Taeniopygia guttata), where sperm size influences sperm swimming velocity, to determine the effect of sperm total length on fertilization success. Sperm competition experiments, in which pairs of males whose sperm differed only in length and swimming speed, revealed that males producing long sperm were more successful in terms of (i) the number of sperm reaching the ova and (ii) fertilizing those ova. Our results reveal that although sperm length is the main factor determining the outcome of sperm competition, complex interactions between male and female reproductive traits may also be important. The mechanisms underlying these interactions are poorly understood, but we suggest that differences in sperm storage and utilization by females may contribute to the outcome of sperm competition.Sperm competition, in which the ejaculates of multiple males compete to fertilize a female's ova, results in strong selection on sperm traits. Although sperm size and swimming velocity are known to independently affect fertilization success in certain species, exploring the relationship between sperm length, swimming velocity and fertilization success still remains a challenge. Here, we use the zebra finch ( The number of sperm inseminated is often important in determining the outcome of sperm competition  longer flagella , providiFaster swimming sperm are often assumed to fertilize more ova because fast sperm may reach the site of fertilization before slow sperm. This relationship between swimming speed and fertilization success is evident in some species of birds ,35 and fTaeniopygia guttata, to clarify the relationship between sperm length and fertilization success. The zebra finch is an ideal species to do this because considerable natural variation in sperm length exists between males  leto 80 μm ,42, and We conducted sperm competition experiments to test the hypothesis that, in a competitive environment, long sperm males fertilize more ova than short sperm males. In a mate-switching experimental design (similar to ), pairs By counting the sperm embedded in the outer perivitelline layer (OPVL) of the avian ovum, it is possible to estimate how many sperm reach the ovum and to determine the likelihood of fertilization following a single insemination . We deve2.(a)n = 18) and one produced short sperm (n = 18). In no case did the sperm of the male pairs overlap in length . Each male pair was allocated to an unrelated  female who originated from either the long (n = 8) or short (n = 10) selection line. The mean relatedness scores (presented as mean ± s.d.) between the male and female pairs, and between pairs of competing males were low . Females were housed singly in a cage (dimensions 0.6 × 0.5 × 0.4 m) with a nest-box half filled with hay. Each female cage had an adjoining cage for use later in the experiment.The zebra finches in this study were part of a domesticated population maintained at the University of Sheffield since 1985. Zebra finch sperm morphology  is highly heritable ,42. We c(b)n = 192) and marked with a unique female code and the egg number. Eggs were artificially incubated at 38°C for 48 h, and stored at 4°C until processing. When the duration of sperm storage for female zebra finches was exceeded—14 days [Sperm competition experiments were conducted using a mate-switching protocol . One mal—14 days —each mat(c)−1)  and incubated in the dark for 2 min. We examined the half of the OPVL that contained the germinal disc (GD) because the majority of sperm are observed around the GD [n = 4420) was measured to the nearest 0.01 μm [r2 = 0.96, t = 15.18, d.f. = 18, p < 0.0001). In cases where the sperm's head was missing, we used flagellum length to identify sperm as long or short .Male competitive success was assessed in two ways: (i) the proportion of sperm from each male that reached each ovum (determined by counting sperm on the OPVL)  and (ii)d the GD . Using f 0.01 μm  (see the(d)At the end of the experiment, all males (fully rested from copulation for at least four weeks) were humanely killed by cervical dislocation and sperm collected from the distal region of the left SG by dissection. The following sperm quality analyses  were carried out to determine whether sperm quality parameters were similar within the male pairs: (i) swimming velocity (the swimming speed of sperm), (ii) viability (the proportion of viable sperm), (iii) morphology , (iv) concentration, and (v) longevity (the length of time sperm remained motile) . Testes mass data were also collected . Data on copulation rate and SG mass were opportunistically collected from long and short sperm males that were not used in the experiment .(e)eneMapper v. 3.7 . Paternity was assigned to embryos (n = 166) using Cervus v. 3.0.3 [DNA was extracted from embryos using the ammonium acetate protocol . DNA wasv. 3.0.3 , at grea(f)All data were analysed in R v. 2.15.1 . Exact bIn order to control for the effects of last male sperm precedence , we then carried out a second GLMM that used the second mating male as the focal male in the analysis. The paternity of each embryo was included as either ‘1’ or ‘0’ . Male mating order (short first/short second), female line (long/short) and the number of days between the male swap and the laying of the focal egg were included as fixed effects. Trio ID was fitted as a random effect. We also modelled all interactions between the three fixed effects. Model simplification was carried out using log-likelihood tests and Akaike information criterion (AIC) values to obtain the minimal adequate models.3.(a)p < 0.0001). Long sperm males sired a greater proportion of embryos (64 ± 8%) than short sperm males  reached the ova than short sperm (43 ± 2%) χ2 = 1.15, p = 0.28)). This means that the patterns of paternity observed in this study cannot be explained simply by the passive loss of sperm from the SSTs, where last male precedence would be expected [Mating order of the males did not determine which male fertilized the egg ; long male second: 60 ± 11% (mean percentage ± s.e.m.); proportion test; expected .(c)z = 3.20, p = 0.001, p = 0.89; see the electronic supplementary material, table S5, for summary data). Taken together, these results demonstrate that in the zebra finch, long sperm males are more successful in a sperm competition scenario than short sperm males.Male fertilization success was also influenced by interacting effects of male mating order and female selection line , they rGallus gallus domesticus) [In a study of domestic fowl (esticus) , it was esticus) ) fertiliesticus) . In thisAlternatively, our results may be accounted for if short sperm are simply less likely to reach and/or enter the SSTs. To reach the uterovaginal junction, where the SSTs are located, sperm must swim through the hostile vaginal region of the oviduct, so it is likely that swimming speed determines success during this phase . Again, first male precedence , and the size and shape of her SR influences a male's fertilization success depending on his sperm length. In an elegant experiment, Miller & Pitnick [Drosophila, artificially selected for divergence in sperm length and SR length, respectively. Long sperm males had a pronounced fertilization advantage when copulating with females with long SRs, possibly due to optimal positioning of long sperm within the SR for fertilization. Given the growing evidence of the pivotal roles of females in determining the outcome of sperm competition, particularly in internally fertilizing species, it is perhaps unsurprising that, in addition to the strong effect of sperm length, we also found some evidence for female effects on competitive fertilization success in the zebra finch.There is increasing evidence that females exert some control over paternity , and tha Pitnick  used pop5.We have experimentally demonstrated that in the zebra finch, long sperm have an advantage in sperm competition compared with short sperm. This long sperm advantage is evident both in the number of sperm that reach the site of fertilization and those that fertilize the ovum. As all other measures of sperm quality, except swimming velocity, were comparable between our long and short sperm males, the competitive success of the long sperm males can clearly be attributed to sperm length. Importantly, however, our results demonstrate that male competitive success is not necessarily the simple outcome of a race between the sperm of rival males. Instead, sperm competitive success appears to be mediated by the female, possibly through as yet unknown mechanisms of differential sperm acceptance or release from sperm storage sites.


