Cloning and spatio-temporal expression of CsKr-h1 encoding the juvenile hormone response gene in Coccinella septempunctata L.

The gene encoding juvenile hormone response (Krüppel homolog1, Kr-hl) in Coccinella septempunctata was investigated by cloning and analysing expression profiles in different developmental stages and tissues by quantitative real-time polymerase chain reaction (PCR). C. septempunctata Kr-hl (CsKr-hl) encoded a 1338 bp open reading frame (ORF) with a predicted protein product of 445 amino acids; the latter showed high similarity to orthologs in other species and contained eight highly-conserved Zn-finger motifs for DNA-binding. CsKr-hl was expressed in different developmental stages of C. septempunctata. The expression levels of CsKr-hl in eggs, 2nd, 3rd, 4th instar larvae, and pupa were 3.31, 2.30, 7.09, 0.58, and 7.48 times the number of 1st instar larvae, respectively. CsKr-hl expression levels in female adults gradually increased at 25-30 days and were significantly higher than expression at 1-20 days. CsKr-hl expression in 20-30 days-old male adults was significantly higher than males aged 1-15 days. CsKr-hl expression levels in heads of male and female adults were significantly higher than expression levels in the thorax, adipose, and reproductive system. Interestingly, CsKr-hl expression levels in the adipose and reproductive system of female adults were significantly higher than in adult male corresponding organs, which suggest that CsKr-hl plays an important role in regulating reproductive development in C. septempunctata.

The CsmiR1579-CsKr-h1 module mediates rice stem borer development and reproduction: An effective target for transgenic insect-resistant rice.

The rice stem borer (RSB, Chilo suppressalis) is a significant agricultural pest that mainly depends on chemical control. However, it has grown to varied degrees of pesticide resistance, which poses a severe threat to rice production and emphasizes the need for safer, more efficient alternative pest management strategies. Here, in vitro and in vivo experiments analyses reveal miR-1579 binds to the critical transcription factor Krüppel homologue 1 (Kr-h1) and negatively regulates its expression. Overexpression of miR-1579 in larvae with significantly lower levels of Kr-h1 was associated with a decline in larval growth and survival. Furthermore, in female pupae, miR-1579 overexpression led to abnormalities in ovarian development, suggesting that targeting miR-1579 could be a potential management strategy against C. suppressalis. Therefore, we generated transgenic rice expressing miR-1579 and screened three lines that had a single copy of highly abundant mature miR-1579 transcripts. Expectedly, fed with transgenic miR-1579 rice lines were significantly lower survival rates in larvae and high levels of resistance to damage caused by C. suppressalis infestation. These findings suggest that miRNA-mediated RNAi could provide an effective and species-specific strategy for C. suppressalis control.

Methoprene-tolerant and Krüppel homolog 1 are actors of juvenile hormone-signaling controlling the development of male sexual behavior in the moth Agrotis ipsilon.

In insects, juvenile hormone (JH) is critical for the orchestration of male reproductive maturation. For instance, in the male moth, Agrotis ipsilon, the behavioral response and the neuronal sensitivity within the primary olfactory centers, the antennal lobes (ALs), to the female-emitted sex pheromone increase with fertility during adulthood and the coordination between these events is governed by JH. However, the molecular basis of JH action in the development of sexual behavior remains largely unknown. Here, we show that the expression of the paralogous JH receptors, Methoprene-tolerant 1 and 2 (Met1, Met2) and of the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) within ALs raised from the third day of adult life and this dynamic is correlated with increased behavioral responsiveness to sex pheromone. Met1-, Met2- and Kr-h1-depleted sexually mature males exhibited altered sex pheromone-guided orientation flight. Moreover, injection of JH-II into young males enhanced the behavioral response to sex pheromone with increased AL Met1, Met2 and Kr-h1 mRNA levels. By contrast, JH deficiency suppressed the behavioral response to sex pheromone coupled with reduced AL Met1, Met2 and Kr-h1 mRNA levels in allatectomized old males and these inhibitions were compensated by an injection of JH-II in operated males. Our results demonstrated that JH acts through Met-Kr-h1 signaling pathway operating in ALs, to promote the pheromone information processing and consequently the display of sexual behavior in synchronization with fertility to optimize male reproductive fitness. Thus, this study provides insights into the molecular mechanisms underlying the hormonal regulation of reproductive behavior in insects.

