Chitin synthase A: a novel epidermal development regulation gene in the larvae of Bombyx mori

Chitin synthase is the key regulatory enzyme for chitin synthesis and excretion in insects, as well as a specific target of insecticides. The chitin synthase A gene (BmChsA) cloned from Bombyx mori, the model species of lepidopteran, is an epidermis-specific expressed gene during the molting stage. Knockdown BmChsA gene in 3rd instar larvae increased the number of non-molting and abnormal molting larvae. Exposure to nikkomycin Z, a chitin synthase inhibitor downregulated the expression of BmChsA and decreased the amount of epidermis chitin during the molting process. The thickness of the new epidermis and its dense structure varied greatly. The exogenous hormones significantly upregulated the expression of BmChsA with low levels of endogenous MH and high levels of endogenous JH immediately after molting. With low levels of endogenous hormones during the mulberry intake process, BmChsA was rarely upregulated by exogenous hormones. With high levels of endogenous MH and low levels of endogenous JH during the molting stage, we did not detect the upregulation of BmChsA by exogenous hormones. The expression of BmChsA was regulated by endocrine hormones, which directly affected the chitin synthesis-dependent epidermal regeneration and molting process.     

The juvenile hormone titer of Trichoplusia ni and its potential role in embryogenesis of the polyembryonic wasp CopidosomaFloridanum

The hemolymph juvenile hormone (JH) titer of third through fifth stadia Trichoplusia ni parasitized by the polyembryonic parasitoid, Copidosoma floridanum, was measured by radioimmunoassay and compared to the titers of unparasitized larvae. The JH titer of parasitized larvae fluctuated from 28 pg/μl to undetectable levels. Maximum levels of hormone were present at ecdysis to the fourth and fifth stadium, and at the prepupal stage. Qualitatively, similar fluctuations were observed in unparasitized larvae. However, the titers in unparasitized larvae were much lower than those of parasitized larvae in the third and early fourth stadia, and the titer fell to undetectable levels in the fifth stadium 24 h earlier (48 h) than in parasitized larvae (72 h). Preventing the JH titer from falling during the fourth and fifth stadia by topical application of (RS)-methoprene or JH II had a juvenilizing effect on parasitized T. ni, and inhibited C. floridanum embryo morphogenesis. The effect of exogenous methoprene and JH on C. floridanum development depended on timing of application and dosage. Application of 100 pmol per day of methoprene beginning at 2 h of the host fourth stadium, prior to the large drop in the endogenous JH titer, inhibited morphogenesis in the majority of C. floridanum embryos. Application of methoprene at later times of host development did not inhibit morphogenesis although other developmental alterations were observed. The potential significance of host JH and ecdysteroid titers on polyembryonic development are discussed.     

Methoprene does not affect juvenile hormone titers in honey bee (Apis mellifera) workers

Methoprene, a juvenile hormone (JH) analog, is a widely used insecticide that also accelerates behavioral development in honey bees (Apis mellifera). JH regulates the transition from nursing to foraging in adult worker bees, and treatment with JH or methoprene have both been shown to induce precocious foraging. To determine how methoprene changes honey bee behavior, we compared JH titers of methoprene‐treated and untreated bees. Behavioral observations confirmed that methoprene treatment significantly increased the number of precocious foragers in 3 out of 4 colonies. In only 1 out of 4 colonies, however, was there a significant difference in JH titers between the methoprene‐treated and control bees. Further, in all 4 colonies, there was no significant differences in JH titers between precocious and normal‐aged foragers. These results suggest that methoprene did not directly affect the endogenous JH secreted by corpora allata. Because methoprene caused early foraging without changing workers’ JH titers, we conclude that methoprene most likely acts directly on the JH receptors as a substitute for JH.     

Hormonally-regulated differential expression of the two duplicated insecticyanin genes,ins-a and ins-b,during development of the tobacco hornworm,Manduca sexta

The effects of juvenile hormone (JH) and 20-hydroxyecdysone (20E) on the developmental expression of the two insecticyanin genes, ins-a and ins-b, were investigated with two gene-specific probes. Removal of the corpora allata (-CA, source of JH) clearly delayed and down-regulated the epidermal expression of these genes but enhanced their expression in the fat body during the early development of the fifth instar. Application of JH I to the -CA larvae at the time of head capsule slippage completely restored the normal epidermal expression pattern of the two genes in the early fifth instar, then INS-a mRNA declined prematurely whereas INS-b mRNA remained similar to that in the intact larvae. By contrast, in the fat body of -CA larvae, the exogenous JH had little effect on the levels of INS-a mRNA, but enhanced expression of INS-b mRNA relative to intact larvae. Culture of epidermis from day 1 fifth instar larvae with 40 ng/ml 20E for up to 24 h accelerated the loss of INS-a mRNA without affecting the levels of INS-b mRNA. Both mRNAs declined in isolated larval abdomens over a 24 h period, and this decline was slowed by 1 g methoprene (a JH analog). Together these results indicate that JH controls the levels of the two mRNAs in both the epidermis and fat body, with additional factors involved in regulating these genes in the fat body during the molt and in the epidermis during the growth phase.     

