Metamorphosis, the change (meta) in form (morphe), is a common phenomenon in the animal kingdom, where it has evolved several times independently. The most dramatic metamorphic changes occur in the most successful group of animals: the insects, which comprise more than 60% of all living animals. Within the group of insects, the Holometabola (e.g. beetles, butterflies, flies and bees) comprise more than 80% of all insect species. Holometabolous insects undergo complete metamorphosis. In a non-feeding pupal life stage, intercalated between the larval and adult stages, their entire anatomy is radically remodelled, including the digestive tract, which undergoes apoptosis and proliferation. The second major group of insects, the Hemimetabola (e.g. grasshoppers, true bugs, and dragonflies), undergo incomplete metamorphosis. Compared to Holometabola, hemimetabolous insects metamorphose more gradually, less drastically and without a pupal stage.
Holometaboly is one of the key evolutionary innovations explaining insects' enormous and unique biodiversity. However, how the evolution of the pupal stage is related to the success of insects is unknown. The remodelling of the larval gut poses a significant challenge to the gut microbiota, as the gut is replaced during pupation, which does not occur in Hemimetabola. It gives holometabolous insects the unique opportunity to drive a change between the larval and adult microbiota, facilitating niche shifts by allowing the insect to acquire specialised symbionts for a life-stage specific diet, ecology and physiology- one barely studied adaptive hypothesis explaining the evolution of the pupa.
In chapter II, using 16S rRNA gene metabarcoding, I studied 18 different herbivorous insect species from five orders of holometabolous and three orders of hemimetabolous insects. Comparing larval and adult specimens, I found a much higher beta-diversity and hence microbiota turnover in holometabolous insects than in hemimetabolous insects. My results support the idea that the pupa offers the opportunity to change the gut microbiota and hence facilitates niche shifts. This possible effect of niche shift facilitation could explain a selective advantage of the evolution of complete metamorphosis.
The unique opportunity to change the microbial composition throughout insect development by gut remodelling during complete metamorphosis also puts holometabolous insects at a higher risk of infections. Holometabola must control their gut microbiota and initiate an immune response to avoid infectious diseases during metamorphosis.
In chapter III, using RNAseq, I compared the expression of immune effector genes in the gut during metamorphosis in two holometabolous and a hemimetabolous insects. I found high read count abundances of differentially expressed immune effectors in the gut at the larval-pupal moult in the two Holometabola; no such high abundances were observed at the nymphal-adult moult in Hemimetabola. My findings confirm that only complete metamorphosis elicits a prophylactic immune response as an adaptive response in holometabolous insects, which controls the microbiota during gut replacement.
Another barely studied and not mutually exclusive hypothesis explaining the success of holometabolous insects could be that intercalating the pupal stage decouples growth and differentiation. Most growth is confined to the larval stages in holometabolous insects, while most development occurs in the pupa, allowing for fast larval growth.
In chapter IV, I conducted a literature review and calculated growth rates and ratios. I compared 33 species from three holo- and seven hemimetabolous insect orders. I found faster larval growth, higher growth ratios, and much higher variances for those traits in holometabolous than hemimetabolous insects. I also found much shorter growth periods of the larval stages in holometabolous than hemimetabolous insects. My results support the decoupling of the growth and differentiation hypothesis in holometabolous insects, allowing fast larval growth.
In this thesis, I investigated two barely studied and not mutually exclusive hypotheses explaining the evolution of the pupa in holometabolous insects, which constitute the majority of animal diversity. I could show a microbiota turnover in holometabolous insects, which is also under the control of the host gut immunity and allows the Holometabola to occupy different niches throughout development. The second hypothesis, which proposes that decoupling growth and differentiation allows for fast larval growth, is supported by my findings of faster larval growth rates in holometabolous than hemimetabolous insects. The facilitation of niche shifts by changes in the gut microbiota could be considered an essential driver of the evolution of the pupa. The microbiota turnover could also be driven by other selective factors such as growth rate. Fast larval growth could be a selective factor for decoupling growth and differentiation, ultimately resulting in the evolution of the pupa in holometabolous insects.
