Noctuid Moth Heliothis Virescens Research Articles

Within-population variability in a moth sex pheromone blend, part 2: selection towards fixation

To understand how variation in sexual communication systems evolves, the genetic architecture underlying sexual signals and responses needs to be identified. Especially in animals where mating signals are important for mate recognition, and signals and responses are governed by independently assorting genes, it is difficult to envision how signals and preferences can (co)evolve. Moths are a prime example of such animals. In the noctuid moth Heliothis virescens, we found within-population variation in the female pheromone. In previous selection experiments followed by quantitative trait locus (QTL) analysis and expression analysis of candidate desaturase genes, we developed a model involving a trans-acting repressor of the delta-11-desaturase. In our current study with new selection lines, we fixed the most extreme phenotype and found a single underlying mutation: a premature stop codon in the first coding exon of delta-11-desaturase, which we could trace back to its origin in the laboratory. Interestingly, we found no pleiotropic effects of this knock-out mutation on the male physiological or behavioural response, or on growth or fertility. This finding is in contrast to Drosophila melanogaster, where a single desaturase gene affects both female pheromone production and male behavioural response, but similar to other Lepidoptera where these traits are under independent genetic control. To our knowledge, this is the first time that a single point mutation has been identified that underlies the phenotypic variation in the pheromone signal of a moth.

Experimental evidence for chemical mate guarding in a moth.

In polyandrous species, males seek to maximize their reproductive output by monopolizing their mate. Often the male transfers substances to the female that suppress her sexual receptivity or antagonize the behavior of competing males; both are usually transferred in seminal fluids and represent forms of chemical mate guarding. In moths, more long-range female sex pheromones have been identified than in any other animal group, and males often display with close-range sex pheromones, yet odor-based post-copulatory mate guarding has not been described in moths so far. We tested the hypothesis that the male sex pheromone in the noctuid moth Heliothis virescens perfumes the female and functions as an anti-aphrodisiac. Indeed, virgin females perfumed with male pheromone extract, or with its main component, mated significantly less than control virgin females, and this effect persisted for two successive nights. This chemical mate guarding strategy was disadvantageous for H. virescens females, because the reproductive output of twice-mated females was significantly higher than that of once-mated females. Since the female and male sex pheromones are biosynthetically related in this and other moth species, chemical mate guarding may also impose selection pressure on the long-range female sex pheromone channel and consequently affect the evolution of sexual communication.

Variability of Bacterial Communities in the Moth Heliothis virescens Indicates Transient Association with the Host.

Microbes associated with insects can confer a wide range of ecologically relevant benefits to their hosts. Since insect-associated bacteria often increase the nutritive value of their hosts' diets, the study of bacterial communities is especially interesting in species that are important agricultural pests. We investigated the composition of bacterial communities in the noctuid moth Heliothis virescens and its variability in relation to developmental stage, diet and population (field and laboratory), using bacterial tag-encoded FLX pyrosequencing of 16S rRNA amplicons. In larvae, bacterial communities differed depending on the food plant on which they had been reared, although the within-group variation between biological replicates was high as well. Moreover, larvae originating from a field or laboratory population did not share any OTUs. Interestingly, Enterococcus sp. was found to be the dominant taxon in laboratory-reared larvae, but was completely absent from field larvae, indicating dramatic shifts in microbial community profiles upon cultivation of the moths in the laboratory. Furthermore, microbiota composition varied strongly across developmental stages in individuals of the field population, and we found no evidence for vertical transmission of bacteria from mothers to offspring. Since sample sizes in our study were small due to pooling of samples for sequencing, we cautiously conclude that the high variability in bacterial communities suggests a loose and temporary association of the identified bacteria with H. virescens.

A Challenge for a Male Noctuid Moth? Discerning the Female Sex Pheromone against the Background of Plant Volatiles.

