Honey Bee Health

Over the past decade, the worldwide decline in honey bee populations has been an important issue due to its implications for beekeeping and honey production. Honey bee pathologies are continuously studied by researchers, in order to investigate the host–parasite relationship and its effect on honey bee colonies. For these reasons, the interest of the veterinary community towards this issue has increased recently, and honey bee health has also become a subject of public interest. Bacteria, such as Melissococcus plutonius and Paenibacillus larvae, microsporidia, such as Nosema apis and Nosema ceranae, fungi, such as Ascosphaera apis, mites, such as Varroa destructor, predatory wasps, including Vespa velutina, and invasive beetles, such as Aethina tumida, are “old” and “new” subjects of important veterinary interest. Recently, the role of host–pathogen interactions in bee health has been included in a multifactorial approach to the study of these insects’ health, which involves a dynamic balance among a range of threats and resources interacting at multiple levels. The aim of this Special Issue is to explore honey bee health through a series of research articles that are focused on different aspects of honey bee health at different levels, including molecular health, microbial health, population genetic health, and the interaction between invasive species that live in strict contact with honey bee populations

New insights into the genome and transmission of the microsporidian pathogen Nosema muscidifuracis

IntroductionNosema is a diverse genus of unicellular microsporidian parasites of insects and other arthropods. Nosema muscidifuracis infects parasitoid wasp species of Muscidifurax zaraptor and M. raptor (Hymenoptera: Pteromalidae), causing ~50% reduction in longevity and ~90% reduction in fecundity.Methods and ResultsHere, we report the first assembly of the N. muscidifuracis genome (14,397,169 bp in 28 contigs) of high continuity (contig N50 544.3 Kb) and completeness (BUSCO score 97.0%). A total of 2,782 protein-coding genes were annotated, with 66.2% of the genes having two copies and 24.0% of genes having three copies. These duplicated genes are highly similar, with a sequence identity of 99.3%. The complex pattern suggests extensive gene duplications and rearrangements across the genome. We annotated 57 rDNA loci, which are highly GC-rich (37%) in a GC-poor genome (25% genome average). Nosema-specific qPCR primer sets were designed based on 18S rDNA annotation as a diagnostic tool to determine its titer in host samples. We discovered high Nosema titers in Nosema-cured M. raptor and M. zaraptor using heat treatment in 2017 and 2019, suggesting that the remedy did not completely eliminate the Nosema infection. Cytogenetic analyses revealed heavy infections of N. muscidifuracis within the ovaries of M. raptor and M. zaraptor, consistent with the titer determined by qPCR and suggesting a heritable component of infection and per ovum vertical transmission.DiscussionThe parasitoids-Nosema system is laboratory tractable and, therefore, can serve as a model to inform future genome manipulations of Nosema-host system for investigations of Nosemosis

Analysis of craniofacial defects in Six1/Eya1-associated Branchio-Oto-Renal Syndrome

Poster Session I - Morphogenesis: 205/B10117th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, 7th Latin American Society of Developmental Biology Meeting and 11th Congreso de la Sociedad Mexicana de Biologia del Desarrollo.Branchio-Oto-Renal (BOR) syndrome patients exhibit craniofacial and renal anomalies as well as deafness. BOR syndrome is caused by mutations in Six1 or Eya1, both of which regulate cell proliferation and differentiation. The molecular mechanism underlying the craniofacial and branchial arch (BA) defects in BOR syndrome is unclear. We have found that Hoxb3 is up-regulated in the second branchial arch (BA2) of Six1-/- mutants. Moreover, Hoxb3 over-expression in transgenic mice leads to BA abnormalities which are similar to the BA defects in Six1-/- or Eya1-/- mutants, suggesting a regulatory relationship among Six1, Eya1 and Hoxb3 genes. The aim of this study is to investigate the molecular mechanism underlying abnormal BA development in BOR syndrome using Six1 and Eya1 mutant mice. Two potential Six1 binding sites were identified on the Hoxb3 gene. In vitro and in vivo Chromatin IP assays showed that Six1 could directly bind to one of the sites specifically. Furthermore, using a chick in ovo luciferase assay we showed that Six1 could suppress gene expression through one of the specific binding sites. On the other hand, in Six1-/- mutants, we found that the Notch ligand Jag1 was up-regulated in BA2. Similarly, in Hoxb3 transgenic mice, ectopic expression of Jag1 could be also detected in BA2. To investigate the activation of Notch signaling pathway, we found that Notch intracellular domain (NICD), a direct indicator of Notch pathway activation, was up-regulated in BAs of Six1-/-; Eya1-/- double mutants. Our results indicate that Hoxb3 and Notch signaling pathway are involved in mediating the craniofacial defects of Six1/Eya1-associated Branchio-Oto-Renal Syndrome.postprin

Sox10 regulates enteric neural crest cell migration in the developing gut

Concurrent Sessions 1: 1.3 - Organs to organisms: Models of Human Diseases: abstract no. 1417th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, VII Latin American Society of Developmental Biology Meeting and XI Congreso de la Sociedad Mexicana de Biologia del Desarrollo. The Conference's web site is located at http://www.inb.unam.mx/isdb/Sox10 is a HMG-domain containing transcription factor which plays important roles in neural crest cell survival and differentiation. Mutations of Sox10 have been identified in patients with Waardenburg-Hirschsprung syndrome, who suffer from deafness, pigmentation defects and intestinal aganglionosis. Enteric neural crest cells (ENCCs) with Sox10 mutation undergo premature differentiation and fail to colonize the distal hindgut. It is unclear, however, whether Sox10 plays a role in the migration of ENCCs. To visualize the migration behaviour of mutant ENCCs, we generated a Sox10NGFP mouse model where EGFP is fused to the N-terminal domain of Sox10. Using time-lapse imaging, we found that ENCCs in Sox10NGFP/+ mutants displays lower migration speed and altered trajectories compared to normal controls. This behaviour was cell-autonomous, as shown by organotypic grafting of Sox10NGFP/+ gut segments onto control guts and vice versa. ENCCs encounter different extracellular matrix (ECM) molecules along the developing gut. We performed gut explant culture on various ECM and found that Sox10NGFP/+ ENCCs tend to form aggregates, particularly on fibronectin. Time-lapse imaging of single cells in gut explant culture indicated that the tightly-packed Sox10 mutant cells failed to exhibit contact inhibition of locomotion. We determined the expression of adhesion molecule families by qPCR analysis, and found integrin expression unaffected while L1-cam and selected cadherins were altered, suggesting that Sox10 mutation affects cell adhesion properties of ENCCs. Our findings identify a de novo role of Sox10 in regulating the migration behaviour of ENCCs, which has important implications for the treatment of Hirschsprung disease.postprin

Imaging polyphenolic therapeutic compounds in a eukaryotic model microbe

Flavonoids are polyphenolic metabolites that have a range of physiological and developmental functions in plants. They are the focus of much work as potential therapeutics, although investigation of specific mode of action remains a notably under-researched area. Monitoring transport and location of flavonoids in cells is difficult because, despite a role in UV-absorption in plants, they emit only low levels of fluorescence. Visualising them in plants is possible using the Naturstoff reagent (NA), reported historically to be a polyphenol-fluorescence-enhancing stain. We explored therefore whether this agent was effective during preclinical assessment of polyphenolic therapeutics in a microbial-model. The eukaryote Dictyostelium discoideum has been shown to be a useful model when identifying novel drug targets for treating various diseases. For example, in the case of polycystic kidney disease, naringenin decreased Dictyostelium cell division whereas a polycystin-2-null Dictyostelium line was resistant to the flavonoid, and, subsequently, naringenin treatment proved to reduce cyst-formation in mammalian-kidney model cell lines1. To monitor transport and site of action of the drugs investigated in such studies, we developed a method using NA-staining in this model organism. A range of polyphenolics were assayed in cells, cell-extracts and the cell-washes, and NA-enhanced imaging was evaluated in parallel with LCMS-quantification. NA-enhanced fluorescence of compounds at therapeutically relevant concentrations proved an effective and qualitative measure of transport and localisation in Dictyostelium, and could be used in concert with localisation dyes. Fluorescence-enhancement is limited to a subset of flavonoids, however, and not more widely applicable in our studies to date

The cyanobacterial rhomboid protease is a regulator of the CCM

Cyanobacteria are aquatic photosynthetic bacteria and useful models for study of the chloroplast and photosynthesis. We are studying a ‘rhomboid’ membrane-located proteases in Synechocystis sp. PCC 6803, which appears to function as a previously undiscovered regulator of the carbon concentrating mechanism (CCM) of this phototroph. Rhomboids are almost ubiquitous across evolution, and are known to activate diverse cellular processes via proteolysis of their specific, membrane-sequestered substrates. Although this well-conserved family has solved crystal structures of bacterial enzymes such as Escherichia coli GlpG, ironically, most work has been carried out on eukaryotic representatives. Following our study of the Arabidopsis thaliana chloroplast RBL10 protease, we identified cyanobacterial orthologues with the aim of discovering if roles might be conserved between these and organellar rhomboids. Molecular biology and reverse-genetics studies were made on slr1461, a mutant in the single rhomboid protease of Synechocystis. When photosynthetic parameters were investigated, it could be seen that inactivation of slr1461 did not affect nonphotochemical quenching, unlike the chloroplast rbl10 mutant, but Slr1461 was required for reduction of photosynthetic activity in mixotrophic conditions. This reduction allows cyanobacteria to avoid expending energy on the uptake of CO2 when an organic carbon source can be utilised: as might be expected, therefore, Slr1461 transcription was linked with downregulation of genes encoding proteins facilitating high-affinity CO2 import under high CO2 and mixotrophic conditions. Quantitative RT-PCR of CCM network genes suggested that Slr1461 is located upstream of known regulators, including another membrane protease, the Slr0228 FtsH, and a central, controlling transcription factor NdhR