Sea star wasting disease pathology in Pisaster ochraceus shows a basal-to-surface process affecting color phenotypes differently

Sea star wasting disease (SSWD) refers to a suite of poorly described non-specific clinical signs including abnormal posture, epidermal ulceration, and limb autotomy (sloughing) causing mortalities of over 20 species of sea stars and subsequent ecological shifts throughout the northeastern Pacific. While SSWD is widely assumed to be infectious, with environmental conditions facilitating disease progression, few data exist on cellular changes associated with the disease. This is unfortunate, because such observations could inform mechanisms of disease pathogenesis and host susceptibility. Here, we replicated SSWD by exposing captive Pisaster ochraceus to a suite of non-infectious organic substances and show that development of gross lesions is a basal-to-surface process involving inflammation (e.g. infiltration of coelomocytes) of ossicles and mutable collagenous tissue, leading to epidermal ulceration. Affected sea stars also manifest increases in a heretofore undocumented coelomocyte type, spindle cells, that might be a useful marker of inflammation in this species. Finally, compared to purple morphs, orange P. ochraceus developed more severe lesions but survived longer. Longer-lived, and presumably more visible, severely-lesioned orange sea stars could have important demographic implications in terms of detectability of lesioned animals in the wild and measures of apparent prevalence of disease.


INTRODUCTION
Sea star wasting disease (SSWD) in Asteroidea (Echinodermata) manifests as variable degrees of abnormal posture, epidermal ulceration, autotomy (sloughing) of rays, or eversion of viscera through the body wall that often leads to death (Hewson et al. 2014). SSWD affects over 20 species of sea stars in the northeastern Pacific and has caused intermittent mass mortality since 2013, leading to large shifts in ecosystem structure (Schultz et al. 2016, Miner et al. 2018, Jaffe et al. 2019, Konar et al. 2019) and functional extirpation of some species like Pycnopodia helianthoides (Montecino-Latorre et al. 2016, Jaffe et al. 2019. SSWD was initially thought to be caused by a densovirus (Hewson et al. 2014) based on molecular evidence of virus in affected animals and an apparent ability to transmit disease using cell-free filtrates that lost activity after heat treatment. However, later studies failed to confirm this hypothesis (Hewson et al. 2018, Jackson et al. 2020b, and the widespread presence of densoviruses in apparently healthy sea stars (Jackson et al. 2020a) argues against densoviruses as a cause of SSWD.
Indeed, challenges with filtered (< 0.2 μm) tissue homogenates (Hewson et al. 2014) do not exclusively point to viruses as candidate etiological agents of SSWD. For instance, arm autotomy in echinoderms is a physiological process in part mediated by sea star proteins and neurotransmitters affecting stiffness of mutable collagenous tissue (MCT), and autotomy can be reproduced by injecting fluids from sea stars undergoing autotomy into normal sea stars (Yamada et al. 2010, Motokawa 2011. Inability to replicate disease using heat-treated filterable extracts from SSWD-affected sea stars (Hewson et al. 2018) could be explained by heat denaturing and inactivation of sea star proteins that mediate stiffness of MCT. So while SSWD is widely assumed to be infectious (Aalto et al. 2020), a non-infectious cause is equally plausible (Aquino et al. 2021). Temperature anomalies , Kohl et al. 2016, Menge et al. 2016, Aalto et al. 2020, meteorological conditions (Hewson et al. 2018), and seawater pCO 2 concentration (Menge et al. 2016) are hypothesized environmental drivers of SSWD disease outbreaks. Finally, the epidemiology of SSWD (a multitude of vastly different asteroid species affected across a broad geographic range) does not seem to support an infectious etiology by densovirus, a DNA virus. Most viruses with broad host ranges are RNA viruses (Bandín & Dopazo 2011). A study of RNA viruses during wasting progression did not yield any genotype associated with lesion margins that could not also be found in scar tissue or grossly normal tissue, or any genotype that was consistent between wasting specimens (Hewson et al. 2020).
