Immune related gene expression in worker honey bee (<i>Apis mellifera carnica</i>) pupae exposed to neonicotinoid thiamethoxam and <i>Varroa</i> mites (<i>Varroa destructor</i>)

<div><p><i>Varroa destructor</i> is one of the most common parasites of honey bee colonies and is considered as a possible co-factor for honey bee decline. At the same time, the use of pesticides in intensive agriculture is still the most effective method of pest control. There is limited information about the effects of pesticide exposure on parasitized honey bees. Larval ingestion of certain pesticides could have effects on honey bee immune defense mechanisms, development and metabolic pathways. Europe and America face the disturbing phenomenon of the disappearance of honey bee colonies, termed Colony Collapse Disorder (CCD). One reason discussed is the possible suppression of honey bee immune system as a consequence of prolonged exposure to chemicals. In this study, the effects of the neonicotinoid thiamethoxam on honey bee, <i>Apis mellifera carnica</i>, pupae infested with <i>Varroa destructor</i> mites were analyzed at the molecular level. <i>Varroa</i>-infested and non-infested honey bee colonies received protein cakes with or without thiamethoxam. Nurse bees used these cakes as a feed for developing larvae. Samples of white-eyed and brown-eyed pupae were collected. Expression of 17 immune-related genes was analyzed by real-time PCR. Relative gene expression in samples exposed only to <i>Varroa</i> or to thiamethoxam or simultaneously to both <i>Varroa</i> and thiamethoxam was compared. The impact from the consumption of thiamethoxam during the larval stage on honey bee immune related gene expression in <i>Varroa</i>-infested white-eyed pupae was reflected as down-regulation of <i>spaetzle</i>, AMPs <i>abaecin</i> and <i>defensin-1</i> and up-regulation of <i>lysozyme-2</i>. In brown-eyed pupae up-regulation of <i>PPOact</i>, <i>spaetzle</i>, <i>hopscotch</i> and <i>basket</i> genes was detected. Moreover, we observed a major difference in immune response to <i>Varroa</i> infestation between white-eyed pupae and brown-eyed pupae. The majority of tested immune-related genes were upregulated only in brown-eyed pupae, while in white-eyed pupae they were downregulated.</p></div

Housekeeping and immune-related primers used in this study.

<p>Housekeeping and immune-related primers used in this study.</p

Heatmap of differential expression of immune-related genes in white-eyed and brown-eyed pupae.

<p>The colors indicate the average mRNA levels compared to average levels of mRNA in control groups: purple indicates lower and green higher levels. Range log₂ value of relative expression ratio is indicated in the legend on the right. Each column corresponds to the expression profile of one treatment (Thiamethoxam; <i>Varroa</i>; Thiamethoxam and <i>Varroa</i>) and each row, to one gene transcript. The immune-related gene names and corresponding immune pathway are indicated on the left. Boxes marked with symbol (*) shows statistically significant effect of treatment on gene expression if the p-value was equal to or less than 0.05.</p

The effect of thiamethoxam on immune related gene expression in <i>Varroa</i> infested bees.

<p>The expression level for each gene is presented as difference in average expression level in <i>Varroa</i> infested group and infested-thiamethoxam treated group. The higher expression levels are indicated with red color, lower expression levels are indicated with blue color. Immune pathway diagram for white-eyed pupae (A) and brown-eyed pupae (B) are presented. The range of relative expression ratio is indicated in the legend (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187079#pone.0187079.s003" target="_blank">S1 Fig</a>). Bluer color in panel indicates higher influence of thiamethoxam on <i>Varroa</i> infested pupae.</p