Mating‐Associated Changes in the Behaviour of Leptothorax gredleri Ant Queens

Throughout their lives, animals adapt their behaviour to environmental fluctuations and to their own requirements. In social insects, behavioural changes are often particularly conspicuous. For example, in many ant species, reproductive sexuals leave their maternal nests and engage in risky mating and dispersal activities. Female sexuals experience, during a short period of time, dramatic changes in terms of behaviour and environmental conditions. But because sexual activities of ants are not easily observed, few studies have quantified in detail how behaviour alters with maturation and mating. We studied how various behavioural traits of Leptothorax gredleri female sexuals, a species in which female sexuals attract males by ‘female calling’, change before and after mating. We tested the hypothesis that behavioural variation reflects the altered requirements of queens to adapt to a particular situation. To this end, we compared geotactic, phototactic and locomotor behaviour across a wide range of life stages from lightly coloured, unmated female sexuals to old, mated queens. The results showed that female sexuals of L. gredleri change conspicuously their geotactic, phototactic and locomotor behavioural traits over their life stages. Three different behavioural states were evident (1) from light to dark female sexuals, individuals have negative phototaxis and reduced locomotor activity; (2) mature female sexuals during the daily period of sexual activity have strong phototaxis, negative geotaxis and an important locomotor activity; and (3) freshly mated and old mated queens avoid light and decrease their locomotor activity. These sharp differences in behaviour between stages match the transition from the relative safety of the nest chamber to the adversary world outside the nest , and back.     

Changes in brain amine levels associated with the morphological and behavioural development of the worker honeybee

Summary Changes in biogenic amine levels associated with the morphological and behavioural development of the worker honeybee are examined.A significant increase in amine levels in the head of the honeybee is associated with transition from the larval to pupal stage. Adult emergence is also accompanied by a significant increase in 5-HT levels in the brain, but no significant change in brain dopamine (DA) levels. NADA (N-acetyldopamine) levels increase during larval and pupal development, but in contrast to both DA and 5-HT, drop significantly during the transition from pupa to adult.Levels of DA in the brain of nectar and pollen forager bees, presumed to be among the oldest adults sampled, were found to be significantly higher than in nurses, undertakers or food storers. These results suggest that an age-dependent change in amine levels occurs in the brain of the worker bee. In the optic lobes, levels of DA and 5-HT were found to be significantly higher in pollen forager bees than in all other behavioural groups. Significant differences in amine levels in the optic lobes of nectar foragers and pollen foragers indicate that some differences in amine levels occur independent of worker age. The functional significance of differences in brain amine levels and whether or not biogenic amines play a direct role in the control of honeybee behaviour has yet to be established.Abbreviations DA dopamine - 5-HT 5-hydroxytryptamine or serotonin - NADA N-acetyldopamine     

Queen reproductive state modulates pheromone production and queen-worker interactions in honeybees

The mandibular glands of queen honeybees produce a pheromone that modulates many aspects of worker honeybee physiology and behavior and is critical for colony social organization. The exact chemical blend produced by the queen differs between virgin and mated, laying queens. Here, we investigate the role of mating and reproductive state on queen pheromone production and worker responses. Virgin queens, naturally mated queens, and queens instrumentally inseminated with either semen or saline were collected 2 days after mating or insemination. Naturally mated queens had the most activated ovaries and the most distinct chemical profile in their mandibular glands. Instrumentally inseminated queens were intermediate between virgins and naturally mated queens for both ovary activation and chemical profiles. There were no significant differences between semen- and saline-inseminated queens. Workers were preferentially attracted to the mandibular gland extracts from queens with significantly more activated ovaries. These studies suggest that the queen pheromone blend is modulated by the reproductive status of the queens, and workers can detect these subtle differences and are more responsive to queens with higher reproductive potential. Furthermore, it appears as if insemination substance does not strongly affect physiological characteristics of honeybee queens 2 days after insemination, suggesting that the insemination process or volume is responsible for stimulating these early postmating changes in honeybee queens.     

