Recombinant yellow protein of the takeout family and albino-related takeout protein specifically bind to lutein in the desert locust.

Yellow protein of the takeout family (YPT) and albino-related takeout protein (ALTO) are involved in body-color polyphenism in Schistocerca gregaria. YPT has been proposed to bind to ß-carotene, whereas the physiological role of ALTO is unclear. Structurally, takeout proteins contain a long continuous tunnel to bind specific ligands. However, the specific ligands of YPT and ALTO have not been fully elucidated. Here, we isolated the full coding cDNAs of these proteins and successfully produced recombinant YPT and ALTO using an Escherichia coli expression system. Absorption spectral analyses of YPT with and without carotenoids revealed that this protein bound to lutein. In contrast, obvious binding of YPT to ß-carotene and astaxanthin was not detected. Similar results were obtained for ALTO. The presence of juvenile hormone only weakly affected the protein/carotenoid interactions. These results suggested that YPT and ALTO specifically bound to lutein in a juvenile hormone-independent manner.

Yellowing and YPT gene expression in the desert locust, Schistocerca gregaria: Effects of developmental stages and fasting.

The yellow protein of the takeout family (YPT) controls the development of yellow body color in the desert locust. This study focused on two aspects related to YPT in the locust. We first examined the expression pattern of YPT during nymphal stages because yellowing was not obvious during the early instars. YPT expression levels were extremely low in the second and third instars compared with the last two nymphal instars. Warm rearing temperature and juvenile hormone (JH) injection, which stimulated YPT expression in the late instars, had little effect in the second instar, suggesting that YPT expression during the early instars was suppressed and could not be stimulated by either of these factors. We also investigated delayed yellowing in fasting male adults, under the hypothesis that fasting decreased the JH titers and delayed the onset of YPT expression. Yellowing was delayed in fasting adults compared with well-fed adults and YPT expression was stimulated by JH injections at Day 15. However, we failed to obtain evidence that fasting significantly influenced the expression levels of YPT and the JH early-inducible gene Krüppel homolog 1 at Days 15 and 20 post-adult emergence. Results suggest that a YPT-independent mechanism possibly induces delayed yellowing in fasting males.

Identification of a transcription factor that functions downstream of corazonin in the control of desert locust gregarious body coloration.

Corazonin (Crz) is a neuropeptide that controls phase-dependent body color polyphenism in locusts. The Crz signaling pathway is responsible for the development of gregarious black patterns in nymphs and determination of the morphometric ratio F/C (F = hind femur length, C = maximum head width) in adults. However, little information is available regarding the mediator and effector proteins regulated by Crz. In this study, we identified a novel transcription factor, Loct, which functions downstream of Crz in Schistocerca gregaria and Locusta migratoria. In S. gregaria, we detected a variant of Loct lacking the N-terminal region. Protein-protein interaction assays showed that both the long and short Loct variants formed a complex with themselves. LOCT knockdown in gregarious nymphs reduced the intensity of their black patterning, but did not affect F/C ratios in adults. LOCT was exclusively expressed in the integument of gregarious nymphs, suggesting that Loct is involved in melanin production. In addition, we found that the melanization-associated protein Yellow (YEL) and the albino-related takeout protein (ALTO) are expressed in the integument and function downstream of Crz. However, Crz injection failed to influence LOCT, YEL, and ALTO expression. Therefore, additional factors probably cooperate with Crz to induce these genes. The gene expression profiles of YEL and ALTO in LOCT-knockdown nymphs suggest that Loct does not directly control the transcription of YEL or ALTO. In summary, we present a working model of the Crz pathway, which is active in crowded S. gregaria nymphs.

Hatching synchrony is controlled by a two-step mechanism in the migratory locust Locusta migratoria (Acrididae: Orthoptera): Roles of vibrational stimuli.

