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Toxins and targets of insecticidal genes of entomopathogenic bacteria: a review.

In modern agricultural system the research on microbial pathogens of insects is increasing considerably to find out environmental friendly alternatives to hazardous chemical pesticides. In this regard, a Science Congress held in Europe entitle "Pesticide use and risk reduction in European farming systems", aimed to reduce the dependence on pesticides in modern agriculture through the implementation of general principles of Integrated Pest Management (IOBC, 2015). The microbials, broadly speaking biopesticides are the biological agents that are usually applied at appropriate formulation and application in a manner similar to synthetic chemical pesticides to achieve desirable pest management (OMICS, 2015; Matthews et al., 2014) in an environmentally friendly way. Whileas, the concept of EcoPesticides is developing, means biological based pesticide and encapsulation technology, designed and aimed to extend the potency and performance of the insect controlling properties of "green" material, especially the naturally occurring bacteria and fungi against to insect pests (Lux, 2015). Currently, the microbial pesticide market is $2 billion and is estimated to double by 2017 and will expectedly reach $5 billion by 2020 (ALBUQUERQUE, 2015). However, the market was assumed to increase to $3.3 billion in 2014 against the pesticide market of $51.1 billion (BCC, 2014) and biopesticides sales in crop protection market is expected to grow by 15% annually until 2020 (Lux, 2015).

The pathogen that cause most diseases in insects and successfully used for insect pest control is bacterium, Bacillus thuringiensis (Bt). Each one of Bt strains produces different mix of toxins and specifically kills one or a few related species of insects as Bt sub. kurstaki and aizawai for lepidopteran larvae and Bt sub. tenebrionis for coleopteran larvae. Bt subspecies israelensis is specific to mosquitoes (Diptera). Research suggests that, in response to bacterial and fungal infections in insects, an innate immune pattern recognition receptors (PRRs) initiate highly complex intracellular signaling cascades, which induce a variety of immune functions that restrain the spread of microbes in the host population (Stokes et al, 2015).In the current year the biopesticide Isaria fumosorosea strain Apopka 97 (Paecilomyces fumosoroseus) have been approved for use (Bird, 2015) as a component in IPM.

The discovery that Bt spore associated toxins are extremely virulent and persist in the environment with high potency (Koch et al., 2015) prompted the development of bacterial spray formulations and also the transgenic (Genetically Modified Plants) pants express the bacterial toxins against the insect pests (Di and Tumer, 2015; Nhan et al., 2015; Kamthan et al., 2015; Katriraee, 2015). The advancement in the characterization of bacterial pathogens, whole genome characterization and comparison has prompted the discovery of novel pest management tools. Insecticidal molecules expressed and secreted by various entomopathogenic bacteria have been targeted for the genetic manipulation to enhance toxicity (Sellami et al., 2015; Lin et al., 2015). Recently other insect pathogenic bacteria with mode of action similar to Bt have been hailed as agriculturally relevant. Currently insect pathogenic bacteria of diverse taxonomic groups and phylogenetic origin have been shown to have striking similarities in the virulence factors which are often encoded on plasmids and bacteriophages and can easily be spread through horizontal gene transfer. For example, Photorhabdus luminescens and Heterorhabditis bacteriophora have been shown to produce virulence factors (Gerdes et al., 2015; Castaneda et al., 2015) similar to that of Bt. B. thuringiensis produce crystal protein. When an insect ingests these proteins they are activated by proteolytic cleavage. The N-terminus is cleaved in all of the proteins and C-terminal extension is cleaved. Once activated the endotoxin binds to the gut epithelium and causes cell lysis by the formation of cation-selective channels leads to insect death.

