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Nosemosis C, a Nosema disease caused by microsporidia parasite Nosema ceranae , is a significant disease burden of the European honey bee Apis mellifera which is one of the most economically important insect pollinators. Nevertheless, there is no effective treatment currently available for Nosema disease and the disease mechanisms underlying the pathological effects of N . ceranae infection in honey bees are poorly understood. Iron is an essential nutrient for growth and survival of hosts and pathogens alike. The iron tug-of-war between host and pathogen is a central battlefield at the host-pathogen interface which determines the outcome of an infection, however, has not been explored in honey bees. To fill the gap, we conducted a study to investigate the impact of N . ceranae infection on iron homeostasis in honey bees. The expression of transferrin, an iron binding and transporting protein that is one of the key players of iron homeostasis, in response to N . ceranae infection was analysed. Furthermore, the functional roles of transferrin in iron homeostasis and honey bee host immunity were characterized using an RNA interference (RNAi)-based method. The results showed that N . ceranae infection causes iron deficiency and upregulation of the A . mellifera transferrin (AmTsf) mRNA in honey bees, implying that higher expression of AmTsf allows N . ceranae to scavenge more iron from the host for its proliferation and survival. The suppressed expression levels of AmTsf via RNAi could lead to reduced N . ceranae transcription activity, alleviated iron loss, enhanced immunity, and improved survival of the infected bees. The intriguing multifunctionality of transferrin illustrated in this study is a significant contribution to the existing body of literature concerning iron homeostasis in insects. The uncovered functional role of transferrin on iron homeostasis, pathogen growth and honey bee’s ability to mount immune responses may hold the key for the development of novel strategies to treat or prevent diseases in honey bees.

Honey bees, the primary managed insect pollinator, suffer considerable losses due to Deformed wing virus (DWV), an RNA virus vectored by the mite Varroa destructor . Mite vectoring has resulted in the emergence of virulent DWV variants. The basis for such changes in DWV is poorly understood. Most importantly, it remains unclear whether replication of DWV occurs in the mite. In this study, we exposed Varroa mites to DWV type A via feeding on artificially infected honey bees. A significant, 357-fold increase in DWV load was observed in these mites after 2 days. However, after 8 additional days of passage on honey bee pupae with low viral loads, the DWV load dropped by 29-fold. This decrease significantly reduced the mites’ ability to transmit DWV to honey bees. Notably, negative-strand DWV RNA, which could indicate viral replication, was detected only in mites collected from pupae with high DWV levels but not in the passaged mites. We also found that Varroa mites contain honey bee mRNAs, consistent with the acquisition of honey bee cells which would additionally contain DWV replication complexes with negative-strand DWV RNA. We propose that transmission of DWV type A by Varroa mites occurs in a non-propagative manner.

The ectoparasitic mite Varroa destructor is one of the most destructive pests of the honey bee ( Apis mellifera ) and the primary biotic cause of colony collapse in many regions of the world. These mites inflict physical injury on their honey bee hosts from feeding on host hemolymph and fat body cells/cellular components, and serve as the vector for deadly honey bee viruses, including Deformed wing virus (DWV) and the related Varroa destructor virus-1 (VDV-1) ( i . e ., DWV-like viruses). Studies focused on elucidating the dynamics of Varroa -mediated vectoring and transmission of DWV-like viruses may be confounded by viruses present in ingested host tissues or the mites themselves. Here we describe a system that includes an artificial diet free of insect tissue-derived components for maintaining Varroa mites for in vitro experimentation. Using this system, together with the novel engineered cDNA clone-derived genetically tagged VDV-1 and wild-type DWV, we demonstrated for the first time that Varroa mites provided an artificial diet supplemented with engineered viruses for 36 hours could acquire and transmit sufficient numbers of virus particles to establish an infection in virus-naïve hosts. While the in vitro system described herein provides for only up to five days of mite survival, precluding study of the long-term impacts of viruses on mite health, the system allows for extensive insights into the dynamics of Varroa -mediated vectoring and transmission of honey bee viruses.

