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During choriogenesis in insects, chorion (eggshell) is formed by surrounding follicular epithelial cells in ovarioles. However, the regulatory endocrine factor(s) activating choriogenesis and the effect of chemical components on eggshell deserve further exploration. In two representative coleopterans, a coccinellid Henosepilachna vigintioctopunctata and a chrysomelid Leptinotarsa decemlineata, genes encoding the 20-hydroxyecdysone (20E) receptor heterodimer, ecdysone receptor (EcR) and ultraspiracle (USP), and two chitin biosynthesis enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP) and chitin synthase (ChS1), were highly expressed in ovaries of the young females. RNA interference (RNAi)-aided knockdown of either HvEcR or Hvusp in H. vigintioctopunctata inhibited oviposition, suppressed the expression of HvChS1, and lessened the positive signal of Calcofluor staining on the chorions, which suggests the reduction of a chitin-like substance (CLS) deposited on eggshells. Similarly, RNAi of LdEcR or Ldusp in L. decemlineata constrained oviposition, decreased the expression of LdUAP1 and LdChS1, and reduced CLS contents in the resultant ovaries. Knockdown of LdUAP1 or LdChS1 caused similar defective phenotypes, i.e., reduced oviposition and CLS contents in the L. decemlineata ovaries. These results, for the first time, indicate that 20E signaling activates choriogenesis in two coleopteran species. Moreover, our findings suggest the deposition of a CLS on the chorions. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is a promising approach to control Leptinotarsa decemlineata . In this study, RNAi efficiency by double-stranded RNA (dsRNA) targeting S-adenosyl-L-homocysteine hydrolase ( LdSAHase ) was compared among L. decemlineata first- to fourth-instar larvae. Ingesting ds LdSAHase successfully decreased the target gene expression, caused lethality, inhibited growth and impaired pupation in an instar- and concentration-dependent manner. To study the role of Dicer2 and Argonaute2 genes in RNAi efficiency, we identified LdDcr2a , LdDcr2b , LdAgo2a and LdAgo2b . Their expression levels were higher in young larvae than those in old ones. Exposure to ds egfp for 6 h significantly elevated LdDcr2a , LdDcr2b , LdAgo2a and LdAgo2b mRNA levels in the first-, second-, third- and fourth-instar larvae. When the exposure periods were extended, however, the expression levels were gradually reduced. Continuous exposure for 72 h significantly repressed the expression of LdAgo2a and LdAgo2b in the first, second and third larval instars, and the four genes in final instars. Moreover, we found that ds LdSAHase -caused LdSAHase suppressions and larval mortalities were influenced by previous ds egfp exposure: 12 h of previous exposure increased LdSAHase silencing and mortality of the final instar larvae, whereas 72 h of exposure reduced LdSAHase silencing and mortality. Thus, it seems the activities of core RNAi-machinery proteins affect RNAi efficiency in L. decemlineata . [ABSTRACT FROM AUTHOR]

Our previous results revealed that RNA interference-aided knockdown of Leptinotarsa decemlineata FTZ-F1 ( LdFTZ-F1 ) reduced 20E titer, and impaired pupation. In this study, we characterized a putative LdHR3 gene, an early-late 20E-response gene upstream of LdFTZ-F1 . Within the first, second and third larval instars, three expression peaks of LdHR3 occurred just before the molt. In the fourth (final) larval instar 80 h after ecdysis and prepupal stage 3 days after burying into soil, two LdHR3 peaks occurred. The LdHR3 expression peaks coincide with the peaks of circulating 20E level. In vitro midgut culture and in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide (Hal) enhanced LdHR3 expression in the final larval instars. Conversely, a decrease in 20E by feeding a double-stranded RNA (dsRNA) against an ecdysteroidogenesis gene Ldshd repressed the expression. Moreover, Hal rescued the transcript levels in the Ldshd -silenced larvae. Thus, 20E peaks activate the expression of LdHR3 . Furthermore, ingesting dsRNA against LdHR3 successfully knocked down the target gene, and impaired pupation. Finally, knockdown of LdHR3 upregulated the transcription of three ecdysteroidogenesis genes ( Ldphm , Lddib and Ldshd ), increased 20E titer, and activated the expression of two 20E-response genes ( LdEcR and LdFTZ-F1 ). Thus, Ld HR3 functions in regulation of pupation in the Colorado potato beetle. [ABSTRACT FROM AUTHOR]

Vacuolar-type H + -ATPases (vATPases) are localized in the apical membranes of nearly all epithelial tissues of insects, energize the membranes to absorb and/or secrete ions and fluids, and play essential roles in many physiological functions. Here we cloned and characterized a 1041-bp full-length vATPase subunit E cDNA (named as LdATPaseE ) that encoded a 226-amino acid protein in Leptinotarsa decemlineata . LdATPaseE mRNA levels were constantly increased from egg to the third- and fourth-instar stages, dropped in wandering and pupal stages and were elevated again in the adult stage. It was highly expressed in ileum and rectum, moderately expressed in Malpighian tubules, midgut and foregut, and lowly expressed in fat body, ventral ganglion, epidermis and haemocytes in the fourth instars. After continuously ingested double-stranded RNAs originated from two LdATPaseE fragments LdATPaseE1 and LdATPaseE2 , the target mRNA levels in the larvae were reduced by 85% and 55%, the larval growth and survival were significantly affected. Furthermore, topical application of fipronil, butane-fipronil, endosulfan and cypermethrin significantly upregulated LdATPaseE expression up to 8.3, 4.2, 2.8 and 6.2-fold 1 day after experiment, and up to 15.8, 3.4, 3.6 and 4.5-fold 2 days after treatment. It seems that depletion of vATPase subunit E is lethal, indicating that targeting vATPases by dsRNA appears a promising means of combating L. decemlineata . Moreover, vATPase subunit E is a pesticide inducible gene and may play a role in pesticide toxicity. [ABSTRACT FROM AUTHOR]

