New components of the honey bee (Apis mellifera L.) queen retinue pheromone

The honey bee queen produces pheromones that function in both releaser and primer roles such as attracting a retinue of workers around her, attracting drones on mating flights, preventing workers from reproducing at the individual (worker egg-laying) and colony (swarming) level, and regulating several other aspects of colony functioning. The queen mandibular pheromone (QMP), consisting of five synergistic components, is the only pheromone chemically identified in the honey bee (Apis mellifera L.) queen, but this pheromone does not fully duplicate the pheromonal activity of a full queen extract. To identify the remaining unknown compounds for retinue attraction, honey bee colonies were selectively bred to have low response to synthetic QMP and high response to a queen extract in a laboratory retinue bioassay. Workers from these colonies were then used in the bioassay to guide the isolation and identification of the remaining active components. Four new compounds were identified from several glandular sources that account for the majority of the difference in retinue attraction between synthetic QMP and queen extract: methyl (Z)-octadec-9-enoate (methyl oleate), (E)-3-(4-hydroxy-3-methoxyphenyl)-prop-2-en-1-ol (coniferyl alcohol), hexadecan-1-ol, and (Z9,Z12,Z15)-octadeca-9,12,15-trienoic acid (linolenic acid). These compounds were inactive alone or in combination, and they only elicited attraction in the presence of QMP. There was still unidentified activity remaining in the queen extract. The queen therefore produces a synergistic, multiglandular pheromone blend of at least nine compounds for retinue attraction, the most complex pheromone blend known for inducing a single behavior in any organism.     

Interaction of cellular and network mechanisms for efficient pheromone coding in moths

Sensory systems, both in the living and in machines, have to be optimized with respect to their environmental conditions. The pheromone subsystem of the olfactory system of moths is a particularly well-defined example in which rapid variations of odor content in turbulent plumes require fast, concentration-invariant neural representations. It is not clear how cellular and network mechanisms in the moth antennal lobe contribute to coding efficiency. Using computational modeling, we show that intrinsic potassium currents (I(A) and I(SK)) in projection neurons may combine with extrinsic inhibition from local interneurons to implement a dual latency code for both pheromone identity and intensity. The mean latency reflects stimulus intensity, whereas latency differences carry concentration-invariant information about stimulus identity. In accordance with physiological results, the projection neurons exhibit a multiphasic response of inhibition-excitation-inhibition. Together with synaptic inhibition, intrinsic currents I(A) and I(SK) account for the first and second inhibitory phases and contribute to a rapid encoding of pheromone information. The first inhibition plays the role of a reset to limit variability in the time to first spike. The second inhibition prevents responses of excessive duration to allow tracking of intermittent stimuli.     

Identification of a pheromone regulating caste differentiation in termites

The hallmark of social insects is their caste system: reproduction is primarily monopolized by queens, whereas workers specialize in the other tasks required for colony growth and survival. Pheromones produced by reining queens have long been believed to be the prime factor inhibiting the differentiation of new reproductive individuals. However, there has been very little progress in the chemical identification of such inhibitory pheromones. Here we report the identification of a volatile inhibitory pheromone produced by female neotenics (secondary queens) that acts directly on target individuals to suppress the differentiation of new female neotenics and identify n-butyl-n-butyrate and 2-methyl-1-butanol as the active components of the inhibitory pheromone. An artificial pheromone blend consisting of these two compounds had a strong inhibitory effect similar to live neotenics. Surprisingly, the same two volatiles are also emitted by eggs, playing a role both as an attractant to workers and an inhibitor of reproductive differentiation. This dual production of an inhibitory pheromone by female reproductives and eggs probably reflects the recruitment of an attractant pheromone as an inhibitory pheromone and may provide a mechanism ensuring honest signaling of reproductive status with a tight coupling between fertility and inhibitory power. Identification of a volatile pheromone regulating caste differentiation in a termite provides insights into the functioning of social insect colonies and opens important avenues for elucidating the developmental pathways leading to reproductive and nonreproductive castes.     

Calreticulin chaperones regulate functional expression of vomeronasal type 2 pheromone receptors

A variety of social behaviors like intermale aggression, fear, and mating rituals are important for sustenance of a species. In mice, these behaviors have been implicated to be mediated by peptide pheromones that are sensed by a class of G protein-coupled receptors, vomeronasal receptor type 2 (V2Rs), expressed in the pheromone detecting vomeronasal organ. Matching V2Rs with their cognate ligands is required to learn what receptors the biologically relevant pheromones are acting on. However, this feat has been greatly limited by the unavailability of appropriate heterologous tools commonly used to study ligand receptor specificity, because this family of receptors fails to traffic to the surface of heterologous cells. Here we show that calreticulin, a housekeeping chaperone commonly expressed in most eukaryotic cells, is sparsely expressed in the vomeronasal sensory neurons (VSNs). Correspondingly, knockdown of calreticulin in commonly available cell lines enables V2Rs to efficiently target to the cell membrane. Using this knowledge, we have now been able to successfully surface express receptors and functionally identify cognate ligands. Additionally, calreticulin4, a homolog of calreticulin shows restricted and enriched expression in the VSNs. Interestingly, in heterologous cells, calreticulin4 does not inhibit surface expression of V2Rs and can in part carry out functions of calreticulin. On the basis of our data, we postulate that V2Rs may use a unique trafficking mechanism whereby an important and more commonly expressed chaperone is deleterious for membrane export and is replaced by a functionally equivalent homolog that does not inhibit export while carrying out its functions.     

