Establishment of Tools for Neurogenetic Analysis of Sexual Behavior in the Silkmoth,Bombyx mori

Background

Silkmoth, Bombyx mori, is an ideal model insect for investigating the neural mechanisms underlying sex pheromone-induced innate behavior. Although transgenic techniques and the GAL4/UAS system are well established in the silkmoth, genetic tools useful for investigating brain function at the neural circuit level have been lacking.

Results

In the present study, we established silkmoth strains in which we could visualize neural projections (UAS-mCD8GFP) and cell nucleus positions (UAS-GFP.nls), and manipulate neural excitability by thermal stimulation (UAS-dTrpA1). In these strains, neural projections and nucleus position were reliably labeled with green fluorescent protein in a GAL4-dependent manner. Further, the behavior of silkworm larvae and adults could be controlled by GAL4-dependent misexpression of dTrpA1. Ubiquitous dTrpA1 misexpression led both silkmoth larvae and adults to exhibit seizure-like phenotypes in a heat stimulation-dependent manner. Furthermore, dTrpA1 misexpression in the sex pheromone receptor neurons of male silkmoths allowed us to control male sexual behavior by changing the temperature. Thermally stimulated male silkmoths exhibited full sexual behavior, including wing-flapping, orientation, and attempted copulation, and precisely approached a thermal source in a manner similar to male silkmoths stimulated with the sex pheromone.

Conclusion

These findings indicate that a thermogenetic approach using dTrpA1 is feasible in Lepidopteran insects and thermogenetic analysis of innate behavior is applicable in the silkmoth. These tools are essential for elucidating the relationships between neural circuits and function using neurogenetic methods.     

Drosophila Ovipositor Extension in Mating Behavior and Egg Deposition Involves Distinct Sets of Brain Interneurons

Oviposition is a female-specific behavior that directly affects fecundity, and therefore fitness. If a fertilized female encounters another male that she has evaluated to be of better quality than her previous mate, it would be beneficial for her to remate with this male rather than depositing her eggs. Females who decided not to remate exhibited rejection behavior toward a courting male and engaged in oviposition. Although recent studies of Drosophila melanogaster identified sensory neurons and putative second-order ascending interneurons that mediate uterine afferents affecting female reproductive behavior, little is known about the brain circuitry that selectively activates rejection versus oviposition behaviors. We identified the sexually dimorphic pC2l and female-specific pMN2 neurons, two distinct classes of doublesex (dsx)-expressing neurons that can initiate ovipositor extension associated with rejection and oviposition behavior, respectively. pC2l interneurons, which induce ovipositor extrusion for rejection in females, have homologues that control courtship behavior in males. Activation of these two classes of neurons appears to be mutually exclusive and each governs hierarchical control of the motor program in the VNC either for rejection or oviposition, contributing centrally to the switching on or off of the alternative motor programs.     

Turning males on: activation of male courtship behavior in Drosophila melanogaster

The innate sexual behaviors of Drosophila melanogaster males are an attractive system for elucidating how complex behavior patterns are generated. The potential for male sexual behavior in D. melanogaster is specified by the fruitless (fru) and doublesex (dsx) sex regulatory genes. We used the temperature-sensitive activator dTRPA1 to probe the roles of fru(M)- and dsx-expressing neurons in male courtship behaviors. Almost all steps of courtship, from courtship song to ejaculation, can be induced at very high levels through activation of either all fru(M) or all dsx neurons in solitary males. Detailed characterizations reveal different roles for fru(M) and dsx in male courtship. Surprisingly, the system for mate discrimination still works well when all dsx neurons are activated, but is impaired when all fru(M) neurons are activated. Most strikingly, we provide evidence for a fru(M)-independent courtship pathway that is primarily vision dependent.     

Differential roles of two major brain structures, mushroom bodies and central complex, for Drosophila male courtship behavior

