Kathy B. Sheehan

STUDIES OF ESTERASE 6 IN DROSOPHILA MELANOGASTER . V. PROGENY PRODUCTION AND SPERM USE IN FEMALES INSEMINATED BY MALES HAVING ACTIVE OR NULL ALLELES

Despite more than a decade of research, the evolutionary significance of molecular variants of enzymes in Drosophila and other organisms is far from clear. Recently several workers have stressed that a new research strategy is needed which will integrate findings on the biochemistry and psysiology of enzyme variants with observed changes in allele frequencies in natural or artificial populations (Clarke, 1973; Koehn, 1978). Our laboratory is engaged in a study of the extent and adaptive significance of molecular variants at the autosomal, esterase 6 (Est 6) locus in D. melanogaster (Cochrane, 1976; Cochrane and Richmond, 1979a, 1979b). Recent work (Sheehan et al., 1979) has shown that the specific activity of esterase 6 (EST 6) reaches its maximum in adult males. (The notation Est 6 will be used throughout to denote the genetic locus; the notation EST 6 refers to the esterase 6 enzyme.) Activity shows a gradual increase through larval until early pupal stages, then remains constant until 12 h post eclosion. At this young adult stage, near the beginning of reproductive fertility, males have about twice the EST 6 activity of females. EST 6 activity remains at a constant low level throughout female adulthood, but rises to five times this level in males by three days post eclosion, and is maintained at these high levels in virgin males. The increase in esterase activity in adult males corresponds to the period when fertility and the frequency of mating is rising in males (Stromnaes and Kvelland, 1962; Fowler, 1973). Further support for the hypothesis that EST 6 has a role in male reproductive fitness is provided by its location in males. Sheehan et al. (1979) and Aronshtam and Kuzin (1974) have shown that the anterior ejaculatory duct (a secretory organ connecting the testes and accessory glands to the ejaculatory bulb) contains EST 6 in concentrations nearly as great as found in whole fly homogenates. Current studies indicate that this enzyme is depleted in males and transferred to females during copulation (Richmond et al., 1980). Does this polymorphic enzyme, transferred to females in significant amounts at copulation, have a clear adaptive role in Drosophila male and/or female reproductive fitness? The availability of D. melanogaster stocks homozygous for null variants of Est 6 provides an excellent tool for examining this question. Many components of the male reproductive phenotype are potentially affected by a reproductive enzyme. These include aspects of courtship, copulation, sperm transfer and storage, sperm viability and use by the female, as well as remating and sperm displacement among males. The absence of an enzyme which contributes to reproductive fitness could be expected to lower the productivity of matings, through an effect on one of these components. Comparisons of the single tnating productivity (progeny per female) of females inseminated by males homozygous for an active Est 6 allele and males homozygous for a null allele were undertaken to test this expectation. Females having a genetic background similar to the two male types, but homozygous for the sex-linked mutation forked, were used to control for female influence on productivity. The complex nature of daily productivity led us to develop a mathematical model

Studies of esterase 6 in Drosophila melanogaster. III. The developmental pattern and tissue distribution

Abstract Developmental changes in the activity of esterase 6 (E.C. 3.1.1.1, carboxylesterase) were studied in both sexes of Drosophila melanogaster . Since a number of esterases are active towards the substrate utilized (β-naphthyl acetate), a spectrophotometric assay largely specific for esterase 6 was developed. Low levels of esterase 6 activity can be detected in newly laid eggs and activity rises slowly until about 36 hr after adults eclose. At this time, esterase 6 activity in males rises sharply and stabilizes at a level 2–3 times that found in adult females. Esterase 6 activity in dissected, adult male tissues was determined by spectrophotometric assay or histochemical staining on starch and acrylamide gels. This analysis shows that esterase 6 in adult males is highly concentrated in the anterior ejaculatory duct. It is suggested that esterase 6 plays a role in male reproduction.

PCR detection and analysis of the free-living amoeba Naegleria in hot springs in Yellowstone and Grand Teton National Parks.

ABSTRACT Free-living thermotolerant amoebae pose a significant health risk to people who soak and swim in habitats suitable for their growth, such as hot springs. In this survey of 23 different hot springs in Yellowstone and Grand Teton National Parks, we used PCR with primer sets specific for Naegleria to detect three sequence types that represent species not previously described, as well as a fourth sequence type identified as the pathogen Naegleria fowleri.

Legionella species diversity in an acidic biofilm community in Yellowstone National Park

ABSTRACT Legionella species are frequently detected in aquatic environments, but their occurrence in extreme, acidic, geothermal habitats has not been explored with cultivation-independent methods. We investigated a predominately eukaryotic algal mat community in a pH 2.7 geothermal stream in Yellowstone National Park for the presence of Legionella and potential host amoebae. Our analyses, using PCR amplification with Legionella-specific primers targeting 16S rRNA genes, detected four known Legionella species, as well as Legionella sequences from species that are not represented in sequence databases, in mat samples and cultivated isolates. The nonrandom occurrence of sequences detected at lower (30°C) and higher (35 to 38°C) temperatures suggests that natural thermal gradients in the stream influence Legionella species distributions in this mat community. We detected only one sequence, Legionella micdadei, from cultivated isolates. We cultured and sequenced partial 18S rRNA gene regions from two potential hosts, Acanthamoeba and Euglena species.