Mating triggers dynamic immune regulations in wood ant queens

Mating can affect female immunity in multiple ways. On the one hand, the immune system may be activated by pathogens transmitted during mating, sperm and seminal proteins, or wounds inflicted by males. On the other hand, immune defences may also be down‐regulated to reallocate resources to reproduction. Ants are interesting models to study post‐mating immune regulation because queens mate early in life, store sperm for many years, and use it until their death many years later, while males typically die after mating. This long‐term commitment between queens and their mates limits the opportunity for sexual conflict but raises the new constraint of long‐term sperm survival. In this study, we examine experimentally the effect of mating on immunity in wood ant queens. Specifically, we compared the phenoloxidase and antibacterial activities of mated and virgin Formica paralugubris queens. Queens had reduced levels of active phenoloxidase after mating, but elevated antibacterial activity 7 days after mating. These results indicate that the process of mating, dealation and ovary activation triggers dynamic patterns of immune regulation in ant queens that probably reflect functional responses to mating and pathogen exposure that are independent of sexual conflict.     

In vivo regulation of dopamine and noradrenaline release by alpha2A-adrenoceptors in the mouse nucleus accumbens

The present study investigated the role of alpha2A-adrenoceptor (alpha2A-AR) subtype in the regulation of noradrenaline (NA) and dopamine (DA) release in the nucleus accumbens (NAc). The effect of locally infused and systemically injected alpha2-AR agonist, dexmedetomidine (DMT), and alpha2-AR antagonist, atipamezole, on NA and DA release was investigated in alpha2A-AR knockout and control mice by using in vivo microdialysis. In addition, we compared the drug effects on DA and NA release in the NAc to their effect on locomotor activity. Baseline NA and DA concentrations did not differ between genotypes. Local infusion of DMT decreased, in a concentration-dependent manner, NA, but not DA, levels in the control mice. However, systemic injection of DMT decreased both NA and DA levels in the control mice. In both cases DMT had no effects on transmitter release in alpha2A-AR knockout mice. Our results suggest that alpha2-ARs regulate the release of NA, but not DA, at the terminal level in the NAc. However, alpha2-ARs regulate DA release in the NAc indirectly by their effect on DA neurones in the ventral tegmental area via an unknown mechanism. In both cases the regulation is mediated by alpha2A-adrenoceptor subtype. Also the modulation of locomotor activity by alpha2-AR agonist and antagonist seems to be mediated via alpha2A-adrenoceptors.     

Reproductive interference between honeybee species in artificial sympatry

Reproductive isolation between closely related species is often incomplete. The Western honeybee, Apis mellifera, and the Eastern hive bee, Apis cerana, have been allopatric for millions of years, but are nonetheless similar in morphology and behaviour. During the last century, the two species were brought into contact anthropogenically, providing potential opportunities for interspecific matings. Hybrids between A. mellifera and A. cerana are inviable, so natural interspecific matings are of concern because they may reduce the viability of A. cerana and A. mellifera populations – two of the world's most important pollinators. We examined the mating behaviour of A. mellifera and A. cerana queens and drones from Caoba Basin, China and Cairns, Australia. Drone mating flight times overlap in both areas. Analysis of the spermathecal contents of queens with species‐specific genetic markers indicated that in Caoba Basin, 14% of A. mellifera queens mated with at least one A. cerana male, but we detected no A. cerana queens that had mated with A. mellifera males. Similarly, in Cairns, no A. cerana queens carried A. mellifera sperm, but one‐third of A. mellifera queens had mated with at least one A. cerana male. No hybrid embryos were detected in eggs laid by interspecifically mated A. mellifera queens in either location. However, A. mellifera queens artificially inseminated with A. cerana sperm produced inviable hybrid eggs or unfertilized drones. This suggests that reproductive interference will impact the viability of honeybee populations wherever A. cerana and A. mellifera are in contact.     

Essential role of NMDA receptor channel ε4 subunit (GluN2D) in the effects of phencyclidine, but not methamphetamine

Phencyclidine (PCP), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, increases locomotor activity in rodents and causes schizophrenia-like symptoms in humans. Although activation of the dopamine (DA) pathway is hypothesized to mediate these effects of PCP, the precise mechanisms by which PCP induces its effects remain to be elucidated. The present study investigated the effect of PCP on extracellular levels of DA (DA(ex)) in the striatum and prefrontal cortex (PFC) using in vivo microdialysis in mice lacking the NMDA receptor channel ε1 or ε4 subunit (GluRε1 [GluN2A] or GluRε4 [GluN2D]) and locomotor activity. PCP significantly increased DA(ex) in wildtype and GluRε1 knockout mice, but not in GluRε4 knockout mice, in the striatum and PFC. Acute and repeated administration of PCP did not increase locomotor activity in GluRε4 knockout mice. The present results suggest that PCP enhances dopaminergic transmission and increases locomotor activity by acting at GluRε4.     

