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.

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.

A look into Colorado potato beetle lipid metabolism through the lens of lipid storage droplet proteins.

The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) inflicts serious damage to potato plants by feeding ravenously on their leaves. Adult L.decemlineata have a photoperiod-induced dormancy response, also known as diapause, which allows them to survive severe winter conditions by digging into soil. Most insects that undergo diapause accumulate abundant lipid reserves prior to diapause and utilize most of them during the diapause. This process is likely to be governed by the interplay of lipid storage droplet proteins (LSDs), also known as perilipins, with the help of other proteins. Here, genes encoding L. decemlineata LSD1 and LSD2 were identified. Both were expressed primarily in the fat body with LdLSD1 and LdLSD2 being primarily expressed in adult and larval stages, respectively. LdLSD1 was up-regulated in starving larvae, while LdLSD2 was primarily expressed in feeding larvae. The expression pattern of LdLSD1 in adults during feeding, diapause and post-diapause contrasted to the total body fat levels, while the expression pattern of LdLSD2 was positively correlated with total body fat levels. RNA interference (RNAi) of LdLSD2 in larvae suggested a core role for LSD2 in the protection/assembly of storage lipids as this treatment reduced overall lipid droplet volume. These data shed light on the functions of these proteins in L. decemlineata and their roles in both diapause and during starvation.

MacoNPV baculovirus midgut-specific gene expression during infection of the bertha armyworm, Mamestra configurata.

Baculoviruses have two forms, occlusion derived virus (ODV) which is responsible for primary infection in host midgut tissue and budded virus (BV), which infects all other host tissues during secondary infection. This study examined the primary infection by ODV of midgut cells of bertha armyworm Mamestra configurata fourth instar larvae and measured the expression of viral genes over a time course of infection. Both digital PCR and RNA sequencing methods showed the profile of transcription to be different from those produced by AcMNPV BV infection of in vitro cell cultures. This included having unique collections of genes expressed early, as well as much greater late gene expression of p6.9 and much reduced expression of polh and p10. These differences likely reflect characteristics unique to the critical step of in vivo midgut cell infection, and provide insights into the processes that regulate viral gene expression in different host tissues.

Identification of the Mamestra configurata (Lepidoptera: Noctuidae) peritrophic matrix proteins and enzymes involved in peritrophic matrix chitin metabolism.

The peritrophic matrix (PM) is essential for insect digestive system physiology as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata (Lepidoptera: Noctuidae), a major pest of cruciferous oilseed crops in North America. Liquid chromatography-tandem mass spectrometry analyses of the PM identified 82 proteins classified as: (i) peritrophins, including a new class with a CBDIII domain; (ii) enzymes involved in chitin modification (chitin deacetylases), digestion (serine proteases, aminopeptidases, carboxypeptidases, lipases and α-amylase) or other reactions (ß-1,3-glucanase, alkaline phosphatase, dsRNase, astacin, pantetheinase); (iii) a heterogenous group consisting of polycalin, REPATs, serpin, C-Type lectin and Lsti99/Lsti201 and 3 novel proteins without known orthologs. The genes encoding PM proteins were expressed predominantly in the midgut. cDNAs encoding chitin synthase-2 (McCHS-2), chitinase (McCHI), and ß-N-acetylglucosaminidase (McNAG) enzymes, involved in PM chitin metabolism, were also identified. McCHS-2 expression was specific to the midgut indicating that it is responsible for chitin synthesis in the PM, the only chitinous material in the midgut. In contrast, the genes encoding the chitinolytic enzymes were expressed in multiple tissues. McCHS-2, McCHI, and McNAG were expressed in the midgut of feeding larvae, and NAG activity was present in the PM. This information was used to generate an updated model of the lepidopteran PM architecture.

Spatial and temporal synthesis of Mamestra configurata peritrophic matrix through a larval stadium.

The structure and synthesis of the Mamestra configurata peritrophic matrix (PM) was examined at various time points during a larval stadium. Bright field and confocal fluorescence microscopy revealed major differences between the PM of feeding and molting larvae. The PM from feeding larvae was thinner and composed of approximately 5-10 layers. In contrast, mid-molt larvae had a chitinaceaous PM composed of multiple thick layers which filled most of the midgut lumen. PM synthesis initiates in the anterior midgut, based on the expression of genes encoding chitin synthase-2 (CHS-2), coincident with the incorporation of the major structural PM proteins (McIIM1, McIIM2 and McPM1). This is followed by reinforcement with other PM proteins (McIIM3 and McIIM4) as it moves toward the posterior of the midgut. Chitin deacetylase (McCDA1) was associated only with the anterior PM. Collectively, these findings indicate that the structural properties of the PM differ along the length of the midgut. Genes encoding chitinolytic enzymes (McCHI and McNAG) were expressed and exochitinase activity was present when the PM had degraded (pre-molt) and when the new PM was forming (mid-molt), indicating that they are involved in either PM turnover and/or maintenance dependent upon the stage.

In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system.

The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix (PM), composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double-stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins (insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1) and the chitin biosynthetic or modifying enzymes (chitin synthase-B and chitin deacetylase 1) in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing (by 24 h) for the gene encoding chitin deacetylase 1 and a slower rate of silencing (by 72 h) for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase-B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double-stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.

