The pathology of embryo death caused by the male-killing Spiroplasma bacterium in Drosophila nebulosa

BACKGROUND: Inherited bacteria that kill male offspring, male-killers, are known to be common in insects, but little is understood about the mechanisms used by male-killing bacteria to kill males. In this paper we describe the tempo and changes that occur during male-killing by Spiroplasma bacteria in the host Drosophila nebulosa. RESULTS: Spiroplasma infected D. nebulosa males were developmentally retarded from 6–8 h into embryonic development at 25°C, and arrested at between stages 12 and 13 of embryogenesis (10–12 h). Dying males were characterized by a failure to form segments, and ultimately disintegration of the normal oval embryonic shape. Prior to death, dying males exhibited widespread apoptosis, as testified by TUNEL staining. CONCLUSION: The Spiroplasma kills male Drosophila in a narrow developmental period, shortly after the formation of the host dosage compensation complex that is required for male-killing. Male death is preceded by widespread apoptosis, but it is uncertain if this is primary or secondary apoptosis

Cytoplasmic incompatibility: a comparative study of Wolbachia strains in Drosophila

The α-proteobacterium Wolbachia pipientis is a very common cytoplasmic symbiont of insects, crustaceans, mites and filarial nematodes. To enhance its transmission, Wolbachia has evolved a large scale of host manipulations: parthenogenesis induction, feminization and male killing. Wolbachia’ s most common effect is a crossing incompatibility between infected males and uninfected females termed Cytoplasmic Incompatibility (CI). Little is known about the genetics and biochemistry of these symbionts because of their fastidious requirements. Such inherited microorganisms are thought to have been major factors for the evolution of sex determination and speciation. Wolbachia isolates are also of interest as vectors for the modification of wild insect populations and biological pest control. The means by which Wolbachia induces CI are currently unknown. However, there is a general consensus that Wolbachia somehow modifies sperm during spermatogenesis and this modification has to be rescued by the same bacterial strain in the egg, for normal development to proceed. In any other case, paternal chromosomes behave abnormally after fertilization and the embryo dies due to asynchronous mitoses. This thesis approached the mechanism of CI by different aspects. Firstly, in preparation for complete genome sequencing project, I estimated the genome sizes of two Wolbachia strains using pulse-field gel electrophoresis and developed a method to purify Wolbachia chromosomal DNA. Secondly, I demonstrated that Wolbachia infections could be simply established, maintained and stored in vitro using standard tissue culture techniques. Finally and most importantly, I monitored the bacterium during germ line development of the fly, where Wolbachia exerts its action. Particularly, I described the growth and distribution of Wolbachia during spermatogenesis, oogenesis and embryogenesis in several different host/symbiont genetic combinations in Drosophila species. Considerable intra- and inter-strain variation in Wolbachia density and tissue distribution was observed. To dissect the relative contribution of host and symbiont factors to the expression of CI I compared the properties of a single Wolbachia strain in different host genetic backgrounds and different Wolbachia strains in the same host background. These experiments demonstrated that the ability to express CI is an intrinsic Wolbachia trait and is not determined by host factors. The level of sperm modification in those lines harboring bacteria capable of modifying sperm however is influenced by host genetic background. Finally, numbers of infected sperm cysts are positively correlated with CI levels. From an evolutionary point of view, it seems that host-symbiont co-evolution is leading to low CI levels, high maternal transmission and low fitness cost of the infection. These three factors are probably linked through a unique feature: bacterial density. Ultimately, a complete understanding of CI will include unraveling the deeper relationships between developmental dynamics of infection and the interplay of host genetic backgrounds with Wolbachia.