Thunnus alalunga) support an economically valuable global fishery, but surprisingly little is known about the population structure of this highly migratory species. Physical tagging data suggest that Albacore from the North and South Pacific Ocean are separate stocks, but results from previous genetic studies did not support this two stock hypothesis. In addition, observed biological differences among juveniles suggest that there may be population substructure in the North Pacific. We used double‐digest restriction site‐associated DNA sequencing to assess population structure among 308 Albacore caught in 12 sample areas across the Pacific Ocean . Since Albacore are highly migratory and spawning areas are unknown, sample groups were not assumed to be equivalent to populations and the genetic data were analyzed iteratively. We tested for putatively adaptive differences among groups and for genetic variation associated with sex. Results indicated that Albacore in the North and South Pacific can be distinguished using 84 putatively adaptive loci, but not using the remaining 12,788 presumed neutral sites. However, two individuals likely represent F1 hybrids between the North and South Pacific populations, and 43 Albacore potentially exhibit lower degrees of mixed ancestry. In addition, four or five cross‐hemisphere migrants were potentially identified. No genetic evidence was found for population substructure within the North Pacific, and no loci appeared to distinguish males from females. Potential functions for the putatively adaptive loci were identified, but an annotated Albacore genome is required for further exploration. Future research should try to locate spawning areas so that life history, demography, and genetic population structure can be linked and spatiotemporal patterns can be investigated.Albacore ( From a practical standpoint, it is hard to comprehensively sample HMMS year‐round across their vast geographic ranges, which typically span the open ocean and extend across numerous international boundaries. As a result, genetic sampling is often temporally and spatially limited, and relies on collaborations across fishery sectors. These sampling challenges often lead to infrequent records of age, sex, and length, which means that ontogenetic, demographic, and phenotypic patterns associated with the genetic samples may not be detected. In addition, the location and timing of reproduction are often unknown for HMMS , panmictic populations with frequent dispersal and high gene flow, and low genetic variation  and the Sailfish Istiophorus platypterus  are ecologically similar HMMS, except that movements of the Sailfish appear to be restricted to coastal waters  revealed six temporally consistent populations, using 59 putatively adaptive loci . Albacore are fast, efficient swimmers capable of traveling over 110 km per day .A total of 361 Albacore were sampled from 12 sample areas in the Pacific Ocean Table . All fisFork length (FL) or total length (TL) was recorded in cm for all samples, except for fish from British Columbia and Tasmania that were not measured Table . Based o2.2A tissue sample  was taken from each individual. Tissue samples were transferred to separate 1.5‐ml microtubes and preserved in either 95% ethanol or RNAlater (Sigma‐Aldrich). All sample processing was conducted at Oregon State University's Center for Genome Research and Biocomputing.PstI‐HF  and SphI‐HF   at 37°C for 2 h. Following digestion, 91 barcoded adapters and a common adapter were ligated to individual samples using T4 Ligase (New England Biolabs) at 22°C for 2 h, and heat‐inactivated at 65°C for 30 min. All samples were pooled and cleaned using QIAquick PCR Purification Kit (Qiagen) and 200‐ to 500‐bp fragments were isolated on a Blue Pippin (Sage Science). Size‐selected fragments were then amplified with Illumina primers under the following conditions: 98°C 30”; 98°C 10”, 68°C 30”, 72°C 30” (15 cycles); and 72°C 5’, 4°C hold. The PCR product was purified, eluted, and quantified using a Qubit fluorometer (Thermo Fisher Scientific). The final library consisted of 361 Albacore organized onto four 96‐well sequencing plates, with 91 or 92 individuals per plate, sequenced on separate lanes. The four library plates were each run on an Illumina HiSeq 3000 lane using 150‐bp paired‐end sequencing chemistry.Genomic DNA was isolated from ~50 mg of tissue using a DNeasy Blood and Tissue Kit (Qiagen) and normalized to 20 ng/µl. Each sample was double‐digested in a 20 µl reaction using the high fidelity restriction enzymes 2.3stacks 2.41 . Population genetic variation was estimated using the populations component of the stacks pipeline. Following the recommendation of Mastretta‐Yanes et al.  based on this error rate, the number of variant loci, and a conservative aim to avoid a high number of potentially erroneous reads.Loci and genotypes were called for mapped reads using the s et al. , we modily Table . As recoly Table , we inveThree datasets were selected for investigation. In the “12 sample area dataset,” all individuals were organized into the 12 sample areas (‐p 12). In the “North and South Pacific dataset,” all individuals were organized into North and South Pacific groups (‐p 2). In the “New Caledonia sex dataset,” all 54 Albacore from New Caledonia were organized into two groups for males and females (‐p 2). The minimum percentage of individuals in a population required to process a locus for a given population was set at either 90% or 95% (‐r 0.9 or 0.95). A minimum allele frequency of 5% was enforced for loci (‐‐min_maf 0.05). Only the first SNP of each locus was included (‐‐write_single_snp), and all SNPs were biallelic.vcftools 0.1.16  in each group  so that these sites were removed from subsequent analyses  in the raw RAD sequencing data that can produce misleading genotypic results  and expected heterozygosity (HE), allelic richness, and the inbreeding coefficient (FIS) were estimated for each tested group  using the adegenet 2.1.1 R package , again implemented in Jombart, . All loc2.7fsthet 1.01  was estimated for groups using putatively adaptive loci, presumed neutral loci, and all loci in each dataset. This index was estimated using the stampp 1.5.1 R package  in structure 2.3.4 . For each value of K, five replications of the admixture model with independent allele frequencies were applied, with an MCMC length of 50,000 generations and a 10% burn‐in. The optimal number of clusters was determined by examining estimates of mean K probability for a given value of K  hybrid individuals were estimated in geneclass 2.0h  and British Columbia (n = 10) were likely influenced by the limited sample sizes for these areas. Hawaii exhibited a lower FIS value relative to NW of Hawaii and other sample areas  (Table In the 12 sample area dataset (6446 loci), slightly fewer heterozygotes were observed than expected for all of the sample areas, except Baja California Table . This sles Table . Resultsas Table . However%) Table . Alterna%) Table . These r%) Table .n = 48) contained a mixture of North and South Pacific individuals  was meaningful based on PC eigenvalues Figure a. The 10s Figure . Memberss Figure . Notablys Figure . These iAlternative patterns of genetic structure were explored within the 12 sample area dataset. Collection year did not reveal any clear pattern among samples Figure a. Likewi3.2.2stacks parameters and group size. There were slightly fewer heterozygotes observed than expected for the North and South Pacific groups, which was reflected by positive FIS values . Genotyping error was again considered to be unlikely to have caused this result, as analysis in whoa estimated a low heterozygote miscall rate (4.3%) for the North and South Pacific dataset  containing a mixture of North and South Pacific individuals  was again the only meaningful axis of variation based on eigenvalues Figure b. As wits Figure . Again, s Figure . These f3.2.3whoa, the estimated heterozygote miscall rate for the dataset was low (3.6%) . Based on analysis in %) Table . Accordi%) Table c, but th%) Table .3.3Since the only prevailing genetic pattern across all three loci datasets was the separation between the North and South Pacific, we focused on the North and South Pacific dataset for subsequent analyses. However, some of these downstream analyses were replicated for the 12 sample area dataset in order to check for consistency in results, which are presented in Appendix Putatively adaptive loci were identified by all four genome scan programs in the North and South Pacific dataset Table . Putativfsthet analysis  for loci are shown in Figure As expected, given the lack of alternative genetic structure, there was a strong relationship between the putatively adaptive loci and PC1, which distinguished samples from the North and South Pacific. This relationship could be observed using the PC1 loadings for loci, where loci with the largest loadings were all identified as putatively adaptive sites Figure . As reco3.4FST value between the North and South Pacific populations was high at 0.4055, whereas the FST estimate for the remaining presumed neutral loci was low at 0.0005 , presumed neutral loci (20 PCs), and all loci  exhibited near‐even assignment (0.4–0.6) between the two clusters. Of these individuals, seven were assigned to Cluster B identified in the PCA plot  in structure (Table ). No other individuals with near‐even assignment in structure were identified as F1 hybrids by geneclass2. Overall, one individual from the Philippine Sea and another from New Caledonia were identified as potential hybrids by all three methods  could be identified as F1 hybrids using the 84 putatively adaptive loci in ic Table . Furtherre Table . No othds Table .3.5blastn, 65 out of 74 of these contigs successfully aligned with fish DNA sequences on GenBank. Of these, 37 contigs aligned with genes, six aligned with microsatellites, and the remaining 22 sequences matched with unannotated contigs for other fish genome sequencing projects  were apparent in the PCA plots using both the 12 sample area and North and South Pacific datasets Figures . Clustert Figure . Furtherns Table . Differegeneclass2  among a priori groups. Collectively, this means an assumption of equivalence between sample areas and populations may bias estimates of genotypic variation and affect the detection of putatively adaptive loci. In this study, we attempted to overcome this challenge by examining numerous groupings within our data, starting from the identification of loci in stacks (Table In this study, we analyzed genetic variation thoroughly by iteratively testing different groupings of individuals and responding to patterns that became apparent in the data. We recommend that this approach be applied to future genetic studies of HMMS, especially for the processing of RADseq data and the detection of putatively adaptive loci. In particular, in analytical pipelines such as ks Table . The ben5The results of this study indicate that Albacore in the North and South Pacific can be distinguished using 84 putatively adaptive loci, but not using the presumed neutral sites. Two individuals likely represent F1 hybrids between the North and South Pacific, and 43 Albacore potentially exhibit lower degrees of mixed ancestry. In addition, four or five migrants were potentially identified between the two populations, but none of these individuals were consistently identified across datasets. Collectively, these genetic results suggest that migration and gene flow occur across the equator, but unknown selective differences in the North and South Pacific have led to strong genetic differentiation for some parts of the Albacore genome. As with studies on other tunas (Grewe et al., The authors have no conflicts of interest to declare.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.Appendix S4Click here for additional data file.


