Mass mortality of collector urchins Tripneustes gratilla in Hawai`i

: As grazers, sea urchins are keystone species in tropical marine ecosystems, and their loss can have important ecological ramifications. Die-offs of urchins are frequently described, but their causes are often unclear, in part because systematic examinations of animal tissues at gross and microscopic level are not done. In some areas, urchins are being employed to control invasive marine algae. Here, we describe the pathology of a mortality event in Tripneustes gratilla in Hawai`i where urchins were translocated to control invasive algae. Although we did not determine the cause of the mortality event, our investigation indicates that animals died from in - flammation of the test and epidermal ulceration, followed by inability to maintain coelomic fluid volume, colonization of coelomic fluid by opportunists (diatom, algae), and inappetence. Para-sites, bacteria, fungi, and viruses were not evident as a primary cause of death. Pathology was suggestive of a toxin or other environmental cause such as lack of food, possibilities that could be pursued in future investigations. These findings highlight the need for caution and additional tools to better assess health when translocating marine invertebrates to ensure maximal biosecurity.


INTRODUCTION
In tropical marine ecosystems, sea urchins play an important role as grazers by helping maintain a balance between fast growing macroalgae and more slowly growing coral reefs (Ogden & Lobel 1978). Because of this, extirpation of urchins from some coral reefs can have profound adverse ecological consequences. A classic illustration of this was unexplained mortalities of Diadema antillarum that led to > 90% declines of urchins over a year throughout the entire Caribbean (Lessios 2016). Concomitant with this urchin mortality, additional insults such as progressive algal overgrowth, widespread coral disease, bleaching events and overfishing led to secular declines over time in the dominant genus of coral Acropora over the ensuing decades; corals and urchins have yet to recover (Aronson & Precht 2001, Jackson et al. 2001, Lessios 2016. The cause of the Diadema mortality was never determined mainly because no samples were taken for laboratory testing (Lessios 2016).
Since that event, several efforts have been taken to better understand causes of mortality in sea urchins (Wang et al. 2013). Most die-offs are assumed to be caused by infectious diseases, usually bacteria, based mainly on presence of clinical signs, microbial cultures, and molecular studies (Tajima et al. 1998, Nagelkerken et al. 1999, Becker et al. 2007, Clemente et al. 2014, Sweet et al. 2016. Other die-offs have been associated with environmental changes such as a mass mortality of Paracentrotus lividus in Spain associated with elevated seawater temperatures (Girard et al. 2012). Studies that focus on care-ful examination of morphology of urchin tissues at the gross and microscopic level provide a systematic framework to exclude potential causes of death in animals (Work & Meteyer 2014); however, such studies are less common. Moreover, understanding changes in host tissues at the cellular level might yield important insights into the anatomic and physiologic processes that leads to mortality. A good example where such approaches have yielded important understanding of etiology, pathogenesis, and pathophysiology of urchin disease is parameobiasis, a parasitic disease of Strongylo centrotus droebachiensis, in Canada that is probably one of the most comprehensively described urchin diseases (Scheibling & Hatcher 2013). Shimizu (1994) used histology to describe an inflammatory response associated with worm infection in the gonads of Strongylocentrotus intermedius in Japan. In some cases, systematic approaches to investigating urchin lesions using microscopic examinations do not al ways yield a cause of death. However, such inves tigations are valuable to help rule out infectious agents as causes of lesions thereby providing more focused avenues of investigations moving forward. They also provide insights on the process leading to death of the animal. For instance, Roberts-Regan et al. (1988) described pathogenesis of test lesions in S. droebachiensis that were sometimes associated with bacteria. In a more recent example, a pathology investigation of a mortality event in Tripneustes ventricosus in St. Kitts showed inflammation of multiple organs that could not be traced to infectious agents leading the authors to suggest perhaps a toxin or virus could be involved (Virwani et al. 2021).
The recognition that sea urchins play an important role in coral reefs has led to their use as a management tool to reduce abundances of invasive algae. Since 2010, the State of Hawai`i has used the abundant native collector urchin Tripneustes gratilla to control several species of invasive algae in patch reefs of Kaneohe Bay (KB), Oahu (Stimson et al. 2001, Westbrook et al. 2015. In this program, invasive algae are mechanically removed from coral reefs, and then urchins (at the time, mostly captured in the wild from other locations on Oahu, with a minority raised in captivity) are released in the newly cleared patches with the aim of keeping algae under control. In 2014, urchins at one of the collection sites on southeast Oahu manifested loss of spines and epidermal ulceration. Subsequently, ur chins at the release site in KB manifested similar clinical signs, and reports of dying urchins were also received from Maui during a similar time frame. Our objective was to describe the temporal course of events, demographics, clinical observations, gross findings, and histopathology of this T. gratilla mortality event.

