The role of grooming in regulating biomass growth of ants and symbiotic fungi under entomopathogenic fungal infection: experiments and mathematical modelling

Social insect pests, particularly leaf-cutting ants, present a considerable challenge in terms of control. Leaf-cutting ants are significant agricultural, forestry, and pasture pests, and understanding their behavior and defense mechanisms is essential for managing their colonies effectively. While toxic ant baits are a primary control method, the limited availability of effective insecticides and concerns over their hazardous nature has spurred the search for alternative solutions, particularly natural compounds, which aligns with the goals of forest certification groups. In the light of previous evidence demonstrating the efficacy of nasturtium leaves (Tropaeolum majus L. (Brassicales: Tropaeolaceae)) in suppressing leaf-cutting ant colonies, this study investigates 2 active components of nasturtium leaf extracts: diphenyl disulfide and lyral. We tested their impact on Atta sexdens (L.) (Hymenoptera: Formicidae), the most prevalent leaf-cutter ant species in Brazil, and their symbiotic fungus, Leucoagaricus gongylophorus (Möller) Singer (Agaricales: Agaricaceae). We conducted experiments with increasing concentrations of diphenyl disulfide and lyral, assessing their effects on the symbiotic fungus and on forager workers and gardeners of A. sexdens colonies. Our findings revealed no fungicidal activity, and ant mortality was minimal in both topical and ingestion bioassays with the exception of gardeners topically exposed to diphenyl sulfide. Furthermore, the compounds did not affect leaf ingestion, but diphenyl disulfide did increase interactions among foragers. These results suggest that neither diphenyl disulfide nor lyral are the primary contributors to the suppression of leaf-cutting ant colonies by nasturtium leaves. However, they may enhance the formicidal activity of other compounds present in nasturtium leaves.

Original grooming data

Metarhizium anisopliae and Trichoderma viride for control of nests of the fungus-growing ant, Atta cephalotes

Monochamus alternatus, the main vector beetles of invasive pinewood nematode, has established a symbiotic relationship with a native ectotrophic fungal symbiont, Sporothrix sp. 1, in China. The immune response of M. alternatus to S. sp. 1 in the coexistence of beetles and fungi is, however, unknown. Here, we report that immune responses of M. alternatus pupae to infection caused by ectotrophic symbiotic fungus S. sp. 1 and entomopathogenic fungus Beauveria bassiana differ significantly. The S. sp. 1 did not kill the beetles while B. bassiana killed all upon injection. The transcriptome results showed that the numbers of differentially expressed genes in M. alternatus infected with S. sp. 1 were 2-fold less than those infected with B. bassiana at 48 hours post infection. It was noticed that Toll and IMD pathways played a leading role in the beetle’s immune system when infected by symbiotic fungus, but upon infection by entomopathogenic fungus, only the Toll pathway gets triggered actively. Furthermore, the beetles could tolerate the infection of symbiotic fungi by retracing their Toll and IMD pathways at 48 h. This study provided a comprehensive sequence resource of M. alternatus transcriptome for further study of the immune interactions between host and associated fungi.

Free-living and symbiotic fungi can interact with, and transform certain uranium species, while general metabolic activities such as decomposition of organic substances and dissolution of rocks and minerals may result in products that indirectly react with uranium. Excretion of organic acids is an important property of many fungi which can result in U-complex formation and U-ore dissolution for example, while fungal biomass can act as an efficient U biosorbent and surface/matrix for U crystallization and biomineralization. Secondary products of organic matter decomposition and mineral dissolution may result in secondary U-complex and U-mineral formation. It is not fully established whether fungi are capable of U(VI) reductive immobilization. Fungal interactions with uranium are relevant to the biogeochemistry of uranium, but also to the applied area of bioremediation. While U biosorption seems not to have a commercial future, some fungal activities may have relevance to contaminated terrestrial habitats. The dependence of almost all land plants on symbiotic mycorrhizal fungi, and the fact that mycorrhizal fungi are capable of uranium transformations may make fungal biogeochemical activity of importance in phyto- or other bioremediation strategies for soils polluted with various forms of uranium.

