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Although muscle development has been widely studied in Drosophila melanogaster, it was a great challenge to apply to developmental processes of other insect muscles. This study was focused on the functional characterization of a basic helix-loop-helix transcription factor gene twist in an herbivorous ladybird Henosepilachna vigintioctopunctata. Its transcript (Hvtwist) levels were detected in all developmental stages. RNA interference (RNAi)-aided knockdown of Hvtwist at the penultimate larval instar stage impaired pupation, and caused a deformed adult in the legs. The tarsi were malformed and did not support the bodies in an upright position. The climbing ability was impaired. Moreover, around 50% of the impaired adults had a malformed elytrum. In addition, they consumed less foliage and did not lay eggs. A hematoxylin-eosin staining of the leg demonstrated that the tibial extensor (TE) and the tibial flexor (TF) muscles were originated from the femurs while levator and depressor muscles of the tarsus (TL and TD) were located in the tibia in the control adults, in which tarsal segments were devoid of muscles. RNAi treatment specific to Hvtwist expression markedly impaired TE and TF muscles in the femurs, and prevented the development of TL and TD muscles in the tibia. Therefore, our findings demonstrate Twist plays a vital role in the myogenesis in H. vigintioctopunctata adult legs., (© 2023 Wiley Periodicals LLC.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Insecticide resistance is a major challenge in successful insect pest control as the insects have the ability to develop resistance to various widely used insecticides. Butene-fipronil is a novel compound with high toxicity to insects and less toxicity to the non-target organisms. In the present study, the effect of butene-fipronil alone and in combination with three enzyme inhibitors, piperonyl butoxide (PBO), diethyl maleate (DEM), and triphenyl phosphate (TPP), was carried out on larvae and adults of Drosophilia melanogaster. Our results indicated that the co-toxicity indices of butene-fipronil + PBO, butene-fipronil + TPP, and butene-fipronil + DEM mixtures were 437.3, 335.0, and 210.3, respectively, in the second-instar larvae, while 186.6, 256.2, and 238.5, respectively, in the adults, indicating synergistic effects. Interestingly, butene-fipronil increased the expression of CYP28A5 in the larvae; CYP9F2, CYP304A1, CYP28A5, and CYP318A1 in the female adults; and CYP303A1 and CYP28A5 in the male adults. Furthermore, high-level expression of Est-7 was observed in the female adults compared to larvae and male adults. Our results suggest that there is no difference in butene-fipronil metabolism in larvae and male and female adults of D. melanogaster.

Rotting fruits offer all of the known resources required for the livelihood of Drosophila melanogaster Meigen (Diptera: Drosophilidae). During fruit fermentation, carbohydrates and proteins are decomposed to produce volatile alcohols and amines, respectively. It is hypothesized that D. melanogaster adults can detect these chemical cues at a distance to identify and locate the decaying fruits. In the present paper, we compared the olfactory responses and movement of male flies varying in mating status and nutritional state to methanol, ethanol, and ammonia sources using a glass Y-tube olfactometer. In general, ethanol vapor at low to moderate concentrations repelled more hungry mated males than satiated ones. In contrast, methanol showed little difference in the attractiveness to males at different nutritional states and mating status. Moreover, ammonia attracted more hungry mated males. The attractiveness increased almost linearly with ammonia concentration from lowest to highest. When ammonia and artificial diet were put together in the odor arm, the responses of male flies to mixed odor mimicked the response to ammonia. Furthermore, odorant concentration, mating status, and nutritional state affected the flies' dispersal. Mated and starved males dispersed at a higher rate than virgin and satiated ones. Thus, our results showed that starved, mated males increased dispersal and preferred ammonia that originated from protein., (This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed.)

