Simple Method for Fluorescence DNA <em>In Situ</em> Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Claremont McKenna, Pitzer, and Scripps Colleges

Distorted sex ratios: a window into RNAi-mediated silencing.

Male killing is caused by diverse microbial taxa in a wide range of arthropods. This phenomenon poses important challenges to understanding the dynamics of sex ratios and host-pathogen interactions. However, the mechanisms of male killing are largely unknown. Evidence from one case in Drosophila suggests that bacteria can target components of the male-specific sex-determination pathway. Here, we investigated male killing by the bacterium Arsenophonus nasoniae in the haplo-diploid wasp Nasonia vitripennis, in which females develop as diploids from fertilized eggs and males develop parthenogenetically as haploids from unfertilized eggs. We found that Arsenophonus inhibits the formation of maternal centrosomes, organelles required specifically for early male embryonic development, resulting in unorganized mitotic spindles and developmental arrest well before the establishment of somatic sexual identity. Consistent with these results, rescue of Arsenophonus-induced male lethality was achieved by fertilization with sperm bearing the supernumerary chromosome paternal sex ratio (PSR), which destroys the paternal genome but bypasses the need for maternal centrosomes by allowing transmission of the sperm-derived centrosome into the egg. These findings reveal a novel mechanism of male killing in Nasonia, demonstrating that bacteria have evolved different mechanisms for inducing male killing in the Arthropods.

A bacterium targets maternally inherited centrosomes to kill males in Nasonia.

Manipulation of host reproduction and efficient maternal transmission have facilitated the global spread of Wolbachia through millions of insect species. Cytological studies of the most common Wolbachia-induced phenotype, cytoplasmic incompatibility (CI), demonstrate that Wolbachia induce CI by altering host cell cycle timing. Cytological analyses also suggest that microtubules and motor proteins may play a role in the maternal and somatic transmission of Wolbachia.

Identification of Wolbachia--host interacting factors through cytological analysis.

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To investigate the role of the host cytoskeleton in the maternal transmission of the endoparasitic bacteria Wolbachia, we have characterized their distribution in the female germ line of Drosophila melanogaster. In the germarium, Wolbachia are distributed to all germ cells of the cyst, establishing an early infection in the cell destined to become the oocyte. During mid-oogenesis, Wolbachia exhibit a distinct concentration between the anterior cortex and the nucleus in the oocyte, where many bacteria appear to contact the nuclear envelope. Following programmed rearrangement of the microtubule network, Wolbachia dissociate from this anterior position and become dispersed throughout the oocyte. This localization pattern is distinct from mitochondria and all known axis determinants. Manipulation of microtubules and cytoplasmic Dynein and Dynactin, but not Kinesin-1, disrupts anterior bacterial localization in the oocyte. In live egg chambers, Wolbachia exhibit movement in nurse cells but not in the oocyte, suggesting that the bacteria are anchored by host factors. In addition, we identify mid-oogenesis as a period in the life cycle of Wolbachia in which bacterial replication occurs. Total bacterial counts show that Wolbachia increase at a significantly higher rate in the oocyte than in the average nurse cell, and that normal Wolbachia levels in the oocyte depend on microtubules. These findings demonstrate that Wolbachia utilize the host microtubule network and associated proteins for their subcellular localization in the Drosophila oocyte. These interactions may also play a role in bacterial motility and replication, ultimately leading to the bacteria's efficient maternal transmission.

Wolbachia utilizes host microtubules and Dynein for anterior localization in the Drosophila oocyte.

