An assessment of the effects of neurokinin1 receptor antagonism against nausea and vomiting: Relative efficacy, sites of action and lessons for future drug development.

A broad-spectrum anti-vomiting effect of neurokinin1 receptor antagonists (NK1 RA), shown in pre-clinical animal studies, has been supported by a more limited range of clinical studies in different indications. However, this review suggests that compared with vomiting, the self-reported sensation of nausea is less affected or possibly unaffected (depending on the stimulus) by NK1 receptor antagonism, a common finding for anti-emetics. The stimulus-independent effects of NK1 RAs against vomiting are explicable by actions within the central pattern generator (ventral brainstem) and the nucleus tractus solitarius (NTS; dorsal brainstem), with additional effects on vagal afferent activity for certain stimuli (e.g., highly emetogenic chemotherapy). The central pattern generator and NTS neurones are multifunctional so the notable lack of obvious effects of NK1 RAs on other reflexes mediated by the same neurones suggests that their anti-vomiting action is dependent on the activation state of the pathway leading to vomiting. Nausea requires activation of cerebral pathways by projection of information from the NTS. Although NK1 receptors are present in cerebral nuclei implicated in nausea, and imaging studies show very high receptor occupancy at clinically used doses, the variable or limited ability of NK1 RAs to inhibit nausea emphasizes: (i) our inadequate understanding of the mechanisms of nausea; and (ii) that classification of a drug as an anti-emetic may give a false impression of efficacy against nausea vs. vomiting. We discuss the potential mechanisms for the differential efficacy of NK1 RA and the implications for future development of drugs that can effectively treat nausea, an area of unmet clinical need.

Review article: An analysis of the pharmacological rationale for selecting drugs to inhibit vomiting or increase gastric emptying during treatment of gastroparesis.

BACKGROUND: Drugs which can inhibit nausea/vomiting and/or increase gastric emptying are used to treat gastroparesis, mostly 'off-label'. Within each category, they act at different targets and modulate different physiological mechanisms. AIMS: Address the questions: In gastroparesis, why should blocking one pathway causing vomiting, be more appropriate than another? Why might increasing gastric emptying via one mechanism be more appropriate than another? METHODS: Drugs used clinically were identified via consensus opinions and reviews, excluding the poorly characterised. Their pharmacology was defined, mapped to mechanisms influencing vomiting and gastric emptying, and rationale developed for therapeutic use. RESULTS: Vomiting: Rationale for 5-HT3 , D2 , H1 or muscarinic antagonists, and mirtazapine, amitriptyline, nortriptyline, are poor. Arguments for inhibiting central consequences of vagal afferent transmission by NK1 antagonism are complicated by doubts over effects on nausea. Gastric emptying: Confusion emerges because of side-effects of drugs increasing gastric emptying: Metoclopramide (5-HT4 agonist, D2 and 5-HT3 antagonist; also blocks some emetic stimuli and causes tardive dyskinesia) and Erythromycin (high-efficacy motilin agonist, requiring low doses to minimise side-effects). Limited trials with selective 5-HT4 agonists indicate variable efficacy. CONCLUSIONS: Several drug classes inhibiting vomiting have no scientific rationale. NK1 antagonism has rationale but complicated by limited efficacy against nausea. Studies must resolve variable efficacy of selective 5-HT4 agonists and apparent superiority over motilin agonists. Overall, lack of robust activity indicates a need for novel approaches targeting nausea (e.g., modulating gastric pacemaker or vagal activity, use of receptor agonists or new targets such as GDF15) and objective assessments of nausea.

Anti-emetic effects of thalidomide: Evidence, mechanism of action, and future directions.