Few studies have reported on the risk factors of dilated Virchow-Robin Spaces (dVRS) in large samples of ischemic stroke patients. Little evidence exists regarding the relationship between dVRS and etiologic subtype of ischemic stroke or lacune. We aimed to investigate the risk factors associated with the severity of dVRS in a large sample of ischemic stroke patients.We consecutively enrolled 1,090 patients who experienced an ischemic stroke within the past seven days and underwent a 3.0 T MRI scan in the Chinese IntraCranial AtheroSclerosis Study (ICAS). Clinical data and cranial MRI information of patients included age, sex, vascular risk factors, dVRS, leukoaraiosis, lacune, and etiologic subtype of ischemic stroke. Analyses were performed regarding the risk factors associated with the severity of dVRS by univariate analysis and multivariable ordinal logistic regression analysis.Through multivariable ordinal logistic regression analysis, age, the severity of leukoaraiosis, lacune, admission National Institutes of Health Stroke Scale (NIHSS) ≤3, and the severity of dVRS in the white matter (WM) and hippocampus (Hip) were correlated with the severity of dVRS in basal ganglia (BG); male, history of hypertension, admission NIHSS ≤3, and the severity of dVRS in BG and Hip were correlated with the severity of dVRS in WM; female, the severity of leukoaraiosis, admission NIHSS >3, small artery occlusion subtype of ischemic stroke, and the severity of dVRS in BG and WM were correlated with the severity of dVRS in Hip.dVRS is an indicator of cerebral small vessel diseases such as leukoaraiosis and lacune. However, the risk factors of dVRS differ in various brain regions. Virchow-Robin spaces (VRS) are perivascular spaces that surround small arteries and arterioles This study was approved by the ethics committee of the Beijing Tian Tan Hospital of Capital Medical University and was performed in accordance with the guidelines of the Helsinki Declaration. After ethical approval of Tian Tan Hospital was obtained and approved by the other 21 participating hospitals , the ethical approval took effect automatically in each center. All patients or their legal representatives provide their written informed consent form.ICAS was a prospective, multicenter, hospital-based study. Clinical and imaging data were collected from consecutive patients with ischemic stroke or transient ischemic attack (TIA) in 22 general hospitals in China. There were 2864 patients with noncardioembolic ischemic cerebrovascular diseases enrolled in ICAS. A total of 1090 subjects with ischemic stroke in seven hospitals underwent 3.0 T MR scanner and were enrolled in our study. The institutional review board of the participating hospitals approved the study. Each participant signed an informed consent statement.Inclusion criteria included onset of symptoms within seven days, and age between 18 and 80 years old. Exclusion criteria were as follows: patients who were clinically unstable , were disabled before admission , and/or were unable to comply with MRI examination. We also excluded patients with cardioembolic risk factors  to rule out stroke patients due to cardioembolism. Full details of the design, and baseline information of the ICAS were previously published We evaluated clinical data  and the information of cranial MRI  and analyzed the relationship between these factors and the severity of dVRS in different brain regions.All 1090 patients in seven hospitals underwent imaging with the 3.0 T magnetic resonance (MR) scanner, including 3-dimensional time-of-flight MR angiography , and axial T2-weighted imaging , T1-weighted imaging , fluid-attenuated inversion recovery sequences , and diffusion-weighted imaging . All above sequences except MRA had 5 mm slice thickness and 1.5 mm interslice gap. MR images were transferred to a personal computer workstation and viewed using software . Rating of dVRS and LA were evaluated. Presence and quantities of lacune were also recorded.Two radiologists who were blinded to patients' clinical information independently evaluated MRI data. Disagreements on the evaluations of dVRS, lacune, Fazekas score, and etiologic subtype of ischemic stroke were resolved through discussions.The definition of dVRS was: 1) signal intensity of dVRS on T2-images being equivalent to that of the cerebrospinal fluid (CSF) ; and 2) round, ovoid, or linear shape with a smooth margin, maximum diameter <3 mm and located in areas supplied by perforating arteries In the basal ganglia (BG), dVRS were rated according to the number of dVRS in the slice containing the maximum amount of dVRS. The grades of dVRS were rated as follows: Grade 1: the number of dVRS <5; Grade 2: the number of dVRS, 5–10; Grade 3: the number of dVRS >10 but still countable; and Grade 4: the number of dVRS was infinite .In the white matter (WM), dVRS were scored as follows: Grade 1 when the number of dVRS <10 in the entire WM slices; Grade 2 when the number of dVRS VRS in the entire WM slices but the number of dVRS <10 in any WM slice; Grade 3 when the number of dVRS ranged from 10 to 20 in the slice containing the maximum number of dVRS; and Grade 4 when the number of dVRS >20 in the slice containing the maximum number of dVRS  [2].In the hippocampus (Hip), dVRS were scored according to number of dVRS in the total sections of hippocampus and parahippocampal gyrus. dVRS were rated as follows: Grade 1 when the number of dVRS <5; Grade 2 when the number of dVRS ranged from 5 to 10; and Grade 3, when the number of dVRS >10 .LA was defined as having a hyperintense lesion on FLAIR and T2 images, which was usually not seen on T1-weighted MRI or shown faint hypointensity at most. We rated LA with the Fazekas method Lacune were defined as lesions of 3 to 15 mm in size with the same signal characteristics as CSF on all sequences, with a hyperintense rim on the FLAIR sequence  Etiologic subtypes of ischemic stroke were classified according to the Stop Stroke Study Trial of Org 10172 in Acute Stroke Treatment (SSS-TOAST) classification criteria The clinical information collected from patients included age; sex; histories of hypertension, hyperlipidemia, ischemic stroke, stroke (including ischemic and hemorrhagic stroke), and coronary heart disease ; hypertension (defined as a history of hypertension or diagnosis at discharge); diabetes (defined as a history of diabetes or diagnosis at discharge); and NIHSS score at admission. Meanwhile, current or former smoking status (continuously smoking ≥1 cigarette a day for 6 months), and heavy drinking status (drinking >2 U/d on average for men or >1 U/d on average for women) were also recorded.2) test was used for comparison of categorical variables. The baseline potential risk factors and their crude distribution were presented according to the severity of dVRS. Multivariable ordinal logistic regression analysis was used to identify predictors of increasing dVRS severity in BG, WM, and Hip. All parameters that were significant in the univariate analysis at P<0.05 level or likely to have pathophysiological influence on dVRS were included in the multivariable ordinal logisitic regression model. Interobserver agreement of the dVRS grading was assessed by using kappa (K) test to quantify the level of agreement. All probability values were 2-tailed; P<0.05 was considered to be statistically significant. All analyses were performed using SAS Version 9.1 .Continuous variables were summarized as mean value (standard deviation) or median (interquartile range) according to whether its distribution conformed to a normal distribution. Categorical variables were presented as percentages and absolute numbers. Analysis of variance was used for comparison of continuous variables with normal distribution, and Kruskal-Wallis test was used for comparison of continuous variables with non-normal distribution. Chi-squared (χBaseline characteristics of the 1090 patients are shown in P<0.0001) between BG and WM dVRS, 0.24 (P<0.0001) between BG and Hip dVRS, and 0.28 (P<0.0001) between WM and Hip dVRS. The proportion (number) of Grade 0–3 of PWMH was 5.5% (60), 46.1% (502), 35.2% (384) and 13.2% (144) respectively, the proportion (number) of Grade 0–3 of DWMH was 15.4% (168), 48.5% (529), 28.0% (305) and 8.1% (88) respectively. Mean Fazekas score was 2.85. Among the 1090 patients, 44 patients' images of lacune could not be evaluated due to the poor quality of MR images. The mean number of lacune was 1.5. Most patients 1069 (98.1%) had symmetric dVRS in bilateral hemisphere, only 21 (1.9%) patients had noticeable inter-hemispheric difference in the severity of dVRS. All these 21 patients had significant stenosis or occlusion of ipsilateral internal carotid artery or middle cerebral artery.All of 1090 patients had dVRS, the percentages and numbers of 0–4 grade dVRS in BG, WM and Hip are demonstrated in K value = 0.68, 0.67, and 0.79 respectively). Interrater agreement from the two assessors for the ratings of PWMH and DWMH were also high .Interrater agreement from the two assessors for the ratings of dVRS in BG, WM, and Hip were high (P<0·0001), Fazekas score ≥3 (P<0·0001), the presence of lacune (P = 0·02), admission NIHSS score ≤3 (P = 0·02), and the severity of dVRS in WM and Hip (P<0·0001) remained statistically significant in association with the severity of dVRS in BG , history of hypertension (P<0·0001), admission NIHSS score ≤3 (P<0·0001), and the severity of dVRS in BG and Hip (P<0·0001) remained significant in association with the severity of dVRS in WM (In order to rule out the influence of white matter hyperintensities (WMH) with regard to the evaluation of dVRS, we carried out univariate and multivariable analyses after removing patients with grade 3 PWMH or grade 3 DWMH, and found that history of hypertension, admission NIHSS score ≤3, the presence of LA, and Fazekas score ≥3 significantly related with more serious dVRS in WM in univariate analyses . In multRS in WM .P = 0.01), admission NIHSS score >3 (P = 0·004), Fazekas score ≥3 (P = 0·003), SAO subtype stroke (P = 0.006), and the severity of dVRS in BG and WM (P<0·0001) remained significant in association with the severity of dVRS in Hip  in noncardioembolic ischemic stroke patients. dVRS most commonly occured in BG, and then in WM and Hip.However, multivariable ordinal logistic regression analysis showed that the risk factors of dVRS varied according to the regions. Increasing age was only significantly associated with dVRS in BG; hypertension was an independent risk factor for dVRS in WM but not in BG and Hip; males had a higher risk for more severe dVRS in WM, whereas females had a higher risk for more severe dVRS in Hip relatively; severe leukoaraiosis (Fazekas score ≥3) was significantly associated with increasing severity of dVRS in BG and Hip, but not with that in WM; the presence of lacune was positively correlated with more severe dVRS in BG, but not with that in WM or Hip; SAO subtype stroke was only significantly related with more severe dVRS in Hip.Risk factors for dVRS in various brain regions were different in our study, several reasons may account for this. First, regional variations in severity have been observed for fibrohyaline thickening, lipohyalinosis, and cerebral amyloid angiopathy (CAA) within the microvasculature during ageing Rouhl et al The association between dVRS and lacune appeared to be stronger in BG than in WM Our study specifically investigated the association between dVRS and SSS-TOAST etiologic subtype of ischemic stroke, and found that SAO subtype stroke was significantly correlated with more severe dVRS in Hip. The result further supports that dVRS is another indicator of small vessel diseases (SVD). The associations between the severity of dVRS and the presence of old lacunes, severity of LA and SAO subtype stroke suggested that the development of dVRS could be the consequence of an underlying SVD. These findings are in agreement with previous pathological and MRI results The strengths of our study included the use of carefully standardized research methods and a large sample size. Our study had several limitations. First, lacune could not be evaluated in 44 patients due to the poor quality of MR images. Second, because some patients' clinical information was incomplete, only 1028 of 1090 patients underwent multivariable logistic regression analysis. Third, this was a cross section study, further prospective studies were needed to confirm the cause and effect relationship between these risk factor and dVRS. Lastly, because the pathological studies about dVRS were very few and limited, further histopathological studies are needed to account for the regional variations in risk factors of dVRS.Overall, our study demonstrated that the severity of dVRS in certain brain regions was significantly correlated with age, history of hypertension, the severity of LA, the presence of lacune, and SAO subtype stroke; and dVRS was another MRI marker of SVD. However, there were some regional variations among the risk factors associated with the severity of dVRS, which indicates that the developmental mechanisms of dVRS may differ in various brain regions.


