Enhancement of an entomopathogenic fungal virulence against the seedcorn maggot, Delia platura, by suppressing immune responses with a bacterial culture broth of Photorhabdus temperata subsp. temperata.

In Korea, there are two maggot species in the Delia genus that commonly infest the roots and stems of the Welsh onion, thus causing serious economic damage on the crop at the seedling stage. In this study, the seedcorn maggot (Delia platura) was detected in onion fields in two different localities in Korea. After overwintering, maggot infestations occurred throughout the entire growing seasons from transplantation to harvest, but their specific patterns of occurrence varied in the two localities examined. Entomopathogenic fungi induced significant virulence against the maggot larvae, in which a strain of Beauveria bassiana was effective, though it exhibited limited mortality in its insecticidal activity. To enhance this insecticidal activity, a culture broth from an entomopathogenic bacterium, Photorhabdus temperata temperata (Ptt), was added to B. bassiana treatment. The addition of Ptt broth significantly increased the insecticidal activity of B. bassiana in a dose-dependent manner. To elucidate this enhancement in insecticidal activity, the immunosuppressive activity of Ptt broth was assessed by identifying the immune responses of the seedcorn maggots. The seedcorn maggots possessed at least three different hemocytes with plasmatocytes, crystal cells, and lamellocytes. These hemocytes exhibited nodule formation in response to the fungal infection. In addition to the cellular immunity, the maggots exhibited inducible expressions of antimicrobial peptide (AMP) genes such as cecropin and defensin. The addition of Ptt broth suppressed the nodule formation and the AMP expressions in response to the fungal infection. Altogether, this study demonstrated the innate immune responses in a non-model insect, D. platura, along with the application of immunosuppression to develop a highly efficient biological control by enhancing the virulence of B. bassiana.

Activity of natural occurring entomopathogenic fungi on nymphal and adult stages of Philaenus spumarius.

The spittlebug Philaenus spumarius (Hemiptera: Aphrophoridae) is the predominant vector of Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae) in Apulia, Italy and the rest of Europe. Current control strategies of the insect vector rely on mechanical management of nymphal stages and insecticide application against adult populations. Entomopathogenic fungi (EPF) are biological control agents naturally attacking spittlebugs and may effectively reduce population levels of host species. Different experimental trials in controlled conditions have been performed to i) identify naturally occurring EPF on P, spumarius in Northwestern Italy, and ii) evaluate the potential for biocontrol of the isolated strains on both nymphal and adult stages of the spittlebug. Four EPF species were isolated from dead P. spumarius collected in semi-field conditions: Beauveria bassiana, Conidiobolus coronatus, Fusarium equiseti and Lecanicillium aphanocladii. All the fungal isolates showed entomopathogenic potential against nymphal stages of P. spumarius (≈ 45 % mortality), except for F. equiseti, in preliminary trials. No induced mortality was observed on adult stage. Lecanicillium aphanocladii was the most promising fungus and its pathogenicity against spittlebug nymphs was further tested in different formulations (conidia vs blastospores) and with natural adjuvants. Blastospore formulation was the most effective in killing nymphal instars and reducing the emergence rate of P, spumarius adults, reaching mortality levels (90%) similar to those of the commercial product Naturalis®, while no or adverse effect of natural adjuvants was recorded. The encouraging results of this study pave way for testing EPF isolates against P, spumarius in field conditions and find new environmentally friendly control strategies against insect vectors of X. fastidiosa.

Antimicrobial peptide cecropin B functions in pathogen resistance of Mythimna separata.

Mythimna separata (Lepidoptera: Noctuidae) is an omnivorous pest that poses a great threat to food security. Insect antimicrobial peptides (AMPs) are small peptides that are important effector molecules of innate immunity. Here, we investigated the role of the AMP cecropin B in the growth, development, and immunity of M. separata. The gene encoding M. separata cecropin B (MscecropinB) was cloned. The expression of MscecropinB was determined in different developmental stages and tissues of M. separata. It was highest in the prepupal stage, followed by the pupal stage. Among larval stages, the highest expression was observed in the fourth instar. Tissue expression analysis of fourth instar larvae showed that MscecropinB was highly expressed in the fat body and haemolymph. An increase in population density led to upregulation of MscecropinB expression. MscecropinB expression was also upregulated by the infection of third and fourth instar M. separata with Beauveria bassiana or Bacillus thuringiensis (Bt). RNA interference (RNAi) targeting MscecropinB inhibited the emergence rate and fecundity of M. separata, and resulted in an increased sensitivity to B. bassiana and Bt. The mortality of M. separata larvae was significantly higher in pathogen plus RNAi-treated M. separata than in controls treated with pathogens only. Our findings indicate that MscecropinB functions in the eclosion and fecundity of M. separata and plays an important role in resistance to infection by B. bassiana and Bt.

