Ecology

The copy numbers for 16S and ITS1 rRNA, and the sequencing depth for all samples are presented in Supplementary File 3 (qPCR data, Sequencing Rarefaction Curves). An average of 14,265.25 reads per housefly sample for the V4 16SrRNA and 16,149.4 reads per housefly sample for the ITS1 were retained after quality filtering. After quality filtering of the egg-laying substrate samples, an average of 10,371.75 reads were retained per sample for the V4 16SrRNA, and an average of 25,479.75 reads were retained per sample for the ITS1 region. The extracted DNA from newly emerged adult houseflies of the Spanish laboratory strain (12 samples in total, newly emerged adults, three replicates from four generations, strain SP100) returned a low copy number for the fungal ITS1 (qPCR data, Supplementary File 3) and a low number of acquired sequencing reads; they were therefore omitted from any further analysis of the fungal microbiota. In addition, the mitochondrial COI phylogeny showed that the Dutch wild-caught strain and the Dutch laboratory strain, which were sampled from the same locality at different times, are in close proximity and form a separate clade from the Spanish lab strain phylotypes (Supplementary File 2).The housefly microbiota alpha-diversity is determined by sampling environmentAbsolute richness (number of ASVs), Shannon index, and Phylogenetic diversity for all housefly strains and developmental stages are shown in Fig. 1. The highest bacterial alpha diversity was observed for the wild-caught housefly population GK0. Strain was an important factor for separating Shannon biodiversity levels both for newly emerged (F = 4.37, P  More

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RESEARCH HIGHLIGHT
01 July 2022

Rock doves on some Scottish islands show almost no sign of having interbred with domestic pigeons.

The relatively long, slender bill of this rock dove from the Outer Hebridean islands of Scotland are characteristic of feral pigeons’ ancestors. Credit: W. J. Smith et al./iScience

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Charles Darwin developed his theory of natural selection in part by studying a form of artificial selection: the nineteenth-century rage for pigeon breeding, which created a wealth of fantastical varieties of pigeon (Columba livia). So widespread was pigeon fancying that it seeded the world with escaped domestic birds and their feral descendants, which then hybridized with their wild ancestors, the rock doves.

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doi: https://doi.org/10.1038/d41586-022-01780-2

