Genomics of alphabaculovirus isolates infecting Mamestra species from North America and Eurasia.

Whole genome sequencing and multiplex PCR analysis were used to characterize previously isolated baculovirus isolates from Mamestra populations in Eurasia. Although these viruses have been previously described as Mamestra brassicae nucleopolyhedrovirus (MbNPV/MabrNPV), we demonstrate here that these isolates represent strains of the baculovirus species Alphabaculovirus maconfiguratae (MacoNPV-A) and Alphabaculovirus altermaconfiguratae (MacoNPV-B). The MabrNPV-Bu and -Uk isolates had 96% nucleotide (nt) identity to the type isolate MacoNPV-A 90/2 at the whole genome level and in addition contained a lef-7 homologue which is found in MacoNPV-A but not MacoNPV-B. MabrNPV-Si, -De and -Nl had 96.6, 96.6 and 98.5% nt identity to the type isolate MacoNPV-B 96/2 at the whole genome level, respectively and contained a helicase-2 homologue. Gene content, synteny and K-2-P lef-8, lef-9 and polh analysis also confirmed the presence of both MacoNPV-A and MacoNPV-B isolates in Eurasia. Thus, both these alphabaculovirus species have wide Holarctic distributions in Mamestra host species. MacoNPV-A and MacoNPV-B have wide host ranges and in addition we showed that MacoNPV-B isolates trended to higher infectivity for T. ni larvae.

The complete genome sequence of a second alphabaculovirus from the true armyworm, Mythimna unipuncta: implications for baculovirus phylogeny and host specificity.

The Mythimna unipuncta nucleopolyhedrovirus isolate KY310 (MyunNPV-KY310) is an alphabaculovirus isolated from a true armyworm (Mythimna unipuncta) population in Kentucky, USA. Occlusion bodies of this virus were examined by electron microscopy and the genome sequence was determined by 454 pyrosequencing. MyunNPV-KY310 occlusion bodies consisted of irregular polyhedra measuring 0.8-1.8 µm in diameter and containing multiple virions, with one to six nucleocapsids per virion. The genome sequence was determined to be 156,647 bp with a nucleotide distribution of 43.9% G+C. 152 ORFs and six homologous repeat (hr) regions were annotated for the sequence, including the 38 core genes of family Baculoviridae and an additional group of 26 conserved alphabaculovirus genes. BLAST queries and phylogenetic inference confirmed that MyunNPV-KY310 is most closely related to the alphabaculovirus Leucania separata nucleopolyhedrovirus isolate AH1, which infects Mythimna separata. In contrast, MyunNPV-KY310 did not exhibit a close relationship with Mythimna unipuncta nucleopolyhedrovirus isolate #7, an alphabaculovirus from the same host species. MyunNPV-KY310 lacks the gp64 envelope glycoprotein, which is a characteristic of group II alphabaculoviruses. However, this virus and five other alphabaculoviruses lacking gp64 are placed outside the group I and group II clades in core gene phylogenies, further demonstrating that viruses of genus Alphabaculovirus do not occur in two monophyletic clades. Potential instances of MyunNPV-KY310 ORFs arising by horizontal transfer were detected. Although there are now genome sequences of four different baculoviruses from M. unipuncta, comparison of their genome sequences provides little insight into the genetic basis for their host specificity.

Autographa californica Multiple Nucleopolyhedrovirus AC83 is a Per Os Infectivity Factor (PIF) Protein Required for Occlusion-Derived Virus (ODV) and Budded Virus Nucleocapsid Assembly as well as Assembly of the PIF Complex in ODV Envelopes.