Field surveys and scoring
On 27 January 2014, urchins were collected from a submerged barge in Maunalua Bay (MB) on SE Oahu for relocation to KB to the northeast (Fig. 1). On 1 February 2014, numbers of urchins were noted to be declining at the barge, but animals were translocated from the barge in MB to KB 2 days later. On 19 February 2014, urchins manifesting loss of spines and epidermal ulceration of the test were seen at the barge in MB, and on 24 February 2014, urchins manifesting lesions similar to those seen at the barge were found in KB (Fig. 1). Two hundred and thirty urchins that had originally been translocated from the barge in MB to KB were removed from KB and placed permanently into flow-through seawater tanks at Sea Life Park Aquarium located on Southwest Oahu on 26 February 2014 and 3 March 2014. Despite this intervention, sick urchins continued to be found in KB to the south of the release site through 6 May 2014 (Fig. 1) A variety of survey techniques were used to assess prevalence of disease and population responses to the mortality event. To assess prevalence (percent) of animals with lesions in KB, teams of divers swam over 3 patch reefs and enumerated animals with and without lesions on 6 and 28 May 2014. To assess long term effects of mortality on urchin populations, surveys were done using one 30 × 2 m transect on SE Oahu at the barge (the site of the initial outbreak) and a location called Z-blocks situated ca. 500 m to the SE. Two surveys were done in 2014 and follow up monthly surveys were done in 2016. Surveys were not done in 2015 due to lack of access to those sites. To assess prevalence of lesions in Honolua Maui, we surveyed a single transect measuring 25 × 2 m at each site during each survey in June 2014. Roving diver surveys were used to assess lesioned urchins in Mahukona, west Hawai`i in June 2014.

