The transmissibility and severity of black spot and wasting disease in sea urchins from laboratory experiments and field studies

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V. Malakooti, Vishnu M. Nair, Ana P. Orloff, Conner M. Hale, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6986455/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Sea urchins can be important in structuring multiple ecosystems, but the causes of population fluctuations including the role of infectious disease are often not well understood. We performed laboratory experiments and field studies with purple sea urchins ( Strongylocentrotus purpuratus ) to investigate the transmissibility and severity of two diseases, wasting and black spot. We found that wasting disease and wasting mortality were equally likely in tanks with and without a wasting urchin. In contrast, the presence of a sea urchin with black spot disease led to faster growth of abrasions and significantly higher (41%) mortality than without a diseased sea urchin (0%). Finally, in the field, the prevalence of black spot disease decreased significantly with distance from a focal urchin with black spot disease but not in plots centered on a healthy reference urchin; black spot prevalence also decreased with sea urchin density. These results suggest that wasting disease likely results from non-transmissible physiological stress, while black spot disease is caused by a pathogen that can be transmitted without contact, is transmitted locally, and can cause substantial mortality. Despite these differences, both diseases can cause mortality and population declines in sea urchin populations with cascading effects on kelp forests. Biological sciences/Ecology/Ecological epidemiology Biological sciences/Ecology/Biooceanography/Fisheries Biological sciences/Ecology/Biooceanography/Microbial biooceanography density dependent transmission Koch’s postulates purple sea urchin (Strongylocentrotus purpuratus) trophic cascade experimental infection injury Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 | Introduction Population declines driven by disease, environmental change, and biotic interactions are increasingly affecting species and ecosystems (Harvell et al. 2002 , Fey et al. 2015 , 2019 , Wong and Daniels 2017 ). However, identifying the cause of a population decline or mass mortality event can be challenging because organisms are often threatened by a combination of stressors (Krebs et al. 2001 , Kiesecker et al. 2001 , Hennon et al. 2012 , Wong and Daniels 2017 , Kock et al. 2018 , Wagner et al. 2021 ). Infectious disease has increasingly been recognized as a factor influencing populations, but identifying the cause of a disease and linking mortality events with a causal agent can be challenging (Hudson et al. 1998 , Rachowicz et al. 2005 , Hewson et al. 2025 ). This is, in part, because other factors like physiological stress can both increase the severity of infectious disease events and cause mortality in its own right (McKechnie and Wolf 2010 , Anderegg et al. 2013 , Duff et al. 2013 , Hing et al. 2016 ). Disentangling the causes of population declines is critical for restoration and management of species and the ecosystems they inhabit. Sea urchin (Echinoidea) population fluctuations are emblematic of these challenges, despite their importance in structuring marine ecosystems including coral reefs, seagrass beds, and kelp forests (Steneck 2020 ). Unchecked growth of sea urchin populations can lead to a dramatic decline in habitat-forming kelp and the formation of urchin barrens, whereas population declines in sea urchins can result in recovery of kelp forests (Steneck 2013 , Filbee-Dexter and Scheibling 2014a , Williams et al. 2021 , Eger et al. 2022 , Miller et al. 2022 ). The factors influencing disease epidemics in sea urchin populations and whether epidemics will reduce sea urchins to low enough densities for kelp to recover are only partly understood. In the northeast Pacific Ocean, two diseases called wasting (or red-spot or bald sea urchin disease) and black spot (or black-ring or spotting disease) have been associated with mortality events or changes in population growth rates in red ( Mesocentrotus franciscanus ) and purple sea urchins ( Strongylocentrotus purpuratus ) for decades (Johnson 1971 , Pearse and Hines 1979 , Gilles and Pearse 1986 , Behrens and Lafferty 2004 , Lafferty 2004 , Lester et al. 2007 , Hendler 2013 , Shaw et al. 2023 , 2024 ). Wasting disease is characterized by shortened and/or lost primary spines; black spot signs include lesions characterized by black glossy or friable tissue and rings of blackened tissue in which the bare test is visible (Fig. 1). Black spot lesions can eventually wear through the urchin internal shell (test) and expose the coelomic cavity (Gilles and Pearse 1986 ). Attempts to identify the causal pathogen for black spot disease have been inconclusive, because the bacteria that were isolated from urchins with black spot disease were able to cause lesions in sea urchins in lab infections, but no mortality was observed (Gilles and Pearse 1986 ). In addition, while multiple studies have induced black spot lesions by transferring diseased tissue to mechanically abraded sea urchins or by holding abraded sea urchins in close contact with diseased urchins, it’s unclear whether the cause of black spot disease can be transmitted without contact and what type of injury is needed for infection (Gilles and Pearse 1986 , Lester et al. 2007 ). The cause of wasting disease in red and purple sea urchins has not been identified (Ritchie et al. 2000 , Clemente et al. 2014 ). Spatial patterns of disease, including correlations with host density, have also been used to examine the transmissibility of pathogens causing disease. Increasing disease prevalence with host density may result from increased contact rates and transmission of an infectious pathogen (Begon et al. 2002 ). However, density dependent patterns of disease prevalence could also result from stressors that increase with increased population density. If higher population density results in lower resource availability and starvation or physiological stress, this could result in density-dependent patterns of disease prevalence for a non-transmissible disease. Past studies of the relationship between urchin disease and density have been mixed. The probability of wasting events increased with sea urchin density in the Channel Islands (Lafferty 2004 ), but in coastal southern California, black spot prevalence declined with sea urchin density and there was no relationship between wasting prevalence or combined wasting and black spot disease prevalence and density (Lester et al. 2007 ). Thus, it is unclear whether declines associated with wasting are caused by infectious pathogens, environmental stress, or some combination of the two. Interest in sea urchin management in the northeast Pacific Ocean increased after the 2014–2016 marine heat wave, which coincided with the loss of predatory sea stars due to sea star wasting disease, subsequent overgrazing by purple sea urchins ( Strongylocentrotus purpuratus ), and the widespread loss of kelp in some regions (Harvell et al. 2019 , Rogers-Bennett and Catton 2019 ). Sea urchin culling has been proposed as a tool for kelp restoration, but it is highly labor intensive and limited in spatial scale and often requires perpetual removals (Eger et al. 2022 , Miller et al. 2022 ). Moreover, the need for culling depends on natural population fluctuations caused by pathogens, competitors, predators, and environmental stressors (James 2024 ). For example, kelp restoration efforts via sea urchin culling in southern California was interrupted by a sea urchin mass mortality event and a few individuals with black spot disease were found (Williams et al. 2021 ). The kelp forests recovered shortly thereafter, demonstrating that naturally occurring sea urchin population declines can sometimes allow recovery of kelp forests (Williams et al. 2021 ). However, it’s unclear if black spot disease was the cause of this decline and what factors might make disease epidemics more severe. More generally, understanding the drivers of spatial and temporal patterns in sea urchin diseases and whether they are caused by transmissible pathogens could improve kelp restoration efforts. Sea urchin removals could be focused on populations less likely to undergo natural population declines and kelp seeding or outplanting projects could take advantage of disease events when urchin densities are lower. Our goal was to determine if wasting and black spot disease were transmissible without contact among sea urchins, what types of injuries (abrasion, spine breakage) were needed for infection, and whether these diseases would lead to mortality. We conducted laboratory experiments and hypothesized that both diseases would be transmissible; tanks that housed diseased urchins would have more disease (spine loss, lesion formation and growth) and higher mortality than tanks without diseased sea urchins. We also conducted subtidal field surveys to determine if spatial patterns of black spot disease prevalence were consistent with local transmission. We hypothesized that prevalence would decrease with distance from diseased sea urchins, but not healthy urchins, and that disease prevalence would increase with sea urchin density, as would be expected for a pathogen transmitted by non-sexual contact or through water (Briggs et al. 2010 ). 2 | Methods Wasting Disease Lab Experiment We collected visually healthy purple sea urchins from an urchin barren in Stillwater Cove, Monterey County, CA (36.563°N, 121.949°W) on SCUBA April 15, 2024 and transported them to the University of California Santa Cruz. We held sea urchins in seawater tables supplied with ambient flow-through seawater (no recirculation) for 17 days. We then randomly assigned visually healthy sea urchins to three “injury” treatment groups:1) Spine clipping, in which we simulated storm damage by shortening primary spines to approximately half their length on half the test area; 2) Abrasion, in which we removed a 5mm diameter patch of spines and epithelium from each sea urchin with a sterile scalpel blade, as might mimic a failed predation attempt on an urchin; 3) Uninjured sea urchins. We placed 10 urchins of a single treatment into each 25.55 L tank fed by chilled seawater (~ 13°C), containing one 300 W heater (Hygger HG-925) and one 80 gph circulating pump (Uniclife-UL016). We placed each sea urchin in a 0.5 L vented bait jar (7.6x11.4x10.2 cm) to prevent physical contact among individuals and allow identification of individual urchins. We also collected sea urchins that had characteristics of wasting disease - significantly shortened or absent primary spines (Fig. 1, panels c and d) from the intertidal zone at Pebble Beach, Sea Ranch, Sonoma County, CA (38.699°N, 123.441°W) on May 1st 2024. We held them in 25.55 L tanks fed by chilled seawater (~ 13°C). We filtered and sterilized the inflow and outflow streams for all tanks with 25 micron filters and 80 watt UV sterilizers (Pentair) to prevent introduction of additional disease-causing microbes into our experimental tanks as well as from our tanks into the ocean. On May 7th, we added one wasting sea urchin to each of 3 Abrasion-Diseased tanks, 3 Spine Clipping-Diseased tanks, and 2 Uninjured-Diseased tanks. There were 2 Abrasion-Control