Entomology 3rd edition - C.Gillott - Chapter 11 pptx

Epidermal cells may form specialized glands that produce components of the cuticle or may develop into particular parts of sense organs.. Structure The innermost component of the integum

Anatomy and Physiology

The Integument

1 Introduction

The integument of insects (and other arthropods) comprises the basal lamina, epidermis, and cuticle It is often thought of as the “skin” of an insect but it has many other functions (Locke, 1974) Not only does it provide physical protection for internal organs but, because

of its rigidity, it serves as a skeleton to which muscles can be attached It also reduces water loss to a very low level in most Insecta, a feature that has been of great significance in the evolution of this predominantly terrestrial class In addition to these primary functions, the cuticular component of the integument performs a number of secondary duties It acts as

a metabolic reserve, to be used cyclically to construct the next stage, or during periods of great metabolic activity or starvation It prevents entry of foreign material, both living and nonliving, into an insect In many insects the waxy outer layer serves as a repository for contact sex pheromones (Chapter 13, Section 4.1.1) The color of insects is also a function

of the integument, especially the cuticular component

The integument is not a uniform structure On the contrary, both its cellular and acellular components may be differentiated in a variety of ways to suit an insect’s needs Epidermal cells may form specialized glands that produce components of the cuticle or may develop into particular parts of sense organs The cuticle itself is variously differentiated according

to the function it is required to perform Where muscles are attached or where abrasion may occur it is thick and rigid; at points of articulation it is flexible and elastic; over some sensory structures it may be extremely thin

2 Structure

The innermost component of the integument (Figure 11.1) is the basal lamina, an amorphous but selectively porous acellular layer that is attached by hemidesmosomes to the epidermal cells It is up to 0.5µm thick and is produced mainly by the epidermis, thoughµµ there are reports that hemocytes also participate The chemical nature of the basal lamina is poorly understood though neutral mucopolysaccharide, glycoproteins, and collagen, similar

to that of vertebrates, have been identified

The epidermis (hypodermis) is a more or less continuous sheet of tissue, one cell thick, responsible for secreting the bulk of the cuticle During periods of inactivity, its

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FIGURE 11.1. Diagrammatic cross-section of mature integument.

cells are flattened and intercellular boundaries are indistinct When active, the cells are more or less cuboidal, and their plasma membranes are readily apparent; one to several nucleoli, extensive rough endoplasmic reticulum, and many Golgi complexes are evi-dent (Locke, 1991, 1998) A characteristic feature of the apical (cuticle-facing) surface

of epidermal cells are the plasma membrane plaques, specialized regions of the plasma membrane at the tips of fingerlike microvilli, from which the cuticulin envelope and new chitin fibers arise (Section 3.1) Electron microscopy has shown that, at metamorphosis, the epidermal cells develop basal processes (“feet”) which can extend to become con-nected with the basal lamina and with other epidermal cells When the feet shorten, the basal lamina is buckled and rearrangement of cells occurs, resulting in a change in the insect’s shape, for example, from a long, thin caterpillar to a short, fat pupa (Locke, 1991, 1998) Epidermal cells also possess the ability to develop various forms of cytoskele-tal extensions which can be used, for example, to draw tracheoles closer to the cell for increased oxygen supply, or to maintain intercellular contact as the cells migrate dur-ing wound healdur-ing and changes in body shape The density of cells in a particular area varies, following a sequence that can be correlated with the molting cycle The cells often contain granules of a reddish-brown pigment, insectorubin, which in some insects con-tributes significantly to their color However, in most insects color is produced by the cuticle (Section 4.3)

Epidermal cells may be differentiated into sense organs or specialized glandular cells Oenocytes are large, ductless, often polyploid cells, up to 100µm inµµ diameter They occur

in pairs or small groups and the cells of each group may be derived from one original epidermal cell Usually they move to the hemocoelic face of the basal lamina, though in some insects they form clusters in the hemocoel or migrate and reassemble within the fat body Oenocytes show signs of secretory activity that can be correlated with the molting cycle, and, on the basis of certain staining reactions, it has been suggested that they produce the lipoprotein component of epicuticle In addition, ultrastructural and biochemical studies have led to the proposal that these cells produce ecdysone (Locke, 1969; Romer, 1991) They also synthesize components of the cuticular wax, including some contact sex pheromones

(Blomquist and Dillwith, 1985; Schal et al., 1998) Dermal glands of various types are also

differentiated In their simplest form the glands are unicellular and have a long duct that penetrates the cuticle to the exterior More commonly, they are composed of several cells

THE INTEGUMENT

The gland cells again exhibit cyclical activity associated with new cuticle production, and

it has been proposed that they secrete the cement layer of epicuticle

The cuticle, which is mainly produced by the epidermal cells, usually includes three

primary layers, the inner procuticle, middle epicuticle, and outer cuticulin envelope (Locke,

2001) (Figure 11.2) In older accounts of the integument the cuticulin envelope is treated as

part of the epicuticle However, Locke (1998, 2001) has argued that, because of its distinct

origin, structure and functions, the cuticulin envelope should be considered separate from

the epicuticle All three primary layers are present over most of the body surface and in the

cuticle that lines major invaginations such as the foregut, hindgut, and tracheae However,

the procuticle is very thin or absent, and certain components of the epicuticle may be

missing, where flexibility or sensitivity is needed, for example, over sensory structures and

the lining of tracheoles Only the cuticulin envelope is universally present, except for the

pores over chemosensilla (Chapter 12, Section 4.1)

The procuticle (= fibrous cuticle) forms the bulk of the cuticle and in most species is

differentiated into two zones, endocuticle and exocuticle, which differ markedly in their

FIGURE 11.2. Electron micrographs showing deposition of the three primary layers of cuticle in Calpodes

ethlius [From M Locke, 2001, The Wigglesworth lecture: Insects for studying fundamental problems in biology,

J Insect Physiol 47:495–507 With permission from Elsevier.]

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FIGURE 11.3. Diagram showing orientation of microfibers in lamellae of endocuticle [From A.C Neville and

S Caveney, 1969, Scarabaeid beetle exocuticle as an optical analogue of cholesteric liquid crystals, Biol Rev 44:

531–562 By permission of Cambridge University Press, London.]

physical properties but only slightly in their chemical composition In some cuticles the border between the two is not clear and an intermediate area, the mesocuticle, is visible Adjacent to the epidermal cells a narrow amorphous layer, the assembly zone, may be seen where chitin microfibers are deposited and oriented

The endocuticle is composed of lamellae (Figure 11.3) Electron microscopy reveals that each lamella is made up of a mass of microfibers arranged in a succession of planes, all fibers in a plane being parallel to each other The orientation changes slightly from plane

to plane making cuticle like plywood with hundreds of layers The exocuticle is the region

of procuticle adjacent to the epicuticle that is so stabilized that it is not attacked by the molting fluid and is left behind with the exuvium at molting (Locke, 1974) Not only is the exocuticle chemically inert, it is hard and extremely strong It is, in fact, procuticle that has been “tanned” (Section 3.3) Exocuticle is absent from areas of the integument where flexibility is required, for example, at joints and intersegmental membranes, and along the ecdysial line In many soft-bodied endopterygote larvae the exocuticle is extremely thin and frequently cannot be distinguished from the epicuticle and cuticulin envelope

Procuticle is composed almost entirely of protein and chitin The latter is a nitrogenous

polysaccharide consisting primarily of N -acetyl- D-glucosamine residues together with a small amount of glucosamine linked in aβ1,4 configuration (Figure 11.4) In other words, chitin is very similar to cellulose, another polysaccharide of great structural significance, except that the hydroxyl group of carbon atom 2 of each residue is replaced by an acetamide group Because of this configuration, extensive hydrogen bonding is possible between adja-cent chitin molecules which link together (like cellulose) to form microfibers Chitin makes

FIGURE 11.4. The chemical structure of chitin.

THE INTEGUMENT

up between 25% and 60% of the dry weight of procuticle but is not found in the epicuticle

and cuticulin envelope It is associated with the protein component, being linked to protein

molecules by covalent bonds, forming a glycoprotein complex Studies have shown that the

epidermis secretes more than a dozen major proteins into the cuticle in a carefully timed

sequence, probably under hormonal control (Suderman et al., 2003) Interestingly, cuticular

proteins of similar molecular weights have been found in a range of insect species suggesting

that the chemical nature of the cuticle has been strongly conserved through evolution The

amino acid composition of the proteins determines their properties For example,

endocu-ticular proteins are generally rich in hydrophobic amino acids with bulky side chains and are

loosely packed (not compact) molecules This provides the endocuticle with flexibility and

will also facilitate “creep” (the ability of layers to slide over each other), hence intrastadial

growth in soft-bodied insects such as caterpillars Conversely, in hard, stiff exocuticle, it is

small, compact amino acids that predominate (Hepburn, 1985)

