Characterization of Heat Shock Protein 60 as an Interacting Partner of Superoxide Dismutase 2 in the Silkworm, Bombyx mori, and Its Response to the Molting Hormone, 20-Hydroxyecdysone

Oxidative stress promotes pupation in some holometabolous insects. The levels of superoxide, a reactive oxygen species (ROS), are increased and superoxide dismutase 1 (BmSod1) and superoxide dismutase 2 (BmSod2) are decreased during metamorphic events in silkworm (Bombyx mori). These observations strongly suggest that pupation is initiated by oxidative stress via the down-regulation of BmSod1 and BmSod2. However, the molecular mechanisms underlying ROS production during metamorphic events in silkworm remain unknown. To investigate these molecular mechanisms, the peripheral proteins of BmSod1 and BmSod2 were identified and characterized using dry and wet approaches in this study. Based on the results, silkworm heat shock protein 60 (BmHsp60) was identified as an interacting partner of BmSod2, which belongs to the Fe/MnSOD family. Furthermore, the present study results showed that BmHsp60 mRNA expression levels were increased in response to oxidative stress caused by ultraviolet radiation and that BmHsp60 protein levels (but not mRNA levels) were decreased during metamorphic events, which are regulated by the molting hormone 20-hydroxyecdysone. These findings improve our understanding of the mechanisms by which holometabolous insects control ROS during metamorphosis.

Concentration-Dependent Feeding Deterrence to 20-Hydroxyecdysone for Three Subterranean Termite Species (Blattodea: Rhinotermitidae)

Effective active ingredients in toxicant bait formulations must be non-deterrent to insect feeding behavior at lethal concentrations. This study evaluated feeding deterrence for Coptotermes formosanus Shiraki, C. gestroi (Wasmann), and Reticulitermes flavipes (Kollar) when provided access to cellulose impregnated with various concentrations of the insect molting hormone, 20-hydroxyecdysone (20E). Termites were exposed to 20E concentrations of 200, 500, 1000 and 2000 ppm and to noviflumuron at 5000 ppm in a 24 h choice-test, and the mass of substrate consumption from treated and untreated media pads was compared for each treatment. 20E feeding deterrence was detected at 500, 1000 and 2000 ppm for C. gestroi, and at 2000 ppm for C. formosanus. No significant differences in consumption of treated and untreated substrate was detected at any concentration for R. flavipes. Potential methods for reducing deterrence are discussed.

20E-mediated regulation of BmKr-h1 by BmKRP promotes oocyte maturation

Background Krüppel homolog 1 (Kr-h1) is a critical transcription factor for juvenile hormone (JH) signaling, known to play a key role in regulating metamorphosis and adult reproduction in insects. Kr-h1 can also be induced by molting hormone 20-hydroxyecdysone (20E), however, the underlying mechanism of 20E-induced Kr-h1 expression remains unclear. In the present study, we investigated the molecular mechanism of Kr-h1 induction by 20E in the reproductive system of a model lepidopteran insect, Bombyx mori. Results Developmental and tissue-specific expression analysis revealed that BmKr-h1 was highly expressed in ovaries during the late pupal and adult stages and the expression was induced by 20E. RNA interference (RNAi)-mediated depletion of BmKr-h1 in female pupae severely repressed the transcription of vitellogenin receptor (VgR), resulting in the reduction in vitellogenin (Vg) deposition in oocytes. BmKr-h1 specifically bound the Kr-h1 binding site (KBS) between − 2818 and − 2805 nt upstream of BmVgR and enhanced the transcription of BmVgR. A 20E cis-regulatory element (CRE) was identified in the promoter of BmKr-h1 and functionally verified using luciferase reporter assay, EMSA and DNA-ChIP. Using pull-down assays, we identified a novel transcription factor B. mori Kr-h1 regulatory protein (BmKRP) that specifically bound the BmKr-h1 CRE and activated its transcription. CRISPR/Cas9-mediated knockout of BmKRP in female pupae suppressed the transcription of BmKr-h1 and BmVgR, resulting in arrested oogenesis. Conclusion We identified BmKRP as a new transcription factor mediating 20E regulation of B. mori oogenesis. Our data suggests that induction of BmKRP by 20E regulates BmKr-h1 expression, which in turn induces BmVgR expression to facilitate Vg uptake and oogenesis.

