Impact of natural and artificial prenatal stimulation on the behavioural profile of Japanese quail.

As the sensory systems of vertebrates develop prenatally, embryos perceive many environmental stimuli that can influence the ontogeny of their behaviour. Whether the nature and intensity of prenatal stimuli affect this ontogeny differently remains to be investigated. In this context, this study aimed to analyse the effects of prenatal auditory stimulation (natural stimulation, NS; predator vocalisations or artificial stimulation, AS; metallic sounds) on the subsequent behaviour of young Japanese quail (Coturnix coturnix japonica). For this, behavioural variables recorded during ethological tests evaluating emotional and social reactivity were analysed using a principal component analysis. This analysis revealed significant differences between the behavioural profile of stimulated chicks and that of non-exposed chicks. Indeed, chicks exposed to NS expressed more intense emotional responses in fearful situations, but less neophobia when exposed to a novel environment or object, whereas chicks exposed to AS appeared more sensitive to social isolation. Our results show that the acoustic environment of embryos can influence the way young birds subsequently interact with their social and physical environment after hatching, and face challenges in changing living conditions.

Awareness of danger inside the egg: Evidence of innate and learned predator recognition in cuttlefish embryos.

Predation can be a very strong selective pressure on prey. Many studies have shown the existence of innate anti-predator responses, mostly in the early developmental stages of juvenile vertebrates. Learning to recognize predators is another possible defensive resource, but such a method involves a high death risk. There is evidence that prenatal learning exists in animals but few studies have explicitly tested for embryonic learning. The aim of this study was to test innate and learned predator recognition in cuttlefish embryos. For this, naïve embryos were exposed to chemical and visual cues emanating from predators, non-predators, and ink. Their response was assessed by measuring their ventilation rate (VR). We first show that VR decreased in response to both visual and chemical predatory cues and ink but not to non-predatory cues. Second, we show that when non-predatory cues (visual or chemical) are paired with predatory cues or ink for several days, embryonic VR significantly decreased. Such a response is likely adaptive, especially in a translucent egg, since it results in reduced movement and hence may lower the risk of detection by visual predators. This freezing-like behavior may also reduce the bioelectric field, thus lessening the predation risk by non-visual foragers. Our results report that cuttlefish embryos had an innate capacity to differentiate between harmless and harmful chemical and visual cues. They were also capable of learning to respond to harmless cues when they were paired with danger (predator or ink) based on conditioning. The combination of these behavioral mechanisms is an example of the early adaptability of cephalopods. Such behavioral plasticity may give the newly hatched cuttlefish a selective advantage when dealing with either known or unfamiliar threats. Nevertheless, more experiments are needed to test the efficiency of the embryos' response faced with known or new predators.

A difference in timing for the onset of visual and chemosensory systems during embryonic development in two closely related cuttlefish species.

Embryos perceive environmental stimuli, thanks to their almost mature sensory systems. In cuttlefish, the embryonic development of Sepia officinalis and Sepia pharaonis is similar but the egg capsule transparency is different. S. officinalis' eggs are black (ink), which provide protection from predators. Conversely, those of S. pharaonis are translucent. The aim of this study was to test the visual and chemosensory perception abilities of these two cuttlefish embryos by observation of the ventilation rate (VR) before and after stimulation. Our results show that S. pharaonis responds to light at stage 22 and S. officinalis at stage 24. Conversely, S. pharaonis responds to predator odor at stage 23 and S. officinalis at stage 22. Both species are able to respond to these stimuli before hatching but do not have the same developmental schedule. Neither are the responses of the two cuttlefish exactly the same. In S. officinalis, VR increases after stimulations. In S. pharaonis, VR increases after light stimulation and decreases following the odor stimulation after stage 25. This result could reveal an ability to recognize stimuli at stage 25. The decrease could be identified as freezing-like behavior which would be more adaptive than an increase, since the embryos are visible.

Behavioral development in embryonic and early juvenile cuttlefish (Sepia officinalis).

