The Drosophila TRPA channel, Painless, regulates sexual receptivity in virgin females.

Transient receptor potential (TRP) channels play crucial roles in sensory perception. Expression of the Drosophila painless (pain) gene, a homolog of the mammalian TRPA1/ANKTM1 gene, in the peripheral nervous system is required for avoidance behavior of noxious heat or wasabi. In this study, we report a novel role of the Pain TRP channel expressed in the nervous system in the sexual receptivity in Drosophila virgin females. Compared with wild-type females, pain mutant females copulated with wild-type males significantly earlier. Wild-type males showed comparable courtship latency and courtship index toward wild-type and pain mutant females. Therefore, the early copulation observed in wild-type male and pain mutant female pairs is the result of enhanced sexual receptivity in pain mutant females. Involvement of pain in enhanced female sexual receptivity was confirmed by rescue experiments in which expression of a pain transgene in a pain mutant background restored the female sexual receptivity to the wild-type level. Targeted expression of pain RNA interference (RNAi) in putative cholinergic or GABAergic neurons phenocopied the mutant phenotype of pain females. However, target expression of pain RNAi in dopaminergic neurons did not affect female sexual receptivity. In addition, conditional suppression of neurotransmission in putative GABAergic neurons resulted in a similar enhanced sexual receptivity. Our results suggest that Pain TRP channels expressed in cholinergic and/or GABAergic neurons are involved in female sexual receptivity.

Application of the gene search system to screen for longevity genes in Drosophila.

We have developed a strategy using Drosophila as a model system to identify genes that are crucial for extension of longevity. A collection of transgenic lines with a P-element based gene search (GS) vector containing UAS (Upstream Activating Sequence) was screened for longevity in combination with an hsp70 promoter-driven GAL4 transgene. Misexpression of the vector-flanking sequence was induced throughout the adult stage to assess its effects on the aging process rather than development. We showed that the longevity was greatly affected by GS inserts, and it was positively correlated with paraquat resistance. Of 646 GS inserts, we selected 23 inserts with relatively longer longevity for further molecular analysis. All of the misexpressed sequences matched either known genes or ESTs (Expressed Sequence Tags). Among 13 genes whose functions are already known or suggested, six were related to stress resistance or redox balance (DmGST2, hsp26, nla, and Drosophila homologs of mammalian TRX, GILT and POSH), suggesting the importance of stress resistance for the extension of longevity. This is the first demonstration that a systematic gain-of-function screen could efficiently detect longevity genes.

Phenotypic association of spontaneous ovulation and sexual receptivity in virgin females of Drosophila melanogaster mutants.

In Drosophila melanogaster females, mating stimulates ovulation/oviposition and switches off receptivity. To investigate the relationship between ovulation and receptivity, we searched for genetic variants in which ovulation occurs in virgins and characterized their behavioral phenotype. Among a collection of 333 P-element insertion lines, we identified eight lines that showed elevated ovulation in virgins. These females show ovipositor extrusion toward courting males, which is normally observed in mated females. To express the amount of rejection behavior, we defined the extrusion index (EI) as a percentage of time that each female extruded the ovipositor within the total time of being courted. There was a positive correlation between ovulation level and EI, suggesting that the two traits are physiologically associated. Genetic analysis of the variants revealed two regions on the third chromosome responsible for the phenotype.

Neural-specific overexpression of drosophila plenty of SH3s (DPOSH) extends the longevity of adult flies.

Extended longevity mutants are extremely useful to understand the molecular mechanism of longevity determination. Here we report identification and characterization of the Drosophila Plenty of SH3s (DPOSH) gene, a candidate that might be associated with the extended longevity phenotype. DPOSH encodes a protein containing a RING finger domain and four SH3 domains. We showed that neural-specific overexpression of DPOSH could extend the mean longevity of adult flies by 14% at 25 degrees C without affecting viability or morphology. In contrast, forced expression of DPOSH in developing imaginal discs produced various phenotypes including lethality and morphological defects such as loss of crossvein, notched wing, and disordered hair polarity. Puckered, a target gene of JNK/SAPK pathway, was activated by overexpression of DPOSH and the forced expression phenotypes were suppressed by introducing a mutation of Drosophila JNK (bsk) or JNKK (hep), suggesting that the JNK/SAPK signaling pathway is one of the critical elements in the determination of longevity.

The gene search system: its application to functional genomics in Drosophila melanogaster.

In Drosophila melanogaster, gain-of-function mutagenesis utilizing the GAL4-UAS system has been established, allowing identification of genes that may not be easily detectable by loss-of-function screening approaches. The conditional features of misexpression systems are especially useful for studying late-stage biological processes, such as those involving adult behavior or lifespan. The gene search system, incorporating a bidirectional misexpression vector, was used to screen for genes critical for longevity determination. We have identified several genes whose misexpression in adulthood extends the fly's lifespan. Phenotypic characterization of fly lines carrying a mis-expression vector, in conjunction with obtaining information about the genomic insertion sites, creates valuable resources for the systematic functional genomics in Drosophila.

