Insect eggs suppress plant defence against chewing herbivores

Plants activate direct and indirect defences in response to insect egg deposition. However, whether eggs can manipulate plant defence is unknown. In Arabidopsis thaliana, oviposition by the butterfly Pieris brassicae triggers cellular and molecular changes that are similar to the changes caused by biotrophic pathogens. In the present study, we found that the plant defence signal salicylic acid (SA) accumulates at the site of oviposition. This is unexpected, as the SA pathway controls defence against fungal and bacterial pathogens and negatively interacts with the jasmonic acid (JA) pathway, which is crucial for the defence against herbivores. Application of P. brassicae or Spodoptera littoralis egg extract onto leaves reduced the induction of insect‐responsive genes after challenge with caterpillars, suggesting that egg‐derived elicitors suppress plant defence. Consequently, larval growth of the generalist herbivore S. littoralis, but not of the specialist P. brassicae, was significantly higher on plants treated with egg extract than on control plants. In contrast, suppression of gene induction and enhanced S. littoralis performance were not seen in the SA‐deficient mutant sid2‐1, indicating that it is SA that mediates this phenomenon. These data reveal an intriguing facet of the cross‐talk between SA and JA signalling pathways, and suggest that insects have evolved a way to suppress the induction of defence genes by laying eggs that release elicitors. We show here that egg‐induced SA accumulation negatively interferes with the JA pathway, and provides an advantage for generalist herbivores.     

Microbes in Helicoverpa armigera oral secretions contribute to increased senescence around plant wounds

1. Plants have long been exposed to insect herbivore attack. Crucial to the plant's ability to defend itself is its ability to identify specific signals associated with attacking insects. Distinctive chemical cues, such as those associated with chewing insect oral secretions (OS), activate targeted defence responses to chewing insect herbivores. 2. Herbivore-associated cues can be complicated by the fact that many herbivores form associations with microbes that produce their own specific signals, which may induce alternative defence processes. 3. Here we report that OS of the global pest, the cotton bollworm (Helicoverpa armigera), induce senescence around wounds in Brachypodium distachyon leaves. Crude OS activate greater levels of senescence than OS with reduced microbial abundance or mechanical wounding alone. Nonetheless, plants closed mechanical wounds more rapidly when treated with H. armigera OS regardless of the microbial component. 4. This study concludes that H. armigera OS can activate senescence and wound closure in plant tissues and that microbes within OS have an important role in shaping plant-herbivore interactions through additional increases in senescence.     

Plants use identical inhibitors to protect their cell wall pectin against microbes and insects

As fundamentally different as phytopathogenic microbes and herbivorous insects are, they enjoy plant‐based diets. Hence, they encounter similar challenges to acquire nutrients. Both microbes and beetles possess polygalacturonases (PGs) that hydrolyze the plant cell wall polysaccharide pectin. Countering these threats, plant proteins inhibit PGs of microbes, thereby lowering their infection rate. Whether PG‐inhibiting proteins (PGIPs) play a role in defense against herbivorous beetles is unknown. To investigate the significance of PGIPs in insect–plant interactions, feeding assays with the leaf beetle Phaedon cochleariae on Arabidopsis thaliana pgip mutants were performed. Fitness was increased when larvae were fed on mutant plants compared to wild‐type plants. Moreover, PG activity was higher, although PG genes were downregulated in larvae fed on PGIP‐deficient plants, strongly suggesting that PGIPs impair PG activity. As low PG activity resulted in delayed larval growth, our data provide the first in vivo correlative evidence that PGIPs act as defense against insects.     

Insect eggs induce a systemic acquired resistance in Arabidopsis

Although they constitute an inert stage of the insect's life, eggs trigger plant defences that lead to egg mortality or attraction of egg parasitoids. We recently found that salicylic acid (SA) accumulates in response to oviposition by the Large White butterfly Pieris brassicae, both in local and systemic leaves, and that plants activate a response that is similar to the recognition of pathogen‐associated molecular patterns (PAMPs), which are involved in PAMP‐triggered immunity (PTI). Here we discovered that natural oviposition by P. brassicae or treatment with egg extract inhibit growth of different Pseudomonas syringae strains in Arabidopsis through the activation of a systemic acquired resistance (SAR). This egg‐induced SAR involves the metabolic SAR signal pipecolic acid, depends on ALD1 and FMO1, and is accompanied by a stronger induction of defence genes upon secondary infection. Although P. brassicae larvae showed a reduced performance when feeding on Pseudomonas syringae‐infected plants, this effect was less pronounced when infected plants had been previously oviposited. Altogether, our results indicate that egg‐induced SAR might have evolved as a strategy to prevent the detrimental effect of bacterial pathogens on feeding larvae.     

