Exploring the contribution of the salivary gland and midgut to digestion in the swede midge (Contarinia nasturtii) through a genomics-guided approach.

The larvae of Contarinia nasturtii (Kieffer) (Diptera: Cecidomyiidae), the swede midge, targets the meristem of brassica crops where they induce the formation of galls and disrupt seed and vegetable production. Previously, we examined the salivary gland transcriptome of newly-hatched first instar larvae as they penetrated the host and initiated gall formation. Here we examine the salivary gland and midgut transcriptome of third instar larvae and provide evidence for cooperative nutrient acquisition beginning with secretion of enzymes and feeding facilitators followed by gastrointestinal digestion. Sucrose, presumably obtained from the phloem, appeared to be a major nutrient source as several α-glucosidases (sucrases, maltases) and ß-fructofuranosidases (invertases) were identified. Genes encoding ß-fructofuranosidases/invertases were among the most highly expressed in both tissues and represented two distinct gene families that may have originated via horizontal gene transfer from bacteria. The importance of the phloem as a nutrient source is underscored by the expression of genes encoding regucalcin and ARMET (arginine-rich mutated in early stages of tumor) which interfere with calcium signalling and prevent sieve tube occlusion. Lipids, proteins, and starch appear to serve as a secondary nutrient sources. Genes encoding enzymes involved in the detoxification of glucosinolates (myrosinases, arylsulfatases, and glutathione-S-transferases) were expressed indicative of Brassicaceae host specialization. The midgut expressed simple peritrophins and mucins typical of those found in Type II peritrophic matrices, the first such description for a gall midge.

Characterization of the swede midge, Contarinia nasturtii, first instar larval salivary gland transcriptome.

Proteins in saliva of gall-forming insect larvae govern insect-host plant interactions. Contarinia nasturtii, the swede midge, is a pest of brassicaceous vegetables (cabbage, cauliflower, broccoli) and canola. We examined the salivary gland (SG) transcriptome of first instar larvae reared on Brassica napus and catalogued genes encoding secreted proteins that may contribute to the initial stages of larval establishment, the synthesis of plant growth hormones, extra-oral digestion and evasion of host defenses. A significant portion of the secreted proteins with unknown functions were unique to C. nasturtii and were often members of larger gene families organized in genomic clusters with conservation patterns suggesting that they are undergoing selection.

Comparative transcriptomic assessment of the chemosensory receptor repertoire of Drosophila suzukii adult and larval olfactory organs.

The spotted wing Drosophila, Drosophila suzukii, has emerged within the past decade as an invasive species on a global scale, and is one of the most economically important pests in fruit and berry production in Europe and North America. Insect ecology, to a strong degree, depends on the chemosensory modalities of smell and taste. Extensive research on the sensory receptors of the olfactory and gustatory systems in Drosophila melanogaster provide an excellent frame of reference to better understand the fundamentals of the chemosensory systems of D. suzukii. This knowledge may enhance the development of semiochemicals for sustainable management of D. suzukii, which is urgently needed. Here, using a transcriptomic approach we report the chemosensory receptor expression profiles in D. suzukii female and male antennae, and for the first time, in larval heads including the dorsal organ that houses larval olfactory sensory neurons. In D. suzukii adults, we generally observed a lack of sexually dimorphic expression levels in male and female antennae. While there was generally conservation of antennal expression of odorant and ionotropic receptor orthologues for D. melanogaster and D. suzukii, gustatory receptors showed more distinct species-specific profiles. In larval head tissues, for all three receptor gene families, there was also a greater degree of species-specific gene expression patterns. Analysis of chemosensory receptor repertoires in the pest species, D. suzukii relative to those of the genetic model D. melanogaster enables comparative studies of the chemosensory, physiology, and ecology of D. suzukii.

Seed choice in ground beetles is driven by surface-derived hydrocarbons.

