Reassessment of the effect of biological rhythm mutations on learning in Drosophila melanogaster

A link between learning deficits and circadian period-lengthening mutations in Drosophila melanogaster previously has been reported. Mutant long-period males performed poorly in two learning assays involving experience-dependent courtship inhibition. In one, normal males that have courted fertilized females subsequently show courtship inhibition with virgin females. In the other, normal males that have courted sexually immature males subsequently fail to court other immature males. Those results have been reassessed in an extended study of genetic variants involving the period gene. 1. Long-period perL1 males demonstrated poor conditioned courtship inhibition when exposed to fertilized females; they showed normal courtship conditioning when exposed to immature males. This could be due to a perL1-associated olfactory deficit with fertilized females, since perL1 males were unable to discriminate behaviorally between fertilized and virgin females. 2. Other long-period males, including perL2 males and transgenic perL1 males bearing a truncated form of the per+ gene, were conditioned normally by fertilized females. Thus, the courtship inhibition defect is specific to the perL1 mutant strain. 3. perL1 (and other per mutant) flies showed normal acquisition and retention of a classically conditioned olfactory avoidance response. 4. Results from a new conditioned courtship inhibition experiment are presented; males exposed to fertilized females during training showed further courtship inhibition during subsequent exposure to fertilized females. From the perspective of learning theory, this can be viewed as a savings experiment.     

Conditioning Mutations in DROSOPHILA MELANOGASTER Affect an Experience-Dependent Behavioral Modification in Courting Males

One aspect of courtship in male Drosophila melanogaster has been reported to be experience dependent. Males that have courted fertilized females are virtually unresponsive to virgin females for 2–3 hr. Here, this response was utilized as an assay for the effects of conditioning mutations on experience-dependent courtship. Seven strains expressing conditioning mutations (previously isolated and characterized for learning or memory defects in an electrical shock-odor association paradigm, independent of courtship) were all found to be mutant in expression of this experience-dependent change in courtship behavior. By comparison, three control strains that were unselected for conditioning defects all expressed normal experience-dependent courtship. Other results indicate that males of the conditioning-defective strains are able to elicit necessary cues from fertilized females, yet do not then modify their courtship with virgin females. Thus, it is suggested that experience-dependent modification of courtship and the previously reported associative olfactory conditioning with electric shock share common elements of processing. The possibility that experience-dependent courtship represents adaptive behavior is discussed.     

The role of chemoreception in sex recognition by male crickets: Acheta domesticus and Teleogryllus oceanicus

ABSTRACT. Contact chemoreception is important in female recognition by Teleogryllus oceanicus (Le Guillou) males. Antennal contact of female conspecifics, body regions, detached antennae and conditioned substrate elicited mostly courtship responses including courtship songs. Aggressive acts were produced only in response to male conspecifics. Male body regions, detached antennae and conditioned substrate elicited very few courtship or aggressive acts and no songs. This suggests that one or several communication modes, in addition to chemical communication, are necessary to elicit aggressive responses. Acheta domesticus (L.) males cannot rely upon chemical cues for recognition of either sex. Responses to conspecifics suggest that A. domesticus males produce aggressive acts immediately after antennal contact with either sex. Aggressive response to males usually persists, but response to females often switches to courtship. Responses to body regions, detached antennae, and conditioned substrate were few, with courtship and aggressive responses elicited by both male- and female-generated stimuli. The importance of contact chemoreception in cricket communication is suggested by (1) failure of hexane-washed antennae to elicit aggressive or courtship acts, and (2) males spending more time in contact with body regions and conditioned substrates than with corresponding controls. Lack of response to male or female odour-laden air suggests that chemical signals are used by males only if directly contacted. Chemical and other signals supplement the obvious use of acoustic signals for intra- and intersexual communication in these crickets. The importance of multimodal communication in sex recognition is discussed.     

