Chun-che Chang, Laboratory for Genetics and Development, Department of Entomology, National Taiwan University, no. 27, Lane 113, Roosevelt Road, Sec. 4, Taipei 106, Taiwan. Tel.: +8862 33665578; fax: +8862 27369366; e-mail: email@example.com
In the dipteran Drosophila, the genes bicoid and hunchback work synergistically to pattern the anterior blastoderm during embryogenesis. bicoid, however, appears to be an innovation of the higher Diptera. Hence, in some non-dipteran insects, anterior specification instead relies on a synergistic interaction between maternally transcribed hunchback and orthodenticle. Here we describe how orthologues of hunchback and orthodenticle are expressed during oogenesis and embryogenesis in the parthenogenetic and viviparous form of the pea aphid, Acyrthosiphon pisum. A. pisum hunchback (Aphb) mRNA is localized to the anterior pole in developing oocytes and early embryos prior to blastoderm formation – a pattern strongly reminiscent of bicoid localization in Drosophila. A. pisum orthodenticle (Apotd), on the other hand, is not expressed prior to gastrulation, suggesting that it is the asymmetric localization of Aphb, rather than synergy between Aphb and Apotd, that regulates anterior specification in asexual pea aphids.
Despite evolutionary divergence in the use of maternal determinants, it has become clear in recent years that insects rely on a common set of toolkit genes for segmentation. For example, although the details of their expression vary, most Drosophila homologues of the gap, pair-rule and segment-polarity genes can be identified in other insects (Peel et al., 2005). In Drosophila, a representative of the long germband mode of segmentation, all segments are specified within a short temporal window at the blastoderm stage (St Johnston and Nüsslein-Volhard, 1992). In contrast to Drosophila, segmentation in intermediate and short germband insects involves the initial specification of only anterior segments in the blastoderm. After gastrulation, a phase of secondary growth occurs in the posterior region, where segments are specified sequentially (French, 2001; Davis & Patel, 2002). The fact that in all insects – long, intermediate, and short germ – the most anterior segments are specified in the blastoderm, raises the possibility that some elements of the molecular mechanisms of anterior specification may be conserved.
In Drosophila, the gap genes hunchback (hb) and orthodenticle (otd) are downstream targets of bcd (Driever & Nüsslein-Volhard, 1989; Gao & Finkelstein, 1998). Synergistic interaction between bcd and hb is required for activating the expression of otd and two other anterior gap genes empty spiracles (ems) and buttonhead (btd) (Simpson-Brose et al., 1994). Loss-of-function alleles for hb and otd show defects in the anterior, including deletions of preantennal and antennal structures (otd), and deletions of gnathal and thoracic segments (hb) (Tautz et al., 1987; Finkelstein & Perrimon, 1990). In insects lacking bcd, such as the long germ wasp Nasonia vitripennis and the short germ beetle Tribolium, orthologues of hb and otd have demonstrably synergistic roles in anterior patterning. Single knockdown of maternal and zygotic hb by parental RNA interference (pRNAi) in both Nasonia and Tribolium results in defects in the head and thorax (Schröder, 2003; Lynch et al., 2006). Similarly, in both species knockdown of maternal and zygotic otd typically results in head defects, ranging from loss of segments to loss of the entire head. Importantly, when subjected to double pRNAi against hb and otd, both Nasonia and Tribolium fail to develop the head, thorax and anterior abdomen (Schröder, 2003; Lynch et al., 2006). This is reminiscent of strong bcd mutants in Drosophila (Frohnhöfer & Nüsslein-Volhard, 1986), suggesting that in these two species synergy between hb and otd substitutes for bcd in anterior specification. Indeed, given that bcd is present only in higher Diptera, anterior specification by hb and otd is likely to be the norm among most insects, and is probably the ancestral condition (Lynch & Desplan, 2003; McGregor, 2006).
To test the hypothesis that hb and otd have conserved and synergistic roles in anterior patterning we sought to clone orthologues of these two genes and describe their expression in A. pisum. In the parthenogenetic and viviparous form of A. pisum, a temporal series of developing oocytes and embryos are enclosed within the ovarian tubules (ovarioles) (Miura et al., 2003; Le Trionnaire et al., 2008). Thus a gene's temporal and spatial expression during oogenesis and embryogenesis can be detected within an ovariole. In light of the developmental continuity from oogenesis to embryogenesis, we explored whether the A-P axis is preformed in the developing oocytes, as it is in Drosophila. We found that A. pisum hunchback (Aphb) mRNA was synthesized in the germaria and anteriorly localized during oogenesis and early embryogenesis. A. pisum orthodenticle (Apotd), by contrast, was not detectable until gastrulation. This result suggests that although the mechanism of anterior patterning in the pea aphid may involve a well-conserved role for hb, it apparently does not involve otd.
Isolation and sequence analysis of Acyrthosiphon pisum hunchback and A. pisum orthodenticle
We amplified, then cloned, Aphb by PCR using gene specific primers based on a putative orthologue found in newly generated genomic sequence available at AphidBase. We cloned Apotd from PCR products amplified using degenerate primers designed to match conserved regions known to flank Otd homeodomains. Extension of our Apotd PCR product downstream of the homeodomain coding region was achieved using 3′ RACE-PCR. Hitherto, except for Aphb and Apotd, we have not found any other paralogues, suggesting that the pea aphid has only one hb and one otd. The gene structures of Aphb and Apotd are shown in Figs 1A and 2A, respectively.
The Aphb orthologue has an open reading frame (ORF) of 1755 base pairs that encodes a protein of 584 amino acids (Fig. 1A). The deduced protein sequence of ApHb contains six zinc finger domains, including four middle zinc fingers (MF 1-4) and two C-terminal zinc fingers (CF 1-2). ApHb also contains a C box and a Basic box (containing basic amino acid residues), both of which are conserved among insect Hunchback proteins (Fig. 1B, C). As with other insect hb genes, the 3′ UTR of the Aphb transcript contains predicted Nanos response elements (NREs), which contain conserved sequences known as Box A and Box B (Fig. 1D) (Gerber et al., 2006). The number of NREs in hb varies among insects: hb in the wasp Nasonia vitripennis contains four NREs (Pultz et al., 2005); at least two species of Drosophila possess a hb orthologue that contains two NREs (Gamberi et al., 2002); while other insects – including Musca domestica (housefly) (Bonneton et al., 1997), Tribolium castaneum (beetle) (Wolff et al., 1995), Schistocerca americana and Locusta migratoria (grasshoppers) (Patel et al., 2001) – possess hb orthologues that contain only one identifiable NRE (Fig. 1D). We have identified two NREs in the 3′ UTR of Aphb; however, because the sequences we cloned likely do not include the entire 3′ UTR, it remains possible that Aphb possesses additional NREs. Pultz et al. (2005) report that the hb NREs in Nasonia have 12–16 nucleotides between Box A and Box B, making the Nasonia NREs longer than other insect NREs, which typically have a 3–4 nucleotide spacing. In A. pisum, the distances between Box A and Box B are atypical – the more proximal NRE has a longer spacing of 8 nucleotides, while the distal NRE spacing is atypically short, consisting of only 1 nucleotides (Fig. 1D).
Both Tribolium and Nasonia have two otd genes: otd-1 and otd-2 (Li et al., 1996; Lynch et al., 2006), and the presence of these two paralogues in various arthropods (e.g. amphipod crustaceans and spiders) suggests that a single ancestral otd gene duplicated prior to or near the base of the arthropod radiation (Browne et al., 2006; Pechmann et al., 2009). Although both paralogues are derived from an ancestral otd (known as Otx), the otd-2 sequence is more similar to extant Otx genes than to the otd-1 sequence. Drosophila has otd-1, but has lost otd-2 (Klein & Li, 1999). Like Drosophila, A. pisum has a single otd gene, but unlike Drosophila, Apotd is more similar to Otx and otd-2 based on the presence of three Otd-2 signature motifs (IWSPA, NMDYL and the C-terminal Box) in the C-terminal region of the ApOtd protein (Fig. 2B). We thus infer that otd-1 has been lost in the aphid lineage.
Developmental expression of Acyrthosiphon pisum hunchback during oogenesis and embryogenesis
In the parthenogenetic and viviparous form of A. pisum, oogenesis and embryogenesis both occur within the ovariole. This allows one to observe the temporal and spatial distribution of a target transcript in germaria, oocytes, and embryos in the same tissue preparation. Expression of Aphb mRNA occurs first in the germarial lumen, which is a central cavity in the germarium located at the anterior tip of the telotrophic ovariole (Fig. 3A, hollow arrowhead). During oogenesis, Aphb mRNA is localized to the anterior region of unsegregated and segregated oocytes (Fig. 3A, B, stage 0 and 1), which are derived from cells in the posterior region of the germarium (Blackman, 1978; Miura et al., 2003). During early embryogenesis, Aphb mRNA remains localized to the anterior pole in egg chambers undergoing syncytial nuclear division (Fig. 3 C–E, stages 3 and 4). During formation of the blastoderm, the aggregation of Aphb transcripts appears to disassociate from the anterior pole such that it becomes positioned just posterior to the anterior blastoderm (Fig. 3K, stage 5). After blastoderm formation, anteriorly localized Aphb transcripts are not detectable in egg chambers bearing newly segregated primordial germ cells (Fig. 3L, stage 6). Apart from the anteriorly localized Aphb mRNA, we did not observe unlocalized Aphb mRNA in early embryos.
In pea aphids reproducing asexually, endosymbiotic bacteria are transferred from the mother during embryogenesis (Braendle et al., 2003). The bacteria begin to invade embryos that are about to gastrulate (stage 7), and when the germ band starts invaginating most bacteria enter into the egg chamber (stage 8) (Miura et al., 2003). During these two developmental stages, we observed weak expression of Aphb mRNA in the anterior region of the egg chamber and in the invaginating germ band at the posterior; Aphb mRNA was neither detectable in the central region, nor in cells housing endosymbiotic bacteria (Fig. 4A). After all the bacteria have been incorporated into the egg chamber, Aphb mRNA is preferentially expressed in the posteriormost region of the egg chamber (Fig. 4B, stage 10, arrowhead), which corresponds to the location of the presumptive gnathos posteriorly adjacent to the cephalic lobe in the embryo (Miura et al., 2003). Further invagination of the germ band into the egg chamber leads to the generation of the ‘S-shaped’ embryo (stage 11), and the anterior part of the embryo turns to face the posterior of the egg chamber until katatrepsis (embryo flip, stage 15). Preferential expression of Aphb mRNA persists in the gnathal region from stage 11 to stage 12 of development (Fig. 4 C–E, arrowheads), during which abdominal segments are extending (Miura et al., 2003).
In embryos with newly formed limb buds (stage 13), expression of Aphb mRNA becomes prominent in the presumptive growth zone (Fig. 4K, asterisk). Moreover, at this stage we also observed Aphb expression in the head, thorax and anterior abdomen (Fig. 4K, arrowheads and arrows). Similar expression patterns occur in embryos at stage 14 of development, just before katatrepsis (Fig. 4L). Compared with the expression of Aphb in stage-13 embryos (Fig. 4K), we found that stronger signals of Aphb occurred in the head region (Fig. 4L, arrowheads) and that the most intense signals were restricted to the optic lobes (Fig. 4M, black arrowheads). Nevertheless, the Aphb signal intensity weakened in the presumptive growth zone of the stage-14 embryos (Fig. 4L, asterisk), and when katatrepsis began Aphb expression in the posteriormost area of the abdomen was almost undetectable (data not shown).
