Transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae.

Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-rearing industry. Whole-transcriptome analyses have revealed non-coding RNAs and their regulatory networks in N. bombycis infected embryos and larvae. However, transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae remains unclear. Here, we simultaneously compared the transcriptomes of N. bombycis and its host B. mori embryos of 5-day and larvae of 1-, 5- and 10-day during congenital infection. For the transcriptome of N. bombycis, a comparison of parasite expression patterns between congenital-infected embryos and larva showed most genes related to parasite central carbon metabolism were down-regulated in larvae during infection, whereas the majority of genes involved in parasite proliferation and growth were up-regulated. Interestingly, a large number of distinct or shared differentially expressed genes (DEGs) were revealed by the Venn diagram and heat map, many of them were connected to infection related factors such as Ricin B lectin, spore wall protein, polar tube protein, and polysaccharide deacetylase. For the transcriptome of B. mori infected with N. bombycis, beyond numerous DEGs related to DNA replication and repair, mRNA surveillance pathway, RNA transport, protein biosynthesis, and proteolysis, with the progression of infection, a large number of DEGs related to immune and infection pathways, including phagocytosis, apoptosis, TNF, Toll-like receptor, NF-kappa B, Fc epsilon RI, and some diseases, were successively identified. In contrast, most genes associated with the insulin signaling pathway, 2-oxacarboxylic acid metabolism, amino acid biosynthesis, and lipid metabolisms were up-regulated in larvae compared to those in embryos. Furthermore, dozens of distinct and three shared DEGs that were involved in the epigenetic regulations, such as polycomb, histone-lysine-specific demethylases, and histone-lysine-N-methyltransferases, were identified via the Venn diagram and heat maps. Notably, many DEGs of host and parasite associated with lipid-related metabolisms were verified by RT-qPCR. Taken together, simultaneous transcriptomic analyses of both host and parasite genes lead to a better understanding of changes in the microsporidia proliferation and host responses in embryos and larvae in N. bombycis congenital infection.

Inactivation of Nosema spp. with zinc phthalocyanine.

Most honey bee pathogens, such as Vairimorpha (Nosema), cannot be rapidly and definitively diagnosed in a natural setting, consequently there is typically the spread of these diseases through shared and re-use of beekeeping equipment. Furthermore, there are no viable treatment options available for Nosema spores to aid in managing the spread of this bee disease. We therefore aimed to develop a new method using novel Zinc Phthalocyanine (ZnPc) as a photosensitizer for the photodynamic inactivation of Nosema spores that could be used for the decontamination of beekeeping equipment. Nosema spores were propagated for in vitro testing using four caged Apis mellifera honey bees. The ZnPc treatment was characterized, encapsulated with a liposome, and then used as either a 10 or 100 µM treatment for the freshly harvested Nosema spores, for either a 30 and or 60-minute time period, under either light or dark conditions, in-vitro, in 96-well plates. In the dark treatment, after 30-min, the ZnPc 100 µM treatment, caused a 30 % Nosema mortality, while this increased to 80 % at the same concentration after the light treatment. The high rate of anti-spore effects, in a short period of time, supports the notion that this could be an effective treatment for managing honey bee Nosema infections in the future. Our results also suggest that the photo activation of the treatment could be applied in the field setting and this would increase the sterilization of beekeeping equipment against Nosema.

A tale of two parasites: Responses of honey bees infected with Nosema ceranae and Lotmaria passim.

Nosema ceranae and Lotmaria passim are two commonly encountered digestive tract parasites of the honey bee that have been associated with colony losses in Canada, the United States, and Europe. Though honey bees can be co-infected with these parasites, we still lack basic information regarding how they impact bee health at the individual and colony level. Using locally-isolated parasite strains, we investigated the effect of single and co-infections of these parasites on individual honey bee survival, and their responsiveness to sucrose. Results showed that a single N. ceranae infection is more virulent than both single L. passim infections and co-infections. Honey bees singly infected with N. ceranae reached < 50% survival eight days earlier than those inoculated with L. passim alone, and four days earlier than those inoculated with both parasites. Honey bees infected with either one, or both, parasites had increased responsiveness to sucrose compared to uninfected bees, which could correspond to higher levels of hunger and increased energetic stress. Together, these findings suggest that N. ceranae and L. passim pose threats to bee health, and that the beekeeping industry should monitor for both parasites in an effort correlate pathogen status with changes in colony-level productivity and survival.

