Development and utility of the FDA 'GutProbe' DNA microarray for identification, genotyping and metagenomic analysis of commercially available probiotics.

AIM: Lactic acid bacteria are beneficial microbes added to many food products and dietary supplements for their purported health benefits. Proper identification of bacteria is important to assess safety as well as proper product labelling. A custom microarray (FDA GutProbe) was developed to verify accurate labelling in commercial dietary supplements. METHODS AND RESULTS: Strain-specific attribution was achieved with GutProbe array which contains genes from the most commonly found species in probiotic supplements and food ingredients. Applied utility of the array was assessed with direct from product DNA hybridization to determine (i) if identification of multiple strains in one sample can be conducted and (ii) if any lot-to-lot variations exist with eight probiotics found on the US market. CONCLUSIONS: GutProbe is a useful tool in identifying a mixture of microbials in probiotics and did reveal some product variations. In addition, the array is able to identify lot-to-lot differences in these products. These strain level attribution may be useful for routine monitoring of batch variation as part of a 'Good Manufacturing Practices' process. SIGNIFICANCE AND IMPACT OF THE STUDY: The FDA GutProbe is an efficient and reliable platform to identify the presence of microbial ingredients and determining microbe differences in dietary supplements. The GutProbe is a fast, rapid method for direct community profiling or food matrix sampling.

Phylogenetic relationships of leopard frogs (Rana pipiens complex) from an isolated coastal mountain range in southern Sonora, Mexico.

Mitochondrial DNA sequence data from the control region and 12S rRNA in leopard frogs from the Sierra El Aguaje of southern Sonora, Mexico, together with GenBank sequences, were used to infer taxonomic identity and provide phylogenetic hypotheses for relationships with other members of the Rana pipiens complex. We show that frogs from the Sierra El Aguaje belong to the Rana berlandieri subgroup, or Scurrilirana clade, of the R. pipiens group, and are most closely related to Rana magnaocularis from Nayarit, Mexico. We also provide further evidence that Rana magnaocularis and R. yavapaiensis are close relatives.

Genetic variation, population structure, and phylogenetic relationships of Triatoma rubida and T. recurva (Hemiptera: Reduviidae: Triatominae) from the Sonoran Desert, insect vectors of the Chagas' disease parasite Trypanosoma cruzi.

Nucleotide and amino acid sequence data from the mitochondrial cytochrome b (Cytb) and cytochrome c oxidase subunit I (COI) gene segments were used to gain insights into the population biology and phylogenetic relationships of two species of hematophagous kissing bugs (Hemiptera: Reduviidae: Triatominae) from the Sonoran Desert of northwestern Mexico and southern Arizona, USA, Triatoma rubida (Uhler, 1894) and T. recurva (Stål, 1868), both of which are vectors of the protozoan parasite Trypanosoma cruzi responsible for Chagas' disease. Analysis of molecular variance of gene sequences indicated significant structure among populations of both species from widely separated geographic localities. Phylogenetic analyses of gene and amino acid sequences employing both Bayesian and parsimony methods showed that T. recurva clustered within the phyllosoma complex of Triatoma species from central and southern Mexico with high statistical support, and that it was closely related to T. longipennis. Triatoma dimidiata also was shown to be closely related to the phyllosoma complex, as was T. sanguisuga which has historically been assigned to the lecticularia complex. Analyses of gene sequences were unable to confidently resolve relationships of T. rubida, although weak support for a T. nitida+T. rubida clade was seen under certain conditions. A provisional calibration of a mitochondrial DNA molecular clock for T. rubida, based on geological dates for the vicariant separation of the Baja California peninsula from mainland Mexico, suggested that pairwise sequence divergences for the Cytb and COI genes were 1.1-1.8% and 0.6-1.0% per million years, respectively. Two highly supported sympatric lineages of T. rubida uhleri from southern Arizona, which are hypothesized to have diverged approximately 550,000-900,000 years ago, were detected in the Cytb gene trees.

Ecology and population genetics of Sonoran Desert Drosophila.

Three species of cactophilic Drosophila endemic to the Sonoran Desert of North America, D. nigrospiracula, D. pachea and D. mettleri, experience marked differences in spatial resource availability, and the first two of these display significant differences in dispersal behaviour. We employed starch gel and cellulose acetate electrophoresis for eight allozyme loci to test for a relationship between these variables and genetic differentiation among geographical populations of each species. No evidence was found for population structure in any of the three species, populations of which were separated by geographical distances of up to 475 km. Allele frequencies for two loci, Mdh-1 and Est-2, in D. nigrospiracula and D. pachea were very similar to those obtained approximately 30 years ago by other workers, indicating that the polymorphisms are remarkably stable under the stressful and variable conditions of the desert environment. High longevity, dispersal and multiple female remating are likely to contribute to the apparent high level of gene flow in all three species.

