Cloning and functional analysis of the ecdysteroid receptor complex in the opossum shrimp Neomysis integer (Leach, 1814).

In this paper, the non-target effects of tebufenozide were evaluated on the estuarine crustacean, the opposum shrimp Neomysis integer (Leach, 1814). Tebufenozide is a synthetic non-steroidal ecdysone agonist insecticide and regarded as potential endocrine-disrupting chemical (EDC). N. integer is the most used crustacean in ecotoxicological research in parallel to Daphnia sp. and has been proposed for the regulatory testing of potential EDCs in the US, Europe and Japan. Major results were: (i) cDNAs encoding the ecdysteroid receptor (EcR) and the retinoid-X-receptor (RXR), were cloned and sequenced, and subsequent molecular phylogenetic analysis (maximum likelihood and neighbor-joining) revealed that the amino acid sequence of the ligand binding domain (LBD) of N. integer EcR (NiEcR) clusters as an outgroup of the Crustacea, while NiRXR-LBD clusters in the Malacostracan clade (bootstrap percentage=75%). (ii) 3D-modeling of ligand binding to NiEcR-LBD demonstrated an incompatibility of the insecticide tebufenozide to fit into the NiEcR-ligand binding pocket. This was in great contrast to ponasterone A (PonA) that is the natural molting hormone in Crustacea and for which efficient docking was demonstrated. In addition, the heterodimerization of NiEcR-LBD with the common shrimp Crangon crangon (Linnaeus, 1758) RXR-LBD (CrcRXR-LBD) was also modeled in silico. (iii) With use of insect Hi5 cells, chimeric constructs of NiEcR-LBD and CrcRXR-LBD fused to either the yeast Gal4-DNA binding domain (DBD) or Gal4-activation domain (AD) were cloned into expression plasmids and co-transfected with a Gal4 reporter to quantify the protein-protein interactions of NiEcR-LBD with CrcRXR-LBD. Investigation of the ligand effect of PonA and tebufenozide revealed that only the presence of PonA could induce dimerization of this heterologous receptor complex. (iv) Finally, in an in vivo toxicity assay, N. integer juveniles were exposed to tebufenozide at a concentration of 100 µg/L, and no effects against the molting process and nymphal development were scored. In conclusion, the in vitro cell reporter assay, based on NiEcR-LBD/CrcRXR-LBD heterodimerization in Hi5 cells and validated with the natural ecdysteroid hormone PonA, represents a useful tool for the screening of putative EDCs. As a test example for non-steroidal ecdysone agonist insecticides, tebufenozide had no negative effects on NiEcR/RXR receptor dimerization in vitro, nor on the molting process and nymphal development of N. integer at the tested concentration (100 µg/L) in vivo.

Colorado potato beetle (Coleoptera) gut transcriptome analysis: expression of RNA interference-related genes.

In the search for new methods of pest control, the potential of RNA interference (RNAi) is being explored. Because the gut is the first barrier for the uptake of double-stranded (ds)RNA, pyrosequencing of the gut transcriptome is a powerful tool for obtaining the necessary sequences for specific dsRNA-mediated pest control. In the present study, a dataset representing the gut transcriptome of the Colorado potato beetle (CPB; Leptinotarsa decemlineata) was generated and analysed for the presence of RNAi-related genes. Almost all selected genes that were implicated in silencing efficiency at different levels in the RNAi pathway (core machinery, associated intracellular factors, dsRNA uptake, antiviral RNAi, nucleases), which uses different types of small RNA (small interfering RNA, microRNA and piwi-RNA), were expressed in the CPB gut. Although the database is of lower quality, the majority of the RNAi genes are also found to be present in the gut transcriptome of the tobacco hornworm [TH; Manduca sexta (19 out of 35 genes analysed)]. The high quality of the CPB transcriptome database will lay the foundation for future gene expression and functional studies regarding the gut and RNAi.

Functional expression of mammalian opioid receptors in insect cells and high-throughput screening platforms for receptor ligand mimetics.