The effect of rhizosphere microbes outweighs host plant genetics in reducing insect herbivory

Rhizosphere microbes affect plant performance, including plant resistance against insect herbivores; yet, a direct comparison of the relative influence of rhizosphere microbes versus plant genetics on herbivory levels and on metabolites related to defence is lacking. In the crucifer Boechera stricta, we tested the effects of rhizosphere microbes and plant population on herbivore resistance, the primary metabolome, and select secondary metabolites. Plant populations differed significantly in the concentrations of six glucosinolates (GLS), secondary metabolites known to provide herbivore resistance in the Brassicaceae. The population with lower GLS levels experienced ~60% higher levels of aphid (Myzus persicae) attack; no association was observed between GLS and damage by a second herbivore, flea beetles (Phyllotreta cruciferae). Rhizosphere microbiome (disrupted vs. intact native microbiome) had no effect on plant GLS concentrations. However, aphid number and flea beetle damage were respectively about three‐ and seven‐fold higher among plants grown in the disrupted versus intact native microbiome treatment. These differences may be attributable to shifts in primary metabolic pathways previously implicated in host defence against herbivores, including increases in pentose and glucoronate interconversion among plants grown with an intact microbiome. Furthermore, native microbiomes with distinct community composition (as estimated from 16s rRNA amplicon sequencing) differed two‐fold in their effect on host plant susceptibility to aphids. The findings suggest that rhizosphere microbes, including distinct native microbiomes, can play a greater role than population in defence against insect herbivores, and act through metabolic mechanisms independent of population.     

Regrowth patterns and rosette attributes contribute to the differential compensatory responses of Arabidopsis thaliana genotypes to apical damage

A plant's compensatory performance refers to its ability to maintain or increase its reproductive output following damage. The ability of a plant to compensate depends on numerous factors including the type, severity, frequency and timing of damage, the environmental conditions and the plant's genotype. Upon apical damage, a cascade of hormonal and genetic responses often produces dramatic changes in a plant's growth, development, architecture and physiology. All else being equal, this response is largely dependent on a plant's genotype, with different regrowth patterns displayed by different genotypes of a given species. In this study, we compare the architectural and growth patterns of two Arabidopsis thaliana genotypes following apical damage. Specifically, we characterise regrowth patterns of the genotypes Columbia‐4 and Landsberg erecta, which typically differ in their compensation to apical meristem removal. We report that Landsberg erecta suffered reductions in the number of stems produced, maximum elongation rate, a delay in reaching this rate, lower average rosette quality throughout the growing period, and ultimately, less aboveground dry biomass and seed production when damaged compared to undamaged control plants. Columbia‐4 had no reductions in any of these measures and maintained larger rosette area when clipped relative to when unclipped. Based on the apparent influence of the rosette on these genotypes' compensatory performances, we performed a rosette removal experiment, which confirmed that the rosette contributes to compensatory performance. This study provides a novel characterisation of regrowth patterns following apical damage, with insights into those measures having the largest effect on plant performance.     

Systematic analysis of rice (Oryza sativa) metabolic responses to herbivory

Plants defend against attack from herbivores by direct and indirect defence mechanisms mediated by the accumulation of phytoalexins and release of volatile signals, respectively. While the defensive arsenals of some plants, such as tobacco and Arabidopsis are well known, most of rice's (Oryza sativa) defence metabolites and their effectiveness against herbivores remain uncharacterized. Here, we used a non‐biassed metabolomics approach to identify many novel herbivory‐regulated metabolic signatures in rice. Most were up‐regulated by herbivore attack while only a few were suppressed. Two of the most prominent up‐regulated signatures were characterized as phenolamides (PAs), p‐coumaroylputrescine and feruloylputrescine. PAs accumulated in response to attack by both chewing insects, i.e. feeding of the lawn armyworm (Spodoptera mauritia) and the rice skipper (Parnara guttata) larvae, and the attack of the sucking insect, the brown planthopper (Nilaparvata lugens, BPH). In bioassays, BPH insects feeding on 15% sugar solution containing p‐coumaroylputrescine or feruloylputrescine, at concentrations similar to those elicited by heavy BPH attack in rice, had a higher mortality compared to those feeding on sugar diet alone. Our results highlight PAs as a rapidly expanding new group of plant defence metabolites that are elicited by herbivore attack, and deter herbivores in rice and other plants.     