While much effort has gone into understanding potential environmental drivers of SSWD (see citations above), there is a lack of a clear case definition of SSWD based on gross and microscopic morphology. Most descriptions of the disease are based on gross lesions which present as a non-specific constellation of clinical signs ranging from focal epidermal ulceration to complete limb autotomy and eversion of gonads through the body wall (Hewson et al. 2014). Our knowledge of the underlying tissue changes associated with lesions of SSWD at the cellular level is limited. For instance, no histology showing convincing cellular pathology associated with viruses was presented in the original description of SSWD (Hewson et al. 2014). While centrifuged tissue homogenates contained viral-like particles compatible in morphology with densovirus, no data were presented showing densoviruses directly associated with cell pathology using methods like transmission electron microscopy, in situ hybridization, or immuno histochemistry (Hewson et al. 2014(Hewson et al. , 2018. This is unfortunate, because understanding disease processes in marine invertebrates at the cellular level using histology can elucidate mechanistic processes and potential causes of cell death that lead to manifestations of gross clinical signs (Work & Meteyer 2014). For instance, in the coral Montipora capitata, tissue loss that appears grossly similar has multiple potential etiologies that can be differentiated at the microscopic level (Work et al. 2012).
A number of studies have attempted to replicate SSWD in controlled environments (Bates et al. 2009, Hewson et al. 2014, Kohl et al. 2016, Jaffe et al. 2019, Kay et al. 2019), but only one study (Bucci et al. 2017) presented histology findings. In that study, captive northwest Atlantic Asterias forbesi developed SSWD lesions which histo logically comprised epidermal vacuolation and edema of connective tissue. Only 2 other studies mentioned sea star pathology, one involving Odontaster validus from South Georgia Island, Antarctica, that showed epidermal ulceration, manifesting microscopically as inflammation of MCT (Núñez-Pons et al. 2018), and one involving various species of sea stars from the North American Pacific coast mortality event in 2014 that showed epidermal ulceration and inflammation (Newton & Smolowitz 2018).
Here, we took advantage of an experiment investigating the impacts of organic matter enrichment on genesis of SSWD in Pisaster ochraceus (Aquino et al. 2021), a species affected by SSWD throughout the Pacific coast of North America, to examine the tissue and hematological changes associated with disease. Specifically, the hypothesis of the experiment was that SSWD lesions could be reproduced by amending surfaces of sea stars with organic matter that would result in proliferation of microorganisms leading to subsequent anoxia near animal surfaces and generation of gross lesions of SSWD. The details of the experiment and microbial and metabolic results are presented elsewhere (Aquino et al. 2021). Our objective here was to document the pathology of experimentally reproduced SSWD lesions, including in specimens that formed lesions in the absence of external stimuli. We show that development of SSWD is a basal-to-surface process starting with inflammation of ossicles and MCT leading to gross clinical signs of epidermal ulceration. Moreover, sea stars manifest increases in a heretofore undescribed cell type in coelomic fluid, i.e. spindle cells, suggesting them as a potential cytological marker of systemic inflammation in this species. Finally, orange sea stars survive longer and develop more severe lesions than purple sea stars, which may affect interpretations of demographic patterns of SSWD in the field.

Treatments and gross exams
Samples for this study were collected during a concurrent study on the impacts of organic matter enrichment on asteroid wasting (Aquino et al. 2021) performed at Bodega Bay, California, USA, in July− August 2019. Pisaster ochraceus (n = 20; mean ± SE mass 303 ± 26 g) were haphazardly collected from the field (Bodega Jetty; 38.3332°N, 123.0481°W) and housed collectively in large (ca. 1400 l at an approximate density of 0.02 sea stars l −1 ) flow-through indoor aquaria for 14 d prior to the start of experiments during which they had ad lib access to a variety of preferred prey (mussels, urchins, and barnacles). Because these animals are invertebrates, Institutional Animal Care and Use Committee (IACUC) approval was not required.