Lepidopteran cell lines have been engineered to constitutively express high levels of mouse delta opioid receptors either alone or in combination with human Galpha16 protein. Biochemical and pharmacological studies demonstrate that these lines contain all the mediator G proteins and downstream effectors required for opioid receptor function, including phospholipase C, and that expression of exogenous Galpha16 does not contribute significantly to increased receptor responses upon activation. The activation of the phospholipase C pathway in the transformed cells upon stimulation with known receptor ligands results in easily and quantitatively measurable increases in free intracellular calcium, which can be monitored by automated fluorescent methods, while the addition of specific antagonists blocks the agonist-induced responses. Therefore, the transformed lepidopteran cell lines can be used as sensitive high-throughput screening platforms for fast detection of delta opioid receptor ligand mimetics (agonists and antagonists) in collections of natural products and synthetic compounds.

The orphan nuclear receptors BmE75A and BmE75C of the silkmoth Bombyx mori: hornmonal control and ovarian expression.

The steroid hormone 20-hydroxyecdysone (20E) plays a key role in the stimulation of ovarian follicle development in the silkmoth, Bombyx mori. To understand better the mechanism by which 20E regulates silkmoth oogenesis, Bombyx homologs of the ecdysone-inducible orphan nuclear receptor E75 (BmE75) were cloned and their expression was analyzed in developing ovaries and staged follicles during metamorphosis. Of the two BmE75 isoforms isolated, only the A-isoform (BmE75A) has been identified previously in lepidopteran insects. BmE75C, on the other hand, shows significant sequence homology in its N-terminus to the Drosophila E75C isoform. Northern blot analysis shows unique expression patterns for each isoform mRNA during ovarian development. While the A-isoform seems to be mainly implicated in the earlier stages of the ecdysone response during previtellogenesis and vitellogenesis, expression of the C-isoform becomes strongly induced in an ecdysteroid-independent fashion at the transition from vitellogenesis to choriogenesis. Our data indicate a complex regulation of the expression of the BmE75 gene during oogenesis and postulate a new role for the BmE75C receptor at the end of vitellogenesis and the beginning of choriogenesis.

The orphan nuclear receptor BmHR3A of Bombyx mori: hormonal control, ovarian expression and functional properties.

Ovarian development in the domesticated silkmoth, Bombyx mori, is induced by the molting hormone 20-hydroxy-ecdysone (20E) shortly after larval-pupal ecdysis. Studies of the ecdysone response in Drosophila and other insects have shown that 20E exerts its effects initially by the induction of a small number of early genes, including the orphan nuclear receptors HR3, that transduce and amplify the hormone signal. Here we show that the silkmoth orphan receptor BmHR3A acts in the 20E-induced regulatory cascade in the ovary during pupal and pharate adult development in a manner different than that observed in the classical ecdysone regulatory hierarchy in Drosophila salivary glands at the end of the third instar. While other isoforms of BmHR3 are induced as early gene products in the ecdysone response, BmHR3A is induced 2 days after 20E administration in the silkmoth ovary and, thus, behaves as late product. The period of accumulation of BmHR3A in ovarian follicular cells occurs during vitellogenesis and coincides with the period of transcriptional expression of the ESP (egg-specific protein) gene, whose product constitutes a major component of the egg yolk, while it is reciprocal to the period of expression of BmGATAbeta, a gene encoding a regulator of late chorion gene expression. Bandshift experiments demonstrate that BmHR3A binds specifically to RORE (Retinoic acid-related Orphan receptor Response Element)-like sequences in the promoters of both genes, thus suggesting a direct role for BmHR3A in regulating the expression of BmGATAbeta and ESP genes during vitellogenesis. Finally, we show that BmHR3A functions as a constitutive transcriptional activator in a B. mori derived cell line. We propose that BmHR3A may function as a regulator of vitellogenesis in the silkmoth ovary.

Mariner (Mos1) transposase and genomic integration of foreign gene sequences in Bombyx mori cells.