The rationale for using thalidomide (THD) as a treatment for nausea and vomiting during pregnancy in the late 1950s appears to have been based on its sedative or hypnotic properties. In contrast to contemporaneous studies on the anti-emetic activity of phenothiazines, we were unable to identify publications reporting preclinical or clinical evaluation of THD as an anti-emetic. Our survey of the literature revealed a clinical study in 1965 showing THD reduced vomiting in cancer chemotherapy which was substantiated by similar studies from 2000, particularly showing efficacy in the delayed phase of chemotherapy-induced nausea and vomiting. To identify the mechanism(s) potentially involved in thalidomide's anti-emetic activity we reviewed its pharmacology in the light of nausea and vomiting mechanisms and their pharmacology with a particular emphasis on chemotherapy and pregnancy. The process identified the following potential mechanisms: reduced secretion of Growth Differentiation Factor 15, suppression of inflammation/prostaglandin production, downregulation of cytotoxic drug induced upregulation of iNOS, and modulation of BK (KCa1.1) channels and GABAA/glutamate transmission at critical points in the emetic pathways (nucleus tractus solitarius, area postrema). We propose ways to investigate these hypothesized mechanisms and discuss the associated challenges (e.g., objective quantification of nausea) in addition to some of the more general aspects of developing novel drugs to treat nausea and vomiting.

Methodological considerations in studying digestive system physiology in octopus: limitations, lacunae and lessons learnt.

Current understanding of cephalopod digestive tract physiology is based on relatively "old" literature and a "mosaic of data" from multiple species. To provide a background to the discussion of methodologies for investigating physiology we first review the anatomy of the cephalopod digestive tract with a focus on Octopus vulgaris, highlighting structure-function relationships and species differences with potential functional consequences (e.g., absence of a crop in cuttlefish and squid; presence of a caecal sac in squid). We caution about extrapolation of data on the digestive system physiology from one cephalopod species to another because of the anatomical differences. The contribution of anatomical and histological techniques (e.g., digestive enzyme histochemistry and neurotransmitter immunohistochemistry) to understanding physiological processes is discussed. For each major digestive tract function we briefly review current knowledge, and then discuss techniques and their limitations for the following parameters: 1) Measuring motility in vitro (e.g., spatiotemporal mapping, tension and pressure), in vivo (labelled food, high resolution ultrasound) and aspects of pharmacology; 2) Measuring food ingestion and the time course of digestion with an emphasis on understanding enzyme function in each gut region with respect to time; 3) Assessing transepithelial transport of nutrients; 4) Measuring the energetic cost of food processing, impact of environmental temperature and metabolic rate (flow-through/intermittent respirometry); 4) Investigating neural (brain, gastric ganglion, enteric) and endocrine control processes with an emphasis on application of molecular techniques to identify receptors and their ligands. A number of major knowledge lacunae are identified where available techniques need to be applied to cephalopods, these include: 1) What is the physiological function of the caecal leaflets and intestinal typhlosoles in octopus? 2) What role does the transepithelial transport in the caecum and intestine play in ion, water and nutrient transport? 3) What information is signalled from the digestive tract to the brain regarding the food ingested and the progress of digestion? It is hoped that by combining discussion of the physiology of the cephalopod digestive system with an overview of techniques and identification of key knowledge gaps that this will encourage a more systematic approach to research in this area.

Synergistic augmentation of rhythmic myogenic contractions of human stomach by arginine vasopressin and adrenaline: Implications for the induction of nausea.

BACKGROUND AND PURPOSE: Nausea is associated with the hormonal secretion of vasopressin and adrenaline, although their actions in inducing nausea is poorly understood. Here, we have investigated their actions on human stomach muscle. EXPERIMENTAL APPROACH: Muscle strips were suspended in tissue baths and neuronal-/non-neuronally-mediated contractions were measured. Custom software analysed eight motility parameters defining spontaneous phasic non-neuronally mediated contractions. Receptor distributions were assessed by qPCR and immunofluorescence. KEY RESULTS: V1A receptors and α1 -adrenoceptors were located on muscle as well as interstitial cells of Cajal (ICCs). Myogenic contractions of human proximal and distal stomach (respectively, 2.6 ± 0.1 and 2.7 ± 0.0 per minute; n = 44) were larger in the distal area (1.1 ± 0.1 and 5.0 ± 0.1 mN), developing relatively slowly (proximal) or rapidly (distal). Vasopressin caused tonic (proximal) or short-lived (distal) increases in muscle tone and increased myogenic contraction amplitude, frequency and rate (acting at V1A receptors; thresholds 10-11 -10-10  M); by contrast, cholinergically mediated contractions were unaffected. Oxytocin acted similarly to vasopressin but less potently, at OT receptors). Adrenaline increased (10-10 -10-5  M; α1 -adrenoceptors) and decreased (≥10-6  M; ß-adrenoceptors) muscle tone and enhanced/reduced myogenic contractions. Cholinergically mediated contractions were reduced (α2 -adrenoceptors). Combined, vasopressin (10-9  M) and adrenaline (10-8  M) increased muscle tone and phasic myogenic activity in a synergistic manner. CONCLUSIONS AND IMPLICATIONS: Vasopressin and adrenaline increased human gastric tone and myogenic contraction amplitude, rate of contraction and frequency. In combination, their actions were further increased in a synergistic manner. Such activity may promote nausea.

