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Nature Video
19 January 2022

Tiny feather wing beetle reveals new way to fly

How a novel flying technique copes with the weird physics of miniature flight

Adam Levy

Adam Levy

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Flying is a tricky business, but when you are less than a millimetre long, things get even tougher. At such tiny scales the physics of flight changes, and yet insects have evolved strategies to fly in miniature. In this Nature video, we zoom in on new research which is revealing the never-before-seen flight tactics of a species of feather wing beetle.Research Article: Novel flight style and light wings boost flight performance of tiny beetles

doi: https://doi.org/10.1038/d41586-022-00144-0

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SamplingEnvironmental samplingSamples were collected from the littoral marine zone at Canoe Cove, Nahant, Massachusetts, USA, on 22 August (ordinal day 222), 18 September (261) and 13 October (286) 2010, during the course of the three month Nahant Collection Time Series sampling11.Bacterial isolation and characterizationBacterial isolationBacterial strains were isolated from water samples using a fractionation-based approach7 as previously described19,20. In brief, seawater was passed first through a 63um plankton net and then sequentially through 5um (Whatman 111113 or Sterlitech PCT5047100), 1um (Whatman 111110 or Sterlitech PCT1047100), and 0.2um (Whatman 111106) hydrophilic polycarbonate filters; material recovered on the filters was resuspended by shaking for 20 min; dilution series of resuspended cells were filtered onto 0.2um polyethersulfone filters (Pall 66234) in a carrier solution of artificial seawater (40 g Sigma Sea Salts, S9883; 0.2um filtered), and filters placed directly onto Vibrio-selective MTCBS plates (BD Difco TCBS Agar 265020, supplemented with with 10 g NaCl per liter to 2% final w/v). Colonies (96) from each of three replicates of each size fraction were selected from the dilution plates with the fewest numbers of colonies (1,152 isolates per isolation day). Colonies were purified by serial passage, first onto TSB-II (Difco Tryptic Soy Broth, 1.5% BD Difco Bacto Agar 214010, amended with 15 g NaCl to 2% w/v), second onto MTCBS, finally onto TSB-II again. Colonies were inoculated into 1 ml of 2216 Marine Broth (BD Difco 279110) in 96-well 2 ml culture blocks and allowed to grow, shaking at room temperature, for 48 h. Glycerol stocks were prepared by combining 100 ul of culture with 100 ul of 50% glycerol (BDH 1172-4LP) in 96-well microtiter plates and sealed with adhesive aluminum foil for preservation at −80 °C.Bacterial hsp60 gene sequencingTo obtain hsp60 gene sequences for isolates, Lyse and Go (LNG) (Pierce, Thermo Scientific 78882) treatments of subsamples of the same overnights cultures used in the bait assay (described below) were used directly as template in PCR amplification reactions. PCR reactions were prepared in 30 ul volumes, as follows: 1 ul LNG template, 3 ul 10x buffer, 3 ul 2 mM dNTPs, 3 ul 2um hsp60-F primer, 3 ul 2um hsp60-R primer, 0.3 ul NEB Taq, 16.7 ul PCR-grade HOH; with hsp60-F (H279) primer sequence: 5′-GAA TTC GAI III GCI GGI GAY GGI ACI ACI AC-3′, and hsp60-R (H280) primer sequence: 5′-CGC GGG ATC CYK IYK ITC ICC RAA ICC IGG IGC YTT-3′61 (Supplementary Table 1). PCR thermocycling conditions were as follows: initial denaturation at 94 °C for 2 min; 35 cycles of 94 °C for 1 min, 37 C for 1 min, 72 °C for 1 min; final annealing at 72 °C for 6 min; hold at 10 °C. PCR products were cleaned up by isopropyl alcohol (IPA) precipitation, as follows: addition of 100 ul 75% IPA to 30 ul PCR reaction product, gentle inversion mixing followed by 25 min incubation at RT, 30 min centrifugation at 2800 rcf, addition of 50 ul 70% IPA with gentle inversion wash, centrifugation at 2000 rcf, inversion on paper towels to remove IPA, 10 min centrifugation at 700 rcf, air drying in PCR hood for 30 min, resuspension in 30 ul PCR HOH. PCR products were Sanger sequenced (Genewiz, Inc.) using hsp60-R primer, as follows: 5 ul of 5 um hsp60-R primer, 7 ul nuclease free water, 3 ul DNA template. For a subset of strains hsp60 sequences were obtained from subsequently determined whole-genome sequences. Hsp60 sequences were aligned to the hsp60 sequence previously published for Vibrio 1S_84 and trimmed to 422 bases using Geneious (https://www.geneious.com/). Accession numbers for these 1287 strains are provided in Supplementary Data 1, where they are identified as baxSet1287.Bacterial hsp60 phylogeniesA phylogenetic tree of relationships among bacterial isolates screened in the bait assay (described below) was produced based on a 422 bp fragment of the hsp60 gene, derived either from Sanger or whole genome sequences; with E. coli K12 serving as the outgroup. Sequences from each of the three days of isolation were aligned using muscle v.3.8.3162 with default settings (muscle -in $seqsALL -out $seqsALL.muscleAln), and a single tree including all 1287 sequences from all the days was generated using FastTree v.2.1.863 (FastTree -gtr -gamma -nt -spr 4 -slow  $seqsALL.muscleAln.fasttree). For presentation in Fig. 1 three sub-trees including only nodes from each day were produced using PareTree v.1.0.264 (java -jar PareTree1.0.2.jar -t O -del notDay222.txt -f $seqsALL.