Histological investigation of organisms with hard skeletons: a case study of siliceous sponges

Siliceous and calcareous sponges commonly are treated with acid to remove the spicules prior to embedding and cutting for histological investigations. Histology of spiculated sponge tissue represents a challenging problem in sponge histotechnology. Furthermore, fluorescence in situ hybridization (FISH), a key method for studying sponge-associated microbes, is not possible after acid treatment. For a broad range of siliceous sponge species, we developed and evaluated methods for embedding in paraffin, methylmethacrylate resins, LR White resin and cryomatrix. Different methods for cutting tissue blocks as well as mounting and staining sections also were tested. Our aim was to enable histological investigations and FISH without prior removal of the spicules. To obtain an overview of tissue and skeleton arrangement, we recommend embedding tissue blocks with LR White resin combined with en bloc staining techniques for large specimens with thick and numerous spicules, but paraffin embedding and subsequent staining for whole small specimens. For FISH on siliceous sponges, we recommend Histocryl embedding if the spicule content is high, but paraffin embedding if it is low. Classical histological techniques are used for detailed tissue examinations.     

Embryonic development of verongid demosponges supports the independent acquisition of spongin skeletons as an alternative to the siliceous skeleton of sponges

Approximately 85% of extant sponges (phylum Porifera) belong to the class Demospongiae, which contains 14 taxonomic orders. In the orders Verongida, Dictyoceratida, and Dendroceratida, jointly referred to as 'keratose demosponges', the skeleton does not contain siliceous spicules but only spongin fibres. This shared trait has encouraged placement of these orders together within Demospongiae, although their relationships remain uncertain. The present study documents for the first time embryo development in the order Verongida ( Aplysina aerophoba ), providing some clues for phylogenetic inference. Spawned eggs were enveloped by a follicle of maternal cells. Embryos and larvae were chimeric organisms, the blastocoel of which was filled with symbionts and maternal cells migrated from the follicle. The ultrastructure of epithelial larval cells revealed: (1) a basal apparatus characterized by a peculiar, angling accessory centriole; (2) a pear-shaped nucleus with a protruding beak connected to the rootlets of the basal body; and (3) a distinctive Golgi apparatus encircling the nuclear apex. Developmental and ultrastructural findings support the concept, in congruence with recent molecular studies, that Verongida are more closely related to Halisarcida (askeletal sponges) and Chondrosida (askeletal sponges + sponges with spongin + spiculate sponges) than to the remaining 'keratose' orders, making a monophyletic 'supra-ordinal unit' equivalent to a subclass (Myxospongia, new subclass). Hence, spongin skeletons have evolved at least twice in Demospongiae. Independent acquisition of 'corneous' materials as an alternative to silica could have been stimulated by the radiation of diatoms at the Cretaceous–Tertiary boundary (approximately 65 Mya), which depleted silicon in the photic zone of the world's ocean.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 427–447.     

First evidence of the presence of chitin in skeletons of marine sponges. Part II. Glass sponges (Hexactinellida: Porifera)

Sponges (Porifera) are presently gaining increased scientific attention because of their secondary metabolites and specific skeleton structures. In contrast to demosponges, whose skeletons are formed from biopolymer spongin, glass sponges (hexactinellids) possess silica-organic composites as the main natural material for their skeletal fibres. Chitin has a crystalline structure and it constitutes a network of organized fibres. This structure confers rigidity and resistance to organisms that contain it, including monocellular (yeast, amoeba, diatoms) and multicellular (higher fungi, arthropods, nematodes, molluscs) organisms. In contrast to different marine invertebrates whose exoskeletons are built of chitin, this polysaccharide has not been found previously as an endogenous biopolymer within glass sponges (Hexactinellida). We hypothesized that glass sponges, which are considered to be the most basal lineage of multicellular animals, must possess chitin. Here, we present a detailed study of the structural and physico-chemical properties of skeletal fragments of the glass sponge Farrea occa. We show that these fibres have a layered design with specific compositional variations in the chitin/silica composite. We applied an effective approach for the demineralization of glass sponge skeletal formations based on an etching procedure using alkali solutions. The results show unambiguously that alpha-chitin is an essential component of the skeletal structures of Hexactinellida. This is the first report of a silica-chitin's composite biomaterial found in nature. From this perspective, the view that silica-chitin scaffolds may be key templates for skeleton formation also in ancestral unicellular organisms, rather than silica-protein composites, emerges as a viable alternative hypothesis.     

