Dimorphic sperm formation by Sex-lethal.

Sex is determined by diverse mechanisms and master sex-determination genes are highly divergent, even among closely related species. Therefore, it is possible that homologs of master sex-determination genes might have alternative functions in different species. Herein, we focused on Sex-lethal (Sxl), which is the master sex-determination gene in Drosophila melanogaster and is necessary for female germline development. It has been widely shown that the sex-determination function of Sxl in Drosophilidae species is not conserved in other insects of different orders. We investigated the function of Sxl in the lepidopteran insect Bombyx mori In lepidopteran insects (moths and butterflies), spermatogenesis results in two different types of sperm: nucleated fertile eupyrene sperm and anucleate nonfertile parasperm, also known as apyrene sperm. Genetic analyses using Sxl mutants revealed that the gene is indispensable for proper morphogenesis of apyrene sperm. Similarly, our analyses using Sxl mutants clearly demonstrate that apyrene sperm are necessary for eupyrene sperm migration from the bursa copulatrix to the spermatheca. Therefore, apyrene sperm is necessary for successful fertilization of eupyrene sperm in B. mori Although Sxl is essential for oogenesis in D. melanogaster, it also plays important roles in spermatogenesis in B. mori Therefore, the ancestral function of Sxl might be related to germline development.

Molecular Characterization of Eye Pigmentation-Related ABC Transporter Genes in the Ladybird Beetle Harmonia axyridis Reveals Striking Gene Duplication of the white Gene.

Many species of ladybird beetles (Coccinellidae) possess vivid body colors. These colors and patterns show diversity between coccinellid species, or even within species. However, the molecular underpinnings of these striking body colors are scarcely understood. One of the candidate pigmentation molecules responsible for ladybird body color is ommochrome pigment, which is well known as the red pigment molecule responsible for the red eyes of Drosophila. Various insects also use ommochrome in body coloration. It is known that ommochrome pigment precursors are imported into appropriate cells by the ATP binding cassette (ABC) transporter proteins White and Scarlet. Thus, these ABC transporter genes are potentially involved in various color and pattern expressions seen in ladybird beetle species. In this study, in order to identify the repertory of ABC transporter genes responsible for such body colors, we performed molecular characterization of pigment-related ABC transporter genes, especially white and scarlet, in the coccinellid Harmonia axyridis. By using whole genome data for H. axyridis and subsequent RACE-PCR, six white orthologs and one scarlet ortholog were successfully identified. According to the results of functional analyses via RNA interference (RNAi), only one of these genes had a major function in eye pigmentation. Specific effects on body color and pattern were not detected by our RNAi experiments of any of these genes. This is the first report of this striking duplication of white genes and their functional analyses in insects.

The role of doublesex in the evolution of exaggerated horns in the Japanese rhinoceros beetle.

Male-specific exaggerated horns are an evolutionary novelty and have diverged rapidly via intrasexual selection. Here, we investigated the function of the conserved sex-determination gene doublesex (dsx) in the Japanese rhinoceros beetle (Trypoxylus dichotomus) using RNA interference (RNAi). Our results show that the sex-specific T. dichotomus dsx isoforms have an antagonistic function for head horn formation and only the male isoform has a role for thoracic horn formation. These results indicate that the novel sex-specific regulation of dsx during horn morphogenesis might have been the key evolutionary developmental event at the transition from sexually monomorphic to sexually dimorphic horns.

Functional analysis of Ultrabithorax in the silkworm, Bombyx mori, using RNAi.

The formation of abdominal appendages in insects is suppressed by the Hox genes Ultrabithorax (Ubx) and abdominal-A (abd-A), but mechanisms of the suppression can differ among species. As the function of Ubx and abd-A has been described in only a few species, more data from various insects are necessary to elucidate the evolutionary transition of regulation on abdominal appendages. We examined the function of Ubx in the silkworm Bombyx mori (Bm-Ubx) by embryonic RNA interference (RNAi). This is the first case in which functional analysis for Ubx is performed in lepidopteran insects. Larvae treated with Bm-Ubx dsRNA displayed an additional pair of thoracic leg-like protuberances in A1, whereas the other abdominal segments had no transformation. Our results suggest that Bm-Ubx is a suppressor of leg development in A1.