Reduced Reproductive Success for a Conditioning Mutant in Experimental Populations of DROSOPHILA MELANOGASTER

Male Drosophila melanogaster that have courted newly-emerged males can modify their subsequent courtship behavior to avoid further courtship with immature males for up to 6 hr (previously reported). Here, it was hypothesized that such an experience-dependent modification would afford a mating advantage to normal males over males that carried a mutation that affects learning and memory. Coisogenic lines were constructed which varied at the dunce gene (dnc+ and dncM14 alleles) in order to test this hypothesis. Whether previously experienced with immature males or not, dnc+ and dncM14 males were indistinguishable in their response and mating efficiency when individually paired with virgin females. However, courtship performance of dnc+ and dncM14 males was different if they were first experienced with immature males and were then individually tested in an artificial population of nine immature males and one virgin female. In this situation, dnc+ males spent much less time in courtship with immature males and achieved copulation in one-third the time required for dncM14 males. As a control, the behavior and mating efficiency of courtship-naive dnc+ and dncM14 males in the artificial population was indistinguishable. In competition for a single virgin female, experienced dncM14 males showed a slight mating advantage over experienced dnc+ males. But when competition by experienced males for a single virgin female took place in the presence of nine immature males, dnc+ males were the successful maters in three-fourths of the trials.     

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.     

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.

     

The behavioural basis of overdominance in competitive mating success at the ebony locus of Drosophila melanogaster

Male files homozygous for the gene ebony¹¹ are partially blind, and at a disadvantage in competitive mating. The courtship of the mutant males is deficient in wing vibration stimulation, which is characterized by a low proportion of sine song and a high intra-pulse frequency. Males heterozygous for ebony have normal vision, but show an increase in courtship song, and are superior in competitive mating to wild type males. The auditory characteristics of courtship song produced by heterozygous males are indistinguishable from those of wild type, and their superiority in competitive mating success is due to overdominance involving this specific element of male courtship behaviour.     

Influence of the White Locus on the Courtship Behavior of Drosophila Males

Since its discovery by Morgan, the Drosophila white gene has become one of the most intensely studied genes and has been widely used as a genetic marker. Earlier reports that over- and misexpression of White protein in Drosophila males leads to male-male courtship implicated white in courtship control. While previous studies suggested that it is the mislocalization of White protein within cells that causes the courtship phenotype, we demonstrate here that also the lack of extra-retinal White can cause very similar behavioral changes. Moreover, we provide evidence that the lack of White function increases the sexual arousal of males in general, of which the enhanced male-male courtship might be an indirect effect. We further show that white mutant flies are not only optomotor blind but also dazzled by the over-flow of light in daylight. Implications of these findings for the proper interpretation of behavioral studies with white mutant flies are discussed.     

Courtship behavior,communicative sound production and resistance to stress in Drosophila mutants with defective <Emphasis Type="Italic">agnostic</Emphasis> gene coding for LIMK1

To elucidate the role of one of the main elements of signal cascade of actin remodeling—LIM-kinase 1 (LIMK1)—in the control of animal behavior we have studied the characteristics of courtship behavior, parameters of acoustic communicative signals and their resistance to heat shock (HS, 37°C, 30 min) in Drosophila melanogaster males with the agn ^ts3 mutation in the agnostic locus which contains CG1848 gene for LIMK1. The data obtained were compared with the results of our previous similar investigation on wild type CS males [1]. Flies were divided into 4 groups. The males of control groups were not subjected to heat shock. Other groups were comprised of males subjected to heat shock either at the beginning of larval development when the mushroom body neuroblasts are predominantly dividing (groups HS1), or at the prepupal stage when the brain central complex is developing (groups HS2), or at the imago stage 1 hour before the test (groups HS). All males were tested at the age of 5 days. Virgin and fertilized CS females were used as courtship objects. Comparison of control groups of the wild type (CS) and mutant strains has shown that the mutation agn ^ts3 does not affect the main parameters of courtship behavior (courtship latency, the rapidity of achieving copulation, courtship efficiency) but leads to: a decrease in sexual activity, an increase in duration of sound trains in the songs and to a slight increase in the rate and stability of singing pacemakers. When compared to wild type, the agn ^ts3 males are more resistant to HS given 1 hour before the test. After HS their courtship intensity does not decrease and the main parameters of their courtship behavior and communicative sound signals either do not change, or appear to be even better stabilized. The frequency of distorted sound pulses (an indicator of frequency of impairments in the activity pattern of neuro-motor circuits of the singing center) decreases, and the working rate and stability of pacemakers of the pulse and the sine songs increases. Therefore, although the sharply elevated LIMK1 and p-cofilin levels detected by immunofluorescent techniques in the cells of agn ^ts3 mutants can lead to dramatic defects in learning and memory [2] accompanied by a decline of motivation of males, they do not appreciably affect the neuro-motor coordination during singing. The higher resistance of characteristics of the mutant behavior and communicative sound signals to heat shock is in agreement with the fact that the extremely high LIMK1 and P-cofilin levels fall down to normal values after HS and both the learning acquisition and memory formation are restored.     

