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A society of bumble bees is primitively eusocial, with an annual life cycle, and can be used as a physiological model of social bees for comparative studies with highly eusocial hymenopterans. We investigated the dynamics of biogenic amine levels in the brain, meso-metathoracic ganglia, terminal abdominal ganglion, and hemolymph in queens 1 day after mating (1DAM), during diapause (Dp), and during colony founding (CF) in the bumble bee, Bombus ignitus . Dopamine levels in the brain of CF queens were significantly lower than in 1DAM and Dp queens, and the levels in the thoracic ganglia and hemolymph in CF queens were lower than in 1DAM queens, but did not differ from other groups in the abdominal ganglion. Octopamine levels in the brains were higher in Dp queens than in 1DAM queens. Serotonin and tyramine levels did not differ between the groups in different compartments of the central nervous system (CNS) that we examined. The dopamine levels in the brains were significantly positively correlated with those in the thoracic ganglia, abdominal ganglion, and hemolymph, suggesting the regulation of dopamine levels among three different compartments of the CNS. In isolated virgin queens, there were no significant correlations between the brain levels of biogenic amines that we examined and the lengths of the largest terminal oocytes, whereas, in isolated workers, the brain dopamine levels were positively correlated with oocyte lengths. These results suggest that dopamine is associated with ovarian activity in reproductive workers, but not in either virgin or mated queens.

Octopamine is an important neurotransmitter in insects with multiple functions. Here, we investigated the role of this amine in a simple form of learning (habituation) in the fruit fly Drosophila melanogaster. Specifically, we asked if octopamine is necessary for normal habituation of a proboscis extension response (PER) to different sucrose concentrations. In addition, we analyzed the relationship between responsiveness to sucrose solutions applied to the tarsus and habituation of the proboscis extension response in the same individual. The Tyramine-beta-hydroxylase (T beta h) mutant lacks the enzyme catalyzing the final step of octopamine synthesis. This mutant was significantly less responsive to sucrose than controls. The reduced responsiveness directly led to faster habituation. Systemic application of octopamine or induction of octopamine synthesis by T beta h expression in a cluster of octopaminergic neurons within the suboesophageal ganglion restored sucrose responsiveness and habituation of octopamine mutants to control level. Further analyses imply that the reduced sucrose responsiveness of T beta h mutants is related to a lower sucrose preference, probably due to a changed carbohydrate metabolism, since T beta h mutants survived significantly longer under starved conditions. These findings suggest a pivotal role for octopamine in regulating sucrose responsiveness in fruit flies. Further, octopamine indirectly influences non-associative learning and possibly associative appetitive learning by regulating the evaluation of the sweet component of a sucrose reward. (C) 2014 Elsevier Ltd. All rights reserved.

Egg-laying in Locusta migratoria involves the control of a variety of complex behavioural patterns including those that regulate digging of the oviposition hole and retention of eggs during digging. These two behavioural patterns are under the control of central pattern generators (CPGs). The digging and egg-retention CPGs are coordinated and integrated with overlapping locations of neural substrate within the VIIth and VIIIth abdominal ganglia of the central nervous system (CNS). In fact, the egg-retention CPG of the VIIth abdominal ganglion is involved in both egg-retention and protraction of the abdomen during digging. The biogenic amine, octopamine, has peripheral effects on oviduct muscle, relaxing basal tension of the lateral and upper common oviduct and enabling egg passage. Here we show that octopamine also modulates the pattern of the egg-retention CPG by altering the motor pattern that controls the external ventral protractor of the VIIth abdominal segment. There is no change in the motor pattern that goes to the oviducts. Octopamine decreased the frequency of the largest amplitude action potential and decreased burst duration while leading to an increase in cycle duration and interburst interval. The effects of octopamine were greatly reduced in the presence of the α-adrenergic blocker, phentolamine, indicating that the action of octopamine was via a receptor. Thus, octopamine orchestrates events that can lead to oviposition, centrally inhibiting the digging behavior and peripherally relaxing the lateral and common oviducts to enable egg-laying.

