Nitrite, ethyl carbamate, and biogenic amines in fermented vegetables are considered harmful compounds. In this study, the concentration of the nitrite, ethyl carbamate, and biogenic amines in four different varieties of fermented vegetables in China was determined. The results show that the nitrite concentration in the fermented cabbage was the highest, followed by fermented mustard, fermented bamboo, and fermented radish. Additionally, nitrite concentration in two fermented cabbage samples and one fermented mustard sample exceeded the maximum allowed residue limit (20 mg/kg) suggested by China’s National Food Safety Standards. However, only one fermented cabbage sample had a very low level of ethyl carbamate (<10 μg/kg). Otherwise, higher biogenic amines were found in the samples of fermented cabbage, fermented bamboo, and fermented mustard. Additionally, the concentration of biogenic amines in some samples exceeded the recommended limit. On the contrary, biogenic amines in fermented radish samples were relatively low. Therefore, the concentration of nitrite and biogenic amine should be closely monitored and controlled during the vegetable fermentation processes, especially for the fermentation processes of bamboo, cabbage, and mustard. Fermentation is a traditional way of preserving vegetables in China. Various vegetables, including celery, mustard, and potherb  can be fNitrite, a potentially cancer-causing compound, is commonly present in fermented vegetables . Hou et Ethyl carbamate, a probable carcinogen, is widely found in fermented foods and beverages, such as bread, spirits, wines, sake, and soy sauce . During Biogenic amines are a group of organic nitrogen compounds with low molecular weight. Most of them are produced via enzymatic decarboxylation of amino acids by microorganisms during the fermentation process . TypicalFermented bamboo, fermented cabbage, fermented mustard, and fermented radish are four popular fermented vegetables in China. Currently, there is little information on the levels of nitrite, ethyl carbamate, and biogenic amines in the four types of fermented vegetables, which raises concerns regarding the potential health risk associated with consuming these fermented vegetables. Therefore, the objective of this research is to investigate the concentration of nitrite, ethyl carbamate, and biogenic amines in fermented vegetable products. The results provide valuable information to food producers, health professionals, and consumers for developing and consuming healthier fermented vegetables.Samples of fermented bamboo, fermented cabbage, fermented mustard, and fermented radish were all purchased on Alibaba.com (accessed on 16 October 2020) . For each type of fermented vegetable, the top 15 commodities from different brands were selected as the samples for our analysis, according to the sales ranking of fermented mustard on Alibaba.com. Before analysis, the samples were stored at −20 °C. Different brands of fermented bamboo, fermented cabbage, fermented mustard, and fermented radish were labeled FB1–FB15, FC1–FC15, FM1–FM15, and FR1–FR15, respectively. All reagents used for the analyses were of analytical grade reagents and were purchased from Sigma-Aldrich.3 solution until the solution became reddish brown (the endpoint of the titration).pH value was measured using a PHS-3G pH meter . Titratable acidity (TA) was measured by titrating the brine of each sample with a 0.10 M NaOH solution until the pH reached 8.2 ± 0.2, and the results were expressed as the concentration of lactic acid according to the method described by Liu et al. . Salinitw/v ZnSO4·7H2O solution and 0.5 mL of 15% (w/v) K4Fe (CN)6·3H2O solution, followed by shaking. After filtration, the sample solution was sequentially mixed with 1 mL of 0.2% sulfanilamide, and 1 mL of 0.1% N-1-naphtyethylene diamine dihydrochloride, which are color development reagents. This color development reaction was carried out at room temperature for 15 min. The optical density (OD) of each sample was obtained at a wavelength of 538 nm with a reagent blank as a reference. The standard curve was obtained by performing the same color development process and OD analysis.The nitrite concentration of each sample was measured using the Griess reaction . Brieflyv/v). Then, the eluent was evaporated to 0.5 mL via N2 flow at room temperature and brought to a volume of exactly 1 mL for testing.The ethyl carbamate concentration in each sample was measured using a gas chromatography–mass spectrometry (GC–MS) method . For eacThe GC–MS analysis was conducted with an Agilent 7890A-5975C equipped with a DB-WAX column . Helium was used as the carrier gas and the flow rate was 1.0 mL/min. Sample aliquots (2.0 µL/sample) were injected under splitless mode. The temperature of the GC oven was programmed as follows: holding at 50 °C for 1 min, ramping to 180 °C at a rate of 8 °C/min, and ramping to 240 °C at a rate of 40 °C/min, followed by holding at 240 °C for 5 min. The mass spectral analysis was performed in a 70 eV electron impact ionization mode; the temperatures of the injector port and mass spectrometer interface lines were 220 °C and 250 °C, respectively. For qualitative analysis, the mass spectrometer was conducted in scan mode, while for quantitative analysis, the mass spectrometer was conducted under the selected ion monitoring (SIM) mode. The ethyl carbamate fragment ions have m/z values of 44, 62, 74, and 89. The calibration curves of ethyl carbamate were constructed using peak area.4 and extracted with ultrasound . Then, the mixture was centrifuged at 10,000× g for 5 min, and the supernatant was collected. Next, 250 μL of the supernatant was combined with 25 μL of NaOH solution (2 M), 75 μL of saturated NaHCO3, and 500 μL of dansyl chloride (5 mg/mL). The mixture was heated at 55 °C for 40 min. Next, 25 μL of NH4OH solution (25%) was added to the mixture, which was incubated at 55 °C for another 10 min. After being filtered by a membrane with a pore size of 0.22 μm, the treated sample was analyzed by an Agilent high-performance liquid chromatography system. The system was equipped with an Eclipse XDB-C18 column  and a 254 nm UV detector. The separation of the amines was achieved using a linear gradient of acetonitrile/H2O mixtures as the mobile phase. The gradient elution program was as follows: 0–4 min, 50% acetonitrile; 4–22 min, 50–90% acetonitrile; 22–30 min, 90–50% acetonitrile; 30–35 min, 50% acetonitrile. The injection volume, flow rate, and column temperature were 10 µL, 0.8 mL/min, and 25 °C, respectively.The concentrations of biogenic amines were analyzed according to the method developed by Zhang et al.  with sli2) were obtained using the linear regression method. Accuracy refers to how close a measurement is to the theoretical value, while precision refers to how close the measurements of the same sample are to each other. Matrix spikes with a nitrite concentration of 50, 20, or 5 mg/kg, an ethyl carbamate concentration of 20, 10, or 5 μg/kg, and a biogenic amine concentration of 100, 50, or 20 mg/kg were employed in the analysis. The accuracy was evaluated based on the recovery rate (RR) of the spiked sample analysis. The intra- and inter-day percent relative standard deviations (RSD %) were determined. The limit of detection (LOD) and limit of quantification (LOQ) were used to evaluate the sensitivity of the methods. LOD is defined as the concentration of an analyte with a signal-to-noise (S/N) ratio of 3:1, while LOQ is defined as the concentration of an analyte with an S/N ratio of 10:1 [The method validation was based on accuracy, precision, and sensitivity. The calibration curves and the related linearities  was adopted for the comparisons between multiple means. The plots were plotted using Origin v2021b .All tests were carried out in triplicate. All data in this manuscript are expressed as mean ± standard deviation (SD). Analysis of variance (ANOVA) testing was conducted using SPSS17.0 , and Duncan’s test , and other fermented vegetable samples had no detectable ethyl carbamate. Similarly, Hasnip et al.  studied As shown in Excess intake of biogenic amines can lead to various health problems. At present, there are no government regulations on the concentration of biogenic amines in fermented vegetables. To the best of our knowledge, histamine is the only biogenic amine that has a government regulation in fish products: 50 mg/kg limit according to the US Food and Drug Administration . SeveralThe concentration of the nitrite, ethyl carbamate, and biogenic amines in four types of Chinese fermented vegetables (15 samples per type of fermented vegetable) were studied. The nitrite concentration in two fermented cabbage samples and one fermented mustard sample exceeded the maximum allowed residue limit (20 mg/kg) suggested by China’s National Food Safety Standards. However, ethyl carbamate was only found in one fermented cabbage sample, and its concentration was below 10 μg/kg. Hence, the risk of excessive ethyl carbamate intake associated with consuming fermented vegetables was extremely low. In addition, the concentrations of biogenic amines in fermented bamboo, fermented cabbage, and fermented mustard samples were high, and the concentration of biogenic amines in some samples exceeded the recommended limit. Therefore, to minimize the health risk, the concentrations of nitrite and biogenic amines should be closely monitored and controlled during the vegetable fermentation processes, especially for the fermentation processes of bamboos, cabbages, and mustard.


