Neuro-endocrine control of reproduction and feeding in Octopus vulgaris

The octopus is renowned for possessing the most complex behavior and cognitive abilities among all invertebrates: they show problem-solving skills such as opening jars and learning behaviors after repeated exposures (Fiorito et al., 1990). Moreover, recently three octopuses’ genomes were sequenced: Octopus bimaculoides (Albertin et al., 2016), Octopus minor (Kim et al., 2018), and Octopus vulgaris (Zarrella et al., 2019), skyrocketing the number of possibilities for further research carried on them. The natural population of O. vulgaris is threatened by pollution of waters and global warming (Ariano et al., 2019; Oliveira et al., 2018; Raimundo et al., 2017; Repolho et al., 2013). Despite this, octopus meat consumption remains extensive: only in Europe, the total consumption in 2017 reached 12.69 million tonnes, which is positioned above the world’s average. Therefore, there is an urgent need to develop methods to establish sustainable O. vulgaris aquaculture, in order to meet the demand from the food industry while preserving its natural population. In addition, the octopus is an iconic invertebrate with exquisite learning ability and intelligence. As a cephalopod, it is protected by the stringent EU directive 2010/63/EU on animal experimentation making adequate conditions for octopus husbandry ethically required. What these conditions precisely entail, is still poorly known. In addition, there is a need to deepen the still juvenile knowledge of the octopus’ young stage physiology. This Ph.D. project will generate new knowledge by investigating the neuroendocrine control of the O. vulgaris’ reproduction and feeding. According to a recently proposed model (Di Cristo, 2013), reproductive and feeding behavior are interlinked thanks to the action of neuropeptides and hormones. The subpedunculate lobe and the olfactory lobe in the O. vulgaris’ brain act on the optic gland by releasing respectively the inhibitory factor FMRFamide and the stimulatory factor octoGnRH (homologous to the vertebrate GnRH). The latter then probably acts as an endocrine gland, releasing a still unknown trophic hormone, controlling gonadal maturation and reproduction behavior. Neuropeptides in the olfactory lobe also control food intake: for example during egg-laying and caring, the female will stop feeding and will die after the embryos hatch. All these processes seem to be controlled by the optic gland hormone, which shifts the balance from feeding and body growth to reproduction. Nonetheless, the major factors inducing oocyte and gonadal maturation, spawning, and fertilization in O. vulgaris remain only roughly described. As a first step, we aim to discover which factors are involved in these processes by analyzing the composition of the hemolymph of female octopuses in different reproductive maturation stages. These factors will be characterized by metabolomics and/or peptidomics. They will be tested in vitro to check whether they induce peristaltic movement of the oviduct, apt for conducting the mature oocyte from the ovary to the oviducal gland, where it’s fertilized, and spawned into the water. These factors will then be administered to the octopus in vivo, by means of injection, implants or brachial respiration, in order to induce the maturation and spawning of adult females. A successful outcome would allow researchers for the first time to have continuous availability of O. vulgaris embryos, improving the quality and quantity of scientific research, in contrast to the current state of the art, where scientists are dependent on the naturally occurring spawning periods, occurring 2 times a year. Moreover, aquaculture facilities would also benefit from this protocol, as obtaining spawning from octopuses in captivity could become less challenging. As a contingency plan, the in vitro fertilization of mature oocytes will be attempted, based on the already successful protocol for oceanic squid (Villanueva et al., 2012) and preliminary observations on O. vulgaris made during my master thesis research. Furthermore, in the last years, our lab optimized and published a protocol for a stand-alone tank system together with a staging atlas for the embryonic developmental stage of O. vulgaris within the egg enclosure. As a second step to achieve sustainable and ethical aquaculture of octopuses, both for scientific and commercial purposes, we aim to do likewise with the embryonic developmental stage following hatching from the egg, namely the free-swimming paralarvae. The optimal rearing conditions (water pH, temperature, oxygen level, light conditions, paralarval density) will be investigated, in order to provide the most favorable environment for them to grow. Comparing behavior, chromatophore reaction and inking will be used as readout for stress. Optimal rearing conditions and feeding are of crucial importance, as the paralarvae, once they reach a certain size, independently of their age, undergo changes in diet and behavior by resting at the bottom of the tanks and crawling instead of swimming (Villanueva, 1995), and thus becoming juveniles. Our hypothesis is that these changes could be comparable to a metamorphic process involving hormones, which will be investigated by means of RNAseq on 2, 5, and 10 days old juveniles, and qPCR for known hormones in metamorphosis in other organisms, like the juvenile hormone and ecdysone in insects and thyroid hormone in vertebrates. Having identified the hormonal pathway, we can then investigate whether this metamorphic process (called settling) can be induced in controlled laboratory conditions. This achievement could be beneficial both for laboratories and aquaculture, as the settlement is still precarious and difficult to achieve.