Background: L. decemlineata is an exotic invasive insect pest, and invaded in Xinjiang Uygur autonomous region in China in the 1990s from Kazakhstan. It is a notorious defoliator of potato throughout most of the northern Xinjiang in current, and often causes extremely large yield losses of potato. Results: The expression stability of nine L. decemlineata house-keeping genes (Actin, ACT1 and ACT2; ADPribosylation factor, ARF1 and ARF4; TATA box binding protein, TBP1 and TBP2; ribosomal protein RP4 and RP18; translation elongation factor 1a EF1a) was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) in seven developmental stages, three larval tissues and two insecticide treatments. The results were analyzed using three software programs: geNorm, NormFinder and BestKeeper. Although there was no consistent ranking observed among the house-keeping genes across the samples, the overall analysis revealed that RP18, RP4, ARF1, and ARF4 were the four most stable house-keeping genes. In contrast, ACT1 and ACT2, two of the most widely used reference genes, had the least stability. Our results suggest that the combined use of the four most stably expressed genes may produce optimal normalization for qRT-PCR. Conclusions: The expression stability of the house-keeping genes varies among different developing stages, in different tissues and under different experimental conditions. Our results will enable a more accurate and reliable normalization of qRT-PCR data in L. decemlineata. [ABSTRACT FROM AUTHOR]

Abstract: A survey of resistance to five conventional insecticides was conducted in 2009 and 2010 for the first generation 4th-instar larvae of Leptinotarsa decemlineata from Urumqi, Changji, Qitai and Qapqal. Compared with the Tekes population, a reference susceptible population, the Changji and Qapqal populations exhibited very high to moderate levels of resistance to cyhalothrin and deltamethrin, moderate to high levels of resistance to carbosulfan and carbofuran, and low levels of resistance to azinphosmethyl. Moreover, the Urumqi and the Qitai populations reached a high and a moderate level of resistance to carbosulfan, respectively. Synergistic effects of triphenyl phosphate, diethylmeleate, and piperonyl butoxide on cyhalothrin and carbosulfan in Changji population revealed that cytochrome P450s were involved in the resistance to cyhalothrin but not carbosulfan. A modified bi-PASA was developed to simultaneously detect point mutations of S291G in the AChE and L1014F in the LdVssc1 genes. The former mutation resulted in the resistance to carbamates and the latter in the resistance to pyrethroids. The rates of homozygous and heterozygous resistant individuals to carbamates (S291G mutation) were 17.6% and 14.7%, 50.6% and 42.2%, 49.9% and 41.7%, 51.3% and 41.4%, and 44.8% and 47.4%; to pyrethroids (L1014F mutation) were 5.8% and 8.7%, 36.1% and 27.0%, 41.8% and 24.8%, 12.2% and 9.7%, and 7.9% and 10.6%, respectively, in samples from Tekes, Changji, Qapqal, Urumqi and Qitai. I392T point mutation in the AChE was detected by RT-PCR among 18 individuals from Changji, Qapqal, Urumqi and Qitai. These results demonstrated that point mutations of S291G in the AChE and L1014F in the LdVscc1 are responsible for, at least partially, the resistance to carbamates and pyrethroids in L. decemlineata in some field populations in northern Xinjiang Uygur autonomous region. [Copyright &y& Elsevier]

Internal gear is an important transmission component. However, it is restricted by its ring−shaped structure, which hinders the finishing process of the tooth surfaces after the heat treatment. Honing is the most commonly used finishing technique for external gear, but it is inconvenient for internal gear. In this paper, a new type of cone−shape honing wheel is proposed and the honing mechanism for improving the honing performance of the internal gear is studied. First, the mathematical model of the honing technology that employs a cone−shape honing wheel is built, and the merits of the new honing method are discussed. Then, the contact conditions between the honing wheel and the internal gear are analyzed. It is established that the tool tilt angle of the honing wheel has a good effect on the honing velocity distributions. Finally, the simulations of honing and profile measuring of the internal gear by employing the cone−shape honing wheel are carried out, by which the feasibility of the new honing technique is verified. [ABSTRACT FROM AUTHOR]

Biomass was used as reducing agent to roast the Baotou low-grade limonite in a high temperature vacuum atmosphere furnace. The effect of calcination temperature, time and ratio of reducing agent on the magnetic properties of calcined ore was studied by VSM. The phase and microstructure changes of limonite before and after calcination were analyzed by XRD and SEM. The results show that in the roasting process the phase transition process of the ferrous material in limonite is first dehydrated at high temperature to formα-Fe2O3, and then it is converted into Fe3O4 by the reduction of biomass. With the increase of calcination temperature, the magnetic properties of the calcined ore first increase and then decrease. When the temperature is higher than 650°C, Fe3O4 will become Fe2SiO4, resulting in reduced the magnetic material in calcined ore and the magnetic weakened. The best magnetization effect was obtained when the roasting temperature is 550°C, the percentage of biomass was 15% and the roasting time was 30min. The saturation magnetization can reach 60.13emu·g-1, the recovery of iron was 72% and the grade of iron was 58%. [ABSTRACT FROM AUTHOR]

