II A natom y and Ph y siolo gy 11 The Inte g umen t 1 . Intr oduc t ion The integument of insects (and other arthropods) comprises the basal lamina, epidermis , and cuticle. It is often thou g ht of as the “skin” of an insect but it has man y other functions ( Loc k e, 1974). Not on ly d oes i t p rov id e phy s i ca lp rotect i on f or i nterna l or g ans b ut, b ecaus e o fi ts r igidi t y , i t serves as a s k e l eton to w hi c h musc l es can b e attac h e d .Ita l so re d uces wate r loss to a very low level in most Insecta, a feature that has been of great significance in the ev olution of this predominantly terrestrial class. In addition to these primary functions, the cuticular com p onent of the inte g ument p erforms a number of secondar y duties. It acts as a meta b o li c reserve, to b e use d c y c li ca lly to construct t h e next sta g e, or d ur i n gp er i o d so f g reat meta b o li c act i v i t y or starvat i on. It p revents entr y o ff ore ig n mater i a l , b ot hli v i n g an d n on li v i ng, i nto an i nsect. In many i nsects t h e waxy outer l ayer serves as a repos i tory f or contact sex pheromones (Chapter 13, Section 4.1.1). The color of insects is also a function of the inte g ument, es p eciall y the cuticular com p onent . T h e i nte g ument i snotaun if orm structure. On t h e contrar y , b ot hi ts ce ll u l ar an d ace ll u l ar com p onents ma yb e diff erent i ate di navar i et y o f wa y stosu i tan i nsect’s nee d s. E pid erma l ce ll s may f orm spec i a li ze d g l an d st h at pro d uce components o f t h e cut i c l e or may d eve l op i nto particular parts of sense organs. The cuticle itself is variously differentiated accordin g t o the function it is re q uired to p erform. Where muscles are attached or where abrasion m a y occur i t i st hi c k an d r igid ;at p o i nts o f art i cu l at i on i t i s fl ex ibl ean d e l ast i c; over some sensor y structures i tma yb e extreme ly t hi n. 2. S tructur e 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 t he e p idermal cells. It is u p to 0.5 µ m thick and is produced mainly by the epidermis, though µ µ th ere are re p orts t h at h emoc y tes a l so p art i c ip ate. T h ec h em i ca l nature o f t h e b asa ll am i na i s p oor ly un d erstoo d t h ou gh neutra l muco p o ly sacc h ar id e, gly co p rote i ns, an d co ll a g en, s i m il a r t o that of vertebrates, have been identified. The epidermis (hypodermis) is a more or less continuous sheet of tissue, one cel l t hick, res p onsible for secretin g the bulk of the cuticle. Durin gp eriods of inactivit y , its 3 5 5 356 CHAPTER 11 F I GU RE 11.1 . D iagrammatic cross-section of mature integument . c ells are flattened and intercellular boundaries are indistinct. When active, the cells are more or l ess cu b o id a l ,an d t h e i r pl asma mem b ranes are rea dily a pp arent; one to severa l nuc l eo li , extens i ve rou gh en d o pl asm i c ret i cu l um, an d man y Go lgi com pl exes are ev i - d ent (Loc k e, 1991, 1998). A c h aracter i st i c f eature o f t h eap i ca l (cut i c l e- f ac i ng) sur f ace o f epidermal cells are the plasma membrane plaques, specialized regions of the plasm a membrane at the tips of fingerlike microvilli, from which the cuticulin envelope and new chi t i nfi b ers ar i se (Sect i on 3.1). E l ectron m i crosco py h as s h own t h at, at metamor ph os i s, t h ee pid erma l ce ll s d eve l o pb asa lp rocesses (“ f eet”) w hi c h can exten d to b ecome con- necte d w i t h t h e b asa ll am i na an d w i t h ot h er ep id erma l ce ll s. W h en t h e f eet s h orten, t h e 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 , 1 998). E p idermal cells also p ossess the abilit y to develo p various forms of c y toskele- ta l extens i ons w hi c h can b e use d , f or exam pl e, to d raw trac h eo l es c l oser to t h ece ll f or i ncrease d ox yg en su pply ,ortoma i nta i n i nterce ll u l ar contact as t h ece ll sm ig rate d ur- ing wound healing and changes in body shape. The density of cells in a particular