C 2013 Wiley Periodicals, Inc. V
genesis 51:803–818 (2013)
ARTICLE
The Embryogenesis of the Tick Rhipicephalus (Boophilus) microplus: The Establishment of a New Chelicerate Model System ria Tobias Santos,1 Lupis Ribeiro,1,2 Amanda Fraga,1,2 Cıntia Monteiro de Barros,2,3 Vito jo,4,5 Eldo Campos,1,2,4 Jorge Moraes,1,2,4 Marcio Ribeiro Fontenele,4,5 Helena Marcolla Arau 1 2,4,6 1,2,4 Natalia Martins Feitosa, Carlos Logullo, and Rodrigo Nunes da Fonseca * 1
rio Integrado de Bioquımica Hatisaburo Masuda (LIBHM), 1 - Nu cio cleo em Ecologia e Desenvolvimento So Laborato NUPEM, Universidade Federal do Rio de Janeiro (UFRJ-Campus Macae ), Brazil Ambiental de Macae
2
s-Graduac¸a ~o em Produtos Bioativos e Biocie ^ncias (PPGPRODBIO), UFRJ, Macae , Rio de Janeiro, Brazil Programa de Po
3
rio Integrado de Morfologia, Nu cio-Ambiental de Macae (NUPEM), UFRJ, cleo em Ecologia e Desenvolvimento So Laborato , Rio de Janeiro, Brazil Macae
4
^ncia e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil Instituto Nacional de Cie
5
rio de Biologia Molecular do Desenvolvimento, Instituto de Cie ^ncias Biome dicas, UFRJ, Rio de Janeiro, Brazil Laborato
6
rio de Quımica e Func¸a ~o de Proteınas e Peptıdeos, Universidade Estadual Norte Fluminense, Campos dos Laborato Goytacazes, RJ, Rio de Janeiro, Brazil
Received 1 April 2013; Revised 3 September 2013; Accepted 26 September 2013
Summary: Chelicerates, which include spiders, ticks, mites, scorpions, and horseshoe crabs, are members of the phylum Arthropoda. In recent years, several molecular experimental studies of chelicerates have examined the embryology of spiders; however, the embryology of other groups, such as ticks (Acari: Parasitiformes), has been largely neglected. Ticks and mites are believed to constitute a monophyletic group, the Acari. Due to their bloodsucking activities, ticks are also known to be vectors of several diseases. In this study, we analyzed the embryonic development of the cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). First, we developed an embryonic staging system consisting of 14 embryonic stages. Second, histological analysis and antibody staining unexpectedly revealed the presence of a population of tick cells with similar characteristics to the spider cumulus. Cumulus cell populations also exist in other chelicerates; these cells are responsible for the breaking of radial symmetry through bone morphogenetic protein signaling. Third, it was determined that the posterior (opisthosomal) embryonic region of R. microplus is segmented. Finally, we identified the presence of a transient ventral midline furrow and the formation and regression of a fourth leg pair; these features may be regarded as hallmarks of late tick embryogenesis. Importantly, most of the aforemen-
tioned features are absent from mite embryos, suggesting that mites and ticks do not constitute a monophyletic group or that mites have lost these features. Taken together, our findings provide fundamental common ground for improving knowledge regarding tick embryonic development, thereby facilitating the establishment of a new chelicerate model system. genesis 51:803–818. C 2013 Wiley Periodicals, Inc. V
Additional Supporting Information may be found in the online version of this article. * Correspondence to: Rodrigo Nunes da Fonseca, Laborat orio Integrado de Bioquımica Hatisaburo Masuda (LIBHM), NUPEM, UFRJ-Macae, Av. S~ao Jose do Barreto, 764, N ucleo de Pesquisas Ecol ogicas de Maca e (NUPEM)—Universidade Federal do Rio de Janeiro (UFRJ), Campus Macae E-mail:
[email protected] Contract grant sponsors: Conselho Nacional de Pesquisa Cientıfica (CNPQ), PRONEX, Instituto Nacional de Ci^encia e Tecnologia em Entomologia Molecular (INCT-EM), and Fundac¸~ao de Amparo a Pesquisa Carlos Chagas Filho (FAPERJ) (RNdF lab); Contract grant sponsor: Conselho de Aperfeic¸oamento de Ensino Superior (CAPES) in the PPG-PRODBIOUFRJ-Macae (L.R. and A.F.) Published online 26 October 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/dvg.22717
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Key words: evo-devo; evolution; development; Toll; BMP/ Dpp; cellularization
BACKGROUND Arthropods are by far the most widely specious phylum of the animal kingdom, and representatives of this phylum are present in nearly all habitats (reviewed in Giribet and Edgecombe, 2012). The subphylum Chelicerata, which includes spiders, ticks, mites, scorpions, and horseshoe crabs, is one of the major subdivisions of the phylum Arthropoda. This subphylum is named for chelicerae, pointed appendages that grasp food and fulfill the role played by mandibles in most other arthropods. Members of Chelicerata typically have adult bodies that may be divided into two parts, or tagmata: the prosoma and the opisthosoma. One pair of chelicerae, one pair of pedipalps and four pairs of locomotory legs are characteristics of organisms in this subphylum (Anderson, 1973). However, Acari (mites and ticks) do not completely conform to this description in that Acari larvae hatch with three rather than four pairs of legs. Recent molecular studies have