Influences of compound age and identity in the effectiveness of insect quinone secretions against the fungus Beauveria bassiana.

Chemical defences against parasites and pathogens can be seen in a wide range of animal taxa, including insect pests such as the red flour beetle Tribolium castaneum. Antimicrobial quinone-based secretions can be used by these beetles to defend against various parasites, particularly the fungal entomopathogen Beauveria bassiana. While quinone secretions can inhibit B. bassiana growth, it is unknown how long they remain effective or how individual secretion compounds contribute to growth inhibition. Here, we tested each individual component of the quinone secretions (methyl-1,4-benzoquinone, ethyl-1,4-benzoquinone, and 1-pentadecene), as well as two mixed solutions that represent the composition range found in natural T. castaneum secretions, after aging for 0, 24, or 72 h. The two quinone compounds equally contributed to B. bassiana inhibition, but their efficacy was significantly reduced after 24 h, with no growth inhibition after 72 h. This indicates that quinones protect insects against B. bassiana for only a limited time, perhaps requiring constant secretion into the environment to effectively defend against this fungal threat. Future investigations may consider the extent to which quinone secretions are effective against other parasites, as well as how their ability to cause parasite damage changes with compound age.

The fungal protease BbAorsin contributes to growth, conidiation, germination, virulence, and antiphytopathogenic activities in Beauveria bassiana (Hypocreales: Cordycipitaceae).

The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), is one of the most destructive agricultural pests. The entomopathogenic fungus Beauveria bassiana (Hypocreales: Clavicipitaceae) is a biopesticide widely used for biocontrol of various pests. Secreted fungal proteases are critical for insect cuticle destruction and successful infection. We have previously shown that the serine protease BbAorsin in B. bassiana has entomopathogenic and antiphytopathogenic activities. However, the contribution of BbAorsin to fungal growth, conidiation, germination, virulence and antiphytopathogenic activities remains unclear. In this study, the deletion (ΔBbAorsin), complementation (Comp), and overexpression (BbAorsinOE) strains of B. bassiana were generated for comparative studies. The results showed that ΔBbAorsin exhibited slower growth, reduced conidiation, lower germination rate, and longer germination time compared to WT and Comp. In contrast, BbAorsinOE showed higher growth rate, increased conidiation, higher germination rate and shorter germination time. Injection of BbAorsinOE showed the highest virulence against S. frugiperda larvae, while injection of ΔBbAorsin showed the lowest virulence. Feeding BbAorsinOE resulted in lower pupation and adult eclosion rates and malformed adults. 16S rRNA sequencing revealed no changes in the gut microbiota after feeding either WT or BbAorsinOE. However, BbAorsinOE caused a disrupted midgut, leakage of gut microbiota into the hemolymph, and upregulation of apoptosis and immunity-related genes. BbAorsin can disrupt the cell wall of the phytopathogen Fusarium graminearum and alleviate symptoms in wheat seedlings and cherry tomatoes infected with F. graminearum. These results highlight the importance of BbAorsin for B. bassiana and its potential as a multifunctional biopesticide.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway contributes to the defense against bacterial infection in the pea aphid.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is activated by infections of bacteria, fungi, viruses and parasites and mediated cellular and humoral immune responses. In the pea aphid Acyrthosiphon pisum little is known about the function of JAK/STAT signaling in its immune system. In this study, we first showed that expression of genes in the JAK/STAT signaling, including the receptors Domeless1/2, Janus kinase (JAK) and transcriptional factor Stat92E, is up-regulated upon bacteria Escherichia coli and Staphylococcus aureus and fungus Beauveria bassiana infections. After knockdown of expression of these genes by means of dsRNA injection, the aphids harbored more bacteria and suffered more death after infected with E. coli and S. aureus, but showed no significant change after B. bassiana infection. Our study suggests the JAK/STAT signaling contributes to the defense against bacterial infection in the pea aphid.

