Purification, characterization and toxicity assessment of PirAB toxins from Photorhabdus akhurstii subsp. akhurstii K-1.

Photorhabdus insect related proteins A & B (PirA, PirB) from Photorhabdus and Xenorhabdus bacteria exhibit both oral and injectable toxicity against lepidopteran and dipteran insect pest. The pirA, pirAt (encoding 6 N-terminal truncated PirA), pirB genes, pirA-pirB (with ERIC sequences), pirA-pirB-mERIC (modified pirA-pirB with mutated ERIC sequences) and polycistronic-pirAB were cloned and expressed in Escherichia coli. However, PirA protein was expressed in insoluble form and therefore the pirA gene was modified to produce PirAt. Moreover, pirA-pirB-mERIC, polycistronic-pirAB and co-transformed pirA/pirB genes were not expressed in the studied prokaryotic expression systems. None of the single purified proteins or mixtures of the individually expressed and purified proteins were toxic to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. However, PirA-PirB protein mixtures purified from pirA-pirB operon plasmid were toxic to A. aegypti and C. quinquefasciatus larvae with LC50 values of 991 and 614 ng/ml, respectively. The presence of ERIC sequences between the two orfs of the pirA-pirB operon could help to obtain the proteins in biologically active form. Further, results confirm that PirA-PirB proteins of P. akhurstii subsp. akhurstii K-1 are binary insecticidal toxins and ERIC sequences could play an important role in expression of Pir proteins. Reports of biophysical characterization of individually purified PirAt, PirB and expressed PirA-PirB toxin mixture could provide the structural insight into these proteins.

Bacterial communities in the gut of wild and mass-reared Zeugodacus cucurbitae and Bactrocera dorsalis revealed by metagenomic sequencing.

BACKGROUND: Insect pests belonging to genus Bactrocera sp. (Diptera: Tephritidae) pose major biotic stress on various fruits and vegetable crops around the world. Zeugodacus and Bactrocera sp. are associated with diverse bacterial communities which play an important role in the fitness of sterile insects. The wild populations of melon fly, Zeugodacus cucurbitae (Coquillett) and Oriental fruit fly, Bactrocera dorsalis (Hendel) were collected from pumpkin and mango fields, respectively. The laboratory populations of Z. cucurbitae and B. dorsalis were mass-reared on bottle gourd and sweet banana, respectively. Bacterial communities present in the gut of wild and mass-reared mature (~ 12 days old) and newly emerged (< 1 h after emergence) male and female adults of Z. cucurbitae and B. dorsalis were assessed. We used Illumina HiSeq next-generation sequencing of 16S rRNA gene to profile the gut bacterial communities of wild and mass-reared mature and newly emerged Z. cucurbitae and B. dorsalis adults. RESULTS: We found diverse bacterial composition in the gut of wild and mass-reared Z. cucurbitae (ZC) and B. dorsalis (BD) with varied relative abundance. Few taxonomic groups were common to both the species. The most dominant phyla in all samples of Z. cucurbitae and B. dorsalis adults were Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. The phylum Proteobacteria occurred more in wild Z. cucurbitae (~ 87.72%) and B. dorsalis (~ 83.87%) as compared to mass-reared Z. cucurbitae (64.15%) and B. dorsalis (~ 80.96%). Higher relative abundance of Phylum Firmicutes was observed in mass-reared fruit fly than wild adults. Cyanobacteria/Chloroplast and Actinobacteria were also present with very low relative abundance in both wild as well as mass-reared melon fly and Oriental fruit fly. Enterobacteriaceae (61.21%) was dominant family in the gut of both wild and mass-reared adults. Providencia and Lactococcus were dominant genera with varied relative abundance in wild as well as in mass-reared mature and newly emerged fruit fly adults of both species. Some of the genera like Morganella and Serratia were only detected in mass-reared mature and newly emerged Z. cucurbitae and B. dorsalis adults. Principal Coordinate Analysis (PCoA) showed that fruit fly adult samples were grouped based on species and age of the adults while no grouping was observed on the basis of sex of the adult fruit fly. CONCLUSIONS: The gut bacterial communities associated with wild and mass-reared mature and newly emerged adults of Z. cucurbitae and B. dorsalis showed variation that depends on species and age of the insects. Understanding the gut microbiota of wild and mass-reared Z. cucurbitae and B. dorsalis using high throughput technology will help to illustrate microbial diversity and this information could be used to develop efficient mass-rearing protocols for successful implementation of sterile insect technique (SIT).

