Keyhole Limpet Megathura Crenulata Research Articles

Genetic Insights into the Giant Keyhole Limpet (Megathura crenulata), an Eastern Pacific Coastal Endemic: Complete Mitogenome, Phylogenetics, Phylogeography, and Historical Demography

Background: The giant keyhole limpet Megathura crenulata is a gastropod mollusk (Fissurella superfamily) that is endemic to the eastern Pacific coast from southern California, USA, to Baja California Sur, Mexico. M. crenulata is socioeconomically important as it produces a potent immune-stimulating protein, called Keyhole Limpet Hemocyanin, which is extracted in vivo and utilized for vaccine development. However, ecological studies are scarce and genetic knowledge of the species needs to be improved. Our objectives were to assemble and annotate the mitogenome of M. crenulata, and to assess its phylogenetic relationships with other marine gastropods and to evaluate its population genetic diversity and structure. Methods: Samples were collected for mitogenome assembly (n = 3) spanning its geographic range, Puerto Canoas (PCA) and Punta Eugenia (PEU), Mexico, and California (CAL), USA. Total DNA was extracted from gills sequenced using Illumina paired-end 150-bp-read sequencing. Reads were cleaned, trimmed, assembled de novo, and annotated. In addition, 125 samples from eight locations were analyzed for genetic diversity and structure analysis at the 16s rRNA and COX1 genes. Results: The M. crenulata mitogenomes had lengths of 16,788 bp (PCA) and 16,787 bp (PEU) and were composed of 13 protein-coding regions, 22 tRNAs, two rRNAs, and the D-Loop region. In terms of phylogeographic diversity and structure, we found a panmictic population that has experienced recent demographic expansion with low nucleotide diversity (0.002), high haplotypic diversity (0.915), and low φST (0.047). Conclusions: Genetic insights into the giant keyhole limpet provides tools for its management and conservation by delimiting fishing regions with low genetic diversity and/or genetically discrete units.

Selective Cytotoxicity and Changes in Protein Expression of T24 Bladder Carcinoma Permanent Cell Line after Treatment with Hemocyanins.

Some molluscan hemocyanins (Hcs) have significant immunological and antitumor potential, enabling their application in oncology. The antitumor activity of Hcs from marine snails Rapana venosa (RvH), giant keyhole limpet Megathura crenulata (KLH) and garden snails Helix lucorum (HlH), as well as their different derivatives, were studied in vitro on a permanent T24 cell line of bladder cancer and normal urothelial cell line HL 10/29 compared to doxorubicin. The antiproliferative activity of the tested Hcs was determined using the WST-1 assay and BrdU ELISA assay. Morphological changes in both urothelial cell lines were confirmed by fluorescence microscopy. The proteomic analysis of a bladder cancer cell line before and after treatment with functional unit (FU) βc-HlH-h using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry revealed differences in the expression of some proteins. Studies prove that the T24 tumor cell line is dose- and time-dependent, sensitive to the action of the tested isoforms, and it glycosylated FU of these hemocyanins. Selective inhibition of T24 cell growth was observed after incubation with structural subunits (βc-HlH, RvHI and RvHII) and FUs (βc-HlH-h and RvHII-e). Additionally, fluorescent microphotographs did not show apoptotic or necrotic alterations in the normal urothelial cell line HL 10/29. The FU βc-HlH-h demonstrated the highest antiproliferative effect (similarly to doxorubicin), in which predominantly apoptotic and less late apoptotic or necrotic changes in the tumor cells were observed. Several downand up-regulated proteins identified by proteome analysis may be associated with the apoptosis pathway. The present study illustrated the selectivity of the cytotoxic effect of Hcs against the Т24 cancer cell line. This is the first report of protein expression in T24 human bladder cancer cells under the influence of FU βc-HlH-h. That is probably due to the specific oligosaccharide structures rich in methylated hexoses exposed on the surface of βc-HlH-h.