placed chelicerates at a basal position in arthropod phylogeny; in particular, chelicerates are either grouped together with myriapods into the clade Myriochelata (Myriapoda 1 Chelicerata) or regarded as a sister group of the clade Mandibulata (which includes Myriapoda, Crustacea, and Hexapoda) (Edgecombe, 2010; Koenemann et al., 2010; Regier et al., 2008; Rota-Stabelli et al., 2011). This basal position of chelicerates in arthropod phylogeny has stimulated the study of the evolution of developmental mechanisms (evo-devo) in chelicerates, particularly spiders and mites. Among spiders, the central American wandering spider, Cupiennius salei, and the common house spider, Parasteatoda (Achaearanea) tepidariorum, are established model organisms (reviewed in Hilbrant et al., 2012; McGregor et al., 2008; Oda and Akiyama-Oda, 2008). Among the Acari, which account for approximately half of the described arachnid diversity, the spider mite Tetranychus urticae has recently been developed as a model system (Grbic et al., 2007). In particular, the genome of this species has been sequenced (Grbic et al., 2011), and several techniques, such as cell ablation, in situ hybridization, and parental RNAi (pRNAi), have been devised for T. urticae experiments. Moreover, clear prospects exist for performing genetic screens in this species. The Acari have traditionally been regarded as a monophyletic group consisting of two distinct lineages, the Actinotrichida (mites-Acariformes) and the Anactinotrichida (ticks-Parasitiformes) (Walter et al., 1996). However, the monophyly of Acari has been questioned by Dunlop and Alberti (2008) in a recent review and by Regier et al. (2010) in a molecular phylogenetic
study. Thus, the Acari may be either a monophyletic or a paraphyletic group (Fig. 1). Importantly, embryogenesis has been used to argue in favor of the monophyletic origin of ticks and mites (Lindquist, 1984), despite the fact that tick embryogenesis has been poorly described (reviewed in Laumann et al., 2010). Studies of tick embryology have been conducted since the 19th century (Bonnet, 1907; Brucker, 1900; Wagner, 1894a,b), although few articles have been published regarding this topic during the prior 50 years. Thus, evo-devo studies of arthropods would benefit from the establishment of a tick model organism. Contradictory interpretations persist regarding key events during tick embryogenesis, particularly cleavage patterns (Aeschlimann, 1958; Campos et al., 2006; Campos et al., 2007; Fagotto et al., 1988; Laumann et al., 2010). Arthropod cleavage patterns have generally been described as superficial by classical texts (Gilbert, 2013), which have clearly been influenced by studies of the model insect Drosophila melanogaster. However, investigations have indicated that during the first four rounds of cleavage, total (holoblastic) cleavage occurs in mites (Dearden et al., 2002), whereas syncytial cleavage (modified total cleavage) occurs in spiders (Kanayama et al., 2010; Suzuki and Konda, 1995). Thus, the acquisition of new data regarding early cleavage patterns in other chelicerate groups, such as ticks, is important to obtaining a greater understanding of the evolution of cleavage patterns among arthropods. Another issue of particular importance is the connection between patterning events during oogenesis and axis formation during embryogenesis. In insects, the initial cues required to determine the anteroposterior (AP) and dorsoventral (DV) axes are established during oogenesis. In the fruit fly D. melanogaster and the beetle Tribolium castaneum, the DV axis is established through maternal interactions among the epidermal growth factor, Toll and bone morphogenetic protein (BMP) pathways (Araujo et al., 2011; Lynch and Roth, 2011; Lynch et al., 2010). In contrast, the AP and DV axes in spiders are established by zygotic gene interactions followed by the migration of the cumulus, a specialized group of cells (Akiyama-Oda and Oda, 2006, 2010; Campos-Ortega and Hartenstein, 1985). In spiders, cumulus mesenchymal cell migration requires the secreted factor decapentaplegic (Dpp), a BMP homolog, and this movement is essential for the determination of the DV axis (Akiyama-Oda and Oda, 2003, 2006). Other important aspects of embryonic specification, such as gastrulation, segmentation, and gut formation, have not been addressed in ticks due to the lack of a suitable model species. To bridge this knowledge gap, we decided to investigate the embryonic development of the southern cattle tick, Rhipicephalus (Boophilus) microplus. R. microplus is an important vector of several diseases; in particular, these diseases include babesiosis, a hemolytic disease produced by a genus of protozoan
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FIG. 1. A schematic representation of the two current hypotheses regarding Acari evolution. (a) The monophyletic and (b) paraphyletic origin hypotheses. Chelicerate species for which embryonic development has been molecularly analyzed are indicated on the right side of each panel. R. microplus—Rhipicephalus microplus.