Detection and characterization of bacterial endosymbionts in Southeast Asian tephritid fruit fly populations.

BACKGROUND: Various endosymbiotic bacteria, including Wolbachia of the Alphaproteobacteria, infect a wide range of insects and are capable of inducing reproductive abnormalities to their hosts such as cytoplasmic incompatibility (CI), parthenogenesis, feminization and male-killing. These extended phenotypes can be potentially exploited in enhancing environmentally friendly methods, such as the sterile insect technique (SIT), for controlling natural populations of agricultural pests. The goal of the present study is to investigate the presence of Wolbachia, Spiroplasma, Arsenophonus and Cardinium among Bactrocera, Dacus and Zeugodacus flies of Southeast Asian populations, and to genotype any detected Wolbachia strains. RESULTS: A specific 16S rRNA PCR assay was used to investigate the presence of reproductive parasites in natural populations of nine different tephritid species originating from three Asian countries, Bangladesh, China and India. Wolbachia infections were identified in Bactrocera dorsalis, B. correcta, B. scutellaris and B. zonata, with 12.2-42.9% occurrence, Entomoplasmatales in B. dorsalis, B. correcta, B. scutellaris, B. zonata, Zeugodacus cucurbitae and Z. tau (0.8-14.3%) and Cardinium in B. dorsalis and Z. tau (0.9-5.8%), while none of the species tested, harbored infections with Arsenophonus. Infected populations showed a medium (between 10 and 90%) or low (< 10%) prevalence, ranging from 3 to 80% for Wolbachia, 2 to 33% for Entomoplasmatales and 5 to 45% for Cardinium. Wolbachia and Entomoplasmatales infections were found both in tropical and subtropical populations, the former mostly in India and the latter in various regions of India and Bangladesh. Cardinium infections were identified in both countries but only in subtropical populations. Phylogenetic analysis revealed the presence of Wolbachia with some strains belonging either to supergroup B or supergroup A. Sequence analysis revealed deletions of variable length and nucleotide variation in three Wolbachia genes. Spiroplasma strains were characterized as citri-chrysopicola-mirum and ixodetis strains while the remaining Entomoplasmatales to the Mycoides-Entomoplasmataceae clade. Cardinium strains were characterized as group A, similar to strains infecting Encarsia pergandiella. CONCLUSIONS: Our results indicated that in the Southeast natural populations examined, supergroup A Wolbachia strain infections were the most common, followed by Entomoplasmatales and Cardinium. In terms of diversity, most strains of each bacterial genus detected clustered in a common group. Interestingly, the deletions detected in three Wolbachia genes were either new or similar to those of previously identified pseudogenes that were integrated in the host genome indicating putative horizontal gene transfer events in B. dorsalis, B. correcta and B. zonata.

PEGylation Enhances Mosquito-Larvicidal Activity of Lysinibacillus sphaericus Binary Toxin.

Toxic strains of Lysinibacillus sphaericus have been used in the field for larval control of mosquito vector diseases. The high toxicity of L. sphaericus is attributed to the binary (BinAB) toxin produced as parasporal crystalline inclusions during the early stages of sporulation. BinA and BinB, the primary components of these spore-crystals, exert high toxicity when administered together. However, instability, short half-lives, and rapid proteolytic digestion can limit their use as an effective insecticide. BinA alone displays larvicidal toxicity, in the absence of BinB, albeit with much reduced activity. Here for the first time, we demonstrate the beneficial effect of PEGylation (covalent attachment of polyethylene glycol) on mosquito-larvicidal activity of BinA. Polymer conjugation was achieved using 750 Da polyethylene glycol (PEG) at two different pH values (pH 7.2 and 8.5). Two different isoforms of the biopolymers, purified to homogeneity, were highly water-soluble and resistant to trypsin and proteinase K. The mono-PEGylated BinA isoforms also displayed preservation of the toxin structure with improved thermal stability by about 3-5 °C, as evident from thermal denaturation studies by circular dichroism and differential scanning fluorimetry. Notably, PEGylation enhanced BinA toxicity by nearly 6-fold. The PEGylated BinA isoforms alone displayed high larvicidal activity (LC50 value of ∼3.4 ng/mL) against the third instar Culex larvae, which compares favorably against LC50 reported for the combination of BinA and BinB proteins. Since BinA can be synthesized easily through recombinant technology and easily PEGylated, the conjugated biopolymers offer a promising opportunity for mosquito control programs.

