Comments on Ekino et al. Cloning and Characterization of a Unique Cytotoxic Protein Parasporin-5 Produced by Bacillus thuringiensis A1100 Strain. Toxins 2014, 6, 1882–1895

Ekino et al. [1] reported the cloning and characterization of a novel cytotoxic protein (Parasporin-5) produced by Bacillus thuringiensis strain A1100. The 33.8-kDa inactive precursor protein exhibited strong cytocidal activity upon proteinase K activation against several mammalian (cancer) cell lines, and showed slight homology with Cry and aerolysin-type -pore-forming toxins. Most research concerning parasporins has mainly been performed in order to demonstrate their use as potential therapeutic agents against cancer, but they are lacking additional research supporting the absence of activity against invertebrates; especially, taking into account that these toxins are not expected to evolve to kill cancer cells. Therefore, it is reasonable to think that they should have another (unknown) target in nature. Despite the fact that in this work, the authors demonstrated the toxic activity of this protein against several types of cancer cells, further complementary studies against a minimum number of insects would be of great interest in order to determine the potential insecticidal activity of this protein and understand its natural role. For example, Palma and collaborators [2] reported the molecular and insecticidal characterization of a novel Cry-related protein closely related to parasporins 2 and 4, (Cry41Aa1 and Cry41Ab1). This protein did not show any toxic activity against five species of Lepidoptera but, after more extensive testing, this protein was found to demonstrate a specific toxic activity against the green-peach aphid Myzus persicae. Nowadays, parasporin proteins are commonly known in the literature to be produced by “non-insecticidal” B. thuringiensis strains and because they exhibit significant and preferential cytocidal activity against cancer cells of various origins [3]. However, the absence of insecticidal activity deserves to be more deeply investigated since a single B. thuringiensis toxin has shown to have a narrow host range while, in general, they are active against a wide range of invertebrates [4,5]. Therefore, the determination of the activity against a minimum number of insects (preferably from different taxonomic orders) is highly desirable and might change the non-insecticidal concept we currently have about parasporins proteins produced by non-insecticidal B. thuringiensis strains.Fil: Palma, Leopoldo. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin

Implications of bio-efficacy and persistence of insecticides when indoor residual spraying and longlasting insecticide nets are combined for malaria prevention.

Bio-efficacy and residual activity of insecticides used for indoor residual spraying (IRS) and long-lasting insecticide nets (LLINs) were assessed against laboratory-reared and wild populations of the malaria vector, Anopheles arabiensis in south eastern Tanzania. Implications of the findings are examined in the context of potential synergies and redundancies where IRS and LLINs are combined. METHODS: Bioassays were conducted monthly for six months on three LLIN types (Olyset(R) PermaNet 2.0(R),and Icon Life(R)) and three IRS treatments (2 g/m2 pirimiphos-methyl, 2 g/m2 DDT and 0.03 g/m2 lambda-cyhalothrin, sprayed on mud walls and palm ceilings of experimental huts). Tests used susceptible laboratory-reared An. arabiensis exposed in cones (nets and IRS) or wire balls (nets only). Susceptibility of wild populations was assessed using WHO diagnostic concentrations and PCR for knock-down resistance (kdr) genes. IRS treatments killed [greater than or equal to] 85% of mosquitoes exposed on palm ceilings and [greater than or equal to] 90% of those exposed on mud walls, but up to 50% of this toxicity decayed within 1-3 months, except for DDT. By 6th month, only 7.5%, 42.5% and 30.0% of mosquitoes died when exposed to ceilings sprayed with pirimiphos-methyl, DDT or lambda-cyhalothrin respectively, while 12.5%, 36.0% and 27.5% died after exposure to mud walls sprayed with the same insecticides. In wire-ball assays, mortality decreased from 98.1% in 1st month to 92.6% in 6th month in tests on PermaNet 2.0(R), from 100% to 61.1% on Icon Life(R) and from 93.2% to 33.3% on Olyset(R) nets. In cone bioassays, mortality reduced from 92.8% in 1st month to 83.3% in 6th month on PermaNet 2.0(R), from 96.9% to 43.80% on Icon Life(R) and from 85.6% to 14.6% on Olyset(R). Wild An. arabiensis were 100% susceptible to DDT, 95.8% to deltamethrin, 90.2% to lambda cyhalothrin and 95.2% susceptible to permethrin. No kdr gene mutations were detected. CONCLUSIONS: In bioassays where sufficient contact with treated surfaces is assured, LLINs and IRS kill high proportions of susceptible An. arabiensis mosquitoes, though these efficacies decay gradually for LLINs and rapidly for IRS. It is, therefore, important to always add intact nets in sprayed houses, guaranteeing protection even after the IRS decays, and to ensure accurate timing, quality control and regular re-spraying in IRS programmes. By contrast, adding IRS in houses with intact LLINs is unlikely to improve protection relative to LLINs alone, since there is no guarantee that unfed vectors would rest long enough on the sprayed surfaces, and because of the rapid IRS decay. However, there is need to clarify these effects using data from observations of free flying mosquitoes in huts. Physiological susceptibility of An. arabiensis in the area remains 100% against DDT, but is slightly reduced against pyrethroids, necessitating caution over possible spread of resistance. The loss of LLIN toxicity, particularly Olyset(R) nets suggests that protection offered by these nets against An. arabiensis may be primarily due to physical bite prevention rather than insecticidal efficacy

