Glue Secretion Research Articles

Gross morphology and adhesion-associated physical properties of Drosophila larval salivary gland glue secretion

One of the major functions of the larval salivary glands (SGs) of many Drosophila species is to produce a massive secretion during puparium formation. This so-called proteinaceous glue is exocytosed into the centrally located lumen, and subsequently expectorated, serving as an adhesive to attach the puparial case to a solid substrate during metamorphosis. Although this was first described almost 70 years ago, a detailed description of the morphology and mechanical properties of the glue is largely missing. Its main known physical property is that it is released as a watery liquid that quickly hardens into a solid cement. Here, we provide a detailed morphological and topological analysis of the solidified glue. We demonstrated that it forms a distinctive enamel-like plaque that is composed of a central fingerprint surrounded by a cascade of laterally layered terraces. The solidifying glue rapidly produces crystals of KCl on these alluvial-like terraces. Since the properties of the glue affect the adhesion of the puparium to its substrate, and so can influence the success of metamorphosis, we evaluated over 80 different materials for their ability to adhere to the glue to determine which properties favor strong adhesion. We found that the alkaline Sgs-glue adheres strongly to wettable and positively charged surfaces but not to neutral or negatively charged and hydrophobic surfaces. Puparia formed on unfavored materials can be removed easily without leaving fingerprints or cascading terraces. For successful adhesion of the Sgs-glue, the material surface must display a specific type of triboelectric charge. Interestingly, the expectorated glue can move upwards against gravity on the surface of freshly formed puparia via specific, unique and novel anatomical structures present in the puparial’s lateral abdominal segments that we have named bidentia.

Apocrine secretion in the salivary glands of Drosophilidae and other dipterans is evolutionarily conserved.

Apocrine secretion is a transport and secretory mechanism that remains only partially characterized, even though it is evolutionarily conserved among all metazoans, including humans. The excellent genetic model organism Drosophila melanogaster holds promise for elucidating the molecular mechanisms regulating this fundamental metazoan process. Two prerequisites for such investigations are to clearly define an experimental system to investigate apocrine secretion and to understand the evolutionarily and functional contexts in which apocrine secretion arose in that system. To this end, we recently demonstrated that, in D. melanogaster, the prepupal salivary glands utilize apocrine secretion prior to pupation to deliver innate immune and defense components to the exuvial fluid that lies between the metamorphosing pupae and its chitinous case. This finding provided a unique opportunity to appraise how this novel non-canonical and non-vesicular transport and secretory mechanism is employed in different developmental and evolutionary contexts. Here we demonstrate that this apocrine secretion, which is mechanistically and temporarily separated from the exocytotic mechanism used to produce the massive salivary glue secretion (Sgs), is shared across Drosophilidae and two unrelated dipteran species. Screening more than 30 species of Drosophila from divergent habitats across the globe revealed that apocrine secretion is a widespread and evolutionarily conserved cellular mechanism used to produce exuvial fluid. Species with longer larval and prepupal development than D. melanogaster activate apocrine secretion later, while smaller and more rapidly developing species activate it earlier. In some species, apocrine secretion occurs after the secretory material is first concentrated in cytoplasmic structures of unknown origin that we name "collectors." Strikingly, in contrast to the widespread use of apocrine secretion to provide exuvial fluid, not all species use exocytosis to produce the viscid salivary glue secretion that is seen in D. melanogaster. Thus, apocrine secretion is the conserved mechanism used to realize the major function of the salivary gland in fruitflies and related species: it produces the pupal exuvial fluid that provides an active defense against microbial invasion during pupal metamorphosis.

Characterisation of a skin secretion with adhesive properties in the ground frog Eupsophus vertebralis (Alsodidae)

Some skin secretions with adhesive properties allow frogs to distract predators and escape; their nature is poorly studied. Here, we report the sticky skin secretion released by the Patagonian frog Eupsophus vertebralis when stressed. This secretion contained ~ 50% proteins spanning 25–250 kDa and required a fast setting time to turn into strong adhesive, which worked well on synthetic and biological materials. Lap-shear assays with Eupsophus glue secretion showed average shear strength of 3.34 MPa, comparable to cyanoacrylate (5.47 MPa). These properties suggest its biotechnological value for practical applications in industrial and medical sectors.