Though a mollusc, the cuttlefish Sepia officinalis possesses a sophisticated brain, advanced sensory systems, and a large behavioral repertoire. Cuttlefish provide a unique perspective on animal behavior due to their phylogenic distance from more traditional (vertebrate) models. S. officinalis is well-suited to addressing questions of behavioral ontogeny. As embryos, they can perceive and learn from their environment and experience no direct parental care. A marked progression in learning and behavior is observed during late embryonic and early juvenile development. This improvement is concomitant with expansion and maturation of the vertical lobe, the cephalopod analog of the mammalian hippocampus. This review synthesizes existing knowledge regarding embryonic and juvenile development in this species in an effort to better understand cuttlefish behavior and animal behavior in general. It will serve as a guide to future researchers and encourage greater awareness of the utility of this species to behavioral science.

Lipids in maternal diet influence yolk hormone levels and post-hatch neophobia in the domestic chick.

We assessed whether the ratio of dietary n-6/n-3 polyunsaturated fatty acids (PUFA) during egg formation engenders transgenerational maternal effects in domestic chicks. We analyzed yolk lipid and hormone concentrations, and HPA-axis activity in hens fed a control diet (high n-6/n-3 ratio) or a diet enriched in n-3 PUFAs (low n-6/n-3 ratio) for 6 consecutive weeks. Their chicks were tested for neophobia during the first week of life. We found higher corticosterone metabolites in droppings of hens fed the diet enriched in n-3 and significantly higher concentrations of yolk progesterone, androstenedione, and estradiol in their eggs compared to controls. Chicks of hens fed the n-3 enriched diet showed a lower body mass at hatch than controls and expressed higher neophobia when exposed to a novel object. These results add support to the hypothesis that the nutritional state of female birds produces variation in yolk hormone levels and engender maternal effects.

Maturation of polarization and luminance contrast sensitivities in cuttlefish (Sepia officinalis).

Polarization sensitivity is a characteristic of the visual system of cephalopods. It has been well documented in adult cuttlefish, which use polarization sensitivity in a large range of tasks such as communication, orientation and predation. Because cuttlefish do not benefit from parental care, their visual system (including the ability to detect motion) must be efficient from hatching to enable them to detect prey or predators. We studied the maturation and functionality of polarization sensitivity in newly hatched cuttlefish. In a first experiment, we examined the response of juvenile cuttlefish from hatching to the age of 1 month towards a moving, vertically oriented grating (contrasting and polarized stripes) using an optomotor response apparatus. Cuttlefish showed differences in maturation of polarization versus luminance contrast motion detection. In a second experiment, we examined the involvement of polarization information in prey preference and detection in cuttlefish of the same age. Cuttlefish preferentially chose not to attack transparent prey whose polarization contrast had been removed with a depolarizing filter. Performances of prey detection based on luminance contrast improved with age. Polarization contrast can help cuttlefish detect transparent prey. Our results suggest that polarization is not a simple modulation of luminance information, but rather that it is processed as a distinct channel of visual information. Both luminance and polarization sensitivity are functional, though not fully matured, in newly hatched cuttlefish and seem to help in prey detection.

General and species-specific recommendations for minimal requirements for the use of cephalopods in scientific research.

Here we list species-specific recommendations for housing, care and management of cephalopod molluscs employed for research purposes with the aim of contributing to the standardization of minimum requirements for establishments, care and accommodation of these animals in compliance with the principles stated in Directive 2010/63/EU. Maximizing their psychophysical welfare was our priority. General recommendations on water surface area, water depth and tank shape here reported represent the outcome of the combined action of the analysis of the available literature and an expertise-based consensus reached - under the aegis of the COST Action FA1301 - among researchers working with the most commonly used cephalopod species in Europe. Information on water supply and quality, environmental conditions, stocking density, feeding and handling are also provided. Through this work we wish to set the stage for a more fertile ground of evidence-based approaches on cephalopod laboratory maintenance, thus facilitating standardization and replicability of research outcomes across laboratories, at the same time maximizing the welfare of these animals.

