Arthropods were treated as gently as possible given the constraints of the experimental design.

2. Materials and Methods

Ethics statement

This study adheres to the guidelines for the treatment of animals in behavioral research and teaching (ASAB/ABS, 2014). All treatments of experimental animals respected the laws of the country (Italy) in which they were performed (D.M. 116192), as well as European Union

regulations (European Commission, 2007). No permits were required by the Italian government for experiments involving arthropod pests. All experiments consisted in behavioral observations.

Arthropods were treated as gently as possible given the constraints of the experimental design.

None were injured or killed during the experiments. The health of every arthropod was constantly assessed by checking that it fed and behaved normally.

2.1. C. vomitoria

2.1.1. Calliphora vomitora rearing and general observations

The larvae of C. vomitoria were purchased in a local reptile food store in Pontedera, Italy.

They were placed in glass pots (diameter 15 cm, length 20 cm) waiting for the pupation. The pots

were singly laid in cylindrical PVC cages (diameter 35 cm, length 60 cm). The aeration in the cages

was guaranteed by a transparent chiffon fabric (mesh size 0.05 mm). Adults were transferred in

cages, each containing 70 flies, with a sex ratio of 1:1, and then maintained under laboratory

conditions [(24±1 °C, 45± 5 % RH, 16:8 L:D photoperiod] at the Department of Agriculture, Food

and Environment, University of Pisa, Italy. The adults were fed with a mixture of yeast extract and

sucrose at a ratio of 1:10 (w:w) with water provided separately on a cotton wick.

All experiments were conducted in March-April 2017 in a room illuminated with fluorescent daylight tubes [16:8 (L:D), lights on at 6:00]. The temperature was 24±1 °C, and the R.H. was 45±

5%. Only insects with intact legs and wings were tested. All observations were focally recorded by an observer.

2.1.2 Innate color preferences

Here, I investigated the innate preference of C. vomitoria adults for colors using a two- choice assay. I tested binary combinations of colors for their ability to elicit innate attractiveness in blowflies. Cardboard models with different colors were used. Tested colors included black, white, yellow, blue and red; acrylic pigments were used to prepare the cardboards (Polycolor®, Maimeri, Italy). The color spectral features are reported in Table 1. The testing arena showed four sections (A, B, C, and C, Figure 1), each section had an equal size. At the end of the A and D sections the colored cardboards were presented. Following our preliminary observations, where we noted that blowflies were attracted by black targets, I tested the following combinations of colors: (i) black vs.

white, (ii) black vs. yellow, (iii) black vs. red, and (iv) black vs. blue. B and C sections were considered as neutral ones. Before the start of each test, food and the water sources were removed from the cages. For each binary combinations of colors tested, flies were observed for 60 min.

During the observations, I recorded the number of the blowflies that showed a preference for a

particular section of the arena at different time intervals (e g. at 5, 15, 30 and 60 min from the

beginning of the experiment). Flies not showing any color preference (e. g. flies stationing in the B

or C sections) were not considered in the data analysis. After each replicate, the arena was rotated

clockwise 90° to avoid orientation bias. 12 replicates were performed for each binary combinations

of colors, in each replicate 70 flies were tested.

2.2. Ixodes ricinus

2.2.1. Ixodes ricinus rearing and general observations

Ixodes ricinus adult females were obtained from fresh carcasses of Vulpes vulpes Linnaeus

at the Department of Veterinary Medicine (2, Viale delle Piagge, Pisa) Ticks were identified relying to the systematic keys by Estrada-Peña et al., (2004), then stored in clean glass vials (diameter 10 mm, length 50 mm) until the testing phase. Each tick was provided with a wet filter paper disc (diameter 10 mm) dipped in tap water. Damaged ticks were discarded and not used for behavioral experiments.

Experiment were conducted in March 2017. All observations were carried out in a Petri dish arena (diameter 100 mm; height: 10 mm) from 11:00 to 19:00 hours, at 25 °C and 65% RH. The arena was carefully washed after each replicate following the method by Carpita et al. (2012). The room was illuminated with fluorescent daylight tubes [16:8 (L:D) photoperiod, lights on at 06:00 hours]. Neon tubes (Philips 30 W/33) were used, and the light intensity in close proximity of the testing arena was 1000 lux, estimated over the 300- to 1100-nm waveband using a LI-1800

spectroradiometer (LI-COR, Lincoln, NE, USA), equipped with a remote cosine receptor (Benelli et al., 2017c). All observations were focally recorded by an observer. In order to avoid visual cues from the observer affecting the behavior of the tested ticks, a white wall of filter paper (Whatman n.1, height 30 cm) surrounded the experimental arena (Romano et al. 2016a).

2.2.2. Lateralized questing

Here, host-seeking behaviors were observed after transferring a tick into the testing arena an

exposing the individuals to a robotic system presenting a host-mimicking cue in close proximity of

the tick. In detail, the robotic stimulus was composed by a servomotor (Hard HS 3004) that was

connected to a rotor (diameter 50 mm) in acrylonitrile butadiene styrene (ABS), designed in SolidWorks and fabricated by rapid prototyping. A tuft of fox fur (60 mm), tied to a hole close to the circumference of the rotor, was placed and it was moved by controlling the servomotor with a microcontroller (Arduino, Mega 2560). This simple system was located on a suspended platform upon the test arena, in order to move the fox fur close to a tested tick.

