Viruses manipulating insect behaviour : prevalence and impact on the structure of host community : example of the association Leptopilina boulardi / LbFV

Les symbioses eucaryotes/micro-organismes constituent une importante source d’innovation évolutive et de diversité écologique. Ces associations sont très répandues chez les insectes, en particulier chez les insectes parasitoïdes (insectes parasites d’autres insectes) qui hébergent en particulier une grande diversité de virus transmis verticalement. Leurs effets directs sur les parasitoïdes ainsi que les effets indirects sur la structure des communautés sont à l’heure actuelle mal compris. Nous avons abordé ces questions au travers l’étude d’un virus héritable (LbFV) ayant la particularité de manipuler le comportement de superparasitisme de son hôte, l’hyménoptère parasitoïde de drosophiles Leptopilina boulardi. La mise au point d’un outil moléculaire diagnostic de l’infection nous a permis de montrer que ce virus, spécifique à L. boulardi, peut atteindre de fortes prévalences dans les populations d’hôtes. Nous avons également mis en évidence un effet de la présence du virus sur les interactions compétitives interspécifiques au sein de la communauté des parasitoïdes de drosophiles. L’approche intégrée de ce travail constitue une étape importante dans la connaissance du rôle des virus héritables sur l’écologie et l’évolution de leurs hôtesEukaryots/microorganisms symbiosis is an important source of evolutionary novelty and ecological diversification. These associations are widespread in insects, particularly in parasitoids (insects that parasitize other insects) where a broad diversity of vertically transmitted viruses are found. However, their direct and indirect effects on host community are poorly understood. In this thesis, we used a system involving a Drosophila parasitoid, Leptopilina boulardi and a heritable virus LbFV that manipulates the behaviour of the parasitoid by increasing its tendency to lay eggs in a host that is already parasitized (superparasitism). Using a viral molecular marker developed in this work, we showed very high prevalences of the virus in L. boulardi populations. Additionally, we found a strong effect of the virus on interspecific competition in the Drosophila parasitoid community. The integrative approach of this work is an important step in understanding the role of heritable viruses in parasitoid ecology and evolutio

Virus manipulateurs du comportement des insectes : prévalence et influence sur la structure des communautés hôtes : exemple de l’association Leptopilina boulardi / LbFV

Eukaryots/microorganisms symbiosis is an important source of evolutionary novelty and ecological diversification. These associations are widespread in insects, particularly in parasitoids (insects that parasitize other insects) where a broad diversity of vertically transmitted viruses are found. However, their direct and indirect effects on host community are poorly understood. In this thesis, we used a system involving a Drosophila parasitoid, Leptopilina boulardi and a heritable virus LbFV that manipulates the behaviour of the parasitoid by increasing its tendency to lay eggs in a host that is already parasitized (superparasitism). Using a viral molecular marker developed in this work, we showed very high prevalences of the virus in L. boulardi populations. Additionally, we found a strong effect of the virus on interspecific competition in the Drosophila parasitoid community. The integrative approach of this work is an important step in understanding the role of heritable viruses in parasitoid ecology and evolutionLes symbioses eucaryotes/micro-organismes constituent une importante source d’innovation évolutive et de diversité écologique. Ces associations sont très répandues chez les insectes, en particulier chez les insectes parasitoïdes (insectes parasites d’autres insectes) qui hébergent en particulier une grande diversité de virus transmis verticalement. Leurs effets directs sur les parasitoïdes ainsi que les effets indirects sur la structure des communautés sont à l’heure actuelle mal compris. Nous avons abordé ces questions au travers l’étude d’un virus héritable (LbFV) ayant la particularité de manipuler le comportement de superparasitisme de son hôte, l’hyménoptère parasitoïde de drosophiles Leptopilina boulardi. La mise au point d’un outil moléculaire diagnostic de l’infection nous a permis de montrer que ce virus, spécifique à L. boulardi, peut atteindre de fortes prévalences dans les populations d’hôtes. Nous avons également mis en évidence un effet de la présence du virus sur les interactions compétitives interspécifiques au sein de la communauté des parasitoïdes de drosophiles. L’approche intégrée de ce travail constitue une étape importante dans la connaissance du rôle des virus héritables sur l’écologie et l’évolution de leurs hôte

Virus manipulateurs du comportement des insectes (prévalence et influence sur la structure des communautés hôtes)

