Crude Extracts of Pleurotus Spp. and the Presence of Their Proteins in the Ovicidal and Larvicidal Activity of Haemonchus contortus.

Mushrooms of the genus Pleurotus have shown nematophagous activity as it produces many chemical compounds and enzymes affecting parasitic nematodes. This study aimed to extract the inhibitory activity of the five strains of the fungus Pleurotus spp. It was evaluated against eggs and larvae of Haemonchus contortus. The extract of P. ostreatus obtained the highest level of inhibition of eggs at 97.6% (1341 µg/mL) followed by P. pulmonarius (EPP) at 81.2% (774 µg/mL). The extract selected for evaluation against larvae was P. pulmonarius, showing no effect for L3 larvae, but for L4 larvae an immobility effect of 56.93% was observed at 900 µg/mL. The protein profile showed the presence of 23 protein bands in the extract. The crude extract of P. pulmonarius showed degradation of tissues both inside the eggs and larvae L1. Metabolites produced by Pleurotus mushrooms can consider using in agriculture sustainable by utilizing in producing of ovicidal and larvicidal against H. contortus instead of chemical compounds.

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%.

Potential control of the infective stage of Taenia pisiformis using Bacillus thuringiensis GP526 strain.

The GP526 strain of Bacillus thuringiensis has been referred as an in vitro helminthicide on various stages of Dipylidium caninum and Centrocestus formosanus. Our study addresses the in vitro ovicidal activity of GP526 strain spore-crystal complex on Taenia pisiformis eggs, evaluating induced damage microscopically. The eggs exposed to the total extract containing spores and crystals show damage after 24 hours, with loss of integrity on the eggshell, and an ovicidal activity of 33% at 1mg/ml. The destruction of the embryophore was observed after 120 h with a 72% of ovicidal activity at 1 mg/ml. The LC50 was 609.6 µg/ml, dose that causes a 50% of lethality on the hexacanth embryo, altering the oncosphere membrane. The spore-crystal proteins were extracted, and the protein profile was obtained by electrophoresis, finding a major band of 100 kDa suggestive of an S-layer protein, since an S-layer was immunodetected in both, spores and extracted proteins. The protein fraction containing the S-layer protein presents adhesion to the T. pisiformis eggs, and 0.4 mg/ml of the protein induces a lethality of 21.08% at 24 h. The characterization of molecular mechanisms of ovicidal activity will be an important contribution, so the characterization of the proteins that make up the extract of the GP526 strain, would be useful to support the biological potential for control of this cestodiasis and other parasitosis. B. thuringiensis is shown as a potent helminthicide on eggs, with useful potential for biological control of this cestodiasis.

Oxalis tetraphylla (Class: Magnoliopsidae) Possess Flavonoid Phytoconstituents with Nematocidal Activity against Haemonchus contortus.

The nematocidal activity of an Oxalis tetraphylla hydroalcoholic extract against the nematode Haemonchus contortus (Hc) was assessed in vitro and the major compounds associated with nematocidal activity were identified. One hydroalcoholic extract was obtained from O. tetraphylla stems and leaves (Ot HE-SLE). The in vitro lethal concentrations (LC50 and LC90) against both eggs and exsheathed Hc infective larvae (L3) were assessed. Ot HE-SLE showed a potent ovicidal activity (LC50 = 0.213 mg/mL; LC90 = 0.71 mg/mL) and larvicidal effect (LC50 = 28.01 mg/mL; LC90 = 69.3 mg/mL). Later on, the extract was bipartitioned to obtain an ethyl acetate phase (EtOAc-Ph) and an aqueous phase (Aq-Ph). Both phases were assessed against Hc eggs at 0.25 and 1.0 mg/mL concentrations. The results with EtOAc-Ph showed 93.6% ovicidal activity, while 96.6% was recorded with Aq-Ph at 48 h post-confrontation (PC). In the case of larvicidal activity, both phases were assessed at 28 mg/mL; Aq-Ph showed >80% larvicidal activity 24 and 72 h PC, while EtOAc-Ph did not show important activity. HPLC analysis showed the presence of coumaric acid and flavonols. Flavonol compounds were the major compounds and were associated with the nematocidal activity. Additionally, the Aq-Ph that showed the highest activity was purified, and the fraction F3 showed the highest nematocidal activity.

