Musical imagery is a relatively unexplored area, partly because of deficiencies in existing experimental paradigms, which are often difficult, unreliable, or do not provide objective measures of performance. Here we describe a novel protocol, the Pitch Imagery Arrow Task (PIAT), which induces and trains pitch imagery in both musicians and non-musicians. Given a tonal context and an initial pitch sequence, arrows are displayed to elicit a scale-step sequence of imagined pitches, and participants indicate whether the final imagined tone matches an audible probe. It is a staircase design that accommodates individual differences in musical experience and imagery ability. This new protocol was used to investigate the roles that musical expertise, self-reported auditory vividness and mental control play in imagery performance. Performance on the task was significantly better for participants who employed a musical imagery strategy compared to participants who used an alternative cognitive strategy and positively correlated with scores on the Control subscale from the Bucknell Auditory Imagery Scale (BAIS). Multiple regression analysis revealed that Imagery performance accuracy was best predicted by a combination of strategy use and scores on the Vividness subscale of BAIS. These results confirm that competent performance on the PIAT requires active musical imagery and is very difficult to achieve using alternative cognitive strategies. Auditory vividness and mental control were more important than musical experience in the ability to perform manipulation of pitch imagery. Musical imagery can be described as “hearing a tune in your head” . It is aMost studies of musical imagery require participants to maintain an image of familiar melodies or scales . These tOther experimental paradigms are designed to induce dynamic musical imagery, and hence require participants to shift from one musical image to another through effortful manipulation. These paradigms have included tasks that require mentally transposing a melody into a different key or imaging the pitches of a melody in reverse (retrograde) order , 5.This distinction—between maintenance and manipulation of mental imagery—has also been the subject of investigation in other modalities, particularly in visual imagery. Studies have shown that the ability to form vivid visual images is psychometrically distinct from the ability to manipulate those images in space. In one study, visual artists were found to excel at maintaining object imagery but had difficulty with spatial manipulation of images, whereas engineers and scientists exhibited the opposite pattern of performance . In the vividness subscale (BAIS-V) and a control subscale (BAIS-C), with the former measuring the subjective clarity of an image and the latter measuring the ease with which participants can change or manipulate an auditory image at will [r ~.30) with musical training or experience [Another area of individual differences that has received relatively little attention is the vividness of auditory imagery among nonclinical populations . The Buc at will . Previou at will , 19, 20.Pitch Imagery Arrow Task (PIAT). Several considerations informed the design of this task. First, a number of paradigms that purport to measure musical imagery do not provide any objective behavioral confirmation that participants have actually used musical imagery [This investigation employed a novel protocol for evaluating musical imagery—the  imagery , 6. For  imagery  had part imagery  had part imagery , 6.Second, although some imagery tasks have an objective behavioural outcome measure, they are so simple or repetitive that musical imagery may be unnecessary to perform the task. For example, Janata and Paroo used familiar ascending scales in a number of their studies, in order to “force listeners to move their mental images in pitch space” . HoweverAt the other extreme are protocols that are so difficult that only expert musicians can perform them, such as the mental reversal of familiar melodies ; see alsFinally it has been said that the study of imagery stands precisely at the intersection of two key branches of cognitive psychology—perception and memory . As suchThe PIAT has a number of advantages over existing protocols for evaluating imagery. Specifically, the task (1) requires a behavioural response to objectively measure accuracy and response times of imagery performance; (2) is extremely difficult to successfully perform using alternative cognitive strategies other than pitch imagery; (3) employs novel rather than familiar sequences of pitches that cannot be anticipated in advance; (4) employs a range of difficulties implemented in a staircase design, such that it can induce imagery in participants with a wide range of musical experience; (5) incorporates a pitch perception control condition, permitting the assessment of differences in strategies and brain mechanism for imagery versus perception; (6) incorporates a mental arithmetic control condition, permitting the assessment of differences in strategies and brain mechanisms for imagery tasks that employ different cognitive computations.Our primary goal was to verify the efficacy of the PIAT in inducing and training pitch imagery in musicians and non-musicians with a wide range of musical experience. We also investigated the role of musical training, imagery vividness, and mental control in predicting performance. We hypothesised that (1) participants who used a pitch imagery strategy would show significantly better performance than participants who employed alternative cognitive strategies; (2) successful task performance should be determined more by an individual’s vividness and control of musical images (as indexed by the BAIS), than one’s history of musical training.24 trained musicians and 16 non-musicians were recruited for the study. All participants self-reported to being right-handed, having normal or corrected-to-normal vision and normal hearing. Only right-handed individuals were recruited. Participants were classified as musicians if they had more than 5 years of continuous formal music lessons and have been actively playing their instrument in the last 2 years. Non-musicians were defined as those with less than 2 years formal training, or those who had been non-active in their instrument for more than 10 years. All participants were classified as either a musician or a non-musician based on these criteria. All participants provided written consent and all procedures were approved by the Macquarie University Human Research Ethics Committee. setup component in which the participant listens to a starting sequence of computer-generated piano notes. Each successive note is immediately up or down the major scale relative to the preceding note, with the scale direction of the next note (ascending or descending) being random and unpredictable from note to note. (2) An imagery (continuation) component in which the piano notes stop while the participant is prompted to imagine a series of 1–5 notes continuing from the last heard note, prompted by vertical up or down arrows which indicate the scale direction for each succeeding note to be imagined; (3) A probe component which presents a heard piano note that either matches or does not match the last of the notes in the imagery sequence. After hearing the probe participants were required to indicate with a button press if the probe is a match or a mismatch to the last note of the imagined sequence.The PIAT, as outlined in The setup component of each trial began with an ascending major scale that started and ended on the tonic, to provide participants with a tonal context that they could use as a frame of reference for their subsequent judgments . FollowiIn the imagery component, one to five arrows were then presented in silence at the same rate as the preceding note / arrow combinations of the setup phase and participants were required to imagine the next scale note up or down from the previous (heard or imagined) note as indicated by the scale direction arrows.In the probe component of each trial, a target screen with an image of a mouse indicating left click for “Correct” and right click for “Incorrect” was displayed for 1 second to alert participants to an impending probe. A probe tone was then played and participants were required to indicate whether the probe tone matched or did not match the final note of the imagery component. If no response was recorded within 4 seconds the trial was coded as a missed trial, and participants received a warning message to respond more quickly on future trials. Feedback was provided on each trial to advise the participant whether their response was accurate or not.The PIAT was programmed with five levels of imagery difficulty, corresponding to the number of sequential notes (from 1–5) in the imagery component. Each imagery level contained three stages that manipulated the complexity of the setup component, in terms of the maximum number of audible notes in the setup sequence, the key signature for the sequence, and whether the starting note was a tonic or dominant. In particular, in stages 2 and 3 the key signatures were randomised from a possible 5 key signatures . These were set so as not to repeat the previous trial to ensure variability in each stage . Table 2The probe tone was a match for 50% of the trials. For mismatch trials the probe tone was always in the same key and within 2 steps of the correct answer. The maximum possible range of notes for each trial was set to 3 scale steps up or down from the starting note. For example, for a trial in C Major, beginning on Middle C (C4), the tones (both heard or to be imagined) were within the range of G below middle C (G3) and F above middle C (F4).The Pitch Perception trials were identical to the imagery trials, except that arrows were always paired with heard notes during the continuation component. In this case the last note in the sequence then became the correct response for the probe.The Mental Arithmetic trial’s start screen showed “Begin Mental Arithmetic”, then a number appeared on the screen which was the starting point of the sequence. The up and down arrows also included a number at their point which indicated how much to increase (up) or decrease (down) the running total by. After a random number of arrows were presented, which varied from a minimum of 4  to a maximum of 11 , the same target screen with the mouse image then appeared. After 1 second, a number appeared at the top of the screen indicating the probe number. Participants then responded in the same way as the musical trials to confirm if the number was correct or incorrect. Incorrect answers were presented on 50% of the trials and were always 1 integer away from the correct number. The mental arithmetic trials also increased in difficulty as the levels progressed, as per In order to assess the role of self-reported auditory vividness and control on pitch imagery performance, the participants also completed the Bucknell Auditory Imagery Scale (BAIS) . The scawww.neurobs.com) was used to control the experiment and to record responses. Acoustic stimuli were generated from the 'Piano' instrument sound by Finale 2012 software  and exported as .wav files for use in Presentation.Presentation software . The Pitch Perception trials were randomly interleaved with the Imagery trials after an initial 10 Imagery trials were presented. The accuracy of response for the Perception trials did not impact on the progression of the participant through the task. Mental Arithmetic trials were presented as participants moved between stages; one trial if moving up a stage or