Female Promiscuity Does Not Lead to Increased Fertility or Fecundity in an Arctiid Moth (<Emphasis Type="Italic">Utetheisa ornatrix</Emphasis>)

Prior work has demonstrated significant phenotypic benefits to female promiscuity in the arctiid moth Utetheisa ornatrix. We were interested in determining whether U. ornatrix females also derive genetic benefits from mating multiply. We specifically tested whether, by mating with several males, females are able to exploit postcopulatory mechanisms that decrease the risk of fertilization by incompatible sperm. We show evidence that U. ornatrix females are not taking multiple mates as fertilization insurance because: (1) females that mate once are as fertile as those that mate three times; and (2) females that take three different mates are no more fertile than those that mate three times with the same male.     

Z LINKAGE OF FEMALE PROMISCUITY GENES IN THE MOTH UTETHEISA ORNATRIX: SUPPORT FOR THE SEXY‐SPERM HYPOTHESIS?

Female preference genes for large males in the highly promiscuous moth Utetheisa ornatrix (Lepidoptera: Arctiidae) have previously been shown to be mostly Z‐linked, in accordance with the hypothesis that ZZ–ZW sex chromosome systems should facilitate Fisherian sexual selection. We determined the heritability of both female and male promiscuity in the highly promiscuous moth U. ornatrix (Lepidoptera: Arctiidae) through parent–offspring and grandparent–offspring regression analyses. Our data show that male promiscuity is not sex‐limited and either autosomal or sex‐linked whereas female promiscuity is primarily determined by sex‐limited, Z‐linked genes. These data are consistent with the “sexy‐sperm hypothesis,” which posits that multiple‐mating and sperm competitiveness coevolve through a Fisherian‐like process in which female promiscuity is a kind of mate choice in which sperm‐competitiveness is the trait favored in males. Such a Fisherian process should also be more potent when female preferences are Z‐linked and sex‐limited than when autosomal or not limited.     

Win,Lose, or Draw: Effects of Residency,Size, Sex,and Kinship on High‐Stakes Larval Contests in a Moth

Many factors can affect the outcome of a competitive interaction. One such factor is the relatedness between competitors as competitive intensity may decrease between kin. Because adult females lay eggs in clusters, larvae of the moth Utetheisa ornatrix are likely to be found in high densities of their siblings. Larval U. ornatrix actively compete for access into seedpods of their host plant (Crotalaria spp.), and successful competitors will reap numerous reproductive benefits during adulthood. The objective of this study was to determine whether residency, size, sex, and relatedness affect competition over seedpods in U. ornatrix larvae. In one‐on‐one trials for access into artificial seedpods, we monitored occupancy and weight change of larvae varying in residency, size, sex, and relatedness. We found that larger larvae have a competitive advantage over smaller larvae. This finding has consequences for the mating system of U. ornatrix in that females, by selecting males based on pheromone levels that are correlated with body size, can rear larger offspring that will have an advantage in competition over seedpods. These data did not support our hypothesis that males would outcompete females, perhaps because the rewards of acquiring a seedpod are substantial for both sexes. Finally, our data show that resident larvae are more likely to maintain control of seedpods over sibling than non‐siblings intruders, which suggests that relatedness affects competitive interactions.     

Female pheromonal chorusing in an arctiid moth, Utetheisa ornatrix

We report an unusual case of communal sexual display in thearctiid moth Utetheisa ornatrix that we designate "female pheromonalchorusing." As in most moths, female U. ornatrix release a long-distancesexual advertisement pheromone during a nightly activity period.We arranged U. ornatrix females in 2 types of signaling conditions:grouped and solitary. When the females were grouped with neighboringsignaling females (grouped), they initiated pheromone releasesooner, continued release with less interruption and over alonger total period, and performed the release with faster abdominalpumping than observed in isolated females (solitary). This differsfrom the usual form of sexual communication in moths: female(chemical) signalers, male receivers, and a general lack ofinteraction among females. At mating, male U. ornatrix transfera large spermatophore that may enhance female reproductive successand which represents either mating effort or paternal investment.This action results in an extended postmating male refractoryperiod leading to a female-biased operational sex ratio. Weargue that this biased sex ratio generates intrasexual competitionamong females, to which they respond by elevating signalingeffort such that the likelihood of at least matching their neighbors'signals is increased. In the field, U. ornatrix are clusteredaround patches of host plants, and we also explore the possibilitythat pheromonal chorusing is driven by cooperation among groupsof related—or nonrelated—females.     