Drosophila male courtship is a complex and robust behavior, the potential for which is genetically built into specific neural circuits in the central nervous system. Previous studies using male-female mosaics and the flies with defects in particular brain structures implicated the critical central regions involved in male courtship behavior. However, their acute physiological roles in courtship regulation still largely remain unknown. Using the temperature-sensitive Dynamin mutation, shibire(ts1), here we demonstrate the significance of two major brain structures, the mushroom bodies and the central complex, in experience-independent aspects of male courtship. We show that blocking of synaptic transmission in the mushroom body intrinsic neurons significantly delays courtship initiation and reduces the courtship activity by shortening the courtship bout length when virgin females are used as a sexual target. Interestingly, however, the same treatment affects neither initiation nor maintenance of courtship toward young males that release courtship-stimulating pheromones different from those of virgin females. In contrast, blocking of synaptic transmission in a central complex substructure, the fan-shaped body, slightly but significantly reduces courtship activity toward both virgin females and young males with little effect on courtship initiation. Taken together, our results indicate that the neuronal activity in the mushroom bodies plays an important role in responding to female-specific sex pheromones that stimulate initiation and maintenance of male courtship behavior, whereas the fan-shaped body neurons are involved in maintenance of male courtship regardless of the nature of courtship-stimulating cues.     

The hector G-protein coupled receptor is required in a subset of fruitless neurons for male courtship behavior

Male courtship behavior in Drosophila melanogaster is controlled by two main regulators, fruitless (fru) and doublesex (dsx). Their sex-specific expression in brain neurons has been characterized in detail, but little is known about the downstream targets of the sex-specific FRU and DSX proteins and how they specify the function of these neurons. While sexual dimorphism in the number and connections of fru and dsx expressing neurons has been observed, a majority of the neurons that express the two regulators are present in both sexes. This poses the question which molecules define the sex-specific function of these neurons. Signaling molecules are likely to play a significant role. We have identified a predicted G-protein coupled receptor (GPCR), CG4395, that is required for male courtship behavior. The courtship defect in the mutants can be rescued by expression of the wildtype protein in fru neurons of adult males. The GPCR is expressed in a subset of fru-positive antennal glomeruli that have previously been shown to be essential for male courtship. Expression of 4395-RNAi in GH146 projection neurons lowers courtship. This suggests that signaling through the CG4395 GPCR in this subset of fru neurons is critical for male courtship behavior.     

Initiation of swimming activity by trigger neurons in the leech subesophageal ganglion

In papers I and II of this series, we described two pairs of interneurons, Tr1 and Tr2, in the leech subesophageal ganglion which can trigger swimming activity in the isolated central nervous system (CNS). In this paper, we describe sensory inputs to these trigger neurons from previously identified mechanosensory neurons. We found that: Weak mechanical stimulation (stroking) of a body wall flap attached to a segmental ganglion in an otherwise isolated CNS excites the contralateral Tr1 slightly. Strong mechanical stimulation (pinching) of a mid-body wall flap evokes a burst of impulses in the contralateral Tr1. For both means of stimulation the effects on the ipsilateral Tr1 and on the Tr2 cell pair were much weaker. Stroking a body wall flap attached to the head ganglion (supra- and subesophageal ganglia) in an otherwise isolated CNS excites both Tr1s and both Tr2s, although the effect is weaker for the Tr2s. Pinching strongly excites both trigger neurons bilaterally. Pressure and nociceptive mechanosensory neurons (P and N cells) in the subesophageal ganglion and the first segmental ganglion appear to make direct excitatory synapses with the contralateral Tr1 and Tr2. Mechanosensory interactions with the ipsilateral trigger neurons appear to be indirect. Functional inactivation of Tr1 by hyperpolarization does not prevent swim initiation either by weak mechanical stimulation of a body-wall flap or by intracellular stimulation of P cells.2+ We conclude that the trigger neurons, Tr1 and Tr2, provide an excitatory pathway by which mechanosensory stimulation can initiate leech swimming activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mushroom body D1 dopamine receptor controls innate courtship drive

Mating is critical for species survival and is profoundly regulated by neuromodulators and neurohormones to accommodate internal states and external factors. To identify the underlying neuromodulatory mechanisms, we investigated the roles of dopamine receptors in various aspects of courtship behavior in Drosophila. Here, we report that the D1 dopamine receptor dDA1 regulates courtship drive in naïve males. The wild‐type naïve males actively courted females regardless their appearance or mating status. On the contrary, the dDA1 mutant (dumb) males exhibited substantially reduced courtship toward less appealing females including decapitated, leg‐less and mated females. The dumb male's reduced courtship activity was due to delay in courtship initiation and prolonged intervals between courtship bouts. The dampened courtship drive of dumb males was rescued by reinstated dDA1 expression in the mushroom body α/β and γ neurons but not α/β or γ neurons alone, which is distinct from the previously characterized dDA1 functions in experience‐dependent courtship or other learning and memory processes. We also found that the dopamine receptors dDA1, DAMB and dD2R are dispensable for associative memory formation and short‐term memory of conditioned courtship, thus courtship motivation and associative courtship learning and memory are regulated by distinct neuromodulatory mechanisms. Taken together, our study narrows the gap in the knowledge of the mechanism that dopamine regulates male courtship behavior.     