Morphology and function of the skin epithelium covering the giant keyhole limpet Megathura crenulata

AbstractMucus‐secreting epithelia protect marine gastropods from abrasive particles and microbes in seawater. We studied the morphology and function of the epithelium in the giant keyhole limpet Megathura crenulata. All exposed surfaces of the limpets were covered by a mucus‐secreting, simple columnar epithelium, in which most cells on the sole of the foot bore cilia, while the majority of cells on the side of the foot and three mantle regions bore microvilli. None of the coatings had antibacterial properties. The epithelium of the foot was distinct from that of other regions, in that the mucus secreted was used for locomotion and was left behind as the limpet moved. The glycocalyx bound to the microvilli of the mantle cells appeared to be clean in visual and SEM examinations, and attempts to enhance the binding of inert particles and bacteria were unsuccessful. Because studies have shown that standard tissue processing may cause artifactual shrinkage of mucous layers, cryostat sections and additives to standard fixatives were tested, but we found no change in the thickness of the mucous layers of the limpets compared to routinely processed tissues. Sloughing of the glycocalyx in vertebrate systems removes bound microbes, and alterations of the glycocalyx layers are associated with disease conditions. Sloughing of the glycocalyx on limpets was rarely observed. In one case, the outer mantle of the limpet was covered with silt and the glycocalyx appeared to be detaching. This process could be experimentally induced by dousing the outer mantle with talc particles. The types of secretory cells producing the mucus in each region of the skin were characterized using standard histological stains and lectin staining. Because analysis was hampered by their small size, classification of the types of secretory cells was based on TEM descriptions of granule morphology, which allowed for comparison to the secretory cells described from skin of the abalone. The possible roles of the numerous secretions, and the mechanisms of mucus production and loss, are compared to what is known in vertebrate systems and to our relative lack of knowledge regarding invertebrate systems.

Trophic Relationships between Two Gastropods and Seaweeds in Subtropical Rocky Reefs Based on Stable Isotope Analyses

ABSTRACT The wavy turban snail Megastraea undosa and keyhole limpet Megathura crenulata, two gastropods with commercial importance, five macroalgae, and one seagrass were collected at Bahia Tortugas, Baja California Sur, Mexico, for analysis of stable isotope ratios. The δ13C and δ15N isotope signatures obtained from gastropod muscle tissue were different between the two gastropods. Isotope signatures of M. crenulata had more enriched δ13C (-14.35‰ ± 1.66‰) and δ15N (15.48‰ ± 0.67‰) than M. undosa isotope signatures for δ13C (-17.59‰ ± 2.43‰) and δ15N (12.42‰ ± 0.61‰). Seaweed isotope signals varied from -21.8‰ to -11.4‰ for δ13C and 9.3‰ to 10.9‰ for δ15N. The δ13C of detritus was -7.93 and of tunicates was -9.3‰ and the δ15N of detritus was 12.4 and of tunicates was 8.1‰. Mixing models showed that the greatest contribution to the diet of M. undosa was from Prionitis cornea and Gelidium robustum. For M. crenulata, it was from detritus and Macrocystis pyrifera. A higher trophic position (3.1) was observed...

Diet of the Keyhole LimpetMegathura crenulata(Mollusca: Gastropoda) in Subtropical Rocky Reefs

ABSTRACT The diet of the keyhole limpet Megathura crenulata was determined from stomach contents of 119 specimens collected from 3 rocky reefs on the west coast of the Baja California Peninsula, Mexico, in July and November 2006 and March 2007. Limpet stomachs contained 78 taxa, including filamentous cyanobacteria, diatoms, brown and red algae, marine seagrass, foraminifera, hydrozoans, bryozoans, nematodes, bivalves, gastropods, crustaceans, and tunicates. The main food items (>10% index of relative importance) consumed by the limpet were a tunicate; red algae of the genera Callophyllis, Coralina, Cryptopleura, Nienburgia, Pterosiphonia, and Rhodymenia; the brown algae Cystoseira osmundacea; the hydrozoan Eudendrium; and the bryozoan Crisia. Despite the differences in composition and abundance of the main items among sites and dates, the food groups of tunicates and algae account for more than 80% index of relative importance of the diet. The trophic niche breadth (Levin's index) was 0.52 ± 0.20. Our fin...

