Accepted: 14 August 2018

DOI: 10.1111/ivb.12230

ORIGINAL ARTICLE

Ultrastructural and elemental characterization of the extracorporeal tube of the sessile conifera (Rotifera: Gnesiotrocha)

Hui Yang1 | Rick Hochberg2

1College of Sciences, University of Massachusetts Lowell, Lowell, Abstract Massachusetts are aquatic microinvertebrates that live in the plankton or in the benthos, 2Biological Sciences, University of which may include a variety of macrophytes. Among these periphytic forms of roti- University Lowell, Lowell, Massachusetts fers, some have taken up a sessile existence and secrete protective tubes around Correspondence their bodies. One type of tube common to species of Floscularia is made of small Hui Yang, College of Sciences, University of Massachusetts Lowell, Lowell, MA. round pellets. To date, the building process and some fine structural details are Email: [email protected] known for , but many questions about the composition of the tube Funding information and its ultrastructure still remain unanswered. Here, we use transmission electron NSF (National Science Foundation), Grant/ microscopy and scanning electron microscopy–energy dispersive X‐ray spectroscopy Award Number: DEB 1257110 (SEM–EDS) to study the ultrastructure of the pellets and their elemental composi- tion, respectively, in the putative sister species, Floscularia conifera. We revealed several new details that add important information about the physiology of tube‐ making in species of Floscularia. First, we note an inner secretory membrane that is thin, electron lucent, and supports the external pellets. The pellets are relatively consistent in size and have a small depression on their inner surface. All pellets are individually wrapped in a secretory membrane that completely encapsulates sus- pended materials collected from the surrounding water. Elemental signatures of pel- lets reveal they consist mostly of carbon (C), nitrogen (N), and oxygen (O), with some silicon (Si) content that is likely the result of diatom shells. Other trace ele- ments such as iron (Fe) and sodium (Na) are also present and likely the result of incorporated bacteria and suspended materials. When larval rotifers are cultured in filtered pond water, the pellets consist mostly of C and O, with little N and no Si; Fe is present in smaller amounts. These new discoveries provide a better under- standing of the physiology of rotifer tube construction and tube composition, and their future utility in understanding if and how changes in freshwater environments might impact these factors.

KEYWORDS allochthonous, pellets, secretion, sessile, silicon

1 | INTRODUCTION well‐studied (Segers, 2007), but the majority of which are benthic periphytic forms. In freshwater systems, rotifers are present from Phylum Rotifera comprises ~2,000 species of mostly freshwater the littoral to the limnetic zones and form an important part of the microinvertebrates, of which the planktonic species are particularly microbial loop (Wallace, 2009). Rotifers are characterized in part by