Transition analysis: a validation study with known-age modern American skeletons

Transition Analysis-a recent skeletal age-estimation procedure (Boldsen et al.: Paleodemography: age distributions from skeletal samples (2002) 73-106)-is evaluated using 252 known-age modern American males and females from the Bass Donated Collection and Mercyhurst forensic cases. The pubic symphysis worked best for estimating age, followed by the sacroiliac joint and cranial sutures. Estimates based on all skeletal characteristics are influenced by the choice of prior distribution, although its effect is dwarfed by both the inaccuracy and imprecision of age estimates. Age intervals are narrowest for young adults, but are surprisingly short in old age as well. When using an informative prior distribution, the greatest uncertainty occurs from the late 40s into the 70s. Transition Analysis estimates do not perform as well as experience-based assessments, indicating the existing procedure is too narrowly focused on commonly used pelvic and cranial structures.     

The bivalve boring Cuenulites amygdaloides nov. isp. in siliceous sponges from the Upper Cretaceous of Germany

Lower Campanian siliceous sponges from epicontinental deposits of the Subhercynian Cretaceous Basin in Germany contain amygdaloidal depressions which are distinguished as a new ichnospecies of the ichnogenus Cuenulites. These bioerosion traces are interpreted as borings of semi-endolithic bivalves, produced without significant rotation movement, probably mostly by chemical action. As there are no signs of tissue reaction in the bored sponges, the structure is considered to be produced post mortem to the sponge, probably in a foreshore-shoreface setting, with redeposition offshore.     

An autopsy case of tuberous sclerosis. Histological and immunohistochemical study.

We report an autopsy case of tuberous sclerosis. A 19-year-old Japanese man had shown facial adenoma sebaceum, intractable convulsive seizures and severe mental retardation. Gross inspection of the brain showed a cortical tuber from the orbital frontal lobe to the rhinencephalon of the left side and a few subependymal nodules. Histological examination revealed many cortical tubers in the cerebral hemispheres, a few subependymal nodules with calcification and multifocal clusters of heterotopic cells in the white matter (white matter nodules). In these lesions, massive giant cells with abundant eosinophilic cytoplasm and without Nissl substances were found. Although the size and shape of the giant cells were variable, the majority of them were gemistcytic, ovoid or polygonal. Immunohistochemistry was employed in these lesions using antibodies against neurofilament protein (NFP), glial fibrillary acidic protein (GFAP), vimentin (VM) and myelin basic protein (MBP). In the cortical tuber, the majority of the giant cells were positive for both NFP and VM, but a few were positive for GFAP. All of them were negative for MBP. In the subependymal nodule and white matter nodule, the majority of the giant cells were positive for NFP, but a few were positive for VM, and none were positive for either GFAP and MBP. These findings suggest that the majority of the giant cells may be immature cells toward neuronal series and a few may be those toward astroglial series. These findings also indicate that the giant cells in the subependymal nodule and white matter nodule may be more differentiated than those in the cortical tuber. The nature of the giant cells in tuberous sclerosis is discussed.     

