Larval Integument Research Articles

Light and scanning electron microscopy of the embryos and glochidia larvae of the Australian freshwater bivalve Hyridella depressa (Hyriidae)

Summary As is characteristic of freshwater Unionacea, the Australian species Hyridella depressa incubates its young within the demibranchs of its modified gills. The development of H. depressa was documented through light and scanning electron microscopic examination of the marsupial pouch in the inner demibranchs of brooding females. Transition from the gastrula to the glochidial stage was accompanied by a split in the larval integument. The glochidia of H. depressa have subtriangular shell valves each having a blunt tooth at the ventral margin. Each glochidium has a pair of hooks, one on each valve internal to the tooth. These hooks are used to attach to its fish host and are structured to interlock when the valves snap shut. One or two tufts of sensory hairs are located on the internal surface of the mantle dorsal to the hook, while a single tuft, encircled by a collar-like structure, is located centrally. The valves are pitted by pores and have concentric lines along the margins. Due to the similarity and phenotypic plasticity of adult Hyridella species, the morphology of their glochidia has potential for use as a taxonomic tool. In comparison to related species, the glochidia of H. depressa are medium-sized with a mean length of 243 μm and a mean height of 249 μm. The number of glochidia present in the gills was measured to determine the reproductive output of H. depressa. The embryos of an unknown mite, Unionicola sp., form cysts within the gill tissue of H. depressa and the adult mites were observed on the surface of the gills.

Integument ultrastructure of Oestrus ovis (L.) (Diptera: Oestridae) larvae: Host immune response to various cuticular components

The nasal bot fly, Oestrus ovis, was investigated to establish which specific cuticular component is most immunogenic to infested sheep and how larval cuticle attains a protective role, if any, against the host immune system. To accomplish these goals, larval cuticle was extracted by a variety of agents and tested against immune sera from infested sheep and experimentally immunized rabbits. The cuticle substructure remaining after extraction was examined to localize various immunogenic components. O. ovis larval integument comprises an inner cellular layer, the epidermis, and an overlying cuticle layer. In 3rd instar larvae, the cuticle comprises 2 additional layers: the procuticle with numerous pore canals and the epicuticle which includes the wax canals. Three additional layers, altogether comprising the cuticulin layer. are present external to the epicuticle. The epicuticle is completed by apposition of an amorphous electrondense material extending for up to 1 μm in thickness. When fixed with ruthenium red, cuticle becomes heavily stained all along the epicuticular surface in larvae of all developmental stages. However, in 3rd instar larvae, ruthenium red deposits are restricted to the cuticulin layer alone. By gel electrophoresis, 3rd instar larval cuticle is shown to contain a number of polypeptides ranging in molecular weight from 180 to 4.5 kDa. The number and relative concentration of low molecular weight polypeptides was shown to vary in relation to the extraction media employed. Cuticular fragments examined after extraction exhibit an altered ultrastructure. When tested by immunoblotting, the cuticular polypeptides most reactive against sheep antisera are in the range of 180-56 kDa. A similar reaction was also detected with sera from rabbits infested experimentally with O. ovis larvae. Results are interpreted in relation to differential polypeptide distribution within the larval cuticle and to accessibility of the host immune system.

Sem study on the invasion of Nomuraea rileyi (Farlow) on silkworm, Bombyx mori Linn. causing green muscardine.

The mature conidia of Nomuraea rileyi (Farlow) germinate on the larval integument of Bombyx mori within 24 h and penetrate the cuticle within 36 h after inoculation at 24.0 +/- 1.0 degrees C temperature and 80.0 +/- 5.0% relative humidity. The penetrating hyphae multiply by budding and septa formation in the hemocoel, and the larva succumbs to the infection 6-7 days post-treatment. The hyphal bodies elongate and become interwoven with other hyphae forming a mycelial complex across different tissues. The ramification of hyphae along the epidermal tissue results in larval mummification in 7-8 days. Numerous conidiophores emerge, producing a confluent white fungal mat over the entire surface of the host larva by 9-10 days. Pale green conidia develop, making the larval body green. Life cycle of the fungus on B. mori is completed in 10-11 days.

