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Research Article
Evidence against a germ plasm in the milkweed bug Oncopeltus fasciatus, a hemimetabolous insect
Ben Ewen-Campen, Tamsin E. M. Jones, Cassandra G. Extavour
Biology Open 2013 2: 556-568; doi: 10.1242/bio.20134390
Ben Ewen-Campen
Department of Organismic and Evolutionary Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
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Tamsin E. M. Jones
Department of Organismic and Evolutionary Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
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Cassandra G. Extavour
Department of Organismic and Evolutionary Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
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  • For correspondence: extavour@oeb.harvard.edu
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Summary

Primordial germ cell (PGC) formation in holometabolous insects like Drosophila melanogaster relies on maternally synthesised germ cell determinants that are asymmetrically localised to the oocyte posterior cortex. Embryonic nuclei that inherit this “germ plasm” acquire PGC fate. In contrast, historical studies of basally branching insects (Hemimetabola) suggest that a maternal requirement for germ line genes in PGC specification may be a derived character confined principally to Holometabola. However, there have been remarkably few investigations of germ line gene expression and function in hemimetabolous insects. Here we characterise PGC formation in the milkweed bug Oncopeltus fasciatus, a member of the sister group to Holometabola, thus providing an important evolutionary comparison to members of this clade. We examine the transcript distribution of orthologues of 19 Drosophila germ cell and/or germ plasm marker genes, and show that none of them localise asymmetrically within Oncopeltus oocytes or early embryos. Using multiple molecular and cytological criteria, we provide evidence that PGCs form after cellularisation at the site of gastrulation. Functional studies of vasa and tudor reveal that these genes are not required for germ cell formation, but that vasa is required in adult males for spermatogenesis. Taken together, our results provide evidence that Oncopeltus germ cells may form in the absence of germ plasm, consistent with the hypothesis that germ plasm is a derived strategy of germ cell specification in insects.

Introduction

In sexually reproducing animals, only germ cells contribute genetic information to future generations. The germ line/soma separation is a cell fate decision shared across Metazoa (Buss, 1987). Despite the fundamental commonality of germ cell function in animals, the molecular mechanisms underlying germ cell specification are remarkably diverse across different taxa (Extavour and Akam, 2003; Extavour, 2007; Ewen-Campen et al., 2010; Juliano et al., 2010).

Primordial germ cells (PGCs) can be specified via different developmental mechanisms; here we call these “cytoplasmic inheritance” and “zygotic induction.” (We and others have previously referred to these mechanisms as “preformation” and “epigenesis” respectively (Nieuwkoop and Sutasurya, 1981; Extavour and Akam, 2003; Extavour, 2007). However, these terms can hold different meanings in other contexts of the history and philosophy of biology (e.g. Callebaut, 2008). We therefore avoid them here in favour of more mechanistically descriptive terms.) Cytoplasmic inheritance is characterised by the asymmetric formation of a specialised cytoplasmic region within the oocyte or early embryo, termed “germ plasm.” Germ plasm contains maternally provided mRNAs and proteins that are individually necessary and collectively sufficient for PGC formation. Cells that inherit germ plasm during embryogenesis acquire germ line fate. The best understood example of cytoplasmic inheritance occurs in Drosophila melanogaster, where germ plasm is maternally synthesised, localised to the posterior of the oocyte during oogenesis, and subsequently incorporated into PGCs (pole cells) during cellularisation. Removing pole cells after their formation, or compromising the molecular components of germ plasm, leads to loss of PGCs and sterility in adulthood (reviewed by Mahowald, 2001). In contrast, zygotic induction of PGCs takes place later in development and requires signalling from neighbouring somatic cells to induce germ line fate. This mode of PGC development is exemplified by Mus musculus, wherein PGCs develop from a subset of presumptive mesodermal cells after the segregation of embryonic and extraembryonic tissues in response to local signalling (reviewed by Magnúsdóttir et al., 2012).

Across Insecta, germ plasm has been almost exclusively reported in taxa nested within Holometabola (“higher” insects, which undergo complete metamorphosis) including D. melanogaster (reviewed by Kumé and Dan, 1968; Anderson, 1973; Nieuwkoop and Sutasurya, 1981), and in only three species belonging to the sister assemblage to the Holometabola (see below). Thus, although the vast majority of our knowledge of insect germ cell development comes from studies of germ plasm in D. melanogaster, this mode of germ cell specification is likely a derived feature of Holometabolous insects and their close sister taxa.

Our present knowledge of PGC specification in basally branching insects (Hemimetabola) is based almost entirely on classical histological studies of insect development conducted over the past 150 years. Nearly all of these report that PGCs arise late in embryogenesis, raising the possibility that they may be specified through inductive mechanisms (Wheeler, 1893; Heymons, 1895; Hegner, 1914; Nelsen, 1934; Roonwal, 1937). Experimental approaches to discovering germ plasm in Hemimetabola are limited, but a study involving destruction of the germ rudiment via irradiation in the cricket Gryllus domesticus (Schwalm, 1965) showed that no specific region of early embryos in this species contains a germ line determinant. Functional tests of genes that may specify germ cells in Hemimetabola have been performed in only one insect, the cricket Gryllus bimaculatus. In this cricket, the conserved germ line markers vasa and piwi are dispensable maternally and zygotically for PGC formation (Ewen-Campen et al., 2013). Most evidence available for the Hemimetabola therefore suggests the absence of germ plasm and the operation of zygotic PGC specification mechanisms.

Exceptions have been reported, however, in some members of the Paraneoptera, an assemblage of insect orders (including Hemiptera [true bugs], Psocoptera [book lice], and Thysanoptera [thrips]) that collectively form the sister group to Holometabola (Yeates et al., 2012). Cytological studies of three paraneopteran species, a book louse (Psocoptera (Goss, 1952)), a thrip (Thysanoptera (Heming, 1979)) and an aphid (Hemiptera (Chang et al., 2009)) suggested the presence of germ plasm in oocytes or early embryos, as did expression studies of vasa, piwi and nanos expression during asexual development of the pea aphid Acyrthosiphon pisum (Chang et al., 2006; Chang et al., 2007; Chang et al., 2009; Lu et al., 2011). However, A. pisum embryogenesis is highly modified relative to that of other hemimetabolous insects and even relative to other members of the same order (Miura et al., 2003). Studies of embryogenesis in most other hemipterans describe absence of germ plasm and PGC origin after cellularisation from the blastopore region at gastrulation stages (Metschnikoff, 1866; Witlaczil, 1884; Will, 1888; Seidel, 1924; Mellanby, 1935; Butt, 1949; Sander, 1956; Kelly and Huebner, 1989; Heming and Huebner, 1994). We therefore wished to examine the expression and function of germ line genes in a hemipteran displaying embryological characteristics more representative of the order.

Here we characterise germ cell formation and migration in the milkweed bug Oncopeltus fasciatus (Hemiptera). We examine the expression of 19 molecular markers including vasa, nanos, and piwi, and test the germ cell function of three of these using RNA interference. We show that in striking contrast to Drosophila, transcripts of none of these genes localise asymmetrically within Oncopeltus oocytes or early embryos. We identify PGCs using multiple criteria, and show that neither vasa nor tudor are required for PGC specification or oogenesis in this species, but that vasa is required for spermatogenesis in adult males. These data show that the PGC specification role of vasa has diverged between Oncopeltus and the Holometabola, and suggest that Oncopeltus PGCs may form in the absence of maternally supplied germ plasm.

Results

Putative germ cells are first detectable in the late blastoderm stage

In contrast to D. melanogaster, classical studies of Oncopeltus fasciatus embryogenesis have not revealed a germ plasm in oocytes or early embryos, and instead first identify cells with cytological characteristics of PGCs at the posterior of the embryo at the end of the cellular blastoderm stage (Butt, 1949). We used semi-thin plastic sectioning and fluorescence microscopy to confirm these observations, and traced the development of these putative PGCs throughout gastrulation and germ band elongation (supplementary material Fig. S1). Our observations of putative PGC formation in Oncopeltus were consistent with historical studies (Butt, 1949), showing that these cells first arise at the blastoderm posterior immediately prior to gastrulation (supplementary material Fig. S1). Unlike pole cells in D. melanogaster, presumptive PGCs in Oncopeltus arise on the basal side of the blastoderm surface, adjacent to the yolk (supplementary material Fig. S1G–H). In order to obtain further evidence that these cells were PGCs and test for the presence of a maternally supplied germ plasm, we examined the expression of conserved germ line markers.

Cloning Oncopeltus germ line markers

We cloned fragments of vasa, nanos, and piwi (Ewen-Campen et al., 2010) and confirmed that each was the best reciprocal BLAST hit to its respective orthologue in D. melanogaster. vasa was cloned using degenerate primers (supplementary material Table S1). nanos and a single piwi gene were recovered from the Oncopeltus transcriptome, in addition to a single AGO-3 orthologue (an additional PIWI family protein belonging to a separate sub-family; not shown). We believe it is unlikely that Oncopeltus possesses additional orthologues of these genes because (1) the Oncopeltus ovarian and embryonic transcriptome, which is nearly saturated for gene discovery and has an average coverage of 23× (Ewen-Campen et al., 2011), contained only one orthologue of each gene; and (2) degenerate PCR for vasa using primers flanking the conserved DEAD box helicase domain (Rocak and Linder, 2004) recovered only a single vasa orthologue.

Phylogenetic reconstruction confirmed that Oncopeltus vasa is nested within other insect vasa genes (supplementary material Fig. S2A), and that Oncopeltus piwi belongs to the PIWI sub-family containing the Drosophila genes piwi and aubergine (which are Drosophila-specific duplications) (supplementary material Fig. S2B). The portion of animal Nanos proteins with conservation sufficient for confident alignment (48 amino acids) is too short to yield significant phylogenetic signal (supplementary material Fig. S2C, note low support values), but Oncopeltus Nanos does contain the diagnostic 2×(CCHC) zinc finger domain found in all Nanos orthologs (supplementary material Fig. S2D).

Our analysis of the Oncopeltus nanos sequence produced an unexpected result: we found that a stop codon is present 771 bp upstream of the first CCHC zinc finger domain, although no methionine is found anywhere in this region. This is unlikely to be a sequencing error, as it was identified with high coverage (22 reads/bp at this position) in the transcriptome (Ewen-Campen et al., 2011) and confirmed using Sanger sequencing of independent clones generated from a different cDNA pool than that used to generate the transcriptome. Furthermore, repeated attempts at 5′ RACE using a third independent DNA pool failed to amplify a start codon. Several lines of evidence confirm that this Oncopeltus nanos sequence represents a highly expressed mRNA and is therefore unlikely to be a pseudogene: it was recovered from a transcriptome made solely from poly(A)-RNA, and is detected via both RT-PCR (Ewen-Campen et al., 2011) and in situ hybridisation (see below). We hypothesise that a large, unspliced intron downstream of the start codon may have been present in our mRNA preparations. Alternatively, given that the length of the predicted translated region upstream of the first CCHC zinc finger domain (266 amino acids) is within the range of known arthropod Nanos orthologues (95 to 332 amino acids) (Wang and Lehmann, 1991; Curtis et al., 1995; Calvo et al., 2005; Lynch and Desplan, 2010), it may be that the Oncopeltus Nanos N terminus has a non-methionine start codon. Although rare, eukaryotic non-AUG translation initiation can occur in nuclear-encoded genes, including developmentally relevant genes (Hellen and Sarnow, 2001), and can be recognized by insect ribosomes (Sasaki and Nakashima, 2000; Jan et al., 2001). In the absence of a complete genome sequence we cannot distinguish between these hypotheses. Despite this uncertainty, we report nanos transcript expression here for the sake of completeness.

vasa, nanos, and piwi transcripts do not localise asymmetrically in ovaries

The distinct cytoplasm inherited by early-specified PGCs in multiple organisms, including D. melanogaster, Caenorhabditis elegans, Xenopus laevis and Danio rerio, contains transcripts of the highly conserved piwi, vasa and nanos gene families. Of these, only nanos mRNA is asymmetrically localised to D. melanogaster germ plasm, while piwi and vasa transcripts are ubiquitous throughout the fly oocyte and embryo. However, in several other organisms vasa orthologue transcripts are asymmetrically localised germ plasm components (reviewed by Ewen-Campen et al., 2010).

