Spatio-Temporal Profiling of Metarhizium anisopliae-Responsive microRNAs Involved in Modulation of Plutella xylostella Immunity and Development.

Metarhizium anisopliae, a ubiquitous pathogenic fungus, regulates a wide array of the insect pest population. The fungus has been employed to control Plutella xylostella, an insecticide-resistant destructive lepidopteran pest, which causes substantial economic losses in crops worldwide. Integration of modern gene-silencing technologies in pest control strategies has become more crucial to counter pesticide-resistant insects. MicroRNAs (miRNA) play essential roles in the various biological process via post-transcriptional gene regulation. In the present study, RNA-seq analysis of control (CK36h, CK72h) and fungal-infected (T36h, T72h) midguts was performed to reveal underlying molecular mechanisms occurring in larval midgut at different time courses. We aimed at exploring M. anisopliae-responsive miRNAs and their target genes involved in development and immunity. After data filtration, a combined set of 170 miRNAs were identified from all libraries. Interestingly, miR-281, miR-263, miR-1, miR-6094 and miR-8 were listed among the most abundantly expressed conserved miRNAs. Furthermore, we experimentally studied the role of differentially expressed miR-11912-5p in regulating corresponding target trypsin-like serine proteinase (Px_TLSP). The luciferase assay (in vitro) revealed that miRNA-11912-5p significantly downregulated its target gene, suggesting it might play a crucial role in defense mechanism of P. xylostella against M.+ anisopliae infection. We used synthetic miRNA mimic/inhibitor (in vivo), to overexpress/silence miRNA, which showed harmful effects on larval duration, survival and adult fecundity. Additionally, fungal application in the presence of mimics revealed enhanced sensitivity of P. xylostella to infection. Our finding provides an insight into the relatively obscure molecular mechanisms involved in insect midgut during the fungal infection.

Gut microbiota mediate Plutella xylostella susceptibility to Bt Cry1Ac protoxin is associated with host immune response.

Insect gut microbiotas have a variety of physiological functions for host growth, development, and immunity. Bacillus thuringiensis (Bt) is known to kill insect pests by releasing insecticidal protoxins, which are activated in the insect midgut. However, the interplay among Bt infection, host immunity, and gut microbiota are still unclear. Here we show that Bt Cry1Ac protoxin interacts with the gut microbiota to accelerate the mortality of P. xylostella larvae. Cry1Ac protoxin was found to cause a dynamic change in the midgut and hemocoel microbiota of P. xylostella, with a significant increase in bacterial load and a significant reduction in bacterial diversity. In turn, loss of gut microbiota significantly decreased the Bt susceptibility of P. xylostella larvae. The introduction of three gut bacterial isolates Enterococcus mundtii (PxG1), Carnobacterium maltaromaticum (PxCG2), and Acinetobacter guillouiae (PxCG3) restored sensitivity to Bt Cry1Ac protoxin. We also found that Cry1Ac protoxin and native gut microbiota can trigger host midgut immune response, which involves the up-regulation of expression of Toll and IMD pathway genes and most antimicrobial peptide genes, respectively. Our findings further shed light on the interplay between insect gut microbiota and host immunity under the Bt toxin killing pressure, and this may provide insights for improving the management of Bt resistance and lead to new strategies for biological control of insect pests.

Metarhizium Anisopliae Challenges Immunity and Demography of Plutella xylostella.

Entomopathogenic fungi are naturally existing microbes, that can serve as a key regulator of insect pests in integrated pest management strategies. Besides having no hazardous effects on the environment, these entomopathogens are alternatives to synthetic insecticides that can control notorious insect-like Plutella xylostella, a destructive pest of cruciferous crops. Three different species of entomopathogenic fungi were evaluated before the selection (high larval mortality and least LC50) of Metarhizum anisopliae. The study was designed to investigate the mortality, development, and immune responses of P. xylostella when challenged with M. anisopliae, a naturally existing soil-borne entomopathogenic fungus. M. anisopliae resulted in high pest mortality by killing 93% of larvae. However, no statistically significant effect on hemocyte concentration was observed. The activity of enzymes (Phenoloxidase and Superoxide dismutase) and immune genes (Defensin, Spaetzle, Cecropin, Lysozyme, and Hemolin) did vary at different time points (24, 48, 72 and 96 h) after exposure to M. anisopliae. Disturbance in the biological cycles of P. xylostella was also detected, significantly shorter adult life span (8.11:6.87, M:F) and reduced fecundity (101 eggs/female) were observed along with disturbed larval and pupal duration. Results suggest that M. anisopliae can efficiently hinder the P. xylostella defense and developmental system, resulting in mortality and disturbed demography.