SCN5A gene using next-generation sequencing (NGS) techniques and Sanger sequencing. Female sportsman (14 years old) underwent genetic counseling and DNA testing due to QT interval prolongation registered during ECG Holter monitoring. Genetic testing of the proband was performed in two independent laboratories. Primary DNA testing was performed by WES using the Ion ProtonTM System. Target panel sequencing of 11 genes was performed using PGM Ion Torrent. Search for variants in non-canonical and canonical exons 6 was performed by Sanger sequencing. The cascade familial screening and control re-sequencing were provided for proband with identified genetic variant p.S216L  in the canonical exon 6 of the SCN5A gene after receiving data from another laboratory. Control Sanger and NGS sequencing revealed the absence p.S216L in the canonical exon 6 and confirmed the presence of p.S216L  in the non-canonical exon 6 of the SCN5A gene. The identified variant was re-interpreted. The non-canonical transcripts of the exon 6 of the SCN5A gene is poorly represented in cardiac tissue (gnomAD). The detected variant was found in proband’s healthy mother. The correct interpretation of genetic data requires close cooperation between clinicians and researchers. It can help to avoid financial costs and stress for proband’s and families.Primary cardiac channelopathies are a group of diseases wherein the role of DNA testing in aiding diagnosis and treatment-based decision-making is gaining increasing attention. However, in some cases, evaluating the pathogenicity of new variants is still challenging. We report an accurate multistage assessment of a rare genetic variant in the  Significant advance has been made in the field of DNA-diagnostic strategies with the development of different massive parallel sequencing methods. These changes are especially pronounced in approaches to perform DNA-based diagnoses of monogenic polyallelic diseases, since it is now possible to simultaneously search for mutations in all candidate genes. Primary cardiac channelopathies are a group of diseases wherein the role of DNA testing in aiding diagnosis and treatment-based decision-making is gaining increasing attention. Although disorders of this class present different electrocardiogram (ECG) phenotypes, they share a high risk of sudden cardiac death (SCD). At present, guidelines prescribed by the American Heart Association, Heart Rhythm Society, European Society of Cardiology, and European Heart Rhythm Association have strongly recommended genetic testing for the diagnosis and treatment of various heart rhythm and conduction disorders .Although next-generation sequencing (NGS) approaches have their obvious advantages, there are certain difficulties in accurately interpreting massive amounts of genetic data in the clinical context; the greatest being the interpretation of new and rare genetic variants with unknown clinical significance (VUS). Standards and guidelines for the interpretation of genetic findings were first published in 2015 by the American College of Medical Genetics and Genomics . In 2019In addition, the growing need for diagnostic tests based on NGS is driving the development of new machine learning technologies for solving a wide range of issues in genetic studies, from selecting targets for sequencing to functional annotation of genes or genetic elements . This haSCN5A gene using NGS techniques and direct Sanger sequencing.In this article, we report an accurate multistage assessment of a rare genetic variant in the Clinical examination and genetic testing were performed in accordance with the principles of the Declaration of Helsinki, and upon the direct request of the patient. Written informed consent from her parents was also obtained. The clinical examination consisted of a general examination, standard ECG at rest and during exercise, and 24-hour Holter monitoring.TM System  using the Ion AmpliSeqTM Exome Kit (Thermo Fisher Scientific). Primary processing of reads was performed using the Ion Proton Software (Thermo Fisher Scientific), and the sequence reads were aligned to the hg19 reference genome using the BWA 0.7.9 software package (Genetic testing was performed in two independent laboratories. Primary DNA testing involved whole exome sequencing (WES) performed on the Ion Proton package .The obtained bam files were filtered using the exome map file, and results were called using the samtools 1.1 package. Finally, variant filtration was completed using the vcf-annotate package and in-house scripts. The vcf file was annotated using the ANNOVAR package . For WES–5, how it was recommended in “Enhancing rare variant interpretation in inherited arrhythmias through quantitative analysis of consortium disease cohorts and population controls”  were filtered by a frequency with a threshold for the maximum allelic contribution of 5.5 × 10ontrols” . Then biontrols” , and verontrols” .KCNQ1, KCNE1, KCNE2, KCNE3, KCNJ2, KCNH2, SNTA1, SCN5A, SCN1B, SCN3B, and SCN4B) using the Ion Torrent semiconductor sequencing platform  was performed. Oligoprimers for this gene panel were designed using the Ion AmpliSeq Designer® online tool (Thermo Fisher Scientific). The reads were preprocessed using Torrent Suite Software 5.6.0, and variant annotation web server Ion Reporter 5.12 (Thermo Fisher Scientific), and 92.15% of the target areas of the genes was covered >20×.An independent target genes panel of 11 genes  tool  with hg1SCN5A gene. Carrier screening for these rare variants was also performed in the parents using capillary electrophoresis-based Sanger sequencing with two oligoprimer pairs  and the canonical exon 6 (chr3: 38655234–38655325) of the er pairs .Pathogenicity evaluation for the identified genetic variants was carried out according to the guidelines .Patient NRF178 was a 14 years old female, competitive athlete (swimming) with 6 years of active sport experience. She had no general health complaints, no history of syncope, no family history of SCD, and she exhibited good exercise tolerance. She was recommended to undergo genetic counseling and testing after an annual cardiac examination.While resting, her ECG showed a sinus rhythm, heart rate of 61 beats/min, QT interval of up to 480–500 ms, and QTc interval of 495 ms. In response to standing up, her ECG showed a sinus rhythm, heart rate of 72 beats/min, QT interval of up to 480 ms, and QTc interval of 535 ms . One monSCN5A gene. This variant was identified in a large number of forward and reverse reads and was automatically recognized by the software. It was classified as a VUS (Class III) and was included in the final genetic report with correct genomic coordinates, changes in the coding sequence, and the corresponding amino acid substitution; however, the reference number (rs201002736) and expression profile  was not mentioned. This final genetic report was directly handed over to the patient’s parents without any genetic counseling. They were then transferred to cardiologists for clinical decision-making. The medical team performed an additional search for information on the genetic variants indicated in the genetic report using “SCN5A,” “p.S216L,” and “primary channelopathies” as keywords. The physicians mainly focused on studies wherein the replacement of p.S216L in the canonical exon 6 of the SCN5A gene was discussed in a clinical context.Primary DNA testing involving WES revealed a rare genetic variant, g.38655522G>A  in the SCN5A gene in an independent laboratory, without providing the initial results. Based on the parent’s formulated request, targeted analysis of exon 6 of SCN5A  was performed by capillary Sanger sequencing, and no genetic abnormalities were reported.However, given the importance of genetic test results in predicting the patient’s health and for deciding whether to continue her sports career, the family requested a second genetic screening of exon 6 of the SCN5A. The rare genetic variant g.38655522G>A was detected in the non-canonical exon 6 of the SCN5A gene  by NGS  by NGS  and Sang) by NGS . Parents) by NGS . The nonc tissue . This vaThe final report of the repeated genetic tests did not contain any variants. During genetic counseling, family members and cardiologists were informed of the reasons for the apparent discrepancies in the final reports.The answers for key questions from the family were formulated as follow:Q1. Is there a discrepancy between the results from two genetic laboratories? A1. No.Q2. Does variant g.38655522G>A really exists in the proband’s DNA? A2. Yes.Q3. Does this mean that the LQTS was confirmed by genetic testing? A3. No.Q4. Does this mean that the LQTS was excluded by genetic testing? A4. No.Q5. Does this mean that competitive sports are safe or prohibited? A5. No/No.Q6. Who and how can they make decisions about sports? A6. A shared decision should be made for this ECG-positive genotype-negative patient taking into account QTc dynamics, patient intentions, possible risks, and current recommendations , and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.AS performed the molecular genetic investigation, data analysis, and drafting the manuscript. LM and VK contributed to the clinical investigation. IK, LS, and EG contributed to the molecular genetic investigation and bioinformatical procession of data. EZ contributed to the genetic counseling, management of the project, and editing and final approval of the manuscript. All authors read, discussed, and approved the manuscript as submitted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The impact on fermented sausages with 25% replacement of the sodium chloride content by 14% potassium chloride, 10% calcium ascorbate and 1% calcium glutamate during fermentation and ripening was evaluated based on the bacterial community composition and physicochemical and sensory characteristic analysis. Our results showed that the use of salt replacement varied the composition of the bacterial community and reduced the diversity of that in sodium-reduced fermented sausages. Moreover, the decrease in pH and the moisture content of fermented sausages with salt replacement accelerated the drying and ripening processes. The texture profile and color analysis did not reveal marked differences between normal fermented sausages and sodium-reduced products with salt replacement; however, salt replacement reduced resilience and lightness of fermented sausages. In addition, as shown in the principal component analysis, the comprehensive parameters of the fermented sausages with salt replacement were similar to those of normal salt products. These results indicate that the complex blends of salt replacement have great potential to be used to produce sodium-reduced fermented sausages. Excessive salt  intake is associated with an increased risk of high blood pressure , cardiovascular disease