Ocean biomass continues to change as climate change progresses (Carozza, Bianchi, & Galbraith, 2019). As some populations of species have decreased over the last decade, cephalopod populations have continued to increase (Pang et al., 2018). Looking at stress and reproductive hormones may provide more insight as to why these highly adaptable animals are successful in the face of these changes in their ecosystems due to climate change. Here, we demonstrate further validation of a minimal invasive technique to measure reproductive and stress hormones from Octopus bimaculoides dermal mucous. Furthermore, we aimed to show changes in stress and reproductive hormone concentrations throughout the reproductive and senescent life stages of this cephalopod.
Cephalopods live in a variety of habitats in the ocean, from the depths of the ocean to its shallow shores (Zylinski & Johnsen, 2014). They also possess the uncanny ability to change the texture and color of their skin (Chubb et al., 2018; Hough, Case, & Boal, 2016; Mather & Alupay, 2016) and problem solve (Kuba, Byrne, Meisel, & Mather, 2006; Richter, Hochner, & Kuba, 2016). Although they tend to be regarded as intelligent animals, cephalopods have rather short life spans, on average living approximately 1.5- 2 years (Anderson, Wood, & Byrne, 2002). Almost all octopuses are semelparous (Anderson, Wood, Byrne, & Octopus, 2002; Hanlon & Messenger, 2018), reproducing once in their life before a swift decline in health that ends in death.
In general, cephalopods invest in growth during the development phase of their life (Hanlon & Messenger, 2018; Huffard, 2013). Growth and sexually maturity are regulated by secretions into the blood from the optic glands (Wells & Wells 1959). Once sexual maturity is reached, the optic gland switches from overall growth of the animal to the growth of the ovary and its ducts in females (O’Dor & Wells, 1978). Similarly, in males the optic gland controls the condition of the testes. Additionally, reproductive hormones including testosterone, estradiol- 17β, and progesterone, have been shown to regulate reproduction in cephalopods, gastropods, and bivalve mollusks (Lafont & Mathieu 2007D’Aniello et al., 1996; Di Cosmo, Di Cristo, & Paolucci, 2001), and are present in the sex organs and blood of octopuses (D’Aniello et al., 1996). Specially, for female reproduction, when preparing for egg laying, females devote all resources to their ovary, restricting all other growth (O’Dor & Wells, 1978). These morphological changes of the reproductive system are paralleled with fluctuations in 17-β estradiol and progesterone (Di Cosmo, Di Cristo, & Paolucci, 2001). Estradiol and progesterone increase during vitellogenesis, or yolk development, and decreased as vitellogenesis is completed (Di Cosmo, Di Cristo, & Paolucci, 2001). In males, testosterone stimulates spermiogenesis and reproductive behaviors (Avila-Poveda et al., 2015; D’Aniello et al., 1996).
Both male and female octopuses go through senescence. In semelparous species of octopus, after egg laying in females, their health declines and they begin to senesce which may be controlled by activation of the optic gland (O’Dor & Wells, 1978). Males, whether they mate or not, see a similar decline in health around the same age as females lay their eggs (O’Dor & Wells, 1978). Senescence can happen quickly (Mather, 2006; Gestal, Pascual, Guerra, Fiorito, & Vieites, 2019) and in octopuses typically presents itself as a decline in feeding, retraction of skin around the eyes, uncoordinated movement, lesions on the body, and increased and undirected activity (Anderson et al., 2002). During this life stage, females typically are brooding eggs, while males show signs of senescence by being more mobile in search of a mate while they cease eating (Anderson et al., 2002; Mather, 2006).
