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Rasoolizadeh A, Goulet MC, Guay JF, Cloutier C, Michaud D. J Insect Physiol. 2018;106:125-133. doi: 10.1016/j.jinsphys.2017.03.001.

Herbivorous insects use complex protease complements to process plant proteins, useful to adjust their digestive functions to the plant diet and to elude the antidigestive effects of dietary protease inhibitors. We here assessed whether basic profiles and diet-related adjustments of the midgut protease complement may vary among populations of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). Two laboratory colonies of this insect were used as models, derived from insect samples collected in potato fields ∼1200 km distant from each other in North America. Synchronized 4th-instar larvae reared on potato were kept on this plant, or switched to tomato or eggplant, to compare their midgut cathepsin activities and content of intestain Cys proteases under different diet regimes. Cathepsin D activity, cathepsin L activity, cathepsin B activity and total intestain content shortly after larval molting on potato leaves were about two times lower in one population compared to the other. By comparison, cathepsin D activity, cathepsin B activity, total intestain content and relative abundance of the most prominent intestain families were similar in the two populations after three days regardless of the plant diet, unlike cathepsin L activity and less prominent intestain families showing population-associated variability. Variation in Cys protease profiles translated into the differential efficiency of a Cys protease inhibitor, tomato cystatin SlCYS8, to inhibit cathepsin L activity in midgut extracts of the two insect groups. Despite quantitative differences, SlCYS8 single variants engineered to strongly inhibit Cys proteases showed improved potency against cathepsin L activity of either population. These data suggest the feasibility of designing cystatins to control L. decemlineata that are effective against different populations of this insect. They underline, on the other hand, the practical relevance of considering natural variability of the protease complement among L. decemlineata target populations, eventually determinant in the success or failure of cystatin-based control strategies on a large-scale basis.

Shi JF, QK Sun, LL Mu, WC Guo, GQ Li. Appl Entomol Zool. 2017;52(1):37-49. doi: 10.1007/s13355-016-0451-2.

Trehalose is used primarily for the metabolic production of ATP energy and carbon sources. Its metabolic availability is regulated by trehalase (TRE). In the present paper, three TRE genes were identified in Leptinotarsa decemlineata (Say), and designated LdTRE1a, LdTRE1b, and LdTRE2 according to their Tribolium homologues. Within the first, second, and third larval instars, the expression levels of LdTREs were high just before and right after the molt, and were low in the mid instar. In the fourth larval instar, two peaks occurred at 24 h after ecdysis and at the wandering stage. In vitro culture of midguts and an in vivo bioassay revealed that 20-hydroxyecdysone (20E) stimulated the expression of the three LdTREs. Conversely, a reduction of 20E by RNA interference (RNAi) of a prothoracicotropic hormone receptor gene LdTorso and an ecdysteroidogenesis gene LdSHD repressed the expression of the three LdTREs. Moreover, disruption of 20E signaling by RNAi of LdEcR, LdE75, and LdFTZ-F1 reduced the transcript levels of the three LdTREs. Similarly, in vitro culture and an in vivo bioassay showed that exogenous juvenile hormone (JH) or JH analogue methoprene and pyriproxyfen activated LdTREs expression. An increase of endogenous JH by RNAi of an allatostatin gene LdAS-C enhanced the transcription. In contrast, a decrease in JH by RNAi of a JH biosynthesis gene LdJHAMT downregulated the transcription. Moreover, knockdown of LdILP2 repressed the expression of the three LdTREs. The content of hemolymph trehalose was increased while the concentration of glucose was decreased. It seems that the transcription of LdTRE1a, LdTRE1b, and LdTRE2 is regulated by 20E, JH, and ILP signaling pathways in Leptinotarsa decemlineata.

Meng Q, Q Xu, P Deng, K. Fu, W Guo, G Li Insect Biochem Mol Biol. 2018;97:1-9. doi: 10.1016/j.ibmb.2018.04.003.

In the tobacco hornworm Manduca sexta, juvenile hormone (JH) is critical for the control of species-specific size. However, whether the basic helix-loop-helix/Per-Arnt-Sim domain receptor methoprene-tolerant (Met) is involved remains unconfirmed. In the present paper, we found that RNA interference (RNAi)-aided knockdown of Met gene (LdMet) lowered the larval and pupal fresh weights and shortened the larval development period in the Colorado potato beetle Leptinotarsa decemlineata. Dietary introduction of JH into the LdMet RNAi larvae rescued neither the decreased weights nor the reduced development phase, even though JH ingestion by control larvae extended developmental time and caused large pupae. Moreover, the transcript levels of five genes involved in prothoracicotropic hormone and cap 'n' collar isoform C/Kelch-like ECH associated protein 1 pathways were upregulated in the LdMet silenced larvae. Ecdysteroidogenesis was thereby activated; 20-hydroxyecdysone (20E) titer was increased; and 20E signaling pathway was elicited in the LdMet RNAi larvae. Therefore, JH, acting through its receptor Met, inhibits PTTH production and release before the attainment of critical weight. Once the critical weight is reached, JH production and release are averted; and the hemolymph JH is removed. The elimination of JH allows the brain to release PTTH. PTTH subsequently stimulates ecdysteroid biosynthesis and release to start larval-pupal transition in L. decemlineata.

