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WOLBACHIA 2014

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8th International Wolbachia Conference · Innsbruck · Austria

6 - 11 June 2014

Igls, Innsbruck, Austria

Abstract book
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# HOST PLANT SPECIALIZATION MATTERS IN THE EPIDEMIOLOGY OF WOLBACHIA ACROSS PHYTOPHAGOUS WASPS (HYMENOPTERA: TORYMIDAE)

T. BOIVIN, H. HENRI, F. VAVRE, C. GIDOIN, P. VEBER, J-N. CANDAU, A. ROQUES & M-A. AUGER-ROZENBERG

Contact: thomas.boivin@paca.inra.fr

## Goal of the study

Focusing on seed wasps of the genus *Megastigmus*, we investigated whether ecological specialization is a likely mechanism of spread of thelytoky within insect communities. In these wasps, arrhenotoky is the dominant and ancestral form of parthenogenesis as thelytoky characterizes only 15 % of species, but both arrhenotokous and thelytokous species can co-occur on similar host-plant families (e.g. Rosaceae or Pinaceae). We explored the joint effects of phylogeny, ecology and endosymbionts on the spread of thelytoky within insects.

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## Association between endosymbiont infection and thelytoky

We tested for thelytoky caused by endosymbionts by screening in a large sample of arrhenotokous and thelytokous species for four endosymbiont candidates, and by performing antibiotic treatments.

* **Wolbachia infection is fixed in all thelytokous species**

* Arrhenotokous species are non-infected

* **Wolbachia curation: male production is reversible**

<table><caption>PCR screening for parthenogenesis-inducing endosymbionts</caption><thead><tr><th>Parthenogenesis and species (N)</th><th>Wolbachia<br>(infection %)</th><th>Cardinium</th><th>Arsenophonus</th><th>Rickettsia</th><th>Arthropod COI</th></tr></thead><tbody><tr><td><strong>Thelytoky</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><em>M. aculeatus</em> (43)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. aculeatus nigrovariegatus</em> (21)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. brevicaudis</em> (1)</td><td>+ (100)</td><td>NA</td><td>NA</td><td>NA</td><td>-</td></tr><tr><td><em>M. boriesi</em> (1)</td><td>+ (100)</td><td>NA</td><td>NA</td><td>NA</td><td>-</td></tr><tr><td><em>M. pictus</em> (30)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. pinsapinis</em> (60)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. pistacae</em> (30)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. rosae</em> (30)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. rosae alba</em> (5)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. suspectus</em> (65)</td><td>+ (100)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><strong>Arrhenotoky</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><em>M. amicorum</em> (20)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. atlanticus</em> (20)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. cryptomeriae</em> (20)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. pinus</em> (20)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. rafni</em> (30)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. spermotrophus</em> (30)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. schimitscheki</em> (30)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><em>M. transvaalensis</em> (20)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>Antibiotic treatments on the thelytokous *M. pinsapinis* (on *Cedrus*, *Pinaceae*)</figcaption></figure>

<table><caption>Figure 1. Phylogenetic placement of the Wolbachia strains infecting *Megastigmus* spp. among other Wolbachia belonging to A and B supergroups. This Bayesian-likelihood inference phylogeny is based on concatenated sequences data set for the four MLST loci coxA, gatB, ftsZ and hcpA (34 strains, 1650 bp).</caption><thead><tr><th colspan="2">Wolbachia infection</th><th colspan="3">Progeny produced by treated females</th></tr><tr><th>Treatment (N*)</th><th>% of wsp positives**</th><th>Number of mothers</th><th>Mean size**</th><th>Mean % of males</th></tr></thead><tbody><tr><td>control (50)</td><td>100</td><td>44</td><td>21.4 ± 5.3<sup>a</sup></td><td>0</td></tr><tr><td>tetracycline (50)</td><td>4.7</td><td>42</td><td>18.7 ± 7.8<sup>a</sup></td><td>92 ± 1.1</td></tr></tbody></table>

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**Horizontal transfer pathways** Seeds are narrow niches favoring highly overlapping breeding sites: opportunities for intimate ecological connections between infected and non-infected species

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Competition for egg-laying sites → Cannibalism or seed tissues as a media for *Wolbachia*

## Evolutionary *Wolbachia*-Megastigmus-host plant associations

We performed phylogenetic reconstructions using *Wolbachia* multilocus sequence typing (MLST) and wasp COI sequences to examine the evolution of *Wolbachia*-mediated thelytoky in *Megastigmus* and its possible connections to host plant specialization.

* We characterized **3 highly homogeneous *Wolbachia* lineages (wA1, wA2 and wB)**, with a strict association with host plant but not with *Megastigmus* (Fig.1)

* Phylogenetic signals of ecological specialization in the wasp COI tree (Fig.2): **1 basal clade on Anacardiaceae, 2 more recent clades on Rosaceae, 1 monophyletic clade on Pinaceae**

* **Thelytoky occurs sporadically** in the *Megastigmus* tree, with 7 independent *Wolbachia* acquisitions (●, Fig.2)

* **Non-random plant-specific distribution** of thelytokous species (P=0.0051)

* **Incongruence between *Wolbachia* and *Megastigmus* phylogenies** (P>0.05) suggests horizontal transmission

<figure><img src="image_6.png" /><figcaption>Figure 2. Bayesian-likelihood inference phylogeny based on cytochrome oxidase I (COI) sequences in *Megastigmus* (26 taxa, 962 bp). Host plant families are indicated at each branch forming a monophyletic group (except for the *Cupressaceae*). *Megastigmus* species infected by Wolbachia are indicated by arrowheads followed by the name of the Wolbachia lineage.</figcaption></figure>

**Conclusion** Ecological specialization in *Wolbachia's* hosts was probably a critical driving force for *Wolbachia* infection and spread of thelytoky, but also a constraint.

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# Host plant specialization matters in the epidemiology of Wolbachia across phytophagous wasps (Hymenoptera: Torymidae)

Thomas Boivin, Hélène Henri, Fabrice Vavre, Cindy Gidoin, Philippe Veber, Jean-Noël Candau, Alain Roques, Marie-Anne Auger-Rozenberg

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## To cite this version:

Thomas Boivin, Hélène Henri, Fabrice Vavre, Cindy Gidoin, Philippe Veber, et al. Host plant spe- cialization matters in the epidemiology of Wolbachia across phytophagous wasps (Hymenoptera: To- rymidae). 8. International Wolbachia Conference, Jun 2014, Innsbruck, Austria. 2014, Wolbachia 2014. 8th International Wolbachia Conference. Abstract Book. hal-01602096

HAL Id: hal-01602096

https://hal.science/hal-01602096

Submitted on 3 Jun 2020

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8th International
Wolbachia Conference

6 - 11 June 2014

Abstract book

Igls, Innsbruck, Austria
Abstract submission for a POSTER PRESENTATION

**Title:** Host plant specialization matters in the epidemiology of *Wolbachia* across phytophagous wasps
(Hymenoptera: Torymidae)

**Authors:** Boivin T¹, Henri H², Vavre F², Gidoin C¹, Veber P², Candau J-N¹,³, Roques A⁴ and Auger-Rozenberg M-A⁴

1. INRA, UR629 Ecologie des Forêts Méditerranéennes, URFM, F-84914 Avignon, France

2. Université de Lyon, F-69000, Lyon ; Université Lyon 1 ; CNRS, UMR5558, Laboratoire de Biométrie et Biologie Evolutive, F-69622, Villeurbanne, France.

3. National Resources Canada, Canadian Forest Service, Great Lakes Forestry Ctr, Sault Ste Marie, ON P6A 2E5, Canada

4. INRA, UR633 Unité de Recherche de Zoologie Forestière, 45075 Orléans, France

Among eukaryotes, sexual reproduction is by far the most predominant mode of reproduction. However, some systems maintaining sexuality appear particularly labile and raise intriguing questions on the evolutionary routes to asexuality. Thelytokous parthenogenesis is a form of spontaneous loss of sexuality leading to strong distortion of sex ratio towards females and resulting from mutation, hybridization or infection by bacterial endosymbionts. We investigated whether ecological specialization is a likely mechanism of spread of thelytoky within insect communities. Focusing on the highly-specialized genus *Megastigmus* (Hymenoptera: Torymidae), we first performed a large literature survey to examine the distribution of thelytoky in these wasps across their respective obligate host plant families. Second, we tested for thelytoky caused by endosymbionts by screening in 15 arrhenotokous and 10 thelytokous species for *Wolbachia*, *Cardinium*, *Arsenophonus* and *Rickettsia* endosymbionts and by performing antibiotic treatments. Finally, we performed phylogenetic reconstructions using multilocus sequence typing (MLST) to examine the evolution of endosymbiont-mediated thelytoky in *Megastigmus* and its possible connections to host plant specialization. We demonstrate that thelytoky evolved from ancestral arrhenotoky through the horizontal transmission and the fixation of the parthenogenesis-inducing *Wolbachia*. We find that ecological specialization in *Wolbachia*'s hosts was probably a critical driving force for *Wolbachia* infection and spread of thelytoky, but also a constraint. Our work further reinforces the hypothesis that community structure of insects is a major driver of the epidemiology of endosymbionts and that competitive interactions among closely related species may facilitate their horizontal transmission.

**Keywords:** parthenogenesis, ecological specialization, thelytoky, multilocus sequence typing, *Megastigmus*
# Imprint

Publisher: © WOLBACHIA 2014 Organising Committee

The authors are responsible for the content of the presentation abstracts.

Editorial work & layout: Wolfgang Arthofer, Birgit C. Schlick-Steiner & Florian M. Steiner
Molecular Ecology Group, Institute of Ecology
University of Innsbruck
Technikerstraße 25, 6020 Innsbruck, Austria

WOLBACHIA 2014 logo: © Wolfgang Arthofer, with contributions by Fred Böhringer, Ron Shimek & Barbara Thaler-Knoflach

Print: Studia, Innsbruck; www.studia.at

Date of publication: June 2014

## Suggested citation:

Simões P. *et al.* (2014) Quantifying the *Wolbachia* turnover. In: Arthofer W., Schlick-Steiner B.C., Steiner F.M. (eds.): Abstract Book of the 8th International Wolbachia Conference.

Published by WOLBACHIA 2014 Organising Committee, Innsbruck, Austria.

# Contents

<table><tr><td>Organising Committee</td><td>3</td></tr><tr><td>Scientific Committee</td><td>3</td></tr><tr><td>Sponsors</td><td>3</td></tr><tr><td>Program</td><td>4</td></tr><tr><td>Abstracts of oral presentations</td><td>9</td></tr><tr><td>Abstracts of poster presentations</td><td>73</td></tr><tr><td>Author index</td><td>118</td></tr><tr><td>List of participants</td><td>121</td></tr></table>
**Organising Committee**

In alphabetical order:

Wolfgang Arthofer (core team), Patrizia Arthofer-Peer, Kostas Bourtzis (core team), Katherina Damisch, Alexandra Grosbusch, Sarina Hammerle, Philipp Kirschner, Bernd Linke, Caroline Pichler, Heike Ritthammer, Sarah Scheld, Birgit C. Schlick-Steiner (core team), Julia Seeber, Barton Slatko, Florian M. Steiner (core team), Iris S. Steiner, Julia S. Steiner, Herbert C. Wagner

**Scientific Committee**

In alphabetical order:

Wolfgang Arthofer, University of Innsbruck, Austria; Seth Bordenstein, Vanderbilt University, USA; Didier Bouchon, Université de Poitiers, France; Kostas Bourtzis, International Atomic Energy Agency, Austria; Jeremy Brownlie, Griffith University, Australia; Sylvain Charlat, Université Lyon, France; Stephen L. Dobson, University of Kentucky, USA; Olivier Duron, Université Montpellier II, France; Horacio Frydman, Boston University, USA; Pierre Grève, Université de Poitiers, France; Peter Hammerstein, Humboldt-Universität zu Berlin, Germany; Abdelaziz Heddi, INSA de Lyon, France; Julie Hotopp, University of Maryland, USA; Martha Hunter, The University of Arizona, USA; John Jaenike, University of Rochester, USA; Lisa Klasson, Uppsala Universitet, Sweden; Wolfgang J. Miller, Medizinische Universität Wien, Austria; Jason Rasgon, The Pennsylvania State University, USA; Markus Riegler, University of Western Sydney, Australia; Barton Slatko, New England Biolabs, USA; William Sullivan, University of California Santa Cruz, USA; Einat Zchori-Fein, Newe Ya'ar Research Center, Israel

**Sponsors**

In alphabetical order:

Austrian Airlines, Vienna, Austria; Entomologie Meier, Munich, Germany; Eurofins MWG Operon, Ebersberg, Germany; Gesellschaft für Medizin- und Labortechnik, Innsbruck, Austria; Innsbruck Alpine Zoo, Innsbruck, Austria; Lactan, Graz, Austria; New England Biolabs, Ipswich, USA; Österreichische Gesellschaft für Entomofaunistik, Vienna, Austria; Qiagen, Hilden, Germany; Swarovski, Wattens, Austria; The Company of Biologists, Cambridge, UK; Thermo Fisher Scientific, Vienna, Austria; Tiroler Landesregierung, Innsbruck, Austria; Universität Innsbruck, Innsbruck, Austria; VWR International, Vienna, Austria
# Program

## Friday, 6 June 2014

1600 - 2000 Registration

1900 - 2200 Welcome reception

## Saturday, 7 June 2014

0814 - 0830 Wakeup call: Steve Reich (* 1936) Music for a large ensemble

0830 - 0900 Official welcome and opening remarks

### Symposium EVOLUTION

Chair: Didier Bouchon

0900 - 0920 Charlat S.: Quantifying the *Wolbachia* turnover

0920 - 0940 Lindsey A.: Experimental evolution of *Wolbachia* in novel hosts

0940 - 1000 Gerth M.: Phylogenomic analyses of *Wolbachia* supergroup relationships

1000 - 1020 Martínez-Rodríguez P.: Understanding codivergence of hosts and their associated bacteria: *Wolbachia* infection in the *Chorthippus parallelus* hybrid zone

1020 - 1050 Coffee break

### Symposium ECOLOGY, DIVERSITY, DYNAMICS

Chair: Sylvain Charlat

1050 - 1110 Truitt A.: Investigations into a case of *Wolbachia* and an endangered butterfly population

1110 - 1130 Ilinsky Y.: Maternal inheritance of long-established laboratory *Drosophila melanogaster* stocks

1130 - 1150 Jeong G.: Endosymbionts-induced microbiome disruption in *Vollenhovia emeryi* (Hymenoptera: Myrmicinae)

1150 - 1210 Bouvaine S.: Diversity of *Wolbachia* in African Cassava whiteflies

1210 - 1230 Hurst G.: Ecological and evolutionary impacts of male-killing bacteria

1230 - 1400 Lunch

### Symposium DISEASE AND PEST CONTROL

Chair: Einat Zchori-Fein

1400 - 1420 Dobson S.: Application of *Wolbachia* as a pesticide against *Aedes albopictus* (Asian Tiger Mosquito) in the USA

1420 - 1440 Calvitti M.: Cytoplasmic incompatibility patterns between naturally and *wPip* infected *Aedes albopictus*: implications for safety and long term effectiveness of a suppression strategy

1440 - 1500 Xi Z.: Interplay of novel *Wolbachia* strains with *Aedes* and *Anopheles* mosquitoes

1500 - 1520 McGraw E.: *Wolbachia* infection lengthens extrinsic incubation period in dengue infected mosquitoes

1520 - 1550 Coffee break
Symposium DISEASE AND PEST CONTROL

Chair: Elisabeth McGraw

1550 - 1610 Telschow A.: *Wolbachia* destabilizes mosquito population dynamics

1610 - 1630 Rasgon J.: *Wolbachia* can enhance pathogen infection in mosquito vectors

1630 - 1650 Martinez J.: Frequency of antiviral protection and correlation with cytoplasmic incompatibility: insights from a comparative analysis of *Wolbachia* strains

1650 - 1710 Bourtzis K.: Medfly - gut microbiota - *Wolbachia* tripartite symbiosis: assessing effects on fitness, mating behavior and pest control

1710 - 1900 Poster session

1900 - 2030 Dinner

2030 - 2200 Social hour

Sunday, 8 June 2014

0801 - 0845 Wakeup call: Adriano Banchieri (1576-1634) Barca di Venezia per Padova

0845 - 0900 Announcements

Symposium CELL BIOLOGY

Chair: Laura Serbus

0900 - 0920 Frydman H.: An integrated approach to dissect the mechanisms of *Wolbachia* tropism

0920 - 0940 Kamath A.: A novel *Wolbachia* tropism: targeting of polar cells in the *Drosophila* follicular epithelium

0940 - 1000 Toomey M.: Mechanisms of *Wolbachia* tropism to the stem cell niche in the *Drosophila* testis

1000 - 1020 Malone C.: Assessing *Wolbachia* dynamics in the *Drosophila* ovary

1020 - 1050 Coffee break

Symposium CELL BIOLOGY

Chair: Horacio Frydman

1050 - 1110 Miller W.: *Wolbachia* in the mind – a mutualistic puppet master orchestrating proper host sexual behaviour

1110 - 1130 Serbus L.: The impact of dietary nutrition on intracellular *Wolbachia* titer

1130 - 1150 Newton I.: Necessity is the mother of invention: actin manipulations by the reproductive parasite *Wolbachia pipientis*

Symposium ECOLOGY, DIVERSITY, DYNAMICS

Chair: Seth Bordenstein

1150 - 1210 Hunter M.: Fitness benefits of a facultative symbiont are influenced by host nuclear genotype

1210 - 1230 Zchori-Fein E.: Beyond single-bacterium symbiosis: factors correlated with variation in symbiotic communities of insects

1230 - 1400 Lunch
Symposium EVOLUTION

Chair: Seth Bordenstein

1400 - 1420 Gottlieb Y.: *Coxiella* endosymbionts in the *Rhipicephalus sanguineus*
brown tick species group

1420 - 1440 Teixera L.: Phylogenomics and evolution of symbiont-mediated
protection to pathogens

1440 - 1500 Husnik F.: Host-symbiont interactions at the symbiotic interfaces of
obligately blood-sucking insects

1500 - 1520 Zug R.: *Wolbachia*-arthropod mutualisms: some comments on pathogen
interference, dependence, tolerance, and resistance

1520 - 1550 Coffee break

Symposium EVOLUTION

Chair: Roman Zug

1550 - 1610 Bordenstein S.: Speciation by symbiosis: what have we learned so far?

1610 - 1630 Hammerstein P.: Dobzhansky-Muller and *Wolbachia*-induced
incompatibilities in a diploid genetic system

1630 - 1650 Riegler M.: Turn up the heat and you'll get more sons! Bacterially
facilitated temperature dependent sex ratio in an Australian
species of thrips

1650 - 1710 Group photo

1710 - 1900 Poster session & informal discussions

1900 - 2030 Dinner

2030 - 2200 Social hour

Monday, 9 June 2014

0802 - 0845 Wakeup call: Motosoma, son of Zeami (1394-1432) Shakkyo

0845 - 0900 Announcements

Symposium DISEASE AND PEST CONTROL

Chair: Jason Rasgon

0900 - 0920 Schneider D.: *Wolbachia* outbreak in tsetse fly hybrids: symbiont titer
regulation, bi-directional CI, and overcoming of male hybrid sterility

0920 - 0940 Kaur R.: What can we infer from symbionts titre in their respective
*Drosophila* host tissues?

0940 - 1000 Makepeace B.: A worm in bacterial clothing: proteomic analysis supports
the hypothesis that *Wolbachia*-driven recruitment of neutrophil
antimicrobial proteins protects *Onchocerca ochengi* against eosinophils

1000 - 1020 Ford L.: A•WOL macrofilaricidal drug discovery and development –
optimisation of anti-*Wolbachia* efficacy

1020 - 1050 Coffee break
Symposium PHENOTYPES

Chair: Markus Riegler

1050 - 1110 Bouchon D.: Functional analysis of the host immune response in the symbiotic association between the pill-bug *Armadillidium vulgare* and the feminising *Wolbachia*

1110 - 1130 Bertaux J.: A bug may hide another: cryptic *Wolbachia* in unfeminized lineages of *Armadillidium vulgare*

1130 - 1150 Kern P.: A new molecular sexing technique for butterflies – does *Wolbachia* really feminise genetic males in *Eurema*?

1150 - 1210 Wang YF.: *Wolbachia*-induced paternal defect in *Drosophila* is likely by interaction with the juvenile hormone pathway

1210 - 1230 Kaltenpoth M.: Symbiotic bacteria affect cuticular hydrocarbon profiles in tsetse flies (*Glossina m. morsitans*)

1230 - 1400 Lunch

1400 - 1900 Excursions

1900 - 2030 Dinner

2030 - 2200 Social hour

Tuesday, 10 June 2014

0829 - 0845 Wakeup call: Lou Reed (1943-2013) Metal machine music – the amine β ring pt. 1

0845 - 0900 Announcements

Symposium ECOLOGY, DIVERSITY, DYNAMICS

Chair: Molly Hunter

0900 - 0920 White J.: Double trouble: multiple endosymbiont infection and multiple manipulations in a linyphiid spider

0920 - 0940 Simhadri R.: Modulation of microbiome of *Drosophila melanogaster* by *Wolbachia*

0940 - 1000 Valiente Moro C.: Are *Wolbachia* and microbiota linked to the genetic structure of invasive and endemic populations of *Aedes albopictus*?