Developmental expression of Acyrthosiphon pisum orthodenticle during oogenesis and embryogenesis
Unlike Aphb, expression of Apotd mRNA was not detected in germaria, developing oocytes or early embryos before stage 10 of development (Fig. 5A and data not shown). The expression of Apotd first becomes visible in the presumptive cephalic region of the stage-10 embryos (Fig. 5B, arrowhead) (Will, 1888; Miura et al., 2003). From stage 11 onward, expression intensifies (Fig. 5 D–G, arrowheads). Miura et al. (2003) report that during stage 10–12 the cephalic lobes migrate toward the posterior region of the egg chamber, where they develop further into the head of the embryo. We found that the migration of Apotd-positive cells corresponds to this route in embryos during these stages (Fig. 5B, D, E), suggesting that Apotd marks the cephalic lobes. Head expression of Apotd mRNA persists in stage 13 and 14 embryos (Fig. 5F, G). From stage 12–14 we observed that the distance between the bilateral clusters of the Apotd-positive cells in the ventral anterior region lessened (Fig. 5 L–O), suggesting movement of these cells or tissue condensation toward the ventral midline. Apotd is also expressed along the ventral midline, in the developing central nervous system (CNS), as it is in other insects and vertebrates (Lichtneckert & Reichert, 2005). This expression first appears at stage 12 (Fig. 5M, arrows) and increases in intensity during stages 13 and 14 (Fig. 5N, O).
In this study we describe the sequence and embryonic expression of Aphb and Apotd, homologues of the Drosophila hunchback (hb) and orthodenticle (otd) genes, in the facultatively asexual pea aphid, Acyrthosiphon pisum. Aphb and Apotd encode highly conserved domains that are common to Hb and Otd proteins in other insects (Figs 1 and 2). However, the Aphb ORF does not contain an A box, a B box, a D box, or N-terminal fingers (NF), all of which are conserved motifs that can be identified in other insect Hb proteins (Fig. 1C). Although we cloned 632 bp cDNA sequences upstream of the ORF (presumably 5′ UTR) and failed to find an A box, B box or NF in any of the three reading frames, we cannot exclude the possibility that there exists an alternative transcript containing exon(s), further upstream, that encode(s) these three missing motifs. Although the specific functions of the A box, B box and NF have not, to our knowledge, been investigated, Drosophila mutations in the MF, C-box, D-box, and CF affect the expression of hb as well as two other gap genes Krüppel (Kr) and knirps (kni), which are known to be regulated by hb (Hülskamp et al., 1990, 1994). Given that MF, C-box, and CF are present in many insect species including A. pisum (Fig. 1C), it is likely that the functions of these three common motifs are conserved.
In Drosophila, the A-P axis of the oocytes and early embryos are established by the anterior and posterior localization, respectively, of the maternally provided bicoid and nanos mRNAs (Driever & Nüsslein-Volhard, 1988; Gavis & Lehmann, 1992). In the pea aphid, maternally provided Aphb mRNA is asymmetrically localized to the anterior pole of developing oocytes and early embryos (Fig. 3 A–E, K), resembling the anterior localization of bicoid (St Johnston et al., 1989). Thus we infer that Aphb is involved in anterior specification in the pea aphid, using a heretofore-undescribed mechanism of hb mRNA localization.