References

Subjects

Conservation biology

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Conservation biology More

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Sample collection from sea turtlesIn total, 45 serum samples from 33 juvenile green turtles (C. mydas), including 6 sea turtles with tumors, 5 juvenile hawksbill turtles (Eretmochelys imbricate), and 7 olive ridley turtles (Lepidochelys olivacea) (juvenile = 5; sub-adult = 2). All turtles were sourced from: eastern Taiwan (n = 24), southern Taiwan (n = 14), central Taiwan (n = 6), and northern Taiwan (n = 1). Among the 45 sea turtle samples, 6 green turtles developed FP (n = 1 with tumor score 1; n = 1 with tumor score 2; n = 4 with tumor score 3)32, while 39 did not have FP. FP tumor tissues were collected from 6 green turtles (from shoulder/flippers/inguinal regions) with FP during surgical procedures. Regarding the collection of normal skin tissues, one normal skin tissue (from shoulder) was collected from one necropsied dead green turtles (stranding and discovered from southern Taiwan) confirmed without FP. All tissue samples were fixed in 10% neutral buffered formalin prior to further analysis. In this study, all sea turtles were discovered and rescued through the official reporting system of the Marine Animal Rescue Network (established by the Ocean Conservation Administration) and admitted to the National Museum of Marine Biology and Aquarium (NMMBA), between 2017 and 2020.Detection of ChHV5 DNA by polymerase chain reaction (PCR)Total DNA was extracted from blood of 45 sea turtles by DNeasy blood & tissue kit (Cat. No. 69504, Qiagen, Valencia, CA, USA) following manufacturer’s instructions. Subsequently, the ChHV5 infection status all 45 sea turtles was determined by PCR using primers targeting on UL18 (capsid protein gene), UL22 (glycoprotein H gene), and UL27 (glycoprotein B gene) regions4. The sequence of primer sets are: UL18F: 5′-CACCACGAGGGGGAAAATGA, UL18R:5′-TCAAATCCCCCGTTCACTCG; UL22F: 5′-ACGGCGTTGGCTAGTGAATC, UL22R: 5′-GCAGTTCGGTACACACCTCT; UL27F: 5′-TAACAAGAAAGAACCGCGCG; UL27R: 5′-ATTTTCCCGGTCAGTGCCAA. PCR amplifications were performed in a total volume of 50 μl. The reaction included 1 μl of the template DNA, 1 μl of each primer (10 μM), 22 μl of distilled water (DDW), and 25 μl of the AmpliTaq Gold® 360 Master Mix (Cat. No. 4398876, Life Technologies, Valencia, CA, USA). The thermocycle for amplification was: Initial denaturing at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s, and then a final extension at 72 °C for 7 min. Results were visualized by gel electrophoresis (2% agarose) with SYBR Safe DNA Gel Stain (Cat. No. S33102, Invitrogen, Carlsbad, CA, USA).Sequence optimization of the UL27 gene for expression of the ChHV5 glycoprotein protein using E. coli
To express large quantities of ChHV5 gB, we adopted the prokaryotic Escherichia coli (E. coli) expression system. The construct (namely UL27/pUC57) containing sequences of the full length UL27 fused with FLAG tag sequence (GenBank accession no. AF035003.3) was synthesized by Allbio Science Co., Ltd, Taiwan. The sequence information of the glycoprotein (gB) datasets used and analyzed for protein expression during the current study was obtained and available from the GenBank repository [https://www.ncbi.nlm.nih.gov/nuccore/AF035003.3]. Considering the difference in tRNA-codon usages between prokaryotes and eukaryotes would possibly affect subsequent protein expression, the optimized UL27 gene sequence, without altering the translated amino acid sequences, to fit the E. coli expression system was synthesized. The codon optimized UL27 gene was further used as the template for amplification of different gene fragments by Polymerase Chain Reaction (PCR).Construction of plasmids expressing partial fragments of ChHV5 gB proteinTo determine the relative antigenicity and also to increase the expression yield, plasmids expressing various regions of gB protein were constructed. Briefly, the five regions covering different fragments of the UL27 gene were amplified from plasmid UL27/pUC57 by PCR using specific primer sets with built-in restriction enzyme sequences shown as underlined in Table 1. The thermal cycling conditions were: 98 °C (5 min) followed by 35 cycles of denaturation (98 °C, 30 s), annealing (58 °C, 1 min), and extension (72 °C, 2 min), and finished with a final extension (72 °C, 10 min). PCR amplicons with expected sizes were isolated from gel and trimmed with the restriction enzymes followed by ligation with vectors either pET24a or pET32b (Novagen, Germany) linearized with the same restriction enzymes. The resulting plasmids with expected insert sizes as confirmed by restriction enzymes were sent for automated DNA sequencing (Mission Biotech, Taipei, Taiwan).Table 1 Information on the constructs expressing the UL27 fragments. The bold characters indicate sequences recognized by restriction enzymes for the ease of further cloning procedure.Full size tableExpression of recombinant gB fragments in E. coli
