Genetic transformation of mosquitoes by microparticle bombardment.

Mosquitoes constitute the major living beings causing human deaths in the world. They are vectors of malaria, yellow fever, dengue, zika, filariases, chikungunya, among other diseases. New strategies to control/eradicate mosquito populations are based on newly developed genetic manipulation techniques. However, genetic transformation of mosquitoes is a major technical bottleneck due to low efficiency, the need of sophisticated equipment, and highly trained personnel. The present report shows the transgenerational genetic transformation of Aedes aegypti, using the particle inflow gun (PIG), by integrating the ecfp gene in the AAEL000582 mosquito gene with the CRISPR-Cas9 technique, achieving a mean efficiency of 44.5% of bombarded individuals (G0) that showed ECFP expression in their tissues, and a mean of 28.5% transformation efficiency measured on G1 individuals. The same transformation technique was used to integrate the egfp/scorpine genes cloned in the Minos transposon pMinHygeGFP into the Anopheles albimanus genome, achieving a mean efficiency of 43.25% of bombarded individuals (G0) that showed EGFP expression in their tissues. Once the technique was standardized, transformation of Ae. aegypti neonate larvae and An. albimanus eggs was achieved when exposed to gold microparticle bombardment. Integration of genes and heterologous protein expression were confirmed by PCR, sequencing, fluorescent microscopy, mass spectrometry, Western blot and dot blot analyses. Transgenerational inheritance of the transgenes was observed only on Ae. aegypti, as all transformed An. albimanus individuals died at the pupal stage of the G0 generation.

Growth and formation mechanism of shape-selective preparation of ZnO structures: correlation of structural, vibrational and optical properties.

A shape-selective preparation method was used to obtain highly crystalline rod-, needle-, nut-, and doughnut-like ZnO morphologies with distinct particle sizes and surface areas. We study the nucleation and growth mechanism of those structures and the influence of physical-chemical parameters, such as the solvent and the pH of the solution, on the morphology, as well as the structural and optical properties. A clear correlation between the growth rate along the c-axis and surface defects was established. Our results suggest that the needle- and rod-like morphologies are formed due to the crystal growth orientation along the c-axis and the occurrence of crystalline defects, such as oxygen vacancies and interstitial Zn2+ located at the surface, whereas nuts and doughnuts are formed due to growth along all crystalline planes except those related to growth along the c-axis. Based on the experimental results, growth mechanisms for the formation of ZnO structures were proposed. We believe this synthetic route will be of guidance to prepare several materials whose shapes will depend on the desired applications.

Translocation of Bacillus thuringiensis in Phaseolus vulgaris tissues and vertical transmission in Arabidopsis thaliana.

AIMS: To demonstrate the ability of Bacillus thuringiensis to penetrate as spore-crystal complex to the internal tissues of bean plants, and keep its insecticidal activity. To test the vertical transmission of the spore-crystal complex in Arabidopsis thaliana. METHODS AND RESULTS: The experimental strain was transformed with the pMUTIN-gfp plasmid which labelled the spores of B. thuringiensis HD-73 with the GFP protein. Once the rhizosphere of the bean plants was inoculated with the labelled strain, the bacterium was recovered from leaves, stems, and petioles. Furthermore, toxicity of treated plants was significantly higher than control plants when bio-assayed on cabbage looper larvae. The labelled strain was recovered from the dead insects. When the rhizosphere of A. thaliana plants was inoculated with the labelled strain, mature seeds from these plants were surface-sterilized and grown under in vitro conditions. The labelled strain was recovered from the seedlings. CONCLUSIONS: We showed that B. thuringiensis subsp. kurstaki (HD-73) in the rhizosphere can translocate to upper tissues of bean plants, and keep its insecticidal activity. Transmission of the labelled B. thuringiensis strain passed to the next generation of A. thaliana. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of B. thuringiensis as a potential facultative endophyte bacterium and the possible biotechnological repercussions are discussed.