Comprehensive Insights into the Remarkable Function and Regulatory Mechanism of FluG during Asexual Development in Beauveria bassiana.

Asexual development is the main propagation and transmission mode of Beauveria bassiana and the basis of its pathogenicity. The regulation mechanism of conidiation and the key gene resources for utilization are key links to improving the conidia yield and quality of Beauveria bassiana. Their clarification may promote the industrialization of fungal pesticides. Here, we compared the regulation of morphology, resistance to external stress, virulence, and nutrient utilization capacity between the upstream developmental regulatory gene fluG and the key genes brlA, abaA, and wetA in the central growth and development pathway. The results showed that the ΔbrlA and ΔabaA mutants completely lost the capacity to conidiate and that the ΔwetA mutant had seriously reduced conidiation capacity. Although the deletion of fluG did not reduce the conidiation ability as much as deletions of brlA, abaA, and wetA, it significantly reduced the fungal response to external stress, virulence, and nutrient utilization, while the deletion of the three other genes had little effect. Via transcriptome analysis and screening the yeast nuclear system library, we found that the differentially expressed genes in the ΔfluG mutants were concentrated in the signaling pathways of ABC transporters, propionate metabolism, tryptophan metabolism, DNA replication, mismatch repair, and fatty acid metabolism. FluG directly acted on 40 proteins that were involved in various signaling pathways such as metabolism, oxidative stress, and cell homeostasis. The analysis indicated that the regulatory function of fluG was mainly involved in DNA replication, cell homeostasis, fungal growth and metabolism, and the response to external stress. Our results revealed the biological function of fluG in asexual development and the responses to several environmental stresses as well as its influence on the asexual development regulatory network in B. bassiana.

Beauveria bassiana rewires molecular mechanisms related to growth and defense in tomato.

Plant roots can exploit beneficial associations with soil-inhabiting microbes, promoting growth and expanding the immune capacity of the host plant. In this work, we aimed to provide new information on changes occurring in tomato interacting with the beneficial fungus Beauveria bassiana. The tomato leaf proteome revealed perturbed molecular pathways during the establishment of the plant-fungus relationship. In the early stages of colonization (5-7 d), proteins related to defense responses to the fungus were down-regulated and proteins related to calcium transport were up-regulated. At later time points (12-19 d after colonization), up-regulation of molecular pathways linked to protein/amino acid turnover and to biosynthesis of energy compounds suggests beneficial interaction enhancing plant growth and development. At the later stage, the profile of leaf hormones and related compounds was also investigated, highlighting up-regulation of those related to plant growth and defense. Finally, B. bassiana colonization was found to improve plant resistance to Botrytis cinerea, impacting plant oxidative damage. Overall, our findings further expand current knowledge on the possible mechanisms underlying the beneficial role of B. bassiana in tomato plants.

Co-application of entomopathogenic fungi with chemical insecticides against Culex pipiens.

Culex pipiens (Diptera: Culicidae) is a vector of many human and animal diseases. Its control is regarded as a preventative approach that is focused on effectively managing such diseases. In this context, dose response assays of two insecticides, bendiocarb and diflubenzuron were performed with two entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae against 3rd instar C. pipiens larvae. The most effective agents, combination experiments as well as enzymatic activities of phenoloxidase (PO) and chitinase (CHI) were also assessed. The results showed that diflubenzuron was more effective at low concentrations (LC50: 0.001 ppm) than bendiocarb (LC50: 0.174 ppm), whereas M. anisopliae was more effective (LC50: 5.2 × 105 conidia/mL) than B. bassiana (LC50: 7.5 × 107 conidia/mL). Synergistic interactions were observed when diflubenzuron was applied at 2- and 4-days post- exposure to M. anisopliae, with the highest degree of synergism observed when diflubenzuron was applied 2 days post-fungal exposure (χ2 = 5.77). In contrast, additive interactions were recorded with all other insecticide-fungal combinations. PO activities significantly (p ≤ 0.05) increased during 24 h after a single diflubenzuron treatment as well as when diflubenzuron was applied prior to M. anisopliae, whereas suppressed after 24 h when M. anisopliae applied prior to diflubenzuron as well as after 48 h from single and combined treatments. CHI activity increased 24 h after both single and combined treatments, the activity remained elevated 48 h after a single diflubenzuron treatment and when diflubenzuron was applied after M. anisopliae. Histological study of the cuticle by transmission electron microscopy revealed abnormalities following single and combined treatments. Germination of the conidia and production of the mycelium that colonizes the lysing cuticle was obvious when diflubenzuron was applied 48 h after M. anisopliae exposure. Overall, these results demonstrate that M. anisopliae is compatible with diflubenzuron at lower concentrations and that combined applications can improve C. pipiens management.

