Knockout of juvenile hormone receptor, Methoprene-tolerant, induces black larval phenotype in the yellow fever mosquito, Aedes aegypti.

The yellow fever mosquito, Aedes aegypti, vectors human pathogens. Juvenile hormones (JH) control almost every aspect of an insect's life, and JH analogs are currently used to control mosquito larvae. Since RNA interference does not work efficiently during the larval stages of this insect, JH regulation of larval development and mode of action of JH analogs are not well studied. To overcome this limitation, we used a multiple single guide RNA-based CRISPR/Cas9 genome-editing method to knockout the methoprene-tolerant (Met) gene coding for a JH receptor. The Met knockout larvae exhibited a black larval phenotype during the L3 (third instar larvae) and L4 (fourth instar larvae) stages and died before pupation. However, Met knockout did not affect embryonic development or the L1 and L2 stages. Microscopy studies revealed the precocious synthesis of a dark pupal cuticle during the L3 and L4 stages. Gene expression analysis showed that Krüppel homolog 1, a key transcription factor in JH action, was down-regulated, but genes coding for proteins involved in melanization, pupal and adult cuticle synthesis, and blood meal digestion in adults were up-regulated in L4 Met mutants. These data suggest that, during the L3 and L4 stages, Met mediates JH suppression of pupal/adult genes involved in the synthesis and melanization of the cuticle and blood meal digestion. These results help to advance our knowledge of JH regulation of larval development and the mode of action of JH analogs in Ae. aegypti.

Double-stranded RNA binding protein, Staufen, is required for the initiation of RNAi in coleopteran insects.

RNA interference (RNAi) is being used to develop methods to control pests and disease vectors. RNAi is robust and systemic in coleopteran insects but is quite variable in other insects. The determinants of efficient RNAi in coleopterans, as well as its potential mechanisms of resistance, are not known. RNAi screen identified a double-stranded RNA binding protein (StaufenC) as a major player in RNAi. StaufenC homologs have been identified in only coleopteran insects. Experiments in two coleopteran insects, Leptinotarsa decemlineata and Tribolium castaneum, showed the requirement of StaufenC for RNAi, especially for processing of double-stranded RNA (dsRNA) to small interfering RNA. RNAi-resistant cells were selected by exposing L. decemlineata, Lepd-SL1 cells to the inhibitor of apoptosis 1 dsRNA for multiple generations. The resistant cells showed lower levels of StaufenC expression compared with its expression in susceptible cells. These studies showed that coleopteran-specific StaufenC is required for RNAi and is a potential target for RNAi resistance. The data included in this article will help improve RNAi in noncoleopteran insects and manage RNAi resistance in coleopteran insects.

The effect of E93 knockdown on female reproduction in the red flour beetle, Tribolium castaneum.

The E93 transcription factor is a member of helix-turn-helix transcription factor family containing a Pip-squeak motif. This ecdysone primary response gene was identified as a regulator of cell death in Drosophila melanogaster where it is involved in ecdysone-induced autophagy and caspase activity that mediate degeneration of larval tissues during metamorphosis from larva to pupa. However, its function in adult insects is not well studied. To study E93 function in the red flour beetle, Tribolium castaneum, double-stranded RNA (dsRNA) targeting E93 (dsE93) was injected into newly emerged adults. Knockdown of E93 caused a decrease in the synthesis of vitellogenin (Vg), oocyte development, and egg-laying. Sequencing of RNA isolated from adults injected with dsE93 and control dsmalE (dsRNA targeting Escherichia coli malE gene) followed by differential gene expression analysis showed upregulation of genes involved in the metabolism of reserved nutrients. E93 knockdown induced changes in gene expression resulted in a decrease in Vg synthesis in the fat body and oocyte maturation in ovaries. Mating experiments showed that females injected with dsE93 did not lay eggs. Knockdown of E93 caused a reduction in the number and size of lipid droplets in the fat body when compared with that in control beetles injected with dsmalE. These data suggest that during the first 2-3 days after the emergence of adult females, E93 suppresses genes coding for enzymes that metabolize reserved nutrients until initiation of vitellogenesis and oogenesis.

RNA interference-mediated control of cigarette beetle, Lasioderma serricorne.

The cigarette beetle (CB; Lasioderma serricorne) is a pest on many stored products including tobacco. Fumigation is the common control method currently used. However, the options for controlling this pest are limited, due to resistance issues and phasing out of currently used chemical insecticides. Here, we evaluated RNA interference (RNAi) as a potential method for controlling the CB. RNA isolated from different stages was sequenced and assembled into a transcriptome. The CB RNA sequences showed the highest homology with those in the red flour beetle, Tribolium castaneum. Orthologs of proteins known to function in RNAi pathway were identified in the CB transcriptome, suggesting that RNAi may work well in this insect. Also, 32 P-labeled double-stranded RNA (dsRNA) injected into CB larvae and adults was processed to small interference RNAs. We selected 12 genes that were shown to be the effective RNAi targets in T. castaneum and other insects and identified orthologs of them in the CB by searching its transcriptome. Injection of dsRNA targeting genes coding for GAWKY, Kinesin, Sec23, SNF7, and 26S proteasome subunit 6B into the CB larvae caused 100% mortality. Feeding dsRNA targeting SNF7 and 26S proteasome subunit 6B by sucrose droplet assay induced more than 90% mortality, which is 1.8 times higher than the mortality induced by dsGFP control (53%). These data demonstrate an efficient RNAi response in CB, suggesting that RNAi could be developed as an efficient method to control this pest.