Ground beetles (Coleoptera: Carabidae) are among the most prevalent biological agents in temperate agroecosystems. Numerous species function as omnivorous predators, feeding on both pests and weed seeds, yet the sensory ecology of seed perception in omnivorous carabids remains poorly understood. Here, we explore the sensory mechanisms of seed detection and discrimination in four species of omnivorous carabids: Poecilus corvus, Pterostichus melanarius, Harpalus amputatus, and Amara littoralis. Sensory manipulations and multiple-choice seed feeding bioassays showed olfactory perception of seed volatiles as the primary mechanism used by omnivorous carabids to detect and distinguish among seeds of Brassica napus, Sinapis arvensis, and Thlaspi arvense (Brassicaceae). Seed preferences differed among carabid species tested, but the choice of desirable seed species was generally guided by the olfactory perception of long chain hydrocarbons derived from the seed coat surface. These olfactory seed cues were essential for seed detection and discrimination processes to unfold. Disabling the olfactory appendages (antennae and palps) of carabid beetles by ablation left them unable to make accurate seed choices compared to intact beetles.

Components of the Female Sex Pheromone of the Newly-Described Canola Flower Midge, Contarinia brassicola.

The canola flower midge, Contarinia brassicola Sinclair (Diptera: Cecidomyiidae), is a newly-described species that induces galls on canola, Brassica napus Linnaeus and Brassica rapa Linnaeus (Brassicaceae). Identification of the sex pheromone of C. brassicola is essential to developing monitoring tools to elucidate the geographic range and hosts of this new pest, and the extent to which it threatens the $30 billion Canadian canola industry. The aim of this study was to identify and synthesize the female-produced sex pheromone of C. brassicola and demonstrate its effectiveness in attracting males to traps in the field. Two peaks were identified through GC-EAG analysis of female-produced volatiles which elicited electrophysiological responses in male antennae. These peaks were initially characterized through GC-MS and synthesis as 2,7-diacetoxynonane (major component) and 2-acetoxynonane (minor component), and the racemic compounds elicited EAG responses in male antennae. All four stereoisomers of 2,7-diacetoxynonane were synthesized and the naturally-produced compound was shown to be primarily the (2R,7S)-isomer by analysis on an enantioselective GC column, with a small amount of (2R,7R)-2,7-diacetoxynonane also present. The configuration of the minor component could not be determined because of the small amount present, but this was assumed to be (2R)-2-acetoxynonane by comparison with the configuration of the other two components. In field trials, none of the four stereoisomers of 2,7-diacetoxynonane, presented individually or as a racemic mixture, was attractive to male C. brassicola. However, dispensers loaded with a 10 µg:1 µg blend of (2R,7S)- and (2R,7R)-2,7-diacetoxynonane caught large numbers of male C. brassicola and significantly more than other blends tested. The addition of 0.5 µg of (2R)-2-acetoxynonane to this blend further increased the number of males caught. In future work, we will seek to identify the optimum trapping protocol for the application of the pheromone in monitoring and surveillance.

De Novo Whole-Genome Assembly of the Swede Midge (Contarinia nasturtii), a Specialist of Brassicaceae, Using Linked-Read Sequencing.

The swede midge, Contarinia nasturtii, is a cecidomyiid fly that feeds specifically on plants within the Brassicaceae. Plants in this family employ a glucosinolate-myrosinase defense system, which can be highly toxic to nonspecialist feeders. Feeding by C. nasturtii larvae induces gall formation, which can cause substantial yield losses thus making it a significant agricultural pest. A lack of genomic resources, in particular a reference genome, has limited deciphering the mechanisms underlying glucosinolate tolerance in C. nasturtii, which is of particular importance for managing this species. Here, we present an annotated, scaffolded reference genome of C. nasturtii using linked-read sequencing from a single individual and explore systems involved in glucosinolate detoxification. The C. nasturtii genome is similar in size and annotation completeness to that of the Hessian fly, Mayetiola destructor, but has greater contiguity. Several genes encoding enzymes involved in glucosinolate detoxification in other insect pests, including myrosinases, sulfatases, and glutathione S-transferases, were found, suggesting that C. nasturtii has developed similar strategies for feeding on Brassicaceae. The C. nasturtii genome will, therefore, be integral to continued research on plant-insect interactions in this system and contribute to effective pest management strategies.

