Entomopathogenic Bacteria Research Articles

A Review on Vector Borne Diseases and Various Strategies to Control Mosquito Vectors

Mosquito vector transmit serious infectious diseases that include dengue, chikungunya, malaria, filariasis, leishmaniasis, Japanese encephalitis, west Nile fever, yellow fever and rift valley fever. Insecticides-based control measures have historically and currently been an important control approach against major mosquito-borne diseases. Chemical pesticides, on the other hand, are non-selective and can harm other beneficial organisms. Controlling mosquitoes with entomopathogenic bacteria is a convincing, ecologically acceptable alternative to chemical pesticides. Bacillus thuringiensis israelensis and Bacillus sphaericus are insecticidal microorganisms which are spore forming and these bacteria are the most extensively utilised alternative mosquito control agents, considering the rapid development of resistance, especially to B. sphaericus by the larvae of Culex spp. Under this circumstances, it is necessary to find an alternative biological control agents from natural resources. The present review focus mainly on the various strategies to control mosquito vectors.

Temperature-modulated host-pathogen interactions between Hermetia illucens L. (Diptera: Stratiomyidae) and Pseudomonas protegens Pf-5

Temperature is an important abiotic factor influencing the survival and fitness of pathogens as well as their hosts. We investigated the effect of three temperatures (18 °C, 27 °C and 37 °C) on survival and performance of black soldier fly larvae (BSFL), Hermetia illucens L., upon infection by an entomopathogenic Gram-negative bacterium, Pseudomonas protegens Pf-5. The effect of different temperatures on pathogen fitness was investigated both in vivo and in vitro. Pathogen performance under exposure to the insect antimicrobial peptide cecropin was investigated at the three temperatures using radial-diffusion plate assays. Higher rearing temperatures resulted in higher larval survival, increased larval weight, and higher inhibitory activity of cecropin against P. protegens Pf-5. At higher temperature, bacterial growth, both in vivo and in vitro, was reduced, resulting in increased BSFL survival. These observations collectively indicate the important effect of rearing temperature on host-pathogen interactions and the possibility to apply temperature treatment in reducing entomopathogen effects in BSFL.

Entomopathogenic Potential of Bacillus velezensis CE 100 for the Biological Control of Termite Damage in Wooden Architectural Buildings of Korean Cultural Heritage.

Biocontrol strategies are gaining tremendous attention in insect pest management, such as controlling termite damage, due to the growing awareness of the irreparable harm caused by the continuous use of synthetic pesticides. This study examines the proteolytic and chitinolytic activities of Bacillus velezensis CE 100 and its termiticidal effect through cuticle degradation. The proteolytic and chitinolytic activities of B. velezensis CE 100 systematically increased with cell growth to the respective peaks of 68.3 and 128.3 units/mL after seven days of inoculation, corresponding with the highest cell growth of 16 × 107 colony-forming units (CFU)/mL. The in vitro termiticidal assay showed that B. velezensis CE 100 caused a rapid and high rate of termite mortality, with a median lethal time (LT50) of >1 h and the highest mortality rates of 91.1% and 92.2% recorded at 11 h and 12 h in the bacterial broth culture and crude enzyme fraction, respectively. In addition to broken setae and deformed sockets, termites treated with the bacterial broth culture exhibited degraded epicuticles, while the crude enzyme fraction caused severe disintegration of both the epicuticle and endocuticle. These results indicate the tremendously higher potential of B. velezensis CE 100 in the biological control of subterranean termites compared to the previously used entomopathogenic bacteria.

A Sporulation-Independent Way of Life for Bacillus thuringiensis in the Late Stages of an Infection.

