Posterior Hox gene reduction in an arthropod: Ultrabithorax and Abdominal-B are expressed in a single segment in the mite Archegozetes longisetosus
© Barnett and Thomas; licensee BioMed Central Ltd. 2013
Received: 30 May 2013
Accepted: 15 July 2013
Published: 30 August 2013
Hox genes encode transcription factors that have an ancestral role in all bilaterian animals in specifying regions along the antero-posterior axis. In arthropods (insects, crustaceans, myriapods and chelicerates), Hox genes function to specify segmental identity, and changes in Hox gene expression domains in different segments have been causal to the evolution of novel arthropod morphologies. Despite this, the roles of Hox genes in arthropods that have secondarily lost or reduced their segmental composition have been relatively unexplored. Recent data suggest that acariform mites have a reduced segmental component of their posterior body tagma, the opisthosoma, in that only two segments are patterned during embryogenesis. This is in contrast to the observation that in many extinct and extant chelicerates (that is, horseshoe crabs, scorpions, spiders and harvestmen) the opisthosoma is comprised of ten or more segments. To explore the role of Hox genes in this reduced body region, we followed the expression of the posterior-patterning Hox genes Ultrabithorax (Ubx) and Abdominal-B (Abd-B), as well as the segment polarity genes patched (ptc) and engrailed (en), in the oribatid mite Archegozetes longisetosus.
We find that the expression patterns of ptc are in agreement with previous reports of a reduced mite opisthosoma. In comparison to the ptc and en expression patterns, we find that Ubx and Abd-B are expressed in a single segment in A. longisetosus, the second opisthosomal segment. Abd-B is initially expressed more posteriorly than Ubx, that is, into the unsegmented telson; however, this domain clears in subsequent stages where it remains in the second opisthosomal segment.
Our findings suggest that Ubx and Abd-B are expressed in a single segment in the opisthosoma. This is a novel observation, in that these genes are expressed in several segments in all studied arthropods. These data imply that a reduction in opisthosomal segmentation may be tied to a dramatically reduced Hox gene input in the opisthosoma.
KeywordsHox Ultrabithorax Abdominal-B, Patched Segment-polarity Arthropod Chelicerate Opisthosoma Acari Acariformes
Hox genes are highly conserved transcription factor-encoding genes that regulate a large suite of transcriptional targets in all bilaterian animals [1, 2]. The conserved role of each Hox gene in specifying distinct body regions along the antero-posterior axis has caused this set of genes to be targets of evolutionary change throughout animal evolution [1, 3–6]. Changes in Hox gene function and expression domains have been shown to have led to a wide array of morphological novelties, for example, the limbless bodies of snakes , the evolution of the tetrapod limb  and the repression of limbs in the insect abdomen . Despite the general observation that changes in Hox gene expression domains correlate with the generation of new morphologies, a relatively less explored phenomenon is how Hox genes are expressed and utilized in body regions that have been secondarily reduced.
The arthropods, including insects, crustaceans, myriapods and chelicerates (arachnids and horseshoe crabs) display a wide degree of morphological variation on their relatively modular and segmented body plan. The origin of this morphological diversity has been due, in large part, to changes in Hox expression domains and targets throughout their evolution [6, 10–12]. In the arthropods, Hox genes act to specify the distinct identities of the developing body segments (for example, head versus abdominal). The relationship of Hox genes to the developing segments is further exemplified in the well-studied model arthropod, Drosophila melanogaster. In D. melanogaster, segments are established via the partitioning of the blastoderm into discrete segmental units by the activation of the gap genes, which subsequently activate the pair-rule and segment polarity genes. The gap and pair-rule gene expression domains are then used to establish the Hox gene domains within each segment [13, 14]. It has also been shown that segment polarity genes directly interact with Hox genes to elicit segmental identity in the D. melanogaster abdomen .
The body plan of chelicerate arthropods is comprised of two main body regions, the anterior prosoma and the posterior opisthosoma. The segments of the prosoma in chelicerates comprise the chelicerae, pedipalps, and four pairs of walking legs. The opisthosoma is more variable and contains the segments bearing the book gills in horseshoe crabs, the chemosensory pectines in scorpions and the book lungs and spinnerets in spiders. Contrary to the morphological variation of the opisthosoma seen throughout many chelicerate groups, the Hox gene Ultrabithorax (Ubx) has been shown to have a conserved early expression boundary in the second opisthosomal, or genital, segment [16–18]. The expression of the Hox gene abdominal-A (abd-A) is expressed in more posterior opisthosomal segments in chelicerates, having an anterior boundary in the third opisthosomal segment, which overlaps with Ubx expression. The most terminally expressed Hox gene in the chelicerate opisthosoma is Abdominal-B (Abd-B), which usually overlaps with the expression of Ubx and abd-A in the posterior opisthosomal segments [16, 18–21].
Previously, we have shown that the mite Archegozetes longisetosus patterns only two segments in the opisthosoma via the expression of orthologues of the segment polarity genes hedgehog (hh) and engrailed (en) , indicating a large degree of segmental fusion or loss in comparison to the ancestral chelicerate opisthosoma, which was likely comprised of twelve segments . To determine whether a reduction in posterior segmentation in A. longisetosus resulted in changes in Hox gene utilization in the mite opisthosoma, the expression patterns of the A. longisetosus orthologues of Ubx and Abd-B (Al-Ubx and Al-Abd-B, respectively) were followed. Also, the expression patterns of the segmentation gene patched (ptc), which encodes an Hh receptor in all other arthropods studied [24–26], were followed to determine if the Al-hh and Al-en expression patterns were unique, or if A. longisetosus truly pattern only two segments. The results of this study suggest that A. longisetosus does only pattern two segments in the opisthosoma during embryonic development, and also that Al-Ubx and Al-Abd-B are both only expressed in the same single segment, a novel observation for any arthropod studied thus far. These data, in conjunction with the observation that acariform mites have lost an abd-A orthologue [22, 27], suggest that Hox gene input in the mite opisthosoma has been reduced either as a cause of or a consequence of segmental reduction.
Archegozetes longisetosus cultures
Mites were reared on a plaster-of-Paris/charcoal substrate in plastic jars to maintain appropriate humidity. Wood chips were added to the jars to promote oviposition. Mites were fed with brewer’s yeast. No ethical approval was needed as A. longisetosus is not subject to any animal care regulations.
Embryo fixation and staining
To collect early-stage embryos (that is, germ band and early segmentation stage), adults were dissected in 1X PBS using a sharpened tungsten needle and sharp forceps. Laid late-stage embryos (that is, post-germ band stage) were collected from the culture chambers with a needle. Embryos of all stages were pooled and dechorionated in 50% bleach for one minute. Fixation occurred in an n-heptane solution over 4% formaldehyde in PBS for 45 minutes. Embryos were devittelinized by placing them into an n-heptane solution chilled on dry ice, subsequently adding room temperature methanol and then shaking vigorously for one minute to rupture the membrane. Embryos were rehydrated in graded methanol/PBS solutions, and placed in PBS with 0.1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) for one minute in the dark. A detailed protocol is available from the authors.
Gene cloning and identification
cDNA was constructed from A. longisetosus total embryonic RNA using the SMARTer RACE cDNA Kit (Clontech, Madison, WI, USA). All gene fragments were amplified using this cDNA as a template in rapid amplification of cDNA ends (RACE) PCR reactions. The A. longisetosus orthologue of the segment-polarity gene patched (ptc) was cloned by using the primer Arlo.ptc.GSP2.1 (GTGTGTGCATTCTTGGCGGCAGCAATTATTCC) in a 3′ RACE reaction. The resulting fragment was subsequently used in a nested 3′ RACE reaction using the primer N.Arlo.ptc.GSP2.1 (AGGTGTTTTGCTCTTCAGGCTGCAATTCTC). Both of these primers were designed against a fragment retrieved from an expressed sequence tag screen. The resulting 2,976 bp sequence consists of a large 1,816 5′ UTR, a 981 bp coding sequence and a 179 bp 3′ UTR (GenBank: KF155150). The coding sequence encodes a 326 amino acid protein, which contains the diagnostic Eukaryotic Sterol Transporter (EST) family domain [see Additional file 1: Figure S1A]. Al-en was cloned and sequenced as described in .
The full-length mRNA sequence of the A. longisetosus Ultrabithorax orthologue (Al-Ubx) was retrieved using both 3′ and 5′ RACE reactions. For the 3′ RACE reactions, the primer Ubx.Rtry.GSP2 (GCTGCAGCTGAAGCACATCAGGCCTACC) was used in an initial RACE PCR reaction. The resulting product was used in a subsequent nested 3′ RACE reaction using the primer N.Ubx.Rtry.GSP2 (CTTTACGACGGAGCGACCAGTCAAGCAT). For the initial 5′ RACE reaction, the primer Ubx.Rtry.GSP1 (CGCCTGTGCCTGTCTCTCCTGTTCGTTT) was used. The resulting product was used in a subsequent nested RACE PCR using the primer N.Ubx.Rtry.GSP1 (CGACCTCTTCGACGCAGACCGTTGGCAC). All four of the aforementioned primers were designed using the deduced Al-Ubx coding sequences of the A. longisetosus Hox cluster (unpublished data, we have dense coverage sequence for the Hox cluster region relevant to this paper). This single Ubx orthologue was the only one retrieved, and matches the genomic sequence in the cluster, thus indicating A. longisetosus likely has only one Ubx orthologue.
The resulting 3′ and 5′ nested RACE PCR reactions were cloned into the pGEM T Easy vector and sequenced. The resulting sequences were assembled using PHRAP to construct the full-length sequence of the Al-Ubx mRNA (GenBank: KF155151). The 1,759 bp AlUbx mRNA sequence consists of a 134 bp 5′ UTR, a 816 bp coding sequence, and a 809 bp 3′ UTR. The deduced amino acid sequence of Al-Ubx has a typical Ubx homeodomain and the diagnostic C-terminal UbdA motif [see Additional file 1: Figure S1B].
Fragments of the A. longisetosus Abd-B orthologue (Al-Abd-B) were cloned from embryonic cDNA using primers developed from genomic Hox cluster sequences. The primers used were AbdB.Rtry.Gsp1 (TAGCCTGTGGAGCACCGGTCCATTCCAG) in the 5′ RACE reaction and the primer AbdB.Rtry.Gsp2 (GGCCAAACACTCCATATCTCAGCAAAGCGG) in the 3′ RACE reaction. The resulting fragments were used in subsequent nested RACE PCR reactions, using the primers N.AbdB.Rtry.Gsp1 (GGTGAGTAGTTGCACCAGGCCGCTGCCG) and N.AbdB.Rtry.Gsp2 (CAGCGGCCTGGTGCAACTACTCACCATA) in the 5′ and 3′ reactions, respectively. These primers are overlapping reverse-compliments of one another. The resulting fragments were cloned into the pGEM T Easy plasmid and sequenced. This single Abd-B orthologue was the only one retrieved, and matches the genomic sequence in the cluster, thus indicating A. longisetosus likely has only one Abd-B orthologue.
The resulting sequences were assembled using PHRAP to construct the full-length sequence of the Al-Abd-B mRNA (GenBank: KF155152). The overlapping 3′ and 5′ RACE Abd-B products resulted in a 2,323 bp mRNA, consisting of a 227 bp 5′ UTR, a 1,191 bp coding sequence, and a 905 bp 3′ UTR. The deduced amino acid sequence of Al-Abd-B consists of a typical Abd-B homeodomain [see Additional file 1: Figure S1C].
patched (Al-Ptc) expression
Al-ptc expression in later stages followed previously reported expression patterns of the A. longisetosus orthologues of hh and en by which the first opisthosomal segment appears initially, followed by the appearance of the segment bearing the anlagen of the fourth pair of walking legs, which is then followed by the appearance of the final second opisthosomal segment. Following the early Al-ptc expression in the early germ-band (Figure 1A-F), Al-ptc expression was expressed in stripes of expression in the cheliceral and pedipalpal segments as well as the segments of the first three pairs of walking legs (Figure 1G-H´); however, the ‘older’ segments, that is, all prosomal segments excluding the third walking leg segments, had a more pronounced stripe of Al-ptc expression in the middle of the segment (Figure 1G), and the posterior stripes were undetected. However, this may be an artifact of our methodology, as in late-stage D. melanogaster embryos, the anterior ptc stripe of expression is much more pronounced than the posterior stripe . At this stage, no opisthosomal expression of Al-ptc was observed, with the posterior-most expression in the posterior limit of the anlagen of the third pair of walking legs (Figure 1H-H´). Following this stage, a broad single stripe of Al-ptc expression was observed in the region of the first opisthosomal segment (O1) (Figure 1I-J), similar to the patterns of Al-hh and Al-en observed in . Also in the same manner as Al-hh and Al-en expression, a stripe of Al-ptc appeared anterior to the O1 stripe in the following stage (Figure 1K). Subsequently, the broad stripe of Al-ptc expression in O1 split into two stripes (Figure 1L), likely to facilitate Al-en expression, as has been observed in a myriapod , a fly  and a spider . Following this stage, Al-ptc was expressed in a broad stripe demarking the second opisthosomal segment. Also at this stage, the Al-ptc expression in the fourth walking leg segment remained in a single stripe (Figure 1M). Whether this is due to the resolution of our images or due to a different patterning mechanism needs to be explored further. In later stages, in which the opisthosoma began to move more anterior forming the caudal bend (see  for morphological movements), Al-ptc expression in the fourth walking leg segment was reduced, with two broad stripes remaining in the opisthosoma demarking the first and second opisthosomal segments, respectively (Figure 1N-O). Observations of expression patterns are complicated in later stages due to the formation of the caudal bend. Therefore, it is unknown when the Al-ptc stripe of the second opisthosomal segment splits nor is it known when the Al-ptc stripe of the fourth walking leg segment splits. Further study into these questions using laser-scanning fluorescent confocal microscopy needs to be conducted, as ptc genes in other arthropod species are expressed in single-cell wide domains [24–26], and the non-fluorescent detection methods prove problematic in ascertaining the small domains in A. longisetosus (personal observations).
Ultrabithorax (Al-Ubx) expression
Abdominal-B (Al-Abd-B) expression
Al-ptc expression provides evidence that the wg/en segmentation pathway is conserved in mites
In the fly D. melanogaster, terminal segmental boundaries are generated by the Engrailed/Wingless auto regulatory loop , whereby en expressing cells activate hh expression and signaling. Hedgehog signaling proteins bind to the Ptc receptor proteins on the anterior adjacent cells to activate the expression of wingless, which encodes a signaling molecule that in turn binds to the Frizzled-2 receptor in the en expressing cells, thereby stabilizing en expression. This signaling pathway has been shown to be conserved in a number of other arthropods (see  for review). patched expression has been observed in three arthropod groups, in the fly D. melanogaster, the millipede G. marginata and the spider Parasteatoda tepidariorum. In D. melanogaster, ptc is initially ubiquitously expressed in the blastoderm. In later stages, ptc is expressed in a segmental manner in cells abutting en expressing cells, via the repression of ptc by en. In subsequent stages, ptc is repressed by an unknown factor, resulting in two thin stripes of ptc expression per segment with en expressing cells in the middle of these two stripes . This two-striped expression pattern of ptc surrounding a stripe of en expression is also seen in the ventral germ band of the millipede G. marginata and also in the developing segments in the spider P. tepidariorum. In all three of these species, ptc is initally expressed in broad stripes prior to the splitting into two stripes per segment. The data presented for Al-ptc expression (Figure 1) suggests that this mode of ptc expression is conserved throughout the arthropods. Previous data on en and hh expression in A. longisetosus indicate that the En/Wg signaling loop acts in A. longisetosus to pattern terminal segmental boundaries [22, 30]. However, the expression patterns of other components of this pathway (for example, wingless, cubitus interruptus and Notum) are needed to confirm this.
Al-Ubx is expressed in a single segment: a novel observation in an arthropod
The above data illustrate that in the mite A. longisetosus, Ubx is expressed in a single segment. The conserved role of Hox genes in specifying segments in arthropods suggests that Al-Ubx specifies the identity to a single segment in the opisthosoma. This is a novel observation in an arthropod in that in all observed arthropod species, Ubx is expressed in several developing posterior segments. In insects, Ubx specifies the abdominal segments, and in some lineages, Ubx is expressed in the second and/or third thoracic segments (for example, [31–35]). In crustaceans, Ubx is also expressed in multiple posterior developing segments [36–38], as is Ubx in myriapods [39–42].
