Astrid T. Groot

Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges

Emergence of polyphagous herbivorous insects entails significant adaptation to recognize, detoxify and digest a variety of host-plants. Despite of its biological and practical importance - since insects eat 20% of crops - no exhaustive analysis of gene repertoires required for adaptations in generalist insect herbivores has previously been performed. The noctuid moth Spodoptera frugiperda ranks as one of the world’s worst agricultural pests. This insect is polyphagous while the majority of other lepidopteran herbivores are specialist. It consists of two morphologically indistinguishable strains (“C” and “R”) that have different host plant ranges. To describe the evolutionary mechanisms that both enable the emergence of polyphagous herbivory and lead to the shift in the host preference, we analyzed whole genome sequences from laboratory and natural populations of both strains. We observed huge expansions of genes associated with chemosensation and detoxification compared with specialist Lepidoptera. These expansions are largely due to tandem duplication, a possible adaptation mechanism enabling polyphagy. Individuals from natural C and R populations show significant genomic differentiation. We found signatures of positive selection in genes involved in chemoreception, detoxification and digestion, and copy number variation in the two latter gene families, suggesting an adaptive role for structural variation.

Allelic variation in a fatty-acyl reductase gene causes divergence in moth sex pheromones

Pheromone-based behaviours are crucial in animals from insects to mammals, and reproductive isolation is often based on pheromone differences. However, the genetic mechanisms by which pheromone signals change during the evolution of new species are largely unknown. In the sexual communication system of moths (Insecta: Lepidoptera), females emit a species-specific pheromone blend that attracts males over long distances. The European corn borer, Ostrinia nubilalis, consists of two sex pheromone races, Z and E, that use different ratios of the cis and trans isomers of acetate pheromone components. This subtle difference leads to strong reproductive isolation in the field between the two races, which could represent a first step in speciation. Female sex pheromone production and male behavioural response are under the control of different major genes, but the identity of these genes is unknown. Here we show that allelic variation in a fatty-acyl reductase gene essential for pheromone biosynthesis accounts for the phenotypic variation in female pheromone production, leading to race-specific signals. Both the cis and trans isomers of the pheromone precursors are produced by both races, but the precursors are differentially reduced to yield opposite ratios in the final pheromone blend as a result of the substrate specificity of the enzymes encoded by the Z and E alleles. This is the first functional characterization of a gene contributing to intraspecific behavioural reproductive isolation in moths, highlighting the importance of evolutionary diversification in a lepidopteran-specific family of reductases. Accumulation of substitutions in the coding region of a single biosynthetic enzyme can produce pheromone differences resulting in reproductive isolation, with speciation as a potential end result.

Transcriptome analysis of the sex pheromone gland of the noctuid moth Heliothis virescens

BackgroundThe chemical components of sex pheromones have been determined for more than a thousand moth species, but so far only a handful of genes encoding enzymes responsible for the biosynthesis of these compounds have been identified. For understanding the evolution of moth sexual communication, it is essential to know which genes are involved in the production of specific pheromone components and what controls the variation in their relative frequencies in the pheromone blend. We used a transcriptomic approach to characterize the pheromone gland of the Noctuid moth Heliothis virescens, an important agricultural pest, in order to obtain substantial general sequence information and to identify a range of candidate genes involved in the pheromone biosynthetic pathway.ResultsTo facilitate identifying sets of genes involved in a broad range of processes and to capture rare transcripts, we developed our majority of ESTs from a normalized cDNA library of Heliothis virescens pheromone glands (PG). Combining these with a non-normalized library yielded a total of 17,233 ESTs, which assembled into 2,082 contigs and 6,228 singletons. Using BLAST searches of the NR and Swissprot databases we were able to identify a large number of putative unique gene elements (unigenes), which we compared to those derived from previous transcriptomic surveys of the larval stage of Heliothis virescens. The distribution of unigenes among GO Biological Process functional groups shows an overall similarity between PG and larval transcriptomes, but with distinct enrichment of specific pathways in the PG. In addition, we identified a large number of candidate genes in the pheromone biosynthetic pathways.ConclusionThese data constitute one of the first large-scale EST-projects for Noctuidae, a much-needed resource for exploring these pest species. Our analysis shows a surprisingly complex transcriptome and we identified a large number of potential pheromone biosynthetic pathway and immune-related genes that can be applied to population and systematic studies of Heliothis virescens and other Noctuidae.

