All species need to reproduce to maintain viable populations, but heat stress kills sperm cells across the animal kingdom and rising frequencies of heatwaves are a threat to biodiversity. Honey bees (Apis mellifera) are globally distributed micro-livestock; therefore, they could serve as environmental biomonitors for heat-induced reductions in fertility. Here, we found that queens have two potential routes of temperature-stress exposure: within colonies and during routine shipping. Our data suggest that temperatures of 15 to 38°C are safe for queens at a tolerance threshold of 11.5% loss of sperm viability, which is the viability difference between failed and healthy queens collected from beekeepers.Heat shock activates expression of specific ATP-independent heat-shock proteins in the spermatheca, which could serve as biomarkers for heat stress. This protein fingerprint may eventually enable surveys .

Background: Queen failure is a persistent problem in beekeeping operations, but in the absence of overt symptoms it is often difficult, if not impossible, to ascertain the root cause. Stressors like heat-shock, cold-shock, and sublethal pesticide exposure can reduce stored sperm viability and lead to cryptic queen failure. Previously, we suggested candidate protein markers indicating heat-shock in queens. Here, we further investigate these heat-shock markers and test new stressors to identify additional candidate protein markers. Results: We found that heat-shocking queens for upwards of 1 h at 40°C was necessary to induce significant changes in the two strongest candidate heat-shock markers, and that relative humidity significantly influenced the degree of activation. In blind heat-shock experiments, we tested the efficiency of these markers at assigning queens to their respective treatment groups and found that one marker was sufficient to correctly assign queens 75% of the time. Finally, we compared cold-shocked queens at 4°C and pesticide-exposed queens to controls to identify candidate markers for these additional stressors, and compared relative abundances of all markers to queens designated as 'healthy' and 'failing' by beekeepers. Queens that failed in the field had higher expression of both heat-shock and pesticide protein markers, but not cold-shock markers. Conclusions: This work offers some of the first steps towards developing molecular diagnostic tools to aid in determining cryptic causes of queen failure. Further work will be necessary to determine how long after the stress event a marker's expression remains elevated, and how accurate these markers will be for field diagnoses.

mRNA turnover is a regulated process that contributes to the steady state level of cytoplasmic mRNA. The amount of each mRNA determines, to a large extent, the amount of protein produced by that particular transcript. In trypanosomes, there is little transcriptional regulation; therefore, differential mRNA stability significantly contributes to mRNA levels in each stage of the parasite life cycle. To investigate the enzymatic activities that contribute to mRNA turnover, we developed a cell-free system for mRNA turnover using the trypanosome Leptomonas seymouri. We identified a decapping activity that removed m(7)GDP from mRNAs that contain an m(7)GpppN cap at their 5' end. In yeast, the release of m(7)GDP by the pyrophosphatase Dcp1p/Dcp2p is a rate-limiting step in mRNA turnover. A secondary enzymatic activity, similar to the human cap scavenger activity, was identified in the trypanosome extracts. Both the human and trypanosome scavenger activities generate m(7)GMP from short capped RNA and are inhibited by addition in trans of m(7)GpppG. A third enzymatic activity uncovered in the parasite extracts functioned as a 3' to 5' exonuclease. Importantly, this exonuclease activity was stimulated by an AU-rich element present in the RNA. In summary, the cell-free system has defined several RNA turnover steps that likely contribute to regulated mRNA decay in trypanosomes.

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Stressful conditions during development can have sub-lethal consequences on organisms aside from mortality. Using previously reported in-hive residues from commercial colonies, we examined how multi-pesticide exposure can influence honey bee (Apis mellifera) queen health. We reared queens in beeswax cups with or without a pesticide treatment within colonies exposed to treated or untreated pollen supplement. Following rearing, queens were open-mated and then placed into standard hive equipment in an “artificial swarm” to measure subsequent colony growth. Our treated wax had a pesticide Hazard Quotient comparable to the average in beeswax from commercial colonies, and it had no measurable effects on queen phenotype. Conversely, colonies exposed to pesticide-treated pollen had a reduced capacity for viable queen production, and among surviving queens from these colonies we observed lower sperm viability. We found no difference in queen mating number across treatments. Moreover, we measured lower brood viability in colonies later established by queens reared in treated-pollen colonies. Interestingly, royal jelly from colonies exposed to treated pollen contained negligible pesticide residues, suggesting the indirect social consequences of colony-level pesticide exposure on queen quality. These findings highlight how conditions during developmental can impact queens long into adulthood, and that colony-level pesticide exposure may do so indirectly.

