RNA Interference-Mediated Suppression of Ecdysone Signaling Inhibits Choriogenesis in Two Coleoptera Species.

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.

Crucial roles of specialized chitinases in elytral and hindwing cuticles construction in Leptinotarsa decemlineata.

BACKGROUND: The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major potato (Solanum tuberosum) pest, infesting over 16 million km2 and causing substantial economic losses. The insect cuticle forms an apical extracellular matrix (ECM) envelope covering exposed organs to direct morphogenesis and confer structural protection. While select chitinase (Cht) genes have proven essential for larval development, their potential activities directing ECM remodeling underlying adult wing maturation remain undefined. RESULTS: We investigated the expression patterns and performed an oral RNA interference (RNAi) screen targeting 19 LdChts in late-instar L. decemlineata larvae. Subsequently, we assessed their effects on adult eclosion and wing characteristics. Knockdown of LdCht5, LdCht7, LdCht10, LdIDGF2, and LdIDGF4, as well as others from Group IV (LdCht15, LdCht12, LdCht17, and LdCht13) and Groups VII-X (LdCht2, LdCht11, LdCht1, and LdCht3), resulting in shrunken, misshapen elytra with reduced areal density, as well as transverse wrinkling and impaired wing-tip folding in hindwings. Scanning electron micrographs revealed eroded elytral ridges alongside thinned, ruptured hindwing veins, indicative of mechanical fragility post-LdCht suppression. Spectroscopic analysis uncovered biomolecular alterations underlying the elytral anomalies, including decreases in peaks representing chitin, proteins, and lipids. This loss of essential ECM components provides evidence for the fragility, wrinkling, and shrinkage observed in the RNAi groups. CONCLUSION: Our findings elucidate the crucial role of chitinases in the turnover of chitinous cuticles on beetle wings, offering insights into RNAi-based control strategies against this invasive pest. © 2024 Society of Chemical Industry.

Identification of chitinase genes and roles in the larval-pupal transition of Leptinotarsa decemlineata.

BACKGROUND: Insect chitinases play crucial roles in degrading chitin in the extracellular matrix, affecting insect development and molting. However, our understanding of the specific functions of various chitinases in Leptinotarsa decemlineata is limited, hindering the deployment of novel gene-targeting technologies as pest management strategies. RESULTS: We identified and characterized 19 full-length complementary DNA (cDNA) sequences of chitinase genes (LdChts) in Leptinotarsa decemlineata. Despite having varying domain architectures, all these chitinases contained at least one chitinase catalytic domain. Phylogenetic analysis classified the chitinase proteins into ten distinct clusters (groups I-X). Expression profiles showed the highest expression in chitin-rich tissues or during specific developmental stages from the larva-to-pupa transition. Gene-specific RNA interference (RNAi) experiments provided valuable insight into chitinase gene function. Silencing of group II LdCht10 prevented larval-larval molting, larval-prepupal, and prepupal-pupal processes. Moreover, our study revealed that LdCht5, LdCht2, LdCht11, LdCht1, and LdCht3 from groups I and VII-X were specifically essential for the transition from prepupal to pupal stage, whereas LdIDGF2 from group V was necessary for the larval-prepupal metamorphic process. The chitinase gene LdCht7 from group III and LdIDGF4 from group V were involved in both the larva-to-prepupa and the prepupa-to-pupa shift. Additionally, our findings also shed light on the exclusive expression of nine chitinase genes within group IV in the digestive system, suggesting their potential role in regulating larval body weight and larva-to-pupa transition. CONCLUSION: Our results provide a comprehensive understanding of the functional specialization of chitinase genes during the molting process of various stages and identify potential targets for RNAi-based management of Leptinotarsa decemlineata. © 2023 Society of Chemical Industry.

RNA interference targeting Ras GTPase gene Ran causes larval and adult lethality in Leptinotarsa decemlineata.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

The actions of neonicotinoid insecticides on nicotinic acetylcholine subunits Ldα1 and Ldα8 of Leptinotarsa decemlineata and assembled receptors.

The insect nicotinic acetylcholine receptor (nAChR) is a pentameric channel protein and also a target for neonicotinoids. There are few reported studies on the molecular interactions of Leptinotarsa decemlineata nAChRs with neonicotinoids. In this study, we analyzed the response of acetylcholine and neonicotinoids (thiamethoxam [TMX], imidacloprid [IMI], and clothianidin [CLO]) on hybrid receptors constructed by nAChR α1 and α8 subunits of L. decemlineata (Ldα1 and Ldα8) co-expressed with rat ß2 subunit (rß2) at different capped RNA (cRNA) ratios in Xenopus oocytes. In addition, we evaluated the expression changes of Ldα1 and Ldα8 after median lethal dose of TMX treatment for 72 h by quantitative polymerase chain reaction (qPCR). The resulting functional nAChRs Ldα1/rß2 and Ldα1/Ldα8/rß2 showed different pharmacological characteristics. The neonicotinoids tested showed lower agonist affinity on Ldα1/Ldα8/rß2 compared to Ldα1/rß2 at same ratios of subunit cRNAs. The sensitivities of neonicotinoids tested for Ldα1/rß2 and Ldα1/Ldα8/rß2 at cRNA ratios of 5:1, 1:1 and 5:5:1, 1:1:1, respectively, were lower than those for nAChRs at ratios of 1:5 and 1:1:5, respectively, whereas the values of maximum response (Imax ) varied. For Ldα1/Ldα8/rß2, a reduction of Lda8 cRNA resulted in increased sensitivity to IMI and decreased sensitivity to TMX. The expression of Ldα1 and Ldα8 significantly decreased in adults by 82.12% and 47.02%, respectively, while Ldα8 was significantly upregulated by 2.44 times in 4th instar larvae after exposure to TMX. We infer that Ldα1 and Ldα8 together play an important role in the sensitivity of L. decemlineata to neonicotinoids.

