Using P. leniusculus, two sets of experiments were completed: the first set tested male dominance and guarding, and female promiscuity, and the second set tested female mate choice. Dominance, guarding and promiscuity experiments took place in September and October 2018, whilst female choice experiments were carried out in 2017 and 2018.
Adult male and female P. leniusculus, for use in the experiments, were collected using baited funnel traps from two adjacent fishing ponds located in Southern England in September 2017 and September 2018. In each year, 200 crayfish (100 M and 100 F) were collected. The size (carapace length) of males ranged from 40 mm to 56 mm, and 38 mm to 46 mm for females. In both years, ≈ 50% of males were sterilised upon capture via removal of the gonopods by either cutting them off with scissors (Stebbing and Rimmer 2014) or pulling them out using a pair of tweezers, with the sterilised/non-sterilised groups being size matched to ensure the groups were of similar carapace lengths. Only crayfish with both chelae intact were kept, although owing to a shortage of adult males, some did have unevenly sized or relatively small chelae in relation to their body size.
On arrival at the laboratory, all crayfish were placed individually into one of five sections within 90 × 30 × 30 cm (80 L) glass tanks. Each section was divided using an opaque plastic partition with a small grille of 2-mm wire mesh at low level (25 mm from bottom of tank) to allow water circulation. Water temperature was initially maintained at 14 °C (to match the field site) with light on a 12:12 h light:dark cycle. Each section (16 L) contained a 20 mm layer of gravel and a shelter made from 50-mm-diameter plastic pipe. The divided tanks were set up in three-tiered flow-through filtration systems, with each system housing 15 crayfish (five per tank). Water was pumped to the topmost tier and then circulated down through the two lower tanks and finally through a filter at ground level. Sterilised males, non-sterilised males and females were housed in different systems, thereby preventing accidental physical contact or semiochemical interaction between individuals of different treatment groups prior to the experiments. The crayfish were fed a diet of fresh carrot twice weekly.
Following a four-day acclimation period in the laboratory, all crayfish carapace lengths were recorded, and for males, chelae length was measured and any abnormalities were recorded. Each individual was given an identifying number (via permanent marker pen on the carapace) that was colour-coded in accordance with sex and sterilisation status. In 2017, temperature was reduced from (± SE) 14 °C to 11 °C ± 0.5 °C, the temperature at which P. leniusculus mate in the UK, in 1 °C daily increments over a period of 3 days. In 2018, temperature was maintained at ambient levels (14 °C ± 0.5 °C) due to the chillers no longer being able to reduce the temperatures any further. For the female choice experiments, males were fitted with a tethering loop a minimum of 24 h prior to being used in an experiment. For this, a hole was made through the central uropod using a sterile needle, then a short length of light-gauge fishing line was threaded through and tied into a loop of approximately 10 mm diameter. Sterilised and non-sterilised males were captured from separate ponds in order to prevent the pairing of males with prior contact experience and therefore potential hierarchical effects.
For experiments, sterilised and non-sterilised males were paired according to carapace and chelae length, with a maximum difference of 2 mm in either characteristic to minimise any size effects. Crayfish with unevenly or unusually sized claws were matched with males with similar attributes in order to prevent any competitive advantage. Females were selected to be a similar size or slightly smaller than the males. Animals in moult or that did not appear to be in good condition were not used.
All subsequent analyses on experimental data were completed using SPSS version 23.0 (IBM 2017). Where error values are presented around means, they represent standard error unless stated. Significance is reported as exact two-tailed unless stated. All data were nonparametric.
Experimental design, data capture and analysis
The set of experiments initially compared sterilised and non-sterilised males’ ability to achieve dominance in an agonistic encounter and to generate dominant and subordinate individuals for the subsequent female choice experiments (2018 only). Here, a female was allowed to choose between a sterilised and non-sterilised male. When mating occurred, guarding and promiscuity were tested via the introduction of a new male, post-copulation.
All experiments were conducted in a 900 × 300 mm tank on a flow-through system (as described above). The water temperature in the experimental tanks also differed between the 2 years (11.0 ± 0.5 °C in 2017; 14.0 ± 0.5 °C in 2018). All experiments were completed in darkness (between 19.00 and 00.00 h), the time when crayfish are normally most active (Franke and Hörstgen-Schwark 2015). The nature of the experiments meant that the males used in individual dominance trials were then reused in female choice trials; the latter took place after a minimum of 24 h and a maximum of 17 days [mean = 12.3 ± 5.8 days (SD)] after the dominance trial. The only illumination was above the tanks to allow filming; this comprised two battery-operated ‘stick-on’ lights positioned 30 cm above the tank in 2017 and LED aquarium lights in 2018. Filming was conducted using a Go-Pro Hero 3 video camera suspended 30 cm above the tank.
The basis for the analysis of the dominance, guarding and promiscuity experiments was a fight ethogram adapted from Bruski and Dunham (1987) by Bergman and Moore (2003). This categorised the different aspects of agonistic behaviour (Table 1) and enabled each animal to be scored by multiplying the length of time (s) spent displaying each behaviour with the score for that behavioural category. For guarding and promiscuity, the ethogram was modified to include relevant behaviours such as sexual activity.
