Optimizing the Application Timing and Dosage of Metarhizium brunneum (Hypocreales: Clavicipitaceae) as a Biological Control Agent of Aedes aegypti (Diptera: Culicidae) Larvae.

Aedes aegypti (Diptera: Culicidae) is the principal vector of dengue and other viruses that cause disease among 100 to 400 million people each year. The recent development of widespread insecticidal resistance has led to the rapid development of biological control solutions aimed at larval control. While the efficacy of Metarhizium brunneum has been shown against Aedes larvae, the impact of larval population dynamics will need to be determined to formulate effective control strategies. In this study, larvae were subjected to four concentrations of M. brunneum (105, 106, 107, 108 conidia ml-1). Larvae were found to be susceptible to M. brunneum with dose-dependent efficacy. When constant larval immigration was added as a parameter, peak mortality was consistently found to occur on the fourth day, before a significant reduction in control efficacy linked to a decline in conidial availability within the water column. This suggests that M. brunneum treatments should be applied at a concentration 1 × 107 conidia ml-1 every four days to effectively control mosquito larvae in the field, regardless of the fungal formulation, water volume, or larval density. Understanding fungal-mosquito dynamics is critical in developing appropriate control programs as it helps optimize the fungal control agent's dose and frequency of application.

Virulence of Metarhizium brunneum (Ascomycota: Hypocreales) Strains Against Stinkbugs Euschistus heros and Dichelops furcatus (Hemiptera: Pentatomidae).

Three strains of fungi belonging to the genus Metarhizium Sorokin (ARSEF 4556, ARSEF 3297, native strain) were assayed against adults and nymphs of the Neotropical brown stinkbug Euschistus heros (F.) and the green-belly stinkbug Dichelops furcatus (F.). The most virulent strain, ARSEF 4556, caused over 90% mortality. The average survival time of the second and fifth instar nymphs and adults following immersion in 1 × 108 conidia ml-1 was 4.8, 5.7, and 5.2 d, respectively. The second instar nymphs were more susceptible than the adults. The LC50 values and median survival times for second instar and adult E. heros were 1.6 × 107 and 3.1 × 107 conidia ml-1 and 6 and 8 d, respectively. Eggs of E. heros and the closely related stinkbug, D. furcatus, were highly susceptible to ARSEF 4556 with the mean mortality of eggs immersed in 1 × 108 conidia ml-1 being 77.4% and 89.7%, respectively. The strain 3297 showed also good aptitudes for stinkbugs control with mortalities higher than 80% against nymphs and adults and eggs mortalities of 75.5% for E. heros and 79.6% for D. furcatus. This study has shown that it is possible to have a two-pronged control strategy, targeting adults and to reduce oviposition and targeting egg clusters to prevent emergence and dispersal of nymphs. Besides early instars of nymphs have been shown to be more susceptible to the fungal strains than late instars and adults.

Differential Pathogenicity of Metarhizium Blastospores and Conidia Against Larvae of Three Mosquito Species.

Biorational insecticides are being increasingly used in integrated pest management programs. In laboratory bioassays, the pathogenicity of blastospores and conidia of the entomopathogenic fungus Metarhizium brunneum ARSEF 4556 was evaluated against larvae of three mosquito species. Three propagule concentrations (1 × 106, 1 × 107, and 1 × 108 spores ml - 1) were used in the bioassays. Results showed that Aedes aegypti had lower survival rates when exposed to blastospores than when exposed to conidia, whereas the converse was true for Culex quinquefasciatus larvae. Anopheles stephensi larvae survival rates were similar when exposed to blastospores and conidia, except at the higher doses, where blastospores were more virulent. Several assays showed little difference in mortalities when using either 1 × 107 or 1 × 108 spores ml - 1, suggesting a threshold above which no higher control levels or economic benefit would be achieved. When tested at the lowest dose, the LT50 of Cx. quinquefasciatus using blastospores, wet conidia, and dry conidia was 3.2, 1.9, and 4.4 d, respectively. The LT50 of Ae. aegypti using blastospores, wet conidia, and dry conidia was 1.3, 3.3, and 6.2 d, respectively. The LT50 of An. stephensi using blastospores, wet conidia, and dry conidia was 2.0, 1.9, and 2.1 d, respectively. These observations suggest that for optimized control, two different formulations of the fungus may be needed when treating areas where there are mixed populations of Aedes, Anopheles, and Culex.

Entomopathogenic Fungi: New Insights into Host-Pathogen Interactions.