Fine infrastructure of released and solidified Drosophila larval salivary secretory glue using SEM

The Golgi-derived large secretory granules of Drosophila salivary glands (SGs) constitute the components of the salivary glue secretion (Sgs). The Sgs represents a highly special and unique extracellular composite glue matrix that has not yet been identified outside of Cyclorrhaphous Dipterans. For over half a century, the only major and unambiguously documented function of the larval salivary glands was to produce a large amount of mucinous glue-containing secretory granules that, when released during pupariation, serves to affix the freshly formed puparia to a substrate. Besides initial biochemical characterization of the Sgs proteins and cloning of their corresponding Sgs genes, very little is known about other properties and functions of the Sgs glue. We report here observations on the fine SEM-ultrastructure of the Sgs glue released into to the lumen of SGs, and after it has been expectorated and solidified into the external environment. Surprisingly, in contrast to long held expectations, it appears to be a highly structured bioadhesive mass with an internal spongious to trabecular infrastructure, reflecting the state of its hydratation. We also found that in addition to its cementing properties, it is highly efficient at glueing and trapping microorganisms, and thus may serve a potentially very important immune and defense role. High hydration capacity, the speed by which this glue can dry, uniqueness of its protein composition and spongious infrastructure can provide inspiration for development of potential biomimetics that can attach completely different or incompatible surfaces with high efficiency and strength.

Localization of Phosphoproteins within the Barnacle Adhesive Interface.

Barnacles permanently adhere to nearly any inert substrate using proteinaceous glue. The glue consists of at least ten major proteins, some of which have been isolated and sequenced. Questions still remain about the chemical mechanisms involved in adhesion and the potential of the glue to serve as a platform for mineralization of the calcified base plate. We tested the hypothesis that barnacle glue contains phosphoproteins, which have the potential to play a role in both adhesion and mineralization. Using a combination of phosphoprotein-specific gel staining and Western blotting with anti-phosphoserine antibody, we identified multiple phosphorylated proteins in uncured glue secretions from the barnacle Amphibalanus amphitrite The protein composition of the glue and the quantity and abundance of phosphoproteins varied distinctly among individual barnacles, possibly due to cyclical changes in the glue secretion over time. We assessed the location of the phosphoproteins within the barnacle glue layer using decalcified barnacle base plates and residual glue deposited by reattached barnacles. Phosphoproteins were found throughout the organic matrix of the base plate and within the residual glue. Staining within the residual glue appeared most intensely in regions where capillary glue ducts, which are involved in cyclical release of glue, had been laid down. Lastly, mineralization studies of glue proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that proteins identified as phosphorylated possibly induce mineralization of calcium carbonate (CaCO3). These results contribute to our understanding of the protein composition of barnacle glue, and provide new insights into the potential roles of phosphoproteins in underwater bioadhesives.

Vacuole dynamics in the salivary glands of Drosophila melanogaster during prepupal development.

A central function of the Drosophila salivary glands (SGs), historically known for their polytene chromosomes, is to produce and then release during pupariation the secretory glue used to affix a newly formed puparium to a substrate. This essential event in the life history of Drosophila is regulated by the steroid hormone ecdysone in the late-larval period. Ecdysone triggers a cascade of sequential gene activation that leads to glue secretion and initiates the developmentally-regulated programmed cell death (PCD) of the larval salivary glands, which culminates 16 h after puparium formation (APF). We demonstrate here that, even after the larval salivary glands have completed what is perceived to be one of their major biological functions--glue secretion during pupariation--they remain dynamic and physiologically active up until the execution phase of PCD. We have used specific metabolic inhibitors and genetic tools, including mutations or transgenes for shi, Rab5, Rab11, vha55, vha68-2, vha36-1, syx1A, syx4, and Vps35 to characterize the dramatic series of cellular changes occurring in the SG cells between pupariation and 7-8 h APF. Early in the prepupal period, they are remarkably active in endocytosis, forming acidic vacuoles. Midway through the prepupal period, there is abundant late endosomal trafficking and vacuole growth, which is followed later by vacuole neutralization and disappearance via membrane consolidation. This work provides new insights into the function of Drosophila SGs during the early- to mid-prepupal period.