Orbivirus (Reoviridae family), causing the notifiable diseases Bluetongue and African horse sickness of ruminants and equids, respectively. Reverse genetics systems for these orbiviruses with their ten-segmented genome of double stranded RNA have been developed. Initially, two subsequent transfections of in vitro synthesized capped run-off RNA transcripts resulted in the recovery of BTV. Reverse genetics has been improved by transfection of expression plasmids followed by transfection of ten RNA transcripts. Recovery of AHSV was further improved by use of expression plasmids containing optimized open reading frames.Bluetongue virus (BTV) and African horse sickness virus (AHSV) are distinct arthropod borne virus species in the genus Plasmids containing full length cDNA of the 10 genome segments for T7 promoter-driven production of full length run-off RNA transcripts and expression plasmids with optimized open reading frames (ORFs) were used. BTV and AHSV were rescued using reverse genetics. The requirement of each expression plasmid and capping of RNA transcripts for reverse genetics were studied and compared for BTV and AHSV.BTV was recovered by transfection of VP1 and NS2 expression plasmids followed by transfection of a set of ten capped RNAs. VP3 expression plasmid was also required if uncapped RNAs were transfected. Recovery of AHSV required transfection of VP1, VP3 and NS2 expression plasmids followed by transfection of capped RNA transcripts. Plasmid-driven expression of VP4, 6 and 7 was also needed when uncapped RNA transcripts were used. Irrespective of capping of RNA transcripts, NS1 expression plasmid was not needed for recovery, although NS1 protein is essential for virus propagation. Improvement of reverse genetics for AHSV was clearly demonstrated by rescue of several mutants and reassortants that were not rescued with previous methods.A limited number of expression plasmids is required for rescue of BTV or AHSV using reverse genetics, making the system much more versatile and generally applicable. Optimization of reverse genetics enlarge the possibilities to rescue virus mutants and reassortants, and will greatly benefit the control of these important diseases of livestock and companion animals. Orbivirus, family Reoviridae. These viruses are spread by specific species of Culicoides biting midges. Outbreaks and geographic expansion of affected areas are associated to various factors, including climate change . Pl. Pl47]. Protein expression was determined by immunoperoxidase monolayer assay (IPMA) according to standard procedures . Transfe5 BSR cells per 2 cm2 were transfected twice with 600 ng RNA containing equimolar amounts of capped run-off RNA transcripts of six segments followed by transfection with a set of all 10 RNA transcripts. After 4 h, transfection medium was replaced by culture medium, and after 2 days culture medium was harvested. Virus recovery was determined by infection of fresh BSR monolayers. Cytopathogenic effect (CPE) and the immunoperoxidase monolayer assay (IPMA) confirmed virus recovery and virus identity. If negative, duplicate transfected wells were passed, screened for CPE and immunostained as described above. Sequential passaging of cells was repeated was repeated with immunostaining serving as indicator of virus replication. If immunostaining was no longer visible, attempts were repeated at least twice in order to conclude that virus recovery was considered unsuccessful.Reverse genetics (RG) using exclusively in vitro synthesized RNAs has been described for BTV , 26. Bri2 wells were first transfected with equimolar amounts of expression plasmids followed by transfection of the set of 10 RNAs as described above. A total of 600 or 300 ng DNA plasmids was transfected using 1.5 or 0.75 μl Lipofectamine™ 2000  in Opti-MEM® I Reduced Serum Medium according to manufacturer’s conditions. The transfection of 10 RNAs was carried out as described above. For AHSV, the RNA transfection was the same but transfection medium was replaced after 22 h, and transfected monolayers were passed once. Medium was harvested after 2 days to infect fresh monolayers in order to confirm virus recovery. If negative, duplicate wells with transfected cells were processed as described for recovery of BTV.Reverse genetics (RG) using expression plasmids and in vitro synthesized RNAs is very similar to the method using exclusively in vitro synthesized RNAs , 26. BriMutants and reassortants of BTV and AHSV were generated according to the described and indicated methods Table . MutatioPlaque morphology (CPE) was determined as previously described , 12. BriInitially, recovery of AHSV4LP by transfecting in vitro synthesized capped run-off RNA transcripts on two subsequent days was studied according to reverse genetics (RG) method for BTV . UnfortuExpression plasmids for ORFs of VP1, 3, 4, 6, 7, and NS1 and 2 were constructed to increase and to extend protein expression. However, the plasmid with VP3-ORF was not stable, and the ORF was successfully optimized for plasmid stability and eukaryotic expression. Results with VP7 expression plasmid showed a stronger expression compared to that from capped RNA (not shown). Transfection of expression plasmids of VP1, 3, 4, 6, 7, and NS1 and 2 followed by transfection of 10 in vitro synthesized capped run-off RNA transcripts resulted in recovery of AHSV4LP  has been developed for viruses of many families including those with double stranded RNA (dsRNA) segmented genomes. The latter includes members of aviridae , 53, ande family . RG for nscripts . Capped nscripts . Howevernscripts . These rPrimary protein expression by the first transfection of RNA transcripts can be replaced by plasmid-driven expression for BTV recovery . A similNS1 expression by the first transfection is not essential for recovery of BTV and AHSV Tables  and 3 4. NS1 incThe minimal requirements of plasmid-driven expression differs between BTV and AHSV Tables  and 3. V, in vitro reconstitution of infectious BTV core particles showed that VP1, VP4 and VP6 form a transcription complex that does not need other proteins for its activity [Similar to the enhancement in recovery by VP7, we suggest that VP3 enhances orbivirus recovery by increase of the initial replication activity by stabilization of premature replication complexes and the formation of subcore particles. Consequently, suboptimal expression of VP1 and NS2 requires expression of VP3 and VP7 to efficiently recovery virus. In full agreement with thisactivity . NS2 recactivity , and is Small amounts of VP4, 6 and 7 expressed from capped RNAs after 18–22 h are sufficient to initiate recovery of AHSV. These capped RNA transcripts are not needed for BTV recovery Tables . Others in trans complementation followed by virus passage on ordinary cells [Summarizing, VP1 and NS2 expression plasmids are essential for an optimized RG system of BTV and AHSV. VP3 expression plasmid is also essential for BTV recovery if uncapped RNAs are used. In contrast, only NS1 expression plasmid could be omitted in combination with uncapped RNAs for AHSV recovery. Generally, we conclude that recovery of BTV is more efficient than of AHSV. In order to increase the success rate for rescue of mutants and reassortants, the most optimal system is the combination of all seven expression plasmids with optimized ORFs and capped RNA transcripts for the second transfection. BTV1 and BTV8 variants were generated using BTV6 based ORF-optimized expression plasmids , and virry cells .Requirements for RG of BTV and AHSV showed many similarities as well as differences, likely caused by the difference in efficiency of virus recovery, but fundamental differences between these orbivirus species cannot be excluded. This comparative analysis of reverse genetics for orbivirus prototypes BTV and AHSV will contribute to development of RG methods for other orbiviruses, such as the OIE listed, notifiable epizootic haemorrhagic disease virus, but also for other orbiviruses and reoviruses. This will increase our understanding of the complex replication cycle and assembly process of these viruses with a segmented double stranded RNA genome.For optimal function of RG systems for the rescue of BTV and AHSV, a limited number of expression plasmids is required. Optimization of reverse genetics enlarge the possibilities to rescue virus mutants and reassortants.AHSV, African horse sickness virus; BTV, bluetongue virus; DISA, Disabled Infectious Single Animal; DISC, Disabled Infectious Single Cycle/Cell; DMEM, Dulbecco’s modified Eagle’s medium; EHDV, epizootic haemorrhagic disease virus; FBS, foetal bovine serum; MOI, multiplicity of infection; mRNA, messenger RNA; ORFs, open reading frames; RG, reverse genetics