The formation of endospores has been considered the unique survival and transmission mode of sporulating Firmicutes due to the exceptional resistance and persistence of this bacterial form. However, nonsporulated bacteria (Spo-) were reported at the early stages following the death of a host infected with Bacillus thuringiensis, an entomopathogenic sporulating bacterium. Here, we investigated the characteristics of the bacterial population in the late stages of an infection in the B. thuringiensis/Galleria mellonella infection model. Using fluorescent reporters and molecular markers coupled to flow cytometry, we demonstrated that the Spo- cells persist and constitute about half of the population 2 weeks post-infection (p.i.). Protein synthesis and growth recovery assays indicated that they are in a metabolically slowed-down state. These bacteria were extremely resistant to the insect cadaver environment, which did not support growth of in vitro-grown vegetative cells and spores. A transcriptomic analysis of this subpopulation at 7 days p.i. revealed a signature profile of this state, and the expression analysis of individual genes at the cell level showed that more bacteria mount an oxidative stress response as their survival time increases, in agreement with the increase of the free radical level in the host cadaver and in the number of reactive oxygen species (ROS)-producing bacteria. Altogether, these data show for the first time that nonsporulated bacteria are able to survive for a prolonged period of time in the context of an infection and indicate that they engage in a profound adaptation process that leads to their persistence in the host cadaver. IMPORTANCE Bacillus thuringiensis is an entomopathogenic bacterium widely used as a biopesticide. It belongs to the Bacillus cereus group, comprising the foodborne pathogen B. cereus sensu stricto and the anthrax agent Bacillus anthracis. Like other Firmicutes when they encounter harsh conditions, these Gram-positive bacteria can form dormant cells called spores. Due to its highly resistant nature, the spore was considered the unique mode of long-term survival, eclipsing any other form of persistence. Breaking this paradigm, we observed that B. thuringiensis was able to persist in its host cadaver in a nonsporulated form for at least 14 days. Our results show that these bacteria survived in the cadaver environment, which proved hostile for actively growing bacteria by engaging in a profound adaptation process. Studying this facet of the life cycle of a sporulating bacterium provides new fundamental knowledge and might lead to the development of strategies to combat sporulating pathogenic species.

Interkingdom Signaling of the Insect Pathogen Photorhabdus luminescens with Plants Via the LuxR solo SdiA.

In bacteria, group-coordinated behavior such as biofilm formation or virulence are often mediated via cell-cell communication, a process referred to as quorum sensing (QS). The canonical QS system of Gram-negative bacteria uses N-acyl homoserine lactones (AHLs) as communication molecules, which are produced by LuxI-type synthases and sensed by cognate LuxR-type receptors. These receptors act as transcriptional regulators controlling the expression of specific genes. Some bacteria harbor LuxR-type receptors lacking a cognate LuxI-type synthases, designated as LuxR solos. Among many other LuxR solos, the entomopathogenic enteric bacterium Photorhabdus luminescens harbors a SdiA-like LuxR solo containing an AHL signal-binding domain, for which a respective signal molecule and target genes have not been identified yet. Here we performed SPR analysis to demonstrate that SdiA acts as a bidirectional regulator of transcription, tightly controlling its own expression and the adjacent PluDJC_01670 (aidA) gene in P. luminescens, a gene supposed to be involved in the colonization of eukaryotes. Via qPCR we could further determine that in sdiA deletion mutant strains, aidA is upregulated, indicating that SdiA negatively affects expression of aidA. Furthermore, the ΔsdiA deletion mutant exhibited differences in biofilm formation and motility compared with the wild-type. Finally, using nanoDSF analysis we could identify putative binding ability of SdiA towards diverse AHLs, but also to plant-derived signals, modulating the DNA-binding capacity of SdiA, suggesting that this LuxR solo acts as an important player in interkingdom signaling between P. luminescens and plants.

Programmable protein delivery with a bacterial contractile injection system

Endosymbiotic bacteria have evolved intricate delivery systems that enable these organisms to interface with host biology. One example, the extracellular contractile injection systems (eCISs), are syringe-like macromolecular complexes that inject protein payloads into eukaryotic cells by driving a spike through the cellular membrane. Recently, eCISs have been found to target mouse cells1–3, raising the possibility that these systems could be harnessed for therapeutic protein delivery. However, whether eCISs can function in human cells remains unknown, and the mechanism by which these systems recognize target cells is poorly understood. Here we show that target selection by the Photorhabdus virulence cassette (PVC)—an eCIS from the entomopathogenic bacterium Photorhabdus asymbiotica—is mediated by specific recognition of a target receptor by a distal binding element of the PVC tail fibre. Furthermore, using in silico structure-guided engineering of the tail fibre, we show that PVCs can be reprogrammed to target organisms not natively targeted by these systems—including human cells and mice—with efficiencies approaching 100%. Finally, we show that PVCs can load diverse protein payloads, including Cas9, base editors and toxins, and can functionally deliver them into human cells. Our results demonstrate that PVCs are programmable protein delivery devices with possible applications in gene therapy, cancer therapy and biocontrol.