Η Wolbachia ανήκει στην α-υποομάδα των πρωτεοβακτηρίων. Είναι ένα υποχρεωτικά ενδοκυττάριο, μητρικά κληρονομούμενο βακτήριο, το οποίο μολύνει αρθρόποδα και νηματώδεις. Προκαλεί μία σειρά αναπαραγωγικών ανωμαλιών στους ξενιστές της, όπως παρθενογένεση, θηλυκοποίηση, θανάτωση αρσενικών εμβρύων και κυτταροπλασματική ασυμβατότητα, οι οποίες ευνοούν τη μετάδοση και εξάπλωσή της. Η Wolbachia την τελευταία δεκαετία έχει προσελκύσει το ενδιαφέρον των ερευνητών, λόγω του ρόλου της σε βασικές εξελικτικές διαδικασίες, όπως η συμβίωση και η ειδογένεση. Επιπρόσθετα, η Wolbachia θα μπορούσε να χρησιμοποιηθεί ως εργαλείο για το γενετικό μετασχηματισμό εντόμων, τα οποία προκαλούν βλάβες στον τομέα της γεωργίας και της υγείας. Η καλύτερα μελετημένη εμπλοκή της Wolbachia στην αναπαραγωγή των εντόμων, είναι η κυτταροπλασματική ασυμβατότητα. Στην πιο απλή της μορφή έχει ως αποτέλεσμα τη θνησιμότητα των εμβρύων, που προέρχονται από τη διασταύρωση ενός μολυσμένου αρσενικού με ένα μη μολυσμένο θηλυκό. Αρκετοί παράγοντες, γενετικοί και περιβαλλοντικοί, έχουν αναγνωριστεί μέχρι σήμερα, οι οποίοι επηρεάζουν τα επίπεδα της ασυμβατότητας (τον αριθμό των αυγών που δεν εκκολάπτονται). Παρόλα αυτά ο μοριακός μηχανισμός της παραμένει άγνωστος. Η Wolbachia κατά γενική παραδοχή τροποποιεί τα πατρικά χρωμοσώματα κατά τη διάρκεια της σπερματογένεσης. Αυτή η τροποποίηση μπορεί να εξουδετερωθεί μόνο αν το ίδιο βακτηριακό στέλεχος είναι παρών στο έμβρυο. Σε διαφορετική περίπτωση διακόπτεται η ανάπτυξη του εμβρύου λόγω της μη κανονικής συμπεριφοράς των πατρικών χρωμοσωμάτων στις πρώτες μιτωτικές διαιρέσεις, που ακολουθούν τη γονιμοποίηση. Μοριακές και βιοχημικές μικροβιολογικές μελέτες, οι οποίες θα βοηθούσαν στην κατανόηση της δράσης του βακτηρίου, είναι δύσκολες λόγω της αδυναμίας του να καλλιεργηθεί σε θρεπτικά μέσα, χωρίς κύτταρα. Για αυτόν το λόγο, η παρούσα διατριβή εστιάστηκε από τη μία στην ανάπτυξη εργαλείων για τον ευκολότερο χειρισμό του βακτηρίου και από την άλλη στη μελέτη της συμπεριφοράς του κατά τη διάρκεια της ανάπτυξης της γαμετικής σειράς της μύγας. Όσον αφορά στον πρώτο τομέα αναπτύχθηκε μία μέθοδος απομόνωσης γενωμικού DNA από Wolbachia, απαλλαγμένου σε ικανοποιητικό ποσοστό από DNA ξενιστή, καθώς και μία μέθοδος in vitro μόλυνσης κυτταρικών σειρών, με το εν λόγω βακτήριο. Η δεύτερη διάσταση της διατριβής αυτής αφορά στη συγκριτική κυτταρολογική μελέτη μιας πληθώρας στελεχών, που εκφράζουν διαφορετικά επίπεδα ασυμβατότητας. Πιο συγκεκριμένα, μελετήθηκε ο πολλαπλασιασμός και η κατανομή του βακτηρίου κατά τη διάρκεια της ανάπτυξης της γαμετικής σειράς της μύγας, σε διαφορετικούς γενετικούς συνδυασμούς ξενιστή-παρασίτου. Παρατηρήθηκε αξιόλογη ετερογένεια, τόσο ανάμεσα στο ίδιο στέλεχος, όσο και ανάμεσα στα διαφορετικά στελέχη. Από τη σύγκριση της συμπεριφοράς ενός συγκεκριμένου βακτηριακού στελέχους σε διαφορετικά γενετικά υπόβαθρα ξενιστή και διαφορετικών στελεχών στον ίδιο ξενιστή, βγαίνουν ενδιαφέροντα συμπεράσματα για τη φύση των παραγόντων, που επηρεάζουν τα επίπεδα της ασυμβατότητας και γενικότερα για την εξέλιξη των σχέσεων ξενιστή-παρασίτου. Έτσι φαίνεται η ικανότητα πρόκλησης ασυμβατότητας να είναι εγγενής ιδιότητα του βακτηρίου, με κάποια στελέχη να την έχουν χάσει. Στα στελέχη που τη διαθέτουν, η ένταση του φαινοτύπου είναι ανάλογη της ποσότητας των βακτηρίων στα σπερματικά κύτταρα, η οποία ελέγχεται από τον ξενιστή. Από εξελικτική σκοπιά, η συνεξέλιξη ξενιστή-παρασίτου φαίνεται να οδηγεί σε χαμηλά επίπεδα ασυμβατότητας, υψηλή μητρική μετάδοση και χαμηλό κόστος της μόλυνσης, παράγοντες αλληλένδετοι με την πυκνότητα των βακτηρίων στους ιστούς-στόχους. Xρειάζεται πλήρης κατανόηση του μοριακού μηχανισμού όμως, για να διαλευκανθούν οι βαθύτερες σχέσεις μεταξύ της αναπτυξιακής δυναμικής των μολύνσεων και της αλληλεπίδρασης ξενιστή-παρασίτου στη ρύθμιση των βακτηριακών αριθμών

Polycomb Repressor Complex 2 in Genomic Instability and Cancer

Polycomb repressor complexes PRC1 and PRC2 regulate chromatin compaction and gene expression, and are widely recognized for their fundamental contributions to developmental processes. Herein, we summarize the existing evidence and molecular mechanisms linking PRC-mediated epigenetic aberrations to genomic instability and malignancy, with a particular focus on the role of deregulated PRC2 in tumor suppressor gene expression, the DNA damage response, and the fidelity of DNA replication. We also discuss some of the recent advances in the development of pharmacological and dietary interventions affecting PRC2, which point to promising applications for the prevention and management of human malignancies