Olfactory expression of a single and highly variable V1r pheromone receptor-like gene in fish species

Sensory neurons expressing members of the seven-transmembrane V1r receptor superfamily allow mice to perceive pheromones. These receptors, which exhibit no sequence homology to any known protein except a weak similarity to taste receptors, have only been found in mammals. In the mouse, the V1r repertoire contains >150 members, which are expressed by neurons of the vomeronasal organ, a structure present exclusively in some tetrapod species. Here, we report the existence of a single V1r gene in multiple species of a non-terrestrial, vomeronasal organ-lacking taxon, the teleosts. In zebrafish, this V1r gene is expressed in chemosensory neurons of the olfactory rosette with a punctate distribution, strongly suggesting a role in chemodetection. This unique receptor gene exhibits a remarkably high degree of sequence variability between fish species. It likely corresponds to the original V1r present in the common ancestor of vertebrates, which led to the large and very diverse expansion of vertebrate pheromone receptor repertoires, and suggests the presence of V1rs in multiple nonmammalian phyla.     

Dynamic functional evolution of an odorant receptor for sex-steroid-derived odors in primates

Odorant receptors are among the fastest evolving genes in animals. However, little is known about the functional changes of individual odorant receptors during evolution. We have recently demonstrated a link between the in vitro function of a human odorant receptor, OR7D4, and in vivo olfactory perception of 2 steroidal ligands—androstenone and androstadienone—chemicals that are shown to affect physiological responses in humans. In this study, we analyzed the in vitro function of OR7D4 in primate evolution. Orthologs of OR7D4 were cloned from different primate species. Ancestral reconstruction allowed us to reconstitute additional putative OR7D4 orthologs in hypothetical ancestral species. Functional analysis of these orthologs showed an extremely diverse range of OR7D4 responses to the ligands in various primate species. Functional analysis of the nonsynonymous changes in the Old World Monkey and Great Ape lineages revealed a number of sites causing increases or decreases in sensitivity. We found that the majority of the functionally important residues in OR7D4 were not predicted by the maximum likelihood analysis detecting positive Darwinian selection.     

From the Cover: Intergenerational transmission of emotional trauma through amygdala-dependent mother-to-infant transfer of specific fear

Emotional trauma is transmitted across generations. For example, children witnessing their parent expressing fear to specific sounds or images begin to express fear to those cues. Within normal range, this is adaptive, although pathological fear, such as occurs in posttraumatic stress disorder or specific phobias, is also socially transmitted to children and is thus of clinical concern. Here, using a rodent model, we report a mother-to-infant transfer of fear to a novel peppermint odor, which is dependent on the mother expressing fear to that smell in pups’ presence. Examination of pups’ neural activity using c-Fos early gene expression and ¹⁴C 2-deoxyglucose autoradiography during mother-to-infant fear transmission revealed lateral and basal amygdala nuclei activity, with a causal role highlighted by pharmacological inactivation of pups’ amygdala preventing the fear transmission. Maternal presence was not needed for fear transmission, because an elevation of pups’ corticosterone induced by the odor of the frightened mother along with a novel peppermint odor was sufficient to produce pups’ subsequent aversion to that odor. Disruption of axonal tracts from the Grueneberg ganglion, a structure implicated in alarm chemosignaling, or blockade of pups’ alarm odor-induced corticosterone increase prevented transfer of fear. These memories are acquired at younger ages compared with amygdala-dependent odor-shock conditioning and are more enduring following minimal conditioning. Our results provide clues to understanding transmission of specific fears across generations and its dependence upon maternal induction of pups’ stress response paired with the cue to induce amygdala-dependent learning plasticity. Results are discussed within the context of caregiver emotional responses and adaptive vs. pathological fears social transmission.Children, including infants, use their parents’ emotions to guide their behavior and learn about safety and danger (1–4). The infant’s ability to regulate behavior in novel situations using the caregiver’s emotional expression is known as social referencing and occurs in humans and nonhuman primates (1). Although parental physical presence itself or particular cues indicating parental presence, such as voice, touch, or smell typically signal safety for the child, infants are especially responsive to the caregiver’s communication during threats (3–5). This social learning is critical for enhancing survival through an adaptation to the environment but also provides transmission of pathological fears, such as occurs in posttraumatic stress disorder (PTSD) or in specific phobias (3–7).Despite existing evidence that children are sensitive to parental fear and anxiety, the neurobiological mechanisms for the transmission of parental specific fear to the offspring have remained elusive (2–7). Animal studies investigating the impact of parental stress on the offspring focused on the history of parental trauma, quality of maternal care, and resultant overall behavioral alterations in the offspring (7, 8). However, to develop efficient survival strategies, progenies must learn about specific environmental threats triggering parental fear (9).Most of what we know about fear learning comes from studies using fear conditioning (FC) (10). In FC, a neutral sensory cue [conditioned stimulus (CS)] is paired with a noxious event [unconditioned stimulus (US)]. Animal studies indicate that the amygdala’s lateral and basal nuclei (LBA) play an important role in FC (10). However, FC in infant rats is naturally attenuated until postnatal day (PND) 10 due to low levels of the stress hormone corticosterone (CORT) during the stress hyporesponsive period (11–15). This fear suppression continues in older pups (until PND 16) in the mother’s presence due to social buffering (attenuation) of the shock-induced CORT increase (15).To study the intergenerational transmission of fear to specific triggers, we developed a mother-to-infant social fear learning paradigm. In social fear learning, an organism learns fear through an exposure to a conspecific expressing fear to a discrete CS. Social fear learning may thus serve as a model explaining how defense responses to specific triggers are transmitted between individuals. Social fear learning has been demonstrated in primates, including humans and in rodents, and involves the amygdala (16–19).     