tanks, 2 Spine Clipping-Control tanks, and 2 Uninjured-Control tanks without diseased urchins added. Whenever wasting sea urchins died in the Disease treatment tanks we replaced them, so that there was always one live wasting sea urchin in each Disease treatment tank. We monitored sea urchins every two days for one week and approximately weekly thereafter, taking photographs and notes on the condition of each sea urchin to assess any indication or progression of wasting disease. On June 16th, 2024, we increased tank temperatures from 13°C to 16°C by approximately 0.5°C per day to increase physiological stress and the potential for disease (Lester et al 2007 ). We fed sea urchins giant kelp ( Macrocystis pyrifera ) ad libitum for 4 days once before the beginning of the experiment (when diseased urchins were added), once for 2 days two weeks after the experiment began (~ 6cm x 3cm per sea urchin), and starved them thereafter. Black Spot Lab Experiment We conducted a similar experiment to assess whether black spot disease was transmissible without physical contact between individuals and whether disease would lead to mortality. We collected visually healthy purple sea urchins from Lovers Point state marine reserve, Monterey County, CA (36.623°N, 121.906°W) in May 2022 and held them in ambient flow-through seawater tables (14°C-16°C) at the University of California Santa Cruz. We also collected purple sea urchins with signs of black spot disease, including one or more dark, glossy patches or patches of visible test with a black ring of tissue (Fig. 1, panels a and b), from the intertidal at White Point Beach, Los Angeles County, CA (33.715°N, 118.320°W) on August 12th 2022. We held urchins overnight in a cooler with moist giant kelp cuttings and ice packs, and then transported them to tanks at the University of California Santa Cruz in a cooler with ice packs, damp towels, and giant kelp cuttings. Each tank was fed by a spigot off of the main water pipe and contained one 200W (Hygger HG-802) heater and one 80 gph circulating pump (Eheim 1046). We filtered and sterilized the inflow and outflow streams with 25 micron filters and 80 watt UV sterilizers (Pentair). We used the same injury treatment groups as in the wasting experiment: Spine Clipping, Abrasion, and Control, and again housed urchins in individual marked 0.5 L vented bait jars (7.6x11.4x10.2 cm) to prevent contact and allow identification of individual urchins. We used 76 L tanks (57.2x36.2x36.8 cm) and placed 17 healthy urchins in each Diseased tank and 18 healthy urchins in each Control tank. We had two tanks each for the Uninjured-Diseased and Uninjured-Control treatments, but only one (unreplicated) tank for each of the remaining four injury-disease treatments (Spine Clipping-Diseased, Spine Clipping Control, Abrasion-Diseased and Abrasion-Control), for a total of eight tanks. We placed a single urchin with symptoms of black spot into Disease treatment tanks on August 15th which survived for 1 to 6 days before dying. Diseased sea urchins were kept in the tanks until they died (2–6 days) and then deteriorated before they were removed; they were in the tanks for 9–16 days total. We examined sea urchins for lesions and mortality every 2–4 days for the first two weeks and weekly thereafter. We measured the diameter of any lesions we observed using calipers (including those we had created in the Abrasion-Diseased and Abrasion-Control treatment groups). To increase the likelihood of disease, we raised water temperature in the tanks by 0.5°C per day starting on August 16, 2022 until temperatures reached ~ 18°C (Lester et al. 2007 ). Black Spot Field Study We conducted subtidal field surveys to quantify spatial and temporal changes in black spot disease prevalence at an urchin barren site near White Point Beach Park, San Pedro, LA County, CA (33.713°N, 118.318°W). We chose this site based on reports of diseased sea urchins by Reef Check and Vantuna Research program SCUBA divers and an exploratory dive. We attempted to avoid bacterial mats and warm water seeps at this site (Miranda et al. 2016 , Roussos 2018 ) in order to preclude their influence on our study, although we occasionally noted small amounts of bacterial filaments on our plots. To begin our surveys, we searched for purple or red sea urchins with characteristic symptoms of black spot disease (Fig. 1). Upon finding a diseased individual, we placed a marker made of steel chain links and flagging tape on the reef to mark the sea urchin’s initial location. We established Control plots by generating random combinations of compass headings and distances (10 m-50m) from each Diseased plot, but constrained their location to the rocky reef (urchins were not present in the surrounding sandy habitats). At each Diseased or Control plot, we quantified number of red and purple sea urchins, disease prevalence (for black spot and wasting separately), depth, and substrate type (bedrock, boulder, cobble, or sand) in 0.5m 2 quadrats centered on the focal disease urchin and along four perpendicular transects at 1, 2, 3, and 4m from the center of the plot (Figure S1 ). We also recorded temperature and relief (the difference between the highest and lowest points in the quadrat, grouped into four categories: 0-10cm, 10cm-1m, 1m-2m, >2m) at the center of each plot. We revisited the site at 2–11 week intervals and resurveyed the plots. When the chain marker was dislodged, we used directions and photographs to relocate it to the original location. For two plots, the chain could not be located and our directions and photographs of the original marker were not sufficient to guarantee overlap with the original plot area, so these plots were not resampled. We surveyed 11 plots between September 2023 through March 2024. In October 2024, we surveyed 6 additional plots, for a total of 9 Diseased and 8 Control plots. Statistical Analyses We analyzed morbidity and mortality in the wasting disease experiment using 3 sets of Cox’s proportional hazard models: one with disease treatment (Disease-Control) only, one with disease treatment (Disease-Control), and injury treatment (Abrasion, Spine Clipping, or Control) and one that also included the interaction of these two treatments as predictors. For the mortality analysis in the wasting experiment, we excluded deaths in the first 30 days because urchins that died during this period appeared to die of acclimation stress, not wasting disease. Urchins dying in the first 30 days had rapid (1–2 days) spine and epithelial tissue loss over expanding regions of their bodies. In contrast, sea urchins dying after the first 30 days lost large primary spines relatively gradually, often over several weeks, and their remaining spines and tube feet were responsive to touch, and their epithelium appeared intact before they died. The symptoms in urchins that died after the first 30 days were consistent with wasting disease, whereas those that died earlier were not (Lester et al. 2007 ). To analyze growth of lesions in the Abrasion injury treatment of the black spot disease laboratory experiments, we fit linear mixed effects models with lesion size on a sea urchin on a day as the response variable, and day, disease treatment, and a day-treatment interaction as fixed effects and individual urchin ID as a random effect. There was only one tank for the Abrasion-Diseased treatment and one for the Abrasion-Control treatment, so treatment and tank effects were confounded. We tested the residuals of the lesion size analysis for normality with a Shapiro-Wilkes test. We compared mortality among treatments using a Cox’s proportional hazard model as well as a Fisher’s exact test. For the black spot field study, we fit a generalized linear model with a binomial distribution and a logit link with the prevalence of black spot disease as the response variable (each individual urchin was a data point) and distance from the center of the plot interacting with plot type (Diseased or Control), urchin density, and julian date as predictors. We initially included survey plot as a random effect to account for repeated measurements, but the random effect variance was estimated to be 0. We used julian date as a predictor because we expected the change in disease prevalence over time to be largely seasonal (Lester et al. 2007 ). We used the lmer and glmer functions in the lme4 package and the coxme and coxph functions in the survival package in R version 4.4.2. 3 | Results Wasting Disease Lab Experiment At the end of the experiment (day 144), 75% of the 126 sea urchins in all treatments had developed symptoms of wasting and > 50% of urchins had wasted in all 14 individual tanks (Fig. 2, S2). There was no effect of putting diseased sea urchins in tanks on wasting in a univariate Disease-Control model (Fig. 2), an additive Injury and Disease-Control model (Control coef.: -0.0016 ± 0.22 Z = -0.01, P = 0.99; Injury treatment: χ 2 = 3.21, df = 2, P = 0.20) or an interactive Cox’s proportional hazard model (Figure S2; Control coef.: 0.28 ± 0.36, Z = 0.79, P = 0.43; Disease-Injury treatment interaction term: χ 2 = 4.42, df = 2, P = 0.11). There was also no effect of putting wasting sea urchins in tanks on sea urchin survival, but 19% (24 of 126) of urchins died (Figure S3). Black Spot Lab Experiment Over the 36 days of the experiment, average lesion sizes in the Abrasion-Control tank decreased significantly (i.e. lesions healed ) over time by 0.021 ± 0.0075 mm per day or approximately 1mm every 50 days (Fig. 3A; Table S1 ). In contrast, in the Abrasion-Diseased tank where diseased black spot urchins were added, average lesion size increased significantly over time by 0.072 ± 0.0093 mm per day or approximately 1mm every two weeks, and approximately two-thirds of urchins had lesions that increased in size before dying or by the end of the experiment (Fig. 3; Table S1 ). Sea urchin mortality was significantly higher in the Abrasion-Diseased tank than in the Abrasion-Control tank (7/17 = 41% vs 0/18 = 0%; Fisher’s exact test P = 0.0029; Cox’s proportional hazard model: likelihood ratio 13.75, df = 1, P = 0.00021; Figure S4). Two urchins in the Spine Clipping-Diseased tank developed lesions by the end of the experiment, whereas no urchins in the Spine Clipping-Control tank developed lesions, but the difference was not significant (Fisher’s exact test: 2/17 vs 0/18; P = 0.23). Only one out of 36 sea urchins in the Uninjured-Diseased treatments died, and no urchins in the Spine Clipping-Diseased, Spine Clipping-Control, or Uninjured-Control treatments died. Black Spot Field Study Black spot disease prevalence in the field declined significantly with julian date, urchin density, and distance from urchins with black spot; the slope of distance was non-significant for Control plots (Figs. 4, 5; Table S2). The slope of distance for Control plots was flatter (less negative) than Diseased plots, and its uncertainty was higher, due to substantial variability among sampling dates (Fig. 4; Table S2). 