DOI: 10.5061/dryad.crjdfn34d
title: Movement data of barnacle Chelonibia testudinaria

description: Movement is a fundamental characteristic of life, yet some taxa, such as
 barnacles, lose their capacity for locomotion as swimming larvae to become
 permanently affixed to a substratum as adults. Barnacles adopted this type
 of sessile life-stage at least 500 million years ago; however, we
 unequivocally confirm a prior report that the epizoic sea-turtle barnacle,
 Chelonibia testudinaria, has the capacity for self-directedlocomotion in
 the adult stage. We used time-series field and laboratory photographs to
 document its movement paired with transplant experiments employing various
 flow conditions and inter-individual configurations to test whether
 foraging or reproduction are ultimate causes of the activity. We also
 examined the barnacle cement microscopically for insight on its role in
 translocation. On loggerhead and green sea turtles, C. testudinaria moved
 distances up to 78.6 mm/year across its host substratum and in traverses
 on laboratory panels, occasionally altered course abruptly by 90o. Our
 findings indicate these movements are behaviourally directed and not
 passively driven by external forces. Barnacles tended to move directly
 against water flow and independent of nearby conspecifics, suggesting that
 movement functions primarily to facilitate foraging, not reproduction.
 While the mechanism enabling this movement remains elusive, we observed
 that trails of cement bore signs of multi-layered, episodic secretion. We
 speculate that proximal causes of movement involve one or a combination of
 rapid shell growth, cement secretion coordinated with basal membrane
 lifting, and directed contraction of basal perimeter muscles.
description: <i>Barnacle movements on captive and wild
 turtles</i> Loggerhead turtles were caught incidentally by
 local fisheries within the Communidad de Valencia, Spain and brought to
 the Área de recuperación y conservación de fauna marina (ARCA), which is
 managed by the Fundación Oceanogràfic. The facility has a permit from the
 Valencian Regional Government for sea turtle rehabilitation. We selected
 five of the turtles admitted to the ARCA between December 2019 and May
 2020 as they each hosted at least 10 <i>C. testudinaria</i>.
 Upon admission to ARCA, each of these animals was suffering from
 decompression sickness and was thus treated and monitored for up to 18
 weeks before being released back into the wild (for details on veterinary
 care, see <sup>42</sup>). Sea turtles were housed in circular
 tanks, ranging from 2 to 6 m in diameter with a water depth of 0.95 m and
 maintained at a water temperature of ~24°C. All animals were fed twice
 daily using a mix of vegetable and fish material.
 While sea turtles were housed at the facility,
 we monitored barnacle movements on each by attaching a 1 cm length fabric
 measuring tape using clear epoxy to within 30 mm of any clusters of
 <i>C. testudinaria </i>or solitary large individuals on the
 carapaces of the selected turtles. We photographed these barnacles every
 two weeks, ensuring that the camera was placed perpendicular to the
 carapace and that the 1 cm tape measure sections were visible in every
 photograph. We measured the growth and movements of 10 haphazardly
 selected barnacles from each of the turtles in ImageJ (v. 1.52p) by using
 the tape measure sections as fixed reference points of known size. The
 barnacle shell diameters (maximum rostro-carinal length) ranged from 14 to
 41 mm (mean 24 mm) at the start of the study and increased to 14 to 45 mm
 (mean 26 mm) by the end of the study (Fig. S1). To mitigate any effect of
 growth on the measurements of barnacle movements, we only considered that
 movement had occurred if the total distance travelled within 14 weeks was
 5 mm as this exceeded the maximum observed growth in barnacle diameter
 over this same period. To assess directional movement of barnacles that
 translocated, we determined whether the barnacles moved in either an
 anterior, posterior, lateral, or medial direction. We additionally noted
 whether each individual <i>C. testudinaria</i> bore attached
 complemental males. To
 make observations of barnacle movements in the wild, we queried
 professional underwater divers at Siao Liu Qiu Island, Taiwan for photos
