Involvement of early embryonic miR‐409‐3p in the establishment of anxiety levels in female mice

Small RNA molecules in early embryos, delivered from sperm to zygotes upon fertilization, are required for normal mouse embryonic development. Even modest changes in the levels of sperm‐derived miRNAs appear to influence early embryos and subsequent development. For example, stress‐associated behaviors develop in mice after injection into normal zygotes sets of sperm miRNAs elevated in stressed male mice. Here, we implicate early embryonic miR‐409‐3p in establishing anxiety levels in adult female, but not male mice. First, we found that exposure of male mice to chronic social instability stress, which leads to elevated anxiety in their female offspring across at least three generations through the paternal lineage, elevates sperm miR‐409‐3p levels not only in exposed males, but also in sperm of their F1 and F2 male offspring. Second, we observed that while injection of a mimic of miR‐409‐3p into zygotes from mating control males was incapable of mimicking this effect in offspring derived from them, injection of a specific inhibitor of this miRNA led to the opposite, anxiolytic effect in female, but not male, and offspring. These findings imply that baseline miR‐409‐3p activity in early female embryos is necessary for the expression of normal anxiety levels when they develop into adult females. In addition, elevated embryo miR‐409‐3p activity, possibly as a consequence of stress‐induced elevation of its expression in sperm, may participate in, but may not be sufficient for, the induction of enhanced anxiety.     

Transgenerational inheritance of chronic adolescent stress: Effects of stress response and the amygdala transcriptome

Adolescent stress can impact health and well‐being not only during adulthood of the exposed individual but even in future generations. To investigate the molecular mechanisms underlying these long‐term effects, we exposed adolescent males to stress and measured anxiety behaviors and gene expression in the amygdala—a critical region in the control of emotional states—in their progeny for two generations, offspring and grandoffspring. Male C57BL/6 mice underwent chronic unpredictable stress (CUS) for 2 weeks during adolescence and were used to produce two generations of offspring. Male and female offspring and grandoffspring were tested in behavioral assays to measure affective behavior and stress reactivity. Remarkably, transgenerational inheritance of paternal stress exposure produced a protective phenotype in the male, but not the female lineage. RNA‐seq analysis of the amygdala from male offspring and grandoffspring identified differentially expressed genes (DEGs) in mice derived from fathers exposed to CUS. The DEGSs clustered into numerous pathways, and the “notch signaling” pathway was the most significantly altered in male grandoffspring. Therefore, we show that paternal stress exposure impacts future generations which manifest in behavioral changes and molecular adaptations.     

Migratory‐stage sea lamprey Petromyzon marinus stop responding to conspecific damage‐released alarm cues after 4 h of continuous exposure in laboratory conditions

This study investigated the length of avoidance response of migratory‐stage sea lamprey Petromyzon marinus exposed continuously to conspecific damage‐released alarm cues for varying lengths of time in laboratory stream channels. Ten replicate groups of P. marinus, separated by sex, were exposed to either deionized water control or to P. marinus extract for 0, 2 or 4 h continuously. Petromyzon marinus maintained their avoidance response to the conspecific damage‐released alarm cue after continuous exposure to the alarm cue for 0 and 2 h but not 4 h. Beyond being one of the first studies in regards to sensory–olfactory adaptation–acclimation of fishes to alarm cues of any kind, these results have important implications for use of conspecific alarm cues in P. marinus control. For example, continuous application of conspecific alarm cue during the day, when P. marinus are inactive and hiding, may result in sensory adaptation to the odour by nightfall when they migrate upstream.     

The ontogeny of chemically mediated antipredator responses of fathead minnows Pimephales promelas

The antipredator responses of adult and larval fathead minnows Pimephales promelas to chemical alarm cues prepared throughout ontogeny were tested using various behavioural assays. Larval epidermis was also examined during ontogeny using standard haematoxylin and eosin staining techniques. Adults elicited an antipredator response to chemical alarm cue made from larvae as young as 8–17 days post‐hatch. Interestingly, larvae did not possess visible club cells until 28–37 days post‐hatch and did not respond to conspecific chemical alarm cue until 48–57 days post‐hatch. These results suggest that chemical alarm cue may not be contained within club cells and that the components of larval and adult chemical alarm cue may be similar throughout ontogeny.     

