Novel insight into lepidopteran phylogenetics from the mitochondrial genome of the apple fruit moth of the family Argyresthiidae.

BACKGROUND: The order Lepidoptera has an abundance of species, including both agriculturally beneficial and detrimental insects. Molecular data has been used to investigate the phylogenetic relationships of major subdivisions in Lepidoptera, which has enhanced our understanding of the evolutionary relationships at the family and superfamily levels. However, the phylogenetic placement of many superfamilies and/or families in this order is still unknown. In this study, we determine the systematic status of the family Argyresthiidae within Lepidoptera and explore its phylogenetic affinities and implications for the evolution of the order. We describe the first mitochondrial (mt) genome from a member of Argyresthiidae, the apple fruit moth Argyresthia conjugella. The insect is an important pest on apples in Fennoscandia, as it switches hosts when the main host fails to produce crops. RESULTS: The mt genome of A. conjugella contains 16,044 bp and encodes all 37 genes commonly found in insect mt genomes, including 13 protein-coding genes (PCGs), two ribosomal RNAs, 22 transfer RNAs, and a large control region (1101 bp). The nucleotide composition was extremely AT-rich (82%). All detected PCGs (13) began with an ATN codon and terminated with a TAA stop codon, except the start codon in cox1 is ATT. All 22 tRNAs had cloverleaf secondary structures, except trnS1, where one of the dihydrouridine (DHU) arms is missing, reflecting potential differences in gene expression. When compared to the mt genomes of 507 other Lepidoptera representing 18 superfamilies and 42 families, phylogenomic analyses found that A. conjugella had the closest relationship with the Plutellidae family (Yponomeutoidea-super family). We also detected a sister relationship between Yponomeutoidea and the superfamily Tineidae. CONCLUSIONS: Our results underline the potential importance of mt genomes in comparative genomic analyses of Lepidoptera species and provide valuable evolutionary insight across the tree of Lepidoptera species.

Large-scale genetic admixture suggests high dispersal in an insect pest, the apple fruit moth.

Knowledge about population genetic structure and dispersal capabilities is important for the development of targeted management strategies for agricultural pest species. The apple fruit moth, Argyresthia conjugella (Lepidoptera, Yponomeutidae), is a pre-dispersal seed predator. Larvae feed on rowanberries (Sorbus aucuparia), and when rowanberry seed production is low (i.e., inter-masting), the moth switches from laying eggs in rowanberries to apples (Malus domestica), resulting in devastating losses in apple crops. Using genetic methods, we investigated if this small moth expresses any local genetic structure, or alternatively if gene flow may be high within the Scandinavian Peninsula (~850.000 km2, 55o - 69o N). Genetic diversity was found to be high (n = 669, mean He = 0.71). For three out of ten tetranucleotide STRs, we detected heterozygote deficiency caused by null alleles, but tests showed little impact on the overall results. Genetic differentiation between the 28 sampling locations was very low (average FST = 0.016, P < 0.000). Surprisingly, we found that all individuals could be assigned to one of two non-geographic genetic clusters, and that a third, geographic cluster was found to be associated with 30% of the sampling locations, with weak but significant signals of isolation-by-distance. Conclusively, our findings suggest wind-aided dispersal and spatial synchrony of both sexes of the apple fruit moth over large areas and across very different climatic zones. We speculate that the species may recently have had two separate genetic origins caused by a genetic bottleneck after inter-masting, followed by rapid dispersal and homogenization of the gene pool across the landscape. We suggest further investigations of spatial genetic similarities and differences of the apple fruit moth at larger geographical scales, through life-stages, across inter-masting, and during attacks by the parasitoid wasp (Microgaster politus).

Attraction of Chrysotropia ciliata (Neuroptera, Chrysopidae) Males to P-Anisaldehyde, a Compound with Presumed Pheromone Function.

In a field-trapping experiment with plant volatiles, we observed notably high attraction of green lacewing (Chrysotropia ciliata) males to the compound p-anisaldehyde. Based on this finding, we initiated the present study to elucidate this phenomenon and to investigate the chemical ecology of C. ciliata. Scanning electron microscopy revealed elliptical glands abundantly distributed on the 2nd to 6th abdominal sternites of C. ciliata males, whereas females of the species completely lacked such glands. No p-anisaldehyde was found in extractions of body parts of C. ciliata. Methyl p-anisate and p-methoxybenzoic acid were identified exclusively in the extract from abdominal segments 2-8 of males. Field-trapping experiments revealed no attraction of C. ciliata to either methyl p-anisate or p-methoxybenzoic acid. In contrast, males showed marked attraction to p-anisaldehyde in the field and antennae showed strong responses to this compound. Headspace collections in the field from living insects in their natural environment and during their main daily activity period indicated that p-anisaldehyde was emitted exclusively by C. ciliata males. Our overall results suggest that p-anisaldehyde might serve as a male-produced pheromone that attracts conspecific C. ciliata males. Here, we discuss hypotheses regarding possible mechanisms involved in regulation of p-anisaldehyde production, including involvement of the compounds methyl p-anisate and p-methoxybenzoic acid, and the potential ecological function of p-anisaldehyde in C. ciliata.