Direct expression patterns of Ubx in chelicerates have been observed in the spiders (Araneae) P. tepidariorum and Cupiennius salei, and also in the harvestman Phalangium opilio. In P. tepidariorum, the single identified Ubx orthologue is expressed in the second opisthosomal segment (O2) through the remaining posterior segments . C. salei has two orthologues of Ubx, in which Ubx-1 is expressed from the anterior portion of O2 through the remaining posterior segments. Ubx-2 is expressed from the posterior half of O2 through the remaining posterior opisthosomal segments . Ubx expression in P. opilio is similar to spider expression patterns, where its anterior border of expression also lies in O2 . Popadić and Nagy (2001) observed expression patterns of the Hox genes Ubx and abd-A simultaneously using the UbdA antibody in the scorpion Paruroctonus mesaensis and the horseshoe crab Limulus polyphemus. The detection of this antibody showed that Ubx has an early expression boundary in O2 in both species, which later moves anteriorly to be expressed in O1. These data indicate that the ancestral chelicerate expression boundary of Ubx lies in the second opisthosomal, or genital, segment. However, the UbdA data should be interpreted with caution, as this antibody detects both Ubx and abd-A expression.
The Al-Ubx expression data indicate that this gene patterns a single segment, the second opisthosomal segment (Figure 2). This adds support to the hypothesis that the second opisthsosomal segment was the ancestral Ubx anterior expression boundary in chelicerates. We, therefore, maintain that the first and second opisthosomal segments are retained in A. longisetosus due to this anterior expression boundary of Al-Ubx. However, unlike the Ubx expression patterns observed in other chelicerates, Al-Ubx was not observed to extend anteriorly or posteriorly in later stages.
Al-Abd-B is expressed in a single segment; a novel observation in an arthropod
The expression data for Al-Abd-B also indicate that this gene is expressed in a single segment as well as in an early domain in the unsegmented telson (Figure 3). This is also a unique observation for arthropods in that in many studied arthropods, Abd-B patterns multiple posterior segments during development (see  for review). In the fly D. melanogaster, Abd-B functions to specify the fourth through the eighth abdominal segments via the expression of the m and r Abd-B isoforms . Abd-B also has a role in specifying the genital region of D. melanogaster embryos [44, 45]. In the beetle Tribolium castaneum, Abd-B also acts to specify the posterior ninth and tenth abdominal segments . In the grasshopper Schistocerca gregaria, Abd-B is expressed in the eighth through the eleventh abdominal segments, as well as in the genital region . In the thysanuran Thermobia domestica, Abd-B is expressed in the eight through the tenth abdominal segments . In the milkweed bug Oncopeltus fasciatus, Abd-B also has a genital-specifying role . In crustaceans, Abd-B is also expressed in the genital segments [50–52] and extends throughout all five segments of the posterior tagma (the pleon) of the isopod Porcellio scaber, but remains in the genital region of Artemia franciscana. In the cirripede Sacculina carcini, Abd-B is expressed throughout the thorax and also in the vestigial abdomen . For myriapods, Abd-B is expressed from the second leg-bearing segment posteriorly to the telson in the centipede Lithobius atkinsoni and is expressed only in the posterior growth zone and the anal valves in the millipede Glomeris marginata; however, as G. marginata undergoes anamorphic growth (that is, more segments are added in post-embryonic stages) Abd-B may be expressed in these posterior segments at later stages . These data, therefore, indicate that Abd-B had an ancestral role in patterning multiple posterior segments in arthropods, as well as a possible ancestral role in specifying the genital region.
In the chelicerates, Abd-B expression has also been observed in the spider C. salei and the harvestman P. opilio. In C. salei, Abd-B has a later expression domain in the cells of the future genital opening of O2, consistent with the hypothesis that Abd-B had a role of genital patterning in the last common ancestor of arthropods, as well as with the hypothesis that Abd-B had a role in patterning the genital region in the last common ancestor of the protostomes and the deuterostomes ( and references therein). Our observations show that Al-Abd-B is expressed in what we interpret as the second opisthosoma segment, due to the expression of Al-Ubx in this segment (see above). However, we are unable to assess when and where the genital rudiments of A. longisetosus form, which would verify that the segment expresses both Al-Ubx and Al-Abd-B. This may be due to our methodology, or due to the complex morphogenesis of the caudal bend during the growth of the opisthosoma. An earlier expression pattern for Al-Abd-B was also not observed in stages earlier than those shown in Figure 3A-D. We, therefore, maintain our interpretation that the two segments observed in the A. longisetosus opisthosoma are the first and second opisthosomal segments.
abdominal-A loss and posterior segmental reductions in arthropods
Like the spider mite Tetranychus urticae, A. longisetosus is likely missing the Hox gene abd-A, as Hox cluster sequencing and PCR surveys have yielded no abd-A orthologue (RH Thomas, unpublished results). T. urticae and A. longisetosus also display a two-segmental pattern of en expression in their opisthosomas, indicating that a loss of abd-A and the reduction of the opisthosoma occurred at the base of the acariform mite lineage [22, 27, 30]. In all arthropods that retain an abd-A orthologue in their genome, abd-A is expressed in posterior regions overlapping Ubx and Abd-B expression (see  for review). In the cirripede crustacean S. carcini, the abdominal segments are never fully developed in the adult. However, an en expression studied indicated that S. carcini patterned five abdominal segments in the developing vestigial abdomen, which are later removed following metamorphosis . Interestingly, hybridization experiments failed to find an expression domain for abd-A in any region during the development of S. carcini, and a subsequent cytogenic analysis found no putative abd-A orthologues in the S. carcini Hox cluster . Pycnogonids (sea spiders) also have a reduced posterior tagma. A PCR survey of Hox genes failed to find an abd-A orthologue in the pycnogonid Endeis spinosa; it also found that the abd-A orthologue in the pycnogonid Nymphon gracile had an unusual degree of sequence divergence in its homeodomain, possibly due to relaxed selection .
The reduction of posterior segmentation and the absence of an abd-A orthologue in these three disparate arthropod groups display a surprising convergent correlation. There may be some trend in arthropod evolution whereby redundant Hox gene functions in segments expressing multiple Hox genes cause the loss of selective pressure retaining one of the redundant genes in the genome. This selective pressure may also be reduced in arthropod lineages in which the posterior segments are reduced. Also, if selection is acting to reduce posterior segments, selection should act to retain only those posteriorly expressed Hox genes with multiple, non-redundant functions that are also highly pleiotropic. Ubx has functions that overlap with those of abd-A in arthropods, (for example, [56, 57]). Therefore, abd-A may have been a target of reduced selection to maintain its presence on the genomes of different arthropod groups during evolution.
As Hox genes act to specify the identities of segments in arthropods, it should follow that the loss of Hox genes (that is, abd-A) followed the loss of segments. In A. longisetosus, it would seem likely that segmental loss was facilitated via repressing the production of posterior segments arising from the posterior growth zone, rather than eliminating anterior segments. However, this is complicated as A. longisetosus patterns posterior segments in a manner that is not currently well-understood, in that it follows an anachronistic delineation pattern (that is, the later appearance of the L4 segment following the delineation of the first opisthosomal segment). Therefore, comparative functional studies are needed to answer these questions surrounding the loss of arthropod Hox genes and posterior segments.
Hox genes and chelicerate tagmosis
In comparison to other chelicerates, A. longisetosus differs in its utilization of Hox genes to pattern segments. Most notably is the single segment of expression of Ubx and Abd-B in the opisthosoma (Figures 2 and 3). Also of note is that Hox3 and Dfd are expressed in both the prosoma and opisthosoma in A. longisetosus (Figure 4A), breaking the tagmatic boundary rule. This observation coupled with the weak expression of Hox3 observed in the opisthosoma of P. tepidariorum and P. opilio may indicate a conserved role of Hox3 in chelicerates, with the expression patterns in C. salei being derived. The remaining Hox genes are expressed in a similar manner to the P. opilio, C. salei and P. tepidariorum, and do not seem to correlate with the borders of the pseudo-tagmata.
The absence of abd-A in A. longisetosus may be tied to the expression patterns of Hox3 and Dfd in the opisthosoma, as they indicate that extra Hox input is needed in these segments. However, functional studies of these genes in A. longisetosus are needed before this can be confirmed. Comparative functional studies of segmentation and posterior Hox gene expression in mites will be necessary to reveal the selection pressures, that is, a reduction in segmentation, miniaturizations, and so on that have led to their loss of abd-A.
Studies are also needed to elucidate how Hox3, Dfd and Antp expression patterns change in the opisthosoma throughout development. Telford and Thomas  show Al-Hox3 expression in the opisthosoma; however, this is at an early stage and its late-stage expression patterns in the opisthosoma are unknown. Likewise, Telford and Thomas  show Al-Dfd expression throughout the opisthosoma; however, its late-stage expression in the telson is unclear. This study also highlights Al-Antp expression in an embryo of the same age as those shown in Figures 2B and 3B. However, whether Al-Antp clears from the telson on later stages is also unclear. Therefore, further study is need into the interactions and dynamic expression patterns of Hox genes in the mite opisthosoma.
The above data illustrate the reduced Hox gene input in the opisthosoma of the mite A. longisetosus by examining the expression of the A. longisetosus orthologues of Ubx and Abd-B. These two Hox genes are restricted in later stages to the same opisthosomal segment, namely the second opisthosomal segment. The reduced segmental composition in the A. longisetosus opisthosoma , coupled with the confirmed absence of abd-A in one acariform mite  and a likely loss of abd-A in A. longisetosus (RH Thomas, unpublished results), calls for further study into the evolution of the mite opisthosoma.
Archegozetes longisetosus Abdominal-B
Archegozetes longisetosus Ultrabithorax
Archegozetes longisetosus engrailed
First walking leg
Second walking leg
Third walking leg
Anlagen of the fourth pair of walking legs
First opisthosomal segment
Second opisthosomal segment
Polymerase chain reaction
Posterior Al-ptc stripe of the third walking leg
Rapid amplification of cDNA ends
Sex combs reduced
This work was funded in part by National Science Foundation grant DEB‒0717389 to RHT, and also in part by the National Science Foundation Evo‒Dev‒Eco Network RCN (IOS # 0955517) to AAB We are indebted to Urs Schmidt‒Ott for the use of his lab facilities at the University of Chicago during portions of this study. We are also indebted to the three anonymous reviewers for their helpful comments.