level, and two trials if moving down a level. The average time taken to complete the task was 53 mins.The task also included a fast exit in which participants who failed to successfully progress through Level 1 of the Imagery Trials on more than 3 attempts  were excused from further trials. These participants were deemed to have found the task too difficult or failed to understand how to complete it. At each point of failing Level 1, the participants were given the opportunity to ask questions and the requirements of the task were reiterated verbally.Upon completion, participants were visually presented with their percent correct scores for each Imagery level, as well as overall percent correct for the Perception and Mental Arithmetic conditions. They were then asked verbally to rate from 1–5 overall how vividly or clearly they formed the musical images during the task  , 25. TheFour participants failed to progress past Level 1 and were excluded from the final analyses. All four excluded participants were non-musicians with an average Musical Experience Index of .04, that is had actively participated in playing music for 4% of their lives (range 0–.12).F = 4.46, p = .01, η2 = .07 but no significant main effect for Group F = 0.04, p = .83. The Group x Condition interaction was also significant F = 6.58, p = .002, η2 = .10, due to the fact that musicians were more accurate than non-musicians on the Imagery  = 2.07, p = .046, d = 0.75) and Perception conditions  = 1.714, p = .096, d = 0.61), while non-musicians were more accurate than musicians in the Mental Arithmetic condition  = 2.351, p = .025, d = 0.86).A 2 x 3 ANOVA of Accuracy  x Condition  revealed a significant main effect for Condition, F = 6.167, p = .02, η2 = .05), with musicians showing slower overall reaction times than non-musicians. There was also a significant main effect of Condition  = 5.034, p = .008, η2 = .08). Post hoc paired t tests showed that reaction times (ms) were not significantly different for the Imagery  and Perception conditions , but differed significantly between Imagery and Mental Arithmetic : (t(35) = 4.92, p < .001, d = 0.82), as well as Perception and Mental Arithmetic: (t(35) = 3.38, p = .002, d = 0.56). All post hoc tests used the Bonferroni correction procedure with a critical alpha of .05/3.A 2 x 3 ANOVA of Mean Hit Reaction Times (Group (Musician / Non-Musician) x Condition  revealed a significant main effect of Group  = 0.589, p = .56, η2 = .01). There was no significant interaction Group x RT interaction . BAIS-C was significantly correlated with overall accuracy for the Imagery  and the Perception condition  as well as significantly negatively correlated with the mean hit reaction times for both the Imagery condition  and the Perception condition .BAIS-V and BAIS-C were correlated across the three conditions for overall accuracy (percent correct) and mean hit reaction times. BAIS-V was significantly correlated with Imagery accuracy , though not with BAIS-C .The BAIS measures were also correlated significantly with a number of other variables. The participant’s debriefing vividness rating was significantly correlated with BAIS-V , but not with BAIS-V . However an independent t-test revealed musicians scored significantly higher than non-musicians only on BAIS-V;  = 2.16, p = .038, d = 0.76), not BAIS-C  = 1.57, p = .125, d = 0.55). The MEI showed a significant positive correlation with BAIS-C  reported hearing the sound in their head or singing the notes in their head thereby following the arrows and hearing the sounds in their minds throughout the imagery (continuation) component of the task. Alternative Strategy users (N = 14) reported a variety of alternative cognitive strategies such as counting arrows, using intuition or visual imagery. These participants were keeping track of the movement of the arrows, but using musical imagery only at the end of the trial, to make a judgement regarding whether the test tone was correct. As the goal of the PIAT is to induce musical imagery throughout the imagery component of the trial, this later group was classified as using an alternative Strategy; with only minimal musical imagery induced.An open-ended question asked participants to describe the strategies they had used for performing the imagery task. Responses fell into two broad categories, In addition, all participants who reached above Level 4 were asked if they possessed absolute pitch (AP), of which two self-reported they did. One reported that although they possessed AP they were not labelling the notes, just imagining the sound in their mind; hence they were categorised as using a musical imagery strategy. The other used an unusual alternative visual-motor strategy. This later individual was the only participant to reach above level 4 on the PIAT without the use of a musical imagery strategy. This participant was excluded from further analysis of strategy use and is considered in more detail in the discussion section. As it could be argued that AP possessors are likely to have clear long term mental categories which are highly likely to influence performance on the task, the following analyses were later re-run excluding both AP possessors, but it had no effect on any of the current results; as such the AP possessor who reported using musical imagery remains included in the results below.t(33) = 4.46, p < .001, d = 1.54) and Perception (t(33) = 4.35, p < .001, d = 1.50) trials. Significantly faster mean hit reaction times were found for Musical imagery strategy users in the Perception condition (t(33) = 2.62, p = .013, d = 0.90), though not the Imagery or Mental Arithmetic condition.Musical imagery strategy users were significantly more accurate on both Imagery (t(33) = 1.61, p = .117, d = 0.56), there was a significant correlation between the MEI and strategy used , such that those with greater musical experience over their lifetime were more likely to report using a musical imagery strategy.While musicians did not differ significantly from non-musicians in strategy use (t(33) = 3.28, p = .002, d = 1.13), though there was no significant difference on BAIS-V  = -1.61, p = .12, d = 0.56). They also had significantly higher debrief vividness scores  = 3.47, p = .001, d = 1.20).Finally, an independent t-test revealed that musical imagery strategy users reported significantly higher BAIS-C  and BAIS-C  were significantly correlated to maximum level attained. Musicians attained a significantly higher maximum level of performance than non-musicians  = 2.08, p = .045, d = 0.73). Maximum level was also correlated significantly with the MEI . Musical imagery strategy users reached a significantly higher level on the PIAT than alternative strategy users  = 5.781, p < .001, d = 1.69). Maximum level reached is a more useful measure of imagery accuracy than Imagery percent correct, which does not account for the variability of difficulty in the levels of the task.The maximum level reached in the PIAT corresponds to the number of tones imagined per trial prior to the test probe. Both BAIS-V (t(34) = 1.92, p = .06). However there was a significant correlation with MEI . Musical imagery strategy users progressed significantly faster than alternative strategy users (t(33) = 4.56, p < .001). Rate of Progression also correlated significantly with both BAIS-V  and BAIS-V .To capture how accurately participants progressed up through the levels , ‘Rate of Progression’ was calculated as the slope of the line of best fit of the level number over the 90 Imagery trials, . An independent t-test between the musicians and non-musicians revealed no significant difference in this Rate of Progression  = 6.24, p = .003, η2 = .11). Post hoc paired t-tests showed that Imagery  was significantly more difficult than Perception : (t(35) = 5.62, p < .001, d = 0.94), but there was no significant difference between the Mental Arithmetic condition  and the other two conditions  = 17.69, p < .001), and accounted for 53% of the variance in the maximum level score . The coefficients from this model are outlined in Regression analyses were conducted to evaluate which variables best predicted accurate performance on the PIAT, taking maximum level reached as the criterion variable. Eleven predictor variables  were included in an initial model and stepwise regression reduced the model to the minimal adequate hierarchical linear model, with only significant predictors. This resulted in a final model containing strategy use and BAIS-V, which significantly predicted the maximum level reached  = 24.11, p < .001), with strategy use alone accounting for 42% of the variance in maximum level . An ANOVA revealed that Model 2 (Strategy Use + BAIS-V) was significantly better than Model 1   = 6.935, p = .013), as seen A linear regression calculated the variance attributable to strategy use alone, given the high correlation between strategy use and maximum level see . The res2 increasing from Model 2 by only .003 and .008 respectively. This suggests that maximum level reached is either not strongly predicted from musical experience (only from Strategy Use and BAIS-V), or that the influence of strategy use or BAIS-V are mediating the relationship between performance of the PIAT and musical training.Additional linear regressions assessed the effect of adding in musical training. Model 3 and 4 on z = 2.817, p = .005), and linear regression confirmed strategy use significantly predicts maximum level . The direct effect of MEI predicting maximum level went from significant  to non-significant when controlling for strategy use , suggesting the mediation was substantial. This result suggests that musical training, though related to the performance on the PIAT (as measured by the maximum level reached), is only predictive of performance due to the impact it has on strategy use. MEI did not significantly predict BAIS-V , and so it can be ruled out as a mediating factor in the relationship between musical training and performance.To test these possibilities, mediation analysis was run using MEI as the predictor, strategy use as the mediating variable and maximum level reached as the outcome variable. Logistic regression confirmed MEI significantly predicts strategy use . The direct effect of BAIS-C predicting maximum level went from significant  to non-significant when controlling for strategy use , again suggesting that strategy use was substantially mediating the relationship between BAIS-C and maximum level reached. Linear regression also confirmed BAIS-C significantly predicted BAIS-V , but when controlling for BAIS-V, both variables were no longer significant in predicting maximum level reached. Finally, the role of BAIS-C was investigated to see how it fits into this model of prediction. Logistic regression showed that BAIS-C significantly predicted strategy use . However there is a significant relationship between BAIS-V and maximum level attained , though not with BAIS-C and maximum level attained .In summary, musicians were significantly more accurate than non-musicians for Imagery but not Perception trials, and there was no significant group difference in reaction times. BAIS-C scores were positively correlated with performance on both Imagery and Perception trials, as well as other measures such as strategy used and MEI. In contrast BAIS-V was positively correlated with Imagery accuracy and debrief vividness. Regression analysis showed that the factors that contributed most to better performance on the PIAT were strategy use and BAIS-V. Participants with more musical experience were more likely to use a musical imagery strategy, and therefore perform better at the task. BAIS-C also