Ethylene modulates gene expression in cells of the marine sponge Suberites domuncula and reduces the degree of apoptosis.

Sponges (phylum Porifera) live in an aqueous milieu that contains dissolved organic carbon. This is degraded photochemically by ultraviolet radiation to alkenes, particularly to ethylene. This study demonstrates that sponge cells (here the demosponge Suberites domuncula has been used), which have assembled to primmorphs, react to 5 microM ethylene with a significant up-regulation of intracellular Ca(2+) concentration and with a reduction of starvation-induced apoptosis. In primmorphs from S. domuncula the expression of two genes is up-regulated after exposure to ethylene. The cDNA of the first gene (SDERR) isolated from S. domuncula encodes a potential ethylene-responsive protein, termed ERR_SUBDO; its putative M(r) is 32,704. Data bank search revealed that the sponge polypeptide shares high similarity (82% on amino acid level) with the corresponding plant molecule, the ethylene-inducible protein from Hevea brasiliensis. Until now no other metazoan ethylene-responsive proteins have been identified. The second gene, whose expression is up-regulated in response to ethylene is a Ca(2+)/calmodulin-dependent protein kinase II. Its cDNA, SDCCdPK, encodes a M(r) 54,863 putative kinase that shares 69% similarity with the corresponding enzyme from Drosophila melanogaster. The expression of both genes in primmorphs from S. domuncula is increased by approximately 5-fold after a 3-day incubation period with ethylene. It is concluded that also metazoan cells, with sponge cells as a model, may react to ethylene with an activation of cell metabolism including gene induction.     

Origin of the integrin-mediated signal transduction. Functional studies with cell cultures from the sponge Suberites domuncula.

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan animals. Recently, from the marine sponge Geodia cydonium a first cDNA encoding a putative integrin receptor molecule was isolated. In the present study basic functional experiments have been conducted to test the hypothesis that in sponges integrin polypeptides also function as adhesion molecules and as outside-in signaling molecules. The sponge Suberites domuncula has been used for the experiments because from this sponge only has a cell culture been established. Here we report that aggregation factor (AF)-mediated cell-cell adhesion is blocked by the RGDS peptide which is known to interact with beta integrin. Both RGDS and AF were found to stimulate DNA synthesis within 24 h. The beta subunit of the integrin receptor was cloned from S. domuncula; the estimated 91-kDa molecule comprises the characteristic signatures. Evolutionary conservation of the beta integrin was assessed by comparison with corresponding beta integrin subunits from evolutionary higher metazoan taxa. Addition of RGDS or of AF to isolated cells of S. domuncula causes a rapid (within 1-2 min) increase in the intracellular Ca2+ concentration which is further augmented in the presence of Ca2+. Furthermore, incubation of the cells with RGDS or AF causes an activation of the GTP-binding protein Ras. In addition it is shown that after a prolonged incubation of the cells with RGDS and AF the expression of the genes coding for Ras and for calmodulin is upregulated. These results suggest that the integrin receptor functions in the sponge system not only as adhesion molecule but also as a molecule involved in outside-in signaling.     

Molecular characterization and structure analysis of RPL10/QM-like protein from the red drum Sciaenops ocellatus (Sciaenidae)

The QM-like gene encodes a ribosomal protein L10. Besides housekeeping roles in protein synthesis, QM-like proteins have multiple extraribosomal functions during cell growth, cell differentiation and apoptosis. We obtained the full-length cDNA of QM-like protein (designated as SoQM) from the salt water game fish Sciaenops ocellatus, using RACE-PCR. The sequence consists of 740 bp, encoding 215-amino acid residues with 24.60 kDa. The AA sequence of the SoQM protein contains a series of functional motifs that belong to the QM family signature, which is conserved among different species. The SoQM gene contains five introns and six exons. The expression pattern of SoQM as determined by RT-PCR indicated that SoQM mRNA was expressed in all tissues tested, including brain, gill, head-kidney, intestine, stomach, heart, spleen, blood, muscle, and gonads. The phylogenetic tree constructed with MEGA 4.0 showed that SoQM clusters together with that of other fish. It was found that the sequences of the SoQM gene are highly conserved, suggesting the fundamental and critical functions of SoQM in S. ocellatus. The three-dimensional structure of the SoQM protein core domain (4~169) was predicted by the Swiss-Model program. Compared with QM proteins in other species, the main structure of SoQM protein was conserved, while the C-terminal domain was different from other QM-like proteins. Prediction of the three-dimensional structure of SoQM would provide valuable insight into the molecular basis of protein function, allowing an effective design of experiments, such as site-directed mutagenesis, studies of disease-related mutations or structure-based design of specific inhibitors.     