A Wind Tunnel for Odor Mediated Insect Behavioural Assays.

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study insect chemical ecology. Insects can locate point sources in a three-dimensional environment through the sensory interaction and sophisticated behavior. The quantification of this behavior is a key element in the development of new tools for pest control and decision support. A wind tunnel with a suitable flight section with laminar air flow, visual cues for in-flight feedback and a variety of options for the application of odors can be used to measure complex behaviour which subsequently may allow the identification of attractive or repellent odors, insect flight characteristics, visual-odor interactions and interactions between attractants and odors lingering as background odors in the environment. A wind tunnel holds the advantage of studying the odor mediated behavioural repertoire of an insect in a laboratory setting. Behavioural measures in a controlled setting provide the link between the insect physiology and field application. A wind tunnel must be a flexible tool and should easily support the changes to setup and hardware to fit different research questions. The major disadvantage to the wind tunnel setup described here, is the clean odor background which necessitates special attention when developing a synthetic volatile blend for field application.

Monitoring of the Apple Fruit Moth: Detection of Genetic Variation and Structure Applying a Novel Multiplex Set of 19 STR Markers.

The apple fruit moth Argyresthia conjugella (Lepidoptera, Yponomeutidae) is a seed predator of rowan (Sorbus aucuparia) and is distributed in Europe and Asia. In Fennoscandia (Finland, Norway and Sweden), rowan fruit production is low every 2-4 years, and apple (Malus domestica) functions as an alternative host, resulting in economic loss in apple crops in inter-mast years. We have used Illumina MiSeq sequencing to identify a set of 19 novel tetra-nucleotide short tandem repeats (STRs) in Argyresthia conjugella. Such motifs are recommended for genetic monitoring, which may help to determine the eco-evolutionary processes acting on this pest insect. The 19 STRs were optimized and amplified into five multiplex PCR reactions. We tested individuals collected from Norway and Sweden (n = 64), and detected very high genetic variation (average 13.6 alleles, He = 0.75) compared to most other Lepidoptera species studied so far. Spatial genetic differentiation was low and gene flow was high in the test populations, although two non-spatial clusters could be detected. We conclude that this set of genetic markers may be a useful resource for population genetic monitoring of this economical important insect species.

The Ratio between Field Attractive and Background Volatiles Encodes Host-Plant Recognition in a Specialist Moth.

Volatiles emitted by plants convey an array of information through different trophic levels. Animals such as host-seeking herbivores encounter plumes with filaments from both host and non-host plants. While studies showed a behavioral effect of non-host plants on herbivore host location, less information is available on how a searching insect herbivore perceives and flies upwind to a host-plant odor plume within a background of non-host volatiles. We hypothesized here that herbivorous insects in search of a host-plant can discriminate plumes of host and non-host plants and that the taxonomic relatedness of the non-host have an effect on finding the host. We also predicted that the ratio between certain plant volatiles is cognized as host-plant recognition cue by a receiver herbivorous insect. To verify these hypotheses we measured the wind tunnel response of the moth Argyresthia conjugella to the host plant rowan, to non-host plants taxonomically related (Rosaceae, apple and pear) or unrelated to the host (Pinaceae, spruce) and to binary combination of host and non-host plants. Volatiles were collected from all plant combinations and delivered to the test insect via an ultrasonic sprayer as an artificial plume. While the response to the rowan as a plant was not affected by the addition of any of the non-host plants, the attraction to the corresponding sprayed headspace decreased when pear or apple but not spruce were added to rowan. A similar result was measured toward the odor exiting a jar where freshly cut plant material of apple or pear or spruce was intermixed with rowan. Dose-response gas-chromatography coupled to electroantennography revealed the presence of seven field attractive and seven background non-attractive antennally active compounds. Although the abundance of field attractive and of some background volatiles decreased in all dual combinations in comparison with rowan alone, an increased amount of the background compounds (3E)-4,8-Dimethyl-1,3,7-nonatriene ((E)-DMNT) and (Z)-3-hexenyl acetate was found in the rowan-apple and rowan-pear but not in the rowan-spruce headspace. A higher ratio between the abundance of each field attractive component and that of (E)-DMNT and (Z)-3-hexenyl acetate was measured for rowan and rowan-spruce in contrast to rowan-pear and rowan-apple headspaces. Our result suggests that the ratio between field attractive and background antennaly active volatiles encodes host-plant recognition in our study system.

Fatal attraction: Male spider mites prefer females killed by the mite-pathogenic fungus Neozygites floridana.

