Adult Integument Research Articles

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum.

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupalnotum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue's dorsal or ventral side.

Dissecting, Fixing, and Visualizing the <em>Drosophila</em> Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue's dorsal or ventral side.

Identification, in silico characterization, and expression analysis of Tenebrio molitor Cecropin‐2

AbstractAntimicrobial peptides (AMPs) are considered to be candidate effectors for eliciting humoral immune responses against infectious pathogens in the host. Cecropins are α‐helical peptides of 30–40 amino acids, which are known to permeabilize bacterial membranes, and play authoritative roles in the innate immune system of insects. In the present study, we identified the full‐length open reading frame (ORF) encoding the Tenebrio molitor cecropin‐2 (TmCec2) gene using the Tribolium castaneum cecropin‐2 (TcCec2) gene to query a T. molitor Rnaseq database. Phylogenetic analysis revealed close identity of TmCec2 with TcCec2. TmCec2 was ubiquitously expressed in the insect during all developmental stages, with the highest expression observed in the adult. Tissue‐specific TmCec2 expression was highest in larval hemocytes and in the adult integument and hemocytes. Microbial challenge experiments revealed that TmCec2 was highly induced in response to gram‐positive and gram‐negative bacteria, and fungi. These data provide credible evidence for a putative role of TmCec2 in insect innate immunity against a plethora of pathogens.

Genomic organization, sequence characterization and expression analysis of Tenebrio molitor apolipophorin-III in response to an intracellular pathogen, Listeria monocytogenes

Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5′-flanking region. BLAST and phylogenetic analyses reveal that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic alpha helices, including a short helix 3′. The ‘helix–short helix–helix’ motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly upregulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor.

Characterization of a Chitin Synthase Encoding Gene and Effect of Diflubenzuron in Soybean Aphid, Aphis Glycines

Chitin synthases are critical enzymes for synthesis of chitin and thus for subsequent growth and development in insects. We identified the cDNA of chitin synthase gene (CHS) in Aphis glycines, the soybean aphid, which is a serious pest of soybean. The full-length cDNA of CHS in A. glycines (AyCHS) was 5802 bp long with an open reading frame of 4704 bp that encoded for a 1567 amino acid residues protein. The predicted AyCHS protein had a molecular mass of 180.05 kDa and its amino acid sequence contained all the signature motifs (EDR, QRRRW and TWGTR) of chitin synthases. The quantitative real-time PCR (qPCR) analysis revealed that AyCHS was expressed in all major tissues (gut, fat body and integument); however, it had the highest expression in integument (~3.5 fold compared to gut). Interestingly, the expression of AyCHS in developing embryos was nearly 7 fold higher compared to adult integument, which probably is a reflection of embryonic molts in hemimetabolus insects. Expression analysis in different developmental stages of A. glycines revealed a consistent AyCHS expression in all stages. Further, through leaf dip bioassay, we tested the effect of diflubenzuron (DFB, Dimilin ®), a chitin-synthesis inhibitor, on A. glycines' survival, fecundity and body weight. When fed with soybean leaves previously dipped in 50 ppm DFB solution, A. glycines nymphs suffered significantly higher mortality compared to control. A. glycines nymphs feeding on diflubenzuron treated leaves showed a slightly enhanced expression (1.67 fold) of AyCHS compared to nymphs on untreated leaves. We discussed the potential applications of the current study to develop novel management strategies using chitin-synthesis inhibitors and using RNAi by knocking down AyCHS expression.

A cuticle protein gene in the honeybee: Expression during development and in relation to the ecdysteroid titer

A cDNA encoding a cuticle protein containing the R&R Consensus was characterized in the honeybee integument. AmelCPR14 developmental expression is distinguished by an on–off–on pattern, the transition from a low to a high level of transcripts occurring as the ecdysteroid titer is declining after the peak that triggers the onset of pharate (pupal and adult) development. The transcript is abundant during cuticle tanning and sclerotization, and persists even in the adult integument, suggesting that the corresponding protein is required for differentiation and maintenance of the adult cuticle. Such developmental pattern suggested that AmelCPR14 gene might be regulated by the titer of ecdysteroids. We confirmed this hypothesis using different experimental strategies. By tying a ligature in early pupae to prevent exposure of abdominal integument to a high ecdysteroid titer, we delayed the accumulation of AmelCPR14 transcripts in the abdominal integument. This is consistent with ecdysteroid priming being required in pupae for the increase in AmelCPR14 expression in pharate adults. By injecting 20-hydroxyecdysone (20E) in early pupae we demonstrated that hormone titer decay after the peak is critical for AmelCPR14 expression induction. Exposure of pupal integument in vitro to a 20E concentration mimicking the pupal ecdysteroid peak repressed AmelCPR14 expression, which was recovered by hormone removal. Taken together, these data are consistent with an ecdysteroid pulse (increase in hormone titer followed by its decline) being critical for a high AmelCPR14 gene expression in pharate adults.