Multiple functions of fire ant Solenopsis invicta mandibular gland products

Alarm pheromones of social insects are best known for their role in the defence and maintenance of colony integrity. Previous studies with the fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae) demonstrate that the mandibular glands of workers (sterile females) and male and female sexuals produce an alarm pheromone, 2‐ethyl‐3,6‐dimethylpyrazine. The function of alarm pheromones in worker ants is well understood and divergent from the function of these compounds in the winged sexual forms. The present study quantifies the amount of pyrazine in the mandibular glands from male and female alate sexuals, as well as queens. Pyrazine production in female alates starts in the late pupal stage and increases until they reach mating flight‐ready maturity; however, after mating flight participation, the pyrazine level declines by >50%. Interestingly, mature male alates lose >85% of their mandibular gland pyrazine during mating flight activity. The results of the present study indicate that male and female sexuals use mandibular gland secretions for mating flight initiation and during mating flights. Furthermore, the ontogeny of mandibular gland products (pyrazine as the marker) from newly‐mated queens to mature colony queens shows a more than two‐fold increase in the amount of pyrazine by 6 months after mating. However, this is followed by a decline to trace amounts in mature colony queens (>2 years old), suggesting a function for mandibular gland products during colony development. Multifunctional use of social insect pheromones is well documented and data are reported in the present study suggesting new roles for mandibular gland products in fire ants.     

Reduced levels of Cacna1c attenuate mesolimbic dopamine system function

Genetic variation in CACNA1C, which codes for the L‐type calcium channel (LTCC) Ca_v1.2, is associated with clinical diagnoses of bipolar disorder, depression and schizophrenia. Dysregulation of the mesolimbic‐dopamine (ML‐DA) system is linked to these syndromes and LTCCs are required for normal DAergic neurotransmission between the ventral tegmental area (VTA) and nucleus accumbens (NAc). It is unclear, however, how variations in CACNA1C genotype, and potential subsequent changes in expression levels in these regions, modify risk. Using constitutive and conditional knockout mice, and treatment with the LTCC antagonist nimodipine, we examined the role of Cacna1c in DA‐mediated behaviors elicited by psychomotor stimulants. Using fast‐scan cyclic voltammetry, DA release and reuptake in the NAc were measured. We find that subsecond DA release in Cacna1c haploinsufficient mice lacks normal sensitivity to inhibition of the DA transporter (DAT). Constitutive haploinsufficiency of Cacna1c led to attenuation of hyperlocomotion following acute administration of stimulants specific to DAT, and locomotor sensitization of these mice to the DAT antagonist GBR12909 did not reach the same level as wild‐type mice. The maintenance of sensitization to GBR12909 was attenuated by administration of nimodipine. Sensitization to GBR12909 was attenuated in mice with reduced Cacna1c selectively in the VTA but not in the NAc. Our findings show that Cacna1c is crucial for normal behavioral responses to DA stimulants and that its activity in the VTA is required for behavioral sensitization. Cacna1c likely exerts these effects through modifications to presynaptic ML‐DA system function.     

Role of medial prefrontal cortex dopamine in age differences in response to amphetamine in rats: Locomotor activity after intra-mPFC injections of dopaminergic ligands

