Gastropods

All of the marine and terrestrial samples were collected in 2001–2013 from a stony shore and a farm in Yugawara (35°08′N, 139°07′E), Japan, respectively. The stony shoreline surveyed represented ∼0.2 hectares and ranged in depth from 0 to 5 m, where brown and red macroalgae are dominant primary producers but seagrass is absent. The farm was also approximately 0.2 hectares with cultivation of fruits and vegetables, all of which were C₃ plants. Green leaves and/or nuts were collected for higher plants, and whole samples of 1–15 individuals within a single stage were collected for the other species. The collected samples were cleaned with distilled water to remove surface contaminants and stored at −20°C. For most terrestrial species and marine macroalgae, whole-organism samples were prepared for isotopic analyses. For the remaining marine specimens, small samples of muscle tissue were taken. Shell samples were taken from several gastropod and lobster specimens, and scales were dissected from most of the fish species (Appendices A1 and A2). There was no substantial effect on the trophic position estimates among these different tissue types within a single animal specimen (e.g., Chikaraishi et al. 2010 , 2011 ; Ogawa et al. 2013 ). The bulk-carbon and bulk-nitrogen isotopic compositions of representative samples (40 coastal marine and 69 terrestrial samples, Appendices A1 and A2) were determined using a Flash EA (EA1112) instrument coupled to a Delta^plusXP IRMS instrument with a ConFlo III interface (Thermo Fisher Scientific, Bremen, Germany). Carbon and nitrogen isotopic compositions are reported in the standard delta (δ) notation relative to the Vienna Peedee Belemnite (VPDB) and to atmospheric nitrogen (AIR), respectively.
The nitrogen isotopic composition of amino acids was determined by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) after HCl hydrolysis and N-pivaloyl/isopropyl (Pv/iPr) derivatization, according to the procedure in Chikaraishi et al. (2009 ) (which are described in greater detail at http://www.jamstec.go.jp/biogeos/j/elhrp/biogeochem/download_e.html). In brief, samples were hydrolyzed using 12 Mol/L HCl at 110°C. The hydrolysate was washed with n-hexane/dichloromethane (3/2, v/v) to remove hydrophobic constituents. Then, derivatizations were performed sequentially with thionyl chloride/2-propanol (1/4) and pivaloyl chloride/dichloromethane (1/4). The Pv/iPr derivatives of amino acids were extracted with n-hexane/dichloromethane (3/2, v/v). The nitrogen isotopic composition of amino acids was determined by GC/C/IRMS using a 6890N GC (Agilent Technologies, Palo Alto, CA) instrument coupled to a Delta^plusXP IRMS instrument via a GC-C/TC III interface (Thermo Fisher Scientific, Bremen, Germany). To assess the reproducibility of the isotope measurement and obtain the amino acid isotopic composition, reference mixtures of nine amino acids (alanine, glycine, leucine, norleucine, aspartic acid, methionine, glutamic acid, phenylalanine, and hydroxyproline) with known δ¹⁵N values (ranging from −25.9‰ to +45.6‰, Indiana University, SI science co.) were analyzed after every four to six samples runs, and three pulses of reference N₂ gas were discharged into the IRMS instrument at the beginning and end of each chromatography run for both reference mixtures and samples. The isotopic composition of amino acids in samples was expressed relative to atmospheric nitrogen (AIR) on scales normalized to known δ¹⁵N values of the reference amino acids. The accuracy and precision for the reference mixtures were always 0.0‰ (mean of Δ) and 0.4–0.7‰ (mean of 1σ) for sample sizes of ≥1.0 nmol N, respectively.
The δ¹⁵N values were determined for the following 10 amino acids: alanine, glycine, valine, leucine, isoleucine, proline, serine, methionine, glutamic acid, and phenylalanine (Appendices A1 and A2). These amino acids were chosen because their peaks were always well separated with baseline resolution in the chromatogram (Chikaraishi et al. 2009 ). Also, it should be noted that glutamine was quantitatively converted to glutamic acid during acid hydrolysis; as a result, the α-amino group of glutamine contributed to the δ¹⁵N value calculated for glutamic acid.
The TP_Glu/Phe value (and its potential uncertainty calculated by taking into account the propagation of uncertainty on each factor in the Eq. (1)) was calculated from the observed δ¹⁵N values (as 1σ = 0.5‰) of glutamic acid and phenylalanine in the organisms of interest, using eq. (1) with the β value of −3.4 ± 0.9‰ for coastal marine and +8.4 ± 1.6‰ for terrestrial samples, and with the TDF value of 7.6 ± 1.2‰ for both ecosystems, according to Chikaraishi et al. (2009 , 2010 , 2011 ). The TP_Tr/Scr values were not calculated, because we did not measure the δ¹⁵N values of lysine and tyrosine for all investigated samples and of serine for approximately a half of samples.