Insecticidal activity of Bacillus thuringiensis towards Agrotis exclamationis larvae-A widespread and underestimated pest of the Palearctic zone.

acillus thuringiensis is an entomopathogenic bacterium commonly used as a bioinsecticide against numerous invertebrate pests. However, the efficacy of this microbe has not yet been determined towards Agrotis exclamationis-a lepidopteran, polyphagous pest, widespread throughout the Palearctic zone. In this work we have detected very low susceptibility of A. exclamationis to B. thuringiensis commercial strains, used as microbial formulations in pest control. To investigate this matter, the biological activity of six selected (Cry1Aa, Cry1Ca, Cry1Ia, Cry2Ab, Cry9Ea and Vip3Aa), heterogously-expressed Bacillus thuringiensis insecticidal proteins has been assessed towards A. exclamationis. Only Cry9Ea and Vip3Aa caused significant mortality in the tested pest species, with LC50 values of 950 and 140 ng/cm2, respectively. The histopathological effects of Cry9Ea and Vip3Aa on A. exclamationis were determined. On the other hand, Cry1- and Cry2-type toxins, which are the main active molecules of the majority of currently-used B. thuringiensis-based biocontrol agents (including the commercial strains tested in this work), did not cause mortality in target insect, but only different levels of growth inhibition. Moreover, in the case of Cry1Ca and Cry1Ia hormesis has been observed-a phenomenon that may be disadvantageous in implementation of these proteins in pest management. The obtained results broaden the existing knowledge regarding B. thuringiensis insecticidal protein target range and depict variable susceptibility of A. exclamationis to different groups of Cry/Vip toxins. This work indicates Cry9Ea and Vip3Aa as good candidates for efficient biological control of A. exclamationis and possibly other Agrotinae and discusses the potential use of Vip3-type and Cry9-type insecticidal proteins as successful bioinsecticides.

The aptness of entompathogenic bacteria against sawtoothed grain beetle (Oryzaephilus surinamensis [L.]) (Coleoptera: Silvanidae) in dates under laboratory conditions

ObjectiveThe sawtoothed grain beetle, Oryzaephilus surinamensis (L). is known as one of the most devastating insect pests in stored date fruits, infrequently found in newly harvested date fruits and raisins but developed a high plenty in dry fruits particularly after long storage. Indiscriminate use of pesticides and fumigants to control insects have resulted in insecticide resistance, environmental hazards, residual toxicity, and pest resurgence so the control trends have been changed and particularly biological control agents like entomopathogenic bacteria Photorhabdus temperata and Xenorhabdus nematophila being used to manage sawtoothed grain beetle. MethodsDuring present study, the pathogenicity of P. temperata and X. nematophila was evaluated against this crucial pest of stored grains. The bacterial concentrations, 1 × 104, 1 × 105, 1 × 106, 1 × 107 and 1 × 108 cells/ml of both bacteria were prepared and used for the experiments. The prepared solutions were applied directly to the date fruits infested with sawtoothed grain beetles and data were recorded. ResultsSignificant results were observed for both bacterial treatments as the highest concentration of 108 cells/ml showed to be very effective against the beetle in terms of fecundity, adult emergence, and mortality. In terms of fecundity, 8.66 to 4.33 mean number of eggs were produced at bacterial P. temperata concentrations of 1 × 104 and 1 × 108 cells/ml, respectively. Similarly, the F1 adult emergence for P. temperata was recorded 15.33 and 9 adults at concentrations of 1 × 104 and 1 × 108 cells/ml, respectively. Results showed a similar trend for the X. nematophila species. ConclusionsThese results indicate that utilizing bacteria as biological control agents can be advantageous for reducing insect populations in storage facilities and preventing the loss of grains and date fruits that have been stored in storage.

XENOFOOD-An Autoclaved Feed Supplement Containing Autoclavable Antimicrobial Peptides-Exerts Anticoccidial GI Activity, and Causes Bursa Enlargement, but Has No Detectable Harmful Effects in Broiler Cockerels despite In Vitro Detectable Cytotoxicity on LHM Cells.

