Genomic characterization of Moraxella bovis and Moraxella bovoculi Uruguayan strains isolated from calves with infectious bovine keratoconjunctivitis.

Infectious bovine keratoconjunctivitis (IBK) is an ocular disease that affects bovines and has significant economic and health effects worldwide. Gram negative bacteria Moraxella bovis and Moraxella bovoculi are its main etiological agents. Antimicrobial therapy against IBK is often difficult in beef and dairy herds and, although vaccines are commercially available, their efficacy is variable and dependent on local strains. The aim of this study was to analyze for the first time the genomes of Uruguayan clinical isolates of M. bovis and M. bovoculi. The genomes were de novo assembled and annotated; the genetic basis of fimbrial synthesis was analyzed and virulence factors were identified. A 94% coverage in the reference genomes of both species, and more than 80% similarity to the reference genomes were observed. The mechanism of fimbrial phase variation in M. bovis was detected, and the tfpQ orientation of these genes confirmed, in an inversion region of approximately 2.18kb. No phase variation was determined in the fimbrial gene of M. bovoculi. When virulence factors were compared between strains, it was observed that fimbrial genes have 36.2% sequence similarity. In contrast, the TonB-dependent lactoferrin/transferrin receptor exhibited the highest percentage of amino acid similarity (97.7%) between strains, followed by cytotoxins MbxA/MbvA and the ferric uptake regulator. The role of these virulence factors in the pathogenesis of IBK and their potential as vaccine components should be explored.

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.

Role of Proteus mirabilis flagella in biofilm formation.

Proteus mirabilis(P. mirabilis) is a common etiological agent of urinary tract infections, particularly those associated with catheterization. P. mirabilis efficiently forms biofilms on different surfaces and shows a multicellular behavior called 'swarming', mediated by flagella. To date, the role of flagella in P. mirabilis biofilm formation has been under debate. In this study, we assessed the role of P. mirabilis flagella in biofilm formation using an isogenic allelic replacement mutant unable to express flagellin. Different approaches were used, such as the evaluation of cell surface hydrophobicity, bacterial motility and migration across catheter sections, measurements of biofilm biomass and biofilm dynamics by immunofluorescence and confocal microscopy in static and flow models. Our findings indicate that P. mirabilis flagella play a role in biofilm formation, although their lack does not completely avoid biofilm generation. Our data suggest that impairment of flagellar function can contribute to biofilm prevention in the context of strategies focused on particular bacterial targets.

Seasonal Dynamics of the Honey Bee Gut Microbiota in Colonies Under Subtropical Climate : Seasonal Dynamics of Honey Bee Gut Microbiota.

Honey bees (Apis mellifera) provide invaluable benefits for food production and maintenance of biodiversity of natural environments through pollination. They are widely spread across the world, being adapted to different climatic conditions. To survive the winter in cold temperate regions, honey bees developed different strategies including storage of honey and pollen, confinement of individuals during the winter, and an annual cycle of colony growth and reproduction. Under these conditions, winter honey bees experience physiological changes, including changes in immunity and the composition of honey bee gut microbiota. However, under tropical or subtropical climates, the life cycle can experience alterations, i.e., queens lay eggs during almost all the year and new honey bees emerge constantly. In the present study, we characterized nurses' honey bee gut microbiota in colonies under subtropical region through a year, combining qPCR, PCR-DGGE, and 16S rDNA high-throughput sequencing. We also identified environmental variables involved in those changes. Our results showed that under the mentioned conditions, the number of bacteria is stable throughout the year. Diversity of gut microbiota is higher in spring and lower in summer and winter. Gradual changes in compositions occur between seasons: Lactobacillus spp. predominate in spring while Gilliamella apicola and Snodgrasella alvi predominate in summer and winter. Environmental variables (mainly precipitations) affected the composition of the honey bee gut microbiota. Our findings provide new insights into the dynamics of honey bee gut microbiota and may be useful to understand the adaptation of bees to different environmental conditions.

Probiotic potential of GABA-producing lactobacilli isolated from Uruguayan artisanal cheese starter cultures.