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.

In Vitro Insecticidal Effect of Commercial Fatty Acids, ß-Sitosterol, and Rutin against the Sugarcane Aphid, Melanaphis sacchari Zehntner (Hemiptera: Aphididae).

ABSTRACT: The sugarcane aphid, Melanaphis sacchari Zehntner (Hemiptera: Aphididae), is the main pest of sorghum, Sorghum bicolor L. Moench (Poaceae), in Mexico. To control this insect, farmers currently use synthetic chemical insecticides, which are toxic to humans and biodiversity. However, natural products are a promising potential source of safer alternative means to control different agricultural pests. The main objective of this study was to evaluate the insecticidal effect of contact by fumigation of pure molecules of four commercial fatty acids (palmitic, stearic, pentadecanoic, and linoleic acids), the phytosterol ß-sitosterol, and the flavonoid rutin. The results showed that fatty acids were the most effective against M. sacchari; the highest mortality rate (85%) was produced by linoleic acid and the mean lethal concentration was 1,181 ppm, followed by stearic and palmitic acids with 74 and 63% mortality, respectively, at a concentration of 2,500 ppm at 72 h. The positive control, imidacloprid, had 100% mortality in 24 h and the Tween 20, negative control, exhibited 4% mortality in 72 h. Our results show that commercial fatty acids are effective against adults of M. sacchari and can be considered an environmentally friendly alternative to the frequent use of synthetic chemical insecticides.

Nematicidal and ovicidal activity of Bacillus thuringiensis against the zoonotic nematode Ancylostoma caninum.

Ancylostoma caninum is a gastrointestinal parasite that affect dogs and humans, considered a worldwide public health problem. The control of these parasitosis is increasingly difficult due to the development of multi-drug resistance. Bacillus thuringiensis is an insecticidal, spore forming bacterium, often species specific. The strain GP526 of B. thuringiensis has toxic effect on the cestode Dipylidium caninum and the trematode Centrocestus formosanus, both of them zoonotic parasites. The high degrees of specificity and environment safe make B. thuringiensis suitable for use against pathogen parasites, especially those resistant to synthetic chemical insecticides. The objective of the current work was to evaluate the in vitro effect of B. thuringiensis on Ancylostoma caninum. Spore-crystal mixture of the strains was co-incubated with 120 adult nematodes (males, non-pregnant females and pregnant females) or with 4800 eggs. GP526 showed a nematicidal effect with an LT50 of 35.8 h and an LC50 of 60 µg/ml. It also showed an ovicidal effect with an LC50 of 94.9 µg/ml. Histological analyses showed detachment of the cuticle and of the uterus in adult females, and vacuolization with destruction of the eggs. The effects of GP526 strain were comparable to those of albendazole, allowing us to propose GP526 for A. caninum control, in both, the adult stage at a gastrointestinal level, and in egg stage in the environment. In addition, GP526 can be proposed as a potential broad spectrum antiparasitic drug.

Oscheius myriophila (Nematoda: Rhabditida) isolated in sugar cane soils in Mexico with potential to be used as entomopathogenic nematode.