At the start of the experiment, 20 individual animals were placed into high-density polyethylene containers measuring 32 × 20 × 6 cm (3600 ml) perforated with 3 mm holes at a density of ~4 holes cm −2 . Containers were placed in 4 flow-through (60 ± 15 ml s −1 ) seawater tables. On Day 0, each of 5 sea stars received the following treatments, administered by applying 0.015 ml g −1 animal (~3−11 ml) with a 3 ml syringe on the surface of the animal's epidermis, the following organic materials: peptone (300 mg ind. −1 ), approx. 10 5 cells of the cultured microalga Dunaliella tertiolecta, and particulate organic matter (POM; > 0.2 μm filtrate) from 120 ml of seawater collected at Bodega Bay; 5 control sea stars received no treatment. These treatments were chosen because it was hypothesized that organic matter (OM), especially that originating from primary production, could result in SSWD signs by producing suboxic microzones through heterotrophic respiration near asteroid surfaces (Aquino et al. 2021). Peptone is a common component of marine bacterial culture media and selects for copiotrophic marine taxa (Moebus 1980). D. tertiolecta culture was used as a unicellular algal source of POM. Coastal POM was used to simulate enrich-ment by organic material > 0.2 μm, which includes phytoplankton, bacterioplankton, and suspended inorganic particulates. The 3 amendments therefore in formed responses to a non-native OM (protein) source, non-native phytoplankton-derived OM, and OM native to the environment from which the specimens were collected. Animals were weighed at the start and end of the experiment. Each day, animals were photographed and subjectively scored (on a scale of 0−4) for severity of lesions as described by Bates et al. (2009) (Table 1, Fig. 1). Animals were further categorized by color (orange or purple). The experiment lasted 16 d.

Histology
Tissues were biopsied using 4 mm sterile biopsy punches comprising epidermis and underlying MCT on the aboral surface or at lesion sites. At biopsy, tissues were classified as lesioned or apparently normal. Prior to histology processing, sections were decalcified in EDTA for 24 h to dissolve ossicles so as to allow sectioning on microtomes. After decalcification, tissues were processed with routine paraffin embedding, sectioning at 5 μm, and staining with hematoxylin and eosin or Masson trichrome (Prophet et al. 1992) to highlight MCT. Tissue changes at the microscopic level were graded (on a scale of 0−3) according to criteria outlined in Table 1. These in cluded inflammation exemplified by infiltrates of coelomocytes, edema exemplified by increased spaces be tween collagen fibrils of MCT, necrosis comprising hyalinization, pyknosis, or karyorrhexis, ulceration of epidermis or lining of coelomic cavity, lysis of ossicles, epidermal atrophy, epidermal vacuolation, epidermal hyperplasia, vacuolation of basement membrane, cleft formation between epidermis and MCT, and increase or decrease of muriform cells as defined by epidermal cells with eosinophilic intracytoplasmic granules (Hyman 1955). Each sea star was then as signed a histology lesion score dependent on the number of lesions and their severity for the section examined (e.g. a sea star with 2 different lesion types, each severe, would have a total score of 6). Higher lesion scores indicate less healthy sea stars. Biopsies (n = 52) were collected comprising 11 controls, 13 POM controls, and 14 each of Dunalliela and peptone treatments sampled intermittently on Days 0 (n = 12), 1 (n = 4), 3 (n = 2), 4 (n = 3), 5 (n = 2), 6 (n = 2), 7 (n = (B) Score 1, purple morph. Note single focal ulcer (arrow). (C) Score 2, orange morph. Note multiple ulcers (arrows) on multiple rays and prominent pale reticulation indicating thinning mutable collagenous tissue over ossicles. (D) Score 3, purple morph. Note prominent pale reticulation as in C and large (≥ 5 mm) ulcer (arrow). (E) Score 4, purple morph. Note autotomy 1), 8 (n = 1), 9 (n = 3), 10 (n = 2), 11 (n = 1), 12 (n = 4), 13 (n = 1), and 14 (n = 5), 15 (n = 9). Aside from sampling on Day 0, timing of sampling of stars was done when new gross lesions arose on the animal. All histology slides were read by a single pathologist with no prior knowledge of the lesion status of the biopsy or the health status of the animal.

Ossicles
To ensure that changes in ossicles seen on histology were not an artefact of decalcification, we did scanning electron microscopy on ossicles of lesioned and normal sea stars. A section of the lateral ray (ca. 1−1.5 cm 3 ) was cut from a fresh frozen (−70°C) star using rongeurs, placed in 15 ml conical tubes containing 10% bleach solution in artificial seawater (Instant Ocean) prepared according to the manufacturer's instructions, and placed on a rocker table for 24 h at room temperature to digest tissues away from calcified structures. Ossicles were then rinsed twice with tap water, once with 70% ethanol, and once with 90% ethanol, and then allowed to dry at 37°C for at least 3 d. Ossicles were placed on adhesive-coated aluminum stubs, sputter-coated with gold/ palladium, and examined with a Hitachi S-4800 field emission scanning electron microscope. Two easily recognized morphologies of ossicles (those derived from pedicellariae, and sellate or saddle-shaped) were enumerated on the entire stub and scored subjectively as not (0), mildly (1), moderately (2), or severely eroded (3) (Fig. 3). This was done for 4 sea stars sampled at termination of the experiment, one for each treatment. Nomenclature of echinoid stereom morphology followed that of Smith (1980).