An oligomeric complex of BinA/BinB is not formed in-situ in mosquito-larvicidal Lysinibacillus sphaericus ISPC-8.

Binary toxin of Lysinibacillus sphaericus is composed of two polypeptides; receptor binding BinB and toxic BinA. Both the polypeptides are required for maximal toxicity. It has been suggested that binary toxin exerts toxicity as a heterotetramer constituted by two copies of each of the component polypeptides. It has also been observed that oligomers consisting of two copies of BinA and BinB are pre-formed in L. sphaericus spore-crystals. However, recombinant proteins from Escherichia coli expression system elute individually as monomers. We purified the likely oligomeric complex from the spore-crystals of highly toxic L. sphaericus ISPC-8 strain and probed it with proteomic tools. The analysis showed that the high molecular mass complex in the toxic spore-crystals is composed of only surface layer protein (SlpC). The purified SlpC from the local isolate exists as a dimer and also showed poor mosquito-larvicidal activity.

The crystal structure of insecticidal protein Txp40 from Xenorhabdus nematophila reveals a two-domain unique binary toxin with homology to the toxin-antitoxin (TA) system.

Txp40 is a ubiquitous, conserved, and novel toxin from Xenorhabdus and Photorhabdus bacteria, toxic to a wide range of insect pests. However, the three-dimensional structure and toxicity mechanism for Txp40 or any of its sequence homologs are not yet known. Here, we are reporting the crystal structure of the insecticidal protein Txp40 from Xenorhabdus nematophila at 2.08 Å resolution. The Txp40 was structurally distinct from currently known insecticidal proteins. Txp40 consists of two structurally different domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), primarily joined by a 33-residue long linker peptide. Txp40 displayed proteolytic propensity. Txp40 gets proteolyzed, removing the linker peptide, which is essential for proper crystal packing. NTD adopts a novel fold composed of nine amphipathic helices and has no shared sequence or structural homology to any known proteins. CTD has structural homology with RNases of type II toxin-antitoxin (TA) complex belonging to the RelE/ParE toxin domain superfamily. NTD and CTD were individually toxic to Galleria mellonella larvae. However, maximal toxicity was observed when both domains were present. Our results suggested that the Txp40 acts as a two-domain binary toxin, which is unique and different from any known binary toxins and insecticidal proteins. Txp40 is also unique because it belongs to the prokaryotic RelE/ParE toxin family with a toxic effect on eukaryotic organisms, in contrast to other members of the same family. Broad insect specificity and unique binary toxin complex formation make Txp40 a viable candidate to overcome the development of resistance in insect pests.

Crystal structures of PirA and PirB toxins from Photorhabdus akhurstii subsp. akhurstii K-1.

PirAB binary toxin from Photorhabdus is toxic to the larvae of dipteran and lepidopteran insect pests. However, the 3-D structures and their toxicity mechanism are not yet fully understood. Here we report the crystal structures of PirA and PirB proteins from Photorhabdus akhurstii subsp. akhurstii K-1 at 1.6 and 2.1 Å, respectively. PirA comprises of eight ß-strands depicting jelly-roll topology while PirB folds into two distinct domains, an N-terminal domain (PirB-N) made up of seven α-helices and a C-terminal domain (PirB-C) consists of ten ß-strands. Despite the low sequence identity, PirA adopts similar architecture as domain III and PirB shared similar architecture as domain I/II of the Cry δ-endotoxin of Bacillus thuringiensis, respectively. However, PirA shows significant structural variations as compared to domain III of lepidopteran and dipteran specific Cry toxins (Cry1Aa and Cry11Ba) suggesting its role in virulence among range of insect pests and hence, in receptor binding. High structural resemblance between PirB-N and domain I of Cry toxin raises the possibility that the putative PirAB binary toxin may mimic the toxicity mechanism of the Cry protein, particularly its ability to perform pore formation. The mixture of independently purified PirA and PirB proteins are not toxic to insects. However, PirA-PirB protein complex purified from expression of pir operon with non-coding Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences found toxic to Galleria mellonella larvae with LD50 value of 1.62 µg/larva. This suggests that toxic conformation of PirA and PirB are achieved in-vivo with the help of ERIC sequences.