Detection of ß-exotoxin synthesis in Bacillus thuringiensis using an easy bioassay with the nematode Caenorhabditis elegans.

UNLABELLED: The insecticidal activity of Bacillus thuringiensis is owing to the action of Cry and Cyt proteins. In addition to the synthesis of insecticidal proteins, some strains are able to synthesize ß-exotoxin, which is highly toxic to humans. In this regard, it is very important to have a simple method to detect ß-exotoxin to avoid the commercial production of this type of strains. In this work, we developed a simple and fast method, using the nematode Caenorhabditis elegans to detect indirectly the synthesis of ß-exotoxin by B. thuringiensis strain. Using this assay, we detected that ~60% of Mexican native strains (i.e. LBIT-471, 491, 492, 497, 507, 511, 515, 536 and 537) were toxic to the nematode (44-97% mortalities) and their ß-exotoxin (ßEx(+) ) production, including a positive control (NRD-12), was confirmed by HPLC. In addition, the negative controls (ßEx(-) ) LBIT-436 (HD-1) and LBIT-438 and also the native strains LBIT-499, 500, 521, 522, 533 and 542, did not show a detrimental effect against nematodes larvae, neither the synthesis of ß-exotoxin as determined by HPLC. Finally, we did not find a correlation between B. thuringiensis strains with similar plasmid patterns and the ß-exotoxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, we implemented a qualitative and fast bioassay using the nematode Caenorhabditis elegans to detect the production of ß-exotoxin in different strains of Bacillus thuringiensis. We show that this assay is useful to detect ß-exotoxin in B. thuringiensis with high reliability, helping to discriminate strains that could not be used as bioinsecticides because of their putative risk to humans. Data show that qualitative bioassay with nematodes is a potential alternative to fly larvae bioassays, and correlated with the determination of ß-exotoxin by HPLC.

Significantly enhancement of sunlight photocatalytic performance of ZnO by doping with transition metal oxides.

In this study we report, the synthesis of ZnO and its doping with Transition Metal Oxides -TMO-, such as Cr2O3, MnO2, FeO, CoO, NiO, Cu2O and CuO. Various characterization techniques were employed to investigate the structural properties. The X-ray diffraction (XRD) data and Rietveld refinement confirmed the presence of TMO phases and that the ZnO structure was not affected by the doping with TMO which was corroborated using transmission Electron microscopy (TEM). Surface areas were low due to blockage of adsorption sites by particle aggregation. TMO doping concentration in the range of 3.7-5.1% was important to calculate the catalytic activity. The UV-Visible spectra showed the variation in the band gap of TMO/ZnO ranging from 3.45 to 2.46 eV. The surface catalyzed decomposition of H2O2 was used as the model reaction to examine the photocatalytic activity following the oxygen production and the systems were compared to bulk ZnO and commercial TiO2-degussa (Aeroxyde-P25). The results indicate that the introduction of TMO species increase significantly the photocatalytic activity. The sunlight photocatalytic performance in ZnO-doped was greater than bulk-ZnO and in the case of MnO2, CoO, Cu2O and CuO surpasses TiO2 (P25-Degussa). This report opens up a new pathway to the design of high-performance materials used in photocatalytic degradation under visible light irradiation.

Detrimental Effects of Induced Antibodies on Aedes aegypti Reproduction.

Aedes aegypti (Linnaeus) (Diptera: Culicidae) is the main vector of viruses causing dengue, chikungunya, Zika, and yellow fever, worldwide. This report focuses on immuno-blocking four critical proteins in the female mosquito when fed on blood containing antibodies against ferritin, transferrin, one amino acid transporter (NAAT1), and acetylcholinesterase (AchE). Peptides from these proteins were selected, synthetized, conjugated to carrier proteins, and used as antigens to immunize New Zealand rabbits. After rabbits were immunized, a minimum of 20 female mosquitos were fed on each rabbit, per replicate. No effect in their viability was observed after blood-feeding; however, the number of infertile females was 20% higher than the control when fed on AchE-immunized rabbits. The oviposition period was significantly longer in females fed on immunized rabbits than those fed on control (non-immunized) rabbits. Fecundity (eggs/female) of treated mosquitoes was significantly reduced (about 50%) in all four treatments, as compared with the control. Fertility (hatched larvae) was also significantly reduced in all four treatments, as compared with the control, being the effect on AchE and transferrin the highest, by reducing hatching between 70 and 80%. Survival to the adult stage of the hatched larvae showed no significant effect, as more than 95% survival was observed in all treatments, including the control. In conclusion, immuno-blocking of these four proteins caused detrimental effects on the mosquito reproduction, being the effect on AchE the most significant.

