Occlusion-derived virions (ODVs) of the nucleopolyhedrovirus of Culex nigripalpus (CuniNPV) were purified by Ludox density gradient ultracentrifugation, and the proteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were identified by using Edman sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry, nanoelectrospray quadrupole time-of-flight mass spectrometry, or a combination of these methods. Half of the 44 polypeptide sequences identified in this analysis were unique open reading frames (ORFs) encoded by the CuniNPV genome and did not show similarity to any other sequences present in protein databases. Of the 22 polypeptides that showed similarities to other baculovirus-encoded proteins, only 17 sequences have previously been identified as structural proteins. The newly identified CuniNPV structural proteins cun058, cun059, cun087, cun106, and cun109 are homologues of Autographa californica nucleopolyhedrovirus (AcMNPV) ORFs 68, 62, 98, 81, and 2, respectively. The products of four genes, namely, lef-1 (cun045), alkaline exonuclease (cun054), helicase (cun089), and DNA polymerase (cun091), were not detected in the CuniNPV ODV preparations. These four genes are conserved among all annotated baculovirus genomes, and their homologues have been detected in the ODV of AcMNPV.

Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in South America has increased the risk of this species invading North America. Morphological similarities make differentiation of H. armigera from the native Helicoverpa zea (Boddie) difficult. Characteristics of adult male genitalia and nucleotide sequence differences in mitochondrial DNA are two of the currently available methods to differentiate these two species. However, current methods are likely too slow to be employed as rapid detection methods. In this study, conserved differences in the internal transcribed spacer 1 (ITS1) of the ribosomal RNA genes were used to develop species-specific oligonucleotide primers that amplified ITS1 fragments of 147 and 334 bp from H. armigera and H. zea, respectively. An amplicon (83 bp) from a conserved region of 18S ribosomal RNA subunit served as a positive control. Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species. In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified. Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs. The high resolution melt analysis combined with rapid DNA extraction could be used as an inexpensive method to genetically differentiate large numbers of H. armigera and H. zea using readily available reagents.

Heliothine pests such as the tobacco budworm, Heliothis virescens (F.), pose a significant threat to production of a variety of crops and ornamental plants and are models for developmental and physiological studies. The efforts to develop new control measures for H. virescens, as well as its use as a relevant biological model, are hampered by a lack of molecular resources. The present work demonstrates the utility of next-generation sequencing technologies for rapid molecular resource generation from this species for which lacks a sequenced genome. In order to amass a de novo transcriptome for this moth, transcript sequences generated from Illumina, Roche 454, and Sanger sequencing platforms were merged into a single de novo transcriptome assembly. This pooling strategy allowed a thorough sampling of transcripts produced under diverse environmental conditions, developmental stages, tissues, and infections with entomopathogens used for biological control, to provide the most complete transcriptome to date for this species. Over 138 million reads from the three platforms were assembled into the final set of 63,648 contigs. Of these, 29,978 had significant BLAST scores indicating orthologous relationships to transcripts of other insect species, with the top-hit species being the monarch butterfly (Danaus plexippus) and silkworm (Bombyx mori). Among identified H. virescens orthologs were immune effectors, signal transduction pathways, olfactory receptors, hormone biosynthetic pathways, peptide hormones and their receptors, digestive enzymes, and insecticide resistance enzymes. As an example, we demonstrate the utility of this transcriptomic resource to study gene expression profiling of larval midguts and detect transcripts of putative Bacillus thuringiensis (Bt) Cry toxin receptors. The substantial molecular resources described in this study will facilitate development of H. virescens as a relevant biological model for functional genomics and for new biological experimentation needed to develop efficient control efforts for this and related Noctuid pest moths.

Seasonal climatic shifts create peripheral habitats that alternate between habitable and uninhabitable for migratory species. Such dynamic peripheral habitats are potential sites where migratory species could evolve high genetic diversity resulting from convergence of immigrants from multiple regionally distant areas. Migrant populations of Helicoverpa zea (Boddie) captured during two different seasons were assessed for genetic structure using microsatellite markers and for host plant type using stable carbon isotope analysis. Individuals (N = 568) were genotyped and divided into 13 putative populations based on collection site and time. Fixation indices (F‐statistics), analysis of molecular variance (AMOVA), and discriminant analysis of principal components (DAPC) were used to examine within and among population genetic variation. Mean number of alleles per locus was 10.25 (± 3.2 SD), and allelic richness ranged from 2.38 to 5.13 (± 3.2 SD). The observed and expected heterozygosity ranged from 0.07 to 0.48 and 0.08 to 0.62, respectively. Low F_ST (0.01 to 0.02) and high F_IS (0.08 to 0.33) values suggest captured migrants originated from breeding populations with different allele frequencies. We postulate that high genetic diversity within migrant populations and low genetic differentiation among migrant populations of H. zea are the result of asymmetrical immigration due to the high dispersal and reproductive behavior of H. zea, which may hinder the adaptation and establishment of H. zea to peripheral habitat. These findings highlight the importance of assessing peripheral population structure in relation to ecological and evolutionary dynamics of this and other highly reproductive and dispersive species.

