Gut Microbiota and a Selectively Bred Taste Phenotype: A Novel Model of Microbiome-Behavior Relationships.

OBJECTIVES: The microbiota-gut-brain axis is increasingly implicated in obesity, anxiety, stress, and other health-related processes. Researchers have proposed that gut microbiota may influence dietary habits, and pathways through the microbiota-gut-brain axis make such a relationship feasible; however, few data bear on the hypothesis. As a first step in the development of a model system, the gut microbiome was examined in rat lines selectively outbred on a taste phenotype with biobehavioral profiles that have diverged with respect to energy regulation, anxiety, and stress. METHODS: Occidental low and high-saccharin-consuming rats were assessed for body mass and chow, water, and saccharin intake; littermate controls had shared cages with rats in the experimental group but were not assessed. Cecum and colon microbial communities were profiled using Illumina 16S rRNA sequencing and multivariate analysis of microbial diversity and composition. RESULTS: The saccharin phenotype was confirmed (low-saccharin-consuming rats, 0.7Δ% [0.9Δ%]; high-saccharin-consuming rats, 28.1Δ% [3.6Δ%]). Regardless of saccharin exposure, gut microbiota differed between lines in terms of overall community similarity and taxa at lower phylogenetic levels. Specifically, 16 genera in three phyla distinguished the lines at a 10% false discovery rate. DISCUSSION: The study demonstrates for the first time that rodent lines created through selective pressure on taste and differing on functionally related correlates host different microbial communities. Whether the microbiota are causally related to the taste phenotype or its correlates remains to be determined. These findings encourage further inquiry on the relationship of the microbiome to taste, dietary habits, emotion, and health.

Au nanoparticle clusters from deposition of a coalescing emulsion.

HYPOTHESIS: Nanoparticle adsorption at the oil-water interface in an unstable, coalescing emulsion leads to cluster formation. EXPERIMENTS: Stable suspensions of clusters are prepared using a facile, two-step procedure involving few reagents and neither thiolated compounds nor chlorinated solvents. First, colloidal gold nanoparticles are assembled at the aqueous-hexanol interface in an emulsion that rapidly coalesces and spontaneously deposits a film on the interior surface of the glass container. The film is dissolved in ethanol with sonication to disperse the clusters. The film and clusters are characterized by transmission electron and atomic force microscopies as well as ultraviolet-visible spectrometry. FINDINGS: Clusters are observed to contain as few as 8 to as many as 24 Au nanoparticles. The clusters are anisotropic and can also be formed from larger nanoparticles. Hydrophobic and hydrophilic interactions are implicated in the formation of these clusters within the interfacial tension gradients of a coalescing emulsion. The clusters can be re-suspended in ethanol and water, maximizing the utility of these clusters with an extinction band in the near-Infrared region of the electromagnetic spectrum.

Three-dimensional microvasculature in rat and human hearts using a non-injection Ca2+-ATPase method on thick and ultra-thick sections.

Currently there are no methods available for staining rat and human myocardial microvasculature on thick sections that would allow for specific staining and differentiation of arterioles, venules, and capillaries. A non-injection technique is described that allows for labeling of the microvascular bed (MVB) in formalin-fixed pieces of the myocardium from humans and the white rat Rattus norvegicus, as well as human full-mount pericardium. Vessel staining is based on the activity of phosphatases (ATPases) and the precipitation of the released phosphate with calcium ions at high pH (pH 10.5-11.5). The resulting precipitate subsequently is converted to black or brown lead sulfide. The specificity of this reaction to vessels of the MVB allows arterioles, venules, capillaries, and pre- and postcapillaries to be clearly visualized in thick (60-100 µm) and ultra-thick (300-500 µm) sections against an unstained background of muscle and connective tissue. In addition, smooth muscle cells of arterioles are also stained allowing for differentiation between arteriolar and venular beds. These observations have not been reported in rat or human myocardium using other methods. This procedure should benefit studies of coronary microcirculation in experimental and pathological conditions, as well as in pharmacological investigations.

Impact of mineral deposition on shrimp, Penaeus monodon in a high alkaline water.