Sensory Integration Regulating Male Courtship Behavior in Drosophila

The courtship behavior of Drosophila melanogaster serves as an excellent model system to study how complex innate behaviors are controlled by the nervous system. To understand how the underlying neural network controls this behavior, it is not sufficient to unravel its architecture, but also crucial to decipher its logic. By systematic analysis of how variations in sensory inputs alter the courtship behavior of a naïve male in the single-choice courtship paradigm, we derive a model describing the logic of the network that integrates the various sensory stimuli and elicits this complex innate behavior. This approach and the model derived from it distinguish (i) between initiation and maintenance of courtship, (ii) between courtship in daylight and in the dark, where the male uses a scanning strategy to retrieve the decamping female, and (iii) between courtship towards receptive virgin females and mature males. The last distinction demonstrates that sexual orientation of the courting male, in the absence of discriminatory visual cues, depends on the integration of gustatory and behavioral feedback inputs, but not on olfactory signals from the courted animal. The model will complement studies on the connectivity and intrinsic properties of the neurons forming the circuitry that regulates male courtship behavior.     

A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship

Propagation in higher animals requires the efficient and accurate display of innate mating behaviors. In Drosophila melanogaster, male courtship consists of a stereotypic sequence of behaviors involving multiple sensory modalities, such as vision, audition, and chemosensation. For example, taste bristles located in the male forelegs and the labial palps are thought to recognize nonvolatile pheromones secreted by the female. Here, we report the identification of the putative pheromone receptor GR68a, which is expressed in chemosensory neurons of about 20 male-specific gustatory bristles in the forelegs. Gr68a expression is dependent on the sex determination gene doublesex, which controls many aspects of sexual differentiation and is necessary for normal courtship behavior. Tetanus toxin-mediated inactivation of Gr68a-expressing neurons or transgene-mediated RNA interference of Gr68a RNA leads to a significant reduction in male courtship performance, suggesting that GR68a protein is an essential component of pheromone-driven courtship behavior in Drosophila.     

Octopamine neuromodulatory effects on a social behavior decision-making network in Drosophila males

Situations requiring rapid decision-making in response to dynamic environmental demands occur repeatedly in natural environments. Neuromodulation can offer important flexibility to the output of neural networks in coping with changing conditions, but the contribution of individual neuromodulatory neurons in social behavior networks remains relatively unknown. Here we manipulate the Drosophila octopaminergic system and assay changes in adult male decision-making in courtship and aggression paradigms. When the functional state of OA neural circuits is enhanced, males exhibit elevated courtship behavior towards other males in both behavioral contexts. Eliminating the expression of the male form of the neural sex determination factor, Fruitless (Fru(M)), in three OA suboesophageal ganglia (SOG) neurons also leads to increased male-male courtship behavior in these same contexts. We analyzed the fine anatomical structure through confocal examination of labeled single neurons to determine the arborization patterns of each of the three Fru(M)-positive OA SOG neurons. These neurons send processes that display mirror symmetric, widely distributed arbors of endings within brain regions including the ventrolateral protocerebra, the SOG and the peri-esophageal complex. The results suggest that a small subset of OA neurons have the potential to provide male selective modulation of behavior at a single neuron level.     

Frequency as a releaser in the courtship song of two crickets,Gryllus bimaculatus (de Geer) and Teleogryllus oceanicus: a neuroethological analysis

