Louses, Crab

The list of species for the vulnerability assessment was based on five different criteria. First, we considered the proportion of each species in the total Portuguese landings between 1989 and 2015, using public landings data from the Direção Geral dos Recursos Marinhos de Portugal (DGRM). The most landed species, accounting for 95% of purse seine, 70% of trawling and 70% of the multigear landings, were included. This selection was carried out separately for each combination of gear and region (Supplementary Table SI1-1). Second, species were chosen in regards of their economic relevance, considering the species representing more than 3% of the total economic revenue of the marine landings within each combination of region and gear (DGRM, Supplementary Table SI1-2). Third, we included the most frequent species in the discards of Portuguese fisheries, according to the work of Leitão et al.^{42 (link)}, where the top-ten discarded species per métier are listed (Supplementary Table SI1-3). Fourth, we included the species of importance for the canning industry, obtained by means of a survey covering the main can enterprises of Portugal (Supplementary Table SI1-5). Fifth, a selection of the species of relevance for the Moroccan fisheries sector was carried out, using the reports from the Department of Marine Fisheries of the Kingdom of Morocco⁴³ and the FAO software FishStatJ (most captured species between 2007 and 2017⁴⁴ ) (Supplementary Table SI1-6). Additionally, due to their importance for specific fleet segments, we included some shark species of interest that were not included by the previous criteria. The selection of shark species was based on reports from the Instituto Português do Mar e as Pescas (IPMA) and included: Galeus melastomus, Prionace glauca, Squalus acanthias, Scyliorhinus canicula, and Hexanchus griseus. Some riverine species were finally removed from the list (Petromyzon marinus, Salmo trutta), as well as cod (Gadus morhua), since it is not captured within the area of study. Finally, some extra species were pointed out by experts during the evaluation process as species with economic interest (Pollicipes pollicipes) or with potential distribution shift into/from the area of study in the context of climate change such as the bivalves Callista chione and Ruditapes philippinarum, and the crabs Callinectes sapidus and Carcinus maenas. The final list of species considered, and their functional group are shown in Table 1.Table 1

Species and functional groups considered during the climate change vulnerability assessment.

Functional group	Species number	Species	Functional group	Species number	Species
Cephalopods	1	Sepia officinalis	Large flatfishes	38	Scophthalmus maximus
	2	Octopus vulgaris	Medium flatfishes	39	Scophthalmus rhombus
	3	Loligo vulgaris		40	Solea solea
	4	Illex coindetii	Large pelagics	41	Sarda sarda
Large bathydemersals	5	Lepidopus caudatus		42	Thunnus thynnus
	6	Lophius piscatorius	Medium pelagics	43	Trachurus trachurus
Medium bathypelagics	7	Brama brama		44	Scomber scombrus
Large benthopelagics	8	Aphanopus carbo		45	Scomber colias
	9	Salmo salar		46	Belone belone
Medium benthopelagics	10	Micromesistius poutassou	Small pelagics	47	Sardina pilchardus
	11	Trisopterus luscus		48	Engraulis encrasicolus
	12	Trachurus picturatus		49	Sardinella spp.
	13	Pagellus erythrinus	Large rays	50	Raja clavata
	14	Spondyliosoma cantharus	Large sharks	51	Centroscymnus coelolepis
	15	Diplodus vulgaris		52	Prionace glauca
	16	Pagellus acarne		53	Squalus acanthias
	17	Lithognathus mormyrus		54	Scyliorhinus canicula
	18	Chelon auratus		55	Hexanchus griseus
Large demersals	19	Merluccius merluccius	Crabs	56	Callinectes sapidus
	20	Conger conger		57	Necora puber
	21	Dicentrarchus labrax		58	Maja squinado
	22	Anguilla anguilla		59	Carcinus maenas
Medium demersals	23	Sparus aurata	Lobsters	60	Homarus gammarus
	24	Mullus surmuletus		61	Palinurus elephas
	25	Diplodus sargus		62	Nephrops norvegicus
	26	Chelidonichthys lucerna	Shrimps	63	Parapenaeus longirostris
	27	Umbrina cirrosa		64	Aristeus antennatus
	28	Boops boops		65	Palaemon serratus
	29	Trachinus draco	Bivalves	66	Callista chione
	30	Chelidonichthys obscurus		67	Cerastoderma edule
	31	Scorpaena notata		68	Ruditapes decussatus
	32	Halobatrachus didactylus		69	Ruditapes philippinarum
	33	Cynoscion regalis		70	Spisula solida
Small demersals	34	Microchirus azevia		71	Ensis siliqua
	35	Macroramphosus scolopax		72	Donax trunculus
	36	Capros aper		73	Mytilus galloprovincialis
	37	Spicara maena	Barnacles	74	Pollicipes pollicipes