Serological diagnosis of canine alveolar echinococcosis

Canine alveolar echinococcosis (AE) is a rare liver disease caused by the larval stage (metacestode) of E. multilocularis. Its diagnosis so far is rather difficult and invasive. Imaging techniques for the diagnosis have been described recently and in this study, the serological diagnosis of canine AE was evaluated in 30 dogs with confirmed AE using ELISA based on seven different antigens ( E. multilocularis-antigens: Em2G11, EmII/3–10, protoscolex (EmP), excretory/secretory and adult integument (EmAd/I); E. granulosus antigens: hydatid fluid (EgHF) and antigen B (EgAgB)) and by the detection of circulating Em2G11-antigen. EmII/3–10 and Em2G11-based tests detected AE in 50 and 53%. The combined serological investigation for specific circulating Em2G11-antigen and specific antibodies against this antigen showed a sensitivity of 77%. This strategy is useful in animals with large parasite masses and ascites. The highest sensitivities for AE were obtained by the EmAd/I and the EmP antigens (97 and 93% sensitivity) with high specificities in the control group of 76 dogs (100 and 98.7%, respectively). Heterologous E. granulosus antigens (EgHF and EgAgB) showed low sensitivities (43 and 50%, respectively) with high numbers of unspecific reactions (>16%) in the control group. In the follow-up of four dogs, the serological reaction patterns against the antigens Em2G11, EmP and EmAd/I showed some correlations with the development of the parasite mass. Intestinal infections cannot be differentiated from AE by most antigens (except EmII/3–10) and have to be excluded by the absence of coproantigens or DNA of E. multilocularis in faecal samples.

Biochemical detection of esterases in the adult female integument of organophosphate-resistant Boophilus microplus (Acari: Ixodidae).

Esterase activity was present in the integument of adult female Boophilus microplus (Canestrini) ticks that are resistant to organophosphates (OP). Three esterases were purified from adult integument, which hydrolyze the substrates p-nitrophenylacetate and beta-naphthyl acetate after comparison of OP-resistant strain and an OP-susceptible strains. The esterases purified by ion-exchange chromatography were characterized using different esterase inhibitors; eserine sulfate, diethyl p-nitrophenyl phosphate (paraoxon), para-hydroxyl-mercuribenzoate (pHMB), and diisopropylphosphofluoridate (DFP). All of the esterases had a molecular mass of 64 Kd (PAGE), but were characterized based on the esterase inhibitor effects as a B-esterase with beta-naphthyl acetate affinity, a carboxylesterase with beta-naphthyl acetate and p-nitrophenyl acetate affinity, and one A-Esterase (nonspecific esterase) with p-nitrophenyl acetate affinity. The described esterases are an important detoxification mechanism in B. microplus ticks at the integument. We describe also a microplate biochemical assay for the detection of esterase activity in the tick integument, potentially a useful tool to detect esterase-mediated OP resistance in B. microplus ticks.

Localization of a cuticular protein during the postembryonal development of Manduca sexta.

The pattern of cuticle protein synthesis by the epidermis of insects changes during the last larval, pupal and adult development, leading to an alteration in cuticular stucture and feature. We have isolated a protein that had an apparent molecular mass of 33.1 kD from larval cuticle of Manduca sexta. Synthesis, transport and accumulation of MsCP33.1 were followed during metamorphosis by immunoblots and immunocytochemical methods using the antibody developed against this protein. Our data prove that the presence of MsCP33.1 in the larval cuticle is general while its appearance in the pupal or adult integument is restricted only in the cuticle of wings and apodemes. We established that the synthesis of 33.1 kD protein is negatively regulated by moulting hormone (20-hydroxyecdysone). Possible roles for this cuticular protein are discussed.