Baculovirus occlusion-derived virus (ODV) initiates infection of lepidopteran larval hosts by binding to the midgut epithelia, which is mediated by per os infectivity factors (PIFs). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes seven PIF proteins, of which PIF1 to PIF4 form a core complex in ODV envelopes to which PIF0 and PIF6 loosely associate. Deletion of any pif gene results in ODV being unable to bind or enter midgut cells. AC83 also associates with the PIF complex, and this study further analyzed its role in oral infectivity to determine if it is a PIF protein. It had been proposed that AC83 possesses a chitin binding domain that enables transit through the peritrophic matrix; however, no chitin binding activity has ever been demonstrated. AC83 has been reported to be found only in the ODV envelopes, but in contrast, the Orgyia pseudotsugata MNPV AC83 homolog is associated with both ODV nucleocapsids and envelopes. In addition, unlike known pif genes, deletion of ac83 eliminates nucleocapsid formation. We propose a new model for AC83 function and show AC83 is associated with both ODV nucleocapsids and envelopes. We also further define the domain required for nucleocapsid assembly. The cysteine-rich region of AC83 is also shown not to be a chitin binding domain but a zinc finger domain required for the recruitment or assembly of the PIF complex to ODV envelopes. As such, AC83 has all the properties of a PIF protein and should be considered PIF8. In addition, pif7 (ac110) is reported as the 38th baculovirus core gene.IMPORTANCE ODV is essential for the per os infectivity of the baculovirus AcMNPV. To initiate infection, ODV binds to microvilli of lepidopteran midgut cells, a process which requires a group of seven virion envelope proteins called PIFs. In this study, we reexamined the function of AC83, a protein that copurifies with the ODV PIFs, to determine its role in the oral infection process. A zinc finger domain was identified and a new model for AC83 function was proposed. In contrast to previous studies, AC83 was found to be physically located in both the envelope and nucleocapsid of ODV. By deletion analysis, the AC83 domain required for nucleocapsid assembly was more finely delineated. We show that AC83 is required for PIF complex formation and conclude that it is a true per os infectivity factor and should be called PIF8.

The complete genome sequence of a third distinct baculovirus isolated from the true armyworm, Mythimna unipuncta, contains two copies of the lef-7 gene.

A baculovirus isolate from a USDA Forest Service collection was characterized by electron microscopy and analysis of its genome sequence. The isolate, formerly referred to as Pseudoletia (Mythimna) sp. nucleopolyhedrovirus #7 (MyspNPV#7), was determined by barcoding PCR to derive from the host species Mythimna unipuncta (true armyworm) and was renamed Mythimna unipuncta nucleopolyhedrovirus #7 (MyunNPV#7). The occlusion bodies (OBs) and virions exhibited a size and morphology typical for OBs produced by the species of genus Alphabaculovirus, with occlusion-derived virions consisting of 2-5 nucleocapsids within a single envelope. The MyunNPV#7 genome was determined to be 148,482 bp with a 48.58% G+C nucleotide distribution. A total of 159 ORFs of 150 bp or larger were annotated in the genome sequence, including the 38 core genes of family Baculoviridae. The genome contained six homologous repeat regions (hrs) consisting of multiple copies of a 34-bp imperfect palindrome. Phylogenetic inference from concatenated baculovirus core gene amino acid sequence alignments placed MyunNPV#7 with group II alphabaculoviruses isolated from other armyworm and cutworm host species of lepidopteran family Noctuidae. MyunNPV#7 could be distinguished from other viruses in this group on the basis of differences in gene content and order. Pairwise nucleotide distances suggested that MyunNPV#7 represents a distinct species in Alphabaculovirus. The MyunNPV#7 genome was found to contain two copies of the late expression factor-7 (lef-7) gene, a feature not reported for any other baculovirus genome to date. Both copies of lef-7 encoded an F-box domain, which is required for the function of LEF-7 in baculovirus DNA replication.

Baculoviruses require an intact ODV entry-complex to resist proteolytic degradation of per os infectivity factors by co-occluded proteases from the larval host.

Baculoviruses orally infect caterpillars in the form of occlusion-derived viruses (ODVs). The ODV-envelope contains a number of proteins which are essential for oral infectivity, called per os infectivity factors (PIFs). Most of these PIFs are involved in the formation of an ODV-entry complex that consists of a stable core, formed by PIF1, PIF2, PIF3 and PIF4, and the more loosely associated PIFs P74 (PIF0) and P95 (PIF8). PIF1, PIF2 and PIF3 are essential for formation of the stable core, whereas deletion of the pif4 gene results in the formation of a smaller complex. P74 is not needed for formation of the stable core. We show here in larva-derived ODVs of the Autographa californica multicapsid nucleopolyhedrovirus that PIF-proteins are degraded by host-derived proteases after deletion of a single pif-gene. Constituents of the stable core-complex appeared to be more resistant to proteases as part of the complex than as monomer, as in ODVs of a p74 deletion mutant only the stable core was found but no PIF monomers. When the stable core lacks PIF4, it lost its proteolytic resistance as the resulting smaller core complex was degraded in a pif4 deletion mutant. We also identified PIF6 as a loosely associated component of the entry complex that appeared nevertheless important for the proteolytic resistance of the stable core, which was degraded after deletion of pif6. We conclude from these results that an intact entry-complex in the ODV-envelope is prerequisite for proteolytic resistance of PIF-proteins under the alkaline conditions of the larval midgut.