Susceptibility of Hyphantria cunea larvae to Beauveria bassiana under Cd Stress: An integrated study of innate immunity and energy metabolism.

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.

The Entomopathogenic Fungi Metarhizium brunneum and Beauveria bassiana Promote Systemic Immunity and Confer Resistance to a Broad Range of Pests and Pathogens in Tomato.

Biocontrol agents can control pathogens by reenforcing systemic plant resistance through systemic acquired resistance (SAR) or induced systemic resistance (ISR). Trichoderma spp. can activate the plant immune system through ISR, priming molecular mechanisms of defense against pathogens. Entomopathogenic fungi (EPF) can infect a wide range of arthropod pests and play an important role in reducing pests' population. Here, we investigated the mechanisms by which EPF control plant diseases. We tested two well studied EPF, Metarhizium brunneum isolate Mb7 and Beauveria bassiana as the commercial product Velifer, for their ability to induce systemic immunity and disease resistance against several fungal and bacterial phytopathogens, and their ability to promote plant growth. We compared the activity of these EPF to an established biocontrol agent, Trichoderma harzianum T39, a known inducer of systemic plant immunity and broad disease resistance. The three fungal agents were effective against several fungal and bacterial plant pathogens and arthropod pests. Our results indicate that EPF induce systemic plant immunity and disease resistance by activating the plant host defense machinery, as evidenced by increases in reactive oxygen species production and defense gene expression, and that EPF promote plant growth. EPF should be considered as control means for Tuta absoluta. We demonstrate that, with some exceptions, biocontrol in tomato can be equally potent by the tested EPF and T. harzianum T39, against both insect pests and plant pathogens. Taken together, our findings suggest that EPF may find use in broad-spectrum pest and disease management and as plant growth promoting agents.

Cellular immune responses of the yellow peach moth, Conogethes punctiferalis (Lepidoptera: Crambidae), to the entomopathogenic fungus, Beauveria bassiana (Hypocreales: Cordycipitaceae).

The yellow peach moth (YPM), Conogethes punctiferalis, is a destructive insect pest of maize in eastern China and adapts to diverse environments, especially against pathogens. In insects, innate immunity comprising both humoral and cellular defense responses, is the primary defense against invading microbial pathogens. In this study, we identified five types of circulating hemocytes from the hemolymph of YPM larvae and analyzed their alterations and functions in immune responses to the infection of Beauveria bassiana, an entomopathogenic fungus infesting many lepidopteran species. The identified hemocytes included prohemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids. Significant decreases of total and differential hemocyte counts were recorded over time in larvae, after they were injected with B. bassiana conidia. Additionally, hemocyte-mediated phagocytosis and nodulation were initiated in the hemolymph of larvae from the B. bassiana conidia challenge. The introduction of DEAE-Sepharose Fast Flow beads stained with Congo red also induced a strong encapsulation response in the larval hemolymph. Our observations unravel the occurrence of phagocytosis, nodulation and encapsulation in the hemocoel of YPM larvae to fight against the fungal infection, and offer the first insight into the YPM immune system.

Entomopathogenic fungi and Schinus molle essential oil: The combination of two eco-friendly agents against Aedes aegypti larvae.

