Localization of RR-1 and RR-2 cuticular proteins within the cuticle of Anopheles gambiae

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Highlights

  • The largest family of cuticular proteins, CPR, can be divided into two groups, RR-1 and RR-2.

  • We used EM immunolocalization to learn how these groups are deployed in the cuticle.

  • RR-1s, with one exception, were found throughout soft cuticle.

  • Some RR-2s were in both endo- and exo-cuticle of hard cuticle.

Abstract

The largest arthropod cuticular protein family, CPR, has the Rebers and Riddiford (R&R) Consensus that in an extended form confers chitin-binding properties. Two forms of the Consensus, RR-1 and RR-2, have been recognized and initial data suggested that the RR-1 and RR-2 proteins were present in different regions within the cuticle itself. Thus, RR-2 proteins would contribute to exocuticle that becomes sclerotized, while RR-1s would be found in endocuticle that remains soft. An alternative, and more common, suggestion is that RR-1 proteins are used for soft, flexible cuticles such as intersegmental membranes, while RR-2s are associated with hard cuticle such as sclerites and head capsules. We used TEM immunogold detection to localize the position of several RR-1 and RR-2 proteins in the cuticle of Anopheles gambiae. RR-1s were localized in the procuticle of the soft intersegmental membrane except for one protein found in the endocuticle of hard cuticle. RR-2s were consistently found in hard cuticle and not in flexible cuticle. All RR-2 antibodies localized to the exocuticle and four out of six were also found in the endocuticle. Hence the location of RR-1s and RR-2s depends more on properties of individual proteins than on either hypothesis.

Introduction

The majority of cuticular proteins (CPs) in every arthropod species examined to date belong to the CPR family named after the R&R Consensus first recognized by Rebers and Riddiford (1988). Subsequent to the first description, the Consensus region has been modified by adding amino acids upstream of a 24 amino acid core and deleting some downstream. The extended Consensus recognized as pfam00379 (Chitin_bind_4) now has about 53 amino acids and has been shown to be necessary and sufficient for chitin binding, first by Rebers and Willis (2001) and subsequently confirmed by several others (Togawa et al., 2004, Togawa et al., 2007, Qin et al., 2009). Other methods have been used to confirm the chitin-binding properties of CPRs (Tang et al., 2010, Dong et al., 2016). Three distinct forms of the Consensus were recognized and named by Andersen, 1998, Andersen, 2000): RR-1, RR-2 and RR-3. RR-3 has been assigned to few proteins, is not clearly defined (Ioannidou et al., 2014), and will not be discussed further. Two Web sites allow one to classify CPRs (Cuticle DB: http://bioinformatics2.biol.uoa.gr/cuticleDB/index.jsp and CutProtFam-Pred: http://aias.biol.uoa.gr/CutProtFam-Pred/home.php). The second site uses the same Hidden Markov Model method for group prediction as the first but provides scores that enable one to assess the accuracy of the assignment as well as to identify additional families of CPs (Magkrioti et al., 2004, Ioannidou et al., 2014). The Consensus region of RR-2s is highly conserved. Fig. 1 provides WebLogos (Crooks et al., 2004) for RR-1 and RR-2 CPs from Anopheles gambiae. It also compares the WebLogos of the RR-2 form of the Consensus from An. gambiae with that from 62 holometabolous insects. Previously published WebLogos (Willis, 2010) showed that the high similarity in the RR-2 Consensus extends to Crustacea and Chelicerata. In contrast, the Consensus region of RR-1s is variable in length and less conserved in sequence in An. gambiae and even more variable when other species are compared (Supplementary File 1A; Willis, 2010). In general, RR-1 proteins have more acidic isoelectric points and fewer histidines, but only the Consensus region is appropriate for group assignment. While it is tempting to speculate that the uniformity among the Consensus of RR-2s contributes to their participation in hard cuticle, it is important to note that the range of lengths of the mature proteins is greater in RR-2s than in RR-1s (Supplementary File 1A). Given the predominance of CPRs in the cuticulome and the consistent differences between proteins classified as either RR-1 or RR-2, we need to learn more about how they participate in cuticle.