The cattle tick Rhipicephalus microplus, is one of the most damaging livestock ectoparasites. Tropical tick infestation limits the introduction of high-yield bovine varieties because they do not have immunity to the diseases transmitted by these ectoparasites. This tick is usually controlled with chemical acaricides but their indiscriminate use has created resistant populations. The discovery of new molecules that can be used for tick control is urgent. Based on the knowledge that octopamine, a biogenic amine analog to epinephrine, is central to the regulation of oviposition in several studied arthropods and that an imbalance in octopamine release causes sterility in a Drosophila model. Tyramine, octopamine and epinastine and 83 adrenergic compounds classified by their effect in the vertebrate systems were screened for their ability to block oviposition in Rhipicephalus microplus. Of these molecules, we found that 10 alpha-agonists, 3 alpha-antagonists, 5 beta-adrenergic agonists, 7 beta-antagonists and Norepinephrine were able to inhibit oviposition in this tick at pharmacological concentrations. Surprisingly, tyramine appears to be more potent than octopamine. The probable physiological causes of this inhibition are discussed. Our results suggest that although there are alpha adrenergic-like receptors in the tick, they do not behave in a manner completely analogous to their vertebrate counterparts.

Butterfly wing color patterns can be modified by the application of temperature shock to pupae immediately after pupation, which has been attributed to a cold-shock-induced humoral factor called cold-shock hormone (CSH). Here, we physiologically characterized CSH and pharmacological action of tungstate, using a nymphalid butterfly Junonia orithya. We first showed that the precise patterns of modification were dependent on the time-point of the cold-shock treatment after pupation, and confirmed that the modification properties induced in a cold-shocked pupa were able to be transferred to another pupa in a parabiosis experiment. Cold-shock application after removal of the head and prothorax together still produced modified wings, excluding major involvement of the brain-retrocerebral neuroendocrine complex. Furthermore, tungstate injection induced modifications even in individuals whose head and prothorax were removed. Importantly, transplantation of tracheae isolated from cold-shocked pupae induced modifications in the recipient wings. We identified a chemical peak in hemolymph of the cold-shocked individuals using HPLC, which corresponded to dopamine, and demonstrated that dopamine and its related biogenic amines have ability to induce small color-pattern changes. Taken together, the present study suggests that CSH is likely to be secreted from trachea-associated endocrine cells upon cold-shock treatment and that tungstate may change color patterns via its direct action on wings.

The biogenic amine octopamine functions as a neuromodulator, neurotransmitter and neurohormone in insect nervous systems. It plays a prominent role in modulating multiple physiological and behavioural processes in invertebrates. Octopamine exerts its effects by binding to specific receptor proteins that belong to the superfamily of G protein-coupled receptors. We found two partial sequences of putative octopamine receptors in the desert locust Schistocerca gregaria (SgOct alpha R and SgOct beta R) and investigated their transcript levels in males and females of both phases and during the transition between long-term solitarious and gregarious locusts. The transcript levels of SgOctaR are the highest in the central nervous system, whereas those of SgOct beta R are the highest in the flight muscles, followed by the central nervous system. Both SgOct alpha R and SgOct beta R show higher transcript levels in long-term gregarious locusts as compared to solitarious ones. The rise of SgOct beta R transcript levels already appears during the first 4 h of gregarisation, during which also the behavioural changes take place.

Precursors of neuroactive substances can be obtained from dietary sources, which can affect the resulting production of such substances in the brain. In social species, an intake of the precursor in food could be controlled by social interactions. To test the effects of dietary tyrosine on the brain dopamine levels in social insect colonies, male and worker honeybees were fed tyrosine or royal jelly under experimental conditions and the brain levels of dopamine and its metabolite were then measured. The results showed that the levels of dopamine and its metabolite in the brains of 4- and 8-day-old workers and 8-day-old males were significantly higher in tyrosine-fed bees than in control bees, but the levels in 4-day-old males were not. The brain levels of dopamine and its metabolite in 4- and 8-day-old males and workers were significantly higher in royal jelly-fed bees than in control bees, except for one group of 4-day-old workers. Food exchanges with workers were observed in males during 1-3 days, but self-feedings were also during 5-7 days. These results suggest that the brain levels of dopamine in males can be controlled by an intake of tyrosine in food via exchanging food with nestmates and by self-feeding.