To test whether any of these transcripts were asymmetrically localised to putative germ plasm in Oncopeltus oocytes, we conducted in situ hybridisation on adult ovaries. The structure of Oncopeltus ovaries is typical of Hemiptera and several other insect orders but differs remarkably from that of Drosophila (Fig. 1A) (Büning, 1994). Rather than each oocyte developing together with its own complement of 15 nurse cells as in Drosophila, all oocytes in Oncopeltus ovarioles share a common pool of syncytial nurse cells located at the anterior of each ovariole in a region termed the “tropharium” (Fig. 1A1). The nurse cell syncytium connects to all oocytes via elongated, microtubule-rich tubes called “nutritive tubes” (Hyams and Stebbings, 1979; Harrison et al., 1991) through which maternal factors, including mRNA, proteins and mitochondria, are transported to developing oocytes (Fig. 1A2,A3) (Stebbings et al., 1985; Stebbings and Hunt, 1987; Anastas et al., 1991; Hurst et al., 1999; Stephen et al., 1999).

Fig. 1.
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Fig. 1. Germ cell markers do not localise asymmetrically during oogenesis.

(A) Overview of a single Oncopeltus ovariole. nc: nurse cells; o: oocytes; nt: nutritive tubes; f: follicle cells. Boxed regions are enlarged in (A1–A3). (A1) Nurse cell syncytium containing polyploid nurse cell nuclei (white) connected by cytoplasmic bridges (green). (A2) Posterior tropharium containing oogonia (arrows) and resting oocytes (arrowheads). Caret indicates polyploid nurse cells in the anterior of this region. (A3) Nutritive tubes (nt) are actin-rich at the end that enters the anterior of each oocyte. Transcripts of vasa (B), nanos (C), piwi (D), tudor (E) and boule (F) are detected in nurse cells, nutritive tubes, and uniformly in oocytes. (G) A representative sense control (for vasa) is shown; sense controls for other genes were similar. Scale bars: 100 µm in A,A3,B (applies to C–G); 25 µm in A1,A2. Anterior to the left in all panels.

vasa, nanos, and piwi were expressed at high levels in Oncopeltus nurse cells and oocytes of all stages, but at no stage of oogenesis did any of these three transcripts localise asymmetrically within oocytes (Fig. 1B–D). Expression was detected in nurse cells, resting oocytes, nutritive tubes, and developing oocytes, suggesting that these transcripts are synthesised in the nurse cells and subsequently transported to oocytes via nutritive tubes (Fig. 1B–D). nanos and piwi were expressed throughout the tropharium (Fig. 1C,D), in contrast to vasa, whose expression was primarily in nurse cells of the posterior tropharium, resting and developing oocytes (Fig. 1B). In late stage oocytes, expression remained ubiquitous (not shown), similar to the expression in just-laid eggs (see below).

In situ screen of conserved Drosophila germ plasm markers fails to reveal a germ plasm in Oncopeltus

The expression of piwi, vasa and nanos suggests that a maternally localised germ plasm containing transcripts of these genes is not present in Oncopeltus oocytes. However, a functional germ plasm that contains gene products other than those encoded by these three genes could be present in oocytes or early embryos. To explore this possibility, we examined the expression of 14 additional genes whose transcripts are enriched in the germ plasm and germ cells of Drosophila (supplementary material Table S2) (Tomancak et al., 2002; Lécuyer et al., 2007; Tomancak et al., 2007) that were also recovered from the Oncopeltus ovarian and embryonic transcriptome (Ewen-Campen et al., 2011) based on best reciprocal BLAST hit analysis with the Drosophila proteome (Zeng and Extavour, 2012). Although several of these genes do not have documented mutant phenotypes for germ cell formation in Drosophila (supplementary material Table S2), all are expressed at high levels in germ plasm and/or pole cells and are therefore molecular markers for germ plasm in Drosophila. We reasoned that if Oncopeltus possessed germ plasm it would likely be revealed by at least one of these genes.

In addition, we examined the expression of boule and tudor, which have widely conserved functions in germ cells across Metazoa (Eberhart et al., 1996; Ewen-Campen et al., 2010; Shah et al., 2010). tudor is one of 23 Tudor domain-containing proteins in Drosophila (Ying and Chen, 2012), but there is no evidence that loss of function of other Tudor domain-containing genes have grandchildless phenotypes in Drosophila (Handler et al., 2011; Pek et al., 2012). We therefore focus only on the expression and function of the orthologue of Drosophila tudor (CG9450). We examined boule and tudor transcript expression throughout oogenesis and embryogenesis through mid-germ band stages.

None of these 16 transcripts localised asymmetrically in ovaries (Fig. 1E,F; supplementary material Fig. S3). Instead, like vasa, piwi and nanos (Fig. 1B–D), all of these genes were expressed ubiquitously throughout oogenesis. Half of the genes examined (sra, CycB, Bsg25D, Uev1A, CG16817, Unr, mael and tud) were expressed, like vasa (Fig. 1B), in nurse cells adjacent to resting oocytes, as well as in the resting and early oocytes themselves (Fig. 2E; supplementary material Fig. S3B–H). Five genes (Gap1, eIF5, bel, orb and bol) were, like piwi and nanos (Fig. 1C,D), strongly expressed in all nurse cells of the tropharium (Fig. 2F; supplementary material Fig. S3I–L). Two genes (cta and Tao) were expressed in resting and early oocytes but barely at all in the tropharium (supplementary material Fig. S3M,N), suggesting that these genes may be transcribed by resting oocyte nuclei rather than by nurse cells. Finally, aret (aka bruno), which is a translational regulator of Oskar in Drosophila (Kim-Ha et al., 1995; Webster et al., 1997), was expressed in nurse cells of the posterior tropharium and in early stages of oogenesis but excluded from resting oocytes (supplementary material Fig. S3O), suggesting that it is transcribed by oocyte nuclei after the onset of oogenesis. In summary, although transcripts of most of these genes are likely to be supplied maternally to oocytes, they are not asymmetrically localised within oocytes of any stage.

Fig. 2.
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Fig. 2. vasa transcript expression first labels PGCs at late blastoderm stages.

(A–D) vasa transcript expression. (A′–D′) Corresponding images of nuclear stains. (A,A′) Immediately following fertilisation vasa is detected ubiquitously. Arrowhead: polar body. (B,B′) In early cleavage stages vasa transcripts are associated with all energid nuclei. (C,C′) During early syncytial blastoderm stages, vasa expression remains ubiquitous. (D,D′) At cellular blastoderm stages (24–28 h AEL), vasa marks putative PGCs at the posterior pit (asterisk). (E–H) End-on perspective of the posterior of Oncopeltus embryos showing vasa expression during PGC formation. (E′–H′) Corresponding images of nuclear stains. (E″–H″) Medial sections of vasa- (purple) and nuclear- (cyan) stained embryos at corresponding time points. (E–E″) In late syncytial blastoderm stages, vasa is expressed ubiquitously. (F–F″) In early cellular blastoderm embryos, vasa expression increases in some posterior cells (arrowheads in F,F″) while levels in the remainder of the blastoderm decrease (arrows in F,F″). (G–G″) At posterior germ band invagination vasa-positive cells (arrowheads) are the first cells to enter the yolk; vasa transcripts continue to be cleared from somatic tissue (arrows). (H–H″) As invagination proceeds vasa expression is largely restricted to PGCs (arrowhead) and nearly cleared from the soma (arrows). Scale bars: 100 µm in A (applies to B–D,A′–D′); 500 µm in E (applies also to F–H′); 50 µm in E″ (applies also to F″–H″). Anterior is to the left in A–D′ and E″–H″.

vasa, boule and tudor transcripts mark PGCs throughout embryogenesis but are not asymmetrically localised in early embryos

Although none of the genes examined showed asymmetric localisation during oogenesis or early embryogenesis, at late blastoderm stages many of the genes appeared enriched at the posterior pit, where PGCs had previously been identified based on cytological criteria (supplementary material Fig. S1) (Butt, 1949). However, because at this stage of development gastrulation begins at the posterior, this region of the blastoderm is multilayered. Upon close examination, we found that the apparent transcript enrichment was an artifact of tissue thickness for all genes except vasa, tudor and boule, whose transcripts appeared truly enriched in putative PGCs at late blastoderm/early gastrulation stages (Fig. 2D–H″, Fig. 4N,S).

Strikingly, we found that vasa, tudor and boule marked PGCs from the time of their formation at cellular blastoderm stages, but that none of these genes' transcripts were asymmetrically localised prior to PGC formation. Immediately after egg laying, vasa transcripts were not localised asymmetrically but rather were ubiquitously distributed throughout the embryo (Fig. 2A). As energid nuclei reached the embryonic surface (Fig. 2B), cytoplasmic islands enriched with these transcripts were distributed evenly across the embryonic surface, remaining there as these energids divided to form the uniform blastoderm (Fig. 2C). Prior to posterior pit formation, vasa expression became restricted to putative PGCs at the embryonic posterior (Fig. 2D).

To visualise vasa expression in the developing PGCs in greater detail, we collected staged embryos in two-hour intervals over the period during which PGCs arise (19 to 27 hours after egg laying (AEL)), performed in situ hybridisation for vasa (Fig. 2E–H), and sectioned the embryos in plastic resin (Fig. 2E″–H″). During this eight-hour period, the blastoderm nuclei undergo two concurrent, dynamic processes: continuing cell divisions increase the nuclear density throughout the blastoderm, and the blastoderm nuclei move towards the posterior pole and ultimately into the yolk (Butt, 1949; Liu and Kaufman, 2004) (Fig. 2E′–H′). From 19–21 hours AEL, the ubiquitous vasa expression seen in early embryos remained unchanged (Fig. 2E–E″). However, from 21–23 hours AEL vasa expression became enriched in a subset of cells at the blastoderm posterior (Fig. 2F–F″). From 23–25 hours AEL, vasa-positive cells increased in density at the blastoderm posterior and began to move into the yolk (Fig. 2G–G″). This movement appeared passive, due to the formation of the posterior pit by invagination of the germ rudiment. However, in the absence of time-lapse data we cannot rule out the possibility of active PGC movement out of the blastoderm epithelium and towards the yolk. From 25–27 hours AEL, as the germ rudiment began its invagination into the yolk, vasa-positive cells formed a distinct mesenchymal clump within the yolk at the posterior of the embryo (Fig. 2H–H″). During this and all following stages, in addition to the marked enrichment in PGCs, vasa transcripts were additionally observed ubiquitously at low levels throughout somatic tissue (Figs 2, 3).

Fig. 3.
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Fig. 3. vasa marks PGCs throughout migration.

(A) vasa transcript expression during progressive stages of germ band development. Arrows indicate vasa-positive PGCs. (B) Medial section of an embryo at 28–32 h AEL, showing vasa in situ hybridisation (purple) and nuclear stain (cyan). Boxed region enlarged in (B′) shows PGCs in contact with ectoderm (Ect.) and the amnion (Amn.). (C) Medial section of an embryo at 36–40 h AEL, when PGCs (arrow) initiate migration along the mesoderm (Mes.). Boxed region enlarged in (C′). Scale bars: 200 µm in A–C; 100 µm in B′,C′. Anterior is up in A, left in B–C′.