The eggs of the migratory locust, Locust migratoria, hatch in synchrony from their pod. In this study, we examined the mechanism controlling hatching synchrony. Two eggs obtained from the same pod hatched in synchrony when kept in contact with one another, whereas those separated by a few millimeters hatched less synchronously. When a screen separated the eggs, the hatching was even more sporadic, indicating that hatching synchrony might be controlled by a two-step mechanism. We hypothesize that in the first step the embryos shortly before hatching control the time to enter a standby stage using some signal from neighboring eggs. The eggs in the standby stage hatch promptly when an additional stimulus is received from neighboring eggs. Before this stage, eggs cannot respond to that stimulus by hatching but may spontaneously hatch later. Introduction of a newly hatched nymph to single eggs 1 or 2 days before hatching advanced hatching of these eggs, but hatching occurred only sporadically. Eggs kept in contact with other eggs that had been killed by freezing shortly before hatching hatched as if they had been kept singly in separate containers, providing no evidence for involvement of chemical stimuli in controlling hatching synchrony. By contrast, eggs separated by several millimeters hatched as synchronously as those kept in contact with one another when they were connected by a piece of wire. Furthermore, vibrational stimulation derived from music greatly advanced hatching of separately kept eggs; however, hatching synchrony was not achieved unless the music started shortly before hatching. These results are consistent with the two-step hypothesis and indicated that locust embryos used vibrational stimuli from neighboring eggs for synchronous hatching.

Environmental and hormonal control of body color polyphenism in late-instar desert locust nymphs: Role of the yellow protein.

Locusts show body color polyphenism that is considered to be an adaptation to various biotic and abiotic environmental changes. In Schistocerca gregaria, wild-type late-instar nymphs growing under crowded conditions (gregarious form) develop yellow and black body coloration, whereas they assume various body colors under isolated conditions (solitarious form). Black and green body colorations are induced by the neuropeptide corazonin (Crz) and juvenile hormone (JH), respectively. To characterize the molecular mechanisms controlling body color polyphenism, we investigated factors influencing body coloration in S. gregaria. We report here that yellow body coloration in the last nymphal instar is caused by the yellow protein of the takeout family (YPT) in this locust. YPT transcription was enhanced under high-temperature conditions during which the nymphs turned bright yellow and had little black patterning. RNAi-mediated YPT knockdown suppressed the appearance of yellow individuals and yellow staining in the exuviae. In albino nymphs, injection of JH induced yellow and green coloration and enhanced the YPT expression levels in both yellow and green individuals. YPT knockdown also suppressed yellow staining in the exuviae but did not prevent the appearance of yellow individuals. Therefore, another factor or pigment may contribute to the observed yellow body color. Injection of Crz into wild-type nymphs caused darkening and suppressed yellowing and YPT expression at high temperatures. Thus, Crz signaling could inhibit yellowing by suppressing YPT expression. Rearing cup substrate color significantly influenced YPT expression in albino nymphs both under isolated and crowded conditions. In contrast, substrate color affected YPT expression in wild-type nymphs only under isolated conditions. From these results, we conclude that YPT is an important factor in the control of body color polyphenism in S. gregaria, and its expression is influenced by temperature, JH, Crz, and substrate color of the growing environment.

RNAi-mediated knockdown of SPOOK reduces ecdysteroid titers and causes precocious metamorphosis in the desert locust Schistocerca gregaria.

The Halloween gene SPOOK (SPO) is involved in the production of the active metabolite of ecdysteroid, 20-hydroxyecdysone (20E), in insects. A previous study showed that RNAi-mediated knockdown of SPO in Schistocerca gregaria last instar nymphs markedly reduced the hemolymph 20E titer, but did not affect metamorphosis. In the present study, the effects of SPO interference on development were re-examined in this locust. Injections of SPO double-stranded RNA (dsSPO) into nymphs at mid and late instars significantly delayed nymphal development and interfered with molting. The 20E levels of dsSPO-treated nymphs were generally low, with a delayed, small peak, suggesting that disturbance of the 20E levels caused the above developmental abnormalities. A small proportion of the dsSPO-injected nymphs metamorphosed precociously, producing adults and adultoids. Precocious adults were characterized by small body size, short wings with abbreviated venation, and normal reproductive activity. Fourth instar nymphs that precociously metamorphosed at the following instar exhibited temporal expression patterns of ecdysone-induced protein 93F and the juvenile hormone (JH) early-inducible gene Krüppel homolog 1 similar to those observed at the last instar in normal nymphs. Adultoids displayed mating behavior and adultoid females developed eggs, but never laid eggs. JH injection around the expected time of the 20E peak in the dsSPO-injected nymphs completely inhibited the appearance of adultoids, suggesting that appearance of adultoids might be due to a reduced titer of JH rather than of 20E. These results suggest that SPO plays an important role in controlling morphogenesis, metamorphosis, and reproduction in S. gregaria.