Involvement of Methoprene-tolerant and Krüppel homolog 1 in juvenile hormone-mediated vitellogenesis of female Liposcelis entomophila (End.) (Psocoptera: Liposcelididae).

Methoprene-tolerant (Met) as an intracellular receptor of juvenile hormone (JH) and the Krüppel-homolog 1 (Kr-h1) as a JH-inducible transcription factor had been proved to contribute to insect reproduction. Their functions vary in different insect orders, however, they are not clear in Psocoptera. In this study, LeMet and LeKr-h1 were identified and their roles in vitellogenesis and ovarian development were investigated in Liposcelis entomophila (Enderlein). Treatment with exogenous JH III significantly induced the expression of LeKr-h1, LeVg, and LeVgR. Furthermore, silencing LeMet and LeKr-h1 remarkably reduced the transcription of LeVg and LeVgR, disrupted the production of Vg in fat body and the uptake of Vg by oocytes, and ultimately led to a decline in fecundity. The results indicated that the JH signaling pathway was essential to the reproductive process of this species. Interestingly, knockdown of LeMet or LeKr-h1 also resulted in fluctuations in the expression of FoxO, indicating the complex regulatory interactions between different hormone factors. Besides, knockdown of both LeMet and LeKr-h1 significantly increased L. entomophila mortality. Our study provides initial insight into the roles of JH signaling in the female reproduction of psocids and provided evidence that RNAi-mediated knockdown of Met or Kr-h1 is a potential pest control strategy.

Reproductive Outbreaks of Sogatella furcifera Mediated by Overexpression of the Nuclear Receptor USP under Pressure from Triflumezopyrim.

Long-term pesticide-driven selection pressure is one of the main causes of insect outbreaks. In this study, we found that low doses of triflumezopyrim could increase the fecundity of white-backed planthoppers (Sogatella furcifera). By continuously screening 20 generations with a low dose of triflumezopyrim, a triflumezopyrim-resistant strain (Tri-strain, resistance ratio = 20.9-fold) was obtained. The average oviposition quantity and longevity of the Tri-strain (208.77 eggs and 21.31 days, respectively) were significantly higher than those of the susceptible strain (Sus-strain) (164.62 eggs and 17.85 days, respectively). To better understand the mechanism underlying the effects on reproduction, we detected the expression levels of several reproduction-related transcription factors in both the Tri- and Sus-strains. Ultraspiracle (USP) was significantly overexpressed in the Tri-strain. Knockdown of USP by RNAi severely inhibited the moulting process of S. furcifera and disrupted the development of female adult ovaries. Among the potential downstream target genes of USP, Kr-h1 (0.19-fold), Cht8 (0.56-fold) and GPCR A22 (0.31-fold) showed downregulated expression after USP-RNAi. In contrast, the expression of EcR (2.55-fold), which forms heterodimers with USP, was significantly upregulated. Furthermore, RNAi was performed on Kr-h1 in the Tri-strain, and the results show that larval moulting and the development of female adult ovaries were inhibited, consistent with the USP-RNAi results in S. furcifera. These results suggest that the transcription factors USP and Kr-h1 play important roles in the reproductive development of S. furcifera, and overexpression of USP and Kr-h1 in the Tri-resistant strain may result in reproductive outbreaks of pests.

Impact of JH Signaling on Reproductive Physiology of the Classical Insect Model, Rhodnius prolixus.