Entomopathogenic bacteria

One of the best modern agricultural defenses against plant eating insects is Bacillus thuringiensis (Ibrahim and Shawer, 2014). In recent times, it has become a source of agriculture innovations, providing a new solution to the age of old problems. Biotechnology is often equated with genetic engineering and the support or opposition to genetically engineered crops is often distilled down to being for or against 'science' (Vogel, 2014). Plant genes are being cloned, genetic regulatory signals deciphered and genes transferred from the entirely unrelated organisms to confer new agriculturally useful traits on crop plants (Josine et al., 2011). Bt protein toxins are highly selective to their target insect and are completely biodegradable. Therefore, Bt is a viable alternative for the control of insect pests in agriculture and disease spreading vectors in public health. Transgenic crops based on insecticidal crystal proteins of Bt are now an international industry with revenues of several billion dollars per year (James, 2011). Classification of Bt strains have been accomplished by H serotyping, the immunological reaction to the bacterial flagellar antigen. Specific flagellin amino acid sequences have been correlated to specific Bt H serotypes and at least 69 H serotypes and 82 serological varieties (serovars) of Bt have been characterized from around the world (Lecadet et al., 1999). After few decades of research on Bacillus thuringiensis (Bt), new novel bacterial species are being discovered and developed into new products especially derived from Brevibacillus laterosporus, Chromobacterium subtsugae and Yersinia entomophaga (Ruiu et al., 2013). It is reported that the entomopathogenic bacteria of diverse taxonomic groups and phylogenetic origin have striking similarities in the virulence factors they produce (Castagnola and Stock, 2014) on infestation.

Insecticidal toxins produced by entomopathogenic bacteria

Insecticidal toxins used in agriculture are predominantly from Gram-positive bacteria and derived mostly from Bacillus thuringiensis. Foliar sprays containing B. thuringiensis represent an organic alternative to synthetic foliar sprays. Entomopathogenic Gram-negative bacteria produce toxins which are categorized into three types based on their target tissues. These are a) neurotoxins b) digestive toxins and c) cytotoxins. According to Ricardo et al. (2015) reported that toxification involves the binding of Cry toxins produced by Bacillus thuringiensis to specific cellular receptors like CADP (cadherin-like protein), a GPI (glycosylphosphatidyl-inositol)-anchored APN (aminopeptidase-N), a GPI-anchored ALP (alkaline phosphatase) and a 270-kDa glycoconjugate. Members in the Enterobacteriaceae such as Photorhabdus, Xenorhabdus, Serratia, and Yersinia spp. produce insecticidal toxins with oral toxicity similar to that of Bt toxins, but have not yet been fully utilized. This section describes the virulence factors associated with both Gram-negative and Gram-positive bacteria as well as their mode of action.

Toxin genes produced by gram positive bacteria Bacillaceae

Bacillus comprised of three species viz., B. thuringiensis, Bacillus cereus, Baccillus anthracis which are the most widely studied taxa in terms of insecticidal toxins (Tahany et al., 2015). Due to their unique pathogenicity properties and the diverse modes of actions of their insecticidal toxins, that support their distinctiveness, therefore, giving each separate species names (Priest et al., 2004). Bacillus thuringiensis upon sporulation forms crystals of proteinaceous endotoxins, called Cry proteins or crystal proteins, which are encoded by cry genes (Hofte and Whiteley, 1989). The Cry toxins, are toxic to may insect of order Lepidoptera, Diptera (Gough, 2002), Coleoptera (Kreig et al., 1984) and Hymenoptera (Rose et al., 1999) and also against nematodes (Hui et al., 2013). The Cry protoxins are first solubilized by the alkaline pH (68) of the insect (Lepidopteran larva) gut, and then proteolytically activated by proteases. The toxins bind to specific receptors on the columnar cells of the larval midgut epithelium causing pore formation and gut cell death. Cry toxins are commonly classified as gut poisons, as they compromise the epithelial-hemocoel layer and ultimately lead to starvation and septicemia and finally the larval death (Schnepf et al., 1998). Crystal toxins after they bind, form pores, and damage midgut epithelial goblet cells in mixed midgut cell cultures (Loeb et al., 2001) and in vivo. The Cry toxins from B. thuringiensis are almost exclusively considered as digestive toxins, however, they have homology to neurotoxins and attacks diverse tissues of lepidopteran larvae as the toxin meet suitable alkaline conditions in gut of larvae. They have been shown to kill larval neurons of the cerebral ganglia of central nervous system in vitro (Cerstiaens et al., 2001), invade liposomes causing morphological deformities to lipid bilayers (Haider and Ellar, 1989), initiate apoptosis in ovary and its derived cell (Zhang et al., 2006) and bind ATP-binding cassette (ABC) transporter (Tanaka et al., 2013). The mutated ABC trasporters are correlated with resistance to Bt toxins in silkworm, Bombyx mori (Atsumi et al., 2012), tobacco budworm, Heliothis virescens (Gahan et al., 2011), diamond back moth Plutella xylostella and cabbage looper Trichoplusia ni (Baxter, 2011). Recently it is reported that recombinant fusants have more efficient and potential toxicity values, compared with insecticidal Bt and the mosquitocidal Bt strains alone against S. littoralis and C. pipiens larvae, respectively (Tahany et., 2015).