Throughout all subsequent stages of germ band elongation and patterning, vasa continued to mark PGCs (Fig. 3). During early stages of germ band elongation prior to limb bud formation (∼28–32 hours AEL) vasa-positive PGCs remained at the embryonic posterior on the dorsal surface of the newly forming mesoderm (Fig. 3A,B,B′). The PGC cluster then became pear-shaped from 32–42 hours AEL, as the anteriormost PGCs began to move towards the anterior of the embryo (Fig. 3A,C,C′). As the head lobes enlarged (36–40 h AEL), PGCs began to migrate anteriorly on the dorsal surface of the embryo and continued their migration during limb bud stages (40–44 h AEL) (Fig. 3A). During appendage elongation stages (44–48 h AEL) PGCs split into distinct clusters spanning the midline in abdominal segments A4–A6, one cluster per segment. As appendage segmentation became morphologically distinct (48–52 h AEL), the segmental clusters split along the ventral midline into bilateral clusters in A4–A6.

tudor and boule were also expressed in PGCs at all stages in a pattern indistinguishable from that of vasa (Fig. 4), providing further evidence that the vasa-positive cells are Oncopeltus PGCs. None of the other genes we examined (supplementary material Fig. S3), including nanos and piwi (Fig. 4), were enriched in PGCs at any stage of embryogenesis.

Fig. 4.
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Fig. 4. Oncopeltus PGCs express tudor and boule, but not nanos or piwi.

In early embryos, expression of all four genes remains ubiquitous during energid proliferation (A,F,K,P) and blastoderm formation (B,C,G,H,L,M,Q,R). During posterior pit formation nanos is expressed throughout the length of the embryo (D), whereas piwi expression is reduced in the presumptive extraembryonic serosal tissue in the anterior of the embryo (I). Apparent posterior staining in (D) and (I) is the result of tissue thickness in that location, and is not specific to PGCs. tudor (N) and boule (S) transcripts become restricted to presumptive PGCs at the time of their specification. In germ band stage embryos, while tudor (O) and boule (T) mark presumptive PGC clusters, nanos is not detected (E), while piwi expression is ubiquitous (J). Scale bars: 500 µm in A (applies also to B–D,F–I,K–N,P–S); 100 µm in E,J; 200 µm in O,T. Anterior to the left.

Neither vasa nor tudor are required for PGC formation

Our gene expression analysis demonstrates that vasa, tudor, and boule are specifically expressed in PGCs beginning at the putative time of their specification at the embryonic posterior just prior to gastrulation. To determine whether these genes were required for PGC formation or development in Oncopeltus, we performed maternal RNAi (mRNAi) for each gene. We confirmed that mRNAi effectively reduced zygotic transcript levels in our experiments using RT-PCR (Fig. 5E). PGC presence or absence was determined with in situ hybridisation against PGC markers at ∼40–54 hours AEL, when germ cells are visible on the dorsal mesoderm.

Fig. 5.
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Fig. 5. vasa and tudor are not required for PGC specification in Oncopeltus.

(A–D) Bright field images of in situ hybridisations for PGC markers in different RNAi conditions; numbers indicate sample sizes and % of embryos with PGCs. Arrowheads indicate PGC clusters in abdominal segments A4–A6. (A′–D′) DIC images of the same embryos shown in (A–D) showing distinct PGC cluster morphology. (A,A′) In control embryos vasa-positive PGCs are visible on the dorsal surface of abdominal segments 4–5. PGCs are present in vasa RNAi (B,B′), tudor RNAi (C,C′), and double vasa + tudor RNAi (D,D′) embryos. (E) RT-PCR validation of RNAi knockdown. Controls are animals injected with DsRed dsRNA. Expression of β-tubulin was analysed to confirm cDNA integrity and allow comparison of amounts of template per lane. Scale bar: 100 µm. Anterior is up in A–D.

RNAi knockdown of vasa or tudor did not disrupt embryonic patterning or germ band development (supplementary material Table S3), despite the widespread expression of these genes at early blastoderm stages (Figs 2, 4), and their persistent low levels of expression in somatic cells even after PGC formation (Figs 3, 4). Strikingly, germ cells were clearly present in both vasa (93.8%, n = 16) and tudor (100%, n = 20) knockdowns, suggesting that neither of these genes is required for PGC specification (Fig. 5A–C′). It is formally possible that residual vasa or tudor transcripts that may have escaped destruction by mRNAi could be sufficient to play an instructive role in PGC formation. However, we note that transcript levels of both genes in the progeny of injected mothers were barely detectable in the case of vasa, and undetectable in the case of tudor, when assessed with RT-PCR even as late as 4 days AEL (Fig. 5E). Moreover, even hypomorphic alleles of tudor (Schüpbach and Wieschaus, 1986) and vasa (Lasko and Ashburner, 1990; Schüpbach and Wieschaus, 1991; Liang et al., 1994) lead to loss of PGCs in Drosophila. We therefore hypothesise that in Oncopeltus, vasa and tudor are required neither maternally nor zygotically for germ cell specification, although they are expressed in the cells specified as PGCs.

To address the possibility of redundancy between these two genes, we performed double knockdown of vasa and tudor, which reduced transcripts of both genes to undetectable levels (Fig. 5E). Eggs laid by vasa + tudor double RNAi females had an increased rate of embryonic lethality relative to controls (supplementary material Table S3; 47.4%, n = 19 vs 10.3%, n = 39), which may mean that these genes work together to play roles in somatic development. However, embryos that escaped this lethality still had PGCs (100%, n = 10) (Fig. 5D,D′).

None of the knockdowns caused any qualitative or quantitative change in egg laying by injected females compared to controls, and ovaries of injected females showed neither morphological abnormalities nor signs of disrupted oogenesis (not shown). This indicates that, in contrast to Drosophila (Schüpbach and Wieschaus, 1991; Styhler et al., 1998; Tomancak et al., 1998; Johnstone and Lasko, 2004), vasa is not required individually or redundantly with tudor for Oncopeltus oogenesis or egg laying.

boule is necessary for Oncopeltus oogenesis and embryonic survival

boule mRNAi caused a complete cessation of egg laying by injected females after four to five clutches (one clutch is laid every one to two days). In contrast, vasa, tudor and control mRNAi females continued to lay up to 12 clutches. Ovaries of boule dsRNA-injected females possessed only a few oocytes at early stages of oogenesis, and few or no mature oocytes (not shown), indicating a requirement for boule in the progression of oogenesis. Eggs laid by boule RNAi females displayed nearly complete embryonic lethality (81.8%, n = 22) in all but the first clutch laid. (The first clutch of Oncopeltus eggs laid following mRNAi typically displays no abnormalities, as these eggs have developed their chorion by the time of injection and are therefore impervious to dsRNA (Liu and Kaufman, 2004).) This was a striking increase in embryonic lethality compared to DsRed controls (26.8%, n = 190), vasa knockdowns (23.2%, n = 198) and tudor knockdowns (5.6%, n = 54). The oogenesis requirement for boule and resulting embryonic lethality thus prevented us from determining whether boule is required for germ cell specification in Oncopeltus, and we do not further report on the role of boule on oogenesis in the present study.

vasa is required for Oncopeltus spermatogenesis

Given that in contrast to Drosophila, vasa is not required for germ cell specification or oogenesis in Oncopeltus, we wished to test for other possible functions of this gene. In mice, despite its expression in the embryonic PGCs of both sexes once they reach the genital ridge (Fujiwara et al., 1994; Diez-Roux et al., 2011). vasa is required not for PGC specification, but rather for gametogenesis in males (Tanaka et al., 2000). Similarly, we recently showed that vasa plays a role in spermatogenesis in the cricket G. bimaculatus (Ewen-Campen et al., 2013). We therefore asked whether vasa also functions during spermatogenesis in Oncopeltus.

The testes of Oncopeltus show an organisation typical of insect testes (Dumser, 1980), with stages of spermatogenesis located in an anterior–posterior progression (supplementary material Fig. S4). Unlike Drosophila, which has a single sperm tubule (testiole) per testis (Hardy et al., 1979), each Oncopeltus testis comprises seven testioles (Bonhag and Wick, 1953). In situ hybridisation for vasa showed that it is strongly expressed in secondary spermatogonia of each testiole, and at lower levels in early primary spermatocytes and post-spermatocyte stages, but not in primary spermatogonia or somatic cells (Fig. 6A). Adult males injected with dsRNA against vasa displayed multiple abnormalities in spermatogenesis. Testioles of vasa RNAi males lacked clearly defined cysts and contained large numbers of small, dense nuclei in the anterior region (Fig. 6H,I,I′), which in controls contained only spermatocytes with large, pale nuclei (Fig. 6C,D,D′; supplementary material Fig. S4E). The primary spermatogonial region of vasa RNAi testioles contained cysts of irregular size (Fig. 6I, arrowheads) with poorly defined cytoplasmic bridges (Fig. 6I′, arrows). In the spermatocyte region vasa RNAi testioles contained large, poorly defined clusters of several hundred cells (Fig. 6J, arrowheads) at varying stages of spermatogenesis (Fig. 6J′). The nuclear morphology of cells in these cysts corresponded to spermatocyte (Fig. 6E; supplementary material Fig. S4E) or early spermatid (Fig. 6F) stages, as well as shell stage-like nuclei (Fig. 6K,K′) typical of the mid-stage spermatids of controls (Fig. 6F′). Cysts of wild type shell stage spermatids are no longer syncytial as the actin-rich cytoplasmic bridges disappear during spermatocyte stages (supplementary material Fig. S4G). In contrast, the anterior shell stage-like nuclei in vasa RNAi testioles remained connected by cytoplasmic bridges (Fig. 6K′, red arrows), consistent with precocious spermatid differentiation. Moreover, although they displayed clear shell stage nuclear morphology (Fig. 6K,K′, red arrowheads), they were larger than wild type shell stage nuclei (Fig. 6F′, red arrowheads), suggesting that they had begun spermatid differentiation as syncytial diploid cells without first proceeding through meiosis as in wild type. Finally, the posteriormost region of vasa RNAi testioles contained irregular groups of cells at mixed stages of late spermatid and spermatozoon differentiation (Fig. 6L), rather than the perfectly synchronised cysts of late spermiogenic stages seen in controls (Fig. 6G). These defects were observed in testes examined 28–29 days following injection of adult males, but are not artefacts of age, as testes of 10 week old wild type adult males showed normal progression through all stages of spermatogenesis (supplementary material Fig. S4B).

Fig. 6.
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Fig. 6. vasa is expressed in adult testes and required for spermatogenesis in Oncopeltus.

Schematics indicate the region of the testis (A–C) or testiole (D–L) shown in each column. (A) vasa in situ of an adult wild-type testiole showing expression in the secondary spermatogonia. PSG: primary spermatogonia; SSG: secondary spermatogonia; PSC: primary spermatocytes; SSC: secondary spermatocytes; ST: spermatids undergoing spermiogenesis; SZ: spermatozoa. (B) vasa sense control probe. DIC optics (C–F,F′) and F-actin (green) and nuclear staining (white) (D′,E′) of control testioles reveals distinct, synchronized spermatogenic cysts separated by clear cyst boundaries (carets) (C), small cysts of spermatogonia (PSG) at the apex (D,D′), larger cysts of secondary spermatocytes (SSC) posterior to the apex (E,E′; arrowhead in (E′) indicates somatic sheath cells associated with cysts of germ cells), early spermatids with round prominent nuclei (early ST) (F) and mid-stage spermatids with smaller, compact round nuclei (mid ST) (F′). (G) Late spermatid cysts in controls are synchronized in spermiogenesis; hollow arrowheads indicate somatic sheath cells. vasa RNAi testioles contain large masses of cells with heterogeneous nuclear morphologies (H; arrowheads). (I) PSG cysts are abnormal in shape and size, contain nuclei of multiple sizes (arrowheads), and (I′) have filamentous actin masses interspersed between nuclei (arrows) rather than clearly defined cytoplasmic bridges (compare with D′, arrows). (J,J′) Abnormal cysts contain clusters of small dense nuclei (arrowheads). (K,K′). Aberrant cysts retain cytoplasmic bridges at spermatid stages (red arrows), and contain nuclei with morphologies corresponding to different spermatogenic stages, including both early (white arrows; compare with F) and mid ST (red arrowheads; compare with F′) stages. (L) vasa RNAi late spermatid cysts are asynchronous, comprising multiple late spermatid and spermatozoon differentiation stages within a single cyst; cysts remain associated with sheath cells (hollow arrowheads). Scale bars: 200 µm in A (applies to B); 100 µm in C,H; 50 µm in D–E′, G, I–J′, L; 25 µm in F,F′,K,K′. Anterior is up in all panels.