In adult females of several insect species, juvenile hormones (JHs) act as gonadotrophic hormones, regulating egg production. JH binds to its nuclear receptor, Methoprene tolerant (Met), triggering its dimerization with the protein Taiman (Tai). The resulting active complex induces transcription of JH response genes, such as Krüppel homolog 1 (Kr-h1). In this study we report for the first time the participation of the isoform JH III skipped bisepoxide (JHSB3) and its signaling pathway in the reproductive fitness of the classical insect model Rhodnius prolixus. The topical application of synthetic JHSB3 increases transcript and protein expression of yolk protein precursors (YPPs), mainly by the fat body but also by the ovaries, the second source of YPPs. These results are also confirmed by ex vivo assays. In contrast, when the JH signaling cascade is impaired via RNA interference by downregulating RhoprMet and RhoprTai mRNA, egg production is inhibited. Although RhoprKr-h1 transcript expression is highly dependent on JHSB3 signaling, it is not involved in egg production but rather in successful hatching. This research contributes missing pieces of JH action in the insect model in which JH was first postulated almost 100 years ago.

Identification of species-specific juvenile hormone response elements in the fall armyworm, Spodoptera frugiperda.

Juvenile hormones (JH) regulate insect development and reproduction. The JH analogs (JHA) are used as insecticides. However, JHAs are rarely used in managing pests such as the fall armyworm, Spodoptera frugiperda that cause damage during larval stages. The insecticides that antagonize JH action and induce stoppage of feeding and precocious metamorphosis might work better to control these pests. Treating insects with JHA insecticides induces the expression of an early JH response gene, Krüppel homolog 1 (Kr-h1) by working through JH response elements (JHRE) present in its promoter. In this study, we identified JHREs present in the promoter of Kr-h1 gene of a global pest, S. frugiperda, and used them to develop a JHRE-reporter cell platform to screen for JH analogs. JHA, methoprene induced the expression of SfKr-h1 both in vitro and in vivo. JHRE present in the promoters of two SfKr-h1 isoforms, SfKr-h1α and SfKr-h1ß were identified. In Sf9 cells, the knockout of isoform-specific JHRE affected JH response in an isoform-specific manner. We also found that S. frugiperda JHRE (SfJHRE) did not function in the mosquito Aedes aegypti Aag2 cells and Tribolium castaneum TcA cells. Similarly, Ae. aegypti AaJHRE and T. castaneum TcJHRE were only functional in cells derived from these insects. The nucleotide sequence at the 3'end to the conserved core JHRE E-box sequence seems to be responsible for the species specificity observed. Two stable cell lines expressing the luciferase and enhanced green fluorescent protein genes under the control of SfJHRE were established. These cell lines responded well to JHA; these two JHRE-reporter cell lines could be used in screening assays to identify insecticides to manage S. frugiperda and other major pests.

Roles of Krüppel Homolog 1 and Broad-Complex in the Development of Dendroctonus armandi (Coleoptera: Scolytinae).

In insects, metamorphosis is controlled by juvenile hormone (JH) and 20-hydroxyecdysone (20E). Krüppel homolog 1 (Kr-h1), a key JH-early inducible gene, is responsible for the suppression of metamorphosis and the regulation of the Broad-Complex (Br-C) gene, which is induced by 20E and functions as a "pupal specifier". In this study, we identified and characterized the expression patterns and tissue distribution of DaKr-h1 and DaBr-C at various developmental stages of Dendroctonus armandi. The expression of the two genes was induced by JH analog (JHA) methoprene and 20E, and their functions were investigated by RNA interference. DaKr-h1 and DaBr-C were predominantly expressed in the heads of larvae and were significantly downregulated during the molting stage. In contrast, the DaKr-h1 transcript level was highest in the adult anterior midgut. DaBr-C was mainly expressed in female adults, with the highest transcript levels in the ovaries. In the larval and pupal stages, both JHA and 20E significantly induced DaKr-h1, but only 20E significantly induced DaBr-C, indicating the importance of hormones in metamorphosis. DaKr-h1 knockdown in larvae upregulated DaBr-C expression, resulting in precocious metamorphosis from larvae to pupae and the formation of miniature pupae. DaKr-h1 knockdown in pupae suppressed DaBr-C expression, increased emergence, caused abnormal morphology, and caused the formation of small-winged adults. These results suggest that DaKr-h1 is required for the metamorphosis of D. armandi. Our findings provide insight into the roles of DaKr-h1 and DaBr-C in JH-induced transcriptional repression and highlight DaKr-h1 as a potential target for metamorphosis suppression in D. armandi.

