetric gene flow.Isolation by distance plots among genetic populations for each lineage using both Euclidean and Erophylla and M. waterhousii in the Greater Antilles and Bahamas. While the estimated magnitude of historic gene flow between genetic populations was generally greater for Erophylla than for M. waterhousii, contemporary gene flow appears to be highly restricted among populations in both lineages. Additionally, parts of our analyses suggested an equally high incidence of asymmetric historic gene flow in both lineages. While this finding could have profound consequences for evolution and conservation of species, we found no evidence that a directional bias of historical gene flow was related to effective population size, island area, sex-biased dispersal, or surface-level trade winds. Results from the IM analysis suggested that these patterns might potentially be a result of shared ancestry rather than gene flow among island populations. Overall, our results support Hedges' The results of this study reveal substantially different patterns of genetic structure for Erophylla exhibited less genetic structure than we had anticipated given its island endemism. Poor dispersal ability may lead to endemism through an inability to colonize distant sites, but this does not appear to be the case for Erophylla. A similarly counterintuitive pattern of genetic structure of an island endemic bat was found by Roberts Erophylla was between bats from the eastern islands of the range of the genus (Hispaniola and Puerto Rico) versus the rest of the Greater Antilles and Bahamas. Fleming et al.Erophylla. Haplotypes were shared extensively within the two clades, and two out of 34 total haplotypes (6%) were shared between clades. Based on these data, the authors rejected the hypothesis of island monophyly, suggesting that gene flow may still be occurring between the two clades. In the current study, we found no evidence of current gene flow between these two clades, consistent with the current taxonomy of the genus Erophylla. Throughout its range, no extirpated populations have been recorded on the islands throughout the Greater Antilles and Bahamas where Erophylla occurs in the fossil record Erophylla but concordant with the results of Fleming et al.M. waterhousii appear to be genetically isolated from one another. The strong genetic subdivisions between island populations may be indicative of this species' sedentary lifestyle and long residence in the Greater Antilles. In contrast to Erophylla, Morgan M. waterhousii has become extinct on six of the 30 islands from which it is included in the fossil record. These findings signify a geographical range contraction since the late Quaternary and suggest that recolonization events between islands are rare in this lineage.Unlike the results for migrate suggest asymmetric historical gene flow among a majority of pairwise comparisons (∼80% in both lineages). Meanwhile, the results of the bayesass analysis suggest that contemporary gene flow is highly restricted among genetic populations for both lineages. It is possible that the high degree of historic gene flow asymmetry inferred from the migrate results is actually an artifact generated from effects of shared ancestry. The results of our im analyses do not generally support an inference of high levels of asymmetric gene flow. There are likely two effects leading to these seemingly discordant results. First, because migrate uses an equilibrium model, effects of shared genetic history among islands, specifically the recent founding of one island population from another, may emerge as asymmetric migration rates in parameter estimation. Second, some actual instances of asymmetry may not be detected in im using the criterion of non-overlapping 95% confidence intervals because the estimates of these values from im are quite large. Recognizing these possibilities, we focus our discussion below on possible explanations for the high levels of asymmetric historical gene flow observed in the migrate analysis.Despite differences in the overall amount of genetic differentiation among populations between these two lineages, results from Previous studies on a variety of taxa provide mixed conclusions on the mechanisms leading to asymmetric gene flow Erophylla appears to 1∶1 Sex-biased dispersal is unlikely to be a contributing factor to asymmetric gene flow in this system. The sex ratio of Erophylla and 2 for M. waterhousii) . The results from this study suggest straightforward dispersal of Erophylla from Jamaica to GBB (presumably first to Cuba) and then to Hispaniola. Our observation of decreasing θ values in Erophylla away from Jamaica along the dispersal pathway also fit this model. The pattern for M. waterhousii is not as clear, but one interpretation of differences in θ's among islands is that Jamaica was colonized first, followed by Hispaniola and then Cuba and the