Pour &#xE9;tudier le r&#xF4;le du cytosquelette d&#x26;#39;accueil dans la transmission de la m&#xE8;re de l&#x26;#39;Wolbachia endoparasite bact&#xE9;ries, nous avons caract&#xE9;ris&#xE9; leur distribution dans la lign&#xE9;e germinale femelle de Drosophila melanogaster. Dans le germarium, Wolbachia sont distribu&#xE9;s &#xE0; toutes les cellules germinales du kyste, l&#x26;#39;&#xE9;tablissement d&#x26;#39;un d&#xE9;but d&#x26;#39;infection dans la cellule destin&#xE9;e &#xE0; devenir l&#x26;#39;ovocyte. Au cours de la mi-ovogen&#xE8;se, Wolbachia pr&#xE9;sentent une concentration distincte entre le cortex ant&#xE9;rieur et le noyau de l&#x26;#39;ovocyte, o&#xF9; de nombreuses bact&#xE9;ries apparaissent &#xE0; contacter l&#x26;#39;enveloppe nucl&#xE9;aire. Apr&#xE8;s r&#xE9;arrangement programm&#xE9; du r&#xE9;seau de microtubules, Wolbachia dissocier de cette position ant&#xE9;rieure et se dispersent tout au long de l&#x26;#39;ovocyte. Ce mod&#xE8;le de localisation est distincte de la mitochondrie et tous les d&#xE9;terminants des axes connus. Manipulation des microtubules et la dyn&#xE9;ine cytoplasmique et Dynactin, mais pas la kin&#xE9;sine-1, perturbe la localisation ant&#xE9;rieure bact&#xE9;rienne dans l&#x26;#39;ovocyte. Dans les chambres d&#x26;#39;&#x153;ufs vivants, le mouvement exposition Wolbachia dans les cellules des infirmi&#xE8;res, mais pas dans l&#x26;#39;ovocyte, ce qui sugg&#xE8;re que les bact&#xE9;ries sont ancr&#xE9;s par des facteurs de l&#x26;#39;h&#xF4;te. En outre, nous identifions la mi-ovogen&#xE8;se comme une p&#xE9;riode dans le cycle de vie de Wolbachia dans laquelle la r&#xE9;plication bact&#xE9;rienne se produit. Total des comptages bact&#xE9;riens montrent que l&#x26;#39;augmentation Wolbachia &#xE0; un taux significativement plus &#xE9;lev&#xE9; dans l&#x26;#39;ovocyte que dans la cellule infirmi&#xE8;re en moyenne, et que les niveaux normaux de Wolbachia dans l&#x26;#39;ovocyte fonction des microtubules. Ces r&#xE9;sultats d&#xE9;montrent que la bact&#xE9;rie Wolbachia utiliser le r&#xE9;seau de microtubules h&#xF4;te et des prot&#xE9;ines associ&#xE9;es pour leur localisation subcellulaire dans l&#x26;#39;ovocyte chez la drosophile. Ces interactions peuvent &#xE9;galement jouer un r&#xF4;le dans la motilit&#xE9; bact&#xE9;rienne et la r&#xE9;plication, conduisant finalement &#xE0; la transmission de la bact&#xE9;rie maternelle efficace.

Wolbachia Utilise microtubules d&#x26;#39;accueil et la dyn&#xE9;ine pour la localisation ant&#xE9;rieure de l&#x26;#39;ovocyte chez la drosophile

Um die Rolle des Zytoskeletts Host in der m&uuml;tterlichen &Uuml;bertragung der endoparasitische Wolbachia-Bakterien zu untersuchen, haben wir ihre Verteilung in der weiblichen Keimbahn von Drosophila Melanogaster gekennzeichnet. In dem Germarium Wolbachia sind verteilt auf alle Keimzellen der Zyste, zur Gr&uuml;ndung einer fr&uuml;hen Infektion in der Zelle bestimmt, um die Eizelle zu werden. W&auml;hrend Mid-oogenesis Wolbachia weisen eine unterschiedliche Konzentration zwischen der vorderen Kortex und der Kern in der Eizelle, wo viele Bakterien erscheinen, die Kernh&uuml;lle zu kontaktieren. Nach programmierten Umlagerung des Netzwerks Microtubule Wolbachia distanzieren von dieser vorderen Position und der Eizelle verstreut werden. Diese Lokalisierung Muster unterscheidet sich von Mitochondrien und alle bekannten Achse Determinanten. Manipulation von Mikrotubuli und cytoplasmatische Dynein und Dctn3, aber nicht Kinesin-1, st&ouml;rt anterior bakterielle Lokalisierung in der Eizelle. Leben Ei Kammern Wolbachia ausstellen Bewegung in Krankenschwester Zellen aber nicht in die Eizelle, was darauf hindeutet, dass die Bakterien durch Host Faktoren verankert sind. Dar&uuml;ber hinaus identifizieren wir Mid-oogenesis als Periode im Lebenszyklus von Wolbachia in die bakterielle Replikation auftritt. Gesamte Keimzahlen zeigen, dass Wolbachia-Anstieg deutlich h&ouml;her in der Eizelle, als in der durchschnittlichen Krankenschwester-Zelle und die normalen Wolbachia Ebenen in der Eizelle von Mikrotubuli abh&auml;ngen. Diese Ergebnisse zeigen, dass Wolbachia die Hostnetzwerk Microtubule und assoziierten Proteinen f&uuml;r ihre subzellulare Lokalisation in der Taufliege-Eizelle zu nutzen. Diese Interaktionen k&ouml;nnen bakterielle Motilit&auml;t und Replikation, letztlich zu den Bakterien effiziente maternale &Uuml;bertragung f&uuml;hrt auch eine Rolle spielen.