Screening of cry gene contents of Bacillus thuringiensis strains isolated from avocado orchards in Mexico, and their insecticidal activity towards Argyrotaenia sp. (Lepidoptera: Tortricidae) larvae.

AIMS: To screen for Bacillus thuringiensis strains from avocado orchards in two Mexican states with lepidopteran-specific cry gene content and evaluate their insecticidal activity against Argyrotaenia sp., an undescribed species present in avocado orchards. METHODS AND RESULTS: Lepidopteran-active cry1, cry2 and cry9 genes were detected by PCR analysis in 37 isolates. cry1 genes were more frequent in Michoacán, but were undetected in Nayarit isolates. cry9 and cry2 genes were detected in isolates from both states, although cry2 genes were less frequent. A variety of crystal shapes were observed among the isolates. According to gene profile, eight isolates were selected and tested against 2-day old Argyrotaenia sp. larvae. Standard strain HD-125 caused the highest mortality followed by strain MR-26 from Michoacán at a concentration of 500 microg ml(-1), respectively. CONCLUSIONS: Bacillus thuringiensis strains isolated from avocado orchards exhibit a low toxic activity towards Argyrotaenia sp. larvae, in spite of their specific cry gene content. SIGNIFICANCE AND IMPACT OF THE STUDY: Toxic activity of B. thuringiensis is not necessarily related to insect pest habitat and neither to specific cry gene content associated to other lepidopterans.

An alternative bioassay employing neonate larvae for determining the toxicity of suspended particles to mosquitoes.

An alternative bioassay using neonate larvae of Aedes aegypti is described for determining the toxicity of suspended particles, particularly insecticidal bacteria, to mosquitoes. This new assay is comparable in precision to the 4th instar bioassay recommended by the World Health Organization, but is quicker and requires much less space. Values obtained using neonate larvae as the test insect showed less variation within and between replicates in regard to LC50 and slope of the regression line, and in general required fewer replicates to fulfill the requirements for a statistically valid bioassay than the W.H.O. assay. The precision of the neonate larval bioassay in combination with its requirements for less time and space may make it especially useful for screening large numbers of samples.

Synergism between the nucleopolyhedroviruses of Autographa californica and Trichoplusia ni.

Previous observations on high virulence of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) acting together led us to test possible synergism between these two nucleopolyhedroviruses (NPVs) on cabbage looper larvae. Because synergism between AcMNPV and the Trichoplusia ni granulovirus (TnGV) has been well established before, these two viruses were included in this study as a positive control. Each virus was assayed separately on first-instar cabbage looper and their LC50s were estimated at 2.33, 0.39 and 462 OB/mm2 diet for AcMNPV, TnSNPV and TnGV, respectively. LC50s of AcMNPV mixed with sub-lethal concentrations of TnSNPV and TnGV increased 8 and 10.7 times, respectively. Synergism between the viruses was analyzed by the ANOVA test for the LC50s, the Plackett and Hewlett's joint-action rate test, and the Tammes-Bakuniak graphic method. All three analyses corroborated the synergism between the viruses. The presence of a putative enhancin in the TnSNPV was analyzed by Southern blot hybridization, using a 1.5 kbp KpnI fragment from the TnGV vef gene as a probe. No hybridization was observed. The occurrence of a new putative synergistic factor in TnSNPV is discussed.