blood parasites, and anaplasmosis, a disease caused by rickettsial parasites of ruminants, Anaplasma spp. (Suarez and Noh, 2011). The blood-sucking activities of R. microplus also cause weight loss and diminished milk production among cattle (Jonsson et al., 1998). R. microplus is a one-host tick; in other words, all life stages of this organism occur on a single host species. The eggs hatch in outdoor environments, and the larvae crawl up grass or other plants to find a host. Several groups have recently reported that the injection of partially engorged R. microplus females with double-stranded (ds) RNA (Fabres et al., 2010; Kocan et al., 2007; Kocan et al., 2011) can cause embryonic lethality in the next generation, suggesting that a parental RNAi (pRNAi) effect occurs in R. microplus that is similar to pRNAi effects that have previously been observed in other species (Bucher et al., 2002). R. microplus is a promising model system for evo-devo studies in ticks due to not only this availability of pRNAi as a potential tool but also the existence of expressed sequence tag libraries for several R. microplus developmental stages and the economic importance of this species. In this study, we establish an embryonic staging system for R. microplus and utilize this knowledge to characterize four important processes during tick development: cumulus formation, transient ventral midline furrow, opisthosomal patterning, and fourth leg regression. METHODS Tick Maintenance Ticks were obtained from a colony at the Universidade Federal do Rio Grande do Sul, Brasil in accordance with previously described procedures (Parizi et al., 2009). These ticks, which were free of Babesia spp., were maintained on calves in an area that lacked other tick species. Completely engorged females were collected. To ensure appropriate egg collection, the females were fixed in metal supports with tape and maintained in an incubator at a constant temperature
(28 C) and humidity level (80%). Oviposition began several days after the females were collected. During oviposition, eggs were collected daily and kept in petri dishes within the same incubator. This collection scheme was utilized to obtain eggs over the course of approximately 1 week. Embryonic Fixation Approximately 100 mg of eggs were separated for fixation and added to a small basket with a nylon membrane (100 mm). The eggs were then washed for 8 min with a solution containing 1.5% sodium hypochlorite and 5% sodium carbonate. Commercial bleach may also produce satisfactory results because bleach treatment causes chorion degradation. Subsequently, the eggs were repeatedly washed with distilled water and transferred to a 2 mL microtube with a fine paintbrush. Approximately 1.7 mL of distilled water was added to the microtube, which was then heated for 2 min at 90 C in a water bath. Immediately following this heating procedure, the microtube containing the eggs was left on ice for 2 min, causing the cracking of the eggshells (ESs). Shortly thereafter, the eggs were removed from the microtube with a paintbrush and added to a fixation solution containing heptane and 4% paraformaldehyde in phosphate-buffered saline (PBS; 1:1). The eggs remained suspended between the phases of the fixation solution, which was mixed at 100 rpm for 40 min or longer at room temperature. The lower phase containing the paraformaldehyde was then removed, one part methanol (100%) was added to the solution, and the flask was vigorously shaken for 2 min. Eggs that had lost their shells sank toward the bottom of the flask, whereas eggs that retained their shells remained at the interface between the phases of the solution. The eggs at the bottom of the flask were collected in a 1.5 mL microtube, washed three times with methanol and stored at 220 C. The above procedure was utilized for the embryonic fixation of embryos at 3 days or more after egg laying (AEL); however, younger embryos (1–2
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days AEL) were more sensitive to bleach treatment and were, therefore, exposed to sodium hypochlorite for only 2–4 min. Nuclear DAPI Staining (40 ,6-diamidino-2phenylindole) and Antibody Staining After eggs were fixed as described above, they were removed from storage at 220 C and gradually transferred to PBST (phosphate buffer solution (PBS) plus 0.1% Tween-20). Shortly thereafter, the embryos were washed three times with PBST to remove all traces of methanol. To block nonspecific epitopes, the embryos were incubated twice (for 20 min per incubation) with Western Block Solution (Roche) diluted 1:5 in PBST. An antirabbit phosphorylated Mothers against dpp (Mad) antibody was purchased from Cell Signaling, and an antirabbit bcatenin monoclonal antibody (C2206) was obtained from Sigma. Staining with Toll polyclonal antibody (Santa Cruz Biotechnology -dY-300: sc-34010, Santa Cruz Biotechnology, Dallas, Texas 75220 U.S.A.) an antibody recognizing phosphorylated tyrosine (pTyr) and 40 ,6-diamidino-2-phenylindole (DAPI) was performed as previously described (Nunes da Fonseca et al., 2008). A table with detailed information regarding the antibodies utilized in this study has been provided in the Supporting Information section of this article. Finally, the embryos were mounted on slides and observed with a fluorescent microscope (Olympus BX2) or a confocal microscope (Leica SP5). Fuchsin Staining Eggs that had been fixed as described above were gradually transferred to PBST, and the fuchsin staining method developed by Wigand et al. (1998) for T. castaneum beetle embryos was applied to these eggs. The stained eggs were sectioned as previously described (Roth et al., 1989) and counterstained with toluidine blue. Cuticle Preparation Exoskeleton preparation was performed as described for Drosophila (Stern and Sucena, 2011). In particular, 100 ml of Hoyer’s solution (Wieschaus and NussleinVolhard, 1986) was added to newly hatched ticks (at 1 day after eclosion), and these ticks were incubated overnight at 60 C. Specimens were collected and photographed under dark-field or differential interference contrast microscopy (Nikon 80i). Images were processed using Adobe Photoshop CS4.
ment was visualized from extremely early stages until hatching, which typically occurs 21 days after egg lay (AEL at 28 C and 80% humidity (Fig. 3). Eggs, which were constantly laid, could readily be obtained from engorged females and were maintained in a simple air incubator. During early embryogenesis, freshly laid eggs were oval shaped (405 mm 3 295 mm) and lacked any obvious AP or DV orientation (Fig. 2f). The examined eggs exhibited a light brown tint during the first day AEL (Fig. 2b) but became darker within 24 h (Fig. 2c). The eggs became more opaque after chorion removal by bleaching. However, current fixation protocols resulted in low levels of shell removal among the examined eggs (Campos et al., 2006). We analyzed several approaches that enhanced embryonic fixation and egg shell (ES) removal. Among these approaches, the combination of bleach treatment with postfixation heat shock and methanol shock (Fig. 2d) caused a high percentage (approximately 80%) of eggs to lose their shells and successfully achieved morphological preservation (Fig. 2f). After methanol shock, removed ES fragments floated above the treated eggs (Fig. 2e). DAPI staining in the absence of an appropriate methanol shock produced bright nuclear fluorescence but indicated that residual ES still surrounded the examined eggs (Fig. 2g). Similar methods involving methanol shock have been described in insect species, such as the hemipteran Oncopeltus fasciatus (Liu and Kaufman, 2004). The successful establishment of an efficient fixation method stimulated us to investigate the morphological events required for R. microplus embryogenesis. The Establishment of an Embryonic Staging System for R. microplus General egg morphology was preserved by our fixation method; therefore, we established an embryological staging system based on DAPI (Fig. 3) and acid fuchsin staining (see Supporting Information Fig. S1 for fuchsin-stained images) that was similar to previously described staging systems for other species, such as the fruit fly D. melanogaster (Campos-Ortega and Hartenstein, 1985) and the spiders P. tepidariorum (Mittmann and Wolff, 2012) and Cupiennius salei (Wolff and Hilbrant, 2011). The major morphological events of this embryonic staging system are discussed below. A table with the characteristics of each stage is provided in the Supporting Information section of this article.