1000 - 1020 Weill M.: Evolution of the interaction *Culex pipiens* | *Wolbachia*

1020 - 1050 Coffee break

Symposium GENETICS & GENOMICS

Chair: Lisa Klasson

1050 - 1110 Fukatsu T.: Functional genomics of beetle-microbe symbioses

1110 - 1130 Comandatore F.: Phylogenomics and analysis of shared genes suggest a single origin of the main lineages of *Wolbachia* in nematodes

1130 - 1150 Chrostek E.: Link between genotype and phenotype in *Wolbachia*

1150 - 1210 Cordaux R.: Impact of *Wolbachia* endosymbionts on the evolution of sex determination in the isopod *Armadillidium vulgare*

1210 - 1230 Badawi M.: Toward the identification of feminizing genes of the bacterial endosymbiont *Wolbachia*

1230 - 1400 Lunch
Symposium GENETICS & GENOMICS

Chair: Takema Fukatsu

1400 - 1420 Nichols R.: *Wolbachia's* Burgess Shale: ancient and modern integration in the genome of *Podisma pedestris* (Orthoptera)

1420 - 1440 Hong X.: Identification of *Wolbachia*-responsive microRNAs in the two-spotted spider mite *Tetranychus urticae*

1440 - 1500 Wang GH. Sex-specific transcription of large proportion of genes in the only cryptic WO prophage genome in a fig wasp species

1500 - 1520 Harumoto T.: *Spiroplasma* infection induces male-specific DNA damage response in *Drosophila melanogaster*

1550 - 1610 Greve P.: *Wolbachia* genome sequencing: phylogenomics, symbiosis-related pan-genome and eukaryote-like proteins involved in host-symbiont interactions

1610 - 1640 Student awards

1640 - 1730 Presentations and discussion about the venue for the Wolbachia Conference 2016

1730 - 1900 Poster session

1900 - 2300 Gala dinner

Wednesday, 11 June 2014

Breakfast at the hotels

Departure
**Abstracts of oral presentations**

7 June 2014

Morning symposia
# Quantifying the *Wolbachia* turnover

**Patricia Simões**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Marc Bailly-Bechet**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Gergely Szöllősi**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Gladys Mialdea**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Marie Cariou**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Marie-France Sagot**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Sylvain Charlat**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France; sylvain.charlat@univ-lyon1.fr

Given its widespread occurrence and often radical phenotypic effects, there is little doubt that *Wolbachia* represents a substantial evolutionary force in arthropods. But the nature and strength of its effects are conditioned by two parameters for which we currently have no estimates: the duration of the typical *Wolbachia* stay in a given host lineage, and the rate at which uninfected lineages acquire a new infection. Assuming the current global incidence of about one third represents a stable equilibrium between transfer and acquisition, one can approximate that infections are lost twice faster than they are acquired. Noting that even closely related host species only rarely share similar, that is, potentially ancestral infections, we can further guess that *Wolbachia* rarely stays more than a few million years in a given host lineage. In an attempt to provide a better-founded estimate of these important parameters, we are currently applying co-phylogenetic methods to estimate the most likely rates of *Wolbachia* acquisition and losses, considering the observed incongruence between *Wolbachia* and arthropod trees. We will present these estimates and discuss their implications regarding the potential evolutionary consequences of *Wolbachia* infections.
# Experimental evolution of *Wolbachia* in novel hosts

**Amelia R. I. Lindsey**

Department of Entomology, University of California Riverside, USA; alind005@ucr.edu

**Richard Stouthamer**

Department of Entomology, University of California Riverside, USA

It has been difficult to undertake detailed studies on new *Wolbachia* infections, because they rarely persist. This is likely due to a lack of co-evolution between host and symbiont. Instead of trying to infect a previously uninfected host with *Wolbachia*, our approach is to manipulate the nuclear genetic background of a host (*Trichogramma* parasitoid wasps) long co-evolved with *Wolbachia*, and then observe how the symbiont adapts to its "new" host background. In *Trichogramma*, unfertilized eggs develop into males, and fertilized eggs develop as female. However, *Wolbachia* induces parthenogenesis, so that unfertilized eggs also develop as female. To challenge *Wolbachia*, recombinant backgrounds were created by exploiting the fact that *Wolbachia*-infected *Trichogramma* will, in rare cases, use sperm from males of uninfected populations to fertilize their eggs. Each recombinant *Wolbachia*-infected daughter can be used to start a unique homozygous colony. This leaves no diversity in the host for selection to act upon. In contrast, *Wolbachia*, with its high replication rate, is free to adapt to the newly created recombinant hosts. We created 10 recombinant lines of *Trichogramma* using this method. At generation three, the *Wolbachia* performance of each line relative to the original infected population, was determined by measuring symbiont titer and host sex ratio. In recombinant lines, we observed lower levels of parthenogenesis and the production of intersex offspring. After 26 generations, we saw improved *Wolbachia* performance, marked by higher proportions of females, and lower proportions of intersex. We conclude that the paternal genome, not originally associated with *Wolbachia*, inhibits the *Wolbachia* phenotype. Over a short period of time, *Wolbachia* will adapt to this new nuclear background, so as to more effectively induce parthenogenesis. This system will be used to identify key physiological and genomic changes occurring during adaptation of *Wolbachia* to a novel host.
# Phylogenomic analyses of *Wolbachia* supergroup relationships

**Michael Gerth**

Institute of Biology, University of Leipzig, Germany; michael.gerth@uni-leipzig.de

**Marie-Theres Gansauge**

Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany

**Anne Weigert**

Institute of Biology, University of Leipzig, Germany

**Christoph Bleidorn**

Institute of Biology, University of Leipzig, Germany

The ubiquitous bacterial intracellular endosymbiont *Wolbachia* is known from many arthropods but is also found in filarial nematodes. Considerable differences in *Wolbachia's* biology have led to the recognition of various supergroups. Nematodes have evolved mutualistic relationships with *Wolbachia* (supergroups C and D) over evolutionary timescales whereas arthropod *Wolbachia* (supergroups A and B) are generally opportunistic, switch hosts frequently and may generate reproductive modifications in their hosts. Further distinct lineages exist, supergroup F being the only strain that was detected in both arthropods and nematodes. Although many *Wolbachia* - host interactions have been studied extensively, the intergroup relationships are only poorly understood. Therefore, *Wolbachia's* evolutionary history, in particular the number and direction of major host and lifestyle transitions, remains elusive. As yet, attempts to reconstruct *Wolbachia's* phylogeny were impeded by insufficient taxon or gene sampling and by long branch artifacts originating from distant outgroups. Here, we attempt to overcome these problems by incorporating whole genome data from so far unsampled supergroups into a large scaled phylogenomic analysis of *Wolbachia* strains. Multiple phylogenetic approaches yielded highly congruent results, placing supergroup F as sistergroup to supergroup C and supergroups E and H at the basis of the *Wolbachia* tree. This suggests that *Wolbachia* has evolved from a specialized ancestor and that major host switches have occurred at least twice. Various genes are shared by all arthropod *Wolbachia* supergroups, implying that the reduced genomes of supergroups C and D have evolved independently. These results will serve as framework for future investigations on *Wolbachia* evolution.
# Understanding codivergence of hosts and their associated bacteria: *Wolbachia* infection in the *Chorthippus parallelus* hybrid zone

**Paloma Martínez-Rodríguez**

Departamento de Biología, Universidad Autónoma de Madrid, Spain; paloma.martinez@uam.es

**Francisca Arroyo-Yebras**

Departamento de Biología, Universidad Autónoma de Madrid, Spain

**José L. Bella**

Departamento de Biología, Universidad Autónoma de Madrid, Spain

Several studies indicate that codivergence of *Wolbachia* strains with their hosts is less common than horizontal transmission from other taxa. However, codivergence processes are of great value in understanding essential aspects of *Wolbachia* biology, including host adaptation, immune system suppression and coevolution.

In this study, we describe the case of the hybrid zone of *Chorthippus parallelus*, a grasshopper extensively studied for evolutionary purposes. Phylogenetic and phylogeographic studies based on previously established host phylogenies and taking a bacterial MLST approach show that the two subspecies of the grasshopper and a number of *Wolbachia* strains codiverged during the last glaciation period. In addition, extrapolation of the divergence time between bacterial strains leads us to propose that *Wolbachia* infected *C. parallelus* during the divergence of the two subspecies that formed the Pyrenean hybrid zone.

This, combined with the cytoplasmic incompatibility induced by this bacterium in natural populations of *C. parallelus*, suggests that the codivergence between bacterium and host partially explains the current status of the hybrid zone and makes it a good model for studying coevolutionary processes.
# Investigations into a case of *Wolbachia* and an endangered butterfly population

**Amy M. Truitt**

Portland State University, USA; amtruitt@pdx.edu

Can population augmentation between populations such as captive rearing and release effectively conserve imperiled populations of insect pollinators, or are these methods too risky for some populations? Augmentation is accomplished by supplementing small unstable populations with individuals of the same species from the same population or from a larger, more stable population. The latter type of augmentation may bring populations together that have historically occurred allopatrically. While augmentation programs are very responsible about not using visibly infected individuals to supplement recipient populations, movement between populations has the potential to spread pathogens. Unknown or unidentified symbiotic bacteria can be introduced when the source population is infected but the recipient population is uninfected or differently infected prior to supplementation. Here we examine the temporal sequence of infection and potential consequences of infection to a butterfly undergoing population supplementation between populations that are infected with symbiotic bacteria, *Wolbachia*, that can decrease population viability.

Despite management actions including habitat restoration, habitat enhancement, and captive rearing and release, some of these populations remain severely susceptible to extirpation. This compelled us to investigate whether CI-inducing *Wolbachia* served as a potential additional variable contributing to the attrition of *Speyeria zerene hippolyta* populations. This study investigates 1) whether the donor populations had different strains of *Wolbachia* from the recipient ones suggesting that the captive rearing and release program accidentally introduced new strains; 2) whether there are differences in reproductive success between infected and uninfected females; and 3) the potential ramifications that introduction of novel *Wolbachia* infections has on populations of pollinator species of conservation concern.
# Maternal inheritance of long-established laboratory *Drosophila melanogaster* stocks

**Yuri Ilinsky**

Institute of Cytology and Genetics, Novosibirsk State University, Russia; paulee@bionet.nsc.ru

**Roman Bykov**

Institute of Cytology and Genetics, Novosibirsk State University, Russia

**Mary Yudina**

Institute of Cytology and Genetics, Novosibirsk State University, Russia

**Nataly Weisman**

Institute of Cytology and Genetics, Novosibirsk State University, Russia

**Ludmilla Zakharenko**

Institute of Cytology and Genetics, Novosibirsk State University, Russia

**Olesya Ignatenko**

Institute of Cytology and Genetics, Novosibirsk State University, Russia

**Nataly Gruntenko**

Institute of Cytology and Genetics, Novosibirsk State University, Russia

**Elena Karpova**

Institute of Cytology and Genetics, Novosibirsk State University, Russia

**Ilya Zakharov**

Institute of Cytology and Genetics, Novosibirsk State University, Russia

*Wolbachia* is widespread in field populations of *Drosophila melanogaster*, however, there is little detailed information on *Wolbachia* infection among long-established laboratory *D. melanogaster* stocks. There have been studied four hundreds of mutant and wild type lines mainly from the collection of the Laboratory of Populations Genetics, Institute of Cytology and Genetics, Novosibirsk, Russia. The stocks are characterized by M-, S- mitotypes, and infection status: a genotype of *Wolbachia* or no infection. The diversity of maternal inheritance can be expressed in cytotypes: M-MEL, M-w-, S-CS, and S-w-.

In scrutinizing the stocks' records genealogy and our results, we observed some cases of bacteria loss in maternal lineages. However, in most cases the cytotypes proved to be inherited stable, with no *Wolbachia* or mitochondria parental transmissions.

In analyzing one-name wide-used stocks from different Stock Centers and Labs by cytotype, mtDNA sequences and mobile element patterns we have revealed facts of great genetic differences that indicate different origin of certain lines.
# Endosymbionts-induced microbiome disruption in *Vollenhovia emeryi* (Hymenoptera: Myrmicinae)

**Gilsang Jeong**

Division of Ecoscience, Ewha Womans University, Seoul, Korea; ecodever@gmail.com

**Soyeon Park**

Division of Ecoscience, Ewha Womans University, Seoul, Korea

**Pureum Noh**

Division of Ecoscience, Ewha Womans University, Seoul, Korea

**Jae Chun Choe**

Division of Ecoscience, Ewha Womans University, Seoul, Korea

The *Vollenhovia emeryi* ant can be categorized by its wing morphology, i.e. long-winged and short-winged. A phylogenetic analysis indicates that short-winged colonies are derived from long-winged colonies in *V. emeryi* suggesting that the short-winged is somehow cured of *Wolbachia* and the host genetic background of the morph may have evolved resistance to *Wolbachia* infection.

This raises a question whether the potentially resistant host genetic background and *Wolbachia* infection affect the bacterial symbiont community diversity in the ant species. For answering the question, the high throughput sequencing method was employed to examine the bacterial community diversity of the long- and the short-winged colonies. The specific aims of this study are (1) to investigate the influence of *Wolbachia* infection and the host genetic background to diversity of bacterial symbionts of the two morphs of the ant, (2) to compare bacterial diversity at the caste level.

We find that there are about 180 bacterial symbionts in the short-winged morph. On the other hand, the long-winged morph harbors only about 20 bacterial symbionts. The bacterial community diversity may be influenced by the existence of endosymbionts including *Wolbachia* but not much, if any, by the host genetic background. The bacterial community comparison among castes within the morph shows that the queen caste has rather dissimilar bacterial community most probably due to food resources. Furthermore, a bacterial strain diversity among castes indicates that there may be labor division even between queens. However, masking by dominant species is a known issue in this method. In this presentation, future research plan to tackle it will also be discussed.
# Diversity of *Wolbachia* in African Cassava whiteflies

**Saptarshi Ghosh**

Natural Resources Institute, University of Greenwich, UK

**Sophie Bouvaine**

Natural Resources Institute, University of Greenwich, UK; bs33@gre.ac.uk

**Maruthi Gowda**

Natural Resources Institute, University of Greenwich, UK

*Bemisia tabaci*, the insect vector of major plant virus diseases across the world, hosts many secondary endosymbionts such as *Wolbachia*, *Rickettsia*, *Arsenophonus*, *Hamiltonella*, *Cardinium* and *Fritschea* which play essential roles in their biology and behaviour. This study was aimed at understanding the prevalence and diversity of endosymbionts associated with *B. tabaci* populations from east and West Africa and the possible role of *Wolbachia* on the biology of its host. Prevalence of secondary endosymbionts in laboratory and field populations of cassava whiteflies from Subsaharan Africa (SSA) collected from 2012 to 2014 and from Cassava mosaic virus disease (CMD) epidemic and non-epidemic areas in Uganda in 1997 were screened by PCR. SSA2 biotype of whiteflies which are associated with CMD epidemic in east Africa collected from epidemic areas in 1997 harboured 100% *Wolbachia* infection. Diversity of *Wolbachia* was determined by multiple locus sequence typing (MLST) which formed two distinct clades in phylogeny of concatenated nucleotide sequences. *Wolbachia* haplotype from SSA1-subgroup2 biotype whiteflies were closer to *Wolbachia* from butterfly species whereas haplotypes from SSA2 and SSA1-subgroup3 whiteflies were clusering together. Quantification of *Wolbachia* from whiteflies by qPCR showed significant variable rates of multiplication of *Wolbachia* in different strains of whiteflies. Whiteflies from SSA2 and SSA1-subgroup2 had high quantities of *Wolbachia*, whereas SSA1-subgroup3 and subgroup1 had intermediate and lower quantities respectively. The temporal transition of whitefly population in CMD pandemic areas of east Africa from SSA2 to SSA1-SG1 implicates a possible role of *Wolbachia* in the biology of whiteflies which affects their population dynamics over time.
# Ecological and evolutionary impacts of male-killing bacteria

**Jon Ryder**

Institute of Integrative Biology, University of Liverpool, UK

**Emily Hornett**

Institute of Integrative Biology, University of Liverpool, UK

**Daria Pastok**

Institute of Integrative Biology, University of Liverpool, UK

**Louise Reynolds**

Institute of Integrative Biology, University of Liverpool, UK

**Sylvain Charlat**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Greg Hurst**

Institute of Integrative Biology, University of Liverpool, UK; g.hurst@liv.ac.uk

The effect symbionts have on host individuals are well known, at least at a phenotypic level. In contrast, our understanding of symbiont impact on host populations is less well established, with a contrast between the extent of theoretical predictions and the degree to which these have been tested in natural populations. In this talk, I will outline a case study in ladybirds relating the impact of male-killers on the dynamics of a ladybird disease, and one in butterflies where the evolutionary impact of male-killing is being resolved. I will argue impacts of symbionts on pathogens require us to understand both demographic and protective effects, and that rare events, such as extreme sex ratio bias associated with male-killing, may drive unusual (and interesting) evolutionary changes.
7 June 2014
Afternoon symposia
# Application of *Wolbachia* as a pesticide against *Aedes albopictus* (Asian Tiger Mosquito) in the USA

**Stephen L. Dobson**

Department of Entomology, University of Kentucky, Lexington, USA; sdobson@uky.edu

**James W. Mains**

MosquitoMate, Inc., Lexington, USA

**Corey L. Brelsfoard**

MosquitoMate, Inc., Lexington, USA

**Robert I. Rose**

Biotechnology Regulatory Consultant, Frederick, USA

The Asian tiger mosquito (*Aedes albopictus*) is an invasive species, biting nuisance and vector of medically important pathogens, including dengue and chikungunya). Despite intensive use of conventional pesticides, its range continues to expand in the USA and globally. A proposed autocidal approach for its control is based on *Wolbachia*, an endosymbiotic bacteria that is common in many insect species. Similar to sterile insect technique, the *Wolbachia* approach is based on the release of *Wolbachia* infected males, which cause a form of conditional sterility, known as Cytoplasmic Incompatibility (CI) in the targeted populations. In July 2013, Experimental Use Permit (No. 89668-EUP-1) was awarded by the US Environmental Protection Agency (EPA) to conduct field trial performance tests of a *Wolbachia* biopesticide approach against the Asian tiger mosquito in the continental US. This presentation will summarize regulatory work with the EPA and early experimental work examining male performance, longevity and dispersal in infested neighborhoods of Lexington, Kentucky, USA. Plans for future work in Kentucky and additional states will be summarized.
# Cytoplasmic incompatibility patterns between naturally and *wPip* infected *Aedes albopictus*: implications for safety and long term effectiveness of a suppression strategy

**Maurizio Calvitti**

Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Rome, Italy;
maurizio.calvitti@enea.it

**Riccardo Moretti**

Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Rome, Italy

**Francesca Marini**

Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Rome, Italy

**Tankeu Nzufo Francine**

Faculty of Medicine and Biomedical Sciences, University of Cameroon

**Angiola Desiderio**

Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Rome, Italy

*Aedes albopictus* is known to be superinfected with two strains of *Wolbachia* (*wAlbA* and *wAlbB*). In previous works we described the generation of a transinfected *Ae. albopictus* line, named "ARwP", by replacing the natural *Wolbachia* double-infection with a single heterologous bacterial strain from *Culex pipiens*. Reported infection data and CI features appeared consistent with a promising use of ARwP line as a tool for *Ae. albopictus* suppression strategies. We also demonstrated that in crosses between wild type males and ARwP females only *wAlbA* causes full CI, while *wAlbB* and *wPip* display an asymmetrical CI pattern.

Data from literature indicate that *Wolbachia* density in natural populations varies within a wide range. In particular *wAlbA* strain, besides being variable among individuals, tends to dramatically decrease with male age. In this work, we confirmed these general findings on mosquitoes collected in Northern and Central Italy. In addition, we evaluated the influence of male *wAlbA* and *wAlbB* loads on CI penetrance in crosses with ARwP transinfected females. To this aim we combined a quantitative PCR approach with egg hatching data from age controlled crossing experiments. We clearly found that the *wAlbA* "mod+" function towards ARwP females is expressed in a density-dependent manner. In fact, young males with very low *wAlbA* load showed a weakened CI, while older males with higher *wAlbA* density were associated to strong CI. Differently, *wAlbB* (a weaker CI inducer towards *wPip* females) showed neither an evident age-dependent density variation in males nor a significant density-dependent impact on CI in crosses with ARwP females.