Andiroba oil containscomponents with anti-inflammatory, cicatrizing and insect-repellant actions. However,virtually nothing is known of the safety of this oil for humans. The aim of this workwas therefore to investigate the hematotoxicity, genotoxicity and mutagenicity ofandiroba oil using the comet and micronucleus assays, and to assess its antioxidantproperties and lipidome as a means of addressing safety issues. For the experiments,andiroba oil was administered by gavage for 14 consecutive days in nulliparous femaleSwiss mice randomly distributed in four groups: negative control and three doses ofoil . These doses were chosen based on recommendationsof the OECD guideline no. 474 (1997). GC/MS was used to investigate the free fattyacid, cholesterol and triterpene content of andiroba oil in a lipidomic analysis. Noclinical or behavioral alterations were observed throughout the period of treatment,and exposure to andiroba oil at the doses and conditions used here did not result inhematotoxic, genotoxic or mutagenic effects. Tests  Carapa guianensis and Carapa procera, of thefamily Meliaceae that occurs in the Amazon  at the Federal University of Pará.After collection, the fruits were stored at 12 °C and transported to the lab wherethe oil was extracted either with an artisanal extraction process . After artisanalextraction, the resulting mass was pressed and squeezed to reduce the oil content to< 5%, as described elsewhere  method. Theprotocol followed was that described by A0 is the absorbance of DPPH, A1 is theabsorbance of DPPH + diluted oils and As is the absorbance ofdiluted oils. The percentages of inhibition were used to calculate theEC50  by non-linear regression. Based on the results of theDPPH assay, the sample with the greatest antioxidant potential (sample SW3) and thatwas available in sufficient quantity was selected for the in vivotests.where in vivo assays, 90-day-old non-pregnant nulliparous femaleSwiss mice (Mus musculus) purchased from the Multidisciplinay Centerfor Biological Investigation (CEMIB) at the State University of Campinas (UNICAMP),Campinas, SP, Brazil, were used. The mice were maintained in the animal house of theDepartment of Genetics and Morphology at the University of Brasília under thefollowing conditions: 23 ± 2 °C, relative humidity 30-70%, and a 12 h light/darkcycle, with 10-15 changes of air/h. The mice had access to drinking water andcommercial rodent chow ad libitum. The animal protocols wereapproved by the Committee for Ethics in Animal Use (CEUA) of the Institute ofBiological Sciences at the University of Brasília (protocol no. 127331/2013).For the ®, Bunge) and thetreated mice received a solution of andiroba oil and corn oil by gavage through agastric tube. The treatments were done for 14 consecutive days and the dose limitcorresponded to that recommended in the Organization for Economic Co-operation andDevelopment (OECD) guidelines for the evaluation of genotoxicity by the micronucleustest : negative control and three doses of oil .The negative control group received corn oil  and xylazine (2%) and, after confirmation of narcosis, bloodsamples were obtained. Blood was collected by cardiac puncture and stored in plasticmicrotubes with 10% EDTA for hematological evaluation and preparation of slides forthe comet assay. During autopsy, the mice were inspected for macroscopic alterationsand the liver, kidneys and spleen were collected and weighed. For the micronucleusand nuclear abnormality tests, slides were prepared using femur bone marrow.On the 15The protocol for this assay was based on the alkaline comet assay (pH > 13)described by The micronucleus test was done using mouse femur bone marrow, as described by For hematological analysis, 370 μL of blood containing 10% disodium EDTA asanticoagulant was analyzed in an automatic veterinary hematocytometer (Sysmex pocH100iV Diff™) calibrated for mice.GC-MS analysis was done with a Thermo GC/MS system equipped with a quadrupole massselective detector operated at 70eV in electron impact (EI) mode. The TRACE-1300 gaschromatograph (GC) was equipped with a RTX-5TG- RESTECK column (30 m x 0.25 mm di x0.25 μm film thickness and the oven was operated at 40-300 °C in increments of 10°C/min with He as the carrier gas (flow: 1 mL/min). The injector was set at 220 °Cand 1 μL of the sample was injected into the GC via an AI/AS-1310 autosampler. Themass spectrometer (MS) was operated in scan mode . The transfer line and ion source were both operated at280 °C. The compounds were identified by comparing their mass spectra with those ofthe NIST-11MS lipid library database.50  was calculated by non-linear regression from the percentage inhibition ofthe free radical in the DPPH assay for the 21 samples of andiroba oil. Thequantitative data were evaluated by parametric or non-parametric statistical methodsbased on the normal distribution of the data. Parametric data were compared byanalysis of variance (ANOVA) followed by Dunnett's test, whereas non-parametric datawere analyzed with the Wilcoxon text followed by the Kruskall-Wallis test. All datacomparisons and statistical analyses were done using the softwares Instat 3.02 andPrism 5.0 , with a value of p ≤ 0.05 indicatingsignificance.The EC50data. Supplementary FiguresS1-S7 show that there was a correlation between thepercentage of DPPH inhibition and the oil concentration needed to cause a 50% reductionof DPPH. Samples with high and low antioxidant properties showed regional variations,e.g., northeastern Pará State, Marajó island and the metropolitanregion of Belém, as well as variations within each region. Two commercial samples ofandiroba oil (Amazon Ervas and Iana® D'amazônia) were also tested for theirantioxidant potential. The Iana sample, diluted in mineral oil as stated on themanufacturer's label, had the lowest antioxidant capacity of all the samples tested,with an EC50 of 8913 μL/mL, compared to 204.8 μL/mL for the Amazon Ervassample, which was similar to the value for the Cametá sample from northeasternBrazil.All of the oil samples showed antioxidant activity towards the DPPH radical, as shown inBased on these results and the availability of each oil, the sample SW3 was chosen forbiological assays. The lipid profiles were determined using the derivatizationtechnique. Saponified compounds represented > 97% of the sample and non-saponifiedcompounds consisted of steroids and triterpenoids derivatives . More thin vivo, the mice wereobserved daily for any alterations during treatment, but no clinical or behavioralalterations were observed and there were no changes in body weight. No macroscopicalterations were seen during autopsy nor were there any alterations in the absolute orrelative weight of the kidneys, liver or spleen   and oil extracted from the fruit ofLitsea cubeba , nor was there a significant increase in MN-PCE frequency or a decrease inthe PCE/NCE ratio for any of the oil samples tested (micronucleus test). The latterfinding indicated that there was no chromosomal structural and/or numerical damage inthe erythroblasts of Swiss mice treated with andiroba oil and there was no cytotoxicityto bone marrow. Together, these findings indicate that andiroba oil is not genotoxic,cytotoxic or mutagenic. Similar results have been reported for other plant oils, such asoil from pequi  may not originate from genetic damage, ina manner similar to that reported for limonoids, substances also found in andiroba oilthat cause cell apoptosis via the mitochondrial route (50) to be > 5g/kg. Since mice, which have a low body mass, were used in the present study, we optedto follow the doses described in the The doses used in this study can be compared with those used by There were no significant changes in body weight or in the absolute and relative weightsfor liver, spleen and the left and right kidneys after treatment. Since the doses of andiroba oil used in this study were similar to those used inphytotherapy, we conclude that the risk of toxicity from this oil when used in humans isvery low. In addition, the DPPH test clearly demonstrated the antioxidant activity ofall 21 samples of andiroba oil, although there was considerable variation among them.This antioxidant activity could protect DNA from oxidative damage. In an analysis of thechemical composition analysis of andiroba oil, Based on the information available at the moment, it is not yet possible to attributeantioxidant activities to a specific compound in andiroba oil. Such identification wouldinitially require separation of the oil components followed by individual analysis ofthe DPPH-reducing activity by thin layer chromatography (TLC) to compare the antioxidantactivity of different classes of substances. The DPPH test was found to be useful fordetecting regional as well as geographic variation in the antioxidant capacity ofandiroba oil samples in the state of Pará.Perilla frutescens from which essential oil is extracted in Chinamay be the result of genetic and/or environmental factors. Similar factors could accountfor the differences in the antioxidant capacity of andiroba oil. The DPPH test wasparticularly useful for screening andiroba oil that was sold diluted in mineral oil, asin the case of the Iana sample. Dilution in mineral oil reduces the oil's antioxidantcapacity and attenuates the therapeutic properties such as acaricidal activity , and the sample used in most of the experiments describedhere, namely, SW3 (EC50 = 59.95), with samples from other studies, such aspequi oil (EC50 = 26.26)  indicated that higher values were obtained with andirobaoil, i.e., this oil had a lower antioxidant potential than pequioil.Comparison of the ECOverall, our data extend our knowledge of the risks of adverse toxic effects and thepotential phytotherapeutic uses of andiroba oil. In the experimental conditions usedhere, andiroba oil was not hematotoxic, genotoxic, mutagenic or cytotoxic. On thecontrary, the antioxidant activity of the oil would tend to protect cellular DNA fromoxidative damage. In conclusion, andiroba oil, which is used in folk medicine amongAmazonian populations, has a low risk of toxicity under the conditions in which it wastested here.


