Plasmodium falciparum parasite population in order to identify foci of transmission, detect outbreaks, and track parasite movement to potentially inform malaria control and elimination strategies.With increasing interest in eliminating malaria from the Caribbean region, Haiti is one of the two countries on the island of Hispaniola with continued malaria transmission. While the Haitian population remains at risk for malaria, there are a limited number of cases annually, making conventional epidemiological measures such as case incidence and prevalence of potentially limited value for fine-scale resolution of transmission patterns and trends. In this context, genetic signatures may be useful for the identification and characterization of the P. falciparum DNA samples extracted from dried blood spots collected from malaria-positive patients reporting to health facilities in three southwestern Haitian departments  in 2016.This study evaluated the genetic signals based on analysis of 21 single-nucleotide polymorphisms (SNPs) from 462 monogenomic (single-genome) Assessment of the parasite genetic relatedness revealed evidence of clonal expansion within Nippes and the exchange of parasite lineages between Nippes, Sud, and Grand'Anse. Furthermore, 437 of the 462 samples shared high levels of genetic similarity–at least 20 of 21 SNPS–with at least one other sample in the dataset.These results revealed patterns of relatedness suggestive of the repeated recombination of a limited number of founding parasite types without significant outcrossing. These genetic signals offer clues to the underlying relatedness of parasite populations and may be useful for the identification of the foci of transmission and tracking of parasite movement in Haiti for malaria elimination. Plasmodium falciparum transmitted mainly in Haiti by Anopheles albimanus mosquitoes . ThePlasmodium populations to help in tracking parasite movement and determining the impact of interventions in shrinking the Plasmodium reservoir. The level of genetic diversity and its distribution could provide insights into parasite transmission and parasite population history. Moreover, genetic variation can be used to uniquely identify parasites that infect individuals in a particular region, establishing clusters of parasite lineages that can be followed over time and space after interventions are applied [In addition to the standard epidemiological measures of program progress, genetic tools offer additional understanding of  applied –8.In this context, the present study assessed the genetic diversity of samples collected during 2016 as a baseline for understanding current population structure in the selected study sites and providing additional information for the future development of a molecular database in Haiti that also includes drug resistance markers. The availability of such a molecular database, with the hope of adding future data collections, may be useful for various programme-related uses, such as tracking parasite movement within the country , evaluation of the efficacy of intervention strategies, and outbreak investigations which help the national programme to apply programme-level applications to progress towards the goal of malaria elimination.Haiti, with an estimated population of 10,788,440 in 2016, occupies the western one-third of the island of Hispaniola located between the Caribbean Sea and the North Atlantic Ocean and shares a border with only the Dominican Republic , 9. The Malaria is transmitted primarily through the rainy seasons in Haiti from May through July and again from October through November. Rainfall is variable, ranging from 400 to more than 3000 mm annually, with the reported incidence of malaria heterogeneously distributed in foci or “hot-spots” primarily in the coastal areas of the Departments located in the southeastern part of the country .The sample sites included four sentinel sites in three departments  from among 11 sites nationwide established for anti-malarial molecular resistance marker surveillance, as described elsewhere [Approximately 200 μL of blood were obtained by fingerprick from each consenting participant, placed on Whatman 903 Protein Saver cards , and allowed to dry overnight before storage in an individual plastic bag with desiccant. To ensure consistency in blood sample collection, all health facility workers had previously been trained on appropriate dried blood spot (DBS) sample preparation. Filter papers were stored at health facilities at room temperature away from sunlight.Genomic DNA was isolated from dried blood spots using QIAamp DNA blood mini kits  according to manufacturer’s protocol except that double the amount of filter paper blood and proteinase K were used. After elution, DNA samples were kept at 4 °C and −40 °C for short- and long-term storage, respectively.P. falciparum molecular barcode genotyping in the 2 × TaqMan PreAmp Master Mix (Applied Biosystems cat# 4391128). The resulting products were cleaned  and used as template in 5-uL TaqMan molecular barcode assays. The genotypes were determined from the raw data reads downloaded from the instrument using an in-house script (available and documented at https://github.com/ndaniels/TaqmanParser).The samples were pre-amplified and genotyped for 24 genome-wide, putatively neutral single-nucleotide polymorphisms (SNPs) using a TaqMan-based barcoding protocol as previously described  on a VIIA total of 648 samples were collected and submitted for genetic analysis. All samples were collected from consenting microscopy or RDT-positive individuals at the clinic sites in Grand’Anse, Sud, and Nippes Fig. . The samMonogenomic 21-SNP barcodes were included in the pairwise comparisons in analysis of identity-by-state (IBS) to identify samples that were identical and highly-related . In thisAdditionally, spatial principal components analysis (sPCA) was performed for comparison to other populations in Central and South America using the adegenet function within the PopGenReport package version 2.0 in R version 3.02  .The number of base differences per sequence from the mean diversity for each site were calculated . StandarThe patient demographics  are shown in Additional file P. falciparum samples collected during 2016 in Haiti for this study, 32 (4.9%) were excluded for 5 or more missing SNP calls. Of the remaining 616 samples, 42 (6.8%) were polygenomic . From the remaining 565 monogenomic (single distinct genome) samples, 462 with complete data for the 21 most informative SNPs were included in the graph analysis  (p < 0.001). In contrast, analysis of the distributions of the number of pairwise differences in 21-SNP barcodes between sites revealed significant differences for all pairwise comparisons, although the magnitude of the difference was greatest between Nippes and the other sites   was 4.881 standard error (SE) 0.824. Comparisons of the genetic diversity between departments revealed similar levels between Grand’Anse and Sud , with significantly lower genetic diversity in Nippes  were identical or highly related to at least one other sample . The relationships between these samples were then graphed. As shown in Fig. The distribution of the number of nodes in each site was as follows: Grand’Anse 26, Nippes 9, and Sud 17. Grand’Anse had two collection sites, Sainte-Hélène (n = 155) and Roseaux (n = 51) with 25 and 10 barcode-identical nodes, respectively. Although the number of samples from each site differed, the same node (N2) was dominant in both sites. This node was unique to Grand’Anse except for one sample found in Sud, which also had the same barcode profile. Additionally, nodes were shared between Grand’Anse and Sud (N2), Grand’Anse and Nippes (N9), Nippes and Sud , and all three sites (N1 and N5). Only 9 nodes were observed in Nippes and 95% of the samples from this department belonged to the same node (N1), which was also included samples from Grand’Anse and Sud , with the next-highest diameters of 2–3.In 2016, an estimated 21,998 cases of malaria were reported in Haiti among a population of 10.8 million . The goaP. falciparum parasite population, to identify foci of transmission, detect outbreaks, and track parasite movement [In addition to the traditional markers of transmission such as epidemiological signals, genetic analysis offers additional information about parasite drug resistance as well as the underlying parasite population structure. Genetic signals can be used for the identification and characterization of the movement , 19.P. falciparum parasites are not commonly imported from neighbouring countries to Haiti and that the residual ancestral Haitian P. falciparum population is responsible for current transmission of malaria.The present study analysed more than 600 samples collected in 2016 from three Departments in Haiti as part of an effort to characterize the parasite population in these regions. Based on its barcodes, Haiti’s parasite population was distinct from those of neighbouring countries in Central and South America, indicating that The low proportion of polygenomic infections supports the historically relatively low transmission rates reported in the country ; howeverThe results also revealed similar and low levels of genetic diversity between Grand’Anse and Sud, each with a number of individual barcode-identical nodes. Nippes, however, had reduced genetic diversity compared to those of the other two sites, with an expanded node of barcode-identical parasites. Although the parasites were similar within Haiti, they were distinct from those sampled from South and Central America.The overall low diversity in this population is suggestive of repeated, long-term inbreeding of parasite types or the expansion of a single clone or limited number of lineages due to differences in reproductive success due to host or vector-related factors, host or vector immune invasion, or other stochastic factors in this region.While the genetic diversity (pi) was comparable between Sud and Grand’Anse, the number of pairwise differences differed significantly between Departments, with a lower mean number of differences in Grand’Anse (2.1077 ± 0.7743) than that in Sud (3.4645 ± 0.9596), suggesting differences between these parasite populations such as focal transmission (hotspots) or higher levels of crossover of parasite types, respectively.In addition to aggregate statistical analysis, graph analysis revealed trends in the sample population consistent with long-term population inbreeding. The high proportion of related nodes within samples from Grand’Anse, Nippes, and Sud suggested that residual ancestral Haitian parasites primarily contribute to malaria transmission in this country. However, the limited number of parasites more distantly or not genetically related to these nodes may also themselves be informative. For example, they may represent evidence of local adaptation or evolution of parasites, which may become newly established lineages within the country over time.The graph property of betweenness centrality revealed only one component . This prP. falciparum in Haiti [. also observed low levels of population structure; however, they also reported high levels of genetic diversity and a lack of evidence of recent parasite population bottlenecks or increased inbreeding, in contrast to the findings of the present study. However, this difference may be due to differences