Feel, smell and see in an egg: emergence of perception and learning in an immature invertebrate, the cuttlefish embryo.

It is now well established that prenatal sensory experience affects development itself and has long-term consequences in terms of postnatal behavior. This study focused on the functionality of the sensory system in cuttlefish in ovo. Embryos of stage 23, 25 and 30 received a tactile, chemical or visual stimulus. An increase of mantle contraction rhythm was taken to indicate a behavioral response to the stimulus. We clearly demonstrated that tactile and chemical systems are functional from stage 23, whereas the visual system is functional only from stage 25. At stage 25 and 30, embryos were also exposed to a repeated light stimulus. Stage 30 embryos were capable of habituation, showing a progressive decrease in contractions across stimulations. This process was not due to fatigue as we observed response recovery after a dishabituation tactile stimulus. This study is the first to show that cuttlefish embryos behaviorally respond to stimuli of different modalities and that the visual system is the last to become functional during embryonic development, as in vertebrate embryos. It also provides new evidence that the memory system develops in ovo in cuttlefish.

Cuttlefish rely on both polarized light and landmarks for orientation.

Cuttlefish are sensitive to linear polarization of light, a sensitivity that they use in predation and possibly in intraspecific communication. It has also been shown that cuttlefish are able to solve a maze using visual landmarks. In this study, cuttlefish were trained to solve a Y-maze with the e-vector of a polarized light and landmarks as redundant spatial information. The results showed that cuttlefish can use the e-vector orientation and landmarks in parallel to orient and that they are able to use either type of cue when the other one is missing. When they faced conflicting spatial information in the experimental apparatus, the majority of cuttlefish followed the e-vector rather than landmarks. Differences in response latencies in the different conditions of testing (training with both types of cue, tests with single cue or with conflicting information) were observed and discussed in terms of decision making. The ability to use near field and far field information may enable animals to interpret the partially occluded underwater light field.

The effect of unexpected rewards on decision making in cuttlefish.

Despite numerous studies demonstrating the cognitive ability of cephalopods, there is currently no study showing an emotion-like behavior in this group of animals. To examine whether cuttlefish have different internal states, we developed a behavioral paradigm to assess if prior surprised events are able to alter the choice made by cuttlefish. By presenting unexpected food rewards to cuttlefish before the test, we investigated whether the reaction time of choosing between two shrimps, an intuitive response toward the prey without previous learning, at three different levels of discriminative tests (easy, difficult, and ambiguous), are different compared to the one without an unexpected reward. This behavioral paradigm serves to demonstrate whether cuttlefish are aware of ambiguous situations, and their choice outcome and reaction time are dependent of their internal states. The results show that the response latency was significantly shortened in the difficult and ambiguous tests when choosing from two shrimps that are either moderately different in size or similar sizes, respectively, when cuttlefish have received unexpected rewards before the test. These results were compared with tests during which the cuttlefish did not receive any reward in advance. Furthermore, this shortening of latency did not result in a difference in choice outcome during the difficult and ambiguous tests. Interestingly, even when cuttlefish have obtained the expected food rewards or simply made tentacular strike without prey capture each time before test, these prior experiences were sufficient to shorten the response latency in the difficult and ambiguous tests. However, different from the result of unexpected rewards, food consumption alone or prey capture failure did affect the choice outcome during the simple and difficult tests. Taken together, our findings suggest that pre-test treatments of unexpected and expected rewards or simply unsuccessful visual attack seem to induce cuttlefish to adopt different foraging behaviors. This context dependent decision making suggests that cuttlefish's foraging strategies are influenced by the previously surprised event and their internal states. It also shows a speed-accuracy tradeoff in difficult and ambiguous situations when foraging for prey. This observation may lead to a future investigation of the presence of emotional state in cephalopods.

