Seagrass as the main food source of Neaxius acanthus (Thalassinidea: Strahlaxiidae), its burrow associates, and of Corallianassa coutierei (Thalassinidea: Callianassidae)

Estuarine, Coastal and Shelf Science 79 (2008) 620–630

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Seagrass as the main food source of Neaxius acanthus (Thalassinidea: Strahlaxiidae), its burrow associates, and of Corallianassa coutierei (Thalassinidea: Callianassidae) Dominik Kneer a, Harald Asmus a, *, Jan Arie Vonk b a

Alfred Wegener Institute for Polar and Marine Research, Wadden Sea Station Sylt, Hafenstraße 43, D-25992 List, Germany Department of Environmental Science, Institute for Wetland and Water Research, Faculty of Science, Radboud University Nijmegen, P.O. Box 9010, 6500 GL Nijmegen, The Netherlands b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 February 2008 Accepted 28 May 2008 Available online 5 June 2008

Burrows of the thalassinidean shrimps Neaxius acanthus and Corallianassa coutierei are striking aspects in tropical seagrass beds of the Spermonde Archipelago, Indonesia. Burrow construction, behaviour, burrow type and associated commensal community were investigated to clarify the ecological role and food requirements of these shrimps and their commensals. Gut content analysis and stable-isotope data were used to unravel the food sources and the trophic interactions among the commensal community. Individuals of Neaxius acanthus were caught on Bone Batang Island. In narrow aquaria filled with sediment they constructed burrows resembling those found in the field. During burrow construction and maintenance only little sediment was brought to the surface, most was sorted and compacted to create a distinct lining. Maintenance work by single shrimps typically took about 5 min, after which the shrimp walked up to the entrance and rested for a similar period of time. There were no differences in behaviour between day and night. Intrasexual encounters inside the burrow were characterised by a high level of aggression and all resulted in one participant being driven out of the burrow. Intersexual encounters led to coexistence with both animals taking turns in burrow maintenance and guarding the entrance. Offered seagrass leaves were pulled underground, cut into pieces and eventually integrated into the lining. Burrows of Corallianassa coutierei resembled a deep U-shape. Chambers branching off halfway down and at the deepest point contained seagrass fragments. All steep parts of the burrow were lined similar to burrows of N. acanthus. No commensals were found associated with Corallianassa coutierei. However, burrows of Neaxius acanthus in the field typically contained a pair of shrimps, up to 8 individuals of the commensal bivalve Barrimysia cumingii and large numbers of gammarid amphipods. Other animals found associated with the burrow were the goby Austrolethops wardi, a palaemonid shrimp species and two species of tubebuilding polychaetes, one of which was also found as an epibiont on N. acanthus. Stable-isotope and gut content analyses indicate that the diet of Neaxius acanthus, its commensal Austrolethops wardi, and Corallianassa coutierei is mainly derived from detrital seagrass leaves, with a potential contribution of sediment organic matter and seagrass epiphytes. In contrast the isotopic signature of Barrimysia cumingii suggests the presence of symbiotic sulphide metabolism bacteria. This study underlines that, besides their interactions with the surrounding ecosystem, thalassinid shrimp burrows play an important role as a sub-habitat with a unique associated fauna. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: burrowing shrimps behaviour tropical seagrasses commensalism stable isotopes feeding

1. Introduction Thalassinidean shrimps are commonly found in coastal ecosystems in the tropical Indo-Pacific region (Mukai et al., 1989; Erftemeijer et al., 1993). One of these shrimp species, the strahlaxiid Neaxius acanthus, excavates large burrows in the sediment of * Corresponding author. E-mail address: [email protected] (H. Asmus). 0272-7714/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecss.2008.05.013

tropical seagrass beds where they live in pairs (Farrow, 1971; Vonk et al., 2008). The callianassid Corallianassa coutierei is another species occurring in the same habitats. These burrowing thalassinideans influence the belowground environment by sediment reworking and the creation of open spaces (burrows). Both shrimp species are observed to catch drifting seagrass leaves which are pulled into the burrows (Farrow, 1971; Abed-Navandi et al., 2005; Vonk et al., 2008). This leaf material is processed inside the burrow. Farrow (1971) assumed that the

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leaves caught by Neaxius acanthus are incorporated into the burrow lining of the shrimps. A study of the underground behaviour of the Caribbean Corallianassa longiventris has shown that caught leaves are cut into pieces and stored in special chambers before consumption (Dworschak et al., 2006). This so-called ‘‘microbial gardening’’ (Abed-Navandi et al., 2005; Atkinson and Taylor, 2005) might also be executed by N. acanthus since the amount of leaves caught is well in excess of the dietary needs of the shrimp (Vonk et al., 2008). The isotopic signature of N. acanthus suggests seagrass as its main food source (Vonk et al., 2008), either direct or via microbial gardening. Both shrimp species therefore also interact with the aboveground ecosystem, by preventing the export of organic matter from the seagrass meadow and promoting nutrient cycling (Vonk et al., 2008). Burrow associates and ectosymbionts are often found in thalassinidean burrows (Atkinson and Taylor, 2005). Farrow (1971) reported a ‘‘commensal grapsid crab’’ and the presence of the ‘‘suspension-feeding’’ bivalve Erycina sp. in burrows of Neaxius acanthus, along with secondary commensals (the mesogastropod Capulus sp.) on the bivalve. An additional commensal, the goby Austrolethops wardi, was found in burrows of N. acanthus in Indonesia, either as single specimens or pairs (Kneer et al., 2008). However, the food sources of the burrow associates of N. acanthus remain unknown. The aim of this study was to elucidate the behaviour of Neaxius acanthus, especially with regard to burrow building, feeding mode and the fate of collected seagrass leaves, to determine symbiotic organisms living in N. acanthus and Corallianassa coutierei burrows, and to construct the food web inside the burrows. Behaviour of N. acanthus was studied in aquaria. In the field a number of burrows of both shrimp species were cast using polyester resin. Organisms living inside the burrows were collected and the food web inside the burrow was analyzed using both gut content and stable-isotope analysis. 2. Materials and methods

