Targeting female reproduction in insects with biorational insecticides for pest management: a critical review with suggestions for future research.

Of the different approaches to pest control, use of juvenile hormone analogs (e.g. methoprene), molting hormone (20-hydroxyecdysone) analogs (e.g. tebufenozide) and chitin synthesis inhibitors (e.g. diflubenzuron) has dominated this field. Since they adversely interfere with the normal growth and development in one way or another, they have been collectively called as 'insect growth regulators' or IGRs. A lesser known fact is that they all have deleterious effects on reproduction as well as act as ovicides. The raison d'être for this review is to summarize what we have learnt during the last 3-4 decades in the use of these IGRs, how they affect insect reproduction and how we can apply this knowledge to control pest insects. Finally, we present, information on the state of the art use of molecular technologies such as RNAi and CRISPR/Cas9 applications for pest management targeting insect reproduction.

Molecular cloning and structural characterization of Ecdysis Triggering Hormone from Choristoneura fumiferana.

At the end of each stadium, insects undergo a precisely orchestrated process known as ecdysis which results in the replacement of the old cuticle with a new one. This physiological event is necessary to accommodate growth in arthropods since they have a rigid chitinous exoskeleton. Ecdysis is initiated by the direct action of Ecdysis Triggering Hormones on the central nervous system. Choristoneura fumiferana is a major defoliator of coniferous forests in Eastern North America. It is assumed that, studies on the ecdysis behavior of this pest might lead to the development of novel pest management strategies. Hence in this study, the cDNA of CfETH was cloned. The open reading frame of the cDNA sequence was found to encode three putative peptides viz., Pre-Ecdysis Triggering Hormone (PETH), Ecdysis Triggering Hormone (ETH), and Ecdysis Triggering Hormone Associated Peptide (ETH-AP). The CfETH transcript was detected in the epidermal tissue of larval and pupal stages, but not in eggs and adults. In order to explore the structural conformation of ETH, ab initio modelling and Molecular Dynamics (MD) Simulations were performed. Further, a library of insecticides was generated and virtual screening was performed to identify the compounds displaying high binding capacity to ETH.

An epidermis-specific chitin synthase CDNA in Choristoneura fumiferana: cloning, characterization, developmental and hormonal-regulated expression.

Chitin synthase catalyzes chitin synthesis in the exoskeleton, tracheal system and gut during insect development. A chitin synthase 1 (CfCHS1) cDNA was identified and cloned from the spruce budworm, Choristoneura fumiferana. The CfCHS1 cDNA is 5,300 bp in length and codes a 1,564-amino acid protein with a molecular mass of 178 kDa. The deduced protein contains 16 transmembrane helixes in its domains A and C. The single copy CfCHS1 gene expressed during each of the larval molts from the 3rd to the 6th instar. The gene expressed highly and periodically in the epidermis during each of molts, whereas no transcripts were detected in the midgut and fat body. 20-hydroxyecdysone and the ecdysone agonist RH5992 suppressed CfCHS1 expression, whereas the juvenile hormone analog methoprene induced CfCHS1 expression. These results implicate that CfCHS1 is involved in the chitin synthase and new chitin formation during molting in the insect.

Diapause disruption with tebufenozide for early-instar control of the spruce budworm, Choristoneura fumiferana.

In North America, the eastern spruce budworm, Choristoneura fumiferana Clem., is an important coniferous pest against which tebufenozide has proven effective as a control product. By acting as an ecdysone agonist, tebufenozide can induce precocious moulting in late (fifth-sixth) instars but can also be carried over to the next generation owing to its persistence on foliage. The authors conducted laboratory experiments on first-instar larvae treated with tebufenozide dissolved in acetone. Larvae exposed to doses equal to or above 0.1 microg cm(-2) displayed precocious moulting in the second instar after hibernaculum spinning, which effectively disrupted diapause. Larger doses induced moulting in first instars. Evidence is provided that this dose-response difference is related to whether or not an effective dose of tebufenozide is ingested by the first instar prior to the peak of moulting hormone (20-hydroxyecdysone) in first instars. Doses ineffective to kill first instars are carried over to the second instar, where they induce a precocious moult. This type of response to tebufenozide is dependent on the presence of a moulting machinery (the EcR-USP receptor complex) that is ready for ecdysone transduction. Interestingly, ecdysone levels are low in second instars, as measured by a radioimmunoassay, which suggests that diapause in spruce budworm is maintained by a suppression of ecdysone production. Thus, diapause disruption by tebufenozide may well provide an alternative control strategy for this important pest.

DNA versus protein immunisation for production of monoclonal antibodies against Choristoneura fumiferana ecdysone receptor (CfEcR).