The anterior restriction of hb function in Drosophila, in the face of maternally provided and unlocalized hb mRNA, is achieved via translational repression by posteriorly localized Nanos. In the grasshopper Schistocerca, Nanos may similarly restrict zygotic hb function, based on expression patterns and the presence of an NRE in the hb 3′ UTR (Lall et al., 2003). In the pea aphid, however, a crossreacting antibody made to Drosophila Nanos reveals that the protein is not localized to the posterior region of developing oocytes (stages 0 and 1; Chang et al., 2006) at the time we observe localization of Aphb mRNA (Fig. 3A, B). Instead, posterior localization of Nanos first occurs in stage-2 egg chambers and persists until blastoderm formation (stage 4). This suggests that the observed anterior restriction of Aphb in the blastoderm is due to localization of the mRNA and is not merely a secondary consequence of translational repression by Nanos. Our finding that the 3′ UTR of Aphb possesses a Nanos response element (Fig. 1D), however, suggests that later translational repression by Nanos may help to maintain the anterior restriction of hb function. A similar duel mechanism of mRNA localization and translational repression is required to achieve the posterior restriction of nanos function in Drosophila, although localization would appear to be more important for Aphb than it is for Drosophila nanos, in which case only 4% of the mRNA is localized to the posterior pole (Bergsten & Gavis, 1999; Dahanukar et al., 1999).
The absence of Aphb expression during germ-cell morphogenesis (Fig. 3L, stage 6) suggests that the Aphb mRNA detected in embryos from the period of bacterial invasion (Fig. 4A, stage 8) onward is zygotic rather than maternal. Our previous studies show that transcripts of either Apvasa or Apnanos are also undetectable during late stage 6 (Chang et al., 2007, 2009), suggesting that a general breakdown of maternally provided mRNAs may occur at this point in development. From stages 10–12, Aphb is preferentially expressed in the anterior part of the embryos (Fig. 4B–E). This Aphb-positive domain is located posteriorly to the presumptive cephalic lobes described by Will (1888) and Miura et al. (2003), and characterized by Apotd expression (Fig. 5B, D). We thus infer that this hb expression domain corresponds to the gnathal and thoracic regions, consistent with the anterior gap domain observed in Drosophila, Tribolium, and Schistocerca at the equivalent stages (Tautz et al., 1987; Wolff et al., 1995; Patel et al., 2001). From stages 13–14, Aphb is also expressed in the presumptive growth zone at the abdominal tip (Fig. 4K, L). Similar hb patterns have been reported in Tribolium (Wolff et al., 1995) and the more closely related Oncopeltus (Liu & Kaufman, 2004). Alternatively, it is possible that, rather than representing persistent expression in the growth zone, the posterior expression we observe instead represents the abdominal gap domain described in Drosophila, Tribolium, and the orthopterans Schistocerca and Gryllus bimaculatus (Tautz et al., 1987; Wolff et al., 1995; Patel et al., 2001; Mito et al., 2005). In summary, our data show that whereas A. pisum is perhaps exceptional in its early anterior localization of Aphb mRNA, it shares with several other insects maternal expression of hb in the anterior blastoderm, as well as later zygotic expression of hb in the gnathos, thorax and growth zone.
In the absence of bcd, anterior specification of the embryo in Nasonia and Tribolium is achieved by a synergistic interaction between maternal hb and otd (Schröder, 2003; Lynch et al., 2006). This has led to the suggestion that anterior specification by maternal hb and otd is the ancestral means of anterior specification among insects (Lynch & Desplan, 2003; McGregor, 2006). In asexual A. pisum, however, co-expression of maternal Aphb and Apotd is not observed in oocytes or early embryos (Figs. 3 and 5). This suggests that whereas Aphb is likely to play a role in anterior specification, this role does not rely on synergy with Apotd. Nevertheless, Apotd is expressed later in the differentiated cephalic lobes at the far anterior of the embryo (Fig. 5B).
