Thoracica

Samples were taken and processed according to the standard operating procedures to ensure maximum comparability. In brief, at least three different specimens of each sponge species were collected into sterile bags and species identities were confirmed by microscopic examination of morphological characters following established protocols (for example, as reviewed in ref. 43 ). Specimens were either processed directly or after freezing, depending on logistical constraints of each sampling event. Specimens were cleaned of external growth (for example, barnacles), washed three times with sterile seawater to remove planktonic or loosely associated microorganisms and cut into small pieces from which a random sub-sample of pieces was used for subsequent DNA extraction. Sediment samples were collected under water in close proximity to sponges. Sediments were scooped into sterile containers using sterile spatulas to avoid laboratory contamination. Seawater was drained from the containers on surfacing and prior to freezing. Sponges and sediment samples were immediately frozen and kept on dry ice or at −80 °C until further processing. DNA was extracted from ∼0.25 g of sponge tissue or sediment using the PowerSoil DNA Extraction kit (MoBio) according to the Earth Microbiome Project standard protocols (http://press.igsb.anl.gov/earthmicrobiome/emp-standard-protocols/dna-extraction-protocol/). Microbial communities in seawater were collected by passing 2 l of seawater through 0.2 μm Sterivex filters and DNA was extracted from the filters as previously described13 (link). Samples were extracted at laboratories at the Australian Institute of Marine Sciences (Townsville, Australia), the University of Wuerzburg (Germany) or the Nova Southeastern University (Dania Beach, FL, USA) to minimize shipment of frozen specimens. Aliquots of the specimens and DNA were kept at the three locations (and are available on request) and an aliquot of the extracted DNA was shipped to the University of Colorado, Bolder, CO, USA for sequencing of the 16S rRNA gene using standard procedures of the Earth Microbiome Project (http://www.earthmicrobiome.org/emp-standard-protocols/16s/). Briefly, the V4 region of the 16S rRNA gene was amplified using the primer 515f–806rB and sequenced using the HiSeq2500 platform (Illumina)44 (link).

Marine heterotardigrades, Echiniscoides cf. sigismundi, were collected from barnacles in the intertidal zone at Lynæs, Zealand, Denmark (see [57 ]). Specimens of a parthenogenetic population of the eutardigrade, Richtersius cf. coronifer, were sampled from moss growing on limestone in Øland, Sweden (see [76 (link)]). Total RNA was extracted from active stage pools of ∼550 E. cf. sigismundi and ∼200 R. cf. coronifer, respectively, using a RNeasy Plus Universal Mini Kit (Qiagen,Hilden, Germany). RNA quantitation and quality analyses were performed using a NanoDrop ND-1000 (Thermo Scientific, Waltham, Massachusetts, USA) and a Bioanalyzer 2100 (Agilent Technologies, Santa Clara).
Cryptic species complexes are common within Tardigrada and the genus Echiniscoides has an exceptionally large genetic variation [19 (link)]. We therefore analyzed both transcriptomes with respect to the often used barcoding sequence of cytochrome c oxidase subunit I (COXI) using BLASTN searches in the NCBI nucleotide non-redundant database. The transcriptome data revealed tardigrade COXI contigs (CL4672.Contig1_Echiniscoides and CL1502.Contig2_Richtersius), which support that the 550 Echiniscoides and 200 Richtersius specimens each constitute single species. BLASTN searches in GenBank returned a 99% identity to the isolate Echiniscoides cf. sigismundi (voucher ZMUC:TAR Esi1; GenBank Accession number: HM193403.1; [77 (link)]. Similarly, the Richtersius cf. coronifer BLASTN search in GenBank returned a 99% identity to the isolate Richtersius cf. coronifer (GenBank Accession number: EU244606.1).