Bacillus cereus has been mostly known for its role in human digestive food poisoning; and the endospore of B. cereus is not insecticidal unlike to Bt. Both B. thuringiensis and B. cereus produce non-proteinous insecticidal exotoxins; in addition, a small proteinous exotoxin is also produce by B. cereus (Perchat et al., 2005). Although, B. cereus grow and proliferate in the insect gut, and is mostly regarded as an opportunistic pathogen with the production of virulence factors that are most effective when titers are high. The Bacillus species known to produce insecticidal toxins are Bacillus circulans (Firmicutes: Bacillaceae) and sphaeriscus (Firmicutes: Bacillaceae) (=Lysinibacillus sphaeriscus). Virulence factors produced by the former species have been shown to affect most dipteran insects and other invertebrates such as nematodes and mollusks (Zwick et al., 2012). During the vegetative growth of the L. sphaeriscus the toxin produced is Sphaericolysin (Berry, 2011). Sphaericolysin toxin has heamoceolic toxicity toward Blattela germanica and S.litura (Nishiwaki et al., 2007).

Clostridiaceae

Clostridium species: These are anaerobic and spore-forming bacteria (Vaishnavi, 2015), produces binary proteinous toxins that are proteolytically activated by serine proteases (Barth et al., 2004). For example, Clostridium bifermentans serovar malaysia, produces larvicidal toxin active against mosquitoes (Nicolas et al., 1993). Clostridium perfringens (Firmicutes: Clostridiaceae) has an Iota toxin which binds actin by ADP-ribosylation and has a C-domain structure like Bt, ultimately targets mammals (Tsuge et al., 2003). The Clostridium difficile produce the exotoxin known as cytotoxin B which cause reorganization of cytoskeletons, similar to Mcf and makes the caterpillars floppy and dull in appearance. The Clostridium difficile has a three domain structure, consisting of receptor binding, translocation, and one catalytic domain (Just et al., 2005). The actin binding C2 toxin of C. botulinum, has four domains of activation for pore formation and receptor recognition. The C2 toxins ribosylate at arginine involves dysfunctioning the actin by inducing actin polymerization (Aktories et al., 2012). The translated product of theplu0822 gene, referred to as Photox toxin, stop actin polymerization by targeting an arginine amino acid with ADP-ribotransferase activity (Visschedyk et al., 2010).

