Determinants of sperm precedence in a noctuid moth Heliothis virescens: a role for male age

1. Females of the noctuid moth Heliothis virescens F. mate more than once. Thus, sperm from two or more males normally compete for fertilisations within the female reproductive tract. The eggs are typically fertilised by sperm from only one male, either the female's last mate or an earlier mate. Twice‐mated females store only one ejaculate's worth of fertilising sperm (eupyrene) but nearly two ejaculates' worth of a nonfertilising sperm morph (apyrene), which is thought to play a role in sperm competition. 2. The mechanism of sperm use in H. virescens was investigated by examining factors that vary with paternity, which was assigned based on allozyme variation. The factors included male and female body masses and ages, male genital characters, the size of the sperm package, and the number of sperm stored by the female. 3. One male typically gained sperm precedence; this was nearly twice as likely to be the second male as it was to be the first. Two factors were found to vary significantly with paternity: female mass and male age. The second male to mate was more likely to gain sperm precedence if the female was larger and if the male was older than the female's first mate. 4. The significance of male age and female mass to several hypothetical models of the mechanism of sperm use is discussed.     

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.     

Vitellogenin and egg production in the moth,Heliothis virescens

The characteristics of vitellogenin (Vg) and the relationship between Vg production and egg production in the tobacco budworm, Heliothis virescens, were studied. The relationship between Vg production and juvenile hormone (JH) and the impact of mating on Vg and egg production were also investigated. Vg appears in the hemolymph of H. virescens about 6 h after moth eclosion. Vg may be separated into two apoproteins (ApoVg-I and ApoVg-II) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights were calculated to be 156,065 ± 800 for ApoVg-I and 39,887 ± 323 for ApoVg-II. SDS-PAGE analysis revealed that the female hemolymph Vg polypeptides appear to be identical to those from eggs but are absent in male hemolymph. Vg concentration was significantly higher in mated females than in virgin females of the same age at 48 h after emergence. Rates of egg production increased as Vg production increased; rates of egg production in mated females were significantly higher than those of virgin females at 48, 72, 96, and 120 h postemergence. Vg production is dependent on JH, because hemolymph from decapitated females lacked Vg while that of decapitated females treated with synthetic JH had Vg at levels comparable to similarly aged, normal H. virescens females. Hemolymph JH titers in mated females were significantly higher compared with those in virgin females at all sampling periods. The high JH level in mated females may explain the high Vg and egg production in mated H. virescens. Arch. Insect Biochem. Physiol. 34:287–300, 1997. © 1997 Wiley-Liss, Inc.     

Sperm storage and use in polyandrous females of the globally invasive fruitfly, Ceratitis capitata

The medfly, Ceratitis capitata, is an invasive species in which polyandry, associated with sperm precedence, is a common behaviour in the wild. In this species, characterized by internal fertilization, we disclose how the sperm from two males are stored in the female storage organs and how they are used in terms of paternity outcome. The experiments were designed to furnish comparable and unbiased estimates of sperm numbers and progeny in twice-mated females. Results are incorporated in a model through which it is possible to relate the amount of stored sperm with the progeny of twice-mated females. The results show that polyandrous medfly females conserve equal amounts of sperm from the two males to fertilize their eggs. However, we observed a clear advantage of the second male's sperm in siring progeny, which interestingly decreases in favor of the first male as ovipositions progress. The results enable us to exclude differential sperm mortality and suggest that it is the mechanics governing the storage organs which causes the initial, but decreasing second male sperm precedence during the female reproductive life. These outcomes allow us to correlate sperm use in polyandrous females with the mating strategies and invasiveness of this fly.     

Two sex pheromone components of the tobacco budworm moth, Heliothis virescens

Two compounds were isolated from female $\text{Heliothis}$$\text{virescens}$ (Lepidoptera: Noctuidae) extracts and identified as $\text{cis}$-9-tetradecenal and $\text{cis}$-ll-hexadecenal. Together they elicit intense male $\text{H}$. $\text{virescnes}$ response in laboratory tests and have attracted males in the field. Although $\text{cis}$-ll-hexadecenal is an $\text{H}$. $\text{zea}$ sex pheromone, no evidence was obtained for $\text{cis}$-9-tetradecenal in $\text{H}$. $\text{zea}$.     

Synthesis and transport of storage proteins by testes in Heliothis virescens.

