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Ring gear is an important transmission component, but restricted by its ring-shape structure, the tooth surfaces are pretty hard to be finished after heat treatment. Honing is the most commonly used finishing technique for external gear, but for ring gear, it is inconvenient. In this paper, a new type of cone-shape honing wheel is proposed and the honing mechanism is studied for improving the honing performance of ring gear. First of all, the mathematical model of the honing technology by use of 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 work gear are analyzed, and it is found that the tool tilt angle of the honing wheel has good influences on the honing mark distribution of the work gear. Finally, the honing simulations for ring gear by use of cone-shape honing wheel are carried out, by which the feasibility of the new honing technique are verified.

RNA interference (RNAi) is a breakthrough technology in pest control. It is highly efficient to Coleopteran pests such as the Colorado potato beetle Leptinotarsa decemlineata, a serious pest defoliator mainly attacking potatoes worldwide. The first step for effective pest control by RNAi is the development of effective and reliable target genes.Our results revealed that continuous ingestion of dsLdRan for 3 days successfully silenced the target gene, inhibited larval growth and killed 100% L. decemlineata larvae. When the bioassay began at the second-, third/fourth-instar larval stages, the larval lethality mainly occurred at the fourth larval instar and prepupal stages, respectively. Importantly, consumption of dsLdRan for 3 days by the newly-emerged males and females effectively knocked down the target transcript, reduced fresh weights and caused 100% of lethality within a week. The LdRan females possessed underdeveloped ovaries.Considering that the larvae, adults and eggs are simultaneously sited on the potato plants, bacterially-expressed dsLdRan is a potential RNAi-based strategy for managing L. decemlineata in the potato field. © 2022 Society of Chemical Industry.

In Drosophila melanogaster, ecdysis-triggering hormone (ETH) and eclosion hormone (EH) mutually enhance the release of each other through a positive feedback loop. Both are involved in the stimulation of ecdysis behavior series and molting-related physiological preparations. However, whether 20-hydroxyecdysone (20E) controls eh expression and whether EH plays a similar role in non-Drosophilid insects remain to be determined. In Leptinotarsa decemlineata, we found that Ldeh mRNA levels were high in the early and/or late stages and low in the intermediate period within each larval instar, indicating that the mRNA peaks positively correlate with 20E pulses. Consistent with the indication, we confirmed that 20E signaling stimulated the transcription of Ldeh. RNA interference (RNAi)-aided knockdown of Ldeh at the final instar stage slightly impaired pupation and significantly affected wing expansion. Around 20% of the Ldeh RNAi larvae remained as prepupae, completely wrapped in the old larval cuticles. These prepupae gradually darkened, dried and withered, and finally died. The remaining (about 80%) Ldeh hypomorphs became pupae and emerged as abnormal adults, bearing smaller and wrinkle elytra and hindwings. The adult tracheae in the Ldeh hypomorphs were full of liquid. In addition, the low pupation rate and high deformed adult proportion cannot be rescued by ingestion of either 20E or juvenile hormone. Thus, EH is critical for ecdysis and trachea clearance in L. decemlineata. The failure of trachea clearance may disenable air-swallowing during larva-pupa-adult transition and thus impacts wing growth and expansion.

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.

In insects, an insulin-like peptide (ILP) triggers the formation of the insulin receptor (InR)/the insulin receptor substrate Chico complex. The complex then recruits downstream of receptor kinase (Drk) and phosphatidylinositol-3-kinase (PI3K) to initiate two signaling branches, i.e., Drk-mitogen-activated protein kinase (MAPK) and Pi3K-protein kinase B subdivisions. Previous findings reveal that RNA interference (RNAi) of LdILP2 or Ldchico, rather than Ldpi3k92E, impairs larval-pupal and pupal-adult molting in the Colorado potato beetle Leptinotarsa decemlineata. It is accordingly hypothesized that the Drk-MAPK branch regulates larval metamorphosis. In the present paper, we first found that silencing LdILP2, Ldchico or Ldpi3k92E did not decrease the expression level of Lddrk, indicating other receptor tyrosine kinases’ signaling except insulin pathway is not affected in the RNAi larvae. Moreover, two InRs and Torso were highly expressed in the final larval instars. Furthermore, RNAi of either Lddrk or Ldchico, or both of them equally affected larval-pupal and pupal-adult molts, and similarly repressed the expression of representative MAPK (Ldras and Ldraf), ecdysteroidogenesis (Ldphm and Ldsad), and 20E signaling (LdEcR, LdUSP, LdHR3 and LdE75) genes. 20E feeding by Lddrk RNAi larvae completely restored the reduced mRNA levels of LdEcR, LdHR3 and LdE75, but did not rescued the decreased Lddrk and LdUSP levels and the lowered pupation and emergence rates. Therefore, our findings suggest that the Drk-MAPK branch is involved in metamorphosis regulation in L. decemlineata.