Host strain specific sex pheromone variation in Spodoptera frugiperda

BackgroundThe fall armyworm Spodoptera frugiperda (Lepidoptera; Noctuidae) consists of two distinct strains with different host plant preferences for corn and rice. To assess whether pheromonal-mediated behavioral isolation accompanies the habitat isolation on different host plants, we compared the sex pheromone composition among females of the two strains. Pheromone glands were extracted with or without injection of pheromone biosynthesis activating neuropeptide (PBAN). To assess the mode of inheritance of this variation, we also analyzed the pheromone composition of F1 hybrid females.ResultsRelative to intra-strain variation, the pheromone composition of the two strains differed significantly. Corn strain females contained significantly more of the second most abundant pheromone compound Z11-16:Ac (m), and significantly less of most other compounds, than rice strain females. When females were injected with PBAN before their glands were extracted, the differences between the strains were less pronounced but still statistically significant. The pheromone composition of hybrid females showed a maternal inheritance of the major component Z9-14:Ac (M) as well as of Z11-16:Ac (m). Most other compounds showed an inheritance indicating genetic dominance of the corn strain. The within-strain phenotypic correlations among the various components were consistent with their hypothesized biosynthetic pathway, and between-strain differences in the correlation structure suggested candidate genes that may explain the pheromone differences between the two strains. These include Δ9- and Δ11 desaturases, and possibly also a Δ7-desaturase, although the latter has not been identified in insects so far.ConclusionThe two host strains of S. frugiperda produce systematically differing female sex pheromone blends. Previously-documented geographic variation in the sexual communication of this species did not take strain identity into account, and thus may be partly explained by different strain occurrence in different regions. The finding of pheromone differences reinforces the possibility of incipient reproductive isolation among these strains, previously shown to differ in the timing of nocturnal mating activity and host plant use. Finding the genetic basis of the pheromone differences, as well as these other biological traits, will help to elucidate the role of premating isolation in the continuing differentiation of these two strains that may eventually lead to speciation.

Sexual isolation of male moths explained by a single pheromone response QTL containing four receptor genes

Long distance sexual communication in moths has fascinated biologists because of the complex, precise female pheromone signals and the extreme sensitivity of males to specific pheromone molecules. Progress has been made in identifying some genes involved in female pheromone production and in male response. However, we have lacked information on the genetic changes involved in evolutionary diversification of these mate-finding mechanisms that is critical to understanding speciation in moths and other taxa. We used a combined quantitative trait locus (QTL) and candidate gene approach to determine the genetic architecture of sexual isolation in males of two congeneric moths, Heliothis subflexa and Heliothis virescens. We report behavioral and neurophysiological evidence that differential male responses to three female-produced chemicals (Z9-14:Ald, Z9-16:Ald, Z11-16:OAc) that maintain sexual isolation of these species are all controlled by a single QTL containing at least four odorant receptor genes. It is not surprising that pheromone receptor differences could control H. subflexa and H. virescens responses to Z9-16:Ald and Z9-14:Ald, respectively. However, central rather than peripheral level control over the positive and negative responses of H. subflexa and H. virescens to Z11-16:OAc had been expected. Tight linkage of these receptor genes indicates that mutations altering male response to complex blends could be maintained in linkage disequilibrium and could affect the speciation process. Other candidate genes such as those coding for pheromone binding proteins did not map to this QTL, but there was some genetic evidence of a QTL for response to Z11-16:OH associated with a sensory neuron membrane protein gene.

The roles and interactions of reproductive isolation mechanisms in fall armyworm (Lepidoptera: Noctuidae) host strains

1. The moth Spodoptera frugiperda presents an interesting opportunity to study the evolution of reproductive isolation, because it consists of two host races that may be in the process of speciation.

Genetics of sex pheromone blend differences between Heliothis virescens and Heliothis subflexa: a chromosome mapping approach.

Males of the noctuid moths, Heliothis virescens and H. subflexa locate mates based on species‐specific responses to female‐emitted pheromones that are composed of distinct blends of volatile compounds. We conducted genetic crosses between these two species and used AFLP marker‐based mapping of backcross families (H. subflexa direction) to determine which of the 30 autosomes in these moths contained quantitative trait loci (QTL) controlling the proportion of specific chemical components in the pheromone blends. Presence/absence of single H. virescens chromosomes accounted for 7–34% of the phenotypic variation among backcross females in seven pheromone components. For a set of three similar 16‐carbon acetates, two H. virescens chromosomes interacted in determining their relative amounts within the pheromone gland and together accounted for 53% of the phenotypic variance. Our results are discussed relative to theories about population genetic processes and biochemical mechanisms involved in the evolution of new sexual communication systems.

