Characterization and functional analysis of BmSR-B1 for phytosterol uptake.

Insects cannot synthesize sterols, such as cholesterol, and require sterols in their diet. Phytophagous insects use dietary phytosterols as a source of cholesterol. Sterols are transported from the midgut by the insect lipoprotein, lipophorin (Lp), although mechanisms for uptake of phytosterols into tissues are unclear. This study characterizes Scavenger Receptor class B type1 (SR-B1) from Bombyx mori (BmSR-B1) as molecules related to phytosterol uptake. According to sterol quantification using LC-MS/MS analysis, the midgut and fat body were phytosterol-rich relative to cholesterol-rich brain and prothoracic glands. Gene expression analysis of Bmsr-b1 in silkworm tissues showed that the genes Bmsr-b1_2, 3, 4, 6, and 10 were expressed in the midgut and fat body. To characterize the function of BmSR-B1, 11 BmSR-B1 homologs expressed in Bombyx ovary-derived BmN cells and Drosophila melanogaster embryo-derived Schneider 2 (S2) cells were incubated with purified Lp. Our analysis showed that BmSR-B1_3 induced the accumulation of campesterol and BmSR-B1_4 induced the accumulation of ß-sitosterol and campesterol in culture cells. BmSR-B1 incorporated specific phytosterols into insect cells by selective uptake across the cell membrane where BmSR-B1 was localized. In conclusion, our study demonstrated that one function of BmSR-B1 is the uptake of phytosterols into silkworm tissues.

Production of the yolk protein precursor vitellogenin is mediated by target of rapamycin (TOR) in the soft tick Ornithodoros moubata (Acari: Argasidae).

Initiation of vitellogenesis by blood feeding is essential for egg maturation in ticks. Nutrients derived from the blood meal are utilized by female ticks to synthesize the yolk protein precursor vitellogenin (Vg). Engorged Ornithodoros moubata ticks can synthesize Vg whether mated or virgin, thus O. moubata is an excellent model for studying the relative roles of blood feeding and mating in tick vitellogenesis. Injection of rapamycin into engorged O. moubata resulted in a reduction of ovarian growth and yolk accumulation in the oocytes of mated females. OmVg expression in the midgut and fat body and protein concentrations in the hemolymph significantly decreased in mated ticks after injection with rapamycin, indicating that inhibition of the nutrient-sensing target of rapamycin (TOR) pathway disrupts egg maturation at the levels of Vg expression and synthesis. These results suggest that the TOR-signaling pathway induces vitellogenesis in response to nutritional stimulation after a blood meal in O. moubata and is functionally independent of the mating-induced pathway.

Twenty-hydroxyecdysone produced by dephosphorylation and ecdysteroidogenesis regulates early embryonic development in the silkmoth, Bombyx mori.

Ecdysteroids are key regulators of embryonic development as well as molting and metamorphosis in insects. Although an active form of ecdysteroids, 20-hydroxyecdysone (20E) is known to be produced through ecdysteroidogenesis from cholesterol and dephosphorylation of 20E-phosphate during embryogenesis in Lepidoptera, the importance of these production mechanisms in embryonic development has been unclear. Here, we investigated the activation timing of ecdysteroidogenesis from cholesterol and 20E-phosphate dephosphorylation during early embryogenesis in non-diapause eggs of the silkmoth Bombyx mori by observing morphological development, quantifying 20E and 20E-phosphate, measuring transcripts of enzymes involved in 20E production, and detecting activity of these enzymes using egg extracts. Stage-dependent 20E fluctuation and changes in mRNA amounts of enzymes suggest that the two 20E-producing mechanisms are activated at different stages during embryogenesis. Furthermore, knockdown of a dephosphorylation enzyme delayed development at early embryogenesis, whereas knockdown of an ecdysteroidogenic enzyme delayed development at early-middle embryogenesis. These results suggest that 20E is primarily produced initially by dephosphorylation of 20E-phosphate, and then by ecdysteroidogenesis from cholesterol to induce progression of embryonic development in B. mori.

Functional analysis of ecdysteroid biosynthetic enzymes of the rice planthopper, Nilaparvata lugens.

Ecdysteroids, insect steroid hormones, play key roles in regulating insect development and reproduction. Hemipteran insects require ecdysteroids for egg production; however, ecdysteroid synthesis (ecdysteroidogenesis) details have not been elucidated. We identified all known genes encoding ecdysteroidogenic enzymes in Nilaparvata lugens and clarified their necessity during nymphal and ovarian development. We confirmed that N. lugens utilized 20-hydroxyecdysone as an active hormone. Assays using heterologous expression of enzymes in Drosophila S2 cells showed conserved functions of enzymes Neverland, CYP306A2, CYP314A1 and CYP315A1, but not CYP302A1. RNA interference and rescue analysis using 20-hydroxyecdysone demonstrated that most of the genes were necessary for nymphal development. The identified N. lugens enzymes showed conserved functions and pathways for ecdysteroidogenesis. Knockdown of ecdysteroidogenic enzyme genes in newly molted females caused failure of egg production: less vitellogenic and mature eggs in ovaries, fewer laid eggs and embryonic development deficiency of laid eggs. Considering the high expressions of ecdysteroidogenic enzyme genes in adults and ovaries, ecdysteroidogenesis in ovaries was critical for N. lugens ovarian development. Our study presents initial evidence that hemipteran insects require ecdysteroidogenesis for ovarian development.

Measurement of 1α hydroxycorticosterone in the Japanese banded houndshark, Triakis scyllium, following exposure to a series of stressors.

An endocrine glucocorticoid response following exposure to a stressor has been well described for many vertebrates. However, despite demonstration of secondary stress responses in a number of elasmobranchs, our understanding of the endocrine control of these responses is lacking. This is largely due to the unusual structure of the dominant corticosteroid in elasmobranch fish, 1α-hydroxycorticosterone (1α-OH-B). Here we describe plasma extraction and HPLC separation procedures that allowed for the measurement of 1α-OH-B and corticosterone from plasma samples in the cannulated, conscious free-swimming Japanese banded houndshark, Triakis scyllium. While patterns of concentration in the plasma for 1α-OH-B and corticosterone were found to be similar in all experiments conducted, circulating levels of 1α-OH-B were consistently 100-fold greater than circulating levels of corticosterone. Immediately following cannulation surgery, circulating levels of 1α-OH-B increased 7-fold compared to pre-surgery levels, while the levels were 11-fold higher than pre-stress levels 30 min post a repeated handling/air-exposure stress. A three week period of fasting resulted in a 22-fold increase in circulating levels of 1α-OH-B in the banded houndshark. This is the first report of direct measurement of changes in circulating levels of the primary corticosteroid in elasmobranch fish, 1α-OH-B, following exposure to a stressor such as handling/air-exposure. Data indicate the steroid may respond similarly to the classic glucocorticoid response, such as cortisol in teleosts.

Sterol Characteristics in Silkworm Brain and Various Tissues Characterized by Precise Sterol Profiling Using LC-MS/MS.

Sterols, especially cholesterol (Chl), are fundamental for animal survival. Insects lacking the ability to synthesize Chl are sterol auxotrophic animals and utilize dietary Chl and phytosterols to survive. The sterols obtained from a diet are distributed to the tissues; however, sterol homeostasis in insect tissues remains to be elucidated. This study sought to understand the sterol characteristics of insect tissues through detailed sterol quantification and statistics. The combination of sterol quantification using liquid chromatography tandem mass spectrometry (LC-MS/MS) and principal component analysis (PCA) revealed tissue-specific sterol characteristics in the silkworm, Bombyx mori, a phytophagous insect. We found that insect tissues have tissue-intrinsic sterol profiles. The brain has a unique sterol composition as compared to other tissues-high concentration of Chl and less accumulation of phytosterols. Other tissues also have intrinsic sterol characteristics, which when defined by dietary sterols or Chl metabolites, indicate preference for a sterol and consistently manage their own sterol homeostasis. Though most tissues never change sterol profiles during development, the brain drastically changes its sterol profile at the wandering stage, indicating that it could alter sterol composition in preparation for metamorphosis. These results suggest the existence of tissue- and sterol-specific systems for sterol homeostasis in insects.

Cholesterol internalization and metabolism in insect prothoracic gland, a steroidogenic organ, via lipoproteins.

Dietary sterols including cholesterol and phytosterols are essential substrates for insect steroid hormone (ecdysteroid) synthesis in the prothoracic glands (PGs). In the silkworm Bombyx mori, one of the model species of insects, the steroidogenesis has been well demonstrated that cholesterol biotransformation into ecdysone in the PG cells. Because insects lack the ability to synthesize cellular sterol de novo, lipoprotein, lipophorin (Lp), has been thought to be the major cholesterol supply source; however, details of cholesterol behavior from Lp to the PG cells has not been analyzed till date. In this report, we developed Lp incorporation method using labeled cholesterols such as 22-NBD-cholesterol and cholesterol-25,26,26,26,27,27,27-d7 (cholesterol-d7), and analyzed the internalization and metabolism of cholesterol in PGs in vitro using the silkworm Bombyx mori. The internalization of cholesterol was visualized using 22-NBD-cholesterol. PGs showed an enriched cellular 22-NBD-cholesterol signal, which dissociated from the Lp localizing at the close area of cell membrane. The distribution pattern observed in the PGs was different from other tissues such as the brain, fat body, and Malpighian tubules, suggesting that the internalization of cholesterol in the PGs was distinct from other tissues. The metabolism of cholesterol was traced using LC-MS/MS methods to detect cholesterol-d7, 7-dehydrocholesterol-d7 (an expected intermediate metabolite), and the final product ecdysone-d6. 7-Dehydrocholesterol-d7 and ecdysone-d6 were detected in the PG culture incubated with labeled Lp, showing that the cholesterol of Lp was utilized for ecdysone synthesis in the PGs. Our results reveal the distinct behavior of cholesterol in the PGs, with the first direct evidence of biochemical fate of lipoprotein cholesterol in insect steroidogenic organ. This will aid in the understanding of the involvement of lipoprotein cholesterol in steroid hormone synthesis in insects.

Deep sequencing of the prothoracic gland transcriptome reveals new players in insect ecdysteroidogenesis.

Ecdysteroids are steroid hormones that induce molting and determine developmental timing in arthropods. In insect larva, the prothoracic gland (PG) is a major organ for ecdysone synthesis and release. Released ecdysone is converted into the active form, 20-hydroxyecdysone (20E) in the peripheral tissues. All processes from ecdysone synthesis and release from the PG to its conversion to 20E are called ecdysteroidogenesis and are under the regulation of numerous factors expressed in the PG and peripheral tissues. Classical genetic approaches and recent transcriptomic screening in the PG identified several genes responsible for ecdysone synthesis and release, whereas the regulatory mechanism remains largely unknown. We analyzed RNA-seq data of the silkworm Bombyx mori PG and employed the fruit fly Drosophila melanogaster GAL4/UAS binary RNAi system to comprehensively screen for genes involved in ecdysone synthesis and/or release. We found that the genes encoding δ-aminolevulinic acid synthase (CG3017/alas) and putative NAD kinase (CG33156) were highly expressed in the PG of both B. mori and D. melanogaster. Neither alas nor CG33156 RNAi-induced larvae could enter into the pupal stage, and they had a lower abundance of the active form ecdysteroids in their prolonged larval stage. These results demonstrated that alas and CG33156 are indispensable for ecdysteroidogenesis.

Identification and expression of nuclear receptor genes and ecdysteroid titers during nymphal development in the spider Agelena silvatica.

Ecdysteroids play an essential role in the regulation of the molting processes of arthropods. Nuclear receptors of the spider Agelena silvatica that showed high homology with other arthropods especially in the functional domains were identified, two isoforms of ecdysone receptor (AsEcRA, AsEcRB), retinoid X receptor (AsRXR) and two isoforms of E75 (AsE75A, AsE75D). AsEcR and AsRXR mRNA did not show major changes in expression but occurred throughout the third instar nymphal stage. AsE75DBD was low or non-existent at first then showed a sudden increase from D7 to D10. On the other hand, AsE75D was expressed in the first half and decreased from D6 to D10. Ecdysteroid titers showed a peak on D6 in A. silvatica third instar nymphs. LC-MS/MS analysis of the ecdysteroid peak revealed only 20-hydroxyecdysone (20E) was present. The 20E peak on D6 and increase in AsE75DBD from D7 is likely a result of ecdysteroids binding to the heterodimer formed with constant expression of the AsEcR and AsRXR receptors. These findings indicate the mechanisms regulating molting widely conserved in insects and other arthropods also similarly function in spiders.

Ligand-dependent responses of the silkworm prothoracicotropic hormone receptor, Torso, are maintained by unusual intermolecular disulfide bridges in the transmembrane region.