Bahamas Estimated relative effective population size (θ) was not correlated with the index of emigration for either species, suggesting that larger populations do not always act as sources in this system. Additionally, island area was not correlated with relative effective population size (θ) or the directional bias of historic gene flow. In fact, despite its ranking as third largest island area, estimates of θ for Jamaica were considerably higher than the group or island with the second largest θ (by a factor of 8 for rhousii) . Samplinon average. Perhaps the spatial and temporal resolution of the wind data used in this study does not adequately capture the overall effect on the movement of bats between islands. However, because the results from the migrate analysis are best viewed as long-term parameter estimates We expected the strongly asymmetric force of wind present in this system to play a role in mediating gene flow among populations. We hypothesized that surface level trade winds may lead to asymmetric gene flow because of their effects on the flight dynamics of volant organisms et al.Erophylla as suited for maneuverable but not fast or efficient flight. Similarly, Valdivieso et al.Erophylla is suited for short bursts of flight based on their study of lactate dehydrogenase isozymes. M. waterhousii has similar wing morphology as ErophyllaErophylla or M. waterhousii. For comparative purposes, Leptonycteris curasoae, a strong flying phyllostomid, commutes between day roost and feeding areas at an average air speed of 8.2 m/s E. sezekorni and M. waterhousii are comparable to those of L. curasoae, then perhaps the mean wind does not represent a large enough additional energetic cost to affect their movement patterns. However, these bats are likely to be slower flyers than L. curasoae, an exceptionally fast flyer . Seasonal variation in wind speed may be important if periods of peak wind speeds correspond to bat migration or dispersal. Repeating our analyses using data from June (the month of peak wind speeds) provided comparable results in that Euclidean distance accounted for genetic differentiation better than effective distance.Wind may not mediate gene flow in these lineages if their flight speeds are greater than the wind speeds they encounter during movements between islands. To investigate seasonal variation in wind speed and direction, we generated time series plots of mean magnitude and direction at three locations within the study area . InferenErophylla, following hurricanes personal observation), but we do not know the exact mechanism leading to these ‘transplants’. Interestingly, the annual peak in hurricane activity in the study area occurs in late summer, which coincides with the time when juvenile bats become volant and are potentially dispersing from their natal colonies .The results of this study do not support our hypothesis that gene flow is mediated by surface-level trade wind for either of these lineages. Wind may still play a role, however, if idiosyncratic events  contribute disproportionately to stochastic, long-distance dispersal of bats. Hurricanes have historically been a major climatological presence in the study area with an average of three hurricane-strength events per year over the past 500 years Ardops nichollsi, Brachyphylla cavernarum, and Artibeus jamaicensis) What differences are responsible for producing the two lineage's different patterns of genetic structure and gene flow observed in this study? Divergent patterns of genetic structure have been reported among other phyllostomid species in the Lesser Antilles Click here for additional data file.


Aedes albopictus, is an anthropophilic aggressive daytime-biting nuisance and an efficient vector of certain arboviruses and filarial nematodes. Over the last 30 years, this species has spread rapidly through human travel and commerce from its native tropical forests of Asia to every continent except Antarctica. In 2011, a population of Asian tiger mosquito  was discovered in Los Angeles (LA) County, California. To determine the probable origin of this invasive species, the genetic structure of the population was compared against 11 populations from the United States and abroad, as well as preserved specimens from a 2001 introduction into California using the mitochondrial cytochrome c oxidase 1 (CO1) gene. A total of 66 haplotypes were detected among samples and were divided into three main groups. Aedes albopictus collected in 2001 and 2011 from LA County were genetically related and similar to those from Asia but distinct from those collected in the eastern and southeastern United States. In view of the high genetic similarities between the 2001 and 2011 LA samples, it is possible that the 2011 population represents in part the descendants of the 2001 introduction. There remains an imperative need for improved surveillance and control strategies