Knock down of <i>edin</i> in the fat body decreases the encapsulation and killing ability of <i>Drosophila</i> larvae.

<p><b>(A)</b> The encapsulation response of two different <i>edin</i> RNAi lines (<i>edin</i>^<i>14289</i> and <i>edin</i>^{<i>109528</i>}) was analyzed 27-29h after a wasp infection. The <i>C564</i>-<i>GAL4</i> (<i>C564>)</i>, <i>Fb-GAL4</i> (<i>Fb></i>) and <i>Hml</i>^<i>Δ</i>;<i>He-GAL4</i> (<i>HH></i>) drivers were used to drive the expression of the RNAi constructs. <i>w</i>^<i>1118</i> (<i>w</i>) was used as control. Data were pooled from one to eight individual experiments, as depicted on each column, each experiment with at least 50 analyzed individual infected larvae. <b>(B)</b> The ability of <i>Drosophila</i> larvae to kill wasp eggs was assessed with two different <i>edin</i> RNAi lines (<i>edin</i>^<i>14289</i> and <i>edin</i>^{<i>109528</i>}) 48-50h after infection. The <i>C564</i>-<i>GAL4</i> (<i>C564></i>) and <i>Fb-GAL4</i> (<i>Fb></i>) drivers were used to drive the expression of the RNAi constructs. <i>w</i>^<i>1118</i> (<i>w</i>) was used as control. Data are pooled from three to sixteen independent experiments, as indicated on each column, and at least 50 infected larvae were scored per experiment. Error bars in A and B show standard deviations. Knocking down the expression of <i>edin</i> in several tissues including the fat body or in the fat body alone caused a significant decrease in the encapsulation activity and killing response of <i>Drosophila</i> larvae compared to controls, whereas knocking down <i>edin</i> in hemocytes had no effect.</p

<i>Edin</i> expression in fat body is dispensable for normal hemocyte attachment to and spreading on glass and wasp eggs, but is necessary to increase blood cell numbers in circulation early after wasp infection.

<p>(<b>A-B”</b>) Hemocytes from infected control larvae (<i>msnCherry</i>,<i>eaterGFP</i>, A-A”) and from infected larvae in which <i>edin</i> was knocked down in the fat body (<i>msnCherry</i>,<i>eaterGFP;Fb>edin</i>^{<i>109528</i>}, B-B”) spread normally on glass 14 hours after wasp infection despite knock down of <i>edin</i> in fat body. The spreading ability of hemocytes was assayed by staining α-Tubulin (blue) and F-actin (magenta). The size bar denotes 10 μm. (<b>C and D</b>). Wasp eggs from infected control larvae (<i>msnCherry</i>,<i>eaterGFP</i>, C) and from infected larvae in which <i>edin</i> was knocked down in the fat body (<i>msnCherry</i>,<i>eaterGFP;Fb>edin</i>^{<i>109528</i>}, D) were stained with the anti-plasmatocyte antibody <i>NimC1</i>. The wasp eggs were dissected 14 hours after parasitization and are still attached to the gut. Plasmatocytes spread normally on the eggs irrespective of <i>edin</i> RNAi in the fat body. Arrows denote examples of plasmatocytes spreading and adhering normally on the surface of the wasp egg. The scale bar depicts 50 μm. (<b>E</b>) <i>Edin</i> RNAi in the fat body (<i>msnCherry</i>,<i>eaterGFP;Fb>edin</i>^{<i>109528</i>}) reduced the number of circulating cells after wasp infection in comparison to control larvae (<i>msnCherry</i>,<i>eaterGFP</i>) 14 hours after infection. Circulating blood cell numbers were obtained with flow cytometry.</p

A schematic presentation of the function of Edin.