Pleiotropy of segregating genetic variants that affect honey bee worker life expectancy

In contrast to many other complex traits, the natural genetic architecture of life expectancy has not been intensely studied, particularly in non-model organisms, such as the honey bee (Apis mellifera L.). Multiple factors that determine honey bee worker lifespan have been identified and genetic analyses have been performed on some of those traits. Several of the traits are included in a suite of correlated traits that form the pollen hoarding syndrome, which was named after the behavior to store surplus pollen in the nest and is tied to social evolution. Here, seven quantitative trait loci that had previously been identified for their effects on different aspects of the pollen hoarding syndrome were studied for their genetic influence on the survival of adult honey bee workers. To gain a more comprehensive understanding of the genetic architecture of worker longevity, a panel of 280 additional SNP markers distributed across the genome was also tested. Allelic distributions were compared between young and old bees in two backcross populations of the bi-directionally selected high- and low-pollen hoarding strain. Our results suggest a pleiotropic effect of at least one of the behavioral quantitative trait loci on worker longevity and one significant and several other putative genetic effects in other genomic regions. At least one locus showed evidence for strong antagonistic pleiotropy and several others suggested genetic factors that influence pre-emergence survival of worker honey bees. Thus, the predicted association between worker lifespan and the pollen hoarding syndrome was supported at the genetic level and the magnitude of the identified effects also strengthened the view that naturally segregating genetic variation can have major effects on age-specific survival probability in the wild.     

Kinetics and molecular properties of pheromone binding and release

Transient kinetic studies have shown that the uptake of the pheromone (bombykol) of the silkworm moth (Bombyx mori), by its pheromone-binding protein (PBP) BmorPBP, proceeds with an "on" rate of 0.068 +/- 0.01 microM(-1).s(-1). With the high concentration of PBP in the sensillar lymph (10 mM), the half-life for the uptake of pheromone in vivo is approximately equal to 1 ms. A pH-dependent conformational change (BmorPBP(B) --> BmorPBP(A)), associated with the release of pheromone, is a first-order reaction (k = 74.1 +/- 0.32 s(-1); t(1/2), 9.3 ms). Under physiological conditions, both reactions proceed with half-life times on the order of milliseconds, as is required for odorant-oriented navigation in insects. Molecular interactions of bombykol with both native and mutated PBPs were analyzed by a novel binding assay. A recombinant protein with the native conformation (BmorPBP) showed high binding affinity (K(D) = 105 nM) at pH 7 but low affinity (K(D) = 1,600 nM) at pH 5, when tested at both low and high KCl concentrations. A protein with a C-terminal segment deleted (BmorPBPDeltaP129-V142) was found to bind bombykol at pH 7 and at pH 5 with the same affinity as the native protein at pH 7, indicating that the C-terminal segment is essential for preventing binding at low pH. Binding studies with three mutated proteins (BmorPBPW37F, BmorPBPW127F, and BmorPBPW37A) showed that replacing Trp-37 (with Phe or Ala) or Trp-127 (with Phe) did not affect the binding affinity to bombykol. Fluorescence studies shed light on the contributions of Trp-37 and Trp-127 emissions to the overall fluorescence.     

