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Circadian rhythms allow animals to coordinate behavioral and physiological processes with respect to one another and to synchronize these processes to external environmental cycles. In most animals, circadian rhythms are produced by core clock neurons in the brain that generate and transmit time-of-day signals to downstream tissues, driving overt rhythms. The neuronal pathways controlling clock outputs, however, are not well understood. Furthermore, it is unclear how the central clock modulates multiple distinct circadian outputs. Identifying the cellular components and neuronal circuitry underlying circadian regulation is increasingly recognized as a critical step in the effort to address health pathologies linked to circadian disruption, including heart disease and metabolic disorders. Here, building on the conserved components of circadian and metabolic systems in mammals and Drosophila melanogaster, we used a recently developed feeding monitor to characterize the contribution to circadian feeding rhythms of two key neuronal populations in the Drosophila pars intercerebralis (PI), which is functionally homologous to the mammalian hypothalamus. We demonstrate that thermogenetic manipulations of PI neurons expressing the neuropeptide SIFamide (SIFa) as well as mutations of the SIFa gene degrade feeding:fasting rhythms. In contrast, manipulations of a nearby population of PI neurons that express the Drosophila insulin-like peptides (DILPs) affect total food consumption but leave feeding rhythms intact. The distinct contribution of these two PI cell populations to feeding is accompanied by vastly different neuronal connectivity as determined by trans-Tango synaptic mapping. These results for the first time identify a non-clock cell neuronal population in Drosophila that regulates feeding rhythms and furthermore demonstrate dissociable control of circadian and homeostatic aspects of feeding regulation by molecularly-defined neurons in a putative circadian output hub.

Human cytomegalovirus (HCMV) modulates numerous cellular pathways to facilitate infection, including key components in cellular iron homeostasis. Iron is essential to many cellular processes but, if present in excess, drives cell death through ferroptosis. Ferroptosis is a process that is dependent upon the accumulation of oxidatively damaged phospholipids (lipid peroxides); when these lipid peroxides accumulate in membranes, this culminates in plasma membrane rupture and eventual cell lysis. Here, we demonstrate that HCMV infection downregulates the expression of a key modulator of lipid peroxidation, glutathione peroxidase 4 (GPX4). HCMV infection also markedly increased levels of lipid peroxides within infected cells. Despite the marked downregulation of GPX4 by HCMV, further inhibition of GPX4 impaired virus replication. Interestingly, overexpression of GPX4 did not reduce the production of lipid peroxides within infected cells. In contrast, lipid peroxide levels were reduced by treatment with ferrostatin-1, a ferrous iron-dependent scavenger of alkoxyl radicals, indicating a role for iron in the production of lipid peroxides. HCMV-infected cells became less sensitive to GPX4 inhibition as infection progressed, requiring substantially higher levels of GPX4 inhibitors to induce ferroptosis compared to uninfected cells. This observed difference in sensitivity to ferroptosis upon infection correlated with a large increase in lipid production by infected cells. Therefore, the marked stimulation of lipid peroxidation by HCMV likely proceeds through a pathway that is independent of GPX4 regulation, but the ability of lipid peroxides to stimulate ferroptosis by modulating plasma membrane rupture is likely blunted by the massive increase in lipid production during HCMV infection., Importance: Human cytomegalovirus (HCMV) infection is intimately linked with countless host cell pathways that are modulated in a coordinated fashion to facilitate infection. Here, we describe HCMV-induced regulation of lipid peroxidation, a precursor of the iron-regulated cell death pathway known as ferroptosis, during human cytomegalovirus infection. These studies reveal hitherto unidentified changes in metabolism mediated by HCMV that decrease sensitivity to ferroptosis, despite increases in lipid peroxidation and transient increases in intracellular iron levels in infected cells.

Human cytomegalovirus (HCMV) modulates numerous cellular pathways to facilitate infection. Iron is essential to many cellular processes and is often incorporated into proteins and enzymes involved in oxidative phosphorylation and DNA synthesis and repair, among others. Despite its prominent role in the cell, little is known about the regulation of iron metabolism during HCMV infection. Herein, we observe modulation of the transferrin receptor (TfR) during infection and a corresponding change in the cellular labile iron pool. TfR and the iron pool are increased early during infection and then return to mock levels at the late stages of infection. We identified the cellular ubiquitin ligase MARCH1 as an important regulator of TfR. MARCH1 plays a proviral role during infection, as its knockdown leads to a decrease in infectious titers. Knockdown of MARCH1 also leads to an increase in ROS, lipid peroxidation, and mitochondrial dysfunction. Inhibiting an early increase in TfR expression during infection also decreases virus production. These findings indicate the importance of tightly regulating iron metabolism during HCMV infection to facilitate efficient virus production. IMPORTANCE Iron is essential for cells, playing important roles in energy generation, DNA replication, and gene expression. During infection, HCMV alters many cellular processes to aid its replication. We found that iron levels are tightly regulated during infection and that dysregulation of iron levels alters the ability to produce infectious virions. We also found that HCMV inactivates many of the cellular safeguards put in place to deal with excess iron. Thus, infected cells become more susceptible to variations in iron levels, which could be exploited as a therapeutic strategy for dealing with HCMV infections.

Honey bees are important pollinators of agricultural crops. Since 2006, US beekeepers have experienced high annual honey bee colony losses, which may be attributed to multiple abiotic and biotic factors, including pathogens. However, the relative importance of these factors has not been fully elucidated. To identify the most prevalent pathogens and investigate the relationship between colony strength and health, we assessed pathogen occurrence, prevalence, and abundance in Western US honey bee colonies involved in almond pollination. The most prevalent pathogens were Black queen cell virus (BQCV), Lake Sinai virus 2 (LSV2), Sacbrood virus (SBV), Nosema ceranae , and trypanosomatids. Our results indicated that pathogen prevalence and abundance were associated with both sampling date and beekeeping operation, that prevalence was highest in honey bee samples obtained immediately after almond pollination, and that weak colonies had a greater mean pathogen prevalence than strong colonies.

Honey bees are critical pollinators of important agricultural crops. Recently, high annual losses of honey bee colonies have prompted further investigation of honey bee infecting viruses. To better characterize the recently discovered and very prevalent Lake Sinai virus (LSV) group, we sequenced currently circulating LSVs, performed phylogenetic analysis, and obtained images of LSV2. Sequence analysis resulted in extension of the LSV1 and LSV2 genomes, the first detection of LSV4 in the US, and the discovery of LSV6 and LSV7. We detected LSV1 and LSV2 in the Varroa destructor mite, and determined that a large proportion of LSV2 is found in the honey bee gut, suggesting that vector-mediated, food-associated, and/or fecal-oral routes may be important for LSV dissemination. Pathogen-specific quantitative PCR data, obtained from samples collected during a small-scale monitoring project, revealed that LSV2, LSV1, Black queen cell virus (BQCV), and Nosema ceranae were more abundant in weak colonies than strong colonies within this sample cohort. Together, these results enhance our current understanding of LSVs and illustrate the importance of future studies aimed at investigating the role of LSVs and other pathogens on honey bee health at both the individual and colony levels.