Ultrastructure and formation of the physogastric termite queen cuticle

The physogastric termite queen is the most striking example in insects of growth in size without cuticular moulting. This phenomenon has been studied with electron microscopy and histochemical tests in two species of higher termites, Cubitermes fungifaber and Macrotermes bellicosus. The abdominal hypertrophy (physogastry) is allowed by growth of the arthrodial membranes of the swarming imago. The growth is slow (over several years) but important: the cuticular dry weight is multiplied by 20 in C. fungifaber, by 100-150 in M. bellicosus. The termite queen cuticle arises from the transformation of the cuticle of the swarming imago or imaginal cuticle (unfolding and growing of the epicuticle, stretching of the endocuticle, resorption of the subcuticle) and from the secretion of a new endocuticle or royal endocuticle. The termite queen is the first example known in insects of epicuticular growth. In the physogastric queen, three cuticular types are observed: the rigid cuticle of the sclerites, the soft cuticle of the arthrodial membranes and the partially rigid cuticle of special structures, the neosclerites, which show both rigidity and growth. The fibrillar architecture varies according to the abdominal zones and the position within the cuticle. It appears to be determined by the forces arising from the musculature and the anisometric abdominal growth. The king does not become physogastric, although its cuticle is also modified.     

Water permeability of plant cuticles: permeance,diffusion and partition coefficients

Summary Using isolated cuticular membranes from ten woody and herbaceous plant species, permeance and diffusion coefficients for water were measured, and partition coefficients were calculated. The cuticular membranes of fruit had much higher permeance and diffusion coefficients than leaf cuticular membranes from either trees or herbs. Both diffusion and partition coefficients increased with increasing membrane thickness. Thin cuticles, therefore, tend to be better and more efficient water barriers than thick cuticles. We compared the diffusion coefficients and the water content of cuticles as calculated from transport measurements with those obtained from water vapor sorption. There is good to fair agreement for cuticular membranes with a low water content, but large discrepancies appear for polymer matrix membranes with high permeance. This is probably due to the fact that diffusion coefficients obtained from transport measurements on membranes with high permeance and water content are underestimated. Water permeabilities of polyethylene and polypropylene membranes are similar to those of leaf cuticular membranes. However, leaf cuticles have much lower diffusion coefficients and a much greater water content than these synthetic polymers. This suggests that cuticles are primarily mobility barriers as far as water transport is concerned.     

Localization of RR-1 and RR-2 cuticular proteins within the cuticle of Anopheles gambiae

The largest arthropod cuticular protein family, CPR, has the Rebers and Riddiford (R&R) Consensus that in an extended form confers chitin-binding properties. Two forms of the Consensus, RR-1 and RR-2, have been recognized and initial data suggested that the RR-1 and RR-2 proteins were present in different regions within the cuticle itself. Thus, RR-2 proteins would contribute to exocuticle that becomes sclerotized, while RR-1s would be found in endocuticle that remains soft. An alternative, and more common, suggestion is that RR-1 proteins are used for soft, flexible cuticles such as intersegmental membranes, while RR-2s are associated with hard cuticle such as sclerites and head capsules. We used TEM immunogold detection to localize the position of several RR-1 and RR-2 proteins in the cuticle of Anopheles gambiae. RR-1s were localized in the procuticle of the soft intersegmental membrane except for one protein found in the endocuticle of hard cuticle. RR-2s were consistently found in hard cuticle and not in flexible cuticle. All RR-2 antibodies localized to the exocuticle and four out of six were also found in the endocuticle. Hence the location of RR-1s and RR-2s depends more on properties of individual proteins than on either hypothesis.     

Studies on proteins in post-ecdysial nymphal cuticle of locust, Locusta migratoria, and cockroach, Blaberus craniifer

