Under fire-simultaneous volatilome and transcriptome analysis unravels fine-scale responses of tansy chemotypes to dual herbivore attack.

BACKGROUND: Tansy plants (Tanacetum vulgare L.) are known for their high intraspecific chemical variation, especially of volatile organic compounds (VOC) from the terpenoid compound group. These VOCs are closely involved in plant-insect interactions and, when profiled, can be used to classify plants into groups known as chemotypes. Tansy chemotypes have been shown to influence plant-aphid interactions, however, to date no information is available on the response of different tansy chemotypes to simultaneous herbivory by more than one insect species. RESULTS: Using a multi-cuvette system, we investigated the responses of five tansy chemotypes to feeding by sucking and/or chewing herbivores (aphids and caterpillars; Metopeurum fuscoviride Stroyan and Spodoptera littoralis Boisduval). Herbivory by caterpillars following aphid infestation led to a plant chemotype-specific change in the patterns of terpenoids stored in trichome hairs and in VOC emissions. The transcriptomic analysis of a plant chemotype represents the first de novo assembly of a transcriptome in tansy and demonstrates priming effects of aphids on a subsequent herbivory. Overall, we show that the five chemotypes do not react in the same way to the two herbivores. As expected, we found that caterpillar feeding increased VOC emissions, however, a priori aphid infestation only led to a further increase in VOC emissions for some chemotypes. CONCLUSIONS: We were able to show that different chemotypes respond to the double herbivore attack in different ways, and that pre-treatment with aphids had a priming effect on plants when they were subsequently exposed to a chewing herbivore. If neighbouring chemotypes in a field population react differently to herbivory/dual herbivory, this could possibly have effects from the individual level to the group level. Individuals of some chemotypes may respond more efficiently to herbivory stress than others, and in a group environment these "louder" chemotypes may affect the local insect community, including the natural enemies of herbivores, and other neighbouring plants.

Volatile, stored and phloem exudate-located compounds represent different appearance levels affecting aphid niche choice.

Intraspecific and intra-individual differences in emitted volatile compounds and in surface and phloem sap-related metabolites do not only affect host plant choice of monophagous aphids but may also guide them to the plant part that provides their ideal niche by maximising their fitness. However, little is known about the variation at these different plant appearance levels. We investigated the preferences of the monophagous aphid species Macrosiphoniella tancetaria and Uroleucon tanaceti for different plant parts (inflorescence stems, young and old leaves) of Tanacetum vulgare plants from two chemotypes, testing their reactions towards volatile, surface and phloem sap-related cues. Furthermore, we studied the variation in leaf glandular trichome density as well as in the composition of volatile, stored and phloem exudate-located specialised (secondary) plant compounds from the different plant parts of these chemotypes. Aphid species showed differences in their preferences. Aphids of M. tanacetaria had to assess the entire plant to choose the stem, whereas U. tanaceti only needed volatile cues to locate the old leaves, which are the plant parts representing their respective niches. Volatiles and stored metabolites varied in their composition and concentration between chemotypes. Stored metabolites additionally differed among plant parts, which was reflected in distinct trichome densities. The composition of phloem exudate-located specialised compounds mostly varied among plant parts. These pronounced differences in plant chemistry on multiple levels provide distinct perception levels for aphids probably driving their niche choice. This study demonstrates the importance to consider these multiple levels to elucidate plant-herbivore interactions with high resolution.

Chemotypic variation in terpenes emitted from storage pools influences early aphid colonisation on tansy.

Tansy plants (Tanacetum vulgare L.) exhibit high chemical variation, particularly in mono- and sesquiterpenes that are stored in specialised glands on the plant surface. In the present work we investigated the effects of terpene chemotypes on Metopeurum fuscoviride, an aphid species specialised on tansy, and their tending ants, at the field scale. Previous studies have chemotyped tansy by assessing dominant compounds; here we propose a method of chemotyping using all volatile compounds that are likely emitted from the storage glands. The analysis is based on two extraction methods: GC-MS analysis of leaf hexane extracts and SBSE analysis of headspace emissions. In an initial screening we identified the subset of compounds present in both chemical patterns, labelled as 'compounds likely emitted from storage'. In a large field survey we could show that the putative chemotypic emission pattern from storage pools significantly affected the early aphid colonisation of tansy. Moreover, the statistical analyses revealed that minor compounds exerted a stronger influence on aphid and tending-ant presence than dominant compounds. Overall we demonstrated that within the enormous chemotypic variation of terpenes in tansy plants, chemical signatures of volatile terpenes can be related to the occurrence of insects on individual plants in the field.