Prenatal maternal stress is associated with behavioural and epigenetic changes in Japanese quail.

Prenatal maternal stress (PMS) influences many facets of offspring's phenotype including morphology, behaviour and cognitive abilities. Recent research suggested that PMS also induced epigenetic modifications. In the present study, we analysed, in the Japanese quail, the effects of PMS on the emotional reactivity and cognitive abilities of the F1 offspring. We also investigated in the hippocampus, the paraventricular hypothalamic nucleus and subnuclei of the arcopallium/amygdala the level of two histone post-translational modifications, H3K4me2 and H3K27me3, known to be impacted by stress. We found that PMS does not affect F1 quail's learning abilities but increases their emotional reactivity. Moreover, we demonstrated that PMS induced an increased density of H3K27me3 positive cells, in the hippocampus, paraventricular hypothalamic nucleus and dorsal nucleus of the amygdala, but not variations of H3K4me2. As these brain regions are involved in the control of vertebrates' emotional responses, the effect of PMS on the epigenetic mark H3K27me3 could possibly be a mechanism involved in the behavioural effects we observed in F1 quail.

Early Exposure to Water Turbidity Affects Visual Capacities in Cuttlefish (Sepia officinalis).

In La Manche (English Channel) the level of turbidity changes, not only seasonally and daily in seawater but also along the coast. As a consequence, vision in marine species is limited when based only on contrast-intensity. It is hypothesized that polarization sensitivity (PS) may help individuals detect preys and predators in turbid environments. In the cuttlefish, Sepia officinalis, to date, all behavioral studies have been conducted on animals reared in clear water. But the cuttlefish sensory system is adapted to a range of turbid environments. Our hypothesis was that rearing cuttlefish in clear water may affect the development of their visual system, and potentially affect their visually guided behaviors. To test this, newly-hatched cuttlefish, from eggs laid by females brought in from the wild, were reared for 1 month under three different conditions: clear water (C group), low turbidity (0.1 g / l of clay, 50-80 NTU, LT group) and high turbidity (0.5 g / l of clay, 300-400 NTU, HT group). The visual capacities of cuttlefish were tested with an optomotor apparatus at 7 days and at 1 month post-hatching. Optomotor responses of juveniles were measured by using three screen patterns (black and white stripes, linearly polarized stripes set at different orientations, and a uniform gray screen). Optomotor responses of juveniles suggest that exposure to turbid water improves the development of their PS when tested in clear water (especially in LT group) but not when tested in turbid water. We suggest that the use of slightly turbid water in rearing systems may improve the development of vision in young cuttlefish with no detrimental effect to their survival rate. Future research will consider water turbidity as a possible factor for the improvement of cuttlefish well-being in artificial rearing systems.

Early exposure to odors changes later visual prey preferences in cuttlefish.

Developmental studies have shown that environmental stimulation received by a developing sensory system can alter the developmental outcome of both that sensory system and other aspects of the nervous system. We investigated the ecologically relevant question of whether prior exposure to prey early in development within one sensory modality could influence later prey choice within a different sensory modality. Cuttlefish are visual predators; they can detect prey odors but attacks on prey cannot be elicited without visual stimulation. Cuttlefish eggs were exposed to the odor of shrimp (preferred prey), crabs (non-preferred prey), mollusks (non-prey), or a seawater control (no prey). Seven days after hatching, prey preferences were tested with a visual choice test between crabs and shrimp. Hatchlings exposed to crabs odors and the seawater control were significantly more likely to attack shrimp. Hatchlings exposed to mollusk odors showed no visual prey preference, while those exposed to shrimp preferentially attacked crabs. These results demonstrate a complex relationship between an early sensory exposure and later prey preference

Human behaviour at the origin of maternal effects on offspring behaviour in laying hens (Gallus gallus domesticus).