3S-(+)-3,7-Dimethyl-1,5-Octadiene-3,7-Diol and Ionone Derivatives from Tea

1-Isobutyl-5-(4-phenoxyphenyl)imidazole (KK-98), an inhibitor of juvenile hormone (JH) biosynthesis in the cockroach, and related imidazole compounds were evaluated against silkworm, Bombyx mori, for their activity to induce precocious metamorphosis. KK-98 induced precocious metamorphosis in the 4th instar larvae at high doses. Replacement of the 4-phenoxy group by a 3-phenoxy or 3-benzyloxy group on the benzene ring increased the activity. Among this series of compounds, 5-(3-benzyloxyphenyl)-1-isopropylimidazole (8) showed the highest activity. The induction of precocious metamorphosis by compound 8 was rescued by the simultaneous application of methoprene, a JH minie. When newly molted 3rd instar larvae were treated with a high dose of compound 8, a few larvae formed larval-pupal intermediates in the 3rd instar stage, which has not been formed by treating of any other imidazoles so far.     

Two Leptinotarsa uridine diphosphate N-acetylglucosamine pyrophosphorylases are specialized for chitin synthesis in larval epidermal cuticle and midgut peritrophic matrix

Uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (UAP) is involved in the biosynthesis of chitin, an essential component of the epidermal cuticle and midgut peritrophic matrix (PM) in insects. In the present paper, two putative LdUAP genes were cloned in Leptinotarsa decemlineata. In vivo bioassay revealed that 20-hydroxyecdysone (20E) and an ecdysteroid agonist halofenozide activated the expression of the two LdUAPs, whereas a decrease in 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD and a 20E signaling gene LdFTZ-F1 repressed the expression. Juvenile hormone (JH), a JH analog pyriproxyfen and an increase in JH by RNAi of an allatostatin gene LdAS-C downregulated LdUAP1 but upregulated LdUAP2, whereas a decrease in JH by silencing of a JH biosynthesis gene LdJHAMT had converse effects. Thus, expression of LdUAPs responded to both 20E and JH. Moreover, knockdown of LdUAP1 reduced chitin contents in whole larvae and integument samples, thinned tracheal taenidia, impaired larval–larval molt, larval-pupal ecdysis and adult emergence. In contrast, silencing of LdUAP2 significantly reduced foliage consumption, decreased chitin content in midgut samples, damaged PM, and retarded larval growth. The resulting larvae had lighter fresh weights, smaller body sizes and depleted fat body. As a result, the development was arrested. Combined knockdown of LdUAP1 and LdUAP2 caused an additive negative effect. Our data suggest that LdUAP1 and LdUAP2 have specialized functions in biosynthesizing chitin in the epidermal cuticle and PM respectively in L. decemlineata.     

Changes in levels of juvenile hormone and molting hormone in larvae and adult females of Chilo suppressalis (Lepidoptera: Pyralidae) after imidacloprid applications to rice

Insect hormones regulate growth and development and fecundity of insects. The current study investigated changes in juvenile hormone (JH) and molting hormone (MH) levels in fourth instars and adult females of Chilo suppressalis (Walker) (Lepidoptera: Pyralidae) after imidacloprid application to rice, Oryza sativa L. The results showed that JH level in fourth instars that developed feeding on Fengyouxiangzhan rice plants sprayed with 15, 30, and 60 ppm imidacloprid was significantly higher than that of larvae that developed on control plants, increasing by 5.04, 6.39, and 4.89 times, respectively. The relationships between JH level and imidacloprid concentrations showed a significant negative correlation. In contrast, molting hormone (MH) level in larvae fed on control plants was significantly higher than that on treated plants. JH:MH values in fourth instars developed from larvae feeding on rice plants treated with 15, 30, 60, 80, and 100 ppm imidacloprid increased by 49.17, 39.43, 13.48, 15.80, and 0.2 times, respectively, compared with control. JH and JH:MH ratio in larvae fed on Wujing 15 plants treated with imidacloprid were significantly lower than those fed on Fengyouxiangzhan under the same treatments. JH level in adult females that developed from larvae feeding on rice plants sprayed with imidacloprid significantly decreased with increase in imidacloprid concentration, but it increased compared with control. JH level in adult females was associated with times of imidacloprid application. JH level in adult females developed from larvae feeding on rice plants after double spray with 30 ppm imidacloprid was significantly higher than control, increasing by 61.6 and 116.5%, respectively, compared with a single spray and the control. Moreover, hormone levels in the larvae were related to the application method of imidacloprid. JH level in fourth instars after root application and topical application of imidacloprid was significantly lower than in control. Thus, the dynamics of JH and MH in insects after insecticide applications are an extremely interesting problem, because hormones are related to insect growth and development.     