Finding a partner is an essential task for members of all species. Like many insects, females of the noctuid moth Heliothis virescens release chemical cues consisting of a species-specific pheromone blend to attract conspecific males. While tracking these blends, male moths are also continuously confronted with a wide range of other odor molecules, many of which are plant volatiles. Therefore, we analyzed how background plant odors influence the degree of male moth attraction to pheromones. In order to mimic a natural situation, we tracked pheromone-guided behavior when males were presented with the headspaces of each of two host plants in addition to the female pheromone blend. Since volatile emissions are also dependent on the physiological state of the plant, we compared pheromone attraction in the background of both damaged and intact plants. Surprisingly, our results show that a natural odor bouquet does not influence flight behavior at all, although previous studies had shown a suppressive effect at the sensory level. We also chose different concentrations of single plant-emitted volatiles, which have previously been shown to be neurophysiologically relevant, and compared their influence on pheromone attraction. We observed that pheromone attraction in male moths was significantly impaired in a concentration-dependent manner when single plant volatiles were added. Finally, we quantified the amounts of volatile emission in our experiments using gas chromatography. Notably, when the natural emissions of host plants were compared with those of the tested single plant compounds, we found that host plants do not release volatiles at concentrations that impact pheromone-guided flight behavior of the moth. Hence, our results lead to the conclusion that pheromone-plant interactions in Heliothis virescens might be an effect of stimulation with supra-natural plant odor concentrations, whereas under more natural conditions the olfactory system of the male moth appears to be well adapted to follow the female pheromone plume without interference from plant-emitted odors.

Effects of immune challenge on the oviposition strategy of a noctuid moth

Infections can have detrimental effects on the fitness of an animal. Reproducing females may therefore be sensitive to cues of infection and be able to adaptively change their oviposition strategy in the face of infection. As one possibility, females could make a terminal investment and shift reproductive effort from future to current reproduction as life expectancy decreases. We hypothesized that females of the noctuid moth Heliothis virescens make a terminal investment and adapt their oviposition timing as well as their oviposition site selectivity in response to an immune challenge. We indeed found that females that were challenged with the bacterial entomopathogen Serratia entomophila laid more eggs than control females one night after the challenge. Additionally, bacteria-challenged females were less discriminating between oviposition sites than control females. Whereas control females preferred undamaged over damaged plants, immune-challenged females did not differentiate between the two. These results indicate that terminal investment is part of the life history of H.virescens females. Moreover, our results suggest that the strategy of terminal investment in H.virescens oviposition represents a fitness trade-off for females: in the face of infection, an increase in oviposition rate enhances female fitness, whereas low oviposition site selectivity reduces female fitness.

Differential response of Trichogramma wasps to extreme sex pheromone types of the noctuid moth Heliothis virescens

1. Chemical espionage in nature may occur when predators or parasitoids home in on animal or plant communication signals. Parasitoid wasps are known to use pheromones emitted by adults hosts to locate host eggs, larvae or pupae. The response of Trichogramma egg parasitoids to a synthetic sex pheromone blend of moths has been shown in a number of studies over the past 40 years.2. Trichogramma pretiosum (Hymenoptera, Trichogrammatidae) is a tiny parasitic wasp, attacking the eggs of the noctuid moth Heliothis virescens (Lepidoptera, Noctuidae). This study investigated whether T. pretiosum homes in on the sex pheromone of H. virescens at close range. The arrestment response of the wasps to sex pheromone gland extracts of two types of female moths, so‐called high and low females, was also tested, referring to two selected extreme pheromone types of H. virescens. The study also investigated whether the wasps would mount females, possibly to hitchhike with them.3. The wasps were arrested by the common, ‘low’ pheromone, but not by the rare, ‘high’ pheromone or by extracts from male hairpencils. The wasps did not show a preference for separate sex pheromone compounds, but when pre‐exposed to the major sex pheromone component of H. virescens before the tests together with H. virescens eggs, they did show a preference, indicating learning behaviour. In the mounting experiments, mated females were mounted significantly more than virgin females or males, suggesting that hitchhiking is a strategy used by these wasps to locate moth eggs.4. This represents the first study to show a differential response of parasitoid wasps to two different sex pheromone types in a single host species. The results warrant further investigations into the potential role of parasitic wasps in the evolution of sexual communication in moths.