predicted both strategy use and BAIS-V. p values remained within the stated significance level). Hence, this second AP participant was included in the analyses.The present results confirm the effectiveness of the PIAT for inducing musical imagery, and provide insights into the roles that musical training, auditory vividness and mental control play in successful imagery performance. The PIAT was highly effective in inducing mental imagery of musical pitch in participants, in that successful performance on the task was highly dependent on the use of a musical imagery strategy, rather than an alternative strategy that used minimal musical imagery. Participants using an alternative cognitive strategy were (bar 1) unsuccessful at reaching above Level 4 on the Imagery task, with musical imagery strategy users significantly outperforming alternative strategy users on all measures of accuracy. The exception, a participant with 10 years of piano training reported, reached level 5 using a visual-motor imagery strategy. This individual reported visualising the keys on a piano and playing them with their hand. However, this individual also possessed AP and knew which pitches and scale degrees were being played. As the only individual to adopt a visual-motor imagery strategy, this participant was considered individually and excluded from the statistical analysis of strategy use and multiple regression analyses. The participant achieved 98% accuracy for the Imagery condition, and was below the sample’s mean for BAIS scores on both subscales. The other participant who also self-reported having AP, reported using pitch imagery. This participant was within one standard deviation of the mean for MEI, BAIS-V and BAIS-C among musical imagery strategy users, and her exclusion from the analyses did not significantly alter any of the findings  and indices of reaction time (mean hit reaction time and rate of change of reaction time per condition). These behavioural measures revealed that better performance on the PIAT was associated with the use of a musical imagery strategy. The measures also showed musicians were only significantly more accurate on the Imagery but not on Perception trials, and were not significantly different in reaction times. This result is consistent with the findings of Aleman et al. , who hadThe second advantage of the PIAT is that it requires participants to actively manipulate a pitch image, rather than just maintain it. This type of manipulative pitch imagery investigation has only up until now been done with more difficult tasks such as mental reversal of melodies or simpler pitch transposition of melodies , 5, 7.Thirdly, unlike previous protocols for inducing imagery, random sequences were manipulated in the imagery component of the trial. This design confers variety and flexibility to help minimise confounds, such as a familiarity with a melody / or familiarity with a probe combination that could be learnt over a task. For example Level 1, Stage 1  had 14 different possible combinations. This number increased dramatically as the participants move through the levels and stages of the task. Not only did the pitch sequence vary randomly, but the length of the initial set-up sequence varied randomly, so that participants unaware when the imagery component of the trial would begin.Fourthly, the staircase design allowed participants to progress through levels at their own rate, while at the same time accommodating individuals with a wide range of musical experience. While 4 non-musicians did fail to progress past Level 1, 3 non-musicians made up the group of 18 who progressed past Level 4 on the PIAT. One of these non-musicians also had a MEI of 0 indicating no musical participation at all. As expected, musicians performed better on the task, with 96% of them getting to Level 3 or above. Nonetheless, 63% of the non-musicians were also able to attain a maximum level of 3 or above, confirming that the PIAT can be used to induce pitch imagery in both musicians and non-musicians.Finally, the basic design of the PIAT allows for very comparable control conditions which do not require the use of musical imagery. In the perception condition, the participants were only made aware when the probe screen appeared that the trial did not require imagery, and therefore was a Perception trial. This ensured participants were actively listening throughout the trial, in anticipation that imagery may be required at any point. It also provided an identical probe presentation for direct comparison to the Imagery trials. Importantly, the mean reaction times for Imagery and Perception trials were not significantly different , due to the use of identical probes in the two conditions.Mental arithmetic provided a second control condition that required no musical imagery, but did require increasing mental capacity as the levels increased and the calculations became longer. The difficulty of the mental arithmetic trials were comparable to the imagery trials, given the similar accuracy measure scores, and decrease in reaction time over the task. These results suggest participants learned the two tasks at a similar rate. Interestingly, musicians performed more poorly than non-musicians on mental arithmetic trials, but the reasons for these differences are unclear. Musical imagery strategy users were generally faster than alternative strategy users  and spatial imagery (manipulation) had differing behavioural and psychometrical properties with visual artists excelling at object imagery and scientists excelling at spatial imagery . A similBoth BAIS subscales, though highly correlated to each other, were significantly correlated with different variables; confirming they index at least partially different aspects of the auditory imagery experience. BAIS-V correlated with the vividness rating participants gave after completing the PIAT (debrief vividness), suggesting that this more abstract auditory scale is associated with the subjective experience of musical imagery vividness during the PIAT. However it was the BAIS-C that correlated most significantly with Imagery and particularly Perception performance, suggesting that being able to manipulate sound images at will may be assisting with the anticipation of the perception of them. Pfordresher & Halpern  also shoA more complete picture of the relationships between these variables emerged through regression analysis, with strategy use proving to be the biggest single predictor of maximum level reached on the PIAT. Therefore while BAIS scores have been shown to be more important than musical experience in performance on this musical imagery task, it is clear the most important factor is the use of a musical imagery strategy. Interestingly, participants who reported using a musical imagery strategy were more accurate and had faster reaction times for Perception trials also, indicating that even when no manipulation of an auditory image was required, performance was facilitated by a musical imagery strategy.It could be argued that the use of up and down arrows in the PIAT reflects a spatial conception of pitch that may encourage the use of a spatial imagery strategy to complete the task. However, arrows were presented merely to indicate which pitch to imagine next, and participants were explicitly instructed to imagine the sounds of the pitches. Indeed, any learned association with pitch height could have been used to guide imagery. We asked participants to describe the strategies they used in completing the musical imagery trials, and the most common and successful of strategy was musical imagery rather than visual imagery. More generally, the PIAT can be readily adapted to other culture-specific schemata. For example, the major scale reflects a western conceptualization of pitch, but the PIAT can easily be modified to alternate musical scales .In this investigation, the PIAT was introduced as a powerful new protocol for assessing musical imagery. We confirmed that the PIAT reliably induces pitch imagery in individuals with a range of musical experience, particularly above Level 4. It entails the active manipulation of an auditory image that most non-expert musicians can readily perform. Our results showed competent performance on the PIAT requires active musical imagery and is very difficult to achieve using alternative cognitive strategies.The PIAT provides a platform in which to address questions of individual differences in musical expertise in imagery performance, as well as the role of auditory vividness and mental control. More musical training, increased self-reported BAIS-V and BAIS-C were associated with better performance on the PIAT. Both BAIS subscales were important, as success in the task required more than the ability to just hear an image in the mind, but involved the ability to successfully manipulate or change that musical image. Our results also support our second hypothesis that both auditory imagery vividness and the ability to control auditory images are more important than musical training in contributing to success in this type of imagery.The task is readily adaptable to neuroimaging studies of the neural correlates of pitch imagery. The basic protocol also lends itself to investigations of aspects of musical imagery including loudness, tempo or rhythm. For example, a future rhythm imagery task could involve the presentation of a simple rhythmical pattern and arrows pointing either upwards/downwards to increase/decrease divisions in the beats or left/right to either mentally reverse or maintain the simple pattern. A future study could compare imagery performance on different types of pitch scales; though it is expected that both musicians and non-musicians would perform poorly when unfamiliar scales are used, and if the task is too difficult it may encourage the use of alternative strategies.Feedback in the PIAT was included to facilitate acquisition of task performance. It could be argued that feedback could have influenced ratings of the vividness of musical imagery, in that participants might assign lower ratings of vividness should their overall imagery performance have been perceived as poor. We acknowledge this possibility but maintain that the benefits of feedback  outweigh the potential disadvantages. More explicit instructions of the types of musical imagery strategies that should be used, as well as the alternatives strategies that should be consciously avoided, may also lead to a higher percentage of participants adopting the desired auditory imagery strategy.Looking forward, the PIAT can also be used to address other theoretical issues surrounding musical imagery. First, unlike the visual domain, where it has been demonstrated that primary visual cortex is employed during visual imagery , there i