4 | Discussion We examined the transmissibility of two sea urchin diseases without contact and their potential to cause mortality. Wasting disease was more likely caused by non-transmissible physiological stress rather than transmission of an infectious pathogen, whereas the cause of black spot disease was transmissible in experiments without contact, but required injury. In addition, patterns of black spot prevalence in the field suggested that transmission occurs locally and is common, despite the possible need for injury to initiate infection. We found that both diseases led to significant (19–41%) mortality in laboratory experiments. The combination of evidence from laboratory experiments and field studies suggesting black spot disease is locally transmitted and can cause substantial mortality supports the possibility that this disease may have been the cause of past mortality events in urchin populations (Johnson 1971 , Williams et al. 2021 ). Overall, these findings suggest that declines in sea urchin populations due to wasting disease may follow physiologically stressful conditions (e.g. high density (Lafferty 2004 ), or marine heatwaves (Lester et al. 2007 )), whereas sea urchin declines due to black spot may increase with partial predation or storm damage combined with the presence of diseased urchins and warmer temperatures (Lester et al. 2007 ). Kelp restoration efforts could take advantage of declines due to both diseases to facilitate kelp restoration, including natural seeding or outplanting of kelp. Our results suggest that wasting disease is not caused by a transmissible pathogen, which is at odds with our hypothesis and some previous research (Lafferty 2004 ). Wasting disease was associated with population declines in the Channel Islands that were positively correlated with sea urchin density (Lafferty 2004 ). This suggested that declines were caused by an infectious pathogen. However, in our experiments, wasting disease occurred at similar rates in all treatments and tanks, whether or not a wasting sea urchin was housed in the tank. This suggests that wasting disease is caused by physiological stress, such as that experienced by our starved urchins in heated tanks. Since higher densities of sea urchins generally occur in nutrient-poor sea urchin barrens (Eger et al. 2024 ), the relationship between high sea urchin densities and epidemics in the Channel Islands (Lafferty 2004 ) could have resulted from low food availability and other stressors, similar to what we observed in the lab. In contrast, we found evidence that the cause of black spot disease was transmissible without contact among sea urchins in our experiments (in both the Abrasion-Diseased treatment and at a lower rate in the Spine Clipping-Disease treatment), is transmitted locally in the field, and, importantly, can cause substantial mortality (41% in the Abrasion-Diseased treatment). The significantly higher mortality we observed when housing abraded sea urchins with a black spot sea urchin is a significant advancement in our knowledge of this disease. Previous studies either found no mortality at all, despite being able to culture bacteria and use them to cause lesions similar to those in the field (Gilles and Pearse 1986 ), or no significant difference in mortality between healthy uninjured sea urchins and abraded sea urchins swabbed with diseased tissue (Lester et al. 2007 : 3/11 vs 1/11 in the 19°C experiment; Fisher’s exact test P = 0.59; no statistical comparison was made in the paper). The difference in mortality among experiments may be due to differences in nutritional stress at the time of exposure. The sea urchins used in previous lab experiments were fed during the experiments, (Gilles and Pearse 1986 , Lester et al. 2007 ), whereas our sea urchins were collected from barrens, starved, and held in warmer water. Finally, it’s worth noting that the abrasions we caused in the Abrasion-Control group healed over time, as in previous studies (Pearse et al. 1977 , Gilles and Pearse 1986 , Lester et al. 2007 ), suggesting that abrasions in apparently healthy sea urchins do not result in black spot lesions without the presence of a diseased individual nearby. Our field study showed evidence of transmission of the pathogen causing black spot disease at a local scale (1–4 m) and declining prevalence from late summer through winter. The former suggests that black spot disease was caused by a pathogen that was more likely to spread to close neighbors. Prevalence was approximately 4% in late summer and varied from 0–20% among quadrats, suggesting that conditions required for black spot (including possible injury) occur relatively frequently at the survey depths at our site and in other locations where black spot disease has been examined (Lester et al. 2007 ). Future studies could determine the importance of injuries for black spot disease in the field by comparing black spot prevalence at sites with differing levels of disturbance due to variation in ocean surge or variation in the abundance of sea urchin predators such as spiny lobsters or sheephead. The decline in black spot prevalence over time with julian date is consistent with a pattern of seasonal variation suggested by previous work (Lester et al. 2007 ), and may be linked to variation in ocean temperatures that follow a similar pattern (Gelpi and Norris 2008 ). We found that black spot prevalence decreased with sea urchin density, which was the opposite of our hypothesis. However, pattern we found is consistent with a previous study on black spot disease in the same region of southern California (Lester et al. 2007 ). A negative relationship between black spot prevalence and density in both studies could be caused by some areas having lower quality or abundance of food or other resources resulting in lower sea urchin density and higher susceptibility to disease. Another possibility is that sea urchins with black spot disease may move away from other urchins or that healthy sea urchins may move away from diseased urchins; disease avoidance behavior has been observed in another marine invertebrate, the Caribbean spiny lobster, Panulirus argus (Behringer et al. 2006 ). Purple sea urchins can respond to conspecific cues in a density-dependent manner (Knight et al. 2022 ), but further study is needed to determine whether they avoid diseased conspecifics. There were four important limitations to our study. First, we used visual criteria to determine whether a sea urchin had wasting disease or black spot because no pathogen has been associated with wasting disease and black spot disease can be caused by multiple bacterial species (Maes and Jangoux 1984 , Gilles and Pearse 1986 ). We only counted sea urchins with clearly identifiable lesions when measuring black spot prevalence in the field which likely biased our prevalence estimates to be lower than the infection prevalence of the pathogen(s) causing black spot at our site. To categorize sea urchins with wasting disease in our lab experiment, we compared photographs taken of the same individual over several months so that differences in spine length and spine presence were clearer. We were conservative in considering a sea urchin as wasting by requiring clear and persistent loss of approximately half its primary spines, so we could have underestimated wasting by excluding less severe cases. Second, in the black spot experiment, we had only one tank in each of the Abrasion-Diseased and Abrasion-Control treatments, so treatment was confounded with tank. Taken by itself, this weakens the strength of the evidence that can be drawn from this experiment, but the results were consistent with past work (Gilles and Pearse 1986 , Lester et al. 2007 ), and, when combined with the results of the field study, suggest that black-spot disease is transmissible without contact, can cause substantial mortality, and transmission occurs locally and frequently in the field. Third, in the black spot lab experiments, we allowed the initial diseased urchins to decompose in the tanks for approximately one week, as we assumed would occur in nature. However, dilution is much higher in the ocean, so sea urchins in our experiments might have been exposed to higher concentrations of the pathogens than they would experience in nature. Fourth, our sea urchins may have been more nutritionally stressed in the lab than in some circumstances in nature, which may have exacerbated disease symptoms and mortality. Sea urchins can subsist off of calcareous coralline algae, bacterial mats, and other organic matter in the ocean when fleshy algae is not available (Filbee-Dexter and Scheibling 2014b). In our experiments, we filtered and sterilized our seawater and held urchins without food for 35 days (black spot experiment) to 144 days (wasting experiment). These limitations suggest caution in overinterpreting the details of our results, but our qualitative conclusions are likely robust to these issues. Kelp forest ecosystems have declined in some regions, including along the northeast Pacific coast (Krumhansl et al. 2016 , Filbee-Dexter and Wernberg 2018 , Rogers-Bennett and Catton 2019 ), and high densities of actively foraging purple sea urchins can prevent kelp recovery (Watson and Estes 2011 , Filbee-Dexter and Scheibling 2014a , Smith and Tinker 2022 ). Occasionally, mass mortalities of sea urchins do occur, but their causes aren’t always clear. We found that black spot disease could cause substantial mortality, and thus could be the cause of these previous mortality events (Johnson 1971 , Pearse et al. 1977 , Gilles and Pearse 1986 , Williams et al. 2021 ). While predicting disease events and associated mortality is difficult, our results suggest conditions that might facilitate wasting and black spot disease epidemics. As sea urchin management is an important element of kelp forest restoration (Eger et al. 2022 , Miller et al. 2022 ), our research on the transmissibility, severity, and drivers of wasting and black spot disease could improve the effectiveness of restoration through better timing and targeting of additional sea urchin removals as well as kelp seeding or planting. Future studies that assess whether partial predation, storm damage, and increased temperature result in higher black spot prevalence and significant mortality in nature would help clarify this, as would experiments that measure the effects of urchin density, nutritional stress, and thermal stress for both diseases. Declarations Acknowledgements Many thanks to Bella Shamoon, Joshua Santiago, Rosie Campbell, Rodrigo Mendez-Arango, Emily Halim, Grace Suh, Ben Walker, and Iris Flores for their assistance in the lab, in the field, and with data entry. This work benefited greatly from discussion with and feedback from Pete Raimondi and Tim Tinker. Author Contributions Statement: NJVM, KJK and AMK contributed to study conception. NJVM, KJK, and AMK contributed to experimental design, and NJVM, CMH, AMK, and KJK contributed to field study design. NJVM, VMN, APO, and CMH collected data. NJVM, KJK, and AMK wrote and edited the manuscript text. NJVM and AMK performed the analyses and created the figures. All authors read and approved the final version of the manuscript. Funding: Funding was provided to NVJM by the Earl H. and Ethel M. Myers Oceanographic and Marine Biology Trust and to AMK by NSF grants DEB-1911853, DEB-1717498. Data Availability Statement We will make data and code available to reviewers in Github. Data will be submitted to Dryad and code to Zenodo upon publication. References Anderegg, W. R. L., J. M. Kane, and L. D. L. Anderegg. 2013. 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Seasonal temperature dynamics of the upper ocean in the Southern California Bight. Journal of Geophysical Research: Oceans 113. Gilles, K. W., and J. S. Pearse. 