A polydnavirus ANK protein acts as virulence factor by disrupting the function of prothoracic gland steroidogenic cells.

Polydnaviruses are obligate symbionts integrated as proviruses in the genome of some ichneumonoid wasps that parasitize lepidopteran larvae. Polydnavirus free viral particles, which are injected into the host at oviposition, express virulence factors that impair immunity and development. To date, most studies have focused on the molecular mechanisms underpinning immunosuppression, whereas how viral genes disrupt the endocrine balance remains largely uninvestigated. Using Drosophila as a model system, the present report analyzes the function of a member of the ankyrin gene family of the bracovirus associated with Toxoneuron nigriceps, a larval parasitoid of the noctuid moth Heliothis virescens. We found that the TnBVank1 expression in the Drosophila prothoracic gland blocks the larval-pupal molt. This phenotype can be rescued by feeding the larvae with 20-hydroxyecdysone. The localization of the TnBVANK1 is restricted to the cytoplasm where it interacts with Hrs and Alix marked endosomes. Collectively, our data demonstrate that the TnBVANK1 protein acts as a virulence factor that causes the disruption of ecdysone biosynthesis and developmental arrest by impairing the vesicular traffic of ecdysteroid precursors in the prothoracic gland steroidogenic cells.

Subcellular localization of the fatty acyl reductase involved in pheromone biosynthesis in the tobacco budworm, Heliothis virescens (Noctuidae: Lepidoptera)

Sex pheromone components are produced in specialized glands of female moths via well-characterized biosynthetic pathways, where a Fatty Acyl Reductase (FAR) is often essential for producing the specific ratio of the different pheromone components. The subcellular localization and membrane topology of FARs is important for understanding how pheromones are synthesized and exported to the exterior for release. We investigated the subcellular localization of HvFAR from the noctuid moth Heliothis virescens by producing recombinant fusion proteins with green fluorescent protein (GFP) in yeast. A C-terminally tagged construct was localized to the endoplasmic reticulum (ER) and retained full reductive activity on a broad range of saturated and unsaturated fatty acyl precursors. In contrast, an N-terminally-tagged construct was poorly expressed in the cytoplasm and was not enzymatically active, indicating that HvFAR requires a free N-terminal for both proper targeting and catalytic activity. A series of truncations of the N-and C-termini of HvFAR was conducted based on in silico-predicted hydrophobic domains and transmembrane regions. The N-terminally truncated protein was found in the cytoplasm and did not retain activity, emphasizing the importance of the N-terminal for FAR function. In addition, the orientation in the membrane of the C-terminus-tagged HvFAR-GFP construct was analyzed using a fluorescence protease protection (FPP) assay, implying that the C-terminal of HvFAR is orientated towards the cytoplasm. These results, together with previous data on the localization of desaturases, confirm the importance of the ER as a subcellular site of pheromone production.

Plant odorants interfere with detection of sex pheromone signals by male Heliothis virescens