$round.muscleAln.fasttree.DAY222). Trees were visualized using iTOL65 and painted with metadata for each of the strains, including: sensitivity to killing in agar overlay by co-occurring phage predators collected on the same day and, for the subset of strains that were genome sequenced and also included in the host range matrix, the bacterial species, based on concatenated ribosomal protein analysis using RiboTree66 as described below. Isolation days for each of the strains included in these analyses are provided in Supplementary Data 1, where these strains are identified as baxSet1287.Bacterial genome sequencing and assignment to populationsTo assign genome-sequenced bacterial isolates used in the host range assay to species, we use the RiboTree tool66 to produce a phylogeny based on concatenated single copy ribosomal proteins as in23. We include strains of previously described Vibrionaceae in preliminary analyses as reference strains and assign species names to new isolates based on clustering with named representatives, as well as provide placeholder names for newly identified clades with no previously described representatives. Trees were visualized using iTOL65 and the representation including only those strains included in the host range assay is shown in Supplementary Fig. 1; population assignments and accession numbers for this set of 294 genomes, which also includes a small number of previously isolated bacterial strains that were included in the host range assay (described below), are provided in Supplementary Data 1, where they are identified as baxSet294.Viral isolation and characterizationWe have previously described features of the viruses of the Nahant Collection20, as well as approaches used for the standardization of their genome assemblies19, additional details are provided below.Viral sample collectionThe iron chloride flocculation approach was used to generate 1000-fold concentrated viral samples from 0.2 um-filtered seawater, as follows. For each isolation day, triplicate 4 L seawater samples were filtered through 0.2 um polyethersulfone cartridge filters (Millipore, Sterivex, SVGP0150) into collection bottles, spiked with 400 uL of FeCl3 solution (10 gL−1 Fe; as 4.83 g FeCl3•6H2O (Mallinckrodt 5029) into 100 ml H2O), and allowed to incubate at room temperature for at least 1 h. Virus-containing flocs were then recovered from the sample by filtration onto 90 mm 0.2 um polycarbonate filters (Millipore, Isopore, GTTP09030) under gentle vacuum in a 90 mm glass cup-frit system (e.g Kontes funnel 953755-0090, fritted base 953752-0090, and clamp 953753-0090); once liquid was fully passed, the funnel was removed and, with the vacuum pump left on, the filters were folded into quarters, removed from the fritted base, and inserted into a 7 ml borosilicate glass vial. A volume of 4 ml of oxalate-EDTA solution (prepared from stock solution as 10 ml 2 M Mg2EDTA (J.T. Baker, JTL701-5), 10 ml 2.5 M Tris-HCl (Promega PAH5123), 25 ml 1 M oxalic acid (Mallinckrodt 2752); adjusted to pH 6 with 10 M NaOH (J.T. Baker, 3722-01); final volume 100 ml; used within 7 days of preparation and maintained at room temperature in the dark) was added to the vial and the sample allowed to dissolve at room temperature for at least 30 min before transfer to storage at 4 °C. A reagent used in this original formulation (JT-Baker 7501 Mg2EDTA) is no longer available and an updated recipe is provided elsewhere67.Bait assay and associated viral plaque archivalIn order to obtain estimates of co-occurring phage predator loads at bacterial strain level resolution, and generate plaques from which to isolate phages, we exposed 1440 purified bacterial isolates to phage concentrates from their same day of isolation (1334 yielded lawns sufficient to evaluate for plaques, and hsp60 sequences could be determined for 1287 of these). Bacterial strains screened included 480 isolates from each ordinal day, representing 120 strains from each of 4 size-fractionation classes (0.2 um, 1.0 um, 5.0 um, 63 um) details of isolation origin are provided for each strain in Supplementary Data 1, and description of naming conventions is as previously described19. For the bait assay each strain was mixed in agar overlay with seawater concentrates containing viruses (15 ul concentrate, equivalent to 15 ml unconcentrated seawater assuming 100% recovery efficiency; derived from pooling of three replicate virus concentrates from each day). We note that recoveries were not tested for individual samples and that previous tests14 of recovery efficiency have shown that resuspension of iron flocculates in oxalate solution yields initial recoveries of approximately 50% (49 ± 3% and 55 ± 11% for a marine sipho- and myo-virus respectively, at 24 h post re-suspension) and shows low decay rate over time (47 ± 5% and 73 ± 16% for a marine sipho- and myo-virus respectively, at 38 days post re-suspension). All of our assays were performed approximately 8–9 months post-sampling from oxalate concentrates stored at 4 °C. Agar overlays were performed based on the previously described Tube-free method13, as follows. Bacterial strains were prepared for agar overlay plating by streaking out from glycerol stocks onto 2216MB agar plates with 1.5% agar (Difco, BD Bacto, 214010), and allowed to grow for 2 days at room temperature. Strains were then inoculated into 1 ml 2216MB in a 96-well culture block and incubated 24 h at room temperature shaking at 275 rpm on a VWR DS500E orbital shaker. Immediately prior to use in direct plating the OD600 was measured in 96-well microtiter plates and subsamples were taken for Lyse and Go (LNG) processing for DNA (10 ul culture, 10 ul LNG). Phage concentrates were prepared for plating by pooling 1.2 ml from each of the concentrate replicates into a 7 ml borosilicate scintillation vial. Cultures were transferred from overnight culture blocks to 96-well PCR plates in 100 ul volume and 15 ul of pooled phage concentrate was added to cultures one row at a time, with each row plated in agar overlay before adding phage concentrate to the next row of bacterial cultures. Mixed samples of 100 ul bacterial overnight culture and 15 ul pooled phage concentrate were transferred to the surface of bottom agar plates (2216MB, 1% agar, 5% glycerol, 125 ml L−1 of chitin supplement [40 g L−1 coarsely ground chitin, autoclaved, 0.2 um filtered]). A 2.5 ml volume of 52 °C molten top agar (2216MB, 0.4% agar, 5% glycerol BDH 1172-4LP) was added to the surface of the bottom agar and swirled around to incorporate and evenly disperse the mixed bacterial and phage sample into an agar overlay lawn. Agar overlay lawns were held at room temperature for 14–16 days and observed for plaque formation. Glycerol was incorporated into this assay to facilitate detection of plaques68. Chitin supplement was incorporated into this assay to facilitate detection of phages interacting with receptors upregulated in response to chitin degradation products. A variety of preliminary tests exploring potential optimizations to agar compositions for direct plating indicated that the addition of chitin did not negatively impact recovery of plaques with control phage strains tested. After approximately 2 weeks, plaques on agar overlay lawns were cataloged and