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Expression of two engrailed genes in the embryo of Vargula hilgendorfii (Müller, 1890) (Ostracoda: Myodocopida)

Expression of two engrailed genes in the embryo of Vargula hilgendorfii (Müller, 1890)... Abstract The interpretation of the segmentation and tagmosis in ostracod crustaceans has remained uncertain. Most ostracods have only seven pairs of anterior limbs, including the cephalic ones. The posterior, limbless trunk region usually has no segmental structures, although there are a few exceptions, such as the platycopid ostracods. Therefore, ostracods are regarded, at least morphologically, as the most oligosegmented crustaceans. The engrailed gene has been known as a segmentation marker in arthropods and their relatives. The expression of two engrailed genes, Vh-en-a and Vh-en-b, was examined in the embryos of the myodocopid ostracod Vargula hilgendorfii (Müller, 1890). Both genes were expressed in the five pairs of anterior embryonic limb buds and segments and in the prospective sixth and seventh limb segments. Vh-en-b expression was also observed in the posterior limbless trunk as two stripes, which should be considered vestiges of trunk segmentation. INTRODUCTION Ostracods are tiny crustaceans wholly enclosed by a bivalved carapace. They are divided into two subclasses, Podocopa and Myodocopa, which consist of the orders Podocopida, Platycopida, and Palaeocopida, and Myodocopida and Halocyprida, respectively (Horne et al., 2002). Most ostracods have only seven pairs of anterior limbs, including the cephalic ones, and a further reduction in the number of limbs to six pairs in platycopids and five pairs in cladocopid halocyprids has been observed (Cohen, 1982; Horne et al., 2002). The homology of ostracod limbs with those of other crustaceans is still being debated. The first four pairs of limbs, namely, the antennules, antennae, mandibles, and maxillules, are undoubtedly cephalic. Because it is uncertain whether the fifth limbs are cephalic or thoracic, the last three pairs of limbs are often called the fifth, sixth, and seventh limbs by many ostracod researchers. When the fifth limbs are cephalic, which should be called the maxillae as in other crustaceans, the sixth and seventh limbs are thoracic. If all of these limbs are considered thoracic, however, the ostracods have only four pairs of cephalic limbs, lacking the maxillae (see reviews by Maddocks, 1982, Cohen et al., 1998, and Horne et al., 2002). Ostracods usually have no visible segments in the trunk region posterior to the last limbs (Cohen, 1982; Horne et al., 2002), although there are a few exceptions, such as the platycopids whose trunk consists of 11 segments (Tsukagoshi et al., 2006; Okada et al., 2008). Several comb-like rows of spines, found on the dorsal surface of the trunk of podocopids, were interpreted as relics of segments (Tsukagoshi & Parker, 2000). In contrast, seven pairs of gills, which were considered a part of the limbs, were observed in the trunk of the cylindrolebelidid myodocopids (Vannier et al., 1996). Most ostracods, however, completely or partly lost their trunk segmentation; moreover, they have no boundaries between the thorax and abdomen. Ostracods could therefore be regarded, at least morphologically, as the most oligosegmented crustaceans. The engrailed gene represents a segmentation marker because it is expressed in the posterior compartment of each forming segment in arthropods and their relatives (Patel et al., 1989; Wedeen et al., 1997; Gabriel & Goldstein, 2007; Eriksson et al., 2009, 2013; Franke & Mayer, 2014). The transverse stripes of the engrailed expression corresponding to the segments have been shown in several crustaceans, such as the brine shrimp Artemia franciscana (Branchiopoda, Anostraca) (Manzanares et al., 1993), the parasitic barnacle Sacculina carcini (Cirripedia, Rhizocephala) (Queinnec et al., 1999; Gibert et al., 2000), woodlouse Porcellio scaber (Malacostraca, Isopoda) (Abzhanov & Kaufman, 2000), and crayfish Cherax destructor and Procambarus clarkii (Malacostraca, Decapoda) (Scholtz, 1995; Abzhanov & Kaufman, 2000). Several additional stripes of the posterior unsegmented abdominal region have been observed and regarded as vestigial segments in S. carcini, C. destructor, and P. clarkii, (Scholtz, 1995; Abzhanov & Kaufman, 2000; Gibert et al., 2000). Two duplicated engrailed genes have been cloned from S. carcini, P. scaber, and P. clarkii, and the expression patterns of these genes were found to be slightly different from each other (Abzhanov & Kaufman, 2000; Gibert et al., 2000). In the present study, the engrailed genes were cloned from the myodocopid marine ostracod Vargula hilgendorfii (Müller, 1890), and the expression patterns of the genes were examined by whole-mount in situ hybridization to unravel the segmentation and body plan of the ostracods. MATERIAL AND METHODS Several hundred specimens of Vargula hilgendorfii were collected using a bait trap (Abe et al., 1995) from Awaji Island, Hyogo prefecture and from Tannowa, Osaka prefecture, Japan. They were kept in an aquarium in the laboratory at approximately 24 °C. Adult females lay eggs on the posterior trunk surface surrounded by the bivalved carapace, which serves as a brood pouch. Embryonic development proceeds synchronously in the brood pouch until the juveniles hatch and leave the pouch. Several tens of embryos at the same developmental stage were collected at once from an adult female. Total RNA was extracted from approximately 100 embryos 5-to-8-day old using RNeasy (Qiagen, Hilden, Germany), and 10 μl of the extract was reverse-transcribed using the First Strand Synthesis Kit (Amersham, Buckinghamshire, UK). For amplification of the conserved homeodomain region of engrailed, nested PCR was performed using the ExTaq kit (Takara, Otsu, Japan), using degenerate primer sets, forward WPAWVYC (5′ TGG CCN GCN TGG GTN TAY TG 3′) and reverse MAQ/EGLYNH (5′ TG RTT RTA NAR NCC YTS NGC CAT 3′) in the first round and forward EKRPRTA (5′ GAR AAR MGN CCN MGN ACN GC 3′) and reverse QIKIWFQN (5′ RTT YTG RAA CCA DAT YTT DAT YTG 3′) in the second round. The first PCR program was as follows: initial denaturation at 94 °C for 10 min; five cycles of denaturation at 94 °C for 30 s, annealing at 37 °C for 30 s, and elongation at 72 °C for 1 min; thirty cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s, followed by elongation at 72 °C for 1 min. The second PCR follows the same program, except for the low-temperature annealing step, which was not performed. The PCR products were cloned using the pGEM-T-Easy vector (Promega, Madison, WI, USA). Based on the DNA sequences of several clones of the homeodomain, exact gene-specific primers were designed for 5′ RACE. To produce longer cDNA suitable for making RNA probes, the 5′ RACE System (Invitrogen, Carlsbad, CA, USA) was used, according to the manufacturer’s instructions. The whole embryos were fixed with 10% formalin in 10 mM phosphate buffered saline (PBS, pH 7.4), containing 50 mM EGTA for 2 h at room temperature with gentle shaking. The fixed specimens were rinsed with PBS containing 0.1% Tween 20 (PBT), passed through a graded methanol series in PBT, dehydrated in 100% methanol, and stored at −20°C until use. The egg membrane of the embryo was removed using a fine needle in 75% methanol in PBT, re-hydrated with PBT, and re-fixed with the same fixative for 20 min. After washing three times with 0.1 M TEA for 5 min, the embryos were treated with 2.5 μl/ml acetic anhydrate in 0.1 M TEA for 20 min and washed twice with PBT. The RNA probes for in situ hybridization were prepared using a DIG RNA Labeling Kit (Roche, Basel, Switzerland), according to the manufacturer's instructions. The fixed embryos were transferred to 1:1 PBT/hybridization buffer (50% formamide, 5× SSC, 5× Denhardt’s solution, 1% DMSO, 0.1% Chaps, and 0.1% Tween 20) and then to a neat hybridization buffer at room temperature. After heating for 30 min at 75 °C, salmon sperm DNA and yeast tRNA were added at a concentration of 100 ng/µg. Prehybridization was performed for 30 min before hybridization overnight with a heat-denatured RNA probe at 60 °C. The embryos were washed four times in a hybridization buffer for 1 h, followed by 2× SSC containing 0.1% Chaps four times for 10 min at 60 °C. After washing with PBT, they were blocked for 1 h in PBT containing 0.2% bovine serum albumin and 1% DMSO at room temperature. They were then incubated overnight at 4 °C with 1:3,000-diluted sheep anti-digoxigenin-AP (Roche) and washed in a blocking buffer before performing BCIP/NBT color reaction (Roche). The colored specimens were washed with PBT and observed using a light and fluorescence (UV excitation) microscope after staining with DAPI. RESULTS Two types of partial cDNA fragments homologous to the engrailed gene were cloned from Vargula hilgendorfii embryos, 1185 bp and 1095 bp long, named Vh-en-a and Vh-en-b, respectively. Both fragments consisted of the 5′-UTR and coding region that includes the conservative engrailed-specific domains, Domain I and II, and the partial homeodomain (Fig. 1, Supplementary material Fig. S1). The difference of these domains between Vh-en-a and Vh-en-b was 82.4% in the amino acid sequences. Figure 1. View largeDownload slide Alignment of functional domains, engrailed-specific domains, Domain I (DI) and II (DII), and partial homeodomain, of Vh-en-a and Vh-en-b