Methanol is among the most common short-chain alcohols in fermenting fruits, the natural food and oviposition sites of the fruit fly Drosophila melanogaster. Our previous results showed that cytochrome P450 monooxygenases (CYPs) were associated with methanol detoxification in the larvae. Catalases, alcohol dehydrogenases (ADHs), esterases (ESTs) and glutathione S-transferases (GSTs) were specifically inhibited by 3-amino-1,2,4-triazole (3-AT), 4-methylpyrazole (4-MP), triphenyl phosphate (TPP) and diethylmeleate (DEM), respectively. CYPs were inhibited by piperonyl butoxide (PBO) and 1-aminobenzotriazole (1-ABT). In the present paper, the involvements of these enzymes in methanol metabolism were investigated in female and male adults by determining the combination indices of methanol and their corresponding inhibitors. When PBO, 1-ABT, 3-AT, 4-MP and TPP were individually mixed with methanol, they exhibited significant synergism to the mortality of the adults after 72h of dietary exposure. In contrast, the DEM and methanol mixture showed additive effects. Moreover, methanol exposure dramatically increased CYP activity and up-regulated mRNA expression levels of several Cyp genes. Bioassays using different strains revealed that the variation in ADH activity and RNAi-mediated knockdown of α-Est7 significantly changed LC50 values for methanol. These results suggest that CYPs, catalases, ADHs and ESTs are partially responsible for methanol elimination in adults. It seems that there are some differences in methanol metabolism between larvae and adults, but not between female and male adults., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Methanol is one of the most common short-chain alcohols in fermenting fruits, the natural food of the fruit fly, Drosophila melanogaster. The larvae cope continuously with methanol at various concentrations in order to survive and develop. In the present article, we found toxicities of dietary methanol and formaldehyde were enhanced by piperonyl butoxide, but not by 3-amino-1, 2, 4-triazole, 4-methylpyrazole, diethylmeleate, and triphenyl phosphate, when assessing by the combination index method. These results reveal that cytochrome P450 monooxygenases (CYPs), rather than catalases, alcohol dehydrogenases, glutathione S-transferases, and esterases, participate in methanol metabolism. Moreover, methanol exposure dramatically increased CYP activity. The ratios of the CYP activities in treated larvae to those in control reached, respectively, up to 3.0-, 3.9-, and 2.7-fold, at methanol concentrations of 22.6, 27.9, and 34.5 mg/g diet. In addition, methanol exposure greatly up-regulated the mRNA expression level of five Cyp genes, which were Cyp304a1, Cyp9f2, Cyp28a5, Cyp4d2, and Cyp4e2. Their resulting proteins were suggested as the candidate enzymes for methanol metabolism in D. melanogaster larvae., (© 2012 Wiley Periodicals, Inc.)

Hydrogen sulfide (H(2)S) has been shown to effect physiological alterations in several animals, frequently leading to an improvement in survival in otherwise lethal conditions. In the present paper, a volatility bioassay system was developed to evaluate the survivorship of Drosophila melanogaster adults exposed to H(2)S gas that emanated from a K(2)S donor. Using this bioassay system, we found that H(2)S exposure significantly increased the survival of flies under arid and food-free conditions, but not under humid and food-free conditions. This suggests that H(2)S plays a role in desiccation tolerance but not in nutritional stress alleviation. To further confirm the suggestion, the mRNA levels of two desiccation tolerance-related genes Frost and Desat2, and a starvation-related gene Smp-30, from the control and treated flies were measured by quantitative real-time PCR. These genes were up-regulated within 2h when the flies transferred to the arid and food-free bioassay system. Addition of H(2)S further increased Frost and Desat2 mRNA levels, in contrast to Smp-30. Thus, our molecular results were consistent with our bioassay findings. Because of the molecular and genetic tools available for Drosophila, the fly will be a useful system for determining how H(2)S regulates various physiological alterations., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

BACKGROUND Uridine diphosphate‐N‐acetylglucosamine (UDP‐GlcNAc) diphosphorylase (UAP) catalyzes the formation of UDP‐GlcNAc, the precursor for the production of chitin in ectodermally derived epidermal cells and midgut, for GlcNAcylation of proteins and for generation of glycosyl‐phosphatidyl‐inositol anchors in all tissues in Drosophila melanogaster. RESULTS: Here, we identified a putative HvUAP gene in Henosepilachna vigintioctopunctata. Knockdown of HvUAP at the second‐, third‐ and fourth‐instar stages impaired larval development. Most resultant HvUAP hypomorphs showed arrested development at the third‐, fourth‐instar larval or prepupal stages, and became paralyzed, depending on the age when treated. Some HvUAP‐silenced larvae had weak and soft scoli. A portion of HvUAP‐depleted beetles formed misshapen pupae. No HvUAP RNA interference pupae successfully emerged as adults. Dissection and microscopic observation revealed that knockdown of HvUAP affected gut growth and food ingestion, reduced cuticle thickness, and negatively affected the formation of newly generated cuticle layers during ecdysis. Furthermore, HvUAP deficiency inhibited development of the tracheal respiratory system and thinned tracheal taenidia. CONCLUSION: The phenotypical defects in HvUAP hypomorphs suggest that HvUAP is involved in the production of chitin. Moreover, our findings will enable the development of a double‐stranded RNA‐based pesticide to control H. vigintioctopunctata. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Simple Summary: Fushi Tarazu Factor-1 (FTZ-F1) plays a crucial regulatory role in molting in insects. It is hypothesized that, by alternative transcription start and splicing, the FTZ-F1 gene generates two isomers (α- and βFTZ-F1) that exert isoform-specific roles in non-Drosophilid insects. In the present paper, we first unveiled that the same post-transcriptional processing in FTZ-F1 occurred in coleopterans, lepidopterans, dipterans and hymenopterans. We then found that αFTZ-F1 and βFTZ-F1 were actively transcribed throughout the development, from embryo to adult, in Henosepilachna vigintioctopunctata. Moreover, by RNA interference, we confirmed that both FTZ-F1 isoforms act as regulators in larval–larval molting and βFTZ-F1 is involved in the regulation of the larval–pupal transition. 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 βFTZ-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 βFTZ-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 βFTZ-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 βFTZ-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Insecticide resistance is a major challenge in successful insect pest control as the insects have the ability to develop resistance to various widely used insecticides. Butene-fipronil is a novel compound with high toxicity to insects and less toxicity to the non-target organisms. In the present study, the effect of butene-fipronil alone and in combination with three enzyme inhibitors, piperonyl butoxide (PBO), diethyl maleate (DEM), and triphenyl phosphate (TPP), was carried out on larvae and adults of Drosophilia melanogaster. Our results indicated that the co-toxicity indices of butene-fipronil + PBO, butene-fipronil + TPP, and butene-fipronil + DEM mixtures were 437.3, 335.0, and 210.3, respectively, in the second-instar larvae, while 186.6, 256.2, and 238.5, respectively, in the adults, indicating synergistic effects. Interestingly, butene-fipronil increased the expression of CYP28A5 in the larvae; CYP9F2, CYP304A1, CYP28A5, and CYP318A1 in the female adults; and CYP303A1 and CYP28A5 in the male adults. Furthermore, high-level expression of Est-7 was observed in the female adults compared to larvae and male adults. Our results suggest that there is no difference in butene-fipronil metabolism in larvae and male and female adults of D. melanogaster. [ABSTRACT FROM AUTHOR]