Non-coding RNAs identification and regulatory networks in pathogen-host interaction in the microsporidia congenital infection.

BACKGROUND: The interaction networks between coding and non-coding RNAs (ncRNAs) including long non-coding RNA (lncRNA), covalently closed circular RNA (circRNA) and miRNA are significant to elucidate molecular processes of biological activities and interactions between host and pathogen. Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-feeding industry. However, little is known about ncRNAs that take place in the microsporidia congenital infection. Here we conducted whole-transcriptome RNA-Seq analyses to identify ncRNAs and regulatory networks for both N. bombycis and host including silkworm embryos and larvae during the microsporidia congenital infection. RESULTS: A total of 4,171 mRNAs, 403 lncRNA, 62 circRNAs, and 284 miRNAs encoded by N. bombycis were identified, among which some differentially expressed genes formed cross-talk and are involved in N. bombycis proliferation and infection. For instance, a lncRNA/circRNA competing endogenous RNA (ceRNA) network including 18 lncRNAs, one circRNA, and 20 miRNAs was constructed to describe 14 key parasites genes regulation, such as polar tube protein 3 (PTP3), ricin-B-lectin, spore wall protein 4 (SWP4), and heat shock protein 90 (HSP90). Regarding host silkworm upon N. bombycis congenital infection, a total of 14,889 mRNAs, 3,038 lncRNAs, 19,039 circRNAs, and 3,413 miRNAs were predicted based on silkworm genome with many differentially expressed coding and non-coding genes during distinct developmental stages. Different species of RNAs form interacting network to modulate silkworm biological processes, such as growth, metamorphosis and immune responses. Furthermore, a lncRNA/circRNA ceRNA network consisting of 140 lncRNAs, five circRNA, and seven miRNAs are constructed hypothetically to describe eight key host genes regulation, such as Toll-6, Serpin-6, inducible nitric oxide synthase (iNOS) and Caspase-8. Notably, cross-species analyses indicate that parasite and host miRNAs play a vital role in pathogen-host interaction in the microsporidia congenital infection. CONCLUSION: This is the first comprehensive pan-transcriptome study inclusive of both N. bombycis and its host silkworm with a specific focus on the microsporidia congenital infection, and show that ncRNA-mediated regulation plays a vital role in the microsporidia congenital infection, which provides a new insight into understanding the basic biology of microsporidia and pathogen-host interaction.

Effect of Chronic Exposure to Sublethal Doses of Imidacloprid and Nosema ceranae on Immunity, Gut Microbiota, and Survival of Africanized Honey Bees.

Large-scale honey bee colony losses reported around the world have been associated with intoxication with pesticides, as with the presence of pests and pathogens. Among pesticides, neonicotinoid insecticides are the biggest threat. Due to their extensive use, they can be found in all agricultural environments, including soil, water, and air, are persistent in the environment, and are highly toxic for honey bees. In addition, infection by different pests and pathogens can act synergistically, weakening bees. In this study, we investigated the effects of chronic exposure to sublethal doses of imidacloprid alone or combined with the microsporidia Nosema ceranae on the immune response, deformed wing virus infection (DWV), gut microbiota, and survival of Africanized honey bees. We found that imidacloprid affected the expression of some genes associated with immunity generating an altered physiological state, although it did not favor DWV or N. ceranae infection. The pesticide alone did not affect honey bee gut microbiota, as previously suggested, but when administered to N. ceranae infected bees, it generated significant changes. Finally, both stress factors caused high mortality rates. Those results illustrate the negative impact of imidacloprid alone or combined with N. ceranae on Africanized honey bees and are useful to understand colony losses in Latin America.

Supplementation of honey bee production colonies with a native beneficial microbe mixture.