Regular visual presence of humans is known to reduce chickens' human-generated stress responses. Here we questioned whether, more than mere visual presence, human behaviour affects laying hen behaviour and subsequently their offspring's behaviour. We hypothesized that human behaviour triggers maternal effects via variations in yolk hormone levels. For five consecutive weeks, two groups of hens were exposed to the same durations of human presence (30 min twice a day, five days a week) but the behaviour of the human differed between groups. The first group (H+) was exposed to predictable arrival of the experimenter, slow movements combined with static presence, stroking during handling and human voice. Whereas the second group of hens (H-) was exposed to unpredictable arrival of the experimenter which remained silent, in motion, and did not provide stroking during handling. At the end of the treatment, we evaluated egg quality and offspring behaviour. We found that avoidance of the experimenter by H+ hens but not by H- hens decreased significantly. Fertility rates and concentrations of yolk progesterone and estradiol in H+ hens' eggs were higher than in H- hens' eggs. Fear of humans, neophobia or the capacity to solve a detour task did not differ significantly between H+ and H- chicks. Social discrimination tests showed that H+ chicks but not H- chicks typically preferred a familiar conspecific to a stranger. These results show that, with the same duration in the presence of the birds, humans through their behaviour engender variations in fertility rates, yolk hormone levels and transgenerational effects on social skills. Rarely explored, our data suggest that maternal effects influence filial imprinting. These data have broad implications for laboratory, commercial systems and conservatory programs where the inevitable presence of humans could trigger maternal effects on offspring phenotype.

Sex differences in spatial cognition in an invertebrate: the cuttlefish.

Evidence of sex differences in spatial cognition have been reported in a wide range of vertebrate species. Several evolutionary hypotheses have been proposed to explain these differences. The one best supported is the range size hypothesis that links spatial ability to range size. Our study aimed to determine whether male cuttlefish (Sepia officinalis; cephalopod mollusc) range over a larger area than females and whether this difference is associated with a cognitive dimorphism in orientation abilities. First, we assessed the distance travelled by sexually immature and mature cuttlefish of both sexes when placed in an open field (test 1). Second, cuttlefish were trained to solve a spatial task in a T-maze, and the spatial strategy preferentially used (right/left turn or visual cues) was determined (test 2). Our results showed that sexually mature males travelled a longer distance in test 1, and were more likely to use visual cues to orient in test 2, compared with the other three groups. This paper demonstrates for the first time a cognitive dimorphism between sexes in an invertebrate. The data conform to the predictions of the range size hypothesis. Comparative studies with other invertebrate species might lead to a better understanding of the evolution of cognitive dimorphism.

Short-distance navigation in cephalopods: a review and synthesis.

This paper provides a short overview of the scientific knowledge concerning short-distance navigation in cephalopods. Studies in laboratory controlled conditions and observations in the field provide converging evidence that cephalopods use visual cues to navigate and demonstrate spatial memory. A recent study also provides the first evidence for the neural substrates underlying spatial abilities in cuttlefish. The functions of spatial cognition in cephalopods are discussed from an evolutionary standpoint.

Incubation temperature affects the expression of young precocial birds' fear-related behaviours and neuroendocrine correlates.

The influence of embryonic microclimate on the behavioural development of birds remains unexplored. In this study, we experimentally tested whether chronic exposure to suboptimal temperatures engendered plasticity in the expression of fear-related behaviours and in the expression of the corticotropin-releasing factor in the brains of domestic chicks (Gallus g. domesticus). We compared the neurobehavioural phenotypes of a control group of chicks incubated in an optimal thermal environment (37.8 °C) with those of a group of experimental chicks exposed chronically in ovo to suboptimal temperatures (27.2 °C for 1 hour twice a day). Chronic exposure to a suboptimal temperature delayed hatching and decreased growth rate and experimental chicks had higher neophobic responses than controls in novel food and novel environment tests. In addition, experimental chicks showed higher expression of corticotropin-releasing factor than did controls in nuclei of the amygdala, a structure involved in the regulation of fear-related behaviours. In this study, we report the first evidence of the strong but underappreciated role of incubation microclimate on the development of birds' behaviour and its neurobiological correlates.