1986. Disease in sea urchins Strongylocentrotus purpuratus: experimental infection and bacterial virulence. Diseases of Aquatic Organisms 1:10. Harvell, C. D., C. E. Mitchell, J. R. Ward, S. Altizer, A. P. Dobson, R. S. Ostfeld, and M. D. Samuel. 2002. Climate warming and disease risks for terrestrial and marine biota. Science (New York, N.Y.) 296:2158–2162. Harvell, C. D., D. Montecino-Latorre, J. M. Caldwell, J. M. Burt, K. Bosley, A. Keller, S. F. Heron, A. K. Salomon, L. Lee, O. Pontier, C. Pattengill-Semmens, and J. K. Gaydos. 2019. Disease epidemic and a marine heat wave are associated with the continental-scale collapse of a pivotal predator ( Pycnopodia helianthoides ). Science Advances 5:eaau7042. Hendler, G. 2013. Recent Mass Mortality of Strongylocentrotus purpuratus (Echinodermata: Echinoidea) at Malibu and a Review of Purple Sea Urchin Kills Elsewhere in California. Bulletin, Southern California Academy of Sciences 112:19–37. Hennon, P. E., D. V. D’Amore, P. G. Schaberg, D. T. Wittwer, and C. S. Shanley. 2012. Shifting Climate, Altered Niche, and a Dynamic Conservation Strategy for Yellow-Cedar in the North Pacific Coastal Rainforest. BioScience 62:147–158. Hewson, I., M. R. Johnson, and B. Reyes-Chavez. 2025. Lessons Learned from the Sea Star Wasting Disease Investigation. Annual Review of Marine Science 17:257–279. Hing, S., E. J. Narayan, R. C. A. Thompson, and S. S. Godfrey. 2016. The relationship between physiological stress and wildlife disease: consequences for health and conservation. Wildlife Research 43:51–60. Hudson, P. J., A. P. Dobson, and D. Newborn. 1998. Prevention of Population Cycles by Parasite Removal. Science 282:2256–2258. James, D. G. 2024. Monarch Butterflies in Western North America: A Holistic Review of Population Trends, Ecology, Stressors, Resilience and Adaptation. Insects 15:40. Johnson, P. T. 1971. Studies on diseased urchins from Point Loma - Kelp habitat improvement project annual report, 1970-1971. California Institute of Technology, Pasadena. Kiesecker, J. M., A. R. Blaustein, and L. K. Belden. 2001. Complex causes of amphibian population declines. Nature 410:681–684. Knight, C. J., R. P. Dunn, and J. D. Long. 2022. Conspecific cues, not starvation, mediate barren urchin response to predation risk. Oecologia 199:859–869. Kock, R. A., M. Orynbayev, S. Robinson, S. Zuther, N. J. Singh, W. Beauvais, E. R. Morgan, A. Kerimbayev, S. Khomenko, H. M. Martineau, R. Rystaeva, Z. Omarova, S. Wolfs, F. Hawotte, J. Radoux, and E. J. Milner-Gulland. 2018. Saigas on the brink: Multidisciplinary analysis of the factors influencing mass mortality events. Science Advances 4:eaao2314. Krebs, C. J., R. Boonstra, S. Boutin, and A. R. E. Sinclair. 2001. What Drives the 10-year Cycle of Snowshoe Hares? BioScience 51:25. Krumhansl, K. A., D. K. Okamoto, A. Rassweiler, M. Novak, J. J. Bolton, K. C. Cavanaugh, S. D. Connell, C. R. Johnson, B. Konar, S. D. Ling, F. Micheli, K. M. Norderhaug, A. Pérez-Matus, I. Sousa-Pinto, D. C. Reed, A. K. Salomon, N. T. Shears, T. Wernberg, R. J. Anderson, N. S. Barrett, A. H. Buschmann, M. H. Carr, J. E. Caselle, S. Derrien-Courtel, G. J. Edgar, M. Edwards, J. A. Estes, C. Goodwin, M. C. Kenner, D. J. Kushner, F. E. Moy, J. Nunn, R. S. Steneck, J. Vásquez, J. Watson, J. D. Witman, and J. E. K. Byrnes. 2016. Global patterns of kelp forest change over the past half-century. Proceedings of the National Academy of Sciences 113:13785–13790. Lafferty, K. D. 2004. Fishing for lobsters indirectly increases epidemics in sea urchins. Ecological Applications 14:1566–1573. Lester, S. E., E. D. Tobin, and M. D. Behrens. 2007. Disease dynamics and the potential role of thermal stress in the sea urchin, Strongylocentrotus purpuratus . Canadian Journal of Fisheries and Aquatic Sciences 64:314–323. Maes, P., and M. Jangoux. 1984. The bald-sea-urchin disease: a biopathological approach. Helgoländer Meeresuntersuchungen 37:217–224. McKechnie, A. E., and B. O. Wolf. 2010. Climate change increases the likelihood of catastrophic avian mortality events during extreme heat waves. Biology Letters 6:253–256. Miller, K. I., C. O. Blain, and N. T. Shears. 2022. Sea Urchin Removal as a Tool for Macroalgal Restoration: A Review on Removing “the Spiny Enemies.” Frontiers in Marine Science 9. Miranda, P. J., N. K. McLain, R. Hatzenpichler, V. J. Orphan, and J. G. Dillon. 2016. Characterization of Chemosynthetic Microbial Mats Associated with Intertidal Hydrothermal Sulfur Vents in White Point, San Pedro, CA, USA. Frontiers in Microbiology 7:1163. Pearse, J. S., D. P. Costa, M. B. Yellin, and C. R. Agegian. 1977. Localized Mass Mortality of Red Sea Urchin, Strongylocentrotus franciscanus, Near Santa Cruz, California. Pearse, J. S., and A. H. Hines. 1979. Expansion of a central California kelp forest following the mass mortality of sea urchins. Marine Biology 51:83–91. Rachowicz, L. J., J.-M. Hero, R. A. Alford, J. W. Taylor, J. A. t. Morgan, V. T. Vredenburg, J. P. Collins, and C. J. Briggs. 2005. The Novel and Endemic Pathogen Hypotheses: Competing Explanations for the Origin of Emerging Infectious Diseases of Wildlife. Conservation Biology 19:1441–1448. Ritchie, K. B., I. Nagelkerken, S. James, and G. W. Smith. 2000. A tetrodotoxin-producing marine pathogen. Nature 404:354–354. Rogers-Bennett, L., and C. A. Catton. 2019. Marine heat wave and multiple stressors tip bull kelp forest to sea urchin barrens. Scientific Reports 9:15050. Roussos, J. M. 2018. Spatial and Temporal Variation in White Point, Palos Verdes Hydrothermal Sulfur Vent Microbial Mat Community Structure. California State University, Long Beach. Shaw, C. G., C. Pavloudi, M. A. Barela Hudgell, R. S. Crow, J. H. Saw, R. A. Pyron, and L. C. Smith. 2023. Bald sea urchin disease shifts the surface microbiome on purple sea urchins in an aquarium. Pathogens and Disease 81:ftad025. Shaw, C. G., C. Pavloudi, R. S. Crow, J. H. Saw, and L. C. Smith. 2024. Spotting disease disrupts the microbiome of infected purple sea urchins, Strongylocentrotus purpuratus. BMC Microbiology 24:11. Smith, J. G., and M. T. Tinker. 2022. Alternations in the foraging behaviour of a primary consumer drive patch transition dynamics in a temperate rocky reef ecosystem. Ecology Letters 25:1827–1838. Steneck, R. S. 2013. Chapter 14 - Sea Urchins as Drivers of Shallow Benthic Marine Community Structure. Pages 195–212 in J. M. Lawrence, editor. Developments in Aquaculture and Fisheries Science. Elsevier. Steneck, R. S. 2020. Chapter 15 - Regular sea urchins as drivers of shallow benthic marine community structure. Pages 255–279 in J. M. Lawrence, editor. Developments in Aquaculture and Fisheries Science. Elsevier. Wagner, D. L., E. M. Grames, M. L. Forister, M. R. Berenbaum, and D. Stopak. 2021. Insect decline in the Anthropocene: Death by a thousand cuts. Proceedings of the National Academy of Sciences 118:e2023989118. Watson, J., and J. A. Estes. 2011. Stability, resilience, and phase shifts in rocky subtidal communities along the west coast of Vancouver Island, Canada. Ecological Monographs 81:215–239. Williams, J., J. Claisse, D. Pondella II, C. Williams, M. Robart, Z. Scholz, E. Jaco, T. Ford, H. Burdick, and D. Witting. 2021. Sea urchin mass mortality rapidly restores kelp forest communities. Marine Ecology Progress Series 664:117–131. Wong, C. M., and L. D. Daniels. 2017. Novel forest decline triggered by multiple interactions among climate, an introduced pathogen and bark beetles. Global Change Biology 23:1926–1941. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6986455","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":496216260,"identity":"b8815439-c411-49de-9b8f-e08150ff0a44","order_by":0,"name":"Nikka J. V. Malakooti","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYDACZjZmEMXDDyITCojXYsAj2QDSYkCUNRAtDAYHGMA0YWDOzpZs8KHij4zx+dWJHx4YMMjzix3Ar8Wyme1w4owzBjxmN95ulgA6zHDm7AT8WgwOszcf5m0DaTm7AaQlweA2sVqMZ5zd/INILWyHk0FaDPh7txFrC1uy4YwzxjwSN3i3WSQYSBDhl/PHjCU+VMjZ8/ef3XzzR4WNPL80AS0IIAFWKUGschDgP0CK6lEwCkbBKBhJAABukT5xdMDz4gAAAABJRU5ErkJggg==","orcid":"","institution":"University of California, Santa Cruz","correspondingAuthor":true,"prefix":"","firstName":"Nikka","middleName":"J. V.","lastName":"Malakooti","suffix":""},{"id":496216261,"identity":"10b512ca-cefa-42af-9d29-6e9f221d3937","order_by":1,"name":"Vishnu M. Nair","email":"","orcid":"","institution":"University of California, Santa Cruz","correspondingAuthor":false,"prefix":"","firstName":"Vishnu","middleName":"M.","lastName":"Nair","suffix":""},{"id":496216262,"identity":"d447cb05-7740-4513-9101-90c063e828ea","order_by":2,"name":"Ana P. Orloff","email":"","orcid":"","institution":"University of California, Santa Cruz","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"P.","lastName":"Orloff","suffix":""},{"id":496216263,"identity":"5fc4a5d3-9a2e-4bc9-849e-8c8ba60d95d3","order_by":3,"name":"Conner M. Hale","email":"","orcid":"","institution":"University of California, Santa Cruz","correspondingAuthor":false,"prefix":"","firstName":"Conner","middleName":"M.","lastName":"Hale","suffix":""},{"id":496216264,"identity":"4c63f34d-20f7-4f73-966e-e8e3515e5cf3","order_by":4,"name":"Kristy J. Kroeker","email":"","orcid":"","institution":"University of California, Santa Cruz","correspondingAuthor":false,"prefix":"","firstName":"Kristy","middleName":"J.","lastName":"Kroeker","suffix":""},{"id":496216265,"identity":"0fd9a96e-27d8-4705-9749-2d140b1ff2b4","order_by":5,"name":"A. Marm Kilpatrick","email":"","orcid":"","institution":"University of California, Santa Cruz","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"Marm","lastName":"Kilpatrick","suffix":""}],"badges":[],"createdAt":"2025-06-26 21:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6986455/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6986455/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88780322,"identity":"662998b0-26ab-406f-9cbd-6491d4a42f6e","added_by":"auto","created_at":"2025-08-11 10:44:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5263458,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotographs of purple sea urchins with and without disease. \u003c/strong\u003e(a) and (b) Lesions indicative of black spot disease. (c) and (d) Sea urchins missing most of their primary spines, indicative of wasting disease; those remaining are mostly shortened. (e) and (f) Healthy purple sea urchins. Photos by N. Malakooti.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6986455/v1/1eca5b0a66d174bf43be49a7.png"},{"id":88780316,"identity":"8cbb7755-4894-4ab3-88d0-57ec07ae32b7","added_by":"auto","created_at":"2025-08-11 10:44:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":23371,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe fraction of urchins remaining healthy (not wasting) plotted against time since diseased urchins were put in the Diseased treatment tanks.\u003c/strong\u003eSea urchins from all tanks in all three injury treatments (spine clipping, abrasion, control) were combined; Figure S2 shows the results for individual treatments. A “+” on a line indicates that a sea urchin died and was “censored” on that day except for the + on days 143-144 which indicate the end of the experiment. There was no effect of Disease treatment on wasting (Cox’s proportional hazard with tank as random effect: Control coef.: -0.012 ± 0.22, Z = -0.06, p = 0.96).