Timothy L. Karr

A newly discovered bacterium associated with parthenogenesis and a change in host selection behavior in parasitoid wasps.

The symbiotic bacterium Wolbachia pipientis has been considered unique in its ability to cause multiple reproductive anomalies in its arthropod hosts. Here we report that an undescribed bacterium is vertically transmitted and associated with thelytokous parthenogenetic reproduction in Encarsia, a genus of parasitoid wasps. Although Wolbachia was found in only one of seven parthenogenetic Encarsia populations examined, the “Encarsia bacterium” (EB) was found in the other six. Among seven sexually reproducing populations screened, EB was present in one, and none harbored Wolbachia. Antibiotic treatment did not induce male production in Encarsia pergandiella but changed the oviposition behavior of females. Cured females accepted one host type at the same rate as control females but parasitized significantly fewer of the other host type. Phylogenetic analysis based on the 16S rDNA gene sequence places the EB in a unique clade within the Cytophaga-Flexibacter-Bacteroid group and shows EB is unrelated to the Proteobacteria, where Wolbachia and most other insect symbionts are found. These results imply evolution of the induction of parthenogenesis in a lineage other than Wolbachia. Importantly, these results also suggest that EB may modify the behavior of its wasp carrier in a way that enhances its transmission.

Genomic and functional evolution of the Drosophila melanogaster sperm proteome

In addition to delivering a haploid genome to the egg, sperm have additional critical functions, including egg activation, origination of the zygote centrosome and delivery of paternal factors. Despite this, existing knowledge of the molecular basis of sperm form and function is limited. We used whole-sperm mass spectrometry to identify 381 proteins of the Drosophila melanogaster sperm proteome (DmSP). This approach identified mitochondrial, metabolic and cytoskeletal proteins, in addition to several new functional categories. We also observed nonrandom genomic clustering of sperm genes and underrepresentation on the X chromosome. Identification of widespread functional constraint on the proteome indicates that sexual selection has had a limited role in the overall evolution of D. melanogaster sperm. The relevance of the DmSP to the study of mammalian sperm function and fertilization mechanisms is demonstrated by the identification of substantial homology between the DmSP and proteins of the mouse axoneme accessory structure.

Stage-specific expression profiling of Drosophila spermatogenesis suggests that meiotic sex chromosome inactivation drives genomic relocation of testis-expressed genes.

In Drosophila, genes expressed in males tend to accumulate on autosomes and are underrepresented on the X chromosome. In particular, genes expressed in testis have been observed to frequently relocate from the X chromosome to the autosomes. The inactivation of X-linked genes during male meiosis (i.e., meiotic sex chromosome inactivation—MSCI) was first proposed to explain male sterility caused by X-autosomal translocation in Drosophila, and more recently it was suggested that MSCI might provide the conditions under which selection would favor the accumulation of testis-expressed genes on autosomes. In order to investigate the impact of MSCI on Drosophila testis-expressed genes, we performed a global gene expression analysis of the three major phases of D. melanogaster spermatogenesis: mitosis, meiosis, and post-meiosis. First, we found evidence supporting the existence of MSCI by comparing the expression levels of X- and autosome-linked genes, finding the former to be significantly reduced in meiosis. Second, we observed that the paucity of X-linked testis-expressed genes was restricted to those genes highly expressed in meiosis. Third, we found that autosomal genes relocated through retroposition from the X chromosome were more often highly expressed in meiosis in contrast to their X-linked parents. These results suggest MSCI as a general mechanism affecting the evolution of some testis-expressed genes.

Intracellular sperm/egg interactions in Drosophila: a three-dimensional structural analysis of a paternal product in the developing egg.

During fertilization in Drosophila, a single 1.75 mm long sperm enters the egg through the anterior end. Using a sperm-specific monoclonal antibody and indirect immunofluorescence of whole fixed eggs and embryos, intracellular interactions between the sperm and egg are examined as they occur inside the fertilized egg. The sperm nucleus remains attached to the axoneme throughout the entire process of fertilization including the stages of pronuclear maturation, pronuclear fusion and karyogamy indicating an intracellular function for the sperm during these stages. Optical sections and three-dimensional reconstructions of whole mount specimens reveal that a stereotypically folded structure forms during fertilization strongly suggesting that this structure positions the male pronucleus in the proper region of the egg in anticipation of pronuclear fusion. This, and the appearance of regional structural changes in the sperm upon entry suggests that sperm are localized via specific interactions with the maternal cytoplasm. Following fertilization and during the ensuing cleavage divisions, the sperm remains intact and localized at the anterior end of the egg. During cellular blastoderm formation the sperm tail is sequestered into the anterior yolk area where it continues to persist well into embryonic development. This structural analysis identifies intracellular sperm/egg interactions as an important aspect of fertilization, and provides a unique model system for the study of sperm/egg interactions not presently available in other systems.

