Alterations of heart development in Xenopus laevis by galactoside-binding lectin or its sugar hapten inhibitor

The early heart anlagen of Xenopus laevis embryos were exposed to purified embryonic galactoside-binding lectin or its potent hapten inhibitor thiodigalactoside (TDG). Heart development was then studied using a variety of microscopical techniques. Conotruncal morphology and positioning with respect to the ventricle are altered in treated animals. In 34% of animals treated with lectin and 35% treated with TDG, the conotruncus leaves the ventricle from an abnormal location. Lectin or TDG treatments are also correlated with altered conotruncal shape, with the conotruncal regions showing greater radii of curvature compared to controls. Conotruncal myocyte differentiation is altered by the test treatments, with lack of development of organized myofibrillar arrays. Conotruncal cushion development is also affected. Changes occur in the shape and size of the primary conotruncal cushion, and alterations of outflow tract septation develop. Less maturation of ventricular myocytes is also observed in test animals. The results suggest that galactose-lectin interactions are important in heart development.     

DECREASED EXPRESSION OF GALECTIN-3 IS ASSOCIATED WITH PROGRESSION OF HUMAN BREAST CANCER

Galectin-3, a member of the β-galactoside-binding lectin family, is involved in several biological events including binding to the basement membrane glycoprotein laminin. Although the exact role of galectin-3 during the interactions between cells and laminin is not yet known, it has recently been observed that its expression is down-regulated at both the protein and the mRNA level in colon cancer tissues in correlation with progression of the disease. This study investigated the possibility that breast cancer cells might also exhibit decreased galectin-3 expression in association with their aggressiveness. The expression of galectin-3 was examined by immunoperoxidase staining, using a polyclonal antibody raised against recombinant galectin-3, in a collection of 98 human breast lesions including 12 fibroadenomas, 15 fibrocystic disease lesions, 22 in situ carcinomas, and 49 infiltrating ductal carcinomas, 19 of which had positive axillary lymph nodes. Normal breast tissue adjacent to the lesions was present in 59 biopsies. Normal breast tissue expressed high levels (3+) of galectin-3. High expression (2+ to 3+) was also found in most benign lesions examined. The expression of galectin-3 was significantly decreased in in situ carcinoma and this down-regulation was more pronounced in invasive ductal carcinoma, particularly when associated with infiltration of axillary lymph nodes. These data constitute the first observation that galectin-3 is down-regulated in breast cancer and suggest the decreased expression of this galactoside-binding lectin is associated with the acquisition of the invasive and metastatic phenotype.     

Ontogeny of central serotonergic neurons in the directly developing frog, Eleutherodactylus coqui

Embryonic development of the central serotonergic neurons in the directly developing frog, Eleutherodactylus coqui, was determined by using immunocytochemistry. The majority of anuran amphibians (frogs) possess a larval stage (tadpole) that undergoes metamorphosis, a dramatic post-embryonic event, whereby the tadpole transforms into the adult phenotype. Directly developing frogs have evolved a derived life-history mode where the tadpole stage has been deleted and embryos develop directly into the adult bauplan. Embryonic development in E. coqui is classified into 15 stages (TS 1–15; 1 = oviposition / 15 = hatching). Serotonergic immunoreactivity was initially detected at TS 6 in the raphe nuclei in the developing rhombencephalon. At TS 7, immunopositive perikarya were observed in the paraventricular organ in the hypothalamus and reticular nuclei in the hindbrain. Development of the serotonergic system was steady and gradual during mid-embryogenesis. However, starting at TS 13 there was a substantial increase in the number of serotonergic neurons in the paraventricular, raphe, and reticular nuclei, a large increase in the number of varicose fibers, and a differentiation of the reticular nuclei in the hindbrain. Consequentially, E. coqui displayed a well-developed central serotonergic system prior to hatching (TS 15). In comparison, the serotonergic system in metamorphic frogs typically starts to develop earlier but the surge of development that transpires in this system occurs post-embryonically, during metamorphosis, and not in the latter stages of embryogenesis, as it does in E. coqui. Overall, the serotonergic development in E. coqui is similar to the other vertebrates.     