and some types of cancer ,2 and isConsequently, much research has been conducted to identify ingredients to replace the functionality of NaCl in meat products, especially focusing on the use of other chloride salts. Potassium chloride KCl, E508) provides one of the most direct salt replacements due to the similarity in the molecular composition and is commonly used in processed meat products without affecting their texture, color and microbiological characteristics  provides,10,11,12Calcium ascorbate  is a possible substitute for salt and is employed as an antioxidant in processed milk and meat products, which also accelerates the curing reaction and develops a stable cured color in cured meat products ,19,20. MHence, in this study, we aim to find out the potential effect of 25% replacement of the NaCl content by 14% KCl, 10% Vc-Ca and 1% calcium glutamate on the bacterial community composition and physicochemical and sensory characteristics of the fermented sausages during fermenting and ripening. This study provides the basis for potential applications of complex blends of salt replacement to be used for low-salt fermented sausage production.Pediococcus pentosaceus and Staphylococcus xylosus  for traditionally fermented meat products. The stuffed sausages with a diameter of 45 mm were hung in a temperature-controlled room for 36 days. Fermentation was performed at 25 °C and 94% relative humidity (RH) for the first 4 days. For the ripening process, the temperature was set at 15 °C, 94% RH for 14 days and 80% RH for 14 days. Samples were taken on days 0, 4 (end of fermentation), 12, 20, 28 and 36 (end of ripening).The fermented sausages were prepared according to Zhang et al.  with somw) was determined using a HygroLab-C1 . The chloride content of the fermented sausages was confirmed by the Volhard method as shown in ISO 1841-1:1996 [The pH was determined by homogenizing 5 g of minced fermented sausages in 50 mL of distilled water and using a pH meter . The weight loss was calculated as a percentage of the reduced weight to the weight of fermented sausages on day 0. The moisture content was measured according to the high-temperature drying method by oven at 105 °C. The water activity  was conducted using a texture analyzer by TAXT software  fitted with a cylindrical probe . Sausages were cut into cylindrical shapes  to fit on the platform. The double compression cycle test was conducted up to 50% of the height of the sample. TPA parameters were set at a pre-test speed of 2 mm/s, test speed of 1 mm/s post-test speed of 2 mm/s, probe return speed of 5 mm/s and contact force of 5 g. The determined parameters were hardness, adhesiveness, cohesiveness, springiness, chewiness and resilience. Color measurement of the fermented sausages was determined using a colorimeter . The values of L* (lightness), a* (redness) and b* (yellowness) were recorded from three different areas of the samples.Staphylococcus and Micrococcus and Enterobacteriaceae were determined by plate count agar, de Man, Rogosa, and Sharpe (MRS) agar, mannitol salt agar (MSA) and violet red bile dextrose agar (VRBDA) at 37 °C for 48 h, respectively. The results were expressed as Log CFU/g.Microbiological analysis was according to the method described by Ravyts et al. . BrieflyBacteria in samples were extracted according to Xiao et al.  and bact® DNA Library Prep Kit . The purified PCR amplicons with the generation of 2 × 300 base pairs (PE300) were sequenced on the Illumina MiSeq platform by Majorbio Bio-Pharm .The universal primers 338F and 806R were used to amplify the V3-V4 regions of bacterial 16S rDNA genes from 30 samples (10 samples × 3 replicates) , and a tThe raw reads obtained were processed using QIIME 1.17 . The quap < 0.05) was analyzed by one-way ANOVA and Tukey’s post hoc test. Principal component analysis (PCA) was performed to visualize differences in multiple parameters of samples among all groups. The first two principal components were used for calculating the PCA models. SPSS 20 was used for all statistical analyses.All samples were carried out in triplicate (three independent productions). The statistical significance . The results showed that salt replacement accelerated the drying and ripening processes of the fermented sausages.The pH, weight loss, moisture content and atermined . As showp < 0.05). The chloride content in the R group was significantly higher than that in the L group (p < 0.05), which showed a proportional increase with the addition of KCl. In comparison with mature normal fermented sausages (N group), the sodium content in f the R group was only 1505.22 ± 11.10%, which was reduced by approximately 25%. These low-salt fermented sausages provide healthier characteristics for reducing sodium intake. In addition, the nitrite content in all groups decreased significantly (p < 0.05) during fermentation and ripening. The results reflected that the nitrite degradation rate in the fermented sausages with salt replacement was significantly faster than that in the other two treatments (p < 0.05), which was mainly related to ascorbate addition [During fermentation and ripening of the fermented sausages, the chloride, sodium and nitrite contents were measured . The chladdition ,36.p < 0.05), which were related to water loss and drying. As reported previously, the replacement of NaCl may change the texture of the fermented sausages [p > 0.05). However, the hardness and chewiness in the final products of the R group showed a significant increase compared with those of the low-salt control (p < 0.05), and there was no significant difference between the R and N groups on day 36 (p > 0.05). In addition, treatment with salt replacement significantly reduced the resilience of the final fermented sausages compared with the normal salt control (p < 0.05).The instrumental texture analysis profile and color analysis of the fermented sausages were shown in sausages ,38,39, wsausages . In thisp < 0.05) and slightly increased redness (a*) and yellowness (b*).Color is an important parameter of the sensory properties that can be affected by NaCl replacement in processed meat ,19,41. TStaphylococcus and Micrococcus and Enterobacteriaceae in fermented sausages were approximately 6.30, 6.25, 6.75 and 4.65 Log CFU/g, respectively. As shown in p < 0.05). Moreover, the maximum Staphylococcus and Micrococcus counts were 7.74 ± 0.04 Log CFU/g in the N group after fermentation, as shown in Staphylococcus and Micrococcus counts in the R group were significantly lower than those in the N and L groups on day 36 (p < 0.05). In addition, significant decreases in Enterobacteriaceae counts in three groups during 36 days of processing (p < 0.05) are shown in Staphylococcus and Micrococcus counts. As previously stated, KCl had a similar evolution of LAB and aerobic mesophile counts as NaCl and increased the growth of Micrococcaceae in fermented sausages [Staphylococcus and Micrococcus can be inhibited by Vc-Ca and calcium glutamate in fermented sausages, and we are working on related research.The microbial counts of the fermented sausages during fermentation and ripening are presented in sausages . We infep > 0.05), which suggests that salt replacement had no effect on bacterial community richness in the fermented sausages. In addition, the Simpson index in the R group was significantly higher and the Shannon index in the R group was significantly lower than that in the normal salt control on day 36 (p < 0.05). The results showed a decrease in the diversity of bacterial community in the sausages fermented with salt replacement.In total, 1,523,684 high-quality and valid sequences were collected across all 30 samples of the fermented sausages, with an average sequence length of 428-bp. Staphylococcus had a dominant position in fermented sausages. However, after fermentation, all treatments in the composition of bacterial community at the phylum level have no obvious difference.The relative abundance of the bacterial community of the fermented sausages at the phyla level was shown in Pediococcus, Staphylococcus, Lactobacillus, Acinetobacter, Psychrobacter and Myroides were the most representative bacteria. Moreover, LAB and Staphylococcus within Firmicutes were abundant genera, accounting for more than 90% in sausages after fermentation, which has been reported previously [Pediococcus composed the vast majority of the bacterial community of the fermented sausages in the R group at the end of processing, which illustrated that salt replacement controlled the growth of almost all bacteria but only slightly inhibited that of Pediococcus. As shown by the above results of microbiological plate colony counting, we inferred that Vc-Ca and calcium glutamate have antibacterial characteristics.The composition analysis of the bacterial community in the fermented sausages at the genus level is shown in eviously ,42. CompPediococcus in all groups increased significantly after fermentation and remained relatively stable until the end of ripening (p < 0.05), while Staphylococcus showed a slow decrease during fermentation and ripening. In fermented sausages, Lactobacillus and Weissella have been considered the principal LAB [Lactobacillus in the L group was higher than that in the other two groups, while the relative abundance of Weissella in the N group was higher after fermentation. These results illustrated that salt replacement inhibited the growth of almost all LAB in the fermented sausages.To understand the diversity of the bacterial community among all samples, a heatmap of the bacterial community structure and composition of the top 25 genera was shown in ipal LAB ,43. As dThe beta diversity among 30 samples (10 groups × 3 replicates) was analyzed and clustered by PCoA . The resStaphylococcus in the N group; Micrococcaceae, Streptococcaceae, Lactococcus and Proteus in the L group; and Lactobacillales, Lactobacillaceae and Pediococcus in the R group were the potential biomarkers in relative abundance for distinguishing among all groups.Bacterial markers among the three groups of the final fermented sausage samples were evaluated and analyzed by LDA . A totalw, moisture, chloride, sodium and nitrite contents, TPA , color analysis  and microbiological analysis (plate colony counting and relative abundance of genera) in fermented sausages during processing . In addition, as shown by the PCA, the comprehensive parameters of the fermented sausages with salt replacement were similar to those of normal salt products. Therefore, complex blends of salt replacement containing KCl, Vc-Ca and calcium glutamate have great potential to be applied to produce sodium-reduced fermented sausages. Future research on the correlations between microbial metabolites and volatile flavor in fermented sausages with salt replacement during fermentation and ripening should be taken.