Behavioral thermoregulation and critical thermal limits of giant keyhole limpet Megathura crenulata(Sowerby 1825) (Mollusca; Vetigastropoda)

The thermoregulatory behavior of the giant keyhole limpet Megathura crenulata was determined in a horizontal thermal gradient during the day at 18.9°C and 18.3°C for the night. The final preferendum determined for giant keyhole limpets was of 18.6±1.2°C.Limpets' displacement velocity was 10.0±3.9cmh−1 during the light phase and 8.4±1.6cmh−1 during the dark phase. The thermotolerance (measured as CTMax at 50%) was determined in a keyhole limpet in three acclimation temperatures 17, 20, and 23°C. Limpets were subjected to water increasing temperatures at a rate of 1°C every 30min, until they detached from the substrate. The critical thermal maximum at 50% was 27.2, 27.9 and 28.3°C respectively.

Production of digestive enzymes along the gut of the giant keyhole limpet Megathura crenulata (Mollusca: Vetigastropoda)

The esophagus and intestine form the longest regions of the digestive tract in the giant keyhole limpet and are lined by epithelial cells sharing a common morphology and releasing materials into the gut lumen by apocrine secretion. The purpose of this study was to determine if these morphologically similar regions release similar digestive enzymes and compare their contributions to digestive enzymes released from other regions of the gut. Principal component analysis of enzymes detected by the API ZYM system for 19 enzymes plus EnzChek assays for protease, α-amylase, lipase, cellulase, and lysozyme identify four distinct regions of the gut: 1) crystalline style and style sac, 2) digestive gland, 3) salivary glands, and 4) esophagus and intestine. Heterogeneity in enzymatic activity was observed in regions of the gut with similar cell morphology (middle and posterior esophagus and intestine) as well as regions with different cell morphology (salivary glands, digestive gland and crystalline style). Enzyme activity in each of these regions is compared to other gastropods, in particular the abalone. Although much of the length of the digestive tract is lined by a morphologically similar epithelium, different regions of the alimentary tract produce a different suite of enzymes which may contribute to the digestive process. These data will help enhance our limited understanding of the digestive physiology of Megathura crenulata and lead to improvement of its culture for clinical research.

Synthesis of 2-aminoethyl 3′-(N-glycolylneuraminyl)-β-actoside and neoglycoconjugates based thereon — a prototype of a cancer vaccine and artificial antigens for the control of immune response

The 2-aminoethyl β-glycoside of the trisaccharide 3′-(N-glycolylneuraminyl)lactose was synthesized in the framework of studies of approaches to the preparation of synthetic tumor vaccines based on carbohydrate chains of tumor-associated gangliosides and converted to the corresponding 2-4-maleimidobutanoylamino)ethyl glycoside. The reaction of the latter with thiolated hemocyanin from the keyhole limpet Megathura crenulata (KLH) gave a carbo-hydrate—protein conjugate, a tumor vaccine prototype containing about 250 trisaccharide ligands attached to a macromolecular KLH complex. A polyvalent conjugate with a polyacryl-amide support was synthesized to be used as a coating antigen in the control of specificity and efficiency of immune response by enzyme-linked immunosorbent assay (ELISA).

Identification and Characterization of Keyhole Limpet Hemocyanin N-Glycans Mediating Cross-reactivity with Schistosoma mansoni

Keyhole limpet hemocyanin (KLH) of the mollusc Megathura crenulata is known to serologically cross-react with Schistosoma mansoni glycoconjugates in a carbohydrate-dependent manner. To elucidate the structural basis for this cross-reactivity, KLH glycans were released from tryptic glycopeptides and fluorescently labeled. Cross-reacting glycans were identified using a polyclonal antiserum reacting with soluble S. mansoni egg antigens, isolated by a three-dimensional fractionation scheme and analyzed by different mass spectrometric techniques as well as linkage analysis and exoglycosidase treatment. The results revealed that cross-reacting species comprise approximately 4.5% of released glycans. They all represent novel types of N-glycans with a Fuc(alpha1-3)GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc motif, which is known to occur also in schistosomal glycoconjugates. The tetrasaccharide unit is attached to the 3-linked antenna of a trimannosyl core, which can be further decorated by galactosyl residues, a xylose residue in 2-position of the central mannose and/or a fucose at the innermost N-acetylglucosamine. This study provides for the first time detailed structural data on the KLH carbohydrate entities responsible for cross-reactivity with glycoconjugates from S. mansoni.

Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and htH.

