The Galleria mellonella-Enteropathogenic Escherichia coli Model System: Characterization of Pathogen Virulence and Insect Immune Responses

The use of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae), an economical insect model, for the study of enteropathogenic Escherichia coli (Migula) (EPEC), a diarrheagenic human pathogen, has been demonstrated previously but remains poorly understood. The present study characterizes the Galleria-EPEC system extensively for future studies using this system. We found that EPEC causes disease in G. mellonella larvae when injected intrahemocoelically but not orally. Disease manifests as increased mortality, decreased survival time, delayed pupation, decreased pupal mass, increased pupal duration, and hemocytopenia. Disease symptoms are dose-dependent and can be used as metrics for measuring EPEC virulence in future studies. The type III secretion system was only partially responsible for EPEC virulence in G. mellonella while the majority of the virulence remains unknown in origin. EPEC elicits insect anti-bacterial immune responses including melanization, hemolymph coagulation, nodulation, and phagocytosis. The immune responses were unable to control EPEC replication in the early stage of infection (≤3 h post-injection). EPEC clearance from the hemocoel does not guarantee insect survival. Overall, this study provided insights into EPEC virulence and pathogenesis in G. mellonella and identified areas of future research using this system.     

Characterization of the Dual Functions of LvCrustinVII from Litopenaeus vannamei as Antimicrobial Peptide and Opsonin

Crustin are a family of antimicrobial peptides that play an important role in protecting against pathogens infection in the innate immune system of crustaceans. Previously, we identified several novel types of crustins, including type VI and type VII crustins. However, their immune functions were still unclear. In the present study, the immune function of type VII crustin LvCrustinVII were investigated in Litopenaeus vannamei. LvCrustinVII was wildly expressed in all tested tissues, with relatively high expression levels in hepatopancreas, epidermis and lymphoid organ. Upon Vibrio parahaemolyticus infection, LvCrustinVII was significantly upregulated in hepatopancreas. Recombinant LvCrustinVII (rLvCrustinVII) showed strong inhibitory activities against Gram-negative bacteria Vibrio harveyi and V. parahaemolyticus, while weak activities against the Gram-positive bacteria Staphylococcus aureus. Binding assay showed that rLvCrustinVII could bind strongly to V. harveyi and V. parahaemolyticus, as well as the cell wall components Glu, LPS and PGN. In the presence of Ca²⁺, rLvCrustinVII could agglutinate V. parahaemolyticus and enhance hemocyte phagocytosis. The present data partially illustrate the immune function of LvCrustinVII, which enrich our understanding on the functional mechanisms of crustins and provide useful information for application of this kind of antimicrobial peptides.     

Prevalence of Endosymbionts in Polish Populations of Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

Colorado potato beetle (CPB, Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) is one of the most serious insect pest feeding on wild and cultivated Solanaceae plants. This pest poses a significant threat to potato crops. CPB originated from North America but has become widespread and has adapted in new localizations. Currently, it is reported in many countries worldwide. Endosymbiotic bacteria might have an influence on insect adaptation to new conditions. They are known to play a role in invasiveness of insect hosts and to facilitate colonization of new niches; however, information on endosymbionts of the CPB is very limited. In this study, we screened CPB populations collected from 20 evenly distributed locations in Poland for the presence of Arsenophonus, Cardinium, Wolbachia, and Flavobacterium. We found the presence of Flavobacterium in the studied insects. Little is known about CPB–endosymbionts interactions, thus this study may provide a reference for future studies in this subject.     

Context-Dependent Viral Transgenerational Immune Priming in Honey Bees (Hymenoptera: Apidae)

Transgenerational immune priming is the process of increased resistance to infection in offspring due to parental pathogen exposure. Honey bees (Apis mellifera L. (Hymenoptera: Apidae)) are hosts to multiple pathogens, and this complex immune function could help protect against overwhelming infection. Honey bees have demonstrated transgenerational immune priming for the bacterial pathogen Paenibacillus larvae; however, evidence for viral transgenerational immune priming is lacking across insects in general. Here we test for the presence of transgenerational immune priming in honey bees with Deformed wing virus (DWV) by injecting pupae from DWV-exposed queens and measuring virus titer and immune gene expression. Our data suggest that there is evidence for viral transgenerational immune priming in honey bees, but it is highly context-dependent based on route of maternal exposure and potentially host genetics or epigenetic factors.     