A survey of entomopathogenic nematodes was conducted in sugar cane crops in a total of 14 soils, and positive results were obtained for strain MC5-2014 in the municipality of Tepalcingo, Morelos, in soil with a sandy loam texture and a pH of 6.4. Species determination was performed via morphological and morphometric techniques by searching for a tubular stoma with a swollen cylindrical pharyngeal body and a metacorpus in the basal part. The range of body length (L) was 750 to 1200 µm in females and 720 to 910 µm in males, while the corresponding maximum widths (W) of the body were 30 to 60 µm and 20 to 30 µm, respectively. Males exhibited bursa with a 1 + 1 + 3 + 3 distribution of papillae, and females exhibited a vulva located at the mid-body. For molecular identification, the ITS region of ribosomal DNA was used. Virulence tests (LC50) were conducted with Galleria mellonella, and a value of 4.732 was obtained for infective juveniles (IJs). Taking taxonomic and molecular characteristics into account, the isolate was determined to be Oscheius myriophila. The isolation of this strain represents the first geographic report of O. myriophila in Mexico, and it should be noted that the cultivation of sugar cane occurs with constant application of insecticides, herbicides, fungicides, and fertilizers as well as harvesting activities such as burning of the crop for harvest. The O. myriophila isolate has the potential to be used in the future as a method of biological control in our country.A survey of entomopathogenic nematodes was conducted in sugar cane crops in a total of 14 soils, and positive results were obtained for strain MC5-2014 in the municipality of Tepalcingo, Morelos, in soil with a sandy loam texture and a pH of 6.4. Species determination was performed via morphological and morphometric techniques by searching for a tubular stoma with a swollen cylindrical pharyngeal body and a metacorpus in the basal part. The range of body length (L) was 750 to 1200 µm in females and 720 to 910 µm in males, while the corresponding maximum widths (W) of the body were 30 to 60 µm and 20 to 30 µm, respectively. Males exhibited bursa with a 1 + 1 + 3 + 3 distribution of papillae, and females exhibited a vulva located at the mid-body. For molecular identification, the ITS region of ribosomal DNA was used. Virulence tests (LC50) were conducted with Galleria mellonella, and a value of 4.732 was obtained for infective juveniles (IJs). Taking taxonomic and molecular characteristics into account, the isolate was determined to be Oscheius myriophila. The isolation of this strain represents the first geographic report of O. myriophila in Mexico, and it should be noted that the cultivation of sugar cane occurs with constant application of insecticides, herbicides, fungicides, and fertilizers as well as harvesting activities such as burning of the crop for harvest. The O. myriophila isolate has the potential to be used in the future as a method of biological control in our country.

Mass production of a S-layer protein of Bacillus thuringiensis and its toxicity to the cattle tick Rhipicephalus microplus.

The most commonly used biopesticides to control agricultural, forest and insect vectors of human diseases are derived from the bacterium Bacillus thuringiensis, which begins to produce Cry and Cyt insecticidal proteins during the onset of the sporulation phase. Some B. thuringiensis strains also produce S-layer proteins that are toxic to certain pests. S-layer proteins are the most abundant proteins in bacteria and archaea. This proteins' key trait to design high performace processes for mass production is their continuous expression during the vegetative phase, unlike Cry and Cyt, which are restricted to the sporulation phase. In this work, a S-layer protein expressed by the GP543 strain of B. thuringiensis that is toxic to the cattle tick Rhipicephalus microplus was mass produced using the batch culture fermentation technique. In addition, the spore-protein complex showed a mortality rate of 75% with a dose of 300 µg·mL-1 on adult females of R. microplus after fourteen days. The lethal concentration 50 was 69.7 µg·mL-1. The treatment also caused a decrease of 13% in the weight of the mass of oviposited eggs with 200 µg·mL-1 of the spore-protein complex and inhibition of the hatching of eggs from 80 to 92%. Therefore, this could be a good option for controlling this parasite. The advantages of S-layer protein synthesis are focused on the production of a new generation of proteins in pest control. This is the first report on the mass production of an S-layer protein that is responsible for toxicity.

Enhanced Tolerance against a Fungal Pathogen and Insect Resistance in Transgenic Tobacco Plants Overexpressing an Endochitinase Gene from Serratia marcescens.