Honey bee colonies form a complex superorganism, with individual and social immune defences that control overall colony health. Sometimes these defences are not enough to overcome infections by parasites and pathogens. For that reason, several studies have been conducted to evaluate different strategies to improve honey bee health. A novel alternative that is being studied is the use of beneficial microbes. In a previous study, we isolated and characterised bacterial strains from the native gut microbiota of honey bees. Four Apilactobacillus kunkeei strains were mixed and administered in laboratory models to evaluate their potential beneficial effect on larvae and adult bees. This beneficial microbe mixture was safe; it did not affect the expression of immune-related genes, and it was able to decrease the mortality caused by Paenibacillus larvae infection in larvae and reduced the Nosema ceranae spore number in infected adult honey bees. In the present study, we aimed to delve into the impact of the administration of this beneficial microbe mixture on honey bee colonies, under field conditions. The mixture was administered in sugar syrup using lyophilised bacterial cells or fresh cultures, by aspersion or sprayed and feeder, once a week for three consecutive weeks, in autumn or spring 2015, 2017 and 2019. Colony strength parameters were estimated before the administration, and one and three months later. Simultaneously different samples were collected to evaluate the infection levels of parasites and pathogens. The results showed that administering the beneficial microbe mixture decreased or stabilised the infection by N. ceranae or Varroa destructor in some trials but not in others. However, it failed to improve the colony's strength parameters or honey production. Therefore, field studies can be a game-changer when beneficial microbes for honey bees are tested, and meticulous studies should be performed to test their effectiveness.

Identification and subcellular localization of NbIAP in the microsporidian Nosema bombycis.

Many organisms go through a process of programmed cell death called apoptosis while newer cells are created. This has the effect of protecting the organism from cellular parasites and is a major line of defense against invading organisms. Apoptosis inhibitors, then, play an important role in aiding infectious agents by inhibiting caspase protease and thus the apoptopic pathway. In this study, we identified an inhibitor of apoptosis protein (IAP) in the microsporidian Nosema bombycis (NbIAP). NbIAP a composed of 218 amino acids containing two overlapping domains; the BIR domain and a zf-C3HC domain. We show, through indirect immunofluorescence, that NbIAP is present throughout the life cycle of N. bombycis and is localized in the nucleus of the parasite and therefor does not act on caspase protease directly. qRT-PCR analysis shows that the expression of the NbIAP gene was the highest on the first day of infection, then decreased to a relatively stable level. In addition, we show that the downregulation of the NbIAP gene directly inhibits the proliferation of N. bombycis. These findings suggest that NbIAP plays an important role in the N. bombycis life cycle.

Effect of amide protoporphyrin derivatives on immune response in Apis mellifera.

The intracellular microsporidian parasite Nosema ceranae is known to compromise bee health by induction of energetic stress and downregulation of the immune system. Porphyrins are candidate therapeutic agents for controlling Nosema infection without adverse effects on honeybees. In the present work, the impact of two protoporphyrin IX derivatives, i.e. PP[Asp]2 and PP[Lys]2, on Apis mellifera humoral immune response has been investigated in laboratory conditions in non-infected and N. ceranae-infected honeybees. Fluorescence spectroscopy analysis of hemolymph showed for the first time that porphyrin molecules penetrate into the hemocoel of honeybees. Phenoloxidase (PO) activity and the expression of genes encoding antimicrobial peptides (AMPs: abaecin, defensin, and hymenoptaecin) were assessed. Porphyrins significantly increased the phenoloxidase activity in healthy honeybees but did not increase the expression of AMP genes. Compared with the control bees, the hemolymph of non-infected bees treated with porphyrins had an 11.3- and 6.1-fold higher level of PO activity after the 24- and 48-h porphyrin administration, respectively. Notably, there was a significant inverse correlation between the PO activity and the AMP gene expression level (r = - 0.61696, p = 0.0143). The PO activity profile in the infected bees was completely opposite to that in the healthy bees (r = - 0.5118, p = 0.000), which was related to the changing load of N. ceranae spores in the porphyrin treated-bees. On day 12 post-infection, the spore loads in the infected porphyrin-fed individuals significantly decreased by 74%, compared with the control bees. Our findings show involvement of the honeybee immune system in the porphyrin-based control of Nosema infection. This allows the infected bees to improve their lifespan considerably by choosing an optimal PO activity/AMP expression variant to cope with the varying level of N. ceranae infection.