Margaritifera margaritifera) for which a number of captive breeding facilities has been established in Europe. The core objective of this study was to compare the genetic constitution of 29 cohorts of captive-bred freshwater pearl mussels from five different breeding facilities in Austria, France, Luxembourg and Germany, with their original 14 source populations from nine major European drainages, based on microsatellite markers. Captive-bred mussels represented 11 different genetic clusters, suggesting an important contribution of the breeding stations to securing the genetic diversity of the species. In almost all cases, the cultured offspring closely resembled the genetic constitution of the source mussels as revealed from the STRUCTURE analysis and the generally high assignment of offspring to the original source populations. The majority of captive-bred cohorts had an increased inbreeding coefficient and decreased genetic variability compared to their source populations as measured by AR and HO. Highest numbers of deformed juveniles coincided with very low levels of HO < 0.05. Since erosion of genetic diversity in captive breeding was mostly evident in individual year-cohorts, long-term breeding over multiple years can minimize such effects. The systematic selection of priority populations for conservation, effective breeding strategies avoiding effects of in- and outbreeding by genetically informed selection of parent individuals, and a network of collaboration among the different breeding facilities would be very useful to increase resilience and effectiveness.Securing genetic integrity is of key importance in conservation-oriented captive breeding programs releasing juveniles into the wild. This is particularly true for species such as the endangered freshwater pearl mussel ( One of the species that receives greatest attention is the freshwater pearl mussel (Margaritifera margaritifera) which simultaneously fulfills the criteria of indicator, keystone, flagship and umbrella species3. Freshwater pearl mussels can reach ages of more than 100 years and have an exceptional life cycle that involves a parasitic life stage on a fish host that can either be Atlantic salmon , brown trout , or both in European populations5. Degradation of pearl mussel habitats, mostly related to siltation and colmation of interstitial spaces in the stream bed where juvenile pearl mussels burrow, have resulted in a lack of recruitment and severe declines of many pearl mussel populations throughout their European range7. Since restoration of stream beds requires consideration of entire catchments and the restoration of natural flow dynamics8, this approach is time-consuming and costly. At the same time, many pearl mussel populations are overaged and expected to die out in the near future. Since the first descriptions of the ongoing threats and population declines9, the downward trend of populations has even become worse7. This development has prompted the creation of a first European CEN standard for a single species10 as well as several captive breeding efforts in different countries14. Captive breeding of freshwater pearl mussel was first established in the Czech Republic by Jaroslav Hruška11 and is mostly based on induced release of glochidia larvae from parent mussels, a subsequent infestation and holding of fish hosts as well as collection and raising of dropped-off juveniles14.Freshwater biodiversity is globally in decline, with freshwater mussels being among the most affected taxonomic groups22 as well as its North American23 distributions, facilitating the consideration of priority populations for conservation in captive breeding. A wise selection of parents or an annual replacement of breeders can help secure the genetic diversity in captive breeding efforts. Still, there are multiple factors that may compromise the genetic constitution of captive-bred mussels compared to their parent populations. For instance, the selection of small numbers of parent mussels that are not representative for the genetic constitution of the entire population can already narrow the basis of genetic variability within the breeding program. During infestation of host fish, the compatibility of the used species and strains among mussel and fish as well as the age of the fish all can have effects on attachment rates, survival and growth25. Physiology of the host fish immune system related to temperature26, the effects of different loading densities of mussel larvae on swimming performance of the host27 as well as the duration of the parasitic phase29 and associated nutrient transfer from the host to the mussel30 all have a potential selective effect on the mussels during the parasitic phase. In the subsequent post-parasitic (i.e. juvenile) phase, stock origin and environmental conditions31, food quantity as well as water and sediment quality34, but also changing of culture systems and the cleaning regimes35 were found to affect survival and growth. Generally, mortalities are highest until the juveniles reach the first millimeter36.Due to the strong conservation interest in freshwater pearl mussel, its genetic constitution has been studied in depth both throughout its European37, consideration of genetic effects during the breeding process and objective evaluation of ongoing breeding activities is needed yet still lacking. In addition to the technical optimization of breeding facilities for freshwater mussels38, such information can be useful in identifying the most suitable captive breeding techniques for retaining a maximum of the genetic-evolutionary potential of mussels3. The core objective of this study thus was to analyze the genetic constitution of captive-bred freshwater pearl mussels from different European breeding facilities and compare their genetic diversity and differentiation with the original populations. These served as a source for the parent mussels and captive-bred juveniles have been, or are intended to be released into the same streams. Specifically, we hypothesized that (1) breeding efforts would be suitable to secure the genetic identity of the captive bred mussels as indicated by low genetic differentiation between original populations and captive-bred juveniles, (2) there would be no decrease of the genetic variability in pearl mussel during the captive breeding as evident from highly similar genetic diversity indices such as Ho and AR between original populations and captive-bred juveniles.In order to make the most of recent advances in freshwater mussel propagation and restoration17 in their natural habitats (based on representative sample collection over multiple locations per stream) or directly from individuals of the broodstock held at the breeding station as practiced in Austria40. Genetic constitution of those mussels was compared to results of 897 propagated juveniles comprising 29 different cohorts which were selected from different age classes ranging from 0 + to ca. 16 years. They were collected either while being held at the breeding facility, or from gravel-filled cage-boxes submerged in the streams of intended release, or from already released and marked individuals.Our study design was based on the comparison of population genetic parameters of natural freshwater pearl mussel populations and corresponding cohorts of juveniles propagated at specialized breeding stations. We included five facilities from four European countries  in our sampling regime. In these, freshwater pearl mussels were propagated from a total of 14 different source populations representing nine main drainage systems: Elbe , Danube , Rhine (Sûre), Maas , Loire (Le Sarthon), Orne (La Rouvre), Sienne (L’Airou), Blavet  and Aulne (L’Elez)  were additionally noted. Haemolymph was transferred into 1.7 mL Eppendorf tubes while juveniles were kept alive in containers filled with water. Both were cooled at 4 °C and subsequently transported to the laboratories of the Technical University of Munich, Germany. Samples were prepared for DNA extraction by centrifuging haemolymph at 14.000 g for 5 min and discarding the supernatant whereas juveniles were transferred to individual reaction tubes. To ensure efficient lysis of the tissue, the smallest individuals (~ 1 mm) were crushed inside the reaction tube using a metal probe, or one shell valve with adhering tissue was used. Genomic DNA was then isolated from cellular pellets and juveniles using the NucleoSpin Tissue Kit  according to the manufacturer’s protocol for tissue samples and eluted in 80 µL of BE buffer.We collected 40–100 µL haemolymph from the foot tissue of adult individuals and juveniles exceeding 30 mm total shell length by following the methodology of Geist & Kuehn15 and Geist & Kuehn17. Polymerase chain reactions (PCRs) were performed in a total volume of 12.5 µL containing 25 ng genomic DNA, 0.2 µM of each primer, 0.2 mM of each dNTP, 3 mM MgCl2 for eight Loci (2 mM MgCl2 for Locus 5280), 1 × FirePol® PCR buffer BD SO4 and 0.5 U FirePol® Taq DNA polymerase  under the cycling conditions described in Geist et al.15 and Geist & Kuehn16. Forward primers were end-labelled with Cy5 fluorescent dye and PCR products were separated on 5% denaturing 19:1 acrylamid:bisacrylamid gels on an ALFexpressII DNA analyser (Amersham Pharmacia Biotech) and allele lengths were scored using ALLELELINKS 1.02 software. To ensure consistent allele scoring between individual lanes and among gels, two internal size standards were included per lane42 as well as 11 size standards and one previously genotyped reference sample in two separate lanes.DNA samples were genotyped at nine standard species-specific microsatellite markers  as described by Geist et al.R) as a standardized measure of the number of alleles corrected for sample size, expected and observed heterzygosities  and inbreeding coefficient (FIS) were calculated using Fstat v. 2.9.343. We used Genepop v. 4.7.344 to test genotypic distributions for conformance with Hardy–Weinberg expectations using the probability test45, to calculate pairwise FST values46 between source populations and offspring cohorts and to estimate the significance of genotypic differentiation between these populations pairs. All probability tests were based on the Markov chain48 method using 10,000 dememorization steps, 100 batches and 5000 iterations per batch. The number of distinct multilocus genotypes (MLG) was determined using the R-package Poppr v. 2.8.349. The R-package Adegenet v. 2.1.150 was used to determine mean individual inbreeding coefficients (Find) for each source population and offspring cohort by calculating for each individual the probability of being homozygous at a locus: p(h) = F + (1 − F) pi refers to the frequency of allele i in a population. Additionally, we estimated relatedness between individuals within populations based on the F-value of the 2mod program51 which provides information on the probability that two genes share a common ancestor within a population and is correlated with effective population size. The Markov chain Monte Carlo (MCMC) simulation was run for 200,000 iterations and the initial 10% of the data were discarded to avoid dependence on starting conditions. We used Structure v. 2.3.4 software52 to determine and visualize the number of genetic clusters (K) present among source populations and to assign probabilities of cluster memberships to propagated individuals. We tested values of K ranging from 2 to 20 under the admixture model and assuming correlated allele frequencies using 20,000 burn-ins, 200,000 MCMC repetitions and 10 iterations per K to assess the convergence of ln P(X│K). The software package Clumpak v. 1.153 was used to infer the most likely number of K based on the ΔK method of Evanno et al.54. The implemented program Clumpp v. 1.155 was used to find the optimal individual alignments of replicated cluster analyses using the LargeKGreedy algorithm and 2000 random input orders which were then visualized using Distruct v. 1.156. Bayesian clustering techniques may produce biased results in terms of cluster recognition when working with unbalanced sample sizes58. We therefore validated Structure results using the multivariate approach of discriminant analysis of principal components (DAPC)59 implemented in the software package Adegenet v. 2.1.150 in R v. 3.6.260 which is less sensitive to uneven sampling58. DAPC first transforms the data using principal component analysis (PCA) and then performs a Discriminant Analysis (DA) on the retained principal components (PC). We retained 13 PCs which explained 85% of the total variation of the data set. Results of the DAPC were visualized by assigning the first three PCs to intensities of the color channels of the RGB system. Similar generated colors thus correspond to similar genetic constitutions of respective individuals or populations59.Microsatellite allele frequencies, the mean number of alleles per locus (A), allelic richness , a 100% assignment to the original population was found, and in 25 out of the 29 cases (86%), more than 80% of individuals were correctly assigned . In most cases, these pronounced differences were not consistently present over different year cohorts, especially in captive breeding situations where glochidia from gravid mussels are annually collected in the wild to infest host fish in the rearing facility and where these source mussels differ from year to year.Most Fps Table . This waO) and allelic richness (AR) in source populations ranged from HO = 0.006 (EL) to HO = 0.460 (WB) and from AR = 1.1 (EL and RU) to AR = 2.9 (WB) while the global means of source populations  were slightly higher than that of juvenile cohorts . In most cohorts of propagated juveniles, remarkably low levels of these two diversity parameters were detected, but also in the majority of the source populations, especially the westernmost ones  as well as different infestation, metamorphosis and rearing success. Variable levels of multiple paternity as previously observed in this species31 as well as the lower risk of genetic erosion and die-offs in the rearing facility would suggest an early release. Colmated stream beds and other adverse habitat conditions to which older and larger mussels are much more resistant than young ones, as well as the absence of a decreased genetic variability of older cohorts compared to younger ones in this study , along with the continuous improvements of survival in rearing facilities66 clearly suggest that stocking with such cohorts is also a feasible option.The age and size at which captive-bred juvenile pearl mussels should be released into the wild still remains controversial. The greater capability of adaptation3. Given the well-established knowledge on the genetic constitution of freshwater pearl mussel throughout its European and North American distribution23 and the large and increasing number of breeding facilities for this species, a more systematic approach of evidence-based conservation and restoration67 can be recommended. Such an approach should also include systematic selection of priority populations for conservation, effective breeding strategies avoiding effects of in- and outbreeding by genetically informed selection of parent individuals, and a network of collaboration among the different facilities.Effective conservation of freshwater pearl mussel will likely depend on a combination of habitat restoration and effective captive breeding to rescue genetically unique populations over time. An integration of scientific findings into the ecology and conservation genetics of the species is thus likely to be most successfulSupplementary Information.