The synthesis of two storage protein subunits, 76,000-Mr and 82,000-Mr polypeptides, by the testes sheath has been studied in Heliothis virescens. Like fat body, which is the primary site of synthesis for the large extratesticular pool, cells of the testes sheath secrete glycosylated storage proteins assembled into hexamers. The testis sheath differed from fat body in several important respects, including the failure to synthesize an abundant (in the hemolymph) 74,000-Mr storage protein, its relatively reduced expression of the 76,000-Mr polypeptide, and the absence of resorption of storage proteins from the lumen of the testis during pupal development. Cyst cells were also shown to import actively the 82,000-Mr storage protein by pinocytosis of testicular fluid and transfer it to the developing spermatids. Unlike other cell types that sequester storage proteins in the form of cytoplasmic granules, their localization within spermatids was exclusively mitochondrial. These observations suggest that expression of the storage protein genes is regulated tissue specifically and reveal novel pathways for their transport and, perhaps, utilization and function during development.     

A substance from Heliothis virescens larvae stimulating larviposition by females of the tachinid,Archytas marmoratus

A chemical present in faeces, haemolymph, and whole bodies of larvae of Heliothis virescens stimulates females of the tachinid Archytas marmoratus to deposit maggots in the vicinity of the host. The larvipositional stimulant is soluble in water and in 0·1 N HCl and 0·1 N acetic acid and is insoluble in apolar solvents. Considerable activity is present in 0·1 N HCl extracts of acetone powders of whole H. virescens larvae. Larvipositional activity is destroyed by alkaline solutions. The activity of acidic extracts is greater than that of aqueous extracts when extracts are stored at 2°C for several days; however, extraction of the larvipositional stimulant is not necessarily more efficient with acidic extracts because there is no difference in the activity of the residues remaining after water and acid extraction. Thus, low pH appears to retard the decomposition of the larvipositional stimulant. Gel filtration, chromatography, and membrane filtration demonstrated that the compound has a molecular weight of 30,000 ± 5000. Rapid loss of activity during incubation with a proteinase (pepsin) indicates that the chemical is a protein. Larvipositional activity is present in larvae, pupae, pharate adults, and newly emerged adults of H. virescens. Most species of noctuids tested are highly active, and some activity is present in pyralid larvae. Very little or no activity is present in larvae of the beetle, Anthonomus grandis, or in Apis mellifera.     

(-)-Germacrene D increases attraction and oviposition by the tobacco budworm moth Heliothis virescens

The sesquiterpene germacrene D (GD) activates a major type of olfactory receptor neuron on the antennae of the heliothine moths. In Heliothis virescens females, 80% of the recordings have shown activity of one neuron type responding with high sensitivity and selectivity to GD. With the aim of determining the behavioural significance of this sesquiterpene, we have used a two-choice wind-tunnel to study the preference of mated H. virescens females for host plants with and without (-)-GD added. Tobacco plants containing dispensers with low release rate of (-)-GD had a greater attractiveness than tobacco plants without this substance. In addition, a significant increase of oviposition was found on the plants with (-)-GD.     

Host plant specialization in the generalist moth Heliothis virescens and the role of egg imprinting

Even though generalist insects are able to feed on many different host plants, local specialization may occur, which could lead to genetic differentiation. In this paper we assessed the level and extent of host plant specialization in the generalist herbivore Heliothis virescens Fabricius (Lepidoptera, Noctuidae). This generalist can grow and survive on many different plant species, belonging to more than 37 families. Previously, two laboratory strains were described that differ in their performance on cotton and chickpea. In this study we explored this phenomenon further. Specifically, we asked the following questions: (1) Do the two strains still differ in their performance on cotton and chickpea? Since we found that the most pronounced difference between the two strains was in their growth on fresh chickpea leaves, we then asked: (2) Does this variation in performance have a genetic basis? In our genetic analysis, we found that growth rates changed over time and that two linkage groups significantly affected the ability to grow on chickpea. One QTL was homologous to Bombyx mori chromosome 15, onto which genes for insecticide resistance and detoxicative enzymes have previously been mapped. (3) Is there a difference in oviposition preference between the two strains? Oviposition experiments revealed no preference in either strain when females were reared on the same artificial diet. However, we did find a maternal inheritance of oviposition preference: daughters collected as eggs from cotton oviposited significantly more eggs on cotton, and daughters collected as eggs from chickpea likewise laid more eggs on chickpea. Thus, Hopkins’ host selection principle seems to holds in this species, although imprinting seems to happen not at the larval but at the egg stage, which is a new finding. This study shows how genetic and nongenetic factors can interact to shape the patterns of local specialization in a generalist herbivore.     