with those of other crustaceans and insects. Dashes indicate identical amino acids. Numbers in parentheses indicate GenBank numbers. Sc, Sacculina carcini (AF057692, AF171074); Af, Artemia franciscana (X70939); Ps, Porcellio scaber (AF254262, AF254263); Pc, Procambarus clarkii (AF254264, AF254265); Td, Thermobia domestica (AF104006, AF104007); Sa, Schistocerca americana (M29262); Pa, Periplaneta americana (AJ243883, AJ243884); Tc, Tribolium castaneum (S73225); Dm, Drosophila melanogaster (P02836, P05527); Bm, Bombyx mori (P27609, P27610). Figure 1. View largeDownload slide Alignment of functional domains, engrailed-specific domains, Domain I (DI) and II (DII), and partial homeodomain, of Vh-en-a and Vh-en-b with those of other crustaceans and insects. Dashes indicate identical amino acids. Numbers in parentheses indicate GenBank numbers. Sc, Sacculina carcini (AF057692, AF171074); Af, Artemia franciscana (X70939); Ps, Porcellio scaber (AF254262, AF254263); Pc, Procambarus clarkii (AF254264, AF254265); Td, Thermobia domestica (AF104006, AF104007); Sa, Schistocerca americana (M29262); Pa, Periplaneta americana (AJ243883, AJ243884); Tc, Tribolium castaneum (S73225); Dm, Drosophila melanogaster (P02836, P05527); Bm, Bombyx mori (P27609, P27610). A few days after the eggs were released, the germband was formed in the embryos in the blastoderm stage, called “blastula” by Wakayama (2007). Subsequently, the synchronous formation of the segment and limb was observed, without the nauplius stage found in podocopid ostracods and other crustaceans. The first juveniles hatched approximately two weeks later. In embryos 5-to-6-day old, the ocular lobe and anterior five pairs of limb buds (antennules, antennae, mandibles, maxillules, and fifth limbs) appeared simultaneously; however, the sixth and seventh limbs were still not formed until just before hatching. A bivalved carapace started to form in this stage. Vh-en-a expression was observed not only in the posterior parts of the ocular, antennular, antennal, mandibular, maxillular, and fifth limb segments but also in the prospective sixth and seventh limb segments of the posterior trunk region; thus, there was a total of eight engrailed stripes (Fig. 2A). In contrast, Vh-en-b expression was observed in the six stripes from the antennular to the fifth limb segment and in the prospective sixth limb segment (Fig. 2D). Figure 2. View largeDownload slide Expression of Vh-en-a (A–C) and Vh-en-b (D–F) in the embryos of Vargula hilgendorfii 5-to-6-day old (A, D) and 7-to-8-day old (B, C, E, F), examined by in situ hybridization (arrowheads). Asterisks indicate Vh-en-b expression in the vestigial trunk segments. Whole-mount observation by light (left) and fluorescence (right) microscopy after DAPI staining. Lateral views (A, B, D, E) are oriented dorsal to the top and anterior to the left. Ventral views (C, F) are oriented anterior to the left. a1, antennule; a2, antenna; md, mandible; mx1, maxillule; 5th, fifth limb. Scale = 100 µm. This figure is available in colour at Journal of Crustacean Biology online. Figure 2. View largeDownload slide Expression of Vh-en-a (A–C) and Vh-en-b (D–F) in the embryos of Vargula hilgendorfii 5-to-6-day old (A, D) and 7-to-8-day old (B, C, E, F), examined by in situ hybridization (arrowheads). Asterisks indicate Vh-en-b expression in the vestigial trunk segments. Whole-mount observation by light (left) and fluorescence (right) microscopy after DAPI staining. Lateral views (A, B, D, E) are oriented dorsal to the top and anterior to the left. Ventral views (C, F) are oriented anterior to the left. a1, antennule; a2, antenna; md, mandible; mx1, maxillule; 5th, fifth limb. Scale = 100 µm. This figure is available in colour at Journal of Crustacean Biology online. In embryos 7-to-8-day old, the five pairs of anterior limb buds were slightly elongated. The carapace was removed from the specimen shown in Fig. 2B, C, E, F. Vh-en-a was expressed as seven stripes from the antennular to the prospective seventh limb segment and from the antennular to the fifth limb bud, although the ocular expression was unclear because of the overlapping of non-specific staining (Fig. 2B, C). In contrast, Vh-en-b expression was not found in the antennular segment but was still observed from the antennal to the fifth limb bud and segment and the prospective sixth limb segment. In addition, Vh-en-b expression was seen not only in the prospective seventh limb segment but also in the two stripes of the non-segmented and limbless trunk region following it (Fig. 2E, F). DISCUSSION Duplicated engrailed genes have been reported among the malacostracan crustaceans, such as in the isopod Porcellio scaber and the decapod Procambarus clarkii, as well as in the rhizocephalan barnacle (Cirripedia) Sacculina carcini (Abzhanov & Kaufman, 2000; Gibert et al., 2000), whereas only one gene has been found in the branchiopod Artemia franciscana (Manzanares et al., 1993). I cloned two engrailed genes, Vh-en-a and Vh-en-b, from the ostracod Vargula hilgendorfii. The phylogenetic tree of engrailed genes traced by Abzhanov & Kaufman (2000) indicates that duplication of engrailed occurs in each of the malacostracan and barnacle lineages. Recent molecular phylogenetic studies have shown that the ostracods belong to the early branching clade, Oligostraca, and they have further evolved, together with the mystacocarids, branchiurans, and pentastomids (Regier et al., 2010; Oakley et al., 2012). It is probable that Vh-en-a and Vh-en-b are derived in the ostracod lineage, independent of the Malacostraca and Cirripedia, but the possibility that the gene duplication events occurred much earlier cannot be dismissed. As in the engrailed genes of other crustaceans and arthropods, Vh-en-a and Vh-en-b were segmentally expressed in the embryonic stage of limb formation in V. hilgendorfii, whereas there were slight differences between the two genes (see below). The first five pairs of limb buds of V. hilgendorfii embryos were formed all at once, as described by Wakayama (2007), and both genes were expressed not only in each of the segments in which these limb buds appear, but also in the prospective sixth and seventh limb segments. Because there is no indication for an additional engrailed stripe between the fourth and fifth limb buds, the five pairs of anterior limbs are considered cephalic and the fifth limbs are the maxillae, as in other crustaceans. Nine engrailed stripes have been detected in the embryonic pleon of the crayfish C. destructor; the anterior six stripes corresponded to the six pleonic segments of adults, whereas the stripes seven to nine appeared in the posterior non-segmented portion of the pleon, which were interpreted as vestiges of ancestral segments (Scholtz, 1995). Similar results have also been reported for another species of crayfish, P. clarkii (Abzhanov & Kaufman, 2000). In contrast, the duplicated engrailed genes of the parasitic barnacle S. carcini, en.a and en.b, have been found to be expressed behind the thorax of naupliar and cyprid larvae, although there were no abdominal segments in both stages (Queinnec et al., 1999; Gibert et al., 2000). In particular, the five fine stripes of en.b observed in the cyprid larvae of S. carcini were considered vestiges of abdominal segments; the number of these stripes is the same as that of the five abdominal segments retained in Ascothoracida, which are thought to preserve the ancestral condition of Thecostraca (Gibert et al., 2000). In the present study of V. hilgendorfii, Vh-en-b was expressed not only in the embryonic and future limb buds but also in the posterior non-segmented and limbless trunk region as two stripes. The last two stripes of Vh-en-b should be regarded as vestigial segments, as in the cases of crayfishes (Scholtz, 1995; Abzhanov & Kaufman, 2000) and the cirriped S. carcini (Gibert et al., 2000). Although the number of these stripes is very few in comparison with the 11 trunk segments in the platycopid ostracods (Tsukagoshi et al., 2006; Okada et al., 2008), this could be considered the derived character in V. hilgendorfii, remarkably decreasing the segmental structures during the ostracod evolution. SUPPLEMENTARY MATERIAL Supplementary material is available at Journal of Crustacean Biology online. S1 Figure. Partial nucleotide and deduced amino acid sequences of Vh-en-a (LC319788) and Vh-en-b (LC319789). Specific domains of engrailed, Domain I (DI) and II (DII), are underlined. Homeodomain is boxed. ACKNOWLEDGEMENTS Special thanks are due to two anonymous reviewers for their critical reviews of the manuscript. This study was funded in part by Grant-in-aid for Young Scientists (B) (16770065 and 18770064) of the Japan Society for the Promotion of Science. REFERENCES Abe, K., Vannier, J.M.C. & Tahara Y. 1995. Bioluminescence of Vargula hilgendorfii (Ostracoda, Myodocopida): its ecological significance and effects of a heart. In: Ostracoda and biostratigraphy  ( J. Ríha, ed.), pp. 11– 18. Balkema, Rotterdam, The Netherlands. Abzhanov, A. & Kaufman, T.C. 2000. Evolution of distinct expression patterns for engrailed paralogues in higher crustaceans (Malacostraca). Development, Genes and Evolution , 210: 493– 506. Google Scholar CrossRef Search ADS   Cohen, A.C. 1982. Ostracoda. Synopsis and classification of living organisms 2  ( S. Parker ed.), pp. 181– 202. McGraw-Hill, New York. Cohen, A.C., Martin, J.W. & Kornicker, L.S. 1998. Homology of Holocene ostracode biramous appendages with those of other crustaceans: the protopod, epipod, exopod and endopod. Lethaia , 31: 251– 265. Google Scholar CrossRef Search ADS   Eriksson, B.J., Tait, N.N., Budd, G.E. & Akam, M. 2009. 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Published by Oxford University Press on behalf of The Crustacean Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Crustacean Biology Oxford University Press

