New reproductive anomalies in fruitless‐mutant Drosophila males: Extreme lengthening of mating durations and infertility correlated with defective serotonergic innervation of reproductive organs

Several features of male reproductive behavior are under the neural control of fruitless (fru) in Drosophila melanogaster. This gene is known to influence courtship steps prior to mating, due to the absence of attempted copulation in the behavioral repertoire of most types of fru‐mutant males. However, certain combinations of fru mutations allow for fertility. By analyzing such matings and their consequences, we uncovered two striking defects: mating times up to four times the normal average duration of copulation; and frequent infertility, regardless of the time of mating by a given transheterozygous fru‐mutant male. The lengthened copulation times may be connected with fru‐induced defects in the formation of a male‐specific abdominal muscle. Production of sperm and certain seminal fluid proteins are normal in these fru mutants. However, analysis of postmating qualities of females that copulated with transheterozygous mutants strongly implied defects in the ability of these males to transfer sperm and seminal fluids. Such abnormalities may be associated with certain serotonergic neurons in the abdominal ganglion in which production of 5HT is regulated by fru. These cells send processes to contractile muscles of the male's internal sex organs; such projection patterns are aberrant in the semifertile fru mutants. Therefore, the reproductive functions regulated by fruitless are expanded in their scope, encompassing not only the earliest stages of courtship behavior along with almost all subsequent steps in the behavioral sequence, but also more than one component of the culminating events. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 121–149, 2001     

What does the fruitless gene tell us about nature vs. nurture in the sex life of Drosophila?

The fruitless (fru) gene in Drosophila has been proposed to play a master regulator role in the formation of neural circuitries for male courtship behavior, which is typically considered to be an innate behavior composed of a fixed action pattern as generated by the central pattern generator. However, recent studies have shed light on experience-dependent changes and sensory-input-guided plasticity in courtship behavior. For example, enhanced male-male courtship, a fru mutant “hallmark,” disappears when fru-mutant males are raised in isolation. The fact that neural fru expression is induced by neural activities in the adult invites the supposition that Fru as a chromatin regulator mediates experience-dependent epigenetic modification, which underlies the neural and behavioral plasticity.     

<Emphasis Type="Italic">fruitless</Emphasis> alternative splicing and sex behaviour in insects: an ancient and unforgettable love story?

Courtship behaviours are common features of animal species that reproduce sexually. Typically, males are involved in courting females. Insects display an astonishing variety of courtship strategies primarily based on innate stereotyped responses to various external stimuli. In Drosophila melanogaster, male courtship requires proteins encoded by the fruitless (fru) gene that are produced in different sex-specific isoforms via alternative splicing. Drosophila mutant flies with loss-of-function alleles of the fru gene exhibit blocked male courtship behaviour. However, various individual steps in the courtship ritual are disrupted in fly strains carrying different fru alleles. These findings suggest that fru is required for specific steps in courtship. In distantly related insect species, various fru paralogues were isolated, which shows conservation of sex-specific alternative splicing and protein expression in neural tissues and suggests an evolutionary functional conservation of fru in the control of male-specific-courtship behaviour. In this review, we report the seminal findings regarding the fru gene, its splicing regulation and evolution in insects.     

Multiple pathways mediate the sex-peptide-regulated switch in female Drosophila reproductive behaviours

Male-derived sex-peptide (SP) induces profound changes in the behaviour of Drosophila females, resulting in decreased receptivity to further mating and increased egg laying. SP can mediate the switch in female reproductive behaviours via a G protein-coupled receptor, SPR, in neurons expressing fruitless, doublesex and pickpocket. Whether SPR is the sole receptor and whether SP induces the postmating switch in a single pathway has not, to our knowledge been tested. Here we report that the SP response can be induced in the absence of SPR when SP is ectopically expressed in neurons or when SP, transferred by mating, can access neurons through a leaky blood brain barrier. Membrane-tethered SP can induce oviposition via doublesex, but not fruitless and pickpocket neurons in SPR mutant females. Although pickpocket and doublesex neurons rely on G(o) signalling to reduce receptivity and induce oviposition, G(o) signalling in fruitless neurons is required only to induce oviposition, but not to reduce receptivity. Our results show that SP''s action in reducing receptivity and inducing oviposition can be separated in fruitless and doublesex neurons. Hence, the SP-induced postmating switch incorporates shared, but also distinct circuitry of fruitless, doublesex and pickpocket neurons and additional receptors.     

