J. Mar. Biol. Ass. U.K. (2004), 84, 629^632 Printed in the United Kingdom
The impact of two species of bioturbating shrimp (Callianassa subterranea and Upogebia deltaura) on sediment denitri¢cation Rebecca L. Howe*O, Andrew P. Rees*P and Stephen Widdicombe* *Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, PL1 3DH, UK. OUniversity of Plymouth, Drake’s Circus, Plymouth, PL4 8AA, UK. PCorresponding author, e-mail:
[email protected]
In a benthic mesocosm experiment, the e¡ects of two species of burrowing Thalassinidean shrimps (Callianassa subterranea and Upogebia deltaura) on rates of sediment denitri¢cation were determined using the isotope pairing technique. Denitri¢cation rate (Dtot) and coupled nitri¢cation^ denitri¢cation (Dn) were shown to be signi¢cantly enhanced by the presence of U. deltaura by 2.9 and 3.3 times respectively, relative to control measurements. For U. deltaura the stimulation of the denitri¢cation rate was found to be signi¢cantly related to the size of the animal (F¼5.81, P¼0.042). No deviation from the rates determined in control cores for either Dtot or Dn was observed for those cores inhabited by C. subterranea. The increase in Dtot with U. deltaura was considered to be the result of a combination of di¡erent factors, including; the direct extension of the sediment ^ water interface and an increase in oxygenation of the sediments and solute transport, as a result of the ventilating activities of the animal itself.
INTRODUCTION Nutrient loading to the marine environment is a cause of concern, in particular with reference to the increasing release of nitrogen from anthropogenic sources over recent decades. Sediments have been shown to have a large capacity to regulate nitrogen from coastal waters, with denitri¢cation playing a particularly important role as a mechanism for the removal of NO37. Denitri¢cation is carried out by facultatively anaerobic bacteria which utilize NO37 as the terminal electron receptor in order to generate energy in the form of ATP. Nitri¢cation is an aerobic process carried out by chemoautotrophic bacteria which require free O2 for the oxidation of NH4+ and NO27. Although both processes have opposing requirements for oxygen, an ecologically signi¢cant relationship is the coupling between nitri¢cation^ denitri¢cation which occurs at the interface between oxic and anoxic layers in sediments. It is considered that for many environments most nitrate is reduced through this nitri¢cation ^ denitri¢cation pathway (Jenkins & Kemp, 1984). The presence of benthic infauna, through their feeding, burrowing and sediment irrigation activities, is known to signi¢cantly modify the biogeochemistry of marine sediments through the promotion of solute exchange between the sediment and the water column (Aller, 1988). Elevated £uxes of O2, TCO2 and dissolved inorganic nitrogen across the sediment ^ water interface between 2.5 and 3.5 times have been observed relative to controls which may act to increase denitri¢cation by aiding in the transport of nitrate from the water column to deeper sediment. Thalassinidean decapods are one of the most active groups of burrow forming benthic organisms present in coastal sediments. The burrows created by Callianassa subterranea (Montagu, 1808), can have a total surface area of 1.5 m2 m72 and a total volume of 6 dm3 m2 (Witbaard Journal of the Marine Biological Association of the United Kingdom (2004)
& Duineveld, 1989). This shrimp occurs in large numbers, a mean density of 38^59 individuals per m2 has been reported for the southern North Sea, and with each individual inhabiting its own burrow there exists the potential for a signi¢cant increase in sediment surface area (Rowden & Jones, 1994). It seems likely that such an organism may have a substantial impact on the rates of sediment denitri¢cation. However, not all thalassinidean shrimps live and feed in the same way. The focus of this study was to investigate the impact upon the rate of sediment denitri¢cation of two Thalassinidean species with contrasting behavioural characters; C. subterranea and Upogebia deltaura (Leach, 1815). Whilst both create extensive permanent burrow systems they di¡er in their burrowing and feeding activities. Callianassa subterranea is a deposit feeder: processing sediments and ingesting particles in the substratum; whilst U. deltaura is a suspension feeder: ¢ltering particles out of the burrow water.
