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An investigation has been made of the junction between the shell and cap in the egg-shell of Rhodnius prolixus. This complex region consists of the thickened rim of the cap connected by a thin sealing bar to the rim of the shell. The secretion of this part of the shell has been followed and compared with the formation of less specialized portions of the shell. The shell has been divided into units, each the product of an individual follicle cell. It has been found that all the seven layers which make up the unspecialized parts of the shell are present in the seal complex; that these consist of five endochorion layers and two exochorion layers in their normal order. The exochorion is secreted around long villi, one from each follicle cell. These give rise to follicular pits in the shell. In this complex region, cells start to secrete at various stages in the seven-phase cycle; their initial secretion is apparently related to the material with which they make contact at that time. After secretion has started, each cell completes the remainder of the cycle. The rim of the cap is the product of four rings of follicle cells; the additional thickness is achieved by an increase in the exochorion layers, secreted around a series of very long follicular pits. The sealing bar, which is produced by one ring of follicle cells, is composed of the inner four layers of the chorion only; the cells do not produce soft endochorion, or exochorion layers. At the cap end of the sealing bar there is the predetermined hatching line. It is apparently produced by the presence in the follicle of cells which are inactive during the secretion of the inner layers, and so prevent co-ordination between the active cells on either side. A weak point is also present at the base of the sealing bar, at the site of other inactive cells, though this fissure is not used at hatching. The rim of the shell is similarly produced by an expansion of the exochorion layers secreted around four rings of follicular villi. Of these, three rings of pits are filled in towards the end of secretion, but the fourth, lying on the upper portion of the rim, remains. These pits become the micropyles and associated structures. There are 200 pits in the completed rim, divided into two groups. About fifteen are micropyles; the remainder are cavities closed at each end, and to which the name ‘pseudomicropyle’ has been given. The pseudomicropyles are formed in a similar way to normal follicular pits, but start in the resistant protein layer, 0.5μ from the inside of the shell. They end in the resistant exochorion, where they are connected to the external surface by small bunches of pore canals. They probably play some part in the respiration of the embryo. The true micropyles form the only free path through the shell. The inner portion of each tube is lined with hydrophilic protein, and the outer portion, which lies slightly posterior to the pseudomicropyles, is composed of hydrophobic lipoprotein. The number of true micropyles is not constant, there being between ten and twenty scattered irregularly around the rim. However, eggs produced by older females contain fewer micropyles; this may account for a higher rate of sterility among these eggs. The cells which form the micropyles and pseudomicropyles are the only ones which do not adhere to the typical cycle of seven secretory products. But in omitting three phases, the attachment of the exochorion to a protein layer is retained. Evidence suggests that the cells forming the micropyles are determined in the earliest stages of secretion by being squeezed out of the pseudomicropylar ring of cells.

The relationship between the temperature of the cuticle and its permeability to water has been determined for a number of different terrestrial arthropods. In most of these animals the waterproofing wax loses its great impermeability to water very abruptly at a particular transition temperature; that temperature varies appreciably with the individual, but appears to be characteristic of any one species at a particular age within a particular instar. No change in permeability with temperature was found in Tipula sp. taken from soil, in which the cuticle was greatly damaged ; above 48° C. the permeability of the cuticle of Schistocerca nymphs changes rapidly with increasing temperature, but this change is not abrupt. In the permeability/temperature curves for larvae of Pieris, Tenebrio, Calliphora and Nematus, pupae of Calliphora and adults of Schistocerca, Ixodes and Ornithodorus only one transition discontinuity is found. In all these animals the wax maintains almost constant permeability up to the point of transition, above which permeability increases very rapidly with temperature. Immediately after moulting, Rhodnius nymphs and pupae of Tenebrio and Pieris also reveal only one transition point; this occurs at a comparatively low temperature. But when cement is secreted over the primary wax layer, a second and higher transition point is found which is independent of the first point. The lower transition temperature seems constant in position during the life of an instar, but the upper point occurs at progressively higher temperatures as the instar ages. The evidence suggests that these animals have two special waterproof layers of wax, the one with high transition temperature occurring on the outer surface of the cement. This outer layer could arise by the migration of wax from the primary layer (with lower transition temperature) through the cement to the outer surface, there to lose volatile components and waterproof the surface. In every animal investigated a permanent increase in permeability is produced by heating a wax layer above its transition point; after such treatment no transition point can be found. From calculations of the absolute permeability of the various wax layers, and from the behaviour of the wax, it is suggested that all epicuticular waxes are laid down with some special molecular arrangement and that in very waterproof insects this may extend throughout the thickness of the wax layers; in less waterproof insects only a portion of the wax layer (as in the cockroach) may be specially organized. Very slight mechanical damage promotes increased transpiration through the cuticle. Stationary adsorptive dusts applied directly to a primary wax layer do not affect permeability; applied to wax above the cement, they promote rates of evaporation in excess of the value exhibited by the same cuticle having a primary wax layer alone. This phenomenon is discussed in relation to the distribution of water within the transpiring cuticle.