The results of this work are an unprecedented contribution for evaluating how strong and stable is the bidirectional CI pattern between wild superinfected and ARwP mosquitoes. Obtained data are discussed in the perspective of developing a bio-ecologically safe and long-term effective area-wide suppression strategy against *Ae. albopictus*.
# Interplay of novel *Wolbachia* strains with *Aedes* and *Anopheles* mosquitoes

**Xiaoling Pan**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA

**Peng Lu**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA

**Guowu Bian**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA

**Deepak Joshi**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA

**Xiao Liang**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA

**Zhiyong Xi**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA;
xizy@msu.edu

Diseases transmitted by blood-feeding arthropod vectors, such as mosquito-borne malaria and dengue fever, cause 1.5 million human deaths every year. The insufficiency of currently available strategies, including vaccines, drugs and pesticides, has led to an increase in vector-borne diseases, making it urgent to develop novel control methods. Due to its ability to induce both cytoplasmic incompatibility and pathogen interference, *Wolbachia* is now widely recognized for its potential to modify mosquito populations such that they become refractory to the human pathogens they transmit. Toward this goal, we have introduced different *Wolbachia* strains into the major disease vectors, including two dengue vectors *Aedes aegypti* and *Ae. albopictus* and one primary malaria vector *Anopheles stephensi*. This provides us the opportunity to compare the interactions of either the same *Wolbachia* with different hosts, or the same host with different *Wolbachia* strains, in terms of pathogen interference, CI and fitness cost. We found *wAlbB* induced resistance to both dengue and malaria, and conferred certain benefits on both *Ae. aegypti* and *An. stephensi*. But *wAlbB* appeared to cause more fitness cost in female fecundity in *An. stephensi* than in *Ae. aegypti*. Two *Ae. albopictus* lines with different tripe infection were generated, and a difference in CI, antiviral effect and fitness is currently investigated. In all the transinfected lines, mosquito basal immunity was boosted by the novel infection, which contributed to *Wolbachia*-mediated pathogen interference. We also explored the interplay of *Wolbachia* with mosquito innate immunity by taking advantage of the currently available reverse genetic tools and transgenic mosquitoes with immune deficiency. The above results will be discussed in relation to developing *Wolbachia*-based strategies for vector-borne disease control.
**Wolbachia** infection lengthens extrinsic incubation period in
dengue infected mosquitoes

**Henry Ye**

School of Biological Sciences, Monash University, Melbourne, Australia

**Scott O'Neill**

School of Biological Sciences, Monash University, Melbourne, Australia

**Beth McGraw**

School of Biological Sciences, Monash University, Melbourne, Australia; beth.mcgraw@monash.edu

The *Wolbachia* strain *wMel*, that has been artificially introduced into the dengue virus mosquito vector *Aedes aegypti*, decreases the susceptibility of mosquitoes to dengue virus. The extrinsic incubation period (EIP), which is the time between when a mosquito takes a dengue infected bloodmeal and the time when the mosquito is capable of infecting a human on a subsequent bite, is one of the key components of disease transmission. We have devised a non-destructive method to repeatedly sample the saliva of mosquitoes to obtain estimates of EIP. We show here that *wMel* (1) delays EIP in dengue infected mosquitos by 1.1 days, (2) narrows the window of infectivity of infected mosquitoes by around 4 days, (3) reduces the amount of dengue copy number in mosquito saliva by approximately 3-fold and (4) reduces the saliva volume produced across the insect's lifespan. Surprisingly, we also found that (5) *wMel* lengthened the lifespan of dengue-infected mosquitoes possibly by lessening their dengue viral burden. These findings allow us to measure the vectorial capacity of *Wolbachia*-infected mosquitoes and more accurately estimate the impact of releasing *Wolbachia* infected mosquitoes as a strategy to reduce dengue transmission.
**Wolbachia destabilizes mosquito population dynamics**

**Arndt Telschow**

Westfalian Wilhelms-University, Munster, Germany; a.telschow@wwu.de

**Florian Grziwotz**

Westfalian Wilhelms-University, Munster, Germany

**Philip Crain**

Westfalian Wilhelms-University, Munster, Germany

**Takeshi Miki**

National Taiwan University, Taiwan

**Jimmy Mains**

University of Kentucky, USA

**George Sugihara**

Scripps Institution of Oceanography, USA

**Steven L. Dobson**

University of Kentucky, USA

**Chi-hao Hsieh**

National Taiwan University, Taiwan

Mosquitoes are important vectors for disease transmission in humans and livestock, causing
worldwide problems for public health and agriculture. Recent efforts in pest control focus on
biological control strategies that incapacitate the pathogen inside the vector by altering the
vector's microbiome. A prominent example is the release of intracellular bacteria *Wolbachia* into natural mosquito populations with the aim to eliminate dengue fever. However, whether such artificial infections can be a successful strategy to control vector-borne diseases and minimize the risks associated with releasing potential disease vectors is still under debate. Here, we demonstrate that *Wolbachia* can destabilize mosquito population dynamics by contrasting *Wolbachia*-infected versus uninfected cage populations of the Asian tiger mosquito (*Aedes albopictus*). We found that the population variability (measured as coefficient of variation of population size through time) of the infected cages is significantly higher than that of the uninfected cages. The elevated population variability is explained by the increasing nonlinear dynamics, as quantified by nonlinear time series analyses (S-Map analysis, state space reconstruction). In conclusion, the results suggest two potential risks for using *Wolbachia* in biological control programs. First, boom-and-bust of the mosquito population size is more likely to occur in *Wolbachia*-infected than in uninfected populations. Second, *Wolbachia*-infected mosquito populations are more difficult to manage because the higher degree of non-linearity makes their population dynamics less predictable. These findings have important management implications for the use of artificial bacterial infections as control of mosquitoes and vector-borne diseases.
*Wolbachia* can enhance pathogen infection in mosquito vectors

**Jason L. Rasgon**

Center for Infectious Disease Dynamics, Penn State University, USA; jlr54@psu.edu

**Brittany L. Dodson**

Center for Infectious Disease Dynamics, Penn State University, USA

**Grant L. Hughes**

Center for Infectious Disease Dynamics, Penn State University, USA

**Laura D. Kramer**

Wadsworth Center, New York State Department of Health, USA

Due to its ability to suppress pathogen infection in vector arthropods, laboratory and field investigations are currently ongoing for the use of *Wolbachia* to control arthropod-borne diseases. However, it has become clear that *Wolbachia* infection does not always lead to pathogen suppression in insects. Multiple studies using rodent and avian malaria models suggest that *Wolbachia* may enhance some *Plasmodium* parasites in mosquitoes, but to this point enhancement has not been observed for any human pathogens. We evaluated the effects of *Wolbachia* on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito *Culex tarsalis*. *Wolbachia* reached high titers and disseminated to numerous tissues including the head, proboscis, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, *Wolbachia* did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in *Wolbachia*-infected mosquitoes compared to controls. Quantitative PCR indicated that REL1 (the activator of the antiviral Toll immune pathway) was downregulated in *Wolbachia*-infected relative to control mosquitoes. This is the first observation of *Wolbachia*-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before using *Wolbachia* as part of a vector-borne disease control program.
# Frequency of antiviral protection and correlation with cytoplasmic incompatibility: insights from a comparative analysis of *Wolbachia* strains

**Julien Martinez**

Department of Genetics, University of Cambridge, UK; jtm35@cam.ac.uk

**Ben Longdon**

Department of Genetics, University of Cambridge, UK

**Simone Bauer**

Department of Genetics, University of Cambridge, UK

**Suzan Ok**

Department of Genetics, University of Cambridge, UK

**Wolfgang J. Miller**

Laboratories of Genome Dynamics, Medical University of Vienna, Austria

**Kostas Bourtzis**

Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna, Austria

**Luis Teixeira**

Instituto Gulbenkian de Ciência, Oeiras, Portugal

**Francis M. Jiggins**

Department of Genetics, University of Cambridge, UK

In the last few years, *Wolbachia* has been shown to play an important role in protecting *Drosophila* and mosquito hosts against viral infection. While the mechanisms remain unclear, it was shown that not all bacterial strains have the ability to confer protection. In order to assess the frequency of such an induced phenotype, we have investigated antiviral protection in 19 *Wolbachia* strains originating from 16 *Drosophila* species after transfer into the same genotype of *D. simulans*. We found that approximately half of the strains protected against two RNA viruses, suggesting that a substantial proportion of insect species may benefit from *Wolbachia*-mediated protection. The level of protection against two distantly related RNA viruses – DCV and FHV – was strongly genetically correlated, which suggests that there is a single mechanism of protection with broad specificity. Furthermore, *Wolbachia* is mainly providing resistance to viral infection, as the increases in survival following infection can be largely explained by reductions in viral titer. Variation in antiviral protection provided by different *Wolbachia* strains is genetically correlated to their density within the host, supporting previous data. Finally, we found no correlation between protection and the ability to induce cytoplasmic incompatibility (CI), showing that the most protective strains do not necessarily have the highest invasive potential by means of CI. Moreover, *Wolbachia* infection was in some cases associated with costs which might represent a limitation for the use of *Wolbachia* as a biocontrol agent to prevent the spread of arboviruses.
**Medfly - gut microbiota - *Wolbachia* tripartite symbiosis:
assessing effects on fitness, mating behavior and pest control**

**George Kyritsis**

Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna,
Austria

**Antonios Augustinos**

Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna,
Austria

**Daniela Schneider**

Laboratories of Genome Dynamics, Medical University of Vienna, Austria

**Vangelis Doudoumis**

Department of Environmental and Natural Resources Management, University of Patras, Agrinio, Greece

**Helias Asimakis**

Department of Environmental and Natural Resources Management, University of Patras, Agrinio, Greece

**Wolfgang J. Miller**

Laboratories of Genome Dynamics, Medical University of Vienna, Austria

**Nikolaos Papadopoulos**

Laboratory of Entomology and Agricultural Zoology, University of Thessaly, N. Ionia Magnisia, Greece

**George Tsiamis**

Laboratories of Genome Dynamics, Medical University of Vienna, Austria

**Carlos Caceres**

Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna,
Austria

**Kostas Bourtzis**

Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna,
Austria; K.Bourtzis@iaea.org

The Mediterranean fruit fly (medfly), *Ceratitis capitata*, is one of the most important agricultural pests worldwide. In many countries, the control of this pest is currently based on the development of Genetic Sexing Strains (GSSs) and their application in programs incorporating the Sterile Insect Technique (SIT), an irradiation-induced male sterility approach. *Wolbachia*-infected lines have also been established and express 100% Cytoplasmic Incompatibility (CI) and could potentially be useful for the development of Incompatible Insect Technique (IIT), a *Wolbachia*-induced male sterility approach, and / or the enhancement of SIT programs. In addition, a number of studies have shown that the gut-associated microbiota of medfly plays a significant role on host life history traits. In this study, we will present data about the effect of *Wolbachia* on host fitness and mating behavior as well on the diversity and structure of the gut-associated microbiota. Our results are of major importance for programs which aim to control pests and diseases using exclusively or combining *Wolbachia*-based approaches.
8 June 2014
Morning symposia
# An integrated approach to dissect the mechanisms of *Wolbachia* tropism

**Horacio Frydman**

Department of Biology, Boston University, USA; hfrydman@bu.edu

**Eva Fast**

Department of Biology, Boston University, USA

**Michelle Toomey**

Department of Biology, Boston University, USA

**Rama Krishna Simhadri**

Department of Biology, Boston University, USA

**Ajit Kamath**

Department of Biology, Boston University, USA

**Mark Deehan**

Department of Biology, Boston University, USA

Tissue tropism is a fundamental aspect of host-microbe interactions. *Wolbachia* have a peculiar tropism for the microenvironment that supports stem cells in the *Drosophila* ovary, known as stem cell niches. This niche tropism facilitates germline colonization promoting successful infection of the next generation. The cellular and molecular basis for niche tropism are not well understood. We are dissecting *Wolbachia* – host interaction using a combination of cellular, molecular, developmental and computational approaches in *Drosophila* cell lines and whole organism. We have measured the dynamics of *Wolbachia* density and proliferation in the developing host, and found that the *Wolbachia* intracellular accumulation is driven mostly by higher division rates of bacteria that infect niche cells. Towards identifying the host molecular pathways responsible for this tropism, we performed a high-throughput sequencing on *Drosophila melanogaster* cell lines that are stably infected with *Wolbachia* and found approximately 1000 genes statistically significantly modified by infection. Host genes that regulate or are part of the stem cell maintenance, cell cycle, metamorphosis, adhesion, sugar metabolism, immunity, proteolysis and antioxidants are transcriptionally affected. Using bioinformatic tools we determined a set of core pathways with potentially relevant biological roles in *Wolbachia* tropism. These candidate pathways are being tested for their functional significance on *Wolbachia* tropism and replication in the host cells.
# A novel *Wolbachia* tropism: targeting of polar cells in the *Drosophila* follicular epithelium

**Ajit Kamath**

Department of Biology, Boston University, USA; ajit9988@bu.edu

**Michelle Toomey**

Department of Biology, Boston University, USA

**Rama Krishna Simhadri**

Department of Biology, Boston University, USA

**Horacio Frydman**

Department of Biology, Boston University, USA

*Wolbachia* propagate mainly by vertical transmission via the female oocyte. They are under a high selective pressure to colonize the female germline and infect the next generation. However *Wolbachia* infection is not just limited to the germline cells, but also several somatic cell populations have been reported to have higher *Wolbachia* loads compared to surrounding tissue, including stem cell niches, neurons, trachea and Malpighian tubes. Here we identify a previously overlooked *Wolbachia* tropism to the polar cells (PC) in the *Drosophila* ovary. PCs are a particular cell type within the follicular epithelium of a *Drosophila* egg chamber which have a role in the patterning of the eggshell, oocyte localization and polarity. During *Drosophila* oogenesis, we can easily follow all the stages of the PC development allowing us to investigate the kinetics of the intracellular accumulation of *Wolbachia* in the PCs and the surrounding follicular epithelia. Surprisingly, *Wolbachia* have been found to co-ordinate their intracellular accumulation with specific developmental events during oogenesis. Our results show that PC tropism of *Wolbachia* is acquired through higher division rates in these cells compared to the surrounding epithelia. In this study, we also investigate various host factors which are responsible for the specific accumulation of *Wolbachia* in these cells. PC tropism provides an additional entry point for *Wolbachia* into the oocyte during egg development. This analysis provides us mechanistic insights into the preferential targeting of *Wolbachia* to specific host cell types.
# Mechanisms of *Wolbachia* tropism to the stem cell niche in the *Drosophila* testis

**Michelle Toomey**

Department of Biology, Boston University, USA; mtoomey5@bu.edu

**Rama Krishna Simhadri**

Department of Biology, Boston University, USA

**Ajit Kamath**

Department of Biology, Boston University, USA

**Horacio Frydman**

Department of Biology, Boston University, USA

*Wolbachia* target the stem cell niches in the *Drosophila* ovary to enhance germline colonization and subsequent vertical transmission. This tropism is pervasive across the *Drosophila* genus, with the pattern of targeting being evolutionarily conserved. We have found that tropism to the stem cell niche in the testis, known as the hub, is not evolutionarily conserved. Using hybrid analysis, we have found that both *Wolbachia* and host derived factors are important for tropism in this system. Towards identifying the cellular and molecular mechanisms of cell tropism, we are investigating hub targeting of the closely related *Wolbachia* strains *wMel* and *wMelPop*, which differ significantly in their frequencies and densities of hub infection. The targeting discrepancy of these two strains of *Wolbachia* indicate that this phenotype is rapidly evolving, as they shared a common ancestor only 8,000 years ago. With the plethora of tools available in *D. melanogaster*, we are using a candidate gene approach to target host proteins enriched in the stem cell niche in the testis for RNAi mediated knockdown in the hub. We have identified *Drosophila* stem cell related signaling pathways that promote *Wolbachia* accumulation. Understanding the cellular and molecular bases of tissue tropism is a fundamental aspect of *Wolbachia*-host interactions.
**Assessing *Wolbachia* dynamics in the *Drosophila* ovary**

**Colin Malone**

Skirball Institute, NYUMC, New York, USA; colin.malone@med.nyu.edu

**Yolande Grobler**

Skirball Institute, NYUMC, New York, USA

**Ruth Lehmann**

Skirball Institute, NYUMC, New York, USA

In order to investigate the molecular relationships underlying *Wolbachia* infection, we have undertaken tissue-specific RNAi screening for host genes that influence *Wolbachia* dynamics. We assayed approximately 1,000 germline RNAi knock-down lines which produce ovarian phenotypes and drove these knock-downs in the presence or absence of *Wolbachia* to ask whether the bacteria would modify the previously determined phenotypes. After measuring egg laying, fertility and ovarian morphology, we observed little difference other than flies laid fewer eggs when infected with *Wolbachia*.

Additionally, we have continued several directed approaches to understand the experience of cells during infection, and how host-cells influence bacterial behavior. We performed gene expression profiling and identified validated numerous host-genes up-regulated during infection. We used this finding to develop a single-molecule RNA FISH assay allowing us to visualize *Wolbachia* transcripts during ovarian development. We have now paired this with tissue-specific gene knock-downs, allowing us to assay bacterial density and gene expression. We have continued selected host-gene knock-downs in immune, metabolic and mitochondrial pathways to assess the dynamics of mitochondrial and bacterial responses within the ovary. Our continued work will be discussed.
*Wolbachia* in the mind – a mutualistic puppet master orchestrating proper host sexual behaviour

**Anton A. Strunov**

Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia

**Daniela Schneider**

Laboratories of Genome Dynamics, Medical University of Vienna, Austria

**Lee Ehrmann**

Natural Sciences, SUNY, Purchase, USA

**Wolfgang J. Miller**

Laboratories of Genome Dynamics, Medical University of Vienna, Austria; wolfgang.miller@meduniwien.ac.at

*Wolbachia* of the neotropical *Drosophila* species cluster *D. paulistorum* present one of the rare examples of conspecificity and obligate mutualism in arthropods. This neotropical species cluster is currently under incipient speciation in nature, consisting of six reproductively isolated semispecies that each harbour closely-related but reciprocally incompatible *Wolbachia* strains. In their native hosts these endosymbionts have evolved fixed vital interactions by affecting fecundity and viability of flies. In mixed genetic backgrounds of F1 hybrids between semispecies however, mutualistic *Wolbachia* transform into pathogens by overreplication via loss of replication control, and thereby cause embryonic mortality and sterility. As recently shown by our group neotropical *Wolbachia* (nPau) also influence host sexual behaviour by affecting female assortative mating, i.e. by orchestrating proper mate recognition, most likely via pheromone perception and interpretation.

Here we have surveyed via FISH analyses in detail the temporal and spatial titer dynamics and tissue tropism of the mutualist in adult fly brains and found that, in contrast to nMel, the presence ofnPau is exclusively restricted to well-defined brain regions which are known for being functionally important for pheromone perception, signal interpretation as well as learning and longterm memory.
# The impact of dietary nutrition on intracellular *Wolbachia* titer

**Laura Serbus**

Separtment of Biological Sciences, Florida International University, Miami, USA; lserbus@fiu.edu

**Pamela White**

MCD Biology, University of California, Santa Cruz, USA

**Roger Albertson**

Biology Department, Albion College, USA

**Amanda Rabe**

MCD Biology, University of California, Santa Cruz, USA

**William Sullivan**

MCD Biology, University of California, Santa Cruz, USA

While a number of studies have identified host factor that influence endosymbiont titer, little is known concerning environmental influences on endosymbiont titer. To address this, we examined nutrient impact on maternally transmitted *Wolbachia* endosymbionts in *Drosophila*. We show that feeding either laboratory-reared or wild-caught *Drosophila* a yeast-enriched diet dramatically suppressed *Wolbachia* titer carried within germline-derived oocytes. This yeast-induced titer reduction is mediated in large part by somatic Tor and insulin signaling pathways. Disrupting Tor with the small molecule Rapamycin dramatically increases oocyte *Wolbachia* titer, whereas hyper-activating somatic TORC1 suppresses oocyte titer. Furthermore, ablation of insulin producing cells located in the *Drosophila* brain eliminated yeast impact on oocyte titer. By contrast, dietary yeast had no impact on *Wolbachia* titer in the somatically derived central nervous system. These findings highlight the interactions between *Wolbachia* and germline cells as distinctive in sensitivity to insulin and TORC1-mediated nutrient responses.
# Necessity is the mother of invention: actin manipulations by the reproductive parasite *Wolbachia* pipientis

**Irene L. G. Newton**

Department of Biology, Indiana University, Bloomington, USA; irnewton@indiana.edu

**Kathy B. Sheehan**

Department of Biology, Indiana University, Bloomington, USA

**Cammie F. Lesser**

Department of Medicine, Harvard Medical School, Cambridge, USA

**Ralph R. Isberg**

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, USA

The obligately intracellular *Wolbachia* pipientis is a ubiquitous α-proteobacterial symbiont of insects and nematodes related to the Rickettsial pathogens *Ehrlichia* and *Anaplasma*. Studies of *Wolbachia* cell biology suggest that this bacterium is capable of manipulating host cortical actin during transmission between somatic cells and the germ line. Here, we identified and characterized *Wolbachia* proteins that localize to or disrupt the eukaryotic actin cytoskeleton. Two of these proteins (WD0830 and WD1171) induce growth defects in yeast upon expression, supporting their role as secreted bacterial effectors. In further support of their interaction with the cytoskeleton, expression of these two proteins abrogates or exacerbates growth defects in the context of the yeast morphogenesis checkpoint kinase mutant (Δswe1). We present an in depth analysis of one of these candidate secreted effectors, WalE1 (WD0830), a protein that promotes actin polymerization through direct interaction with actin and a reduction in the critical concentration of actin. This bacterial protein is unique, having no homology to previously identified formins or actin nucleating domain structures and is the first identification of any *Wolbachia* protein that alters actin polymerization dynamics. Finally, we analyze effects of WalE1 expression on the actin cytoskeleton and the *Wolbachia*-Drosophila symbiosis in the context of cell lines and germ line stem cell niche tropism. We suggest these *Wolbachia* proteins, and WalE1 specifically, may be utilized by the bacterium during invasion of the stem cell niche.
# Fitness benefits of a facultative symbiont are influenced by host nuclear genotype

