Wu JJ, Mu LL, Chen ZC, Fu KY, Guo WC, Li C, Li GQ. 2019. Journal of Asia-Pacific Entomology. 22(2):443–452.

Chitin deacetylases (CDAs) catalyze N-deacetylation of chitin, a crucial process for chitin modification. In the present paper, LdCDA1 was identified in Leptinotarsa decemlineata. It was copiously expressed in larval foregut, hindgut and epidermis. Just before the molt in the first, second and third larval instars, the mRNA levels of LdCDA1 were high. In the fourth (final)-instar larvae, a peak occurred 4 days after ecdysis. In vivo results revealed that LdCDA1 transcriptionally responded, positively and negatively respectively, to 20-hydroxyecdysone and juvenile hormone titers. Moreover, knockdown of LdCDA1 significantly reduced foliage consumption, lengthened developing period and prevented growth in the final instar larvae. Three distinct lethal phenotypes were noted in the LdCDA1 RNAi larvae. About 30% of the RNAi larvae became moribund and finally died; approximately 50% of deformed pupae died as pharate adults; and around 20% of LdCDA1 depleted pupae finally emerged as abnormal adults and eventually died within 1 week after emergence. Furthermore, chitin content was low and the mRNA levels of five chitin biosynthesis transcripts (LdUAP1, LdUAP2, LdChSAa, LdChSAb and LdChSB) were significantly declined in the LdCDA1 RNAi larvae. In addition, glucose, trehalose and glycogen contents were increased in the LdCDA1 depleted hypomorphs, along with highly expressed genes coding for trehalose and glycogen synthesis enzymes. The findings provide a compelling piece of evidence that CDA1 is critical for chitin deposition in L. decemlineata. Moreover, LdCDA1 may be a potential target for control of the larvae.

Rainio MJ, Margus A, Virtanen V, Lindström L, Salminen JP, Saikkonen K, Helander M. 2020. Chemosphere. 258:127254.

Glyphosate is the most used herbicide worldwide, targeting physiological pathways in plants. Recent studies have shown that glyphosate can also cause toxic effects in animals. We investigated the glyphosate-based herbicide (GBH)-induced changes in potato (Solanum tuberosum) plant chemistry and the effects of a GBH on the survival rate and oxidative status of the Colorado potato beetle (Leptinotarsa decemlineata). The beetles were reared on potato plants grown in pots containing soil treated with a GBH (Roundup Gold, 450 g/l) or untreated soil (water control). The 2nd instar larvae were introduced to the potato plants and then collected in 2 phases: as 4th instar larvae and as adults. The main glycoalkaloids of the potato plants, α-solanine and α-chaconine, were measured twice during the experiment. The α-solanine was reduced in potato plants grown in GBH-treated soil, which can be detrimental to plant defenses against herbivores. GBH treatment had no effect on the survival rate or body mass of the larvae or the adult beetles. In the larvae, total glutathione (tGSH) concentration and the enzyme activity of catalase (CAT), superoxide dismutase, and glutathione-S-transferase were increased in the GBH treatment group. In the adult beetles, CAT activity and tGSH levels were affected by the interactive effect of GBH treatment and the body mass. To conclude, environmentally relevant concentrations of a GBH can affect the potato plant's glycoalkaloid concentrations, but are not likely to directly affect the survival rate of the Colorado potato beetle, but instead, modify the antioxidant defense of the beetles via diet.

Máximo WPF, Howell JL, Mogilicherla K, Basij M, Chereddy SCRR, Palli SR. 2020. Archives of Insect Biochemistry and Physiology. 104:e21685.

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.

He W, Xu W, Fu K, Guo W, Zhang J. 2020. Insects. 11(7):1–9.