Fucoidan Independently Enhances Activity in Human Immune Cells and Has a Cytostatic Effect on Prostate Cancer Cells in the Presence of Nivolumab

Fucoidan compounds may increase immune activity and are known to have cancer inhibitory effects in vitro and in vivo. In this study, we aimed to investigate the effect of fucoidan compounds on ex vivo human peripheral blood mononuclear cells (PBMCs), and to determine their cancer cell killing activity both solely, and in combination with an immune-checkpoint inhibitor drug, Nivolumab. Proliferation of PBMCs and interferon gamma (IFNγ) release were assessed in the presence of fucoidan compounds extracted from Fucus vesiculosus, Undaria pinnatifida and Macrocystis pyrifera. Total cell numbers and cell killing activity were assessed using a hormone resistant prostate cancer cell line, PC3. All fucoidan compounds activated PBMCs, and increased the effects of Nivolumab. All fucoidan compounds had significant direct cytostatic effects on PC3 cells, reducing cancer cell numbers, and PBMCs exhibited cell killing activity as measured by apoptosis. However, there was no fucoidan mediated increase in the cell killing activity. In conclusion, fucoidan compounds promoted proliferation and activity of PBMCs and added to the effects of Nivolumab. Fucoidan compounds all had a direct cytostatic effect on PC3 cells, as shown through their proliferation reduction, while their killing was not increased.     

Sex Specificity in Innate Immunity of Insect Larvae

The innate immunity of insects has been widely studied. Although the effect of sex on insect immunity has been extensively discussed, differences in immunity between the sexes of larvae insects remain largely unstudied. Studying larval sex differences in immunity may provide valuable information about the mechanisms underlying the insect immune system, which, in turn, can be valuable for the development and improvement of pest management. Here we compared the antibacterial activity in both the midgut tissue and cell-free hemolymph of Lymantria dispar L. (Lepidoptera: Erebidae) females and males at the larval stage without and after a challenge by entomopathogenic bacterium Bacillus thuringiensis Berliner. We also evaluated the sex-specific mortality of L. dispar induced by B. thuringiensis infection. We find that antibacterial activity in the midgut is activated by infection, but only in females. Thus, sex differences in immunity can have important effects even before sexual differentiation at adulthood.     

Analysis of Inter-Individual Bacterial Variation in Gut of Cicada Meimuna mongolica (Hemiptera: Cicadidae)

Intestinal bacterial community plays a crucial role in the nutrition, development, survival, and reproduction of insects. When compared with other insects with piercing-sucking mouthparts, the habitats of cicada nymphs and adults are totally different. However, little is known about the differences in the gut bacterial communities in the nymphs and adults within any cicada species. The diversity of bacteria in the gut of nymphs and adults of both genders of Meimuna mongolica (Distant) was studied using the denaturing gradient gel electrophoresis (DGGE) method. Few inter-individual variations among gut microbiota were observed, suggesting that M. mongolica typically harbors a limited and consistent suite of bacterial species. Bacteria in the genera Pseudomonas and Enterobacter were the predominant components of the gut microflora of M. mongolica at all life stages. Bacteria of Pantoea, Streptococcus, and Uruburuella were also widespread in the cicada samples but at relatively lower concentrations. The relative stability and similarity of the PCR-DGGE patterns indicate that all individuals of this cicada species harbor a characteristic bacterial community which is independent from developmental stages and genders. Related endosymbionts that could be harbored in bacteromes of cicadas were not detected in any gut samples, which could be related to the cicada species and the distribution of these endosymbionts in the cicada cavity, or due to some of the possible limitations of PCR-DGGE community profiling. It is worthwhile to further address if related cicada endosymbiont clades distribute in the alimentary canals and other internal organs through diagnostic PCR using group-specific primer sets.     

Survival of two varieties ofErwinia carotovora onDrosophila melanogaster meigen andDrosophila busckii Coquillett, (Diptera:Drosophilidae) vectors of potato blackleg in Colorado

Survival time ofErwinia carotovora var.carotovora (Ecc) andE.c. var.atroseptica (Eca), causal agents of potato blackleg and bacterial soft rot, was determined both internally and externally on two known insect vectors,Drosophila melanogaster Meig. andD. busckii Coq. The results indicate that the two bacterial varieties survived internally on both vectors for 48 hours and in some cases up to 72 hours.Ecc andEca survived externally on both vectors for at least 24 hours and in some cases at low levels for 48 hours. The two varieties of bacteria declined in number with elapsed time both internally and externally. We suggest that the vectors enhance survival of the non-spore formingErwinia cells outside the plant host. The enhanced survival, combined with the dispersal ability of the vectors, increases the destructive potential of these bacteria. There appeared to be no differential survival of the two bacterial varieties on the vectors studied that might account for the differential transmission reported by previous workers.     

