Evaluation of reference genes for quantitative expression analysis in Mylabris sibirica (Coleoptera, Meloidae).

Mylabris sibirica is a hypermetamorphic insect whose adults feed on oilseed rape. However, due to a shortage of effective and appropriate endogenous references, studies on molecular functional genes in Mylabris sibirica, have been tremendously limited. In this study, ten internal reference genes (ACT, ARF1, AK, EF1α, GAPDH, α-TUB, RPL6, RPL13, RPS3 and RPS18) were tested and assessed under four selected treatments including adult ages, adult tissues, temperatures, and sex by RT-qPCR based on five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). Our findings showed that RPL6 and RPL13 were the most optimal internal reference gene combination for gene expression during various adult ages and under diverse temperatures; The combination of RPL6 and RPS18 was recommended to test gene transcription levels under different adult tissues. AK and RPL6 were the best reference genes in male and female adults. RPL6 and RPL13 were the most appropriate reference gene pair to estimate gene expression levels under four different tested backgrounds. The relative transcript levels of a uridine diphosphate (UDP)-N-acetylglucosamine-pyrophosphorylase (MsUAP), varied greatly according to normalization with the two most- and least-suited reference genes. This study will lay the basis for further molecular physiology and biochemistry studies in M. sibirica, such as development, reproduction, sex differentiation, cold and heat resistance.

Dusky-like Is Critical for Morphogenesis of the Cellular Protuberances and Formation of the Cuticle in Henosepilachna vigintioctopunctata.

Dusky-like (Dyl) is a transmembrane protein containing a zona pellucida domain. Its physiological roles during metamorphosis have been well explored in Drosophila melanogaster and have also been documented in Tribolium castaneum. However, Dyl has undergone a functional shift between Diptera and Coleoptera insects. Further investigation of Dyl in other insects will be helpful to further clarify its function in insect growth and development. Henosepilachna vigintioctopunctata is an important Coleoptera that causes enormous economic losses in agriculture in China. In this study, we found that the expression of Hvdyl was detectable in embryos, larvae, prepupae, pupae, and adults. We knocked down Hvdyl in third- and fourth-instar larvae and pupae with RNA interference (RNAi). RNAi of Hvdyl mainly caused two phenotypic defects. Firstly, the growth of epidermal cellular protuberances was suppressed. Injection of dsdyl (double-stranded dusky-like RNA) at the third-instar larval stage truncated the scoli throughout the thorax and abdomen and shortened the setae on the head capsules and mouthparts of the fourth-instar larvae. Introduction of dsdyl at the third- and fourth-instar stages led to misshapen pupal setae. The setae were shortened or became black nodules. Treatment with dsdyl at the larval and pupal stages resulted in deformed adults with completely suppressed wing hairs. Moreover, the knockdown of Hvdyl at the third-instar stage caused deformed larval mouthparts at the fourth-instar period. As a result, foliage consumption was inhibited, and larval growth was slowed. The results indicate that Dyl is associated with the growth of cellular protuberances throughout development and with the formation of the cuticle in H. vigintioctopunctata.

Knockdown of Ecdysone-Induced Protein 93F Causes Abnormal Pupae and Adults in the Eggplant Lady Beetle.

Ecdysone-induced protein 93F (E93) plays triple roles during post-embryonic development in insects whose juvenile instars are more than four. However, it only acts as a specifier of adult structures in Drosophila flies whose larval instars are fixed at three. In this study, we determined the functions of E93 in the eggplant lady beetle (Henosepilachna vigintioctopunctata), which has four larval instars. We uncovered that E93 was abundantly expressed at the prepupal and pupal stages. A precocious inhibition of the juvenile hormone signal by RNA interference (RNAi) of HvKr-h1 or HvHairy, two vital downstream developmental effectors, at the penultimate instar larval stage increased the expression of E93, Conversely, ingestion of JH by the third-instar larvae stimulated the expression of HvKr-h1 but repressed the transcription of either HvE93X1 or HvE93X2. However, disturbance of the JH signal neither drove premature metamorphosis nor caused supernumerary instars. In contrast, depletion of E93 at the third- and fourth-instar larval and prepupal stages severely impaired pupation and caused a larval-pupal mixed phenotype: pupal spines and larval scoli were simultaneously presented on the cuticle. RNAi of E93 at the pupal stage affected adult eclosion. When the beetles had suffered from a dsE93 injection at the fourth-instar larval and pupal stages, a few resultant adults emerged, with separated elytra, abnormally folded hindwings, a small body size and short appendages. Taken together, our results suggest the larval instars are fixed in H. vigintioctopunctata; E93 serves as a repressor of larval characters and a specifier of adult structures during the larval-pupal-adult transition.