are a v aries, following a sequence that can be correlated with the molting cycle. The cells ofte n c ontain g ranules of a reddish-brown p i g ment, insectorubin, which in some insects con- tr ib utes s ig n i ficant ly to t h e i rco l or. However, i n most i nsects co l or i s p ro d uce dby t h e cut i c l e (Sect i on 4.3). E p id erma l ce ll s may b e diff erent i ate di nto sense organs or spec i a li ze d g l an d u l ar ce ll s . O enocytes are large, ductless, often polyploid cells, up to 10 0 µ min µ µ d iameter. They occu r in p airs or small g rou p s and the cells of each g rou p ma y be derived from one ori g ina l e pid erma l ce ll . Usua lly t h e y move to t h e h emocoe li c f ace o f t h e b asa ll am i na, t h ou gh in some i nsects t h e yf orm c l usters i nt h e h emocoe l or m ig rate an d reassem bl ew i t hi nt h e f a t b o d y. Oenocytes s h ow s i gns o f secretory act i v i ty t h at can b e corre l ate d w i t h t h emo l t i n g c ycle, and, on the basis of certain staining reactions, it has been suggested that they produc e the li p o p rotein com p onent of e p icuticle. In addition, ultrastructural and biochemical studie s h ave l e d to t h e p ro p osa l t h at t h ese ce ll s p ro d uce ec dy sone (Loc k e, 19 6 9; Romer, 1991). T h e y a l so s y nt h es i ze com p onents o f t h e cut i cu l ar wax, i nc l u di n g some contact sex ph eromone s (Blomquist and Dillwith, 198 5 ; Schal et a l. , 1998). Derma l g l an d so f var i ous types are a l s o differentiated. In their simplest form the glands are unicellular and have a long duct tha t p enetrates the cuticle to the exterior. More commonly, they are composed of several cells. 3 5 7 THE INTE GU MEN T The gland cells again exhibit cyclical activity associated with new cuticle production, an d i t has been p ro p osed that the y secrete the cement la y er of e p icuticle. T h e cut i c l e, w hi c hi sma i n ly p ro d uce dby t h ee pid erma l ce ll s, usua lly i nc l u d es t h re e p r i mar yl a y ers, t h e i nner p rocut i c l e, m iddl ee pi cut i c l e, an d outer cut i cu li nenve l o p e (Loc k e, 2001) (F i gure 11.2). In o ld er accounts o f t h e i ntegument t h e cut i cu li nenve l ope i s treate d a s part of the epicuticle. However, Locke (1998, 2001) has argued that, because of its distinc t ori g in, structure and functions, the cuticulin envelo p e should be considered se p arate from th ee pi cut i c l e. A ll t h ree p r i mar yl a y ers are p resent over most o f t h e b o dy sur f ace an di nt h e cut i c l et h at li nes ma j or i nva gi nat i ons suc h as t h e f ore g ut, hi n dg ut, an d trac h eae. However , th e procut i c l e i s very t hi nora b sent, an d certa i n components o f t h eep i cut i c l e may b e m issing, where flexibility or sensitivity is needed, for example, over sensory structures and t he lining of tracheoles. Only the cuticulin envelope is universally present, except for the p ores over c h emosens ill a(C h a p ter 12, Sect i on 4.1) . T h e p rocut i c l e( = fi b rous cut i c l e) f orms t h e b u lk o f t h e cut i c l ean di n most s p ec i es i s diff erent i ate di nto two zones, en d ocut i c l ean d exocut i c l e, w hi c h diff er mar k e dl y i nt h e i r F I GU RE 11.2. El ectron m i cro g ra ph ss h ow i n gd e p os i t i on o f t h et h ree p r i mar yl a y ers o f cut i c l e in C a l po d e s ethliu s . [From M. Locke, 2001, The Wigglesworth lecture: Insects for studying fundamental problems in biology, J. Insect P h ysio l . 47 :495–507. With permission from Elsevier. ] 358 CHAPTER 11 F I GU RE 11.3 . Diagram showing orientation of microfibers in lamellae of endocuticle. [From A.C. Neville and S. Caveney, 19 6 9, Scara b ae id b eet l e exocut i c l easanopt i ca l ana l ogue o f c h o l ester i c li qu id crysta l s, B io l .Rev . 