In the current study, the recombinant gB protein is a key reagent that served as antigen for seroprevalence of ChHV5 as well as for the generation of ChHV5 gB antibody (conducted by Yao-Hong Biotech Inc., Taiwan). The plasmids expressing individual gB fragment were transformed into E. coli host cells, strain BL21 (DE3), Rosetta. Expression of all the recombinant gB fragments was induced by 0.8 mM of isopropyl β-d-1-thiogalactopyranoside (IPTG) at 28 °C for 16 h. As all the gB fragments cloned into the pET series vectors were expressed as a fusion protein with a 6-histidine tag at C-terminus end, they could be further purified by Ni–NTA column chromatography using the chelating Sepharose Fast Flow (GE Healthcare) following the method described in one previous study33. The yield and purity of recombinant gB proteins were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, 6 M urea and 0.4 M imidazole contained in the purified protein were depleted by step-wise dialysis against 1 × PBS buffer (0.02 M phosphate, 0.15 M NaCl) with gradually decreased concentrations of urea at 4 °C. The concentration of recombinant proteins were then estimated by National Institutes of Health ImageJ software (https://imagej.nih.gov/ij/, 1997–2018.) using the standard curve established by bovine serum albumin (BSA) with known concentrations42.Western blot analysisRecombinant gB fragments were separated by 12.5% or 15% SDS-PAGE and electrotransferred to PVDF membrane by using Mini Proten III apparatus (Cat. No. 165-3301, BioRad). The filters were blocked in PBS-T buffer (0.02 M phosphate, 0.15 M NaCl, 0.05% Tween-20) containing 5% skimmed milk and reacted with mouse anti-his tag antibody (1:5,000, Cat. No. GTX40628, GeneTex) at 4 °C for overnight. After six-time wash with PBS-T buffer, the PVDF filter was then incubated with the secondary antibody, 1:5000 diluted goat anti-mouse IgG conjugated with horseradish peroxidase (HRP), or 1:500 diluted Protein A/G-HRP (Cat. No. 32400, Thermo fisher scientific™, United States) for sea turtle antibody detection, at room temperature for 1 h followed by PBS-T wash to remove the unbound antibodies. Ultimately, the signal was detected by ECL reagents (Thermo Fisher Scientific, United States) and the image was acquired by ImageQuant LAS 4000 Mini (GE Healthcare).Immunohistochemical (IHC) analysisTo establish IHC protocol, normal skin tissue from PCR-negative sea turtles served as the negative control. In total, the FP on skin tissue from six individual sea turtles that were detected positive for ChHV5 DNA (positive tissue samples), and one normal tissue detected negative (the negative tissue) were included in the IHC analysis.IHC procedure was conducted as reported in our previous study34. In brief, sections of formalin-fixed and wax-embedded skin tissues of sea turtles were made using a rotary microtome (Leica RM2245, Leica Biosystems, Germany) and were further deparaffinized and rehydrated. Antigen retrieval was carried out by heat-induced epitope retrieval method: slides immersed into boiled sodium citrate buffer (0.01 M, pH 6.0), which was preheated up to 100 °C, for 20 min and cooled at room temperature for 20 min. Subsequently, the slides were incubated with peroxidase-blocking reagent (Cat. No. S200389, Dako, Denmark) for 30 min, and then treated with or without primary antibodies (the anti-gB serum prepared from this study). In each interval of the following procedures, sections were rinsed with a mixture of TBST buffer. Tissue sections were then reacted with secondary antibody (HRP anti-rabbit/mouse, DAKO, Denmark), followed by incubation of DAB and chromogen (dilution 1 μL in 100 μL) from a commercial ChemMate EnVision detection kit (Cat. No. K5007, Dako, Denmark). Ultimately, tissue sections were counterstained with Mayer’s hematoxylin reagents (Code S3309, Dako, Denmark) for 2 min followed by wash with DDW, and reacted with 37 mM ammonia water for 5 s and rinsed with DDW.Immunofluorescent assay (IFA)Human 293 T cells were transfected with plasmids expressing full-length ChHV5 gB protein fused with FLAG tag at its C-terminus. At 24 h post transfection, 293 T cells (CRL-3216, ATCC, USA) were fixed with 2% formaldehyde for 10 min, followed by permeabilization with 0.1% Triton X-100 for another 10 min. Subsequently, cells were incubated with anti-FLAG antibody (1:500) (F7425; Sigma-Aldrich), or antisera (F1, F2, F3, F2–3) at the dilution of 1:500 for 1 h at room temperature. After six times of washes with PBS containing1% bovine serum, goat anti-mouse IgG (1:2,000 fold diluted) (Cat. No. A28175, Alexa Fluor® 488, Invitrogen) was used as secondary antibody. After one-hour incubation, nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI, Cat. No. D9542, Sigma-Aldrich) for 10 min, followed by confocal microscopy (FV1000, Olympus, Tokyo, Japan) with Olympus FV10-ASW 1.3 viewer software.Statistical analysisTo evaluate the association between seropositivity and FP or viremia tested by PCR of UL27 gene, Fisher’s exact test was applied due to very limited number of sea turtles with FP. The statistical significance was determined by p  More