Chelonia mydas and the frequencyof haplotypes in feeding areas can aid understanding of the genetic andecological diversity, since with this information it is possible to identify theorigin of the individuals. The current study aims to characterize and comparegenetic diversity along the coast of Paraná with 17 other feeding areas in theAtlantic Ocean. A total of 285 samples from juveniles were DNA sequenced in thecontrol region, resulting in the identification of 12 haplotypes, with apredominance of the CMA8 haplotype (69%) and the first registration of CMA23.For the study subjects, haplotypic and nucleotide diversity were 0.469 ± 0.032and 0.00189 ± 0.00020, respectively, and comparisons with other feeding areaspresented significant values for the majority of FST and ΦST. The results pointto the importance of this region and provide evidence that over the years amixed stock has used the region as a feeding area. This variation could berelated to sea currents, climatic changes, and oceanographic characteristicsthat may alter the availability of food, water temperature, and the presence ofturtles. The current results can be considered in conservation plans forChelonia mydas.Mixed stocks are described for  Chelonia mydas, is affected by multiple threats,and thousands of juveniles are found stranded dead along the Brazilian coastannually  and genetic origin. The species uses pelagic and oceanic areas during,approximately, its first 2 or 3 years of life, and then moves to demersal andneritic areas in its juvenile and adult phases . IndividBetween 2-4 years of age, juvenile green turtles are recruited from oceanic tocoastal zones, and different factors  influencemigratory patterns .Ontogenhotspot , comprises a remnant of Atlantic Forest and a UNESCO Natural WorldHeritage site . It istEstuarine areas are crucial because of their ecological role and the ParanáEstuarine Complex is an important foraging and migratory area for juvenile seaturtles. However, the region presents intensive human activity , exposing thespecies to multiple threats .The individuals were found either stranded dead, debilitated, or accidentallycaptured in fishing nets. Samples of liver, epithelial or muscular tissues fromjuvenile green turtles were collected along the Paraná coast throughout the yearfrom 2007 to 2014 .The collected tissues were identified according to the animal’s stage ofdecomposition, adapted from 2, 0.5 μM of each primer, 3 U of Taq DNA polymerase[Invitrogen] and 5-50 ng of the DNA sample). The mixture was incubated at 94°Cfor 5 min; followed by 35 cycles of 40 s at 94°C, 30 s at 51°C, and 1 min at72°C; and finally, a further 9 min at 72°C. The samples amplified successfullywere read in an ABI PRISM 3500xL automatic sequencer (Applied Biosystems).Genomic DNA was extracted according to the phenol-chloroform protocol describedby h),nucleotide diversity (π), and haplotype counts were calculated using DnaSP 5.1software , Espírito Santo/BR , Ubatuba/BR , Paraná/BR , Arvoredo Island/BR  the North Atlanticgroup, comprising the areas of North Carolina/USA NC, , Floridad/BR AI,, CassinoST) and the Kimura 2P genetic distanceparameters (ST), was calculated using Arlequin 3.5.2.2 software , wsoftware . The samIn the sampling period, approximately 1500 sea turtles were recorded along 40 km ofthe Paraná coast and estuarine areas according to researchers at Laboratório deEcologia e Conservação (CEM-UFPR). Tissue samples for genetic analysis werecollected from 436 green turtles and DNA samples were sequenced in 285 individuals(~65%). From the mtDNA samples analyzed, 12 haplotypes were defined according to a485pb region, and 11 of these haplotypes presented 14 polymorphic sites betweenthem, whereas one haplotype (CMA42) had a 4pb insertion/deletion (indel).Haplotype CMA8 was the most frequently observed haplotype, identified in 195individuals (69%), followed by CMA5 in 52 individuals (18%). The other 10 haplotypesoccurred with a frequency of less than 5%, with only haplotype CMA8 being identifiedin all years .p =0.809), and no differences were detected among the mean CCLs of individualsidentified as having different haplotypes (p = 0.114).The CCLs of the analyzed individuals ranged from 27.5 to 47.6 cm (mean±SD = 39.95 ±7.32 cm), indicating that all individuals are classified as juveniles (<65 cmCCL) (h) and nucleotide (π) diversities were 0.469 ±0.032 and 0.00189 ± 0.00020, respectively  had the greatest number ofsamples, with seven (7) identified haplotypes, whereas 2007 (n = 4) had the smallestnumber of samples, with only two (2) identified haplotypes . On the ectively .ST comparisons and 73 of 120 ΦST comparisons werestatistically significant, considering p ≤ 0.05 , the AMOVAresults indicated that the greatest difference is actually between the groups,whereas the smallest difference is between feeding areas within the groups . Comparip ≤ 0.05 . Particu, and PB .A total of 12 haplotypes were identified among the 285 juvenile green turtles sampledalong the Paraná coast, Southern Brazil. A predominance of the haplotype CMA8,concerning both number of individuals and continuous presence over the years,reinforces the genetic characteristics recorded in different regions over theSouthwestern Atlantic Ocean. The number of haplotypes and the genetic diversityvalues found for the individuals sampled between 2007 and 2014 highlight therelevance of this region as a feeding and development ground for multiple geneticstocks of juvenile green turtles from the South Atlantic Ocean.Considering the individuals from the Paraná coast by sampling year, the second mostfrequent haplotype (CM-A5) was observed for all years except 2007, and the other 10haplotypes occurred with a relatively low frequency (≤ 5%). Three haplotypesoccurred more frequently on the Paraná coast than in the other previously describedAtlantic feeding areas; haplotype CMA42 was identified in six individuals, whereashaplotypes CMA39 and CMA46 were identified only in Brazil, once in Arvoredo Island(Santa Catarina state) and once in Ubatuba (São Paulo state), respectively . These dThe haplotypes analyzed in this study are identified mainly from nesting areas in theSouth Atlantic (coast of Brazil and Africa)  may be associated with the geographical distances between the feeding areasand the influence of sea currents; the relatively warm currents that flow off theBrazilian coast  form a direct frontagainst the cold-water Malvinas Current . These dIn studies on South Atlantic feeding areas, non-significant values were observed whencomparing the data with other areas geographically close to the Paraná coast, whichwas expected because these regions have similar conditions in terms of the influenceof sea currents and other environmental factors. Similar results were observed forfeeding areas of the Province of Buenos Aires on the coast of Argentina, whichpresented significant differences when compared with the five feeding areas in theNortheastern Atlantic, but also with one northern area in the Southwestern AtlanticOcean . The comST and ΦST values are significant when considering thecomparisons between the Paraná coast and North Atlantic areas, and the comparisonswith genetic difference values are either high or very high. Comparing the Paranácoast with other South Atlantic areas, FST had significant values for AL,FN, and RA, and ΦST had significant values for AL, but genetic differencevalues were either moderate or small. The genetic difference values obtained betweenthe Paraná coast and other regions could be the result of oceanographic parameters,considering that the sea currents may favor individuals following the direction ofthe south coast of Brazil or move them away from Brazil with other predominantcurrents  . The occCheloniamydas from multiple origins, and evidence its considerable geneticvariability. The study also highlights the presence of different haplotypes indifferent years and the importance of developing systematic and continuousmonitoring of feeding areas to understand connectivity and migratory patterns. Thisvariation over the years may be related to the sea currents, climatic issues, orother oceanographic characteristics; these and other factors should also beinvestigated more carefully in the future. Furthermore, future studies shouldinclude analysis using different molecular markers to improve knowledge on turtleorigins and biogeography. In general, the results contribute crucial information toimprove management and conservation plans for Chelonia mydas andits habitats.Collectively, the results of the included samples from 2007 to 2014 along the Paranácoast, support the use of this specific foraging ground by juvenile


The spatial inequilibrium phenomenon is apparent during China’s rapid urbanization in the past four decades. As the fertility rate decreases and the population ages, this phenomenon will become more critical. To accurately forecast the future economic development of China, it is necessary to quantify the attractiveness of individual cities. This study introduces web search data to quantify the attractiveness of cities with a fine spatial scale (prefecture-level city) and relatively long-term span (nine years). Results confirm that the estimated city attractiveness can unravel a city’s capability to attract labor force, and suggest that tourism and health care functions of a city have a positive effect to the city’s attractiveness. Additionally, China’s north-south gap in economic development has been widened in the past decade, and 11 cities with nationwide influence have prosperous development potential. This study provides a new lens for predicting China’s economic development, as well as its spatial patterns.The online version contains supplementary material available at 10.1140/epjds/s13688-022-00332-y. China has experienced a rapid economic growth and urbanization in the last 40 years , during At the national scale, the geography of Chinese cities’ attractiveness has been constantly changing, although the pace was not too fast. Such change is reasonable and expectable because the attractiveness of a city is a synthetic indicator of many factors including wage, house price, culture, and climate conditions. Even a subtle factor such as local cuisine or air quality might lead a person to move to or flee from a city. Examples include that many of the young generation have been fleeing from Beijing and Shanghai, the two most attractive cities in China about ten years ago, due to unaffordable living expenditures . ConsequTo understand the attractiveness of cities, traditional approach mainly relies on survey data such as socio-economic statistics indicators , which oBased on longitudinal web search volumes, this study estimates the attractiveness of individual cities in China over the last decade by reversely fitting the gravity model, and investigates its spatio-temporal patterns. The estimated city attractiveness is yielded from a novel directed gravity model with particle swarm optimization, and can provide us with reasonable evidences to foresee the future development of the nation’s urban systems.Measuring the attractiveness of cities has been a central topic in geography and regional studies for a long time . PreviouAlternatively, reversely fitting the gravity model on city interactions becomes a commonly used approach to reconstruct the attractiveness of cities. The gravity model  is an important method for urban and regional analysis. There is a rich body of literature on the model and its various extensions, termed the family of gravity models , 23. In The existing methods for reversely fitting gravity models can be divided into two categories, exact methods and heuristic methods. Based on the mathematical expression of the reverse gravity model, exact methods includes three existing approaches: linear regression, linear programming (LP), and the algebraic method. O’Kelly et al.  applied There is much to learn from understanding the discrepancies between the estimated gravitational attractions  and actual city sizes. City attractiveness reported in previous studies heavily relies on a single type of spatial interactions in the physical space , 24–27. Baidu Index  releasedThe aim is to estimate the attractiveness of cities in China for each year from 2011 to 2019, and the seasonal variation is not our focus. For each city pair, the daily search index data from 2011 to 2019 are collected, and averaged annually to avoid the influence of seasonal variation. To leave out the influence of COVID-19 on web search since 2020, we concentrated on city attractiveness before 2020 for the sake of reliability. To cover mainland China  as much as possible, we try to include all prefecture-level cities as well as county-level cities directly under provincial government, except for those whose names have not been included as keywords or not available as user areas. As a result, our city list covers 333 prefecture-level cities in mainland China, excluding Sansha in Hainan province due to lack of data. We also cover all four municipalities directly under the central government, namely Beijing, Tianjin, Shanghai and Chongqing as well as 20 county-level cities in our study. A total of 357 cities are considered from 2017 to 2019, while from 2011 to 2016, only 322 cities are covered due to data incompleteness. From the Baidu Index data between city pairs, we construct a directed network to depict the searching behaviors among Chinese cities for each year. An example of 2019 is presented in Fig. In addition, we evaluate two general issues concerning the web search activity. The first is toponym ambiguity . Cities It is also worth noting that, according to China Internet Network Information Center (CNNIC), the Internet penetration rate in China is 64.5% in total and 76.5% in urban areas in 2019 . In 2018As aforementioned, the gravity model can be applied to estimate the theoretical sizes of the cities, known as gravitational attractions, given the interaction flows and distances between each city pair. To solve the problem of asymmetric flows, we introduce a directed modification of the gravity model, including two parameters, propulsion and attraction, for each city. Based on web search volumes and the distance between each pair of cities, we reversely fit the gravity model with a particle swarm optimization (PSO) approach. The derived values of attraction parameters stand for attractiveness of cities. Subsequent analyses are performed to explore the spatial pattern and temporal evolution of city attractiveness. Moreover, multivariate linear regression models are built to investigate the association between city attractiveness and socio-economic factors. Finally, the analysis of spheres of influence (SOI) provides another perspective to understand the change of major cities’ attractiveness. The proposed analytic framework is illustrated in Fig. i to city j, i and j, β is the distance decay coefficient, k is a constant.As an analogy to the law of universal gravitation, the gravity model was proposed for the modeling of spatial interactions. The simplified gravity model  can be eNote that Eq.  is not s studies  and the  studies , we intrmentclass2pt{minimmentclass2pt{minimi to city j; i in that year, respectively; i and j. The topological distance of two cities is defined as their shortest path length on the adjacent graph. Two cities are considered adjacent if and only if they share a border as areal units. Hence, the distance between a city and its neighbor is 1, and between two non-adjacent cities with a common neighbor is 2 and so on. For cities on islands, we make Haikou linked to Zhanjiang (by Yuehai Railway) and Zhoushan to Ningbo (by Zhoushan Sea-crossing Bridge) so that the adjacent graph is connected. Topological distances are used instead of Euclidean distances for the following reasons. Regarding Euclidean distances, a point coordinate needs to be determined for each city area, which involves ambiguity [The reversely fitting approach can be performed on the directed model as well, enabling us to use the search index data to estimate city propulsions and attractions. In specific, the model is fitted for search index data each year, where entclass1pt{minimambiguity . Additiombiguity . MoreovePbest, while the swarm records the best position reached by all particles as Gbest. In a minimization context, for example, a position is better than another if lower value of the objective function is obtained. During each iteration, each particle flies to a new position, which is usually generated under the guidance of its Pbest and Gbest with randomness. After the movement, each Pbest and Gbest are updated. The algorithm exits when it reaches the maximum number of iterations, or the objective function falls below a predefined value. PSO is derivative-free, simple to implement, and intrinsically parallelizable. Similar to many other intelligent optimization methods, the theoretical foundation of PSO is relatively lack, yet its practical performance has been widely recognized [Among existing methods for reversely fitting the gravity model, PSO is capable of handling large spatial interaction networks, and is applicable to the directed model. Therefore, we adopt PSO as our model solver. Particle swarm optimization (PSO) is a swarm intelligence approach to optimize non-linear continuous functions, proposed by Kennedy and Eberhart . In PSO,cognized .N is the number of cities, i to city jβ as an input parameter and try different value at step 0.05. The β that produces the lowest RMSE is selected afterwards. Without loss of generality, let This study uses PSO to minimize the root mean square error of the directed gravity model, as the objective function The challenge of applying PSO to the optimization of a high-dimensional problem like this is nontrivial. In our experiment, the canonical PSO based on velocity update failed to find the optimal solution. To improve the algorithm performance, we incorporated features of two PSO variants, bare bones PSO with jumps (BBJ) and cooperative hybrid particle swarms (CPSO-H).D-dimensional solution vector, α is a hyperparameter to control the exploration range. Besides, each dimension of each particle disobeys this formula at a probability jump. When this happens, the new coordinate is generated from uniform distribution in Based on bare bones particle swarms proposed by Kennedy , BBJ appCooperative PSO  is propoWe implemented and tested four algorithms on average search index data in 2018, namely Canonical PSO, BBJ, CPSO-H + Canonical PSO and CPSO-H + BBJ. To reduce the computation time, we utilized GPU-based parallel computing with CUDA Toolkit 10.0. In our implementation, the objective function computations for each particle run in parallel on GPU cores, while the main thread does all the other work. Swarms with up to 4096 particles are used in our high-dimensional problem, which is greatly larger than the common setting of 20-50 particles . ResultsGiven a city, the attractiveness reflects search behavior of individuals, including potential visitors and people interested in the city. It is related to many attributes and functions of the city, including economic development, traffic convenience, health care, educational services, employment, recreation and shopping services, ecological quality, administrative services, and housing . As urbaChina City Statistical Yearbook 2019 [The influencing factors, the corresponding economic indicators, and their sources are given in Table ook 2019 , and theook 2019 . Two of ook 2019 , includiA total of 254 cities are considered in the OLS regression, while other cities are excluded due to lack of data. Before the regression, the correlations between all statistical variables and city attractiveness are examined with Pearson correlation coefficient. Results show that all the variables are related at 0.01 significance . People in city Z may be interested in a number of cities, including A, which can be obtained based on search volumes from city Z to other cities. For example, if the search volume from Z to A is greater than those from Z to any other city, city A ranks first in the list of cities in which city Z is interested, and city A has the strongest influence on city Z. However, if A’s position is not so high, say 20, A has a relatively weak impact on Z. Hence, given a rank threshold N, the N-SOI of city A can be defined as A, Z are cities and A’s rank in the list of cities in which Z is interested according to search volumes. Figure A and B. We can use the flow of web searches originated from other cities to a city By reversely fitting a directed gravity model for web search index data, we obtained the attractions and propulsions of 357 Chinese cities in 2019. The ce decay , yet thece decay . CompareThe overall spatial distribution of the Chinese cities’ attractiveness Fig. a was higNote that Chengdu and Xi’an outperform traditional metropolises and become the most attractive cities Fig. b. In addThe estimated attractiveness index can be cross validated by other city attractiveness indicators, such as the Top 100 cities for talent attraction in 2019 . These searches are not counted in the search index, thus not reflected in our estimated city attractiveness. As most cities encounter the same problem, we assume that the impact of this issue is tolerable. Second, high search volume might come from negative public emergency rather than city attractiveness. By filtering out abnormal values, this effect could be largely mitigated. Third, the interests of those who seldom or even never surf the Internet is overlooked due to the sample bias of the data. As a result, the search index might reflect interests of young people more than the elderly, for example. Recent statistical data  show a rThose limitations imply that a one-size-fits-all solution for measuring inter-city interactions as well as the derived city attractiveness might be impossible. Nonetheless, as the access to data set capturing the interactions between cities in both physical space and virtual space increases, it opens a possibility to synergize different interactions in the two spaces to fully understand city attractiveness. Furthermore, incorporating them is not straightforward, and the relationship between interactions needs to be considered. We may conjecture that the interplay between interactions in physical space and virtual space is either mutually complementary or exclusive. Moreover, there might exist spatially and/or temporally lagged effects between population movements in physical space and web search activities in virtual space. Empirical verification using spatio-temporal models will be a direction of further investigation. In a sense, our study introduces web search data as a promising (while being largely ignored) data source for the analysis of inter-city interactions, and appeals that more attentions should be paid to understanding city attractiveness in a coupled and multi-faceted fashion. As the big data are increasingly accessible, we see a lot of opportunities as well as challenges on this topic.Including supplementary data, as well as detailed discussions on toponym ambiguity, comparison experiments on gravity model forms and distance metrics, implementation details and comparison experiments of particle swarm algorithms. (DOCX 49 kB)Below is the link to the electronic supplementary material.


Genetic testing is a tool used in a variety of settings for medical and nonhealth related purposes. The goal of this analysis was to better understand the awareness and use of genetic testing in the United States.Data from the 2020 Health Information National Trends Survey 5 cycle 4 were used to assess the awareness and use of genetic testing by demographic characteristics, personal cancer history, and family cancer history.Overall, 75% of participants were aware of genetic testing and 19% of participants had genetic testing. Ancestry testing was the most common type of testing that the participants were aware of and had received. Non-Hispanic Asian, Non-Hispanic Black, and Hispanic respondents and participants with incomes less than $20,000 were less likely to be aware of and have received any type of genetic testing than the Non-Hispanic White participants and participants with higher income, respectively. Participants with a family history of cancer were more likely to be aware of cancer genetic testing than those without, and participants with a personal history of cancer were more likely to have had cancer genetic testing.It appears awareness of genetic testing is increasing in the United States, and differences in awareness persist by race/ethnicity and income. Genetic testing, which assesses the genetic variations inherited from each parent (germline variation), has multiple potential applications. In health care settings, genetic testing is used for predicting and managing the risk of developing several disease conditions, such as certain cancers, heart diseases, and a variety of inherited diseases. Genetic testing is commonly used in prenatal settings for preconception and prenatal screening. Results from these tests can have substantial effects for both individuals and their families. In addition to clinical settings, genetic testing for personal use has become increasingly popular. These tests can be used to determine ancestry, characteristic testing , and identifying certain health-related traits .1 Despite almost half of the Americans reporting being aware of genetic testing, awareness appears to differ across communities in the United States. Previous works have shown differences in awareness of genetic testing by race and ethnicity and income4 and differences in uptake specifically for cancer genetic testing by race and ethnicity.6The Health Information National Trends Survey (HINTS) is a nationally representative survey of adults aged 18 years and older in the United States and is designed to assess public awareness and use of health-related information. Previous research using the 2017 HINTS estimated that 57% of Americans were aware of genetic testing.7 Because of this relationship between awareness and receipt of genetic testing, it is critical to better understand public’s awareness of genetic testing. Differences in awareness of genetic testing may exacerbate existing disparities in health outcomes for low income and racial and ethnic minority groups.Understanding the differences in awareness is important because lack of awareness of genetic testing represents one potential barrier to receiving appropriate genetic testing. Those who are aware of genetic testing may be more interested in undergoing genetic testing and discuss it with their health care provider.The purpose of this study was to examine the association between current state of awareness and use of a variety of genetic tests and demographic characteristics, including race and ethnicity, family history of cancer, and personal cancer history.8 There were 3865 respondents to the HINTS 5 cycle 4, with a response rate of 37%. The analytical sample for this study included the 3767 participants who responded to at least one of the genetic testing questions (section F) on the HINTS. The 98 respondents who were excluded from the analysis were significantly more likely to be older, not employed, and widowed/divorced/separated than those who were included in the analysis; however, they did not differ significantly in terms of race, ethnicity, income, or personal/family cancer history.Data were analyzed from the 2020 HINTS 5 cycle 4 study, a probability-based nationally representative survey of adults aged 18 years and older in the United States designed to assess public awareness and use of health-related information. HINTS 5 cycle 4 was a self-administered mailed questionnaire. The HINTS instrument, methodology, and data are accessible to the public for analysis online.BRCA 1/2 or Lynch syndrome), not sure, and/or other. For the applicable outcome variable (heard of or received genetic testing), participants who selected either ancestry, genetic health-risk testing, high-risk cancer testing, or other were categorized as heard of or had any genetic test overall.Participants were asked separately whether they had ever heard of or had specific types of genetic tests (questions F1 and F3). For each question, participants were told to “mark all that apply” with the following response options: ancestry , genetic health-risk testing , high-risk cancer testing  what “other” type of test they had heard of or had received. Each “other” free text response was reviewed to determine whether the response belonged to one of the prespecified answer choices , should remain in the “other” category , or was not a genetic test. Participants who were missing a response to heard of genetic testing (F1) and reported having a genetic test (F3) were recoded as hearing of a genetic test. Respondents who responded “not sure” for heard of genetic testing (F1) and “not sure” for having a genetic test (F3) were recoded as not hearing of/having genetic testing. Participants missing for having a genetic test (F3) who reported not hearing of a genetic test (F1) were recoded as not having a genetic test (F3). In this analysis, “hearing” about genetic testing was interpreted as awareness of genetic testing.Demographic variables of interest included sex, race/ethnicity, age, income level, employment level, and marital status and were categorized on the basis of self-report from survey responses. A combined race and ethnicity variable was created with the following categories Non-Hispanic White, Non-Hispanic Black, Non-Hispanic Asian, Non-Hispanic other and Hispanic. Because of the small number of responses, respondents who identified as Alaska Native/American Indian, Pacific Islander, or as multiple races were combined into an “other” race category for the analysis. The counts for each of these groups are reported separately in N = 116: n = 105 with breast cancer and n = 11 with ovarian cancer) compared with those without a personal history of cancer because there were sufficient numbers to examine this subgroup, and there are clinical guidelines for discussing genetic testing. In addition, awareness of and receipt of genetic tests were examined looking at subgroups of those with and those without a personal history of cancer and those with and those without a family history of cancer. The results of these analyses were similar to the overall analysis. In compliance with the HINTS analytical recommendations, data were analyzed using the final sample weight to obtain population-level point estimates and the set of jackknife replicate weights to obtain correct started errors.9 A comparison of the weighted and unweighted percentages can be found in P value of <.05 was considered statistically significant. All analyses were performed in STATA/SE 16.1 (StataCorp).Associations between the demographic and cancer history variables with awareness of or had genetic testing were examined using multivariable logistic regression. Odds ratios (ORs) and 95% CI were calculated for each variable, adjusting for the other variables in the model. For awareness of and had high-risk cancer genetic testing, additional analyses were conducted to calculate adjusted ORs for participants with a personal history of breast or ovarian cancer ; however, only 19% of reported having genetic testing. The most common type of genetic test that participants were aware of and had received was ancestry testing. Furthermore, this study identified lower awareness and use of genetic testing in Non-Hispanic Asian and Non-Hispanic Black participants, Hispanic participants, and individuals with an annual income less than $20,000. Participants with a family history of cancer were more likely to be aware of cancer genetic testing and participants with a personal history of cancer were more likely to have received cancer genetic testing than their counterparts without a family or personal history of cancer.1 In the 2014 HINTS, 38% of respondents were aware of “genetic tests that analyzey our DNA, diet, and lifestyle for potential health risks are currently being marketed by companies directly to consumers.”7 Comparing the results of this analysis to the HINTS in 2017 and 2014, it appears that genetic testing awareness has increased over time. However, despite the high awareness of genetic testing in 2020, this study identified a lower percentage of respondents being aware of genetic testing for health risk and cancer purposes compared with ancestry genetic testing. Because of the high number of respondents being aware of ancestry testing, the change in awareness of genetic testing could be attributed to an increase in advertisements of at-home genetic testing kits.10 More work is still needed to better educate the public on genetic testing for health and cancer purposes.Compared with the results of a previous HINTS mailed in 2017, it appears that awareness of genetic testing is increasing, from 57% of respondents reporting that they were aware of genetic testing in 2017 to 75% in 2020.1 These differences are persistent across the different types of genetic tests. Racial, ethnic, and income disparities have previously been reported in awareness of genetic testing,15 and these differences have remained consistent over time. Differences in awareness of genetic testing represent 1 potential barrier for receiving genetic testing and may further exacerbate health disparities for racial and ethnic communities. In addition to being aware of the test, other barriers previously reported included concerns about effect of findings for family members, the potential for negative emotional responses, and mistrust and concerns over misuse.17 Although these groups are experiencing multiple barriers to genetic testing, previous work has suggested that racial and ethnic minority groups are interested in participating in genetic testing.18 Because germline genetic testing may have significant implications for health, more work is needed to understand barriers and identify methods to address them to improve awareness among racial and ethnic minority communities.Consistent with findings from previous HINTSs, this study found racial, ethnic, and income differences in awareness of genetic testing.10 and previous work using HINTS 5 cycle 4, which found that those with a history of breast, ovarian or colorectal cancer were more likely to have cancer genetic testing.5 In this analysis, results may have been driven by breast cancer cases among those with a personal history of