Introduction
In the first edition of this series, Silvert (1985) outlined several major areas of uncertainty regarding cuticular proteins. The questions raised were: Were proteins extracted from cuticle authentic cuticular proteins, or might some be contaminants of adhering cells and hemolymph? Was the epidermis the sole site of synthesis of cuticular proteins, or were some synthesized in other tissues and transported to the cuticle? What was the relation among cuticular proteins of various developmental stages? Did cuticular proteins share common structural features?
That review presented all the cuticular protein sequence data then available — four complete and three partial sequences from Drosophila melanogaster, and one partial sequence from Sarcophaga bullata. The considerable sequence similarity seen with those limited data indicated that cuticular protein genes belonged to multi-gene families, and the even more limited genomic information revealed that similar genes were adjacent on a chromosome.
Progress over the next two decades was impressive, but not surprising, given the advances in relevant techniques. Elegant immunolocalization analyses solved the problem of the sources of cuticular proteins. The 2005 version of this article reported that over 300 cuticular protein sequences had been recognized, from 6 orders and over 20 species of insects. Progress since 2005 has been predominantly in identifying new cuticular protein sequences. Since then, several whole genomes have been annotated and hundreds of new sequences have been posted, based on EST (expressed sequence tag) analyses. It is no longer possible to list the available sequences. Rather, this topic will summarize what was learned from these data. Some areas of analysis have not progressed far since the 2005 version, and will be repeated here without revision.
Cuticle Structure and Synthesis
Cuticle Morphology
Terminology The descriptive terms used here to describe the regions of cuticle have been simplified according to Locke’s (2001) cogent suggestions for new nomenclature. He proposes the use of the term "envelope" to describe the outermost layer of cuticle, rather than the previous term "cuticulin." At the start of each molt cycle, the smooth apical plasma membrane forms microvilli with plaques at their tips where the new envelope assembles. This discrete layer of 10—30 nm serves not only to protect the underlying epidermis from molting fluid enzymes that begin to digest the old cuticle, but also, as Locke points out, affects "resistance to abrasion and infection, penetration of insecticides, permeability, surface reflectivity, and physical colors." The sequences and properties of its constituent proteins remain unknown.
Next formed is the epicuticle, about 1 pm in thickness. This chitin-free layer (but see Section 5.2.1.3) is stabilized by quinones. It was formerly referred to as the "inner epi-cuticle," with cuticulin being the outer.
Former arguments about the precise distinction between exo- and endo-cuticle are eliminated by Locke’s lumping together of the inner regions of the cuticle under the term "procuticle," encompassing both pre-ecdysial and post-ecdysial secretions. The procuticle, then, is the region that combines chitin and cuticular proteins in various combinations, and becomes sclerotized and pigmented to varying degrees. This is the region depicted in electron micrographs showing stacks of precisely oriented lamellae. According to Locke (2001), it is the secretion of chitin fibers by apical microvilli that apparently bend in concert across the epithelial sheet to orient the laminae that gather into lamellae (Neville, 1975; Locke, 1998). Alternative mechanisms have been proposed by Moussian (2010), involving movement of the "chitin synthesis complex" across the cell surface or merely self-assembly. While knowledge of the process of secreting and assembling such a highly ordered structure is limited, details about the proteins associated with the lamellae are now voluminous.
Growth of the cuticle within an instar Central to the issue of cuticle structure is the important fact that considerable cuticle growth can occur during an intermolt period (Williams, 1980), some of it by a smoothing out of macro- and microscopic folds and pleats (Carter and Locke, 1993). During intra-instar growth, new cuticular proteins are interspersed among the old, necessitating a model of chitin-protein and protein-protein interactions that will permit such intussusception (Condoulis and Locke, 1966; Wolfgang and Riddiford, 1986).
Localization of cuticular proteins within the cuticle Precise localization of cuticular proteins within the cuticle, and even within cellular organelles, has been made possible with immunogold labeling of electron-microscopic sections. Here, a specific primary antibody is bound to the sections and visualized with a secondary antibody conjugated to colloidal gold particles.