RESULTS AND DISCUSSION
The Investigation of Early Tick Development and Cell Membrane Formation
The Establishment of a Fixation Method to Observe R. microplus Embryos: Heat Shock is Essential for Cracking the ES
One important difference among various arthropods with respect to embryo development is the timing of cellularization. In the dipteran D. melanogaster, a syncytial stage occurs that theoretically enables the diffusion of transcription factors to occur during early
Each R. microplus female can lay up to 3,000 eggs during adulthood (Fig. 2a). Tick embryonic develop-
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FIG. 2. The establishment of a R. microplus embryonic fixation method. (a) R. microplus females during egg laying. (b) Freshly laid eggs and (c) eggs at 2 days after egg laying (AEL). (d) A graphic indicating that heating is essential for ES removal and embryonic fixation. (e) After methanol shock, ESs have largely been removed and remain floating above the eggs. (f) Several residual ES fragments (arrow) may remain that can easily be removed with forceps. (g) DAPI staining of the nuclei of embryos subjected to an inappropriate methanol shock procedure. The autofluorescent ES (white arrows) is observed.
embryogenesis. This diffusion results in the formation of protein gradients that are an important component of the Drosophila segmentation cascade. Direct confirmation of cell membrane formation may only be obtained through the injection of fluorescent dyes that diffuse in a syncytial environment but remain restricted to a single cell in a cellularized environment. Fluores-
cent dye injection was recently performed in the spider P. tepidariorum. In this spider, cellularization was already evident by the 16-cell stage (Kanayama et al., 2010). The spider mite T. urticae exhibited cellularization at an even earlier time point, prior to the 8-cell stage (Dearden et al., 2002); therefore, it is unlikely that similar transcription factor diffusion mechanisms occur
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in spiders and spider mites (reviewed in Damen, 2007). The investigation of the precise time point of cellularization in ticks is important for understanding early patterning events. Unfortunately, the injection of tick eggs has been described as a remarkably difficult undertaking (Laumann et al., 2010; Santos and Fonseca, unpublished observations); therefore, we stained early embryos (stage 1) with an antibody against pTyr. This approach, which has previously been utilized in other arthropods (Nunes da Fonseca et al., 2008), should demarcate cell outlines. In accordance with observations from other tick species (Fagotto et al., 1988), R. microplus embryos are difficult to fix during their first hours of development. The initial rounds of cleavage occurred within the yolk (data not shown). Later in the first day of embryonic development, shortly after these cleavages had been completed (stage 1), a small number of nuclei could be visualized at the egg periphery by DAPI staining (Fig. 3a). In addition, three dimensional (3D) confocal reconstruction revealed strong pTyr staining around each nucleus. Narrow projections that connected nuclei were also observed (as indicated by the arrows in Fig. 4a–c). Antibody staining using a polyclonal antibody against the Toll membrane receptor produced staining patterns that exhibited large overlap with pTyr staining patterns at stage 3, suggesting that pTyr stains cell membranes (Fig. 4d–f). Negative controls at the same stage displayed no Toll or pTyr staining (data not shown). The Toll receptor is known to be involved in early DV patterning in all holometabolous insect species that have been studied to date (Buchta et al., 2013; Lynch and Roth, 2011; Nunes da Fonseca et al., 2008); however, a role for Toll signaling during early chelicerate embryogenesis has not previously been reported. Our data suggest that a Toll-like protein is present during early R. microplus embryogenesis (Fig. 4d–g). Our results suggest that cellularization occurs early in tick development and that the major differences between stages 1 and 3 are caused by intense cell prolif-
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eration (Fig. 3a0 –c0 , see also Fig. 4). These findings are consistent with the results of previous electron microscopic analyses of eggs from other tick species, which have suggested that cellularization occurs prior to the 16-cell stage of development (Fagotto et al., 1988; Laumann et al., 2010), although this issue remains under debate (e.g., see Campos et al., 2006 for an examination of the syncytial blastoderm stage). Axis Establishment During Tick Embryogenesis: The Identification of a Cell Population That Resembles a Cumulus In insects, the AP and DV axes of the egg correspond to the long and short axes of egg symmetry, respectively (reviewed in Lynch and Roth, 2011), which are established during oogenesis. In contrast, in the chelicerate spider Parasteatoda tepidariorum, newly laid embryos are radially symmetric, and the establishment of the DV axis depends on the appropriate regulation of BMP signaling by a specific group of cells known as cumulus cells. Cumulus mesenchymal cells express the secreted BMP homolog dpp and induce BMP activity in overlaying epithelial cells during migration. BMP activity has been observed in several arthropods through antibody staining against pMAD, the