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sandy shoal with a surrounding reef flat and was covered by a barrier reef on the wave exposed side. An extensive multi-species seagrass meadow covered the reef flat, which consisted of coarse carbonate sand and coral rubble comparable to the nearby Barrang Lompo Island (93–100% CaCO3; Erftemeijer, 1994). The most common macrobenthic fauna species in the meadow were the shrimp Neaxius acanthus, large bivalves like Pinna muricata and the sea urchin Tripneustes gratilla (Vonk et al., 2008). The shrimp Corallianassa coutierei also occured, but the densities were much lower (ca. 0.1 opening m2, personal observation) compared to N. acanthus (ca. 2 openings m2, Vonk et al., 2008). 2.2. Behaviour of Neaxius acanthus In January 2006 the behaviour of Neaxius acanthus was observed in narrow aquaria (49.0  4.5  49.5 cm; l  w  h) filled with sediment (about 40 cm depth) from the study site, water (about 10 cm above sediment) and living seagrass shoots. All aquaria were individually supplied with running seawater and two weeks after filling one shrimp was introduced. The progress of burrow construction was monitored daily for two weeks. Once the burrow resembled those in the field the behaviour of four single animals was observed for 15 min at four time points (noon, evening, night and morning) during two consecutive days. The time spent on 12 defined behavioural states was measured using a stop watch (Table 1). These behavioural states were integrated into 5 behavioural classes and the time the animals were not visible was added. Intraspecific behaviour was observed by introducing another shrimp into these aquaria. Four enforced encounters were observed: resident female vs. female, resident male vs. male, and resident male vs. female (2). We offered the shrimps in the aquaria seagrass leaves to determine their fate after being pulled into the burrow. Over a period of 15 min three leaves were offered to the shrimps, based on the frequency of leaf-catching events observed in the field. This was repeated 6 times with both single shrimps and couples.

2.1. Study area 2.3. Burrow characteristics The experiments were carried out in the Spermonde Archipelago, which consists of a large group of coral islands and submerged reefs on the continental shelf along the west coast of South Sulawesi, Indonesia (see Stapel et al., 2001 for map and details). For the research location we chose Bone Batang (5 0100400 S; 119190 4400 E), an uninhabited coral island located 15 km offshore and 2 km north of Barrang Lompo Island. The island consisted of an intertidal

In the field casts were made of burrows of Neaxius acanthus (n ¼ 5; see also Vonk et al., 2008) and Corallianassa coutierei (n ¼ 3) using polyester resin. The material was mixed on the spot and poured into the burrows using a funnel and tube. After two days hardening the casts were dug out and attached sediment was removed. The location of collected seagrass material, burrow

Table 1 All behaviour of Neaxius acanthus observed were ascribed to one of the 12 behavioural states which were integrated into 5 behavioural classes Behaviour Class

State

Description

Construction

Bulldozering Carrying Digging Manipulating objects Scratching Stirring Tamping

Pushing sediment in front of the carapace Sediment is carried in a basket formed by maxillipeds 3 and pereopods 1 and 2. Setal rows on the pereopods close the basket Picking up sediment which is then carried somewhere else Working stones and shell fragments out of or into the ground or cutting seagrass leaves into pieces Stirring on the glass wall or on a shell fragment to remove algal layers Sorting sediment by stirring movements of maxillipeds 3 and pereopods 2; pereopods 1 can be included to enhance effects Tamping sediment and plant fragments into the burrow walls using stabbing movements and mucus secretetion from maxillipeds 3 and/or pereopods 2, while this mixture is secured by pereopods 1 and 3.

Grooming

Cleaning

Pereopods 4 are used to clean the setal rows on pereopods 2, maxillipeds 3 clean the antennae and pereopods 5 clean the ventral side of the carapace

Feeding

Feeding

Manipulating sediment and litter with the mouth parts

Locomotion

Turning Walking

Performing a forward somersault followed by a half roll along the body axis or by rotating 180 Moving forwards or backwards

Resting

Resting

No activity except for breathing movements of scaphognathite and pleopods 3–5

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dimensions and the number and position of commensal organisms were determined. The influence of burrow openings of Neaxius acanthus (see also Vonk et al., 2008) and Corallianassa coutierei on sediment composition was analyzed. Six cores (diameter 16 cm, depth 30 cm) were taken from the meadow at undisturbed locations, and six cores each were taken encircling N. acanthus and C. coutierei burrow entrances. From the cores without burrows (undisturbed), sediment sub samples were taken from the surface, 15 and 30 cm depth. From the cores encircling burrows, burrow lining material was collected from the same depths. The thickness of the burrow lining was recorded. All visible animals and plant fragments were removed from the sediment samples. Wet weight, dry weight (48 h at 70  C) and ash-free dry weight (4 h at 500  C) were determined. Grain size composition of sediment and lining material was analyzed after shaking dried sediment samples for 10 min on a series of test sieves (JEL 200 T, J. Engelmann AG; mesh sizes 2000, 1000, 500, 250, 125 and 63 mm). 2.4. Food-web analysis Samples were collected of shrimps, commensals (amphipods, fish and bivalves), seagrasses, seagrass epiphytes, lining material and sediment organic matter for stable-isotope food-web analysis and additional stomach-content analysis of the shrimps and fish. All material was collected within a square of approximately 5  5 m in the intertidal zone within two weeks in October 2005. 2.4.1. Primary producers and sediment organic matter Six leaf samples of fresh (from the centre of the shoot and free of epiphytes) and six samples of senescent (from the margin of the shoot, heavily overgrown with epiphytes and easily detachable) material were collected from the three most common seagrass species (Thalassia hemprichii, Halodule uninervis and Cymodocea rotundata) around the sampled burrows. Three samples of a mixture of detached leaves were collected from the sediment surface. Epiphytes were removed from the senescent and detached leaves and pooled. Burrow lining was obtained from six burrows of Neaxius acanthus and six burrows of Corallianassa coutierei by separating all lining from sediment in cores (30 cm depth). Sediment organic matter was collected from two sediment cores not containing living seagrass rhizomes (10 cm depth). All macroscopic organic particles were picked out by hand and regarded as the big fraction of sediment particulate organic matter (sPOM > 1 mm). The resuspendable fraction of the remainder was obtained by shaking a mixture of the sediment sample and filtered seawater in a glass jar and decanting the supernatant which was then filtered using a GF-F (0.7 mm) filter. This was then regarded as small fraction of sediment particulate organic matter (sPOM < 1 mm). All samples were dried at 60  C for 48 h. 2.4.2. Consumers Six individuals of Neaxius acanthus and three individuals of Corallianassa coutierei were caught by luring them close to the entrance of their burrows with a seagrass leaf and blocking the retreat with a metal spike. Six individuals of Austrolethops wardi and two individual amphipods were caught by injecting ca. 20 ml of 5.25% NaClO into the burrows using a flexible tube and catching animals leaving the burrow with a handheld net. One commensal cleaner shrimp was caught by hand in the burrow entrance of N. acanthus. Six commensal clams were dug out of the burrows. All animals were killed by putting them in a freezer overnight. Muscle tissue was separated and dried for 48 h at 60  C (except for the amphipods which were dried whole). The rest of the animals was then conserved in 4% seawater formalin and gut contents of

