The Hox genes Ultrabithorax and abdominal-A specify three different types of abdominal appendage in the springtail Orchesella cincta (Collembola)
© Konopova and Akam; licensee BioMed Central Ltd. 2014
Received: 16 September 2013
Accepted: 20 November 2013
Published: 7 January 2014
In Drosophila and many other insects, the Hox genes Ultrabithorax (Ubx) and abdominal-A (abd-A) suppress limb formation on most or all segments of the abdomen. However, a number of basal hexapod lineages retain multiple appendages on the abdomen. In the collembolans or springtails, three abdominal segments develop specialized organs that originate from paired appendage primordia which fuse at the midline: the first abdominal segment bears the collophore (ventral tube), involved in osmoregulation; the fourth segment bears the furca, the leaping organ, and the third segment bears the retinaculum, which retains the furca at rest. Ubx and abd-A are known to be expressed in the springtail abdomen, but what role they play in specifying these distinct abdominal appendages is not known. This is largely because no genetic model has been established in collembolans or any other non-insect hexapod.
We have developed a convenient method for laboratory culture of the collembolan Orchesella cincta on defined media, a method for in-situ hybridization to embryos and a procedure for gene knockdown by parental injection of double-stranded RNA (RNAi). We show that Orchesella Ubx transcripts are detectable in the first to third abdominal segments, and abd-A transcripts in the second to fourth segments. Knockdown of Oc-Ubx leads to the homeotic transformation of the collophore into a pair of walking legs (a more anterior identity) but the retinaculum into a furca (a more posterior identity). Knockdown of Oc-abd-A leads to the transformation of the retinaculum into a collophore and of the furca into legs (both anterior transformations). Simultaneous silencing of both Oc-Ubx and Oc-abd-A transformed all three of these appendages into paired legs, but did not cause appendages to develop on the second, or on the most posterior abdominal segments.
We conclude that, in Orchesella, Oc-Ubx alone specifies the collophore on the first and Oc-abd-A alone specifies the furca on the fourth abdominal segment. Oc-Ubx and Oc-abd-A function together, apparently combinatorially, to specify the retinaculum on the third segment. The efficiency of RNAi in Orchesella makes this an attractive model for further genetic studies of development and physiology in basal hexapods.
Keywordsabdominal-A Appendage specification Collembola Evolution Hox Homeosis RNA interference Segment identity Ultrabithorax
Springtails (Collembola), Protura and Diplura, together with the insects proper, form a clade of arthropods called the Hexapoda. A defining feature of hexapods is that their body has six legs, a pair growing from each of the three thoracic segments [1, 2]. In larvae and adults of the more basal hexapod lineages, such as the springtails, Protura, Diplura and wingless insects, small limbs or other appendages also develop on the abdomen. In this respect, these basal lineages resemble many crustaceans, from some lineage of which the Hexapoda originated [2–4]. In the winged insects (Pterygota), abdominal appendages may develop in larvae, but are lost during metamorphosis to the adult.
In general, the distinct identities of segments in insects are specified by Hox genes : in the absence of Hox gene function, all segments of the trunk develop similarly (reviewed in ). The Hox genes encode transcription factors that modulate many aspects of segment development, by interacting with a large number of downstream targets . They are expressed from head to tail in partially overlapping domains, but typically, the more posteriorly expressed gene dominates phenotypically over the more anterior one. Changes in the domains of expression of these genes, and changes in the set of downstream targets that they regulate, have been shown to play a significant role in the evolution of arthropod diversity [15–24].
In insects, the distinct identities of different segments in the pre-genital abdomen are specified by the Hox genes Ultrabithorax (Ubx) and abdominal-A (abd-A) (reviewed in [25, 26]), and by Abdominal-B, which plays a major role in the genital segments, and a minor role more anteriorly [27–29]. We do not consider Abd-B further here.
In Drosophila, and most other insects studied, Ubx is initially expressed within A1 (strictly speaking, from the parasegment 5/6 boundary back) while abd-A is expressed from A2 (parasegment 6/7 boundary) back [26, 30–33]. It is relatively well understood how this early Ubx and abd-A expression represses limbs in Drosophila[34, 35]. Slightly later in development, Ubx expression extends anteriorly into the thorax. This later expression is unable to repress limb development, but it modifies the identity of the limbs that form [22, 35, 36].
While both Ubx and abd-A suppress appendages in Drosophila, in the beetle Tribolium, and probably also in many other less derived insects, Ubx does not repress appendage development in A1. Instead, it specifies on A1 the development of a pair of glandular appendages called pleuropods, which mature in the embryo and are shed at or before hatching [37, 38]. Knockdown of Ubx in embryos results in homeotic transformation of the pleuropods towards the phenotype of a walking leg.
How Ubx and abd-A might function to specify the multiple distinct abdominal appendages of basal hexapods is not known. An antibody that detects both Ubx and Abd-A was used by Palopoli and Patel  to show that either one or both of these proteins is present in each of the abdominal appendages of the springtails Folsomia and Xenylla, consistent with the hypothesis that these proteins play some role in appendage specification, but this reagent cannot discriminate between the two Hox proteins.
Despite their phylogenetic position at the base of the Hexapoda, no springtail species has been established for wide use in comparative developmental genetics, perhaps because most species are very small, the development of the larger species is often slow, and in many cases their embryos are either inaccessible or difficult to work with [40, 41]. The twin aims of our research were therefore: (1) to find a springtail that would be amenable for developmental and functional genetic experiments, and (2) to find out how Ubx and abd-A specify collembolan abdominal appendages.
Here we present our study on Ubx and abd-A function by parental RNAi in the springtail Orchesella cincta (Entomobryomorpha). Orchesella is a surface-dwelling soil springtail that reaches about 4 mm in size. It has sexual reproduction. It has previously been used for ecotoxicological experiments , and its genome has recently been sequenced (D. Roelofs, personal communication). We have generated an embryonic transcriptome. We show here that Orchesella can be raised on cultured algae and yeast as a food source and that the embryos are accessible for in-situ hybridization, albeit with some difficulty. We also show that parental RNAi is simple and effective for gene knockdown in this species. Using this technique, we show that both Oc-Ubx and Oc-abd-A have unique limb modulatory functions: Oc-Ubx on its own specifies the collophore, while Oc-abd-A on its own specifies the furca. Both Oc-Ubx and Oc-abd-A jointly specify the retinaculum.