for this species.The Asian tiger mosquito,  Aedes albopictus (Skuse) is currently the most invasive mosquito species in the world. Over the last 30 years, this species has spread rapidly through human travel and commerce from its native tropical forests of Asia to every continent except Antarctica Ae. albopictus is a less competent vector of arboviruses than its congeneric species Ae. aegypti (L.) Ae. albopictus further demonstrate the increasing public health impact of this species worldwide.The Asian tiger mosquito Ae. albopictus in the continental United States (U.S.) was discovered in Harris County, Texas, during the summer of 1985 Ae. albopictus expanded to much of the eastern U.S., including at least 911 counties in 25 states Ae. albopictus at least four times between 1946 and 2004 Ae. albopictus was imported from mainland China in trans-oceanic shipments of “lucky bamboo” (Dracaena spp.) packaged in standing water. Distribution of these plants to wholesale nurseries throughout California resulted in 15 localized infestations in six counties Ae. albopictus had survived the winter at some of those sites, but no evidence was subsequently found to suggest that populations had become established The first clearly established population of Ae. albopictus was discovered in the City of El Monte, Los Angeles County, following a request from a citizen for vector control service Ae. albopictus, the population survived and reemerged throughout the infested area in May of 2012. The source of this inland population of Ae. albopictus was uncertain since a connection to international commerce was lacking. Possible sources of the infestation included an introduction from other regions of the U.S. mainland where Ae. albopictus is established, importation from Hawai’i or abroad, or surviving mosquitoes from past introductions. Elucidating the origin of these mosquitoes could identify new invasion pathways and the potential for continued expansion. We compared samples of this newly-discovered population of Ae. albopictus to preserved specimens from past introductions and populations from other regions of the U.S. and other countries. We also assessed the potential impact to public health in California of an established population of Ae. albopictus.In early September 2011, No permits were required for this study. Residents consented at all properties where mosquitoes were collected. No sites were protected by law and this study did not involve endangered or protected species.Aedes albopictus were collected as larvae or adults from 11 localities in the U.S. and around the world in 2011  and 18S rDNA regions  in 2011 . SamplesGGTCAACAAATCATAAAGATATTGG 3′) and 2160R (5′ TAAACTTCTGGATGACCAAAAAATCA 3′); 2027F (5′ CCCGTATTAGCCGGAGCTAT 3′) and 2886R (5′ ATGGGGAAAGAAGGAGTTCG 3′). A total of 23 µl reaction mix containing 3 µl of template DNA, 0.5 mM MgCl2, 0.2 mM dNTP, 10 pmol of each primer and 0.5 U of Taq polymerase (Qiagen) was used in each PCR, and amplification was performed in a Bio-Rad MyCycler Thermal Cycler. PCR cycles involved an initial denaturing step at 94°C for 3 min, then 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. An additional extension was performed at 72°C for 6 min. Products of PCR were visualized on 1% agarose gels and then purified and sequenced directly using primers 2160R and 2027F with ABI Big Dye Terminator Cycle Sequencing Kit.Total DNA was extracted from a single mosquito leg of each specimen using the Fast Tissue-to-PCR Kit  following the methods of Zhong et al. Hd), and nucleotide diversity (π) within each of the sites were determined using DnaSP version 4.10.1 The CO1 gene sequences from 346 mosquitoes were aligned using BioEdit, and then edited manually with Sequence Alignment Editor version 1.d1 (SE-Al) K) was determined by simulating a range of K values from 1  to 12 . The posterior probability of each value was then used to detect the modal value of ΔK, a quantity related to the second order rate of change with respect to K of the likelihood function et al. K was run five times with the ‘independent allele frequency’ option that allows individuals with ancestries in more than one group to be assigned into one cluster. Individuals were partitioned into multiple groups according to the membership coefficient (Q) that ranges from 0 (lowest affinity to a group) to 1 (highest affinity to a group) across the K groups. The partitioning of clusters was visualized using the program DISTRUCT To examine genetic structuring of individuals, a model-based Bayesian analysis was performed on the basis of nucleotide variation using STRUCTURE version 2.2 under varying assumptions on Hardy–Weinberg (HW) and linkage equilibriums FSTFs statistics DHri) Genetic distance matrices were calculated using Kimura two-parameter distance dH) ranged from 0.37 in the laboratory strain from Jiangsu, China to 0.83 in preserved specimens from Los