MATERIALS AND METHODS Experimental set-up
This experiment was conducted in the benthic mesocosm of the Plymouth Marine Laboratory. The system consists of four interconnected 1m3 plastic tanks, ¢lled with 1 mm ¢ltered seawater. A closed circulation system via an external 1 mm ¢lter provides £ow through to each cell. Each cell also contains an internal pump, to facilitate mixing of the water, and to promote laminar £ow across the surface of the cell. On 10 June 2003 sandy-mud sediment cores were collected by box corer from a site 100 m north of the breakwater in Plymouth Sound (50.338N 04.148W). The cores were transferred from the box corer to 30 cm diameter,
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40 cm deep plastic buckets with the minimum amount of disturbance to the sediment structure as was possible. The cores were then transferred to the mesocosm and allowed to settle. Any organisms found on the surface of the cores after 24 hours were carefully removed by hand. The cores were left to settle for a period of ten days before the shrimps were introduced. Using a box corer Callianassa subterranea and Upogebia deltaura were collected on 17 June 2003 from Jennycli¡ Bay in Plymouth Sound. Shrimps were placed individually into small, plastic mesh containers which were immersed in continuously £owing seawater. Each shrimp was weighed, returned to its container and kept within the mesocosm system until they were introduced to the sediment. On 20 June 2003, 18 cores were selected that showed no obvious signs of bioturbator activity. These cores were consolidated in two mesocosm cells with each cell containing nine cores. Four cores, two in each cell, were randomly assigned as controls (no shrimp added). A single shrimp was added to each of the remaining cores. The experiment used eight C. subterranea, ranging in size from 0.8 g to 2.9 g, and six U. deltaura, 1.8 g to 14.1g. The di¡erent shrimps were randomly assigned to buckets, constrained to ensure an even distribution of species and sizes between each of the cells. Throughout the experiment the cores were kept in darkened conditions and the water temperature was maintained at 15 18C with a salinity of 35. Measuring denitri¢cation
Denitri¢cation was determined using an adaptation of the isotope pairing technique (Nielsen, 1992). Water overlying the sediments was removed and carefully replaced to minimize disturbance of the sediment surface with 0.2 mm ¢ltered seawater containing 510 nmol l1 NO3 collected from the oligotrophic North Atlantic. Once any resuspended material had settled each core was isolated from the atmosphere with an air-tight Perspex lid which was seated into the top of each bucket taking care to exclude air bubbles. The lids were ¢tted with an inlet and outlet gland which was controlled by a 3-way Luer valve and a £ow of water was created over the sediment surface using a peristaltic pump. 15N-NO3 (99 atom%) was added to each core to a ¢nal concentration of approximately 50 mmol l1; 40 ml of seawater was collected and ¢ltered (GF/F, Whatman Inc.) for determination of NO3 concentration before and after addition of 15N. The amended cores were incubated in the dark at 158C for four hours, after which time denitri¢cation was terminated by mixing the sediment and overlying water with a clean metal stirring bar to create an homogenous slurry. Triplicate samples were collected into acid-cleaned 50 ml amber bottles to which 1.2 ml of ZnCl2 (50% w/v) was added to halt microbial activity. Mass spectrometric analysis was usually started within four hours of sample collection, when this was not possible samples were stored (24 h maximum) at 48C. Once samples had reached room temperature, a 12 ml headspace of helium was introduced to each bottle and using an orbital shaker, dissolved N2 was equilibrated with the helium. Determination of the 14 N : 15N isotopic ratios as 29N2 and 30N2 was then made Journal of the Marine Biological Association of the United Kingdom (2004)
using continuous £ow, stable-isotope mass spectrometry (PDZ-Europa Ltd, 20:20) using a (TGII) gas inlet system. Each sample analysis was performed immediately following an analysis of atmospheric N2 to which it was referenced. Rates of denitri¢cation (Dtot) and coupled nitri¢cation^ denitri¢cation (Dn) were then estimated using the equations of Steingruber et al. (2001). Data analysis
Statistical analyses of these indices were performed using the MINITAB 13 for Windows computer package. Relationships between shrimp biomass and Dtot were examined using regression analysis. One-way analysis of variance was used to test for di¡erences in the rates of Dn between control and shrimp treatments.
RESULTS Rates of denitri¢cation determined during this study were comparable with those recorded for other areas (e.g. Steingruber et al., 2001), although due to the period and means of incubation the values presented are not intended to be taken as directly representative of naturally occurring rates that are to be found in Plymouth Sound. However, the use of laboratory based experiments is a well established method of conducting comparative studies of ecosystem processes (e.g. Widdicombe & Austen, 1998). Rates of Dtot (Figure 1) for sediment incubated in the presence of Callianassa subterranea (10.06 6.46 mol m72 h71; mean 1 SD) show no measurable di¡erence from the rates determined in the control sediments, 10.07 3.72 mmol m72 h71. The presence of Upogebia deltaura, however, creates an apparent increase in the mean rate of Dtot (29.38 16.87 mmol m72 h71) by approximately three times relative to both control and C. subterranea treatments.
Figure 1. Mean rate of denitri¢cation for control, Upogebia deltaura and Callianasa subterranea treatments, with 95% con¢dence intervals.
Thalassinideans and denitri¢cation
R.L. Howe et al. 631
Figure 2. E¡ect of Upogebia deltaura body size on the rate of denitri¢cation.
U. deltaura in sediments, however, resulted in a signi¢cant elevation in the rate of Dn relative to Dtot so that 87.6 4.7% of the denitri¢ed NO37 was supplied via this route, and that rates of Dn were signi¢cantly higher than those observed in either the controls (F=36.7, P=0.000) or the C. subterranea treatments (F=12.4, P=0.004).