Liopholis kintorei), which has a patchy, but widespread distribution in the western region of the Australian arid zone. As a species of cultural importance to local Aboriginal groups and nationally listed as Vulnerable, it is a conservation priority for numerous land managers in central Australia. Analysis of mitochondrial ND4 sequence data and ten nuclear microsatellite loci across six sampling localities through the distribution of L. kintorei revealed considerable differentiation among sites, with mitochondrial STF and microsatellite STF′ ranging from 0.047-0.938 and 0.257-0.440, respectively. The extent of differentiation suggests three main regions that should be managed separately, in particular the southeastern locality of Uluru. Current genetic delineation of these regions should be maintained if future intervention such as translocation or captive breeding is to be undertaken.Knowledge of genetic structure and patterns of connectivity is valuable for implementation of effective conservation management. The arid zone of Australia contains a rich biodiversity, however this has come under threat due to activities such as altered fire regimes, grazing and the introduction of feral herbivores and predators. Suitable habitats for many species can be separated by vast distances, and despite an apparent lack of current geographical barriers to dispersal, habitat specialisation, which is exhibited by many desert species, may limit connectivity throughout this expansive region. We characterised the genetic structure and differentiation of the great desert skink ( The Australian arid zone occupies 70% of the continent’s landmass and supports an extraordinary biodiversity, including among the world’s richest assemblages of lizards , 2]. De. De2]. DEgernia inornata , an ariThere are also additional benefits derived from knowledge of connectivity; for example, it is established that parts of a species distribution that experience prolonged isolation may become sufficiently genetically differentiated that they are worthy of separate management –16. FurtLiopholis kintorei, is a species endemic to the arid-zone of Australia, currently listed as ‘Vulnerable’ [2, it is known to be patchily distributed, with its presence recorded at fewer than 100 localities [The great desert skink, nerable’ . It is anerable’ . Althougcalities . Great dcalities ), excavacalities . It is acalities , and thicalities ,19,20,24ru-Kata-Tjuta National Park (NT), Newhaven Wildlife Sanctuary (NT), Karlamilyi National Park (WA), Ngaanyatjarra Indigenous Protected Area , and in the Watarru IPA within the Anangu Pitjantjatjara Yankunytjatjara Lands ; and in these a number of monitoring and management actions have previously been undertaken. The extent of isolation or genetic differentiation between these regions is unknown, but likely to be high given the apparent disjunct distribution and low dispersal of the species. Here we use mtDNA sequence data (ND4) and ten microsatellite loci to characterise genetic structure and divergence across the range of L. kintorei. We aim to identify any regions where the genetic distinctiveness of L. kintorei heightens the conservation value and influences the management options.Areas containing great desert skinks are known to occur in a number of geographically distant regions within declared conservation areas: UluAll animals were handled in accordance with a protocol considered and approved by Macquarie and Charles Darwin University Animal Ethics committee recommendations (ARA 2008/025 and ARA 2011/037). Sample collection was licensed by the Northern Territory Parks and Wildlife Commission and the South Australian Department of Environment and Heritage.L. kintorei samples were collected from six locations throughout the distribution of the species (n = 30) and Sangster’s Bore in the northeast of their distribution , Petalu-Docker River toward the centre of their distribution , Uluru-Kata-Tjuta National Park to the east , Warburton to the west within the Ngaanyatjarra IPA  and Watarru within the IPA of the APY Lands at the southern extent of the distribution . Samples from Warburton were provided by the Western Australian Museum. Tissue was obtained via tail-tip biopsy and preserved in 90% ethanol. Liopholis kintorei is a species that exhibits kin-based social living, and high natal philopatry [Ninety-four  species : Australilopatry . To ensuWhole genomic DNA was extracted from tissue using a modified salting-out protocol . For eac2, 0.2 μM of each dNTP, 0.125 μM of each primer (ND4 and tRNA-leu) and 1 U Taq Polymerase (Promega). Thermocycling began with an initial denaturation for 5 min at 94°C, followed by four touchdown cycles with 94°C denaturation for 30 sec, annealing temperatures  for 30 sec, and 72°C extension for 45 sec. An additional 35 cycles were carried out at an annealing temperature of 47°C, followed by a final 72°C extension step for 10 min.Polymerase chain reactions (PCR) for all markers were carried out using a PTC-100 Thermocycler . Mitochondrial ND4 was amplified using previously developed primers . PCRs weBX6, CKD, FQR, J3F; see Est1, Est2, Est9, Est12 [Ecu2, Ecu3 [2, 0.2 μM of each dNTP, 0.02 μM of forward primer, 0.1 μM reverse primer, 0.1 μM of fluoro-labelled tag  and 1 U Taq Polymerase (Promega). Thermocycling began with an initial denaturation for 3 min at 94°C, followed by five touchdown cycles with 94°C denaturation for 30 sec, annealing temperatures  for 30 sec, and 72°C extension for 45 s. An additional 35 cycles were carried out at an annealing temperature of 50°C, followed by a final 72°C extension step for 10 min. PCR products were visualized by electrophoresis on 2% agarose gel. All PCR purification, sequencing and fragment separation was performed by Macrogen (Korea).Microsatellite PCRs were carried out in 10 μL volumes containing ~50 ng of DNA. A -29 M13 sequence was added to the 5’ end of each forward primer to allow for the incorporation of a complimentary M13 fluorescent-labelled tag, following the protocol of Schuelke [9, Est12 ; Ecu2, Eu2, Ecu3 ). All miND4 sequences were checked by eye and aligned with ClustalW, implemented in MEGA 5.0 , and subΦST, an analogue of FST [A minimum-spanning network of ND4 haplotypes was constructed in TCS 1.21 . Global  of FST , were ca of FST  with 100Microsatellite alleles were visualised and scored using Peak Scanner 1.0 (Applied Biosystems). To ensure amplification and scoring consistency, at least 10% of samples at each locus were independently rerun and genotyped. Summary statistics, including exact tests for Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were conducted in GenAlEx 6.4  and GENEFST analogues from highly polymorphic data such as microsatellites, within-population variance can often approach the level of the total variance, resulting in very low FST values even when the populations share no alleles [F′ST) were standardised using the program RECODEDATA 0.1 [When calculating  alleles ,41. Foll alleles  and MeirK = 1–8 , with 10 replicate runs for each, 105 MCMC iterations burn-in and 104 main iterations. Hubisz et al. [K) was determined using the ΔK method of Evanno et al. [STRUCTURE v.2.3  analysisz et al.  developeo et al. .Discriminant analysis of principal components (DAPC) was used to describe the genetic relationship between sampling localities. DAPC is a multivariate analysis that first uses principal components analysis (PCA) to transform data into uncorrelated components. These components are then analysed using a linear discriminant method, minimising within-group variance while maximising among-group variance . Furtherwww.r-project.org), with K selected using the find.clusters function and Bayesian Information Criterion (BIC). We also ran DAPC using sample locations as groups (K = 4) to assess the differentiation of our sample sites. PCA was performed in R using the dudi.pca function in the package ade4 [DAPC was carried out in the R package adegenet , implemeage ade4 . Missingage ade4 ). Determage ade4 . AnalyseMitochondrial ND4 sequences of 585 bp were successfully amplified from 72 individuals sampled from the six localities. Sequences contained 23 (3.9%) variable sites, of which 18 (3.1%) were parsimony informative, revealing a total of 17 unique haplotypes . HaplotyP < 0.05; IS ranged from 0.053–0.036, with these positive values probably reflecting a spatial Wahlund effect [One of the four remaining sample localities was out of HWE , were high and significant  and microsatellites (F′ST) was substantial  when no location information was used, however the BIC score for K = 4 was only slightly higher . For K the rest . Howeverthe rest . The optLiopholis kintorei. Each of the localities from which L. kintorei was sampled contained similar levels of genetic variation, and individuals from the Uluru-Kata-Tjuta region were most genetically distinctive ; howeveL. kintorei is distributed, it is not surprising that there are environmental differences across our sampling localities. For example, mean monthly minimum and maximum temperatures are consistently and significantly cooler at Uluru than the northernmost locality of Sangster’s Bore , though rainfall patterns differ with Uluru receiving more winter rain. Sangster’s Bore experiences more days above 35°C and 40°C than Uluru . Given their ectothermic physiology, life history traits in reptiles have been demonstrated to be linked to altitudinal or latitudinal variability in climate [L. kintorei, and may have led to adaptive differences in, for example, thermal tolerance or seasonal activity.Furthermore, there are environmental differences between the sites that may contribute to localised adaptation, another factor that flags the possibility of outbreeding depression from translocation according to Frankham et al. . Similar climate . As suchL. kintorei is found in these areas varies also. The Sangster’s Bore and Newhaven localities occur along and adjacent to palaeodrainage lines, with the species’ preferred habitat in semi-saline spinifex plains dominated by soft spinifex (Triodia pungens) and inland tea tree . At Watarru, L. kintorei were found within open mulga woodland or Eremophila and woolybutt (Eragrostis eriopoda) grass shrubland, and at Uluru they occur on sand plains and flat swales dominated by either hard (Triodia basedowi) or soft spinifex (T. pungens and T. schinzii) [The habitat in which chinzii) .F′ST = 0.285), but were not significantly differentiated at mitochondrial ND4 (FST = 0.047). This discrepancy, taking into account the different inheritance and mutation rates for these genetic markers [Uluru was the most genetically distinct in all analyses, and shared no haplotypes with any other site; Watarru was also highly differentiated from the other areas, but shared haplotypes with the other two southwestern localities (Docker River and Warburton). More sampling at Docker River and Warburton is required to determine the true extent of differentiation among these localities. Sangster’s Bore and Newhaven to the north were highly differentiated based on the microsatellite data set , and geat Uluru . Transloat Uluru ,59. In tL. kintorei should prioritise the preservation of suitable habitat, in particular addressing recent and localised changes in fire regimes and predation pressure to reduce the risk of further localised population declines and thus erosion of genetic diversity. While further sampling needs to be conducted at Watarru, Docker River and Warburton, the evidence suggests three main delineations for management: (1) Uluru to the southeast, (2) Newhaven and Sangster’s Bore to the north, and (3) Watarru, Docker River and Warburton to the southwest. Uluru in particular should be considered separately for management, and this distinctiveness should be recognised if intervention such as translocation or captive breeding is to be undertaken.Knowledge of connectivity combined with landscape management of biological processes is needed to conserve biodiversity . ConservS1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 FigΔK values for each potential number of genetic clusters (K) examined, showing a best-fit value of K = 2. (b) Bar plot showing population assignment of individuals from each sample locality. (c) DAPC plot showing K = 2 genotypic clusters. The y-axis represents the density of individuals along the given discriminant function. One cluster (red) comprised the Uluru samples, and the other (green) clumped Newhaven, Sangster’s Bore and Watarru together.(a) (TIFF)Click here for additional data file.


