Immuno-physiological adaptations confer wax moth Galleria mellonella resistance to Bacillus thuringiensis

The Greater wax moth, Galleria mellonella, is a pest of beehives and is gaining a reputation as an important organism for modelling host-pathogen interactions. A G. mellonella population was selected for enhanced resistance to Bacillus thuringiensis (Bt), which is a widely-used entomopathogenic biological pesticide. Resistance and defence mechanisms were investigated in this insect line, and compared with a non-selected (suspectible) line. We also investigated the possible cost of those survival strategies. In the uninfected state, resistant insects exhibited enhanced basal expression of genes related to regeneration and amelioration of Bt toxin activity in the midgut. In addition, these insects also exhibited elevated activity of genes linked to inflammation/stress management, and fat body immune defences. Following oral infection with Bt, several of these genes wwere more highly expressed in resistant insect than in the susceptible line. Gene expression analysis reveals a pattern of resistance mechanisms targeted to anatomical sites predominantly attacked by Bt. The resistant insect concentrates its response towards tissue repair. Unlike the susceptible insects, Bt infection significantly reduced the diversity and richness (abundance) of the gut microbiota in the resistant insects. These observations suggest that the resistant line not only has a more intact midgut but is secreting antimicrobial factors into the gut lumen which not only mitigate Bt activity but also affect the viability of other gut bacteria. Remarkably the resistant line employs these multifactorial adaptations for resistance to Bt without any detectable negative trade-off, since the insects also exhibited higher fecundity

Fungal infection dynamics in response to temperature in the lepidopteran insect Galleria mellonella

This is an investigation of how temperature modulates the dynamics of fungus – insect interactions. The study focusses on the ability of the entomopathogenic fungus Metarhizium robertsii to infect the wax moth, Galleria mellonella, which is a well-studied and important model insect. Wax moth larvae in the wild develop optimally at around 34°C in beehives. However, surprisingly little research on wax moths has been conducted at temperatures relevant to the wild. Metarhizium robertsii was considerably more pathogenic at a constant temperature of 24°C compared with 34°C. The cooler temperature allowed greater condial adhesion to the cuticle, germination and haemocoel invasion. The wax moth larvae immune responses also altered with the temperature, and with the infective dose of the fungus. Enzyme-based immune defenses (lysozyme and phenoloxidase) exhibited enhanced activity at the warmer temperature. The predominant insect epicuticular fatty acids (palmetic, oleic and linoleic acids) were synthesised in greater quantities in larvae maintained at 24°C compared with those at 34°C, but these failed to exert fungistatic effects on topically applied fungus. It is clearly important to choose environmental conditions that are relevant to the habitat of the insect host when investigating the dynamics and outcome of insect / fungus interactions. The study has particular significance for the application of entomopathogens as biocontrol agents

Effect of Juvenile Hormone on Resistance against Entomopathogenic Fungus Metarhizium robertsii Differs between Sexes

Juvenile hormone has been suggested to be a potential mediator in the trade-off between mating and insects' immunity. Studies on various insect taxons have found that juvenile hormone interferes with humoral and cellular immunity. Although this was shown experimentally, studies using highly virulent parasites or pathogens are lacking so far. In this study, we tested if juvenile hormone administration affected resistance against entomopathogenic fungi, Metarhizium robertsii, in the mealworm beetle, Tenebrio molitor. In previous studies with T. molitor, juvenile hormone has been found to reduce a major humoral immune effector-system (phenoloxidase) in both sexes and decrease the encapsulation response in males. Here, we found that juvenile hormone administration prolonged survival time after infection with M. robertsii in males but reduced survival time in females. This study indicates that the effects of juvenile hormone on insect immunity might be more complicated than previously considered. We also suggest that there might be a trade-off between specific and non-specific immunity since, in males, juvenile hormone enhances specific immunity but corrupts non-specific immunity. Our study highlights the importance of using real parasites and pathogens in immuno-ecological studies