Robust cross-links in molluscan adhesive gels: Testing for contributions from hydrophobic and electrostatic interactions

The cross-linking interactions that provide cohesive strength to molluscan adhesive gels were investigated. Metal-based interactions have been shown to play an important role in the glue of the slug Arion subfuscus (Draparnaud), but other types of interactions may also contribute to the glue's strength and their role has not been investigated. This study shows that treatments that normally disrupt hydrophobic or electrostatic interactions have little to no effect on the slug glue. High salt concentrations and non-ionic detergent do not affect the solubility of the proteins in the glue or the ability of the glue proteins to stiffen gels. In contrast, metal chelation markedly disrupts the gel. Experiments with gel filtration chromatography identify a 40 kDa protein that is a central component of the cross-links in the glue. This 40 kDa protein forms robust macromolecular aggregations that are stable even in the presence of high concentrations of salt, non-ionic detergent, urea or metal chelators. Metal chelation during glue secretion, however, may block some of these cross-links. Such robust, non-specific interactions in an aqueous environment are highly unusual for hydrogels and reflect an intriguing cross-linking mechanism.

Localized Adhesion of Nongerminated Venturia inaequalis Conidia to Leaves and Artificial Surfaces

Adhesion to the host surface is the first step for successful plant pathogen development and has been reported to be associated with both passive and active processes. For conidia of Venturia inaequalis, which depend on leaf wetness for germination, this process has not yet been described. Conidia of V. inaequalis adhered to wet hydrophobic surfaces immediately after contact to the surface, hours before initiation of germination. Attachment of nongerminated conidia was much better on hydrophobic surfaces, such as apple leaves and polystyrene, than on hydrophilic glass. Conidia released adhesive material localized in a droplet named spore tip glue (STG) at the spore apex which interacted with a contact surface only when water was present. Histochemical investigations indicated the presence of proteins and carbohydrates in STG, lectin labeling the presence of beta-galactose and N-acetylglucosaminyl residues. Transmission electron microscopy revealed two phases in the STG at the tip of dry mature conidia; as STG was present on the outer side of the intact fungal cell wall its formation should be associated with the secretion of glue through pores of the conidial wall. Surface-active substances affected the adhesion of conidia to hydrophobic surfaces stressing the importance of hydrophobic interactions. The use of protein biosynthesis inhibitors did not affect adhesion of conidia indicating that the adhesive material was preformed. It is concluded that the coincidence of STG, contact to a hydrophobic surface, and free water are essential for the adhesion of V. inaequalis conidia.

Ecdysone-regulated puff genes 2000

The Ashburner model for the hormonal control of polytene chromosome puffing has provided a strong foundation for understanding the basic mechanisms of steroid-regulated gene expression (Cold Spring Harbor Symp. Quant. Biol. 38 (1974) 655). According to this model, the steroid hormone 20-hydroxyecdysone (referred here as ecdysone) directly induces the expression of a small set of early regulatory genes. These genes, in turn, induce a much larger set of late target genes that play a more direct role in controlling the biological responses to the hormone. The recent characterization of two early puff genes, E63-1 and E23, and three late puff genes, D-spinophilin, L63, and L82, provide further confirmation of the Ashburner model. In addition, these studies provide exciting new directions for our understanding of ecdysone signaling. Overexpression studies of E63-1 implicate this gene in directing calcium-dependent salivary gland glue secretion. In contrast, overexpression of E23 indicates that this ABC transporter family member may negatively regulate ecdysone signaling by actively transporting the hormone out of target cells. Finally, genetic studies of the L63 and L82 late genes reveal unexpected possible functions for ecdysone in controlling developmental timing and growth. This review surveys the recent characterization of these ecdysone-inducible genes and provides an overview of how they expand our understanding of ecdysone functions during development.

Glue Secretion in the Drosophila Salivary Gland: A Model for Steroid-Regulated Exocytosis

Small hydrophobic hormones like steroids control many tissue-specific physiological responses in higher organisms. Hormone response is characterized by changes in gene expression, but the molecular details connecting target-gene transcription to the physiology of responding cells remain elusive. The salivary glands of Drosophila provide an ideal model system to investigate gaps in our knowledge, because exposure to the steroid 20-hydroxyecdysone (20E) leads to a robust regulated secretion of glue granules after a stereotypical pattern of puffs (activated 20E-regulated genes) forms on the polytene chromosomes. Here, we describe a convenient bioassay for glue secretion and use it to analyze mutants in components of the puffing hierarchy. We show that 20E mediates secretion through the EcR/USP receptor, and two early-gene products, the rbp+ function ofBR-C and the Ca2+-binding protein E63-1, are involved. Furthermore, we demonstrate that 20E treatment of salivary glands leads to Ca2+ elevations by a genomic mechanism and that elevated Ca2+ levels are required for ectopically produced E63-1 to drive secretion. The results presented establish a connection between 20E exposure and changes in Ca2+ levels that are mediated by Ca2+-effector proteins, and thus establish a mechanistic framework for future studies.