This study compares water quality parameters, shrimp growth and mortality rates, and biomass at harvest in two ponds of equal size, seeded with the same density (7 m2) of White Spot Syndrome Virus (WSSV) and Monodon Baculo Virus (MBV) negative post-larvae (PL)-20 of shrimp, Penaeus monodon in the Vellar estuary of South India. The primary difference between the ponds was the water source; one was filled from the estuary and the second with water from bore wells with high alkalinity. Temperature in both ponds was similar and reached 320C after 185 days of culture. Dissolved oxygen (DO) levels were within the acceptable range although levels in the alkaline pond were near the lower limit for the last 90 days before harvest. Salinity levels were similar in both ponds, above optimal levels, and increased over the 185 days. Alkalinity in the estuarine water was typically <50 ppm and again 200-320 ppm in the alkaline pond. In the alkaline pond, beginning on the 75th day mineral deposits was observed covering all parts of the shrimp including the eye and the inner gill chambers, and by harvest, 42% of the shrimp showed this coating. Elemental analysis identified the major constituents as calcium, phosphorus and manganese. Survival rates in the estuarine-water-fed pond was 92% with a total pond biomass at harvest of 1.65 tons ha-1 compared to survival of 79% in the alkaline pond and a biomass at harvest of 1.020 tons ha-1. When well water must be used, its alkalinity should be monitored and diluted with water from other sources.

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.

Morphology of epithelial cells lining the digestive tract of the giant keyhole limpet, Megathura crenulata (Mollusca; Vetigastropoda).

To understand the digestive functions in the giant keyhole limpet, it is important to know the types of cells present in each region of the gut and their roles in the secretion of digestive enzymes and absorption of nutrients. This study describes the morphology of cells lining the entire gut and identifies sites that may be secreting materials to aid digestion. Previous studies involving electron microscopy and enzyme analysis have focused on the salivary and digestive glands of several gastropods. Studies on the rest of the gut tract typically include only histological descriptions of the epithelia and although several types of cells have been described, they appear very similar. The purpose of this study is to determine if electron microscopy can provide better insights into the functions of cells in these poorly studied regions of the gut. Our ultrastructural observations suggest that only two types of cells, mucus secreting cells and apocrine secretory cells make up the epithelium in the esophagus, style sac, and intestine. These regions account for 85% of the length of the entire digestive tract. Apocrine secretory cells contain pigment granules, bear cilia, and/or microvilli at their apices, and release product into the gut lumen via apocrine secretion. This suggests that the secretory processes involved with digestion are occurring in most regions of the gut and that apocrine secretion is the primary mode by which materials are introduced into the gut lumen. The lips, salivary glands, stomach, and digestive gland lack apocrine secretory cells and the epithelial cells are similar to those described in other gastropods. J. Morphol. 271:1134-1151, 2010. (c) 2010 Wiley-Liss, Inc.

Venom kinematics during prey capture in Conus: the biomechanics of a rapid injection system.

Cone snails use an extensile, tubular proboscis as a conduit to deliver a potent cocktail of bioactive venom peptides into their prey. Previous studies have focused mainly on understanding the venom's role in prey capture but successful prey capture requires both rapid physiological and biomechanical mechanisms. Conus catus, a fish-hunting species, uses a high-speed hydraulic mechanism to inject its hollow, spear-like radular tooth into prey. We take an integrated approach to investigating the biomechanics of this process by coupling kinematic studies with morphological analyses. Taking advantage of the opaque venom and translucent proboscis of a mollusc-hunting juvenile cone snail, Conus pennaceus, we have determined that a high-speed prey capture mechanism is not unique to cone species that hunt fish prey. Two morphological structures were found to play crucial roles in this process. A constriction of the lumen near the tip of the proboscis, composed of tall epithelial cells densely packed with microfilaments, impedes forward movement of the radular tooth prior to its propulsion. Proximal to the constriction, a muscular sphincter was found to regulate venom flow and pressurization in the proboscis. In C. pennaceus, the rapid appearance and flushing of venom within the proboscis during prey capture suggests a mechanism involving the delivery of a discrete quantity of venom. The interplay between these elements provides a unique and effective biomechanical injection system for the fast-acting cone snail venom peptides.

Peritrophic membrane of the penaeid shrimp Sicyonia ingentis: structure, formation, and permeability.

