Xu, Q.-Y., Q.-W. Meng, P. Deng, K.-Y. Fu, W.-C. Guo, and G.-Q. Li. Bulletin of Entomological Research https://doi.org/10.1017/S0007485318001050

Dietary delivery of bacterially expressed double-stranded RNA (dsRNA) has a great potential for management of Leptinotarsa decemlineata. An important first step is to discover possible RNA-interference (RNAi)-target genes effective against larvae, especially the old larvae. In the present paper, five putative Broad-Complex (BrC) cDNAs (Z1-Z4, and Z6) were identified in L. decemlineata. The expression of the five LdBrC isoforms was suppressed by juvenile hormone signaling, whereas the transcription was upregulated by 20-hydroxyecdysone signaling at the fourth (final) instar larval stage. Feeding of bacterially expressed dsBrC (derived from a common fragment of the five LdBrC variants) in the third- and fourth-instar larvae successfully knocked down the target mRNAs. For the fourth-instar LdBrC RNAi hypomorphs, they had a higher larval mortality compared with the controls. Moreover, most dsBrC-fed beetles did not pupate normally. After removal of the apolysed larval cuticle, a miniature adult was found. The adult head, compound eyes, prothorax, mesothorax, metathorax were found on the dorsal view. Distinct adult cuticle pigmentation was seen on the prothorax. The mouthparts, forelegs, midlegs, and hindlegs could be observed on the ventral view of the miniature adults. For the third-instar LdBrC RNAi specimens, around 20% moribund beetles remained as prepupae and finally died. Therefore, LdBrC is among the most attractive candidate genes for RNAi to control the fourth-instar larvae in L. decemlineata.

Naqqash, M. N., A. Gökçe, E. Aksoy, A. Bakhsh. Chemosphere, https://doi.org/10.1016/j.chemosphere.2019.124857

Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), is the important pest of potato all over the world. This insect pest is resistant to more than 50 active compounds belonging to various chemical groups. Potential of RNA interference (RNAi) was explored to knock down transcript levels of imidacloprid resistant genes in Colorado potato beetle (CPB) under laboratory conditions. Three important genes belonging to cuticular protein (CP), cytochrome P450 monoxygenases (P450) and glutathione synthetase (GSS) families encoding imidacloprid resistance were targeted. Feeding bio-assays were conducted on various stages of imidacloprid resistant CPB lab population by applying HT115 expressing dsRNA on potato leaflets. Survival rate of insects exposed to CP-dsRNA decreased to 4.23%, 15.32% and 47.35% in 2nd, 3rd and 4th instar larvae respectively. Larval weight and pre-adult duration were also affected due to dsRNAs feeding. Synergism of RNAi with imidacloprid conducted on the 2nd instar larvae, exhibited 100% mortality of larvae when subjected to reduced doses of GSS and CP dsRNAs along with imidacloprid. Utilization of three different dsRNAs against imidacloprid resistant CPB population reveal that dsRNAs targeting CP, P450 and GSS enzymes could be useful tool in management of imidacloprid resistant CPB populations.

García-Robles, I., De Loma, J., Capilla, M., Roger, I., Boix-Montesinos, P., Carrión, P., Vicente, M., López-Galiano, MJ, Real, MD, Rausell, C. Developmental & Comparative Immunology DOI: 10.1016/j.dci.2019.103525

Bacillus thuringiensis (Bt) toxins constitute effective, environmentally safe biopesticides. Nevertheless, insects' tolerance to Bt is influenced by environmental factors affecting immunity. To understand larval immune response in the devastating coleopteran insect pest Colorado potato beetle (CPB), we undertook a proteomic analysis of hemolymph of non-treated control larvae and larvae consuming non-lethal doses of spore-crystal mixtures containing the coleopteran-active Cry3Aa toxin. Results revealed lower amount of proteins involved in insect growth and higher amount of immune response-related proteins in challenged insects, sustaining the larval weight loss observed. Additionally, we found a potential regulatory role of the evolutionary conserved miR-8 in the insect's immune response relying on antimicrobial peptides (AMPs) production. Upon toxin challenge, different patterns of hemolymph AMPs expression and phenoloxidase activity were observed in CPB larvae reared on different Solanaceae plants. This suggests that diet and diet-associated insect midgut microbiota might modulate this insects' tolerance to non-lethal doses of Bt.