Identification of N‐Acetylgalactosamine in Carbohydrates of Xenopus laevis Testis

Identification of glycans in amphibian testis has shown the existence of N‐acetylgalactosamine (GalNAc)‐containing carbohydrates. Labeling of the sperm acrosome with GalNAc‐binding lectins has allowed the identification of GalNAc‐containing glycans in this organelle. Futhermore, this specific labeling of the acrosome has allowed the study of acrosomal biogenesis by lectin histochemistry. However, the testis of Xenopus laevis has never been analyzed by lectin histochemistry to locate GalNAc‐containing glycoconjugates. The aim of this work was to elucidate the expression of GalNAc in glycoconjugates of Xenopus testis using five specific lectins. The results showed that most of the lectins labeled the interstitium with variable intensity. However, labeling of the different spermatogenetic germ cell types showed different labeling patterns. Some lectins produced weak or very weak staining in germ cells, for example, horse gram Dolichos biflorus agglutinin, which labeled most of the germ cell types, and lima bean Phaseolus lunatus agglutinin, which weakly labeled only spermatogonia, but did not stain other germ cells. By contrast, Maclura pomifera lectin (MPL) moderately labeled all germ cell types, except mature sperm. Labeling with other lectins was seen only at later stages, suggesting variations involved in the spermatogenetic development. Thus, snail Helix pomatia agglutinin labeled spermatids, but neither spermatogonia nor spermatocytes, while soybean Glycine max agglutinin (SBA) labeled from preleptotene spermatocytes to later stages. The periphery of the acrosome was labeled with MPL and SBA, but no specific labeling of the acrosomal content was seen with any lectin. Thus, the GalNAc‐binding lectins that have been used as acrosomal markers in some amphibians cannot be used in Xenopus testis, suggesting that acrosomal glycoconjugates in amphibians are species specific. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

Appl1 is essential for the survival of Xenopus pancreas,duodenum, and stomach progenitor cells

An understanding of the molecular mechanisms governing the survival of organ progenitor cells in vivo is crucial for in vitro tissue regeneration. Here, we have found that Xenopus appl1 and akt2 share a similar embryonic expression pattern, showing characteristic expression in the central nervous system as well as in the pancreas and part of the stomach/duodenum (SD) at tadpole stages of development. Specific knockdown of appl1 in endoderm or inhibition of akt activity did not affect the formation of endodermal organ primordia at tail bud stages of development, but led to a gut‐coiling defect, strong apoptosis in endodermal organs, and pancreas and SD hypoplasia or even aplasia at tadpole stages of development. Furthermore, appl1 is required for akt phosphorylation and akt2 in turn can rescue appl1 knockdown phenotypes. Together, our data suggest that appl1‐akt signaling is specifically required for the survival of pancreas and SD progenitor cells in Xenopus laevis embryos. Developmental Dynamics 239:2198–2207, 2010. © 2010 Wiley‐Liss, Inc.     

Prenatal development of coronary arteries in the rat: morphologic patterns

The aim of this work was to address spatiotemporal and morphologic patterns of coronary artery development in rats, based on immunohistochemical and ultrastructural studies of hearts at different stages of prenatal development. Griffonia simplicifolia I lectin and α-smooth muscle antibody were used to demonstrate endothelial cells and/or their precursors and smooth muscle cells, respectively. Ultrastructural examination was performed on ED14–16 hearts to study the morphology of the developing coronary arteries in different regions of the truncus arteriosus and adjacent myocardium. On ED14 endothelial-like cells present within the mesenchyme surrounding the outflow tract penetrated the aortic wall and the truncoconal proximal myocardium. On ED15 these penetrating cells formed vascular clusters, which were the first signs of presumptive vascular channels. Development of the coronary artery proceeded by coalescence of discontinous vascular clusters, formation of the lumen (vascular channels) and establishing a connection of the proximal part with the aorta. The second layer of cells around vascular channels (embryonic media) consisted of mesenchymal cells that were attracted to the immature vessel and were first seen on ED15. At this time no lumenized connection of the coronary artery with the aorta has been seen. After the lumenized connection of the coronary artery with the aorta had been established perivascular cells of the media started to differentiate into vascular smooth muscle, as was shown by α-smooth muscle actin-staining. Further development and differentiation of the media and adventitia proceeded distally (towards the apex). Accepted: 30 April 1999     

A method for the selection of mutants of Phycomyces blakesleeanus defective in germination

Summary A simple method for the physical separation of dormant and germinated cells of Phycomyces blakesleeanus has been designed. Separation was attained by isopycnic centrifugation in a preformed discontinuous density gradient of Urografin. In this gradient, dormant spores and active cells at two different stages of germination were separated in three homogeneous and clearly distinct bands. The method has been used to follow the kinetics of the variation of cellular density during germination of wild type spores and to select for mutants temperature-sensitive for heat-shock induced germination. Sixty-nine mutants have been isolated by various procedures which include a separation step in the described Urografin gradient.     

Attenuated recombinant Yersinia as live oral vaccine carrier to protect against amoebiasis

Various attenuated Yersinia enterocolitica strains expressing different sections of the Entamoeba histolytica surface lectin via the type III protein secretion system (T3SS) were assessed for their use to orally vaccinate rodents against invasive amoebiasis. The T3SS was found to efficiently express and secrete or translocate subfragments as well as the entire heavy subunit of the lectin. Oral vaccination with recombinant Yersinia conferred significant protection against amoebic liver abscess formation when the antigen was expressed as a fusion molecule with the translocation domain of Yersinia outer protein E. However, effectiveness of vaccination was dependent on gender and the rodent species used. Protection was mediated primarily by cellular immune mechanisms as it was independent from the antibody titre against the amoeba lectin but correlated with an antigen-specific Th1-cytokine response. The results suggest that Gram-negative bacteria expressing E. histolytica antigens via T3SS may constitute a suitable oral vaccine carrier against amoebiasis and that an effective IFN-γ response is required for protection against invasive amoebiasis.     

β-Galactoside-binding protein secreted by activated T cells inhibits antigen-induced proliferation of T cells

We have used mRNA differential display PCR to search for genes induced in activated T cells and have found the LGALS1 (lectin, galactoside-binding, soluble) gene to be strongly up-regulated in effector T cells. The protein coded by the LGALS1 gene is a β-galactoside-binding protein (βGBP), which is released by cells as a monomeric negative growth factor but which can also associate into homodimers (galectin-1) with lectin properties. Northern blot analysis revealed that ex vivo isolated CD8⁺ effector T cells induced by a viral infection expressed high amounts of LGALS1 mRNA, whereas LGALS1 expression was almost absent in resting CD8⁺ T cells. LGALS1 expression could be induced in CD4⁺ and CD8⁺ T cells upon activation with the cognate peptide antigen and high levels of LGALS1 expression were found in concanavalin A-activated T cells but not in lipopolysaccharide-activated B cells. Gel filtration and Western blot analysis revealed that only monomeric βGBP was released by activated CD8⁺ T cells and in vitro experiments further showed that recombinant βGBP was able to inhibit antigen-induced proliferation of naive and antigen-experienced CD8⁺ T cells. Thus, these data indicate a role of βGBP as an autocrine negative growth factor for CD8⁺ T cells.     

N-CAM, polysialic acid and chick tail bud development

Summary We have previously shown that the binding of the lectin wheat germ agglutinin (WGA) to developing tail buds results in a range of caudal axial defects, which were most likely due to the affinity of the lectin for sialic acid residues. In the present study, we examined the distribution and role of a sialic acid-containing glycoprotein, N-CAM, in chick tail bud development. In the early tail bud, anti N-CAM, staining was found in the medullary cord. However, there was no uptake of an antibody specific to N-CAM containing moderate to long chains of polysialic acid (5A5 monoclonal antibody). At later stages, while N-CAM localized throughout the neural tube, staining with the 5A5 antibody was restricted to the floor plate. Sub-blastodermal injection of the anti N-CAM antibody beneath the tail bud region of HH stages 13–14 embryos produced caudal axial malformations. These malformations included the presence of accessory segments of neural tube and/or notochord, and fusion between the neural tube and underlying segment of notochord. Our results suggest that N-CAM is present during the development of the secondary neuraxis from the tail bud, although the highly sialylated form of this molecule could not be visualized until relatively late stages. N-CAM probably plays a role in the normal course of tail bud development, since perturbation of the molecule with an antibody resulted in malformations. Since these malformations were similar to those we have previously reported when we treated similarly staged chick embryos with WGA, there is a possibility that the sialic acid residues recognized and bound by the lectin are those associated with the N-CAM molecule.     