Cardiac responses to sensory stimulation in the adult tobacco budworm moth, Heliothis virescens

Electrocardiogram recordings were performed on intact Heliothis virescens moths using Ag-AgCl electrodes positioned on the body cuticular surface. Regular heart activity and its changes in response to mechanical and olfactory stimulations were analysed. Moths were also tested in their anemotactic orientation in response to olfactory stimulation. Results show that regular cardiac activity in Heliothis is cyclical and consists of the alternation of a high and a low spike-frequency period. This activity pattern is strongly influenced by sensory stimulation. Both mechanical stimulations at various intensities and olfactory stimulations with sex pheromone and 1-hexanol evoked tachycardiac effects when applied during low spike-frequency cardiac activity. In contrast, they did not affect high spike-frequency cardiac activity. It is concluded that the cardiac response in Heliothis is an effective indicator of sensory reception. It would therefore appear to represent a valid tool for testing insect reactivity. Accepted: 30 August 1997     

Immunolocalization of a candidate pheromone receptor in the antenna of the male moth, Heliothis virescens

Pheromone recognition in insects is thought to involve distinct receptor proteins in the dendritic membrane of antennal sensory neurons. We have generated antibodies directed against a peptide derived from the sequence of the candidate pheromone receptor HR13 from Heliothis virescens. The antibodies specifically labelled the cell bodies of a distinct neuron population housed in male-specific pheromone-sensitive sensilla. Combining antibody staining with in situ hybridization the reactive cells were found to express the HR13 gene. In addition, dendrites projecting into sensilla hairs as well as the axonal processes of immunoreactive cells were labelled. Labelling of axons has allowed visualization of their fasciculation within antennal segments and permits tracking of axons as they merge into the antennal nerve. The HR13 protein was first detected 1 day before eclosion. Thus, the distribution of HR13 protein in the antennal neurons of the male moth strongly suggests a role of the HR13 receptor in recognition of pheromones.     

Electrophysiological characterization of responses from gustatory receptor neurons of sensilla chaetica in the moth Heliothis virescens

Discrimination of edible and noxious food is crucial for survival in all organisms. We have studied the physiology of the gustatory receptor neurons (GRNs) in contact chemosensilla (insect gustatory organs) located on the antennae of the moth Heliothis virescens, emphasizing putative phagostimulants and deterrents. Sucrose and the 2 bitter substances quinine and sinigrin elicited responses in a larger proportion of GRNs than inositol, KCl, NaCl, and ethanol, and the firing thresholds were lowest for sucrose and quinine. Variations in GRN composition in individual sensilla occurred without any specific patterns to indicate specific sensillum types. Separate neurons showed excitatory responses to sucrose and the 2 bitter substances quinine and sinigrin, implying that the moth might be able to discriminate bitter substances in addition to separating phagostimulants and deterrents. Besides being detected by separate receptors on the moth antennae, the bitter tastants were shown to have an inhibitory effect on phagostimulatory GRNs. Sucrose was highly appetitive in behavioral studies of proboscis extension, whereas quinine had a nonappetitive effect in the moths.     

The fate of topically applied fatty acids in the sex pheromone gland of the moth Heliothis virescens

Deuterium-labeled hexadecanoic acid (D4-16:COOH), a sex pheromone biosynthetic intermediate, and heptadecanoic acid (D3-17:COOH), an acid that cannot be converted to sex pheromone, were topically applied to the pheromone gland of female Heliothis virescens, and the fate of the label determined. Both acids were incorporated similarly into the glycerolipids, with by far the greatest amount found in the triacylglycerols (TGs), and relatively small amounts found in other neutral and polar classes. For D4-16:COOH, the labeled pheromone precursor, (Z)-11-hexadecenoate, was also found predominantly in the TGs but relatively (compared to labeled hexadecanoate) high amounts were also found in the phospholipids. Within the TGs, both acids, as well as the pheromone precursor, were found almost exclusively on the sn-3 position of the glycerol backbone. This demonstrates that the major fate, in the glycerolipids, of free fatty acids is addition to 1,2-diacylglycerols. A relatively large amount of the applied acid was also found in the gland in the form of the acyl-CoA thioester. In a 24-h time-course study, this form remained at a relatively high level for the duration of the assay, and decreased at a rate comparable to the titer of this acid in the TGs, suggesting that titers of fatty acids in the glycerolipids and acyl-CoA thioesters may be in equilibrium. A time-course assay with D4-16:COOH demonstrated that peak pheromone titer after application was reached before peak titers of both total hexadecanoate and hexadecanoyl-CoA. Combined with a dose-response experiment, which showed that labeled pheromone titer did not increase above an applied concentration of 20 mg/ml, these data suggest that the final step in pheromone biosynthesis, reduction of Z11-16:Acyl-CoA, may be inhibited by increased acyl-CoA titers in the gland. Overall, our data are consistent with the glycerolipids modulating acyl-CoA concentrations in the pheromone gland.     