Evidence for tetrodotoxin-resistant spontaneous myogenic contractions of mouse isolated stomach that are dependent on acetylcholine.

BACKGROUND AND PURPOSE: Gastric pacemaker cells, interstitial cells of Cajal (ICC), are believed to initiate myogenic (non-neuronal) contractions. These become damaged in gastroparesis, associated with dysrhythmic electrical activity and nausea. We utilised mouse isolated stomach to model myogenic contractions and investigate their origin and actions of interstitial cells of Cajal modulators. EXPERIMENTAL APPROACH: Intraluminal pressure was recorded following distension with a physiological volume; tone, contraction amplitude and frequency were quantified. Compounds were bath applied. KEY RESULTS: The stomach exhibited regular large amplitude contractions (median amplitude 9.0 [4.7-14.8] cmH2 O, frequency 2.9 [2.5-3.4] c.p.m; n = 20), appearing to progress aborally. Tetrodotoxin (TTX, 10-6 M) had no effect on tone, frequency or amplitude but blocked responses to nerve stimulation. ω-conotoxin GVIA (10-7 M) ± TTX was without effect on baseline motility. In the presence of TTX, (1) atropine (10-10 -10-6 M) reduced contraction amplitude and frequency in a concentration-related manner (pIC50 7.5 ± 0.3 M for amplitude), (2) CaCC channel (previously ANO1) inhibitors MONNA and CaCCinh-A01 reduced contraction amplitude (significant at 10-5 , 10-4 M respectively) and frequency (significant at 10-5 M), and (3), neostigmine (10-5 M) evoked a large, variable, increase in contraction amplitude, reduced by atropine (10-8 -10-6 M) but unaffected (exploratory study) by the H1 receptor antagonist mepyramine (10-6 M). CONCLUSIONS AND IMPLICATIONS: The distended mouse stomach exhibited myogenic contractions, resistant to blockade of neural activity by TTX. In the presence of TTX, these contractions were prevented or reduced by compounds blocking interstitial cells of Cajal activity or by atropine and enhanced by neostigmine (antagonised by atropine), suggesting involvement of non-neuronal ACh in their regulation.

COVID-19, nausea, and vomiting.

Exclusion of nausea (N) and vomiting (V) from detailed consideration as symptoms of COVID-19 is surprising as N can be an early presenting symptom. We examined the incidence of NV during infection before defining potential mechanisms. We estimate that the overall incidence of nausea (median 10.5%), although variable, is comparable with diarrhea. Poor definition of N, confusion with appetite loss, and reporting of N and/or V as a single entity may contribute to reporting variability and likely underestimation. We propose that emetic mechanisms are activated by mediators released from the intestinal epithelium by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) modulate vagal afferents projecting to the brainstem and after entry into the blood, activate the area postrema (AP) also implicated in anorexia. The receptor for spike protein of SARS-CoV-2, angiotensin 2 converting enzyme (ACE2), and transmembrane protease serine (for viral entry) is expressed in upper gastrointestinal (GI) enterocytes, ACE2 is expressed on enteroendocrine cells (EECs), and SARS-CoV-2 infects enterocytes but not EECs (studies needed with native EECs). The resultant virus-induced release of epithelial mediators due to exocytosis, inflammation, and apoptosis provides the peripheral and central emetic drives. Additionally, data from SARS-CoV-2 show an increase in plasma angiotensin II (consequent on SARS-CoV-2/ACE2 interaction), a centrally (AP) acting emetic, providing a further potential mechanism in COVID-19. Viral invasion of the dorsal brainstem is also a possibility but more likely in delayed onset symptoms. Overall, greater attention must be given to nausea as an early symptom of COVID-19 and for the insights provided into the GI effects of SARS-CoV-2.