AIMS: In this study, we sought to identify and characterize a collection of 101 lactobacilli strains isolated from natural whey starters used in Uruguayan artisan cheese production, based on their capacity to produce gamma-aminobutyric acid (GABA) and their probiotic potential. METHODS AND RESULTS: The probiotic potential was assessed using low pH and bile salt resistance assays; bacterial adhesion to intestinal mucus was also evaluated. Selected strains were then identified by 16S sequencing, and their GABA-producing potential was confirmed and quantified using a UHPLC-MS system. Twenty-five strains were identified and characterized as GABA-producing lactobacilli belonging to the phylogenetical groups Lactiplantibacillus (n = 19) and Lacticaseibacillus (n = 6). Fifteen strains of the Lactiplantibacillus group showed a significantly higher GABA production than the rest. They showed the predicted ability to survive the passage through the gastrointestinal tract, according to the in vitro assays. CONCLUSIONS: A set of promising candidate strains was identified as potential probiotics with action on the gut-brain axis. Further studies are needed to assess their possible effects on behaviour using in vivo assay. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the potential of strains isolated from local natural whey starters as probiotics and for biotechnological use in functional GABA-enriched foods formulation.

Virulence genes of Escherichia coli in diarrheic and healthy calves.

Escherichia coli ETEC, EPEC, NTEC and STEC/EHEC pathotypes are often isolated from bovine feces. The objective of this study was to detect 21 E. coli virulence genes in feces from 252 dairy calves in Uruguay (149 with neonatal diarrhea - NCD - and 103 asymptomatic). Genes iucD, f17A, afa8E, papC, clpG and f17G(II) were the most prevalent (81.3%; 48.4%; 37.3%; 35.7%; 34.1%; 31.3%, respectively). Genes eae, stx1and stx2 were poorly represented; 13/252 animals harbored one or a combination of these genes. The prevalence of the cnf gene was 4.4%, while that of cdt-IV and cdt-III genes was 24.2% and 12.7% respectively. This study reports updated data about the virulence profiles of E. coli in dairy calves in Uruguay. A large number of adhesins and toxin genes were detected. Our results demonstrate that E. coli from bovine feces has diarrheagenic and extraintestinal profiles although other NCD risks factors may contribute to the disease outcome.

Detection of Lotmaria passim in Africanized and European honey bees from Uruguay, Argentina and Chile.

Trypanosomatids affecting honey bees, Crithidia mellificae and Lotmaria passim, have been poorly studied in South America. We therefore analyzed their presence in Africanized and European honeybees from Uruguay, Argentina and Chile collected between 1990 and 2011 and assessed their association with other bee parasites and pathogens. Crithidia mellificae was not detected while L. passim was wide-spread. This report shows that L. passim has been present in this region at least since 2007 and it infects both Africanized and European honey bees. L. passim infected colonies showed high V. destructor parasitization levels, suggesting an association between them.

Phenotypic, molecular and pathological characterization of motile aeromonads isolated from diseased fishes cultured in Uruguay.

Information about motile aeromonads from aquaculture systems of the Neotropical region is scarce. The aim of this study was to characterize motile Aeromonas isolated from ornamental and consumable fishes cultured in Uruguay. Biochemical and molecular methods were used for species identification. Antimicrobial susceptibility and the presence of virulence genes were evaluated. Genetic diversity was analysed by rep-PCR, and virulence of the most representative isolates was determined by calculating the fifty lethal dose in experimentally challenged fish (Australoheros facetus). Aeromonas hydrophila and A. veronii were the most prevalent identified species (38.2% and 32.4%, respectively), whereas A. allosacharophila, A. bestiarium, A. caviae and A. punctata were less prevalent. This study constitutes the first report of these last four species in Uruguay. All isolates were resistant to at least three antimicrobials, and 82.3% of them showed multidrug resistance. Virulence genotypes were correlated with the Aeromonas species and haemolytic activity. The genotype act+/alt+/ast+/ela+/lip+ was the most prevalent (26.5%). A correlation between virulence genotypes and Aeromonas species was found. A. punctata showed a clonal structure according to rep-PCR analysis, whereas other species showed high genetic diversity. The number of virulence genes of the isolates was related with virulence according to the experimental challenge assays.

Sublethal effects of acaricides and Nosema ceranae infection on immune related gene expression in honeybees.

