Reduction of embryonic E93 expression as a hypothetical driver of the evolution of insect metamorphosis

Abstract

The early embryo of the cockroach Blattella germanica exhibits high E93 expression. In general, E93 triggers adult morphogenesis during postembryonic development. Here we show that E93 is also crucial in early embryogenesis in the cockroach, as a significant number of E93-depleted embryos are unable to develop the germ band under maternal RNAi treatment targeting E93. Moreover, transcriptomic analysis indicates that E93 depletion results in important gene expression changes in the early embryo, and many of the differentially expressed genes are involved in development. Then, using public databases, we gathered E93 expression data in embryo and preadult stages, finding that embryonic expression of E93 is high in hemimetabolan species (whose juveniles, or nymphs, are similar to the adult) and low in holometabolans (whose juveniles, or larvae, are different from the adult). E93 expression is also low in Thysanoptera and in Hemiptera Sternorrhyncha, hemimetabolans with postembryonic quiescent stages, as well as in Odonata, the nymph of which is very different from the adult. In ametabolans, such as the Zygentoma Thermobia domestica, E93 transcript levels are very high in the early embryo, whereas during postembryonic development they are medium and relatively constant. We propose the hypothesis that during evolution, a reduction of E93 expression in the embryo of hemimetabolans facilitated the larval development and the emergence of holometaboly. Independent decreases of E93 transcripts in the embryo of Odonata, Thysanoptera, and different groups of Hemiptera Sternorrhyncha would have allowed the development of modified juvenile stages adapted to specific ecophysiological conditions.

Keywords: E93; complex life cycles; embryogenesis; insect metamorphosis.

Fig. 1.

Effects of E93 depletion on embryo development in B. germanica. (A) E93 expression in embryos of different ages from day 0 (ED0) to day 16 (ED16). Note that the MZT takes place between ED0 and ED2. (B) E93 expression after maternal RNAi at ED0 in control (C) and dsE93-treated (E93-i) insects. (C) Phenotypes resulting from E93 depletion. Phenotype A (PA): embryos that interrupted development around the formation of germ-band anlage; Phenotype B (PB) (14.4%): embryos that interrupted development between Tanaka stages 10 and 15; Phenotype C (PC) (19.4%): apparently well-formed late embryos, with dark cuticle; and Phenotype D (PD) (40.6%): well-formed late embryos without malformations, which would be close to hatching. (D) Accumulation of energids in the ventral side of the embryo (white asterisks) in control and E93-i specimens, in ED2. In A and B, each qRT-PCR expression measurement represents three biological replicates, and results are expressed as copies of the examined transcript per 1,000 copies of BgActin-5c mRNA; data are represented as mean ± SEM. In B, the asterisk indicates statistically significant differences with respect to controls (P-value < 0.05), calculated on the basis of the Relative Expression Software Tool (REST) (16). (Scale bars in C, 500 µm; in D, 200 µm.)

Fig. 2.

Transcriptomic changes resulting from the depletion of E93 in the embryo of B. germanica. (A) PCA of the 11 RNA-seq libraries after batch correction (six from controls and five from E93-depleted embryos). (B) Volcano plot showing the DEGs in control and E93-depleted embryos. A total of 98 genes were significantly down-regulated (blue dots) and 47 were up-regulated (red dots) (adjusted P-value < 0.05). (C) GSEA showing the top significantly enriched Gene Ontology (GO) terms of biological processes down-regulated after depleting E93 in early embryos. GO terms containing the word “morphogenesis” are in blue, with “development” in green, and “metamorphosis” in red.

Fig. 3.

Expression of a selection of genes in E93-depleted (E93-i) embryos of B. germanica. The following nine genes were selected among the 98 found to be significantly down-regulated (adjusted P-value < 0.05) in E93-i samples according to differential expression analysis of transcriptomic data: Krüppel (Kr), even-skipped (eve), fushi tarazu (ftz), neverland (nvd), shade (shd), thisbe (ths), Partner of paired (Ppa), knirps-like (knrl), and Bger_23358. The following five genes were selected among the 47 found to be significantly up-regulated according to the same analysis: seven in absentia (sina), starvin (stv), Bger_02424, Bger_15646, Bger_20604, and Bger_27721. The expression profile in 0-, 1-, 2-, 6-, and 13-d-old embryogenesis, according to the transcriptomes reported previously (13) (Left bar diagram), and the qRT-PCR expression measurements (Right bar diagram) in controls (C, dsMock-treated) and E93-i, are shown for each gene. Each qRT-PCR expression measurement represents three to five biological replicates, and results are expressed as copies of the examined transcript per 1,000 copies of BgActin-5c mRNA; data are represented as mean ± SEM; the asterisk indicates statistically significant differences with respect to controls (P-value < 0.05), calculated on the basis of the REST (29).

Fig. 4.

Ratio in log2 scale between the E93 expression levels in the PA/E ratio in different species of ametabolan (F. candida, T. domestica), hemimetabolan (Aphis gossypii, Acyrthosiphon pisum, B. germanica, C. dipterum, Gryllus bimaculatus, I. senegalensis, N. lugens, Zootermopsis nevadensis), “neometabolan” (B. tabaci, D. citri, F. occidentalis, H. brevitubus, P. kraunhiae), and holometabolan (Apis mellifera, Anopheles stephensi, Bactrocera dorsalis, B. mori, Camponotus floridanus, Culex quinquefasciatus, D. melanogaster, Harpegnathos saltator, Hermetia illucens, Leptopilina heterotoma, Manduca sexta, Monomorium pharaonis, Odontomachus brunneus, Papilio polytes, Pieris rapae, Plutella xylostella, Spodoptera exigua, Tribolium castaneum, Trichomalopsis sarcophagae, Vanessa tameamea, Zeugodacus cucurbitae) species. Data are from publicly available RNA-seq data (dots), qRT-PCR measurements (triangles), and colored according to the order to which the species belong. Ratios were calculated as a log2 of the quotient of TPM means for the highest expression sample in preadult (nymph or pupa) to embryo stages for each species analyzed. The asterisk in the boxplots indicates statistically significant differences (two-sided Wilcox test, P-value < 0.05) between different types of metamorphosis.

Fig. 5.

Expression of E93 in T. domestica, and hypothesis to explain the evolution of different types of insect metamorphosis in relation to embryonic E93. (A). E93 expression pattern in embryos, nymphs, and adults of T. domestica, compared with Kr-h1 and Br-C expression. The early expression of E93 and Br-C (0-12 h) is probably of maternal origin. The expression was measured by qRT-PCR, and each measurement represents three biological replicates, except for ninth instar nymphs (12 biological replicates); in the case of Br-C, we used primers designed in the core region, thus measuring the set of all possible isoforms; mRNA levels were normalized to those of rp49 and data are represented as mean ± SEM; in the nymphal period, the nine nymphal instars (N1 to N9) are indicated; in the adult (female), two periods were studied: that of fecundation (Fec.) and that of oviposition (Ovip.). (B) The evolution of different postembryonic morphologies and types of insect metamorphosis can be explained on the basis of the levels of embryonic E93 expression. The decrease of E93 expression in the hemimetabolan embryo may have unconstrained the nymphal developmental program, thus facilitating the development of morphologically divergent juveniles, and the emergence of new types of metamorphoses and life cycles.