Proteins were extracted from the cuticle of mid-instar nymphs of locusts, Locusta migratoria, and cockroaches, Blaberus craniifer. Seven proteins were purified from the locust extract and five from the cockroach extract, and their amino acid sequences were determined. Polyacrylamide gel electrophoresis indicates that the proteins are present only in the post-ecdysially deposited layer of the nymphal cuticles. One of the locust and one of the cockroach nymphal proteins contain a 68-residue motif, the RR-2 sequence, which has been reported for several proteins from the solid cuticles of other insect species. Two of the cockroach proteins contain a 75-residue motif, which is also present in a protein from the larval/pupal cuticle of a beetle, Tenebrio molitor, and in proteins from the exoskeletons of a lobster, Homarus americanus, and a spider, Araneus diadematus. The motif contains a variant of the Rebers-Riddiford consensus sequence, and is called the RR-3 motif. One of the locust and three of the cockroach post-ecdysial proteins contain one or more copies of an 18-residue motif, previously reported in a protein from Bombyx mori pupal cuticle. The nymphal post-ecdysial proteins from both species have features in common with pre-ecdysial proteins (pharate proteins) in cuticles destined to be sclerotised; they show little similarity to the post-ecdysial cuticular proteins from adult locusts or to proteins from soft, pliable cuticles. Possible roles for post-ecdysial cuticular proteins are discussed in relation to the reported structures.     

Monitoring and visualising plant cuticles by confocal laser scanning microscopy

The present work shows the visualisation of phenolics and flavonoids of plant cuticles by confocal laser scanning microscopy (CLSM). Selected isolated fruit and leaf cuticles were monitored on the basis of autofluorescent phenolics and flavonoids which, in most cases, permitted us to obtain three-dimensional images of the cuticular membranes. The utility of this technique in investigations of cuticular translocation and diffusion of exogenous applied chemicals and cuticle degradation has also been explored.     

Ultrastrucuture of the fat body of the reproductive pair in higher termites

The fine structure of the fat body of the higher termite king and queen has been studied both in species with (Macrotermes bellicosus, M. subhyalinus) and without (Cubitermes fungifaber) tracheal rosettes. There is a very pronounced sexual dimorphism. The adipocytes of the queen are highly specialized for protein synthesis and secretion; they store only a small quantity of reserves. The adipocytes of the king are not specialized in protein synthesis, but accumulate large amounts of reserve substances. The previously proposed different functions of the termite queen's fat body are discussed; it appears to be mainly concerned with vitellogenesis.     

Investigations on epiphytic living Pseudomonas species from Malus domestica with an antagonistic effect to Venturia inaequalis on isolated plant cuticle membranes

In order to understand better the survival and mutual interaction of epiphytic bacteria and fungi on apple plants, bacteria collected from these plants were cultivated on intact adaxial, stoma free cuticle membranes originally obtained from apple. The bacteria were labelled with luciferase genes from Vibrio harveyi in order to follow up their development and activity on the isolated cuticles. Our finding was that the epiphytic bacteria can have access to nutrients below the cuticle without causing damage to these cuticular membranes. Bacterial proteins may enable this nutrient mobilization and we found, indeed, that more than 46 proteins that must have been delivered by the bacteria in response to interaction with the cuticles as they could be found below the cuticle membrane. Eight major representatives of this group of external proteins have been sequenced with electron spray quadrupole time of flight mass spectrometry and subsequently identified by data base homology search as a flagellin, a porin type protein and proteins that are involved in amino acid recruitment and metabolism.     

Characterization of the diffusion of non-electrolytes across plant cuticles: properties of the lipophilic pathway

Systemic crop protection products are commonly sprayed onto foliage, whereupon the active substances must penetrate into the leaves in order to become biologically active. Penetration of the plant cuticle is the rate-limiting step. The diffusion of organic non-electrolytes within cuticles is a purely physical process that can be described and analysed in the same way as is done for diffusion in synthetic polymer membranes. Solute mobilities in cuticles vary considerably between plant species. For a given species they decrease with increasing solute size, and this size selectivity holds for all of the plant species investigated so far. Wax extraction from leaf cuticles increases the mobility of solutes tremendously, but size selectivity is not affected. Furthermore, diffusion within plant cuticles is extremely temperature dependent. An analogous increase in solute mobility can be achieved by using accelerators, which enhance the fluidity of the polymer matrix and of the waxes. The effects of temperature and plasticizers on the diffusion of non-electrolytes in wax and the cutin matrix have been used to characterize the nature of the lipophilic pathway. The 'free volume' theory can be used to explain the influence of the size and shape of the solute, and its dependence on temperature. The physico-chemical nature of the diffusion pathway has been shown, by thermodynamic analysis, to be identical for a wide range of solute lipophilicities. This approach also explains the mode of action and the intrinsic activity of plasticizers.     