Among various genome editing tools available for functional genomic studies, reagents based on clustered regularly interspersed palindromic repeats (CRISPR) have gained popularity due to ease and versatility. CRISPR reagents consist of ribonucleoprotein (RNP) complexes formed by combining guide RNA (gRNA) that target specific genomics regions and a CRISPR associated nuclease (Cas). The gRNA targeting specific gene sequences may be delivered as a plasmid construct that needs to be transcribed or as a synthetic RNA. The Cas nuclease can be introduced as a plasmid construct, mRNA, or purified protein. The efficiency of target editing is dependent on intrinsic factors specific to each species, the target gene sequence, and the delivery methods of CRISPR gRNA and the Cas nuclease. Although intrinsic factors affecting genome editing may not be altered in most experiments, the delivery method for CRISPR/Cas reagents can be optimized to produce the best results. In this study, the efficiency of genome editing by CRISPR/Cas system in the bollworm, Helicoverpa zea (Boddie), was evaluated using ribonucleoprotein (RNP) complexes assembled by binding synthetic gRNA with purified Cas9 nuclease engineered with nuclear localization signals to target the vermillion (eye color) gene. Mutation rates of adults emerging from embryos microinjected with 1, 2, or 4 μM RNP complexes were compared using replicated experiments. Embryos injected with 2 or 4 μM RNP complexes displayed significantly higher mutation rates (>88%) in surviving adults compared to those injected with 1 μM. The hatch rate in embryos injected with RNP complexes and with injection buffer only (mock injections) was reduced by 19.8(±5.2)% compared to noninjected control embryos, but did not differ significantly between injected embryos. Evaluation of potential off-target sites in H. zea genome did not identify any mutations. This study demonstrates that in vitro assembled synthetic RNP complexes can be used to obtain high genome editing rates in a reproducible manner in functional genomics or genetic manipulation studies.

Interpreting variable laboratory measurements of Helicoverpa zea Boddie susceptibility to toxins from Bacillus thuringiensis Berliner (Bt) has been challenging due to a lack of clear evidence to document declining field control. Research that links laboratory measurements of susceptibility to survival on Bt crops is vital for accurate characterization and any subsequent response to the occurrence of an implied H. zea resistance event. In this study, H. zea survival and the resultant damage to plant fruiting structures of non-Bt, Bollgard II, and Bollgard III cottons from two insect colonies with differing levels of laboratory susceptibility to Bt toxins were evaluated in large field cages. Laboratory bioassays revealed resistance ratios of 2.04 and 622.14 between the two H. zea colonies for Dipel DF and Cry1Ac, respectively. Differences between the two H. zea colonies measured via bioassays with Bollgard II and Bollgard III cotton leaf tissue in the laboratory were not statistically discernable. However, there was 17.6% and 5.3% lower larval mortality in Bollgard II and Bollgard III for the feral relative to the laboratory colony of H. zea, respectively. Although H. zea larval numbers in cages infested with the laboratory susceptible colony did not differ between the two Bt cottons, there were fewer larvae per 25 plants in Bollgard III than in Bollgard II cotton in cages containing tolerant insects. Cages infested with tolerant H. zea moths had higher numbers of total larvae than those containing the laboratory susceptible colony in both Bollgard II and Bollgard III cottons. Bollgard II and Bollgard III cottons received 77.4% and 82.7% more total damage to total plant fruiting structures in cages infested with tolerant insects relative to those containing the laboratory susceptible colony. The damage inflicted to fruiting structures on Bollgard III cotton by a feral H. zea colony with decreased measurements of laboratory susceptibility to Dipel DF and Cry1Ac indicate that the addition of Vip3A to third generation Bt cottons may not provide sufficient control in situations where infestations levels are high.