Smell, learn and live: The role of chemical alarm cues in predator learning during early life history in a marine fish

The speed with which individuals can learn to identify and react appropriately to predation threats when transitioning to new life history stages and habitats will influence their survival. This study investigated the role of chemical alarm cues in both anti-predator responses and predator identification during a transitional period in a newly settled coral reef damselfish, Pomacentrus amboinensis. Individuals were tested for changes in seven behavioural traits in response to conspecific and heterospecific skin extracts. Additionally, we tested whether fish could learn to associate a previously novel chemical cue (i.e. simulated predator scent) with danger, after previously being exposed to a paired cue combining the conspecific skin extract with the novel scent. Fish exposed to conspecific skin extracts were found to significantly decreased their feeding rate whilst those exposed to heterospecific and control cues showed no change. Individuals were also able to associate a previously novel scent with danger after only a single previous exposure to the paired conspecific skin extract/novel scent cue. Our results indicate that chemical alarm cues play a large role in both threat detection and learned predator recognition during the early post-settlement period in coral reef fishes.     

Paternal care in a fish: epigenetics and fitness enhancing effects on offspring anxiety

In many animals, including humans, interactions with caring parents can have long-lasting effects on offspring sensitivity to stressors. However, whether these parental effects impact offspring fitness in nature is often unclear. In addition, despite evidence that maternal care can influence offspring behaviour via epigenetic alterations to the genome, it remains unclear whether paternal care has similar effects. Here, we show in three-spined sticklebacks, a fish in which fathers are the sole provider of offspring care, that the direct care provided by fathers affects offspring anxiety and the potential for epigenetic alterations to the offspring genome. We find that families are differentially vulnerable to early stress and fathers can compensate for this differential sensitivity with the quality of their care. This variation in paternal care is also linked to the expression in offspring brains of a DNA methyltransferase (Dnmt3a) responsible for de novo methylation. We show that these paternal effects are potentially adaptive and anxious offspring are unlikely to survive an encounter with a predator. By supplying offspring care, fathers reduce offspring anxiety thereby increasing the survival of their offspring—not in the traditional sense through resource provisioning but through an epigenetic effect on offspring behavioural development.     

Increasing paternal age alters anxiety‐related behaviour in adult mice

Advanced paternal age (APA) is associated with an increased risk of adverse health outcomes in offspring, including autism and schizophrenia. In the present study, we investigated the behaviour of young (3‐month‐old; Control), middle aged (12 to 15‐month‐old; APA1) and old (24‐month‐old; APA2) C57BL/6J sires and their adult offspring. Male and female mice were tested at 10 weeks of age on a behavioural test battery including the elevated plus‐maze, hole board, light/dark emergence, forced swim test, novelty‐suppressed feeding and for prepulse inhibition of the acoustic startle response. Increasing the APA sire age to 24 months was shown to be associated with increased anxiety‐related behaviour in the offspring, and indicated that increasing APA sire age produced a more robust hypoexplorative phenotype. Thus, increasing paternal age was associated with an increase in severity of an anxiogenic phenotype in their adult offspring. Ultimately, the results of these studies show that mouse models of APA are valuable for elucidating the mechanisms by which APA influences brain‐related outcomes.     

Habitat degradation disrupts neophobia in juvenile coral reef fish

Habitat degradation not only disrupts habitat‐forming species, but alters the sensory landscape within which most species must balance behavioural activities against predation risk. Rapidly developing a cautious behavioural phenotype, a condition known as neophobia, is advantageous when entering a novel risky habitat. Many aquatic organisms rely on damage‐released conspecific cues (i.e. alarm cues) as an indicator of impending danger and use them to assess general risk and develop neophobia. This study tested whether settlement‐stage damselfish associated with degraded coral reef habitats were able to use alarm cues as an indicator of risk and, in turn, develop a neophobic response at the end of their larval phase. Our results indicate that fish in live coral habitats that were exposed to alarm cues developed neophobia, and, in situ, were found to be more cautious, more closely associated with their coral shelters and survived four‐times better than non‐neophobic control fish. In contrast, fish that settled onto degraded coral habitats did not exhibit neophobia and consequently suffered much greater mortality on the reef, regardless of their history of exposure to alarm cues. Our results show that habitat degradation alters the efficacy of alarm cues with phenotypic and survival consequences for newly settled recruits.     

