Beauveria bassiana (Bal.-Criv.) is an important entomopathogenic fungus being used for the management of various agricultural pests worldwide. However, all strains of B. bassiana may not be effective against whitefly, Bemisia tabaci, or other pests, and strains show diversity in their growth, sporulation, virulence features, and overall bioefficacy. Thus, to select the most effective strain, a comprehensive way needs to be devised. We studied the diversity among the 102 strains of B. bassiana isolated from 19 insect species based on their physiological features, virulence, and molecular phylogeny, to identify promising ones for the management of B. tabaci. Strains showed diversity in mycelial growth, conidial production, and their virulence against B. tabaci nymphs. The highest nymphal mortality (2nd and 3rd instar) was recorded with MTCC-4511 (95.1%), MTCC-6289 (93.8%), and MTCC-4565 (89.9%) at a concentration of 1 × 106 conidia ml-1 under polyhouse conditions. The highest bioefficacy index (BI) was in MTCC-4511 (78.3%), MTCC-4565 (68.2%), and MTCC-4543 (62.1%). MTCC-4511, MTCC-4565, and MTCC-4543 clustered with positive loading of eigenvalues for the first two principal components and the cluster analysis also corresponded well with PCA (principal component analysis) (nymphal mortality and BI). The molecular phylogeny could not draw any distinct relationship between physiological features, the virulence of B. bassiana strains with the host and location. The BI, PCA, and square Euclidean distance cluster were found the most useful tools for selecting potential entomopathogenic strains. The selected strains could be utilized for the management of the B. tabaci nymphal population in the field through the development of effective formulations. KEY POINTS: • 102 B. bassiana strains showed diversity in growth and virulence against B. tabaci. • Bioefficacy index, PCA, and SED group are efficient tools for selecting potential strains. • MTCC-4511, 4565, and 4543 chosen as the most virulent strains to kill whitefly nymphs.

Biological control is widely used for integrated pest management. However, there are many abiotic factors that can affect the biocontrol efficiency. In this study, we investigated the susceptibility of Hyphantria cunea larvae to Beauveria bassiana under Cd stress, and the corresponding mechanism was analyzed around innate immunity and energy metabolism. The results showed that mortality of H. cunea larvae treated with Cd and B. bassiana was significantly higher than those treated with B. bassiana alone, and the combined lethal effect exhibited a synergistic effect. Compared with the single fungal treatment group, the total hemocyte count in the combined Cd and fungal treatment group decreased significantly, accompanied by a decrease in phagocytosis, encapsulation, and melanization activity. The expression levels of three phagocytosis-related genes, one encapsulation-promoting gene, and one melanization-regulating gene were significantly lower in the combined treatment group than those in the single fungal treatment group. Furthermore, pathogen recognition ability, signal transduction level, and immune effector expression level were weaker in the combined treatment group than those in the single fungal treatment group. The expression levels of 14 key metabolites and 7 key regulatory genes in glycolysis and tricarboxylic acid cycle pathways were significantly lower in the combined treatment group than those in the single fungal treatment group. Taken together, the weakness of innate immunity and energy metabolism in response to pathogen infection resulted in an increased susceptibility of H. cunea larvae to B. bassiana under Cd pre-exposure. Microbial insecticide is a preferred strategy for pest control in heavy metal-polluted areas. AVAILABILITY OF DATA AND MATERIAL: All the data that support the findings of this study are available in the manuscript.