in the types of markers used (microsatellites versus SNPs in the present study) [The present study used genomic data from a set of SNPs to assess both aggregate data (genetic diversity and pairwise distances) as well as individual data  to assess the characteristics of the parasite population in this region of Haiti. Previous studies have also used different genomic markers (microsatellites) to characterize the population structure of in Haiti , 22. Thet study) .Pfcrt, the genetic marker associated with resistance to chloroquine, the drug primarily administered in Haiti [Pfdhfr S108N (associated with antifolate resistance) was found in 47% of the samples. Other unknown parasite characteristics could have contributed to the differences in relative fitness of this parasite type, including those related to human hosts and mosquito vectors. Finally, this finding may simply be a stochastic event in which a single parasite type is propagated throughout the population. Previous studies have made similar observations of clonal types in other countries in which P. falciparum transmission was reduced to low levels, including Peru, Ecuador, and Thailand [While some of these discordant findings may be due to differences between studies, including study sites, collection years, patient demographics, sampling and genetic markers used, to our knowledge, few studies have reported a very large number of highly related parasites as was observed in Nippes. There are several possible explanations for this finding. First, this could indicate the emergence and spread of a specific parasite type more reproductively successful than other parasites in the population, such as the emergence of drug resistance or reduced drug sensitivity. However, a previous study monitoring drug resistance alleles in these samples  showed tin Haiti . AdditioThailand –25.The limitations of this study include potential biases due to the passive case detection and the limited number of sites, which prevent generalization of the results even within Departments without additional sampling. Moreover, not all available samples were included in the analysis.In addition, epidemiological data, particularly longitudinal data, would be useful to place these genetic patterns in context with incidence trends and to differentiate stochastic population effects from those caused by control efforts. Furthermore, evaluation of these samples using technologies other than SNP genotyping of a limited number of markers, including whole-genome sequencing or microsatellite typing, are warranted in future studies to potentially differentiate sub-sets of samples with lower levels of relatedness or shared regions of the genome and those evolving on a shorter evolutionary timescale.P. falciparum genetic diversity throughout Haiti will improve the field’s understanding of the parasite population structure and help to track movement of parasites as malaria elimination efforts progressively reduce ongoing malaria transmission in pursuit of malaria elimination in this region. These data are informative for the development of a molecular database in Haiti that includes drug resistance and other molecular markers from sites nationwide. This database, with the addition of data from future collections, may be useful for program-related activities such as tracking parasite movement within the country that could inform the prioritization and sequence of high transmission areas to be targeted, evaluation of the efficacy of intervention strategies, and outbreak investigations to understand the epidemiology and inform a response.The use of the population genetic approaches relies on the routine collection of parasite genetic data since the discriminatory power of the genotyping method depends on the local epidemiology and transmission setting. Since genetic signals can change over time, continued characterization of Population genetic data can inform these targeted control efforts, particularly when resources are limited. For instance, surveillance based on the genetic relatedness of parasites may show the emergence of a highly related or clonal population, as observed in the present study in Nippes. This genetic evidence is suggestive of local transmission, for which vector control efforts would likely be most effective. In contrast, a lack of clonality or the presence of higher proportions of polygenomic samples may suggest importation or a mobile or human population for which mass test-and-treat or other (human) drug-based measured may be warranted.The selection of appropriate tools for genetic and genomic analysis is also based on the main goals of the national control programmes as well practical concerns such as budget. While declining costs for whole-genome sequencing appear to make this technology more attractive, as it offers ‘all the data’, the infrastructure required to support instruments and to perform analysis remain a hurdle in many settings, especially those interested in near-real-time data analysis for decision making. Furthermore, the whole-genome sequencing analysis tools for polygenomic samples require additional development to provide quantitative data on the number of genomes or the predominant alleles present in a sample. The fine-scale genetic relationships revealed by whole-genome sequencing are best suited for contexts with relatively fewer cases such that reactive case detection and follow-up can reveal more distantly related samples to track parasite movement and inbreeding across regions.Thus, there remains a role for technologies such as the molecular barcode and microsatellite typing as more general and more easily accessible tools to identify larger-scale changes in parasite populations warranting further investigation and resources for infection control and elimination. Both SNP genotyping as used by the molecular barcode and microsatellite typing offer comparable information, although microsatellites vary on different evolutionary timescales due to their higher rates of mutation  which, dThese programmatic considerations and uses of parasite genomic information will become more important as the number of malaria cases decline, corresponding with a decrease in genetic diversity, as the country moves towards elimination.Additional file 1: Table S1. All barcodes, samples, cluster numbers for monogenomic and polygenomic sample.


Anastrepha fraterculus (Diptera: Tephritidae) is a major agricultural pest attacking several fruit commodities. This impedes international trade and has a significant negative impact on the local economies. Given the importance of sterile male-only releases, the development of a GSS for A. fraterculus would facilitate the implementation of an efficient and cost-effective SIT operational program against this insect pest species.Area-wide integrated pest management programs (AW-IPM) incorporating sterile insect technique (SIT) have been successful in suppressing populations of different fruit fly species during the last six decades. In addition, the development of genetic sexing strains (GSS) for different fruit fly species has allowed for sterile male-only releases and has significantly improved the efficacy and cost effectiveness of the SIT applications. The South American Fruit Fly A. fraterculus sp. 1, including the black pupae (bp) gene located on chromosome VI. The black pupa phenotype was used as a selectable marker to develop genetic sexing strains by linking the wild type allele (bp+) to the Y-chromosome -via irradiation to induce a reciprocal Y-autosome translocation. Four GSS were established and one of them, namely GSS-89, showed the best genetic stability and the highest fertility. This strain was selected for further characterization and cytogenetic analysis.For potential use in a GSS, three new morphological markers (mutants) were isolated in a laboratory strain of A. fraterculus sp. 1, using as a selectable marker the black pupae genetic locus.We herein report the development of the first genetic sexing strain of a major agricultural pest,  The sterile insect technique (SIT) is a species-specific and environmentally friendly genetic method to control populations of major insect pests. This method involves the rearing of the target pest species, the induction of lethal mutations and atrophy of reproductive organs to induce reproductive sterilization through the exposure to ionizing radiation, in the hope that the release of sterile insects in the wild and their mating with the wild population will result in infertile eggs .The possibility of sterile male-only releases in some species has made the SIT application more efficient and cost effective in several ways. As the probability of mating between sterile males and wild females is increased, the damage of fruits due to the stinging by sterile females is avoided and, moreover, the overall costs associated with releasing and monitoring are drastically reduced , 3. MaleCeratitis capitata being the model species  were developed and evaluated in respect to their genetic stability and productivity. From this, the strain designated as GSS-89 was chosen as being the most genetically stable and productive. As the selection is based on the pupal color, using this strain a robust sex separation system can also be established by using a color sorting machine. This would allow for male-only releases and would greatly facilitate the development and implementation of large scale operational SIT programmes against this important pest in South America.The present study reports on three novel morphological mutations in A. fraterculus sp. 1 population at the Insect Pest Control Laboratory (IPCL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Seibersdorf, Vienna, Austria . The Tukey’s HSD test was used as a post-hoc method to compare means between strains on significant factors. In order to normalize the data distribution and stabilize the variances, the data in percentages were transformed following csinex+1 . All dat


Our ddRAD dataset showed three genetic clusters in the sampled Canadian range, congruent with previous studies based on microsatellites across the same regions; however, due to a lack of sampling in Norwegian Bay, we were unable to confirm the existence of a unique cluster in that subpopulation. These data on the genetic structure of polar bears using SNPs provide a detailed baseline against which future shifts in population structure can be assessed, and opportunities to develop new noninvasive tools for monitoring polar bears across their range.Predicting the consequences of environmental changes, including human‐mediated climate change on species, requires that we quantify range‐wide patterns of genetic diversity and identify the ecological, environmental, and historical factors that have contributed to it. Here, we generate baseline data on polar bear population structure across most Canadian subpopulations ( With ongoing rapid sea ice loss and environmental change in the Arctic, there is the potential for rapid changes in polar bear population structure. Here, we generated baseline data on polar bear population structure across 12 of 13 Canadian subpopulations using genome‐wide SNPs. These data on the genetic structure of polar bears using SNPs provide a detailed baseline against which future shifts in population structure can be assessed, and opportunities to develop new noninvasive tools for monitoring polar bears across their range. Predicting how species might respond to future environments requires that we identify the factors that have shaped present‐day intraspecific genetic structure  population structure has emerged over time through population genetic studies based on mitochondrial sequences and microsatellite genotypic data  sequencing. We compare our results based on analyses of the ddRAD dataset to previous studies of polar bear population structure.22.1The pan‐Arctic geographic range of polar bears is divided into 19 subpopulations or “management units” . Only extractions with high molecular weight DNA were used for library preparation. Extracts were quantified using a NanoDrop ND_1000 Spectrophotometer (NanoDrop Technologies Inc.).Genomic DNA was extracted from these samples using a modified salt extraction protocol . Briefly, the script removes duplicates when the degenerate region in the P2 index matches between reads to ensure PCR duplicates are removed. Once this is done, libraries were demultiplexed using the “process_radtags” tool included as part of Stacks v2.2  [PMID: 24813606] using the BWA‐MEM v0.7.17 aligner  were used in a second round of “bcftools mpileup” for all individuals.Finally, the alignments were used to call SNPs. To reduce the computation time, we used an initial representative group of 327 individuals from the first set of libraries sequenced for variant detection using BCFTOOLS v1.9 .Filtering of the resulting vcf files was done in two rounds using VCFTOOLS v0.1.15 , with ten iterations of each, using a run length of 300,000 Markov chain Monte Carlo replicates after a burn‐in period of 100,000 and correlated allele frequencies under an admixture model with alpha set to 0.5. The most likely number of clusters was determined by plotting the log probability of the data (ln Pr(X|K)) across the range of K values tested and selecting the K value where the value of ln Pr(X|K) plateaued, as suggested in the STRUCTURE manual, and by calculating the deltaK statistic . The chosen value of K was based on the minimum number of clusters after which the BIC decreased by a negligible amount.Evidence for population substructure was assessed using multiple methods. We used Bayesian clustering analysis, as implemented in STRUCTURE 2.3.4  to evaluate the proportions of total genetic variation that were contained within and among subpopulations, and within and among the genetic clusters suggested by population structure analyses.bed2diffs_v2, which imputes missing data based on the average genotype for that locus. We ran EEMS initially using the default values for proposal variances and other parameters and changed the values in subsequent runs until the frequency of accepting proposals of each type was between 10% and 40%, as suggested in the software documentation. Using the optimized set of parameters, we ran five independent chains for 10,000,000 MCMC iterations, with a burn‐in of 2,000,000, and thinning every 9,999 iterations. We assessed convergence of these chains and plotted the combined EEMS results using the R package rEEMSplots. The resulting contour plot was overlaid with a map of Canada using the R packages rworldmap and rworldxtra  for STRUCTURE plateaued around K = 3 , we cannot confirm its existence.In this study, we used thousands of genome‐wide SNP markers to assess population structure across the range of polar bears in Canada. Our results clearly show differentiation within Canada, and the membership patterns and geographic ranges of the “Hudson Complex,” “Arctic Archipelago,” and “Polar Basin” genetic groups match closely with the results of previous studies using microsatellite loci  and the associated differences in polar bear life history  and Baffin Bay (BB). Although imperfect, our ability to correctly assign individual bears to the subpopulation of origin may have management implications . Levels of genetic diversity, as measured by observed and expected heterozygosity, do not vary markedly across the subpopulations  studies has been minimized in favor of aerial genetic biopsy CMR to estimate abundance . New methods using single nucleotide polymorphism (SNP) markers may allow a higher rate of successful genotyping (Campbell, Harmon, & Narum, 5Within the Canadian range of polar bears, subpopulation structure is present and has been consistently recovered across datasets and genetic markers. There is congruence between our results based on thousands of genome‐wide SNPs and previous studies using 16–21 microsatellite or ~3,000 SNP loci. Our samples have not been included in any previous studies (except for three which were also used by Peacock et al., None declared.E.L.J., P.V.C.D.G., and S.L.C. conceived and designed the study; M.B. and M.D. facilitated access to the tissue samples and provided insight on polar bear ecology and management; E.L.J., C.T., R.C.C., and K.M.H. collected genomic data and performed analyses; and E.L.J., C.T., and S.C.L. wrote the first version of the manuscript with all authors contributing to revisions.Supplementary MaterialClick here for additional data file.Table S1Click here for additional data file.


Apis nigrocincta, a native bee species from Sulawesi and the Philippines, plays a vital role in pollinating flowering plants in local ecosystem and agriculture. In this study, we assessed the intraspecific genetic variation of A. nigrocincta using the sequence of cytochrome c oxidase subunit I (COI). Molecular phylogenetic analysis showed that there were three main clades in A. nigrocincta specimens from Sulawesi based on their respective locations . Genetic distance analysis using the Kimura 2-parameter (K2P) model showed that the intraspecific genetic distance in Sulawesi specimens ranged from 0.000 to 0.055. There are 26 nucleotide polymorphic sites within Sulawesi A. nigrocincta. The variation was dominated by transition T ↔ C. The molecular identification result was supported by morphological identification. The results of the two methods agree that the specimen under study was A. nigrocincta. The result of genetic distance calculation indicated that although the tested specimens were derived from remote locations, the genetic variation was still within the range of intraspecific variation.Asian cavity-nesting honey bee  Apis. These insects play a significant role in the pollination of important crops. Currently, there are nine species of honey bees known to inhabit the world: Apis dorsata, A. laboriosa, A. mellifera, A. florea, A. andreniformis, A. cerana, A. koschevnikovi, A. nigrocincta , and A. nuluensis , with approximately 44 subspecies [There are about 30,000 bee species distributed worldwide. About 17,000 of them have been described . Apidae,A. nigrocincta has been poorly studied. In recent years, molecular methods have been applied to study intraspecific genetic variation, species identification, and phylogenetic relationship among close-related taxa [A. nigrocincta to facilitate species identification and analyze their genetic distances.The honey bees are prone to extinction due to their haplodiploid sex-determining mechanism, limited population because of anthropogenic activities , and lowted taxa . One tected taxa . For aniSphagneticola trilobata (L.) Pruski, Tachytarpheta jamaicensis (L.) Vahl., and Mikania micrantha Kunth.The adult worker bees were captured using a swingnet from an abandoned local garden in Mahakeret village, city of Manado, North Sulawesi province, Indonesia (1°28'53”N and 124°50'21”E). The location was situated at an altitude of about 40 meters above sea level. The average temperature at sample collection place (March 2019) was between 29°C and 32°C with humidity of 85–89%. The percentage of vegetation cover was 80%. The vegetation consisted mainly of bushes, grasses, and wild flowers such as The observed morphological characters included length of following parts: head (HD), antenna (AT), proboscis (PB), thorax (TO), abdomen (AB), fore-wing (FW), hind-wing (FL), midleg (ML), and hind-leg (HL) using a caliper .Total DNA was extracted from coxa connected to the abdomen using ZR Tissue & Insect DNA MiniPrep™ (Zymo Research). The genomic DNA was cleaned with DNA Clean & Concentrator™-5 (DCC™-5) (Zymo Research) for the generation of high-quality DNA for PCR. The DNA barcode of COI region was amplified using Toyobo KOD FX Neo PCR Master Mix with primer pairs LCO1490 and HCO2198 . The conhttp://www.ncbi.nlm.nih.gov). Identification was performed using BLAST identity search provided by the same platform. The clean sequences were aligned using Clustal O (1.2.1) multiple sequence alignment  with other allied honey bee COI sequences from different parts in Sulawesi retrieved from GenBank. The evolutionary history was inferred by the Maximum Likelihood (ML) method based on the Kimura 2-parameter (K2P) model [Chromatograms were subjected to the procedure as was done previously . The cleP) model . EvolutiP) model .A. nigrocincta provided by Hadisoesilo et al. [The photograph of honey bee specimen obtained from Manado Mahakeret is shown in o et al. . The moro et al. .A. nigrocincta CAL01 has been deposited in GenBank with accession number MK880239. The complete BLAST search is presented in The cytochrome oxidase I (COI) sequence of A. nigrocincta specimens. Estimation of genetic distance amongst A. nigrocincta is shown in A. nigrocincta. Variation of intraspecific COI gene showed that transition T ↔ C dominated the polymorphic pattern.The molecular phylogenetic analysis by the ML method based on the K2P model is shown in v was equal to 100. The number of transition substitution was 86.89, while the number of substitution was 13.11. Therefore, the transition/transversion ratio (ti/tv) was 6.63. However, sampling size strongly influences the ti/tv. The estimated maximum likelihood ti/tv bias (R) was 9.05 under the K2P model [Estimation of substitution using the maximum composite likelihood method can be seen in Apis nigrocincta . This phenomenon was supported by studies conducted by Damus and Otis [A. cerana and A. cerana japonica were confirmed as being distinct from the rest of A. cerana. This indicates that Apis nigrocincta found in Pakistan was most likely the part of the group A. cerana japonica. Another finding showed that A. nigrocincta from Sulawesi mainland and Sangihe Island were embedded in the A. cerana group [A. nigrocincta had similarities with A. cerana [The specimen KY834222 from Islamabad Pakistan was observed to be out of the Sulawesi group. In the phylogenetic tree created by involving its sister species, and Otis  which stna group . The phy. cerana , 22.A. nigrocincta maintained a high specific genetic diversity on Sulawesi Island.The closest genetic distance was 0.000, between haplotype CAL01 and DQ020233 from Bogani Nani Wartabone National Park, Gorontalo (Indonesia). The farthest genetic distance was 0.082, between haplotype CAL01 and KY834222 from Islamabad (Pakistan), and 0.052 with DQ020223 from Bantimurung, South Sulawesi (Indonesia). The genetic distance was 0.055 between NC038114 from Sangihe Island North Sulawesi (Indonesia) and DQ020223. With this value, Takahasi et al. [A. mellifera in 10 locations across China and Pakistan was 0.039 [A. cerana was detected between Flores' and Taiwan's haplotypes [The result of genetic distance calculation indicated that although the tested specimens were derived from remote locations, it was still within the range of intraspecific variation, except with specimen from Pakistan. Maximum pairwise distance of as 0.039 . The resplotypes . The cloplotypes . Differeplotypes , 27. Theplotypes , 29. Theplotypes , 31.A. nigrocincta. However, six polymorphic sites were detected in Bombus koreanus in China [Apis mellifera in United States [A. cerana in India [No information was provided earlier on the polymorphic sites of in China  and Apisd States . The train India . Due to in India .A. nigcrocinta group from different locations in Sulawesi has been demonstrated in this study. This molecular marker was also able to identify A. nigrocincta successfully based on the similarity sequence with other A. nigrocincta specimens. This finding provides valuable information on the effective use of COI as molecular marker in research on phylogenetic studies.The ability of COI gene to differentiate between A. nigrocincta using COI gene sequences. The results showed that there were three main clades of Sulawesi specimen, namely, North, Central, and South Sulawesi. This finding suggested that genetic variation in A. nigrocincta in Sulawesi is still in the category of intraspecific variation.The current study assessed the intraspecific genetic variation in


The influence of the drying technique on certain quality attributes of dried grapes was analysed. Five varieties of grape were used in this study . All the varieties were dried using four methods: drying at 40 °C, combined drying , drying at 70 °C, and lyophilisation. The quality attributes such as total soluble solids, water activity, content of vitamin C, the colour parameters , and microbiological  and sensory  analysis were determined, depending on the method of drying. Lyophilised grapes from the Jakubské variety had the highest vitamin C content. They contained 0.58 mg of vitamin C per 100 g of dried grapes. However, no significant differences between varieties in vitamin C content were found. This confirmed concerns about the negative impact on the nutritional quality of dried grapes with increasing temperature. The lowest total count of microorganisms (CFU), moulds, and yeasts were determined after using highest drying temperature (70 °C). Higher CFU values were determined in lyophilised grapes, and then followed by grapes dried at 40 °C. Lyophilised grapes and grapes dried at 40 °C achieved the best sensory rating in general. The final product was intended to have a unique appearance while maintaining the evaluated quality parameters. Consumption of grapes and raisins dates to prehistoric times, which is proven by rock paintings from the Mediterranean region . It is aGrapes and products made from grapes are among the most important horticultural products in the world. They are grown on all the inhabited continents on Earth ,4,5,6. ADrying is one of the oldest and simplest methods of food conservation. It is a gentle method of preservation by which beneficial aromatic and content properties of the food are retained. After drying, food gains a longer storage life, becomes lighter, and its volume decreases ,9,10. GrDried grapes have valuable nutritional properties because of high mineral, vitamin, and other bioactive substance content. Therefore, current research indicates that grape consumption has many health benefits such as the reduction of the risk of cancer, type 2 diabetes, stroke, obesity, cardiovascular diseases, and other chronic diseases ,10,14,15Dried grapes are excellent sources of health-promoting bioactive compounds, but their quality can be negatively affected during drying. The aim of this study was to compare several drying techniques and decide, whether there are differences between the drying techniques in selected nutritional (vitamin C content), microbial, and sensory quality parameters for five varieties typical from the region.Five varieties of grapes were used in this research: Jakubské (JA), Beauty seedless (BT), Vrboska (VR), Bezsemenné (BS), and Perlette (PE). The varieties are described in For the experiments, 7 kg of ripened grapes on the bunch stem from each variety was carefully taken and randomly divided equally among each method of drying.Before drying, samples were rinsed under running tap water, and the bunch stems were not separated from the berries, so the entire bunch of grapes was dried. The grape bunches were dried using 4 methods: three with air at 40 °C, at 70 °C and using a combined method  in an SO 1070 fruit drier , and one method using lyophilisation at a temperature of −42 °C and a pressure of 0.22 mbar using an Alpha 1–2 LD Plus lyophilising machine .w = 0.6. Water activity was determined using an electrolytic sensor on the LabSwift-aw device  in triplicates and with a sensor stabilisation time of 6 min. The preparation of dried samples included cutting to pieces of diameter about 4 mm.The samples were dried until they reached the water activity value of apH was determined on fresh grapes in triplicates at a temperature of 25 °C using a Portavo 907 MULTI pH meter  equipped with a puncture probe.Total soluble solids content (TSS) was measured in the manually pressed juice from the fresh grapes using a pocket optical refractometer , with the procedure repeated three times at a temperature of 25 °C.Vitamin C content was determined on 10 g of crushed, homogenised, and filtered samples of fresh and dried grapes in duplicates using a solution of oxalic acid. Vitamin C was determined using a 1260 Infinity II liquid chromatograph  on a Luna Omega Polar C18 column, 5 µg, 250 × 4.6 mm . Oxalic acid (0.005 M) was used as a mobile phase and the flow-through rate of the mobile phase was 0.8 mL per minute. Ten microlitres of the sample was injected. The concentration of vitamin C was determined using a DAD (Diode-Array Detection) detector at a wavelength of 254 nm using a calibration curve. The calibration curve points were 0.1, 0.2, 0.5, 1, and 10 µg/mL. The limit of detection was 0.01 µg/mL and the limit of quantification was 0.02 µg/mL. The measurements were taken at a temperature of 25 °C.ab in the following formula) from fresh berries to dried raisins was computed using the equation, where parameters with subscript L*0, a*0, b*0 are values for the fresh grapes and L*1, a*1, and b*1 are values of the dried raisins, respectively. Overall total colour change ΔE*ab is an important, widely accepted method of evaluating the significance of colour difference [Instrumental colour measurement was determined on fresh and dried raisins. Colour was measured using a CM 3500d spectrophotometer , which was connected to a computer with CMs-100w SpectraMagic NX software . Colour was measured by using the CIE L*a*b* tristimulus colour space at two different sides of the grape berry. Grape colour measurements were performed in triplicates on 10 berries of grapes, on a slot with a measurement area of 8 mm, specular component excluded (SCE), and an observer with an angle of 8°, CIE standard illuminant D65. Results were expressed as L*, a*, and b* values (CIELab coordinates). The L* represents lightness (100 for white and 0 black), chromatic coordinate a* represents the colour space between red (+a*) and green (−a*), chromatic coordinate b* represents the space between yellow (+b*) and blue (−b*). The total colour change or difference . The following descriptors were evaluated: smoothness, gloss , colour, aroma, chewability, juiciness , taste (hedonic: pleasantness of taste) and overall impression (hedonic). Sensory analysis was performed in two sessions.Single-factor ANOVA with standard deviation, weighted average, and Tukey’s HSD test were used for statistical evaluation using STATISTICA 12 software . Principal component analysis (PCA) on correlation matrices with the Varimax rotation was used to find the relationships between vitamin C content, sensory descriptors, instrumental colour attributes, and water activity among raisins of different varieties and drying treatments .p < 0.05) pH values than the grapes of Bezsemenné and Jakubské varieties. Grapes of the Vrboska variety had significantly higher TSS content than those of the Perlette variety, which had the lowest TSS content (The Perlette variety had the highest pH (3.42), and the Bezsemenné variety had the lowest pH (3.14). It was found that the grapes of Perlette, Vrboska, and Beauty seedless varieties had significantly higher  belonged to the genus Aspergillus. According to Hakobyan et al. [2. This is an effective preservative and has a significant inhibitory effect on the development of moulds during drying and storage, including the species producing toxins. In this study, the grapes were not treated with any preservative, which can be an explanation for the more extensive development of moulds in lyophilised grapes and dried grapes at 40 °C. Zemni et al. [Hakobyan et al.  analysedi et al.  analysedp ˂ 0.05 was found between the samples.After yeast enumeration, it was established that the samples contained much higher numbers of yeasts than moulds , and lyoThe total number of microorganisms in dried grapes of different varieties and drying methods is shown on It can be concluded that higher temperature during drying had a more detrimental effect on total number of microorganisms, moulds, and yeasts in dried raisins.McCoy et al.  examinedp ˂ 0.05) depending on the colour parameter.Assessment of surface colour for quality assessment is a common practice in dried grape production. Evaluation of dried grape berries is mostly based on visual assessment by trained humans, a process that is subjective. To eliminate errors, it is necessary to combine the results and supplement them with instrumentally based methods with an effort to objectify the results, such as colour measurement . Colour ab was more pronounced in white grapes varieties. The lowest total colour change (ΔE*ab = 2.24) was in the Jakubské grape variety, while the other blue variety Vrboska had very prominent colour difference after lyophilisation. It can be concluded that white grape varieties were characterised by higher colour change after drying, than the blue or pink grape varieties.The white grape varieties, Perlette and Bezsemenné, had increased L* and b* values especially after lyophilisation. This was not the case with the pink variety (Beauty seedless). The colour change ΔE*Just like sensory analysis, instrumental colour may not be essential for expressing differences depending on the processing method and expression for overall quality. The overall quality of food must be co-created by comprehensive results, and it is therefore desirable to create an appropriate combination of available methods for food evaluation ,30. It iThe appearance, texture, taste, and odour, along with the nutritional value, are properties that have the greatest impact on the consumer when choosing a food product. The results of sensory analysis are shown in In general, we can state that grapes dried using lyophilisation had the best results for evaluation of the smoothness of the surface. There is growing interest in drying using lyophilisation in the food industry, because these products have noticeable quality compared to other methods of drying. Lyophilisation takes place at low temperatures, which maintains flavour, colour, and appearance. This also minimises thermal damage to heat-sensitive nutrients. Because the entire process takes place in a solid state, shrinkage