Data analyses
Data were evaluated for normality with the Shapiro-Wilk test (Shapiro & Wilk 1965), which dictated the use of parametric or non-parametric statistics. For gross exams, percent weight loss from start and end of the experiment and mean gross lesion severity scores for all animals and time points were compared between treatment groups by 2-way ANOVA with treatment and color as factors. We chose to incorporate color into the analysis because orange P. ochraceus appeared more susceptible to de veloping gross lesions of SSWD in the field  (Menge et al. 2016). In cases of significant differences, pairwise comparisons were made with t-tests using a Bonferroni adjustment of p-values to account for multiple tests (Rice 1989). We also plotted cumulative survivorship (Kaplan & Meier 1958) and mean gross lesion scores of purple vs. orange morphs over time. For hematology, box plots of microscope field counts vs. treatment or lesion score for all animals and all time periods or simple presence/ absence of lesions were examined. Based on the plot that appeared to show a difference (presence/absence of lesions), a Wilcoxon rank sum test was used to compare the 5 cell types (multinucleated, spindle, vibratile, stippled, spherule) between 2 sea stars with and without lesions with a Bonferroni adjustment of alpha (0.05/5 or 0.01). Phagocytes were not included, because they were not counted due to their overwhelming predominance in microscope fields (see Section 2.2). Histology lesion scores among the 4 treatment groups and gross lesion scores for all animals and time points were compared using 2-way ANOVA. In case of significant differences, post hoc comparisons were made as above. All analyses were done using R 3.5.3 (R Core Team 2017).

Cytology of coelomic fluid
Box plots of cell counts vs. treatment or severity score showed no clear differences, but those with lesions showed significantly elevated numbers of spindle cells (W = 325, p = 4 × 10 -5 ) ( Table 3). Cell morphology did not differ in sick vs. healthy sea stars save for the occasional diatom seen in the coelomic fluid of sick sea stars.

Ossicles
Four sea stars were processed for ossicle examination (1 for each treatment). Erosive lesions were seen in both sellate (Fig. 3A) and pedicellaria (Fig. 3B) ossicles. Those on sellate ossicles were seen on the surface of trabeculae and appeared to start as focal linear lesions (Fig. 3C) that eventually merged (Fig. 3E) and resulted in large serpiginous erosions (Fig. 3G). For pedicellariae, lesions appeared to originate around pits (Fig. 3D) that progressed to the smooth part of the stereom (Fig. 3F), with severe cases showing massive erosion leaving elevated pit walls (Fig. 3H).

DISCUSSION
The lesions and clinical progression seen here were grossly and microscopically indistinguishable from those described in wild sea stars with SSWD from the Atlantic (Bucci et al. 2017) and the Pacific (Hewson et al. 2014). This finding indicates that while SSWD is commonly assumed to be caused by an infectious agent (Fuess et al. 2015, Aalto et al. 2020, non-infectious etiologies are equally possible, or this type of response is a non-specific sequela to general insults in this animal group. The animals used here originated from the wild, so it is possible that lesions could have originated from stress of captivity. Supporting this possibility is that control animals also developed lesions, albeit less severe but otherwise indistinguishable from the treatment groups. This invites a need to refine management of captive sea stars to reliably interpret experimental results and minimize the occurrence of gross and microscopic lesions. We saw no evidence that infectious agents were the cause of the lesions seen here, at least those visible at the light microscope level such as bacteria, parasites, or fungi. The sea stars were not fed during the 16 d of the study, and given that all animals lost weight over that time, it is tempting to conclude that starvation was at least in part responsible for the development of gross lesions. However, several experimental studies of starvation in sea stars have not reported lesions of SSWD, with effects of starvation limited to autolysis of pyloric ceca and shorten- ing of rays (Hancock 1958, Lawrence 1971, Féral 1985, St-Pierre & Gagnon 2015. Our finding of orange Pisaster och ra ceus developing more severe gross lesions of SSWD than purple individuals accorded with findings of Menge et al. (2016) in wild P. ochraceus in Oregon (USA) affected by SSWD; there, orange sea stars appeared disproportionately more affected by the disease even though they comprised only about 20% of the population (Raimondi et al. 2007). Based on our findings, it appears that orange sea stars not only developed more severe lesions but survived longer than purple sea stars. This has implications for field surveys in that lesioned orange sea stars with their ability to survive longer with more severe, and presumably more visible, lesions may be more readily visible in field surveys than purple sea stars, thereby introducing potential detection biases. Marking sea stars (Lamare et al. 2009) and monitoring them over time might provide valuable insights on important demographic parameters of disease such as species-specific case fatality rate. Color in P. ochraceus is thought to be driven mainly by diet (Fox & Scheer 1941) rather than genetics (Harley et al. 2006). Differential susceptibility to disease based on color for marine invertebrates is not limited to sea stars. For in stance, an analogous situation exists for corals in Hawaii. There, red phenotypes of Montipora capitata are more abundant than orange phenotypes, and red phenotypes are more likely to develop bleaching while orange phenotypes are more susceptible to tissue loss diseases, presumably due to host factors such as endogenous pigments or types of symbiotic algae (Shore-Maggio et al. 2018). Understanding the physiological mechanisms responsible for such differences might explain community assemblages of sessile marine invertebrates and the role of disease in population structures.