The CO2 vent found at Levante Bay  also releases high concentrations of trace elements to its surrounding seawater, and is therefore a unique site to examine the effects of long-term exposure of nearby organisms to high pCO2 and trace element enrichment in situ. The sea anemone Anemonia viridis is prevalent next to the Vulcano vent and does not show signs of trace element poisoning/stress. The aim of our study was to compare A. viridis trace element profiles and compartmentalization between high pCO2 and control environments. Rather than examining whole anemone tissue, we analyzed two different body compartments—the pedal disc and the tentacles, and also examined the distribution of trace elements in the tentacles between the animal and the symbiotic algae. We found dramatic changes in trace element tissue concentrations between the high pCO2/high trace element and control sites, with strong accumulation of iron, lead, copper and cobalt, but decreased concentrations of cadmium, zinc and arsenic proximate to the vent. The pedal disc contained substantially more trace elements than the anemone’s tentacles, suggesting the pedal disc may serve as a detoxification/storage site for excess trace elements. Within the tentacles, the various trace elements displayed different partitioning patterns between animal tissue and algal symbionts. At both sites iron was found primarily in the algae, whereas cadmium, zinc and arsenic were primarily found in the animal tissue. Our data suggests that A. viridis regulates its internal trace element concentrations by compartmentalization and excretion and that these features contribute to its resilience and potential success at the trace element-rich high pCO2 vent.Ocean acidification (OA) is not an isolated threat, but acts in concert with other impacts on ecosystems and species. Coastal marine invertebrates will have to face the synergistic interactions of OA with other global and local stressors. One local factor, common in coastal environments, is trace element contamination. CO Recent  volume) . This unTrace elements are bioconcentrated in marine invertebrates by at least one order of magnitude when compared to seawater . Some trAnemonia viridis is a temperate Mediterranean species, which occurs naturally throughout Levante Bay. The anemones show no apparent signs of stress to the prevalent environmental conditions close to the primary vent  and animal tissues.The simultaneous exposure of 2 vents create a natural CO2/pH gradient along the north-easterly side of the bay ranging from pH 6.05 to 8.29 at >350 m from the vent site (pCO2) was in proximity to the CO2 vents (∼260 m) in accordance with previous studies , part of the Aeolian Island chain, NE Sicily  in May 2ent site . The venent site . Two sitent site ; Site 1  studies .A. viridis, a dominant benthic organism in Levante Bay, was prevalent throughout the study area  and then converted from lux to µmol quanta m−2 s−1  and temperature were measured several times a day for 4 days using a pH meter . Water samples for total alkalinity (TA) analyses were collected from each site, cooled and stored in the dark until analysis. TA was quantified with a Metrohm 862 compact titrosampler . The pCOsalinity , using tsalinity , selecti m−2 s−1 .MΩ; MQ Academic) and double distilled acids. All test tubes were trace-element clean certified (Stardest) and other lab ware was thoroughly cleaned with acids  for 24 h following a thorough rinse in DDW water). Solutions were prepared with DDW water and analytical grade or higher purity salts.Special care was taken to minimize sample contamination during handling. Samples were treated in a trace element clean facility  equipped with three class 100 laminar flow hoods, using double distilled water  and the pedal disc (n = 10 per site) of each anemone. The subsamples were placed in pre-weighed trace element clean 15 ml test-tubes and their wet weight recorded with a sensitive scale .To examine the distribution of trace elements in the anemone body, two distinct body compartments were excised and analyzed separately . Duplican = 5 per site). The subsamples were placed immediately in a trace element clean pre-weighed 15 ml test tube and their wet weight recorded. One duplicate was digested in acid to yield the total tentacle trace element concentration. The other duplicate received 5 ml NaCl solution (35 g L−l) and was homogenized for 3 min with an electric homogenizer . The homogenate was centrifuged for 5 min at 5,000 rpm  to separate the Symbiodinium (pellet) and the host tissue (supernatant). Visual inspections revealed no crossover of material between these components, but both were washed carefully. The host supernatant was homogenized and centrifuged for 10 min at 13,500 rpm. Then, the pelleted host material was washed three times by means of re-suspension in 3 ml NaCl solution (20 g L−l) and centrifuged for 5 min at 13,500 rpm. The Symbiodinium pellet was washed three times by means of re-suspension in 2 ml NaCl solution (20 g L−l) and centrifuged for 3 min at 5,000 rpm.To further examine the partitioning of trace elements between the symbiotic algae and anemone host tissue, duplicate subsamples of ∼1 gr were excised from the tentacles of each anemone  was carried out using an Agilent 7500cx ICP-MS (Agilent Inc.). A set of calibration standards was added to the analysis together with appropriate blanks. An internal standard solution containing Sc, Re and Rh was introduced during the analyses to all standards and samples. The reported values are blank subtracted and averaged over duplicate analysis, with difference in % between duplicate analyses typically smaller than 10%. Results are given in ng trace element per mg dry weight (wt) of anemone.Samples were digested within the test tubes in the presence of 5–10 mL double distilled 9.8 N HNOSymbiodinium and anemone tissue was analyzed using the Kruskal–Wallis test. Post-hoc Mann–Whitney U-tests were run for separation of significant factors. Differences between factors were considered significant for a p value <0.05. Unless otherwise specified, mean values are presented ± SEM. All analyses were performed using SPSS version 20 .Differences between pedal disc and tentacles in each site, and differences between sites for each body compartment were tested with one-way ANOVA and a multiple comparison test of means (Tukey). Prior to each analysis, all data were checked for normality using the Kolmogorov–Smirnov test and for homogeneity of variances using Cochran’s test. The effect of the site on trace element concentrations in tentacle −2), consistent with findings by pCO2 site, anemones appeared to be healthy with their tentacles fully extended and without any excess amounts of mucus  was strongly elevated compared to typical seawater conditions that were recorded at the control site (−2 s−1) were similar between sites (1–2 m depth), while TA was elevated at the high pCO2 site  next to the vent than at the control site (Next to the vent the pH was significantly lowered and the rol site . SeawateCO2 site . The susrol site  and S2. rol site . Macro eOur sampling scheme provided us with a paired data set of trace element concentrations (per unit of dry weight) in the pedal disc and the tentacles of each individual anemone. Plotting the concentrations of each of the 12 tested trace elements in the pedal disc vs. that in the tentacles (10 per site), we can visualize differences among sampling sites and body compartments, as well as variability among individual anemones  and S1. p < 0.05; p < 0.05; pCO2 site, all trace elements except Sr, were more concentrated in the pedal disc than in the tentacles , (2) Group II: Trace elements that are accumulated in the pedal disc but are slightly depleted in the tentacles at the high pCO2 site , and (3) Group III: Trace elements that are depleted in both body compartments at the high pCO2 site . Statistical differences between sites are found for most trace elements mentioned above (p < 0.05), except Co and Group II elements in the tentacles, and Cd and As in the pedal disc , whereas there was a significant decrease from 3,162 ± 168 to 2,201 ± 197 ng/mg dry wt in the tentacles (p < 0.05).Macro element concentrations  did not change in both body compartments between sites. P, however, significantly increased in the pedal disc at the high Having analyzed the algae and host fractions as well as the whole tentacle, we can probe for contamination or trace element loss in the separation procedure, by comparing the sum of the separated fractions to the whole tentacle . Six of the trace elements  were screened out due to contamination or detection problems. In three trace elements , that were mostly associated with the algae, the sum of the separated fractions balanced out adequately with the total. In the other trace elements , which were mostly associated with the host, the total greatly exceeded the sum of the separated fractions, indicative of significant metal loss. Hence, we chose not to include the measured host fraction. Instead, it was calculated from the difference between the total and algal fraction ; Table 3p < 0.05 for whole tentacle trace element concentrations; p < 0.05). Cd, however, was lost predominantly from the host tissue and the low remaining Cd was evenly distributed between the algae (45%) and host tissue (55%)  .2 vents at Levante Bay of Vulcano Island are intensively studied within the frame of OA research. Given the intrinsic geochemical complexity of the location, we studied the combined effects of trace element exposure and elevated pCO2 on the anemone A. viridis. The present study provides important insights into the performance of A. viridis under natural (control) and enriched trace element conditions. Collectively, our data shows that trace elements are differentially accumulated in A. viridis between ambient (control) and low pH/high pCO2 environments, which confirms our hypothesis . The trace element accumulation pathways consist of: absorbance across the body surface, ingestion of particles, and/or active predation  increased in both body compartments at the high Group I; , while fII; pCO2  may contII; pCO2 , which dII; pCO2 . It is cII; pCO2 . Our meaII; pCO2  and S2.pCO2 site in our study) is responsible for enhanced Fe solubility of 40%, causing an increase in Fe bioavailability in the water column. Accordingly, a high seawater concentration of dissolved Fe was reported close to the primary CO2 vent , although there was enhanced accumulation in the leaves . Similar to Cd and Zn, As is associated with the host tissue  light intensities at stations 1 (control) and 2 (high pCO2). Both sites were at 1–2 m depth. Data represent the mean value (± SD).Measurements of pH, TA, temperature, calculated Click here for additional data file.10.7717/peerj.538/supp-2Table S2A. viridis. Plankton hand nets  were towed at each site for 15 min tocollect plankton and other suspended particulate material from seawater. The measured concentrations (ppb) are presented per tow.Suspended particulate material was sampled to assess the trace element content in the potential prey of Click here for additional data file.10.7717/peerj.538/supp-3Figure S1Ten anemones were sampled from each site and analyzed for trace element concentrations (ng/mg dry wt) in the tentacles and pedal disc. (A) Manganese, (B) barium, (C) nickel, and (D) chromium.Click here for additional data file.