 of turtles with barnacles. We received photos of three green turtles with
 barnacles that were repeatedly photographed over a 16-week period. We used
 the shape of the post-ocular scutes to confirm the identity of the turtles
 and then inspected the photos visually to determine the movements of the
 barnacles<sup>43</sup>.   <i>Barnacle cementation and translocation on
 synthetic substrata </i> We
 collected <i>Charybdis</i> crabs from the shores of Taiwan using crab
 traps and selected those individuals that had
 <i>C. testudinaria
 </i>barnacles
 attached to their carapaces (Fig. S3A). To remove the barnacles, the crabs
 were euthanized and then each crab’s carapace was carefully trimmed to the
 edge of each barnacle’s base. Within 3-4 days, the remaining carapace
 adhering to the barnacles’ bases had degraded (~3-4 days) without damaging
 the membranous base of the barnacles. The “cleared” barnacles were laced
 onto 15x15 cm
 acyclic plates until they
 reattached (Fig. S3B). Successful reattachment of the barnacles was
 confirmed by the
 appearance of white cement<sup>10</sup>, which was visible around the periphery of the
 base (Fig. S3C). In total,
 15 specimens successfully reattached. We
 monitored the movements of these 15 C.<i> testudinaria</i>
 using time-series photographs for up to one year in an aerated tank. Each
 barnacle was photographed once per week at the apical, lateral and basal
 view using a digital camera (Panasonic, Lumix DMC-G1). On each acrylic
 plate, four yellow
 marker dots were
 placed on the plate as
 reference points and empty shells of <i>C. testudinaria
 </i>were attached to the plate as calibration markers. During this experiment,
 the barnacles were cultured in a Polyethylene tray (70 x 20 x 10 cm) with closed
 circulation and continuous aeration. The specimens were fed live adult <i>Artemia</i> once per day and the shell surfaces were cleaned by brushing every three days to
 avoid algal overgrowth. Sea water was changed daily.
 This same experiment was repeated a second time
 but this time, we used three <i>C. testudinaria</i> that were
 obtained from the carapaces of dead stranded green sea turtles. These
 barnacles were also photographed in apical, lateral and basal view but
 instead of recording photos weekly they were recorded daily. Each of these
 barnacles were monitored for 3, 5, and 8 months,
 respectively.   <i>Barnacle
 cement trail analysis</i> After observing that
 moving barnacles often left behind a trail of cement, we visually
 inspected the cement to see if it would provide insights into the
 mechanism enabling barnacle locomotion. This cement could be readily
 detached after air-drying for more than one week. We attempted further
 air-drying (up to a month) but this resulted in cracking of the material
 (see Fig. 3A). We also attempted cutting the cement with a razor blade,
 but this destroyed the microstructure along the cut edge (data not shown).
 Thus, the edges of natural cracks that ran almost perpendicular to the
 longitudinal axis of the cement strip were examined under SEM (FEI Quanta
 200) to obtain cross-section views of the internal microstructure of the
 cement trail. Pieces of cement trail were mounted on SEM stubs and gold
 coated prior to observation.  
 <i>Barnacle detachment force</i>
 To compare the attachment strength among barnacles that were
 either experimentally reattached to acrylic plates or naturally attached
 to crab carapaces, a shear force was applied to each test individual
 parallel to the substratum following methods specified in ASTM
 (2005)<sup>44</sup> and the force requires to detach them was
 measured with a force gauge (FG-20G, Taiwan). Barnacles on acrylic plates
 were divided into four groups for analysis based on their reattachment
 time and nested by size (1-10 (n = 10), 11-20 (n = 5), 21-30 (n =10) and
 &gt;365 days (n = 5)). Individuals from the latter cohort were similar
 in size to those naturally attached to crabs to which they were compared.
 Before detachment with the force gauge, the underside of each transparent
 acrylic plate was photographed and the area of cement secreted was
 measured using the image analysis software Sigma Scan Pro. Variations in
 the force needed to detach the barnacles were analysed using one-way
 analysis of variance (ANOVA) after the data passed a homogeneity of
 variance test<sup>45</sup>.  