In this study we cloned a chitinase gene (SmchiC), from Serratia marcescens isolated from the corpse of a Diatraea magnifactella lepidopteran, which is an important sugarcane pest. The chitinase gene SmchiC amplified from the S. marcescens genome was cloned into the transformation vector p2X35SChiC and used to transform tobacco (Nicotiana tabacum L. cv Petit Havana SR1). The resistance of these transgenic plants to the necrotrophic fungus Botrytis cinerea and to the pest Spodoptera frugiperda was evaluated: both the activity of chitinase as well as the resistance against B. cinerea and S. frugiperda was significantly higher in transgenic plants compared to the wild-type.

Effects on Meccus pallidipennis (Hemiptera: Reduviidae) Eggs Exposed to Entomopathogenic Fungi: Exploring Alternatives to Control Chagas Disease.

Meccus pallidipennis Stål is a vector for Chagas disease. The extensive use of pyrethroid insecticides to control triatomines in Mexico has resulted in the development of resistant populations. As an alternative control approach, the effects on M. pallidipennis eggs of two entomopathogenic fungi, Isaria fumosorosea Wize (Hypocreales: Cordycipitaceae) EH-511/3 and Metarhizium anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) EH-473/4, were examined. Egg mortality was estimated 1 mo after egg infection, based on hyphal growth and unsuccessful hatching as proxies for infection and death. Sporulation and conidial production rates were also recorded. Mortality rates caused by I. fumosorosea and Me. anisopliae were 92% ± 3.1 and 88% ± 3.7, respectively. Sporulation rate and conidial production were greater in I. fumosorosea than in Me. anisopliae. Transmission electron microscopy revealed hyphal penetration by both fungal species and damage to embryonic epidermal and cuticular cells. Our results demonstrated that I. fumosorosea and Me. anisopliae are promising candidates for controlling M. pallidipennis eggs and offer alternatives to control the transmission of Chagas disease under natural conditions.

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.

Therapeutic use of Bacillus thuringiensis in the treatment of psoroptic mange in naturally infested New Zealand rabbits.

Bacillus thuringiensis is a bacteria known for its bioinsecticidal toxins and it has been proposed as an alternative in the treatment of several parasites that infect domestic animals (helminths, ticks, mites). In this work, we evaluated the clinical efficiency of the Bacillus thuringiensis GP532 strain in the treatment of six rabbits naturally infested with the P. cuniculi mite. GP532 extract (10mg/ml) was applied by aspersion in both pinna, with a second application after seven days, and the therapeutic effect was measured in both qualitative and quantitative manner. GP532 application resulted in a decreased infestation rate, which was observed as early as 3days post-treatment. At day 14, a decrease from 4.66±0.61 to 0.50±0.10 in the left pinna and from 1.66±0.21 to 0.66±0.16 (P<0.05) in the right pinna was observed. This response was comparable to the commercial drug Ivermectin, which induced a decreased infestation rate from 4.00±0.51 to 0.16±0.10 in the left pinna and from 4.66±0.80 to 0.25±0.11 in the right pinna (P<0.05). At day 30 post-treatment, GP532 decreased the total infested area by 76.80±16.06%, whereas Ivermectin resulted in a 97.41±0.99% decrease. Neither treatment produced irritation or macroscopic lesions. Our results show that the B. thuringiensis GP532 strain has a therapeutic potential in the treatment of psoroptic mange in rabbits.

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.

Acaricidal effect and histological damage induced by Bacillus thuringiensis protein extracts on the mite Psoroptes cuniculi.

