Sexual Experience Enhances Drosophila melanogaster Male Mating Behavior and Success

Competition for mates is a wide-spread phenomenon affecting individual reproductive success. The ability of animals to adjust their behaviors in response to changing social environment is important and well documented. Drosophila melanogaster males compete with one another for matings with females and modify their reproductive behaviors based on prior social interactions. However, it remains to be determined how male social experience that culminates in mating with a female impacts subsequent male reproductive behaviors and mating success. Here we show that sexual experience enhances future mating success. Previously mated D. melanogaster males adjust their courtship behaviors and out-compete sexually inexperienced males for copulations. Interestingly, courtship experience alone is not sufficient in providing this competitive advantage, indicating that copulation plays a role in reinforcing this social learning. We also show that females use their sense of hearing to preferentially mate with experienced males when given a choice. Our results demonstrate the ability of previously mated males to learn from their positive sexual experiences and adjust their behaviors to gain a mating advantage. These experienced-based changes in behavior reveal strategies that animals likely use to increase their fecundity in natural competitive environments

Regulation of the vapBC-1 Toxin-Antitoxin Locus in Nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) are human-adapted commensal bacteria that can cause a number of chronic mucosal infections, including otitis media and bronchitis. One way for these organisms to survive antibiotic therapy and cause recurrent disease is to stop replicating, as most antimicrobials target essential biosynthetic pathways. Toxin-antitoxin (TA) gene pairs have been shown to facilitate entry into a reversible bacteriostatic state. Characteristically, these operons encode a protein toxin and an antitoxin that associate following translation to form a nontoxic complex, which then binds to and regulates the cognate TA promoter. Under stressful conditions, the labile antitoxin is degraded and the complex disintegrates, freeing the stable toxin to facilitate growth arrest. How these events affected the regulation of the TA locus, as well as how the transcription of the operon was subsequently returned to its normal state upon resumption of growth, was not fully understood. Here we show that expression of the NTHi vapBC-1 TA locus is repressed by a complex of VapB-1 and VapC-1 under conditions favorable for growth, and activated by the global transactivator Factor for Inversion Stimulation (Fis) upon nutrient upshift from stationary phase. Further, we demonstrate for the first time that the VapC-1 toxin alone can bind to its cognate TA locus control region and that the presence of VapB-1 directs the binding of the VapBC-1 complex in the transcriptional regulation of vapBC-1

The Effects of Genetic and Environmental Factors on the Reproductive Behaviors of Drosophila melanogaster

The behavioral responses to varying environmental conditions and social interactions are multifaceted and require the coordination of complex neural circuits. Behaviors in animals are continuously affected by varying factors including, but not limited to, environment, genetic makeup, physiological state, or experience. Understanding the fundamental interactions between genotype, environment and phenotype is essential in understanding how evolutionary pressures shape behavior. In this dissertation, I used D. melanogaster to investigate various environmental and genetic components regulating the adult male and female mating behaviors. I explored the genetic components regulating different mating behaviors in adults by investigating the role of p24 proteins in male and female mating behaviors. p24 proteins comprise a family of type-I transmembrane proteins of ~24kDa that are present in yeast and plants as well as metazoans ranging from Drosophila to humans. These proteins are most commonly localized to the endoplasmic reticulum (ER)-Golgi interface and are incorporated in anterograde and retrograde transport vesicles. Drosophila melanogaster expresses nine p24 genes, grouped into four subfamilies. Based upon our mRNA and protein expression data, Drosophila p24 family members are expressed in a variety of tissues. To identify biological functions for particular Drosophila p24 proteins, we used RNA interference (RNAi) to reduce p24 expression. Ubiquitous reduction of most p24 genes resulted in complete or partial lethality during development. Reducing p24 levels in adults caused defects in female fecundity (egg laying) and reduced male fertility. We showed that reduced female fecundity is related to decreased neural p24 expression. These results provide the first genetic analysis of all p24 family members in a multi-cellular animal and indicate vital roles for Drosophila p24s in development and reproduction, implicating neural expression of p24s in the regulation of female behavior. Reproductive behaviors are also modified by social and environmental factors. Particularly, optimizing behavioral strategies that increase mating success are important, and prior sexual experiences as well as the current social environment can potentially affect an animal’s strategy for obtaining mates. Therefore, I investigated two separate scenarios, one where adult males were placed in a male-dense environment and their mating behaviors were quantified post male-male social interaction, and another scenario where the postmating behaviors of males were evaluated after achieving a successful sexual experience. Males reared in male-dense environments increased their mating durations with females, but do not affect the egg laying behavior or fecundity of the females. I found that although majority of the females remated with males within 24 hrs of the first mating, the prior social experience of the male did not influence the female remating latency. Males exposed to other males during early adulthood also did not have a competitive advantage against males raised in isolation. On the other hand, males with prior sexual experience changed their courtship dance and out competed sexually naïve males in the same mating arena. Females also preferred sexually experienced males by employing their auditory abilities to listen to the male’s modified courtship dance and responded positively by mating with them. Our findings have helped highlight the different behavioral responses shown by flies towards various environmental conditions

Gel shift substrates.^a

a<p>Sense strand sequence of the 50 bp substrate shown with substitutions underlined.</p

Competition binding of 50TIR and 50US by Vap proteins.

