Patterns of fatty acid usage in two nocturnal insectivores: the Mediterranean house gecko (Hemidactylus turcicus) and the Etruscan pygmy shrew (Suncus etruscus).

Dietary fatty acids (FAs) have been demonstrated to be differentially stored or used as a metabolic fuel, depending on carbon chain length or saturation level. However, intestinal absorption also differs among FAs, potentially biasing conclusions on functional differences and their subsequent implications. We tested dietary FA usage in a nocturnal insectivorous reptile and a nocturnal insectivorous mammal of similar size: the gecko Hemidactylus turcicus and the shrew Suncus etruscus. We compared the relative presence of 13C isotopes in breath and feces following ingestion of three isotopically enriched fatty acids: linoleic acid (a polyunsaturated FA), oleic acid (monounsaturated) and palmitic acid (saturated). Both species oxidized linoleic and oleic acids at much higher levels than palmitic acid. Egestion of palmitic acid in feces was much higher than that of linoleic and oleic acids. The major difference between geckos and shrews was that the latter digested fatty acids much faster, which was best explained by the difference in the metabolic rates of the species. Circadian differences were evident for gecko metabolic and FA oxidation rates, peaking at night; for shrews, peak oxidation was achieved faster at night but rates did not differ. Our study is among the first to integrate oxidation and absorption patterns, as well as metabolic rates and their rhythms, providing important insights into the utilization of different dietary FAs in different species.

Squamate metabolic rates decrease in winter beyond the effect of temperature.

The reptilian form of hibernation (brumation) is much less studied than its mammalian and insect equivalents. Hibernation and brumation share some basic features but may differ in others. Evidence for hypometabolism in brumating reptiles beyond the effect of temperature is sporadic and often ignored. We calculated the standard metabolic rates (SMR, oxygen uptake during inactivity), in winter and/or summer, of 156 individuals representing 59 species of Israeli squamates across all 17 local families. For 32 species, we measured the same individuals during both seasons. We measured gas exchange continuously in a dark metabolic chamber, under the average January high and low temperatures (20°C and 12°C), during daytime and nighttime. We examined how SMR changes with season, biome, body size, temperature and time of day, using phylogenetic mixed models. Metabolic rates increased at sunrise in the diurnal species, despite no light or other external cues, while in nocturnal species the metabolic rates did not increase. Cathemeral species shifted from a diurnal-like diel pattern in winter to a nocturnal-like pattern in summer. Regardless of season, Mediterranean species SMRs were 30% higher than similar-sized desert species. Summer SMR of all species together scaled with body size with an exponent of 0.84 but dropped to 0.71 during brumation. Individuals measured during both seasons decreased their SMR between summer and winter by a 47%, on average, at 20°C and by 70% at 12°C. Q10 was 1.75 times higher in winter than in summer, possibly indicating an active suppression of metabolic processes under cold temperatures. Our results challenge the commonly held perception that squamate physiology is mainly shaped by temperature, with little role for intrinsic metabolic regulation. The patterns we describe indicate that seasonal, diel and geographic factors can trigger remarkable shifts in metabolism across squamate species.

Passive heat diffusion in nests with downward-facing cells: Implications for early colony development in social wasps.

Social insects employ a variety of active and passive mechanisms for nest thermoregulation. Many social wasp species exhibit a particular nest-architecture by building their nests with cells facing downward. By using thermal imaging to characterize the heat diffusion throughout Oriental hornet nests from different angular positions, we show that the heat diffusion along the vertical gradient of nests is more efficient when the cell openings face downward than when facing sideways or upward, demonstrating the efficiency of this specific architecture in increasing the nest temperature. This passive thermoregulation mechanism could be especially important during the initial stage of the colony, when the queen is alone to rear her first brood. Among the social insects that build cells to raise their brood, we suggest that wasps can take advantage of the thermal benefits of this particular architecture of their cells as, unlike bees, they do not usually store food in them.

Insect flight metabolic rate revealed by bolus injection of the stable isotope 13C.