Although many insects successfully live in dangerous environments exposed to diverse communities of microbes, they are often exploited and killed by specialist pathogens. Studies of host-pathogen interactions (HPI) provide valuable insights into the dynamics of the highly aggressive coevolutionary arms race between entomopathogenic fungi (EPF) and their arthropod hosts. The host defenses are designed to exclude the pathogen or mitigate the damage inflicted while the pathogen responds with immune evasion and utilization of host resources. EPF neutralize their immediate surroundings on the insect integument and benefit from the physiochemical properties of the cuticle and its compounds that exclude competing microbes. EPF also exhibit adaptations aimed at minimizing trauma that can be deleterious to both host and pathogen (eg, melanization of hemolymph), form narrow penetration pegs that alleviate host dehydration and produce blastospores that lack immunogenic sugars/enzymes but facilitate rapid assimilation of hemolymph nutrients. In response, insects deploy an extensive armory of hemocytes and macromolecules, such as lectins and phenoloxidase, that repel, immobilize, and kill EPF. New evidence suggests that immune bioactives work synergistically (eg, lysozyme with antimicrobial peptides) to combat infections. Some proteins, including transferrin and apolipophorin III, also demonstrate multifunctional properties, participating in metabolism, homeostasis, and pathogen recognition. This review discusses the molecular intricacies of these HPI, highlighting the interplay between immunity, stress management, and metabolism. Increased knowledge in this area could enhance the efficacy of EPF, ensuring their future in integrated pest management programs.

More than a colour change: insect melanism, disease resistance and fecundity.

A 'dark morph' melanic strain of the greater wax moth, Galleria mellonella, was studied for its atypical, heightened resistance to infection with the entomopathogenic fungus, Beauveria bassiana. We show that these insects exhibit multiple intraspecific immunity and physiological traits that distinguish them from a non-melanic, fungus-susceptible morph. The melanic and non-melanic morphs were geographical variants that had evolved different, independent defence strategies. Melanic morphs exhibit a thickened cuticle, higher basal expression of immunity- and stress-management-related genes, higher numbers of circulating haemocytes, upregulated cuticle phenoloxidase (PO) activity concomitant with conidial invasion, and an enhanced capacity to encapsulate fungal particles. These insects prioritize specific augmentations to those frontline defences that are most likely to encounter invading pathogens or to sustain damage. Other immune responses that target late-stage infection, such as haemolymph lysozyme and PO activities, do not contribute to fungal tolerance. The net effect is increased larval survival times, retarded cuticular fungal penetration and a lower propensity to develop haemolymph infections when challenged naturally (topically) and by injection. In the absence of fungal infection, however, the heavy defence investments made by melanic insects result in a lower biomass, decreased longevity and lower fecundity in comparison with their non-melanic counterparts. Although melanism is clearly correlated with increased fungal resistance, the costly mechanisms enabling this protective trait constitute more than just a colour change.

Effects of successive subculturing on stability, virulence, conidial yield, germination and shelf-life of entomopathogenic fungi.

AIMS: To determine the stability and conidial yield of two strains of the entomopathogenic fungus Metarhizium anisopliae and one strain of M. brunneum, being developed for the control of insect pests. METHODS AND RESULTS: The conidial yields and the shelf-life of the conidia of two commercially viable strains of M. anisopliae V275 (=F52) and ARSEF 4556 and one strain of M. brunneum (ARSEF 3297) were determined after harvesting conidia from in vitro subcultures on Sabouraud dextrose agar (SDA) and broken basmati rice. The strains were stable and showed no decline in virulence against Tenebrio molitor, even when subcultured successively 12 times on SDA. Conidia-bound Pr1 protease activity decreased in conidia harvested from SDA and mycosed cadavers after the 1st subculture, but increased in conidia produced on rice. The C:N ratio of conidia from mycosed cadavers was lower than that of conidia from rice or SDA. Irrespective of the number of subcultures, strain ARSEF 4556 produced significantly higher conidial yields than ARSEF 3297 and V275. The 12th subculture of V275 and ARSEF 3297 produced the lowest conidial yield. Shelf-life studies showed that conidia of strain ARSEF 4556 had a higher conidial viability than V275 and ARSEF 3297 after 4 months, stored at 4°C. CONCLUSIONS: The current study shows that determining strain stability and conidial yield through successive subculturing is an essential component for selecting the best strain for commercial purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to compare quality control parameters in the production of conidia on rice, and it shows that the level of Pr1 is comparatively high for inoculum produced on rice.

Susceptibility of Culicoides biting midge larvae to the insect-pathogenic fungus, Metarhizium anisopliae: prospects for bluetongue vector control.