RNA interference (RNAi)-based technology has been proven as a novel approach for insect pest control. However, whether insects could evolve resistance to RNAi and the underlying mechanism is largely unknown. The target gene mutations were thought to be one of the potential ways to develop the resistance. Here we predicted the effective siRNA candidates that could be derived from dsRNA against the Colorado potato beetle (CPB) β-Actin gene (dsACT). By site-directed mutagenesis, we synthesized the dsRNAs with the defect in generation of effective siRNAs (and thus were supposed to have comparable low RNAi efficacy). We showed that, with mismatches to the target gene, all the dsRNA variants caused similar levels of silencing of target gene, mortality and larval growth retardation of CPB. Our results suggest that when the mismatch rate of dsACT and target β-Actin mRNA is less than 3%, the RNAi efficiency is not impaired in CPB, which might imply the low possibility of RNAi resistance evolving through the sequence mismatches between dsRNA and the target gene.

Gui S, Taning CNT, Smagghe G. 2020. Insect Science: Early View.

Insect neuropeptides regulate various physiological processes, such as reproduction, feeding, growth and development, and have been considered as viable targets in the development of alternative strategies for pest control. Amongst these neuropeptides is myosuppressin (MS), a very conserved neuropeptide that has been reported to regulate cardiac and skeletal muscle contractility, feeding and pupal diapause in insects. In this study, we investigated the involvement of MS in fecundity in a notorious defoliator of potato and other solanaceous plants, the Colorado potato beetle (CPB), Leptinotarsa decemlineata. We identified an MS-precursor-encoding transcript in the L. decemlineata transcriptomic database and then evaluated its transcript levels in various CPB tissues. MS transcript levels were found to be highest in the central nervous system, gut and muscle of CPB males and females. To investigate the role of MS in fecundity, MS was silenced in adult CPBs through RNA interference (RNAi). This resulted in a significant reduction in oviposition (over 80%) and oocyte size (69%) in the treated beetles compared to the controls. Also, the reduction in oviposition in treated females was confirmed to be dependent on MS knockdown and independent of male fertilization. Furthermore, MS-knockdown in females resulted in decreased levels of ecdysteroid hormone titers and the transcript levels of its receptor. Interestingly, the injection of 20-hydroxyecdysone into females following MS knockdown could rescue ovary development. Altogether, this study highlights the important role played by MS in regulating fecundity in CPB.

Ashouri S, Farshbaf Pourabad R. 2021. Gene. 766:145159.

Considering the relevance of insect α-amylases and natural α-amylase inhibitors present in plants to protect against insect damage, we investigated the effect of white bean and rapeseed protein extracts on digestive α-amylase gene expression of the Colorado potato beetle, Leptinotarsa decemlineata (Say). For this purpose, in vitro and in vivo trials were performed to determine the inhibitory activity of seed proteins on the third and fourth instar larvae. In both trials, the significant inhibitory effect of each extracts on the third and fourth instar larval α-amylase activity and considerable mortality in treatments were observed compared to control trials. In the RT-qPCR, expression ratio demonstrated that the α-amylase gene of two different larval stages grown on both proteins treated leaves had significantly differentiated expression and was up-regulated in third instar larvae and down-regulated in fourth instar larvae compared to control. Results suggest that the hyper-production of α-amylase in third instar larvae is elicited to compensate for the enzyme activity inhibition at an earlier stage and also down-regulation suggests the existence of a negative feedback of plant proteins on the last instar larvae via impaired food intake and digestive α-amylase activity in Colorado potato beetle. Therefore, disruption of the insect's digestive physiology by plant defensive proteins can be considered in the development of innovative controlling methods of this crucial potato pest.

Favell, G.; McNeil, J.N.; Donly, C. Insects 2020, 11, 135.

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a significant agricultural pest that has developed resistance to many insecticides that are used to control it. Investigating the mechanisms of insecticide detoxification in this pest is important for ensuring its continued control, since they may be contributors to such resistance. Multidrug resistance (MDR) genes that code for the ABCB transmembrane efflux transporters are one potential source of insecticide detoxification activity that have not been thoroughly examined in L. decemlineata. In this study, we annotated the ABCB genes found in the L. decemlineata genome and then characterized the expression profiles across midgut, nerve, and Malpighian tubule tissues of the three full transporters identified. To investigate if these genes are involved in defense against the macrocyclic lactone insecticide ivermectin in this insect, each gene was silenced using RNA interference or MDR protein activity was inhibited using a chemical inhibitor, verapamil, before challenging the insects with a dose of ivermectin. Survival of the insects did not significantly change due to gene silencing or protein inhibition, suggesting that MDR transporters do not significantly contribute to defense against ivermectin in L. decemlineata.