44 : 5 31– 5 62. B yp ermission of Cambrid g e Universit y Press, London. ] p h y sical p ro p erties but onl y sli g htl y in their chemical com p osition. In some cuticles th e b or d er b etween t h etwo i s not c l ear an d an i nterme di ate area , t h e mesocut i c l e ,i sv i s ibl e. Adj acent to t h ee pid erma l ce ll s a narrow amor ph ous l a y er, t h e assem bly zone, ma yb e see n w here chitin microfibers are deposited and oriented . T he endocuticle is composed of lamellae (Figure 11.3). Electron microscopy reveal s that each lamella is made u p of a mass of microfibers arran g ed in a succession of p lanes, all fib ers i na pl ane b e i n gp ara ll e l to eac h ot h er. T h eor i entat i on c h an g es s ligh t ly f rom pl ane to pl ane ma ki n g cut i c l e lik e ply woo d w i t hh un d re d so fl a y ers. T h e exocut i c l e i st h ere gi on of procut i c l ea dj acent to t h eep i cut i c l et h at i ssosta bili ze d t h at i t i s not attac k e db yt he molting fluid and is left behind with the exuvium at molting (Locke, 1974). Not only i s the exocuticle chemicall y inert, it is hard and extremel y stron g . It is, in fact, p rocuticle tha t h as b een “tanne d ” (Sect i on 3.3). Exocut i c l e i sa b sent f rom areas o f t h e i nte g ument w h er e fl ex ibili t yi sre q u i re d , f or exam pl e, at j o i nts an di nterse g menta l mem b ranes, an d a l on g t h e ec d ys i a lli ne. In many so f t- b o di e d en d opterygote l arvae t h e exocut i c l e i s extreme l yt hin and frequently cannot be distinguished from the epicuticle and cuticulin envelope . P rocuticle is composed almost entirely of protein and chitin. The latter is a nitrogenous p ol y saccharide consistin gp rimaril y of N - acet y l - D -gl ucosam i ne res id ues to g et h er w i t ha s ma ll amount o fgl ucosam i ne li n k e di n a β 1,4 confi g urat i on (F ig ure 11.4). In ot h er wor d s , chi t i n i s very s i m il ar to ce ll u l ose, anot h er po l ysacc h ar id eo f great structura l s i gn i ficance, e x cept that the hydroxyl group of carbon atom 2 of each residue is replaced by an acetamid e g rou p . Because of this confi g uration, extensive h y dro g en bondin g is p ossible between ad j a - c ent c hi t i nmo l ecu l es w hi c hli n k to g et h er ( lik ece ll u l ose) to f orm m i crofi b ers. C hi t i nma k e s F I GU RE 11.4 . T h ec h em i ca l structure o f c hi t i n . 3 5 9 THE INTE GU MEN T u p between 2 5 % and 60% of the dry weight of procuticle but is not found in the epicuticle and cuticulin envelo p e. It is associated with the p rotein com p onent, bein g linked to p rotein m o l ecu l es by cova l ent b on d s, f orm i n g a gly co p rote i n com pl ex. Stu di es h ave s h own t h at t h e epid erm i s secretes more t h an a d ozen ma j or p rote i ns i nto t h e cut i c l e i n a care f u lly t i me d sequence, pro b a bl yun d er h ormona l contro l (Su d erman e ta l. , 2003). Interest i ng l y, cut i cu l a r proteins of similar molecular weights have been found in a range of insect species suggesting t hat the chemical nature of the cuticle has been stron g l y conserved throu g h evolution. The am i no ac id com p os i t i on o f t h e p rote i ns d eterm i nes t h e i r p ro p ert i es. For exam pl e, en d ocu - ti cu l ar p rote i ns are g enera lly r i c hi n hyd ro ph o bi cam i no ac id sw i t hb u lky s id ec h a i ns an d ar e l oose l y pac k e d (not compact) mo l ecu l es. T hi s prov id es t h een d ocut i c l ew i t hfl ex ibili ty an d w ill also facilitate “creep” (the ability of layers to slide over each other), hence intrastadial g rowth in soft-bodied insects such as caterpillars. Conversely, in hard, stiff exocuticle, it i s small, com p act amino acids that p redominate (He p burn, 1985). I nt h e exocut i c l e, a dj acent p rote i nmo l ecu l es are li n k e d to g et h er by a q u i none mo l ecu l e, an d t h e cut i c l e i ssa id to b e tanne d (Sect i on 3.3). T h e tanne d (sc l erot i ze d ) prote i n, w hi c hi s known as “sclerotin,” comprises several different molecules. Resilin is a rubberlike material found in cuticular structures that undergo springlike movements, for example, wing hinges, t he p roboscis of Le p ido p tera, the hind le g s of fleas (Cha p ter 