A novel fusion protein that functions as an enhanced green fluorescent protein reporter and a tetracycline-controlled transcriptional activator.

Binary expression systems are widely employed to analyze gene function in vivo using transgenic organisms. The tetracycline-off (Tet-Off) system, which is a binary expression system that uses a tetracycline-controlled transactivator protein (tTA) and its tetracycline operator sequence (tetO) binding site, was developed as a method for temporally controlling gene expression. To facilitate the use of the Tet-Off system in animal species other than the model organisms that are widely used for genetic analysis, we constructed two different fusion proteins containing enhanced green fluorescent protein (EGFP) as the reporter gene and tTA as the transactivator, in different configurations. We assessed the utility of these fusion proteins designated as tTA-EGFP and EGFP-tTA in transgenic fruit flies. We showed that, although EGFP of both fusion proteins was efficiently fluoresced, transcriptional activation occurred only by the tTA-EGFP fusion protein. Furthermore, tetracycline (Tc) and doxycycline (Dox) both effectively inactivated tTA-EGFP, repressing gene expression under tetO control in a concentration-dependent manner. Additionally, the removal of Tc or Dox from the diet can recover the transactivator activity of tTA-EGFP in a concentration- and time-dependent manner. The tTA-EGFP fusion protein will therefore be useful in the analysis of gene function in a wide range of animal species.

Germ cell specification and early embryonic patterning in Bombyx mori as revealed by nanos orthologues.

In the silkmoth Bombyx mori, the germ cells first appear from the posterior ventral side of the egg (from within the mesodermal primordium) after blastoderm formation. This is in contrast to Drosophila, where germ cells appear at the posterior pole before cellular blastoderm formation. To date, germ plasm has not been found in B. mori. In this study, we describe the identification and expression pattern of nanos from B. mori, in which we recovered four nanos orthologues. One orthologue showed strong expression in embryonic germ cells, which was traced back to periplasmic granules dispersed on the ventral midline of the egg from the posterior-ventral focus of preblastoderm embryos. This suggests that, in B. mori, as in dipterans, germ cell formation depends on a localized determinant in the egg. The expression of another orthologue was observed in the posterior of the germ band. We speculate that nanos has dual functions; one in germ cell formation and the other in posterior body patterning, which is conferred by one nanos gene in Drosophila, but is assigned to different genes in B. mori.

Changes in the expression of soluble and integral-membrane trehalases in the midgut during metamorphosis in Bombyx mori.

To elucidate the relationship between soluble trehalase (Treh1) and integral-membrane trehalase (Treh2) in the Bombyx mori midgut, expression profiles for both proteins and mRNAs were examined during metamorphosis by using Western-blotting and quantitative real-time PCR analyses. Two bands of Treh2 (about 74 kDa) were detected in the midgut of 0-day-old 5th (last) instar larvae. Levels of Treh2 decreased as the developing larvae approached spinning (8 days old). In contrast, towards the onset of the spinning stage, Treh1 (68 kDa) was clearly observed, and levels increased until the middle of the pupal stage. Treh2 mRNA expression relative to Bmrp49 mRNA expression was almost constant, although fluctuations were detected. Treh1 mRNA expression relative to Bmrp49 mRNA increased sharply just after spinning. To further examine the expression mechanism of the Treh1 gene in midgut, actively feeding larvae (4 days old) were starved or ligated between the 4th and 5th segments. Injection of a molting hormone into the larval-isolated abdomen led to activation of Treh1, demonstrating that molting hormone acts on the midgut and activates this gene.

FXPRL-amide peptides induce ecdysteroidogenesis through a G-protein coupled receptor expressed in the prothoracic gland of Bombyx mori.

The FXPRL-amide peptide family (pyrokinin/PBAN family) consists of insect peptides that function broadly in insect life processes and are characterized by a conserved C-terminal motif. In the silkworm, Bombyx mori, sex pheromone biosynthesis and induction of embryonic diapause are regulated by peptides from this family. To elucidate other functions of Bombyx FXPRL-amide peptides, we analyzed the tissue expression patterns of two known Bombyx G-protein coupled receptors for these peptides. We found that the Bombyx diapause hormone receptor (BmDHR), is expressed in the prothoracic gland (PG), the organ which synthesizes and releases the insect molting hormones, ecdysteroids. Furthermore, diapause hormone (DH), a member of the Bombyx FXPRL-amide peptides, increases both intracellular Ca(2+) and cAMP concentrations and induces ecdysteroidogenesis in late fifth instar PGs coincident with BmDHR expression in the PGs. DH also has the highest prothoracicotropic activity among the FXPRL-amide peptides, which corresponds well to the ligand specificity of heterologously expressed BmDHR. These results demonstrate that FXPRL-amide peptides can function as prothoracicotropic factors through the activation of BmDHR and may play an important role in controlling molting and metamorphosis.

ERK/MAPK regulates ecdysteroid and sorbitol metabolism for embryonic diapause termination in the silkworm, Bombyx mori.

The eggs of the silkworm Bombyx mori undergo a state of suspended overt development and diminished metabolism called "diapause" to escape adverse environmental conditions. Termination of Bombyx embryonic diapause requires 2-3 months of low temperature (5 degrees C), but the molecular mechanisms underlying diapause termination are unknown. Diapause termination requires a decrease in the sorbitol concentration, which arrests embryonic development, and the secretion factors from yolk cells that promote embryonic development. In the present study, we report that 20-hydroxyecdysone promoted the development of denuded embryos and that ecdysteroid-phosphate phosphatase (EPPase), which is a key enzyme for active ecdysteroid production, was induced by incubation of diapausing eggs at 5 degrees C. In dechorionated egg cultures, extracellular signal-regulated kinase (ERK), which is activated by incubating diapausing eggs at 5 degrees C, regulated sorbitol-glycogen conversion, ecdysteroid secretion via gene transcription of key enzymes, sorbitol dehydrogenase-2, and EPPase, suggesting that ERK has a key role in diapause termination.

Molecular cloning and functional characterization of the sex-determination gene doublesex in the sexually dimorphic broad-horned beetle Gnatocerus cornutus (Coleoptera, Tenebrionidae).

Various types of weapon traits found in insect order Coleoptera are known as outstanding examples of sexually selected exaggerated characters. It is known that the sex determination gene doublesex (dsx) plays a significant role in sex-specific expression of weapon traits in various beetles belonging to the superfamily Scarabaeoidea. Although sex-specific weapon traits have evolved independently in various Coleopteran groups, developmental mechanisms of sex-specific expression have not been studied outside of the Scarabaeoidea. In order to test the hypothesis that dsx-dependent sex-specific expression of weapon traits is a general mechanism among the Coleoptera, we have characterized the dsx in the sexually dimorphic broad-horned beetle Gnatocerus cornutus (Tenebrionidea, Tenebirionidae). By using molecular cloning, we identified five splicing variants of Gnatocerus cornutus dsx (Gcdsx), which are predicted to code four different isoforms. We found one male-specific variant (GcDsx-M), two female-specific variants (GcDsx-FL and GcDsx-FS) and two non-sex-specific variants (correspond to a single isoform, GcDsx-C). Knockdown of all Dsx isoforms resulted in intersex phenotype both in male and female. Also, knockdown of all female-specific isoforms transformed females to intersex phenotype, while did not affect male phenotype. Our results clearly illustrate the important function of Gcdsx in determining sex-specific trait expression in both sexes.

Myocyte enhancer factor 2 (MEF2) is a key modulator of the expression of the prothoracicotropic hormone gene in the silkworm, Bombyx mori.

