Exploration of Some Heterocyclic Compounds Containing Trifluoromethylpyridine Scaffold as Insecticides Toward Aphis gossypii Insects.

Five types of heterocyclic compounds containing trifloromethylpyridine scaffold namely; 3-cyano-2-(N-phenyl)carbamoylmethylthio-6-(thiophen-2-yl)-4-trifluoromethyl-pyridine (6a), thieno[2,3-b]pyridines 3-5 and 7a-c, pyrido[3',2':4,5]thieno[3,2-d] pyrimidines 8-13 and 15a-c, pyrido[3',2':4,5]thieno[3,2-d][1,2,3]triazines 16a,b, and 9-(thiophen-2-yl)-7-(trifluoromethyl) pyrido [3',2':4,5]thieno[2,3-e][1,2,4]triazolo[1,5-c]pyrimidine (14) were synthesized in excellent yields and very pure state. The structures of these compounds were confirmed by elemental and spectral analyses. Most of the synthesized compounds were evaluated as insecticidal agents toward Aphis gossypii insects and promising results obtained. Among all tested compounds, only 6, 7a, 7c and 15c being the most potent compounds against nymphs and adults of Aphis gossypii and their activities are nearly to that of acetamiprid as a reference. The effect of 6a compounds 7a, 7c and 15c on the Aphis digestive system from histological point of view was also included.

The Potential of Metarhizium anisopliae Blastospores to Control Aedes aegypti Larvae in the Field.

Entomopathogenic fungi are promising as an environmentally benign alternative to chemical pesticides for mosquito control. The current study investigated the virulence of Metarhizium anisopliae blastospores against Aedes aegypti under both laboratory and field conditions. Virulence bioassays of conidia and blastospores were conducted in the laboratory, while field simulation bioassays were conducted under two conditions: totally shaded (TS) or partially shaded (PS). In the first bioassay (zero h), the larvae were added to the cups shortly after the preparation of the blastospores, and in the subsequent assays, larvae were added to the cups 3, 6, 9, and 12 days later. The survival of the larvae exposed to blastospores in the laboratory was zero on day two, as was the case for the larvae exposed to conidia on the sixth day. Under TS conditions, zero survival was seen on the third day of the bioassay. Under PS conditions, low survival rates were recorded on day 7. For the persistence bioassay under PS conditions, low survival rates were also observed. Metarhizium anisopliae blastospores were more virulent to Ae. aegypti larvae than conidia in the laboratory. Blastospores remained virulent under field simulation conditions. However, virulence rapidly declined from the third day of field bioassays. Formulating blastospores in vegetable oil could protect these propagules when applied under adverse conditions. This is the first time that blastospores have been tested against mosquito larvae under simulated field conditions, and the current study could be the basis for the development of a new biological control agent.

Potential of 3-octanone as a lure and kill agent for control of the Brown garden snail.

The brown garden snail (Cornu aspersum) is a major agricultural pest, causing damage to a wide range of economically important crops. Withdrawal or restricted use of pollutant molluscicides like metaldehyde has prompted a search for more benign control products. This study investigated the response of snails to 3-octanone; a volatile organic compound (VOCs) produced by the insect pathogenic fungus Metarhizium brunneum. Concentrations of 1 - 1000 ppm of 3-octanone were first assessed in laboratory choice assays to determine behavioural response. Repellent activity was found at 1000 ppm whereas attractance was found for the lower concentrations of 1, 10 and 100 ppm. These three concentrations of 3-octanone were carried forward in field evaluations to assess potential for use in "lure and kill" strategies. The highest concentration (100 ppm) was the most attractive to the snails but also the most lethal. Even at the lowest concentration this compound proved toxic effects making 3-octanone an excellent candidate for the development as a snail attractant and molluscicide.

Stress-Mediated Responses of Aedes aegypti (Diptera: Culicidae) Larvae When Exposed to Metarhizium brunneum (Hypocreales: Clavicipitaceae) and Toxorhynchites brevipalpis (Diptera: Culicidae).

Aedes aegypti mosquitoes are capable of vectoring a wide range of diseases including dengue, yellow fever, and Zika viruses, with approximately half of the worlds' population at risk from such diseases. Development of combined predator-parasite treatments for the control of larvae consistently demonstrates increased efficacy over single-agent treatments, however, the mechanism behind the interaction remains unknown. Treatments using the natural predator Toxorhynchites brevipalpis and the entomopathogenic fungus Metarhizium brunneum were applied in the laboratory against Ae. aegypti larvae as both individual and combined treatments to determine the levels of interaction between control strategies. Parallel experiments involved the removal of larvae from test arenas at set intervals during the course of the trial to record whole body caspase and phenoloxidase activities. This was measured via luminometric assay to measure larval stress factors underlying the interactions. Combined Metarhizium and Toxorhynchites treatments were seen to drastically reduce lethal times as compared to individual treatments. This was accompanied by increased phenoloxidase and caspase activities in combination treatments after 18 h (p < 0.001). The sharp increases in caspase and phenoloxidase activities suggest that combined treatments act to increase stress factor responses in the larvae that result in rapid mortality above that of either control agent individually. This work concludes that the underlying mechanism for increased lethality in combined parasite-predator treatments may be related to additive stress factors induced within the target host larvae.

