Arthropods Crustaceans Research Articles

Halorina cryptica nov. ichnogen., nov. ichnosp., mass-occurrence of Upper Triassic crustacean microcoprolites from neptunian dikes and sills cutting the Dachstein-type carbonate platform and their paleoenvironmental significance (Northern Apuseni Mountains, Romania)

The new crustacean microcoprolite Halorina cryptica nov. ichnogen., nov. ichnosp., is reported from cryptic cavities cutting the Upper Triassic (Carnian to lower Rhaetian) Dachstein-type limestones from the Northern Apuseni Mountains, Romania. The new ichnotaxon is extremely abundant in cavities, neptunian dikes and sills filled with red ferruginous carbonate sediment. The associated microfauna consists of ostracods and rare foraminifera. The microfacies is represented by bioclastic coprolite-bearing wackestone-packstone to grainstone. The red ferruginous carbonate fillings are strongly bioturbated. The neptunian sill located at the top of the studied section contains a rich ichnofauna associated with brachiopod accumulations. It is dominated by the dimerelloid rhynchonellid Halorella, indicating a late Norian-early Rhaetian age. Although neptunian dikes and sills are rather common in the Dachstein-type carbonate platform that extended on the northwestern Tethyan margin, we report here the first record of mass-occurrence of crustacean microcoprolites in neptunian dikes and sills filled with red carbonate sediments of marine origin. They are interpreted here as cryptic cavities with specific palaeoenvironmental features (e.g., lack of light, abundant nutrients, intensive microbial activity) where crustacean arthropods thrived.

Efficiency of invertebrate animals for risk assessment and biomonitoring of hazardous contaminants in aquatic ecosystem, a review and status report.

Invertebrate animals exhibited successful detoxification and storage of hazardous inorganic contaminants. Moreover, waterflea Daphnia sp., other crustacean arthropods and molluscan species were implied as sentinel organisms for aquatic contaminants. Benthic invertebrates might be used as biological monitors for heavy metal pollution. They spend most of their lives at the contaminated site. They can determine the concentration of existed heavy metal pollution. A variation in the efficiency of heavy metal accumulation was shown between different invertebrate groups. Molluscan animals specially gastropods showed the greatest capability to bioaccumulate heavy metals within their digestive cells. They acquired higher bioaccumulation factor relative to arthropods and annelids. At the cellular level, heavy metals might be stored after detoxification as granules of different shapes and sizes or as lipid droplets. The present review discusses the capability of different invertebrate groups to detoxify and store inorganic contaminants and their efficiency as possible biomonitor organisms for aquatic ecosystem.

Ostracods: The ultimate survivors

Ostracods are tiny crustacean arthropods just a few millimetres long, with a bivalved carapace made of calcium carbonate that covers the whole body, and into which the animal can retreat from the world outside. Because of their diminutive size they are largely overlooked as fossils, but they have a fascinating history. Silent witnesses to life in the seas since the time of trilobites, they have a fossil record extending back to the Early Ordovician, and possibly the Cambrian. Ostracods have survived nearly 500 million years of Earth history including the ‘big five’ mass extinctions of the Phanerozoic Eon; they are true survivors. They are almost perfectly adapted for the aquatic environments in which they live, and can be found from the ocean abyssal plains to damp leaf litter. The ostracod carapace is a triumph of biological engineering that has been re‐configured into myriad different morphologies according to environment. Streamlined and agile species plough through the ocean water column, sometimes reaching a ‘giant’ size of a centimetre in length, whilst their tinier sea bottom cousins make elaborately ornamented carapaces to withstand the pressures of living at the seabed, or shape their carapaces into forms that facilitate burrowing into sediment. Ostracods are key components of aquatic ecosystems. As primary consumers they are food for larger animals both in seabed and planktonic habitats, and they recycle much of the organic detritus produced by larger animals and plants. Delve into the history of ostracods and it is possible to find pioneers who triumphed in the plankton, early colonisers of terrestrial aquatic ecosystems, and ostracods that literally conquered the land. And in more recent times, ostracods have even hitched rides on rockets into space.