Small non‐coding RNA and colorectal cancer

Colorectal cancer (CRC) is the third most common malignance. Although great efforts have been made to understand the pathogenesis of CRC, the underlying mechanisms are still unclear. It is now clear that more than 90% of the total genome is actively transcribed, but lack of protein‐coding potential. The massive amount of RNA can be classified as housekeeping RNAs (such as ribosomal RNAs, transfer RNAs) and regulatory RNAs (such as microRNAs [miRNAs], PIWI‐interacting RNA [piRNAs], tRNA‐derived stress‐induced RNA, tRNA‐derived small RNA [tRFs] and long non‐coding RNAs [lncRNAs]). Small non‐coding RNAs are a group of ncRNAs with the length no more than 200 nt and they have been found to exert important regulatory functions under many pathological conditions. In this review, we summarize the biogenesis and functions of regulatory sncRNAs, such as miRNAs, piRNA and tRFs, and highlight their involvements in cancers, particularly in CRC.     

Separation between maternal and paternal effects on offspring following exposure of adult red flour beetles to two stressors

1. The contribution of non‐genetic maternal effects to offspring performance is well established and the evidence for paternal effects has also been increasing recently. Studies determining the relative contributions of the two parents to offspring success are, however, still rare. 2. In this study, two stressors were applied to adult red flour beetles (Tribolium castaneum) – starvation and cold stress – and a full‐factorial design was used to distinguish between maternal and paternal reproductive decisions and their effects on the offspring. 3. Starvation had a stronger negative effect than cold stress on both males and females, and the likelihood of starved females producing offspring was very low. Furthermore, starved fathers led to lower offspring mass at the larval stage, probably leading to impaired starvation tolerance of the offspring. 4. Cold‐stressed fathers were less likely than unstressed fathers to reproduce, whereas cold‐stressed mothers demonstrated a similar effect by producing fewer offspring. 5. Applying stress probably led to energy saving that came at the expense of reproduction intensity. It is suggested that the smaller offspring mass is a negative consequence of the parental exposure to stress. 6. The differences between the consequences of the two stressors applied and between the relative contribution of each parent could perhaps be explained by the distinct physiological responses of each sex to each of the stressors.     

The effects of ultraviolet radiation on a freshwater prey fish: physiological stress response,club cell investment,and alarm cue production

Recent anthropogenic activities have caused deleterious effects to the stratospheric ozone layer, resulting in a global increase in the level of ultraviolet radiation (UVR). Understanding the way that organisms respond to such stressors is key to predicting the effects of anthropogenic activities on aquatic ecosystems and the species that inhabit them. The epidermal layer of the skin of fishes is not keratinized and acts as the primary interface between the fish and its environment. The skin of many species of fishes contains large epidermal club cells (ECCs) that are known to release chemicals (alarm cues) serving to warn other fishes of danger. However, the alarm role of the cells is likely secondary to their role in the immune system. Recent research suggests that ECCs in the epidermis may play a role in protecting the fish from damage caused by UVR. In the present study, we examined the effects of in vivo exposure to UVR on fathead minnows (Pimephales promelas), specifically investigating ECC investment, physiological stress responses, and alarm cue production. We found that fish exposed to UVR showed an increase in cortisol levels and a substantive decrease in ECC investment compared to non‐exposed controls. Unexpectedly, our subsequent analysis of the behavioural response of fish to alarm cues revealed no difference in the potency of the cues prepared from the skin of UV‐exposed or non‐exposed minnows. Our results indicate that, although nonlethal, UVR exposure may lead to secondary mortality by altering the fish immune system, although this same exposure may have little influence on chemically‐mediated predator–prey interactions. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 832–841.     