To assess the vertical distribution and concentration of microplastic particles, high volumes of seawater were filtered in situ at discrete depths from the greater Monterey Bay pelagic ecosystem off the central California coast. A series of remotely operated vehicle (ROV) dives were conducted with the ROV Ventana, in April 2017 on the R/V Rachel Carson (see Fig. 1A). Two collection sites were chosen based on: (i) their proximity to outflow sources on land, and (ii) bottom depth within the submarine canyon (see SI for detailed map). The nearshore site, closest to potential land-based waste sources, was located at the mouth of Moss Landing Harbor (36.8°N, 121.82°W), where seawater collections reflect drainage from the Elkhorn Slough and surrounding agricultural and residential areas. The second, offshore site, where the majority of seawater samples were filtered for microplastic particles is a time-series site continuously visited since 1989, located approximately 25 km offshore in 1600 m of water (36.7°N, 122.05°W). Single ROV dives resulted in 1–2 depth-discrete samples filtered onto sterile nylon mesh (100 µm mesh) (see SI for sampler configuration, implementation, and limitations). However, given (i) the low sample sizes typical of deep-sea research, and (ii) our overarching objective to quantify microplastic concentrations across the water column, we combined concentration measurements for similar sampling depths across the two sites.
Water column samples were carefully collected over a total of three ship days (42 working hours) and ten individual ROV dives. The following selected depths were sampled for microplastic particles: 5 m (n = 3), 25 m (n = 5), 50 m (n = 2), 75 m (n = 1), 100 m (n = 1), 200 m (n = 1), 400 m (n = 1), 600 m (n = 1), 800 m (n = 1), 1000 m (n = 1). This overall depth range was selected to encompass full sampling across epipelagic (~0–200 m) and mesopelagic (~200–1000 m) zones. Discrete depths were chosen in a manner that balanced sampling effort (individual ROV dives) with even sampling across depths, while also targeting finer sampling resolution in the physically and biologically dynamic epipelagic zone. One of the 25 m samples was discarded due to challenges with flow and pressure. One additional ROV collection sampled water depths obliquely from 25 to 200 m; we used the median value of 112.5 m for this sample. Water samples were filtered in situ by purpose-built samplers and coupled pumps on the ROV, wherein the ROV moved forward at a select depth during sample collection as particles larger than 100-µm were collected onto sterile mesh. An integrated flowmeter recorded the volume of water filtered from each discrete depth. Filtered volumes of seawater ranged from 1,007 to 2,378 m³ per depth horizon. Field-blanks mirroring the exact avenues of the water sample collection process at depth were not feasible, but strict measures were taken to minimize contamination (details provided in SI).
We collected discarded giant larvacean particle-filtering houses (Bathochordaeus spp.) known as “sinkers” from discrete depths using detritus samplers on ROVs Ventana and Doc Ricketts after Robison et al.^{33 (link)}. Briefly, we collected eight individual sinkers across a range of depths (251 to 2,967 m), aiming to both encompass and exceed the depth range of our water samples during January, February, and April of 2017. After ROV retrieval, sinker samples were filtered onto sterile mesh with a vacuum pump system within a controlled shipboard environment (sealed cold room with an isolated ventilation system). Sinker material was pulled onto glass fiber filters for subsequent Raman analysis to quantify microplastic particles and associated material composition.
Toward the end of the 2014–2016 El Niño event (September 2016), we collected beach-cast pelagic red crabs (Pleuroncodes planipes) from two proximate locations in Monterey, California. We randomly selected freshly-dead crabs, collecting the maximum permitted amount per location (n = 35), and preserved specimens at 0 °C. For a random subset of these samples, we measured basic morphometrics (carapace length, carapace width) and recorded the whole-body mass and removed the gastrointestinal tracts with solvent-cleaned dissection tools in a controlled laboratory environment (see SI for further details). A total of 24 individuals were examined to quantify microplastic ingestion and associated material composition.

Extracellular vesicles (EVs) were isolated from the haemolymph of Hematodinium-positive (n = 6) and -negative (n = 6) crabs. Three samples were pooled to achieve ~900 µL for each group (parasitized and non-parasitized males/females). For EV isolation, differential centrifugation was performed as described by Bowden et al. [39 (link)]. Per group, 100 µL of haemolymph was added to 400 µL Dulbecco’s Phosphate Buffered Saline (DPBS), centrifuged at 4000 x g for 30 min, the supernatant was collected and re-centrifuged at 100,000 x g for 1 h at 4°C for retrieval of total EVs. The EV enriched pellet was resuspended in 500 µL DPBS and centrifuged again at 100,000 x g for 1 h at 4°C, discarding the supernatant and diluting the EV pellet in 100 µL DPBS. For EV quantification by nanoparticle tracking analysis (NTA), 10 µL of diluted EV pellet was added to 990 µL DPBS and applied onto an NS300 Nanosight (Malvern, UK) using a syringe pump at a flow rate 50. Particles were recorded at camera level 13 with four-times 1 min recordings, and post-analysis was carried out at threshold level 3 with 30–40 particles per window. The four readings were averaged per sample, using the in-built NTA software (version 3, Malvern, UK). EVs were further analyzed by Western blotting for two surface markers, CD63 (ab68418, Abcam, 1/1000) and Flotillin-1 (ab41927, Abcam 1/1000), as well as visualized for morphological assessment using transmission electron microscopy (TEM) as described in Bowden et al. [39 (link)] in accordance with the minimum requirements for EV characterization set by the International Society for Extracellular Vesicle Research [45 (link)].

Un-paired t-tests were used to compare EV datasets (numbers and modal size) between parasitized and control crabs, in Graph Pad Prism v7. Statistical significance was regarded as P < 0.05. NTA analysis was carried out using the in-built software (v3) and is based on four reads per pooled sample and presented as average reads (black line) with standard error (red line).