Distance chemoreception and the detection of conspecifics in Octopus bimaculoides

Octopuses are solitary predators that typically use their arms to grope into crevices to find food. They detect odours on contact using chemosensory cells on their lips and suckers (Budelmann, 1996). They can also detect water-borne odours (distance chemoreception) using receptors in their olfactory organs (olfactory pits; Budelmann, Schipp & Boletzky, 1997). Behavioural experiments have demonstrated increased arousal (Boyle, 1983), activity (Boyle, 1986) and attraction (Chase & Wells, 1986; Lee, 1992) in response to food-related odours, but responses to odours of conspecifics have not previously been investigated. Chemical communication has been demonstrated previously in cephalopods, such as Nautilus (Basil et al., 2002), Loligo (Gilly & Lucero, 1992; Buresch et al., 2003; King, Adamo, & Hanlon, 2003) and Sepia (Boal, 1997; Boal & Marsh, 1998; Boal et al., 2010). Here we investigate behavioural evidence for detection and attraction to conspecific odours by Octopus. Subjects were wild-caught Octopus bimaculoides Pickford & McConnaughey, 1949 (30–90 g) of unknown age and maturity (not correlated with physical size within this size range), trapped off the coast of Southern California and shipped overnight to Millersville, PA, USA. Differences in physical size did not explain any patterns in experimental results and will not be discussed; sex was confirmed by necropsy. Housing and experimental tanks were interconnected within a 57,000-l marine system of recirculating artificial seawater (ASW) made from Instant Ocean brand salts (salinity 34+2 ppt, temperature 17.5+ 1.58C) (see Hvorecny et al., 2007). Trials were conducted between 2004 and 2009. All subjects were acclimated to the laboratory for 3 weeks to 6 months prior to being used in experiments. Subjects were fed thawed frozen shrimp or live fiddler crabs (Uca spp.) each day after experimental trials. Odour samples were collected as follows. Crab odour samples were collected from a holding tank (c. 1,000 l) housing 80–100 fiddler crabs (Uca pugilator). Shrimp odour samples were obtained by thawing one tail segment of a large (c. 9.5 g) frozen shrimp in 10 ml of ASW; seaweed odours were obtained by soaking 3 g of dried seaweed (Porphyra yezoensis; Julian Sprung’s Sea Veggies) in 800 ml of ASW for 10 min. For both shrimp and seaweed, the particulate was removed and the remaining liquid served as the odour source. Conspecific odour and intact egg odour samples were collected by placing an octopus or clutch of about 50 eggs (63+ 1 days postlaying) into a small aquarium filled with ASW to a total volume of 2 l, with aeration (air stone), for 20 min (initial trials) or 30 min (later trials). After removal of the octopus or eggs, the remaining liquid was used as the odour source. For egg extracts, clutches of conspecific eggs of unknown age (not recently laid) were collected from newly wild-caught females (in California), frozen on dry ice, shipped to Texas and stored at 2808C until extraction. Eggs were individually extracted (2004) or extracted in batches of 10 eggs (2005), centrifuged and purified using separate C18 Sep-Pak cartridges (Waters Corp., Milford, MA, USA) as described previously (Buresch et al., 2003); C18 Sep-Pak purification primarily retains small molecules, peptides and small proteins. Small molecules such as these have been demonstrated to serve as signal molecules in other molluscs (Susswein & Nagle, 2004). The resulting pellets were dissolved in ASW to a concentration of 1.5 eggs/15 ml for use in trials. To assess distance odour detection, we measured changes in the ventilation rate of octopuses in response to odours of seawater (negative control), food (positive control), conspecifics (same and opposite sex) and conspecific eggs (intact and purified extracts). Ventilation-rate trials were conducted in the octopus’s individual housing tanks (38.1 19.6 26.7 cm, 18.5 l) that were provided with flow rates of 91+ 4 ml/s (Boal & Golden, 1999). The ventilation rate of an individual octopus was measured by counting ventilation cycles (inspiration and expiration) for the first 20 s (food, seawater) or 30 s (conspecifics, conspecific eggs) of each min. Ten minutes after the start of observations (baseline), a 5 ml (food, seawater, purified egg extracts) or 500 ml (conspecific, conspecific eggs) odour sample was added to the water supply line and ventilation cycles were counted for another 15 min. For conspecific odours, each octopus was exposed to the odour of each of the other octopuses once. For all other trials, each octopus received each odour treatment twice and responses were averaged. Resting ventilation rates (cycles min) differed substantially among octopuses (23–42 cycles min). Consequently, the change in ventilation rate for each octopus was used to evaluate response: peak ventilation rate (maximum recorded ventilation rate in the 5 min immediately after odour addition) minus mean prestimulus ventilation rate (5 min immediately prior to odour addition). Peak response was used because responses tended to be short-lived. Octopuses increased their ventilation rates significantly in response to water-borne odours from foods (Kruskal–Wallis one-way analysis of variance by ranks, KW 1⁄4 14.32, n 1⁄4 30, df 1⁄4 2, P, 0.01; Fig. 1A). A pair-wise comparison showed that the responses to crab and shrimp odours were significantly different from the responses to the control (P, 0.05), but the two food odours were not different from each other. Octopuses also increased their ventilation rates significantly in response to water-borne odours from conspecifics (Fig. 1B). The sex of the odour-donor had a significant effect on responses (Wilcoxon signed-ranks test, same vs opposite sex, z 1⁄4 2.11, n 1⁄4 17, P, 0.05). Females increased their ventilation rates significantly in response to odours from males but not to odours from other females (Friedman two-way analysis of variance by ranks, Fr 1⁄4