In the exocuticle, adjacent protein molecules are linked together by a quinone molecule,

and the cuticle is said to be tanned (Section 3.3) The tanned (sclerotized) protein, which is

known as “sclerotin,” comprises several different molecules Resilin is a rubberlike material

found in cuticular structures that undergo springlike movements, for example, wing hinges,

the proboscis of Lepidoptera, the hind legs of fleas (Chapter 14, Section 3.1.2.), and the

wing-hinge ligament that stretches between the pleural process and second axillary sclerite

(Chapter 14, Sections 3.3.1 and 3.3.3) (Neville, 1975) Like rubber, resilin, when stretched,

is able to store the energy involved When the tension is released, the stored energy is used

to return the protein to its original length

In addition to these structural proteins, enzymes also exist in the cuticle, including

phenoloxidases, which catalyze the oxidation of dihydric phenols used in the tanning process

(Section 3.3) These enzymes appear to be located in or just beneath the epicuticle

A variety of pigments have been found in the cuticle (or in the epidermis) which may

give an insect its characteristic color (Section 4.3) Also, in a few beetles and larvae and

pupae of some Diptera, mineralized calcium (as the carbonate) is deposited, presumably to

increase rigidity (Leschen and Cutler, 1994)

Certain processes occur at the surface of the cuticle after it has been formed, for

example, secretion and repair of the wax layer and tanning of the outer procuticle Thus,

a route of communication must remain open between the epidermis and cuticular surface

This route takes the form of pore canals which are formed as the new procuticle is deposited

(Section 3.1), and which may or may not contain a cytoplasmic process Most often, the

canals do not contain an extension of the epidermal cell but have at least one “filament”

produced by the cell Locke (1974) suggested that the filament(s) might keep a channel open

in the newly formed cuticle until the latter hardens, and anchor the cells to the cuticle In

some insects the pore canals become filled with cuticular material once epicuticle formation

(including tanning) is complete The pore canals terminate immediately below the epicuticle

Running from the tips of the pore canals to the outer surface of the epicuticle are lipid-filled

channels known as wax canals

The epicuticle is a composite structure produced partly by epidermal cells and partly

by specialized glands It ranges in thickness from a fraction of a micrometer to several

micrometers and generally comprises three layers The layers are, from outside to inside,

cement, wax (these are secreted outside the cuticulin envelope), and the so-called protein

epicuticle The nature of cement varies, though it is likely to be approximately similar to

shellac The latter is a mixture of laccose and lipids The cement is undoubtedly a hard,

protective layer in some insects In others it appears to be more important as a sponge that

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soaks up excess wax The latter could quickly replace that lost, for example, by surface abrasion The wax is a complex mixture whose composition varies both among and within species, sometimes over different body regions of the same insect, and in some species seasonally Generally, long-chain hydrocarbons and fatty acid esters predominate, though varied proportions of alcohols, fatty acids, and sterols may also occur In some species the

mixture has relatively few different components, whereas in others, for example, Musca, more than 100 compounds have been identified (Blomquist and Dillwith, 1985; Jacob et al.,

1997) According to Locke (1974), within the wax layer three regions can be distinguished Adjacent to the cuticulin envelope is a monolayer of tightly packed molecules in liquid form that gives the cuticular surface its high contact angle with water and its resistance to water loss (but see Section 4.2.) Most wax is in the middle layer, which is less ordered and permeates the cement The outer wax layer, which comprises crystalline wax blooms, is not present in all insects The innermost layer of the epicuticle, the protein epicuticle, lies beneath the cuticulin envelope It may be several micrometers thick and like the cuticulin envelope it covers almost all of the surface of the insect It is absent from tracheoles and parts of some sense organs It comprises dense, amorphous protein tanned in a manner similar to the protein of the exocuticle (Section 3.3) but contains no chitin