Identification of a pheromone that increases anxiety in rats

Chemical communication plays an important role in the social lives of various mammalian species. Some of these chemicals are called pheromones. Rats release a specific odor into the air when stressed. This stress-related odor increases the anxiety levels of other rats; therefore, it is possible that the anxiety-causing molecules are present in the stress-related odorants. Here, we have tried to identify the responsible molecules by using the acoustic startle reflex as a bioassay system to detect anxiogenic activity. After successive fractionation of the stress-related odor, we detected 4-methylpentanal and hexanal in the final fraction that still possessed anxiogenic properties. Using synthetic molecules, we found that minute amounts of the binary mixture, but not either molecule separately, increased anxiety in rats. Furthermore, we determined that the mixture increased a specific type of anxiety and evoked anxiety-related behavioral responses in an experimental model that was different from the acoustic startle reflex. Analyses of neural mechanisms proposed that the neural circuit related to anxiety was only activated when the two molecules were simultaneously perceived by two olfactory systems. We concluded that the mixture is a pheromone that increases anxiety in rats. To our knowledge, this is the first study identifying a rat pheromone. Our results could aid further research on rat pheromones, which would enhance our understanding of chemical communication in mammals.Chemical communication plays an important role in the social lives of various mammalian species. Some of these chemicals are called “pheromones.” Because the term pheromone was coined and defined based on findings in insects (1), there is still a debate as to whether the original definition can be applied to mammals (2). Researchers have proposed revised definitions by modifying the original definition and/or specifying additional requirements (3–6). On the basis of the original and revised definitions, we set a working definition of pheromone within this study as (i) substances that are secreted to the outside by an individual and received by a second individual of the same species, in which they cause a specific reaction; (ii) substances that are effective in minute amounts; (iii) substances that are released from living individuals; and (iv) substances that mediate communication for an evolutionarily adaptive function.Rats release a specific odor into the air when stressed (7). This stress-related odor increases anxiety levels (8, 9) and induces a variety of anxiety-related responses depending on their situation with other rats (9–18). Rats respond to their own stress-related odor in a similar manner to odor released from the other rats, suggesting that the odor has general effects (10, 19). In addition, the stress-related odor appears to be effective in minute amounts (20, 21). Therefore, the molecules responsible for increasing anxiety levels should exist in the stress-related odor.The responsible molecules appear to meet the definition of pheromones because they (i) were released from rats and increased anxiety in other rats and (ii) were effective in minute amounts. In addition, (iii) rats were alive while releasing the stress-related odor that included the responsible molecules. Furthermore, (iv) the communication mediated by the responsible molecules appears to have an evolutionarily adaptive function. First, the communication mediated by the responsible molecules might be evolutionarily conserved. Studies have shown that the stress-related odors are released by variety of mammalian species in addition to rats; examples include mice (22, 23), deer (24), cattle (25), swine (26), and humans (27). Second, increasing anxiety levels appear to be an adaptive response for highly developed animals. Although the odor released by mice evokes stereotypical avoidance responses, deer, cattle, and swine show cautious behavior rather than an avoidance response per se. In humans, the odor has been shown to increase anxiety levels (28). The higher the level of development of a particular organism, the more complex its life is. As a result, increased anxiety, rather than stereotypical avoidance responses, would enable developed animals to cope with a variety of dangerous stimulus appropriately, depending on the situation and the type of the stimulus, which may increase inclusive fitness. Therefore, the communication mediated by the responsible molecules can be suggested to have an evolutionarily adaptive function.To identify the responsible molecules, the acoustic startle reflex (ASR) was used as a bioassay system for assessing the anxiogenic activity of the molecules. The ASR pertains to rapid contractions of facial and body muscles evoked by sudden and intense acoustic stimulus, which are observed in a variety of mammals, including humans (29). Earlier studies involving animals and humans have revealed that the amplitude of the ASR increases with increased level of anxiety (29). In rats, the amplitude of the ASR is expressed as a voltage change in the output of an accelerometer, which is displaced by the movement of a rat in an animal holder. Rats show an enhanced ASR when levels of anxiety are increased (8, 30, 31). Therefore, we defined molecules as positive (+) for anxiogenic activity when we observed a significant increase in the amplitude of the ASR following exposure.In the present study, we first collected the stress-related odor by applying electrical stimulations to the perianal region of anesthetized rats (32). Because the anal glands are located immediately inside the anal verge (33) (Fig. S1), these simulations induce muscle contractions around the anus, which may squeeze the odor out of the anal glands. Then, we successively fractionated the collected odor, leading to the discovery of 4-methylpentanal and hexanal in the final small fraction that retained anxiogenic properties. Subsequent analyses with synthetic molecules revealed that the mixture of these molecules, but not either individual molecule, increased a specific type of anxiety, even in minute amounts. We further investigated the communication by behavioral and neuroscience analyses. The neural circuit related to anxiety [specifically the bed nucleus of stria terminalis (BNST)] appeared to be activated only when the two molecules were simultaneously detected, possibly via two separate nasal chemosensory systems. The mixture also evoked anxiety-related behavioral responses in a different experimental model to the ASR test.     