Antifungal activity of "HO21-F", a formulation based on Olea europaea plant extract, in honey bees infected with Nosema ceranae.

Nosema ceranae is a microsporidium parasite that silently affects honey bees, causing a disease called nosemosis. This parasite produces resistant spores and germinates in the midgut of honey bees, extrudes a polar tubule that injects an infective sporoplasm in the host cell epithelium, proliferates, and produces intestinal disorders that shorten honey bee lifespan. The rapid extension of this disease has been reported to be widespread among adult bees, and treatments are less effective and counterproductive weakening colonies. This work aimed to evaluate the antifungal activity of a prototype formulation based on a non-toxic plant extract (HO21-F) against N. ceranae. In laboratory, honey bees were infected artificially, kept in cages for 17 days and samples were taken at 7 and 14 days post infection (dpi). At the same time, in field conditions we evaluated the therapeutic effect of HO21-F for 28 days in naturally infected colonies. The effectiveness of the treatment has been demonstrated by a reduction of 83.6 % of the infection levels observed in laboratory conditions at concentrations of 0.5 and 1 g/L without affecting the survival rate. Besides, in-field conditions we reported a reduction of 88 % of the infection level at a concentration of 2.5 g/L, obtaining better antifungal effectiveness in comparison to other commercially available treatments. As a result, we observed that the use of HO21-F led to an increase in population size and honey production, both parameters associated with colony strength. The reported antifungal activity of HO21-F against N. ceranae, with a significant control of spore proliferation in worker bees, suggests the promising commercial application use of this product against nosemosis, and it will encourage new research studies to understand the mechanism of action, whether related to the spore-inhibition effect and/or a stimulating effect in natural response of colonies to counteract the disease.

Heterologous expression of scFv fragment against Vairimorpha (Nosema) ceranae hexokinase in Sf9 cell culture inhibits microsporidia intracellular growth.

Secretion of hexokinase (HK) by microsporidia into infected cells suggests an important role for this enzyme for the intracellular development of parasites. To verify whether the expression of HK-specific antibodies in the host cell cytoplasm can suppress the growth of microsporidia, we constructed an immune library of recombinant scFv fragments against the enzyme of the honey bee pathogen Vairimorpha (Nosema) ceranae (VcHK) with a representativeness of about 5 million bacterial transformants. Two variants of VcHK-specific recombinant antibodies were selected by library panning and expressed in lepidopteran Sf9 cell line. Infecting of cells expressing two selected and control scFv fragments with V. ceranae spores was followed by their cultivation for 4 days. Analysis of parasite ß-tubulin as well as spore wall protein SWP32 transcripts in infected cultures by reverse transcription PCR and real-time qPCR showed (1) V. ceranae growth in cells heterologous to bee pathogens, (2) its inhibition by one of the selected VcHK-specific recombinant antibodies. The latter result once again emphasizes an important role of microsporidia hexokinases in their relationships with infected host cells and suggests further focusing on the mechanisms of such suppression, as well as on the search for new V. ceranae - inhibiting scFv fragments.

Natural extracts as potential control agents for Nosema ceranae infection in honeybees, Apis mellifera.

Nosema disease is one factor that can cause colony decline in honeybees (Apis mellifera L.) worldwide. Nosema ceranae has outcompeted Nosema apis in the Western honeybee (A. mellifera) which is its original host. Fumagilin is an effective antibiotic treatment to control Nosema infection but currently it is forbidden in many countries. In this study, 12 plant extracts were evaluated for their toxicity to adult bees and antimicrosporidian activity under laboratory and field conditions. N. ceranae-infected adult bees were fed ad libitum with 50% sucrose solution containing 1% and 5% (w/v) of each plant extract. Bee mortality in N. ceranae-infected groups fed with plant extracts was higher than that in the control group treated with fumagilin. The results demonstrated that 9 of 12 extracts had high antimicrosporidian activity against N. ceranae and their efficacies were comparable to fumagilin. Spore reduction in infected bees was 4-6 fold less after extract treatment. Following laboratory screening, Annona squamosa, Ocimum basilicum, Psidium guajava and Syzygium jambos were tested in honeybee colonies. Plant extracts of 2% concentration (w/v) inhibited the development of Nosema spores after 30 days of treatment. At the end of experiment (90 days), spores in the plant extract treated groups were lower than in group treated with fumagilin but there was no significant difference. Although, extracts tested in this study showed high toxicity to bee in laboratory cages, they did not show negative affects on bees under whole colony conditions. Therefore, the effectiveness of plant extracts tested in this study was notable and warrants further study as potential Nosema control agents in honey bees. Plant extracts would offer a non-antibiotic alternative for Nosema control and help reduce the overuse of antibiotics in livestock.