Colonization of formulated substrates of different composition by benthic macroinvertebrate community in an estuarine ecosystem: a case study of response of benthic invertebrates to substrate alteration

Colonization of formulated artificial substrate was investigated in a tropical estuarine ecosystem for six months to determine possible response of benthic macroinvertebrates to disturbance arising from substrate alteration. Five different formulated substrates which served as content for ten cages were used for this experiment. A total of 406 individuals comprising 18 macroinvertebrate species colonized the cages. In overall, cage 1 which is the control recorded the highest number of individuals and average number (8) of colonizing species at a time, while the least number (4) was observed in cage 3. Cages 2, 4, and 5 recorded average of five species each. In terms of number of individuals, cage 1 which is the control, had the highest number (153), followed by cage 2 which recorded 122 individuals. Cages 3, 4 and 5, recorded 41, 61 and 80 individuals respectively. Analysis of temporal variability in the number of species observed in the experimental cages shows that there was a progressive decrease in the number of colonizing species after the second month. Colonizing macroinvertebrate groups were not different from the known resident species in the estuarine lagoon. However, pattern of variation in the number of individuals of colonizing species were remarkably different. The assemblage recorded in this experiment was defined by the populations of two bivalve mollusc; Macoma cumana and Tellina nymphalis , two gastropod mollusc; Pachymelina aurita and Tympanotonus fuscatus and two crustacean arthropods; Clibanarius africanus and Penaeus notialis . Among the FFGs recorded, collector-filterers dominated in number of individuals and species. Nutrient concentrations of formulated substrate had strong effects on number of colonizing individuals and species.

Novel membrane-associated prostaglandin E synthase-2 from crustacean arthropods.

Prostaglandins (PG) have been shown to play important physiological roles in insects and marine invertebrates, yet the knowledge of their biosynthetic pathways is often lacking. Recently, we described cyclooxygenases in two amphipod crustaceans, Gammarus sp. and Caprella sp. In the present study, we report the cloning and characterization of prostaglandin E synthases (PGES) from the same organisms. The amphipod membrane-bound PGES-2-type enzymes share about 40% of the amino acid sequence identity with human mPGES-2, contain a conserved Cys110-x-x-Cys113 motif and have very low heme-binding affinity. The recombinant enzymes purified in the absence of dithiothreitol specifically catalyze the isomerization of PGH2 into PGE2. The PGES activity is increased in the presence of reduced glutathione and inhibited with a sulfhydryl group inhibitor. We assume that the amphipod mPGES-2, unlike in their mammalian counterparts, is responsible for PGE2 synthesis, not only in vitro but also in vivo.

Is the species flock concept operational? The Antarctic shelf case.

There has been a significant body of literature on species flock definition but not so much about practical means to appraise them. We here apply the five criteria of Eastman and McCune for detecting species flocks in four taxonomic components of the benthic fauna of the Antarctic shelf: teleost fishes, crinoids (feather stars), echinoids (sea urchins) and crustacean arthropods. Practical limitations led us to prioritize the three historical criteria (endemicity, monophyly, species richness) over the two ecological ones (ecological diversity and habitat dominance). We propose a new protocol which includes an iterative fine-tuning of the monophyly and endemicity criteria in order to discover unsuspected flocks. As a result nine « full » species flocks (fulfilling the five criteria) are briefly described. Eight other flocks fit the three historical criteria but need to be further investigated from the ecological point of view (here called « core flocks »). The approach also shows that some candidate taxonomic components are no species flocks at all. The present study contradicts the paradigm that marine species flocks are rare. The hypothesis according to which the Antarctic shelf acts as a species flocks generator is supported, and the approach indicates paths for further ecological studies and may serve as a starting point to investigate the processes leading to flock-like patterning of biodiversity.