14, Section 3.1.2.), and the wi n g - hi n g e lig ament t h at stretc h es b etween t h e pl eura lp rocess an d secon d ax ill ar y sc l er i t e ( Chapter 14, Sections 3.3.1 and 3.3.3) (Neville, 197 5 ). Like rubber, resilin, when stretched, i s able to store the energy involved. When the tension is released, the stored energy is use d t o return the protein to its original length. I n addition to these structural p roteins, enz y mes also exist in the cuticle, includin g ph eno l ox id ases, w hi c h cata ly ze t h eox id at i on o f dihyd r i c ph eno l s use di nt h e tann i n gp roces s ( Sect i on 3.3). T h ese enz y mes a pp ear to b e l ocate di nor j ust b eneat h t h ee pi cut i c l e. Avar i ety o f p i gments h ave b een f oun di nt h e cut i c l e (or i nt h eep id erm i s) w hi c h ma y g ive an insect its characteristic color (Section 4.3). Also, in a few beetles and larvae an d p u p ae of some Di p tera, mineralized calcium (as the carbonate) is de p osited, p resumabl y t o i ncrease r igidi t y (Lesc h en an d Cut l er, 1994). C erta i n p rocesses occur at t h e sur f ace o f t h e cut i c l ea f ter i t h as b een f orme d , f o r e xamp l e, secret i on an d repa i ro f t h ewax l ayer an d tann i ng o f t h e outer procut i c l e. T h us , 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 ma y or ma y not contain a c y to p lasmic p rocess. Most often, the cana l s d o not conta i n an extens i on o f t h ee pid erma l ce ll b ut h ave at l east one “fi l ament” pro d uce db yt h ece ll . Loc k e (1974) suggeste d t h at t h efi l ament(s) m i g h t k eepac h anne l ope n i n 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 ( includin g tannin g )iscom p lete. The p ore canals terminate immediatel y below the e p icuticle. R unn i n gf rom t h et ip so f t h e p ore cana l stot h e outer sur f ace o f t h ee pi cut i c l e are lipid -fi ll e d c h anne l s k nown as wax cana l s . The epicuticle is a composite structure produced partly by epidermal cells and partl y b y specialized glands. It ranges in thickness from a fraction of a micrometer to several m icrometers and g enerall y com p rises three la y ers. The la y ers are, from outside to inside , cement, wax (t h ese are secrete d outs id et h e cut i cu li nenve l o p e), an d t h e so-ca ll e dp rote i n epi cut i c l e. T h e nature o f cement var i es, t h ou gh i t i s lik e ly to b ea pp rox i mate ly s i m il ar to s h e ll ac. T h e l atter i sam i xture o fl accose an dli p id s. T h e cement i sun d ou b te dl ya h ar d , protective layer in some insects. In others it appears to be more important as a sponge tha t 360 CHAPTER 11 soaks up excess wax. The latter could quickly replace that lost, for example, by surface abrasion. The wax is a com p lex mixture whose com p osition varies both amon g and within s p ec i es, somet i mes over diff erent b o dy re gi ons o f t h e same i nsect, an di n some s p ec i es seasona ll y. Genera ll y, l ong-c h a i n h y d rocar b ons an df atty ac id esters pre d om i nate, t h oug h v aried proportions of alcohols, fatty acids, and sterols may also occur. In some species th e mixture has relatively few different components, whereas in others, for example , M u s c a , more than 100 com p ounds have been identified (Blom q uist and Dillwith, 1985; Jaco b et al. , 1 997). Accor di n g to Loc k e (1974), w i t hi nt h ewax l a y er t h ree re gi ons can b e di st i n g u i s h e d. Adj acent to t h e cut i cu li nenve l o p e i s a mono l a y er o f t igh t ly p ac k e d mo l ecu l es i n liq u id f orm that gives the cuticular surface its high contact angle with water and its resistance t o w ater loss (but see Section 4.2.). Most wax is in the middle layer, which is less ordered an d p ermeates the cement. The outer wax la y er, which com p rises cr y stalline wax blooms, i s not p resent i na ll i nsects. T h e i nnermost l a y er o f t h ee pi cut i c l e, t h e p rote i ne pi cut i c l e, li e s b eneat h t h e cut i cu li nenve l o p e. It ma yb e severa l m i crometers