Mke 400

On May 29, 2017, in the Ribeira Porco area (1°33.03'N, 7°22.29'E, Fig. 1), one individual was captured using mist-nets (Fig. 2A), measured and a blood sample collected. This individual was euthanised by inhaling an Isoflurane 1 mL/5 L solution, dissected, fixed with absolute EtOH, and preserved in 80% EtOH. Afterwards, the specimen was prepared as a study skin and spread wing, and the partial skeleton was prepared following a modified procedure (Cataldo 2017 ) from that described by Davis and Payne (1992) and Baker et al. (2003) . Previously to being captured, recordings of its vocalisations and of those of a second bird were obtained using a recorder (Edirol R-09HR, Roland, Japan) and a microphone (MKE 400, Sennheiser, Germany); these were elicited by playing back previously recorded vocalisations of this taxon to attract it into the nets. The voucher specimen was photographed to document life colouration and appearance (Fig. 2A). The voucher was deposited in the ornithological collection of the Natural History and Science Museum of the University of Porto (Table 1).

The test was conducted as previously described (Santangelo et al. 2016 (link)). Briefly, the subjects were separated from their partner and divided into the right-hand upper quadrant of the home cage (separated phase, Fig. 2A). The subject’s behavior was recorded using a GoPro camera and a Sennheiser MKE 400 microphone, which were placed at a short distance from the cage (Fig. 2B). After 8 min, an unfamiliar experimenter, the “HI,” entered the room and stood 40 cm away in front of the cage. The HI was disguised using a realistic looking human mask (Masks Direct) and wearing familiar scrubs and gown. When possible, the intruder made direct eye contact with the subject for 2 min (intruder phase). The intruder then quietly left the room, and the subject’s behavior was recorded for a further 5 min (postintruder phase).

Video stimuli used in our mate choice trials were filmed with a camera (D7100, Nikon, Tokyo, Japan), mounted to a tripod, connected to a directional microphone (MKE400, Sennheiser, Hanover, Germany). The videos were recorded at day in order to obtain better frames, although little torrent frogs can breed and communicate with visual (limb movements) and acoustic signals (calls) day and night. Moreover, they were recorded in front of focal frogs (N=20 males) from a distance of 0.3–0.5 m. We identified four types of limb movement that were often evoked by interactions with parasites. For each of these types (i.e. AW, HFL, W, and LSA), we selected three representative videos, from three different frogs, containing a sequence in which a limb movement was accompanied by a nearby flying midge, and a sequence in which only a flying midge occurred (but with no limb movement). Both sequences were in absence of vocal sac movement. There were two reasons for the exclusion of vocal sac. First, the movement of vocal sac is flexibly coupled with limb displays in natural conditions. Second, the role of limb movements may be masked by vocal sac movement because it has a strong sexual attractiveness and can play a role in mate choice when coupled with advertisement calls (Zhao, 2021 ). TT, LS, and FF displays were not produced during parasite interactions, and thus were not included in the test.