Toxin genes produced by gram negative bacteria Photorhabdus

Photorhabdus species: Like Bacillus, three species of Photorhabdus have been identified namely, P luminescens, P. temperate and P. asymbiotica (Fischer et al., 1990). All photorhabdus species have a strong mutualistic association with Heterorhabditis nematodes which are parasitic to insects. However, the species P asymbiotica has also been found associated with skin injuries in human (Gerrard et al., 2006) and is considered as an emerging human pathogen model system (Garrard et al., 2004). Photorhabdus spp. are facultative anaerobes and cannot live freely in the soil environment unlike to B. thuringiensis. Almost all the Photorhabdus species are vectored by the nematodes and together with it form an insecticidal complex that kills the insect in general and use the carcass for various life processes like reproduction and nutrition. Once the bacteria are delivered by the nematodes in the insect hemocoel, it first invades insect immune system and then produces toxins which break its epithelial tissues and finally kills the insect. The Photorhabdus genome contains a multitude of lump like pathogenicity islands with an abundance of toxin genes (Duchaud et al., 2003). The major virulence factors produced by Photorhabdus consist of mcf1 and mcf2 (=makes caterpillar floppy) genes, the Tc (toxin complex) genes, Pir (Photorhabdus insect related) operon, and a multitude of other virulence factors associated with Photorhabdus virulence cassettes (PVC) (Rodou et al., 2010). The Mf toxins are responsible for both rearrange actin cytoskeletons and induce apoptosis in both insect hemocytes and epithelial tissue, leading to tissue damage to the extent that there is a complete loss of turgor pressure throughout the infected insect (Daborn et al., 2002). The Tc toxin factors, similar to Bt Cry toxins, are orally ingested toxic compounds that have been known to be insecticidal to the insect taxa including coleopteran, lepidoptera, dipteral and hemiptera. Experiments has shown that P. luminescens is highly insecticidal and pathogenic when injected into hosts such as African cotton leafworm, S. littoralis (Lepidoptera: Noctuidae) and P. xylostella (Lepidoptera: Pluttidae). As the Tc are orally toxic compounds; therefore, are active in the lumen side (inside the lumen) of the insects' midgut epithelium and not in the basal side of this tissue, which is common rout for a hemocoel pathogen (Waterfield et al., 2005a). Pir proteins are other group of Photorhabdus toxins, known to have hemolymph (Waterfield et al., 2005b) and oral (Blackburn et al., 2006) toxicity. The Pir proteins have similarity in certain aspects to neurotoxin, leptinotarsin (Blackburn et al., 2006) and are binary (Rodou et al., 2010) in structure. The binding and destructive effects to insect neural tissue are a major factor in toxicity when Photorhabdua are injected into susceptible host. There are yet many more toxins to be characterized from the Photorhabdus genome which are responsible for hemolymph-based insect toxicity. For example, the Txp40 protein has been identified in 59 different strains of both Photorhabdus and Xenorhabdus species, cause injectable toxicity to many lepidopteran pests, like greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae), Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae), corn earworm, Helicoverpa armigera (Lepidoptera: Noctuidae), and Australian sheep blowfly, Lucilia cuprina (Diptera: Calliphoridae) (Brown et al., 2006). Escherichia coli when expresses txp40 gene, it has been shown to be insecticidal to P xylostella (Park et al., 2012). The midgut and the body cell lines of dipteral and lepidopteral insects are damaged by Txp40 protein in vitro (Brown et al., 2006) while as, hemolymph (Waterfield et al., 2005b) and oral (Blackburn et al., 2006) toxicity were exhibited by Pir toxin proteins and Photorhabdus toxins. The Xenorhabdua species of bacteria in this genus are also non-free living; though, they are symbiotically associated with nematodes of genus Steinernema (Brown et al, 2004). Like Heterorhabditis, Steinernema nematodes are play a key role in vectoring the Photorhabdus and Xenorhabdus from one insect host to another, thereby dispersing the bacteria and finally mange the pest population. Xenorhabdus also produce a large number of insecticidal toxins and one example of toxin is from Xenorhabdus nematophila (Proteobacteria: Enterobacteriaceae) called A24tox, which kills G mellonella and H. armigera. However, this toxin has a hypothetical homology in Photorhabdus, but without a significant match outside of this group (Sicard et al., 2003).

The xenocin operon consists of two genes, xciA and ximB, when it is expressed; these proteinous molecules get secreted through flagellar type II secretion pathway. Xenocin xciA gene has RNAse activity and cytotoxicity; once these proteins are co-expressed it has an antimicrobial effect killing competing microbes in insect larvae (Sing et al., 2013). Xenorhabdus bacteria also produce another insecticidal protein called HIP57 that is similar to chaperonins like GroEL produce by E. coli. GroEL chaperonins help to combat problems such as aggregation when nascent proteins have hydrophobic residues exposed before reaching a fully folded native state (Ellis, 2005). The HIP57 have injectable toxicity to G.mellonella, and the insecticidal property of it is a novel function for the GroEL proteins.

