Transcriptional control of a metabolic switch regulating cellular methylation reactions is part of a common response to stress in divergent bee species.

Recent global declines in bee health have elevated the need for a more complete understanding of the cellular stress mechanisms employed by diverse bee species. We recently uncovered the biomarker lethal (2) essential for life [l(2)efl] genes as part of a shared transcriptional program in response to a number of cell stressors in the western honey bee (Apis mellifera). Here, we describe another shared stress-responsive gene, glycine N-methyltransferase (Gnmt), which is known as a key metabolic switch controlling cellular methylation reactions. We observed Gnmt induction by both abiotic and biotic stressors. We also found increased levels of the GNMT reaction product sarcosine in the midgut after stress, linking metabolic changes with the observed changes in gene regulation. Prior to this study, Gnmt upregulation had not been associated with cellular stress responses in other organisms. To determine whether this novel stress-responsive gene would behave similarly in other bee species, we first characterized the cellular response to endoplasmic reticulum (ER) stress in lab-reared adults of the solitary alfalfa leafcutting bee (Megachile rotundata) and compared this with age-matched honey bees. The novel stress gene Gnmt was induced in addition to a number of canonical gene targets induced in both bee species upon unfolded protein response (UPR) activation, suggesting that stress-induced regulation of cellular methylation reactions is a common feature of bees. Therefore, this study suggests that the honey bee can serve as an important model for bee biology more broadly, although studies on diverse bee species will be required to fully understand global declines in bee populations.

Bleomycin reduces Vairimorpha (Nosema) ceranae infection in honey bees with some evident host toxicity.

Microsporidia cause disease in many beneficial insects, including honey bees, yet few pathogen control tools are available for protecting these important organisms against infection. Some evidence suggests that microsporidia possess a reduced number of genes encoding DNA repair proteins. We hypothesized that microsporidia would thus be susceptible to treatment with DNA-damaging agents and tested this hypothesis using a novel, rapid method for achieving robust and homogenous experimental infection of large numbers of newly emerged honey bees with one of its microsporidia pathogens, Vairimorpha (Nosema) ceranae. In carrying out these experiments, we found this novel V. ceranae inoculation method to have similar efficacy as other traditional methods. We show that the DNA-damaging agent bleomycin reduces V. ceranae levels, with minimal but measurable effects on honey bee survival and increased expression of midgut cellular stress genes, including those encoding SHSP. Increased expression of UpdlC suggests the occurrence of epithelial regeneration, which may contribute to host resistance to bleomycin treatment. While bleomycin does reduce infection levels, host toxicity issues may preclude its use in the field. However, with further work, bleomycin may provide a useful tool in the research setting as a potential selection agent for genetic modification of microsporidia.IMPORTANCEMicrosporidia cause disease in many beneficial insects, yet there are few tools available for control in the field or laboratory. Based on the reported paucity of DNA repair enzymes found in microsporidia genomes, we hypothesized that these obligate intracellular parasites would be sensitive to DNA damage. In support of this, we observed that the well-characterized DNA damage agent bleomycin can reduce levels of the microsporidia Vairimorpha (Nosema) ceranae in experimental infections in honey bees. Observation of slightly reduced honey bee survival and evidence of sublethal toxicity likely preclude the use of bleomycin in the field. However, this work identifies bleomycin as a compound that merits further exploration for use in research laboratories as a potential selection agent for generating genetically modified microsporidia.

Colonization of Honey Bee Digestive Tracts by Environmental Yeast Lachancea thermotolerans Is Naturally Occurring, Temperature Dependent, and Impacts the Microbiome of Newly Emerged Bees.

Honey bees are critical pollinators in both agricultural and ecological settings. Recent declines in honey bee colonies in the United States have put increased strain on agricultural pollination. Although there are many environmental stressors implicated in honey bee disease, there has been intensifying focus on the role of microbial attacks on honey bee health. Despite the long-standing appreciation for the association of fungi of various groups with honey bees and their broader environment, the effects of these interactions on honey bee health are incompletely understood. Here, we report the discovery of colonization of the honey bee digestive tract by the environmental yeast Lachancea thermotolerans. Experimental colonization of honey bee digestive tracts by L. thermotolerans revealed that this yeast species maintains high levels in the honey bee midgut only at temperatures below the typical colony temperature. In newly eclosed bees, L. thermotolerans colonization alters the microbiome, suggesting that environmental yeasts can impact its composition. Future studies should be undertaken to better understand the role of L. thermotolerans and other environmental yeasts in honey bee health. IMPORTANCE Although many fungal species are found in association with honey bees and their broader environment, the effects of these interactions on honey bee health are largely unknown. Here, we report the discovery that a yeast commonly found in the environment can be found at high levels in honey bee digestive tracts. Experimentally feeding this yeast to honey bees showed that the yeast's ability to maintain high levels in the digestive tract is influenced by temperature and can lead to alterations of the microbiome in young bees. These studies provide a foundation for future studies to better understand the role of environmental yeasts in honey bee health.

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

Paromomycin is a naturally occurring aminoglycoside antibiotic that has effects on both prokaryotic and eukaryotic microbes. However, previous reports have indicated that it has little effect on microsporidia, including Vairimorpha (Nosema) ceranae, in cell culture models. V. ceranae is one of a number of microsporidia species that cause disease in honey bees and substantial efforts to find new treatment strategies for bees that are infected with these pathogens are ongoing. When testing compounds for potential activity against V. ceranae in whole organisms, we found that paromomycin reduces the infection intensity of this parasite. Critically, the necessary doses of paromomycin have high activity against the bacteria of the honey bee microbiome and cause evident stress in bees. Microsporidia have been shown to lack an essential binding site on the ribosome that is known to allow for maximal inhibition by paromomycin. Thus, it is possible that paromomycin impacts parasite levels through non-cell autonomous effects on microsporidia infection levels via effects on the microbiome or midgut cellular function. As paromomycin treatment could cause widespread honey bee health issues in agricultural settings, it does not represent an appropriate anti-microsporidia agent for use in the field.