Extrafloral nectaries as a deterrent mechanism against seed predators in the chemically protected weed Crotalaria pallida (Leguminosae)

Abstract In several plants, extrafloral nectaries (EFN) are located close to the reproductive structures, suggesting that ants may act as a defence against specialized seed predators that overcome chemical defences. Alternatively, ants may also deter herbivores in a generalized manner, thereby protecting the whole plant. In this work, we examined the relationship between the chemically protected weed Crotalaria pallida Ait. (Leguminosae) that bears EFN, its specialized seed predator, the larvae of the arctiid moth Utetheisa ornatrix L. (Arctiidae) and ants. We tested two hypotheses related to the type of deterrence caused by ants. The Seed Predator Deterrence Hypothesis predicts that ant deterrence is directed primarily towards herbivores that destroy seeds and other reproductive structures, without attacking herbivores on vegetative structures. The General Deterrence Hypothesis states that ants are general in their effects, equally deterring herbivores in vegetative and reproductive structures. Our results supported the predictions of the Seed Predator Deterrence Hypothesis, namely, that (i) ant activity on EFN was related to the vulnerability of reproductive structures to attack by U. ornatrix; (ii) ant patrolling was restricted almost entirely to racemes; (iii) ants removed termites used as baits more frequently on racemes than on leaves; and (iv) U. ornatrix larvae were often expulsed from the racemes. These results indicate that EFN can act as another deterrent mechanism in chemically protected plants by promoting the expulsion of specialist seed predators.     

Parental Body Mass as a Determinant of Egg Size and Egg Output in an Arctiid Moth (Utetheisa ornatrix)

Females of the moth Utetheisa ornatrix (family Arctiidae) mate preferentially with larger males. As a consequence, females have larger sons and daughters, which have been shown to be more acceptable in courtship and more fecund, respectively. We demonstrate that mating with larger males results in accelerated oviposition by the female on the day after mating and that females are intrinsically prone to lay larger eggs in the first days after mating. Both these additional size-dependent effects have potential adaptive consequences.     

Native or nonnative host plants: What is better for a specialist moth?

The enemy release hypothesis (ERH) predicts that the lack of natural enemies, such as herbivores, contributes to the success of nonnative plants as colonizers. Larvae of the Neotropical specialist moth Utetheisa ornatrix (Erebidae: Arctiinae) can feed on unripe seeds and leaves of both native and nonnative Crotalaria species (Fabaceae). Despite some species being able to eat nonnative plants, such behavior can impair the herbivore, as they are not adapted to the alien plant, and still contribute to the success of the nonnative species via enemy release. We tested the performance of the moth from hatching to adulthood fed on two native (C. micans and C. paulina) and two nonnative (C. pallida, C. juncea) host plants. Utetheisa ornatrix performed better (lower development time, heavier pupae and more eggs) on the native host plants than in the nonnative. However, larva performance in nonnative C. pallida was similar to that in the native host plants. Using the larval weight 7 days after hatching from the eggs as a proxy for performance in twelve Crotalaria species (five Neotropical natives, four nonnatives from Afrotropical region, and three nonnatives from India), we found similar results. Crotalaria nutritional compounds, the defensive pyrrolizidine alkaloids and Crotalaria phylogeny did not explain moth performance. Our results give some support to the ERH. The good moth performance in nonnative C. pallida may be related to its high availability as host plant for U. ornatrix, and its longer time since their introduction in Neotropics which would provide opportunity for the moth to adapt.     

Ten polymorphic microsatellite markers developed in the masson pine moth Dendrolimus punctatus Walker (Lepidoptera: Lasiocampidae)

A set of 10 polymorphic di‐ and trinucleotide microsatellite loci were developed for the forestry pest insect, masson pine moth, Dendrolimus punctatus Walker. The expected heterozygosity at these loci ranges from 0.285 to 0.859, and the observed allele numbers from five to 19. Cross species amplification of these loci in four other congeneric pine moth species indicates variable levels of loci conservation and thus cross‐applicability. Therefore, the microsatellite loci reported here should be useful for population genetic and other related studies in the masson pine moth and other closely related species.     