described with respect to plaque morphology and plaques were picked for storage based on the previously described Archiving Plaques method13, as follows. All plaques were archived from plates containing less than 25 plaques, on plates with larger numbers of plaques a random subsample of plaques from each distinct morphology were archived. A polypropylene 96-well PCR plate was filled with 200 ul aliquots of 0.2 um filtered 2216MB, agar plugs were collected from plates using a 1 ml barrier pipette tip and ejected into the 2216MB, skipping one well between each sample to minimize potential for cross-contamination, for a final count of 48 phage plugs per plate. Plaque plugs were soaked at 4 °C for several hours to allow elution of phage particles into the media. After soaking, 96-well plates were centrifuged at 2,000 rcf for 3 min before proceeding to the next step. Plug soaks were then processed for two independent storage treatments. For storage at 4 °C, plates were processed by transferring 150 ul of eluate from each well to a 0.2 um filtration plate (Millipore, Multiscreen HTS GV 0.22um Filter Plate MSGVS22) and gently filtered under vacuum to remove bacteria, the cell-free filtrates containing eluted phage particles from each plaque plug were stored at 4 °C. For storage at −20 °C, 50 ul of 50% glycerol was added to the residual ~50 ul of the plug elution, often still containing the agar plug. In this way all plaques were characterized and many plaques from each strain were archived in two independent sets of conditions. Total plaque counts for all strains included in the bait assay are represented in Fig. 1, and provided in Supplementary Data 1, where they are identified as baxSet1287. Notes on limitations to the assay: Water temperatures on each of the three isolations days were 13.8 °C, 16.3 °C, and 14.2 °C, for days 222, 261, and 286; as bait assays were performed at room temperature (approximately 22 °C) some phages requiring lower temperatures may not have yielded plaques. The majority of plates were evaluated for plaque formation twice, on day 1 and day 13, thus any plaques appearing after day 1 and disappearing before day 13 – for example as a result of overgrowth of lysogens—are likely to have been missed in these assays.Viral purificationA subset of plaques archived during the bait assay was selected for phage purification, genome sequencing, and host range characterization. This subset included single randomly-selected representatives from each plaque-positive bacterial strain. Minor details of the purification and lysate preparation varied across samples but were largely as follows. Phages were purified from inocula derived primarily from −20 °C plaque archives, and secondarily from 4 °C archives when primary attempts with −20 °C stocks failed to produce plaques. Three serial passages were performed using Molten Streaking for Singles13 method. Agar overlay lawns for passages were prepared by aliquoting 100 ul of host overnight culture (4 ml 2216MB, colony inoculum from streak on 2216MB with 1.5% Bacto Agar, shaken overnight at RT at 250 rpm on VWR DS500E orbital shaker) onto a standard size bottom agar plate and adding 2.5 ml of molten 52 °C top agar as in the bait assay, swirling to disperse the host into the top agar and form a lawn, and streaking-in phage with a toothpick either from the plaque archive or directly from well-separated plaques in overlays from the previous step in serial purification. Following plaque formation on the third serial passage plate plaque plugs were picked using barrier tip 1 ml pipettes and ejected into 250 ul of 2216MB to elute overnight at 4 °C. Plaque eluates were spiked with 20 ul of host culture and grown with shaking for several hours to generate a primary small-scale lysate. Small scale primary lysates were centrifuged to pellet cells and titered by drop spot assay to estimate optimal inoculum volume to achieve confluent lysis in a 150 mm agar overlay plate lysate. Plate lysates were generated by mixing 250 ul of overnight host culture with primary lysate and plating in 7.5 ml agar overlay. After development of confluent lysis of lawns as compared against negative control without phage addition, the lysates were harvested by addition of 25 ml of 2216MB, shredding of the agar overlay with a dowel, and collection of the broth and top agar. Freshly harvested lysates were stored at 4 °C overnight for elution of phage particles, the following day lysates were centrifuged at 5,000 rcf for 20 min and the supernatant filtered through a 0.2 um Sterivex filter into a 50 ml tube and stored at 4 °C.Viral genome sequencingSequencing of Nahant Collection viruses was described in previous work19, and was performed as follows. For DNA extraction approximately 18 ml of phage lysate was concentrated using a 30 kD centrifugal filtration device (Millipore, Amicon Ultra Centrifugal Filters, Ultracel 30 K, UFC903024) and washed with 1:100 2216MB to reduce salt concentrations inhibitory to downstream nuclease treatments. Concentrates were brought to approximately 500 ul using 1:100 diluted 2216MB and then treated with DNase I and RNase A (Qiagen RNase A 100 mg mL−1) for 65 min at 37 °C to digest unencapsidated nucleic acids. Nuclease treated concentrates were extracted using an SDS, KOAc, phenol-chloroform extraction and resuspended in EB Buffer (Qiagen 19086) for storage at 20 °C. Phage genomic DNA was sheared by sonication in preparation for genome library preparation. DNA concentrations of extracts were determined using PicoGreen (Invitrogen, Quant-iT PicoGreen dsDNA Reagent and Kits P7589) in a 96-well format and samples brought to 5 ug in 100 ul final volume of PCR-grade water diluent for sonication. Samples were sonicated in batches of 6 for 6 cycles of 5 min each, at an interval of 30 s on/off on the Low Intensity setting of the Biogenode Bioruptor to enrich for a fragment size of ~300 bp. Illumina constructs were prepared from sheared DNA as follows: end repair of sheared DNA (NEB, Quick Blunting Kit, E1201L), 0.72×/0.21× dSPRI (AMPure XP SPRI Beads) size selection to enrich for ~300 bp sized fragments, ligation (NEB, Quick Ligation Kit, M2200L) of Illumina adapters and unique pairs of forward and reverse barcodes for each sample, SPRI (AMPure XP SPRI Beads) clean up, nick translation (NEB, Bst DNA