Time-course analysis of <i>Drosophila suzukii</i> interaction with endoparasitoid wasps evidences a delayed encapsulation response compared to <i>D</i>. <i>melanogaster</i>

<div><p><i>Drosophila suzukii</i> (the spotted-wing Drosophila) appears to be unsuitable for the development of most Drosophila larval endoparasitoids, be they sympatric or not. Here, we questioned the physiological bases of this widespread failure by characterizing the interactions between <i>D</i>. <i>suzukii</i> and various parasitoid species (<i>Asobara japonica</i>, <i>Leptopilina boulardi</i>, <i>Leptopilina heterotoma</i> and <i>Leptopilina victoriae</i>) and comparing them with those observed with <i>D</i>. <i>melanogaster</i>, a rather appropriate host. All parasitoids were able to oviposit in L1 and L2 larval stages of both hosts but their propensity to parasitize was higher on <i>D</i>. <i>melanogaster</i>. <i>A</i>. <i>japonica</i> and, to a much lesser extent, <i>L</i>. <i>heterotoma</i>, were the two species able to successfully develop in <i>D</i>. <i>suzukii</i>, the failure of the parasitism resulting either in the parasitoid encapsulation (notably with <i>L</i>. <i>heterotoma</i>) or the host and parasitoid deaths (especially with <i>L</i>. <i>boulardi</i> and <i>L</i>. <i>victoriae</i>). Compared to <i>D</i>. <i>melanogaster</i>, encapsulation in <i>D</i>. <i>suzukii</i> was strongly delayed and led, if successful, to the production of much larger capsules in surviving flies and, in the event of failure, to the death of both partners because of an uncontrolled melanization. The results thus revealed a different timing of the immune response to parasitoids in <i>D</i>. <i>suzukii</i> compared to <i>D</i>. <i>melanogaster</i> with a lose-lose outcome for parasitoids (generally unsuccessful development) and hosts (high mortality and possible reduction of the fitness of survivors). Finally, these results might suggest that some European endoparasitoids of Drosophila interact with this pest in the field in an unmeasurable way, since they kill their host without reproductive success.</p></div

Parasitoid propensity to parasitize (PP) and infestation rate (IR).

<p>Parasitoid propensity to parasitize (PP) and infestation rate (IR).</p

Suitability of <i>D</i>. <i>suzukii</i> L1 and L2 larval stages for the parasitoid species/strains.

<p>Suitability of <i>D</i>. <i>suzukii</i> L1 and L2 larval stages for the parasitoid species/strains.</p

Time-course of the wasp-host interaction in parasitized <i>D</i>. <i>suzukii</i> larvae.

<p>Live larvae of <i>D</i>. <i>suzukii</i> parasitized by the different parasitoid strains or <i>D</i>. <i>melanogaster</i> YR (YR) parasitized by <i>L</i>. <i>boulardi</i> ISy were dissected at different time (0h-240h) and the main observed steps of the encapsulation response are reported. E, free parasitoid egg; EE, encapsulated parasitoid egg; FL, free parasitoid larva; LC, free parasitoid larva with a thin coat of light-colored cells; DL, dead parasitoid larva; EL, encapsulated parasitoid larva; W, developing wasp. Aj, <i>Asobara japonica</i>; Lv, <i>Leptopilina victoriae</i>; LhGoth, <i>L</i>. <i>heterotoma</i> Gotheron; LhJapan, <i>L</i>. <i>heterotoma</i> Japanese strain; Lbm, <i>L</i>. <i>boulardi</i> ISm strain; Lby, <i>L</i>. <i>boulardi</i> ISy strain; Lb16, <i>L</i>. <i>boulardi</i> strain 16.</p

Sequence analysis of LbGAPy, the LbGAP homolog from the ISy <i>L. boulardi</i> line.

<p>(<b>A</b>) Schematic representation of LbGAP and LbGAPy amino acid sequences. The signal peptide (SP) and the RhoGAP domain are shown as black and gray rectangles, respectively. (<b>B</b>) Sequence alignment of LbGAP and LbGAPy amino acid sequences. Residues identical or similar are highlighted in black and grey, respectively. The signal peptide is indicated by a dotted line. The RhoGAP domain is underlined. Stars identify LbGAP residues involved in the interaction with Rac GTPases.</p

The RacGAP protein is necessary for virulence of ISm females.

<p>(<b>A</b>) <i>D. melanogaster</i> resistant L2 larvae were injected with ISm venom incubated either with the pre-immune serum as a control or a specific polyclonal antibody against LbGAP, then parasitized with the ISy avirulent line. The encapsulation rate was recorded after 48 h. In brackets are the numbers of injected larvae. (<b>B</b>) <i>D. melanogaster</i> resistant L2 larvae were injected with the pre-immune serum as a control or the specific antibody against LbGAP, and then parasitized with the ISm virulent line. The encapsulation rate was recorded after 48 h. In brackets are the numbers of injected larvae.</p

<i>LbGAP</i> expression in ISm females is higher than <i>LbGAP</i>y expression in ISy females.

<p>(<b>A</b>) Relative expression of <i>LbGAP</i> (ISm line) and <i>LbGAP</i>y (ISy line) in venom-producing tissues compared to the rest of the body without venom-producing tissues. (<b>B</b>) Ratio of relative expression of <i>LbGAP</i> (ISm line) compared to <i>LbGAP</i>y (ISy line) in venom-producing tissues and in the rest of the body without venom-producing tissues. For each value, error bars represent the standard error of three measurements.</p

Variation of expression of <i>LbGAP</i> and <i>LbGAPy</i>.

<p>PCR experiments were performed on genomic DNA (+) to assess the specificity of the <i>LbGAP</i>- and <i>LbGAPy</i>-specific primers, and on serial dilutions of cDNA templates (1, 1/10, 1/100) to determine whether the variation of expression of <i>LbGAP</i> and <i>LbGAPy</i> is under the control of <i>cis</i>- or <i>trans</i>-acting elements. The <i>ITS2</i> ribosomal sequence was used as control to assess the quantity of the DNA and RNA samples. (−) Negative control.</p

Functions of spherulocytes and wax cells.

<p>(A–C) Early alterations of spherulocytes after hemolymph collection: (A) loss of spherules and fibrils' formation (arrowhead). <i>Ba</i>: <i>B. aphidicola</i>. (B) Unstable cytoplasmic blebs (arrow) derive from spherulocytes while spherules remain intact (arrowhead). (C) From blebs (arrow), long stable strands like strings of pearls extend (asterisk; see also movie 1). (D) Low magnification of a large coagulum stained by neutral red showing a granular aspect and the presence of an intact spherulocyte (<i>Sp</i>). (E–F) Wax cells (<i>Wx</i>) are localized at the base and inside the cornicles (<i>Co</i>), secretory appendices localized at the posterior end of aphids' bodies (inset in (E)). (E) Red lipid staining showing large accumulation of lipid-containing cells at the base of the cornicle. (F) Wax cell containing a large neutral lipidic inclusion (pink staining) that almost fills entirely the cytoplasm. Scale bars: 15 µm (A), 10 µm (B, C, D, F) and 100 µm (E and inset in (E)).</p

Origin and characteristics of the aphid lines.

*<p>: artificial lines.</p