The cuticulin envelope (about 20 nm thick) extends over the entire body surface and ectodermal invaginations, including the most minute tracheoles, but is absent from specific areas of sense organs and from the tips of certain gland cells It may be considered the most important layer of the cuticle for the following reasons (Locke, 1974, 2001) (1) It is a selectively permeable barrier During breakdown of the old cuticle, it allows the “activating factor” for the molting gel to move out and the products of cuticular hydrolysis to enter, yet ff

it is impermeable to the enzymes in the molting fluid It is permeable to waxes (as these are deposited only after the cuticulin layer has formed) and, in some insects, it permits the entry

of water (2) It is inelastic and, therefore, serves as a limiter of growth (3) It provides the base on which the wax monolayer sits The nature of the cuticulin envelope will therefore determine whether the wax molecules are oriented with their polar or nonpolar groups facing outward and, therefore, the surface properties of the cuticle (4) It plays a role in determining the surface pattern of the cuticle (5) It is resistant to abrasion and helps prevent infection (6) It is involved in production of physical colors Despite the importance of the cuticulin envelope, its composition is largely unknown

3 Cuticle Formation

Formation of new cuticle (Figure 11.5) may be viewed largely as a succession of syntheses by epidermal cells, with dermal glands and oenocytes adding their products at the appropriate moment (Locke, 1974) It must be realized, however, that other, related processes such as dissolution of old cuticle are going on concurrently and that cuticle formation is partly a preecdysial and partly a postecdysial event; that is, much endocuticle formation, tanning of the outer procuticle, wax secretion, and other processes occur after the remains of the old cuticle are shed

3.1 Preecdysis

In most species the onset of a molting cycle is marked by an increase in the volume

of the epidermal cells and/or by epidermal mitoses These events are soon followed by

THE INTEGUMENT

FIGURE 11.5. Summary of cuticle formation during the molt/intermolt cycle Individual components are not

drawn to scale The numbers in Figure 11.5B indicate the sequence of actions resulting in plaque digestion (A)

Secretion of ecdysial droplets (B) Pinocytosis and apolysis of plasma membrane (C) Redifferentiation of plaques

and cuticulin envelope deposition (D) Cuticulin envelope complete and digestion of old cuticle (E) Secretion of

inner epicuticle and bucking of cuticulin envelope (F) Beginning of procuticle secretion (G) Cuticle immediately

after ecdysis (H) Cuticle after tanning.

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apolysis, the detachment of the epidermis from the old cuticle The epidermal cells, at this time, show signs of preparation for future synthetic activity One or more nucleoli become prominent, the number of ribosomes increases, and the ribonucleic acid content of the cells

is elevated Two components of the epidermal cells are especially important, namely, the Golgi complexes and the plasma membrane plaques, whose activities alternate to create the new cuticle Just prior to apolysis, Golgi complex activity increases, and the vesicles pro-duced migrate to the apical plasma membrane where they release their contents—ecdysial droplets—between the epidermal microvilli (Figure 11.5A) The ecdysial droplets contain proteinases and chitinases for cuticle digestion, though the enzymes remain in an inactive form until after formation of the new cuticulin envelope when the epidermal cells secrete an

“activation factor.” In Calpodes ethlius large quantities of an amidase are generated by the

epidermis and fat body during the intermolt The amidase (in its inactive form) accumulates

in the hemolymph until the molt cycle begins, when it moves into the molting fluid and is ac-tivated, enabling precise initiation of cuticle breakdown (Marcu and Locke, 1999) Between 80% and 90% of the old cuticle is digested and may be reused in the production of new cuticle In earlier accounts it was assumed that the molting fluid, including the breakdown products, were resorbed across the body wall However, recent studies have demonstrated that most of the molting fluid is recovered by both oral and anal drinking, reentering the

body cavity by absorption across the midgut wall (Yarema et al., 2000) The exocuticle,

muscle insertions, and sensory structures in the integument are not degraded by molting fluid Thus, an insect is able to move and receive information from the environment more

or less to the point of ecdysis

After release of the ecdysial droplets, the microvilli are withdrawn and their plaques are pinocytosed and digested in multivesicular bodies (Figure 11.5B) New microvilli, with plaques at their tips, then differentiate The first layer of new cuticle deposited is the cuticulin envelope Minute convex patches of cuticulin appear above the plaques (Figure 11.5C), the patches eventually fusing together to form a continuous but buckled layer (Figure 11.5D) The buckling permits expansion of the cuticle after molting and is also important in the formation of annuli and taenidia in tracheae and tracheoles (Chapter 15, Section 2.1) Other buckling patterns determine the specific surface structure of scales, bristles, and microtrichia Oenocytes are maximally active at this time, and it is possible that they are involved in cuticulin formation, perhaps by synthesizing a precursor for the epidermal cells When the envelope is complete, it becomes tanned The Golgi complexes then show renewed activity, their vesicles discharging their contents to form the inner (protein) epicuticle (Figure 11.5E)