Octopamine plays important neuromodulatory roles in the honeybee brain. Accordingly, mRNA from a recently identified honeybee octopamine receptor (AmOA1) is distributed throughout the brain. We have evaluated the occurrence of AmOA1 in the antennal lobe (AL) as well as rest of the brain (RB) by western blotting using an antiserum raised against a peptide selected from AmOA1 sequence. In addition to an expected band (78 kDa in the AL), one additional band (72 kDa) was identified from the AL and four bands (48, 60, 72 and 78 kDa) were observed in the RB. These bands were also recognized with antiserum against a different peptide segment from an octopamine receptor ortholog from the fruitfly (OAMB). Significant sequence identity with the peptide segment used to generate the antiserum was only found with OAMB and its splice variants in fruitfly; it was less conserved in other biogenic amine receptors from honeybee and other insects. Furthermore, western blot analysis performed on brains with dsRNA-treated antennal lobes showed a decrease in the intensity of all four bands. This suggests that AmOA1 antiserum specifically recognizes one or more types of AmOA1 receptors in the honeybee brain. We extend our earlier study of RNAi to quantify the rate of spread of dsRNA from a localized injection to other neuropils.

The biogenic amine octopamine and the pentapeptide proctolin are two important neuroactive chemicals that control contraction of the oviducts of the African locust Locusta migratoria. The physiological responses and signal transduction pathways used by octopamine and proctolin have been well characterized in the locust oviducts and this therefore provides the opportunity to examine the interaction between these two pathways. Octopamine, via the intracellular messenger adenosine 3',5'-cyclic monophosphate (cyclic AMP), inhibits contraction of the oviducts, while proctolin, via the phosphoinositol pathway, stimulates contraction. We have examined the physiological response of the oviducts to combinations of octopamine and proctolin and also looked at how combinations of these affect one of the main intracellular mediators of the octopamine response, namely cyclic AMP. It was found that application of octopamine to the oviducts led to a dose-dependent reduction in tonus of the muscle and also a decrease in the amplitude and frequency of spontaneous phasic contractions. Octopamine-induced relaxation was enhanced in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Octopamine was also able to inhibit proctolin-induced contractions of the oviducts in a dose-dependent manner. A 10(-9) M proctolin-induced contraction was inhibited by 83% in the presence of 10(-5) M octopamine, and was completely inhibited in the presence of 10(-5) M octopamine plus 5x10(-4) M IBMX. Octopamine led to a dose-dependent increase in cyclic AMP content as measured by radioimmunoassay. In the presence of 10(-9) M proctolin, this octopamine-induced increase in cyclic AMP was reduced by as much as 60%. Proctolin also caused a dose-dependent decrease in the cyclic AMP elevation produced by 5x10(-6) M octopamine. These results indicate that octopamine and proctolin can antagonize each other's physiological response when added in combination, and that proctolin is able to modulate the response of the oviducts to octopamine by influencing cyclic AMP levels.

The time course of changes in dopamine content during development was studied in Drosophila virilis under normal and stressful conditions. When development was normal, there were two dopamine peaks, one of which appeared during puparium formation, and the other during fly emergence. Short-term exposure to heat (60 min, 38°C) was found to be without influence on the dopamine content in pupae, but produced a rise in adult flies. Mechanical stress also produced an increase in dopamine content. D. virilis lines differing in dopamine content under normal conditions and in stress response of the dopamine system were established. The results of genetic analysis of these differences are presented. The gene (or genes) controlling differences in dopamine content under normal conditions is located on the X-chromosome, and the gene(s) controlling the differences in responsiveness of the dopamine system to stress was found to be autosomal.

The effects of octopamine and some selected octopamine agonists on neurally-evoked contrations and cyclic AMP levels of the lateral oviducts of the locust, Locusta migratoria, have been examined. Octopamine caused reversible, dose-dependent decreases in both the basal tonus and amplitude of neurally-evoked contractions of the lateral oviducts, and inhibited myogenic contractions. The 2-aminooxazoline, Ac6 [2-(4-chloro-o-toluidino)-2-oxazoline], and the substituted phenyliminoimidazolidines (PIIs), NC5 (2,6-diethyl-PII) and NC7 (2-methyl-4-chloro-PII), were each capable of eliciting similar responses to octopamine on neurally-evoked contractions. The vertebrate α-adrenergic receptor antagonist phentolamine blocked the physiological effects of all agonists tested. The effect of these agonists on cyclic AMP levels was also examined. Octopamine and the three agonists were able to increase the cyclic AMP content of the lateral oviducts in a dose-dependent manner. The increases in cyclic AMP were inhibited in the presence of various vertebrate receptor antagonists. The results of this study indicate that AC6, NC5, and NC7 all act as agonists to the octopamine2-like receptors present on locust oviduct and confirm previous studies for the agonistic properties of these agents.