In many insects, mate finding relies on female-released sex pheromones, which have to be deciphered by the male olfactory system within an odorous background of plant volatiles present in the environment of a calling female. With respect to pheromone-mediated mate localization, plant odorants may be neutral, favorable, or disturbing. Here we examined the impact of plant odorants on detection and coding of the major sex pheromone component, (Z)-11-hexadecenal (Z11-16:Ald) in the noctuid moth Heliothis virescens. By in vivo imaging the activity in the male antennal lobe (AL), we monitored the interference at the level of olfactory sensory neurons (OSN) to illuminate mixture interactions. The results show that stimulating the male antenna with Z11-16:Ald and distinct plant-related odorants simultaneously suppressed pheromone-evoked activity in the region of the macroglomerular complex (MGC), where Z11-16:Ald-specific OSNs terminate. Based on our previous findings that antennal detection of Z11-16:Ald involves an interplay of the pheromone binding protein (PBP) HvirPBP2 and the pheromone receptor (PR) HR13, we asked if the plant odorants may interfere with any of the elements involved in pheromone detection. Using a competitive fluorescence binding assay, we found that the plant odorants neither bind to HvirPBP2 nor affect the binding of Z11-16:Ald to the protein. However, imaging experiments analyzing a cell line that expressed the receptor HR13 revealed that plant odorants significantly inhibited the Z11-16:Ald-evoked calcium responses. Together the results indicate that plant odorants can interfere with the signaling process of the major sex pheromone component at the receptor level. Consequently, it can be assumed that plant odorants in the environment may reduce the firing activity of pheromone-specific OSNs in H. virescens and thus affect mate localization.

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.

γ-Aminobutyric acid immunostaining in the antennal lobe of the moth Heliothis virescens and its colocalization with neuropeptides

The antennal lobe is the primary processing center for olfactory information in insects. To understand further the neural circuitry of this brain area, we have investigated the distribution of gamma-aminobutyric acid (GABA) and its colocalization with neuropeptides in the antennal lobe of the noctuid moth Heliothis virescens. Immunocytochemical experiments with an antiserum against GABA showed a large number of labeled somata in the antennal lobe; these somata were located exclusively in the lateral cell cluster. Stained neurites innervating all antennal-lobe glomeruli, including the male-specific macroglomerular complex, suggested a prominent role of GABA in processing olfactory information, including signals from pheromones, interspecifically acting odors, and plant odors. Fibers in two antennocerebral tracts (the middle and dorsal antennocerebral tract) exhibited prominent GABA immunoreactivity. Double-labeling experiments revealed that immunostaining for three neuropeptides, viz., A-type allatostatin, Manduca sexta allatotropin, and FMRFamide-related peptides, was largely colocalized with GABA in cell bodies of the lateral cell cluster. The general absence of peptide immunostaining in the antennocerebral tracts strongly indicated that these peptides were colocalized with GABA in local interneurons of the antennal lobe. In contrast, tachykinin-related peptides occurred in a distinct population of local antennal-lobe neurons that did not exhibit GABA immunostaining. Thus, local interneurons that were not GABAergic were present in the moth antennal lobe.

Immunocytochemical localization of an allatotropin in developmental stages of Heliothis virescens and Apis mellifera

Juvenile hormone biosynthesis by the corpora allata is regulated by stimulatory neuropeptides called allatotropins and inhibitory neuropeptides called allatostatins. This study localized Manduca sexta allatotropin-like material in developmental stages of the noctuid moth Heliothis virescens and the honeybee Apis mellifera. Immunocytochemical methods using both fluorescence-tagged antibodies and enzyme-coupled antibodies were used to stain the central nervous tissue of both species. H. virescens contains M. sexta allatotropin (Manse-AT)-like material consistently throughout larval development. The distribution patterns of Manse-AT immunoreactive cell bodies in the CNS persisted from one larval instar to the next. It will be discussed how larval Manse-AT distribution patterns differed from those in adults. The total number of AT-containing cells in brain and subesophageal ganglion gradually increased during larval development, whereas in the thoracic and abdominal ganglia, the number of AT-containing neurons remained constant. In the honeybee A. mellifera, Manse-AT immunoreactive cells were only found in a few brains from late last instar larvae (prepupae). Manse-AT-like material was present in a group of 6–8 cells in the pars intercerebralis. However, we did not find any Manse-AT-like material in brains of early last instar larvae, whose corpora allata (CA) are more sensitive to in vitro stimulation by Manse-AT than prepupal CA.