Antiparasitic effects of Elettaria cardamomum L. essential oil and its main compounds, 1-8 Cineole alone and in combination with albendazole against Echinococcus granulosus protoscoleces.

Background: The present investigation aims to determine the chemical structure and protoscolicidal effects of Elettaria cardamomum L. essential oil (ECEO) and its main compounds 1-8 cineole alone and along with albendazole (ALZ) against Echinococcus granulosus protoscoleces in vitro and ex vivo. We also decided to evaluate some cellular mechanisms such as the apoptotic activity and the permeability of plasma membrane of protoscoleces treated with ECEO and 1-8 cineole. Methods: Hydatid cyst protoscoleces were divided into seven groups including protoscoleces treated with ECEO 50 µl/mL (T1), protoscoleces treated with ECEO 100 µl/mL (T2), protoscoleces treated with ECEO 200 µl/mL (T3), protoscoleces treated with 1-8 cineole 100 µg/mL (T4), protoscoleces treated with 1-8 cineole 200 µg/mL (T5), protoscoleces treated with 1-8 cineole 100 µg/mL + albendazole 50 µg/mL (T6), and protoscoleces treated with 1-8 cineole 200 µg/mL + albendazole ALZ-50 µg/mL (T7). The viability of protoscoleces were recorded by eosin staining examination. Moreover, the induction of apoptosis and the plasma membrane permeability of the protoscoleces treated with ECEO and 1-8 cineole were evaluated. Results: The highest protoscolicidal effect of ECEO was observed at the dose of 200 µl/ml (T3). 1,8-Cineole alone and combined with ALZ, particularly at the dose of 200 µg/ml (T5 and T7), destroyed the 100% protoscolices after 10 min incubation. The ECEO (T1-T3) and 1-8 cineole alone (T4 and T5) and in combination with ALZ (T6 and T7) took longer to display their protoscolicidal effect ex vivo. The obtained results of relative fuorescent items exhibited that the protoscoleces incubated with ECEO and 1,8-Cineole, alter the permeability of plasma membrane by Sytox Green with increasing the concentration. The findings revealed exhibited that ECEO and 1,8-Cineole increasingly and dose-dependently induced activation of caspase-3 enzyme ranging from 6.8 to 23.3%. Conclusion: Our obtained results revealed that ECEO and its main compound, 1,8-Cineole exhibited the potent protoscolicidal in vitro and ex vivo; and if more research is done on their efficacy and toxicity in animal models and even clinical setting, it can be suggested as a protoscolicidal agent to use during hydatid cyst surgery.

Population Genetic Analysis of Aedes aegypti Mosquitoes From Sudan Revealed Recent Independent Colonization Events by the Two Subspecies.

Increases in arbovirus outbreaks in Sudan are vectored by Aedes aegypti, raising the medical importance of this mosquito. We genotyped 12 microsatellite loci in four populations of Ae. aegypti from Sudan, two from the East and two from the West, and analyzed them together with a previously published database of 31 worldwide populations to infer population structure and investigate the demographic history of this species in Sudan. Our results revealed the presence of two genetically distinct subspecies of Ae. aegypti in Sudan. These are Ae. aegypti aegypti in Eastern Sudan and Ae. aegypti formosus in Western Sudan. Clustering analysis showed that mosquitoes from East Sudan are genetically homogeneous, while we found population substructure in West Sudan. In the global context our results indicate that Eastern Sudan populations are genetically closer to Asian and American populations, while Western Sudan populations are related to East and West African populations. Approximate Bayesian Computation Analysis supports a scenario in which Ae. aegypti entered Sudan in at least two independent occasions nearly 70-80 years ago. This study provides a baseline database that can be used to determine the likely origin of new introductions for this invasive species into Sudan. The presence of the two subspecies in the country should be consider when designing interventions, since they display different behaviors regarding epidemiologically relevant parameters, such as blood feeding preferences and ability to transmit disease.

Demographic expansion and high level of matrilineal diversity in two populations of East African Baggara cattle.