Peritrophic membranes (PTMs) are secreted acellular layers that separate ingested materials from the gut epithelium in a variety of invertebrates. In insects and crustaceans, PTMs are produced in the midgut trunk (MGT, or intestine), but the MGT in decapod crustaceans, unlike that of insects, is not involved with digestion or absorption of food. We demonstrate that the PTM in the penaeid shrimp Sicyonia ingentis is similar to that in other crustaceans that have been studied and is primarily composed of chitin. The lectin WGA binds only to the PTM and glycocalyx along the microvilli of the midgut cells, which is consistent with the suggestion that the chitin is synthesized along the microvilli. The PTM is only permeable to inert particles smaller than 20 nm. We also describe the secretion of granules, which fill the apices of the epithelial cells, into the ectoperitrophic space. Although their function is not clear, they do not contribute to the PTM.

The three-dimensional detection of microvasculatory bed in the brain of white rat Rattus norvegicus by a Ca2+-ATPase method.

A procedure is described which allows for the selective and non-injectional staining of the three-dimensional microvasculatory bed (MVB) in thick sections (60-140 microm) of formalin-fixed brain tissue of white rats Rattus norvegicus. This histochemical method detects ATPase activity and takes place between pH 10.5 and 11.2. Calcium ion is used to capture inorganic phosphate, calcium phosphate is converted to lead phosphate, and subsequently converted to black or dark brown lead sulfide. All vessels are revealed due to a precipitate on the endothelium and smooth muscle cells of arterioles. In some vessels, red blood cells also stain. The background is transparent with no staining of neurons, nerve fibers, glial cells, or nuclei. This allows for clear identification of arterioles, venules, and capillaries, which is difficult using other methods. New observations are described including the presence of webs connecting branching parts of arterioles and constrictions along vessels. This procedure should be useful in investigations of the MVB in rat brain.

Vibrio parahaemolyticus and V. harveyi cause detachment of the epithelium from the midgut trunk of the penaeid shrimp Sicyonia ingentis.

Shrimp Sicyonia ingentis were either injected with Vibrio parahaemolyticus (10(4) CFU) or V. harveyi (10(6) CFU) or immersed in ASW containing either species at 10(5) CFU ml(-1). These densities were shown in preliminary experiments to kill approximately half the population by 7 d. On Day 7, surviving shrimp were classified as either diseased or apparently healthy, and their midgut trunks (MGT) were examined by light and electron microscopy. All shrimp immersed in ASW containing either species of Vibrio showed detachment of the epithelium in the MGT. In shrimp injected with either species of Vibrio, epithelial detachment was common in diseased shrimp but not in apparently healthy animals. Experiments with live shrimp were supported by in vitro experiments where MGTs were removed, tied off at both ends, and injected with either pathogenic bacteria (V. parahaemolyticus or V. harveyi), non-pathogenic bacteria (Bacillus subtilis or Escherichia coli), or ASW. After 2 h incubations in ASW at 15 degrees C, the MGTs were processed and examined. The epithelium consistently detached from isolated MGTs injected with either species of Vibrio, but not from MGTs injected with non-pathogenic bacteria or ASW. Because the MGT epithelium secretes the peritrophic membrane, loss of the epithelium eliminates 2 layers that may restrict penetration of ingested pathogens into the shrimp body and may disrupt the osmoregulatory function of the MGT. A second finding was that fixed, large-granule hemocytes associated with the basal lamina degranulated in the presence of the 2 species of Vibrio, but not with the non-pathogenic bacteria or ASW. These blood cells may help fight specific bacteria penetrating the MGT.

Morphology of the midgut trunk in the penaeid shrimp, Sicyonia ingentis, highlighting novel nuclear pore particles and fixed hemocytes.

The morphology of the midgut trunk (MGT) in the penaeid shrimp Sicyonia ingentis was examined by light and scanning and transmission electron microscopy. Although the function of the MGT is poorly understood, it is not involved with the digestion and absorption of nutrients, and it appears to be the surface of a shrimp least protected from penetration by potential pathogens. As described for other decapod crustaceans, the MGT in shrimp is composed of a simple columnar epithelium separated from a layer of connective tissue by a thick basal lamina. Beneath the basal lamina is a previously unreported layer of hemocytes, exclusively of the granulocyte variety, embedded in a matrix continuous with the basal lamina and extending into the connective tissue. This layer was observed in four other species of penaeid shrimp. Granulocytes in circulation can phagocytose and encapsulate foreign material and the granules contain antibacterial molecules, lysosomal enzymes, and prophenoloxidase. We suggest that the granulocytes associated with the basal lamina have matured at this site and are well positioned to fight potential pathogens that have penetrated the epithelial layer of the MGT. A second observation is the presence of clusters of cylinders bound to the nuclear pores of the epithelial cells. The possibility that these clusters are viruses, organelles, or abnormal organelles induced by disease or toxic materials is discussed. These unique particles were observed in S. ingentis but none of the other penaeid shrimp we examined.

