Phenotypic screen and transcriptomics approach complement each other in functional genomics of defensive stink gland physiology.

BACKGROUND: Functional genomics uses unbiased systematic genome-wide gene disruption or analyzes natural variations such as gene expression profiles of different tissues from multicellular organisms to link gene functions to particular phenotypes. Functional genomics approaches are of particular importance to identify large sets of genes that are specifically important for a particular biological process beyond known candidate genes, or when the process has not been studied with genetic methods before. RESULTS: Here, we present a large set of genes whose disruption interferes with the function of the odoriferous defensive stink glands of the red flour beetle Tribolium castaneum. This gene set is the result of a large-scale systematic phenotypic screen using RNA interference applied in a genome-wide forward genetics manner. In this first-pass screen, 130 genes were identified, of which 69 genes could be confirmed to cause phenotypic changes in the glands upon knock-down, which vary from necrotic tissue and irregular reservoir size to irregular color or separation of the secreted gland compounds. Gene ontology analysis revealed that many of those genes are encoding enzymes (peptidases and cytochromes P450) as well as proteins involved in membrane trafficking with an enrichment in lysosome and mineral absorption pathways. The knock-down of 13 genes caused specifically a strong reduction of para-benzoquinones in the gland reservoirs, suggesting a specific function in the synthesis of these toxic compounds. Only 14 of the 69 confirmed gland genes are differentially overexpressed in stink gland tissue and thus could have been detected in a transcriptome-based analysis. However, only one out of eight genes identified by a transcriptomics approach known to cause phenotypic changes of the glands upon knock-down was recognized by this phenotypic screen, indicating the limitation of such a non-redundant first-pass screen. CONCLUSION: Our results indicate the importance of combining diverse and independent methodologies to identify genes necessary for the function of a certain biological tissue, as the different approaches do not deliver redundant results but rather complement each other. The presented phenotypic screen together with a transcriptomics approach are now providing a set of close to hundred genes important for odoriferous defensive stink gland physiology in beetles.

Odoriferous Defensive stink gland transcriptome to identify novel genes necessary for quinone synthesis in the red flour beetle, Tribolium castaneum.

Chemical defense is one of the most important traits, which endow insects the ability to conquer a most diverse set of ecological environments. Chemical secretions are used for defense against anything from vertebrate or invertebrate predators to prokaryotic or eukaryotic parasites or food competitors. Tenebrionid beetles are especially prolific in this category, producing several varieties of substituted benzoquinone compounds. In order to get a better understanding of the genetic and molecular basis of defensive secretions, we performed RNA sequencing in a newly emerging insect model, the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae). To detect genes that are highly and specifically expressed in the odoriferous gland tissues that secret defensive chemical compounds, we compared them to a control tissue, the anterior abdomen. 511 genes were identified in different subtraction groups. Of these, 77 genes were functionally analyzed by RNA interference (RNAi) to recognize induced gland alterations morphologically or changes in gland volatiles by gas chromatography-mass spectrometry. 29 genes (38%) presented strong visible phenotypes, while 67 genes (87%) showed alterations of at least one gland content. Three of these genes showing quinone-less (ql) phenotypes - Tcas-ql VTGl; Tcas-ql ARSB; Tcas-ql MRP - were isolated, molecularly characterized, their expression identified in both types of the secretory glandular cells, and their function determined by quantification of all main components after RNAi. In addition, microbe inhibition assays revealed that a quinone-free status is unable to impede bacterial or fungal growth. Phylogenetic analyses of these three genes indicate that they have evolved independently and specifically for chemical defense in beetles.

Cockchafer larvae smell host root scents in soil.

In many insect species olfaction is a key sensory modality. However, examination of the chemical ecology of insects has focussed up to now on insects living above ground. Evidence for behavioral responses to chemical cues in the soil other than CO(2) is scarce and the role played by olfaction in the process of finding host roots below ground is not yet understood. The question of whether soil-dwelling beetle larvae can smell their host plant roots has been under debate, but proof is as yet lacking that olfactory perception of volatile compounds released by damaged host plants, as is known for insects living above ground, occurs. Here we show that soil-dwelling larvae of Melolontha hippocastani are well equipped for olfactory perception and respond electrophysiologically and behaviorally to volatiles released by damaged host-plant roots. An olfactory apparatus consisting of pore plates at the antennae and about 70 glomeruli as primary olfactory processing units indicates a highly developed olfactory system. Damage induced host plant volatiles released by oak roots such as eucalyptol and anisol are detected by larval antennae down to 5 ppbv in soil air and elicit directed movement of the larvae in natural soil towards the odor source. Our results demonstrate that plant-root volatiles are likely to be perceived by the larval olfactory system and to guide soil-dwelling white grubs through the dark below ground to their host plants. Thus, to find below-ground host plants cockchafer larvae employ mechanisms that are similar to those employed by the adult beetles flying above ground, despite strikingly different physicochemical conditions in the soil.