Compensatory Abilities of Canola in Response to Swede Midge (Diptera: Cecidomyiidae) Damage.

Swede midge (Contarinia nasturtii Kieffer) is an invasive, economic pest of canola (Brassica napus L.) that threatens production throughout Canada. Swede midge has up to four overlapping generations, placing canola at risk of multiple infestations in the field. The relationship between single and multiple swede midge infestations at different canola stages, and the resulting impacts on yield, are unknown. Laboratory experiments were conducted to evaluate the impact of single and multiple infestations of swede midge on four plant stages of canola: three-leaf, seven-leaf, primary bud, and secondary bud. Previously, the seven-leaf and primary bud stages were considered vulnerable to swede midge, but we determined that the secondary bud stage is also vulnerable. Evidence of compensation by canola in response to herbivory by swede midge was discovered. Compensation occurred mainly through increased production of tertiary racemes and pods and was maximized with exposure of 4.5-6.5 female midges per plant. Although compensation may increase potential yield, it also results in uneven crop maturation resulting in delayed harvest and yield loss. Consequently, to prevent delays in maturation and harvest, insecticides should be applied before these densities are reached.

Disjunction between canola distribution and the genetic structure of its recently described pest, the canola flower midge (Contarinia brassicola).

Population genomics is a useful tool to support integrated pest management as it can elucidate population dynamics, demography, and histories of invasion. Here, we use a restriction site-associated DNA sequencing approach combined with whole-genome amplification (WGA) to assess genomic population structure of a newly described pest of canola, the diminutive canola flower midge, Contarinia brassicola. Clustering analyses recovered little geographic structure across the main canola production region but differentiated several geographically disparate populations at edges of the agricultural zone. Given a lack of alternative hypotheses for this pattern, we suggest these data support alternative hosts for this species and thus our canola-centric view of this midge as a pest has limited our understanding of its biology. These results speak to the need for increased surveying efforts across multiple habitats and other potential hosts within Brassicaceae to improve both our ecological and evolutionary knowledge of this species and contribute to effective management strategies. We additionally found that use of WGA prior to library preparation was an effective method for increasing DNA quantity of these small insects prior to restriction site-associated DNA sequencing and had no discernible impact on genotyping consistency for population genetic analysis; WGA is therefore likely to be tractable for other similar studies that seek to randomly sample markers across the genome in small organisms.

Examining population structure of a bertha armyworm, Mamestra configurata (Lepidoptera: Noctuidae), outbreak in western North America: Implications for gene flow and dispersal.

The bertha armyworm (BAW), Mamestra configurata, is a significant pest of canola (Brassica napus L. and B. rapa L.) in western North America that undergoes cyclical outbreaks every 6-8 years. During peak outbreaks millions of dollars are spent on insecticidal control and, even with control efforts, subsequent damage can result in losses worth millions of dollars. Despite the importance of this pest insect, information is lacking on the dispersal ability of BAW and the genetic variation of populations from across its geographic range which may underlie potential differences in their susceptibility to insecticides or pathogens. Here, we examined the genetic diversity of BAW populations during an outbreak across its geographic range in western North America. First, mitochondrial cytochrome oxidase 1 (CO1) barcode sequences were used to confirm species identification of insects captured in a network of pheromone traps across the range, followed by haplotype analyses. We then sequenced the BAW genome and used double-digest restriction site associated DNA sequencing, mapped to the genome, to identify 1000s of single nucleotide polymorphisms (SNP) markers. CO1 haplotype analysis identified 9 haplotypes distributed across 28 sample locations and three laboratory-reared colonies. Analysis of genotypic data from both the CO1 and SNP markers revealed little population structure across BAW's vast range. The CO1 haplotype pattern showed a star-like phylogeny which is often associated with species whose population abundance and range has recently expanded and combined with pheromone trap data, indicates the outbreak may have originated from a single focal point in central Saskatchewan. The relatively recent introduction of canola and rapid expansion of the canola growing region across western North America, combined with the cyclical outbreaks of BAW caused by precipitous population crashes, has likely selected for a genetically homogenous BAW population adapted to this crop.

