Plant volatiles and priority effects interactively determined initial community assembly of arthropods on multiple willow species.

Plant traits, which are often species specific, can serve as environmental filtering for community assembly on plants. At the same time, the species identity of the initially colonizing arthropods would vary between plant individuals, which would subsequently influence colonizing arthropods and community development in the later stages. However, it remains unclear whether interindividual divergence due to priority effects is equally important as plant trait-specific environmental filtering in the initial stages. In this study, we propose that plant volatile organic compounds (PVOCs) may play a crucial role as an environmental filter in the initial stages of community assembly, which can prevent the community assembly process from being purely stochastic. To test this hypothesis, we conducted short term but highly frequent monitoring (19 observations over 9 days) of arthropod community assembly on intact individuals of six willow species in a common garden. PVOC compositions were analyzed before starting the experiment and compared with arthropod compositions occurring on Days 1-2 of the experiment (earliest colonizer community) and those occurring on Days 8-9 of the experiment (subsequent colonizer community). Unintentionally, deer herbivory also occurred at night of Day 2. Distance-based statistics demonstrated that PVOC compositions were plant species specific, but neither the earliest colonizer nor the subsequent colonizer community composition could be explained by plant species identity. Rather, Procrustes analysis showed that both the PVOC composition and that of the earliest colonizer community can be used to explain the subsequent colonizer community. In addition, the linkage between PVOCs and the subsequent colonizer community was stronger on individuals with deer herbivory. These findings indicate that PVOCs have widespread effects on initial community assembly, as well as priority effects brought on by stochastic immigration, and that plant species identity only has weak and indirect effects on the actual composition of the community.

Non-destructive collection and metabarcoding of arthropod environmental DNA remained on a terrestrial plant.

Reliable survey of arthropods is a crucial for their conservation, community ecology, and pest control on terrestrial plants. However, efficient and comprehensive surveys are hindered by challenges in collecting arthropods and identifying especially small species. To address this issue, we developed a non-destructive environmental DNA (eDNA) collection method termed "plant flow collection" to apply eDNA metabarcoding to terrestrial arthropods. This involves spraying distilled or tap water, or using rainfall, which eventually flows over the surface of the plant, and is collected in a container that is set at the plant base. DNA is extracted from collected water and a DNA barcode region of cytochrome c oxidase subunit I (COI) gene is amplified and sequenced using a high-throughput Illumina Miseq platform. We identified more than 64 taxonomic groups of arthropods at the family level, of which 7 were visually observed or artificially introduced species, whereas the other 57 groups of arthropods, including 22 species, were not observed in the visual survey. These results show that the developed method is possible to detect the arthropod eDNA remained on plants although our sample size was small and the sequence size was unevenly distributed among the three water types tested.

Effects of Prohydrojasmon on the Number of Infesting Herbivores and Biomass of Field-Grown Japanese Radish Plants.

Prohydrojasmon (PDJ), an analog of jasmonic acid (JA), was found to induce direct and indirect defenses against herbivores in non-infested plants. To test whether PDJ can be used for pest control in crop production, we conducted experiments in pesticide-free Japanese radish fields from October 4 to December 12 in 2015. Twenty-four Japanese radish plants in three plots were treated with a 100 times-diluted commercial formulation (5%) of PDJ (treated plants), and 24 plants in three different plots were treated with water (control plants) until November 29 every week. Throughout the observation period, the number of aphids, leaf-mining fly larvae, vegetable weevils, and thrips was significantly lower on the treated plants than on the control plants. In contrast, the number of lepidopteran larvae was not significantly different between the treated and control plants throughout the study period. Parasitized aphids (mummies) were also observed in both plots. Poisson regression analyses showed that a significantly higher number of mummies was recorded on the treated plants as compared to that on the control plants when the number of aphids increased. This suggested that PDJ application to Japanese radish plants attracted more parasitoid wasps on the treated plants than on the control plants. We also identified eight terpenoids and methyl salicylate as the PDJ-induced plant volatiles in the headspace of the treated plants. Some of these volatiles might be responsible for attracting aphid-parasitoid wasps in the field. However, for other insect pests, we did not find any natural enemies. Interestingly, the genes of the JA and salicylic acid signaling pathways were differentially upregulated in the treated plants. We also observed that the PDJ treatments induced the expression of the genes related to glucosinolate biosynthesis and the subsequent isothiocyanate formation. Additionally, the weights of both the aboveground and belowground parts of the treated plants were significantly lower than those of the respective parts of the control plants. These results indicated that the treatment of Japanese radish plants with a 100 times-diluted commercial formulation of PDJ induced their direct and indirect defenses against several insect pest species to reduce their numbers, and negatively affected their biomass.