Orchesella cincta culture
A culture of the springtail of Orchesella cincta was obtained from the Vrije Universiteit Amsterdam (kindly provided by Nico van Straalen, Dick Roelofs and Janine Mariën). Springtails were kept at 25°C with a photoperiod of 18 h light and 6 h dark in Petri dishes on a solid base made from plaster of Paris mixed with charcoal, covered with a layer of the alga Pleurococcus sp. (obtained from CCAP: the Culture Collection of Algae and Protozoa, Argyll, Scotland, UK) and a little baker’s yeast to serve as food. The cultures were moistened with algal liquid culture twice a week. Algae were grown in conical flasks with 3 N-BBM + V medium (recipe from CCAP website, http://www.ccap.ac.uk) standing on a windowsill. To prepare the dishes for springtails, the grown algal culture was poured onto Petri dishes with a solid base. Algae were left to settle for a few days, after which time, the liquid medium was poured out and soaked up with a piece of tissue.
Primers for cloning
Oc-Ubx full-length protein
Oc-abd-A RNAi and probe
Oc-abd-A full-length protein
Females lay eggs individually on the surface of the culture media. Eggs were collected from the Petri dishes using a fine soft paintbrush, fixed overnight in 4% formaldehyde in PBS + 0.1% Tween 20 and stored in methanol at -20°C. Eggshells (the blastodermic cuticles)  were manually removed using forceps. Samples were processed according to a published protocol  with the modification that PBS + 0.1% Tween 20 was used for washes and hybridization was at 55°C. The probe lengths were 1,251 bases for Oc-Ubx and 1,230 bases for Oc-abd-A.
dsRNAs at lengths of 725 bases for Oc-Ubx and 1,230 bases for Oc-abd-A were synthesized using the T3 and T7 Megascript kits (Ambion, Austin, TX). Orchesella females were anesthetized on a carbon dioxide plate and injected with dsRNAs at a concentration of 5 μg/μl until the abdomen was obviously inflated. After an overnight recovery, the females were kept together with males of a similar age. To check that the RNAi lowered levels of the targeted mRNAs, we performed in-situ hybridization on the Oc-Ubx(RNAi) and Oc-abd-A(RNAi) embryos and compared the intensity of staining with that in control embryos (Additional file 1). We detected a substantial reduction in staining intensity.
Samples for expression analyses were observed on a Zeiss Axioskop2 MOT plus compound microscope with a Zeiss AxioCam MRm camera, a Zeiss Axiophot compound microscope with Leica DFC300FX camera and a Leica TCS SP5 confocal microscope. For scanning electron microscopy, larvae were fixed in 80% ethanol, post-fixed with osmium tetroxide, dehydrated through an ethanol series, critical point dried, gold coated and observed on a FEI/Philips XL30 FEGSEM microscope. The photos were adjusted using Adobe Photoshop (version CS5) and Fiji.
Laboratory culture of Orchesella cincta
In Amsterdam, the source culture of Orchesella was maintained on twigs from trees overgrown by algae and sterilized by freezing (J. Mariën, personal communication). The algae and fungi on the twigs are a vital source of food [45, 46]. Because this method does not enable quick and easy inspection of the culture and timed egg collections, we developed a method of culturing on Petri dishes with defined media. In our culturing method we feed Orchesella the alga Pleurococcus (syn. Desmococcus), because this alga was previously found to be that most consumed by Orchesella in its natural habitat. Baker’s yeast forms the fungal component in the diet. All individuals in the dishes can easily be inspected, anesthetized by filling the dish with CO2, and tapped into a fresh dish.
Sequences of the Orchesella Ubx and abd-A genes
No full-length Hox protein sequences have previously been reported for springtails, though short Hox fragments have previously been isolated from the springtail Folsomia candida. We isolated cDNAs for the full protein-coding regions of Ubx and abd-A from embryos of Orchesella. Both genes contain the expected conserved sequence signatures, which include the homeodomain (HD), hexapeptide motif (HX) and UbdA peptide (Additional file 2 and Additional file 3). Two isoforms were recovered for both Oc-Ubx and Oc-abd-A. The isoforms differ in the length of the linker region (LR) between the hexapeptide and the homeodomain (8 vs 12 amino acids in Oc-Ubx; 27 vs 49 in Oc-abd-A). The linker in the long isoform of Oc-abd-A is noticeably long compared with that in other arthropod abd-A sequences.
A common feature of insect Ubx sequences is the presence of two motifs C-terminal to the UbdA peptide; the QAQA motif and the poly-alanine stretch, which were both shown to be important for the limb repressive role of Ubx in insects [47, 48]. The QAQA motif, but not the poly-alanine stretch, was found in Folsomia Ubx. Similarly, Oc-Ubx contains the QAQA motif, but lacks the poly-alanine stretch suggesting that this is a common feature of springtail Ubx. The C-termini of Ubx from diverse arthropods, but not the insects, contain several phosphorylation sites; the phosphorylation at these sites was shown to block the general ability of Ubx to repress appendages . The C-terminus of Folsomia Ubx contains one phosphorylation site. We did not find any predicted phosphorylation sites in Oc-Ubx C-terminal to the UbdA peptide, suggesting that the single site in Folsomia Ubx is not a common feature of springtail Ubx. The C-termini of the insect abd-A sequences are typically enriched in the amino acid glutamine, but we did not find any glutamine residues in the C-terminus of Oc-abd-A. The TD motif, which is a site for interaction with cofactors, and is present in insect but not other arthropod abd-A sequences , is missing in Oc-abd-A.
In summary, the Oc-Ubx sequence shows a combination of ancestral and novel (insect) features, while the Oc-abd-A is more similar to abd-A sequences from arthropods other than insects.
