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.

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.

Naqqash, M. N., A. Gökçe, E. Aksoy, A. Bakhsh. Chemosphere,

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.

Cingel A, Savić J, Lazarević J, et al. Insect Science. 2017;24(5):768-780. doi: 10.1111/1744-7917.12364.

Colorado potato beetle (CPB; Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II (OCI and OCII) have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato (Solanum tuberosum L.) cultivars Desiree, Dragačevka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition of cysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long-term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.

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.

Kryukov VY, Tomilova OG, Luzina OA, et al. Pest Manag Sci. 2018;74(3):598-606.

BACKGROUND: The search for compounds that interact synergistically with entomopathogenic fungi is aimed at enhancing the efficacy and stability of biological products against pest insects, for example, against the Colorado potato beetle (CPB). We hypothesized that fluorine-containing derivatives of usnic acid (FUA) might be candidates for the development of multicomponent bio-insecticides. The aim of this study was to analyze the co-influence of FUA and Beauveria bassiana on the survival and immune-physiological reactions of CPB larvae. RESULTS: Synergy between FUA and B. bassiana was observed after treatment of second, third and fourth larvae instars under laboratory conditions. Furthermore, synergy was observed in field trials in continental climate conditions in southeastern Kazakhstan. In a field experiment, the median lethal time was shortened three-fold, and cumulative mortality for 15 days increased by 36% in the combined treatment compared with a fungal infection alone. FUA treatment delayed larval development, decreased the total hemocyte count, and increased both the phenoloxidase activity in integuments and the detoxification enzyme rate in hemolymph. A combined treatment with fungus and FUA led to increases in the aforementioned changes. CONCLUSION: Toxicosis caused by FUA provides a stable synergistic effect between FUA and B. bassiana. The combination can be promising for the development of highly efficient products against CPB.

Meng, Q.W., J.J. Wang, J.F. Shi, W.C. Guo, and G.Q. Li. 2018. American Journal of Potato Research. 95(5):463-472. doi: 10.1007/s12230-018-9646-0.

The potential of teflubenzuron was assessed in a series of laboratory studies in order to achieve consistent, long-term, integrated management of the Colorado potato beetle, Leptinotarsa decemlineata (Say). Teflubenzuron exhibited excellent stomach toxicity to the larvae. Its larvicidal activity was comparable with those of cyhalothrin, chlorantraniliprole and spinosad. Moreover, the teflubenzuron-fed larvae consumed less foliage, grew slower, and needed a longer period to develop, in a dose dependent manner. Most of these larvae died during larval-larval molting, larval-pupal ecdysis or adult emergence. Furthermore, chitin contents in body carcass (without midgut) and integument specimen of the teflubenzuron-fed larvae significantly decreased, whereas the chitin amount in the midgut peritrophic matrix was not affected. In addition, uridine diphosphate-N-acetylglucosamine-pyrophosphorylase gene (LdUAP1), which was mainly responsible for chitin biosynthesis in ectodermally-derived tissues, was suppressed after teflubenzuron ingestion, in contrast to its partner LdUAP2 for chitin formation in the midgut peritrophic matrix. In a word, by inhibition of chitin production in ectodermally-derived tissues, teflubenzuron is an effective benzoylurea insecticide to L. decemlineata larvae. It can be a valuable tool in effective integrated pest management and insecticide resistance management programs against L. decemlineata.

Guo, W., et al. 2018 J Agric Food Chem. 66(45):11990-11999. doi: 10.1021/acs.jafc.8b03914.

RNA interference (RNAi) has been developed for plant pest control. In this study, hairpin-type double-stranded RNA (dsRNA) targeting the juvenile hormone (JH) acid methyltransferase (JHAMT) gene (dsJHAMT) was introduced in potato plants via Agrobacterium-mediated transformation. The results indicated that the transcriptional RNA of dsJHAMT accumulated in the transgenic plants. The transcripts and proteins of the L. decemlineata JHAMT gene were significantly reduced in larvae feeding on dsJHAMT transgenic foliage. The dsJHAMT had a significant negative effect on the growth and development of L. decemlineata, especially resulting in less oviposition. Importantly, in the field trials, transgenic plants are high-efficiently protected from insect damage mainly because surviving insects laid fewer or no eggs. Even full protection from beetle damage can be acquired by continuously lowering insect population size at large scale in the field over the years. Therefore, the transgenic plants expressing dsJHAMT successfully provided an additional option for plant pest control.

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.