Controlling sap-sucking insect pests with recombinant endophytes expressing plant lectin

We developed a novel pest management strategy, which uses endophytes to express anti-pest plant lectins. Fungal endophyte of Chaetomium globosum YY-11 with anti-fungi activities was isolated from rape seedlings, and bacterial endophytes of SJ-10 (Enterobacter sp.) and WB (Bacillus subtilis) were isolated from rice seedlings. Pinellia ternate agglutinin gene was cloned into SJ-10 and WB for expression by a shuttle vector, and YY-11 was mediated by Agrobacterium tumefaciens. Positive transformants were evaluated using PCR and Western blot assay. Recombinant endophytes colonized most of crops, and resistance of rice seedlings, which were inoculated with the recombinant endophytic bacteria, to white backed planthoppers was dramatically enhanced by decreasing the survival and fecundity of white backed planthoppers. Rape inoculated with recombinant endophytic fungi significantly inhibited the growth and reproduction of aphids. Recombinant endophytes expressing PTA may endow hosts with resistance against sap-sucking pests

Investigations on Tuta absoluta (Lepidoptera: Gelechiidae): larval infestation on the tomato cultivated in open field and evaluation of five essential oils against larvae in laboratory

Abstract The tomato miner Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) is a Neotropical species, which cause serious damages to tomato in spread areas. It appeared in 2008 in Italy, where it seriously threatened tomato production both in greenhouse and open field cultivations. In this work a study on infestation by this leafminer on leaves was conducted in an organic cultivation of tomato (Lycopersicon esculentum Mill.) "Pizzutello" variety in Sicily (Italy), and the insecticidal activity of 5 essential oils (EOs) on T. absoluta was evaluated in laboratory. EOs of basil, cypress, laurel, peppermint and Spanish oregano were used against 3rd- and 4th-instar larvae to assess contact effects and, only for oregano and laurel, inhalation effects. Results showed that the infestation in field had a variable trend showing peaks in two periods, in the months of July and October. Larvae of T. absoluta developed on leaves for most of the growing season of tomato showing a peak of 1.5 larva per leaf in the first part of July decreasing in the subsequent months. Results on the use of essential oils against the leafminer showed a good larvicidal effects confirming their use as potential alternative for pest control. Basil, cypress and laurel oils showed major contact efficacy. Inhalation effects of the oils of oregano and laurel increased with increasing their doses with a positive correlation. Oregano oil was effective at lower doses than laurel

A new family of diverse skin peptides from the microhylid frog genus phrynomantis

A wide range of frogs produce skin poisons composed of bioactive peptides for defence against pathogens, parasites and predators. While several frog families have been thoroughly screened for skin-secreted peptides, others, like the Microhylidae, have remained mostly unexplored. Previous studies of microhylids found no evidence of peptide secretion, suggesting that this defence adaptation was evolutionarily lost. We conducted transcriptome analyses of the skins of Phrynomantis bifasciatus and Phrynomantis microps, two African microhylid species long suspected to be poisonous. Our analyses reveal 17 evolutionary related transcripts that diversified from to those of cytolytic peptides found in other frog families. The 19 peptides predicted to be processed from these transcripts, named phrynomantins, show a striking structural diversity that is distinct from any previously identified frog skin peptide. Functional analyses of five phrynomantins confirm the loss of a cytolytic function and the absence of insecticidal or proinflammatory activity, suggesting that they represent an evolutionary transition to a new, yet unknown function. Our study shows that peptides have been retained in the defence poison of at least one microhylid lineage and encourages research on similarly understudied taxa to further elucidate the diversity and evolution of skin defence molecules

Cytotoxicity of the Bacillus thuringiensis Crystal Protein against Mammalian Cells