Chinmo is the larval member of the molecular trinity that directs Drosophila metamorphosis.

The molecular control of insect metamorphosis from larva to pupa to adult has long been a mystery. The Broad and E93 transcription factors, which can modify chromatin domains, are known to direct the production of the pupa and the adult, respectively. We now show that chinmo, a gene related to broad, is essential for the repression of these metamorphic genes. Chinmo is strongly expressed during the formation and growth of the larva and its removal results in the precocious expression of broad and E93 in the first stage larva, causing a shift from larval to premetamorphic functions. This trinity of Chinmo, Broad, and E93 regulatory factors is mutually inhibitory. The interaction of this network with regulatory hormones likely ensures the orderly progression through insect metamorphosis.

Molecular Identification and Functional Characterization of Methoprene-Tolerant (Met) and Krüppel-Homolog 1 (Kr-h1) in Harmonia axyridis (Coleoptera: Coccinellidae).

Juvenile hormone (JH) plays a key role in regulating insect reproductive processes. Methoprene-tolerant (Met), as a putative JH receptor, transduces JH signals by activating the transcription factor krüppel homolog 1 (Kr-h1). To understand the effects of Met and Kr-h1 genes on female reproduction of natural enemy insects, the Met and Kr-h1 were identified and analyzed from Harmonia axyridis Pallas (HmMet and HmKr-h1). The HmMet protein belonged to the bHLH-PAS family with bHLH domain, PAS domains, and PAC domain. HmMet mRNA was detected in all developmental stages, and the highest expression was found in the ovaries of female adults. The HmKr-h1 protein had eight C2H2-type zinc finger domains. HmKr-h1 mRNA was highly expressed from day 7 to day 9 of female adults. The tissue expression showed that HmKr-h1 was highly expressed in its wing, leg, and fat body. Knockdown of HmMet and HmKr-h1 substantially reduced the transcription of HmVg1 and HmVg2, inhibited yolk protein deposition, and reduced fecundity using RNA interference. In addition, the preoviposition period was significantly prolonged after dsMet-injection, but there was no significant difference after dsKr-h1-silencing. However, the effect on hatchability results was the opposite. Therefore, we infer that both HmMet and HmKr-h1 are involved in female reproduction of H. axyridis, and their specific functions are different in certain physiological processes. In several continents, H. axyridis are not only beneficial insects, but also invasive pests. This report will provide basis for applying or controlling the H. axyridis.

Krüppel homolog 1 regulates photoperiodic reproductive plasticity in the cabbage beetle Colaphellus bowringi.

Many insects exhibit reproductive plasticity where the photoperiod determines whether the insect becomes reproductively active or enters diapause. Adult reproductive diapause is a strategy that allows insects to survive harsh environmental conditions. A deficiency in juvenile hormone (JH) leads to reproductive diapause. However, little is known about the molecular mechanisms by which JH signaling regulates reproductive diapause. In this study, we used the cabbage beetle Colaphellus bowringi, a serious pest, to investigate the role of Krüppel homolog 1 (Kr-h1) in controlling photoperiodic plasticity of female reproduction. We focused on Kr-h1, since it acts as a key mediator of JH signaling. We show here that JH-Methoprene-tolerant signaling upregulated the expression of Kr-h1 in reproductively active C. bowringi females when reared under short day conditions. In the long day-treated diapausing females, Kr-h1 transcripts decreased dramatically. Interfering with Kr-h1 function repressed reproductive development by blocking vitellogenesis and ovarian growth. Further, Kr-h1 depletion induced other diapause-like traits, including elevated lipid accumulation and high expression of diapause-related genes. RNA-Seq showed that Kr-h1 played both activating and repressive roles, depending on whether downstream genes were acting in reproduction- or diapause pathways, respectively. Finally, we identified the DNA replication gene mini-chromosome maintenance 4 and two triacylglycerol lipase genes as critical downstream factors of Kr-h1 that are critical for reproductive plasticity in C. bowringi. These results reveal that Kr-h1 is a key component of the regulatory pathway that coordinates reproduction and diapause in insects in response to photoperiodic input.

