QTL analysis of sex pheromone blend differences between two closely related moths: Insights into divergence in biosynthetic pathways

To understand the evolution of premating signals in moths, it is important to know the genetic basis of these signals. We conducted Quantitative Trait Locus (QTL) analysis by hybridizing two noctuid moth species, Heliothis virescens (Hv) and Heliothis subflexa (Hs), and backcrossing the F₁ females to males of both parental species. One of these backcrosses (F₁ × Hs) was a biological replicate of our previous study (Sheck et al., 2006) and served to test the robustness of our previous findings. The backcross to Hv was designed to reveal QTL with recessive inheritance of the Hv character state. This study confirms previously discovered QTL, but also reports new QTL. Most importantly, we found relatively large QTL affecting Z9-16:Ald, the critical sex pheromone component of Hs. For Z9-14:Ald, the critical sex pheromone component of Hv, as well as for the minor pheromone compound 14:Ald, we found QTL in which the change in pheromone ratio was opposite-to-expected. Linking QTL to the biosynthetic pathways of the pheromone compounds of Hv and Hs implicates several candidate genes in the divergence of these premating signals, the most important of which are acetyl transferase, one or more desaturase(s), and a fatty acyl reductase or alcohol oxidase.     

Phenotypic plasticity in sexual communication signal of a noctuid moth

Variability within sex pheromone signalling systems is generally believed to be low because of strong stabilizing selection; yet the noctuid moth Heliothis subflexa (Hs) shows significant intraspecific variation. One possible explanation is that females may alter their sex pheromone blend depending on prevailing olfactory cues in the habitat, which we termed the 'experience hypothesis'. This could be adaptive if Hs females experiencing the pheromone of another species, Heliothis virescens (Hv), responded to reduce the frequency of heterospecific matings. We exposed Hs females to no pheromone, Hs pheromone or Hv pheromone in the first 3 days of their adult lives. Hs females in the latter treatment produced significantly more of the acetate Z11-16:OAc, which inhibits the attraction of Hv males. To our knowledge, this is the first study showing adaptive phenotypic plasticity in a moth sex pheromone and suggests that behavioural differentiation may precede genetic divergence in the sexual signals of moths.     

Communication interference in sympatrically occurring moth species

In moth species, females emit a species‐specific sex pheromone that is perceived over long distance by conspecific males. The species‐specificity in the chemical communication channel is achieved by a combination of unique components in specific ratios and sometimes also by interspecific behavioural antagonists to deter sympatrically occurring heterospecific males. In this study, we determined possible antagonistic effects in Helicoverpa gelotopoeon Dyar (Lepidoptera: Noctuidae) males to the major sex pheromone component of sympatrically occurring heliothine moths, Z11‐16:Ald, as well as to the sex pheromone of the sympatrically occurring Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) (Z11‐16:Ald and Z9‐14:Ald). We also explored whether other co‐occurring species are attracted to these pheromone blends. Our field experiments showed that the addition of Z11‐16:Ald alone or in combination with Z9‐14:Ald inhibited trap catches of H. gelotopoeon males and that this inhibition depended on the concentration of these compounds. In addition, other moth species were attracted to the blends. Together, our results confirm the antagonistic effect of heterospecific sex pheromone compounds of H. virescens to H. gelotopoeon.     

The role of alcohols in pheromone biosynthesis by two noctuid moths that use acetate pheromone components

Primary alcohols varying in chain length from C₁₃ to C₁₆, and in number, position, and geometric configuration of double bonds, were applied in dimethyl sulfoxide to the surface of the female sex pheromone glands of Heliothis subflexa (Gn.) and Hydraecia micacea (Esper). Capillary gas chromatographic analysis of extracts of the treated glands indicated that the alcohols were converted to the corresponding aldehydes by H. subflexa females and to the acetates by H. micacea females. Conversions of the alcohols showed no preferences for molecular weight, number, position, or geometry of the double bonds in either species. Application of the acetates of the primary alcohols to the gland surface of H. subflexa females resulted in the production of both the corresponding alcohols and aldehydes, while neither alcohols nor aldheydes were produced when acetates were applied to the glands of H. micacea. In addition, application of the acetates to the gland surface of Heliothis virescens (F.) resulted in the production of both the corresponding alcohols and aldehydes. However, no evidence was found to indicate that acetates are ever produced by the pheromone gland of females of H. virescens.     