| Invertebrate Biology. 2018;1–10. wileyonlinelibrary.com/journal/ivb © 2018, The American Microscopical Society, Inc. 1 2 | YANG AND HOCHBERG a ciliated region (corona) on top of the head, which functions for 2 | METHODS both locomotion and food gathering (Wallace, 2009). While most rotifers use the corona for these functions, females in a subset of Specimens of F. conifera were collected from submerged plants (spe- rotifer species have evolved to be sessile during their adult life stage, cies of Utricularia) in Flint Pond in Tyngsboro, MA (42°40′19.2″N, which limits the use of the corona to locomotion in the larval stage 71°25′55.2″W) and Naticook Lake in Merrimack, NH (42.819914, and food gathering in the adult stage (Edmondson, 1944, 1945; Wal- −71.525776). Individual and colonies were identified with lace, Snell, & Ricci, 2006). These sessile species belong to the three brightfield and DIC microscopy on a Zeiss Axioskop A1, and pho- families in the superorder Gnesiotrocha: Atrochidae, Collothecidae, tographs were taken with a Sony digital Handycam camera. After and . In these rotifers, larvae are strictly nonfeeding identification, specimens were anesthetized with drops of 0.5% bupi- dispersal stages; upon settlement and permanent attachment, larvae vacaine in a finger bowl of pond water (~5 ml). metamorphose to the adult morphology and produce a tubular For SEM, anaesthetized specimens were fixed in 2.5% glutaralde- sheath of extracellular secretions around their bodies that may func- hyde in 0.1 M phosphate buffer (PB; pH 7.3) for 24 hr and rinsed in tion in defense, camouflage, or both (Wallace, 2009). The morphol- 0.1 M PB for 1 hr. The specimens were transferred to BEEM cap- ogy of these tubes can be divided into three broad categories: a sules with 42‐μm mesh ends and dehydrated in an ethanol series transparent mucus sheath (hydrogel), known from several species in (50%, 70%, 90%, 100%, 10 min each) followed by critical point dry- all three families; a somewhat hardened tube constructed of pseudo‐ ing in a Tousimis SamDri PVT‐3D (Hochberg et al., 2015). Dried fecal pellets deposited on a thin gelatinous layer, as in Floscularia rin- specimens were subsequently placed on carbon‐coated SEM stubs gens LINNAEUS 1758 (Cubitt, 1872; Wright, 1950); and a hardened and approximately half of them were carefully pried open by OOO hyaline tube with a uniquely ringed pattern, which is characteristic insect pins to expose the morphology of the inner side of the tube, of melicerta WEISSE 1848 (Cubitt, 1871; Wright, 1954; Yang as well as the that lives inside the tube. Samples were then & Hochberg, 2018). coated with gold using a Leica SCD500 Sputter Coater before Wright (1950) described the tubes of individuals of F. ringens inspection on a JEOL JSM 7401 Field‐emission Scanning Electron and their building process, which was later elaborated on by Fon- Microscope. SEM‐EDS analysis was conducted on the specimens taneto, Melone, and Wallace (2003). In short, free‐swimming larvae using the EDAX Genesis XM2 Imaging System, which includes a settle on submerged plants, attach with a permanent adhesive, 10‐mm2 Si(Li) detector with SUTW window. Images and spectra and then secrete a translucent gelatinous base around their foot. were collected at 15 kV with a probe current of 15 and emission The larvae grow gradually and soon begin making small pellets current of 10 μA. inside a specialized organ on their corona called the modulus Adult specimens were cultured in glass bowls of pond water for (Wright, 1950). The pellets appear to be a combination of secre- 5 days to produce eggs and swimming larvae. The culture was tions from the rotifer and suspended materials from the surround- observed every 8 hr and individual larvae were collected and placed ing water; all pellets are of a similar size and packed tightly in small bowls (~10 ml) of filtered pond water (filter size 0.22 μm) to together to form a tubular monolayer in the shape of a chimney test whether larvae without access to suspended (allochthonous) that surrounds the animal. The tube is not directly connected to particles were capable of building a tube. Juvenile tubes were the animal, so the rotifer is free to move within the tube without collected and examined by SEM and SEM‐EDS following the same affecting tube shape or size. procedure as with the adults. The most recent study on F. ringens revealed features that For TEM, specimens were originally fixed for SEM and then were previously undetected by the light microscopical research of post‐fixed in 1% OsO4 in 0.1 M PB for 1 hr. The specimens were Wright (1950): an underlying membrane that supports the pellets, rinsed in PB for 1 hr, dehydrated in an ethanol series as in SEM, and a small depression on the back of each pellet, and a potential slowly infiltrated by an ethanol : resin mixture (Low Viscosity secretory groove on the animal (Fontaneto et al., 2003). Since Embedding Kit by Dr. Spurr; Electron Microscopy Sciences) in ratios the study of Fontaneto et al. (2003), there has been no further of 2:1, 1:1, 1:2 for 2 hr, respectively, and then in pure resin over- research on the exquisite tubular architecture of pellet‐forming night. The specimens were then removed and embedded in pure rotifers, and so it remains unknown if related species make simi- resin, cured at 60°C for 24 hr, and sectioned at 70–90 nm on a Leica lar tubes. In this study, we examine a closely related species, EM UC6 Cryo‐Ultramicrotome. Sections were stained with uranyl