Involvement of Methoprene-tolerant and Krüppel homolog 1 in juvenile hormone-signaling regulating the maturation of male accessory glands in the moth Agrotis ipsilon.

Male accessory glands (MAGs) produce seminal fluid proteins that are essential for the fertility and also influence the reproductive physiology and behavior of mated females. In many insect species, and especially in the moth Agrotis ipsilon, juvenile hormone (JH) promotes the maturation of the MAGs but the underlying molecular mechanisms in this hormonal regulation are not yet well identified. Here, we examined the role of the JH receptor, Methoprene-tolerant (Met) and the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) in transmitting the JH signal that upregulates the growth and synthetic activity of the MAGs in A. ipsilon. We cloned two full length cDNAs encoding Met1 and Met2 which are co-expressed with Kr-h1 in the MAGs where their expression levels increase with age in parallel with the length and protein content of the MAGs. RNAi-mediated knockdown of either Met1, Met2, or Kr-h1 resulted in reduced MAG length and protein amount. Moreover, injection of JH-II into newly emerged adult males induced the transcription of Met1, Met2 and Kr-h1 associated to an increase in the length and protein content of the MAGs. By contrast, JH deficiency decreased Met1, Met2 and Kr-h1 mRNA levels as well as the length and protein reserves of the MAGs of allatectomized old males and these declines were partly compensated by a combined injection of JH-II in operated males. Taken together, our results highlighted an involvement of the JH-Met-Kr-h1 signaling pathway in the development and secretory activity of the MAGs in A. ipsilon.

Crucial Role of Juvenile Hormone Receptor Components Methoprene-Tolerant and Taiman in Sexual Maturation of Adult Male Desert Locusts.

Currently (2020), Africa and Asia are experiencing the worst desert locust (Schistocerca gregaria) plague in decades. Exceptionally high rainfall in different regions caused favorable environmental conditions for very successful reproduction and population growth. To better understand the molecular mechanisms responsible for this remarkable reproductive capacity, as well as to fill existing knowledge gaps regarding the regulation of male reproductive physiology, we investigated the role of methoprene-tolerant (Scg-Met) and Taiman (Scg-Tai), responsible for transducing the juvenile hormone (JH) signal, in adult male locusts. We demonstrated that knockdown of these components by RNA interference strongly inhibits male sexual maturation, severely disrupting reproduction. This was evidenced by the inability to show mating behavior, the absence of a yellow-colored cuticle, the reduction of relative testes weight, and the drastically reduced phenylacetonitrile (PAN) pheromone levels of the treated males. We also observed a reduced relative weight, as well as relative protein content, of the male accessory glands in Scg-Met knockdown locusts. Interestingly, in these animals the size of the corpora allata (CA), the endocrine glands where JH is synthesized, was significantly increased, as well as the transcript level of JH acid methyltransferase (JHAMT), a rate-limiting enzyme in the JH biosynthesis pathway. Moreover, other endocrine pathways appeared to be affected by the knockdown, as evidenced by changes in the expression levels of the insulin-related peptide and two neuroparsins in the fat body. Our results demonstrate that JH signaling pathway components play a crucial role in male reproductive physiology, illustrating their potential as molecular targets for pest control.

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.

Effects of methyl farnesoate on Krüppel homolog 1 (Kr-h1) during vitellogenesis in the Chinese mitten crab (Eriocheir sinensis).