Linking intra‐ and interspecific assortative mating: Consequences for asymmetric sexual isolation

Assortative mating is of interest because of its role in speciation and the maintenance of species boundaries. However, we know little about how within‐species assortment is related to interspecific sexual isolation. Most previous studies of assortative mating have focused on a single trait in males and females, rather than utilizing multivariate trait information. Here, we investigate how intraspecific assortative mating relates to sexual isolation in two sympatric and congeneric damselfly species (genus Calopteryx). We connect intraspecific assortment to interspecific sexual isolation by combining field observations, mate preference experiments, and enforced copulation experiments. Using canonical correlation analysis, we demonstrate multivariate intraspecific assortment for body size and body shape. Males of the smaller species mate more frequently with heterospecific females than males of the larger species, which showed less attraction to small heterospecific females. Field experiments suggest that sexual isolation asymmetry is caused by male preferences for large heterospecific females, rather than by mechanical isolation due to interspecific size differences or female preferences for large males. Male preferences for large females and male–male competition for high quality females can therefore counteract sexual isolation. This sexual isolation asymmetry indicates that sexual selection currently opposes a species boundary.     

The genetics of mating recognition between Drosophila simulans and D. sechellia

During courtship, visual and chemical signals are often exchanged between the sexes. The proper exchange of such signals ensures intraspecific recognition. We have examined the genetic basis of interspecific differences in male mating behaviour and pheromone concentration between Drosophila simulans and D. sechellia by using Drosophila simulans/D. sechellia introgression lines. Our results show a majority of quantitative trait loci (QTLs) explaining variation in both male mating behaviour and pheromone concentration to be located on the third chromosome. One QTL found on the third chromosome explains variation in time needed to start courtship and copulation as well as time spent courting. The position of such QTL (approximately 84A-88B) with effects on courtship and copulation aspects of mating includes the candidate sex determination gene doublesex (84E5-6) and Voila (86E1-2), a gene that affects male courtship in D. melanogaster. One additional third chromosome QTL explained variation in 7-tricosene pheromone concentrations among males. The interval mapping position of this QTL (approximately 68E-76E) did not overlap with the position detected for differences in mating behaviour and the intervals did not include candidate genes previously identified as having an effect on D. melanogaster cuticular hydrocarbon production. We did not detect any directionality of the effect of Drosophila sechellia allele introgressions in male mating recognition.     

Fine-scale genetic analysis of species-specific female preference in Drosophila simulans

Behavioural differences are thought to be the first components to contribute to species isolation, yet the precise genetic basis of behavioural isolation remains poorly understood. Here, we used a combination of behaviour assays and genetic mapping to provide the first refined map locating candidate genes for interspecific female preference isolating Drosophila simulans from D. melanogaster. First, we tested whether two genes identified as affecting D. melanogaster female intraspecific mate choice also affect interspecific mate choice; neither of these genes was found to contribute to species‐specific female preference. Next, we used deficiency mapping to locate genes on the right arm of the third chromosome for species‐specific female preference and identified five small significant regions that contain candidate genes contributing to behavioural isolation. All five regions were located in areas that would have low interspecific recombination, which mirrors the results of other behavioural isolation studies that used quantitative trait locus (QTL) mapping, but without the potential concern of bias towards regions of low recombination that QTL mapping may have. As this model system may be refined to the individual gene level using the same methodology, this initial map we provide may potentially serve as a ready template for the identification and characterization of the first behavioural isolation genes.     

