Aspergillus niger Culture Filtrate (ACF) Mediated Biocontrol of Enteric Pathogens in Wastewater

Robust control of pathogens in sewage facilitates safe reuse of wastewater rich in valuable nutrients for potential valorization through biological means. Aspergillus niger is widely reported in bioremediation of wastewater but studies on control of enteric pathogens in sewage are very sparse. So, this study aimed at exploring the antibacterial and nematicidal activity of A. niger culture filtrate (ACF). Antibacterial activity of ACF on enteric pathogens (Klebsiella pneumoniae, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella enterica, Shigella dysenteriae, Escherichia coli, Staphylococcus aureus, Klebsiella variicola) was determined by spectrophotometric growth analysis, resazurin based viability assay and biofilm formation assay. ACF showed inhibition against all enteric pathogens except Pseudomonas aeruginosa. Nematicidal studies on Caenorhabditis elegans showed 85% egg hatch inhibition and 52% mortality of L1 larvae. Sewage treatment with ACF at 1:1 (v/v) showed 2–3 log reduction in coliforms, Klebsiella, Shigella, Salmonella, S. aureus and Vibrio except Pseudomonas, indicating significant alteration of complex microbial dynamics in wastewater. Application of ACF can potentially be used as a robust biocontrol strategy against infectious microbes in wastewater and subsequent valorization by cultivating beneficial Pseudomonas.

Extending the Storage Time of Clanis bilineata tsingtauica (Lepidoptera; Sphingidae) Eggs through Variable-Temperature Cold Storage

Clanis bilineata tsingtauica Mell, 1922 (Lepidoptera, Sphingidae), also known as “Doudan” in China, is an important pest in legume crops. As an edible insect, it is most commonly consumed in Jiangsu, Shandong, and Henan Provinces. Mass rearing requires access to large numbers of eggs. This stage, however, is of short duration and supplies are frequently not sufficient for insect production. Therefore, we identified the cold storage conditions for C. bilineata tsingtauica that can effectively prolong the storage time of the eggs, to make supplies more readily available. We found that when stored at 4 °C, only 7.5% of the eggs hatched after 7 days, while at 10 °C the hatch rate was 78.3%. At 15 °C, the egg hatch rate remained at this same level (77.8% even after 14–20 days). Considering various combinations, we found that optimal egg hatch occurred if eggs were stored at 15 °C for 11 days, and then held at 15–20 °C under dark conditions. Stored as described above, the egg hatch rate was not significantly different from the control group (at 28 °C). These conditions allow for easier mass rearing of C. bilineata tsingtauica by providing a stable supply of eggs.

Development of a nondiapausing strain of northern corn rootworm with rearing techniques for both diapausing and nondiapausing strains

The northern corn rootworm, Diabrotica barberi Smith & Lawrence, has a univoltine life cycle that typically produces one generation a year. When rearing the northern corn rootworm in the laboratory, in order to break diapause, it is necessary to expose eggs to a five month cold period before raising the temperature. By selective breeding of the small fraction of eggs that hatched without cold within 19–32 days post oviposition, we were able to develop a non-diapausing colony of the northern corn rootworm within five generations of selection. Through selection, the percentages of adult emergence from egg hatch without exposure to cold treatment significantly increased from 0.52% ± 0.07 at generation zero to 29.0% ± 2.47 at generation eight. During this process, we developed an improved method for laboratory rearing of both the newly developed non-diapausing strain as well as the diapausing strain. The development of the non-diapausing colony along with the improvements to the rearing system will allow researchers to produce up to six generations of the northern corn rootworm per year, which would facilitate research and advance our knowledge of this pest at an accelerated rate.

Anthelminthic effects of extracts of indigenous browses from mid rift valley of Ethiopia