Analyses of proximate mechanisms that control mating and oviposition behaviours in insects are important because they link behavioural ecology and physiology. Recently, seed beetles have been used as models to study evolution of female multiple mating and cost of reproduction including mating. In the present study, we investigated the effects of biogenic amines into the abdomens of females of two Callosobruchus species, Callosobruchus chinensis and Callosobruchus maculatus, on mating receptivity and oviposition behaviour. In C. chinensis, injection of octopamine and tyramine reduced receptivity to mating and tyramine and serotonin increased the number of eggs laid. Similarly, injection of tyramine reduced the receptivity of females and increased the number of eggs laid by females of C. maculatus. These results show the possibility that biogenic amines control mating receptivity and oviposition behaviour in females of two Callosobruchus species.

The importance of visual experience, during the early period after emergence of a fly, for the neural development of visual pattern discrimination has been demonstrated through behavioural experiments. As a first step to determine the molecular mechanisms underlying such plasticity in neural development, a study was made using HPLC with electrochemical detection of biogenic amines, their precursors and metabolites which are present in the optic lobes of the fly. Seven, optic lobe-dominant substances were detected. Among these, two substances were found to increase transiently during the first several days after emergence when flies experienced visual stimuli, while such an increase was not observed when kept in constant darkness. The transient increase in the two substances was not seen when flies were reared in constant darkness during the first 5 days only after emergence, and also when they were injected with an inhibitor of protein synthesis. In flies having a unilateral compound eye covered, the transient increase in the two substances occurred only in the optic lobe of the intact side and not in the covered side. These results support the previous findings suggesting that two “experience-dependent” substances, the features of which have not yet been identified, are contributory to the plasticity in the development of visual function.

Dopamine (DA) is a major functional biogenic amine in insects and has been suggested to regulate reproduction in female honeybees. However, its function has not been investigated in male drones. To clarify developmental changes of DA in drones, brain DA levels were investigated at various ages and showed a similar pattern to the previously reported juvenile hormone (JH) hemolymph titer. The DA level was lowest at emergence and peaked at day 7 or 8, followed by decline. Application of JH analog increased brain DA levels in young drones (2-4-days-old), suggesting regulation of DA by JH in drones. In young drones, maturation of male reproductive organs closely matched the increase in brain DA. The dry weight of testes decreased and that of seminal vesicles increased from emergence to day 8. The dry weight of mucus glands increased up to day 4. Consequently, DA regulated by JH might have reproductive behavior and/or physiological functions in drones.

Results in this study are consistent with those of Murdock and his colleagues who clearly demonstrated that clonidine, an agonist of octopaminergic receptors in some insects, significantly increases sucrose feeding. Their studies, however, did not examine the effect of clonidine on protein feeding. Injection of a 20 microg/microl/fly dose of clonidine significantly reduces protein feeding in both sexes of Phormia regina, instead of stimulating feeding as is observed with carbohydrate feeding. The manner in which the flies are fed prior to starvation and the method of testing influences the amounts of diet consumed. It is proposed that the biogenic amines influence the state of hunger (i.e., protein versus carbohydrates) while other chemicals and neural mechanisms (i.e., such as sulfakinins and stretch receptors, respectively) affect satiety.

The storage lobe of the locust corpus cardiacum consists primarily of neurosecretory cell axons and terminals and serves as a neurohaemal organ for the release of several neuropeptides. Biogenic amines have previously been identified in the storage lobe of the adult corpus cardiacum, and interest in the functional roles of these amines has arisen due to their modulatory actions on numerous physiological processes in insects. It has been suggested that these compounds may be released from the storage lobe as neurohormones, however an alternative possibility is that they serve as neurotransmitters or neuromodulators which act at sites within the corpus cardiacum proper. The present studies were undertaken to determine if specific amine receptor-adenylate cyclases exist in the storage lobe. Octopamine increases the cAMP content of intact cells in the storage lobes of adult and 5th-instar locusts. Forskolin and 5-HT also enhance cAMP levels in the storage lobe however additivity experiments suggest that 5-HT may be interacting with octopamine binding sites. Dopamine is a poor agonist and norepinephrine has no effect on cAMP content. In adult storage lobes octopamine acts on receptors that have pharmacological similarities to octopamine receptors of identified octopaminergic systems. There thus appears to be an octopamine receptor-adenylate cyclase complex in this tissue. These results suggest that octopamine may be involved in regulating some aspect of neurosecretion in the storage lobe of the locust corpus cardiacum. In addition, significantly higher levels of cGMP were found in the storage lobe compared to those reported for the neighbouring glandular lobe, indicating a possible functional role for this cyclic nucleotide.

