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

Accepted Manuscript Identification and expression of nuclear receptor genes and ecdysteroid titers during nymphal development in the spider Agelena silvatica Yoshiko Honda, Wataru Ishiguro, Mari H. Ogihara, Hiroshi Kataoka, DeMar Taylor PII: DOI: Reference:

S0016-6480(17)30079-5 http://dx.doi.org/10.1016/j.ygcen.2017.01.032 YGCEN 12580

To appear in:

General and Comparative Endocrinology

Received Date: Revised Date: Accepted Date:

23 July 2016 26 January 2017 28 January 2017

Please cite this article as: Honda, Y., Ishiguro, W., Ogihara, M.H., Kataoka, H., Taylor, D., Identification and expression of nuclear receptor genes and ecdysteroid titers during nymphal development in the spider Agelena silvatica, General and Comparative Endocrinology (2017), doi: http://dx.doi.org/10.1016/j.ygcen.2017.01.032

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Title. Identification and expression of nuclear receptor genes and ecdysteroid titers during nymphal development in the spider Agelena silvatica Author names and affiliation. Yoshiko Hondaa,b, Wataru Ishiguroa, Mari H. Ogiharac, Hiroshi Kataokac, DeMar Taylord a. Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan Research and Development Department, Fumakilla Limited, Hatsukaichi, Hiroshima, Japan c, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan d. Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan b.

[email protected] (DeMar Taylor) [email protected] (Yoshiko Honda) [email protected] (Mari. H. Ogihara) Corresponding author.

DeMar Taylor

Abstract 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.

Highlights. ・Isolation and expression of nuclear receptor EcR, RXR, E75 from the spider Agelena silvatica ・ Identification of ecdysteroid hormone in the spider Agelena silvatica Keywords. molting, ecdysone, hormone, EcR, E75, RXR, 20E Acknowledgment. This research was supported by funds to faculty members from the University of Tsukuba

1. Introduction Molting is a common phenomenon necessary for arthropod development and progresses with complex sequential hormonal regulation. Mechanisms of molting have been well studied in holometabolous insects and ecdysteroids are the main hormones that regulate molting in most arthropods. In most insects, ecdysone secreted into the hemolymph from a secretory tissue is oxidized to the active form 20-hydroxyecdysone (20E) and interacts with nuclear receptors in the target tissues to prepare the arthropod for molting (reviewed by Nakagawa and Henrich, 2009). Spider molting also requires ecdysteroids. Ecdysteroids in nymphs of Pisaura mirabilis were previously measured and characterized as 20E by RIA and TLC (Bonaric and De Reggi, 1977). The 8th nymphal stage lasted for 22 days and ecdysteroid titers remained at low levels during the intermolting phase. However, the ecdysteroid titers rapidly peaked before ecdysis. A rapid large peak of titers before ecdysis is similar to that seen in numerous other arthropods (Suganthi and Anilkumar, 1999; Langelan et al., 2000; Mizoguchi et al., 2001; Palmer et al., 2002; Cruz et al., 2003; Sullivan and Thummel, 2003; Ogihara et al., 2007). Injection of 20E into the spiders Araneus cornutus, Dugesiella hentzi and P. mirabilis induced apolysis, deposit of new cuticle for molting (Krishnakumaran and Schneiderman, 1968, 1970; Bonaric, 1976). Timing of injection is another important factor. Injection of 20E into P. mirabilis during the early stages of the 7-9th instar nymphs showed low concentrations of ecdysteroids induce a prolonged instar, whereas injection into spiders during the middle stages induce premature molting (Bonaric, 1976). Based on these early studies, ecdysteroids are expected to induce spider molting. The mechanisms regulating molting in insects have been elucidated through

studies on ecdysteroid functions at the molecular level. Ecdysteroids regulate molting through gene transcription of target genes required for molting. For the ecdysteroid function, two nuclear receptors, the ecdysone receptor (EcR) and retinoid X receptor (RXR) or insect ultraspiracle (USP), a homologue of RXR (Yao et al., 1992, 1993; Thomas et al., 1993) are required. A heterodimer composed of EcR and RXR binds ecdysteroids to form a functional complex (ecdysteroid/EcR/RXR) for the induction of transcription in target genes. The ecdysteroid/EcR/RXR complex binds to the gene regulatory region, ecdysone response element (EcRE), in the promoter region of a target gene to trigger gene transcription. Therefore, ecdysteroid dependent regulation of molting requires ecdysone, EcR and RXR. Ecdysteroid dependent molting is common in arthropods and identification of EcR and RXR are reported from numerous other arthropods

(Nakagawa

and

Henrich,

2009).

Genes

directly

regulated

by

ecdysteroid/EcR/RXR are called ecdysone early genes and function as regulation factors for indirect regulation of gene expression. The E75 gene, a nuclear receptor identified from ecdysone inducible early puff E75B of Drosophila, is one ecdysone early gene involved in insect molting (Segraves and Hogness, 1990). These receptors contain a gene structure common to nuclear receptors. The amino (N) terminus contains an A/B domain variable region that is responsible for the occurrence of numerous isoforms through alternative splicing. The DNA binding domain (DBD) and ligand binding domain (LBD) are highly conserved. The EcR gene produces several splicing variants with different A/B domains resulting in several EcR isoforms. Up to three isoforms (A, B1 and B2) have been isolated from arthropods and each isoform shows specific expression patterns in different tissues and stages for regulation of target genes (Koelle et al., 1991; Talbot et

al., 1993; Kamimura et al., 1997; Jindra et al., 1996). The ecdysone early gene E75 produces variants namely A, B, C, D and E isoforms with different A/B domains reported from several arthropods (Segraves and Hogness, 1990; Mané-Padrós et al., 2008; Dubrovskaya et al., 2004). The structure of E75 A, C and E isoforms contain domains conserved in other arthropod nuclear receptors. However, the B isoform lacks the full DNA binding domain and the D isoforms have no DNA binding domain residues. In Manduca sexta, E75B suppresses expression of dopa decarboxylase (DDC) that functions in sclerotization of cuticular formation (Hiruma and Riddiford, 2009). While E75 B and D isoforms do not contain DBD, they can regulate transcription by heterodimerization. For example, Drosophila E75B that lacks a complete DBD, shows inhibition of the inductive function of DHR3 by forming a complex on the target gene promoter region (White et al., 1997). These studies suggest E75 isoforms and domains function as important transcriptional factors to regulate the ecdysteroid signals in various ways. In the Chelicerates, EcR and RXR have been reported from the hard tick Amblyomma americanum (Guo et al., 1997, 1998), soft tick Ornithodoros moubata (Horigane et al., 2007, 2008) and scorpion Liocheles australasiae (Nakagawa et al., 2007), and functions have been studied by Guo et al. (1998) and Nakagawa et al. (2007). Although a partial EcR sequence was isolated from the spider Nephila clavata (Watanabe et al., 2010), there are no reports of the EcR, RXR and E75 full length ORFs and their expression patterns in any spiders to date. Moreover several behaviors such as pheromone secretion, sexual behaviors and vitellogenesis of spiders have been reported to be regulated by ecdysteroids. Identification and analysis of these transcriptional factors is required for understanding the regulation of molting and other behaviors by

ecdysteroids in spiders. This study is the first report of the identification, expression analysis and comparison with ecdysteroid titers of these nuclear regulatory factors from a spider.

2. Materials and Methods 2.1. Spiders Egg sacs including eggs and first or second instar nymphs of A. silvatica were collected from the University of Tsukuba campus (Tsukuba, Ibaraki, Japan) in September to November 2008 and 2010. In this study, freshly hatched larvae were defined as first instar. Second instar nymphs were obtained from first instar nymphs and individually kept in screw cap vials (27.5 × 61 ㎜) at 25°C ± 2 °C under a L/D 16:8 photoperiod. Third instar nymphs molting from these second instar nymphs were used in all experiments. All nymphal spiders were fed first or second instar armyworm larvae (Mythimna separata) daily to insure a food source was always available. Most third instar nymphs molted into fourth instar nymphs 11 days after molting from second instar nymphs.

2.2. Isolation of RNA and cDNA synthesis The eggs and whole body of nymphs of A. silvatica were frozen in liquid nitrogen and total RNA extracted with TRIzol reagent as described by the manufacturer (Invitrogen, Carlsbad). Total RNA (2 μg) was treated with DNase I Amp grade (Invitrogen) as described by the manufacturer and used for cDNA synthesis with the Superscript III First Strand Synthesis System (Invitrogen). The reverse transcription reaction was performed at 50°C for 50 min followed by heating at 85°C for 5 min with Oligo (dT)20 nucleotides according to the manufacturer.

2.3. Identification of AsEcR, AsRXR and AsE75 partial fragments by degenerate PCR Partial fragments of AsEcR and AsRXR were amplified by PCR using

degenerate primers from the study on the scorpion L. australasiae (Nakagawa et al., 2007) (Table 1). Primers for AsEcR B isoform (EcRBdF) were designed from the EcRB isoform specific sequences of the spider N. clavata (AB490022), crustacean Daphnia magna (AB274824), myriapod Thereuopoda clunifera (AB490023), and several insects including Ctenolepisma villosa (AB536931), Ephemera strigata (AB490025), Periplaneta

fuliginosa

(AB490031),

Sympetrum infuscatum

(AB490026) and

Thermobia domestica (AB490024). The degenerate primers for AsE75 (Table 1) were designed from the DNA binding domain sequences of Ixodes scapularis putative mRNA (XM_002410005), Gecarcinus lateralis (DQ058409), Daphnia magna (EF369510), B. germanica E75A (AM238653), A. mellifera (AB264313) and B. mori E75A (AB024904) (Table 1). Nested PCR for partial cDNA of AsEcR, AsRXR and AsE75 were performed with Platinum Taq DNA Polymerase (Invitrogen). First PCR reaction was performed under conditions of 2 min at 94°C followed by 35 cycles at 92°C, 40°C and 72°C for 60 sec each and elongation at 72°C for 10 min using primers EcRdF1 and EcRdR1. A second PCR was performed using primers EcRdF2 and EcRdR2 and a third PCR using EcRdF3 and EcRdR3. To determine if other isoforms of AsEcR exist, nested PCR was performed using degenerate primer EcRBdF and a reverse primer from the DNA binding domain (EcRCR). For AsRXR, RXRdF1 was used as a forward primer and RXRdR1 as the reverse primer for the first PCR. Second PCR was performed using primers RXRdF2 and RXRdR2. For E75, the first PCR was performed under conditions of 2 min at 94°C followed by 5 cycles at 92°C, 40°C and 72°C for 60 sec each, 25 cycles at 92°C, 45°C and 72°C for 60 sec each and elongation at 72°C for 7 min. Second PCR was performed for 2 min at 94°C followed by 5 cycles at 92°C, 45°C and 72°C for 60 sec each, 25 cycles at 92°C, 50°C and 72°C for 60 sec each and elongation

at 72°C for 7 min. E75dF1 was used as the forward primer and E75dR1 as the reverse primer for the first PCR then E75dF2 and E75dR2 used for nested PCR.

2.4. Determination of AsEcR, AsRXR and AsE75 sequences by rapid amplification of cDNA ends (RACE) To determine the full length actin sequence, 5’ and 3’ rapid amplification of cDNA ends (RACE) was performed with First Choice RACE® – Ready cDNA Kit as described by the manufacturer (Ambion, Tokyo). Nested PCR for RACE products was performed with BD AdvantageTM 2 PCR Enzyme System (BD Clontech, Shiga). The 5’ RACE-PCR for AsEcR, 5’ RACE outer primer and 5’ RACE inner primer were used as forward primers and AsEcRrR as the reverse primer for 2 min at 95°C, 25 cycles for 30 sec at 95°C, 10 sec at 65°C and 2 min at 68°C followed by 10 min at 68°C. For 3’ RACE-PCR, AsEcRrF1, AsEcRrF2, AsEcRrF3, AsEcRrF4 and AsEcRrF5 were used as forward primers and 3’ RACE outer primer and 3’ RACE inner primer as reverse primers (Table 1). In a similar way for AsRXR and AsE75, 5’ RACE outer primer and 5’ RACE inner primer were used as forward primers, AsRXRrR1 and AsRXRrR2, or E75dR1, AsE75rR1 and AsE75rR2 as reverse primers. For 3’ RACE, AsRXRrF1, AsRXRrF2, AsRXRrF3 and AsRXRrF4 or E75dF1 and AsE75rF1 were used as forward primers and 3’ RACE outer primer and 3’ RACE inner primer as reverse primers (Table 1). PCR products were subcloned into a pGEM®-T Easy Vector System (Promega, Tokyo). The sequence reactions of purified plasmids were performed with Big Dye ® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Tokyo) using T7 and sp6 primers (Table 1).

2.5. Analysis of nucleotides and amino acids of AsEcR, AsRXR and AsE75 For phylogenetic analysis the complete gene sequences were determined and then converted to putative amino acids. Isolated nucleotide sequences and amino acid sequences were compared with EcR, RXR or USP and E75 genes of other arthropods. Phylogenetic trees were profiled by neighbor-joining method and created by Clustal X program. Bootstrap values were assessed with 1000 replicates. Amino acid sequences for comparison were obtained from GenBank. The sequences used are shown in Table S1.

2.6. Expression analysis of AsEcR, AsRXR and AsE75 The expression patterns of AsEcR, AsRXR and AsE75 were determined from third instar nymphs and fourth instar nymphs. Three nymphs were sampled each day from the new molt up to 10 days after ecdysis because most nymphs molted to fourth instar nymphs 11 days after ecdysis under the laboratory conditions of this study. Nymphs that molted on the eleventh day were used for day 0 of the fourth instar. RNA extraction and cDNA synthesis were performed as described previously. Real-time PCR was performed with SYBR®GreenER™ qPCR SuperMix Universal Kit (Invitrogen). Primers EcRC were designed for the sequences of the DBD to detect total AsEcR expression (EcRcom) and EcRA from isoform specific domain (A/B domain) to detect AsEcRA-specific expression. Similarly, primers E75DBD and E75D were designed from E75 DBD and D domains to detect the expression of each isoform. E75DBD primers will detect E75 isoforms with a complete DBD (two zinc fingers). Therefore, not only will E75A (isolated in this study) be detected, but other isoforms such as E75C (not isolated in this study) may be detected if expressed. Actin F and R were used for the

internal control AsActin5C (Table 1). Final concentrations of each primer were 0.1 μM/well for internal control actin and 0.2 μM for target genes. Amplifying PCR reactions were performed for 2 min at 50°C, 10 min at 95°C, and 40 cycles at 95°C for 15 sec and 60°C for 60 sec, followed by 95°C for 15 sec, 60°C for 15 sec and 95°C for 15 sec for the dissociation curve. Triplicate reactions were run on an ABI Prism 7900 HT (Applied Biosystems). Analysis of data was performed with SDS 2.1 (Applied Biosystems) and Excel 2008 (Microsoft) according to ABI PRISM® 7700 Sequence Detection System User Bulletin #2 (Applied Biosystems). The amplification efficiencies of primers were 97.2% and 98.2% so the comparative CT method was used for expression analysis of EcRCOM and RXR. The expression patterns of EcRA, E75DBD and E75D were determined by standard curve methods.

2.7. Measurement of ecdysteroid titers during the 3rd instar molting cycle Spiders for ecdysteroid analysis were collected each day after the second molting throughout the third instar until the second day of the fourth instar. Spiders were stored in tubes containing 2 ml 100% methanol and stored at -80℃ until ecdysteroid analysis. Procedures for ecdysteroid extraction from the whole body samples followed the methods of Ogihara et al. (2007) with the following modifications; methanol supernatant was dried under a N2 gas stream after centrifugation or under reduced pressure; chloroform treatment to remove lipids was conducted at a 1:1 chloroform : supernatant ratio for each whole body sample. Chloroform treatment was repeated 3 times in the same tube for each sample. Each sample was finally dissolved in 250 μl of Tris-buffered saline (TBS, pH 7.4, 24.76 mM Tris, 136.9 mM NaCl and 2.683 mM KCl) containing 0.5% bovine serum albumin

(BSA, BSA-TBS) and 0.1% sodium azide. Ecdysteroid titers of the samples were determined by the Enzyme Linked ImmunoSorbent Assay (ELISA) described by Mizoguchi et al. (2013). 20E conjugated with ovalbumin (OVA, 20E-OVA) and anti-20E rabbit serum were gifts from Dr. Akira Mizoguchi, Nagoya University. 20E for standard curve was purchased from Sigma (Tokyo). Each sample was measured in duplicate and absorbance was measured at 490 nm with an Ultramark Microplate Imaging System (BIO-RAD). A standard curve of 20E titers was used as the standard hormone for all assays, so the ecdysteroids are expressed as 20E equivalents.

2.8. Determination of ecdysteroids using LS-MS/MS Steroids were extracted with the same methods for ELISA. Samples were dried and redissolved in 50 μl of methanol. The samples were vortexed 10 min and centrifuged 10 min at 3,000 xg. Supernatant was transferred to a glass tube and 10 μl of the solution loaded for LC-MS/MS analysis. Spider ecdysteroids were determined with the multiple-reaction-monitoring (MRM) detection method on LC-MS/MS as described by Ogihara et al. (2015). Three ecdysteroids (Ecdysone, 20E, and Ponasterone A) were used as standards. Ecdysone and 20E were purchased from Sigma Aldrich (Tokyo). Ponasterone A was purchased from Santa Cruz Biotechnologies (Santa Cruz).

3. Results 3.1. Identification of AsEcR, AsRXR and AsE75 cDNA sequence and characterization of amino acid sequences Partial AsEcR (386 nucleotides) was amplified with degenerate primers and the full length AsEcR (GenBank accession number GQ281317) determined by RACE using gene specific primers based on partial fragments. The presence of a specific A box (NGxSPSxxSSYDxxYSP) in the A/B domain common to EcRA isoforms indicates the AsEcR is an EcRA isoform (Fig. 1A). A second AsEcR with a different A/B domain was also identified. This partial fragment (350 bp) contained 116 amino acid residues (Genbank accession number LC128752). The sequence was the same as AsEcRA in the latter part of the A/B domain and DBD, but the anterior part of the A/B domain (DLEFWDLDINESSNANTAASVS) differed. Multiple alignment of the partial A/B domain with EcRB isoforms of other arthropods showed the partial AsEcR amino acid sequence was similar to EcRB isoforms, suggesting this fragment is an AsEcRB isoform (Fig. 1B). In a similar manner to AsEcRA, AsRXR (2017 nucleotides and 406 amino acids, GQ281318), AsE75A (3262 nucleotides and 916 amino acids, HM102368) and AsE75D (2924 nucleotides and 861 amino acids, HM102369) were isolated. AsE75D had a different A/B domain when compared with AsE75A. In addition, the sequence lacked the DBD and had only a partial D domain. This sequence is similar to the D isoform of insect E75s, so the sequence was designated the AsE75D isoform. Determined AsEcRA, AsRXR and AsE75A showed all conserved domains of nuclear receptors (Fig. 1 A, C, D). The homology of amino acids was compared with other arthropods (Table 2-5). The A/B domain of AsEcR showed a slightly higher identity with the EcRA

isoform of the arachnids

L. australasiae (LaEcRA)(49%), A. americanum

(AamEcRA1)(48%) and O. moubata (OmEcRA)(55%). The DBD of nuclear receptors identified from A. silvatica were highly conserved when compared with other receptors. The DBD of AsEcRA was identical to EcRA of L. australasiae, B. germanica and Tribolium castaneum. Also the DBD of AsE75A was identical to all other sequences. The homology of the LBD showed great variability between species. The LBD of AsEcRA was highly similar to the arachnids, crustaceans and insects. Similarly the LBD of AsRXR also showed the highest homology with LaRXR (74%) and homologies were higher (40-66%) for the other arachnids AamRXR and OmRXR, the crustaceans UpRXR and MjRXR, the insects BgRXR, AmUSP, TcUSP and the Human RXR α. On the other hand, the LBD of MsUSP (41%) and DmUSP (40%) showed low homology with AsRXR. These results indicate AsRXR is similar to other RXR receptors and not insect USP receptors. In the LBD, BgE75 showed the highest homology with AsE75 (71%) and the homologies of E75s were high (64-69%) when compared to another arachnid OmE75, crustaceans MeE75 and DmagE75, and insects TcE75 and AmE75. On the other hand, the LBD of MsE75 and DmE75 showed low homology (52%) with AsE75. The identities of the A/B domain were low in both the E75A and D isoforms. Multiple alignments compared nuclear receptor sequences from A. silvatica with other animals (Fig. 1A-C). The DBD of all receptors contained identical or similar P and D boxes for recognition of DNA. The T and A boxes in the D domain for dimerization and DNA binding were conserved in AsEcRA and AsRXR. The T and A boxes were also conserved. The LBD of AsEcRA and AsRXR showed conserved helices (H1 to H12) for ligand binding and the AF-2 region for heterodimerization similar to other nuclear receptors. AsE75A and AsE75D contained all His and Cys

residues that function to bind the heme moiety in the LBD (Reinking et al., 2005; de Rosny et al., 2006). Therefore, AsE75 is likely able to bind heme and DNA to regulate gene transcription as reported in some insects. Phylogenetic trees of the LBDs of EcR, RXR and E75 are shown in Fig. 2. AsEcR is included in the arachnida group with LaEcR, AamEcR, OmEcR and TuEcR. The arachnid branch is separated from the insects and crustaceans. The LBD of AsRXR is included in the arachnida branch with LaRXR, AamRXR and OmRXR, and the branch is widely separated from the RXR and USP of insects. On the other hand, the branch of the arachnida RXRs is close to the RXR of human RXRα and crustacean RXRs. The homology of E75 LBD was widely divided into four branches. AsE75 was included in the arachnida branch with OmE75 and TuE75, but separated from the E75 sequences of the insect and crustacean branches. These results suggest the ligand binding functions of arachnid EcR and E75 is different than other arthropods. Conversely the RXR tree suggests ligand binding function of AsRXR is more similar to crustaceans and vertebrates than insects.

3.2. Analysis of AsEcR, AsRXR and AsE75 gene expression during nymphal molting cycle E75 is one ecdysone early gene that has been shown to be important for ecdysis in insects. Therefore, the expression patterns of AsEcR, AsRXR and AsE75 during the 11 days of the third instar nymphs and the first day after molting in fourth instar nymphs were measured by real-time PCR. AsActin5C (Gene accession number; FJ554633) was used as the internal control. The total AsEcR mRNA expression patterns were measured with primers designed in the DBD and expression levels of AsEcRA

were determined with primers designed from the A/B isoform specific domain and compared with other EcR isoforms. The expression levels of total AsEcR increased on D1, D4 and D9, but decreased on D7 and D10 (Fig. 3A). A large standard error in D4 through D8 may have occurred because there are variations in the days that spiders molt. On the other hand, expression levels of AsEcRA reached a peak on D1 but decreased after D2 then became low on D7 and D8 (Fig. 3B). The different expression patterns of total AsEcR and AsEcRA suggest other AsEcR isoforms may be expressed during the third instar. Constant expression of total AsEcR during the third instar nymphs indicates the receptor is waiting for an ecdysteroid signal. The expression levels of AsRXR mRNA were similar except for what may be a peak from D7 to D10, especially when compared with the decrease in expression on D11 (Fig. 3C). The expression of AsRXR occurs constantly throughout the third instar nymph with a possible peak on D7 to D10, indicating AsRXR is primed to regulate gene transcription. The expression patterns of AsE75DBD and AsE75D were measured with specific primers designed for each isoform (Fig. 3D, E). The expression level of AsE75DBD was extremely low on D0 to D5, but significantly increased from D6 to D10, then rapidly decreased on D11. On the other hand, AsE75D expressed in the first half and decreased from D6 to D10, the level returned on D11. These expression patterns suggest AsE75 isoforms include DBD and AsE75D functions in different manners during the nymphal molting cycle.

3.3. Analysis of ecdysteroid titer during nymphal molting cycle Daily changes in total ecdysteroid titers in the whole body samples of spiders during the third instar to early fourth instar were determined by ELISA (Fig. 3F).

Ecdysteroid titers remained at low concentrations, approximately 0.1 ng during day 0 to day 4 of the third instar, but showed a steep rise on day 5 and day 6, reaching a peak of 1.47±0.605 ng. After this peak, ecdysteroid titers immediately decreased, but remained a little higher than during the first 4 days of the third instar. The ecdysteroid peak occurs one day before the expression of AsE75DBD indicating ecdysteroids stimulate the expression of this gene.

3.4. Identification of ecdysteroids in Agelena silvatica Ecdysteroids in A. silvatica were analyzed using LC-MS/MS. A 20E peak with the same retention time as the 20E standard (Fig. 4A and B) was detected. In addition, the major MS/MS fragments of 20E (Fig. 4C and D) were also observed. A small peak of ecdysone was detected from A. silvatica, but the MS/MS fragments could not be obtained because of the low amount. Ponasterone A was not detected from A. silvatica (Fig. S1). These results indicate the major ecdysteroid of A. silvatica is 20E converted from ecdysone.