polymerase, M0275L), and final SPRI (AMPure XP SPRI Beads) clean up (Rodrigue et al., 2010). Constructs were enriched by PCR using PE primers following qPCR-based normalization of template concentrations. Enrichment PCRs were prepared in octuplicate 25 ul volumes, with the recipe: 1 ul Illumina construct template, 5 ul 5x Phusion polymerase buffer (NEB, 5X Phusion HF Reaction Buffer, B0518S), 0.5 ul 10 mM dNTPs (NEB, dNTP Mix (1 mM; 0.5 ml), N1201AA), 0.25 ul 40 uM IGA-PCR-PE-F primer, 0.25 ul 40 uM IGA-PCR-PE-R primer, 0.25 ul Phusion polymerase (NEB, Phusion High Fidelity DNA Pol, M0530L), 17.75 ul PCR-grade water. PCR thermocycling conditions were as follows: initial denaturation at 98 °C for 20 sec; batch dependent number of cycles (range of 12–28) of 98 °C for 15 sec, 60 °C for 20 see, 72 °C for 20 sec; final annealing at 72 °C for 5 min; hold at 10 °C. For each sample 8 replicate enrichment PCR reactions were pooled and purified by 0.8x SPRI beads (AMPure XP) clean up. Each sample was then checked by Bioanalyzer (2100 expert High Sensitivity DNA Assay) to confirm the presence of a unimodal distribution of fragments with a peak between 350–500 bp. Sequencing of phage genomes was distributed over 4 paired-end sequencing runs as follows: HiSeq library of 18 samples pooled with 18 external samples, 3 MiSeq libraries each containing ~100 multiplexed phage genomes. Accession numbers for all sequenced phage genomes are provided in Supplementary Data 1, where they are identified as phageSet283; the subset of phages used in the majority of analyses in this work are identified as phageSet248 and exclude non-independent isolates derived from the same plaque, as well as well as identical phages isolated from multiple independent plaques from the same host strain in the bait assay.Viral protein clusteringTo characterize and annotate groups of proteins in assembled viral genomes in the Nahant Collection19, proteins were clustered using MMseqs2 v. 2.2339469 with default parameter settings, the 21,937 proteins reported in the GenBank files associated with each of the 283 Nahant Collection phage genomes were clustered into 5,929 clusters including 2,978 singletons. MMseqs2 cluster assignments for each protein sequence are provided in Supplementary Data 6.Viral protein cluster annotationAll proteins were annotated using InterProScan70 v.5.39–77.0; eggNOG-mapper71,72 v.2 using both automated and viral HMM selection options; Meta-iPVP73; and with best matches to 9518 Viral Orthologous Groups74 HMM profiles (obtained at http://dmk-brain.ecn.uiowa.edu/pVOGs/downloads.html); search was performed with hmmer, requiring a bitscore of 50 or greater (highest e-value 5.80E-13), as follows: hmmsearch -o $out_dir/$hmm_group.$hmmfile.$prots_short_name.hmm.out -tblout $out_dir/$hmm_group.$hmmfile.$prots_short_name.hmm.tbl.out -noali -T 50 $hmmfile $prots_dir/$prots_file. Annotations for viral protein clusters are provided in Supplementary Data 6.Receptor binding proteins (RBPs) were annotated as follows. RBPs were defined here to include both globular and fibrous host interacting proteins and general protein annotations were reviewed for similarity to known phage receptor binding proteins and supplemented with Phyre275, HHpred, and literature review76. Annotated RBPs were mapped onto phage genome diagrams and additional RBPs were annotated based on gene order conservation with phages in the same genus for which RBPs were already identified; annotated RBPs were then used to iteratively search against all Nahant Collection phage proteins using the jackhmmer search tool in the HMMER77 v.3.2.1 package (jackhmmer -cpu 16 -N 3 -E 0.00001 -incE 0.01 -incdomE 0.01 -o $run.$1.vs.$2.jackhmmer.iters-$iters.out -tblout $run.$1.vs.$2.jackhmmer.iters-$iters.tbl.out -domtblout $run.$1.vs.$2.jackhmmer.iters-$iters.dom.tbl.out $queryFASTAS $subjectFASTAS) and new hits were manually reviewed. All annotations were performed on a protein-cluster level and annotations of proteins and protein clusters as “adsorption – RBP” are indicated in Supplementary Data 6.Recombinases were annotated as follows: Homologs of single strand annealing protein recombinases in the Rad52, Rad51 and Gp2.5 superfamilies in the Nahant Collection phages were identified as described below. First, iterative HMM searches were performed against the Nahant Collection phage proteins using as seeds 194 recombinases identified in Lopes et al.44 (excluding RecET fusion protein YP_512292.1; http://biodev.extra.cea.fr/virfam/table.aspx), these represent 6 families of SSAP recombinases (UvsX, Sak4, Sak, RedB, ERF, and Gp2.5); searches were performed using the jackhmmer function of HMMER v.3.1.2 (jackhmmer -cpu 16 -N 5 -E 0.00001 -incE 0.01 -incdomE 0.01 -o $run.$1.vs.$2.jackhmmer.out -tblout $run.$1.vs.$2.jackhmmer.tbl.out -domtblout $run.$1.vs.$2.jackhmmer.dom.tbl.out $queryFASTAS $subjectFASTAS) – this yielded 156 proteins. Second, all hits were plotted onto genome diagrams for all phages in the collection and additional candidate recombinases identified based on gene neighborhood comparisons (Supplementary Data 9) – this step identified 4 additional protein clusters (mmseqs 297, 149, 2211, and 600), totaling 224 proteins. Third, all proteins clusters were curated by manual review of annotations made using InterProScan70, EggNOG-mapper71, and Phyre275 (annotations provided in Supplementary Data 6) to identify potential false positives (none identified), and references to recombinases in annotations. Where these annotation methods did not provide additional support, sequences were evaluated for additional support using HHpred78 (hhsearch -cpu 8 -i../results/full.a3m -d /cluster/toolkit/production/databases/hh-suite/mmcif70/pdb70 -o../results/2058109.hhr -oa3m../results/2058109.a3m -p 20 -Z 250 -loc -z 1 -b 1 -B 250 -ssm 2 -sc 1 -seq 1 -dbstrlen 10000 -norealign -maxres 32000 -contxt /cluster/toolkit/production/bioprogs/tools/hh-suite-build-new/data/context_data.crf) as implemented on the MPI Bioinformatics Toolkit webserver (mmseq 2896 and 5138 both gave >99% probability hits to DNA repair protein Rad52 with PDB ID 5JRB_G), or JackHMMER (-E 1 -domE 1 -incE 0.01 -incdomE 0.03 -mx BLOSUM62 -pextend 0.4 -popen 0.02 -seqdb uniprotkb) as implemented on the EMBL-EBI webserver (mmseq 2990 showed hits to diverse RedB family