Feeding on Host Plants with Different Concentrations and Structures of Pyrrolizidine Alkaloids Impacts the Chemical-Defense Effectiveness of a Specialist Herbivore

Sequestration of chemical defenses from host plants is a strategy widely used by herbivorous insects to avoid predation. Larvae of the arctiine moth Utetheisa ornatrix feeding on unripe seeds and leaves of many species of Crotalaria (Leguminosae) sequester N-oxides of pyrrolizidine alkaloids (PAs) from these host plants, and transfer them to adults through the pupal stage. PAs confer protection against predation on all life stages of U. ornatrix. As U. ornatrix also uses other Crotalaria species as host plants, we evaluated whether the PA chemical defense against predation is independent of host plant use. We fed larvae from hatching to pupation with either leaves or seeds of one of eight Crotalaria species (C. incana, C. juncea, C. micans, C. ochroleuca, C. pallida, C. paulina, C. spectabilis, and C. vitellina), and tested if adults were preyed upon or released by the orb-weaving spider Nephila clavipes. We found that the protection against the spider was more effective in adults whose larvae fed on seeds, which had a higher PA concentration than leaves. The exceptions were adults from larvae fed on C. paulina, C. spectabilis and C. vitellina leaves, which showed high PA concentrations. With respect to the PA profile, we describe for the first time insect-PAs in U. ornatrix. These PAs, biosynthesized from the necine base retronecine of plant origin, or monocrotaline- and senecionine-type PAs sequestered from host plants, were equally active in moth chemical defense, in a dose-dependent manner. These results are also partially explained by host plant phylogeny, since PAs of the host plants do have a phylogenetic signal (clades with high and low PA concentrations in leaves) which is reflected in the adult defense.     

Ants visiting extrafloral nectaries and pyrrolizidine alkaloids may shape how a specialist herbivore feeds on its host plants

The presence of extrafloral nectaries (EFNs) attracts predators and parasitoids, and protects the plant against herbivorous insects. By improving plant defences, EFNs reduce the fitness of herbivores. The use of similar host plants with no EFNs or adaptations in response to predators and parasitoids may enhance herbivore fitness. In this context, we studied the feeding habit (on leaves or on unripe seeds inside the pods) of larvae of the specialist moth Utetheisa ornatrix in two Crotalaria host plant species in which EFNs are present (C. micans) or absent (C. paulina). We hypothesized that the moths’ feeding habit was influenced by its natural enemies via their presence on EFNs. In C. micans, we found more larvae feeding inside the pods rather than on the leaves, while in C. paulina, larvae were found in both parts of the plant. There was greater activity of natural enemies in C. micans than in C. paulina. The moth sequesters enough pyrrolizidine alkaloid (PAs) to defend against predators in the leaves and seeds of C. paulina, but only in seeds of C. micans. Therefore, a change in the feeding habit in U. ornatrix larvae is a plastic response that depends on whether EFNs are present or not, or whether PA concentrations are low or high. This change does not affect overall moth performance. However, other factors, such as pod hardness, predation by organisms other than those visiting EFNs or even parasitoids cannot be ruled out as being responsible for the change in feeding habit. To date, both the EFNs and PAs in Crotalaria species are a parsimonious explanation of how larvae of U. ornatrix use different species of Crotalaria for feeding.     

A free lunch? No cost for acquiring defensive plant pyrrolizidine alkaloids in a specialist arctiid moth (Utetheisa ornatrix)

Many herbivorous insects sequester defensive chemicals from their host plants. We tested sequestration fitness costs in the specialist moth Utetheisa ornatrix (Lepidoptera: Arctiidae). We added pyrrolizidine alkaloids (PAs) to an artificial diet at different concentrations. Of all the larval and adult fitness components measured, only development time was negatively affected by PA concentration. These results were repeated under stressful laboratory conditions. On the other hand, the amount of PAs sequestered greatly increased with the diet PA concentration. Absence of a detectable negative effect does not necessarily imply a lack of costs if all individuals express the biochemical machinery of detoxification and sequestration constitutively. Therefore, we used qPCR to show that expression of the gene used to detoxify PAs, pyrrolizidine‐alkaloid‐N‐oxygenase (pno), increased 41‐fold in our highest PA treatment. Nevertheless, fitness components were affected only slightly or not at all, suggesting that sequestration in this species does not incur a strong cost. The apparent lack of costs has important implications for our understanding of the evolution of ecological interactions; for example, it implies that selection by specialist herbivores may decrease the levels of certain chemical defences in plant populations.     

