Honey bee sHSP are responsive to diverse proteostatic stresses and potentially promising biomarkers of honey bee stress

Abstract

The pollination services provided by the honey bee are critical in both natural and agricultural ecosystems. Honey bee colonies in the United States have suffered from an increased rate of die-off in recent years, stemming from a complex set of interacting stresses that remain poorly described. Defining specific common cellular processes and cellular stress responses impacted by multiple stressors represent a key step in understanding these synergies. Proteotoxic stresses negatively impact protein synthesis, folding, and degradation. Diverse proteotoxic stresses induce expression of genes encoding small heat shock proteins (sHSP) of the expanded lethal (2) essential for life (l(2)efl) gene family. In addition to upregulation by the Integrated Stress Response (ISR), the Heat Shock Response (HSR), and the Oxidative Stress Response (OSR), our data provide first evidence that sHSP genes are upregulated by the Unfolded Protein Response (UPR). As these genes appear to be part of a core stress response that could serve as a useful biomarker for cellular stress in honey bees, we designed and tested an RT-LAMP assay to detect increased l(2)efl gene expression in response to heat-stress. While this assay provides a powerful proof of principle, further work will be necessary to link changes in sHSP gene expression to colony-level outcomes, to adapt our preliminary assay into a Point of Care Testing (POCT) assay appropriate for use as a diagnostic tool for use in the field, and to couple assay results to management recommendations.

Paromomycin Reduces Vairimorpha (Nosema) ceranae Infection in Honey Bees but Perturbs Microbiome Levels and Midgut Cell Function

“…The quantification of cellular stress responses via the measuring of HSP has been used as a surrogate to identify general organismal stress in a variety of settings, including the honey bee (reviewed in [63]). Based on the robust induction of some of the l(2)efl genes in response to a broad array of stressors, the quantification of these sHSP genes has been proposed as an optimal biomarker for honey bee stress [25][26][27], although the levels of these genes have not been directly linked to individual bee or colony-level health, as has been achieved for other biomarkers [64]. Some of the sHSP genes discussed here have been shown to be a part of the antiviral response in honey bees and bumble bees [65,66], suggesting that they may possess anti-Vairimorpha properties.…”

“…RNA was prepared from the midgut tissue of the bees, as previously described [25]. Midgut tissue was manually crushed with a disposable pestle in Trizol Reagent (Invitrogen, San Diego, CA, USA) and RNA was then extracted as per the manufacturer's instructions.…”

“…Reactions were run in a LightCycler 480 thermal cycler (Basel, Switzerland) or Bio-Rad CFX Opus (Bio-Rad, Hercules, CA, USA) using the PCR conditions stated above. The primer sequences targeting the transcripts of the gene of interest were from [25]. The difference between the threshold cycle number for β-actin and that of the gene of interest was used to calculate the level of that gene relative to β-actin using the typical 2 (−∆CT) method [23].…”

“…In light of the reduced survival of honey bees after paromomycin treatment, we wished to examine whether sublethal effects of paromomycin were observed in the honey bee cells. We measured the expression of selected shsp genes of the l(2)efl family, which have been identified as useful stress biomarker genes in honey bees [25][26][27] that respond to diverse types of stress (Figure 4A). Using qPCR, we found that the expression of 724367 and 410087a increased in the midguts of uninfected bees that were treated with paromomycin relative to the control bees after 4 days of feeding (Figure 4B).…”

Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response

“…While transcriptional upregulation has been studied under a number of stress conditions (Guan et al 2017; Gonen et al 2019; Mahat et al 2016; Shih et al 2021), the interplay between transcriptional activation, translation status, and turnover of the resulting transcripts during the acute and chronic phases of the mammalian UPR is less understood. Recent advances in nascent RNA metabolic labeling methods in studies of mRNA synthesis and degradation dynamics (Rabani et al 2011; Battich et al 2020; Eisen et al 2020) now allow for the analysis of the translation and turnover of newly synthesized, stress-induced mRNAs during the progression from the acute to chronic UPR, which can be compared to global translation and mRNA turnover to reveal differences in the regulatory programs of those pools of mRNAs.…”