Antibodies have been raised against extracts of whole cuticle or isolated electrophoretic bands, and the specificity of each antibody ascertained with Western blots. While each polyclonal antibody raised against a single band was specific for the immunizing protein, monoclonals raised against cuticular extracts frequently reacted with more than one electrophoretic band.
One concern with immunolocalization is that as cutic-ular proteins become modified in the cuticle by binding to chitin or by becoming sclerotized, the immunizing epi-topes might become masked – a problem that should be more serious with monoclonal than with polyclonal antibodies. All groups recognized that while the presence of an antigen is significant, its absence may reflect no more than such masking.
This concern is significant when one considers results of immunolocalization in the assembly zone, the region of cuticle directly above the microvilli. It is here that chi-tin secreted from the tips of the microvilli interacts with cuticular proteins secreted into the perimicrovillar space. Immunolocalization studies revealed only a few of the cuticular proteins within the perimicrovillar space, but the same ones and others were abundant in the assembly zone directly above it (Locke et al, 1994; Locke, 1998). The authors’ conclusion was that the assembly zone "is where we should expect proteins to unravel and expose most epitopes in preparation for assuming a new configuration as they stabilize in the maturing cuticle." Wolfgang et al. (1986, 1987) found two Drosophila melanogaster cuticular proteins exclusively in this zone, and suggested they might function in cuticle assembly. Locke et al. (1994) point out that it was common for antibodies raised against Calpodes ethilus proteins to react more strongly with the assembly zone than with more mature regions of cuticle, where sclerotization and chitin binding might mask epi-topes. Thus, further substantial evidence than the failure to detect a protein in more mature regions is needed to confirm that it belonged exclusively to the assembly zone.
It was known from earlier work on protein and mRNA distribution that cuticles from different metamorphic stages and different anatomical regions had different cuticular proteins, and that there may be a change in cuticular proteins synthesized by a single cell within a molt cycle (for review, see Willis, 1996). Such a transition in proteins synthesized is especially apparent at the time of ecdysis, and, in some insects, late in the instar. Consistent with this, immunolocalization revealed different proteins in morphologically distinct early and late lamellae in D. melanogaster pupae, and Tenebrio molitor and Manduca sexta larvae (Doctor et al., 1985; Fristrom et al, 1986; Wolfgang and Riddiford, 1986; Wolfgang et al., 1986; Lemoine et al., 1989, 1993; Bouhin et al., 1992a, 1992b; Rondot et al, 1998). Only two proteins with known sequence are among this group: TMACP22 (P26968.1) and TMLPCP22 (P80686.2).
Csikos and colleagues (1999) have used immunohisto-chemistry to follow some of Manduca’s cuticular proteins throughout the molt cycle. These proteins are obviously in a dynamic state as they move from epidermis to cuticle to molting fluid to fat body, and then apparently back to cuticle via the hemolymph. More detailed studies are needed to learn if the same molecules make the return trip, and whether their initial passage from molting fluid into the hemolymph is solely via uptake and then basal secretion by the epidermis, or whether the midgut plays a role, since lepidopteran larvae drink their molting fluid (Cornell and Pan, 1983).
The findings with epicuticle, the first region to be secreted beneath the envelope, were complex. None of the monoclonal antibodies that recognized Tenebrio cuticular proteins reacted with epicuticle (Lemoine et al., 1990). On the other hand, arylphorin from Calpodes has been localized to epicuticle and no other cuticular region (Leung et al., 1989), and several proteins, of unknown sequence, were found both in the epicuticle and in the lamellar regions of the procuticle in D. melanogaster (Fristrom et al., 1986) and Calpodes (Locke et al, 1994). This finding of cuticular proteins in both epicuticle and lamellar regions was surprising, since the epicuticle had always been described as lacking chitin (cf. Fristrom et al., 1986, Fristrom and Fristrom, 1993) and thus was expected to have unique proteins. A study of moth olfactory sensilla detected chitin in the procuticle with gold-conjugated wheat germ agglutinin, but it was not found in the epicuticle (Steinbrecht and Stankiewicz, 1999).
