Unveiling chemical defense in the rice stalk stink bug against the entomopathogenic fungus Metarhizium anisopliae

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Highlights

  • Differential susceptibility of T. limbativentris to M. anisopliae is reported.

  • Eggs comprised the most susceptible stage to M. anisopliae.

  • Adults and older nymphs displayed the highest resistance to M. anisopliae.

  • Aldehydes from cuticular and gland extracts impaired fungal infection.

  • (E)-2-decenal exhibited the strongest antifungal activity on M. anisopliae.

Abstract

Eggs, nymphs (1st–5th instar) and adults of Tibraca limbativentris were challenged by conidial suspensions of its major fungal pathogen Metarhizium anisopliae in order to assess their susceptibility. The role of chemical defensive compounds from exocrine secretions produced by both nymphs and adults were examined for their participation on M. anisopliae infection. Although insect susceptibility to M. anisopliae followed a dose-dependent manner, adults followed by older nymphs displayed the highest resistance. Eggs were highly susceptible showing >96% fungal infection. Crude extracts isolated from metathoracic scent gland and dorsal abdominal glands of adults and nymphs, respectively, showed fungistatic effects by impairing spore germination, vegetative growth and sporulation. Gas chromatography–mass spectrometry analysis of these extracts revealed that the major components were short-chain hydrocarbons (C10–13) and unsaturated aldehydes. In vitro tests with the corresponding synthetic standards indicated compounds with greater antifungal activity including (E)-2-hexenal, (E)-2-octenal, and (E)-2-decenal, with the latter being the most deleterious to fungal fitness. We demonstrated that differential susceptibility of the rice stalk stink bug to M. anisopliae infection is age-specific and partly mediated by fungistatic properties of aldehydes, which are produced by scent glands of both nymphs and adults.

Introduction

The rice stalk stink bug, Tibraca limbativentris Stal. (Heteroptera: Pentatomidae), is an economic important pest of rice crops (Oryza sativa L.) in South America, including Brazil (Pantoja, 1997). Although both nymphs and adults of T. limbativentris feed on the developing stalks, the main damage occurs during pre-flowering and panicle formation that consequently leads to rice yield losses by 10–80% (Costa and Link, 1992, Ferreira et al., 1986). In Brazil, the major control method relies primarily on the use of synthetic chemical insecticides (Quintela et al., 2013). However, due to the short-lived effects of synthetic insecticides, the repopulation through migration from non-treated areas, and primarily by the emerging of insecticide resistance in related neotropical stink bugs in Brazilian soybean fields (Sosa-Gómez et al., 1997), there is pressing need to develop alternative control strategies. The use of biological control agents, such as entomopathogenic fungi, represents a practical and ecologically friendly tactic to manage agricultural pests. The entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorok. (Hypocreales: Clavicipitaceae) strain CG168 was first isolated (causing epizootics) from naturally infected T. limbativentris; an oil-based formulation of hydrophobic aerial conidia of this isolate is underway to be registered to use in integrated management programs for this pest (Quintela et al., 2013).