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 dsTai 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.

Chitin deacetylases (CDAs) catalyze N-deacetylation of chitin, a crucial process for chitin modification. In the present paper, LdCDA1 was identified in Leptinotarsa decemlineata. It was copiously expressed in larval foregut, hindgut and epidermis. Just before the molt in the first, second and third larval instars, the mRNA levels of LdCDA1 were high. In the fourth (final)-instar larvae, a peak occurred 4 days after ecdysis. In vivo results revealed that LdCDA1 transcriptionally responded, positively and negatively respectively, to 20-hydroxyecdysone and juvenile hormone titers. Moreover, knockdown of LdCDA1 significantly reduced foliage consumption, lengthened developing period and prevented growth in the final instar larvae. Three distinct lethal phenotypes were noted in the LdCDA1 RNAi larvae. About 30% of the RNAi larvae became moribund and finally died; approximately 50% of deformed pupae died as pharate adults; and around 20% of LdCDA1 depleted pupae finally emerged as abnormal adults and eventually died within 1 week after emergence. Furthermore, chitin content was low and the mRNA levels of five chitin biosynthesis transcripts (LdUAP1, LdUAP2, LdChSAa, LdChSAb and LdChSB) were significantly declined in the LdCDA1 RNAi larvae. In addition, glucose, trehalose and glycogen contents were increased in the LdCDA1 depleted hypomorphs, along with highly expressed genes coding for trehalose and glycogen synthesis enzymes. The findings provide a compelling piece of evidence that CDA1 is critical for chitin deposition in L. decemlineata. Moreover, LdCDA1 may be a potential target for control of the larvae.

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.

Bemisia tabaci (Gennadius) cryptic complex has invaded Xinjiang, China, since 1998. The distribution of Mediterranean (MED) and Middle East-Asia Minor 1 (MEAM1) B. tabaci substrains has been gradually identified due to the development of molecular technology. In this study, the distribution of MED and MEAM1 in Xinjiang was determined by cleaved amplified polymorphic sequence (CAPs). Results showed that MED dominated in northern Xinjiang (84%), whereas MEAM1 was dominant in southern Xinjiang (72%). Five pairs of simple sequence repeat (SSR) primers were used to analyze the genetic diversity of B. tabaci among 36 geographic populations. The genetic diversity of MED and MEAM1was low and varied little among populations in Xinjiang (0.09 ± 0.14 and 0.09 ± 0.13, respectively). Based on ∆K statistic, 13 populations of MEAM1 could be classified into two subgroups at K = 2, whereas the 23 populations of MED could be classified into four subgroups at K = 4. However, Mantel t-test demonstrated no correlation between geographical and genetic distances among B. tabaci complex (R = 0.42, P = 1.00). Neighbor-joining and principal coordinate analysis showed that geographical isolation and interspecific differences were the main causes of the genetic variation. Gene flow predicted that MEAM1 was most likely introduced from Urumqi to the southern Xinjiang. Meanwhile, a large proportion of MED in Kashi region came from Changji and Yining. To block ongoing dispersal, strict detection and flower quarantine regulations need to be enforced.

