Investigating trade-offs between ovary activation and immune protein expression in bumble bee (Bombus impatiens) workers and queens.

Evidence for a trade-off between reproduction and immunity has manifested in many animal species, including social insects. However, investigations in social insect queens present a conundrum: new gynes of many social hymenopterans, such as bumble bees and ants, must first mate, then transition from being solitary to social as they establish their nests, thus experiencing confounding shifts in environmental conditions. Worker bumble bees offer an opportunity to investigate patterns of immune protein expression associated with ovary activation while minimizing extraneous environmental factors and genetic differences. Here, we use proteomics to interrogate the patterns of immune protein expression of female bumble bees (Bombus impatiens) by (i) sampling queens at different stages of their life cycle, then (ii) by sampling workers with different degrees of ovary activation. Patterns of immune protein expression in the haemolymph of queens are consistent with a reproduction-immunity trade-off, but equivalent samples from workers are not. This brings into question whether queen bumble bees really experience a reproduction-immunity trade-off, or if patterns of immune protein expression may actually be due to the selective pressure of the different environmental conditions they are exposed to during their life cycle.

In Vivo Delivery of Spherical and Cylindrical In Vitro Reconstituted Virus-like Particles Containing the Same Self-Amplifying mRNA.

The dramatic effectiveness of recent mRNA (mRNA)-based COVID vaccines delivered in lipid nanoparticles has highlighted the promise of mRNA therapeutics in general. In this report, we extend our earlier work on self-amplifying mRNAs delivered in spherical in vitro reconstituted virus-like particles (VLPs), and on drug delivery using cylindrical virus particles. In particular, we carry out separate in vitro assemblies of a self-amplifying mRNA gene in two different virus-like particles: one spherical, formed with the capsid protein of cowpea chlorotic mottle virus (CCMV), and the other cylindrical, formed from the capsid protein of tobacco mosaic virus (TMV). The mRNA gene is rendered self-amplifying by genetically fusing it to the RNA-dependent RNA polymerase (RdRp) of Nodamura virus, and the relative efficacies of cell uptake and downstream protein expression resulting from their CCMV- and TMV-packaged forms are compared directly. This comparison is carried out by their transfections into cells in culture: expressions of two self-amplifying genes, enhanced yellow fluorescent protein (EYFP) and Renilla luciferase (Luc), packaged alternately in CCMV and TMV VLPs, are quantified by fluorescence and chemiluminescence levels, respectively, and relative numbers of the delivered mRNAs are measured by quantitative real-time PCR. The cellular uptake of both forms of these VLPs is further confirmed by confocal microscopy of transfected cells. Finally, VLP-mediated delivery of the self-amplifying-mRNA in mice following footpad injection is shown by in vivo fluorescence imaging to result in robust expression of EYFP in the draining lymph nodes, suggesting the potential of these plant virus-like particles as a promising mRNA gene and vaccine delivery modality. These results establish that both CCMV and TMV VLPs can deliver their in vitro packaged mRNA genes to immune cells and that their self-amplifying forms significantly enhance in situ expression. Choice of one VLP (CCMV or TMV) over the other will depend on which geometry of nucleocapsid is self-assembled more efficiently for a given length and sequence of RNA, and suggests that these plant VLP gene delivery systems will prove useful in a wide variety of medical applications, both preventive and therapeutic.

Candidate stress biomarkers for queen failure diagnostics.

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.

Common viral infections inhibit egg laying in honey bee queens and are linked to premature supersedure.

With their long lives and extreme reproductive output, social insect queens have escaped the classic trade-off between fecundity and lifespan, but evidence for a trade-off between fecundity and immunity has been inconclusive. This is in part because pathogenic effects are seldom decoupled from effects of immune induction. We conducted parallel, blind virus infection experiments in the laboratory and in the field to interrogate the idea of a reproductive immunity trade-off in honey bee (Apis mellifera) queens and to better understand how these ubiquitous stressors affect honey bee queen health. We found that queens injected with infectious virus had smaller ovaries and were less likely to recommence egg-laying than controls, while queens injected with UV-inactivated virus displayed an intermediate phenotype. In the field, heavily infected queens had smaller ovaries and infection was a meaningful predictor of whether supersedure cells were observed in the colony. Immune responses in queens receiving live virus were similar to queens receiving inactivated virus, and several of the same immune proteins were negatively associated with ovary mass in the field. This work supports the hypothesized relationship between virus infection and symptoms associated with queen failure and suggests that a reproductive-immunity trade-off is partially, but not wholly responsible for these effects.