SNAP-24, a Drosophila SNAP-25 homologue on granule membranes, is a putative mediator of secretion and granule-granule fusion in salivary glands.

Fusion of vesicles with target membranes is dependent on the interaction of target (t) and vesicle (v) SNARE (soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor) proteins located on opposing membranes. For fusion at the plasma membrane, the t-SNARE SNAP-25 is essential. In Drosophila, the only known SNAP-25 isoform is specific to neuronal axons and synapses and additional t-SNAREs must exist that mediate both non-synaptic fusion in neurons and constitutive and regulated fusion in other cells. Here we report the identification and characterization of SNAP-24, a closely related Drosophila SNAP-25 homologue, that is expressed throughout development. The spatial distribution of SNAP-24 in the nervous system is punctate and, unlike SNAP-25, is not concentrated in synaptic regions. In vitro studies, however, show that SNAP-24 can form core complexes with syntaxin and both synaptic and non-synaptic v-SNAREs. High levels of SNAP-24 are found in larval salivary glands, where SNAP-24 localizes mainly to granule membranes rather than the plasma membrane. During glue secretion, the massive exocytotic event of these glands, SNAP-24 containing granules fuse with one another and the apical membrane, suggesting that glue secretion utilizes compound exocytosis and that SNAP-24 mediates secretion.

Effect of bisacylhydrazine ecdysteroid mimics (RH-5849 and RH-5992) on chromosomal puffing, imaginal disc proliferation and pupariation in larvae of Drosophila melanogaster

Two bisacylhydrazine insecticides with ecdysone-mimetic action, RH-5849 and RH-5992, have been subjected to several bioassay procedures that are prerequisites for ecdysone action in Drosophila larvae: (a) induction of early ecdysone-specific puffs on the polytene chromosomes of the larval salivary glands; (b) secretion of glycoprotein glue into the lumen of the salivary glands; (c) evagination of imaginal discs of adult wings and legs; and (d) partial rescue of wild-type phenotypic expression in ecdysone-deficient mutants ( ecdysoneless 1 ( ecd 1 ) and suppressor of forked ts67g ( su(f) ts67g ). In all these bioassays on Drosophila larvae, the two purely synthetic hydrazines exhibited similar dose–response relationships as did the natural steroid hormone, 20-hydroxyecdysone. In assays involving induction of early chromosomal puffs (74EF, 75B) or regression of the preexisting puffs (25AC, 68C), the dosages required for induction of standard ED-50 effects were one order of magnitude larger for the hydrazines in comparison with 20-hydroxyecdysone. In the assays related to glycoprotein glue secretion, evagination of imaginal discs, or rescue of phenotypic expression in ecdysone-deficient mutants, 20-hydroxyecdysone was two orders of magnitude more active than RH-5849 and RH-5992. We conclude that, in spite of these quantitative differences, the two hydrazine compounds studied are able to duplicate in Drosophila larvae the complex of qualitative biological effects that are a prerequisite for ecdysteroid hormones. The hormonomimetic stimulus of RH-compounds has been given at very low, intracellular, chromosomal level.

Glue Secretion and Adhesion by Larvae of Sailfin Shiner (Pteronotropis hypselopterus)

Pteronotropis hypselopterus (sailfin shiner) larvae attached to submerged objects with an elastic glue secreted from two pores located on their heads. The cement pores were located ventrally in hatchlings and migrated in an anterior and dorsal direction through a subterminal to a terminal position as larvae grew. Larvae were adhesive and sedentary during the developmental interval from hatching until swimup. Pteronotropis hypselopterus offspring were supplied solely by an endogenous energy source (yolk) throughout this entire developmental interval. During this interval, concurrently, yolk was depleted; the yolksac became more stream lined; and gill structures, mouth parts, lining of the gut, and posterior swimbladder all formed. Development of these features while larvae were adhesive and the subsequent inflation of the posterior swimbladder and movement into the water column were precursors to initial feeding and transition to a solely exogenous energy source. A brief period of overlap between endogenous and exogenous nourishment frequently occurred after swim-up. Adhesion and the sedentary behavior may help the young, poorly developed larvae to maintain their position in flowing water, while conserving endogenous energy stores for growth and development. Benefits may also include avoidance of predators and anoxic sediments.