DNA barcoding of Arctic Ocean holozooplankton for species identification and recognition

Zooplankton species diversity and distribution are important measures of environmental change in the Arctic Ocean, and may serve as ‘rapid-responders’ of climate-induced changes in this fragile ecosystem. The scarcity of taxonomists hampers detailed and up-to-date monitoring of these patterns for the rarer and more problematic species. DNA barcodes (short DNA sequences for species recognition and discovery) provide an alternative approach to accurate identification of known species, and can speed routine analysis of zooplankton samples. During 2004–2008, zooplankton samples were collected during cruises to the central Arctic Ocean and Chukchi Sea. A ∼700 base-pair region of the mitochondrial cytochrome oxidase I (mtCOI) gene was amplified and sequenced for 82 identified specimens of 41 species, including cnidarians (six hydrozoans, one scyphozoan), arthropod crustaceans (five amphipods, 24 copepods, one decapod, and one euphausiid); two chaetognaths; and one nemertean. Phylogenetic analysis used the Neighbor-Joining algorithm with Kimura-2-Parameter (K-2-P) distances, with 1000-fold bootstrapping. K-2-P genetic distances between individuals of the same species ranged from 0.0 to 0.2; genetic distances between species ranged widely from 0.1 to 0.7. The mtCOI gene tree showed monophyly (at 100% bootstrap value) for each of the 26 species for which more than one individual was analyzed. Of seven genera for which more than one species was analyzed, four were shown to be monophyletic; three genera were not resolved. At higher taxonomic levels, only the crustacean order Copepoda was resolved, with bootstrap value of 83%. The mtCOI barcodes accurately discriminated and identified known species of 10 taxonomic groups of Arctic Ocean holozooplankton. A comprehensive DNA barcode database for the estimated 300 described species of Arctic holozooplankton will allow rapid assessment of species diversity and distribution in this climate-vulnerable ocean ecosystem.

Reduced Fitness of Daphnia magna Fed a Bt-Transgenic Maize Variety

Genetically modified (GM) maize expressing the Bt-toxin Cry1Ab (Bt-maize) was tested for effects on survival, growth, and reproduction of the water flea Daphnia magna, a crustacean arthropod commonly used as a model organism in ecotoxicological studies. In three repeated experiments, D. magna were fed 100% ground maize in suspension, using either GM or isogenic unmodified (UM) maize. D. magna fed GM-maize showed a significantly reduced fitness performance: The mortality was higher, a lower proportion of females reached sexual maturation, and the overall egg production was lower compared to D. magna fed UM isogenic maize. We conclude that the tested variety of Bt-maize and its UM counterpart do not have the same quality as food sources for this widely used model organism. The combination of a reduced fitness performance combined with earlier onset of reproduction of D. magna fed Bt-maize indicates a toxic effect rather than a lower nutritional value of the GM-maize.

영암호 저서동물군집에 미친 하구둑 건설의 영향

To assess the macrobenthic community of oligohaline Youngam Lake, which is located at the Youngsan Watershed on the southwest part of Korea, macrobenthic fauna were collected at 45 stations during May, 2002. A total of 16 species of macrofauna were recorded with a mean density of 240 individuals per <TEX>$m^2$</TEX> and a mean biomass of 7.07 g wet weight per <TEX>$m^2$</TEX>. Major dominant faunal groups were crustacean arthropods in terms of the number of species and abundance, and polychaete annelids in terms of biomass. The mean grain size was <TEX>$5.7\;{\phi}$</TEX> which was dominated by silt fraction. The hydrological environment of the lake was characterised as an oligohaline environment with a mean surface water temperature of <TEX>$17.8^{\circ}C$</TEX> and a mean salinity of 2.08 psu. The major dominant species were amphipods, Corophium sp. (<TEX>$31\%$</TEX>) and Jesogammarus sp. (<TEX>$25\%$</TEX>). Lowe. values of species diversity (H') with a mean of 0.81 (less than 1.0 from most stations) reflected the overall poor faunal diversity in this area. Multivariate analysis suggested that this benthic faunal community could be divided into four sub-regions such as the area from lake proper to water channel to the south, the stations located at the entrance and northern water channel, the stations near the dike, and the lake proper area. Freshwater and brackish water species which occurred in each station group were corresponded to the oligohaline salinity regime. Bottom hypoxia appeared in the entrance part of the lake between dyke and lake proper on May, which was resulted from stratification from spring season. These facts imply that marine macrobenthos were severely impacted by low salinity and a consequent hypoxia after embankment of the lake due to the restriction of water circulation.