Hybridization studies in the planthopper genus Ribautodelphax (Homoptera,Delphacidae)

Seven known Ribautodelphax species and four recently discovered taxa were crossed. Forty-three percent of the interspecific crosses were successful. Interspecific crosses generally resulted in low numbers of hybrids. In some combinations prolonged larval development of the hybrid males was observed. Backcrosses showed that both male and female hybrids were fertile. Reproductive isolation was mainly maintained by premating barriers. Insemination tests revealed that prolonged confinement of males and females lowered mating thresholds. The taxonomic status of the known species and new taxa was shown to be in need of revision. No indications for a hybrid origin of the pseudogamous triploid Ribautodelphax females were found.     

Genetic analysis and population survey of sex pheromone variation in the adzuki bean borer moth, Ostrinia scapulalis

Sexual communication in many moths occurs between females emitting a sex pheromone and males responding to it. Females of Ostrinia scapulalis (Lepidoptera: Crambidae) show a large variation in blend ratios of the two sex pheromone components (E)‐ and (Z)‐11‐tetradecenyl acetates. E type females produce a pheromone with a high percentage of (E)‐11‐tetradecenyl acetate, whereas Z type females produce the opposite blend. We established laboratory cultures of E and Z types. Females of the F₁ generation produced an intermediate blend (I type) in both reciprocal crosses of the E and Z cultures. Results of further crossing experiments suggested that the three pheromone types are primarily controlled by a single autosomal locus with two alleles. Also, analyses of the variation in pheromone blend within F₁, backcross and F₂ families suggested that other genetic factors modify the pheromone blend of the I and Z types. Investigation of the pheromone variation in natural populations at 14 localities in Japan has shown that the E type was predominant in northern Japan, whereas the pheromone was highly polymorphic in central Japan. At a locality in central Japan, the pheromone was constantly polymorphic for several years, and the pheromone type frequencies did not deviate from Hardy–Weinberg expectations, providing no evidence of selection or assortative mating between the pheromone types. Analyses of pheromone variation within families derived from feral females indicated that matings between a pair with different genotypes for pheromone production was occurring in natural populations. Overall, this study showed that the genetic basis of the pheromone variation in O. scapulalis is very similar to that in its sibling species Ostrinia nubilalis although the state of pheromone polymorphisms in natural populations appears to differ between the two species. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84 , 143–160.     

Do quantitative trait loci (QTL) for a courtship song difference between Drosophila simulans and D. sechellia coincide with candidate genes and intraspecific QTL?

The genetic architecture of traits influencing sexual isolation can give insight into the evolution of reproductive isolation and hence speciation. Here we report a quantitative trait loci (QTL) analysis of the difference in mean interpulse interval (IPI), an important component of the male courtship song, between Drosophila simulans and D. sechellia. Using a backcross analysis, we find six QTL that explain a total of 40.7% of the phenotypic variance. Three candidate genes are located in the intervals bounded by two of the QTL and there are no significant QTL on the X chromosome. The values of mean IPI for hybrid individuals imply the presence of dominant alleles or epistasis. Because unisexual hybrid sterility prevents an F(2) analysis, we cannot distinguish dominant from additive genetic effects at the scale of QTL. A comparison with a study of QTL for intraspecific variation in D. melanogaster shows that, for these strains, the QTL we have identified for interspecific variation cannot be those that contribute to intraspecific variation. We find that the QTL have bidirectional effects, which indicates that the genetic architecture is compatible with divergence due to genetic drift, although other possibilities are discussed.     

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.     

