Erratum to: Genomic innovations, transcriptional plasticity and gene loss underlying the evolution and divergence of two highly polyphagous and invasive Helicoverpa pest species

Upon publication of the original article [1], it was noticed that Dr Papanicolaou’s surname was spelt incorrectly. The correct spelling is “Papanicolaou”, as shown in the author list of this erratum.Additional co-authors: A. Anderson, S. Asgari, P. G. Board, A. Bretschneider, P. M. Campbell, T. Chertemps, J. T. Christeller, C. W. Coppin, S. J. Downes, G. Duan, C. A. Farnsworth, R. T. Good, L. B. Han, Y. C. Han, K. Hatje, I. Horne, Y. P. Huang, D. S. T. Hughes, E. Jacquin-Joly, W. James, S. Jhangiani, M. Kollmar, S. S. Kuwar, S. Li, N-Y. Liu, M. T. Maibeche, J. R. Miller, N. Montagne, T. Perry, J. Qu, S. V. Song, G. G. Sutton, H. Vogel, B. P. Walenz, W. Xu, H-J. Zhang, Z. Zou, P. Batterham, O. R. Edwards, R. Feyereisen, R. A. Gibbs, D. G. Heckel, A. McGrath, C. Robin, S. E. Scherer, K. C. Worley, Y. D. W

Identification Of The Western Tarnished Plant Bug (\u3ci\u3eLygus Hesperus\u3c/i\u3e) Olfactory Co-Receptor Orco: Expression Profile And Confirmation Of Atypical Membrane Topology

Lygus hesperus (western tarnished plant bug) is an agronomically important pest species of numerous cropping systems. Similar to other insects, a critical component underlying behaviors is the perception and discrimination of olfactory cues. Consequently, the molecular basis of olfaction in this species is of interest. To begin to address this issue, we utilized homology-based PCR as a commonly accepted abbreviation but if necessary it is polymerase chain reaction methods to identify the L. hesperus olfactory receptor co-receptor (Orco) ortholog, a receptor that has been shown to be essential for olfaction. The L. hesperus Orco (LhOrco) shares significant sequence homology with known Orco proteins in other insects. Parallel experiments using the sympatric sister species, Lygus lineolaris (tarnished plant bug), revealed that the Lygus Orco gene was completely conserved. Surprisingly, a majority of the membrane topology prediction algorithms used in the study predicted LhOrco to have both the N and C terminus intracellular. In vitro immunofluorescent microscopy experiments designed to probe the membrane topology of transiently expressed LhOrco, however, refuted those predictions and confirmed that the protein adopts the inverted topology (intracellular N terminus and an extracellular C terminus) characteristic of Orco proteins. RT-PCR analyses indicated that LhOrco transcripts are predominantly expressed in adult antennae and to a lesser degree in traditionally nonolfactory chemosensory tissues of the proboscis and legs. Expression is not developmentally regulated because transcripts were detected in all nymphal stages as well as eggs. Taken together, the results suggest that LhOrco likely plays a critical role in mediating L. hesperus odorant perception and discrimination

Entomopathogenicity of Ascomycete Fungus <i>Cordyceps militaris</i> on the Cotton Bollworm, <i>Helicoverpa zea</i> (Boddie) (Lepidoptera: Noctuidae)

This study investigated the exposure of the cotton bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) to a novel pathogenic fungal agent historically associated with human medicinal value, a commercial strain of Cordyceps militaris ((L.) Fr.) Vuill. (Hypocreales). A series of comparative studies were conducted to evaluate the efficacy of two different exposure methods using four concentrations (n × 109, n × 108, n × 107, n × 106) of C. militaris, where n × 109 provided a concentration of approximately 420 ± 37 spores per mm2 with 398 ± 28 viable spores. Survival of cotton bollworms of all stages was not affected by C. militaris at any concentration 1 d post-exposure. The greatest reduction in survival and highest sporulation rates were observed primarily on or after 7 d post-exposure for early instars (first and second). Significant declines in the survival of early instars were observed for all concentrations at 7 d, and 95% mortality by 10 d, with the exception of the fifth instars that experienced a less severe reduction in survival (35%) when exposed to any concentrations used in the study. Survival of late instars (third to fifth) ranged from 44% to 68% on day 10, while adult survival was near 99% across the duration of the experiment. The relatively narrow range observed for both the lethal concentration and sporulation of second, third, and fifth instar cotton bollworms exposed to the C. militaris strain may demonstrate potential field application for control of larval populations of cotton bollworms

Unique venom proteins from Solenopsis invicta x Solenopsis richteri hybrid fire ants