Two types of albino mutants in desert and migratory locusts are caused by gene defects in the same signaling pathway.

Albinism is caused by mutations in the genes involved in melanin production. Albino nymphs of Locusta migratoria and Schistocerca gregaria reared under crowded conditions are uniformly creamy-white in color. However, nothing is known about the molecular mechanisms underlying this phenomenon in locusts. The albino strain of L. migratoria is known to lack the dark-color-inducing neuropeptide corazonin (Crz). In this study, we report that this albino strain has a 10-base-pair deletion in the gene LmCRZ, which encodes Crz. This mutation was found to cause a frame-shift, resulting in a null mutation in Crz. On the other hand, the albino strain of S. gregaria is known to have an intact Crz. This strain was found to possess a single-nucleotide substitution in the middle of the Crz receptor-encoding gene, SgCRZR, which caused a nonsense mutation, resulting in a truncated receptor. Silencing of SgCRZR in wild-type S. gregaria nymphs greatly reduced the area and intensity of their black patterning, suggesting that the functional defect of SgCRZR likely causes the albinism. The expression level of SgCRZR in the albino S. gregaria was comparable to that in the wild type. Unlike the wild type, the albino strain of this locust did not show a phase-dependent shift in a morphometric trait controlled by Crz. From these results, we conclude that the mutations in LmCRZ and SgCRZR are responsible for the albinism in L. migratoria and S. gregaria, respectively, indicating that the two types of albinism are caused by different genetic defects in the same Crz signaling pathway.

Identification of amino acid residues of mammalian mitochondrial phosphate carrier important for its functional expression in yeast cells, as achieved by PCR-mediated random mutation and gap-repair cloning.

The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate "revertants" viable on the glycerol plate by expressing randomly mutated ΔNrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of ΔNrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of ΔNhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria.

Geographic variation in RNAi sensitivity in the migratory locust.

The RNA interference (RNAi) technology has been widely used in basic and applied research. It is known that RNAi works in some species but not in others, although the cause for this difference remains unclear. Here, we present inter- and intra-populational variations in RNAi sensitivity in the migratory locust Locusta migratoria, and provide information on the genetic background of such variations. In the four strains analyzed, originating from different Japanese localities, most individuals from two of the strains were sensitive to injections of double-stranded RNA (dsRNA) against the corazonin (CRZ) and ecdysone receptor genes, whereas those from the other two strains were resistant. Selection for individuals sensitive to dsCRZ produced a dramatic increase in the RNAi sensitivity in the following generations, although phenotypes also varied in the selected line, suggesting that several genes might control RNAi sensitivity. Reciprocal crosses between a sensitive and a resistant strain suggested that the resistant phenotype is dominant. The expression levels of nine RNAi-associated genes known for other organisms were not correlated with the variation in RNAi sensitivity observed in L. migratoria. Variations in RNAi sensitivity as the ones observed in this study should be considered when using RNAi in basic and applied research as well as in pest management.

The mechanism controlling phenotypic plasticity of body color in the desert locust: some recent progress.

Schistocerca gregaria exhibits density-dependent body color polyphenism. Nymphs occurring at low population densities show green-brown polyphenism. They show phase polyphenism and develop black patterns at high population densities. Recent studies suggest a third type of polyphensim, that is, homochromy, a response to background color. Laboratory experiments that considered homochromy suggest that humidity is not directly involved in green-brown polyphenism and that odor from other individuals does not induce black patterns. Black patterns can be induced in isolated nymphs by video images of locusts and tadpoles. Juvenile hormone and [His7]-corazonin control body color in locusts. The gene encoding the latter has been identified for S. gregaria and Locusta migratoria, and its key role in controlling black patterning has been demonstrated.

Structural characterization of an aldo-keto reductase (AKR2E5) from the silkworm Bombyx mori.

We report a new member of the aldo-keto reductase (AKR) superfamily in the silkworm Bombyx mori. Based on its amino acid sequence, the new enzyme belongs to the AKR2 family and was previously assigned the systematic name AKR2E5. In the present study, recombinant AKR2E5 was expressed, purified to homogeneity, and characterized. The X-ray crystal structures were determined at 2.2 Å for the apoenzyme and at 2.3 Å resolution for the NADPH-AKR2E5 complex. Our results demonstrate that AKR2E5 is a 40-kDa monomer and includes the TIM- or (ß/α)8-barrel typical for other AKRs. We found that AKR2E5 uses NADPH as a cosubstrate to reduce carbonyl compounds such as DL-glyceraldehyde, xylose, 3-hydroxy benzaldehyde, 17α-hydroxy progesterone, 11-hexadecenal, and bombykal. No NADH-dependent activity was detected. Site-directed mutagenesis of AKR2E5 indicates that amino acid residues Asp70, Tyr75, Lys104, and His137 contribute to catalytic activity, which is consistent with the data on other AKRs. To the best of our knowledge, AKR2E5 is only the second AKR characterized in silkworm. Our data should contribute to further understanding of the functional activity of insect AKRs.

Characterization of two adenine nucleotide translocase paralogues in the stink bug, Plautia stali.

Adenine nucleotide translocase (ANT) is a nuclear-coded mitochondrial protein that exchanges ATP for ADP across the mitochondrial inner membrane. Most organisms possess several ANT paralogues, and functional differences among these paralogues remain largely unknown. In the present study, we identified ANT paralogue genes in hemipteran species: the stink bug, bean bug, pea aphid, and Japanese mealybug. The ANT paralogues of the stink bug, Plautia stali, are encoded by two genes, PsANTI1 and PsANTI2. PsANTI1 was constantly expressed at all developmental stages and in all tissues analyzed. In contrast, the expression levels of PsANTI2 were undetectable in first instar nymphs and adult antennae. Gene silencing of each paralogue in P. stali revealed that PsANTI1 plays an important role in homeostasis, whereas the depletion of PsANTI2 failed to result in lethality. Thus, we concluded that PsANTI1 is a good target gene for developing novel pesticides.

Trichopolyn VI: a new peptaibol insecticidal compound discovered using a recombinant Saccharomyces cerevisiae screening system.

In the course of searching for insecticides from soil microorganisms, we found that a fermentation broth of the fungus, Trichoderma brevicompactum FKI-6324, produced Trichopolyn VI, a new peptaibol, which possessed significant insecticidal potential. Spectroscopic analysis showed the compound to be a new trichopolyn I derivative. This paper describes the isolation, structure elucidation and biological activity of trichopolyn VI.

Knockdown of the corazonin gene reveals its critical role in the control of gregarious characteristics in the desert locust.

The two plague locusts, Schistocerca gregaria and Locusta migratoria, exhibit density-dependent phase polyphenism. Nymphs occurring at low population densities (solitarious forms) are uniformly colored and match their body color to the background color of their habitat, whereas those occurring at high population densities (gregarious) develop black patterns. An injection of the neuropeptide, corazonin (Crz) has been shown to induce black patterns in locusts and affect the classical morphometric ratio, F/C (F, hind femur length; C, maximum head width). We herein identified and cloned the CRZ genes from S. gregaria (SgCRZ) and L. migratoria. A comparative analysis of prepro-Crz sequences among insects showed that the functional peptide was well conserved; its conservation was limited to the peptide region. Silencing of the identified SgCRZ gene in gregarious S. gregaria nymphs markedly lightened their body color and shifted the adult F/C ratio toward the value typical of solitarious forms. In addition, knockdown of the gene in solitarious nymphs strongly inhibited darkening even after a transfer to crowded conditions; however, these individuals developed black patterns after being injected with the Crz as a rescue treatment. SgCRZ was constitutively expressed in the brains of S. gregaria during nymphal development in both phases. This gene was highly expressed not only in the brain in both phases, but also in the corpora allata in the gregarious phase. This conspicuous phase-dependent difference in SgCRZ gene expression may indicate a functional role in the control of phase polyphenism in this locust.