In Leptinotarsa decemlineata, a final-instar wandering larva typically undergoes an ontogenetic niche shift (ONS), from potato plant during the foraging stage to its pupation site below ground. Using high-throughput sequencing of the bacterial 16S ribosomal RNA gene, we determined the hypothesis that the L. decemlineata pupae harbor stage-specific bacteria to meet the physiological requirements for underground habitat. We identified 34 bacterial phyla, comprising 73 classes, 208 orders, 375 families, and 766 genera in the collected specimens. Microbes across phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were enriched in the pupae, while those in the phylum Proteobacteria, Tenericutes, Firmicutes, and Bacteroidetes dominated in the larvae and adults. A total of 18 genera, including Blastococcus, Corynebacterium_1, Gordonia, Microbacterium, Nocardia, Nocardioides, Rhodococcus, Solirubrobacter, Tsukamurella, Enterococcus, Acinetobacter, Escherichia_Shigella, Lysobacter, Pseudomonas, and Stenotrophomonas, were specifically distributed in pupae. Moreover, soil sterilizing removed a major portion of bacteria in pupae. Specifically, both Enterococcus and Pseudomonas were eliminated in the soil sterilizing and antibiotic-fed beetle groups. Furthermore, the pupation rate and fresh pupal weight were similar, whereas the emergence rate and adult weight were decreased in the antibiotic-fed beetles, compared with controls. The results demonstrate that a switch of bacterial communities occurs in the pupae; the pupal-specific bacteria genera are mainly originated from soil; this bacterial biodiversity improves pupa performance in soil. Our results provide new insight into the evolutionary fitness of L. decemlineata to different environmental niches.

In Drosophila melanogaster, ecdysis triggering hormone (ETH) is the key factor triggering ecdysis behavior and promoting trachea clearance. However, whether ETH plays the dual roles in non-dipteran insects is unknown. In this survey, we found Ldeth mRNA levels were positively correlated with circulating 20-hydroxyecdysone (20E) titers in Leptinotarsa decemlineata. Ingestion of an ecdysteroid agonist halofenozide or 20E stimulated the transcription of Ldeth, whereas RNA interference (RNAi) of ecdysteroidogenesis (LdPTTH or LdSHD) or 20E signaling (LdEcR, LdUSP, or LdFTZ-F1) genes inhibited the expression, indicating ETH acts downstream of 20E. RNAi of Ldeth at the final instar stage impaired pupation. More than 80% of the Ldeth depleted beetles remained as prepupae, completely wrapped in the old larval cuticles. These prepupae became withered, dried and darkened gradually, and finally died in soil. The remaining Ldeth hypomorphs pupated and emerged as abnormal adults, bearing smaller and wrinkle elytrum and hindwing. Moreover, the tracheae in the Ldeth hypomorphs were full of liquid. We accordingly proposed that the failure of trachea clearance disenabled air-swallowing after pupa-adult ecdysis and impacted wing expansion. Our results suggest that ETH plays the dual roles, initiation of ecdysis and motivation of trachea clearance, in a coleopteran. This article is protected by copyright. All rights reserved.

A heterodimer of ultraspiracle (USP) and ecdysone receptor (EcR) mediates 20-hydroxyecdysone (20E) signalling cascade to regulate insect moulting and metamorphosis. However, at least two questions remain to be addressed in terms of the molecular importance of USP in insect species. First, is USP involved in both regulation of ecdysteroidogenesis and mediation of 20E signalling in non-drosophilid insects, as in Drosophila melanogaster? Second, does USP play any role in larval metamorphosis except as the partner of heterodimeric receptor to activate the downstream 20E signalling genes? In this paper, we found that RNA interference (RNAi) of LdUSP in the final (fourth) instar larvae reduced the messenger RNA levels of four ecdysteroidogenesis genes (Ldspo, Ldphm, Lddib and Ldsad) and 20E titre, and repressed the expression of five 20E signal genes (EcRA, HR3, HR4, E74 and E75) in Leptinotarsa decemlineata. The LdUSP RNAi larvae remained as prepupae, with developing antennae, legs and discs of forewings and hindwings. Dietary supplement with 20E restored the expression of the five 20E signal genes, but only partially alleviated the decreased pupation rate in LdUSP RNAi beetles. Knockdown of LdUSP at the penultimate (third) instar larvae did not affect third-fourth instar moulting. However, silencing LdUSP caused similar but less severe impairments on pupation. Accordingly, we propose that USP is undoubtedly necessary for ecdysteroidogenesis, for mediation of 20E signalling and for initiation of metamorphosis in L. decemlineata.