Lectins and saccharides inLymnaea stagnalis haemocyte recognition

Lectins as important non-self-recognition factors are present on molluscan haemocytes as membrane receptors or in cell-free plasma as soluble recognition factors. The recognition mediated by them can be operational in Ca²⁺-dependent and-independent ways. In our phagocytosis system,Lymnaea stagnalis haemocytes and rabbit erythrocytes served as effector and target cells, respectively. The Ca²⁺-dependent phagocytosis was inhibited by aminosaccharides and their acetylated forms,l-fucose, laminarin (a β-1,3-glucan) and mannan. By omitting calcium from the buffer, the phagocytosis was reduced nearly to zero. However, certain heterologous (commercial) lectins stimulated the process to levels detected in Ca²⁺-containing buffer. Knowing lectin targets on haemocytes and erythrocytes, lectin specificities and glycosylation, we could infer the role of heterologous lectins in recognition: some lectins created bridges between carbohydrates on effector and target cells whereas the others functioned as opsonins, i.e. they bound to saccharides on target cells and created in this way attractive epitopes for haemocyte receptors; both lectin types enabled subsequent phagocytosis. Thus, Ca²⁺-dependent and-independent lectin-mediated recognition has been demonstrated. Infection byTrichobilharia szidati did not substantially impair the recognition process. The role of soluble lectins ofL. stagnalis origin (homologous lectins) remains to be solved.     

The morphology of tadpole (Rana catesbeiana) liver under varying conditions of organ culture

Primary explants from Rana catesbeiana tadpole liver have been maintained in organ culture for six days using methods of cultivation which are described in detail. The organization and morphology of the cells in culture have been defined by light and electron microscopy. By adjustment of the experimental conditions of culture, after six days, the cells are morphologically identical to those freshly excised from tadpole liver. Soon after being put into culture, however, the chromatin of the nuclei becomes condensed, and only after about 30 hours in culture do the original euchromatic characteristics of the nucleus reappear. The effects of changes in the culture conditions on these morphological characteristics are presented. The system that has been developed is discussed with a view toward elucidating the basis of the cytological changes which occur during anuran metamorphosis.     

Role of TrkA signaling during tadpole tail regeneration and early embryonic development in Xenopus laevis

Neurotrophic signaling regulates neural cell behaviors in development and physiology, although its role in regeneration has not been fully investigated. Here, we examined the role of neurotrophic signaling in Xenopus laevis tadpole tail regeneration. After the tadpole tails were amputated, the expression of neurotrophin ligand family genes, especially ngf and bdnf, was up‐regulated as regeneration proceeded. Moreover, notochordal expression of the NGF receptor gene TrkA, but not that of other neurotrophin receptor genes TrkB and TrkC, became prominent in the regeneration bud, a structure arising from the tail stump after tail amputation. The regenerated tail length was significantly shortened by the pan‐Trk inhibitor K252a or the TrkA inhibitor GW‐441756, but not by the TrkB inhibitor ANA‐12, suggesting that TrkA signaling is involved in elongation of regenerating tails. Furthermore, during Xenopus laevis embryonic development, TrkA expression was detected in the dorsal mesoderm at the gastrula stage and in the notochord at the neurula stage, and its knockdown led to gastrulation defects with subsequent shortening of the body axis length. These results suggest that Xenopus laevis TrkA signaling, which can act in the mesoderm/notochord, plays a key role in body axis elongation during embryogenesis as well as tail elongation during tadpole regeneration.     

Chronotopographical distribution patterns of cell death and of lectin‐positive macrophages/microglial cells during the visual system ontogeny of the small‐spotted catshark Scyliorhinus canicula