Campylopterus curvipennis) is correlated with genetic divergence, and examined processes that explain best the origin of intraspecific song variation. We contrasted estimates of genetic differentiation, genetic structure, and gene flow across leks from microsatellite loci of wedge-tailed sabrewings with measures for acoustic signals involved in mating derived from recordings of males singing at leks throughout eastern Mexico. We found a strong acoustic structure across leks and geography, where lek members had an exclusive assemblage of syllable types, differed in spectral and temporal measurements of song, and song sharing decreased with geographic distance. However, neutral genetic and song divergence were not correlated, and measures of genetic differentiation and migration estimates indicated gene flow across leks. The persistence of acoustic structuring in wedge-tailed sabrewings may thus best be explained by stochastic processes across leks, in which intraspecific vocal variation is maintained in the absence of genetic differentiation by postdispersal learning and social conditions, and by geographical isolation due to the accumulation of small differences, producing most dramatic changes between populations further apart.Hummingbirds have developed a remarkable diversity of learned vocalizations, from single-note songs to phonologically and syntactically complex songs. In this study we evaluated if geographic song variation of wedge-tailed sabrewings ( Understanding the processes and mechanisms by which phenotypic diversity arises is fundamental in evolutionary biology. Although most of research has focused in the role that natural selection can play in the processes of population divergence, an interest to study the role of stochastic processes involved in phenotypic and population divergence has raised lately e.g., e.g., Song divergence among populations can be driven by three non-mutually exclusive processes: (1) selection of particular acoustic traits due to environmental or habitat characteristics, where different frequencies of sound travel best in different environments or because ambient noise differences between environments Campylopterus curvipennis , is a sexually monochromatic, size dimorphic hummingbird with a wide distribution through the cloud forest and humid tropical forest of eastern Mexico The wedge-tailed sabrewing, Campylopterus curvipennis is a species complex composed of three allopatrically distributed lineages corresponding to subspecies C. c. curvipennis along the Sierra Madre Oriental, C. c. excellens in the Sierra de los Tuxtlas, and C. c. pampa in the Yucatan Peninsula. Second, morphological and acoustic data along with information about the habitat (climate and topography) indicated that these subspecies are morphologically and acoustically divergent, independently of habitat-related variation, and that the genetic divergence among subspecies was coupled with song divergence. Third, a coalescent analysis to evaluate whether song divergence could be attributed to drift or selection, suggested that the fixation of song types, assumed to be encoded by nuclear genes, has occurred faster than expected by genetic drift, suggesting that selection may have played a role in driving song evolution in the past. However, the assumption that fixation of song types is encoded by nuclear genes needs to be taken with caution in such a dynamic cultural system. Furthermore, the observed positive relationship between genetic and acoustic distances in that study, independent of geographic distances, is expected under a drift model of song evolution, where song divergence is higher between allopatric populations that have been isolated for the longest time (i.e. subspecies) with low potential for hybridization  for the field study described. This collecting permit specifically allowed for the collection of tail feathers from the birds. Manipulation of birds in the field was minimal. Birds were captured at leks with mist nets and their two outermost tail feathers were removed for genetic analyses before they were released. All procedures with birds were carried out in accordance with the Guidelines for the Use of Wild Birds in Research proposed by the North American Ornithological Council and the ethics of experimental procedures were revised and authorized by the Animal Care and Use Committee under the Graduate Studies Committee (Doctorado en Ciencias Biomédicas) of the Universidad Nacional Autónoma de México (UNAM), the Instituto de Ecología, A.C. (INECOL), and CONACyT (61710). While the field studies involve a non-threatened or protected species, no specific permits are required for song recording or field studies as the one described here.Table S1), with 3–27 individuals sampled per lek. Sampled leks along the Sierra Madre Oriental (SMO) were categorized in three geographic areas: (i) northern limit of their distribution, from southern Tamaulipas and north of San Luis Potosí ; (ii) central, from south of San Luis Potosí ; and (iii) southern limit of the distribution, from Puebla and Veracruz  (Table S1).Feather samples were collected from a total of 105 wedge-tailed sabrewings captured during the 2006–2008 breeding seasons  according to Morin et al. STST parameters of genetic differentiation due to genetic structure in RSTCALC v2.2 v2.9.3 K) based on microsatellite data. We ran two independent STRUCTURE analyses, with the LOCPRIOR or the default mode, both under the admixture model with correlated allele frequencies K, from K = 1 to K = 12. The length of the burn-in was 500,000 and the number of Markov chain Monte Carlo (MCMC) replications after the burn-in was 1,000,000. To determine an accurate number of clusters we calculated the statistic ΔK based on the rate of change in the log probability of data between successive K values following Evanno et al. To investigate lek genetic structure we calculated global and pairwise RemN) between leks and between groups of leks  was estimated with a maximum likelihood coalescent approach using genotypic data and MIGRATE v3.0 ST calculations. We ran five short and three long chains  or 20 short and six long chains  for estimation between leks and between groups of leks, respectively, after discarding the first 5,000 genealogies as a burn-in.Gene flow (4c. 9 recordings per bird). Some of these recordings were used in previous studies www.birds.cornell.edu/raven).Songs were recorded from territory-holding males at the same leks in which feather samples were taken. Between 3 and 13 birds were recorded at each lek, with 5 to 19 song recordings obtained from each of 56 males . For the introductory syllable we quantified the total number of elements, and the number of different elements. For the introductory and the shared syllable, we measured the duration (s), minimum frequency (kHz), bandwidth , and peak frequency . Because the shared syllable has several elements (notes), we took the same measures on two of their elements  and the duration of another element . In total six measures were taken on the introductory and 13 on the shared syllable.Based on the digitized songs we constructed three different data sets for acoustic analyses: (1) presence/absence of syllable types, (2) relative frequency of each syllable per song, and (3) acoustic traits of two comparable syllables. For the first two data sets, we used syllabic units throughout the wedge-tailed sabrewing repertoire. Syllables were visually classified by CG based on the structure observed on the printed spectrograms, and assigned a letter or combination of letters for further identification. A syllable was defined as an element or several elements always grouped together in a fixed composition. The visual classification of syllable types was not difficult, as syllables are discrete units composed of one or more elements that in general differ consistently in their acoustic structure in obvious ways (for details see K). The ploidy option was set at 1, and ten independent chains were run for each K, from K = 1 to K = 12. Length of the burn-in was 250,000 and the number of Markov chain Monte Carlo (MCMC) replications after the burn-in was 500,000. To determine the accurate number of clusters, ΔK was also calculated.To evaluate song divergence among leks, we performed a hierarchical cluster analysis using the unweight pair-group method with arithmetic mean (UPGMA) and the Euclidean distances to construct a dendrogram in SPSS (SPSS Inc.) based on the binary presence/absence matrix of syllable types. We additionally ran STRUCTURE v2.2.3 We used a principal components analysis (PCA) with the measurements of the introductory and the shared syllable to reduce the number and intercorrelation of the variables. The resulting PC scores were tested for significant differences among leks and between groups of leks (see Results) performing multivariate analyses of variance (MANOVA) followed by one-way ANOVAs. Mensural data and counts were log- and square root transformed, respectively, to produce normality in the data. Finally, we used the relative frequency of syllables, and the measurements of the introductory and shared syllable as predictors in a discriminant function analysis (DFA) for each of the three data sets to examine whether individuals could be classified according to their lek of origin.ST and FST distances. These relationships were controlled for potential effects of geographic distance through partial Mantel tests. To test for the effects of isolation by distance we looked at the relationships between acoustic and geographic distance, controlling for genetic distance. Finally, we tested the