4. Discussion Spiders require ecdysteroids for their development and molting. Although the necessity of ecdysteroids has been reported over decades, the detailed regulatory mechanisms of ecdysteroids have not yet been clarified, because of little information on the essential regulatory factors. In this study, the complete ORFs of nuclear receptors AsEcRA, AsRXR, AsE75A and AsE75D were identified from the spider A. silvatica. In addition, the major ecdysteroid of A. silvatica was shown to be 20E. All nuclear receptors contained structures commonly conserved in the nuclear receptor superfamily and showed high identities especially in the DBDs. The DBDs of AsEcR and AsRXR showed greater than 88% and 92% identities with other arthropods. AsEcR and AsRXR showed the highest homologies with EcRs and RXRs of the hard tick A. americanum and scorpion L. australasiae, for which binding affinity to Drosophila EcRE were confirmed (Guo et al., 1998; Palmer et al., 2002; Nakagawa et al., 2007). These results indicate that a 20E/AsEcR/AsRXR complex can bind to the EcRE in the promoter region and regulate similar gene sets required for molting. The LBD of AsEcR also showed high identities with those of other arthropods, especially arachnids and hemimetabolous insects. This study showed the major ecdysteroid of A. silvatica was 20E, similar to other arachnids and arthropods. LBD of AsEcR appears to accept 20E as a molting hormone. On the other hand, the phylogenetic tree of EcR LBD also showed AsEcR separated from insect EcRs and was included in an arachnid clade. These results suggest the function of the LBD of AsEcR is similar to that of other arachnids but may be different from insects and crustaceans. Even in insects that have high homologies in the LBD, the binding affinities to ecdysteroids and their analogs are different between different orders, such as Diptera,

Lepidoptera and Hemiptera (Carmichael et al., 2005). These differences originate from the structures of the ligand binding pockets. Therefore, arachnid EcRs may have ligand affinities different from insects based on the three dimensional structure. The phylogenetic tree of LBDs (Fig. 2B) shows AsRXR is included in a different clade than insects and crustaceans. Recent studies indicate RXR and USP can also function by themselves. RXR of mammals can bind RAs (Mangelsdorf and Evans, 1995). Similarly, RXR of crustacean and ancient insects also can bind RAs (Chung et al., 1998; Hopkins et al., 2008, Nowichyj et al., 2008; Wang and LeBlanc, 2009). Therefore, spider RXR may recognize RA as a ligand. Further studies on the ligands that interact with EcR, RXR and functions of EcR and RXR are needed to clarify the EcR/RXR gene regulatory mechanisms and their evolution in spiders. In addition to direct regulation of gene expression by the 20E/EcR/RXR complex, genes can be indirectly regulated through ecdysone early genes such as E75, E74 and broad complex. The ecdysone early gene E75 was isolated and analyzed from A. silvatica in this study. The DBD of AsE75 was completely identical to other arthropods indicating AsE75 can also bind to DNA as a transcriptional factor to regulate target gene expression. RNA interference of E75 isoforms in B. germanica showed negative feedback of E75 in different E75 isoforms (Mané-Padrós et al., 2008). Even though E75 B and D isoforms do not contain the DBD region, they also can regulate transcription by heterodimerization. Studies on B. mori E75 showed the E75 F domain is necessary for repression of BmHR3 (Swevers et al., 2002). This suggests AsE75D may function with the LBD or F domains. E75 is an orthologue of the vertebrate orphan nuclear receptor Rev-Erbα, but E75 of D. melanogaster shows heme binding in the LBD and repressor activity for HR3 is suppressed to allow the transcriptional activity of

HR3 for gene regulation (Reinking et al., 2005; de Rosny et al., 2006). AsE75 shows high identities with other arthropods in the LBD and also has the conserved heme binding residues, so AsE75 may upregulate gene transcription involved in the regulation of numerous processes related to molting by ecdysteroids. However, further studies are required to determine the actual functions of AsE75 and other E75s in the regulation of gene expression in arthropods including spiders. The expression patterns of AsEcR, AsRXR and AsE75 mRNA were measured by real-time PCR. The expression of AsEcR and AsRXR mRNA did not show major changes but occurred throughout the third instar nymphal stage (Fig. 3A-C). On the other hand, AsE75DBD expression was low or non-existent at first, then showed a sudden increase from D7 to D10 (Fig. 3D). The AsE75D isoform showed different expression patterns (Fig. 3E) with similar levels from D0 to D5 and then a decrease on D6 to D10. AsRXR also showed a slight increase during the same period. This period concurred with the ecdysteroid titer peak of A. silvatica on D6 (Fig. 4). Both 20E and ecdysone were detected from A. silvatica. Ecdysone is the prohormone of 20E, so 20E in A. silvatica appears to be converted from ecdysone. Quantification of ecdysteroids by ELISA also indicates mainly 20E is present. Ponasterone A was not detected from A. silvatica despite reports that Ponasterone A is the major ecdysteroid in a mite (Grbic et al., 2011). Arachnids such as the scorpion and tick also use 20E not Ponasterone A (Miyashita et al., 2011; Ogihara et al., 2015). Other natural ecdysteroids such as Makisterone A were not detected from A. silvatica (data not shown). Therefore, the major ecdysteroid in Arachnids appears to be 20E. Based on these results, an ecdysteroid peak on D6 in A. silvatica, an increase in AsE75DBD on D7 is likely a result of 20E binding to the AsEcR/AsRXR heterodimer to upregulate

expression of the ecdysone early gene AsE75. On the other hand, the AsE75D decrease on D6 suggests early gene E75 isoforms show different responses to ecdysterioids. Another spider P. mirabilis showed an ecdysteroid peak on D17 in a 22 day molting cycle (Bonaric and De Riggi, 1977), and apolysis and synthesis of new cuticle also occurred during this period (Bonaric, 1980). The interval of the molting cycle of P. mirabilis and A. silvatica is different, but the peak of ecdysteroids and apolysis in P. mirabilis similarly occurs a few days before molting. In this study, a complete EcRA isoform, partial EcRB sequence, one type of RXR, complete E75A and D isoforms were identified from the spider A. silvatica. This is the first report of identification of EcR and RXR and E75 from Araneae. Expression patterns of EcR, RXR, USP and E75 isoforms are reported to differ depending on developmental stages, different tissues and different species of arthropods. EcRA and RXR of ticks show high expression levels once or twice before ecdysteroid titers peak in the final nymphal stages (Palmer et al., 2002; Horigane et al., 2007, 2008). On the other hand, the expression of EcRA, RXR-L and RXR-S in B. germanica and USP in Drosophila were constant throughout the immature stages (Maestro et al., 2005; Cruz et al., 2006; Sullivan and Thummel, 2003). To date we have only isolated the AsEcRA and a partial AsEcRB isoform and one isoform of AsRXR, subsequent studies will be carried out to identity other isoforms and determine the separate expression patterns of each isoform to better understand the more specific regulation of molting by ecdysteroids in spiders. E75 is an ecdysone early gene and the numbers of isoforms differ in species (Segraves and Hogness, 1990; Zhou et al., 1998; Pierceall et al., 1999; Matsuoka and Fujiwara, 2000; Mané-Padrós et al., 2008; Hannas and LeBlanc, 2010). The E75

isoforms of B. germanica (Mané-Padrós et al., 2008) and E75A of M. sexta (Zhou et al., 1998) show expression without ecdysteroids or before the ecdysteroid titer peak and their expressions are suppressed by ecdysone early genes. The nuclear receptors HR3, HR4 and E75 isoforms themselves are known as target genes for E75 in the regulation of molting (Horner et al., 1995; White et al., 1997; Hiruma and Riddiford, 2004, 2009; Mané-Padrós et al., 2008). Therefore, further studies are needed to clarify the functions of AsE75A and D isoforms and determine whether other AsE75 isoforms exist in spiders. Continual expression of AsEcR and AsRXR in A. silvatica indicates these receptors are waiting for an ecdysteroid signal. When ecdysteroids enter the target cell, AsEcR can form a heterodimer with AsRXR, and upregulate the expression of the ecdysone early gene AsE75 as well as other genes are important in molting. Analyses of these transcriptional factors are required to fully understand the regulation of molting by ecdysteroids in spiders. As well as molting, ecdysteroids have been reported to regulate pheromone secretion, venom production, feeding, web spinning, sexual behaviors and vitellogenesis of spiders (Pourié and Trabalon, 2003; Trabalon et al., 1992, 1998, 2005; Herzig, 2010). AsEcR, RXR, and E75 determined in this study may be players for these physiological processes and behaviors. Information on spider EcR, RXR and E75 can provide a better understanding of the regulation mechanisms in spiders and help with understanding the evolution of these mechanisms from a broader view in the arthropods.

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Fig.1. Multiple alignment of deduced amino acids of EcRA(A), EcRB(B), RXR(C) and E75A(D). The specific A box in the A/B domain, P and D boxes in the DNA binding domain, T and A boxes in the D domain, and the AF-2 region in the ligand binding domain are enclosed with solid line boxes. Putative DBD and LBD positions are indicated. # in multiple alignment of E75 (C) shows His and Cys required for heme binding. Helixes in the ligand binding domain of EcRs follow Carmichael et al. (2005) and those of RXRs follow Sasorith et al. (2002). Species abbreviations: LaEcRA, LaRXR (Liocheles australasiae); OmEcRA, OmRXR (Ornithodoros moubata); NcEcRB (Nephila clavata); DmagEcRA, DmagEcRB (Daphnia magna); UpEcRB, UpRXR (Uca pugilator); GlE75 (Gecarcinus lateralis); BgEcRA, BgRXR-L, BgE75A (Blattella germanica); BmEcRB1 (Bombyx mori), LmEcRB (Locusta migratoria); Human RXR (Homo sapiens); MsUSP (Manduca sexta); AmE75A (Apis mellifera); AaE75A (Aedes aegypti); DmEcRA, DmUSP, DmE75A (Drosophila melanogaster); AsEcR, AsRXR, AsE75A (Agelena silvatica) (this study). See Table S1 for accession numbers. Fig. 2. Phylogenetic tree of LBD of EcR(A), RXR(B) and E75(C). Phylogenetic trees were constructed by the Neighbor-joining Method with Clustal X Program. Numbers at the branches indicate bootstrap values with 1000 replications. Human LXRα, HumanRXRα and Human Rev-erb were used as outgroup. Species abbreviation of (A): AamEcR (Amblyomma americanum), OmEcR (Ornithodoros moubata), LaEcR (Liocheles australasiae), TuEcR (Tetranychus urticae), UpEcR (Uca pugilator), MjEcR (Marsupenaeus japonicus), CcEcR (Crangon crangon), DmagEcR (Daphnia magna), AmEcR (Apis mellifera), BgEcR (Blattella germanica), LmEcR (Locusta migratoria), TcEcR (Tribolium castaneum), BmEcR (Bombyx mori), MsEcR (Manduca sexta), DmEcR (Drosophila melanogaster), AaEcR (Aedes aegypti) and AsEcR (Agelena silvatica) (this study). Species abbreviations for (B): AamRXR (Amblyomma americanum), OmRXR (Ornithodoros moubata), LaRXR (Liocheles australasiae), UpRXR (Uca pugilator), MjRXR from (Marsupenaeus japonicus), DmagRXR (Daphnia magna), AmUSP (Apis mellifera), BgRXR-L and BgRXR-S (Blattella germanica), LmRXR (Locusta migratoria), TcUSP (Tribolium castaneum), BmUSP (Bombyx mori), MsUSP (Manduca sexta), DmUSP (Drosophila melanogaster), AaUSP (Aedes aegypti), Human RXR (Homo sapiens), Ascidian RXR (Polyandrocarpa misakiensis), Zebrafish RXR (Danio rerio), Snail RXR (Lymnaea stagnalis) and AsRXR (Agelena silvatica) (this study). Species abbreviations for (C): OmE75 (Ornithodoros moubata), TuE75 (Tetranychus urticae), GlE75 (Gecarcinus lateralis),

MeE75 (Marsupenaeus ensis, DmagE75 (Daphnia magna, AmE75 (Apis mellifera), BgE75 (Blattella germanica), TcE75 (Tribolium castaneum), BmE75 (Bombyx mori), MsE75 (Manduca sexta), DmE75 (Drosophila melanogaster), AaE75 (Aedes aegypti) and AsE75 (Agelena silvatica) (this study). See Table S1 for gene accession numbers. Fig. 3. Temporal gene expression pattern of (A) AsEcRDBD, (B) AsEcRA, (C) AsRXR, (D) AsE75DBD and (E) AsE75D quantified by real-time PCR. Relative expression levels are standardized with day 0 after molting of third instar nymphs. Values presented are means ± SE (n=3). (F) Ecdysteroid titers in whole bodies of spiders at day ages 3rd instar Day 0 to 4th instar Day 2 measured by ELISA (n = 4, 5-15 replications per each sample). Data are presented as means ± SEM. Fig. 4. Ecdysteroids in A. silvatica. Chromatograms of 20E and ecdysone detected using MRM methods on LC-MS/MS. (A) Chromatogram of 20E and ecdysone with standard chemicals (A) and A. silvatica (B). Arrowheads indicate peaks of 20E and ecdysone. MS/MS fragments of 20E of standard (C) and A. silvatica (D).