The patterns of distribution of TUNEL‐positive bodies and of lectin‐positive phagocytes were investigated in the developing visual system of the small‐spotted catshark Scyliorhinus canicula, from the optic vesicle stage to adulthood. During early stages of development, TUNEL‐staining was mainly found in the protruding dorsal part of the optic cup and in the presumptive optic chiasm. Furthermore, TUNEL‐positive bodies were also detected during detachment of the embryonic lens. Coinciding with the developmental period during which ganglion cells began to differentiate, an area of programmed cell death occurred in the distal optic stalk and in the retinal pigment epithelium that surrounds the optic nerve head. The topographical distribution of TUNEL‐positive bodies in the differentiating retina recapitulated the sequence of maturation of the various layers and cell types following a vitreal‐to‐scleral gradient. Lectin‐positive cells apparently entered the retina by the optic nerve head when the retinal layering was almost complete. As development proceeded, these labelled cells migrated parallel to the axon fascicles of the optic fiber layer and then reached more external layers by radial migration. In the mature retina, lectin‐positive cells were confined to the optic fiber layer, ganglion cell layer and inner plexiform layer. No evident correlation was found between the chronotopographical pattern of distribution of TUNEL‐positive bodies and the pattern of distribution of lectin‐labelled macrophages/microglial cells during the shark′s visual system ontogeny.     

Agglutination of murine and guinea pig peritoneal cells by α-L-fucose-binding lectin: Evonymus europaea

Among lectins from Lotus tetragonolobus, Ulex europaeus I and Evonymus europaea, agglutinating cells with blood group H determinants containing L -fucose α1→2-linked to subterminal D -galactose, only the last lectin agglutinates thioglycolate- and paraffin oil-stimulated murine and guinea pig peritoneal exudate cells (PEC). The agglutination is inhibited by specific inhibitors of Evonymus lectin only: lacto-N-fucopentaose I and lactose. These results suggest the presence of a determinant on the surface of PEC, containing L -fucose α1-linked at the nonreducing end which is different from blood group H determinants. Nonstimulated murine peritoneal cells (PC) are not agglutinated by the lectin but become agglutinable after neuraminidase treatment. Unstimulated guinea pig PC from different animals are agglutinated to a different extent by the same lectin concentration and show increased agglutinability after neuraminidase digestion. These results show that receptor for Evonymus lectin also exists on the nonstimulated PC but access to it is hindered by sialic acid. Trypsin- and pronase-digested PEC show increased agglutinability with Evonymus lectin. These results suggest that the lectin receptor is a glycolipid. Since α-linked L -fucose has been suggested as a part of the macrophage receptor for migration inhibitory factor in the guinea pig (Remold, J. Exp. Med. 1973. 138: 1065), the effect of Evonymus europaea lectin on the migration of PEC was studied. It was found that lectin inhibits the migration of PEC in the capillary tube assay up to 80%.     

Induction of hyperpigmentation and heat shock protein 70 response to the toxicity of methomyl insecticide during the organ development of the Arabian toad,Bufo arabicus (Heyden,1827)

ABSTRACT

Methomyl (MET) is a carbamate insecticide which is used as a substitute for organophosphorus compounds to protect crops against insects. The present study aims to evaluate the cytoprotection response of pigment cells and heat shock protein 70 (HSP70) after exposure to MET during the tadpole developmental stages of the Arabian toad, Bufo arabicus. Three developmental larval stages of the toad were selected and divided into two groups; Control and MET-exposed (MET-EX) tadpoles (10ppm). MET-EX tadpoles showed an increased number of pigment cells in the liver, kidney, anterior eye chamber, and skin tissues as compared to the control. The glycogen content in the developing liver and muscles (myotomes) of MET-EX tadpoles was decreased as compared to the control. In the MET-EX tadpoles, immunohistochemical staining showed an increase of HSP70 expression in the liver hepatocytes, the nucleated red blood cells (nRBC) in kidney glomeruli, the iridocorneal angle of anterior eye chamber, and the skin as compared to the control. The current study concluded that pigment cells and HSP70 represented a cytoprotecting response against MET insecticide during the organ development of B. arabicas tadpoles. Therefore, MET use should be regularly monitored in the environment to protect animals and human from exposure to this insecticide.     

小鼠动情周期、妊娠和哺乳过程中凝集素PHA-E与UEA的受体在子宫内膜的分布

目的 探讨子宫内膜菜豆凝集素(PHA-E)和荆豆凝集素(UEA)的受体与胚泡植入的关系。方法 应用亲合细胞化学和图像分析方法检测PHA-E与UEA的受体在昆明小鼠动情周期、妊娠和哺乳过程中子宫内膜的分布状况和变化规律。结果 以上2种凝集素受体均存在于不同阶段的小鼠子宫内膜，但2种凝集素受体的数量和分布存在差异。PHA-E受体在孕早期，尤其是围植入期的水平显著高于动情周期组和哺乳期组；其广泛分布于围植入期子宫内膜腔上皮和腺上皮游离缘、胚胎组织、蜕膜细胞表面及其周围的细胞外基质(ECM)。UEA受体则呈现动情期水平最高，孕期逐渐下降的趋势，其主要分布于子宫内膜腺上皮游离缘。结论凝集素PHA-E受体与小鼠胚泡植入过程密切相关，对UEA受体与胚胞植入的关系尚难做出满意地解释。     