RecT-like sequences at e-value ≤1e-05). Following this third step, there were 3 protein clusters for which support was limited, these were included in the final dataset as putative SSAP recombinases but are highlighted here. Protein cluster mmseq 297 (present in 21 phages in 6 genera): was always encoded by genes adjacent to genes in protein cluster mmseq 3923, which was itself a recombinase associated exonuclease that was found either adjacent to mmseq 297 or to the well-supported putative SSAP recombinase mmseq 3721 (sometimes separated by one gene from mmseq 3721). Protein cluster mmseq 600 (present in 2 phages in 2 genera): was encoded adjacent to a protein cluster annotated as a recombination associated exonuclease; iterative HHMER searches of a mmseq 600 cluster representative (AUR82881.1) against Viruses in UniProtKB using jackhmmer yielded hits to proteins in mmseq 297 in iteration 3. Protein cluster mmseq 2990 (present in 1 phage): was encoded adjacent to two small proteins encoding putative recombination associated exonucleases and was in the same genomic position relative to neighboring genes as putative recombinases in related phages in the genus. Finally, all putative SSAP recombinase genes were assigned to a recombinase family by clustering based on 2 iterations of all-by-all HMM jackhmmer sequence similarity searches of all candidates and the reference seed set of Lopes44 (jackhmmer -cpu 16 -N 2 -E 0.00001 -incE 0.01 -incdomE 0.01 -o $run.$1.vs.$2.jackhmmer.out -tblout $run.$1.vs.$2.jackhmmer.tbl.out -domtblout $run.$1.vs.$2.jackhmmer.dom.tbl.out $queryFASTAS $subjectFASTAS); similarities were were visualized using Cytoscape v.3.3.0 using the “Edge-weighted Spring Embedded Layout” based on jackhmmer score, clusters were identified using the ClusterMaker2 v.1.2.1 Cytoscape plugin with the MCL cluster option and all settings at default and Granularity=2.5. Proteins in 3 mmseq clusters (149, 297, 600) did not fall into MCL clusters with recombinases from the annotated seed set and therefore are described as “unknown” rather than being assigned to a family of recombinases. All final assignments of genes to a recombinase superfamily and family, as well as all associated annotations, are provided in Supplementary Data 6 (sheet A.prots_overview column anno_Recombinase_manual). Additional details regarding seed sequences and MCL cluster assignments associated with recombinase analyses are provided in Supplementary Data 7 which contains a main descriptor sheet (00.readme), an overview of the 224 Nahant phages with recombinases (sheet 01.NahantPhageRecombinases_224), a table of InterPro domains associated with each of the reference and Nahant recombinases, with specific mmseqs and MCL clusters (sheet 02.IPR_annos_Lopes+Nahant), a list of all references used (sheet 03.List1_LOPES_ALL.noETfusion), the output of the iterative jackhmmer search with seeds against all Nahant Collection proteins (sheet 04.List1_vs_NahantProts), the output of the all-by-all jackhmmer search for 194 references and 224 putative Nahant recombinases (sheet 05.Lopes+Nahant224_v_self2iter), and information on the assignment of all Nahant and reference proteins to MCL clusters as shown in Fig. 6 (06.Recombinase_assign_by_MCL).All proteins were assigned to one of three broad categories – structural, other (non-structural), or no prediction – based on manual review of annotations derived from: NCBI product ID, Virfam21, PhANNs79, pVOGs74, eggNOG-mapper72, Phyre275, the MPI Bioinformatics implementation of HHpred78, and targeted annotations of predicted receptor binding proteins and recombinases (see descriptions for targeted annotations in Methods, above). Protein clusters (mmseq groups) were reviewed for conflicting calls and ultimately all proteins within each protein cluster (mmseqsID) were assigned to a single category. All assignments, and annotations on which they were based, are provided in Supplementary Data 6.The approach for assigning annotations to these broad categories was as follows: Step 1) All genes identified as putative recombinases through targeted annotations were assigned as “other”. Step 2) All genes identified as putative receptor binding proteins through targeted annotations were assigned as “structural”. Step 3) Genes not assigned to a category in steps 1 and 2, and which were identified by Virfam as “head-neck-tail” associated were assigned as follows: Genes annotated by Virfam as a terminase (TerL) were assigned as “other”; genes annotated by Virfam as a major capsid protein (MCP), portal (portal), adaptor (Ad1, Ad2, Ad3), head-closure (Hc1, Hc2, Hc3), tail completion (Tc1, Tc2), major tail protein (MTP), neck (Ne), or sheath (sheath) were assigned as “structural”. Step 4) Genes not assigned to a category in steps 1–3, were assigned as “structural” or “other” (non-structural) if identified as such by PhANNs with a confidence of ≥95%. Cases where conflicting annotations were observed between PhANNs and other annotations were flagged for review in subsequent steps. Step 5) Genes with annotations of VOG0263 (DNA transfer protein); terminal protein, any reference to internal virion protein, DNA circularization protein, and MuF-like proteins were assigned as “other”; in the case of conflict the Step 5 annotation superseded the prior annotations. Step 6) Genes with annotation as a terminase (large subunit, small subunit, and unspecified) by any of the tools (requiring ≥ 90% confidence if based on Phyre2) were assigned as “other”. Step 7) All genes lacking support across annotations were assigned as “no prediction”, high confidence Phyre2 predictions qualitatively judged as inappropriate were disregarded. Step 8) Genes flagged in Step 4 were reviewed and assigned as “structural” when containing any structural related genes (i.e. those listed in Step 3 and any others identifiable as structural based on words in the annotations and consensus across tools, e.g. containing the word baseplate, capsid, coat, head, spike, tail, whisker, fibritin). Additional targeted annotation by HHpred was used to facilitate assignment to “structural” (known structural proteins as described for Step 3 and in the aforementioned list), “other” (non-structural), “no prediction” (e.g. no assignable function based on available annotations and a PhANNs confidence of More