Taken together, these data suggest that vasa is required for the maintenance of synchrony within cysts at multiple stages of spermatogenesis. In addition, vasa may be required for secondary spermatogonia to enter correct meiotic progression as spermatocytes, in the absence of which germ cells are nonetheless able to continue with subsequent stages of spermatogenesis.

Discussion

Oncopeltus germ cell formation

In several cases, analyses of molecular markers such as vasa mRNA have revealed the presence of a cryptic germ plasm that had eluded prior histological studies (Yoon et al., 1997; Tsunekawa et al., 2000; Wu et al., 2011). In Oncopeltus, we have shown that none of the transcripts of an extensive suite of conserved germ cell markers localise asymmetrically within oocytes or in early embryos (Figs 1, 2, 4; supplementary material Fig. S3), including transcripts of genes that localise to and are required for the function of germ plasm in Drosophila. Gene products of at least one of these conserved germ line markers have been found in the germ plasm of every species where a germ plasm is known to exist (Ewen-Campen et al., 2010), although we note the important caveat that in Drosophila, several of these genes (vasa, piwi, and tudor) are localized as proteins rather than mRNAs. Thus, the lack of localisation of transcripts of any of these 19 genes during oogenesis or early embryogenesis suggests that Oncopeltus lacks germ plasm. Instead, our data support the hypothesis that Oncopeltus germ cells form in the absence of germ plasm, and are not present prior to the onset of posterior invagination at the end of the cellular blastoderm stage. We cannot, however, rule out the possibility that untested molecular markers, including protein products of the genes examined here, could be asymmetrically localised to a putative germ plasm in Oncopeltus.

While we provide multiple markers of PGCs, further experiments could be useful to confirm the identity of these cells. However, demonstration that these cells are functional PGCs via ablation experiments is complicated by the fact that they arise at the inner face of the blastoderm at the gastrulation center, so that their physical disruption would likely also compromise mesoderm formation and subsequent embryogenesis. Moreover, we note that while pole cell removal experiments in Drosophila result in sterility, pole cell removal in another insect with germ plasm, the wasp Pimpla turionellae, yields fertile adults despite the fact that these pole cells are bona fide PGGs in wild type embryos (Bronskill, 1959; Achtelig and Krause, 1971; Fleischmann, 1975). Further, we are currently unable to genetically ablate these cells and determine their effect on fertility, as our vasa, tudor and vasa + tudor RNAi double RNAi experiments do not disrupt their formation (Fig. 5). Lineage tracing techniques that would permit tracking of the putative PGCs over the six-week period between PGC formation and sexual maturity are not currently available for Oncopeltus. These caveats notwithstanding, the molecular and morphological evidence that the cells we identify in this report are bona fide Oncopeltus PGCs is comparable to that available for PGC identification in most studied animal species: (1) three conserved germ line genes, vasa, tudor, and boule, are specific germ cell markers in Oncopeltus (Figs 1–4); (2) transcripts of these genes first become enriched in germ cells specifically at the time that these cells were previously reported to arise based on morphological and cytological criteria (Figs 2–4) (Butt, 1949); and (3) cells with these molecular markers undergo migration and primordial gonad occupation (supplementary material Fig. S1; Figs 3, 4) consistent with the well-documented behavior of PGCs in many other hemipterans (Seidel, 1924; Mellanby, 1935; Butt, 1949; Sander, 1956; Kelly and Huebner, 1989; Heming and Huebner, 1994).

Although the posterior location of germ cells at the time of their specification is superficially similar to that of pole cells in Drosophila and other Diptera, PGC specification and development in Oncopeltus differs in several important ways. First, while Drosophila pole cells form on the exterior of the posterior syncytial blastoderm before somatic cellularisation, Oncopeltus germ cells appear on the yolk side of the cellular blastoderm. Second, while Drosophila pole cells are the first cells in the embryo to cellularise (Huettner, 1923), Oncopeltus germ cells arise after blastoderm cellularisation is complete (Butt, 1949). Third, because Oncopeltus is an intermediate-germ insect, only the gnathal and thoracic segments have been specified at the time that germ cells arise (Liu and Kaufman, 2004), whereas in the long-germ insect Drosophila, pole cells form posterior to the abdominal embryonic segments. Lastly, Oncopeltus germ cells form on the dorsal surface of the embryo, and remain on the yolk-facing surface of the mesoderm during their migration to the gonad primordium in anterior abdominal segments (Fig. 3). As a result, they do not undergo a transepithelial migration through the hindgut epithelium as in Drosophila (reviewed by Richardson and Lehmann, 2010).

The function of “germ line genes” in Oncopeltus

Our functional analysis led to the surprising discovery that neither vasa nor tudor play instructive roles in germ cell specification in Oncopeltus. Both of these genes are required for germ cell specification in Drosophila (Boswell and Mahowald, 1985; Schüpbach and Wieschaus, 1986) and other species (Sunanaga et al., 2007; Spike et al., 2008). However, vasa has widely divergent roles across Metazoa (reviewed by Yajima and Wessel, 2011), and in many cases is dispensable for PGC specification (Tanaka et al., 2000; Braat et al., 2001; Li et al., 2009; Özhan-Kizil et al., 2009). In several organisms it plays a role in adult gametogenesis (Tanaka et al., 2000; Ohashi et al., 2007; Salinas et al., 2007; Fabioux et al., 2009; Salinas et al., 2012; Ewen-Campen et al., 2013).

Intriguingly, we find that similar to the mouse and the cricket, vasa is required for spermatogenesis in adult Oncopeltus (Fig. 6), but not for oogenesis. This sex-specific function may relate to a putative role in stem cell function. As in other hemimetabolous insects (Büning, 1994), in Oncopeltus germ line stem cells are likely active in the apex of the testes (Schmidt and Dorn, 2004) but are not thought to be present in adult ovaries. One caveat to this hypothesis is that vasa transcript was not detected by in situ hybridisation in the primary spermatogonia (Fig. 6A), although it may be present at very low levels in those stem cells. Alternatively, given its strong expression in secondary spermatocytes and the defects in cyst integrity and synchrony caused by vasa RNAi (Fig. 6), Oncopeltus vasa may play a male-specific role in the onset or synchrony of meiosis. Consistent with a conserved role for vasa in bilaterian meiosis, male germ cells in vasa knockout mice arrest just prior to meiosis onset (Tanaka et al., 2000), and in human stem cell-derived germ cells vasa overexpression enhances meiotic progression (Medrano et al., 2012). Oncopeltus vasa RNAi leads to premature spermatid differentiation by some diploid secondary spermatocytes within a cyst, resulting in cyst asynchrony. In Drosophila, mutations are known that disrupt meiosis but do not prevent sperm formation (Davis, 1971), consistent with the hypothesis that spermiogenesis can be decoupled from meiotic status.

The evolutionary origins of germ plasm in insects

Together with recent molecular and classical histological data on germ cell specification in other insects, our results are consistent with the hypothesis that germ plasm is a derived mode of germ cell specification that arose in the ancestor to holometabolous insects (Fig. 7) (Lynch et al., 2011; Ewen-Campen et al., 2012). The only other functional genetic analysis of germ line specification in a hemimetabolous insect to date (Ewen-Campen et al., 2013) has also provided evidence that maternally supplied posterior germ plasm is absent, and that vasa is dispensable maternally and zygotically for germ cell formation. Our data thus support the notion that germ plasm-driven germ cell specification mechanisms operative in Drosophila melanogaster and Nasonia vitripennis are derived relative to the Hemimetabola (Fig. 7).

Fig. 7.
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Fig. 7. Phylogenetic distribution of germ cell specification mechanisms and migration patterns across insects.

Species shown are those for which data on the expression and/or function of molecular markers for germ cells during oogenesis and embryogenesis are available. Molecular data suggest absence of germ plasm in oocytes (circles) and early embryos (squares) of some Holometabola (Tribolium, Apis) and Hemimetabola (Oncopeltus and Gryllus), and somatic expression of vasa at post-blastoderm stages of development (diamonds) is not uncommon. In most species, PGCs undergo extensive migration from the site of specification to the gonad primordia (triangles). Data from this study (also Nakao, 1999; Mahowald, 2001; Donnell et al., 2004; Zhurov et al., 2004; Chang et al., 2006; Dearden, 2006; Juhn and James, 2006; Nakao et al., 2006; Schröder, 2006; Chang et al., 2007; Juhn et al., 2008; Zhao et al., 2008; Chang et al., 2009; Khila and Abouheif, 2010; Lynch et al., 2011; Ewen-Campen et al., 2013; C. von Levetzow, Konservierte und divergente Aspekte der twist-, snail- und concertina-Funktion im Käfer Tribolium castaneum, PhD thesis, Universität zu Köln, 2008). Phylogenetic relationships from Yeates et al. (Yeates et al., 2012).

The ubiquitous distribution of germ cell markers in early Oncopeltus embryos and their subsequent enrichment in presumptive germ cells at the blastoderm posterior is reminiscent of vasa expression in the beetle Tribolium (Fig. 7) (Schröder, 2006; C. von Levetzow, Konservierte und divergente Aspekte der twist-, snail- und concertina-Funktion im Käfer Tribolium castaneum, PhD thesis, Universität zu Köln, 2008). Further taxonomic sampling, and functional studies in Tribolium, will be needed to determine whether the PGC specification mechanisms in these two species may be the result of common ancestry (Fig. 7).

A large number of transcripts that localise to germ plasm in Drosophila are expressed ubiquitously in Oncopeltus oocytes and early embryos. This suggests that the evolution of germ plasm in Holometabolous insects involved a large-scale change in the localisation of many transcripts in the oocyte. We propose that this likely resulted from a change in the localisation of an upstream component capable of recruiting many downstream transcripts, rather than via the sequential evolution of distinct localisation mechanisms for individual transcripts. Studies on the genetic mechanism of evolutionary redeployments of multiple downstream genes have largely focused on transcription factors, as individual transcription factors are capable of regulating large numbers of target genes (Hoekstra and Coyne, 2007; Moczek, 2008; Stern and Orgogozo, 2008; Craig, 2009). Interestingly, in the case of germ plasm, transcription factors are unlikely to have been key players in the mechanisms of evolutionary change for a number of reasons. First, regulation of germ line determinants is largely post-transcriptional (Arkov and Ramos, 2010; Richter and Lasko, 2011; Sengupta and Boag, 2012; Nousch and Eckmann, 2013). Second, germ plasm transcript function relies on their subcellular localisation (often mediated via signals in their 3′UTRs) rather than their presence or absence (Rangan et al., 2009). Finally, unlike key transcription factors identified as largely sufficient to induce specific somatic cell fates (e.g. Akam, 1998; Kozmik, 2005; Baena-Lopez and García-Bellido, 2006), there is no single conserved gene that is sufficient to confer germ cell fate across metazoans. The evolution of germ plasm may therefore serve as an example of how a novelty (asymmetrically localized germ plasm in the oocyte) arose via changes in RNA localisation rather than transcriptional regulation.