THE GENETICS OF AN ISOLATING MECHANISM BETWEEN TWO SIBLING SPECIES OF DROSOPHILA

Matings between Drosophila simulans females and males of the sibling species D. mauritiana are of abnormally short duration. These rapid matings interrupt the transfer of sperm, leading to substantial reproductive isolation in interspecific as compared to intraspecific copulations. Genetic analysis of this behavior shows that it is influenced much more by the male than the female genotype, with genes from D. simulans being dominant. In males, shortened copulation is caused by interspecific divergence at a minimum of three loci, with one gene on each of the major chromosomes. This is an underestimate of the true number of loci affecting the trait, which could be much larger. The two autosomes have the largest effect, whereas that of the X chromosome is much smaller. The genetic architecture of copulation duration and the larger effect of male than female genotype suggest that females can detect and discriminate against differences in male genitalia.     

THE GENETIC BASIS OF SEXUAL ISOLATION BETWEEN DROSOPHILA MELANOGASTER AND D. SIMULANS

The genetic analysis of sexual isolation between the closely-related species Drosophila melanogaster and Drosophila simulans involved two experiments with no-choice tests. The efficiency of sexual isolation was measured by the frequency of courtship initiation and interspecific mating. We first surveyed the variation in sexual isolation between D. melanogaster strains and D. simulans strains of different geographic origin. Then, to investigate variation in sexual isolation within strains, we made F₁ diallel sets of reciprocal crosses within strains of D. melanogaster and D. simulans. The F₁ diallel progeny of one sex were paired with the opposite sex of the other species. The first experiment showed significant differences in the frequency of interspecific mating between geographic strains. There were more matings between D. simulans females and D. melanogaster males than between D. melanogaster females and D. simulans males. The second experiment uncovered that the male genotypes in the D. melanogaster diallel significantly differed in interspecific mating frequency, but not in courtship initiation frequency. The female genotypes in the D. simulans diallel were not significantly different in courtship initiation and interspecific mating frequency. Genetic analysis reveals that in D. melanogaster males sexual isolation was not affected by either maternal cytoplasmic effects, sex-linked effects, or epistatic interaction. The main genetic components were directional dominance and overdominance. The F₁ males achieved more matings with D. simulans females than the inbred males. The genetic architecture of sexual isolation in D. melanogaster males argues for a history of weak or no selection for lower interspecific mating propensity. The behavioral causes of variation in sexual isolation between the two species are discussed.     

Do allopatric male Calopteryx virgo damselflies learn species recognition?

There is a growing amount of empirical evidence that premating reproductive isolation of two closely related species can be reinforced by natural selection arising from avoidance of maladaptive hybridization. However, as an alternative for this popular reinforcement theory, it has been suggested that learning to prefer conspecifics or to discriminate heterospecifics could cause a similar pattern of reinforced premating isolation, but this possibility is much less studied. Here, we report results of a field experiment in which we examined (i) whether allopatric Calopteryx virgo damselfly males that have not encountered heterospecific females of the congener C. splendens initially show discrimination, and (ii) whether C. virgo males learn to discriminate heterospecifics or learn to associate with conspecifics during repeated experimental presentation of females. Our experiment revealed that there was a statistically nonsignificant tendency for C. virgo males to show initial discrimination against heterospecific females but because we did not use sexually naïve individuals in our experiment, we were not able to separate the effect of innate or associative learning. More importantly, however, our study revealed that species discrimination might be further strengthened by learning, especially so that C. virgo males increase their association with conspecific females during repeated presentation trials. The role of learning to discriminate C. splendens females was less clear. We conclude that learning might play a role in species recognition also when individuals are not naïve but have already encountered potential conspecific mates.     

