bioRxiv preprint doi: https://doi.org/10.1101/2020.06.27.175059; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Colloblasts act as a biomechanical sensor for suitable prey in Pleurobrachia Townsend, JPa, 1, *, Merces, GOTb, c, *, Castellanos, GPd, and Pickering, Mb, c aDepartment of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104 bSchool of Medicine, University College Dublin, Belfield, Dublin 4, Ireland, D04 V1W8 cUCD Centre for Biomedical Engineering, University College Dublin, Belfield, Dublin 4, Ireland, D04 V1W8 dDepartment of Biology, University of Puerto Rico at Cayey, Cayey, Puerto Rico 00736 1Corresponding author *These authors contributed equally to this work Summary statement Most species in the phylum fall within the class Tentacu- Ctenophore colloblast adhesive is found to be strong, but few lata (Haddock, 2007), characterized by the presence of ten- colloblasts are simultaneously active, producing a weakly- tacles whose surface is covered with a type of cell unique adhering system. A physical model demonstrates how such a to ctenophores—colloblasts (Franc, 1978; Haddock, 2007; system may filter unsuitable prey. Tamm, 2014; von Byern et al., 2010). The present study focuses on Pleurobrachia bachei and Pleurobrachia pileus, two closely related and morphologically similar ctenophores Abstract of the order Cydippida found in the inshore waters of the Pa- Ctenophores are a group of largely-planktonic, gelatinous cific and Atlantic oceans, respectively. Members of this or- carnivores whose most common method of prey capture is der possess a pair of tentacles with numerous, evenly-spaced, nearly a phylum-defining trait. Tentaculate ctenophores re- smaller tentilla extending perpendicularly off the main tenta- lease an unknown proteinaceous adhesive from specialized cle body. While hunting, cydippid ctenophores tend to keep colloblast cells lining their tentacles following prey contact their tentacles and tentilla extended, waiting for prey to drift with the tentacles. There exist no extant studies of the me- into the resulting dragnet (Haddock, 2007; Tamm, 2014). chanical properties of colloblast adhesive. We use live mi- When prey make contact with the tentacles, the colloblasts croscopy techniques to visualize adhesion events between release their adhesive and bond to the prey, tethered by “spi- Pleurobrachia pileus colloblasts and probes of different sur- ral filaments” to the tentacle body (Franc, 1978; von Byern face chemistries in response to probing with varying contact et al., 2010). Colloblasts have a bouquet-shaped morphol- areas. We further define two mechanisms of adhesion ter- ogy with an apical enlargement protruding from the tentacle mination upon probe retraction. Adapting a technique for surface. On contact with prey, the colloblast adhesive is re- measuring surface tension, we examine the adhesive strength leased from its storage place, likely a collection of internal of tentacles in the ctenophore Pleurobrachia bachei under vesicles, bonding the colloblast to the prey. This discharg- varying pH and bonding time conditions, and demonstrate ing action presumably destroys the colloblasts, so these one- the destructive exhaustion of colloblast adhesive release. We time use cells may be continually replaced by differentiation find that colloblast-mediated adhesion is rapid, and that the from epithelial stem cells. (Alié et al., 2011; Franc, 1978; bonding process is robust against shifts in ambient pH. How- Hernandez-Nicaise, 1991) ever, we find that the Pleurobrachia colloblast adhesive sys- Ctenophores’ hunting technique is reminiscent of the am- tem is among the weakest biological adhesive systems yet de- bush strategy of orb weaver spiders (Greene et al., 1986) scribed. We place this surprising observation into a broader and the release of an adhesive from colloblasts in response ecophysiological context by modeling prey capture for prey to prey-contact is superficially similar to the harpoon-like of a range of sizes. We find that limited use of colloblast stinging cells in cnidarians, called nematocytes or cnidocytes adhesive with high surface area contact is suitable both for (Holstein and Tardent, 1984; Kass-Simon and Scappaticci, capturing appropriately sized prey and rejecting, by detach- Jr., 2002; Nüchter et al., 2006; Tamm, 2014). Cnidocytes ment, prey above a certain size threshold. This allows Pleuro- and colloblasts do not share a common evolutionary origin brachia, lacking a mechanism to directly “see” potential prey (Babonis et al., 2018), and the use of adhesive-, rather than they are interacting with, to invest