Временная составляющая аналитической модели действий диспетчера управления воздушным движением

It has been considered the mathematical apparatus, allowing to find time characteristics of air traffic control dispatcher actions with the interface of an automated workplace of the regional dispatching centre of Uniform system of air movement organisation of Republic of Belarus.Рассмотрен математический аппарат, позволяющий находить временные характеристики действий диспетчера управления воздушным движением с интерфейсом автоматизированного рабочего места районного диспетчерского центра Единой системы организации воздушного движения Республики Беларусь

Влияние микробного препарата БакСиб на озимый ячмень и состояние чернозема южного

In the article, the authors showed the effectiveness of bacterization of winter barley variety Basalt with a complex microbial preparation BakSib. The authors used a semi-dry method of seed treatment (10 l/t of working solution) and a single treatment of vegetative plants on the southern chernozem together with chemical pesticides and without them. After treatment with the drug was observed: 1) a decrease in the number of microscopic fungi in the soil up to 3 times in the tillering phase and by 21% in the full ripeness phase in comparison with the control; 2) an increase in the number of ammonifiers from 44 to 81.4%; 3) an increase in the number of bacteria that assimilate mineral nitrogen from 79 to 116%. The authors revealed the stimulating effect of BakSib on the height of barley plants in all growth phases by 7.9–13.3% compared to the control. The yield of barley increased to 9%, and the number of grains per ear increased by 9.6% compared to the control and by 6.4% when comparing the full technology of plant protection of the farm and additional bacterization with BakSib. The authors also determined the compatibility of BakSib with the insecticide Clothianidin Pro (clothianidin 350 g/l) and the fungicide Sphinx (tebuconazole 60 g/l) for seed dressing. During the study, the authors found that under the action of chemical protectants, the initial titer of the biological product (1010–1012 cells) is reduced by 3-4 times. The maximum decrease noted for individual groups of Baksiba microorganisms is two orders of magnitude. The drug contributed to a prolonged increase in copiotrophic bacteria in the southern chernozem and replenishment of its nitrogen fund during the heading period, which is important for grain productivity due to increased mineralization of organic residues in the soil.Показана эффективность бактеризации озимого ячменя Базальт комплексным микробным препаратом БакСиб полусухим методом обработки семян (10 л/т рабочего раствора) и однократной обработкой вегетирующих растений на черноземе южном совместно с химическими пестицидами и без них. После обработки препаратом наблюдалось снижение численности микроскопических грибов в почве до 3 раз в фазе кущения и на 21% в фазе полной спелости в сравнении с контролем, увеличение числа аммонификаторов от 44 до 81,4%, увеличение численности бактерий, усваивающих минеральный азот, от 79 до 116%. Выявлено стимулирующее действие БакСиба на высоту растений ячменя во все фазы роста на 7,9–13,3% в сравнении с контролем. Урожайность ячменя повысилась до 9%, увеличилось количество зерен в колосе на 9,6% по сравнению с контролем и на 6,4% при сравнении полной технологии защиты растений хозяйства и дополнительной бактеризации препаратом БакСиб. Определена совместимость БакСиба с инсектицидом для протравливания семян Клотианидином Про (клотианидин 350 г/л) и фунгицидом Сфинксом (тебуконазол 60 г/л). Показано, что под действием химических протравителей исходный титр биопрепарата (1010–1012 клеток) снижается в 3–4 раза; максимальное снижение, отмеченное для отдельных групп микроорганизмов БакСиба, составляет два порядка. Препарат способствовал пролонгированному увеличению в черноземе южном копиотрофных бактерий и пополнению ее азотного фонда в важный для зерновой продуктивности период колошения за счет повышенной минерализации органических остатков в почве

Metarhizium anisopliae Pathogenesis of Mosquito Larvae: A Verdict of Accidental Death