Materials and Methods

Animal culture

Oncopeltus fasciatus were cultured at 28°C as previously described (Ewen-Campen et al., 2011). Timing of embryonic events reported here may differ from that reported in other studies using lower rearing temperatures (e.g. Liu and Kaufman, 2004).

Cloning and phylogenetic analysis

Total RNA was extracted from mixed-stage embryos and ovaries using TRIzol (Invitrogen) and used for first strand cDNA synthesis with qScript cDNA SuperMix (Quanta BioSciences). An Oncopeltus vasa fragment was cloned using degenerate primers (supplementary material Table S1). nanos and piwi fragments were obtained from the Oncopeltus transcriptome (Ewen-Campen et al., 2011). Fragments were extended using RACE PCR (SMART RACE cDNA kit, Clontech), and used as templates for DIG-labeled in situ probes and dsRNA fragments following sequence verification (supplementary material Table S1). Genes for the in situ hybridisation screen (supplementary material Tables S1, S2) were obtained from the Oncopeltus transcriptome (Ewen-Campen et al., 2011; Zeng and Extavour, 2012) and amplified using primers containing linker sequence (5′-CCCGGGGC-3′) enabling direct addition of a T7 site to the 3′ end in a subsequent PCR reaction. Extended sequences are available from ASGARD (http://asgard.rc.fas.harvard.edu) (Zeng and Extavour, 2012). All coding sequences reported in this study have been submitted to GenBank [accession numbers KC261571–KC261587] except for orb and Uev1A, for which we obtained only 3′ UTR sequence.

Maximum-likelihood phylogenetic analysis was performed for vasa, piwi, and nanos as previously described (Ewen-Campen et al., 2012).

Tissue fixation and gene expression analysis

Embryos were fixed and stained as previously described (Liu and Kaufman, 2004; Erezyilmaz et al., 2009; Kainz et al., 2011). Adult gonads were dissected in 1× PBS and fixed in 4% formaldehyde in 1× PBS for at least one hour. Antibodies used were mouse anti-alpha tubulin DM1A (Sigma) 1:50 and goat anti-mouse Alexa Fluor 568 (Invitrogen) 1:500–1:1000, and counterstains were FITC-phalloidin (Invitrogen) 0.5–1 µl and Hoechst 33342 (Sigma) 0.1–0.5 µg/ml.

Plastic sectioning

In situ hybridisation and/or Sytox Green (Invitrogen) staining were performed prior to embedding embryos in Durcupan ACM Fluka (Sigma), mixed at a ratio of 32:27:1:0.6  =  components A:B:C:D. Embryos were dehydrated through 10-minute washes in each of 50%, 70%, 90%, 2× 100% ethanol and 100% acetone, transferred to a 1:1 mixture of acetone: catalysed Durcupan, and left uncovered in a fume hood overnight. Embryos were individually transferred to fresh Durcupan in silicone molds (Electron Microscope Sciences NO. 70903) and oriented following a 30-minute initial hardening at 65°C. Resin blocks were baked for 24 hours at 65°C.

Block fronts were trimmed with a razor blade and sectioned at 5–6 µm on a Leica RM2255 microtome with a high-profile knife holder using High-Profile disposable “diamond-edge” steel knives (C.L. Sturkey NO. D554D50). Sections were collected on water droplets on charged slides, dehydrated on a heat block, and mounted in Permount (Fisher Scientific).

Parental RNAi

dsRNA for all genes (supplementary material Table S1) was prepared as previously described (Kainz et al., 2011) and resuspended in injection buffer (5 mM KCl, 0.1 mM NaH2PO4) to a concentration of 2 µg/uL. Male and female adults were injected three days after final molt with 5 µL of 2 µg/uL dsRNA using a Hamilton syringe and size 26 needles. Testes were collected from injected males 27–29 days after injection.

Reverse-transcription PCR

Half of each clutch laid by injected females was fixed for in situ hybridisation, and the other half was homogenised in TRIzol (Invitrogen) and stored at −80°C before isolation of total RNA. RNA was isolated separately from late blastoderm (24–29 hours AEL), early germ band (24–48 hours AEL) and late germ band (72–96 hours AEL) embryos laid by injected mothers. Genomic DNA was treated with Turbo DNase (Ambion) at 37°C for 30 minutes, followed by DNase heat-inactivation and phenol/chloroform extraction. cDNA was synthesised from 120 ng of each RNA sample using Superscript III Supermix (Invitrogen). PCR was performed using Advantage 2 DNA Polymerase from 1 µL of cDNA template and primers indicated in supplementary material Table S1 at 60°C annealing temperature with 35 PCR cycles. RT-PCR results for samples of all three embryonic ages tested yielded indistinguishable results, indicating that maternal RNAi was effective at reducing zygotic transcripts in embryos at least up to four days AEL.

Acknowledgements

We thank Omar Delannoy Bruno for conducting additional in situ hybridisations, Ian Dunn for assistance collecting embryos, and Jeremy Lynch for providing the T7-linker PCR probe synthesis protocol. This work was partially supported by National Science Foundation (NSF) awards [grant numbers IOS-0817678 and IOS-1257217] to C.G.E., an NSF Predoctoral Fellowship to B.E.-C., and a Frank Knox Memorial Fellowship to T.E.M.J.

Footnotes

  • Author Contributions B.E.-C. and C.G.E. designed the research; B.E.-C., T.E.M.J. and C.G.E. performed experiments, collected and analysed data and wrote the manuscript; C.G.E. obtained funding for the research.

  • Competing interests The authors have no competing interests to declare.

  • Received February 11, 2013.
  • Accepted March 1, 2013.
  • © 2013. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0/).