relationships between genetic and geographic distances.Acoustic distance matrices regarding song sharing were built by estimating the pairwise Jaccard similarity coefficient between individuals among leks. This coefficient was calculated in ESTIMATES v8.0.0 The number of alleles per locus varied from 3 to 15, and the observed heterozygosity values indicate no consistent deviations from H-W equilibrium. After Bonferroni corrections, only four leks deviated from H-W equilibrium in locus CACU13-2, two in locus CACU5-7, and one lek in locus CACU13-7 Table 1.ST estimate ± SE, –0.032±0.0017, p = 0.455; global FST estimate ± SE, 0.021±0.002, p = 0.972). Pairwise RST among leks ranged from –0.053 to 0.082, and pairwise FST ranged from –0.0138 to 0.249. Results of STRUCTURE analysis showed no genetic clustering when population information was not provided, whereas a weak genetic structure among leks was revealed when using the LOCPRIOR function. When ΔK was calculated the break in slope of the distribution of L(K) was at K = 2 . In addition, we observed a geographic pattern in song sharing: leks from the northern , central  and southern limit of the distribution  are clustered together in different groups, except three individuals from the Orduña lek, which are basal in the dendrogram. On average, the proportion of syllables shared between individuals within leks was 0.60±0.18 (mean±SD), whereas the proportion of syllables shared among leks was 0.15±0.04, these correspond to 20.4 and 5.1 syllables respectively  was at K = 3 when ΔK is calculated  and among groups of leks along the Sierra Madre Oriental . Regarding measurements of the shared syllable, the 13 variables were reduced to three principal components that accounted for 81% of the variation. The first PC (52.1%) was mainly explained by the peak frequency of the syllable, PC2 (18.8%) by the peak frequency of the second element, and PC3 (10%) was explained by duration, minimum frequency, and bandwidth of the syllable and their elements. A MANOVA with the PC scores also showed significant variation among leks  but the variation among groups of leks was not significant .The six measurements of the introductory syllable were reduced to a single principal component that accounted for 85.7% of the total variation. A one-way ANOVA with the PC scores showed that spectral and temporal measurements of the introductory syllables varied significantly among leks . Likewise, the correlations between FST values and acoustic measures were no statistically significant . In contrast, the correlation between acoustic and geographic distances was positive and significant when using the song sharing distance matrix  or the shared syllable . Removing the effects of genetic distance did not significantly alter the positive relationship between geographic and song sharing distances . Finally, genetic and geographic distances were not significantly correlated .We found that the correlation between Rcurvipennis subspecies studied here are not genetically structured along the Sierra Madre Oriental. Measures of migration rates, genetic differentiation and population structure indicated gene flow among leks and low levels of genetic structure. The relationships between acoustic and genetic distances were sharply discordant with previous results comparing acoustic and genetic data of all three wedge-tailed sabrewing subspecies. González et al. ST and RST values) and acoustic distances (song sharing and common syllable), and the significance of these relationships was not altered when the effects of geographic distance were removed. These analyses indicate that genetically divergent subspecies shared fewer syllable types, and differed acoustically in the common syllable, independently of distance, which might be the result of long isolation after divergence in allopatry. However, the relationships between acoustic and genetic distances were not statistically significant within the curvipennis subspecies studied here . Although song traits play a potential role in increasing genetic polymorphisms and generating reproductive isolation and speciation in parapatry  were more divergent between than within leks.Our study revealed that lek members of the curvipennis subspecies yielded a non-significant correlation between genetic and acoustic distance. Studies using rapidly evolving genetic markers in a wide variety of song learners, mainly songbirds and parrots, have shown similar patterns of genetic differentiation (FST), and have also failed to find genetic divergence coupled with song divergence Both the genetic adaptation and epiphenomenon hypotheses predict that dialects do affect genetic structure  showed that individuals changed their song-type at leks during a four-year period, and that all song replacements occurred after a crystallized song was already produced, which indicates that song learning may well extend throughout their lifespan The decoupled patterns between the neutral genetic and acoustic distance highlights the role that learning might play in the formation of lek-level vocal structure in wedge-tailed sabrewings. As in songbirds and parrots, song learning in hummingbirds is culturally transmitted through imitation Table S2), indicating that the observed geographic structure of curvipennis songs can not be explained by founder effects after colonization of northern areas K = 3; curvipennis songs along the Sierra Madre Oriental lend support to the idea that song sharing between lek members , could be a selective force operating to promote convergence within wedge-tailed sabrewings leks, where song sharing among neighbors confers social benefits that could possibly increase their reproductive success The decreased syllable sharing with increased geographic distance (isolation-by-distance) suggests that song divergence is also a result of neutral processes, where individuals within leks copy syllables randomly from each other and small differences in song accumulate along a geographical gradient due to the reduced probability of interaction among individuals from more distant leks (i.e. between geographic areas with no apparent barriers to gene flow). Then, the accumulated syllabic differences in song are probably distributed within a lek by behavioral matching between its members, which would explain why lek members share a high proportion of syllables. Regardless of the distance between leks, the song of each lek had a characteristic introductory syllable with strong differences in structure. This finding is consistent with previous observations in which members of song neighborhoods within leks shared the introductory syllable In summary, our study reveals independence between acoustic and neutral genetic divergence in wedge-tailed sabrewings, contrary to predictions of neutral evolution hypotheses. Along the continuous distribution of this subspecies, with no apparent geographic barriers, genetic divergence appears to be restricted by homogenizing gene flow, and geographical structure of vocal variation corresponds with restricted south-to-north gene flow between geographic areas of the Sierra Madre Oriental. Finally, the highly structured and marked song divergence between leks suggests that the evolution of song elaboration in wedge-tailed sabrewing resulted from a combination of processes probably linked to postdispersal learning, isolation by distance, and social factors associated with male-male interactions. As these birds are seemly open-ended learners, we interpret our results as that cultural variation appears more plausible than genetic variation in modifying songs differently at different leks and that social selection is involved in inducing this divergence. With negligible geographic barriers and no behavioral checks on dispersal, little genetic differentiation has developed in wedge-tailed sabrewings along the Sierra Madre Oriental despite the apparent intense social  selection acting on the elaborate acoustic traits see also . AlthougFigure S1Measurements taken from the (A) introductory, and (B) shared syllable emitted by every recorded individual. Numbers in (B) refer to three elements where the same measures as the complete syllable were taken. A fragment of a song bout is shown in (C) indicating the introductory syllable with an asterisk and the shared syllable with arrows.(TIF)Click here for additional data file.Table S1Localities, geographic location and altitude of wedge-tailed sabrewing sampled leks. Regions correspond to north, central and south of the Sierra Madre Oriental .(DOC)Click here for additional data file.Table S2Mean ± SD of spectral and temporal measurements of introductory syllables across leks of wedge-tailed sabrewings. N = number of individuals, n = number of songs.(DOC)Click here for additional data file.Table S3Mean ± SD of spectral and temporal measurements of the shared syllable and three of its elements, across leks of wedge-tailed sabrewings see . N = num(DOC)Click here for additional data file.Audio S1Song of El Cielo lek.(WAV)Click here for additional data file.Audio S2Song of Gómez Farías lek.(WAV)Click here for additional data file.Audio S3Song of El Naranjo lek.(WAV)Click here for additional data file.Audio S4Song of Aquismón lek.(WAV)Click here for additional data file.Audio S5Song of Xilitla lek.(WAV)Click here for additional data file.Audio S6Song of Cuetzalan lek.(WAV)Click here for additional data file.Audio S7Song of Macuiltépetl lek.(WAV)Click here for additional data file.Audio S8Song of La Orduña lek.(WAV)Click here for additional data file.Audio S9Song of Ursulo Galván lek.(WAV)Click here for additional data file.


