BACKGROUND Leptinotarsa decemlineata is an able disperser by flight. Novel control strategies must be explored to control the damage and inhibit the dispersal efficiently. Proline is a major energy ...substrate during flight. Delta -Pyrroline-5-carboxylate dehydrogenase (P5CDh) catalyses the second step of proline degradation for the production of ATP. RESULTS A full-length Ldp5cdh cDNA was cloned. Ldp5cdh was ubiquitously expressed in the eggs, the first through fourth larval instars, wandering larvae, pupae and adults. In the adults, Ldp5cdh mRNA was widely distributed in thorax muscles, midgut, foregut, hindgut, Malpighian tubules, ventral ganglion, fat body and epidermis, with the expression levels from the highest to the lowest. Two double-stranded RNAs (dsRNAs) (ds Ldp5cdh 1 and ds Ldp5cdh2 ) targeting Ldp5cdh were constructed and bacterially expressed. Ingestion of ds Ldp5cdh 1 and ds Ldp5cdh2 successfully silenced Ldp5cdh , significantly increased the contents of proline, arginine and alanine, but strongly decreased the contents of asparate, asparagine, glutamate and glutamine in the haemolymph. Moreover, knocking down Ldp5cdh significantly reduced ATP content, decreased flight speed, shortened flight distance and increased adult mortality. CONCLUSIONS It seems that identified Ldp5cdh encodes a functional P5CDh enzyme, and Ldp5cdh may serve as a potential target for dsRNA-based pesticide for controlling the damage and dispersal of L. decemlineata adults.
Delta 1-pyrroline-5-carboxylate synthetase (P5CS) catalyzes the conversion of glutamate (Glu) to Glu semialdehyde (GSA). GSA spontaneously cyclizes to form P5C. P5C is then reduced to proline (Pro) ...or is converted to ornithine, the intermediate for arginine (Arg) biosynthesis. In the present study, a full-length Ldp5cs complementary DNA was cloned from the Colorado potato beetle Leptinotarsa decemlineata, a notorious insect defoliator of potato in most potato-growing regions of the world. Ldp5cs encodes a 792-amino-acid protein which shares high identity to homologues from other insect species. Quantitative reverse transcription polymerase chain reaction revealed that Ldp5cs was ubiquitously expressed in the eggs, first to fourth-instar larvae, wandering larvae, pupae and sexually mature adults. In the adults, Ldp5cs mRNA levels were higher in the fat body, foregut, midgut and hindgut, moderate in the ventral ganglion, lower in the thorax muscles, epidermis and Malpighian tubules. Two double-stranded RNAs (dsRNAs) (dsLdp5cs1 and dsLdp5cs2) targeting Ldp5cs were constructed and bacterially expressed. Ingestion during 3 consecutive days of dsLdp5cs1 or dsLdp5cs2 successfully silenced Ldp5cs, significantly reduced the contents of Pro and Arg in the hemolymph, decreased flight speed and shortened flight distance of the resulting adults. Furthermore, knocking down Ldp5cs significantly increased adult mortality. Thus, our results suggest that identified Ldp5cs encodes a functional P5CS enzyme that is involved in the biosynthesis of Pro and Arg in L. decemlineata.
RNA interference (RNAi) is being used to develop methods to control pests and disease vectors. RNAi is robust and systemic in coleopteran insects but is quite variable in other insects. The ...determinants of efficient RNAi in coleopterans, as well as its potential mechanisms of resistance, are not known. RNAi screen identified a double-stranded RNA binding protein (StaufenC) as a major player in RNAi. StaufenC homologs have been identified in only coleopteran insects. Experiments in two coleopteran insects, Leptinotarsa decemlineata and Tribolium castaneum, showed the requirement of StaufenC for RNAi, especially for processing of double-stranded RNA (dsRNA) to small interfering RNA. RNAi-resistant cells were selected by exposing L. decemlineata, Lepd-SL1 cells to the inhibitor of apoptosis 1 dsRNA for multiple generations. The resistant cells showed lower levels of StaufenC expression compared with its expression in susceptible cells. These studies showed that coleopteran-specific StaufenC is required for RNAi and is a potential target for RNAi resistance. The data included in this article will help improve RNAi in noncoleopteran insects and manage RNAi resistance in coleopteran insects.
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•Knockdown of LdHR38 inhibits ecdysteroidogenesis in third instar larvae.•Silencing LdHR38 intensify the expression of early ecdysone genes but represses LdFTZ-F1.•Depletion of LdHR38 ...impairs larval-pupal ecdysis.•Silencing LdHR38 at the fourth instar causes similar but less severe defects.
In insects, nuclear receptors (NRs) including EcR (NR1H1), USP (NR2B4), E75 (NR1D3), HR3 (NR1F), HR4 (NR6) and FTZ-F1 (NR5A3) mediate the 20-hydroxyecdysone (20E) signaling cascade to play a critical role during larval metamorphosis. In this present paper, we focused on hormone receptor 38 (HR38) in Leptinotarsa decemlineata, the only insect homolog of the NR4A subclass. RNA interference (RNAi) of LdHR38 in the penultimate (third) instar larvae reduced the expression of an ecdysteroidogenesis gene and declined the titer of 20E. Knockdown of LdHR38 intensified the expression of LdUSP, LdE75, LdE74, LdE93, LdBroad and LdHR3, whereas repressed the transcription of LdFTZ-F1. Disruption of 20E signaling inhibited chitin biosynthesis in the larval cuticle. Approximately 25% of the LdHR38 RNAi larvae died, around 40% of the resultant larvae remained as prepupae or become deformed pupae. The body surface of the HR38 depleted abnormal prepupae and pupae looked wet, just like the cuticle being covered with a layer of liquid. Moreover, the increase of larval mortality, and the impairment of pupation and emergence exhibited dose-dependent manners. Furthermore, silencing LdHR38 at the final (fourth) instar caused similar but less severe impairment of pupation. Dietary supplement with 20E for the third instar larvae did not rescue the high larval death and only slightly alleviated the low pupation rate in the LdHR38 RNAi hypomorphs. Accordingly, we propose that HR38 is necessary for tune of ecdysteroidogenesis and for mediation of 20E signaling during metamorphosis in L. decemlineata.