Angeles County, California, collected in 2001 (LA01). Nucleotide diversity (π) ranged from 0.06 in specimens from Guangzhou and Taiwan to 0.30 in specimens collected in Singapore. Overall, samples from southern China (XM) and southern California (LA01) showed the highest diversity, whereas the laboratory strain (JS) showed the lowest diversity, followed by specimens from the City of El Monte in Los Angeles County, California, collected in 2011 (LA11) and from Monmouth County, New Jersey (NJ).The mitochondrial CO1 gene gave a total aligned length of 1,433 bp in which 35 variable sites were observed and 33 of them were parsimony informative . HaplotySixty-six haplotypes were detected . SamplesK = 6 with structure analysis. The pie charts in Ae. albopictus populations studied. The largest membership coefficient value (Q), or the proportion of individuals assigned to a cluster, were high for New Jersey and Texas samples (Q >0.74), suggesting a strong affinity to be included in a single cluster (cluster 4). This cluster was shared with some of the individuals from Italy, consistent with the results obtained in the network analysis . The majority of genetic variation (65%) was within-populations, whereas approximately 35% was among-population. Pairwise FST values ranged from 0.09 (JS and IT) to 0.69 (GZ and NJ) . Samples collected in 2001 and 2011 from Los Angeles County exhibited a large genetic differentiation from samples from New Jersey and Texas (p<0.001), suggesting they are not likely the origin of the Los Angeles County samples. Tajima’s D values for LA01 and LA11 are 0.22 and −0.26, respectively, and these values are not statistically significant (p>0.05). Tajima’s D value for all other studied localities, with the exception of TW , are also not statistically significant (Fs test for the two Los Angeles populations was not significant and rejected the population expansion/bottleneck model (Hri for LA11 was not significant (P>0.05)  . No genenificant , indicatck model . The bim(P>0.05) .Aedes albopictus is one of the most invasive and widespread mosquito species in the world. Technological advances in mass production, overseas trade, and global travel have accelerated its movement within and across continents. One of the factors contributing to the spread of Ae. albopictus is its close association with human activities and dwellings, which has facilitated the movement of eggs around the world and resulted in the invasion of new lands with suitable climates. In California, it has been speculated that the hot, dry summer period typical throughout most of the state provides a hostile environment for the rapid population growth and establishment of this species Ae. albopictus to permanently establish despite numerous opportunities, particularly during the extensive introductions from China discovered during the summer of 2001 Ae. albopictus was discovered in Los Angeles County in early September of 2011 in an area comprised primarily of single-family suburban homes Ae. albopictus worldwide. The mitochondrial genome of Aedes mosquitoes is maternally inherited and very rarely undergoes recombination Anopheles and Aedes mosquitoes Ae. albopictus samples, which was much higher than that observed in other Aedes populations (≤4 haplotypes) previously reported in Cameroon Ae. albopictus into the country or the inclusion of samples from a relatively narrow geographical range Ae. albopictus mosquitoes recently discovered in Los Angeles County.The CO1 gene is a valuable diagnostic tool to study genetic diversity and spread of Ae. albopictus included in this study. While most of the genetic variation was found within populations, these populations appear to have reached equilibrium and that population size remains stable through time  and Taiwan, suggesting a temperate Asian origin , but plants were also grown and exported from Taiwan, Thailand, Indonesia, Cambodia, and Vietnam Ae. albopictus. Multiple introductions have been critically important for invasive species such as Ae. albopictus to become established and in forming their population genetic structure D and Fu’s Fs tests  implied multiple origins from different parts of Asia. Their genetic structures further suggested links to both temperate and tropical origins. Multiple introductions and origins of LA01 mosquitoes were likely since a tremendous number of Fs tests  and the Fs tests  indicateAe. albopictus in 2001. The aggressive eradication campaign centered on infested wholesale nurseries was regarded a success at most locations; however, Ae. albopictus apparently survived the winter to reemerge in 2002 at two nursery locations and up to 500 m away in surrounding residential neighborhoods Ae. albopictus had been eradicated throughout the region. Two of the 15 wholesale nurseries discovered in 2001 were located in the City of El Monte 2Ae. albopictus with available surveillance equipment. The