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6986455/v1/a0c57114d517bf06bc31095a.png"},{"id":88780321,"identity":"d3479336-9ca6-44ac-b331-b9e1a12381fa","added_by":"auto","created_at":"2025-08-11 10:44:35","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":530655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLesion diameter (mm) over time in the abrasion-control (A) and abrasion-black spot tanks. \u003c/strong\u003eEach colored line represents one individual purple sea urchin. Solid black lines and gray ribbons show the fitted model and 95% CI, and X’s indicate death of that sea urchin.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6986455/v1/3b1e59b9de447d9510f30a55.jpeg"},{"id":88780320,"identity":"a6d24151-80dd-4c6d-b7e4-3cc7787d46b3","added_by":"auto","created_at":"2025-08-11 10:44:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":228121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlackspot prevalence on a square-root transformed scale plotted against the distance from the center of the transect.\u003c/strong\u003e Points show the prevalence (±95% CI) grouped across all survey plots (because the estimated variance for plot_ID was 0) for plots centered around a Diseased (red) or Control (blue) urchin. Lines show the fitted model (solid red line for Diseased and dashed blue line for Control plots, with urchin density set to the mean value), and ribbons show ±1SE. Panels show individual survey dates arranged by julian date, irrespective of year, starting in September. For the 10-10-2023 survey, there was no estimate of prevalence at the control point at 0 m because only a single plot was surveyed on this date and no urchins were found in the 0m quadrat on that plot.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6986455/v1/136a0dcebf8172f728364bc5.png"},{"id":88781270,"identity":"33542d23-7b52-48f6-8959-fe6ce4763b5b","added_by":"auto","created_at":"2025-08-11 10:52:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":229426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlackspot prevalence (square-root transformed) plotted against sea urchin density.\u003c/strong\u003e Points show the prevalence across all surveys with a given sea urchin density, and point size shows the total number of urchins examined for that point. The line and 95% CI ribbon shows a univariate fitted generalized linear model with a binomial distribution and logit link that has a similar slope and uncertainty as the full model in Table S2 (slope: -0.046 ± SE = 0.018).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6986455/v1/e97b12313c97a8be049db418.png"},{"id":96251412,"identity":"148f2685-6778-4b1e-9cc1-30513a01b743","added_by":"auto","created_at":"2025-11-19 07:39:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8215983,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6986455/v1/b3de342a-830e-4d2c-9c48-fe3903a70f59.pdf"},{"id":88781269,"identity":"e49629ad-e885-4f90-be22-b449f9e99df3","added_by":"auto","created_at":"2025-08-11 10:52:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":277658,"visible":true,"origin":"","legend":"","description":"","filename":"SciReptsSupplement.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6986455/v1/39a2bc72f7664346b784a891.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The transmissibility and severity of black spot and wasting disease in sea urchins from laboratory experiments and field studies","fulltext":[{"header":"1 | Introduction","content":"\u003cp\u003ePopulation declines driven by disease, environmental change, and biotic interactions are increasingly affecting species and ecosystems (Harvell et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Fey et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Wong and Daniels \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, identifying the cause of a population decline or mass mortality event can be challenging because organisms are often threatened by a combination of stressors (Krebs et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Kiesecker et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Hennon et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Wong and Daniels \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Kock et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Wagner et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Infectious disease has increasingly been recognized as a factor influencing populations, but identifying the cause of a disease and linking mortality events with a causal agent can be challenging (Hudson et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, Rachowicz et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Hewson et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This is, in part, because other factors like physiological stress can both increase the severity of infectious disease events and cause mortality in its own right (McKechnie and Wolf \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Anderegg et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Duff et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Hing et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Disentangling the causes of population declines is critical for restoration and management of species and the ecosystems they inhabit.\u003c/p\u003e\u003cp\u003eSea urchin (Echinoidea) population fluctuations are emblematic of these challenges, despite their importance in structuring marine ecosystems including coral reefs, seagrass beds, and kelp forests (Steneck \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Unchecked growth of sea urchin populations can lead to a dramatic decline in habitat-forming kelp and the formation of urchin barrens, whereas population declines in sea urchins can result in recovery of kelp forests (Steneck \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Filbee-Dexter and Scheibling \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e, Williams et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Eger et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Miller et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The factors influencing disease epidemics in sea urchin populations and whether epidemics will reduce sea urchins to low enough densities for kelp to recover are only partly understood. In the northeast Pacific Ocean, two diseases called wasting (or red-spot or bald sea urchin disease) and black spot (or black-ring or spotting disease) have been associated with mortality events or changes in population growth rates in red (\u003cem\u003eMesocentrotus franciscanus\u003c/em\u003e) and purple sea urchins (\u003cem\u003eStrongylocentrotus purpuratus\u003c/em\u003e) for decades (Johnson \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1971\u003c/span\u003e, Pearse and Hines \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1979\u003c/span\u003e, Gilles and Pearse \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Behrens and Lafferty \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Lafferty \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Hendler \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Shaw et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Wasting disease is characterized by shortened and/or lost primary spines; black spot signs include lesions characterized by black glossy or friable tissue and rings of blackened tissue in which the bare test is visible (Fig.\u0026nbsp;1). Black spot lesions can eventually wear through the urchin internal shell (test) and expose the coelomic cavity (Gilles and Pearse \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Attempts to identify the causal pathogen for black spot disease have been inconclusive, because the bacteria that were isolated from urchins with black spot disease were able to cause lesions in sea urchins in lab infections, but no mortality was observed (Gilles and Pearse \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). In addition, while multiple studies have induced black spot lesions by transferring diseased tissue to mechanically abraded sea urchins or by holding abraded sea urchins in close contact with diseased urchins, it\u0026rsquo;s unclear whether the cause of black spot disease can be transmitted without contact and what type of injury is needed for infection (Gilles and Pearse \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The cause of wasting disease in red and purple sea urchins has not been identified (Ritchie et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Clemente et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSpatial patterns of disease, including correlations with host density, have also been used to examine the transmissibility of pathogens causing disease. Increasing disease prevalence with host density may result from increased contact rates and transmission of an infectious pathogen (Begon et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). However, density dependent patterns of disease prevalence could also result from stressors that increase with increased population density. If higher population density results in lower resource availability and starvation or physiological stress, this could result in density-dependent patterns of disease prevalence for a non-transmissible disease. Past studies of the relationship between urchin disease and density have been mixed. The probability of wasting events increased with sea urchin density in the Channel Islands (Lafferty \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), but in coastal southern California, black spot prevalence declined with sea urchin density and there was no relationship between wasting prevalence or combined wasting and black spot disease prevalence and density (Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Thus, it is unclear whether declines associated with wasting are caused by infectious pathogens, environmental stress, or some combination of the two.\u003c/p\u003e\u003cp\u003eInterest in sea urchin management in the northeast Pacific Ocean increased after the 2014\u0026ndash;2016 marine heat wave, which coincided with the loss of predatory sea stars due to sea star wasting disease, subsequent overgrazing by purple sea urchins (\u003cem\u003eStrongylocentrotus purpuratus\u003c/em\u003e), and the widespread loss of kelp in some regions (Harvell et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Rogers-Bennett and Catton \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Sea urchin culling has been proposed as a tool for kelp restoration, but it is highly labor intensive and limited in spatial scale and often requires perpetual removals (Eger et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Miller et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, the need for culling depends on natural population fluctuations caused by pathogens, competitors, predators, and environmental stressors (James \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). For example, kelp restoration efforts via sea urchin culling in southern California was interrupted by a sea urchin mass mortality event and a few individuals with black spot disease were found (Williams et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The kelp forests recovered shortly thereafter, demonstrating that naturally occurring sea urchin population declines can sometimes allow recovery of kelp forests (Williams et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, it\u0026rsquo;s unclear if black spot disease was the cause of this decline and what factors might make disease epidemics more severe. More generally, understanding the drivers of spatial and temporal patterns in sea urchin diseases and whether they are caused by transmissible pathogens could improve kelp restoration efforts. Sea urchin removals could be focused on populations less likely to undergo natural population declines and kelp seeding or outplanting projects could take advantage of disease events when urchin densities are lower.