Prosopis juliflora leaves collection and processing for RibotypingProsopis juliflora species of the genus Prosopis, family of Fabaceae had its genetic variation in Doha evaluated. Seven samples of P. juliflora leaves were collected from six different locations in Doha, Qatar, during five field trips. Plant leaves were collected after proper permissions and all methods were carried out in accordance with relevant guidelines and regulations. Trees in all locations were naturally growing around urbanization areas in their normal arid habitat without artificial irrigation, samples were collected from fully mature trees. Table 1 shows the samples details. Figure 1 shows the location sites of where the samples were collected on the map of Qatar, Doha. Leaf samples were kept in sterile labeled bags until having reached the laboratory where few leaflets were washed with sterile distilled water and sterilized using 70% ethanol to be used for DNA extraction.Table 1 Location details of the collection sites of P. juliflora leaves.Full size tableFigure 1Location map of collection sites of P. juliflora leaf samples (ArcGIS software).Full size imageRibotyping analysisThe leaflets of each sample were transferred into a sterile mortar previously cooled at -20 ˚C and used for DNA extraction following the kit manufacturer instructions (DNeasy Plant Mini Kit-QIAGEN-USA).Extracted DNA of each sample were subject to PCR using ITS1 and ITS4 primers. PCR products obtained were purified using the Invitrogen Quick PCR Purification Kit (QIAGEN, Germany) as indicated by the manufacturer and sequenced using Sanger sequencer (3130/3130xl DNA Analyzers, Thermofisher Scientific, USA) as previously described22.Sanger sequencer raw data was read using BioEdit software. Basic Local Alignment Search Tool (BLAST) network services of the National Centre for Biotechnology Information (NCBI) database were used to compare the obtained sequences to the existing sequences. Sequences were submitted to NCBI for accession numbers. The various P. juliflora ribosomal sequences obtained were also uploaded on MEGA-X software and the phylogeny tree was generated using the neighbor-joining algorithm26.Minimum inhibitory concentrations of PJ-WS-LE extracts prepared using leaf samples collected from various locations against A. alternata and C. gloeosporioides
Preparation of PJ-WS-LE extractFresh, young full leaves of P. juliflora were collected from various locations as indicated in Fig. 1. Samples were washed, dried and ground into powder to be used in the preparation of PJ-WS-LE extract as previously described22. Briefly, every 20 g of the leaf powder were incubated in 200 mL of 70% ethanol for 48 h. The supernatant has its solvent evaporated, the extract was then re-dissolved in sterile distilled water. Only water-soluble phytochemicals were tested by centrifuging the final preparation tubes and excluding the pellet. Stock solution of 100 g L−1 was stored at 4 °C to be used for later experiments. PJ-WS-LE extract concentration used in treatments was 8 g L−1 which is double the highest minimum inhibitory concentration of the extract against spoiling microorganisms as previously determined22.Determination of minimum inhibitory concentrationThe MIC test was conducted in a sterile 96-well plate, with each well containing 100 μl of potato dextrose broth (PDB) (HIMEDIA-India). Every four wells made one replication, nine different concentrations of the crude extracts were tested (1:1 dilutions) ranging from 42 to 0.16 g L−1. Wells were then inoculated with one of the two tested microorganisms’ spore suspensions (A. alternata and C. gloeosporioides). The last three rows are control rows: no spores and no extract control wells, negative control with spores but no extract wells, and positive control with spores and 10 µl of the fungicidal Clatrimazole (1%) wells.Fungal spore suspensions were adjusted to the range of 104 spores L−1 using a 10 day old fungal plate and sterile distilled water, the spore concentration was calculated using a heamatocytometer.Fungal growth in each well was monitored using Resazurin (HIMEDIA-India) dye. Upon cells division, Resazurin changes its color from blue to pink and fluorescent27. Results were taken within 48 h of incubation at 25 °C. MIC was recorded as the last extract concentration that shows no change in the color of Resazurin within the incubation period.Curative and preventive effects of PJ-WS-LE extract against A. alternata and C. gloeosporioides induced infection in mangoesPathogensThe two fungal strains used C. gloeosporioides and A. alternata were obtained from our laboratory collection, Department of Biological and