Schwann cell (SC) transplantation exhibits significant potential for spinal cord injury (SCI) repair and its use as a therapeutic modality has now progressed to clinical trials for subacute and chronic human SCI. Although SC implants provide a receptive environment for axonal regrowth and support functional recovery in a number of experimental SCI models, axonal regeneration is largely limited to local systems and the behavioral improvements are modest without additional combinatory approaches. In the current study we investigated whether the concurrent delivery of the polyamine putrescine, started either 30 min or 1 week after SCI, could enhance the efficacy of SCs when implanted subacutely (1 week after injury) into the contused rat spinal cord. Polyamines are ubiquitous organic cations that play an important role in the regulation of the cell cycle, cell division, cytoskeletal organization, and cell differentiation. We show that the combination of putrescine with SCs provides a significant increase in implant size, an enhancement in axonal (sensory and serotonergic) sparing and/or growth, and improved open field locomotion after SCI, as compared to SC implantation alone. These findings demonstrate that polyamine supplementation can augment the effectiveness of SCs when used as a therapeutic approach for subacute SCI repair. To date no treatment can completely reverse the clinical outcomes associated with spinal cord injury (SCI). Tissue injury following SCI occurs in two phases: the first involves the primary mechanical injury itself, whereas the second is progressive, mediated over the course of days to weeks after SCI by a variety of cytotoxic factors present within the injury environment. Host cellular efforts to restrict progressive tissue injury, including glial reactivity, extracellular matrix deposition, and scar formation around the lesion site, subsequently give rise to a nonpermissive environment for axon regeneration and the endogenous recovery of function . This ex arginase 1 expression and polyamine synthesis and was associated with favorable M2 macrophage responses and axonal regeneration [One encouraging adjunctive strategy for both surmounting the growth-inhibitory environment of the lesion and stimulating intrinsic regeneration has been the elevation of cyclic adenosine monophosphate (cyclic AMP), a second messenger molecule . Sustainneration , 9. Addineration . Based uneration , 4, 6, wSCs were obtained from the sciatic nerves of adult female Fischer rats  as previously described , 12. SCsN = 34; 180–200 g) were housed according to the guidelines recommended by the NIH and the Guide for the Care and Use of Animals. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Miami. Prior to surgery, rats were anesthetized  by intraperitoneal injection.Adult female Fischer rats  contusion injury . A laminn = 11), (ii) SC implant with acute putrescine , and (iii) SC implant with delayed putrescine . Putrescine (100 mM) was delivered at a rate of 0.5 μL/hr using two sequentially implanted subcutaneous Alzet minipumps  for a 2-week administration period; emptied pumps were replaced after 1 week [Prior to SCI induction, animals were randomly assigned into one of the following three treatment groups: (i) SC implant with 0.9% physiological saline (vehicle) control , which was held in a micromanipulator with an attached microinjector . The injection pipette was held in place for an additional 3 min postinjection to minimize leakage upon withdrawal. One animal was excluded from the study due to a malfunction of the microinjector during cell transplantation. Following the injection, the muscle layers and the skin were closed separately and animals received postoperative care as described above.Before implantation, SCs were trypsinized, harvested, counted, resuspended in DMEM/F12 media as aliquots and placed on ice prior to surgery. One week after injury the rats were anesthetized with 2% halothane and the injured spinal cord was reexposed. Rats then received fluid implants of SCs as described elsewhere . Cells wμm into five series free-floating.Rats were euthanized at 10 weeks after injury  and transcardially perfused with 4% paraformaldehyde . The tissue was then dissected and sectioned with a freezing microtome for immunohistochemical analysis according to previous procedures . Spinal NTR; 1 : 5,000; Abcam, Cambridge, MA), serotonin , or calcitonin gene related peptide  for identifying the implanted SCs or delineating the growth of specific descending or ascending axonal populations, respectively, within the p75+ SC implants. Sections were then washed and incubated with corresponding fluorescent secondary antibodies . Sections were mounted onto Snowcoat X-tra slides  and cover-slipped with Vectashield mounting medium  containing the nuclear dye Hoechst for storage at 4°C.As previously described, every fifth sagittal section was immunochemically stained for subsequent fluorescent microscopic analysis using a double labeling procedure involving monoclonal and polyclonal antibodies . These a+ area for each of the three sections that comprised the center of the SC implant was traced under a 20x objective, quantified using Neurolucida, and then the SC implant area calculated for each animal as the average p75+ area across the three sections [In one series of sections, the average area of p75-positive cellular immunoreactivity was quantified across three sections containing the center of the SC implant. Expression of p75 permits delineation of the implant and identification of the implant-host cord interface. For this analysis, stained sections were visualized and quantitatively assessed using an unbiased method employing computer-assisted fluorescence microscopy and Neurolucida software . The p75sections .μm rostral to the center of the implant (as identified using p75 immunoreactivity) was performed on the second series of sections using 63x objective under oil immersion. Calcitonin gene related peptide (CGRP) labeled axons were quantified similarly, but at 1000, 500, 100, and 0 μm caudal to the center of the implant. The average number of fibers per section was obtained by counting those immunostained axons that crossed imaginary lines perpendicular to the rostral-caudal axis at the 1000, 500, 100, and 0 μm intervals from the center of the implant [Quantification of 5-hydroxytryptophan (5HT) labeled axon growth along the rostral-caudal axis of the spinal cord at distances of 1000, 500, 100, and 0  implant . The tot implant .The open field BBB locomotor test developed by Basso et al. was employed to assess weekly gross locomotor performance after SCI for 10 weeks . In addip < 0.05 ∗,#, <0.01∗∗,##, and <0.001∗∗∗,###, compared to the SC only control or the other temporal treatment group, as indicated. All errors are given as standard errors of the mean.A one-way ANOVA and subsequent Bonferroni post hoc test were used to compare counts of immunostained axons among groups. For analysis of weekly functional recovery patterns following implantation (BBB and BBB subscore), a mixed factorial (repeated measures) ANOVA followed by the Tukey-Kramer posttest was employed. Differences were acknowledged to be statistically significant at F2,31 = 5.784, p < 0.05). Compared to the implant size in the SC only group (1.23 ± 0.07 mm2), those animals receiving putrescine administration beginning at the time of implantation exhibited 1.8-fold increase in implant area .Statistical comparison of SC implant size among control and treatment cohorts showed a significant effect of treatment . Acute putrescine supplementation significantly increased the number of serotonergic fibers within the proximal SC implant compared to the vehicle control: an average of 20 ± 1.2 5HT+ fibers per section (f/s) at 500 μm  and 9 ± 0.6 f/s at 100 μm  was quantified rostral to the center of the SC implant, while in vehicle controls 13 ± 1.8 5HT+ f/s and 2 ± 0.4 f/s, respectively, were observed at these distances .Serotonergic axons descending from the reticular formation and raphe nuclei of the brainstem were identified penetrating the SC implant using immunostaining for 5HT  and within the center of the SC implant . At 100 μm caudal to the center of the SC implant, acute and delayed putrescine supplementation resulted in 2.3-fold  and 1.7-fold  increases in CGRP+ axon numbers, respectively, compared to the vehicle control (8.3 ± 0.77 f/s). In addition, acute putrescine administration (11 ± 1.2 f/s) significantly increased CGRP+ axon ingrowth within the center of the SC implant compared to the vehicle control . Acute putrescine was also significantly more effective in promoting CGRP+ axon growth than delayed delivery at both 100 μm caudal to  and within the center of the SC implant .Small diameter sensory axons originating from dorsal root ganglia that were immunoreactive for CGRP were quantified within the SC implant  and at various time points after implantation  and delayed  putrescine with SC implant treatment groups. Statistical comparison of BBB subscores among groups showed a significant effect of treatment at all weeks after implantation  and delayed  putrescine with SC implant treatment groups. Statistical comparison of the number of footfall errors on the grid walk among groups showed a significant effect of treatment  and the SC implant, vehicle controls .Locomotor performance and functional recovery following SCI, SC implantation, and putrescine treatment were assessed using the BBB score, BBB subscore, and the irregularly spaced grid walk test. Statistical comparison of BBB scores among groups showed a significant treatment effect both at endpoint  from which responding DRGNs are located.The growth of primary sensory afferents, immunoreactive for CGRP, which have terminations in the superficial lamina of the spinal cord and are associated with transmission of pain, also benefitted from the delivery of putrescine, with enhanced axon numbers within the center of the SC implant. CGRP-positive neurons have also been implicated in the modulation of fine motor control and the control of posture and thus their growth could be indicative of changes in locomotor function . PrimaryPutrescine supplementation of SC implants also resulted in significant functional improvement, as measured in the open field with the BBB score and on the grid walk test. Irrespective of the timing of putrescine delivery, enhanced locomotor ability, as measured by using the BBB score and subscore, was obtained with treatment compared to SC implanted, vehicle controls. Overall, both acute and delayed putrescine administration generated similar improvements in hind limb performance and tail posturing, including a one-point higher BBB score and five-point higher BBB subscore. Only the acute delivery of putrescine, however, reduced footfall errors on the horizontal grid walk test, by 25% compared to the vehicle control. The effectiveness of acute putrescine only on this outcome measure would indicate that tissue preservation would be a likely mechanistic explanation for this effect. The observed functional improvements following supplementation of SC implants with putrescine are in addition to those that are known to be provided when SC implants alone are used after SCI , 13. TheIn conclusion, we supply evidence that modulating polyamines in conjunction with cell implantation is an effective approach for enhancing the functionality of the implant as well as locomotor recovery after SCI. Future research should seek to gain a greater understanding of the mechanisms underlying putrescine's effects on SC function, migration, and survival, as well as its ability to promote axon sparing, growth, and/or sprouting.