Characterization of aqueous pores in plant cuticles and permeation of ionic solutes

Plant cuticles are lipid membranes with separate diffusion paths for lipophilic non-electrolytes and hydrated ionic compounds. Ions are lipid insoluble and require an aqueous pathway across cuticles. Based on experimental data, the aqueous pathway in cuticles has been characterized. Aqueous pores arise by hydration of permanent dipoles and ionic functional groups. They can be localized using ionic fluorescent dyes, silver nitrate, and mercuric chloride. Aqueous pores preferentially occur in cuticular ledges, at the base of trichomes, and in cuticles over anticlinal walls. Average pore radii ranged from 0.45 to 1.18 nm. Penetration of ions was a first order process as the fraction of the salt remaining on the cuticle surface decreased exponentially with time. Permeability of cuticles to ions depended on humidity and was highest at 100% humidity. Wetting agents increased rate constants by factors of up to 12, which indicates that the pore openings are surrounded by waxes. The pores in cuticular ledges of Helxine soleirolii allowed passage of berberine sulphate, which has a molecular weight of 769 g mol(-1). Increasing the molecular weight of solutes from 100 to 500 g mol(-1) decreased the rate constants of penetration by factors of 7 (Vicia faba) and 13 (Populus canescens), respectively. Half-times of penetration of inorganic salts and organic ions across Populus cuticles and Vicia leaf surfaces varied between 1 and 12 h. This shows that penetration of ionic compounds can be fairly rapid, and ions with molecular weights of up to 800 g mol(-1) can penetrate cuticles that possess aqueous pores.     

Characterization of proteins from arthrodial membranes of the lobster, Homarus americanus

A total of six proteins from the abdominal arthrodial membrane (intersegmental membrane) of the lobster, Homarus americanus, were purified and their amino acid sequences were determined by a combination of mass spectrometry and Edman degradation. The proteins are acidic with pI-values close to 4 and they all have molecular masses approximately 12 kDa. The sequences of five of the proteins differ in only a few residues, while the sixth protein differs from the others in more than half of the positions. Only little similarity is observed between the sequences of the arthrodial membrane proteins and those of proteins purified from the calcified parts of the exoskeleton of H. americanus. The arthrodial membrane proteins contain the Rebers-Riddiford consensus sequence common in proteins from insect cuticles. Comparison of the complete sequences to the sequences available in databases shows that the lobster membrane proteins are more closely related to proteins from insect pliant cuticles than to proteins derived from cuticles destined for sclerotization. Characteristic features in the protein sequences are discussed, and it is suggested that the various sequence regions have specific roles in determining the mechanical properties of arthrodial membranes.     

Social and genetic structure of paper wasp cofoundress associations: tests of reproductive skew models

Recent models postulate that the members of a social group assess their ecological and social environments and agree a "social contract" of reproductive partitioning (skew). We tested social contracts theory by using DNA microsatellites to measure skew in 24 cofoundress associations of paper wasps, Polistes bellicosus. In contrast to theoretical predictions, there was little variation in cofoundress relatedness, and relatedness either did not predict skew or was negatively correlated with it; the dominant/subordinate size ratio, assumed to reflect relative fighting ability, did not predict skew; and high skew was associated with decreased aggression by the rank 2 subordinate toward the dominant. High skew was associated with increased group size. A difficulty with measuring skew in real systems is the frequent changes in group composition that commonly occur in social animals. In P. bellicosus, 61% of egg layers and an unknown number of non-egg layers were absent by the time nests were collected. The social contracts models provide an attractive general framework linking genetics, ecology, and behavior, but there have been few direct tests of their predictions. We question assumptions underlying the models and suggest directions for future research.     

Characterization of proteins from arthrodial membranes of the lobster, Homarus americanus

A total of six proteins from the abdominal arthrodial membrane (intersegmental membrane) of the lobster, Homarus americanus, were purified and their amino acid sequences were determined by a combination of mass spectrometry and Edman degradation. The proteins are acidic with pI-values close to 4 and they all have molecular masses ≈12 kDa. The sequences of five of the proteins differ in only a few residues, while the sixth protein differs from the others in more than half of the positions. Only little similarity is observed between the sequences of the arthrodial membrane proteins and those of proteins purified from the calcified parts of the exoskeleton of H. americanus. The arthrodial membrane proteins contain the Rebers-Riddiford consensus sequence common in proteins from insect cuticles. Comparison of the complete sequences to the sequences available in databases shows that the lobster membrane proteins are more closely related to proteins from insect pliant cuticles than to proteins derived from cuticles destined for sclerotization. Characteristic features in the protein sequences are discussed, and it is suggested that the various sequence regions have specific roles in determining the mechanical properties of arthrodial membranes.     