 <i>Barnacle locomotion relative to current
 flow</i> To study the effect of unidirectional
 water flow on barnacles, six individuals were reattached to the centre of
 replicate 60 x 60 mm acrylic plates. Each plate was placed in a 60 x 50 x
 28 cm tank and positioned in the flow of an underwater pump (flow rate 10
 l min<sup>-1</sup>) directed out of a cylindrical PVC funnel,
 made by cutting the bottom out of a 250 ml commercial PVC bottle. The
 internal diameter of the outflow tube of the underwater pump was 9.24 mm
 and thus the flow velocity was 2.48 m s<sup>-1</sup> (Velocity
 = flow rate x tube cross section area). The experimental design included
 six replicate plates for three different treatments: (1) barnacles with
 the rostrum (cirral net) facing towards the flow (i.e., facing the narrow
 opening of the funnel), (2) barnacles with the rostrum (cirral net) facing
 away from the flow (i.e., facing the wide opening of the funnel), and (3)
 barnacles on control plates inserted into the funnel but with the water
 pump shut off. Water temperatures were maintained at 25 °C for all
 experiments (Fig. 5A, B). The distances traversed and the angles of
 movement of barnacles were measured after three months (Fig. 5D) using the
 intersection point of the paired scutum and tergum opercular plates of the
 barnacle as the center point. All barnacles were fed with
 <i>Artemia</i> nauplii, dispersed and recirculated for a
 duration of six hours per day, after which the tank water was
 exchanged.   <i>Barnacle
 locomotion relative to conspecifics</i> To
 investigate whether barnacles move to reduce mating distance, we attached
 six pairs of barnacles at inter-individual distances of 5 cm, 10 cm, and
 15 cm. Each pair of barnacles was laced onto acrylic plates 16 x 16 cm (5
 cm and 10 cm pair treatment) and 16 x 24 cm (15 cm pair treatment). The
 plates were maintained in 25<sup>o</sup>C aerated seawater.
 Barnacles were fed six hours per day and the experiment was monitored for
 10 months. We analyzed variation in inter-individual distances in each
 treatment using paired t-tests. This experiment was repeated again but
 this time using five barnacles each laced on to 16 x 16 cm acrylic plates,
 at an inter-individual distance of 5 cm (Fig. S6E). We ran five replicates
 for this experiment and each experiment lasted for 3 months. To assess
 whether barnacles were clustering, separating, or moving randomly we
 calculated the area of the minimum convex polygon needed to encompass the
 area occupied by the barnacles both at the start and end of the
 experiment. The differences in areas were analysed using paired t-tests
 (Fig. S4E). To further assess whether the nearby
 presence of conspecifics affects the movement patterns of <i>C.
 testudinaria</i>, we reattached 29 and 31 individuals<i>
 </i>onto separate acrylic plates 36 cm x 36 cm. The barnacles were
 placed at inter-neighbour distances ranging between 30-50 mm. Barnacles
 were allowed to reattach for three weeks prior to taking any measurements.
 Photographs of barnacles were collected weekly over 12 weeks. In each
 photograph, the distance to the nearest neighbour for each barnacle was
 measured using image analysis software (Sigma Scan Pro 5) and a nearest
 neighbour index (NNI) was used to assess the
 distribution<sup>46</sup>. The value was calculated using the
 following formula: R<sub>n</sub><i>=</i>D(Obs)<i>0.5 </i>an<i>   </i> where ‘R<sub>n</sub>’ is the NNI value, ‘D(Obs)’ is the average observed nearest neighbour distance in cm between the barnacles, ‘a’ is the spatial area in cm<sup>2</sup> occupied by the barnacles, and ‘n’ is the total number of individual barnacles. Values of the R<sub>n</sub> index range in a continuum from 0 to 2.15. Rn values close to 0 indicate a clustered distribution, a value of 1 indicates a random distribution and a value near 2.15 indicates a regular distribution. During all laboratory movement experiments, barnacles were kept at 25 <sup>o</sup>C water temperature, which is typical of seawater temperatures during summer in Taiwan when most barnacles produce mature gonads for reproduction. <i>Chelonibia testudinaria</i> had mature ovary and testis upon dissection of dead individuals in the experiments, suggesting that individuals were reproductively active during all movement experiments.