Marine Biology (2018) 165:40 https://doi.org/10.1007/s00227-018-3293-3 SCCWRP #1037 SHORT NOTES Infuence of bacteria on shell dissolution in dead gastropod larvae and adult Limacina helicina pteropods under ocean acidifcation conditions Alexandra R. Bausch1 · M. Angeles Gallego2 · Januar Harianto3 · Patricia Thibodeau4 · Nina Bednaršek5 · Jonathan N. Havenhand6 · Terrie Klinger7 Received: 5 July 2017 / Accepted: 15 January 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract Ocean acidifcation (OA) increases aragonite shell dissolution in calcifying marine organisms. It has been proposed that bacteria associated with molluscan shell surfaces in situ could damage the periostracum and reduce its protective function against shell dissolution. However, the infuence of bacteria on shell dissolution under OA conditions is unknown. In this study, dissolution in dead shells from gastropod larvae and adult pteropods (Limacina helicina) was examined following a 5-day incubation under a range of aragonite saturation states (Ωarag; values ranging from 0.5 to 1.8) both with and without antibiotics. Gastropod and pteropod specimens were collected from Puget Sound, Washington (48°33′19″N, 122°59′49″W and 47°41′11″N, 122°25′23″W, respectively), preserved, stored, and then treated in August 2015. Environmental scanning electron microscopy (ESEM) was used to determine the severity and extent of dissolution, which was scored as mild, severe, or summed (mild + severe) dissolution. Shell dissolution increased with decreasing Ωarag. In gastropod larvae, there was a signifcant interaction between the efects of antibiotics and Ωarag on severe dissolution, indicating that microbes could medi- ate certain types of dissolution among shells under low Ωarag. In L. helicina, there were no signifcant interactions between the efects of antibiotics and Ωarag on dissolution. These fndings suggest that bacteria may diferentially infuence the response of some groups of shelled planktonic gastropods to OA conditions. This is the frst assessment of the microbial–chemical coupling of dissolution in shells of either gastropod larvae or adult L. helicina under OA. Responsible Editor: H.-O. Pörtner. Introduction Reviewed by J. M. Hall-Spencer and an undisclosed expert. The oceanic uptake of anthropogenic carbon dioxide (CO 2) * Alexandra R. Bausch signifcantly afects seawater carbon chemistry and causes [email protected] decreases in pH and aragonite saturation state—a process known as ocean acidifcation (OA). OA represents a sig- 1 Department of Earth and Environmental Sciences, Lamont-Doherty Earth Observatory, Columbia University, nifcant threat to calcifying marine organisms, particularly Palisades, NY 10964, USA shelled molluscs (Gazeau et al. 2013), some of which serve 2 Department of Oceanography, University of Hawaii, important roles in pelagic food webs and the export of car- Honolulu, HI 96822, USA bon to the deep ocean (Berner and Honjo 1981; Hunt et al. 3 Discipline of Anatomy and Histology, School of Medicine, 2008). Planktonic, highly motile gastropod larvae and adult The University of Sydney, Sydney, NSW 2006, Australia thecosome pteropods may be especially sensitive to changes 4 Department of Biological Sciences, Virginia Institute in ocean chemistry due to their thin shells and incomplete of Marine Science, Gloucester Point, VA 23062, USA protection of the outer organic layer of their shells (peri- 5 Southern California Coastal Waters Research Project, ostracum), resulting in rapid dissolution of the aragonite Costa Mesa, CA 92626, USA shell matrix under low aragonite saturation state (Ωarag; e.g., 6 Department of Marine Sciences, Tjärnö, Gothenburg Bednaršek et al. 2012b, 2016; Gazeau et al. 2013). Pteropods University, 45296 Strömstad, Sweden in particular serve as one of the frst bioindicators of OA in 7 School of Marine and Environmental Afairs, University pelagic marine ecosystems (Bednaršek et al. 2012a, 2014a, of Washington, Seattle, WA 98105, USA 2016). Vol.:(0123456789)1 3 40 Page 2 of 9 Marine Biology (2018) 165:40 Extensive dissolution of the exterior of larval and adult antibiotic treatments (due to presumably higher numbers pteropod shells has been demonstrated under OA in both of shell-associated bacteria). field studies and laboratory experiments. In naturally undersaturated waters in the feld, severe shell dissolu- tion was observed among live juveniles of the pteropod Materials and methods Limacina helicina antarctica (Bednaršek et al. 2012b). In laboratory incubation experiments, signifcant changes Specimen collection and sorting in shell size and degradation were also observed in the pteropod Limacina helicina under simulated OA (e.g., Gastropod veliger larvae and adult Limacina helicina ptero- Lischka et al. 2011; Lischka and Riebesell 2012; Busch pods were collected from Puget Sound, Washington, USA et al. 2014), and complete shell loss was observed in larvae (Fig. 1), preserved, stored, and later treated. We examined of the pteropod Cavolinia infexa under OA conditions the