phosphorylated form of MAD (the arthropod SMAD protein) (AkiyamaOda and Oda, 2003, 2006, 2010; Nunes da Fonseca et al., 2010). Freshly laid R. microplus eggs exhibited no clear asymmetries along either the long or short axes; therefore, we were unable to identify AP or DV axes from stage 1 to 3. Interestingly, nuclear DAPI staining at stage 4 revealed an unusual group of cells at one side of the short axis of the egg (indicated by an arrow in Fig. 3d0 ). We observed that this cell population appeared to migrate cortically toward the long axis of the egg during stage 5, when the number of nuclei in the embryo has considerably increased (Fig. 3e0 ). To investigate the morphology of this cell population, we performed acid fuchsin staining on whole mounts to stain nuclei, followed by the staining of histological
FIG. 3. A developmental staging system for R. microplus. Schematic drawings (a–t) and their corresponding nuclear DAPI-stained images (a0 –t0 ). Stages 1–14 are illustrated in this figure. (a,a0 ) Stage 1 includes the first 24 h of development; the embryo has likely become cellularized during this time. (b,b0 ) At stage 2, cell numbers greatly increase (to a total of up to 512 cells), and the cells migrate to the egg periphery. (c,c0 ) At stage 3, cell number almost quadruples, although no distinct cellular regions can be observed. (d,d0 ) At stage 4, a distinct cell group may be observed on one side of the egg, along the egg’s short axis (white arrow). (e,e0 ) At stage 5, these cells appear to migrate toward the long axis of the egg. (f,f0 ) At stage 6, the egg may be divided into two regions, including one area with large nuclei and a second area with small nuclei; the latter area is the prospective germ band. (g,g0 ) Early in stage 7, the distinction between the germ band and the remaining cells with large nuclei becomes even more evident. (h,h0 ) Later in stage 7, the head (Hd), thorax (Th), and growth zone (GZ) become evident, and segmental grooves may be distinguished. (i,i0 ) Early in stage 8, three pairs of leg primordia (L1–L3) become apparent, and later in stage 8, the fourth leg (L4) stops growing in size. (k,k0 ,l,l0 ) During the early and middle portions of stage 9, a ventral furrow (VF) may be observed, and L4 is distinctly shorter than the other leg pairs. (m,m0 ) Late in stage 9, the VF begins to close, and both embryonic halves fuse again at stage 10 (n,n0 ). (o,o0 ) The later portions of stage 10 are characterized by opisthosomal retraction, which may be observed from an examination of changes in the red region. (p,p0 ) During stage 11, the embryo retracts to the anterior-ventral region. (q,q0 ,r,r0 ) During stage 12, dorsal closure begins with opisthosomal and prosomal migration (arrows). (s,s0 ) At stage 13, dorsal closure continues to occur, and chelicera positioning is altered in the head. (t,t0 ) At stage 14, dorsal closure has been completed, and the deposition of the cuticle impairs DAPI staining. Lb, labium; Ch, chelicera; P, pedipalp; Pg, pedipalp gnathostome; VF, ventral furrow; L1–L4, leg 1 to leg 4; Hd, Head; Th, thorax; GZ, growth zone. The scale bar corresponds to 100 mm.
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FIG. 4. Membrane formation and Toll signaling occur early in R. microplus embryogenesis. 3D confocal reconstructions of stage 1 (a–c) and stage 3 (d–g) embryos. (a–c) An embryo stained with the nuclear marker DAPI (a) and an antiphospho-tyrosine (pTyr) antibody (b). The merged image is displayed in panel C. Note the thin projections (arrows) that connect each nucleus. (d–g) At stage 3, cell numbers have greatly increased relative to stage 1, and Toll and pTyr are detectable at the cell outlines. The scale bar corresponds to 100 mm.
cross sections with toluidine blue, a general cationic dye. Whole-mount fuchsin staining clearly revealed an unusual cell group that may correspond to cumulus cells (as indicated by the Cm label in Fig. 5a). The examination of transverse cross sections at stage 4 revealed two cell populations at the egg periphery (Fig. 5b,c). One of these cell populations (indicated by the arrow in Fig. 5c) was continuous along the entire DV axis and possessed small oval nuclei that were surrounded by yolk mass (indicated by Yk), whereas the second cell population possessed large nuclei and a large quantity of cytoplasm (as indicated by the arrowhead in Fig. 4c). These peripheral cells migrated during stage 5 (Fig. 5d), and the two cell populations could be identified by longitudinal cross sections (Fig. 5e,f). We did not observe the presence of a blastopore at the center of the germ disc, although this blastopore is typically present after cumulus migration in spiders. To investigate whether these cell populations are comparable to the spider cumulus, we characterized these cells on a molecular level. Spider cumulus cells are characterized by the expression of several specific transcription factors and signaling molecules (AkiyamaOda and Oda, 2006, 2010). At stage 4, BMP activity (as determined by pMad staining) was detected at a specific cell population located along the short axis of symmetry (Fig. 6a–c). These cells with BMP activity appeared to undergo cell migration along the long axis of symmetry from stage 4 to 5 (Fig. 6d–f). Importantly, BMP activity was not observed in cells at the surface of the embryo but rather in cells underneath the superficial epithelial cells (Fig. 6a–c).