General principles of attraction and competitive attraction as revealed by large-cage studies of moths responding to sex pheromone

Knowledge of how insects are actually affected by sex pheromones deployed throughout a crop so as to disrupt mating has lacked a mechanistic framework sufficient for guiding optimization of this environmentally friendly pest-control tactic. Major hypotheses are competitive attraction, desensitization, and camouflage. Working with codling moths, Cydia pomonella, in field cages millions of times larger than laboratory test tubes and at substrate concentrations trillions of times less than those typical for enzymes, we nevertheless demonstrate that mating disruption sufficiently parallels enzyme (ligand) –substrate interactions so as to justify adoption of conceptual and analytical tools of biochemical kinetics. By doing so, we prove that commercial dispensers of codling moth pheromone first competitively attract and then deactivate males probably for the remainder of a night. No evidence was found for camouflage. We generated and now validate simple algebraic equations for attraction and competitive attraction that will guide optimization and broaden implementation of behavioral manipulations of pests. This analysis system also offers a unique approach to quantifying animal foraging behaviors and could find applications across the natural and social sciences.     

Unusual macrocyclic lactone sex pheromone of Parcoblatta lata, a primary food source of the endangered red-cockaded woodpecker

Wood cockroaches in the genus Parcoblatta, comprising 12 species endemic to North America, are highly abundant in southeastern pine forests and represent an important prey of the endangered red-cockaded woodpecker, Picoides borealis. The broad wood cockroach, Parcoblatta lata, is among the largest and most abundant of the wood cockroaches, constituting >50% of the biomass of the woodpecker's diet. Because reproduction in red-cockaded woodpeckers is affected dramatically by seasonal and spatial changes in arthropod availability, monitoring P. lata populations could serve as a useful index of habitat suitability for woodpecker conservation and forest management efforts. Female P. lata emit a volatile, long-distance sex pheromone, which, once identified and synthesized, could be deployed for monitoring cockroach populations. We describe here the identification, synthesis, and confirmation of the chemical structure of this pheromone as (4Z,11Z)-oxacyclotrideca-4,11-dien-2-one [= (3Z,10Z)-dodecadienolide; herein referred to as "parcoblattalactone"]. This macrocyclic lactone is a previously unidentified natural product and a previously unknown pheromonal structure for cockroaches, highlighting the great chemical diversity that characterizes olfactory communication in cockroaches: Each long-range sex pheromone identified to date from different genera belongs to a different chemical class. Parcoblattalactone was biologically active in electrophysiological assays and attracted not only P. lata but also several other Parcoblatta species in pine forests, underscoring its utility in monitoring several endemic wood cockroach species in red-cockaded woodpecker habitats.     

Detection and avoidance of a carnivore odor by prey

Predator-prey relationships provide a classic paradigm for the study of innate animal behavior. Odors from carnivores elicit stereotyped fear and avoidance responses in rodents, although sensory mechanisms involved are largely unknown. Here, we identified a chemical produced by predators that activates a mouse olfactory receptor and produces an innate behavioral response. We purified this predator cue from bobcat urine and identified it to be a biogenic amine, 2-phenylethylamine. Quantitative HPLC analysis across 38 mammalian species indicates enriched 2-phenylethylamine production by numerous carnivores, with some producing >3,000-fold more than herbivores examined. Calcium imaging of neuronal responses in mouse olfactory tissue slices identified dispersed carnivore odor-selective sensory neurons that also responded to 2-phenylethylamine. Two prey species, rat and mouse, avoid a 2-phenylethylamine odor source, and loss-of-function studies involving enzymatic depletion of 2-phenylethylamine from a carnivore odor indicate it to be required for full avoidance behavior. Thus, rodent olfactory sensory neurons and chemosensory receptors have the capacity for recognizing interspecies odors. One such cue, carnivore-derived 2-phenylethylamine, is a key component of a predator odor blend that triggers hard-wired aversion circuits in the rodent brain. These data show how a single, volatile chemical detected in the environment can drive an elaborate danger-associated behavioral response in mammals.     

Genetic dissection of pheromone processing reveals main olfactory system-mediated social behaviors in mice

