Microbial associates of the elm leaf beetle: uncovering the absence of resident bacteria and the influence of fungi on insect performance.

Microbial symbionts play crucial roles in the biology of many insects. While bacteria have been the primary focus of research on insect-microbe symbiosis, recent studies suggest that fungal symbionts may be just as important. The elm leaf beetle (ELB, Xanthogaleruca luteola) is a serious pest species of field elm (Ulmus minor). Using culture-dependent and independent methods, we investigated the abundance and species richness of bacteria and fungi throughout various ELB life stages and generations, while concurrently analyzing microbial communities on elm leaves. No persistent bacterial community was found to be associated with the ELB or elm leaves. By contrast, fungi were persistently present in the beetle's feeding life stages and on elm leaves. Fungal community sequencing revealed a predominance of the genera Penicillium and Aspergillus in insects and on leaves. Culture-dependent surveys showed a high prevalence of two fungal colony morphotypes closely related to Penicillium lanosocoeruleum and Aspergillus flavus. Among these, the Penicillium morphotype was significantly more abundant on feeding-damaged compared with intact leaves, suggesting that the fungus thrives in the presence of the ELB. We assessed whether the detected prevalent fungal morphotypes influenced ELB's performance by rearing insects on (i) surface-sterilized leaves, (ii) leaves inoculated with Penicillium spores, and (iii) leaves inoculated with Aspergillus spores. Insects feeding on Penicillium-inoculated leaves gained more biomass and tended to lay larger egg clutches than those consuming surface-sterilized leaves or Aspergillus-inoculated leaves. Our results demonstrate that the ELB does not harbor resident bacteria and that it might benefit from associating with Penicillium fungi.IMPORTANCEOur study provides insights into the still understudied role of microbial symbionts in the biology of the elm leaf beetle (ELB), a major pest of elms. Contrary to expectations, we found no persistent bacterial symbionts associated with the ELB or elm leaves. Our research thus contributes to the growing body of knowledge that not all insects rely on bacterial symbionts. While no persistent bacterial symbionts were detectable in the ELB and elm leaf samples, our analyses revealed the persistent presence of fungi, particularly Penicillium and Aspergillus on both elm leaves and in the feeding ELB stages. Moreover, when ELB were fed with fungus-treated elm leaves, we detected a potentially beneficial effect of Penicillium on the ELB's development and fecundity. Our results highlight the significance of fungal symbionts in the biology of this insect.

Pine Response to Sawfly Pheromones: Effects on Sawfly's Oviposition and Larval Growth.

Insect pheromones have been intensively studied with respect to their role in insect communication. However, scarce knowledge is available on the impact of pheromones on plant responses, and how these in turn affect herbivorous insects. A previous study showed that exposure of pine (Pinus sylvestris) to the sex pheromones of the pine sawfly Diprion pini results in enhanced defenses against the eggs of this sawfly; the egg survival rate on pheromone-exposed pine needles was lower than that on unexposed pine. The long-lasting common evolutionary history of D. pini and P. sylvestris suggests that D. pini has developed counter-adaptations to these pine responses. Here, we investigated by behavioral assays how D. pini copes with the defenses of pheromone-exposed pine. The sawfly females did not discriminate between the odor of pheromone-exposed and unexposed pine. However, when they had the chance to contact the trees, more unexposed than pheromone-exposed trees received eggs. The exposure of pine to the pheromones did not affect the performance of larvae and their pupation success. Our findings indicate that the effects that responses of pine to D. pini sex pheromones exert on the sawfly eggs and sawfly oviposition behavior do not extend to effects on the larvae.

The impact of insect egg deposition on Pinus sylvestris transcriptomic and phytohormonal responses to larval herbivory.