Monte Carlo simulation method for forecasting the timing of pest insect attacks

The timing of attack by pest insects can vary greatly both from region to region and from year to year because the rates at which insects complete their life cycles depend mainly on temperature. In addition, variation between individual insects in their rate of development can lead to a spread of activity. A simulation method, based on rates of insect development, has been produced for forecasting the timing of attack by pest insects. Variability is incorporated using the ‘same-shape property’ which implies that the coefficient of variation of the rate of insect development is independent of temperature. The method is feasible because it uses a fixed number of individuals from one generation to the next and simulates the timing of events rather than the population dynamics of the insects. The method is sufficiently flexible to allow insect resting phases to occur at the appropriate instant for each individual. Its effectiveness in predicting the behaviour of the cabbage root fly (Delia radicum), the carrot fly (Psila rosae), the bronzed blossom beetle (Meligethes aeneus) and the large narcissus fly (Merodon equestris) is demonstrated for one site in one season. To be of practical use, the method has been designed to use standard meteorological data. Estimation of soil temperatures at 6 cm and the derivation of equations for estimating diurnal variation are described in two appendices. The basis of the method is the integration of a temperature-dependent rate function over time. A third appendix describes a quicker and more accurate method of integration than that usually employed in pest forecasting work.     

Insect transmission ofErwinia carotovora var.carotovora andErwinia carotovora var.atroseptica to potato plants in the field

Adult fruit flies (Drosophila melanogaster Meig.) artificially contaminated withErwinia carotovora var.carotovora (Jones) Dye (Ecc) and/orE. carotovora var.atroseptica (van Hall) Dye (Eca) readily transmitted the bacteria to plants in the field that had been injured by crushing the stem. Injured, inoculated plants developed disease symptoms when maintained at high relative humidities.Erwinia was transmitted to ten-hour-old injuries and they became infected as frequently as freshly made wounds. Insect transmission ofEca, Ecc, and mixtures was greatest during the afternoon, which was the warmest part of the day.Ecc was transmitted significantly less frequently during the cold morning than during afternoon or evening hours. A potato cull pile placed in a commercial potato field attracted a natural insect population which increased during the season. BothEcc andEca were isolated from uninoculated rotting tubers in the cull pile and from insects associated with the pile from May through September. These naturally infested insects transmittedEcc and/orEca from the cull pile to artificially injured field plants during July and August at distances as great as 183 m from the cull pile. NoErwinia was isolated from injured plants in a neighboring control field, which lacked a cull pile, further than 6 m upwind from the cull pile. We suggest that insects are important agents in the epidemiology of potato blackleg and soft rot even in areas with low relative humidities.     

Identifying an Optimal Screen Mesh to Enable Augmentorium-Based Enhanced Biological Control of the Olive Fruit Fly Bactrocera oleae (Diptera: Tephritidae) and the Mediterranean Fruit Fly Ceratitis capitata (Diptera: Tephritidae)

The augmentorium is a cost-effective screened enclosure designed to receive fruits infested with insect pests, retain the pests inside but let their natural enemies escape to enhance biological control of pest populations. Screen selection is critical to ensure that an augmentorium is effective for a particular system. Here we tested five types of screens with a mini-augmentorium design and measured the escape of four insect species under laboratory conditions: the pests olive fruit fly Bactocera oleae (Rossi) (Diptera: Tephritidae) and Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), and the parasitoids Psyttalia lounsburyi (Silvestri) (Hymenoptera: Braconidae) and Psyttalia ponerophaga (Silvestri) (Hymenoptera: Braconidae). The sex ratio of insects that escaped the screens was compared to the sex ratio of insects that could not escape. Results showed that one screen type (‘crystal mesh’) was the best for the purpose of designing a functional augmentorium: it retained 90% of B. oleae adults and 100% of C. capitata adults while letting 72% of Psyttalia lounsburyi adults and 94% of P. ponerophaga adults escape. The other screen types tested were suboptimal, either because they let too many flies freely escape or because they retained too many parasitoids. Sex ratio was almost always similar for insects that managed to escape the screens and insects that were retained, except for P. ponerophaga and the screen type ‘light mesh’. These results are promising for the development of a functional augmentorium against the olive fruit fly and further implementation against Mediterranean fruit fly.     

