N.melanotragus is a herbivore, actively grazing on microalgae, cyanobacteria, and diatoms attached to intertidal substrates. N.melanotragus only feeds when submerged, hence the incoming tide is most favourable as the intertidal zone is inundated at this time (Davey 2000). Once the tide inundates the intertidal zone enough, this species moves from sheltered rock platforms out into the exposed pools. The incoming tide (high tide) also is thought to replenishes food resource deposits within this environment (Chapperon et al. 2011), meaning more microalgae for the Black Nerite when feeding during this period. Like many other marine snails, two cephalic feeding tentacles are constantly touching and feeling the substrate. The extension of the oral hood from the apical end of the animal acts like a vacuum cleaner, constantly attached and sliding across the substrate scrapping up microalgae using its radula covered in rows of teeth (Borell 2004; Rohde 1981).

N.melanotragus is a dioecious (separate sexes) species which spawns almost all-year round in small rock pools on the intertidal zones of eastern Australia, with peak spawning observed throughout summer (Przeslawski 2008; Przeslawski 2011). N.melanotragus becomes sexual active at 20 months of age. After internal fertilisation occurs, females N.melanotragusproduces white, elliptical egg capsules filled with colourless jelly (each containing 20-50 eggs), attaching it to the underside of rocks within a sheltered rock pool (Figure 5). Eggs hatch after 14-15 days after attached to the substrate (Anderson 1962). N.melanotragus larvae can emerge as both lecithotrophic veliger and crawling juveniles; however, crawling juveniles are less frequent (Przeslawski 2011). Intertidal environmental factors such as temperature, salinity, and wave exposure are thought to affect the time in which the N.melanotragus larvae is released. Hence, enabling the species to develop and use multiple modes of larvae development through hatching plasticity. Yet, it remains unknown if N.melanotragus truly displays both of larval forms (Przeslawski 2011). N.melanotragus’ juveniles take two years to reach full adult size (Underwood 1975).

Recent morphological and genetic evidence has suggested that what was previously known as Nerita atramentosa may have been a combination of two quite distinct species within Australia. Nerita melanotragus and Nerita atramentosahave been consistently confused within past ecological and taxonomic literature. N.melanotragus is located predominately on the eastern coast of Australia, northern New Zealand, and Norfolk Island. Whereas, N.atramentosa is distributed along southern Australia and is often confused with its warmer water relation due to their extremely similar biology (Spencer et al. 2007). One distinction between these animal’s biology is the colour of the operculum. N.melanotragus has an orange/tan operculum, and N.atramentosa has a black operculum.

Introduction
This experimental investigation aims to identify the behavioural response of N.melanotragus individuals towards abnormal thermal stress. It is hypothesised that N.melanotragus individuals under increased thermal condition will show closer aggregation than under normal conditions. N.melanotragus is said to aggregate in large groups during warmer conditions such as a midday low tide. Displaying such behaviour is quite common within intertidal gastropods, and is thought to reduce dehydration, and body temperature of the animals (Chapperon et al. 2012; Chapperon et al.2013).

Method
N.melanotragus individuals were collected from Coolum Beach, Queensland, Australia (26º53’97”S, 153º097’378”E). Ethics approval and collection permits/licenses were not required for this species. Twenty-four individuals were collected during low tide, and identified as N.melanotragus owing to their orange/tan operculum. Only individuals that were between the sizes of 24-28mm in size were collected, this measurement was taken from the anterior to posterior ends of the shell. Specimens were then transported to the closed-circuit marine aquarium at the University of Queensland St Lucia within 2 hours of collection. N.melanotragus specimens were given four days to adjust and settle into the new environment before the first experiment. Three experiments were conducted over a two-week period; experimental set up for all experiments can be seen in Figures 6 & 7.

The preliminary experiment included six specimens evenly placed out (14cm apart) over one smaller tray, inside a larger tray hold hot freshwater (40-50ºC). Specimens were added to the experimental tray once seawater inside the tray hit 39ºC. Experiment was conducted for 10 minutes recording observations on the behaviour of the specimens. After the experiment, these specimens were then transported back into the marine aquarium.

The first of the main experiments was the treatment experiment, the specimen’s response to thermal stress. Treatment and control experiments had both three replicates; however, only two trays could fit inside the larger tray, meaning two replicates were done, and then the third replicate was done after the first two (under same starting condition). Two plastic trays holding a small amount of seawater in each within the larger tray containing hot freshwater (40-50ºC). Hot freshwater was refreshed between the preliminary and the first experiment. Once the seawater within the smaller trays was heated to 36ºC, 12 new specimens were added to the two trays (6 per tray, 14cm apart), and another 6 specimens were added to the third replicate (18 specimens across 3 replicates). Experiment was conducted for 35 minutes, where observations, distance to the nearest neighbour for each of the six specimens, and the percentage of the specimens that were aggregated at each 5-minute time interval were recorded over the three trays. An aggregation was considered formed when direct contact between two or more specimens occurred (Chapperon et al. 2013); number within the aggregation was recorded and put into a percentage of the sample population (aggregation of 2, 2/6= 33%). Two thermometers were used to observe the water temperature of the trays, and if water was becoming too hot/too cold, warm/cold water was added to the larger tray. The treatment experiment held the seawater temperature at a range between 33-35ºC. After the experiment, the specimens were transported back to the marine aquarium. One week between the treatment and control experiments were given, ensuring that thermal stress endured the treatment experiment did not carry over to the control experiment.