cancer. Although these results are encouraging that there is an increased awareness of genetic testing for those with a family history or personal history of cancer, we were unable to confirm that those reporting of having a genetic test did so in accordance with current clinical care guidelines.In addition to identifying racial, ethnic, and income disparities, this analysis identified differences in awareness of genetic testing by cancer history. Notably, respondents with a family history of cancer were more likely to be aware of cancer genetic testing than those without a family history of cancer, and respondents with a personal history of cancer were more likely to have cancer genetic testing than those without a personal history of cancer. This is consistent with the findings from previous HINTS iterationsAlthough a strength of the study is the nationally representative nature of the survey, the response rate was low (37%). However, of note, weighted responses were similar to unweighted values. Second, because HINTS is a cross-sectional study, we are unable to comment on the temporal relationship between awareness and receipt of genetic testing in our study population. Another limitation is that some demographic groups were not well represented, including those with lower education and without health insurance. This limited our ability to examine association by these characteristics and limited generalizability. In addition, we were unable to examine other types of clinical genetic testing besides cancer testing . The absence of detailed information on personal and family history of cancer also limited the ability to evaluate in accordance with clinical guidelines. Because there were small number of individuals who had a personal history of cancer, we were unable to look at the differences across different cancer types. In addition, there were a small number of individuals with a personal history of cancer and who had cancer genetic testing, limiting our ability to evaluate the relationship between these variables. Future work is needed to examine uptake of genetic testing in those populations in which genetic testing is advised on the basis of clinical guidelines. Finally, in this analysis, we analyzed awareness of genetic testing, but we did not assess understanding of genetic testing among respondents.The results of this analysis suggest that most Americans are aware of genetic testing, but differences by race, ethnicity, and income persist for both awareness and use of genetic testing overall and by the different test types. Because of the increasing importance of genetic testing in the health care setting, it is critical to assess and address barriers to both awareness and access to genetic testing.1


Vitis vinifera L.) are a variety preferred by consumers owing to their exciting flavour and widely cultivated in Aegean Region in Turkey. The aim was to identify the biogenic amines of these table grapes during berry ripeness. The biogenic amines were analyzed by HPLC in six different berry phenological stages. Italia grapes presented lower biogenic amine content than Bronx Seedless table grapes. The concentration of most of the biogenic amines analyzed linearly raised from the beginning of berry touch to when berries ripen for harvest stages. The most common biogenic amines in grape varieties were putrescine, followed by histamine, agmatine, and tyramine. There was also a positive correlation between all biogenic amines of the two grape varieties. The weakest correlation was found between spermine and cadaverine, whereas the strongest correlation was found among dopamine, trimethylamine, norepinephrine, tyramine, and histamine amines. The present study is the first report of a synthesis study regarding the effect of B.A.s on quality characteristics throughout berry ripeness in grape varieties containing foxy and muscat tastes. The concentration and composition of biogenic amines identified for both varieties might provide helpful information regarding human health and the vintage.Bronx Seedless and Italia ( Vitis sp.) one of the most cultivated fruit crops worldwide [Global grape production reaches roughly 75 million tons annually, making grapes  and muscat  tastes are cultivated in different regions of Turkey, such as Central Anatolia Aegean, Southeastern Anatolia, and Marmara [Vitis vinifera grape cultivars and knowledge of the variation of BAs content in these varieties may be key for consumers. However, our knowledge of the dynamics of changes in the B.A.s profiles of Bronx Seedless and Italia table grapes throughout berry ripeness is limited. Monitoring B.A.s levels in these grape varieties can be an important marketing advantage and allow the establishment of B.A profiles for the safety and quality control of fresh consumption or by-products of these grapes. Therefore, this paper aims to present the changes in biogenic amines in Bronx Seedless and Italia table grapes throughout berry ripeness. Grape varieties containing foxy . Both putrescine (Put) and histamine (His) were mostly amines, whereas less common BAs were spermine (Spn) and norepinephrine (Nor). Generally, berries collected at BBCH-89 and BBCH-85 stages showed higher B.A. content than berries collected at other phenological stages for both grape cultivars. Put and his content reached the highest level at BBCH-89, whereas lower rates of spermine, norepinephrine, and spermidine were found at BBCH-77 when compared to BBCH-89, BBCH-85, and BBCH-83 for both grape cultivars. Considering the phenological stages, there was a linear increase in B.A.s contents of both grape varieties (p ≤ 0.05) between the two grape varieties. Total biogenic amine contents were higher in Italy grapes than in Bronx Seedless grapes  and serotonin (Ser). For both grape cultivars, the most common B.A.s in grape varieties was putrescine (Put), followed by histamine (His), agmatine (Agm), and tyramine (Tyr). There was wide variation in the B.A profiles, with values ranging from 0.83 (norepinephrine for Bronx Seedless) to 12.24 µg · Larieties . On the s grapes .p ≤ 0.01), whereas there was no correlation between Spn and Cad  appear in  and Cad . Apart f and Cad . PC1  (Nor). In addition, Put, His, Agm, and Try were the majority amine, coinciding with the results of Gomez et al. [Experimental observations on highly complex systems for B.A.s in grape varieties are mainly based on measurements performed in wine and must be rationalized in terms of elementary article science. The present study is, in this context, the first report of a synthesis study regarding the effect of B.A.s on quality characteristics throughout berry ripeness in grape varieties containing foxy and muscat tastes. Based on our results, we can state that there was wide variation in the total B.A.s between grape varieties, with values ranging from 12.24 (Put) to 0.83 µg · Lz et al. . Moreovez et al. ,28. On the other hand, the Bronx Seedless grape variety showed lower content of all B.A.s than the Italia grape variety, except for Spn and Spd . This waAs far as phenological stages are concerned, as the berry development progresses, individual B.A.s concentrations also rise, and there was a linear increase in all B.A.s contents of both grape varieties. The berries of BBCH-89 and BBCH-85 stages reached higher B.A. content than the berries of other phenological stages. The most common B.A.s in grape varieties were Put, His, Agm, and Try, whereas among these amines, Spn, Nor, and Spd were the lowest . It has p ≤ 0.05) was obtained among B.A.s (p ≤ 0.05), except for His, Dop, Tyr, Spn, Agm, and Nor ; between Cad and other B.A.s, except for Spn, Ser, and Tyr . The strongest correlation was found among Dop, Tma, Nor, Try, and His amines, whereas the weakest correlation was found between Spn and Cad  of B.A.s, on the other hand, efforts must be made to control B.A.s in grape products and regulatory limits for B.A.s have not yet been established by the OIV  for fresh grape, raisins, and wines; however, Lehtonen , stated The study was conducted in 2021 on twenty-year-old Bronx Seedless (New York 8536 × Sultanina) and Italia (Bicane × muscat Hamburg) vines grafted onto 5 B.B. root-stock in the Manisa Province (Manisa Viticulture Research Institute), Aegean Region, Turkey  cordon trellis system with 12–15 shoots per vine and one cluster per shoot at a northwest orientation, and vines were spur-pruned. Healthy berries (450 per cultivar) were randomly harvested in triplicate from the cluster’s bottom, middle, and top parts. Samplings were carried out on July 27 , and the latest sampling was August 28 , for a total of six selections. Based on BBCH scale published by Lorenz et al. , clusterStandard solutions of spermine (in the 1 to 30 mg/L range), agmatine, spermidine, serotonin, histamine, tryptamine, dopamine, norepinephrine, cadaverine, trimethylamine, putrescine, and tyramine were obtained from Sigma-Aldrich Chemie, Steinheim, Germany. Berries (5 g) were homogenized using an Ultra-turax homogenizer, including 0.5 mL of 70% perchloric acid. Homogenate (X g) was centrifuged at 10,000 rpm for 10 min. The supernatant was recovered, filtered over a 0.22 mm membrane, and diluted with 10% perchloric acid to the initial homogenate weight. The sample was then filtered over 0.45 µm and injected into the HPLC.−1 fresh weight.Biogenic amines (B.A.) were separated and quantified according to the method of Nagy et al. , with mop ≤ 0.05). The Pearson correlation coefficients were evaluated as scatterplot matrices using Analyse-it statistical software, and they were significant at the 0.01 (**) and 0.05 (*) levels.Descriptive statistics for the studied variables were analyzed considering a completely randomized design with the factorial arrangement, accounting for two grape varieties involving six phenological stages. The variables were subjected to a variety analysis (ANOVA) that was performed using SPSS 21.0 . Duncan’s test detected the significance of the differences in data (Although scientific advances have been achieved in understanding the biochemical, molecular, and physiological aspects of berry ripening in grapes, there is no previous research on the change of B.A.s throughout berry ripeness. Therefore, this study is the first report on B.A.s. The findings showed that Bronx Seedless table grapes presented higher contents of Put, Cad, Agm, Spd, His, Try, Ser, Tyr, Tma, Dop, Nor, and lower contents of Spn than Italia table grapes. The analyzed B.A.s concentration increased linearly as the berry ripened, and there reached the highest level in BBCH-77 and BBCH-89 stages. In addition, a significant positive correlation was detected between B.A.s and a strong correlation between Dop, Tma, Nor, Try, and His amines. Given the therapeutic value and importance of some amines of these grapes for humans, the pharmaceutical industry may capitalize on the potential human health benefits of pharmaceutical preparations for plants, animals, and humans. Indeed, it is likely that soon, we will be able to see the processing of pre-and post-harvest food crops and the production of fruits and vegetables with higher amine levels by combining traditional and modern growing approaches.