in capturing only prey of venom-loaded cells distinguishes them further. The attach- an appropriate size, decreasing the risk of injury. ment of colloblasts to an organ such as the tentacles as op- ctenophore | colloblast | adhesion | prey | filter posed to an external structure like a spider’s web, in addition Correspondence: [email protected] to the unique cellular physiology of the colloblast, have made them a quasi-phylum-defining trait for ctenophores (Dunn et Introduction al., 2015). Ctenophores are a phylum of gelatinous zooplankton known Despite the conspicuousness of colloblast-covered adhesive for being voracious and efficient ambush predators (Bishop, tentacles as a trait, many physical and biochemical questions 1968; Greene et al., 1986; Haddock, 2007; Tamm, 2014). about colloblasts remain open: What do adhesion and dead- Townsend et al. | bioRχiv | June 27, 2020 | 1–15 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.27.175059; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. hesion events look like? How strong is colloblast adhesive? shore-hardness = 10A). The silicone arenas were placed onto How quickly does it bond to a target? Is adhesion affected clean, dry, charged microscope slides (Superfrost® Plus, by ambient water conditions? Understanding the answers to Thermo Scientific), and pressed firmly to ensure adhesion to these questions would aid our understanding of colloblast ad- the glass. Anchoring slits were cut into the two corners of hesive as a unique biomaterial and inform the potential limi- the arena. P. pileus were used for all imaging experiments. tations it puts on ctenophore predation. A single portion of severed P. pileus tentacle was transferred In this study, we apply live microscopy techniques to visual- into the arena with ~300 µl of natural sea water. The tentacle ize adhesion between probes and tentacles of Pleurobrachia was orientated within the arena using clean fine-tip forceps, pileus, assessing the fates of individual colloblasts engaging and each end of the tentacle fragment was manipulated into in adhesion. We assess the impact of contact area on ad- an anchor slit to maintain tentacle position. hesion, in addition to comparing probes of different surface Probes were generated from 1.75mm diameter filaments chemistries. We adapt instrumentation for measuring sur- of polylactic acid polymer (PLA; Yellow PLA filament, face tension to measure the adhesive force exerted by the col- Prusa, FLM-PLA-175-YEL) by heating segments and loblast adhesive system of Pleurobrachia bachei. Using our pulling to create a tapered strand. The strand was then cut method, we can control what region of the tentacle is probed perpendicularly to the probe to generate a flat, circular tip. as well as the ambient water conditions that the tentacle and Lysine probes were generated by immersing PLA probes in its colloblasts experience. 50 mg · ml−1 poly-D-lysine in PBS until the solution was Our data demonstrate that ctenophore prey capture, medi- fully evaporated to coat the probe tips in lysine. Copepod ated by colloblast adhesion, is a robust mechanism that acts probes were generated by coating the tip of a PLA probe in a quickly to ensnare prey under a variety of conditions. Un- small amount of clear nail varnish. A single copepod carcass derstanding of the adhesion strength of the system, and of in- (Calanoida sp. Seahorse Aquariums, Dublin, Ireland) dividual colloblasts, is gained. Furthermore, the burgeoning was pressed into the nail varnish, and left to dry. A clean understanding of colloblasts is itself integral to our under- probe was brought into contact with the tentacle fragment standing of ctenophore ecology in a rapidly changing marine (velocity = 20 mm · min−1) under 20X magnification video environment as well as the role of this early-diverging animal microscopy using an open source microscopy system (Court- lineage in the larger story of animal cell type evolution (Ryan ney et al., 2020). Contact/compression was maintained et al., 2013). for 5 seconds, followed by probe retraction (velocity = 5 mm · min−1) (Fig. 1). Methods Probing videos were manually analyzed in ImageJ (Schin- delin et al., 2012) to assess the area of contact between Sample collection probe and tentacle, the total number of colloblast adhesion Pleurobrachia pileus were collected from the Irish Sea at events, the number of colloblasts showing adhesive failure Howth Harbor, Dublin, Ireland (53.393060, -6.066148) (colloblast head detaching from probe), the distance at using plankton nets with an associated collection chamber, which the individual colloblast