Ultrastructural and Elemental Characterization of the Extracorporeal Tube of the Sessile Rotifer Floscularia Conifera (Rotifera: Gnesiotrocha)
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Accepted: 14 August 2018 DOI: 10.1111/ivb.12230 ORIGINAL ARTICLE Ultrastructural and elemental characterization of the extracorporeal tube of the sessile rotifer Floscularia conifera (Rotifera: Gnesiotrocha) Hui Yang1 | Rick Hochberg2 1College of Sciences, University of Massachusetts Lowell, Lowell, Abstract Massachusetts Rotifers 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 Floscularia ringens, 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 animals 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 Flosculariidae. 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 Limnias melicerta WEISSE 1848 (Cubitt, 1871; Wright, 1954; Yang as well as the animal 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.