Floscularia conifera (HUDSON 1886), which also makes extracorpo- acetate and lead citrate before examination on a Phillips CM10 elec- real tubes of pellets. We use scanning electron microscopy tron microscope with Philips Electron Optics chromatically corrected (Klusemann, Kleinow, & Peters, 1990) to characterize tube mor- objective lens at the University of Massachusetts Medical School in phology, transmission electron microscopy (Hochberg, Hochberg, Worcester, MA. Brightness and contrast were adjusted in Adobe & Chan, 2015) to examine pellet ultrastructure, and energy dis- Photoshop CS6 without any further graphic change. Measurements persive X‐ray spectroscopy (SEM‐EDS) (Spada et al., 2001) to were made on digital photos using IMAGEJ 1.51h. Two SEM pictures examine the elemental signatures of the individual pellets. This were used to measure general tube size. Three SEM pictures of the study is the first examination of both pellet ultrastructure and tube near the top, middle, and base were used to measure pellet elemental composition. diameter. Three TEM pictures (top, middle, and base of the tube) YANG AND HOCHBERG | 3 were used to measure the thickness of the underlying membrane. layers were artifacts of preparation or were natural variation in the SEM‐EDS was conducted at 45 sites on an adult tube and 30 sites tube that perhaps showed a specific pattern along the tube's length. on a juvenile tube. For each measurement, average value and stan- Pellet shapes vary from approximately elliptical (Figure 2C) to dard deviation were calculated using MS Office Excel. rectangular (Figure 3C, outlined), with one end attached to the most external layer of the fibrous membrane (layer 2). The pellets them- selves were also encased in a dense secretion (Figure 3B,D, layer 3). 3 | RESULTS Each pellet was composed of contents with different staining prop- erties: some materials were electron lucent, while others showed 3.1 | Light microscopical observations various levels of electron density (Figure 3D, transparent regions and Specimens of F. conifera were present as colonies on submerged dark regions). Most pellets appeared to be fibrous or spongy in tex- vegetation (Figure 1A). Colonies reached to 3–4 mm long and con- ture. Some particles resembled phytoplankton based on the presence sisted of numerous individuals, some of which were juveniles that of extensive plastids (Figure 3C inset), while others appeared to be settled close to the top of adult tubes (i.e., type II colonies of Wal- bacteria that were present in an empty space that was itself encap- lace, 1987) (Figure 1B). The pellets of all tubes had a variable shape, sulated by pellet matrix (Figure 3E–G). These identifications are pre- with some appearing almost hexagonal and others rectangular in liminary and only based on ultrastructural observations. shape (Figure 1C). All appeared to have a depression in their center (Figure 1C). The pellets formed a monolayer and appeared to rest on 3.4 | Elemental composition of the tube a membrane on the inside of the tube (Figure 1D). Our SEM‐EDS analyses are based on nonpolished samples with irreg- ular surface topography, which translates into less precise quantitative 3.2 | Surface structure of the tube measurements (Goldstein et al., 2018). Data are reported as normal- Extracorporeal tubes were roughly cylindrical in shape (mean and SD ized percentages (weight% or atom%) of the total elements being 933 ± 351 μm in length and 92 ± 17 μm width, n = 5) and com- quantified; we considered elements >1% to be of significance posed of numerous pellets. Each pellet was 11.8 ± 0.7 μm in diame- because they were above noise levels (Gallant & Hochberg, 