Changes in medial prefrontal cortex (mPFC) dopamine receptor expression and in mPFC projections to the nucleus accumbens in adolescence suggest that there may be age differences in the regulation of drug‐related behavior by the mPFC. The age‐specific role of prelimbic D1 dopamine receptors on amphetamine‐induced locomotor activity was investigated. In experiment 1, rats aged postnatal day 30 (P30), P45, and P75, corresponding to early and late adolescence and adulthood, were given an injection of D1 and D2 antagonists into the prelimbic mPFC before a systemic injection of 1.5 mg/kg of amphetamine and locomotor activity was recorded. In experiment 2, effects of intra‐prelimbic injections of a D1 agonist and antagonist on locomotor activity produced by a lower dose (0.5 mg/kg) of amphetamine were investigated. D2 receptor antagonist did not alter amphetamine‐induced activity, whereas the D1 receptor antagonist reduced activity produced by 1.5 mg/kg of amphetamine more in P30 than in P45 and P75 rats. In addition, D1 agonist enhanced the locomotor activating effects of 0.5 mg/kg of amphetamine in adolescent rats and decreased activity in adult rats. These results suggest that insufficient activation of mPFC D1 receptors may underlie the reduced activity at the low dose of amphetamine in early adolescent compared to adult rats. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Dopamine enhances locomotor activity for mating in male honeybees (Apis mellifera L.)

Dopamine plays multiple roles in the regulation of reproduction in female honeybees where it appears to act independently of juvenile hormone (JH). In males the role of dopamine and its relationship to JH control have not been elucidated. In the present study we determined hemolymph levels of dopamine and its metabolite (N-acetyldopamine) in males at post-emergence days 0-16. The development of locomotor and flight activities were recorded over the same period. Hemolymph levels of dopamine and N-acetyldopamine were found to increase at the time of onset of mating flight activity and those of dopamine decreased thereafter. Both locomotor and flight activities increased in parallel with hemolymph dopamine levels but the increased activity levels were maintained following decline of dopamine levels. Brain and meso-metathoracic ganglia levels of dopamine showed a similar developmental profile to hemolymph dopamine levels. Locomotor activities were temporarily inhibited by injection of a dopamine-receptor antagonist (cis(Z)-flupenthixol) into the thorax, and were enhanced by injection of a dopamine-receptor agonist (6,7-ADTN). These results suggest that dopamine regulates locomotor activities for mating and plays a role downstream of JH in premature males in honeybees.     

Sex and age related changes in the locomotor activity and phototactic behaviors of two closely related species of Camponotus ants

A virgin ant queen has only one opportunity in her lifetime to realize her reproductive fitness when she leaves her nest for a mating flight. After successful mating she sheds her wings, excavates a nest and starts laying eggs to initiate her own colony. Here we report the results of our study on two related species of Camponotus ants - day active Camponotus paria and night active Camponotus compressus - aimed at investigating (i) if there exist inter-species differences in the activity and phototactic behaviors of males and queens, (ii) whether these behaviors in the queen change after mating, and (iii) whether the activity rhythm of queens changes with age. We find that while activity profiles differ between C. paria and C. compressus virgin males and queens, such differences in queens disappear after mating. Once mated, the activity rhythm of queens shows little change with age; the rhythm in virgin queens, on the other hand, changes considerably. As virgins, C. paria queens are positively phototactic, while C. compressus queens are negatively phototactic. After mating, C. paria queens become less phototactic, particularly during the subjective night, while C. compressus queens remain negatively phototactic. These results indicate that there are considerable differences in the activity and phototactic behaviors of virgin queens of the two related species of Camponotus ants. Most of these differences disappear after mating, which suggests that these behaviors may have evolved primarily for the proper execution of pre-mating events.     

Making a queen: an epigenetic analysis of the robustness of the honeybee (Apis mellifera) queen developmental pathway

Specialized castes are considered a key reason for the evolutionary and ecological success of the social insect lifestyle. The most essential caste distinction is between the fertile queen and the sterile workers. Honeybee (Apis mellifera) workers and queens are not genetically distinct, rather these different phenotypes are the result of epigenetically regulated divergent developmental pathways. This is an important phenomenon in understanding the evolution of social insect societies. Here, we studied the genomic regulation of the worker and queen developmental pathways, and the robustness of the pathways by transplanting eggs or young larvae to queen cells. Queens could be successfully reared from worker larvae transplanted up to 3 days age, but queens reared from older worker larvae had decreased queen body size and weight compared with queens from transplanted eggs. Gene expression analysis showed that queens raised from worker larvae differed from queens raised from eggs in the expression of genes involved in the immune system, caste differentiation, body development and longevity. DNA methylation levels were also higher in 3‐day‐old queen larvae raised from worker larvae compared with that raised from transplanted eggs identifying a possible mechanism stabilizing the two developmental paths. We propose that environmental (nutrition and space) changes induced by the commercial rearing practice result in a suboptimal queen phenotype via epigenetic processes, which may potentially contribute to the evolution of queen–worker dimorphism. This also has potentially contributed to the global increase in honeybee colony failure rates.     