Perennial ryegrass (Lolium perenne L.) possesses a high level of nutritional quality and is widely used as a forage species to establish permanent pastures in southern Chile. However, the productivity of most such pastures is limited by various environmental agents, such as insect pests and drought. In this context, our work stresses the need for elucidating the ability of fungal endophytes to establish interactions with plants, and to understand how these processes contribute to plant performance and fitness. Therefore, we evaluated the colonization and impact of two native strains of the endophytic insect-pathogenic fungus (EIPF) group isolated from permanent ryegrass pastures in southern Chile. Roots and seeds of ryegrass and scarabaeid larvae were collected from nine different ryegrass pastures in the Los Ríos region of southern Chile to specifically isolate EIPFs belonging to the genera Beauveria and Metarhizium. Fungal isolations were made on 2% water agar with antibiotics, and strains were identified by analyzing the entire internal transcribed spacer (ITS) 1-5.8S-ITS2 ribosomal DNA region. Four strains of Beauveria and 33 strains of Metarhizium were isolated only in scarabaeid larvae from ryegrass pastures across four sites. Experimental mini-pastures that were either not inoculated (control) or co-inoculated with conidia of the strains Beauveria vermiconia NRRL B-67993 (P55_1) and Metarhizium aff. lepidiotae NRRL B-67994 (M25_2) under two soil humidity levels were used. Ryegrass plants were randomly collected from the mini-pastures to characterize EIPF colonization in the roots by real-time PCR and fluorescence microscopy. Aboveground biomass was measured to analyze the putative impact of colonization on the mini-pastures\' aboveground phenotypic traits with R software using a linear mixed-effects model and the ANOVA statistical test. Seasonal variation in the relative abundance of EIPFs was observed, which was similar between both strains from autumn to spring, but different in summer. In summer, the relative abundance of both EIPFs decreased under normal moisture conditions, but it did not differ significantly under water stress. The aboveground biomass of ryegrass also increased from autumn to spring and decreased in summer in both the inoculated and control mini-pastures. Although differences were observed between moisture levels, they were not significant between the control and inoculated mini-pastures, except in July (fresh weight and leaf area) and October (dry weight). Our findings indicate that native strains of B. vermiconia NRRL B-67993 (P55_1) and M. aff. lepidiotae NRRL B-67994 (M25_2) colonize and co-exist in the roots of ryegrass, and these had little or no effect on the mini-pastures\' aboveground biomass; however, they could have other functions, such as protection against root herbivory by insect pests.

Entomopathogenic fungi (EPF) now a possible safer microbial control measure that could be considered as a substitute for chemical control of insect pests. Three EPF viz., Metarihizium anisopliae, Isaria furnosoroseus and Beauveria bassiana were evaluated for their virulence against the grubs of Khapra beetle, Trogoderma granarium (Everts) under laboratory conditions. The isolates were applied by two methods viz., diet incorporation and an immersion method with 3rd instar 20 grubs of T. granarium for each. The virulence of EPF was determined using percent mortality. Significantly higher mortality was observed in M. anisopliae applied through immersion (98.33%) and diet incorporation (93.33%) methods followed by B. bassiana (90.83 and 85.83%, respectively). The mortality caused by I. furnosoroseus was statistically lower in immersion and diet incorporation methods i.e. 81.67 and 73.33%, respectively. Based on the immersion method, all EPF were studied for multiple conidial concentration i.e., 1×104, 1×105, 1×106, 1×107 and 1×108 under the same in-vitro conditions. All the isolates were pathogenic to grub of T. granarium at the highest conidial concentration. M. anisopliae was proved the most effective virulent resulting in 98.33% mortality of the pest with LT50 4.61 days at 1 × 108 conidial concentration followed by 90.83 and 81.67 percent mortality with 5.07 and 8.01 days LT50, in the application of B. bassiana and I. furnosoroseus, respectively. M. anisopliae showed higher efficacy and could be considered as promising EPF for the development of myco-insecticides against effective biocontrol of T. granarium.

