Fig 1.
Representative images of the separation of larvae carried out in this work.
Fourth instar larva were aged and sexed based on the development of genital and thoracic imaginal discs, according to [33], who divided the fourth instar into nine phases (phase I to phase IX). In order to reduce stressful conditions to larvae, phases were grouped into five broader categories (I-II; III-IV; V-VI; VII-VIII and IX, respectively). A. (I-II); B. (III-male); C. (IV-female); D. (V-male); E. (VI-female); F. (VII-male); G. (VIII-female); H. (IX-male) and I. (IX-female).
Table 1.
Primers used for Real-Time PCR amplification of the genes studied in C. riparius.
Fig 2.
Ontogenetic variations in the expression pattern of the genes analyzed.
Transcriptional levels of genes from C. riparius involved in the ecdysone-related pathway (EcR, usp, E74 and vg), the folding and maturation of steroid hormone receptors (hsp70 and hsc70) and the synthesis of the ribosomal protein L13. Gene expression was measured from embryo to pupa stages of development. The mRNA values were calculated relative to actin, GAPDH and 26s as reference genes. Each bar is the mean ± SE obtained from four independent samples, each with three experimental replicates. A total of 4 egg masses and 20 larvae of each stage or phase were used. All the analyzed genes showed significant differences among the different stages (Fig 3).
Fig 3.
Significant differences in the expression pattern of the genes analyzed.
All the analyzed genes showed significant differences among the different stages according to Kruskal-Wallis Post hoc test. Three p values were tested in all cases.—(no differences), + (p < 0.05), ++ (p < 0.01), +++ (p < 0.001).
Fig 4.
Hierarchical clustering dendrogram of the groups established for the expression analysis of ecdysone related genes.
Cluster analysis arranges biological samples into groups based on the expression levels measured in female (A) and male (B) larvae. Relationships among samples are represented by a dendrogram whose branch lengths reflect the degree of similarity between them as assessed by pairwise comparisons of gene expression profiles. Two groups of developmental stages or phases are clearly separated with slight differences between sexes: 1) 1st, 2nd, 3rd instar larvae and early 4th instar phases; and 2) late 4th instar larvae, pupa and embryo. The root represents the whole data set and a leaf corresponds to a single object in it. An internal node represents the union of all objects in its sub-tree. The weight of an internal node represents the distance between its two child nodes.
Fig 5.
Ecdysteroid titers determination throughout C. riparius development.
Ecdysteroid levels in the whole organism were analysed from embryo to pupa. Two EIA assays were performed and results are expressed as mean ± SE. A total of six egg masses and eight individuals of each developmental category were used in this study. Data corresponding the 4th instar larvae are shown separately by sex. Results are shown in 20E equivalents (relative levels; pg/larva).
Fig 6.
Profiles for EcR, usp, E74, vg and hsp70 in late developmental stages in C. riparius.
Schematic drawing of the life cycle of a non-biting midge (Diptera: Chironomidae, Chironomus) based on the results obtained in this work. Larval instars were identified by measuring head capsule widths. Age and sex of fourth instar larvae were established from the development of genital and thoracic imaginal discs according to [33]. Lines corresponding to hormone titers (20E and JH) are based on previous works [1,43–45].