How insects conquered land

How insects conquered land

Three quarters of all species are insects and this enormous diversity is attributed to the incredible ability of insects to adapt to any circumstance. Insects are one of the first land animals; an important adaptation to terrestrial life was the evolution of a waterproof cuticle (skin). Once on land, other adaptations such as flight and their co-evolution with plants, made insects the most successful and diverse group on the planet.



There is however one aspect of terrestrial life which has gotten little attention, the insect egg. Although the waterproof cuticle of adult insects made it possible for them to live on land, as long as the eggs are not able to resist desiccation, they would still be constrained to areas with water close-by to lay their eggs in. In a recent publication in the journal “Proceedings of the Royal Society B” we show how insect eggs are protected against desiccation, which was crucial for a true terrestrial lifestyle.

The type of development of the egg coincides with their habitat

Insects are part of the group Arthropoda, of which the Crustacea (Crabs etc.), Myriapoda (millipedes etc.) and Chelicerata (spiders etc.) are also part. In order to find out why insect eggs are able to resist desiccation but many other Arthropod eggs are not, we looked at the type of development in relation to their phylogeny and habitat. The phylogeny is shown in the figure below, above each group is a schematic of the type of development, and below each group is the description of the habitat in which they live/lay their eggs in. Habitats which are moist are indicated in blue, dry habitats are indicated in yellow. Spiders can survive well in dry habitats, but in this group the mothers take care of the eggs and protect them (by for example spinning their eggs in silk). The Myriapoda (millipedes etc.), Crustacea (Crabs etc.) and Enthognatha (springtails etc.) generally live in moist environments. The eggs in these groups only develop one extraembryonic membrane, which covers the top side of the egg, but never envelopes the embryo. Insects on the other hand, develop 2 extraembryonic membranes, of which the serosa completely enfolds the embryo (you can find more information about extraembryonic development here). The development of 2 extraembryonic membranes coincides with their ability to survive in dry environments. Surprisingly, one group of flies reverted to one extraembryonic membrane. This single extraembryonic membrane covers the top of the egg, much like in Crustacea etc., but will never envelope the embryo. The eggs of this group of flies are also restricted to humid environments for their development. This relation between the ability to live in dry environments and the presence of the extraembryonic serosa led us to hypothesize that the serosa might protect against desiccation.

Phylogeny
The egg of the flour beetle as model for desiccation resistance

To assess whether it is indeed the serosa which protects the egg against desiccation we needed a suitable insect to test our hypothesis. We chose for the flour beetle, Tribolium castaneum. This beetle has 2 extraembryonic membranes. This is however not the reason we chose this species, the reason is that in this beetle we are able to prevent the development of the serosa by knocking down the gene Tc-zen1 with RNAi. Knocking down this gene prevents the development of the serosa but these serosa-less eggs are able to survive under normal conditions (see figure below).

zen1knockdown

Furthermore, we discovered that the serosa also secretes a cuticle, which consists in large part of chitin. This is exactly the same mechanism by which adult insects resist desiccation. In the figure below you can see the serosa and the cuticle it secretes (20.000x magnification).

 

serosal cuticle

Not only could we show that the serosa secretes a cuticle, we could also prevent the secretion of this cuticle by knocking down the gene Tc-chs1 with RNAi. In this way we could not only compare eggs with and without a serosa, but also eggs without a cuticle (but still with serosa).

Does the serosa protect against desiccation?



To test whether the serosa protects against desiccation, we collected eggs and kept these at different humidities and scored how many eggs survived. We did this for eggs with a serosa, eggs without a serosa and eggs without a cuticle. To check for an effect of the RNAi treatment we also performed RNAi on a gene which does not knock anything down in the flour beetle (control). These eggs have both the serosa and the cuticle and should behave like normal eggs (more info about RNAi can be found here). We performed this experiment at different temperatures and the results from 35 degrees are in the figure below. As you can see, of both the normal eggs (blue line) and the control eggs (green line), about 80% survive at all humidities. However, when we look at the serosa-less eggs (red line), we see that their survival decreases dramatically at low humidities. This indicates that they desiccate. To see whether it is the serosa itself or the cuticle it secretes that protects against desiccation, we also tested eggs without a cuticle (purple line). The survival of eggs without a cuticle is also dramatically decreased at low humidity, indicating that it is the cuticle secreted by the serosa which protects against desiccation. What might have caught your attention is the strong decrease in survival at high humidity for serosa-less eggs (red line). This decrease is due to another function of the serosa.

hatchingrates35
The serosa at high humidity

Apparently, the serosa is also crucial at high humidity. But why? The cuticle seems not to be involved as eggs without a cuticle (but with serosa) are fine at high humidity. To find out what is killing the eggs at high humidity we looked at eggs, which have been at high humidity, under the microscope (figure below, a-c). We noticed that both eggs without a serosa (b) and eggs without a cuticle (c) looked swollen. To quantify this we measured how round the eggs are by dividing the width of the egg by the length of the egg. This gives a low number for oval shapes (0.55) and a larger number for round eggs (0.65). From this data we could clearly see that eggs without a serosa (figure below, red line) and eggs without a cuticle (purple line) are rounder at high humidity than eggs with serosa and cuticle (blue and green lines).Although eggs without a cuticle swell up, they survive just fine at high humidity. This cannot be said of eggs without a serosa, so swelling up seems to be a problem only when you don’t have a serosa. The serosa is also involved in a process which is called “dorsal closure”. This is the process at the end of development when the embryo closes its body at the backside (for more information check here). By staining embryos that were kept at high humidity and looking at them under the microscope we could see that eggs without a serosa (figure below, e) often had an open back. Eggs without a cuticle (but with serosa) did not have this problem (figure below, f). The number of eggs with an open back correlated well with the number of eggs that did not hatch. This shows that the lack of a serosa can be compensated normally, but in bad conditions (a swollen body increases the distance towards the back side to be covered) many embryos do not survive.

opzwellen
H = head, Y = yolk, L = legs

Conclusion

Insects are incredibly successful and being one of the first land animals has certainly played a major role in their success. Adult insects were able to survive on land by their waterproof cuticle. This is also true for spiders, but spiders protect their eggs against desiccation by for example wrapping them in silk. Insects generally do not protect their eggs. We show for the first time that an cuticle produced by the egg itself protects the insect egg against desiccation. The combination of a waterproof cuticle in both the adult and the egg ensured survival of insects in any place on land, and has certainly helped the insects becoming as successful as they are today.

Reference:

Jacobs, C. G. C., Rezende, G.L., Lamers, G.E.M. and van der Zee, M. The extraembryonic serosa protects the insect egg against desiccation. Proceedings of the Royal Society B. http://dx.doi.org/10.1098/rspb.2013.1082

 

Leave a Reply

This site uses Akismet to reduce spam. Learn how your comment data is processed.