Embryonic expression of Oc-Ubx and Oc-abd-A
In early stages of expression both Oc-Ubx and Oc-abd-A were detected as two pairs of spots, which we identified as buds on A1 and A3 for Oc-Ubx and A3 and A4 for Oc-abd-A (Figure 2A,E). Expression of both genes was also visible in A2, but was less intense (Figure 2A,C). In slightly older embryos, the expression spread to other parts of the segments and became more intense (Figure 2B,F). In embryos with well-defined abdominal appendages, Oc-Ubx was evident in A1 to A3 and Oc-abd-A in A2 to A4 segments (Figure 2D,G). We could not see whether the expression domains extended also to the posterior part of the next most anterior segment (that is, T3p for Oc-Ubx; A1p for Oc-abd-A), as they do in many insects.
Knockdown of Oc-Ubx leads to homeotic transformation of the A1 and A3 appendages
To understand the function of Oc-Ubx and Oc-abd-A in Orchesella, we reduced the levels of gene function in embryos by injecting double-stranded RNA for each gene into the abdomens of female parents (a process colloquially known as 'knock down by parental RNAi’) . In Tribolium, and an increasing number of other insect species tested, this has been shown to reduce gene function, though there is considerable variability in both the degree and duration of knockdown for different genes, and in different species [51, 52].
Efficiency of Oc-Ubx and Oc-abd-A silencing by parental RNAi in Orchesella
Oc-Ubx + MalE
Oc-abd-A + MalE
Oc-Ubx + Oc-abd-A (injected once)
Oc-Ubx + Oc-abd-A (injected twice)
Larvae from the females injected by Oc-Ubx dsRNA also hatched normally, but 76.5% of the larvae that hatched from eggs collected within the first 10 days after the injections died at the end of the first instar and showed homeotic transformation of the A1 and A3 appendages (Table 2). The collophore on A1 was transformed into a pair of walking legs and the retinaculum on A3 into a structure resembling the furca (Figure 1B,E).
Additional file 4: The Oc-Ubx(RNAi) larvae move with their homeotic legs on A1 when they walk . The movie shows a larva slowly walking by using all four pairs of legs. The black arrow marks the A1 leg, the outlined arrows mark the thoracic legs. The first pair of legs is less visible, because it is kept just under the head. These larvae have problems with walking because of the voluminous furca on A3, which is the homeotically transformed retinaculum. As they do not have the retinaculum (the 'holder’ of the furca), the natural furca on A4 is not attached to the ventral side as normally, but hangs down and is usually dragged behind. The object in the front is an empty eggshell. The movie was captured by Olympus μ digital camera pointed at a computer screen projecting pictures from a Leica MZIII stereomicroscope adapted with a Leica DFC500 camera. (MOV 9 MB)
The ectopic furca on A3 resembled the normal furca on A4, except that even in the most strongly transformed individuals it was smaller (Figure 1E, Figure 3H; compare with Figure 3G). In summary, Oc-Ubx in Orchesella is required for giving the appendage buds on A1 the collophore identity and those on A3 the retinaculum identity; a general function of Oc-Ubx is to promote the fusion of paired appendage primordia.
Knockdown of Oc-abd-A leads to homeotic transformation of the A3 and A4 appendages
We next repeated the injections with Oc-abd-A dsRNA. As with Oc-Ubx(RNAi), injection of Oc-abd-A(RNAi) had no effect on hatching. In over 75 per cent of the resulting larvae, the retinaculum on A3 was transformed into a collophore and the furca on A4 was transformed into walking legs (Table 2). Strong transformations are illustrated in (Figure 1C and F). These larvae fed and walked until the end of the first instar, when most of them died. Only a few of the more weakly affected individuals started ecdysis to the second instar; these died during shedding the cuticle or soon after.
The collophore on A3 of the most strongly transformed individuals was similar in morphology and size to the endogenous collophore on A1 (Figure 1C,F; compare with Figure 1D; Figure 3F; compare with Figure 3E). The ectopic legs on A4 were segmented like legs; their proximal parts were separate (Figure 1C,F) and they had a claw at their tips (Figure 3D; compare with Figure 3A,C). They were not moved during walking. The ventral groove was extended from its normal ending at the collophore in A1 throughout A2 to the tip of the ectopic collophore on A3 and even behind it (Figure 4C; compare with Figure 4A,B); his suggests that in normal animals one role of Oc-abd-A is to inhibit development of the ventral groove in segments posterior to A1.
In summary, Oc-abd-A is required for specification of the retinaculum on A3 and the furca on A4. It induces fusion of the appendages (similarly to Oc-Ubx) and in the cuticle it represses the ventral groove. In the absence of Oc-abd-A, both the A3 and the A4 appendages transform to a more anterior fate.
Simultaneous knockdown of Oc-Ubx and Oc-abd-A transforms all Orchesella abdominal appendages into leg-like structures but does not induce appendages on A2
The experiments above show that when Orchesella abdominal appendages are missing the function of both Oc-Ubx and Oc-abd-A they have a walking leg identity. We observed this in the A1 appendages in Oc-Ubx(RNAi) and A4 appendages in Oc-abd-A(RNAi). We next wanted to test whether simultaneous silencing of Oc-Ubx and Oc-abd-A by double RNAi would transform all Orchesella abdominal appendages into legs, and whether it would allow the development of legs on A2, which normally makes no appendage.
Effective knockdown of two genes is difficult, because the maximum amount of each type of dsRNA that can be injected is reduced to one half compared with the single-gene RNAi. To probe the strength of double RNAi in Orchesella, we first co-injected mothers with dsRNA against one of the Hox genes mixed with control dsRNA. The penetrance of the phenotype was slightly reduced for both Oc-Ubx and Oc-abd-A, but the strong transformation was still observed in more than 38 percent of larvae (Table 2).
In summary, these experiments show that double RNAi is possible in Orchesella, because we achieved a novel transformation compared with single-gene RNAi.
In this study we have investigated the role of the Hox genes Ubx and abd-A in the specification of abdominal appendages in the springtail Orchesella. The results of our RNAi experiments are summarized diagrammatically in Figure 7. We conclude that:
The activity of Oc-Ubx alone causes appendage buds to develop as a collophore, because (i) when Oc-Ubx is knocked down, the buds on A1, which express Oc-Ubx but not Oc-abd-A, develop into legs, not a collophore; (ii) when Oc-abd-A is knocked down, the buds on A3, which normally express both Oc-Ubx and Oc-abd-A, develop into a collophore, presumably because depletion of Oc-abd-A leaves Oc-Ubx alone expressed in these buds.
