Behavioural Toxicity of Arsenic Trioxide: Alteration in Auto-Grooming Behaviour of a Freshwater Prawn, Macrobrachium lamarrei

TABLECREATED

Table 1: Several environmental contaminants induced behavioural effects on M. lamarrei.

Methodology

Prior to an organized experiment, to evaluate the alterations of auto-grooming behaviour in M. lamarrei, preliminary laboratory observations revealed a specific auto-grooming behaviour pattern in this species. Prawns were sampled from ponds (natural habitat) and were maintained in the laboratory in clear tap water. Twenty prawns were acclimatised for seven days in arsenic free tap water; the toxicity range was predetermined; 1.72 ppm arsenic trioxide was found to be non toxic in an exposure regimen of 15 days. Thereafter prawns were transferred to thisnon-lethal dose for 24 hours for videography. Assessment of the behaviour pattern was undertaken by recording the behaviour of prawns executed in both clear tap water (control) and arsenic dosed water.The entire study and experiments were done in glass aquaria. Temperature was maintained between 25-270 C. Food (flour globules) was given ad libitum, once every day at 4pm considering their nocturnal habit. The recording was done in a Canon EOS DSLR camera (High Definition mode), using 18-55 mm lens, manual focusing, without a tripod. The video-graphic evaluation of auto-grooming in both arsenic contaminated and arsenic free water was conducted followed by manual quantification from the recorded videos. The quantification was done in terms of time spent for grooming.

Scanning electron microscopy

While analysing the behaviour pattern, the thoracic appendages were identified to be used effectively for the purpose of grooming. A prawn was sacrificed, and the appendages were dissected out and fixed in 3% glutaraldehyde overnight, to see the adaptive modifications by scanning electron microscopy (SEM). Samples for SEM were then dehydrated using 30% - 50% - 70% - 90% - 100% ethanol (30 minutes at each step). Critical Point Drying (CPD) was done to eliminate the alcohol and the super dried samples were then gold coated to observe under a SEM which was done at the University of Burdwan, West Bengal, India.

Results and Discussion

Analysis of auto-grooming patterns in Macrobrachium lamarrei

The body of Macrobrachium lamarrei is roughly divided into two parts; the outer hard exoskeleton (non-living part) and soft organs under the exoskeleton (like, gills mouth, eyes, abdomen, eggs). Grooming or cleaning of these parts is specifically executed by efficient use of the first pair of chelipeds and the fifth pair of pereiopods.

Depending on the intricate observation study of the grooming pattern, we have categorised the phenomenon as anterior grooming and posterior grooming, depending on the body regions. The anterior grooming broadly includes carapace grooming (exoskeleton) and cephalothorax (living part) grooming. Folding of the jointed first pairs of chelipeds enable them to approach inside the carapace to groom and clean the branchial chamber (Figure1a,b) and also to groom and clean the top and sides of the carapace.

Posterior grooming includes abdomen cleaning. Abdomen of a prawn is a segmented part with discrete joints, with relatively small postural degree of freedom. Apparently, a prawn�s abdomengrooming posture would depend on the relative angles of the abdomen. These angles make a favourable posture for the animal to clean the abdomen, both dorsal and ventral regions. The abdomen is covered by a hard dorsal exoskeleton. M. lamarrei cleans the ventral part of the abdomen with the first pair of chelipeds (Figure 2a) .Very rarely they use their second chelipeds to clean the abdomen. The fifth pair of pereiopods mainly cleans the dorsal part of the abdomen (Figure 2b).

Adaptive modifications in the grooming appendages of Macrobrachium lamarrei

Adaptive modifications of the grooming appendages have been described in different species [6,17]. Accordingly SEM analyses demonstrated in the present investigation intricate modifications in the key grooming appendages.

The above figures demonstrate the intricate, finer adaptations (microscopic structures) on the grooming appendages. The first pair of chelipeds is the major grooming appendage. Electron micrographs of this appendage show different types of setation on its anterior portion (Figure 3a). Higher magnifications show long filamentous grooming structures which originate in tufts or bunch from a socket like structure (Figure 3b). The filaments have human palm like structures with pointed finger like microstructure (Figure 3c). The propodus have two separate varieties of setae (Figures 4a & 5a). The first type on the propodus is comb like microstructures with blunt blades (Figure 4d). The comb blades have human palm like microstructures with pointed finger like projections (Figure 4e). The last type of setae on the propodus has bilayers of leafy structures with pointed ends revealed at the higher SEM magnification (Figures 5f,g).