Can Cephalopods Vomit? Hypothesis Based on a Review of Circumstantial Evidence and Preliminary Experimental Observations.

In representative species of all vertebrate classes, the oral ejection of upper digestive tract contents by vomiting or regurgitation is used to void food contaminated with toxins or containing indigestible material not voidable in the feces. Vomiting or regurgitation has been reported in a number of invertebrate marine species (Exaiptasia diaphana, Cancer productus, and Pleurobranchaea californica), prompting consideration of whether cephalopods have this capability. This "hypothesis and theory" paper reviews four lines of supporting evidence: (1) the mollusk P. californica sharing some digestive tract morphological and innervation similarities with Octopus vulgaris is able to vomit or regurgitate with the mechanisms well characterized, providing an example of motor program switching; (2) a rationale for vomiting or regurgitation in cephalopods based upon the potential requirement to void indigestible material, which may cause damage and ejection of toxin contaminated food; (3) anecdotal reports (including from the literature) of vomiting- or regurgitation-like behavior in several species of cephalopod (Sepia officinalis, Sepioteuthis sepioidea, O. vulgaris, and Enteroctopus dofleini); and (4) anatomical and physiological studies indicating that ejection of gastric/crop contents via the buccal cavity is a theoretical possibility by retroperistalsis in the upper digestive tract (esophagus, crop, and stomach). We have not identified any publications refuting our hypothesis, so a balanced review is not possible. Overall, the evidence presented is circumstantial, so experiments adapting current methodology (e.g., research community survey, in vitro studies of motility, and analysis of indigestible gut contents and feces) are described to obtain additional evidence to either support or refute our hypothesis. We recognize the possibility that further research may not support the hypothesis; therefore, we consider how cephalopods may protect themselves against ingestion of toxic food by external chemodetection prior to ingestion and digestive gland detoxification post-ingestion. Reviewing the evidence for the hypothesis has identified a number of gaps in knowledge of the anatomy (e.g., the presence of sphincters) and physiology (e.g., the fate of indigestible food residues, pH of digestive secretions, sensory innervation, and digestive gland detoxification mechanisms) of the digestive tract as well as a paucity of recent studies on the role of epithelial chemoreceptors in prey identification and food intake.

Microplastics presence in cultured and wild-caught cuttlefish, Sepia officinalis.

Amongst cephalopods microplastics have been reported only in jumbo squid gut. We investigated microplastics in the digestive system of wild cuttlefish (Sepia officinalis) as they are predators and prey and compared the stomach, caecum/intestine and digestive gland (DG) of wild and cultured animals, exposed to seawater from a comparable source. Fibers were the most common type (≈90% of total count) but were ≈2× higher in relation to body weight in wild vs. cultured animals. Fibers were transported to the DG where the count was ≈2× higher /g in wild (median 1.85 fibers/g) vs. cultured. In wild-caught animals the DG was the predominant location but in cultured animals the fibers were more evenly distributed in the digestive tract. The potential impact of microplastics on health of cuttlefish is discussed. Cuttlefish represent a previously unrecognized source of microplastic trophic transfer to fish and finding fibers in cultured animals has implications for aquaculture.

Functional brain networks and neuroanatomy underpinning nausea severity can predict nausea susceptibility using machine learning.