Morin MD, Lyons PJ, Crapoulet N, Boquel S, Morin PJ. International Journal of Molecular Sciences. 2017;18(12):2728. doi: 10.3390/ijms18122728.

The Colorado potato beetle (Leptinotarsa decemlineata (Say)) is a significant pest of potato plants that has been controlled for more than two decades by neonicotinoid imidacloprid. L. decemlineata can develop resistance to this agent even though the molecular mechanisms underlying this resistance are not well characterized. MicroRNAs (miRNAs) are short ribonucleic acids that have been linked to response to various insecticides in several insect models. Unfortunately, the information is lacking regarding differentially expressed miRNAs following imidacloprid treatment in L. decemlineata. In this study, next-generation sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were used to identify modulated miRNAs in imidacloprid-treated versus untreated L. decemlineata. This approach identified 33 differentially expressed miRNAs between the two experimental conditions. Of interest, miR-282 and miR-989, miRNAs previously shown to be modulated by imidacloprid in other insects, and miR-100, a miRNA associated with regulation of cytochrome P450 expression, were significantly modulated in imidacloprid-treated beetles. Overall, this work presents the first report of a miRNA signature associated with imidacloprid exposure in L. decemlineata using a high-throughput approach. It also reveals interesting miRNA candidates that potentially underly imidacloprid response in this insect pest.

Wang Y., Y. Li, G. Li, P. Wan, C. Li. J Econ Entomol. 2019;112(2):912-923. doi: 10.1093/jee/toy396.

Structural cuticular proteins (CPs) are the primary components of insect cuticle, linings of salivary gland, foregut, hindgut and tracheae, and midgut peritrophic membrane. Variation of CPs in insect cuticle can cause penetration resistance to insecticides. Moreover, depletion of specific CP by RNA interference may be a suitable way for the development of potential pest control traits. Leptinotarsa decemlineata (Say) CPs are poorly characterized at present, and therefore, we mined the genome and transcriptome data to better annotate and classify L. decemlineata CPs in this study, by comparison with the annotated CPs of Tribolium castaneum Browse (Coleoptera: Tenebrionidae). We identified 175 CP genes. Except one miscellaneous CP with an 18-amino acid motif, these CPs were classified into 7 families based on motifs and phylogenetic analyses (CPs with a Rebers and Riddiford motif, CPR; CPs analogous to peritrophins, CPAP3 and CPAP1; CPs with a tweedle motif, TWDL; CPs with a 44-amino acid motif, CPF; CPs that are CPF-like, CPFL; and CPs with two to three copies of C-X5-C motif, CPCFC). Leptinotarsa decemlineata CPRs could be categorized into three subfamilies: RR-1 (50), RR-2 (85), and RR-3 (2). The RR-1 proteins had an additional motif with a conserved YTADENGF sequence. The RR-2 members possessed a conserved RDGDVVKG region and three copes of G-x(3)-VV. Few genes were found in TWDL (9), CPAP1 (9), CPAP3 (8), CPF (5), CPFL (4), and CPCFC (2) families. The findings provide valuable information to explore molecular modes of penetration resistance to insecticides and to develop dsRNA-based control method in L. decemlineata.

Wu JJ, Chen ZC, Wang YW, Fu KY, Guo WC, Li GQ. Insect Mol Biol. 2019;28(1):52-64.

Insect chitin deacetylases (CDAs) are carbohydrate esterases that catalyze N-deacetylation of chitin to generate chitosan, a process essential for chitin organization and compactness during the formation of extracellular chitinous structure. Here we identified two CDA2 splice variants (LdCDA2a and LdCDA2b) in Leptinotarsa decemlineata. Both splices were abundantly expressed in larval foregut, rectum, and epidermis; their levels peaked immediately before ecdysis within each instar. In vivo results revealed that the two isoforms transcriptionally responded, positively and negatively respectively, to 20-hydroxyecdysone and juvenile hormone signaling pathways. RNA interference (RNAi)-aided knockdown of the two LdCDA2 variants (hereafter LdCDA2) or LdCDA2b, rather than LdCDA2a, resulted in three negative effects. First, foliage consumption was significantly reduced, larval developing period was lengthened, and larval growth was retarded. Second, chitin contents were reduced, whereas glucose, trehalose, and glycogen contents were increased in the LdCDA2 and LdCDA2b RNAi larvae. Third, approximately 20% of LdCDA2 and LdCDA2b RNAi larvae were trapped within the exuviae and finally died. About 60% of the abnormal pupae died as pharate adults. Around 20% of the RNAi pupae emerged as deformed adults, with small size and wrinkled wings. These adults eventually died within 1 week after molting. Our results reveal that knockdown of CDA2 affects chitin accumulation. Consequently, LdCDA2 may be a potential target for control of L. decemlineata larvae.

Kaplanoglu E, Chapman P, Scott IM, Donly C. Scientific Reports. 2017;7(5):1762. doi: 10.1038/s41598-017-01961-4.