Repetitive flanking sequences (ReFS): novel molecular markers from microsatellite families

Although microsatellite markers have become exceedingly popular in molecular studies of wild organisms, their development in some taxonomic groups is challenging. This is partly because of repetitive flanking sequences, which lead to the simultaneous amplification of alleles from multiple loci. Until now, these microsatellite DNA families have been considered unsuitable for population genetics studies, but here we describe our development of these repetitive flanking sequences (ReFS) as novel molecular markers. We illustrate the utility of these markers by using them to address an outstanding taxonomic question in the moth genus Schrankia.     

Isolation of nine microsatellite loci in Dolichogenidea homoeosomae (Hymenoptera) a parasitoid of the sunflower moth Homoeosoma electellum (Lepidoptera)

Nine microsatellite loci were isolated from the insect Dolichogenidea homoeosomae (Hymenoptera: Braconidae), an important parasitoid of the sunflower moth Homosoeosoma electellum (Lepidoptera: Pyralidae), and assayed for polymorphism. All nine loci were polymorphic within the five populations tested, with two to 14 alleles per locus. Expected and observed heterozygosities ranged from 0.39 to 0.90 and 0.25 to 0.72 respectively. These are the first microsatellite primers developed for D. homeosomae and will be useful for studies of population dynamics and connectivity.     

Isolation and characterization of microsatellite markers from Zale galbanata (Lepidoptera: Noctuidae) and amplification in other members of the genus

The noctuid moth Zale galbanata is described as feeding on maples (Acer sp.) in general, but its primary food plant is box elder (Acer negundo). We isolated five microsatellite loci to investigate the association of food plant use and the genetic similarity of populations in these moths. These markers are polymorphic to various degrees (5–13 alleles), and are presented here. Our initial tests show that some of these loci also work on other Zale species.     

Microsatellites isolated from diamondback moth,Plutella xylostella (L.), for studies of dispersal in Australian populations

Diamondback moth, Plutella xylostella (L.), is a worldwide agricultural pest that has developed resistance to many insecticides used for its control. Population structure and gene flow are yet to be determined for P. xylostella in Australia, but are important factors for the design of effective control strategies. We have isolated six polymorphic microsatellite markers: three from a partial genomic library, two from an Expressed Sequence Tagged library and one from an aminopeptidase intron of P. xylostella. These microsatellites will be used to determine population structure and gene flow in Australian populations of P. xylostella to improve insecticide resistance management.     

Genetic differentiation of the pine processionary moth at the southern edge of its range: contrasting patterns between mitochondrial and nuclear markers

The pine processionary moth (Thaumetopoea pityocampa) is an important pest of coniferous forests at the southern edge of its range in Maghreb. Based on mitochondrial markers, a strong genetic differentiation was previously found in this species between western (pityocampa clade) and eastern Maghreb populations (ENA clade), with the contact zone between the clades located in Algeria. We focused on the moth range in Algeria, using both mitochondrial (a 648 bp fragment of the tRNA‐cox2) and nuclear (11 microsatellite loci) markers. A further analysis using a shorter mtDNA fragment and the same microsatellite loci was carried out on a transect in the contact zone between the mitochondrial clades. Mitochondrial diversity showed a strong geographical structure and a well‐defined contact zone between the two clades. In particular, in the pityocampa clade, two inner subclades were found whereas ENA did not show any further structure. Microsatellite analysis outlined a different pattern of differentiation, with two main groups not overlapping with the mitochondrial clades. The inconsistency between mitochondrial and nuclear markers is probably explained by sex‐biased dispersal and recent afforestation efforts that have bridged isolated populations.     