\u003c/p\u003e\u003cp\u003eOur goal was to determine if wasting and black spot disease were transmissible without contact among sea urchins, what types of injuries (abrasion, spine breakage) were needed for infection, and whether these diseases would lead to mortality. We conducted laboratory experiments and hypothesized that both diseases would be transmissible; tanks that housed diseased urchins would have more disease (spine loss, lesion formation and growth) and higher mortality than tanks without diseased sea urchins. We also conducted subtidal field surveys to determine if spatial patterns of black spot disease prevalence were consistent with local transmission. We hypothesized that prevalence would decrease with distance from diseased sea urchins, but not healthy urchins, and that disease prevalence would increase with sea urchin density, as would be expected for a pathogen transmitted by non-sexual contact or through water (Briggs et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e"},{"header":"2 | Methods","content":"\u003cp\u003e\u003cem\u003eWasting Disease Lab Experiment\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe collected visually healthy purple sea urchins from an urchin barren in Stillwater Cove, Monterey County, CA (36.563\u0026deg;N, 121.949\u0026deg;W) on SCUBA April 15, 2024 and transported them to the University of California Santa Cruz. We held sea urchins in seawater tables supplied with ambient flow-through seawater (no recirculation) for 17 days. We then randomly assigned visually healthy sea urchins to three \u0026ldquo;injury\u0026rdquo; treatment groups:1) Spine clipping, in which we simulated storm damage by shortening primary spines to approximately half their length on half the test area; 2) Abrasion, in which we removed a 5mm diameter patch of spines and epithelium from each sea urchin with a sterile scalpel blade, as might mimic a failed predation attempt on an urchin; 3) Uninjured sea urchins. We placed 10 urchins of a single treatment into each 25.55 L tank fed by chilled seawater (~\u0026thinsp;13\u0026deg;C), containing one 300 W heater (Hygger HG-925) and one 80 gph circulating pump (Uniclife-UL016). We placed each sea urchin in a 0.5 L vented bait jar (7.6x11.4x10.2 cm) to prevent physical contact among individuals and allow identification of individual urchins. We also collected sea urchins that had characteristics of wasting disease - significantly shortened or absent primary spines (Fig.\u0026nbsp;1, panels c and d) from the intertidal zone at Pebble Beach, Sea Ranch, Sonoma County, CA (38.699\u0026deg;N, 123.441\u0026deg;W) on May 1st 2024. We held them in 25.55 L tanks fed by chilled seawater (~\u0026thinsp;13\u0026deg;C). We filtered and sterilized the inflow and outflow streams for all tanks with 25 micron filters and 80 watt UV sterilizers (Pentair) to prevent introduction of additional disease-causing microbes into our experimental tanks as well as from our tanks into the ocean.\u003c/p\u003e\u003cp\u003eOn May 7th, we added one wasting sea urchin to each of 3 Abrasion-Diseased tanks, 3 Spine Clipping-Diseased tanks, and 2 Uninjured-Diseased tanks. There were 2 Abrasion-Control tanks, 2 Spine Clipping-Control tanks, and 2 Uninjured-Control tanks without diseased urchins added. Whenever wasting sea urchins died in the Disease treatment tanks we replaced them, so that there was always one live wasting sea urchin in each Disease treatment tank. We monitored sea urchins every two days for one week and approximately weekly thereafter, taking photographs and notes on the condition of each sea urchin to assess any indication or progression of wasting disease. On June 16th, 2024, we increased tank temperatures from 13\u0026deg;C to 16\u0026deg;C by approximately 0.5\u0026deg;C per day to increase physiological stress and the potential for disease (Lester et al \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). We fed sea urchins giant kelp (\u003cem\u003eMacrocystis pyrifera\u003c/em\u003e) \u003cem\u003ead libitum\u003c/em\u003e for 4 days once before the beginning of the experiment (when diseased urchins were added), once for 2 days two weeks after the experiment began (~\u0026thinsp;6cm x 3cm per sea urchin), and starved them thereafter.\u003c/p\u003e\u003cp\u003e\u003cem\u003eBlack Spot Lab Experiment\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe conducted a similar experiment to assess whether black spot disease was transmissible without physical contact between individuals and whether disease would lead to mortality. We collected visually healthy purple sea urchins from Lovers Point state marine reserve, Monterey County, CA (36.623\u0026deg;N, 121.906\u0026deg;W) in May 2022 and held them in ambient flow-through seawater tables (14\u0026deg;C-16\u0026deg;C) at the University of California Santa Cruz. We also collected purple sea urchins with signs of black spot disease, including one or more dark, glossy patches or patches of visible test with a black ring of tissue (Fig.\u0026nbsp;1, panels a and b), from the intertidal at White Point Beach, Los Angeles County, CA (33.715\u0026deg;N, 118.320\u0026deg;W) on August 12th 2022. We held urchins overnight in a cooler with moist giant kelp cuttings and ice packs, and then transported them to tanks at the University of California Santa Cruz in a cooler with ice packs, damp towels, and giant kelp cuttings. Each tank was fed by a spigot off of the main water pipe and contained one 200W (Hygger HG-802) heater and one 80 gph circulating pump (Eheim 1046). We filtered and sterilized the inflow and outflow streams with 25 micron filters and 80 watt UV sterilizers (Pentair).\u003c/p\u003e\u003cp\u003eWe used the same injury treatment groups as in the wasting experiment: Spine Clipping, Abrasion, and Control, and again housed urchins in individual marked 0.5 L vented bait jars (7.6x11.4x10.2 cm) to prevent contact and allow identification of individual urchins. We used 76 L tanks (57.2x36.2x36.8 cm) and placed 17 healthy urchins in each Diseased tank and 18 healthy urchins in each Control tank. We had two tanks each for the Uninjured-Diseased and Uninjured-Control treatments, but only one (unreplicated) tank for each of the remaining four injury-disease treatments (Spine Clipping-Diseased, Spine Clipping Control, Abrasion-Diseased and Abrasion-Control), for a total of eight tanks. We placed a single urchin with symptoms of black spot into Disease treatment tanks on August 15th which survived for 1 to 6 days before dying. Diseased sea urchins were kept in the tanks until they died (2\u0026ndash;6 days) and then deteriorated before they were removed; they were in the tanks for 9\u0026ndash;16 days total. We examined sea urchins for lesions and mortality every 2\u0026ndash;4 days for the first two weeks and weekly thereafter. We measured the diameter of any lesions we observed using calipers (including those we had created in the Abrasion-Diseased and Abrasion-Control treatment groups). To increase the likelihood of disease, we raised water temperature in the tanks by 0.5\u0026deg;C per day starting on August 16, 2022 until temperatures reached\u0026thinsp;~\u0026thinsp;18\u0026deg;C (Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eBlack Spot Field Study\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe conducted subtidal field surveys to quantify spatial and temporal changes in black spot disease prevalence at an urchin barren site near White Point Beach Park, San Pedro, LA County, CA (33.713\u0026deg;N, 118.318\u0026deg;W). We chose this site based on reports of diseased sea urchins by Reef Check and Vantuna Research program SCUBA divers and an exploratory dive. We attempted to avoid bacterial mats and warm water seeps at this site (Miranda et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Roussos \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) in order to preclude their influence on our study, although we occasionally noted small amounts of bacterial filaments on our plots. To begin our surveys, we searched for purple or red sea urchins with characteristic symptoms of black spot disease (Fig.\u0026nbsp;1). Upon finding a diseased individual, we placed a marker made of steel chain links and flagging tape on the reef to mark the sea urchin\u0026rsquo;s initial location. We established Control plots by generating random combinations of compass headings and distances (10 m-50m) from each Diseased plot, but constrained their location to the rocky reef (urchins were not present in the surrounding sandy habitats). At each Diseased or Control plot, we quantified number of red and purple sea urchins, disease prevalence (for black spot and wasting separately), depth, and substrate type (bedrock, boulder, cobble, or sand) in 0.5m\u003csup\u003e2\u003c/sup\u003e quadrats centered on the focal disease urchin and along four perpendicular transects at 1, 2, 3, and 4m from the center of the plot (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We also recorded temperature and relief (the difference between the highest and lowest points in the quadrat, grouped into four categories: 0-10cm, 10cm-1m, 1m-2m, \u0026gt;2m) at the center of each plot. We revisited the site at 2\u0026ndash;11 week intervals and resurveyed the plots. When the chain marker was dislodged, we used directions and photographs to relocate it to the original location. For two plots, the chain could not be located and our directions and photographs of the original marker were not sufficient to guarantee overlap with the original plot area, so these plots were not resampled. We surveyed 11 plots between September 2023 through March 2024. In October 2024, we surveyed 6 additional plots, for a total of 9 Diseased and 8 Control plots.\u003c/p\u003e\u003cp\u003e\u003cem\u003eStatistical Analyses\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe analyzed morbidity and mortality in the wasting disease experiment using 3 sets of Cox\u0026rsquo;s proportional hazard models: one with disease treatment (Disease-Control) only, one with disease treatment (Disease-Control), and injury treatment (Abrasion, Spine Clipping, or Control) and one that also included the interaction of these two treatments as predictors. For the mortality analysis in the wasting experiment, we excluded deaths in the first 30 days because urchins that died during this period appeared to die of acclimation stress, not wasting disease. Urchins dying in the first 30 days had rapid (1\u0026ndash;2 days) spine and epithelial tissue loss over expanding regions of their bodies. In contrast, sea urchins dying after the first 30 days lost large primary spines relatively gradually, often over several weeks, and their remaining spines and tube feet were responsive to touch, and their epithelium appeared intact before they died. The symptoms in urchins that died after the first 30 days were consistent with wasting disease, whereas those that died earlier were not (Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo analyze growth of lesions in the Abrasion injury treatment of the black spot disease laboratory experiments, we fit linear mixed effects models with lesion size on a sea urchin on a day as the response variable, and day, disease treatment, and a day-treatment interaction as fixed effects and individual urchin ID as a random effect. There was only one tank for the Abrasion-Diseased treatment and one for the Abrasion-Control treatment, so treatment and tank effects were confounded. We tested the residuals of the lesion size analysis for normality with a Shapiro-Wilkes test. We compared mortality among treatments using a Cox\u0026rsquo;s proportional hazard model as well as a Fisher\u0026rsquo;s exact test.