Fish sampling, laboratory housing and experimental design
Orsinigobius croaticus was sampled using electrofishing (Hans
Grassl, model: EL65 IIGI, power: 13 kW) from a boat during spring 2019.
Sampling was performed on the Matica River in Croatia (near the village
Vina 43°10’30.33” N, 17°23’12.36” E). In total, we collected 25
individuals (15 males and 10 females) from the main river channel at a
depth of 0.5 – 2 m. Fish were transferred to large plastic water
containers equipped with aerators and transported to the laboratory. At
the laboratory, fish were sexed based on urogenital papilla and body
coloration (Miller, 1984) and housed in appropriate community
rectangular tanks (120 L). After an acclimatization period of 5 – 8
days, prospective soniferous males were chosen for subsequent laboratory
acoustic-visual recordings based on coloration (complete or partially
darken body, fins and head; Zanella et al., 2011), aggressiveness and
territoriality. Eight males (x̄ ± s.d. = 49.21 ± 0.8 mm
total length, LT; range: 48.07 – 50.06 mm; 41.07 ± 1.01
mm standard length, LS; range: 38.96 – 42.29 mm; 1.24 ±
0.11 g weight, W; range: 1.10 – 1.40 g) exhibiting typical reproductive
behaviour were chosen for the experiments. Females (N = 5; 36.75 ± 5.10
mm LS; range: 28.75 – 41.27 mm) were chosen for the
recording sessions according to yellow belly coloration, luminescent
green spot on the first dorsal fin and dark eyes, all indicators of
female readiness for spawning (Zanella et al., 2011; Bloom et al.,
2016). Measurements were made using digital callipers CD-15APX with a
precision of 0.01 mm (Mitutoyo, Japan) and a digital scale (0.1 g
precision). As a metric of male body condition, we calculated the
condition factor [Fulton’s K , where K =
(W/LS3) *105].
We followed the acoustic-visual recording protocol established by
previous authors (Amorim & Neves, 2007; Amorim et al., 2013; Pedroso et
al., 2013), where experimental tanks were divided into three
compartments separated by removable partitions. Each lateral compartment
housed one territorial male with a nest (artificial pvc shelter), while
the middle section (“arena”) was occupied by a ripe female. The female
compartment was not provided with a nest. Briefly, our experimental
rectangular tanks were 120 L (60 x 48 x 35 cm) in volume and were
divided into three unequal sections by means of two opaque removable
acrylic partitions to prevent intersexual (visual) interactions. The
tanks had a 5 cm thick layer of fine sand or gravel and each male in
each section was provided with a water pump system and aeration. The
experiments were performed from mid-April to October, at random times.
The reproductive behaviour of resident males was elicited by introducing
one ripe female into the “arena”. Before experiments, each female was
left 12 – 24 h in the experimental tank for acclimatization. Eight
territorial males were kept in the experimental tanks until the end of
the recording sessions in October, after which they were returned to
male community tanks. Prior to recordings (approx. 15 min), electricity,
water pumps and aeration were switched off to minimise ambient noise.
Between recording sessions of the same resident male, 15 – 30 min
pauses were included. In addition, to reduce substrate-born noise, the
experimental tanks were placed onto three cm thick rubber foam shock
absorbers. Fish were kept at natural photoperiod and fed daily ad
libitum with Daphnia . Water temperature, monitored with a
thermometer (AQUATERRA, Garešnica, Croatia), was maintained between 18
– 22°C. The male-female trials lasted approximately 30 min, and began
by removing one of the lateral partitions, allowing intersexual
interaction. To eliminate the noise from the room light system,
recording sessions were performed in the dark, with the experimental
tank illuminated by LED light from the side of the aquarium. This
procedure had no noticeable effects on fish behaviour.