Wolbachia nutzt Host Mikrotubuli und Dynein anterior Lokalisierung in der Taufliege-Eizelle.

Per indagare il ruolo del citoscheletro ospite nella trasmissione materna dei batteri endoparasitic Wolbachia, noi abbiamo caratterizzato loro distribuzione nella linea germinale femminile di Drosophila melanogaster. In germarium, Wolbachia sono distribuiti a tutte le cellule di germe della cisti, che istituisce un&#39;infezione precoce nella cella destinata a diventare l&#39;ovocita. Durante mid-oogenesis, Wolbachia esibiscono una concentrazione distinta tra la corteccia anteriore e il nucleo in ovocita, dove molti batteri appaiono a contatto con la membrana nucleare. A seguito di riorganizzazione programmata della rete dei microtubuli, Wolbachia dissociare da questa posizione anteriore e si disperdano in tutto l&#39;ovocita. Questo modello di localizzazione &egrave; distinto da mitocondri e tutti i fattori determinanti noto asse. Manipolazione dei microtubuli e dineina citoplasmatica e Dctn1, ma non chinesina-1, sconvolge la localizzazione batterica anteriore nell&#39;ovocita. Nell&#39;uovo live chambers, Wolbachia esibiscono movimento nelle cellule infermiera ma non nell&#39;ovocita, suggerendo che i batteri sono ancorati da fattori di host. Inoltre, ci identifichiamo mid-oogenesis come un periodo nel ciclo di vita di Wolbachia nel quale replica batterica si verifica. Conta batterica totale Visualizza che Wolbachia aumento ad un tasso significativamente pi&ugrave; alto nell&#39;ovocita che nella cella infermiere medio e quello normale Wolbachia livelli nell&#39;ovocita dipendono da microtubuli. Questi risultati dimostrano che Wolbachia utilizzano la rete host dei microtubuli e proteine associate per la loro localizzazione sottocellulare nell&#39;ovocita della drosofila. Queste interazioni possono anche svolgere un ruolo nella motilit&agrave; batterica e replica, che in definitiva porta efficiente trasmissione materna dei batteri.

Wolbachia utilizza host microtubuli e dineina per localizzazione anteriore nell&#39;ovocita della drosofila.

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B;&#x3001;&#x7D30;&#x83CC;&#x306E;&#x904B;&#x52D5;&#x6027;&#x3068;&#x30EC;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306E;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3059;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x30DC;&#x30EB;&#x30D0;&#x30AD;&#x30A2;&#x306F;&#x3001;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8;&#x306E;&#x5375;&#x6BCD;&#x7D30;&#x80DE;&#x3067;&#x306F;&#x524D;&#x65B9;&#x306E;&#x30ED;&#x30FC;&#x30AB;&#x30EA;&#x30BC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306E;&#x305F;&#x3081;&#x306E;&#x30DB;&#x30B9;&#x30C8;&#x306E;&#x5FAE;&#x5C0F;&#x7BA1;&#x3068;&#x30C0;&#x30A4;&#x30CB;&#x30F3;&#x3092;&#x6D3B;&#x7528;