Les symbioses eucaryotes/micro-organismes constituent une importante source d innovation évolutive et de diversité écologique. Ces associations sont très répandues chez les insectes, en particulier chez les insectes parasitoïdes (insectes parasites d autres insectes) qui hébergent en particulier une grande diversité de virus transmis verticalement. Leurs effets directs sur les parasitoïdes ainsi que les effets indirects sur la structure des communautés sont à l heure actuelle mal compris. Nous avons abordé ces questions au travers l étude d un virus héritable (LbFV) ayant la particularité de manipuler le comportement de superparasitisme de son hôte, l hyménoptère parasitoïde de drosophiles Leptopilina boulardi. La mise au point d un outil moléculaire diagnostic de l infection nous a permis de montrer que ce virus, spécifique à L. boulardi, peut atteindre de fortes prévalences dans les populations d hôtes. Nous avons également mis en évidence un effet de la présence du virus sur les interactions compétitives interspécifiques au sein de la communauté des parasitoïdes de drosophiles. L approche intégrée de ce travail constitue une étape importante dans la connaissance du rôle des virus héritables sur l écologie et l évolution de leurs hôtesEukaryots/microorganisms symbiosis is an important source of evolutionary novelty and ecological diversification. These associations are widespread in insects, particularly in parasitoids (insects that parasitize other insects) where a broad diversity of vertically transmitted viruses are found. However, their direct and indirect effects on host community are poorly understood. In this thesis, we used a system involving a Drosophila parasitoid, Leptopilina boulardi and a heritable virus LbFV that manipulates the behaviour of the parasitoid by increasing its tendency to lay eggs in a host that is already parasitized (superparasitism). Using a viral molecular marker developed in this work, we showed very high prevalences of the virus in L. boulardi populations. Additionally, we found a strong effect of the virus on interspecific competition in the Drosophila parasitoid community. The integrative approach of this work is an important step in understanding the role of heritable viruses in parasitoid ecology and evolutionLYON1-BU.Sciences (692662101) / SudocSudocFranceF

An inherited virus influences the coexistence of parasitoid species through behaviour manipulation.

International audienceThe potential role of pathogens or parasites in maintaining species coexistence is well documented. However, the impact of vertically transmitted symbionts, that can markedly modify their host's biology, is largely unknown. Some females of the Drosophila parasitoid Leptopilina boulardi are infected with an inherited virus (LbFV). The virus forces females to lay supernumerary eggs in already parasitised hosts, thus allowing its horizontal transmission. Using two independent experimental procedures, we found that LbFV impacts inter-specific competition between L. boulardi and the related L. heterotoma. While L. boulardi rapidly outcompetes L. heterotoma in the absence of the virus, L. heterotoma was able to maintain or even to eliminate L. boulardi in the presence of LbFV. By forcing females to superparasitise, LbFV induced egg wastage in L. boulardi thus explaining its impact on the competition outcome. We conclude that this symbiont whose transmission is L. boulardi-density-dependant may affect the coexistence of Leptopilina species

Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

International audiencePore-forming toxins are potent virulence factors secreted by a large array of bacteria. Here, we deciphered the action of ExlA from Pseudomonas aeruginosa and ShlA from Serratia marcescens on host cell-cell junctions. ExlA and ShlA are two members of a unique family of pore-forming toxins secreted by a two-component secretion system. Bacteria secreting either toxin induced an ExlA- or ShlA-dependent rapid cleavage of E-cadherin and VE-cadherin in epithelial and endothelial cells, respectively. Cadherin proteolysis was executed by ADAM10, a host cell transmembrane metalloprotease. ADAM10 activation is controlled in the host cell by cytosolic Ca2+ concentration. We show that Ca2+ influx, induced by ExlA or ShlA pore formation in the plasma membrane, triggered ADAM10 activation, thereby leading to cadherin cleavage. Our data suggest that ADAM10 is not a cellular receptor for ExlA and ShlA, further confirming that ADAM10 activation occurred via Ca2+ signalling. In conclusion, ExlA- and ShlA-secreting bacteria subvert a regulation mechanism of ADAM10 to activate cadherin shedding, inducing intercellular junction rupture, cell rounding and loss of tissue barrier integrity

ADAM10 requirement for ExlA-dependent cadherin cleavage.