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.

Bacteria of the genus Stenotrophomonas are opportunistic and have been documented in the guts of several insect species. Here, we present the complete genome sequence of S. maltophilia strain CPBW01, isolated from the wings of the Colorado potato beetle, Leptinotarsa decemlineata, collected from potato fields in Urumqi (43.71N, 87.39E), Xinjiang, China.

Broad-Complex (BrC) is a downstream target of both 20-hydroxyecdysone and juvenile hormone signalling. BrC regulates morphogenetic changes between nymphal instars in hemimetabolans, whereas it controls pupal commitment, pupal morphogenesis and inhibits adult differentiation in holometabolans. Among five BrC cDNAs (Z1-Z4 and Z6) identified in the Colorado potato beetle, we found in this work that Z1, Z2 and Z6 were mainly expressed at the last (fourth) instar and prepupal stages, whereas the levels of Z3 and Z4 increased during the penultimate (third) instar stage, peaked at the last instar larval phase and gradually decreased at the prepupal and pupal periods. When knocking down all BrC isoforms by RNA interference (RNAi) at the penultimate instar stage, around 20% of the resultant larvae remained as moribund beetles. These moribund BrC RNAi larvae were completely or partially wrapped in old cuticle. Likewise, a portion of larvae treated for a single double-stranded RNA of Z3, Z4 or Z6 displayed a degree of similar aberrancies, increasing in the order of isoforms Z6

Insect chitin deacetylases (CDAs) are carbohydrate esterases that catalyze N-deacetylation of chitin to generate chitosan, a process essential for chitin organization and compactness during the formation of extracellular chitinous structure. Here we identified two CDA2 splice variants (LdCDA2a and LdCDA2b) in Leptinotarsa decemlineata. Both splices were abundantly expressed in larval foregut, rectum, and epidermis; their levels peaked immediately before ecdysis within each instar. In vivo results revealed that the two isoforms transcriptionally responded, positively and negatively respectively, to 20-hydroxyecdysone and juvenile hormone signaling pathways. RNA interference (RNAi)-aided knockdown of the two LdCDA2 variants (hereafter LdCDA2) or LdCDA2b, rather than LdCDA2a, resulted in three negative effects. First, foliage consumption was significantly reduced, larval developing period was lengthened, and larval growth was retarded. Second, chitin contents were reduced, whereas glucose, trehalose, and glycogen contents were increased in the LdCDA2 and LdCDA2b RNAi larvae. Third, approximately 20% of LdCDA2 and LdCDA2b RNAi larvae were trapped within the exuviae and finally died. About 60% of the abnormal pupae died as pharate adults. Around 20% of the RNAi pupae emerged as deformed adults, with small size and wrinkled wings. These adults eventually died within 1 week after molting. Our results reveal that knockdown of CDA2 affects chitin accumulation. Consequently, LdCDA2 may be a potential target for control of L. decemlineata larvae.

Some insect growth disruptors (IGDs), such as pyriproxyfen and halofenozide, may be used to control Leptinotarsa decemlineata. However, their mechanism of action remains elusive. Methoprene-tolerant (Met) mediates juvenile hormone (JH) signal to control numerous essential physiological processes. In the present paper, we identified a Met gene (LdMet). LdMet protein was a typical basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) transcription factor with a bHLH domain, two PAS domains (PAS-A and PAS-B) and a region called PAS associated C terminal (PAC). Eight conserved amino acids critical for JH binding were located in PAS-B and PAC domains. The temporal expression pattern of LdMet was in accordance with the variation of circulating JH titers. Feeding of juvenoid methoprene or pyriproxyfen, or provide for JH dose-dependently stimulated the expression of LdMet. RNA interference-mediated knockdown of two JH degradation genes increased the transcription of LdMet, while silencing of a JH biosynthesis gene repressed the transcription. Conversely, ingestion of an ecdysteroid agonist halofenozide or 20-hydroxyecdysone (20E) reduced the mRNA levels of LdMet, in a dose-dependent manner, whereas knockdown of either ecdysteroidogenesis or 20E signaling genes increased the mRNA accumulation. Providing that the expression of LdMet can be disturbed by methoprene, pyriproxyfen and halofenozide, LdMet may be a potential target of these IGDs in L. decemlineata larvae.