Endoparasitic &#xBC15;&#xD14C;&#xB9AC;&#xC544; Wolbachia&#xC758; &#xBAA8;&#xC131; &#xC804;&#xC1A1;&#xC5D0;&#xC11C; &#xD638;&#xC2A4;&#xD2B8; cytoskeleton&#xC758; &#xC5ED;&#xD560;&#xC744; &#xC870;&#xC0AC;&#xD560; &#xC6B0;&#xB9AC; Drosophila melanogaster&#xC758; &#xC5EC;&#xC131; &#xC810; &#xB77C;&#xC778;&#xC5D0;&#xC11C; &#xADF8;&#xB4E4;&#xC758; &#xBD84;&#xD3EC;&#xB294; &#xD2B9;&#xC9D5;&#xC785;&#xB2C8;&#xB2E4;. Germarium&#xC5D0;&#xC11C; Wolbachia &#xC218;&#xB9BD; &#xB41C; oocyte&#xC774; &#xB420; &#xC6B4;&#xBA85; &#xC138;&#xD3EC;&#xC5D0; &#xCD08;&#xAE30; &#xAC10;&#xC5FC;, &#xB0AD;&#xC885;&#xC758; &#xBAA8;&#xB4E0; &#xC138;&#xADE0; &#xC138;&#xD3EC;&#xC5D0; &#xBC30;&#xD3EC; &#xB429;&#xB2C8;&#xB2E4;. Mid-oogenesis, &#xB3D9;&#xC548; Wolbachia &#xC55E;&#xCABD;&#xC5D0; &#xB300;&#xB1CC; &#xD53C; &#xC9C8;&#xC5D0; &#xB9CE;&#xC740; &#xBC15;&#xD14C;&#xB9AC;&#xC544;&#xB294; &#xD575; &#xBD09;&#xD22C;&#xC5D0; &#xC5F0;&#xACB0;&#xD560; &#xB098;&#xD0C0;&#xB098;&#xB294; oocyte &#xD575; &#xC0AC;&#xC774; &#xB69C;&#xB837;&#xD55C; &#xB18D;&#xB3C4; &#xC804;&#xC2DC;. Microtubule &#xB124;&#xD2B8;&#xC6CC;&#xD06C;&#xC758; &#xD504;&#xB85C;&#xADF8;&#xB7A8; &#xB41C; &#xC7AC;&#xBC30;&#xCE58; Wolbachia&#xC774; &#xC55E;&#xCABD;&#xC5D0; &#xC704;&#xCE58;&#xC5D0;&#xC11C; &#xD574;&#xB9AC;&#xB97C; &#xD569;&#xB2C8;&#xB2E4; oocyte&#xC5D0; &#xAC78;&#xCCD0; &#xBD84;&#xC0B0; &#xB420;. &#xC774; &#xC9C0;&#xC5ED;&#xD654; &#xD328;&#xD134;&#xC740; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;&#xC640; &#xBAA8;&#xB4E0; &#xC54C;&#xB824;&#xC9C4;&#xB41C; &#xCD95; &#xACB0;&#xC815; &#xAD6C;&#xBD84; &#xB429;&#xB2C8;&#xB2E4;. &#xC870;&#xC791; microtubules &#xC138;&#xD3EC;&#xC9C8; &#xD68C; Dynactin, &#xD558;&#xC9C0;&#xB9CC; &#xD558;&#xC9C0; Kinesin 1&#xC758; &#xC55E;&#xCABD;&#xC5D0; &#xC138;&#xADE0;&#xC131; &#xC9C0;&#xC5ED;&#xD654; &#xB41C; oocyte &#xBC29;&#xD574;. &#xB77C;&#xC774;&#xBE0C; &#xACC4;&#xB780; &#xCC54;&#xBC84; Wolbachia &#xBC15;&#xD14C;&#xB9AC;&#xC544; &#xD638;&#xC2A4;&#xD2B8; &#xC694;&#xC778;&#xC5D0; &#xC758;&#xD574; &#xACE0;&#xC815; &#xC81C;&#xC548; &#xC6B4;&#xB3D9; &#xAC04;&#xD638;&#xC0AC; &#xC140;&#xB9CC; oocyte, &#xC804;&#xC2DC;. &#xB610;&#xD55C; &#xC6B0;&#xB9AC; Wolbachia&#xC758; &#xB77C;&#xC774;&#xD504; &#xC0AC;&#xC774;&#xD074; &#xAE30;&#xAC04; mid-oogenesis &#xC2DD;&#xBCC4; &#xC138;&#xADE0; &#xBCF5;&#xC81C;&#xAC00; &#xBC1C;&#xC0DD; &#xD569;&#xB2C8;&#xB2E4;. &#xCD1D; &#xC138;&#xADE0; &#xC218;&#xB294; oocyte &#xC218;&#xC900; microtubules &#xC758;&#xC874; &#xD3C9;&#xADE0; &#xAC04;&#xD638;&#xC0AC; &#xC140;&#xC5D0;&#xC11C; &#xADF8; &#xC815;&#xC0C1;&#xC801;&#xC778; Wolbachia &#xBCF4;&#xB2E4;&#xB294; oocyte&#xC5D0;&#xC11C; &#xC0C1;&#xB2F9;&#xD788; &#xB192;&#xC740; &#xBE44;&#xC728;&#xB85C; Wolbachia &#xC99D;&#xAC00; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; &#xBCF4;&#xC5EC; Wolbachia &#xD638;&#xC2A4;&#xD2B8; microtubule &#xB124;&#xD2B8;&#xC6CC;&#xD06C; &#xBC0F; &#xCD08;&#xD30C;&#xB9AC; oocyte&#xC5D0;&#xC11C; &#xADF8;&#xB4E4;&#xC758; subcellular &#xC9C0;&#xC5ED;&#xD654; &#xAD00;&#xB828;&#xB41C; &#xB2E8;&#xBC31;&#xC9C8; &#xC774;&#xC6A9;. &#xB610;&#xD55C; &#xC774;&#xB7EC;&#xD55C; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xC138;&#xADE0;&#xC758; &#xC6B4;&#xB3D9; &#xC131; &#xBC0F; &#xBCF5;&#xC81C;, &#xAD81;&#xADF9;&#xC801;&#xC73C;&#xB85C; &#xBC15;&#xD14C;&#xB9AC;&#xC544;&#xC758; &#xD6A8;&#xC728;&#xC801;&#xC778; &#xBAA8;&#xC131; &#xC804;&#xC1A1;&#xC5D0; &#xC120;&#xB3C4; &#xC5ED;&#xD560;&#xC744; &#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

