Sclerotin and lipid in the waterproofing of the insect cuticle

In all the cuticles studied waterproofing is effected by extracuticular material, a mixture of sclerotin precursors and lipids, exuded from the tubular filaments of the pore canals. In Rhodnius larval abdomen it is a layer of thickness similar to the outer epicuticle, believed to be composed of 'sclerotin' and wax, in Schistocerca larval sternal cuticle and in Carausius sternal cuticle it is similar. In Tenebrio adult sternal cuticle of the abdomen, in both the extracuticular exudation and the contents of the distal endings of the tubular filaments, the wax component is obscured by hard 'sclerotin'. In Manduca larva a very thin layer of 'sclerotin' and wax is covered by an irregular wax layer, average 0.75 micron, twice the thickness of the inner epicuticle. In Periplaneta and Blattella the abdominal cuticle is covered by a soft waxy layer, often about 1 micron thick, which is mixed with argentaffin material. Below this is a very thin waterproof layer of wax and 'sclerotin' continuous with the contents of the tubular filaments, which is readily removed by adsorptive dusts. In Apis adult abdominal terga free wax plus sclerotin precursors form a thin layer which is known to be removed by adsorptive dusts. In Calliphora larva there is a very thin layer of the usual mixed wax and sclerotin and below this a thick (0.5 micron) layer, lipid staining and strongly osmiophil, likewise extracuticular and exuded from the epicuticular channels. This material (which is often called 'outer epicuticle') has the same staining and resistance properties as the true outer epicuticle on which it rests. In the abdomen of Calliphora adult the waterproofing wax-sclerotin mixture forms a thin layer over the entire cuticle including the surface of the microtrichia. There is also a thin detachable layer of free wax on the surface.     

An osmoregulatory syncytium and associated cells in a freshwater mosquito

In embryological terms the anal papillae are the product of eversion of the hindgut tissues. The rectum and the anal papillae have the same origin and have a marked structural similarity. The insect hindgut is very labile being able to produce salt transporting or ‘chloride cells’ from any of the tissues of which it is composed.The hindgut consists of four distinct regions: the ileum and part of the anal canal have a mechanical function, the rectum and the posterior anal canal contain transporting cells. Two new cell types, ‘interstitial’ and ‘tertiary’ are reported in the rectum.The structure of the anal papillae changes with increased salinity. Changes in the plasma membranes alter the surface area for transport. Changes in the number of mitochondria are not accompanied by changes in oxygen consumption. If mitochondria are the site of oxidative metabolism then their number docs not control the level of oxygen consumption.In Aedes aegypti the papillary epithelium appears to be a syncytium. Across the lumen of the papillae there are cellular sheets supporting the tracheoles. At the base of the papillae there is a cellular transition zone; circular muscles in this region may be used to occlude the papillae. The control of salt transport may be hormonal.     

Changes in the mechanical properties of the extensible cuticle of Rhodnius through the fifth larval instar

The ultimate tensile strength (σ_UT) and the modulus of elasticity (E) of Rhodnius extensible cuticle are σ_UT = 2.20 × 10⁷ Nm^?2, E = 2.43 × 10⁸ Nm^?2 (unplasticised); σ_UT = 1.43 × 10⁷ Nm^?2, E = 9.45 × 10⁶ Nm^?2 (plasticised with 5HT) and σ_UT = 9.05 × 10⁶ Nm, E = 2.46 × 10⁶ Nm^?2 (plasticised in pH 5 buffer).The mechanical properties of cuticle from insects which have deposited additional layers of cuticle after they have been fed differ from those of cuticle from unfed insects. This is possibly due to the different composition of the additional cuticle: it is suggested that the post-feeding cuticle is providing protection and a template for the next instars cuticle.The maximum strain of extensible cuticle from starved insects is related to the amount of matrix protein present.     

Giant sperm cells with accessory macrotubules in a neuropteran insect

The flagellar axoneme of the atypical spermatozoa (paraspermatozoa) of Mantispa perla (Neuroptera, Planipennia) contains accessory microtubules or rather macrotubules that are 55 nm in diameter and that has a wall consisting of about 40 protofilaments. The sperm tail further contains two giant mitochondrial derivatives, which during spermiogenesis store an electron dense material. The mature spermatozoon has a flattened acrosome and a elliptical nucleus. These giant spermatozoa may furnish nutrients to the functional spermatozoa (euspermatozoa) when they reach the female genital tracts or/and they function in sperm competition filling the spermatheca.     