The microsporidium Nosema pyrausta as a potent microbial control agent of the beet webworm Loxostege sticticalis.

The microsporidium Nosema pyrausta is an important mortality factor of the European corn borer, Ostrinia nubilalis. The present study was aimed at N. pyrausta virulence testing to the beet webworm (BW), Loxostege sticticalis. This agricultural pest, L. sticticalis, was highly vulnerable to N. pyrausta. The parasite's spores were located in salivary glands, adipose tissue, and Malpighian tubules of the infected specimens. Infection was transmitted transovarially through at least 3 laboratory generations, in which BW fitness indices were lower than in the control, and moth emergence and fertility decreased prominently. Transovarial infection was most detrimental to female egg-laying ability, resulting in zero fertility in F3. When propagated in BW, the microsporidium tended to increase its virulence to L. sticticalis, as compared to the Ostrinia isolates. The parasite's ability to infect this host at low dosages and transmit vertically should guarantee its effective establishment and spread within BW populations. In conclusion, N. pyrausta is a promising agent against BW, which is a notorious polyphagous pest in Eurasia.

The pathological effects of a Nosema ceranae infection in the giant honey bee, Apis dorsata Fabricius, 1793.

Nosema ceranae is an intracellular microsporidian pathogen that lives in the midgut ventricular cells of all known honey bee Apis species. We suspect that N. ceranae may also cause energetic stress in the giant honey bee because this parasite is known to disrupt nutrient absorption resulting in energetic stress in the honey bee species Apis mellifera. To understand how N. ceranae impacts the energetic stress of the giant honey bee, A. dorsata, we measured the hemolymph trehalose levels of experimentally infected giant honey bees on days three, five, seven, and fourteen post infection (p.i.). We also measured the hypopharyngeal gland protein content, the total midgut proteolytic enzyme activity, honey bee survival, infection ratio, and spore loads comparing infected and uninfected honey bees across the same time frame. Nosema ceranae-infected honey bees had significantly lowered survival, trehalose levels, hypopharyngeal gland protein content, and midgut proteolytic enzyme activity. We found an increasing level of parasitic loads and infection ratio of N. ceranae-infected bees after inoculation. Collectively, our results suggest that the giant honey bee suffers from energetic stress and limited nutrient absorption from a N. ceranae infection, which results in lowered survival in comparison to uninfected honey bees. Our findings highlight that other honey bee species besides A. mellifera are susceptible to microsporidian pathogens that they harbor, which results in negative effects on health and survival. Therefore, these pathogens might be transmitted at a community level, in the natural environment, resulting in negative health effects of multiple honey bee species.

Effect of feeding chitosan or peptidoglycan on Nosema ceranae infection and gene expression related to stress and the innate immune response of honey bees (Apis mellifera).

Nosema ceranae is a microsporidian parasite that causes nosema disease, an infection of the honey bee (Apis mellifera) midgut. Two pathogen-associated molecular patterns (PAMPs), chitosan and peptidoglycan, and N. ceranae spores were fed to worker bees in sucrose syrup and compared to non-inoculated and N. ceranae-inoculated bees without PAMPs. Both chitosan and peptidoglycan significantly increased bee survivorship and reduced spore numbers due to N. ceranae infection. To determine if these results were related to changes in health status, expression of the immune-related genes, hymenoptaecin and defensin2, and the stress tolerance-related gene, blue cheese, was compared to that of control bees. Compared to the inoculated control, bees with the dose of chitosan that significantly reduced N. ceranae spore numbers showed lower expression of hymenoptaecin and defensin2 early after infection, higher expression mid-infection of defensin2 and lower expression of all three genes late in infection. In contrast, higher expression of defensin2 early in the infection and all three genes late in the infection was observed with peptidoglycan treatment. Changes late in the parasite multiplication stage when mature spores would be released from ruptured host cells are less likely to have contributed to reduced spore production. Based on these results, it is concluded that feeding bees chitosan or peptidoglycan can reduce N. ceranae infection, which is at least partially related to altering the health of the bee by inducing immune and stress-related gene expression.