The Solenopsis venom protein 2 transcript was amplified, sequenced, probed, and analyzed from Solenopsis invicta x Solenopsis richteri hybrid ant colonies (hybrids) collected from across Tennessee to determine the extent of introgression of each parent allele (Solenopsis invicta venom protein 2 [Soli2] and Solenopsis richteri venom protein 2 [Solr2]). Chemotaxonomic analyses of venom alkaloids and cuticular hydrocarbons were used to categorize hybrid colonies and their relative relatedness to each parent species. Hybrid colonies were chosen randomly from each chemotaxonomic hybridization category, including “very near S. richteri,” “near S. richteri,” “near S. invicta,” and “very near S. invicta.” Lateral flow immunoassays for detection of the Soli2 and Solr2 venom proteins were largely in agreement with the chemotaxonomic analyses for the very near S. richteri (100% Solr2) and very near S. invicta (80% Soli2, 20% Soli2 + Solr2 detected in the sample) groups, while Soli2 and Solr2 were reported in 60% and 40% in the near S. invicta and near S. richteri chemotaxonomic groups. Analysis of transcripts from the hybrid colonies revealed a sequence with 100% identity to Soli2 (GenBank Accession L09560) and three unique sequences, which we identify as Solenopsis hybrid venom protein 2 (Solh2; GenBank Accession MT150127), Solenopsis hybrid truncated venom protein 2 (Solh2Tr97; Genbank Accession MT150129), and Solenopsis richteri venom protein 2, D to A change at position 69 (Solr2A69; GenBank Accession MT150128). The predicted open reading frame for Solh2 and Solh2Tr97 revealed sequences unique to hybrid ants, with Solh2Tr97an alternatively spliced form. A third unique sequence, Solr2A69, is likely the correct sequence for Solr2, which appears to have been published previously with a sequencing error (GenBank Accession P35776)

CRISPR/Cas9 mediated high efficiency knockout of the eye color gene <i>Vermillion</i> in <i>Helicoverpa zea</i> (Boddie)

<div><p>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, <i>Helicoverpa zea</i> (Boddie), was evaluated using ribonucleoprotein (RNP) complexes assembled by binding synthetic gRNA with purified Cas9 nuclease engineered with nuclear localization signals to target the <i>vermillion</i> (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 <i>H</i>. <i>zea</i> genome did not identify any mutations. This study demonstrates that <i>in vitro</i> assembled synthetic RNP complexes can be used to obtain high genome editing rates in a reproducible manner in functional genomics or genetic manipulation studies.</p></div

Average number pf eggs injected, average hatch count, corrected hatch count, percentage of adults emerged from larvae, and the percentage of mutants from control, mock injected (with injection buffer), and 1, 2, and 4 μM ribonucleoprotein complexes targeting exon 6 of the tryptophan 2,3-dioxygenase gene in <i>Helicoverpa zea</i>.

<p>Average number pf eggs injected, average hatch count, corrected hatch count, percentage of adults emerged from larvae, and the percentage of mutants from control, mock injected (with injection buffer), and 1, 2, and 4 μM ribonucleoprotein complexes targeting exon 6 of the tryptophan 2,3-dioxygenase gene in <i>Helicoverpa zea</i>.</p

Genetic Knockouts Indicate That the ABCC2 Protein in the Bollworm Helicoverpa zea Is Not a Major Receptor for the Cry1Ac Insecticidal Protein

Members of the insect ATP binding cassette transporter subfamily C2 (ABCC2) in several moth species are known as receptors for the Cry1Ac insecticidal protein from Bacillus thuringiensis (Bt). Mutations that abolish the functional domains of ABCC2 are known to cause resistance to Cry1Ac, although the reported levels of resistance vary widely depending on insect species. In this study, the function of the ABCC2 gene as a putative Cry1Ac receptor in Helicoverpa zea, a major pest of over 300 crops, was evaluated using CRISPR/Cas9 to progressively eliminate different functional ABCC2 domains. Results from bioassays with edited insect lines support that mutations in ABCC2 were associated with Cry1Ac resistance ratios (RR) ranging from 7.3- to 39.8-fold. No significant differences in susceptibility to Cry1Ac were detected between H. zea with partial or complete ABCC2 knockout, although the highest levels of tolerance were observed when knocking out half of ABCC2. Based on &gt;500–1000-fold RRs reported in similar studies for closely related moth species, the low RRs observed in H. zea knockouts support that ABCC2 is not a major Cry1Ac receptor in this insect

cDNA sequence, gene organization, and the CRISPR RNA (crRNA) designed for editing targets in the exon 6 of the tryptophan 2,3-dioxygenase (TO) gene in <i>Helicoverpa zea</i>.

<p><b>(A)</b> Open reading frame of the cDNA sequence is in upper case letters and the amino acids of the tryptophan dioxygenase domain of the TO are indicated in bold text. Target sequences (protospacer) for two crRNA are shown in bold red text. Protospacer adjacent motif (PAM) and double-strand DNA break site for each target site are shown by underlined text and green arrows, respectively (accession number MG976796). (B) The TO gene consists of 10 exons (red boxes) ranging from 73 to 199 bp and introns ranging from 475 to 4323 bp in size, spanning 17,396 bp. The nucleotide sequences of crRNA targets are shown in red text and protospacer adjacent motifs (PAM) are underlined (accession numberMF598173).</p

A representative sample of nucleotide sequences of the eye color mutants and wild type eye color insects from CRISPR injection experiments.

<p>Sequences IM 1 through IM 24 and IW 1 through IW 24 are from insects with mutant and wild type eye color phenotype, respectively, from CRISPR/Cas9 injections. Partial nucleotide sequences of exon 6 of the TO gene from the reference sequence (Ref) and uninjected wild type (WT) are also shown. Nucleotides identical to the exon 6 of the reference sequence (accession: MF598173) are indicated by a dot (.) and alignment gaps are shown by a hyphen (-). Nucleotides that differ from the reference are indicated on each sequence. Target (protospacer) sequences for which crRNA were designed are underlined with dotted lines and protospacer adjacent motifs (PAM) are underlined with a solid line.</p