Most mammals have two major olfactory subsystems: the main olfactory system (MOS) and vomeronasal system (VNS). It is now widely accepted that the range of pheromones that control social behaviors are processed by both the VNS and the MOS. However, the functional contributions of each subsystem in social behavior remain unclear. To genetically dissociate the MOS and VNS functions, we established two conditional knockout mouse lines that led to either loss-of-function in the entire MOS or in the dorsal MOS. Mice with whole-MOS loss-of-function displayed severe defects in active sniffing and poor survival through the neonatal period. In contrast, when loss-of-function was confined to the dorsal MOB, sniffing behavior, pheromone recognition, and VNS activity were maintained. However, defects in a wide spectrum of social behaviors were observed: attraction to female urine and the accompanying ultrasonic vocalizations, chemoinvestigatory preference, aggression, maternal behaviors, and risk-assessment behaviors in response to an alarm pheromone. Functional dissociation of pheromone detection and pheromonal induction of behaviors showed the anterior olfactory nucleus (AON)-regulated social behaviors downstream from the MOS. Lesion analysis and neural activation mapping showed pheromonal activation in multiple amygdaloid and hypothalamic nuclei, important regions for the expression of social behavior, was dependent on MOS and AON functions. Identification of the MOS-AON–mediated pheromone pathway may provide insights into pheromone signaling in animals that do not possess a functional VNS, including humans.Most mammals have two major olfactory subsystems―the main olfactory system (MOS) and vomeronasal system (VNS). The MOS comprises the main olfactory epithelium (MOE), in which olfactory sensory neurons detect odorants, and their projection target, the main olfactory bulb (MOB) (Fig. S1A). Although the MOS is thought to detect volatile odorants and the VNS is thought to be important for the detection of nonvolatile pheromones, evidence shows that the MOS is also involved in pheromone detection (1–8). Surgical blocking of odorant access to the MOE, but not surgical ablation of the vomeronasal epithelium (VNE), eliminates preference to odors from the opposite sex in ferrets (9, 10). In mice, chemical ablation of the MOE impairs male and female sexual behaviors (11, 12). In these experiments in which the MOE was ablated, the function of the VNS is not directly disrupted, because the VNS is activated by direct application of urine to the nostril. Thus, these results indicate that the MOS also contributes to pheromone processing and related behaviors.Nonconditional disruption of genes encoding signal transduction proteins that are required for activation of olfactory neurons, such as cyclic nucleotide-gated channel (Cnga2) or adenylyl cyclase 3, impairs several social behaviors (11, 13–15). However, complete loss of MOS function causes anosmia and severely impairs sniffing and chemoinvestigatory behaviors (11, 13, 14, 16). Chemoinvestigation and the accompanying physical contact of the nose and pheromone source are essential for VNS activation; therefore, the loss of MOS function indirectly impairs the VNS, because the VNS becomes behaviorally isolated from pheromone cues in mice (16–19).Therefore, it is useful to establish a novel animal model, in which functions of the MOS in controlling social behavior are genetically dissociated from those of the VNS to answer several fundamental questions, such as which social behaviors are regulated by the MOS or what is the difference between the MOS and VNS in regulating social behaviors? A further question is whether the MOS has its own pheromone pathway in the brain, separate from the VNS. These questions are also important for understanding pheromone-induced social behaviors in animals that do not possess a functional VNS, such as humans.In this study, we established two Cnga2 conditional knockout mouse lines with regional differences in the extent of loss-of-function in the MOS: (i) MOS-specific conditional Cnga2 knockout mice, the ΔMOS(cng) line, which had severe defects in active sniffing and poor survival through the neonatal period; and (ii) Cnga2 conditional knockout mice with loss-of-function confined to the olfactory sensory neurons in the dorsal zone, the ΔD(cng) line. The ΔD(cng) mice and the previously generated ΔD(dta) mice, in which the dorsal part of the MOB is deleted by targeted expression of the diphtheria toxin fragment-A (dta) gene (20) (Fig. 1A and Fig. S1B), maintained the ability to smell and chemoinvestigate their conspecifics. These mice therefore showed preserved pheromonal activity in the VNS, but had defects in sexual, maternal, aggressive, and risk-assessment behaviors. This functional dissection of the MOS and VNS enabled us to demonstrate social behaviors that are regulated by the MOS in a VNS-independent manner and to further characterize functional roles for the MOS-mediated pheromone pathway.Open in a separate windowFig. 1.Preserved detection of odors and pheromones in ΔD mice. (A) Strategy for generating ΔMOS(cng) mice, ΔD(dta) mice, and ΔD(cng) mice. (B) Schematic diagram for generating Cnga2 conditional knockout mice. Following Cre recombination, exon 6 is deleted resulting in a frameshift of exon 7, which contains the channel pore and cAMP binding domains. (C) Verification of homologous recombination by Southern blot analysis. The 9.1-kb band is from the wild-type allele, and the 2.7-kb band is from the targeted allele. (D) Most of the ΔMOS(cng) mice die before weaning (3–4 wk old), whereas ΔD(dta) or ΔD(cng) mice do not. The number of surviving mice at the time of weaning was divided by the expected birth number to calculate survival rate. (E) ΔD(dta), ΔD(cng), and control mice spend similar amounts of time sniffing eugenol (n = 6 for each genotype). (F) Urine habituation-dishabituation test. Graphs present total amount of time (in seconds, s) that male control (n = 8), ΔD(dta) (n = 7), and ΔD(cng) (n = 8) mice spend sniffing a piece of filter paper spotted with water or urine from male or female mice. A decrease or increase in sniffing time indicates that the mice perceive the presented odor to be same as, or different from, the previously presented odor, respectively. O-MACS^cre/+ mice were used as controls. NSE, neuron-specific enolase, pan-neuronal promoter; dta, diphtheria toxin A. Data are presented as mean + SEM or mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ns, P > 0.05 (Student’s t test).     