KEY POINTS: Nausea is an adverse experience characterised by alterations in autonomic and cerebral function. Susceptibility to nausea is difficult to predict, but machine learning has yet to be applied to this field of study. The severity of nausea that individuals experience is related to the underlying morphology (shape) of the subcortex, namely of the amygdala, caudate and putamen; a functional brain network related to nausea severity was identified, which included the thalamus, cingulate cortices (anterior, mid- and posterior), caudate nucleus and nucleus accumbens. Sympathetic nervous system function and sympathovagal balance, by heart rate variability, was closely related to both this nausea-associated anatomical variation and the functional connectivity network, and machine learning accurately predicted susceptibility or resistance to nausea. These novel anatomical and functional brain biomarkers for nausea severity may permit objective identification of individuals susceptible to nausea, using artificial intelligence/machine learning; brain data may be useful to identify individuals more susceptible to nausea. ABSTRACT: Nausea is a highly individual and variable experience. The central processing of nausea remains poorly understood, although numerous influential factors have been proposed, including brain structure and function, as well as autonomic nervous system (ANS) activity. We investigated the role of these factors in nausea severity and if susceptibility to nausea could be predicted using machine learning. Twenty-eight healthy participants (15 males; mean age 24 years) underwent quantification of resting sympathetic and parasympathetic nervous system activity by heart rate variability. All were exposed to a 10-min motion-sickness video during fMRI. Neuroanatomical shape differences of the subcortex and functional brain networks associated with the severity of nausea were investigated. A machine learning neural network was trained to predict nausea susceptibility, or resistance, using resting ANS data and detected brain features. Increasing nausea scores positively correlated with shape variation of the left amygdala, right caudate and bilateral putamen (corrected P = 0.05). A functional brain network linked to increasing nausea severity was identified implicating the thalamus, anterior, middle and posterior cingulate cortices, caudate nucleus and nucleus accumbens (corrected P = 0.043). Both neuroanatomical differences and the functional nausea-brain network were closely related to sympathetic nervous system activity. Using these data, a machine learning model predicted susceptibility to nausea with an overall accuracy of 82.1%. Nausea severity relates to underlying subcortical morphology and a functional brain network; both measures are potential biomarkers in trials of anti-nausea therapies. The use of machine learning should be further investigated as an objective means to develop models predicting nausea susceptibility.

A History of Drug Discovery for Treatment of Nausea and Vomiting and the Implications for Future Research.

The origins of the major classes of current anti-emetics are examined. Serendipity is a recurrent theme in discovery of their anti-emetic properties and repurposing from one indication to another is a continuing trend. Notably, the discoveries have occurred against a background of company mergers and changing anti-emetic requirements. Major drug classes include: (i) Muscarinic receptor antagonists-originated from historical accounts of plant extracts containing atropine and hyoscine with development stimulated by the need to prevent sea-sickness among soldiers during beach landings; (ii) Histamine receptor antagonists-searching for replacements for the anti-malaria drug quinine, in short supply because of wartime shipping blockade, facilitated the discovery of histamine (H1) antagonists (e.g., dimenhydrinate), followed by serendipitous discovery of anti-emetic activity against motion sickness in a patient undergoing treatment for urticaria; (iii) Phenothiazines and dopamine receptor antagonists-investigations of their pharmacology as "sedatives" (e.g., chlorpromazine) implicated dopamine receptors in emesis, leading to development of selective dopamine (D2) receptor antagonists (e.g., domperidone with poor ability to penetrate the blood-brain barrier) as anti-emetics in chemotherapy and surgery; (iv) Metoclopramide and selective 5-hydroxytryptamine3(5-HT3) receptor antagonists-metoclopramide was initially assumed to act only via D2 receptor antagonism but subsequently its gastric motility stimulant effect (proposed to contribute to the anti-emetic action) was shown to be due to 5-hydroxytryptamine4 receptor agonism. Pre-clinical studies showed that anti-emetic efficacy against the newly-introduced, highly emetic, chemotherapeutic agent cisplatin was due to antagonism at 5-HT3 receptors. The latter led to identification of selective 5-HT3 receptor antagonists (e.g., granisetron), a major breakthrough in treatment of chemotherapy-induced emesis; (v) Neurokinin1receptor antagonists-antagonists of the actions of substance P were developed as analgesics but pre-clinical studies identified broad-spectrum anti-emetic effects; clinical studies showed particular efficacy in the delayed phase of chemotherapy-induced emesis. Finally, the repurposing of different drugs for treatment of nausea and vomiting is examined, particularly during palliative care, and also the challenges in identifying novel anti-emetic drugs, particularly for treatment of nausea as compared to vomiting. We consider the lessons from the past for the future and ask why there has not been a major breakthrough in the last 20 years.