The permeability to water of the cuticles of a number of aquatic insects has been measured under conditions of evaporation. The range of rates of evaporation amongst these animals is very great; some adult aquatic beetles are quite as waterproof as more permeable terrestrial insects (caterpillars for example), whereas their larvae would seem to be without specific mechanism for restricting the passage of water. Two methods are described, whereby local differences in permeability may be detected, and from one of these methods it is possible to calculate the permeability/ temperature characteristics of different areas of cuticle of an intact insect. The more impermeable insects have uniform permeability over their entire surfaces; these include adult beetles, and both mature and immature stages of Hemiptera. Larvae of mayflies and of some beetles, which are very permeable to water, likewise show no local differences in permeability. The gills of all the species examined are very permeable to water; those of zygopteran dragonflies are more permeable than the rest of the cuticle at lower temperatures, those of Sialis are much more permeable at all temperatures. The effect of temperature upon permeability has been measured. The more waterproof insects reveal transition phenomena characteristic of organized monolayers of lipid, such as are found in terrestrial insects, but by comparison with them the temperatures at which transition occurs are very low. In particular, the temperature at which adults of Dytiscus and Gyrinus become suddenly more permeable (ca. 25° C.) could obtain in natural circumstances, and the evidence suggests that these animals die through osmotic invasion of water when placed at higher temperatures. Other beetles and aquatic Hemiptera, with higher transition temperatures, survive temperatures above 25° C. These ecological implications are discussed. A transition phenomenon occurs in organized lipid over the main cuticle of Sialis, whereas the more permeable gill shows no evidence of this. There is no evidence for organized lipid on the main cuticle of zygopteran larvae, but the temperature/permeability relations of the general cuticle and of the gill are very different. The absolute permeability, and the shape of the permeability/temperature function, of the cuticle of those insects which have very hydrofuge areas of surface suggest that their waterproofing system consists of only one monolayer of organized lipid, which may be either grease or wax.

The ability of insects to withstand desiccation was shown by Kühnelt (1928) to be due to the epicuticular layer of the cuticle. This outermost part was found to be highly resistant to strong acids and was shown, by chemical tests, to contain fatty acids and cholesterol type molecules. Wigglesworth (1933) described a complex fatty or waxy substance in the upper layers of the cuticle which he called ‘cuticulin’ ; Pryor (1940) found that this lipoid is entirely confined to the epicuticular layer, suggesting that it is used to impregnate a tanned protein. Lipoid material has also been extracted from the exuviae of the silkworm Bombyx mori (Bergmann, 1938). Bergmann found that hot alkali merely fused the epicuticle into oily droplets suggesting that saponification was taking place. The extract so obtained was a brown waxy substance, forming about 4% of the total weight of the cuticle, and it consisted of paraffins, saturated aliphatic acids and esters. A more detailed study of the waxes of certain insects specialising in wax formation has been given by Chibnail, Piper, Pollard, Williams & Sahai (1934b) ; Chibnall, Latner, Williams & Ayre (1934a), and Blount, Chibnall & el Mangouri (1937). All these substances were complicated mixtures of alcohols, acids, paraffins and esters with chain length of the order of C30. Hurst (1941) has suggested that the asymmetry of the insect cuticle is regulated by the ‘outer free lipoid’. He postulates a cuticle surface in the form of a lipoprotein mosaic. It is the object of this study to investigate the distribution and properties of these lipoid materials, to obtain evidence of the structure of the epicuticle by comparison of in vitro phenomena with those of living insect cuticle, and to indicate the function of lipoids in resisting the action of certain insecticides.