Transverse cuticular structures arise above actin filament bundles in the epidermis of Calpodes ethlius (Stöll) (Lepidoptera : Hesperiidae)

Rhodaminyl phalloin labelling of larval epidermal cells in Calpodes ethlius (Stöll) (Lepidoptera : Hesperiidae) shows dorsal areas with apical bundles of F-actin. The bundles are present only during the first 36 hr of the 5th stadium. Most cells have only one or 2, rarely 3, 4 or 5. The bundles extend into the overlying cuticle as the cores of large helical microvilli that continue on as transverse cuticle components, resembling very large helical pore canals. The transverse structures are like those seen in extensible insect cuticles that may allow cuticular stretching during larval growth. Neither the bundles nor the transverse structures are easily resolvable by conventional stains for LM or EM. The results suggest that transverse fibrillar structures may be a more common component of soft cuticles than has been generally realized.     

Determination of the covalent structure of an N- and C-terminally blocked glycoprotein from endocuticle of Locusta migratoria. Combined use of plasma desorption mass spectrometry and Edman degradation to study post-translationally modified proteins

The complete structure of protein isolated from endocuticle of sexually mature locusts, Locusta migratoria, has been determined by a combination of automatic Edman degradation and plasma desorption mass spectrometry. The protein is extensively post-translationally modified. The N-terminal is 5-oxoproline (pyroglutamic acid) and the C-terminal proline residue is amidated. Furthermore, the protein is glycosylated by a single N-acetyl-galactosamine residue at one, two or three threonines. The N-terminal sequence was obtained by analysing the N-acetylated N,O-permethylated derivative using plasma desorption mass spectrometry. The position and type of carbohydrate were determined by combining an HPLC-based carbohydrate analysis with the peak pattern of the phenylthiohydantoin derivative in automatic sequencing and with mass information on peptides. The protein has pronounced similarity to cuticular proteins from larvae of diptera and lepidoptera, but only slight resemblance to the previously sequenced locust exocuticular proteins. This indicates a similarity between soft larval cuticles and locust endocuticle, a similarity which may extend to their mechanical properties.     

Farming termites determine the genetic population structure of Termitomyces fungal symbionts

Symbiotic interactions between macrotermitine termites and their fungal symbionts have a moderate degree of specificity. Consistent with horizontal symbiont transmission, host switching has been frequent over evolutionary time so that single termite species can often be associated with several fungal symbionts. However, even in the few termite lineages that secondarily adopted vertical symbiont transmission, the fungal symbionts are not monophyletic. We addressed this paradox by studying differential transmission of fungal symbionts by alate male and female reproductives, and the genetic population structure of Termitomyces fungus gardens across 74 colonies of Macrotermes bellicosus in four west and central African countries. We confirm earlier, more limited, studies showing that the Termitomyces symbionts of M. bellicosus are normally transmitted vertically and clonally by dispersing males. We also document that the symbionts associated with this termite species belong to three main lineages that do not constitute a monophyletic group. The most common lineage occurs over the entire geographical region that we studied, including west, central and southern Africa, where it is also associated with the alternative termite hosts Macrotermes subhyalinus and Macrotermes natalensis. While Termitomyces associated with these alternative hosts are horizontally transmitted and recombine freely, the genetic population structure of the same Termitomyces associated with M. bellicosus is consistent with predominantly clonal reproduction and only occasional recombination. This implies that the genetic population structure of Termitomyces is controlled by the termite host and not by the Termitomyces symbiont.     

Is beta-pleated sheet the molecular conformation which dictates formation of helicoidal cuticle?