Cephalopod Brains: An Overview of Current Knowledge to Facilitate Comparison With Vertebrates.

Cephalopod and vertebrate neural-systems are often highlighted as a traditional example of convergent evolution. Their large brains, relative to body size, and complexity of sensory-motor systems and behavioral repertoires offer opportunities for comparative analysis. Despite various attempts, questions on how cephalopod 'brains' evolved and to what extent it is possible to identify a vertebrate-equivalence, assuming it exists, remain unanswered. Here, we summarize recent molecular, anatomical and developmental data to explore certain features in the neural organization of cephalopods and vertebrates to investigate to what extent an evolutionary convergence is likely. Furthermore, and based on whole body and brain axes as defined in early-stage embryos using the expression patterns of homeodomain-containing transcription factors and axonal tractography, we describe a critical analysis of cephalopod neural systems showing similarities to the cerebral cortex, thalamus, basal ganglia, midbrain, cerebellum, hypothalamus, brain stem, and spinal cord of vertebrates. Our overall aim is to promote and facilitate further, hypothesis-driven, studies of cephalopod neural systems evolution.

Curcumin and derivatives function through protein phosphatase 2A and presenilin orthologues in Dictyostelium discoideum.

Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a complex molecule possessing wide-ranging biological activities, cellular mechanisms and roles in potential therapeutic treatment, including Alzheimer's disease and cancer. Here, we investigate the physiological effects and molecular targets of curcumin in Dictyostelium discoideum We show that curcumin exerts acute effects on cell behaviour, reduces cell growth and slows multicellular development. We employed a range of structurally related compounds to show the distinct role of different structural groups in curcumin's effects on cell behaviour, growth and development, highlighting active moieties in cell function, and showing that these cellular effects are unrelated to the well-known antioxidant activity of curcumin. Molecular mechanisms underlying the effect of curcumin and one synthetic analogue (EF24) were then investigated to identify a curcumin-resistant mutant lacking the protein phosphatase 2A regulatory subunit (PsrA) and an EF24-resistant mutant lacking the presenilin 1 orthologue (PsenB). Using in silico docking analysis, we then showed that curcumin might function through direct binding to a key regulatory region of PsrA. These findings reveal novel cellular and molecular mechanisms for the function of curcumin and related compounds.

Gastric myoelectric activity during cisplatin-induced acute and delayed emesis reveals a temporal impairment of slow waves in ferrets: effects not reversed by the GLP-1 receptor antagonist, exendin (9-39).

Preclinical studies show that the glucagon-like peptide-1 (GLP-1) receptor antagonist, exendin (9-39), can reduce acute emesis induced by cisplatin. In the present study, we investigate the effect of exendin (9-39) (100 nmol/24 h, i.c.v), on cisplatin (5 mg/kg, i.p.)-induced acute and delayed emesis and changes indicative of 'nausea' in ferrets. Cisplatin induced 37.2 ± 2.3 and 59.0 ± 7.7 retches + vomits during the 0-24 (acute) and 24-72 h (delayed) periods, respectively. Cisplatin also increased (P<0.05) the dominant frequency of gastric myoelectric activity from 9.4 ± 0.1 to 10.4 ± 0.41 cpm and decreased the dominant power (DP) during acute emesis; there was a reduction in the % power of normogastria and an increase in the % power of tachygastria; food and water intake was reduced. DP decreased further during delayed emesis, where normogastria predominated. Advanced multifractal detrended fluctuation analysis revealed that the slow wave signal shape became more simplistic during delayed emesis. Cisplatin did not affect blood pressure (BP), but transiently increased heart rate, and decreased heart rate variability (HRV) during acute emesis; HRV spectral analysis indicated a shift to 'sympathetic dominance'. A hyperthermic response was seen during acute emesis, but hypothermia occurred during delayed emesis and there was also a decrease in HR. Exendin (9-39) did not improve feeding and drinking but reduced cisplatin-induced acute emesis by ~59 % (P<0.05) and antagonised the hypothermic response (P<0.05); systolic, diastolic and mean arterial BP increased during the delayed phase. In conclusion, blocking GLP-1 receptors in the brain reduces cisplatin-induced acute but not delayed emesis. Restoring power and structure to slow waves may represent a novel approach to treat the side effects of chemotherapy.