The crystal proteins produced by Bacillus thuringiensis subsp, israelensis (Bti) and subsp. coreanensis A1519 strain were examined for the cytotoxicity against MOLT-4 and HeLa cells by MTT assay and LDH assay, The A1519 crystal proteins processed by proteinase K exhibited the specific cell-killing activity toward MOLT-4 with little damage to the cell membrane, On the other hand, the Bti crystal proteins processed by proteinase K caused the substantial damage to the cell membrane of both MOLT-4 and HeLa, leading to the cell lysis. The non-digested crystal proteins of both strains exhibited no cytotoxicity, These data suggested that while the Bti crystal proteins caused the colloid-osmotic swelling and cell lysis of MOLT-4 and HeLa, the proteinase K-digested A1519 crystal proteins induced the specific cell death of MOLT-4 through a mechanism other than that of Bti

Isolation of multiple subspecies of Bacillus thuringiensis from a population of the European sunflower moth, Homoeosoma nebulella

Five subspecies of #Bacillus thuringiensis# were isolated from dead and diseased larvae obtained from a laboratory colony of the European sunflower moth, #Homoeosoma nebulella#. The subspecies isolated were #B. thuringiensis# subspp. #thuringiensis# (H la), #kurstaki# (H 3a3b3c), #aizawai# (H 7), #morrisoni# (H 8a8b), and #thompsoni# (H 12). Most isolates produced typical bipyramidal crystals, but the #B. thuringiensis#. subsp. #thuringiensis# isolate produced spherical crystals and the #B. thuringiensis# subsp. #thompsoni# isolate produced a pyramidal crystal. Analysis of the parasporal crystals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the crystals from the #B. thuringiensis# subsp. #kurstaki# and #aizawai# isolates contained a protein of 138 kDa whereas those from #B. thuringiensis# subsp. #morrisoni# contained a protein of 145 kDa. The crystals from #B. thuringiensis# subsp. #thuringiensis# contained proteins of 125, 128, and 138 kDa, whereas those from #B. thuringiensis# subsp. #thompsoni# were the most unusual, containing proteins of 37 and 42 kDa. Bioassays of purified crystals conducted against second-instar larvae of #H. nebulella# showed that the isolates of #B. thuringiensis# subspp.#aizawai#, #kurstaki#, and #thuringiensis# were the most toxic, with 50% lethal concentrations (LCS 50s) of 0.15, 0.17, and 0.26 ug/ml, respectively. The isolates of #B. thuringiensis# subspp. #morrisoni# and #thompsoni# had LC 50s of 2.62 and 37.5 ug/ml, respectively. These results show that a single insect species can simultaneously host and be affected by a variety of subspecies of #B. thuringiensis# producing different insecticidal proteins. (Résumé d'auteur

An experimental hut evaluation of PermaNet(®) 3.0, a deltamethrin-piperonyl butoxide combination net, against pyrethroid-resistant Anopheles gambiae and Culex quinquefasciatus mosquitoes in southern Benin.

PermaNet 3.0 is a long-lasting combination net with deltamethrin present on the sides and a mixture of deltamethrin and piperonyl butoxide (PBO), an oxidase synergist, on the top panel. An experimental hut trial comparing unwashed and 20 times washed PermaNet 3.0 and PermaNet 2.0, Olyset Net and a conventional deltamethrin-treated net washed three times was conducted in southern Benin. Anopheles gambiae and Culex quinquefasciatus from this area are highly resistant to pyrethroids through kdr and cytochrome P450 mechanisms. The unwashed PermaNet 3.0 killed slightly more A. gambiae (52%) than the unwashed PermaNet 2.0 (44%) (P=0.036), indicating only partial synergism of resistance. After washing there was significant loss of activity to a similar level, with PermaNet 3.0 killing 31%, PermaNet 2.0 killing 29% and the conventional net killing 26%. Blood-feeding rates were partially inhibited for unwashed PermaNet 3.0 and Olyset Net (27% inhibition). Personal protection against A. gambiae derived from PermaNet 3.0 was similar to that from PermaNet 2.0 before washing (50% vs. 47%), and after 20 washes it decreased to 30%. Against C. quinquefasciatus, no treatment killed >24% entering the huts. The synergism from unwashed PermaNet 3.0 was lower than expected, probably due to an unidentified resistance mechanism unaffected by PBO

Effects of insecticidal ketones present in mint plants on GABA A receptor from mammalian neurons

The genus Mentha, an important member of the Lamiaceae family, is represented by many species commonly known as mint. The insecticidal activity of Mentha oil and its main components has been tested and established against various insects/pests. Among these, the ketone monoterpenes that are most common in different Mentha species demonstrated insect toxicity, with pulegone being the most active, followed by carvone and menthone. Considering that the GABAA receptor (GABAA-R) is one of the main insecticide targets on neurons, and that pulegone would modulate the insect GABA system, it may be expected that the insecticidal properties of Mentha ketones are mediated by their interaction with this receptor.Fil: Sánchez, Mariela Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaFil: Delgado Marín, Leticia Ester. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaFil: Garcia, Daniel Asmed. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentin