Enterococcus innesii sp. nov., isolated from the wax moth Galleria mellonella

Four bacterial strains were isolated from two different colony sources of the wax moth Galleria mellonella. They were characterized by a polyphasic approach including 16S rRNA gene sequence analysis, core-genome analysis, average nucleotide identity (ANI) analysis, digital DNA–DNA hybridization (dDDH), determination of G+C content, screening of antibiotic resistance genes, and various phenotypic analyses. Initial analysis of 16S rRNA gene sequence identities indicated that strain GAL7T was potentially very closely related to Enterococcus casseliflavus and Enterococcus gallinarum , having 99.5–99.9 % sequence similarity. However, further analysis of whole genome sequences revealed a genome size of 3.69 Mb, DNA G+C content of 42.35 mol%, and low dDDH and ANI values between the genomes of strain GAL7T and closest phylogenetic relative E. casseliflavus NBRC 100478T of 59.0 and 94.5 %, respectively, indicating identification of a putative new Enterococcus species. In addition, all novel strains encoded the atypical vancomycin-resistance gene vanC-4. Results of phylogenomic, physiological and phenotypic characterization confirmed that strain GAL7T represented a novel species within the genus Enterococcus , for which the name Enterococcus innesii sp. nov. is proposed. The type strain is GAL7T (=DSM 112306T=NCTC 14608T).

Evaluation Efficacy and Persistence of Some Volatile Plant Oils on Immature Stages of Galleria mellonella (L.)

The Experiment was carried out to measure the effect of four natural plant oils (namely: Cinnamonium zeylanicum L., Syzygium aromatic L., Citrus aurantium amara, and Lavandula spica) on controlling the greater wax moth and their ability to persist. The methodology included collecting and rearing the greater wax moth under laboratory conditions to be able to isolate the target tested stages. The immature stages (eggs and larvae) of the experiment moth were exposed to fumes of tested oils (50 µl/500 ml) to calculate hatchability and mortality percentage. The results were clear that fumes of C. zeylanicum and S. aromatic were caused 100% unhatching eggs after zero time as the same recorded by comparable compound (paradix) and able to persist their effect at the closed and limited zone for 60 days. On another side, C. zeylanicum and S. aromat were affected on last larval instars and emerged adults. The accumulative effect was appeared in the case of treatment by C. zeylanicum which was caused sterility to emerged adults and reduced eggs deposition to zero. The data indicated that some natural product compounds could be used as an alternative way of control insect pests.

Identification of a Novel Secreted Metabolite Cyclo(Phenylalanyl-Prolyl) from Batrachochytrium Dendrobatidis and its effect on Galleria Mellonella.

The fungus, Batrachochytrium dendrobatidis, is the causative agent of chytridiomycosis and a leading cause of global decline in amphibian populations . The first stages of chytridiomycosis include: inflammation, hyperkeratosis, lethargy, loss of righting reflex, and disruption of internal electrolyte levels leading to eventual death of the host. Previous work indicates that B. dendrobatidis can produce immunomodulatory compounds and other secreted molecules that regulate the growth of the fungus. In this study, filtrates of the fungus grown in media and water were subjected to ultra performance liquid chromatography-mass spectrometry and analyzed using Compound Discoverer 3.0. Identification of cyclo(phenylalanyl-prolyl), chitobiose, and S-adenosylmethionine were verified by their retention times and fragmentation patterns from B. dendrobatidis supernatants. Previous studies have analyzed the effects of B. dendrobatidis on amphibian models, in vitro, or in cell culture. We studied the effects of live B. dendrobatidis cells, spent culture filtrates containing secreted metabolites, and cyclo(pheylalanyl-prolyl) on wax moth larvae ( Galleria mellonella) . Concentrated filtrates caused melanization within 24 hours, while live B. dendrobatidis caused melanization within 48 hours. Our results indicate B. dendrobatidis produces secreted metabolites previously unreported. These findings provide another alternative for the use of a non-amphibian model system to study pathogenicity traits in this fungus.

A Klebsiella pneumoniae DedA family membrane protein is required for colistin resistance and for virulence in wax moth larvae

Ineffectiveness of carbapenems against multidrug resistant pathogens led to the increased use of colistin (polymyxin E) as a last resort antibiotic. A gene belonging to the DedA family encoding conserved membrane proteins was previously identified by screening a transposon library of K. pneumoniae ST258 for sensitivity to colistin. We have renamed this gene dkcA (dedA of Klebsiella required for colistin resistance). DedA family proteins are likely membrane transporters required for viability of Escherichia coli and Burkholderia spp. at alkaline pH and for resistance to colistin in a number of bacterial species. Colistin resistance is often conferred via modification of the lipid A component of bacterial lipopolysaccharide with aminoarabinose (Ara4N) and/or phosphoethanolamine. Mass spectrometry analysis of lipid A of the ∆dkcA mutant shows a near absence of Ara4N in the lipid A, suggesting a requirement for DkcA for lipid A modification with Ara4N. Mutation of K. pneumoniae dkcA resulted in a reduction of the colistin minimal inhibitory concentration to approximately what is found with a ΔarnT strain. We also identify a requirement of DkcA for colistin resistance that is independent of lipid A modification, instead requiring maintenance of optimal membrane potential. K. pneumoniae ΔdkcA displays reduced virulence in Galleria mellonella suggesting colistin sensitivity can cause loss of virulence.

DEVELOPMENT OF A PLANT FOR THE INTRODUCTION OF ENERGY-SAVING LIGHT TECHNOLOGY FOR GROWING GREATER WAX MOTH IN AN INDUSTRIAL SCALE

The larvae of the greater wax moth (Galleria mellonella L.) are not only a serious pest of bee colonies, but also a valuable raw material for the production of biologically active substances widely used in pharmaceuticals, cosmetology, food industry, animal feed, as well as a source of high-quality protein. We have developed an energy-saving installation for industrial insect cultivation, consisting of two modules: the first for butterflies (adults), the second for larvae, pupae and eggs. To implement energy-saving light technology, experiments with a large wax moth were carried out in the laboratory of Udmurt Federal Research Center of the Ural Branch of the Russian Academy of Sciences. The experiments were repeated 4 times. There were 20 specimens of G. mellonella in each experiment. The air temperature was maintained at 28° C, air humidity 50%. The influence of the duration and wavelength of radiation (400 nm, 491 nm, 546 nm and 577 nm) on the number of adults that moved to the module for butterflies and on the mass of laid eggs was studied. The radiation of 400 nm turned out to be the most effective, since 45% of butterflies moved to it and the mass of laid eggs was 32%, which is significantly higher than that of the radiation of 491, 546 and 577 nm. The duration of the radiation was 10 minutes, 30 minutes and 60 min. With an experiment duration of 60 min. 45% of the large wax moth is transferred to the butterfly module, with an experiment duration of 30 minutes. - 44%, and with an experiment duration of 10 minutes, 43% of the large wax moth moved. Therefore, it is recommended to maintain the operating time of LEDs with a wavelength of 400 nm for 30 minutes. The aim of the research is to develop an installation for the implementation of energy-saving light technology for the cultivation of larvae of the greater wax moth (Galleria mellonella L.) on an industrial scale