2017; ter (Figure 2, n = 30). The pellets were packed tightly together on Gallant, Hochberg, & Ada, 2016). Because of time and cost con- the external surface of the tubes. Each pellet was a mixture of parti- straints, our studies were restricted to observations of multiple pellets cles of variable size and shape: some appeared fibrous, others on only two tubes: an adult tube that was secreted in native pond appeared crystalline (Figure 2B left inset, arrow), and some resem- water (isolated in a glass bowl) and a juvenile tube that was secreted bled fragments of diatom frustules (Figure 2C left inset, arrow). Pel- in 0.22‐μm filtered pond water (isolated in a glass bowl) (Figures 4 lets that composed juvenile tubes had no sign of incorporated and 5). Pellets in both tubes were mostly composed of carbon (C) and particles and had a less smooth surface (Figure 2B right inset). On oxygen (O) (Figure 5). Weight percentage of C ranged 22%–39% the inner side of the tube, there was a thin membrane that appeared (28.6 ± 4.7%) in the adult tube and 46%–63% (54.6 ± 4.6%) in the to support the pellets (Figure 2C,D). This membrane had a fibrous juvenile tube. The O weight percentage was 31%–37% (33.7 ± 2%) in texture under high magnification (Figure 2D). There was also a deep the adult tube and 20%–26% (23 ± 1.7%) in the juvenile tube. Nitro- hollow on the backside of each pellet (Figure 2D inset). gen (N) weight ranged 3%–5% in the adult tube (4.1 ± 0.6%) and 5%– 13% in the juvenile tube (9.1 ± 3%). Silicon (Si) was present only in the adult tube (range 16%–27%, mean 22.3 ± 3.9%). A low amount of 3.3 | Ultrastructure of the tube sodium (Na) was detected in both tubes: 1%–2% (1.6 ± 0.2%) in the Cross‐sections of adult tubes revealed a thick (116.3–1,486 nm), adult tube and 2%–5% (3.5 ± 1%) in the juvenile tube. Iron (Fe) was inner gelatinous matrix (layer 1, Figure 3A, arrowhead) beneath a present in the adult tube at 4%–14% (8.2 ± 3.1%) and in the juvenile thinner outer membrane that was connected to the overlying pellets tube at 6%–13% (9.7 ± 2.2%). There was also a low amount of arsenic (Figure 3A, arrow). The thick gel matrix was mostly observed near (As) in the adult tube (1.4 ± 0.8%). These metal elements appeared in the foot of the animal and did not appear to be present throughout a relatively wide range of values around a low mean percentage, sug- the entire tube, although complete serial sections of tubes were not gesting that they may come from the debris on the tube rather than examined. The gel appeared fibrous and had a consistent electron from the pellets themselves. Aluminum (Al) was not included in the density throughout (Figure 3A inset). The external membrane that weight percentage calculation since it amounted to <1% of element abutted the pellets was electron dense (Figure 3B,D; layer 2). It was composition and was thus considered to be background noise. Both continuous throughout the entire tube and had a thickness of 97.7– samples were coated in gold (Au), so Au signals were also excluded 586.5 nm (mean 202.2 ± 79.2 nm; n = 62). The inner layer (layer 1) from the element percentage evaluation. Composition of adult and was more electron lucent and also highly variable in thickness juvenile tubes differed significantly in percentages of C, N, O, Na, Si, (116.3–1,485.8 nm). This layer was excessively thick in some regions and As (p < 0.01, two‐tailed type 2 Student's t‐test); there was no sig- (e.g., Figure 3C, layer 1) and absent in some other regions (e.g., Fig- nificant difference between adult and juvenile tubes in percentage of ure 3D). We could not determine whether the thicknesses of these Fe (p=0.22). 4 | YANG AND HOCHBERG