Figure 1A Click here to download high resolution image

Figure 1B Click here to download high resolution image

Figure 1C Click here to download high resolution image

Figure 1D Click here to download high resolution image

Figure 2A

HumanLXR

A TuEcR AamEcR

1000

1000

OmEcR

Arachnida

979

AsEcR 894

LaEcR DmagEcR 774

MjEcR Crustacea 858

CcEcR 1000

UpEcR AaEcR

432 1000

DmEcR 999

BmEcR Insecta

1000

MsEcR

570

AmEcR 1000

TcEcR

789

BgEcR 954

LmEcR 0.1

Fig. 2.

Chordata

Figure 2B

B

HumanRXR ZeblafisRXR Chordata AscidianRXR SnailRXR MjRXR

998 1000

UpRXR

Crustacea

967

604

CcRXR TuRXR 398 AsRXR 959

811 LaRXR 427

Arachnida AamRXR

998 OmRXR 494 DmagRXR

Crustacea AaUSP

877 DmUSP

772 1000

MsUSP 1000 BmUSP

556 TcUSP 797

AmUSP

946

LmUSP 995

0.1

Fig. 2. (Continued)

BgRXR-L 1000 BgRXR-S

Insecta

Human Rev-erb

Figure 2C

C

AaE75 997 DrnE75 1000

Insecta BmE75 1000 MsE75

OmE75 968 TuE75

Arachnida

666 AsE75 828 GlE75 1000 MeE75

Crustacea

897 DmagE75

791

AmE75 999 BgE75 901 TcE75

0.1

Fig. 2. (Continued)

Insecta

Figure 3 Click here to download high resolution image

Figure 4

B

A

20E 1.10e5

8.0e4

1.00e5 Intensity (counts per min)

Intensity (counts per min)

20E 9.0e4

7.0e4 6.0e4

Ecdysone

5.0e4 4.0e4 3.0e4 2.0e4 1.0e4

9.00e4 8.00e4 7.00e4 6.00e4 5.00e4 4.00e4 3.00e4 2.00e4

Ecdysone

1.00e4

0.0

0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0

Time (min)

Time (min)

D

C 481.2

481.2

3.0e5

1.8e4

1.4e4

Intensity (counts per min)

Intensity (counts per min)

1.6e4 445.3

1.2e4 1.0e4 371.2

8000.0 6000.0

303.2

4000.0

2.4e5 445.2 1.8e5

1.2e5

371.2 303.2

6.0e4

427.4 463.3

2000.0

313.0

0.0 50

Fig. 4.

100

150

200

250 300 m/z (Da)

350

400

450

500

0.0 50

100

150

200

250 300 m/z (Da)

350

427.4 463.3 409.2 400

450

500

Table 1. List of all primers Primers for degenerate PCR EcRdF1

5’-WSNGGNTAYCAYTAYAAYGC-3’

EcRdF2

5’-GARGGNTGYAARGGNTTYTT-3’

EcRdF3

5’-TGMGNMGNAARTGYCARGARTG-3’

EcRdR1

5’-TCNSWRAADATNRCNAYNGC-3’

EcRdR2

5’-CATCATNACYTCNSWNSWNSWNGC-3’

EcRdR3

5’-AAYTCNACDATNARYTGNACNGT-3’

EcRBdF

5’-GAYCTSGAATTCTGGGACCTCGA-3’

EcRC R

5’-GGCATTTCTTTAACCGACACTC-3’

RXRdF1

5’-ATHTGYGGNGAYMGNGC-3’

RXRdF2

5’-GGNAARCAYTAYGGNGTNTA-3’

RXRdR1

5’-TCYTCYTGNACNGCYTC-3’

RXRdR2

5’-CAYTTYTGRTANCKRCARTA-3’

E75dF1

5’-AGGCCWSMGGYTTCCACTAY-3’

E75dF2

5’-YTTCCACTAYGGYGTKCAYT-3’

E75dR1

5’-GCAYTTYTTSAGSCKRCART-3’

E75dR2

5’-CARTAYTGGCAYCGGTTSCK-3’

Gene-specific primers for RACE AsEcRrF1

5’-CCCCCTCAGAAGAGGATTTC-3’

AsEcRrF2

5’-AAAGGGTTCCTGGTTTCGATA-3’

AsEcRrF3

5’-GCGTCCTCAAAGTGGACAAT-3’

AsEcRrF4

5’-CCCTCAAGGTCCAGAACAAA-3’

AsEcRrF5

5’-CAGGTTGGGATCATGGTTTC-3’

AsEcRrR

5’-ACACATTCTGGCCTCATTCC-3’

AsRXRrF1 AsRXRrF2

5’-AGGCTGCAAAGGTTTCTTCA-3’ 5’-ACGGACTGTGCGAAAAGATT-3’

AsRXRrF3

5’-ATGGACAAGACGGAGCTAGG-3’

AsRXRrF4

5’-TTGGTAACCCTTAGCCATGC-3’

AsRXRrR1

5’-GCAACGGTTCCTCTGTCTTT-3’

AsRXRrR2

5’-GAAACCTTTGCAGCCTTCAC-3’

AsE75rF1

5’-AGCAATGTTCCATCCTACGG-3’

AsE75rF2

5’-TGGAAGTGAGTGGAACACCA-3’

AsE75rF3

5’-ACCTTCGCACCTTGAACACT-3’

AsE75rF4

5’-AGCTCCTCCTTTGGTGAACA-3’

AsE75rR1

5’-CCGTAGGATGGAACATTGCT-3’

AsE75rR2

5’-GCGAATCGCTCAGAGAAATC-3’

Gene-specific primers for expression analysis EcRC F

5’-CTCACCTGTGAAGGATGCAA-3’