A new method of cycloid disc tooth modification is presented in this paper. Its main idea is to design the modification clearance curves to adapt to different modification targets. A detailed procedure of the new modification clearance curve, which can be defined by adjusting the position of 5 key points is developed. Numerical experiments are carried out to verify the effectiveness of the new method. A Multi-DOF nonlinear dynamic model of cycloidal speed reducer is established, then the cycloid disc rotational displacement and rotational velocity versus time of different modification clearance are solved by the Runge-kutta numerical method. The results show that this method can improve the carrying capability of cycloidal drive, eliminate noise and vibration and develop the transmission accuracy. [ABSTRACT FROM AUTHOR]

Five insulin-like peptide LdILP genes were identified in Leptinotarsa decemlineata . All of them contained three exons and two introns, with three genes tandemly arrayed and well separated from the other two . Phylogenetic analysis revealed that the three Ld ILPs from three tandemly-arrayed genes grouped with Tc ILP1, whereas the other two resembled with Tc ILP2 and Tc ILP4 from Tribolium castaneum. Thus, the five LdILP genes were provisionally named LdILP1a , LdILP1b , LdILP1c , LdILP2 and LdILP4 . LdILP2 was widely expressed in several tissues such as the brain–corpora cardiaca–corpora allata (BR–CC–CA) complex, gut and fat body. In contrast, LdILP1a and LdILP1b were only transcribed in BR–CC–CA, LdILP4 was in ovaries, and LdILP1c was in both BR–CC–CA and ovaries. Ingestion of double-stranded RNAs (dsRNAs) targeting LdILP2 (ds LdILP2 -1 and ds LdILP2 -2) specifically knocked down LdILP2 and upregulated the transcription of both LdInR and Ld4EBP , indicating insulin/insulin-like growth factor signaling pathway (IIS) was inhibited. Approximately 50% of the LdILP2 RNAi larvae did not normally pupate and about 50% of the LdILP2 RNAi pupae did not emerge. Moreover, silencing LdILP2 reduced the expression of a juvenile hormone (JH) biosynthesis gene, lowered JH titer and disturbed JH signaling. Finally, knocking down LdILP2 inhibited an ecdysteroidogenesis gene, decreased 20-hydroxyecdysone (20E) titer, and repressed the expression of two 20E-response genes LdHR3 and LdFTZ-F1 . Thus, the IIS pathway is involved in larval–pupal metamorphosis by modification of both JH and 20E signaling in L. decemlineata . [ABSTRACT FROM AUTHOR]

In Drosophila melanogaster prothoracic gland (PG) cells, Torso mediates prothoracicotropic hormone (PTTH)-triggered mitogen activated protein kinase (MAPK) pathway (consisting of four core components Ras, Raf, MEK and ERK) to stimulate ecdysteroidogenesis. In this study, LdTorso , LdRas , LdRaf and LdERK were cloned in Leptinotarsa decemlineata . The four genes were highly or moderately expressed in the larval prothoracic glands. At the first- to third-instar stages, their expression levels were higher just before and right after the molt, and were lower in the mid instars. At the fourth-instar stage, their transcript levels were higher before prepupal stage. RNA interference-mediated knockdown of LdTorso delayed larval development, increased pupal weight, and impaired pupation and adult emergence. Moreover, knockdown of LdTorso decreased the mRNA levels of LdRas , LdRaf and LdERK , repressed the transcription of two ecdysteroidogenesis genes ( LdPHM and LdDIB ), lowered 20E titer, and downregulated the expression of several 20E-response genes ( LdEcR , LdUSP , LdHR3 and LdFTZ-F1 ). Furthermore, silencing of LdTorso induced the expression of a JH biosynthesis gene LdJHAMT , increased JH titer, and activated the transcription of a JH early-inducible gene LdKr-h1 . Thus, our results suggest that Torso transduces PTTH-triggered MAPK signal to regulate ecdysteroidogenesis in the PGs in a non-drosophiline insect. [ABSTRACT FROM AUTHOR]

In insect, juvenile hormone (JH) titers are tightly regulated in different development stages through synthesis and degradation pathways. During JH degradation, JH epoxide hydrolase (JHEH) converts JH to JH diol, and hydrolyses JH acid to JH acid diol. In this study, two full length LdJHEH cDNAs were cloned from Leptinotarsa decemlineata , and were provisionally designated LdJHEH1 and LdJHEH2 . Both mRNAs were detectable in the thoracic muscles, brain–corpora cardiaca–corpora allata complex, foregut, midgut, hindgut, ventral ganglia, Malpighian tubules, fat bodies, epidermis, and hemocytes of the day 2 fourth-instar larvae, and in female ovaries as well as male reproductive organs of the adults. Moreover, both LdJHEH1 and LdJHEH2 were expressed throughout all larval life, with the highest peaks occurring 32 h after ecdysis of the final (fourth) instar larvae. Four double-stranded RNAs (dsRNAs) (ds JHEH1 -1, ds JHEH1 -2, ds JHEH2 -1, ds JHEH2 -2) respectively targeting LdJHEH1 and LdJHEH2 were constructed and bacterially expressed. Ingestion of ds JHEH1 -1, ds JHEH1 -2, ds JHEH2 -1, ds JHEH2 -2, and a mixture of ds JHEH1 -1 + ds JHEH2 -1 successfully knocked down corresponding target gene function, and significantly increased JH titer and upregulated Krüppel homolog 1 ( LdKr-h1 ) mRNA level. Knockdown of either LdJHEH1 or LdJHEH2 , or both genes slightly reduced larval weight and delayed larval development, and significantly impaired adult emergence. Therefore, it is suggested that knockdown LdJHEH1 and LdJHEH2 affected JH degradation in the Colorado potato beetle. [ABSTRACT FROM AUTHOR]