Entomopathogenic bacteria are obligate symbionts of entomopathogenic nematode (EPN) species. These bacteria biosynthesize and release non-ribosomal-templated hybrid peptides (NR-AMPs), with strong, and large-spectral antimicrobial potential, capable of inactivating pathogens belonging to different prokaryote, and eukaryote taxa. The cell-free conditioned culture media (CFCM) of Xenorhabdus budapestensis and X. szentirmaii efficiently inactivate poultry pathogens like Clostridium, Histomonas, and Eimeria. To learn whether a bio-preparation containing antimicrobial peptides of Xenorhabdus origin with accompanying (in vitro detectable) cytotoxic effects could be considered a safely applicable preventive feed supplement, we conducted a 42-day feeding experiment on freshly hatched broiler cockerels. XENOFOOD (containing autoclaved X. budapestensis, and X. szentirmaii cultures developed on chicken food) were consumed by the birds. The XENOFOOD exerted detectable gastrointestinal (GI) activity (reducing the numbers of the colony-forming Clostridium perfringens units in the lower jejunum. No animal was lost in the experiment. Neither the body weight, growth rate, feed-conversion ratio, nor organ-weight data differed between the control (C) and treated (T) groups, indicating that the XENOFOOD diet did not result in any detectable adverse effects. We suppose that the parameters indicating a moderate enlargement of bursas of Fabricius (average weight, size, and individual bursa/spleen weight-ratios) in the XENOFOOD-fed group must be an indirect indication that the bursa-controlled humoral immune system neutralized the cytotoxic ingredients of the XENOFOOD in the blood, not allowing to reach their critical cytotoxic concentration in the sensitive tissues.

Sublethal effects of Brevibacillus laterosporus an emerging novel entomopathogenic bacterium with potential role to trigger defense response in chili pepper Capsicum annuum L. against potato aphid Macrosiphum euphorbiae

The emerging elicitor protein PeBL1, extracted from Brevibacillus laterosporus A60, was tested against the potato aphid (Macrosiphum euphorbiae) for its biocontrol potential. Its effects on M. euphorbiae survival, lifespan, immature development, adult reproductive performance, and pest–pathogen interaction were assessed using electrical penetration graphs (EPGs), The levels of jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) were significantly increased in the B. laterosporus-treated C. annuum seedlings, indicating substantial accumulation. Brevibacillus laterosporus improved the height of the C. annuum L. plant and the surface structure of the leaves, by reducing M. euphorbiae reproduction and preventing colonization, according to the findings of this study. The activation of pathways was also part of the defensive response (JA, SA, and ET). The use of B. laterosporus for integrated pest management in the agroecosystem appears to be suitable based on these findings against M. euphorbiae.

HETERORHABDITIS BACTERIOPHORA NEMATODES ARE SENSITIVE TO THE BACTERIAL PATHOGEN PHOTORHABDUS ASYMBIOTICA.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora infects a wide range of insect hosts with the aid of its mutualistic bacteria Photorhabdus luminescens. While the mutualistic relationship between H. bacteriophora and P. luminescens and the infectivity of the nematode-bacteria complex have been characterized, how nematode fitness is affected by entomopathogenic bacteria existing in association with other EPN species remains poorly understood. In this study, the survival of H. bacteriophora infective juveniles containing or lacking P. luminescens was tested against the entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus asymbiotica as well as the non-pathogenic Escherichia coli. While X. nematophila and E. coli did not significantly affect the survival of H. bacteriophora, P. asymbiotica exerted a significant effect on nematode survival, particularly on those lacking P. luminescens. These results imply that P. asymbiotica encodes factors that are pathogenic to EPNs. Future efforts will focus on the identification of the bacterial molecular components that induce these effects. This study makes an important contribution to a growing body of research aimed at exploiting the full potential of nematode-bacterial complexes for eliminating noxious insect pests and treating infectious diseases caused by parasitic nematodes.

Genetic improvement of some microorganisms to increase the effect of bio-control on the potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)