Hypothalamic GABAergic modulation of respiratory responses to baroreceptor stimulation

Little is known about possible hypothalamic modulation of the respiratory response to baroreceptor activation. The purpose of this study was to determine if the respiratory depression associated with the baroreceptor reflex is modulated by neurons in the posterior hypothalamus. Breathing frequency and tidal diaphragmatic activity were derived from diaphragmatic electromyographic recordings in anesthetized rats. Respiratory responses to baroreceptor activation were analyzed before and after unilateral microinjections of GABA antagonists (picrotoxin, bicuculline methiodide) or a GABA synthesis inhibitor (3-mercaptopropionic acid, 3-MP) into the posterior hypothalamus. Baroreceptor stimulation prior to microinjections elicited a decrease in both breathing frequency and tidal diaphragmatic activity. Microinjection of picrotoxin elicited an increase in respiratory activity. The decrease in tidal diaphragmatic activity evoked by baroreceptor stimulation was blocked after the microinjection. Furthermore, the baroreceptor-induced fall in breathing frequency was converted to an increase in breathing frequency. These effects of picrotoxin were reversed by microinjections of a GABA agonist (muscimol) into the same site. Microinjections of 3-MP also blocked the decrease in breathing frequency associated with baroreceptor stimulation. The GABA antagonist bicuculline methiodide elicited similar effects. These results indicate that a GABAergic mechanism in the posterior hypothalamus modulates the respiratory responses to baroreceptor stimulation.     

Regulation of behavioral maturation by a primer pheromone produced by adult worker honey bees

Previous research showed that the presence of older workers causes a delayed onset of foraging in younger individuals in honey bee colonies, but a specific worker inhibitory factor had not yet been identified. Here, we report on the identification of a substance produced by adult forager honey bees, ethyl oleate, that acts as a chemical inhibitory factor to delay age at onset of foraging. Ethyl oleate is synthesized de novo and is present in highest concentrations in the bee's crop. These results suggest that worker behavioral maturation is modulated via trophallaxis, a form of food exchange that also serves as a prominent communication channel in insect societies. Our findings provide critical validation for a model of self-organization explaining how bees are able to respond to fragmentary information with actions that are appropriate to the state of the whole colony.     

Evolution of moth sex pheromone composition by a single amino acid substitution in a fatty acid desaturase

For sexual communication, moths primarily use blends of fatty acid derivatives containing one or more double bonds in various positions and configurations, called sex pheromones (SPs). To study the molecular basis of novel SP component (SPC) acquisition, we used the tobacco hornworm (Manduca sexta), which uses a blend of mono-, di-, and uncommon triunsaturated fatty acid (3UFA) derivatives as SP. We identified pheromone-biosynthetic fatty acid desaturases (FADs) MsexD3, MsexD5, and MsexD6 abundantly expressed in the M. sexta female pheromone gland. Their functional characterization and in vivo application of FAD substrates indicated that MsexD3 and MsexD5 biosynthesize 3UFAs via E/Z14 desaturation from diunsaturated fatty acids produced by previously characterized Z11-desaturase/conjugase MsexD2. Site-directed mutagenesis of sequentially highly similar MsexD3 and MsexD2 demonstrated that swapping of a single amino acid in the fatty acyl substrate binding tunnel introduces E/Z14-desaturase specificity to mutated MsexD2. Reconstruction of FAD gene phylogeny indicates that MsexD3 was recruited for biosynthesis of 3UFA SPCs in M. sexta lineage via gene duplication and neofunctionalization, whereas MsexD5 representing an alternative 3UFA-producing FAD has been acquired via activation of a presumably inactive ancestral MsexD5. Our results demonstrate that a change as small as a single amino acid substitution in a FAD enzyme might result in the acquisition of new SP compounds.Sex pheromones (SPs) are a diverse group of chemical compounds that are central to mate-finding behavior in insects (1). Variation in SP composition between closely related species and among populations is well documented. Despite this variation, SPs are presumed to be under strong stabilizing selection, and thus the genetic mechanisms driving SP diversification represented an enigma (2). Research on SPs in moths (Insecta: Lepidoptera) helped establish the hypothesis of asymmetric tracking as a major driving force in SP diversification. In this scenario, abrupt changes in female SP composition via a shift in component ratio or the inclusion or loss of a component result in a distinct SP that attracts males with more broadly or differentially tuned SP preference (3). Assortative mating, the preferential mating of females producing a novel SP with males attracted to this SP, restricts gene flow between subpopulations with differing SP compositions. This can ultimately lead to speciation and fixation of novel communication channels (4). Work in insect models such as wasps (5), fruit flies (6), and especially moths (7–9) is helping uncover the genetic basis of SP diversification.In the majority of moth species, females use species-specific mixtures of SP components (SPCs) consisting of volatile fatty acid (FA) derivatives to attract conspecific males at long range. These SPCs are predominantly long-chain aliphatic (C12–C18) acetates, alcohols, or aldehydes containing zero to three double bonds of various configurations at different positions along the carbon backbone (10). Pheromone biosynthesis involves modifications of fatty acyl substrates, such as chain shortening and elongation, reduction, acetylation, oxidation, and desaturation (11). SP biosynthetic enzymes [i.e., FA reductases (8), FA chain-shortening enzymes (12, 13), and particularly FA desaturases (FADs) (7, 9, 14–17)] are the most commonly discovered traits underlying SP divergence in moths.Manduca sexta females attract males by releasing an SP containing in addition to mono- and diunsaturated aldehydes, which are typical structural themes in SPs of Bombycoidea moths (10), also uncommon conjugated triunsaturated aldehydes. The production of triunsaturated SPCs represents an easily traceable phenotype, thus making M. sexta a convenient yet unexploited model organism for unraveling the mechanisms of chemical communication evolution via novel SPC recruitment. In our previous attempts to decipher the desaturation pathway leading to triunsaturated SPC FA precursors (3UFAs), we identified the MsexD2 desaturase, which exhibits Z11-desaturase and conjugase (1,4-dehydrogenase) activity and participates in stepwise production of monounsaturated (1UFA) and diunsaturated (2UFA) SPC precursors. The terminal desaturation step resulting in the third conjugated double bond remained, however, elusive (18, 19).Here, we isolated and functionally characterized FAD genes abundantly and specifically expressed in the pheromone gland (PG) capable of producing 3UFA pheromone precursors and demonstrated the biosynthesis of 3UFAs from 2UFAs. We used site-directed mutagenesis of M. sexta FADs and identified a minimal structure motif leading to acquisition of new desaturase specificities. The reconstructed evolutionary relationship of moth FADs demonstrated that the 3UFA pheromone precursors in M. sexta were acquired via (i) activation of a presumably inactive ancestral FAD gene and/or (ii) duplication of an ancestral FAD gene producing 1UFA and 2UFA SPC precursors followed by functional diversification of an FAD duplicate.     