There were 26 male dominance trials completed and analysed. One sterilised male and one non-sterilised male were placed at each end of the tank, being separated by an opaque plastic partition placed half way along its length and extending to the top of the tank. Following an acclimatisation period (10 min), the partition was removed and the behaviour of both males filmed (15 min). The starting position in the tank (left/right) was alternated for the two categories of male to avoid positional bias. The males were not tethered during this experiment, and the fitted tethering loop was considered unlikely to interfere with normal behaviour as the animals had become accustomed to its presence, plus any disturbance effect would be equal as both animals had tethers. In the analyses, an encounter was deemed to have started when one crayfish approached another and ended when the crayfish moved more than one body length away and reverted to behavioural intensity level 0 (Table 1). The frequency and intensity of each behaviour was multiplied together to give each male a dominance score in accordance with the fight ethogram. These data were then tested between using a Mann–Whitney U test using the dominance score as the test variable and sterilised or non-sterilised (S or M) as the grouping variable. Position in arena (left or right side) was tested in the same way in order to ascertain positional bias. The distribution of sterilised and non-sterilised males as dominance contest winners was tested for goodness of fit using a Chi-squared test (χ2).
Female choice experiment
In this experiment, one sterilised and one non-sterilised male were tethered and placed into equal-sized arenas delineated by a clear Perspex partition (enabling the female to have sight of both males) secured half way across the width of the tank (at 150 mm), extending for 300 mm into the tank and being 300 mm high (Fig. 1). In 2018, the same pairs used in the previous dominance experiments were selected, meaning the relative dominance status of each male was known prior to the experiment. Each male was tethered by attaching the loop through the uropod to a 500 g fishing weight via a metal clip (in 2017) or a safety pin (in 2018) and length of fishing line, the length of line being sufficient to maintain them within the area delineated by the partition and prevent them from interacting with each other. The relative position of the two categories of male was alternated between experiments in order to prevent left–right bias.
The males were allowed to acclimatise (10 min) prior to the introduction of the female at the opposite end of the tank (Fig. 1). Interactions between the three individuals were filmed either until mating took place or for 30 min in the absence of mating. Videos where mating did not occur within 30 min were discarded. A nominal ‘territory’ for each male covering two-thirds of their half of the tank was devised for video analysis purposes (Fig. 1). The videos were subsequently analysed to record the amount of time the female spent in the ‘territory’ of each male (Fig. 1), recorded as starting when half of the female’s carapace crossed the line. To ensure that the female had the opportunity to make a choice, only videos where the female had sight of both males prior to copulation were analysed. A total of 50 trials were completed, of which 28 resulted in copulation between the female and one of the males. However, of these 28 copulations, 9 were considered as not being appropriate for analysis as the female appeared not to have sight of both males prior to the start of copulation or the video was not of sufficient quality for analysis, reducing the sample size to 19 (9 in 2017, 10 in 2018).
The total time spent by the female in the territory of each male was expressed as the percentage of total interaction time, i.e. of total time spent in their territory. Owing to the differences in temperature between 2017 and 2018, the data were tested for difference between years using a Mann–Whitney ‘U’ Test. These differences were not significant (U = 180.0; P = 1.0) so the data were combined to enable a single test. The mate choice by the female (sterilised or non-sterilised) and (in 2018) the dominance status of males (dominant or subordinate) were tested using a Chi-squared goodness-of-fit test.
Post-copulatory guarding experiment
When copulation occurred in the previous experiment, the mated pair were moved post-copulation to a separate 900 × 300 m tank containing two shelters to minimise stress. After 5 min, a new male of haphazardly chosen size and sterilisation status was introduced and the interaction between the three animals filmed for up to 10 min. The original mate was classified as the ‘mate’, whilst the new male was the ‘challenger’. The videos generated were analysed by categorising the different aspects of agonistic and guarding behaviour using the modified version of the fight ethogram (Table 1). Due to video recording issues, a total of 11 trials were analysed for post-copulatory guarding.
The frequency [as time (s)] and intensity of each recorded behaviour was multiplied together for mate and challenger, giving them a ‘guarding’ score. As the resulting data had high variance, they were log transformed then tested for differences between ‘mate’ and ‘challenger’ scores using a Mann–Whitney U Test. Additionally the animals with the highest scores in each bout were tested against their sterilisation status using Mann–Whitney U and Chi-squared goodness-of-fit tests.
At the end of the guarding experiment, the original mate was removed and interactions between the female and male challenger were filmed for up to 10 min. Ten experiments were completed, one being discontinued as the male’s attempts to copulate risked harming the female. The videos were analysed using the ‘willingness ethogram’ modified from the fight ethogram used in the dominance and guarding experiments (Table 2, modifications marked with*). The frequency and intensity of each behaviour were multiplied together to generate a ‘willingness score’ for each male and female. The difference in willingness scores between males (challengers) and females, and between sterilised and non-sterilised males, was tested using Mann–Whitney U and Chi-squared goodness-of-fit tests.