Resistance of Apanteles eggs to the haemocytic encapsulation by their habitual host, Pieris

The calyx fluid in the lateral oviduct of a gregarious parasitoid, Apanteles glomeratus contained ellipsoid particles of ca. 130 × 200 nm. These calyx fluid particles did not appear to be embedded in a fibrous outer layer on the surface of eggs in the lateral oviduct. They were not observed on the surfaces of the eggs 3 to 4 hr after being deposited into the host haemocoele. Oviposition experiments indicated that the occurrence of haemocytic defence reactions of the late 2nd instar larvae of the Pieris rapae crucivora against 1 st instar larvae of the parasitoid increased with a decreasing number of the parasitoid eggs introduced into a host, and that more than 5 to 9 parasitoid eggs were needed for suppressing the ability of the host to encapsulate its parasitoid larvae immediately after hatching. When eggs with calyx fluid obtained from egg reservoir were injected into the host, they were found to be encapsulated 1 to 2 days after the injection. They could not start their embryonic development. When calyx fluid-free 3-hr-old eggs were injected in a number of more than 5 eggs into a 5th instar larva of Pieris, 58% of 31 eggs injected had normally hatched without evoking encapsulation reactions by the host. Both electron microscopic observations of parasitoid eggs in the host haemocoele and the experimental results suggested that calyx fluid or calyx fluid particles of the parasitoid might not be involved in the encapsulation-inhibiting activity of the parasitoid eggs. Rather it was anticipated that a substance (or substances) might be secreted by the parasitoid eggs into the haemocoele of the host, which suppressed defence reactions of the host.     

How discriminating are cabbage butterflies?

The egg-laying responses of cabbage butterflies Pieris rapae L. to differences in the size, quality and spatial distribution of available oviposition sites, were studied in the laboratory (Australia) and the field (Australia and Canada). Differences on three scales were investigated: between leaves within the same plant, between plants within a patch, and between patches of plants. The butterflies lay most eggs on the larger, older leaves of plants, though the oldest are sometimes avoided. Larger plants also receive more eggs than smaller ones of the same cultivar, but cultivar preferences may override the response to size. The butterflies do not discriminate against plants already bearing eggs or larvae, unless larval feeding damage to the plant is severe. They lay more eggs on young plants than old ones of the same size; plants grown in the lower of two light intensities also received more eggs. Australian butterflies discriminate not only between different species of crucifers, but also between varieties of the same species. This discrimination against less acceptable varieties is just as strong when plants of preferred varieties are not present, as when both varieties occur in the same patch. The observed responses are discussed in relation to the butterflies’ host-finding behaviour and the adaptive significance of the lack of a response to eggs and larvae is considered.     

Chemical-based resistance of Brassica oleracea and Rorippa dubia in response to Spodoptera litura attack

A plant's chemical-based resistance against insects can be characterized by an accumulation of phytochemicals that can severely harm or deter the generalist and non-adapted specialist insects. In the absence of an herbivore threat, plants maintain a basic concentration of defensive compounds as a constitutive resistance mechanism. On the other hand, plants are capable of sharply increasing their production of defensive compounds as induced defence in response to herbivore attacks. In this study, we highlight the role of chemical-based defence by comparing the constitutive and herbivore-induced resistances of cauliflower (Brassica oleracea var. botrytis L.) and yellow cress (Rorippa dubia (Persoon)) with respect to time-dependent changes of phytochemicals that directly influence the performance of the generalist herbivore insect, Spodoptera litura (Fabricius ) (Lepidoptera: Noctuidae). The results of the study showed that both plant species were able to switch, albeit different degrees, between constitutive and induced resistances to combat herbivore attack. Brassica oleracea amassed lesser defensive compounds of constitutive resistance (trypsin inhibitor, polyphenol oxidase and sinigrin) than R. dubia. This led to a higher relative growth rate (RGR) of the S. litura larvae fed on cauliflower than in those fed on yellow cress. Brassica oleracea also demonstrated a lower induced response capability than R. dubia in its production of allelochemicals in herbivore-induced resistances. This was shown by the decrease of the RGR of larvae fed on induced foliage of the cauliflower compared to those fed on yellow cress. These results of the study proved and added to the belief that modern crop variety is not only lower in constitutive resistance but also weaker in induced resistance capability against herbivores, which is likely to be an effect of domestication even Rorippa and Brassica are relatively far from each other in Brassicaceae phylogeny.     