Mitigating Nosema ceranae infection in western honey bee (Apis mellifera) workers using propolis collected from honey bee and stingless bee (Tetrigona apicalis) hives.

Beekeepers need sustainable control options to treat Nosema ceranae infection in colonies of western honey bees (Apis mellifera L.) they manage. Propolis is a natural product derived from plant resins and contains chemical compounds with potential antimicrobial activity against N. ceranae. Here, we determined the efficacy of propolis from A. mellifera (USA) and Tetrigona apicalis (stingless bees, Thailand) colonies as treatments for N. ceranae infection in honey bee workers. Newly emerged bees were individually fed 2 µL of 50% (w/v) sucrose solution containing 1 × 105N. ceranae spores. Following this, the infected bees were treated with 50% propolis extracted from A. mellifera or T. apicalis hives and fed in 50% sucrose solution (v/v). All bees were maintained at 34 ± 2 °C and 55 ± 5% RH. Dead bees were counted daily for 30 d to calculate survival. We also determined infection rate (# infected bees/100 bees), infectivity (number of spores per bee) and protein content in the hypopharyngeal glands and hemolymph on 7, 14, and 21 d post infection as measures of bee health. Propolis from both bee species significantly reduced bee mortality, infection rate and infectivity compared with those of untreated bees and led to significantly greater protein contents in hypopharyngeal glands and hemolymph in treated bees than in untreated ones (p < 0.0001). In conclusion, propolis from A. mellifera and T. apicalis colonies shows promise as a control against N. ceranae infection in honey bees.

CCTδ colocalizes with actin and ß-tubulin: Insight into its involvement in the cytoskeleton formation of the intracellular parasite Nosema bombycis.

The chaperonin-containing t-complex polypeptide 1 (CCT) is a molecular chaperone protein that is widely present in eukaryotic cytoplasm and can assist in the folding of newly synthesized proteins. The CCT complex consists of eight completely different subunits, among which the δ subunit plays an extremely important role in the folding and assembly of cytoskeleton proteins as an individual or complex with other subunits. In this study, we identified the CCTδ in the microsporidian Nosema bombycis (NbCCTδ) for the first time. The NbCCTδ gene contains a complete ORF of 1497 bp in length that encodes a 498 amino acid polypeptide. NbCCTδ is expressed throughout the entire lifecycle of N. bombycis and rather higher in early stage of proliferation. Indirect immunofluorescence results showed that NbCCTδ was colocalized with actin and ß-tubulin during the proliferative and sporogonic phases of N. bombycis. RNA interference down-regulated the expression of the NbCCTδ gene. These results imply that NbCCTδ may participate in cytoskeleton formation and proliferation of N. bombycis.

Septin homologs cooperating in the Proliferative Stage of Microsporidia Nosema bombycis.

The single-celled pathogen Nosema bombycis, that can infect silkworm Bombyx mori and other lepidoptera including Spodoptera, is the first identified Microsporidia which has diplokaryotic nuclei throughout the life cycle. Septin proteins can form highly ordered filaments, bundles or ring structures related to the cytokinesis in fungi. Here, three septin proteins (NbSeptin1, NbSeptin2 and NbSeptin3) from Nosema bombycis CQ I are described. These proteins, appear to be conserved within the phylum Microsporidia. NbSeptins transcripts were detected throughout the pathogen developmental cycle and were significantly enhanced from second days of infection, which lead to our hypothesis that NbSeptins play a role in merogony. Immunofluorescence assay (IFA) revealed a broad distribution of NbSeptins in meronts and partly co-localization of NbSeptins. Interestingly, in some of meronts, NbSeptin2 and NbSeptin3 showed localization between the nuclei of the diplokaryon. Yeast two-hybrid and co-immunoprecipitation analysis verified that NbSeptins can interact with each other. Our findings suggest that NbSeptins can cooperate in the proliferation stage of Nosema bombycis and contribute towards the understanding of the rols of septins in microsporidia development.