Nosema ceranae is an obligate intracellular parasite and the etiologic agent of Nosemosis that affects honeybees. Beside the stress caused by this pathogen, honeybee colonies are exposed to pesticides under beekeeper intervention, such as acaricides to control Varroa mites. These compounds can accumulate at high concentrations in apicultural matrices. In this work, the effects of parasitosis/acaricide on genes involved in honeybee immunity and survival were evaluated. Nurse bees were infected with N. ceranae and/or were chronically treated with sublethal doses of coumaphos or tau-fluvalinate, the two most abundant pesticides recorded in productive hives. Our results demonstrate the following: (1) honeybee survival was not affected by any of the treatments; (2) parasite development was not altered by acaricide treatments; (3) coumaphos exposure decreased lysozyme expression; (4) N. ceranae reduced levels of vitellogenin transcripts independently of the presence of acaricides. However, combined effects among stressors on imagoes were not recorded. Sublethal doses of acaricides and their interaction with other ubiquitous parasites in colonies, extending the experimental time, are of particular interest in further research work.

Seasonal Variation of Honeybee Pathogens and its Association with Pollen Diversity in Uruguay.

Honeybees are susceptible to a wide range of pathogens, which have been related to the occurrence of colony loss episodes reported mainly in north hemisphere countries. Their ability to resist those infections is compromised if they are malnourished or exposed to pesticides. The aim of the present study was to carry out an epidemiological study in Uruguay, South America, in order to evaluate the dynamics and interaction of honeybee pathogens and evaluate their association with the presence of external stress factors such as restricted pollen diversity and presence of agrochemicals. We monitored 40 colonies in two apiaries over 24 months, regularly quantifying colony strength, parasite and pathogen status, and pollen diversity. Chlorinated pesticides, phosphorus, pyrethroid, fipronil, or sulfas were not found in stored pollen in any colony or season. Varroa destructor was widespread in March (end of summer-beginning of autumn), decreasing after acaricide treatments. Viruses ABPV, DWV, and SBV presented a similar trend, while IAPV and KBV were not detected. Nosema ceranae was detected along the year while Nosema apis was detected only in one sample. Fifteen percent of the colonies died, being associated to high V. destructor mite load in March and high N. ceranae spore loads in September. Although similar results have been reported in north hemisphere countries, this is the first study of these characteristics in Uruguay, highlighting the regional importance. On the other side, colonies with pollen of diverse botanical origins showed reduced viral infection levels, suggesting that an adequate nutrition is important for the development of healthy colonies.

Intracellular bacteria in the pathogenesis of Escherichia coli urinary tract infection in children.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is the most common agent of urinary tract infection (UTI). The classic model of pathogenesis proposes the ascent of UPEC by the urethra and external adherence to the urothelium. Recently, the ability of UPEC to invade urothelial cells and to form intracellular bacterial communities (IBCs) has been described. METHODS: The objective of the present study was to determine the presence of intracellular bacteria (IB) in children with UTI caused by E. coli and to characterize its virulence attributes and its relation with clinical outcomes. One hundred thirty-three children with E. coli UTI who attended a reference children's hospital between June and November 2012 were included. Urine samples were analyzed by optical and confocal microscopy looking for exfoliated urothelial cells with IB. Phylogenetic group and 24 virulence factors of UPEC were determined using multiplex polymerase chain reaction. Medical records were analyzed. RESULTS: The presence of IB was detected in 49 of 133 (36.8%) samples by confocal microscopy, in 30 cases as IBC, and in 19 as isolated intracellular bacteria (IIB). Only 50% of these cases could be detected by light microscopy. Seventy-four medical records were analyzed, 34 with IBC/IIB, 40 without IB. Any virulence gene was associated with IBC/IIB. The presence of IBC/IIB was associated with recurrent UTI (odds ratio [OR], 3.3; 95% confidence interval [CI], 1.3-9; P = .017), especially in children without urinary tract functional or morphological abnormalities (OR, 8.0; 95% CI, 2.3-27.4; P = .000). IBCs were associated with lower urinary tract syndrome (OR, 3.6; 95% CI, 1.1-11.8; P = .05) and absence of fever (P = .009). CONCLUSIONS: IBCs/IIB could explain a high proportion of children with recurrent UTI.

Native flagellin does not protect mice against an experimental Proteus mirabilis ascending urinary tract infection and neutralizes the protective effect of MrpA fimbrial protein.