Peripheral chemoreceptor control of fetal renin responses to hypoxia and hypercapnia

The renin response to hypoxia in late gestation fetal sheep has been well characterized. However, the renin response to asphyxia--the combination of hypoxia and hypercapnia--has not been extensively studied. The purpose of this study was to determine 1) the interaction of hypoxia and hypercapnia in the control of renin secretion in late gestation fetal sheep and 2) the role of peripheral arterial chemoreceptors therein. Chronically catheterized fetal sheep (intact or sinoaortic denervated) were exposed to hypoxia and/or hypercapnia for 30 minutes. Hypercapnia alone had no effect on plasma renin activity or aldosterone but did result in a significant increase in angiotensin II. Hypercapnia combined with hypoxia resulted in a significant increase in renin activity, angiotensin II, and aldosterone. Sinoaortic denervation attenuated the renin and angiotensin II responses to hypercapnia plus hypoxia. The increase in renin and angiotensin II in response to hypercapnia with or without concomitant hypoxia strongly correlated with the magnitude of the decrease in arterial pH in intact fetuses only. Hypoxia alone and in concert with hypercapnia increased mean arterial pressure and decreased heart rate in intact but not sinoaortic denervated fetuses. We conclude that 1) hypercapnia more potently increases plasma renin activity than does hypoxia in late gestation fetal sheep, 2) arterial pH may be the relevant signal perceived by the peripheral arterial chemoreceptors for the control of the renin-angiotensin system during asphyxia, and 3) the cardiovascular response to hypoxia is mediated, in part, by peripheral arterial chemoreceptors.     

Insulin signaling is involved in the regulation of worker division of labor in honey bee colonies

It has been proposed that one route of behavioral evolution involves novel regulation of conserved genes. Age-related division of labor in honey bee colonies, a highly derived behavioral system, involves the performance of different feeding-related tasks by different groups of individuals. Older bees acquire the colony's food by foraging for nectar and pollen, and the younger "nurse" bees feed larvae processed foods. The transition from hive work to foraging has been shown to be socially regulated and associated both with decreases in abdominal lipid stores and with increases in brain expression of genes implicated in feeding behavior in Drosophila melanogaster. Here we show that division of labor is influenced by a canonical regulator of food intake and energy balance in solitary species, the insulin/insulin-like growth factor signaling (IIS) pathway. Foragers had higher levels of IIS gene expression in the brain and abdomen than did nurses, despite their low lipid stores. These differences are likely nutritionally mediated because manipulations that induced low lipid stores in young bees also up-regulated these genes. Changes in IIS also causally influenced the timing of behavioral maturation: inhibition of the insulin-related target of rapamycin pathway delayed the onset of foraging in a seasonally dependent manner. In addition, pathway analyses of microarray data revealed that nurses and foragers differ in brain energy metabolism gene expression, but the differences are opposite predictions based on their insulin-signaling status. These results suggest that changes in the regulation of the IIS pathway are associated with social behavior.