Stimulation of protein (collagen) synthesis in sponge cells by a cardiac myotrophin-related molecule from Suberites domuncula.

The body wall of sponges (Porifera), the lowest metazoan phylum, is formed by two epithelial cell layers of exopinacocytes and endopinacocytes, both of which are associated with collagen fibrils. Here we show that a myotrophin-like polypeptide from the sponge Suberites domuncula causes the expression of collagen in cells from the same sponge in vitro. The cDNA of the sponge myotrophin was isolated; the potential open reading frame of 360 nt encodes a 120 aa long protein (Mr of 12,837). The sequence SUBDOMYOL shares high similarity with the known metazoan myotrophin sequences. The expression of SUBDOMYOL is low in single cells but high after formation of primmorph aggregates as well as in intact animals. Recombinant myotrophin was found to stimulate protein synthesis by fivefold, as analyzed by incorporation studies using [3H] lysine. In addition, it is shown that after incubation of single cells with myotrophin, the primmorphs show an unusual elongated, oval-shaped appearance. It is demonstrated that in the presence of recombinant myotrophin, the cells up-regulate the expression of the collagen gene. The cDNA for S. domuncula collagen was isolated; the deduced aa sequence shows that the collagenous internal domain is rather short, with only 24 G-x-y collagen triplets. We conclude that the sponge myotrophin causes in homologous cells the same/similar effect as the cardiac myotrophin in mammalian cells, where it is involved in initiation of cardial ventricular hypertrophy. We assume that an understanding of sponge molecular cell biology will also contribute to a further elucidation of human diseases, here of the cardiovascular system.     

Origin of metazoan stem cell system in sponges: first approach to establish the model (Suberites domuncula)

It is established that Porifera (sponges) represent the earliest phylum which branched off from the common ancestor of all multicellular animals, the Urmetazoa. In the present study, the hypothesis is tested if, during this transition, pluripotent stem cells were formed which are provided-similar to the totipotent cells (archaeocytes/germ cells)-with a self-renewal capacity. As a model system, primmorphs from the sponge Suberites domuncula were used. These 3D-cell aggregates were cultivated in medium (RPMI 1640/seawater) either lacking silicate and ferric iron or in medium which was supplemented with these 'morphogenetic' factors. As molecular markers for the potential existence of stem cells in primmorphs, two genes which encode proteins found in stem cells of higher metazoan species, were cloned from S. domuncula. First, the noggin gene, which is present in the Spemann organizer of amphibians and whose translation product acts during the formation of dorsal mesoderm derivatives. The second gene encodes the mesenchymal stem cell-like protein. Both cDNAs were used to study their expression in primmorphs in dependence on the incubation conditions. It was found that noggin expression is strongly upregulated in primmorphs kept in the presence of silicate and ferric iron, while the expression of the mesenchymal stem cell-like protein was downregulated. These data are discussed with respect to the existence of stem cells in sponges.     

The molecular basis for the evolution of the metazoan bodyplan: extracellular matrix-mediated morphogenesis in marine demosponges