Determinants of Sperm Transfer by Males of the Noctuid Moth Heliothis virescens

Ejaculate composition can be an important determinant of male reproductive success in the face of sperm competition, which varies with the mating history of the female. Here we examine the effect of various male and female mating histories and morphological traits on ejaculate sperm numbers in the polyandrous moth Heliothis virescens. We show that when mating with nonvirgin females, males passed larger sperm packages (spermatophores) but did not alter either the sperm count or the ratio of nucleated-to-nonnucleated sperm. Males also passed fewer sperm in their second ejaculates. Finally, older males passed more sperm than did younger males. Earlier research found that females store more sperm from older males and that older males are more likely to gain sperm precedence over younger rivals. These earlier results, taken together with the present results, indicate that the advantage enjoyed by older males is due to an increased sperm count.

Sperm storage by females of the polyandrous noctuid moth Heliothis virescens

Female tobacco budworm moths, Heliothis virescens, generally mate with more than one male, receiving from each mate both fertilizing sperm (eupyrene) and nonfertilizing anucleate sperm (apyrene), which is thought to play a role in sperm competition. One male typically gains sperm precedence, but it is not consistently the last or the first male to mate. I investigated the mechanism of this variable pattern of paternity by examining the patterns of storage of both types of sperm in the female's spermatheca as a function of multiple mating and male phenotype. The number of stored apyrene sperm varied with mating history, being greatest in twice-mated females and least in females mated to one nonvirgin male. In contrast, only one ejaculate's worth of eupyrene sperm was stored regardless of female mating history (once or twice mated). Thus, while they store two complements of apyrene sperm, twice-mated females apparently store only one ejaculate's worth of eupyrene sperm. This biased pattern of sperm storage may contribute to the variable pattern of paternity observed in this species. Eupyrene sperm storage also correlated positively with female size, male age and spermatophore size. Finally, a new sperm storage site was identified and described. It is a bulged region in the seminal duct.

A female nervous system is necessary for normal sperm storage in Drosophila melanogaster: a masculinized nervous system is as good as none

A male Drosophila melanogaster deposits many more sperm in a female's bursa copulatrix than are stored in her ventral receptacle or paired spermathecae soon after copula has ended. The remaining sperm are expelled by the female. These observations suggest a sexual conflict over the processes involved in sperm storage. We used genetically manipulated flies to study the role of the central nervous system in sperm storage. Flies with female bodies but masculinized nervous systems, or isolated female abdomens, stored significantly fewer sperm than did control females. Furthermore, compared with control flies, there were relatively more sperm in the ventral receptacle and relatively fewer in the spermathecae. These results suggest that the female nervous input counteracts the male's attempts to force sperm into the ventral receptacle during copula and promotes active transport of sperm to the spermathecae during and after copula. The female is clearly a very active partner in influencing processes involved in sperm competition, especially as only stored sperm can be used later to fertilize eggs. To our knowledge, this is the first study to show directly the involvement of the female nervous system in sperm storage.

Neuronal control of post-coital pheromone production in the mothHeliothis virescens

The mechanism involved in bringing about post-coital suppression of pheromone production, pheromonostasis, was studied in the noctuid moth Heliothis virescens. Mating results in a transient suppression in pheromone production, the signal for which appears to originate in the testes and other components of the male's reproductive system. The mating-induced pheromonostasis is due to an ascending signal via the central nervous system that appears to inhibit the release of the pheromonotropin, pheromone biosynthesis activating neuropeptide (PBAN), or other potential pheromonotropic substances, and is not due to a refractoriness in response of the sex pheromone glands to PBAN in the female. A similar mechanism is operative in several species of moths where post-coital pheromonostasis has been observed. Sperm quality is not important for pheromonostasis in H. virescens, because males with apyrene or eupyrene sperm elicit similar pheromonostatic responses. The pheromonostatic activity of the ecdysteroid 20-OH-ecdysone appears to be the result of a direct effect on the sex pheromone glands. © 1996 Wiley-Liss, Inc.

