In Vivo Production, Development and Storage of Oscheius myriophila (Nematoda: Rhabditida) in Galleria mellonella (Lepidoptera: Pyralidae).

Entomopathogenic nematodes have been used in biological control for some time and are an alternative for the control of insect pests, but during their implementation, situations have arisen that can be improved. These vary with each species and include their production and storage. Oscheius myriophila, an entomopathogenic nematode (EPN), was monitored for its performance when produced in vivo, as well as its development using Galleria mellonella larvae, using the MC5-2014 strain isolated from soil samples in the municipality of Tepalcingo, Morelos, México. For a study with native strains of EPNs, a wide range of tests must be conducted because the required conditions can be very specific. In vivo production was quantified at initial infective juvenile (IJ) inocula of 50, 100 and 500, and we obtained the same production for the three inocula. The life cycle of the EPNs lasted 12 days, and two generations were observed in which adults were found at days 5 and 9. Both evaluations were performed at a temperature of 27 °C in G. mellonella larvae. In addition, the temperatures of 8, 12, 20 and 24 °C were evaluated for their storage, and we observed that the EPNs can be kept for at least 6 months, maintaining a survival rate of 58.67% and a good infective capacity at a temperature of 12 °C, remaining above 60%.

Identification and Venom Characterization of Two Scorpions from the State of Chihuahua Mexico: Chihuahuanus coahuliae and Chihuahuanus crassimannus.

Chihuahua is the largest state in Mexico. The ecosystem of this region is composed of large area of bushes, forests, and grasslands, which allows for a specific diversity of fauna; among them are interesting species of non-lethal scorpions. Most of the Chihuahuan scorpions have been previously morphologically and molecularly described; however, this manuscript could be the first to describe the composition of those venoms. This work aimed at the collection of two scorpion species from the region of Jiménez (Southwest of the State of Chihuahua), which belong to the species Chihuahuanus cohauilae and Chihuahuanus crassimanus; the two species were taxonomically and molecularly identified using a 16S DNA marker. Reverse-phase high-performance liquid chromatography (RP-HPLC) of C. coahuilae and C. crassimanus venoms allowed the identification of three fractions lethal to mice. Additionally, three fractions of each scorpion displayed an effect on house crickets. In the end, three new fractions from the venom of C. coahuilae were positive for antimicrobial activity, although none from C. crassimanus venom displayed growth inhibition. Despite being a preliminary study, the venom biochemical analysis of these two uncharacterized scorpion species opens the opportunity to find new molecules with potential applications in the biomedical and biotechnological fields.

Characterization of insecticidal Cry1Cb2 protein from Bacillus thuringiensis toxic to Myzus persicae (Sulzer).

The toxins produced by Bacillus thuringiensis (Bt) are well known for their insecticidal activity against Lepidoptera, Diptera and Coleoptera; however, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. We describe the aphicidal effect of Cry toxin from Bt strain GP919 against one of the most pernicious hemipterans in the agricultural environment, Myzus persicae. The mortality bioassay shows that the strain cause mortality rates above 80% at concentration of 10 ng/µl with a LC50 of 9.01 ng/µl; whereas it showed no lethal toxicity against the lepidopteran Spodoptera frugiperda. The mayor protein (∼130 kDa) expressed by this strain was subjected to purification, solubilization and trypsin digestion, the band of âˆ¼ 65 kDa which was obtained from trypsin digestion was purified by ion-exchange chromatography and was used to feed the aphid. The bioassay shows mortality rates above 85% at concentration of 10 ng/µl and the LC50 was 6.58 ng/µl. The resulting fragment from the digestion was identified by mass spectrometry and the candidate protein showed an overall 100% amino acid sequence identity to the reported Cry1Cb2 (WP 033698561.1) protein from Bt. Koch's postulated also was carried out with the GP919 strain and also, we document the signs of infection caused by this strain. This is the first report of a Cry1Cb2 protein that is toxic to a sucking insect and this protein may become a promising environmentally friendly tool for the control of M. persicae and possible also for other sap sucking insect pests.

Biophysical characterization of the insertion of two potent antimicrobial peptides-Pin2 and its variant Pin2[GVG] in biological model membranes.