Isolation of a relatively nontoxic 65-kilodalton protein inclusion from the parasporal body of Bacillus thuringiensis subsp. israelensis.

Ultrastructural studies of the mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis revealed that the parasporal body contained three major inclusion types, designated types 1, 2, and 3, which could be differentiated on the basis of electron opacity and size and, to some extent, shape. The type-2 inclusion, which was of moderate electron density and often appeared as a bar-shaped polyhedral body, was isolated on NaBr gradients from purified parasporal bodies and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transmission electron microscopy, and bioassays against neonate larvae of Aedes aegypti. Purified inclusions averaged 150 to 200 nm by 500 to 700 nm in transverse sections and consisted almost exclusively of a 65-kilodalton (kDa) protein contaminated with minor quantities of 38- and 28-kDa proteins. Lethal concentration values at the 50% level for preparations of the purified parasporal body and the type-2 inclusion were, respectively, 0.66 and 43 ng/ml, indicating that the 65-kDa protein is only slightly toxic to mosquitoes in comparison to the intact parasporal body. Analysis of the type-2 polyhedral inclusion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioassays during different stages of purification demonstrated a positive correlation between the toxicity of the preparation and the degree of contamination with the 28-kDa protein. These results indicate that the 65-kDa protein is not the primary larvicidal toxin, although it may act in conjunction with other parasporal body proteins to produce the high mosquitocidal toxicity characteristic of this bacterium.

Site-directed mutagenesis of Autographa californica nucleopolyhedrovirus (AcNPV) polyhedrin: effect on polyhedron structure.

Amino acids Lys34, His36, and Phe37 were substituted by PCR-mediated, site-directed mutagenesis for three Trp's in the AcNPV polyhedrin sequence. Phase contrast microscopy revealed refringent, amorphous polyhedra in the nuclei of infected cells. Electron microscopy confirmed a great variation in form and size of the mutated polyhedra. Although crystallization of the mutated polyhedrin occurred, it was irregular within each polyhedron. Virion occlusion was also severely affected. Virions were partially occluded, or only one virion was occluded per polyhedron. Results suggest that the substitution of these three amino acids affected the morphology of polyhedra, the uniformity of crystallization within each polyhedron, and the virion occlusion.

Characterization of a Novel Strain of Bacillus thuringiensis.

Bacillus thuringiensis is a well-known species of entomopathogenic bacteria that is widely used as a biopesticide against many insect pests. Insecticidal proteins, coded for by genes located in plasmids, form typical parasporal, crystalline inclusions during sporulation. In this report, an unusual strain of B. thuringiensis subserovar oyamensis (LBIT-113), isolated from living larvae of Anopheles pseudopunctipennis in Mexico, was characterized by its ultrastructure, the protein composition of its parasporal crystal, plasmid pattern, and toxicological properties against several insect and noninsect targets. The parasporal crystal is enclosed within the spore's outermost envelope (exosporium), as determined by transmission electron microscopy, and exhibits a square, flat shape. Its main components are two proteins with sizes of 88 and 54 kDa. Despite some crystal morphology resemblance, both proteins are immunologically unrelated to the Cry IIIA protein, as shown by immunoblot analysis, when probed with antisera raised against the 88-kDa protein and the Cry IIIA protein. Partial N-terminal sequence of the 88-kDa protein revealed a unique amino acid arrangement among the Cry proteins. Solubilization of the crystal proteins was achieved at 3.3 M NaBr, and its digestion with trypsin showed only one ca. 60-kDa peptide, as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The patterns of three plasmids of strain LBIT-113 were considerably different from those of B. thuringiensis subspp. kurstaki, tenebrionis, and israelensis. Parasporal crystals showed no toxicity to larvae of four species of caterpillar, three species of mosquito, two species of beetle, one species of cricket, one species of ant, one species of aphid, one species of nematode, one species of ostracod, one species of ameba, and one species of rotifer.

Sublethal iridovirus disease of the mosquito Aedes aegypti is due to viral replication not cytotoxicity.