Widespread occurrence in insects and the capacity to transpose in the absence of host-derived factors means that mariner-like elements are considered to be attractive candidates for the development of a universal insect genetic transformation system. Here we show that the Mos1 mariner element of Drosophila mauritiana is capable of mediating excision and transposition events in a silkmoth (Bombyx mori) derived tissue culture cell line (Bm5 cells). Plasmid rescue assays, in combination with Southern hybridization and polymerase chain reaction (PCR) analyses, confirm that the Mos1 transposase can mediate excision of DNA sequences, inserted between terminally repeated sequences recognized by the transposase, and integration into the chromosomal DNA of the Bm5 cells. In addition to chromosomal integration events, inter- and intraplasmid transposition and target element excision events were also detected. Approximately 50% of the plasmids recovered from plasmid rescue assays were found to contain the 'signature' of Mos1-specific excision and/or integration events, indicating that the mariner transposase functions efficiently in the Bombyx cells. Because mariner-induced excision and integration events are strictly dependent on the presence of a co-transfected Mos1 transposase expression vector, it is clear that the multiple copies of endogenous mariner-like elements (Bmmar1) that exist in the Bombyx genome are neither functional nor do they interfere with the efficiency of the transposition process. Thus, the Mos1 element and, probably, mariner elements, in general, hold great promise for the development of genetic transformation systems for lepidopteran insects.

The p95 gene of Bombyx mori nuclear polyhedrosis virus: temporal expression and functional properties.

As part of our effort to identify baculovirus proteins acting as transcriptional regulators, we have characterized a gene, p95, of Bombyx mori nuclear polyhedrosis virus (BmNPV) that encompasses an open reading frame for a putative 95-kDa polypeptide (P95). The N-terminal half of the conceptually translated P95 contains two zinc finger-type DNA-binding motifs, and its C terminus contains a proline-rich region reminiscent of transcriptional activation regions. Northern blot analysis indicates that two mRNA species, 3.5 and 1.7 kb in size, are transcribed from the p95 gene at different times postinfection. These two mRNA species are produced by differential polyadenylation site usage. While the longer transcript can encode the P95 protein, the shorter one may encode a prematurely terminated version of the P95 polypeptide produced by ribosome frameshifting occurring at heptanucleotide "slippage" sites located near the relevant polyadenylation site. Transcription of the p95 gene is initiated at a proximal site located 70 nucleotides upstream of the translation start codon of P95, a middle site located 170 nucleotides from the start codon, and a set of three closely spaced distal sites located 385, 390, and 409 nucleotides from the translation start codon. The middle and distant initiation sites are utilized before and after BmNPV DNA replication, while transcripts initiated at the proximal site occur largely during the late and very late stages of viral infection. Transient-expression assays indicate that P95 can stimulate gene expression driven by the promoter of its own gene and the promoter of the cytoplasmic actin gene of B. mori. The P95-mediated trans activation can be further augmented by BmIE1, an immediate-early gene product of BmNPV. In contrast to the case with the actin promoter, however, the promoter of the p95 gene can be trans activated by the product of its own gene only in the presence of BmIE1. Our data suggest that proteins P95 and BmIE1 of BmNPV and, by analogy, those of other baculoviruses may interact with each other and synergize to potentiate transcription.

The orphan receptor BmHNF-4 of the silkmoth Bombyx mori: ovarian and zygotic expression of two mRNA isoforms encoding polypeptides with different activating domains.

Two silkmoth nuclear receptor isoforms, BmHNF-4a and BmHNF-4b, that are related to the mammalian orphan receptor HNF-4, were characterized. Their characterization revealed that they differ from each other only in their 5' UTR and N-terminus of the predicted polypeptides. In ovarian tissue, the two receptors are expressed as a delayed response to 20-hydroxy-ecdysone and their expression increases during vitellogenesis. BmHNF-4 mRNA is localized in the cytoplasm of follicular cells and a binding activity that recognizes a mammalian HNF-4 response element is present in follicular cell nuclear extracts. BmHNF-4 mRNA is also present in the oocyte, the unfertilized egg and the early embryo, thus displaying a behavior reminiscent of maternal mRNA. Both mRNA isoforms are found in the embryo following fertilization and their abundance is modulated during ensuing embryogenesis. In contrast to the rather limited distribution of HNF-4 in mammalian tissues, BmHNF-4 is expressed in most larval and pharate adult tissues of the silkmoth.

Bombyx EcR (BmEcR) and Bombyx USP (BmCF1) combine to form a functional ecdysone receptor.

The Drosophila ecdysone receptor (DmEcR) is a member of the nuclear receptor superfamily; it functions as an obligate heterodimer with another nuclear receptor, DmUSP. EcR homologs have now been cloned from several other insects. We report here that one such homolog, BmEcR from the commercial silkmoth, Bombyx mori, is a functional ecdysone receptor. Upon dimerization with BmCF1, the silkmoth homology of DmUSP, BmEcR binds the radiolabeled steroid ligand 125I-iodoponasterone A with Kd = 1.1 nM, indistinguishable from that exhibited by DmEcR/DmUSP. BmEcR/BmCF1 forms a specific complex with an ecdysone response element (EcRE) derived from the heat shock protein 27 (hsp27) gene promoter of Drosophila; and, as with DmEcR/DmUSP, formation of this complex is stimulated by the presence of 20-hydroxyecdysone. Finally, BmEcR can substitute for DmEcR in an EcR-deficient Drosophila tissue culture line, stimulating trans-activation of an ecdysone-inducible reporter gene construct. Thus, BmEcR and BmCF1 are the functional counterparts of DmEcR and DmUSP, respectively and, despite considerable sequence divergence between the Drosophila and Bombyx proteins, the counterparts are--at least qualitatively--functionally equivalent.

The silkmoth homolog of the Drosophila ecdysone receptor (B1 isoform): cloning and analysis of expression during follicular cell differentiation.

To understand the role that 20-hydroxy-ecdysone (20E) plays during ovarian development in Bombyx mori, we have undertaken the cloning of the silkworm ecdysone receptor (EcR) and a study of its expression during follicular cell differentiation. We have cloned a cDNA that contains a complete open reading frame for a 68.1 kDa polypeptide that shares extensive similarities with the B1 isoform of the Drosophila EcR. The presumed silkmoth EcR (BmEcR) is encoded by a single copy gene whose length is in excess of 23 kb. A portion of this gene encompassing seven exons that constitute the cloned BmEcR cDNA was also characterized. Employment of monoclonal antibodies, directed against the DNA binding domain of the Drosophila EcR, in Western blot analyses revealed the presence of a major 70 kDa polypeptide in extracts of follicular cells and other silkmoth tissues. The mRNA and protein encoded by BmEcR are present in constant amounts in follicular cells throughout vitellogenesis but disappear transiently at the onset of choriogenesis and reappear during the later stages of choriogenesis. The down-regulation of BmEcR in follicular cells during oogenesis suggest a complex relationship between 20E, the induction of the program of chorion gene expression in follicular cells during mid-vitellogenesis and the execution of this program at the end of vitellogenesis.

Developmental regulation of a silkworm gene encoding multiple GATA-type transcription factors by alternative splicing.

Gene BmGATA beta of the silkworm Bombyx mori was previously shown to encode factor BCFI, which regulates the expression of a class of chorion genes expressed during the late stages of choriogenesis. We now show that the expression of the BmGATA beta gene is spatially and temporally regulated by alternative splicing that generates two major (BmGATA beta 1 and BmGATA beta 2) and one minor (BmGATA beta 3) mRNA isoforms of non-identical tissue distribution. The three isoforms differ in the organization of the DNA-binding domains of the corresponding polypeptides. While all three isoforms are expressed in ovarian follicular cells and in testes, only one of them, BmGATA beta 1, is gonad-specific. BmGATA beta 2 is expressed in a variety of other larval and pupal tissues, while BmGATA beta 3 is detected in some pupal but none of the larval tissues. Analysis of RNA isolated from follicular cells of developing ovarian follicles has shown that the onset of ovarian transcription for all three mRNA isoforms occurs during late vitellogenesis, and that the level of accumulated mRNA declines significantly at the onset of choriogenesis. Coincident with the onset of late chorion gene expression, we have observed a significant change in the preference of splice site selection in favour of the one that results in the generation of BmGATA beta 1 mRNA. The transcriptional activation of the BmGATA beta gene in follicular cells during late vitellogenesis correlates with the previously demonstrated initial accumulation of factor BCFI in the cytoplasm of follicular cells and its appearance in follicular cell nuclei only during the late stages of choriogenesis. The relationship between factor BCFI and the different polypeptides encoded by the three BmGATA beta mRNA isoforms is discussed.

Protein phosphorylation and control of chorion gene activation through temporal mobilization of a promoter DNA binding factor from the cytoplasm into the nucleus.

The transcriptional activation of high cysteine chorion genes in the follicular cells of the silkworm Bombyx mori occurs at the end of oogenesis and coincides with the appearance of a chorion promoter DNA binding factor, BCFI, in follicular cell nuclei. Follicular cells of vitellogenic and choriogenic follicles that do not express high cysteine chorion genes contain high levels of a latent form of BCFI in their cytoplasm. The abundance of the cytoplasmic factor, termed cBCFI, is dramatically reduced during late choriogenesis, coincident with the appearance of factor BCFI in the nucleus and the transcriptional activation of high cysteine genes. Mobility shift assays performed with partially proteolyzed nuclear and cytoplasmic extracts of follicular cells, DNA binding assays carried out in the presence of anti-BCFI antibodies, and electrophoretic analyses of the proteins present in the nuclear and cytoplasmic fractions of follicular cells and recognized by the same antibodies suggest that factor cBCFI represents a covalently modified version of BCFI. The DNA-binding sites of BCFI and cBCFI include a core sequence, AGATAA, but, while this sequence is sufficient for specific binding of BCFI, it only constitutes part of the DNA-binding site of cBCFI. Dephosphorylation of cBCFI results in a change of its binding specificity to that of BCFI. The cytoplasmic sequestration of cBCFI appears to be mediated by a phosphorylation-dependent, reversible association of this factor with an ancillary cytoplasmic factor.

On the origin of vertebrate-type steroids present in Locusta migratoria: do they originate from the food?

1. This study investigates the origin of vertebrate-type steroids which were reported to be present in Locusta migratoria: are the steroids synthesized by the locust or are they derived from the diet, i.e. grass and rolled oats? 2. It is unlikely that the steroids are synthesized by locust tissues. In vitro incubations of eleven different tissues with labeled pregnenolone or androstenedione did not result in androgen or estrogen synthesis respectively. 3. Steroid synthesis was also not detected when tissues were incubated in the presence of the early precursors cholesterol and isopentenyl pyrophosphate. 4. It is unlikely that the steroids are derived from the diet. Feeding experiments indicate that only low amounts of steroids are capable of crossing the gut-body barrier. 5. Injection of testosterone in the hemolymph also resulted in rapid excretion, instead of storage in tissues. 6. Moreover, radioimmunological measurements indicate that vertebrate-type steroids are absent in the food of locusts. 7. Specificity studies indicate that substances other than vertebrate-type steroids are detected by radioimmunoassay in locust tissue extracts. 8. Because vertebrate-type steroids are absent in locust tissues, it can be concluded that vertebrate-type steroids do not have a physiological function in Locusta.

Early establishment and autonomous implementation of a developmental program controlling silkmoth chorion gene expression.