DISCUSSION
Figure 3. Mean values of denitri¢cation^nitri¢cation coupling for control, Upogebia deltaura and Callianasa subterranea treatments, with 95% con¢dence intervals.
Con¢dence intervals in Dtot for U. deltaura inhabited sediments initially appear relatively large. Further analysis of this dataset (Figure 2) revealed a signi¢cant relationship (r2 ¼0.42, F¼5.81, P¼0.042) between the shrimp biomass and that of Dtot so that the presence of the larger shrimp were found to result in elevated rates of denitri¢cation. The two medium sized shrimps (7 g and 9 g) appeared not to have a¡ected the rate of denitri¢cation and fall below the projected regression line. As no assessment of shrimp activity during the incubation period was made, it is possible that the shrimps in these two treatments were either inactive or dead. Coupled nitri¢cation ^ denitri¢cation is recognized for a large number of sediments, particularly for those underlying an oxic water-column and with enhanced penetration of O2 into the sediments to account for a large percentage of denitri¢cation (e.g. Nielsen, 1992). During this study, Dn in the control cores (Figure 3) accounted for a mean of 76.7 10.7% of Dtot, whilst cores inhabited by C. subterranea again showed little deviation from this; 75.8 15.2% (F¼0.55, P¼0.474). The presence of Journal of the Marine Biological Association of the United Kingdom (2004)
The current study has shown that the presence of Upogebia deltaura signi¢cantly increases the rate of Dtot compared with unbioturbated sediments, but the mechanisms by which this increase occurs may not be simple. It is important to recognize throughout any such discussion that any observed e¡ect on denitri¢cation rate is not a direct consequence of macrofaunal bioturbation, but is a result of an altered environment on the activity of the bacterial population i.e. bioturbators may indirectly impact on microbially driven sediment processes. It has been suggested that the extension of the sediment ^ water interface, resulting from the creation of burrows, may be responsible for a¡ecting denitri¢cation rates in a number of ways. Firstly, by simply enlarging the sediment-water interface and increasing the surface area available for di¡usive solute exchange, burrows will promote exchange between the sediment and the water column (Aller, 1988). An increased supply of NO37 to anoxic sediments is thus likely to promote rates of denitri¢cation. However, the current study has shown that the increase in denitri¢cation caused by U. deltaura is not altogether driven by increased NO37 exchange; instead it is largely fuelled by NO37 generated by nitri¢cation from within the sediment. Extension of surface area produced by burrows has been proposed to result in an increase in bacterial numbers and metabolic activity. As both nitri¢cation and denitri¢cation are bacterially mediated, any e¡ects upon bacterial numbers or activity are likely to be re£ected in the rate of these processes. The U. deltaura burrows by generating an increased surface area for bacterial growth may, therefore, be resulting in an increase in bacterial numbers and activity. However, if the e¡ect of U. deltaura was purely due to an increase in the substrate available for bacterial growth a similar increase in rates of denitri¢cation would have been expected for Callianassa subterranea. This was not the case. It must be noted however, that any discussions concerning the relative burrow size and burrowing activity
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of di¡erent shrimps are based on published accounts of burrowing activity. This was because an accurate assessment of burrow volumes was not possible during the current study as the slurrying technique used for the estimation of denitri¢cation results in the complete destruction of the burrow. It is evident that there is no guarantee that simply by building burrows an organism will increase the rate of denitri¢cation. It is vital we consider the morphology of the burrow and the activity of the animal within it. Callianassa subterranea constructs a deep, complex series of temporary or semi-permanent tunnels (Nickell & Atkinson, 1995), whilst U. deltaura generally construct fairly simple U- or Y-shaped burrows (Nickell & Atkinson, 1995). These U. deltaura burrows are considered to be fairly permanent structures with walls hardened by a glandular secretion. This di¡erence in burrow morphology between U. deltaura and C. subterranea has been attributed to the di¡erent feeding mechanisms employed by these two species of shrimp. Callianassa subterranea is a deposit feeder, whilst U. deltaura is essentially a suspension feeder. This di¡erence in feeding mode not only results in di¡erent burrow morphology, it also a¡ects the rate at which these species ventilate their burrows. Published values for measured mean burrow £ow rates show that U. deltaura draws approximately three times as much water through its burrow than does C. subterranea, 149.5 35.5 ml h71 compared with 50.3 33.6 ml h71 (Nickell, 1992). The irrigation of the burrows is an important factor and animals which continuously irrigate their burrows have been seen to impact