Ultrastructural changes of Drosophila larval and prepupal salivary glands cultured in vitro with ecdysone.

Alterations in the ultrastructure of in vitro cultured larval salivary glands of Drosophila melanogaster in response to the steroid hormone ecdysone were studied in relation to complex changes in puffing patterns. We found that the changes in the fine structure of cultured glands reflected progression of the puffing pattern, and they paralleled those seen in vivo. We observed that glue secretion by exocytosis, the main function of salivary glands, took place between puff stage 5 (PS5) and PS7. Glue could not be expectorated under culture conditions but was slowly released from the lumen through a duct into the medium. After the cultured glands reached PS13/PS14, further progress of puffing and fine structural alterations required that the ecdysteroid titer be transiently extremely low or absent. Under in vitro conditions we did not observe the putative new secretory program(s) described for glands in vivo after PS12. However, ultrastructural changes which unambiguously indicated that an autohistolytic process had begun in vitro started to appear after PS17. Many salivary gland cells developed numerous features of progressive self-degradation between PS18 and PS21. Actual degradation of salivary glands in vivo seemed to be rapid, but in vitro degradation was never completed, probably due to a lack of exogenous factors from the hemolymph. Manipulations of ecdysone titer in vitro in the culture medium, known during the larval puffing cycle to cause premature induction of developmentally specific puffing patterns, did not affect the normal development of ultrastructural features of the cytoplasm and nucleus.

Adhesion and reattachment of compound ascidians to various substrata: weak glue can prevent tissue damage.

Sessile, soft-bodied, compound ascidians are successful competitors for substrata in crowded benthic and epibiotic marine communities and can be effective colonists of new sites, through adult rafting and reattachment. Adhesion to the substratum is essential for these ecologically important functions; we therefore studied the material properties of colony attachment to various substrata in the rafting ascidians Botrylloides sp. We found that, compared with the strength of the colony tissues, the glue attaching Botrylloides sp. to the substratum is very weak. This relative weakness may protect the soft-bodied colonies from damage if they are ripped from their host. For sessile animals, such a weak-glue 'strategy' is only effective if the animals can later reattach to a substratum. By detaching Botrylloides sp. colonies from host eelgrass blades and allowing them to reattach, before measuring peel strengths, we learned that the initial reattachment of a colony depends upon rapid new growth of the colony rather than on fresh secretion of glue beneath old zooids. We also found that the propagation peel force necessary to remove Botrylloides sp. from different substrata (e.g. mussel shells, barnacle basal plates or eelgrass blades) depends upon the surface texture of the host. Thus, the overall tenacity of a colony is affected by the types of substrata that it overgrows.

Feeding behaviour and prey choice in Macroperipatus torquatus (Onychophora)

Macroperipatus torquatus feeds nocturnally on crickets and a few other invertebrates on the floor of the Trinidadian rain forest. Prey are inspected by gentle application of the antennae and, if suitable, are captured by entangling them in proteinaceous glue squirted from the oral papillae. Entangled prey are bitten through an arthrodial membrane and immobilized by injected saliva, which may also partly digest the flesh. Ingestion of the flesh takes several hours, comprising some 90% of total handling time, and normally only one prey item is eaten per night. The deplected carcass is discarded. Fully charged glue reserves amount to about 11% of body mass and after exhaustion are replenished in about 24 days. The quantity of glue used in an attack increases up to about 80% of reserve capacity for larger prey. Glue adhering to the prey is ingested, but some attached to the substratum is always lost. Squirting glue may therefore be costly for two reasons. Firstly, depleted glue reserves render peripatus less capable of attacking further prey or of defending themselves; secondly, unrecovered glue together with the metabolic cost of glue secretion will detract from the energetic yield of the prey. Small prey will scarcely repay the cost of glue used whereas larger ones are more likely to escape; consequently the energetically optimal prey are relatively large, but somewhat smaller than those potentially available. Accordingly, adult peripatus preferred larger prey and grew better when fed on them in the laboratory, whereas juveniles grew better on smaller prey. The size distribution of prey in the forest was heavily biased towards smaller types and it seemed likely that the productivity of large peripatus would be limited by the availability of profitable prey.