The Colorado beetle, Leptinotarsa decemlineata (Say), is one of the most important pests in many potato‐producing regions. Colorado beetle infestations are normally kept under economic damage ...thresholds by applying insecticides such as pyrethroids. Pyrethroids are known to act on voltage‐gated sodium channels and have been used for several decades to control L. decemlineata. Their continuous and widespread use has resulted in the development of resistance, which is often linked to a L1014F target‐site mutation in the voltage‐gated sodium channel, known as knockdown resistance (kdr). Since pyrethroids are used in many potato‐growing regions in Germany, more than 140 L. decemlineata samples were collected and analysed for the presence of the kdr allele by pyrosequencing diagnostics. Results showed that kdr is present in many German L. decemlineata populations, but even its homozygous presence does not substantially compromise the efficacy of recommended label rates of pyrethroids, as demonstrated by bioassays and crossing experiments. The implications of these findings for resistance management are briefly discussed.
Incidence et propagation de la résistance knockdown (kdr) dans les populations allemandes de doryphores de la pomme de terre (Leptinotarsadecemlineata Say)
L'un des organismes nuisibles les plus importants dans de nombreuses régions productrices de pommes de terre est le doryphore, Leptinotarsadecemlineata (Say). Les infestations de doryphores sont normalement maintenues sous les seuils de dégâts économiques en appliquant des insecticides tels que les pyréthrinoïdes. Les pyréthrinoïdes sont connus pour agir sur les canaux sodiques et ont été utilisés pour la lutte contre L. decemlineata depuis plusieurs décennies. Leur utilisation continue et généralisée a entraîné le développement d'une résistance, qui est souvent liée á une mutation du site‐cible L1014F dans le canal sodique, connue sous le nom de résistance knockdown (kdr). Dans la mesure oú les pyréthrinoïdes sont utilisés dans de nombreuses régions de culture de pommes de terre en Allemagne, plus de 140 échantillons de L. decemlineata ont été prélevés et analysés pour la diagnostiquer la présence de l'alléle kdr par pyroséquençage. Les résultats ont montré que kdr est présent dans de nombreuses populations allemandes de L. decemlineata, cependant même sa présence homozygote ne compromet pas considérablement l'efficacité des pyréthrinoïdes aux doses recommandées sur l'étiquette comme démontré par des essais biologiques et des expériences de croisement. Les implications de ces résultats pour la gestion de la résistance sont discutées briévement.
Встречаемость и распространенность резистентности ‘kdr’ в немецких популяциях колорадского жука (Leptinotarsa decemlineata Say)
Один из наиболее серьезных вредителей во многих производящих картофель районах – колорадский жук Leptinotarsa decemlineata (Say). Зараженность колорадским жуком обычно поддерживается ниже порогов вредоносности за счет применения инсектицидов, таких как пиретроиды. Пиретроиды, как известно, воздействуют на управляемый импульсами напряжения натриевый канал, и использовались для борьбы с L. decemlineata в течение нескольких десятилетий. Их непрерывное и широкое использование привело к развитию резистентности, которая часто связана с мутацией целевого сайта L1014F в управляемом импульсами напряжения натриевом канале, известной как резистентность ‘kdr’. В связи с тем, что пиретроиды используются во многих районах Германии, где выращивается картофель, больше 140 образцов L. decemlineata были собраны и проанализированы на наличие аллелей ‘kdr’ методом диагностики пиросеквенирования. Результаты исследования показали, что ‘kdr’ присутствует во многих немецких популяциях L. decemlineata, но даже его гомозиготное присутствие существенно не ставит под вопрос эффективность рекомендуемых доз применения пиретроидов, что доказывается биоанализами и перекрестными экспериментами. В статье кратко рассматривается значение этих результатов для управления резистентностью.
The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), is considered as one of the most important pests of potato plants. Control of CPB is mainly based on ...chemical insecticides. However, control failure is often reported because of the rapid resistance development ability of CPB; therefore, there is an urgent need for integrated control methods. In this study, we evaluated the combined usage of insecticides chlorantraniliprole, spinosad, and spinetoram, which are registered for CPB control, with the entomopathogenic nematode Steinernema feltiae (Rhabditida: Steinernematidae).
In addition, the efficacy of S. feltiae on a chlorantraniliprole-resistant CPB population exposed to synergistic chemicals PBO (Piperonyl butoxide), DEM (Diethyl maleate) and DEF (S,S,S-tributylphorotrithioate) inhibiting certain detoxification enzyme groups was determined. The tested insecticides showed no adverse effect on the survival and infectivity of S. feltiae and the isolate was considered compatible with integrated CPB control. This study also showed that pre-treatment with synergistic chemicals (particularly PBO and DEM) increased the efficacy of S. feltiae against CPB. To our knowledge, this is the first study determining the usage of entomopathogenic nematode + synergist combination. Therefore, further studies on the potential use of synergistic chemicals in combination with entomopathogenic nematodes should be performed under both laboratory and field conditions.
•Leptinotarsa decemlineata is one of the major pests affecting potato production worldwide.•Insecticide resistance in Leptinotarsa decemlineata indicates an urgent need to develop sustainable control methods.•Entomopathogenic nematode Steinernema feltiae is a potential biological control agent of Leptinotarsa decemlineata.
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