Aedes aegypti transmits arbovirus, which is a public health concern. Certain filamentous fungi have the potential to control the disease. Here, the effects of Metarhizium anisopliae s.l. CG 153, Beauveria bassiana s.l. CG 206 and Schinus molle L. were investigated against Aedes aegypti larvae. In addition, the effect of essential oil on fungal development was analyzed. Fungal germination was assessed after combination with essential oil at 0.0025 %, 0.0075 %, 0.005 %, or 0.01 %; all of the oil concentrations affected germination except 0.0025 % (v/v). Larvae were exposed to 0.0025 %, 0.0075 %, 0.005 %, or 0.01 % of the essential oil or Tween 80 at 0.01 %; however, only the essential oil at 0.0025 % achieved similar results as the control. Larvae were exposed to fungi at 107 conidia mL-1 alone or in combination with the essential oil at 0.0025 %. Regardless of the combination, M. anisopliae reduced the median survival time of mosquitoes more than B. bassiana. The cumulative survival of mosquitoes exposed to M. anisopliae alone or in combination with essential oil was 7.5 % and 2 %, respectively, and for B. bassiana, it was 75 % and 71 %, respectively. M. anisopliae + essential oil had a synergistic effect against larvae, whereas B. bassiana + essential oil was antagonistic. Scanning and transmission electron microscopy, and histopathology confirmed that the interaction of M. anisopliae was through the gut and hemocoel. In contrast, the mosquito's gut was the main route for invasion by B. bassiana. Results from gas chromatography studies demonstrated sabinene and bicyclogermacrene as the main compounds of S. molle, and the in-silico investigation found evidence that both compounds affect a wide range of biological activity. For the first time, we demonstrated the potential of S. molle and its interaction with both fungal strains against A. aegypti larvae. Moreover, for the first time, we reported that S. molle might be responsible for significant changes in larval physiology. This study provides new insights into host-pathogen interplay and contributes to a better understanding of pathogenesis in mosquitoes, which have significant consequences for biological control strategies.

Biological responses of Tetranychus urticae (Acari: Tetranychidae) to sub-lethal concentrations of the entomopathogenic fungus Beauveria bassiana.

Tetranychus urticae (Acari: Tetranychidae) is one of the most important pests of agricultural crops with worldwide distribution causing considerable damage to different products. Application of chemical acaricides is one of the most important strategies used for the control of this pest. Entomopathogenic fungi, however, have been proposed as alternative control agents. In this study, sub-lethal effects (LC10 = 6.76 × 102, LC20 = 8.74 × 103, and LC30 = 55.38 × 103 conidia ml-1) of Beauveria bassiana strain TV on the life table parameters of T. urticae were evaluated under laboratory conditions. The results demonstrated that by increasing the concentration, a significant decline was observed in adult longevity of both male and female individuals. Total fecundity of T. urticae was calculated as 45.16, 36.28, 23.98, and 18.21 eggs in control, LC10, LC20, and LC30 treatments, respectively. Sub-lethal concentrations drastically affected the population parameters of this mite pest. The intrinsic rate of increase (r) ranged from 0.1983 to 0.1688 day-1 for the mites treated with distilled water and LC20 treatments, respectively. The net reproductive rate (R0) was affected by the sub-lethal concentrations (lower value at LC30 concentration: 11.19 offspring/individual). Considering the detrimental effects of B. bassiana on some biological parameters of T. urticae, it can be concluded that this product can be used to develop targeted interventions aimed at integrated pest management of this pest.

A pair of native fungal pathogens drives decline of a new invasive herbivore.

Two North American fungal pathogens caused a coepizootic leading to localized collapse of an outbreak population of the newly invasive planthopper pest, the spotted lanternfly (Lycorma delicatula), in the eastern United States. The pathogens partitioned the habitat, with the majority of L. delicatula on tree trunks killed by Batkoa major, while cadavers of L. delicatula killed by Beauveria bassiana were usually on the ground. The future will show whether these pathogens will be drivers in boom-bust cycles or will result in recurrent low population densities of this new invasive species.

Comparative analysis of the immune system and expression profiling of Lymantria dispar infected by Beauveria bassiana.