Selection for feed efficiency is one of the best ways to decrease poultry production costs and environmental impacts. While literature on its genetic determinism is abundant, it is limited to one or a few periodic values over the animals’ lifespans. With the development of new phenotyping tools, kinetics of growth and feed intake are now available, providing access to daily data on feed efficiency. In this study, over the course of 6 weeks, we described the kinetics of body weight (BW), average daily weight gain (ADG), feed intake (FI), and daily cumulative feed conversion ratio (DCFCR) using electronic feed stations. We then estimated the genetic parameters of daily data in two fast growing lines of chicken divergently selected for breast meat ultimate pH (heritability and genetic correlations with breast meat yield and pH).Birds from the line selected to have a more acidic meat (pHu-), were more efficient than those selected to have a less acidic meat (pHu+), with a 4.3% higher BW from d7 to d29 and 5.0% better feed efficiency from d12 to slaughter. The line effect for ADG and DCFCR appeared to be significant as early as d5, which is consistent with the early age at which metabolic differences between the two lines appear. Genetic parameters estimated within each line revealed different genetic determinisms of growth and feed efficiency, with a higher impact of maternal effects on BW during the growing phase (d10 to d20) in the pHu+ line and much higher heritability values of DCFCR during the finishing phase (d26-d42) in the pHu- line.Genetic profiles of daily performance highlighted the difference between both lines. Their behavior during dietary transitions reinforced the already known impact of these periods in the animals’ lives. Based on the profiles of genetic parameters within each line, it seems feasible to identify early criteria for selecting feed efficiency, but they must be defined for each line, as the genetic determinism of these traits is line-dependent.The online version contains supplementary material available at 10.1186/s12863-022-01035-z. Diversifying feedstuffs is another way to reduce the social impact of poultry production by increasing the proportion of local or less concentrated feedstuffs and agricultural by-products in poultry diets, and reducing importations of soybean into Europe [Feed efficiency has been one of the most important criteria of selection in poultry for decades because of its economic importance and benefits for the environmental impact of poultry production . In the t et al.  estimateo Europe –6.Literature on the genetic determinism of feed efficiency is abundant. Estimates of heritability vary largely depending on the periods studied and the strains. For example, heritability estimates for feed conversion ratio at 42 d vary from 0.13 to 0.41 depending on the strains and on the duration of data collection –10. EstiThanks to recent developments in the phenotyping of feed consumption and animal weight, it is now possible to have access to the kinetics of animal growth and feed efficiency. Thus, using the BIRD-e electronic feeder that provides daily data of feed intake and body weight on individuals raised on the floor , 12, BerThese new phenotyping tools make it possible to revisit the genetic study of feed efficiency in chickens and to optimize the search for selection criteria by considering the kinetics of daily data. The objective of this study was thus to describe the evolution profile of genetic parameters of body weight, feed intake and feed conversion ratio in fast-growing chickens, in a context of feedstuffs diversification. Animals were thus fed with a diet containing the same amount of energy and proteins than usual diets, but including alternative feedstuffs to soybean, produced locally and less in competition with human food. Feed efficiency depends on many factors such as basal metabolism, protein turnover, body composition, and digestive efficiency . A link P= 0.001). Breast meat yield (BMY) was also 3.5% lower in the pHu- line than in the pHu+ line .As expected from the selection performed on these divergent lines, breast meat ultimate pH at 42 d was much lower in the pHu- line than in the pHu+ line .Heritability estimates of daily performance are presented in Fig. Heritability of ADG and DFI showed comparable kinetics Figs. b, c. BefFinally, for BW, heritability increased until 14 d for pHu- and 20 d for the pHu+ line, estimates being 14 to 146% higher for pHu-  and daily traits are reported in Fig. 42 and body weight were low throughout the whole period in both lines (0.17 in pHu+ and -0.02 in pHu- on average).Genetic correlations between final cumulative feed conversion ratio DCFCR2 and dai42 varied more between the two lines. Two periods of stability occurred for pHu+. The first one occurred between day 5 and day 13 and showed values around 0.62. The second one was between day 15 and slaughter, and showed values around 0.4. The correlation between DFI and DCFCR42 in pHu- was more variable, increasing to 0.7 before the first change of diet and decreasing to 0.2 the day after. After this change, the correlation remained stable until 22 days. Then it increased strongly to 0.72 at 29 days and finally decreased to 0.2 in the last days. The line difference could also be seen in the correlations between DCFCR42 and ADG. Thus, the correlation within the pHu+ line remained stable and low during the whole life of the chickens (0.10 on average), whereas this correlation showed a decrease after 27 days in the pHu- line. Average correlation values were equal to 0.04 before 27 d and -0.32 after 27 d, indicating that in pHu- a lower efficiency was associated with a decreased growth rate at later ages.Profiles of correlations between DFI and DCFCRGenetic correlations between the criteria of selection, breast meat ultimate pH, and daily traits are reported in Fig. The correlation between DCFCR and pHu increased in both lines from hatch to the first diet change (to +0.38 for pHu+ and +0.80 for pHu-). After this event, the correlation for pHu+ decreased down to -0.6 at 13 days and remained stable until the second diet change. Finally, the correlation increased slowly up to -0.15 at 28 days. From 29 d until the end, it decreased to reach -0.89. The correlation for pHu- was more stable, decreasing after the first change of diet and being stable from 15 to 30 days, at around +0.53. After this day, it increased and reached +0.71 at 42 days.Figure This study made it possible to give a detailed description of the kinetics of growth and feed efficiency in the two divergent lines for breast meat ultimate pH. While the weight of chicks was higher at hatching in the pHu+ line , their daily feed intake was lower in the first days of life so that BW became similar between the two lines at 5 d. As ultimate pH and muscle glycogen stores are highly genetically correlated at slaughter age (-0.97) , selectiThe current study also provided original results on the genetic determinism of the kinetics of growth and feed efficiency in chickens. While heritability of BW evolved in a similar way in the two lines from 20 days onwards, decreasing from high values of 0.7 to moderate values of 0.35-0.40, genetic controls of BW differed between the two lines in the first part of life. In particular, BW heritability was much lower between 10 d and 20 d in the pHu+ line. This may partly be due to maternal effects that greatly influenced BW variability during this period in the pHu+ line, while they remained low in the pHu- line. In the current standardized conditions of poultry production, maternal effects are mainly mediated by egg characteristics. Factors such as egg size and nutrient content  or maternal hormones can impact the development and the phenotypes linked to the fitness of the offspring . The higThe range of estimates of heritability of feed conversion ratio available in the literature was 0.11 to 0.54 , 24–29. 42) to the indirect selection for the same indicator measured earlier. Given our experimental results, selection for DCFCR could be envisaged as soon as 20 d in pHu+ birds and 29 d in pHu- birds with an expected response amounting to 80% of the maximum response that would be obtained by selecting for DCFCR42. Whatever the scenario, the expected response would be higher in the pHu- line than in the pHu+ line . Moreover, given the profiles of correlations with BMY and meat pHu, improved FE would lead to an increased pH value and a decreased breast yield in the pHu+ line, but a more acidic meat without any correlated response on BMY in the pHu- line. This illustrated again the different genetic determinism of FE in the two lines.Access to daily data can offer new opportunities to improve selection strategies for feed efficiency. At first, the profiles of genetic parameters can lead to identifying earlier predictors of the final feed efficiency. By combining information on heritability and genetic correlations, we compared expected response to a direct selection for the cumulative feed conversion ratio measured at slaughter age  the improvement of FCR performance with a high economic impact or 2) at the same FCR, the spare of resources that could be used in human food. In a former study, we already showed that rapid growing chickens could reach similar performances with a classical corn-soybean diet than with an alternative diet , the comOur study highlighted differences in the kinetics of feed intake, growth rate, and feed efficiency between the two broiler lines diverging for breast meat ultimate pH in relationship with their early metabolic differences. The genetic determinism of these traits varied to some extent between the two lines, and showed that the respective contributions of growth rate and feed intake on genetic determinism of FCR differed in the two metabolic contexts. Having access to daily FCR data allowed for the identification of early relevant predictors, and opens the way to new strategies or methodologies for selecting feed efficiency.Pectoralis major muscle during fourteen generations. These lines came from a fast-growing grandparental female broiler line selected for both growth and reproduction traits. A complete description of the breeding scheme and performance of these lines can be found in Alnahhas et al. [In this study, we used animals from two experimental broiler lines divergently selected for a high (pHu+) or a low (pHu-) ultimate pH of the s et al. . Two batThe temperature was set at 32°C at hatch and decreased progressively to reach 20-23°C from 29 d on. The light program was 24D:0N at d0, 23D:1N at 1 d and 18D:6N from 3 to 42 d. At hatch, the animals were identified with a wing band and an electronic Radio frequency identification device (RFID) chip, then weighed and placed in one pen on a floor covered with wooden chips. The RFID chip was placed at the base of the neck and secured with a plastic string passing under the skin.ad libitum with a diet containing a high proportion of sunflower, rapeseed, and fava bean in order to reduce the soybean meal proportion in the diet. The detailed composition is shown in Table -1 DM; 21.5 % CP), a grower diet from 9 to 19 d , and a finisher diet from 20 to 42 d . Diet was formulated using the PORFAL software v.4.0.17  in order to follow Hubbard broiler nutrition recommendations [The animals were fed ndations . Potentindations .Table 1CBody weight and feed intake were individually and continuously recorded throughout the experiment thanks to electronic feed stations  and calcij) and daily cumulative feed conversion ratio (DCFCRij) were calculated as:Body weight (BW), daily feed intake (DFI), average daily gain (ADG), and daily cumulative feed conversion ratio (DCFCR) were calculated as in Berger et al. . Brieflyij and BWij are the daily feed intake and body weight of animal i for day j, respectively. DCFCR42 stands for the cumulative feed conversion ratio over the whole rearing period.where DFIAt 42 d, the animals were weighed after 8 hours of feed withdrawal and transferred to the slaughterhouse of the PEAT INRAE poultry experimental facility.Pectoralis major and Pectoralis minor were cut and weighed. Breast meat yield (BMY) was calculated as:After 24 hours of chilling, the right Pectoralis major (pHu) was measured with a portable pH meter .The ultimate pH of the All phenotypes were first analyzed using the PROC GLM procedure of SAS 9.4  with theijkl being the trait for animal l of line i, sex j and hatch k and eijkl the residual for animal l.with yHeritability and genetic correlations between traits were estimated using VCE6.0 software , 34. In N=2) and batch (N=31) and the direct genetic effect of animal . When the maternal permanent environment effect  was not null, it was also included in the model of analysis.Genetic analyses were conducted separately for each line. The animal model included the fixed effects of the sex (Additional file 1.