region’s large population of immigrants who are native to countries where Ae. albopictus or other day-biting species are common and the patchy distribution of Ae. albopictus in neighborhoods lends credence to how this established population may have escaped detection for potentially over a decade The premise that LA11 may represent a carryover population is supported by empirical evidence and surveillance data collected in Los Angeles County following the initial detection of Ae. albopictus invading California. The genetic data presented herein combination with earlier shipping records and local surveillance strongly support the notion that the population in the cities of El Monte and South El Monte was founded by individuals imported during the late 1990 s or early 2000 s from Asia. The size of the infested area suggests a relatively slow rate of spread that could be a result of the short dispersal range typical of this species Ae. albopictus. The inconsistent presence of adults observed throughout the infested neighborhoods creates a weak foundation for transmission of dengue or chikungunya viruses. Nonetheless, an established population of Ae. albopictus leaves open the potential for endemic transmission of these viruses in the area. The re-discovery of Ae. albopictus in Los Angeles County in 2011 provides convincing evidence that this species can become established in California and could become a severe nuisance and competent vector of disease. Local mosquito and vector control agencies should strongly consider including proactive surveillance and control programs for Ae. albopictus to maximize the potential for early detection and eradication.This study represents the first genetic analysis of Table S1Sixty-six COI gene haplotypes identified in the 12 Aedes albopictus populations examined. Identity with the reference sequence (Genbank accession no. JQ004525) is denoted by a dot and substitution by a different base letter. GZ: Guangzhou; XM: Xiamen; JS: Jiangsu; TW: Taiwan; JP: Japan; SG: Singapore; IT: Italy; LA01: Los Angeles 2001; LA11: Los Angeles 2011; NJ: New Jersey; TX: Texas; and HW: Hawai’i.(XLSX)Click here for additional data file.


Musicians use different kinds of imagery. This review focuses on kinesthetic imagery, which has been shown to be an effective complement to actively playing an instrument. However, experience in actual movement performance seems to be a requirement for a recruitment of those brain areas representing movement ideation during imagery. An internal model of movement performance might be more differentiated when training has been more intense or simply performed more often. Therefore, with respect to kinesthetic imagery, these strategies are predominantly found in professional musicians. There are a few possible reasons as to why kinesthetic imagery is used in addition to active training; one example is the need for mental rehearsal of the technically most difficult passages. Another reason for mental practice is that mental rehearsal of the piece helps to improve performance if the instrument is not available for actual training as is the case for professional musicians when they are traveling to various appearances. Overall, mental imagery in musicians is not necessarily specific to motor, somatosensory, auditory, or visual aspects of imagery, but integrates them all. In particular, the audiomotor loop is highly important, since auditory aspects are crucial for guiding motor performance. All these aspects result in a distinctive representation map for the mental imagery of musical performance. This review summarizes behavioral data, and findings from functional brain imaging studies of mental imagery of musical performance. Mental imagery of a piece of music in one's mind is commonly used by professional musicians for instance to rehearse difficult parts of an already executed musical passage , motor imagery and imagery of temporal processes of tapping frequency. A typical instruction for this kind of kinesthetic imagery (or sensorimotor imagery) would be to first perform a certain movement repetitively and then go on doing it internally with prevention of actual movement. This example also illustrates that imagery following a period of prior experience of actual movement is widely used , but also auditory elements. Auditory aspects of imagery in musicians have been the topic of research for decades already , the premotor cortex (PMC), for parietal areas and for the cerebellum. In particular the posterior SMA and the PMC (BA 6) seem to be the predominant areas responsible for movement imagery. Neurons in the SMA are involved in the preparation of movements and it is reasonable that preparatory aspects of a movement may be closely related to motor imagery. The PMC, can be subdivided into a dorsal (dPMC) and a ventral (vPMC) area. Whereas the vPMC lies adjacent to the posterior part (BA44) of