Microscopic investigation of AcMNPV infection in the Trichoplusia ni midgut.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the type species for the genus Alphabaculovirus in the family Baculoviridae. In nature, AcMNPV infection begins with ingestion of viral occlusion bodies (OBs) from which occlusion-derived viruses (ODV) are released to infect midgut cells. This study explored the early stages of Trichoplusia ni midgut infection using recombinant viruses expressing green fluorescent protein (GFP) and/or a VP39-mCherry fusion protein under the control of early and late promoters, respectively. Using a recombinant ie1:GFP virus, the anterior midgut region was identified as the predominant site for primary infection. Infection of midguts using the GFP-VP39mCherry-dual labelled recombinant virus revealed that active viral replication and cell-to-cell spread was required for the formation of infection foci and the subsequent spread to uninfected midgut cells and tracheoblasts. The spread of the infection from primary infected cells to secondary cells within the midgut was shown to be dependent upon the membrane fusion protein GP64.

Combining RNA-seq and proteomic profiling to identify seminal fluid proteins in the migratory grasshopper Melanoplus sanguinipes (F).

BACKGROUND: Seminal fluid proteins control many aspects of fertilization and in turn, they play a key role in post-mating sexual selection and possibly reproductive isolation. Because effective proteome profiling relies on the availability of high-quality DNA reference databases, our knowledge of these proteins is still largely limited to model organisms with ample genetic resources. New advances in sequencing technology allow for the rapid characterization of transcriptomes at low cost. By combining high throughput RNA-seq and shotgun proteomic profiling, we have characterized the seminal fluid proteins secreted by the primary male accessory gland of the migratory grasshopper (Melanoplus sanguinipes), one of the main agricultural pests in central North America. RESULTS: Using RNA sequencing, we characterized the transcripts of ~ 8,100 genes expressed in the long hyaline tubules (LHT) of the accessory glands. Proteomic profiling identified 353 proteins expressed in the long hyaline tubules (LHT). Of special interest are seminal fluid proteins (SFPs), such as EJAC-SP, ACE and prostaglandin synthetases, which are known to regulate female oviposition in insects. CONCLUSIONS: Our study provides new insights into the proteomic components of male ejaculate in Orthopterans, and highlights several important patterns. First, the presence of proteins that lack predicted classical secretory tags in accessory gland proteomes is common in male accessory glands. Second, the products of a few highly expressed genes dominate the accessory gland secretions. Third, accessory gland transcriptomes are enriched for novel transcripts. Fourth, there is conservation of SFPs' functional classes across distantly related taxonomic groups with very different life histories, mating systems and sperm transferring mechanisms. The identified SFPs may serve as targets of future efforts to develop species- specific genetic control strategies.

Mamestra configurata nucleopolyhedrovirus-A transcriptome from infected host midgut.

Infection of an insect by a baculovirus occurs in two distinct phases, an initial infection of host midgut by occlusion-derived virions (ODVs) and subsequent systemic infection of other tissues by budded virions (BV). A vast majority of investigations of the infection process have been restricted to cell culture studies using BV that emulate the systemic phase of infection. This is one of the first studies to investigate baculovirus gene expression in ODV infected midgut cells. We have focused on the critical first phase of in vivo infection by Mamestra configurata nucleopolyhedrovirus-A in M. configurata larvae, using qPCR and RNAseq mass sequencing to measure virus gene expression in midgut cells. The earliest genes detected by each method had significant overlap, including known early genes as well as genes unique to MacoNPV-A and genes of unknown function. The RNAseq data also revealed a large range of expression levels across all ORFs, which could not be measured using qPCR. This dataset provides a first whole genome transcriptomic analysis of viral genes required for virus infection in vivo and will provide the basis for functionally analyzing specific genes that may be critical elements in baculovirus midgut infectivity and host range.

Analysis of the autographa californica multiple nucleopolyhedrovirus overlapping gene pair lef3 and ac68 reveals that AC68 is a per os infectivity factor and that LEF3 is critical, but not essential, for virus replication.

Autographa californica multiple nucleopolyhedrovirus ac68 is a core gene that overlaps lef3 which encodes the single-stranded DNA binding protein. A knockout (KO) virus lacking both lef3 and ac68 was generated (lef3-ac68 2×KO) to enable the functional study of ac68. To produce an ac68KO virus that did not impact lef3 expression, the lef3-ac68 2×KO virus was repaired with a DNA fragment containing lef3 and ac68, in which ac68 contained point mutations so that only LEF3 was expressed. Repair of lef3-ac68 2×KO with just ac68 generated an lef3KO virus. Analysis of the ac68KO virus showed that viral DNA replication and budded virus (BV) levels were unaffected compared to levels in the double-repair or wild-type (WT) control virus. Bioassay analyses of Trichoplusia ni larvae injected with BV directly into the hemolymph, bypassing the gut, showed no difference in mortality rates between the ac68KO and the WT viruses. However, in oral bioassays the ac68KO occlusion bodies failed to kill larvae. These results show that the core gene ac68 encodes a per os infectivity factor (pif6). The lef3KO virus was also analyzed, and virus replication was drastically reduced compared to WT virus, but very low levels of lef3KO virus DNA replication and BV production could be detected. In addition, in transfected cells P143 was transported to the nucleus in the absence of LEF3. This study therefore shows for the first time that even though the loss of LEF3 severely impairs virus replication, it is not absolutely essential for P143 nuclear import or viral replication.