Traslucent pheromone traps increase trapping efficiency of ambrosia beetle Megaplatypus mutatus

Megaplatypus mutatus (=Platypus mutatus) is an ambrosia beetle native to South America. Unlike most ambrosia beetles, it attacks only living trees, penetrating the xylem of its host by boring long tunnels. These galleries cause severe stem-breakage and mortality in commercial poplar plantations.In previous studies we showed that volatile emissions from pioneer male M. mutatus are composed of (+)-6-methyl-5-hepten-2-ol, 6-methyl-5-hepten-2-one and 3-pentanol. Recently, we developed reservoir type controlled release dispensers and tested the efficacy of various trap designs in the field. Results showed that black cross-vane traps (CIPEIN-CV) captured significantly more insects than other traps designs baited with the same dispensers. In the present study we modified the colour, the construction material and the size of traps with the aim of increasing trapping efficacy and reducing the final trap cost. The results showed that significantly more female and male M. mutatus were captured in small and large traslucent traps (CIPEIN-CVt) than in black CIPEIN-CV trap. Furthermore, traslucent CIPEIN-CVt trap costs were significantly lower than costs for CIPEIN-CV traps. Significantly improved trapping efficacy with an important decrease in final costs will allow detection of considerably lower populations of M. mutatus in monitoring programs.     

Helicoverpa zea (Lepidoptera: Noctuidae) and Spodoptera frugiperda (Lepidoptera: Noctuidae) Responses to Sorghum bicolor (Poales: Poaceae) Tissues From Lowered Lignin Lines

The presence of lignin within biomass impedes the production of liquid fuels. Plants with altered lignin content and composition are more amenable to lignocellulosic conversion to ethanol and other biofuels but may be more susceptible to insect damage where lignin is an important resistance factor. However, reduced lignin lines of switchgrasses still retained insect resistance in prior studies. Therefore, we hypothesized that sorghum lines with lowered lignin content will also retain insect resistance. Sorghum excised leaves and stalk pith Sorghum bicolor (L.) Moench (Poales: Poaceae) from near isogenic brown midrib (bmr) 6 and 12 mutants lines, which have lowered lignin content and increased lignocellulosic ethanol conversion efficiency, were examined for insect resistance relative to wild-type (normal BTx623). Greenhouse and growth chamber grown plant tissues were fed to first-instar larvae of corn earworms, Helicoverpa zea (Boddie) and fall armyworms Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), two sorghum major pests. Younger bmr leaves had significantly greater feeding damage in some assays than wild-type leaves, but older bmr6 leaves generally had significantly less damage than wild-type leaves. Caterpillars feeding on the bmr6 leaves often weighed significantly less than those feeding on wild-type leaves, especially in the S. frugiperda assays. Larvae fed the pith from bmr stalks had significantly higher mortality compared with those larvae fed on wild-type pith, which suggested that bmr pith was more toxic. Thus, reducing lignin content or changing subunit composition of bioenergy grasses does not necessarily increase their susceptibility to insects and may result in increased resistance, which would contribute to sustainable production.     

Effect of Infection by Beauveria bassiana and Metarhizium anisopliae on the Feeding of Uvarovistia zebra

To identify the susceptibility of long-horned grasshoppers to entomopathogenic fungi, the effect of infection with the fungi Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae) and Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) on food consumption by Uvarovistia zebra (Uvarov) (Orthoptera: Tettigoniidae) was investigated. Preliminary results showed that both fungi had a negative effect on food consumption of the insects. For both fungi a significant reduction of food consumption and faeces production by insects were observed between the highest spore concentration (5 × 10⁶ spores/ml) and other treatments. Compared with control insects, the insects treated with 5 × 10⁶ spores/ml of B. bassiana and M. anisopliae showed 60 and 63% reduction in mean food consumption/insect, respectively. The corrected cumulative percent mortality of the insects treated with the highest concentration of B. bassiana and M. anisopliae were 57.7 and 55.5%, respectively. This was the first account of these entomopathogenic fungi being used against a species from this family, therefore based on the results obtained from this research, it could be said that the fungi have pathogenicity effect on U. zebra as a long-horned grasshopper.     

Pheromone traps for the pest management of phycitid moths

Pheromone traps of different designs, suitable for monitoring stored-product phycitids were: (1) a commercially made fly strip; (2) a bottle trap with insecticide or water for killing captured insects and (3) a cylinder trap with half-closed entrances. The optimum dose of (Z)-9, (E)-12-tetradecadienyl acetate was 1·0 mg via a rubber dispenser. Release and recapture experiments performed in empty rooms demonstrated that a fly-strip pheromone trap can consistently capture well over 50% of released males of Ephestia kuehniella, and that synthetic pheromone is a significantly more efficient bait than six live virgin females. A 2-year mass trapping trial performed in a chocolate factory demonstrated the applicability of the pheromone method for maintenance of an acceptably low population density of E. cautella suppressed initially by insecticides.     

Yellow sticky traps for decision-making in whitefly management: What has been achieved?