Culicoides biting midges (Diptera: Ceratopogonidae) are responsible for the spread of several arboviruses of livestock and humans that are of international importance. This study assesses the virulence of 18 insect-pathogenic fungal strains from the genera Metarhizium, Beauveria, Isaria and Lecanicillium to larval stages of Culicoides nubeculous Meigen as a means of examining their potential as biocontrol agents. In initial screening, six strains of M. anisopliae (ERL700, CA1, V275, LRC181A, ARSEF 3291 and ARSEF 4556) outperformed the other tested genera and were found to cause between 90% and 100% larval mortality in all larval instars of this species at 72 h post inoculation. The virulence of the most effective strain, M. anisopliae V275, was then further tested by exposing larvae to doses which ranged from 10(4)-10(8) conidia/ml and recording mortality at 24, 48 and 72 h in a 24-multi-well plate with each well containing 600 microl of water and at 24 and 48 h in 250 ml plastic cups containing 50 ml of water. Sensitivity of larvae was extremely high in the multi-well plates, with LC(50) values of 4.3-4.5 x 10(3)conidia/ml and no significant differences between larval instars. In the 250 ml cups, M. anisopliae V275 caused mortalities of between 70% and 100% to larvae and later instars exhibited higher mortality rates. The results are discussed in relation to incorporation of M. anisopliae into biocontrol programmes to control arboviruses vectored by Culicoides.

Molecular monitoring and evaluation of the application of the insect-pathogenic fungus Beauveria bassiana in southeast China.

AIMS: To monitor the fate of inundatively applied strains of Beauveria bassiana against Masson's pine caterpillar, Dendrolimus punctatus, in Magushan Forest, southeast China, and evaluate the potential environmental risks of biocontrol introduction. METHODS AND RESULTS: Filed samplings of mycosed cadavers were carried out continuously for more than one year after the inundative applications of two exotic B. bassiana strains. By employing four complementary molecular markers, the purified cultures from collections were characterized and the two released strains Bb17 and Bb13 were recovered with respective frequencies of 9.1 and 5.2%. The released strains were isolated from nontarget hosts. The genetic diversity of the population of B. bassiana in the trial plot changed dynamically over time and no single genotype strain dominated throughout the sampling period or at different seasons. The indigenous strains were observed to be predominant in the local environment. Co-infection and/or genetic recombination might occur between strains under natural conditions. CONCLUSION: Field applications of the biocontrol agent B. bassiana appear to pose no major risk to the environment. There was no evidence of displacement of indigenous strains of B. bassiana. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that the introduction of exotic B. bassiana strains can persist in local environment and infect nontarget insect hosts, highlighting the importance of safety assessment of the large-scale application of fungal biocontrol agents.

Molecular studies of co-formulated strains of the entomopathogenic fungus, Beauveria bassiana.

A 28S rDNA intron was used as a molecular marker to distinguish between two single spore strains of Beauveria bassiana, Bb123 and Bb151. When co-formulated and assayed against larvae of Galleria mellonella these strains exhibited no synergistic increase in virulence, rather Bb123 usually dominated. This study shows that the success of any strain to infect Galleria is dependent on the dose and method of inoculation (injection versus immersion). The result of co-formulated strains grown on solid culture also showed that usually one strain dominated, i.e., strain displacement could happen both in vivo and in vitro. The speed by which one strain was displaced following successive sub-culturing on PDA partly depended on the ratio of Bb151 and Bb123. The co-formulated inoculum could widen the window over which parent strains would be active on different water activity media. Co-infection did result in heterokaryosis within the Galleria host. Molecular studies also showed that the heterokaryon was not stable and could revert back to the parent strain.

Effects of efrapeptin and destruxin, metabolites of entomogenous fungi, on the hydrolytic activity of a vacuolar type ATPase identified on the brush border membrane vesicles of Galleria mellonella midgut and on plant membrane bound hydrolytic enzymes.

The brush border membrane of the insect midgut is an initial site for interaction of insecticidal proteins. We have investigated the possibility that it may contain a target site for two insecticidal fungal toxins, destruxin and efrapeptin, both of which are ATPase inhibitors. We have studied the effects of the toxins on the hydrolytic activity of a vacuolar type ATPase (V-ATPase) that we have identified from Galleria mellonella midgut columnar cell brush border membrane vesicles (BBMV) by its cation and pH dependence, sensitivity to proton pump inhibitors and K(m) (0.49 mM ATP). Efrapeptin strongly inhibited the BBMV V-ATPase but destruxin had little effect. We compared the effects of the inhibitors on known plant membrane hydrolytic enzymes, and although the vacuolar pyrophosphatase and plasma membrane ATPase were not inhibited by the toxins, the V-ATPase from mung bean, but not barley, was inhibited (50%) by 10 microM concentrations of both compounds. Different forms of the toxins were tested on the ATPases and destruxin B and efrapeptin F were the most effective. Kinetic analysis showed that the purified forms of both compounds inhibited the V-ATPases uncompetitively and modelling of data for inhibition of the BBMV V-ATPase by efrapeptin at concentrations of 0.06--12 microM yielded a K(i) of 0.125 microM.