We developed a honey bee RNA-virus vector based on the genome of a picorna-like Deformed wing virus (DWV), the main viral pathogen of the honey bee (Apis mellifera). To test the potential of DWV to be utilized as a vector, the 717 nt sequence coding for the enhanced green fluorescent protein (eGFP), flanked by the peptides targeted by viral protease, was inserted into an infectious cDNA clone of DWV in-frame between the leader protein and the virus structural protein VP2 genes. The in vitro RNA transcripts from egfp-tagged DWV cDNA clones were infectious when injected into honey bee pupae. Stable DWV particles containing genomic RNA of the recovered DWV with egfp inserts were produced, as evidenced by cesium chloride density gradient centrifugation. These particles were infectious to honey bee pupae when injected intra-abdominally. Fluorescent microscopy showed GFP expression in the infected cells and Western blot analysis demonstrated accumulation of free eGFP rather than its fusions with DWV leader protein (LP) and/or viral protein (VP) 2. Analysis of the progeny egfp-tagged DWV showed gradual accumulation of genome deletions for egfp, providing estimates for the rate of loss of a non-essential gene an insect RNA virus genome during natural infection.

Microbial communities residing within the midgut of insect vectors play a critical role in the response to various zoonotic and human pathogens, and can directly alter the development and survival of the insects. Sand flies are the primary vector of Leishmania, the causative pathogen of leishmaniasis, a neglected tropical disease. Sand flies acquire many microbes from the soil where immature stages develop until emergence as adults. Gram-negative Pantoea agglomerans and gram-positive Bacillus subtilis are two bacteria commonly associated with sand fly populations. Here, I demonstrated that an EGFP- and a GFP-expressing version of these two bacteria localize to different compartments of the midgut; a phenomenon that is achieved, in part, to pH differences found across the length of the gut. Additionally, P. agglomerans is able to selectively induce midgut epithelial apoptosis while B. subtilis does not. This is accompanied by differential immune and homeostasis responses to both bacteria highlighted by immune pathway suppression via the Poor Immune Response upon Knock-in (Pirk) gene. These effects may actually be representative of a broader type of response to bacterial infection that might be present across several insect species. Finally, I demonstrated that during metamorphosis the sand fly relies, at least in part, upon the activation of multiple genes from the autophagy pathway to aid in generating adult tissues. More specifically, I demonstrate, using microscopy, the presence of ATG6 in the cytoplasm of developing midgut epithelial cells of the sand fly pupae.

We developed a honey bee RNA-virus vector based on the genome of a picorna-like Deformed wing virus (DWV), the main viral pathogen of the honey bee (Apis mellifera). To test the potential of DWV to be utilized as a vector, the 717 nt sequence coding for the enhanced green fluorescent protein (eGFP), flanked by the peptides targeted by viral protease, was inserted into an infectious cDNA clone of DWV in-frame between the leader protein and the virus structural protein VP2 genes. The in vitro RNA transcripts from egfp-tagged DWV cDNA clones were infectious when injected into honey bee pupae. Stable DWV particles containing genomic RNA of the recovered DWV with egfp inserts were produced, as evidenced by cesium chloride density gradient centrifugation. These particles were infectious to honey bee pupae when injected intra-abdominally. Fluorescent microscopy showed GFP expression in the infected cells and Western blot analysis demonstrated accumulation of free eGFP rather than its fusions with DWV LP and/or VP2 proteins. Analysis of the progeny egfp-tagged DWV showed gradual accumulation of genome deletions for egfp, providing estimates for the rate of loss of a non-essential gene an insect RNA virus genome during natural infection.