Eight sequence clusters, groups of genes, generally linked, that code for proteins that have a high degree of similarity account for 66 of the 101 RR-2 genes in An. gambiae (Cornman et al., 2008, Cornman and Willis, 2008). There are 42 CPRs that have been classified as RR-1.

We will follow the terminology proposed by Locke (2001) for naming the regions of cuticle. From the apical surface: the envelope, 10–30 nm; epicuticle, about 1 μm in thickness and chitin-free; the procuticle, the region that combines chitin and CPs. The procuticle has been further subdivided into exo- and endo-cuticle. Between the procuticle and epidermis, sometimes one sees an assembly zone where chitin and proteins are presumed to first interact. Definitions of these divisions differ. Some call the pigmented, sclerotized region exocuticle and the region closest to the epidermis endocuticle. Others, including the authors of this paper, prefer to use the term exocuticle for the region secreted prior to ecdysis with the endocuticle being synonymous with post-ecdysial cuticle.

We did not see distinguishing morphological characteristics between the pre- and post-ecdysial cuticle of the intersegmental membranes or the soft cuticle of the adult head, so we just call this region procuticle. We also could not distinguish between envelope and epicuticle in sclerites (Fig. 2).

The first paper to define the RR-1 and RR-2 groups of CPRs (Andersen, 1998) noted the difference between proteins used in the exo- (pre-ecdysial) and endo- (post-ecdysial) cuticles. Prior to their work, several studies had found a marked change in protein composition between pre- and post-ecdysial cuticle (e.g. Roberts and Willis, 1980, Andersen and Højrup, 1987, Lemoine et al., 1989, Lemoine et al., 1990, Nøhr and Andersen, 1993, Jensen et al., 1997). Andersen (1998) built on these findings by isolating spots on 2D gels that differed between samples harvested before and after ecdysis and sequencing them via MALDI-MS (Matrix-assisted laser ionization desorption mass spectrometry) and PDMS (plasma desorption mass spectrometry). Only RR-1 proteins were found in spots unique to animals harvested after ecdysis. Subsequent studies have tried to use the timing of mRNAs to predict whether a protein would be used in exo- or endo-cuticle. Togawa et al. (2008) looked at transcript levels (with RT-qPCR) of more than 150 CPRs from An. gambiae at 19 precisely timed points from hatching to adult eclosion. The majority of the genes had transcripts that appeared first in a pharate stage and persisted into the next stage, making it impossible even to speculate about the localization of the corresponding proteins. More recently, Shahin et al. (2016) carried out an extensive RT-qPCR analysis on wing discs and pupal wings of Bombyx mori, once again finding that most CPR genes had transcripts that appeared before pupation, although transcripts from RR-1s persisted longer. Thus, they concluded: “From the present results, RR-2 CP appears to be involved in the exocuticle and RR-1 in exo- and endocuticle layers.” A complication of using transcript appearance to predict protein localization is the evidence that newly secreted CPs can be interspersed in older regions of the cuticle, a phenomenon called intussusception (Carter and Locke, 1993). Obviously transcript data are only a very indirect way of assessing the location of CPR proteins.

EM immunolocalization of an RR-2 protein from Tribolium castaneum (TcCPR27) localized it in both laminae and vertical pore canals of the elytra's hard cuticle (Noh et al., 2014). Since only cuticle from pharate adults was examined, no information is available about whether this protein might also be found in the post-ecdysial endocuticle.

The second hypothesis about the deployment of RR-1s and RR-2s was that RR-1s and RR-2s would be used to build soft (flexible) and hard (rigid) cuticle, respectively (Andersen, 1998). This generalization came from earlier studies with protein extracts from carefully dissected samples, in situ hybridization, immunolocalization, and Northern analyses with RNA extracted from tissues taken from specific regions from six species (A summary of some of these data can be found in Table 1 of Willis et al., 2012). Perhaps the most impressive data came from in situ hybridization where abrupt borders were seen between intersegmental membranes and sclerites with mRNA for RR-1 proteins in the former, and RR-2 in the latter (Charles et al., 1992, Rebers et al., 1997, Mathelin et al., 1998). Yet, Gu and Willis (2003) presented quantitative data on transcript levels that indicated that message from a spatially “inappropriate” mRNA might be present, but from 25- to 3125-fold less abundant.