Serratia

Serratia species: These bacteria often possess fungicidal properties, but are facultatively associate with insect (Lamelas et al., 2011) and nematodes (Abebe et al., 2011). Genome studies have found several insecticidal genes in the Serratia genome and few species are responsible for causing amber disease in grass grubs, Costelytra zealandica (Coleoptera: Scarabaeidae) (Jackson et al., 2001). However, contrastingly Serratia marcescens (Proteobacteria: Enterobacteriaceae) infects other host such as poorly reared H. virescens (Sikorowski et al., 2001). Whileas, pADAP plasmid from Serratia entomophila contains the genes sepA, sepB and sepC, which are similar to the Tc genes described in P luminescens and the xpt genes observed from X. nematophila. There is no need for the entire pADAP plasmid to be associated with the sep genes to cause death. However, when only sep genes are expressed without the entire plasmid, the scarab beetles do not cease feeding (Hurst et al., 2000) which is one symptom of amber disease. Actually the virulence factor of pADAP that stops feeding in amber disease is the antifeeding prophage (Afp) (Hurst et al., 2007). Therefore, it is concluded that both sep genes and Afp are needed for full virulence of Serratia in grass grubs that leads to its death.

Yersinia

Yersinia species: Yersinia pestis, the causative agent of bubonic disease is associated with fleas (UNC, 2015; CDCP, 2014) humans and rodent intermediates. Whileas, the two other species Yersinia enterocolitica and Yersinia pseudotuberculosis often cause diarrheal disease and fever with inflammation in human beings. Yersinia entomophaga (Proteobacteria: Enterobacteriaceae) and Yersinia frederiksenii (Dodd et al., 2006), cause disease in grass grubs and plague in humans (Gonzalez et al., 2015).

Targets of insecticidal genes and toxins produced by bacteria

This section summarizes the current knowledge of hemolymph based toxicity caused by various entomopathogenic bacteria in relation to the neurobiology of insect pests.

Hemolymp as a Novel Target

The genomic organization of P. luminescens and asymbiotica consists of Pir operon with promoter region, Pir toxin i.e, PirA and PirB (Waterfield et al, 2005a), but it is not known how PirA and Pir B are differentially expressed when targeting an insect host. Pir toxin is a binary protein that may have an interesting mode of action based upon its homology profile, while PirB is homologous endotoxins consisting of a poreforming domain unit and leptinotarsin. Leptinotarsin has been obtained from the infected Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) and has homology to juvenile hormone esterase which is a regulatory protein present mainly in insect immature. However, Pir toxin does not disturb insect metamorphosis (Ffrench and Waterfield, 2005) but it (Leptinotarsin) is a neurotoxin that stimulates and activates the release of acetylcholine at the presynaptic nerve terminal (McClure et al., 1980). That is why, the mode of action of Pir toxin and its potential relationship to the insect nervous system is yet to be elucidated. Serratia and Photorhabdus have phage related loci, and in Serratia these loci are pADAP, which causes decreased feeding in infected insects. Both Serratia and Photorhabdus phage related loci confer hemolymph based injectable toxicity on Galleria larvae, with hemolymph-circulating phagocytes (Yang, 2006) as a virulence factor. There are also the cycle inhibiting factors (cif) produced by P. luminescens, but it is not known how Cif interacts with an insect host, when the cif gene is incorporated into Spodoptera derived Sf9 cells. The cells once infected undergo apoptosis and thereby, the cell cycle arrests (Chavez et al., 2014).

Hemolymph based toxicity and ecofriendly insect pest control

The insecticides lack pest specificity therefore, promotes development of pest resistance (Davies et al., 2012) in ecosystem, often leads to a problem referred to as the pesticide treadmill (Knight, 1989). One of the best examples is Bt applied as foliar sprays which are organic have high specificity and negligible environmental impact compared to their synthetic counterparts (Castagnola and Fuentes, 2012). The toxins produced by mites, spiders and other venomous organisms have been known to have neurotoxic effects on insects (Windley et al., 2012) without deteriorating the environment. Some of the genes encoding these toxins can be expressed in transgenic plants, thereby contribute to decrease of target insect population (Khan et al., 2006).