Leptinotarsa decemlineata adults exhibit a season-dependent activity. In spring, post-diapause beetles often fly a long distance from overwintering sites to potato fields. In summer and autumn, the flight ability is sharply reduced. Proline is the main energy substrate of L. decemlineata during flight and proline dehydrogenase (ProDH) catalyzes the first step in proline catabolism. Here we identified a putative LdProDH gene; it had three cDNA isoforms which shared the same 5′UTR and coding region, but differed in the lengths of 3′UTRs (515, 1 092 and 1 242 bp for isoforms-1, -2 and -3, respectively). LdProDH encoded a 616 amino acid protein that showed high sequence similarity to ProDH-like proteins from other insect species. LdProDH was expressed in the third and fourth instars larvae and adults, but not in pupae. Dietary ingestion of bacterially expressed LdProDH-dsRNA by adults significantly decreased its messenger RNA (mRNA) level, and caused an elevation of free proline content in the hemolymph. Further observation revealed that three canonical polyadenylation signals (AATAAA) were tandemly located in the 3′UTR of isoform-3. The first, second and third polyadenylation sites gave rise to isoforms-1, -2 and -3, respectively. Analysis of the genomic DNA uncovered that the three isoforms resulted from alternative polyadenylation. The mRNA level of isoform-1, which expressed at low levels in pre-diapause adults, became abundant in post-diapause beetles. It is indicated that the LdProDH expression is fine-tuned through 3′UTR to control proline catabolism for the season-dependent activity of L. decemlineata adults. [ABSTRACT FROM AUTHOR]

Tuta absoluta is a devastating invasive pest worldwide, causing severe damage to the global tomato industry. It has been recorded recently in the northwestern border areas of China, posing a significant threat to tomato production. It was presumed that the region's winter-related low temperatures would avert the alien species from successfully overwintering. In this study, the supercooling capacity and low-temperature tolerance of this pest were examined under laboratory conditions and its overwintering potential in Xinjiang was estimated. The results showed that the lowest supercooling point was recorded in the adult stage (− 19.47 °C), while the highest (− 18.11 °C) was recorded in the pupal stage. The supercooling points of pupae and adults were not influenced by gender. The Ltemp50 and Ltemp90 of female and male adults were the lowest when exposed to cold for 2 h. However, when the duration of exposure extended from 4 to 10 h, the Ltemp50 and Ltemp90 of female and male pupae were the lowest. Comparison of the lowest Ltemp50 and Ltemp90 with temperatures in January indicated that T. absoluta might not be able to overwinter in most of the northern and central regions of Xinjiang. However, in the southern regions, the extremely low temperature was higher than the Ltemp90, suggesting that T. absoluta has a higher overwintering potential in these regions. These results form a basis for predicting the dispersal potential and possible geographic range of this pest in Xinjiang. In addition, our findings provide guidance for the control of this pest by reducing overwintering shelters. [ABSTRACT FROM AUTHOR]

A heterodimer of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle, mediates 20‐hydroxyecdysone (20E) signaling to modulate many aspects in insect life, such as molting and metamorphosis, reproduction, diapause and innate immunity. In the present paper, we intended to determine the isoform‐specific roles of EcR during larval–pupal–adult transition in the Colorado potato beetle. Double‐stranded RNAs (dsRNAs) were prepared using the common (dsEcR) or isoform‐specific (dsEcRA, dsEcRB1) regions of EcR as templates. Ingestion of either dsEcR or dsEcRA, rather than dsEcRB1, by the penultimate (3rd) and final (4th) instar larvae caused failure of larval–pupal and pupal–adult ecdysis. The RNA interference (RNAi) larvae remained as prepupae, or became deformed pupae and adults. Determination of messenger RNA (mRNA) levels of EcR isoforms found that LdEcRA regulates the expression of LdEcRB1. Moreover, silencing the two EcR transcripts, LdEcRA or LdEcRB1 reduced the mRNA levels of Ldspo and Ldsad, and lowered 20E titer. In contrast, the expression levels of HR3, HR4, E74 and E75 were significantly decreased in the LdEcR or LdEcRA RNAi larvae, but not in LdEcRB1 depleted specimens. Dietary supplement with 20E did not restore the expression of five 20E signaling genes (USP, HR3, HR4, E74 and E75), and only partially alleviated the pupation defects in dsEcR‐ or dsEcRA‐fed beetles. These data suggest that EcR plays isoform‐specific roles in the regulation of ecdysteroidogenesis and the transduction of 20E signal in L. decemlineata. [ABSTRACT FROM AUTHOR]