Western Baggara cattle breed (WBCB) is an East African zebu inhabiting Sudan, well-known as beef-producing cattle. We investigated herein two phenotypically and geographically distinct populations of this breed, namely Nyalawi and Daeinawi, which are renowned for their unique meat production capabilities and adaptation attributes, with the aim to contribute to our understanding of their maternal genetic diversity and demography dynamics. Genetic polymorphism analysis of the full-length D-loop mtDNA region revealed 44 and 35 polymorphic sites defining 28 and 24 distinct haplotypes in the Nyalawi and the Daeinawi, respectively. Observed genetic diversity is high within the population with a low level of genetic differentiation between populations. Approximate Bayesian computation via the calculation of Bayesian skyline plots and neutrality tests support past expansion with a higher maternal effective population size (Ne ) in Nyalawi compared with the Daeinawi population and a population expansion beginning around 4,500 YBP and 3,500 YBP, respectively, before the arrival of zebu into the continent.

Assessment and Validation of Globodera pallida as a Novel In Vivo Model for Studying Alzheimer's Disease.

BACKGROUND: Whole transgenic or non-transgenic organism model systems allow the screening of pharmacological compounds for protective actions in Alzheimer's disease (AD). AIM: In this study, a plant parasitic nematode, Globodera pallida, which assimilates intact peptides from the external environment, was investigated as a new potential non-transgenic model system of AD. Methods: Fresh second-stage juveniles of G. pallida were used to measure their chemosensory, perform immunocytochemistry on their neurological structures, evaluate their survival rate, measure reactive oxygen species, and determine total oxidized glutathione to reduced glutathione ratio (GSSG/GSH) levels, before and after treatment with 100 µM of various amyloid beta (Aß) peptides (1-40, 1-42, 17-42, 17-40, 1-28, or 1-16). Wild-type N2 C. elegans (strain N2) was cultured on Nematode Growth Medium and directly used, as control, for chemosensory assays. RESULTS: We demonstrated that: (i) G. pallida (unlike Caenorhabditis elegans) assimilates amyloid-ß (Aß) peptides which co-localise with its neurological structures; (ii) pre-treatment with various Aß isoforms (1-40, 1-42, 17-42, 17-40, 1-28, or 1-16) impairs G. pallida's chemotaxis to differing extents; (iii) Aß peptides reduced survival, increased the production of ROS, and increased GSSG/GSH levels in this model; (iv) this unique model can distinguish differences between different treatment concentrations, durations, and modalities, displaying good sensitivity; (v) clinically approved neuroprotective agents were effective in protecting G. pallida from Aß (1-42) exposure. Taken together, the data indicate that G. pallida is an interesting in vivo model with strong potential for discovery of novel bioactive compounds with anti-AD activity.

BoLA-DRB3 gene haplotypes show divergence in native Sudanese cattle from taurine and indicine breeds.

Autochthonous Sudanese cattle breeds, namely Baggara for beef and Butana and Kenana for dairy, are characterized by their adaptive characteristics and high performance in hot and dry agro-ecosystems. They are thus used largely by nomadic and semi-nomadic pastoralists. We analyzed the diversity and genetic structure of the BoLA-DRB3 gene, a genetic locus linked to the immune response, for the indigenous cattle of Sudan and in the context of the global cattle repository. Blood samples (n = 225) were taken from three indigenous breeds (Baggara; n = 113, Butana; n = 60 and Kenana; n = 52) distributed across six regions of Sudan. Nucleotide sequences were genotyped using the sequence-based typing method. We describe 53 alleles, including seven novel alleles. Principal component analysis (PCA) of the protein pockets implicated in the antigen-binding function of the MHC complex revealed that pockets 4 and 9 (respectively) differentiate Kenana-Baggara and Kenana-Butana breeds from other breeds. Venn analysis of Sudanese, Southeast Asian, European and American cattle breeds with 115 alleles showed 14 were unique to Sudanese breeds. Gene frequency distributions of Baggara cattle showed an even distribution suggesting balancing selection, while the selection index (ω) revealed the presence of diversifying selection in several amino acid sites along the BoLA-DRB3 exon 2 of these native breeds. The results of several PCA were in agreement with clustering patterns observed on the neighbor joining (NJ) trees. These results provide insight into their high survival rate for different tropical diseases and their reproductive capacity in Sudan's harsh environment.

Highly specific host-pathogen interactions influence Metarhizium brunneum blastospore virulence against Culex quinquefasciatus larvae.