Invertebrate iridescent viruses (Iridoviridae) possess a highly cytotoxic protein. In mosquitoes (Diptera: Culicidae), invertebrate iridescent virus 6 (IIV-6) usually causes covert (inapparent) infection that reduces fitness. To determine whether sublethal effects of IIV-6 are principally due to cytotoxicity of the viral inoculum (which inhibits macromolecular synthesis in the host), or caused by replication of the virus larvae of the mosquito Aedes aegypti (L) were exposed to untreated IIV-6 virus that had previously been deactivated by heat or ultraviolet light. Control larvae were not exposed to virus. Larval development time was shortest in control larvae and extended in larvae exposed to untreated virus. Covertly infected mosquitoes laid significantly fewer eggs, produced between 20 and 35% fewer progeny and had reduced longevity compared to other treatments. Wing length was shortest in mosquitoes exposed to heat-deactivated virus. Multivariate analysis of the same data identified fecundity and progeny production as the most influential variables in defining differences among treatments. Overall, viral infection resulted in a 34% decrease in the net reproductive rate (R0) of covertly infected mosquitoes, vs. only 5-17% decrease of R0 following treatments with deactivated virus, compared to controls. Sublethal effects of IIV-6 in Ae. aegypti appear to be mainly due to virus replication, rather than cytotoxic effects of the viral inoculum.

Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. morrisoni isolate PG-14 toxins.

The parasporal bodies of the mosquitocidal isolates of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 were compared with regard to their hemolytic and cytolytic activities and the immunological relatedness of the 28- and 65-kilodalton (kDa) proteins that occur in both subspecies. The alkali-solubilized parasporal bodies of B. thuringiensis subsp. israelensis caused 50% lysis of human erythrocytes at 1.14 micrograms/ml, whereas those of B. thuringiensis subsp. morrisoni caused similar lysis at 1.84 micrograms/ml. Preincubation of solubilized parasporal bodies with dioleolyl phosphatidylcholine significantly inhibited the hemolytic activity of both supspecies. In cytolytic assays against Aedes albopictus cells, the toxin concentrations causing 50% lysis for B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni were 1.87 and 11.98 micrograms/ml, respectively. Polyclonal antibodies raised separately against the 25-kDa protein (a tryptic digest of the 28-kDa protein) or the 65-kDa protein of B. thuringiensis subsp. israelensis cross-reacted, respectively, with the 28- and the 65-kDa proteins of B. thuringiensis subsp. morrisoni. However, neither of these antibodies cross-reacted with the 135-kDa protein of either subspecies. These results indicate that the mosquitocidal and hemolytic properties of B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 are probably due to the biologically related proteins that are present in the parasporal bodies of both subspecies. The lower hemolytic activity of the B. thuringiensis subsp. morrisoni may be due to the presence of lower levels of the 28-kDa protein in that subspecies.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of INTA 51-3, a new atypical strain of Bacillus thuringiensis from Argentina.

Several isolates of Bacillus thuringiensis native to Argentina obtained in a nationwide screening program showed atypical crystal morphology. One of these strains, INTA 51-3, was further characterized in order to determine other features like protein composition of its parasporal crystal, plasmid pattern, identification of cry genes and toxicological properties. B. thuringiensis INTA 51-3 (serovar tohokuensis) had an amorphous inclusion containing a major protein component of ca. 130 kDa. After trypsin digestion of solubilized crystals, SDS-PAGE resolved a unique protease-resistant peptide of ca. 90 kDa. The plasmid pattern from INTA 51-3 resembled that of the standard strain HD-1. However, Southern analysis showed no hybridization to fragments of cry1Aa, cry2Aa, cry3A, and cry11A genes. Degenerate primers were used for identification of the cry1 genes by PCR. Nevertheless, the presence of cry1 type gene(s) in B. thuringiensis INTA 51-3 was confirmed. Highly concentrated crystal suspensions showed to be weakly toxic only to lepidopteran species.