Pinctada maxima is a bivalve mollusc widely distributed throughout the Indo-Pacific and is the main species targeted for cultured pearl and pearl shell production in Australia. Pearl production in Australia relies heavily on wild-caught individuals, the majority of which come from the Eighty Mile Beach region near Broome in Western Australia. In this study, we used a genotyping by sequencing approach to explore fine-scale patterns of genetic connectivity among inshore shallow and offshore deep populations of P. maxima near Eighty Mile Beach. Our results revealed high-levels of gene flow among inshore and offshore sites and no differences in genetic diversity between depths. Global estimates of genetic differentiation were low (FST = 0.006) but significantly different from zero, and pairwise estimates of genetic differentiation among sites were significant in only 3% of comparisons. Moreover, Bayesian clustering detected no separation of inshore and offshore sample sites, and instead showed all samples to be admixed among sites, locations and depths. Despite an absence of any clear spatial clustering among sites, we identified a significant pattern of isolation by distance. In a dynamic environment like Eighty Mile Beach, genetic structure can change from year-to-year and successive dispersal and recruitment events over generations likely act to homogenize the population. Although we cannot rule out the null hypothesis of panmixia, our data indicate high levels of dispersal and connectivity among inshore and offshore fishing grounds.An understanding of stock recruitment dynamics in fisheries is fundamental to successful management.  Samples collected offshore  included one deep fishing ground (Compass Rose) and three other areas where moderate densities of pearl oysters had been recorded during oyster habitat towed video and multibeam sonar surveys  by commercial divers on surface-supplied air operating from Industry fishing vessels during neap tides on two sampling trips; shallow inshore samples (<10 m) were collected in May/June 2018, and offshore deep samples (>30 m) collected in July 2019 . Samplin surveys . Tissue fastq files were processed for poor quality (>Q20) and identical reads. These filtered reads were used in the secondary pipeline for DArT PL’s proprietary SNP calling algorithms (DArTsoft14).We generated reduced representation libraries using Pstl-HpaII restriction enzymes at Diversity Arrays Technology (DArT). DArTseq is conceptually similar to RADseq methods and uses enzymes to fragment DNA for sequencing. In this case, two enzymes were used to increase the number of fragments for SNP calling. The PstI-compatible adapter included Illumina flowcell attachment sequence, sequencing primer sequence and sample barcode region. Reverse adapter contained flowcell attachment region and HpaII-compatible overhang sequence. Libraries are amplified in 30 rounds of PCR using the following reaction conditions: PCR conditions consisted of an initial denaturation at 94 °C for 1 min followed by 30 cycles of 94 °C for 20 s, 58 °C for 30 s and 72 °C for 45 s, with a final extension step at 72 °C for 7 min. Equimolar amounts of amplification product are sequenced on an Illumina Hiseq2500 (single end 77 cycles). FASTQ files were processed for poor quality (>Q20) and identical sequences are collapsed into “fastqcall files”, which are groomed using DArT’s proprietary algorithm that corrects low quality bases from singleton tags using collapsed tags with multiple members as a template. These files are used in the secondary pipeline for DArT PL’s proprietary SNP calling algorithms (DArTsoft14). All tags from all libraries were clustered using DArT PL’s C++ algorithm at the threshold distance of three, followed by parsing of the clusters into separate SNP loci using a range of technical parameters, especially the balance of read counts for the allelic pairs. Additional selection criteria were added to the algorithm based on analysis of approximately 1,000 controlled cross populations. Testing for Mendelian distribution of alleles in these populations facilitated selection of technical parameters discriminating true allelic variants from paralogous sequences and contaminating sequences that has been achieved through training DArTsoft14 proprietary software’s capacity to perform this “filtering” of viral and/or bacterial sequences based on analysis of thousands of control crosses in large diversity of organisms. Equimolar amounts of 744 libraries were sequenced across nine lanes on an Illumina Hiseq2500 (single end 77 cycles). Raw dartR . Outliers were identified with fsthet using the fthetboot function and based on an alpha value of 0.05 and 1,000 reps. Loci identified as FST outliers under either approach were removed from the dataset prior to exploring patterns of population genetic structure.All statistical analyses were implemented using R software, version 3.0.1 unless noted . We filtdartR . We alsodartR , so it ioutflank  and fsthoutflank . Outflanpoppr (aov) to test for the significance of depth on heterozygosity. Overall levels of inbreeding (FIS) were calculated using hierfstat , correlated allele frequencies, a burn-in of 200,000 MCMC iterations and 500,000 iterations for each run. The number of K ranged from 1 to 10 (number of sampled sites), with five replicate analyses for each K value. The appropriate number of K was identified by comparing the likelihood of the data for different values of K and using the ∆K method in structure harvester  among sites for each species using PopGenReport  in ith ade4  using Eutructure  and Bayeadegenet . Structuarvester . Results , we observed a strong and significant (p < 0.001) heterozygote deficit in the total population, with only 18% of all loci that passed QC (559 SNPs) to be in strict HWE before adjusting for multiple comparisons at the 0.05 significance level (fastq files are available via the Open Science Framework (https://osf.io/v9u38/).Approximately 2M reads per sample  were used for variant calling in DArTsoft14, which returned 11,280 binary SNPs . After ftructure . DespiteIS = 0.211) and significantly different from zero at all sample sites (P < 0.001), we did not detect any differences in genetic diversity (as HE) between depths , ruling out the null hypothesis of panmixia. Despite a significant global FST value, only 3% of pairwise comparisons among sites (n = 12) were significant after correcting for multiple comparisons (n = 8), which was the most northerly of our inshore sample locations, or an Offshore four site (n = 7), which was the most southerly of our offshore sample sites. None of these comparisons, however, remained significant when restricting our analyses to the subset of loci in strict HWE. Similarly, hierarchical analysis of molecular variance (AMOVA) indicated that a small but significant portion of genetic variation was attributed to differences between sites within location  and depth  but not offshore  sites. Finally, spatial autocorrelation analysis showed no significant positive autocorrelation (greater than random genetic similarity) at most spatial scales in the dataset (Consistent with low levels of differentiation under the AMOVA framework, there were no obvious spatial clustering of samples by depth based on PCA , all samas K = 2 , but admas K = 2 . These pas K = 2 . Despite dataset , reconfiPinctada maxima from inshore and offshore sites near Eighty Mile Beach in Western Australia reflects one of high gene flow. Levels of genetic differentiation among sites were low and Bayesian clustering analyses indicated that all samples formed a single genetic population with no obvious separation of inshore and offshore sites. These patterns of genetic homogeneity across out study site are consistent with Pinctada species from other regions across similar spatial scales (The overall pattern of genetic structure in the silverlip pearl oyster l scales , and of l scales .P. maxima larvae tens of kilometres (P. maxima in the region. Oysters collected from offshore sites were larger in size than inshore oysters, but these differences may reflect our different sampling strategies rather than a result of the gauntlet fishing strategy of the industry. Pearl oyster collection inshore was conducted during normal commercial diving operations, and it is possible that divers preferentially selected smaller shells for our study that were not size for culture and retained larger animals to be sold to the boat for profit. The collections offshore were only for scientific purposes and therefore size was not confounded by fishing preferences.The patterns of high gene flow identified in this study are not surprising considering the limited spatial extent of our study, the moderate larval duration of the species , and thelometres . Particllometres . DespiteP. maxima in northwest Australia using allozymes and microsatellite markers (P. maxima near Eighty Mile Beach could be the result of inbreeding and assortative mating driven by subtle differences in the timing of spawning of individuals and/or patchiness in larval recruitment (Pinctada, and considering the strength of the inbreeding signal, it is possible that allele dropout is causing the observed deficits in heterozygosity observed across most of the loci.Our initial screening for deviations from HWE based on an adjusted significance threshold revealed a strong heterozygote deficit across thousands of loci. Heterozygote deficits are common in marine bivalves , and hav markers . An exce markers , or due ruitment . HoweverFST based on a small subset of loci in strict HWE, cautioning interpretations of the significant genetic differentiation observed here. Nevertheless, populations of highly fecund marine invertebrates often reflect a ‘sweepstakes’ chance of reproductive success, where a small group of individuals can account for a large proportion of the successful recruits to a given area (Estimates of genetic differentiation among sample sites showed that approximately 3% of pairwise comparisons were significant, most of which included a Cape Bossut site or an Offshore 4 site. It may be the case that complex currents and oceanographic features that form around the Cape act to entrap larvae and isolate nearby populations. Pearl oyster habitat in the Cape Bossut area is likely seeded by larvae to the north of Eighty Mile Beach region, possibly explaining the patterns of genetic differentiation observed here . These pven area . This reven area , 1984. I10.7717/peerj.13323/supp-1Supplemental Information 1Click here for additional data file.