Clearance of bacteria injected into the hemolymph of the ridgeback prawn, Sicyonia ingentis (Crustacea: Decapoda): Role of hematopoietic tissue.

In an earlier investigation, radiolabelled bacteria injected into the hemolymph of the shrimp Sicyonia ingentis were cleared rapidly from circulation. Most of the bacteria were localized in the gills, followed by other organs including the heart, abdominal musculature, and hematopoietic nodules (HPN). We determined the organ-specific effectiveness of bacterial clearance and found the HPN to be the most effective on a per gram basis. Light and electron microscopy were used to determine the mechanism of clearance in the HPN. The HPN are readily permeable to individual bacteria, which then are recognized and phagocytosed exclusively by small granule hemocytes. Within the first hour, the bacteria are degraded, leaving only whorls of membrane in the phagocytic vacuoles. Subsequently, the hemocytes that ingested bacteria lyse, as has been observed in vitro. These observations support earlier suggestions that hemocytes in the HPN are functionally mature and held in reserve until they are released into circulation. © 1996 Wiley-Liss, Inc.

Organization of hematopoietic tissue in the intermolt lobster, Homarus americanus.

Hematopoiesis in the American lobster Homarus americanus, as in most decapod crustaceans, occurs in a thin tissue covering the dorsal surface of the foregut. This tissue is composed of loosely attached, ovoid lobules containing the hematopoietic precursors and maturing hemocytes. Release of hemocytes into the dorsal hemocoel is accomplished by rupture of a portion of the connective tissue capsule covering the lobule. Cross sections of the lobules contain between 6 and 40 hematopoietic cells, of which approximately 90% constitute stages in granulocyte maturation and 10% are intermediates in hyaline cell maturation. Hematopoietic precursors in these two lines are similar to those recently described in a penaeid shrimp Sicyonia ingentis. The mitotic rate averaged 5.1% (range = 0.7% to 15.8%) in intermolt lobsters, 90% comprised granulocyte precursors. © 1993 Wiley-Liss, Inc.

Morphological comparison of major arteries in the ridgeback prawn, Sicyonia ingentis.

The structure of the major vessels conducting hemolymph away from the heart of the ridgeback prawn, Sicyonia ingentis, was examined using light and electron microscopy. Three varieties of vessel wall morphology were observed. In most vessels the wall is composed of the following four layers, proceeding from lumen to exterior: (1) thick basal lamina, which selectively stains with Verhoffapos;s Van Giesonapos;s stain for elastin, (2) a continuous layer of cells rich in microtubules, (3) a loose connective tissue layer with highly branched cells widely separated by a fibrillar matrix, which also stains for elastin, and (4) a thin basal lamina covering the outer surface of the vessel. The dorsal abdominal artery shows the second type of vessel wall construction and differs from the previous vessels in the following ways: (1) the inner basal lamina is thinner, (2) a layer of striated muscle replaces the unspecialized endothelial layer, and (3) the connective tissue layer includes two well-organized bands of elastin-like material. The hematopoietic vessels that branch off the ophthalmic arteries display the third type of vessel wall morphology. The lumen of the vessel is lined by a very thin basal lamina, which completely encircles the endothelial cells. The rest of the wall is composed of hemocytes, presumably in various stages of maturation, embedded in a matrix of fibroblast-like cells and collagen fibrils. The morphology of the vessel wall is the same as that previously described for the tubules of the hematopoietic nodules. The functional significance of these different vessel walls is discussed.

CYTOCHEMICAL FEATURES OF SHRIMP HEMOCYTES.