Characterization of Wolbachia Host Cell Range via the In Vitro Establishment of Infections

Maternally transmitted bacteria of the genus Wolbachia are obligate, intracellular symbionts that are frequently found in insects and cause a diverse array of reproductive manipulations, including cytoplasmic incompatibility, male killing, parthenogenesis, and feminization. Despite the existence of a broad range of scientific interest, many aspects of Wolbachia research have been limited to laboratories with insect-rearing facilities. The inability to culture these bacteria outside of the invertebrate host has also led to the existing bias of Wolbachia research toward infections that occur in host insects that are easily reared. Here, we demonstrate that Wolbachia infections can be simply established, stably maintained, and cryogenically stored in vitro using standard tissue culture techniques. We have examined Wolbachia host range by introducing different Wolbachia types into a single tissue culture. The results show that an Aedes albopictus (Diptera: Culicidae) cell line can support five different Wolbachia infection types derived from Drosophila simulans (Diptera: Drosophilidae), Culex pipiens (Culicidae), and Cadra cautella (Lepidoptera: Phycitidae). These bacterial types include infection types that have been assigned to two of the major Wolbachia clades. As an additional examination of Wolbachia host cell range, we demonstrated that a Wolbachia strain from D. simulans could be established in host insect cell lines derived from A. albopictus, Spodoptera frugiperda (Lepidoptera: Noctuidae), and Drosophila melanogaster. These results will facilitate the development of a Wolbachia stock center, permitting novel approaches for the study of Wolbachia infections and encouraging Wolbachia research in additional laboratories

Polycomb-mediated silencing of miR-8 is required for maintenance of intestinal stemness in Drosophila melanogaster

Abstract Balancing maintenance of self-renewal and differentiation is a key property of adult stem cells. The epigenetic mechanisms controlling this balance remain largely unknown. Herein, we report that the Polycomb Repressive Complex 2 (PRC2) is required for maintenance of the intestinal stem cell (ISC) pool in the adult female Drosophila melanogaster. We show that loss of PRC2 activity in ISCs by RNAi-mediated knockdown or genetic ablation of the enzymatic subunit Enhancer of zeste, E(z), results in loss of stemness and precocious differentiation of enteroblasts to enterocytes. Mechanistically, we have identified the microRNA miR-8 as a critical target of E(z)/PRC2-mediated tri-methylation of histone H3 at Lys27 (H3K27me3) and uncovered a dynamic relationship between E(z), miR-8 and Notch signaling in controlling stemness versus differentiation of ISCs. Collectively, these findings uncover a hitherto unrecognized epigenetic layer in the regulation of stem cell specification that safeguards intestinal homeostasis

Veneti et al_Heredity_raw data_30may2012

Raw data associated with all tables presented in the manuscrip

Multiple Rescue Factors Within a Wolbachia Strain

Wolbachia-induced cytoplasmic incompatibility (CI) is expressed when infected males are crossed with either uninfected females or females infected with Wolbachia of different CI specificity. In diploid insects, CI results in embryonic mortality, apparently due to the the loss of the paternal set of chromosomes, usually during the first mitotic division. The molecular basis of CI has not been determined yet; however, several lines of evidence suggest that Wolbachia exhibits two distinct sex-dependent functions: in males, Wolbachia somehow “imprints” the paternal chromosomes during spermatogenesis (mod function), whereas in females, the presence of the same Wolbachia strain(s) is able to restore embryonic viability (resc function). On the basis of the ability of Wolbachia to induce the modification and/or rescue functions in a given host, each bacterial strain can be classified as belonging in one of the four following categories: mod+ resc+, mod− resc+, mod− resc−, and mod+ resc−. A so-called “suicide” mod+ resc− strain has not been found in nature yet. Here, a combination of embryonic cytoplasmic injections and introgression experiments was used to transfer nine evolutionary, distantly related Wolbachia strains (wYak, wTei, wSan, wRi, wMel, wHa, wAu, wNo, and wMa) into the same host background, that of Drosophila simulans (STCP strain), a highly permissive host for CI expression. We initially characterized the modification and rescue properties of the Wolbachia strains wYak, wTei, and wSan, naturally present in the yakuba complex, upon their transfer into D. simulans. Confocal microscopy and multilocus sequencing typing (MLST) analysis were also employed for the evaluation of the CI properties. We also tested the compatibility relationships of wYak, wTei, and wSan with all other Wolbachia infections. So far, the cytoplasmic incompatibility properties of different Wolbachia variants are explained assuming a single pair of modification and rescue factors specific to each variant. This study shows that a given Wolbachia variant can possess multiple rescue determinants corresponding to different CI systems. In addition, our results: (a) suggest that wTei appears to behave in D. simulans as a suicide mod+ resc− strain, (b) unravel unique CI properties, and (c) provide a framework to understand the diversity and the evolution of new CI-compatibility types

Veneti et al_Heredity_raw data_30may2012

Raw data associated with all tables presented in the manuscrip