Methyl farnesoate (MF), a de-epoxidized form of juvenile hormone (JH) Ⅲ in insects, may regulate developmental processes such as reproduction and ovarian maturation in crustaceans. Krüppel homolog 1 (Kr-h1) is a target response gene for the methoprene-tolerant (Met) protein that is a component of the JH signaling pathway in insects. In the present study, Es-Kr-h1 was cloned from E. sinensis and characterized to ascertain whether JH/MF signaling in insects is conserved in crustaceans. The findings with molecular structure analysis indicated Es-Kr-h1 contains seven zinc finger motifs (Zn2-Zn8) commonly conserved in other crustaceans, but the Zn1 motif was not detected to be present. The PCR results indicated that relative abundance of Es-Kr-h1 mRNA transcript in the hepatopancreas was greatest in the Stage Ⅱ, followed by the Stage Ⅳ ovarian developmental categories. The relative abundance of Es-Kr-h1 mRNA transcript in vitro was greater after MF addition to the hepatopancreas, however, not the ovarian tissues. The results from in vivo and eyestalk ablation experiments indicated the relative abundance of Es-Kr-h1 mRNA transcript was greater after MF treatment and bilateral eyestalk removal in the hepatopancreas, however, not ovarian tissues. Notably, there were effects of MF on relative abundance of Es-Kr-h1 mRNA transcript pattern. The Es-Kr-h1 protein, therefore, may be involved in MF-mediated vitellogenesis resulting from the response to Es-Met in E. sinensis, and the JH/MF signaling pathway is potentially conserved in crustaceans.

Transcriptomic analysis reveals the regulation network of BmKrüppel homolog 1 in the oocyte development of Bombyx mori.

Krüppel homolog 1 (Kr-h1), a zinc finger transcription factor, is involved in the metamorphosis and adult reproduction of insects. However, the role of Kr-h1 in reproduction of holometabolic insects remains to be elucidated. The regulation network of Kr-h1-associated genes in the reproduction in Bombyx mori was investigated in this study. The higher expression level of BmKr-h1 in the ovaries was detected during the late pupal stage and adults. RNA interference (RNAi)-mediated depletion of BmKr-h1 in the female at day 6 of pupae resulted in abnormal oocytes at 48 h post-double-stranded RNA treatment, which showed less yolk protein deposition and partially transparent chorion. RNA-seq and subsequent differentially expressed transcripts analysis showed that knockdown of BmKr-h1 caused a decrease in the expression of 2882 genes and an increase in the expression of 2565 genes in the oocytes at day 8 of pupae. Totally, 27 genes coding for transcription factors were down-regulated, while six genes coding for other transcription factors were up-regulated. BmKr-h1 bound to the Kr-h1 binding site of the transcription factors AP-1 (activating protein-1) and FOXG1 to increase their messenger RNA transcripts in the BmN cells, respectively. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses of that positively co-expressed with AP-1 and FOXG1 transcripts showed mainly enrichment in the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes. These data suggested that BmKr-h1 might directly regulate the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes or probably through AP-1 and /or FOXG1 to regulate oocyte development.

Krüppel homolog 1 and E93: The doorkeeper and the key to insect metamorphosis.

Insect metamorphosis is regulated by two main hormones: ecdysone (20E), which promotes molting, and juvenile hormone (JH), which inhibits adult morphogenesis. The transduction mechanisms for the respective hormonal signals include the transcription factors Krüppel homolog 1 (Kr-h1) and E93, which are JH- and 20E-dependent, respectively. Kr-h1 is the main effector of the antimetamorphic action of JH, while E93 is a key promoter of metamorphosis. The ancestral regulatory axis of metamorphosis, which operates in insects with hemimetabolan (gradual) metamorphosis and is known as the MEKRE93 pathway, is based on Kr-h1 repression of E93. In the last juvenile stage, when the production of JH dramatically decreases, Kr-h1 expression is almost completely interrupted, E93 becomes upregulated and metamorphosis proceeds. The holometabolan (complete) metamorphosis mode of development includes the peculiar pupal stage, a sort of intermediate between the final larval instar and the adult stage. In holometabolan species, Broad-Complex (BR-C) transcription factors determine the pupal stage and E93 stimulates the expression of BR-C in the prepupa. The MEKRE93 pathway is conserved in holometabolan insects, which have added the E93/BR-C interaction loop to the ancestral (hemimetabolan) pathway during the evolution from hemimetaboly to holometaboly.