Metarhizium anisopliae, a fungal pathogen of terrestrial arthropods, kills the aquatic larvae of Aedes aegypti, the vector of dengue and yellow fever. The fungus kills without adhering to the host cuticle. Ingested conidia also fail to germinate and are expelled in fecal pellets. This study investigates the mechanism by which this fungus adapted to terrestrial hosts kills aquatic mosquito larvae. Genes associated with the M. anisopliae early pathogenic response (proteinases Pr1 and Pr2, and adhesins, Mad1 and Mad2) are upregulated in the presence of larvae, but the established infection process observed in terrestrial hosts does not progress and insecticidal destruxins were not detected. Protease inhibitors reduce larval mortality indicating the importance of proteases in the host interaction. The Ae. aegypti immune response to M. anisopliae appears limited, whilst the oxidative stress response gene encoding for thiol peroxidase is upregulated. Cecropin and Hsp70 genes are downregulated as larval death occurs, and insect mortality appears to be linked to autolysis through caspase activity regulated by Hsp70 and inhibited, in infected larvae, by protease inhibitors. Evidence is presented that a traditional host-pathogen response does not occur as the species have not evolved to interact. M. anisopliae retains pre-formed pathogenic determinants which mediate host mortality, but unlike true aquatic fungal pathogens, does not recognise and colonise the larval host

Highly specific host-pathogen interactions influence Metarhizium brunneum blastospore virulence against Culex quinquefasciatus larvae

Entomopathogenic fungi are potential biological control agents of mosquitoes. Our group observed that not all mosquitoes were equally susceptible to fungal infection and observed significant differences in virulence of different spore types. Conidiospores and blastospores were tested against Culex quinquefasciatus larvae. Blastospores are normally considered more virulent than conidia as they form germ tubes and penetrate the host integument more rapidly than conidia. However, when tested against Cx. quinquefasciatus, blastospores were less virulent than conidia. This host-fungus interaction was studied by optical, electron and atomic force microscopy (AFM). Furthermore, host immune responses and specific gene expression were investigated. Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores did not readily adhere to Culex larval integument and the main route of infection was through the gut. Adhesion forces between blastospores and Culex cuticle were significantly lower than for other insects. Larvae challenged with blastospores showed enhanced immune responses, with increased levels of phenoloxidase, glutathione-S-transferase, esterase, superoxide dismutase and lipid peroxidase activity. Interestingly, M. brunneum pathogenicity/stress-related genes were all down-regulated in blastospores exposed to Culex. Conversely, when conidia were exposed to Culex, the pathogenicity genes involved in adhesion or cuticle degradation were up-regulated. Delayed host mortality following blastospore infection of Culex was probably due to lower adhesion rates of blastospores to the cuticle and enhanced host immune responses deployed to counter infection. The results here show that subtle differences in host-pathogen interactions can be responsible for significant changes in virulence when comparing mosquito species, having important consequences for biological control strategies and the understanding of pathogenicity processes

Creating a tobacco line with a weaker antifeedant property against colorado potato beetle