The mite Varroa destructor is one of the most destructive parasites of the honey bee (Apis mellifera) and the primary cause of colony collapse in most regions of the world. These mites cause serious injury to their hosts, especially during the larval and pupal stages, and serve as the vector for several viruses, which affect honey bee health causing colony death. Attempts by beekeepers to control these mites have yielded limited success. The inability to rear populations of mites in vitro that excludes contact with their honey bee hosts has stymied research of Varroa biology. Previous attempts to rear and/or maintain Varroa mites in vitro by feeding them on artificial diets have had limited success. Several methods were plagued by mechanical failures including leaking membranes and, thus far, none have been widely adopted. Here we report a robust system for maintaining Varroa mites that includes an artificial diet, which does not contain honey bee tissue-derived components, thus making it particularly valuable in studying mite vectoring of honey bee viruses. With our system we demonstrated for the first time that Varroa mites maintained on an artificial diet supplemented with the particles of honey bee viruses, cDNA clone-derived genetically tagged Varroa destructor virus-1 and wild-type Deformed wing virus, can acquire and later transmit these viruses to recipient honey bee pupae. Along with providing an opportunity to study parasites and pathogens in the absence of honey bee hosts, this in vitro system for Varroa mite maintenance is both scalable and consistent. These features can be used to better understand mite nutritional needs, metabolic activity, responses to chemicals and other biological functions.

Honey bees, the primary managed insect pollinator, suffer considerable losses due to Deformed wing virus (DWV), an RNA virus vectored by the mite Varroa destructor. Mite vectoring has resulted in the emergence of virulent DWV variants. The basis for such changes in DWV is poorly understood. Most importantly, it remains unclear whether replication of DWV occurs in the mite. In this study, we exposed Varroa mites to DWV type A via feeding on artificially infected honey bees. A significant, 357-fold increase in DWV load was observed in these mites after 2 days. However, after 8 additional days of passage on honey bee pupae with low viral loads, the DWV load dropped by 29-fold. This decrease significantly reduced the mites' ability to transmit DWV to honey bees. Notably, negative-strand DWV RNA, which could indicate viral replication, was detected only in mites collected from pupae with high DWV levels but not in the passaged mites. We also found that Varroa mites contain honey bee mRNAs, consistent with the acquisition of honey bee cells which would additionally contain DWV replication complexes with negative-strand DWV RNA. We propose that transmission of DWV type A by Varroa mites occurs in a non-propagative manner.

The synergistic interactions between the ectoparasitic mite Varroa destructor and Deformed wing virus (DWV) lead to the reduction in lifespan of the European honey bee Apis mellifera and often have been implicated in colony losses worldwide. However, to date, the underlying processes and mechanisms that form the multipartite interaction between the bee, mite, and virus have not been fully explained. To gain a better understanding of honey bees’ defense response to Varroa mite infestation and DWV infection, the DWV titers and transcription profiles of genes originating from RNAi, immunity, wound response, and homeostatic signaling pathways were monitored over a period of eight days. With respect to DWV, we observed low viral titers at early timepoints that coincided with high levels of Toll pathway transcription factor Dorsal, and its downstream immune effector molecules Hymenoptaecin, Apidaecin, Abaecin, and Defensin 1. However, we observed a striking increase in viral titers beginning after two days that coincided with a decrease in Dorsal levels and its corresponding immune effector molecules, and the small ubiquitin-like modifier (SUMO) ligase repressor of Dorsal, PIAS3. We observed a similar expression pattern for genes expressing transcripts for the RNA interference (Dicer/Argonaute), wound/homeostatic (Janus Kinase), and tissue growth (Map kinase/Wnt) pathways. Our results demonstrate that on a whole, honey bees are able to mount an immediate, albeit, temporally limited, immune and homeostatic response to Varroa and DWV infections, after which downregulation of these pathways leaves the bee vulnerable to expansive viral replication. The critical insights into the defense response upon Varroa and DWV challenges generated in this study may serve as a solid base for future research on the development of effective and efficient disease management strategies in honey bees.