Catabolism of isoxanthopterin during the development of the silkworm, Bombyx mori

7-Oxylumazine (deaminated isoxanthopterin) was discovered in the silkworm, Bombyx mori. Isoxanthopterin deaminase, the enzyme responsible for the synthesis of 7-oxylumazine from isoxanthopterin, was most active in larvae and adults while absent from the egg and pupal stages. A correlation was found between the occurrence of the enzyme and the localization of 7-oxylumazine in larval and adult integument. 7-Oxylumazine is synthesized from isoxanthopterin and stored in situ in larval and adult integument, whereas 7-oxylumazine in the egg and pupal tissues is a transported and stored form arising from isoxanthopterin metabolized in the larval and adult stages.

The emission and form of spermatophores and the fine structure of adultEriophyes sheldoniEwing (Acarina, Eriophyoidea)

AbstractThe emission and form of the spermatophores ofEriophyes sheldoniEwing are described and illustrated. Glued to the substratum by a drop-like pad which varies in shape, they consist of a stalk with a basal stiff section and a distal flexible part ending in a strongly convex lens-like head, in which the sperm mass is kept. The fine structures of the adult integument, characteristic of this species, are described and illustrated.

Developmental capacities of benign and malignant neoplasms ofDrosophila.

The developmental properties of three neoplasms found in lethal mutants ofDrosophila melanogaster were studied and compared with the development of the corresponding wild-type organs. Two of these neoplasms are found in the late larval lethal mutant,lethal (2) giant larvae 4 (l(2)gl 4) and its allele,lethal (2) giant larvae (l(2)gl). The third neoplasm occurs in the hemizygous lethal male embryos of the mutantNotch 8 (Df(1)N 8).The mutantsl(2)gl 4 andl(2)gl were shown to possess defects involving imaginal primordia of ectodermal origin, such as the imaginal discs of the adult integument and the imaginal optic primordia in the larval brain. They also have enlarged lymph glands and abnormal gonads, salivary and ring glands. Thel(2)gl 4 andl(2)gl mutations transform the imaginal discs into noninvasive, lethal neoplasms and the imaginal optic primordia in the larval brain into an invasive and lethal neuroblastoma. Both neoplasms were serially subcultured in female adult hosts where they grew rapidly and killed their hosts in 7-14 days. The neoplastic development of thel(2)gl 4 imaginal discs and brain proved autonomous at all stages that were investigated from 10 hour old embryos to mature larvae. When exposed to the hormonal conditions of metamorphosis, the neoplastic tissues,in situ as well as those that had been culturedin vivo, ceased to grow but failed to metamorphose into parts of the adult integument or brain. Furthermore, in almost all cases they failed to resume their growth in the adult fly after metamorphosis.Thel(2)gl 4 brain neuroblastoma changed after prolonged subculturein vivo so that although the cells stopped dividing temporarily at the time of metamorphosis of the host, they resumed their growth shortly thereafter and continued to divide throughout adult development and in the emerged adult. Many of thel(2)gl 4 neuroblastoma cells showed abnormal karyotypes, shapes and sizes after prolonged culturein vivo.Thel(2)gl 4 imaginal disc tissue resembles, in both structure and behavior, certain atelotypic tissue sublines which arise from wild-type imaginal discs after prolonged culturein vivo: the ultrastructure of both types of cells are similar and both cease to grow when exposed to hormonal conditions of metamorphosis. Apparently the epigenetic processes which transform wild-type imaginal discs afterin vivo subculture into atelotypic neoplasms have the same phenotypic expression as the genetic processes at work inl(2)gl 4 imaginal discs.An analysis of several other late larval and larval-pupal mutants with defective imaginal discs, such asl(2)gd andl(1)d.lg.-1 revealed that they also had defects in parts of the brain destined to form adult structures. This observation indicates that mutations that affect imaginal discs of the adult integument also affect the imaginal primordia of the adult brain.The neoplasm in the hemizygous male embryo of the embryonic lethalNotch 8 is a teratoma-like growth which is lethal and invasive; when implanted into female adult hosts it gives rise to a mass of tissue which consists of most cell types present in the larval nervous system and a number of other unidentified cell types. It too can be subculturedin vivo for many transfer generations.The relative infrequency of neoplasms in insects is analyzed and an attempt is made to explain this infrequency and the occurrence of neoplasms in specific tissues of insects at specific developmental stages. Genetic and epigenetic neoplasms ofDrosophila are compared with neoplasms of other insects and of vertebrates and it is concluded that both genetic and epigenetic neoplasms ofDrosophila share the essential features of vertebrate neoplasms.