Background. Genetic modification of plants is one of the promising strategies to increase their resistance to insect pests. The development of metabolic or RNA interference systems for plant protection requires appropriate models of host-insect interactions. Nicotiana tabacum L. is a classical model plant used in molecular and metabolic engineering. We consider tobacco as a model for developing protective strategies against Colorado potato beetle (Leptinotarsa decemlineata Say, CPB). Normally, tobacco is toxic for CPB due to high content of nicotine and related alkaloids in leaves. Modification of the tobacco genome could provide tobacco genotypes with altered metabolism suitable for CPB feeding. It is known that different mutations in Berberine Bridge-Like (BBL) genes cause different alterations in tobacco leaf alkaloid levels. In the current study, the Cas9/gRNA system targeting members of the BBL gene family of tobacco was used to create a line which can serve as a diet for CPB. Results. In order to obtain tobacco with modified alkaloid content, two gRNAs matching target sequences in six BBL genes were selected. Each gRNA was cloned into a gRNA/Cas9 generic vector. The created constructs were mixed and used for biolistic transformation of tobacco leaf explants together with the pBI121 plasmid harboring the kanamycin resistance gene nptII and the reporter E.coli betaglucuronidase (GUS) gene. Regenerants were selected on 100 mg/l of kanamycin and checked for transgene presence by histochemical GUS-assay. Unexpectedly, the regenerated plants displayed a variety of adverse phenotypic effects including different degree of growth and rooting inhibition, early flowering, increased number of internodes, changes in leaf shape, fusion of flowers, longostyly, and partial sterility. Only one from seven obtained calli produced a population of regenerated plants without severe phenotypic abnormalities. The NtaBBL5-14 line of clonally propagated plants was selected from this population and used for a CPB feeding experiment. It was shown that CPB larvae consume the leaves of NtaBBL5-14 line ten times more efficiently than the leaves of control plants (97±0.5% vs. 9±3% in 24 h respectively). Conclusion. The NtaBBL5-14 tobacco line is suitable for CPB feeding and can be further used as a model for studies in plant-pest interaction. The modification of other genes regulating nicotine metabolism can be a promising strategy to obtain tobacco plants edible for CPB with less pleiotropic effects

Transgenerational Effects of Heavy Metal Pollution on Immune Defense of the Blow Fly Protophormia terraenovae

Recently environmental conditions during early parental development have been found to have transgenerational effects on immunity and other condition-dependent traits. However, potential transgenerational effects of heavy metal pollution have not previously been studied. Here we show that direct exposure to heavy metal (copper) upregulates the immune system of the blow fly, Protophormia terraenovae, reared in copper contaminated food. In the second experiment, to test transgenerational effects of heavy metal, the parental generation of the P. terraenovae was reared in food supplemented with copper, and the immunocompetence of their offspring, reared on uncontaminated food, was measured. Copper concentration used in this study was, in the preliminary test, found to have no effect on mortality of the flies. Immunity was tested on the imago stage by measuring encapsulation response against an artificial antigen, nylon monofilament. We found that exposure to copper during the parental development stages through the larval diet resulted in immune responses that were still apparent in the next generation that was not exposed to the heavy metal. We found that individuals reared on copper-contaminated food developed more slowly compared with those reared on uncontaminated food. The treatment groups did not differ in their dry body mass. However, parental exposure to copper did not have an effect on the development time or body mass of their offspring. Our study suggests that heavy metal pollution has positive feedback effect on encapsulation response through generations which multiplies the harmful effects of heavy metal pollution in following generations

An Eye to a Kill: Using Predatory Bacteria to Control Gram-Negative Pathogens Associated with Ocular Infections

Ocular infections are a leading cause of vision loss. It has been previously suggested that predatory prokaryotes might be used as live antibiotics to control infections. In this study, Pseudomonas aeruginosa and Serratia marcescens ocular isolates were exposed to the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus. All tested S. marcescens isolates were susceptible to predation by B. bacteriovorus strains 109J and HD100. Seven of the 10 P. aeruginosa isolates were susceptible to predation by B. bacteriovorus 109J with 80% being attacked by M. aeruginosavorus. All of the 19 tested isolates were found to be sensitive to at least one predator. To further investigate the effect of the predators on eukaryotic cells, human corneal-limbal epithelial (HCLE) cells were exposed to high concentrations of the predators. Cytotoxicity assays demonstrated that predatory bacteria do not damage ocular surface cells in vitro whereas the P. aeruginosa used as a positive control was highly toxic. Furthermore, no increase in the production of the proinflammatory cytokines IL-8 and TNF-alpha was measured in HCLE cells after exposure to the predators. Finally, injection of high concentration of predatory bacteria into the hemocoel of Galleria mellonella, an established model system used to study microbial pathogenesis, did not result in any measurable negative effect to the host. Our results suggest that predatory bacteria could be considered in the near future as a safe topical bio-control agent to treat ocular infections. © 2013 Shanks et al