Insect susceptibility to entomopathogenic microorganisms under heavy metal stress, as well as its regulatory mechanism is still poorly understood. This study aims to investigate the susceptibility of gypsy moth larvae to Beauveria bassiana under cadmium (Cd) stress (at 3.248 or 44.473 mg Cd/kg fresh food), and reveal the potential molecular mechanisms underlying the Cd effect on the larval susceptibility to B. bassiana via combined transcriptome and proteome analyses. Our results showed that pre-exposure to Cd increased the susceptibility of gypsy moth larvae to B. bassiana, and there was an additive effect between Cd exposure and B. bassiana infection on the larval mortality. Under the Cd stress at low and high concentrations, 138 and 899 differentially expressed genes (DEGs), as well as 514 and 840 differentially expressed proteins (DEPs) were identified, respectively. Immunotoxic effects induced by Cd exposure at the transcription level increased in a negative dose-response manner, with no immunity-related DEGs obtained at the low Cd concentration and a high number of immunity-related DEGs down-regulated at the high Cd concentration. In contrast, a potentially suppressed or stimulated trend in the Toll and Imd signaling pathway at protein level was revealed under low or high concentration of Cd treatment. Analysis of xenobiotics biodegradation-related pathways at both transcription and translation levels revealed that the gypsy moth larvae possessed an efficient homeostasis regulatory mechanism to the low-level Cd exposure, but exhibited a reduced xenobiotics biodegradation capability to the Cd stress at high levels. Together, these findings demonstrate Cd contamination promote the microbial-based biocontrol efficacy, and unravel the molecular regulatory network of heavy metal exposures that affects susceptibility of insects to pathogenic diseases.

BACKGROUND: Mosquito-borne viruses are imposing an ever increasing health burden worldwide. In addition to the recent Zika and chikungunya virus epidemics, dengue viruses have become the fastest growing problem with a 40-fold increase in the number of reported cases over the past five decades. Current mosquito control techniques involving larval source reduction, larviciding, and space spray of adulticides are costly, laborious, and of debatable efficacy. There remains an urgent need for the development of intervention methods that can be reasonably implemented in the context of modern day urbanisation. Auto-dissemination (AD) of insecticide by adult mosquitoes offers a potentially practical and useful tool in an integrated vector control programme. Recently, an immediately employable AD device, the In2Care® mosquito trap, has been commercialised and shows promise as an effective tool. However, there remains a lack of demonstration of epidemiological efficacy.
METHODS: This trial aims to assess the extent to which implementation of In2Care® mosquito traps can reduce vector Aedes (Stegomyia) spp. adult mosquito densities and dengue virus transmission as measured by sequential sero-conversion rates in children 6-16 years of age in a dengue endemic location: Lipa City, Philippines. To achieve this, we will carry out a parallel, two-armed cluster randomised trial evaluating AD efficacy for reducing the incidence of dengue over a 2-year period with 4 consecutive months of vector control during peak dengue transmission each year.
CONCLUSIONS: For decades, it has been commonly accepted that an integrated approach to mosquito control is required. The World Health Organization (WHO) Global Strategic Framework for Integrated Vector Management recommends a range of interventions, in combination, to increase control impact to reduce transmission. This efficacy trial of the first commercial product using the AD approach will be informative in assessing the general utility of AD in reducing not only adult vector densities but, more importantly, reducing the incidence of dengue. The AD technique may complement source reduction and larviciding campaigns by more efficiently targeting the most productive containers and those beyond human reach. If successful, this mosquito control strategy could prove an invaluable tool in the fight against urban mosquito vectors and a reduction in the burden of associated disease.
BACKGROUND: ISRCTN44272773 . Registered on 31 January 2019.

Solenopsis invicta (Buren; Hymenoptera: Formicidae) is a species that has invaded China in recent years. Currently, S. invicta is mainly controlled by chemical treatment, though long-term use of chemical pesticides can cause serious environmental pollution. In this study, a microbial insecticide formulated for the control of S. invicta was screened for laboratory toxicity and field efficacy. The co-toxicity coefficients (CTCs) of the combination of Beauveria bassiana and thiacloprid at various mass ratios were 356.53, 251.20, 182.50, 215.03, and 143.19. When B. bassiana powder and thiacloprid were mixed at a mass ratio of 8:2, the CTC was 356.53, demonstrating a very significant synergistic effect. According to a field efficacy test, at 3 d after treatment, the efficacy of mound injection was significantly better than that of mound drenching. In this study, the insecticidal activity of pathogenic microorganisms against S. invicta was markedly enhanced by using a self-designed apparatus for mound injection of the tested preparation of a complex containing a pathogenic microbe. The results show that S. invicta can be sustainably controlled while ensuring the safety of the environment. The findings are a good reference for the promotion and application of safe control of S. invicta in the future.