Methoxyfenozide, a Molting Hormone Agonist, Affects Autogeny Capacity, Oviposition, Fecundity, and Fertility in Culex pipiens (Diptera: Culicidae)

The current study aimed to evaluate the effects of methoxyfenozide (RH-2485), an insect growth disrupter (IGD) belonging to molting hormone agonist class, against female adults of Culex pipiens L. under laboratory conditions. Lethal concentrations (LC50 = 24.54 µg/liter and LC90 = 70.79 µg/liter), previously determined against fourth instar larvae, were tested for adult female fertility, fecundity and oviposition after tarsal contact before mating and any bloodmeal. Methoxyfenozide was found to alter negatively their autogeny capacity and oviposition. A strong reduction of 56% and 72% (P < 0.001) in females’ autogeny capacity was observed in both treated series, respectively. Alteration in oviposition were found to be higher with LC90 (OAI-LC90 = −0.62) than with the LC50 (OAI-LC50 = −0.42). Also fecundity and hatching rate (fertility) were significantly reduced in treated series as compared to controls. A significant reduction of 37.65 and 28.23% in fecundity and decrease of 56.85 and 71.87% in fertility were found, respectively in LC50 and LC90 treated series. Obtained data clearly demonstrated that methoxyfenozide have significant depressive effect on reproductive potential against medically important vector with minimizing ecotoxicological risks in mosquitoes management.

E93-depleted adult insects preserve the prothoracic gland and molt again

ABSTRACTInsect metamorphosis originated around the middle Devonian, associated with the innovation of the final molt; this occurs after histolysis of the prothoracic gland (PG; which produces the molting hormone) in the first days of adulthood. We previously hypothesized that transcription factor E93 is crucial in the emergence of metamorphosis, because it triggers metamorphosis in extant insects. This work on the cockroach Blattella germanica reveals that E93 also plays a crucial role in the histolysis of PG, which fits the above hypothesis. Previous studies have shown that the transcription factor FTZ-F1 is essential for PG histolysis. We have found that FTZ-F1 depletion towards the end of the final nymphal instar downregulates the expression of E93, whereas E93-depleted nymphs molt to adults that retain a functional PG. Interestingly, these adults are able to molt again, which is exceptional in insects. The study of insects able to molt again in the adult stage may reveal clues about how nymphal epidermal cells definitively become adult cells, and whether it is possible to reverse this process.

E93-depleted adult insects preserve the prothoracic gland and molt again

ABSTRACTInsect metamorphosis originated around the middle Devonian, associated with the innovation of the final molt; this occurs after the histolysis of the prothoracic gland (PG; which produces the molting hormone) in the first days of adulthood. We previously hypothesized that transcription factor E93 was crucial in the emergence of metamorphosis, since it triggers metamorphosis in extant insects. This work on the cockroach Blattella germanica reveals that E93 also plays a crucial role in the histolysis of PG, which fits the above hypothesis. Previous studies have shown that the transcription factor FTZ-F1 is essential for PG histolysis. We have found that FTZ-F1 depletion, towards the end of the final nymphal instar, downregulates the expression of E93, while E93-depleted nymphs molt to adults that retain a functional PG. Interestingly, these adults are able to molt again, which is exceptional in insects. The study of insects able to molt again in the adult stage may reveal clues as to how nymphal epidermal cells definitively become adult cells, and if it is possible to revert this process.Summary statementThe prothoracic gland disintegrates after insect metamorphosis. It was believed that the factor FTZ-F1 determines this disintegration. This work reveals that FTZ-F1 action is mediated by the factor E93.