In contrast to otd-1, otd-2 is not expressed in the blastoderm stage, at least not in the two arthropods thus far examined, Tribolium (Li et al., 1996) and the amphipod crustacean Parhyale hawaiensis (Browne et al., 2006). Such is also the case for Apotd (Fig. 5A), whose product is more similar to Otd-2 (Fig. 2B) and whose expression during early development is more reminiscent of otd-2. During gastrulation and germband extension, however, Apotd expression does not resemble otd-2 expression in either Tribolium or Parhyale, where the gene is expressed in only limited subsets of cells in the head (Tribolium) or along the ventral midline (Parhyale) (Li et al., 1996; Browne et al., 2006). Instead, Apotd is expressed in broad domains in both of these tissues (Fig. 5 E–G, M–O), in a manner more reminiscent of otd-1. Apotd thus appears to function like otd-2 in early development and like otd-1 in later development.
In this work we describe the expression patterns of Aphb and Apotd during the parthenogenetic and viviparous mode of development in A. pisum, but we would also like to understand better the roles these genes play in the sexual and oviparous mode. It remains an open question, for example, whether the early anterior localization of Aphb mRNA and the lack of Apotd expression in the blastoderm that we find in viviparous development is also found in oviparous development, or whether these sexual embryos specify their anterior regions using the apparently more conserved mechanisms of translational repression of hb mRNA in the posterior and synergy between hb and otd in the anterior.
Pea aphids (A. pisum) were reared on garden pea plants Pisum sativum at 20 °C in a growth chamber, under a 16 h/8 h light/dark cycle so as to maintain their parthenogenetic state. The A. pisum used in this study had been in continuous culture over one hundred generations. Staging of oogenesis and embryogenesis follows the developmental scheme described in Miura et al. (2003).
Cloning of Acyrthosiphon pisum hunchback and A. pisum orthodenticle
Total RNA was extracted from dissected ovarioles using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed using poly dT18 primers and StrataScript Reverse Transcriptase (Stratagene, La Jolla, CA, USA). The 1755 base pairs (bp) of the ORF of Aphb were cloned using primers flanking the putative start and stop codons (forward, 5′-GATCGGATCCATGTTTTTGGAAAACGAACACCAG-3′ (MFLENEHQ); reverse, 5′-AGCTAAGCTTCTAACTGTGCGGCACGCGCGCTAT-3′ (IARVPHS)), respectively. A cDNA fragment containing 632 bp upstream of the putative start codon and 351 bp of the ORF were isolated with a forward primer in the presumptive 5′untranslated region (UTR; 5′-GGTGGATTCCAATTTCCAAC -3′) and a reverse primer (5′-GCACTCCAGCCCACAGTGTTTGCACT-3′ (CKHCGLEC)) in the ORF. A partial PCR fragment of Aphb containing 990 bp was cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) using gene specific primers (forward, 5′-AAAACCTGAACAGTTGCTGGAGTG-3′ (KPEQLLEC); reverse, 5′-ACACCGACTT AGGCTGCGATTC-3′ (ESQPKSV)), and subsequently used as a template for in vitro transcription. Cloning of the Apotd homeobox sequences was performed with degenerate primers (forward: 5′-AAGCAGCGGCGGARMGNACNAC-3′ (KORRERTT); reverse: 5′-GCCCGCCGGTTCTTRAACCANAC-3′ (VWFKNRRA)), followed by 3′ rapid amplification of the cDNA ends (RACE)-PCR using the GeneRacer kit (Invitrogen, Carlsbad, CA, USA). Outer primer: 5′-CAGTTGGACGTGCTGGAAACATTG-3′ (QLDVLETL); nested primer: 5′-ACATTGTTCGCCAAGACTCG-3′ (TLFAKTR). An 895-bp portion of the Apotd sequence, which covers the homeobox and the 3′-RACE fragment, was then amplified for use as a template for in vitro transcription using gene-specific primers (forward: 5′-CAGTTGGACGTGCTGGAAACATTG-3′ (QLDVLETL); reverse: 5′-CCAGTTGTTTACTTGCGTTCCC-3′ (ERK plus non-coding nucleotides)). Two presumptive NREs were identified in the PCR fragments (627 bp) amplified using cDNA templates synthesized with a reverse primer (5′-GACCACGCGTATCGATGTCGACT16V-3′; V = A, C or G), two outer primers (forward: 5′-ATCGCGTGCAAACTCGAGCGACCGGCA-3′ (SRANSSDR); reverse: 5′-GACCACGCGTATCGATGTCGAC-3′), and two nested primers (forward: 5′-CGAGGAATAAAAGCCATCGCC-3′; reverse: 5′-CTCACATCA AGTAGAATCCTGC-3′) located in the potential 3′ UTR (accession number: GQ415688). Alignments of sequences were constructed using MacVector version 7.2.2 (Accelrys Inc., San Diego, CA, USA), and MUSCLE (Edgar, 2004).
