The CPCFC cuticular protein family: Anatomical and cuticular locations in Anopheles gambiae and distribution throughout Pancrustacea

Abstract

Arthropod cuticles have, in addition to chitin, many structural proteins belonging to diverse families. Information is sparse about how these different cuticular proteins contribute to the cuticle. Most cuticular proteins lack cysteine with the exception of two families (CPAP1 and CPAP3), recently described, and the one other that we now report on that has a motif of 16 amino acids first identified in a protein, Bc-NCP1, from the cuticle of nymphs of the cockroach, Blaberus craniifer (Jensen et al., 1997). This motif turns out to be present as two or three copies in one or two proteins in species from many orders of Hexapoda. We have named the family of cuticular proteins with this motif CPCFC, based on its unique feature of having two cysteines interrupted by five amino acids (C-X(5)-C). Analysis of the single member of the family in Anopheles gambiae (AgamCPCFC1) revealed that its mRNA is most abundant immediately following ecdysis in larvae, pupae and adults. The mRNA is localized primarily in epidermis that secretes hard cuticle, sclerites, setae, head capsules, appendages and spermatheca. EM immunolocalization revealed the presence of the protein, generally in endocuticle of legs and antennae. A phylogenetic analysis found proteins bearing this motif in 14 orders of Hexapoda, but not in some species for which there are complete genomic data. Proteins were much longer in Coleoptera and Diptera than in other orders. In contrast to the 1 and occasionally 2 copies in other species, a dragonfly, Ladona fulva, has at least 14 genes coding for family members. CPCFC proteins were present in four classes of Crustacea with 5 repeats in one species, and motifs that ended C-X(7)-C in Malacostraca. They were not detected, except as obvious contaminants, in any other arthropod subphyla or in any other phylum. The conservation of CPCFC proteins throughout the Pancrustacea and the small number of copies in individual species indicate that, when present, these proteins are serving important functions worthy of further study.

Keywords: Arthropod phylogeny; Cuticle; EM immunolocalization; In situ hybridization; RT-qPCR.

Fig. 1

RT-qPCR analysis of AgamCPCFC1 transcripts in Anopheles gambiae. L48 and P24 are actually pharates of the next stage. See Text and Supplementary File 1 for methods.

Fig. 2

In situ hybridization of AgamCPCFC1 on sections of 4^th instar larvae. A. Photograph of larva with arrows showing location of lateral setae on thorax and abdomen and a double arrow indicating the grid and fringe at the posterior end. B. Head capsule and bit of prothoracic segment. Note the presence of hybridization in the small cells that form setae at the anterior edge of the prothorax. C. Section of the abdomen showing cells that are forming setae. D. Grid and accompanying fringe at posterior end of a larva. E. Section showing cells secreting large and small setae. (B,D 3’ probe; C,E coding region probe).

Fig. 3

In situ hybridization of AgamCPCFC1 on sections of pupae less than 1 hour after pupation. A. Section of abdomen showing epidermal hybridization in sclerites (Scl) and only in intersegmental membrane (IsM) where muscles (Mus) are inserting into the cuticle. B. Lateral surface of pupal abdomen with setae-forming cells. C. Developing antenna in pupa. Structure was recognized because it is similar to that shown in Fig. 76a of Harbach and Knight (1980). D. Limb with developing scales showing hybridization. E. Muscle insertion zone with strong hybridization. (B,D 3’ probe; A,C,E coding region probe.)

Fig. 4

In situ hybridization of AgamCPCFC1 on sections of pharate adults Animals were fixed 24 hours after pupation, which are a few hours before ecdysis to the adult. A. Hybridization to epidermis of sclerites (Scl), but not intersegmental membranes (IsM). B. Hybridization in muscle attachment region. C. Hybridization in spermatheca (Sp). D. Hybridization under basal plate (BP) of Johnston's organ, the surrounding pedicel (Ped) and the flagellum (Fl). E. Hybridization to part of cervical sclerite. (D,E 3’ probe; A,B,C coding region probe.)

Fig. 5

In situ hybridization of AgamCPCFC1 on adults less than 12 hours after eclosion. A. Antenna with Johnston's organ (JO) and flagellum (Fl) showing strong hybridization. B. cerci at the terminal end of the male abdomen. C and D. Hybridization in appendages. (All coding region probe.)

Fig. 6

EM Immunolocalization of AgamCPCFC1 on legs from adults of various ages. In these sections label is restricted to endocuticle. A. Leg from adult one day after eclosion. B. Apodeme from same animal. Exocuticle is interior in the apodemes. C. Section of leg from animal 8 days after eclosion. D. Pharate adult with only exocuticle and no labeling visible. ex, exocuticle; en, endocuticle; ep, epidermis. Scale bars are 500 nm.

Fig. 7

EM immunolocalization of AgamCPCFC1 in both exo- and endo-cuticle. A. Leg of a pharate adult (P24) showing areas of lamellar exocuticle with labeling near a joint. Insert lower power of relevant region. B. Labeling in exocuticle of P24 pedicel. C. Both exo- and endo-cuticle labeled in flagellum of adult <12 h after eclosion. Abbreviations as in Fig. 6. Scale bars are 500 nm.

Fig. 8

WebLogos constructed for CPCFC motifs highlighted in Supplementary Files 7 and 9.