Introduction
The meagre (Argyrosomus regius ) is an emerging species for the Mediterranean aquaculture industry and its reproductive dysfunctions have been resolved using gonadotropin releasing hormone agonist (GnRHa) -based spawning induction protocols. However, to eventually be able to obtain fertilized eggs spontaneously, more information on the environmental requirements of the species and its breeding behavior is needed. Although sound production has been described so far in little more than 1100 fish species, recent studies demonstrate that this communication modality is shared by nearly two-thirds of actinopterygian species. The meagre is a member of the Sciaenidae family -called “drums” or “croakers” - and has a wide vocal repertoire , emitting sounds made from a single pulse up to more than 100 pulses . The calling rate and sound temporal features have been suggested as indicative of reproductive events. The aim of this study was to describe the reproductive behavior and parental contribution of meagre in aquaculture conditions, after spawning induction with (GnRHa), and document sound production during spawning and investigate how sounds may relate with specific behaviors in aquaculture facilities.
Materials and Methods
Two meagre broodstocks (n=2 females and 3 males, each) were utilized for two consecutive years (2021 and 2022 ). At the expected spawning period, fish were induced to spawn with a GnRHa injection (15 μg kg -1 for females) or implant (50 μg kg-1 for males). Each breeder was externally tagged for individual identification , and their behavior was monitored using underwater cameras . Egg production and quality was evaluated in terms of fecundity, fertilization success, 24-h embryo survival, hatching and 5-d larval survival. A sub-sample of the eggs from each spawn was analyzed for parental contribution. In 2022 only, t wo HTI- 96-Min hydrophones were also connected to the audio input of the cameras . Using Adobe Premiere, audio files were extracted from the videos before and after spawning induction (5–13 May, 2022). All audio files were analyzed by audio and visual assessment (Raven Pro 64 1.4). S ounds produced by the fish were manually labelled on the basis of the number of pulses as knocks (1-3 pulses) , or short (4-7 pulses) , intermediate (8-29 pulses) and long grunts (≥30 pulses) . When a sound could be associated with one of the identified stereotype behaviors -such as “male(s) trailing a female” , “darting” , “male-to-male agonistic behavior” or “spawning” the occurrence was annotated from the video observations.
Results
Spawning induction led to three consecutive daily spawns, 2 days after GnRHa treatment, with maximum fecundity on day 3 . Spawning usually took place with all three males following a female (Fig. 1), and after numerous male-to-male agonistic interactions. Mean daily fecundity (n=4 years x broodstock) was 159,743±23,315 eggs kg-1 , and mean fertilization success and 24-h embryo survival were 83-92% and 93-100%, respectively .
Of the eggs produced in both years, 77±3% were spawned by one of the females in each tank , while 77±9% of the eggs from each tank were fertilized by one of the three males in each broodstock . In broodstock 1 (Fig. 2), t he larger male fertilized 68±9% of the eggs in both years and i n broodstock 2, the most aggressive male fertilized 86±8% of the eggs.
After GnRHa treatment, t he number of sounds produced increased significantly during spawning days, especially during the evening hours when spawning took place. Long grunts, in particular, were emitted only during spawning days. Knocks, on the other hand, were associated with all identified behaviors and were the only sound type associated with male-to-male agonistic behavior . Grunts, on the other hand, were associated with “male(s) trailing a female” during spawning days .
Discussion
The study demonstrated that hierarchies developed within meagre broodstocks, and even after exogenous GnRHa stimulation of all individuals in the broodstock, spawning success was not equal among breeders. In both years, the same male and female dominated the produced progeny. It is not clear at this stage what factor contributes to dominance of the two sexes, especially in females , but in males it may be size or aggressiveness. The video and sound production results agree with previous studies that recorded long grunts during spawning nights, as well as with studies conducted in the wild, which reported long choruses of knocks. Knocks and long grunts are, therefore, suggested as “carriers of information” during spawning in meagre. In the future, we will examine the potential of sound “play-back” as an environmental inducer of spawning in cultured meagre and a disruptor of male hierarchies.
The work was funded by the project NewTechAqua (EU H2020, GA 862658).