Measuring metabolic rate (MR) poses a formidable challenge in free-flying insects who cannot breathe into masks or be trained to fly in controlled settings. Consequently, flight MR has been predominantly measured on hovering or tethered insects flying in closed systems. Stable isotopes such as labelled water allow measurement of MR in free-flying animals but integrates the measurement over long periods exceeding the average flight duration of insects. Here, we applied the 'bolus injection of isotopic 13C Na-bicarbonate' method to insects to measure their flight MR and report a 90% accuracy compared to respirometry. We applied the method on two beetle species, measuring MR during free flight and tethered flight in a wind tunnel. We also demonstrate the ability to repeatedly use the technique on the same individual. Therefore, the method provides a simple, reliable and accurate tool that solves a long-lasting limitation on insect flight research by enabling the measurement of MR during free flight.

Juvenile hormone functions as a metabolic rate accelerator in bumble bees (Bombus terrestris).

Juvenile hormone (JH) is a modulator of many physiological transitions in insects, including molting, metamorphosis, diapause, and reproduction. These processes often include metabolic changes. Here we show that JH accelerates metabolic rate in bumble bees (Bombus terrestris). We reduced JH levels in worker bumble bees by removing their corpora allata (allatectomy) and elevated JH levels in queens through a topical application of JH-III. Natural and high JH levels increased the metabolic rate in both workers and queens and triggered an increased protein turnover rate. Following the treatments, JH also caused an increase in food consumption and a reduction in lipid levels and flight muscle mass of queens, and a reduction in lipids levels in workers. Furthermore, the topical application of a JH analog to queens prior to their diapause caused a decline in their survival of diapause. These findings support the hypothesis that JH acts as a metabolic rate accelerator, initiating a resource shift in bumble bees, and thereby reducing diapause survival in queens. Based on previous studies on JH we suggest that, additional to its behavioral or physiological effects, JH's function as an accelerator of metabolic processes is conserved across different life stages and insect species.

More than just sugar: allocation of nectar amino acids and fatty acids in a Lepidopteran.

The ability to allocate resources, even when limited, is essential for survival and fitness. We examine how nutrients that occur in minute amounts are allocated among reproductive, somatic, and metabolic demands. In addition to sugar, flower nectars contain two macronutrients-amino acids and fatty acids. We created artificial nectars spiked with 13C-labelled amino acids and fatty acids and fed these to adult moths (Manduca sexta: Sphingidae) to understand how they allocate these nutrients among competing sinks (reproduction, somatic tissue, and metabolic fuel). We found that both essential and non-essential amino acids were allocated to eggs and flight muscles and were still detectable in early-instar larvae. Parental-derived essential amino acids were more conserved in the early-instars than non-essential amino acids. All amino acids were used as metabolic fuel, but the non-essential amino acids were oxidized at higher rates than essential amino acids. Surprisingly, the nectar fatty acids were not vertically transferred to offspring, but were readily used as a metabolic fuel by the moth, minimizing losses of endogenous nutrient stores. We conclude that the non-carbohydrate components of nectar may play important roles in both reproductive success and survival of these nectar-feeding animals.

Sex differences in the utilization of essential and non-essential amino acids in Lepidoptera.

The different reproductive strategies of males and females underlie differences in behavior that may also lead to differences in nutrient use between the two sexes. We studied sex differences in the utilization of two essential amino acids (EAAs) and one non-essential amino acid (NEAA) by the Carolina sphinx moth (Manduca sexta). On day one post-eclosion from the pupae, adult male moths oxidized greater amounts of larva-derived AAs than females, and more nectar-derived AAs after feeding. After 4 days of starvation, the opposite pattern was observed: adult females oxidized more larva-derived AAs than males. Adult males allocated comparatively small amounts of nectar-derived AAs to their first spermatophore, but this allocation increased substantially in the second and third spermatophores. Males allocated significantly more adult-derived AAs to their flight muscle than females. These outcomes indicate that adult male and female moths employ different strategies for allocation and oxidation of dietary AAs.