Expression of two engrailed genes in the embryo of Vargula hilgendorfii (Müller, 1890) (Ostracoda: Myodocopida)

Ikuta, Kyosuke
The Journal of Crustacean Biology , Volume 38 (1) – Jan 1, 2018
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Abstract

Abstract The interpretation of the segmentation and tagmosis in ostracod crustaceans has remained uncertain. Most ostracods have only seven pairs of anterior limbs, including the cephalic ones. The posterior, limbless trunk region usually has no segmental structures, although there are a few exceptions, such as the platycopid ostracods. Therefore, ostracods are regarded, at least morphologically, as the most oligosegmented crustaceans. The engrailed gene has been known as a segmentation marker in arthropods and their relatives. The expression of two engrailed genes, Vh-en-a and Vh-en-b, was examined in the embryos of the myodocopid ostracod Vargula hilgendorfii (Müller, 1890). Both genes were expressed in the five pairs of anterior embryonic limb buds and segments and in the prospective sixth and seventh limb segments. Vh-en-b expression was also observed in the posterior limbless trunk as two stripes, which should be considered vestiges of trunk segmentation. INTRODUCTION Ostracods are tiny crustaceans wholly enclosed by a bivalved carapace. They are divided into two subclasses, Podocopa and Myodocopa, which consist of the orders Podocopida, Platycopida, and Palaeocopida, and Myodocopida and Halocyprida, respectively (Horne et al., 2002). Most ostracods have only seven pairs of anterior limbs, including the cephalic ones, and a further reduction in the number of limbs to six pairs in platycopids and five pairs in cladocopid halocyprids has been observed (Cohen, 1982; Horne et al., 2002). The homology of ostracod limbs with those of other crustaceans is still being debated. The first four pairs of limbs, namely, the antennules, antennae, mandibles, and maxillules, are undoubtedly cephalic. Because it is uncertain whether the fifth limbs are cephalic or thoracic, the last three pairs of limbs are often called the fifth, sixth, and seventh limbs by many ostracod researchers. When the fifth limbs are cephalic, which should be called the maxillae as in other crustaceans, the sixth and seventh limbs are thoracic. If all of these limbs are considered thoracic, however, the ostracods have only four pairs of cephalic limbs, lacking the maxillae (see reviews by Maddocks, 1982, Cohen et al., 1998, and Horne et al., 2002). Ostracods usually have no visible segments in the trunk region posterior to the last limbs (Cohen, 1982; Horne et al., 2002), although there are a few exceptions, such as the platycopids whose trunk consists of 11 segments (Tsukagoshi et al., 2006; Okada et al., 2008). Several comb-like rows of spines, found on the dorsal surface of the trunk of podocopids, were interpreted as relics of segments (Tsukagoshi & Parker, 2000). In contrast, seven pairs of gills, which were considered a part of the limbs, were observed in the trunk of the cylindrolebelidid myodocopids (Vannier et al., 1996). Most ostracods, however, completely or partly lost their trunk segmentation; moreover, they have no boundaries between the thorax and abdomen. Ostracods could therefore be regarded, at least morphologically, as the most oligosegmented crustaceans. The engrailed gene represents a segmentation marker because it is expressed in the posterior compartment of each forming segment in arthropods and their relatives (Patel et al., 1989; Wedeen et al., 1997; Gabriel & Goldstein, 2007; Eriksson et al., 2009, 2013; Franke & Mayer, 2014). The transverse stripes of the engrailed expression corresponding to the segments have been shown in several crustaceans, such as the brine shrimp Artemia franciscana (Branchiopoda, Anostraca) (Manzanares et al., 1993), the parasitic barnacle Sacculina carcini (Cirripedia, Rhizocephala) (Queinnec et al., 1999; Gibert et al., 2000), woodlouse Porcellio scaber (Malacostraca, Isopoda) (Abzhanov & Kaufman, 2000), and crayfish Cherax destructor and Procambarus clarkii (Malacostraca, Decapoda) (Scholtz, 1995; Abzhanov & Kaufman, 2000). Several additional stripes of the posterior unsegmented abdominal region have been observed and regarded as vestigial segments in