Wolbachia &#xD65C;&#xC6A9; &#xD638;&#xC2A4;&#xD2B8; microtubules &#xD68C; Drosophila oocyte &#xC55E;&#xCABD;&#xC5D0; &#xC9C0;&#xC5ED;&#xD654;&#xC5D0; &#xB300; &#xD55C;.

Para investigar el papel del citoesqueleto de acogida en la transmisi&#xF3;n de la madre de la Wolbachia endopar&#xE1;sitos bacterias, se han caracterizado su distribuci&#xF3;n en la l&#xED;nea germinal femenina de Drosophila melanogaster. En el germario, Wolbachia se distribuyen a todas las c&#xE9;lulas germinales del quiste, el establecimiento de una infecci&#xF3;n temprana en la celda destinada a convertirse en el ovocito. A mediados de la ovog&#xE9;nesis, Wolbachia presentan una concentraci&#xF3;n distinta entre la corteza anterior y el n&#xFA;cleo en el ovocito, donde muchas bacterias parecen ponerse en contacto con la envoltura nuclear. Despu&#xE9;s de reorganizaci&#xF3;n programada de la red de microt&#xFA;bulos, Wolbachia se disocian de esta posici&#xF3;n anterior y se dispersan a lo largo de los ovocitos. Este patr&#xF3;n de localizaci&#xF3;n es distinta de las mitocondrias y todos los factores determinantes de los ejes conocidos. La manipulaci&#xF3;n de los microt&#xFA;bulos y dine&#xED;na citoplasm&#xE1;tica y dynactin, pero no Kinesin-1, interrumpe localizaci&#xF3;n anterior bacteriana en el ovocito. En c&#xE1;maras de huevos en vivo, el movimiento de Wolbachia exposici&#xF3;n en las c&#xE9;lulas de la enfermera, pero no en el ovocito, lo que sugiere que las bacterias est&#xE1;n delimitadas por los factores del hu&#xE9;sped. Adem&#xE1;s, identificamos a mediados de la ovog&#xE9;nesis como un per&#xED;odo en el ciclo de vida de la Wolbachia en el que se produce la replicaci&#xF3;n bacteriana. Total de los recuentos bacterianos muestran que el aumento de Wolbachia a una tasa significativamente m&#xE1;s alta en el ovocito que en la c&#xE9;lula nodriza promedio, y que los niveles normales de Wolbachia en el ovocito depende de los microt&#xFA;bulos. Estos hallazgos demuestran que la Wolbachia utilizar la red de microt&#xFA;bulos de acogida y las prote&#xED;nas asociadas a su localizaci&#xF3;n subcelular en el ovocito de Drosophila. Estas interacciones tambi&#xE9;n pueden desempe&#xF1;ar un papel en la motilidad bacteriana y la replicaci&#xF3;n, conduciendo finalmente a la transmisi&#xF3;n de las bacterias materna eficiente.