We placed all patients on standard non-invasive monitors. Anesthesia was induced according to the standard operating procedures [SOP] of our hospital using 1.5–2.5 mg/kg body weight (BW) Propofol, 0.2–0.5 µg/kg BW Sufentanil and 0.5–0.6 mg/kg ideal body weight (IBW) Atracurium or 0.6 mg/kg IBW Rocuronium at the discretion of the attending anesthesiologist. Maintenance of anesthesia was accomplished using Sevoflurane adjusted to age-corrected mean alveolar concentration. Analgesia was adapted to meet patient individual demands using additional Sufentanil boluses of 5–10 µg. A non-opioid analgetic was administered 30 min before the end of the procedure for early post-operative analgesia.
An arterial cannula was placed in the radial artery for continuous, invasive blood pressure monitoring (Philips IntelliVue MX700, Philips Medizin Systeme GmbH, Boeblingen, Germany). Invasive blood pressure monitoring was calibrated by placing the pressure transducer at the level of the right atrium and venting the transducer to atmospheric pressure for reference. ∆PP was calculated as $$\mathrm{\Delta }\mathrm{PP}[\%](({\mathrm{PP}}_{\max }-{\mathrm{PP}}_{\min })/([{\mathrm{PP}}_{\max }+{\mathrm{PP}}_{\min }]/2))\times 100$$
where PP_max and PP_min are the maximum and minimum pulse pressure during one respiratory cycle. ΔPP measurements were calculated from beat-to-beat arterial pressure values and reported as the average value of the last 32 s.
Stroke volume was measured by esophageal doppler monitoring (CardioQ-ODM®, Deltex Medical Ltd, Chichester, UK). The tip of the doppler probe was placed in the pars thoracica of esophagus at the level of the descending aorta. Stroke volume was calculated by the product of stroke distance (area of the doppler derived velocity–time waveform) and aortic cross-sectional area [12 (link)].
Patients were mechanically ventilated with a tidal volume ≥ 8 ml/kg IBW using pressure-controlled ventilation. Positive end-exspiratory pressure was kept between 5–8 mbar, respiratory rate was set to 12–16 min⁻¹ and fraction of inspired oxygen (F_iO₂) was set to keep oxygen saturation above 95% according to departmental standard operating procedures. All settings and target values were continuously adapted to patient characteristics and clinical situation using repetitive blood gas analyses.
In case of possible hypovolemia a fluid bolus of 7 ml/kg IBW was administered at the discretion of the attending anesthesiologist.
Trigger for fluid bolusing were:

Difference in pulse pressure [∆PP] ≥ 9% [13 (link), 14 (link)] and/or

corrected flow time [FTc] ≤ 350 ms [15 (link)]

An increase in stroke volume ≥ 10% following the fluid bolus was considered ‘fluid responsive’.