Egg load is a cue for offspring sex ratio adjustment in a fig‐pollinating wasp with male‐eggs‐first sex allocation

Fig‐pollinating wasps (Agaonidae) only reproduce within fig tree inflorescences (figs). Agaonid offspring sex ratios are usually female‐biased and often concur with local mate competition theory (LMC). LMC predicts less female‐bias when several foundresses reproduce in a fig due to reduced relatedness among intra‐sexually competing male offspring. Clutch size, the offspring produced by each foundress, is a strong predictor of agaonid sex ratios and correlates negatively with foundress number. However, clutch size variation can result from several processes including egg load (eggs within a foundress), competition among foundresses and oviposition site limitation, each of which can be used as a sex allocation cue. We introduced into individual Ficus racemosa figs single Ceratosolen fusciceps foundresses and allowed each to oviposit from zero to five hours thus variably reducing their eggs‐loads and then introduced each wasp individually into a second fig. Offspring sex ratio (proportion males) in second figs correlated negatively with clutch size, with males produced even in very small clutches. Ceratosolen fusciceps lay mainly male eggs first and then female eggs. Our results demonstrate that foundresses do not generally lay or attempt to lay a ‘fixed’ number of males, but do ‘reset to zero’ their sex allocation strategy on entering a second fig. With decreasing clutch size, gall failure increased, probably due to reduced pollen. We conclude that C. fusciceps foundresses can use their own egg loads as a cue to facultatively adjust their offspring sex ratios and that foundresses may also produce more ‘insurance’ males when they can predict increasing rates of offspring mortality.     

Anti‐predator response of naïve and experienced common bully to chemical alarm cues

Histological analysis of the skin of common bully Gobiomorphus cotidianus , a New Zealand native eleotrid fish, revealed the presence of club cells in the epidermis. Epidermal club cells are frequently associated with the production of alarm substance (Schreckstoff). The behavioural responses of perch‐naïve and perch‐experienced common bullies to either conspecific skin extract or chemical cues from an introduced predator, perch Perca fluviatilis , were then examined. Both perch‐naïve and perch‐experienced common bullies exhibited a behavioural response when exposed to conspecific skin extract, indicating the probable presence of an alarm substance. In contrast, only perch‐experienced common bullies recognized and exhibited a subsequent behavioural response to the odour of perch. This study is the first to document the presence of epidermal club cells and a behavioural response to a conspecific chemical alarm signal for fishes in the Eleotridae. The results indicate that common bully can learn to recognize perch odour as a threat, and that this ability may be a result of previous predator labelling involving a conspecific alarm substance.     

Predator‐induced phenotypic plasticity in an arid‐adapted tropical tadpole

Adaptive phenotypic plasticity is widespread and involves diverse phenotypes. Key environmental stressors, such as predation risk, can simultaneously induce changes in multiple traits, but the magnitude of response is dependent upon the environmental conditions. Species that utilize temporary ponds are expected to exhibit stronger predator‐induced responses in the form of morphology than behaviour (i.e. reduced activity) to meet the demands of rapid development by maintaining high foraging activity while reducing predation risk via morphologically plastic traits. In a laboratory experiment, I examined the effects of predator chemical cues and conspecific alarm cues on activity, development and morphology on Leptodactylus bufonius tadpoles. This species has terrestrial oviposition and completes the early part of its development outside of ephemeral and temporary ponds in the Gran Chaco ecoregion of South America. Tadpoles in the predator treatments exhibited both behavioural and morphological predator‐induced plastic responses. Tadpoles tended to possess shorter, deeper tails when exposed to predators. The greatest reduction in activity was observed in tadpoles exposed to both predator and conspecific alarm cues, which subsequently resulted in the slowest development. Temporary and ephemeral pond adapted species with terrestrial oviposition may capitalize on a head start in development by being able to afford reduced growth rates via a reduction in activity. This may occur when the constraints imposed by pond hydroperiod (e.g. risk of pond drying) are relaxed when compared with species with aquatic oviposition, which must undergo all stages of development during the pond's hydroperiod. Thus, in addition to the predator and hydroperiod gradients, examining phenotypically plastic responses along a ‘terrestriality gradient’ in a comparative framework would provide insights as to the costs and benefits of increasing terrestriality in anuran reproductive modes to environmental stressors.     

