Requirement of Myoglianin for metamorphosis in the beetle Henosepilachna vigintioctopunctata.

Henosepilachna vigintioctopunctata is a serious defoliating beetle attacking Solanaceae and Cucurbitaceae plants in many Asian countries. In the present paper, we identified a putative myoglianin (myo) gene. Hvmyo was actively transcribed throughout development, from embryo to adult. RNA interference (RNAi)-aided knockdown of Hvmyo delayed larval development by more than 2 days, reduced larval body size, inhibited the growth of antennae, wings and legs and disturbed gut purge. Knockdown of Hvmyo impaired the larval-pupal transition. All the Hvmyo RNAi larvae arrested at the larval stage or formed misshapen pupae or adults. The deformed pupae and adults were partially wrapped with exuviae, bearing separated wings. Moreover, the expression levels of five ecdysteroidogenesis genes (Hvspo, Hvphm, Hvdib, Hvsad and Hvshd), a prothocicotropic hormone (PTTH)/Torso pathway gene (Hvtorso), two 20E receptor genes (HvEcR and HvUSP), and two 20E signalling genes (HvE93 and HvFTZ-F1) were as a result of HvMyo RNAi significantly lowered. Conversely, the expression of a JH biosynthesis gene (Hvjhamt), a JH receptor gene HvMet and a JH signalling gene HvKr-h1 was greatly enhanced. Although ingestion of 20E and Hal rescued the 20E signal, it could not alleviate larval performance and defective phenotypes. Our results suggest that Myo exerts four distinctive roles in ecdysteroidogenesis, JH production, organ growth and larva-pupa-adult transformation in H. vigintioctopunctata.

Disruption of kynurenine pathway reveals physiological importance of tryptophan catabolism in Henosepilachna vigintioctopunctata.

Kynurenine pathway is critically important to catabolize tryptophan, to produce eye chromes, and to protect nervous system in insects. However, several issues related to tryptophan degradation remain to be clarified. In the present paper, we identified three genes (karmoisin, vermilion and cardinal) involved in kynurenine pathway in Henosepilachna vigintioctopunctata. The karmoisin and cardinal were highly expressed in the pupae and adults having compound eyes. Consistently, high-performance liquid chromatography result showed that three ommochrome peaks were present in adult heads rather than bodies (thoraces, legs, wings and abdomens). RNA interference (RNAi)-aided knockdown of vermilion caused accumulation of tryptophan in both adult heads and bodies, disappearance of ommochromes in the heads and a complete loss of eye color in both pupae and adults. Depletion of cardinal brought about excess of 3-hydroxykynurenine and insufficient ommochromes in the heads and decolored eyes. RNAi of karmoisin resulted in a decrease in ommochromes in the heads, and a partial loss of eye color. Moreover, a portion of karmoisin-, vermilion- or cardinal-silenced adults exhibited negative phototaxis, whereas control beetles showed positive phototaxis. Furthermore, dysfunctions of tryptophan catabolism impaired climbing ability. Our findings clearly illustrated several issues related to kynurenine pathway and provided a new insight into the physiological importance of tryptophan catabolism in H. vigintioctopunctata.

Characterization of candidate odorant-binding proteins and chemosensory proteins in the tea geometrid Ectropis obliqua Prout (Lepidoptera: Geometridae).

Insects rely heavily on their sophisticated chemosensory systems to locate host plants and find conspecific mates. Although the molecular mechanisms of odorant recognition in many Lepidoptera species have been well explored, limited information has been reported on the geometrid moth Ectropis obliqua Prout, an economically important pest of tea plants. In the current study, we first attempted to identify and characterize the putative olfactory carrier proteins, including odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). By analyzing previously obtained transcriptomic data of third-instar larvae, five OBPs and 14 CSPs in E. obliqua were identified. Sequence alignment, conserved motif identification, and phylogenetic analysis suggested that candidate proteins have typical characteristics of the insect OBP or CSP family. The expression patterns regarding life stages and different tissues were determined by quantitative real-time PCR. The results revealed that four transcripts (OBP2, OBP4 and CSP8, CSP10) had larvae preferential expression profiles and nine candidate genes (PBP1, OBP1 and CSP2, CSP4, CSP5, CSP6, CSP7, CSP11, and CSP13) were adult-biased expressed. Further specific tissue expression profile evaluation showed that OBP1, OBP2, OBP4, and PBP1 were highly expressed at olfactory organs, implying their potential involvement in chemical cue detection, whereas CSPs were ubiquitously detected among all of the tested tissues and could be associated with multiple physiological functions. This study provided a foundation for understanding the physiological functions of OBPs and CSPs in E. obliqua and will help pave the way for the development of a new environmental friendly pest management strategy against the tea geometrid moth.

Disruption of tetrahydrobiopterin (BH4) biosynthesis pathway affects cuticle pigmentation in Henosepilachna vigintioctopunctata.