S. carcini, C. destructor, and P. clarkii, (Scholtz, 1995; Abzhanov & Kaufman, 2000; Gibert et al., 2000). Two duplicated engrailed genes have been cloned from S. carcini, P. scaber, and P. clarkii, and the expression patterns of these genes were found to be slightly different from each other (Abzhanov & Kaufman, 2000; Gibert et al., 2000). In the present study, the engrailed genes were cloned from the myodocopid marine ostracod Vargula hilgendorfii (Müller, 1890), and the expression patterns of the genes were examined by whole-mount in situ hybridization to unravel the segmentation and body plan of the ostracods. MATERIAL AND METHODS Several hundred specimens of Vargula hilgendorfii were collected using a bait trap (Abe et al., 1995) from Awaji Island, Hyogo prefecture and from Tannowa, Osaka prefecture, Japan. They were kept in an aquarium in the laboratory at approximately 24 °C. Adult females lay eggs on the posterior trunk surface surrounded by the bivalved carapace, which serves as a brood pouch. Embryonic development proceeds synchronously in the brood pouch until the juveniles hatch and leave the pouch. Several tens of embryos at the same developmental stage were collected at once from an adult female. Total RNA was extracted from approximately 100 embryos 5-to-8-day old using RNeasy (Qiagen, Hilden, Germany), and 10 μl of the extract was reverse-transcribed using the First Strand Synthesis Kit (Amersham, Buckinghamshire, UK). For amplification of the conserved homeodomain region of engrailed, nested PCR was performed using the ExTaq kit (Takara, Otsu, Japan), using degenerate primer sets, forward WPAWVYC (5′ TGG CCN GCN TGG GTN TAY TG 3′) and reverse MAQ/EGLYNH (5′ TG RTT RTA NAR NCC YTS NGC CAT 3′) in the first round and forward EKRPRTA (5′ GAR AAR MGN CCN MGN ACN GC 3′) and reverse QIKIWFQN (5′ RTT YTG RAA CCA DAT YTT DAT YTG 3′) in the second round. The first PCR program was as follows: initial denaturation at 94 °C for 10 min; five cycles of denaturation at 94 °C for 30 s, annealing at 37 °C for 30 s, and elongation at 72 °C for 1 min; thirty cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s, followed by elongation at 72 °C for 1 min. The second PCR follows the same program, except for the low-temperature annealing step, which was not performed. The PCR products were cloned using the pGEM-T-Easy vector (Promega, Madison, WI, USA). Based on the DNA sequences of several clones of the homeodomain, exact gene-specific primers were designed for 5′ RACE. To produce longer cDNA suitable for making RNA probes, the 5′ RACE System (Invitrogen, Carlsbad, CA, USA) was used, according to the manufacturer’s instructions. The whole embryos were fixed with 10% formalin in 10 mM phosphate buffered saline (PBS, pH 7.4), containing 50 mM EGTA for 2 h at room temperature with gentle shaking. The fixed specimens were rinsed with PBS containing 0.1% Tween 20 (PBT), passed through a graded methanol series in PBT, dehydrated in 100% methanol, and stored at −20°C until use. The egg membrane of the embryo was removed using a fine needle in 75% methanol in PBT, re-hydrated with PBT, and re-fixed with the same fixative for 20 min. After washing three times with 0.1 M TEA for 5 min, the embryos were treated with 2.5 μl/ml acetic anhydrate in 0.1 M TEA for 20 min and washed twice with PBT. The RNA probes for in situ hybridization were prepared using a DIG RNA Labeling Kit (Roche, Basel, Switzerland), according to the manufacturer's instructions. The fixed embryos were transferred to 1:1 PBT/hybridization buffer (50% formamide, 5× SSC, 5× Denhardt’s solution, 1% DMSO, 0.1% Chaps, and 0.1% Tween 20) and then to a neat hybridization buffer at room temperature. After heating for 30 min at 75 °C, salmon sperm DNA and yeast tRNA were added at a concentration of 100 ng/µg. Prehybridization was performed for 30 min before hybridization overnight with a heat-denatured RNA probe at 60 °C. The embryos were washed four times in a hybridization buffer for 1 h, followed by 2× SSC containing 0.1% Chaps four times for 10 min at 60 °C. After washing with PBT, they were blocked for 1 h in PBT containing 0.2% bovine serum albumin and 1% DMSO at room temperature. They were then incubated overnight at 4 °C with 1:3,000-diluted sheep anti-digoxigenin-AP (Roche) and washed in a blocking buffer before performing BCIP/NBT color reaction (Roche). The colored specimens were washed with PBT and observed using a light and fluorescence (UV excitation) microscope after staining with DAPI. RESULTS Two types of partial cDNA fragments homologous to the engrailed gene were cloned from Vargula hilgendorfii embryos, 1185 bp and 1095 bp long, named Vh-en-a and Vh-en-b, respectively. Both fragments consisted