In addition to temporal differences in the secretion of cuticular proteins by single cells, there may be regional differences in the cuticle secreted by single cells. Individual epidermal cells of the articulating membranes (intersegmental membranes) in Tenebrio secrete a cuticle with sclerotized cones embedded in softer cuticle. Two of the classes of monoclonal antibodies raised against Tenebrws larval and pupal cuticular proteins recognized proteins in these cones. The same antibodies recognized proteins in cuticles in other regions that were destined to be sclerotized. Different antibodies recognized the proteins in the softer cuticle (Lemoine et al., 1990, 1993).
Locke et al. (1994) were able, using carefully reconstructed sections of Calpodes larval cuticle, to distinguish one protein (C36) that was found with the same distribution as the chitin microfibrils that had been visualized with wheat germ agglutinin (WGA), a lectin that recognizes N-acetylglucosamine, while other antigens failed to show this distribution. Notably, only C36 isolated from cuticle reacted with WGA on lectin blots. Based on this evidence, Locke et al. (1994) suggest that the isolated protein may have obtained its N-acetylglucosamine from chitin.
Cuticles formed following disruption of normal metamorphosis Treatment of many insects with juvenile hormone (JH) causes them to resynthesize a cuticle with a morphology characteristic of the current metamorphic stage, rather than the next. Thus, in Tenebrio, treatment of pupae with JH prior to pupal—adult apolysis causes the formation of a second pupa rather than an adult. Earlier work revealed that these second pupae had proteins with the same electrophoretic mobility as those extracted from normal pupae (Roberts and Willis, 1980; Lemoine et al., 1989). A combination of Northern analysis and in situ hybridization demonstrated that second pupae have the same cuticular protein mRNAs and protein localization as normal pupae (Lemoine et al., 1993; Rondot et al., 1998). Adult cuticular proteins are not deposited in these cuticles, and the adult mRNAs do not appear (Lemoine et al., 1989, 1993; Bouhin et al., 1992a, 1992b; Charles et al., 1992). Some JH-treated Tenebrio pupae form two cuticles, the first pupal-like in morphology and the second with adult features. The adult-like cuticle was shown with immunolocalization to have TMACP22 (P26968.1) (Bouhin et al., 1992a). IfJH is applied too late to form a perfect second pupa, the next cuticle formed will be a composite with morphological features of two metamorphic stages (Willis et al., 1982). Bouhin et al. (1992b) found that all the epidermal cells laying down such a composite cuticle had mRNAs for TMACP22.
Zhou and Riddiford (2002) used Northern analysis to characterize the somewhat nondescript cuticles made by D. melanogaster that had been manipulated by mis-expressing the gene br (broad ), which codes for a transcription factor that first appears before the larval/pupal molt in flies and moths. By following mRNAs for the adult cuticular protein ACP65A (CG10297) or the pupal cuticular protein Edg78E (CG7673), they were able to demonstrate the essential role of br in directing pupal development, and thereby clarified the perplexing action of juvenoids in the higher Diptera.
The Site of Synthesis of Cuticular Proteins
One of the unresolved issues addressed in Silvert’s (1985) review was the site of synthesis of cuticular proteins. This might appear to be a trivial issue, for one would expect that the epidermis that underlies the cuticle would synthesize the cuticular proteins. There are, however, reports in the literature that proteins found in the hemolymph were present in cuticle, and even that labeled proteins injected into the hemolymph would appear in cuticle. Sil-vert discussed the possibility that the injected protein had been broken down and resynthesized so that the cuticular protein was labeled solely because its constituent amino acids had come from a labeled pool.
Five methods have now provided data that address the site of synthesis of cuticular proteins. The most common method is to learn in what tissues and at which stages mRNA is present for a particular cuticular protein by detecting its presence via Northern analysis, RT-PCR, or qRT-PCR. This method is so common that specific examples will not be given. The second method is to incubate epidermis or integument in vitro with radioactive amino acids, separate the proteins, and compare the electropho-retic mobility of the labeled proteins to proteins isolated from cleaned cuticles. A third method is to isolate mRNAs from tissues and translate these in vitro with commercially available wheat germ extracts or rabbit reticulocytes, and compare the translation products to known cuticular proteins. The fourth method is in situ hybridization, and the fifth is immunolocalization to visualize proteins within the endoplasmic reticulum and Golgi apparatus.