Ontogeny of Crustacean Respiratory Proteins

The respiratory proteins hemoglobin and hemocyanin share the function of oxygen transport, but the proteins belong to separate gene families, and their active sites and the metal ions that bind the oxygen differ. Either hemoglobin or hemocyanin, but not both, is expressed in the hemolymph of many arthropod crustaceans. Hemoglobin is present in Branchiopoda, Ostracoda, Copepoda, rhizocephalan Cirripedia and one suborder of amphipodan Malacostraca, while hemocyanin has been described in Malacostraca. Recent work by several laboratories have provided new information on the gene structure, exon-intron patterns, site of synthesis and expression of hemoglobins in the branchiopods Artemia and Daphnia. These studies suggest the branchiopods are excellent model organisms for studies of oxygen sensors and hypoxia inducible transcription factors during developmental and adult stages. The focus in our laboratory on the ontogeny of hemocyanin in the Dungeness crab, Cancer magister, has demonstrated that both structure and function of hemocyanin change from megalopa to adult crab. The hemocyanin of an oceanic megalopa contains four subunits. Another subunit appears about the time of metamorphosis to first juvenile instar, and expression of a sixth subunit begins four or five molts later. The timing of onset of adult hemocyanin can be altered experimentally by food levels and temperature. Gene expression and functional properties of both red and blue oxygen transport proteins of crustaceans change during ontogeny to insure oxygen delivery appropriate for each developmental stage.

Ontogeny of Crustacean Respiratory Proteins1

Abstract The respiratory proteins hemoglobin and hemocyanin share the function of oxygen transport, but the proteins belong to separate gene families, and their active sites and the metal ions that bind the oxygen differ. Either hemoglobin or hemocyanin, but not both, is expressed in the hemolymph of many arthropod crustaceans. Hemoglobin is present in Branchiopoda, Ostracoda, Copepoda, rhizocephalan Cirripedia and one suborder of amphipodan Malacostraca, while hemocyanin has been described in Malacostraca. Recent work by several laboratories have provided new information on the gene structure, exon-intron patterns, site of synthesis and expression of hemoglobins in the branchiopods Artemia and Daphnia. These studies suggest the branchiopods are excellent model organisms for studies of oxygen sensors and hypoxia inducible transcription factors during developmental and adult stages. The focus in our laboratory on the ontogeny of hemocyanin in the Dungeness crab, Cancer magister, has demonstrated that both ...

Production and characterization of monoclonal antibodies against Panulirus interruptus hemocyanin.

Since the primary and higher-order structures of hemocyanin from the crustacean arthropod Panulirus interruptus have been elucidated completely, it should be possible to determine which regions of this immunogenic molecule are recognized most often by antibodies. Monoclonal antibodies were raised against subunits a and b of this hemocyanin, and fourteen of them were further characterized. The produced antibodies were of class IgG, subclasses 1 or 2a. Most of them had dissociation constants on the order of magnitude 10(-8)-10(-10), a few had lower affinities. Most clones showed no or negligible cross-reactivity with other crustacean hemocyanins. The reactivity of most other clones diminished with increasing sequence difference between the investigated hemocyanins. However, in a few instances a stronger reactivity with other hemocyanins was observed than with that from Panulirus interruptus. After complete denaturation of the hemocyanin there was no reaction with the monoclonal antibodies, indicating that the latter recognize conformational epitopes. Only one monoclonal antibody reacted with denatured hemocyanin. This antibody was also the only one which reacted with a CNBr digest, which means that it recognizes a sequential epitope. Several antibodies showed a faint reaction on Western blots, indicating the presence of some refolded native structure. Limited proteolysis of the hemocyanin molecule results in the formation of a 18 kDa fragment, representing domain 1, and a 55 kDa fragment representing domains 2 and 3. It was determined on Western blots of the digest on which fragment epitopes for eleven of the monoclonal antibodies were located.