**Martha S. Hunter**

Department of Entomology, University of Arizona, Tucson, USA; mhunter@ag.arizona.edu

**Anna G. Himler**

Department of Entomology, University of Arizona, Tucson, USA

**Peter Asiimwe**

Department of Entomology, University of Arizona, Tucson, USA

**Suzanne E. Kelly**

Department of Entomology, University of Arizona, Tucson, USA

**Bodil N. Cass**

Department of Entomology, University of Arizona, Tucson, USA

**Netta Mozes-Daube**

Newe Ya'ar Research Center, Agricultural Research Organization, Ramat Yishay, Israel

**Einat Zchori-Fein**

Newe Ya'ar Research Center, Agricultural Research Organization, Ramat Yishay, Israel

Some arthropod-associated symbionts such as *Wolbachia*, known previously only as reproductive parasites, have recently been shown to benefit their hosts through nutrition and/or defense. Multiple roles of symbionts create more possibilities for genetic conflict among host and symbiont genes. The global whitefly species complex *Bemisia tabaci* has several facultative bacterial symbionts, among which *Rickettsia* sp. nr. *bellii* is common worldwide. *Rickettsia* swept rapidly to near fixation in the invasive *B. tabaci* "B" species in the southwestern USA in the early 2000s. In a laboratory line established from a local population, *Rickettsia* causes fitness benefits such as greater fecundity, developmental success and shorter development times as well as reproductive manipulation in the form of strongly female-biased sex ratios. We found differences in the phenotype of *Rickettsia*, however, in a second whitefly line collected from the same population. While *Rickettsia* appears to increase performance in both lines, the magnitude of benefit varies. We used introgression between the lines to separate the contributions to the *Rickettsia* phenotype of the host cytotype (including the primary symbiont *Portiera*, the fixed symbiont *Hamiltonella*, and mitochondrial genes) from those of the nuclear genotype. Our results suggest whitefly nuclear genotype is more important than cytotype in influencing *Rickettsia* expression. Furthermore, field surveys in Israel show a decline in *Rickettsia* frequency in whiteflies from 2000-2012, where earlier work found few fitness benefits associated with *Rickettsia* infection. In general, our results suggest that differences in the *Rickettsia* phenotype in whiteflies underlie the variable frequencies of *Rickettsia* in whiteflies worldwide, and raise the question: if *Rickettsia* is so good for whiteflies, why is there host control of the *Rickettsia* phenotype?
**Beyond single-bacterium symbiosis: factors correlated with variation in symbiotic communities of insects**

**Einat Zchori-Fein**

Newe Ya'ar Research Center, Agricultural Research Organization, Ramat Yishay, Israel; einat@agri.gov.il

**Tamar Lahav**

Newe Ya'ar Research Center, Agricultural Research Organization, Ramat Yishay, Israel

**Shiri Freilich**

Newe Ya'ar Research Center, Agricultural Research Organization, Ramat Yishay, Israel

The target of natural selection is suggested to be the holobiont – the organism together with its associated symbiotic microorganisms. The well-defined endosymbiotic communities of insects make them a useful model for exploring the role of symbiotic interactions in shaping the functional repertoire of plants and animals. Here, we studied the variations in the symbiotic communities of the whitefly *Bemisia tabaci* by compiling a dataset of over 2,000 individuals derived from several independent screenings. The facultative bacteria harbored by each individual were clustered into entities termed secondary symbiont community (SSCs), each representing a natural assemblage of co-occurring bacterial species. The association of SSCs with whitefly individuals stratified the otherwise homogeneous population into holobiont units. We both identified community structures that are specific to whitefly groups sharing unique genetic backgrounds, and characterized the SSC variations within these groups. The analysis revealed that community variations are typically associated with ecological preferences, and that SSC complexity is positively correlated with both distance from the equator and specificity of the insect host interaction. Our findings point to the role of host-community and intra community interactions in shaping community structure, and encourage further exploration of the functional significance of community variations.
8 June 2014
Afternoon symposia
**Coxiella endosymbionts in the *Rhipicephalus sanguineus* brown tick species group**

**Yuval Gottlieb**

Koret School of Veterinary Medicine, Hebrew University, Rehovot, Israel; yuvalgd@yahoo.com

**Itai Lalzar**

Koret School of Veterinary Medicine, Hebrew University, Rehovot, Israel

**Lisa Klasson**

Department of Cell and Molecular Biology, University of Uppsala, Sweden

Obligatory interactions between arthropods and microbial symbionts stem from a stressful environment or restricted resources, such as diet. Ticks, which are pestiferous obligatory blood feeders that have to cope with long periods of starvation between blood meals, are therefore expected to be inhabited by primary obligatory symbionts. Although evidence for the presence of obligatory symbiotic bacteria in ticks has accumulated over the years, little is known about specific tick-symbiont interactions. We recently demonstrated a possible obligatory association between a symbiotic bacterium of the genus *Coxiella* and two of the brown tick species group: *Rhipicephalus sanguineus* and *R. turanicus*. *Coxiella* was prevalent in all individuals examined, did not interact quantitatively with other secondary symbionts, was specifically localized to the Malpighian tubules and female gonad tissues, and was maternally transmitted. The genome of the symbiotic *Coxiella*, however, demonstrates genomic characterizations of both primary and secondary known symbionts of insects, as it has a 1.7 Mbp chromosome in which there are only 922 protein-coding genes, and only 48.7% of the genome content is coding. The possible evolution route and function of *Coxiella* in *Rhipicephalus* spp. will be discussed in light of these findings.
# Phylogenomics and evolution of symbiont-mediated protection to pathogens

## Luís Teixeira

Instituto Gulbenkian de Ciência, Oeiras, Portugal; lteixeira@igc.gulbenkian.pt

*Wolbachia* protect several insects from viral infection. This tripartite interaction was initially described in *Drosophila melanogaster* carrying *wMel*, its natural *Wolbachia* strain. *wMel* has been shown to be genetically polymorphic and there has been a recent change in variant frequencies in natural populations.

We have compared the antiviral protection conferred by different *wMel* variants, their titres and influence on host longevity. The phenotypes cluster the variants into two groups - *wMelCS*-like and *wMel*-like. *wMelCS*-like variants give stronger protection against viruses, reach higher titres and often shorten the host lifespan. We have sequenced and assembled the genomes of these *Wolbachia*, and shown that the two phenotypic groups are two monophyletic groups. Our results indicate that the more protective *wMelCS*-like variants, which may have a cost, were replaced by the less protective but more benign *wMel*-like variants in natural populations.

We are currently analyzing how *wMel* variants frequencies change under experimental evolution on an outbred *D. melanogaster* population infected with *Drosophila* C virus. This will elucidate to what extent selection can act on maternally transmitted symbionts variation when a host phenotype is under selection.

Our work helps to understand the natural variation in *wMel* and its evolutionary dynamics and informs the use of *Wolbachia* in arthropod-borne disease control.
# Host-symbiont interactions at the symbiotic interfaces of obligately blood-sucking insects

**Filip Husnik**

Department of Parasitology, University of South Bohemia, Ceske Budejovice, Czech Republic; filip@paru.cas.cz

**Eva Novakova**

Department of Parasitology, University of South Bohemia, Ceske Budejovice, Czech Republic

**Eva Sochova**

Department of Parasitology, University of South Bohemia, Ceske Budejovice, Czech Republic

**Alistair Darby**

University of Liverpool, UK

**Vaclav Hypsa**

Department of Parasitology, University of South Bohemia, Ceske Budejovice, Czech Republic

Insects living exclusively on vertebrate blood, such as lice, bedbugs, Triatominae bugs or blood-sucking flies (louse flies, bat flies and tsetse flies), rely on symbiotic bacteria to supplement the nutrients missing from their blood diet (B-vitamins and cofactors). Although these insects are common vectors of pathogens, data on their interactions with the host, as well as the interactions among the mutualistic and parasitic members of the microbiome, are scarce. We characterize a simple but specific symbiotic system within the sheep parasite *Melophagus ovinus* (Diptera: Hippoboscidae). The system is composed of obligatory mutualists *Arsenophonus melophagi* and four additional facultative microorganisms, three bacteria (*Sodalis melophagi*, *Bartonella melophagi* and *Wolbachia*), and one kinetoplastid (*Trypanosoma melophagium*). Using combination of microscopy, genome sequencing and RNA-seq, we untangle interactions within the system and compare it to the functionally convergent, but phylogenetically independent symbiotic systems of other blood-sucking insects.
# *Wolbachia*-arthropod mutualisms: some comments on pathogen interference, dependence, tolerance, and resistance

**Roman Zug**

Institute for Theoretical Biology, Humboldt-Universität zu Berlin, Germany; roman.zug@biologie.hu-berlin.de

**Peter Hammerstein**

Institute for Theoretical Biology, Humboldt-Universität zu Berlin, Germany

*Wolbachia* are the most abundant bacterial endosymbionts among arthropods. They are notorious for their reproductive parasitism which, although lowering host fitness, ensures their spread. However, even for reproductive parasites it can pay to enhance host fitness. Indeed, there is increasing evidence of *Wolbachia*-associated fitness benefits, suggesting that the symbionts can also behave as mutualists, both of the facultative and obligate type. In particular, many studies report *Wolbachia*-induced anti-pathogenic effects, e.g. against several RNA viruses, different *Plasmodium* species, and bacteria. Here we critically assess the biological relevance of such *Wolbachia*-induced pathogen interference. To this end, it is crucial to check whether pathogen interference does occur in nature and, if yes, whether it is associated with a fitness benefit to the host. Based on a literature survey, we find that, so far, there is only limited support for a fitness-enhancing effect of anti-pathogenic effects in natural *Wolbachia*-host interactions.

In contrast to facultative mutualisms such as protection against pathogens, there are other *Wolbachia*-arthropod interactions where the host strictly depends on symbiont presence for reproduction or survival. We show that, frequently, such cases of host dependence (obligate mutualisms) arise through the evolution of tolerance (or "compensatory evolution") in the host. Originally, tolerance evolved as a means to cope with the harmful effects of *Wolbachia* infection. However, tolerance mechanisms are costly in the absence of infection and thus tend to render the host dependent on *Wolbachia*. Once the host depends on its symbiont, evolution of resistance (another host strategy to cope with infection) is no longer an option. This might explain why host resistance to *Wolbachia* has been found only so rarely, given that selection would act on hosts to suppress reproductive parasites.
**Speciation by symbiosis: what have we learned so far?**

**Seth Bordenstein**

Department of Biological Sciences, Vanderbilt University, Nashville, USA; s.bordenstein@vanderbilt.edu

**Rob Brucker**

Department of Biological Sciences, Vanderbilt University, Nashville, USA

In the early 20th century, Ivan Wallin conceived a significant role for microorganisms in the origin of species because he discovered the bacterial nature of mitochondria. The idea was subsequently resurrected and popularized by Lynn Margulis. Since then, several studies on *Wolbachia* helped ground the evidence for speciation by symbiosis and "moved the needle" from a controversy to a reality. I will consolidate this history and discuss how *Wolbachia*-based studies were just a beginning for blending microbial symbionts and nuclear genes, i.e. the hologenome, into a unified theory on the origin of species.
# Dobzhansky-Muller and *Wolbachia*-induced incompatibilities in a diploid genetic system

**Peter Hammerstein**

Institute for Theoretical Biology, Humboldt-Universität zu Berlin, Germany; p.hammerstein@biologie.hu-berlin.de

**Arndt Telschow**

Institute for Evolution and Biodiversity, Westfalian Wilhelms-University, Munster, Germany

Genetic incompatibilities are supposed to play an important role in speciation. A general theoretical problem is to explain the persistence of genetic diversity after secondary contact. Previous work has pointed out that Dobzhansky-Muller incompatibilities (DMI) are not stable in the face of migration unless local selection acts on the alleles involved in incompatibility. With local selection, genetic variability exists up to a critical migration rate but is lost when migration exceeds this threshold value. Here, we investigate the effect of intracellular *Wolbachia* on the stability of hybrid zones formed after the Dobzhansky Muller model. *Wolbachia* are known to cause a cytoplasmic incompatibility (CI) within and between species. Incorporating *Wolbachia* into speciation scenarios can lead to a significant increase of critical migration rates and maintenance of divergence, primarily because *Wolbachia*-induced incompatibility acts to reduce frequencies of F1 hybrids. *Wolbachia* infect up to two-thirds of all insect species and it is therefore likely that CI co-occurs with DMI in nature. The results indicate that both isolating mechanisms strengthen each other and under some circumstances act synergistically. Thus they can drive speciation processes more forcefully than either when acting alone. Moving from the abstract to the concrete, however, a number of puzzles remain. While two North American fruit fly species (*Drosophila recens* and *D. subquinaria*) serve as a showcase of CI-aided speciation, this fascinating system remains difficult to grasp theoretically.
# Turn up the heat and you'll get more sons! Bacterially facilitated temperature dependent sex ratio in an Australian species of thrips

**Duong Nguyen**

Hawkesbury Institute for the Environment, University of Western Sydney, Penrith, Australia

**Robert Spooner-Hart**

School of Science and Health, University of Western Sydney, Penrith, Australia

**Markus Riegler**

Hawkesbury Institute for the Environment, University of Western Sydney, Penrith, Australia;
m.riegler@uws.edu.au

Kelly's citrus thrips, *Pezothrips kellyanus* (Thysanoptera: Thripidae), is an economically important pest of citrus in Australasia and the Mediterranean region. We investigated the genetic diversity of *P. kellyanus* populations collected from its current range in order to detect its most likely origin. In this way, we also detected *Cardinium* and *Wolbachia* for the first time and studied their roles in the reproduction of this species.

Based on genetic markers and infection diversity we conclude that *P. kellyanus* is more likely to originate from Australasia. Australian populations are infected with both *Wolbachia* and *Cardinium*. Antibiotic treatment resulted in the establishment of uninfected populations and populations singly infected with *Cardinium*, while establishment of populations singly infected with *Wolbachia* failed. In combination, both infections caused cytoplasmic incompatibility in crosses between uninfected females and infected males. This resulted in embryonic mortality of fertilised diploid females but survival of unfertilised haploid males, and thus in all-male offspring. This is the first evidence of CI in the order Thysanoptera, potentially induced by either *Cardinium* or *Wolbachia*, or both, and we are currently testing their relative roles. We were surprised to find that not all fertilised females delivered the expected all-female offspring, suggesting male fertility problems in the absence of *Cardinium* and *Wolbachia*. Intriguingly, bacterial infections appear to restore fertility in infected individuals at lower temperature, resulting in more female-biased sex ratios at 20°C while at 25°C the sex ratio is comparable to that of uninfected thrips. Such a combined temperature and bacterial effect on sex ratio in *P. kellyanus* is unique, as sex in most insects is genetically determined and not influenced by temperature. This interaction could potentially enable this species to adapt its reproductive output to seasonal temperature changes.
9 June 2014
Morning symposia
**Wolbachia** outbreak in tsetse fly hybrids: symbiont titer regulation, bi-directional CI, and overcoming of male hybrid sterility

**Daniela I. Schneider**

Laboratories of Genome Dynamics, Medical University of Vienna, Austria; daniela.schneider@meduniwien.ac.at

**Andrew G. Parker**

Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna, Austria

**Adly M. M. Abd-Alla**

Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna, Austria

**Drion G. Boucias**

Entomology and Nematology Department, University of Florida, Gainesville, USA

**Kostas Bourtzis**

Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna, Austria

**Wolfgang J. Miller**

Laboratories of Genome Dynamics, Medical University of Vienna, Austria

Tsetse flies (*Glossina*) are vectors of *Trypanosoma*, causative agent of Human African Sleeping sickness. They have evolved an intimate relationship with three microbial endosymbionts: *Wigglesworthia glossinida*, *Sodalis glossinidus* and *Wolbachia*. They impact essential host traits like female fertility, larval development, reproduction and parasite immunity. It was suggested earlier to exploit cytoplasmic incompatibility (CI) to induce natural reproductive sterility in tsetse populations and consequently block transmission of *Trypanosoma*. This idea is currently revived as strong unidirectional CI was observed among crosses between *Wolbachia*-infected and antibiotic-treated *Glossina morsitans* group members. Natural hybridization between *Glossina* species has been reported repeatedly, suggesting weak pre-mating barriers to hybrid formation. However, hybrids exhibit reduced female fecundity and male sterility under laboratory conditions.

Here, we show that besides uni-directional CI, *Wolbachia* also trigger bi-directional CI in the *Glossina morsitans* group. We further demonstrate intensive *Wolbachia* increase in tsetse inter-species hybrids and speculate that disturbance of the native host-symbiont equilibrium in hybrids can transform *Wolbachia* into pathogens by loss of replication control. This might consequently trigger hybrid incompatibilities between tsetse flies and thereby host speciation as a side effect. In addition, we discuss the regulation of the other key bacterial endosymbionts *Wigglesworthia* and *Sodalis* in mixed hybrid backgrounds. Finally, we demonstrate that male hybrid sterility can be successfully overcome by knock-down of male *Wolbachia* titer.
**What can we infer from symbionts titre in their respective
*Drosophila* host tissues?**

**Rupinder Kaur**

Department of Agriculture and Sustainable Bioresources, Fondazione Edmund Mach, San Michele all'Adige, Italy;
rupinderkaur@fmach.it

**Thomas Hummel**

Department of Neurobiology, University of Vienna, Austria

**Omar Rota-Stabelli**

Department of Agriculture and Sustainable Bioresources, Fondazione Edmund Mach, San Michele all'Adige, Italy

**Wolfgang J. Miller**

Laboratories of Genome Dynamics, Medical University of Vienna, Austria

The endosymbiotic bacterium *Wolbachia* is considered as a potential biological tool for controlling invasive insect pest populations such as *Drosophila suzukii* (*Dsuz*), which has recently invaded European countries and come out as an emerging threat for fresh fruit cultivation. Recent reports have shown that European *Dsuz* harbors a new *Wolbachia* strain named *wSuzi*, related to the strong CI-causing *wRi* strain of *D. simulans*. Nothing is known behind this novel host-microbial integrative biology as yet. Thus, to get more insights into this symbiotic relationship, we have examined *Wolbachia* tissue tropism in adult *Dsuz* individuals sampled according to different age status. Quantitative Real Time PCR (qRT-PCR) was used to calculate density of *Wolbachia* in reproductive as well as in different somatic tissues of 1, 7, 14 and 21 days old *Dsuz* flies of both sexes. We detected significant differences in *Wolbachia* titre 1) between reproductive organs of males and females, 2) among different somatic tissues, 3) based on different age groups. Notably, in male gonads, *Wolbachia* density substantially decreases with age, suggesting the possible use of *Wolbachia*-rich young males to evaluate their CI-inducing capability. Results will be further discussed with those obtained from *D. simulans* and *D. melanogaster* harboring their respective *wRi* and *wMel* strains. Thereby this approach will help in evaluating the potential use of *wSuzi* to control *Dsuz* population in future.
# A worm in bacterial clothing: proteomic analysis supports the hypothesis that *Wolbachia*-driven recruitment of neutrophil antimicrobial proteins protects *Onchocerca ochengi* against eosinophils

**Germanus S. Bah**

Department of Infection Biology, University of Liverpool, UK

**Stuart D. Armstrong**

Department of Infection Biology, University of Liverpool, UK

**Vincent N. Tanya**

Institut de Recherche Agricole pour le Développement, Regional Centre of Wakwa, Ngaoundéré, Cameroon

**Jonathan M. Wastling**

Department of Infection Biology, University of Liverpool, UK

**Benjamin L. Makepeace**

Department of Infection Biology, University of Liverpool, UK; blm1@liv.ac.uk

Onchocerciasis is currently controlled by annual mass drug administration of a single anthelminthic, ivermectin. Although this drug is highly effective at reducing disease symptoms, it does not kill the long-lived adult filarial worms (*Onchocerca volvulus*), necessitating repeated treatments for >15 years. Both *O. volvulus* and the closely related bovine parasite *O. ochengi* have a mutualistic relationship with *Wolbachia*. Clearance of this symbiont using tetracycline leads to killing of adult *Onchocerca* spp. approximately one year post-treatment. However, the precise mechanism of action remains unclear. In this study, we treated *O. ochengi*-infected cattle with a short, ineffective oxytetracycline regimen or prolonged, adulticidal therapy. Female worms were removed from nodules in bovine skin at three time-points (0, 12 and 36 weeks post-treatment), and protein extracts were subjected to label-free, quantitative proteomics on an Orbitrap Velos mass spectrometer. Approximately 1,500 proteins were quantifiable per sample, with 30% derived from the worm, 70% from the bovine host, and <1% from *Wolbachia*. Around 100 proteins were differentially regulated, of which the vast majority were host-derived, although *Wolbachia* protein levels were significantly reduced in the prolonged treatment group as expected. Conversely, there was little impact on the expression of worm proteins, except for a glutathione S-transferase and small number of intracellular signalling proteins. The largest group of downregulated bovine proteins were neutrophil-derived antimicrobial proteins, particularly cathelicidins, azurocidin and cathepsin G. In parallel, a bovine homologue of eosinophil major basic protein was upregulated. These data support the hypothesis that *Wolbachia* induces an ineffective neutrophilic response that is disrupted by antibiotic therapy, ultimately leading to immune clearance of the worms by eosinophils.
**A•WOL macrofilaricidal drug discovery and development –
optimization of anti- *Wolbachia* efficacy**