Current control of insect pests relies on chemical insecticides, however, insecticide resistance development by pests is a growing concern in pest management. The main mechanisms for insecticide resistance typically involve elevated activity of detoxifying enzymes and xenobiotic transporters that break-down and excrete insecticide molecules. In this study, we investigated the molecular mechanisms of imidacloprid resistance in the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), an insect pest notorious for its capacity to develop insecticide resistance rapidly. We compared the transcriptome profiles of imidacloprid-resistant and sensitive beetle strains and identified 102 differentially expressed transcripts encoding detoxifying enzymes and xenobiotic transporters. Of these, 74 were up-regulated and 28 were down-regulated in the resistant strain. We then used RNA interference to knock down the transcript levels of seven up-regulated genes in the resistant beetles. Ingestion of double-stranded RNA successfully knocked down the expression of the genes for three cytochrome P450s (CYP6BQ15, CYP4Q3 and CYP4Q7), one ATP binding cassette (ABC) transporter (ABC-G), one esterase (EST1), and two UDP-glycosyltransferases (UGT1 and UGT2). Further, we demonstrated that silencing of CYP4Q3 and UGT2 significantly increased susceptibility of resistant beetles to imidacloprid, indicating that overexpression of these two genes contributes to imidacloprid resistance in this resistant strain.

Morin MD, Frigault JJ, Lyons PJ, et al. Insect Mol Biol. 2017;26(5):574-583. doi: 10.1111/imb.12320.

The Colorado potato beetle [Leptinotarsa decemlineata (Say)] is an important insect pest that can inflict considerable damage to potato plants. This insect can survive extended periods of cold exposure, and yet the molecular switches underlying this phenomenon have not been fully elucidated. A better characterization of this process would highlight novel vulnerabilities associated with L. decemlineata that could serve as targets for the management of this devastating pest. Using high-throughput sequencing, the current work reveals a cold-associated signature group of microRNAs (miRNAs) in control (15°C) and -5°C-exposed L. decemlineata. The results show 42 differentially expressed miRNAs following cold exposure including miR-9a-3p, miR-210-3p, miR-276-5p and miR-277-3p. Functional analysis of predicted targets associated with these cold-responsive miRNAs notably linked these changes with vital metabolic and cellular processes. Overall, this study highlights the miRNAs probably responsible for facilitating cold adaptation in L. decemlineata and implicates miRNAs as a key molecular target to consider in the development of novel pest management strategies against these insects.

Clements J, Sanchez-Sedillo B, Bradfield CA, Groves RL. PLoS ONE. 2018;13(10):e0205881. doi: 10.1371/journal.pone.0205881.

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is an agricultural pest of commercial potatoes in parts of North America, Europe, and Asia. Plant protection strategies within this geographic range employ a variety of pesticides to combat not only the insect, but also plant pathogens. Previous research has shown that field populations of Leptinotarsa decemlineata have a chronological history of resistance development to a suite of insecticides, including the Group 4A neonicotinoids. The aim of this study is to contextualize the transcriptomic response of Leptinotarsa decemlineata when exposed to the neonicotinoid insecticide imidacloprid, or the fungicides boscalid or chlorothalonil, in order to determine whether these compounds induce similar detoxification mechanisms. We found that chlorothalonil and imidacloprid induced similar patterns of transcript expression, including the up-regulation of a cytochrome p450 and a UDP-glucuronosyltransferase transcript, which belong to protein families associated with xenobiotic metabolism. Further, transcriptomic responses varied among individuals within the same treatment group, suggesting individual insects' responses vary within a population and may cope with chemical stressors in a variety of manners.

Ruiz-Arroyo V, García-Robles I, Ochoa-Campuzano C, et al. Insect Mol Biol. 2017;26(2):204-214. doi: 10.1111/imb.12285.

Bacillus thuringiensis parasporal crystal proteins (Cry proteins) are insecticidal pore-forming toxins that bind to specific receptor molecules on the brush border membrane of susceptible insect midgut cells to exert their toxic action. In the Colorado potato beetle (CPB), a coleopteran pest, we previously proposed that interaction of Cry3Aa toxin with a CPB ADAM10 metalloprotease is an essential part of the mode of action of this toxin. Here, we annotated the gene sequence encoding an ADAM10 metalloprotease protein (CPB-ADAM10) in the CPB genome sequencing project, and using RNA interference gene silencing we demonstrated that CPB-ADAM10 is a Cry3Aa toxin functional receptor in CPB. Cry3Aa toxicity was significantly lower in CPB-ADAM10 silenced larvae and in vitro toxin pore-forming ability was greatly diminished in lipid planar bilayers fused with CPB brush border membrane vesicles (BBMVs) prepared from CPB-ADAM10 silenced larvae. In accordance with our previous data that indicated this toxin was a substrate of ADAM10 in CPB, Cry3Aa toxin membrane-associated proteolysis was altered when CPB BBMVs lacked ADAM10. The functional validation of CPB-ADAM10 as a Cry3Aa toxin receptor in CPB expands the already recognized role of ADAM10 as a pathogenicity determinant of pore-forming toxins in humans to an invertebrate species.