Development of Trichogramma australicurn Girault (Hymenoptera: Trichogrammatidae) on Helicoverpa armigera (Hubner) Eggs (Lepidoptera: Noctuidae)

ABSTRACT Trichograrnma australicum were reared on eggs of Helicoverpa arrnigera. Immature T. australicurn were studied with a confocal laser scanning microscope and a light microscope. Development of egg, larva, prepupa and pupa required about 24‐26, 24‐26, 42‐47 and 84‐92 h, respectively, at 28°, 45% RH and 12L:12D photoperiod. The eggs of T. australicurn increased significantly in size during incubation. The larval mandibles were gradually revealed as the larva increased in size. Exuviae were not found during the larval stage, and we conclude that T. australicurn has one larval instar. Staining larvae and pre‐pupae with 1% aniline blue dissolved in Neisenheimer's solution induced fluorescence. Stained larvae and pre‐pupae gave a clear image of anatomy when observed with a confocal laser scanning microscope. Urate bodies appeared at the onset of the prepupal stage. The vitelline membrane of host egg gradually blackened. Legs, wings, antennae, body segmentation, genitalia, colour of compound eyes and ocelli became distinct during the prepupal stage. Larval integument and mandibles were shed at the onset of pupation. The adult wasps emerged 182.40 ± 0.40 h (n = 10) after oviposition, 2.80 ± 0.25 (n = 10) wasps emerged from a host egg, and the sex ratio was 1 ♀: 3 ♂♂.

Larval integument and its differentiations in Micropterix calthella L. (Lepidoptera : Micropterigidae): Anatomy and ultrastructure

The outer layer of the integument of the larva of Micropterix calthella (Lepidoptera : Micropterigidae) shows numerous modifications shaped like crests, nipples, and cones. Sections made at the level of these modifications or structures show that internal and external layers of the exocuticle are separate and form intracuticular cavities filled with a fluid. Every segment on the thorax and abdomen bears 3 disc-shaped structures, one dorsally and 2 laterally. These discs correspond to muscle attachments. Leaf-shaped structures, measuring up to 180 μm high in the last-instar larva, are located along longitudinal lines on all segments. Despite their great anatomical diversity, these structures have the same ultrastructural organization. Although larvae appear to lack spiracles, they have an internal tracheal system, which does not seem to be linked with the intracuticular cavities.

Ultrastructure of Ceratitis capitata Wiedemann Larval Integument and Changes Induced by the IGI Cyromazine

Cyromazine inhibits larval growth and mobility and decreases the pupation and adult emergence rates in Ceratitis capitata Wiedemann. This work details changes induced by cyromazine in the integument and muscles of third-instar larvae. Severely affected larvae show a conspicuous swelling of the integument in the anterior region of the body, with a local thinness of the cuticle due to the gap between the epicuticle and the procuticle. In other areas, a discontinuity between procuticular layers with many empty spaces, inclusion bodies, and an abnormal thickening of the cuticle is also observed. The epidermic layer is folded and disorganized and its cells have elongated to invade the adjacent space. The epicuticle is the only layer unaffected by the treatment. Muscle attachments to the cuticle are not broken, but a great inhibition of locomotion is detected and no popped pupae are recorded. However, under the electron microscope, muscles with altered mitochondria, distorted area of fiber arrangement, and a less fiber packing can be observed and may account for the lack of movement. Ultrastructural observations confirm the hypothesis that cyromazine acts at the hormonal level, as previously stated, but its effects on skeletal muscles are also indicative of protein involvement in the mode of action of this product, and warrant further investigation.

Identification of ommin in the integument of the silkworm, Bombyx mori.

An ommin was isolated from the integument of the quail mutant of the silkworm, Bombyx mori, using column chromatography on SP-Sephadex and cellulose powder. As a standard, ommin was isolated from diapause eggs of the normal silkworm. The red pigment from the larval integument of quail mutants was identical to the standard compound with respect to absorption spectrum, infrared spectrum and RF values in thin-layer chromatography (TLC). After acid hydrolysis of the pigment, 3-hydroxykynurenine was detected by TLC. This is the first report of an ommin in a lepidopteran larval integument.

Sequestration of pyrrolizidine alkaloids in several arctiid moths (Lepidoptera: Arctiidae)

Sequestration of dietary pyrrolizidine alkaloids (PA) by larvae and adults of six European arctiid moth species (Spilosoma lubricipeda, Arctia caja, Phragmatobia fuliginosa, Callimorpha dominula, Diacrisia sannio, andTyria jacobaeae) was investigated for comparison with the well-studied Asian arctiidCreatonotos transiens. Larvae of all species metabolized free PA bases into the respectiveN-oxides. Only adults ofA. caja, P. fuliginosa, andS. lubricipeda, but not their larvae, converted dietary 7(S)-heliotrine to 7(R)-heliotrine, a direct precursor of a male pheromone in some arctiids, 7(R)-hydroxydanaidal. The larval integument figures as the main storage site for resorbed alkaloids; only minor amounts were found in other tissues. In addition, a significant amount of alkaloid is deposited in the cocoon ofArctia caja; only traces of alkaloids could be found in the meconium and the exuviae of this species. A substantial part of the alkaloids fed was degraded to unknown, nonalkaloidal products.