Previous methods for determining the temperature characteristics of the permeability to water of insect cuticle are criticized on five fundamental points, and in particular it is shown that they do not measure the temperature of the cuticle which will be substantially different from that of the surrounding air. The physical principles for accurate measurement are outlined, involving the accurate control of air temperature and air circulation, the measurement of cuticle temperature and of water loss within a sealed enclosure, and the assessment of true saturation deficiency. Apparatus for meeting these requirements and making these measurements is described. Determinations have been made of the permeability of the cuticle of Periplaneta americana nymphs. When the permeability is plotted against cuticle temperature a sudden and substantial increase in permeability is seen to take place at around 30° C. The temperature at which this change occurs is higher the longer the time which has elapsed since the nymph previously moulted. A method of measuring the permeability of the cuticular grease, when spread as a layer over an isolated water droplet, is described. This layer, whose thickness is of molecular dimensions, has much greater absolute impermeability to water than the thick grease on the insect. It also exhibits a sudden increase in permeability at about 30° C., and the order of change is considerably greater than that found with intact cuticle. The findings are quantitatively compatible with the postulate that, at lower temperatures, the grease on the cockroach consists of an orientated ‘monolayer’ organized by the attraction of its polar groupings for the aqueous cuticle surface underlying it, together with a much greater thickness of unorganized grease above it. In absolute units, the grease is about five times as permeable to water as is the ‘monolayer’, but at the transition temperature the measurements agree with the supposition that the molecular organization of the ‘monolayer ‘breaks down into the disorganized state of the major part of the grease with a corresponding increase in permeability. The suggestion that films of grease on water, and the lowermost portion of the grease on the animal, have orientated molecular organization is supported by some observations on the effect of dust particles on the films and by information previously published.

The production of massive quantities of waxy materials by bees and similar insects has been known for a very long time, but it was not until 1938 that Bergmann showed the existence of smaller quantities of wax in the cuticle of Bombyx. Ramsay (1935) was the first to demonstrate that a greasy material on the surface of the cuticle was responsible for waterproofing the cockroach, and subsequently the work of Wigglesworth (1945) and Beament (1945) produced evidence for believing that the existence of a thin layer of wax in the epicuticle was widespread throughout the insects, and was the chief mechanism for restricting water-loss. It is now known that wax layers are also present in ticks (Lees, 1947) and in many arthropod eggshells (Beament, 1946, 1951, etc.), while Chibnall, Piper, Pollard, Williams & Sahai (1934) have carried out a detailed analysis of beeswax and of the wax of the white pine chermes. These waxes consist of long chain alcohols, paraffins and acids ; the aliphatic chains always appear to be saturated. The similarity of the physicochemical behaviour of cuticular waxes with beeswax, as outlined by Beament (1945), seems to leave little doubt that the minute but essential amounts of wax in typical insect cuticle are composed of the same type of chemical components as those found in beeswax. The range of melting-points of these materials shows that relative changes in the chain length of the component would be sufficient to account for the differences between the specific properties of waxes derived from different species.