Analysis of the roof failure characteristics of coal seamBefore mining, fracturing was conducted on a portion of gritstone in the lower section of the Naoro Formation and then entered the mining stage. Figure 9 shows the influence law of coal roof rupture under different periodic pressures. With mining of the #2 coal seam working face, the direct roof of the coal seam partially broke and collapsed, forming gangue in the goaf. There is a clear separation between the direct and basic roof. When the working face advanced to 228.2 mm, the old roof ruptured, and the working face started to enter the periodic pressure-bearing stage. As the working face advanced to 592.9 mm, the roof exhibited the fourth periodic pressure. The overlying layer roof in the excavation area was affected by the upper bearing arch pressure, leading to the collapsed rock to not completely contact the upper roof. With the increasing distance of coal seam mining, the roof developed significant subsidence, and the influence range of the bedrock boundary caused by the mining was still in the isolation layer fracturing zone. The bedrock influence boundary angle reached 73.57°, and the rock fracture angle was 56.95°. When the working face advanced to 726.5 mm, the fifth periodic pressure on the roof occurred. The bedrock layer in the upper right of the workings was near the right boundary of the first isolated coal seam rupture. Then, coal mining was suspended, and a second isolated seam fracturing process was conducted. The bedrock influence boundary angle reached 73.57°, and the rock rupture angle was 56.95°.Figure 9Influence law of coal roof rupture during different periodic pressure.Full size imageWhen the processing was advanced to 798.4 mm, the bedrock layer in the upper right of the processed area became close to the right boundary of the second isolated seam fracturing. After the third isolated layer fracturing process, the rock impact boundary angle reached 75.33°, and the rock fracture angle was 50.39°. Proceeding to 1031.6 mm, eighth periodic pressure was generated on the roof. The falling gangue in the mined-out area was in contact with the roof, with the bedrock impact boundary angle reaching 74.77° and the rock fracture angle reaching 57.06°. Thereafter, the bedrock layer of the roof gradually entered the full-scale mining stage. As the working face continues to advance, the bedrock impact boundary caused by coal seam mining should be in isolated coal seam fractures. When the bedrock layer at the working face is close to the right boundary of the isolation layer fracturing, the next isolation layer fracturing should be performed.Analysis of roof stress evolution lawFigure 10 illustrates the change law of the roof support pressure when mining of the working face, in which the roof support pressure curve is the stress change minus the initial value of the sensor before mining. After the excavation of the working face, the surrounding rock will exhibit stress redistribution. The increase in tangential stress in front of the working face or on both sides is called the support pressure. The peak value of the support pressure generally occurs on the front of the working face. As the working face advanced to 228.2 mm, the direct roof gradually broke and collapsed with mining. Due to the redistribution of surrounding rock stress, the stress fluctuation at the open cut was clear. In front of the working face, the overlying rock stress was redistributed due to mining, and the vertical pressure peak area appeared, with a stress increment of 0.03 MPa. When the working face advanced to 360.8 mm, the first cycle pressure on the roof occurred. The falling gangue in the mine-out area gradually approached its upper strata, and the peak support pressure increments reached 0.05 MPa. During the advancement of the working face to 592.9 mm, the direct roof continued to collapse. The gangue at the cuttings was gradually compacted with the roof, and the stresses gradually restored to stability. Coal seam mining led to the decompression of the floor, and the vertical stress maximum reduction at the working face was 0.045 MPa. The peak vertical pressure in front of the working face shifted to the right as mining progressed. When degradation reached 726.5 mm, the fifth periodic pressure on the roof occurred. Figure 10b shows that the fracture of the isolation layer had no apparent effect on the change in roof stress. Within 560 mm from the open excavation, the mine-out area gangue gradually compacted with the roof. Vertical pressure changes between the fourth and fifth periodic pressures are slight and practically nonsignificant.Figure 10Vertical pressure variation law with coal mining. (a) First pressure and First periodic pressure difference. (b) Fourth and First periodic pressure difference. (c) Eighth and Ninth periodic pressure difference. (d) Eleventh and Twelfth periodic pressure difference. (e) Variation laws of vertical pressure with mining.Full size imageWhen the mining reached 1031.6 mm, the directly caving gangue completely filled the goaf and was compacted with the roof. The upper roof of the caving rock was supported again, and the compaction range of the mining area extended to 821 mm. As the working face advanced to 1338.9 mm, the peak vertical pressure appeared at 1400 mm, with a maximum increment of 0.375 MPa. The compaction range of the mining area extends to 1200 mm. Then, the fractured isolation layer can be grouted. The subsequent working face advances until the end of mining, and the rock movement above the mine-out zone will exhibit a periodic “falling-filling-cutting-compaction” process. Fracture grouting of the flexible isolation layer has no significant effect on the vertical stress changes, and the stress unloading area and the peak vertical pressure will continue to change with mining. Nevertheless, consideration needs to be given to the adequacy of the gangue falling from the roof for isolation layer grouting.Roof displacement and development pattern of water-conducting fracture zoneFigure 11 shows the development law of the roof water-conducting fissures in the roof of the coal seam during different pressure periods, where the illustration shows the von Mises equivalent strain. Figure 12 shows the development trend of the water-conducting fracture zone height. From the whole observation, although the isolation layer is treated by fracturing before back mining, it has less influence on the displacement and deformation of the overlying rock layer because it is restricted by the surrounding rock of the model. When the working face was mined to 228.2 mm, the upper roof of the mining face collapsed, and the first periodic pressure occurred on the roof. The roof displacement reached the Yan’an Group mudstone layer, and the roof collapse height was only 104.3 mm. As the mining advanced, the roof fractures in the mining-out area continued to develop upwards. When the working face was mined to 360.8 mm, the first cycle pressure on the roof occurred, and the roof collapse height extended upwards to the siltstone of the Yan’an Formation, with a collapse range of 117.6 mm. At this point, only a small displacement change occurred around the direct roof, and the flexible isolation layer was basically not affected by any impact.Figure 11Development regularity of roof water-conducting fissures during different period pressure.Full size imageFigure 