Scaeva pyrastri and Scaeva selenitica to celestial cues during their autumn migration. Hoverflies oriented southwards when they could see the sun and shifted this orientation westward following a 6 h advance of their circadian clocks. Our results demonstrate the use of a time-compensated sun compass as the primary navigational mechanism, consistent with field observations that hoverfly migration occurs predominately under clear and sunny conditions.The sun is the most reliable celestial cue for orientation available to daytime migrants. It is widely assumed that diurnal migratory insects use a ‘time-compensated sun compass’ to adjust for the changing position of the sun throughout the day, as demonstrated in some butterfly species. The mechanisms used by other groups of diurnal insect migrants remain to be elucidated. Migratory species of hoverflies (Diptera: Syrphidae) are one of the most abundant and beneficial groups of diurnal migrants, providing multiple ecosystem services and undergoing directed seasonal movements throughout much of the temperate zone. To identify the hoverfly navigational strategy, a flight simulator was used to measure orientation responses of the hoverflies  They pr argante ,13. To t argante ,13. The  argante . The neu argante ,16, with argante ,18.Drosophila have demonstrated that the sun and patterns of polarized light in the sky can be used to maintain a constant heading [Drosophila adopt arbitrary headings with respect to a simulated sun with no evidence of time compensation [Drosophila [Episyrphus balteatus  and Eupeodes corollae (vagrant hoverfly) travel over the southern region of Britain annually [Tethered flight experiments in  heading ,20. Howeensation . While sosophila ,22. Hoveosophila ,23–27 anannually . In addiannually ,28.et al. in the 1960s made use of these low-level flights to carry out mark-recapture experiments on insects during the autumn migration period in the Swiss Alps [Eristalis tenax, Ep. balteatus and Syrphus vitripennis were able to cover 3 km in 10–15 min (12 to 18 km h−1). The most distant recoveries were made 111 km away with a moderate tailwind of 6–8 m s−1 and the authors suggest that hoverflies covered this distance in a single flight [−1 (36 km h−1) aided by the selection of favourable winds [Patterns of hoverfly migration are best understood in Europe where seasonal influxes into northern regions begin in spring and are followed by a broad front southward migration during autumn ,22,23,29iss Alps . These ee flight . More rele winds , suggestle winds ,36. Meanle winds .Scaeva pyrastri (pied hoverfly) and Scaeva selenitica (yellow-clubbed hoverfly), caught at ground level during their southward journey over the Pyrenees. We predict that hoverflies would continue to orientate southwards in a seasonally favourable direction when given a view of the sky including the sun but excluding geographical features, and that when subjected to the clock-shift procedure, hoverflies would predictably shift their orientation in accordance with the solar azimuth at their perceived time.To investigate whether hoverflies use a time-compensated sun compass to orientate, we undertook a series of tethered flight experiments on actively migrating . 2 (a)S. selenitica and S. pyrastri to be the most consistent fliers during tethered flight experiments. We therefore selected these species for further investigation, hereafter referring to them as ‘hoverflies’. During September and October of 2018 and 2019, migrating hoverflies were caught using hand nets as they traversed the Pyrenees, 2273 m above sea level at the mountain pass of Bujaruelo on the French–Spanish border . Permission to conduct experiments was obtained from the Parc national des Pyrénées  and the Gobierno de Aragon . Flies were housed in 30 cm cubed mesh cages (Watkins & Doncaster) and flown immediately or transferred to the village of Gavarnie for further experiments. Flies that were stored for later experiments were kept outside to maintain their circadian rhythm to natural daylight hours and supplied with 40% honey solution, pollen and water ad libitum.Initial investigation of flight behaviour in a range of species of actively migrating hoverflies was carried out in Switzerland from August to October 2017 and revealed  (b)b). We constructed the flight simulator from a white 200 mm diameter × 250 mm high PVC cylinder, commonly used as ducting. This contained a large Neodymium ring magnet . The magnet was supported centrally on a transparent acrylic shelf above a 20 mm central hole. A raspberry Pi camera (Pi v. 2.1 8MP 1080p camera module) mounted to the underside of the shelf filmed flight behaviour from below. The image was manually focused by screwing the lens fitting. A button attached to the raspberry Pi (Pi 3 Model B running Raspbian) initiated a custom Python script  that recorded flight behaviour for 5 min, with recording status indicated by an illuminated LED. A transparent acrylic strip (20 mm wide) resting on top of the flight simulator supported two 15 × 6 mm neodymium magnets (Magnet Expert Ltd), one above and one below, positioned directly above the lower ring magnet. Visual cues were obscured in the flight simulator with a lid that allowed a 116° visual field of the sky and sun but obscured the surrounding landscape. In preliminary experiments, hoverflies were found to orientate directly towards sunlight illuminating the opposite wall inside the flight simulator. To prevent this, a layer of the photographic diffuser  was used to cover the opening which diffused the light more equally within the flight simulator . The reduction in the degree of polarization was calculated from Stokes' parameter measurements, using a Glan Thompson polarizer, of the light both before and after passing through the diffuser [To study the hoverflies response to celestial cues, flight direction was measured using a portable flight simulator with magnetic tethering b. We condiffuser . (c)c). Tethered hoverflies were then placed inside the flight simulator with the needle touching the upper magnet, where they were held in place but free to rotate in the horizontal plane. The video recording was then initiated, filming the hoverfly for 5 min.Sun compass experiments were undertaken in the flight simulator between 10.37 and 16.34 local time. Immediately prior to experimentation, flies were restrained on a sponge using a weighted mesh and glued by the thorax to a truncated sewing needle  with ultraviolet curing cement Bondic™; c. TetherTo exclude the sun but still include intensity and polarized-light gradient cues, a lid with a smaller circular opening was used to restrict the field of view to 70°. These experiments were undertaken in the evenings between 16.17 and 18.45 when levels of overhead polarization were greatest and, as the sun had passed behind the mountains, without the photographic diffuser. Flies used in this experiment came from the same stock as the sun compass experiment which were exposed to normal daylight hours.Finally, to investigate time compensation, a separate batch of flies was subjected to a 6 h advanced clock shift according to the mountain daylight time . To achieve this, hoverflies were stored for 10 days in a larger 90 × 60 × 60 cm mesh cage (Watkins & Doncaster), blacked out and illuminated on a timer by a 7.5 W, 4000 k colour-temperature LED bulb. Hoverflies were fed ad libitum as before, but owing to larger cage dimensions, food and water was additionally attached to the sides of the cage and branches were added to provide shelter. The experiments were undertaken with the same methodology as the sun compass, but later in the day to allow a minimum of 1 h after the artificial sunrise before experimentation began. The difference in the sun's azimuth between the time of the experiment and the insect's expected time, 6 h earlier, ranged from 91.1° to 104.5° depending upon the date and time of the experiment (mean = 98.1°). With full time compensation for the sun position, flies would be expected to alter their heading by an angle within this range. Experiments were carried out between 7 and 11 October 2019 between 14.29 and 16.42, overlapping with a subset of the sun compass and restrictor experiments. (d)−1) using Total Video Audio Converter [mageJ [figure 1d; electronic supplementary material, file S2 for ImageJ script and file S4 for raw outputs).Analysis of the videos of hoverfly flight was first undertaken by hand, noting the timings of the initiation and cessation of flight as well as any perturbations (such as a shadow or the fly spinning out of control). Only recordings that included 70 s or more of uninterrupted flight were selected for analysis, with the first 10 s discarded to allow time for acclimatization and orientation. Video files were converted into jpg images at a frequency of 5 Hz  angles were rectified to be relative to the sun's azimuth so that the sun was in the 180° position. To achieve this, for each recording, every angle was rotated by the difference between geographical south and the sun's azimuth when the experiment was undertaken. To enable calculation of the angle difference, nonlinear least-squares models were fitted using the dose–response model (function L.4) from the R package drc (v. 3.0-1)  to azimutudio 1.3 and the packages circular (0.4-93) [R* is an arbitrarily scaled alternative to r of circular regression. This is compared to a statistical table of probabilities to test the null hypothesis. The corresponding rank-weighted mean direction is also given, for which an additional script was created to bootstrap confidence intervals. All custom scripts are available in the electronic supplementary material, file S3.Statistical analysis and graphing were carried out in R v. 3.5  using R S(0.4-93) , plotrix(0.4-93)  and glmm(0.4-93) . To test(0.4-93) ,45. The  (e)The activity pattern of dipteran migrants crossing the mountain pass of Bujaruelo in 2019 between 6 September and 11 October was extracted from video footage. A video camera sampled a 2 × 2 m cross-section of the mountain pass for 1 min every 15 min between 9.00 and 17.00. The number of individuals crossing the midpoint of the frame southwards were counted manually for each timepoint and averaged across all the recorded days. Temperatures were taken from a temperature logger located on the pass.. 3θ = 188.2°, n = 30, R* = 1.294, p < 0.01; figure 2a). Diurnal activity patterns of migrants crossing the mountain pass show a marked increase beginning after 14.00, coinciding with increasing temperatures (figure 2b). Splitting the data of the sun compass experiment in half, hoverflies flown before 13.57 orientated randomly , whereas hoverflies flown after this time, hereafter referred to as the ‘last 15’, displayed a more directed group heading to the south-southwest . Traditional Rayleigh tests produced comparable results  test:  results .Figure R* = 0.634, n = 42, p < 0.5; figure 2e). To account for the ambiguous nature of the polarized-light cue, where opposite directions along an axis cannot be distinguished, we tested for a bidirectional line-up response by bidirectionally transforming the angles and repeating the test; however, the results remained non-significant .To explore the role of celestial cues other than the sun, hoverflies were flown with a restricted view of the sky, which excluded the sun but provided an unobscured view of light intensity, chromatic gradients and polarized light. These hoverflies orientated randomly . A Mardia-Watson-Wheeler test confirmed that the mean directions of hoverflies under clock-shifted conditions were significantly different from the sun compass group both in its entirety  and for the last 15 flown . The mean direction of the time-shift experiment was 68.9° clockwise from that of the sun compass experiment and 62.7° as compared to the last 15, representing 64–70% of the 98.1° adjustment required to fully compensate for the mean 6 h azimuth change and 70–77% of the adjustment required under a linear compensation scenario of 15° h−1. However, confidence intervals , while back transforming the angle of the clock-shifted hoverflies based on their perceived time results in a group heading that is not significantly different from the sun compass . Together this indicates that the hoverflies were indeed clock-shifted, flying in different directions with respect to the sun and compensating for its position rather than just following its course throughout the day.As the clock-shifted experiments were undertaken later in the day when the sun would naturally be in a more clockwise position, to rule out simple phototaxis, the analyses were repeated on angles that were rectified to be relative to the direction of the sun, so that the direction of the sun (instead of south) is in the 180° position . The rectified mean directions of hoverflies under normal and clock-shifted conditions still differed significantly, as confirmed by a Mardia-Watson-Wheeler test  and 34 were S. pyrastri , with sex and species ratios representative of the capture rate in the field. To explore whether there were behavioural differences between sexes or species, we pooled the vector data from the different experiments together and investigated two parameters of the recorded flights: flight directedness  and deviation from the experimental mean direction . There was no significant effect of sex (p = 0.477) or species (p = 0.657) on flight directedness (r), and a lower relative model fit than the intercept only model Akaike information criterion (AIC) . To investigate deviation from the experimental mean direction, the potential deviation of −180° to +180° was normalized to 0–1 by dividing by 180 and changing negatives to their additive inverse. For this, only experiments with significant mean directions were used (sun compass and clock-shifted sun compass). There was also no significant effect of sex (p = 0.461) or species (p = 0.593) on deviation from the experimental mean direction and a lower relative model fit than the intercept only model . See the electronic supplementary material, table S1 for effect estimates.Mixed sexes and species of S. selenitica and S. pyrastri for the time-shift experiment . As 10 individuals in each group are required for the chi-squared comparison, insufficient S. pyrastri (n = 9) prevented a comparison for the sun compass experiment. Low numbers of males prevented an inter-sex comparison; however, males had on average a lower deviation from the group mean direction than females, making excess leverage owing to male outliers unlikely. For these reasons, we are satisfied that any behavioural differences between sexes and among species are minor enough to justify pooling the data.A Mardia-Watson-Wheeler test confirmed the lack of significant directional bias by species of . 