Before the inner epicuticle is fully formed production and deposition of the new procu-ticle begin In contrast to the epicuprocu-ticle, whose layers are produced sequentially from inside

to outside, the new procuticle is produced with the newest layers on the inside Again, it

is the plasma membrane plaques that are involved, new chitin fibers arising on their outer surface (Figure 11.5F,G) However, details of the mechanism by which new procuticle is produced remain sketchy The epidermal cells contain the enzymes necessary for synthesis

of acetylglucosamine from trehalose Acetylglucosamine units perhaps are then secreted into the apolysial space, polymerization into chitin being promoted by the enzyme chitin synthetase attached to the plasma membrane plaques Some procuticular proteins are

syn-thesized by the epidermal cells while others are acquired from the hemolymph (Sass et al., 1993; Suderman et al., 2003) How the proteins become incorporated into the procuticle

remains unclear

THE INTEGUMENT

Deposition of the wax layer of the epicuticle begins some time prior to ecdysis For

example, in Blattella germanica oenocytes associated especially with the integument of

abdominal sternites 3–6 become major producers of hydrocarbons early in the molt

cy-cle The hydrocarbons are stored in fat body, then transported to the epidermis bound to

lipophorin a few days before molting occurs (Schal et al., 1998; Young et al., 1999) The

wax is secreted by the epidermal cells, probably as lipid-water liquid crystals, and passes

along the pore canals to the outside Wax production continues after ecdysis and, in some

insects, throughout the entire intermolt period and in the adult stage

3.2 Ecdysis

At the time of ecdysis, the old cuticle comprises only the original exocuticle and

epicuticle In many insects it is separated from the new cuticle by an air space and a

thin ecdysial (apolysial) membrane that is formed from undigested inner layers of the

endocuticle These layers are not digested because they became tanned along with the new

cuticulin envelope Shortly before molting an insect begins to swallow air (or water, if

aquatic), thereby increasing the hemolymph pressure by as much as 12 kPa Hemolymph

is then localized in the head and thorax following contraction of intersegmental abdominal

muscles In many insects these muscles become functional only at the time of ecdysis and

histolyze after each molt The local increase in pressure in the anterior part of the body

causes the old cuticle to split along a weak ecdysial line where the exocuticle is thin or

absent An insect continues to swallow air or water after the molt in order to stretch the new

cuticle prior to tanning

3.3 Postecdysis

Several processes are continued or initiated after ecdysis As noted wax secretion

con-tinues, and the major portion of the endocuticle is deposited at this time Indeed, endocuticle

production in some insects appears to be a more or less continuous process throughout the

intermolt period It is also at this time that the dermal glands release the cement

The most striking postecdysial event, however, is the differentiation of the exocuticle,

that is, the hardening of the outer procuticle (Figure 11.5H) This results from a biochemical

process known as tanning (sclerotization), in which proteins become covalently bound to

each other (and hence stabilized) by means of quinones Hardening is usually accompanied

by darkening (melanization), though the two may be distinct processes; that is, some species

have pale but very hard cuticles Though tanning is discussed here in the context of the

cuticle, it should be noted that it also an important process in the final structure of insect

egg shells, egg cases (oothecae) and protective froths, cocoons, puparia and various silk

structures Indeed, much of the basic understanding of tanning came from studies using

the cockroach ootheca and the fly puparium (Andersen, 1985; Hopkins and Kramer, 1992)

More recently, the cuticles of the Manduca sexta pupa and of locusts and grasshoppers have

proved to be excellent models for study of this process Though the details may differ, it

is now possible to provide a basic scheme for the events that culminate in a tanned cuticle

(Figure 11.6)

Before tanning begins, the level of the amino acid tyrosine in the hemolymph increases

The tyrosine is mostly bound to glucose, phosphate, or sulphate, forming water-soluble

conjugates This is thought to increase the amount of tyrosine that can be carried in the