Ji-Feng Shi

Functions of nuclear receptor HR3 during larval-pupal molting in Leptinotarsa decemlineata (Say) revealed by in vivo RNA interference

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, LdHR3 functions in regulation of pupation in the Colorado potato beetle.

Nuclear receptor ecdysone-induced protein 75 is required for larval–pupal metamorphosis in the Colorado potato beetle Leptinotarsa decemlineata (Say)

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.

RNA interference of chitin synthase genes inhibits chitin biosynthesis and affects larval performance in Leptinotarsa decemlineata (Say).

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.

IDENTIFICATION AND HORMONE INDUCTION OF PUTATIVE CHITIN SYNTHASE GENES AND SPLICE VARIANTS IN Leptinotarsa decemlineata (SAY)

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.

Novaluron ingestion causes larval lethality and inhibits chitin content in Leptinotarsa decemlineata fourth-instar larvae

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.

Effect of Teflubenzuron Ingestion on Larval Performance and Chitin Content in Leptinotarsa 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.ResumenPara lograr el manejo integral consistente, a largo plazo, del escarabajo de la papa de Colorado, Leptinotarsa decemlineata (Say), se evalúa el potencial de teflubenzuron en el laboratorio. El producto exhibió excelente toxicidad estomacal en la larva. Su actividad larvicida fue comparable con la de cyhalothrin, chlorantraniliprole y spinosad. Observamos los efectos negativos de la ingestión de teflubenzuron en la larva. La larva alimentada con este producto consumió menos follaje, creció más lentamente, y tuvo un período más largo para su desarrollo de una manera dosis dependiente. La mayoría de estas larvas murieron durante la muda larva-larva, larva-ecdicis pupal, o emergencia de adulto. Aún más., los contenidos de quitina en el cuerpo (sin el intestino medio) y el integumento del espécimen de la larva resultante disminuyeron significativamente, mientras que no se afectó la cantidad de quitina en la matriz peritrófica en el intestino medio. Adicionalmente, el gen uridina difosfato-N-acetilglucosamina-pirofosforilasa (LdUAP1) que fue el principalmente responsable de la biosíntesis de quitina en tejidos derivados ectodermalmente, fue suprimido después de la ingestión de teflubenzuron, en contraste a su complemento LdUAP2 para la formación de quitina en la matriz peritrófica del intestino medio. En una palabra, mediante la inhibición de la formación de quitina, teflubenzuron puede ser una herramienta valiosa en programas efectivos de IPM contra L. decemlineata.