5). A no-probe control verified the specific fluorescence of the endosymbionts, as no fluorescence was MLN2238 nmr observed. Figure 4 FISH of infected and uninfected M. pygmaeus

ovarioles (60 x objective). All images were acquired using identical settings and the contrast has been adapted equally. A: Maximum intensity projection of 20 confocal sections of an infected M. pygmaeus ovariole, B: Optical section of an infected M. pygmaeus ovariole, C: Optical section of a cured M. pygmaeus ovariole. 1: Bright field channel, 2: Rickettsia Cy3 channel, 3: Wolbachia Cy5 channel, 4: overlay of Rickettsia and Wolbachia channel. Green: Rickettsia, Red: Wolbachia. Figure 5 Volume rendered view of an infected ovariole, showing the colocalization of Rickettsia (green) and Wolbachia (red). The picture was made in NIS-viewer (Nikon Instruments Inc., Badhoevedorp, The Netherlands) based on 21 confocal slices. Scale bar = 10µm. Fitness effects Bio-assays were carried out to examine potential fitness effects of the endosymbionts on their Macrolophus host. In a first experiment, nymphal development was

compared between infected and uninfected individuals of M. pygmaeus, revealing positive effects of the infection on some developmental traits (Table 4). Infected M. pygmaeus males developed significantly faster than cured males (P<0.001). Smoothened antagonist Moreover, infected females were significantly heavier at emergence than uninfected ones (P=0.011). In a second experiment, fecundity was compared between infected and uninfected M. pygmaeus females. Infection status had no effect on the amount of eggs laid (P=0.575), nor on the oocyte counts of dissected females (P=0.069). Table 4 Nymphal developmental time, adult weight, sex ratio, number of eggs laid in the first week and oocyte counts of infected and uninfected M. pygmaeus. Cross Pazopanib solubility dmso Developmental time (days) Adult weight (mg) Sex ratio (♂ : ♀) No. of eggs laid Weighted sum of oocytes   Males (n) Females (n) Males (n) Females (n)       I♂ x I♀ 17.61 ± 0.13 a (28) 18.04 ± 0.20 a (23) 0.82

± 0.02 a (28) 1.31 ± 0.02 a (23) 1 : 0.8 12.33 ± 1.60 a (30) 15.02 ± 0.97 a (30) U♂ x U♀ 18.54 ± 0,19 b (26) 18.60 ± 0.30 a (15) 0.83 ± 0.02 a (26) 1.19 ± 0.04 b (15) 1 : 0.6 10.96 ± 1.20 a (22) 12.44 ± 0.94 a (28) Mean values (±SE) within a column followed by the same letter are not significantly different (P>0.05, One-Way ANOVA or Mann-Whitney U test) Discussion In the present study, the microbial community of check details various populations of two predators of the mirid genus Macrolophus was investigated. The bacterial diversity of Macrolophus spp. was explored by cloning 16S rRNA sequences and PCR-DGGE. The cloning experiment was executed on the laboratory strain of M. pygmaeus, revealing the presence of bacteria from the Alpha-proteobacteria, Beta-proteobacteria, Gamma-proteobacteria and Firmicutes classes (Table 3). Three bacteria -R. limoniae, R. bellii and Wolbachia- can be considered as endosymbionts.