on denitri¢cation. U-shaped burrows facilitate the movement of water which intuitively should promote solute exchange between the sediments and overlying water. However in this study increased denitri¢cation was primarily fuelled by nitri¢cation. This would suggest that ventilation of the burrow for denitri¢cation is not as important for NO37 exchange as is the supply of NH4+ and the £ux of O2 to maintain elevated rates of nitrifying bacteria. A number of authors have highlighted a link between a stimulation of denitri¢cation and an increased supply of NH4+ in macrofauna burrows (Webb & Eyre, 2004; and references therein). The site within the burrow where NH4+ is produced may have important consequences. Upogebia deltaura primarily operates in the oxic sections of its burrow, so that excreted NH4+ may be available to and actively utilized by nitrifying bacteria. In contrast, C. subterranea spends much of its time deep in its burrow where conditions are commonly anoxic and nitrifying activity is low or negligible. NH4+ produced here is likely to either accumulate or be anaerobically oxidized to N2 through the anammox pathway. Further evidence for the importance of burrow morphology and animal activity in setting rates of denitri¢cation is provided by comparing the results of the current study with those of Webb & Eyre (2004). These authors examined the biogeochemical impact of the burrowing shrimp Trypaea australiensis and found that this species stimulated dentri¢cation through a tight coupling between nitri¢cation and denitri¢cation. This species is reported to build a ‘U’ shaped respiratory loop within the upper 30^40 cm of sediment. From a turning chamber at the bottom of this loop a multi-branched network descends Journal of the Marine Biological Association of the United Kingdom (2004)
to over 1m deep. From the current study and that of Webb & Eyre (2004) in would appear that a simple ‘U’ shaped burrow that is well ventilated and oxygenated is instrumental in the stimulation of nitri¢cation and the subsequent increase in denitri¢cation. There is a large body of evidence to suggest that bioturbation of marine sediments impacts on the cycling of nitrogenous material therein. This study reveals a signi¢cant relationship between the activities of Upogebia deltaura and sediment denitri¢cation rate, but found that under the conditions of this experiment no such relationship existed between Callianassa subterranea and the rate of sediment denitri¢cation. It is considered that the enhancement of denitri¢cation is not simply a product of increased surface area following the establishment of burrows, but is reliant on the maintenance of suitable environmental conditions to support the microbial population. Our thanks go to Katie Chamberlain, Joanna Dagnan, John Spicer, Mike Townsend, Hester Willson and the crew of RV ‘Squilla’ for their contributions to this project. This work contributes to the Core Science Programme of the Plymouth Marine Laboratory and was funded in part by the Natural Environment Research Council.
REFERENCES Aller, R.C., 1988. Benthic fauna and biogeochemical processes in marine sediments: the role of burrow structures. In Nitrogen cycling in coastal marine environments (ed. T.H. Blackburn and J. Sorensen), pp. 301^338. SCOPE, New York: John Wiley & Sons Ltd. Jenkins, M.C. & Kemp, W.M., 1984. The coupling of nitri¢cation and denitri¢cation in two estuarine sediments. Limnology and Oceanography, 29, 609^619. Nielsen, L.P., 1992. Denitri¢cation in sediment determined from nitrogen isotope pairing. FEMS Microbiology Ecology, 86, 357^362. Nickell, L.A., 1992. Deep bioturbation in organically enriched marine sediments. PhD thesis, University of London, UK. Nickell, L.A. & Atkinson, R.J.A., 1995. Functional morphology of burrows and trophic modes of three thalassinidean shrimp species, and a new approach to the classi¢cation of thalassinidean burrow morphology. Marine Ecology Progress Series, 128, 181^197. Rowden, A.A. & Jones, M.B., 1994. A contribution to the biology of the burrowing mud shrimp, Callianassa subterranea (Decapoda: Thalassinidea). Journal of the Marine Biological Association of the United Kingdom, 74, 623^635. Steingruber, S.M., Friedrich, J., Gachter, R. & Wehrli, B., 2001. Measurement of denitri¢cation in sediments with the 15N isotope pairing technique. Applied and Environmental Microbiology, 67, 3771^3778. Webb, A.P. & Eyre, B.D., 2004. The e¡ect of natural populations of the burrowing thalassinidean shrimp Trypea austaliensis on sediment irrigation, benthic metabolism, nutrient £uxes and denitri¢cation. Marine Ecology Progress Series, 268, 205^220. Witbaard, R. & Duineveld, G.C.A., 1989. Some aspects of the biology and ecology of the burrowing shrimp Callianassa subterranea (Montagu) (Thalassinidea) from the southern North Sea. Sarsia, 74, 209^219. Widdicombe, S. & Austen, M.C., 1998. Experimental evidence for the role of Brissopsis lyrifera (Forbes, 1841) as a critical species in the maintenance of benthic diversity and the modi¢cation of sediment chemistry. Journal of Experimental Marine Biology and Ecology, 228, 241^255. Submitted 4 November 2003. Accepted 26 March 2004.