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.

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.

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.

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.

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.

Sublethal effects of insecticides on insects have been widely documented. However, the underlining mechanisms remain largely unclear. Insecticides may be as ligands and disruptors to affect the expression and function of tyrosine kinase receptors (RTKs). Based on the transcriptome and the genomic data of Leptinotarsa decemlineata , 15 novel RTK members were identified and annotated in the present paper. These RTKs showed distinct 1:1 orthology relationships with Tribolium castaneum ones, suggesting functional conservation of RTKs in insects. The expression responses of the 16 RTKs to sublethal exposure to two juvenile hormone analogs (methoprene and pyriproxyfen) and three γ-aminobutyric acid receptor blockers (fipronil, butene-fipronil and endosulfan) were determined. Each of the 5 insecticides changed the expression levels of a specific subset of RTK genes, demonstrating that RTK genes respond transcriptionally to insecticides by a mode-of-action independent way. Our results raise the possibilities that insecticides may exert their sublethal effects through affecting the expression of RTK genes.

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.

Dietary introduction of bacterially expressed double-stranded RNA (dsRNA) has great potential for management of Leptinotarsa decemlineata. Identification of the most attractive candidate genes for RNA interference (RNAi) is the first step. In the present paper, three complete chitin synthase cDNA sequences (LdChSAa, LdChSAb and LdChSB) were cloned. LdChSAa and LdChSAb, two splicing variants of LdChSA gene, were highly expressed in ectodermally-derived epidermal cells forming epidermis, trachea, foregut and hindgut, whereas LdChSB was mainly transcribed in midgut cells. Feeding bacterially expressed dsChSA (derived from a common fragment of LdChSAa and LdChSAb), dsChSAa, dsChSAb and dsChSB in the second- and fourth-instar larvae specifically knocked down their target mRNAs. RNAi of LdChSAa+LdChSAb and LdChSAa lowered chitin contents in whole body and integument samples, and thinned tracheal taenidia. The resulting larvae failed to ecdyse, pupate, or emerge as adults. Comparably, knockdown of LdChSAb mainly affected pupal-adult molting. The LdChSAb RNAi pupae did not completely shed the old larval exuviae, which caused failure of adult emergence. In contrast, silencing of LdChSB significantly reduced foliage consumption, decreased chitin content in midgut sample, damaged midgut peritrophic matrix, and retarded larval growth. As a result, the development of the LdChSB RNAi hypomorphs was arrested. Our data reveal that these LdChSs are among the effective candidate genes for an RNAi-based control strategy against L. decemlineata.

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.

Trehalose metabolism is critical for production of ATP, provision of carbon source, and facilitation of carbohydrate absorption. Trehalose-6-phosphate synthase (TPS) is a rate-limiting enzyme in trehalose biosynthesis. In the present paper, a TPS gene ( LdTPS ) was cloned in Leptinotarsa decemlineata . At young larval instars, the expression levels of LdTPS were high just before and right after ecdysis, and were low at the mid instar stages. In the fourth-instar larvae, two peaks occurred at 24 h after ecdysis and at the wandering stage. In vitro midgut culture and an in vivo bioassay revealed that 20E stimulated LdTPS transcription. Conversely, a reduction of 20E by RNA interference (RNAi) of a prothoracicotropic hormone receptor gene LdTorso and an ecdysteroidogenesis gene LdSHD repressed LdTPS expression. Moreover, disruption of 20E signaling by knockdown of LdEcR , LdE75 and LdFTZ- F1 reduced LdTPS transcript levels. Similarly, in vitro culture and an in vivo bioassay showed that exogenous JH or JH analog methoprene and pyriproxyfen activated LdTPS expression. An increase in endogenous JH by RNAi of an allatostatin gene LdAS-C enhanced the transcription. In contrast, a decrease in JH by silencing of a JH biosynthesis gene LdJHAMT downregulated LdTPS expression. Moreover, knockdown of LdILP 2 repressed LdTPS transcription. Therefore, LdTPS transcription is tuned by 20E, JH and ILP signaling pathways in L. decemlineata .