<p><b>A</b>. A549 cells were either treated with 0.1 μg/mL PMA or 5 μM ionomycin (Iono) for 30 min, or infected with CLJ1 (90 min), or left untreated/uninfected (NI). Cellular extracts were analysed by Western blot using E-cadherin and β-actin antibodies. FL, full-length; CTF, C-terminal fragment. The experiment was performed twice. <b>B</b>. A549 cells (left) or HUVECs (right) were pre-treated with DMSO, the general metalloprotease inhibitor GM6001 (10 μg/mL) or the specific ADAM10 inhibitor GI254023X (5 μM) and then incubated with CLJ1 or IHMA87 (90 min), or uninfected (NI). Cellular extracts were analysed as above. The experiment was performed twice for A549 and 3 times for HUVECs. <b>C</b>. A549 or ADAM10-deficient A549 (A549 <i>ADAM10</i>^<i>-/-</i>) cells were incubated with either CLJ1 or IHMA87. Cellular extracts were prepared at different time points post-infection as indicated and analysed by Western blot (left). The right panel shows the FACS analysis of ADAM10 surface expression of both cell lines, as well as the negative control. The experiment was performed 3 times. <b>D</b>. Similar experiment with HUVECs, either transfected with ADAM10 siRNA or untreated. The experiment was performed twice.</p

Intracellular calcium elevation generated by ExlA-secreting bacteria.

<p>Intracellular Ca²⁺ content was analysed by videomicroscopy using the permeant Fluo3-AM fluorescent probe. Plasma membrane rupture was evaluated using the non-permeant Draq7 fluorescent probe. A549 cells (<b>A,C,E,G,I,K</b>) or HUVECs (<b>B,D,F,H,J,L</b>) were either non-infected (<b>A,B</b>) or infected with PAO1F (<b>C,D</b>), CLJ1 (<b>E,F</b>), IHMA87 (<b>G,H</b>), IHMA87Δ<i>exlA</i> (<b>I,J</b>) or IHMA87Δ<i>exlA</i>::<i>exlBA</i> (<b>K,L</b>). The fluorescence intensities of five cells were analysed in each case; the Fluo3 intensities are represented by solid lines and the Draq7 intensities by dashed lines, using the same colour code for one cell. The data are representative of 4–7 experiments. Uninfected conditions were performed in each experiment.</p

Effects of S. marcescens ShlA on cadherin cleavage and calcium influx.

<p><b>A</b>. A549 cells (left) or HUVECs (right) were incubated with the <i>S</i>. <i>marcescens</i> ShlA-secreting strain Db11, or with the non-ShlA-secreting mutant 21C4. Cellular extracts were analysed for their E- or VE-cadherin contents. The experiment was performed twice for the left panel and once for the right panel. <b>B</b>. Similar analysis using A549 <i>ADAM10</i>^<i>-/-</i>. The experiment was performed once. <b>C</b>. Similar analysis using A549 cells, in presence/ absence of BAPTA-AM. <b>D-G</b>. Intracellular Ca²⁺ contents and plasma membrane permeability were measured using Fluo3-AM and Draq7 fluorescent probes, respectively. A549 cells (<b>D,F</b>) and HUVECs (<b>E,G</b>) were infected with Db11 (<b>D,E</b>) or 21C4 (<b>F,G</b>) and fluorescence was recorded on both channels by videomicroscopy. Five cells were analysed in each case; the Fluo3 intensities are represented by straight lines and the Draq7 intensities by dashed lines, using the same colour code for one cell. Data are representative of 8 and 5 independent experiments for A549 and HUVECs, respectively.</p

Mechanisms of ExlA/ShlA-induced cadherin cleavage.

<p>In uninfected cells, pro-ADAM10 is associated with calmodulin, preventing its maturation and export to the plasma membrane. Pore formation by ExlA or ShlA induces a massive Ca²⁺ influx in host cells. Intracellular Ca²⁺ interacts with the Ca²⁺-binding protein calmodulin, which detaches from pro-ADAM10, allowing its maturation to m-ADAM10. m-ADAM10 cleaves E- and VE-cadherin in epithelial and endothelial cells, respectively, provoking intercellular junction rupture.</p

ExlA necrotizing activity is preserved in ADAM10-deficient cells.

<p>Plasma membrane rupture was monitored by LDH release in the supernatant. A549 or A549<i>ADAM10</i>^<i>-/-</i> cells were incubated for 5 hours with IHMA87, IHMA87Δ<i>exlA</i> or IHMA87Δ<i>exlA/exlA</i> strains. The supernatants were the tested for LDH activity. The histograms show the mean ± s.d. of triplicates. The data are representative of 3 experiments.</p