\u003c/p\u003e\u003cp\u003eFor the black spot field study, we fit a generalized linear model with a binomial distribution and a logit link with the prevalence of black spot disease as the response variable (each individual urchin was a data point) and distance from the center of the plot interacting with plot type (Diseased or Control), urchin density, and julian date as predictors. We initially included survey plot as a random effect to account for repeated measurements, but the random effect variance was estimated to be 0. We used julian date as a predictor because we expected the change in disease prevalence over time to be largely seasonal (Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). We used the \u003cem\u003elmer\u003c/em\u003e and \u003cem\u003eglmer\u003c/em\u003e functions in the \u003cem\u003elme4\u003c/em\u003e package and the \u003cem\u003ecoxme\u003c/em\u003e and \u003cem\u003ecoxph\u003c/em\u003e functions in the \u003cem\u003esurvival\u003c/em\u003e package in R version 4.4.2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"3 | Results","content":"\u003cp\u003e\u003cem\u003eWasting Disease Lab Experiment\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAt the end of the experiment (day 144), 75% of the 126 sea urchins in all treatments had developed symptoms of wasting and \u0026gt;\u0026thinsp;50% of urchins had wasted in all 14 individual tanks (Fig.\u0026nbsp;2, S2). There was no effect of putting diseased sea urchins in tanks on wasting in a univariate Disease-Control model (Fig.\u0026nbsp;2), an additive Injury and Disease-Control model (Control coef.: -0.0016\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 Z = -0.01, P\u0026thinsp;=\u0026thinsp;0.99; Injury treatment: χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;3.21, df\u0026thinsp;=\u0026thinsp;2, P\u0026thinsp;=\u0026thinsp;0.20) or an interactive Cox\u0026rsquo;s proportional hazard model (Figure S2; Control coef.: 0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36, Z\u0026thinsp;=\u0026thinsp;0.79, P\u0026thinsp;=\u0026thinsp;0.43; Disease-Injury treatment interaction term: χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;4.42, df\u0026thinsp;=\u0026thinsp;2, P\u0026thinsp;=\u0026thinsp;0.11). There was also no effect of putting wasting sea urchins in tanks on sea urchin survival, but 19% (24 of 126) of urchins died (Figure S3).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eBlack Spot Lab Experiment\u003c/em\u003e\u003c/p\u003e\u003cp\u003eOver the 36 days of the experiment, average lesion sizes in the Abrasion-Control tank decreased significantly (i.e. lesions healed\u003cb\u003e)\u003c/b\u003e over time by 0.021\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0075 mm per day or approximately 1mm every 50 days (Fig.\u0026nbsp;3A; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In contrast, in the Abrasion-Diseased tank where diseased black spot urchins were added, average lesion size increased significantly over time by 0.072\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0093 mm per day or approximately 1mm every two weeks, and approximately two-thirds of urchins had lesions that increased in size before dying or by the end of the experiment (Fig.\u0026nbsp;3; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Sea urchin mortality was significantly higher in the Abrasion-Diseased tank than in the Abrasion-Control tank (7/17\u0026thinsp;=\u0026thinsp;41% vs 0/18\u0026thinsp;=\u0026thinsp;0%; Fisher\u0026rsquo;s exact test P\u0026thinsp;=\u0026thinsp;0.0029; Cox\u0026rsquo;s proportional hazard model: likelihood ratio 13.75, df\u0026thinsp;=\u0026thinsp;1, P\u0026thinsp;=\u0026thinsp;0.00021; Figure S4). Two urchins in the Spine Clipping-Diseased tank developed lesions by the end of the experiment, whereas no urchins in the Spine Clipping-Control tank developed lesions, but the difference was not significant (Fisher\u0026rsquo;s exact test: 2/17 vs 0/18; P\u0026thinsp;=\u0026thinsp;0.23). Only one out of 36 sea urchins in the Uninjured-Diseased treatments died, and no urchins in the Spine Clipping-Diseased, Spine Clipping-Control, or Uninjured-Control treatments died.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eBlack Spot Field Study\u003c/em\u003e\u003c/p\u003e\u003cp\u003eBlack spot disease prevalence in the field declined significantly with julian date, urchin density, and distance from urchins with black spot; the slope of distance was non-significant for Control plots (Figs.\u0026nbsp;4, 5; Table S2). The slope of distance for Control plots was flatter (less negative) than Diseased plots, and its uncertainty was higher, due to substantial variability among sampling dates (Fig.\u0026nbsp;4; Table S2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"4 | Discussion","content":"\u003cp\u003eWe examined the transmissibility of two sea urchin diseases without contact and their potential to cause mortality. Wasting disease was more likely caused by non-transmissible physiological stress rather than transmission of an infectious pathogen, whereas the cause of black spot disease was transmissible in experiments without contact, but required injury. In addition, patterns of black spot prevalence in the field suggested that transmission occurs locally and is common, despite the possible need for injury to initiate infection. We found that both diseases led to significant (19\u0026ndash;41%) mortality in laboratory experiments. The combination of evidence from laboratory experiments and field studies suggesting black spot disease is locally transmitted and can cause substantial mortality supports the possibility that this disease may have been the cause of past mortality events in urchin populations (Johnson \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1971\u003c/span\u003e, Williams et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Overall, these findings suggest that declines in sea urchin populations due to wasting disease may follow physiologically stressful conditions (e.g. high density (Lafferty \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), or marine heatwaves (Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e)), whereas sea urchin declines due to black spot may increase with partial predation or storm damage combined with the presence of diseased urchins and warmer temperatures (Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Kelp restoration efforts could take advantage of declines due to both diseases to facilitate kelp restoration, including natural seeding or outplanting of kelp.\u003c/p\u003e\u003cp\u003eOur results suggest that wasting disease is not caused by a transmissible pathogen, which is at odds with our hypothesis and some previous research (Lafferty \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Wasting disease was associated with population declines in the Channel Islands that were positively correlated with sea urchin density (Lafferty \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). This suggested that declines were caused by an infectious pathogen. However, in our experiments, wasting disease occurred at similar rates in all treatments and tanks, whether or not a wasting sea urchin was housed in the tank. This suggests that wasting disease is caused by physiological stress, such as that experienced by our starved urchins in heated tanks. Since higher densities of sea urchins generally occur in nutrient-poor sea urchin barrens (Eger et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), the relationship between high sea urchin densities and epidemics in the Channel Islands (Lafferty \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) could have resulted from low food availability and other stressors, similar to what we observed in the lab.\u003c/p\u003e\u003cp\u003eIn contrast, we found evidence that the cause of black spot disease was transmissible without contact among sea urchins in our experiments (in both the Abrasion-Diseased treatment and at a lower rate in the Spine Clipping-Disease treatment), is transmitted locally in the field, and, importantly, can cause substantial mortality (41% in the Abrasion-Diseased treatment). The significantly higher mortality we observed when housing abraded sea urchins with a black spot sea urchin is a significant advancement in our knowledge of this disease. Previous studies either found no mortality at all, despite being able to culture bacteria and use them to cause lesions similar to those in the field (Gilles and Pearse \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1986\u003c/span\u003e), or no significant difference in mortality between healthy uninjured sea urchins and abraded sea urchins swabbed with diseased tissue (Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e: 3/11 vs 1/11 in the 19\u0026deg;C experiment; Fisher\u0026rsquo;s exact test P\u0026thinsp;=\u0026thinsp;0.59; no statistical comparison was made in the paper). The difference in mortality among experiments may be due to differences in nutritional stress at the time of exposure. The sea urchins used in previous lab experiments were fed during the experiments, (Gilles and Pearse \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), whereas our sea urchins were collected from barrens, starved, and held in warmer water. Finally, it\u0026rsquo;s worth noting that the abrasions we caused in the Abrasion-Control group healed over time, as in previous studies (Pearse et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1977\u003c/span\u003e, Gilles and Pearse \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), suggesting that abrasions in apparently healthy sea urchins do not result in black spot lesions without the presence of a diseased individual nearby.