Isolation and Sequencing of cDNA Clones Coding for Juvenile Hormone Esterase from Heliothis virescens: Evidence for a catalytic mechanism for the serine carboxylesterases different from that of the serine proteases

Three cDNA clones containing the entire coding sequence for JH esterase from the noctuid moth Heliothis virescens have been isolated from an expression library using specific antibodies and oligonucleotide probes. The complete sequence of one of the clones shows a 2989-base pair insert that is approximately the length of the single 3.0-kilobase pair mRNA transcript detected by Northern blotting. The clone has an open reading frame of 1714 base pairs that predicts a mature protein (minus signal peptide 19 residues long) of 61,012 Da. The 3'-nontranslated region has three signals for polyadenylation although only one apparently is used. Edman degradation of purified juvenile hormone lesterase indicates two slightly different proteins (one being 75% of the total) while the three cDNA clones differ at three bases in their 5' region causing their predicted sequence to differ at one or two residues of the 35 amino acids sequenced from the major form. Comparison of the predicted protein sequence to other carboxylesterase sequences indicates extensive similarity in the NH2-terminal half of the protein and the active site serine to be at position 201. The lack of conserved histidine and aspartate residues and the presence of conserved arginine and glutamate residues in appropriate positions in the NH2-terminal half of the homologous serine carboxylesterases suggests a catalytic mechanism for the serine carboxylesterases that is different from that of the serine proteases.

Interneurones responding to sound in the tobacco budworm mothHeliothis virescens (Noctuidae): morphological and physiological characteristics

1. A group of 7 acoustically activated interneurones has been identified in the central nervous system of the noctuid mothHeliothis virescens. The neurones have their cell bodies and major processes in either the mesoor metathoracic neuromeres of the fused pterothoracic ganglion, and axons contralateral to the cell body (Figs. 1, 2). All neurones except one project via the contralateral connective to at least the prothoracic ganglion. 2. The frequency-response characteristics and auditory thresholds for most interneurones were similar to that of the A1 receptor (40–45 dB SPL at 16 kHz) (Fig. 3A). Interneurone intensity-response characteristics were also generally of the sigmoid type displayed by the A1 and A2 receptors (Fig. 3B). 3. Each interneurone responded to a tone with a compound EPSP of characteristic shape (Figs. 2, 4, 5). In two neurones with very similar responses, the shapes of the rising and plateau phases of their EPSPs, and the suggestion of a delayed input to one neurone, were sufficient to distinguish them (Fig. 5). 4. The subthreshold responses of all identified interneurones considerably outlasted the stimulus duration, but spiking patterns reflected stimulus duration more accurately (Figs. 4, 5, 6). Most neurones responded in a tonic or phasic/tonic manner, suggesting they might be ‘repeater’ type neurones; whereas one local (Fig. 2) and one unidentified interneurone (Fig. 9) responded like ‘pulse-coder’ neurones. 5. All responses to sound by identified interneurones were excitatory, and no sound-related IPSPs were seen (Figs. 2, 3, 4). Further, little habituation of the response occurred in any interneurone for stimulus rates up to 20/s (Fig. 7A, B). 6. The latency of the EPSP from stimulus onset in several interneurones (501, 503, 504) was in each case equivalent to a synaptic delay of less than 1 ms compared with the response latency of the A1 receptor, suggesting direct connectivity (Table 1). The input-output curves and EPSP of neurone 501 changed in such a way as to suggest a possible additional input from the A2 receptor at higher intensities (Figs. 3 B, 4). 7. Electrical stimulation of the tympanal nerve and sound stimulation of the ear evoked a similar EPSP and spike pattern in interneurone 502 (Fig. 8A, B). However, the latency of the EPSP in neurone 502 elicited by electrical stimulation was too long (9 ms) for the connection between neurone 502 and either A1 or A2 receptors to be direct. 8. The neuronal morphologies and response characteristics described above provided the basis for a simple model of the neural circuitry mediating the phonotactic response of a moth to a bat call (Fig. 10).