Morphological studies suggest that there are several types of decapod hemocytes; however, distinguishing criteria based on conventional staining techniques are often subtle or ambiguous. Cytochemical features of ridgeback prawn (Penaeidae: Sicyonia ingentis) hemocytes were studied using specific stains for lysosomes, cytoplasmic contents, and granule enzymes. This approach facilitates the differentiation of cell types in the ridgeback prawn and provides information on the functions of and relation ships among different cell types. Agranular hemocytes and a subgroup of small granule hemocytes contain extensive cytoplasmic glycoprotein deposits which display smudgy, intense staining with Sudan black B. As previously shown, coagulogen-the clotting material in decapods-stains with Sudan black B when extracted from lysed hemocytes. Other hemocyte types display light staining limited to granule membranes. Lysosomes are not observed in agranular cells and are rarely present in small granule hemocytes with glycoprotein deposits. Small granule hemocytes without deposits and large granule hemocytes contain numerous lysosomes as shown by the presence of acid phosphatase, ß-glucuronidase, and nonspecific esterase. Acid phosphatase is observed in the Golgi body of these cells, within small vesicles, and in small granules. The granules in large granule hemocytes rarely show acid phosphatase reaction, yet small acid phosphatase-positive vesicles fuse with the large granules. The acid phosphatase in the large granules may exist in an inactive form. Prophenoloxidase activity is localized only in large granules. The physiological significance of hemocyte cytochemistry is also discussed.

Fine structure and histochemistry of the freshly extruded and hardened spermatophore of the spiny lobster, Panulirus interruptus.

Freshly extruded and hardened spermatophores of the spiny lobster, Panulirus interruptus, were compared using light and electron microscopy (EM). The spermatophore is composed of a sperm tube embedded in an acellular matrix. The sperm tube consists of tightly packed spherical cavities in an acellular material within which the sperm lie. The extruded spermatophore is white, soft, and sticky on all surfaces. The highly coiled sperm tube can be seen near the surface of the foot of the spermatophore, which is the side that will attach to the exoskeleton of the female. The opposite surface, the cap, will harden and darken after exposure to seawater. In the soft spermatophore, the matrix surrounding the sperm tube and extending from foot to cap is composed of small (2-µm) granules embedded in a loose weave of filaments. In the hardened spermatophore, the matrix is composed of small (4-µm) empty spheres. At the cap region the matrix darkens, and at the foot the granules dissolve to form a thick layer characterized by vertical striations. The structure of this spermatophore is compared to those spermatophores of other decapods that have been described at the EM level. The chemical composition and possible function(s) of the various components are discussed.

Structure of hematopoietic nodules in the ridgeback prawn, Sicyonia ingentis: Light and electron microscopic observations.

The architecture and fine structure of the epigastric hematopoietic nodules of the ridgeback prawn, Sicyonia ingentis, are described. The nodules consist of a highly branched series of tubules that contain the maturing hemocytes within a connective tissue stroma. Hemocytes can exit the hematopoietic nodules by penetrating through fenestrations in the endothelial cell layer into the central hemal space or by migrating through the outer later of capsular cells and associated collagen fibrils. Four hemocyte categories were observed: agranular, small granule with cytoplasmic deposits, small granule without cytoplasmic deposits, and large granule hemocytes. This classification was based upon the presence, size, and type of cytoplasmic granules and the presence of cytoplasmic deposits. Only agranular cells and small granule hemocytes without cytoplasmic deposits appeared capable of division. Intermediate stages were observed between agranular hemocytes and small granule hemocytes with deposits and between small granule hemocytes without deposits and large granule hemocytes, suggesting existence of two distinct hemocyte lines.

Fine structure and classification of shrimp hemocytes.

The structure of hemocytes from two species of penaeid shrimp was examined by light and electron (TEM) microscopy. Hemocytes from the two species are indistinguishable and are classified as either agranular, small-granule, or large-granule hemocytes. Agranular hemocytes are the smallest of the hemocytes, lack granules, compose only 5-10% of the circulating hemocytes, and are nonrefractile when examined by light microscopy. Small-granule hemocytes are the most abundant type of hemocyte (75% of all hemocytes), appear nonrefractile, and contain a variable number (1-40) of granules (0.4 µm diameter). Large-granule hemocytes compose 10-20% of the hemocytes. They are filled with granules (0.8 µm in diameter) that are highly refractile when examined by light microscopy and are electron-dense when examined by TEM. Our classification scheme is based solely on the absence or presence and relative size of granules. Features used by other researchers, such as cell size, shape, and staining properties, were not used because these features are subtle and/or subjective. The proposed classification is compared with schemes developed for other decapods, and its usefulness and limitations are discussed. This scheme will serve as a basis for further studies on the maturation and physiological function(s) or crustacean hemocytes.