Hematological studies in sea stars appear uncommon. One recent study looking at sea star coelomic fluid relative to SSWD found electrolyte imbalances, but the authors were unable to effectively evaluate coelomocytes, probably in part due to finding too few cells in standard coelomic fluid smear preparations (Wahltinez et al. 2020). We found that concentrating the cells using syringe barrels (Moore 2017, Work et al. 2020b) allowed visualization and enumeration of cell types. The few studies that have looked at hematological responses of sea stars to insults involved responses to amputation. Pinsino et al. (2007) examined the hematological response of Asterias rubens to traumatic amputation and found modest increases of circulating coelomocytes in coelomic fluid and time-dependent expression of heat shock proteins. Silva & Peck (2000) also found increased numbers of circulating coelomocytes after amputation in Odontaster validus. Although we did not do total coelomocyte counts, we, like Pinsino et al. (2007), also found that phagocytes comprised > 95% of cell populations in P. ochraceus. On wet mounts, Pinsino et al. (2007) identified macrophages, phagocytes, vibratile cells, and hemocytes in A. rubens. In the same species, Sharlaimova et al. (2014) used azure stains to document 7 types of coelomocytes, including small coelomocyte types 1 and 2, small and large petaloid agranulocytes, eosinophilic granulocytes, fusiform coelo mocytes, and bi-or trinucleate cells. Pinsino et al. (2007) did not see a shift in relative populations of cells after amputation, whereas Sharlaimova et al. (2014) found a transient increase in the proportion of small circulating coelomocytes. An increase in spindle cells circulating in coelomic fluid of lesioned sea stars has not been documented before. Spindle-like cells have been seen in underlying coelomic epithelium in A. rubens (Gorshkov et al. 2009), and these cells are known to recruit to wound repair sites in the same sea star species (Ben Khadra et al. 2015a) and are apparently important in regeneration of muscle in asteroids (García-Arrarás & Dolmatov 2010). Sorting out the origin and role of spindle cells in P. och raceus might provide a tool to better understand host response to injuries and could be a potential marker of inflammation in this species. We ruled out the spindle structures as diatoms, because no characteristic refractile silica skeleton was seen on light microscopy.
Based on the frequency of occurrence of histological lesions, we suspect that the pathogenesis of epidermal ulceration in P. ochraceus starts with a combination of lysis of ossicles followed by or closely associated with inflammation of MCT and edema. This leads to a collapse of MCT, causing cleft formation between MCT and epidermis, with eventual sloughing and ulceration of the epidermis (Fig. 2). The changes seen here have similarities and differences from simple wound repair, a process that has been well characterized in sea stars and that consists of an orderly progression of repair, early, and advanced regenerative phases (Candia Carnevali 2006, Ben Khadra et al. 2015a,b, 2017. In repair, there is contraction of MCT to close off the wound with edema and inflammation of MCT, clot formation, and re-epithelialization that occurs from the edge of the wound. Early regeneration involves genesis of organs such as water vascular canals and nervous tissues. Late regeneration in -volves formation of tube feet and other adnexa. Our comparisons to these studies were limited, because examinations of tissues were constrained to 4 mm biopsies, whereas wound repair studies in volved examination of entire rays. However, inflammation and edema of MCT were a commonality we saw with wound repair. In contrast, ossicle lysis, contraction of MCT with cleft formation, and epidermal ulceration have not been described in wound repair, nor did we see clot formation or centripetal re-epithelialization ). Our pathology findings fit with a model of loss of turgor with collapse of MCT and ossicles, followed by epidermal ulceration seen in field outbreaks of SSWD in California in 1999 (Eckert et al. 2000). Given the importance of the nervous system in mediating stiffness of MCT (Motokawa 2011), perhaps SSWD is an uncontrolled form of autotomy originating in neuronal dysfunction, and understanding parallels or differences of autotomy vs. SSWD at the cellular level might shed further light on the pathogenesis of SSWD. Collapse of the body wall would also fit with metatranscriptomic analyses of SSWD in Pycnopodia helianthoides, which showed decreases in expression of genes associated with cytoskeletal integrity and tissue remodeling (Guden kauf & Hewson 2015).