BackgroundDuring the last five decades, chemical synthetic pesticides have been used extensively to control pests and protect crops. Use of synthetic pesticide has caused some unfortunate consequences like environmental pollution, pest resistance and toxicity to other non-target organisms. Due to the hazardous effects of their chemical residues to human and animal health, several studies have been carried out to determine effective alternative control methods. One of methods is the usage of entomopathogens such as bacteria, virus and fungi. Entomopathogenic bacteria have unique insecticidal properties mainly due to the production of larvicidal proteins that accumulate as parasporal crystalline inclusions within the cell. The bio-insecticidal bacterium Bacillus thuringiensis (Bt) has been widely used in agriculture for the control of pest insects which attack crops.ResultsThirteen genetically stable fusants strains were obtained as a result of protoplast fusion technique between a local Bacillus thuringiensis (Bt) and each of B. subtilis subsp. subtilis strain (Bs1), Bacillus licheniformis strain (Bl) or B. subtilis subsp. spizizeniie (Bs2). Thirteen fusants were obtained, including three fusants (group B) from Bt::Bs1 fusion, six fusants (group C) from Bt::Bl fusion and four fusants (group D) from Bt::Bs2 fusion. All fusants were chosen for bioassay treatments against the potato tuber moth (PTM) Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) which recorded a high mortality percentage of PTM ranged from 75 to 80% in F1 and F2 of first attempt B (Bt::Bs1). The accumulative larval mortality was notation the highest percentage reached to 90% in case of treatment by fusants F4 followed by F7, F8 and F9 which gave 75, 70 and 80% larval mortality, respectively, for second attempt C (Bt::Bl). The third attempt D (Bt::Bs2) fusants F10, F11, F13 achieved the highest mortality percentage up to 60, 60 and 70%, respectively. Expression of apoptosis-related encoding genes in PTM was determined in three fusants B (Bt::Bs1), C (Bt::Bl) and D (Bt::Bs2) and compared with A (Bt) and Control. The results showed a high expression of gene apoptosis to fusants D (Bt::Bs2) to Caspase-16 gene, Dronc and Dredd genes in tissues of the (PTM) treated with different biological pesticides.ConclusionsThe study used protoplast fusion technique between a local Bacillus thuringiensis (Bt) and B. subtilis subsp. subtilis strain (Bs1), B. licheniformis strain (Bl) and B. subtilis subsp. spizizeniie (Bs2). Thirteen fusants were chosen for bioassay treatments against PTM. Bacterial fusant, F1, F2, F4, F7, F9 and F13, achieved the highest mortality rates against PTM ranged 75–90% under laboratory conditions. The highest expression of gene apoptosis to fusants D (Bt::Bs2) to Caspase-16 gene, Dronc and Dredd genes was recorded in insect tissues treated with different bio-insecticides. As a result of the effected on the genes responsible for expression the vital processes (genes of apoptosis) in the insect as a result of treatment with bacteria, this led to deformities and death of PTM.

Isolation, Identification and Antimicrobial Activities of Four Bacteria Associated with Entomopathogenic Nematodes, Steinernema spp.

Recently, the search for novel products derived from natural sources has become necessary due to the decreasing effectiveness of current antibiotics in treating bacteria that are antibiotic-resistant. In this context, it is well known that entomopathogenic bacteria (EPBs) produce a broad range of secondary metabolites with antibacterial activities. Therefore, an in-vitro trial was conducted to isolate and identify non-symbiotic bacteria associated with entomopathogenic nematodes, Steinernema spp. and evaluate the antibacterial activity against four antibiotic-resistant bacteria. Four bacterial isolates, i.e. Pseudomonas alcaligenes ST-1, Paenibacillus barcinonensis ST-2, Bacillus mojavensis ST-3, and Bacillus megaterium ST-4 were non-symbiotically isolated from the heamolymph of dead Steinernema-infected Galleria mellonella larvae and molecularly characterized. The bacterial cells and filtrates from P. alcaligenes ST-1 strongly inhibited the growth of Staphylococcus aureus through disk diffusion (43 mm), minimum inhibitory concentration (2.5 µL/mL), and minimal bactericidal concentration (5 µL/mL) assay. Conclusively, the direct application of endogenous Steinernema-associated EPB as an antibacterial agent for antibiotic-resistant bacteria looked promising.

Virulent and necrotrophic strategies of Bacillus thuringiensis in susceptible and resistant insects, Galleria mellonella.