Broca's area in the left hemisphere and Broca's analog in the right hemisphere, the dPMC is more associated with anterior parts of the primary motor hand area (BA 4a). Different imagery strategies involve different parts of the PMC; where kinesthetic imagery involves the dorsal PMC, visual strategies involve more ventral parts  -effects magnitude] during imagery than during execution of the same movement. Most of the studies describing primary motor cortex activation during kinesthetic imagery used an instruction method for mental imagery, with the executed movement preceding imagined movement. The studies controlled the execution of the movements via the use of electromyography (EMG) of the effector muscles , is increased , Broca's area and its homologue  and the superior and inferior parietal lobe. Additionally, subcortical motor areas in the cerebellum and basal ganglia, and the midbrain were involved. In contrast to studies with instrumentalists, we observed significant primary motor and somatosensory cortex activation and thalamus activation during imagined singing. Interestingly, areas processing emotions also showed intense activation . This is quite remarkable, since to our knowledge no functional imaging study on professional instrumentalists has ever reported emotional areas being active in processing musical performance or kinesthetic imagery. Comparable to instrumentalists imagining playing a musical piece, singers showed no activation in the primary auditory cortex or in the auditory belt area, but in the temporo-parietal lobe bilaterally. This result is in line with several observations on sound and music imagery, which found only activity in auditory association cortices, but not the primary auditory cortex  could not be demonstrated during imagined musical performance  involved in recalling a musical piece Halpern, . AdditioModern data evaluation strategies enable us to record real world data in groups of musicians interacting, as has been nicely demonstrated in the kinematic studies of ensembles in the Keller laboratory Keller, . These rSystematic research into the neurophysiological correlates of mental rehearsal of musical motor performance is in a very basic state. In order to suggest practical applications for training with kinesthetic imagery from the experience of neurophysiologic studies, more behavioral protocols on the effects on different imagery strategies for improving musical performance have to be tested. To develop strategies for improving the use of imagery training in musicians or for testing and selecting appropriate imagery techniques for training, an interaction of experienced musicians and neuroscientific research is essential. The most fruitful advances might be those, applying cognitive tests to identify characteristic performance gains dependent on different training procedures. Additionally, the control of mental processes performed during mental imagery is essential. The following questions might lead us: How can we identify good imagers? Why and how do subjects profit from imagery training? Is the temporal duration of imagined performance and vividness rating enough or shouldn't we apply more sophisticated neurophysiological tests  to control and investigate the quality of imagery? All these questions might help to understand the fascinating processes associated with experienced performance of musicians.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Population differentiation in environments without well-defined geographical barriers represents a challenge for wildlife management. Based on a comprehensive database of individual sighting records (1988–2009) of blue whales from the winter/calving Gulf of California, we assessed the fine-scale genetic and spatial structure of the population using individual-based approaches. Skin samples of 187 individuals were analyzed for nine microsatellite loci. A single population with no divergence among years and months and no isolation by distance  were found. We ran two Bayesian clustering methods using Structure and Geneland softwares in two different ways: 1) a general analysis including all individuals in which a single cluster was identified with both softwares; 2) a specific analysis of females only in which two main clusters  were revealed by Geneland program. This study provides information indicating that blue whales wintering in the Gulf of California are part of a single population unit and showed a fine-scale structure among females, possibly associated with their high site fidelity, particularly when attending calves. It is likely that the loss of genetic variation is minimized by male mediated gene flow, which may reduce the genetic drift effect. Opportunities for kin selection may also influence calf survival and, in consequence, have a positive impact on population demography in this small and endangered population. Detecting population