Effects of cyromazine on the mechanical properties of the larval integument ofLucilia cuprina(Diptera: Calliphoridae)

AbstractThe effect of cyromazine on the mechanical properties of the larval integument ofLucilia cuprina(Wiedemann) was measured using third instar larvae 2–3 h after moulting placed on liver containing 1 ppm cyromazine. Cuticle stiffness (Δ force (N)/δ strain) was approximately 20–30% higher in treated larvae (relative to controls) after 6–10 h treatment, after which time stiffness decreased to levels well below controls. Larvae placed on 1 ppm cyromazine early in the second instar also showed a short period of slightly elevated cuticle stiffness (relative to controls) after 3–5 h treatment. Third instar larvae placed on cyromazine-treated diet for 8 h then transferred to normal diet showed the same short period of elevated stiffness (at 6–10 h) followed by rapid decrease in stiffness to levels well below controls and greatly reduced growth as larvae fed cyromazine continually. A slight increase in cuticle stiffness appears to be only an early and temporary effect of cyromazine poisoning inL. cuprina. These results are discussed in relation to the previously documented effects of cyromazine on the mechanical properties of the larval cuticle ofManduca sexta(Linnaeus).

Role of the integument in insect immunity: epicuticular abrasion and induction of cecropin synthesis in cuticular epithelial cells.

When the epicuticle of a silkworm larva, Bombyx mori, was lightly abraded in the presence of live Bacillus licheniformis, Enterobacter cloacae, or bacterial cell wall components, cecropin mRNAs were detected in the underlying epithelial cells and in fat body cells remote from the abraded area. Antibacterial activity due to cecropin was detected in the matrix of the lightly abraded cuticle but not in nonabraded portions of the cuticular matrix or in the hemolymph surrounding the fat body, unless a more severe cuticular abrasion was administered. A light abrasion to a larva of the giant silkworm moth, Hyalophora cecropia, in the presence of E. cloacae also induced antibacterial activity in the abraded cuticle. These data illustrate that the ectodermally derived lepidopteran larval integument, when challenged by live bacteria or their cell wall components, mounts an immune response. Hence, the insect exoskeleton, which is often considered as an inert protective armor, is indeed actively participating in defense.

Cuticular and non-cuticular substrate influence on expression of cuticle-degrading enzymes from conidia of an entomopathogenic fungus,Nomuraea rileyi

Larval cuticle fromTrichoplusia ni, Helicoverpa (=Heliothis)zea, andHeliothis virescens and a cellulose substrate were used to quantify release of proteolytic, chitinolytic, and lipolytic enzymes by germinating conidia of the entomopathogenic fungus,Nomuraea rileyi. There was no significant difference in conidial viability incubated withT. ni, H. zea or cellulose substrates. Conidial viability onH. virescens cuticle, however, was significantly lower (ca. 19–25%) than the other three substrates. The presence of cuticle substrates, especially cuticle ofT. ni, stimulated germination. The nature of the substrate influenced both the time and quantity of the enzymes expressed. Specific proteases (aminopeptidase, chymoelastase, trypsin) generally were expressed earlier and/or in greater quantities on cuticular than on the cellulose substrate. Although both chitinolytic enzymes (endochitinase, N-acetylglucosaminidase) were detected on all three cuticular substrates, their activity was substantially lower than that of the proteolytic enzymes. Lipase activity was only minimally present. Early concurrent release of both proteases and chitinases suggested that both may be important in the penetration of the larval integument by germinating conidia ofN. rileyi. Expression of proteases and chitinases, especially aminopeptidase and endochitinase was probably a specific response to cuticle, because little or no activity was expressed on the non-host, cellulose substrate.