On-board recordings reveal no jamming avoidance in wild bats.

Animals often deal with situations in which vast sensory input is received simultaneously. They therefore must possess sophisticated mechanisms to select important input and ignore the rest. In bat echolocation, this problem is at its extreme. Echolocating bats emit sound signals and analyse the returning echoes to sense their environment. Bats from the same species use signals with similar frequencies. Nearby bats therefore face the difficulty of distinguishing their own echoes from the signals of other bats, a problem often referred to as jamming. Because bats commonly fly in large groups, jamming might simultaneously occur from numerous directions and at many frequencies. Jamming is a special case of the general phenomenon of sensory segregation. Another well-known example is the human problem of following conversation within a crowd. In both situations, a flood of auditory incoming signals must be parsed into important versus irrelevant information. Here, we present a novel method, fitting wild bats with a miniature microphone, which allows studying jamming from the bat's 'point of view'. Previous studies suggested that bats deal with jamming by shifting their echolocation frequency. On-board recordings suggest otherwise. Bats shifted their frequencies, but they did so because they were responding to the conspecifics as though they were nearby objects rather than avoiding being jammed by them. We show how bats could use alternative measures to deal with jamming instead of shifting their frequency. Despite its intuitive appeal, a spectral jamming avoidance response might not be the prime mechanism to avoid sensory interference from conspecifics.

Subtropical mouse-tailed bats use geothermally heated caves for winter hibernation.

We report that two species of mouse-tailed bats (Rhinopoma microphyllum and R. cystops) hibernate for five months during winter in geothermally heated caves with stable high temperature (20°C). While hibernating, these bats do not feed or drink, even on warm nights when other bat species are active. We used thermo-sensitive transmitters to measure the bats' skin temperature in the natural hibernacula and open flow respirometry to measure torpid metabolic rate at different ambient temperatures (Ta, 16-35°C) and evaporative water loss (EWL) in the laboratory. Bats average skin temperature at the natural hibernacula was 21.7 ± 0.8°C, and no arousals were recorded. Both species reached the lowest metabolic rates around natural hibernacula temperatures (20°C, average of 0.14 ± 0.01 and 0.16 ± 0.04 ml O2 g(-1) h(-1) for R. microphyllum and R. cystops, respectively) and aroused from torpor when Ta fell below 16°C. During torpor the bats performed long apnoeas (14 ± 1.6 and 16 ± 1.5 min, respectively) and had a very low EWL. We hypothesize that the particular diet of these bats is an adaptation to hibernation at high temperatures and that caves featuring high temperature and humidity during winter enable these species to survive this season on the northern edge of their world distribution.

Gamma-aminobutyric acid in flower nectar and its possible physiological and behavioral effects on insect pollinators.

Floral nectar, a vital nutrition source for pollinators, contains diverse chemical compounds, including γ-aminobutyric acid (GABA), a prevalent nonproteinogenic amino acid. While GABA's physiological role is known and well-studied, its ecological significance in plant-pollinator interactions remains unclear. Recent studies on GABA's effects on pollinators' preference, consumption, survival, physiology, and behavior show varying outcomes according to the species, indicating a complex relationship. GABA consumption impacts motor function and cognitive abilities, potentially influencing pollination efficiency. Future research addressing diverse concentrations, species, and behavioral aspects is crucial for comprehensively understanding GABA's ecological role in plant-pollinator interactions.

Seasonal remodeling of visceral organs in the invasive desert gecko Tarentola annularis.