Wolbachia Utiliza microt&#xFA;bulos de acogida y dine&#xED;na para la localizaci&#xF3;n anterior en el ovocito Drosophila

Cytoplasmic incompatibility (CI) is a reproductive sterility found in arthropods that is caused by the endoparasitic bacteria Wolbachia. In CI, host progeny fail to develop during early embryogenesis if Wolbachia-infected males fertilize uninfected females. It is widely accepted that this lethality is caused by some unknown Wolbachia-induced modification of the paternal nuclear material in the host testes. However, the direct means by which this modification leads to early embryonic death are currently unresolved. Results from previous studies suggested that CI lethality occurs as a result of asynchrony in cell cycle timing between the paternal and maternal pronuclei. This hypothesis can be tested experimentally by the prediction that the Wolbachia-modified paternal pronucleus should support androgenetic development (i.e., from the paternal pronucleus only). Using specific mutations in Drosophila melanogaster that produce androgenetic progeny, we demonstrate that the Wolbachia-induced modification inhibits this type of development. This result suggests that CI occurs independently of the maternal pronucleus and argues against pronuclear asynchrony as the primary cause of CI lethality. We propose that CI occurs instead as the result of either a developmentally incompetent paternal pronucleus or asynchrony between the paternal pronucleus and the cell cycle of the egg cytoplasm.

A genetic test of the role of the maternal pronucleus in Wolbachia-induced cytoplasmic incompatibility in Drosophila melanogaster.

A longstanding enigma has been the origin of maternal centrosomes that facilitate parthenogenetic development in Hymenopteran insects. In young embryos, hundreds of microtubule-organizing centers (MTOCs) are assembled completely from maternal components. Two of these MTOCs join the female pronucleus to set up the first mitotic spindle in unfertilized embryos and drive their development. These MTOCs appear to be canonical centrosomes because they contain gamma-tubulin, CP190, and centrioles and they undergo duplication. Here, we present evidence that these centrosomes originate from accessory nuclei (AN), organelles derived from the oocyte nuclear envelope. In the parasitic wasps Nasonia vitripennis and Muscidifurax uniraptor, the position and number of AN in mature oocytes correspond to the position and number of maternal centrosomes in early embryos. These AN also contain high concentrations of gamma-tubulin. In the honeybee, Apis mellifera, distinct gamma-tubulin foci are present in each AN. Additionally, the Hymenopteran homolog of the Drosophila centrosomal protein Dgrip84 localizes on the outer surfaces of AN. These organelles disintegrate in the late oocyte, leaving behind small gamma-tubulin foci, which likely seed the formation of maternal centrosomes. Accessory nuclei, therefore, may have played a significant role in the evolution of haplodiploidy in Hymenopteran insects.

The origin of centrosomes in parthenogenetic hymenopteran insects.

The insect order of Hymenoptera (ants, bees, sawflies, and wasps) consists almost entirely of haplodiploid species. Under haplodiploidy, males develop from unfertilized eggs and are haploid, whereas females develop from fertilized eggs and are diploid. Although diploid males commonly occur, haploid females have never been reported. In analyzing the phenomenon of gynandromorphism in the parasitoid wasp Nasonia vitripennis, we found a line that generates complete phenotypic females from unfertilized eggs. These females have ovaries, can lay eggs, and are haploid, as shown by cytological and flow cytometric analyses. The data show that diploidy is not necessary for female development.

Haploid females in the parasitic wasp Nasonia vitripennis.

Postzygotic reproductive barriers such as sterility and lethality of hybrids are important for establishing and maintaining reproductive isolation between species. Identifying the causal loci and discerning how they interfere with the development of hybrids is essential for understanding how hybrid incompatibilities (HIs) evolve, but little is known about the mechanisms of how HI genes cause hybrid dysfunctions. A previously discovered Drosophila melanogaster locus called Zhr causes lethality in F1 daughters from crosses between Drosophila simulans females and D. melanogaster males. Zhr maps to a heterochromatic region of the D. melanogaster X that contains 359-bp satellite repeats, suggesting either that Zhr is a rare protein-coding gene embedded within heterochromatin, or is a locus consisting of the noncoding repetitive DNA that forms heterochromatin. The latter possibility raises the question of how heterochromatic DNA can induce lethality in hybrids. Here we show that hybrid females die because of widespread mitotic defects induced by lagging chromatin at the time during early embryogenesis when heterochromatin is first established. The lagging chromatin is confined solely to the paternally inherited D. melanogaster X chromatids, and consists predominantly of DNA from the 359-bp satellite block. We further found that a rearranged X chromosome carrying a deletion of the entire 359-bp satellite block segregated normally, while a translocation of the 359-bp satellite block to the Y chromosome resulted in defective Y segregation in males, strongly suggesting that the 359-bp satellite block specifically and directly inhibits chromatid separation. In hybrids produced from wild-type parents, the 359-bp satellite block was highly stretched and abnormally enriched with Topoisomerase II throughout mitosis. The 359-bp satellite block is not present in D. simulans, suggesting that lethality is caused by the absence or divergence of factors in the D. simulans maternal cytoplasm that are required for heterochromatin formation of this species-specific satellite block. These findings demonstrate how divergence of noncoding repetitive sequences between species can directly cause reproductive isolation by altering chromosome segregation.