Developmental expression and hormonal regulation of the nuclear 29 kDa juvenile hormone‐binding protein in Manduca sexta larval epidermis

AbstractWe have utilized a high specific activity photoaffinity analog of methoprene, [3H]‐methoprene diazoketone (MDK), to study the developmental expression and hormonal regulation of the 29 kDa juvenile hormone (JH)‐binding protein. The 29 kDa MDK‐binding protein was detected in the epidermis of the feeding 4th and 5th instar larvae but decreased in abundance during the molts to the 5th instar and the pupa. This protein was also detected in day 1 pupal epidermis. Culturing of day 2 5th instar larval integument in the presence of 2 × 10−6 M 20‐hydroxyecdysone (20HE) caused a threefold decrease in the 29 kDa MDK‐binding protein. Presence of 3 × 10−6 M JH I prevented this loss. Allatectomy of molting 4th instar larvae after the critical period for JH for a larval molt resulted in the absence of the 29 kDa MDK‐binding protein in epidermis isolated 25 hr later or from either day 1 5th instar larvae or day 1 pupae. Application of 5 μ of JH I or methoprene at the time of allatectomy and every 12 hr thereafter until dissection was necessary for the presence of this protein in day 1 5th instar larval epidermis. Application of 5 μg of JH I on the day of wandering and the next day caused the appearance of the 29 kDa MDK‐binding protein in the epidermis of day 1 pupae formed by allatectomized larvae.

Alterations of insect epicuticular hydrocarbons during infection with Beauveria bassiana or B. brongniartii

Scanning electron microscopy allowed us to distinguish the successive phases induced on the integument by virulent and nonvirulent strains. By chemical analyses, the epicuticular hydrocarbons alterations were observed on Ostrinia nubilalis and Melolontha melolontha larval integument. More precisely, only within the first 6 hr or so, even before the spore germination, several monomethylalkanes completely disappeared, even if the strain was not able to specifically penetrate in the host. Finally, between 32 and 51% of the cuticular hydrocarbons of M. melolontha and 77 and 86% of those of O. nubilalis might be affected 96 hr after a cuticular application of conidia of Beauveria bassiana or B. brongniartii.

Scanning Electron Microscopy of soft-bodied tephritid larvae

The soft body of dipteran larva has been posing a difficulty for entomologist to go for scanning electron microscopy. Dehydration by the conventional methods results in crumpling of the larval integument thereby distorting the minute details. The cuticle is impermeable to various types of fixatives and its puncturing with needle causes severe distortion of structure due to haemoiymphloss. To avoid this a new method was developed to prepare specimen for scanning electron microscopy. The specimen was kept in doubled is tilled deionized water at 60°C for half an hour before further treatment. It was sonicated in mild detergent (Sodium bicarbonate) for 30 sec. It was again washed with deionized water and specimen was placed in modified super skiper's solution (Grodowitz et al., 1982) and then rinsed twice in Carl's solution, after dehydration through ethanol series, critical point drying was done. Specimen was coated with gold pulludium and photographed by using JEM - 1200 EX (Jeol) electron microscope and Indu Panchromatic 125 ASA black and white film.

Ultrastructure of the anal organ of Musca domestica larvae (Insecta, Diptera) in relation to ion transport

The anal organ of full-grown, third-instar larvae of Musca domestica is situated ventrally in the last segment and consists of two symmetrical epithelial plates with saddle-shaped features, encircling the anus. Structural studies using light and electron microscopy have revealed that in the area of the anal organ the epidermal cells and the overlying cuticle differ markedly from those of the adjacent normal larval integument. The fine structure of the cells in the region of the anal chloride epithelium indicates that these cells have to be regarded as epidermal cells specialized for transport. Considering the electron microscopy results in connection with the histochemical location of chloride exclusively in this epithelial area, it has been suggested that the larval anal organ of Musca domestica is involved in the active transport of ions from the medium to the haemocoel, and that the epithelium is ion-absorbing. The results of the study are compared with what is known about comparable structures and these comparisons are discussed in detail.

Development of the Nervous System of Sea Urchin Embryos: Formation of Ciliary Bands and the Appearance of Two Types of Ectoneural Cells in the Pluteus: (ectoneural cell/sea urchin pluteus/ciliary band/axoneme).

An ultrastructural study of the larval integument of the sea urchin, Hemicentrotus pulcherrimus, was conducted with special emphasis on the development of the nervous system in relation to the formation of ciliary bands. In the integument of 4-armed pluteus larvae, cells associated with the ciliary band, which have 200 nm-thick projections at their apices, and cells in the squamous epithelium, which have a cilium and long, fine radiating processes in the apical region, were observed. Both cell types have axons at their basal ends that form nerve bundles beneath the ciliary bands, where the axons make contact with ectodermal effector cells with motile cilia. The cilia and other apical projections of these ectoneural cells run parallel to the surface of the cells, and are under the hyaline layer. The axoneme of the cilium has a typical "9 + 2" microtubular arrangement, but generally has no dynein arms. These ectoneural cells are more frequent on the oral surface than on the antioral surface.