Purification, characterization and proteolytic processing of mosquito larvicidal protein Cry11Aa from Bacillus thuringiensis subsp. israelensis ISPC-12.

Cry11Aa is the most potent mosquito larvicidal protein of Bacillus thuringiensis subsp. israelensis (Bti). Development of resistance against insecticidal proteins including Cry11Aa is known but no field resistance was observed with Bti. The phenomenon of increasing resistance in insect pests necessitates the development of new strategies and techniques to enhance efficacy of insecticidal proteins. Recombinant technology offers better control over the molecule and allows modification of protein to achieve maximal effect against target pests. In this study, we standardised protocol for recombinant purification of Cry11Aa. Recombinant Cry11Aa found active against larvae of Aedes and Culex mosquito species and LC50 were estimated. Detailed biophysical characterization provides crucial insights into stability and in-vitro behaviour of the recombinant Cry11Aa. Moreover, trypsin hydrolysis doesn't improve overall toxicity of recombinant Cry11Aa. Proteolytic processing suggests domain I and II are more prone to proteolysis in comparison to domain III. Significance of structural features for proteolysis of Cry11Aa was observed after performing molecular dynamics simulations. Findings reported here are contributing significantly in method for purification, understanding in-vitro behaviour and proteolytic processing of Cry11Aa which could facilitate in efficient utilisation of Bti for insect pests and vectors control.

Transgenic banana plants expressing small interfering RNAs targeted against viral replication initiation gene display high-level resistance to banana bunchy top virus infection.

The banana aphid-transmitted Banana bunchy top virus (BBTV) is the most destructive viral pathogen of bananas and plantains worldwide. Lack of natural sources of resistance to BBTV has necessitated the exploitation of proven transgenic technologies for obtaining BBTV-resistant banana cultivars. In this study, we have explored the concept of using intron-hairpin-RNA (ihpRNA) transcripts corresponding to viral master replication initiation protein (Rep) to generate BBTV-resistant transgenic banana plants. Two ihpRNA constructs namely ihpRNA-Rep and ihpRNA-ProRep generated using Rep full coding sequence or Rep partial coding sequence together with its 5' upstream regulatory region, respectively, and castor bean catalase intron were successfully transformed into banana embryogenic cells. ihpRNA-Rep- and ihpRNA-ProRep-derived transgenic banana plants, selected based on preliminary screening for efficient reporter gene expression, were completely resistant to BBTV infection as indicated by the absence of disease symptoms after 6 months of viruliferous aphid inoculation. The resistance to BBTV infection was also evident by the inability to detect cDNAs coding for viral coat protein, movement protein and Rep protein by RT-PCR from inoculated transgenic leaf extracts. Southern analysis of the two groups of transgenics showed that ihpRNA transgene was stably integrated into the banana genome. The detection of small interfering RNAs (siRNAs) derived from the ihpRNA transgene sequence in transformed BBTV-resistant plants positively established RNA interference as the mechanism underlying the observed resistance to BBTV. Efficient screening of optimal transformants in this vegetatively propagated non-segregating fruit crop ensured that all the transgenic plants assayed were resistant to BBTV infection.

Txp40, an insecticidal toxin protein from Xenorhabdus nematophila: Purification, toxicity assessment and biophysical characterization.

