Vol. 572: 117–128, 2017 MARINE ECOLOGY PROGRESS SERIES Published May 31 https://doi.org/10.3354/meps12155 Mar Ecol Prog Ser OPEN ACCESS Osculum dynamics and filtration activity in small single-osculum explants of the demosponge Halichondria panicea Lars Kumala1,2,3,*, Hans Ulrik Riisgård1, Donald Eugene Canfield4 1Marine Biological Research Centre, University of Southern Denmark, 5300 Kerteminde, Denmark 2Max-Planck Odense Center on the Biodemography of Aging and Department of Biology, University of Southern Denmark, 5230 Odense M, Denmark 3Max Planck Institute for Demographic Research, 18057 Rostock, Germany 4Nordic Center for Earth Evolution, Department of Biology, University of Southern Denmark, 5230 Odense M, Denmark ABSTRACT: Contraction−inflation behavior, including the closure and opening of the exhalant opening (osculum), is common among sponges. This behavior may temporally affect filtration activity, making it difficult to study and understand sponge feeding biology. To examine the inter- play between osculum dynamics and filtration activity, small (18 mm3) single-osculum explants of the demosponge Halichondria panicea were studied. Time-lapse video stereo-microscope record- ings of the osculum cross-sectional area (OSA) were made simultaneously with measurements of the filtration rate (~15°C, ~20 PSU) using the clearance method. Osculum dynamics, as expressed by temporal variation of the OSA, including osculum contraction and expansion, correlated with variability in the explant filtration rate, and no water pumping was observed during periods of osculum closure. A linear relationship between filtration rate (FR) and OSA revealed a constant −1 exhalant jet velocity: vjet = FR/OSA = 2.3 ± CI95% 0.13 cm s . The mean filtration rate of explants −1 −1 was 0.28 ± CI95% 0.06 ml min , corresponding to a volume-specific filtration rate of 15 ml min (cm3 sponge)−1, which is 2 to 3 times higher than that reported for larger individuals of H. panicea with multiple oscula. This is the first demonstration of a direct relationship between filter feeding and osculum dynamics in a sponge. KEY WORDS: Filtration · Osculum · Contraction · Pumping activity · Sponge explant INTRODUCTION sponge surface (exopinacoderm; Gaino et al. 1994) and along the inhalant canal system (Kilian 1952, Sponges are sessile filter-feeding invertebrates Reiswig 1971a, Bergquist 1978). In demosponges, the that actively pump seawater through their water pumping power is generated by choanocyte cham- canal system to obtain suspended food particles bers in which 80 to 100 beating flagella impel water (Bergquist 1978). The basic element for both water to enter the sponge body through numerous pores pumping and filtration is the choanocyte, with a (ostia), creating flow through a complex aquiferous beating flagellum to induce water flow and a collar of system composed of inhalant and exhalant canals microvilli that sieves free-living bacteria and other that merge into one or more exhalant opening particles down to ~0.1 µm for the purpose of feeding (oscula) on the sponge chimney or surface (Larsen & (Fjerdingstad 1961, Brill 1973, Riisgård & Larsen Riisgård 1994). The size (Wilkinson 1978) and density 2000, Leys et al. 2011). Feeding on phytoplankton (Massaro et al. 2012) of such pumping units (i.e. cells (>5 µm) is accomplished by phagocytosis at the choanocyte chambers) allow demosponges, such as © The authors 2017. Open Access under Creative Commons by *Corresponding author: [email protected] Attribution Licence. Use, distribution and reproduction are un - restricted. Authors and original publication must be credited. Publisher: Inter-Research · www.int-res.com 118 Mar Ecol Prog Ser 572: 117–128, 2017 Halichondria panicea, to filter a volume of water of at ships between osculum dynamics and filtration activ- least 6 times their own body volume per minute ity. Therefore, in the present study, we use video-mi- (Weisz et al. 2008, Riisgård et al. 2016). croscope observations of small single-osculum sponge Temporal variations in filtration activity appear to explants to explore the relationship between osculum be common among sponges under both laboratory opening dynamics and filtration rate, with implications and natural conditions (Frost 1978, Willenz et al. for elucidating patterns in sponge feeding. 1986, Osinga et al. 2001, Stabili et al. 2006, Riisgård et al. 2016). The intertidal sponge H. panicea, com- monly found in the red algae zone of the Western MATERIALS AND METHODS Baltic Sea and the North Sea (Barthel 1986, 1988, 1989), has shown significant variations in filtration Preparation and cultivation of sponge explants rates over a period of 6 d in the laboratory (Riisgård et al. 2016). Similar observations of variable filtration Specimens of Halichondria panicea were collected rates were made during short-term (<2 h) in situ in the inlet to Kerteminde Fjord, Denmark, and trans- measurements on the freshwater sponge Spon gilla ported to the nearby Marine Biological Research lacustris (Frost 1978). Such variation in filtration Center in Kerteminde where sponges