Determining Temperature-Dependent Development and Mortality Parameters of the Swede Midge (Diptera: Cecidomyiidae).

The swede midge (Contarinia nasturtii Kieffer) is an invasive insect in North America whose feeding has caused a decline of over 60% of total canola acreage in Ontario, Canada since 2011. Temperature-dependent development and mortality information are important to develop an effective pest management strategy for this insect; as the most comprehensive study on C. nasturtii development was completed on populations from the United Kingdom in the 1960s, new geographically relevant information is needed. Contarinia nasturtii eggs, larvae, pupae, and adults were reared from wild populations collected from Elora, Ontario, and allowed to develop at different temperatures. Resulting development rates were fit to a series of growth models and the model with the best relative goodness-of-fit was selected to represent the given life stage. Eggs from Ontario populations developed more quickly than their UK counterparts at temperatures below approximately 17°C, but more slowly at temperatures above 17°C. The same phenomenon was observed in larvae at 20°C. Pupae from both populations had similar development rates, and adult longevity was similar as well. This information will inform the management of C. nasturtii, and may help prevent its spread to other canola-producing regions of North America.

Challenges of Mating Disruption Using Aerosol-Emitting Pheromone Puffers in Red Clover Seed Production Fields to Control Coleophora deauratella (Lepidoptera: Coleophoridae).

Sex pheromone-mediated mating disruption using pheromone puffer dispensers was evaluated to control Coleophora deauratella (Lepidoptera: Coleophoridae) at three red clover seed production fields in Alberta, Canada. The objectives of the study were to determine aspects of the biology of C. deauratella which may affect successful mating disruption, evaluate the ability of aerosol-emitting pheromone puffers to reduce male moth catch in small-plot trials, and evaluate the ability of puffers to reduce male capture in traps, larval numbers and damage in large-plot trials. The median longevity of male and female C. deauratella was 6 d in the laboratory where males emerged in larger numbers earlier than females (protandry). Male response to pheromone peaked at sunrise; thus, puffers were programmed to dispense pheromone throughout this time period. Small-plot (0.25 ha) mating disruption trials indicated that pheromone released from puffers could reduce male C. deauratella orientation to traps by 60.7 ± 18.6% compared with that in untreated control plots. Reduction of male orientation to traps in large-plot (5 ha) trials over the course of the season was also successful (93.7 ± 1.6%). However, there was no corresponding decrease in larval numbers or increase in seed yield in pheromone-treated plots. Challenges of mating disruption of C. deauratella appear to be immigration of mated females combined with high population densities.

Mating disruption of Coleophora deauratella (Lepidoptera: Coleophoridae) using laminate flakes in red clover seed production fields.