Lysis of ossicles associated with coelomocyte infiltrates was a signal feature of experimentally induced SSWD. This has not been described in histological examinations of wound repair in other sea stars, including A. rubens (Ben Khadra et al. 2015b) Leptasterias hexactis (Mladenov et al. 1989), A. rollestoni (Fan et al. 2011), Amphiura filiformis and Echinaster sepositus , Antedon mediterranea (Candia Carnevali et al. 1995), or Odontaster validus (Núñez-Pons et al. 2018, where, as in our study, tissues were decalcified with EDTA prior to processing for histology. Lysis of ossicles was reported in wild sea stars with epidermal ulceration from the Atlantic (Bucci et al. 2017) but not in those from the Pacific (Newton & Smolowitz 2018). In the case of Atlantic sea stars, lysis of ossicles was noted in both normal and lesioned sea stars, with the latter having more severe inflammation, and the authors were unclear about the role that decalcification could play in the lysis of ossicles in normal sea stars (Bucci et al. 2017). We doubt that the lesions seen here were an artefact of tissue processing. However, to confirm this, we looked at ossicles that were not decalcified and showed a range of lesions by electron microscopy (Fig. 3), again suggesting that our findings were not a processing artefact. What prompts these ossicle lesions is unclear; however, the presence of cellular infiltrates indicates that inflammation could play a role. For example, urchins have calcite phagocytizing cells that remodel ossicles during spine formation (Märkel & Röser 1983a,b). Holland & Grimmer (1981) examined syzygies (specific areas where arms autotomize) in the crinoid Florometra serratissima, and showed, via scanning electron microscopy, areas of lysis in ossicles very similar to the mild ossicular lesions seen here (Fig. 3C). The initiation of lytic lesions near perforations in the stereom (where tissues and cells are located) in pedicellariae of P. ochra ceus seen here was intriguing and might, along with histology, indicate that cellular reabsorption processes are responsible for ossicle lesions. Understanding the cell types responsible for this would be important.
In summary, within the constraints herein, SSWD in P. ochraceus appears to be a basal-to-tissue surface process starting with the collapse of ossicles and MCT, leading to epidermal ulceration and visible clinical signs of wasting that appears to affect orange morphs more severely than purple morphs. Given the similarity of gross lesions among different species of sea stars and regions, it is likely that, like other animals, sea stars have a limited host response repertoire when it comes to manifestation of gross lesions. There is thus a clear need to use additional tools like microscopic and clinical pathology to better refine case definitions of SSWD, because like other animals, clinical manifestation of SSWD in sea stars will likely differ between species and potentially regions. A reasonable analogy is fibropapillomatosis, a tumor disease of green turtles Chelonia mydas that has different pathologic (Work et al. 2004) and immunologic (Work et al. 2020a) manifestations between the Pacific and Atlantic. Careful examination of sick sea stars at the cellular level could go a long way towards illuminating the causes of SSWD and the genesis of management interventions to stem disease in this ecologically important group of animals. Data availability. Data associated with this article are available at https://doi.org/10.5066/P9LGH5ZF.
Acknowledgements. Any use of trade, firm, or product names is for descriptive purposes only and does not imply en dorsement by the US Government. We thank Karl Menard and the staff at the Bodega Bay Marine Lab (University of California, Davis) for assistance with sample collection, and anonymous reviewers for providing constructive comments.

LITERATURE CITED
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