Entomopathogenic fungal infection following immune gene silencing decreased behavioral and physiological fitness in Aedes aegypti mosquitoes

Entomopathogenic fungi as biological control agents for the vector of the laurel wilt disease, the redbay ambrosia beetle, Xyleborus glabratus (Coleoptera: Curculionidae)

The pecan weevil, Curculio caryae (Horn), is a major pest of pecans in the Southeastern United States. Entomopathogenic nematodes and fungi are potential alternatives to chemical insecticides for C. caryae control. Our objective was to survey pecan orchards in the southeastern United States for entomopathogenic nematodes and fungi and determine the virulence of the new isolates to C. caryae larvae. Soil was collected from 105 sites in 21 orchards in Arkansas, Georgia, Louisiana, and Mississippi. Entomopathogens were isolated by exposing soil to C. caryae and greater wax moth larvae, Galleria mellonella, (L.). We isolated entomopathogenic fungi and nematodes from 16 and 6 of the 21 orchards surveyed, respectively. The entomopathogenic fungi included Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin, and nematodes included Heterorhabditis bacteriophora Poinar, Steinernema carpocapsae (Weiser), Steinernema glaseri (Steiner), and Steinernema rarum (Doucet). This is the first report of Steinernema rarum in the United States. Soil characteristics in orchards were analyzed for pH, organic matter, and nutrients; we detected a negative relationship between fungal occurrence and manganese levels in soil and a positive relationship between M. anisopliae occurrence and calcium or magnesium levels. In laboratory assays, virulence of 15 nematode and 22 fungal isolates to C. caryae larvae was tested in small plastic cups containing soil. Results indicated poor susceptibility of the C. caryae larvae to entomopathogenic nematodes. Several fungal isolates that caused significantly higher mortality in C. caryae larvae than other strains (including a commercial strain of B. bassiana) should be investigated further as potential control agents of C. caryae.

Herbivory by leaf‐cutting ants can lead to serious problems for agricultural and forest systems in the Neotropics. Chemical insecticides are the major, if not only, control tactic, but the efficiency of this approach is questionable. Moreover, continued reliance on insecticidal control leads to environmental damage and risks to human and animal health. Developing alternative management tactics for leaf‐cutting ants is thereby a justifiable and sensible need. Recent findings suggest that the Mexican sunflower, Tithonia diversifolia and the jackbean, Canavalia ensiformis, have detrimental effects on leaf‐cutting ant populations under laboratory and field conditions. It is unclear, however, whether those effects involve the development and growth of the ant’s symbiotic fungus, the ants or both. To address this question, an experiment was conducted on 18 artificial Atta cephalotes colonies maintained with three feeding treatments: a control (mango, Mangifera indica), T. diversifolia and C. ensiformis. Six repetitions of each treatment were assigned to the colonies in a completely randomized design and evaluated over a period of 2 months under laboratory conditions. Both T. diversifolia and C. ensiformis negatively altered growth of the symbiotic fungus Leucoagaricus gongilophorus and thereby affected the survival of A. cephalotes. The greatest reduction in fungus size (up to 100%) and the highest ant mortality (83.3%) were observed in colonies fed on T. diversifolia, but not until 45 days after the start of the experiment. In colonies fed on C. ensiformis, the greatest reduction in fungus size (95.3%) and the highest ant mortality (66.6%) were reached at 57 days. During the course of the study, no negative effects were observed in the control colonies, which displayed a consistently healthy symbiotic fungus and low rate of ant mortality (16.6%). Based on these results, plants of both T. diversifolia and C. ensiformis offer promise for the biological control of A. cephalotes populations.