structure in taxa characterized by continuous distribution in environments without well-defined geographical barriers is one of the most challenging problems in population genetics. This applies to the conceptual definition of populations, but also represents a challenge for conservation and wildlife management. Following the evolutionary paradigm proposed by Many aspects of life history, as well as several environmental factors, can influence the structure of populations and, therefore, the individual’s dispersal potential alone does not always allow predictions about the gene flow among populations. This is especially true for cetaceans, for which movements are not limited by physical barriers. Although most cetacean species show wide distributions and high potential for dispersal, their populations usually exhibit some kind of structure Megaptera novaeangliaeEubalaena glacialis and E. australis respectively Eschrichtius robustusPhyseter macrocephalus, Balaenoptera musculus intermedia), that showed divergence in mitochondrial DNA and microsatellite markers among the six feeding grounds in the Antarctic Female philopatry and migration patterns are known to have a strong influence on population structure in large mammals Balaenoptera musculus) is a cosmopolitan species, with both coastal and oceanic distribution. Like most baleen whales, this species migrates between feeding grounds, located in areas at high latitudes, and calving grounds at mid and low latitudes The blue whale  were occupied by distinct populations, and in each of them no structure was found probably due to small sample sizes In this paper we investigated the population structure of blue whales in the GC, the only studied calving ground for this species using an extensive data base of individual sighting histories obtained during the last 22 years The GC is a narrow internal sea that separates Baja California Peninsula from the Mexican mainland, located between 23° to 31°N and between 107° to 115°W in the Northeast Pacific Ocean . It is 1We conducted 2–6 day-long surveys between January and May 1988–2009, covering the area between the cities of La Paz and Loreto . MoreoveFrom 1997 to 2009 skin-blubber biopsies were collected with a similar system as described in Lambersten We avoided duplicating biopsy samples within and between seasons by comparing newly photographed whale  with those included in a field catalogue of biopsied whales. The biopsies collected from 1997 to 2001 were preserved in 20% dimethylsulfoxide (DMSO) saturated with table salt Photo-identification and biopsy samples were collected under the annual research permits  issued by the Secretaria de Medio Ambiente Recursos Naturales y Pesca  from 1997 to 2001, and by the Dirección General de Vida Silvestre, Secretaría de Medio Ambiente y Recursos Naturales  from 2002 to 2009, which now represents the only approved Government institution to issue research permits on endangered species in Mexico. By issuing these annual research permits they approved the number of blue whale skin-blubber biopsies collection for each year of the permit. Our institution does not have an Animal Ethics Committee; therefore no information is given on that aspect.Taq Polymerase (Invitrogen™), 0.5 µM of each primer , 0.2 mM dNTPs and MgCl2 concentration as specified in 1  and the expected (HE) heterozygosity and with Arlequin 3.1 IS coefficient in Fstat 2.9.3.2 We calculated the number of alleles per locus, the observed (H”). As a result of this criterion, the 1997–2009 period for the “years” category, and the 1999–2009 period for the “month-year” category were analyzed  according to field season when individuals were sighted (hereafter “years”), and to the month and year of sightings  were tested for departure from our population sex ratio (see results) with chi-square test. We tested deviation from HWE and calculated the fixation indices- RQGRLTo verify the presence of isolation by distance we carried out an individual-based Mantel test, both on the complete dataset  and on females only, using the kinship coefficients R6 iterations, following a burn-in period of 60000 iterations. The mean values of the 10 runs for each K were reported, and we chose the K value with the highest probability (Ln P(D)) as the best estimated number of clusters. In the second analysis, we used the mixture model with correlated allele frequency and free Voronoi tessellation 6 iterations and Kmax = 10. Each set of 25 runs were ranked using the posterior probability of each, and we post-processed the best 10 runs. We obtained the K value from the mode value of these 10 runs, and reported the best posterior probabilities of this mode. For post-processing a burn-in period of 60000 iterations and a 800×200 pixel resolution for x and y axis were used respectively. The geo-referencing of each individual used in this analysis was the same as in the individual-IBD