In winter, many reptiles have a period of inactivity ("brumation"). During brumation there is no energetic intake, therefore there would be an advantage to reducing energetic expenditure. The size of energetically costly organs, a major determinant of metabolic rate, is known to be flexible in many tetrapods. Seasonal plasticity of organ size could serve as both an energy-saving mechanism and a source of nutrients for brumating reptiles. We studied a population of an invasive gecko, Tarentola annularis, to test for seasonal changes in activity, metabolic rate, and mass of various organs. The observed period of inactivity was December-February. Standard metabolic rates during the activity season were 1.85 times higher than in brumating individuals. This may be attributed to decreased organ mass during winter: heart mass decreased by 37%, stomach mass by 25%, and liver mass by 69%. Interestingly, testes mass increased by 100% during winter, likely in preparation for the breeding season, suggesting that males prioritize breeding over other functions upon return to activity. The size of the kidneys and lungs remained constant. Organ atrophy occurred only after geckos reduced their activity, so we hypothesize that organ mass changes in response to (rather than in anticipation of) cold winter temperatures and the associated fasting. Degradation of visceral organs can maintain energy demands in times of low supply, and catabolism of the protein from these organs can serve as a source of both energy and water during brumation. These findings bring us closer to a mechanistic understanding of reptiles' physiological adaptations to environmental changes.

Intraspecific scaling and early life history determine the cost of free-flight in a large beetle (Batocera rufomaculata).

The scaling of the energetic cost of locomotion with body mass is well documented at the interspecific level. However, methodological restrictions limit our understanding of the scaling of flight metabolic rate (MR) in free-flying insects. This is particularly true at the intraspecific level, where variation in body mass and flight energetics may have direct consequences for the fitness of an individual. We applied a 13C stable isotope method to investigate the scaling of MR with body mass during free-flight in the beetle Batocera rufomaculata. This species exhibits large intraspecific variation in adult body mass as a consequence of the environmental conditions during larval growth. We show that the flight-MR scales with body mass to the power of 0.57, with smaller conspecifics possessing up to 2.3 fold higher mass-specific flight MR than larger ones. Whereas the scaling exponent of free-flight MR was found to be like that determined for tethered-flight, the energy expenditure during free-flight was more than 2.7 fold higher than for tethered-flight. The metabolic cost of flight should therefore be studied under free-flight conditions, a requirement now enabled by the 13C technique described herein for insect flight.

Metabolomics analysis of larval secretions reveals a caste-driven nutritional shift in a social wasp colony.

Social wasps exhibit a unique nutritional cycle in which adults feed larvae with prey, and larvae provide adults with larval secretions (LS). LS serves as a vital nutritional source for adults, contributing to the colony's health and reproductive success. The LS nutrient composition has been previously reported in various wasp species, yet these analyses focused solely on worker-destined larvae, overlooking the potential caste designation effects on LS composition. Using metabolomics techniques, we analysed and compared the metabolite and nutrient composition in LS of queen- and worker-destined larvae of the Oriental hornet. We found that queen-destined LS (QLS) contain greater amounts of most metabolites, including amino acids, and smaller amounts of sugars compared to worker-destined LS (WLS). The amino acid-to-sugar ratio in QLS was approximately tenfold higher than in WLS. Thus, as the colony transitions from the production of workers to the production of reproductives, it gradually experiences a nutritional shift that may influence the behaviour and physiology of the adult nest population. This caste-specific metabolite profile and nutrient composition of LS reflect the differences in the diet and physiological requirements of worker- and queen-destined larvae and may play a critical role in caste determination in social wasps.

Distance from human settlements favors wild-type appearance of feral cats (Felis catus) in Mediterranean woodland.

Camouflage is a common trait enabling animals to avoid detection by predators and prey. Patterns such as spots and stripes are convergent across carnivore families, including felids, and are hypothesized to have adaptive value through camouflage. House cats (Felis catus) were domesticated thousands of years ago, but despite artificial selection for a wide variety of coat colors, the wild-type pattern of tabby cats is very common. We aimed to determine whether this pattern grants an advantage over other morphs in natural environments. We collected cat images taken with camera traps in natural areas near and far from 38 rural settlements in Israel, to compare the habitat use by feral cats of different colors. We tested the effect of proximity to villages and habitat vegetation (normalized difference vegetation index, NDVI) on the probability of space use by the tabby morph compared to the others. NDVI had a positive effect on site use in both morphs, but non-tabby cats had a 2.1 higher probability of using the near sites than the far sites, independent of NDVI. The wild-type tabby cats' probability of site use were equally likely to be unaffected by proximity, or have an interaction of proximity with NDVI whereby the far transects are used with increasing probability in sites of denser vegetation. We hypothesize that the camouflage of tabby cats, more than other colors and patterns, confers an advantage in roaming the woodland habitats for which this pattern evolved. This has both theoretical implications as rare empirical evidence of the adaptive value of fur coloration, and practical implications on managing the ecological impact of feral cats worldwide.