The first three methods suffer from the possibility that tracheae and adhering tissues, fat body, muscles, and hemocytes contribute to the mRNA pool. Both labeling methods suffer from the problem that cuticular proteins are notoriously sensitive to solubilizing buffer and gel conditions (pH, urea concentration) (Cox and Willis, 1987a), and, unless cuticular protein standards and labeled translation products are mixed prior to electrophoresis, they may not show identical electrophoretic mobility even in adjacent lanes. Some workers have precipitated labeled translation products with antibodies raised against extracts of cuticle or individual cuticular proteins, then solublized the precipitate, run it on a gel, and detected the labeled product with fluorography. Csikos et al. (1999) used Western blots of translation products to identify cuticular proteins. Since cuticular proteins are destined for secretion from cells, they have a signal peptide that is cleaved before the protein is secreted into the cuticle. Hence, translation products made in vitro will be larger than the protein extracted from cuticle. There are two methods to circumvent this problem. The translation products can have their signal peptides cleaved by adding a preparation of canine microsomes, or antibodies against cuticular proteins (specific or against an extract) can be used to precipitate the translation products before they are solubilized and run on a gel. Either method allows some certainty in the comparison of these in vitro translation products with authentic cuticular proteins. It was also found that some commercial preparations of wheat germ extract have endogenous signal peptide processing activity (Binger and Willis, 1990).
Frequently, 35S-methionine was used for metabolic labeling of integument and for in vitro translation. This is an unfortunate choice, as most mature cuticular proteins lack methionine residues (see section 5.3.2.1). The initiator methionine will be lost, along with the entire signal pep-tide. Clear differences in labeling patterns with 35S-methi-onine and 3H-leucine have been found, with none of the major proteins from pharate adult cuticle of D. melanogaster or from larval cuticles of H. cecropia showing methionine labeling (Roter et al.,1985; Willis, 1999). Why, then, did several studies find all of the known cuticular proteins labeled with methionine? Perhaps the finding that 35S-methionine can donate its label to a variety of amino acids in preformed proteins (Browder et al.,1992; Kalin-ich and McClain, 1992) explains its appearance, and suggests that it needs to be used with caution for such studies with cuticular proteins.
The fourth method is in situ hybridization, where specific mRNAs can be identified in the epidermis. In situ hybridization allows one to be somewhat more discerning about the site of synthesis of a cuticular protein, because it is possible to monitor the presence or absence of a particular mRNA at the level of an individual cell. With this technique, integument is fixed and sectioned, and then probed with a labeled cDNA or cRNA, allowing the identification of particular regions of the epidermis by examining the morphology of the overlying cuticle. With most detection methods, contaminating tissues and precise regions of the epidermis can be identified, and the presence of the particular mRNA in them can be assessed. Thus, this technique identifies the location of the mRNAs recognized by the specific probe used. It was this technique that revealed the precision with which mRNAs are produced, for abrupt boundaries of expression occur between sclerites and intersegmental membranes (Rebers et al., 1997), or at muscle insertion zones (Horodyski and Riddiford, 1989), or next to specialized epidermal cells (Horodyski and Riddiford, 1989; Rebers et al., 1997). This technique even revealed the presence of mRNA for cuticular proteins in epithelia of imaginal discs from young larvae (Gu and Willis, 2003). A limitation of the technique is that some cRNA probes bind to the cuticle itself, possibly obscuring detection of mRNA in the underlying epidermis (Fechtel et al., 1989, Gu and Willis, 2003). Fechtel et al. (1989) found this artifact to be cuticle-type- as well as strand- and probe-specific. Results from several species are summarized in Table 1.