Plants can improve their resistance to feeding damage by insects if they have perceived insect egg deposition prior to larval feeding. Molecular analyses of these egg-mediated defence mechanisms have until now focused on angiosperm species. It is unknown how the transcriptome of a gymnosperm species responds to insect eggs and subsequent larval feeding. Scots pine (Pinus sylvestris L.) is known to improve its defences against larvae of the herbivorous sawfly Diprion pini L. if it has previously received sawfly eggs. Here, we analysed the transcriptomic and phytohormonal responses of Scots pine needles to D. pini eggs (E-pine), larval feeding (F-pine) and to both eggs and larval feeding (EF-pine). Pine showed strong transcriptomic responses to sawfly eggs and-as expected-to larval feeding. Many egg-responsive genes were also differentially expressed in response to feeding damage, and these genes play an important role in biological processes related to cell wall modification, cell death and jasmonic acid signalling. EF-pine showed fewer transcriptomic changes than F-pine, whereas EF-treated angiosperm species studied so far showed more transcriptional changes to the initial phase of larval feeding than only feeding-damaged F-angiosperms. However, as with responses of EF-angiosperms, EF-pine showed higher salicylic acid concentrations than F-pine. Based on the considerable overlap of the transcriptomes of E- and F-pine, we suggest that the weaker transcriptomic response of EF-pine than F-pine to larval feeding damage is compensated by the strong, egg-induced response, which might result in maintained pine defences against larval feeding.

Drought induces moderate, diverse changes in the odour of grassland species.

Plants react to drought stress with numerous changes including altered emissions of volatile organic compounds (VOC) from leaves, which provide protection against oxidative tissue damage and mediate numerous biotic interactions. Despite the share of grasslands in the terrestrial biosphere, their importance as carbon sinks and their contribution to global biodiversity, little is known about the influence of drought on VOC profiles of grassland species. Using coupled gas chromatography-mass spectrometry, we analysed the odorants emitted by 22 European grassland species exposed to an eight-week-lasting drought treatment (DT; 30% water holding capacity, WHC). We focused on the odorants emitted during the light phase from whole plant shoots in their vegetative stage. Emission rates were standardised to the dry weight of each shoot. Well-watered (WW) plants (70% WHC) served as control. Drought-induced significant changes included an increase in total emission rates of plant VOC in six and a decrease in three species. Diverging effects on the number of emitted VOC (chemical richness) or on the Shannon diversity of the VOC profiles were detected in 13 species. Biosynthetic pathways-targeted analyses revealed 13 species showing drought-induced higher emission rates of VOC from one, two, three, or four major biosynthetic pathways (lipoxygenase, shikimate, mevalonate and methylerythritol phosphate pathway), while six species exhibited reduced emission rates from one or two of these pathways. Similarity trees of odorant profiles and their drought-induced changes based on a biosynthetically informed distance metric did not match species phylogeny. However, a phylogenetic signal was detected for the amount of terpenoids released by the studied species under WW and DT conditions. A comparative analysis of emission rates of single compounds released by WW and DT plants revealed significant VOC profile dissimilarities in four species only. The moderate drought-induced changes in the odorant emissions of grassland species are discussed with respect to their impact on trophic interactions across the food web. (294 words).

Plant defensive responses to insect eggs are inducible by general egg-associated elicitors.

Egg deposition by herbivorous insects is well known to elicit defensive plant responses. Our study aimed to elucidate the insect and plant species specificity of these responses. To study the insect species specificity, we treated Arabidopsis thaliana with egg extracts and egg-associated secretions of a sawfly (Diprion pini), a beetle (Xanthogaleruca luteola) and a butterfly (Pieris brassicae). All egg extracts elicited salicylic acid (SA) accumulation in the plant, and all secretions induced expression of plant genes known to be responsive to the butterfly eggs, among them Pathogenesis-Related (PR) genes. All secretions contained phosphatidylcholine derivatives, known elicitors of SA accumulation and PR gene expression in Arabidopsis. The sawfly egg extract did not induce plant camalexin levels, while the other extracts did. Our studies on the plant species specificity revealed that Solanum dulcamara and Ulmus minor responded with SA accumulation and cell death to P. brassicae eggs, i.e. responses also known for A. thaliana. However, the butterfly eggs induced neoplasms only in S. dulcamara. Our results provide evidence for general, phosphatidylcholine-based, egg-associated elicitors of plant responses and for conserved plant core responses to eggs, but also point to plant and insect species-specific traits in plant-insect egg interactions.