A reevaluation of the role of cis-vaccenyl acetate, cis-vaccenol and esterase 6 in the regulation of mated female sexual attractiveness in Drosophila melanogaster

Abstract Gas-liquid chromatography was used to investigate the role of cis -vaccenyl acetate, cis -vaccenol and esterase 6 in the inhibition of male courtship in D. melanogaster . Results indicate that (1) cis -vaccenyl acetate is not converted to cis -vaccenol, (2) esterase 6 has no effect on the rate of cis -vaccenyl acetate loss from the reproductive tracts of mated females, (3) in vivo concentrations of cis -vaccenyl acetate transferred to females during copulation fall below effective courtship-inhibitory levels within 4 h, (4) cis -vaccenyl acetate is not translocated from the female reproductive tract to the abdominal cuticle, and that (5) mutant male flies that do not reduce the post-mating sexual attractiveness of females contain and transfer normal amounts of cis -vaccenyl acetate. Therefore, direct chemical analyses do not support the contention that esterase 6, cis -vaccenyl acetate and its hydrolysis product, cis -vaccenol, act in concert as an anti-aphrodisiac pheromone system.

Studies of esterase 6 inDrosophila melanogaster. VII. Remating times of females inseminated by males having active or null alleles

Esterase 6 (EST 6) inDrosophila melanogaster is a male reproductive enzyme transferred to females as a component of the seminal fluid [Richmond, R. C., Gilbert, D. G., Sheehan, K. B., Gromko, M. H., and Butterworth, F. W. (1980).Science207:1483–1485]. Here we report investigations into the relation between EST 6 and remating by females. EST 6 activity in a strain selected for decreased time to remating is increased over control levels. Inseminated females remated to males carrying null or active alleles show no differences in the timing of remating. However, females inseminated by EST 6-active males remate significantly sooner than females inseminated by null males. Interrupted copulation experiments demonstrate that the remating effect is not due to EST 6 alone but requires other components of the ejaculate. Other evidence suggests that sperm stored in the ventral receptacle respond to EST 6 levels and control remating time. As the first mate of a female who will remate, null-EST 6 males have, under laboratory conditions, a significantly higher fitness than males carrying active alleles. Thus the absence of null alleles of EST 6 in natural populations presents a dilemma suggesting that the remating effect of EST 6 may be balanced by other effects on reproduction.

Localization and longevity of seminal-fluid esterase 6 in mated female Drosophila melanogaster.

Abstract Esterase 6 is a non-specific carboxylesterase (EC 3.1.1.1) and is a component of the seminal fluid of Drosophila melanogaster. Previous studies showed the presence of this enzyme in seminal fluid is associated with a variety of effects on female reproductive behaviour and physiology. In the present experiments, male-derived esterase 6 was initially transferred into the females reproductive tract but was translocated within minutes of the beginning of mating into the haemolymph. Seminal-fluid esterase 6 was detected by Western blot analyses in mated-female haemolymph for as long as 4 days after mating. These results show that the enzyme remains in females for a period long enough to account for many of its behavioural and physiological effects.

Random Mating in Two Species of Drosophila

Data are presented which show a very high frequency (between 40% and 100%) of repeated mating by female Drosophila athabasca, Eastern A. No deviations from random mating, either as disproportionate representation of male genotypes in the mating population or as deviations from random choice of mates by members of the mating population, were evident in any of the D. athabasca or D. melanogaster populations sampled. The data do not rule out the possibility of frequency-dependent mate selection in a nonequilibrium population. We have compared the magnitudes of the deviations from random mating reported in the literature to the magnitude of the deviations we could have detected with our genetic system and sample sizes. Deviations from random mating might well have been found had they occurred to the same degree as previously detected.

Wolbachia Utilize Host Actin for Efficient Maternal Transmission in Drosophila melanogaster

Wolbachia pipientis is a ubiquitous, maternally transmitted bacterium that infects the germline of insect hosts. Estimates are that Wolbachia infect nearly 40% of insect species on the planet, making it the most prevalent infection on Earth. The bacterium, infamous for the reproductive phenotypes it induces in arthropod hosts, has risen to recent prominence due to its use in vector control. Wolbachia infection prevents the colonization of vectors by RNA viruses, including Drosophila C virus and important human pathogens such as Dengue and Chikungunya. Here we present data indicating that Wolbachia utilize the host actin cytoskeleton during oogenesis for persistence within and transmission between Drosophila melanogaster generations. We show that phenotypically wild type flies heterozygous for cytoskeletal mutations in Drosophila profilin (chic221/+ and chic1320/+) or villin (qua6-396/+) either clear a Wolbachia infection, or result in significantly reduced infection levels. This reduction of Wolbachia is supported by PCR evidence, Western blot results and cytological examination. This phenotype is unlikely to be the result of maternal loading defects, defects in oocyte polarization, or germline stem cell proliferation, as the flies are phenotypically wild type in egg size, shape, and number. Importantly, however, heterozygous mutant flies exhibit decreased total G-actin in the ovary, compared to control flies and chic221 heterozygous mutants exhibit decreased expression of profilin. Additionally, RNAi knockdown of profilin during development decreases Wolbachia titers. We analyze evidence in support of alternative theories to explain this Wolbachia phenotype and conclude that our results support the hypothesis that Wolbachia utilize the actin skeleton for efficient transmission and maintenance within Drosophila.

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine.

UNLABELLED Bacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4At) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4At and the TraJ/VirD4At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ΔCTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4Ats CTD to the VirE2 effector, although other VirD4At domains bound this substrate in vitro We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains. IMPORTANCE Bacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through the Escherichia coli pKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.