- Hueber SD, Lohmann I: Shaping segments: Hox gene function in the genomic age. Bioessays. 2008, 30: 965-979. 10.1002/bies.20823.View ArticlePubMedGoogle Scholar
- Pearson JC, Lemons D, McGinnis W: Modulating Hox gene functions during animal body patterning. Nat Rev Genet. 2005, 6: 893-904. 10.1038/nrg1726.View ArticlePubMedGoogle Scholar
- Mallo M, Wellik DM, Deschamps J: Hox genes and regional patterning of the vertebrate body plan. Dev Biol. 2010, 344: 7-15. 10.1016/j.ydbio.2010.04.024.PubMed CentralView ArticlePubMedGoogle Scholar
- Pick L, Heffer A: Hox gene evolution: multiple mechanisms contributing to evolutionary novelties. Ann NY Acad Sci. 2012, 1256: 15-32. 10.1111/j.1749-6632.2011.06385.x.View ArticlePubMedGoogle Scholar
- Lee PN, Callaerts P, de Couet HG, Martindale MQ: Cephalopod Hox genes and the origin of morphological novelties. Nature. 2003, 424: 1061-1065. 10.1038/nature01872.View ArticlePubMedGoogle Scholar
- Hughes CL, Kaufman TC: Hox genes and the evolution of the arthropod body plan. Evol Dev. 2002, 4: 459-499. 10.1046/j.1525-142X.2002.02034.x.View ArticlePubMedGoogle Scholar
- Cohn MJ, Tickle C: Developmental basis of limblessness and axial patterning in snakes. Nature. 1999, 399: 474-479. 10.1038/20944.View ArticlePubMedGoogle Scholar
- Schneider I, Aneas I, Gehrke AR, Dahn RD, Nobrega MA, Shubin NH: Appendage expression driven by the Hoxd Global Control Region is an ancient gnathostome feature. Proc Natl Acad Sci USA. 2011, 108: 12782-12786. 10.1073/pnas.1109993108.PubMed CentralView ArticlePubMedGoogle Scholar
- Ronshaugen M, McGinnis N, McGinnis W: Hox protein mutation and macroevolution of the insect body plan. Nature. 2002, 415: 914-917. 10.1038/nature716.View ArticlePubMedGoogle Scholar
- Damen WGM: Hox genes and the body plans of chelicerates and pycnogonids. Hox Genes: Studies from the 20th to the 21st Century. 2010, Berlin: Springer-Verlag Berlin, 689: 125-132. 10.1007/978-1-4419-6673-5_9.View ArticleGoogle Scholar
- Averof M: Arthropod Hox genes: insights on the evolutionary forces that shape gene functions. Curr Opin Genet Dev. 2002, 12: 386-392. 10.1016/S0959-437X(02)00314-3.View ArticlePubMedGoogle Scholar
- Cook CE, Smith ML, Telford MJ, Bastianello A, Akam M: Hox genes and the phylogeny of the arthropods. Curr Biol. 2001, 11: 759-763. 10.1016/S0960-9822(01)00222-6.View ArticlePubMedGoogle Scholar
- Carroll SB, DiNardo S, O’Farrell PH, White RAH, Scott MP: Temporal and spatial relationships between segmentation and homeotic gene-expression in Drosophila embryos - distributions of the Fushi-tarazu, Engrailed, Sex combs reduced, Antennapedia, and Ultrabithorax proteins. Gene Dev. 1988, 2: 350-360. 10.1101/gad.2.3.350.View ArticlePubMedGoogle Scholar
- McGinnis W, Krumlauf R: Homeobox genes and axial patterning. Cell. 1992, 68: 283-302. 10.1016/0092-8674(92)90471-N.View ArticlePubMedGoogle Scholar
- Gebelein B, McKay DJ, Mann RS: Direct integration of Hox and segmentation gene inputs during Drosophila development. Nature. 2004, 431: 653-659. 10.1038/nature02946.View ArticlePubMedGoogle Scholar
- Damen WGM, Hausdorf M, Seyfarth EA, Tautz D: A conserved mode of head segmentation in arthropods revealed by the expression pattern of Hox genes in a spider. Proc Natl Acad Sci USA. 1998, 95: 10665-10670. 10.1073/pnas.95.18.10665.PubMed CentralView ArticlePubMedGoogle Scholar
- Popadic A, Nagy L: Conservation and variation in Ubx expression among chelicerates. Evol Dev. 2001, 3: 391-396. 10.1046/j.1525-142X.2001.01049.x.View ArticlePubMedGoogle Scholar
- Sharma PP, Schwager EE, Extavour CG, Giribet G: Hox gene expression in the harvestman Phalangium opilio reveals divergent patterning of the chelicerate opisthosoma. Evol Dev. 2012, 14: 450-463. 10.1111/j.1525-142X.2012.00565.x.View ArticlePubMedGoogle Scholar
- Damen WGM, Tautz D: Abdominal-B expression in a spider suggests a general role for Abdominal-B in specifying the genital structure. J Exp Zool. 1999, 285: 85-91. 10.1002/(SICI)1097-010X(19990415)285:1<85::AID-JEZ10>3.0.CO;2-N.View ArticlePubMedGoogle Scholar
- Damen WGM, Tautz D: Comparative molecular embryology of arthropods: the expression of Hox genes in the spider Cupiennius salei. Invertebr Reprod Dev. 1999, 36: 203-209. 10.1080/07924259.1999.9652701.View ArticleGoogle Scholar
- Khadjeh S, Turetzek N, Pechmann M, Schwager EE, Wimmer EA, Damen WGM, Prpic N-M: Divergent role of the Hox gene Antennapedia in spiders is responsible for the convergent evolution of abdominal limb repression. Proc Natl Acad Sci USA. 2012, 109: 4921-4926. 10.1073/pnas.1116421109.PubMed CentralView ArticlePubMedGoogle Scholar
