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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.

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 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.

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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.

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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.

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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.

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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.

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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.

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Andrés MF, Rossa GE, Cassel E, et al. Food and Chemical Toxicology. 2017;109:1086-1092.

In this study we evaluated the effect of a pressure gradient (1-2 atm) in the extraction and composition of the essential oil (EO) of Piper hispidinervum by steam distillation. We also evaluated the insect antifeedant effects (Spodoptera littoralis, Leptinotarsa decemlineata, Myzus persicae and Rhopalosiphum padi) and nematicidal activity (Meloidogyne javanica) of the oils, their major components and their synergistic interactions. Safrole was the major component (78-81%) followed by terpinolene (5-9%). The EOs tested were effective insect antifeedants. Safrole, explained most of the insect antifeedant action of P. hispidinervum EOs. When safrole and terpinolene were tested in binary combinations, low ratios of safrole improved the antifeedant effects of terpinolene. P. hispidinervum EOs caused higher mortality of M. javanica juveniles than their major components. In binary combinations, low ratios of terpinolene increased the nematicidal effects of safrole. The EO treatment strongly suppressed nematode egg hatching and juvenile infectivity. P. hispidinervum EOs affected the germination of S. lycopersicum and L. sativa mostly at 24 h of treatment, being L. sativa the most sensitive. Safrole moderately affected germination and root growth of L. sativa, S. lycopersicum and L. perenne. Terpinolene only affected S. lycopersicum root growth.

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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.

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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.