efects of OA and antibiotics on shells of dead speci- (Comeau et al. 2010). Based on similar shell structure and mens for several reasons: (1) to describe the response of mineralogical composition, it is presumed that the pelagic shell dissolution to bacteria in seawater; (2) to isolate the larvae of benthic gastropods may also be particularly sus- role of bacteria in shell dissolution under OA conditions, ceptible to dissolution under OA (Gazeau et al. 2013). independent of the physiological efects of live molluscs (as Aragonite dissolution in shelled molluscs such as L. in Manno et al. 2012); and (3) to avoid the logistical chal- helicina occurs over the surface of the entire shell rather lenges of maintaining healthy specimens in culture (Manno than simply at the oldest, inner whorls or along the new- et al. 2017), particularly under low Ωarag conditions unsuit- est, growing edge (Bednaršek et al. 2012b). The periostra- able for pteropod maintenance. cum, an outer organic layer of the shell, exists as a bar- Live gastropod larvae were collected from San Juan rier between the mineral layers of the shell and ambient Channel (48°33′19″N, 122°59′49″W) on August 6, 2015 seawater (Harper 2000; Peck et al. 2016), but does not by plankton tow (310 μm mesh) at a maximum depth of provide sufcient protection against chemical dissolution 50 m (T = 12.8 ± 0.2 °C, S = 29.8, Ωarag = 1.08 ± 0.03; under OA conditions (Bednaršek et al. 2016). Although mean ± SD). These were the planktonic larvae of benthic shell dissolution can occur despite an intact periostracum, the local thinning or degradation of this protective veneer can further exacerbate dissolution damage at discrete sites on the shell surface (Gazeau et al. 2013; Bednaršek et al. 020 40 km 2016)—a process similar to that seen in nassariid gastro- 49°N pods (Garilli et al. 2015). It has been recognized for some time that bacteria living in association with mollusc shells could decrease the pH of the shell microenvironment via respiration, resulting in shell damage and potential loss of G integrity of the organic matrix (Glover and Kidwell 1993; 48.5°N Clark 1999). However, a search of the literature found no information on the infuence of bacteria on the extent of dissolution among gastropod shells, especially under OA. In this study, we examined dissolution of dead shells from gastropod veliger larvae and adult thecosome 48°N pteropods resulting from a 5-day exposure to a range of Ωarag both with and without antibiotics. To examine the efects of bacteria on dissolution under simulated OA, G the research objectives of this study were threefold: (1) N to determine the infuence of Ω on shell dissolution; arag W E (2) to determine the infuence of bacteria on shell dis- 47.5°N solution under a range of Ωarag; and (3) to examine the S dissolution responses among gastropod larvae and adult L. helicina collected from Puget Sound, Washington, USA, 123.5°W123°W122.5°W 122°W where waters are seasonally subjected to corrosive con- ditions (Feely et al. 2008, 2010). We hypothesized that: Fig. 1 Locations of plankton tows to collect gastropod larvae (red) (1) shell dissolution would increase with decreasing Ωarag and adult Limacina helicina specimens (blue) in Puget Sound, Wash- and (2) shell dissolution would increase in the absence of ington, USA 1 3 Marine Biology (2018) 165:40 Page 3 of 9 40 gastropod species. Specimens were sorted under a dissect- inoculated with 25 mg L−1 each of ampicillin and strepto- ing microscope, rinsed with fltered seawater (0.22 μm), and mycin, as implemented by Maas et al. (2015). The addition preserved by air-drying for 24 h (as in Peck et al. 2016). of antibiotics slightly decreased the seawater pH T (pH on Inspection showed that all gastropod veliger larval speci- the total hydrogen scale) by 0.04 ± 0.02 pH units, which mens had dextrally coiled shells and, therefore, were not was insignifcant compared to the diferences in treatment opisthobranchs (the taxonomic group that includes Pter- conditions. We assumed that the antibacterial activity of the opoda). We could not classify the larval specimens any fur- ampicillin and streptomycin was similar to that reported in ther solely based on morphological characteristics. These Maas et al. (2015). Ampicillin and streptomycin are known non-pteropod gastropod larvae are hereafter referred to as to be synergistically bactericidal for some Gram-positive “gastropod larvae”. and Gram-negative bacteria by inhibiting cell-wall synthesis Limacina helicina specimens were collected from Puget and protein synthesis (Brunton et al. 2011); however, we did Sound Station P28 of the Puget Sound Regional Synthesis not count or otherwise assess bacterial number or density. Model monitoring program (47°41′11″N, 122°25′23″W) Shells of up to fve gastropod larvae were selected hap- on October 30, 2014 using a Bongo net (335 μm mesh) hazardly and placed together into each treatment in 960 mL at a maximum depth of 188 m (T = 12.2 ± 0.2 °C, borosilicate glass jars with CO2-manipulated seawater.