Substrate preference in Southern Pygmy Squid, Idiosepius notoides: does background colour affect attachment behaviour?

by Samantha Reynolds

Abstract

The Southern Pygmy Squid, Idiosepius notoides, is a coleoid cephalopod closely related to the cuttlefish (family Sepiidae). It is endemic to Australian coastal waters and is typically found in seagrass or macroalgal beds attached to fronds by a dorsal adhesive gland. Studies on cuttlefish have found that they are able to display substrate preferences for both artificial and natural substrates and that different substrates elicit different behavioural responses. This study investigates the ability of I. notoides to display a similar substrate preference and whether substrate has an effect on the attachment behaviour of this species. It was found that I. notoides displays a significant preference for grey over white substrate and that attachment to vegetation was significantly faster on white than on grey substrates. The current study was limited to only two experimental animals, however from these results it can be tentatively suggested that I. notoides is also able to display a preference for substrates and that substrate colour may affect behavioural responses.

Introduction

The ability of cephalopods to change the colour and even the shape of their skins is one of the most intriguing aspects of the biology of these active and intelligent molluscs. The camouflage capabilities of octopus (e.g. Hanlon et al. 1999; Josef et al. 2012) and cuttlefish (e.g. Barbosa et al. 2004; Allen et al. 2010; Chiao et al. 2010) have been extensively studied, showing that they are able to camouflage themselves rapidly in a huge variety of diverse environments.

The speed with which cephalopods are able to change their body patterning is achieved by having direct neural control over the muscles controlling the chromatophores, the organs of pigmentation in the skin, allowing them to be rapidly expanded and contracted, producing changes in body colour and pattern. Many cephalopods, particularly octopus and cuttlefish are also able to change their shape and texture using papillae in the skin (Hanlon 2007).

Idiosepius notoides is commonly known as the Southern Pygmy Squid, however Idiosepius spp. are more closely related to cuttlefish and their allies, belonging to order Sepioidea, rather than the ‘true squid’, order Teuthoidea (Hylleberg and Nateewathana 1991) and therefore may show affinities with this group. There have been many studies of cuttlefish camouflage that have particularly focused on the effect of substrate colour and texture on body pattern and behaviour (e.g. Mäthger et al. 2007; Barbosa et al. 2008; Allen et al. 2010). The cuttlefish, Sepia officinalis, was shown by Allen et al. (2010) to have the ability to distinguish between, and show preferences for, different substrates, both artificial and natural. In one of a series of experiments, they found that cuttlefish disliked white or light coloured backgrounds and preferred uniform grey backgrounds. It was also found that when given a choice of natural substrates, cuttlefish preferred ones in which they were able to bury themselves.

Comparatively little study has been done on I. notoides in general, let alone on the camouflage ability and substrate preferences of this species. I. notoides inhabits shallow coastal waters, from southern Queensland to southern Western Australia and Tasmania, and is known to attach to seagrass and macroalgal fronds, for protection and to lay eggs, via a special adhesive gland on their dorsal surface (Norman and Reid 2000). This is in contrast to cuttlefish, which tend to be benthic and settle on or in the substrate (Allen et al. 2010). In preliminary observations for this study, it was noted that if vegetation was available, I. notoides would generally come to rest attached to it rather than settle on the bottom or attach to the sides of the tank or container.

This study aims to investigate whether I. notoides has the ability to display a preference for different substrates and how these different substrates may affect their behaviour in attaching to available vegetation. The hypothesis that I. notoides will display a preference for uniform grey backgrounds over white backgrounds, similar to that found in cuttlefish (Allen et al. 2010), is tested. The effect that the two substrate colours has on the attachment by I. notoides to vegetation is also examined and it is hypothesised that if indeed white backgrounds are disfavoured, the time taken to attach to the vegetation will be shorter on these backgrounds than on uniform grey backgrounds.

Methods

Animals were collected, housed and studied under the University of Queensland’s (UQ) Animal Welfare Permit No. 158/09. Subjects came from the wild population of I. notoides found in Moreton Bay, Queensland. They were collected by dip netting on 6 May, 2014, from the seagrass, Zostera marina, beds near Dunwich Harbour on North Stradbroke Island and housed in the marine aquariums at UQ for the duration of the study. Unfortunately only two subjects were available at the time of the experiments, so they were used in more than one replicate for each experiment, creating some pseudoreplication. The test animals were rested between trials and experiments in holding containers in which stimulus was limited and vegetation cover was plentiful, in order to minimise their stress.

Experiment 1 – Substrate Preference

A clear petri dish with a diameter of 14cm containing seawater from the tank in which the subjects were housed was placed on a divided background so that half the bottom of the dish was uniform grey and the other half was bright white. One animal at a time was introduced to the dish and allowed to acclimate for one minute. A five minute trial period was then started in which the animal was observed at 30 second intervals. A photograph was taken at the start of the trial period and every 30 seconds up to the end of the trial at 5 minutes and the animal’s position, on either the white or grey background, was noted. Each of the two test animals was observed for two five minute trials. This yielded 11 observations in each trial, 44 in total, which were placed into two categories: grey or white. Using a Chi-squared test for goodness of fit, the observed numbers of occurrences of the animals on each background were compared to those expected under the null hypothesis, i.e. if I. notoides does not display a preference for either grey or white substrate, the probability of choosing each background would be 0.5.