N. acanthus, C. coutierei and A. wardi were analyzed under a stereomicroscope. 2.4.3. Stable-isotope analysis All food-web samples were ground to a fine powder in a ball mill (Retsch MM 2000), decalcified (except for muscle tissue and seagrass leaves), weighed into tin capsules and subjected to d13C/ d15N and C/N analysis using continuous-flow gas isotope ratio mass spectrometry (CF-IRMS) (Isoprime, Micromass, UK). The results are expressed in the standard d unit notation as dX ¼ [(R samples/R reference)  1]  1000, where X represents 13C or 15N, and R represents the 13C:12C or 15N:14N ratios. The standard reference materials were Pee Dee Belemnite standard (PDB) for carbon and atmospheric N2 for nitrogen. Sample analytical precision on the machine was 0.1& for d13C and 0.15& for d15N. 2.5. Statistical analysis One-way ANOVA was used to compare the mean percentage of time spent in the different behavioural classes at the four time points, to compare the percentage of different grain sizes from different depths and from the burrow lining, and to compare the 13 C and 15N signatures of fresh and senescent seagrass leaves. Sediment grain size data were arcsin root transformed and seagrass isotope data were root transformed prior to analysis to achieve normal distribution. Seagrass leaf litter, burrow lining and sPOM 13 C values were compared using a Kruskal–Wallis test. 3. Results 3.1. Behaviour of Neaxius acanthus Within two weeks after introduction of the shrimps in the aquaria they all had constructed a burrow resembling those cast in the field. The animals first created a depression in the ground. The moment this depression had grown to a tunnel, the whole animal would slide tail first into this and tamp the sediment around the entrance. The tunnel was then enlarged, dumping the sediment on the surface. At about 15 cm depth a chamber was created to permit turning around. Thereafter, the entrance was only approached head first and the burrow was never left again. Now only a small amount of the sediment was brought to the surface and most was reworked inside the burrow. Small fragments were sorted out by stirring, resulting in a small pile of sediment which was carried in a basket (see Table 1 for extended behaviour descriptions). This fraction was incorporated into the burrow wall by tamping behaviour to create a distinct lining. A tunnel was then dug from the floor of the first chamber and a second chamber was created about 15 cm below the first one. Blind tunnels of about 10 cm length were frequently dug starting from the chambers. Excavated sediment was dumped on the chamber floor, sorted and then used to refill the tunnel. These tunnels never lasted for more than 12 h but already showed the typical burrow lining. Maintenance work by single shrimps typically took about 5 min, after which the shrimp walked up to the entrance and rested for a similar period of time. The mean percentage of time spent in the different behavioural classes by single shrimps in aquaria (32 observations of 15 min) at the four time points was normally distributed with the exception of the behavioural class ‘‘feeding’’. No significant differences between day and night time activities were detected for the other behavioural classes, including the time the animals were not visible (ANOVA, all p > 0.05). Data from the different time points were therefore pooled (Fig. 1). Construction (43%) was the main activity for Neaxius acanthus followed by resting (32%), which was almost exclusively performed in the burrow entrance, where the animals were positioned 81% of the total time spent resting and 28% of the

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The seagrass leaves offered to the shrimps were dragged into the burrow. Most leaves were not directly consumed, but rather shredded into pieces using their chelae and maxillipeds and scattered on the chamber floor where this material was buried. The fragments were then included in the sorted sediment and incorporated into the burrow walls.

3.2. Burrow characteristics

Fig. 1. Time budget of single individuals of Neaxius acanthus heltered in aquaria as estimated from 32 observations of 15 min on 4 animals. Bars are standard errors (SE).

total time spent on construction. Exclusively walking (walking while carrying or stirring was ascribed to construction) took 15% of the animal’s time. Feeding made up only a small part of the time (1%), but this probably also occurred while the shrimp was tamping and stirring. They performed brushing movements on the burrow wall, indicating this might play some role in nutrition. Animals were not visible during 6% of the time. When we introduced a new shrimp into an aquarium, it instantly tried to descend into the existing burrow. Both intrasexual encounters were characterised by a high level of aggression, up to breaking off the opponent’s appendages and collapsing the roof to form a barrier. This resulted in one participant being driven out of the burrow which started to construct a new burrow. In contrast, the two intersexual encounters led to coexistence, with the newcomer walking past the resident and immediately starting to maintain the burrow structure. From thereon, both shrimps took turns in resting in the entrance and burrow maintenance in the deeper parts. Exchanges of position took place approximately every 5 min and were typically initiated by the shrimp in the deeper parts by touching the tail fan or pleopods with the antennae. The entrance was never unattended for a prolonged period of time, as observed in the field.