Molecular analysis of a gene in a developmentally regulated puff of Drosophila melanogaster.

An increase in the concentration of the steroid hormone ecdysone in late larval life triggers a profound change in the pattern of polytene chromosome puffs in the Drosophila melanogaster salivary gland. One of the preexisting puffs that regress as the ecdysone concentration increases is located at the 3C11-12 bands, the site of the Sgs-4 gene, which codes for the sgs-4 protein, one of the proteins in the salivary glue secretion. We have isolated cloned segments of chromosomal DNA that define a 60-kilobase region containing the 0.9-kilobase Sgs-4 gene, and we have determined its position and orientation within this region. Fine structure restriction endonuclease mapping shows that approximately 45% of this gene consists of tandemly repeated sequences of 21 base pairs that occupy most of its 5' half, indicating that most of the amino-terminal half of the sgs-4 protein consists of tandemly repeated amino acid sequences of seven residues. We also report on the amount of the Sgs-4 mRNA as a function of developmental stage and in nine different strains, four of which produce little or no sgs-4 protein. Three of the null strains produce minute amounts of the mRNA and one yields none, whereas the five sgs-4 producing strains yield abundant amounts. The mRNAs frm these strains exhibit different lengths, which correlate with different gene lengths that appear to result from different numbers of the repeated sequences in their tandem arrays.

Drosophila: the genetics of two major larval proteins

A series of irradiation-induced deficiencies covering 62 polytene chromosome bands in chromosome arm 3L of Drosophila melanogaster includes the loci of two abundant developmentally regulated larval proteins. The structural gene for larval serum protein 2 (LSP 2) lies at 68E3 or 4, and that for salivary glue secretion protein 3 between 68A8 and 68C11, coincident with a major intermoult puff active in the salivary gland at the time of glue synthesis. The structural genes for esterase 6 and four visible recessive loci lie within the same region.

Synthesis and secretion of mucoprotein glue in the salivary gland ofDrosophila melanogaster.

The processes of involved in the accumulation and secretion of adhesive mucoprotein ("glue") in third instar larvae ofD. melanogaster are described. The RNA templates for glue protein seem to be synthesized within 72-85 hours after oviposition, because treatment of larvae at this age with actinomycin D inhibits the accumulation of the secretion. The secretory granules appear in the salivary gland cells of 90 hour larvae and are discharged into the gland lumen of 118 hour larvae.Secretion of mucoprotein material by salivary gland cells into the gland lumen depends on high ecdysone concentration. Mucoproteins are synthesized, but not discharged, by these cells in ecdysone-deficient homozygousl (2) gl larvae as well as in normal larvae isolated from ecdysone by ligatures or transplantation of the salivary glands into adult abdomens. Ecdysterone injection into normal 100 hour larvae, 7 dayl (2) gl larvae or into adult imaginal abdomens into which salivary glands have previously been stimulates mucoprotein secretion into the gland lumen.Determination of total protein content in the salivary gland by Lowry's method has shown that "glue" proteins make up 21-32% of the total protein content in the gland.The glands of thel (3) tl mutant do not give a PAS-positive reaction, nor are the granules of the secretion observed under the phase contrast microscope.

Time sequence in the expression of various developmental characters induced by ecdysterone in Drosophila hydei

The effect of injection of different ecdysterone concentrations into third instar larvae of Drosophila hydei at various stages was studied with regard to changes in genome activity, secretion of mucopolysacharides by the salivary gland cells, and pigmentation. It is found that the level and period of activity of puff 78B, a representative of the first series of changes in genome activity, depends upon the concentration of the steroid applied. For the second series of changes in genome activity, represented by puff 77BC, it is established that not only the level and period of its activity, but also its time of appearance depends on the age of the larvae and the steroid concentration administered. Younger animals are less responsive to the steroid than older ones. Glue secretion and pigmentation occur with a longer delay in young than in old larvae. This delay can be reduced by raising the steroid concentration. The possible relationships between the reaction of the various processes upon experimental administration of the steroid are discussed.