Observation of an anisotropic texture inside the wax layer of insect cuticle

The outermost part of insect cuticles is very often covered with wax, which prevents desiccation and serves for chemical communication in many species. Earlier studies on cuticular waxes have mainly focused on their chemical composition revealing complex mixtures of lipids. In the absence of information on their physical organization, cuticular waxes have been considered isotropic. Here we report the presence of parallel stripes in the wax layer of the carapace of the scarab beetle, Chrysina gloriosa, with a textural periodicity of ca. 28 nm, as revealed by electron microscopy of transverse sections. Observations at oblique incidence argue for a layered organization of the wax, which might be related to a layer-by-layer deposition of excreted wax. Our findings may lay the foundation for further studies on the internal structure of cuticular waxes for other insects.     

Consideration of the importance of hydrophobic interactions in stabilizing insect cuticle

Protein fractions of insect cuticles with different mechanical properties have related values of polarity and hydrophobicity. Hydrophobicity is important for the self-assembly of cuticle which is produced prior to the moult and in plasticization of cuticle. The cuticles of soft-bodied fly larvae are quite distinct from those of exopterygotes (e.g. locusts) and this can be related to the chemistry and mode of tanning. The properties of cuticular proteins are compared: the proteins of the pliant cuticles most closely resemble globulins, and the proteins in stiff cuticles are more like fibrous and hydrophobic structural proteins. Changes in the environment of the proteins may alter their shape and hence the mechanical properties of the cuticle.     

Observations on the osmoregulatory system of pentastomids: The tegumental chloride cells

The chitinous procuticle of pentastomids is permeated by small pores which communicate with specialized epidermal cells previously termed cuticular cells. Ultrastructural evidence is presented which suggests that these cells are concerned with the regulation of the hydromineral balance of the haemolymph. Probably all pentastomids regulate hypo-osmotically: that is they maintain a blood concentration which is lower than that of their host. Since pentastomids feed on host blood or lymph which is rich in sodium and chloride, it suggested that chloride cells secrete excess ions. The extent to which chloride cells must concentrate ions depends upon the nature of the food, the rate of exosmosis and the rate of salt gain by diffusion.     

Superhydrophobic cuticle with a "pinning effect" in the larvae of the iris sawfly, Rhadinoceraea micans (Hymenoptera, Tenthredinidae)

The integument of some sawfly larvae can be easily damaged causing haemolymph bleeding (easy bleeding phenomenon). In the present study, we investigated intact cuticle, cuticle without wax coverage and cuticle replicas of sawfly larvae by using cryo-scanning electron microscopy and contact angle (CA) measurements. The easy bleeder Rhadinoceraea micans was compared to the non-easy bleeder Nematus pavidus (both Tenthredinidae), and the examination showed that the surface structures of R. micans were hierarchically arranged, whereas the cuticle surface of N. pavidus appeared much smoother. The intact cuticle proved to be superhydrophobic in R. micans (CAs: 156° dorsally, 120° ventrally), but not in N. pavidus (CAs: 67° dorsally, 47° ventrally). The wettability of R. micans increased significantly after the waxes had been peeled off. Replicas of the intact cuticle surface were hardly wettable in both species. It is suggested that wax crystals in combination with cuticle microsculptures are the most important features for rendering the integument of the easy bleeder species superhydrophobic. The wax-free tips of the cuticle microsculptures in R. micans are sites where haemolymph droplets can adhere. The integument surface of easy bleeders is assumed to be adapted to their particular defence strategy, simultaneously contributing to the maintenance and "reuse" of released haemolymph droplets on the superhydrophobic surface due to this "pinning effect".     