An exploration of novel control strategies for Leptinotarsa decemlineata is becoming more pressing given rapid evolution of insecticide resistance and rise of production loss of potato. Dietary delivery of bacterially expressed double-stranded RNA (dsRNA) is a promising alternative for management. An important first step is to uncover possible RNA-interference (RNAi)-target genes effective against both young and old larvae. Taiman (Tai) is a basic-helix-loop-helix/Per-Arnt-Sim transcription factor that is involved in the mediation of both juvenile hormone (JH) and 20-hydroxyecdysone (20E) signaling. In the present paper, we found that continuous ingestion of ds Tai for three days by third (penultimate)-instar larvae caused approximately 20% larval mortality and 80% pupation failure. The larval lethality resulted from failed cuticle and tracheae shedding, which subsequently reduced foliage consumption and nutrient absorption, and depleted lipid stores. In contrast, pupation failure derived from disturbed JH and 20E signals, and disordered nutrient homeostasis including, among others, inhibition of trehalose metabolism and reduction of chitin content. Knockdown of LdTai caused similar larval lethality and pupation impairment in second and fourth (final) larval instars. Therefore, LdTai is among the most attractive candidate genes for RNAi to control L. decemlineata larvae. Unlabelled Image • DsRNA of LdTaiman (ds Tai) inhibits pupation in 3rd instar larvae. • Ds Tai leads to similar pupation impairment in 2nd and 4th larval instars. • Defects by ds Tai result from disturbed metabolism, and JH and 20E signals. • LdTaiman is a potential target for RNAi to control L. decemlineata larvae. [ABSTRACT FROM AUTHOR]

Many animals exploit several niches sequentially during their life cycles, a fitness referred to as ontogenetic niche shift (ONS). To successfully accomplish ONS, transition between development stages is often coupled with changes in one or more primitive, instinctive behaviors. Yet, the underlining molecular mechanisms remain elusive. We show here that Leptinotarsa decemlineata larvae finish their ONS at the wandering stage by leaving the plant and pupating in soil. At middle wandering phase, larvae also switch their phototactic behavior, from photophilic at foraging period to photophobic. We find that enhancement of juvenile hormone (JH) signal delays the phototactic switch, and vise verse. Moreover, RNA interference (RNAi)-aided knockdown of LdPTTH (prothoracicotropic hormone gene) or LdTorso (PTTH receptor gene) impairs avoidance response to light, a phenotype nonrescuable by 20-hydroxyecdysone. Consequently, the RNAi beetles pupate at the soil surface or in shallow layer of soil, with most of them failing to construct pupation chambers. Furthermore, a combination of depletion of LdPTTH/LdTorso and disturbance of JH signal causes no additive effects on light avoidance response and pupation site selection. Finally, we establish that TrpA1 (transient receptor potential (TRP) cation channel) is necessary for light avoidance behavior, acting downstream of PTTH. We conclude that JH/PTTH cascade concomitantly regulates metamorphosis and the phototaxis switch, to drive ONS of the wandering beetles from plant into soil to start the immobile pupal stage. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract Image 1 Highlights • Knockdown of chico or pi3k92E postpones larval development and inhibits larval growth in Leptinotarsa decemlineata. • Knockdown of Ldchico , rather than Ldpi3k92E , impairs larval-pupal and pupal-adult ecdysis. • Chico is critical for the regulation of metamorphosis by a PI3K-independent pathway. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To accomplish consistent, long-term, integrated management (IPM) of the Colorado potato beetle, Leptinotarsa decemlineata (Say), research assessing the potential of novel, IPM-compatible insecticides is essential. Novaluron is a potent benzoylurea insecticide. In the present paper, we found that novaluron ingestion by the fourth-instar larvae inhibited foliage consumption, reduced larval fresh weight, and delayed development period, in a dose dependent manner. Most of the resulting larvae fail to pupate, and died at prepupae stage, with larvicidal activity comparable with those of cyhalothrin and spinosad but lower than those of fipronil and abamectin. Moreover, many surviving pupae that fed novaluron failed to emerge as adults, in a dose dependent pattern. Furthermore, feeding of novaluron significantly decreased chitin contents in body carcass (without midgut) and integument specimen, whereas the chitin concentration in the midgut peritrophic matrix was not affected. Furthermore, uridine diphosphate- N -acetylglucosamine-pyrophosphorylase gene ( LdUAP1 ) and chitin synthase Aa ( LdChSAa ), which were mainly responsible for chitin biosynthesis in ectodermally-derived tissues, were surpressed and activated respectively after novaluron ingestion. Therefore, novaluron is an effective benzoylurea insecticide to L. decemlineata fourth-instar larvae. It inhibited chitin biosynthesis in ectodermally-derived tissues, disrupted ecdysis, impaired pupation and adult emergence, and led to death in juvenile life stages. [ABSTRACT FROM AUTHOR]

Drosophila cap ‘n’ collar isoform C (CncC) and Kelch-like ECH associated protein 1 (Keap1) regulate metamorphosis by transcriptional control of a subset of genes involved in ecdysteroidogenesis, 20-hydroxyecdysone (20E) signaling, and juvenile hormone (JH) degradation. In the present paper, we found that prothoracicotropic hormone signal was required for the activation of LdCncC and LdKeap1 in Leptinotarsa decemlineata . Moreover, RNA interference of LdCncC or LdKeap1 in the fourth-instar larvae delayed development. As a result, the treated larvae obtained heavier larval and pupal fresh weights and had larger body sizes than the controls. Furthermore, knockdown of LdCncC or LdKeap1 significantly reduced the mRNA levels of four ecdysone biosynthetic genes ( Ldspo , Ldphm , Lddib and Ldsad ), lowered 20E titer and decreased the transcript levels of five 20E response genes ( LdEcR , LdUSP , LdE75 , LdHR3 and LdFTZ-F1 ). However, the expression of two JH epoxide hydrolase genes and JH contents were not affected in the LdCncC and LdKeap1 RNAi larvae. Dietary supplementation with 20E shortened the developmental period to normal length, rescued the larval and pupal body mass rises, and recovered or even overcompensated the expression levels of the five 20E response genes in either LdCncC or LdKeap1 RNAi hypomorphs. Therefore, Ld CncC/ Ld Keap1 signaling regulates several ecdysteroidogenesis genes, and consequently 20E pulse, to modulate the onset of metamorphosis in L. decemlineata . [ABSTRACT FROM AUTHOR]