**Louise Ford**

Parasitology Department, Liverpool School of Tropical Medicine, UK; louise.ford@liverpool.ac.uk

**Gemma L. Nixon**

Parasitology Department, Liverpool School of Tropical Medicine, UK

**Kelly L. Johnston**

Parasitology Department, Liverpool School of Tropical Medicine, UK

**Joseph D. Turner**

Parasitology Department, Liverpool School of Tropical Medicine, UK

**Neil G. Berry**

Department of Chemistry, University of Liverpool, UK

**Paul M. O'Neill**

Department of Chemistry, University of Liverpool, UK

**Stephen A. Ward**

Parasitology Department, Liverpool School of Tropical Medicine, UK

**Mark J. Taylor**

Parasitology Department, Liverpool School of Tropical Medicine, UK

There is an urgent need to develop a novel treatment for filariasis, and targeting *Wolbachia* provides safe macrofilaricidal activity with superior therapeutic outcomes compared to standard anti-filarial treatments. The Anti-*Wolbachia* (A•WOL) Consortium has developed both in vitro and in vivo assays, to screen chemical libraries for anti-*Wolbachia* activity. The outputs from the A•WOL program are now being pursued as part of A•WOL II Macrofilaricide Drug Discovery & Development programs. Screening of >10,000 compounds from the BioFocus library and chemoinformatic analysis have generated six independent lead series chemotypes with the potential to enter a medicinal chemistry "hit-to-lead" and lead optimization program. A•WOL Drug Discovery is now progressing these lead series through a rigorous lead optimisation and candidate selection process, using iterative cycles of medicinal chemistry and biological testing in order to deliver at least one novel pre-clinical candidate and a chemically distinct back-up, aligned with our target product profiles for an anti-*Wolbachia* macrofilaricide. In addition, ongoing screening of large diversity-based libraries (150 - 500 k compounds) aims to provide additional, chemically diverse hits, with one-order improvement in absolute potency or significant shortening of treatment time, in order to expand the structural diversity of anti-*Wolbachia* chemotypes. A•WOL Drug Development is optimising regimens of anti-*Wolbachia* monotherapy and combination treatment of registered anti-*Wolbachia* and anti-filarial drugs in vivo using an adult *Brugia malayi* mouse model. This efficacy testing is driven by a rational PK/PD modelling approach which supports dosage regimens, in order to identify the best treatment regimens to test in field trials.
# Functional analysis of the host immune response in the symbiotic association between the pill-bug *Armadillidium vulgare* and the feminising *Wolbachia*

**Cyril Noël**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Yves Moné**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Marie-Christine Carpentier**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Fabrice Vavre**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Pierre Grève**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Frédéric Chevalier**

Texas Biomedical Research Institute, San Antonio, USA

**Christine Braquart-Varnier**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Bouziane Moumen**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Didier Bouchon**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France; didier.bouchon@univ-poitiers.fr

Today, there is a wide consensus on the essential role of microbial associations to eukaryote evolution. In most cases, the relationship between host and symbiont is so close that the microorganisms cannot be cultured, making them difficult to study. However, high-throughput sequencing has offered new opportunities for symbiosis research.

Due to their widespread association with the *Wolbachia* endosymbionts, terrestrial isopods represent a model system to understand intimate symbioses. *Wolbachia* are vertically transmitted facultative bacteria acting as reproductive parasites in isopods, inducing the feminisation of genetic males in the pill bug *Armadillidium vulgare*. Among the three feminising *Wolbachia* identified in this host, two strains (*w*VulC and *w*VulM) vary in their prevalence and extended phenotypes. *w*VulC, the most prevalent strain exhibiting the strongest feminising effect, is also the most virulent strain inducing various fitness costs.

To decipher the conflicting associations between *w*VulC, *w*VulM and *A. vulgare*, RNA-seq experiments were conducted on cDNA libraries prepared from ovaries and from whole animals challenged by pathogenic intracellular bacteria according to their *Wolbachia* infection status. Reference transcriptome was constructed by *de novo* assembly and annotated. Immune genes were identified and compared to the immune repertoire of *Daphnia pulex*, the only one crustacean genome published today. Identification of differentially expressed immune genes (DE) was then carried out using the R package DESeq. Interestingly the highest number of DE genes was recorded in the animals infected by the less virulent strain *w*VulM. In most treatments, these DE genes could be assigned to GO categories that are underrepresented when *Wolbachia* are on board. This study is part of the widest program ImmunSymbArt granted by the French National Research Agency which aims to determine the symbiotic syndrome in four arthropod systems.
# A bug may hide another: cryptic *Wolbachia* in unfeminized lineages of Armadillidium vulgare

**Lise-Marie Genty**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Maryline Raimond**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Didier Bouchon**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Joanne Bertaux**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France; joanne.bertaux@univ-poitiers.fr

Among the *Wolbachia*-host symbioses a growing number of unexpected low titer infections has been described in bark beetles, *Drosophila paulistorum* complex of species, aphids, *D. simulans*, tse-tse fly and cherry fly. For 30 years our laboratory has bred distinct lineages of the terrestrial isopod *Armadillidium vulgare*, steadily infected or uninfected with *Wolbachia*. However, using Fluorescence In Situ Hybridization (FISH) and nested PCR, we showed that females from laboratory lineages regarded as uninfected were actually infected with cryptic *Wolbachia*. While *Wolbachia* have a feminizing effect in *A. vulgare*, the crypto-infected lineages have an equilibrated sex ratio. Moreover, *Wolbachia* remain undetected with PCR and TEM, the former standard methods to discriminate between infected and uninfected lineages.

Even more surprisingly, we also discovered a serendipitous crypto-*Wolbachia* in males of infected lineages, whereas they are considered as the individuals who escaped from the infection. In laboratory lineages both infected with *Wolbachia* and crypto-infected, males presented an opposite pattern of infection compared to crypto-infected females, with a huge infection in gonads and a low infection in the nerve chord. Additionally, we put in relation *Wolbachia* and crypto-*Wolbachia* localization patterns with the host phenotype, under the hypothesis that *Wolbachia* presence in some specific cells of the nerve chord is required to feminize woodlice.
# A new molecular sexing technique for butterflies – does *Wolbachia* really feminise genetic males in *Eurema*?

**Peter Kern**

Hawkesbury Institute for the Environment, University of Western Sydney, Penrith, Australia; p.kern@uws.edu.au

**Daisuke Kageyama**

National Institute of Agrobiological Sciences, Tsukuba, Japan

**James M. Cook**

Hawkesbury Institute for the Environment, University of Western Sydney, Penrith, Australia

**Markus Riegler**

Hawkesbury Institute for the Environment, University of Western Sydney, Penrith, Australia

The endosymbiont *Wolbachia* can bias sex ratios and modify host sex determination through cytoplasmic incompatibility, parthenogenesis, male-killing or feminisation. Feminisation is the least reported phenotype, mostly detected in terrestrial crustaceans. It also occurs in three insect species, including two closely related butterfly species in the genus *Eurema*. Feminisation cause genetic males to develop into phenotypic and functional females. So far the main evidence of feminised male butterflies has been the absence of the W chromatin body that can be detected in true females. However, its detection is not reliable for all Lepidoptera and a molecular method based on sex chromosomal markers may be better. We developed a quantitative PCR (qPCR) assay, comparing relative gene dose ratios of Z-chromosomal genes with an autosomal gene, to differentiate directly between true genotypic females and *Wolbachia* feminised males. This test correctly genotyped the sex of butterflies that lacked the feminising *Wolbachia* strain *wFem*. However, all *wFem*-infected females that were negative in the W chromatin body assay (thus considered feminised males) also had female gene dose ratios. These results conflict with the current model of feminisation of males in *Eurema* and suggest re-assessment of the role of *Wolbachia* as feminising agent in this host.
*Wolbachia*-induced paternal defect in *Drosophila* is likely by
interaction with the juvenile hormone pathway

Chen Liua

College of Life Sciences, Central China Normal University, Wuhan, China

Jia-Lin Wanga

College of Life Sciences, Central China Normal University, Wuhan, China

Ya Zheng

College of Life Sciences, Central China Normal University, Wuhan, China

En-Juan Xiong

College of Life Sciences, Central China Normal University, Wuhan, China

Jing-Jing Li

College of Life Sciences, Central China Normal University, Wuhan, China

Lin-Ling Yuan

College of Life Sciences, Central China Normal University, Wuhan, China

Xiao-Qiang Yu

School of Biological Sciences, University of Missouri, Kansas City, USA

Yu-Feng Wang

College of Life Sciences, Central China Normal University, Wuhan, China; yfengw@mail.ccnu.edu.cn

*Wolbachia* are endosymbionts that infect many insect species. They can manipulate the host's reproduction to increase their own maternal transmission. Cytoplasmic incompatibility (CI) is one such manipulation, which is expressed as embryonic lethality when *Wolbachia*-infected males mate with uninfected females. However, matings between males and females carrying the same *Wolbachia* strain result in viable progeny. The molecular mechanisms of CI are currently not clear. We have previously reported that the gene Juvenile hormone-inducible protein 26 (JhI-26) exhibited the highest upregulation in the 3rd instar larval testes of *Drosophila melanogaster* when infected by *Wolbachia*. This is reminiscent of an interaction between *Wolbachia* and juvenile hormone (JH) pathway in flies. Here we first found that the expressions of Jhamt and Met, which play key roles in JH pathway, were significantly increased in the presence of *Wolbachia*, suggesting an interaction of *Wolbachia* with the JH signaling pathway. Then we found that overexpression of JhI-26 in *Wolbachia*-free transgenic male flies caused significantly decrease in hatch rate. Surprisingly, *Wolbachia*-infected females could rescue the egg hatch. Overexpression of JhI-26 caused upregulation of the male accessory gland protein (Acp) gene CG10433, but not vice versa, suggesting that JhI-26 may function at the upstream of CG10433. Likewise, overexpression of CG10433 also led to paternal-effect lethality. Both JhI-26 and CG10433 overexpressing males resulted in nuclear division defects in the early embryos. Finally, *Wolbachia*-infected males decreased the propensity of the mated females to remating, a phenotype also caused by both JhI-26 and CG10433 overexpressing males. Our results provide a working hypothesis whereby *Wolbachia* induce paternal defects in *Drosophila* probably by interaction with the JH pathway via JH response genes JhI-26 and CG10433.

This work was supported by the National Natural Science Foundation of China (31172155).
# Symbiotic bacteria affect cuticular hydrocarbon profiles in tsetse flies (Glossina m. morsitans)

**Martin Kaltenpoth**

Research Group Insect Symbiosis, Max Planck Institute for Chemical Ecology, Jena, Germany;
mkaltenpoth@ice.mpg.de

**Tobias Engl**

Research Group Insect Symbiosis, Max Planck Institute for Chemical Ecology, Jena, Germany

**Veronika Michalkova**

Division of Epidemiology of Microbial Diseases, Yale University School of Medicine, New Haven, USA

**Brian Weiss**

Division of Epidemiology of Microbial Diseases, Yale University School of Medicine, New Haven, USA

**Daniela Schneider**

Laboratories of Genome Dynamics, Medical University of Vienna, Austria

**Wolfgang J. Miller**

Laboratories of Genome Dynamics, Medical University of Vienna, Austria

**Serap Aksoy**

Division of Epidemiology of Microbial Diseases, Yale University School of Medicine, New Haven, USA

Microbial symbionts play important roles for the ecology and evolution of many insects, with their most well-known effects being nutritional supplementation, reproductive manipulation, and defense against antagonists. However, recent studies indicated that symbionts can also alter the chemical profile of the host and thereby affect mate choice decisions, which can ultimately result in reproductive isolation and speciation. Here, we investigated the effect of bacterial symbionts on the host's cuticular hydrocarbon profiles in the multipartite symbiosis between tsetse flies (Glossina morsitans morsitans) and their associated microbial symbionts, i.e. *Wigglesworthia, Sodalis*, and *Wolbachia*. We manipulated symbiont infection status by treatment with ampicillin (reducing *Wigglesworthia* and to a lesser extent *Sodalis* titers) and tetracycline (reducing titers of all symbionts, i.e. *Wigglesworthia, Sodalis* and *Wolbachia*), respectively, and subsequently analyzed CHC profiles of male and female offspring ten days after eclosion. The results revealed that ampicillin treatment did not affect CHC amount or composition in female offspring, but changed the CHC profile of male offspring. Tetracycline treatment, on the other hand, strongly affected CHC composition of both female and male offspring, and it significantly reduced the overall amount of CHCs in males. Furthermore, the relative amount of the females' contact sex pheromone 15,19,23-trimethyl-heptatriacontane decreased upon tetracycline treatment. These results provide evidence for an influence of *Wolbachia* and possibly *Sodalis*, as well as to a lesser extent – *Wigglesworthia* on the amount and composition of CHC profiles in *G. m. morsitans*, which may have important implications for reproductive success and mate choice in tsetse flies.
10 June 2014
Morning symposia
# Double trouble: multiple endosymbiont infection and multiple manipulations in a linyphiid spider

**Meghan M. Curry**

Department of Entomology, University of Kentucky, Lexington, USA

**Jennifer A. White**

Department of Entomology, University of Kentucky, Lexington, USA; jenawhite@uky.edu

Spiders are host to a plethora of heritable endosymbiotic bacteria, but symbiont function within spider hosts has rarely been determined. We screened an assemblage of 60 linyphiid spiders in the genus *Mermessus*, collected from a single locale, and found 12 different endosymbiotic strains in the genera *Wolbachia*, *Rickettsia*, and *Cardinium*. In an additional screen of a single species, *M. fradeorum*, we detected one *Rickettsia* and two *Wolbachia* strains in multiple combinations. We established laboratory populations of two *M. fradeorum* matrilineés: one doubly infected with *Rickettsia* and *Wolbachia* and one singly infected with the other *Wolbachia* strain. The *Rickettsia*-*Wolbachia* infected mothers produced extremely female-biased offspring. Antibiotic treatment successfully eliminated both endosymbionts and restored the sex ratio to the expected 1:1 in subsequent generations. Fitness assays and preliminary chromosomal examination suggest a feminizing mechanism. The other *Wolbachia* strain induced cytoplasmic incompatibility. In a two-way factorial mating assay between *Wolbachia* infected and cured spiders, we found that cured females mated with infected males produced 73% fewer offspring than all other pairings. Interestingly, in a separate assay we also found that the *Rickettsia*-*Wolbachia* females exhibited CI when mated with males with this alternative *Wolbachia*, but at a much lower level, suggesting partial rescue. These results show that populations of *M. fradeorum* are composed of individuals with contrasting reproductive manipulations, and that symbiont infection frequencies are likely quite dynamic, potentially influencing multiple aspects of spider evolution and ecology.
# Modulation of microbiome of *Drosophila melanogaster* by *Wolbachia*

**Rama Krishna Simhadri**

Department of Biology, Boston University, USA; ramaks@bu.edu

**Eva Fast**

Department of Biology, Boston University, USA

**Horacio Frydman**

Department of Biology, Boston University, USA

The gut microbiome and *Wolbachia* affect several aspects of their host's biology, including development, fecundity, lifespan, immunity, metabolism, stem cell activity and even mate choice. The gut microbiome has also been recently implicated in altering disease transmitting capabilities of mosquitos. However, the potential interactions of *Wolbachia* with commensal bacteria are mostly unknown. Here we show that *Wolbachia* can alter the composition of the microbiome of the *Drosophila* host. We initially observed the changes in the pH of the food of the flies in a *Wolbachia*-dependent fashion. By 16S ribosomal sequencing, we identified differences in the commensal population of infected and non-infected flies as a potential source of the pH changes in the food. We further characterized the effect of *Wolbachia* on the microbiome composition dynamics of flies differentially infected with *Wolbachia* for several generations by metagenomic sequencing. We hypothesize that *Wolbachia* induced alterations in the immunity, oxidative stress, and metabolism could favor the selection of certain microbial populations by the host. We are testing these hypotheses by using *Drosophila* cell line and whole organism assays. This study raises the possibility that *Wolbachia*-host interactions could contribute to host phenotypes that were previously attributed to *Wolbachia* alone.
# Are *Wolbachia* and microbiota linked to the genetic structure of invasive and endemic populations of *Aedes albopictus*?

**Claire Valiente Moro**

University of Lyon 1, France; claire.valiente-moro@univ-lyon1.fr

**Guillaume Minard**

University of Lyon 1, France

**Florence Helene Tran**

University of Lyon 1, France

**Van Tran Van**

University of Lyon 1, France

**Christophe Bellet**

Entente Interdépartementale Rhône-Alpes pour la Démoustication, Chindrieux, France

**Khanh Ly Huynh Kim**

Institut Pasteur, Ho Chi Minh City, Vietnam

**Trang Huynh Thi Thuy**

Institut Pasteur, Ho Chi Minh City, Vietnam

**Patrick Mavingui**

University of Lyon 1, France

Above and beyond their ecological impact, invasive mosquitoes are a source of worldwide concern because of their utmost importance as vectors of a wide range of pathogens to humans and animals. Originated from South-East Asia, *Aedes albopictus* is considered as one of the most invasive species worldwide. As insect associated microbiota is recognized to play a significant role in host biology, we hypothesized that bacterial microbiota could be parts of the puzzle of understanding the driving forces of *Ae. albopictus* adaptation and subsequent invasiveness. In this context, we performed a comparative analysis of genetic (11 microsatellite markers and COI haplotype) and bacterial diversity (metagenomics on V5-V6 region of the 16S rRNA gene) of field-caught mosquito populations from Vietnam (area of origin) and France (recently colonized area). In whole mosquito body, bacterial microbiota was largely dominated by *Wolbachia pipientis* (64 to 89% of the whole bacterial content) with a conserved proportion of both *wAlbA* and *wAlbB* clades. However, analysis of gut microbiota revealed a higher diversity, in terms of species richness and evenness that was dominated by genera *Dysgonomonas* sp. and *Chryseobacterium* sp. Two important bacterial correlation networks were identified revealing possible interactions between these groups. The gut microbiota of French populations displayed a lower diversity in comparison to the Vietnamese ones as well as a lower host genetic diversity estimated through allelic richness. A new cryptic species living in sympatry with *Ae. albopictus* was identified in sylvatic Vietnamese site. Interestingly, this species did not harbor *Wolbachia* but showed a bacterial diversity similar to other ones. Our ecological genomics approach highlighted the presence of a mosquito core microbiota regardless of the geographical origin of the populations or host species and raises the question of its role in the mosquito biology and ecology.
# Evolution of the interaction *Culex pipiens* | *Wolbachia*

**Célestine Atyame**

Institut des Sciences de l'Évolution, Université Montpellier 2, France

**Emilie Dumas**

Institut des Sciences de l'Évolution, Université Montpellier 2, France

**Anna-Sophie Fiston-Lavier**

Institut des Sciences de l'Évolution, Université Montpellier 2, France

**Arnaud Berthomieu**

Institut des Sciences de l'Évolution, Université Montpellier 2, France

**Patrick Makoundou**

Institut des Sciences de l'Évolution, Université Montpellier 2, France

**Olivier Duron**

Institut des Sciences de l'Évolution, Université Montpellier 2, France

**Mathieu Sicard**

Institut des Sciences de l'Évolution, Université Montpellier 2, France

**Mylène Weill**

Institut des Sciences de l'Évolution, Université Montpellier 2, France; mylene.weill@univ-montp2.fr

Almost all *Culex pipiens* mosquitoes are infected with *wPip* strains and this interaction exhibits the greatest variation in cytoplasmic incompatibility (CI) crossing types reported in any insect. This diversity appears to be under the control of *Wolbachia* uniquely, since neither the nuclear background nor other bacterial symbionts have been reported to influence the outcome of the crossings.

After developing a specific MLST, we were able to distinguish five distinct *wPip* genetic groups, which all share a monophyletic origin. An extensive worldwide screening of *wPip* infections in 118 natural populations of the *C. pipiens* complex evidenced a clear geographic structuration. Each population was found mostly monomorphic for the *wPip* infection group. However, no clear association between *Wolbachia* and the taxons of the *C. pipiens* complex was demonstrated, showing no implication of *Wolbachia* in their differentiation.

Additionally, we crossed many *C. pipiens* lines from different geographic origins, infected with strains from distinct *wPip* groups. This large dataset showed that most crosses involving *wPip* strains from the same genetic group were compatible. The only intragroup CI observed were unidirectional, between *C. pipiens* from different geographic origins. These results support the theoretical studies predicting that only compatible *Wolbachia* strains can stably coexist in panmictic host populations.