As mentioned, many neurotoxins have been discovered from invertebrates. For example, leptinotarsin, isolated and identified from the Colorado potato beetle, has been shown to disrupt the release of acetylcholine at the presynaptic nerve terminal of rat synaptosomes (Yoshino et al., 1980). Although peptides of leptinotarsin have shown homology to both juvenile hormone esterase (JHE) of insects and Cry toxins of Bt, there is no evidence that leptinotarsin has JHE activity. Because of the way proteinous neurotoxins interact and disrupt neural tissues function, they can be used to study the physiological consequences of the nervous system dysfunction (Khan et al., 2006). Understanding their mode of action and interactions with the insect nervous system, Bt can be a powerful tool with application in insect pest management.

Insect Paralysis by B.thuringensis

Whether a particular Bt protein bind to insect gut receptor or not, it is necessary to be determined (OMICS, 2015) at first. Once the Bt Cry toxins are accepted by sensory receptors only then these are ingested by larvae of lepidopteran, coleopteran and dipteran insect and causes a number of toxic effects viz. paralysis, cessation of feeding and reduced movements. Paralysis of midgut is a predominant characteristic caused by Bt, and is a preliminary way to discriminate among different insecticidal Bacillus species and strains (Heimpel and Anguset 1958). Paralysis induced by Bt can be categorized by insect type. Type I insects are characterized by the symptom of whole body paralysis; larvae become inactive and fall off their host plant. The increase of pH in the insect hemolymph is the main cause for this type. Unlike Type I insects, type II insects involves paralysis symptoms limited to gut movement; however, increased pH once again plays a key role in toxicity intensification. Paralysis is thought to be caused by the breakdown of the epithelial integument, characterized by inhibiting insect physiological function and movement (Heimpel and Anguset, 1959). Gut paralysis in Type II insects involves the cessation of feeding and frass production. Second generation Bt crops (B.thuringiensis genes) used for the management of crop pests by combined action of more than one genes exhibited synergism and antagonism between Vip3A and Cry 1 Proteins in response to the damage done by Heliothis virescens, Diatraea saccharalis and Spodoptera frugiperda (Lemes et al., 2014)

Knoweldege about the potential role of the insect nervous system, brain, or neuromuscular junction in insect gut or whole body paralysis is limited; however in general the peristalsis in insects is controlled by the stomatogastric nervous system (Hartenstein, 1997). The frontal ganglion especially the frontal connective controls the peristaltic movements, thereby enabling the foregut to empty food into the midgut. So, it is clear that if digestion has ceased then this aspect of the nervous system may have been targeted. However, larva regurgitation is a common physiological defense response during ingestion of plant defense molecules. In depth we can say that toxins of insect pathogens which have both oral and injectable toxicity could selectively silence specific insect nervous tissues involved in digestion and midgut muscle movement. Understanding this nature of toxicity of microbial toxins, we may judge the function of frontal connective tissues (nervous tissues) in insect behavior, especially the tissues involved in food consumption (Rodriguez et al., 2008). One Cry toxin of Bt, Cry1C, targets the tissues of nervous system (Cerstiaens et al., 2001) and digestive system especially the gut epithelium (Aronson and Shai, 2001) of various lepidopteran species.

Role of other Bt strains in Neurotoxicity of insects

Neurotoxic symptoms have been observed when Bt var. israelensis (Bt) are injected into Cabbage semiloopers. Trichoplusi ni. When injected into hemolymph at higher does Bti stops heart activity. Moreover, symptoms like loss of motor activity, paralysis and flaccidity were observed. The proteins conferring neurotoxicity in lepidopteran insects were components of the crystal endotoxin from Bti (Cheung et al., 1985). It is assumed that the presynaptic nerve terminal function were blocked; whereas, the postsynaptic membranes and axons in the ventral nerve cord remained unaffected. The symptoms like 6th abdominal ganglion transmitter release, calcium uptake and complete blockage of transmitters were observed. However, in rat muscle degeneration were observed when treated with crystal protein of Bti. In short, the mode of action of crystal toxins involve [Na.sup.+]/[K.sup.+] ATPase damage and [K.sup.+] levels decrease; whileas, the [Na.sup.+] levels increase within muscles cells with increasing [Ca.sup.2+] influx (Cahan et al, 1985).