Alterations of heart development in Xenopus laevis by galactoside-binding lectin or its sugar hapten inhibitor

The early heart anlagen of Xenopus laevis embryos were exposed to purified embryonic galactoside-binding lectin or its potent hapten inhibitor thiodigalactoside (TDG). Heart development was then studied using a variety of microscopical techniques. Conotruncal morphology and positioning with respect to the ventricle are altered in treated animals. In 34% of animals treated with lectin and 35% treated with TDG, the conotruncus leaves the ventricle from an abnormal location. Lectin or TDG treatments are also correlated with altered conotruncal shape, with the conotruncal regions showing greater radii of curvature compared to controls. Conotruncal myocyte differentiation is altered by the test treatments, with lack of development of organized myofibrillar arrays. Conotruncal cushion development is also affected. Changes occur in the shape and size of the primary conotruncal cushion, and alterations of outflow tract septation develop. Less maturation of ventricular myocytes is also observed in test animals. The results suggest that galactose-lectin interactions are important in heart development.     

Cloning and characterization of voltage‐gated calcium channel alpha1 subunits in Xenopus laevis during development

Voltage‐gated calcium channels play a critical role in regulating the Ca²⁺ activity that mediates many aspects of neural development, including neural induction, neurotransmitter phenotype specification, and neurite outgrowth. Using Xenopus laevis embryos, we describe the spatial and temporal expression patterns during development of the 10 pore‐forming alpha1 subunits that define the channels' kinetic properties. In situ hybridization indicates that Ca_V1.2, Ca_V2.1, Ca_V2.2, and Ca_V3.2 are expressed during neurula stages throughout the neural tube. These, along with Ca_V1.3 and Ca_V2.3, beginning at early tail bud stages, and Ca_V3.1 at late tail bud stages, are detected in complex patterns within the brain and spinal cord through swimming tadpole stages. Additional expression of various alpha1 subunits was observed in the cranial ganglia, retina, olfactory epithelium, pineal gland, and heart. The unique expression patterns for the different alpha1 subunits suggests they are under precise spatial and temporal regulation and are serving specific functions during embryonic development. Developmental Dynamics 238:2891–2902, 2009. © 2009 Wiley‐Liss, Inc.     

Immunohistochemical localization and biological activity of 3β-hydroxysteroid dehydrogenase and 5α-reductase in the brain of the frog,Rana esculenta,during development

The occurrence of several enzymes responsible for the biosynthesis of neurosteroids in the brain of adult frogs is now firmly established but the expression of these enzymes during ontogenesis has not yet been investigated. In the present report, we describe the immunohistochemical distribution and biological activity of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 5α-reductase (5α-R) in the brain of the European green frog, Rana esculenta, during larval development. The spatio-temporal distribution of 3β-HSD and 5α-R immunoreactivities in the tadpole brain was generally different, although these two enzymes were occasionally detected in the same areas such as the olfactory bulbs and cerebellum. Identification of neurons based on their morphological aspect as well as labeling of astrocytes with an antiserum against glial fibrillary acidic protein (GFAP) revealed that, in the tadpole brain, 3β-HSD- and 5α-R-immunoreactive materials were contained in both neurons and glial cells. Incubation of tadpole brain explants with [³H]-pregnenolone resulted in the formation of several tritiated steroids including progesterone, 17-hydroxyprogesterone, androstenedione, 5α-dihydroprogesterone and 5α-dihydrotestosterone. The present study provides the first immunocytochemical mapping of two key steroidogenic enzymes in the developing frog brain. The data also indicate that neurosteroid biosynthesis occurs in the brain of tadpoles, as previously shown for adult amphibians, birds and mammals. The transient expression of steroidogenic enzymes in several regions of the tadpole brain suggests that, in amphibians, neurosteroids may be implicated in neurotrophic activities during larval development.