Pathology
Live moribund urchins, as determined by visible movement of teeth in lantern or active movement of spines, were collected by hand using SCUBA or snorkel. Prior to necropsy, animals were photographed orally and aborally and were subjectively assigned a gross lesion score based on severity of spine loss as 0 (healthy), 1 (mild), 2 (moderate) or 3 (severe) ( Fig. 2A−H). Specifically, Score 0 urchins were those with vigorously moving spines with white tips, extended tube feet, and often with objects (pieces of rubble, shell fragments) held on top of the urchin with pedicellariae (characterizing these animals as collector urchins). Score 1 urchins had dull spines often flattened or attenuated with absence of extended tube feet. Score 2 urchins were as in Score 1 with evidence of mild focal to multifocal ulceration. Score 3 urchins had diffuse denudation or attenuation of spines with ulceration. Test diameter was measured to the nearest 0.1 cm using calipers and animals were weighed (gross weight) to the nearest gram. Coelomic fluid was drained by puncturing the peristomial membrane, and animals were again weighed (net weight). Percent weight as coelomic fluid was calculated as follows: (gross -net)/gross. Fill of the gut with algae was scored subjectively as 2 (large amounts of algae), 1 (trace amounts of algae) or 0 (no visible contents) ( Fig. 2J−P). Drops of coelomic fluid were placed on microscope slides, allowed to air dry, stained with Wright/Giemsa (Hema 3, Thermo-Fisher, Waltham, MA) according to manufacturer instructions, and the smears examined for presence of intracellular or extracellular organisms. Ceolomocytes were not quantified, because standardized methods to evaluate hematology for T. gratilla were not established until after this outbreak (Work et al. 2020). Sections of the gonad, gut, peristomial membrane, lantern and associated muscles, oral and aboral test comprising ambulacral and interambulacral regions were saved in 10% formalin. For histology processing, calcified tissues were decalcified over night in formic acid (Cal ex II, Thermo-Fisher) prior to embedding in paraffin, sectioning at 5 μm, and staining with hematoxylin and eosin (HE; Thermo-Fisher). For the test, severity of coleomocyte in filtrates were subjectively scored as 0, 1, 2, or 3 (none, mild, moderate, severe) ( Fig. 2M−P). All histology slides were read blind with no knowledge of gross lesion score category. Special stains (Thermo-Fisher) used in cluded Giemsa to detect protozoa, and periodic-acid-Schiff's reaction (PAS) to see if the large granules in the large eosinophilic coelomocytes consisted of polysaccharides (Prophet et al. 1992).
For electron microscopy, tests showing infiltrates of large eosinophilic cells were preserved in modified Karnovsky's fixative (Karnovsky 1965) made with artificial seawater and decalcified in EDTA. Tissues were rinsed in 0.1 M sodium cacodylate buffer containing 0.35 M sucrose and postfixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. Tissue was dehydrated in a graded ethanol series, then embedded in LX112 epoxy resin. Epoxy-embedded tissues were cut into 1 μm thick sections and stained with Richardson's stain for light microscopy. Ultrathin (60 to 80 nm) sections were obtained on an RMC Power tome ultramicrotome, double stained with uranyl acetate and lead citrate, viewed on a Hitachi HT7700 transmission electron microscope (TEM) at 100 kV, and photographed with an AMT XR-41B 2k-by-2k charge-coupled device camera. Electron microscopy was done at the University of Hawai`i Biological Electron Microscope Facility, Honolulu, Hawai`i.
Electron-microscopy findings were suggestive of intracellular structures compatible with either filamentous viral-like particles or tubulin. Feulgen's stain and methyl green pyronin were used to see if these structures contained DNA or RNA, respectively, with urchin gut as positive controls (Prophet et al. 1992).
We assayed for tubulin using immunohistochemistry (IHC) and western blot containing rabbit polyclonal anti-tubulin ab15568 antibodies (Abcam, Boston, MA). For IHC, tissues were deparaffinized with xylene and re hydrated in ethanol series 100%, 95%, followed by equilibration in Tris buffer. Antigens were retrieved by heating tissues at 90°C for 10 min in 10 mM citrate buffer pH 6 and endogenous peroxidases blocked with 3% H 2 O 2 for 5 min. After washing with Tris buffer, tissues were blocked with 10% goat serum in phosphate-buffered saline for 1 h at room temperature. Tissues were then incubated with anti-tubulin antibody at 1:200, 1:500, and 1:1000 overnight. After 3 washes in Tris buffer, secondary antibodies conjugated to horseradish peroxidase (HRP) were applied (Vector Labs, Newark, CA), and tissues washed again 3 times. Color development was done with diamino benzidine, tissues counterstained with hematoxylin, and mounted for microscopic examination. Urchin testes were used as positive controls because sperm are a rich source of tubulin (Mohri 1968). Negative controls were treated as above, minus incubation with primary anti-tubulin antibodies. For Western blot, 0.3 g tests were homogenized in 1 ml lysis buffer (0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, 5% glycerol, pH 7.4) on ice with 3.6 μl protease inhibitor (Sigma), centrifuged at 13 000 × g for 10 min in a refrigerated centrifuge, decanted, and proteins quantified with Pierce BCA assay (Thermo-Fisher). Homogenates (20 μg protein per well) were resolved on denaturing reducing electrophoresis on 10% gels, and proteins transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat dried milk in Tris buffer for 1 h, washed 3 times, probed with rabbit anti-tubulin (Abcam, Waltham, MA) at 1:1000 in 3% milk-Tris buffer overnight at 4°C, washed 3 times, and incubated with HRP-conjugated goat-anti-rabbit polyclonal antibodies (Southern Biotech) diluted 1:1000 in 3% milk-Tris buffer for 1 h. After 3 washes, membrane was incubated with diaminobenzidine. Positive control was HeLa cell lysates as recommended by the antibody manufacturer (Abcam).

Statistics
We used chi-square test to compare test inflammation scores between oral and aboral tests for animals where both regions were sampled. We used ordinal regression to see if test inflammation score, gut fill score, test diameter, and percent coelomic fluid volume differed significantly (p < 0.05) among gross lesion severity score categories. All analyses were done with R Version 4.0.5 (R Core Team 2017) using the MASS package (Venables & Ripley 2002).

Field surveys and scoring
On 6 May 2014, 60 of 2070 (3%) Tripneustes gratilla had lesions on 3 patch reefs surveyed in KB. Follow up surveys on 28 May 2014 revealed 16 of 1520 (1%) urchins with lesions. Numbers of urchins at the original outbreak site (barge) declined during the outbreak and numbers remained low thereafter, whereas numbers at nearby Z-blocks were invariant in 2016 (Fig. 3)

Testes
Presence of algae 0 (0) 0 (0) 0 (0) 2 (9) coelomocytes in gut mucosa, and invasion of testes by algae (Fig. 6F) (Table 1). Electron microscopy of large eosinophilic inflammatory cells in the test showed them to be distended with intracytoplasmic accumulations of variably sized tubules without a core and of variable electron density (Fig. 7A), measuring about 90 nm in diameter, and often associated with membranes (possibly ly sosomes) (Fig. 7B). Inclusions in nuclei of gut cells (Fig. 7C) were square or rhomboid, and composed of arrays of small dots (Fig. 7D).