CONCLUSION

Insecticidal toxins are important options for the biological control of insect pests. Their use in the genetic engineering of plants could provide a new generation of resistant crops. The Bt whether incorporated into a foliar spray or toxins expressed in transgenic plants, is regarded as the premier entomopathogens used in pest management. Once the Bt Cry toxins are accepted by sensory receptors only then these are ingested by larvae of insect pests and cause a number of toxic effects viz. paralysis, cessation of feeding and reduced movements. Paralysis of midgut is a predominant characteristic caused by Bt, and is a preliminary way to discriminate among different insecticidal Bacillus species and strains. Recently, similar toxins to that of Bt have been identified throughout bacterial kingdom. The use of Bt for insect control help to gain further knowledge of the origin of entomopathogens and their associated virulence factors. As it would be interesting to investigate if the combination of virulence factors of the two different entomopathogenic bacteria such as Photorhabdus and Bacillus delay resistance. Combining toxins with different modes of actions, may delay the onset of resistance by forcing insects to develop two separate mechanisms of resistance. Combination of toxins could result in a more lethal insecticide that would result in a better control tactic for a problematic insect pest. Furthermore the transfer and delivery mechanism of Photorhabdus into the haemocoel suggests to existence of virulence factors with novel tissues as targets in the lepidopteran pests that can be further investigated.

The neurotoxic effects of Bti were investigated in American Cockroach, Periplaneta americana (Blattodea: Blattidae). The presynaptic nerves terminal function was suspected to be blocked were as the postsynaptic membranes and axons in the ventral nerve cord remained unaffected. The sixth abdominal ganglion transmitter release calcium uptake and complete blockage of transmitters were observed. The mode of action were related to [Na.sup.+]/[K.sup.+]- ATPase damage upon incubation and [K.sup.+] level decrease while [Na.sup.+] level increase within muscle cells with increasing [Ca.sup.2+] influx. In general the entomopathogens have history of horizontal gene transfer shuffling the toxin containing plasmids resulting in Pathogenicity Island between each other. The acquisition of insecticidal genes may be a strategy that may develop to onset virulence once bacteria were ingested; thereby broadening the availability of possible sources and helps in insect pest management.

ACKNOWLEDGEMENTS

Authors are highly thankful to Department of Science and Technology, Delhi for the financial support to compile this project.

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Showket Ahmad Dar [1] and S.A. Padder [2]

[1] Department of Entomology, [2] Microbiology Sher-e-Kashmir University of Agricultural Science and Technology, Shalimar, J&K - 190 025, India.

(Received: 21 July 2015; accepted: 02 September 2015)

* To whom all correspondence should be addressed. E-mail: showketdar43@gmail.com
Table 1. Paralytic effects of ingested Bt on various lepidopteran
families and species (Castagnola and Stock, 2014)

Family         Species            Bt component     Response

Noctuidae      Spodoptera         Not              No paralysis
               spp.               specified

               H. virescens       Bt var.          Midgut paralysis
                                  kurstaki

               T. ni              Not              Type I paralysis
                                  specified

Saturniidae    Philosamia         Bt var.          Whole body
               ricini             sotto            paralysis
                                  crystals         (type I)

Crambidae      Ostrinia           Bt var.          Gut paralysis
               nubilalis          thuringiensis
                                  crystalline
                                  paraspores

Pyralidae      Phlegathontius     Thuricide        Abnormally
                                  (International   quiescent,
                                  Minerals and     cessation of
                                                   feeding

               G mellonella       Spores and       No paralysis,
                                  crystals         Type III most
                                  derived from     susceptible
                                  Thuricide

               Ephestia           Not specified    Type II
               cautella                            paralysis

Sphingidae     quinqueaculatus    Chemical         No paralytic
                                  Corp.,           effect
                                  Libertyville,
                                  IL, USA)

Erebidae       L. dispar          Not specified    Type II paralysis

Plutellidae    P. xylostella      Bt biological    Decreased
                                  products         movement with
                                                   subsequent
                                                   paralysis