A brief re-examination of the function and regulation of extracellular magnesium and its relationship to activity in crustacean arthropods

1. 1. The magnesium ion [Mg 2+] plays an important role as a co-factor in enzyme systems and as a modulator of the haemocyanin of crustacean arthropods. 2. 2. Mg 2+ is actively regulated in most decapod crustaceans via the antennal gland. The degree of regulation can be correlated to some extent with the “activity” of a particular species although there are “exceptions to the rule”. 3. 3. Intraspecific studies indicate that there is a clear relationship between haemolymph [Mg 2+] and the level of activity in particular crustacean species. 4. 4. A plea is made for the investigation of temporal changes in the [Mg 2+] of the haemolymph of a number of crustaceans and for more studies of Mg 2+ homoeostasis in general.

Isolation and characterisation of arthropod gap junctions

Gap junctions have been isolated from the hepatopancreas of the crustacean arthropod, Nephrops norvegicus (Norway lobster). SDS-PAGE of these preparations shows two major protein bands, mol. wt. 18 000 (18 K) and mol. wt. 28 000 (28 K). The 18-K and 28-K proteins are interconvertible, cannot be distinguished by two dimensional tryptic and chymotryptic peptide mapping, and therefore appear to be different (most likely monomeric and dimeric) forms of the same protein. The protein can also aggregate to higher multimeric forms mol. wt. 38 000 (presumed trimer), and mol. wt. 52 000 (presumed tetramer). The buoyant density of the isolated gap junctions in continuous potassium iodide gradients is 1.260 g/cm. The junctions are progressively solubilized in increasing SDS concentrations, mostly between 0.1% and 0.2% SDS, and this is accompanied by the release of the 18-K and 28-K forms of the junctional protein. The Nephrops hepatopancreas 18-K junctional protein has antigenic determinants in common with the vertebrate 16-K junctional protein as shown by cross-reactivity with two different affinity purified antibody preparations. However, no detectable similarity can be seen between the major I-labelled tryptic and chymotrytpic peptides of the Nephrops hepatopancreas 18-K protein and the mouse liver 16-K protein.

Species and tissue distribution of gholecystokinin/gastrin-like substances in some invertebrates

Twenty-six species of invertebrates representing eight phyla were surveyed for the presence of cholecystokinin/gastrin-like (CCK/gastrin-like) peptides by radioimmunoassay of various tissue extracts. This is the first report of the presence of CCK/gastrin-like peptides in representatives of the phylum Ectoprocta, the arthropodan classes Crustacea and Merostomata, and in the nervous systems of the gastropod mollusc Aplysia californica and the oligochaete annelid Lumbricus terrestris. It has been proposed that CCK/gastrin evolved in the invertebrates as a neural peptide and was subsequently exploited by the vertebrates as a regulatory peptide in both the nervous system and the gastrointestinal endocrine system. The present results indicate that some gastropod molluscs, a merostomatan arthropod, and an annelid have detectable CCK/gastrin in both nervous and gut tissue. However, extractable CCK/gastrin was found only in gut tissue and not in the central nervous system of a crustacean arthropod. The tissue origin of the extracted CCK/gastrin in Bugula (phylum Ectoprocta) was not determined. Final resolution of the question of the nervous versus gut endocrine cellular origin of CCK/gastrin in invertebrates awaits further investigation. CCK/gastrin-like peptides are widely distributed among the invertebrates, which thus provide a rich source of comparative material for study of these regulatory substances.

