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.

Rainio MJ, Margus A, Lehmann P, Helander M, Lindström L. Comparative Biochemistry and Physiology C, Toxicology & Pharmacology. 2019;215:47-55. doi: 10.1016/j.cbpc.2018.09.005.

Glyphosate is the globally most used herbicide against a wide range of weeds. Glyphosate has been considered safe to animals as it mainly targets physiological pathways in plants. However, recent toxicological studies have revealed that glyphosate can cause various toxic effects also on animals. In this study, we investigated the direct toxic effects of a glyphosate-based herbicide (GBH, Roundup® Bio) on (1) survival and (2) oxidative status of a non-target herbivore by using Colorado potato beetles (Leptinotarsa decemlineata), originating from Poland and USA, as model species. Larvae were randomly divided into three groups: (1) high concentration (100% Roundup Bio, 360 g/l), (2) low concentration (1.5% Roundup Bio) and (3) control group (water). Larvae were exposed to Roundup for different time periods: 2 h, 24 h, 48 h, 72 h and 96 h. Larval survival decreased in the group treated with high concentration of GBH compared to controls, whereas the low concentration group did not differ from the control group. GBH treatment had no association with oxidative status biomarkers (i.e. catalase, superoxide dismutase, glutathione-S-transferase, glutathione and glutathione related enzymes), but increased lipid hydroperoxide levels after 2 h exposure, suggesting increased oxidative damage soon after the exposure. Larvae of different origin also differed in their oxidative status, indicating population-dependent differences in antioxidant defence system. Environmentally relevant concentrations of GBH are not likely to affect larval survival, but high concentrations can reduce survival and increase oxidative damage of non-target herbivores. Also, populations of different origin and pesticide usage history can differ in their tolerance to GBH.

Yaroslavtseva ON, Dubovskiy IM, Khodyrev VP, Duisembekov BA, Kryukov VY, Glupov VV. J Insect Physiol. 2017;96:14-20. doi: 10.1016/j.jinsphys.2016.10.004.

The synergistic effect between the entomopathogenic fungus Metarhizium robertsii and a sublethal dose of the bacterium Bacillus thuringiensis ssp. morrisoni var. tenebrionis was studied in terms of immune defense reactions and detoxification system activity of the Colorado potato beetle, Leptinotarsa decemlineata, fourth instar larvae. Bacterial infection led to more rapid germination of fungal conidia on integuments. We found a significant decrease of cellular immunity parameters, including total hemocyte count and encapsulation response, under the influence of bacteria. Phenoloxidase activity in integuments was increased under bacteriosis, mycosis and combined infection compared to controls. However, phenoloxidase activity in the hemolymph was enhanced under bacteriosis alone, and it was decreased under combined infection. Activation of both nonspecific esterases and glutathione-S-transferases in the hemolymph was shown at the first day of mycosis and third day of bacteriosis. However, inhibition of detoxification enzymes was detected under combined infection. The suppression of cellular immunity and detoxification reactions in Colorado potato beetle larvae with a sublethal dose of bacteria is discussed as a reason for synergy between B. thuringiensis and M. robertsii.

Hussain T, Aksoy E, Çalișkan ME, Bakhsh A. Transgenic Res. 2019;28(1):151-164. doi: 10.1007/s11248-018-0109-7.

Most of the commercialized insect resistant transgenic crops express cry gene(s) isolated from Bacillus thuringiensis; however, intensive cultivation of Bt crops over almost two decades has been questioned regarding its sustainability and durability in pest management. The present study focused on silencing of highly specific molting-associated Ecdysone receptor (EcR) gene of Colorado potato beetle (CPB) using RNA interference (RNAi) approach. The partial cDNA of EcR gene of CPB was amplified using specific primers in sense and anti-sense orientations, and cloned in pRNAi-GG vector flanked by an intronic sequence (pdk). Leaf and internodal explants of Agria and Lady Olympia potato cultivars were infected with Agrobacterium strain LBA4404 harboring constructs under the control of CaMV 35S promoter. Standard molecular analysis of primary transformants showed proper integration of T-DNA in plant genome. The transgenic plants of both cultivars were evaluated for their efficacy against first, second and third instar CPB larvae. The leaf biotoxicity assays revealed 15-80% of CPB mortality. A significantly lower fold-change (0.87-4.14×) in larval weight was observed in insects fed on transgenic plants compared to the ones fed on control plants (1.87-6.53×). Furthermore, CPB larvae fed on transgenic plants exhibited reduced EcR transcripts, indicating the functionality of dsRNA EcR in silencing EcR gene expression. This study is an excellent example of the integration of an alternative, effective and reliable method to cope with potato insect pests that incur significant losses to potato production in the world.

Molnár I, Besenyei E, Thieme R, et al. Pest Manag Sci. 2017;73(7):1428-1437.