The toxicities of butene-fipronil to Drosophila melanogaster were evaluated, in comparison to nine conventional insecticides. According to the mean LD values of the larvae from CS, w and Oregan strains, butene-fipronil, abamectin, spinosad and chlorpyrifos exhibited high levels of toxicity. Imidacloprid, α-cypermethrin and clothianidin showed middle levels of toxicity. And acephate, methomyl and acetamiprid had low levels of toxicity. To the adults, butene-fipronil, α-cypermethrin, spinosad, and chlorpyrifos were relatively more toxic, whereas acephate, methomyl, acetamiprid, imidacloprid, clothianidin and abamectin were relatively less toxic. Butene-fipronil and abamectin were more toxic to larvae. In contrast, spinosad, α-cypermethrin, and clothianidin were more toxic to adults. Moreover, the toxic risk assessment using a risk quotient value revealed that butene-fipronil was safe to D. melanogaster adults. Thus, our results suggested that butene-fipronil is harmful to D. melanogaster larvae, but it is relatively safe to the adults. [ABSTRACT FROM AUTHOR]

To accommodate growth, insects must periodically shed their exoskeletons. In Manduca sexta , Drosophila melanogaster and Tribolium castaneum , Bursicon (Burs)/ Partner of bursicon (Pburs)-LGR2 signal is an indispensable component for the proper execution of ecdysis behavior during adult eclosion. Nevertheless, the behavioral events and the roles of bursicon signaling in other insects deserve further exploration. In the current paper, we found that the pupal-adult ecdysis in Henosepilachna vigintioctomaculata could be divided into three distinct stages, preecdysis, ecdysis and postecdysis. Preecdysis behavioral sequences included abdomen twitches, dorsal-ventral contractions and air filling that function to loosen the old cuticle. Ecdysis events began with anterior-posterior contractions that gradually split the old integument along the dorsal body midline, followed by freeing of legs and mouthparts, and culminated in detachment from pupal cuticle. Postecdysis behavioral processes contained three actions: perch selection and stretching of elytra and hindwings. RNA interference for HvBurs , HvPburs or Hvrk (encoding LGR2) strongly impaired wing expansion actions, and slightly influenced preecdysis and ecdysis behaviors. The RNAi beetles failed to extend their elytra and hindwings. In addition, injected with ds rk also caused kinked femurs and tibia. Our findings establish that bursicon pathway is involved in regulation of adult eclosion behavior, especially wing expansion motor programs. Given that wings facilitate food foraging, courtship, predator avoidance, dispersal and migration, our results provide a potential target for controlling H. vigintioctomaculata. [Display omitted] • Adult eclosion can be divided into preecdysis, ecdysis and postecdysis in Henosepilachna vigintioctomaculata. • Knockdown of bursicon genes impairs wing expansion. • The RNAi beetles fail to extend their elytra and hindwings. • Bursicon way triggers wing expansion motor programs. [ABSTRACT FROM AUTHOR]

[Display omitted] • RNAi of Hvsmox inhibits larval growth and impairs larval development. • Depleting Hvsmox delays the remodel of guts and Malpighian tubules. • Knockdown of Hvsmox disrupts JH and 20E signaling. • RNAi of HvMet and Hvsmox partially restores negative phenotypes. 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-β (TGFβ) signaling. TGFβ 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. [ABSTRACT FROM AUTHOR]