The activity of Oc-abd-A alone causes appendage buds to develop as a furca, because (i) when Oc-abd-A is knocked down, the buds on A4, which express only Oc-abd-A, develop into legs, not the furca; (ii) when Oc-Ubx is knocked down, so that presumably Oc-abd-A alone is expressed in A3, the A3 buds develop into a furca.
If both Oc-Ubx and Oc-abd-A are expressed in a pair of limb buds, it develops as a retinaculum.
Oc-Ubx specifies a unique identity for A1 in hexapods
In collembolans, as in insects, A1 is the only abdominal segment that is specified by Ubx in the absence of abd-A. Silencing of Oc-Ubx in Orchesella replaces the collophore with a pair of walking legs. Similarly in insects, depletion of Ubx alone results in the appearance of leg structures on A1 [12, 26, 38, 53, 54]. In insects, in the absence of Ubx, expression of the more anterior Hox gene Antennapedia persists in A1, where it is normally repressed by Ubx, resulting in the development of a leg-like appendage. It is likely that the same happens in collembolans, although this has not been tested.
In dipterans and some other endopterygote insects, for example, the lepidopteran Manduca, Ubx functions as a limb repressor, and A1 bears no appendages. In many other species though, including the beetle Tribolium, the milkweed bug Oncopeltus and the cricket Acheta, Ubx specifies the embryonic glandular appendages on A1 called pleuropods [26, 37, 38, 53]. Like the collophore of springtails, pleuropods are vesicular structures that are likely involved in secretion or excretion, but unlike the collophore, they remain paired rather than forming a single midline organ, and they degenerate before hatching [57, 58]. It seems possible that the specification of this derived type of glandular appendage is a conserved ancestral role of Ubx in hexapods, and that the collophore in collembolans is a homologue of the insect pleuropods. Ubx presumably targets a unique set of genes in A1 to specify these organs. These targets have presumably been lost in Drosophila, concomitant with the acquisition of a limb suppression role, but they may have been conserved throughout most of hexapod evolution, as pleuropods are present in some advanced lineages of Endopterygota, such as Lepidoptera and Coleoptera.
Oc-abd-A does not repress appendage development in springtails
We argue that the role of Oc-abd-A expressed alone in the limb buds of Orchesella is to specify appendage buds to develop as the furca, and that in combination with Oc-Ubx it specifies the development of the retinaculum. It has been shown that the middle section of the furca expresses the gene dac, which is normally expressed in the middle part of the thoracic leg ; this suggests that the furca is homologous to the full-length leg. Thus, Oc- abd-A does not repress formation of any part of the leg, but modifies it. This is quite different from the general role of abd-A in the pterygote insects, such as Drosophila, Tribolium and Oncopeltus, where abd-A represses appendage bud formation from an early stage in development, by repressing the expression of the distal-less (dll) gene [26, 34, 60, 61]. The observation that the Orchesella abd-A sequence lacks several of the derived features seen in insects is consistent with this very different role.
There is a precedent for abd-A promoting specific appendage development in pterygote insects. In the larvae of the silkworm Bombyx, abd-A specifies the abdominal locomotory appendages known as prolegs, present on A3 to A6 [62, 63]. However, parsimony suggests that this is a secondary adaptation that evolved long after abd-A acquired a role in limb repression.
It is probably premature to speculate whether limb repression or appendage specification is the ancestral role of abd-A in hexapods until further studies of appendage specification are carried out in basal hexapods and apterygote insects. In Diplura, and in the primitively wingless insects Archaeognatha (jumping bristletails) and Zygentoma (silverfish), abdominal segments up to A9 may develop small appendages called styli and ventral sacs (for embryogenesis see, for example, ). The fossil record has shown that abdominal styli were also present in the adults of Palaeozoic pterygotes . It is clear, though, from studies of crustaceans and spiders [24, 66] that abd-A specifies unique appendage types in other arthropod groups, for example, in crustaceans that have distinct appendages on the pereon and pleon.
Combinatorial specification of the A3 appendage by both Oc-Ubx and Oc-abd-A
The limb buds on A3 express both Oc-Ubx and Oc-abd-A. Both are required for the development of a morphologically and functionally unique appendage, the retinaculum . Interestingly, when Oc-abd-A is removed, the A3 appendage acquires a more anterior identity (collophore), as is commonly the case in Hox knockdowns, but when Oc-Ubx is removed, the A3 appendage acquires a more posterior identity; that of the furca. In this case, the more posterior Hox gene expressed in A3, Oc-abd-A, does not show posterior prevalence [67, 68], but acts combinatorially with Oc-Ubx to specify the A3 identity.
It remains to be seen whether the combinatorial regulation of A3 appendage morphology by Oc-Ubx and Oc-abd-A is indeed a true combinatorial role of these two Hox proteins functioning together in individual limb bud cells, or whether distinct cell populations in the bud express one or the other of these two proteins at different times, and the combinatorial function is at the supracellular level. The limited resolution of our in-situ analysis cannot distinguish between these possibilities. This would require specific antibodies for Oc-Ubx and Oc-abd-A.
Why do no appendages develop on A2?
All body segments of arthropods have the potential to develop appendages. If appendages are absent in the segment, it is generally because they are suppressed by a Hox gene, which frequently acts by repressing dll expression, a developmental gene that acts in the early stages of limb initiation. The regulation of dll may either be direct or indirect, for example, via btd[34, 59, 69].
Springtails never have appendages on A2, and in those species tested, this segment does not express dll. We might therefore expect that one of the Hox genes expressed in the abdomen represses limb development in A2.
We have shown in Orchesella that both Oc-Ubx and Oc-abd-A are expressed in A2, although the expression is at lower levels, and perhaps initiated later, than in other segments. However, neither Oc-Ubx nor Oc-abd-A seems to repress limbs, because silencing either or both of these genes, sufficiently to transform other limb identities, never leads to the development of appendages on A2. What does suppress appendage primordia in A2 remains unknown.