Trehalose is proposed to serve multiple physiological roles in insects. However, its importance remains largely unconfirmed. In the present paper, we knocked down either a trehalose biosynthesis gene (trehalose-6-phosphate synthase, LdTPS ) or each of three degradation genes (soluble trehalases LdTRE1a , LdTRE1b or membrane-bound LdTRE2 ) in Leptinotarsa decemlineata by RNA interference (RNAi). Knockdown of LdTPS decreased trehalose content and caused larval and pupal lethality. The LdTPS RNAi survivors consumed a greater amount of foliage, obtained a heavier body mass, accumulated more glycogen, lipid and proline, and had a smaller amount of chitin compared with the controls. Ingestion of trehalose but not glucose rescued the food consumption increase and larval mass rise, increased survivorship, and recovered glycogen, lipid and chitin to the normal levels. In contrast, silencing of LdTRE1a increased trehalose content and resulted in larval and pupal lethality. The surviving LdTRE1a RNAi hypomorphs fed a smaller quantity of food, had a lighter body weight, depleted lipid and several glucogenic amino acids, and contained a smaller amount of chitin. Neither trehalose nor glucose ingestion rescued these LdTRE1a RNAi defects. Silencing of LdTRE1b caused little effects. Knockdown of LdTRE2 caused larval death, increased trehalose contents in several tissues and diminished glycogen in the brain-corpora cardiaca-corpora allata complex (BCC). Feeding glucose but not trehalose partially rescued the high mortality rate and recovered glycogen content in the BCC. It seems that trehalose is involved in feeding regulation, sugar absorption, brain energy supply and chitin biosynthesis in L. decemlineata larvae. [ABSTRACT FROM AUTHOR]

The mevalonate pathway is responsible for the biosynthesis of many essential molecules important in insect development, reproduction, chemical communication and defense. Based on Leptinotarsa decemlineata transcriptome and genome data, we identified ten genes that encoded acetoacetyl-CoA thiolase ( LdAACT1 and LdAACT2 ), hydroxymethylglutaryl (HMA)-CoA synthase ( LdHMGS ), HMG-CoA reductase ( LdHMGR1 and LdHMGR2 ), mevalonate kinase ( LdMevK ), phospho-mevalonate kinase ( LdPMK ), mevalonate diphosphate decarboxylase ( LdMDD ), isopentenyl-diphosphate isomerase ( LdIDI ) and farnesyl pyrophosphate synthetase ( LdFPPS ). Nine of these genes (except for LdAACT1 ) were mainly expressed in the larval brain-corpora cardiaca-corpora allata complex, and adult ovary and testis. The 9 genes were transcribed at high levels right after each ecdysis, and at low levels in the mid instar. Therefore, the 9 genes were indicated to be involved in JH biosynthesis. Moreover, knockdown of a JH biosynthesis gene LdJHAMT to lower JH titer significantly downregulated the transcription of the 9 genes. Ingestion of JH to activate JH signaling also significantly suppressed the expression of the 9 genes. It appears that the accumulation of JH precursors in LdJHAMT RNAi larvae and a high JH titer in JH-fed specimens may cause negative feedbacks to repress the expression of the 9 mevalonate enzyme-encoding genes (excluding LdAACT1 ) to balance the enzyme quantity in L. decemlineata . [ABSTRACT FROM AUTHOR]

Uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (UAP) is involved in the biosynthesis of chitin, an essential component of the epidermal cuticle and midgut peritrophic matrix (PM) in insects. In the present paper, two putative LdUAP genes were cloned in Leptinotarsa decemlineata . In vivo bioassay revealed that 20-hydroxyecdysone (20E) and an ecdysteroid agonist halofenozide activated the expression of the two LdUAP s, whereas a decrease in 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD and a 20E signaling gene LdFTZ-F1 repressed the expression. Juvenile hormone (JH), a JH analog pyriproxyfen and an increase in JH by RNAi of an allatostatin gene LdAS-C downregulated LdUAP1 but upregulated LdUAP2 , whereas a decrease in JH by silencing of a JH biosynthesis gene LdJHAMT had converse effects. Thus, expression of LdUAP s responded to both 20E and JH. Moreover, knockdown of LdUAP1 reduced chitin contents in whole larvae and integument samples, thinned tracheal taenidia, impaired larval–larval molt, larval-pupal ecdysis and adult emergence. In contrast, silencing of LdUAP2 significantly reduced foliage consumption, decreased chitin content in midgut samples, damaged PM, and retarded larval growth. The resulting larvae had lighter fresh weights, smaller body sizes and depleted fat body. As a result, the development was arrested. Combined knockdown of LdUAP1 and LdUAP2 caused an additive negative effect. Our data suggest that Ld UAP1 and Ld UAP2 have specialized functions in biosynthesizing chitin in the epidermal cuticle and PM respectively in L. decemlineata . [ABSTRACT FROM AUTHOR]