Table 1 Evidence for the Association of Location or Type of Cuticle and Sequence Class of Some Cuticular Proteins
|
|
Sequence Class |
|
Nature of Evidenceb |
When Deposited |
|
|
|
Species |
Protein |
Localization |
Reference |
|||
|
Bombyx mori |
BMLCP18 |
RR-1 |
Imaginal discs |
EST |
Gu and Willis (2003) |
|
|
Drosophila melanogaster |
EDG-78 |
RR-1 |
Larval and imaginal cells of prepupa |
ISH |
Fechtel et al. (1989) |
|
|
Drosophila melanogaster |
EDG-84 |
RR-2 |
Imaginal disc cells |
ISH |
Fechtel et al. (1989) |
|
|
Drosophila melanogaster |
PCP |
RR-1 |
Prepupal thorax and abdomen |
ISH |
Henikoff et al. (1986) |
|
|
Hyalophora cecropia |
HCCP12 |
RR-1 |
Soft cuticle; imaginal discs |
CD and ISH |
Cox and Willis (1985), Gu and Willis (2003) |
|
|
Hyalophora cecropia |
HCCP66 |
RR-2 |
Hard cuticle |
CD and ISH |
Cox and Willis (1985), Gu and Willis (2003) |
|
|
Locusta migratoria |
LM-ACP7 |
RR-2 |
Hard cuticle |
CD |
Andersen et al. (1995) |
|
|
Locusta migratoria |
LM-ACP8 |
RR-2 |
Hard cuticle |
CD |
Andersen et al. (1995) |
|
|
Locusta migratoria |
LM-ACP19 |
RR-2 |
Hard cuticle |
CD |
Andersen et al. (1995) |
|
|
Manduca sexta |
CP14.6 |
RR-1 |
Soft cuticle |
ISH |
Rebers et al. (1997) |
|
|
Manduca sexta |
LCP 16/17 |
RR-1 |
Soft cuticle |
ISH |
Horodyski and Riddiford (1989) |
|
|
Tenebrio molitor |
ACP17 |
Glycine-rich |
Hard cuticle |
ISH |
Strongest post-ecdysis |
Mathelin et al. (1995, 1998) |
|
Tenebrio molitor |
ACP20 |
RR-2 |
Hard cuticle |
ISH |
Primarily pre-ecdysis |
Charles et al. (1992) |
|
Tenebrio molitor |
ACP-22 |
RR-2 |
Hard cuticle |
ISH, mAB |
Pre-ecdysis |
Bouhin et al. (1992a,1992b) |
|
Tenebrio molitor |
TMLPCP22 |
51 aa motif |
Hard and soft cuticle pre-ecdysis, then only soft cuticle |
ISH, mAB |
Primarily pre-ecdysis |
Rondot et al. (1998) |
|
Tenebrio molitor |
TMLPCP23 |
51 aa motif |
Hard and soft cuticle |
ISH |
Only pre-ecdysis |
Rondot et al. (1998) |
|
Tenebrio molitor |
TMLPCP29 |
RR-3 and 18-residue motif |
Hard and soft cuticle, except not posterior borders of sclerites |
ISH |
Post-ecdysis |
Mathelin et al. (1998) |
aFor in situ hybridization, cuticle type was determined by nature of cuticle overlying the epidermis.
bCD, careful dissection prior to extraction of proteins; ISH, in situ hybridization used to to localize mRNA; mAB, monoceonal antibody immunolo-calization; EST, from Bombyx EST project (Mita et al. 2002).
The fifth method, immunolocalization, was described earlier in conjunction with localization of specific proteins within the cuticle, but it can also be used to identify the site of synthesis by looking for a particular protein within the endoplas-mic reticulum or Golgi apparatus (Sass et al., 1994a, 1994b).
The results from mRNA detection, metabolic tissue labeling, and in vitro translations reveal that all cuticu-lar proteins with known sequences or for which specific probes are available are synthesized by the integumental preparations. Different proteins are synthesized at different times in a molt cycle, and in different anatomical regions, and there are some cuticular proteins whose synthesis is stage-specific. Differences in the presence of mRNA parallel the appearance of labeled proteins, indicating that much of the temporal and spatial control of cuticular protein synthesis is at the level of transcription. As mentioned above, however, all three of these methods are limited by the possible contamination of tissues by non-epidermal cells, and by their inability to address heterogeneity of cell types within the epidermis.