Larvicidal, Ovicidal, Synergistic, and Repellent Activities of Sophora alopecuroides and Its Dominant Constituents Against Aedes albopictus.

In the current study, to combat insecticide resistance, we explored larvicidal, ovicidal, synergistic, and repellent activities of Sophora alopecuroides extract and its dominant constituents against Aedes albopictus. The results of the toxicity bioassays demonstrated that the extract of S. alopecuroides exerted significant larvicidal activity (16.66-86.66%) against the third-instar larvae of Ae. albopictus at different concentrations (5-50 ug/mL) and low hatchability of eggs (2.32-75%) at 5-50 ug/mL. The constituents of S. alopecuroides showed a synergistic effect when applied as a mixture (LC30 + LC30) against larvae, while no synergistic effect was observed against the eggs of Ae. albopictus. S. alopecuroides extract provided 93.11% repellency in the first 90 min and gradually decreased to 53.14% after 240 min, while the positive control DEET (N,N-diethyl-3-methylbenzamide) showed 94.18% in the first 90 min and 55.33% after 240 min. All of the results exhibited a concentration-dependent effect. To the best of our knowledge, this is the first time that a study has identified a highly effective extract of S. alopecuroides, which could be used as an alternative agent to control larvae and eggs and to repel adults of Ae. albopictus.

Assessment of Lethal, Sublethal, and Transgenerational Effects of Beauveria Bassiana on the Demography of Aedes Albopictus (Culicidae: Diptera).

Dengue fever is one of the most rapidly spreading arthropod-borne diseases. Diurnal vectorial properties of Aedes albopictus contribute to the dispersion of the dengue viruses. Frequent and injudicious use of synthetic insecticides led to the evolution of resistant phenotypes in Ae. albopictus which necessitates the search for an alternative of current control strategies. Developing a long-lasting and environmentally safe tactic based on knowledge of ecology and population dynamics of Ae. albopictus is critical. Therefore, with a view towards biological control and ecology, the effect of entomopathogenic fungi (EPF) Beauveria bassiana on filial and first filial generations of Ae. albopictus were studied. Investigations showed 87.5% adulticidal activity leading to altered fecundity and adult longevity in a filial generation. The lethal (LC50) and sublethal (LC20) concentrations of B. bassiana were applied to filial generation (F0) to study demographic parameters in the first filial generation (F1). Results showed reduced net reproductive rates (Ro) intrinsic rate of increase (r), and mean generation time (T) compared to uninfected controls. Prolonged larval and pupal duration were observed followed by reduced longevity of male and female adults. Fecundity in the first filial generation was significantly changed with the lethal and sublethal concentrations of B. bassiana. Thus, it is concluded that B. bassiana has the potential to play a vital role in integrated mosquito management strategies.

Pathogenicity and Transgenerational Effects of Metarhizium anisopliae on the Demographic Parameters of Aedes albopictus (Culicidae: Diptera).

Dengue fever is a vector-borne infectious disease that spreads swiftly and threatens human lives in several tropical countries. Most of the strategies employed for the control of Aedes albopictus (Skuse) involve synthetic chemicals. The indiscriminate use of synthetic chemicals has led to the development of resistance and is unsafe for human and environmental health. Therefore, there is a need to develop ecologically safe tactics, such as the use of the entomopathogenic fungus (EPF) Metarhizium anisopliae (Metchnikoff 1879) (Met-11.1). The following study investigated the effectiveness of EPF-Met-11.1 on different demographic parameters of Ae. albopictus. Mortality bioassays showed 92.5% mortality when adult Ae. albopictus were treated with M. anisopliae. Metarhizium anisopliae absorbs the hemolymph sugar which results in retarded development. Metarhizium anisopliae LC50 not only affected the parental generation (F0) but also affected the demographic parameters of the offspring (F1). Transgenerational results (F1) with Met-11.1 showed decreased net reproductive rates (Ro), intrinsic rates of increase (r), and mean generation times (T) compared to those of uninfected controls. The larval developmental duration in the treatment group was 8.22 d, compared to 8.00 d in the control. There was a significant decrease in mean fecundity in the treated group (208.87 eggs) compared to that of the control group (360.27 eggs), and adult longevity was also significantly reduced in the treated group. Therefore, it is concluded that M. anisopliae can have lasting effects on the developmental parameters of Ae. albopictus, indicating that it can be an integral part of mosquito control strategies.