and other changes to structure are reduced substantially . This faAccording to many studies, a linear relationship between wrinkling, the degree of moisture, and temperature has been observed during drying. The reason for this is the fact that drying leads to changes to the food on a micro-structural level, and this subsequently influences its macroscopic properties. Loss of water and segregation of components cause damage and disruption to cellular walls and even disintegration of cellular tissue. In general, these changes are linked to a reduction of the volume of the product . Angulo p < 0.05. The brown colour of dried grapes originates from the effects of enzymatic reactions (the effects of the polyphenol oxidase enzyme) and non-enzymatic browning (Maillard reaction). The colour of dried raisins is subsequently darker than the colour of fresh grapes [The colour of dried grapes was generally rated positively, lyophilised grapes were again rated with higher values. The Jakubské variety was also highly rated when dried by combined methods and at a temperature of 40 °C. Within most methods of drying, a downward tendency was noticed from samples of the Jakubské variety through Beauty seedless, Vrboska, and Bezsemenné to Perlette, which scored smaller values. When evaluating the colour, no statistically significant difference was found at a level of significance of h grapes ,29. Cherh grapes  stated th grapes  noticed,When evaluating odour, it is not clear which method of drying or which variety was better, because the results were very similar. According to the assessors, the lyophilised grapes of the Jakubské variety had the most intense aroma (79.9). The grapes of the Bezsemenné variety dried at 40 °C were rated as the second most intense odour (75.8). No statistically significant differences between the samples were found. According to literature, there are three ways that aromatic substances can originate in the dried grapes. Most of them come from the fresh grapes, other metabolites such as furans and pyrazines are formed during drying as a result of the Maillard reactions, and the third is origination of aromatic substances  by oxidation of unsaturated fatty acids .Lyophilised grapes of the Jakubské variety had the highest chewability followed by grapes of the Beauty seedless variety dried at 40 °C. Raisins from the Vrboska variety dried at 70 °C were rated with smaller values. Grapes dried at 70 °C were generally rated as difficult to chew and tough. Xiao et al.  examinedThe juiciness of the grapes was generally rated uniformly, but the Beauty seedless variety dried at 40 °C had the highest value; in contrast, grapes that were nearly totally dry and sticky were grapes from the Vrboska variety dried at 70 °C.According to the assessors, the Bezsemenné variety dried at 40 °C had the most intense taste, followed by lyophilised grapes of the same variety. Grapes of the Jakubské variety dried at 70 °C had the lowest score, meaning the most unpleasant and atypical taste. No statistically significant differences in taste between drying treatment within varieties were found. Taste and odour are considered important attributes of quality, which, along with appearance, influence the saleability of the product. While volatile components influence both taste and odour, non-volatile compounds only influence taste. Odour and taste are probably the parameters that are the most difficult to determine because they are very subjective. Their evaluation mostly depends on the consumer’s personal preferences as stated by Khiari et al. .Angulo et al.  examinedAt the end, the assessors were also required to record the overall impression in a sensory questionnaire. Generally, it was established that overall impression values correspond with the taste values. The Beauty seedless variety dried at 40 °C had the best overall impression, immediately followed by the Bezsemenné variety prepared by lyophilisation. The Jakubské variety dried at 70 °C scored the smallest values. No statistically significant differences in overall impression were found between drying treatment within varieties.In general, it can be stated that panellists rated samples of raisins dried at 40 °C and lyophilised samples with higher scores. Samples of grapes dried at the highest temperature, 70 °C, were rated with smaller values. Angulo et al.  found thw value. PC2 was mostly defined by sensory variables, although this link was weaker because of smaller squared cosines. Nevertheless, PC2 was correlated with overall impression, taste, and juiciness on the positive side, and with colour on the negative side. It was found that sensory traits such as chewability, odour, and gloss on one hand, and the content of vitamin C on the other had low impact on raisin characterisation. As expected, overall impression was close to taste, meaning high correlation. Although those traits had high contribution to PC2 definition, none of the observations were close to them. Therefore, it can be concluded that the taste and overall impression were not key determinants of any variety or drying treatment.The principal component analysis (PCA) was conducted with the aim of determining the most suitable variables for elaboration and description of dried raisins. The first two principal components (PCs) accounted for 50.82% of variance (PC1 = 30.06%, PC2 = 20.76%; There was no clear separation of drying treatments and grape varieties, and a lot of overlapping was observed . HoweverThis study examined the impact of drying methods on the quality of five varieties of grape dried at different temperatures or lyophilised on the bunch stem, it could affect processing companies. We can state that sensory traits of lyophilised raisins were rated with higher values, followed by grapes dried at 40 °C. Raisins dried at the highest temperature, this being 70 °C, scored smaller values. Similar results were achieved when evaluating vitamin C content, where the highest content was recorded in lyophilised grapes and in grapes dried at 40 °C, while the lowest content was found in grapes dried at 70 °C. No significant differences between varieties in vitamin C content were found. With regard to microbiological analysis, the results showed the opposite trend to sensory evaluation and vitamin C content, which means that the lowest level of contamination by microorganisms was recorded in grapes dried at 70 °C, which was expected with regard to the fact that the grapes were not treated with any preservative. Nevertheless, lyophilisation as a gentle method for vitamin C content affected microbial quality, especially count of moulds and yeasts. PCA analysis revealed that colour traits, smoothness, juiciness, taste, and overall impression were the most important variables for principal components definition. Although observations were not clearly separated, several conclusions could be stated, which corresponds to the discussion on the analysis performed. A pattern of attribute change with increase in temperature was perceived, from visual appreciation at lyophilisation temperature, through juiciness at mild temperatures, to odour and colour appreciation at higher temperatures. All selected varieties have achieved adequate quality parameters. Varieties Vrboska and Bezsemenné appear to be the positive rated.


Extraordinary states of highly localised pressure and temperature can be generated upon the collapse of impulsively driven cavities. Direct observation of this phenomenon in solids has proved challenging, but recent advances in high-speed synchrotron radiography now permit the study of highly transient, subsurface events in real time. We present a study on the shock-induced collapse of spherical cavities in a solid polymethyl methacrylate medium, driven to shock states between 0.49 and 16.60 GPa. Utilising multi-MHz phase contrast radiography, extended sequences of the collapse process have been captured, revealing new details of interface motion, material failure and jet instability formation. Results reveal a rich array of collapse characteristics dominated by strength effects at low shock pressures and leading to a hydrodynamic response at the highest loading conditions. Subsequent investigations revealed that, if the collapse occurs near a solid boundary, a jet forms which impacts the solid boundary3. For both the jetting and non-jetting cases, deterioration of the propeller surface is caused by the accumulation of the actions of many individual collapsing cavities. In the modern context, cavity collapse plays an important role in several applications, including the in-situ destruction of kidney and gall stones through shock lithotripsy5, targeted drug delivery8, and the cleaning of surfaces exposed to ultrasonic fields in liquids11.The importance of the cavity collapse phenomenon was first recognised by Lord Rayleigh, among others, who in 1917 attempted to explain the progressive damage experienced by screw propellers in water. By assuming the cavity is sufficiently far from the propeller surface, Rayleigh analytically modelled a spherical void collapsing under hydrostatic pressure. The results revealed an extraordinary increase in pressure at the instant of collapse, far in excess of the strength of materials used at the time14. Although more stable under ambient fluctuations, these cavities may be driven to collapse under intense pressure waves, such as those produced under shock loading. Depending on the magnitude of the shock relative to the strength of the solid, the dynamics of the collapse process vary significantly. For the strongest shocks, hydrodynamic collapse is expected15, and energy is localised during the formation of a high-speed jet and toroidal vortex that forms after jet impact16. Other mechanisms become dominant at lower shock pressures, when the deviatoric strength of the solid medium is significant relative to the stresses generated during the impact15. This also corresponds to an increase in the complexity of the collapse process, due to presence of failure and other strength-related phenomena. Simulations have been performed to model cavity collapse in a solid, in both the hydrodynamic18 and strength20 shock pressure regimes, but the precise effect that strength has on the collapse process and the mechanisms of energy dissipation is not known.More recently, there has been interest in the collapse of cavities in solid media, particularly in relation to hot spot formation and ignition mechanisms in energetic materials22 and liquid media25, direct observation in solid media has thus far proved challenging, primarily due to the opacity of most solids to visible light. Even for optically transparent solids, large refractive effects coupled with severe deformations in shock loading serve to either distort or obscure entirely details of the evolving sub-surface interfaces. A recent optical imaging study by Ma et al.26, looking at weak shock-cavity interactions in polymethyl methacrylate (PMMA), partly addressed this issue by cutting cylindrical holes from blocks of PMMA. This provided a two-dimensional (2D) cavity geometry that avoided the issues of optical refraction. Comparisons to 2D simulations gave predictions for the temperature field around the cavity during hot spot formation. Stronger shock loading, sufficient to induce collapse of a solid cavity, has yet to be observed.While hydrodynamic shock-induced