Physiological effects of the interaction between Nosema ceranae and sequential and overlapping exposure to glyphosate and difenoconazole in the honey bee Apis mellifera.

Pathogens and pollutants, such as pesticides, are potential stressors to all living organisms, including honey bees. Herbicides and fungicides are among the most prevalent pesticides in beehive matrices, and their interaction with Nosema ceranae is not well understood. In this study, the interactions between N. ceranae, the herbicide glyphosate and the fungicide difenoconazole were studied under combined sequential and overlapping exposure to the pesticides at a concentration of 0.1 µg/L in food. In the sequential exposure experiment, newly emerged bees were exposed to the herbicide from day 3 to day 13 after emerging and to the fungicide from day 13 to day 23. In the overlapping exposure experiment, bees were exposed to the herbicide from day 3 to day 13 and to the fungicide from day 7 to day 17. Infection by Nosema in early adult life stages (a few hours post emergence) greatly affected the survival of honey bees and elicited much higher mortality than was induced by pesticides either alone or in combination. Overlapping exposure to both pesticides induced higher mortality than was caused by sequential or individual exposure. Overlapping, but not sequential, exposure to pesticides synergistically increased the adverse effect of N. ceranae on honey bee longevity. The combination of Nosema and pesticides had a strong impact on physiological markers of the nervous system, detoxification, antioxidant defenses and social immunity of honey bees.

Immunity and physiological changes in adult honey bees (Apis mellifera) infected with Nosema ceranae: The natural colony environment.

Nosema ceranae is a microsporidium that infects Apis mellifera, causing diverse physiological and behavioral alterations. Given the existence of individual and social mechanisms to reduce infection and fungal spread in the colony, bees may respond differently to infection depending on their rearing conditions. In this study, we investigated the effect of N. ceranae in honey bee foragers naturally infected with different fungal loads in a tropical region. In addition, we explored the effects of N. ceranae artificially infected young bees placed in a healthy colony under field conditions. Honey bees naturally infected with higher loads of N. ceranae showed downregulation of genes from Toll and IMD immune pathways and antimicrobial peptide (AMP) genes, but hemolymph total protein amount and Vitellogenin (Vg) titers were not affected. Artificially infected bees spread N. ceranae to the controls in the colony, but fungal loads were generally lower than those observed in cages, probably because of social immunity. Although no significant changes in mRNA levels of AMP-encoding were observed, N. ceranae artificially infected bees showed downregulation of miR-989 (an immune-related microRNA), lower vitellogenin gene expression, and decreased hemolymph Vg titers. Our results demonstrate for the first time that natural infection by N. ceranae suppresses the immune system of honey bee foragers in the field. This parasite is detrimental to the immune system of young and old bees, and disease spread, mitigation and containment will depend on the colony environment.

Managed bumble bees acquire parasites from their foraging environment: A case study on parasite spillback.

The use of commercially reared bumble bees in agricultural environments has been recognized as a potential threat to wild pollinators due to competition, genetic contamination, and most notably, disease transmission. Higher parasite prevalence near greenhouses where managed bumble bees are used has been linked to parasite spillover from managed to wild bees. However, pathogen transmission is not unidirectional, and can also flow from wild to managed bees. These newly infected managed bees can subsequently re-infect (other) wild bees, in a process known as spillback, which is an alternative explanation for the increased parasite prevalence near greenhouses. Reducing parasite prevalence in managed bees is key to controlling host-parasite dynamics in cases of spillover; in spillback, producing managed bees that are resilient to infection is important. Here we establish that the managed bumble bee Bombus terrestris can acquire parasites from their foraging environment, which is the major infection route for Apicystis spp. and Crithidia spp., but not for Nosema spp.. Managed B. terrestris were found to have a higher prevalence of Crithdia and a higher load of Apicystis than local wild conspecifics, showing that for these parasites, spillback is a possible risk scenario.