The benefits of male ejaculate sex peptide transfer in Drosophila melanogaster

The accessory gland protein (Acp) ejaculate molecules of male Drosophila melanogaster mediate sexual selection and sexual conflict at the molecular level. However, to date no studies have comprehensively measured the timing and magnitude of fitness benefits to males of transferring specific Acps. This is an important omission because without this information it is not possible to fully understand the strength and form of selection acting on adaptations such as Acps. Here, we measured the fitness benefits to males of ejaculate sex peptide (SP) transfer. SP is of interest because it is a candidate for mediating sexual conflict: its frequent receipt reduces female fitness. In single matings with virgin females SP is known to increase egg laying and decrease receptivity. Hence, we predicted that SP could: (i) boost a male’s absolute paternity by increasing offspring production and delaying female remating and/or (ii) boost relative paternity share. We tested these predictions using two different lines of SP‐lacking males, in both two‐mating and free‐mating assay conditions. SP transfer conferred higher absolute, but not relative, male reproductive success. In matings with virgin females, SP transfer increased mating productivity and delayed remating and hence the onset of sperm competition. In already mated females, SP transfer did not elevate absolute progeny production, but did increase intermating intervals and hence the period over which a male could gain paternity. Consistent with this, under free‐mating conditions over an extended period, we detected a ‘per‐mating’ fitness benefit for males transferring SP. These benefits are consistent with a role for SP in mediating conflict, with SP acting to maximize short‐term fitness benefits for males.     

A Small Subset of Fruitless Subesophageal Neurons Modulate Early Courtship in Drosophila

We show that a small subset of two to six subesophageal neurons, expressing the male products of the male courtship master regulator gene products fruitless^Male (fru^M), are required in the early stages of the Drosophila melanogaster male courtship behavioral program. Loss of fru^M expression or inhibition of synaptic transmission in these fru^M(+) neurons results in delayed courtship initiation and a failure to progress to copulation primarily under visually-deficient conditions. We identify a fru^M-dependent sexually dimorphic arborization in the tritocerebrum made by two of these neurons. Furthermore, these SOG neurons extend descending projections to the thorax and abdominal ganglia. These anatomical and functional characteristics place these neurons in the position to integrate gustatory and higher-order signals in order to properly initiate and progress through early courtship.     

Genotypes and their interaction effects on reproduction and mating‐induced immune activation in Drosophila melanogaster

Mating causes considerable alterations in female physiology and behaviour, and immune gene expression, partly due to proteins transferred from males to females during copulation. The magnitude of these phenotypic changes could be driven by the genotypes of males and females, as well as their interaction. To test this, we carried out a series of genotype‐by‐genotype (G × G) experiments using Drosophila melanogaster populations from two distant geographical locations. We expected lines to have diverged in male reproductive traits and females to differ in their responses to these traits. We examined female physiological and behavioural post‐mating responses to male mating traits, that is behaviour and ejaculate composition, in the short to mid‐term (48 hr) following mating. We then explored whether a sexually transferred molecule, sex peptide (SP), is the mechanism behind our observed female post‐mating responses. Our results show that the genotypes of both sexes as well as the interaction between male and female genotypes affect mating and post‐mating reproductive traits. Immune gene expression of three candidate genes increased in response to mating and was genotype‐dependent but did not show a G × G signature. Males showed genotype‐dependent SP expression in the 7 days following eclosion, but female genotypes showed no differential sensitivity to the receipt of SP. The two genotypes demonstrated clear divergence in physiological traits in short‐ to mid‐term responses to mating, but the longer‐term consequences of these initial dynamics remain to be uncovered.     