were pro- activity may be associated with cyclic contraction cessed within 24 h after collection. During this time, and inflation of the sponge body as well as the sponges were kept in a flow-through aquarium (30 l) inhalant (ostia) and exhalant (oscula) openings with aerated seawater (~12–15°C, ~18–22 PSU). To (Reiswig 1971b, Simpson 1984, Gaino et al. 1991, generate ex plants, the upper 2.0 to 2.5 cm of a Nickel 2004, Leys & Meech 2006, Nickel et al. 2006, sponge chimney was cut with a razor in 2 mm thick Elliott & Leys 2007). Although sponges lack a nerv- slices, and these were further cut into ~18 mm3 ous system and true muscles (Pavans de Ceccatty pieces (= 0.018 ml × 0.07 g dry wt ml−1 = 1.26 × 10−3 g 1986, 1989), actin microfilaments, myocytes and dry wt; cf. Tho mas sen & Riisgård 1995) (Fig. 1A) actinocytes abundantly located in the pinacoderm, comprising both ectosomal (peripheral) and canal system and osculum (Prosser et al. 1962, Elliott choa no somal (inner) parts of the donor sponge (cf. de & Leys 2007, Nickel et al. 2011) allow for contractile Caralt 2003). The cuttings were placed on numbered behavior. This behavior seems to be modulated by glass slides submerged in an aquarium (30 l) with neuro-active substances such as gamma-amino - flow-through of aerated bio- filtered seawater (~15°C, butyric acid and glutamate (Ellwanger et al. 2004, ~18–22 PSU; Fig. 1B) to reduce microbial growth on 2007, Ellwanger & Nickel 2006, Elliott & Leys 2010). cut surfaces (bacterial infection) preventing success- Recent studies suggest the osculum senses external ful explant culture (Hummel et al. 1988, de Caralt et stimuli by means of primary cilia that line its inner al. 2003). The seawater was pre-filtered by Mytilus epithelium (Ludeman et al. 2014, Leys 2015). Oscu- edulis mussels. These mussels show retention effi- lum contractions appear to be stimulated by air expo- ciencies of up to 100% for bacteria and phytoplank- sure (Parker 1910), electrical, mechanical and chem- ton (Møhlenberg & Riisgård 1978), thus allowing the ical stimuli (Parker 1910, Prosser et al. 1962, Elliott & sponge explants to live under controlled food condi- Leys 2007), and changes in light intensity (Reiswig tions. The cut surfaces healed within 5 d as seen from 1971b) and water flow dynamics (Riisgård et al. smoothing and rounding of the explants’ periphery 2016). Exposure to clay also triggers oscular contrac- and edges (Fig. 1C). After this 5 d period, glass slides tion (Gerrodette & Flechsig 1979, Bannister et al. with attached explants (Fig. 1C) were placed in glass 2012), indicating that sponges can protect and clean slide holders and these were transferred to a feeding their filter-feeding apparatus from unwanted parti- tank (9 l) with continuous flow (80 to 100 ml min−1) of cles (Elliott & Leys 2007, Tompkins-MacDonald & bio-filtered seawater (~15°C, ~20 PSU). Explants Leys 2008, Leys 2015). Furthermore, opening and were regularly fed with the alga Rhodomonas salina closure of oscula in a tropical demosponge indicate (about 6.3 µm diameter) added with a peristaltic an intrinsic species-specific rhythm possibly coordi- pump (Ole Dich) to establish a steady-state concen- nated by changes in the flagellar activity of choano - tration of about 4000 cells ml−1 (equivalent to 5 µg cytes (Reiswig 1971b). chl a l−1; Clausen & Riisgård 1996). These algae have Sponges with several exhalant openings typically previously been used in growth and filtration studies display asynchronous osculum closure and opening on explants of H. pani cea, as well as other sponge (Parker 1910, Pfannkuchen et al. 2009, Riisgård et al. species (Thomassen & Riisgård 1995, Osinga et al. 2016), making it difficult to unravel possible relation- 1999), demonstrating both ingestion and digestion of Kumala et al.: Osculum dynamics and filtration activity 119 Fig. 1. Halichondria panicea. Preparation and cultivation of sponge explants. (A) Cuttings (18 mm3) from sponge chimneys, (B) cuttings placed on submerged glass slides in a flow-through aquarium with bio-filtered seawater, (C) sponge explant (5 d old) after attachment on a glass slide, and (D) example of an explant (62 d old) with single osculum (arrow). Scale bars: (A) 10 mm (C,D) 1 mm R. salina by sponges (Osinga et al. 1999). Attached next 80 to 120 min by taking water samples (10 ml) explants developed a single osculum during the next at fixed time intervals (20 min) and measuring algal 5 d, initiating active filter feeding (Fig. 1D). These concentration with an electronic particle counter explants were used for experiments. (Elzone 5380), which removed 1.5 to 2.0 ml of the sample per counting. The remainder of the sample was returned to the experimental chamber to avoid Measurement of filtration rate significant reduction in water volume and algal con- centration due to sampling during the experiment. To investigate the pumping activity of sponge ex - The filtration rate (FR, ml min−1) was determined plants, we repeatedly measured the filtration rate from the exponential decrease in algal concentration using the clearance method (cf.