Txp40 is a ubiquitous toxin from Xenorhabdus and Photorhabdus bacteria, exhibits insecticidal activity against a wide range of insect pests belonging to Lepidoptera and Diptera orders. Initially, Txp40 affects midgut of the target insect and further damages some other tissues like fat bodies but the detailed mode of action is not known. Txp40 shares no significant sequence match to any proteins with known structure or function, suggesting that it is a novel type of insecticidal toxin. Here, we report purification, toxicity and biophysical characterization of the Txp40b toxin from X. nematophila (ATCC, 19061). The recombinant Txp40b was found toxic to Galleria mellonella larvae with LD50 of 30.42 ng larva-1. Circular dichroism spectroscopy revealed that purified Txp40b is an α-helix rich protein with a relatively lower melting temperature of 45 °C. In-silico model generated suggests two domain structure of Txp40b toxin. Detailed structural analysis of Txp40b will provide new insights about the mode of action and possibly it would illustrate a new domain and/or motif in the area of insecticidal proteins.

Purification and characterization of mosquitocidal Bacillus sphaericus BinA protein.

Certain strains of Bacillus sphaericus produce a highly toxic mosquito-larvicidal binary toxin during sporulation. The binary toxin is composed of toxic BinA (41.9kDa) and receptor binding BinB (51.4kDa) polypeptides and is active against vectors of filariasis, encephalitis and malaria. The toxin has been tested with limited use for the control of vector mosquitoes for more than two decades. The binA gene from a local ISPC-8 strain of B. sphaericus that is highly toxic to Culex and Anopheles mosquito species was cloned into pET16b and expressed in Escherichia coli. The purified BinA protein differs by one amino acid (R197M) from BinA of the highest toxicity strains 1593/2362/C3-41. Majority of the expressed protein was observed in inclusion bodies. BinA inclusions alone from E. coli did not show toxic activity, like reported previously. However, the active form of BinA could be purified to homogeneity from the soluble fraction of E. coli cell lysate, grown at reduced temperature after isopropyl beta-d-thiogalactopyranoside induction. The purified BinA protein with and without poly-histidine tag showed LC(50) dose of 82.3 and 66.9ngml(-1), respectively, at 48h against Culex quinquefasciatus larvae. The secondary structure of BinA is expected to be mainly beta strands as estimated using far-UV circular dichroism. The estimates matched well with the secondary structure predictions using amino acid sequence. This is the first report of large-scale purification and accurate toxicity estimation of soluble B. sphaericus BinA. This can help in design and synthesis of improved bacterial insecticide.

Diversity of bacterial communities in the midgut of Bactrocera cucurbitae (Diptera: Tephritidae) populations and their potential use as attractants.

BACKGROUND: The microbiota plays an important role in insect development and fitness. Understanding the gut microbiota composition is essential for the development of pest management strategies. Midgut bacteria were isolated from nine wild B. cucurbitae populations collected from different agroecological zones of India. These isolates were further studied for attractant potential of fruit fly adults, and the chemical constituents in the supernatants of gut bacteria were analysed. RESULTS: Twenty-six bacterial isolates belonging to the families Enterobacteriaceae, Bacillaceae, Micrococcaceae and Staphylococcaceae were isolated and identified on the basis of 16S rRNA gene sequence analysis. The dominant species in the midgut of melon fly were from the genera Enterobacter (34.6%), Klebsiella (19.2%), Citrobacter (7.7%), Bacillus (15.4%) and Providencia (7.7%), and 3.8% each of Micrococcus, Staphylococcus, Leclercia and Exiguobacterium. Bactrocera cucurbitae and B. dorsalis adults were significantly attracted to bacterial whole cell cultures and their supernatants in the fruit fly attraction bioassays. Bacillus cereus, Enterobacter, Klebsiella, Citrobacter and Providencia species attracted both male and females of Bactrocera species. The supernatants of Klebsiella, Citrobacter and Providencia species attracted a significantly greater number of females than males. The most abundant chemical constituents in supernatants of K. oxytoca and C. freundii were 3-methyl-1-butanol, 2-phenylethanol, butyl isocyanatoacetate, 2-methyl-1-propanol and 3-hydroxy-2-butanone, as identified by gas chromatography-mass spectrometry. CONCLUSIONS: The bacterial endosymbionts associated with melon fly exhibited attractant potential which could facilitate eco-friendly insect control strategies. © 2015 Society of Chemical Industry.