Molecular characterization of the Krüppel-homolog 1 and its role in ovarian development in Sogatella furcifera (Hemiptera: Delphacidae).

Juvenile hormone (JH) plays a pivotal role in insect reproduction. The Krüppel-homolog 1 (Kr-h1) is a JH-inducible zinc finger transcription factor that has also been found to play a role in insect reproduction, however, its function varies across species. In this study, we cloned SfKr-h1 from Sogatella furcifera and investigated its role in ovarian development. The open reading frame of SfKr-h1 is 1 800 bp encoding 599 amino acids. The putative amino acid sequence of SfKr-h1 contains eight putative C2H2-type zinc finger domains and is highly homologous with the Kr-h1s of other hemipteran species. Expression of SfKr-h1 peaked 96 h after adult emergence and was highest in the ovary. RNA interference (RNAi) knockdown of SfKr-h1 substantially reduced the transcription of SfVg, and arrested ovarian development. These results suggest that SfKr-h1 is critical for normal ovarian development in S. furcifera.

The evolution of insect metamorphosis: a developmental and endocrine view.

Developmental, genetic and endocrine data from diverse taxa provide insight into the evolution of insect metamorphosis. We equate the larva-pupa-adult of the Holometabola to the pronymph-nymph-adult of hemimetabolous insects. The hemimetabolous pronymph is a cryptic embryonic stage with unique endocrinology and behavioural modifications that probably served as preadaptations for the larva. It develops in the absence of juvenile hormone (JH) as embryonic primordia undergo patterning and morphogenesis, the processes that were arrested for the evolution of the larva. Embryonic JH then drives tissue differentiation and nymph formation. Experimental treatment of pronymphs with JH terminates patterning and induces differentiation, mimicking the processes that occurred during the evolution of the larva. Unpatterned portions of primordia persist in the larva, becoming imaginal discs that form pupal and adult structures. Key transcription factors are associated with the holometabolous life stages: Krüppel-homolog 1 (Kr-h1) in the larva, broad in the pupa and E93 in the adult. Kr-h1 mediates JH action and is found whenever JH acts, while the other two genes direct the formation of their corresponding stages. In hemimetabolous forms, the pronymph has low Broad expression, followed by Broad expression through the nymphal moults, then a switch to E93 to form the adult. This article is part of the theme issue 'The evolution of complete metamorphosis'.

Cross-talk between juvenile hormone and ecdysone regulates transcription of fibroin modulator binding protein-1 in Bombyx mori.

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) are the most important hormones in silkworm and play vital roles in silkworm development, metamorphosis, and silk protein synthesis. Fibroin modulator binding protein-1 (FMBP-1) is a novel transcription factor regulating fibroin heavy chain (fib-H) transcription in Bombyx mori. The roles of JH and 20E on FMBP-1 transcription are less known. Here, we show FMBP-1 transcription is repressed by juvenile hormone analog (JHA) and activated by 20E. We identify two Krüppel homolog 1 (Kr-h1) binding sites (KBS1 and KBS2) and an E74A binding site (EBS) in the promoter of FMBP-1. We demonstrate Kr-h1 directly binds to KBS1 and KBS2 to repress FMBP-1 transcription, and 20E promotes FMBP-1 transcription through E74A. In the presence of JH and 20E, E74A abolishes the repression of Kr-h1 and activates FMBP-1 transcription through direct binding to EBS between KBS1 and KBS2 in FMBP-1 promoter. Further, JHA and 20E treatment and RNA interference confirm the effects of JH and 20E on FMBP-1 transcription in vivo, thus affecting fib-H transcription. Our results reveal the molecular mechanism of FMBP-1 transcription regulated by the cross-talk between JH and 20E in Bombyx mori, and provide novel insights into FMBP-1 transcriptional regulation and silk protein synthesis.