To address the question of whether prechoriogenic follicles of the silkmoth Bombyx mori have the capacity to enter choriogenesis in organ culture and define the stage at which choriogenesis becomes established as a follicle-autonomous program, we have cultured immature ovarioles dissected from developing pupae and examined the protein synthetic profiles of follicular cells of individual follicles at the end of the culture period. The protein synthetic profiles of the cultured follicles were also correlated with corresponding profiles of chorion mRNA accumulation. Our results demonstrate that the last 17 (+/- 2) vitellogenic follicles of Day 5 to 7 pupae are capable of initiating choriogenesis in organ culture. The earliest vitellogenic stage to enter choriogenesis in vitro does so after 34 (+/- 4) hr in culture and follicles entering choriogenesis in vitro are capable of proceeding through all choriogenic stages at a speed comparable to that occurring in vivo. Therefore, once the choriogenic program becomes established in follicular cells, it can be implemented autonomously in the absence of extrafollicular factors. Earlier vitellogenic stages lack this capacity, presumably because they require additional hemolymph factors to establish the choriogenic potential. Our results demonstrate that the choriogenic potential of cultured vitellogenic follicles cannot be influenced by addition of 20-hydroxyecdysone to the culture medium.

Lack of essential enzymes for the biosynthesis of C19 and C18 steroids in gonads of the migratory locust, Locusta migratoria.

Ovaries and testes of the African migratory locust, Locusta migratoria migratorioides, were incubated in vitro with six tritiated steroid precursors. Three developmental stages were investigated--1 day, 14 days, and 6 weeks after adult moulting. 20 alpha-Hydroxysteroid dehydrogenase (HSD), 20 beta-HSD, 17 beta-HSD, 3 beta-HSD/isomerase, C17-C20 lyase, glucuronyl-transferase, sulfotransferase, and acyltransferase were identified in both sexes. A synthesis of androgens or estrogens comparable to the vertebrate type, however, was not apparent in the locust gonads. 20 alpha-HSD, 20 beta-HSD, and 17 beta-HSD activities were high, while more important steps in steroid synthesis such as 3 beta-HSD and C17-C20 lyase were far less intense. Ovarian 17 alpha-hydroxylase activity was slight. Aromatase activity was not demonstrated. Water-soluble conjugate formation was high in the incubations of "14th-day" and "6th-week" gonads but was absent in "1st-day" ovaries and testes. Active ester formation of pregnenolone was demonstrated in "6th-week" testes. The other steroid conversions were similar in all developmental stages investigated. Major differences between testes and ovaries were not observed. The gonads of the migratory locust are concluded not to produce androgens or estrogens.

Synthesis and metabolism of vertebrate-type steroids by tissues of insects: a critical evaluation.

This review covers the synthesis and the metabolism of vertebrate-type steroids (progesterone, testosterone, estradiol, corticosteroids) by insect tissues and discusses the significance of the reactions for insect physiology. Biosynthesis of vertebrate-type steroids from cholesterol hitherto has been demonstrated in only two insect species, i.e. the water beetle Acilius sulcatus (Coleoptera) and the tobacco hornworm Manduca sexta (Lepidoptera). In Acilius, steroid synthesis is associated with exosecretion (chemical defense). Nothing, however, is known about a physiological role of the C21 steroid conjugate present in ovaries and eggs of Manduca. No synthesis of vertebrate-type steroids was observed in any other insect investigated to date. Most metabolic conversions of steroids by insects concerned oxidoreduction of oxygen groups (hydroxysteroid dehydrogenase activity) and (polar and apolar) conjugate formation. All important enzymatic steps involved in synthesis and catabolism, as known from studies with tissues of vertebrates, were not, or hardly observed. The conclusion is drawn that typical vertebrate-type (C21, C19 and C18) steroids probably do not act as physiologically active substances in insects.

The presence of a pregnenolone-binding factor in the copulatory organ of the migratory locust, Locusta migratoria migratorioides R. & F.

The presence of binding sites for nonecdysteroid steroids was investigated in the cytosol of several tissues of the migratory locust, Locusta migratoria migratorioides. Binding of androgens was not observed. Most tissues, however, showed nonsaturable binding of estrogens and in some tissues saturable progestin binding could be demonstrated. A pregnenolone binder, that was found to be present in the male copulatory organ, was further studied. It showed a dissociation constant of 4.4 (+/- 1.6) X 10(-8) M. This is the first report of a nonecdysteroid steroid-binding factor in an insect tissue.