Experiment 2 – Effect of Substrate on Attachment Time

A study animal was introduced into a clear petri dish of 8.5cm diameter that was on either a grey or white background and contained seawater and a strand of vegetation (Caulerpa sp.) from the tank in which they were housed. The length of time taken for the animal to attach itself to the seaweed after introduction to the dish was measured (see Predator Avoidance Strategies tab for a video of this). Three trials on each background were conducted for each of the two test animals, resulting in six data points for each substrate colour. The average attachment times for grey and white backgrounds were calculated and compared using a Student’s t-Test.

Results

Experiment 1 – Substrate Preference

Test animals were found on the grey background in 37 out of the 44 total observations, and on the white background in only seven out of the 44 observations. The observations showed a temporal bias with most observations of animals on the white background occurring in the first minute (N = 6), rather than in the rest of the trial (N = 1) (Figure 1).

Experiment 2 – Effect of Substrate on Attachment Time

The colour of the substrate on which the test animals were placed was found to have a significant effect on the time it took them to attach to seaweed fronds (t-Test_{5, 6} = 4.23, P = 0.008). On the white background, attachment times were significantly shorter than on the grey backgrounds. Subjects took on average 18.2 seconds (SE ± 3.7) to attach to the vegetation, whereas on the grey background, the average attachment time was just over eight times longer at 146.5 seconds (SE ± 30.1) (Figure 2).

Discussion

Although the conclusions that can be drawn from this study are limited by the fact that pseudoreplication occurred in the experiments as a result of having only two test subjects available, it is still interesting to discuss the implications of the findings in light of previous research on related species.

The ability of cephalopods to interpret and respond to cues in their environment is well documented and requires complex visual and neural systems that can rapidly assess visual stimuli and produce the appropriate camouflage response (Hanlon 2007). The substrate or background on which the animal finds itself will therefore be likely to have a profound effect on its behaviour.

The current study, in which I. notoides was observed significantly more often than would be expected by chance on a uniform grey substrate as opposed to a bright white one, suggests that it is able to distinguish between the two backgrounds and that there is a preference for the grey one. This is similar to the results obtained by Allen et al. (2010) in a study on S. officinalis, and may be a response to the fact that bright white substrates are rarely found in the natural environments of I. notoides, which typically inhabit seagrass beds (Norman and Reid 2000). Certainly the substrate of the seagrass beds from which the study animals were collected in Moreton Bay is a muddy grey-brown colour. I. notoides may simply feel more ‘at home’ against a grey background than against a white one.

The results obtained in this study which showed that the substrate colour affected the attachment time of I. notoides to seaweed fronds may also support this suggestion. The time taken by subjects to attach themselves to the seaweed was significantly longer on the grey substrate than on the white. During preliminary observations it was seen that I. notoides were inquisitive when placed in a new environment. When no vegetation cover was available in the animals’ containers, they would explore their environment extensively, perhaps looking for cover and determining the extent of their new habitat. That they continued to do this when containers that held available cover were on the grey background suggests that they were comfortable enough in this environment to continue their explorations, however on the white background they preferred to find and use cover quickly. The fact that more observations of animals on the white background occurred at the start of the trials may be interpreted as meaning that they were moving around more at this time, perhaps exploring their environment, discovering the differences in substrate and then choosing to remain on the grey coloured side, lending support to the idea that this is where they are most comfortable. This may mean that this species is more able to use camouflage effectively on more naturalistic substrates but this was not tested in the current study. Future research could explore the effects of substrates on the camouflage techniques of I. notoides.

This species is thought to be a nocturnal predator, using its adhesive gland to attach to vegetation for cover during the day, emerging at night to hunt prey such as small crustaceans (Norman and Reid 2000). The fact that all experiments were carried out during the day in well lit conditions in the lab may have had an influence on attachment times, but does not provide an explanation as to why these were longer on the grey background.

In their study, Allen et al. (2010) also showed that S. officinalis preferred a deep substrate in which they could bury themselves. Changing body colour and pattern may have large energetic costs for cephalopods (Hanlon et al. 1999), and perhaps this preference for burying themselves in the substrate reflects this, i.e. it is less costly to hide than to produce camouflage patterning. The attachment behaviour of I. notoides could be interpreted as a similar strategy to the burying behaviour of cuttlefish and may have developed as a less costly alternative to camouflage.

Of course there may be many other factors that affect the attachment times of I. notoides in their natural environment. Predation pressure, density of vegetation cover and intraspecific competition may all play a role in determining when animals will attach to available cover. It may also be interesting to investigate whether I. notoides displays territoriality and whether this has an influence on where females attach their eggs.

Very little is known of the ecology and behaviour of this intriguing species and although the current study has its limitations, it suggests that I. notoides may be able to display substrate preference and that substrate may have an effect on behaviour. Further study in this area could confirm this and investigate the energetic costs of the different predator avoidance strategies used, to determine if a preference for one over the other is displayed.

Acknowledgements

Grateful acknowledgement of the help of lecturers Bernie and Sandie Degnan, the tutors of BIOL3211 and the staff of the Biological Sciences Laboratories at the University of Queensland is made.

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