Cryptosporidium.Cryptosporidiosis is a gastrointestinal disease affecting many people worldwide. Disease incidence is often unknown and surveillance of human cryptosporidiosis is installed in only a handful of developed countries. A genetic marker that mirrors disease incidence is potentially a powerful tool for monitoring the two primary human infected species of Cryptosporidium isolates from ZoopNet, which currently contains more than 1400 records with their sampling nations, and the names of the host species from which the isolates were obtained. Based on 296 C. hominis and 195 C. parvum GP60 sequences from human origin, the genetic diversities of Cryptosporidium was estimated for several nations. Notified cases of human cryptosporidiosis were collected from statistics databases for only four nations.We used the molecular epidemiological database with C. hominis were estimated in 10 nations in 5 continents, and that of C. parvum of human origin were estimated in 15 nations. Correlation with reported incidence of human cryptosporidiosis in four nations  was positive and significant. A linear model for testing the relationship between the genetic diversity and incidence produced a significantly positive estimate for the slope .Genetic diversities of C. hominis and C. parvum may therefore be an independent and complementary measure for quantifying disease incidence, for which only a moderate number of stool samples from each nation are sufficient data input.The hypothesis that genetic diversity at GP60 locus mirrors notification rates of human cryptosporidiosis was not rejected based on the data presented. Genetic diversity of  Cryptosporidium is one of the most common diarrhea-causing parasitic genera in the world [he world -3. Humanhe world  where crhe world .Cryptosporidium parasites that are important for humans are classified into the two main species, C. hominis and C. parvum, and further into subtype families based on sequence variability in the GP60 gene [Cryptosporidium is a parasite that has a sexual lifecycle, with most of the stages of the lifecycle being haploid while the zygote is diploid and the oocyst contains four sporozoites that should represent two different sets of identical recombinant genes if parental isolates are genetically different. This means that sexual recombination is likely to be common if strains from different genetic heritages infect the same animal or person. Mallon et al. argued that the populations of C. parvum in the UK were panmictic, based on genetic diversity whereas those of C. hominis are more clonal [Cryptosporidium isolates from humans and from animals in various countries have been sequenced and compiled into a database [Giardia and Cryptosporidium). In particular, sampled countries cover parts of the globe extending to the continents of Asia and Africa in addition to America, Europe, and Australia. The broad geographic range is a strong asset of the database, if we can infer the population risk of human cryptosporidiosis from the regional sequence data.P60 gene  that encP60 gene . Cryptose clonal . Cryptosdatabase , 11. ThiCryptosporidium at the GP60 locus from human isolates positively correlates with the population risk. In other words, the exposure to infectious oocysts in a human population could be indirectly measured by quantifying the genetic diversities of C. hominis and C. parvum DNA sequences, as found in human fecal samples. Each cell division is a potential event for a novel genetic variant. Hence, a higher number of cell divisions per unit time should drive genome to a greater diversity. The rate of cell divisions for Cryptosporidium species that infect humans could be quantified using an annual incidence rate as a proxy. We refer by genetic diversity to an estimate for the population size based on the coalescent theory in molecular evolution literature [Borrelia burgdorferi sensu lato in ticks. It presents a precedent case validating the application of molecular evolution approach to estimating the extent to which humans are exposed to the infectious microorganism, where no alternative approach is possible.Here, we test the hypothesis that the genetic diversity of terature . A similterature , where iCryptosporidium sequence database are empirical input into the molecular evolutionary analysis to test our hypothesis.For the current study, national public health statistics and the existing Notification rates of human cryptosporidiosis were obtained from the literature (summarized in Table Giardia and Cryptosporidium isolates. The current database (February 2013) contains more than 1400 records of GP60 sequences, sampled countries, and host species from which isolates were collected. Cryptosporidium isolates of human (n = 882), animal (n = 374) or unknown origin (n = 199) present in the ZoopNet-database [Cryptosporidium GP60 DNA sequences were also downloaded and frequently updated from the Entrez Nucleotide Database (GenBank) at the National Center for Biotechnology Information and processed as described [C. hominis and 195 C. parvum sequences were stored and analysed in Bionumerics . DNA sequences and data used for this study are available upon request.A network of European public health and veterinary institutions (ZoopNet) was established to generate and collect molecular epidemiological data from database  were columan n = 82, animaCryptosporidium sequences of known species and subtype families were retrieved from GenBank using the accession numbers AY738184 to AY738196 [Subtype families of the isolates were identified based on amino acid sequence similarity. C. hominis and C. parvum. For each species, GP60 sequences were grouped per nation and the sequences were aligned using the multiple alignment software MAFFT [Genetic diversity of human isolates was estimated separately for re MAFFT . An obsere MAFFT . ReconstCryptosporidium species. Hence, we combined the genetic diversities estimated separately for each species into one measure. We performed all statistical computations described in this section using the software Mathematica, version 9 .Notification rates account for the two major C. hominis and C. parvum in the total number of isolates per nation  and significant . Spearman rank was positive (0.8) and significant .Pearson correlation between the P-value < 0.05) between the estimated mean genetic diversity and the estimated mean notification rate  and significant . The slope of the generalized linear model was significantly greater than zero . We repeated the additional analyses using the C. parvum dataset alone. Spearman rank was not significant . The slope of the generalized linear model was not significantly different from zero .The relationship between estimated genetic diversity and the notification rates could be specific only to the combination of A study made a suggested observation that genetic variation at GP60 locus might be essential for the parasite’s long-term success . It is tC. parvum from human isolates might directly originate from one or more animal reservoirs. Extensive zoonotic transmission would change the estimated genetic diversity in either upward direction or downward direction, depending on the genetic diversity of zoonotic isolates in animal reservoirs. Hence, one might expect only a weak relationship between estimated genetic diversities with human cryptosporidiosis notification rates, when extensive transmission from animal reservoirs would occur. A significant and positive slope of the linear model indicated however, that zoonotic transmission interfered little on the relationship between the estimated genetic diversity and the epidemiology in those countries. Second, some infections might have been acquired abroad. Over-represented, foreign acquired infection is expected to level the estimated genetic diversities between nations. Contrary to this expectation, the slope of the linear model testing the relationship between the genetic diversity and incidence was positive and significant, in the developed nations where the residents might travel more frequently compared to those in developing nations, indicating that the overall Cryptosporidium genetic diversity was predominantly determined by autochthonous cases. It is possible that, due to social and environmental circumstances different from the nations analyzed in this study, human cryptosporidiosis acquired from livestock animals or from abroad alters the genetic diversity and its relationship to epidemiology in those nations.Nonetheless, we like to comment on some factors that might affect the result of this study. First, some Cryptosporidium could reflect either a relative difference in the estimates for the Cryptosporidium population sizes between nations, or a relative difference in mutation rate. We postulate that mutation rate is a less-likely source of variation and by eliminating the possibility, we opt for the idea that the Cryptosporidium population size reflects the national notification rate of human cryptosporidiosis.In the molecular evolutionary approach that we have adopted, genetic diversity is proportional to the product of the mutation rate and the estimate for population size based on the coalescent theory . Thus, aRandom sampling is an assumption in the molecular evolution approach, and selectively sampled sequences are expected to influence the estimate on genetic diversity. One might expect for example that two isolates collected from an outbreak in a single site are more closely related than e.g., two isolates collected in two distant provinces in China. This sampling bias might change the estimates on genetic diversity in each nation in terms of absolute value. However, we expect such bias to influence independently the reported disease incidence in corresponding nations, and therefore, aligning genetic diversity and disease incidence in a number of nations by a spurious mechanism such as sampling bias appears unlikely.In theory, our hypothesis could be tested using any genetic elements, including MLST and microsatellite. GP60 sequence is rich in repeats of tri-nucleotides . The length of the tri-nucleotide repeats can change by a deletion and insertion events, and this process is not accounted for by a model of DNA evolution by a point mutation in the molecular evolutionary approach . The dupA longitudinal study in a single nation is an additional test case to our hypothesis regarding the population exposure and the population risk. Seasonal incidence and dated molecular sequences from those affected in the same season in the same nation are empirical input to test this hypothesis based upon independent observations.Cryptosporidium genetic diversity mirrors the notification rates of human cryptosporidiosis could not be rejected based on the datasets from the four developed countries. This inference from the molecular sequences may be an independent and alternative approach to estimate the risk of human cryptosporidiosis where collection of the samples is an economically suitable option.Hypothesis that