Serotonin-producing pancreatic endocrine tumour. Histological, ultrastructural and immunohistochemical study of a case

Serotonin-producing pancreatic endocrine tumours are rare neoplasms which in most cases exhibit malignant biological behaviour. These tumours, in the majority of the well-documented cases, are composed of argyrophil- and argentaffin-positive cells which contain large pleomorphic neurosecretory granules. In contrast, argyrophilic non-argentaffin pancreatic endocrine tumours with tumour cells containing round neurosecretory granules are exceptional. In this study we describe such a tumour not associated with clinical evidence of carcinoid syndrome in a 60-year-old woman. Histological examination revealed tumour extension in pancreatic lymphatic vessels and veins but no evidence of locoregional or distant metastases. Ten months after surgery the patient showed no recurrence of the disease. Immunohistochemistry revealed cytoplasmic serotonin production in the tumour cells which were negative for anti-gastrin, insulin, glucagon, somatostatin, pancreatic polypeptide (PP), vasoactive intestinal peptide (VIP) and ACTH. This study emphasizes the usefulness of combined ultrastructural and immunohistochemical investigations in order to identify and characterize the rare pancreatic endocrine tumours with serotonin production.     

Ocean acidification and calcifying reef organisms: a mesocosm investigation

A long-term (10 months) controlled experiment was conducted to test the impact of increased partial pressure of carbon dioxide (pCO₂) on common calcifying coral reef organisms. The experiment was conducted in replicate continuous flow coral reef mesocosms flushed with unfiltered sea water from Kaneohe Bay, Oahu, Hawaii. Mesocosms were located in full sunlight and experienced diurnal and seasonal fluctuations in temperature and sea water chemistry characteristic of the adjacent reef flat. Treatment mesocosms were manipulated to simulate an increase in pCO₂ to levels expected in this century [midday pCO₂ levels exceeding control mesocosms by 365 ± 130 μatm (mean ± sd)]. Acidification had a profound impact on the development and growth of crustose coralline algae (CCA) populations. During the experiment, CCA developed 25% cover in the control mesocosms and only 4% in the acidified mesocosms, representing an 86% relative reduction. Free-living associations of CCA known as rhodoliths living in the control mesocosms grew at a rate of 0.6 g buoyant weight year⁻¹ while those in the acidified experimental treatment decreased in weight at a rate of 0.9 g buoyant weight year⁻¹, representing a 250% difference. CCA play an important role in the growth and stabilization of carbonate reefs, so future changes of this magnitude could greatly impact coral reefs throughout the world. Coral calcification decreased between 15% and 20% under acidified conditions. Linear extension decreased by 14% under acidified conditions in one experiment. Larvae of the coral Pocillopora damicornis were able to recruit under the acidified conditions. In addition, there was no significant difference in production of gametes by the coral Montipora capitata after 6 months of exposure to the treatments.     

Optimization of AFLP fingerprinting of organisms with a large-sized genome: a study on Alstroemeria spp.

 The recently introduced PCR-based DNA fingerprinting technique AFLP (amplified fragment length polymorphism) allows the selective amplification of subsets of genomic restriction fragments. AFLP has been used for multiple purposes such as the construction of linkage maps, marker saturation at specific genomic regions, analysis of genetic diversity and molecular phylogeny and cultivar identification. AFLP can be tailored by varying the number of selective nucleotides added to core primers and can allow accurate amplification, even in complex template mixtures generated from plant species with very large genomes. In this study Alstroemeria, a plant species with a very large genome, was tested for adapting the AFLP protocol. The results indicated that the estimated number of amplification products was close to the observed number when eight selective nucleotides were used but that seven selective nucleotides did not increase the number of amplification products fourfold. However, we found reproducibility in both +7 and +8 fingerprints. Various distributions of selective nucleotides over the various rounds of preamplifications were tested. Preamplification with four selective nucleotides followed by final amplification with eight selective nucleotides produced clear and reproducible AFLP patterns. The effects of GC content of primers and multiple preamplification steps were also discussed. Received: 16 March 1998 / Accepted: 14 July 1998     

Proteomics as a tool to improve investigation of substantial equivalence in genetically modified organisms: the case of a virus-resistant tomato

At present, the so-called "substantial equivalence" is the only widely accepted criterion for deciding whether or not a transgenic food is, from an alimentary point of view, to be considered totally correspondent to the "traditional" one from which it derives. Although never exactly defined, it deals with a comparison between the chemical composition of the two foods. A more in-depth analysis can be performed by one of the most suitable methods that allows for the simultaneous screening of many components without prior identification, the analysis of the proteome. As a model for testing this kind of approach, we compared protein expression of two types of tomato plants, having the same genetic background, except for a virus resistance trait introduced by genetic engineering. When proteins extracted from seedlings of the two types were analyzed by two-dimensional electrophoresis, no significant differences, either qualitative or quantitative, were detected, indicating that in this case the expression of major proteins was unmodified by the genetic manipulation. Fifteen proteins were identified by peptide mass fingerprinting.     

