ABSTRACT The nuchal organ of the embryos and neonates of the cladoceran, Daphnia magna , has been shown to be a site of Na + influx and H + , NH 4 + and Cl − efflux. This study combines the scanning ion-selective electrode technique with application of inhibitors of specific transporters to assess the mechanisms of Na + transport across the nuchal organ. Na + influx across the nuchal organ was inhibited both by inhibitors of the Na + /K + -ATPase (ouabain, bufalin) and by inhibitors of the vacuolar H + -ATPase (bafilomycin, N -ethylmaleimde, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, KM91104, S -nitrosoglutathione). Na + influx was unaffected by the epithelial Na + channel blocker benzamil, but was sensitive to ethylisopropyl amiloride and elevated external ammonium concentrations, consistent with roles for Na + /H + and Na + /NH 4 + exchangers in the apical membrane but not Na + channels. Transport across the basolateral membrane into the haemolymph is proposed to involve the Na + /K + -ATPase and a thiazide-sensitive Na + /Cl − cotransporter.

ABSTRACT Some species of durophagous moray eels (Muraenidae) have been documented emerging from the marine environment to capture intertidal crabs but how they consume prey out of water is unknown. Here, we trained snowflake morays, Echidna nebulosa , to undulate out of the aquatic environment to feed on land. On land, snowflake morays remove prey from the substrate by biting and swallow prey using pharyngeal jaw enabled transport. Although snowflake morays exhibit smaller jaw rotation angles on land when apprehending their prey, transport kinematics involving dorsoventral flexion of the head to protract the pharyngeal jaws and overall feeding times did not differ between terrestrial and aquatic treatments. We suggest that their elongate body plan, ability to rotate their heads in the dorsoventral and lateral directions, and extreme pharyngeal movements all contribute to the ability of durophagous morays to feed in the terrestrial environment.

ABSTRACT Aversive learning – avoiding certain situations based on negative experiences – can profoundly increase fitness in animal species, yet no studies have systematically quantified its repeatability. Therefore, we assessed the repeatability of aversive learning by conditioning approximately 100 zebrafish ( Danio rerio ) to avoid a colour cue associated with a mild electric shock. Across eight different colour conditions, zebrafish did not show consistent individual differences in aversive learning ( R= 0.04). Within conditions, when zebrafish were conditioned to the same colour, blue conditioning was more repeatable than green conditioning ( R =0.15 and R =0.02). Overall, aversive learning responses of zebrafish were weak and variable. We speculate that the effect of aversive learning might have been too weak to quantify consistent individual differences, or directional selection might have eroded additive genetic variance. We also discuss how confounded repeatability assays and publication bias could have inflated estimates of repeatability in the literature.

ABSTRACT Endotherms at high altitude face the combined challenges of cold and hypoxia. Cold increases thermoregulatory costs, and hypoxia may limit both thermogenesis and aerobic exercise capacity. Consequently, in comparisons between closely related highland and lowland taxa, we might expect to observe consistent differences in basal metabolic rate (BMR), maximal metabolic rate (MMR) and aerobic scope. Broad-scale comparative studies of birds reveal no association between BMR and native elevation, and altitude effects on MMR have not been investigated. We tested for altitude-related variation in aerobic metabolism in 10 Andean passerines representing five pairs of closely related species with contrasting elevational ranges. Mass-corrected BMR and MMR were significantly higher in most highland species relative to their lowland counterparts, but there was no uniform elevational trend across all pairs of species. Our results suggest that there is no simple explanation regarding the ecological and physiological causes of elevational variation in aerobic metabolism.

In wild animals, telomere attrition during early development has been linked with several fitness penalties throughout life. Telomerase enzyme can elongate telomeres, but it is generally assumed that its activity is suppressed in most somatic tissues upon birth. However, recent evidence suggests that this may not be the rule for long-lived bird species. We have therefore investigated whether telomerase activity is maintained during the postnatal growth period in a wild yellow-legged gull ( Larus michahellis ) population. Our results indicate that telomerase activity is not negligible in the blood cells, but activity levels sharply decline from hatching to fledging following a similar pattern to that observed in telomere length. Our results further suggest that the observed variation in telomere length may be the result of a negative effect of fast growth on telomerase activity, thus providing a new mechanism through which growth rates may affect telomere dynamics and potentially life-history trajectories.