The insect membrane-protein, Torso, is a member of the receptor-tyrosine-kinase family, and is activated by its ligand, prothoracicotropic hormone (PTTH). Although PTTH is one of the most important regulators of insect development, the mechanism of Torso activation by the hormone has remained elusive. In this study, using heterologous expression in cultured Drosophila S2 cells, we detected ligand-independent dimerization of silkworm Torso, and found that the receptor molecules in the dimer were linked by intermolecular disulfide bridges. By examining the oligomerization states of several truncation and substitution mutants of Torso, atypical cysteine residues in the transmembrane region were identified as being responsible for the intermolecular linkage in the dimer. The replacement of all of the cysteines in the region with phenylalanines abolished the disulfide-bond-mediated dimerization; however, non-covalent dimerization of the mutant was detected using a cross-linking reagent, both with and without ligand stimulation. This non-covalent dimerization caused apparent receptor autophosphorylation independently of the ligand stimulation, but did not promote the ERK phosphorylation in the downstream signaling pathway. The unique Torso structure with the intermolecular disulfide bridges in the transmembrane region is necessary to maintain the ligand-dependent receptor functions of autophosphorylation and downstream activation.

Negative regulation of juvenile hormone analog for ecdysteroidogenic enzymes.

Disruption of the appropriate balance between juvenile hormone (JH) and ecdysteroids causes abnormal insect development. The application of a JH analog (JHA) during the early days of the final (fifth) instar induces dauer larvae with low ecdysteroid titers in insects, but the mechanism that underlies the action of JHA remains unclear. In this study, we clarified the negative effects of JHA on ecdysteroidogenic enzymes. JHA application to Bombyx mori larvae during the early stage of the fifth instar suppressed the expression of four enzymes, i.e., neverland (nvd), spook, phantom, and disembodied but not non-molting glossy and shadow. Furthermore, JHA application reduced the amount of 7-dehydrocholesterol, a metabolite produced by Nvd, in both the prothoracic glands and hemolymph, indicating JHA can disrupt ecdysteroidogenic pathway from the first step. Neck ligation resulted in increased nvd expression, whereas JHA application reversed this increase. These results suggest that the endogenous JH represses ecdysteroidogenesis during the early days in final instar larvae. Neck ligation and JHA application had no substantial effects on the expression of a transcription factor, ftz-f1, or a prothoracicotropic hormone receptor, torso; therefore, the inhibitory regulation of JHA may not involve these factors. Further analysis is required to clarify the regulation of JHA in ecdysteroidogenesis, but this study showed that JHA, and probably endogenous JH, can suppress the transcription of four of six ecdysteroidogenic enzymes. This regulation may be essential for maintaining the appropriate balance between JH and ecdysone during insect development.

Simultaneous quantification of individual intermediate steroids in silkworm ecdysone biosynthesis by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring.

The concentration changes of endogenous ecdysteroids are closely related to the regulation of insect growth and development. Although they are frequently measured by immunoassays with anti-steroid antibodies, the separate estimations of the individual concentrations of ecdysone and other ecdysteroids with similar chemical structures are quite difficult to accomplish. In this study, an efficient method for the simultaneous, individual quantification of intermediate steroids in ecdysone biosynthesis was developed, using LC-MS/MS. By employing multiple reaction monitoring (MRM) in the MS detection, the selectivity and sensitivity of the method were greatly enhanced, allowing the estimation of trace amounts of steroids in biological samples from silkworm prothoracic glands and hemolymph.

A highly specific and sensitive quantification analysis of the sterols in silkworm larvae by high performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

The biochemical quantification of sterols in insects has been difficult because only small amounts of tissues can be obtained from insect bodies and because sterol metabolites are structurally related. We have developed a highly specific and sensitive quantitative method for determining of the concentrations of seven sterols-7-dehydrocholesterol, desmosterol, cholesterol, ergosterol, campesterol, stigmasterol, and ß-sitosterol-using a high performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC/APCI-MS/MS). The sterols were extracted from silkworm larval tissues using the Bligh and Dyer method and were analyzed using HPLC/APCI-MS/MS with selected reaction monitoring, using cholesterol-3,4-(13)C(2) as an internal standard. The detection limits of the method were between 12.1 and 259 fmol. The major sterol in most silkworm larval tissues was cholesterol, whereas only small quantities of the dietary sterols were detected. Thus, a simple, sensitive, and specific method was successfully developed for the quantification of the sterol concentrations in each tissue of an individual silkworm larva. This method will be a useful tool for investigating to molecular basis of sterol physiology in insects, facilitating the quantification of femtomole quantities of sterols in biological samples.