Prothoracicotropic hormone (PTTH) plays a central role in controlling molting, metamorphosis, and diapause termination in insects by stimulating the prothoracic glands to synthesize and release the molting hormone, ecdysone. Using Autographa californica nucleopolyhedrovirus (AcNPV)-mediated transient gene transfer into the central nervous sytem (CNS) of the silkworm, Bombyx mori, we identified two cis-regulatory elements that participate in the decision and the enhancement of PTTH gene expression in PTTH-producing neurosecretory cells (PTPCs). The cis-element mediating the enhancement of PTTH gene expression binds the transcription factor Bombyx myocyte enhancer factor 2 (BmMEF2). The BmMEF2 gene was expressed in various tissues including the CNS. In brain, the BmMEF2 gene was expressed at elevated levels in two types of lateral neurosecretory cells, namely PTPCs and corazonin-like immunoreactive lateral neurosecretory cells. Overexpression of BmMEF2 cDNA caused an increase in the transcription of PTTH. Therefore, BmMEF2 appears to be particularly important in the brain where it is responsible for the differentiation of lateral neurosecretory cells, including the enhancement of PTTH gene expression. This is the first report to identify a target gene of MEF2 in the invertebrate nervous system.

Establishment of a sandwich ELISA system to detect diapause hormone, and developmental profile of hormone levels in egg and subesophageal ganglion of the silkworm, Bombyx mori.

In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.

Wing serial homologs and the origin and evolution of the insect wing.

The origin and evolution of insect wings has been the subject of extensive debate. The issue has remained controversial largely because of the absence of definitive fossil evidence or direct developmental evidence of homology between wings and a putative wing origin. Recent identification of wing serial homologs (WSHs) has provided researchers with a potential strategy for identifying WSHs in other species. Future comparative developmental analyses between wings and WSHs may clarify the important steps underlying the evolution of insect wings.

Establishment of transgenic lines for jumpstarter method using a composite transposon vector in the ladybird beetle, Harmonia axyridis.

In this post-genomic era, genome-wide functional analysis is indispensable. The recent development of RNA interference techniques has enabled researchers to easily analyze gene function even in non-model organisms. On the other hand, little progress has been made in the identification and functional analyses of cis-regulatory elements in non-model organisms. In order to develop experimental platform for identification and analyses of cis-regulatory elements in a non-model organism, in this case, the ladybird beetle, Harmonia axyridis, we established transgenic transposon-tagged lines using a novel composite vector. This vector enables the generation of two types of insertion products (jumpstarter and mutator). The jumpstarter portion carries a transposase gene, while the mutator segment carries a reporter gene for detecting enhancers. The full-composite element is flanked by functional termini (required for movement); however, the mutator region has an extra terminus making it possible for the mutator to remobilize on its own, thus leaving an immobile jumpstarter element behind. Each insertion type is stable on its own, but once crossed, jumpstarters can remobilize mutators. After crossing a jumpstarter and mutator line, all tested G2 females gave rise to at least one new insertion line in the next generation. This jumping rate is equivalent to the P-element-mediated jumpstarter method in Drosophila. These established transgenic lines will offer us the ideal experimental materials for the effective screening and identification of enhancers in this species. In addition, this jumpstarter method has the potential to be as effective in other non-model insect species and thus applicable to any organism.

Cloning of cDNA encoding a Bombyx mori homolog of human oxidation resistance 1 (OXR1) protein from diapause eggs, and analyses of its expression and function.

To better understand the molecular mechanisms of diapause initiation, we used the sensitive cDNA subtraction (selective amplification via biotin- and restriction-mediated enrichment) method and isolated a novel gene expressed abundantly in diapause eggs of the silkworm, Bombyx mori, which encodes a homolog of the human oxidation resistance 1 (OXR1) protein. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analyses confirmed that BmOXR1 mRNA and its 140-kDa protein were differentially expressed in diapause eggs compared to non-diapause eggs. OXR1 double-stranded RNA (dsRNA) was injected into diapause-destined eggs before the cellular blastoderm stage, and 4days later, when untreated eggs reached the diapause stage, the OXR1 protein disappeared; however, these eggs remained in diapause, suggesting that BmOXR1 is not essential for diapause initiation and/or maintenance. To further investigate the in vivo function of BmOXR1 apart from its role in diapause, we overexpressed BmOXR1 in Drosophila melanogaster. The fruit fly male adult life-span was significantly extended in the 50%-survival time when adults were reared on diets both with and without H2O2 solution under 25°C incubation. These results suggest that BmOXR1 functions in D. melanogaster via a possible antioxidant effect. As BmOXR1 was expressed mainly in the nuclei of D. melanogaster cells, the mechanism underlying its antioxidation effect appears to be different from that in humans where it is expressed mainly in the mitochondria. Taken together, these results suggest that BmOXR1 might serve as an antioxidant regulator during the early diapause stage.

