This post is part of a series on the insect cuticle as a biological material that can inspire novel engineered materials. The characteristics of the cuticle, setting it apart from most synthetic/engineered materials, will be discussed in this series. The introduction to the series can be found here.

Structural hierarchy, and thus strength, is intrinsic

The insect cuticle can have great strength and flexibility, much greater than if it were just made up of stacked sheets of chitin and protein. The cuticle also exhibits energy absorption properties. This is all due to its ingenious structure. It has a complex hierarchical micro-architecture that spans multiple length scales from the nanoscale to the macroscale, which exhibit a remarkable combination of stiffness, ability for crack deflection, low weight and strength. The cuticle’s special self-assembly characteristics, as those of other biological materials, have attracted interest from materials scientists for the development of laminated composite materials and from bioengineers interested in molecular scale self-assembly.

Many biological materials exhibit structural hierarchy, and some have been studied in more detail than insect cuticle: nacre (mother-of-pearl) and bone. There is often hierarchy within hierarchy, at the nano-, micro-, all the way to the meso-level.  Hierarchical structure is primarily due to the outcome of developmental pathways of biological systems that create the material. Having hierarchy within a biological material is intrinsic. The only forces acting during material synthesis are intermolecular, and are thus very weak and work on only a short range.

This can result in very beneficial properties.

• areas and layers that are softer than the rest can greatly affect the failure properties of a material because these interfaces can stop cracks, or divert them.
• The hierarchical material’s microstructures can exhibit dramatic increases in compressive strength compared with that of solids of similar density with conventional structure.

Since human-made materials are often made using a top down approach, by forcing structure and shape onto a material, we do not often see structural hierarchies in engineered materials, or only to a limited extend.

Characteristic figure from “Insects Did It 1st” book by Akre, Paulson and Catts. This illustration by E. Paul Catts accompanied the chapter on plywood/cuticle. Plywood is a human-made material that is layered – if not quite in a hierarchical manner.

The mechanical properties of biological materials, such as the insect cuticle, may give insights into the design of more advanced engineered composites.

The layers

Meso-level hierarchy

Strictly speaking the insect’s integument is comprised of the epidermal cell layer (epidermis) and the cuticle. The Integument itself is build upon a basement membrane (basal lamina).

The insect integument. Let’s call this meso-level hierarchy. The integument is comprised of an epidermal cell layer and cuticle, all on top of basement membrane. (Drawing by Marianne Alleyne)

The basement membrane is only 0.5 μm thick and separates the epidermal cells from the circulating hemolymph (=insect blood).  The membrane can some proteins through, and nerves and trachea also penetrate it, but when this membrane gets breached alarm bells go off in the insect’s body. Often an immune response is mounted by blood cells in response to the breach and the epidermal cells will help prepare it.

The epidermis is comprised of living cells arranged in a single layer. The cells can be modified into structures such as dermal glands and sensory receptors.

The cuticle is secreted by the epidermal cells and can be incredibly thin (e.g. larval endoparasitoids) or incredibly thick (e.g. adult rhinoceros beetles). The cuticle is non-living. It lines the external surface of the body as well as the lining of the trachea and the anterior and posterior sections of the alimentary canal and even parts of the reproductive system. This means that often even these structures undergo a re-lining during a molt.

Meso/Micro-level hierarchy

Several horizontal divisions of the cuticle are obvious, when using an electron microscope, giving the cuticle at this micro/meso-level also a hierarchical structure. These divisions came about because the sublayers were produced in a certain sequence.

The meso/micro-level hierarchy of the insect integument. The cuticle is comprised of the epicuticle and procuticle. The procuticle is in turn subdivided into the endo-, meso- and exocuticle. (Drawing by Marianne Alleyne)