Cytological analysis of fertilization and early embryonic development in incompatible crosses of Drosophila simulans.

Cytoplasmic incompatibility (CI) is a unique form of male sterility found in numerous insect species that harbor a bacterial endosymbiont Wolbachia. CI is characterized by severe reduction in the progeny produced when infected males are crossed to uninfected females. The reduction in progeny correlates with developmental defects that arise during and immediately following fertilization, suggesting that sperm function is disrupted. To investigate the nature of the cellular defects associated with CI, fertilization and early embryonic development were examined in normal and incompatible crosses of Drosophila simulans using anti-sperm, anti-tubulin and anti-chromatin antibodies. Although pleiotropic, defects associated with CI can be classified into five broad categories: (1) sperm defects in the egg; (2) aberrant morphology of the mitotic apparatus; (3) defects in chromatin structure; (4) proliferation of centrosomes in the absence of nuclear division; and (5) loss of mitotic synchrony. Although mitosis and chromosome behavior are severely disrupted in CI crosses during early development, centrosome duplication and migration appear to continue unabated. The available cytological data suggest that the primary defects observed in incompatible crosses are due to defects in chromosome replication/segregation and in associated centrosome/microtubule-based processes.

Heads or Tails: Host-Parasite Interactions in the Drosophila-Wolbachia System

ABSTRACT Wolbachia strains are endosymbiotic bacteria typically found in the reproductive tracts of arthropods. These bacteria manipulate host reproduction to ensure maternal transmission. They are usually transmitted vertically, so it has been predicted that they have evolved a mechanism to target the hosts germ cells during development. Through cytological analysis we found that Wolbachia strains display various affinities for the germ line of Drosophila. Different Wolbachia strains show posterior, anterior, or cortical localization in Drosophila embryos, and this localization is congruent with the classification of the organisms based on the wsp (Wolbachia surface protein) gene sequence. This embryonic distribution pattern is established during early oogenesis and does not change until late stages of embryogenesis. The posterior and anterior localization of Wolbachia resembles that of oskar and bicoid mRNAs, respectively, which define the anterior-posterior axis in the Drosophila oocyte. By comparing the properties of a single Wolbachia strain in different host backgrounds and the properties of different Wolbachia strains in the same host background, we concluded that bacterial factors determine distribution, while bacterial density seems to be limited by the host. Possible implications concerning cytoplasmic incompatibility and evolution of strains are discussed.

Origin and neofunctionalization of a Drosophila paternal effect gene essential for zygote viability

BACKGROUND Although evolutionary novelty by gene duplication is well established, the origin and maintenance of essential genes that provide entirely new functions (neofunctionalization) is still largely unknown. Drosophila is a good model for the search of genes that are young enough to allow deciphering the molecular details of their evolutionary history. Recent years have seen increased interest in genes specifically required for male fertility because they often evolve rapidly. A special class of genes affecting male fertility, the paternal effect genes, have also become a focus of study to geneticists and reproductive biologists interested in fertilization and sperm-egg interactions. RESULTS Using molecular genetics and the annotated Drosophila melanogaster genome, we identified CG14251 as the Drosophila paternal effect gene, ms(3)K81 (K81). This assignment was subsequently confirmed by P-element rescue of K81. A search for orthologous K81 sequences revealed that the distribution of K81 is surprisingly restricted to the 9 species comprising the melanogaster subgroup. Phylogenetic analyses indicate that K81 arose through duplication, most likely retroposition, of a ubiquitously expressed gene before the radiation of the melanogaster subgroup, followed by a period of rapid divergence and acquisition of a critical male germline-specific function. Interestingly, K81 has adopted the expression profile of a flanking gene suggesting that transcriptional coregulation may have been important in the neofunctionalization of K81. CONCLUSION We present a detailed case history of the origin and evolution of a new essential gene and, in so doing, provide the first molecular identification of a Drosophila paternal effect gene, ms(3)K81 (K81).