Insect morphological diversification through the modification of wing serial homologs.

Fossil insects living some 300 million years ago show winglike pads on all thoracic and abdominal segments, which suggests their serial homology. It remains unclear whether winglike structures in nonwinged segments have been lost or modified through evolution. Here, we identified a ventral lateral part of the body wall on the first thoracic segment, the hypomeron, and pupal dorsolateral denticular outgrowths as wing serial homologs in the mealworm beetle Tenebrio molitor. Both domains transform into winglike structures under Hox RNA interference conditions. Gene expression and functional analyses revealed central roles for the key wing selector genes, vestigial and scalloped, in the hypomeron and the denticular outgrowth formation. We propose that modification, rather than loss, of dorsal appendages has provided an additional diversifying mechanism of insect body plan.

A Baculovirus immediate-early gene, ie1, promoter drives efficient expression of a transgene in both Drosophila melanogaster and Bombyx mori.

Many promoters have been used to drive expression of heterologous transgenes in insects. One major obstacle in the study of non-model insects is the dearth of useful promoters for analysis of gene function. Here, we investigated whether the promoter of the immediate-early gene, ie1, from the Bombyx mori nucleopolyhedrovirus (BmNPV) could be used to drive efficient transgene expression in a wide variety of insects. We used a piggyBac-based vector with a 3xP3-DsRed transformation marker to generate a reporter construct; this construct was used to determine the expression patterns driven by the BmNPV ie1 promoter; we performed a detailed investigation of the promoter in transgene expression pattern in Drosophila melanogaster and in B. mori. Drosophila and Bombyx belong to different insect orders (Diptera and Lepidoptera, respectively); however, and to our surprise, ie1 promoter-driven expression was evident in several tissues (e.g., prothoracic gland, midgut, and tracheole) in both insects. Furthermore, in both species, the ie1 promoter drove expression of the reporter gene from a relatively early embryonic stage, and strong ubiquitous ie1 promoter-driven expression continued throughout the larval, pupal, and adult stages by surface observation. Therefore, we suggest that the ie1 promoter can be used as an efficient expression driver in a diverse range of insect species.

Heat shock factor binds to heat shock elements upstream of heat shock protein 70a and Samui genes to confer transcriptional activity in Bombyx mori diapause eggs exposed to 5°C.

To understand the molecular mechanisms of how 5°C-incubation activates mRNA expression of Hsp70a and Samui genes in Bombyx mori diapause eggs, we first searched the 5'-upstream regions of the Hsp70a and Samui genes for heat shock elements (HSEs) and found two regions [Hsp70aHSE-1 (-95 to -58) and -2 (-145 to -121), and SamuiHSE-1 (-84 to -55) and -2 (-304 to -290)] corresponding to HSEs (repeats of nGAAn and/or nTTCn). We cloned four cDNAs encoding heat shock factor (HSF)-a2 (627 amino acids), -b (685 aa), -c (682 aa) and -d (705 aa), which were produced by alternative splicing. When we exposed diapause eggs to 5°C beginning at 2 day post-oviposition to break diapause, HSFd mRNA only increased after chilling for 6-8 days, a pattern very similar to those of Hsp70a and Samui mRNAs. To examine further whether HSFd binds to the respective HSEs, we carried out a gel shift assay using HSFd protein expressed in a cell-free system and the isolated HSEs; migration of the respective digoxigenin(DIG)-labeled HSE-1 and -2 of Hsp70a and Samui was retarded by addition of HSFd; the retarded bands disappeared after addition of the corresponding unlabeled HSE-1 and -2 as competitors, but were not affected by addition of the respective mutated unlabeled HSE-1 and -2. These results indicated that HSFd protein binds to the respective HSEs and may activate mRNA expression of Hsp70a and Samui genes upon exposure of diapause eggs to 5°C.