The water in cuticles excised from living cockroaches and locusts is not in osmotic equilibrium with their blood. It represents on average an osmotic pressure 15 atmospheres greater than the blood osmotic pressure. In the locust a cuticle in equilibrium with the blood would contain 60% more water. This is cited as evidence for the continuous expenditure of energy by the epidermal cell in regulating the water balance of the cuticle.

The firebrat, Thermobia, is very impermeable to the outward flow of water, losing only 0·013 mg. of water/mm. Hg saturation deficit/hr./standard animal of 30 mg. between 19 and 26° C. The permeability increases to a higher level at a critical temperature just below 30° C. and well below the optimal temperature for the insect as a whole. The waterproofing is mobile, for it regains its lower permeability on cooling below the transition temperature and is also restored a short time after abrasion. Extractions support the hypothesis that the impermeable layer is a stiff, epicuticular grease but further suggest that an imperforate lamella, perhaps a cement, lies over the grease. The live firebrat can absorb water from moist air, reaching an equilibrium between absorption and transpiration at a saturation deficit of approximately 10 mm. Hg at a temperature of 21° C. Although abrasion of the integument decreases the net rate of water uptake and lowers the saturation deficit at which equilibrium is possible, it may act only by increasing temporarily the rate of transpiration.

The main regions of the Rhodnius prolixus egg-shell have been defined; a concise definition has been obtained for the term ‘chorion’. The formation and structure of the unspecialized chorion has been followed from the time of differentiation of the follicle cells, up to the completion of the shell, and an assessment made of the chemistry and permeability of each component shell layer. The follicle cells are binucleate ; changes in morphology and histology prior to secretion of the shell are outlined. The secretory products of the follicle cells fall naturally into an endoehorion and an exochorion; the endoehorion consists of five modifications of a proteinaceous substance. They are, in order of secretion: The Inner Polyphenol Layer, which consists of a series of tanned granules of average diameter 2 μ, containing large quantities of polyphenols. The layer is discontinuous and has no effect on permeability. The Resistant Protein Layer, a tanned and possibly vulcanized layer of protein, 1 to 2 μ thick, containing diffuse polyphenols. It is resistant to strong acids and bases, and permeable to water, ions, and large water-soluble molecules. The Outer Polyphenol Layer, which is similar to the inner layer, but has more minute granules. The Amber Layer.—This is the only coloured layer of the shell, and is less than 0-1 thick. It consists of tanned protein to which oil is added after secretion. It is therefore a lipidized protein, which is excessively resistant to acids and alkalies, and permeable to oils and oil-soluble material and to small ions and water. 5. The Soft Protein Layer.—This is a thick laminated layer some 8 ft thick, similar to, but less resistant than, the resistant protein layer. It contains polyphenols and tyrosine. The layer is freely permeable to -water-soluble substances. Throughout the secretion of the endochorion, the follicle cells stain deeply and appear to be filled with the protein components of the shell. The exochorion consists of two layers of the lipoprotein ‘chorionin ‘. 6. The Soft Exochorion Layer is a lipoprotein which is soluble in potash but not in strong acids ; the layer is permeable to lipoid solvents and to water and small ions, but not to larger particles. It is 8 p. thick at its maximum thickness, but contains follicular pits which, during secretion, are filled by long processes from the follicle cells. 7. The Resistant Exochorion Layer is a more resistant form of chorionin. It lines the pits and covers the surface of the shell, giving rise to the polygonal markings corresponding to the follicle cells, each with a pit at its centre. The follicle cells contain quantities of lipoprotein during this phase of secretion, and are difficult to stain. A method of staining is described which shows that pore canals of two varieties are present in the exochorion layers only. They run from the walls of the pits but do not reach the endochorion. None of the layers of the chorion waterproofs the shell. In the rear end of the shell, the outer polyphenol layer is displaced towards the exochorion, thus increasing the resistant protein layer and reducing the soft protein layer. It is shown that all seven layers are present in the neck, and in the central region of the cap, and that the order of secretion is the same. Modifications are produced by variations in the thickness of the various layers. In the neck, the soft protein layer is reduced ; in the cap, the resistant protein layer is reduced while the amber layer is 2/z. thick, giving the cap a brown appearance. The soft protein layer is extremely thin and irregular while the exochorion layers are 16/z, thick. Pore canals are again present in two varieties. Some analysis is made of the formation of follicular pits; this appears to be correlated with the thickness of the exochorion and endochorion layers.