12Development height curve of water-conducting fracture zone.Full size imageFrom the second cycle pressure onwards, the development trend accelerated significantly, and the collapsed height rose rapidly to 210.9 mm. When the working face advanced to 537.1 mm, the third cycle pressure occurred on the roof. The collapsed Yan’an Formation mudstone layer was further pressurized by its upper layers and collapsed to a height of 344.7 mm. The roof displacement had spread to the coarse sandstone of the Naoro Formation, but the height of the water-conducting fracture zone had not reached the bottom of the isolation layer. When the workings reached 592.9 mm, the roof collapsed again, showing the fourth periodic pressure. The water-conducting fissure zone continues to develop upwards to 355.3 mm, which passes through the fissure isolation layer and reaches the gritstone at the top of the isolation layer. The fractured isolation layer is in an “activated” state.When the working face reached 1031.6 mm, fallen gangue completely filled the mining-out area and compacted with the roof, and eighth periodic pressure occurred on the roof. The height of the water-conducting fracture zone developed to 496.8 mm, which was lower than the height of the water-conducting fracture zone of 565.8 mm at the seventh periodic pressure. After that, the old roof collapsed as a cantilevered beam. The development height of the water-conducting fracture zone was allegedly less than 565.8 mm. Afterwards, the roof fracturing direction was consistent with the direction of working face advancement, from left to right. Displacement and fracture of the overlying rock layer were mainly caused by the overall downwards sliding of the upper rock seam due to the collapse of the bottom rock seam. At different heights of the coal seam roof, the degree of displacement damage decreased with increasing height.When the working face reached 1178.7 mm, the roof covering the open cut stabilized. The fractured isolation layers in the 1st ~ 13th groups were grouted, and then the coal was mined only after the slurry had completely solidified and reached a certain strength. The eleventh periodic pressure occurred on the roof, with a water-conducting fracture height of 367.6 mm at this time. When the working face was advanced to 1471.9 mm and 1645.2 mm, the roof had twelfth and fourteenth periodic pressures, and the heights of the water-conducting fracture zone were 332.0 mm and 416.0 mm, respectively. Then, the 14th ~ 15th and 16th ~ 17th group isolation layers of the upper coal seam were grouted while fracturing the right isolation layer. However, the disruption of displacement towards the extent of the development had a relatively small impact, mainly on the roof rock layer above the mining face. Table 2 indicates the development height of the water-conducting fracture zone and the fracture and grouting sequence of the isolated layer.Table 2 Development pattern of water-conducting fracture zone and fracture and grouting sequence of isolated layer.Full size tableDuring the mining process, damage to the water-conducting fissure zone was always a major factor in the displacement of the roof slab. Nonetheless, after fracturing and grouting measures, the effects of the damage were significantly reduced such that the damage to the roof rock was contained within the flexible isolation layer. After grouting, the enhanced strength of the isolation layer ensured that mining was carried out normally. During the mining period, four grouting reforms were made, and the isolation layer was fractured six times, with the maximum development height of the water-conducting fracture zone located at the seventh periodic pressure, reaching 565.8 mm.Analysis of water flow evolution law of overburden roofTo analyse the seepage law of the overburden roof, seven water flow monitoring lines were arranged from the top of the flexible isolation layer to the direct roof of the coal seam. The No. 1 water flow monitoring line was placed in the position of the third group of the isolation layer, which is initially located outside the deformation range of bedrock disturbed by mining and outside the stop line. The flow line was mainly used to monitor the influence of the rock disturbance boundary above the open cut on isolated seam fracturing and grouting. No 2–3 water flow monitoring lines were placed at the isolation layer positions of Group 12 and Group 14, which were initially located near the maximum height of the water-conducting fracture zone and were mainly used to monitor the change laws of the water-conducting fracture zone with mining impact. Monitoring Lines 4–6 were placed in isolation layers No. 17, No. 22 and No. 26 to study the impact of water flow changes with mining disturbance and the advanced influence scope. Water flow monitoring line No. 7 was placed in the thirtieth group of isolated layers, which was originally outside the cut-off line. As shown in Fig. 13, white arrows are water flow vectors in mL/min. Fracturing the 1–18 isolation layers before mining, the water tank hot water was injected into the flexible isolation layer such that the iodized salt in the flexible isolation layer was completely dissolved, and the infrared monitor showed the yellow area in the image. At this point, the water flow monitoring Lines 1–3 and 5–7 show yellow status, indicating that after the fracturing of the isolation layer, the aquifer water flows downwards along the fracture. The lower part of monitoring Line 4 was compacted at the top of the coal seam, indicating that the cracks between the roof and the aquifer had not been communicated. Therefore, the water flow rate was 0 mL/min until the sixth periodic pressure. Mining was then undertaken on the working face. The No. 1 monitoring line was therefore less affected by mining due to its layout outside the stop line, and there was no significant change in water flow before the first grouting.Figure 13Water flow evolution of the overburden roof with coal mining.Full size imageAs shown in Fig. 13, when the working face progressed to second periodic pressure, with the collapse of the coal seam, the stress of the surrounding rock was redistributed, the height of the water flowing fractured zone of the roof increased, and the water flow of the No. 2 monitoring line increased from the initial 9.1 mL/min to 14.0 mL/min. As the working face was advanced above the No. 2 monitoring line, the fifth periodic of pressure were generated in the roof. The development height of the roof water flowing fractured zone reached 504.4 mm. The roof was separated and collapsed, the cracks in the monitoring line communicated with each other, and the rock stress was released. The water flow in the No. 2 monitoring line increased significantly. Monitoring line No. 3 was affected by advanced mining, resulting in the coal seam roof’s increased rock fissures, the water flow path and resistance were reduced, and the water flow reached 48.3 mL/min. At the same time, the influence range of working face bedrock was close to the boundary of the first fracturing of the flexible isolation layer, and Groups 20–22 of isolation lays had been fractured.When mining started at the sixth periodic pressure, the roof water-conducting fracture zone gradually reached the maximum height and penetrated the fractured isolation layer, and the fracture of the roof rock increased. Lines No. 2 and No. 3 reached 