Representation of primary plant odorants in the antennal lobe of the moth Heliothis virescens using calcium imaging

The primary olfactory centre, the antennal lobe of Heliothis virescens moths, contains 62 glomeruli which process plant odour information and four male-specific glomeruli which form the macroglomerular complex, involved in processing information about pheromone and interspecific signals. Using calcium imaging, we recorded the spatio-temporal activity pattern of the glomeruli in the anterior antennal lobe during stimulation with odorants produced by plants or insects. Each odorant elicited specific excitatory responses in one or a few glomeruli: the major pheromone component did so exclusively in the large glomerulus of the macroglomerular complex and the plant odours exclusively in the ordinary glomeruli. Eight glomeruli, with corresponding plant odour responses and positions, were identified within each sex. Glomeruli responded specifically to linalool, beta-ocimene/beta-myrcene or germacrene D/alpha-farnesene. Responses to two essential plant oils covered the response areas of their major constituents, as well as activating additional glomeruli. Stronger activation in the AL due to increased odour concentration was expressed as increased response strength within the odorant-specific glomeruli as well as recruitment of less sensitive glomeruli.     

Genetic differentiation across North America in the generalist moth Heliothis virescens and the specialist H. subflexa

The two moth species Heliothis virescens (Hv) and H. subflexa (Hs) are closely related, but have vastly different feeding habits. Hv is a generalist and an important pest in many crops in the USA, while Hs is a specialist feeding only on plants in the genus Physalis. In this study, we conducted a comparative population genetic analysis to assess whether and how generalist and specialist life styles are reflected in differences in population structures. In Hv 98% of the total variation occurred within populations. The overall differentiation (F(ST) ) between regions was 0.006 and even lower between years (0.0039) and hosts (0.0028). Analyses of population structure suggest that all individuals form one genetically homogeneous population, except for at most 12 individuals (6%) that diverged from this cluster. Population homogeneity likely results from the high mobility of Hv and its generalist feeding behaviour. Hs exhibited substantially more population structure. Even though 96% of the total variation was attributable to within-population variability, F(ST) -values between Hs populations were 10 times higher than between Hv populations. Hs populations showed significant isolation by distance. Analyses of Hs population structure suggest at least two subpopulations and thus some degree of metapopulation structure. We speculate that the patchy distribution of Physalis- the exclusive food source of Hs - contributes to differences in population structure between these closely related species. The finding that the specialist shows more population differentiation than the generalist corroborates the notion that host specialization is not an evolutionary dead end but a dynamic trait.     

Aldehyde-oxidizing enzymes in an adult moth: in vitro study of aldehyde metabolism in Heliothis virescens

The conversion of pheromonal aldehydes to carboxylic acids in vitro in tissue extracts of Heliothis virescens is catalyzed by both aldehyde dehydrogenase and aldehyde oxidase enzymes. The aldehyde-oxidizing activity in antennae, heads, legs, and hemolymph from male and female moths was examined by radiochromatographic and spectroscopic assays. First, the enzymatic activity was measured in the presence or absence of added NAD+ using either (Z)-9-tetradecenal or (Z)-11-hexadecenal as tritiated substrate. Second, substrate specificity was determined spectroscopically by (i) indirect measurement of the AO-released hydrogen peroxide through the coupled AO-horseradish peroxidase reaction and by (ii) direct measurement of the ALDH-produced NADH. Both aldehyde-oxidizing activities were associated with soluble enzymes in the antennal extracts, and these enzymes degraded pheromone and nonpheromonal aldehydes. Both AO and ALDH activities were present in male and female tissues. AO activity was exhibited primarily in the antennal extracts and to a lesser degree in the leg extracts. Moreover, ALDH activity was distributed in the antenna, head, and leg extracts. A vinyl ketone analog of (Z)-11-hexadecenal preferentially inhibited the ALDH activity over the AO activity.     

Metabolic inhibitors: Effects on metamorphosis and flight muscle mitochondrial development in the moth,Heliothis virescens

Acriflavin, aminotriazole, chloramphenicol, and cycloheximide were tested for their effects on the initiation of metamorphosis, adult morphogenesis and mitochondriogenesis in Heliothis virescens. The experiments evaluated the contributions of mitochondrial and cytoplasmic biosynthetic processes to metamorphosis.Acriflavin had little effect on metamorphosis. All other agents were inhibitory with cycloheximide having the greatest effect. All affected morphogenesis at doses lower than those preventing the initiation of metamorphosis. Moths which appeared to emerge normally after treatment with aminotriazole died within 24 hr.Respiratory development was not affected by chloramphenicol or aminotriazole. Acriflavin prevented cytochrome c oxidase development but had no effect on cytochrome c reductase.