Keyhole limpet hemocyanin (KLH) is a well-established immune stimulant and hapten carrier, and Haliotis tuberculata hemocyanin (HtH) is a related product. Biologically, KLH and HtH are blue copper proteins which serve as oxygen carriers in the blood of the keyhole limpet Megathura crenulata and the abalone H. tuberculata, respectively, two marine gastropods. Both hemocyanins occur as two distinct isoforms, termed KLH1 KLH2, HtH1, and HtH2. Each of these molecules is based on a very large polypeptide chain, the subunit (molecular mass ca 400 kDa), which is folded into a series of eight globular functional units (molecular mass ca 50 kDa each). Twenty copies of this subunit form a cylindrical quaternary structure (molecular mass ca 8 MDa). This article reviews the recent data on the biosynthesis, quaternary structure, subunit architecture, amino acid sequence, gene structure, and recombinant production of KLH and HtH.

Controlled cleavage of KLH1 and KLH2 by the V8 protease from Staphylococcus aureus reassociation, electrophoretic and transmission electron microscopy study of peptide fragments.

The reassociation behaviour of protease V8-cleaved peptides from KLH1 and KLH2, the two hemocyanin isoforms from the giant keyhole limpet Megathura crenulata, has been studied by transmission electron microscopy of negatively stained specimens and SDS/PAGE. Reassociation of the complete mixture of protease cleavage products and of combinations of peptide fragments purified by HPLC was performed in the presence of 100 mm CaCl2 and 100 mm MgCl2 at pH 7.4, over a period of 1 to 4 weeks. The V8 protease splits KLH1 into peptide fragments containing the functional units abc, def, defg, defgh, g and h. This mixture of peptide fragments reassociated to form helical tubular polymers, with a diameter of approximately 25 nm. The single functional units g and h were not incorporated into the polymer. An essentially identical polymer was formed from the re-mixed HPLC-purified fragments abc, def and defg alone. As with uncleaved subunit, the tubular polymer of V8-cleaved KLH1 forms bundles. The combination of peptides def and defg led to the formation of short arc-like filamentous structures, which aggregated but showed little tendency to associate into larger polymers. The KLH1 peptide fragments abc and def alone, did not reassociate and in combination their potential to form polymers was very low. With KLH2, the V8 protease generated peptide fragments containing the functional units abc, defg, defgh and h, which in combination slowly reassociated to form a tubular polymer significantly different to that obtained from the KLH1 V8 fragments. The three-functional unit fragment abc from KLH2 showed no tendency to polymerize and the combination of peptides defg + defgh generated only disordered aggregates, with some indication of malformed tubules. The combination of biochemical and electron microscopical methods enabled the characterization of these polymers with respect to peptide composition and higher order structure.

Negative Stains Containing Trehalose: Application to Tubular and Filamentous Structures

Several examples are presented that show the successful application of uranyl acetate and ammonium molybdate negative staining in the presence of trehalose for TEM studies of filamentous and tubular structures. The principal benefit to be gained from the inclusion of trehalose stems from the considerably reduced flattening of the large tubular structures and the greater orientational freedom of single molecules due to an increased depth of the negative stain in the presence of trehalose. Trehalose is likely to provide considerable protection to protein molecules and their assemblies during the drying of negatively stained specimens. Some reduction in the excessive density imparted by uranyl acetate around large assemblies is also achieved. Nevertheless, in the presence of 1% (w/v) trehalose, it is desirable to increase the concentration of negative stain to 5% (w/v) for ammonium molybdate and to 4% for uranyl acetate to produce satisfactory image contrast. In general, the ammonium molybdate-trehalose negative stain is more satisfactory than the uranyl acetate-trehalose combination, because of the greater electron beam sensitivity of the uranyl negative stain. Reassembled taxol-stabilized pig brain microtubules, together with collagen fibrils, sperm tails, helical filaments, and reassociated hemocyanin (KLH2), all from the giant keyhole limpet Megathura crenulata, have been studied by negative staining in the presence of trehalose. In all cases satisfactory TEM imaging conditions were readily obtained on the specimens, as long as regions of excessively deep stain were avoided.

Electron microscopy and biochemical characterization of a 350-kDa annular hemolymph protein from the keyhole limpet Megathura crenulata.