GenBank data was parsed using a combination of command-line and custom Perl scripts using BioPerl modules [22 (link)]. Tabular data was formatted using Python and plotted in R [23 ]. We use the terminology from Nilsson et al., (2005) and refer to taxa identified to the species rank as ‘fully identified’ and all other taxa as ‘insufficiently identified’ [24 (link)]. We also focused on NCBI nucleotide data deposited from 2003, the year COI barcoding was first introduced to the community, to present (2017) [25 (link)].
The names and taxonomic identifications for all Eukaryotes annotated to the species rank were retrieved from the NCBI taxonomy database using the Entrez query "Eukaryota[ORGN]+AND+species[RANK]" with an ebot script [Accessed November 3, 2017] [26 ]. Taxa were filtered according to the contents of the species field so that only fully identified taxa with a complete Latin binomial (genus and species) were retained. Entries that contained the abbreviations sp., nr., aff., or cf. were discarded. The remaining species names were formatted for use in the next query [species list]. For each year from 2003–2017 [year], records in the NCBI nucleotide database containing COI sequences were retrieved using the Entrez query "("CO1"[GENE] OR "COI"[GENE] OR "COX1"[GENE] OR "COXI"[GENE]) AND "Eukaryota"[ORGN] AND [year][PDAT]) AND [species list]” [2003–2016, accessed November 2017; 2017, accessed April 2018]. GenBank records were parsed, retaining information on year of record deposition and number of fully identified records. For fully identified records, sequence length as well as country and/or latitude-longitude fields were parsed.
We also assessed the number of high quality COI sequences that meet the standards developed between the INSDC and the Consortium for the Barcode of Life by looking for the BARCODE keyword in the GenBank record [11 (link)]. For each year from 2003–2017 [year], records in the NCBI nucleotide database containing COI BARCODE sequences were retrieved using the Entrez query "("CO1"[GENE] OR "COI"[GENE] OR "COX1"[GENE] OR "COXI"[GENE]) AND "Eukaryota"[ORGN] AND [year][PDAT] AND “BARCODE”[KYWD]) AND [species list]”. Fully identified and geotagged records were parsed as described above.
For our application example on freshwater biomonitoring, we retrieved a high-level list of relevant groups from Elbrecht and Leese (2017) to facilitate comparisons across studies [27 (link)]. Target freshwater taxa included: Annelida classes Clitellata and Polychaeta; Insecta (Arthropoda) orders Coleoptera, Diptera, Ephemeroptera, Megaloptera, Odonata, Plecoptera, and Trichoptera; Malacostraca (Arthropoda) orders Amphipoda and Isopoda; Mollusca classes Bivalvia and Gastropoda; and Platyhelminthes class Turbellaria. Within these groups there are likely to be non-freshwater taxa included, however, this method allowed us to quickly gauge the representation of freshwater taxa contained therein. These are also the same groupings often used to summarize results from COI freshwater biomonitoring assessments. A detailed look at specific freshwater taxa at finer taxonomic levels is beyond the scope of this paper and will be published elsewhere. For each freshwater target group we queried the NCBI taxonomy database for records identified to the species rank as described above. These taxon ids were concatenated and used to query the NCBI nucleotide database as described above. We assessed the representation of freshwater indicator taxa in the NCBI nucleotide database and level of annotation as described above.
For our application example on IUCN endangered animal species, we retrieved a list of endangered species names from http://www.iucnredlist.org from all available years (1996, 2000, 2002–2004, 2006–2017) filtering the results for native Animalia species [Accessed Dec. 12, 2017]. We excluded insufficiently identified species containing the terms ‘affinis’, ‘sp.’, or ‘sp. nov.’, leaving us with a list of 4,289 endangered animal species as well as 2,089 synonyms. We submitted this combined list of species names to the ‘NCBI Taxonomy name/id Status Report Page’ (https://www.ncbi.nlm.nih.gov/Taxonomy/TaxIdentifier/tax_identifier.cgi) and retrieved a list of 2,613 taxon ids. For each taxon id, we queried the NCBI taxonomy and nucleotide databases as described above.
To assess the number of COI records unique to the BOLD database compared with the NCBI nucleotide database, we also retrieved records from the BOLD Application Programming Interface (API) as well as from the data releases. Since the BOLD database contains records from several DNA barcode markers such as ITS rDNA for fungi and COI mtDNA for animals, it was necessary to target just the COI records. COI sequences were retrieved from the BOLD API (http://www.boldsystems.org/index.php/API_Public/sequence?) using the terms ‘marker = COI-3P|COI-5P&taxon = ‘ for each Eukaryote phylum except for Arthropoda which was queried separately for each class, and Insecta which was queried separately for each order to enable the download of complete files [Accessed Apr. 26, 2018]. Lists of Eukaryote phyla, Arthropoda classes, and Insecta orders were retrieved from the BOLD taxonomy browser (http://www.boldsystems.org/index.php/TaxBrowser_Home). COI records were also retrieved from the BOLD data releases (http://www.boldsystems.org/index.php/datarelease). All available releases of animal COI records up to and including Release 6.50v1 were individually downloaded and parsed. Note that the records retrieved from the data releases may not be as current as those retrieved through the BOLD API.