FIGURE 1 Light microscopy of live specimens of Floscularia conifera.A,Colony growing on a lily pad (upside down) next to a species of Hydra (Cnidaria). B, Close up of a portion of a different colony isolated from its plant substratum. A newly settled juvenile is indicated with an arrow. C, Pellets of an adult's tube showing their variable shape and apparently hollow center. D, Focus through an adult's tube revealing the monolayer of pellets against a thick membrane (arrowhead). The adult foot (ft) and egg (eg) are seen inside the hollow of the tube. Scale bars: A = 1.5 mm; B = 100 μm; C = 45 μm; D = 40 μm YANG AND HOCHBERG | 5

FIGURE 2 External structure of a tube from an individual of Floscularia conifera. A, SEM of an adult tube with three juvenile tubes attached to it. B, Higher magnification of the tube pellets. Inset (left): cracked pellet showing the particles wrapped inside. Inset (right): juvenile tube that was cultured in filtered water. The pellets have relatively rough surface and no sign of encapsulated material. C, Inside view of the tube, showing the membrane under the pellets. Inset: higher magnification of the membrane and pellet. D, Inside view of the tube under higher magnification, showing the fibrous membrane. Inset: backside of a single pellet, showing a big hollow space. Scale bars: A = 100 μm; B,C,D = 10 μm; all insets = 2 μm

4 | DISCUSSION environment (Fenaux, 1986; Flood, 2003). In other animals, such as

the sessile freshwater bryozoan Pectinatella magnifica LEIDY 1851 The secretion of extracorporeal tubes is not exclusive to rotifers. (Ectoprocta: Phylactolaemata), organic materials are not actively Many aquatic invertebrates produce extracorporeal secretions incorporated but rather settle on or in the secretion matrix. For around their bodies that function for protection and feeding (Bone, example, cyanobacteria and algae often use the thick gelatinous Carré, & Chang, 2003; Flood, 2003; Stewart, Wang, Song, & Jones, matrix as a source of nutrients (Šetlíková, Skácelová, Šinko, Rajchard, 2017). In some cases, these secretions include a variety of endoge- & Balounová, 2013). Some groups of animals and their secretions nous proteins (Stewart et al., 2017) that function as a cement for have been well‐studied, but to date, there are few studies on the allochthonous materials collected from the environment. For exam- extracorporeal secretions of rotifers (e.g., Fontaneto et al., 2003; ple, in the sessile marine annelid Sabellaria alveolata LINNAEUS 1767 Yang & Hochberg, 2018), and we have little understanding of the (Annelida: Canalipalpata), inorganic materials such as sand grains and function of these secretions or their ecological significance. shell pieces are normally included to add rigidity and strength to the The presence of tubular secretions in rotifers has been known tubular secretion (Dubois, Barillé, Cognie, & Beninger, 2005). In spe- for more than a century (Cubitt, 1872), and some tubes have been cies of Oikopleura (Chordata: Tunicata), the gelatinous house is made observed in detail under light microscopy (Hudson & Gosse, 1886; of cellulose and functions for filter feeding, resulting in the accumu- Wright, 1950, 1954). Despite the grand diversity of their morpholo- lation of suspended inorganics and organics from the planktonic gies, all tubes can be classified by their general appearance or 6 | YANG AND HOCHBERG

FIGURE 3 TEM of tube pellets from an individual of Floscularia conifera. A, Longitudinal section of a tube. A membrane (arrow) and a gel matrix (arrowhead) were observed. Inset: high magnification of the gel matrix. B, Higher magnification of the gel matrix, the underlying membrane, and the encasing membrane. C, Cross‐section of pellets. The outlined region is an individual pellet. Inset: higher magnification of the embedded particle that resemble a dinoflagellate cell. D, Higher magnification view of the region where the pellet is attached on the membrane. The irregular edge between the pellet and the membrane marks the surface of the pellet. E, F, G, Particles that resemble bacteria. Scale bars: A,C = 2 μm; B,D,E‐G = 200 nm; all insets = 200 nm. ex, exterior; in, interior; ly1, layer 1; ly2, layer 2; ly3, layer 3; pt, pellet YANG AND HOCHBERG | 7 composition into three main types: tubes composed of a gelatinous present in species with purely gelatinous tubes. Perhaps what makes hydrogel‐like matrix (e.g., Stephanoceros fimbriatus GOLDFUSS 1820); the pellet tubes of species of Floscularia so unusual (relative to the hardened pipe‐like tubes (e.g., L. melicerta); and tubes composed of a two other forms) is that they represent an amalgamation of native mixture of pellets from adhesive secretions and allochthonous parti- secretions and non‐native (allochthonous) particles that are built into cles (e.g., F. ringens). Without any knowledge of the phylogenetic the tube like bricks on top of a prelaid gel base (Wright, 1950). relationships among these tube builders, and with limited knowledge Based on the light microscopical observations of Hudson and of tube composition and secretion in general, it is impossible to Gosse (1886) and Wright (1950), and with more recent details pro- know whether the different tubes represent different grades or vided by the SEM observations of Fontaneto et al. (2003) and the clades of tube evolution. In spite of that uncertainty, at least two of SEM/TEM observations of our current study, we find that the pellet the tube types, the hardened pipe and the pellet tube, appear to tubes of F. ringens and F. conifera share several fine morphological require a level of behavioral sophistication that is not obviously details: all pellets appear to consist of native secretions and