<p>(1.) <i>Edin</i> is induced in the fat body shortly after wasp infection and secreted into the hemolymph. There, Edin directly or indirectly induces the release of plasmatocytes from the sessile hemocyte compartment. These cells go into circulation, find the wasp egg and participate in forming the capsule around the parasitoid egg. (2.) If the expression of <i>edin</i> is knocked down in the fat body in the context of a wasp infection, plasmatocytes are retained in the sessile compartment instead of being released into circulation, causing a defect in the encapsulation of the wasp egg.</p

Six different hemocyte classes.

<p>(A) Hemocyte gates. Plasmatocytes (GFP^himCh^-) and lamelloblasts (GFP^lomCh^-) expressed only GFP. Activated plasmatocytes (GFP^himCh^lo) were equal in size to plasmatocytes and had varying amounts of mCherry-positive punctae in their cytoplasm. Similar to activated plasmatocytes, type II lamellocytes expressed both markers (GFP^hi mCh^hi). Type II lamellocytes were larger than plasmatocytes, similar in shape to lamellocytes, and with strong expression of mCherry in their cytoplasm. Prelamellocytes (GFP^lomCh^lo) had low GFP expression while increasing their mCherry expression. Type I lamellocytes (GFP^-mCh^hi) expressed only mCherry. The negative population expressed neither GFP nor mCherry. (B-B”‘) Representative images of hemocyte types. (B) plasmatocytes (filled arrowheads) and lamelloblasts (open arrowheads). (B’) Activated plasmatocytes (filled arrowheads). (B”) Type II lamellocyte (star). (B”‘) Lamellocytes type I (arrows), prelamellocyte (open arrowhead), activated plasmatocyte (filled arrowhead), and lamellocyte type II (star). Scale bars 10 μm. (C) Flow cytometry plots at representative time points after a wasp infection. <i>Me/w</i> larvae were uninfected or infected by <i>L</i>. <i>boulardi</i>, <i>L</i>. <i>clavipes</i>, and <i>L</i>. <i>heterotoma</i>. The time points were chosen to cover major changes in the composition of the hemocyte population in the course of the time line experiment. In uninfected animals mainly plasmatocytes were present in the circulation. The cellular immune response after infection by <i>L</i>. <i>boulardi</i> and <i>L</i>. <i>clavipes</i> proceeded in a stereotypical way. At 10–12 h after infection, two plasmatocyte-like populations, plasmatocytes and lamelloblasts, were present in the circulation. At 18–20 h after infection, lamelloblasts developed into prelamellocytes. Plasmatocytes started to appear already 8–10 h after infection, and were the dominant cell type at 48–50 h. The first type I lamellocytes were seen in the circulation 20–22 h after infection. Large numbers of type I lamellocytes were in the circulation 28–30 h and 48–50 h after infection. A <i>L</i>. <i>heterotoma</i> infection induced a similar immune response until 18–20 h after infection. Then the numbers of prelamellocytes and lamellocytes type I were reduced in comparison to <i>L</i>. <i>boulardi</i> and <i>L</i>. <i>clavipes</i>-infected larvae. 48–50 h after a <i>L</i>. <i>heterotoma</i> infection, plasmatocytes and activated plasmatocytes were the dominant hemocyte types present. They were accompanied by only very few type I lamellocytes.</p

Model.

<p>(A) Steady-state hematopoiesis: Hemocytes of the plasmatocyte lineage prevail. Plasmatocytes self-renew, proliferate, and give rise to a small percentage of activated plasmatocytes. (B) Demand-adapted hematopoiesis. After wasp infection, hemocytes of the plasmatocyte lineage increase their proliferation rate. Plasmatocytes give rise to activated plasmatocytes with large and abundant mCherry-positive foci. And plasmatocytes transdifferentiate into type II lamellocytes on the wasp egg. A wasp infection induces the lamellocytes lineage. Putative prohemocytes that reside within the sessile hemocyte bands, and which are double-negative for the dual reporter constructs, give rise to circulating vigorously dividing lamelloblasts that transiently express GFP. These cells develop into type I lamellocytes via an intermediate prelamellocyte. All cell types are able to divide with the exception of type I lamellocytes.</p