Developmental genetics of Drosophila imaginal discs.

The aim of developmental biology is to discover how the genetic instructions for development are implemented by the epigenetic processes of development. In recent years Drosophila has been increasingly used to answer many questions concerned with development, and the imaginal discs have received special attention. These small nests of cells are set aside early in development for the formation of the adult integument. The entire surface of the adult fly is formed from imaginal discs so that any mutation that affects the cuticle of an adult fly directly or indirectly alters the development of imaginal discs. Many such mutations have been detected, described, and analyzed(180). This review exam­ ines the genetic basis of the development of the imaginal discs of Drosophila. Other aspects of Drosophila developmental genetics such as chromosome puffing (6), behavioral genetics (16a), genetics of embryogenesis (48, 351), spermatoge­ nesis (131, 156), oogenesis (159), and biochemical genetics (64, 307) have recently been reviewed.

Intercellular adhesivity and pupal morphogenesis inDrosophila melanogaster.

Leg and wing imaginal discs of mature larvae ofDrosophila melanogaster when treated with 0.1% trypsin for 5-10 min underwent a change in shape that closely resembled normal pupal morphogenesis. Simultaneously, the cells of the disc epithelium changed in shape from tall columnar to cuboidal. Colcemid eliminated microtubules but was without effect on the shape of the imaginal discs or their cells. Tryptic digestion reduced non-junctional intercellular adhesivity but septate desmosomes and gap junctions remained intact.It is proposed that the structure of imaginal discs permits the packaging of the anlagen of the adult integument so that they can change shape and replace the larval structures in a brief period. Apparently most of the definitive form of the pupal leg is built into the disc and becomes visible within a few minutes as intercellular adhesivity is changed.

The skin and hair of the Southern Elephant Seal Mirounga leonina (L.) III. morphology of the adult integument

The histological structure and certain histochemical features of the skin, hair follicles, and associated glands have been studied in 57 male and 49 female elephant seals. The epidermis comprises only three layers, stratum germinativum, stratum spinosum, and stratum corneum. The absence of granular and clear layers leads to a parakeratotic condition of the horny layer which is made up of large cornified sheets joined to lateral strands of the pelage hairs. Although pigment abounds in the interfollicular epidermis, it does not extend into the external root sheath cells of the hair follicles. There are no arrector pili muscles attached to the pelage hair follicles which are simple, unbranched, and contain only one fully developed lanceolate hair. Hairs are firmly anchored in their follicles by means of lateral filaments insinuated between external root sheath cells. A single, small, apocrine-type sweat gland opens into the hair canal below the common duct of the large, bilobed, sebaceous gland on the ental side of the follicle. The lipid-rich sebum is secreted within the lamellae of the stratum corneum and its function appears to be the maintenance of the outer horny layers in a pliable and perhaps water-resistant condition. The walls of the sebaceous duct are replete with glycogen. The dermis comprises a network of collagenous fibres and a decreasing number of elastic fibres in aging seals, as well as histiocytes and dermal fat cells. Problems of thermoregulation arising from loss of hair in seals exemplified by M. leonina and compensatory features apparent in the morphology of the skin and hair are discussed.

The hormonal control of the development of hairs and bristles in the milkweed bug, Oncopeltus fasciatus, Dall.

ABSTRACT As in Rhodnius, the larval Oncopeltus has bristles which are supplemented at each moult. However, at metamorphosis a dense population of non-innervated hairs develops. Implantation of corpora allata into 5th-stage larvae showed that the development of these hairs can be inhibited universally or locally by the juvenile hormone (JH). Transplantations of integument between 5th-stage larvae of different stages in the moult cycle gave some information about the power of the host to synchronize the graft to its own moult cycle. Transplantations between different larval stages showed that the grafted integument responded to the hormonal milieu of the host. Adult integument was transplanted onto larvae to study the reversal of metamorphosis. It was found that the development of a supernumerary population of hairs depended on the integument passing through a moult cycle in the presence of JH. After two moults in the presence of JH, reversal of metamorphosis was found to vary over the surface of the transplant, being further advanced at the margin. At the edge of the graft properly formed larval bristles developed, while at the centre adult hairs were formed in adult cuticle. Intermediately formed bristles were found in the intervening areas. It is suggested that reactions associated with wounding are the cause of this heterogeneous result. The significance of these results in relation to other work and to theories concerning the mode of action of the juvenile hormone is discussed.