Chemosensitivity and role of swimming legs of mud crab, Scylla paramamosain, in feeding activity as determined by electrocardiographic and behavioural observations

Crabs and holding condition

A total of 20 female and male specimens of adult mud crab, S. paramamosain, (9–11 cm in carapace width, 80–100 g in body weight) caught from the wild population were obtained from a local market, Kota Kinabalu, Sabah. They were stocked in a plastic tank (122 cm ×148.5 cm, 55 cm high) filled with 55 L of seawater (30 cm deep) and placed in the roofed Shrimp Hatchery, Borneo Marine Research Institute, Universiti Malaysia Sabah. Half of the tank bottom was covered with 5 cm thick coral rubble to function as a biofilter substrate. Five pipes of polyvinyl chloride, 11 cm diameter and 20 cm long, were placed at the tank bottom as shelters for the crab. Tank water was aerated to ensure adequate oxygen level and the temperature, salinity, pH, and concentration of dissolved oxygen ranged from 25.8 to 28.9 °C, 19.47–20.05 ppt, 6.89–7.29, and 5.6–6.8 mg L⁻¹, respectively. These parameters were measured using a pH/ORD/EC/DO tester (Hanna Instruments, HI 9828, Washington, USA). The crabs were fed with fresh squid (Loligo sp.) in the morning and fish (Decapterus sp.) in the afternoon.

Electrocardiography

Three crabs (one male and two females) were used for the electrocardiography experiment. The specimens were excitable in response to the movement of the experimenter, so their eyes were painted with white nail polish to temporarily block their vision. The crabs were cold-anesthetised followed by drilling a small hole of about 0.5 mm diameter at each cardiac lobe of the carapace to expose the pericardial membrane. Two active electrodes (enamel-coated copper wire, 0.05 mm diameter) were implanted through the holes and glued with cyanoacrylate. Crab specimens with implanted electrodes were left for a day to recover in separate tanks with well-aerated seawater at a temperature 28 °C, pH 8.1, and salinity 20 ppt.

To record the electrocardiogram (ECG), a test crab with two implanted electrodes was placed in a plastic test chamber (17 cm × 30 cm × 9.5 cm) filled with filtered seawater to 5 cm depth. The water level was enough to submerge the carapace. A third electrode was placed in the chamber as a ground reference. The recording wires were of sufficient length to allow the freedom of movement of the crab. However, the test specimens seldom moved during the ECG recordings. The test chamber water was kept at a temperature of 28 °C, pH of 8.1, and salinity of 20 ppt. The ECG was linked to an amplifier (MEG-1200, Nihon Kohden, Tokyo, Japan) aligned to a digital converter (FTT3P7PVB, ALS Controls, and Instruments, Nishinomiya, Japan), which converted analogue signals to digital signals. The signals were then processed with EasyLogger software (Easy SYNC Ltd., Glasgow, Scotland) and displayed on a laptop monitor. Stable ECGs were obtained 15–30 min after the crab specimens were placed in the test chamber (Fig. 1). Subsequently, a stimulus (touch or sugarcane juice) was applied to the appendages. The ECGs were recorded during 25 s of stimulation.

Stimuli

Each appendage of the crab was stimulated thrice in two ways (touch and chemical). The appendages were touched three times at intervals of 15–30 min with a hand-held micropipette (heat-pulled glass capillary with a 50 µm tip). For each crab, touch stimulation was conducted at the swimming leg dactyls, walking leg dactyls, claws, antennae (tip and middle part), antennules (lateral and medial flagella), and mouthpart (third maxilliped). Although the lateral and medial flagella are close to each other and frequently in motion, it was possible to touch them with the micropipette separately.

For the chemical stimulation, fresh sugarcane juice was selected because it contains 9.6–10.9% sucrose and 0.22–0.33% glucose and fructose, which are more effective feeding stimulants than amino acids for the fiddler crab Uca pugilator (Robertson, Fudge & Vermeer, 1981). The sugarcane juice measuring 0.1 mL (diluted to 50% or 10% with test chamber water, and filtered through paper Whatman No.1) was applied to the appendages by a hand-held Teflon pipette. Pipetting is one of the common methods to deliver a chemical solution to test crustaceans (Cericola & Daniel, 2010; Aggio et al., 2012). During the chemical stimulation, the threshold concentration of the sugarcane juice at the mouthparts and walking legs were maintained to comply with previous reports. According to Liew et al. (2020), the threshold concentration of the sugarcane juice at the mouthparts and walking legs was between 1% and 10% in S. tranquebarica. The movement and dispersion of the sugarcane juice around the appendages were conspicuous due to its greenish colouration. Thereafter, the crab was removed and the test chamber was emptied. A control test was performed by replacing the sugarcane juice with the tank water (0.1 mL) before the application of the sugarcane juice, which was presented in triplicates.