\u003c/p\u003e\u003cp\u003e Our field study showed evidence of transmission of the pathogen causing black spot disease at a local scale (1\u0026ndash;4 m) and declining prevalence from late summer through winter. The former suggests that black spot disease was caused by a pathogen that was more likely to spread to close neighbors. Prevalence was approximately 4% in late summer and varied from 0\u0026ndash;20% among quadrats, suggesting that conditions required for black spot (including possible injury) occur relatively frequently at the survey depths at our site and in other locations where black spot disease has been examined (Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Future studies could determine the importance of injuries for black spot disease in the field by comparing black spot prevalence at sites with differing levels of disturbance due to variation in ocean surge or variation in the abundance of sea urchin predators such as spiny lobsters or sheephead. The decline in black spot prevalence over time with julian date is consistent with a pattern of seasonal variation suggested by previous work (Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), and may be linked to variation in ocean temperatures that follow a similar pattern (Gelpi and Norris \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe found that black spot prevalence decreased with sea urchin density, which was the opposite of our hypothesis. However, pattern we found is consistent with a previous study on black spot disease in the same region of southern California (Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). A negative relationship between black spot prevalence and density in both studies could be caused by some areas having lower quality or abundance of food or other resources resulting in lower sea urchin density and higher susceptibility to disease. Another possibility is that sea urchins with black spot disease may move away from other urchins or that healthy sea urchins may move away from diseased urchins; disease avoidance behavior has been observed in another marine invertebrate, the Caribbean spiny lobster, \u003cem\u003ePanulirus argus\u003c/em\u003e (Behringer et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Purple sea urchins can respond to conspecific cues in a density-dependent manner (Knight et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), but further study is needed to determine whether they avoid diseased conspecifics.\u003c/p\u003e\u003cp\u003eThere were four important limitations to our study. First, we used visual criteria to determine whether a sea urchin had wasting disease or black spot because no pathogen has been associated with wasting disease and black spot disease can be caused by multiple bacterial species (Maes and Jangoux \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1984\u003c/span\u003e, Gilles and Pearse \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). We only counted sea urchins with clearly identifiable lesions when measuring black spot prevalence in the field which likely biased our prevalence estimates to be lower than the infection prevalence of the pathogen(s) causing black spot at our site. To categorize sea urchins with wasting disease in our lab experiment, we compared photographs taken of the same individual over several months so that differences in spine length and spine presence were clearer. We were conservative in considering a sea urchin as wasting by requiring clear and persistent loss of approximately half its primary spines, so we could have underestimated wasting by excluding less severe cases. Second, in the black spot experiment, we had only one tank in each of the Abrasion-Diseased and Abrasion-Control treatments, so treatment was confounded with tank. Taken by itself, this weakens the strength of the evidence that can be drawn from this experiment, but the results were consistent with past work (Gilles and Pearse \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Lester et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), and, when combined with the results of the field study, suggest that black-spot disease is transmissible without contact, can cause substantial mortality, and transmission occurs locally and frequently in the field. Third, in the black spot lab experiments, we allowed the initial diseased urchins to decompose in the tanks for approximately one week, as we assumed would occur in nature. However, dilution is much higher in the ocean, so sea urchins in our experiments might have been exposed to higher concentrations of the pathogens than they would experience in nature. Fourth, our sea urchins may have been more nutritionally stressed in the lab than in some circumstances in nature, which may have exacerbated disease symptoms and mortality. Sea urchins can subsist off of calcareous coralline algae, bacterial mats, and other organic matter in the ocean when fleshy algae is not available (Filbee-Dexter and Scheibling 2014b). In our experiments, we filtered and sterilized our seawater and held urchins without food for 35 days (black spot experiment) to 144 days (wasting experiment). These limitations suggest caution in overinterpreting the details of our results, but our qualitative conclusions are likely robust to these issues.\u003c/p\u003e\u003cp\u003eKelp forest ecosystems have declined in some regions, including along the northeast Pacific coast (Krumhansl et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Filbee-Dexter and Wernberg \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Rogers-Bennett and Catton \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and high densities of actively foraging purple sea urchins can prevent kelp recovery (Watson and Estes \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Filbee-Dexter and Scheibling \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e, Smith and Tinker \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Occasionally, mass mortalities of sea urchins do occur, but their causes aren\u0026rsquo;t always clear. We found that black spot disease could cause substantial mortality, and thus could be the cause of these previous mortality events (Johnson \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1971\u003c/span\u003e, Pearse et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1977\u003c/span\u003e, Gilles and Pearse \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Williams et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). While predicting disease events and associated mortality is difficult, our results suggest conditions that might facilitate wasting and black spot disease epidemics. As sea urchin management is an important element of kelp forest restoration (Eger et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Miller et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), our research on the transmissibility, severity, and drivers of wasting and black spot disease could improve the effectiveness of restoration through better timing and targeting of additional sea urchin removals as well as kelp seeding or planting. Future studies that assess whether partial predation, storm damage, and increased temperature result in higher black spot prevalence and significant mortality in nature would help clarify this, as would experiments that measure the effects of urchin density, nutritional stress, and thermal stress for both diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMany thanks to Bella Shamoon, Joshua Santiago, Rosie Campbell, Rodrigo Mendez-Arango, Emily Halim, Grace Suh, Ben Walker, and Iris Flores for their assistance in the lab, in the field, and with data entry. This work benefited greatly from discussion with and feedback from Pete Raimondi and Tim Tinker.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNJVM, KJK and AMK contributed to study conception. NJVM, KJK, and AMK contributed to experimental design, and NJVM, CMH, AMK, and KJK contributed to field study design. NJVM, VMN, APO, and CMH collected data. NJVM, KJK, and AMK wrote and edited the manuscript text. NJVM and AMK performed the analyses and created the figures. All authors read and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding was provided to NVJM by the Earl H. and Ethel M. Myers Oceanographic and Marine Biology Trust and to AMK by NSF grants DEB-1911853, DEB-1717498.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe will make data and code available to reviewers in Github. Data will be submitted to Dryad and code to Zenodo upon publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnderegg, W. R. L., J. M. 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Lawrence, editor. Developments in Aquaculture and Fisheries Science. Elsevier.\u003c/li\u003e\n\u003cli\u003eWagner, D. L., E. M. Grames, M. L. Forister, M. R. Berenbaum, and D. Stopak. 2021. Insect decline in the Anthropocene: Death by a thousand cuts. Proceedings of the National Academy of Sciences 118:e2023989118.\u003c/li\u003e\n\u003cli\u003eWatson, J., and J. A. Estes. 2011. Stability, resilience, and phase shifts in rocky subtidal communities along the west coast of Vancouver Island, Canada. Ecological Monographs 81:215\u0026ndash;239.\u003c/li\u003e\n\u003cli\u003eWilliams, J., J. Claisse, D. Pondella II, C. Williams, M. Robart, Z. Scholz, E. Jaco, T. Ford, H. Burdick, and D. Witting. 2021. Sea urchin mass mortality rapidly restores kelp forest communities. Marine Ecology Progress Series 664:117\u0026ndash;131.\u003c/li\u003e\n\u003cli\u003eWong, C. M., and L. D. Daniels. 2017. Novel forest decline triggered by multiple interactions among climate, an introduced pathogen and bark beetles. Global Change Biology 23:1926\u0026ndash;1941.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"density dependent transmission, Koch’s postulates, purple sea urchin (Strongylocentrotus purpuratus), trophic cascade, experimental infection, injury","lastPublishedDoi":"10.21203/rs.3.rs-6986455/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6986455/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSea urchins can be important in structuring multiple ecosystems, but the causes of population fluctuations including the role of infectious disease are often not well understood. We performed laboratory experiments and field studies with purple sea urchins (\u003cem\u003eStrongylocentrotus purpuratus\u003c/em\u003e) to investigate the transmissibility and severity of two diseases, wasting and black spot. We found that wasting disease and wasting mortality were equally likely in tanks with and without a wasting urchin. In contrast, the presence of a sea urchin with black spot disease led to faster growth of abrasions and significantly higher (41%) mortality than without a diseased sea urchin (0%). Finally, in the field, the prevalence of black spot disease decreased significantly with distance from a focal urchin with black spot disease but not in plots centered on a healthy reference urchin; black spot prevalence also decreased with sea urchin density. These results suggest that wasting disease likely results from non-transmissible physiological stress, while black spot disease is caused by a pathogen that can be transmitted without contact, is transmitted locally, and can cause substantial mortality. Despite these differences, both diseases can cause mortality and population declines in sea urchin populations with cascading effects on kelp forests.\u003c/p\u003e","manuscriptTitle":"The transmissibility and severity of black spot and wasting disease in sea urchins from laboratory experiments and field studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-11 10:44:31","doi":"10.21203/rs.3.rs-6986455/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"384ab7ca-63e7-4ca6-b8dd-02f9bf33f4d1","owner":[],"postedDate":"August 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52703898,"name":"Biological sciences/Ecology/Ecological epidemiology"},{"id":52703899,"name":"Biological sciences/Ecology/Biooceanography/Fisheries"},{"id":52703900,"name":"Biological sciences/Ecology/Biooceanography/Microbial biooceanography"}],"tags":[],"updatedAt":"2025-11-18T09:39:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-11 10:44:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6986455","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6986455","identity":"rs-6986455","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}