Burrow characteristics were obtained from casts. The single entrance of the Neaxius acanthus burrow was usually situated in a shallow funnel-shaped depression (Fig. 2A). Aggregations of coral rubble around the opening were common. The following vertical shaft was cylindrical (diameter 1.7  0.1 cm) and stabilized with a lining consisting of a mixture of small sediment grains and fragments of multi-cellular plants as observed using light microscopy. Up to three small chambers were positioned between the opening and the large basal chamber (50  4 cm depth), which contained fragments of seagrass leaves and from which short protuberances extended into the surrounding sediment. All walls with an inclination of more than about 45% were lined. Total burrow volume was 1.8  0.6  103 cm3 and surface area was 1.8  0.3  103 cm2 (Table 2). Burrows of Corallianassa coutierei started with a small funnel, followed by a cylindrical steep shaft (diameter 9.5  1.3 mm) with a chamber-like extension situated approximately halfway down (Fig. 2B). At the deepest part (69  15 cm) the shaft branched into several tunnels leading in all directions, which ended in swollen extensions filled with seagrass debris. All burrow walls were lined comparable to Neaxius acanthus. Lined shafts filled with sediment were found, probably leading to the surface. Total burrow volume was 0.24  0.10  103 cm3 and surface area was 0.07  0.02  103 cm2 (Table 2). Only one cast contained an individual of C. coutierei, pointing at the existence of further tunnels which were not filled with resin in the other two. Several different commensals were found in Neaxius acanthus burrows (Table 2). The goby Austrolethops wardi (Teleostei: Gobiidae) was found as a single specimen or as pairs in the burrows. Up to 8 individuals per burrow of the bivalve Barrimysia cumingii (Bivalvia: Galeommatidae, Fig. 3A) were associated with the casts.

Fig. 2. Burrow morphology drawn from casts of Neaxius acanthus (A) and Corallianassa coutierei (B) showing also the position of symbiotic species, collected seagrass material and accumulations of coarse sediment. The biggest and the smallest (volume) casts per shrimp species are depicted.

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Table 2 Burrow properties measured from the burrow casts and number of some of the animals (host shrimp Neaxius acanthus or Corallianassa coutierei, goby Austrolethops wardi, bivalve Barrimysia cumingii) trapped in the resin Species

Shaft

Maximum extension

Surface area

Volume

B

h

4

mm

cm

N. acanthus

15 16 16.5 17.5 18

C. coutierei

7 10.5 11

cm

cm2

cm3

43 63 51 39 53

54 65.5 37.5 71 55.5

1506 1416 1429 1273 3105

42.5 69 96

27.5 31.5 27

308 633 1048

At least one species of gammarid amphipod was found in large numbers within each burrow (Fig. 3B). A spirorbid polychaete was found growing on the carapace of N. acanthus (Fig. 3C) or on the burrow lining (Fig. 3D), where another tube-building polychaete species was also found (Fig. 3E). Crabs (Fig. 3F) and juvenile fish (genera Dischistodus and Apogon) sought refuge in the entrance at low tide. A palaemonid shrimp species (Fig. 3G) was occasionally observed on the burrow lining (Fig. 3H) or sitting on its host shrimp. We did not find any commensals associated with burrows of Corallianassa coutierei. The lining of Neaxius acanthus (2–10 mm) burrows was thicker than the lining of Corallianassa coutierei (2–4 mm) burrows. Sediment analysis showed a significant difference in organic content and grain size composition between undisturbed sediments and burrow linings, but it did not show differences in grain size composition and organic content with depth (0, 15 and 30 cm) in each of these structures. The organic content of the lining of the burrows of N. acanthus (16% of DW) and C. coutierei (5% of DW) was higher compared to the organic content of the surrounding sediment (3% of DW) and consisted of fragments of multi-cellular plants (observed using light microscopy). The burrow linings showed a significantly higher proportion of small grain sizes (classes between 0 and 63 mm and 63 and 125 mm) compared to the surrounding sediment (ANOVA, F(28, 70) ¼ 10.259, p < 0.001; Fig. 4). 3.3. Food web 3.3.1. Primary producers and sediment organic matter Of all primary producers and organic matter, the epiphytes were most depleted in d13C (13.36&) and most enriched in d15N (4.17&). Epiphytes also had the lowest C/N ratio (10.10). Seagrass leaf stable-isotope signatures differed between species and changed with leaf age. Values ranged from 12.26& (senescent Halodule uninervis) to 8.58& (fresh Thalassia hemprichii) for d13C, and from 1.97& (detached leaves, mixture of all species) to 3.19& (fresh T. hemprichii) for d15N. Senescent seagrass leaves in general show a trend towards depletion in both 13C and 15N compared to fresh material (Fig. 6). Significant differences were measured between both d13C (ANOVA, F (5, 30) ¼ 16.63, p < 0.01) and d15N signatures (ANOVA, F (5, 30) ¼ 4.69, p < 0.01) of fresh and senescent seagrass leaves. Detached leaves were not included in the statistical analysis due to the small number of replicates. A Tukey HSD post hoc test revealed significant differences between the d13C signatures of senescent Halodule uninervis leaves compared to all other groups (p < 0.01) and fresh H. uninervis leaves compared to fresh Thalassia hemprichii (p < 0.05). For d15N signatures significant differences were found between fresh T. hemprichii and senescent T. hemprichii and C. rotundata leaves (Tukey HSD post hoc test, all p < 0.01; Table 3, Fig. 6). Seagrass C/N ratios increase with age