cavity collapse has been observed experimentally in gaseous33. These techniques have been used to study shockwave dynamics and jet formation in shock-compressed polymer lattices by Branch et al.34, and shock-induced collapse of hollow spherical glass cavities in silicone and TNT by Armstrong et al.35. The latter study found evidence of higher temperatures in the collapsed cavity region. However, due to the sub-millimetre cavity sizes and constraints imposed by the electron bunch spacing of the synchrotron, only a single frame could be captured per collapse.For the probing of 3D cavities fully embedded in a solid, X-ray imaging (XRI) offers distinct advantages, as the higher transmission of X-rays provides direct access to the sub-surface details of the collapse process. Furthermore, compared to high-speed optical imaging, XRI benefits from reduced spatial distortion due to the negligible refraction of X-rays. Recent advancements in XRI techniques at synchrotron facilities mean it is now possible to image the sub-surface dynamics of materials at ultra-high-speed (UHS) rates of over 1 million frames per second (Mfps), with each image created from a single 100 ps X-ray pulse32. Fluid-dominated dynamics were studied, as well as the previously unobserved strength-dominated and transition regimes, by exploring a wide range of stress states.Motivated by the dearth of currently available experimental data — in both the hydrodynamic and strength regimes — this study presents direct observations of shock-cavity interactions in a solid medium. Single-stage and two-stage gas gun plate impact experiments were performed at the European Synchrotron Radiation Facility (ESRF) to generate planar shocks in PMMA targets containing spherical cavities. A bespoke UHS XRI system was employed to capture dynamic radiographs of collapsing cavities at rates exceeding 1 MfpsThe experiments were performed at beamline ID19 of the ESRF synchrotron. A range of dynamic stress states were investigated by employing both a single-stage and two-stage gas gun, producing shock pressures from 0.49 to 16.60 GPa. Dynamic compression at beamline ID19 is a recent development, and this work presents the first use of a two-stage gas gun at the facility. As such, the experimental setup is described here in full.29). The apparatus consists of a breech and target chamber, connected by a 25 mm bore barrel. A projectile (consisting of a sabot fronted by a flyer plate) is accelerated down the barrel with high pressure helium gas initially contained within the breech (t1). After the projectile is launched from the breech (t2), a pair of light gates records its velocity before it reaches the target, which is mounted within the evacuated target chamber. These light gates also serve to trigger the imaging system. The entire gun apparatus is oriented to place the impact axis perpendicular to the propagation direction of the synchrotron X-rays, giving a side-on radiographic projection of the event. Low-Z Mylar windows in the target chamber maintain the vacuum whilst minimising X-ray attenuation.A schematic diagram of the single-stage gun system can be seen in Fig. Figure A diagram of the flyer impacting the target is shown in Fig. 2/0.1%bw/s as a function of the photon energy in keV. The softer X-rays below 10 keV were absorbed by diamond and aluminium filters, reducing the heat load on the scintillator and optics. The result is a mean absorbed X-ray energy of ~30 keV. Based on the camera resolution and the size of the features of interest, the imaging system was placed 7 m downstream from the experiment such that the radiographs were recorded in the near-field Fresnel diffraction regime. Such a standoff was sufficient to introduce ‘phase contrast’ interference fringes in the radiographs, which are pronounced at positions where there is a sudden change in the refractive index of the material. The result is enhanced contrast of material edges37, which allows for the in-situ tracking of cavity collapse features, such as the cavity interface position, shock position and new interfaces (cracks).Real-time UHS XRI was performed using the ESRF’s 16-bunch filling mode, which provides a continuous train of ~100 ps X-ray pulses separated by 0.176 μs. The maximum frame-rate of a radiograph sequence is thus limited by this inter-pulse time. A plot of the undulator X-ray source spectrum can be seen in Fig. 1.8Y0.2SiO5, Hilger Crystals, UK), which absorbs X-ray photons and re-emits visible photons at a peak wavelength of 420 nm38. The visible light emitted both down- and up-range of the scintillator is folded away from the X-ray beam path by a pellicle beam splitter and mirror, prior to being captured by two Shimadzu Hyper Vision HPV-X2 cameras. The HPV-X2 has a CMOS burst image sensor in a (250 × 400) pixel array format with 32 × 32 μm pixel size. The lens system provided a 1:1 magnification, producing a 8 × 12 mm field of view. For the single stage experiments, one HPVX-2 camera was used to record every third X-ray bunch, giving an inter-frame time of 0.528 μs and frame-rate of 1.89 Mfps. This three-bunch minimum frame separation time was imposed by maintaining synchronisation of the camera exposure and X-ray bunch structure. Two cameras were used for the two-stage experiments, with the images interleaved to produce a single image sequence with an effective frame rate of 3.79 Mfps for images separated by 0.176 and 0.352 μs. A more detailed explanation of the imaging system, including further modifications to include a third camera and second in-line scintillator, is given by Escauriza et al.32.A schematic diagram of the imaging system can be seen in Fig. Optical imaging of the experiments was also performed to reveal the stages of tori formation during cavity collapse in the high-pressure regime. An Invisible Vision UHSi-24 framing camera was employed for this purpose, imaging the rear of the target (opposite the impact) using a mirror. The rear surface of the PMMA target was polished to allow capture of the toroidal plasma emission arising from rapid compression of trapped gas inside the collapsing cavity.39. As the code does not include material strength, these simulations provided a baseline understanding of the collapse within a fluid medium, allowing the effects of strength to be more clearly contrasted to fully hydrodynamic behaviour. Hytrac is based on an earlier front-tracking code FronTier40, which has been successfully applied to shock-cavity interactions in a liquid, in both 2D and 3D42. In the present work, a cell-based adaptive mesh refinement (AMR) scheme was used to balance computational resources with resolution requirements.Two-dimensional axisymmetric simulations of the experiments were performed in Hytrac, a multi-material front-tracking Eulerian hydrocode which has been used in earlier studies to model dynamic flow instabilities in PMMA under shock compression43. Two sets of simulations were performed, for the single-stage and two-stage experiments, with initial cell sizes of 100 μm and 137.5 μm, respectively. For both sets, 4 levels of AMR were used, giving minimum cell sizes one eighth of the initial value. An axisymmetric half-domain was modelled with a central reflective boundary and transmissive boundaries along the other three edges. For the simulations of the two-stage experiments the whole flyer and target geometries were simulated because of the small size of the target and flyer. This ensured that effects from rarefaction waves at the edge of the projectile were accounted for. Due to the larger target and flyer sizes used in the single-stage experiments, release effects were not a concern. This allowed the simulation setup to simply be a uniform domain of PMMA with a central cavity located a fixed distance from the loading surface. To replicate the impact event, the loading surface was assigned the particle velocity corresponding to the shock state of interest.For the PMMA targets and flyer materials a Frankfurt equation of state package was used to relate the pressure and specific internal energy to the density and temperature, producing a tabulated equation of statep generated upon impact is calculated from the Rankine-Hugoniot jump conditions, where the shock pressure is given by the product of the shock impedance, up, such that,ρ0 is the initial density of the material and Us is the shock velocity15. From the conservation of momentum jump condition, p and up must be continuous at the interface between the flyer and target at the instant after impact. This allows respective values to be calculated at the intercept between the p-up Hugoniot projections for the target and flyer, with the initial particle velocity of the flyer given by the impact velocity vi. The values of Us in Eq. Us-up relationships found in the literature , we can define the collapse time et al.19, which included the interaction between a 9.4 GPa shock and 1 μm cavity in HMX. They also noted high levels of deviatoric stresses around the cavity, which cause highly localised temperature regions in the form of shear bands. The surface perturbations and crack formation visible in Fig. Complete collapse was observed at shock pressures of 1.25 GPa and above. Jet formation, jet impact and tori formation were observed at shock pressures greater than 8.49 GPa Fig. . Betweenp was observed. Such non-linearity was predicted in the analysis of Rayleigh collapse, which showed the dimensionless collapse time scales inversely with the square root of the pressure difference between the fluid pressure in the liquid medium far from the cavity and the vapour pressure of the gas inside the cavity53. A similar scaling is found for the hydrostatic collapse of a cavity near a solid boundary, with an additional prolongation factor to account for the non-spherical collapse54. An inverse square root dependency has also been found for numerical simulations of shock-induced collapse, showing remarkable similarities between Rayleigh collapse and shock-induced collapse, despite their notable differences55. However, a deviation from this exponent was found by Swantek & Austin, who looked at the collapse times of shock and stress wave induced hydrodynamic collapse of cylindrical cavities in agarose-GGB gel25. As in the present study, millimetre-sized cavities were used. Including data from a similar Dear & Field study56, they found that the dimensionless minimum volume time against the pressure ratio p/p0, where p0 is the initial pressure of the surrounding medium, follows the power-law relationship,25.For the experiments in which total collapse was observed, a non-linear relationship between the dimensionless collapse time r*,In the present work, the dimensionless collapse time c allows for cases when the area does not completely fall to zero. After comparing functional forms to hydrocode simulations run in Hytrac, the effective radius that most closely matched the simulation data was a, b and c were fitting parameters, yielding the following expression for normalised projected area,The constant p0 value of p/p0 vs. R2 value of 0.99 determined by the fitting. The two closed-symbol points in the top left of the figure show the collapse times estimated from the radiographs, for the 1.21 GPa and 1.25 GPa experiments, which were not used in the power-law fitting. This behaviour away from the power law is possibly explained either by a change in failure