t hi c k an d lik et h e cut i cu li n enve l ope i t covers a l most a ll o f t h e sur f ace o f t h e i nsect. It i sa b sent f rom trac h eo l es an d p arts of some sense organs. It comprises dense, amorphous protein tanned in a manne r similar to the p rotein of the exocuticle (Section 3.3) but contains no chitin. Th e cut i cu li nenve l o p e(a b out 20 nm t hi c k ) exten d s over t h e ent i re b o dy sur f ace an d ecto d erma li nva gi nat i ons, i nc l u di n g t h e most m i nute trac h eo l es, b ut i sa b sent f rom s p ec i fic areas o f sense organs an df rom t h et i ps o f certa i ng l an d ce ll s. It may b e cons id ere d t he most important layer of the cuticle for the following reasons (Locke, 1974, 2001). (1) It is a selectivel yp ermeable barrier. Durin g breakdown of the old cuticle, it allows the “activatin g f actor” for the molting gel to move out and the products of cuticular hydrolysis to enter, yet ff i t i s i m p ermea bl etot h e enz y mes i nt h emo l t i n gfl u id .It i s p ermea bl e to waxes (as t h ese are d epos i te d on l ya f ter t h e cut i cu li n l ayer h as f orme d )an d , i n some i nsects, i t perm i ts t h e entry o f water. (2) It is inelastic and, therefore, serves as a limiter of growth. (3) It provides th e base on which the wax monolayer sits. The nature of the cuticulin envelope will therefore determine whether the wax molecules are oriented with their p olar or non p olar g rou p s f ac i n g outwar d an d ,t h ere f ore, t h e sur f ace p ro p ert i es o f t h e cut i c l e. (4) It pl a y saro l e 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 th e c uticulin envelope, its composition is largely unknown. 3. C ut i cle Format i o n F ormation of new cuticle (Figure 11. 5 ) may be viewed largely as a succession of s y ntheses b y e p idermal cells, with dermal g lands and oenoc y tes addin g their p roducts at t h ea pp ro p r i ate moment (Loc k e, 1974). It must b e rea li ze d , h owever, t h at ot h er, re l ate d p rocesses suc h as di sso l ut i on o f o ld cut i c l e are go i ng on concurrent l yan d t h at cut i c l e f ormation is partly a preecdysial and partly a postecdysial event; that is, much endocuticl e f ormation, tanning of the outer procuticle, wax secretion, and other processes occur after the remains of the old cuticle are shed. 3 .1. Preecd y sis In most species the onset of a molting cycle is marked by an increase in the volume o f the e p idermal cells and/or b y e p idermal mitoses. These events are soon followed b y 36 1 THE INTE GU MEN T F IGURE 11.5 . Summary o f cut i c l e f ormat i on d ur i ng t h emo l t /i ntermo l tcyc l e. In di v id ua l components are no t drawn to scale. The numbers in Fi g ure 11. 5 B indicate the se q uence of actions resultin g in p la q ue di g estion. (A) S ecretion of ecdysial droplets. (B) Pinocytosis and apolysis of plasma membrane. (C) Redifferentiation of plaques an d cut i cu li nenve l ope d epos i t i on. (D) Cut i cu li nenve l ope comp l ete an ddi gest i on o f o ld cut i c l e. (E) Secret i on o f i nner e pi cut i c l ean db uc ki n g o f cut i cu li nenve l o p e. (F) Be gi nn i n g o fp rocut i c l e secret i on. (G) Cut i c l e i mme di ate ly after ecdysis. (H) Cuticle after tanning . 362 CHAPTER 11 apolysis, the detachment of the epidermis from the old cuticle. The epidermal cells, at this time, show si g ns of p re p aration for future s y nthetic activit y . One or more nucleoli become p rom i nent, t h e num b er o f r ib osomes i ncreases, an d t h er ib onuc l e i cac id content o f t h ece ll s i se l evate d . Two com p onents o f t h ee pid erma l ce ll s are es p ec i a lly i m p ortant, name ly ,t he Go l g i comp l exes an d t h ep l asma mem b rane p l aques, w h ose act i v i t i es a l ternate to create t h e new cuticle. Just prior to apolysis, Golgi complex activity increases, and the vesicles pro- duced mi g rate to the a p ical p lasma membrane where the y release their contents—ecd y sial dro p lets—between the e p idermal microvilli (Fi g ure 