Exploring prospective mates can be risky. Males of the spider mite Tetranychus urticae approach and guard immobile (quiescent) female nymphs to increase their chances of fathering offspring, this being a first-male sperm priority species. We investigated the behaviour of male T. urticae towards females killed by the mite pathogenic fungus Neozygites floridana, letting them choose between a fungal killed and a healthy quiescent female. The dead female (called cadaver) was in one of three stages: (1) non-sporulating; (2) sporulating with primary conidia (non-infective); (3) surrounded/partly covered by secondary capilliconidia (infective). When the cadaver was in stage 1 or 2, males were significantly more often observed near the cadaver than near the healthy female. When the cadaver was in stage 3 (infective capilliconidia), males preferred the vicinity of healthy females. The frequency of two male behaviours, touching and guarding, was also recorded. Touching the cadaver tended to decrease as cadaver developed, whereas touching the healthy females increased. Guarding of non-sporulating cadavers and healthy females was equally common, while guarding of sporulating cadavers was only observed once (stage 2) or not at all (stage 3). To differentiate between the effect of fungal infection and sex, we also let males choose between a non-sporulating cadaver of each sex. Males then preferred to approach the female cadaver. Touching behaviour followed the same pattern, and guarding of male cadavers was not observed. Our results indicate that T. urticae males are more attracted to non-infective female cadavers than to healthy females, only detecting their mistake when very close. Moreover, males approach and explore cadavers surrounded by infective conidia. Whether the results of host manipulation by the pathogen or just sensory constraints in the host, this inability to detect unsuitable and indeed infective mates promotes transmission of the pathogen.

Attraction of pea moth Cydia nigricana to pea flower volatiles.

The pea moth Cydia nigricana causes major crop losses in pea (Pisum sativum) production. We investigated attraction of C. nigricana females to synthetic pea flower volatiles in a wind tunnel and in the field. We performed electroantennogram analysis on 27 previously identified pea plant volatiles, which confirmed antennal responses to nine of the compounds identified in pea flowers. A dose-dependent response was found to eight of the compounds. Various blends of the nine pea flower volatiles eliciting antennal responses were subsequently studied in a wind tunnel. A four-compound blend comprising hexan-1-ol, (E)-2-hexen-1-ol, (Z)-ß-ocimene and (E)-ß-ocimene was equally attractive to mated C. nigricana females as the full pea flower mimic blend. We conducted wind-tunnel tests on different blends of these four pea flower compounds mixed with a headspace sample of non-flowering pea plants. By considering the effects of such green leaf background odour, we were able to identify (Z)- and (E)-ß-ocimene as fundamental for host location by the pea moths, and hexan-1-ol and (E)-2-hexen-1-ol as being of secondary importance in that context. In the field, the two isomers of ß-ocimene resulted in trap catches similar to those obtained with the full pea flower mimic and the four-compound blend, which clearly demonstrated the prime significance of the ß-ocimenes as attractants of C. nigricana. The high level of the trap catches of female C. nigricana noted in this first field experiment gives a first indication of the potential use of such artificial kairomones in pea moth control.

Efficient mass trapping: catching the pest, Calliphora vicina, (Diptera, Calliphoridae), of Norwegian stockfish production.

Attracting high numbers of adult females, extensive trapping prior to damage, and a pest species with a predominantly univoltine lifecycle, are important factors contributing to the success of mass-trapping programs. We conducted a mass-trapping experiment over 4 years, using yellow sticky traps and odor-baited funnel traps for control of the blowfly Calliphora vicina in stockfish production areas in Loften, Norway. Blowfly-related damage to fish in five mass-trapping sites was compared with damage at five control sites. Population monitoring established a connection between blowfly activity and damage. Blowfly activity in the period from mid-April to mid-May was correlated with damage to fish in the same year. Interception (sticky) traps, based on visual cues, caught blowflies during the early drying period, whereas odor-baited funnel traps were more effective during the later drying period, and continued to catch flies throughout the year until late autumn. In the funnel traps, 80% of the captured flies were females. Compared to control sites, mass trapping reduced overall damage to stockfish by 8-31%. In the final year of the experiment, we compared mass-trapping and control treatments, controlling for drying time period and sun exposure (which both contribute to damage), and found a damage reduction of 63%. Thus, mass-trapping with a female-attracting kairomone, and using traps of different functionality to extend the critical trapping period, in accord with pest phenology, is an efficient method for control of C. vicina in stockfish production.

Optimization of a phenylacetaldehyde-based attractant for common green lacewings (Chrysoperla carnea s.l.).

In field trapping tests, the catch of Chrysoperla carnea sensu lato (Neuroptera: Chrysopidae) increased when acetic acid was added to lures with phenylacetaldehyde. The addition of methyl salicylate to the binary mixture of phenylacetaldehyde plus acetic acid increased catches even further. The ternary blend proved to be more attractive than beta-caryophyllene, 2-phenylethanol, or 3-methyl eugenol (compounds previously described as attractants for chrysopids) on their own, and no influence on catches was recorded when these compounds were added as fourth components to the ternary blend. There were minimal changes in activity when (E)-cinnamaldehyde or methyl anthranylate (both evoking large responses from female or male antennae of C. carnea in this study) were added, although both compounds showed significant attraction on their own when compared to unbaited traps. In subtractive field bioassays with the ternary mixture, it appeared that the presence of either phenylacetaldehyde or methyl salicylate was important, whereas acetic acid was less so in the ternary mixture. The ternary blend attracted both female and male lacewings at sites in southern, central, and northern Europe. Possible applications of a synthetic attractant for lacewings are discussed.