Characterization of novel components of the baculovirus per os infectivity factor complex.

Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, and SF58, which might work in concert with previously known PIFs to facilitate ODV infection. In order to understand the ODV entry mechanism, the identification of all components of the PIF complex is crucial. Hence, the aim of this study was to identify additional components of the PIF complex. Coimmunoprecipitation (CoIP) combined with proteomic analysis was used to identify the components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex. PIF4 and P95 (AC83) were identified as components of the PIF complex while PIF5 was not, and this was confirmed with blue native PAGE and a second CoIP. Deletion of the pif4 gene impaired complex formation, but deletion of pif5 did not. Differentially denaturing SDS-PAGE further revealed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. P95 and P74 are more loosely associated with this complex. Three other proteins, AC5, AC68, and AC108 (homologue of SF58), were also found by the proteomic analysis to be associated with the PIF complex. Finally the functional significance of the PIF protein interactions is discussed.

The genome of a baculovirus isolated from Hemileuca sp. encodes a serpin ortholog.

The genome sequence of a baculovirus from Hemileuca sp. was determined. The genome is 140,633 kb, has a G+C content of 38.1 %, and encodes 137 putative open-reading frames over 50 amino acids. 126 of these ORFs showed similarity to other baculovirus genes in the database including all 37 core genes. Of the remaining 11 predicted genes, one is related to a lepidopteran serpin gene. This is the first report of a baculovirus encoding a member of this family of serine protease inhibitors, and to our knowledge the first report of a viral serpin outside the Poxviridae. The genome also contained three homologous repeat sequences. Phylogenetic analysis indicated that the virus is a group II Alphabaculovirus and belongs to a lineage that includes Orgyia leucostigma, Ectropis obliqua, Apocheima cinerarium, and Euproctis pseudoconspersa nucleopolyhedroviruses.

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.

Role of enhancin in Mamestra configurata nucleopolyhedrovirus virulence: selective degradation of host peritrophic matrix proteins.

To infect per os, baculovirus virions cross the peritrophic matrix (PM) to reach the midgut epithelium. Insect intestinal mucins (IIMs) are PM proteins that protect the PM and aid passage of the food bolus through the gut. Some baculoviruses, including Mamestra configurata nucleopolyhedrovirus (MacoNPV-A), encode metalloproteases, known as enhancins, that facilitate infection by degrading IIMs. We examined the interaction between MacoNPV-A enhancin and M. configurata IIMs both in vivo and in vitro. Per os inoculation of M. configurata larvae with MacoNPV-A occlusion bodies (OBs) resulted in the degradation of McIIM4 within 4 h of OB ingestion, while McIIM2 was unaffected. The PM recovered by 8 h post-inoculation. To investigate whether enhancin was responsible for the degradation of IIM, a recombinant Autographa californica multiple nucleopolyhedrovirus expressing MacoNPV enhancin (AcMNPV-enMP2) was constructed. Enhancin was found to be a component of occlusion-derived virions in AcMNPV-enMP2 and MacoNPV-A. In in vitro assays, McIIM4 was degraded after MacoNPV-A and AcMNPV-enMP2 treatments. Degradation of McIIM4 was inhibited by EDTA, a metalloprotease inhibitor, indicating that the degradation was due to enhancin activity. Thus, MacoNPV-A enhancin is able to degrade major structural PM proteins, but exhibits target substrate specificity.

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.

Horizontally transmitted parasitoid killing factor shapes insect defense to parasitoids.

Interkingdom competition occurs between hymenopteran parasitoids and insect viruses sharing the same insect hosts. It has been assumed that parasitoid larvae die with the death of the infected host or as result of competition for host resources. Here we describe a gene family, parasitoid killing factor (pkf), that encodes proteins toxic to parasitoids of the Microgastrinae group and determines parasitism success. Pkfs are found in several entomopathogenic DNA virus families and in some lepidopteran genomes. We provide evidence of equivalent and specific toxicity against endoparasites for PKFs found in entomopoxvirus, ascovirus, baculovirus, and Lepidoptera through a mechanism that elicits apoptosis in the cells of susceptible parasitoids. This highlights the evolutionary arms race between parasitoids, viruses, and their insect hosts.