Synchronous Occurrences of the Diamondback Moth (Lepidoptera: Plutellidae) and its Parasitoid Wasp Cotesia vestalis (Hymenoptera: Braconidae) in Greenhouses in a Satoyama Area.

We characterized the correlation between the occurrences of diamondback moth, Plutella xylostella (L.), larvae and their dominant native parasitoid wasp, Cotesia vestalis (Haliday), in commercial greenhouses in a satoyama area, called Miyama, in Kyoto, Japan. In the three greenhouses used in this study, cruciferous 'mizuna' (Brassica rapa var. laciniifolia [Brassicales: Brassicaceae]) crops were grown. Pesticides against diamondback moth were not routinely applied in the greenhouses. We confirmed that populations of diamondback moth and C. vestalis were maintained on the wild crucifer plant Rorippa indica in the surrounding area from March to December. In the greenhouses, we observed several occurrences of diamondback moth larvae that were, in most cases, followed by occurrences of C. vestalis. We found that C. vestalis females were attracted by volatiles emitted from mizuna plants that were lightly infested with second-stadium diamondback moth larvae under laboratory conditions. The synchronous appearance of diamondback moth larvae and C. vestalis could be explained by the latter being attracted by the volatiles emitted from mizuna plants infested by diamondback moth larvae in the greenhouses.

Parasitoid wasps' exposure to host-infested plant volatiles affects their olfactory cognition of host-infested plants.

Using Cotesia vestalis, a parasitoid wasp of diamondback moth larvae and three crucifer plant species (cabbage, komatsuna, and Japanese radish), we examined the effects of exposure to host-infested plant volatiles from one plant species on a newly emerged wasp's subsequent olfactory cognition of host-infested plant volatiles from the same or different plant species. The preference of C. vestalis between infested and uninfested plant volatiles was tested in a choice chamber. Volatile-inexperienced wasps significantly preferred infested cabbage and infested radish volatiles, but not infested komatsuna volatiles. After exposure to infested cabbage volatiles, wasps showed a significant preference for infested cabbage volatiles, while the significant preference for infested radish volatiles that had been observed in inexperienced wasps was no longer observed. After exposure to infested komatsuna volatiles, wasps significantly preferred infested komatsuna volatiles, and the pre-exposure significant preferences for infested cabbage volatiles and infested radish volatiles remained. After exposure to infested radish volatiles, the significant preferences for infested cabbage and infested radish volatiles did not change. Furthermore, wasps showed a significant preference for infested komatsuna volatiles. The compound compositions of the volatile blends from the three infested plant species were grouped separately in a principal coordinates analysis. The experience-based cognition of C. vestalis for host-infested plant volatiles of three plant species is discussed.

Egg laying of cabbage white butterfly (Pieris brassicae) on Arabidopsis thaliana affects subsequent performance of the larvae.

Plant resistance to the feeding by herbivorous insects has recently been found to be positively or negatively influenced by prior egg deposition. Here we show how crucial it is to conduct experiments on plant responses to herbivory under conditions that simulate natural insect behaviour. We used a well-studied plant--herbivore system, Arabidopsis thaliana and the cabbage white butterfly Pieris brassicae, testing the effects of naturally laid eggs (rather than egg extracts) and allowing larvae to feed gregariously as they do naturally (rather than placing single larvae on plants). Under natural conditions, newly hatched larvae start feeding on their egg shells before they consume leaf tissue, but access to egg shells had no effect on subsequent larval performance in our experiments. However, young larvae feeding gregariously on leaves previously laden with eggs caused less feeding damage, gained less weight during the first 2 days, and suffered twice as high a mortality until pupation compared to larvae feeding on plants that had never had eggs. The concentration of the major anti-herbivore defences of A. thaliana, the glucosinolates, was not significantly increased by oviposition, but the amount of the most abundant member of this class, 4-methylsulfinylbutyl glucosinolate was 1.8-fold lower in larval-damaged leaves with prior egg deposition compared to damaged leaves that had never had eggs. There were also few significant changes in the transcript levels of glucosinolate metabolic genes, except that egg deposition suppressed the feeding-induced up-regulation of FMOGS-OX2 , a gene encoding a flavin monooxygenase involved in the last step of 4-methylsulfinylbutyl glucosinolate biosynthesis. Hence, our study demonstrates that oviposition does increase A. thaliana resistance to feeding by subsequently hatching larvae, but this cannot be attributed simply to changes in glucosinolate content.