No. of shrimps

No. of gobies

No. of bivalves

1347 1182 1290 1209 4083

2 1 1 2 2

0 2 0 1 1

4 8 5 5 7

76 216 413

1 0 0

0 0 0

0 0 0

(fresh < senescent < detached) and range from 13.12 (fresh T. hemprichii) to 27.73 (detached leaves, mixture of all species). The d13C and d15N signatures of the burrow lining of both shrimp species and sPOM < 1 mm were comparable. The d13C and d15N signatures of Neaxius acanthus lining and sPOM < 1 mm were positioned within the variation of the detached seagrass leaves (Kruskal–Wallis test, p > 0.05). Corallianassa coutierei lining and sPOM > 1 mm, however, were within this range only with their d15N signature, while the d13C signature was more depleted (C. coutierei lining, Kruskal–Wallis test p < 0.01) or enriched (sPOM > 1 mm, Kruskal–Wallis test p < 0.05) (Fig. 7). Sediment organic matter C/N ratios are very similar, with a range from 9.65 for sPOM < 1 mm to 10.67 for N. acanthus lining, only the value measured for sPOM > 1 mm (16.75) stands out from the rest. 3.3.2. Consumers The gut analysis revealed that the shrimps Neaxius acanthus (6 individuals) and Corallianassa coutierei (3 individuals) had only ingested seagrass fragments besides sediment particles (Fig. 5A,B). Guts of the goby Austrolethops wardi (6 individuals) were literally filled with seagrass fragments, some of them bearing cutmarks probably caused by N. acanthus, while lower quantities were found of cyanobacteria (1 out of 6), fragments of small red algae (4 out of 6) and harpacticoid copepods (2 out of 6; Fig. 5C–H). The cyanobacteria and red algae are species found as epiphytes on seagrass leaves (personal observation). The stable-isotope food-web analysis showed that the animal most depleted in 13C was the bivalve Barrimysia cumingii (17.32&), followed by the cleaner shrimp (13.65&), Austrolethops wardi (10.77&), the amphipods (9.19&), Neaxius acanthus (8.77&) and Corallianassa coutierei, which was most enriched (7.58&). Barrimysia cumingii was also most depleted (0.92&) in 15N, with the amphipods (3.7&), C. coutierei (4.42&), N. acanthus (4.87&), A. wardi (6.51&) and the cleaner shrimp (6.67&) being more enriched (Table 3, Fig. 7). 4. Discussion 4.1. Behaviour and burrow morphology The only activities observable in the field were tamping sediment in the walls next to the entrance, ejecting sediment or rubble parts, catching drifting leaves or collection of leaves growing next to the entrance. Burrows in the field were usually occupied by pairs (Vonk et al., 2008) and the partners took turns in guarding the entrance about every 5 min, which is about the interval between two successful leaf-catching events (personal observation). Aquarium observations showed that Neaxius acanthus reworks sediment inside the burrow by sorting sediment to increase compaction. Almost no sediment was expelled outside once the

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Fig. 3. Commensals of Neaxius acanthus: bivalve Barrimysia cumingii (A), gammarid amphipod (B), spirorbid polychaetes on tail fan of host shrimp (C) and on burrow lining (D), tube-building polychaete (E); and animals occasionally found in burrow entrance of N. acanthus: crab (F), palaemonid shrimp (G, H). All scale bars are 5 mm.

burrow was established. The shrimp used fine sediment and seagrass litter to produce a burrow lining. We observed that N. acanthus burrow lining is agglutinated, most likely using mucus excreted from mucus glands as shown before in Pestarella (formerly

Callianassa) tyrrhena and Pestarella candida (Dworschak, 1998). This type of lining was also created by Corallianassa coutierei, probably by using seagrass material and/or faecal pellets as described for Corallianassa longiventris (Dworschak et al., 2006). Lining was

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Fig. 4. Mean grain size composition of sediment (A), and lining material from burrows of Neaxius acanthus (B) and Corallianassa coutierei (C) (SE). Significant differences per grain size are denoted (s ¼ significant, p < 0.05; hs ¼ highly significant, p < 0.01).

produced on all walls with steep angles. The plant fragments and the excreted mucus gave the lining its high organic content and provided a high stability of the burrow structure. The morphology of N. acanthus burrows cast in the field corresponds to the burrows built in aquaria: a more or less vertical shaft connects one or more turning chambers to a large basal chamber, from which finger-like extensions (‘‘mining operations’’) protrude into the surrounding sediment (Dworschak et al., 2006). The animals might mine for sPOM for feeding, for small sediment particles to build their burrow lining, or both. This similarity in burrow structure indicates that the behaviour of the shrimps inside the aquaria was representative of their behaviour under natural conditions. Two shrimps of different sexes quickly cooperate within one burrow (observed in aquaria) and take turns in ‘‘guarding’’ the burrow entrance (observed in the field and in aquaria). The shrimps benefit from pair bonding by dividing the labour of territorial defence and maintenance (Mathews, 2002). This is also an effective way to ensure reproduction at low densities (Shimoda et al., 2005). Since they are vulnerable outside, this can ensure reproduction

without leaving their burrows. As observed for Neaxius vivesi (Berrill, 1975), Neaxius acanthus never completely left its burrow, neither in the field nor in aquaria. Animals placed in the open are vulnerable to predators. Three individual N. acanthus shrimps were quickly consumed by the bream Pentapodus trivittatus (Teleostei: Nemipteridae), when placed outside their burrow (personal observation). The high level of intersexual aggression observed, which could lead to serious damage, and the voluminous burrow structure (up to 4l), makes it unlikely that this species exists at much higher densities than the maximum 9 burrows m2, as observed in these meadows (Vonk et al., 2008) and reported for N. vivesi (Leija-Tristan, 1994). If burrows become connected, individuals will be killed or driven out of their burrow. However, Neaxius spp. have no territory outside their burrows, since burrow openings can be as close as 5 cm together (Berrill, 1975, personal observation). The types of behaviour observed in this study are very similar to those described by Stamhuis et al. (1996) for Callianassa subterranea and Dworschak et al. (2006) for Corallianassa longiventris and Pestarella tyrrhena. Dworschak et al. (2006) distinguish between a ‘‘sediment only’’, a ‘‘catching seagrass’’ (continuous supply of leaves) and a subsequent ‘‘with debris in burrow’’ mode in their behavioural study. Since the aquaria used in the present study were put with living seagrass plants there always was a small amount of seagrass available in the burrows. This excluded the observation of behaviour in a pure ‘‘sediment only’’ mode sensu Dworschak et al. (2006) and the time budget in Fig. 1 more represents a ‘‘with debris in burrow’’ mode. Dworschak et al. (2006) observed that C. longiventris spent considerably more time on debris manipulation in the ‘‘catching seagrass’’ mode compared to the other modes. In this study, no detailed records of the time spent on behavioural states were made while offering the shrimps seagrass leaves, and only one leaf was offered every 5 min. The leaves were, however, cut into pieces rather quickly, scattered on the burrow floor and subsequently treated like sediment. Some of the time Neaxius acanthus spends on sorting sediment is therefore also debris manipulation. In accordance with Stamhuis et al. (1996) we suspect that thalassinidean behaviour is very stereotypical. Neaxius acanthus do not seem to be really influenced by the presence or absence of a partner since they perform the same routine (e.g. switching between resting in the entrance and construction work in the deeper parts of the burrow at 5-min intervals) whether they are alone or not. As observed both in the field and in aquaria they obviously also are not influenced by the presence or absence of daylight. They do, however, strongly respond to floating objects near the burrow openings and to shrimps of the opposite sex. Collection of seagrass litter (Vonk et al., 2008), and processing of this material inside the burrow, appeared to be important for the shrimp. We frequently observed the manipulation of plant debris and the burrow wall with the mouth parts, indicating that microbial gardening may be important for the nutrition of the shrimp. Based on our observations we exclude suspension feeding as proposed by Farrow (1971) for Neaxius acanthus because the animals lack specialized filtering appendages as in Upogebia spp. and the burrows have only one opening and therefore do not permit an unidirectional filtration flow (Griffis and Suchanek, 1991). We confirm the bivalve Barrimysia cumingii, which we assume to be equivalent to Erycina sp. in Farrow (1971), as a commensal in the Neaxius burrows, but did not find the mesogastropod Capulus sp. as a secondary commensal on the bivalve (Farrow, 1971). However, a number of additional, previously undescribed commensals were found: the goby Austrolethops wardi (cf. Kneer et al., 2008), amphipods and two polychaete species. The bivalve B. cumingii, the goby A. wardi and the polychaetes were never observed outside the burrows during this study, we therefore assume that they truly depend on Neaxius acanthus burrows. We also found B. cumingii, but not A. wardi or the polychaetes, in cores