References

  1. ↵
    1. Achtelig M.,
    2. Krause G.
    (1971). Experimente am ungefurchten ei von Pimpla turionellae L. (Hymenoptera) zur funktionsanalyse des oosombereichs. Wilhelm Roux Arch. Entwickl. Mech. Org. 167, 164–182. doi:10.1007/BF00577038
    OpenUrlCrossRef
  2. ↵
    1. Akam M.
    (1998). Hox genes: from master genes to micromanagers. Curr. Biol. 8, R676–R678. doi:10.1016/S0960-9822(98)70433-6
    OpenUrlCrossRefPubMedWeb of Science
  3. ↵
    1. Anastas A.,
    2. Hunt C.,
    3. Stebbings H.
    (1991). Characterization of a nucleotide-sensitive high molecular weight microtubule-associated protein in the ovary of a hemipteran insect. Cell Motil. Cytoskeleton 19, 37–48. doi:10.1002/cm.970190106
    OpenUrlCrossRef
  4. ↵
    1. Anderson D. T.
    (1973). Embryology And Phylogeny In Annelids And Arthropods. Oxford: Pergamon Press.
  5. ↵
    1. Arkov A. L.,
    2. Ramos A.
    (2010). Building RNA-protein granules: insight from the germline. Trends Cell Biol. 20, 482–490. doi:10.1016/j.tcb.2010.05.004
    OpenUrlCrossRefPubMedWeb of Science
  6. ↵
    1. Baena–Lopez L. A.,
    2. García–Bellido A.
    (2006). Control of growth and positional information by the graded vestigial expression pattern in the wing of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 103, 13734–13739. doi:10.1073/pnas.0606092103
    OpenUrlAbstract/FREE Full Text
    1. Bardsley A.,
    2. McDonald K.,
    3. Boswell R. E.
    (1993). Distribution of tudor protein in the Drosophila embryo suggests separation of functions based on site of localization. Development 119, 207–219.
    OpenUrlAbstract
  7. ↵
    1. Bonhag P. F.,
    2. Wick J. R.
    (1953). The functional anatomy of the male and female reproductive systems of the milkweed bug, Oncopeltus fasciatus (Dallas) (Heteroptera: Lygaeidae). J. Morphol. 93, 177–283. doi:10.1002/jmor.1050930202
    OpenUrlCrossRef
  8. ↵
    1. Boswell R. E.,
    2. Mahowald A. P.
    (1985). tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster. Cell 43, 97–104. doi:10.1016/0092-8674(85)90015-7
    OpenUrlCrossRefPubMedWeb of Science
  9. ↵
    1. Braat A. K.,
    2. van de Water S.,
    3. Korving J.,
    4. Zivkovic D.
    (2001). A zebrafish vasa morphant abolishes vasa protein but does not affect the establishment of the germline. Genesis 30, 183–185. doi:10.1002/gene.1060
    OpenUrlCrossRefPubMedWeb of Science
  10. ↵
    1. Bronskill J. F.
    (1959). Embryology of Pimpla turionellae (L.) (Hymenoptera: Ichneumonidae). Can. J. Zool. 37, 655–688. doi:10.1139/z59-068
    OpenUrlCrossRef
  11. ↵
    1. Büning J.
    (1994). The Insect Ovary: Ultrastructure, Previtellogenic Growth And Evolution. London: Chapman and Hall.
  12. ↵
    1. Buss L. W.
    (1987). The Evolution Of Individuality. Princeton, NJ: Princeton University Press.
  13. ↵
    1. Butt F. H.
    (1949). Embryology Of The Milkweed Bug: Oncopeltus Fasciatus (Hemiptera), pp. 2–43. Ithaca, NY: Cornell University Agricultural Experiment Station.
  14. ↵
    1. Callebaut M.
    (2008). A review: Historical evolution of preformistic versus neoformistic (epigenetic) thinking in embryology. Belg. J. Zool. 138, 20–35.
    OpenUrl
  15. ↵
    1. Calvo E.,
    2. Walter M.,
    3. Adelman Z. N.,
    4. Jimenez A.,
    5. Onal S.,
    6. Marinotti O.,
    7. James A. A.
    (2005). Nanos (nos) genes of the vector mosquitoes, Anopheles gambiae, Anopheles stephensi and Aedes aegypti. Insect Biochem. Mol. Biol. 35, 789–798. doi:10.1016/j.ibmb.2005.02.007
    OpenUrlCrossRefPubMedWeb of Science
  16. ↵
    1. Chang C. C.,
    2. Lee W. C.,
    3. Cook C. E.,
    4. Lin G. W.,
    5. Chang T.
    (2006). Germ-plasm specification and germline development in the parthenogenetic pea aphid Acyrthosiphon pisum: Vasa and Nanos as markers. Int. J. Dev. Biol. 50, 413–421. doi:10.1387/ijdb.052100cc
    OpenUrlCrossRefPubMedWeb of Science
  17. ↵
    1. Chang C. C.,
    2. Lin G. W.,
    3. Cook C. E.,
    4. Horng S. B.,
    5. Lee H. J.,
    6. Huang T. Y.
    (2007). Apvasa marks germ-cell migration in the parthenogenetic pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea). Dev. Genes Evol. 217, 275–287. doi:10.1007/s00427-007-0142-7
    OpenUrlCrossRefPubMedWeb of Science
  18. ↵
    1. Chang C. C.,
    2. Huang T. Y.,
    3. Cook C. E.,
    4. Lin G. W.,
    5. Shih C. L.,
    6. Chen R. P.
    (2009). Developmental expression of Apnanos during oogenesis and embryogenesis in the parthenogenetic pea aphid Acyrthosiphon pisum. Int. J. Dev. Biol. 53, 169–176. doi:10.1387/ijdb.082570cc
    OpenUrlCrossRefPubMed
  19. ↵
    1. Craig L. R.
    (2009). Defending evo-devo: a response to Hoekstra and Coyne. Philos. Sci. 76, 335–344. doi:10.1086/649808
    OpenUrlCrossRef
  20. ↵
    1. Curtis D.,
    2. Apfeld J.,
    3. Lehmann R.
    (1995). nanos is an evolutionarily conserved organizer of anterior-posterior polarity. Development 121, 1899–1910.
    OpenUrlAbstract
  21. ↵
    1. Davis B. K.
    (1971). Genetic analysis of a meiotic mutant resulting in precocious sister-centromere separation in Drosophila melanogaster. Mol. Gen. Genet. 113, 251–272. doi:10.1007/BF00339546
    OpenUrlCrossRefPubMedWeb of Science
  22. ↵
    1. Dearden P. K.
    (2006). Germ cell development in the honeybee (Apis mellifera); vasa and nanos expression. BMC Dev. Biol. 6, 6. doi:10.1186/1471-213X-6-6
    OpenUrlCrossRefPubMed
  23. ↵
    1. Diez–Roux G.,
    2. Banfi S.,
    3. Sultan M.,
    4. Geffers L.,
    5. Anand S.,
    6. Rozado D.,
    7. Magen A.,
    8. Canidio E.,
    9. Pagani M.,
    10. Peluso I.
    et al. (2011). A high-resolution anatomical atlas of the transcriptome in the mouse embryo. PLoS Biol. 9, e1000582. doi:10.1371/journal.pbio.1000582
    OpenUrlCrossRefPubMed
  24. ↵
    1. Donnell D. M.,
    2. Corley L. S.,
    3. Chen G.,
    4. Strand M. R.
    (2004). Caste determination in a polyembryonic wasp involves inheritance of germ cells. Proc. Natl. Acad. Sci. USA 101, 10095–10100. doi:10.1073/pnas.0403625101
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Dumser J. B.
    (1980). The regulation of spermatogenesis in insects. Annu. Rev. Entomol. 25, 341–369. doi:10.1146/annurev.en.25.010180.002013
    OpenUrlCrossRefWeb of Science
  26. ↵
    1. Eberhart C. G.,
    2. Maines J. Z.,
    3. Wasserman S. A.
    (1996). Meiotic cell cycle requirement for a fly homologue of human Deleted in Azoospermia. Nature 381, 783–785. doi:10.1038/381783a0
    OpenUrlCrossRefPubMed
    1. Economopoulos A. P.,
    2. Gordon H. T.
    (1971). Growth and differentiation of the testes in the large milkweed bug, Oncopeltus fasciatus (Dallas). J. Exp. Zool. B 177, 391–405. doi:10.1002/jez.1401770402
    OpenUrlCrossRef
  27. ↵
    1. Erezyilmaz D. F.,
    2. Rynerson M. R.,
    3. Truman J. W.,
    4. Riddiford L. M.
    (2009). The role of the pupal determinant broad during embryonic development of a direct-developing insect. Dev. Genes Evol. 219, 535–544. doi:10.1007/s00427-009-0315-7
    OpenUrlCrossRefPubMed
  28. ↵
    1. Ewen–Campen B.,
    2. Schwager E. E.,
    3. Extavour C. G.
    (2010). The molecular machinery of germ line specification. Mol. Reprod. Dev. 77, 3–18. doi:10.1002/mrd.21091
    OpenUrlCrossRefPubMedWeb of Science
  29. ↵
    1. Ewen–Campen B.,
    2. Shaner N.,
    3. Panfilio K. A.,
    4. Suzuki Y.,
    5. Roth S.,
    6. Extavour C. G.
    (2011). The maternal and early embryonic transcriptome of the milkweed bug Oncopeltus fasciatus. BMC Genomics 12, 61. doi:10.1186/1471-2164-12-61
    OpenUrlCrossRefPubMed
  30. ↵
    1. Ewen–Campen B.,
    2. Srouji J. R.,
    3. Schwager E. E.,
    4. Extavour C. G.
    (2012). oskar predates the evolution of germ plasm in insects. Curr. Biol. 22, 2278–2283. doi:10.1016/j.cub.2012.10.019
    OpenUrlCrossRefPubMed
  31. ↵
    1. Ewen–Campen B.,
    2. Donoughe S.,
    3. Clarke D. N.,
    4. Extavour C. G.
    (2013). Germ cell specification requires zygotic mechanisms rather than germ plasm in a basally branching insect. Curr. Biol. (in press).
  32. ↵
    1. Extavour C. G.
    (2007). Evolution of the bilaterian germ line: lineage origin and modulation of specification mechanisms. Integr. Comp. Biol. 47, 770–785. doi:10.1093/icb/icm027
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Extavour C. G.,
    2. Akam M. E.
    (2003). Mechanisms of germ cell specification across the metazoans: epigenesis and preformation. Development 130, 5869–5884. doi:10.1242/dev.00804
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Fabioux C.,
    2. Corporeau C.,
    3. Quillien V.,
    4. Favrel P.,
    5. Huvet A.
    (2009). In vivo RNA interference in oyster – vasa silencing inhibits germ cell development. FEBS J. 276, 2566–2573. doi:10.1111/j.1742-4658.2009.06982.x
    OpenUrlCrossRefPubMed
  35. ↵
    1. Fleischmann V. G.
    (1975). Origin and embryonic development of fertile gonads with and without pole cells of Pimpla turionellae L. (Hymenoptera, Ichneumonidae). Zoologische Jahrbücher. Abteilung für Anatomie und Ontogenie der Tiere. 94, 375–411.
    OpenUrl
  36. ↵
    1. Fujiwara Y.,
    2. Komiya T.,
    3. Kawabata H.,
    4. Sato M.,
    5. Fujimoto H.,
    6. Furusawa M.,
    7. Noce T.
    (1994). Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage. Proc. Natl. Acad. Sci. USA 91, 12258–12262. doi:10.1073/pnas.91.25.12258
    OpenUrlAbstract/FREE Full Text
    1. Golumbeski G. S.,
    2. Bardsley A.,
    3. Tax F.,
    4. Boswell R. E.
    (1991). tudor, a posterior-group gene of Drosophila melanogaster, encodes a novel protein and an mRNA localized during mid-oogenesis. Genes Dev. 5, 2060–2070. doi:10.1101/gad.5.11.2060
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Goss R. J.
    (1952). The early embryology of the book louse, Liposcelis divergens Badonnel (Psocoptera; liposcelidae). J. Morphol. 91, 135–167. doi:10.1002/jmor.1050910107
    OpenUrlCrossRef
  38. ↵
    1. Handler D.,
    2. Olivieri D.,
    3. Novatchkova M.,
    4. Gruber F. S.,
    5. Meixner K.,
    6. Mechtler K.,
    7. Stark A.,
    8. Sachidanandam R.,
    9. Brennecke J.
    (2011). A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors. EMBO J. 30, 3977–3993. doi:10.1038/emboj.2011.308
    OpenUrlCrossRefPubMedWeb of Science
  39. ↵
    1. Hardy R. W.,
    2. Tokuyasu K. T.,
    3. Lindsley D. L.,
    4. Garavito M.
    (1979). The germinal proliferation center in the testis of Drosophila melanogaster. J. Ultrastruct. Res. 69, 180–190. doi:10.1016/S0022-5320(79)90108-4
    OpenUrlCrossRefPubMedWeb of Science
  40. ↵
    1. Harrison A.,
    2. Stebbings H.,
    3. Hyams J. S.
    (1991). Different patterns of α-tubulin post-translational modification in ovarian nutritive tubes of two hemipteran insects. J. Cell Sci. 100, 501–507.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Hegner R. W.
    (1914). Studies on germ cells. I. The history of the germ cells in insects with special reference to the Keimbahn-determinants. II. The origin and significance of the Keimbahn-determinants in animals. J. Morphol. 25, 375–509. doi:10.1002/jmor.1050250302
    OpenUrlCrossRefWeb of Science
  42. ↵
    1. Hellen C. U.,
    2. Sarnow P.