One possibility is that a specific limb repressing Hox gene is expressed in A2. We have no evidence that other copies of Oc-Ubx or Oc-abd-A exist in the collembolan genome. No other copies are present in our embryonic transcriptome, and none was identified in an extensive screen of Folsomia Hox fragments by PCR. It is possible that Abd-B plays a role in A2, because in some arthropods its expression domain extends anteriorly (for example, [70, 71]). We consider this less likely, because Abd-B typically specifies the genital segments and in Orchesella the genital opening is localized between segments A5 and A6. This remains to be tested.
Alternatively, the development of limb buds on A2 might be suppressed not by a Hox gene, but by some upstream factor in the segmentation hierarchy, analogous to the gap genes in Drosophila. Such factors distinguish different regions along the anteroposterior axis before and during segment specification (reviewed in ). There is no reason a priori why they should not locally regulate dll or other early expressed limb specification factors, though we are aware of no precedents for such effects.
A single specimen of a collembolan having a retinaculum on A2 has been found in nature (cited in ). Clearly therefore, there is some gene whose malfunction can lead to the development of an appendage on A2. The fact that this appendage developed as a retinaculum is consistent with our observation that A2 normally expresses both Oc-Ubx and Oc-abd-A, as does the normal A3, which develops the retinaculum.
The role of Oc-Ubx and Oc-abd-A genes in structures other than the appendage primordia
Apart from its role in specification of the appendages, our results clearly show that Oc-abd-A has a role in the ventral epidermis, where it normally represses the ventral groove posterior to the collophore, on A2, A3 and A4.
It is possible that other ectodermal structures are modified by Oc-Ubx and Oc-abd-A knockdown, and therefore that whole segments are homeotically transformed. Apart from the ventral groove, however, there are no obvious characteristics of the cuticle morphology that are segment specific in the abdomen, and so might reveal these roles. We have not examined in detail either the size of cuticular regions in the different segments, or the precise distribution of bristles and other sensillae in the cuticle that might reveal such effects.
Orchesella as a springtail model
Because of their small size, springtails are often overlooked, but they are widespread and abundant soil-living organisms. They play an important role in soil decomposition and serve as models for ecotoxicological research [5, 73, 74]. Compared with other springtails Orchesella has several features that make it well suited for genetic experiments. It is conveniently large (for a springtail: 4 mm!), it has a relatively short generation time (1 month at 25°C), and it can be kept continuously in laboratory conditions. Orchesella is a surface-dwelling springtail adapted to heterogeneous moisture conditions and so is relatively resistant against desiccation [75, 76]. In natural conditions, Orchesella suffers from high mortality by predation, which is compensated by high fecundity ; in the culture, about 30 eggs are laid by a female every 2 to 3 days.
The homeotic transformations presented in this paper demonstrate that gene knockdown by parental RNAi functions well in Orchesella. It is likely that RNAi will work equally well for many other genes, allowing functional studies of both development and physiology. The current disadvantage of Orchesella is that the early embryonic stages (before the appendage buds are visible) are not accessible, because a tough egg cuticle is secreted at a very early stage of development. This problem applies to other collembolans that have been used for embryological studies . However, we hope that a way will be found to circumvent it.
The springtail Hox genes Ubx and abd-A each specify a distinct appendage type when acting on their own; together, they specify a third, novel, appendage identity.
RNAi functions well in Orchesella; the phylogenetic position of this species at the base of the Hexapoda, its ease of culture in the laboratory and the available genetic resources, suggest that it will be of further use for comparative genetic studies.
A2, A3…: first, second, third … abdominal segments
polymerase chain reaction
third thoracic segment.
We thank researchers at the Vrije Universiteit Amsterdam, Janine Mariën, Nico van Straalen and Dick Roelofs for providing us with Orchesella and advice. For further advice on springtails, we thank Frans Janssens (University of Antwerp) and Peter Shaw (University of Roehampton). We are grateful to Alison Smith and Maria Zori (University of Cambridge) for help with algal cultures, and The Eastern Sequence and Informatics Hub (EASIH, Cambridge, UK) for sequencing the Orchesella embryonic transcriptome. We thank Jeremy Skepper (University of Cambridge) for assistance with scanning electron microscopy. This work was supported by a Long-Term Human Frontier Science Program postdoctoral fellowship LT000733/2009-L (to BK) and BBSRC grant BB/K009133/1 (to MA).