The Leptinotarsa forkhead transcription factor O exerts a key function during larval-pupal-adult transition.

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.

Crustacean cardioactive peptide as a stimulator of feeding and a regulator of ecdysis in Leptinotarsa 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 switch of microbial flora coupled with ontogenetic niche shift in Leptinotarsa decemlineata.

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.

Two Splice Isoforms of Leptinotarsa Ecdysis Triggering Hormone Receptor Have Distinct Roles in Larva-Pupa Transition.

Insect ecdysis triggering hormone (ETH) receptors (ETHRs) are rhodopsin-like G protein-coupled receptors. Upon binding its ligand ETH, ETHR initiates a precisely programed ecdysis behavior series and physiological events. In Drosophila melanogaster, the ethr gene produces two functionally distinct splicing isoforms, ethra and ethrb. ETH/ETHRA activates eclosion hormone (EH), kinin, crustacean cardioactive peptide (CCAP), and bursicon (burs and pburs) neurons, among others, in a rigid order, to elicit the behavioral sequences and physiological actions for ecdysis at all developmental stages, whereas ETH/ETHRB is required at both pupal and adult ecdysis. However, the role of ETHRB in regulation of molting has not been clarified in any non-drosophila insects. In the present paper, we found that 20-hydroxyecdysone (20E) signaling triggers the expression of both ethra and ethrb in a Coleopteran insect pest, the Colorado potato beetle Leptinotarsa decemlineata. RNA interference (RNAi) was performed using double-stranded RNAs (dsRNAs) targeting the common (dsethr) or isoform-specific (dsethra, dsethrb) regions of ethr. RNAi of dsethr, dsethra, or dsethrb by the final-instar larvae arrested larva development. The arrest was not rescued by feeding 20E. All the ethra depleted larvae stopped development at prepupae stage; the body cavity was expanded by a large amount of liquid. Comparably, more than 80% of the ethrb RNAi larvae developmentally halted at the prepupae stage. The remaining Ldethrb hypomorphs became pupae, with blackened wings and highly-expressed burs, pburs and four melanin biosynthesis genes. Therefore, ETHRA and ETHRB play isoform-specific roles in regulation of ecdysis during larva-pupa transition in L. decemlineata.

Complete Genome Sequence of Stenotrophomonas maltophilia Strain CPBW01, Isolated from the Wings of the Colorado Potato Beetle in Xinjiang, China.

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.

Involvement of Leptinotarsa hormone receptor 38 in the larval-pupal transition.

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.

Ecdysone receptor isoforms play distinct roles in larval-pupal-adult transition in Leptinotarsa 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.

Silencing Taiman impairs larval development in Leptinotarsa 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.

Hormonal signaling cascades required for phototaxis switch in wandering Leptinotarsa decemlineata larvae.

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.

RNA interference against the putative insulin receptor substrate gene chico affects metamorphosis in Leptinotarsa 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.

Involvement of methoprene-tolerant (Met) in the determination of the final body size in Leptinotarsa decemlineata (Say) 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.

Knockdown of a putative insulin-like peptide gene LdILP2 in Leptinotarsa decemlineata by RNA interference impairs pupation and adult emergence.

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 LdILPs from three tandemly-arrayed genes grouped with TcILP1, whereas the other two resembled with TcILP2 and TcILP4 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 (dsLdILP2-1 and dsLdILP2-2) 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.

Knockdown of juvenile hormone acid methyl transferase severely affects the performance of Leptinotarsa decemlineata (Say) larvae and adults.

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 Krüppel 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.

Knockdown of a nutrient amino acid transporter gene LdNAT1 reduces free neutral amino acid contents and impairs Leptinotarsa decemlineata pupation.

A Leptinotarsa decemlineata SLC6 NAT gene (LdNAT1) was cloned. LdNAT1 was highly expressed in the larval alimentary canal especially midgut. LdNAT1 mRNA levels were high right after the molt and low just before the molt. JH and a JH analog pyriproxyfen activated LdNAT1 expression. RNAi of an allatostatin gene LdAS-C increased JH and upregulated LdNAT1 transcription. Conversely, silencing of a JH biosynthesis gene LdJHAMT decreased JH and reduced LdNAT1 expression. Moreover, 20E and an ecdysteroid agonist halofenozide repressed LdNAT1 expression, whereas a decrease in 20E by RNAi of an ecdysteroidogenesis gene LdSHD and disruption of 20E signaling by knockdown of LdE75 and LdFTZ-F1 activated LdNAT1 expression. Thus, LdNAT1 responded to both 20E and JH. Moreover, knockdown of LdNAT1 reduced the contents of cysteine, histidine, isoleucine, leucine, methionine, phenylalanine and serine in the larval bodies and increased the contents of these amino acids in the larval feces. Furthermore, RNAi of LdNAT1 inhibited insulin/target of rapamycin pathway, lowered 20E and JH titers, reduced 20E and JH signaling, retarded larval growth and impaired pupation. These data showed that LdNAT1 was involved in the absorption of several neutral amino acids critical for larval growth and metamorphosis.

Instar-dependent systemic RNA interference response in Leptinotarsa decemlineata larvae.

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.