Heavy-metal toxicity in an insect cell line. Effects of cadmium chloride, mercuric chloride and methylmercuric chloride on cell viability and proliferation in Aedes albopictus cells

We evaluated the toxicity of CdCl2, HgCl2, and MeHgCl on the C6/36 cell line of Aedes albopictus. This cell line proved to be a suitable tool for studying heavy-metal toxicity in insect cells. Since data on heavy-metal toxicity in invertebrate cell cultures are almost nonexistent, our results are discussed in relation to in vivo invertebrate and in vitro vertebrate studies. Viability and proliferation were assessed by dye exclusion and DNA quantification, respectively. Viability tests were carried out with and without 5% fetal calf serum in the medium. The three metal species decreased viability to different extents (MeHgCl>HgCl2>CdCl2), and fetal calf serum had a protective effect. In serum-deprived cultures, LD50 values were 140.20, 2.51, and 2.08 µmol/L for CdCl2, HgCl2, and MeHgCl, respectively. For cultures with fetal calf serum, LD50 values were 149.71, 12.01, and 5.47 µmol/L, respectively. The viability curve for CdCl2 under serum-free conditions suggests the induction of a cell defense system. The three metal species also inhibited cell proliferation (MeHgCl> CdCl2> HgCl2). The IC50 values were 1.75, 18.36, and 0.96 µmol/L for CdCl2, HgCl2, and MeHgCl, respectively. In summary, low MeHgCl concentrations caused both cell death and inhibition of cell proliferation; HgCl2 primarily disrupted the plasma membrane, whereas CdCl2 primarily inhibited cell proliferation.     

External macro- and micromorphology of the male of the stick insect Hermarchus leytensis (Insecta: Phasmatodea) with phylogenetic considerations

External morphological features of adult males are described in the stick insect Hermarchus leytensis from the Philippines, a species belonging to the little-known euphasmatodean lineage Stephanacridini. Mouthparts are characterized by some likely specialized features: (1) a dentate dorsal cutting edge on the mandibles; (2) distinct differences in size and shape between the galeae; (3) absence of an apical field of trichomes on the galeae; and (4) lacinial setae not protruding from the mesal margin, which features three bearing-like protuberances. The latter character state represents a very unusual condition, not known in other phasmatodeans or even in polyneopteran insects. A distinctive characteristic of attachment devices is that each euplantula is divided into two separated pads with a smooth microstructure, as it also occurs in some members of the clade Schizodecema. Male terminalia exhibit character states previously unknown in Stephanacridini, including (1) a symmetrical type of vomer and (2) claspers equipped with ∼70 very minute ventral teeth on each thorn pad. Potential implications for the systematic placement of H. leytensis are discussed. The results also underline the importance of microanatomical investigations as source of substantial characters for future analyses on phasmatodean systematics.     

Loss of genetic diversity in the North American mayfly Ephemerella invaria associated with deforestation of headwater streams

1. Terrestrial dispersal by aquatic insects increases population connectivity in some stream species by allowing individuals to move outside the structure of the stream network. In addition, individual survival and reproductive success (as well as dispersal) are tightly linked to the quality of the terrestrial habitat. 2. In historically forested catchments, deforestation and altered land use have the potential to interfere with mayfly dispersal or mating behaviours by degrading the quality of the terrestrial matrix among headwater streams. We hypothesised that loss of tree cover in first‐order catchments would be associated with an increase in population substructure and a decrease in genetic diversity of mayfly populations. 3. To test this hypothesis, we investigated spatial patterns of genetic variation in the common mayfly Ephemerella invaria across a gradient of deforestation in the central piedmont region of eastern United States. Intraspecific genetic diversity and population substructure were estimated from data obtained using fluorescent amplified fragment length polymorphism (AFLP) markers. 4. We found that mayfly populations had low population substructure within headwater stream networks and that genetic diversity was strongly negatively correlated with mean deforestation of the first‐order catchments. The large‐scale pattern of population substructure followed a pattern of isolation by distance (IBD) in which genetic differentiation increases with geographical distance, but assignment tests placed a few individuals into populations 300 km away from the collection site. 5. Our results show that loss of genetic diversity in this widespread aquatic insect species is co‐occurring with deforestation of headwater streams. 6. Most arguments supporting protection of headwater streams in the United States have centred on the role of these streams as hydrological and biogeochemical conduits to downstream waters. Our work suggests that headwater stream land use, and specifically tree cover, may have a role in the maintenance of regional genetic diversity in some common aquatic insect species.     