Based on the Leptinotarsa decemlineata transcriptome dataset and the GenBank sequences, 70 novel carboxylesterases and 2 acetylcholinesterases were found. The 72 members belong to a multifunctional carboxylesterase/cholinesterase superfamily (CCE). A phylogenetic tree including the 72 Ld CCEs and the CCEs from Tribolium castaneum, Drosophila melanogaster and Apis mellifera revealed that all CCEs fell into three main phylogenetic groups: dietary/detoxification, hormone/semiochemical processing, and neurodevelopmental classes. Numbers of L. decemlineata CCEs in the three classes were 52, 12 and 8, respectively. The dietary/detoxification class includes two clades: coleopteran xenobiotic metabolizing and α-esterase type CCEs. CCEs in the two clades have independently expanded in L. decemlineata . The hormone/semiochemical processing class has three clades: integument CCEs, β- and pheromone CCEs and juvenile hormone CCEs. Integument CCEs in L. decemlineata have also expanded. The neurodevelopmental CCEs are implicated the most ancient class, containing acetylcholinesterase, neuroligin, neurotactin, glutactin, gliotactin and others. Among the 70 novel CCE genes, KM220566, KM220530, KM220576, KM220527 and KM220541 were fipronil-inducible, and KM220578, KM220566, KM220542, KM220564, KM220561, KM220554, KM220527, KM220538 and KM220541 were cyhalothrin-inducible. They were the candidates involving in insecticide detoxification. Moreover, our results also provided a platform to understand the functions and evolution of L. decemlineata CCE genes. [ABSTRACT FROM AUTHOR]

Alanine aminotransferase (ALT) plays important physiological and biochemical roles in insect. In this study, a full-length Ldalt cDNA was cloned from Leptinotarsa decemlineata. It was ubiquitously expressed in the eggs, larvae, pupae and adults. In the adults, Ldalt mRNA was widely distributed in thorax muscles, fat body, midgut, foregut, hindgut, Malpighian tubules, ventral ganglion and epidermis, with the expression levels from the highest to the lowest. Two double-stranded RNAs (dsRNAs) (ds Ldalt1 and ds Ldalt2) targeting Ldalt were constructed and bacterially expressed. After adults fed on ds Ldalt1- and ds Ldalt2-immersed foliage for 3 day, Ldalt mRNA abundance was significantly decreased by 79.5 and 71.1 %, and ALT activities were significantly reduced by 64.5 and 67.6 %, respectively. Moreover, silencing Ldalt affected free amino acid contents. Lysine was decreased by 100.0 and 100.0 %, and arginine was reduced by 87.5 and 89.4 %, respectively, in the hemolymph from ds Ldalt1- and ds Ldalt2-ingested beetles, compared with control ones. In contrast, proline was increased by 88.7 and 96.4 %. Furthermore, ingestion of ds Ldalt1 and ds Ldalt2 significantly decreased flight speed, shortened flight duration time and flight distance. In addition, knocking down Ldalt significantly increased adult mortality. These data imply that LdALT plays important roles in amino acid metabolism and in flight in L. decemlineata. [ABSTRACT FROM AUTHOR]

Juvenile hormone III (JH III) plays primary roles in regulation of metamorphosis, reproduction and diapause in Leptinotarsa decemlineata , a notorious defoliator of potato. The neurosecretory cell-borne substance(s) negatively affects the final two steps in JH biosynthesis, catalyzed respectively by an epoxidase CYP15A1 and a juvenile hormone acid methyltransferase (JHAMT). In a few insect species other than L. decemlineata , the inhibitory substance is allatostatin (AS) neuropeptide. In this study, two putative AS genes encoding LdAS-C and LdAS-B precursors were cloned. Both LdAS - C and LdAS - B were expressed in the egg, larvae, pupae and adults, and highly expressed in the brain and the gut. Dietary introduction of double-stranded RNAs (dsRNAs) targeting LdAS - C and LdAS - B successfully knocked down respective target genes. Ingestion during 3 and 6 consecutive days of ds LdAS - C significantly increased the LdJHAMT mRNA levels by 3.8 and 9.9 fold respectively. In contrast, ingestion of ds LdAS - B only slightly increased the LdJHAMT expression level by 1.1 and 1.7 fold. Moreover, after one, two and three days' ingestion of ds LdAS - C , the relative JH levels in the hemolymph of treated larvae were 2.5, 4.2 and 1.9 fold higher than those in control beetles. Furthermore, ingestion of ds LdAS - C and ds LdAS - B significantly affected larval growth and delayed larval development. Thus, we provide a line of experimental evidence in L. decemlineata to support the concept that AS-C acts as an allatostatin and inhibit JH biosynthesis. [ABSTRACT FROM AUTHOR]

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) (ds Ldp5cs 1 and ds Ldp5cs 2) targeting Ldp5cs were constructed and bacterially expressed. Ingestion during 3 consecutive days of ds Ldp5cs 1 or ds Ldp5cs 2 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 . [ABSTRACT FROM AUTHOR]

Highlights: [•] A total of 74 cytochrome P450 genes (Cyps) were identified. [•] Nine Cyp genes were highly expressed in cyhalothrin-treated larvae. [•] Our results facilitate further researches. [Copyright &y& Elsevier]