The stability of mRNAs is an important point in the regulation of gene expression in eukaryotes. The mRNA turnover pathways have been identified in yeast and mammals. However, mRNA turnover pathways in trypanosomes have not been widely studied. Deadenylation is the first step in the major mRNA turnover pathways of yeast and mammals. To better understand mRNA degradation processes in these organisms, we have developed an in vitro mRNA turnover system that is functional for deadenylation. In this system, addition of poly(A) homopolymer activates the deadenylation of poly(A) tails. The trypanosomal deadenylase activity is a 3→ 5 exonuclease specific for adenylate residues, generates 5-AMP as a product, is magnesium dependent, and is inhibited by neomycin B sulfate. These characteristics suggest similarity with other eukaryotic deadenylases. Furthermore, this activity is cap independent, indicating a potential difference between the trypanosomal activity and PARN, but suggesting similarity to Ccr4p/Pop2p activities. Extracts immunodepleted of Pab1p required the addition of poly(A) competition to activate deadenylation. Trypanosomal Pab1p functions as an inhibitor of the activity under in vitro conditions. Pab1p appears to be one of several mRNA stability proteins in trypanosomal extracts.

Deadenylation is often the rate-limiting event in regulating the turnover of cellular mRNAs in eukaryotes. Removal of the poly(A) tail initiates mRNA degradation by one of several decay pathways, including deadenylation-dependent decapping, followed by 5 to 3 exonuclease decay or 3 to 5 exosome-mediated decay. In trypanosomatids, mRNA degradation is important in controlling the expression of differentially expressed genes. Genomic annotation studies have revealed several potential deadenylases. Poly(A)-specific RNase (PARN) is a key deadenylase involved in regulating gene expression in mammals, Xenopus oocytes, and higher plants. Trypanosomatids possess three different PARN genes, PARN-1, -2, and -3, each of which is expressed at the mRNA level in two life-cycle stages of the human parasite Trypanosoma brucei. Here we show that T. brucei PARN-1 is an active deadenylase. To determine the role of PARN-1 on mRNA stability in vivo, we overexpressed this protein and analyzed perturbations in mRNA steady-state levels as well as mRNA half-life. Interestingly, a subset of mRNAs was affected, including a family of mRNAs that encode stage-specific coat proteins. These data suggest that PARN-1 functions in stage-specific protein production. Regulation of gene expression in the protozoan parasiteTrypanosoma brucei allows the organism to adapt and survive during its life cycle in two very different environments, the mammalian bloodstream and the tsetse fly. Expression of numerous protein-coding genes is regulated posttranscriptionally, particularly at the level of mRNA stability (4,11,25). For example, differential mRNA stability accounts for the stagespecific expression of procyclins, hexose transporters, and phosphoglycerate kinases (6,20,27,28,53).In the well-studied Saccharomyces and mammalian systems, mRNA decay is a tightly controlled, multistep, and multipathway process. Various cis-acting elements, embedded in specific mRNAs, are recognized by RNA-binding proteins (7,52,55), which stabilize mRNAs or recruit RNases to carry out mRNA degradation and inhibit translation (8,34,39,47). The removal of the mRNA 3Ј poly(A) tail by 3Ј to 5Ј exoribonucleases (deadenylases) is often the rate-limiting step in mRNA degradation in vertebrates and thus a key point in regulation of mRNA turnover (19,40).A number of different deadenylases exist in eukaryotes, including poly(A)-specific RNase (PARN), the CCR4/CAF1/ NOT complex, and the PAN2/PAN3 complex (reviewed in reference 23). The specific role of each of these proteins remains largely unknown, although evidence suggests that each enzyme may recognize a particular set of mRNA substrates (46). PARN functions in the targeted degradation of specific mRNAs in humans, Xenopus, and higher plants (1,3,22,31,32,36,58). To date, no PARN-encoding genes have been characterized in any single-cell eukaryote (56). In humans, PARN initiates decay of mRNAs containing AU-rich elements or nonsense codons. In Xenopus, PARN regulates oocyte maturation, whereas in Arabidopsis, PARN regulates embryogen...

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