Another important characteristic of the spider cumulus population is its expression of b-catenin/Armadillo, an essential downstream component of the Wnt pathway (Oda et al., 2007). However, b-catenin also works in conjunction with cadherins to mediate cell adhesion in several metazoans (reviewed in Lilien and Balsamo, 2005). To test whether the b-catenin monoclonal antibody would specifically label regions of cell adhesion in R. microplus embryos, we analyzed these embryos at stage 8, when a germ band with a head, a thorax, and a growth zone can be identified (Fig. 7g). At this stage, bcatenin expression was specifically observed in a rosette-like pattern in large cells that were located laterally to the germ band (Fig. 7g,h—arrows). This antibody staining was specific; therefore, we used this antibody to assess other embryonic stages. b-catenin was detected in cumulus cells at stage 5 (Fig. 7a–d) as these cells underwent migration (see Fig. 5). At higher magnification levels (Fig. 7a0 –d0 ), it was evident that b-catenin was not detected in the nucleus and that b-catenin expression did not overlap with pTyr expression (Fig. 7b,b0 ). Thus, cell adhesion mediated by b-catenin could potentially be lost during cumulus migration. This hypothesis is supported by previous results indicating that tyrosine phosphorylation inhibits cadherin function, leading to the loss of cell–cell adhesion (Lilien and Balsamo, 2005). Thus, it is possible that at stage 5, the cumulus includes two cell populations with different cell adhesion properties. In combination, these results suggest that the loss of cell adhesion and BMP signaling may occur in the cumulus. Our observations suggest that this cumulus population disappeared
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FIG. 5. A cumulus cell population is evident along the short axis of R. microplus embryos. (a,d) Whole-mount fuchsin staining of stage 4 (a) and stage 5 (d) embryos. (b,c) Transverse cross sections indicate blastoderm cells surrounding the egg cell, with large cells (the cumulus cells, Cm) lying on top of the blastoderm. (e,f) Longitudinal cross sections at stage 5 indicate the displacement of the cumulus cell population toward the long axis of the egg. The Cm cell population is heterogeneous and includes large cells with round nuclei (arrow). Occasionally, several Cm cells are observed on top of the yolk (Yk). Differences in the color of the nuclei cannot be interpreted as differences in cell populations. The scale bar corresponds to 100 mm.
FIG. 6. BMP activity is detected specifically among the cumulus cell population. (a–c) Ventral views of a stage 4 embryo, illustrating the results of staining with DAPI and pMAD (which indicates BMP activity). pMAD staining is restricted to the cumulus cell population. (d,e,f) Lateral views of a stage 5 embryo, revealing that cumulus cells and pMAD staining have shifted toward the long axis of symmetry. The scale bar corresponds to 100 mm.
when the germ rudiment was first observed at stage 6 (Fig. 3e,f); however, only detailed time-lapse analysis can establish the final fate of these cells. In ticks, classical studies have demonstrated that the entire germ disc migrates over the surface yolk mass and that a broad germ band is formed by the aggregation and proliferation of blastoderm cells in front of the
germ disc (Aeschlimann, 1958). Anderson (1973) proposed that the axis of bilateral symmetry of the Acari metastigmatid germ band is established by the direction of the migration of the germ disc, similarly to the way in which the bilateral symmetry axis is established by the migrating posterior cumulus in the spider Agelena labyrinthica (Holm, 1952). Thus, it appears that the
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FIG. 7. b-catenin and phospho-tyrosine (pTyr) may be detected in the cumulus cell population and during germ band formation. DAPI (a,a0 ,e—blue), pTyr (b,b0 ,f—green) and b-catenin (c,c0 ,g—red) staining results and their merged images (d,d0 ,g) at stages 5 and 8. (a,b,c,d) Late in stage 5, pTyr and b-catenin are detectable in migrating cumulus cells. (a0 ,b0 c0 ,d0 ) Higher magnification illustrates that pTyr and bcatenin are expressed in different regions of the cumulus. (e,f,g,h) At stage 8, during germ band elongation, b-catenin may be detected at the edges of certain cells (arrows), particularly large cells. Hd, head; Th, thorax; GZ, growth zone. The scale bar corresponds to 100 mm.
lack of any prior recognition of a cumulus population in ticks might have been caused by the small size of ticks relative to other arthropods. Opiliones possess a cumulus (Juberthie, 1964, referenced in Sharma et al., 2012), whereas no obvious cumulus population is evident in the spider mite T. urticae (Dearden et al., 2002). Cumulus formation may, therefore, be a general feature of chelicerates, as emphasized in a recent review (Hilbrant et al., 2012; Oda and Akiyama-Oda, 2008). The absence of a cumulus in spider mites suggests that the cumulus was lost during Acari evolution but most likely present in the common ancestor of Actinotrichida (mites) and Anactinotrichida (ticks); alternatively, this distinction between mites and ticks may provide evidence to support recent suggestions that Actinotrichida and Anactinotrichida do not constitute a monophyletic group (Pepato et al., 2010).
sponded to extraembryonic cells (Fig. 3g0 ). The head, thorax, and posterior growth zone could be identified by nuclear DAPI staining (as indicated by Hd, Th, and GZ, respectively, in Fig. 3h,h0 ). At stage 8, four leg pair primordia were visible in the thoracic region; all these primordia were of comparable length along the proximal-distal axis (Fig. 3i). Late in stage 8, the three anteriormost leg pairs began to proliferate, whereas the fourth leg pair stopped increasing in size (Fig. 3j,j0 ). This fourth leg pair subsequently regressed at stage 9 and eventually disappeared by stage 11 (Figs. 3k–p and 8a–f). Interestingly, tick larvae hatch with three pairs of legs, and four pairs of legs are observed only during the nymphal stage. It has not yet been determined whether the generation of a fourth leg pair among nymphs occurs through the stimulation of a small group of cells that persist throughout embryogenesis.