In addition to its health benefits, exercise training has been pointed out as modulator of the gut microbiota. However, the effects of resistance training (RT) on gut microbiota composition remain unknown. Wistar rats underwent 12 weeks of RT. Body weight, glucose tolerance test, visceral body fat, triglyceride concentration, and food consumption were evaluated. The gut microbiota was analyzed by 16S rRNA gene sequencing. Rats that underwent RT showed lower body weight (p=0.0005), lower fat content ( p = 0.02 ), and better glucose kinetics ( p = 0.047 ) when compared to the control. Improvements in the diversity and composition of the gut microbiota were identified in the RT group. The relative abundance of Pseudomonas, Serratia, and Comamonas decreased significantly after 12 weeks of RT ( p < 0.001 ). These results suggest that RT has the potential to enhance the diversity of the gut microbiota and improve its biological functions.

ABSTRACT Relatively little effort has been directed towards elucidating the role of physiological stress pathways in mediating avian responses to global heating. For free-ranging southern pied babblers, Turdoides bicolor , daily maximum air temperatures ( T max ) between ∼35 and ∼40°C result in reduced foraging efficiency, loss of body mass and compromised breeding success. We tested the hypothesis that very hot days are experienced as stressors by quantifying relationships between T max and faecal glucocorticoid metabolite (fGCM) levels in naturally excreted droppings. On days when T max <38°C, fGCM levels were independent of T max (mean±s.d. 140.25±56.92 ng g −1 dry mass). At T max >38°C, however, fGCM levels increased linearly with T max and averaged 190.79±70.13 ng g −1 dry mass. The effects of T max on fGCM levels did not carry over to the following morning, suggesting that very hot days are experienced as acute stressors.

ABSTRACT We investigated age-related changes to fascicle length, sarcomere length and serial sarcomere number (SSN), and how this affects passive force. Following mechanical testing to determine passive force, the medial gastrocnemius muscle of young ( n= 9) and old ( n= 8) Fisher 344BN hybrid rats was chemically fixed at the optimal muscle length for force production; individual fascicles were dissected for length measurement, and laser diffraction was used to assess sarcomere length. Old rats had ∼14% shorter fascicle lengths than young rats, which was driven by a ∼10% reduction in SSN, with no difference in sarcomere length (∼4%). Passive force was greater in the old than in the young rats at long muscle lengths. Shorter fascicle lengths and reduced SSN in the old rats could not entirely explain increased passive forces for absolute length changes, owing to a slight reduction in sarcomere length in old rats, resulting in similar sarcomere length at long muscle lengths.

Environmental pollutants can exert sublethal deleterious effects on animals. These include disruption of cognitive functions underlying crucial behaviours. While agrochemicals have been identified as a major threat to pollinators, metal pollutants, which are often found in complex mixtures, have so far been overlooked. Here we assessed the impact of acute exposure to field-realistic concentrations of three common metal pollutants, lead, copper, arsenic, and their combinations, on honey bee appetitive learning and memory. All treatments involving single metals slowed down learning and disrupted memory retrieval at 24 h. Combinations of these metals had additive negative effects on both processes, suggesting common pathways of toxicity. Our results highlight the need to further assess the risks of metal pollution on invertebrates.

ABSTRACT Honey bees estimate distances to food sources using image motion experienced on the flight path and they use this measure to tune the waggle phase duration in their dance communication. Most studies on the dance-related odometer are based on experiments with Apis mellifera foragers trained in small tunnels with black and white patterns, which allows the creation of quantifiable changes in optic flow. In this study, we determined the waggle phase duration-distance curves of two Asian honey bee species, Apis florea and Apis cerana , in two different natural environments with clear differences in the vegetation conditions and hence visual contrast. We found that the dense vegetation condition (with higher contrast) elicited a more rapid increase in the waggle phase duration with distance than the sparse vegetation condition in A. florea but not in A. cerana . Our findings suggest that contrast sensitivity of the waggle dance odometer might vary among honey bee species.