Dickeya dadantii pectic enzymes necessary for virulence are also responsible for activation of the Arabidopsis thaliana innate immune system

Soft‐rot diseases of plants attributed to Dickeya dadantii result from lysis of the plant cell wall caused by pectic enzymes released by the bacterial cell by a type II secretion system (T2SS). Arabidopsis thaliana can express several lines of defence against this bacterium. We employed bacterial mutants with defective envelope structures or secreted proteins to examine early plant defence reactions. We focused on the production of AtrbohD‐dependent reactive oxygen species (ROS), callose deposition and cell death as indicators of these reactions. We observed a significant reduction in ROS and callose formation with a bacterial mutant in which genes encoding five pectate lyases (Pels) were disrupted. Treatment of plant leaves with bacterial culture filtrates containing Pels resulted in ROS and callose production, and both reactions were dependent on a functional AtrbohD gene. ROS and callose were produced in response to treatment with a cellular fraction of a T2SS‐negative mutant grown in a Pels‐inducing medium. Finally, ROS and callose were produced in leaves treated with purified Pels that had also been shown to induce the expression of jasmonic acid‐dependent defence genes. Pel catalytic activity is required for the induction of ROS accumulation. In contrast, cell death observed in leaves infected with the wild‐type strain appeared to be independent of a functional AtrbohD gene. It was also independent of the bacterial production of pectic enzymes and the type III secretion system (T3SS). In conclusion, the work presented here shows that D. dadantii is recognized by the A. thaliana innate immune system through the action of pectic enzymes secreted by bacteria at the site of infection. This recognition leads to AtrbohD‐dependent ROS and callose accumulation, but not cell death.     

Elm leaves ‘warned’ by insect egg deposition reduce survival of hatching larvae by a shift in their quantitative leaf metabolite pattern

Plants may take insect eggs on their leaves as a warning of future herbivory and intensify their defence against feeding larvae. Responsible agents are, however, largely unknown, and little knowledge is available on this phenomenon in perennial plants. We investigated how egg deposition affects the anti‐herbivore defence of elm against the multivoltine elm leaf beetle. Prior egg deposition caused changes in the quality of feeding‐damaged leaves that resulted in increased larval mortality and reduced reproductive capacity of the herbivore by harming especially female larvae. Chemical analyses of primary and secondary leaf metabolites in feeding‐damaged, egg‐free (F) and feeding‐damaged, egg‐deposited (EF)‐leaves revealed only small differences in concentrations when comparing metabolites singly. However, a pattern‐focused analysis showed clearly separable patterns of (F) and (EF)‐leaves because of concentration differences in especially nitrogen and phenolics, of which robinin was consumed in greater amounts by larvae on (EF) than on (F)‐leaves. Our study shows that insect egg deposition mediates a shift in the quantitative nutritional pattern of feeding‐damaged leaves, and thus might limit the herbivore's population growth by reducing the number of especially female herbivores. This may be a strategy that pays off in a long run particularly in perennial plants against multivoltine herbivores.     

Herbicide residues in soil decrease microbe-mediated plant protection

• The residues of glyphosate are found to remain in soils longer than previously reported, affecting rhizosphere microbes. This may adversely affect crop and other non-target plants because the plant's resilience and resistance largely rely on plant-associated microbes. Ubiquitous glyphosate residues in soil and how they impact mutualistic microbes inhabiting the aboveground plant parts are largely unexplored.
• We studied the effects of herbicide residues in soil on Epichloë sp., which are common endophytic symbionts inhabiting aerial parts of cool-season grasses. In this symbiosis, the obligate symbiont subsists entirely on its host plant, and in exchange, it provides alkaloids conferring resistance to herbivores for the host grass that invests little in its own chemical defence.
• We first show decreased growth of Epichloë endophytes in vitro when directly exposed to two concentrations of glyphosate or glyphosate-based herbicides. Second, we provide evidence for a reduction of Epichloë-derived, insect-toxic loline alkaloids in endophyte-symbiotic meadow fescue (F. pratensis) plants growing in soil with a glyphosate history. Plants were grown for 2 years in an open field site, and natural herbivore infestation was correlated with the glyphosate-mediated reduction of loline alkaloid concentrations.
• Our findings indicate that herbicides residing in soil not only affect rhizosphere microbiota but also aerial plant endophyte functionality, which emphasizes the destructive effects of glyphosate on plant symbiotic microbes, here with cascading effects on plant–pest insect interactions.