Finally, we showed that compatible *Wolbachia* strains from the same *wPip* group, with identical MLST genotypes, could display distinct CI patterns when crossed with genetically distant lines. Their complete genomes were thus sequenced. A comparative genomic approach revealed surprisingly high genome dynamics at such a small evolutionary scale, essentially driven by repeated elements. These genomic analyses combined with our large dataset of CI relationships in *C. pipiens* provide a unique opportunity to finally identify the genes responsible for CI.
# Functional genomics of beetle-microbe symbioses

## Takema Fukatsu

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan; t-fukatsu@aist.go.jp

Beetles represent the most species-rich animal order Coleoptera, embracing over 400,000 described species in the world and accounting for almost 25% of all known animal forms. The insects are generally armored with sclerotized elytra and exoskeleton, comprising a considerable portion of the biodiversity in the terrestrial ecosystem, occupying important ecological niches as herbivores, xylovores, fungivores or carnivores, and often regarded as notorious agricultural, forestry or medical pests. Reflecting their diversity, microbial symbiotic partners of the beetles are also diverse: some are bacteria while others are fungi; some are stored in external pouches called mycangia, some are residing in the gut cavity, and others are harbored in specialized cells called bacteriocytes; some are cultured by the host beetles and utilized as food sources, some help digestion of woody materials, some provide protection against natural enemies, and others synthesize essential nutrients. Here we present our recent research progresses in the field of beetle-microbe symbiotic associations, with special emphasis on their genomic and functional aspects.
# Phylogenomics and analysis of shared genes suggest a single origin of the main lineages of *Wolbachia* in nematodes

## Francesco Comandatore

DiVET, University of Milan, Italy; francesco.comandatore@unimi.it

While in arthropods *Wolbachia* is often associated with distortion of host reproduction, in filarial nematodes it appears to be essential for the survival of the host, and this symbiotic relationship is considered mutualistic. Phylogenetic analyses highlighted that most of the *Wolbachia* strains are symbionts of filarial nematodes belonging to the C and D supergroups. The origin of this mutualistic endosymbiosis is of interest for both basic and applied reasons. To understand how and when the symbiosis between *Wolbachia* and filarial nematodes originated, a robustly rooted tree is required. The genetic distance between *Wolbachia* and the nearest outgroups and the current limited number of *Wolbachia* genomes hamper this objective. To address these issues, we sequenced the genome of the D supergroup *Wolbachia* endosymbiont of *Litomosoides sigmodontis*, thus obtaining two genomes for each of the C and D filarial *Wolbachia* supergroups.

All the high quality *Wolbachia* genomes available in database were retrieved (from A, B, C and D supergroups). A gene database was generated selecting orthologues on the basis of homology and annotation, and removing duplicated or possibly recombined or saturated genes. For these selected genes, and for the respective amino acid sequences, the best evolutionary models were found and then used in Maximum Likelihood and Bayesian phylogenetic analyses, performed on both the nucleotide and amino acid partitioned concatenates. The *Wolbachia* phylogenies obtained were congruent, and presented a highly supported root between a (A + B) clade and a (C + D) clade. This analysis supports a scenario wherein the symbiosis between *Wolbachia* and filarial nematodes originated from a single transition event. This result leaves open many questions about the mechanism of this transition. Comparative genomic analyses are currently being performed, with the goal of trying to elucidate how this transition happened.
# Link between genotype and phenotype in *Wolbachia*

**Ewa Chrostek**

Instituto Gulbenkian de Ciência, Oeiras, Portugal; echrostek@igc.gulbenkian.pt

**Luís Teixeira**

Instituto Gulbenkian de Ciência, Oeiras, Portugal

Symbiotic bacteria *Wolbachia* live within the cells of numerous insect species. Our work aims at understanding the interaction of the endosymbionts of *Drosophila melanogaster*, *wMel*, with their hosts. Although natural *wMel* variants are mutualistic, the laboratory variant *wMelPop* proliferates massively in the hosts, causing degeneration of tissues which culminates in insets early death. Sequencing and assembly of the genomes of *wMelPop* and very closely related *wMelCS* allowed us to identify genetic differences between the two variants. We demonstrate that the identified differences are responsible for the pathogenic phenotype of *wMelPop*. Therefore we provide the first link between genes and phenotypes in *Wolbachia*.
# Impact of *Wolbachia* endosymbionts on the evolution of sex determination in the isopod *Armadillidium vulgare*

**Sébastien Leclercq**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Julien Thézé**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Isabelle Giraud**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Lise Ernenwein**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Bouziane Moumen**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Pierre Grève**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Clément Gilbert**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Richard Cordaux**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France; richard.cordaux@univ-poitiers.fr

In the isopod *Armadillidium vulgare*, genetic sex determination follows female heterogamety (ZZ males and ZW females). However, many *A. vulgare* populations harbor maternally-inherited *Wolbachia* bacterial endosymbionts which can convert genetic males into phenotypic females, leading to populations with female-biased sex ratios. The W sex chromosome has been lost in lines infected by *Wolbachia* and all individuals are ZZ genetic males. The female sex is determined by the inheritance of *Wolbachia* by the *A. vulgare* individual, thereby leading to a shift from genetic to cytoplasmic sex determination. Surprisingly, some *A. vulgare* lines exhibit female-biased sex ratios despite the lack of *Wolbachia*. In these lines, female individuals are ZZ genetic males carrying an unknown feminizing factor. To elucidate the genetic basis of female sex determination in these lines, we sequenced the genome of a female by Illumina technology. After *de novo* genome assembly, we identified a large piece of the *Wolbachia* genome transferred into the *A. vulgare* nuclear genome. The transferred genomic fragment shows non-Mendelian inheritance and co-segregates perfectly with the female sex in pedigrees, in agreement with observed biased sex ratios. These results suggest that sex determination in these *A. vulgare* lines is under the control of nuclear gene(s) of bacterial origin. Overall, our results indicate that *Wolbachia* bacteria can drive shifts in sex determination mechanisms in *A. vulgare*. More generally, they emphasize that bacterial endosymbionts can be powerful sources of evolutionary novelty for fundamental biological processes in eukaryotes, such as sex determination.

This research is funded by an ERC Starting Grant (EndoSexDet) to RC.
# Toward the identification of feminizing genes of the bacterial endosymbiont *Wolbachia*

**Myriam Badawi**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France; myriam.badawi@univ-poitiers.fr

**Isabelle Giraud**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Bouziane Moumen**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Pierre Grève**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Richard Cordaux**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

Symbiotic interactions are a major driver of evolution. The symbiont genotype is able to alter the host phenotype, and the other way round: it is called "extended phenotype". In this respect, *Wolbachia* endosymbiosis is remarkable. This intracellular bacterium is a well-known reproductive parasite able to induce feminization of genetic males or cytoplasmic incompatibility in its terrestrial isopod crustacean hosts. Currently, no molecular genetic basis of these reproductive manipulations has been described. In order to identify genes involved in feminization, we used a comparative genomics approach of two closely related strains of *Wolbachia*: one inducing feminization of its host *Armadillidium vulgare* (*wVulC*) and the other one inducing cytoplasmic incompatibility in *Cylisticus convexus* (*wCon*). The effect induced by these strains is considered strain-specific as it is conserved when *Wolbachia* is cross-transfected to the other host. First, we sequenced the genome of *wCon* by 454 pyrosequencing. Genome assembly of *wCon* resulted in ~200 contigs for a total genome length of ~2 Mb and ~2,000 predicted genes. Next, a home-made bioinformatics pipeline comparing the genome of *wCon* with the previously sequenced genome of *wVulC* enabled us to determine 326 candidate genes for feminization. Then, we investigated expression patterns of these candidate genes during host sexual differentiation in order to pinpoint genes involved in the manipulation of host sex determination.

This work is funded by an ERC grant (EndoSexDet) to RC, which aims to identify the genetic factors implicated in the sex determination of *A. vulgare*, thereby contributing to evaluate the evolutionary impact of endosymbionts on sex determination of their hosts.
10 June 2014
Afternoon symposia
# *Wolbachia's Burgess Shale: ancient and modern integration in the genome of *Podisma pedestris* (Orthoptera)*

**Paloma Martínez-Rodríguez**

Departamento de Biología, Universidad Autónoma de Madrid, Spain

**José L. Bella**

Departamento de Biología, Universidad Autónoma de Madrid, Spain

**Richard A. Nichols**

SBCS, Queen Mary University of London, UK; r.a.nichols@qmul.ac.uk

Evidence is accumulating for lateral gene-transfer of *Wolbachia* into invertebrate host genomes: Approximately one-third of sequenced invertebrate genomes contain *Wolbachia* gene insertions, which range in size from short segments to nearly the entire *Wolbachia* genome. NGS skimming (very low coverage sequencing) of the grasshopper *Podisma pedestris* suggests massive repeated introgression – and a well persevered record of ancient events. We use 454 amplicon sequencing of nuclear inserts to survey the same integrated sequence from several wild populations.

Because *Podisma* has been a model species for hybrid-zone studies, its postglacial colonisation of the French Alps has been worked out in detail. Making use of the known timings for the isolation of different populations, we have begun to draw inferences about the insertions, and the post-insertion fate of integrated sequences.
# Identification of *Wolbachia*-responsive microRNAs in the two-spotted spider mite, *Tetranychus urticae*

**Xiao-Yue Hong**

Department of Entomology, Nanjing Agricultural University, China; xyhong@njau.edu.cn

**Xia Rong**

Department of Entomology, Nanjing Agricultural University, China

**Yan-Kai Zhang**

Department of Entomology, Nanjing Agricultural University, China

The two-spotted spider mite, *Tetranychus urticae* Koch, is infected with *Wolbachia*, which have the ability to manipulate host reproduction. *T. urticae*, like other organisms, produces several microRNAs (miRNAs), which are a type of small RNA (sRNA) that have the ability to regulate genes at the post-transcriptional level. To understand the role of miRNAs in the response of *T. urticae* to *Wolbachia* infection, we constructed sRNA libraries of infected and uninfected *T. urticae* for both sexes (four libraries). Illumina sequencing led to the identification of 83 known miRNAs and 112 novel miRNAs in the four libraries. We identified 91 miRNAs that were differentially expressed between infected and uninfected females and 20 miRNAs that were differentially expressed between infected and uninfected males. The four transcriptomes also provided information on protein-encoding genes that were differentially expressed in response to *Wolbachia* infection. A comparison of the miRNA and the mRNA data suggested that the miRNAs were regulating genes related to amino acid metabolism, sphingolipid metabolism, lysosome function and apoptosis. The differential expression of 14 miRNAs and one target gene were confirmed by qRT-PCR. These findings not only help to understand *Wolbachia*-mediated host miRNA variations but also provide molecular targets for further functional studies.
# Sex-specific transcription of large proportion of genes in the only cryptic WO prophage genome in a fig wasp species

**Guan-Hong Wang**

Institute of Zoology, Chinese Academy of Sciences, Beijing, China; wangguanhong@ioz.ac.cn

**Jin-Hua Xiao**

Institute of Zoology, Chinese Academy of Sciences, Beijing, China

**Da-Wei Huang**

Institute of Zoology, Chinese Academy of Sciences, Beijing, China

Temperate bacteriophage WO is a model system for studying tripartite interactions among viruses, bacteria, and eukaryotes, especially investigations of the genomic stability of obligate intracellular bacteria. Few WO genomes exist because of the difficulty in isolating viral DNA from eukaryotic hosts, and most reports are by-products of *Wolbachia* sequencing; only one partial genome of a WO phage has been determined directly from isolated particles. We successfully determined the complete genome sequence of prophage WO (WOSol) in *Wolbachia* strain *wSol*, which is the only *Wolbachia* strain infecting the fig wasp *Ceratosolen solmsi* (Hymenoptera: Chalcidoidea), by high-efficiency thermal asymmetric interlaced PCR. We further used real-time qPCR to prove WOSol that is a cryptic prophage, which are simply regarded as genetic remnants.

Only three open reading frames (ORFs) in cryptic WO prophages have previously been reported to be actively transcribed. We comprehensively examined the transcription of the only cryptic WOSol prophage in *Wolbachia* strain *wSol* by conventional reverse transcription PCR and real-time qPCR. Our major findings were: i) a high percentage of ORFs are actively transcribed (59%, 17/29); ii) the expression of these ORFs is highly sex-specific, with a strong male bias (three in females, 15 in males); iii) an ank (ankyrin-domain-containing) gene actively transcribed in both wasp sexes is more highly expressed in males. Our results suggest that cryptic WO may play roles in *Wolbachia* biology, particularly through the males.
*Spiroplasma* infection induces male-specific DNA damage response in *Drosophila melanogaster

Toshiyuki Harumoto

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan; t-harumoto@aist.go.jp

Hisashi Anbutsu

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

Takema Fukatsu

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

In *Drosophila*, two bacteria, *Wolbachia* and *Spiroplasma* are known as heritable symbionts. These bacteria are remarkable in their ability to manipulate host reproduction like cytoplasmic incompatibility and male-killing. Molecular mechanisms underlying these symbiont-induced reproductive phenotypes are of great interest but remain poorly understood. Interaction between *Drosophila melanogaster* and its native *Spiroplasma* symbiont strain MSRO is one of the best model systems for exploring how the host's molecular and cellular pathways are involved in the symbiont-induced male-killing. Previous studies showed that *Spiroplasma* infection induces ectopic apoptosis and severe neural defects only in male individuals during embryogenesis. In this study, we performed RNA-seq analysis for further understanding the molecular mechanisms of these pathogenic phenotypes. By analyzing differently expressed genes, we identified a group of genes specifically up-regulated in *Spiroplasma*-infected male embryos. These genes include regulators involved in apoptotic pathway and DNA damage response pathway. In this presentation, we would like to show our detailed molecular genetic analyses on male-specific DNA damage, and discuss the mechanistic insight into how *Spiroplasma* selectively kills male embryos in *Drosophila*.
**Wolbachia** genome sequencing: phylogenomics, symbiosis-
related pan-genome and eukaryote-like proteins involved in
host-symbiont interactions

**Sandrine Geniez**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Bouziane Moumen**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Sanjay Kumar**

New England Biolabs Inc., Ipswich, USA

**Didier Bouchon**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Jeremy M. Foster**

New England Biolabs Inc., Ipswich, USA

**Barton E. Slatko**

New England Biolabs Inc., Ipswich, USA

**Pierre Grève**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France; pierre.greve@univ-poitiers.fr

*Wolbachia* endosymbionts are widespread in arthropods and are considered reproductive parasites, inducing a variety of phenotypes including cytoplasmic incompatibility, parthenogenesis, feminization and male killing, which serve to promote their spread through populations. In contrast, *Wolbachia* infecting filarial nematodes that cause human diseases, including elephantiasis and river blindness, are obligate mutualists. These *Wolbachia* are subjects of drug discovery initiatives, but purification methods for efficient sequencing of these unculturable bacteria have proven difficult using a variety of techniques including chemical gradients, PFG purification, library construction followed by gene walking, etc. To examine the biology of symbiosis in worldwide natural populations, we have developed a novel targeted-genome enrichment procedure that allows specific isolation of endosymbiotic DNA from host mitochondrial and nuclear DNA. We assayed the robustness of this method on phylogenetically distant *Wolbachia* strains demonstrating that this procedure can be used to enrich target DNA from unculturable microbes over large evolutionary distances.

The combination of this approach with Next Generation Sequencing technologies (Illumina HiSeq and MiSeq) allowed us to successfully sequence six new strains of *Wolbachia* that induce either host feminization or cytoplasmic incompatibility in terrestrial isopods. Comparative genomics were then performed using these newly sequenced genomes together with 16 publically available genomes to establish a pan-genome of *Wolbachia* and phylogenomics studies. We then investigated the composition of this pan-genome in order to look at the insight of the basis of *Wolbachia* symbiosis. A conversed motif recognition analysis using HMMER3 also allowed us to identify eukaryote-like proteins such as ankyrin repeats containing proteins that are known to be involved in host-symbiont interactions, including the virulence of the bacteria.
**Abstracts of poster presentations**

(in alphabetical order of the presenting author)
# Quantitative analysis of proliferation and gene expression of bacteriophages infecting male-killing and non-male-killing spiroplasmas in *Drosophila*

**Hisashi Anbutsu**

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan; h-anbutsu@aist.go.jp

**Naruo Nikoh**

The Open University of Japan, Chiba, Japan

**Kohjiro Tanaka**

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

**Toshiyuki Harumoto**

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

**Takema Fukatsu**

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

Spiroplasmas are endosymbiotic bacteria infecting a variety of plants and arthropods. These bacteria cause male-killing in *Drosophila* and are also known as SRO (sex ratio organisms). It was reported that SRO Spiroplasmas are infected with short-tailed bacteriophages, but few studies have been done on the SRO phages, mainly because the SRO spiroplasmas are difficult to culture. In this study, we determined the whole genome sequences of SRO phages infecting *Spiroplasma* strains MSRO (SRO from *D. melanogaster*), NSRO (SRO from *D. nebulosa*) and NSRO-A (non-male-killing variant of NSRO), and then investigated the density dynamics of SRO phages and the expression of phage genes. In general, density dynamics of the three SRO phages in terms of the copy numbers of phage ORFs per host EF1-alpha copy were similar except for a short period immediately after the emergence. However, the density dynamics of three ORFs on NSRO-A phage showed different patterns from the other phages. On the other hand, density dynamics of the SRO phages in terms of phage ORF copies per spiroplasma dnaA copy were different among the *Spiroplasma* strains. Upon adult emergence, densities of all the three SRO phages were similar at 10 - 20 copies per *Spiroplasma*. After that, the densities of MSRO phage were almost constant throughout the adult life. In contrast, the densities of NSRO and NSRO-A phages increased around tenfold during the first week and then kept the same level. However, the densities of the three ORFs mentioned above did not increase so much in NSRO-A infected files. Interestingly, the expression levels of a gene encoded by one of the tree ORFs were about ten times higher in adult flies infected with male-killing MSRO and NSRO than in those infected with non-male-killing NSRO-A. These results represent the confusing and complex nature of NSRO-A phage, which is different from the other two phages infecting male-killing spiroplasmas and may related with the loss of pathogenicity.
# Neutralization of a redundant gene involved in homologous recombination caught in the act in *Wolbachia* endosymbionts

**Myriam Badawi**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France; myriam.badawi@univ-poitiers.fr

**Isabelle Giraud**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Fabrice Vavre**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Pierre Grève**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Richard Cordaux**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

As obligate intracellular bacteria, *Wolbachia* endosymbionts exhibit reduced genomes. This is caused by the process of genomic reduction, occurring through large genomic deletions and the accumulation of mutations. The latter process of genomic reduction involves successive steps including gene neutralization, pseudogenization and gradual erosion until complete loss. While many examples of pseudogenes at various levels of degradation have been reported in endosymbiont genomes, neutralization cases are scarce because of the transient nature of the process. Gene neutralization may occur due to relaxation of selection in non-essential genes, e.g. those involved in redundant functions. In endosymbiont genomes, the homologous recombination (HR) pathway is commonly depleted, whereas it seems intact in some (but not all) *Wolbachia* strains. The HR pathway is implicated in DNA repair and generates genome plasticity. In order to evaluate the evolutionary importance of the HR pathway for *Wolbachia* genomes, we analysed 13 major HR genes. They all have been globally under strong purifying selection during the evolution of *Wolbachia* strains hosted by arthropods. However, we uncovered the recent neutralization of the RuvA gene in a subset of *Wolbachia* strains, which may be related to an ancestral, clade-specific amino acid change that impaired DNA binding activity. Strikingly, RuvA is part of the RuvABC complex involved in branch migration and resolution of Holliday junctions, a function which may be fulfilled by the analagous RecG helicase. While RuvA is experiencing neutralization, RecG is under strong purifying selection. Thus, our high phylogenetic resolution enabled us to characterize a rare example of targeted neutralization of a gene involved in a redundant function in an endosymbiont genome.
# Exploring the influence of *Wolbachia* on *Chorthippus parallelus* mating in a Pyrenean hybrid zone

**Mar Pérez-Ruiz**

Departamento de Biología, Universidad Autónoma de Madrid, Spain

**Francisca Arroyo-Yebras**

Departamento de Biología, Universidad Autónoma de Madrid, Spain

**Paloma Martínez-Rodríguez**

Departamento de Biología, Universidad Autónoma de Madrid, Spain

**Gunter Koehler**

Institut für Ökologie, Universität Jena, Germany

**José L. Bella**

Departamento de Biología, Universidad Autónoma de Madrid, Spain; bella@uam.es

The Pyrenean hybrid zone between *Chorthippus parallelus parallelus* and the Iberian endemite *Chorthippus parallelus erythropus* (Orthoptera) was originally described as a "secondary contact zone", maintained by the balance between dispersion and the presumed reduced fitness of the natural hybrids. This is supported by the close adherence to Haldane's rule (whereby heterogametic males are sterile) of F1 laboratory hybrids of pure individuals of the two subspecies, their mating behaviour and the homogamy detected in studies of females' sperm preference.

However, recent biogeographical, molecular, cytogenetic and other findings suggest a need to complement this view with data on the influence of *Wolbachia* on various aspects of the biology of these organisms and their hybrid zone. These include cytoplasmic uni- and bidirectional incompatibility, different patterns of infection by the bacterial endosymbiont in distinct populations and areas inside and outside the hybrid zone, and the induction of a higher proportion of abnormal spermatids in natural hybrids.