Prey Capture, Ingestion, and Digestion Dynamics of Octopus vulgaris Paralarvae Fed Live Zooplankton.

Octopus vulgaris is a species of great interest in research areas such as neurobiology, ethology, and ecology but also a candidate species for aquaculture as a food resource and for alleviating the fishing pressure on its wild populations. This study aimed to characterize the predatory behavior of O. vulgaris paralarvae and to quantify their digestive activity. Those processes were affordable using the video-recording analysis of 3 days post-hatching (dph), mantle-transparent paralarvae feeding on 18 types of live zooplanktonic prey. We show for the first time in a live cephalopod that octopus paralarvae attack, immobilize, drill, and ingest live cladocerans and copepods with 100% efficiency, which decreases dramatically to 60% on decapod prey (Pisidia longicornis). The majority (85%) of successful attacks targeted the prey cephalothorax while unsuccessful attacks either targeted the dorsal cephalothorax or involved prey defensive strategies (e.g., juvenile crab megalopae) or prey protected by thick carapaces (e.g., gammaridae amphipods). After immobilization, the beak, the buccal mass and the radula were involved in exoskeleton penetration and content ingestion. Ingestion time of prey content was rapid for copepods and cladocerans (73.13 ± 23.34 s) but much slower for decapod zoeae and euphausiids (152.49 ± 29.40 s). Total contact time with prey was always <5 min. Contrary to the conventional view of crop filling dynamics observed in adult O. vulgaris, food accumulated first in the stomach of paralarvae and the crop filled after the stomach volume plateaued. Peristaltic crop contractions (~18/min) moved food into the stomach (contractions ~30/min) from where it passed to the caecum. Pigmented food particles were seen to enter the digestive gland, 312 ± 32 s after the crop reached its maximum volume. Digestive tract contents passed into the terminal intestine by peristalsis (contraction frequency ~50/min) and defaecation was accompanied by an increased frequency of mantle contractions. Current results provide novel insights into both, O. vulgaris paralarvae-live prey capture strategies and the physiological mechanisms following ingestion, providing key information required to develop an effective rearing protocol for O. vulgaris paralarvae.

The Digestive Tract of Cephalopods: a Neglected Topic of Relevance to Animal Welfare in the Laboratory and Aquaculture.

Maintenance of health and welfare of a cephalopod is essential whether it is in a research, aquaculture or public display. The inclusion of cephalopods in the European Union legislation (Directive 2010/63/EU) regulating the use of animals for scientific purposes has prompted detailed consideration and review of all aspects of the care and welfare of cephalopods in the laboratory but the information generated will be of utility in other settings. We overview a wide range of topics of relevance to cephalopod digestive tract physiology and their relationship to the health and welfare of these animals. Major topics reviewed include: (i) Feeding cephalopods in captivity which deals with live food and prepared diets, feeding frequency (ad libitum vs. intermittent) and the amount of food provided; (ii) The particular challenges in feeding hatchlings and paralarvae, as feeding and survival of paralarvae remain major bottlenecks for aquaculture e.g., Octopus vulgaris; (iii) Digestive tract parasites and ingested toxins are discussed not only from the perspective of the impact on digestive function and welfare but also as potential confounding factors in research studies; (iv) Food deprivation is sometimes necessary (e.g., prior to anesthesia and surgery, to investigate metabolic control) but what is the impact on a cephalopod, how can it be assessed and how does the duration relate to regulatory threshold and severity assessment? Reduced food intake is also reviewed in the context of setting humane end-points in experimental procedures; (v) A range of experimental procedures are reviewed for their potential impact on digestive tract function and welfare including anesthesia and surgery, pain and stress, drug administration and induced developmental abnormalities. The review concludes by making some specific recommendations regarding reporting of feeding data and identifies a number of areas for further investigation. The answer to many of the questions raised here will rely on studies of the physiology of the digestive tract.