FIGURE 4 SEM‐EDS analysis of tubes from individuals of Floscularia conifera. A, SEM of an adult tube. Different spots were examined for elemental components (arrows). B, SEM of the juvenile tube showing the spot that was analyzed (arrows). C, SEM‐EDS spectrum of a pellet near the middle of the adult tube, showing the element signals detected at the spot. Scale bars: A = 100 μm; B = 10 μm 8 | YANG AND HOCHBERG

FIGURE 5 Average weight percentages of elements in pellets from tubes made by individuals of Floscularia conifera. Both juvenile and adult tubes mainly consisted of C, N, O, Na, and Fe. Si and Al were only detected in the adult tube. Other than the presence of a large amount of Si, there was also an increase in O percentage in the adult tube, while the C:N ratio remained unchanged between the two tubes (~8:1). Al and Au were not included in the element quantification analysis suspended particles encased in a secreted membrane; all pellets here is not to make specific comparisons to detailed studies of eco- have a central depression of unknown origin on their inner side; logical stoichiometry, but to get a better understanding of the gen- and all pellets adhere to a gelatinous base. However, we note eral composition of these pellets relative to what might be present some minor differences between the species. Compared to those in in surrounding waters (if these waters were to be measured). Per- F. ringens (Fontaneto et al., 2003), the pellets in individuals of F. haps more interesting than these putative ratios are the following conifera are more elongate and with a rougher surface that some- three observations: (a) we detected low levels of several metals, times reveals the identities of the encapsulated foreign materials including Fe, As, and Na; (b) there was no Si (metalloid) detected in (Figure 2B,C). Importantly, these materials were not observed in the juvenile tube from filtered water; (c) the amount of O increased the juvenile tube pellets when individuals of F. conifera were cul- along with the appearance of Si in the adult tube; and (d) Fe was tured in filtered water. Also, the inner gelatinous base of tubes in present in both adult and juvenile tubes, although slightly less in the F. conifera is composed of two layers; without TEM observations, adult. it is unknown how many layers makes up the base of tubes in Without knowledge of elemental compositions of tubes from F. ringens. other rotifers (e.g., specimens collected from other sites, or other In addition to these morphological observations, we also species such as F. ringens), it is difficult to know the reasons behind applied TEM and SEM–EDS to further explore the potential chem- our observations, but we offer the following hypotheses. (a) The low ical and elemental composition of the tubes respectively. Based percentages of Na, As, and, potentially, Al (although in amounts not on observations with the double contrast method of staining (ura- significantly different from background noise) on the tubes, and their nyl acetate and lead citrate) and osmium post‐fixation, we note high variability across pellets, suggest that they are not permanently that all native secretions are distinctly fibrous and likely to consist incorporated in the pellets, but instead merely attached to the sur- of glycoproteins (Bansil & Turner, 2006, 2018; Lang et al., 2016). face. The source of these metals may be dissolved compounds in And while there are clear differences between the inner and outer the native waters (adult tubes) or sediments that can readily pass matrices (layer 1 and layer 2), we cannot distinguish them any fur- through the 0.22‐μm filter to accumulate on the juvenile tubes as ther without differential (single) staining, which was not performed well (e.g., Mandal & Suzuki, 2002). (b) Si is associated with O and in this study. The composition of the pellets based on staining is exists in the form of silica. It is attributed to the diatoms we even more difficult to determine because of the highly variable observed in many pellets of adult tubes, but we cannot rule out nature of the contents. Immediately beneath the encasing mem- other potential sources such as sand grains (Figure 2B) that appeared brane of each pellet (Figure 3D, layer 3), there is generally an to be present in some pellets. (c) The higher percentage of Fe com- electron‐lucent region with lightly stained fibers surrounding a pared to other metals suggest at least two possible origins. One large amount of amorphous, electron‐dense, flocculent material, in source might be particulate (<0.22 μm) or colloidal iron (0.025–0.22 which may be embedded bacteria, diatoms, and perhaps other μm), the latter of which is apparently reactive and can coagulate or (previously) living organisms, as well as suspended organic and become soluble (Xing & Liu, 2011). Other sources could be the phy- inorganic debris (Figure 3E–G). toplankton, which can sequester iron (Xing & Liu, 2011), or bacterial The SEM‐EDS analyses provided some additional resolution on cells, which electrostatically bind metals and serve as nucleation sites the composition of the pellets, at least at the elemental (but not for crystal growth (Konhauser, 1998). With these hypotheses in compound) level. The pellets of both adult and juvenile tubes are mind, we think that future studies of F. conifera collected from dif- mainly composed of C, N, and O, with similar C : N ratios (8:1 in ferent environments and cultured under different laboratory condi- adults and 8.3:1 in juveniles). Importantly, these ratios are based on tions might be one way to parcel out the real sources of these relatively few observations, and moreover, the quantitative accuracy elemental signals and provide further insights into the potential size of SEM‐EDS based on nonpolished surfaces is prone to error and and chemical selection of allochthonous materials made by tube‐ should be considered inexact (Goldstein et al., 2018). Our intention building rotifers. YANG AND HOCHBERG | 9

