Kai-Yun Fu

Validation of reference genes for expression analysis by quantitative real-time PCR in Leptinotarsa decemlineata (Say)

BackgroundL. decemlineata is an exotic invasive insect pest, and invaded in Xinjiang Uygur autonomous region in China in the 1990s from Kazakhstan. It is a notorious defoliator of potato throughout most of the northern Xinjiang in current, and often causes extremely large yield losses of potato.ResultsThe expression stability of nine L. decemlineata house-keeping genes (Actin, ACT1 and ACT2; ADP-ribosylation factor, ARF1 and ARF4; TATA box binding protein, TBP1 and TBP2; ribosomal protein RP4 and RP18; translation elongation factor 1α EF1α) was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) in seven developmental stages, three larval tissues and two insecticide treatments. The results were analyzed using three software programs: geNorm, NormFinder and BestKeeper. Although there was no consistent ranking observed among the house-keeping genes across the samples, the overall analysis revealed that RP18, RP4, ARF1, and ARF4 were the four most stable house-keeping genes. In contrast, ACT1 and ACT2, two of the most widely used reference genes, had the least stability. Our results suggest that the combined use of the four most stably expressed genes may produce optimal normalization for qRT-PCR.ConclusionsThe expression stability of the house-keeping genes varies among different developing stages, in different tissues and under different experimental conditions. Our results will enable a more accurate and reliable normalization of qRT-PCR data in L. decemlineata.

Colorado Potato Beetle Leptinotarsa decemlineata (Say)

Colorado potato beetle (CPB) was naturally dispersed from Kazakhstan into Xinjiang Autonomous Region of China in 1993. Since then, it has been widely distributed all over the southern region of the Tianshan Mountain. Chinese scientists focused on its monitoring and invasion risk management in China, as well as its invasion biology and ecology related to rapid dispersal, such as developmental threshold and cumulative temperature, diapause condition, and influence factors for flight. In invaded regions of China, several techniques such as improved crop cultivation techniques, friendly environmental chemical control (low or none toxic insecticides), biological control, physical techniques, ecological regulations etc. can be combined into an integrated pest management of CPB. However, some questions still remain in theses fields, for example, the genetic variations, environment (hosts, habitats, climates, soil etc.) adaptabilities and geographical populations of CPB are still unclear, and mechanisms of CPB’s rapid resistance development to pesticides have not been well understood. The concerned diapause mechanisms are still unknown. Moreover, the interactions between CPB, hosts, pathogens, predators and parasitoids, environments have not been studied, and the resistance or tolerance of potato plants to CPB and their related mechanisms all need to be understood in order to breed CPB-resistant potato crops.

Involvement of FTZ-F1 in the regulation of pupation in Leptinotarsa decemlineata (Say).

During the final instar larvae of holometabolous insects, a pulse of 20-hydroxyecdysone (20E) and a drop in juvenile hormone (JH) trigger larval-pupal metamorphosis. In this study, two LdFTZ-F1 cDNAs (LdFTZ-F1-1 and LdFTZ-F1-2) were cloned in Leptinotarsa decemlineata. Both LdFTZ-F1-1 and LdFTZ-F1-2 were highly expressed just before or right after each molt, similar to the expression pattern of an ecdysteroidogenesis gene LdSHD. Ingestion of an ecdysteroid agonist halofenozide (Hal) enhanced LdFTZ-F1-1 and LdFTZ-F1-2 expression in the final larval instar. Conversely, a decrease in 20E by feeding a double-stranded RNA (dsRNA) against LdSHD repressed the expression. Moreover, Hal rescued the expression levels in LdSHD-silenced larvae. Thus, 20E peaks seem to induce the transcription of LdFTZ-F1s. Furthermore, ingesting dsLdFTZ-F1 from a common fragment of LdFTZ-F1-1 and LdFTZ-F1-2 successfully knocked down both LdFTZ-F1s, and impaired pupation. Finally, knocking down LdFTZ-F1s significantly repressed the transcription of three ecdysteroidogenesis genes, lowered 20E titer, and reduced the expression of two 20E receptor genes. Silencing LdFTZ-F1s also induced the expression of a JH biosynthesis gene, increased JH titer, but decreased the mRNA level of a JH early-inducible gene. Thus, LdFTZ-F1s are involved in the regulation of pupation by modulating 20E and JH titers and mediating their signaling pathways.

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.

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.

Response of the vacuolar ATPase subunit E to RNA interference and four chemical pesticides in Leptinotarsa decemlineata (Say)

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

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.

Characterization of two juvenile hormone epoxide hydrolases by RNA interference in the Colorado potato beetle

In insect, juvenile hormone (JH) titers are tightly regulated in different development stages through synthesis and degradation pathways. During JH degradation, JH epoxide hydrolase (JHEH) converts JH to JH diol, and hydrolyses JH acid to JH acid diol. In this study, two full length LdJHEH cDNAs were cloned from Leptinotarsa decemlineata, and were provisionally designated LdJHEH1 and LdJHEH2. Both mRNAs were detectable in the thoracic muscles, brain-corpora cardiaca-corpora allata complex, foregut, midgut, hindgut, ventral ganglia, Malpighian tubules, fat bodies, epidermis, and hemocytes of the day 2 fourth-instar larvae, and in female ovaries as well as male reproductive organs of the adults. Moreover, both LdJHEH1 and LdJHEH2 were expressed throughout all larval life, with the highest peaks occurring 32h after ecdysis of the final (fourth) instar larvae. Four double-stranded RNAs (dsRNAs) (dsJHEH1-1, dsJHEH1-2, dsJHEH2-1, dsJHEH2-2) respectively targeting LdJHEH1 and LdJHEH2 were constructed and bacterially expressed. Ingestion of dsJHEH1-1, dsJHEH1-2, dsJHEH2-1, dsJHEH2-2, and a mixture of dsJHEH1-1+dsJHEH2-1 successfully knocked down corresponding target gene function, and significantly increased JH titer and upregulated Krüppel homolog 1 (LdKr-h1) mRNA level. Knockdown of either LdJHEH1 or LdJHEH2, or both genes slightly reduced larval weight and delayed larval development, and significantly impaired adult emergence. Therefore, it is suggested that knockdown LdJHEH1 and LdJHEH2 affected JH degradation in the Colorado potato beetle.

Identification of carboxylesterase genes and their expression profiles in the Colorado potato beetle Leptinotarsa decemlineata treated with fipronil and cyhalothrin.

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

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.