Mass Spectrometry Analysis of Shark Skin Proteins.

The mucus layer covering the skin of fish has several roles, including protection against pathogens and mechanical damage in which proteins play a key role. While proteins in the skin mucus layer of various common bony fish species have been explored, the proteins of shark skin mucus remain unexplored. In this pilot study, we examine the protein composition of the skin mucus in spiny dogfish sharks and chain catsharks through mass spectrometry (NanoLC-MS/MS). Overall, we identified 206 and 72 proteins in spiny dogfish (Squalus acanthias) and chain catsharks (Scyliorhinus retifer), respectively. Categorization showed that the proteins belonged to diverse biological processes and that most proteins were cellular albeit a significant minority were secreted, indicative of mucosal immune roles. The secreted proteins are reviewed in detail with emphasis on their immune potentials. Moreover, STRING protein-protein association network analysis showed that proteins of closely related shark species were more similar as compared to a more distantly related shark and a bony fish, although there were also significant overlaps. This study contributes to the growing field of molecular shark studies and provides a foundation for further research into the functional roles and potential human biomedical implications of shark skin mucus proteins.

The genetic basis of novel trait gain in walking fish.

A major goal in biology is to understand how organisms evolve novel traits. Multiple studies have identified genes contributing to regressive evolution, the loss of structures that existed in a recent ancestor. However, fewer examples exist for genes underlying constructive evolution, the gain of novel structures and capabilities in lineages that previously lacked them. Sea robins are fish that have evolved enlarged pectoral fins, six mobile locomotory fin rays (legs) and six novel macroscopic lobes in the central nervous system (CNS) that innervate the corresponding legs. Here, we establish successful husbandry and use a combination of transcriptomics, CRISPR-Cas9 editing, and behavioral assays to identify key transcription factors that are required for leg formation and function in sea robins. We also generate hybrids between two sea robin species with distinct leg morphologies and use allele-specific expression analysis and gene editing to explore the genetic basis of species-specific trait diversity, including a novel sensory gain of function. Collectively, our study establishes sea robins as a new model for studying the genetic basis of novel organ formation, and demonstrates a crucial role for the conserved limb gene tbx3a in the evolution of chemosensory legs in walking fish.

Identification of Novel Glycans in the Mucus Layer of Shark and Skate Skin.

The mucus layer covering the skin of fish has several roles, including protection against pathogens and mechanical damage. While the mucus layers of various bony fish species have been investigated, the composition and glycan profiles of shark skin mucus remain relatively unexplored. In this pilot study, we aimed to explore the structure and composition of shark skin mucus through histological analysis and glycan profiling. Histological examination of skin samples from Atlantic spiny dogfish (Squalus acanthias) sharks and chain catsharks (Scyliorhinus retifer) revealed distinct mucin-producing cells and a mucus layer, indicating the presence of a functional mucus layer similar to bony fish mucus albeit thinner. Glycan profiling using liquid chromatography-electrospray ionization tandem mass spectrometry unveiled a diverse repertoire of mostly O-glycans in the mucus of the two sharks as well as little skate (Leucoraja erinacea). Elasmobranch glycans differ significantly from bony fish, especially in being more sulfated, and some bear resemblance to human glycans, such as gastric mucin O-glycans and H blood group-type glycans. This study contributes to the concept of shark skin having unique properties and provides a foundation for further research into the functional roles and potential biomedical implications of shark skin mucus glycans.

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.

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.

Anesthetic Efficacy of Magnesium Chloride and Ethyl Alcohol in Temperate Octopus and Cuttlefish Species.

Cephalopods are important in biologic and biomedical research, yet relatively little objective information is available to guide researchers and veterinarians regarding the best methods for anesthetizing these animals for various experimental procedures. Recent studies demonstrate that ethyl alcohol and magnesium chloride are effective at depressing efferent and afferent neural signals in some tropical cephalopod species when measured via the pallial nerve. Here we used similar methods to test 2 temperate species (Octopus bimaculoides and Sepia officinalis) and demonstrate that (1) ethyl alcohol and magnesium chloride were effective at reversibly depressing evoked activity in the pallial nerve, (2) ethyl alcohol generally had shorter induction and recovery times compared with magnesium chloride, (3) both agents were associated with a latency between the behavioral and neural effects, and it was longer with magnesium chloride, and (4) senescent animals generally had longer induction or recovery times than young animals. Both agents successfully anesthetized both life stages; however, our data show that assessing anesthesia based solely on behavior may lead to premature commencement of invasive procedures. We conclude that temperate cephalopods can be humanely, effectively, and completely anesthetized by using these 2 agents and that the loss of neural signal we show here is consistent with true anesthesia and not merely paralysis. This relatively simple, nondestructive nerve recording technique can be applied to the study of other prospective anesthetic agents in cephalopods.

A Simple Microbiome in the European Common Cuttlefish, Sepia officinalis.