Annotation of Acyrthosiphon pisum hunchback and A. pisum orthodenticle
We identified hb and otd genes in AphidBase by looking for sequences encoding the Hb and Otd homeodomains, respectively. For methods of searching homologues of hb and otd in the pea aphid genome please see Shigenobu et al. (2010). Candidate sequences were then verified via BLAST searches against GenBank to ensure the gene identities. Furthermore, in order not to miss any paralogues of hb and otd in the pea aphid genome, we used TBLASTN to search the unassembled genome sequences.
Whole mount in situ hybridization and microscopy
The Aphb and Apotd riboprobes, including sense and antisense strands, were transcribed from linearized plasmids containing the sequences described above. DNA templates of Aphb and Apotd included sequences encoding the conserved hb zinc-finger domains and the otd homeodomain, respectively. Digoxigenin (DIG)-labelled uridine triphosphates (UTPs) were incorporated into the Aphb and Apotd probes using the DIG RNA Labeling Kit (SP6/T7) (Roche, Basel, Switzerland). An Apvasa riboprobe for double in situ hybridization was synthesized with the Fluorescein (Flu) Labeling Mix (Roche). All DIG/Flu labeled riboprobes were detected with anti-DIG/Flu Fab fragments conjugated with alkaline phosphatase (AP). For single in situ assays (e.g. Fig. 3), we used the substrate Nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Roche). For double in situs, substrate combinations were as follows: (1) NBT/BCIP for detecting the expression of Aphb; 2-[4-Iodophenyl]-3-[4-nitrophenyl]-5-phenyl-tetrazolium chloride (INT)/BCIP (Sigma, St. Louis, MO, USA) for Apvasa (Fig. 4). (2) NBT/BCIP for Apotd; 4-benzoylamino-2,5-diethoxybenzenediazonium chloride hemi[zinc chloride] (Fast Blue BB) salt/naphthol-AS-MXphosphate (NAMP) (Sigma) for Apvasa (Fig. 5). Concentrations for each substrate and procedures followed our recent study (Chang et al., 2008). Nuclear staining was performed with DAPI (2 ng/µl) (Sigma). Nomarski images of samples were produced by a Leica DMR (Leica, Wetzlar, Germany) connected to a Fuji FinePix S2 Pro digital camera (Fujifilm, Tokyo, Japan). Photos of nuclear staining were taken with the C-Apo 40X/1.2 water lens linked to a Zeiss LSM510 META laser-scanning microscope (Carl Zeiss, Jena, Germany).
We are grateful to The Human Genome Sequencing Center at the Baylor College of Medicine for providing the sequences of the pea aphid genome and to Gee-Way Lin, Hsiao-Ling Lu, Te-pin Chang and Jou-Han Chen for manuscript proofreading. C.C. would like to thank Sue-Ping Lee (Institute of Molecular Biology, Academia Sinica) for technical support on confocal microscopy. This work was supported by the National Science Council (95-2313-B-002-097-MY2; 97-2313-B-002-035-MY3) and the Bureau of Animal and Plant Health Inspection and Quarantine (BAPHIQ) of the Agricultural Council (97-1.1.1-B1(6); 98-1.1.1-B1(2)) in Taiwan.