Highlights: [•] A putative LdSAHase gene was cloned from L. decemlineata. [•] dsLdSAHase ingestion caused negative effects on larval performance. [•] Pyriproxyfen application partially rescued the negative effects. [•] This suggests that LdSAHase plays a critical role in JH biosynthesis. [ABSTRACT FROM AUTHOR]

The Colorado potato beetle ( Leptinotarsa decemlineata [Say]) in the north Xinjiang Uygur autonomous region in China has evolved resistance to pyrethroids and carbamates. Mutations resulting in target site insensitivity, namely L1014F in LdVssc1 voltage-sensitive sodium channel and S291G in acetylcholine esterase, confer, at least partially, the resistance. Insecticides with different modes of action may serve as likely replacements. Endosulfan and fipronil are GABA-gated chloride channel-blocking insecticides. In this article, we found that the contact toxicities of the two compounds were among the highest ever estimated to the 4th-instar larvae and the adults and affirmed that they were useful for L. decemlineata control in north Xinjiang. Regarding stomach toxicities of the two compounds, adult beetles were less sensitive than 2nd-, 3rd-, and 4th-instar larvae, suggesting that the appropriate timing for spraying is the early larval stage. Mixtures of endosulfan and α-cypermethrin at 1:160, 1:80, and 1:40 ratios, of endosulfan and phoxim at 1:24 ratio, and of endosulfan and isocarbophos at 1:72 and 1:288 ratios, significantly increased toxicity in a field population. The combination indices were significantly below 1 at both LD and LD levels, revealing synergistic effects. Our results demonstrated that endosulfan and fipronil could be applied alone and endosulfan may also be used in binary mixtures to restore pyrethroid susceptibility. These findings may have considerable practical implications for L. decemlineata resistance management. [ABSTRACT FROM AUTHOR]

[Display omitted] • 20E stimulates the expression of LdFoxO. • Silencing LdFoxO represses 20E signaling and reduces larval weight. • RNAi of LdFoxO impairs larva-pupa-adult transition. • 20E or JH consumption cannot restore defects in LdFoxO depleted animals. Forkhead box O (FoxO) protein, a major downstream transcription factor of insulin/insulin-like growth factor signaling/target of rapamycin pathway (IIS/TOR), is involved in the regulation of larval growth and the determination of organ size. FoxO also interacts with 20-hydroxyecdysone (20E) and juvenile hormone (JH) signal transduction pathways, and hence is critical for larval development in holometabolans. However, whether FoxO plays a critical role during larval metamorphosis needs to be further determined in Leptinotarsa decemlineata. We found that 20E stimulated the expression of LdFoxO. RNA interference (RNAi)-aided knockdown of LdFoxO at the third-instar stage repressed 20E signaling and reduced larval weight. Although the resultant larvae survived through the third-fourth instar ecdysis, around 70% of the LdFoxO depleted moribund beetles developmentally arrested at prepupae stage. These LdFoxO depleted beetles were completely wrapped in the larval exuviae, gradually darkened and finally died. Moreover, approximately 12% of the LdFoxO RNAi beetles died as pharate adults. Ingestion of either 20E or JH by the LdFoxO depletion beetles excessively rescued the corresponding hormonal signals, but could not alleviate larval performance and restore defective phenotypes. Therefore, FoxO plays an important role in regulation of larval-pupal-adult transformation in L. decemlineata , in addition to mediation of IIS/TOR pathway and stimulation of ecdysteroidogenesis. [ABSTRACT FROM AUTHOR]

Crustacean cardioactive peptide (CCAP), a highly conserved amidated neuropeptide, stimulates feeding in Drosophila melanogaster and Periplaneta americana , and regulates pupa-adult transition in Tribolium castaneum and Manduca sexta. In the present paper, we intended to address whether CCAP plays the dual roles in the Colorado potato beetle Leptinotarsa decemlineata. We found that the levels of Ldccap were high in the dissected samples of brain-corpora cardiaca-corpora allata complex and ventral nerve cord, midgut and hindgut in the final (fourth)-instar larvae. A pulse of 20-hydroxyecdysone triggered the expression of Ldccap in the central nervous system but decreased the transcription in the midgut. In contrast, juvenile hormone intensified the expression of Ldccap in the midgut. RNA interference (RNAi)-aided knockdown of Ldccap at the penultimate instar stage inhibited foliage consumption, reduced the contents of trehalose and chitin, and lowered the mRNA levels of two chitin biosynthesis genes (LdUAP1 and LdChSAb). Moreover, around 70% of the Ldccap RNAi larvae remained as prepupae, completely wrapped in the old larval exuviae, and finally died. The remaining RNAi beetles continually developed to severely-deformed adults: most having wrinkled and smaller elytra and hindwings, and shortened legs. Therefore, CCAP plays three distinct roles, stimulating feeding in foraging larval stage, regulating ecdysis, and facilitating wing expansion and appendage elongation in a coleopteran. In addition, Ldccap can be used as a potential target gene for developing novel management strategies against this coleopteran pest. [Display omitted] • 20E triggers the expression of Ldccap in neural tissues. • JH intensifies the transcription of Ldccap in the midgut. • Knockdown of Ldeth in larvae affects foliage consumption. • Silencing Ldeth arrests larval development. • The RNAi adults have wrinkled and smaller wings and shortened legs. [ABSTRACT FROM AUTHOR]

• 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. [ABSTRACT FROM AUTHOR]