The fifth pairs of pereiopods are secondary appendages used for grooming but not as frequently as the first pairs of chelipeds. It is used only for the grooming of the exoskeleton of the abdomen. The anterior region of the fifth pereiopods has dense setation bearing leafy or comb like microstructures with pointed blades (Figures 6a-c).

Microscopic study of the appendages clearly indicates that they are appropriately modified to increase the efficacy of the auto-grooming act. The first chelipeds are found to be responsible for cleaning most of the body; for this reason, they have diverse setation on them, which probably helps to efficiently clean the interior corners of the branchial chamber and the genital region. The fifth pereiopods are not used for interior cleaning as evidenced by a simple pattern of setation.

Macrobrachium lamarrei is a sensitive species and have a prominent grooming performance. Auto-grooming pattern is described in several other prawn species, however, unlike other prawns, M. lamarrei does not show prominent antennal grooming mechanisms. However, antennal grooming is very essential as the antennule and the antennal flagellum are sensory organs for chemoreception and olfaction.

Comparative profile of auto-grooming in arsenic free and arsenic contaminated water

Quantification of grooming activity in terms of time spent is a convenient way to distinguish between patterns. (Figure 7) demonstrates that in water contaminated with 1.72 ppm of arsenic trioxide noticeably increase in grooming activity in this species. This concentration of arsenic trioxide is not acute for this species. Depending on the auto-grooming pattern (described earlier) the process can be divided into four major groups CG = Carapace Grooming, where the carapace is groomed; VCG = Ventral Cephalothorax Grooming, where the inner part of cephalothorax is groomed through the ventral region; DAG = Dorsal Abdomen Grooming, where the dorsal part of the abdomen is groomed and VAG = Ventral Abdomen Grooming, where the ventral abdomen is groomed. The division is made considering the distribution of vital organs and body position. CG and VCG belong to anterior grooming (AG) and DAG and VAG represent posterior grooming (PG).

Subtypes of auto-grooming in M. lamarrei also demonstrate the same scenario (Figure 8). All the subtypes have enhanced grooming activity in the contaminated environment as compared to the arsenic free water. The mean rate of VCG, DAG, VAG in arsenic contaminated ambience is more than double the rate of VCG, DAG, VAG in arsenic free water (Figure 8).

Analysis of the subtypes of AG suggests that the mean rate of CG and VCG in arsenic free water are approximately same. However, in arsenic contaminated water, the mean rate of VCG is more or less double the mean rate of CG. Similarly, in PG, mean rates of DAG and VAG in arsenic free water do not differ much. However, in arsenic contaminated water, the mean rate of VAG is approximately twice the mean rate of DAG. The ventral region of the body, which is not covered by the exoskeleton, is groomed more in the arsenic contaminated water and arsenic is found to induce a higher rate of grooming activity in the soft visceral part of the body (Figure 8).

In normal arsenic free condition the average rate of AG is considerably higher than the rate of PG; probably, the anterior region of the body have relatively vital organs, morphologically more complex and susceptible to accumulation of more unwanted matters than the posterior region. In arsenic contaminated water, the mean rate of AG and PG increased significantly against the rate of grooming in arsenic free condition (Figure 9).

Conclusion

Arsenic is a known neurotoxicant, which can modulate neuronal activities in animals. Our study establishes a fundamental approach to consider grooming behaviour as an index or marker of sensitivity and stress caused by toxicants in aquatic invertebrates using M. lamarrei as a model. Studying robust behaviours can give us indications of the status of neuronal activities in an organism. This study further reveals a significant change in the pattern of grooming activity in Macrobrachium lamarrei due to a metalloid toxicity. We conclude that a non-lethal concentration of arsenic trioxide can trigger neuronal activity in such a way that it can induce changes in the grooming pattern of the study organism, within only 24 hours of exposure.

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