This study was conducted to evaluate the potential anthelminthic properties of extracts of leaves of indigenous browses (Acacia seyal, Acacia senegal, Acacia tortilis, Millettia ferruginea, and Vernonia amygadalina) based on three in vitro assays. Acetone extracts of browses at different concentrations (75 to 1200 μg/ml, for egg and larvae and 100mg/ml for an adult) were tested on three developmental stages of Haemonchus contortus (eggs, infective larvae, and adult worms) using egg hatch assay (EHA), larval migration inhibition assay (LMIA) and adult worm motility inhibition assay (AMIA). Significant effects were obtained with all five browses but differences were observed depending on the parasitic stages. The effects of five browse extracts on egg hatching were concentration-dependent, the highest (P<0.05) egg hatch inhibition rate was observed at 1200 μg/ml concentration for all browses. All extracts had a higher effect (P<0.01) than that of the negative control, phosphate buffer saline (PBS). In contrast, no concentration-response relationship was found for infective larvae and adult worms, although more potent effects were observed with the highest concentrations. The LMI rate (70%) induced by Vernonia amygadalina extract, at a concentration of 300 μg/ml, was the highest (P<0.05) of all other browses, even at higher concentrations. The highest LMI rate (62%) induced by Acacia senegal extract at higher concentration, was lower than that of LMI rate (70%) induced by Vernonia amygadalina, at 300 μg/ml concentration. Vernonia amygadalina was found to be highly and rapidly effective against adult worms inducing the highest mortality rate (90%) as soon as 4 hrs after incubation. Overall, the in vitro results suggest that these five browses do possess anti-parasitic properties and Vernonia amygadalina showed the most effective anti-parasitic property. These effects remain to be confirmed through in vivo study.

In Vitro Assessment of N-Phenyl Imides in the Management of Meloidogyne Incognita

The infestation with root knot nematode Meloidogyne spp. is a key issue in agriculture. Conventional control methods are based on the use of synthetic nematicides, which comes with severe environmental problems. In this study, n-phenyl imide and n-phenyl phthalamic acid were synthesized and reacted independently with Enantia chlorantha crude extract–manganese chloride complex. The effects of the resulting organic compounds were appraised against the root knot nematode Meloidogyne incognita (Kofoid and White 1919) juveniles and eggs in two laboratory experiments. The most active compound was n-phenyl phthalamic acid (PN/TLMA) with 4% egg hatch over a 9-day observation after treatment as against distilled water which recorded 100% egg hatch at 9 days after treatment. n-Phenyl phthalamic acid showed 100% juvenile mortality at 10 days of observation compared to carbofuran dissolved in water (CBFN/water) and carbofuran dissolved in hydroxypropyl-β-cyclodextrin (CBFN/HPCD) while no outstanding (P < 0.05) difference was recorded between the effects of other organic compounds and carbofuran in both solvents. The different rates of treatment applications were not appreciably (P<0.05) dissimilar on percentage juvenile mortality and egg hatch. The nematicidal test results indicated that the synthesized imide compounds with manganese complex moiety are a promising basis for developing new nematicidal compounds with less environmental hazard.

Metabolomics provide new insights into mechanisms of Wolbachia-induced paternal defects in Drosophila melanogaster

Wolbachia is a group of intracellular symbiotic bacteria that widely infect arthropods and nematodes. Wolbachia infection can regulate host reproduction with the most common phenotype in insects being cytoplasmic incompatibility (CI), which results in embryonic lethality when uninfected eggs fertilized with sperms from infected males. This suggests that CI-induced defects are mainly in paternal side. However, whether Wolbachia-induced metabolic changes play a role in the mechanism of paternal-linked defects in embryonic development is not known. In the current study, we first use untargeted metabolomics method with LC-MS to explore how Wolbachia infection influences the metabolite profiling of the insect hosts. The untargeted metabolomics revealed 414 potential differential metabolites between Wolbachia-infected and uninfected 1-day-old (1d) male flies. Most of the differential metabolites were significantly up-regulated due to Wolbachia infection. Thirty-four metabolic pathways such as carbohydrate, lipid and amino acid, and vitamin and cofactor metabolism were affected by Wolbachia infection. Then, we applied targeted metabolomics analysis with GC-MS and showed that Wolbachia infection resulted in an increased energy expenditure of the host by regulating glycometabolism and fatty acid catabolism, which was compensated by increased food uptake. Furthermore, overexpressing two acyl-CoA catabolism related genes, Dbi (coding for diazepam-binding inhibitor) or Mcad (coding for medium-chain acyl-CoA dehydrogenase), ubiquitously or specially in testes caused significantly decreased paternal-effect egg hatch rate. Oxidative stress and abnormal mitochondria induced by Wolbachia infection disrupted the formation of sperm nebenkern. These findings provide new insights into mechanisms of Wolbachia-induced paternal defects from metabolic phenotypes.