- Barnett AA, Thomas RH: The delineation of the fourth walking leg segment is temporally linked to posterior segmentation in the mite Archegozetes longisetosus (Acari: Oribatida, Trhypochthoniidae). Evol Dev. 2012, 14: 383-392. 10.1111/j.1525-142X.2012.00556.x.View ArticlePubMedGoogle Scholar
- Westheide W, Regier R: Spezielle Zoologie, Erster Teil: Einzeller und Wirbellose Tiere. 1996, Verlag, Stuttgart: Gustav FischerGoogle Scholar
- Janssen R, Budd GE, Damen WGM, Prpic N-M: Evidence for Wg-independent tergite boundary formation in the millipede Glomeris marginata. Dev Genes Evol. 2008, 218: 361-370. 10.1007/s00427-008-0231-2.View ArticlePubMedGoogle Scholar
- Akiyama-Oda Y, Oda H: Cell migration that orients the dorsoventral axis is coordinated with anteroposterior patterning mediated by Hedgehog signaling in the early spider embryo. Development. 2010, 137: 1263-1273. 10.1242/dev.045625.View ArticlePubMedGoogle Scholar
- Hidalgo A, Ingham P: Cell patterning in the Drosophila segment - spatial regulation of the segment polarity gene patched. Development. 1990, 110: 291-301.PubMedGoogle Scholar
- Grbić M, Van Leeuwen T, Clark RM, Rombauts S, Rouze P, Grbić V, Osborne EJ, Dermauw W, Ngoc PCT, Ortego F, Hernandez-Crespo P, Diaz I, Martinez M, Navajas M, Sucena E, Magalhaes S, Nagy L, Pace RM, Djuranovic S, Smagghe G, Iga M, Christiaens O, Veenstra JA, Ewer J, Villalobos RM, Hutter JL, Hudson SD, Velez M, Yi SV, Zeng J: The genome of Tetranychus urticae reveals herbivorous pest adaptations. Nature. 2011, 479: 487-492. 10.1038/nature10640.View ArticlePubMedGoogle Scholar
- Sanson B: Generating patterns from fields of cells - examples from Drosophila segmentation. EMBO Rep. 2001, 2: 1083-1088. 10.1093/embo-reports/kve255.PubMed CentralView ArticlePubMedGoogle Scholar
- Peel AD, Chipman AD, Akam M: Arthropod segmentation: beyond the Drosophila paradigm. Nat Rev Genet. 2005, 6: 905-916.View ArticlePubMedGoogle Scholar
- Telford MJ, Thomas RH: Expression of homeobox genes shows chelicerate arthropods retain their deutocerebral segment. Proc Natl Acad Sci USA. 1998, 95: 10671-10675. 10.1073/pnas.95.18.10671.PubMed CentralView ArticlePubMedGoogle Scholar
- Kelsh R, Weinzierl ROJ, White RAH, Akam M: Homeotic gene-expression in the locust Schistocerca - an antibody that detects conserved epitopes in Ultrabithorax and Abdominal-A proteins. Dev Genet. 1994, 15: 19-31. 10.1002/dvg.1020150104.View ArticlePubMedGoogle Scholar
- Weatherbee SD, Nijhout HF, Grunert LW, Halder G, Galant R, Selegue J, Carroll S: Ultrabithorax function in butterfly wings and the evolution of insect wing patterns. Curr Biol. 1999, 9: 109-115. 10.1016/S0960-9822(99)80064-5.View ArticlePubMedGoogle Scholar
- Warren RW, Nagy L, Selegue J, Gates J, Carroll S: Evolution of homeotic gene regulation and function in flies and butterflies. Nature. 1994, 372: 458-461. 10.1038/372458a0.View ArticlePubMedGoogle Scholar
- Zheng Z, Khoo A, Fambrough D, Garza L, Booker R: Homeotic gene expression in the wild-type and a homeotic mutant of the moth Manduca sexta. Dev Genes Evol. 1999, 209: 460-472. 10.1007/s004270050279.View ArticlePubMedGoogle Scholar
- Bennett RL, Brown SJ, Denell RE: Molecular and genetic analysis of the Tribolium Ultrabithorax ortholog, Ultrathorax. Dev Genes Evol. 1999, 209: 608-619. 10.1007/s004270050295.View ArticlePubMedGoogle Scholar
- Liubicich DM, Serano JM, Pavlopoulos A, Kontarakis Z, Protas ME, Kwan E, Chatterjee S, Tran KD, Averof M, Patel NH: Knockdown of Parhyale Ultrabithorax recapitulates evolutionary changes in crustacean appendage morphology. Proc Natl Acad Sci USA. 2009, 106: 13892-13896. 10.1073/pnas.0903105106.PubMed CentralView ArticlePubMedGoogle Scholar
- Pavlopoulos A, Kontarakis Z, Liubicich DM, Serano JM, Akam M, Patel NH, Averof M: Probing the evolution of appendage specialization by Hox gene misexpression in an emerging model crustacean. Proc Natl Acad Sci USA. 2009, 106: 13897-13902. 10.1073/pnas.0902804106.PubMed CentralView ArticlePubMedGoogle Scholar
- Averof M, Patel NH: Crustacean appendage evolution associated with changes in Hox gene expression. Nature. 1997, 388: 682-686. 10.1038/41786.View ArticlePubMedGoogle Scholar
- Janssen R, Damen WGM: The ten Hox genes of the millipede Glomeris marginata. Dev Genes Evol. 2006, 216: 451-465. 10.1007/s00427-006-0092-5.View ArticlePubMedGoogle Scholar
- Janssen R, Budd GE: Gene expression suggests conserved aspects of Hox gene regulation in arthropods and provides additional support for monophyletic Myriapoda. EvoDevo. 2010, 1: 4-10.1186/2041-9139-1-4.PubMed CentralView ArticlePubMedGoogle Scholar
- Grenier JK, Garber TL, Warren R, Whitington PM, Carroll S: Evolution of the entire arthropod Hox gene set predated the origin and radiation of the onychophoran/arthropod clade. Curr Biol. 1997, 7: 547-553. 10.1016/S0960-9822(06)00253-3.View ArticlePubMedGoogle Scholar