Proteus mirabilis expresses several virulence factors including MR/P fimbriae and flagella. Bacterial flagellin has frequently shown interesting adjuvant and protective properties in vaccine formulations. However, native P. mirabilis flagellin has not been analyzed so far. Native P. mirabilis flagellin was evaluated as a protective antigen and as an adjuvant in co-immunizations with MrpA (structural subunit of MR/P fimbriae) using an ascending UTI model in the mouse. Four groups of mice were intranasally treated with either MrpA, native flagellin, both proteins and PBS. Urine and blood samples were collected before and after immunization for specific antibodies determination. Cytokine production was assessed in immunized mice splenocytes cultures. Mice were challenged with P. mirabilis, and bacteria quantified in kidneys and bladders. MrpA immunization induced serum and urine specific anti-MrpA antibodies while MrpA coadministered with native flagellin did not. None of the animals developed significant anti-flagellin antibodies. Only MrpA-immunized mice showed a significant decrease of P. mirabilis in bladders and kidneys. Instead, infection levels in MrpA-flagellin or flagellin-treated mice showed no significant differences with the control group. IL-10 was significantly induced in splenocytes of mice that received native flagellin or MrpA-flagellin. Native P. mirabilis flagellin did not protect mice against an ascending UTI. Moreover, it showed an immunomodulatory effect, neutralizing the protective role of MrpA. P. mirabilis flagellin exhibits particular immunological properties compared to other bacterial flagellins.

Insights into the effects of sublethal doses of pesticides glufosinate-ammonium and sulfoxaflor on honey bee health.

Insect pollinators are threatened worldwide, being the exposure to multiple pesticides one of the most important stressor. The herbicide Glyphosate and the insecticide Imidacloprid are among the most used pesticides worldwide, although different studies evidenced their detrimental effects on non-target organisms. The emergence of glyphosate-resistant weeds and the recent ban of imidacloprid in Europe due to safety concerns, has prompted their replacement by new molecules, such as glufosinate-ammonium (GA) and sulfoxaflor (S). GA is a broad-spectrum and non-selective herbicide that inhibits a key enzyme in the metabolism of nitrogen, causing accumulation of lethal levels of ammonia; while sulfoxaflor is an agonist at insect nicotinic acetylcholine receptors (nAChRs) and generates excitatory responses including tremors, paralysis and mortality. Although those molecules are being increasingly used for crop protection, little is known about their effects on non-target organisms. In this study we assessed the impact of chronic and acute exposure to sublethal doses of GA and S on honey bee gut microbiota, immunity and survival. We found GA significantly reduced the number of gut bacteria, and decreased the expression of glucose oxidase, a marker of social immunity. On the other hand, S significantly increased the number of gut bacteria altering the microbiota composition, decreased the expression of lysozyme and increased the expression of hymenoptaecin. These alterations in gut microbiota and immunocompetence may lead to an increased susceptibility to pathogens. Finally, both pesticides shortened honey bee survival and increased the risk of death. Those results evidence the negative impact of GA and S on honey bees, even at single exposition to a low dose, and provide useful information to the understanding of pollinators decline.

Shiga Toxin-Producing Escherichia coli (STEC) Associated with Calf Mortality in Uruguay.

In Uruguay, the mortality of dairy calves due to infectious diseases is high. Escherichia coli is a natural inhabitant of the intestinal microbiota, but can cause several infections. The aim of the work was to characterize E. coli isolates from intestinal and extraintestinal origin of dead newborn calves. Using PCR, virulence gene characteristics of pathogenic E. coli were searched. The pathogenic E. coli were molecularly characterized and the phylogroup, serogroup and the Stx subtype were determined. Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method and plasmid-mediated quinolone resistance (PMQR) genes with PCR. Finally, clonal relationships were inferred using PFGE. Gene characteristics of the Shiga toxin-producing E. coli (STEC), Enteropathogenic E. coli (EPEC) and Necrotoxigenic E. coli (NTEC) were identified. The prevalence of the iucD, afa8E, f17, papC, stx1, eae and ehxA genes was high and no f5, f41, saa, sfaDE, cdtIV, lt, sta or stx2 were detected. The prevalence of STEC gene stx1 in the dead calves stood out and was higher compared with previous studies conducted in live calves, and STEC LEE+ (Enterohemorrhagic E. coli (EHEC)) isolates with stx1/eae/ehxA genotypes were more frequently identified in the intestinal than in the extraintestinal environment. E. coli isolates were assigned to phylogroups A, B1, D and E, and some belonged to the O111 serogroup. stx1a and stx1c subtypes were determined in STEC. A high prevalence of multi-resistance among STEC and qnrB genes was determined. The PFGE showed a high diversity of pathogenic strains with similar genetic profiles. It can be speculated that EHEC (stx1/eae/ehxA) could play an important role in mortality. The afa8E, f17G1 and papC genes could also have a role in calf mortality. Multidrug resistance defies disease treatment and increases the risk of death, while the potential transmissibility of genes to other species constitutes a threat to public health.