ABSTRACT The ability of crocodilian haemoglobins to bind HCO 3 – has been appreciated for more than half a century, but the functional implication of this exceptional mechanism has not previously been assessed in vivo . Therefore, the goal of the present study was to address the hypothesis that CO 2 primarily binds to haemoglobin, rather than being accumulated in plasma as in other vertebrates, during diving in caimans. Here, we demonstrate that CO 2 primarily accumulates within the erythrocyte during diving and that most of the accumulated CO 2 is bound to haemoglobin. Furthermore, we show that this HCO 3 – binding is tightly associated with the progressive blood deoxygenation during diving; therefore, crocodilians differ from the classic vertebrate pattern, where HCO 3 – accumulates in the plasma upon excretion from the erythrocytes by the Cl – /HCO 3 – exchanger.

ABSTRACT Mantis shrimp commonly inhabit seafloor environments with an abundance of visual features including conspecifics, predators, prey and landmarks used for navigation. Although these animals are capable of discriminating color and polarization, it is unknown what specific attributes of a visual object are important during recognition. Here, we show that mantis shrimp of the species Neogonodactylus oerstedii are able to learn the shape of a trained target. Further, when the shape and color of a target that they had been trained to identify were placed in conflict, N. oerstedii tended to choose the target of the trained shape over the target of the trained color. Thus, we conclude that the shape of the target was more salient than its color during recognition by N. oerstedii , suggesting that the shapes of objects, such as landmarks or other animals, are important for their identification by the species.

ABSTRACT Animals must selectively attend to relevant stimuli and avoid being distracted by unimportant stimuli. Jumping spiders (Salticidae) do this by coordinating eyes with different capabilities. Objects are examined by a pair of high-acuity principal eyes, whose narrow field of view is compensated for by retinal movements. The principal eyes overlap in field of view with motion-sensitive anterior-lateral eyes (ALEs), which direct their gaze to new stimuli. Using a salticid-specific eyetracker, we monitored the gaze direction of the principal eyes as they examined a primary stimulus. We then presented a distractor stimulus visible only to the ALEs and observed whether the principal eyes reflexively shifted their gaze to it or whether this response was flexible. Whether spiders redirected their gaze to the distractor depended on properties of both the primary and distractor stimuli. This flexibility suggests that higher-order processing occurs in the management of the attention of the principal eyes.

ABSTRACT We quantified drinking behavior in three species of North American watersnakes: Nerodia clarkii , which is a marine or brackish water amphibious species, and Nerodia fasciata and Nerodia taxispilota , both freshwater amphibious species. All three species have relatively small and similar thresholds of dehydration (TH, approximately −4% loss of body mass) that elicit thirst and drinking of fresh water. These species have higher thirst sensitivity than several species of hydrophiine and laticaudine sea snakes, which are characterized by much lower TH (greater dehydration, −9% to <−20%). Nerodia clarkii , which is often found in coastal oceanic water, refused to drink seawater, but drank fresh water when dehydrated. In separate trials involving dehydration of N. clarkii and N. fasciata that were concurrently fed fish at regular intervals, snakes eventually refused to eat at TH of approximately −12% of original body mass, but resumed eating after they were allowed to drink fresh water and rehydrate. The drinking behaviors of Nerodia corroborate previous data on the importance of fresh water for drinking, and they complement growing evidence that dietary water does not itself mitigate dehydration in snakes. These new data increase understanding of water relationships in the context of evolutionary transitions from land to sea, and they emphasize the importance of fresh water resources in the conservation of coastal and marine species of reptiles.

ABSTRACT Gecko substrate use is likely influenced by adhesive performance, yet few studies have demonstrated this empirically. Herein, we examined the substrate use, adhesive performance and vertical clinging behaviour of Gekko gecko in captivity to investigate whether adhesive performance influences patterns of substrate use. We found that geckos were observed significantly more often on the substrate (glass) that elicited maximal adhesive performance relative to its availability within our experimental enclosures, indicating that geckos preferentially use substrates on which their adhesive performance is maximal. Our work here provides additional, yet crucial data establishing connections between adhesive performance and patterns of substrate use in captivity, suggesting the hypothesis that substrate preferences of free-ranging geckos should be correlated with adhesive performance. Clearly, further experimental and field research is necessary to test this hypothesis and identify other parameters that individually and/or collectively influence the habitat use of free-ranging geckos.