Antennal and behavioral responses of Cis boleti to fungal odor of Trametes gibbosa.

Cis boleti (Coleoptera: Ciidae) preferentially colonizes fungi from the genus Trametes that are known as important wood decomposers. The aim of our research was to investigate if C. boleti uses the chemical volatile composition of its fungal host, Trametes gibbosa, as a key attraction factor. Therefore, the T. gibbosa fruiting body volatiles were analysed by using gas chromatography-mass spectrometry, with parallel electroantennographic detection (GC-MS/EAD) using adults of C. boleti. Furthermore, we examined the behavioral responses of C. boleti to the T. gibbosa volatile compounds. The dominant component of the T. gibbosa fruiting body bouquet was 1-octen-3-ol. Other volatiles, like the aldehydes hexanal, nonanal, and (E,E)-2,4-decadienal and the terpene alpha-bisabolol, were present in minor quantities. 1-Octen-3-ol was released with a ratio of the (R)- and (S)-enantiomers of 93:7, respectively. Electroantennography (EAG) employing C. boleti antennae yielded consistently dominant responses to 1-octen-3-ol. GC-EAD and EAG responses to pure standard compounds showed that C. boleti also perceived other host fungal volatiles. A highly significant attraction to 1-octen-3-ol was observed in behavioral tests. Female beetles were significantly attracted to the (S)-(+)- enantiomer at 10 times lower doses than male beetles. Our finding is the first direct proof that ciid beetles use 1-octen-3-ol as a key cue for host finding.

Volatile emissions from Aesculus hippocastanum induced by mining of larval stages of Cameraria ohridella influence oviposition by conspecific females.

Larval stages of the horse chestnut leafminer Cameraria ohridella can completely destroy the surface of horse chestnut leaves, Aesculus hippocastanum. This study investigated the effect of the degree of leaf browning caused by the insect's larvae on olfactory detection, aggregation, and oviposition of C. ohridella adults. The influence of A. hippocastanum flower scent on oviposition of the first generation was also evaluated. Utilizing gas chromatography coupled with parallel detection by mass spectrometry and electroantennography (GC-MS/EAD), more than 30 compounds eliciting responses from antennae of C. ohridella were detected. Oviposition and mining by C. ohridella caused significant changes in the profile of leaf volatiles of A. hippocastanum. After oviposition and subsequent mining by early larval stages (L1-L3), substances such as benzaldehyde, 1,8-cineole, benzyl alcohol, 2-phenylethanol, methyl salicylate, (E)-beta-caryophyllene, and (E,E)-alpha-farnesene were emitted in addition to the compounds emitted by uninfested leaves. Insects were able to detect these compounds. The emitted amount of these substances increased with progressive larval development. During late larval stages (L4, L5) and severe loss of green leaf area, (E,E)-2,4-hexadienal, (E/Z)-linalool oxide (furanoid), nonanal, and decanal were also released by leaves. These alterations of the profile of volatiles caused modifications in aggregation of C. ohridella on leaves. In choice tests, leaves in early infestation stages showed no significant effect on aggregation, whereas insects avoided leaves in late infestation stages. Further choice tests with leaves treated with single compounds led to the identification of substances mediating an increase or decrease in oviposition.

Linalool and lilac aldehyde/alcohol in flower scents. Electrophysiological detection of lilac aldehyde stereoisomers by a moth.

The stereoisomers of linalool and lilac aldehyde/alcohol were determined in the flower scent of 15 plant species using enantioselective multidimensional gas chromatography/mass spectrometry (enantio-MDGC/MS). Both linalool and all 8 stereoisomers of lilac alcohol and lilac aldehyde were detected, and there was a species-specific pattern. Single stereoisomers were collected by micropreparative-enantio-MDGC and were electrophysiologically tested on antennae of the noctuid moth Hadena bicruris, a species known to rely on lilac aldehyde for finding its host plant. The moth responded to all 8 stereoisomers, though only four stereoisomers were found in the scent of its host plant. The moth was less sensitive to some isomers than to others.

Gas chromatography with mass spectrometric and electroantennographic detection: analysis of wood odorants by direct coupling of insect olfaction and mass spectrometry.

A gas chromatography-mass spectrometry-electroantennographic detection (GC-MS/EAD) setup has been designed by adapting a commercially available "Olfactory Detector Port" to the use with an insect antenna. Measurements were performed with antennae of the old house borer Hylotrupes bajulus, a widespread insect pest of coniferous timbers. Headspace volatiles from timber of Pinus sylvestris were collected and analysed by GC-MS. About 30 compounds were identified in the Kovacs range from 500 to 1200, especially terpenoids and aliphatic alcohols and aldehydes. The antennae of H. bajulus responded to nearly half of the detected volatiles with a peculiar sensitivity for alpha-pinene among the terpenes and for hexanal among the aldehydes.