analysis (see above). The range of some blue whale movements can extend as much as the whole study area 95% was 3197.6 km2, thus the uncertainty value used was 57 Km.We carried out two Bayesian clustering analysis, both on the complete dataset  and on females only. In the first, we used the admixture model with correlated allele frequency implemented in Structure 2.3 ST, FST and FIS between and within clusters respectively. For both models we performed 10 preliminary runs in order to know the maximum K value and the length of the chain that should be used so as not to affect the results.In order to verify if clusters obtained were artifacts of the models we analyzed HWE and divergence between them, using clusters with at least five individuals 2 = 28.8, p = 0.053). No pair of loci was in linkage disequilibrium . The inbreeding coefficient was close to zero , and the mean observed (HO) and expected (HE) heterozygosity were equal   .A total of 187 blue whale samples were analyzed for 9 microsatellite loci. We obtained a 100% match in genotyping of 5 duplicated samples that were classified as different individuals by photo-ID. These 10 samples were excluded and we carried out all further analyses on 177 whales . In accordance to a previous study ST = 0–0.01, p>0.05, FST = 0–0.03, p>0.05).The number of individuals observed ranged from 16, in 1998, to 61 in 2009 . We founST and FST in nine comparisons, but not after we applied the Benjamini-Yekutieli correction , March (n = 11) and April (n = 2). We found a significant deviation from the 1.41/1 population sex ratio towards females in February , nor in females only data set .No evidence of IBD was found in individual-based Mantel tests performed on the complete data set  in both analyses  was similar to values reported for this species in the South Pacific (Ho = 0.72) and the Antarctic (Ho = 0.75) Oceans O = 0.66 in Perth Canyon, Western Australia; Ho = 0.59 in Bonney Upwelling, Southern Australia) IS close to zero) suggest a panmictic population.The mean observed heterozygosity of blue whales in the GC  ST = 0.005 p = 0.031) ST = 0.01, p = 0.01, FST = 0.008, p = 0.02). The spatial distribution of the genetic clusters was not completely homogeneous, as expected in very mobile animals, suggesting that the genetic segregation is only partially matched by ecological segregation. Moreover, the low values of RST and FST indicate that gene flow is maintained between clusters, allelic frequencies are correlated among them, and the recognition of clusters may be difficult As mentioned above, it has been reported that Structure models fail to find structure when FThe uncertainty in spatial position is an important parameter of the Geneland model, and allows to take into consideration both the measurement error , and the process error . Our uncertainty value was calculated mainly from females’ individual utilization areas and, therefore, the higher power in detecting clusters with data on females may just be an effect of the better representation of their spatial structure.Fine-scale population structure has been reported in various mammalian species. In most cases, female natal philopatry generates aggregated grouping patterns, and this produced some level of genetic differentiation N. simplex have been reported N. simplex during the winter From an ecological point of view, blue whale spatial distribution is linked to the distribution of euphausiids Eubalaena australis), mother-calf pair congregates in specific nursery grounds, and are segregated from males in winter grounds, possibly to reduce the occurrence of male harassment which can be fatal to the calves From a behavioural point of view, blue whale female-calf pairs prefer coastal waters, more protected from the strong dominant North wind and swell Female fine-scale genetic structure has potentially important consequences for conservation and management. Opportunities for kin selection may have a positive effect on calf survival. Blue whale females with calves, that need to implement efficient foraging strategies due to heavy energetic demands, may take advantage of the high seasonal productivity and calm waters of the southwestern GC islands, and share these areas with relatives. Mature females and calves survival has a dramatic impact on population demography, and, therefore, management policies tailored to the areas where mother-calf pairs are frequently sighted could be greatly beneficial for population viability. Moreover, female fine-scale genetic structure may influence the rate at which genetic diversity is lost through genetic drift. Our results suggest that in blue whales, the loss of genetic variation may be minimized by male mediated gene flow. In this context, a socially structured population, with at least two main groups with high levels of heterozygosity, may lead to a reduction of inbreeding, and of the effects of genetic drift.