1. There is a thick inner PROCUTICLE. This is the only layer that contains chitin, it also contains proteins.  As the molting cycle progresses the procuticle becomes horizontally subdivided.
   a)    Exocuticle – Is the first portion of procuticle to become synthesized, and then is pushed outward and becomes the outer layer of the procuticle.  Contains heavily cross-linked (insoluble) proteins and chitin.  This layer cannot be reused and is shed during the molt.
   b)    Endocuticle – This layer is formed just above the epidermis.  Consists of several lamellar layers of protein and chitin.  In soft-bodied insects and in areas of flexibility it is this layer that comprises most of the cuticle.  It is flexible because there is little cross-linking of proteins.  It can also be reabsorbed.
   c)    Mesocuticle – This layer cannot always be identified.  It appears to be a transitional layer.  Proteins are yet untanned (like endocuticle) but impregnated with lipids and proteins (like exocuticle), may also contain chitin.
2. Thin outer EPICUTICLE consists of several layers, which are produced by the epidermal cells and dermal glands.  This layer does not contain chitin.  Since it is only 1 to 4 um thick it has been incredibly difficult to study. It is lipids in the wax layer on top of the epicuticle that play protective and communicative roles in the life of insects.

Nano/Micro-level hierarchy

The cuticle is a composite consisting of chitin fibers within proteinaceous matrix, all arranged in a layered structure. Several horizontal divisions can be observed. Each layer (or lamina) consists of a layer of parallel chitin chains but the laminae are stacked in such a way that the chitin molecules are arranged in an antiparallel manner.

So what does this mean? Lets see if we can build a cuticle graphically from the nano- to micro-scale.

• Chitin is one of the two major components of the procuticle, it can make up almost half of the total dry weight of the cuticle – but it probably most likely to be less than 20% for some of the most familiar insects, such as caterpillars. Chitin is a very stable molecule: insoluble in water, dilute acids, concentrated alkali, alcohol, and organic solvents. These features make the molecule a great biological building block, but very difficult to study. Proteins are the other major component of the insect cuticle. It is the interaction between proteins and chitin that provides the mechanical function of the cuticle – giving it strength. (In the future I will come back to interesting proteins, such as resilin.)

• Chitin is very similar to cellulose. It is an acetylated polysaccharide. A ribbon-like chitin chain is created when N-acetyl-D-glucosamines and glucosamines link together.

A portion of a chitin chain. In most insect cuticles these chains are arranged in an anti-parallel manner.

• Adjacent chains of chitin are linked together through hydrogen bonds. Most commonly the orientation of the chitin chains relative to each other is anti-parallel. This arrangement (already at this nano-level hierarchical scale) allows for tighter packaging and contributes to the strength and stability of the cuticle. The grouping of 18-25 chitin molecule chains then creates chitin microfibrils that are about 3nm thick. These microfibrils are wrapped in protein.

Nanofibrils are created when groups of chitin molecules link by way of hydrogen bonds and are wrapped in protein.

• At the next hierarchical level the nanofibrils cluster together into long chitin-protein fibers.

Nanofibrils cluster together to form chitin-protein fibers.

• The microfibrils are laid down in an almost parallel pattern within a single layer. This creates a network or matrix of chitin and proteins, and other components such as minerals. The chitin microfibrils are covalently linked to the proteins.

Chitin-protein fibers are woven into a matrix.

• These layers are stacked on top each other in successive layers that are rotated by a constant angle to produce a helicoidal arrangement that further contributes to the strength of the overall cuticle.

The helicoidal stacking of chitin-protein layers (matrix) creates a twisted arrangement. This creates an arching pattern in the cuticle as a whole. (Here layers were created by stacking sushi mats in a twisted manner. Pictures by Marianne Alleyne)

It is the hierarchical structure created at the nano-scale, when chitin molecules link together and form chains in close association with proteins, all the way up to the meso-scale, where different cuticular layers are stacked in non-random orientations, that makes the insect cuticle such an inspiring biological material. It is because of this hierarchical structure that the insect cuticle can take on all the different functions that it has. And ultimately the cuticle is one of the main reasons why insects can be found to be thriving in so many different habitats – air, water, land, even inside horse guts. Modern analysis of small-scale composites and self-assembly-type manufacturing techniques will enable us to make materials equally as versatile as the insect cuticle.

Further reading:

Design and mechanical properties of insect cuticle. J. F. V. Vincent and U. G. K. Wegst (2004) Arthropod Structure and Development V33 pp-187-199. DOI: 10.1016/j.asd.2004.05.006

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Side note about those sushi mats:

Wild&crazy Sat night at home: making insect cuticle models out of sushi mats. #ActiveLearning #IB427 pic.twitter.com/ND0I2XhHxH

— Marianne Alleyne (@Cotesia1) January 18, 2014