     

How insects overcome two‐component plant chemical defence: plant β‐glucosidases as the main target for herbivore adaptation

Insect herbivory is often restricted by glucosylated plant chemical defence compounds that are activated by plant β‐glucosidases to release toxic aglucones upon plant tissue damage. Such two‐component plant defences are widespread in the plant kingdom and examples of these classes of compounds are alkaloid, benzoxazinoid, cyanogenic and iridoid glucosides as well as glucosinolates and salicinoids. Conversely, many insects have evolved a diversity of counter‐adaptations to overcome this type of constitutive chemical defence. Here we discuss that such counter‐adaptations occur at different time points, before and during feeding as well as during digestion, and at several levels such as the insects' feeding behaviour, physiology and metabolism. Insect adaptations frequently circumvent or counteract the activity of the plant β‐glucosidases, bioactivating enzymes that are a key element in the plant's two‐component chemical defence. These adaptations include host plant choice, non‐disruptive feeding guilds and various physiological adaptations as well as metabolic enzymatic strategies of the insect's digestive system. Furthermore, insect adaptations often act in combination, may exist in both generalists and specialists, and can act on different classes of defence compounds. We discuss how generalist and specialist insects appear to differ in their ability to use these different types of adaptations: in generalists, adaptations are often inducible, whereas in specialists they are often constitutive. Future studies are suggested to investigate in detail how insect adaptations act in combination to overcome plant chemical defences and to allow ecologically relevant conclusions.     

Hydrocarbon biosynthesis in Triatoma infestans eggs

Triatoma infestans eggs are shown to synthesize hydrocarbons. Radio-gas chromatography was used to demonstrate metabolism of [1-¹⁴C]propionate into precursor methyl-branched fatty acids and into methyl-branched hydrocarbons in T. infestans eggs. These reactions have not been demonstrated previously in insect eggs. An in vivo study showed that hydrocarbons are also transported to eggs by the hemolymph. Inhibition of hydrocarbon synthesis by sodium trichloroacetate (NaTCA) was correlated with reduced oviposition, reduced hatchability, and reduced insect survival. Scanning electron microscopy showed impoverishment of the eggs' epicuticular waxes following NaTCA treatment. © 1994 Wiley-Liss, Inc.     

A simple method to disperse eggs from lepidopteran scalelike egg masses and to observe embryogenesis

Various insect species lay tiny, thin- and soft-shelled eggs in a connected “egg mass”. Especially in several lepidopteran species, the structure of such clustered eggs is covered with complicated scale-like secretion, which has so far prevented analysis of individual embryos. However, few studies on methods to disperse egg clusters of such insects and to compare different methods have been carried out. To overcome these problems, we developed methods to separate egg masses into individual eggs, using two Tortricidae pests, Homona magnanima Diakonoff and Adoxophyes honmai Yasuda (Lepidoptera: Tortricidae). The eggs were successfully separated from each other using potassium hydroxide and sodium hypochlorite. Although the separated eggs no longer continued their embryogenesis, fixation with heptane–paraformaldehyde, permeabilization with heptane–methanol, and staining with several dyes enabled easy observation of embryogenesis. This protocol is expected to be applicable to other insect taxa and will facilitate further morphological and genetic studies in insects that lay egg masses.     

Development of eggs and larvae of Stolephorus commersonnii and taxonomic key to fish eggs of the Clupeidae and Engraulidae off China

Fish eggs of successive stages of embryonic development, which were identified as belonging to specimens of Commerson's anchovy Stolephorus commersonnii through comparative molecular techniques, were collected from Leqing Bay, Zhejiang, China. Some eggs were reared artificially to obtain samples of successive developmental stages of larvae. The fertilized eggs of S. commersonnii are ellipsoidal and non-adhesive. The surface of the egg membrane is smooth and the perivitelline space is narrow. There is a single oil globule in the irregularly segmented yolk. Newly hatched larvae are transparent and devoid of pigments. Development of the larvae occurs in the following sequence: 8?h after hatching, the anus unfolds; 12?h after hatching, the pectoral fins emerge; 24?h after hatching, the liver and branchial arches emerge, and the alimentary canal differentiates into the oesophagus and intestinal canal; 30?h after hatching, the eyes become pigmented; 36?h after hatching, the upper and lower jaws become distinct; 42?h after hatching, the stellate melanophores emerge; 72?h after hatching, the postlarval developmental stage begins with the emergence of the dorsal fin. Based on the morphology of eggs and larvae of S. commersonnii, a taxonomic key to fish eggs of the Clupeidae and Engraulidae off China is established to provide an efficient and convenient way to identify the egg specimens. The fish eggs of the Clupeidae and Engraulidae off China could be identified to some extent by the buoyancy, shape and diameter of eggs, the number of layers in the egg membrane, the size of the perivitelline space, the number and diameters of oil globules, and the distribution of pigments. Meanwhile, their prelarvae could be identified by the number and diameters of oil globules, the distribution of pigments, the location of the anus and the number of myomeres.