11.5A). The ecd y sial dro p lets contai n p rote i nases an d c hi t i nases f or cut i c l e dig est i on, t h ou gh t h e enz y mes rema i n i nan i nact i ve f orm unt il a f ter f ormat i on o f t h e new cut i cu li nenve l ope w h en t h eep id erma l ce ll s secrete a n “activation factor.” I n C a lp o d es et hl iu s large quantities of an amidase are generated by th e epidermis and fat body during the intermolt. The amidase (in its inactive form) accumulates i nt h e h emo ly m ph unt il t h emo l tc y c l e b e gi ns, w h en i t moves i nto t h emo l t i n gfl u id an di sac - t i vate d , ena bli n gp rec i se i n i t i at i on o f cut i c l e b rea kd own (Marcu an d Loc k e, 1999). Betwee n 8 0% an d 90% o f t h eo ld cut i c l e i s di geste d an d may b e reuse di nt h e pro d uct i on o f new c uticle. In earlier accounts it was assumed that the molting fluid, including the breakdow n p roducts, were resorbed across the body wall. However, recent studies have demonstrated that most of the moltin g fluid is recovered b y both oral and anal drinkin g , reenterin g the b o dy cav i t yby a b sor p t i on across t h em idg ut wa ll (Yarema et al. , 2000). T h e exocut i c l e , musc l e i nsert i ons, an d sensory structures i nt h e i ntegument are not d egra d e db ymo l t i n g fl uid. Thus, an insect is able to move and receive information from the environment more o r less to the point of ecdysis . After release of the ecd y sial dro p lets, the microvilli are withdrawn and their p la q ue s are p inoc y tosed and di g ested in multivesicular bodies (Fi g ure 11.5B). New microvilli , wi t hpl a q ues at t h e i rt ip s, t h en diff erent i ate. T h e first l a y er o f new cut i c l e d e p os i te di st h e c ut i cu li nenve l ope. M i nute convex patc h es o f cut i cu li n appear a b ove t h ep l aques (F i gure 1 1. 5 C), the patches eventually fusing together to form a continuous but buckled layer (Fi g ure 11.5D). The bucklin gp ermits ex p ansion of the cuticle after moltin g and is also im p ortant in the formation of annuli and taenidia in tracheae and tracheoles (Cha p ter 15 , S ect i on 2.1). Ot h er b uc kli n gp atterns d eterm i ne t h es p ec i fic sur f ace structure o f sca l es , b r i st l es, an d m i crotr i c hi a. Oenocytes are max i ma ll y act i ve at t hi st i me, an di t i s poss ible 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 complexe s then show renewed activit y , their vesicles dischar g in g their contents to form the inne r ( p rotein) e p icuticle (Fi g ure 11.5E) . B e f ore t h e i nner ep i cut i c l e i s f u ll y f orme d pro d uct i on an dd epos i t i on o f t h e new procu- ticle begin. In contrast to the epicuticle, whose layers are produced sequentially from inside to outside, the new procuticle is produced with the newest layers on the inside. Again, i t is the p lasma membrane p la q ues that are involved, new chitin fibers arisin g on their oute r surface (Fi g ure 11.5F,G). However, details of the mechanism b y which new p rocuticle i s p ro d uce d rema i ns k etc hy .T h ee pid erma l ce ll s conta i nt h e enz y mes necessar yf or s y nt h es is o f acetylglucosamine from trehalose. Acetylglucosamine units perhaps are then secreted into the apolysial space, polymerization into chitin being promoted by the enzyme chiti n s y nthetase attached to the p lasma membrane p la q ues. Some p rocuticular p roteins are s y n- t h es i ze dby t h ee pid erma l ce ll sw hil eot h ers are ac q u i re df rom t h e h emo ly m ph (Sas s et al. , 1 993 ; Su d erma n e ta l. , 2003). How t h e p rote i ns b ecome i ncor p orate di nto t h e p rocut i c l e rema i ns unc l ear . 