BACKGROUND: Colorado potato beetle (CPB) has become the biggest enemy of cultivated potato worldwide. One of the most effective sources of resistance to CPB is Solanum chacoense, an accession with a high leptine glycoalkaloid content. The aim of our study was to assay the repellence and toxicity of S. chacoense, its somatic hybrids (SHs) and their backcross progenies (BC1) with potato for CPB adults and larvae. Transgenic S. chacoense, deficient in DNA mismatch repair (MMR), was also used to produce SHs, in order to increase homeologous recombination and hence introgression of wild-species DNA into the potato gene pool. RESULTS: Wild-type SH was highly resistant to CPB. Resistance to CPB of BC1 progenies showed a 1:3 inheritance pattern. MMR-deficient SHs performed better in the resistance analysis. Most MMR-deficient SHs had a similar toxicity as S. chacoense and an intensely repellent effect on CPB adults. Resistance of SHs and BC1 clones may be attributed to leptine biosynthesis, which was confirmed using a RAPD marker. CONCLUSION: This is the first report of SHs and their progenies exhibiting both antibiosis and antixenosis against CPB. Resistant SHs are an important step forward in combating this voracious pest of potato.

 Boiteau G, MacKinley P. Can Entomol. 2017;149(2):174-190. doi: 10.4039/tce.2016.52.

This laboratory study confirmed that the strategy of adult terrestrial Colorado potato beetle (Leptinotarsa decemlineata (Say); Coleoptera Chrysomelidae) to survive the threat of drowning in water is based on avoidance of water crossings. It also showed that beetles at the surface of a body of water after failing to avoid it, long considered limited to passive floating and phoretic transport were in fact likely to rely on a complex fight or flee response. Beetles showed capacity to swim in a pattern similar to land foraging beetles. Beetles also tolerated submergence and walked underwater. These active behaviours should improve their probability of finding shore or refuge for longer survival. Results confirmed that Colorado potato beetles are likely to accumulate near water features in the potato agro-ecosystem landscape but suggest that successful crossings and colonisation of crops on the other side are more likely than previously expected. On a larger scale, new information provided by this study combined with our knowledge of dominant winds and currents should make it possible for future research to better predict the probability of surviving encounters with water and the orientation of invasive Colorado potato beetle colonisers dispersing at the surface of bodies of water.

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.

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.

QingYu X, QingWei M, Pan D, WenChao G, GuoQing L. Insect Biochem Mol Biol. 2018;94:50-60. doi: 10.1016/j.ibmb.2017.09.012.

Hormone receptor 4 (HR4) is involved in the regulation of 20-hydroxyecdysone (20E) biosynthesis and the mediation of 20E signaling during larval-pupal transition in a holometabolan Drosophila melanogaster, whereas it acts as a repressor in 20E-responsive transcriptional cascade in a hemimetabolan, Blattella germanica. Here we characterized two HR4 splicing variants, LdHR4X1 and LdHR4X2, in a coleopteran Leptinotarsa decemlineata. LdHR4X1 was highly expressed in the prothoracic gland and epidermis while LdHR4X2 was abundantly transcribed in the nervous system. In vivo results showed that both prothoracicotropic hormone and 20E pathways transcriptionally regulated LdHR4, in an isoform-dependent pattern. RNA interference of LdHR4 at the final (fourth) larval instar, in contrast to the second- and third-instar periods, enhanced the expression of two ecdysteroidogenesis genes, increased 20E titer, upregulated transcription of five 20E-response genes, and reduced the mRNA level of Fushi tarazu-factor 1 (FTZ-F1). As a result, the fourth-instar LdHR4 RNAi larvae exhibited accelerated development and reduced body weight. Moreover, knockdown of LdHR4 at the fourth instar resulted in larval lethality and impaired pupation. Feeding of pyriproxyfen (a mimic of juvenile hormone) or silencing of a juvenile hormone degrading enzyme gene restored the normal course of ecdysteroidogenesis, duration of larval development, and body weight in fourth-instar LdHR4 RNAi larvae. The treatment partially suppressed the larval mortality but not the failure to pupate. The dual role of HR4 during larval-pupal metamorphosis appears to be evolutionarily conserved among holometabolans.

Malik RJ, Ali JG, Bever JD.  Pedobiologia. 2018;66:29-35. doi: 10.1016/j.pedobi.2017.12.004.

While arbuscular mycorrhizal (AM) fungi may have a prominent role in trophic ecology, mycorrhizal improvement or reduction on herbivore growth and survival may also be dependent on herbivore's stage of development. Solanum lycopersicon (tomato) was grown on sterile background soil treated with either mycorrhizal inoculant (AM+) or non-mycorrhizal control (AM-). Mycorrhizal treatments included four single species of AM-fungi (Entrophospora infrequens, Funneliformis mosseae, Claroideoglomus claroideum, and Racocetra fulgida) and a mixture of all four species (fungal community). To determine if mycorrhizal treatment indirectly alters the ability of beetle larvae (Leptinotarsa decemlineata) to access plant resources, plant damage and trichome density were quantified as plants were infested with a single neonate (early-stage) for 96 hours (h). In a second experiment, beetle growth rate was assessed as plants were infested with a single third-instar (late-stage). After 72 h of late-stage beetle infestation, beetle mass was measured. It was found that early-stage beetles inflicted more damage on AM+ tomatoes. Interestingly, this corresponds with fewer trichomes on AM+ tomatoes, as well as higher early-stage beetle survivorship. Specifically, AM taxon, C. claroideum increases herbivory and thereby reduces beetle mortality. Among late-stage beetles, C. claroideum does not improve beetle growth nor rate of survival. This suggests that AM taxa that are beneficial to early-stage beetles may not necessarily provide an advantage to late-stage beetles. Taken together, these findings highlight potential dependencies of AM-fungal effects on herbivory and herbivore life history, including growth and life-stage specific survival.