Aedes aegypti. Alternative vector control strategies consist in releasing artificially modified mosquitos in the wild, but knowledge on their dispersal ability is necessary for a successful implementation. Despite being documented that Ae. aegypti can be passively transported for long distances, no study to date has been aimed at understanding whether human marine transportation can substantially shape the migration patterns of this mosquito. With thousands of islands connected by a dense network of ships, the Philippines is an ideal environment to fill this knowledge gap.Dengue virus (DENV) is an extraordinary health burden on global scale, but still lacks effective vaccine. The Philippines is endemic for dengue fever, but massive employment of insecticides favored the development of resistance mutations in its major vector, Ae. aegypti from 15 seaports in seven major islands of central-western Philippines were collected and genotyped at seven microsatellite loci. Low genetic structure and considerable gene flow was found in the area. Univariate and multivariate regression analyses suggested that anthropic factors  can explain the observed population structure, while geographical distance was not correlated. Interestingly, cargo shipments seem to be more efficient than passenger ships in transporting Ae. aegypti. Bayesian clustering confirmed that Ae. aegypti from busy ports are more genetically similar, while populations from idle ports are relatively structured, regardless of the geographical distance that separates them.Larvae of Ae. aegypti. Hopefully corroborated by further research, the present findings could assist the design of more effective vector control strategies.The results confirmed the pivotal role of marine human-mediated long-range dispersal in determining the population structure of  Aedes aegypti. To control the spread of the virus, insecticides have been abundantly used but Ae. aegypti has developed a genetic resistance to them. Currently, alternative methods are being tested wherein artificially modified mosquitos are released in the wild to interfere with the mating of natural populations. It is important then to understand how the mosquito spreads in the environment. It is known that Ae. aegytpi can be passively transported for long distances by human vehicles, but it was not clear how common this event is, especially in case of marine transportation. In population genetics, a basic assumption says that if populations frequently exchange migrants, they become genetically more similar than relatively isolated populations. We estimated the genetic similarity between Ae. aegypti collected in the Philippines from 15 seaports of different sizes and ship connectivity. The mosquitos from busy ports, even distant ones, were genetically similar, while in the small ports, even close ones, Ae. aegypti were relatively differentiated. It was also suggested that Ae. aegypti's dispersal is affected by cargo shipments more than passenger ships.Dengue fever threatens the health of millions in the tropics and its causative agent, dengue virus, is mainly transmitted by the mosquito  Aedes aegypti is the major vector of dengue virus (DENV). An estimated 96 million dengue cases and 300 million asymptomatic infections were reported globally in 2010 [Wolbachia [The dispersal ability of the vector should be taken into account when implementing any of the above-mentioned population suppression techniques. If a vector can travel long distances it can quickly and effectively spread insecticide resistance mutations, symbiotic bacteria or deleterious mutations.Ae. aegypti is narrow  [Ae. aegypti collected from ships right after docking [Ae. aegypti immatures, both by land and sea [Ae. aegypti have been for the first time collected at Narita Airport . It is strongly suspected that they were transported via airplane from a foreign tropical country [Ae. aegypti's migration flows are mainly driven by active dispersal, the genetic differentiation between populations should increase with the geographical distance; this is called Isolation By Distance (IBD) [The flight range of to 500m) –5 and itto 500m) –8. There docking  and the  and sea . Recentl country . If Ae. ce (IBD) . OtherwiAe. aegypti in many regions of the world. The genetic structure of this important vector mosquito has been analyzed at micro and macro geographical scales with different methods and purposes. Long-distance migration has been hypothesized and frequently related to human movements, but this association was not addressed in a quantitative way and only briefly brought up in the final discussion as a possible explanation of the results [Ae. aegypti's dispersal; yet, only the land transportation was taken into account [Ae. aegypti's migration [Ae. aegypti at a macro-geographical scale.A remarkable amount of population genetics studies have been conducted on  results –15. A fe account –20. One igration . TherefoAe. aegypti in the Philippines [The Philippines is an archipelago located in Southeast Asia and is endemic for dengue fever. According to the World Health Organization, the annual reported cases of dengue fever increased around 17-fold in the decade 2000–2010 . Unlike lippines –26. A relippines ,28. FurtAe. aegypti among different islands of the Philippines. Specific objectives were: (1) to analyze the genetic structure of Ae. aegypti among different islands; (2) to quantitatively determine if natural  or anthropic (port size and intensity of marine transportation) factors affect the observed genetic structure.For these reasons we decided to undertake a population genetics study aimed at understanding if and how human marine transportation affects the movements of Ae. aegypti, seaport locations on several islands were sampled, as populations of mosquitos residing near the seaports are more likely to end up resting or laying eggs on the ships (either cargo or passenger). Each site corresponded to one seaport and all individuals from a site were considered as one separate population, therefore the terms "site", "population" and "port" will be used interchangeably, hereinafter. The survey was conducted between September and October 2013 in the central-western area of the Philippines (Region IV-A and IV-B). Larvae were collected from 15 ports of different levels of connectivity and development, across seven islands of different sizes and degrees of urbanization , Ae. aegypti's flight range and its so-called "skip oviposition" behavior were taken into account [In order to analyze the effect of human sea transportation on the migration of nization . At each account  and a miTM Tissue PCR Kit  following the manufacturer's protocol. Seven microsatellite loci were scored for each individual. Each PCR reaction (10μl) contained 400nM of M13 fluorescent primer (FAM-labeled) [2+ 2mM included), 200nM of each dNTP, 0.25U of Takara ExTaq  and 1μl of sample. Thermocycling conditions were: (5' at 94°C) for one cycle,  for 35 cycles, (5' at 72°C) for 1 cycle and 4°C as holding T. T of annealing (Ta) was specifically determined for each pair of primers .Genomic DNA was extracted from individual larvae with RedExtract-N-Amplabeled) , 200nM o primers . After Pa were conducted in order to find the most suitable for each primer pair  on the AVs. As the data showed considerable heteroscedasticity they were log-transformed before PCA .a+b was used, were a and b are the corresponding value for each two ports. Pearson correlation coefficients were computed in R with the command "rcorr". To detect collinearity, the variance inflation factors (VIF) between eight predictors were computed (command "vif"). The command "lm" was used to run the multivariate linear regression models. The best model was chosen with the command "step", which performs a stepwise exclusion of each predictor and calculates the Akaike Information Criterion. Simple and partial Mantel tests were run using the online software "Isolation by distance web service" (IBDWS) [As the AVs were expressed as a single value for each port , while Fst values were pairwise , in order to quantitatively compare them, the values for the AVs were transformed to pairwise; the formula  (IBDWS) . One-taiAe. aegypti to genetic clusters based on the microsatellite allelic frequencies. Admixture was selected as ancestry model with LOCPRIOR function. Alpha was allowed to vary and was separately calculated for each population. A correlated allele frequency model was chosen, with lambda set to one. Ten independent replicates were run with a burn-in step of 300,000 iterations followed by 500,000 replications. Average L(K), ΔK [The Bayesian clustering method implemented in the software STRUCTURE v. 2.3.4  was usedL(K), ΔK  and propAe. aegypti larvae per site were collected . At each site between six and 15 containers were inspected and between four and eight were positive. One out of five larvae was randomly selected from each container and an average of 43 per population was genotyped  (An average of 211 l = 642) .The test for deviation from HWE calculated 105 Fis values , one forThe test for LD calculated a total of 315 values (21 locus-by-locus pairs for each population), of which 28 (8.9%) remained significant after Bonferroni sequential correction. However, no locus-by-locus pair showed significance consistently across populations. Therefore this significance was unlikely due to linkage. None of the markers was excluded from the analysis.For most loci between four and six alleles were detected; only AG5 had 11. The Fst for all loci and populations was 0.056, while the pairwise Fst ranged from 0.005 and 0.147; after Bonferroni correction 79/105 (75.2%) remained significant (P<0.0005) .2 was 0.24 (P<0.001). The regression coefficients for Density and Cargo indicated a negative association with pairwise Fst (P<0.001)  . TonnageP<0.001) . The squThe PCA on the seven AVs returned a PC1 able to explain 74% of the total variance of the dataset; the directions of the vectors representing the AVs were almost opposite to the PC1 . It is tSTRUCTURE analysis could detect no genetic structure unless the option LOCPRIOR was chosen. This function uses information on sampling locations to assist the clustering in case the signal of structure is too weak . A consiAe. aegypti.The results suggested low genetic structure and considerable gene flow. Pairwise Fst values were generally low (0.005~0.147), in accordance with other studies conducted in Florida 0.004~0.171), Pakistan (0.013~0.17) and Southeast Asia (0.02~0.185) 1, Pakist,48. In tBoth univariate and multivariate regression models excluded IBD. In fact, Distance/Fst Pearson's r was almost zero  and Distance was dropped by multiple linear regression stepwise analysis . On the Ae. aegypti to be accidentally transported, decreasing the genetic differentiation and consequently the Fst. Similarly, more densely populated municipalities have likely busier ports, with more congested ship traffic and offer more opportunities to Ae. aegypti to be transported.Our interpretation is that increasing cargo shipments increase the probability for Ae. aegypti more effectively than passenger ships. If we consider that cargo ships (like barges) are usually bigger in size than passenger ships, they may offer a higher number of breeding sites (i.e. collections of water). Moreover, the cargo storage compartments are usually kept closed during the navigation, while passenger ships' decks are often not enclosed; this could cause more adult mosquitos to enter and being effectively transported by the cargo ships. Another hypothesis (not excluding the previous ones) is that cargo shipment routes are implemented earlier than passenger ships' routes during the development of the ports. In other words, ports of small and intermediate sizes could be connected by more cargo than passenger ships, while busiest ports operate a similar amount of the two ship types. This hypothesis is supported by the scatter plots between the four "ship connectivity" predictors ; all of them are highly correlated, but only Cargo shows a non-linear relation with the others. Values of Cargo increase earlier than values of the other three predictors  to remarkably isolated (port 1) . At bothAe. aegypti's population structure at broader spatial scales ; in these studies the ancestry of individual mosquitos was more definite, but mosquitos from the same geographical populations were assigned to different genetic clusters  [Interestingly, the individuals had mixed ancestry while the populations as a whole were homogeneous  . This coar plot) ,50. A reMain limitation of this study could be the sample size , as someThe data from the Philippines Statistics Authority used to elaborate Vessel, Tonnage, Cargo and Passenger was incomplete and it was necessary to estimate the corresponding values for four ports.However, we believe that these limitations did not undermine the general meaning of our findings.Ae. aegypti in the central-western part of the Philippines. Regardless of their geographical location, large ports in highly-populated municipalities exchange individuals of Ae. aegypti at a faster rate than small-sized ports in less developed municipalities. Insecticide spraying, release of sterile males, release of Wolbachia symbionts: whichever vector control strategy will from now on be applied in the Philippines and other archipelagoes, these results should be taken into account.Our overall interpretation is that the human ship transportation substantially shapes the dispersal patterns of S1 FigPeaks of ΔK were found at K3, K6, K9. L(K) were averaged over 10 independent replicates.(PDF)Click here for additional data file.S2 FigDensity is non-linearly correlated to predictors related to port size (i.e. Inhabitant and Dock), while Cargo is non-linearly correlated to predictors related to ship connectivity . Then, Density and Cargo are totally non-correlated between each other. For detailed description of the predictors refer to (PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 Dataset(ZIP)Click here for additional data file.