Lymantria dispar is one of the most devastating forest pests worldwide. Fungal biopesticides have great potential as alternatives owing to their high lethality to pests and eco-friendly feature, which is, however, often severely compromised by the pests' innate immunity. A better understanding of the antifungal immune system in L. dispar would significantly facilitate the development of the biopesticide. Here, we investigated phylogenetic characteristics of immunity-related genes as well as the tissue expression patterns in L. dispar after the infection of an entomopathogen Beauveria bassiana using RNA-sequencing data. Results showed most immune genes remain at a low level of response after 24 h post-infection (HPI). Almost all genes in the Toll pathway were significantly up-regulated at 48 HPI, and SPH1, SPN6, Toll6, Toll12, Myd88, pelle, and Drosal were significantly down-regulated at 72 HPI. Immunoblotting analysis revealed that the protein levels of ßGRP3 and PPO1 were significantly upregulated at 24 and 48 HPI, while Myd88 was downregulated at 24 HPI, which was further confirmed by Quantitative real-time PCR experiments. Moreover, the relative content of H2O2, a potent reactive oxygen species (ROS), was significantly increased with the decrease of the total antioxidant capacity, indicating that oxidative stress system positively participates in the clearance of the pathogenic fungus. Together, our study provides detailed genetic characteristics of antifungal immunity as well as profiling of the host defense against entomopathogenic infection, and comprehensive insight into molecular interaction between L. dispar and the entomopathogen.

Structure-function analysis of PGRP-S1 from the oriental armyworm, Mythimna separata.

Peptidoglycan recognition proteins (PGRPs) are well known for their abilities to recognize or hydrolyze peptidoglycan (PGN), one of the major bacterial cell wall components. However, much less is known about their antifungal activities. PGRP-S1 was previously identified from a crop pest, Mythimna separata (Walker) (Lepidoptera: Noctuidae). PGRP-S1 showed bacteriolytic activities against Gram-positive and Gram-negative bacteria. In this study, tissue expression analysis showed that PGRP-S1 was mainly expressed in the midgut of naïve larvae. The induction analysis showed that it was significantly induced in the larval midgut 12 h post the injection of Beauveria bassiana conidia. To identify the key residues that are related to its microbicidal activities, the structure of PGPR-S1 was predicted for structural comparison and molecular docking analysis. Six residues (H61, H62, Y97, H171, T175, and C179) were mutated to Ala individually by site-directed mutagenesis. The recombinant wild-type (WT) and mutant proteins were expressed and purified. The recombinant proteins bound to different polysaccharides, PGNs, and bacteria. H61A, Y97A, H171A, and C179A lost amidase activity. Accordingly, antibacterial assay and scanning electron microscopy confirmed that only H62A and T175A retained bacteriolytic activities. The germination of B. bassiana conidia was significantly inhibited by WT, H61A, Y97A, T175A, and C179A mutants. Electron microscopy showed that some conidia became ruptured after treatment. The growth of hyphae was inhibited by the WT, H61A, H62A, and T175A. In summary, our data showed that different residues of PGRP-S1 are involved in the antibacterial and antifungal activities.

Blood deprivation and heat stress increase mortality in bed bugs (Cimex lectularius) exposed to insect pathogenic fungi or desiccant dust.

Bed bugs (Cimex lectularius L.) have returned as a nuisance pest in the last 20 years. Different bed bug control measures in combination have not been thoroughly studied, although induction of multiple stressors may improve extermination. The effects of heat stress only, heat stress followed by exposure to insect pathogenic fungi, and heat stress followed by exposure to desiccant dust on starved and blood-fed bed bugs were investigated. Five days at 22 °C (control), 32 °C, 34 °C, or 36 °C (heat stress) did not cause mortality in adults. However, their starved first instar nymphs produced after heat stress suffered mortalities of 33%, 56% and 100%, respectively. Exposure to insect pathogenic fungi after heat stress increased the mortality of adults and their progeny compared to exposure to fungi without heat stress. The beneficial effects of heat stress were not observed in blood-fed bed bugs. Desiccant dust killed all nymphs within 2 days and all adults within 3 days regardless of previous heat stress, but survival time was prolonged by access to blood. This study highlights the advantage of combining different methods in pest management, and points to heat stress combined with blood deprivation as possible management elements to increase the control success.

Earthworms and their cutaneous excreta can modify the virulence and reproductive capability of entomopathogenic nematodes and fungi.