The European common cuttlefish, Sepia officinalis, is used extensively in biological and biomedical research, yet its microbiome remains poorly characterized. We analyzed the microbiota of the digestive tract, gills, and skin in mariculture-raised S. officinalis using a combination of 16S rRNA amplicon sequencing, quantitative PCR (qPCR), and fluorescence spectral imaging. Sequencing revealed a highly simplified microbiota consisting largely of two single bacterial amplicon sequence variants (ASVs) of Vibrionaceae and Piscirickettsiaceae. The esophagus was dominated by a single ASV of the genus Vibrio. Imaging revealed bacteria in the family Vibrionaceae distributed in a discrete layer that lines the esophagus. This Vibrio was also the primary ASV found in the microbiota of the stomach, cecum, and intestine, but occurred at lower abundance, as determined by qPCR, and was found only scattered in the lumen rather than in a discrete layer via imaging analysis. Treatment of animals with the commonly used antibiotic enrofloxacin led to a nearly 80% reduction of the dominant Vibrio ASV in the esophagus but did not significantly alter the relative abundance of bacteria overall between treated versus control animals. Data from the gills were dominated by a single ASV in the family Piscirickettsiaceae, which imaging visualized as small clusters of cells. We conclude that bacteria belonging to the Gammaproteobacteria are the major symbionts of the cuttlefish Sepia officinalis cultured from eggs in captivity and that the esophagus and gills are major colonization sites. IMPORTANCE Microbes can play critical roles in the physiology of their animal hosts, as evidenced in cephalopods by the role of Vibrio (Aliivibrio) fischeri in the light organ of the bobtail squid and the role of Alpha- and Gammaproteobacteria in the reproductive system and egg defense in a variety of cephalopods. We sampled the cuttlefish microbiome throughout the digestive tract, gills, and skin and found dense colonization of an unexpected site, the esophagus, by a microbe of the genus Vibrio, as well as colonization of gills by Piscirickettsiaceae. This finding expands the range of organisms and body sites known to be associated with Vibrio and is of potential significance for understanding host-symbiont associations, as well as for understanding and maintaining the health of cephalopods in mariculture.

Pharmacokinetics of buprenorphine following intravenous and oral transmucosal administration in dogs.

Pharmacokinetic analysis of buprenorphine administered to six healthy dogs via the oral transmucosal (OTM) route at doses of 20 and 120 microg/kg was conducted using liquid chromatography-electrospray ionization-tandem mass spectroscopy (LC-ESI-MS/MS). Bioavailability was 38% plus or minus 12% for the 20 microg/kg dose and 47%+/-16% for the 120 microg/kg dose. Maximum plasma concentrations were similar for buprenorphine doses of 20 microg/kg IV and 120 microg/kg OTM. Sedation and salivation were common side effects, but no bradycardia, apnea, or cardiorespiratory depressive effects were seen. When the two OTM dosing rates were normalized to dose, LC-ESI-MS/MS analysis of buprenorphine and its metabolites detected no significant difference (P>.05), indicating dose proportionality. The results of this study suggest that OTM buprenorphine may be an alternative for pain management in dogs.

Effect of orally administered tramadol alone or with an intravenously administered opioid on minimum alveolar concentration of sevoflurane in cats.

OBJECTIVE-To compare the effect of oral administration of tramadol alone and with IV administration of butorphanol or hydromorphone on the minimum alveolar concentration (MAC) of sevoflurane in cats. DESIGN-Crossover study. ANIMALS-8 Healthy 3-year-old cats. PROCEDURES-Cats were anesthetized with sevoflurane in 100% oxygen. A standard tail clamp method was used to determine the MAC of sevoflurane following administration of tramadol (8.6 to 11.6 mg/kg [3.6 to 5.3 mg/lb], PO, 5 minutes before induction of anesthesia), butorphanol (0.4 mg/kg [0.18 mg/lb], IV, 30 minutes after induction), hydromorphone (0.1 mg/kg [0.04 mg/lb], IV, 30 minutes after induction), saline (0.9% NaCl) solution (0.05 mL/kg [0.023 mL/lb], IV, 30 minutes after induction), or tramadol with butorphanol or with hydromorphone (same doses and routes of administration). Naloxone (0.02 mg/kg [0.009 mg/lb], IV) was used to reverse the effects of treatments, and MACs were redetermined. RESULTS-Mean +/- SEM MACs for sevoflurane after administration of tramadol (1.48 +/- 0.20%), butorphanol (1.20 +/- 0.16%), hydromorphone (1.76 +/- 0.15%), tramadol and butorphanol (1.48 +/- 0.20%), and tramadol and hydromorphone (1.85 +/- 0.20%) were significantly less than those after administration of saline solution (2.45 +/- 0.22%). Naloxone reversed the reductions in MACs. CONCLUSIONS AND CLINICAL RELEVANCE-Administration of tramadol, butorphanol, or hydromorphone reduced the MAC of sevoflurane in cats, compared with that in cats treated with saline solution. The reductions detected were likely mediated by effects of the drugs on opioid receptors. An additional reduction in MAC was not detected when tramadol was administered with butorphanol or hydromorphone.

A comparison of anesthetic and cardiorespiratory effects of tiletamine-zolazepam-butorphanol and tiletamine-zolazepam-butorphanol- medetomidine in cats.

Using a randomized crossover design, this study compared the anesthetic and cardiorespiratory effects of three intramuscular anesthetic combinations in seven 2-year-old cats: tiletamine-zolazepam (8 mg/kg) and butorphanol (0.2 mg/kg) (TT); tiletamine-zolazepam (3 mg/kg), butorphanol (0.15 mg/kg), and medetomidine (15 microg/kg) (TTD); or the TTD protocol plus atipamezole (75 microg/kg IM) given 20 minutes later to reverse medetomidine. Analgesia was assessed using algometry and needle pricking. All three combinations effectively induced anesthesia suitable for orotracheal intubation within 5 minutes after injection. Hemoglobin oxygen saturation was lower than 90% at least once in all three groups between 5 and 15 minutes after drug administration. Blood pressure and heart and respiratory rates were within normal ranges. Both TT and TTD appeared to be effective injectable anesthetic combinations. TTD provided significantly better analgesia with a longer duration than did TT. Atipamezole administration shortened the duration of analgesia and decreased blood pressure but did not shorten total recovery time.