- Snodgrass RE: Principles of Insect Morphology. 1993, Ithaca: Cornell University PressGoogle Scholar
- Grimaldi DA: 400 million years on six legs: on the origin and early evolution of Hexapoda. Arthropod Struct Dev. 2010, 39: 191-203. 10.1016/j.asd.2009.10.008.View ArticlePubMedGoogle Scholar
- Regier JC, Shultz JW, Zwick A, Hussey A, Ball B, Wetzer R, Martin JW, Cunningham CW: Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences. Nature. 2010, 463: 1079-1083. 10.1038/nature08742.View ArticlePubMedGoogle Scholar
- Rota-Stabelli O, Daley AC, Pisani D: Molecular timetrees reveal a Cambrian colonization of land and a new scenario for ecdysozoan evolution. Curr Biol. 2013, 23: 392-398. 10.1016/j.cub.2013.01.026.View ArticlePubMedGoogle Scholar
- Hopkin SP: Biology of the Springtails (Insecta: Collembola). 1997, Oxford: Oxford University PressGoogle Scholar
- Uemiya H, Ando H: Embryogenesis of a springtail, Tomocerus ishibashii (Collembola, Tomoceridae): external morphology. J Morphol. 1987, 191: 37-48. 10.1002/jmor.1051910105.View ArticleGoogle Scholar
- Nutman SR: Function of the ventral tube in Onychiurus armatus (Collembola). Nature. 1941, 148: 168-169.View ArticleGoogle Scholar
- Noble-Nesbitt J: A site of water and ionic exchange with the medium in Podura aquatica L. (Collembola, Isotomidae). J Exp Biol. 1963, 40: 701-711.Google Scholar
- Eisenbeis G: Physiological absorption of liquid water by Collembola: absorption of ventral tube at different salinities. J Insect Physiol. 1982, 28: 11-20. 10.1016/0022-1910(82)90017-8.View ArticleGoogle Scholar
- Verhoef HA, Bosman C, Bierenbroodspot A, Boer HH: Ultrastructure and function of the labial nephridia and the rectum of Orchesella cincta (L.) (Collembola). Cell Tissue Res. 1979, 198: 237-246.View ArticlePubMedGoogle Scholar
- Verhoef HA, Witteveen J, van der Woude HA, Joosse ENG: Morphology and function of the ventral groove of Collembola. Pedobiologia. 1983, 25: 3-9.Google Scholar
- Lewis EB: A gene complex controlling segmentation in Drosophila. Nature. 1978, 276: 565-570. 10.1038/276565a0.View ArticlePubMedGoogle Scholar
- Robertson LK, Mahaffey JW: Insect homeotic complex genes and development, lessons from Drosophila and beyond. Insect Development. Edited by: Gilbert LI. 2009, San Diego: Elsevier, 1-57.Google Scholar
- Pavlopoulos A, Akam M: Hox gene Ultrabithorax regulates distinct sets of target genes at successive stages of Drosophila haltere morphogenesis. Proc Natl Acad Sci U S A. 2011, 108: 2855-2860. 10.1073/pnas.1015077108.PubMed CentralView ArticlePubMedGoogle Scholar
- Averof M, Akam M: Hox genes and the diversification of insect and crustacean body plans. Nature. 1995, 376: 420-423. 10.1038/376420a0.View ArticlePubMedGoogle Scholar
- Warren RW, Nagy L, Selegue J, Gates J, Carroll S: Evolution of homeotic gene regulation and function in flies and butterflies. Nature. 1994, 372: 458-461. 10.1038/372458a0.View ArticlePubMedGoogle Scholar
- Averof M, Patel NH: Crustacean appendage evolution associated with changes in Hox gene expression. Nature. 1997, 388: 682-686. 10.1038/41786.View ArticlePubMedGoogle Scholar
- Stern DL: A role of Ultrabithorax in morphological differences between Drosophila species. Nature. 1998, 396: 463-466. 10.1038/24863.PubMed CentralView ArticlePubMedGoogle Scholar
- Weatherbee SD, Nijhout HF, Grunert LW, Halder G, Galant R, Selegue J, Carroll S: Ultrabithorax function in butterfly wings and the evolution of insect wing patterns. Curr Biol. 1999, 9: 109-115. 10.1016/S0960-9822(99)80064-5.View ArticlePubMedGoogle Scholar
- Tomoyasu Y, Wheeler SR, Denell RE: Ultrabithorax is required for membranous wing identity in the beetle Tribolium castaneum. Nature. 2005, 433: 643-647. 10.1038/nature03272.View ArticlePubMedGoogle Scholar
- Brena C, Chipman AD, Minelli A, Akam M: Expression of trunk Hox genes in the centipede Strigamia maritima: sense and anti-sense transcripts. Evol Dev. 2006, 8: 252-265. 10.1111/j.1525-142X.2006.00096.x.View ArticlePubMedGoogle Scholar
- Khila A, Abouheif E, Rowe L: Evolution of a novel appendage ground plan in water striders is driven by changes in the Hox gene Ultrabithorax. PLoS Genet. 2009, 5: e1000583-10.1371/journal.pgen.1000583.PubMed CentralView ArticlePubMedGoogle Scholar
- Pavlopoulos A, Kontarakis Z, Liubicich DM, Serano JM, Akam M, Patel NH, Averof M: Probing the evolution of appendage specialization by Hox gene misexpression in an emerging model crustacean. Proc Natl Acad Sci USA. 2009, 106: 13897-13902. 10.1073/pnas.0902804106.PubMed CentralView ArticlePubMedGoogle Scholar
- Khadjeh S, Turetzek N, Pechmann M, Schwager EE, Wimmer EA, Damen WG, Prpic NM: Divergent role of the Hox gene Antennapedia in spiders is responsible for the convergent evolution of abdominal limb repression. Proc Natl Acad Sci U S A. 2012, 109: 4921-4926. 10.1073/pnas.1116421109.PubMed CentralView ArticlePubMedGoogle Scholar
- Duncan I: The bithorax complex. Annu Rev Genet. 1987, 21: 285-319. 10.1146/annurev.ge.21.120187.001441.View ArticlePubMedGoogle Scholar
- Angelini DR, Liu PZ, Hughes CL, Kaufman TC: Hox gene function and interaction in the milkweed bug Oncopeltus fasciatus (Hemiptera). Dev Biol. 2005, 287: 440-455. 10.1016/j.ydbio.2005.08.010.View ArticlePubMedGoogle Scholar
- Casanova J, Sánchez-Herrero E, Morata G: Identification and characterization of a parasegment specific regulatory element of the Abdominal-B gene of Drosophila. Cell. 1986, 47: 627-636. 10.1016/0092-8674(86)90627-6.View ArticlePubMedGoogle Scholar