Izzo VM, Chen YH, Schoville SD, Cong W, Hawthorne DJ. J Econ Entomol. 2018;111(2):868-878. doi: 10.1093/jee/tox367.

Colorado potato beetle (Leptinotarsa decemlineata Say [Coleoptera: Chrysomelidae]) is a pest of potato throughout the Northern Hemisphere, but little is known about the beetle's origins as a pest. We sampled the beetle from uncultivated Solanum host plants in Mexico, and from pest and non-pest populations in the United States and used mitochondrial DNA and nuclear loci to examine three hypotheses on the origin of the pest lineages: (1) the pest beetles originated from Mexican populations, (2) they descended from hybridization between previously divergent populations, or (3) they descended from populations that are native to the Plains states in the United States. Mitochondrial haplotypes of non-pest populations from Mexico and Arizona differed substantially from beetles collected from the southern plains and potato fields in the United States, indicating that beetles from Mexico and Arizona did not contribute to founding the pest lineages. Similar results were observed for AFLP and microsatellite data. In contrast, non-pest populations from the states of Colorado, Kansas, Nebraska, New Mexico, and Texas were genetically similar to U.S. pest populations, indicating that they contributed to the founding of the pest lineages. Most of the pest populations do not show a significant reduction in genetic diversity compared to the plains populations in the United States. We conclude that genetically heterogeneous beetle populations expanded onto potato from native Solanum hosts. This mode of host range expansion may have contributed to the abundant genetic diversity of contemporary populations, perhaps contributing to the rapid evolution of climate tolerance, host range, and insecticide resistance.

Xu QY, Meng QW, Deng P, Fu KY, Guo WC, Li GQ. Insect Mol Biol. 2018;27(4):439-453. doi: 10.1111/imb.12384.

Two Drosophila melanogaster E-twenty-six domain transcription factor isoforms (E74A and E74B) act differentially at the start of the 20-hydroxyecdysone (20E) signalling cascade to regulate larval-pupal metamorphosis. In the present paper, we identified the two isoforms (LdE74A and LdE74B) in Leptinotarsa decemlineata. During the larval development stage, the mRNA transcript levels of the two LdE74 isoforms were correlated with circulating 20E titres. In vitro midgut culture and in vivo dietary supplementation with 20E revealed that the presence of 20E induced expression peaks of both LdE74A and LdE74B, with similar patterns observed for the two isoforms. Moreover, the mRNA transcript levels of both LdE74A and LdE74B isoforms were significantly downregulated in the L. decemlineata ecdysone receptor RNA interference (RNAi) specimens, but not in the LdE75 RNAi beetles. Ingestion of 20E reduced the larval fresh weights and shortened the larval development period, irrespective of knockdown of LdE74 or not. RNAi of LdE74 did not affect 20E-induced expression of the Ecdysone induced protein 75-hormone receptor 3-fushi tarazu factor 1 (E75-HR3-FTZ-F1) transcriptional cascade. Thus, it seems that LdE74 mediates 20E signalling independent of the E75-HR3-FTZ-F1 transcriptional cascade. Furthermore, silencing of both LdE74 isoforms caused failure of ecdysis. Most of the LdE74 RNAi beetles remained as prepupae. The LdE74 RNAi prepupae exhibited adult character-like forms underneath after removal of the apolysed larval cuticle. Their appendages such as antennae, legs and wings were shorter than those of control larvae. Only a few LdE74 RNAi larvae finally became deformed pupae, with shortened antennae and legs. Therefore, LdE74 is required for larval-pupal metamorphosis and appendage growth in L. decemlineata.

Crossley MS, Rondon SI, Schoville SD. American Journal of Potato Research. 2018;95(5):495-503. doi: 10.1007/s12230-018-9654-0.