Entomopathogenic fungi are potential biological control agents of mosquitoes. Our group observed that not all mosquitoes were equally susceptible to fungal infection and observed significant differences in virulence of different spore types. Conidiospores and blastospores were tested against Culex quinquefasciatus larvae. Blastospores are normally considered more virulent than conidia as they form germ tubes and penetrate the host integument more rapidly than conidia. However, when tested against Cx. quinquefasciatus, blastospores were less virulent than conidia. This host-fungus interaction was studied by optical, electron and atomic force microscopy (AFM). Furthermore, host immune responses and specific gene expression were investigated. Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores did not readily adhere to Culex larval integument and the main route of infection was through the gut. Adhesion forces between blastospores and Culex cuticle were significantly lower than for other insects. Larvae challenged with blastospores showed enhanced immune responses, with increased levels of phenoloxidase, glutathione-S-transferase, esterase, superoxide dismutase and lipid peroxidase activity. Interestingly, M. brunneum pathogenicity/stress-related genes were all down-regulated in blastospores exposed to Culex. Conversely, when conidia were exposed to Culex, the pathogenicity genes involved in adhesion or cuticle degradation were up-regulated. Delayed host mortality following blastospore infection of Culex was probably due to lower adhesion rates of blastospores to the cuticle and enhanced host immune responses deployed to counter infection. The results here show that subtle differences in host-pathogen interactions can be responsible for significant changes in virulence when comparing mosquito species, having important consequences for biological control strategies and the understanding of pathogenicity processes.

Combined use of the entomopathogenic fungus, Metarhizium brunneum, and the mosquito predator, Toxorhynchites brevipalpis, for control of mosquito larvae: Is this a risky biocontrol strategy?

Mosquitoes transmit several diseases, which are of global significance (malaria, dengue, yellow fever, Zika). The geographic range of mosquitoes is increasing due to climate change, tourism and trade. Both conidial and blastospore formulations of the entomopathogenic fungus, Metarhizium brunneum ARSEF 4556, are being investigated as mosquito larvicides. However, concerns have been raised over possible non-target impacts to arthropod mosquito predators such as larvae of Toxorhynchites brevipalpis which feed on larvae of mosquito vector species. Laboratory-based, small container bioassays showed, that T. bevipalpis larvae are susceptible to relatively high concentrations (i.e. ≥107 spores ml-1) of inoculum with blastospores being significantly more virulent than conidia. At lower concentrations (e.g. <107 spores ml-1), it appears that M. brunneum complements T. brevipalpis resulting in higher control than if either agent was used alone. At a concentration of 105 spores ml-1, the LT50 of for conidia and blastospores alone was 5.64 days (95% CI: 4.79-6.49 days) and 3.89 days (95% CI: 3.53-4.25 days), respectively. In combination with T. brevipalpis, this was reduced to 3.15 days (95% CI: 2.82-3.48 days) and 2.82 days (95% CI: 2.55-3.08 days). Here, combined treatment with the fungus and predator was beneficial but weaker than additive. At 107 and 108 blastospores ml-1, mosquito larval mortality was mostly due to the fungal pathogen when the predator was combined with blastospores. However, with conidia, the effects of combined treatment were additive/synergistic at these high concentrations. Optimisation of fungal concentration and formulation will reduce: (1) risk to the predator and (2) application rates and costs of M. brunneum for control of mosquito larvae.

Metarhizium brunneum Blastospore Pathogenesis in Aedes aegypti Larvae: Attack on Several Fronts Accelerates Mortality.

Aedes aegypti is the vector of a wide range of diseases (e.g. yellow fever, dengue, Chikungunya and Zika) which impact on over half the world's population. Entomopathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have been found to be highly efficacious in killing mosquito larvae but only now are the underlying mechanisms for pathogenesis being elucidated. Recently it was shown that conidia of M. anisopliae caused stress induced mortality in Ae. aegypti larvae, a different mode of pathogenicity to that normally seen in terrestrial hosts. Blastospores constitute a different form of inoculum produced by this fungus when cultured in liquid media and although blastospores are generally considered to be more virulent than conidia no evidence has been presented to explain why. In our study, using a range of biochemical, molecular and microscopy methods, the infection process of Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores was investigated. It appears that the blastospores, unlike conidia, readily adhere to and penetrate mosquito larval cuticle. The blastospores are readily ingested by the larvae but unlike the conidia are able infect the insect through the gut and rapidly invade the haemocoel. The fact that pathogenicity related genes were upregulated in blastospores exposed to larvae prior to invasion, suggests the fungus was detecting host derived cues. Similarly, immune and defence genes were upregulated in the host prior to infection suggesting mosquitoes were also able to detect pathogen-derived cues. The hydrophilic blastospores produce copious mucilage, which probably facilitates adhesion to the host but do not appear to depend on production of Pr1, a cuticle degrading subtilisin protease, for penetration since protease inhibitors did not significantly alter blastospore virulence. The fact the blastospores have multiple routes of entry (cuticle and gut) may explain why this form of the inoculum killed Ae. aegypti larvae in a relatively short time (12-24hrs), significantly quicker than when larvae were exposed to conidia. This study shows that selecting the appropriate form of inoculum is important for efficacious control of disease vectors such as Ae. aegypti.