Clustering of loci controlling species differences in male chemical bouquets of sympatric Heliconius butterflies

The degree to which loci promoting reproductive isolation cluster in the genome—that is, the genetic architecture of reproductive isolation—can influence the tempo and mode of speciation. Tight linkage between these loci can facilitate speciation in the face of gene flow. Pheromones play a role in reproductive isolation in many Lepidoptera species, and the role of endogenously produced compounds as secondary metabolites decreases the likelihood of pleiotropy associated with many barrier loci. Heliconius butterflies use male sex pheromones to both court females (aphrodisiac wing pheromones) and ward off male courtship (male‐transferred antiaphrodisiac genital pheromones), and it is likely that these compounds play a role in reproductive isolation between Heliconius species. Using a set of backcross hybrids between H. melpomene and H. cydno, we investigated the genetic architecture of putative male pheromone compound production. We found a set of 40 significant quantitative trait loci (QTL) representing 33 potential pheromone compounds. QTL clustered significantly on two chromosomes, chromosome 8 for genital compounds and chromosome 20 for wing compounds, and chromosome 20 was enriched for potential pheromone biosynthesis genes. There was minimal overlap between pheromone QTL and known QTL for mate choice and color pattern. Nonetheless, we did detect linkage between a QTL for wing androconial area and optix, a color pattern locus known to play a role in reproductive isolation in these species. This tight clustering of putative pheromone loci might contribute to coincident reproductive isolating barriers, facilitating speciation despite ongoing gene flow.     

Inter- and Intraspecific Effects of Volatile and Nonvolatile Sex Pheromones on Males, Mating Behavior, and Hybridization in Eretmocerus mundus and E. eremicus (Hymenoptera: Aphelinidae)

Eretmocerus species (Hym. Aphelinidae) are solitary parasitoids of Bemisia tabaci (Gennadius). Mate finding and mating behavior of two species, E. mundus and E. eremicus, were studied under laboratory conditions. We used three populations of Eretmocerus: typical arrhenotokous populations of E. eremicus (from USA) and E. mundus (from Spain), and an atypical thelytokous population of E. mundus (from Australia). We studied the intra- and interspecific responses of males to volatile and nonvolatile components of the female sex pheromones, mating behavior, and hybridization between populations and species. In both arrhenotokous populations, males reacted to volatile pheromones by walking toward conspecific virgin females. Males also reacted to nonvolatile pheromones by spending more time on and around patches on leaves of poinsettia plants that had been exposed to virgin females. Males of E. eremicus showed the same reaction to the nonvolatile sex pheromone of E. mundus females, but E. mundus males did not show any reaction to the nonvolatile sex pheromone of E. eremicus. There was no response of males of both species to thelytokous females of E. mundus. In both species three phases were distinguished in the mating behavior: premating, mating, and postmating. The duration of the phases differed between the three populations. Successful copulation between the two Eretmocerus species did not occur. In contrast, we recorded some successful copulations between Australian males and Spanish females of E. mundus, but they did not produce any hybrid females.     

Within-population variability in a moth sex pheromone blend: genetic basis and behavioural consequences

Evolutionary diversification of sexual communication systems in moths is perplexing because signal and response are under stabilizing selection in many species, and this is expected to constrain evolutionary change. In the moth Heliothis virescens, we consistently found high phenotypic variability in the female sex pheromone blend within each of four geographically distant populations. Here, we assess the heritability, genetic basis and behavioural consequences of this variation. Artificial selection with field-collected moths dramatically increased the relative amount of the saturated compound 16:Ald and decreased its unsaturated counterpart Z11–16:Ald, the major sex pheromone component (high line). In a cross between the high- and low-selected lines, one quantitative trait locus (QTL) explained 11–21% of the phenotypic variance in the 16:Ald/Z11–16:Ald ratio. Because changes in activity of desaturase enzymes could affect this ratio, we measured their expression levels in pheromone glands and mapped desaturase genes onto our linkage map. A delta-11-desaturase had lower expression in females producing less Z11–16:Ald; however, this gene mapped to a different chromosome than the QTL. A model in which the QTL is a trans-acting repressor of delta-11 desaturase expression explains many features of the data. Selection favouring heterozygotes which produce more unsaturated components could maintain a polymorphism at this locus.     