Double-stranded RNAs targeting inhibitor of apoptosis gene show no significant cross-species activity.

RNA interference (RNAi) has become an integral part of mainstream research due to its versatility and ease of use. However, the potential nontarget effects associated with double-stranded RNAs (dsRNA) are poorly understood. To explore this, we used dsRNAs targeting the inhibitor of apoptosis (iap) gene from nine insect species and assayed their possible nontarget effects. For each assay, we used a control (dsRNA targeting the gene coding for green fluorescent protein, GFP) and a species-specific dsRNA targeting nine iap genes in insect species to evaluate target gene knockdown efficiency, apoptosis phenotype in cells and mortality in insects. Our results revealed that dsIAP efficiently knocks down iap gene expression and induces apoptosis phenotype and mortality in target insect species. In contrast, no significant knockdown of the iap gene expression, apoptosis phenotypes, or mortality were detected in cell lines developed from nontarget insects or nontarget insects treated with dsIAPs. Interestingly, even among closely related insects such as stink bugs, Nezara viridula, Halyomorpha halys, and Murgantia histrionica, with substantial sequence similarity among iap genes from these insects, no significant nontarget effects of dsIAP were observed under the conditions tested. These data demonstrate no significant nontarget effects for dsIAPs and suggest that the threat of nontarget effects of RNAi technology may not be substantial.

Transport of orally delivered dsRNA in southern green stink bug, Nezara viridula.

The southern green stink bug (SGSB, Nezara viridula) is an emerging polyphagous pest in many regions of the world. RNA interference (RNAi) is a valuable method for understanding gene function and holds great potential for pest management. However, RNAi efficiency is variable among insects and the differences in transport of double-stranded RNA (dsRNA) are one of the major factors that contribute to this variability. In this study, Cy3 labeled dsRNA was used to track the transport of dsRNA in SGSB tissues. Cy3_dsRNA was detected in the hemocytes, fat body (FB), epidermis, and midgut tissues at 24-72 hr after injection. Orally delivered Cy3_dsRNA or Cypher-5E labeled dsRNA was mostly detected in the midgut and a few signals were detected in parts of the FB and epidermis. Both injected and fed Cy3_dsRNA showed stronger signals in SGSB tissues when compared to Cy3_siRNA (small interfering RNA) or Cy3_shRNA (short hairpin RNA). dsRNA targeting the gene for a vacuolar-sorting protein, SNF7, induced higher knockdown of the target gene and greater SGSB mortality compared to siRNA or shRNA targeting this gene. 32 P-labeled dsRNA injected into SGSB was processed into siRNA, but fed 32 P-labeled dsRNA was not efficiently processed into siRNA. These data suggest that transport of orally delivered dsRNA across the midgut epithelium is not efficient in SGSB which may contribute to variable RNAi efficiency. Targeting genes expressed in the midgut rather than other tissues and using dsRNA instead of siRNA or shRNA would be more effective for RNAi-mediated control of this pest.

Inhibitor of apoptosis is an effective target gene for RNAi-mediated control of Colorado potato beetle, Leptinotarsa decemlineata.

The Colorado potato beetle (CPB; Leptinotarsa decemlineata) is one of the most notorious and difficult to control pests of potato and other solanaceous crops in North America. This insect has evolved a remarkable ability to detoxify both plant and synthetic toxins, allowing it to feed on solanaceous plants containing toxic alkaloids and to develop resistance to synthetic chemicals used for its control. RNA interference (RNAi) is a natural mechanism that evolved as an immune response to double-stranded RNA (dsRNA) viruses where dsRNA triggers silencing of target gene expression. RNAi is being developed as a method to control CPB. Here, we evaluated four CPB-specific genes to identify targets for RNAi-mediated control of this insect. Out of the four dsRNAs evaluated in CPB larvae and adults, dsIAP (dsRNA targeting inhibitor of apoptosis, iap gene) performed better than dsActin, dsHSP70, and dsDynamin in inducing larval mortality. However, in adults, the mortality induced by dsActin is significantly higher than the mortality induced by dsIAP, dsHSP70, and dsDynamin. Interestingly, a combination of dsIAP and dsActin performed better than either dsIAP or dsActin alone by inducing feeding inhibition in 24 hr and mortality in 48 hr in larvae. When the dsIAP and dsActin were expressed in the Escherichia coli HT115 strain and applied as a heat-killed bacterial spray on potato plants, it protected the plants from CPB damage. These studies show that the combination of dsIAP and dsActin shows promise as an insecticide to control CPB.

