Determining the effectiveness of using dermal swabs to evaluate the stress physiology of laboratory cephalopods: A preliminary investigation.

To better comprehend the physiology of cephalopods, we used a minimal invasive technique of skin mucus swabs to measure immunoreactive corticosteroids in three cephalopod species commonly kept in captivity and promoted as new model organisms: Euprymna berryi, Sepia bandensis, and Octopus chierchiae. We compared results between sexes and age classes and then evaluated their stress responses during acclimation to a new habitat. To better understand glucocorticoid production, we conducted an adrenocorticotropic hormone, using Cosyntropin (an adrenocorticotropin (ACTH) analogue) challenge with a saline control and swabbed their mantles at 15-minute intervals for 2 h. Results showed cortisol was higher for younger individuals. Additionally, cortisol and corticosterone concentrations decreased by 2-fold after 2 to 4 days of acclimation to a new habitat. We were able to successfully measure 2-fold increase in immunoreactive corticosteroids which reacted with cortisol and corticosterone assays for all the species following ACTH injection, although not all individuals responded similarly. With further investigation, this technique can increase our understanding and management of cephalopods in captivity.

Exploring the Effect of Age on the Reproductive and Stress Physiology of Octopus bimaculoides Using Dermal Hormones.

Our goal was to validate the use of dermal swabs to evaluate both reproductive and stress physiology in the California two-spot octopus, Octopus bimaculoides. Our objectives were to (1) use dermal swabs to evaluate glucocorticoids and reproductive hormones of O. bimaculoides; (2) determine the influence of life stage on hormone production (glucocorticoids in all individuals; testosterone, estrogen, and progesterone in females; and testosterone in males) of reproductive (n = 4) and senescent (n = 8) individuals to determine the effect of age on hormonal patterns; and (3) determine whether these hormones change significantly in response to an acute stressor. For the stress test, individuals were first swabbed for a baseline and then chased around the aquarium with a net for 5 min. Afterward, individuals were swabbed for 2 h at 15 min intervals to compare to the pre-stress test swab. Reproductive individuals responded to the stressor with a 2-fold increase in dermal cortisol concentrations at 15 and 90 min. Six of the eight senescent individuals did not produce a 2-fold increase in dermal cortisol concentrations. Reproductive individuals had significantly higher sex hormone concentrations compared to senescent individuals (progesterone and estradiol measured in females, and testosterone for both sexes). After the stressor, only reproductive males produced a 2-fold increase in dermal testosterone concentrations, while sex hormones in females showed no change. The stress hormone cortisol was significantly higher in senescent than in reproductive individuals, independent of sex. Dermal corticosterone concentrations were highest in senescent females followed by senescent males, and lowest in reproductive individuals regardless of sex. Dermal swabs provide an effective and noninvasive means for evaluating octopus hormones. Application of these indicators may be imperative as cephalopods are more commonly cultured in captivity for experimentation, display, and consumption.

Creation of an albino squid line by CRISPR-Cas9 and its application for in vivo functional imaging of neural activity.

Cephalopods are remarkable among invertebrates for their cognitive abilities, adaptive camouflage, novel structures, and propensity for recoding proteins through RNA editing. Due to the lack of genetically tractable cephalopod models, however, the mechanisms underlying these innovations are poorly understood. Genome editing tools such as CRISPR-Cas9 allow targeted mutations in diverse species to better link genes and function. One emerging cephalopod model, Euprymna berryi, produces large numbers of embryos that can be easily cultured throughout their life cycle and has a sequenced genome. As proof of principle, we used CRISPR-Cas9 in E. berryi to target the gene for tryptophan 2,3 dioxygenase (TDO), an enzyme required for the formation of ommochromes, the pigments present in the eyes and chromatophores of cephalopods. CRISPR-Cas9 ribonucleoproteins targeting tdo were injected into early embryos and then cultured to adulthood. Unexpectedly, the injected specimens were pigmented, despite verification of indels at the targeted sites by sequencing in injected animals (G0s). A homozygote knockout line for TDO, bred through multiple generations, was also pigmented. Surprisingly, a gene encoding indoleamine 2,3, dioxygenase (IDO), an enzyme that catalyzes the same reaction as TDO in vertebrates, was also present in E. berryi. Double knockouts of both tdo and ido with CRISPR-Cas9 produced an albino phenotype. We demonstrate the utility of these albinos for in vivo imaging of Ca2+ signaling in the brain using two-photon microscopy. These data show the feasibility of making gene knockout cephalopod lines that can be used for live imaging of neural activity in these behaviorally sophisticated organisms.