The aim of this study was to investigate the factors that govern the activity and selectivity of two potent antimicrobial peptides (AMPs) using lipid membrane models of bacterial, erythrocyte and fungal cells. These models were used in calcein liposome leakage experiments to explore peptide efficiency. The AMPs (Pin2 and its variant Pin2[GVG]) showed highest affinity towards the bacterial models in the nanomolar range, followed by the erythrocyte and fungal systems. The presence of sterols modulated the variant's selectivity, while the wild type was unaffected. Liposome leakage experiments with Fluorescein Isothiocyanate-dextran (FITC)-dextran conjugates indicated that pore size depended on peptide concentration. Dynamic Light Scattering revealed peptide aggregation in aqueous solution, and that aggregate size was related to activity. The interacting peptides did not alter liposome size, suggesting pore forming activity rather than detergent activity. Atomic Force Microscopy showed differential membrane absorption, being greater in the bacterial model compared to the mammalian model, and pore-like defects were observed. Electrophysiological assays with the Tip-Dip Patch Clamp method provided evidence of changes in the electrical resistance of the membrane. Membrane potential experiments showed that liposomes were also depolarized in the presence of the peptides. Both peptides increased the Laurdan Generalized Polarization of the bacterial model indicating increased viscosity, on the contrary, no effect was observed with the erythrocyte and the fungal models. Peptide membrane insertion and pore formation was corroborated with Langmuir Pressure-Area isotherms and Brewster Angle Microscopy. Finally, molecular dynamics simulations were used to get an insight into the molecular mechanism of action.

Antibacterial activity and phospholipid recognition of the recombinant defensin J1-1 from Capsicum genus.

The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with isopropyl thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisXarJ1-1 product obtained from the affinity chromatography step showed single main peptide fraction of molecular masses of 7050.6 Da and after treatment with DTT a single fraction of 7, 042.6 Da corresponding to the reduced peptide was observed. An in vitro folding step of the HisXarJ1-1 generated a distinct profile of oxidized forms of the peptide this oxidized peptide was capable of binding phosphatidic acid in vitro. Possible dimer and oligomer of HisXarJ1-1 were visible in gel electrophoresis and immunodetected with anti-His antibodies. Pure recombinant defensin HisXarJ1-1 exhibited antibacterial activity against Pseudomonas aeruginosa.

Effects of Bacillus thuringiensis strains virulent to Varroa destructor on larvae and adults of Apis mellifera.

The sublethal effects of two strains of Bacillus thuringiensis, which were virulent in vitro to Varroa destructor, were measured on Apis mellifera. The effects of five concentrations of total protein (1, 5, 25, 50 and 100µg/mL) from the EA3 and EA26.1 strains on larval and adult honey bees were evaluated for two and seven days under laboratory conditions. Based on the concentrations evaluated, total protein from the two strains did not affect the development of larvae, the syrup consumption, locomotor activity or proboscis extension response of adults. These same parameters were also tested for the effects of three concentrations (1, 10 and 15µg/kg) of cypermethrin as a positive control. Although no significant differences were observed after two days of treatment with cypermethrin, a dose-response relationship in syrup consumption and locomotor activity was observed. A significant reduction in the proboscis extension response of the bees treated with cypermethrin was also observed. Therefore, in contrast to cypermethrin, our results indicate that the EA3 and EA26.1 strains of B. thuringiensis can be used in beehives to control V. destructor and reduce the negative effects of this mite on colonies without adverse effects on the larvae and adults of A. mellifera. Additionally, the overuse of synthetic miticides, which produce both lethal and sublethal effects on bees, can be reduced.

Anthelmintic Effect of Bacillus thuringiensis Strains against the Gill Fish Trematode Centrocestus formosanus.

Parasitic agents, such as helminths, are the most important biotic factors affecting aquaculture, and the fluke Centrocestus formosanus is considered to be highly pathogenic in various fish species. There have been efforts to control this parasite with chemical helminthicides, but these efforts have had unsuccessful results. We evaluated the anthelmintic effect of 37 strains of Bacillus thuringiensis against C. formosanus metacercariae in vitro using two concentrations of total protein, and only six strains produced high mortality. The virulence (CL50) on matacercariae of three strains was obtained: the GP308, GP526, and ME1 strains exhibited a LC50 of 146.2 µg/mL, 289.2 µg/mL, and 1721.9 µg/mL, respectively. Additionally, these six B. thuringiensis strains were evaluated against the cercariae of C. formosanus; the LC50 obtained from the GP526 strain with solubilized protein was 83.8 µg/mL, and it could be considered as an alternative control of the metacercariae and cercariae of this parasite in the productivity systems of ornamental fishes.