The fall armyworm, Spodoptera frugiperda (Noctuidae; Lepidoptera), is a serious threat to food security as it has the potential to feed on over 353 plant species. To control this insect pest, endophytic colonization of entomopathogenic fungi (EPF) in plants is being considered as a safer and more effective alternative. This study evaluated the efficacy of two EPFs, Beauveria bassiana and Metarhizium anisopliae, for endophytic colonization using foliar spray and seed treatment methods on maize plants, and their impact on the survival, development, and fecundity of S. frugiperda. Both EPF effectively colonized the maize plants with foliar spray and seed treatment methods, resulting in 72–80% and 50–60% colonization rates, respectively, 14 days after inoculation. The EPF negatively impacted the development and fecundity of S. frugiperda. Larvae feeding on EPF-inoculated leaves had slower development (21.21 d for M. anisopliae and 20.64 d for B. bassiana) than the control treatment (20.27 d). The fecundity rate was also significantly reduced to 260.0–290.1 eggs/female with both EPF applications compared with the control treatment (435.6 eggs/female). Age-stage-specific parameters showed lower fecundity, life expectancy, and survival of S. frugiperda when they fed on both EPF-inoculated leaves compared with untreated leaves. Furthermore, both EPFs had a significant effect on population parameters such as intrinsic (r = 0.127 d−1 for B. bassiana, and r = 0.125 d−1 for M. anisopliae) and finite rate (λ = 1.135 d−1 for B. bassiana, and λ = 1.1333 d−1 for M. anisopliae) of S. frugiperda compared with the control (r = 0.133 d−1 and λ = 1.146 d−1). These findings suggest that EPF can be effectively used for the endophytic colonization of maize plants to control S. frugiperda. Therefore, these EPFs should be integrated into pest management programs for this pest.

High rainfall with increase in humidity (68% to 75%) along with a decrease in temperature ( 3 to 5°C less than usual ) during June, July, and August months favor the emergence of white grub beetles, its egg laying which in turn resulted in severe sugarcane clump damage by white grubs in July, August and September months. Seasonal incidence of white grub in sugarcane growing areas in Andhra Pradesh state showed that damage of sugarcane clumps caused by white grubs infestation was significantly high in march ratoon compared to February ratoon crop; January plant crop and low in January seedling plant crop. Grub damage recorded high in 4 months age sugarcane ratoon crop and low in 6 months and 7 months age sugarcane plant crop. Field efficacy of entomopathogenic nematode and entomopathogenic fungus in the management of white grub, Holotrichia consanguinea in sugarcane was investigated and promoted biocontrol in white grub endemic areas during 2017-18 to 2019-20. Entomopathogenic nematode (Heterorhabditis indica NBAII-H38), entomopathogenic fungi (Metarhizium anisopliae (NBAIR Ma4 )along with a chemical Chlorantraniliprole were evaluated through soil application. The results indicated that Heterorhabditis indica NBAII-H38 (12 kg a.i./ha) soil application two times at monthly interval resulted in higher reduction of white grub damage. Metarhizium anisopliae NBAIR Ma4 (2.5 kg a.i./ha) treated plots caused higher percentage reduction of white grub than chlorantraniliprole treatment. Entomopathogenic nematode, Heterorhabditis indica and entomopathogenic fungus, Metarhizium anisopliae were found effective than the insecticidal application in the management of sugarcane white grub. Cost benefit of biocontrol agents, Metarhizium anisopliae and H. indica was proved superior to insecticidal application.
 Graphical Abstract

Leaf-cutting ants (LCAs) are dominant herbivores of the Neotropics, as well as economically important pests. Their foraging ecology and patterns/mechanisms of food selection have received considerable attention. Recently, it has been documented that LCAs exhibit a delayed rejection of previously accepted food plants following treatment with a fungicide that makes the plants unsuitable as substrate for their symbiotic fungus. Here, we investigated whether LCAs similarly reject plants with induced chemical defenses, by combining analysis of volatile emissions with dual-choice bioassays that used LCA subcolonies (Atta sexdens L.). On seven consecutive days, foraging ants were given the choice between leaf disks from untreated control plants and test plants of Vitis vinifera ssp. vinifera L. treated with the phytohormone jasmonic acid (JA) to mimic herbivore attack. Chemical analysis revealed the emission of a characteristic set of herbivore-induced volatile organic compounds (VOC) from JA-induced plants. Dual-choice experiments indicated that workers did not show any preference initially, but that they avoided JA-treated plants from day five onwards. Our finding that A. sexdens foragers learn to avoid VOC-emitting plants, which are likely detrimental to their symbiotic fungus, represents the first evidence for avoidance learning in attine ants toward plants with induced defenses.