Earthworms are ecological engineers that can contribute to the displacement of biological control agents such as the entomopathogenic nematodes (EPNs) and fungi (EPF). However, a previous study showed that the presence of cutaneous excreta (CEx) and feeding behavior of the earthworm species Eisenia fetida (Haplotaxida: Lumbricidae) compromise the biocontrol efficacy of certain EPN species by reducing, for example, their reproductive capability. Whether this phenomenon is a general pattern for the interaction of earthworms-entomopathogens is still unknown. We hypothesized that diverse earthworm species might differentially affect EPN and EPF infectivity and reproductive capability. Here we investigated the interaction of different earthworm species (Eisenia fetida, Lumbricus terrestris, and Perionyx excavatus) (Haplotaxida) and EPN species (Steinernema feltiae, S. riojaense, and Heterorhabditis bacteriophora) (Rhabditida) or EPF species (Beauveria bassiana and Metarhizium anisopliae) (Hypocreales), in two independent experiments. First, we evaluated the application of each entomopathogen combined with earthworms or their CEx in autoclaved soil. Hereafter, we studied the impact of the earthworms' CEx on entomopathogens applied at two different concentrations in autoclaved sand. Overall, we found that the effect of earthworms on entomopathogens was species-specific. For example, E. fetida reduced the virulence of S. feltiae, resulted in neutral effects for S. riojaense, and increased H. bacteriophora virulence. However, the earthworm P. excavates increased the virulence of S. feltiae, reduced the activity of H. bacteriophora, at least at specific timings, while S. riojaense remained unaffected. Finally, none of the EPN species were affected by the presence of L. terrestris. Also, the exposure to earthworm CEx resulted in a positive, negative or neutral effect on the virulence and reproduction capability depending on the earthworm-EPN species interaction. Concerning EPF, the impact of earthworms was also differential among species. Thus, E. fetida was detrimental to M. anisopliae and B. bassiana after eight days post-exposure, whereas Lumbricus terrestris resulted only detrimental to B. bassiana. In addition, most of the CEx treatments of both earthworm species decreased B. bassiana virulence and growth. However, the EPF M. anisopliae was unaffected when exposed to L. terrestris CEx, while the exposure to E. fetida CEx produced contrasting results. We conclude that earthworms and their CEx can have positive, deleterious, or neutral impacts on entomopathogens that often coinhabit soils, and that we must consider the species specificity of these interactions for mutual uses in biological control programs. Additional studies are needed to verify these interactions under natural conditions.

Antifungal roles of adult-specific cuticular protein genes of the red flour beetle, Tribolium castaneum.

The insect cuticle is a composite structure that can further be divided into a few sub-structural layers. Its large moiety comprises a lattice of chitin fibrils and structural proteins, both of which are stabilized by covalent bonding among them. The cuticle covers the whole surface of insect body, and thus has long been suggested for the involvement in defense against entomopathogens, especially entomopathogenic fungi that infect percutaneously. We have been addressing this issue in the past few years and have so far demonstrated experimentally that chitin synthase 1, laccase2 as well as benzoquinone synthesis-related genes of Tribolium castaneum have indispensable roles in the antifungal host defense. In the present study we focused on another major component of the insect cuticular integument, structural cuticular proteins. We chose three genes coding for adult-specific cuticular proteins, namely CPR4, CPR18 and CPR27, and examined their roles in forming immunologically sound adult cuticular integuments. Analyses of developmental expression revealed that the three genes showed high level expression in the pupal stage. These results are consistent with their proposed roles in constructing cuticle of adult beetles. The RNA interference-mediated gene knockdown was employed to silence these genes, and the administration of double strand RNAs in pupae resulted in the adults with malformed elytra. The single knockdown of the three genes attenuated somewhat the defense of the resulting adult beetles against Beauveria bassiana and Metarhizium anisopliae, but statistical analyses indicated no significant differences from controls. In contrast, the double or triple knockdown mutant beetles displayed a drastic disruption of the host defense against the two entomopathogenic fungal species irrespective of the combination of targeted cuticular protein genes, demonstrating the important roles of the three cuticular protein genes in conferring robust antifungal properties on the adult cuticle. Scanning electron microscopic observation revealed that the germination of conidia attached on the adult body surface was still suppressed after the gene knockdown as in the case of wild-type beetles, suggesting that the weakened antifungal phenotypes resulted from the combined knockdown of the adult-specific cuticular protein genes could not be accounted for by the disfunction of secretion/retention of fungistatic benzoquinone derivatives.