Ultrastructure changes associated with osmoregulation in the hindgut cells of a saltwater insect, Ephydrella sp. (Ephydridae: Diptera)

Ephydrella larvae strictly regulate their blood osmotic pressure and Na⁺ content over a wide range of environmental salinities (7 mM to 3000 mM NaCl). They can survive in distilled water and 6000 mM NaCl for several days. In the hindgut the ileum is concerned with the regulation of urine composition whilst the rectum has a purely mechanical function. In the ileum there are large cells which have long basal channels and short apical microvilli, and small cells which have long apical leaflets and short basal channels. It is suggested that the large cells reabsorb water and that the small cells either reabsorb or secrete ions. The configurations of the channels, and the spacing of leaflets and microvilli change with alterations in environmental salinity. Fixation experiments using fixatives of different osmotic pressures show that the configuration of extracellular space in the cells has a marked dependence on both the osmotic pressure of the fixative and upon the environmental salinity. It is suggested that the osmotic response of the cells to fixatives indicates that the osmotic pressure of the cells increases with increasing environmental salinity. It is suggested, in general, that correlation of changes in the volume of extracellular space with changes in ion and water transport must be regarded with caution.     

Fine structure of chloride cells in freshwater- and seawater-adapted Oreochromis niloticus (Linnaeus) and Oreochromis mossambicus (Peters)

Ultrastructural features of branchial chloride cells in Oreochromis niloticus (Linnaeus) and O. mossambicus (Peters) adapted to both fresh water and sea water were compdred. In freshwater adapted fish of both species chloride cells showed similar morphological features. Multicellular complexes made of a mature chloride cell and one or more accessory cells sharing a single apical crypt have been observed. Whereas high percentages of 0. mossambicus survived at maximum salinity only a few individuals of 0. niloticus showed the capacity to adapt to sea water. In the seawater-adapted individuals of 0. niloficus and 0. rnossambicus the chloride cells showed a two- and three-fold increase in sizei. respectively. Most chloride cells are organized in large multi-cellular complcxcs with apical interdigitations of accessory cells and 'leaky junctions'. These results indicate that the difference in euryhalinity of the species studied is related to functional rather than structural differences.     

Role of betaine in the control of respiration and osmoregulation of a halotolerant bacterium

Abstract This review deals with work on some aspects of halotolerance carried out in our laboratory. The organism used throughout these investigations is a halotolerant, obligate aerobic rod, designated as Ba₁. Attention is focused on the effect of osmotic stress on respiratory rate, since under the conditions tested the latter is rate-limiting for growth. Under hyperosmotic conditions there is a steep drop in the rate of oxygen uptake, but the inhibition can be relieved by betaine. Two major factors seem to be involved in this effect of betaine: (a) Iso-osmotic adaptation due to its accumulation in the cytosol (deplasmolysis); (b) facilitation of the penetration of Na⁺ into plasmolysed cells. Na⁺ is required for the proper functioning of the respiratory chain. Na⁺ probably enters the cells in symport with betaine, whereas an excess of this cytotoxic cation is extruded by a powerful primary Na⁺ pump which is found to operate in Ba₁. Such a pump has also been identified in another halotolerant organism, Vibrio alginolyticus . In both micro-organisms the site of Na⁺-stimulation corresponds to the site of Na⁺ translocation.     

The transfer of lipid in insects from the epidermal cells to the cuticle

The structure of the pore canals and the tubular filaments they contain are described in a series of insects and types of cuticle. In all these cuticles the tubular filaments arise from the plasma membrane of the epidermal cells and they contain argentaffin material, regarded as sclerotin precursors, and lipid-staining material, regarded as wax precursors. These materials are transferred to the inner epicuticle and are exuded over the surface of the outer epicuticle to form the waterproofing layer as described in the preceding paper. They are also transported to those parts of the endocuticle destined to form hard exocuticle. There are no terminations of tubular filaments in the soft cuticle of Manduca larva, in the soft expanding cuticle of Rhodnius, and in the non-sclerotized post-ecdysial endocuticle of Tenebrio. Apis. etc. In the puparium of Calliphora lipid appears to be added by the epidermal cells directly and not by way of tubular filaments. It is confirmed that lipid is a component of sclerotized cuticle.     