Dissecting the Isoform-Specific Roles of FTZ-F1 in the Larval-Larval and Larval-Pupal Ecdyses in Henosepilachna vigintioctopunctata.

Fushi Tarazu Factor 1 (FTZ-F1), a member of the nuclear receptor superfamily, is the downstream factor of 20-hydroxyecdysone signaling. In Drosophila melanogaster, alternative transcription start and splicing in the FTZ-F1 gene generate αFTZ-F1 and ßFTZ-F1 isoforms, which are vital for pair-rule segmentation in early embryogenesis and post-embryonic development, respectively. However, whether the same mRNA isoforms are present and exert the conservative roles remains to be clarified in other insects. In the present paper, we first mined the genomic data of representative insect species and unveiled that the same post-transcriptional processing in FTZ-F1 occurred in coleopterans, lepidopterans, dipterans and hymenopterans. Our expression data in Henosepilachna vigintioctopunctata, a serious polyphagous defoliator damaging a wide range of crops in Solanaceae and Cucurbitaceae, showed that both αFTZ-F1 and ßFTZ-F1 were actively transcribed throughout the development, from embryo to adult. The RNA interference-aided knockdown of both isoforms completely arrested larval ecdysis from the third to the fourth instar, in contrast to the depletion of either isoform. In contrast, silencing ßFTZ-F1, rather than αFTZ-F1, severely impaired the larval-pupal transformation. We accordingly propose that both FTZ-F1 isoforms are essential but mutually interchangeable for larval-larval molting, while ßFTZ-F1 is necessary for the larval-pupal transition and sufficient to exert the role of both FTZ-F1s during larval-pupal metamorphosis in H. vigintioctopunctata.

Reference Genes for Expression Analyses by qRT-PCR in Phthorimaea operculella (Lepidoptera: Gelechiidae).

Due to a lack of effective internal references, studies on functional genes in Phthorimaea operculella, a serious Lepidopteran pest attacking potatoes worldwide, have been greatly limited. To select suitable endogenous controls, ten housekeeping genes of actin (ACT), α-tubulin (α-TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), 18S and 28S ribosomal RNA (18S, 28S), ribosomal protein genes RPL4, RPL13 and RPL27 and superoxide dismutase (SOD) were tested. Their expression levels were determined under three different experimental conditions (developmental stages, tissues/organs and temperatures) using qRT-PCR technology. The stability was evaluated with five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). The results clarified that RPL13, EF1α and RPL27 are ranked as the best reference gene combination for measuring gene expression levels among different developing stages and under various temperatures; EF1α and RPL13 are recommended to normalize the gene expression levels among diverse tissues. EF1α and RPL13 are the best reference genes in all the experimental conditions. To validate the utility of the selected reference pair, EF1α and RPL13, we estimated the tissue-biased expression level of chitin synthase A gene (PoChSA). As expected, PoChSA was abundantly expressed in ectodermally derived epidermal cells, and lowly transcribed in the midgut. These findings will lay the foundation for future research on the molecular physiology and biochemistry of P. operculella.

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.

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.

RNA interference targeting ecdysone receptor blocks the larval-pupal transition in Henosepilachna vigintioctopunctata.

Henosepilachna vigintioctopunctata is a serious insect pest which attacks a large number of nightshades and cucurbits in Asian countries, Brazil and Australia. Prolonged application of traditional pesticides has caused environmental pollution and exerted deleterious effects on human health. Finding new approaches with high target specificity and low environmental contamination has become an urgent task. RNA interference (RNAi) induced by double-stranded RNA (dsRNA) is expected to be applicable to managing this pest. Here we evaluated the effects of Escherichia coli-expressed dsRNAs targeting ecdysone receptor (EcR) gene via dietary delivery in laboratory and foliar spraying in a greenhouse. The target transcript was successfully knocked down when the 4th-instar larvae had fed on potato foliage dipped with dsEcR in a laboratory bioassay. Around 85% of the HvEcR RNAi larvae remained as prepupae or became abnormal pupae, and failed to emerge into adults. Ingestion of dsEcR-immersed foliage by the 3rd-instar larvae effectuated a comparable RNAi response and brought about more severe defects: all the resultant larvae arrested development, remained as prepupae and finally died. For assay in the greenhouse, a dsEcR-contained E. coli suspension was directly sprayed to the foliage of greenhouse-growing potato plants and the 3rd- and 4th-instar larvae were transferred to the leaves. High RNAi efficacy was obtained and identical RNAi phenotypes were observed in treated larvae. In addition, spraying dsEcR reduced leaf damage. Our results indicate a possibility of practical application of dsEcR as an environmentally friendly RNA pesticide to control H. vigintioctopunctata larvae.

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.

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.