Drosophila von Hippel-Lindau tumor suppressor complex possesses E3 ubiquitin ligase activity.

Mutations of the von Hippel-Lindau (VHL) tumor suppressor gene predispose individuals to a variety of human tumors, including renal cell carcinoma, hemangioblastoma of the central nervous system, and pheochromocytoma. Here we report on the identification and characterization of the Drosophila homolog of VHL. The predicted amino acid sequence of Drosophila VHL protein shows 29% identity and 44% similarity to that of human VHL protein. Biochemical studies have shown that Drosophila VHL protein binds to Elongins B and C directly, and via this Elongin BC complex, associates with Cul-2 and Rbx1. Like human VHL, Drosophila VHL complex containing Cul-2, Rbx1, Elongins B and C, exhibits E3 ubiquitin ligase activity. In addition, we provide evidence that hypoxia-inducible factor (HIF)-1alpha is the ubiquitination target of both human and Drosophila VHL complexes.

Disruption of the microsomal glutathione S-transferase-like gene reduces life span of Drosophila melanogaster.

Microsomal glutathione S-transferase-I (MGST-I) has been thought to be important for protecting the cell from oxidative damages and/or xenobiotics. We have previously identified the Microsomal glutathione S-transferase-like (Mgstl) gene, a Drosophila homologue of human MGST-I. To investigate the function of the enzyme using Drosophila as a model system, we examined the expression pattern of Mgstl during development, and generated loss-of-function mutants to assess its in-vivo function. Mgstl was expressed in all developmental stages. It is expressed ubiquitously with the highest expression in the larval fat body, an insect organ thought to be functionally corresponding to mammalian liver, while relatively low in the central nervous system. This tissue distribution is consistent with that of MGST-I in humans or Rats. Mgstl null mutants generated from a P element insertion line showed no obvious defects in morphology, indicating that it is not essential for the development. However, their life span was significantly reduced compared to control flies, suggesting that the MGSTL protein is involved in processes somehow contributing to aging. We found an Mgstl pseudogene, which is apparently derived through the reverse transcription of Mgstl mRNA and subsequent integration into the genome.

The gene search system. A method for efficient detection and rapid molecular identification of genes in Drosophila melanogaster.

We have constructed a P-element-based gene search vector for efficient detection of genes in Drosophila melanogaster. The vector contains two copies of the upstream activating sequence (UAS) enhancer adjacent to a core promoter, one copy near the terminal inverted repeats at each end of the vector, and oriented to direct transcription outward. Genes were detected on the basis of phenotypic changes caused by GAL4-dependent forced expression of vector-flanking DNA, and the transcripts were identified with reverse transcriptase PCR (RT-PCR) using the vector-specific primer and followed by direct sequencing. The system had a greater sensitivity than those already in use for gain-of-function screening: 64% of the vector insertion lines (394/613) showed phenotypes with forced expression of vector-flanking DNA, such as lethality or defects in adult structure. Molecular analysis of 170 randomly selected insertions with forced expression phenotypes revealed that 21% matched the sequences of cloned genes, and 18% matched reported expressed sequence tags (ESTs). Of the insertions in cloned genes, 83% were upstream of the protein-coding region. We discovered two new genes that showed sequence similarity to human genes, Ras-related protein 2 and microsomal glutathione S-transferase. The system can be useful as a tool for the functional mapping of the Drosophila genome.

Ovulation stimulating substances in Drosophila biarmipes males: their origin, genetic variation in the response of females, and molecular characterization.

Ovulation responses of Drosophila biarmipes females to an injection of methanolic extract from conspecific males vary with the strains of females. This strain difference seems to be controlled by a small number of autosomal genes, with low responsiveness being recessive. Strangely, all D. biarmipes strains show a high level of ovulation after mating. We pursued the reason for this discrepancy and found that D. biarmipes males produce two different substances with ovulation-inducing activity. One of them is derived from the accessory glands and effective in females of all strains. Another originates in the ejaculatory duct and is inactive in some strains. In an active HPLC fraction of the ejaculatory duct extract, we found a peptide consisting of 32 amino acids. Its C-terminal region has a striking similarity to the sex-peptide of D. melanogaster, but the N-terminal region was entirely different. Evolutionary implications of these findings are discussed.

Ectopic expression of sex-peptide in a variety of tissues in Drosophila females using the P[GAL4] enhancer-trap system.