Polymeric macroporous formulations for the control release of mosquitocidal Bacillus sphaericus ISPC-8.

Bio-polymeric mosquitocidal formulations were developed for the control release of Bacillus sphaericus ISPC-8 by the immobilization of its spore-crystal complex onto the macroporous polymeric matrices. The biodegradable formulations were synthesized at sub-zero temperature using natural polymeric substrates like agarose, alginate, cellulose, non-adsorbent cotton, wooden cork powder and also magnetite nanoparticles. The obtained polymeric matrices were morphologically characterized, which showed 85-90% porosity, uniform pores distribution, high permeability and controlled degradation (19-30%) in 4 weeks depending upon the composition of formulations. Further, the polymeric macroporous formulations were tested for persistence of mosquitocidal activity against Culex quinquefasciatus larvae. Unformulated B. sphaericus ISPC-8 spores retained 54% of larvicidal activity after 7 days, which completely reduced after 35 days of treatment. However, the immobilized B. sphaericus spores in agarose-alginate formulations showed high larvicidal activity on day 7 and retained about 45% activity even after 35 days of treatments. Studies on UV-B and pH dependent inactivation of toxins and spore viability showed that these formulations were significantly protecting the spores as compared to the unformulated spores, which suggest its potential application for the mosquito control program.

Interaction between mosquito-larvicidal Lysinibacillus sphaericus binary toxin components: analysis of complex formation.

The two components (BinA and BinB) of Lysinibacillus sphaericus binary toxin together are highly toxic to Culex and Anopheles mosquito larvae, and have been employed world-wide to control mosquito borne diseases. Upon binding to the membrane receptor an oligomeric form (BinA2.BinB2) of the binary toxin is expected to play role in pore formation. It is not clear if these two proteins interact in solution as well, in the absence of receptor. The interactions between active forms of BinA and BinB polypeptides were probed in solution using size-exclusion chromatography, pull-down assay, surface plasmon resonance, circular dichroism, and by chemically crosslinking BinA and BinB components. We demonstrate that the two proteins interact weakly with first association and dissociation rate constants of 4.5×10(3) M(-1) s(-1) and 0.8 s(-1), resulting in conformational change, most likely, in toxic BinA protein that could kinetically favor membrane translocation of the active oligomer. The weak interactions between the two toxin components could be stabilized by glutaraldehyde crosslinking. The cross-linked complex, interestingly, showed maximal Culex larvicidal activity (LC50 value of 1.59 ng mL(-1)) reported so far for combination of BinA/BinB components, and thus is an attractive option for development of new bio-pesticides for control of mosquito borne vector diseases.

Characterization of highly toxic indigenous strains of mosquitocidal organism Bacillus sphaericus.

Three indigenous isolates of Bacillus sphaericus (ISPC-5, ISPC-6 and ISPC-8), along with standard 2362 and 1593 strains, were evaluated for spore viability and mosquitocidal activity. Among these, ISPC-8 was the most viable and virulent isolate, exhibiting a significantly higher total viability count (TVC) and lower LC(50) values. The TVC of the standard strains ranged from 4.0 to 9.2 x 10(8) spores mL(-1), whereas it was 1.3 x 10(9) spores mL(-1) for ISPC-8. The LC(50) values of ISPC-8, 2362 and 1593 against Culex quinquefasciatus were 0.68 x 10(3), 1.22 x 10(3) and 1.85 x 10(3) spores mL(-1), respectively. The ISPC-8 was further assessed for host spectrum and found to be more active against C. quinquefasciatus, followed by Culex tritaeniorhynchus, Aedes albopictus and Aedes aegypti. The ISPC-8 strain was thus found to be a promising isolate for developing biopesticides. Among the indigenous strains, only ISPC-8 was found to have binary toxin genes (binA and binB). Comparative sequence analysis revealed that the BinA (41.9 kDa) protein of ISPC-8 differs by one amino acid (R197M), whereas BinB (51.4 kDa) differs by two amino acids (H99P, P174S) as compared with 1593 and 2362 strains. The purified binary proteins of ISPC-8 showed an LC(50) value of 6.32 ng mL(-1) against C. quinquefasciatus larvae after 48 h.