of the 5′-UTR and coding region that includes the conservative engrailed-specific domains, Domain I and II, and the partial homeodomain (Fig. 1, Supplementary material Fig. S1). The difference of these domains between Vh-en-a and Vh-en-b was 82.4% in the amino acid sequences. Figure 1. View largeDownload slide Alignment of functional domains, engrailed-specific domains, Domain I (DI) and II (DII), and partial homeodomain, of Vh-en-a and Vh-en-b with those of other crustaceans and insects. Dashes indicate identical amino acids. Numbers in parentheses indicate GenBank numbers. Sc, Sacculina carcini (AF057692, AF171074); Af, Artemia franciscana (X70939); Ps, Porcellio scaber (AF254262, AF254263); Pc, Procambarus clarkii (AF254264, AF254265); Td, Thermobia domestica (AF104006, AF104007); Sa, Schistocerca americana (M29262); Pa, Periplaneta americana (AJ243883, AJ243884); Tc, Tribolium castaneum (S73225); Dm, Drosophila melanogaster (P02836, P05527); Bm, Bombyx mori (P27609, P27610). Figure 1. View largeDownload slide Alignment of functional domains, engrailed-specific domains, Domain I (DI) and II (DII), and partial homeodomain, of Vh-en-a and Vh-en-b with those of other crustaceans and insects. Dashes indicate identical amino acids. Numbers in parentheses indicate GenBank numbers. Sc, Sacculina carcini (AF057692, AF171074); Af, Artemia franciscana (X70939); Ps, Porcellio scaber (AF254262, AF254263); Pc, Procambarus clarkii (AF254264, AF254265); Td, Thermobia domestica (AF104006, AF104007); Sa, Schistocerca americana (M29262); Pa, Periplaneta americana (AJ243883, AJ243884); Tc, Tribolium castaneum (S73225); Dm, Drosophila melanogaster (P02836, P05527); Bm, Bombyx mori (P27609, P27610). A few days after the eggs were released, the germband was formed in the embryos in the blastoderm stage, called “blastula” by Wakayama (2007). Subsequently, the synchronous formation of the segment and limb was observed, without the nauplius stage found in podocopid ostracods and other crustaceans. The first juveniles hatched approximately two weeks later. In embryos 5-to-6-day old, the ocular lobe and anterior five pairs of limb buds (antennules, antennae, mandibles, maxillules, and fifth limbs) appeared simultaneously; however, the sixth and seventh limbs were still not formed until just before hatching. A bivalved carapace started to form in this stage. Vh-en-a expression was observed not only in the posterior parts of the ocular, antennular, antennal, mandibular, maxillular, and fifth limb segments but also in the prospective sixth and seventh limb segments of the posterior trunk region; thus, there was a total of eight engrailed stripes (Fig. 2A). In contrast, Vh-en-b expression was observed in the six stripes from the antennular to the fifth limb segment and in the prospective sixth limb segment (Fig. 2D). Figure 2. View largeDownload slide Expression of Vh-en-a (A–C) and Vh-en-b (D–F) in the embryos of Vargula hilgendorfii 5-to-6-day old (A, D) and 7-to-8-day old (B, C, E, F), examined by in situ hybridization (arrowheads). Asterisks indicate Vh-en-b expression in the vestigial trunk segments. Whole-mount observation by light (left) and fluorescence (right) microscopy after DAPI staining. Lateral views (A, B, D, E) are oriented dorsal to the top and anterior to the left. Ventral views (C, F) are oriented anterior to the left. a1, antennule; a2, antenna; md, mandible; mx1, maxillule; 5th, fifth limb. Scale = 100 µm. This figure is available in colour at Journal of Crustacean Biology online. Figure 2. View largeDownload slide Expression of Vh-en-a (A–C) and Vh-en-b (D–F) in the embryos of Vargula hilgendorfii 5-to-6-day old (A, D) and 7-to-8-day old (B, C, E, F), examined by in situ hybridization (arrowheads). Asterisks indicate Vh-en-b expression in the vestigial trunk segments. Whole-mount observation by light (left) and fluorescence (right) microscopy after DAPI staining. Lateral views (A, B, D, E) are oriented dorsal to the top and anterior to the left. Ventral views (C, F) are oriented anterior to the left. a1, antennule; a2, antenna; md, mandible; mx1, maxillule; 5th, fifth limb. Scale = 100 µm. This figure is available in colour at Journal of Crustacean Biology online. In embryos 7-to-8-day old, the five pairs of anterior limb buds were slightly elongated. The carapace was removed from the specimen shown in Fig. 2B, C, E, F. Vh-en-a was expressed as seven stripes from the antennular to the prospective seventh limb segment and from the antennular to the fifth limb bud, although the ocular expression was unclear because of the overlapping of non-specific staining (Fig. 2B, C). In contrast, Vh-en-b expression was not found in the antennular segment but was still observed