Species-specific heterochromatin prevents mitotic chromosome segregation to cause hybrid lethality in Drosophila.

Satellites are one of the most enigmatic parts of the eukaryotic genome. These highly repetitive, noncoding sequences make up as much as half or more of the genomic content and are known to play essential roles in chromosome segregation during meiosis and mitosis, yet they evolve rapidly between closely related species. Research over the last several decades has revealed that satellite divergence can serve as a formidable reproductive barrier between sibling species. Here we highlight several key studies on Drosophila and other model organisms demonstrating deleterious effects of satellites and their rapid evolution on the structure and function of chromosomes in interspecies hybrids. These studies demonstrate that satellites can impact chromosomes at a number of different developmental stages and through distinct cellular mechanisms, including heterochromatin formation. These findings have important implications for how loci that cause postzygotic reproductive isolation are viewed.

How can satellite DNA divergence cause reproductive isolation? Let us count the chromosomal ways.

B chromosomes are centric chromosomal fragments present in thousands of eukaryotic genomes. Because most B chromosomes are non-essential, they can be lost without consequence. In order to persist, however, some B chromosomes can impose strong forms of intra-genomic conflict. An extreme case is the paternal sex ratio (PSR) B chromosome in the jewel wasp Nasonia vitripennis. Transmitted solely via the sperm, PSR 'imprints' the paternal chromatin so that it is destroyed during the first mitosis of the embryo. Owing to the haplo-diploid reproduction of N. vitripennis, PSR-induced loss of the paternal chromatin converts embryos that should become females into PSR-transmitting males. This conversion is key to the persistence of PSR, although the underlying mechanisms are largely unexplored. We assessed how PSR affects the paternal chromatin and then investigated how PSR is transmitted efficiently at the cellular level. We found that PSR does not affect progression of the paternal chromatin through the cell cycle but, instead, alters its normal Histone H3 phosphorylation and loading of the Condensin complex. PSR localizes to the outer periphery of the paternal nucleus, a position that we propose is crucial for it to escape from the defective paternal set. In sperm, PSR consistently localizes to the extreme anterior tip of the elongated nucleus, while the normal wasp chromosomes localize broadly across the nucleus. Thus, PSR may alter or bypass normal nuclear organizational processes to achieve its position. These findings provide new insights into how selfish genetic elements can impact chromatin-based processes for their survival.

Impact of a selfish B chromosome on chromatin dynamics and nuclear organization in Nasonia.

A widespread phenomenon in nature is sex ratio distortion of arthropod populations caused by microbial and genetic parasites. Currently little is known about how these agents alter host developmental processes to favor one sex or the other. The paternal sex ratio (PSR) chromosome is a nonessential, paternally transmitted centric fragment that segregates in natural populations of the jewel wasp, Nasonia vitripennis. To persist, PSR is thought to modify the hereditary material of the developing sperm, with the result that all nuclear DNA other than the PSR chromosome is destroyed shortly after fertilization. This results in the conversion of a fertilized embryo--normally a female--into a male, thereby insuring transmission of the "selfish" PSR chromosome, and simultaneously leading to wasp populations that are male-biased. To begin to understand this system at the mechanistic level, we carried out transcriptional profiling of testis from WT and PSR-carrying males. We identified a number of transcripts that are differentially expressed between these conditions. We also discovered nine transcripts that are uniquely expressed from the PSR chromosome. Four of these PSR-specific transcripts encode putative proteins, whereas the others have very short open reading frames and no homology to known proteins, suggesting that they are long noncoding RNAs. We propose several different models for how these transcripts could facilitate PSR-dependent effects. Our analyses also revealed 15.71 MB of novel transcribed regions in the N. vitripennis genome, thus increasing the current annotation of total transcribed regions by 53.4%. Finally, we detected expression of multiple meiosis-related genes in the wasp testis, despite the lack of conventional meiosis in the male sex.

Transcriptome profiling of Nasonia vitripennis testis reveals novel transcripts expressed from the selfish B chromosome, paternal sex ratio.