Bacillus thuringiensis (Bt) is one of the most common entomopathogenic bacteria used as a biopesticide, and source of endotoxin genes for generating insect-resistant transgenic plants. The mechanisms underpinning an insect's susceptibility or resistance to B. thuringiensis are diverse. The bacterial lifecycle does not end with the death of a host, they continue to exploit the cadaver to reproduce and sporulate. Herein, we studied the progression of B. thuringiensis subsp. galleriae infection in two populations of wax moth larvae (Galleria mellonella) to gain further insight into the "arms race" between B. thuringiensis virulence and insect defences. Two doses of B. thuringiensis subsp. galleriae (spore and crystalline toxin mixtures) were administered orally to compare the responses of susceptible (S) and resistant (R) populations at ∼30% mortality each. To investigate B. thuringiensis-insect antibiosis, we used a combination of in vivo infection trials, bacterial microbiome analysis, and RNAi targeting the antibacterial peptide gloverin. Within 48h post-inoculation, B. thuringiensis-resistant insects purged the midgut of bacteria, i.e., colony forming unit numbers fell below detectable levels. Second, B. thuringiensis rapidly modulated gene expression to initiate sporulation (linked to quorum sensing) when exposed to resistant insects in contrast to susceptible G. mellonella. We reinforce earlier findings that elevated levels of antimicrobial peptides, specifically gloverin, are found in the midgut of resistant insects, which is an evolutionary strategy to combat B. thuringiensis infection via its main portal of entry. A sub-population of highly virulent B. thuringiensis can survive the enhanced immune defences of resistant G. mellonella by disrupting the midgut microbiome and switching rapidly to a necrotrophic strategy, prior to sporulation in the cadaver.

Entomopathogenic potential of bacteria associated with soil-borne nematodes and insect immune responses to their infection.

Soil-borne nematodes establish close associations with several bacterial species. Whether they confer benefits to their hosts has been investigated in only a few nematode-bacteria systems. Their ecological function, therefore, remains poorly understood. In this study, we isolated several bacterial species from rhabditid nematodes, molecularly identified them, evaluated their entomopathogenic potential on Galleria mellonella larvae, and measured immune responses of G. mellonella larvae to their infection. Bacteria were isolated from Acrobeloides sp., A. bodenheimeri, Heterorhabditis bacteriophora, Oscheius tipulae, and Pristionchus maupasi nematodes. They were identified as Acinetobacter sp., Alcaligenes sp., Bacillus cereus, Enterobacter sp., Kaistia sp., Lysinibacillus fusiformis, Morganella morganii subsp. morganii, Klebsiella quasipneumoniae subsp. quasipneumoniae, and Pseudomonas aeruginosa. All bacterial strains were found to be highly entomopathogenic as they killed at least 53.33% G. mellonella larvae within 72h post-infection, at a dose of 106 CFU/larvae. Among them, Lysinibacillus fusiformis, Enterobacter sp., Acinetobacter sp., and K. quasipneumoniae subsp. quasipneumoniae were the most entomopathogenic bacteria. Insects strongly responded to bacterial infection. However, their responses were apparently little effective to counteract bacterial infection. Our study, therefore, shows that bacteria associated with soil-borne nematodes have entomopathogenic capacities. From an applied perspective, our study motivates more research to determine the potential of these bacterial strains as biocontrol agents in environmentally friendly and sustainable agriculture.

Steinernema carpocapsae

Steinernema and Heterorhabditis are not closely related phylogenetically but they share many characteristics via convergent evolution. Over 100 species of Steinernema have been described. Here we focus on Steinernema carpocapsae. All entomopathogenic nematode (EPN) species are symbiotically associated with entomopathogenic bacteria: S. carpocapsae retains Xenorhabdus nematophila. The bacteria kill the insect host along with EPN-produced factors, provide nutrition for the EPNs, and produce metabolites that protect the infection by inhibiting opportunistic colonization by other soil organisms. S. carpocapsae is a common species isolated from soil from every continent except Antarctica and was the first EPN-produced in vitro and commercially developed for pest control. To date, 12 species of EPNs have been commercialized for biological control of soil insect pests for agriculture, municipalities, and homeowners.

Test of Pathogenicity of Bacillus thuringensis (Berliner) in Biological Control for the Cotton Leaf Worm, Spodoptera littoralis (Boisd.)

The study was carried out to evaluate the pathogenicity of the bacteria, Bacillus thuringensis of the cotton leafworm, Spodoptera littoralis (Boisd) at the rate of 1x106 spores/ml Laboratory results revealed that egg hatching percentage, at the age of 1-2 days were 0.0, 0.0 and 8.2 % 2 and 4 days after application respectively. While the hatching percentage of the 3-4 days old eggs was 6.8% at the age of 2-4 days after 2 days' treatment. The mortality rate of larval stages (2, 4, 6 instars) was 100% 14 days after application. When pupae were treated with the bacteria adult emergence percentage was 51.3 % 15 days after treatment compared to 100% emergence for the control treatment. The results in greenhouse show that the entomopathogenic bacteria also achieved mortality rate for 2, 4, 6 larval instars of the pest were 94.4, 91.8, 100, 88.4, 85.6 and 81.3 % for the soil and plant treatments respectively, 14 days after application.
 