BACKGROUND: The mite Psoroptes cuniculi is a common worldwide ectoparasite and the most frequently found in rabbit farms. It causes significant economic losses on commercial rabbit breeding associated with poor leather quality, reduced conception rates, weight loss, poor growth and death. Several strategies have been proposed for the treatment of mange caused by this mite, ranging from the use of acaricides, entomopathogenic fungi, essential oils and vaccines. However, therapy and control of both human scabies and animal mange are still based mainly on the use of drugs and chemicals such as ivermectin, which involves disadvantages including genotoxic and cytotoxic effects, resistance and environmental damage. Bacillus thuringiensis is a bacterium, innocuous for human being, domestic animals and plants that produces highly biodegradable proteins, and has been used worldwide for biological control. The aim of this work was to find an alternative treatment based on biological control for scabies caused by Psoroptes cuniculi, using protein extracts from strains of Bacillus thuringiensis. METHODS: P. cuniculi mites were obtained from naturally infected New Zealand rabbits, and different doses of protein from B. thuringiensis were added to the mites. We measured mortality and obtained the median lethal concentration and median lethal times. For histological analysis, the mites were fixed in 10% formalin, processed according to the paraffin embedded tissue technique. Sections were stained with hematoxylin-eosin to observe the general histological structure. RESULTS: We report here for the first time evidence about the in vitro acaricidal effect caused by the strain GP532 of B. thuringiensis on the mite Psoroptes cuniculi, with an LC50 of 1.3 mg/ml and a LT50 of 68 h. Histological alterations caused by B. thuringiensis on this mite, included the presence of dilated intercellular spaces in the basal membrane, membrane detachment of the peritrophic matrix and morphological alterations in columnar cells of the intestine. CONCLUSIONS: Since this mite is an obligate ectoparasite that affects rabbits, goats, horses, cows and sheep, B. thuringiensis protein extracts are proposed as a potential treatment for biological control of mange in farm animals.

In vitro and in vivo Nematocidal Activity of Allium sativum and Tagetes erecta Extracts Against Haemonchus contortus.

OBJECTIVE: In the Mexican ethno-medicine, a number of plants have shown a successful anthelmintic activity. This fact could be crucial to identify possible green anti-parasitic strategies against nematodes affecting animal production. This research evaluated the in vitro and in vivo nematocidal effects of two single and combined plant extracts: bulbs of Allium sativum (n-hexane) and flowers of Tagetes erecta (acetone). The in vivo assay evaluated the administration of extracts either individually or combined against Haemonchus contortus in experimentally infected gerbils. METHODS: The in vitro larvicidal activity percentage (LAP) of A. sativum and T. erecta extracts against H. contortus (L3) was determined by means of individual and combined usage of the extracts. Similarly, the extracts were evaluated in terms of reduction in the parasitic population in gerbils infected with H. contortus by individual and combined usage. RESULTS: The LAP at 40 mg/mL was 68% with A. sativum and 36.6% with T. erecta. The combination caused 83.3% mortality of parasites. The oral administration of A. sativum and T. erecta extracts at 40 mg/mL, caused 68.7% and 53.9% reduction of the parasitic burden, respectively. Meanwhile, the combined effect of both extracts shown 87.5% reduction. CONCLUSION: This study showed evidence about the effect of A. sativum and T. erecta plant extracts by means of individual and combined usage against H. contortus in in vitro and in vivo bioassays in artificially H. contortus-infected gerbils as a model.

In vitro ovicidal and cestocidal effects of toxins from Bacillus thuringiensis on the canine and human parasite Dipylidium caninum.

Bacillus thuringiensis is a gram-positive soil-dwelling bacterium that is commonly used as a biological pesticide. This bacterium may also be used for biological control of helminth parasites in domestic animals. In this study, we evaluated the possible ovicidal and cestocidal effects of a total protein extract of B. thuringiensis native strains on the zoonotic cestode parasite of dogs, Dipylidium caninum (D. caninum). Dose and time response curves were determined by coincubating B. thuringiensis proteins at concentration ranging from 100 to 1000 µ g/mL along with 4000 egg capsules of D. caninum. Egg viability was evaluated using the trypan blue exclusion test. The lethal concentration of toxins on eggs was 600 µ g/ml, and the best incubation time to produce this effect was 3 h. In the adult stage, the motility and the thickness of the tegument were used as indicators of damage. The motility was inhibited by 100% after 8 hours of culture compared to the control group, while the thickness of the cestode was reduced by 34%. Conclusively, proteins of the strain GP526 of B. thuringiensis directly act upon D. caninum showing ovicidal and cestocidal effects. Thus, B. thuringiensis is proposed as a potential biological control agent against this zoonosis.