American Foulbrood in Uruguay: twelve years from its first report.

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a deleterious disease that affects honeybees. In Uruguay it was first reported in 1999. In 2001 the bacterium was spread all over the country, and its prevalence in honey was estimated in 51%. Two P. larvae genotypes were found; ERIC I - BOX A, worldwide distributed and ERIC I - BOX C, exclusively detected in Argentina until then. In the present manuscript we analyzed the evolution of AFB outbreaks from 1999 to 2009, presented a new nation-wide survey carried out during 2011 when a prevalence of 2% was found and discuss national strategies for prevention of the disease. Since Uruguay is a small country where almost all beekeepers are registered, Uruguayan experience can be useful to be applied in other countries.

Probiotic Lactobacilli Administration Induces Changes in the Fecal Microbiota of Preweaned Dairy Calves.

Early microbial colonization is a determinant factor in animal health, and probiotic administration has been demonstrated to modulate intestinal microbiota and promote health in dairy calves. The objective of this study was to evaluate changes in calves' fecal microbiota after the administration of two probiotic lactobacilli strains that had previously exhibited beneficial effects in calves' health in relation to neonatal calf diarrhea. An in vivo assay was performed with 30 newborn male Holstein calves that were divided into three groups. Two groups were orally administered with two different lactobacilli strains (Lactobacillus johnsonii TP1.6 or Limosilactobacillus reuteri TP1.3B), and the third was the control group. Calves (5 to 9 days old) were administered with freeze-dried bacteria once a day for 10 consecutive days. Feces samples were taken before the first administration (day 0) and then again on days 10 and 21, and the V4 region of the bacterial 16S ribosomal gene was sequenced with an Illumina MiSeq 250 paired-end platform. The administration of both strains significantly affected the total bacterial community composition, and the effect lasted for 11 days after the last dose. In particular, amplicon sequence variants related to Bifidobacterium and Akkermansia genera were significantly higher in both treated groups. Therefore, modulation of the intestinal microbiota is a potential mechanism of action behind the beneficial effects of these probiotic strains.

Subclinical bovine mastitis associated with Staphylococcus spp. in eleven Uruguayan dairy farms.

INTRODUCTION: Bovine mastitis is the most common disease affecting the dairy industry, with staphylococci being considered as one of the most significant and prevalent causes. This study aimed to assess the presence of staphylococcal subclinical mastitis (SCM) in Uruguayan dairy farms and to identify Staphylococcus aureus (SA) and non-aureus staphylococci (NAS) in milking cows. In addition, the antibiotic susceptibility of isolated staphylococci was evaluated. METHODOLOGY: We tested 546 apparently healthy milking cows from 11 farms for detecting SCM using the California Mastitis Test (CMT). The cows were not treated with antibiotics. CMT-positive samples were cultured, and colonies compatible with Staphylococcus spp. were further identified through molecular techniques. The susceptibility of the Staphylococcus spp. isolates against thirteen antibiotics was determined using the disk diffusion method. RESULTS: Subclinical staphylococcal mastitis was present in almost all (82%) farms. SA (n = 39) was more common than NAS (n = 9) in the 48 samples tested. Isolates exhibited resistance to one, two, and even three different antibiotics. Resistance to penicillin was the most frequent among SA (23/39) and NAS (4/9). No staphylococci isolates exhibited resistance to cefoxitin, vancomycin, trimethoprim-sulfamethoxazole, erythromycin, or clindamycin. CONCLUSIONS: Staphylococcal SCM is one of the most common diseases in Uruguayan dairy farms. SA was the prevalent pathogen, however SA and NAS mastitis coexisted in many farms. NAS were identified and its distribution was similar to other countries. Penicillin had the highest and most frequent percentage of resistance.