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Table 3 Number of replicates (n), carbon and nitrogen stable-isotope signatures (mean  SD), C, N contents and C/N ratios of Neaxius acanthus, Corallianassa coutierei, commensals of N. acanthus, and potential food sources. Animal species identification numbers as in Fig. 7 are shown in parentheses n

d13C (&)

d15N (&)

C (%)

N (%)

C/N

6 3 1 2

8.77  0.4 7.58  0.32 13.65 9.45 to 8.94

4.87  0.27 4.42  0.12 6.67 3.21 to 4.20

42.91  2.65 40.85  2.74 32.45 34.05 to 45.39

12.03  0.85 11.67  1.13 8.07 6.89 to 9.11

3.57  0.08 3.50  0.12 4.02 4.94 to 4.98

6

10.77  0.87

6.51  0.32

40.20  5.69

11.83  1.92

3.40  0.08

6

17.32  0.98

0.92  0.72

36.80  2.75

8.88  0.64

4.14  0.13

Primary producers Thalassia hemprichii fresh T. hemprichii old Halodule uninervis fresh H. uninervis old Cymodocea rotundata fresh C. rotundata old Seagrass leaf litter (all species) Epiphytes

6 6 6 6 6 6 3 1

8.58  0.39 8.97  0.49 10.13  0.74 12.27  1.66 9.26  0.51 9.49  0.33 10.23  2.05 13.36

3.19  0.67 2.01  0.66 2.77  0.34 2.49  0.49 2.37  0.26 2.05  0.37 1.97  0.84 4.17

36.03  1.25 37.22  0.51 35.44  0.88 36.01  2.18 41.52  0.85 37.42  0.82 32.67  4.00 16.63

2.75  0.37 2.21  0.27 2.20  0.18 1.75  0.15 2.91  0.14 2.39  0.16 1.22  0.31 1.65

13.12  1.45 16.84  2.57 16.08  1.3 20.59  1.72 14.25  0.42 15.67  0.88 27.73  6.55 10.10

Sediment organic matter Lining Neaxius Lining Corallianassa sPOM < 1 mm sPOM > 1 mm

6 6 2 2

11.13  0.04 12.64  0.25 12.08 to 11.98 7.86 to 8.25

1.70  0.15 1.36  0.27 0.53 to 1.76 1.44 to 2.96

7.30  1.04 0.86  0.11 2.02 to 3.79 6.74 to 9.34

0.68  0.11 0.09  0.01 0.20 to 0.39 0.37 to 0.62

10.67  0.17 10.10  0.79 9.65 to 9.99 15.12 to 18.39

Consumers Crustacea (1) N. acanthus (2) C. coutierei (3) Cleaner shrimp (4) Amphipod Teleostei (5) Austrolethops wardi Bivalvia (6) Barrimysia

from a sandy area with high densities of Glypturus martensi (Thalassinidea: Callianassidae) next to the seagrass meadows studied, indicating its association with more than one shrimp species. Amphipods are abundant anywhere in the seagrass meadow, the species found in the burrow might also occur outside. The palaemonid shrimp tended to leave the burrow when disturbed. We therefore consider it an opportunistic commensal. The same applies for the crabs and juvenile fishes because they were only observed seeking refuge in the burrow entrances at low tide when they represented small tidal pools. The co-occurring shrimp Corallianassa coutierei constructs burrows very similar to those described for Corallianassa longiventris by Dworschak et al. (2006), e.g. a U-shaped shaft with chambers branching off at different depths. The behaviour is also remarkably similar: C. coutierei keeps the burrow openings closed with sediment plugs most of the time. We observed that these plugs were removed in response to elevated water movement (e.g. waves or researcher activities). Corallianassa coutierei was also observed to catch drifting seagrass leaves, but commensal organisms were absent in its burrows. It cannot be excluded, however, that small animals like amphipods, which are hard to see when enclosed in the resin, were overlooked. The absence of larger commensals might be due to the much narrower burrow structure without large chambers. Another likely factor is the smaller amount of seagrass available in the burrow. Intake rates have never been quantified for C. coutierei but seem to be much lower than for Neaxius acanthus, as reflected in the lower organic content of the burrow lining (5% against 16%) and its lower thickness (2–4 mm against 2–10 mm). No pairs of C. coutierei have been found inhabiting the same burrow. 4.2. Food web The seagrass species had different stable-isotope signatures. This difference was statistically significant for the d13C signature of fresh Thalassia hemprichii and fresh Halodule uninervis. Fresh leaf material of larger seagrass species seems to be more enriched in d13C compared to smaller species from the same site, as shown before (Vonk et al., 2008). With increasing age, leaves become more

