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, 280 (1764), 20131082

The Extraembryonic Serosa Protects the Insect Egg Against Desiccation

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The Extraembryonic Serosa Protects the Insect Egg Against Desiccation

Chris G C Jacobs et al. Proc Biol Sci.

Abstract

Insects have been extraordinarily successful in occupying terrestrial habitats, in contrast to their mostly aquatic sister group, the crustaceans. This success is typically attributed to adult traits such as flight, whereas little attention has been paid to adaptation of the egg. An evolutionary novelty of insect eggs is the serosa, an extraembryonic membrane that enfolds the embryo and secretes a cuticle. To experimentally test the protective function of the serosa, we exploit an exceptional possibility to eliminate this membrane by zerknüllt1 RNAi in the beetle Tribolium castaneum. We analyse hatching rates of eggs under a range of humidities and find dramatically decreasing hatching rates with decreasing humidities for serosa-less eggs, but not for control eggs. Furthermore, we show serosal expression of Tc-chitin-synthase1 and demonstrate that its knock-down leads to absence of the serosal cuticle and a reduction in hatching rates at low humidities. These developmental genetic techniques in combination with ecological testing provide experimental evidence for a crucial role of the serosa in desiccation resistance. We propose that the origin of this extraembryonic membrane facilitated the spectacular radiation of insects on land, as did the origin of the amniote egg in the terrestrial invasion of vertebrates.

Keywords: Tribolium castaneum; chs (chitin synthase); cuticle; desiccation resistance; zen (zerknüllt).

Figures

Figure 1.
Figure 1.
The serosa is an evolutionary novelty of insects. (a) Phylogeny of the main arthropod groups [10,11]. The bar under the groups indicates whether species in this class are generally aquatic or restricted to humid environments for reproduction (blue), or terrestrial (yellow), or if species of this class live in very different environments concerning humidity (yellow diagonal stripes). Above the groups, schematic cross-section drawings of the embryo (green) and extraembryonic membranes are shown (open black circles, extraembryonic membrane; red open circles, serosa; blue closed circles, amnion). Jura [12], Machida [13] and Machida & Ando [14] call the extraembryonic membrane in Entognatha a serosa. We, however, adopted the terminology of Anderson [15]. Although parallel evolution of two extraembryonic membranes took place in the scorpions, a serosa completely enveloping the embryo and secreting a cuticle is an evolutionary novelty of the insects. In the Schizophoran flies, a secondary reduction took place. (b) Schematic drawing of Tribolium wild-type and Tc-zen1 RNAi development. In wild-type eggs, the serosa completely envelops yolk and embryo. After Tc-zen1 RNAi, an amnion covers the yolk dorsally; the serosa is absent.
Figure 2.
Figure 2.
Eggs without a serosa or a serosal cuticle become desiccation-susceptible. (a) Hatching rates of control (grey squares), wild-type (black circles), Tc-zen1 RNAi (grey triangles) and Tc-chs1 RNAi (black diamonds) eggs at 35°C. Error bars indicate standard error among three to 10 replicates of 96 eggs. (bd) Heat maps summarizing hatching rates of (b) control eggs, (c) Tc-zen1 RNAi eggs and (d) Tc-chs1 RNAi eggs.
Figure 3.
Figure 3.
Tribolium castaneum produces a chitinized serosal cuticle. (a,b) TEM pictures of an approximately 37 h old (a) wild-type and (b) Tc-chs-1 RNAi egg. Note the absence of the chitinous layers in the Sc after Tc-chs1 RNAi. The vitelline membrane no longer sticks to the cuticle. The serosal cells might have a slightly aberrant appearance because they lost contact with the extracellular matrix, similar to the chitin-secreting epidermal cells in Drosophila chs1 (kkv) mutants [52]. (c) Nuclear DRAQ5 stain (purple) of the egg shown in (d). The serosal nuclei are widely spaced. Ventrally, the dense nuclei of the embryo proper (headlobes to the left) are prominently visible. (d) Tc-chs1 in situ hybridization during gastrulation. Tc-chs1 mRNA (red) is detected around the serosal nuclei and not in the embryo. (e,f) A single confocal plane showing an optical cross-section of the embryo shown in (c) and (d). a, anterior; p, posterior; d, dorsal; v, ventral. HL, head lobes; E, embryo; A, amnion; S, serosa; Sc, serosal cuticle; V, vitelline membrane.
Figure 4.
Figure 4.
Tc-zen1 RNAi embryos display defects in dorsal closure at high humidity. (a) Width/length ratio (1 = perfect sphere) of control (green squares), wild-type (blue circles), Tc-zen1 RNAi (red triangles) and Tc-chs1 RNAi (purple diamonds) eggs measured in their vitelline membrane using ImageJ [53]. (bd) Phase contrast images of a 65–75 h old (b) wild-type, (c) Tc-zen1 RNAi and (d) Tc-chs1 RNAi embryo at 90% RH. (eg) DAPI (blue) label cell nuclei and WGA-FITC [36] (green) label chitin of the larval cuticle in (e) a dorsally closed wild-type embryo, (f) a Tc-zen1 RNAi embryo that has not completed dorsal closure, and (g) a dorsally closed Tc-chs1 RNAi embryo. a, anterior; p, posterior; d, dorsal; v, ventral. In (e), a small piece of vitelline membrane is stuck between the legs. (h) Overview TEM pictures of 65–75 h old wild-type egg during dorsal closure with dorsally condensed serosa. (i) Magnification of area boxed in (h). A layered serosal cuticle can be detected (Sc), but the serosa has condensed dorsally. No epidermal cuticle is found 65 h AEL. H, head; Y, yolk; L, legs; E, embryo; Sc, serosal cuticle; V, vitelline membrane; S, serosa; A, amnion.

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