Whole brains from Rhodnius prolixus treated with glyoxylic acid display catecholamine histofluorescence in eleven bilaterally distributed clusters of neurones, and in three circumscribed neuropile regions. This simple method offers resolution of some features not detected in studies on sectioned insect tissue; cephalic neurosecretory cells in chronically starved animals are histofluorescent and diverse catecholamine-handling fibres converge within the brain of this insect.

Lipid synthesis from leucine by fat cells of Glossina morsitans in vitro was inhibited by dopamine, adrenaline, noradrenaline and octopamine. Noradrenaline and octopamine were most active with maximal response occurring at 10 −4 and 10 −5 M respectively. The release of free fatty acids and the synthesis of proline from alanine by fat cells were stimulated by octopamine but not by the other amines. Maximal release of material from fat cells occurred at an octopamine concentration of 10 −2 M but at higher concentrations the response was diminished. The inhibition of lipid synthesis by octopamine was blocked by the α-adrenergic antagonist phentolamine but not by the β-adrenergic antagonist propranolol. Neither receptor-blocking-agent affected the action of corpora cardiaca extracts upon fat cells indicating that separate receptors are present for amines and the peptide hormones.

The effects of 5-hydroxytryptamine, noradrenalin, adrenalin, octopamine and dopamine on the transepithelial voltage and fluid secretion of Malpighian tubules of Aedes aegypti were studied. Of these biogenic amines, only 5-hydroxytryptamine altered the transepithelial voltage. When applied in short pulses to superfused tubules, the transepithelial voltage depolarized rapidly and then hyperpolarized. The transepithelial resistance was reduced during both the depolarizing and hyperpolarizing phases of the response to 5-hydroxytryptamine. When added to the bathing saline at a concentration of 10−6 M, the transepithelial voltage showed a more complex response with both depolarizing and hyperpolarizing elements. 5-Hydroxytryptamine was also the only biogenic amine tested that increased fluid secretion. The maximal increase of fluid secretion in response to 5-hydroxytryptamine was small compared to that of crude head extract, containing the putative diuretic hormones of this species. The concentration of potassium in secreted fluid of Malpighian tubules stimulated by 5-hydroxytryptamine was decreased by 20mM, while the sodium concentration showed an increase of similar magnitude.

The adult brain octopamine content (2.7 ng) of Acheta domesticus was about 50% greater than that of last-instar larvae (1.7 ng). There were no significant peaks in brain octopamine during larval or adult development. The condition of being mated or virgin had no effect on octopamine levels. The octopamine concentration in the brain is 100 times greater than in the blood. 2 days before the final moult the blood octopamine concentration increased from the larval level (22 pg/μl) to nearly adult levels (38 pg/μl), perhaps in response to preparation for the moult. A broad peak of elevated blood and brain octopamine occurred in adult crickets during the 4 h after lights off when entrained to a 14 h light-10 h dark photoregime. Newly emerged adults placed in constant darkness for 9 days showed no octopamine rhythm, but if newly emerged adults were exposed to 14 h light-10 h dark for 2 days, followed by 7 days of constant darkness, then a free running octopamine rhythm in the brain developed. The brain rhythm in Acheta appeared to be endogenous with a period of about 24.4 h. This is the first evidence of a free running rhythm of a biogenic amine in insects. A free running blood rhythm was indicated but not statistically demonstrable. 5 min of running stress doubled the blood octopamine concentration which did not increase with an additional 10 min of running stress. Recovery to control levels of octopamine after 15 min of running was rapid, requiring only 15 min of rest.

A differential transformer displacement transducer recorded the activity of the visceral muscle of Blaberus giganteus (L.) with a high degree of sensitivity. The denervated foregut proved to have myogenic properties with certain specific pacemaker regions. Nevertheless, stimulation of the oesophageal nerve innervating the region of the crop caused a slow type of graded contraction in the longitudinal muscles. Preparations of both the innervated and the denervated foregut were sensitive to 5-hydroxytryptamine (10−7 M), noradrenaline (10−6 M), and acetylcholine (5 × 10−6 M). Also, Factor S, a biogenic amine of uncertain structure found in arthropods, caused an excitatory response in innervated foreguts and an inhibitory response in denervated foreguts. Denervated foreguts had a greater sensitivity to l -glutamic acid than innervated preparations. The neurochemical divergence between the control of visceral and somatic musculature of insects is discussed in relation to the pharmacology of these biogenic amines.