Molecular data on development/differentiation and on comparative genomics allow insights into the genetic basis of the evolution of a bodyplan. Sponges (phylum Porifera) are animals that are the (still extant) stem group with the hypothetical Urmetazoa as the earliest common ancestor of all metazoans; they possess the basic features of the characteristic metazoan bodyplan also valid for the animals of the crown taxa. Here we describe three homeobox genes from the demosponge Suberites domuncula whose deduced proteins (HOXa1_SUBDO, HOXb1_SUBDO, HOXc1_SUBDO) are to be grouped with the Antennapedia class of homeoproteins (subclasses TIx-Hox11 and NK-2). In addition, a cDNA encoding a LIM/homeobox protein has been isolated which comprises high sequence similarity to the related LIM homeodomain (HD) proteins in its LIM as well as in its HD domains. To elucidate the potential function of these proteins in the sponge a new in vitro system was developed. Primmorphs which are formed from dissociated cells were grown on a homologous galectin matrix. This galectin cDNA was cloned and the recombinant protein was used for the preparation of the matrix. The galectin/polylysine matrix induced in primmorphs the formation of channels, one major morphogenetic process in sponges. Under such conditions the expression of the gene encoding the LIM/homeobox protein is strongly upregulated, while the expression of the other homeobox genes remains unchanged or is even downregulated. Competition experiments with galactosylceramides isolated from S. domuncula were performed. They revealed that a beta-galactosylceramide, named Sdgal-1, prevented the expression of the LIM gene on the galectin matrix, while Sdgal-2, a diglycosylceramide having a terminal alpha-glycosidically linked galactose, caused no effect on the formation of channels in primmorphs or on LIM expression. This study demonstrates for the first time that an extracellular matrix molecule, galectin, induces a morphogenetic process in sponges which is very likely caused by a LIM/homeobox protein. Furthermore, a new model is introduced (galectin-caused channel formation in sponge primmorphs) to investigate basic pathways, thus allowing new insights into the functional molecular evolution of Metazoa.     

Emergence and disappearance of an immune molecule,an antimicrobial lectin,in basal metazoa. A tachylectin-related protein in the sponge Suberites domuncula

Sponges (phylum Porifera) represent the evolutionarily oldest metazoans that comprise already a complex immune system and are related to the crown taxa of the protostomians and the deuterostomians. Here, we demonstrate the existence of a tachylectin-related protein in the demosponge Suberites domuncula, termed Suberites lectin. The MAPK pathway was activated in response to lipopolysaccharide treatment of the three-dimensional cell aggregates, the primmorphs; this process was abolished by the monosaccharide D-GlcNAc. The cDNA encoding the S. domuncula lectin was identified and cloned; it comprises 238 amino acids (26 kDa) in the open reading frame. The deduced protein has one potential transmembrane region, three characteristic Cys residues, and six internal tandem repeats; it shares the highest sequence similarity with lectins from the horseshoe crab Tachypleus trunculus. The steady-state level of expression of the Suberites lectin rises in primmorphs in response to lipopolysaccharide, an effect that was prevented by co-incubation with D-GlcNAc. The natural sponge lectin was purified by affinity chromatography; it has a size of 27 kDa and displays antibacterial activity against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus. The putative protein, deduced from the cloned gene, is identical/similar to the purified natural protein, as demonstrated by immunological cross-reactivity with specific antibodies. We conclude that the S. domuncula lectin acts as an antibacterial molecule involved in immune defense against bacterial invaders.     

Okadaic acid, an apoptogenic toxin for symbiotic/parasitic annelids in the demosponge Suberites domuncula

The role of okadaic acid (OA) in the defense system of the marine demosponge Suberites domuncula against symbiotic/parasitic annelids was examined. Bacteria within the mesohyl produced okadaic acid at concentrations between 32 ng/g and 58 ng/g of tissue (wet weight). By immunocytochemical methods and by use of antibodies against OA, we showed that the toxin was intracellularly stored in vesicles. Western blotting experiments demonstrated that OA also existed bound to a protein with a molecular weight of 35,000 which was tentatively identified as a galectin (by application of antigalectin antibodies). Annelids that are found in S. domuncula undergo apoptotic cell death. OA is one candidate inducer molecule of this process, since this toxin accumulated in these symbionts/parasites. Furthermore, we identified the cDNA encoding the multifunctional prosurvival molecule BAG-1 in S. domuncula; it undergoes strong expression in the presence of the annelid. Our data suggest that sponges use toxins (here, OA) produced from bacteria to eliminate metazoan symbionts/parasites by apoptosis.     

Bruton tyrosine kinase-like protein, BtkSD, is present in the marine sponge Suberites domuncula

Sponges, the simplest and most ancient phylum of Metazoa, encode in their genome complex and highly sophisticated proteins that evolved together with multicellularity and are found only in metazoan animals. We report here the finding of a Bruton tyrosine kinase (BTK)-like protein in the marine sponge Suberites domuncula (Demospongiae). The nucleotide sequence of one sponge cDNA predicts a 700-aa-long protein, which contains all of the characteristic domains for the Tec family of protein tyrosine kinases (PTKs). The highest homology (38% identity, 55% overall similarity) was found with human BTK and TEC PTKs. Sponge PTK was therefore named BtkSD. Human BTK is involved in the maturation of B cells and mutations in the BTK gene cause X-linked agammaglobulinemia. Kinases from the Tec family are not present in Caenorhabditis elegans and, until now, they were found only in insects and higher animal taxa. Our finding implies that the BTK/TEC genes are of a very ancient origin.     