Statistics
Although the total histology inflammation severity score for oral test (71) was about 50% higher than that for aboral test (48), the relationship between severity scores between aboral and oral test by Fischer's exact test was not significant (p > 0.05). Ordinal regression revealed that gross lesion severity score significantly influenced percent weight coelomic fluid and gut fill score, whereas no significant relationship existed for test inflammation score or test diameter (Fig. 8, Table S1). With increasing gross lesion score, more animals with empty guts were ob served, and percent coelomic fluid increased (Fig. 8).

DISCUSSION
The causes of most urchin mortalities published in the literature remain enigmatic. Although microorganisms like bacteria are commonly implicated as causes of urchin deaths (Wang et al. 2013), diagnostic efforts to confirm this in the form of systematic exams of tissues at the microscopic level are uncommon. Williams et al. (1996) documented unusual mortalities of Tripneustes ventricosus in Puerto Rico in 1995 with gas-filled urchins floating at the ocean surface and losing their spines. An earlier mortality of the same species in the same region in 1993 manifesting loss of spines was attributed to increased water temperature (Colon-Jones 1993) and in 1968, again in Puerto Rico, Glynn (1968 documented mortalities of T. ventricosus associated with low tides and excessive high water temperature. In   (2021), we were unable to determine the ultimate cause of the 2014 T. gratilla die-off despite extensive examinations of tissues. How ever, our exercise was important because it sheds insights on host responses of T. gratilla and pathology in echinoderms from mortality events. Balding and spine loss in sea urchins are non-specific clinical signs illustrating that urchins, like other echinoderms, have a limited host response re pertoire, a point made by others (Vir wani et al. 2021). For instance, sea star wasting disease manifests as epidermal ulceration, a symptom also associated with temperature anomalies (Aalto et al. 2020) and deposition of organic matter on the surface of the starfish leading to anaerobic conditions and tissue wasting (Aquino et al. 2021, Work et al. 2021. Urchins with more severe gross lesions had higher likelihood of having empty guts and higher volumes of coelomic fluid. Whether empty guts and increased coelomic fluid volume lead to test lesions or vice versa cannot be determined from this study. Without monitoring and sampling sick urchins over time, conclusions on how morbidity progressed and the sequence of lesion development are limited. Moreover, findings in this study did not reveal in - fectious agents (viruses, bacteria, parasites, fungi), at least visible on light and electron microscopy, as responsible for this outbreak, but rather suggests an environmental or potentially toxic cause.
Most of the urchins in this die-off had reduced stomach contents indicating that food limitation might have contributed to this mortality event. In the field, this would presumably be manifested by high density of urchins followed by population crashes. For instance, in kelp forest ecosystems, grazing urchins go through boom-bust cycles with populations increases, overgrazing of kelp, and subsequent population crashes (Scheibling 1986). However, this seems to be an exception, and in general, many echinoderms can maintain very high spatial densities in absence of food for long periods (Andrew 1989). Conversely, in seagrass beds in the Caribbean, T. ventricosus converged on a food-limiting density of 1 urchin m -2 (Keller 1983). On the island of Hawai`i, densities of T. gratilla did not exceed 1 urchin m -2 (Ebert 1971). The surveys of the outbreak site and a nearby site in 2014 and 2016 showed a steep decline of urchins at one site (Z blocks) during the time of the outbreak, indicating that the mortality had some measurable demographic effect with no evident recovery of populations. However, densities were well below 0.5 urchins m -2 . No survey data was collected prior to the epizootic, so it is im possible to definitively Urchins with more severe gross lesions had a higher volume of coelomic fluid relative to apparently normal animals. This indicates that sick urchins were unable to modulate coelomic fluid volume. Some aspects of coelomic fluid regulation in echinoderms are known. In the sea star Pisaster ochraceus, the madreporite, a specialized structure through which an interchange between seawater and coelomic fluid oc curs, is important in maintenance of coelomic fluid volume (Ferguson 1992). When the sea urchins Lytechinus variegatus, Echinometra lucunter, and Arbacia lixula are immersed in freshwater, the coelomic fluid osmolality decreases (Santos et al. 2013); unfortunately, fluid volume was not measured in that study. In the urchin L. variegatus, the movement of the lantern, jaws, and associated muscle are important in circulation of coelomic fluids (Hanson & Gust 1986). Studying Strongylocentrotus purpuratus, Ellers & Tel ford (1992) found that coelomic fluid pressure fluctuated rhythmically and that pressure did not differ be tween starved and fed urchins; again, fluid volume was not measured. Presumably, higher volume of coelomic fluid in sick urchins would lead to greater pressure, but coelomic fluid pressure was not measured in this study and therefore, no conclusions could be made. Given the epidermal ulceration and inflammation of the test seen in T. gratilla in the current study, and considering the aforementioned studies, a combination of madreporite dysfunction and loss of epidermal barrier likely conspired to prevent adequate coelomic fluid regulation in sick urchins. Future studies could examine the madreporite microscopically to see if additional clues can be gained as to the nature of this inability to maintain coelomic fluid balance in sick T. gratilla.