BACKGROUND: The red clover casebearer, Coleophora deauratella, is a significant pest in red clover (Trifolium pratense) seed production regions throughout the world. The internal feeding nature of C. deauratella larvae makes infestations difficult to control with insecticide. We test the ability of Hercon Disrupt Micro-Flakes(®) releasing the complete pheromone blend of C. deauratella to disrupt communication and mating in red clover seed production fields. RESULTS: Initial small-plot (0.25 ha) trials found a significant reduction (93.6 ± 2.9%) of male C. deauratella captured in pheromone-treated plots compared with untreated controls. Subsequent large-plot (5 ha) mating disruption trials found a significant reduction (72.3 ± 5.7%) in male C. deauratella captured in pheromone-treated plots compared with untreated control plots over the growing season. Furthermore, larval numbers were significantly reduced and seed yield was increased in pheromone-treated plots compared with untreated control plots. In a concurrent small-plot (0.0625 ha) trial with various flake densities, disruption increased with pheromone flake density, and the resulting graphical disruption profiles matched the theoretical predictions of mating disruption by competitive attraction. CONCLUSION: Pheromone-mediated mating disruption with laminate flakes has the potential to suppress C. deauratella populations and may help to reduce damage even at high pest densities. © 2014 Society of Chemical Industry.

Efficacy and mechanisms of communication disruption of the red clover casebearer moth (Coleophora deauratella) with complete and partial pheromone formulations.

The red clover casebearer, Coleophora deauratella Leinig and Zeller (Lepidoptera: Coleophoridae), is a major pest of red clover (Trifolium pratense L.) grown for seed in Canada and parts of Europe. Insecticides are ineffective against C. deauratella, and other control methods, such as pheromone-mediated mating disruption, need to be explored. The efficacy and mechanisms of communication disruption were evaluated in small-plot trials (0.25 ha) with reservoir-type rope dispensers loaded with either the complete pheromone blend [10:1 ratio of (Z)-7-dodecenyl acetate: (Z)-5-dodecenyl acetate] or the major component alone [(Z)-7-dodecenyl acetate]. Both formulations reduced catches of male C. deauratella in pheromone traps (>99.6 %). In pheromone-treated plots, more males were caught on yellow sticky cards near dispensers that released the complete pheromone blend, than on cards near dispensers releasing the major component. In the laboratory, after pre-exposure to either the complete blend or the major component for 1 h, male C. deauratella antennae became adapted, as measured by electroantennograms conducted 5 min. after treatment. Adaptation due to pre-exposure to either formulation resulted in a shift in the pheromone response threshold; antennae from pre-exposed moths responded more strongly to high pheromone dosages (5-50 µg) than did antennae from untreated control moths. Antennae from moths held in clean air for 24 h after pre-exposure recovered and responded similarly to pheromone as antennae from control moths. These results suggest that both formulations have the potential to disrupt pheromone communication in C. Deauratella, but that the disruption mechanisms of the two formulations likely differ.

Factors affecting pheromone-baited trap capture of male Coleophora deauratella, an invasive pest of clover in Canada.

Coleophora deauratella Leinig and Zeller (Lepidoptera: Coleophoridae) is an invasive pest of red clover, Trifolium pratense L. (Fabeles: Fabaceae), grown for seed in Canada. A pheromone-based monitoring program to determine the presence and seasonal activity of adult C. deauratella would be a valuable tool for growers because larvae are difficult to sample due to their internal feeding behavior. We conducted field experiments to evaluate several pheromone-baited trap characteristics, including pheromone lure substrate, trap type, trap color, lure longevity, trap height, and field position. The type of substrate that pheromone was released from and lure age did not affect trap capture of male C. deauratella. Moth capture in nonsaturating green Unitraps was significantly higher than Diamond or Wing traps when inspected at 2-wk intervals. Multi-colored Unitraps caught significantly more male C. deauratella than Diamond, and Wing traps when inspected at weekly intervals. Trap color did not influence C. deauratella capture, but by-catch of bumblebees, Bombus spp., was greatest in yellow and white colored Unitraps. Traps placed 35 cm above the soil surface captured more male C. deauratella than those placed at 1 m above or at ground level regardless of trap position within the field. Green Unitraps baited with either gray or red rubber septa lures placed 35 cm above the soil surface and 5 m from the field edge should be used to monitor C. deauratella. These characteristics optimize pheromone-baited trap performance and reduce by-catch of beneficial insects, and should be incorporated into a C. deauratella pheromone-monitoring program.