depleted in both d13C and d15N. The signatures of detached leaves fall within the range defined by the senescent leaves, and all three species are assumed to contribute equally to this pool since their production is about the same in the meadow studied (Vonk et al., 2008). As observed in the field, the shrimp species rarely have access to (fresh or senescent) attached leaves. Only detrital leaves are likely to end up in the shrimp burrows in large quantities. Other accessible food sources are the burrow linings and sPOM. Compared to detrital leaf material, Corallianassa coutierei lining is slightly more depleted and sPOM > 1 mm slightly more enriched. The values for Neaxius acanthus lining and sPOM < 1 mm are within the variation of the detrital leaves (no significant differences). Since seagrass roots are more enriched in 13C than leaf material (Vonk et al., 2008) it is assumed that their presence within sPOM > 1 mm causes this relative enrichment. sPOM and burrow lining particles seem to be increasingly more depleted with decreasing size of organic particles since N. acanthus lining contains the biggest leaf fragments (up to several millimeters, personal observation). sPOM < 1 mm and burrow lining probably have similar signatures because they essentially consist of the same material, e.g. seagrass-derived detritus. The majority of sPOM in the seagrass meadow originates from seagrass plants, while the organic part of the burrow linings of the two shrimp species represents a mixture of sPOM, which is mined for and sorted by the shrimps, macerated detrital seagrass leaves which are caught at the burrow entrance, and mucus secreted by the shrimps. This would also explain the almost identical C/N ratios of sPOM < 1 mm and the burrow lining of both shrimp species. The isotopic fractionation (D) of decapod muscle tissue not treated with HCl ranges from 1.1 to 3.1& for Dd13C (Stephenson et al., 1986) and from 2.4 to 3.7& for Dd15N (Parker et al., 1989; Yokoyama et al., 2005). For whole amphipods treated with HCl, the diet-tissue fractionation ranges from 1.5 to 0.4& for Dd13C and from 0.7 to 2.7& for Dd15N (Macko et al., 1982). The diet-tissue fractionation of non-acidified teleost muscle tissue is between 0.39 and 3.7& for Dd13C and from 0.77 to 5.6& for Dd15N (Tominaga et al., 2003; Gaston and Suthers, 2004). Fractionation rates seem to be species-specific, so controlled feeding experiments with consumers are desirable. Before this is accomplished, applying an

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Fig. 5. Seagrass fragments and sediment grains recovered from guts of Neaxius acanthus (A), Corallianassa coutierei (B) and Austrolethops wardi (C, cutmarks caused by N. acanthus); recognizable fragments of seagrass epiphytes like cyanobacteria (D) and red algae (E, F, G) were only found in guts of A. wardi, besides harpacticoid copepods (H). Scale bars are 1 mm (A–G) and 250 mm (H).

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Fig. 6. Carbon and nitrogen stable-isotope signatures (SD) of fresh (f) and senescent (s) leaves of Thalassia hemprichii (Th), Cymodocea rotundata (Cr) and Halodule uninervis (Hu).

isotope source mixing model (Phillips and Gregg, 2001) might lead to a significant over- or underestimation of an individual source because of small errors in fractionation values. If, however, an isotopic fractionation of 0–1& for Dd13C and 3–4& for Dd15N is assumed, in spite of the above-mentioned uncertainties, Neaxius acanthus, Corallianassa coutierei and Austrolethops wardi could all be considered primary consumers of detrital seagrass leaves (Fig. 7). This is confirmed by the gut content analysis: the dominant food item in guts of N. acanthus, C. coutierei and A. wardi was seagrass. Amphipods are commonly considered to be important consumers of seagrass detritus (Tomascik et al., 1997). If an isotopic diet-tissue fractionation <3 for Dd15N is assumed for the amphipods, they would also join the group of seagrass consumers. The treatment of the amphipods has been different from the treatment of the other animal samples due to their small size (acidified whole animals vs. non-acidified muscle tissue). Yokoyama et al. (2005) state that Dd13C and Dd15N can vary within one species depending on the treatment. Their values measured for acidified whole crustaceans were smaller than for untreated muscle tissue, which is reflected in Macko et al. (1982).

629

We consider the combined evidence from gut content and stable-isotope analyses sufficient to identify senescent seagrass leaves as important food sources for Neaxius acanthus and Corallianassa coutierei. Additional uptake of the burrow lining, organic debris embedded within the sediment (sPOM) and small sediment grains is also considered very likely, based on our observations. Burrow lining consists of detached seagrass leaves and small sediment grains, and N. acanthus was observed to perform stirring behaviour to collect parts of this material. sPOM and burrow lining had C/N ratios which were lower compared to seagrass leaf material, indicating that they might be a superior food source (Abed-Navandi and Dworschak 2005). The importance of the burrow lining for nutrition is also addressed by Koller et al. (2007), who measured elevated microbial and meiofaunal numbers in burrow walls of Pestarella (formerly Callianassa) tyrrhena. Neaxius acanthus and C. coutierei may also consume epiphytes and meiofauna (both of which have lower C/N ratios compared to seagrass), but these items are probably ground beyond recognition by their mandibles and gastric mills. Austrolethops wardi and Amblygobius sp. (Vonk et al., 2008) are the first gobies for which a seagrass diet could be established. In addition, seagrass epiphytes and crustaceans were present in the stomach of A. wardi. In another study on the gut contents of A. wardi, seagrass leaves were the most dominant item but seagrass epiphytes and small crustaceans were also found (Liu et al., 2008). The cleaner shrimp is most depleted in 13C and most enriched in 15 N of all organisms analyzed from the shrimp burrows. This suggests a food mixture of algae and small animals. We did, however, only analyze a single specimen. The bivalve Barrimysia cumingii is an exception compared to the other burrow associates, because it is strongly depleted in both 13C and 15N. Carlier et al. (2007) likewise found that the bivalve Loripes lacteus was strongly depleted in both 13C and 15N compared to the food sources present in the food web analyzed (the Lapalme Lagoon in the northwestern Mediterranean). Loripes lacteus is known to harbour sulphide-metabolising symbiotic bacteria which are considered responsible for the isotopic signature of their host (Carlier, personal communication). Felder (2001) reports that the bivalve Phacoides pectinatus, which harbours sulphide-metabolising bacteria, is associated with sulphide-rich burrows of Axianassa australis. We have not measured sulphide but found high concentrations of ammonium and reactive phosphate in burrows of Neaxius acanthus (Vonk et al., 2008). The high concentrations of dissolved nutrients resulting from the decay of seagrass material in burrows of N. acanthus could explain the presence of sulphidemetabolising symbionts. The two polychaete species found associated with Neaxius acanthus or its burrow were not analyzed. Their food requirements remain unknown. 5. Conclusion