EcRC R

5’-GGCATTTCTTTAACCGACACTC-3’

RXR F

5’-AGGCTGCAAAGGTTTCTTCA-3’

RXR R E75DBD F

5’-GCAACGGTTCCTCTGTCTTT-3’ 5’-CAAAGGCTTCTTCCGACGTA-3’

E75DBD R

5’-GCCCACAGCGATACACTTTT-3’

E75D F

5’-TGCTGGTGCATCTCTCTGTT-3’

E75D R

5’-GTACCGACCACGGCTTATCA-3’

Actin F

5’-TCCTCTGAACCCCAAAGCTA-3’

Actin R

5’-TGCGTACAAGGACAGGACAG-3’

5’ RACE outer primer

5’-GCTGATGGCGATGAATGAACACTG-3’ 5’-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3

5’ RACE inner primer

’

3’ RACE outer primer

5’-GCGAGCACAGAATTAATACGACT-3’

3’ RACE inner primer

5’-CGCGGATCCGAATTAATACGACTCACTATAGG-3’

T7

5’-TAATACGACTCACTATAGGG-3’

sp6

5’-TATTTAGGTGACACTATAG-3’

Table 2. Homology of AsEcRA domains compared with other arthropod EcRs. % Identity (similarity) of each domain Amino acids

A/B

C

100 (100) 71 (84)

LaEcRA

Scorpion

49 (59)

OmEcRA

Tick

55 (62)

AamEcRA1 Tick

48 (57)

MjEcRA

Prawn

33 (40)

DmagEcR

Daphnia

BgEcRA

D

99

E 87 (94)

63 (73)

82 (91)

99 (100)

58 (63)

82 (92)

96 (100)

53 (60)

58 (74)

29 (36)

96 (99) 43 (50)

69 (83)

Orthoptera

36 (42)

100 (100) 57 (68)

67 (80)

TcEcRA

Coleoptera

42 (61)

100 (100) 69 (79)

69 (80)

AmEcRA

Hymenoptera 33 (41)

99 (100)

61 (75)

71 (80)

89 (97)

66 (75)

57 (72)

88 (96)

55 (65)

59 (76)

MsEcRA

35 (44) Lepidoptera

MsEcRB1

30 (42)

DmEcRA

30 (38) Diptera

DmEcRB1

39 (48)

(100)

Amino acids identity (similarity) with AsEcR is given for Liocheles australasiae, Ornithodoros moubata, Amblyomma americanum, Marsupenaeus japonicas, Daphnia magna, Blattella germanica, Tribolium castaneum, Apis mellifera, Manduca sexta, Drosophila melanogaster. See Table S1 for accession numbers.

Table 3. Homology of AsRXR domains compared with RXRs and USPs from other arthropods and human % Identity (similarity) of each domain Amino acids

A/B

C

D

E

LaRXR

Scorpion

37 (53)

92 (96)

68 (79)

74 (85)

OmRXR

Tick

38 (51)

94 (99)

87 (91)

61 (75)

AamRXR1

Tick

23 (37)

96 (97)

74 (96)

64 (79)

UpRXR

Crab

31 (47)

94 (99)

54 (75)

57 (69)

MjRXR

Prawn

43 (55)

96 (97)

42 (62)

66 (80)

DmagRXR

Daphnia

35 (45)

92 (97)

65 (91)

66 (80)

BgRXR-S

Orthoptera

33 (42)

97 (100)

83 (91)

66 (80)

HsRXR alfa

Human

27 (36)

85 (97)

83 (96)

66 (81)

TcUSP

Coleoptera

37 (45)

97 (100)

74 (87)

58 (76)

AmUSP

Hymenoptera 35 (43)

96 (100)

78 (91)

66 (81)

MsUSP

Lepidoptera

58 (62)

94 (99)

57 (65)

41 (60)

DmUSP

Diptera

33 (48)

94 (97)

18 (24)

40 (58)

Amino acids identity (similarity) with AsRXR is given for Liocheles australasiae, Ornithodoros moubata, Amblyomma americanum, Uca pugilator, Marsupenaeus japonicas, Daphnia magna, Blattella germanica, Homo sapiens, Tribolium castaneum, Apis mellifera, Manduca sexta, Drosophila melanogaster. See Table S1 for accession numbers.

Table 4. Homology of AsE75A domains compared with E75s from other arthropods. % Identity (similarity) of each domain Amino acids

A/B

C

D

100 (100) 83 (89)

E

F

NA

NA

IsE75

Tick

63 (82)

OmE75

Tick

NA

MeE75A

Prawn

30 (49)

100 (100) 73 (85)

64 (79)

DmagE75

Daphnia

16 (20)

100 (100) 69 (82)

62 (80) 23 (32)

BgE75A

Orthoptera

41 (55)

100 (100) 72 (87)

71 (81) 28 (40)

TcE75

Coleoptera

49 (56)

100 (100) 71 (84)

69 (80) 24 (33)

AmE75A

Hymenoptera 39 (55)

100 (100) 72 (87)

69 (84) 29 (40)

MsE75A

Lepidoptera 36 (55)

100 (100) 62 (79)

52 (74) 19 (27)

DmE75A

Diptera

100 (100) 59 (79)

52 (74) 22 (31)

7 (9)

NA

82 (87)

69 (83) NA 13 (19) 21 (21) *

Amino acids identity (similarity) with AsE75A is given for Ixodes scapularis, Ornithodoros moubata, Metapenaeus ensis, Daphnia magna, Blattella germanica, Tribolium castaneum, Apis mellifera, Manduca sexta, Drosophila melanogaster. See Table S1 for accession numbers. * in MeE75A is from the deduced amino acid edited by Kim et al. (2005).

Table 5. Homology of AsE75D domains compared with E75Ds from insects. % Identity domain Amino acids

A/B

D

(similarity)

of

E

F

each

BgE75D

Orthoptera

11 (14) 70 (86) 71 (81) 28 (40)

AmE75D

Hymenoptera 3 (10)

70 (86) 69 (84) 29 (40)

MsE75D

Lepidoptera 7 (15)

59 (78) 52 (74) 19 (27)

DmE75D

Diptera

56 (78) 52 (74) 22 (31)

6 (19)

Amino acids identity (similarity) with AsE75D is given for Blattella germanica, Apis mellifera, Manduca sexta, Drosophila melanogaster. See Table S1 for accession numbers.