The isolation and biochemical characterization of an annular non-hemocyanin hemolymph protein from a marine gastropod, the Californian giant keyhole limpet (Megathura crenulata) is presented. By analytical ultracentrifugation, the protein has a sedimentation coefficient of 12S and molecular mass of approximately 350 kDa. The subunit mass, obtained by SDS/PAGE in the presence of -SH reagent and 8 M urea, is approximately 35 kDa, thereby indicating the presence of 10 subunits in the native molecule. By negative staining, the protein is revealed in one predominant image projection as a pentagonal approximately 8 nm ring-like structure with an approximately 2-nm stain-filled centre and, in another image projection, as a dimeric rectangular structure, approximately 8 nm x 14 nm. In deep stain, each of the components of the side-on dimer also shows an indication of stain penetration within a central channel. Unlike the hemolymph protein limulin/C-reactive protein from the horseshoe crab Limulus polyphemus, this Megathura protein does not possess any potential for the aggregation of phosphatidylcholine liposomes although it has an affinity for this phospholipid and causes bilayer destruction. It now appears likely that the previously reported lipid-bilayer ionic-conductance species in keyhole limpet hemolymph, attributed to hemocyanin, could indeed by due to the presence of this annular protein.

Electron microscopy of a double helical tubular filament in keyhole limpet (Megathura crenulata) hemolymph.

A approximately 25 nm hollow double helical filament has been detected ultrastructurally in the cell-free supernatant from hemolymph of the keyhole limpet Megathura crenulata (Gastropoda: Prosobranchia: Fissurellidae). Subsequently, much higher concentrations of this material were found in the cell pellet from hemolymph. Both negative staining and thin sectioning have been performed in an attempt to obtain a preliminary structural characterization of this new filament. It is proposed that the filaments are released or secreted from blood hemocytes in response to bleeding, but it has not been possible to define absolutely an intracellular organelle containing this material. It is shown that the hollow filaments, which have fine fibrillar surface extensions, are readily distinguishable from collagen fibrils and from tubular (multi-decameric) keyhole limpet hemocyanin (KLH).

Two-dimensional crystallization, transmission electron microscopy and image processing of keyhole Limpet Haemocyanin (KLH)

KLH, the respiratory protein haemocyanin of the keyhole limpet Megathura crenulata, has been obtained as a high g pellet from the cell-free haemolymph and purified by gel permeation chromatography on a Biogel A15m column. The leading major protein peak eluted has been found to contain haemocyanin multi-decamers, followed by a second major peak containing single di-decamers, with small amounts of decamer and partly dissociated material following in the later fractions. The purified KLH di-decamer has been for two- dimensional crystallization studies with the negative staining-carbon film technique, in the presence of polyethylene glycol. In the side-on orientation, KLH has been found to produce two-dimensional crystals with a half-molecule linear displacement in consecutive rows. This is shown to be due to a specific association and two-dimensional crystal nucleation of the molecules in this arrangement. When oriented end-on, KLH has been found to form close- packed hexagonal monomolecular arrays which are not truly crystalline. This is because the five-fold rotational symmetry of the cylindrical macromolecule is not readily compatible with the hexagonal molecular packing. Computer-processed averaged images have been produced from the side-on KLH two-dimensional crystals and the end-on arrays, which reveal the principal molecular features of this homo-oligomeric protein complex to a resolution of ca 2.7 nm.

The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. I. Chemical composition and structural studies.

The egg vitelline envelope of the marine invertebrate Megathura crenulata is a glycoprotein composed of 37.3 mol % protein and 62.7 mol % carbohydrate. Of the total amino acid content, 61 mol % consists of a single amino acid, threonine. The carbohydrate content includes galactosamine, galactose, and fucose. The molar ratio of threonine to galactosamine is about 1:1. Most of the threonine residues are linked to galactosamine residues via O-glycosidic bonds. A single peptide that was purified following alkaline borohydride treatment of the vitelline envelope had the structure: Abu-Pro-Abu-(Abu6, Pro1, Thr1), where Abu is 2-aminobutyric acid. Several sugar residues have been isolated following the alkaline hydrolysis of the vitelline envelope that include an octasaccharide Gal4Fu4, an hexasaccharide Gal3Fu3, a trisaccharide Gal3, fucose, and galactose. It is proposed that the vitelline envelope of Megathura crenulata eggs is composed of polypeptide chains built to a large extent of closely spaced threonine residues. Almost every threonine residue is linked to a saccharide moiety.