Environmental Sciences, Qatar University, Qatar. Both fungal isolates were previously isolated from locally collected fruit samples. Isolates were molecularly identified by sequencing the Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) that was amplified by PCR. Identified fungal isolates were given the strains code of AaltQU17 for A. alternata and CgloQU17 for C. gloeosporioides22. Preserved cultures were sub-cultured on potato dextrose agar (PDA) plates and incubated at 25 °C for 10 days. Plates were then flooded with 10 mL of sterile distilled water each, to prepare the needed spores suspension solutions. The concentrations of spores suspensions were adjusted to 106 spores L−1 using a heamatocytometer18.FruitThe mango (Mangifera indica) type known as Neelam imported from India was used in the experiments. Fruit were bought from the whole sale market upon their arrival to the country. Only undamaged mature fruit were used in the experiment. Fruits chosen were ripen but not yet soft with firmness average of 20 ± 5.1 N, weight average of 177.61 ± 0.2 g and TSS average of 70 ± 5.3%. Fruit were first washed with sink water and sterilized twice with 70% ethanol to be then washed with sterile distilled water and left to air dry.Preventive and curative effects of PJ-WS-LE extractWounded mango fruit were used during the experiment, the wounds were made through three needle pricks (2 mm deep) in three different places for each plant using a sterile syringe. A completely randomized design was used and each treatment was made of a triplicate of 10 fruit each. The experiment was repeated twice.PJ-WS-LE extract of leaves collected from Qatar university field was first tested for its efficacy in preventing fungal contamination in wounded mango fruit (preventive effect). Therefore, the wounded zone of each fruit was sprayed with 8 g L−1 PJ-WS-LE extract and then left to air-dry. Once dry the fruit were sprayed again with the extract at the same concentration and left to dry. Control fruit were only treated with sterile distilled water without the plants extract. After two hours all wounds were inoculated with 20 μL of conidia aqueous solution (106 spores mL−1) of one of the tested fungi. The extract was then tested for its ability to cure fungal contamination in wounded fruit. Therefore, wounds were inoculated first with 20 μL of conidia aqueous solution (106 spores mL−1) and left to dry. Wounds were then sprayed twice with 8 g L−1 PJ-WS-LE extract.All mangoes were stored in sterilized plastic trays inside an incubator at 25 °C and 75% humidity. Fruit were observed every 24 h for 5 days for C. gloeosporioides inoculated fruit and for 10 days for A. alternata inoculated fruit. Three parameters were recorded at the end of the experiment: disease incidence (DI), disease severity (DS), and percent plant extract efficacy (%EE). To calculate disease severity, the diameter of the infected area of each fruit was measured in two perpendicular directions and mean diameter mycelial growth was calculated28,29.$$mathrm{DI}=frac{(mathrm{Number, of, rotten, fruit})times 100}{mathrm{Total, number, of, fruit}}$$$$mathrm{DS }=frac{(mathrm{Average, lesion, diameter, of, treated, plants})times 100}{mathrm{Average, Lesion, diameter, of, control, plants})}$$$$mathrm{%EE}=frac{(mathrm{Disease, incidence, in, Control, batch}-mathrm{Disease, incidence, in, treated, batch})times 100}{mathrm{Disease, incidence, in, Control, batch}}$$End of the trial samples firmnessAt the end of the trial, remaining mango fruit were tested for their flesh quality using a penetrometer (Agriculture Solutions, USA) to test the flesh firmness. Fruit were peeled, then the stainless steel probe of the instrument was inserted in three different points towards the equator of the fruit. Firmness in Newton was recorded and compared with standard fruit firmness to judge fruit quality18.Effectiveness of PJ-WS-LE extract as long-term coating material and the preservative value of its chitosan-embedded formCoating solutions preparationChitosan solution of 1% concentration was prepared by stirring chitosan powder (CAS 9012-76-4, Himedia, India) in 1% glacial acetic acid (IsoLab, Germany) overnight. The final chitosan solution pH was adjusted to 5.6 using 0.1 M NAOH (Sigma-Aldrich, Germany). To prepare PJ-WS-LE extract chitosan-embedded coating material, filter-sterilized PJ-WS-LE extract stock solution was added to 1% chitosan to achieve a final concentration of 8 g L−130.Samples preparationEighty-four mango samples chosen as described above, were divided into four groups of 18 samples each. Samples were divided into four treatment batches and treated as following:

Batch A: non-treated fruit.

Batch B: PJ-WS-LE extract at 8 g L–1 was used to spray the fruit.

Batch C: 1% chitosan was used to spray the fruit.

Batch D: 8 g L−1 PJ-WS-LE extract embedded in 1% chitosan was used to spray the fruit.

Every experimental replicate was made up of three mango samples that were stored together in one sterile bag at 4 °C. The number of replications per treatment was six. The experiment was repeated twice31.Evaluation of sensory qualityA five-points scale was used for the evaluation of the sensory quality of the samples for overall quality, smell, and color change. The attributes were evaluated weekly using the fruit of one experimental replicate. Scores were given using the following scale: 5 points indicate “extremely liked”, 4 points indicate “liked”, 3 points indicate “acceptable” 2 points indicate “disliked” and 1 point indicates “extremely disliked”. The weekly average score per batch was also calculated32.Estimation of weight lossUpon treatment at day zero, all mango samples were weighed and their weights were recorded as initial weights. Weights of all remaining samples were measured at the end of every week. The variation between the start weight and weekly weights is calculated as weekly weight loss. The average percent of weekly weight loss of each batch was calculated32.Determination of samples firmnessThe samples of each experimental replicate evaluated on a weekly basis had their firmness measured as previously described. The weekly average samples firmness (N) of every treatment batch was also calculated33.pH measurementMango fruit of each experimental replicate were blended weekly into juice, after filtration, a digital pH meter (Jenway, UK) was used to measure pH. The weekly average fruit pH of every treatment batch was also calculated. The pH meter was calibrated using a buffer solution of pH 734.Total soluble solids (TSS) measurementTotal soluble solids of the prepared mango juice samples were measured in percent brix using a refractometer (ANTAHI, New Zealand). The weekly average fruit TSS (%) for each treatment batch was also calculated. The refractometers was calibrated using distilled water35.DPPH radical scavenging assayA 1/10 mango juice dilution was prepared using sterile distilled water. 100 μL of each dilution was mixed with 1 mL of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (100 mg L−1) to be incubated in the dark at 37 °C for 45 min. After incubation, samples were centrifuged and the pellet was discarded. The intensity of the change in color of the supernatant was measured by spectrophotometry at 517 nm using methanol as a blank. 100 μL of methanol in 1 mL DPPH was used as the control for the experiment. Percent radical scavenging activity was calculated as per the below formula:$$ % {text{ radical scavenging activity}}, = ,left( {{text{absorbance of the control solution}} – {text{ absorbance of the juice sample}}} right)*{1}00/{text{absorbance of the control solution}}. $$The weekly average % radical scavenging activity for each treatment batch was finally calculated31.Statistical analysisThe experimental design used was Completely Randomized Design (CRD). One-way ANOVA followed by Tukey Post-Hoc test was used to evaluate the significance of the weekly percent change in weight among treatment batches at P ≤ 0.05. The significances of pH and TSS variation within different treatment batches were evaluated using One-way ANOVA test at P ≤ 0.05. Data was presented as average ± standard error of the Means (SEM). SPSS (Ver. 27, SPSS Inc. Chicago, USA) was used to perform the statistical analysis tests. More

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