The fossil specimens dealt with herein were discovered at and around a sand quarry in the hinterland of Arcille (Campagnatico, Grosseto Province, Tuscany, central Italy). Arcille is located in the Baccinello–Cinigiano basin (Figure 1A), one of the post-collisional basins of the northern Apennines, whose Neogene infill comprises both continental and marine deposits [14 ]. The sedimentary succession cropping out at this site (Figure 1B) consists of terrigenous deposits dominated by yellowish, fossiliferous, shallow-marine shoreface sandstones with minor fluvial conglomeratic intercalations capped by greyish, open-shelf offshore mudstones [15 (link),16 ] (Figure 2). These sediments have been referred by Dominici et al. [17 (link)] to their S2 Synthem, a lithologically diverse, Lower Pliocene depositional unit that includes fluvial conglomerates, fluvio-deltaic and shoreface sandstones, and shelf mudstones. Biostratigraphic analyses of the planktic foraminiferal assemblage from the mudstone division cropping out at Arcille indicate the lower part of the Zanclean, i.e., the Mediterranean Pliocene (=MPl) zone 2, which has been referred by Lourens et al. [18 ] to the 5.08–4.52 Ma time span [19 (link)].
Palaeontological highlights of the Arcille quarry include: (i) various specimens of Metaxytherium subapenninum, the latest sirenian of the Mediterranean Sea, which on the whole comprise a reference record for reconstructing the osteoanatomy, phylogenetic relationships and palaeoecological habits of this halitheriine dugongid species [15 (link),19 (link)]; (ii) the holotype and referred specimen of Casatia thermophila, which represents one of the geologically oldest monodontid taxa, as well as the first and only representative of this odontocete family in the Mediterranean Basin [21 (link),22 (link)]; (iii) the holotype and referred specimens of Nebriimimus wardi, an idiosyncratic rajiform batoid whose unusual multicuspid tooth morphology is currently unparalleled [23 (link)]; and (iv) some teeth assigned to the extant requiem shark species Carcharhinus limbatus, which represent the first occurrence of the blacktip shark as a fossil from both Europe and the Mediterranean Basin [24 (link)]. Other remarkable vertebrate fossils from the sandy strata exposed at Arcille include two partial skeletons of a marlin (cf. Makaira sp.), as well as abundant and diverse elasmobranch teeth and spines [19 (link),23 (link),25 ,26 (link),27 (link)]. All things considered, the taxonomic composition of the marine vertebrate assemblage from Arcille indicates a warm-water, shallow-marine palaeoenvironment placed close to the coastline. In the same deposits, the remains of macro-invertebrates are also abundant, being dominated by bivalves (mainly pectinids and venerids, including the extinct large-sized clam Pelecyora gigas) with subordinate gastropods, scaphopods, echinoids and corals [25 ]. Given the presence of P. gigas, the molluscan assemblage can be referred to a stock of tropical or near-tropical taxa, categorised as the Mediterranean Pliocene Molluscan Unit (=MPMU) 1, whose most thermophilic members did not survive the cooling episode that affected the Mediterranean region around 3 Ma [28 ,29 (link)].
The three M. subapenninum specimens studied herein (GAMPS 62M, GAMPS 63M and MSNUP I-15892) originate from the highest portion of the sandstone division cropping out at Arcille. Such skeletons were discovered at two different horizons, resting upon as many shell beds [16 ,25 ]. The same stratigraphic intervals have yielded the holotype of N. wardi and the referred specimen of C. thermophila, as well as teeth of C. limbatus and fragmentary postcrania of cf. Makaira sp. [22 (link),23 (link),24 (link)]. The molluscan assemblage includes Glycymeris nummaria, Limopsis aurita, Venus nux, Procardium indicum, Helminthia triplicata, Oligodia spirata, Thetystrombus coronatus and Neverita olla [16 ]; scaphopods, barnacles and solitary corals (flabellids) are also present [25 ]. Macroscopic evidence of bioencrustation and bioerosion of the shell remains is apparently largely absent [25 ].

In 2020, the Ocean Affairs Council in Taiwan announced a “Major Wildlife Habitat of Indo-Pacific Humpback Dolphin” as an area within 1–3 nautical miles offshore from the west coast of Taiwan, from 24°42′ N to 23°26′ N. The major wildlife habitat involves four counties: Miaoli, Taichung, Changhua, and Yunlin. This study focused on two counties located within the central habitat area: Taichung County (24°58′ N~24°11′ N) and Changhua County (24°11′ N~23°51′ N) (Figure 1). The study area included waters from the shoreline to shallow waters with depths < 15 m. Boat-based photo-ID surveys were conducted in 2018 (May to September), 2019 (April to September), and 2021 (April to November) by traveling at an average speed of 6 to 9 knots (13.89 km/h), with an average of 2 to 3 knots (4.63 km/h) when approaching animals using 10~12-meter-long CT-1 fishing boats. Dolphins were photographed using digital single-lens reflex cameras (Canon, Olympus, Pentax, or Nikon with 70–300 mm zoom lenses or 400 mm prime lenses). Every image was cataloged and cut into shape using PhotoImpact 11software, and individuals were further identified from those images by their distinctive scars and markings.
Identified individuals were grouped into five coloration stages based on changes in body color [21 ]: gray and unspotted (calf), gray with some white spotting (mottled), white with lots of black spotting (speckled), white with less black spotting (spotted adult), and white with a pinkish tint and no spotting (unspotted adult) (Figure 2). In this study, markings that possibly resulted from conspecifics or sharks were excluded from the analysis. Injuries were classified into five categories: dorsal fin/fluke mutilation, narrow-spaced linear marks, wide-spaced linear marks, back indentation, and others (Table 1). Skin lesions were classified into seven categories: nodules, hypertrophic scars, barnacles, pale, black patch, red patch, and orange/yellow patch (Table 2).