Spatial distribution of tree species governs the spatio-temporal interaction of leaf area index and soil moisture across a forested landscape.

Quantifying coupled spatio-temporal dynamics of phenology and hydrology and understanding underlying processes is a fundamental challenge in ecohydrology. While variation in phenology and factors influencing it have attracted the attention of ecologists for a long time, the influence of biodiversity on coupled dynamics of phenology and hydrology across a landscape is largely untested. We measured leaf area index (L) and volumetric soil water content (θ) on a co-located spatial grid to characterize forest phenology and hydrology across a forested catchment in central Pennsylvania during 2010. We used hierarchical Bayesian modeling to quantify spatio-temporal patterns of L and θ. Our results suggest that the spatial distribution of tree species across the landscape created unique spatio-temporal patterns of L, which created patterns of water demand reflected in variable soil moisture across space and time. We found a lag of about 11 days between increase in L and decline in θ. Vegetation and soil moisture become increasingly homogenized and coupled from leaf-onset to maturity but heterogeneous and uncoupled from leaf maturity to senescence. Our results provide insight into spatio-temporal coupling between biodiversity and soil hydrology that is useful to enhance ecohydrological modeling in humid temperate forests.

Expression patterns of genes encoding proteins with peritrophin A domains and protein localization in Mamestra configurata.

Genes encoding three proteins (McPPAD1-3) with peritrophin A chitin-binding domains (PADs) were identified from a Mamestra configurata larval midgut cDNA library. In addition to midgut, McPPAD1-3 and a previously identified gene encoding the peritrophin, McPM1, were expressed in foregut, hindgut, Malpighian tubules, tracheae, fat body and cuticle; however, the corresponding McPPAD proteins exhibited different localization patterns. McPPAD1 was restricted to the digestive tract and Malpighian tubules, McPPAD2 to Malpighian tubules, and McPPAD3 to the foregut, midgut, hindgut, tracheae and cuticle. Protein fold recognition analysis using tachycitin as a guide structure modelled the McPPAD1 PADs, but not McPPAD2 or McPPAD3 PADs. The McPPAD1 PADs were predicted to contain three anti-parallel ß-sheets and a hevein-like fold that form a chitin-binding pocket containing two hydrophobic R-groups in a sandwich-like orientation.

Isolation and characterization of plaque-purified strains of Malacosoma disstria Nucleopolyhedrovirus.

Seven plaque-purified genotypic variants or strains, derived from a previously described field isolate of the Malacosoma disstria Nucleopolyhedrovirus (MadiNPV) from Alberta populations of forest tent caterpillar, were characterized based on distinctive restriction endonuclease fragment patterns. Two strains, MadiNPV-pp3 and MadiNPV-pp11, were selected for further characterization, as they represented strains producing high and low budded virus (BV) titres, respectively, in the M. disstria cell line UA-Md203. Analysis of restriction endonuclease fragment profiles indicated the genomes differed significantly in size, 133.8 +/- 2.4 kb for MadiNPV-pp3 and 118.1 +/- 3.5 kb for MadiNPV-pp11. These strains were characterized based on their BV production in three different cell lines derived from M. disstria haemocytes. Compared with MadiNPV-pp11, MadiNPV-pp3 produced two- to three-fold more BVs in UA-Md203 and 210 other cell lines; however, BV production was only marginally higher for MadiNPV-pp3 in the UA-Md221 cell line. Similarly, the yield of polyhedral inclusion bodies was significantly higher for MadiNPV-pp3 in UA-Md203 and 210 cell lines than for MadiNPV-pp11 but not in the UA-Md221 cell line. This data, although derived from a limited number of cell lines, suggested MadiNPV-pp3 may have a broader tissue tropism than MadiNPV-pp11.

Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: characterization, cDNA cloning, and expressed sequence tag analysis.

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.

Molecular characterization of Brassica napus NAC domain transcriptional activators induced in response to biotic and abiotic stress.

Subtractive expressed sequence tag analysis and screening of cDNA libraries derived from Brassica napus leaves subjected to mechanical wounding, flea beetle feeding or cold temperatures revealed eight genes encoding NAC-domain transcription factors. The genes were found to be differentially regulated in response to biotic and abiotic stresses including wounding, insect feeding, Sclerotinia sclerotiorum infection, cold shock and dehydration. Five BnNAC proteins were orthologous to Arabidopsis thaliana ATAF1 or ATAF2 and gave rise to developmental abnormalities similar to the A. thaliana nam and cuc mutants when expressed ectopically in A. thaliana. Transgenic lines expressing BnNAC14, exhibited large leaves, thickened stems and hyper-developed lateral root systems similar to that observed with A. thaliana NAC1, but also were delayed in bolting and lacked an apical dominant tap root. Several of the BnNAC proteins were capable of activating gene expression in yeast and recognized an element within the CaMV35S promoter. A yeast two-hybrid screen revealed that BnNAC14 interacted with other select BnNAC proteins in vitro and identified an additional BnNAC gene, BnNAC485. The protein interaction and transcriptional activation domains were mapped by deletion analysis.