Geographic and temporal variation in moth chemical communication

In moth pheromone communication signals, both quantitative and qualitative intraspecific differences have been found across geographic regions. Such variation has generally been hypothesized to be due to selection, but evidence of genetic control of these differences is largely lacking. To explore the patterns of variation in pheromone signals, we quantified variation in the female sex pheromone blend and male responses of two closely related noctuid moth species in five different geographic regions for 2–3 consecutive years. We found significant variation in the ratios of sex pheromone blend components as well as in male response, not only between geographic regions but also within a region between consecutive years. The temporal variation was of a similar magnitude as the geographic variation. As far as we know, this is the first study reporting such temporal variation in moth chemical communication systems. The geographic variation seems to at least partly be controlled by genetic factors, and to be correlated with the quality of the local chemical environment. However, the pattern of temporal variation within populations suggests that optimization of the pheromonal signal also may be driven by within-generation physiological adjustments by the moths in response to their experience of the local chemical environment.

Semi-Selective Fatty Acyl Reductases from Four Heliothine Moths Influence the Specific Pheromone Composition

Background Sex pheromones are essential in moth mate communication. Information on pheromone biosynthetic genes and enzymes is needed to comprehend the mechanisms that contribute to specificity of pheromone signals. Most heliothine moths use sex pheromones with (Z)–11–hexadecenal as the major component in combination with minor fatty aldehydes and alcohols. In this study we focus on four closely related species, Heliothis virescens, Heliothis subflexa, Helicoverpa armigera and Helicoverpa assulta, which use (Z)–11–hexadecenal, (Z)–9–tetradecanal, and (Z)–9–hexadecenal in different ratios in their pheromone blend. The components are produced from saturated fatty acid precursors by desaturation, β–oxidation, reduction and oxidation. Results We analyzed the composition of fatty acyl pheromone precursors and correlated it to the pheromone composition. Next, we investigated whether the downstream fatty–acyl reduction step modulates the ratio of alcohol intermediates before the final oxidation step. By isolating and functionally characterizing the Fatty Acyl Reductase (pgFAR) from each species we found that the pgFARs were active on a broad set of C8 to C16 fatty acyl substrates including the key pheromone precursors, Z9–14, Z9–16 and Z11–16:acyls. When presenting the three precursors in equal ratios to yeast cultures expressing any of the four pgFARs, all reduced (Z)–9–tetradecenoate preferentially over (Z)–11–hexadecenoate, and the latter over (Z)–9–hexadecenoate. Finally, when manipulating the precursor ratios in vitro, we found that the pgFARs display small differences in the biochemical activity on various substrates. Conclusions We conclude that a pgFAR with broad specificity is involved in heliothine moth pheromone biosynthesis, functioning as a semi–selective funnel that produces species–specific alcohol product ratios depending on the fatty–acyl precursor ratio in the pheromone gland. This study further supports the key role of these in pheromone biosynthesis and emphasizes the interplay between the pheromone fatty acyl precursors and the Lepidoptera specific pgFARs in shaping the pheromone composition.

Sex-Related Perception of Insect and Plant Volatiles in Lygocoris pabulinus

We recorded electroantennograms of male and female Lygocoris pabulinus antennae to 63 insect and plant volatiles. EAGs were between 100 and 500 μV. Overall, male EAGs were about twice the size of female EAGs. In both sexes, largest EAGs were recorded to (E)-2-hexenyl butanoate and (E)-2-hexen-1-ol. Response profiles were similar in both sexes. However, male antennae were more sensitive to a number of esters, especially the butanoates and pentanoates. Female antennae were more sensitive to nine of the 19 plant volatiles, i.e., to hexan-1-ol, heptan-1-ol, 1-octen-3-ol, 2-heptanone, (R)-carvone, linalool, geraniol, nerol, and methyl salicylate. Sexual differences in responses suggest that males are more sensitive to insect-produced pheromone-type compounds, whereas females are more sensitive to plant compounds for their orientation towards oviposition sites.