1. The courtship behavior of male field crickets, Gryllus bimaculatus (De Geer) and Teleogryllus oceanicus, is a complex, multimodal behavioral act that involves acoustic signals (a courtship song; Fig. 1A,B). The dominant frequency is 4.5 kHz for T. oceanicus song (Fig. 1A) and 13.5 kHz for G. bimaculatus (Fig. IB).
2. When courting males are deprived of their courtship song by wing amputation, their courtship success declines markedly but is restored when courting is accompanied by tape-recordings of their courtship songs or a synthetic courtship song with only the dominant frequency of the natural song; other naturally occurring frequency components are ineffective for restoring mating success (Figs. 4, 5).
3. It has been suggested that an identified auditory interneuron, AN2, plays a critical role in courtship success. Chronic recordings of AN2 in an intact, tethered female show that AN2's response to the natural courtship song and synthesized songs at 4.5 and 13.5 kHz is similar in T. oceanicus. By contrast, in G. bimaculatus, AN2's response to the natural courtship song and synthesized song at 13.5 kHz, but not at 4.5 kHz, is similar (Figs. 2,3).
4. In behavioral experiments, playback of a 30 kHz synthetic courtship song in G. bimaculatus does not restore courtship success, yet this same stimulus elicits as strong a response from AN2 as does the normal courtship song (Fig. 6). Thus, contrary to earlier work by others, we conclude AN2 is not, by itself, a critical neural link in the courtship behavior of these two species of crickets.

     

Drosophila Pheromone-Sensing Neurons Expressing the ppk25 Ion Channel Subunit Stimulate Male Courtship and Female Receptivity

As in many species, gustatory pheromones regulate the mating behavior of Drosophila. Recently, several ppk genes, encoding ion channel subunits of the DEG/ENaC family, have been implicated in this process, leading to the identification of gustatory neurons that detect specific pheromones. In a subset of taste hairs on the legs of Drosophila, there are two ppk23-expressing, pheromone-sensing neurons with complementary response profiles; one neuron detects female pheromones that stimulate male courtship, the other detects male pheromones that inhibit male-male courtship. In contrast to ppk23, ppk25, is only expressed in a single gustatory neuron per taste hair, and males with impaired ppk25 function court females at reduced rates but do not display abnormal courtship of other males. These findings raised the possibility that ppk25 expression defines a subset of pheromone-sensing neurons. Here we show that ppk25 is expressed and functions in neurons that detect female-specific pheromones and mediates their stimulatory effect on male courtship. Furthermore, the role of ppk25 and ppk25-expressing neurons is not restricted to responses to female-specific pheromones. ppk25 is also required in the same subset of neurons for stimulation of male courtship by young males, males of the Tai2 strain, and by synthetic 7-pentacosene (7-P), a hydrocarbon normally found at low levels in both males and females. Finally, we unexpectedly find that, in females, ppk25 and ppk25-expressing cells regulate receptivity to mating. In the absence of the third antennal segment, which has both olfactory and auditory functions, mutations in ppk25 or silencing of ppk25-expressing neurons block female receptivity to males. Together these results indicate that ppk25 identifies a functionally specialized subset of pheromone-sensing neurons. While ppk25 neurons are required for the responses to multiple pheromones, in both males and females these neurons are specifically involved in stimulating courtship and mating.     

Neural circuitry that governs Drosophila male courtship behavior

Male-specific fruitless (fru) products (Fru(M)) are both necessary and sufficient to "hardwire" the potential for male courtship behavior into the Drosophila nervous system. Fru(M) is expressed in approximately 2% of neurons in the male nervous system, but not in the female. We have targeted the insertion of GAL4 into the fru locus, allowing us to visualize and manipulate the Fru(M)-expressing neurons in the male as well as their counterparts in the female. We present evidence that these neurons are directly and specifically involved in male courtship behavior and that at least some of them are interconnected in a circuit. This circuit includes olfactory neurons required for the behavioral response to sex pheromones. Anatomical differences in this circuit that might account for the dramatic differences in male and female sexual behavior are not apparent.     

Testosterone response to courtship predicts future paternal behavior in the California mouse, Peromyscus californicus

In the monogamous and biparental California mouse (Peromyscus californicus), paternal care is critical for maximal offspring survival. Animals form pair bonds and do not engage in extrapair matings, and thus female evaluation of paternal quality during courtship is likely to be advantageous. We hypothesized that male endocrine or behavioral response to courtship interactions would be predictive of future paternal behavior. To test this hypothesis, we formed 20 pairs of California mice, and evaluated their behavior during the first hour of courtship interactions and again following the birth of young. We also collected blood from males at baseline, 1 hr after pairing, 3 weeks paired, and when young were 4 days old to measure testosterone (T). We found that male T-response to courtship interactions predicted future paternal behavior, specifically the amount of time he huddled over young when challenged by the temporary removal of his mate. Males that mounted T increases at courtship also approached pups more quickly during this challenge than males who had a significant decrease in T at courtship. Proximity of the male and female during courtship predicted paternal huddling during a 1-hr observation, and a multiple regression analysis revealed that courtship behavior was also predictive of birth latency. We speculate that male T-response to a female in P. californicus is an honest indicator of paternal quality, and if detectable by females could provide a basis for evaluation during mate choice.     