- Celniker SE, Sharma S, Keelan DJ, Lewis EB: The molecular genetics of the bithorax complex of Drosophila: cis-regulation in the Abdominal-B domain. EMBO J. 1990, 9: 4277-4286.PubMed CentralPubMedGoogle Scholar
- Kelsh R, Dawson I, Akam M: An analysis of Abdominal-B expression in the locust Schistocerca gregaria. Development. 1993, 117: 293-305.PubMedGoogle Scholar
- Akam ME, Martinez-Arias A: The distribution of Ultrabithorax transcripts in Drosophila embryos. EMBO J. 1985, 4: 1689-1700.PubMed CentralPubMedGoogle Scholar
- White RA, Wilcox M: Distribution of Ultrabithorax proteins in Drosophila. EMBO J. 1985, 4: 2035-2043.PubMed CentralPubMedGoogle Scholar
- Karch F, Bender W: Weiffenbach B: abdA expression in Drosophila embryos. Genes Dev. 1990, 4: 1573-1587. 10.1101/gad.4.9.1573.View ArticlePubMedGoogle Scholar
- Macias A, Casanova J, Morata G: Expression and regulation of the abd-A gene of Drosophila. Development. 1990, 110: 1197-1207.PubMedGoogle Scholar
- Vachon G, Cohen B, Pfeifle C, McGuffin ME, Botas J, Cohen SM: Homeotic genes of the Bithorax complex repress limb development in the abdomen of the Drosophila embryo through the target gene Distal-less. Cell. 1992, 71: 437-450. 10.1016/0092-8674(92)90513-C.View ArticlePubMedGoogle Scholar
- Castelli-Gair J, Akam M: How the Hox gene Ultrabithorax specifies two different segments: the significance of spatial and temporal regulation within metameres. Development. 1995, 121: 2973-2982.PubMedGoogle Scholar
- Mahfooz NS, Li H, Popadić A: Differential expression patterns of the hox gene are associated with differential growth of insect hind legs. Proc Natl Acad Sci U S A. 2004, 101: 4877-4882. 10.1073/pnas.0401216101.PubMed CentralView ArticlePubMedGoogle Scholar
- Bennett RL, Brown SJ, Denell RE: Molecular and genetic analysis of the Tribolium Ultrabithorax ortholog, Ultrathorax. Dev Genes Evol. 1999, 209: 608-619. 10.1007/s004270050295.View ArticlePubMedGoogle Scholar
- Lewis DL, DeCamillis M, Bennett RL: Distinct roles of the homeotic genes Ubx and abd-A in beetle embryonic abdominal appendage development. Proc Natl Acad Sci U S A. 2000, 97: 4504-4509. 10.1073/pnas.97.9.4504.PubMed CentralView ArticlePubMedGoogle Scholar
- Palopoli MF, Patel NH: Evolution of the interaction between Hox genes and a downstream target. Curr Biol. 1998, 8: 587-590.View ArticlePubMedGoogle Scholar
- Krzysztofowicz A: Ultrastructural studies on embryonic development of Tetrodontophora bielanensis (Waga) (Collembola). Formation of the 1st and 2nd blastodermal cuticles. Acta Biol Cracov. 1986, 28: 19-26.Google Scholar
- Schaeper ND, Wimmer EA, Prpic NM: Appendage patterning in the primitively wingless hexapods Thermobia domestica (Zygentoma: Lepismatidae) and Folsomia candida (Collembola: Isotomidae). Dev Genes Evol. 2013, 223: 341-350. 10.1007/s00427-013-0449-5.View ArticlePubMedGoogle Scholar
- Roelofs D, Janssens TKS, Timmermans MJTN, Nota B, Mariën J, Bochdanovits Z, Ylstra B, van Straalen NM: Adaptive differences in gene expression associated with heavy metal tolerance in the soil arthropod Orchesella cincta. Mol Ecol. 2009, 18: 3227-3239. 10.1111/j.1365-294X.2009.04261.x.View ArticlePubMedGoogle Scholar
- Uemiya H, Ando H: Blastodermic cuticles of a springtail, Tomocerus ishibashii Yosii (Collembola: Tomoceridae). Int J Insect Morphol Embryol. 1987, 16: 287-294. 10.1016/0020-7322(87)90001-8.View ArticleGoogle Scholar
- Schinko J, Posnien N, Kittelmann S, Koniszewski N, Bucher G: Single and double whole-mount in situ hybridization in red flour beetle (Tribolium) embryos. Cold Spring Harb Protoc. 2009, doi:10.1101/pdb.prot5258Google Scholar
- Verhoef HA, Prast JE, Verweij RA: Relative importance of fungi and algae in the diet and nitrogen nutrition of Orchesella cincta (L.) and Tomocerus minor (Lubbock) (Collembola). Funct Ecol. 1988, 2: 195-201. 10.2307/2389695.View ArticleGoogle Scholar
- Joosse ENG, Testerink GJ: The role of food in the population dynamics of Orchesella cincta (Linné) (Collembola). Oecologia. 1977, 29: 189-204. 10.1007/BF00345694.View ArticleGoogle Scholar
- Galant R, Carroll SB: Evolution of a transcriptional repression domain in an insect Hox protein. Nature. 2002, 415: 910-913. 10.1038/nature717.View ArticlePubMedGoogle Scholar
- Ronshaugen M, McGinnis N, McGinnis W: Hox protein mutation and macroevolution of the insect body plan. Nature. 2002, 415: 914-917. 10.1038/nature716.View ArticlePubMedGoogle Scholar
- Merabet S, Litim-Mecheri I, Karlsson D, Dixit R, Saadaoui M, Monier B, Brun C, Thor S, Vijayraghavan K, Perrin L, Pradel J, Graba Y: Insights into Hox protein function from a large scale combinatorial analysis of protein domains. PLoS Genet. 2011, 7 (10): e1002302-10.1371/journal.pgen.1002302.PubMed CentralView ArticlePubMedGoogle Scholar
- Bucher G, Scholten J, Klingler M: Parental RNAi in Tribolium (Coleoptera). Curr Biol. 2002, 12: R85-R86. 10.1016/S0960-9822(02)00666-8.View ArticlePubMedGoogle Scholar
- Belles X: Beyond Drosophila: RNAi in vivo and functional genomics in insects. Annu Rev Entomol. 2010, 55: 111-128. 10.1146/annurev-ento-112408-085301.View ArticlePubMedGoogle Scholar
- Miller SC, Miyata K, Brown SJ, Tomoyasu Y: Dissecting systemic RNA interference in the red flour beetle Tribolium castaneum: parameters affecting the efficiency of RNAi. PLoS One. 2012, 7: e47431-10.1371/journal.pone.0047431.PubMed CentralView ArticlePubMedGoogle Scholar