A microarray analysis of isolated hemocytes from An. gambiae revealed the presence of mRNA for nine cuticular proteins (Baton et al., 2009). Transcripts for AgamCPR26 and AgamCPR90 were significantly higher in adults challenged with heat-killed Micrococcus luteus than in naive individuals. A massive study on hemocytes in D. melanogaster found significant levels of transcript for DmelLCP1-4 in hemocytes from both naive and bacteria-challenged larvae (Irving et al., 2005).
Studies that have combined tissue labeling or in vitro translations with immunolocalization have at last clarified the relationship between hemolymph and cuticular proteins with identical electrophoretic and immunological properties. The most comprehensive studies of protein trafficking were carried out in Calpodes, and revealed four classes of exported proteins that are handled by the epidermis.
These findings are so important that the experimental methodology is worth discussing. The first approach used was to seal sheets of final instar integument into a bathing chamber so there could be no leakage from the cut edges of the tissue, and then find what proteins were made in a 2-hour exposure to 35S-methionine. Three classes of proteins were identified with this procedure; one was secreted exclusively into the cuticle (C class), a second appeared in the bathing fluid and hence had been secreted basally (B class), while the third was secreted in both directions (BD class) (Palli and Locke, 1987). Immunolocaliza-tion of numerous other Calpodes proteins (of unknown sequence) confirmed the existence of these three routing classes of epidermal proteins. A fourth class, the T class, was identified for proteins transported into cuticle but not synthesized by the epidermis. Its presence eliminated any concerns that the classes might be artifacts from labeling with 35S-methionine (Sass et al., 1993).
One member of the T class (T66) was studied in more detail. It was localized by immunogold throughout the cuticle, and, although found in epidermal cells, was not found in association with the Golgi apparatus, confirming its transcellular transport, rather than synthesis by the epidermis. A subsequent study identified the exclusive site of its synthesis as spherulocytes (Sass et al., 1994a).
Whether the BD proteins are secreted from both apical and basal borders of epidermal cells is still not clear. Locke (1998, 2003) now favors the possibility that all secretion is apical, where the Golgi are concentrated, and that the secreted proteins are subsequently taken back into the cell from perimicrovillar space and transported in vesicles to the basal surface, where the contents are released into the hemolymph.
In conclusion, it is now clear that the epidermis can synthesize both cuticular and hemolymph proteins. It can also transport proteins made in tissues other than epidermis from hemolymph to cuticle.
Tracheal Cuticular Proteins
An often-neglected source of cuticle in insects is the tra-cheal system. Since tracheae are associated with all insect tissues, caution is needed in interpreting the significance of the presence of mRNAs or cuticular proteins from non-integumental tissues. Cox and Willis (1985) recognized that some of the proteins from tracheae had the same isoelectric points as proteins isolated from integumentary cuticle. A further study was carried out a decade later by Sass et al. (1994b), combining electrophoretic analysis with immunogold labeling. Chitin was localized with wheat germ agglutinin, and found in all regions of tracheae and tracheoles except the taenidial cushion. Antibodies that had been raised against individual electro-phoretic bands from integumentary extracts represented proteins from all four classes of integumentary peptides. Some C proteins, those from the surface cuticle, were found associated with chitin, but only in taenidia; other C proteins were in the general matrix, with and without chitin. The B and BD peptides were only found in the taenidial cushion, the region lacking chitin. It appears that hemolymph peptides that are synthesized by the epidermis may be tracheal cuticle precursors. The one T protein studied (T66, made in spherulocytes) was also found in the general matrix. An important insight from this study was the conclusion that: "The extremely thin tracheal epithelium suggests that transepithelial transport might supply proteins to the tracheal cuticle more evenly than Golgi complex secretions" (Sass et al., 1994b). Analysis of tracheal morphogenesis is an active field that has recently been reviewed (Centanin et al., 2010; Moussian, 2010; see also Ghabrial et al., 2003). Little information is available about the proteins that contribute to tracheae. Gasp (a member of the CPAP-3 family) was found to be restricted to tracheae in D. melanogaster (Barry et al., 1999), but transcripts from its clear ortholog in the lepidopteran Choristoneura fumierana were associated primarily with the body surface epidermis (Nisole et al., 2010).