The Colorado potato beetle, Leptinotarsa decemlineata Say, is a serious pest of potato, Solanum tuberosum L., worldwide. Leptinotarsa decemlineata has a history of repeated adaptation to insecticides, and exhibits a geographic pattern of decreasing insecticide resistance from east to west in the USA. Imidacloprid is one of the most widely used insecticide in western states. In this study, we measured imidacloprid resistance among larval and adult L. decemlineata from ten locations in the Columbia Basin (southeastern Washington and northeastern Oregon) using topical LD50 bioassays, and compared them to estimates from ten locations in Minnesota and Wisconsin. Larval and adult imidacloprid LD50's and mean percent mortality were generally lowest in Washington and Oregon, but some sites exhibited reductions in mortality comparable to those observed at some Wisconsin sites. Adult LD50's suggest L. decemlineata in the Columbia Basin may be evolving in response to selection by neonicotinoid insecticides, but larval data suggest high susceptibility to imidacloprid remains in most populations. Future work should expand resistance monitoring efforts to include more regions in the West and other insecticide modes of action.

Crossley MS, Rondon SI, Schoville SD. Evolutionary Applications. 2019;12(4):804-814. doi: 10.1111/eva.12757.

Changing landscape heterogeneity can influence connectivity and alter genetic variation in local populations, but there can be a lag between ecological change and evolutionary responses. Temporal lag effects might be acute in agroecosystems, where land cover has changed substantially in the last two centuries. Here, we evaluate how patterns of an insect pest's genetic differentiation are related to past and present agricultural land cover change over a 150-year period. We quantified change in the amount of potato, Solanum tuberosum L., land cover since 1850 using county-level agricultural census reports, obtained allele frequency data from 7,408 single-nucleotide polymorphism loci, and compared effects of historic and contemporary landscape connectivity on genetic differentiation of Colorado potato beetle, Leptinotarsa decemlineata Say, in two agricultural landscapes in the United States. We found that potato land cover peaked in Wisconsin in the early 1900s, followed by rapid decline and spatial concentration, whereas it increased in amount and extent in the Columbia Basin of Oregon and Washington beginning in the 1960s. In both landscapes, we found small effect sizes of landscape resistance on genetic differentiation, but a 20× to 1,000× larger effect of contemporary relative to historic landscape resistances. Demographic analyses suggest population size trajectories were largely consistent among regions and therefore are not likely to have differentially impacted the observed patterns of population structure in each region. Weak landscape genetic associations might instead be related to the coarse resolution of our historical land cover data. Despite rapid changes in agricultural landscapes over the last two centuries, genetic differentiation among L. decemlineata populations appears to reflect ongoing landscape change. The historical landscape genetic framework employed in this study is broadly applicable to other agricultural pests and might reveal general responses of pests to agricultural land-use change.

Deng, P., Q.Y. Xu, K.Y. Fu, WC.. Guo, G.Q. Li. 2018. Insect Biochem Mol Biol. 103:1-11. doi: 10.1016/j.ibmb.2018.10.001.

It is noted that insect insulin/insulin-like growth factor/target of rapamycin signaling is critical for the regulation of metamorphosis in holometabolous insects. However, the molecular mechanism remains undetermined. Our previous findings reveal that RNA interference (RNAi)-mediated knockdown of an insulin gene (LdILP2) in Leptinotarsa decemlineata disturbs both 20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling, and impairs pupation. In the present paper, we further observed that the expression of the insulin receptor substrate gene chico (Ldchico) and the phosphoinositide-3-kinase gene pi3k (Ldpi3k92E) was repressed in LdILP2 depleted larvae. Moreover, RNAi of Ldchico or Ldpi3k92E decreased food consumption, affected absorption and metabolism of amino acids and sugars, and reduced expression of several 20E (LdEcR, LdHR3 and LdE75) and JH (LdJHAMT, LdKr-h1 and LdHairy) signaling genes. As a result, larval development was postponed and larval growth was inhibited. Intriguingly, knockdown of Ldchico, rather than Ldpi3k92E, impaired larval-pupal and pupal-adult ecdysis, and specifically repressed transcription of another 20E signaling gene LdUSP. Ingestion of 20E rescued the expression of LdEcR, LdHR3 and LdE75, whereas 20E feeding restored neither the decreased LdUSP mRNA level, nor the reduced pupation and adult emergence rates in Ldchico RNAi larvae. Therefore, Chico is critical for the regulation of larval-pupal-adult transition by a PI3K-independent pathway, perhaps through activation of USP in L. decemlineata.