Dumas, P. , M. Sambou , J. D. Gaudet , M. D. Morin , C. E. Moffat , S. Boquel , P. Morin. Archives of Insect Biochemistry and Physiology https://doi.org/10.1002/arch.21642

The Colorado potato beetle (Leptinotarsa decemlineata [Say]) is an insect pest that can significantly harm potato plants worldwide. Control of this insect relies heavily on chemical insecticides such as chlorantraniliprole. Nevertheless, the complete molecular signature associated with response to this compound is lacking in L. decemlineata. In this study, amplification and quantification by qRT‐PCR (quantitative reverse transcription‐polymerase chain reaction) of targets relevant to chlorantraniliprole were undertaken in insects exposed to this chemical. This approach showed modulation of numerous cytochrome P450s, such as CYP350D1 and CYP4Q3, as well as upregulation of microRNAs (miRNAs), including miR‐1‐3p and miR‐305‐5p, in chlorantraniliprole‐exposed insects. Functional assessment of transcript targets predicted to be regulated by these miRNAs was performed and revealed their likely impact on transcriptional regulation. RNAi‐based targeting of CYP350D1 notably provided preliminary evidence of its underlying implication for chlorantraniliprole response in L. decemlineata. Overall, this study strengthens the current knowledge of the molecular changes linked to chlorantraniliprole response in L. decemlineata and provides novel targets with potential relevance to chlorantraniliprole susceptibility in this insect pest of global relevance.

Tomilova, O. G., O. N. Yaroslavtseva, M. D. Ganina, M. V. Tyurin, E. I. Chernyak, I. V. Senderskiy, Y. A. Noskov, O. V. Polenogova, Y. B. Akhanaev, V. Yu. Kryukov, V. V. Glupov, S. V. Morozov. Journal of Insect Physiology 116: 106-117. https://doi.org/10.1016/j.jinsphys.2019.05.003

Susceptibility to the fungus Metarhizium robertsii and changes in host defences were evaluated in different stages of the intermoult period (4–6 h, 34–36 h and 84–86 h post moult in IV larval instars) of the Colorado potato beetle. A significant thickening of the cuticle during larval growth was accompanied by decreases in cuticle melanization, phenoloxidase activity and epicuticular hydrocarbon contents (C28-C32). At the same time, a decrease in the conidial adhesion rate and an increase in resistance to the fungus were observed. In addition, we recorded significant elevation of the encapsulation rate and total haemocyte counts in the haemolymph during the specified period. The activity of detoxification enzymes decreased in the haemolymph but increased in the fat body during larval growth. No significant differences in the fatty acid content in the epicuticle were observed. The role of developmental disorders in susceptibility to entomopathogenic fungi is also discussed.

Margus, A., Piiroinen, S., Lehmann, P. et al. Scientific Reports 9, 11320 (2019) doi:10.1038/s41598-019-47473-1

Stress tolerance and adaptation to stress are known to facilitate species invasions. Many invasive species are also pests and insecticides are used to control them, which could shape their overall tolerance to stress. It is well-known that heavy insecticide usage leads to selection of resistant genotypes but less is known about potential effects of mild sublethal insecticide usage. We studied whether stressful, sublethal pyrethroid insecticide exposure has within-generational and/or maternal transgenerational effects on fitness-related traits in the Colorado potato beetle (Leptinotarsa decemlineata) and whether maternal insecticide exposure affects insecticide tolerance of offspring. Sublethal insecticide stress exposure had positive within-and transgenerational effects. Insecticide-stressed larvae had higher adult survival and higher adult body mass than those not exposed to stress. Furthermore, offspring whose mothers were exposed to insecticide stress had higher larval and pupal survival and were heavier as adults (only females) than those descending from control mothers. Maternal insecticide stress did not explain differences in lipid content of the offspring. To conclude, stressful insecticide exposure has positive transgenerational fitness effects in the offspring. Therefore, unsuccessful insecticide control of invasive pest species may lead to undesired side effects since survival and higher body mass are known to facilitate population growth and invasion success.