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.

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.

Chitin synthase (ChS) plays a critical role in chitin synthesis and excretion. In this study, two ChS genes (LdChSA and LdChSB) were identified in Leptinotarsa decemlineata. LdChSA contains two splicing variants, LdChSAa and LdChSAb. Within the first, second, and third larval instars, the mRNA levels of LdChSAa, LdChSAb, and LdChSB coincide with the peaks of circulating 20-hydroxyecdysone (20E) and juvenile hormone (JH). In vitro culture of midguts and an in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide stimulated the expression of the three LdChSs. Conversely, a reduction of 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD repressed the expression of these LdChSs, and ingestion of halofenozide by LdSHD RNAi larvae rescued the repression. Moreover, disruption of 20E signaling by RNAi of LdEcR, LdE75, LdHR3, and LdFTZ-F1 reduced the expression levels of these genes. Similarly, in vitro culture and an in vivo bioassay showed that exogenous JH and a JH analog methoprene activated the expression of the three LdChSs, whereas a decrease in JH by RNAi of a JH biosynthesis gene LdJHAMT downregulated these LdChSs. It seems that JH upregulates LdChSs at the early stage of each instar, whereas a 20E pulse triggers the transcription of LdChSs during molting in L. decemlineata.

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 LdUAPs, 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 LdUAPs 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 LdUAP1 and LdUAP2 have specialized functions in biosynthesizing chitin in the epidermal cuticle and PM respectively in L. decemlineata.

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.

20-hydroxyecdysone (20E) and juvenile hormone (JH) are key regulators of insect development. In this study, three Leptinotarsa decemlineata Ecdysone-induced protein 75 (LdE75) cDNAs (LdE75A, B and C) were cloned from L. decemlineata. The three LdE75 isoforms were highly expressed just before or right after each moult. Within the fourth larval instar, they showed a small rise and a big peak 40 and 80 h after ecdysis. The expression peaks of the three LdE75s coincided with the peaks of circulating 20E levels. In vitro midgut culture and in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide (Hal) enhanced LdE75 expression in the day 1 final larval instars. Conversely, a decrease in 20E by feeding a double-stranded RNA (dsRNA) against an ecdysteroidogenesis gene, Shade (LdSHD), repressed the expression of LdE75. Moreover, Hal upregulated the expression of the three LdE75s in LdSHD-silenced larvae. Thus, 20E pulses activate the transcription of LdE75s. Furthermore, ingesting dsE75-1 and dsE75-2 from a common fragment of the three isoforms successfully knocked down these LdE75s, and caused developmental arrest. Finally, knocking down LdE75s significantly repressed the transcription of three ecdysteroidogenesis genes, lowered the 20E titre and affected the expression of two 20E-response genes. Silencing LdE75s also induced the expression of a JH biosynthesis gene, increased JH titre and activated the transcription of a JH early-inducible gene. Thus, Ld E75s are required for larval-pupal metamorphosis and act mainly by modulating 20E and JH titres and mediating their signalling pathways.