Cheaters sometimes prosper: targeted worker reproduction in honeybee (Apis mellifera) colonies during swarming

Kin selection theory predicts that honeybee (Apis mellifera) workers should largely refrain from producing their own offspring, as the workers collectively have higher inclusive fitness if they rear the sons of their mother, the queen. Studies that have quantified levels of ovary activation and reproduction among workers have largely supported this prediction. We sampled pre‐emergent male pupae and adult workers from seven colonies at regular intervals throughout the reproductive part of the season. We show that the overall contribution of workers to male (drone) production is 4.2%, nearly 40 times higher than is generally reported, and is highest during reproductive swarming, when an average of 6.2% of the males genotyped are worker‐produced. Similarly, workers in our samples were 100 times more likely to have active ovaries than previously assumed. Worker reproduction is seasonally influenced and peaks when colonies are rearing new queens. Not all worker subfamilies contribute equally to reproduction. Instead, certain subfamilies are massively over‐represented in drone brood. By laying eggs within the period in which many colonies produce virgin queens, these rare worker subfamilies increase their direct fitness via their well‐timed sons.     

Catechols involved in sclerotization of cuticle and egg pods of the grasshopper,Melanoplus sanguinipes,and their interactions with cuticular proteins

N‐Acetyldopamine (NADA) is the major catechol in the hemolymph of nymphal and adult grasshoppers, Melanoplus sanguinipes (F.), and mainly occurs as an acid‐labile conjugate indicated to be a sulfate ester. Its concentration increases in last instar nymphs and peaks during adult cuticle sclerotization. Dopamine (DA), the precursor of NADA and melanic pigments, is about 10 times lower in concentration than NADA, but shows a similar pattern of accumulation. NADA also predominates in cuticle, but its concentration is lowest during the active period of sclerotization, reflecting its role as a precursor for quinonoid tanning agents. Two other catechols, 3,4‐dihydroxybenzoic acid (DOBA) and 3,4‐dihydroxyphenylethanol (DOPET), also occur in hemolymph and cuticle, and their profiles suggest a role in cuticle stabilization. Solid‐state NMR analysis of sclerotized grasshopper cuticle (fifth instar exuviae) estimated the relative abundances of organic components to be 59% protein, 33% chitin, 6% catechols, and 2% lipid. About 99% of the catechols are covalently bound in the cuticle, and therefore are involved in sclerotization of the protein‐chitin matrix. To determine the types of catechol covalent interactions in the exocuticle, samples of powdered exuviae were heated in Hcl under different hydrolytic conditions to release adducts and cross‐linked products. 3,4‐Dihydroxyphenylketoethanol (DOPKET) and 3,4‐dihydroxyphenylketoethylamine (arterenone) are the major hydrolysis products in weak and strong acid, respectively, and primarily represent NADA oligomers that apparently serve as cross‐links and filler material in sclerotized cuticle. Intermediate amounts of norepinephrine (NE) are released, which represent N‐acetylnorepinephrine (NANE), a hydrolysis product of NADA bonded by the b‐carbon to cuticular proteins and possibly chitin. Small quantities of histidyl‐DA and histidyl‐DOPET ring and side‐chain C‐N adducts are released by strong acid hydrolysis. Therefore, grasshopper cuticle appears to be sclerotized by both o‐quinones and p‐quinone methides of NADA and dehydro‐NADA, which results in a variety of C‐O and C‐N covalent bonds linked primarily through the side‐chain carbons of the catechol moiety to amino acid residues in cuticular proteins. The primary catechol extracted from both the female accessory glands/calyx and the proteinaceous frothy material of the egg pod is DOBA, which also commonly occurs in cockroach accessory glands and oothecae, presumably as a tanning agent precursor. 3,4‐Dihydroxyphenylalanine (DOPA) was also detected in extracts of the accessory glands/calyx of grasshoppers, and may serve as a precursor for DOBA synthesis. Arch. Insect Biochem. Physiol. 40:119–128, 1999. © 1999 Wiley‐Liss, Inc.     