Structures of Invertebrate Hemoglobins

Hemoglobin is widely distributed among the invertebrates. Intracellular hemoglobins consist of relatively small molecules with mol wts of 15–17,000 or dimeric, tetrameric or octameric aggregates of 15–17,000 mol wt subunits. Sequence homology is present but not extensive in those pigments which have been studied and the characteristic myoglobin fold of vertebrate hemoglobin occurs in at least two invertebrate hemoglobins. The wide array of aggregation states among invertebrate hemoglobins provides some simple models for understanding homotropic functional properties exhibited by many of these pigments. Polymeric extracellular hemoglobins are present in annelids molluscs crustacean arthropods and nematodes. Annelid extracellular hemoglobins and chlorocruorins consist of 3 × 106 mol wt two-tiered hexagonal arrays of submultiples which in turn are based on polypeptide chain subunits of mol wt 14–16 000. Molluscan extracellular hemoglobins are constructed from a different subunit arrangement. In the planorbid snail and clam extracellular hemoglobins the subunits appear to be 175 000 and 300 000 mol wt linear series of 15–17 000 dalton oxygen binding domains respectively. Planorbid snail native hemoglobin presents circular structures 200 Å in diameter in the electron microscope with 10-fold symmetry in at least one view, and clam extracellular hemoglobins are huge 345 by 1200 Å rodlike structures. Crustacean extracellular hemoglobins are also polymeric pigments and at least in a few species appear to have subunits which are tandemly linked oxygen binding domains. The polymeric hemoglobins of nematodes have molecular weights of about 330 000. The subunit molecular weight and heme content suggest a value of 40,000 daltons which set the nematode pigments apart from all other hemoglobins so far studied. An overview of invertebrate hemoglobin structures and some of the questions they pose are presented in this paper.

Origin and evolution of the Class Rostroconchia

The pseudobivalved Rostroconchia, first recognized as a separate class of molluscs in 1972, may be the only extinct molluscan class. Until recently, primitive rostroconchs (ribeirioids) were generally thought to be the carapace of crustacean arthropods and advanced rostroconchs (conocardioids) were considered to be unusual pelecypods. In fact, rostroconchs were a diverse class of molluscs (2 orders, 8 families, 31 genera, 400 + species known) that grew a bivalved adult shell from a univalved larval and juvenile shell. They were bilaterally symmetrical animals that probably had an anterior mouth, posterior anus, a pair of lateral gills, and a pelecypod-like foot. Most are believed to have been deposit feeders that used enlarged anterior mantle tissue to collect food, but some were clearly suspension feeders. They lived on top of the sea floor (rarely) or partly buried within it (commonly). Rostroconchs are known only from Palaeozoic rocks and range in age from earliest Cambrian to latest Permian (approximately 575-245 Ma ago). They evolved from untorted univalved molluscs (helcionellacean monoplacophorans) in the late Precambrian, remained an inconspicuous component of the biota through the Cambrian, and then radiated rapidly in the palaeotropical seas of the Early Ordovician. Relatively few genera survived the Ordovician, possibly because of competition by the Pelecypoda, but many species of these are found in younger Palaeozoic rocks. The last refuge of the class seems to have been the cool-temperature regions of the Permian Earth. By the middle Early Cambrian, the first pelecypod Fordilla had evolved from a primitive rostroconch. Rostroconchs were preadapted to exploit the pelecypod form, and the appearance of Fordilla may have been a relatively insignificant step. Somewhat later, probably in the Late Cambrian or Early Ordovician, the Scaphopoda were also derived from the Rostroconchia. This evolutionary event seems to have required the ventral fusion of an elongate rostroconch shell at the post-larval stage of development. Animals resembling primitive rostroconchs were required as theoretical links between monoplacophorans and pelecypods before the Class Rostroconchia was well studied. These hypothetical intermediates differ from the real thing in only one important respect; it was predicted that such forms would have many pedal muscle insertions on each valve, which they do not. The single anterior and posterior pedal muscle insertions of primitive rostroconchs therefore indicate a secondary simplification of the molluscan stock; this probably occurred after the anatomy of Neopilina was attained, but before the main radiation of the phylum.

Some Phylogenetical Aspects on the Occurrence of Somatostatin in the Gastro-Entero-Pancreatic Endocrine System