    (2001). Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 15, 1593–1612. doi:10.1101/gad.891101
    OpenUrlFREE Full Text
  43. ↵
    1. Heming B. S.
    (1979). Origin and fate of germ cells in male and female embryos of Haplothrips verbasci (Osborn) (Insecta, Thysanoptera, Phlaeothripidae). J. Morphol. 160, 323–343. doi:10.1002/jmor.1051600305
    OpenUrlCrossRef
  44. ↵
    1. Heming B. S.,
    2. Huebner E.
    (1994). Development of the germ cells and reproductive primordia in male and female embryos of Rhodnius prolixus Stål (Hemiptera, Reduviidae). Can. J. Zool. 72, 1100–1119. doi:10.1139/z94-148
    OpenUrlCrossRef
  45. ↵
    1. Heymons R.
    (1895). Die Embryonalentwicklung Von Dermapteren Und Orthopteren. Jena: G. Fischer.
  46. ↵
    1. Hoekstra H. E.,
    2. Coyne J. A.
    (2007). The locus of evolution: evo devo and the genetics of adaptation. Evolution 61, 995–1016. doi:10.1111/j.1558-5646.2007.00105.x
    OpenUrlCrossRefPubMedWeb of Science
  47. ↵
    1. Huettner A. F.
    (1923). The origin of the germ cells in Drosophila melanogaster. J. Morphol. 37, 385–423. doi:10.1002/jmor.1050370204
    OpenUrlCrossRef
  48. ↵
    1. Hurst S.,
    2. Talbot N. J.,
    3. Stebbings H.
    (1999). A staufen-like RNA-binding protein in translocation channels linking nurse cells to oocytes in Notonecta shows nucleotide-dependent attachment to microtubules. J. Cell Sci. 112, 2947–2955.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Hyams J. S.,
    2. Stebbings H.
    (1979). The formation and breakdown of nutritive tubes – massive microtubular organelles associated with cytoplasmic transport. J. Ultrastruct. Res. 68, 46–57. doi:10.1016/S0022-5320(79)90141-2
    OpenUrlCrossRefPubMed
  50. ↵
    1. Jan E.,
    2. Thompson S. R.,
    3. Wilson J. E.,
    4. Pestova T. V.,
    5. Hellen C. U.,
    6. Sarnow P.
    (2001). Initiator Met-tRNA-independent translation mediated by an internal ribosome entry site element in cricket paralysis virus-like insect viruses. Cold Spring Harb. Symp. Quant. Biol. 66, 285–292. doi:10.1101/sqb.2001.66.285
    OpenUrlAbstract/FREE Full Text
  51. ↵
    1. Johnstone O.,
    2. Lasko P.
    (2004). Interaction with eIF5B is essential for Vasa function during development. Development 131, 4167–4178. doi:10.1242/dev.01286
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Juhn J.,
    2. James A. A.
    (2006). oskar gene expression in the vector mosquitoes, Anopheles gambiae and Aedes aegypti. Insect Mol. Biol.15, 363–372. doi:10.1111/j.1365-2583.2006.00655.x
    OpenUrlCrossRefPubMedWeb of Science
  53. ↵
    1. Juhn J.,
    2. Marinotti O.,
    3. Calvo E.,
    4. James A. A.
    (2008). Gene structure and expression of nanos (nos) and oskar (osk) orthologues of the vector mosquito, Culex quinquefasciatus. Insect Mol. Biol. 17, 545–552. doi:10.1111/j.1365-2583.2008.00823.x
    OpenUrlCrossRefPubMed
  54. ↵
    1. Juliano C. E.,
    2. Swartz S. Z.,
    3. Wessel G. M.
    (2010). A conserved germline multipotency program. Development 137, 4113–4126. doi:10.1242/dev.047969
    OpenUrlAbstract/FREE Full Text
  55. ↵
    1. Kainz F.,
    2. Ewen–Campen B.,
    3. Akam M.,
    4. Extavour C. G.
    (2011). Notch/Delta signalling is not required for segment generation in the basally branching insect Gryllus bimaculatus. Development 138, 5015–5026. doi:10.1242/dev.073395
    OpenUrlAbstract/FREE Full Text
    1. Kaye J. S.,
    2. McMaster–Kaye R.
    (1966). The fine structure and chemical composition of nuclei during spermiogenesis in the house cricket. I. Initial stages of differentiation and the loss of nonhistone protein. J. Cell Biol. 31, 159–179. doi:10.1083/jcb.31.1.159
    OpenUrlAbstract/FREE Full Text
  56. ↵
    1. Kelly G. M.,
    2. Huebner E.
    (1989). Embryonic development of the hemipteran insect Rhodnius prolixus. J. Morphol. 199, 175–196. doi:10.1002/jmor.1051990205
    OpenUrlCrossRef
  57. ↵
    1. Khila A.,
    2. Abouheif E.
    (2010). Evaluating the role of reproductive constraints in ant social evolution. Philos. Trans. R. Soc. B 365, 617–630. doi:10.1098/rstb.2009.0257
    OpenUrlAbstract/FREE Full Text
  58. ↵
    1. Kim–Ha J.,
    2. Kerr K.,
    3. Macdonald P. M.
    (1995). Translational regulation of oskar mRNA by Bruno, an ovarian RNA-binding protein, is essential. Cell 81, 403–412. doi:10.1016/0092-8674(95)90393-3
    OpenUrlCrossRefPubMedWeb of Science
  59. ↵
    1. Kozmik Z.
    (2005). Pax genes in eye development and evolution. Curr. Opin. Genet. Dev. 15, 430–438. doi:10.1016/j.gde.2005.05.001
    OpenUrlCrossRefPubMedWeb of Science
  60. ↵
    1. Kumé M.,
    2. Dan K.
    (1968). Invertebrate Embryology. Belgrade: Prosveta.
  61. ↵
    1. Lasko P. F.,
    2. Ashburner M.
    (1990). Posterior localization of vasa protein correlates with, but is not sufficient for, pole cell development. Genes Dev. 4, 905–921. doi:10.1101/gad.4.6.905
    OpenUrlAbstract/FREE Full Text
  62. ↵
    1. Lécuyer E.,
    2. Yoshida H.,
    3. Parthasarathy N.,
    4. Alm C.,
    5. Babak T.,
    6. Cerovina T.,
    7. Hughes T. R.,
    8. Tomancak P.,
    9. Krause H. M.
    (2007). Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131, 174–187. doi:10.1016/j.cell.2007.08.003
    OpenUrlCrossRefPubMedWeb of Science
  63. ↵
    1. Li M.,
    2. Hong N.,
    3. Xu H.,
    4. Yi M.,
    5. Li C.,
    6. Gui J.,
    7. Hong Y.
    (2009). Medaka vasa is required for migration but not survival of primordial germ cells. Mech. Dev. 126, 366–381. doi:10.1016/j.mod.2009.02.004
    OpenUrlCrossRefPubMed
  64. ↵
    1. Liang L.,
    2. Diehl–Jones W.,
    3. Lasko P.
    (1994). Localization of vasa protein to the Drosophila pole plasm is independent of its RNA-binding and helicase activities. Development 120, 1201–1211.
    OpenUrlAbstract
  65. ↵
    1. Liu P. Z.,
    2. Kaufman T. C.
    (2004). hunchback is required for suppression of abdominal identity, and for proper germband growth and segmentation in the intermediate germband insect Oncopeltus fasciatus. Development 131, 1515–1527. doi:10.1242/dev.01046
    OpenUrlAbstract/FREE Full Text
  66. ↵
    1. Lu H. L.,
    2. Tanguy S.,
    3. Rispe C.,
    4. Gauthier J. P.,
    5. Walsh T.,
    6. Gordon K.,
    7. Edwards O.,
    8. Tagu D.,
    9. Chang C. C.,
    10. Jaubert–Possamai S.
    (2011). Expansion of genes encoding piRNA-associated argonaute proteins in the pea aphid: diversification of expression profiles in different plastic morphs. PLoS ONE 6, e28051. doi:10.1371/journal.pone.0028051
    OpenUrlCrossRefPubMed
  67. ↵
    1. Lynch J. A.,
    2. Desplan C.
    (2010). Novel modes of localization and function of nanos in the wasp Nasonia. Development 137, 3813–3821. doi:10.1242/dev.054213
    OpenUrlAbstract/FREE Full Text
  68. ↵
    1. Lynch J. A.,
    2. Ozüak O.,
    3. Khila A.,
    4. Abouheif E.,
    5. Desplan C.,
    6. Roth S.
    (2011). The phylogenetic origin of oskar coincided with the origin of maternally provisioned germ plasm and pole cells at the base of the Holometabola. PLoS Genet. 7, e1002029. doi:10.1371/journal.pgen.1002029
    OpenUrlCrossRefPubMed
  69. ↵
    1. Magnúsdóttir E.,
    2. Gillich A.,
    3. Grabole N.,
    4. Surani M. A.
    (2012). Combinatorial control of cell fate and reprogramming in the mammalian germline. Curr. Opin. Genet. Dev. 22, 466–474. doi:10.1016/j.gde.2012.06.002
    OpenUrlCrossRefPubMed
  70. ↵
    1. Mahowald A. P.
    (2001). Assembly of the Drosophila germ plasm. Int. Rev. Cytol. 203, 187–213. doi:10.1016/S0074-7696(01)03007-8
    OpenUrlCrossRefPubMedWeb of Science
  71. ↵
    1. Medrano J. V.,
    2. Ramathal C.,
    3. Nguyen H. N.,
    4. Simon C.,
    5. Reijo Pera R. A.
    (2012). Divergent RNA-binding proteins, DAZL and VASA, induce meiotic progression in human germ cells derived in vitro. Stem Cells 30, 441–451. doi:10.1002/stem.1012
    OpenUrlCrossRefPubMedWeb of Science
  72. ↵
    1. Mellanby H.
    (1935). The early embryonic development of Rhodnius prolixus (Hemiptera, Heteroptera). Q. J. Microsc. Sci. 78, 71–90.
    OpenUrl
  73. ↵
    1. Metschnikoff E.
    (1866). Embryologische studien an insekten. Z. Wiss. Zool. 16, 389–500.
    OpenUrl
  74. ↵
    1. Miura T.,
    2. Braendle C.,
    3. Shingleton A.,
    4. Sisk G.,
    5. Kambhampati S.,
    6. Stern D. L.
    (2003). A comparison of parthenogenetic and sexual embryogenesis of the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea). J. Exp. Zool. B Mol. Dev. Evol. 295, 59–81.
    OpenUrlCrossRefPubMed
  75. ↵
    1. Moczek A. P.
    (2008). On the origins of novelty in development and evolution. Bioessays 30, 432–447. doi:10.1002/bies.20754
    OpenUrlCrossRefPubMedWeb of Science
  76. ↵
    1. Nakao H.
    (1999). Isolation and characterization of a Bombyx vasa-like gene. Dev. Genes Evol. 209, 312–316. doi:10.1007/s004270050257
    OpenUrlCrossRefPubMed
  77. ↵
    1. Nakao H.,
    2. Hatakeyama M.,
    3. Lee J. M.,
    4. Shimoda M.,
    5. Kanda T.
    (2006). Expression pattern of Bombyx vasa-like (BmVLG) protein and its implications in germ cell development. Dev. Genes Evol. 216, 94–99. doi:10.1007/s00427-005-0033-8
    OpenUrlCrossRefPubMed
  78. ↵
    1. Nelsen O. E.
    (1934). The segregation of the germ cells in the grasshopper, Melanoplus differentialis (Acrididae; Orthoptera). J. Morphol. 55, 545–575. doi:10.1002/jmor.1050550306
    OpenUrlCrossRef
  79. ↵
    1. Nieuwkoop P. D.,
    2. Sutasurya L. A.
    (1981). Primordial Germ Cells In The Invertebrates: From Epigenesis To Preformation. Cambridge: Cambridge University Press.
  80. ↵
    1. Nousch M.,
    2. Eckmann C. R.
    (2013). Translational control in the Caenorhabditis elegans germ line. Adv. Exp. Med. Biol. 757, 205–247. doi:10.1007/978-1-4614-4015-4_8
    OpenUrlCrossRefPubMed
  81. ↵
    1. Ohashi H.,
    2. Umeda N.,
    3. Hirazawa N.,
    4. Ozaki Y.,
    5. Miura C.,
    6. Miura T.
    (2007). Expression of vasa (vas)-related genes in germ cells and specific interference with gene functions by double-stranded RNA in the monogenean, Neobenedenia girellae. Int. J. Parasitol. 37, 515–523. doi:10.1016/j.ijpara.2006.11.003
    OpenUrlCrossRefPubMed
  82. ↵
    1. Özhan–Kizil G.,
    2. Havemann J.,
    3. Gerberding M.
    (2009). Germ cells in the crustacean Parhyale hawaiensis depend on Vasa protein for their maintenance but not for their formation. Dev. Biol. 327, 230–239. doi:10.1016/j.ydbio.2008.10.028
    OpenUrlCrossRefPubMedWeb of Science
    1. Panfilio K. A.
    (2008). Extraembryonic development in insects and the acrobatics of blastokinesis. Dev. Biol. 313, 471–491. doi:10.1016/j.ydbio.2007.11.004
    OpenUrlCrossRefPubMed
  83. ↵
    1. Pek J. W.,
    2. Anand A.,
    3. Kai T.
    (2012). Tudor domain proteins in development. Development 139, 2255–2266. doi:10.1242/dev.073304
    OpenUrlAbstract/FREE Full Text
  84. ↵
    1. Rangan P.,
    2. DeGennaro M.,
    3. Jaime–Bustamante K.,
    4. Coux R.–X.,
    5. Martinho R. G.,
    6. Lehmann R.
    (2009). Temporal and spatial control of germ-plasm RNAs. Curr. Biol. 19, 72–77. doi:10.1016/j.cub.2008.11.066
    OpenUrlCrossRefPubMed
  85. ↵
    1. Richardson B. E.,
    2. Lehmann R.