Male Wing Fanning Performance During Successful and Unsuccessful Mating in the Parasitic Wasp Lariophagus distinguendus Förster (Hymenoptera: Pteromalidae)

Lariophagus distinguendus Förster (Hymenoptera: Pteromalidae) is a generalist solitary ectoparasitoid that parasitizes the immature stages of at least 11 foodstuff beetles from five families, including species developing in the seeds of Poaceae, Fabaceae and in dried natural products. In L. distinguendus, male wing fanning is a key phase in the courtship sequence and is elicited by a female-borne sex pheromone. However, studies aimed at understanding the role of wing vibration in L. distinguendus during courtship are lacking. In this research, the wing fanning performances of the L. distinguendus male towards other females and males were analyzed through high-speed video recordings and examined in relation to mating success. Only small differences were found between the fanning characteristics displayed toward females and young males, highlighting that young males are really perceived as females by older males. Male mating success was affected by the quality of the wing fanning in the courtship phase. Indeed, both immediately after the female perception and during mating attempts, fanning before a successful courtship differed compared to wing fanning performed prior to an unsuccessful courtship in terms of their mean frequency, but not their amplitude. Overall, our work increases knowledge of the courtship behavior of L. distinguendus and highlights the crucial importance of wing fanning among the range of sensory modalities used in the sexual communication of this parasitic wasp.     

Diversification of takeout, a male-biased gene family in Drosophila

The display of courtship behavior has evolved in response to sexual selection driven by competition to obtain mates. Sexually dimorphic mate selection rituals are likely controlled at least in part by genes with sex-biased patterns of expression. In Drosophila melanogaster, male courtship behavior has been well described and consists of a series of stereotyped behaviors. The takeout gene is predominantly expressed in males and affects male courtship behavior. In this study, we examine the patterns of expression and evolution in takeout and the family of related proteins. We show that a number of genes in the takeout gene family show male-biased expression in D. melanogaster, largely in non-reproductive tissues. Phylogenetic analysis reveals that this gene family is conserved across insects. As expected for genes with male-biased expression, we also find evidence of positive selection in some lineages. Our results suggest that the genes in this family may have evolutionarily conserved sex specific roles in male mating behavior across insects.     

Comparative study of four P-insertional memory-deficient Drosophila mutants

The previously suggested physiological model of conditioned courtship suppression in Drosophila was experimentally supported through investigation into the behaviour of the mutant males in two tests: the memory retention test and the retraining test. The mutations disrupted both types of the conditioned connections predicted by the model: association between conditioned and unconditioned stimuli and association between the conditioned stimulus and the aversive motivational system mediating the response to the unconditioned stimulus. The third category of mutants (P95, P124) appears to disrupt the function of the aversive motivational system.     