Increased gene expression of a cytokine-related molecule and profilin after activation of Suberites domuncula cells with xenogeneic sponge molecule(s)

Porifera (sponges) constitute the lowest metazoan phylum. Experiments examined whether sponges can recognize self/nonself molecules. Cells from the marine sponge Suberites domuncula were incubated with membranes from either S. domuncula or another marine sponge, Geodia cydonium, as well as with recombinant alpha-integrin from G. cydonium. The cells responded immediately with a rise of intracellular Ca2+ ([Ca2+i]) if they were treated with membranes from G. cydonium but not after treatment by those from S. domuncula. This change of [Ca2+i] was also recorded with G. cydonium alpha-integrin. In parallel, the expression of two genes was strongly upregulated; one codes for a cytokine-related molecule, pre-B-cell colony-enhancing factor, and the other for profilin. These genes have previously been found to be highly expressed in human or echinoderm cells in the presence of xenogeneic proteins. Our data support the hypothesis that a primordial immune response system is present in sponges.     

The complete set of ribosomal proteins from the marine sponge Suberites domuncula

The siliceous marine sponge Suberites domuncula is a member of the most ancient and simplest extant phylum of multicellular animals-Porifera, which have branched off first from the common ancestor of all Metazoa. We have determined primary structures of 79 ribosomal proteins (r-proteins) from S. domuncula: 32 proteins from the small ribosomal subunit and 47 proteins from the large ribosomal subunit. Only L39 and L41 polypeptides (51 and 25 residues long in rat, respectively) are missing. The sponge S. domuncula is, after nematode Caenorhabditis elegans and insect Drosophila melanogaster the third representative of invertebrates with known amino acid sequences of all r-proteins. The comparison of S. domuncula r-proteins with r-proteins from D. melanogaster, C. elegans, rat, Arabidopsis thaliana and Saccharomyces cerevisiae revealed very interesting findings. The majority of the sponge r-proteins are more similar to their homologues from rat, than to those either from invertebrates C. elegans and D. melanogaster, or yeast and plant. With few exceptions, the overall sequence conservation between sponge and rat r-proteins is 80% or higher. The phylogenetic tree of concatenated r-proteins from 6 eukaryotic species (rooted with archaeal r-proteins) has the shortest branches connecting sponge and rat. Both model invertebrate organisms experienced recently accelerated evolution and therefore sponge r-proteins very probably better reflect structures of proteins in the ancestral metazoan ribosome, which changed only little during metazoan evolution. Furthermore, r-proteins from the plant A. thaliana are significantly closer to metazoan r-proteins than are those from the yeast S. cerevisiae.     

Isolation and characterization of a cDNA encoding a potential morphogen from the marine sponge Geodia cydonium that is conserved in higher metazoans.

Species belonging to the lowest metazoan phylum, the sponges (Porifera), exhibit a surprisingly complex and multifaceted Bauplan (body plan). Recently, key molecules have been isolated from sponges which demonstrate that the cells of these animals are provided with characteristic metazoan adhesion and signal transduction molecules, allowing tissue formation. In order to understand which factors control the spatial organization of these cells in the sponge body plan, we screened for a cDNA encoding a soluble modulator of the behaviour of endothelial cells. A cDNA encoding a putative protein, which is highly similar to the human and mouse endothelial monocyte-activating polypeptide (EMAP) II has been isolated from a library of the marine sponge Geodia cydonium. The sponge EMAP-related polypeptide (EMAPR) has been termed EMAPR1_GC. The full-length cDNA clone, GCEMAPR1, has a size of 592 nucleotides (nt) and contains a 447 nt-long potential open reading frame; the molecular weight (MW) of the deduced amino acid sequence, 16,499 Da, is close to that of mature mammalian EMAP II (ca. 18 kDa). The sponge polypeptide is also closely related to a deduced polypeptide from the cosmid clone F58B3 isolated from Caenorhabditis elegans. A phylogenetic analysis revealed that the sponge and the nematode EMAPR molecules form a cluster which is significantly separated from the corresponding mammalian EMAP molecules. The function of the first cloned putative soluble modulator of endothelial cells in sponges remains to be determined.     