Clements J, Schoville S, Peterson N, Huseth AS, Que L, Groves RL. Pestic Biochem Physiol. 2017;135:35-40. doi: 10.1016/j.pestbp.2016.07.001.

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a major agricultural pest of potatoes in the Central Sands production region of Wisconsin. Previous studies have shown that populations of L. decemlineata have become resistant to many classes of insecticides, including the neonicotinoid insecticide, imidacloprid. Furthermore, L. decemlineata has multiple mechanisms of resistance to deal with a pesticide insult, including enhanced metabolic detoxification by cytochrome p450s and glutathione S-transferases. With recent advances in the transcriptomic analysis of imidacloprid susceptible and resistant L. decemlineata populations, it is possible to investigate the role of candidate genes involved in imidacloprid resistance. A recently annotated transcriptome analysis of L. decemlineata was obtained from select populations of L. decemlineata collected in the Central Sands potato production region, which revealed a subset of mRNA transcripts constitutively up-regulated in resistant populations. We hypothesize that a portion of the up-regulated transcripts encoding for genes within the resistant populations also encode for pesticide resistance and can be suppressed to re-establish a susceptible phenotype. In this study, a discrete set of three up-regulated targets were selected for RNA interference experiments using a resistant L. decemlineata population. Following the successful suppression of transcripts encoding for a cytochrome p450, a cuticular protein, and a glutathione synthetase protein in a select L. decemlineata population, we observed reductions in measured resistance to imidacloprid that strongly suggest these genes control essential steps in imidacloprid metabolism in these field populations.

Du X., Fu K., Xu Q., Ahmat T., Ding X., He J., Guo W. Acta Entomol Sin. 2018;61(6):637-645.

Aim: This study aims to clarify the function of N-β-alanyl-dopamine (NBAD) hydrolase gene important in melanin biosynthesis in the Colorado potato beetle, Leptinotarsa decemlineata by RNA interference. Methods: The NBAD hydrolase gene in L. decemlineata was characterized by data mining based on its transcriptome, its cDNA was cloned by RT-PCR, and the gene completeness and phylogeny were determined by multiple alignment and phylogenetic analysis, respectively. The expression levels of NBAD hydrolase gene in different developmental stages, tissues of the 4th instar larvae and male gonad and ovary of adults of L. decemlineata were detected by qPCR. The color change during larval growth was observed after RNAi, and the mechanism how the expression of NBAD hydrolase gene was influenced by juvenile hormone (JH) and molting hormone (MH) was assayed. Results: An NBAD hydroxylase gene was cloned from L. decemlineata and named Ldtan (GenBank accession no.: KY221866). Its encoded protein shows the highest amino acid sequence identity with the homologous proteins from Tribolium castaneum and Dendroctonus ponderosae and clustered into the same clade with them. The spatial expression profiles showed that Ldtan were highly expressed in ventral nerve cord, hindgut and cuticle of L. decemlineata, with the relative expression levels of 99.36±0.95, 17.79±3.11 and 9.21±0.12, respectively, while the temporal expression profiles showed that its expression level increased along with larval growth and reached the peak at the adult stage. Knockdown of Ldtan gene by feeding dsLdtan to the 2nd instar larvae not only led to tanned color, but also a degree of lethal effect. Knocking down the expression of JH synthesis and signal-related genes by RNAi downregulated the expression of Ldtan, while knocking down the expression of MH synthesis and signal-related genes by RNAi upregulated the expression of Ldtan. Conclusion: The results suggest that Ldtan is involved in melanin synthesis in L. decemlineata, and JH and MH probably regulate its expression.