Entomopathogenic fungi start the infection process mainly by penetration through the insect cuticle. The outermost surface layer of the cuticle is the epicuticle, mostly composed of lipids. The epicuticle exhibits characteristic water barrier properties and protect insects against predators and pathogens (Borges and Aldrich, 1992, Pareja et al., 2007). Interspersed within the cuticle barrier there are biochemical components such as antimicrobial lipids and phenols, enzyme inhibitors, proteins, and other defensive compounds that entomopathogens must overcome for successful virulence (Pedrini et al., 2013). Thus, fungal virulence is frequently correlated with rapid germination and growth rates on the cuticle, and higher germination rates might help increase the probability of infection before spores are removed from the cuticle (Altre et al., 1999). The epicuticle composition is also associated with the insect susceptibility to entomopathogenic fungi. For example, insects containing a blend of saturated straight and branched chains lipids are more susceptible to fungi that those insects with a predominance of unsaturated chains and/or the presence of some compounds showing an antibiotic role, such as quinone derivatives or short chain lipids (Pedrini et al., 2007). It is known that allomones produced by stink bugs from the family Pentatomidae comprise an assortment of hydrocarbons, saturated and α,β-unsaturated aldehydes and esters (Aldrich, 1988). These compounds are firstly synthesized in specialized exocrine scent glands and then released embedding the cuticle. Nymphs, for instance, produce exocrine secretions of odoriferous compounds from dorsal abdominal glands (DAGs) that are shed along with the exuviae during molting. Thus, extraction of exuviae consists of a suitable method to obtain and identify compounds synthesized in DAGs (Borges and Aldrich, 1992). In adults, defensive secretions constitute allomones produced in the metathoracic scent gland (MTG), which can be easily extracted by dissection (Aldrich, 1988, Fávaro et al., 2011). It is also known that ratio and composition of these compounds vary across nymphal instars and physiological ages in adults (Borges and Aldrich, 1992, Fávaro et al., 2011, Fávaro et al., 2012). Although some of them are known to constitute the alarm pheromone, their antimicrobial role (Borges and Aldrich, 1992, Sosa-Gómez et al., 1997) is often related with the low susceptibility of pentatomid stink bugs to entomopathogenic fungi observed at field conditions (Sosa-Gómez and Moscardi, 1998, Moraes et al., 2008).

Little is known about the mechanisms underlying the low susceptibility of T. limbativentris to its major fungal pathogen M. anisopliae. Here, we have hypothesized that defensive compounds derived from scent glands of nymphs and adults of this stink bug could be involved in this natural resistance to fungal infection. Therefore, our goal in this communication is to describe the susceptibility of eggs, nymphs and adults of T. limbativentris to M. anisopliae (strain CG168), and at the same time to understand in what extent the chemical compounds isolated from both MTG and DAGs affect infection by this pathogen.

Section snippets

Insect colony

Rice stink bugs were reared on 5-L potted rice plants (Oryza sativa L., cv. BR-IRGA-409) under screenhouse conditions. Insect founders were natural and annually collected from rice fields free of chemical pesticides at the National Rice and Beans Research Center of the Brazilian Agricultural Research Corporation (EMBRAPA Rice and Beans) located in St. Antônio de Goiás, GO, Brazil. Insect density per pot consisted of 100 adults (sex ratio 1♀:1♂) at the same age. This insect colony was free of

Egg susceptibility to M. anisopliae

Egg masses inoculated with M. anisopliae at concentrations of 5 × 106 and 5 × 107 conidia mL−1 exhibited a remarkable decrease in viability (3.8% hatchability) in comparison to the control group (93.8% egg viability) (χ2 = 75.81; df = 2; P < 0.0001) (Fig. 1). The remaining newly hatched nymphs were further infected by M. anisopliae resulting in no survivors. In fungal-treated eggs, it was evident that fungal outgrowth caused mycosis.

Susceptibility of nymphs and adults to M. anisopliae

The susceptibility of nymphal stages and adults of T. limbativentris to

Discussion

This report provides relevant evidence of differential susceptibility of T. limbativentris life stages toward its major fungal pathogen M. anisopliae, which is associated with the antifungal properties found in the crude extracts from exocrine scent glands of both nymphs and adults. Although there is a wealth body of literature concerning the elucidation of chemical compounds of stink bugs from the family Pentatomidae (Sosa-Gómez et al., 1997, Borges and Aldrich, 1992, Adams, 2007, Marques et

Acknowledgments

We thank Embrapa (Brasilia, DF, Brazil) for providing financial support to perform this work. We appreciate Edmar de Moura Cardoso and José Francisco da Silva e Arruda for their outstanding technical support in rearing the insect colony. Our sincere gratitude to three anonymous reviewers for their helpful and insightful comments on an earlier draft of this article.

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