Two adenine nucleotide translocase paralogues involved in cell proliferation and spermatogenesis in the silkworm Bombyx mori.

Mitochondrial adenine nucleotide translocase (ANT) specifically acts in ADP/ATP exchange through the mitochondrial inner membrane. This transporter protein thereby plays a significant role in energy metabolism in eukaryotic cells. Most mammals have four paralogous ANT genes (ANT1-4) and utilize these paralogues in different types of cells. The fourth paralogue of ANT (ANT4) is present only in mammals and reptiles and is exclusively expressed in testicular germ cells where it is required for meiotic progression in the spermatocytes. Here, we report that silkworms harbor two ANT paralogues, the homeostatic paralogue (BmANTI1) and the testis-specific paralogue (BmANTI2). The BmANTI2 protein has an N-terminal extension in which the positions of lysine residues in the amino acid sequence are distributed as in human ANT4. An expression analysis showed that BmANTI2 transcripts were restricted to the testis, suggesting the protein has a role in the progression of spermatogenesis. By contrast, BmANTI1 was expressed in all tissues tested, suggesting it has an important role in homeostasis. We also observed that cultured silkworm cells required BmANTI1 for proliferation. The ANTI1 protein of the lepidopteran Plutella xylostella (PxANTI1), but not those of other insect species (or PxANTI2), restored cell proliferation in BmANTI1-knockdown cells suggesting that ANTI1 has similar energy metabolism functions across the Lepidoptera. Our results suggest that BmANTI2 is evolutionarily divergent from BmANTI1 and has developed a specific role in spermatogenesis similar to that of mammalian ANT4.

Differential contribution of the Fanconi anemia-related proteins to repair of several types of DNA damage in cultured silkworm cells.

The silkworm Fanconi anemia (FA) pathway is required for normal cellular resistance to mitomycin C (MMC) in silkworms, but little is known about the requirement for repair of other types of DNA damage. Here we report that silkworm cells deficient for FA proteins FancD2 and FancM exhibit normal sensitivities to hydroxyurea (HU) and camptothecin (CPT), although FancM-dependent FancD2 monoubiquitination is induced upon these treatments. Similar results were observed in cells depleted for Rmi1 and Mhf1, which interact with the FancM protein. We also found that Rad51-knockdown cells exhibited normal sensitivity to HU despite induction of double-strand breaks by HU treatment.

Monoubiquitination-dependent chromatin loading of FancD2 in silkworms, a species lacking the FA core complex.

The Fanconi anemia (FA) pathway is required for activation and operation of the DNA interstrand cross-link (ICL) repair pathway, although the precise mechanism of the FA pathway remains largely unknown. A critical step in the FA pathway is the monoubiquitination of FANCD2 catalyzed by a FA core complex. This modification appears to allow FANCD2 to coordinate ICL repair with other DNA repair proteins on chromatin. Silkworm, Bombyx mori, lacks apparent homologues of the FA core complex. However, BmFancD2 and BmFancI, the putative substrates of the complex, and BmFancL, the putative catalytic E3 ubiquitin ligase, are conserved. Here, we report that the silkworm FancD2 is monoubiquitinated depending on FancI and FancL, and stabilized on chromatin, following MMC treatment. A substitution of BmFancD2 at lysine 519 to arginine abolishes the monoubiquitination, but not the interaction between the FancD2 and FancI. In addition, we demonstrated that depletion of BmFancD2, BmFancI or BmFancL had effects on cell proliferation in the presence of MMC. These results suggest that the FA pathway in B. mori works in the same manner as that in vertebrates.

Analysis of protein interactions with two-hybrid system in cultured insect cells.

Many biological processes are usually coupled to the formation of protein complexes. The yeast two-hybrid system is a powerful tool for analyzing protein-protein interactions. Different patterns of protein modifications, such as glycosylation, phosphorylation, and acetylation, may affect the ability of proteins to interact. In this study, we developed the two-hybrid system that can be used in insect cells. To validate the insect two-hybrid (I2H) system, we analyzed and confirmed the known oligomer or dimer formation of silkworm Rad51 or RPA2-RPA3, respectively. The results established the feasibility of the I2H system for efficient analysis of protein interaction under conditions that closely reflect the normal physiological environment.