    (2010). Mechanisms guiding primordial germ cell migration: strategies from different organisms. Nat. Rev. Mol. Cell Biol. 11, 37–49. doi:10.1038/nrm2815
    OpenUrlCrossRefPubMedWeb of Science
  86. ↵
    1. Richter J. D.,
    2. Lasko P.
    (2011). Translational control in oocyte development. Cold Spring Harb. Perspect. Biol. 3, a002758. doi:10.1101/cshperspect.a002758
    OpenUrlAbstract/FREE Full Text
  87. ↵
    1. Rocak S.,
    2. Linder P.
    (2004). DEAD-box proteins: the driving forces behind RNA metabolism. Nat. Rev. Mol. Cell Biol. 5, 232–241. doi:10.1038/nrm1335
    OpenUrlCrossRefPubMedWeb of Science
  88. ↵
    1. Roonwal M. L.
    (1937). Studies on the embryology of the african migratory locust, Locusta migratoria migratoides Reiche and Frm. (Orthoptera, Acrididae). II. Organogeny. Philos. Trans. R. Soc. B 227, 175–244. doi:10.1098/rstb.1937.0003
    OpenUrlFREE Full Text
  89. ↵
    1. Salinas L. S.,
    2. Maldonado E.,
    3. Macías–Silva M.,
    4. Blackwell T. K.,
    5. Navarro R. E.
    (2007). The DEAD box RNA helicase VBH-1 is required for germ cell function in C. elegans. Genesis 45, 533–546. doi:10.1002/dvg.20323
    OpenUrlCrossRefPubMed
  90. ↵
    1. Salinas L. S.,
    2. Franco–Cea A.,
    3. Láscarez–Lagunas L. I.,
    4. Villanueva–Chimal E.,
    5. Maldonado E.,
    6. Navarro R. E.
    (2012). Germ cell survival in C. elegans and C. remanei is affected when the DEAD box RNA helicases VBH-1 or Cre-VBH-1 are silenced. Genesis 50, 801–818. doi:10.1002/dvg.22043
    OpenUrlCrossRefPubMed
  91. ↵
    1. Sander K.,
    2. Mirza M. B.
    (1956). The Early Embryology Of Pyrilla Perpusilla Walker (Homoptera), Including Some Observations On The Later Development (On Indian Insect Types, no. 4), p. 61. Aligarh: Aligarh Muslim University Publications.
  92. ↵
    1. Sasaki J.,
    2. Nakashima N.
    (2000). Methionine-independent initiation of translation in the capsid protein of an insect RNA virus. Proc. Natl. Acad. Sci. USA 97, 1512–1515. doi:10.1073/pnas.010426997
    OpenUrlAbstract/FREE Full Text
  93. ↵
    1. Schmidt E. D.,
    2. Dorn A.
    (2004). Structural polarity and dynamics of male germline stem cells in the milkweed bug (Oncopeltus fasciatus). Cell Tissue Res. 318, 383–394. doi:10.1007/s00441-004-0983-6
    OpenUrlCrossRefPubMed
  94. ↵
    1. Schröder R.
    (2006). vasa mRNA accumulates at the posterior pole during blastoderm formation in the flour beetle Tribolium castaneum. Dev. Genes Evol. 216, 277–283. doi:10.1007/s00427-005-0054-3
    OpenUrlCrossRefPubMed
  95. ↵
    1. Schüpbach T.,
    2. Wieschaus E.
    (1986). Maternal-effect mutations altering the anterior-posterior pattern of the Drosophila embryo. Roux. Arch. Dev. Biol. 195, 302–317. doi:10.1007/BF00376063
    OpenUrlCrossRef
  96. ↵
    1. Schüpbach T.,
    2. Wieschaus E.
    (1991). Female sterile mutations on the second chromosome of Drosophila melanogaster. II. Mutations blocking oogenesis or altering egg morphology. Genetics 129, 1119–1136.
    OpenUrlAbstract/FREE Full Text
  97. ↵
    1. Schwalm F. E.
    (1965). Zell – und mitosenmuster der normalen und nach rötgenbestrahlung regulierenden keimanlage von Gryllus domesticus. Zeitschrift für Morphologie und Ökologie der Tiere 55, 915–1023. doi:10.1007/BF00407848
    OpenUrlCrossRef
  98. ↵
    1. Seidel F.
    (1924). Die geschlechtsorgane in der embryonalentwicklung von pyrrhocoris apterus L. Zeitschrift für Morphologie und Ökologie der Tiere 1, 429–506. doi:10.1007/BF00407470
    OpenUrlCrossRef
  99. ↵
    1. Sengupta M. S.,
    2. Boag P. R.
    (2012). Germ granules and the control of mRNA translation. IUBMB Life 64, 586–594. doi:10.1002/iub.1039
    OpenUrlCrossRefPubMed
  100. ↵
    1. Shah C.,
    2. Vangompel M. J. W.,
    3. Naeem V.,
    4. Chen Y.,
    5. Lee T.,
    6. Angeloni N.,
    7. Wang Y.,
    8. Xu E. Y.
    (2010). Widespread presence of human BOULE homologs among animals and conservation of their ancient reproductive function. PLoS Genet. 6, e1001022. doi:10.1371/journal.pgen.1001022
    OpenUrlCrossRefPubMed
  101. ↵
    1. Spike C.,
    2. Meyer N.,
    3. Racen E.,
    4. Orsborn A.,
    5. Kirchner J.,
    6. Kuznicki K.,
    7. Yee C.,
    8. Bennett K.,
    9. Strome S.
    (2008). Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins. Genetics 178, 1973–1987. doi:10.1534/genetics.107.083469
    OpenUrlAbstract/FREE Full Text
  102. ↵
    1. Stebbings H.,
    2. Hunt C.
    (1987). The translocation of mitochondria along insect ovarian microtubules from isolated nutritive tubes: a simple reactivated model. J. Cell Sci. 88, 641–648.
    OpenUrlAbstract/FREE Full Text
  103. ↵
    1. Stebbings H.,
    2. Sharma K.,
    3. Hunt C.
    (1985). Protein turnover in the cytoplasmic transport system within an insect ovary – a clue to the mechanism of microtubule-associated transport. FEBS Lett. 193, 22–26. doi:10.1016/0014-5793(85)80071-5
    OpenUrlCrossRefPubMed
  104. ↵
    1. Stephen S.,
    2. Talbot N. J.,
    3. Stebbings H.
    (1999). Poly(A) mRNA is attached to insect ovarian microtubules in vivo in a nucleotide-sensitive manner. Cell Motil. Cytoskeleton 43, 159–166. doi:10.1002/(SICI)1097-0169(1999)43:2<159::AID-CM7>3.0.CO;2-U
    OpenUrlCrossRefPubMedWeb of Science
  105. ↵
    1. Stern D. L.,
    2. Orgogozo V.
    (2008). The loci of evolution: how predictable is genetic evolution? Evolution 62, 2155–2177. doi:10.1111/j.1558-5646.2008.00450.x
    OpenUrlCrossRefPubMedWeb of Science
  106. ↵
    1. Styhler S.,
    2. Nakamura A.,
    3. Swan A.,
    4. Suter B.,
    5. Lasko P.
    (1998). vasa is required for GURKEN accumulation in the oocyte, and is involved in oocyte differentiation and germline cyst development. Development 125, 1569–1578.
    OpenUrlAbstract
  107. ↵
    1. Sunanaga T.,
    2. Watanabe A.,
    3. Kawamura K.
    (2007). Involvement of vasa homolog in germline recruitment from coelomic stem cells in budding tunicates. Dev. Genes Evol. 217, 1–11. doi:10.1007/s00427-006-0112-5
    OpenUrlCrossRefPubMedWeb of Science
  108. ↵
    1. Tanaka S. S.,
    2. Toyooka Y.,
    3. Akasu R.,
    4. Katoh–Fukui Y.,
    5. Nakahara Y.,
    6. Suzuki R.,
    7. Yokoyama M.,
    8. Noce T.
    (2000). The mouse homolog of Drosophila Vasa is required for the development of male germ cells. Genes Dev. 14, 841–853. doi:10.1101/gad.14.7.841
    OpenUrlAbstract/FREE Full Text
  109. ↵
    1. Tomancak P.,
    2. Guichet A.,
    3. Zavorszky P.,
    4. Ephrussi A.
    (1998). Oocyte polarity depends on regulation of gurken by Vasa. Development 125, 1723–1732.
    OpenUrlAbstract
  110. ↵
    1. Tomancak P.,
    2. Beaton A.,
    3. Weiszmann R.,
    4. Kwan E.,
    5. Shu S.,
    6. Lewis S. E.,
    7. Richards S.,
    8. Ashburner M.,
    9. Hartenstein V.,
    10. Celniker S. E.
    et al. (2002). Systematic determination of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 3, research0088.1–research0088.14. doi:10.1186/gb-2002-3-12-research0088
    OpenUrlCrossRef
  111. ↵
    1. Tomancak P.,
    2. Berman B. P.,
    3. Beaton A.,
    4. Weiszmann R.,
    5. Kwan E.,
    6. Hartenstein V.,
    7. Celniker S. E.,
    8. Rubin G. M.
    (2007). Global analysis of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 8, R145. doi:10.1186/gb-2007-8-7-r145
    OpenUrlCrossRefPubMed
  112. ↵
    1. Tsunekawa N.,
    2. Naito M.,
    3. Sakai Y.,
    4. Nishida T.,
    5. Noce T.
    (2000). Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development 127, 2741–2750.
    OpenUrlAbstract
  113. ↵
    1. Wang C.,
    2. Lehmann R.
    (1991). Nanos is the localized posterior determinant in Drosophila. Cell 66, 637–647. doi:10.1016/0092-8674(91)90110-K
    OpenUrlCrossRefPubMedWeb of Science
  114. ↵
    1. Webster P. J.,
    2. Liang L.,
    3. Berg C. A.,
    4. Lasko P.,
    5. Macdonald P. M.
    (1997). Translational repressor bruno plays multiple roles in development and is widely conserved. Genes Dev. 11, 2510–2521. doi:10.1101/gad.11.19.2510
    OpenUrlAbstract/FREE Full Text
  115. ↵
    1. Wheeler W. M.
    (1893). A contribution to insect embryology. J. Morphol. 8, 1–161. doi:10.1002/jmor.1050080102
    OpenUrlCrossRef
  116. ↵
    1. Will L.
    (1888). Entwicklungsgeschichte der viviparen aphiden. Zoologische Jahrbücher 3, 201–280.
    OpenUrl
  117. ↵
    1. Witlaczil E.
    (1884). Entwicklungsgeschichte der aphiden. Z. Wiss. Zool. 40, 559–696.
    OpenUrl
  118. ↵
    1. Wu H.–R.,
    2. Chen Y.–T.,
    3. Su Y.–H.,
    4. Luo Y.–J.,
    5. Holland L. Z.,
    6. Yu J.–K.
    (2011). Asymmetric localization of germline markers Vasa and Nanos during early development in the amphioxus Branchiostoma floridae. Dev. Biol. 353, 147–159. doi:10.1016/j.ydbio.2011.02.014
    OpenUrlCrossRefPubMed
  119. ↵
    1. Yajima M.,
    2. Wessel G. M.
    (2011). The multiple hats of Vasa: its functions in the germline and in cell cycle progression. Mol. Reprod. Dev. 78, 861–867. doi:10.1002/mrd.21363
    OpenUrlCrossRefPubMed
  120. ↵
    1. Yeates D. K.,
    2. Cameron S. L.,
    3. Trautwein M.
    (2012). A view from the edge of the forest: recent progress in understanding the relationships of the insect orders. Aust. J. Entomol. 51, 79–87. doi:10.1111/j.1440-6055.2012.00857.x
    OpenUrlCrossRef
  121. ↵
    1. Ying M.,
    2. Chen D.
    (2012). Tudor domain-containing proteins of Drosophila melanogaster. Dev. Growth Differ. 54, 32–43. doi:10.1111/j.1440-169X.2011.01308.x
    OpenUrlCrossRefPubMed
  122. ↵
    1. Yoon C.,
    2. Kawakami K.,
    3. Hopkins N.
    (1997). Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells. Development 124, 3157–3165.
    OpenUrlAbstract
  123. ↵
    1. Zeng V.,
    2. Extavour C. G.
    (2012). ASGARD: an open-access database of annotated transcriptomes for emerging model arthropod species. Database 2012, bas048. doi:10.1093/database/bas048
    OpenUrlAbstract/FREE Full Text
  124. ↵
    1. Zhao G.,
    2. Chen K.,
    3. Yao Q.,
    4. Wang W.,
    5. Wang Y.,
    6. Mu R.,
    7. Chen H.,
    8. Yang H.,
    9. Zhou H.
    (2008). The nanos gene of Bombyx mori and its expression patterns in developmental embryos and larvae tissues. Gene Expr. Patterns 8, 254–260. doi:10.1016/j.gep.2007.12.006
    OpenUrlCrossRefPubMed
  125. ↵
    1. Zhurov V.,
    2. Terzin T.,
    3. Grbić M.
    (2004). Early blastomere determines embryo proliferation and caste fate in a polyembryonic wasp. Nature 432, 764–769. doi:10.1038/nature03171
    OpenUrlCrossRefPubMed
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  • vasa
  • tudor
  • boule
  • Germ line
  • RNA interference
  • Spermatogenesis

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Research Article
Evidence against a germ plasm in the milkweed bug Oncopeltus fasciatus, a hemimetabolous insect
Ben Ewen-Campen, Tamsin E. M. Jones, Cassandra G. Extavour
Biology Open 2013 2: 556-568; doi: 10.1242/bio.20134390
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Research Article
Evidence against a germ plasm in the milkweed bug Oncopeltus fasciatus, a hemimetabolous insect
Ben Ewen-Campen, Tamsin E. M. Jones, Cassandra G. Extavour
Biology Open 2013 2: 556-568; doi: 10.1242/bio.20134390

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