Regulation of Sex-Specific Selection of fruitless 5′ Splice Sites by transformer and transformer-2

In Drosophila melanogaster, the fruitless (fru) gene controls essentially all aspects of male courtship behavior. It does this through sex-specific alternative splicing of the fru pre-mRNA, leading to the production of male-specific fru mRNAs capable of expressing male-specific fru proteins. Sex-specific fru splicing involves the choice between alternative 5′ splice sites, one used exclusively in males and the other used only in females. Here we report that the Drosophila sex determination genes transformer (tra) and transformer-2 (tra-2) switch fru splicing from the male-specific pattern to the female-specific pattern through activation of the female-specific fru 5′ splice site. Activation of female-specific fru splicing requires cis-acting tra and tra-2 repeat elements that are part of an exonic splicing enhancer located immediately upstream of the female-specific fru 5′ splice site and are recognized by the TRA and TRA-2 proteins in vitro. This fru splicing enhancer is sufficient to promote the activation by tra and tra-2 of both a 5′ splice site and the female-specific doublesex (dsx) 3′ splice site, suggesting that the mechanisms of 5′ splice site activation and 3′ splice site activation may be similar.     

Courtship-stimulating volatile compounds from normal and mutant Drosophila

Volatile compounds from Drosophila melanogaster males and females dramatically affect male courtship behaviour. These substances, which have been extracted from flies of different ages and genotypes, have been analysed by gas chromatography (GC) and in behavioural assays. Extracts from virgin females and males have different gas chromatographic profiles, which may reflect the fact that extract from virgin females stimulates high levels of courtship between males over short distances, while extract from mature wild-type males does not affect sexual behaviour. However, volatile compounds from very young males or males expressing the fruitless (fru) mutation do stimulate courtship between males, and chromatographic profiles of young male and fru male extracts differ from the GC profile of extracts from mature wild-type males.     

In this issue of Fly

Previous studies in Drosophila have demonstrated that whether flies fight like males or females can be switched by selectively manipulating genes of the sex determination hierarchy in male and female nervous systems. Here we extend these studies by demonstrating that changing the sex of cholinergic neurons in male fruit fly nervous systems via expression of the transformer gene increases the levels of aggression shown by the flies without altering the way the flies fight. transformer manipulation in this way does not change phototaxis, geotaxis, locomotion or odor avoidance of the mutant males compared to controls. Cholinergic neurons must be feminized via this route during the late larval/early pupal stages of development to show the enhanced aggression phenotype. Other investigators have shown that this is the same time period during which sexually dimorphic patterns of behavior are specified in flies. Neurons that co-express fruitless and choline acetyl transferase are found in varying numbers within different clusters of fruitless-expressing neurons: together they make up approximately 10% of the pool of fruitless-expressing neurons in the brain and nerve cord.     

New tools for mitochondrial genetics of Chlamydomonas reinhardtii: manganese mutagenesis and cytoduction

Summary A novel and efficient genetic procedure is described for generating mitochondrial mutants of the green alga Chlamydomonas reinhardtii. The development of a mutagenesis procedure using manganese cations and the application of cytoduction techniques resulted in a combined approach for the generation and analysis of mitochondrial mutants. Although mitochondrial mutations are inherited in sexual crosses from the minus mating type parent, the cytoduction technique can be used to transfer mitochondrial mutations into recipient strains with different genetic backgrounds, irrespective of their mating type. Cytoduction allows the transfer of mitochondrial markers from diploid to haploid cells also, which is of great benefit since diploid cells do not germinate in C. reinhardtii. We report here the isolation and characterisation of eight mutants, which are resistant to the antibiotics myxothiazol and mucidin. The mutants all have point mutations in the mitochondrial gene for apocytochrome b. Using in vitro-amplified cytb gene fragments as probes for direct DNA sequencing, three different types of single base pair substitutions were revealed in all mutants tested. In particular, amino acid substitutions in the mutant apocytochrome b polypeptide have been identified at residues 129, 132 and 137, which have been implicated in forming part of an antibiotic-binding niche. The amino acid substitution at position 132 has not been so far described for mutant apocytochrome b in any other organism, prokaryotic or eukaryotic. The genetic approach presented here confirms C. reinhardtii as a model system that is unique among plant cells.