Preference for high concentrations of plant pyrrolizidine alkaloids in the specialist arctiid moth Utetheisa ornatrix depends on previous experience

Secondary metabolites are one the most pervasive defensive mechanisms in plants. Many specialist herbivores have evolved adaptations to overcome these defensive compounds. Some herbivores can even take advantage of these compounds by sequestering them for protection and/or mate attraction. One of the most studied specialist insects that sequesters secondary metabolites is the arctiid moth Utetheisa ornatrix. This species sequesters pyrrolizidine alkaloids (PAs) from its host plant, the legume Crotalaria spp. The sequestered PAs are used as a predator repellent and as a mating pheromone. We used this species to test larval preference for different concentrations of PAs. We purified PAs from plant material and added them at different concentrations to an artificial diet. Larvae of U. ornatrix previously feeding on low and high PA concentration artificial diets were allowed to choose between two new artificial diets with different PA concentrations. The amount of PAs sequestered and larval preference were dependent on their previous exposure to low or high PA content in the diet. Larvae that were pretreated with a low PA diet significantly consumed more diet with the high PA concentration, while larvae that were pretreated with a high PA diet showed no discrimination between future feeding of different PA concentration diets. We discuss our results using mechanistic and evolutionary approaches. Finally, we discuss how these results have important implications on the evolution of plant herbivore interactions and how specialist herbivores may decrease the levels of chemical defenses on plant populations.     

Why do larvae of Utetheisa ornatrix penetrate and feed in pods of Crotalaria species? Larval performance vs. chemical and physical constraints

Larvae of Utetheisa ornatrix (L.)(Lepidoptera: Arctiidae) are found mainly inside unripe pods of several alkaloid‐bearing Crotalaria (Fabaceae) species. Although eggs are laid on the leaves, the larvae are usually found feeding on unripe seeds in the pods. In this work, we investigated the selective pressures that could explain why U. ornatrix larvae feed primarily on unripe pods with seeds and not on leaves. Our results showed that larval survivorship in the laboratory was unaffected by feeding on leaves or unripe seeds, and that larval development up to the pupal stage was better in larvae that fed on unripe seeds, although perforating unripe pods to reach seeds was costly in terms of survivorship. Females were also heavier when fed on unripe seeds, but there was no significant difference in the fecundity of females fed either of the two diets. Feeding on unripe seeds in pods had other benefits for U. ornatrix, including a lower predation rate for larvae that fed inside compared to larvae that fed outside the pods. Similarly, adults derived from larvae that fed on unripe seeds were preyed upon less frequently by the orb‐weaving spider Nephila clavipes than were adults that fed on leaves. The latter benefit may be closely related to the high concentration of pyrrolizidine alkaloids in unripe seeds, which is about five times more than in leaves. These alkaloids are sequestered by the larvae and transferred to adults, which then become chemically protected. However, this chemical defence does not protect the larvae against ants such as Ectatomma quadridens and Camponotus crassus. Pods with unripe seeds that confer physical protection to larvae and pyrrolizidine alkaloids that confer chemical protection to adults limit the use of leaves by U. ornatrix larvae.     

Microsatellite markers for testing host plant‐related population differentiation in the moth genus Yponomeuta

Nine microsatellite primers were developed for Yponomeuta padellus (Lepidoptera: Yponomeutidae) and tested for their applicability in analysing genetic population structure. Eight of the nine loci were highly polymorphic with on average 11.4 alleles. Cross‐species amplification of the nine primer pairs was tested in five other moth species. Primer pairs amplified in Y. cagnagellus, Y. malinellus, Y. evonymellus, and Y. rorellus but not in Y. sedellus and Plutella xylostella.     

Cross-amplification of microsatellites from the codling moth Cydia pomonella to three other species of the tribe Grapholitini (Lepidoptera: Tortricidae)

This study examined cross-species amplification of 33 microsatellite markers, previously developed for Cydia pomonella, in three related fruit moth species of the same tribe (Grapholitini), namely Grapholita molesta, Grapholita funebrana and Grapholita lobarzewskii. Eight microsatellite loci yielded polymorphic products for G. molesta, nine for G. funebrana and 11 for G. lobarzewskii. At all these loci, the number of alleles ranged between four and 11 in G. molesta, and between four and nine in G. funebrana and G. lobarzewskii each. The successful cross-amplified loci can be used for research on population genetics and gene flow of the three target species.