The Drosophila melanogaster sperm proteome-II (DmSP-II)

Advances in mass spectrometry technology, high-throughput proteomics and genome annotations have resulted in significant increases in our molecular understanding of sperm composition. Using improved separation and detection methods and an updated genome annotation, a re-analysis of the Drosophila melanogaster sperm proteome (DmSP) has resulted in the identification of 956 sperm proteins. Comparative analysis with our previous proteomic dataset revealed 766 new proteins and an updated sperm proteome containing a total of 1108 proteins, termed the DmSP-II. This expanded dataset includes additional proteins with predicted sperm functions and confirms previous findings concerning the genomic organization of sperm loci. Bioinformatic and protein network analyses demonstrated high quality and reproducibility of proteome coverage across three replicate mass spectrometry runs. The use of whole-cell proteomics in conjunction with characterized phenotypes, functional annotations and pathway information has advanced our systems level understanding of sperm proteome functional networks.

Organization of Wolbachia pipientis in the Drosophila fertilized egg and embryo revealed by an anti-Wolbachia monoclonal antibody

Cytoplasmic incompatibility (CI) in Drosophila is related to the presence of Wolbachia, an intracellular microorganism found in many species of insects. In order to study the intracellular localization of Wolbachia in eggs and embryos, we have purified the bacteria from fly embryos and subsequently generated a monoclonal antibody (Mab Wol-1) specific for Wolbachia. Indirect immunofluorescence staining using Wol-1 reveals that during mitosis, Wolbachia are localized near spindle poles and centrosomes. Double label immunofluorescence experiments using anti-tubulin and anti-Wolbachia antibodies show that Wolbachia co-localize with centrosomal microtubules throughout the cell cycle. Direct interactions between the bacteria and centrosome-organized microtubules are implied from seven observations: (1) throughout the mitotic cycle, the position and movement of Wolbachia precisely mimic the behavior of the centrosome and apparently associated with centrosome-organized microtubules; (2) Wolbachia segregate equally to each spindle pole during mitosis; (3) Wolbachia do not associate with spindle microtubules during mitosis; (4) Wolbachia located in the egg cortex localize to the domains of cytoplasm organized by microtubules during blastoderm formation; (5) polar body nuclei that lack centrosomes but contain associated microtubules do not contain Wolbachia; (6) Wolbachia no longer associated with yolk nuclei, following differentiation and loss of centrosomes; (7) during pole cell formation, Wolbachia co-localize with the centrosome on the apical side of the nucleus as pole cells form. Quantitative data indicates that no Wolbachia growth occurs during the preblastoderm period even though rapid nuclear, and subsequent cellular, proliferation takes place during this same period. This indicates that Wolbachia are under strict growth regulation by the host suggesting that host factors play a role in regulating growth of Wolbachia in the egg. Further cellular and molecular studies of the extensive, global interactions between host and symbiont observed in this egg should provide important new insights into the evolution of host/symbiosis and the cell biology of cytoplasmic incompatibility.

Sperm Proteomics Reveals Intensified Selection on Mouse Sperm Membrane and Acrosome Genes

Spermatozoa are a focal point for the impact of sexual selection due to sperm competition and sperm-female interactions in a wide range of sexually reproducing organisms. In-depth molecular investigation of the ramifications of these selective regimes has been limited due to a lack of information concerning the molecular composition of sperm. In this study, we utilize three previously published proteomic data sets in conjunction with our whole mouse sperm proteomic analysis to delineate cellular regions of sperm most impacted by positive selection. Interspecific analysis reveals robust evolutionary acceleration of sperm cell membrane genes (which include genes encoding acrosomal and sperm cell surface proteins) relative to other sperm genes, and evidence for positive selection in approximately 22% of sperm cell membrane components was obtained using maximum likelihood models. The selective forces driving the accelerated evolution of these membrane proteins may occur at a number of locations during sperm development, maturation, and transit through the female reproductive tract where the sperm cell membrane and eventually the acrosome are exposed to the extracellular milieu and available for direct cell-cell interactions.