4Scaeva spp. hoverflies to orientate in a southerly direction during autumn migration when given a view of the sky including the sun but excluding geographical features. This result is supported by previous radar studies showing southward migration headings of Ep. balteatus and Eu. corollae hoverflies over southern Britain in the autumn  but high directness later in the day for the last 15 flown (r = 0.66). Despite this, these values remain lower than in other systems and may represent a less directed strategy employed by autumn migrating Scaeva spp. hoverflies, experimental noise, or a combination of both factors. Our methodology differed from butterfly experiments in the use of a magnetic tethering system, attenuation of celestial cues with a diffuser layer and a lack of laminar airflow that may have each contributed to poorer orientation responses. We note however that monarch butterflies navigate to a specific location during their autumn migration, so might be more committed to maintaining the appropriate direction when flown in a flight simulator. Hoverflies migrating to warmer climates without a specific destination could be more flexible and adapt their flight headings more readily to the prevailing conditions. This might result in a lower concentration of observed directions as alternative vectors might come at very little cost to fitness.The sun is thought to be the most important cue for diurnal migrants. Here, we demonstrate the ability of  heading  versus amigrants . These s 0.83 in  and 0.8  0.83 in ). We obsS. pyrastri migration typically reaches its peak earlier in the season than in the Pyrenees, and with most individuals active between 10.00 and 16.00 in relatively even numbers. As migration is a physiologically demanding activity, it may be that in the Pyrenees, hoverflies optimize their flight time to be later in the day, which would not be necessary at lower elevations and earlier in the season owing to the warmer temperatures. The roles of temperature, photoperiod and solar elevation warrant further investigation into their potential roles in modifying migratory flight times.We observed a lack of directness in flights undertaken earlier in the day that may be directly controlled by circadian behavioural patterns such as early day feeding or indirectly by the effect of low temperatures on activity. Temperature appears to be an important motivating factor for migratory flight in Monarch flight simulator experiments  and rateet al. [As has been demonstrated in migrating butterflies ,13, our et al.  set out et al. . With ouet al. ,34.Scaeva spp. hoverflies use a time-compensated sun compass in their autumn migration and, together with data from radar studies in other hoverfly species, we suggest that the time-compensated sun compass may be used by the numerous other species of hoverflies found co-migrating through this pass and at other sites around the globe [Our results show that he globe ,29,47,48he globe . To the r-values, but these are arbitrary with respect to the celestial cues and may represent an ancestral mechanism in insects for covering large distances [Drosophila, dung beetles and locusts to orientate to a vector for efficient dispersal [In the field, we observed a pattern of surges of migratory activity during sunny periods, with little activity when the sun was obscured. This observation, together with the loss of group orientation seen in our restricted view experiment, suggest that the sun acts as the primary celestial cue for orientation and that hoverflies wait for favourable navigational conditions, namely a visible sun, to migrate rather than relying on other cues. However, individuals flown with a restricted view are still able to maintain directed flights, as indicated by high individual istances ,51. To fispersal –54, and ispersal ,56. Foraispersal . Howeverispersal , but seeispersal . FinallyClick here for additional data file.


Elaeis guineensis) germplasm is exclusively maintained as ex situ living collections in the field for genetic conservation and evaluation. However, this is not for long term and the maintenance of field genebanks is expensive and challenging. Large area of land is required and the germplasms are exposed to extreme weather conditions and casualty from pests and diseases. By using 107 SSR markers, this study aimed to examine the genetic diversity and relatedness of 186 palms from a Nigerian-based oil palm germplasm and to identify core collection for conservation. On average, 8.67 alleles per SSR locus were scored with average effective number of alleles per population ranging from 1.96 to 3.34 and private alleles were detected in all populations. Mean expected heterozygosity was 0.576 ranging from 0.437 to 0.661 and the Wright’s fixation index calculated was -0.110. Overall moderate genetic differentiation among populations was detected  and this was further confirmed by AMOVA analysis. UPGMA dendogram and Bayesian structure analysis concomitantly clustered the 12 populations into eight genetic groups. The best core collection assembled by Core Hunter ver. 3.2.1 consisted of 58 palms accounting for 31.2% of the original population, which was a smaller core set than using PowerCore 1.0. This core set attained perfect allelic coverage with good representation, high genetic distance between entries, and maintained genetic diversity and structure of the germplasm. This study reported the first molecular characterization and validation of core collections for oil palm field genebank. The established core collection via molecular approach, which captures maximum genetic diversity with minimum redundancy, would allow effective use of genetic resources for introgression and for sustainable oil palm germplasm conservation. The way forward to efficiently conserve the field genebanks into next generation without losing their diversity was further discussed.Oil palm ( Elaeis guineensis Jacq.) is the most productive oil crop, accounting for 40% of the global production of vegetable oils, and producing 3–8 times more oil per hectare as compared to other oil crops with planting on only 0.4% of global agricultural land [Oil palm  and Costa Rica [Elaeis oleifera) since 1970s [ex situ living collections in the field and Malaysia currently has the largest oil palm field genebank in the world [Despite being originated from Africa, oil palm is mostly cultivated in Southeast Asia with large plantations established in Malaysia and Indonesia due to favourable tropical climates . Commercndonesia . Seeds fsta Rica . In viewEx situ field genebanks provide an easy and ready access to the resources for utilization, characterization and evaluation. However, they require large land area and are expensive and labour intensive to maintain, especially for perennial crops i.e., oil palm. The living collections are also threatened by extreme weather conditions such as flood, drought as well as various pest and diseases in the field that may lead to potential loss of the invaluable genetic resources. In order to efficiently conserve and exploit the genetic resources, understanding of the genetic diversity and population structure of the oil palm germplasm is essential. Conservation of minimal number of accessions with maximal genetic diversity/ minimal genetic redundancy, namely the core collection, would reduce the maintenance cost of oil palm germplasm  and allow for sustainable germplasm conservation. Genetic diversity of germplasm can be investigated using various techniques such as morphological assessment, biochemical markers (isozymes) and molecular markers. With rapid development of molecular technology, genetic evaluation of oil palm populations has been conducted using various markers available, for example, isozymes [Conservation of genetic resources is crucial because it provides a reservoir of genes for development of novel traits associated with yield enhancement, adaptability to climate change as well as pest and diseases resistance/tolerance in crop plantation. Hence plants genetic resources are important and useful for improvement of productivity and sustainability of the agricultural production systems . Ex situisozymes , restricisozymes , 12, ampisozymes –18, and isozymes . The advisozymes  have furisozymes , 22. Seqi.e., molecular-assisted germplasm conservation.In AAR, exotic germplasms were collected from different sources via collaboration and are currently being maintained as field genebanks. One of the collections are Nigerian-based germplasm which represents PORIM ’s selection for short stature and high yielding materials for productions of their PS1 DxP materials . The PS1The Nigerian-based germplasm collection, which descended from the MPOB Nigerian population 12, was planted in 1995 at AAR oil palm breeding research station in Paloh Estate, Johor, Malaysia. A total of 186 progenies from 12 controlled-crosses were assayed in the present study .® DNeasy Plant Mini kit according to manufacturer’s instruction . The concentration and quality of the DNA samples were examined using a Nanodrop OneC Spectrophotometer  and agarose gel electrophoresis. The stock DNA samples were diluted to a working concentration of 10 ng/μl using sterile water.The leaflets of the first fully-opened frond were harvested for genomic DNA isolation using the QIAGENE. guineensis microsatellite markers were used for genotyping in this study, comprising 103 markers developed at CIRAD  [5’-TGT AAA ACG ACG GCC AGT-3’ (18 bp) at the 5’-end. The PCR amplification was performed in a 10 μl reaction mixture containing 20 ng DNA, 5 μl HotStarTaq®Plus Master Mix , 4 μM M13-tagged forward primer, 4 μM reverse primer, and 0.4 μM IRDye®-labelled M13 (-21) primer. PCR reactions were performed with an initial denaturation of 95°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 1 min., extension at 72°C for 1 min., with a final extension of 72°C for 10 min. The thermocycling was performed with Veriti™ Thermal Cycler . The PCR products were mixed with 7 μl of loading buffer (98% formamide (v/v), 0.1% bromophenol blue (w/v) and 0.01 M EDTA pH 8.0) and heat-denatured. The PCR products were then separated on 6% polyacrylamide gels using the infrared dye detection system NEN 4300 DNA Analyzer .A set of 107 lopment) , 25 and lopment) , 27 (S1 A), number of effective alleles (Ae), number of private alleles (PA), Shannon’s Information Index (I), observed and expected heterozygosity (Ho and He), and fixation index (F) for each locus and population.Clearly visualised fragments detected using NEN4300 DNA Analyzer were examined and data were scored manually in a binary format (1 or 0). Software GenAlEx version 6.503 , 29 was FST value were also computed. Analysis of Molecular Variance (AMOVA) was performed to calculate the molecular variance among and within the populations with the number of permutations = 999.Cluster analysis based on the unweighted pair-group with arithmetic averaging (UPGMA) was performed using the multivariate statistical package MVSP  to construct a dendogram. Principal