- Hughes CL, Kaufman TC: Exploring the myriapod body plan: expression patterns of the ten Hox genes in a centipede. Development. 2002, 129: 1225-1238.PubMedGoogle Scholar
- Kuziora MA, McGinnis W: Different transcripts of the Drosophila Abd-B gene correlate with distinct genetic sub-functions. EMBO J. 1988, 7: 3233-3244.PubMed CentralPubMedGoogle Scholar
- Celniker SE, Lewis EB: Transabdominal, a dominant mutant of the bithorax complex, produces a sexually dimorphic segmental transformation in Drosophila. Gene Dev. 1987, 1: 111-123. 10.1101/gad.1.2.111.View ArticlePubMedGoogle Scholar
- Estrada B, Sanchez-Herrero E: The Hox gene Abdominal-B antagonizes appendage development in the genital disc of Drosophila. Development. 2001, 128: 331-339.PubMedGoogle Scholar
- Beeman RW, Stuart JJ, Haas MS, Denell RE: Genetic analysis of the homeotic gene complex (HOM-C) in the beetle Tribolium castaneum. Dev Biol. 1989, 133: 196-209. 10.1016/0012-1606(89)90311-4.View ArticlePubMedGoogle Scholar
- Kelsh R, Dawson I, Akam M: An analysis of Abdominal-B expression in the locust Schistocerca gregaria. Development. 1993, 117: 293-305.PubMedGoogle Scholar
- Peterson MD, Rogers BT, Popadic A, Kaufman TC: The embryonic expression pattern of labial, posterior homeotic complex genes and the teashirt homologue in an apterygote insect. Dev Genes Evol. 1999, 209: 77-90. 10.1007/s004270050230.View ArticlePubMedGoogle Scholar
- Aspiras AC, Smith FW, Angelini DR: Sex-specific gene interactions in the patterning of insect genitalia. Dev Biol. 2011, 360: 369-380. 10.1016/j.ydbio.2011.09.026.View ArticlePubMedGoogle Scholar
- Averof M, Akam M: Hox genes and the diversification of insect and crustacean body plans. Nature. 1995, 376: 420-423. 10.1038/376420a0.View ArticlePubMedGoogle Scholar
- Brena C, Liu PZ, Minelli A, Kaufman TC: Abd-B expression in Porcellio scaber Latreille, 1804 (Isopoda : Crustacea): conserved pattern versus novel roles in development and evolution. Evol Dev. 2005, 7: 42-50. 10.1111/j.1525-142X.2005.05005.x.View ArticlePubMedGoogle Scholar
- Blin M, Rabet N, Deutsch JS, Mouchel-Vielh E: Possible implication of Hox genes Abdominal-B and abdominal-A in the specification of genital and abdominal segments in cirripedes. Dev Genes Evol. 2003, 213: 90-96.PubMedGoogle Scholar
- Gibert JM, Mouchel-Vielh E, Queinnec E, Deutsch JS: Barnacle duplicate engrailed genes: divergent expression patterns and evidence for a vestigial abdomen. Evol Dev. 2000, 2: 194-202. 10.1046/j.1525-142x.2000.00059.x.View ArticlePubMedGoogle Scholar
- Geant E, Mouchel-Vielh E, Coutanceau J-P, Ozouf-Costaz C, Deutsch JS: Are Cirripedia hopeful monsters? Cytogenetic approach and evidence for a Hox gene cluster in the cirripede crustacean Sacculina carcini. Dev Genes Evol. 2006, 216: 443-449. 10.1007/s00427-006-0088-1.View ArticlePubMedGoogle Scholar
- Manuel M, Jager M, Murienne J, Clabaut C, Le Guyader H: Hox genes in sea spiders (Pycnogonida) and the homology of arthropod head segments. Dev Genes Evol. 2006, 216: 481-491. 10.1007/s00427-006-0095-2.View ArticlePubMedGoogle Scholar
- Casares F, Calleja M, SanchezHerrero E: Functional similarity in appendage specification by the Ultrabithorax and abdominal-A Drosophila HOX genes. EMBO J. 1996, 15: 3934-3942.PubMed CentralPubMedGoogle Scholar
- Ueno K, Hui CC, Fukuta M, Suzuki Y: Molecular analysis of the deletion mutants in the E-homeotic complex of the silkworm Bombyx mori. Development. 1992, 114: 555-563.PubMedGoogle Scholar
- Abzhanov A, Popadic A, Kaufman TC: Chelicerate Hox genes and the homology of arthropod segments. Evol Dev. 1999, 1: 77-89. 10.1046/j.1525-142x.1999.99014.x.View ArticlePubMedGoogle Scholar
- Telford MJ, Thomas RH: Of mites and zen: expression studies in a chelicerate arthropod confirm zen is a divergent Hox gene. Dev Genes Evol. 1998, 208: 591-594. 10.1007/s004270050219.View ArticlePubMedGoogle Scholar
- Telford MJ: Evidence for the derivation of the Drosophila fushi tarazu gene from a Hox gene orthologous to lophotrochozoan Lox5. Curr Biol. 2000, 10: 349-352.View ArticlePubMedGoogle Scholar
- Schwager EE, Schoppmeier M, Pechmann M, Damen WGM: Duplicated Hox genes in the spider Cupiennius salei. Front Zool. 2007, 4: 1-10. 10.1186/1742-9994-4-1.View ArticleGoogle Scholar
- Damen WGM, Tautz D: A Hox class 3 orthologue from the spider Cupiennius sale i is expressed in a Hox-gene-like fashion. Dev Genes Evol. 1998, 208: 586-590. 10.1007/s004270050218.View ArticlePubMedGoogle Scholar
- Damen WGM, Janssen R, Prpic NM: Pair rule gene orthologs in spider segmentation. Evol Dev. 2005, 7: 618-628. 10.1111/j.1525-142X.2005.05065.x.View ArticlePubMedGoogle Scholar
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