Conserved Genes in Highly Regenerative Metazoans Are Associated with Planarian Regeneration.

Metazoan species depict a wide spectrum of regeneration ability which calls into question the evolutionary origins of the underlying processes. Since species with high regeneration ability are widely distributed throughout metazoans, there is a possibility that the metazoan ancestor had an underlying common molecular mechanism. Early metazoans like sponges possess high regenerative ability, but, due to the large differences they have with Cnidaria and Bilateria regarding symmetry and neuronal systems, it can be inferred that this regenerative ability is different. We hypothesized that the last common ancestor of Cnidaria and Bilateria possessed remarkable regenerative ability which was lost during evolution. We separated Cnidaria and Bilateria into three classes possessing whole-body regenerating, high regenerative ability, and low regenerative ability. Using a multiway BLAST and gene phylogeny approach, we identified genes conserved in whole-body regenerating species and lost in low regenerative ability species and labeled them Cnidaria and Bilaterian regeneration genes. Through transcription factor analysis, we identified that Cnidaria and Bilaterian regeneration genes were associated with an overabundance of homeodomain regulatory elements. RNA interference of Cnidaria and Bilaterian regeneration genes resulted in loss of regeneration phenotype for HRJDa, HRJDb, DUF21, DISP3, and ARMR genes. We observed that DUF21 knockdown was highly lethal in the early stages of regeneration indicating a potential role in wound response. Also, HRJDa, HRJDb, DISP3, and ARMR knockdown showed loss of regeneration phenotype after second amputation. The results strongly correlate with their respective RNA-seq profiles. We propose that Cnidaria and Bilaterian regeneration genes play a major role in regeneration across highly regenerative Cnidaria and Bilateria.

Identification and characterization of highly active promoters from the fall armyworm, Spodoptera frugiperda.

The cell lines derived from the fall armyworm (FAW), Spodoptera frugiperda, have been widely used for production of recombinant proteins for applications in both basic research and applications in medicine and agriculture. Promoters from the nucleopolyhedrovirus (NPV) are commonly used in these expression systems. These promoters have some limitations, which may be overcome by using promoters of genes from S. frugiperda. However, information on these promoters is not available. We identified several highly expressed genes from the transcriptomes of S. frugiperda midgut, fat body, epidermis, ovarian cell line (Sf9), and a midgut cell line (Sf17). The activity of potential promoters of 21 highly expressed genes was evaluated in Sf9 and Sf17 cells. Two of these promoters, SfHSC70-P1780 and SfPub-P2009, showed higher activity than commonly used hr5/ie1 (baculovirus enhancer element, hr5 and immediate early gene 1, ie1) promoter. Interestingly, the activity of these two promoters increased after adding hr5 enhancer element. The hr5/SfPub-P2009 promoter performance was evaluated by expressing an exogenous P450 protein in Sf9 cells using a plasmid-based expression system. The activity of this promoter was also evaluated in the FAW by expressing green fluorescence protein using the baculovirus expression system. In both cases, the hr5/SfPub-P2009 promoter performed better than the commonly used hr5/ie1 promoter. These strong endogenous promoters will be useful for studies in S. frugiperda and other lepidopteran insects for multiple applications, including protein expression, genome editing, and transgenic insects.

Genome editing in the fall armyworm, Spodoptera frugiperda: Multiple sgRNA/Cas9 method for identification of knockouts in one generation.

The CRISPR/Cas9 system is an efficient genome editing method that can be used in functional genomics research. The fall armyworm, Spodoptera frugiperda, is a serious agricultural pest that has spread over most of the world. However, very little information is available on functional genomics for this insect. We performed CRISPR/Cas9-mediated site-specific mutagenesis of three target genes: two marker genes [Biogenesis of lysosome-related organelles complex 1 subunit 2 (BLOS2) and tryptophan 2, 3-dioxygenase (TO)], and a developmental gene, E93 (a key ecdysone-induced transcription factor that promotes adult development). The knockouts (KO) of BLOS2, TO and E93 induced translucent mosaic integument, olive eye color, and larval-pupal intermediate phenotypes, respectively. Sequencing RNA isolated from wild-type and E93 KO insects showed that E93 promotes adult development by influencing the expression of the genes coding for transcription factor, Krüppel homolog 1, the pupal specifier, Broad-Complex, serine proteases, and heat shock proteins. Often, gene-edited insects display mosaicism in which only a fraction of the cells are edited as intended, and establishing a homozygous line is both costly and time-consuming. To overcome these limitations, a method to completely KO the target gene in S. frugiperda by injecting the Cas9 protein and multiple sgRNAs targeting one exon of the E93 gene into embryos was developed. Ten percent of the G0 larvae exhibited larval-pupal intermediates. The mutations were confirmed by T7E1 assay, and the mutation frequency was determined as >80%. Complete KO of the E93 gene was achieved in one generation using the multiple sgRNA method, demonstrating a powerful approach to improve genome editing in lepidopteran and other non-model insects.