To prevent the diffusion of the sugarcane juice from the antennule to the antennae, the antennules were cauterised using an electric cauterizer (Gemini BC00314, Cellpoin Scientific, Gaithersburg, Germany) after applying the sugarcane juice. Thereafter, the sugarcane juice was applied to the antennae. For the cauterization, the crab was taken out of the test chamber, cold-anesthetised, and gently held with a wet towel, and the tip of the cauterizer was touched on the distal segment bearing the outer and inner flagellum. After the cauterisation, the crab was placed back into the chamber and left for 20 to 30 min to recover to its stable and uniform ECG. The antennae were stimulated with 10% and 50% sugarcane juice. Each stimulation was delivered at an interval of 20–30 min to minimise residual responses to a previous stimulus and to avoid desensitization.

Recording and measuring electrocardiograms

Fifty-four stable ECGs were obtained from three test crabs: constant interbeat intervals (IBIs) of 0.5 −0.8 s and constant amplitude of 0.3 −0.4 mV. During the stimulus application, the ECGs were examined for the IBIs, measured to 0.1 s. The first post-stimulus IBI was considered as the relevant response. The IBI values were normalised by logarithmic transformation (Nishi, Kawamura & Matsumoto, 2004): normalised interval= log ₁₀(T+1), where T is the raw value of the IBI (s). Each test IBI was then compared with the mean pre-test IBI (the test-beat is out of the 95% confidence interval calculated using 10 pre-test IBIs).

Behavioural bioassay

During the stimulation at each appendage, the motion of other appendages was observed visually and combined with video recordings using CCD cameras with temporal resolutions of 0.017 s, corresponding to 60 frames per second (Olympus Tough TG-3, Olympus Corporation, Tokyo, Japan). A total of 63 video recordings were analyzed, which were played back to examine the feeding behaviour on a computer screen.

After the electrocardiography experiment, the feeding behaviour of each crab was observed in the test chamber. The antennule was stimulated with a tweezered small piece of salted fish flesh (Decapterus sp.) (about 10 mm ×5 mm, two mm thick) obtained from a local market. To stimulate the walking legs or swimming legs, a small piece of salted fish flesh was placed close to their dactyl. As described by Liew et al. (2020), the feeding behaviour in response to the stimulation was evaluated based on three appendage movements: (i) increase in antennular flicking rate, (ii) remarkable movement of the third maxillipeds, and (iii) probing the test chamber bottom with the claws. These behaviours were visually observed, video-recorded, and a total of 30 video recordings were analyzed.

Electrocardiography

The electrocardiogram showed typical heartbeat response, slower heartbeat, or wider IBI of the crab, after the 10% or 50% sugarcane juice stimulation was presented to the swimming leg dactyl (Fig. 2A). However, no response was observed by mechanical touch stimulation (Fig. 2B). The crab with the antennules showed the heartbeat response, while those with the cauterised antennules showed no response to the stimulation by the sugarcane juice application given to the antennae. A statistically significant (P <  0.05) cardiac response to the sugarcane juice was recorded for the swimming legs, walking legs, claws, antennules (both the lateral and medial flagella), and mouthparts (Table 1). Regarding the touch test, statistically significant responses (P <  0.05) were observed for the walking legs, claws, antennae, and mouthparts of three specimens (Table 2). Both the lateral and medial flagella were not touch-sensitive. In the control test, the crab exhibited neither the cardiac response nor the feeding behaviour in response to tank water that was applied to all appendages.

Behaviour change in response to stimuli

Stimulation with the sugarcane juice elicited both the cardiac and feeding response of the crab. During electrocardiography, the mud crab exhibited an increase in the antennule flicking rate, claw probing, and movement of the third maxillipeds. These responses occurred following the stimulation of the antennules and dactyls (both swimming and walking) with sugarcane juice. Such feeding behaviour was not observed since the antennae were touch-sensitive (Table 2), but not chemosensitive (Table 1) . The touch stimulus alone did not elicit the feeding behaviour at all.

In response to a small piece of salted fish flesh placed on the swimming leg dactyl, the mud crab immediately exhibited movement of mouthpart appendages and kicked the flesh to the claw under its body (Fig. 3). Other exhibited features include pushing the flesh to the walking leg dactyl and grabbing the piece with the claw to bring it to the mouth. There was a clear involvement of the swimming legs in feeding.