The control experiment had the same experimental set up and parameters as the treatment; however, room temperature freshwater was added to the large tray instead of the hot freshwater. Seawater within the smaller trays were held at 24ºC (room temperature). The same 18 Black Nerite specimens were used in both treatment and control experiments. 

Results
Data collected from the experiments were analysed using statistical t-tests, revealing insignificant results for both percentage aggregated (df= 10, t= 0.68, p= 0.509), and distance to nearest neighbour (df= 14, t= 1.72, p= 0.106). Although the data was found to be insignificant, an obvious relationship between the percentage of aggregate specimens within the treatment environment, evident through Figure 8. R² for the treatment and control calculated by the graph’s trend line as .918 and .0406, showing a substantial difference. The relationship between the treatment environment and the distance of the nearest neighbour was not seen evident within Figure 9.

Discussion
Within the preliminary experiment, it was observed that all six specimens did not move from their original positions, yet did stay attach to the substrate. Under such conditions (39ºC), it is common for these animals to enter a heat coma, restricting movement but also continuing to attach to the substrate, reflecting this animal's robust nature (Chapperon et al. 2013).  

The statistical analysis showed the experimental results as insignificant, and therefore the original hypothesis cannot be supported. Despite these insignificant results, the R² values for specimen’s aggregated (%) do reflect a substantial difference between the treatment and control environments evident though figure 7. Aggregation behaviour is thought to maintain the moisture and reduce the evaporation rate by decreasing the surface-to-volume ratio in contact with the atmosphere (Chase et al. 1980). This similar behaviour is seen within other molluscs such as mussels, by which aggregated mussels exhibit lower body-temperatures by 4-5ºC than solitary mussels (Helmuth 1998). The aggregation behaviour of N.melanotragus does significantly increase water content, also assisting in decreasing the body temperature of the aggregated animals. Not only is this behaviour essential in regulating thermal stress, but aggregations of N.melanotragus could potentially reduce the risk of dislodgement by waves, and effect predation by crustaceans (Chapperon et al. 2013). Within the preliminary experiment, it was observed that all six specimens did not move from their original positions, yet did stay attach to the substrate. Under such conditions (39ºC), it is common for these animals to enter a heat coma, restricting movement but also continuing to attach to the substrate (Chapperon et al. 2013). Investigating such thermoregulatory behaviour could suggest that mobile, intertidal invertebrates like the Black Nerite may be less vulnerable than previously thought to the warming climate.

N.melanotragus circulatory system is analogous to other mollusc circulatory systems, yet, owing to torsion, the pericardial cavity and the heart has moved to the anterior visceral mass. Vetigastropods such as N.melanotragus are diotocardian, and only hold one atrium within its circulatory system (this can be seen within Figure 10).

N.melanotragus excretory system is assumed to be similar to other intertidal gastropods. These animals switch between two modes of excretion, depending upon specific environmental conditions; uricotelic during the outgoing tide and ammonotelic (ammonia is end product) on the incoming tide (Ruppert et al. 2004). Nephridium is a blind sac surrounded by hemocoel located on the anterior end of the visceral mass owing to torsion. Kidneys of the animal are located at the end of the sac, connecting to the pericardial cavity with a renopericardial canal. Nephridiopore within the excretory system opens at the rear of the aniaml’s mantle cavity, allowing waste product to be removed by the respiratory water current (Ruppert et al. 2004; Taylor et al. 1988).

The digestive system of N.melanotragus includes: a mouth, buccul cavity, esophagus, stomach, intestine, rectum, and anus (Figure 12). Digestion within this animal is both intercellular and extracellular. Extracellular digestion requires numerous enzymes to efficiently break down microalgae. These enzymes are produced by a combination of several digestive organs such as the salivary glands, esophageal pouches, and digestive ceca. Extracellular digestion occurs within the stomach of the digestive tract, whereas intercellular digestion occurs in the digestive ceca. Owing to the torsion of the stomach 180º, the esophasgus and intestine are connected posteriorly and anteriorly respectively (Ruppert et al. 2004).

The muscular foot is responsible for the movement of the Black Nerite, assisting them in feeding, prey capture, and reproduction (Figure 13 shows semi-extended foot). The columellar muscle is connected to this foot, allowing for efficient extension and retraction. Within the sole of the foot are tarsos muscles which also contribute to the gastropod’s locomotion (Ruppert et al. 2004). This sole is extremely flat and broad in N.melanotragus, reflecting the ability to move around a variety of different substrates. The power for locomotion in the Black Nerite is provided by muscular waves moving along the ventral surface of the foot, where these waves are attached to the substrate surface by pedal mucus. This mucus adheres to the substrate, allowing the animal to move/crawl (Denny 1980). An advantage of this mucus is that the animal can chemically change the consistency of the mucus depending upon the animal’s needs (Ruppert et al. 2004). Wave-exposed areas require stickier gel to prevent waves washing them off the substrate, whereas the mucus may be a less adhesive liquid when the animal is situated in sheltered areas.