The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. II. Products formed by lysis with sperm enzymes and dithiothreitol.

The egg vitelline envelope of the marine invertebrate, Megathura crenulata, was lyzed either by sperm lysins A, B, C or by dithiothreitol. In each case the lysis mixture consisted of two major fractions, I and II, that could be separated by hydroxylapatite chromatography and had different electrophoretic mobilities on cellulose acetate strips. The amino acid, amino sugar, and neutral sugar compositions of fractions I and II were similar and resembled that of the intact vitelline envelope. Fractions I and II of each lysis mixture emerged in the exclusion volume of a Sepharose 6B column. A vitelline envelope fragment enzymatically formed by lysin was further degraded by dithiothreitol to form smaller fragments. A model of the vitelline envelope of the Megathura crenulata egg is suggested whereby the envelope is composed of polypeptide chains cross-linked by disulfide bonds and built to a large extent of closely spaced threonine residues. Most of the threonine residues are linked to carbohydrate units. Dithiothreitol dissolves the envelope by reducing disulfide bonds, whereas lysins most likely dissolve the envelope by degrading polypeptide chains.

Some Effects of Sperm Extracts on the Eggs of Mytilus

Next article No AccessConference on Problems of General and Cellular Physiology Relating to Fertilization. IISome Effects of Sperm Extracts on the Eggs of MytilusWilliam E. BergWilliam E. Berg Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The American Naturalist Volume 83, Number 812Sep. - Oct., 1949 Published for The American Society of Naturalists Article DOIhttps://doi.org/10.1086/281601 Views: 6Total views on this site Citations: 5Citations are reported from Crossref PDF download Crossref reports the following articles citing this article:A.N. Sastry PELECYPODA (EXCLUDING OSTREIDAE), (Jan 1979): 113–292.https://doi.org/10.1016/B978-0-12-282505-7.50012-9Charles B. Metz Gamete Surface Components and Their Role in Fertilization**Contribution No. 063 from the Institute of Molecular Evolution. During preparation of this manuscript, support was received from the National Aeronautics and Space Administration (NsG 689), the National Science Foundation (GB 3899), and the National Institutes of Health (5T1 HD 26-04)., (Jan 1967): 163–236.https://doi.org/10.1016/B978-1-4832-2776-4.50011-3S.K. Wada, J.R. Collier, J.C. Dan Studies on the acrosome, Experimental Cell Research 10, no.11 (Feb 1956): 168–180.https://doi.org/10.1016/0014-4827(56)90083-0Jean C. Dan The Acrosome Reaction, (Jan 1956): 365–393.https://doi.org/10.1016/S0074-7696(08)62576-0Max Krauss Lytic agents of the sperm of some marine animals. I. The egg membrane lysin from sperm of the giant keyhole limpet Megathura crenulata, Journal of Experimental Zoology 114, no.22 (Jul 1950): 239–278.https://doi.org/10.1002/jez.1401140203

EVIDENCE FOR THE PROTEIN NATURE OF THE SPERM AGGLUTININS OF THE KEYHOLE LIMPET AND THE SEA-URCHIN

Some of the chemical and physical properties of the sperm agglutinin (fertilizin) of the keyhole limpet Megathura crenulata and of the sea-urchin Strongylocentrotus purpuratus were investigated. In both species the agglutinins were found to be non-dialyzable. They precipitate completely without loss of activity in nearly saturated ammonium sulfate. They are adsorbed by CaCO3, Al2O3, charcoal and kaolin. The agglutinins are insoluble in alcohol and ether. Active concentrates give, contrary to the findings of earlier investigators, the common color tests for proteins and are found to contain nitrogen. Solutions of crystallized proteinases (trypsin and chymotrypsin) inactivate the agglutinins. They are also inactivated by heat as a function of pH. From the evidence it is concluded that in both species the agglutinating principle is either protein or very closely associated with protein.The keyhole limpet agglutinin is much more resistant than is that of the sea-urchin to inactivation by heat and by proteolytic enzymes. It is suggested that the difference between the two species in the duration of the agglutination reaction may be related to the difference in stability of the respective agglutinins.