Multiple S-Layer Proteins of Brevibacillus laterosporus as Virulence Factors against Insects.

S-layers are involved in the adaptation of bacteria to the outside environment and in pathogenesis, often representing special virulence factors. Vegetative cells of the entomopathogenic bacterium Brevibacillus laterosporus are characterized by an overproduction of extracellular surface layers that are released in the medium during growth. The purpose of this study was to characterize cell wall proteins of this bacterium and to investigate their involvement in pathogenesis. Electron microscopy observations documented the presence of multiple S-layers, including an outermost (OW) and a middle (MW) layer, in addition to the peptidoglycan layer covering the plasma membrane. After identifying these proteins (OWP and MWP) by mass spectrometry analyses, and determining their gene sequences, the cell wall multilayer-released fraction was successfully isolated and used in insect bioassays alone and in combination with bacterial spores. This study confirmed a central role of spores in bacterial pathogenicity to insects but also detected a significant virulence associated with fractions containing released cell wall multilayer proteins. Taken together, S-layer proteins appear to be part of the toxins and virulence factors complex of this microbial control agent of invertebrate pests.

Analysis of the Toll and Spaetzle Genes Involved in Toll Pathway-Dependent Antimicrobial Gene Induction in the Red Flour Beetle, Tribolium castaneum (Coleoptera; Tenebrionidae).

Insects rely only on their innate immune system to protect themselves from pathogens. Antimicrobial peptide (AMP) production is the main immune reaction in insects. In Drosophila melanogaster, the reaction is regulated mainly by the Toll and immune deficiency (IMD) pathways. Spaetzle proteins, activated by immune signals from upstream components, bind to Toll proteins, thus, activating the Toll pathway, which in turn, induces AMP genes. Previous studies have shown the difference in immune systems related to Toll and IMD pathways between D. melanogaster and Tribolium castaneum. In T. castaneum, nine Toll and seven spaetzle (spz) genes were identified. To extend our understanding of AMP production by T. castaneum, we conducted functional assays of Toll and spaetzle genes related to Toll-pathway-dependent AMP gene expression in T. castaneum under challenge with bacteria or budding yeast. The results revealed that Toll3 and Toll4 double-knockdown and spz7 knockdown strongly and moderately reduced the Toll-pathway-dependent expression of AMP genes, respectively. Moreover, Toll3 and Toll4 double-knockdown pupae more rapidly succumbed to entomopathogenic bacteria than the control pupae, but spz7 knockdown pupae did not. The results suggest that Toll3 and Toll4 play a large role in Toll-pathway-dependent immune reactions, whereas spz7 plays a small part.

<i>Attacus atlas</i> (L.) sericin extract as an effective UV Protectant of <i>Bacillus thuringiensis</i> serotype <i>kurstaki</i> for controlling <i>Spodoptera litura</i> (Fab.)

Bacillus thuringiensis serotype kurstaki is an entomopathogenic bacteria commonly used to control the cutworm Spodoptera litura (Fab.). However, B. thuringiensis has disadvantage of being easily degraded due to exposed sunlight. The objective of this research was to determine the effectiveness of adding A. atlas (L.) cocoon extract as UV protectant B. thuringiensis to the mortality of S. litura. This research formulated 2.5% of the original substance of A. atlas cocoon extract and B. thuringiensis serotype kurstaki strain HD-7 applied from commercial product DiPel-WP®. The formulation was exposed to sunlight for 0, 1, 2, and 3 weeks. The suspension treated for 20 individuals of first instar larvae S. litura shifted into the artificial diet using 3-5 replicates. The scanning electron microscope (SEM) method began from a sample that was vacuumed, sample coated, and observed on SEM with the electron in a certain level probe. This research showed that the mortality of S. litura decreased with the growth of S. litura. The mortality of S. litura achieved 20%-100% mortality after treatments. The A. atlas cocoon extract was effective as UV protectant B. thuringiensis for three weeks of exposure to sunlight. The SEM analysis represented that formulation of B. thuringiensis and A. atlas cocoon extract sunlight exposure for one week has harsher surface than exposed during three weeks.