An apparent trade-off between direct and signal-based induced indirect defence against herbivores in willow trees.

Signal-based induced indirect defence refers to herbivore-induced production of plant volatiles that attract carnivorous natural enemies of herbivores. Relationships between direct and indirect defence strategies were studied using tritrophic systems consisting of six sympatric willow species, willow leaf beetles (Plagiodera versicolora), and their natural predators, ladybeetles (Aiolocaria hexaspilota). Relative preferences of ladybeetles for prey-infested willow plant volatiles, indicating levels of signal-based induced indirect defence, were positively correlated with the vulnerability of willow species to leaf beetles, assigned as relative levels of direct defence. This correlation suggested a possible trade-off among the species, in terms of resource limitation between direct defence and signal-based induced indirect defence. However, analyses of volatiles from infested and uninfested plants showed that the specificity of infested volatile blends (an important factor determining the costs of signal-based induced indirect defence) did not affect the attractiveness of infested plant volatiles. Thus, the suggested trade-off in resource limitation was unlikely. Rather, principal coordinates analysis showed that this 'apparent trade-off' between direct and signal-based induced indirect defence was partially explained by differential preferences of ladybeetles to infested plant volatiles of the six willow species. We also showed that relative preferences of ladybeetles for prey-infested willow plant volatiles were positively correlated with oviposition preferences of leaf beetles and with the distributions of leaf beetles in the field. These correlations suggest that ladybeetles use the specificity of infested willow plant volatiles to find suitable prey patches.

Can insect egg deposition 'warn' a plant of future feeding damage by herbivorous larvae?

Plant anti-herbivore defence is inducible by both insect feeding and egg deposition. However, little is known about the ability of insect eggs to induce defences directed not against the eggs themselves, but against larvae that subsequently hatch from the eggs. We studied how oviposition (OP) by the sawfly Diprion pini on Pinus sylvestris foliage affects the plant's defensive potential against sawfly larvae. Larvae that initiated their development on P. sylvestris twigs on which they hatched from eggs gained less weight and suffered higher mortality than those fed on egg-free twigs. The poor performance of these larvae also affected the next herbivore generation since fecundity of resulting females was lower than that of females which spent their larval development on egg-free pine. Transcript levels of P. sylvestris sesquiterpene synthases (PsTPS1, PsTPS2) were increased by D. pini OP, reached their highest levels just before larval hatching, and decreased when larvae started to feed. However, concentrations of terpenoid and phenolic metabolites presumed to act as feeding deterrents or toxins for herbivores did not change significantly after OP and feeding. Nevertheless, our performance data suggest that insect egg deposition may act to 'warn' a plant of upcoming feeding damage by larvae.

Specialist leaf beetle larvae use volatiles from willow leaves infested by conspecifics for reaggregation in a tree.

Young, gregariously living larvae of the willow leaf beetles Plagiodera versicolora are known to exhibit characteristic aggregation-dispersion-reaggregation behavior and local fidelity to a host tree. In this study, we investigated whether plant volatiles induced by feeding P. versicolora larvae were involved in the reaggregation behavior. Under laboratory conditions, we conducted dual-choice bioassays and found that the first and second instars discriminated between volatiles from leaves infested by larvae and volatiles from uninfested leaves. The discriminative behavior was dependent on both the time leaves were infested and the age of discriminating larvae. First and second instars preferred odor from 1-d-infested leaves to odor from uninfested leaves, whereas third instars (solitary stage) did not discriminate between these volatile blends. Odor from 2-d-infested leaves was preferred to odor from 1-d-infested leaves by first instars, whereas odor from leaves infested for 3 d was not attractive to these very young larvae. Neither was odor of leaves infested for 1 d and then left uninfested for 1 or 2 d attractive to young larvae. The data suggest that the first and second instars use volatiles from a leaf newly infested by conspecific larvae as one of the reaggregation cues. We detected several herbivore-induced compounds in the headspace of the attractive leaves. Among those, a mixture of synthetic (E)-beta-ocimene, (Z)-beta-ocimene, allo-ocimene, and linalool was found to attract the larvae.