The fa(c)ts that matter: Bumble bees differentially allocate and oxidate three common fatty acids in pollen.

Pollen serves as a crucial source of protein and lipids for numerous insects. Despite the importance of pollen lipids for nutrient regulation in bees, the digestibility and absorption of different fatty acids (FAs) by bees remain poorly understood. We used 13C labeled fatty acids (FAs) to investigate the absorption and allocation of three common dietary FAs in pollen by bumble bees. Palmitic acid, the most common saturated FA in pollen, was poorly absorbed, even when supplied as tripalmitate, emulsified, or mixed in vegetable oil. In contrast, the essential linoleic acid was absorbed and allocated at the highest rate among the three FAs tested. Oleic acid, a non-essential monounsaturated FA, was absorbed and oxidized at lower rates than linoleic acid. Notably, a feeding rate experiment revealed that different fatty acids did not affect the consumption rate of pollen. This results suggests that the specific FA's absorption efficiency and allocation differ in bumble bees, impacting their utilization. These findings demonstrate the importance of considering the digestibility and absorption of different FAs. Furthermore, the study highlights the influence of pollen lipid composition on the nutritional content for pollinators and raises questions about the utilization of polyunsaturated FAs in insect metabolism.

Metabolic cost of flight and aerobic efficiency in the rose chafer, Protaetia cuprea (Cetoniinae).

Rose chafer beetles (Protetia cuprea) are pollinators as well as agricultural pests, flying between flowers and trees while foraging for pollen and fruits. Calculating the energy they expend on flying during foraging activity faces the challenge of measuring the metabolic rate (MR) of free-flying insects in an open space. We overcame this challenge by using the bolus injection of 13 C Na-bicarbonate technique to measure their metabolic energy expenditure while flying in a large flight arena. Concurrently, we tracked the insects with high-speed cameras to extract their flight trajectory, from which we calculated the mechanical power invested in flying for each flight bout. We found that the chemical (metabolic) energy input converted to mechanical flight energy output at a mean efficiency of 10.4% ± 5.2%, with a trend of increased efficiency in larger conspecifics (efficiency scaled with body mass to the power of 1.4). The transition in the summer from a diet of pollen to that of fruits may affect the energy budget available for foraging. Starved P. cuprea, feeding on apples ad libitum, increased their body mass by an average of 6% in 2 h. According to our calculations, such a meal can power a 630-m flight (assuming a carbohydrate assimilation efficiency of 90%). Pollen, with a low water and carbohydrate content but rich in proteins and lipids, has a higher caloric content and should assimilate differently when converting food to flight fuel. The high cost of aerial locomotion is inherent to the foraging behavior of rose chafers, explaining their short flight bouts followed by prolonged feeding activity.

Continuous exchange of nectar nutrients in an Oriental hornet colony.

Nutritional exchanges play a fundamental role in the evolution of animal societies. In higher animal societies, while adult individuals can be both food donors and receivers, the offspring usually only receive food from the adults. Hornets and wasps are fierce insect hunters that feed their larvae with prey. However, although the adults also consume floral nectar, the role of nectar in vespid nutrition has remained largely unknown. We provided experimental colonies of the Oriental hornet with artificial nectar enriched with a 13C-labeled amino acid, and found that a continuous cycle of nutrition took place, in which nectar nutrients were used and exchanged back and forth between adults and larvae. We posit that this continuous cycle of nutrients constitutes a mechanism contributing to social cohesion. In an additional experiment, we found that nectar consumption was essential for adult and larval survival, suggesting the importance of wasps and hornets as pollinators in natural ecosystems.