The controversy, potential and roles of spermatozoal RNA

The majority of cellular and molecular andrologists endorse the view that the sperm is a vessel for transporting the paternal genome to the waiting egg and nothing more. Any requirement for additional spermatozoal components that enter the ooplasm apart from the paternal centriole and the soluble egg-activating factor is generally dismissed. Many studies, however, have reported RNAs in ejaculate spermatozoa and we now know that mRNAs are delivered to the egg on fertilisation. The function and utility of sperm mRNA remains essentially unexplored. Here, we examine the controversy surrounding spermatozoal mRNA carriage, the evidence refuting its presence as an artefact and how spermatozoal mRNA is leading us to suspect that, quite apart from its undoubted diagnostic potential, it might have an important role in the establishment and maintenance of a viable paternal genome.     

Maternal predator‐exposure affects offspring size at birth but not telomere length in a live‐bearing fish

The perception of predation risk could affect prey phenotype both within and between generations (via parental effects). The response to predation risk could involve modifications in physiology, morphology, and behavior and can ultimately affect long‐term fitness. Among the possible modifications mediated by the exposure to predation risk, telomere length could be a proxy for investigating the response to predation risk both within and between generations, as telomeres can be significantly affected by environmental stress. Maternal exposure to the perception of predation risk can affect a variety of offspring traits but the effect on offspring telomere length has never been experimentally tested. Using a live‐bearing fish, the guppy (Poecilia reticulata), we tested if the perceived risk of predation could affect the telomere length of adult females directly and that of their offspring with a balanced experimental setup that allowed us to control for both maternal and paternal contribution. We exposed female guppies to the perception of predation risk during gestation using a combination of both visual and chemical cues and we then measured female telomere length after the exposure period. Maternal effects mediated by the exposure to predation risk were measured on offspring telomere length and body size at birth. Contrary to our predictions, we did not find a significant effect of predation‐exposure neither on female nor on offspring telomere length, but females exposed to predation risk produced smaller offspring at birth. We discuss the possible explanations for our findings and advocate for further research on telomere dynamics in ectotherms.     

Paternal body size affects reproductive success in laboratory-held zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

Across many fish species, large females tend to exhibit higher individual reproductive success due to elevated fecundity and the provisioning of better conditioned eggs and offspring compared to small females. By contrast, effects of paternal body size on reproductive success are less well understood. We disentangled the maternal- and paternal-size dependent effects on reproductive output and early life history in zebrafish (Danio rerio). In the laboratory, females and males from four size categories (small, medium-sized, large and very large) were allowed to spawn freely in a full factorial design with 10 replicates per size combination. As expected, larger females produced more eggs and better conditioned offspring compared to smaller females. Male body size further contributed to zebrafish reproductive success: offspring sired by large males exhibited higher hatching probability and these offspring also hatched earlier and larger than offspring fertilized by small males. However, the largest males experienced lower mating success and received fewer eggs than males of the smaller size classes. While male body size substantially affected reproductive success in zebrafish, it remained unclear whether and to what degree direct paternal effects (e.g., related to sperm quality) or indirect paternal effects stemming from differential allocation patterns by females were the mechanism behind our findings. Answering this question constitutes an important future research topic.     

Alarm cue induces an antipredator morphological defense in juvenile Nicaragua cichlids Hypsophrys nicaraguensis

Olfactory cues that indicate predation risk elicit a number of defensive behaviors in fishes, but whether they are sufficient to also induce morphological defenses has receivedlittle attention. Cichlids are characterized by a high level of morphological plasticity during development, and the few species that have been tested do exhibit defensive behaviors when exposed to alarm cues released from the damaged skin of conspecifics. We utilized young juvenile Nicaragua cichlids Hypsophrys nicaraguensis to test if the perception of predation risk from alarm cue (conspecific skin extract) alone induces an increased relative body depth which is a defense against gape-limited predators. After two weeks of exposure, siblings that were exposed to conspecific alarm cue increased their relative body depth nearly double the amount of those exposed to distilled water (control) and zebrafish Danio rerio alarm cue. We repeated our measurements over the last two weeks (12 and 14) of cue exposure when the fish were late-stage juveniles to test if the rate of increase was sustained; there were no differences in final dimensions between the three treatments. Our results show that 1) the Nicaragua cichlid has an innate response to conspecific alarm cue which is not a generalized response to an injured fish, and 2) this innate recognition ultimately results in developing a deeper body at a stage of the life history where predation risk is high [Current Zoology 56 (1): 36-42, 2010].