Sex pheromone of the iris gelechiid moth Monochroa divisella

The iris gelechiid moth, Monochroa divisella (Douglas) (Lepidoptera: Gelechiidae), is a serious pest in iris horticulture. The overuse of pesticides for conventional control has a great risk of water pollution because irises are often planted in damp areas. To minimize pesticide applications, it is essential to monitor moth emergence by using pheromone traps. The female pheromone of M. divisella was analyzed by means of gas chromatography‐electroantennographic detection (GC‐EAD) and GC‐mass spectrometry. Two male EAD‐active compounds were detected and identified as (Z)‐3‐dodecenyl acetate (Z3‐12:Ac) and (Z)‐5‐tetradecenyl acetate (Z5‐14:Ac), the amounts of which were ca. 40 and 6 ng per female (100:15), respectively. Synthetic Z3‐12:Ac alone attracted only a few males in field trap experiments, but the attractiveness was significantly enhanced when 15% (wt/wt) Z5‐14:Ac was added; traps baited with their binary mixtures captured 21.3‐106.4 males/trap/day, as compared with 2.3 males/trap/day attracted by a crude extract of 10 virgin females. We conclude that Z3‐12:Ac and Z5‐14:Ac are M. divisella pheromone components, which can be offered as an attractant in monitoring traps. Although dodecenyl acetates are a major class of lepidopteran pheromone components, those with the double bond at the 3 position are rare and appear to be unique to gelechiids.     

Intraspecific variation in sexual isolation in the jewel wasp Nasonia

Abstract.— Divergence in mate recognition systems can lead to reproductive isolation. In this study, we investigate patterns of intraspecific variation that contribute to premating isolation within and between two haplodiploid species, Nasonia vitripennis and N. longicornis . In a broad-scale survey of 17 North American isofemale lines encompassing the two species, we report strong asymmetric sexual isolation between species and a dramatic level of intraspecific variation for mate discrimination between species. A general lack of incipient speciation was found, with the exception of low levels of interpopulational sexual isolation within N. vitripennis . Regression analysis shows that the degree of intraspecific variation for within-species mating frequency is not associated with the degree for between-species mating frequency. Reinforcement or reproductive character displacement may be involved in some of the variation in inter-species premating isolation.     

Sex-linked control of sex pheromone behavioral responses in European corn-borer moths (Ostrinia nubilalis) confirmed with TPI marker gene

In two races of European corn-borer moths (ECB), the E-race females emit and males respond to 99:1 sex pheromone blend of (E)/(Z)-11-tetradecenyl acetates, whereas the Z-race females and males produce and respond to the opposite 3:97 pheromone blend of (E)/(Z)-11-tetradecenyl acetates, respectively. We previously have shown that female production of the final blend ratio is under control of a major autosomal locus but that the sequence of male upwind flight responses to the blend is controlled by a sex-linked (Z-linked) locus. This sex-linked control of behavioral responses in crosses of E and Z ECB now is confirmed by use of sex-linked TPI (triose phosphate isomerase) allozyme phenotypes to determine the origin of the sex chromosomes in F₂ populations. F₁ males from reciprocal E × Z crosses generate similar behavioral-response profiles in wind-tunnel studies, with moderate numbers responding to the Z pheromone and intermediate blends (35%–65% Z), but very few responding to the E pheromone. The F₂ behavioral-response profiles indicate that they are composed of 1:1 mixtures of hybrids and paternal profiles. Analysis of TPI allozyme differences allowed us to separate male F₂ populations into individuals whose Z chromosomes both originated from their grandfathers, and individuals who had one Z chromosome originating from each grandparent. With these partitioned F₂s, the TPI homozygotes exhibited behavioral-response profiles very much like their grandfathers, whereas the TPI hybrids produced response profiles similar to their heterozygous F₁ fathers. These results demonstrate incontrovertibly that the response to sex pheromone in male ECB is controlled by a sex-linked gene that is tightly linked to the TPI locus and therefore is independent of the locus controlling pheromone blend production in females.