In the first assay, I presented the host-mimicking cue to the ticks frontally. Once the tick extended the forelegs exhibiting the ”questing posture” (Lees 1948), the robotic stimulus was frontally brought about 5 mm from the tick palps and the leg used to transfer itself on the mimicking host was noted. 30 replicate were performed for each tick. Ticks that did not display questing posture or were constrained to the side of the arena were not considered for laterality observations.

In the second assay, a tick was placed in the testing arena and the stimulus was swung normally to the body axis of the tick, 5 mm from its palps, after the tick questing posture occurred.

For each tick we recorded (i) the number of climbing attempts occurred when the stimulus was provided from the left and from the right of the tick, (ii) the first leg used to attempt a climbing, as well as (iii) the success in climbing (e.g. when a tick intercepted and fixed itself on the stimulus).

Furthermore, the duration of the successful anchored ticks on the stimulus was recorded, evaluating the number of ticks fixed on the mimicking host for more than 30 seconds. Ticks that were not involved in any seeking behavior or that were constrained to the side of the arena were discarded.

30 stimulus swings were proposed to each tick.

2.3. L. sericata

2.3.1. L. sericata rearing and general observations

The pupae of L. sericata were kindly provided by Bioplanet (Cesena, Italy). Pupae were located in glass pots (diameter 15 cm, length 20 cm) containing 500 pupae for each one. The glass pots were situated individually in cylindrical PVC cages (diameter 35 cm, length 60 cm). The aeration was ensured by a side lined with transparent chiffon fabric (mesh size: 0.05 mm). To obtain coeval virgin individuals, L. sericata adults were sorted by sex within 8 h from emergence and stored separately in the Plexiglas cages described above until the testing phase. Each cage contained 150 individuals. The adults were feed with a mixture of yeast extract and sucrose at a ratio of 1:10 (w:w), while water provided separately on a cotton wick. Canned veal was also provided since a protein source is necessary to obtain receptive females in the genus Lucilia (Browne et al., 1976;

Wall 1993). Adults were maintained under laboratory conditions [(21±1 °C, 55± 5 % RH, 16:8 (light (L)/dark (D)) photoperiod] in University of Pisa laboratories.

The experiment were conducted in a Plexiglas testing arena (diameter: 400 mm; length: 250 mm). An entrance hole, with a diameter of 50 mm, was made on the top, in the central part of the arena. A transparent chiffon fabric (mesh size: 0.05 mm) covered both ends of the testing arena.

The arena contained canned veal on a Petri dish (diameter 50 mm) that was changed at each replicate. All experiments were carried out in April-May 2017 in a room illuminated with

fluorescent daylight tubes [16:8 (L/D) photoperiod, lights on at 06:00]. The temperature was 21±1

°C, and the R.H. was 55± 5%.

2.3.2. Courtship and mating behavior

Here I investigated the courtship and mating behavior of L. sericata, as well as if mating pairs have any lateral bias during these interactions. Furthermore, I evaluated if lateralization has any impact on the copula duration and the mating success of this species. Three L. sericata males were transferred in the cylindrical arena 10 minutes before introducing the females. Then, seven females were released in the arena and mating interactions were visually tracked for 60 minutes or until the end of the mating. During preliminary observations, we noted that the courtship and mating behavior of L. sericata consists of three phases: pre-mounting, mounting and copula phase.

For each male courting a female I recorded; (i) the number of the head contacts (i.e., when the male intercepts the female and touches her with his aristae), (ii) the number of the foreleg tapping (i.e., when the male recognizes the female and starts palpating her body with his forelegs), (iii) the number of copula attempts (i.e., the swinging movements of the male attempting copula), (iv) the number of foreleg tapping during the copula phase (i.e., when the male starts beating the body of female with the forelegs), as well as (v) the male mating success (i.e., if the copula was successful or if the female reject the mounting male, avoiding genital contact). I also quantified (vi) the duration of the pre-mounting phase, (vii) the duration of the mounting phase (i.e., from the mounting of the male on the female until the copula beginning), (viii) the duration of hind legs contacts (i.e., when a female try to dismount a male with their hind legs), and (ix) duration of copula (i.e., from the intromission of the aedeagus to genital disentanglement after copulation)

Furthermore, the side of the female approached by the male, as well as the first leg used during the foreleg tapping, both in the pre-mounting and the copula phase, were noted.

A total of 247 mating pairs of flies were observed. 117 mating pairs were analyzed. Flies not involved in any courtship and mating interaction were discarded. Female in a constraining

localization were not considered for laterality observations (Romano et al., 2016, Benelli et al.,

2016).

Data analysis

Data on innate color preferences in C. vomitoria were transformed into arcsine√proportion, and analyzed with a general linear model (JMP 9® SAS) with one fixed effect (i.e., the tested combination of cues): yj = μ + Cj + ej in which yj is the observation, μ is the overall mean, Oj the tested combination of cues (j = 1–2), and ej the residual error. A probability level of P < 0.05 was used for the significance of differences between means.

Concerning tick lateralized questing data, population-level differences in the overall number of ticks displaying a lateralized responses was analyzed using a likelihood ratio X

tests, with Yates correction (Sokal and Rohlf 1981). Individual-level lateralization in the tested ticks was computed calculating the laterality index (LI) following the method by Frasnelli et al., (2012).

Differences in courtship and mating traits of L. sericata occurring after lateralized acts were

analyzed using the general linear model described above with one fixed factor (i.e. laterality). A

probability level of P < 0.05 was used for the significance of differences between means.