Polarity factor 'Frizzled' in the demosponge Suberites domuncula: identification, expression and localization of the receptor in the epithelium/pinacoderm(1)

Until recently, it was assumed that polarity and axis formation have evolved only in metazoan phyla higher than Cnidaria. One key molecule involved in the signal transduction causing tissue polarity is Frizzled, a seven-transmembrane receptor that is activated by the Wnt family of secreted proteins. We report the isolation and characterization of a Frizzled gene from the demosponge Suberites domuncula (Sd-Fz). The deduced polypeptide comprises all characteristic domains known from Frizzled receptors of higher metazoans. In situ hybridization studies show that Sd-Fz is expressed in cells close to the surface of the sponges and in the pinacocytes of some canals. Northern blot analysis demonstrates its upregulation during the formation of three-dimensional sponge cell aggregates in culture. These data provide for the first time experimental evidence that already in the lowest metazoan phylum (Porifera) genes are present which are very likely involved in tissue polarity.     

Tissue-/stage-dependent expression of a cloned Bombyx mandarina QM homologue

QM, a novel gene that was firstly isolated as a putative tumor suppressor gene from Wilms' tumor cell line. Although it is well known that the QM gene product plays an important role within the tumor cells, the precise role of QM in the non-tumor cells has remained elusive. With in this mind we isolated a cDNA encoding QM homologue from Bombyx mandarina to understand the function of QM. The 596 bp cDNA has an open reading frame of 219 amino acids and a predicted mol. wt. of 25 kDa. The protein has more than 88% amino acid sequence identity to the QM protein from Drosophila melanogaster. mRNA expression gradually increased from 1-2 days after egg laying to 2 days of finial instar, while very low expressions were detected for either the pupae and the moth stages. The organs, posterior/middle division of silkgland, midgut, fat body and malpighian tubes, also show relatively high mRNA expression levels, respectively. The high degree of conservation and expression of the B. mandarina QM homologous suggest that it has a selectively conserved amino acid sequence due, presumably, to an important biological role which is associated with pupae formation.     

Identification of highly conserved genes: SNZ and SNO in the marine sponge Suberites domuncula: their gene structure and promoter activity in mammalian cells(1)

Recently, we reported that cells from the sponge Suberites domuncula respond to ethylene with an increase in intracellular Ca(2+) level [Ca(2+)](i), and with an upregulation of the expression of (at least) two genes, a Ca(2+)/calmodulin-dependent protein kinase and the potential ethylene-responsive gene, termed SDSNZERR (A. Krasko, H.C. Schr?der, S. Perovic, R. Steffen, M. Kruse, W. Reichert, I.M. Müller, W.E.G. Müller, J. Biol. Chem. 274 (1999)). Here, we describe for the first time that also mammalian (3T3) cells respond to ethylene, generated by ethephon, with an immediate and transient, strong increase in [Ca(2+)](i). Next, the promoter for the sponge SDSNZERR gene was isolated from S. domuncula. It was found that the SDSNZERR gene is positioned adjacent to the SNZ-related gene (SNZ-proximal open reading frame) (SDSNO) and linked, as in Saccharomyces cerevisiae, in a head-to-head manner. Until now, neither homologues nor orthologues of these two genes have been identified in higher metazoan phyla. The full-length genes share a bidirectional promoter. 3T3 cells were transfected with this promoter; the activity of the SDSNZERR promoter was strong and twice as high as that of the SV40 promoter, while the SDSNO promoter was less active. Surprisingly, the activity of the SDSNZERR promoter could not be modulated by ethylene or salicylic acid while it is strongly upregulated, by 4-fold, under serum-starved conditions. It is concluded that the modulation of the level of [Ca(2+)](i) by ethylene in mammalian cells is not correlated with an upregulation of the ethylene-responsive gene SDSNZERR. The data indicate that in mammalian cells, the activity of the SDSNZERR promoter is associated with the repression of serum-mediated growth arrest.     