BackgroundThe fall armyworm (FAW) Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is a newly introduced pest that damages maize production in Indonesia. To control this pest in maize fields, better solution is to use the egg parasitoid, such as Telenomus remus Nixon (Hymenoptera: Scelionidae), as another better option to apply topically entomopathogenic fungi (EPF). Therefore, it is necessary to study the effect of the EPF on the egg parasitoid of T. remus. The objective of this research was to evaluate susceptibility of immature T. remus to the EPFs, Beauveria bassiana, Chaetomium sp., Curvularia lunata, Penicillium citrinum, and Metarhizium anisopliae. The EPFs (1 × 106 conidia mL−1) were sprayed topically on one-day-old mummies (immature T. remus) in post-parasitism periods.ResultsThe results showed that the cumulative percentage of T. remus adult emergence from the mummies treated with EPF on 11 days after treatment ranged 54–100% and was non-significantly different than those of control (untreated with EPF) (90.48%). Therefore, the immature stage of T. remus was not susceptible to the EPF topical application. The EPFs were harmless to the immature stage of T. remus. Percentage of aborted mummies (embryonic death) of T. remus after treated with the EPF was also non-significantly different than those of control. However, the EPFs could significantly affect developmental times of immatures stages of T. remus. The EPF also could shorten the adult longevity of the egg parasitoid.ConclusionsThe immature T. remus is less sensitive to the EPFs; B. bassiana, Chaetomium sp., C. lunata, P. citrinum, and M. anisopliae. It can be considered integrating the EPF with T. remus inundation in maize field. However, it is necessary to limit the topical application of the EPF to avoid negative effects on the adult longevity of the egg parasitoid. Thus, it needed to be further investigated that the application of the endophytic EPFs by inoculating the fungi within the plant tissue could be harmless to the egg parasitoids.

Microbial control of mosquitoes via the use of symbiotic or pathogenic microbes, such as Wolbachia and entomopathogenic fungi, are promising alternatives to synthetic insecticides to tackle the rapid increase in insecticide resistance and vector-borne disease outbreaks. This study evaluated the susceptibility and host responses of two important mosquito vectors, Ae. albopictus and Cx. pipiens, that naturally carry Wolbachia, to infections by entomopathogenic fungi. Our study indicated that while Wolbachia presence did not provide a protective advantage against entomopathogenic fungal infection, it nevertheless influenced the bacterial / fungal load and the expression of select anti-microbial effectors and phenoloxidase cascade genes in mosquitoes. Furthermore, although host responses from Ae. albopictus and Cx. pipiens were mostly similar, we observed contrasting phenotypes with regards to susceptibility and immune responses to fungal entomopathogenic infection in these two mosquitoes. This study provides new insights into the intricate multipartite interaction between the mosquito host, its native symbiont and pathogenic microbes that might be employed to control mosquito populations.

Alternative methods of mosquito control are needed to tackle the rising burden of mosquito-borne diseases while minimizing the use of synthetic insecticides, which are threatened by the rapid increase in insecticide resistance in mosquito populations. Fungal biopesticides show great promise as potential alternatives because of their ecofriendly nature and ability to infect mosquitoes on contact. Here we describe the temporospatial interactions between the mosquito Aedes aegypti and several entomopathogenic fungi. Fungal infection assays followed by the molecular assessment of infection-responsive genes revealed an intricate interaction between the mosquito immune system and entomopathogenic fungi. We observed contrasting tissue and time-specific differences in the activation of immune signaling pathways and antimicrobial peptide expression. In addition, these antifungal responses appear to vary according to the fungal entomopathogen used in the infection. Enzyme activity-based assays coupled with gene expression analysis of prophenoloxidase genes revealed a reduction in phenoloxidase (PO) activity in mosquitoes infected with the most virulent fungal strains at 3 and 6d post-fungal infection. Moreover, fungal infection led to an increase in midgut microbiota that appear to be attributed in part to reduced midgut reactive oxygen species (ROS) activity. This indicates that the fungal infection has far reaching effects on other microbes naturally associated with mosquitoes. This study also revealed that despite fungal recognition and immune elicitation by the mosquito, it is unable to successfully eliminate the entomopathogenic fungal infection. Our study provides new insights into this intricate multipartite interaction and contributes to a better understanding of mosquito antifungal immunity.