1. During the oviposition of ticks a glandular organ—the organ of Gêné— is everted and touches the egg. If it is prevented from everting most of the eggs shrivel rapidly; few hatch even in a humid atmosphere. 2. The waterproofing properties of the normal egg are conferred by a superficial coating of wax, 0 · 5 – 2 ·0 μ in thickness. In Ornithodorusmoubata the wax is secreted and applied solely by Géné’s organ. In Ixodes ricinus waterproofing takes place in two stages: an incomplete covering of wax, probably secreted by the lobed accessory glands, is first smeared over the egg during its passage down the vagina; waterproofing is then completed by a further application of wax from Géné’s organ after the egg has been laid. Owing to its superficial position on the egg the wax layer is readily attacked by solvents and emulsifiers. 3. The morphology of Géné’s organ in O. moubata is described. The gland is a proliferation of the epidermis which lies detached from the cuticle. Its secretion, a watery refractile liquid containing the wax precursor, accumulates between the gland and the cuticle in two horn-like extensions. The wax is probably secreted through pore canals distributed over a narrow zone of cuticle below the horns; the cement covering-layer of the epicuticle does not extend to this zone. 4. The transparent, heat-stable material isolated from the horns of Géné’s organ is regarded as the wax precursor. Solubility in water is probably conferred by chemical linkage with protein. The precursor is taken up from the horns, where it is stored, and is presumably broken down within the gland cells. The wax is then secreted through the pore canals while the protein moiety is retained by the cell. 5. The critical temperatures of the eggs of Ixodidae range from 35° C. in I. ricinus to 44° C. in Hyalomma savignyi; only slightly higher critical temperatures were recorded for Argasidae (45° C. in O. moubata). Eggs with lower critical temperatures are more susceptible to desiccation. The susceptibility of the eggs of a given species is of the same order as that of the parent species; but whereas in Ixodidae the critical temperatures of the egg and the cuticle of the female tick are approximately the same, in Argasidae the critical temperatures of the cuticle are much higher (62° C. in O. moubata). These differences are related to the physical properties of the waxes. The cuticular wax in O. moubata is hard and crystalline (m.p. 65° C.), whereas the egg wax is soft and viscous (m.p. 50-54° C.). 6. The natural wax from Géné’s organ has definite powers of spreading on the surface of the egg and so completing the waterproofing layer. 7. The material extracted with boiling chloroform from egg-shells or from nymphal cuticles separates spontaneously into two fractions, a hard white wax (c. 85 per cent, by weight) and a soft yellow grease (c. 15 per cent.). The properties of these two lipoids differ conspicuously from those of the natural wax. Attempts to deposit the extracted materials on membranes in the form of a waterproofing layer were unsuccessful. 8. Ovulation is described in O. moubata. The shell of the tick egg is secreted by the oocyte itself and not by follicle cells. Three layers can be distinguished in the 24-hour egg: (i) an outer wax layer; (ii) an incomplete layer of granules which reduce ammoniacal silver nitrate; (iii) a shell layer. A fourth layer, the inner membrane (iv), is secreted by the oocyte after incubation for 2-3 days. 9. Both the shell layer and the inner membrane are composed of resistant, elastic protein and are devoid of chitin. The shell layer of the unwaterproofed egg is highly permeable to water and to large molecules with either hydrophilic or lipophilic affinities. The inner membrane is at first freely permeable to water and to inorganic ions. During the course of incubation the wax gradually migrates into the shell material and may reach the inner membrane. As this occurs, the effectiveness of abrasive dusts and of chloroform in promoting increased transpiration through the shell is notably reduced.