Tetrahydrobiopterin (BH4) is produced from guanosine triphosphate (GTP) under catalyzation of GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS) and sepiapterin reductase (SR), among others. In Drosophila melanogaster, BH4 and other pteridines are required for cuticle tanning and eye pigmentation. In this study, two Hvgtpch (Hvgtpch-a and Hvgtpch-b), an Hvptps and an Hvsr transcripts were identified in a serious defoliator Henosepilachna vigintioctopunctata. Hvgtpch-a and Hvgtpch-b were highly expressed just before and/or right after the molt, in contrast to Hvptps and Hvsr. RNA interference (RNAi) by injection of a dsgtpch targeting the common fragment of Hvgtpch-a and Hvgtpch-b into the third instar larvae caused albino fourth-instar larvae and pupae. Around 80% of the Hvgtpch RNAi larvae failed to pupate. The remaining 20% of Hvgtpch RNAi pupated beetles did not completely remove the larval/pupal exuviae after emerged as adults and eventually died. Depletion of Hvgtpch at the fourth instar stage resulted in under-pigmented pupae and adults, with significantly low pupation and emergence rates. The Hvgtpch RNAi adults rarely moved and fed on plant leaves; they died within a week after emergence. Silence of Hvptps or Hvsr at the third- and fourth-instar stages led to similar but less serious phenotypes, with lowest influence in the Hvsr RNAi ladybirds. Moreover, RNAi of Hvgtpch, Hvptps or Hvsr did not affect coloration of the larval ocelli and pupal/adult compound eyes. Therefore, our results demonstrated that pteridines are involved in melanin formation but not in eye pigmentation in H. vigintioctopunctata. Moreover, our findings will enable the development of a dsgtpch-based pesticide to control H. vigintioctopunctata larvae.

Smad on X is vital for larval-pupal transition in a herbivorous ladybird beetle.

Insect development is regulated by a combination of juvenile hormone (JH) and 20-hydroxyecdysone (20E). Production of both JH and 20E is regulated by transforming growth factor-ß (TGFß) signaling. TGFß can be classified into two branches, the Activin and Bone Morphogenetic Protein (BMP) pathways. In Drosophila melanogaster, BMP signaling is critical for JH synthesis, whereas Activin signal is required to generate the large pulse of 20E necessary for entering metamorphosis. However, to which extent the roles of these signals are conserved remains unknown. Here we studied the role of an Activin component Smad on X (Smox) in post-embryonic development in a defoliating ladybird Henosepilachna vigintioctopunctata. RNA interference (RNAi)-aided knockdown of Hvsmox inhibited larval growth, and impaired larval development. All Hvmyo RNAi larvae arrested at the fourth-instar larval stage. Moreover, knockdown of Hvsmox delayed gut and Malpighian tubules remodeling. Furthermore, the expression of a JH biosynthesis gene (Hvjhamt), a JH receptor gene HvMet and a JH response gene HvKr-h1 was greatly enhanced. Conversely, the expression levels of an ecdysteroidogenesis gene (Hvspo), a 20E receptor gene (HvEcR) and six 20E response genes (HvBrC, HvE74, HvE75, HvE93, HvHR3 and HvHR4) were significantly lowered. Knockdown of HvMet partially restored the negative phenotypes in the Hvsmox RNAi beetles. Our results suggest that Smox exerts regulative roles in JH production, ecdysteroidogenesis and organ remodeling, thus contributing to modulate the larva-pupa-adult transformation in H. vigintioctopunctata.

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.

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.

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.

[Application of Raman spectra feature extraction in chemical fiber component qualitative identification].

According to the characteristics of the textile fibers Raman spectra, a qualitative identification method based on Raman feature extraction is proposed. This fast method consists of spectrum measurement and spectral data processing algorithm, including spectrum preprocessing, feature extraction and matching recognition. It can be used to identify the components of fibers or fabrics, especially chemical fibers, which is an inspective difficulty in daily analytic work for its remarkable Raman feature. The authors performed an experiment to analyze 4 typical and widely used kinds of fibers as algorithm verification. They are terylene fiber, acrylic fiber, nylon fiber and rayon fiber. To identify the components of one test sample, first the authors set up feature tables of these 4 standard samples, which describe the features of their preprocessed spectra containing both position information and intensity information, then extract features of the test sample. The authors match these features with the tables and calculate the matching confidence coefficients of the results, which can be used to filter the unexpected matching results caused by accident and attain the final qualitative identification result. The experimental results confirm that this method is effective, efficient and expansible, which means it can be used to identify more actual fiber types by adding more standard spectra to the feature table database. In addition, it is a pure optical method, which needs only a small quantity of sample without any pretreatment. The whole identification process is damage-free, pollution-free and suitable for various kinds of fabrics. Compared to all existing methods, this Raman spectrum identification method can solve the limitation of efficiency, pollution, universality, and fill a gap in fabric inspection field.