Origin of insulin receptor-like tyrosine kinases in marine sponges

One autapomorphic character restricted to all Metazoa including Porifera [sponges] is the existence of transmembrane receptor tyrosine kinases (RTKs). In this study we screened for molecules from one subfamily within the superfamily of the insulin receptors. The subfamily includes the insulin receptors (InsR), the insulin-like growth factor I receptors, and the InsR-related receptors--all found in vertebrates--as well as the InsR-homolog from Drosophila melanogaster. cDNAs encoding putative InsRs were isolated from the hexactinellid sponge Aphrocallistes vastus, the demosponge Suberites domuncula, and the calcareous sponge Sycon raphanus. Phylogenetic analyses of the catalytic domains of the putative RTKs showed that the sponge polypeptides must be grouped with the InsRs. The relationships revealed that all sponge sequences fall into one branch of this group, whereas related sequences from mammals (human, mouse, and rat), insects and molluscs, and polypeptides from one cephalochordate, fall together into a second branch. We have concluded that (i) the InsR-like molecules evolved in sponges prior to the "Cambrian Explosion" and contributed to the rapid appearance of the higher metazoan phyla; (ii) the sponges constitute a monophyletic taxon, and (iii) epidermal growth factor (EGF)-like domains are present in sponges, which allows the insertion of this domain into potential receptor and matrix molecules.     

Expression of one sponge Iroquois homeobox gene in primmorphs from Suberites domuncula during canal formation

Sponges (Porifera) represent the evolutionary oldest multicellular animals. They are provided with the basic molecules involved in cell-cell and cell-matrix interactions. We report here the isolation and characterization of a complementary DNA from the sponge Suberites domuncula coding for the sponge homeobox gene, SUBDOIRX-a. The deduced polypeptide with a predicted Mr of 44,375 possesses the highly conserved Iroquois-homeodomain. We applied in situ hybridization to localize Iroquois in the sponge. The expression of this gene is highest in cells adjacent to the canals of the sponge in the medulla region. To study the expression of Iroquois during development, the in vitro primmorph system from S. domuncula was used. During the formation of these three-dimensional aggregates composed of proliferating cells, the expression of Iroquois depends on ferric iron and water current. An increased expression in response to water current is paralleled with the formation of canal-like pores in the primmorphs. It is suggested that Iroquois expression is involved in the formation of the aquiferous system, the canals in sponges and the canal-like structures in primmorphs.     

Sphingosine-induced c-jun expression: differences between sphingosine- and C2-ceramide-mediated signaling pathways

Sphingolipids such as ceramide and sphingosine are putative intracellular signal mediators in cell differentiation, growth inhibition and apoptosis. Previously, we reported that C2-ceramide induced c-jun expression in apoptosis of human leukemia HL-60 cells. Here we report that sphingosine also induced c-jun expression in apoptosis of HL-60 cells. Sphingosine-induced c-jun expression was stimulated by H-89, a protein kinase A inhibitor, whereas C2-ceramide-induced c-jun expression was inhibited by protein kinase C inhibitors. Furthermore, H-89 potentiated sphingosine-induced but not C2-ceramide-induced growth inhibition. These results suggest that sphingosine and C2-ceramide might induce c-jun expression and apoptosis in distinct signaling pathways.     

Tissue-/stage-dependent expression of a cloned Bombyx mandarina QM homologue

QM, a novel gene that was firstly isolated as a putative tumor suppressor gene from Wilms’ tumor cell line. Although it is well known that the QM gene product plays an important role within the tumor cells, the precise role of QM in the non-tumor cells has remained elusive. With in this mind we isolated a cDNA encoding QM homologue from Bombyx mandarina to understand the function of QM. The 596 bp cDNA has an open reading frame of 219 amino acids and a predicted mol. wt. of 25 kDa. The protein has more than 88% amino acid sequence identity to the QM protein from Drosophila melanogaster. mRNA expression gradually increased from 1–2 days after egg laying to 2 days of finial instar, while very low expressions were detected for either the pupae and the moth stages. The organs, posterior/middle division of silkgland, midgut, fat body and malpighian tubes, also show relatively high mRNA expression levels, respectively. The high degree of conservation and expression of the B. mandarina QM homologous suggest that it has a selectively conserved amino acid sequence due, presumably, to an important biological role which is associated with pupae formation.