               P. xylostella      Bt var.          Reduction of
                                  kurstaki-HDl     movements

Papilionidae   Papilio            Bti Berliner     Fairly rapid
               demoleus           spore            paralysis
                                                   followed by
                                                   alkalinity

Gelechiidae    Pectinophora       Delta-           Evidence of gut
               gossypella         endotoxin        paralysis gut
                                  endotoxin        muscles
                                                   surrounding the
                                                   disorganized
                                                   epithelium
                                                   relaxed

Bombycidae     B. mori            Bacillus sotto   Paralysis within
                                                   four hours

               B. mori            Bt var. sotto    Paralysis

               B. mori            Not specified    Type I paralysis

               Quinquemaculata    Bt               General paralysis

               P.                 Not specified    Type I paralysis
               quinquemaculata

               Protoparce         Bt crystals      General paralysis
               sexta

               Antheraea          Bt crystals      General paralysis
               pernyi

Pieridae       Colias             Bt var.          No paralysis
               eurytheme          thuringiensis

               Pieris rapae       Not specified    Type II paralysis

Hesperiidae    Urbanus            Bt var.          no general
               acawoios           kurstaki         paralysis
                                  wettable
                                  powder

Tortricidae    Urbanus            Bt Dipel         Interruption of
               acawoios           foliar spray     feeding due to
               fumiferana                          gut paralysis
                                  Bt Dipel         resulted in
                                  foliar spray     reducing rate
                                                   of development

Table 2. Susceptibility of Helicoverpa virescens neonate
larvae to the combinations of the insecticidal proteins
Vip3A and Cry1 crystals protoxins (Lemes et al., 2014)

                                              Per cent larval
                                              mortality

S.    Crystal and            Respective       Observed
No    vegetative             concentrations   frequency
      insecticidal protein   (a)              (b)
      combination

1     Cry1Aa                 3.50             44
2     Cry1Ac                 0.04             52
3     Cry1Ca                 3.10             42
4     Vip3Aa                 1.65             52
5     Vip3Ae                 0.95             50
6     Vip3Af                 0.87             50
7     Vip3Aa+Cry1Aa          1.65+3.50        69
8     Vip3Aa+Cry1Ca          1.65+0.04        67
9     Vip3Ae+Cry1Ca          1.65+3.10        33
10    Vip3Ae+Cry1Aa          0.946+3.50       73
11    Vip3Ae+Cry1Ac          0.946+0.04       63
12    Vip3Ae+Cry1Ca          0.946+3.10       31
13    Vip3Af+Cry 1Aa         0.874+3.50       50
13    Vip3Af+Cry1Ac          0.874=0.04       50
14    Vip3Af+Cry1Ca          0.874-3.10       37

      Per cent larval
      mortality

S.    Expected          Fisher's          Chi ([u.sup.2])
No    frequency         test (d)          square
      (c)               (T-test)          Test (P) (e)

1     50                No significance   No significance
2     50                do                do
3     50                do                do
4     50                do                do
5     50                do                do
6     50                do                do
7     73                0.4113            0.2017 (0.6534) *
8     77                0.1823            1.2882 (0.2554) *
9     72                0.00009 **        15.101(0.0001) ***
10    72                0.5907 **         0.0000 (1.0000) *
11    76                0.1354 **         1.7455 90.1864) *
12    17                0.0001 **         15.048 (0.0001) *
13    72                0.0177 **         5.3211 (0.0211) *
13    76                0.0149 **         5.7501 (0.0165) *
14    71                0.0009 **         10.741 (0.001) **

Concentrations of proteins were chosen such as to equal their
respective LC50 values. Values are expressed as mg/[cm.sup.2].

(b) Each value represents the mean from three replicates of 16 larvae
per replicate (n = 48).

(c) Expected mortality considering simple independent action.

(d) Asterisks indicate significant differences at P, 0.05, and two
asterisks at P,0.001.

(e) Chi-square and P values.
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Author:Dar, Showket Ahmad; Padder, S.A.
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Jun 1, 2016
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