Humoral responses are downregulated in spaceflight larvae.

<p>Gene Ontology (GO) categories of defense response show altered gene expression in flight larvae compared to ground-reared larvae: 30 out of the 37 classified as defense response are downregulated in flight. These include the majority of response to bacteria and in particular humoral response genes. Six experimental repeats of each of the samples (flight and ground) were used to extract RNA for microarray analysis and are represented in individual columns in the figure. Rows represent levels of individual gene expression in each of the experimental repeats. Data is shown following Z-score transformation. Red colors indicate Z-scores >0 (above mean), blue colors indicate Z-scores <0 (below mean). The gene names can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015361#pone-0015361-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015361#pone-0015361-t002" target="_blank">2</a>.</p

The ability to clear bacterial infections is maintained in adult flies after spaceflight.

<p>Adults were infected with <i>E. coli</i> HB101 immediately after return from spaceflight. The overall capacity to clear the bacterial infection was measured by quantifying the live bacteria (CFU) remaining in the animal up to 3 days following infection. Flight adults cleared bacteria as effectively as controls immediately after flight, and more efficiently at days 2 and 3 post infection (<i>p-</i>value <0.05).</p

The expression of genes involved in hemocyte maturation and pathogen recognition and binding is downregulated in spaceflight larvae.

<p>Among the major categories of genes that were affected in larvae that developed in microgravity were the genes that mark the differentiation and maturation of plasmatocytes (l(3)mbn, Pxn, Pvf2,) and crystal cells (lz); genes involved in <i>E. coli</i> binding and phagocytosis; and the pattern recognition receptors GNBP3 and PGRPs which activate the humoral pathways. Gene expression level in spaceflight samples compared to ground controls is expressed as fold change. The statistical significance of the gene changes is shown by the adjusted <i>p</i>-value. The false discovery rate (FDR) cut-off was less than 0.05 for all genes.</p

Spaceflight alters phagocytosis.

<p>A. The number of actively phagocytosing cells is reduced by spaceflight. At 15, 25, and 35 minutes, a significantly lower number of cells engulfed <i>E. coli</i> in hemocyte cultures from spaceflight larvae. The calculated <i>p</i>-values were 0.32, 2.16E-07, 3.38E-05, 3.86E-08, and 5.8E-04 for the 5, 15, 25, 35 and 45-minute time points respectively. The data points are tight and give calculated standard error values in the 10⁻² range, not visible on the graph. B. The phagocytic capacity of larval plasmatocytes is stunted. The number of Alexa594-labeled <i>E. coli</i> engulfed by larval plasmatocytes in culture was compared in samples from flight and ground up to 45 minutes after infection. Cells from both space- and ground-reared larvae had similar levels of activity up to 25 minutes post-infection, while after 35 minutes space larvae plasmatocytes had significantly reduced phagocytic capacity.</p

The constitutive expression of humoral immunity genes is altered in spaceflight larvae.

<p>Specific classes of genes involved in the humoral immune response that were altered by spaceflight included: the AMP response pathway, the stress factors turandot A and C, the lysozyme genes and the melanization gene black cells. Although some of the genes upstream of AMPs were upregulated, the target AMP effectors were all downregulated. The statistical significance of gene changes is shown by the adjusted <i>p</i>-value at an FDR threshold of less than 0.05.</p

The humoral response to <i>E. coli</i> infection in adults after spaceflight.

<p>The time course of gene expression after bacterial infection was compared between flight and ground-reared adult females using qPCR. Data was averaged from two experimental samples, with the exception of relish. Mock- infected flight RNA levels were normalized against ground control to obtain the pre-infection level, and all time course points for ground and flight were normalized against their respective pre-infection level.</p

Space-reared larvae have fewer plasmatocytes and are smaller.

<p>A. The average number of plasmatocytes per larva was significantly reduced in flight vs ground (<i>p</i>-value <0.008, n = 20). B. Larvae reared in space were significantly smaller than ground-reared larvae (<i>p</i>-value <0.0085, n = 10).</p