CO2 narcosis induces a metabolic shift mediated via juvenile hormone in Bombus impatiens gynes.

Carbon dioxide (CO2) has pleiotropic effects on insect physiology and behavior. Although diverse, many impacts are related to changes in metabolism and reallocation of macronutrients. Here we examined the metabolic shift induced by CO2 and its regulation using Bombus impatiens. CO2 applied to bumble bee gynes induces bypass of diapause and transition into reproduction. We analyzed ovary activation and macronutrient amounts in four tissues/body parts (fat body, thorax, ovaries, and crop) at three timepoints following CO2 administration. To tease apart the effects of CO2 on reproduction and metabolism, we monitored the metabolic changes in gynes following ovary removal and CO2 narcosis. We also explored the role of juvenile hormone in mediating CO2 impact by feeding queens with a JH antagonist (Precocene). Gynes ovary activation was increased following CO2 treatment. Additionally, CO2-treated gynes showed lower lipid amount in the fat body and higher glycogen and protein amount in the ovary ten days after the treatment. CO2 treatment following ovary removal also resulted in decreased fat body lipids, suggesting that CO2 operates by inducing a metabolic shift independent of reproduction. Lastly, gynes fed with precocence did not show a metabolic shift following CO2, suggesting CO2 impact is mediated via juvenile hormone. Overall, these data suggest that CO2 induces transfer of macronutrients and utilization of stored reserved by accelerating metabolism. The proposed mechanism of CO2 may explain many of the pleiotropic effects of CO2 across species and can aid in understanding how this common anastatic influences insect physiology.

The effects of dietary proline, ß-alanine, and γ-aminobutyric acid (GABA) on the nest construction behavior in the Oriental hornet (Vespa orientalis).

Adult wasps primary food resource is larval saliva. This liquid secretion consists mainly of amino acids and carbohydrates processed from the prey brought to the colony by the foragers. However, adults also regularly consume floral nectar. The nectar's most abundant proteinogenic amino acid is proline, and the two most abundant non-proteinogenic amino acids are ß-alanine and GABA. These three amino acids are also common in larval saliva. Here, we study the effect of these dietary amino acids on the physiology and nest construction behavior of the Oriental hornet. Our results reveal their deleterious effects, especially at high concentrations: ß-alanine and GABA consumption reduced the hornets' lifespan and completely inhibited their construction behavior; while proline induced a similar but more moderate effect. At low concentrations, these amino acids had no effect on hornet survival but did slow down the nest construction process. Using carbon isotopically labeled amino acids, we show that, unlike proline, ß-alanine is stored in most body tissues (brain, muscles, and fat body), suggesting that it is rapidly metabolized after consumption. Our findings demonstrate how a single amino acid can impact the fitness of a nectarivore insect.

Nutrient Utilization during Male Maturation and Protein Digestion in the Oriental Hornet.

Males of social Hymenoptera spend the first days following eclosion inside the nest before dispersing to find a young queen to mate with. During this period, they must acquire enough nutrients to enable their sexual maturation and store energy to sustain them through their nuptial journey. It was previously argued that adult hornets are unable to process dietary proteins and rely on the larvae to supply them with free amino acids and carbohydrates that they secrete via trophallaxis. Using isotopically enriched diets, we examined nutrient allocation and protein turnover in newly-emerged males of the Oriental hornet during their maturation period and tested the protein digestion capability in the presence and absence of larvae in both males and worker hornets. The results indicated that protein turnover in males occurs during the first days following eclosion, while carbohydrates are incorporated into body tissues at higher rates towards the end of the maturation period. Additionally, we found that males cannot digest protein and depend on larval secretions as a source of nutrition, while workers, in contrast to previous reports, can metabolize protein independently. Our findings demonstrate the contribution of adult male nutrition and larval secretions to colony fitness.