44.4 mL/min and 85.6 mL/min, respectively. In fact, the encounter may indicate that the confined water of the gritstone aquifer was released, and the water flow of the working face increased. Then, the working face progressed, and the collapsed gangue above the mining-out area was compacted into the bedrock roof. The stress in the goaf did not change significantly, and the cracks in the strata decreased. The No. 2 and No. 3 water flows of the monitoring line gradually dropped. During this period, the change law of monitoring Lines 4–7 was similar to that of No. 2 and No. 3. During coal seam mining, the roof underwent a process of fracture, collapse, compaction and full mining, and the water flow monitoring line also went through a process of rising and then falling.When the working face was advanced to the eleventh periodic pressure, the grouting transformation of isolation layers 1–12 was conducted. The slurry was injected into the flexible isolation layer by hand pressure pump along the grouting pipe. After the slurry solidified, the colour of the No. 1 and No. 2 monitoring lines gradually became shallow, and the water flow gradually decreased under infrared observation. As the extraction of the coal seam progressed and the flexible insulation layer was broken and grouted, the colour of observation Lines 1–4 turned black in the infrared observation until the fourth grouting of insulation layer 18–19, and the water flow rate all showed 0 mL/min. However, the lower strata of the flexible isolation layer were not yet stabilized, so monitoring Lines 5–7 did not undergo any grouting transformation and still had a large water flow until the end of mining. Flow metre and infrared observations show that the destruction and grouting of the flexible isolation layer had a noticeable effect on the seepage characteristics of the overburden. In particular, after the grouting of the isolation layer, the slurry filled and solidified rapidly, the water flow decreased rapidly, and the water plugging effect of flexible isolation layer grouting was remarkable.Discussion and analysisDuring coal seam mining, the fracturing of the flexible isolation layer should be based on the premining overtopping influence range; that is, when the boundary line of bedrock influence extends to the range of the flexible isolation layer reached by the fracturing area of the flexible isolation layer, the next fracturing should continue. The average boundary angle range of the bedrock was 76.7°, and the field angle should not be less than 73.57°. The grouting of the flexible isolation layer considers the full mining degree of the coal seam. When there is no significant change in stress in the mined area, grouting of the flexible isolation layer at the top of the goaf is conducted. According to the simulation experiment in this paper, the full mining distance of the working face is 1338.9 mm, and the actual distance on site is 187.446 m. It is calculated that the distance between the fracture of the flexible isolation layer should be no less than 854.8 mm away from the working face, and the actual distance on site is 119.672 m. After the working face enters full mining, the shortest distance between the fracturing grouting range of the flexible isolation layer and the working face is not less than 242.6 mm, and the actual distance on site is 33.964 m.As seen from the previous analysis, with the advancement of the working face, the bedrock influence boundary angle of the coal seam does not change significantly, which only plays a guiding role in the fracturing sequence of the flexible isolation layer. The fracturing of the flexible isolation layer had an clear influence on the seepage of water-rich bedrock at the bottom of the Zhiluo Formation. The water-flowing fractured zone formed in the process of coal seam mining promoted the release of fractured water in the water-rich bedrock at the bottom of the Zhiluo Formation. The higher the height of the water-flowing fractured zone is, the greater the seepage of the water-rich bedrock. Coal seam mining had little effect on the seepage characteristics of the water-rich bedrock layer at the bottom of the Zhiluo Formation in the range of not disturbed by mining and advanced influence.In accordance with the stress sensor data, when the working face passed a certain distance, the bottom plate of the extraction area was compacted by the falling gangue, and the sensor pressure data did not change with the mining face. At this time, the grouting of the fracturing area of the flexible isolation layer corresponding to the above goaf was not affected by the mining face. For example, the stress in the goaf of 1200 mm had no clear change. Therefore, the first grouting was conducted in the fracturing area. After the solidification of the grouting slurry, the water flow of monitoring lines No. 1 and No. 2 decreased significantly. This minimized the impact on the original geological environment and at the same time reduced the goaf water drainage of the working face. The sealing effect of the isolation layer has an important influence on promoting water-retaining coal mining.The experimental application of the flexible isolation layer has realized its feasibility from the physical simulation test method in this paper. The realization of a flexible isolation layer requires premining fracturing and postmining isolation grouting. At present, premining fracturing can be achieved by directional drilling technology. There are also examples of roof separation grouting for postmining flexible isolation layer grouting28,29. Therefore, there is no technical bottleneck in field applications. Moreover, there is still a certain distance from the specific engineering application. According to the results of this study, it is predicted that the implementation of a flexible isolation layer will have great significance for water conservation coal mining in western China, which can reduce soil erosion and protect surface ecology. More

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18 January 2022

Portugal leads with Europe’s largest marine reserve

Filipe Alves

 ORCID: http://orcid.org/0000-0003-3752-2745

0
,

João G. Monteiro

 ORCID: http://orcid.org/0000-0002-3401-6495

1
,

Paulo Oliveira

2
&

João Canning-Clode

 ORCID: http://orcid.org/0000-0003-2143-6535

3

Filipe Alves

MARE Marine and Environmental Sciences Centre, Madeira, Portugal.

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João G. Monteiro

MARE Marine and Environmental Sciences Centre, Madeira, Portugal.

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Paulo Oliveira

Institute of Forests and Nature Conservation (IFCN, IP-RAM), Funchal, Madeira, Portugal.

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João Canning-Clode

Smithsonian Environmental Research Center, Maryland, USA.

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Marine conservation is central to the United Nations’ Sustainable Development Goals 13 (climate action) and 14 (life below water). Portugal has now created the largest marine reserve with full protection in Europe and the North Atlantic, an achievement that other nations could follow.

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Nature 601, 318 (2022)
doi: https://doi.org/10.1038/d41586-022-00093-8

Competing Interests
The authors declare no competing interests.

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