Like reproductive hormones, glucocorticoid production vary throughout an animal’s life span due to life events and metabolic differences between the life stages (Azevedo et al., 2019; Monfort, Mashburn, Brewer, & Creel, 1988; Romero, 2004; Schell, Young, Lonsdorf, & Santymire, 2013; Touma & Palme, 2005). The Hypothalamic-pituitary-adrenal (HPA) axis activation is well known in vertebrates. Briefly, a perceived stressor results in the hypothalamus releasing corticotropin releasing hormone, which then stimulates the pituitary to release adrenocorticotrophic hormone (ACTH) (Moberg, 2000; Palme, Rettenbacher, Touma, El-Bahr, & Möstl, 2005). Once ACTH is released it stimulates the adrenal glands to release glucocorticoids, including cortisol and corticosterone. These steroids cause an increase in the release and production of glucose, which is used by the body to alleviate the stressor.
Although the stress pathways for cephalopods are unknown (Ottaviani, Caselgrandi, Petraglia, & Franceschi, 1992), octopus stress hormones apparently respond to stressors, or changes in homeostasis, similarly to vertebrates (Larson & Anderson, 2010; Chancellor, et al., Under Review). Juvenile cephalopods produce a higher concentration of glucocorticoids compared to older individuals (Chancellor, et al., Under Review). Both glucocorticoids can be measured in cephalopods; however, cortisol was determined to be the predominate stress hormone found in dermal secretions (Chancellor et al., Under Review). Understanding octopus stress physiology would provide a more robust understanding of cephalopod’s life history along with providing insight to better diagnosis of health issues.
The challenge with studying cephalopod endocrinology is the sampling method, which often uses invasive techniques. For example, animals were euthanized so that the reproductive organs could be harvested at different life stages and analyzed for hormone concentration within the tissue (D’Aniello et al., 1996; Di Cosmo et al., 2001). Other studies used blood draws to measure hormones (Malham, Lacoste, Gélébart, Cueff, & Poulet, 2002). Non-invasive techniques for measuring hormones while being able to repeatedly sample the same individual non-destructively are essential to protecting the welfare of octopuses. While sex and stress hormones have been measured non-invasively by feces (Avila-Poveda, Montes-Pérez, Benitez-Villalobos, & Rosas, 2013; Larson & Anderson, 2010), due to water circulation and group cultures in aquaria it can be difficult to collect feces in a standardized manner without constant monitoring. Mucus swabs of the octopus’ skin surface may provide a non-invasive alternative for measuring hormones. Skin swabs have been used previously in freshwater fish (De Mercado, Larrán, Pinedo, & Tomás-Almenar, 2018; Schultz et al., 2005), amphibians (Santymire, Manjerovic, & Sacerdote-Velat, 2018), and recently validated for use with various cephalopods (Chancellor et al., Under Review). The swabs allow for collection of samples from target individuals. Sampling can be repeated as needed. Ideally, swabbing exerts less stress than other techniques, and can be integrated into specific experiments.
Our goal is to expand the knowledge of cephalopod endocrinology by using dermal mucous swabs to measure glucocorticoid and reproductive hormones in reproductive and senescent life stages in the Two Spot Octopus (Octopus bimaculoides). O. bimaculoides was the first cephalopod to have their genome sequenced (Albertin et al., 2015) and further used extensively in studies (Albertin & Simakov, 2020). The lifespan of O. bimaculoides is 15–17 months. Their three life history stages include juveniles (Boyle 1983), reproductive adults, and post-reproductive senescent adults (Robin et al., 2014). Death typically occurs within approximately 2 months after egg spawning (Forsythe & Hanlon, 1988). Males senesce around the same time as the females (Anderson, Wood, Byrne, & Octopus, 2002). Upon reaching adulthood, even in the absence of opportunities to mate, O. bimaculoides will senesce in 12–14 months.
Our objectives are to: 1) validate the use of dermal swabs to evaluate glucocorticoids and reproductive hormones; 2) determine the influence of life stage on hormone production; 3) examine the relationship among glucocorticoids and reproductive hormones; and 4) determine whether these hormones change significantly in response to an acute stress. We predict that senescent individuals will produce lower stress and reproductive hormones than reproductive individuals. We expect a larger and more immediate increase in stress hormones than reproductive hormones in response to an acute stressor. We also expect the increase in stress hormones will be higher in reproductive adults than in senescent adults.