Proteasome Inhibition Is an Effective Treatment Strategy for Microsporidia Infection in Honey Bees.

The microsporidia Nosema ceranae is an obligate intracellular parasite that causes honey bee mortality and contributes to colony collapse. Fumagillin is presently the only pharmacological control for N. ceranae infections in honey bees. Resistance is already emerging, and alternative controls are critically needed. Nosema spp. exhibit increased sensitivity to heat shock, a common proteotoxic stress. Thus, we hypothesized that targeting the Nosema proteasome, the major protease removing misfolded proteins, might be effective against N. ceranae infections in honey bees. Nosema genome analysis and molecular modeling revealed an unexpectedly compact proteasome apparently lacking multiple canonical subunits, but with highly conserved proteolytic active sites expected to be receptive to FDA-approved proteasome inhibitors. Indeed, N. ceranae were strikingly sensitive to pharmacological disruption of proteasome function at doses that were well tolerated by honey bees. Thus, proteasome inhibition is a novel candidate treatment strategy for microsporidia infection in honey bees.

Adult stem cells and niche cells segregate gradually from common precursors that build the adult Drosophila ovary during pupal development.

Production of proliferative follicle cells (FCs) and quiescent escort cells (ECs) by follicle stem cells (FSCs) in adult Drosophila ovaries is regulated by niche signals from anterior (cap cells, ECs) and posterior (polar FCs) sources. Here we show that ECs, FSCs, and FCs develop from common pupal precursors, with different fates acquired by progressive separation of cells along the AP axis and a graded decline in anterior cell proliferation. ECs, FSCs, and most FCs derive from intermingled cell (IC) precursors interspersed with germline cells. Precursors also accumulate posterior to ICs before engulfing a naked germline cyst projected out of the germarium to form the first egg chamber and posterior polar FC signaling center. Thus, stem and niche cells develop in appropriate numbers and spatial organization through regulated proliferative expansion together with progressive establishment of spatial signaling cues that guide adult cell behavior, rather than through rigid early specification events.

Actin polymerization plays a significant role in asbestos-induced inflammasome activation in mesothelial cells in vitro.

Asbestos exposure leads to malignant mesothelioma (MM), a deadly neoplasm of mesothelial cells of various locations. Although there is no doubt about the role of asbestos in MM tumorigenesis, mechanisms are still not well explored. Recently, our group demonstrated that asbestos causes inflammasome priming and activation in mesothelial cells, which in part is dependent on oxidative stress. Our current study sheds light on yet another mechanism of inflammasome activation by asbestos. Here we show the role of actin polymerization in asbestos-induced activation of the nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome. Using human mesothelial cells, we first demonstrate that asbestos and carbon nanotubes induced caspase-1 activation and high-mobility group box 1, interleukin 1 beta and interleukin 18 secretion was blocked by Cytochalasin D (Cyto D) an actin polymerization inhibitor. Next, to understand the mechanism, we assessed whether phagocytosis of fibers by mesothelial cells is affected by actin polymerization inhibition. Transmission electron microscopy showed the inhibition of fiber uptake by mesothelial cells in the presence of Cyto D. Furthermore, localization of components of the inflammasome, apoptotic speck-like protein containing a CARD domain (ASC) and NLRP3, to the perinuclear space in mitochondria or endoplasmic reticulum in response to fiber exposure was also interrupted in the presence of Cyto D. Taken together, our studies suggest that actin polymerization plays important roles in inflammasome activation by fibers via regulation of phagocytosis and/or spatial localization of inflammasome components.

DR4004, a putative 5-HT(7) receptor antagonist, also has functional activity at the dopamine D2 receptor.

The tetrahydrobenzindole, 2a-(4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl)-2a,3,4,5-tetrahydrobenzo[cd]indol-2(1H)-one (DR4004) has been described as a highly selective antagonist for the 5-hydroxytryptamine(7) (5-HT(7)) receptor [J. Med. Chem. 42 (1999) 533]. Consistent with original data, DR4004 bound to rat hypothalamic membranes with an affinity of 7.3+/-0.2 (pK(i)+/-S.E.M.) for the 5-HT(7) receptor. However, competition binding studies showed that DR4004 had poor receptor selectivity with the following affinity profile; dopamine D2 receptor, alpha(1)-adrenoceptor > or =5-HT(7) receptor>histamine H(1) receptor, alpha(2)-adrenoceptor>dopamine D1 receptor>beta-adrenoceptor, muscarinic and 5-HT(2A/C) receptors. In conscious rats DR4004 (1, 5 or 10 mg/kg i.p.) produced a dose-dependent hyperglycaemia and hypothermia, but the former was reduced by the dopamine D2 receptor antagonist raclopride. Another 5-HT(7) receptor antagonist, (R)-3-(2-(2-(4-methylpiperidin-1-yl)-ethyl)pyrrolidine-1-sulfonyl)phenol (SB-269970) produced hypothermia but no hyperglycaemia. This study confirms that DR4004 has high affinity for the 5-HT(7) receptor but suggests that dopamine D2 receptor activity contributes to some of the in vivo effects.