About 10% of urchins had foreign organisms in their coelomic fluid including bacteria, diatoms, or other algae visible on light microscopy. Evidence for the sterility of coelomic fluid in echinoderms is mixed. For instance, Unkles (1977) cultured a few bacteria from coelomic fluid of Echinus esculentus but judged those to be contaminants from gut flora,  For instance, microscopic lesions were seen in apparently normal sea stars when epidermal lesions were experimentally induced (Work et al. 2021). The most substantial microscopic lesions were seen in the test and manifested in varying degrees of inflammation, occasionally associated with algal incursions into urchin tissues. The test comprises 52-67% of the body weight of T. gratilla (Bangi & Juinio-Meñez 2019), explaining why pathology is most often seen in this tissue in urchins (Virwani et al. 2021). Roberts-Regan et al. (1988) observed in S. droebachiensis more epidermal abrasion in the oral test but had no explanation for this. Although inflammation score in oral test was almost 2 times higher than aboral test, no significant difference was detected. The oral and aboral test of urchins show anatomical and physiological differences. In S. intermedius, calcification occurs more rapidly in aboral test (Koba yashi & Taki 1969). In S. droebachiensis, oral podia used for walking are thicker than aboral podia which are used for capturing debris (Leddy & Johnson 2000). How these differences could explain tropism for inflammation in oral test of T. gratilla is unclear.
In this study, the large granular inflammatory cells seen in T. gratilla were distended with large (90 nm diameter) intracytoplasmic tubules that stained negative for DNA and RNA, indicating they were not viruses, but stained positive with PAS, showing presence of polysaccharides (Prophet et al. 1992). The diameter of these tubules (ca. 90 nm) was larger than microtubules (25 nm) (Gall 1966) composed of the protein tubulin, which we found in T. gratilla spermatozoa. Whether tubulin was present in inflammatory cells of this study is unclear; however, the protein has been detected in coelomocytes of other urchin species (Edds 1984, D'Andrea-Winslow & Novitski 2008. Intracytoplasmic tubules have been seen by electron microscopy in coelomocytes of other invertebrates, including snails (Matricon-Gondran & Letocart 1999, Martin et al. 2007) and limpets (Harris & Markl 1992), but these were far smaller (25 nm diameter) than what was observed here and were more compatible with microtubules. Electron microscopy of coelomocytes from apparently normal T. gratilla sampled after this mortality event failed to reveal such tubules, indicating they are not normal (Work et al. 2020). Given that no evidence of viruses was seen on electron microscopy, future studies might focus on the role of toxins or metabolic abnormalities as the cause of tubular structure accumulation in urchin cells.
Other microscopic changes such as ciliates in the gut and intracytoplasmic inclusions were considered incidental findings. Virwani et al. (2021) saw similar findings in T. ventricosus in St. Kitt's that they also considered as incidental. Although it was not confirmed with special stains (Virwani et al. 2021), it is likely that these inclusions are iron crystals, which have been seen in urchins elsewhere (Borges et al. 2010, Branco et al. 2012. Unlike Virwani et al. (2021), no commensal bacterial aggregates were observed in urchins examined in this study.
Although an infectious etiology was not found in this mortality event, and the event did not lead to landscape-scale declines in urchins, it highlights the current lack of knowledge about the health of this important group of organisms. From a disease and biosecurity standpoint, managers might reconsider the practice of translocating urchins with more careful health as sessments to ensure that urchins being translocated are as healthy as possible. Gross exami-nation of animals alone may not be sufficient to assess urchin health as evidenced by the presence of cellular pathology in apparently normal urchins seen here, a pheno menon previously observed in urchins (Virwani et al. 2021) and other marine invertebrates like corals (Lands berg et al. 2020). In addition to potentially moving infectious agents, translocating urchins could lead to introduction of invasive marine algae because algal spores can survive digestion in sea urchins (Santelices et al. 1983). Comprehensive guidelines exist to enhance biosecurity during trans location of invertebrates (Cunningham 1996, Hartley & Sainsbury 2017. Applying these to marine in vertebrates in might lead to more successful reintroductions.