Biology and morphology of immature stages of Adosomus roridus (Coleoptera: Curculionidae: Lixinae).

Mature larva and pupa of Adosomus (s. str.) roridus (Pallas, 1781) (Curculionidae: Lixinae: Cleonini) are described and compared with ten other taxa of Cleonini with known larvae. This weevil is an oligophagous species on the Asteraceae family. From our observations in Slovakia, we found active adults from April to September in dry sunny places within extensively used or fallow vineyards and in ruderal vegetation with host plants. The weevil is a root borer--larvae, pupae and fresh adults were collected from the root necks and roots of Common Tansy (Tanacetum vulgare L.) and rarely from Mugwort (Artemisia vulgaris L.). Each plant was usually occupied by one larva, or more rarely with two or three larvae. The new generation of adult individuals appeared from early summer to autumn. Both larvae and some of the adults overwinter, which is quite unique within Cleonini.

How aphids decide what is good for them: experiments to test aphid feeding behaviour on Tanacetum vulgare (L.) using different nitrogen regimes.

Leaf-chewing herbivores select food with a protein/carbohydrate ratio of 0.8-1.5, whereas phloem sap, which aphids feed on, has a ratio of approximately 0.1. Enhanced N fertilization increases the amino acid concentration in phloem sap and elevates the N/C ratio. The study examines: (1) whether aphids select between plants of different N nutrition, (2) whether feeding time correlates with the amino acid composition of phloem sap, and (3) at which stage of probing aphids identify the quality of the plant. Uroleucon tanaceti (Mordvilko) and Macrosiphoniella tanacetaria (Kaltenbach), specialist aphids feeding on tansy (Tanacetum vulgare L.), were reared on this host plant grown essentially hydroponically (in Vermiculite) in the greenhouse on 1, 3, 6, or 12 mM NH(4)NO(3). One and 3 mM NH(4)NO(3) corresponds to the situation found in natural tansy stands. Aphid stylet penetration was monitored by electrical penetration graphs whilst phloem sap was sampled by stylectomy. Both aphid species settled 2-3 times more frequently on plants fertilized with 6 or 12 mM NH(4)NO(3). The phloem sap of these plants contained up to threefold higher amino acid concentrations, without a change in the proportion of essential amino acids. No time differences were observed before stylet penetration of plant tissue. After the first symplast contact, most aphids penetrated further, except M. tanacetaria on low-N plants, where 50% withdrew the stylet after the first probing. The duration of phloem feeding was 2-3 times longer in N-rich plants and the time spent in individual sieve tubes was up to tenfold longer. Aphids identified the nutritional quality of the host plant mainly by the amino acid concentration of phloem sap, not by leaf surface cues nor the proportion of essential amino acids. However, U. tanaceti infestation increased the percentage of methionine plus tryptophan in phloem tenfold, thus manipulating the plants nutritional quality, and causing premature leaf senescence.

Metapopulation structure of the specialized herbivore Macrosiphoniella tanacetaria (Homoptera, Aphididae).

We investigated population dynamics, genetic diversity and spatial structure in the aphid species Macrosiphoniella tanacetaria, a specialist herbivore feeding on tansy, Tanacetum vulgare. Tansy plants (genets) consist of many shoots (ramets), and genets are grouped in sites. Thus, aphids feeding on tansy can cluster at the level of ramets, genets and sites. We studied aphid population dynamics in 1997 and 2001 and found that within sites: (i). at any time, aphids used only a fraction of the available ramets and genets; (ii). at the level of ramets, most aphid colonies survived only one week; (iii). at the level of genets, mean survival time was less than 4 weeks; and (iv). colonization and extinction events occurred throughout the season. We sampled aphids in seven sites in the Alsace region, France (4-45 km apart) and two sites in Germany in 1999 to study genetic structure within and between populations. Genetic analyses using nine microsatellite loci showed that: (i). genotypic variability was high, (ii). none of the populations was in Hardy-Weinberg equilibrium, (iii). heterozygote deficits and linkage disequilibria were frequent, and (iv). all populations were genetically differentiated, even at a small geographical scale. Renewed sampling of the Alsace sites in 2001 showed that three populations had become extinct and significant genetic changes had occurred in the remaining four populations. The frequencies of extinction and colonization events at several spatial scales suggest a hierarchical metapopulation structure for M. tanacetaria. Frequent population turnover and drift are likely causes for the genetic differentiation of M. tanacetaria populations.