Visual Ecology and the Development of Visually Guided Behavior in the Cuttlefish.

Cuttlefish are highly visual animals, a fact reflected in the large size of their eyes and visual-processing centers of their brain. Adults detect their prey visually, navigate using visual cues such as landmarks or the e-vector of polarized light and display intense visual patterns during mating and agonistic encounters. Although much is known about the visual system in adult cuttlefish, few studies have investigated its development and that of visually-guided behavior in juveniles. This review summarizes the results of studies of visual development in embryos and young juveniles. The visual system is the last to develop, as in vertebrates, and is functional before hatching. Indeed, embryonic exposure to prey, shelters or complex background alters postembryonic behavior. Visual acuity and lateralization, and polarization sensitivity improve throughout the first months after hatching. The production of body patterning in juveniles is not the simple stimulus-response process commonly presented in the literature. Rather, it likely requires the complex integration of visual information, and is subject to inter-individual differences. Though the focus of this review is vision in cuttlefish, it is important to note that other senses, particularly sensitivity to vibration and to waterborne chemical signals, also play a role in behavior. Considering the multimodal sensory dimensions of natural stimuli and their integration and processing by individuals offer new exciting avenues of future inquiry.

Going Up or Sideways? Perception of Space and Obstacles Negotiating by Cuttlefish.

While octopuses are mostly benthic animals, and squid prefer the open waters, cuttlefish present a special intermediate stage. Although their body structure resembles that of a squid, in many cases their behavior is mostly benthic. To test cuttlefish's preference in the use of space, we trained juvenile Sepia gibba and Sepia officinalis cuttlefish to reach a shelter at the opposite side of a tank. Afterwards, rock barriers were placed between the starting point and the shelter. In one experiment, direct paths were available both through the sand and over the rocks. In a second experiment the direct path was blocked by small rocks requiring a short detour to by-pass. In the third experiment instead, the only direct path available was over the rocks; or else to reach the goal via an exclusively horizontal path a longer detour would have to be selected. We showed that cuttlefish prefer to move horizontally when a direct route or a short detour path is available close to the ground; however when faced with significant obstacles they can and would preferentially choose a more direct path requiring a vertical movement over a longer exclusively horizontal path. Therefore, cuttlefish appear to be predominantly benthic dwellers that prefer to stay near the bottom. Nonetheless, they do view and utilize the vertical space in their daily movements where it plays a role in night foraging, obstacles negotiation and movement in their home-range.

Maternal and Embryonic Stress Influence Offspring Behavior in the Cuttlefish Sepia officinalis.

Stress experienced during prenatal development-either applied to reproducing females (maternal stress), directly to developing offspring (embryonic stress) or in combination-is associated with a range of post-natal behavioral effects in numerous organisms. We conducted an experiment to discern if maternal and embryonic stressors affect the behavior of hatchlings of the cuttlefish Sepia officinalis, a species with features that allow for the examination of these stress types in isolation. Separating the impact of stress transmitted through the mother vs. stress experienced by the embryo itself will help clarify the behavioral findings in viviparous species for which it is impossible to disentangle these effects. We also compared the effect of a naturally-occurring (predator cue) and an "artificial" (bright, randomly-occurring LED light) embryonic stressor. This allowed us to test the hypothesis that a threat commonly faced by a species (natural threat) would be met with a genetically-programmed and adaptive response while a novel one would confound innate defense mechanisms and lead to maladaptive effects. We found that the maternal stressor was associated with significant differences in body patterning and activity patterns. By contrast, embryonic exposure to stressors increased the proportion of individuals that pursued prey. From these results, it appears that in cuttlefish, maternal and embryonic stressors affect different post-natal behavior in offspring. In addition, the effect of the artificial stressor suggests that organisms can sometimes react adaptively to a stressor even if it is not one that has been encountered during the evolutionary history of the species.