Efficacy of levonorgestrel when administered as an irradiated,slow‐release injectable matrix for feline contraception

Reliable and safe methods of reversible contraception are needed for use in zoo felids. The efficacy of levonorgestrel (LNG) as a contraceptive, when delivered as a cesium‐irradiated, slow‐release, injectable matrix, was tested in domestic cats as a model for exotic cats. An increase (P = 0.0017) in body weight was observed in treated but not control queens (P = 0.2146). All control queens (n = 6), which received injections of matrix only, but none of the LNG‐treated queens (n = 6) became pregnant during the trial. Levonorgestrel was effective in preventing pregnancy for at least 36 weeks after two injections of drug‐loaded formulations (40 mg/kg body weight), administered 68 days apart. Throughout the study, all control queens displayed luteal activity and fluctuating fecal estradiol concentrations, whereas the LNG‐treated queens displayed lower estradiol concentrations and no luteal activity after treatment. We conclude that LNG, when delivered as a cesium‐irradiated, slow‐release, injectable matrix, is an effective contraceptive in domestic cats, reducing follicular activity, and thus, preventing mating and luteal activity. Zoo Biol 20:407–421, 2001. © 2001 Wiley‐Liss, Inc.     

Biphasic alterations in serotonin-1B (5-HT1B) receptor function during abstinence from extended cocaine self-administration

Alterations in 5‐HT_1B receptor function during cocaine abstinence were evaluated in rats given either limited‐ or extended access (LA and EA, respectively) to cocaine self‐administration. The locomotor response to the 5‐HT_1B/1A agonist RU24969 was significantly reduced in cocaine‐experienced animals relative to cocaine‐naïve controls following 6 h of abstinence but became sensitized over the subsequent 14 days of abstinence. Both the early phase subsensitivity and later phase supersensivity to RU 24969‐induced activity were greater in EA versus LA animals. Intra‐nucleus accumbens administration of the 5‐HT_1B agonist CP 93, 129 produced significantly greater increases in dialysate dopamine levels in EA versus control animals following 14 days of abstinence. However, there was no difference between EA and cocaine‐naïve control animals in the augmentation of cocaine‐induced increases in nucleus accumbens DA produced by intra‐VTA CP 93, 129. Collectively these findings demonstrate that 5‐HT_1B receptor function is persistently altered by cocaine self‐administration.     

Mating in the rain? Climatic variance for polyandry in the honeybee (Apis mellifera jemenitica)

We tested the impact of colony density and climatic conditions on the level of queen polyandry in different wild populations of the honeybee (Apis mellifera jemenitica). We identified the mating frequency of queens by genotyping worker offspring (n = 672) of 28 wild colonies with microsatellite DNA markers. The populations sampled in different climatic regions in Sudan showed a high variance for the queen mating frequency ranging from 9 to 23 estimated matings with an average of 14.00 ± 3.94 (13.16 ± 4.87 effective matings). The degree of polyandry was highly significantly correlated with the rainfall in the various regions. In general, more rainfall resulted in lower mating frequencies. Polyandry did not correlate with colony density or with genetic diversity of the local sample populations. This suggests that variation in polyandry in wild honeybee populations of Sudan is primarily driven by climatic differences among ecosystem variation rather than by the absolute or effective local honeybee population size.     

Lobster locomotor activity as a measure of GABAAreceptor modulation

Gamma‐aminobutyric acid (GABA) is a well‐known neurotransmitter. A crustacean behavioural assay was developed for the examination of the effects of a benzodiazepine agonist and an antagonist of GABA_A‐type receptors. Both adult and juvenile male and female lobsters, Homarus americanus, were treated with single (or follow‐up) empirically determined doses of the agonist, diazepam, one of its metabolites (desmethyldiazepam), the antagonist, N‐methyl‐ß‐carboline‐3‐carboxamide (MBC), or vehicle alone. The effects of the compounds were monitored in a submerged circular open‐field for differences in locomotor activity, which was significantly inhibited by diazepam. Treatment with MBC following injection of diazepam reversed the latter's effects on locomotion. These results suggest that benzodiazepines may affect crustacean GABA receptors in a fashion similar to the GABA_A type found in the vertebrates, and that they may be involved in the regulation of locomotor behaviour.