Yellow sticky traps (YSTs) are a key component of IPM programmes for several greenhouse pests. The development of YST-based decision-making tools, e.g. sampling protocols and economic thresholds (ETs), however, has been limited. This review assesses to what extent YST-counts comply with the four criteria of effective sampling (reliability, representativeness, relevance and practicality) as described by Binns et al. (2000) in an attempt to understand the feasibility of designing YST-based decision-making tools for managing whiteflies in greenhouse crops, particularly tomato (Lycopersicon esculentum Mill) and cucumber (Cucumis sativus L.). Many factors are known to affect whitefly flight behaviour and thus, trap catches. The possibility of manipulating such factors to improve YST efficiency and reliability or of interpreting whitefly catches in YSTs with automated tools is discussed. A few studies have shown the correlation between trap and direct visual pest counts from plants. These studies are discussed in the context of whiteflies and trap densities to enhance the representativeness of sampling with YSTs. Relevance implies that the results of sampling reflect crop loss to a sufficient degree. Only few YSTs-based action thresholds have been suggested in the literature, particularly for use with chemical control. There are a number of approaches and technological innovations that can improve the practicality of YSTs by decreasing the effort and time associated with counting insects, a method to facilitate the identification of species in mixed populations on the trap, and sampling methods such as sequential sampling for calculating appropriate sample size, which have been already put in practice to develop YSTs-based sampling protocols. Knowledge gaps are identified and discussed, and a route map for further research to advance YSTs as a decision-making tool is outlined, with geostatistical methods as the recommended approach for further increasing the usefulness of YSTs-based decision-making.     

Crop hosts and genotypic resistance influence the biological activity of Bacillus thuringiensis towards Helicoverpa armigera

Studies on the influence of genotypic resistance on biological activity of a commercial formulation of Bacillus thuringiensis (Bt) and pure Bt toxin Cry1Ac were carried out to develop appropriate strategies for pod borer, Helicoverpa armigera management in chickpea, sorghum, pigeonpea and cotton. The interaction effects of host plant resistance and biological activity of commercial Bt/Cry1Ac were studied by incorporating the lyophilized tissues of chickpea leaves, milk stage sorghum grain, pigeonpea pods and cotton squares into the artificial diet with and without Bt formulation or Cry1Ac. The H. armigera larval weights were significantly lower in insects reared on diets with square powder of the insect - resistant Bt-cotton RCH 2 + Bt/Cry1Ac and pod powder of insect - resistant pigeonpea genotype, ICPL 332WR + Bt/Cry1Ac as compared to the larvae reared on diets with leaf powder of H. armigera susceptible chickpea genotype, ICCC 37 and the standard artificial diet. Pupation and adult emergence were significantly lower in insects reared on diets with tissues of pod borer-resistant genotypes + Bt/Cry1Ac as compared to insects reared on diets with tissues of the insect susceptible genotypes + Bt/Cry1Ac. Insects reared on diets containing insect-resistant and -susceptible genotypes of sorghum, pigeonpea and cotton and pod borer-resistant genotype of chickpea (ICC 506EB) + Bt/Cry1Ac did not lay any eggs. However, eggs were laid by the insects reared on diets containing pod borer-susceptible genotype of chickpea, ICCC 37 and on the standard artificial diet + Bt/Cry1Ac. The insects reared on diets with sorghum genotype, ICSV 745, and Bt-cotton, RCH 2 without Bt/Cry1Ac also did not lay eggs. The results suggested that Bt/Cry1Ac is more effective for management of H. armigera when deployed in combination with insect-resistant genotypes of cotton, chickpea, pigeonpea and sorghum.     

螺旋粉虱体内共生菌的研究

采用变性梯度凝胶电泳技术[denaturing gradient gel electrophoresis（DGGE）]、16S rDNA文库构建技术,以及使用Wolbachia菌特异引物研究在不同寄主上取食繁殖的螺旋粉虱自然种群体内共生菌。结果显示,在不同寄主植物取食的新入侵螺旋粉虱体内共生菌均稳定存在有初级共生菌（Candidatus Portiera aleyrodidarum）,属于变形菌门（Proteobacteria）,γ-变形菌纲,共生菌多样性较少;研究同时发现取食不同寄主植物的螺旋粉虱个体间共生菌群落多样性有一定差异,一些螺旋粉虱个体内还生存有葡萄球菌（Staphylococcus sp.）;此外,研究还发现新入侵的螺旋粉虱体内尚未检测到Wolbachia菌。表明取食不同寄主的螺旋粉虱体内均稳定存在有共生菌,同时取食不同的寄主其共生菌群落也会发生变化;Wolbachia菌尚未在新入侵海南的螺旋粉虱中建立共生关系。