Evidence for spicule homology in calcareous and siliceous sponges: biminerallic spicules in Lenica sp. from the Early Cambrian of South China

Botting, J.P., Muir, L.A., Xiao, S., Li, X. & Lin, J.‐P. 2012: Evidence for spicule homology in calcareous and siliceous sponges: biminerallic spicules in Lenica sp. from the Early Cambrian of South China. Lethaia, Vol. 45, pp. 463–475. The relationships of the extant sponge classes, and the nature of the last common ancestor of all sponges, are currently unclear. Early sponges preserved in the fossil record differ greatly from extant taxa, and therefore information from the fossil record is critical for testing hypotheses of sponge phylogenetic relationships that are based on modern taxa. New specimens of the enigmatic sponge Lenica sp., from the Early Cambrian Hetang Biota of South China, exhibit an unusual spicule structure. Each spicule consists of a siliceous core with an axial canal, an organic outer layer and a middle layer interpreted to have been originally calcium carbonate. This finding confirms previous work suggesting the existence of biminerallic spicules in early sponges. Combined with data from other early sponges, the new findings imply that the two fundamental spicule structures of modern sponges were derived from a compound, biminerallic precursor. Spicules are therefore homologous structures in Calcarea and Silicea, and if sponges are paraphyletic with respect to Eumetazoa, then spicules may also have been a primitive feature of Metazoa. □Calcarea, Early Cambrian, Hetang Biota, phylogeny, Silicea, taphonomy.     

The effect of benzo[a]pyrene on sponges as model organisms in marine pollution

The majority of the investigations were performed with the marine sponge Tethya lyncurium at concentrations of 2 X 10(-8) to 1 X 10(-11) g/ml of benzo[a]pyrene (BaP). Sea-pollution was characterized as BaP equivalent activity in the Ames test. Increased activity of ornithine decarboxylase (ODC) was observed when sponges were artificially exposed at polluted marine areas for 3 weeks. In contrast to the situation in higher animals no ODC induction of the fast type was observed. Mixed function oxygenases (MFO) were not detected in sponges nor could they be induced as in vertebrates. BaP was absorbed by Tethya and concentrated 30--60-fold. In live, but not dead, artificially perfused sponges [3H]- and [14C]BaP-radiolabeled became firmly associated with DNA, RNA and protein of the sponges. The association persisted in isolated fractions, in nucleotides, in nucleosides and in protein hydrolysates. The BaP binding ratio to DNA was found to be strongly correlated to the concentration of BaP. Light modifies BaP and thus enables binding. In the dark only very low association, if any, is observed. The possible consequences of these findings are discussed.     

Soft sponges with tricky tree: On the phylogeny of dictyoceratid sponges

Keratose (horny) sponges constitute a very difficult group of Porifera in terms of taxonomy due to their paucity of diagnostic morphological features. (Most) keratose sponges possess no mineral skeletal elements, but an arrangement of organic (spongin) fibers, with little taxonomic or phylogenetic information. Molecular phylogenetics have targeted this evolutionary and biochemically important lineage numerous times, but the conservative nature of popular markers combined with ambiguous identification of the sponge material has so far prevented any robust phylogeny. In the following study, we provide a phylogenetic hypothesis of the keratose order Dictyoceratida based on nuclear markers of higher resolution potential (ITS and 28S C-region), and particularly aim for the inclusion of type specimens as reference material. Our results are compared with previously published data of CO1, 18S, and 28S (D3-D5) data, and indicate the paraphyly of the largest dictyoceratid family, the Thorectidae, due to a sister group relationship of its subfamily Phyllospongiinae with Family Spongiidae. Irciniidae can be recovered as monophyletic. Results on genus level and implications on phylogenetic signals of the most frequently described morphological characters are discussed.     