Down‐regulation of a chitin synthase a gene by RNA interference enhances pathogenicity of Beauveria bassiana ANU1 against Spodoptera exigua (HÜBNER)

Chitin synthase (CHS) is an important enzymatic component, which is required for chitin formation in the cuticles and cuticular linings of other tissues in insects. CHSs have been divided into two classes, classes A and B, based on their amino acid sequence similarities and functions. Class A CHS (CHS‐A) is specifically expressed in the epidermis and related ectodermal cells such as tracheal cells, while class B CHS (CHS‐B) is expressed in gut epithelial cells that produce peritrophic matrices. In this study, we cloned the CHS‐A gene from the beet armyworm, Spodoptera exigua (SeCHS‐A). The SeCHS‐A contains an open reading frame of 4,698 nucleotides, encoding a protein of 1,565 amino acids with a predicted molecular mass of approximately 177.8 kDa. The SeCHS‐A mRNA was expressed in all developmental stages and specifically in the epidermis and tracheae tissue by quantitative real‐time‐PCR analysis. Expression of SeCHS‐A gene was suppressed by feeding double‐stranded RNA (dsCHS‐A, 400 ng/larva) in the third instar larvae of S. exigua. Suppression of the SeCHS‐A gene expression significantly increased 35% of mortality on pupation of S. exigua. Also, the third instar larvae fed with dsCHS‐A significantly increased susceptibility to entomopathogenic fungi, Beauveria bassiana ANU1 at 3 days after treatment. These results suggest that the SeCHS‐A gene plays an important role in development of S. exigua and RNA interference may apply to effective pest control with B. bassiana.     

Genome‐wide identification of chitin‐binding proteins and characterization of BmCBP1 in the silkworm,Bombyx mori

The insect cuticle plays important roles in numerous physiological functions to protect the body from invasion of pathogens, physical injury and dehydration. In this report, we conducted a comprehensive genome-wide search for genes encoding proteins with peritrophin A-type (ChtBD2) chitin-binding domain (CBD) in the silkworm, Bombyx mori. One of these genes, which encodes the cuticle protein BmCBP1, was additionally cloned, and its expression and location during the process of development and molting in B. mori were investigated. In total, 46 protein-coding genes were identified in the silkworm genome, including those encoding 15 cuticle proteins analogous to peritrophins with one CBD (CPAP1s), nine cuticle proteins analogous to peritrophins with three CBD (CPAP3s), 15 peritrophic membrane proteins (PMPs), four chitinases, and three chitin deacetylases, which contained at least one ChtBD2 domain. Microarray analysis indicated that CPAP-encoding genes were widely expressed in various tissues, whereas PMP genes were highly expressed in the midgut. Quantitative polymerase chain reaction and western blotting showed that the cuticle protein BmCBP1 was highly expressed in the epidermis and head, particularly during molting and metamorphosis. An immunofluorescence study revealed that chitin co-localized with BmCBP1 at the epidermal surface during molting. Additionally, BmCBP1 was notably up-regulated by 20-hydroxyecdysone treatment. These results provide a genome-level view of the chitin-binding protein in silkworm and suggest that BmCBP1 participates in the formation of the new cuticle during molting.     

Localization of molting chitinase in insect cuticle

Chitinase activity in molting larvae of Manduca sexta is localized in old cuticle; it is not quantitatively extracted during homogenization, has good activity at the pH of molting fluid, and preferentially utilizes endogenous cuticle chitin as substrate. It is concluded that cuticle chitinase is the physiologically active molting enzyme in Manduca.     

Simultaneous determination of molting and juvenile hormone titers of the greater wax moth

A simple and rapid extraction procedure was developed to determine simultaneously the molting hormone (MH) and juvenile hormone (JH) activity in a single insect tissue sample. From the onset of the last larval stage to adult eclosion of the greater wax moth, Galleria mellonella, three JH peaks were noted: at the time of the sixth larval ecdysis, 1 day before the seventh larval ecdysis, and at the time of adult eclosion. Three MH peaks were recorded for the male: at 1 day before the sixth larval ecdysis, 1 day before the seventh larval ecdysis, and 2 days after pupation. In the female, a fourth peak was shown at the time of adult eclosion. This fourth peak exhibits the highest molting hormone activity of all samples, 1600 Musca units/g of fresh tissue or an equivalent of 5.6 μg/g of ecdysterone. Eighty per cent of this MH accumulated in the ovary. The significance of MH and JH titers as related to the endocrine regulation of development is discussed in the light of this finding.