mode from cracking to flow, or because of release effects at later times. In terms of the power relationship, this is in strong agreement with the Swantek & Austin finding in Eq. 25. This is perhaps unexpected given the differences between the experimental arrangements: in the present study spherical cavities were used, as opposed to cylindrical; the pressure ratios explored are an order of magnitude greater; and the initial state of the cavity medium is a solid, rather than a liquid. The closely matched exponents thus suggest a universal mechanism underlying the collapse of cylindrical and spherical cavities, in both liquid and solid media.For two of the experiments the cavities did not collapse. These can be seen as the two dashed lines in the top of the figure that increase in area after reaching a minimum. For the next two highest pressures, although the cavities did collapse, the functional form did not agree with the collapse times observed in the radiographs. These curves, which show the 1.21 GPa and 1.25 GPa experiments, are shown as the two blue dashed lines in the middle of Fig. 53, which applies to the hydrostatic compression of a bubble in an infinite fluid, predicts a collapse time based on the density ρ and pressure p of the fluid, given by,Also shown in Fig. To plot the dimensionless Rayleigh collapse time, the same scaling was used as for the data, such that,This gives respective expressions for PMMA and agarose-GG gel of,Despite the strong agreement in the exponents of experimental scaling laws for the present work and the Swantek & Austin study, there is a significant offset between the two lines, caused by a large difference in their multiplicative factors. Interestingly, for both shock-induced collapse (solid lines) and Rayleigh collapse (dashed lines), the same ratio is observed between the factors for PMMA and the gel: 617/305 = 137/69 = 2.0. This ratio can be explained by the difference in sound speed and density of the two materials, which are used to calculate the scaled collapse time for the Rayleigh expressions: x. By introducing a dimensionless interface displacement x′ = x/D0, the dimensionless acceleration a′, assuming constant acceleration, is determined by fitting the quadratic relationship,x′ vs. t′ curves extracted from hydrodynamic Hytrac simulations over the full range of shock pressures studied experimentally, shown in Fig. R2 values in excess of 0.99, confirming constant jet acceleration is a characteristic of cavity collapse in fluids. The plots from the experimental data are shown in Fig. p. Contrasted to the simulation plots, for the weaker shocks below p = 4.80 GPa the curves are not well-fitted by constant acceleration. The trajectories of these displacements are instead shown as dashed lines with piece-wise linear interpolation between the points. This demonstrates the assumption of constant acceleration is not valid in the strength regime.Recalling the right-hand image in Fig. a′ as a function of shock pressure p, for both the Hytrac simulations and the data extracted from the radiographs above p = 4.80 GPa. The simulation data is best fitted by a quadratic relationship given by,R2 value of 0.99. However, the experimental data is adequately described by a linear relationship,R2 value of 0.98. Notwithstanding the difference in dimensionality between the simulations and experiments , the clearest source of the model deviating from the radiographic results is the lack of a strength model in the simulations. If the effects of strength were to abruptly disappear above the transition to fluid-like behaviour, we would expect the experimental curve to quickly converge with the simulations. Instead we see a consistent offset in the acceleration values, suggesting the effects of strength persist and influence the dynamics of cavity collapse in PMMA at shock pressures in excess of 16.60 GPa. These observations will be investigated in future work using simulations that include a strength model.Figure 25 suggests that cavity collapse in this regime can at least in part be described in terms of hydrodynamic scaling and points to an underlying universality of shock-induced cavity collapse, even for spherical cavities in solid media and at pressures ratios an order of magnitude greater. Furthermore, the offset between the PMMA data and the gel data can be explained by the differences in sound speed and density, for both the analytic Rayleigh collapse times and the experimental shock-induced data. Hydrodynamic simulations of the experiments, which didn’t include a strength model, predicted a constant acceleration of the driven cavity interface. Analysis of cavity kinematics from the data showed that the motion is not well described by constant acceleration until 4.80 GPa. This indicates that a transition to a hydrodynamic response occurs between 3.08 GPa and 4.80 GPa.There is a visible transition in the radiographs of Figs. Us-up Hugoniot equation of state, from a cubic to linear relationship, at a shock pressure of ~2 GPa44. Drawing upon Pastine’s equation of state model for polyethylene57, Carter & Marsh explain the non-linear region of the curve in terms of the molecular structure of amorphous polymers generally: the forces along the backbone of the polymer chain are an order of magnitude greater than the forces between neighbouring chains. The initial compression of the polymer is thus dominated by the inter-molecular forces in the planes orthogonal to the polymer backbone46. By ignoring the spatial dimension along the backbone, the P(V) relationship is modelled in two dimensions, where V is the volume, and then transformed into the Us-up plane. This reproduced the observed cubic shape in the low-pressure region of the Hugoniot. Using the same arguments, Carter & Marsh explain that the linear region above the cubic region in the Us-up Hugoniot is initiated when the initial shock compression reaches a level at which the inter-molecular forces approach a significant value with respect to the forces along the chain. At this point the polymer behaves in the expected manner of a shocked isotropic solid46.Previous studies identify a change in the et al.44 observed a change in the characteristic relaxation time constant in the rounding of the up wavefronts, which increased to a maximum value at 1 GPa before decreasing rapidly until 2 GPa; (ii) A sudden increase in the dielectric constant above 2 GPa was observed by Hauver58 and Graham59; (iii) A similar increase in the temperature of shocked PMMA at this pressure was observed by Bloomquist & Sheffield61; (iv) Jordan et al.62 measured the dependence of the strength on impact pressure, combining their data with that of an earlier Millet & Bourne study63. They observed the start of an approximate plateau in shear strength with increasing shock pressure above 2 GPa, indicative of a transition to hydrodynamic behaviour15. Although the precise mechanisms which underlie these phenomena have yet to be established, these studies point to a significant transition in the shock response of PMMA at shock pressures of ~2 GPa. In the present work, this transition is manifest as a change in the collapse characteristics of cavities in PMMA, from a strength-dominated mode to a hydrodynamic mode in which the collapse energy is either distributed throughout the cavity periphery, or concentrated within the jet. Interestingly, this is the approximate pressure at which the collapse times begin to fall on the power-law relationship curve (1.84 GPa and above), which extends into the hydrodynamic regime, suggesting this may correspond to a change in collapse dynamics and the onset of a transition to fluid-like behaviour. This transition demarcates distinct regimes of dynamic material behaviour differing in how shock energy is internalised, which furthermore can play an important role in driving processes that depend upon the precise distribution of energy.It is interesting to note that different transitions have been observed within this pressure region in other experiments on the shock response of PMMA: (i) Lacina et al.64 revealed similar collapse dynamics as observed in the present study: (i) the presence of perturbations and shear localisation in the weak shock regime; (ii) intermediate strength shocks producing weak jetting; and (iii) strong shocks generating pronounced jets and high levels of vorticity and mixing after collapse. The results also predicted a dependence between shock loading pressure and the size and shape of hot spots, key to yielding a sustained reaction, with the reaction threshold further linked to the ratio of shock strength to the yield stress of HMX. In the present work, the presence of tori in the experiments for shock pressures 8.49 GPa and above also indicate high levels of vorticity (see the tori in the images of Fig. 22 and liquid24 media, indicates a high level of material mixing, which is necessary for prompt, non-diffusive mass transport of reactant species65.One example of the importance of shock energy distribution lies in the impact initiation of energetic materials. Mesoscale simulations of shock-cavity interactions in HMX by Rai High-speed radiography was used to reveal the time evolution of sub-surface features during shock-induced cavity collapse in a solid. These included surface perturbations and crack propagation for weaker shocks, and jet and tori formation for strong shocks. A transition from brittle fracture to material flow was observed, and, consistent with studies that have looked at shock-cavity interactions in liquids, analysis of the normalised projected cavity area vs. dimensionless collapse time revealed a power law relationship between the collapse time and shock pressure at 1.84 GPa and above. Surprisingly, this relationship holds across the shock regimes, from strength-dominated to hydrodynamic. Strength-free simulations performed in Hytrac exhibited constant jet acceleration during hydrodynamic collapse, suggesting this to be a common characteristic of fluids. The present experimental measurements however reveal more complex collapse kinematics, with the onset of jet formation and constant acceleration behaviour occurring at pressures in excess of 4.80 GPa. Taken alone, these observations imply a complete loss of strength at this critical pressure. The persistence of an offset between the measurements and the ideal fluid behaviour rather indicates the prevailing effects of strength well above this transition.We have presented previously unobserved features of shock-induced cavity collapse in a solid medium. The results showcase the latest developments in UHS synchrotron imaging capabilities, which allow sub-surface dynamics of transient events to be captured to an unprecedented level of detail. The quality of the data and its broad scope will be essential for the development and improvement of new models governing the dynamic behaviour of solids in both the strength and hydrodynamic regimes, as well the less-explored transition regime. Specifically, we expect the direct observations of hydrodynamic phenomena such as jet formation, impact and tori formation to be an invaluable resource for research into medical treatments where cavity collapse plays a central role. Additionally, new details on the richness and complexity of strength-mediated collapse will help guide the design of improved materials for harnessing and releasing energy.


