Abstract

Background

   Plant defense against herbivores begins with perception. The earlier
   plant detects the harm, the greater plant will benefit in its arm race
   with the herbivore. Before feeding, the larvae of the rice pest
   Cnaphalocrocis medinalis, initially spin silk and fold up a leaf. Rice
   can detect and protect itself against C. medinalis feeding. However,
   whether rice could perceive C. medinalis leaf rolling behavior is
   currently unknown. Here, we evaluated the role of leaf rolling by C.
   medinalis and artificial leaf rolling in rice plant defense and its
   indirect effect on two important C. medinalis parasitoids (Itoplectis
   naranyae and Apanteles sp.) through a combination of volatile
   profiling, gene-transcriptional and phytohormonal profiling.

Results

   Natural leaf rolling by C. medinalis resulted in an increased
   attraction of I. naranyae when compared to the undamaged plant after
   12 h. Volatile analysis revealed that six out of a total 22 components
   significantly increased in the headspace of C. medinalis rolled plant
   when compared to undamaged plant. Principal component analysis of these
   components revealed similarities in the headspace of undamaged plant
   and artificially rolled plant while the headspace volatiles of C.
   medinalis rolled plant deferred significantly. Leaf rolling and feeding
   by C. medinalis up-regulated the plant transcriptome and a series of
   jasmonic acid (JA) and salicylic acid (SA) related genes. While feeding
   significantly increased JA level after 12 to 36 h, rolling
   significantly increased SA level after 2 to 12 h. Compared to
   artificial rolling, natural rolling significantly increased JA level
   after 36 h and SA level after 2 and 12 h.

Conclusions

   Our findings suggest that natural leaf rolling by C. medinalis can be
   perceived by rice plant. The detection of this behavior may serve as an
   early warning signal in favor of the rice plant defenses against C.
   medinalis.

   Keywords: Cnaphalocrocis medinalis, Jasmonic acid, Salicylic acid,
   Volatiles, Transcriptome, Early detection

Background

   Throughout their life cycle, plants experience biotic and abiotic
   stresses, which can severely affect their growth and yield
   [[43]1–[44]3]. Globally, herbivore insects are among the most serious
   biotic problems and have close relationships with plants [[45]4]. While
   insects have evolved ways to find hosts, plants are under selection
   pressure to evade detection or defend themselves when attacked
   [[46]4–[47]6].

   To fend off insect herbivores, plants have evolved intricate and
   dynamic defense systems. Phytohormones play an important role in
   regulating plant defenses [[48]7–[49]9]. The phytohormones, jasmonic
   acid (JA) and salicylic acid (SA), and their derivatives play
   predominant roles in signal transduction of plant defenses against
   pathogens and herbivorous insects [[50]10, [51]11]. Plant hormones can
   mediate downstream regulation of plant defenses, including direct
   defense mechanisms such as the production of defensive proteins and
   enzymes, and indirect defense mechanisms involving the production of
   volatile blends [[52]12–[53]14].

   Plant volatiles are released naturally, and their emission can change
   in response to different stress factors [[54]15, [55]16]. With
   plant–insect co-evolution, herbivore-induced plant volatiles (HIPVs)
   have become cost-saving weapons for plant defenses, and are useful in
   attracting natural enemies of herbivores [[56]17–[57]19]. Host
   recognition by natural enemies is specific, as they identify volatile
   blends from various plant species. However, natural enemies differ in
   their ability to distinguish HIPVs source depending on the distance of
   the natural enemy to the cues [[58]20–[59]22].

   Conflicts in interactions of plants with herbivores begin with
   perception. Interestingly, plants can identify attacks from different
   insect species and mount several defenses [[60]23, [61]24]. Plants
   perceive insect herbivores’ activities like oviposition, feeding,
   walking on the leaf surface, as well as chemical cues from insect oral
   secretion and frass [[62]25–[63]27]. Spinning is an important activity
   of silk-producing insects. The ability to produce silk has evolved in
   many groups of insects, and is used to accomplish a wide array of
   activities that enhance survival [[64]28–[65]30]. Several insects’ silk
   has been reported to attract parasitic wasps. For instance, Apanteles
   melanoscelus (Ratzeburg) (Hymenoptera: Braconidae) has a positive
   response to gypsy moth silk, and silk extracts of Plodia interpunctella
   (Hübner) (Lepidoptera: Pyralidae) larvae also attract parasitic wasps
   [[66]31, [67]32]. However, no research has shown whether rolling
   behaviors following spinning can mediate plant defenses.

   In Asia, the rice leaf folder, Cnaphalocrocis medinalis Guenée
   (Lepidoptera: Pyralidae), is one of the most important insect pest of
   paddy rice [[68]33]. Severe feeding by this pest often affects the
   growth of rice plant leading to yield loss [[69]34, [70]35]. Various
   wasp parasitoids of C. medinalis commonly exist in rice fields, such as
   Itoplectis naranyae (Ashmead) (Hymenoptera: Ichneumonidae) and
   Apanteles sp. (Hymenoptera: Braconidae) [[71]36, [72]37]. The
   characteristic behavior of C. medinalis larvae is to roll a leaf
   longitudinally, by spinning silk before feeding [[73]38, [74]39]. There
   are reports indicating that the feeding behavior of C. medinalis can
   induce rice defenses, but there is no evidence to indicate whether leaf
   rolling alone prior to feeding by the pest, can be perceived by the
   plant [[75]40, [76]41].

   Early detection of herbivores is beneficial for plants to develop
   effective and sustainable defenses. It is therefore important to
   determine whether rice plant can detect the early threat of C.
   medinalis leaf rolling, by initiating defenses against the pest
   [[77]42]. In this study, we investigated the dynamics of rice plant
   responses to C. medinalis leaf rolling, C. medinalis leaf rolling and
   feeding and artificial leaf rolling, and compared with undamaged plant.
   To achieve this goal, we integrated results from wasps’ behavioral
   assays, plant volatile profiles, plant transcriptional and
   phytohormonal profiles.

Results

Behavior of natural enemies

   We hypothesized that leaf rolling behavior of C. medinalis larvae can
   trigger rice plant defenses. Therefore, we tested the preferences of