- Mahfooz N, Turchyn N, Mihajlovic M, Hrycaj S, Popadić A: Ubx regulates differential enlargement and diversification of insect hind legs. PLoS One. 2007, 2: e866-10.1371/journal.pone.0000866.PubMed CentralView ArticlePubMedGoogle Scholar
- Masumoto M, Yaginuma T, Niimi T: Functional analysis of Ultrabithorax in the silkworm, Bombyx mori, using RNAi. Dev Genes Evol. 2009, 219: 437-444. 10.1007/s00427-009-0305-9.View ArticlePubMedGoogle Scholar
- Hafen E, Levine M, Gehring WJ: Regulation of Antennapedia transcript distribution by the bithorax complex in Drosophila. Nature. 1984, 307: 287-289. 10.1038/307287a0.View ArticlePubMedGoogle Scholar
- Booker R, Truman JW: Octopod, a homeotic mutation of the moth Manduca sexta, influences the fate of identifiable pattern elements within the CNS. Development. 1989, 105: 621-628.PubMedGoogle Scholar
- Wheeler WM: On the appendages of the first abdominal segment of embryo insects. Trans Wiscontin Acad Sci Arts Lett. 1889, 8: 87-140.Google Scholar
- Heming BS: Origin and fate of pleuropodia in embryos of Neoheegeria verbasci (Osborn) (Thysanoptera: Phalaeothripidae). Advances in Thysanopterology. Edited by: Bhatti JS. 1993, New Delhi: Scientia Publishing, 205-223.Google Scholar
- Angelini DR, Kaufman TC: Insect appendages and comparative ontogenetics. Dev Biol. 2005, 286: 57-77. 10.1016/j.ydbio.2005.07.006.View ArticlePubMedGoogle Scholar
- Sánchez-Herrero E, Vernós I, Marco R, Morata G: Genetic organization of Drosophila bithorax complex. Nature. 1985, 313: 108-113. 10.1038/313108a0.View ArticlePubMedGoogle Scholar
- Stuart JJ, Brown SJ, Beeman RW, Denell RE: The Tribolium homeotic gene Abdominal is homologous to abdominal-A of the Drosophila bithorax complex. Development. 1993, 117: 233-243.PubMedGoogle Scholar
- Ueno K, Hui CC, Fukuta M, Suzuki Y: Molecular analysis of the deletion mutants in the E homeotic complex of the silkworm Bombyx mori. Development. 1992, 114: 555-563.PubMedGoogle Scholar
- Tomita S, Kikuchi A: Abd-B suppresses lepidopteran proleg development in posterior abdomen. Dev Biol. 2009, 328: 403-409. 10.1016/j.ydbio.2009.01.040.View ArticlePubMedGoogle Scholar
- Machida R: External features of embryonic development of a jumping bristletail, Pedetontus unimaculatus Machida (Insecta, Thysanura, Machilidae). J Morphol. 1981, 168: 339-355. 10.1002/jmor.1051680310.View ArticleGoogle Scholar
- Kukalova-Peck J: Fossil history and the evolution of hexapod structures. The Insects of Australia. Volume 1. 2nd edition. Edited by: CSIRO. 1991, Melbourne: Melbourne University Press, 41-179.Google Scholar
- Abzhanov A, Kaufman TC: Crustacean (malacostracan) Hox genes and the evolution of the arthropod trunk. Development. 2000, 127: 2239-2249.PubMedGoogle Scholar
- González-Reyes A, Morata G: The developmental effect of overexpressing a Ubx product in Drosophila embryos is dependent on its interactions with other homeotic products. Cell. 1990, 61: 515-522. 10.1016/0092-8674(90)90533-K.View ArticlePubMedGoogle Scholar
- Duboule D, Morata G: Colinearity and functional hierarchy among genes of the homeotic complexes. Trends Genet. 1994, 10: 358-364. 10.1016/0168-9525(94)90132-5.View ArticlePubMedGoogle Scholar
- Estella C, Rieckhof G, Calleja M, Morata G: The role of buttonhead and Sp1 in the development of the ventral imaginal discs of Drosophila. Development. 2003, 130: 5929-5941. 10.1242/dev.00832.View ArticlePubMedGoogle Scholar
- Damen WG, Tautz D: Abdominal-B expression in a spider suggests a general role for Abdominal-B in specifying the genital structure. J Exp Zool. 1999, 285: 85-91. 10.1002/(SICI)1097-010X(19990415)285:1<85::AID-JEZ10>3.0.CO;2-N.View ArticlePubMedGoogle Scholar
- Barnett AA, Thomas RH: Posterior Hox gene reduction in an arthropod: Ultrabithorax and Abdominal-B are expressed in a single segment in the mite Archegozetes longisetosus. EvoDevo. 2013, 4: 23-10.1186/2041-9139-4-23.PubMed CentralView ArticlePubMedGoogle Scholar
- Peel AD, Chipman AD, Akam M: Arthropod segmentation: beyond the Drosophila paradigm. Nat Rev Genet. 2005, 6: 905-916.View ArticlePubMedGoogle Scholar
- Fountain MT, Hopkin SP: Folsomia candida (Collembola): a 'standard’ soil arthropod. Annu Rev Entomol. 2005, 50: 201-222. 10.1146/annurev.ento.50.071803.130331.View ArticlePubMedGoogle Scholar
- van Straalen NM, Timmermans MJTN, Roelofs D, Berg MP: Apterygota in the spotlights of ecology, evolution and genomics. Eur J Soil Biol. 2008, 44: 452-457. 10.1016/j.ejsobi.2008.07.003.View ArticleGoogle Scholar
- Joosse ENG: Population structure of some surface dwelling Collembola in a coniferous forest soil. Neth J Zool. 1969, 19: 621-634.View ArticleGoogle Scholar
- Verhoef HA, Witteween J: Water balance in Collembola and its relation to habitat selection; cuticular water loss and water uptake. J Insect Physiol. 1980, 26: 201-208. 10.1016/0022-1910(80)90081-5.View ArticleGoogle Scholar
- van Straalen NM: Comparative demography of forest floor Collembola populations. Oikos. 1985, 45: 253-265. 10.2307/3565712.View ArticleGoogle Scholar
- Noro B, Lelli K, Sun L, Mann RS: Competition for cofactor-dependent DNA binding underlies Hox phenotypic suppression. Genes Dev. 2011, 25: 2327-2332. 10.1101/gad.175539.111.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.