Thunnus albacares , covers majority of the Philippines’ tuna catch, one of the major fisheries commodities in the country. Due to its high economic importance sustainable management of these tunas has become an imperative measure to prevent stock depletion. Currently, the Philippine yellowfin tuna is believed to be part of a single stock of the greater WCPO though some reports suggest otherwise. This study therefore aims to establish the genetic stock structure of the said species in the Philippines as compared to Bismarck Sea, Papua New Guinea using nine (9) DNA microsatellite markers.The yellowfin tuna, DNA microsatellite data revealed significant genetic differentiation between the Philippine and Bismarck Sea, Papua New Guinea yellowfin tuna samples. , which is further supported by multilocus distance matrix testing (PCoA) and model-based clustering (STRUCTURE 2.2).With these findings, this study posits that the yellowfin tuna population in the Philippines is a separate stock from the Bismarck Sea population. These findings add evidence to the alternative hypothesis of having at least 2 subpopulations of yellowfin tuna in the WCPO and calls for additional scientific studies using other parameters to investigate this. Accurate population information is necessary in formulating a more appropriate management strategy for the sustainability of the yellowfin tuna not only in the Philippines but also in the WCPO. Globally, tuna production has constantly been an important source of annual total marine production for coastal countries. In the Western and Central Pacific Ocean (WCPO), tuna species, primarily skipjack, yellowfin and bigeye tunas, have been the leading source of fishery catch and production. These three species alone contributed an approximate 2.2 million mt in the region’s fishery catch in 2011, representing 79% of the total Pacific Ocean catch .Sustaining tuna resources in the WCPO and in the Philippines is not only important economically but more so to the ecosystem. The Coral Triangle, a region considered to be the global center of marine biodiversity and one of the world’s top priorities for marine conservation, spanning eastern Indonesia, Malaysia, Papua New Guinea, Timor-Leste and the Solomon Islands, and the Philippines, has become a priority for monitoring and conservation, especially the marine environment . ConservThunnus albacares , is widely believed to be panmictic within and between oceans. YFT population between the Atlantic and the Pacific Oceans show low levels of genetic differentiation indicating a very slow genetic drift due to the species’ large population size .TheObe231, Obe294, Obe652, Obe467, Obe157, Obe674, Obe527, Obe237, Obe218, and Obe236) isolated from bigeye tuna were cross-amplified using the prescribed protocol [st BASE), 0.08U standard Taq polymerase (KAPA), and 1μl DNA template. The mix was aliquoted to a 12-μl reaction and was run using the following PCR parameters: initial denaturation at 95°C for 2min; 30 and 35 cycles of amplification with denaturation at 94°C for 30s, annealing at 58°C for 30s, and extension at 72°C for 30s; and a final extension at 72°C for 2min. Resulting PCR products were confirmed by running them in gel electrophoresis using 3% agarose gel. Fragment length analysis of the samples was outsourced to Macrogen Inc., Korea using the 3730XL DNA analyzer  and Big Dye Terminator v3.1 Cycle Sequencing Kit  with size standards 400-HD and 500-LIZ.Muscle tissues were extracted from the left posterior part of the fresh or frozen YFT samples of various sizes ranging from 15 to 89 cm in fork length . The musprotocol . Primer Prior to statistical analyses, the samples were identified as YFTs by running phylogenetic trees  in MEGA5  using paObe652, was found to significantly deviate from HWE, thus was not used in the rest of the analysis. The allelic richness observed from each sample site using these nine loci ranges from two to 13 alleles. Observed heterozygosities range from 0.182 to 0.867 as compared to the sample population’s expected heterozygosities ranging from 0.355 to 0.861. Basic descriptive statistics of each locus in each sample site are shown in Yellowfin tuna samples of sizes ranging from 15 to 89 cm in fork length  were cola priori as two groups, the pooled Philippine sites and the Bismarck Sea, Papua New Guinea site. Significant genetic differentiation was observed between these two groups with FST = 0.034 (P = 0.016). This finding between the pooled Philippine samples and the Bismarck Sea samples is considered moderate variation based on Wright’s qualitative guideline [Hierarchical variations using distance method based on the number of different alleles for the whole population are presented in uideline .Genetic differentiation estimates were also determined between pairs of sites using distance method based on the number of different alleles. Samples from the Philippine sites were compared to those from Bismarck Sea, Papua New Guinea to confirm whether these sites exhibit structuring, in an attempt to support the variation observed between the two groups.P = 0.00000).On the other hand, Significant FSTs were observed in Bismarck Sea, Papua New Guinea when paired to the four Philippine sites with FST ranging from 0.2233 to 0.2582, exhibiting moderate to great variation  using a multilocus distance matrix as represented in Further support on the distinct clustering of YFT was obtained upon using the model-based clustering method, STRUCTURE, on our multilocus genotype data. Ten values of K , with 10Fisheries conservational management has been a main concern for coastal countries like the Philippines in the past few decades. Strategies have been implemented over the years in sustaining marine stocks especially the commercially important organisms. Technologies have also been enhanced in an effort to aid in creating and improving existing management strategies especially in the wider marine systems. Among these technologies, the advent of the more stable genetic markers in inferring marine system connectivities has been one of the greatest breakthroughs in population studies. Though not an absolute deciding factor in delineating subpopulations among organisms, identifying an organism’s genetic stock structure has become a key in determining other equally important factors like gene flow, migration and dispersal with which a stock may be concretely determined. Specifically, these genetic markers can provide hints on the connectivity of stocks of marine organisms that can be further used in designing and redesigning sustainable management strategies . StudiesAnalyses of molecular variance using suitable genetic distance methods were conducted to confirm whether the Philippine stock is separate from the widely believed single WCPO stock. There was no significant differentiation observed among the YFT samples from the four Philippine sites based on DNA microsatellite data albeit inconclusive due to sampling limitations. Philippine was then compared to Bismarck Sea YFTs which represents part of the WCPO. Moderate genetic differentiation was observed between the two areas based on both genetic distances and pairwise differences. Similarly, comparison between the Bismarck Sea YFTs and each group of samples from different Philippine sites yielded significant variation based on both genetic distances and pairwise differences. The Zambales-Bismarck Sea pair, which has the highest degree of differentiation observed, was the most significant among the pairwise estimates due perhaps to the Zambales fishing site which is within the West Philippine Sea and is geographically separated from the Pacific Ocean by the Philippine archipelago. These evidences further strengthen the hypothesis that the Philippines might have a single stock that is separate from the greater Western and Central Pacific stock, contrary to the current assumption that the region only has a single stock of yellowfin tuna. To further support this assumption, clustering into two YFT stocks were observed in both PCoA using distance matrix and model-based clustering method, STRUCTURE. Both analyses clearly delineated the two distinct groups observed in both tests for genetic distances and pairwise differences.Having a Philippine stock of yellowfin tuna separate from the Western and Central Pacific is possible because of the presence of biogeographic barriers such as eddies and upwellings as well as strong ocean currents like the North Equatorial Current on Philippine borders. Jackson et al. , suggestKatsuwonus pelamis), also support this divergent tuna stocks scenario, as they revealed a possible stock delineation in the Western and Central Pacific Ocean using serum esterase & transferrin system allozymes [A recent study on yellowfin tuna also reported a possible admixture of Taiwan stock to the Philippine Sea stock, although only one geographic location was sampled . Populatllozymes ,35.The analysis of these YFT samples using DNA microsatellite markers exhibited moderate variation between the pooled Philippine samples and the Bismarck Sea samples. This strongly suggests the existence of a distinct YFT stock in the Philippines different from the YFT stock found in the Bismarck Sea, Papua New Guinea.To further support the findings of this study, it is recommended that further studies should include additional sampling sites from both areas of Papua New Guinea and the Philippines and eventually the rest of the Western and Central Pacific region. Additionally, larval sampling may be added as the source of genetic material. Other parameters to test the stock structure of the yellowfin tuna in the WCPO could be included such as the use of length frequencies, reproductive biology, otolith, parasites, tagging etc. The accuracy of such population structure approach will greatly help in assessing stock status and determining the most suitable management strategy for the region’s yellowfin tuna.S1 FigT. albacares partial D-loop fragment sequences of Pedrosa-Gerasmio et al. (2012) to confirm the correct species identification of the sampled individuals. Representative bigeye tuna sequences from the same study were used as outgroup.A subsample (n = 73) was analyzed with the (TIF)Click here for additional data file.S2 FigT. albacares partial D-loop fragment sequences of Pedrosa-Gerasmio et al. (2012) to confirm the correct species identification of the sampled individuals. Representative bigeye tuna sequences from the same study were used as outgroup.A subsample (n = 73) was analyzed with the (TIF)Click here for additional data file.S3 FigPlots for values of K = 1 to K = 10 were constructed in STRUCTURE 2.2, with 10 replicate runs for each K value.(TIF)Click here for additional data file.S1 FileT. albacares(Table A). Allele size frequencies of Locus Obe236 for all populations of T. albacares(Table B). Allele size frequencies of Locus Obe231 for all populations of T. albacares(Table C). Allele size frequencies of Locus Obe294 for all populations of T. albacares(Table D). Allele size frequencies of Locus Obe652 for all populations of T. albacares(Table E). Allele size frequencies of Locus Obe467 for all populations of T. albacares(Table F). Allele size frequencies of Locus Obe157 for all populations of T. albacares(Table G). Allele size frequencies of Locus Obe674 for all populations of T. albacares(Table H). Allele size frequencies of Locus Obe527 for all populations of T. albacares(Table I). Allele size frequencies of Locus Obe237 for all populations of T. albacares(Table J).Allele size frequencies of Locus Obe218 for all populations of (XLSX)Click here for additional data file.S1 TableFork length of each sample was measured in cm (±1.0).(XLSX)Click here for additional data file.S2 TableAllele sizing was done using Peak Scanner software v1.0. Samples were coded according to their sampling location sites: YFG—General Santos; YFS—Samar; YFZ—Zambales; YFP—Palawan; YFBS—Bismarck Sea, Papua New Guinea.(XLSX)Click here for additional data file.


