Muscular Foot Background
A characteristic of all molluscs is the present of a muscular foot. This foot is primarily used for locomotion, but can have other functions such as defense, capture, or reproduction depending on the organism (Ruppert et al. 2004). In gastropods, this foot is mostly used for locomotion and reproduction. As P. ebeninus organisms are not carnivorous, they do not utilize their foot for the capture of prey. The foot is flat and broad to allow locomotion on a variety of substrates. Although some smaller snails in muddy environments use ciliary to create propulsion, P. ebeninus rely strictly on muscles with propelling waves and the production of mucus to maintain contact with the substrate. The type of wave used for motion determine the how much of the foot is used and where the contraction occurs. The columellar muscles present allow for the retraction and extension of the head and foot.
A fine bundle of muscles present at the sole of the foot is known as the tarsos muscle that is mostly responsible for the locomotion and molding of egg cases in females. The sole may also be divided into 3 sections: the anterior propodium, middle mesopodium locomotary region, and the posterior metapodium containing the operculum. The connection of the muscular foot is considered to vary between different substrates and organisms. It is hypothesized that P. ebeninus will have greater muscular foot strength measured with an increased foot diameter and shell size on sandy substrates, the most similar substrate to their natural habitat. The aims of this study are to test the muscular foot strength of P. ebeninus on different substrates including: 1) hard substrate 2) sandy substrate and 3) rocky substrate through the analysis of A) foot diameter and B) shell length. 


Study Site & Collection 
This study was conducted at the University of Queensland in St. Lucia during the month of May in 2014. At the low morning tide on North Stradbroke Island at approximately 10:30 am, 21 specimens of Pyrazus ebeninus were collected on the mudflat about 1.5 km from the Dunwich Stradbroke Flyer Jetty.

Specimens were placed in a large collection container partially filled with water and mudflat substrate. (Fig. 1) The organisms were then carried back to the laboratory through multiple public transportations with about 18 Morula marginalba specimen. In the lab, both specimens were then added to the aquarium in a single enclosure isolated from other organisms.

Figure 1. Specimens after collection in container with mud substrate prior to adding water and Morula marginalba specimen.

Methods 

The specimens were removed from the aquarium 6 days later into a shallow tray filled partially with water. Upon removing the specimens from the aquarium, it was noted that the M. marginabla samples were seen preying on the P. ebeninus specimens. Three P. ebeninus specimens experienced mortality identified by empty shells with boring holes in the upper region of the teleoconch. The remaining living specimens were fitted with a slip knot to the dorsal region of its shell with additional line at the end using fishing line. Specimens were left approximately 1 hour to become adapted to the fishing line attachment and specimen tray. The specimens, once seen mobile and attached to the hard substrate, were then clipped to a Pesola precision spring scale using the additional line from the slip knot. Each specimen was tested for muscular foot attachment strength using the spring scale measuring up to 50 grams of force. The testing process consisted of a continuously increasing gentle upward pull until the organism released from the test substrate (Fig. 2). The specimens, once tested, were then measured for shell length and foot diameter using a ruler out of the specimen tray and were then recorded into an excel spreadsheet (Fig. 3). The organisms were then placed in a separate tray filled with water to avoid pseudo-replication. Due to the discovery of the M. marginabla feeding habits, the P. ebeninus specimens were moved to an isolated aquarium partition to avoid further predation of any other organisms after the first week. This method was repeated for sandy and rocky substrates. The forces for the individual specimens was recorded for each substrate and used for comparison in statistical analyses upon the completion of the trail over a 2 week time span. With each substrate test, new slip knots were tied for every individual specimen and proper adaptation periods between trials were administered of about 1 hour. The specimens were returned to North Stradbroke Island upon the completion of the trial period.  


Figure 2: Specimens of Pyrazus ebeninus being tested on hard substrate using the Pesola precision scale.


Figure 3: Pyrazus ebeninus specimen being measured for shell length from base to tip of teleoconch post substrate test.


Results
Upon completing the 3 substrate tests, the data was analyzed using linear regressions to compare the different substrates. I analyzed each data set for shell length and foot diameter in correlation with foot strength measured. The highest correlation of foot strength occurred in the rocky substrate with shell length (R²=0.735; Fig 4). Subsequently, the highest correlation of foot strength for foot diameter also occurred on the rocky substrate (R²=0.552; Fig. 5). The lowest correlation for foot diameter compared to muscular foot strength occurred on the sandy substrate (R²=0.375; Fig. 5), while the lowest correlation of shell length to muscular foot strength occurred on the hard substrate (R²=0.390; Fig 4). The average foot strength was highest on the hard substrate (28.38±3.28), followed by sandy substrate (11.76±1.44), with the lowest found on rocky substrate (10.44±1.08). 


Figure 4: Correlation comparisons between shell length (cm) and muscular foot strength (g) sandy, rocky and hard substrates. 


Figure 5: Correlation comparisons between foot diameter (mm) and muscular foot strength (g) sandy, rocky and hard substrates.

Discussion
Throughout the study, it was noted that the snails became less attached as the substrate changed. The greatest variability was seen in the hard substrate due to the large range of snails and whether or not they attempted to stay attached to the tray. Some of the specimens as a natural defense, close their corneous operculum off to avoid predation rather than stay attached to the substrate, which led to inaccurate results on foot strength (Heckley 2005). For the hard substrate, the foot diameter experienced greater correlation likely because it gave a more accurate representation of the actual foot strength in comparison to the approximate weight provided by the shell length data. In the subsequent substrate samples, the specimens did not attach to either of the substrates, but rather roamed the surface and released upon attachment of the scale, which may have created a bias in the sample. This finding would explain the higher correlations in the shell length (weight) rather than in foot diameter for these two substrate samples. The specimens tended to have only slightly high muscular foot strength on the sand substrate rather than rocky.
 As these organisms are typically not found in rocky substrates, it would be valid to assume that this may have led to the the smallest average foot strength. Muddy intertidal areas where P. ebeninus are commonly found should have been similar to the fine particle sandy test substrate, but had significantly less average foot strength than hard substrates. As these organisms are found near the mostly dry high tide line, the muddy environment be closer to the hard substrate than the sandy substrate (Edwards 2009). As these specimens are also not in contact with high wave action, it is a consideration that they may not need great foot strength, indicated by the low average foot strength on sandy substrates. In future studies, I would suggest using a more sensitive scale with a wider range due to that there were two organisms who reached the end of the scale on the hard substrate and could not provide an accurate reading. I would also remove the specimens that did not provide accurate readings from the data when they released at the beginning of the pulling motion to obtain more conclusive data. I would also collect a muddy (dry and wet) substrate to test the muscular foot strength in their given environment. There was a significant difference found in the average foot strength measured on hard substrate compared to rocky and sandy substrates, although the low correlation between the foot strength and foot diameter/shell length renders this data inconclusive on the effects of substrates on muscular foot strength.