The issue of differential spatial expression of RR-1 vs. RR-2 has recently been addressed in T. castaneum (Dittmer et al., 2012). Microarray data revealed that elytra (rigid, hard cuticle) and hind wings (flexible, soft cuticle) of T. castaneum did not have an exclusive presence of one type of CPR; rather the distinguishing feature was transcript abundance with significantly higher levels of RR-1s and RR-2s in hindwing and elytra, respectively (Dittmer et al., 2012).

Despite the many studies focused on this topic, resolution of whether RR-1 and RR-2 CPs can be distinguished by anatomical or intra-cuticular location has not yet had a clear answer. There is a technique, EM immunolocalization, which could provide direct information about the intra-cuticular localization of these two groups. For at least 30 years, this technique has been employed by others to study the location of CPs in the cuticle, first with frozen ultrathin sections (Wolfgang et al., 1986), and shortly thereafter with conventionally embedded tissue (Leung et al., 1989). Most often the antibodies used were raised against proteins from cuticle extracts or extracted from electrophoretic gels and, with few exceptions, their sequences were not known. The only CPR to be localized was an RR-2 protein from Tenebrio molitor, TmACP20, localized to the exocuticle of the adult sclerite (Bouhin et al., 1992, Bouhin et al., 1993). Our previous studies with this technique (Vannini et al., 2014, Vannini et al., 2015), did not investigate CPRs.

In this study, we have taken advantage of the wealth of information about sequences, transcript levels, and anatomical distribution of RR-1 and RR-2 proteins in An. gambiae to learn directly about their distribution by examining their presence within the cuticle itself. We now report the precise localization of CPRs within the cuticle of An. gambiae by immunogold labeling of electron microscopic sections. At the same time, we obtained information about anatomical locations.

Section snippets

Mosquito rearing

The colony of An. gambiae (G3 strain) is maintained in an insectary at the University of Georgia Entomology Department. Newly hatched first instar larvae were kept at 27 °C in a 12/12 h L/D photoperiod. Larvae were fed ground Koi Food Staple Diet (Foster and Smith Aquatics, Rhinelander, WI USA), and adults had access to an 8% fructose solution. To obtain developmentally synchronized animals, larvae were collected when they showed signs of a recent molt because of a pale head capsule; pupae were

Validation of technique

EM immunolocalization of CPs is an established technique, but this is the first time it has been used to address the problem of deployment of RR-1 and RR-2 CPRs within the cuticle. We selected stages and tissues to examine based on earlier work from our laboratory, especially an extensive RT-qPCR analysis of transcript levels throughout development (Togawa et al., 2008).

Several controls were used: preimmune serum, flow through from the affinity columns, and no primary antibody. Some of the

Conclusions

At last we have direct evidence for the location of RR-1 and RR-2 proteins in the cuticle. While generalization awaits comparable analyses in other species, in An. gambiae we found support for both anatomical and intra-cuticular specialization for these two groups of CPRs. For both RR-1 and RR-2, a subset of the proteins deviated from the others in its properties and negated the universality of one of the hypotheses. RR-1s, with one exception, were not found in hard cuticle such as sclerites

Acknowledgments

We thank Mark R. Brown and Anne Elliot for maintaining the mosquito facility from which the animals were obtained and M.R. Brown for advice on immunolabeling. We thank Yihong Zhou for advice on protein isolation, John Hunter Bowen for help with the sequence analyses and both for comments on the MS. We also thank Mary B. Ard and John P. Shield of the Center for Advanced Ultrastructural Research at the University of Georgia for technical support. Two anonymous reviewers provided helpful

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