In a further experiment to evaluate the role of *Wolbachia* in the hybrid zone, we have analysed the infection status of both members of copulating pairs of hybrid individuals collected in the field. Here we discuss the results of a preliminary survey of 79 pairs of hybrids using PCR with primers for *Wolbachia*'s specific wsp gene, and analysed with the Jmating, a program that examines mating frequency data to address sexual selection and sexual isolation.
**Incidence of *Wolbachia* sp., *Spiroplasma* sp. and other bacterial endosymbionts in certain parthenogenetic and non-parthenogenetic species of phasmids (Phasmatodea)**

**Mar Pérez-Ruiz**

Departamento de Biología, Universidad Autónoma de Madrid, Spain

**Paloma Martínez-Rodríguez**

Departamento de Biología, Universidad Autónoma de Madrid, Spain

**Jesús Herranz**

Departamento de Ecología, Universidad Autónoma de Madrid, Spain

**José L. Bella**

Departamento de Biología, Universidad Autónoma de Madrid, Spain; bella@uam.es

Phasmatodea reproductive biology is partially determined by poorly understood ecological and genetic mechanisms. This order includes standard sexual species, but also many others that display distinct types of parthenogenesis (tychoparthenogenesis, automixis, apomixis, etc.), or both systems facultatively. In a preliminary survey we have analysed *Wolbachia* and *Spiroplasma* infection in close to 250 individuals from around 25 species of stick-insects by bacterial 16S rRNA gene amplification. Our objective was to determine whether some of the bacterial endosymbionts involved in distinct reproductive alterations in other arthropods, including parthenogenesis and male killing, are present in phasmids.

Our results demonstrate that there was no *Wolbachia* infection in any of the phasmid species analysed, but confirm the presence of *Spiroplasma* in some sexual, mixed and asexual species. Phylogenetic analysis identifies these bacterial strains as belonging to the Ixodetis clade. Other bacteria genera were also detected. The possible role of these bacteria in Phasmatodea biology is discussed.
# The diversity of bacterial endosymbionts within bees
## (Anthophila)

**Michael Gerth**

Molecular Evolution and Systematics of Animals, University of Leipzig, Germany

**Christoph Bleidorn**

Molecular Evolution and Systematics of Animals, University of Leipzig, Germany; bleidorn@uni-leipzig.de

Several evolutionary phenomena in arthropods were shown to be due to the presence of inherited symbionts, e.g., sex-ratio-distortion, cytoplasmic incompatibility, conferring resistance to pathogens, nutritional mutualism and others. All these phenomena may be induced by *Wolbachia*, an alpha-proteobacterium that is estimated to occur in about two thirds of all terrestrial arthropods. Other endosymbionts are comparatively understudied and known only from a few model systems. Although *Wolbachia* is by far the most frequently found endosymbiont in arthropods, detailed screenings for endosymbionts of lower-ranked taxa are lacking. Furthermore, potential interactions of various endosymbionts within one species are not well understood.

In the present study, we screened 307 individuals belonging to 170 bee (Anthophila) species for the presence of the endosymbionts *Wolbachia*, *Rickettsia*, *Spiroplasma*, *Cardinium* and *Arsenophorus*. Because *Wolbachia* is present in about 66% of all bees, we tested if species carrying this infection are more or less likely to carry other endosymbionts. Interestingly, we found very different *Wolbachia* infection frequencies across bee families. Furthermore, we find that ecological and phylogenetic host background determines endosymbiont communities in bees.
# Detection of *Wolbachia* driven reduction of mitochondrial diversity using RADseq

**Marie Cariou**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France; marie.cariou@univ-lyon1.fr

**Laurent Duret**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Sylvain Charlat**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

Maternally inherited intracellular symbionts can spread within populations and across closely related species following hybridization events. Because of co-transmission with other cytoplasmic elements, this spread can induce indirect selection on mitochondrial genomes, driving selective sweeps on mitochondria or favoring mitochondrial introgression. Using a large sample of Arthropods from French Polynesia, we aim at estimating the frequency of this phenomenon and the contribution of *Wolbachia*. Selection on mitochondrial genomes can be detected through discordances between nuclear and mitochondrial coalescence times. Mitochondrial data have previously been obtained; we envisage to use RADseq to estimate nuclear diversity within and between specimens.

Estimation of genetic diversity using RADseq data can be hindered by several problems. First, polymorphism on restriction sites may lead to a biased sampling of markers toward shorter coalescent times and to an over-estimation of monomorphic loci in diploid specimens, both processes inducing an underestimation of genetic diversity. Second, the incomplete sampling of alleles in diploid specimens, when the coverage is limiting, may also induce an over-estimation of monomorphic loci and accentuate the under-estimation of polymorphism.

Here we present an ABC approach to overcome these biases by estimating jointly population parameters and parameters associated with the RADseq experiment. Large sets of sequences are simulated under different population models and RADseq data are derived from these sequences with different coverage parameters. Summary statistics are calculated on each simulated data set corresponding to each parameter set. Then, parameters that would best explain the observed summary statistics are estimated. This ABC approach should allow us to estimate nuclear coalescence time between specimens and thus to detect significantly reduced mitochondrial coalescence times, potentially driven by *Wolbachia*.
**Wolbachia shaping symbiotic communities? Insights from the
terrestrial isopod microbiome**

**Jessica Dittmer**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France; jessica.dittmer@univ-poitiers.fr

**Jérôme Lesobre**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

**Didier Bouchon**

Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, France

Terrestrial isopods represent an excellent model system for multipartite animal-bacteria
symbioses due to their well-characterized association with the endosymbiotic reproductive
parasite *Wolbachia*. To date, three different feminizing *Wolbachia* strains have been
identified in *Armadillidium vulgare*, presumably representing different co-evolutionary
histories with their common host. However, the microbiome of terrestrial isopods has never
been analysed on a large scale and the role of *Wolbachia* within the bacterial community
remains unknown. In order to fill this gap, quantitative PCR and 16S rRNA gene amplicon
pyrosequencing were combined to characterize the microbiota of *A. vulgare* on multiple
levels: (i) In field vs. lab populations, (ii) in different host tissues, and (iii) depending on
*Wolbachia* infection status, i.e. presence/absence of *Wolbachia* as well as infection with
different *Wolbachia* strains. This integrative approach allowed us to identify the major factors
shaping the microbiota associated with *A. vulgare*. *Wolbachia* infection was an important
factor influencing bacterial community structure. Furthermore, *Wolbachia* represented the
predominant member of the bacterial community in infected individuals. These findings
indicate that *Wolbachia* plays an important role within the terrestrial isopod microbiome.
However, *Wolbachia* was not the only major player: *Candidatus* Hepatoplasma
crinochetorum, a facultative symbiont previously reported from the midgut caeca, was for
the first time observed to be highly abundant in all tested host tissues. The potential
interactions of *Wolbachia* and *Ca*. H. crinochetorum constitute an interesting example for
symbiont-symbiont relationships between two highly abundant members of a diverse
bacterial community.
**Stable *Wolbachia* infection rate in the parasitoid wasp**
***Hyposoter horticola***

**Anne Duplouy**

Metapopulation Research Group, University of Helsinki, Finland; anne.duplouy@helsinki.fi

**Christelle Couchoux**

Metapopulation Research Group, University of Helsinki, Finland

**Ilkka Hanski**

Metapopulation Research Group, University of Helsinki, Finland

**Saskya van Nouhuys**

Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, USA

Despite a recent growing interest for the study of *Wolbachia* and its consequences on insect populations, our understanding of its persistence in structured and complex natural systems remains poor. We identified a new *Wolbachia* infection, strain called *wHho*, present at a low and stable rate in *Hyposoter horticola* the parasitoid wasp of the Glanville fritillary butterfly, *Melitaea cinxia*. Within the last two decades, the Glanville fritillary butterfly has become a model organism for the study of metapopulation. On the Åland archipelago between Finland and Sweden, the butterfly and its parasitoid wasp follow a similar population dynamic, with yearly local population turnover. After describing the new bacterial strain present in the wasp species, we investigated its effects on several life-history traits of its host in order to try and explain its stable persistence at a low prevalence in the Åland metapopulation system.
# Evolutionary origin of insect-*Wolbachia* nutritional mutualism

**Naruo Nikoh**

The Open University of Japan, Chiba, Japan

**Takahiro Hosokawa**

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

**Minoru Moriyama**

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

**Kenshiro Oshima**

The University of Tokyo, Japan

**Masahira Hattori**

The University of Tokyo, Japan

**Takema Fukatsu**

National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan; t-fukatsu@aist.go.jp

Obligate insect-bacterium nutritional mutualism is among the most sophisticated forms of symbiosis wherein the host and the symbiont are integrated into a coherent biological entity and unable to survive without the partnership. Originally, however, such obligate symbiotic bacteria must have been derived from free-living bacteria. How highly specialized obligate mutualisms have arisen from less specialized associations is of interest. Here we address this evolutionary issue by focusing on an exceptional insect-*Wolbachia* nutritional mutualism. While *Wolbachia* endosymbionts are ubiquitously found in diverse insects and generally regarded as facultative / parasitic associates for their insect hosts, a *Wolbachia* strain associated with the bedbug *Cimex lectularius*, designated as *wCle*, was shown to be essential for host's growth and reproduction via provisioning of B vitamins. We determined the 1,250,060 bp genome of *wCle*, which was generally similar to the genomes of insect-associated facultative *Wolbachia* strains, except for the presence of an operon encoding the complete biotin synthetic pathway that was acquired via lateral gene transfer presumably from a co-infecting endosymbiotic *Cardinium* or *Rickettsia*. Nutritional and physiological experiments, in which *wCle*-infected and *wCle*-cured bedbugs of the same genetic background were fed on B vitamins-manipulated blood meals via an artificial feeding system, demonstrated that *wCle* certainly synthesizes biotin and the *wCle*-provisioned biotin significantly contributes to the host fitness. These findings strongly suggest that acquisition of a single gene cluster consisting of biotin synthesis genes underlies the bedbug-*Wolbachia* nutritional mutualism, uncovering an evolutionary transition from facultative symbiosis to obligate mutualism facilitated by lateral gene transfer in an endosymbiont lineage.
# Cytological mechanism of *Cardinium*-induced cytoplasmic incompatibility in *Encarsia pergandiella* (Hymenoptera: Aphelinidae): preliminary results

**Marco Gebiola**

Department of Entomology, The University of Arizona, Tucson, USA; marco.gebiola@gmail.com

**Suzanne E. Kelly**

Department of Entomology, The University of Arizona, Tucson, USA

**Massimo Giorgini**

Istituto per la Protezione delle Piante, Consiglio Nazionale delle Ricerche, Portici, Italy

**Martha S. Hunter**

Department of Entomology, The University of Arizona, Tucson, USA

Cytoplasmic incompatibility (CI) between closely related populations occurs frequently in arthropods as a consequence of infections by *Wolbachia* and *Cardinium* endosymbionts. CI causes sterility in matings between infected males and uninfected or differently-infected females. CI-bacteria infect a large number of arthropods and strongly influence the ecology and evolution of their hosts. CI may also affect the invasiveness of alien genotypes and the effectiveness of biocontrol agents. Cytological analyses of *Wolbachia* CI crosses in *Drosophila* and *Nasonia* have provided insights into the mechanism of CI. These studies have shown that incompatible embryos die in the first mitotic division of the zygote, and implicate paternal chromatin remodeling and asynchrony of male and female pronuclei entering the first mitotic division. The delay leads to abnormal division or the exclusion of male chromatids. So far, nothing is known about the sequence of events in the embryogenesis of incompatible CI *Cardinium* crosses.

We conducted a cytological analysis of reproductive incompatibility caused by CI *Cardinium* in *Encarsia pergandiella*. We set up incompatible and control matings, dissected eggs from whitefly hosts, and fixed and stained eggs with DAPI during early embryogenesis. DAPI-stained eggs were imaged with a confocal microscope. We also attempted live-staining with the nucleic acid stain Syto-11 to allow for visualization of embryogenesis in real time. This technique involves dissection in buffer followed by a short incubation before imaging with a deconvolution microscope.

We observed the formation of chromatin bridges after the first mitotic division of the zygote, which is a common feature of *Wolbachia*-induced CI. Our preliminary results suggest a common mechanism of CI induced by the two bacterial lineages. Identifying parallels between CI caused by *Wolbachia* and *Cardinium* will shed light on fundamental mechanisms that underlie this reproductive incompatibility.
# Bacterial endosymbionts may induce female-biased sex ratio in the invasive mitochondrial type Q2 of *Bemisia tabaci*

**Massimo Giorgini**

Istituto per la Protezione delle Piante, Consiglio Nazionale delle Ricerche, Portici, Italy; giorgini@ipp.cnr.it

**Anna Giulia Nappo**

Istituto per la Protezione delle Piante, Consiglio Nazionale delle Ricerche, Portici, Italy

**Giuseppe Parrella**

Istituto per la Protezione delle Piante, Consiglio Nazionale delle Ricerche, Portici, Italy

The whitefly *Bemisia tabaci* (Gennadius) is a complex of cryptic species some of which, namely the Mediterranean (MED) and the Middle East-Asia Minor 1 (MEAM1), are highly invasive, and injurious crop pests worldwide, and able to displace local genotypes. Invasiveness of *B. tabaci* may correlate with the phenotype of inherited bacterial endosymbionts. In a greenhouse crop area of South Italy Q1 and Q2 mitochondrial types of MED, currently the only species found in this area, coexist in the field. Here, the introduction of Q2 (eastern Mediterranean origin) has been very recent and in few years (less than five years) Q2 largely outnumbered Q1 (the indigenous mitotype of western Mediterranean origin). Now 70% of individuals in the field are Q2. Each mitochondrial type is characterized by a specific endosymbiont composition. *Hamiltonella* and *Rickettsia* are at near fixation in Q1 and Q2 respectively; *Arsenophonus*, *Cardinium* and *Wolbachia* infect both types although at different frequencies. Contrarily to Q1, with an even sex ratio, Q2 shows a significant female-biased sex ratio. Although different agro-ecological conditions may have favored the invasion of Q2 in South Italy, the female-biased sex ratio may strongly affect the invasion biology of this mitotype. Endosymbionts (i.e. *Rickettsia*) may have a role in Q2 invasiveness acting as sex-ratio manipulators.
# Can *Wolbachia* destabilize the population dynamics of their hosts? – A mathematical analysis

**Florian Grziwotz**

Institute for Evolution and Biodiversity, Westfalian Wilhelms-University, Munster, Germany; f_grzi01@wwu.de

**Arndt Telschow**

Institute for Evolution and Biodiversity, Westfalian Wilhelms-University, Munster, Germany

Understanding the population dynamics of an arthropod species is key to design biological programs that aim to either conserving an endangered species (e.g., butterflies like the Oregon silverspot) or to controlling pests and disease vectors (e.g., mosquitoes of the genus *Aedes*). An important aspect is the stability of the system. For example, destabilized dynamics are typically associated with increased booms and busts of the population size, which could lead (1) to an increased extinction risk of endangered populations, or (2) to unpredictable and severe outbreaks of insect pests. *Wolbachia* is well known for naturally infecting new host species, and also some biological control programs involve artificial infections with *Wolbachia*. It is therefore an important question to what extend *Wolbachia* influences the population dynamics of their host. Here, we extended classical models for insect population dynamics (e.g., Dye model, Ricker model) by incorporating process noise and *Wolbachia*. Each model parameter was tested for its effect on the non-linearity of the dynamics. We found that the non-linear signature is most sensitive to parameters of density dependent regulation. Interestingly, larger variance of the parameters is more likely to destabilize the dynamics, but not their absolute values. These findings suggest that *Wolbachia*-strains, which affect intra-host dynamics, are most likely to destabilize host population dynamics. The results are discussed with respect to the Asian tiger mosquito *Aedes albopictus*, for which *Wolbachia* was shown to affect larval competition.
# Habitat-specific fitness dynamics among *Wolbachia* clades in *Drosophila melanogaster*

**Paul Herrera**

Institut für Populationsgenetik, Vetmeduni Vienna, Austria; paulherrera88@gmail.com

A natural *Drosophila melanogaster* population infected with multiple *Wolbachia* clades was allowed to evolve in two different laboratory environments (hot and cold). Pronounced habitat-specific evolutionary dynamics were observed for the different *Wolbachia* clades: the infection spread to fixation in both temperature regimes. One *Wolbachia* clade increased more than 50% in frequency within 15 generations of evolution in the cold environment (10°C / 20°C daily cycles), while the other two clades decreased in frequency. In contrast, the composition of the *Wolbachia* population remained stable over 37 generations in the hot environment (18°C / 28°C daily cycles). This population of *D. melanogaster* has continued evolving in the two environments and I will report results from more advanced generations. The causative factor(s) behind these habitat-specific patterns remain to be elucidated, however. *Wolbachia* is known to increase host fitness through protection against viral infection, improved metabolic processes or by increasing the fertility of the host. The observed patterns may also be a result of cytoplasmic incompatibility (CI) or fecundity differences among the different clades. To test this hypothesis, crosses between individuals carrying different *Wolbachia* clades are being performed among flies from both environments, allowing the direct quantification of CI. I will discuss the CI values obtained from the various crosses carried out.
# Host phenotype-associated *Wolbachia* infection polymorphism in *Vollenhovia emeryi* (Hymenoptera: Myrmicinae)

**Pureum Noh**

Division of Ecoscience, Ewha Womans University, Seoul, Korea

**Soyeon Park**

Division of Ecoscience, Ewha Womans University, Seoul, Korea

**Jae Chun Choe**

Division of Ecoscience, Ewha Womans University, Seoul, Korea

**Gilsang Jeong**

Division of Ecoscience, Ewha Womans University, Seoul, Korea; ecodever@gmail.com

The ant species, *Vollenhovia emeryi* Wheeler (Hymenoptera: Myrmicinae), is polymorphic in wing morphology of the queen caste; long-winged queen and short-winged queen. Recent studies show that they are clonally reproduced regardless of the wing morphology. Consequently, this study was conducted to investigate the *Wolbachia* infection status of the ant that might potentially be involved in its bizarre reproduction in the ant.

Either individuals or colonies of *V. emeryi* were collected during 2010 - 2013 from 80 locations, encompassing 68 locations in South Korea, 11 in Japan, and one in USA. All of the long-winged morphs were invariably infected with *Wolbachia*. On the other hand, all of the short-winged morphs were devoid of *Wolbachia* collected in South Korea. However, the Japanese short-winged morphs were geographically partially infected. Therefore we tentatively concluded that *Wolbachia* has no role in the host's clonal reproduction. Nevertheless, this may be the first case of the *Wolbachia* infection status linked to host phenotype. The phylogenetic analysis reveals that the short-winged is derived from the long-winged. This suggests the possibility of linkage between the short-wing trait and the evolution of resistance to *Wolbachia* infection in the short-winged.
**High Content Imaging: more than a pretty picture**

**Kelly L. Johnston**

Department of Parasitology, Liverpool School of Tropical Medicine, UK; johnkel@liverpool.ac.uk

**Rachel Clare**

Department of Parasitology, Liverpool School of Tropical Medicine, UK

**Darren A. Cook**

Department of Parasitology, Liverpool School of Tropical Medicine, UK

**Andrew Cassidy**

Department of Parasitology, Liverpool School of Tropical Medicine, UK

**Vera T. Unwin**

Department of Parasitology, Liverpool School of Tropical Medicine, UK

**Louise Ford**

Department of Parasitology, Liverpool School of Tropical Medicine, UK

**Stephen A. Ward**

Department of Parasitology, Liverpool School of Tropical Medicine, UK

**Mark J. Taylor**

Department of Parasitology, Liverpool School of Tropical Medicine, UK

The A•WOL consortium aims to find a novel macrofilaricidal drug to treat the debilitating diseases lymphatic filariasis and onchocerciasis, through targeting the *Wolbachia* bacteria that reside within these parasitic nematodes. Typically, screening chemical libraries directly against parasitic nematodes is cumbersome, low throughput and relies on animal reservoirs. By focusing on the endosymbiont, we have been able to utilise an insect cell-based screening approach that originally relied upon a quantitative polymerase chain reaction readout, but now employs a High Content Imaging readout running on the Perkin Elmer Operetta® platform. This assay uses texture analysis of cells stained with SYTO®11 (fluorescent DNA stain) as a direct measure of bacterial load and allows the consortium to screen up to 10× 384 well plates per day; a radical increase in throughput from the qPCR screen. Further to its use as a screening tool, the Operetta® is also being used for more fundamental biological investigations including experiments surrounding the infection dynamics within host cells as well as the development of a filarial nematode cell line as part of a Grand Challenges Explorations grant funded by the Bill and Melinda Gates Foundation. The nature of the Anti- *Wolbachia* screening approach will be presented in addition to preliminary data from other High Content Imaging investigations surrounding *Wolbachia* and host cells.
*Wolbachia* strain *wAlbB* confers both fitness cost and benefit to *Anopheles stephensi*

**Deepak Joshi**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA;
djoshi@msu.edu

**Mike Mcfadden**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA

**David Bevins**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA

**Fengrui Zhang**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA

**Zhiyong Xi**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA

*Wolbachia* is a maternally transmitted intracellular bacterium that infects up to 65% of insect species. However, it is not naturally present in *Anopheles* malaria vectors. This provides an opportunity to develop *Wolbachia*-based strategies for disease control either through population replacement to reduce vector capacity or through population suppression to reduce mosquito population. We previously generated *Anopheles stephensi* carrying a stable *wAlbB Wolbachia* infection, and demonstrated its ability to invade into the wild type laboratory populations and confer mosquitoes resistant to *Plasmodium*. The result of the undergoing fitness experiment revealed that infection of *wAlbB* caused a reduction in female fecundity and had a minor negative impact on male mating competiveness. We also observed that *wAlbB* increased the life span of both male and female mosquitoes when they were maintained solely on sugar meals; however, there was no impact on the life span of blood-fed females. In addition, *wAlbB* did not influence immature development and survivorship, and adult body sizes. Additional experiments were conducted to study the interactions of *wAlbB* causing low egg hatch with the blood sources, food nutrition and oxidative stress. We discuss these findings in possible deployment of *Wolbachia*-based strategies for malaria vector control.
# Molecular mechanism of *Wolbachia*-induced feminization in Lepidoptera