from the antennal to the fifth limb bud and segment and the prospective sixth limb segment. In addition, Vh-en-b expression was seen not only in the prospective seventh limb segment but also in the two stripes of the non-segmented and limbless trunk region following it (Fig. 2E, F). DISCUSSION Duplicated engrailed genes have been reported among the malacostracan crustaceans, such as in the isopod Porcellio scaber and the decapod Procambarus clarkii, as well as in the rhizocephalan barnacle (Cirripedia) Sacculina carcini (Abzhanov & Kaufman, 2000; Gibert et al., 2000), whereas only one gene has been found in the branchiopod Artemia franciscana (Manzanares et al., 1993). I cloned two engrailed genes, Vh-en-a and Vh-en-b, from the ostracod Vargula hilgendorfii. The phylogenetic tree of engrailed genes traced by Abzhanov & Kaufman (2000) indicates that duplication of engrailed occurs in each of the malacostracan and barnacle lineages. Recent molecular phylogenetic studies have shown that the ostracods belong to the early branching clade, Oligostraca, and they have further evolved, together with the mystacocarids, branchiurans, and pentastomids (Regier et al., 2010; Oakley et al., 2012). It is probable that Vh-en-a and Vh-en-b are derived in the ostracod lineage, independent of the Malacostraca and Cirripedia, but the possibility that the gene duplication events occurred much earlier cannot be dismissed. As in the engrailed genes of other crustaceans and arthropods, Vh-en-a and Vh-en-b were segmentally expressed in the embryonic stage of limb formation in V. hilgendorfii, whereas there were slight differences between the two genes (see below). The first five pairs of limb buds of V. hilgendorfii embryos were formed all at once, as described by Wakayama (2007), and both genes were expressed not only in each of the segments in which these limb buds appear, but also in the prospective sixth and seventh limb segments. Because there is no indication for an additional engrailed stripe between the fourth and fifth limb buds, the five pairs of anterior limbs are considered cephalic and the fifth limbs are the maxillae, as in other crustaceans. Nine engrailed stripes have been detected in the embryonic pleon of the crayfish C. destructor; the anterior six stripes corresponded to the six pleonic segments of adults, whereas the stripes seven to nine appeared in the posterior non-segmented portion of the pleon, which were interpreted as vestiges of ancestral segments (Scholtz, 1995). Similar results have also been reported for another species of crayfish, P. clarkii (Abzhanov & Kaufman, 2000). In contrast, the duplicated engrailed genes of the parasitic barnacle S. carcini, en.a and en.b, have been found to be expressed behind the thorax of naupliar and cyprid larvae, although there were no abdominal segments in both stages (Queinnec et al., 1999; Gibert et al., 2000). In particular, the five fine stripes of en.b observed in the cyprid larvae of S. carcini were considered vestiges of abdominal segments; the number of these stripes is the same as that of the five abdominal segments retained in Ascothoracida, which are thought to preserve the ancestral condition of Thecostraca (Gibert et al., 2000). In the present study of V. hilgendorfii, Vh-en-b was expressed not only in the embryonic and future limb buds but also in the posterior non-segmented and limbless trunk region as two stripes. The last two stripes of Vh-en-b should be regarded as vestigial segments, as in the cases of crayfishes (Scholtz, 1995; Abzhanov & Kaufman, 2000) and the cirriped S. carcini (Gibert et al., 2000). Although the number of these stripes is very few in comparison with the 11 trunk segments in the platycopid ostracods (Tsukagoshi et al., 2006; Okada et al., 2008), this could be considered the derived character in V. hilgendorfii, remarkably decreasing the segmental structures during the ostracod evolution. SUPPLEMENTARY MATERIAL Supplementary material is available at Journal of Crustacean Biology online. S1 Figure. Partial nucleotide and deduced amino acid sequences of Vh-en-a (LC319788) and Vh-en-b (LC319789). Specific domains of engrailed, Domain I (DI) and II (DII), are underlined. Homeodomain is boxed. ACKNOWLEDGEMENTS Special thanks are due to two anonymous reviewers for their critical reviews of the manuscript. This study was funded in part by Grant-in-aid for Young Scientists (B) (16770065 and 18770064) of the Japan Society for the Promotion of Science. REFERENCES Abe, K., Vannier, J.M.C. & Tahara Y. 1995. 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The Journal of Crustacean Biology – Oxford University Press

Published: Jan 1, 2018

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