Autographa californica multiple nucleopolyhedrovirus core gene ac96 encodes a per Os infectivity factor (PIF-4).

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac96 is a core gene, but its role in virus replication is still unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac96-null virus (vAc(96)(null)). Our analyses showed that the absence of ac96 does not affect budded virus (BV) production or viral DNA replication in infected Sf9 cells. Western blotting and confocal immunofluorescence analysis showed that AC96 is expressed in both the cytoplasm and the nucleus throughout infection. In addition, AC96 was detected in the envelope fractions of both BV and occlusion-derived virus. Injection of vAc(96)(null) BV into the hemocoel killed Trichoplusia ni larvae as efficiently as repaired and control viruses; however, vAc(96)(null) was unable to infect the midgut tissue of Trichoplusia ni larvae when inoculated per os. Therefore, the results of this study show that ac96 encodes a new per os infectivity factor (PIF-4).

Characterization of the Mamestra configurata (Lepidoptera: Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet, Brassica species, and protease inhibitor.

The midgut protease profiles from 5th instar Mamestra configurata larvae fed various diets (standard artificial diet, low protein diet, low protein diet with soybean trypsin inhibitor [SBTI], or Brassica napus) were characterized by one-dimensional enzymography in gelatin gels. The gut protease profile of larvae fed B. napus possessed protease activities of molecular masses of approximately 33 and 55 kDa, which were not present in the guts of larvae fed artificial diet. Similarly, larvae fed artificial diet had protease activities of molecular masses of approximately 21, 30, and 100 kDa that were absent in larvae fed B. napus. Protease profiles changed within 12 to 24 h after switching larvae from artificial diet to plant diet and vice versa. The gut protease profiles from larvae fed various other brassicaceous species and lines having different secondary metabolite profiles did not differ despite significant differences in larval growth rates on the different host plants. Genes encoding putative digestive proteolytic enzymes, including four carboxypeptidases, five aminopeptidases, and 48 serine proteases, were identified in cDNA libraries from 4th instar M. configurata midgut tissue. Many of the protease-encoding genes were expressed at similar levels on all diets; however, three chymoptrypsin-like genes (McSP23, McSP27, and McSP37) were expressed at much higher levels on standard artificial diet and diet containing SBTI as was the trypsin-like gene McSP34. The expression of the trypsin-like gene McSP50 was highest on B. napus. The adaptation of M. configurata digestive biochemistry to different diets is discussed in the context of the flexibility of polyphagous insects to changing diet sources.

Role of the peritrophic matrix in insect-pathogen interactions.

The peritrophic matrix (PM) is an acellular chitin and glycoprotein layer that lines the invertebrate midgut. The PM has long been considered a physical as well as a biochemical barrier, protecting the midgut epithelium from abrasive food particles, digestive enzymes and pathogens infectious per os. This short review will focus on the latter function, as a barrier to pathogens infectious per os. We focus on the evidence confirming the role of the PM as protective barrier against pathogenic microorganisms of insects, mainly bacteria and viruses, as well as the evolution of a variety of mechanisms used by pathogens to overcome the PM barrier.

Proteomics analysis of Trichoplusia ni midgut epithelial cell brush border membrane vesicles.

The insect midgut epithelium is composed of columnar, goblet, and regenerative cells. Columnar epithelial cells are the most abundant and have membrane protrusions that form the brush border membrane (BBM) on their apical side. These increase surface area available for the transport of nutrients, but also provide opportunities for interaction with xenobiotics such as pathogens, toxins and host plant allelochemicals. Recent improvements in proteomic and bioinformatics tools provided an opportunity to determine the proteome of the T. ni BBM in unprecedented detail. This study reports the identification of proteins from BBM vesicles (BBMVs) using single dimension polyacrylamide gel electrophoresis coupled with multi-dimensional protein identification technology. More than 3000 proteins were associated with the BBMV, of which 697 were predicted to possess either a signal peptide, at least one transmembrane domain or a GPI-anchor signal. Of these, bioinformatics analysis and manual curation predicted that 185 may be associated with the BBMV or epithelial cell plasma membrane. These are discussed with respect to their predicted functions, namely digestion, nutrient uptake, cell signaling, development, cell-cell interactions, and other functions. We believe this to be the most detailed proteomic analysis of the lepidopteran midgut epithelium membrane to date, which will provide information to better understand the biochemical, physiological and pathological processes taking place in the larval midgut.

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.