Our study of the tubes of two individuals of F. conifera has Cubitt, C. (1872). Remarks on the homological position of the members revealed novel details about tube structure and composition, but we constituting the thecated section of the class Rotatoria. Monthly Microscopical Journal, 8,5–12. https://doi.org/10.1111/j.1365-2818. did not attempt to identify the source(s) of the underlying secretions, 1872.tb02160.x which would be a more significant undertaking. It is known that the Dubois, S., Barillé, L., Cognie, B., & Beninger, P. G. (2005). Particle capture modulus organ of the corona is the source of at least some of the and processing mechanisms in Sabellaria alveolata (Polychaeta: Sabel- secretion (perhaps layer 3), but the source of the thick gelatinous lariidae). Marine Ecology Progress Series, 301, 159–171. https://doi. org/10.3354/meps301159 matrix remains unknown. Fontaneto et al. (2003) speculated that the Edmondson, W. T. (1944). 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Invertebrate Biology, 136, 345– 359. https://doi.org/10.1111/ivb.12187 ACKNOWLEDGMENTS Gallant, J., Hochberg, R., & Ada, E. (2016). Elemental characterization of the cuticle in the marine intertidal pseudoscorpion, Halobisium occi- The authors thank the editor and reviewers for comments that sig- dentale. Invertebrate Biology, 135, 127–137. https://doi.org/10.1111/ nificantly improved this manuscript. We also acknowledge funding ivb.12123 by the National Science Foundation to support this research (DEB Goldstein, J. I., Newbury, D. E., Michael, J. R., Ritchie, N. W. M., Scott, J. 0918499 to R. Hochberg). Any opinions, findings, and conclusions or H. J., & Joy, D. C. (2018). Scanning electron microscopy and X-ray microanalysis (4th edn). New York, NY: Springer-Verlag. https://doi. recommendations expressed in this material are those of the authors org/10.1007/978-1-4939-6676-9 and do not necessarily reflect the views of the National Science Hochberg, R., Hochberg, A., & Chan, C. 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