Male killing bacteria such as Spiroplasma are widespread pathogens of numerous arthropods including Drosophila melanogaster. These maternally transmitted bacteria can bias host sex ratios toward the female sex in order to 'selfishly' enhance bacterial transmission. However, little is known about the specific means by which these pathogens disrupt host development in order to kill males. Here we show that a male-killing Spiroplasma strain severely disrupts nervous tissue development in male but not female D. melanogaster embryos. The neuroblasts, or neuron progenitors, form properly and their daughter cells differentiate into neurons of the ventral nerve chord. However, the neurons fail to pack together properly and they produce highly abnormal axons. In contrast, non-neural tissue, such as mesoderm, and body segmentation appear normal during this time, although the entire male embryo becomes highly abnormal during later stages. Finally, we found that Spiroplasma is altogether absent from the neural tissue but localizes within the gut and the epithelium immediately surrounding the neural tissue, suggesting that the bacterium secretes a toxin that affects neural tissue development across tissue boundaries. Together these findings demonstrate the unique ability of this insect pathogen to preferentially affect development of a specific embryonic tissue to induce male killing.

Male killing Spiroplasma preferentially disrupts neural development in the Drosophila melanogaster embryo.

Some circularized X-Y chromosomes in Drosophila melanogaster are mitotically unstable and induce early embryonic lethality, but the genetic basis is unknown. Our experiments suggest that a large region of X-linked satellite DNA causes anaphase bridges and lethality when placed into a new heterochromatic environment within certain circularized X-Y chromosomes. These results reveal that repetitive sequences can be incompatible with one another in cis. The lethal phenotype also bears a remarkable resemblance to a case of interspecific hybrid lethality.

Heterochromatin position effects on circularized sex chromosomes cause filicidal embryonic lethality in Drosophila melanogaster.

Hybrid incompatibilities (HIs) cause reproductive isolation between species and thus contribute to speciation. Several HI genes encode adaptively evolving proteins that localize to or interact with heterochromatin, suggesting that HIs may result from co-evolution with rapidly evolving heterochromatic DNA. Little is known, however, about the intraspecific function of these HI genes, the specific sequences they interact with, or the evolutionary forces that drive their divergence. The genes Hmr and Lhr genetically interact to cause hybrid lethality between Drosophila melanogaster and D. simulans, yet mutations in both genes are viable. Here, we report that Hmr and Lhr encode proteins that form a heterochromatic complex with Heterochromatin Protein 1 (HP1a). Using RNA-Seq analyses we discovered that Hmr and Lhr are required to repress transcripts from satellite DNAs and many families of transposable elements (TEs). By comparing Hmr and Lhr function between D. melanogaster and D. simulans we identify several satellite DNAs and TEs that are differentially regulated between the species. Hmr and Lhr mutations also cause massive overexpression of telomeric TEs and significant telomere lengthening. Hmr and Lhr therefore regulate three types of heterochromatic sequences that are responsible for the significant differences in genome size and structure between D. melanogaster and D. simulans and have high potential to cause genetic conflicts with host fitness. We further find that many TEs are overexpressed in hybrids but that those specifically mis-expressed in lethal hybrids do not closely correlate with Hmr function. Our results therefore argue that adaptive divergence of heterochromatin proteins in response to repetitive DNAs is an important underlying force driving the evolution of hybrid incompatibility genes, but that hybrid lethality likely results from novel epistatic genetic interactions that are distinct to the hybrid background.

The Hmr and Lhr hybrid incompatibility genes suppress a broad range of heterochromatic repeats.

In eukaryotes, abnormally circularized chromosomes, known as 'rings,' can be mitotically unstable. Some rings derived from a compound X-Y chromosome induce mitotic abnormalities during the embryonic cleavage divisions and early death in Drosophila melanogaster, but the underlying basis is poorly understood. We recently demonstrated that a large region of 359-bp satellite DNA, which normally resides on the X chromosome, prevents sister ring chromatids from segregating properly during these divisions. Cytogenetic comparisons among 3 different X-Y rings with varying levels of lethality showed that all 3 contain similar amounts of 359-bp DNA, but the repetitive sequences surrounding the 359-bp DNA differ in each case. This finding suggests that ring misbehavior results from novel heterochromatin position effects on the 359-bp satellite. The purpose of this view is to explore possible explanations for these effects with regard to heterochromatin formation and replication of repetitive sequences. Also discussed are similarities of this system to a satellite-based hybrid incompatibility and potential influences on genome evolution.

Patrick M. Ferree

W. M. Keck Science Department

Claremont McKenna, Pitzer, and Scripps Colleges

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Patrick M. Ferree

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Claremont McKenna, Pitzer, and Scripps Colleges

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

W. M. Keck Science Department