Twenty-four well-assembled lophotrochozoan genomes were selected for phylogenetic analysis, include one annelid (Helobdella robusta) as outgroup, 21 bivalves (Archivesica marissinica, Argopecten concentricus, Argopecten irradians, Conchocele bisecta, Crassostrea gigas, Crassostrea virginica, Cyclina sinensis, Gigantidas platifrons, Lutraria rhynchaena, Mactra quadrangularis, Mercenaria mercenaria, Mizuhopecten yessoensis, Modiolus philippinarum, Mytilus coruscus, Pecten maximus, Pinctada fucata, Pinctada imbricata, Ruditapes philippinarum, Saccostrea glomerata, Scapharca broughtonii, Sinonovacula constricta), 5 gastropods (Aplysia californica, Chrysomallon squamiferum, Lottia gigantea, Haliotis rufescens, Pomacea canaliculata), and 2 cephalopods (Octopus bimaculoides and Octopus vulgaris) [22 (link), 26 (link), 52 (link), 113 (link)–132 ]. SonicParanoid v1.3.0 was used to define gene family clusters among different species [133 (link)]. The amino acid sequences of one-to-one single-copy orthologous genes were used to reconstruct their phylogenetic topology. The protein sequences were aligned using MAFFT v7.407 under default settings [134 (link)], and then were concatenated for phylogenetic analysis using a maximum-likelihood method implemented in IQ-TREE v 2.0.6 with the “-m MFP” parameter was applied to each protein partition [135 (link)]. To estimate divergence times, the rooted maximum-likelihood tree, along with a concatenated fourfold degenerate site sequence extracted from single-copy CDS (coding sequence), was used as the input of MCMCtree software implemented in PAML v4.8 [136 (link)]. For calibration, nine nodes were constrained by either fossil records obtained from website of TimeTree.

The studied site is a private habitation located in the Parisian suburb (4°5’0” N; 2°47’24” E). Access was permitted by the homeowner. In the Parisian region, the climate is described as hot temperate (Cf. Koppen and Geiger classification) with the presence of rainfall in driest months. The annual average temperature is 11.6°C and annual average rainfall is 693.6 mm for 1981–2010 period (infoclimat.fr).
Two campaigns of measurement were carried out: the first one between May 7th 2020 and June 18th 2020 (spring) and the second campaign between November 5th 2020 and December 30th 2020 (fall-winter). During both campaigns, climatic data (daily mean relative humidity (%), daily mean temperature (°C) and precipitation (mm)) were collected from the Roissy-en-France meteorological station (infoclimat.fr).
Different zones of measurements were selected (Fig 1) to assess the role of different parameters: the position, the orientation and the sheltered by a tree or not situation. Vertical zones (wall surfaces) are made out roughcast that is a mix between coarse sand (millimetric clasts) and concrete (Fig 1A and 1B). The horizontally surfaces are composite tiles composed by concrete, pebble clasts (millimetric to centimetric clast size) and bioclasts like gastropods (Fig 1C and 1D). Even if the nature of materials is different, the comparison is interesting to assess the role of the position. Indeed, horizontal position can potentially retain more water than vertical position, which influences the microbial diversity [18 ].
For the vertical surface, two areas were selected: one close to a chestnut (sheltered situation), that limits the direct impact of rainfall (VA with 3 measurement zones, VA1-2-3, at 100, 143 and 182 cm high, respectively, Fig 1A) and favors a high humidity and one not sheltered by the tree (VNA with also 3 zones, VNA4-5-6, at 200, 150 and 15 cm high, respectively, Fig 1B). Vertical surfaces display some colour changes and weathering forms. On VA1, there are reddish runs compared to VA2. VA3 is located in a humid area due to the proximity of a waste bin. The non-sheltered vertical surfaces display less color change. VA2 did not show significant color change, unlike VA1 and VA3 with reddish and greyish coloration, respectively. VA position are located on north oriented facades and VNA on south oriented façade.
For the horizontal position, two zones were also chosen, sheltered (HA1) and not sheltered (HNA2) by the tree. Moreover, in order to understand biological colonization behavior, two tiles were cleaned with bleach: one on the soil (HBS, Fig 1F) and one raised on a bucket to avoid lateral contamination (HBP Fig 1E). Measurements on HBP (Fig 1E) was realized only during spring season.

Fifth-instar larvae of H. cunea (at the ventral segment of the third gastropod) were injected with 5 μL of protein per larva using a microsampler. Forty larvae were injected with CJPRB (1 mg/mL) and GFP (1 mg/mL) each, and 0.1 g was sampled after 6 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h.