Evolutionary significance of courtship conditioning in Drosophila melanogaster

Males of the fly Drosophila melanogaster initially court mated, unreceptive females but later develop an avoidance reaction against them and even become temporally unresponsive to virgin females. This conditioned inhibition has been described as an associative process, the conditioned stimulus being a mixture of pheromones on the female's cuticle. To assess the evolutionary significance of courtship conditioning we recorded and analysed the male's behaviour during the conditioning process. The time traces of individual males were marked by an abrupt behavioural change. The time he spent courting suddenly decreased, and the frequency of ‘turn-away’ events at the same time sharply increased. Thus, the gradual decline of courtship observed as a group average does not reflect a slow change in motivation of the individual male but rather the interindividual differences of an active, experience-guided all-or-none decision to stop courting and to avoid the female. Three recently collected D. melanogaster strains were each maintained under two distinct mating conditions. Males were kept together with females for either ca. 2 weeks or 18 h. After 21 generations males of the two regimes differed markedly in their behaviour towards mated females. With long interaction periods between males and females, selection favoured courtship conditioning, while with short periods no such selection was observed. Slowly recovering receptivity of mated females may be needed for the maintenance of courtship conditioning. Courtship conditioning in D. melanogaster appears to be a fitness-relevant behaviour adapted to high-density populations with females mating a second time.     

A meal or a male: the ‘whispers’ of black widow males do not trigger a predatory response in females

Introduction

Female spiders are fine-tuned to detect and quickly respond to prey vibrations, presenting a challenge to courting males who must attract a female’s attention but not be mistaken for prey. This is likely particularly important at the onset of courtship when a male enters a female’s web. In web-dwelling spiders, little is known about how males solve this conundrum, or about their courtship signals. Here we used laser Doppler vibrometry to study the vibrations produced by males and prey (house flies and crickets) on tangle webs of the western black widow Latrodectus hesperus and on sheet webs of the hobo spider Tegenaria agrestis. We recorded the vibrations at the location typically occupied by a hunting female spider. We compared the vibrations produced by males and prey in terms of their waveform, dominant frequency, frequency bandwidth, amplitude and duration. We also played back recorded male and prey vibrations through the webs of female L. hesperus to determine the vibratory parameters that trigger a predatory response in females.

Results

We found overlap in waveform between male and prey vibrations in both L. hesperus and T. agrestis. In both species, male vibrations were continuous, of long duration (on average 6.35 s for T. agrestis and 9.31 s for L. hesperus), and lacked complex temporal patterning such as repeated motifs or syllables. Prey vibrations were shorter (1.38 - 2.59 s), sporadic and often percussive. Based on the parameters measured, courtship signals of male L. hesperus differed more markedly from prey cues than did those of T. agrestis. Courtship vibrations of L. hesperus males differed from prey vibrations in terms of dominant frequency, amplitude and duration. Vibrations of T. agrestis males differed from prey in terms of duration only. During a playback experiment, L. hesperus females did not respond aggressively to low-amplitude vibrations irrespective of whether the playback recording was from a prey or a male.

Conclusions

Unlike courtship signals of other spider species, the courtship signals of L. hesperus and T. agrestis males do not have complex temporal patterning. The low-amplitude ‘whispers’ of L. hesperus males at the onset of courtship are less likely to trigger a predatory response in females than the high-amplitude vibrations of struggling prey.     

REQUIREMENT OF THE TEC FAMILY TYROSINE KINASE BTK29A FOR COURTSHIP MEMORY IN Drosophila MALES

A male Drosophila that is not successful in courtship will reduce his courtship efforts in the next encounter with a female. This courtship suppression persists for more than 1 h in wild‐type males. The Btk29A^ficP mutant males null for the Btk29A type 2 isoform, a fly homolog of the nonreceptor tyrosine kinase Btk, show no courtship suppression, while Btk29A hypomorphic males exhibit a rapid decline in courtship suppression, leading to its complete loss within 30 min. The males of a revertant stock or Btk29A^ficP males that are also mutant for parkas, a gene encoding the presumptive negative regulator of Btk29A, exhibit normal courtship suppression. Since another behavioral assay has shown that Btk29A^ficP mutants are sensitization‐defective, we hypothesize that the mutant flies are unable to maintain the neural excitation state acquired by experience, resulting in the rapid loss of courtship suppression.     