Sex-peptide (SP), which is secreted by the accessory gland of Drosophila males, is transferred to the female during copulation, thereby reducing her sexual appetite (receptivity to males) and stimulating ovulation/oviposition. SP is known to be taken up into the hemolymph of mated females, but it is not clear whether there are two separate target tissues, for behavioral changes and ovulation or only one target for both responses. We have employed the GAL4-UAS system to express SP transgene constructs, both in different tissues and in different cellular components of virgin females. A cytoplasmic form of SP lacking a signal sequence did not evoke any responses, even when expressed ubiquitously. In contrast, a membrane-bound form of SP induced typical post-mating behavior, indicating that SP must be outside the cell in order to exert its biological effects. A total of 204 randomly selected P[GAL4] enhancer-trap lines were screened for their ability to induce SP responses in combination with the membrane-bound SP expressed under GAL4 control. Thirty-three lines were associated with both behavioral change and stimulated ovulation. No line was associated with only one of the two responses, implying that the SP target(s) mediating the two responses are either identical, very closely located, or present in two distinct tissues with a common set of genetic determinants. Western blot analysis of head, thorax, and abdominal extracts revealed that the biological activity was correlated with expression in the head fraction.

Purification and partial amino acid sequence of initiatorin, a prostatic endopeptidase of the silkworm, Bombyx mori.

We have purified initiatorin, a prostatic endopeptidase that initiates the protein-arginine degradation cascade in the spermatophore of Bombyx mori. Purification of the enzyme from spermatophores was monitored by measuring BAEE (N alpha-benzoyl-L-arginine-ethyl ester) hydrolyzing activity. Spermatophores were used as a source for this enzyme. Of several isoforms the major form (MW, 29 kDa) was purified over 200-fold. The N-terminal sequence of initiatorin showed strong homology with those of serine-type of endopeptidases.

Ectopic expression of sex peptide alters reproductive behavior of female D. melanogaster.

Sex peptide, a secreted component of the male accessory glands, has been shown to induce behavioral and physiological changes in mated Drosophila. We transformed flies with a hybrid gene containing an hsp70 promoter fused to a cDNA encoding sex peptide. Heat-induced ectopic expression of the peptide in transgenic virgin females altered their reproductive behavior, in the presence of courting males, to that observed in mated females. This demonstrates that the peptide is functional as expected. Time course studies revealed that the behavioral change appeared earlier than the stimulated ovulation. We have also introduced a modified sex peptide gene that is driven by the yp1 enhancer, conferring expression in adult females, and shown that these flies refuse mating constitutively in the presence of courting males and lay unfertilized eggs at the rate of mated females.

A male accessory gland peptide that regulates reproductive behavior of female D. melanogaster.

The adult male accessory glands of D. melanogaster synthesize and secrete a peptide that represses female sexual receptivity and stimulates oviposition. Normally, this peptide is transferred to females during copulation; however, the peptide shows the same biological activity after purification and subsequent injection into the abdominal cavity of female virgins. Amino acid sequencing of the purified peptide and oligonucleotide-directed cDNA cloning established that the peptide consists of 36 amino acids. It appears to be synthesized as a precursor with a hydrophobic signal sequence of 19 residues at its N-terminal end. The precursor peptide is encoded by a short mRNA that accumulates exclusively in the male accessory gland. The gene has been localized by in situ hybridization to polytene chromosomes at 70A.

Individual analysis of age-associated changes in reproductive activity and lifespan of Drosophila virilis.

In the TK strain of Drosophila virilis, age-associated changes in reproductive activities (copulating activity and fertility in males and egg-laying activity in females) and lifespan were examined in individual flies. The mean lifespan of individually aged flies was about 11 weeks for both males and females. Copulating activity was maximum in two-week old males, then decreased gradually and finally ended at the age of 13 weeks at which about half of males were still surviving. Females also had a peak in egg-laying activity at the age of two weeks while no eggs were laid by females older than ten weeks, although more than half of females were alive at the stage. Analyses of the relationship between reproductive activity and lifespan of individual flies revealed that shorter lived males exhibited a higher copulating activity in the early stage of their lives than longer lived males. In males whose lifespan was less than ten weeks the reproductive period increased with the lifespan while the post-reproductive period was almost constant (one to two weeks). In males living longer than ten weeks, the reproductive period remained constant (about eight weeks) while the post-reproductive period increased in parallel with the total lifespan. Similar tendencies were also observed in the egg-laying activity of female flies.

Effect of mating status on Drosophila virilis lifespan.

Mating status markedly affected the lifespan of Drosophila virilis flies but the effects of virginity on lifespan were different between the sexes. In all cases lengthening of female lifespan and shortening of male lifespan were observed as an effect of virginity. Lifespan was also affected either by the population density or by the sex ratio per culture. At a sex ratio (proportion of females) of 0.5, lifespan was constant in both sexes irrespective of the population density and females lived significantly longer than males. At high (0.9) and low (0.1) sex ratios mean lifespan did not differ between sexes, but it was longer at high sex ratios than at low ones. A negative correlation between mean lifespan and the number of males per vial was revealed for both sexes. Effects of mating on egg-laying activity were also analyzed. Although the number of eggs laid during the first 20 days period after eclosion was greater in mated females than in unmated virgin females, lifetime egg production was not always larger in the former than in the latter. No particular relationship between egg-laying activity and female lifespan was proven. The lifespan of D. virillis flies seemed to be determined by complex interactions between both sexes, in which the sexual activity of males may play the most important role.