Comparative study of the choanosome of porifera: II. The keratose sponges

The aquiferous system of representatives of the orders Dictyocer-atida, Dendroceratida, and Verongida has been studied to note its relevance to the systematics of the groups. The volume of the choanocyte chamber, the size and shape of the choanocytes, the number of choanocytes per chamber, the relative development of the mesohyl, and the features of endopinacocytes are estimated from scanning and transmission electron microscopic observations of representatives of most families of the three orders. Although the Dysideidae have a reticulate skeleton and were classified in the order Dictyoceratida, they are actually closer to the Aplysillidae (Dendroceratida) than to dictyoceratids. The anatomy and cytology of the Halisarcidae differ profoundly from those of these three orders and are clearly more closely related to nonkeratose sponges. Some changes in classification lead to a pattern with highly homogeneous orders that clearly differ in their anatomic and cytologic features, which does not support the hypothesis of a common origin of the “keratose” sponges.     

Synthesis of novel triplet drugs with 1,3,5-trioxazatriquinane skeletons and their pharmacologies. 1: Synthesis of triplet drugs with morphinan skeletons

We synthesized symmetrical and nonsymmetrical triplet drugs with 1,3,5-trioxazatriquinane skeletons. The isolation of key intermediates, oxazoline dimers, made it possible to effectively produce nonsymmetrical triplets. Among the synthesized triplets, KNT-93, composed of three identical opioid μ receptor agonists, showed dose-dependent antinociception via the μ receptor. The effect was 56-fold more potent than that of morphine, a representative μ agonist. The profound analgesic effect induced by KNT-93 might result from simultaneous occupation of three μ opioid receptors.     

Looking where it's hard to see: a case study documenting rare Eucyclogobius newberryi presence in a California lagoon

Environmental DNA (eDNA) analysis is increasingly used for biomonitoring and research of fish populations and communities by environmental resource managers and academic researchers. Although managers are much interested in expanding the use of eDNA as a survey technique, they are sceptical about both its utility (given that information is often limited to presence/absence of a species) and feasibility (given the need for proper laboratory facilities for sample processing). Nonetheless, under the right circumstances, eDNA analysis is cost-effective compared to many traditional aquatic survey methods and does not disturb habitat or harm the animals being surveyed. This article presents a case study in which eDNA analysis was successfully used to document the presence of a rare fish species in a waterway earmarked for restoration. The authors discuss the conditions that allowed this study to occur quickly and smoothly and speculate on how the goals of researchers and managers can be integrated for efficient and informative use of this tool.     

The interactions of neuropeptides with membrane model systems: a case study.

The interactions between the positively charged neuropeptides substance P (SP), bradykinin (BK), and zwitterionic Met-enkephalin (ME) neuropeptides, and negatively charged SDS and zwitterionic lysophosphatidylcholine (LPC) membrane model systems, have been investigated using one- and two-dimensional nmr experiments. Proton longitudinal relaxation studies were used to characterize these interactions as intrinsic or extrinsic. An extrinsic interaction are similar to those observed for extrinsic membrane proteins. An intrinsic interaction are similar to those observed for intrinsic membrane proteins, and would require that the hydrophobic residues penetrate or insert into the hydrophobic core of the membrane. The interactions between both SP and BK and SDS, based on nmr results, may be characterized as intrinsic, and the interaction between ME and SDS may be characterized as extrinsic. Two-dimensional nuclear Overhauser enhancement spectroscopy experiments proved the insertion of the phenylalanine residues on both SP and BK into the hydrophobic core of SDS micelles. The interaction between SP and BK with LPC based on nmr results are characterized as extrinsic, with the interaction between ME and SDS characterized as weakly intrinsic.