Coordinate Analysis (PCoA) was carried out using GenAlEx version 6.503 to further analyse the genetic relationship among individual palms of all the populations. The genetic distance between populations according to  and pairK set from 1 to 12. The most likely number of populations (K) was determined via the ad hoc statistic ΔK [Population structure of the Nigerian-based germplasm was analysed using the Bayesian model-based clustering programme STRUCTURE version 2.3.4 . The modistic ΔK .https://cran.r-project.org/package=corehunter). Core Hunter was applied to maximize allelic coverage index (CV) in order to retain all unique alleles in the original populations for current work.PowerCore 1.0  and CoreThe different core sets established were compared based on (i) proportions of alleles retained in the core collection; (ii) average genetic distance between each accession in the original collection and the nearest entry in the core set (A-NE); (iii) average distance between entries (E-E); (iv) minimum and average distance between each entry and the nearest neighbouring entry (E-NE), as proposed by . A-NE inThe evaluated 107 SSRs detected a total of 928 alleles across the 186 Nigerian-based germplasm, with an average of 8.67 alleles per loci . Both thA) and effective alleles (Ae) within each population was 4.290 and 2.777, respectively. Population 606 with the highest samples size (N = 31) recorded the highest number of alleles (A = 6.365) whereas pop. 599 exhibited the highest number of effective alleles (Ae = 3.343). Population 604 with the lowest number of palms (N = 5) and a selfed-crossing nature revealed the lowest in number of different alleles (A = 2.654), effective alleles (Ae = 1.964), Shannon’s Information Index (I = 0.729) and expected heterozygosity (He = 0.437). Private alleles were detected in all 12 populations with the lowest and highest in pop. 598 (PA = 1) and 606 (PA = 33), respectively. Population 606 recorded the highest Shannon’s Information Index (I = 1.322). The heterozygosity in the Nigerian-based populations was generally high with an average observed (Ho) and expected heterozygosity (He) of 0.644 and 0.576, respectively. Except for pop. 605 (F = 0.161), Ho consistently exceeded He leading to negative fixation index (F) in the studied populations, which reflected excess of heterozygotes.The genetic variability parameters of each Nigerian-based population are summarised in FST analysis were performed to investigate the genetic variations among the populations. High genetic differentiation was observed among pop. 604 with pop. 597, 598, 599, 600, 601, 603 and 607 (FST = 0.153–0.202) as well as among pop. 607 with pop. 597 and 601  (FST = 0.177) while the remaining populations have moderate genetic differentiation (FST = 0.066–0.150). The average genetic differentiation among populations was 0.120 and the overall gene flow value (Nm) was estimated at 1.117, indicating certain level of alleles migration among populations. AMOVA analysis further disclosed that 71% of molecular variance observed was due to the genetic difference within population, whereas 29% genetic variation was partitioned among populations.The genetic distance calculated among the Nigerian-based populations revealed close genetic relationship (mean = 0.466). The largest genetic distance was observed between pop. 601 and 597 (0.698) whereas the closest genetic relationship was between pop. 598 and 603 (0.242) . Both anctively) . PopulatA total of eight genetic clusters were discerned from UPGMA clustering for the 186 Nigerian palms . MixtureK, reached its maximum value when K = 8, classifying the Nigerian-based populations into eight genetic groups . Core Hunter outperformed PowerCore with lesser number of palms shortlisted. Expected heterozygosity (He) and average genetic distance between entries (E-E) were comparable for both core collections  and 58 (31.2%) palm identified, respectively Tables . The higlections . PowerCo2 = 0.952 and 0.948 for core sets obtained by PowerCore and Core Hunter, respectively)  showed that there was no significant difference in the genetic diversity indexes calculated between the germplasm genotyped and the identified core collections using PowerCore and Core Hunter , suggestctively) . UPGMA cctively) . A totalPlant breeding still relies, mainly, on the exploitation of existing natural variation to broaden the genetic base of current commercial planting materials. Oil palm is being considered as orthodox seed and not recalcitrant, but the seeds can only be stored for 2 years with gradual reduction in germination rate . Oil palAn average of 8.67 alleles per microsatellite locus was scored in the present study with an effective number of marker alleles per locus of 2.78. This is lesser than the average number of alleles  revealed by molecular characterization of oil palm germplasm collected from Africa and Latin America . The difElaeis guineensis oil palm [He = 0.576), which was higher than those reported for MPOB Nigerian germplasm based on isozymes (He = 0.15–0.244 with He of population 12 = 0.187 by [He = 0.228 by [He = 0.442 by [He = 0.534 by [He = 0.712–0.803 with He of population 12 = 0.736). The number of effective alleles calculated from our collection  was also lower than those reported by [Ae = 2.9–3.6 with Ae of population 12 = 3.2). Despite being descendants of MPOB Nigerian population 12, high genetic variation and prevalence of private alleles in all population in the current AAR’s Nigerian-based germplasm indicate that they are invaluable resources for introgression into commercial planting material. These results also suggest that the set of SSR used is very polymorphic and robust for diversity studies of oil palm.Extensive molecular studies have been conducted on the MPOB oil palm germplasm with comparatively high variation noticed in Nigerian collection, indicating that it is a possible centre of diversity for oil palm , 10, 11.0.534 by  using SSorted by  , indicating inbreeding due to self-mating nature. Negative F values were also observed in MPOB Nigerian germplasm [FST ranging from 0.066 to 0.202 and gene flow Nm = 1.117) as well as close genetic relationship (mean Nei’s genetic distance = 0.466) were revealed in our collection. This was further evidenced by the 71% of variation attributed by genetic differences within population from AMOVA analysis. Significant genetic variations among individual within population was reported in various studies of oil palm, as expected for an allogamous and long-lived perennial species [FST value of 0.15 among 51 oil palm genotypes from Congo using 7 randomly amplified microsatellite markers RAM indicating substantial genetic difference among populations. Mating among closely-related individuals may have contributed to some degree of gene flow and thus moderate differentiation among population analyzed.Fixation index, ermplasm . Despiteermplasm , 44, aga species , 43, 46. species  whereas  species  reportedHT = 0.759) [E. oleifera collection with origins from South and Central America  with overall mean He = 0.221) [Maintenance of living collections, common for perennial tree crops such as oil palm, is expensive and labour intensive. Establishment of core collections is an efficient approach for germplasm manipulation, reducing the cost while keeping the maximum genetic diversity of the entire germplasm with minimum repetitiveness . Various= 0.759)  but larg= 0.221) . The fin= 0.221) , 55. In = 0.221) .Genetic distance-based criteria were proposed to evaluate core collections as they are intuitive to understand and both representativeness of accessions and diversity within core collections were explored . CriteriA, Ae, I, Ho, He, F) between both core collections and the original Nigerian populations with increased negative value of F, albeit insignificant. This was expected because the diversity increases with elimination of genetically similar accessions during core set development [Additionally, both core sets obtained were also validated and concurred with the selection criteria presented in other studies –60. Firselopment . Third, elopment . CollectGanoderma boninense [Field genebank allows an easy and ready access for utilization. However, living collection is not the best route for long term conservation as potential loss may occur due to extreme weather conditions and pest and diseases such as basal stem rot in oil palm caused by the fungal pathogen oninense , 65. Besin vitro culture conditions [The focus of germplasm conservation for oil palm can be shifted from whole collection to the identified core set. One of the possible ways to conserve germplasm is to clone the identified core set via tissue culture. This allows precise conservation of maximum genetic diversity with minimum redundancy and zero genetic erosion. The number of ramets to be planted in the field can be lower, more manageable and cost-saving than progeny trials. However, oil palm tissue culture is not widely implemented, making up only 2% of commercial planting material, due to its long and labour intensive process, low efficiency of culture amenability and high risk of somaclonal variation , 67. Depnditions , 69. ManA more practical breeding approach is to re-create the germplasm through selfing or intercrossing of the core set that was identified through molecular analysis. With known genetic diversity and relationship of the core set, this approach stands a better chance to capture the diversity of the original germplasm collection. However, there is little disadvantage in term of land size and expenditure required to maintain the living collection in the field. The intercrossing of 58 core palms into 29 populations within each genetic cluster via punnet square design for the present study would require 13.4 ha of land for planting.  suggesteThe use of molecular markers for the development of core collection is advantageous as they can accurately reflect the genetic diversity regardless of plant growth status, developmental and environment effects . PopulatThe present study reported SSR marker-based molecular assessment on genetic diversity and population structure of a Nigerian-based oil palm field genebank in AAR. High levels of genetic heterozygosity were detected despite being descendants of MPOB Nigerian germplasm population 12, indicating this material is invaluable for introgression into current breeding programme. Two different software were compared for their effectiveness to develop core collection of Nigerian population and Core Hunter version 3.2.1 outperformed PowerCore 1.0. Core Hunter constructed a core collection of 58 palms, accounting for 31.2% of the total population. This is the first report describing the molecular characterization and validation of core set establishment for oil palm germplasm material. The core set captures all the alleles with genetically distinct entries while maintaining intact genetic diversity and structure of the original population. This core collection would be of great interest to breeders for exploitation of the prevalent diversity in crop improvement programs as well as effective conservation of the field genebank: a step towards molecular-assisted germplasm conservation.