Fig. 7. Mean d13C and d15N signatures of Neaxius acanthus (1), Corallianassa coutierei (2), cleaner shrimp (3), amphipods (4), Austrolethops wardi (5), Barrimysia cumingii (6) and their potential food sources. The area surrounded by dotted lines includes all primary consumers of detrital seagrass leaves, provided that the isotopic diet-tissue fractionation is between 0 and 1& (d13C) and between 3 and 4& (d15N): a ¼ d15N of detrital seagrass leaves  1 SD þ 3, b ¼ d15N of detrital seagrass leaves þ 1 SD þ 4, c is parallel to the y-axis and defined as d13C of detrital seagrass leaves  1 SD, d has a slope of 3 and its extension intersects the point (d13C of detrital seagrass leaves þ 1 SD/d15N of detrital seagrass leaves  1 SD).

By collecting detached leaves Neaxius acanthus and Corallianassa coutierei prevent the export of organic material, and their individual behaviour promotes nutrient recycling and may therefore tighten the pathway between seagrass production and decomposition (Vonk et al., 2008). This study provides further evidence for the central role of thalassinidean shrimps in tropical seagrass ecosystems: in constructing voluminous and stable burrows N. acanthus creates a sub-habitat within seagrass meadows which is used by a variety of commensal organisms. The shrimps take turns in ‘‘guarding’’ the entrance and catching leaves, therefore predators are kept out and a continuous supply of detrital seagrass leaves is maintained. The bivalve Barrimysia cumingii, the goby Austrolethops wardi, the two polychaete species and possibly the amphipods were never observed outside

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a burrow, we therefore consider them to be obligatory commensals. Neaxius acanthus and Corallianassa coutierei mainly feed on detrital seagrass leaves. Only a fraction of this leaf material is consumed immediately after collection, the rest is stored in special chambers, buried as sPOM during mining activities, or incorporated into the burrow lining. All these activities lead to improved C/N ratios. We found evidence that the commensal goby Austrolethops wardi, and probably the amphipods as well, rely on the seagrass accumulated by their host shrimp for nutrition. The cleaner shrimp isotopic signature might be caused by the uptake of benthic microalgae, and the bivalve Barrimysia cumingii seems to profit from elevated sulphide levels in the burrows, caused by decaying plant matter, via symbiotic bacteria. Corallianassa coutierei shows a similar behaviour to Neaxius acanthus but commensal organisms are absent from its much narrower burrows which are closed most of the time. Whether the striking behavioural similarities between Neaxius spp. and Corallianassa spp. are an evolutionary basal trait, as suggested by Dworschak et al. (2006), and are therefore homologous, or rather are products of the convergent evolution of infaunal deposit feeders as adaptations to survival in nutrient-poor sediments remains an interesting question. The function of commensals found in N. acanthus burrows also remains unknown. However, it appears that symbiotic organisms might not only be attracted by the shelter created, but also by the food available. Acknowledgements Saido, Japri and Sayuri Sarinita are acknowledged for assistance in the field. Jamaluddin Jompa and Magdalena Litaay at the CCRR (Center for Coral Reef Research) within UnHas (Hasanuddin University) provided valuable administrative support in Indonesia. The authors also thank Pierre Richard and Gael Guillou at CRELA, UMR 6217 CNRS-Ifremer-ULR in L’Houmeau/France for their help with the stable-isotope analysis. The investigation by D.K. on Neaxius acanthus was part of a diploma thesis within the SPICEproject (Science for Protection of Indonesian Coastal Ecosystems) financed by the German Federal Ministry of Education and Research (BMBF). J.A.V. was supported by NWO-WOTRO grant W86-168. References Abed-Navandi, D., Dworschak, P.C., 2005. Food sources of tropical thalassinidean shrimps: a stable-isotope study. Marine Ecology Progress Series 291, 159–168. Abed-Navandi, D., Koller, H., Dworschak, P.C., 2005. Nutritional ecology of thalassinidean shrimp constructing burrows with debris chambers: the distribution and use of macronutrients and micronutrients. Marine Biology Research 1, 202–215. Atkinson, R.J.A., Taylor, A.C., 2005. Aspects of the physiology, biology and ecology of thalassinidean shrimps in relation to their burrow environment. Oceanography and Marine Biology: An Annual Review 43, 173–210. Berrill, M., 1975. The burrowing, aggressive and early larval behavior of Neaxius vivesi (Bouvier) (Decapoda, Thalassinidea). Crustaceana 29, 92–98. Carlier, A., Riera, P., Amouroux, J.M., Bodiou, J.Y., Escoubeyrou, K., Desmalades, M., Caparros, J., Gre´mare, A., 2007. A seasonal survey of the food web in the Lapalme Lagoon (northwestern Mediterranean) assessed by carbon and nitrogen stable isotope analysis. Estuarine, Coastal and Shelf Science 73, 299–315. Dworschak, P.C., 1998. The role of tegumental glands in burrow construction by two Mediterranean callianassid shrimp. Senckenbergiana Maritima 28, 143–149. Dworschak, P.C., Koller, H., Abed-Navandi, D., 2006. Burrow structure, burrowing and feeding behaviour of Corallianassa longiventris and Pestarella tyrrhena (Crustacea, Thalassinidea, Callianassidae). Marine Biology 148, 1369–1382.

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