Secretory hyperactivity and mitochondrial changes in neurosecretory cells of an insect

Summary The neurosecretory cells of the corpus cardiacum of the desert locust Schistocerca gregaria secrete their hormonal products by exocytosis. The insecticide lindane is known to cause release of hyperglycaemic and adipokinetic neurohormones. Electron microscopy of lindane-poisoned corpora cardiaca reveals many exocytotic omega figures. Mitochondria are affected either directly by the poison or by the consequences of secretory hyperactivity. They occur in increased number, divide, acquire dense mitochondrial granules larger than in the controls, and sometimes line up along plasma membranes, mitochondria in two neighbouring cells forming pairs in juxtaposition. Between two such mitochondria the plasma membranes form a junction-like structure. It is suggested that these effects reflect an excessive calcium entry caused by lindane.This work was supported by a Wellcome-Carlsberg Travelling Research Fellowship awarded to T.N. and by a Commonwealth Scholarship for Postgraduate Research awarded to M.S.     

Ultrastructure of an integumental organ with probable sensory function in Paragordius varius (nematomorpha)

The cuticle of late parasitic stages of Paragordius varius (Leidy, 1851) is composed of a layer with large fibres and a second layer (often named the areolar layer) distal from it. In this paper, organs are described that start at the basal side of the epidermis, pass the epidermis and the fibrous layer of the cuticle and merge with large, cushion‐like structures in the distal layer of the cuticle. The epidermal part of the organs is composed of darkly stained cells, which are probably in contact with the basi‐epidermal nervous system. Up to four processes of this cell traverse the cuticle. These processes might include cilia, because they contain microtubule‐like structures. The probable connection to nerve cells and the connection to the cushion‐like structures in the outer cuticular layer make it likely that the organs described here are sensory in function.     

Elaborations of the basal surface of the cells of the Malpighian tubules of an insect

Electrical measurements of membrane potential and resistance using intracellular microelectrodes showed that the fluid-secreting parts of the Malpighian tubules of Rhodnius have superficial regions different in electrical properties from the main body of the cell. The membrane potential in these superficial regions was smaller by 30-40 mV, but showed a standard depolarization on changing the potassium concentration of the bathing medium. The response to changes in the external chloride concentration also differed in the two regions, a finding that was reinforced by different responses to the drug, furosemide. Electron microscopy of the basal regions of the cells revealed many long cellular projections that run parallel to the cell surface and interdigitate with similar projections from neighbouring cells. The degree of interdigitation was examined by marking individual cells with alcian blue or by horseradish peroxidase injection. A survey of the published micrographs of insect Malpighian tubules shows that most have similar projections on their basal surfaces and not the simple basal infoldings previously supposed.     

Differentiation of the oocyte and nurse cells in an apterygote insect (Campodea)

Differentiated complexes of cystocytes in an apterygote insect (Diplura: Campodea sp.) are arranged in unbranched chains. Cystocyte lying approximately in the centre of a chain differentiate into the oocyte: two cells adjoining the oocyte and connected with it via cytoplasmic bridges develop into the 'intermediate cells'. Other cystocytes become typical 'nurse cells'. The intermediate cells are structurally transitional between oocytes and nurse cells. In this account, factors controlling the differentiation of oocytes and nurse cells are discussed.     

Ultrastructural evidence for paracellular fluid flow in the Malpighian tubules of a larval mayfly

The ultrastructure of the Malpighian tubules of larvae of the Mayfly Ecdyonurus dispar (Ephemeroptera) is described. There are about 60 tubules, which consist of four distinct regions. The most proximal section (region I) appears to be responsible for fluid secretion. A unique feature is the presence of channels leading off the main lumen, which end close to the basal border of the cells. Microvilli are confined to these channels in region I. Region II is a short spiral region, the cells of which possess long basal folds and associated mitochondria. Region III is a simple conducting tube leading to one of six collecting ducts (region IV) arranged radially around the gut. In each collecting duct there are two cell types present. Type 2 cells are relatively simple, but give rise to numerous, long, microvilli-like projections. Type 1 cells possess long basal folds, and curious membrane whorls in the apical zone. Evidence is presented which suggest that water movements into region I takes place via the paracellular route. Region II is probably a reabsorptive region, but the function of region IV, based on ultrastructural evidence is more difficult to elucidate.