363 THE INTE GU MEN T Deposition of the wax layer of the epicuticle begins some time prior to ecdysis. For e xam p le, i n B lattella germanica oenoc y tes associated es p eciall y with the inte g ument of a bd om i na l stern i tes 3– 6b ecome ma j or p ro d ucers o f hyd rocar b ons ear ly i nt h emo l tc y- c l e. T h e hyd rocar b ons are store di n f at b o dy ,t h en trans p orte d to t h ee pid erm i s b oun d t o li pop h or i na f ew d ays b e f ore mo l t i ng occurs (Sc h a l et a l. , 1998; Youn g e ta l. , 1999). T he wa xis s ecreted by the epidermal cells, probably as lipid-water liquid crystals, and passes alon g the p ore canals to the outside. Wax p roduction continues after ecd y sis and, in some i nsects, t h rou gh out t h e ent i re i ntermo l t p er i o d an di nt h ea d u l t sta g e . 3.2. Ecdys i s At the time of ecdysis, the old cuticle comprises only the original exocuticle and ep icuticle. In man y insects it is se p arated from the new cuticle b y an air s p ace and a thi nec dy s i a l (a p o ly s i a l ) mem b rane t h at i s f orme df rom un dig este di nner l a y ers o f t h e e n d ocut i c l e. T h ese l ayers are not di geste db ecause t h ey b ecame tanne d a l ong w i t h t h ene w 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 i s then localized in the head and thorax followin g contraction of interse g mental abdominal m usc l es. In man yi nsects t h ese musc l es b ecome f unct i ona l on ly at t h et i me o f ec dy s i san d hi sto ly ze a f ter eac h mo l t. T h e l oca li ncrease i n p ressure i nt h e anter i or p art o f t h e b o dy causes the old cuticle to split along a weak ecdysial line where the exocuticle is thin o r absent. An insect continues to swallow air or water after the molt in order to stretch the ne w cuticle p rior to tannin g . 3.3. Postecd y sis S everal processes are continued or initiated after ecdysis. As noted wax secretion con- t inues, and the ma j or p ortion of the endocuticle is de p osited at this time. Indeed, endocuticle p ro d uct i on i n some i nsects a pp ears to b e a more or l ess cont i nuous p rocess t h rou gh out t h e i ntermo l t p er i o d .It i sa l so at t hi st i me t h at t h e d erma lgl an d sre l ease t h e cement. T h e most str iki ng postec d ys i a l event, h owever, i st h e diff erent i at i on o f t h e exocut i c l e, t hat is, the hardening of the outer procuticle (Figure 11. 5 H). This results from a biochemical process known as tanning (sclerotization), in which proteins become covalently bound to e ach other (and hence stabilized) b y means of q uinones. Hardenin g is usuall y accom p anied by d ar k en i n g (me l an i zat i on), t h ou gh t h etwoma yb e di st i nct p rocesses; t h at i s, some s p ec i es h ave pa l e b ut very h ar d cut i c l es. T h oug h tann i ng i s di scusse dh ere i nt h e context o f t h e 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 sil k structures. Indeed, much of the basic understandin g of tannin g came from studies usin g t he cockroach ootheca and the fl yp u p arium (Andersen, 1985; Ho p kins and Kramer, 1992) . More recent ly ,t h e cut i c l es o f t he Man d uca s exta p u p aan d o fl ocusts an dg rass h o pp ers h av e prove d to b e exce ll ent mo d e l s f or stu d yo f t hi s process. T h oug h t h e d eta il s may diff er, i t i s now possible to provide a basic scheme for the events that culminate in a tanned cuticl e ( Fi g ure 11.6) . Be f ore tann i n gb e gi ns, t h e l eve l o f t h eam i no ac id t y ros i ne i nt h e h emo ly m ph i ncreases. T h et y ros i ne i s most ly b oun d to gl ucose, ph os ph ate, or su lph ate, f orm i n g water-so l u bl e con j ugates. T hi s i st h oug h tto i ncrease t h e amount o f tyros i ne t h at can b e carr i e di nt he [...]...364 CHAPTER 11 FIGURE 11. 6 Summary of the tanning process hemolymph, to protect it from oxidation, and to prevent its use in competing metabolic pathways In a few species the tyrosine accumulates within fat-body vacuoles, particularly as the dipeptide N - -alanyltyrosine The tyrosine is converted to “dopa” (dihydroxyphenylalanine) which is then decarboxylated forming dopamine, N - -alanyldopamine,... (Lepidoptera, Hesperiidae), J Insect Physiol 45:861–870 Neville, A C., 1975, Biology of the Arthropod Cuticle, Springer-Verlag, Berlin 371 THE INTEGUMENT 372 CHAPTER 11 Okot-Kotber, B M., Morgan, T D., Hopkins, T L., and Kramer, K J., 