Over 100 sequences for cuticular proteins are now available, but there have been no formal analyses of how these sequences might contribute to the helicoidal architecture of cuticle or to the interaction of these proteins with chitin. A secondary structure prediction scheme (Hamodrakas, S.J., 1988. A protein secondary structure prediction scheme for the IBM PC and compatibles. CABIOS 4, 473-477) that combines six different algorithms predicting alpha-helix, beta-strands and beta-turn/loops/coil has been used to predict the secondary structure of chorion proteins and experimental confirmation has established its utility (Hamodrakas, S.J., 1992. Molecular architecture of helicoidal proteinaceous eggshells. In: Case, S.T. (Ed.), Results and Problems in Cell Differentiation, Vol. 19, Berlin-Heidelberg, Springer Verlag, pp. 116-186 and references therein). We have used this same scheme with eight cuticular protein sequences associated with hard cuticles and nineteen from soft cuticles. Secondary structure predictions were restricted to a conserved 68 amino acid region that begins with a preponderance of hydrophilic residues and ends with a 33 amino acid consensus region, first identified by Rebers and Riddiford (Rebers, J.F., Riddiford, L.M., 1988. Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes. J. Mol. Biol. 203, 411-423). Both classes of sequences showed a preponderance of beta-pleated sheet, with four distinct strands in the proteins from 'hard' cuticles and three from 'soft'. In both cases, tyrosine and phenylalanine were found on one face within a sheet, an optimal location for interaction with chitin. We propose that this beta-sheet dictates formation of helicoidal cuticle.     

Exoskeletal proteins from the crab, Cancer pagurus.

Twelve proteins from calcified regions and five from flexible regions (arthrodial membranes) of the exoskeleton of Cancer pagurus have been purified and sequenced. One of the proteins from calcified exoskeleton is identical to one of the arthrodial membrane proteins. Several of the proteins from the calcified regions resemble proteins from corresponding regions of the exoskeleton of the lobster, Homarus americanus, in containing either two or four copies of an 18-residue sequence motif, which so far has been found only in crustacean calcified exoskeletons. The proteins obtained from the flexible arthrodial membranes resemble the proteins from lobster arthrodial membranes, and the similarities are shared with a number of proteins from flexible cuticles in insects, indicating that the common features in these proteins may be important for the mechanical properties of the materials in which they occur.     

Leaf cuticles behave as asymmetric membranes : evidence from the measurement of diffusion potentials

Cuticles were isolated enzymatically from the leaves of two maple species (Acer saccharum Marsh and A. platanoides L.) and from orange (Citrus aurantium L.). The cuticles were placed in a plastic cuvette and different concentrations of KCl were perfused over the physiological inner and outer surfaces while the electrical potential (E¹⁰) that developed across the cuticles and was caused by ion diffusion was measured. E¹⁰ was always positive, indicating that the permeability of K⁺ was always greater than that of Cl^-. Measured E¹⁰ in cuticles did not fit the Goldman equation, whereas, E¹⁰ measured during KCl diffusion across selected artificial membranes fit the equation. The magnitude of E¹⁰ in cuticles and artificial membranes also was dependent on ionic strength, decreasing as ionic strength increased. These observations are explained by combining classical transport equations with equations that describe the equilibrium ion distribution between ionic double layers in the cuticle or membranes and the bathing solution.     

137Cs penetration by contact exchange through isolated plant cuticles: cuticles as asymmetric transport membranes

The penetration of ¹³⁷Cs by contact exchange through cuticular membranes from the adaxial surface of leaves of Pyrus communis and Prunus cerasus has been investigated. The resistance of the cuticles to the caesium penetration was dependent on the counter-ions associated with the fixed negative ion exchange sites in the membrane. The mobility of hydrated potassium ions and their tenuous connection to -COOH^?-groups in the membrane encouraged caesium permeation in contrast to cuticular membranes with predominantly protonized ion exchange sites. Divalent calcium ions caused a strong reduction (4–20 times) of the caesium permeability which is decisive for the calculation of the caesium uptake by the intact leaf. Under these conditions, a penetration rate of the deposited caesium of 0·11±0·05% h^?1 for pear cuticles and of 0·036±0·025% h^?1 for cherry cuticles was measured after the adjustment to steady state conditions. Approximately 12–24% and 4·5–7·5% of initially retained caesium could be absorbed by the leaves of pear and cherry, respectively, in a rain-free period of 7 d in the area of Munich after wet deposition of fallout from the Chernobyl reactor accident. Furthermore, the caesium penetration from the physiological inside to the outside of the membrane was found to be smaller by a factor of 100–150 compared with that of the opposite direction.