A w AlbB Wolbachia transinfection displays stable phenotypic effects across divergent Aedes aegypti mosquito backgrounds

Aedes mosquitoes harboring intracellular Wolbachia bacteria are being released in arbovirus and mosquito control programs. With releases taking place around the world, understanding the contribution of host variation to Wolbachia phenotype is crucial. We generated a Wolbachia transinfection ( w AlbB Q ) in Aedes aegypti and performed backcrossing to introduce the infection into Australian or Malaysian nuclear backgrounds. Whole Wolbachia genome sequencing shows that the w AlbB Q transinfection is near-identical to the reference w AlbB genome, suggesting few changes since the infection was first introduced to Ae. aegypti over 15 years ago. However, these sequences were distinct from other available w AlbB genome sequences, highlighting the potential diversity of w AlbB in natural Ae. albopictus populations. Phenotypic comparisons demonstrate effects of w AlbB infection on egg hatch and nuclear background on fecundity and body size, but no interactions between w AlbB infection and nuclear background for any trait. The w AlbB infection was stable at high temperatures and showed perfect maternal transmission and cytoplasmic incompatibility regardless of host background. Our results demonstrate the stability of w AlbB across host backgrounds and point to its long-term effectiveness for controlling arbovirus transmission and mosquito populations. Importance Wolbachia bacteria are being used to control the transmission of dengue and other arboviruses by mosquitoes. For Wolbachia release programs to be effective globally, Wolbachia infections must be stable across mosquito populations from different locations. In this study, we transferred Wolbachia (strain w AlbB) to Aedes aegypti mosquitoes with an Australian genotype and introduced the infection to Malaysian mosquitoes through backcrossing. We found that the phenotypic effects of Wolbachia are stable across both mosquito backgrounds. We sequenced the genome of w AlbB and found very few genetic changes despite spending over 15 years in a novel mosquito host. Our results suggest that the effects of Wolbachia infections are likely to remain stable across time and host genotype.

Impact of feeding experiences on oviposition and sex allocation of Neoseiulus californicus (Acari: Phytoseiidae)

Feeding experiences of predators during immature and adult stages may impact females’ reproduction. In the present study, we investigated reproductive performances of Neoseiulus californicus when both parents had different feeding experiences on Tetranychus urticae and Frankliniella occidentalis. Female and male immatures fed on either prey species. Each newly emerged female adult individually mated with a male had either the same or different feeding experience. Prey for mated females were either same as or different from that consumed during their immature stages. Therefore, eight reproductive treatments were created. The highest cumulative fecundity (57.5±3.3 eggs/female) was observed when both female and male fed on T. urticae with the lowest fecundity (34.2±2.7 eggs/female) observed when the parents fed on F. occidentalis. Daily fecundity and oviposition duration were mainly affected by prey of mated females. Mated females preyed on T. urticae had 56.6% higher daily fecundity and 22.3% shorter oviposition duration than those preyed on F. occidentalis. No significant difference in offspring sex ratio and egg hatch rate was detected between treatments. About 88% of the first-laid eggs developed to males. Impact of prey species consumed by male immatures was only observed on pre-oviposition duration. The average pre-oviposition duration of females who mated with males fed on T. urticae in their immaturity was 33.0% shorter than those females who mated with males fed on F. occidentalis in their immaturity. Results of the present study are valuable in optimizing N. californicus field release strategies, and will help further investigations into the nutritional requirements of this species.

New Insights into Anthelmintic Mechanisms of Action of a Synthetic Peptide: An Ultrastructural and Nanomechanical Approach

Resistant nematodes are not affected by the most common drugs commercially available. In the search for new anthelmintics, peptides have been investigated. Here, a linear synthetic peptide named RcAlb-PepIII bioinspired from the antimicrobial protein Rc-2S-Alb was designed, synthesized, and tested against barber pole worm Haemonchus contortus. The physicochemical properties of the peptide, the 3D structure model, the egg hatch inhibition, and larval development inhibition of H. contortus were carried out. Additionally, the ultrastructure of the nematode after treatment with the peptide was evaluated by atomic force microscopy. The RcAlb-PepIII inhibited the larval development of H. contortus with an EC50 of 90 µM and did not affect egg hatch. Atomic force microscopy reveals the high affinity of RcAlb-PepIII with the cuticle of H. contortus in the L2 stage. It also shows the deposition of RcAlb-PepIII onto the surface of the cuticle, forming a structure similar to a film that reduces the roughness and mean square roughness (Rq) of it. In conclusion, the bioinspired RcAlb-PepIII has the potential to be used as a new anthelmintic compound to control gastrointestinal nematode parasites.