Sander vitreus). Lake Winnipeg walleye represent the largest component of the second‐largest freshwater fishery in Canada. In the present study, large female walleye were sampled via nonlethal gill biopsy over two years at three spawning sites representing a latitudinal gradient in the lake. Genetic variation from sequenced mRNA was analyzed for neutral and adaptive markers to investigate population structure and possible adaptive variation. We find low population divergence (FST = 0.0095), possible northward gene flow, and outlier loci that vary latitudinally in transcripts associated with cell membrane proteins and cytoskeletal function. These results indicate that Lake Winnipeg walleye may be effectively managed as a single demographically connected metapopulation with contributing subpopulations and suggest genomic differences possibly underlying observed phenotypic differences. Despite its high cost relative to other genotyping methods, RNA sequencing data can yield physiological in addition to genetic information discussed here. We therefore argue that it is useful for addressing diverse molecular questions in the conservation of freshwater species.RNA sequencing is an effective approach for studying aquatic species yielding both physiological and genomic data. However, its population genetic applications are not well‐characterized. We investigate this possible role for RNA sequencing for population genomics in Lake Winnipeg, Manitoba, Canada, walleye  called from RNA seq data, 386 showed signatures of selection across a latitudinal gradient in Lake Winnipeg, Manitoba, Canada. Lake Winnipeg is characterized by a north and a south basin connected by a narrow channel . With n = 16 individuals per site over both years, sample sizes can predict mean population allele frequency by site, because sample sizes as low as n = 12 have been shown to be sufficient with RNA sequencing data  in accordance with approved animal use protocols of Fisheries and Oceans Canada , the University of Manitoba (F2018‐019), and the University of Nebraska‐Lincoln (Project ID: 1208). Fish were sampled nonlethally for gill tissue, where 2–3 mm of the terminal ends of 3–4 filaments from the left side each fish were collected and placed in RNAlater (Thermo Fisher Scientific) that was kept at 4°C for 24 hr prior to storage at −80°C. Total RNA extractions were performed on gill tissue using RNeasy Plus Mini Prep Kits (QIAGEN) following manufacturer's protocols, with minor modifications (provided in the Gill tissue was collected from large ≥1.2 kg) predominately female  Table  walleye .2 kg prehttp://gqinnovationcenter.com) for cDNA library preparation and sequencing. A mRNA isolation approach was performed on 250 nanograms of total RNA using the NEBNext Poly(A) Magnetic Isolation Module (New England BioLabs). From mRNA, stranded cDNA libraries were created using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). Each library was individually barcoded with NEBNext dual adaptors (New England Biolabs) prior to sequencing 100 base pair paired‐end reads. All 48 fish were sequenced on a single lane of a NovaSeq 6,000 (Illumina). 2.17 billion reads total were sequenced, with an average of 45,225,548 reads per sample collected   and electrophoresis on a 1% agarose gel, respectively. Total RNA was normalized to 80 ng/µl and sent to the McGill University and Génome Québec Innovation Centre sequencing facility (D) Table .2.2https://www.westgrid.ca/). Read files were checked for quality using FastQC version 0.11.8  using information from Corset and the original transcriptome, where 263,272 unique gene features were identified using Trinotate   Table .p‐value < .005 . Ten replicates of K = 2–5 were tested.To investigate population structure using the 52,372 putatively neutral SNPs, we used a combination of exploratory analyses, either with no prior information or with sampling location provided as priors, and population reassignment and differentiation tests to find genetic clusters despite possible signals of admixture or gene flow. Structure version 2.3.4  were identified as having a large effect between years and were thus removed, leaving 38,732 SNPs. Analyses for population structure were then rerun with this smaller set of SNPs. FST was calculated both between sites and between two reassigned clusters described by Adegenet . The Colony command‐line input file was then generated to run the program with updated allele frequencies, dioecy, inbreeding possible, polygamy allowed, no clones, full sibship scaling, no sibship prior, unknown population allele frequencies, ten runs of medium length, full likelihood inference, and high precision. This Colony input file was generated using a script originally written by M. Ackerman  to that transcript's putative function and gene ID from the annotated reference transcriptome, a database of transcripts which diverged by sampling location or year was created for the Lake Winnipeg walleye in the present study. From this database, a list of transcripts relevant to either sampling location or year was used for gene set enrichment analysis using EnrichR , while the Dauphin River fish were the most genetically distinct group sampled   and twelve in the other , ten fish were in another group (Cluster 2), and 19 fish in a final group (Cluster 3). Clusters 1 and 3 were characterized by a year effect, where every individual in Cluster 1 was captured in 2018 and every individual in Cluster 3 was captured in 2017. Both Clusters 1 and 3 had 16 out of 19 fish coming from the Red River or Matheson Island sites. Meanwhile, all ten fish in Cluster 2 were from the Dauphin River with five fish each collected in 2017 and 2018 Table .3.3FST estimates overlapped over both sampling years, indicating consistent patterns of between‐site divergence in 2017 and 2018. However, values between the Dauphin River and Matheson Island varied the most, with an estimate of 0.0044 (0.0035–0.0052) in 2017, and 0.0060 (0.0051–0.0070) in 2018 , where a kinship coefficient of approximately 0.5 would indicate full‐siblings.We found no evidence of kinship using either Colony or the method of moments. Over ten replicate runs in Colony, individuals belonged to separate families with inclusive and exclusive probabilities of 1.0000 each. Using the method of moments implemented in SNPRelate  in the pcadapt analysis Figure . In totar Figure . For ther Figure , 120 uni05 Table . By filtp‐value < .05. For transcripts with ≥2 PC2 outlier SNPs, 17 uniquely annotated genes were identified of which six were either transposons, transposable elements, or fragments of transposons , which varied by year Figure , 130 unins Table . Two genns Table .4FST of 0.0095, but with gene flow between them primarily at the channel connecting the two basins. Consistent with results in the present study, a study using microsatellites found a similar weak, but significant, differentiation between Lake Winnipeg walleye from some sites in the north and south basins , the greatest pairwise FST confidence interval difference between years was between walleye collected at Matheson Island and the Dauphin River, where confidence intervals for FST estimates overlapped by only 0.0001 between 2017 and 2018. Following Amrhein, Greenland, and McShane . Nevertheless, confidence intervals for FST overlapped between years between the Matheson Island and Dauphin River sites; therefore, patterns of environment‐dependent spawning site fidelity must be corroborated with other data, such as by telemetry.While pairwise site  McShane , we inteNotably, gene flow appears to be one way from the southern Red River, northward. Going by capture location, no fish caught in the Red River showed a genetic background consistent with the Dauphin River fish, while with the Adegenet‐reassigned clusters, no fish assigned to the mostly Dauphin River group was found in the Red River. On the other hand, fish which showed a genetic background consistent with the Red River group were found in the Dauphin River, both based on capture location and population reassignment.4.2Osmerus mordax), after which walleye body condition has decreased since 2010  or Atlantic salmon  ; Data curation ; Formal analysis (lead); Methodology ; Software (lead); Validation ; Visualization ; Writing‐original draft (lead); Writing‐review & editing . Jennifer D. Jeffrey: Investigation (supporting); Validation ; Writing‐review & editing (supporting). Jason R. Treberg: Funding acquisition ; Investigation (supporting); Methodology (supporting); Project administration ; Supervision ; Writing‐review & editing (supporting). Douglas A. Watkinson: Investigation ; Methodology ; Resources ; Supervision ; Writing‐review & editing (supporting). Eva C. Enders: Conceptualization ; Funding acquisition (lead); Investigation ; Project administration (lead); Resources (lead); Supervision ; Writing‐review & editing (supporting). Ken M. Jeffries: Conceptualization ; Data curation ; Formal analysis (supporting); Funding acquisition ; Investigation (lead); Methodology ; Project administration ; Resources ; Software ; Supervision (lead); Validation (supporting); Visualization ; Writing‐original draft (supporting); Writing‐review & editing (lead).https://www.ncbi.nlm.nih.gov/sra/PRJNA596986; https://github.com/BioMatt/Walleye_RNAseq.This article has been awarded Open Materials, Open Data Badges. All materials and data are publicly accessible via the Open Science Framework at Supplementary MaterialsClick here for additional data file.