Metalloprotease production by Paenibacillus larvae during the infection of honeybee larvae.

American foulbrood is a bacterial disease of worldwide distribution that affects larvae of the honeybee Apis mellifera. The causative agent is the Gram-positive, spore-forming bacterium Paenibacillus larvae. Several authors have proposed that P. larvae secretes metalloproteases that are involved in the larval degradation that occurs after infection. The aim of the present work was to evaluate the production of a metalloprotease by P. larvae during larval infection. First, the complete gene encoding a metalloprotease was identified in the P. larvae genome and its distribution was evaluated by PCR in a collection of P. larvae isolates from different geographical regions. Then, the complete gene was amplified, cloned and overexpressed, and the recombinant metalloprotease was purified and used to generate anti-metalloprotease antibodies. Metalloprotease production was evaluated by immunofluorescence and fluorescence in situ hybridization. The gene encoding a P. larvae metalloprotease was widely distributed in isolates from different geographical origins in Uruguay and Argentina. Metalloprotease was detected inside P. larvae vegetative cells, on the surface of P. larvae spores and secreted to the external growth medium. Its production was also confirmed in vivo, during the infection of honeybee larvae. This protein was able to hydrolyse milk proteins as described for P. larvae, suggesting that could be involved in larval degradation. This work contributes to the knowledge of the pathogenicity mechanisms of a bacterium of great economic significance and is one step in the characterization of potential P. larvae virulence factors.

Teaching during the COVID-19 Pandemic: Sharing Results and Data Obtained from the Ames Test.

We present a resource for instructors that contains results and data sets from the Ames test. Our aim is to share the results we have collected in previous semesters with other instructors, so they will be able to "conduct" the Ames test without the need to set foot in a laboratory classroom. Instructors will be able to use our online resource to perform the test remotely, as a supplement to their laboratory classroom, or even under hybrid circumstances. The coronavirus disease 2019 (COVID-19) pandemic brought many changes, including the way we, as instructors, were able to carry out our educational curricula, since access to laboratory classrooms was not always possible. While COVID-19 restrictions are still in place, and thus access to laboratory classrooms is limited or null, instructors can use our online resource, without the need to set foot in a laboratory classroom. When COVID-19 restrictions are lifted and access to laboratory classrooms is permitted, instructors can follow the procedures we describe and compare their results with ours, which appear in Results and Discussion, or use our data sets as take-home assignments for their students. In addition to its use in detecting the potential mutagenicity of different samples, we have found the Ames test to be extremely useful for developing problem-solving skills by means of exercises like the ones included in this resource. Furthermore, the potential of this test as a starting point for problem-based learning is remarkable. Some suggestions for its use in active learning settings are provided.

Impact of Chronic Exposure to Sublethal Doses of Glyphosate on Honey Bee Immunity, Gut Microbiota and Infection by Pathogens.

Glyphosate is the most used pesticide around the world. Although different studies have evidenced its negative effect on honey bees, including detrimental impacts on behavior, cognitive, sensory and developmental abilities, its use continues to grow. Recent studies have shown that it also alters the composition of the honey bee gut microbiota. In this study we explored the impact of chronic exposure to sublethal doses of glyphosate on the honey bee gut microbiota and its effects on the immune response, infection by Nosema ceranae and Deformed wing virus (DWV) and honey bee survival. Glyphosate combined with N. ceranae infection altered the structure and composition of the honey bee gut microbiota, for example by decreasing the relative abundance of the core members Snodgrassella alvi and Lactobacillus apis. Glyphosate increased the expression of some immune genes, possibly representing a physiological response to mitigate its negative effects. However, this response was not sufficient to maintain honey bee health, as glyphosate promoted the replication of DWV and decreased the expression of vitellogenin, which were accompanied by a reduced life span. Infection by N. ceranae also alters honey bee immunity although no synergistic effect with glyphosate was observed. These results corroborate previous findings suggesting deleterious effects of widespread use of glyphosate on honey bee health, and they contribute to elucidate the physiological mechanisms underlying a global decline of pollination services.