Aedes aegypti has become one of the most abundant and dangerous arbovirus vectors in Asia. In this study, we assessed the invasion pattern of Aedes aegypti by investigating its distribution, relative abundance with its closest competitor Aedes albopictus, and genetic diversity in Vietnam. Our results revealed that the distribution and abundance of this species are significantly influenced by climate region, larval habitat, and the presence of Aedes albopictus. Furthermore, we found a high level of genetic diversity in Aedes aegypti, with two major genetic lineages. The results of this study have significant potential for optimizing vector control strategies regarding future mosquito invasion and outbreak prediction in Asia.Over the last few decades, the yellow fever mosquito Aedes aegypti has coexisted with the native species Ae. albopictus and has been reported to transmit several infectious diseases. However, the development of efficient disease prevention and vector control is hindered by the relatively poor understanding of the biogeography and the genetic diversity of Ae. aegypti in the region. This study aimed to determine the invasion patterns of Ae. aegypti by evaluating the distribution and abundance of Ae. aegypti and Ae. albopictus in different climatic regions  and habitats . We further analyzed the genetic diversity and phylogenetic relationships of Ae. aegypti populations in Vietnam using mitochondrial COI gene sequences. Both Aedes species were observed at most of the study sites, but only Ae. albopictus thrived in northern mountainous areas. In sympatric ranges, the individual abundance of the species was influenced by regional climate and habitats. The tropical climate and availability of domestic containers facilitated the dominance of Ae. aegypti, whereas temperate climates and natural breeding sites facilitated that of Ae. albopictus. In addition, many genetic polymorphisms were detected in the Ae. aegypti populations, which formed two distinct genetic groups; however, this genetic diversity is unlikely to be relevant to the invasive success of Ae. aegypti. These findings provide insights into the mechanisms and patterns of Ae. Aegypti invasion, which depend on the climate and reproductive strategies in the native range of Ae. albopictus in Asia.Since its introduction to Asia,  Aedes aegypti Linnaeus 1762 and Ae. albopictus (Skuse 1894), the two most invasive mosquito vectors native to Africa and Southeast Asia, respectively, have attracted increasing attention [Recently, interest in researching methods of biological invasions has increased significantly, especially with respect to mosquito species that are capable of transmitting vector-borne diseases . Particuttention . InteresSkuse 189, the twottention ,6. TheirSkuse 189, the twottention ,11. Howettention .Ae. albopictus and Ae. aegypti, results in biological invasion leading to interspecific competition, affecting the distribution and abundance of the resident species. For example, Ae. albopictus has shown a competitive advantage over Ae. aegypti in the native range of Ae. aegypti in Africa [Ae. aegypti habitats in southeastern USA [Ae. aegypti by Ae. albopictus, including interspecific competition [Ae. albopictus [Aedes abundance in southern Asian cities have suggested that Ae. aegypti could displace Ae. albopictus [The reliance of native and invasive species on similar ecological niches, as in the case of n Africa ,14,15 antern USA  and Braztern USA ,18. A vapetition ,20, matibopictus , differebopictus , and tolbopictus . In contbopictus ,25. HoweAe. aegypti. Thus, a combination of biogeographic distribution and population genetic analyses of invasive species is needed to fully understand the invasion and evolutionary processes of invasive species in novel habitats and regions. Mitochondrial COI genes are widely used as molecular markers for population genetic studies in mosquitoes [Population genetics significantly contributes to the study of mosquito invasions by providing critical information for tracking invasion routes and putative source populations, measuring and comparing the genetic diversity of native and introduced populations, and investigating the evolution of introduced populations in novel environments ,27,28. Asquitoes ,31,32,33Ae. aegypti invasion was first reported in a central province (Hue) in the 1910s [Ae. albopictus, which is indigenous to Vietnam, is considered a secondary vector of both dengue and chikungunya [Aedes in Vietnam was conducted over 10 years ago. In addition, the study only sampled used tires, which may not provide a comprehensive evaluation of the relative abundance of the native species or the progress of Ae. aegypti invasion because both species utilize water stagnated in various types of containers in urban areas [Ae. aegypti populations in Vietnam has not yet been determined by tracing the maternal genetic relationship using the COI gene.In Vietnam, he 1910s . After ahe 1910s ,36,37. Dhe 1910s  and has he 1910s ,40. Intekungunya . Despitean areas ,38,39,42Ae. albopictus and the invasive species Ae. aegypti in Vietnam and to evaluate the genetic diversity of Ae. aegypti in Vietnam. The outcomes of this study may provide insights into the colonization process of Ae. aegypti in the native range of Ae. Albopictus, clarify the roles of regional climate and habitat types in determining the distribution and abundance of Aedes species, and elucidate the poorly understood genetic aspects.The aims of this study were to compare the geographic distribution of the native species Vietnam is part of the eastern Indochinese peninsula. The land area extends from 8 to 24° N in latitude, encompassing a great variety of terrain. The South China monsoon also contributes to climatic heterogeneity between the northern and southern areas of the country. In general, Vietnam includes two main climatic regions (northern and southern Vietnam). More specifically, the northern region is characterized by a temperate/subtropical climate with a cold-dry winter, especially in the northern mountainous regions, which often experience subzero temperatures. The southern region is characterized by a tropical climate with two hot seasons (hot-rainy and hot-dry) .Aedes mosquitoes, and frequently reported dengue transmission  , peri-domestic , and natural  habitats . Aedes albopictus samples from several locations were also included in our previous study, which investigated the genetic diversity and population structure of Ae. albopictus in Vietnam [We chose one study site per city such that all study sites had similar habitat conditions and included a variety of potential sampling points, such as residential houses, recreational parks, playgrounds, car repair stations, pagodas, and schools. Each study site was a circular area 5 km in diameter or approximately 78.5 kmn total) . The areg points . Furtherhabitats c. The moat types . Multi-w Vietnam .Ae. aegypti populations in both north and south Vietnam. Only one or two specimens from each breeding habitat were included in the genetic analysis to prevent sibling bias; these specimens were preserved in absolute ethanol at −20 °C.The representative study sites that harbored specimens from both species were used to investigate the genetic diversity and population structure of the COI gene was PCR-amplified from each DNA sample using the AccuPower PCR PreMix kit  and a previously published universal primer pair  [Genomic DNA was extracted from the legs of adult specimens and abdominal muscles of larval specimens using the DNAeasy Blood & Tissue kit , by following the manufacturer’s recommendations. A 658-bp fragment of the mtDNA ATCA-3′) . The ampATCA-3′) . D and Fu’s Fs were estimated for each population using ARLEQUIN v. 3.5.2.2 to establish non-neutral evolution and deviation from mutation-drift equilibrium, with population expansion signatures inferred using significance values that were calculated by generating 1000 random samples [CT), among populations within regions (ΦSC), and within populations (ΦST) using permutation tests with 1000 random permutations.The number of haplotypes, number of polymorphic sites (S), haplotype diversity (Hd), and nucleotide diversity (π) were calculated using DnaSP v. 6.12.03 . Tajima’ samples . To invehttp://popart.otago.ac.nz/index.shtml, accessed on 23 September 2021) [A haplotype network was inferred using the nexus file from DnaSP and PopArt v. 1.7. software (er 2021) .Ae. aegypti in Vietnam, the phylogenetic relationship between haplotypes from Vietnam and those from native and invasive Ae. aegypti populations worldwide  of the study sites, followed by natural  and domestic habitats  with standing water were identified in the 18 study sites along a north–south transect in Vietnam ; most coAedes albopictus and Ae. aegypti accounted for 58.7 and 41.3%, respectively, of the 17,178 immature specimens collected during this study . In contrast, peri-domestic containers had a higher frequency of 68.9% (310 containers) for Ae. albopictus compared with 31.1% (153 containers) for Ae. aegypti. Similar results were observed for natural breeding types, with 106 containers containing Ae. albopictus larvae, whereas the Ae. aegypti larvae were found in 12 containers . Furthermore, Ae. aegypti were dominant over Ae. albopictus in almost all domestic container types at sites where both species coexisted (S7–S18) and in peri-domestic containers at the southern sites , Culex quinquefasciatus Say, 1823, Toxorhynchites splendens , Tripteroides aranoides , and Aedes annandalei , were also observed in Aedes habitats, but were observed to occur only in a few containers at very low densities, suggesting the dominance of Aedes species in such breeding habitats.Other species, including COI sequences from 122 Ae. aegypti specimens yielded 29 haplotypes, which were distinguished by 34 variable sites (4.7%); of these, 25 were parsimony-informative  yielded insignificant results (p > 0.05) for the population as a whole or each local population individually, indicating that the observed polymorphisms were consistent with the neutral mutation model ; however, most of the variance still occurred within the populations  sites, whereas the native species Ae. albopictus accounted for a larger proportion of immature mosquitoes observed at the northern  sites  sites a. The inbopictus ,53,54. Mow 17 °C . In the ow 17 °C ,56. The pable of . Consequ in 2010 , which snvasions . Aedes species. Ae. aegypti generally prefers indoor containers, whereas Ae. albopictus prefers outdoor water sources near vegetated areas in distributional ranges [Ae. albopictus by Ae. aegypti in Vietnam. In particular, Ae. aegypti was dominant in peri-domestic water sources in southern Vietnam and in most domestic water sources in areas where both species occurred, whereas Ae. albopictus maintained its dominance in natural water sources and peri-domestic habitats of northern Vietnam . Furthermore, when alien species encounter environments that differ from their native ranges, they need to adapt to local environments [Ae. aegypti that enable it to be a successful invader, such as: (1) house entry (endophily), (2) preference for oviposition in human-made domestic containers, and (3) preference for blood-feeding from humans [Ae. aegypti were dominant in domestic containers in locations where close contact with humans was more likely to occur . Among the domestic containers, flowerpots and ceramic jars were the most frequent oviposition containers for Ae. aegypti. Meanwhile, both Aedes species utilized used tires regardless of the region. This habitat information could be important for Aedes surveillance and its control in urban areas in Asian countries, especially in Vietnam. Additional laboratory and field investigations focusing on the interactions of the Aedes species and effects of environmental factors  on the outcomes of their competition are needed to elucidate the mechanisms underlying species displacement and facilitate the prediction of future range expansion of Aedes in Asia. Furthermore, because this study was conducted in urban areas across Vietnam, investigations must be undertaken in other areas, such as rural or forest areas, to examine the invasion pattern of Ae. aegypti, especially in the southern ranges.The preference of breeding habitat could also affect the relative abundance of l ranges ,55. Inde Vietnam . A multironments . The finm humans . Indeed,to occur . This beAe. aegypti using genetic analyses. Interestingly, multiple genetic lineages were detected  being similar to that at the southern sites , its density was significantly lower in the northern sites , which accounted for 60.7% of all specimens analyzed in this study, have been reported in many invasive populations in the Americas, Asia, and Europe. Therefore, these major haplotypes are highly adaptive to novel environments and could be primary invasion sources, not just in Vietnam, but worldwide. Moreover, the phylogenetic tree revealed two deeply divergent groups (I and II), with Group II further divided into subgroups IIa and IIb (Ae. aegypti is likely ongoing, and different genetic lineages have invaded the country, perhaps at different times and possibly through different routes. The phylogenetic tree constructed in this study was unable to strongly support the origin of Ae. aegypti lineages in Vietnam; however, most of the detected haplotype groups have also been associated with invasion in other parts of the world. Taken together with the major haplotype similarity results, the Ae. aegypti population in Vietnam is likely to have been introduced through other invasive populations. Future studies using high-resolution multilocus genomic data, a larger scale of sampling, and sampling in both native and invasive regions are needed to elucidate the invasion history of Ae. aegypti in Vietnam. Such studies could help determine whether genetic clusters were introduced in a series of invasions or in a single event.This study used the phylogenetic analysis of  and IIb . The pre and IIb . This suAe. aegypti in the native range of its closest competitor Ae. albopictus in Vietnam. This study highlights the success of Ae. aegypti invasion in a part of Asia that has significant variation in local climate and habitat preference. Furthermore, the contrasting distribution and abundance patterns of the two Aedes species indicate an ongoing displacement process in the region. The sources of Ae. aegypti in Vietnam are likely to be genetically similar to other invasive Ae. aegypti species worldwide. Furthermore, we observed a high level of genetic diversity throughout the country. However, the level of genetic diversity is not likely to be responsible for the invasive success of this species.By combining biogeographic and population genetic approaches, this study provides novel insights into the introduction patterns, expansion, evolutionary history, and genetic structure of the invasive mosquito