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Then the above-mentioned influencing factors on CCOPSPTT and DOPSPTT were analyzed separately in Hubei and other provinces. The results showed that CCOPSPTT and DOPSPTT were significantly different among five city types outside Hubei province (p < 0.05), but not obviously different in Hubei province (p > 0.05). The CCOPSPTT had significant correlation with socio-economic determinants (GDP and population), DW, climate and time after the outbreak of COVID-19 outside Hubei province (p < 0.05), while was only significantly related with GDP in Hubei province (p < 0.05). The DOPSPTT showed significant correlation with socio-economic determinants, DW, time and CCOPSPTT outside Hubei province (p < 0.05), while was significantly correlated with GDP and CCOPSPTT in Hubei province (p < 0.05). Compared with other factors, socio-economic determinants have the largest relative contribution to variance of CCOPSPTT in all studied cities (> 78%). The difference of DOPSPTT among cities was mainly affected by CCOPSPTT. Our results showed that influences of city types on the confirmed cases and death differed between Hubei and other provinces. Socio-economic determinants, especially GDP, have higher impact on the change of COVID-19 transmission compared with other factors.This study is to assess the influences of climate, socio-economic determinants, and spatial distance on the confirmed cases and deaths in the raise phase of COVID-19 in China. The positive confirmed cases and deaths of COVID-19 over the population size of 100,000 over every 5 consecutive days  covered from 25th January to 29th February, 2020 in five city types ( Due to i.e., small-, medium-, large-, very large- and super large-sized cities) [The confirmed cases and deaths of COVID-19 may vary between city types ( cities) –12. Some cities) , 14. The cities) , 16. Aff cities) , 18. Bec cities) , 16, 20, cities) , 21. ThiConfirmed cases and deaths of COVID-19 among different cities may be affected by socio-economic determinants , 22. VirAt present, however, most studies on the impacts of socio-economic determinants, climate factors and spatial distance on the confirmed cases and deaths of COVID-19 only investigated a single type of factors , 29. ForAfter large-scale outbreak of COVID-19 in Wuhan , China’s government implemented many control measures in order to minimize the infection of virus to local residents , 36. Morth January to 29th February 2020, the data for above-mentioned four factors in all China’s cities were collected. Then relationships among them were analyzed using generalized linear mixed models (GLMMs) and hierarchical partitioning analysis (HPA). The aims of this study are to answer the following three questions: (1) Are there any differences in the confirmed cases and deaths of COVID-19 among different city types? (2) Are there any differences in relative contribution of city sizes, socio-economic determinants, climate and spatial distance to variability of the confirmed cases and deaths of COVID-19? and (3) Are these relative contributions different between Hubei province and other regions? Our results will provide a useful insight in explaining the variability of COVID-19 transmission (confirmed cases and deaths), the relevant influencing factors as well as the relative contribution of different factors. These results will give a practical guidance for drawing up the prevention and control measures of COVID-19 at present and in the future.In this study, in order to explore the relationship between COVID-19 transmission and city sizes, socio-economic determinants, climate factors as well as spatial distance, the confirmed cases and deaths of COVID-19 from 25No specific permissions were required for the described studies, because we did not carry out any experiments. All data used in this study are issued by China’s National Health Commission, China Meteorological Administration, Global Climate Dataset and Local City Weather Bureaus see . All datth January to 29th February 2020 as our studied period because China National Health Commission (NHC) began to collect and release epidemic data across the whole country after 25th January [In this study, we chose 29 January . It was  January , 5. Chin January , 26, 29.Socio-economic determinants including GDP and population, for which the data at the end of 2018 were collected from the China’s National Statistical Yearbook because data for year 2019 and 2020 have not been released. GDP and population were chosen because they have decisive effects on factors that directly affected transmission and prevention of COVID-19, such as population mobility, education level and medical conditions , 39. AssThe number of cities selected in this study is 334, which is far less than the total number of cities in China (approximately 661 in 2020). This is because the cities selected in this study are all cities where the conformed case of COVID-19 was reported from NHC, while the cities without the conformed case were not selected. All cities (324) were categorized into five types: small-, medium-, large-, very large- and super large-sized cities, as suggested from land transaction website of China . The citBased on the investigative data of 324 cities, GLMMs were used to test the multiple-influences of city sizes, socio-economic determinants, climate factors, spatial distance and time on CCOPSPTT and DOPSPTT of COVID-19. In GLMMs, city sizes were set as a random variable in order to reduce the dependencies of epidemic intensity on population scale. In addition, previous studies have suggested that variances of CCOPSPTT and DOPSPTT can be affected by time . After tC, SD and SED represent climate factors, spatial distance and socio-economic determinants, respectively. Their sets are respectively expressed in Formula 3–5. c and t represent city size and time respectively. SRI, AT and RAH are solar radiation intensity, average temperature and relative air humidity, respectively. ε is the random error; which is formed by the random action of many factors in the process of measurement and statistics. If the significance of regression coefficients, such as α and β, is less than 0.05, suggesting that they have the significantly impacts on CCOPSPTT or DOPSPTT.In the models, e.g., much data of CCOPSPTT and DOPSPTT in different city types did not conformed to the normal distribution, see Tables SD > Mean indicates non-normal distribution). As COVID-19 control conditions were much more complicated in Hubei compared with other provinces, like lockdown and mass screening, data analysis of Hubei and other province were performed separately. The use and explanation of GLMMs and HPA see Dichmont [Finally, we used HPA to compare the relative contributions of these factors on CCOPSPTT and DOPSPTT. Here, the relative contribution was the proportion of each independent variable from the goodness-of-fit measures across all variable combinations in a hierarchy, which indicated weight of one variable versus other independent variables that obviously contributed to COVID-19 transmission. When performing GLMMs and HPA, all data were standardized by logarithmic transformation in order to avoid the trivial influence of the non-normal data on statistical results  and p-values. High R2 and p-values <0.05 indicated that statistical results were credible. Besides, we also tested the robustness of the model to ensure the credibility of the research results. In this study, since independent variables, especially climate factors, are highly correlated between adjacent time observation points, their values at time t not only have an impact on CCOPSPTT and DOPSPTT, but also have a significant impact at time t+1. Therefore, our model is time-delayed. According to the results of Jiang et al [i.e., lag regression or time-delay model). When the regression parameters, like p-value, R2 and estimate coefficients, of time-delay and baseline  models are close to each other, it indicates that our model has high robustness. All data processing was conducted in R. 3.4.3 software.The creditability of GLMMs and HPA results was determined by determinate coefficient  , medium-sized cities (5×E-4) and small-sized cities (1×E-4). Wuhan was more than 12 times higher than other cities on CCOPSPTT , 2×E-5 (average) and 2×E-5 (average), respectively. There was over 200 times higher CCOPSPTT in Wuhan and in other cities (p > 0.05)  . In most < 0.05) . Within  < 0.05) . For exaCCOPSPTT . On 29thr cities . In all  > 0.05) .th January and 29th February, 2020 (p > 0.05) (p < 0.05). DOPSPTT showed no significant difference among other four  types of cities outside Hubei province (p > 0.05) (p > 0.05) (DOPSPTT showed no significant difference among five city types outside Hubei province on 25 > 0.05) . But dur > 0.05) . In Hube > 0.05) .R2 values (> 0.10) and significant p-values (< 0.001) in all analysis . In contrast, DLPC had no significant correlation (p> 0.05) with CCOPSPTT (p < 0.05), whereas other factors did not (p > 0.05) , population (positive), DW (negative), relative air humidity (negative), solar radiation intensity (positive), average temperature (positive) and time (positive) had a significant regression relationship with CCOPSPTT (CCOPSPTT . In Hube > 0.05) .p < 0.01) (p < 0.05), but not related to other factors (p > 0.05)  . However > 0.05) .R2 values  and significant p-values (< 0.001) in all analysis, indicating that HPA can be good used to decompose the relative contribution of each influencing factor to CCOPSPTT and DOPSPTT. This also suggested our results have high credibility (The HPA results showed high dibility . Among cdibility . All clidibility . In Hubedibility . The reldibility . The reldibility .The relative contributions to DOPSPTT were higher from CCOPSPTT (44.06%) and socio-economic determinants (44.06%) than from spatial distance (2.10%), climate (1.83%) and time (2.84%) in cities outside Hubei province . ParticuThe difference of CCOPSPTT among five city types outside Hubei province  suggestei.e., a higher GDP) might promote higher population mobility, e.g., potentially favoring the spread of epidemics. But at the same time, a richer region should have larger amount of resources to put into place more effective control interventions. On the other hand, during a sudden and rapid period of COVID-19 outbreak, a larger GDP implying a potentially more educated population; which might allow the adoption of individual protective measures at a higher rate. This suggests that the potential role of GDP on COVID-19 can be complicated between different city types. This would also explain the insignificant difference of CCOPSPTT between super large- and very large-sized cities, as well as between large- and medium-sized cities.Among five city types, CCOPSPTT showed no significant difference between super large- and very large-sized cities, as well as between large- and medium -sized cities . This math January, while traffic controls in other cities of Hubei province started after 28th January, 2020. Home quarantine and traffic control could block the transmission of coronavirus [Except for Wuhan, CCOPSPTT among large-, medium- and small-sized cities in Hubei province was not significantly different . This inonavirus . Thus, conavirus , 48. Theonavirus , 32. Sucth January to 29th February). Government disease controls and the spread patterns of COVID-19 were different between Wuhan and other cities [The DOPSPTT in super large-sized cities were higher than that in other cities, whereas there was no significant difference between very large-, large-, medium- and small-sized cities both in Hubei and other provinces . This mar cities , 20. Sper cities , 19. Wuhr cities . Shutdowr cities , 36, 37.r cities , 33, 37.It is the most noteworthy that we studied the rise period of COVID-19 when China implementing the strictest control. The impact of city types on confirmed cases and deaths through GDP, population and spatial distance may be the extension of their original impact before the implementation of government’s control measures. More specifically, nearly 5 million people left Wuhan for other cities before its shutdown . After tOur results showed that socio-economic determinants have the positive and greatest contribution to variability of CCOPSPTT among cities outside Hubei province , Table 4There was a subtle difference in the contributions of socio-economic determinants, spatial distance, climate and time to CCOPSPTT between cities in Hubei and other provinces . This miThe confirmed cases was the main contributor to the deaths, which accounted 40.61% and 44.06% for the variance of DOPSPTT in Hubei and other provinces, respectively , Table 4e.g., social distancing, lockdown, etc) were implemented; (ii) the promptness  and intensity of such interventions measures; and (iii) the extent of further measures such as testing and isolation of asymptomatic cases [Outside Hubei province, population, DW and time have significant correlation with DOPSPTT . This maic cases , 22, 31.Our results found that city sizes had a significant impact on the confirmed cases and deaths of COVID-19 over the population size of 100,000 , but this effect differed between Hubei and other provinces. The results of GLMMs and HPA showed that the confirmed cases and deaths among different city types were affected by socio-economic determinants, climate and spatial distance and time. Socio-economic determinants were the dominating factor affecting COPSPTT among different cities, while the deaths were mainly determined by the confirmed cases and GDP. Socio-economic determinants and time had positive effect on while spatial distance negative correlated to the confirmed cases and death among different cities. In addition, our results showed that relative air humidity had negative influence on CCOPSPTT while average temperature and solar radiation intensity displayed positive influences. The different influences of socio-economic determinants, climate factors and spatial distance on the confirmed cases and deaths between Hubei and other provinces may be due to China’s strong control measures (more strict in Wuhan and Hubei province) that reduced the spread of novel coronavirus. In conclusion, our study indicated that large cities have a higher risk of COVID-19 infection than small cities. Home quarantine and restriction of population mobility are effective ways to control virus transmission. Therefore, minimization of the population mobility can effectively protect people from COVID-19 infection during its rise phase.S1 Data(ZIP)Click here for additional data file.