To estimate existing within-breed genetic variability in Marwari goats under field conditions and the generated data that can be used to determine genetic relationships with other breed of goats.A total of 146 blood samples of goats of Marwari breed were randomly collected from genetically unrelated animals from different villages of Bikaner Districts of Rajasthan, India. Genomic DNA was extracted from whole blood using proteinase K-digestion followed by standard phenol–chloroform extraction procedure at room temperature and confirmed through horizontal electrophoresis on 0.8% agarose gel containing ethidium bromide. Fifteen caprine microsatellite markers were used to estimate genetic variability among the goats of Marwari breed in terms of allelic and genotype frequencies, heterozygosities and polymorphism information content (PIC) value.A total of 74 alleles were contributed by Marwari goat across all 15 microsatellite loci. The number of alleles per locus varied from two (ILSTS-087) to 9 (ILSTS-058) alleles, with a mean of 4.93 whereas the effective number of allele varied from 1.35 (ILSTS-005) to 3.129 (ILSTS011) with a mean of 2.36. The effective number of allele is lesser than observed number at all the loci. Allelic sizes ranged from 125 bp (ILSTS-028 and ILSTS-033) to 650 bp (ILSTS-011 and ILSTS-019). The expected heterozygosity ranged from 0.240 (locus ILSTS-005) to 0.681 (locus ILSTS-011), with an average value of 0.544. The observed heterozygosity (Ho) ranged from 0.1428 (locus ILSTS-087) to 0.9285 (locus ILSTS-034), with an average value of 0.5485 indicates substantial and very good number of heterozygotes, in the population. The highest PIC value (1.1886) was observed at ILSTS-044 locus and least (0.0768) at ILSTS-065 locus for Marwari goat.Microsatellite analysis revealed a high level of polymorphism across studied microsatellite markers and informativeness of the markers for genetic diversity analysis studies in Marwari goats. This high level of polymorphism can be utilized to plan future biodiversity studies to exploit the uniqueness and adaptability of this breed to Western Rajasthan. Most studied microsatellite markers proving to be good candidates for genetic characterization and diversity analysis of this breed of goat. Marwari goat, a major meat breed of Rajasthan (India), is well adapted to the arid environment, grows faster, bred efficiently, can tolerate higher salt loads, and requires less water than many other species of this region . These uAmong the various molecular marker systems for genetic characterization, microsatellites markers have been widely used as genetic markers in bovine population studies and pedigree verification, mainly because of their large polymorphism information content (PIC), widespread distribution in the eukaryotic genome  and robust methodology. Microsatellites have been effective in evaluating differences within cattle breeds and in determining population substructures . More thMost of the studies using microsatellites have concentrated on cattle, sheep and pigs while the information available about the genetic characterization of goats is limited . TherefoAll essential procedures of sample collection were performed strictly as specified by Institutional Ethics Committee with minimal stress to animals.The study was conducted at the College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Science, Bikaner, Rajasthan, India, located at 27°29′ North latitude and 77°40′ East latitude (ms l-174 m). The climate of the study area is classified as tropical (semiarid zone). The temperature in this region varies from 49°C (May and June) to −2°C (winter). Low and erratic rainfall (39-392 mm) is a common feature. The soil is sandy, and vegetation is composed of natural pasture and bushes.The Marwari goat, a native breed of arid and semi-arid desert region of West Rajasthan, is predominantly found in Jodhpur, Pali, Nagour and Bikaner districts of West Rajasthan. They are primarily used for meat purpose. Goats were allowed free range grazing on the natural pasture from 08.00-17.00 h daily except during the summer (April to June) when split grazing during cooler hours of the day was observed from 06.00 to 12.00 and 15.00 to 19.00 h. Supplementary feeding to pregnant and lactating ewes and young lambs in the form of a concentrate and harvested fodder were provided.Blood samples were randomly collected from 146 genetically unrelated animals of Marwari goat from different villages of Bikaner district of Rajasthan in line with MoDAD recommendations . GenomicA panel of 15 microsatellite markers was selected from the list as recommended by International Society for Animal Genetics and FAO’s (DAD-IS) for Caprine, based on their level of polymorphism, allele size range and reliability of allele calling to characterized and reveal the extent of genetic diversity in Marwari goats as follow:2, 200 μM dNTPs, 1.0 μl of each primer, ~3.0 μl of template DNA and 0.25 μl of Taq DNA polymerase  using PX-2 Thermocycler . PCR cycling conditions were: 5 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min at annealing temperature (52–58°C) of each primer, 45 s at 72°C, and final extension of 30 s at 72°C. Gradient PCR was attempted to determine the exact annealing temperature. Annealing temperatures as mentioned in literature and optimized for the present study are given in 2 concentration; hence 1.5 mM concentration already present in the assay buffer was used for all amplifications. Taq DNA polymerase was initially used at 5 U  but later reduced to 1.25 U  per reaction.PCR was carried out in 50 μl reaction volume containing 1.5 mM MgClPCR-amplified products were resolved on 6% urea polyacrylamide gel electrophoresis (PAGE) denaturing sequencing gel at 75 W  and visualized by silver staining. Allele sizes were estimated using a 100 bp ladder . The genotype of each individual animal at 25 different loci was recorded by direct counting.et al. [Genotype of each individual animal was determined and recorded from the silver-stained gels for each microsatellite locus. Different measures of within-breed genetic variations, namely number of alleles, allele frequencies, effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), were estimated to evaluate variability at DNA level. PIC for each locus was calculated according to Botstein et al. .The various parameters of genetic diversity in Marwari goat such as allele number, effective number of allele, PIC, observed and expected heterozygosity within population are furnished in et al. [All the loci were amplified successfully and exhibited substantial levels of genetic diversity estimates  and 2. Aet al.  suggesteThe average number of observed alleles found in Marwari goats was comparable with Assam Hill goats, which ranged from 2 to 10 with an overall mean of 4.9 . It was The allele sizes ranged from 125 bp (ILSTS-028 and ILSTS-033) to 650 bp (ILSTS-011 and ILSTS-019). The number of alleles available in literature was 4 for ILSTS059, 6 for ILSTS022, ILSTS065 and ILSTS087, 7 for ILSTS019 and ILSTS028, 8 for ILSTS044 and ILSTS058 in Barbari goats from India . It has 0) ranged from 0.1428 (locus ILSTS-087) to 0.9285 (locus ILSTS-034), with an average value of 0.5485, while the expected heterozygosity ranged from 0.240 (locus ILSTS-005) to 0.681 (locus ILSTS-011), with an average value of 0.544 across the 15 microsatellite markers for the Marwari goats population.The heterozygosity is an appropriate measure of genetic variability within a population because genetic diversity can be measured as the amount of actual or potential heterozygosity. The observed heterozygosity (H0=0.55) observed in the present study are higher than that of in Assam Hill goats, 0.43 [The average genetic variation  obtained in this study was higher than other studies in Barberi goats, 0.6208-0.8509 , and Assχ2) test revealed that 15 microsatellite loci in the Marwari goat population are in equilibrium. These results established that the samples were drawn from the large random mating population.Locus ILSTS-034 exhibits the highest level of observed heterozygosity and ILSTS-087 locus exhibits the lowest observed heterozygosity. The low observed heterozygosity 0.1428 (ILSTS-087) was observed in the present study may be due to the presence of more homozygote individual in the samples analyzed. Though few loci exhibited lower heterozygosity values, most of the loci showed relatively higher expected heterozygosity, which reflects the existence of differentiation in the population. The locus with the highest level of heterozygosity is the most informative locus for Marwari goat breed. The Chi-square  to as high as 1.2057 (ILSTS-019) for polymorphic markers with mean PIC of 0.78096, which is regarded slightly informative (<0.5). Reported PIC values for these markers in other goat breeds have shown that they are well suited for genetic diversity analysis in goats ,26. HoweThe average value 0.78096) of PIC estimated in the present study are comparable with those values obtained in Chinese goat breeds, which ranged from 0.746 to 0.800 8096 of P in Tali Microsatellite analysis revealed a high level of polymorphism across studied microsatellite markers and informativeness of the markers for genetic diversity analysis studies in Marwari goats. This high level of polymorphism can be utilized to plan future biodiversity studies to exploit the uniqueness and adaptability of this breed to Western Rajasthan. Based on the PIC values, the microsatellite primers used in the present study are proved to be highly polymorphic in nature and hence can be well utilized for molecular characterization of Marwari goat germplasm.ASY, KG designed the work plan, collected, processed the blood samples, carried out PCR and electrophoresis. MA helps to carried out PCR and PAGE electrophoresis. GCG and ASY compiled, tabulated, transformed and analyzed the data. MA and GCG interpreted the results. ASY, KG and MLY prepared the manuscript. All authors read and approved the final manuscript.