CRISPR-induced DNA reorganization for multiplexed nucleic acid detection.

Nucleic acid sensing powered by the sequence recognition of CRIPSR technologies has enabled major advancement toward rapid, accurate and deployable diagnostics. While exciting, there are still many challenges facing their practical implementation, such as the widespread need for a PAM sequence in the targeted nucleic acid, labile RNA inputs, and limited multiplexing. Here we report FACT (Functionalized Amplification CRISPR Tracing), a CRISPR-based nucleic acid barcoding technology compatible with Cas12a and Cas13a, enabling diagnostic outputs based on cis- and trans-cleavage from any sequence. Furthermore, we link the activation of CRISPR-Cas12a to the expression of proteins through a Reprogrammable PAIRing system (RePAIR). We then combine FACT and RePAIR to create FACTOR (FACT on RePAIR), a CRISPR-based diagnostic, that we use to detect infectious disease in an agricultural use case: honey bee viral infection. With high specificity and accuracy, we demonstrate the potential of FACTOR to be applied to the sensing of any nucleic acid of interest.

Fertility costs of cryptic viral infections in a model social insect.

Declining insect populations emphasize the importance of understanding the drivers underlying reductions in insect fitness. Here, we investigated viruses as a threat to social insect reproduction, using honey bees as a model species. We report that in two independent surveys (N = 93 and N = 54, respectively) of honey bee (Apis mellifera) queens taken from a total of ten beekeeping operations across British Columbia, high levels of natural viral infection are associated with decreased ovary mass. Failed (poor quality) queens displayed higher levels of viral infection, reduced sperm viability, smaller ovaries, and altered ovary protein composition compared to healthy queens. We experimentally infected queens with Israeli acute paralysis virus (IAPV) and found that the ovary masses of IAPV-injected queens were significantly smaller than control queens, demonstrating a causal relationship between viral infection and ovary size. Queens injected with IAPV also had significantly lower expression of vitellogenin, the main source of nutrition deposited into developing oocytes, and higher levels of heat-shock proteins, which are part of the honey bee's antiviral response. This work together shows that viral infections occurring naturally in the field are compromising queen reproductive success.

Trade-offs between sperm viability and immune protein expression in honey bee queens (Apis mellifera).

Queens of many social hymenoptera keep sperm alive within their specialized storage organ, the spermatheca, for years, defying the typical trade-off between lifespan and reproduction. However, whether honey bee (Apis mellifera) queens experience a trade-off between reproduction and immunity is unknown, and the biochemical processes underlying sperm viability are poorly understood. Here, we survey quality metrics and viral loads of honey bee queens from nine genetic sources. Queens rated as 'failed' by beekeepers had lower sperm viability, fewer sperm, and higher levels of sacbrood virus and black queen cell virus. Quantitative proteomics on N = 123 spermathecal fluid samples shows, after accounting for sperm count, health status, and apiary effects, five spermathecal fluid proteins significantly correlating with sperm viability: odorant binding protein (OBP)14, lysozyme, serpin 88Ea, artichoke, and heat-shock protein (HSP)10. The significant negative correlation of lysozyme-a conserved immune effector-with sperm viability is consistent with a reproduction vs. immunity trade-off in honey bee queens.

A death pheromone, oleic acid, triggers hygienic behavior in honey bees (Apis mellifera L.).

Eusocial insects live in teeming societies with thousands of their kin. In this crowded environment, workers combat disease by removing or burying their dead or diseased nestmates. For honey bees, we found that hygienic brood-removal behavior is triggered by two odorants - ß-ocimene and oleic acid - which are released from brood upon freeze-killing. ß-ocimene is a co-opted pheromone that normally signals larval food-begging, whereas oleic acid is a conserved necromone across arthropod taxa. Interestingly, the odorant blend can induce hygienic behavior more consistently than either odorant alone. We suggest that the volatile ß-ocimene flags hygienic workers' attention, while oleic acid is the death cue, triggering removal. Bees with high hygienicity detect and remove brood with these odorants faster than bees with low hygienicity, and both molecules are strong ligands for hygienic behavior-associated odorant binding proteins (OBP16 and OBP18). Odorants that induce low levels of hygienic behavior, however, are weak ligands for these OBPs. We are therefore beginning to paint a picture of the molecular mechanism behind this complex behavior, using odorants associated with freeze-killed brood as a model.