**Daisuke Kageyama**

National Institute of Agrobiological Sciences, Ibaraki, Japan; kagymad@affrc.go.jp

Sexual differentiation in the silkworm *Bombyx mori* is controlled by sex-specific splicing of doublesex (dsx). In *B. mori*, the sex-specific splicing of dsx is influenced by the insulin-like growth factor II mRNA-binding protein (Imp). *Wolbachia*-induced feminization is known in the butterfly *Eurema mandarina*. In *E. mandarina*, naturally-occurring double infection of distinct *Wolbachia* strains (*w*CI and *wFem*) is associated with feminization, while naturally-occurring single infection (*wCI*) is not. Female-specific splicing of dsx manifested in feminized *E. mandarina* can partially be reverted to male-specific splicing by treating the larvae with tetracycline, suggesting that the target of *wFem* is located in the upstream of dsx. When *wCI* and *wFem* were transferred together into cell culture derived from male embryos of *B. mori*, both Imp and dsx showed female-like pattern. On the other hand, *wCI* alone did not affect the expression of both Imp and dsx. These results may suggest that the *wFem* has a feminizing effect on *B. mori* cells. To gain more insight into the molecular mechanism of *Wolbachia*-induced feminization, transcriptome analysis (RNA-seq) was performed on uninfected cell lines derived from female *B. mori* and uninfected, single (*wCI*) infected and double (*wCI* and *wFem*) infected cell lines derived from male *B. mori*. Nearly 46,000 mRNAs were surveyed for candidate genes involved in sex determination / differentiation, *Wolbachia*-induced feminization, and / or *Wolbachia* infection / endosymbiosis.
# Evidence for horizontal *Wolbachia* transmission within *Eurema* butterflies

**Peter Kern**

Hawkesbury Institute for the Environment, University of Western Sydney, Penrith, Australia; p.kern@uws.edu.au

**Darrell Kemp**

Department of Biological Sciences, Macquarie University, North Ryde, Australia

**James M. Cook**

Hawkesbury Institute for the Environment, University of Western Sydney, Penrith, Australia

**Markus Riegler**

Hawkesbury Institute for the Environment, University of Western Sydney, Penrith, Australia

Individuals of Japanese *Eurema hecabe* and *Eurema mandarina* (Lepidoptera: Pieridae) carry either a single *Wolbachia* infection (wCI) or a double infection of wCI and wFem. wCI causes cytoplasmic incompatibility (CI), while wFem has been reported to feminise genetic males into functional females. These two *Wolbachia* strains have distinct wsp and MLST gene sequences and both strains occur in both *Eurema* species. This is probably due to *Wolbachia* introgression through hybridisation, because infected individuals in the different host species share mitochondrial haplotypes. While *E. mandarina* is restricted to temperate regions of eastern Asia, *E. hecabe* is widely distributed in Africa, Asia and Australia. In Australia, it occurs in the tropical and subtropical coastal regions of Queensland and New South Wales, and the tropical top end of the Northern Territory. Besides *E. hecabe*, Australia is home to six other *Eurema* species, which vary in their geographic ranges, life histories and host plant choice.

We sampled and screened populations of six Australian *Eurema* species (including *E. hecabe*) for *Wolbachia*. We found *Wolbachia* in five of the species, although at different prevalences. The MLST profile was identical to the wCI strain in four of the species, and we found a new MLST strain in a fifth species. However, we did not find wFem. We then COI-barcoded infected individuals and did not find any evidence of shared mitochondrial haplotypes between species, suggesting that the shared *Wolbachia* strain has not spread through hybridisation across Australian *Eurema* species. Clear differentiation at mitochondrial and nuclear loci of individuals from different species infected with the same *Wolbachia* strain also argues against *Wolbachia*-host co-divergence. We conclude that horizontal transmission by an as yet unknown route has played a key role in the evolution of *Wolbachia*-host associations in this group of butterflies.
# Supergroup J: the less-known supergroup in filarial nematode

**Emilie Lefoulon**

Muséum National d'Histoire Naturelle, Paris, France; elefoulon@mnhn.fr

**Jerome Bourret**

Muséum National d'Histoire Naturelle, Paris, France

**Ricardo Guerrero**

Laboratorio de Biología de Vectores y Parásitos, Universidad Central de Venezuela, Caracas, Venezuela

**Jorge Cárdenas**

Asociación Peruana de Helminthología e Invertebrados Afines, Lima, Peru

**Kerstin Junker**

ARC-Onderstepoort Veterinary Institute, Onderstepoort, South Africa

**Coralie Martin**

Muséum National d'Histoire Naturelle, Paris, France

*Wolbachia* are vertically transmitted intracellular bacteria which exhibit a broad host spectrum, including numerous families of arthropods and only one family of nematodes, the Onchocercidae filariae, including species that cause human filarial diseases, e.g. lymphatic filariasis and onchocerciasis. The different *Wolbachia* lineages have been classified in supergroups which show an asymmetric distribution within the large host range: supergroup A, B, E, H, I and K are commonly found in arthropods; supergroups C, D and J are limited to filariae. Only the supergroup F encompasses arthropod and filarial hosts. The present study focused on the poorly studied supergroup J. This supergroup has been defined on only the lineage found in the filarial nematode *Dipetalonema gracile*. New samples of Onchocercidae species were analyzed using PCR and immunohistochemical staining to detect *Wolbachia* and describe its strains. Here, the supergroup J *Wolbachia* was newly identified in two filaria species isolated from capybaras (Caviidae), e.g. *Yatesia hydrochoerus* and *Cruorifilaria tuberocauda*, and in four Dipetalonema species from monkeys, all originated from South America. In the adult filariae, *Wolbachia* infect the female germline and the hypodermis, with a various density in the different *Dipetalonema* species. The phylogenetic analyses confirmed the monophyly of the clade composed of *Wolbachia* from *Dipetalonema*, *Yatesia* and *Cruorifilaria* genera. The supergroup J was positioned as a sister taxon of supergroup C. Finally, this study pinpoints a specific geographic area for the supergroup J.
# Developing and characterizing *Aedes albopictus* with triple *Wolbachia* infections for disease control

**Xiao Liang**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA;
liangxiao0611@gmail.com

**Guowu Bian**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA

**Deepak Joshi**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA

**David Bevins**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA

**Zhiyong Xi**

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, USA

*Aedes albopictus* is a competent vector for at least 22 arboviruses, notably dengue and Chikungunya virus. The dramatic global expansion of *Ae. albopictus*, displacing *Ae. aegypti* in some areas, has led to increased public health concern that this mosquito would lead to serious outbreaks of arbovirus diseases. This is supported by the observation that *Ae. albopictus* is the primary cause of dengue epidemics in a number of areas including China. The ability of *Wolbachia* to both block virus transmission and spread into mosquito populations has led to an international effort to develop *Wolbachia*-based population replacement to eliminate dengue, with successful demonstrations of its potential for area-wide implementation. Different to *Ae. aegypti*, *Ae. albopictus* is naturally carrying two strains of *Wolbachia*, *wAlbA* and *wAlbB*. In order to utilize the unidirectional cytoplasmic incompatibility (CI) to spread of *Wolbachia* into the population, we introduced the third strain *wMel* into *Ae. albopictus*, resulting in a stably infected HM line carrying triple *Wolbachia* infections. The HM line is able to maintain *wMel* at a 100% maternal transmission rate. Cross experiments are conducted to determine the CI pattern between HM, the wild type, and tetracycline treated aposymbiotic lines. We also examined a potential competition among the three strains of *Wolbachia* by comparing their density in different tissues of HM and the wild type mosquito. We will discuss the above results in relation to the HM vector competence for dengue virus and its potential for use in *Wolbachia*-based vector control strategies.
# A fast multiplex PCR test for three important insect endosymbionts

**Bernd Linke**

Molecular Ecology Group, University of Innsbruck, Austria; bernd.linke@student.uibk.ac.at

**Florian M. Steiner**

Molecular Ecology Group, University of Innsbruck, Austria

**Birgit C. Schlick-Steiner**

Molecular Ecology Group, University of Innsbruck, Austria

**Wolfgang Arthofer**

Molecular Ecology Group, University of Innsbruck, Austria

The need to test for *Wolbachia* infections whenever mitochondrial diversity or sex bias in insect populations is assessed is broadly accepted today. In contrast, the contribution of other strictly endosymbiotic, sexual-parasitic proteobacteria to the current mitochondrial landscape of Hexapoda remains heavily understudied. This situation is, to a large part, caused by a lack of routine testing. Here, we present a simple one-tube test for the presence of *Wolbachia*, *Cardinium*, and *Spiroplasma*, the three most prominent bacteria causing sexual alterations in insects. The test is based on a multiplex PCR targeting the IS5 repeat region of *Wolbachia* and specific 16S regions of *Cardinium* and *Spiroplasma*, followed by a High Resolution Melting Analysis. The presence of any of the three endosymbionts will be indicated by a specific peak in the first derivative of the amplicon's melting curve. The test will provide results within two hours, without need of gel electrophoresis or any other post-PCR wetlab step. So far, we have assessed this test system with plasmids derived from host model organisms. A variety of field samples will be used in a next step to verify and fine-tune the test's diagnostic performance.
# Investigation of the putative SAM transporter in the REIS Island of *wMel*

**Eric D. LoVullo**

Agricultural Research Service, United States Department of Agriculture, Gainesville, USA;
eric.lovullo@ars.usda.gov

**Richard B. Furlong**

Agricultural Research Service, United States Department of Agriculture, Gainesville, USA

**Paul D. Shirk**

Agricultural Research Service, United States Department of Agriculture, Gainesville, USA

S-adenosylmethione (SAM) is essential for methylation in all cells. The accepted paradigm for the methylation cycle is that SAM is first produced by S-adenosylmethionine synthetase, which is encoded by metK. Subsequently, the cell utilizes an S-adenosylhomocysteinase, encoded by sahH, to detoxify the S-adenosylhomocysteine (SAH) that is produced after the transfer of the methyl group from SAM to the methylation substrate. However, some obligate intracellular bacteria have lost metK and sahH. These bacteria have replaced these functions with a SAM transporter that imports SAM as it exports the methylation inhibitor SAH.

The *wMel* genome contains a genetic island that is bounded on both ends by WoMelB prophage sequences. This island encodes for ten ORFs including a putative SAM transporter (WD0621) which appear to have been laterally transferred from a *Rickettsia* Endosymbiont of *Ixodes scapularis* (REIS) like organism. Structurally, SAM transporters share several conserved motifs including an EamA domain, a RhaT domain, and ten transmembrane domains. *In silico* analysis suggests that WD0621 fulfills all these requirements as well as having strong homology to the SAM transporter of *Rickettsia prowazekii* strain Madrid E. (RP076). The *wMel* genome also encodes a metK (WD0136), however no putative sahH has been identified.

Using a ΔmetK conditionally lethal strain of *E. coli* we analyze the function of WD0621 and determine whether the *wMel* metK can complement this deletion. We also assess the levels of transcript in *wMel* for WD0621 and metK.

We finally discuss the implications of SAM transport for an intracellular bacterium, and whether a *Wolbachia* that contains the REIS island has a selective advantage, due to the export of the methylation inhibitor SAH over *Wolbachia* strains that do not contain a SAM transporter.
# Low-input transcriptomics of *Wolbachia* and their filarial nematode hosts

**Ashley N. Luck**

New England Biolabs, Inc., Ipswich, USA; luck@neb.com

**Jeremy M. Foster**

New England Biolabs, Inc., Ipswich, USA

**Barton E. Slatko**

New England Biolabs, Inc., Ipswich, USA

*Wolbachia* is present in most human parasitic filarial nematodes, where it is an obligate endosymbiotic bacterium. In *Brugia malayi* (a filarial nematode responsible for human lymphatic filariasis, a debilitating disease affecting nearly 100 million people worldwide) depletion of *Wolbachia* through tetracycline antibiotic treatment leads to decreased fertility and eventual death in adult worms. A better understanding of how *Wolbachia* contributes to overall nematode fitness is necessary for the development of enhanced drug targeting of filarial nematodes and their *Wolbachia*. The advent of RNA-Seq has provided a method to investigate this complicated symbiosis. Numerous studies have examined the transcriptomics of filarial nematodes in response to *Wolbachia* depletion however; few have specifically examined changes in gene expression of the bacterial endosymbiont. The preparation of RNA-seq samples using the NEBNext® Ultra RNA Library Prep Kit for Illumina® (New England BioLabs, Inc.) has enabled the use of very low input amounts of total RNA extracted from filarial nematodes to examine gene expression profiles of filarial worms and *Wolbachia* simultaneously. Using this technology, we have been able to identify a number of *wBm* (*Wolbachia* from *Brugia malayi*) genes that are differentially expressed in response to the critical metabolite, heme. As heme biosynthesis occurs in the symbiont but not in the nematode, these heme-responsive genes in both *Wolbachia* and *Brugia malayi* may be critical for the symbiotic relationship and thus a target for drug discovery.
# Genetic basis of *Cardinium*-caused cytoplasmic incompatibility

**Evelyne Mann**

Institute for Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, Austria;
evelyne.mann@vetmeduni.ac.at

**Corinne M. Stouthamer**

Department of Entomology, University of Arizona, Tucson, USA

**Suzanne E. Kelly**

Department of Entomology, University of Arizona, Tucson, USA

**Martha S. Hunter**

Department of Entomology, University of Arizona, Tucson, USA

**Stephan Schmitz-Esser**

Institute for Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, Austria

Endosymbionts that infect arthropods and cause cytoplasmic incompatibility (CI) manipulate the reproduction of their hosts through "modification" of sperm and "rescue" of affected sperm in symbiont-infected eggs. The recently published genome of *Cardinium hertigii* provided first perspectives on potential CI-candidate genes. In this planned project, i.) additional genomes of *Cardinium* strains from *Encarsia* spp. causing different phenotypes (parthenogenesis, CI and one asymptomatic), will be sequenced. ii.) genes involved in host interactions and CI will be identified, iii.) differences in gene expression of *Cardinium* in male and female wasps will be examined. For i.), genome comparisons of *Cardinium* strains causing different phenotypes will greatly aid in the identification of CI candidate genes. For ii.) and iii.), an RNA extraction protocol yielding sufficient amounts of *Cardinium* RNA was established. RNA will be isolated from 1 - 3 day old male and female parasitoid wasps (*Encarsia pergandiella*). A symbiont-host RNASeq protocol already established for the amoeba symbiont *Amoebophilus asiaticus*, the closest relative of *Cardinium*, will be used. The metatranscriptome of *Cardinium* and *Encarsia pergandiella* will be analyzed, focusing on the expression of potential candidate proteins for CI and eukaryotic cell cycle regulation (e.g. ankyrin repeats, the putative antifeeding prophage secretion system or interference with the host ubiquitin system). We expect that the identification of sex-specific expression of CI-candidate genes and the comparison of *Cardinium* genomes causing different phenotypes will reveal the first insights into the reproductive manipulation of these elusive endosymbionts.
# Widespread presence of *Wolbachia* in an Alpine population of the viviparous leaf beetle *Oreina cacaliae* (Coleoptera: Chrysomelidae)

**Elena Martin**

Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano, Italy;
elena.martin@unimi.it

**Alessandra Cafiso**

Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano, Italy

**Valeria Mereghetti**

Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano, Italy

**Bessem Chouaia**

Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente, Università degli Studi di Milano, Italy

**Chiara Bazzocchi**

Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano, Italy

**Claudio Bandi**

Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano, Italy

**Sara Epis**

Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano, Italy

**Matteo Montagna**

Dipartimento di Scienze Agrarie e Ambientali, Università degli Studi di Milano, Italy

*Oreina cacaliae* (Coleoptera: Chrysomelidae) is a rare example of viviparous insect, able to feed on toxic plants and sequester toxic compounds. Here we present the results of a study on the microbiota associated with *O. cacaliae*, based on 16S rRNA bacterial gene sequencing. *Wolbachia* resulted as the dominant bacterium, both in males (100%) and in females (91.9%). Based on multilocus sequence typing, the detected *Wolbachia* was described as a new sequence type (*Wolbachia Ocac_A_wD0). Phylogenetic analyses assigned *Wolbachia Ocac_A_wD0* to supergroup-A. In situ hybridization and electron microscopy confirmed the presence of *Wolbachia* within *O. cacaliae* oocytes, indicating its transovarial transmission. PCR specific for *Wolbachia* was performed on representatives of six species of *Oreina*; the presence / absence of *Wolbachia* was then mapped on a cladogram representing the phylogeny of the insect host. Finally, since viviparous species of *Oreina* were either infected or non-infected by *Wolbachia*, we cannot derive any conclusion about the possibility that this symbiont played some role in the evolution of viviparity.
# Oxidative homeostasis and the evolution of insect / *Wolbachia* symbioses

**David Monnin**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France; david.monnin@univ-lyon1.fr

**Clément Berny**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Natacha Kremer**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Emmanuel Desouhant**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

**Fabrice Vavre**

Biometry and Evolutionary Biology Lab, University of Lyon 1, France

Reactive oxygen species (ROS) play a crucial role in insect immune defence. They also contribute to oxidative stress, a state that can lead to severe damages to cell components. Because of such antagonist effects, the oxidative homeostasis has been recently recognized as a key factor underlying trade-offs between life-history traits. Since the immune processes of insects can be triggered against any type of microorganism, a disturbance of oxidative homeostasis is expected to occur following the establishment of a symbiosis. In cases where the symbiont is highly prevalent, this disturbance is likely to constrain the host's life-history and could drive its adaptation to the symbionts (tolerance). An interaction between oxidative stress and symbiosis has been shown in associations involving *Wolbachia*. As a dramatic example, the dependence of *Asobara tabida* (Hymenoptera) to *Wolbachia* for oogenesis may be due to a breakdown of oxidative homeostasis resulting from tolerance evolution. This scenario implies both that the infection is costly for the host, and that a modulation of oxidative homeostasis can lessen this cost. We addressed these questions using *Drosophila melanogaster*, where *Wolbachia* infection is facultative, as a model. In order to assess the phenotypic effects of the infections, we measured the life-history traits of three strains of *D. melanogaster* differing only by their infection status: uninfected or infected by either *wMel* or *wMelPop*. Experimental manipulations of the oxidative environment differentially affect the life-history traits of the different strains, suggesting that the phenotypic effects of *Wolbachia* can be altered by a modulation of oxidative homeostasis. *Wolbachia* density measurements by qPCR suggest that this alteration is achieved by two independent means, one, but not the other, being related to symbiont density. These results suggest that oxidative homeostasis may often be a target of selection following symbiosis establishment.
# Transinfection of *Aedes vexans* with *wAlbB Wolbachia* by adult microinjection

**Riccardo Moretti**

Italian National Agency for New Technologies, Energy and Sustanaible Economic Development, Rome, Italy

**Francesca Marini**

Istituto Zooprofilattico Sperimentale del Lazio e Toscana, Rome, Italy

**Claudio De Liberato**

Istituto Zooprofilattico Sperimentale del Lazio e Toscana, Rome, Italy

**Maurizio Calvitti**

Italian National Agency for New Technologies, Energy and Sustanaible Economic Development, Rome, Italy

*Aedes* (*Aedimorphus) *vexans* (Meigen) is a mosquito species widely distributed all over the northern hemisphere but also present in various parts of Africa, Central America, Australia and the Pacific. In Europe, it can be considered one of the most common mosquito species. It is a known vector of various viruses among which West Nile, Rift Valley Fever, Eastern and Western Equine Encephalitis, Tahyna. Common to other species of *Aedes*, *Ae. vexans* may play a significant role in virus enzootic cycles, due to the ascertained capacity to transmit transovarially certain viruses, maintaining them in nature during inter-epidemic periods. West Nile and RVFV have already shown their potential to establish and spread in newly infected areas. Due its abundance and high potential as vector, *Ae. vexans* could be a key species in determining new outbreaks outside the present geographical range of these important viruses.

Certain *Wolbachia* strains are known to interfere with pathogen transmission causing the viremia titer in the infected hosts to not reach levels compatible with the infection of potential vectors.

*Wolbachia* transinfection is considered a valuable tool to attempt the production in laboratory of lines with a desired phenotype and having the potential to spread in the wild populations thanks to the driving force of the CI phenomenon. This strategy may lead to the replacement of vector populations with populations with reduced vector competence reducing the risk of epidemics of important diseases. Unfortunately, not all the vector species are susceptible to *Wolbachia* transinfection.

In this work, we report about the use of three different *Wolbachia* strains, *wAlbA* and *wAlbB* from *Aedes albopictus* (Skuse) and *wMel* from *Drosophila melanogaster* (Meigen) to transinfect *Ae. vexans* (naturally uninfected), by embryonic and adult transinfection. Transinfected adults were obtained by the latter method. *wAlbB* infection was transmitted vertically and established.