The subtilisins of the invertebrate mycopathogens Verticillium chlamydosporium and Metarhizium anisopliae are serologically and functionally related.

The major extracellular proteases from the nematophagous fungus Verticillium chlamydosporium and the entomophagous fungus Metarhizium anisopliae, VCP1 and Pr1, respectively, are closely related both functionally and serologically. Antibodies raised against either enzyme cross-reacted with both antigens, suggesting that they have common epitopes. The VCP1 and Pr1 antisera labelled bovine pancreatic elastase and proteinase K, respectively. Neither antiserum reacted with commercial chymotrypsin. An antiserum to a serine protease from the closely related V. suchlasporium also cross-reacted with VCP1 and Pr1. In contrast, a polyclonal antibody to an isoform of Pr1 exclusive to M. anisopliae isolate ME1 failed to recognize Pr1 from M. anisopliae V245 or VCP1. The N-terminal amino acid sequence of VCP1 revealed similarities with subtilisin-like enzymes from other fungi, but the closest match was with Pr1. The pure enzymes, VCP1 and Pr1, failed to hydrolyse mono-aminoacyl-naphthylamide substrates but demonstrated dipeptidyl peptidase activity against Gly-Pro-beta NA and Leu-Ala-beta NA, respectively. These results are discussed in the context of specificity of invertebrate mycopathogens.

The nematophagous fungus Verticillium chlamydosporium produces a chymoelastase-like protease which hydrolyses host nematode proteins in situ.

The nematophagous fungus Verticillium chlamydosporium secreted several proteases in submerged culture in which soya peptone was the sole carbon and nitrogen source. One protease, VCP1 (M(r) 33,000, pI 10.2), was purified 14-fold from culture filtrates to apparent homogeneity using preparative isoelectric focusing in free solution, and shown to rapidly hydrolyse the chymotrypsin substrate Suc-(Ala)2-Pro-Phe-pNA and elastin. VCP1 had a Km for Suc-(Ala)2-Pro-Phe-pNA of 4.3 x 10(-5) M and a kcat of 5.8 s-1. It was highly sensitive to PMSF and TPCK, but only moderately sensitive to chicken egg-white and soya bean trypsin inhibitors. VCP1 degraded a wide range of polymeric substrates, including Azocoll, hide protein, elastin, casein and albumin, and accounted for most of the non-specific protease activity detected in culture filtrates. The purified enzyme hydrolysed proteins in situ from the outer layer of the egg shell of the host nematode Meloidogyne incognita and exposed its chitin layer. VCP1 was secreted by several isolates of V. chlamydosporium and V. lecanii, pathogens of nematodes and insects respectively, but not plant-pathogenic species of Verticillium. These observations suggest that VCP1 or similar enzyme(s) may play a role in the infection of invertebrates.

The changing distribution of actin and nuclear behavior during the cell cycle of the mite-pathogenic fungus Neozygites sp.

We have analyzed the behavior of nuclei and actin during the cell cycle of Neozygites sp. with mithramycin and rhodamine-labeled phalloidin. This fungus is an entomophagous zygomycete which grows as a rod-shaped fission yeast containing 2 to 12, mostly 3 to 4, nuclei per cell. The cell cycle is regulated such that there is not a constant nucleus-to-cytoplasmic volume ratio, and mitosis is initiated slightly asynchronously from one end of the cell. During interphase, detected actin occurs exclusively as peripheral plaques, which are most abundant at growing cell tips, and as perinuclear shells. Because the shells disperse and reform concomitantly with the formation and breakdown of a new septum-associated actin array, we infer that they are a novel form of actin storage. Intranuclear mitosis occurs in the absence of detectable spindle actin which suggests that actin is not a universal feature of mitotic systems and may be a cytoplasmic contaminant in open spindles of plant cells. Actin is involved in septum synthesis in previously unreported ways. Prior to morphologically detectable septum initiation, a peripheral equatorial band of longitudinal actin filaments assembles and then shortens to a transverse belt at the future site of septum synthesis. We suggest that this actin array recruits and organizes cell wall synthetic complexes for subsequent septum growth. During detectable septum synthesis, the invaginating plasmalemma bears plaques at a similar concentration to those at growing cellular tips.