Effect of social condition on behavioral development during early adult phase in <Emphasis Type="Italic">Drosophila prolongata</Emphasis>

Behavioral changes during early development provide useful insights into the internal mechanisms that generate complex behavior expressed by mature individuals. At the same time, social conditions during early adult phase can influence behavior in later stages of development even in holometabolous insects. In this study, age-dependent changes in courtship behavior and the effect of social conditions were examined in a fruit fly, Drosophila prolongata. Younger males showed lower mating activity and simpler courtship behavior. Mating activity reached a maximum level by 5 days after eclosion, whereas expression of complex courtship behavior was not yet fully developed at that time, suggesting that they are controlled by different mechanisms. When two males were maintained in the same vial, not only mating activity but also courtship complexity was reduced, demonstrating for the first time that preceding social experience, not current social conditions, influenced the complexity of male courtship. The effect of social experience was completely erased by 1 day of isolation, however, showing that social experience did not suppress or promote behavioral development itself. Rather, these results suggest that the observed effect of social experience was a plastic response of males that reduced investment in courtship effort by anticipating increased male–male competition.     

Exploring the influence of individual courtship behaviors on male mating success in a blow fly

Male courtship behavior and displays influence female mating decisions, and therefore affect mating success in a diverse range of organisms. While there is substantial evidence confirming that females prefer males who invest more in courtship, less attention has been paid to the relative importance of individual behaviors, and the discrete sequences of courtship that result in mating success. The small hairy maggot blow fly, Chrysomya varipes, performs stereotyped courtship behaviors, involving orienting, tapping, waving, arching, wing vibration and mounting. This study aimed to quantify male investment in specific courtship behaviors, and compare courtship investment and behavioral transitions between males who gained mating success (successful males) and those that did not (unsuccessful males). Our results show that mating success was influenced by the behaviors orienting, tapping, arching and mounting. Behavioral transitions revealed a distinct pattern of behaviors leading to a mating attempt, and some differences were observed between successful and unsuccessful males. Overall, our findings suggest that female mating decisions were based on differences in specific male courtship behaviors. This detailed observational study has quantified multiple courtship behaviors for the first time in C. varipes, and highlights the importance of considering multiple behavioral traits when exploring the influence of male courtship on mating success.     

Behavior and Cytogenetics of Fruitless in Drosophila Melanogaster: Different Courtship Defects Caused by Separate, Closely Linked Lesions

The fruitless (fru) courtship mutant was dissected into three defects of male reproductive behavior, which were separable as to their genetic etiologies by application of existing and newly induced chromosomal aberrations. fru itself is a small inversion [In(3R) 90C; 91B] on genetic and cytological criteria. Uncovering the fru distal breakpoint with deletions usually led to males with two of the fru courtship abnormalities: no copulation attempts with females (hence, behavioral sterility) and vigorous courtship among males, including the formation of "courtship chains." However, certain genetic changes involving region 91B resulted in males who formed courtship chains but who mated with females. Uncovering the fru proximal breakpoint led to males that passively elicit inappropriately high levels of courtship. This elicitation property was separable genetically from the sterility and chain formation phenotypes and provisionally mapped to the interval 89F-90F, which includes the fru proximal breakpoint. Behavioral sterility and chaining were also observed in males expressing certain abnormal genotypes, independent of the fru inversion. These included combinations of deficiencies, each with a breakpoint in 91B, and a transposon inserted in 91B.     

Linyphid spider courtship: Releaser and attractant functions of a contact sex pheromone

Experiments with male bowl-and-doily spiders (Frontinella communis) on methanol-washed and unwashed female webs indicate that the courtship of males is chemically released. Parallel experiments on the webs of females of another linyphiid species and on immature F. communis webs indicate that the releaser pheromone is species specific and that it is produced only by mature females. The pheromone that releases male courtship also functions as an attractant, changing the male's orientation from negatively to positively geotactic. The presence or absence of a female on the test web has only a slight effect on the male's behaviour.