It is assumed that social life can lead to the rapid spread of infectious diseases and outbreaks. In ants, disease outbreaks are rare and the expression of collective behaviors is invoked to explain the absence of epidemics in natural populations. Here, we address the ecological approach employed by many studies that have notably focused (89% of the studies) on two genera of generalist fungal parasites (Beauveria and Metarhizium). We ask whether these are the most representative models to study the evolutionary ecology of ant-fungal parasite interactions. To assess this, we critically examine the literature on ants and their interactions with fungal parasites from the past 114years (1900-2014). We discuss how current evolutionary ecology approaches emerged from studies focused on the biological control of pest ants. We also analyzed the ecological relevance of the laboratory protocols used in evolutionary ecology studies employing generalist parasites, as well as the rare natural occurrence of these parasites on ants. After a detailed consideration of all the publications, we suggest that using generalist pathogens such as Beauveria and Metarhizium is not an optimal approach if the goal is to study the evolutionary ecology of disease in ants. We conclude by advocating for approaches that incorporate greater realism.

BACKGROUND: Fungal biopesticides are of great interest to vector control scientists as they provide a novel and environmentally friendly alternative to insecticide use. The aim of this study was to determine whether genes associated with pyrethroid resistance in Anopheles arabiensis from Sudan and South Africa are further induced following exposure to the entomopathogenic fungus, Beauveria bassiana (strain GHA).
METHODS: Following B. bassiana bioassays, RNA was extracted from infected mosquitoes and the transcription of four important insecticide resistance genes, CYP9L1, CYP6M2 and CYP4G16 (cytochrome P450s) and TPX4 (thioredoxin peroxidase) was investigated using quantitative real-time PCR.
RESULTS: Beauveria bassiana strain GHA was highly infective and virulent against An. arabiensis. In terms of changes in gene transcription, overall, the fold change (FC) values for each gene in the infected strains, were lower than 1.5. The FC values of CYP9L1, CYP6M2 and TPX4, were significantly lower than the FC values of the same genes in uninfected resistant An. arabiensis.
CONCLUSIONS: These data suggest that B. bassiana does not enhance the pyrethroid resistant phenotype on a molecular level as the two An. arabiensis strains used here, with different pyrethroid resistance mechanisms, revealed no increase in pre-existing metabolic transcripts. This supports the fact that fungal pathogens are suitable candidates for vector control, particularly with regard to the development of novel vector control strategies.

Insect prophenoloxidase (PPO) is essential for physiological functions such as melanization of invading pathogens, wound healing and cuticle sclerotization. The insect PPO activation pathway is well understood. However, it is not very clear how PPO is released from hemocytes and how PPO takes part in cellular immunity. To begin to assess this, three Drosophila melanogaster PPO genes were separately fused with GFP at the C-terminus (rPPO-GFP) and were over-expressed in S2 cells. The results of staining and morphological observation show that rPPO-GFP expressed in S2 cells has green fluorescence and enzyme activity if Cu(2+) was added during transfection. Each rPPO-GFP has similar properties as the corresponding rPPO. However, cells with rPPO-GFP over-expressed are easier to trace without PO activation and staining. Further experiments show that rPPO1-GFP is cleaved and activated by Drosophila serine protease, and rPPO1-GFP binds to Micrococcus luteus and Beauveria bassiana spores as silkworm plasma PPO. The above research indicates that the GFP-tag has no influence on the fusion enzyme activation and PPO-involved innate immunity action in vitro. Thus, rPPO-GFP may be a convenient tool for innate immunity study in the future if it can be expressed in vivo.