Discrimination of conspecific individuals via cuticular pheromones by males of the cricket Gryllus bimaculatus

Cuticular substances on the body surface of crickets serve as pheromones that elicit a variety of different behaviors in male crickets. Antennal contact between males and females resulted in courtship behavior, and that between two males resulted in aggressive displays. As a first step in elucidating how crickets recognize and discriminate individuals, behavioral responses of male individuals to cuticular substances of conspecific males or females were investigated. The behavioral responses of males to antennal or palpal stimulation with an isolated antenna from a male or a female were recorded. To both antennal and palpal stimulation with female antennae, the majority of males responded with courtship behavior; to stimulation with male antennae, males responded with aggressive displays. To gain insight into the chemical nature of the behaviorally relevant components, isolated antennae were washed in either n-hexane, acetone or ethanol before behavior assays. Washed antennae no longer elicited courtship or aggressive responses in males. Next, polypropylene fibers were smeared with substances from the body surface of females and used for antennal stimulation. This experiment showed that the quality and quantity of cuticular substances appear to be highly age-dependent. Significantly more males responded with courtship behavior to cuticular substances from younger females. Isolated males generally showed higher levels of aggression than males reared in groups. Grouped males also were more likely to display courtship behavior towards antennae from younger females, and aggressive behavior towards antennae from older females. These results suggest that male discrimination of mating partners depends on the nature of female cuticular substances.     

Drosophila female precopulatory behavior is modulated by ecdysteroids

The effect of ecdysteroid signaling on Drosophila female precopulatory behavior was investigated using two types of mutants with either globally reduced ecdysteroid availability or reduced expression of ecdysone receptors in fruitless neurons, known to control sexual behavior. While being courted by males, mutant females performed significantly less full ovipositor extrusion behavior to reject male copulation attempts. Ecdysteroid depleted females (ecdysoneless(1)) performed male-like courtship behaviors, including unilateral wing extension and song production with patterns very similar to male courtship song. These results support the hypothesis that ecdysteroids modulate female sexual behavior, perhaps acting as a regulator of sexual motivation, and as a component affecting the performance of sex specific behavior patterns.     

The octopamine-containing buccal neurons are a new group of feeding interneurons in the pond snail Lymnaea stagnalis

In the pond snail, Lymnaea stagnalis, the paired buccal ganglia contain 3 octopamine-immunoreactive neurons, which have previously been shown to be part of the feeding network. All 3 OC cells are electrically coupled together and interact with all the known buccal feeding motoneurons, as well as with all the modulatory and central pattern generating interneurons in the buccal ganglia. N1 (protraction) phase neurons: Motoneurons firing in this phase of the feeding cycle receive either single excitatory (depolarising) synaptic inputs (B1, B6 neurons) or a biphasic response (hyperpolarisation followed by depolarisation) (B5, B7 motoneurons). Protraction phase feeding interneurons (SO, N1L, NIM) also receive this biphasic synaptic input after OC stimulation. All of protraction phase interneurons inhibit the OC neurons. N2 (retraction) phase neurons: These motoneurons (B2, B3, B9, B10) and N2 interneurons are hyperpolarised by OC stimulation. N2 interneurons have a variable (probably polysynaptic) effect on the activity of the OC neurons. N3 (swallowing) phase: OC neurons are strongly electrically coupled to both N3 phase (B4, B4cluster, B8) motoneurons and to the N3p interneurons. In case of the interneuronal connection (OC<->N3) the electrical synapse is supplemented by reciprocal chemical inhibition. However, the synaptic connections formed by the OC neurons or N3p interneurons to the other members of the feeding network are not identical. CGC: The cerebral, serotonergic CGC neurons excite the OC cells, but the OC neurons have no effect on the CGC activity. In addition to direct synaptic effects, the OC neurons also evoke long-lasting changes in the activity of feeding neurons. In a silent preparation, OC stimulation may start the feeding pattern, but when fictive feeding is already occurring, OC stimulation decreases the rate of the fictive feeding. Our results suggest that the octopaminergic OC neurons form a sub-population of N3 phase feeding interneurons, different from the previously identified N3p and N3t interneurons. The long-lasting effects of OC neurons suggest that they straddle the boundary between central pattern generator and modulatory neurons.