Major Landmarks of Tick Embryogenesis: A Germ Band With a Growth Zone and Four Leg Pairs
Opisthosomal Segmentation is Observed During the Germ Band Stage
At 7 days AEL, two cell populations could be distinguished via DAPI staining of nuclei (Fig. 3g0 ). Small nuclei were found in the prospective embryonic primordium on the ventral side of each egg, whereas the sparse large nuclei on the dorsal side of each egg corre-
One of the most important questions regarding arthropod evolution involves the segmentation of the chelicerate body plan (Damen, 2007). The chelicerate opisthosoma is evolutionarily a far more labile tagma than the prosoma in both embryos and adults. Mites
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FIG. 8. Segmentation at the opisthosoma is highlighted by Antp antibody staining. (a), DAPI staining of a stage 9 R. microplus embryo. (b) Antp antibody staining. (c) The merged image of panels a and b.
and ticks have been traditionally viewed as a monophyletic group (Acari) that has lost opisthosomal segmentation (Lindquist, 1984). In mites, it has been demonstrated that changes in posterior Hox genes are associated with losses in opisthosomal segmentation (Barnett and Thomas, 2012; Grbic et al., 2011; Telford and Thomas, 1998). Developmental studies of two mite species have revealed that only two anterior opisthosomal segments express the conserved segment polarity gene engrailed, suggesting a reduction in opisthosomal segmentation in mites. Interestingly, the posterior Hox gene abd-A has been lost from the Hox complex of two mite species (Barnett and Thomas, 2012; Grbic et al., 2011). The genome of the tick Ixodes scapularis contains abd-A (Barnett and Thomas, 2012); therefore, it appears likely that a more typical Hox complex has been retained in ticks than in mites. Thus, we decided to investigate opisthosomal patterning in R. microplus. As a first step toward achieving this goal, we performed DAPI staining at the germ band stage (stage 9). Several segmental grooves were observed at the opisthosoma, suggesting that this region is segmented (Figs. 3j0 and 9a). Second, we analyzed the expression of the Antennapedia (Antp) protein. A monoclonal antibody has successfully been utilized to detect Antp in the grasshopper Schistocerca (Hayward et al., 1995) and in the beetle Tribolium (Supporting Information Fig. S2). As indicated in Figure 9b, Antp antibody staining in R. microplus revealed strong segmental expression in the entire opisthosoma (indicated by an O) but not the walking leg segments L1–L4. Although the expression of only one Hox gene was analyzed in this study, it appears that the tick opisthosoma may exhibit a complex segmentation pattern. Antp protein distribution in R. microplus appeared more similar to the distribution of this protein in spiders (Khadjeh et al., 2012) and opiliones (Sharma et al., 2012) than to Antp protein distribution in mites (Grbic et al., 2011; Telford and Thomas, 1998). Future studies using other cross-reactive antibodies against Hox genes in R. microplus may help further understanding regarding the evolution of opisthosomal patterning in arthropods.
The Fourth Leg Pair is Generated From the Thoracic Region but Regresses Prior to Hatching At stages 9 and 10, most morphological changes occur at the head and the thorax; these changes include an increase in appendage size and the generation of a transient ventral furrow (Fig. 8a–f). Interestingly, a recent analysis of the formation of the fourth leg pair in the mite Archegozetes longisetosus (Acari: Oribatida, Trhypochthoniidae) demonstrated that the formation of the fourth pair of locomotory legs is linked to posterior (opisthosomal) segmentation (Barnett and Thomas, 2012). In spiders and other chelicerates, the fourth leg pair arises from the thoracic region rather than the opisthosoma (Prpic and Damen, 2004). Our morphological analysis (Figs. 3 and 8) indicated that the fourth leg pair in R. microplus was generated in the most posterior portion of the thoracic region; therefore, we favor the hypothesis that the common ancestor of Acariformes (mites) and Parasitiformes (ticks) possessed four thoracic legs. Thus, the generation of the opisthosomal fourth locomotory leg pair of mites (Barnett and Thomas, 2012) would be a derived condition in Acari evolution (and therefore, in chelicerate evolution). In addition, our results imply that the hexapodal larvae observed in tick and mite larvae may not share a common morphogenetic basis and, therefore, cannot be used as evidence for Acari monophyly. A Transient Furrow at the Ventral Midline is a Characteristic of R. microplus Embryonic Development From Stages 8 to 11 The formation of a furrow in the ventral midline embryonic region is a common feature of several chelicerates (McGregor et al., 2008; Regier et al., 2010). In spiders, the embryo splits in two halves that migrate dorsally and subsequently fuse during the process of dorsal closure (Wolff and Hilbrant, 2011). Interestingly, a furrow at the ventral midline was also observed in ticks between stages 8 and 10 (indicated by an arrow in Fig. 8b,b0 ,c,c0 ). As embryonic development proceeded, the furrow became more evident
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FIG. 9. Ventral furrow formation is a transient process for R. microplus embryos. (a–f) The top row of panels provides schematic drawings of ventral regions of R. microplus embryos. (a0 ,f0 ) The lower row of panels highlights the corresponding nuclear DAPI staining results obtained from stage 8 to 11. Head segments, the thorax, and the opisthosoma are displayed in blue, green, and red, respectively. (a0 ) At stage 8, a furrow begins to be observed at the ventral midline. (b0 ,c0 ) At stage 9, the furrow begins to form in a segmental fashion (arrows); shortly thereafter, a complete furrow is observed along the AP axis (c0 -arrowhead). (d0 ,e0 ) At stage 10, the ventral sulcus begins to close, which is concomitant with an increase in leg size. (f) At stage 11, the thoracic segments retract toward the anteriormost region of the egg, and the prospective fourth leg and the ventral furrow disappear. The scale bar corresponds to 100 mm.