ABSTRACT Locusts have been reported to elevate metabolic rate in response to high carbohydrate diets; this conclusion was based on metabolic rates calculated from CO 2 production, a common practice for insects. However, respiratory exchange ratio (RER, CO 2 production divided by O 2 consumption) can rise above 1 as a result of de novo lipid synthesis, providing an alternative possible explanation of the prior findings. We studied the relationship between macronutrient ingestion, RER and lipid synthesis using South American locusts ( Schistocerca cancellata ) reared on artificial diets varying in protein:carbohydrate (p:c) ratio. RER increased and rose above 1 as dietary p:c ratio decreased. Lipid accumulation rates were strongly positively correlated with dietary carbohydrate content and ingestion. RERs above 1 were only observed for animals without food in the respirometry chamber, suggesting that hormonal changes after a meal may drive lipid synthesis. Schistocerca cancellata does not elevate metabolic rate on low p:c diets; in fact, the opposite trend was observed.

ABSTRACT Poison frogs sequester chemical defenses from their diet of leaf litter arthropods for defense against predation. Little is known about the physiological adaptations that confer this unusual bioaccumulation ability. We conducted an alkaloid-feeding experiment with the Diablito poison frog ( Oophaga sylvatica ) to determine how quickly alkaloids are accumulated and how toxins modify frog physiology using quantitative proteomics. Diablito frogs rapidly accumulated the alkaloid decahydroquinoline within 4 days, and dietary alkaloid exposure altered protein abundance in the intestines, liver and skin. Many proteins that increased in abundance with decahydroquinoline accumulation are plasma glycoproteins, including the complement system and the toxin-binding protein saxiphilin. Other protein classes that change in abundance with decahydroquinoline accumulation are membrane proteins involved in small molecule transport and metabolism. Overall, this work shows that poison frogs can rapidly accumulate alkaloids, which alter carrier protein abundance, initiate an immune response, and alter small molecule transport and metabolism dynamics across tissues.

ABSTRACT We examined the effects of epicatechin (Epi), a flavonoid abundant in green tea and cocoa, on long-term memory (LTM) formed following conditioned taste aversion (CTA) training in Lymnaea stagnalis . In CTA training, the snails learnt to avoid a food that initially they liked (i.e. sucrose). Twenty-four hours after CTA training, 67% of the trained snails showed a significant decrease in the feeding behaviour elicited by sucrose. Placing snails in the Epi solution in CTA training did not alter the percentage of snails exhibiting LTM, but it significantly increased LTM persistence. We also examined changes following Epi exposure in spontaneous activity of the cerebral giant cells (CGCs) that modulate feeding behaviour and are necessary for CTA-LTM. Our data suggest that Epi causes a decrease in CGC activity and increases LTM persistence, possibly via a GABAergic mechanism.

ABSTRACT Fast escape responses to a predator threat are fundamental to the survival of mobile marine organisms. However, elasmobranchs are often underrepresented in such studies. Here, we measured the escape latency (time interval between the stimulus and first visible reaction) of mechanically induced escape responses in the Pacific spiny dogfish, Squalus suckleyi , and in two teleosts from the same region, the great sculpin, Myoxocephalus polyacanthocephalus , and the pile perch, Rhacochilus vacca . We found that the dogfish had a longer minimum latency (66.7 ms) compared with that for the great sculpin (20.8 ms) and pile perch (16.7 ms). Furthermore, the dogfish had a longer latency than that of 48 different teleosts identified from 35 different studies. We suggest such long latencies in dogfish may be due to the absence of Mauthner cells, the giant neurons that control fast escape responses in fishes.

ABSTRACT Marine mammals rely on oxygen stored in blood, muscle and lungs to support breath-hold diving and foraging at sea. Here, we used biomedical imaging to examine lung oxygen stores and other key respiratory parameters in living ringed seals ( Pusa hispida ). Three-dimensional models created from computed tomography (CT) images were used to quantify total lung capacity (TLC), respiratory dead space, minimum air volume and total body volume to improve assessment of lung oxygen storage capacity, scaling relationships and buoyant force estimates. The results suggest that lung oxygen stores determined in vivo are smaller than those derived from postmortem measurements. We also demonstrate that, whereas established allometric relationships hold well for most pinnipeds, these relationships consistently overestimate TLC for the smallest phocid seal. Finally, measures of total body volume reveal differences in body density and net vertical forces in the water column that influence costs associated with diving and foraging in free-ranging seals.