Trehalase-2 protein contributes to trehalase activity enhanced by diapause hormone in developing ovaries of the silkworm, Bombyx mori.

Diapause hormone (DH) targets developing ovaries in female pupae to induce embryonic diapause immediately after completion of mesoderm segregation of the silkworm, Bombyx mori. At the same time, DH enhances trehalase activity on the oolemma, which leads to higher concentrations of glycogen in oocytes through the stimulated incorporation of hemolymph trehalose. In B. mori, the treh-1 and -2 genes encoding soluble trehalase (68 kDa) and integral-membrane trehalase (74kDa) have been isolated. DH stimulates mRNA expression of both of these genes. In this study, we aimed to clarify whether ovarian trehalase originates from Treh-1 or Treh-2. Western blotting of the developing ovaries showed positive bands in the membrane-bound fraction, containing trehalase activity, only with antibodies against Treh-1&2 and Treh-2, but not Treh-1, irrespective of nondiapause or diapause egg-producers. The intensities of the positively stained 74 kDa bands were increased approximately 4-fold in ovaries from pupae with intact subesophageal ganglion (SG, a unique DH-biosynthesizing organ), and from pupae that were injected with DH at the middle pupal stage after their SGs were removed on the day of pupation. Furthermore, quantitative real-time PCR data showed that in developing ovaries, copy number of treh-2 mRNA per one copy of rp49 mRNA was approximately 1000-fold higher than that of treh-1 mRNA. These results demonstrate that trehalase activities enhanced by DH originate mainly from treh-2 protein regulated at the transcriptional level.

Cloning of cDNAs encoding sorbitol dehydrogenase-2a and b, enzymatic characterization, and up-regulated expression of the genes in Bombyx mori diapause eggs exposed to 5 °C.

We previously cloned a cDNA for sorbitol dehydrogenase (SDH1) from Bombyx mori. In the present study we cloned two additional cDNAs encoding SDHs (designated as SDH2a and SDH2b). The amino acid sequences of SDH2ab were almost the same and had higher similarity to the SDHs of other organisms than to B. mori SDH1. The SDH2ab and SDH1genes were located in tandem within about 40 kbp on chromosome 21. SDH2ab mRNAs increased after exposing diapause eggs to 5 °C for 40 days, beginning at 2 days post-oviposition, to break diapause. However, they were at very low levels in diapausing eggs incubated at 25 °C continuously from oviposition. These changes in expression pattern of SDH2ab mRNA were almost the same as that of SDH1 mRNA. To understand whether SDH1 and SDH2 were responsible for the SDH activity seen in diapause eggs exposed to 5 °C for more than 60 days, we expressed a His-tagged SDH2a fusion protein in Escherichia coli and examined its enzymatic parameters. The maximum activity of SDH2a observed at pH 8.4∼9.0, and the Km value for sorbitol was 12.6 mM, similar to the kinetic properties of other SDHs. Due to the significantly higher similarity between SDH2a and b, they were thought to have similar kinetic properties. Therefore, we purified SDH from B. mori diapause-terminated eggs exposed to 5 °C for 300 days which showed higher SDH activity using two-step affinity chromatography. The highly purified SDH showed a higher Km value (125 mM) for sorbitol, being similar to the value (136 mM) determined previously from Eadie-Hofstee plots using egg crude extract as an enzyme source; additionally, the plots showed one slope indicating one Km value. Moreover, in silico analysis indicated that no SDH genes other than SDH1 and 2ab are present in B. mori genomic DNA. These results suggest that SDH1 activity may be responsible for the majority of the increased SDH activity seen in diapause eggs after acclimation to 5 °C rather than SDH2ab. Further, the relative sequence divergence among these genes is consistent with the idea/hypothesis that the original SDH gene was first duplicated into SDH1 and SDH2, and then SDH2 was duplicated into the SDH2a and SDH2b genes.