1994, Characterization of two high molecular weight catechol-containing proteins from pharate pupal cuticle of the tobacco hornworm, Manduca sexta, Insect Biochem Molec Biol... speciesand/or caste-recognition pheromone (See also Chapter 13, Section 4.1.2.) Interestingly, some beetles that live in termite colonies produce the same hydrocarbon profile as the host, enabling them to remain unmolested in the nest The species-specific nature of the lipids has been turned to advantage by some parasitic Hymenoptera who use these chemical cues (known as kairomones [Chapter 13, Section... decreasing) concentrations, others requiring higher (or increasing) titers for their expression In this way, varying amounts of β-ecdysone throughout 365 THE INTEGUMENT 366 CHAPTER 11 the molt cycle serve to synchronize the many steps and processes that occur It must be noted that β-ecdysone does not act directly on genes; rather, it binds with receptors in the nuclear membrane causing release of second... to mean a surface-supporting structure that is thin in relation to total size) is about three times as strong as a solid rod of the same material having the same cross-sectional area as the shell (i.e., they both contain the same amount of skeletal material) The force required to distort the shell is proportional to the thickness of the shell and inversely proportional to the cross-sectional area of... calculations that show the abrupt permeability changes at the so-called transition point to be artifactual (Blomquist and Dillwith, 1985) Some insects that are normally found in extremely dry habitats and may go for long periods without access to free water, for example, Tenebrio molitor and prepupae of fleas, 367 THE INTEGUMENT 368 CHAPTER 11 are able to take up water from an atmosphere in which the humidity... Though autotanning has never been demonstrated, “dopa” has been found in small amounts in certain cuticular proteins of Manduca sexta (Okot-Kotber et al., 1994) and presumably could engage in cross-linking reactions The color of tanned cuticle depends on the amount of o-quinone that is present When this molecule is present in small quantities the cuticle is pale; if it is in excess, and especially if it... M., 1998, Epidermis, in: Microscopic Anatomy of Invertebrates, Vol 11A (Insecta) (F W Harris and M V Locke, eds.), Wiley-Liss, New York Locke, M., 2001, The Wigglesworth lecture: Insects for studying fundamental problems in biology, J Insect Physiol 47:495–507 Marcu, O., and Locke, M., 1999, The origin, transport and cleavage of the molt-associated cuticular protein CECP22 from Calpodes ethlius (Lepidoptera,... with the terminal amino group or a sulfhydryl group of the protein to form an N -catechol protein, which in the presence of excess quinone is oxidized to an N -quinonoid protein The latter is able to react with the terminal amino group or sulfhydryl group of another protein to link the two molecules In a number of species β-alanine is incorporated into the cuticle during tanning, and it is speculated... environments or stages with water-conservation problems, for example, eggs and pupae, are covered with “hard” wax, whose transition temperature is high (in most species above the thermal death point of the insect) More recent studies have questioned the validity of Beament’s ordered monolayer model Evidence against it includes the observation that hydrocarbons (non-polar molecules) are the dominant . e pi cut i c l ean db uc ki n g o f cut i cu li nenve l o p e. (F) Be gi nn i n g o fp rocut i c l e secret i on. (G) Cut i c l e i mme di ate ly after ecdysis. (H) Cuticle after tanning . 362 CHAPTER 11 apolysis,. molting cycle. The cells ofte n c ontain g ranules of a reddish-brown p i g ment, insectorubin, which in some insects con- tr ib utes s ig n i ficant ly to t h e i rco l or. However, i n most i nsects. cyclical activity associated with new cuticle production, an d i t has been p ro p osed that the y secrete the cement la y er of e p icuticle. T h e cut i c l e, w hi c hi sma i n ly p ro d uce dby t h ee pid erma l ce ll s,