(arrowhead in Fig. 8c,c0 ), particularly in the thoracic region. In R. microplus embryos, this ventral midline furrow (ventral sulcus) was transient (Fig. 8d,d0 ,e,e0 ) and began to fade as embryonic development continued (Fig. 8d,d0 ,e,e0 ,f,f0 ). Classical studies have demonstrated that several tick species exhibit a transient ventral furrow but that this furrow is typically absent in small acarine embryos (Reuter, 1909; reviewed in Anderson, 1973). In spiders, a second movement known as ventral closure occurs after dorsal closure. During spider ventral closure, embryonic tissue envelops the yolk (Wolff and Hilbrant, 2011). In insects, dorsal closure involves cells from the extraembryonic membranes, the amnion and the serosa (Panfilio, 2008). However, both membranes are absent in chelicerates. In R. microplus, ventral furrow formation is transient, and neither embryonic half exhibits dorsal migration similar to the migration that occurs in spiders; therefore, the tick embryo only undergoes dorsal closure at later stages of embryogenesis. The Final Stages of R. microplus Embryogenesis: Dorsal Closure Occurs Through Cell Movements of the Opisthosoma and Prosoma As development continued, limb buds increased in size (Fig. 3o); in addition, the germ band became short and broad by stage 11, particularly in the opisthosoma (Figs. 3p and 8f0 ). Shortly thereafter, during stage 12, the opisthosomal cells began to migrate dorsally until they directly surrounded the yolk mass (Fig. 3q–s). At stage 13, dorsal closure was nearly complete; this clo-
sure appeared to occur more quickly in the opisthosoma than in the prosoma. At stage 14, cuticle development impaired whole-mount staining (Fig. 3t0 ), and the prosoma completely enclosed the egg. Therefore, in R. microplus, dorsal closure movements appear to involve the surrounding of the yolk mass by active embryonic cells. These dorsal closure movements appear more similar to the dorsal closure processes of opilionids than to the dorsal closure processes of spiders and other chelicerates (Sharma et al., 2012; Wolff and Hilbrant, 2011). Cuticle Formation Analysis Enabled the Identification of Major Structures in R. microplus Larvae A straightforward method to analyze embryonic structure in arthropods involves the preservation of the exoskeleton structure and the chemical digestion of organs and tissues. This technique has been widely used to analyze embryos from the fly D. melanogaster (Stern and Sucena, 2011), the beetle T. castaneum (e.g., van der Zee et al., 2006) and the wasp Nasonia vitripennis (Pultz et al., 2000; van der Zee et al., 2006). This method may be utilized to visualize several important features of tick larvae, such as the rectal sac (RS), three pairs of legs (L1, L2, and L3), the abdomen, the thorax, and the head (Fig. 10a). Several characteristics were observed from detailed analyses of the larval legs (Fig. 10b). The tarsus, located in the distalmost part of the leg, was large and exhibited several specializations, such as claws and pads (Fig. 10b). Bristles with possible sensory functions were also identified at every
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FIG. 10. Analyses of cuticle formation in freshly emerged R. microplus larvae. R. microplus larvae (ventral view) exhibit typical features of the Acari (chelicerates). However, three leg pairs are observed instead of the four leg pairs that are common among the body plans of various chelicerates, such as spiders. P, pedipalp; Hy, hypostome; Ch, chelicera. Leg features observed among these larvae include the trochanter 1 (Tr1), trochanter 2 (Tr2), coxa, femur, patella, and tibia. The rectal sac (RS) is a structure responsible for the storage of debris. (b) A higher-magnification image of the distalmost regions of two legs. Claws are used to attach to the host, and the tarsus is extremely long relative to the other leg segments. (c) A higher-magnification image of the head region. Hy, hypostome; Ch, chelicera; TCh, teeth of the chelicera; P, pedipalp. The scale bar corresponds to 500 mm.
appendage. In addition, hard ticks, including R. microplus, possess a dorsal shield and mouthparts that protrude forward if observed from above (Fig. 10c). Typical characteristics of ticks could be identified by cuticle analysis, including not only, pedipalps and chelicera (labeled with P and Ch, respectively) but also the teeth of the chelicera (labeled with TCh), which are used to puncture the host. The hypostome is a harpoon-like structure that anchors the ticks firmly on a host mammal (Fig. 10c). Several morphological features at the terminal regions of the appendages appeared to be largely adapted to attach to host tissues. Thus, on the whole, cuticle analysis enabled the identification of major external structures of R. microplus and will prove to be an extremely useful method for analyzing the effects of gene knockdowns or drug treatments during embryogenesis. CONCLUSIONS The analysis of R. microplus embryonic development has established this tick as a useful model system for developmental biology studies of chelicerates. The ability to visualize embryonic development and the definition of a staging system based on morphological characteristics is important for future studies using pRNAi followed by phenotypic analysis. The staging system established in this study was used as the basis for revealing at least three remarkable characteristics of R. microplus development. First, we utilized molecular markers to identify a cell population resembling the spider cumulus. Second, we observed the formation of both a transient fourth leg pair arising from the thoracic
regions and a transient furrow occurring at the ventral midline. Finally, we observed opisthosomal segmentation. We anticipate that these embryonic features may render ticks more similar to other chelicerate groups than to mites (Acari) because mites do not exhibit the aforementioned embryonic characteristics. Thus, our data suggest either that mites and ticks do not constitute a monophyletic group or that mites have lost features present in certain tick species, such as R. microplus. These features and other important characteristics of Acari embryogenesis will be revealed through future functional studies using this tick species. ACKNOWLEDGMENTS Vit oria Santos is currently a CNPq fellow and previously received a FUNEMAC undergraduate scholarship. The authors would also like to thank Jose Garcia Abreu and Nipam Patel for providing us with the anti-b-catenin antibody and the Antp antibody, respectively. Moreover, they are grateful to Simone Gomes for technical assistance and to Itabajara Vaz (UFRGS) for providing helpful comments regarding the manuscript. Tick adults were a kind gift from Emerson Pontes (UFRRJ) and Itabajara Vaz (UFRGS). LITERATURE CITED Aeschlimann A. 1958. Developpement embryonnaire d’Ornithodorus moubata (Murray) et transmission transovarienne de Borrelia dutton^ı. Acta Trop 15: 15–62.
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