BACKGROUND Juvenile hormone (JH) plays a critical role in the regulation of metamorphosis in Leptinotarsa decemlineata, a notorious defoliator of potato. JH acid methyltransferase (JHAMT) is involved in one of the final steps of JH biosynthesis. RESULTS A putative JHAMT cDNA (LdJHAMT) was cloned. Two double-stranded RNAs (dsRNAs) (dsJHAMT1 and dsJHAMT2) against LdJHAMT were constructed and bacterially expressed. Experiments were conducted to investigate the effectiveness of RNAi in both second- and fourth-instar larvae. Dietary introduction of dsJHAMT1 and dsJHAMT2 successfully knocked down the target gene, lowered JH titre in the haemolymph and reduced the transcript of Kruppel homologue 1 gene. Ingestion of dsJHAMT caused larval death and weight loss, shortened larval developmental period and impaired pupation. Moreover, the dsJHAMT-fed pupae exhibited lower adult emergence rates. The resulting adults weighed an average of 50 mg less than the control group, and the females did not deposit eggs. Application of pyriproxyfen to the dsJHAMT-fed insects rescued all the negative effects. CONCLUSIONS LdJHAMT expresses functional JHAMT enzyme. The RNAi targeting LdJHAMT could be used for control of L. decemlineata. © 2015 Society of Chemical Industry

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 dsLdSAHase 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 dsegfp 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 dsLdSAHase-caused LdSAHase suppressions and larval mortalities were influenced by previous dsegfp 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.

Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH degradation. In the present article, a putative JHDK cDNA (LdJHDK) was cloned from the Colorado potato beetle Leptinotarsa decemlineata. The cDNA consists of 814 bp, containing a 555 bp open reading frame encoding a 184 amino acid protein. LdJHDK reveals a high degree of identity to the previously reported insect JHDKs. It possesses three conserved purine nucleotide-binding elements, and contains three EF-hand motifs (helix-loop-helix structural domains). LdJHDK mRNA was mainly detected in hindgut and Malpighian tubules. Besides, a trace amount of LdJHDK mRNA was also found in thoracic muscles, brain-corpora cardiaca-corpora allata complex, foregut, midgut, ventral ganglia, fat body, epidermis, and hemocytes. Moreover, LdJHDK was expressed throughout all developmental stages. Within the first, second, and third larval instar, the expression levels of LdJHDK were higher just before and right after the molt, and were lower in the intermediate instar. In the fourth larval instar, the highest peak of LdJHDK occurred 56 h after ecdysis. Ingestion of double-stranded RNA (dsRNA) against LdJHDK successfully knocked down the target gene, increased JH titer, and significantly upregulated LdKr-h1 mRNA level. Knockdown of LdJHDK significantly impaired adult emergence. Thus, we provide a line of experimental evidence in L. decemlineata to support that LdJHDK encodes function protein involved in JH degradation.

The basic helix–loop–helix (bHLH) transcription factors possess crucial functions in cell proliferation, determination, differentiation, cell cycle maintenance and homeostasis or stress response pathways. Since a few juvenile hormone analogues such as hydroprene, methoprene and pyriproxyfen targeting a bHLH member Met are already registered for pest management, there is a potential to develop more insecticides targeting various bHLHs. The identification of bHLH members is the first step. Based on the transcriptome and the genome of the Colorado potato beetle Leptinotarsa decemlineata, the most important pest in potato, 49 bHLH members were identified. All LdbHLH members were defined by their names and families according to various phylogenetic analyses with bHLH homologues of Drosophila melanogaster, Apis mellifera, Bombyx mori and Tribolium castaneum. Among these LdbHLHs, 17, 10, 10, 1, 10 and 1 members belonged to A, B, C, D, E and F high-order groups, respectively. The results would provide useful background information for future studies on functions of bHLH proteins in the regulation of L. decemlineata development. Moreover, the bHLHs could serve as potential insecticide targets. These proteins could be used in small molecule screen, or in the development of RNAi-based pest management methods.

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 dsLdAS-C significantly increased the LdJHAMT mRNA levels by 3.8 and 9.9 fold respectively. In contrast, ingestion of dsLdAS-B only slightly increased the LdJHAMT expression level by 1.1 and 1.7 fold. Moreover, after one, two and three days' ingestion of dsLdAS-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 dsLdAS-C and dsLdAS-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.

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.

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. Δ-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) (dsLdp5cdh1 and dsLdp5cdh2) targeting Ldp5cdh were constructed and bacterially expressed. Ingestion of dsLdp5cdh1 and dsLdp5cdh2 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. © 2014 Society of Chemical Industry

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.