Cuticle secretion during larval growth in Drosophila melanogaster

The moulting cycle and growth of the larval integument of Drosophila melanogaster has been studied by light and electron microscopy. Growth during the first, second and third larval instars is accompanied by 3.0-, 3.4- and 3.7-fold increases in surface area, respectively. Growth in surface area occurs continuously during the larval stages, with no detectable relationship to the moulting cycle. Measurements of the thickness of the cuticular layers show that the endocuticle grows in thickness by apposition and in surface area by stretching. The pre-apolytic epicuticle remains at fairly constant thickness during the increase in surface area, indicating that it grows by intussusception of new components. Post-apolytic epicuticle becomes thinner and increases in surface area by stretching. The epicuticle and pre-ecdysial endocuticle are traversed by filaments, but these do not penetrate the endocuticle secreted after ecdysis. We suggest that the filaments transport breakdown products from the old cuticle inward to the epidermis for reutilization. The growth and deposition of cuticle in two larval growth mutants, lethal (2) giant larvae and Chubby Tubby, involves mechanisms similar to those found in wild-type larvae, but in Chubby Tubby the endocuticle contains inclusions which are ultrastructurally similar to dense epicuticle.

Chiton integument: Metamorphic changes in Mopalia muscosa (Mollusca, Polyplacophora)

The larval integument and juvenile girdle integument of Mopalia muscosa (Mollusca: Polyplacophora) were studied by light microscopy. Within 24 h of settlement, eight distinctive changes occur that characterize metamorphosis: loss of the functional prototroch and apical tuft, secretion of a cuticle over the mantle field followed by the secretion of calcareous shell plates and the extrusion of spicules into the cuticle, a 20% decrease in length, secretion of chitinous hairs and the incorporation of the lateral ciliated bands into the pallial grooves. Similar changes which were often not recognized as metamorphic have been reported for other species. Evidence for metamorphosis being a common developmental feature of chitons is presented.

In vivo development and pathogenicity of Ascosphaera proliperda (Ascosphaeraceae) to the alfalfa leafcutting bee, Megachile rotundata

First instar larvae of the leafcutting bee, Megachile rotundata, were fed on either artificial or natural provisions containing spores of Ascosphaera proliperda. Two isolates were used as a source of inocula: one originated from in vitro isolates obtained while culturing what was thought to be pure spores of A. aggregata, the second originated from in vitro cultures from Denmark. Histological and scanning electron microscopy studies revealed that the spores germinated in the gut lumen and the developing hyphae invaded all tissues, after which they penetrated through larval integument and began the sexual phase of the life cycle aerially. Virtually all fungus-exposed larvae developed symptoms of disease regardless of source of inoculum, type of provision, and spore dose (1.5 × 10 3 to 3 × 10 6) per insect. It was concluded that the fungus was pathogenic to the alfalfa leafcutting bee under laboratory conditions and future studies should be conducted to determine its etiology, cross infectivity, and natural distribution in other bee taxa.

Juvenile hormone control of larval colouration in the silkworm, Bombyx mori: Characterization and determination of epidermal brown colour induced by the hormone

Larval colouration in the silkworm, Bombyx mori, is under hormonal control. Juvenile hormone injection at certain critical times during the moulting cycle induces a colour change in larval epidermis from greyish white to yellowish brown at ecdysis. This epidermal brown pigment was isolated from larvae injected with the juvenile hormone analogue (JHA) methoprene. Based on redox behaviour, visible and near ultraviolet absorption spectra and R f values after thin layer chromatography in various solvents, the brown pigment was identified as xanthommatin. Epidermal xanthommatin was assayed by the use of high pressure liquid chromatography (HPLC). 3-Hydroxykynurenine, the direct precursor of the pigment, and other epidermal pigments such as sepialumazine and riboflavin were also determined by HPLC. The higher the dose of injected hormone, the greater were the quantities of xanthommatin and 3-hydroxykynurenine found in larval integument. However, the amounts of sepialumazine and riboflavin were little affected by the hormone. These facts indicate that JHA acts on tryptophan metabolism and that xanthommatin is yet another pigment which causes characteristic colouration of silkworm larval integument.