<p>Gel shift products from addition of 50US substrate into samples containing 50TIR substrate and either (A) VapBC-1 or (B) VapC-1 at a 150∶1 molar ratio of VapC-1 protein to DNA. VapB-1 and VapC-1 are at a 3∶1 molar ratio in the VapBC-1 samples, but VapC-1∶DNA molar ratios are reported since VapC-1 is the DNA binding protein and the actual amount of VapBC-1 complexes cannot be determined. The gels show the products of the following samples: 50TIR without protein (<i>lane 1</i>), protein with only 50TIR (<i>lane 2</i>), the addition of a 1∶1, 5∶1, 10∶1 or 50∶1 molar ratio of cold 50US:50TIR (<i>lanes 3–6</i>), and DNA only at 50∶1 molar ratio of 50US:50TIR (<i>lane 6</i>). The identity of each band is noted at the right of each gel. Each gel represents one of two independent experiments.</p

Bacteria and plasmids used in this study.

<p>Bacteria and plasmids used in this study.</p

VapB-1 targets VapC-1 to the <i>vapB-1</i> translation initiation region.

<p>Gel shift products from a titration of VapB-1 into samples following VapC-1 binding to (A) 50TIR or (B) 50US at a 100∶1 molar ratio of protein to DNA. The gels show the products of the following samples: VapC-1 and DNA without VapB-1 (<i>lane 1</i>), the addition of Vap B-1 at a 50, 100, 200 or 300 molar ratio with the DNA substrate (<i>lanes 2–5</i>), and VapB-1 only at a 300∶1 protein to DNA ratio (<i>lane 6</i>). The identity of each band is noted at the right of each gel. Each gel represents one of two independent experiments.</p

Fis, VapC-1 and VapBC-1 bind the <i>vapB-1</i> TIR.

<p>(A) The sequence of the 50TIR indicating the putative Fis binding site (<i>underline</i>) and the inverted repeat regions (<i>arrows</i>). (B) Gel shift products from samples containing 50TIR alone (<i>lane 1</i>) and 10, 30, 150 or 600 molar ratios of Fis to DNA. (C) Products from samples containing 50TIR alone (<i>lane 1</i>), VapC-1 (<i>lane 2</i>), VapC-1 followed by VapB-1 (<i>lane 3</i>), VapB-1 (<i>lane 4</i>), or the reconstituted VapBC-1 complex (<i>lane 5</i>). VapB-1 and VapC-1 are present in a 150∶1 molar ratio to 50TIR. In VapBC-1 samples, VapB-1 and VapC-1 are at a 3∶1 molar ratio, with a VapC-1∶DNA molar ratio of 150∶1. This ratio is reported since VapC-1 is the DNA binding protein and the actual amount of VapBC-1 complexes cannot be determined. The identity of each band is noted at the right of each gel. Each gel represents one of two independent experiments.</p

Model for the regulation of the <i>vapBC-1</i> locus.

<p>(A) During colonization under favourable conditions, the VapBC-1 complex binds to and autorepresses TA operon transcription. (B) Stress induces Lon and Clp proteases that degrade VapB-1, releasing active VapC-1 toxin. (C) The ribonuclease activity of VapC-1 facilitates a state of bacteriostasis, resulting in nonspecific antibiotic tolerance. (D) Upon improved conditions, Fis activates <i>vapBC-1</i> operon transcription, displacing any bound VapC-1. Fis levels decrease in early exponential growth, allowing the VapBC-1 complex to bind and restore transcriptional equilibrium.</p

DNase I protection of the <i>vapBC-1</i> locus control region by Fis and Vap proteins.

<p>(A) The ³²P-labeled sense strand of 153 bp DNA substrate containing <i>vapB-1</i> TIR and upstream sequence in the <i>vapBC-1</i> locus control region, is shown with numbers indicating the distance from the 5′-labeled end. The putative Fis site (<i>underline</i>), inverted repeat regions (<i>arrows</i>), <i>vapB-1</i> translation start ATG (<i>italics</i>), and G cleavage products (<i>*</i>) seen in (D, <i>lane G</i>) are noted. On each gel shown, a 10 bp DNA ladder (<i>lane M</i>), 153 bp substrate without protein (<i>lane 1</i>), and DNase I digest of the substrate (<i>lane 2</i>) are indicated. Gels show DNase I cleavage products from samples containing: (B) a Fis∶DNA molar ratio of 2∶1, 7.5∶1, 15∶1, or 30∶1 (<i>lanes 3–6</i>), (C) a VapBC-1∶DNA molar ratio of 2∶1, 7.5∶1, 15∶1, or 30∶1 (<i>lanes 3–6</i>) or 40∶1 VapB-1∶DNA (<i>lane 7</i>), and (D) VapC-1∶DNA molar ratio of 40∶1 or 80∶1 (<i>lanes 3</i> and <i>4</i>). Vertical bars indicate the DNase I footprint from protein binding. Arrows (<) in panel <i>D</i> indicate DNase I hypersensitive sites. The gels each represent one of two independent experiments.</p