Rodioimmunoassayable somatostatin (SRIF) was found in acid ethanol extracts from various parts of the gastro-entero-pancreatic (GEP) endocrine system in reptiles, amphibians, teleost bony fish, cartilaginous fish, and jawless fish, as well as in a deuterostomian invertebrate, the tunicate, Ciona intestinalis. The cellular sites could, as a rule, be easily visualized light-microscopically by the peroxidase-anti-peroxidase (PAP) immunocytochemical procedure, using guinea-pig and rabbit antisera against synthetic SRIF. The standard Hellerström-Hellman technique, used to detect argyrophi SRIF-storing D cells, failed to visualize the SRIF cells in teh GEP endocrine system of the tumicate and of the jaw-less fish. Moreover, the results comfirmed the previous description that this technique only exceptionally (and sometimes only after further modifications) gave positive results when applied to the GEP endocrine system of bony fish, amphibians, and reptiles. In cartilaginous fish, however, it worked adequately and confirmed the radio-immunological and immunocytochemical observations. In the mucosa of the alimentary tract and in the parenchyma of its associated glands of one echinoderm and two pelecypod molluscs and one crustacean arthropod no sgns of the occurrence of SRIF-storing cells were observed using the three correlated procedures. In several of these tissues, signs of the occurrence of insulin-producing cells had perviously been observed. Thus, SRIF seems to appear at a later evolutionary stage than insulin. The principal islets (Brockmann corpusles) of the marine teleost fish, Cottus scorpius, had the highest concentrations of radioimmunoassayable SRIF of all the GEP organs and tissues investigated, viz. about 200 ng/mg wet weight. Nevertheless, it was only 1/5 of the actual insulin content.

Insulin production in vertebrates and invertebrates

From a comparative point of view the cyclostome islet parenchyma offers several interesting histophysiological aspects on the evolution of insulin production. Differences observed between the results of trials to evoke diabetes by alloxan administration and by surgical isletectomy indicated the occurrence of extrainsular insulin-producing β-cells in the hagfish. They seemed to occur in the mucosa of the gastrointestinal tract. With the working hypothesis that the β-cells had evolved as a functional specialization of the epithelium of the digestive mucosa, a search for insulin production was made in deuterostome and protostome invertebrates. Insulin production was found in the mucosa of the alimentary tract of a tunicate, an echinoderm, a gastropod mollusc, a cephalopod molluse, and possibly also in the hepatopancreas of a decapod crustacean arthropod. It was suggested that goblet cell-associated epithelial cells with small cytoplasmic secretion granules of “endocrine” type might represent the cellular site of this insulin production. Based on these observations and on the peculiar structure of cyclostome islet parenchyma, current hypotheses on the evolution of insulin production are considered to have obtained some structural and functional support. The role of insulin in invertebrates is briefly examined in relation to some results of current experiments.

Insulin assays and light microscopical studies of digestive organs in protostomian and deuterostomian species and in coelenterates

Acid alcohol extracts of the digestive tract of the deuterostomian chordate Ciona intestinalis (a tunicate) and that of the protostomian invertebrates Buccinum undatum, Pecten maximus, and Eledone cirrosa (three molluscs), as well as of the hepatopancreas of Carcinus maenas (a crustacean arthropod) showed insulin activity when tested by the mouse hemidiaphragm insulin assay. The activity in the extracts from Ciona, Buccinum, and Eledone was neutralized by antisera to ox and chicken insulins, but not by antiserum to cod insulin, proving that insulin is present in these three species. The cellular site of this insulin production could not be demonstrated with certainty by conventional histological and histochemical procedures for light microscopical analysis of vertebrate islet parenchyma. Ordinary goblet cells and adjacent coarsely granulated cells seemed to be the only kinds of cells in the mucosa of the digestive canal that showed β-cell staining reactions with aldehyde-fuchsin, chromehematoxylin, and pseudoisocyanin, and that occurred in quantities roughly proportional to the insulin yields. In the intestinal submucosa of Pectan maximus and in the hepatopancreas of the two crustacean arthropods Homarus gammarus and Carcinus maenas, however, scattered dark epithelial cells showed β-cell staining characteristics without any obvious association to cells with typical goblet cell features. Possibly except for an acinar gland in the stomach wall of the whelk and crab, no α 1- or α 2-cells were found in the mucosa of the alimentary tract and in the parenchyma of associated glands in any of the species studied, and the sulfide-silver procedure for detecting heavy metals in these structures gave consistently negative results. Tissues from two cnidarian species, Aurelia aurita and Metridium senile, were devoid of cells that resembled mammalian α- or β-cells, and acid alcohol extracts of these tissues did not contain detectable insulin activity.