The full-length ecdysone receptor cDNA of Choristoneura fumiferana (CfEcR-B) was cloned into bacterial expression systems and the recombinant protein was expressed either with a His-tag (His-EcR-B) or glutathione-S-transferase (GST) fusion (GST-EcR-B). The His-EcR-B was expressed mostly as insoluble aggregates, while the GST-EcR-B was partially soluble and could be purified using affinity chromatography. Mice were then immunised with the purified GST-EcR-B protein. Due to the time-consuming protein expression and purification procedures and the solubility problem of the recombinant protein, we also inserted the full-length CfEcR-B cDNA into the mammalian DNA vaccine expression vector, pVAC1-mcs for DNA immunisation. In vitro expression of CfEcR-B in mammalian cells transfected with the pVAC-EcR-B plasmid was confirmed prior to the delivery of the DNA vaccine into mice. The anti-CfEcR-B MAbs generated from both DNA and protein vaccines were characterised and shown to recognise native CfEcR-B protein induced by 20E in CF-203 insect cells. DNA immunisation was shown to overcome the solubility problem of the bacterial expressed EcR and created a more direct route for monoclonal antibody production for this receptor protein obviating the need for EcR expression and purification to generate the antigen.

Heterodimerization of ecdysone receptor and ultraspiracle on symmetric and asymmetric response elements.

Heterodimerization of nuclear receptors is facilitated by the interaction of two dimerization interfaces: one spanning the DNA-binding (C domain) region and the adjacent hinge (D domain) region, and the other in the ligand-binding (E domain) region. Ultraspiracle (USP) heterodimerizes with ecdysone receptor (EcR) and this complex participates in ecdysone signal transduction. The natural ecdysone response elements (EcREs) discovered so far are asymmetric elements composed of either imperfect palindromes or direct repeats. However, gel mobility shift assays have shown that both symmetric (perfect palindromes) and asymmetric (imperfect palindromes and direct repeats) elements can bind to the EcR/USP complex. Therefore, we analyzed EcR/USP domains involved in heterodimerization on different types of response elements (RE). Gel shift assays using full-length and truncated EcR and USP proteins showed that heterodimerization of these two proteins in the presence of asymmetric RE (DR4 and the natural EcRE hsp27) requires both dimerization interfaces present in CD and E domains of both proteins. In contrast, the dimerization interface present in the E domain of either EcR or USP was not essential for heterodimerization on symmetric RE such as PAL1 or IR1. We conclude that the use of heterodimerization interfaces present in CD and E domains of EcR/USP depends on the nature of response elements they bind to.

Sequence and organization of the Neodiprion lecontei nucleopolyhedrovirus genome.

All fully sequenced baculovirus genomes, with the exception of the dipteran Culex nigripalpus nucleopolyhedrovirus (CuniNPV), have previously been from Lepidoptera. This study reports the sequencing and characterization of a hymenopteran baculovirus, Neodiprion lecontei nucleopolyhedrovirus (NeleNPV), from the redheaded pine sawfly. NeleNPV has the smallest genome so far published (81,755 bp) and has a GC content of only 33.3%. It contains 89 potential open reading frames, 43 with baculovirus homologues, 6 identified by conserved domains, and 1 with homology to a densovirus structural protein. Average amino acid identity of homologues ranged from 19.7% with CuniNPV to 24.9% with Spodoptera exigua nucleopolyhedrovirus. The conserved set of baculovirus genes has dropped to 29, since NeleNPV lacks an F protein homologue (ac23/ld130). NeleNPV contains 12 conserved lepidopteran baculovirus genes, including that for DNA binding protein, late expression factor 11 (lef-11), polyhedrin, occlusion derived virus envelope protein-18 (odv-e18), p40, and p45, but lacks 21 others, including lef-3, me53, immediate early gene-1, lef-6, pp31, odv-e66, few polyhedra 25k, odv-e25, protein kinase-1, fibroblast growth factor, and ubiquitin. The lack of identified baculovirus homologues may be due to difficulties in identification, differences in host-virus interactions, or other genes performing similar functions. Gene parity plots showed limited colinearity of NeleNPV with other baculoviruses, and phylogenetic analysis indicates that NeleNPV may have existed before the lepidopteran nucleopolyhedrovirus and granulovirus divergence. The creation of two new Baculoviridae genera to fit hymenopteran and dipteran baculoviruses may be necessary.

Identification and characterization of a juvenile hormone (JH) response region in the JH esterase gene from the spruce budworm, Choristoneura fumiferana.

Using a differential display of mRNA technique we discovered that the juvenile hormone (JH) esterase gene (Cfjhe) from Choristoneura fumiferana is directly induced by juvenile hormone I (JH I), and the JH I induction is suppressed by 20-hydroxyecdysone (20E). To study the mechanism of action of these two hormones in the regulation of expression of this gene, we cloned the 1270-bp promoter region of the Cfjhe gene and identified a 30-bp region that is located between -604 and -574 and is sufficient to support both JH I induction and 20E suppression. This 30-bp region contains two conserved hormone response element half-sites separated by a 4-nucleotide spacer similar to the direct repeat 4 element and is designated as a putative juvenile hormone response element (JHRE). In CF-203 cells, a luciferase reporter placed under the control of JHRE and a minimal promoter was induced by JH I in a dose- and time-dependent manner. Moreover, 20E suppressed this JH I-induced luciferase activity in a dose- and time-dependent manner. Nuclear proteins isolated from JH I-treated CF-203 cells bound to JHRE and the binding was competed by a 100-fold excess of the cold probe but not by 100-fold excess of double-stranded oligonucleotides of unrelated sequence. JH I induced/modified nuclear proteins prior to their binding to JHRE and 20E suppressed this JH I induction/modification. These results suggest that the 30-bp JHRE identified in the Cfjhe gene promoter is sufficient to support JH induction and 20E suppression of the Cfjhe gene.

Morphological and molecular effects of 20-hydroxyecdysone and its agonist tebufenozide on CF-203, a midgut-derived cell line from the spruce budworm, Choristoneura fumiferana.

The morphological and molecular responses of a midgut-derived cell line of the spruce budworm, Choristoneura fumiferana, to 20-hydroxyecdysone (20E) and the nonsteroidal ecdysone agonist, tebufenozide (RH-5992), were investigated. The cells responded to these compounds by clumping, generating filamentous extensions, increased mortality and expression of the transcription factor, Choristoneura hormone receptor 3 (CHR3). This cell line can be used as a model system to study the mode of action of ecdysone and its agonists. With subsequent passaging in ecdysteroid-containing medium, the degree of clumping increased and the clumping could not be reversed by subculturing in ecdysteroid-free medium. Cell numbers of the adapted cell lines in 20E and RH-5992 containing media were not significantly decreased, compared to the control, but both cell lines accumulated less (14)C-labeled RH-5992 and lost the capability of expressing CHR3 in response to these compounds. Taken together, the cell lines appeared to develop a mechanism to adapt to the toxic effects of these compounds. Arch. Insect Biochem. Physiol. 55:68-78, 2004.

Ecdysone agonists: mechanism and importance in controlling insect pests of agriculture and forestry.

Molting is the result of the expression of a cascade of genes that is sequentially both up and down-regulated by the molting hormone, 20-hydroxyecdysone (20E), which is secreted as a pulse during each instar. Benzoyl hydrazine analogs of 20E act like the native molting hormone at the molecular level by binding with the ecdysone receptor complex and transactivating a succession of molt initiating transcription factors that, in turn, induce the expression of a group of molt-related genes. As a result of the expression of these up-regulated genes, the larva undergoes apolysis and head capsule slippage and takes on the appearance of the pharate larva. However, unlike 20E, which is cleared at this juncture, allowing the down-regulated genes to be expressed, these synthetic analogs bind strongly to the receptors and remain in place and repress all the down-regulatory genes such as the ones necessary for cuticle elaboration, sclerotization, and ecdysis resulting in a developmental arrest in this state. As a result, the treated larva goes into a precocious incomplete molt that is lethal. Two of the analogs, tebufenozide and methoxyfenozide, are lepidopteran specific and have good control potential for open feeding larvae that ingest this material while a third one, halofenozide, acts on coleopteran larvae. Since they specifically act through an insect receptor complex, they have little or no effect on non-target species, making them environmentally attractive pest control agents. Some insects, however, show resistance to these analogs and this could be, inter alia, due to an ATP Binding Cassette Transporter like system that selectively pumps out the analogs.

Development of SCAR markers for the DNA-based detection of the Asian long-horned beetle, Anoplophora glabripennis (Motschulsky).

DNA markers were identified for the molecular detection of the Asian long-horned beetle (ALB), Anoplophora glabripennis (Mot.), based on sequence characterized amplified regions (SCARs) derived from random amplified polymorphic DNA (RAPD) fragments. A 2,740-bp DNA fragment that was present only in ALB and not in other Cerambycids was identified after screening 230 random primers in a PCR-based assay system. Three pairs of nested 22-mer oligonucleotide primers were designed on the basis of the sequence of this fragment and were used to perform diagnostic PCR. The first pair of primers (SCAR1) amplified a single 745-bp fragment of ALB DNA, but this did not differentiate ALB from other species. The other two pairs of SCAR primers (SCAR2 and SCAR3) amplified bands of 1,237- and 2,720-bp, respectively, that were capable of differentiating ALB from other closely related non-native and native Cerambycids, such as A. chinensis (Forster), A. malasiaca (Thomson), A. nobilis (Ganglbauer), Monochamus scutellatus (Say), Plectrodera scalator (Fab), Saperda tridentata (Olivier), and Graphisurus fasciatus (Degeer). The latter two SCAR markers could be amplified using DNA extracted from body parts of ALB such as the wing, the leg, and the antennae as well as tissues from all the developmental stages including the egg, larva, pupa, and adult. These markers were also capable of identifying ALB using the DNA extracted from frass. Our results demonstrate that the SCAR markers we have identified can be used for unambiguously identifying ALB from other closely related Cerambycids using a simple PCR procedure.