The evolutionary enigma of bonefishes (Albula spp.): cryptic species and ancient separations in a globally distributed shorefish.

Many examples of cryptic marine species have been demonstrated with biochemical and molecular studies. In most cases, a broadly distributed taxon is actually a group of sibling species that can be distinguished (upon closer examination) by ecological or morphological characters. Fishes of the family Albulidae constitute a notable exception. Bonefish (Albula spp.) morphology and ecology are highly conserved around the globe, and their extended pelagic larval stage could allow population connections on a vast geographic scale. Based on this perceived homogeneity, bonefishes were classified as a single pantropical species, A. vulpes. However, allozyme studies of Hawaiian populations indicated that two sympatric species (A. glossodonta and A. neoguinaica) are included in the synonymy of A. vulpes. To ascertain the number and distribution of evolutionary partitions in Albula, we surveyed 564 bp of mitochondrial DNA (mtDNA) cytochrome b from 174 individuals collected at 26 locations. Sequence comparisons reveal eight deep lineages (d = 5.56-30.6%) and significant population structure within three of the four lineages that could be tested (phiST = 0.047-0.678). These findings confirm the genetic distinctiveness of the three species noted above and invoke the possibility of five additional species. Clock estimates for mtDNA indicate that these putative species arose 4-20 million years ago. Distinct evolutionary lineages coexist in several sample locations, yet show little morphological or ecological differentiation in sympatry. Thus, bonefish species seem to defy the evolutionary conventions of morphological differentiation over time and ecological displacement in sympatry. Despite multiple cases of sympatry, sister-taxa relationships inferred from mtDNA indicate that divergence in allopatry has been the predominant speciation mechanism in Albula. Stabilizing selection in the homogeneous habitat occupied by bonefishes (tropical sand flats) could promote the retention of highly conserved morphology and ecology.

Structure of keratan sulfate from bonefish (Albula sp.) larvae deduced from NMR spectroscopy of keratanase-derived oligosaccharides.

Structural details of keratan sulfate (KS) glycosaminoglycan, isolated from early-metamorphosing larvae (leptocephali) of bonefish (Albula sp.), are described. Bonefish KS was analyzed by first hydrolyzing the purified compound with KS endo-beta-galactosidase (keratanase) from Pseudomonas spp., and then examining the resulting oligosaccharides with reversed-phase high-performance liquid chromatography (HPLC) and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy at 400 MHz. Spectral analyses were performed by COSY and HMQC. The results showed that a single oligosaccharide was produced whose structure is consistent with that of a tetrasaccharide containing two, beta-linked, N-acetyllactosamine units. Enzymic evidence indicated that the internal galactose of the tetrasaccharide was O-sulfated at C-6, and that the reducing-end galactose was unsulfated. Spectral data for C-1 of the two galactose residues were consistent with the proposed sulfation pattern. In addition, spectral evidence confirmed that a C-6 on one of the sugars was sulfated: this sulfate was tentatively assigned to the internal galactose. Chemical studies have shown that an additional sulfate group is present, but its assignment could not be confirmed, owing to the complexity of the spectral data. The known specificities of keratanase, and the production of a single tetrasaccharide, however, require that the additional sulfate reside on C-6 of either of the two available N-acetylglucosamine (GlcNAc) moieties, and that it cannot alternate between the two. The inability of beta-N-acetylglucosaminidase from beef kidney to liberate GlcNAc from the tetrasaccharide provided preliminary support for the view that this sulfate is located on the nonreducing-end GlcNAc. We conclude that the native, high molecular weight (M(r) = 55,000) KS polymer from bonefish larvae consists of a disulfated disaccharide alternating with an unsulfated disaccharide in the adjacent N-acetyllactosamine unit, with this pattern repeating itself in a regular fashion along most, or all, of the chain. This structure could provide an explanation for the ability of bonefish KS chains to self-associate into dimers. Although the N-acetyllactosamine repeat is characteristics of KS in general, the sulfation pattern is different from that postulated for the well-characterized KS chains of lower molecular weight obtained from mammalian cornea and cartilage. An additional difference was the inability to demonstrate sialic acid in bonefish KS.

Alcohol dehydrogenase polymorphism in barrel cactus populations of Drosophila mojavensis.

Starch gel electrophoresis revealed that the alcohol dehydrogenase (ADH-2) locus was polymorphic in two populations (from Agua Caliente, California and the Grand Canyon, Arizona) of cactophilic Drosophila mojavensis that utilize barrel cactus (Ferocactus acanthodes) as a host plant. Electromorphs representing products of a slow (S) and a fast (F) allele were found in adult flies. The frequency of the slow allele was 0.448 in flies from Agua Caliente and 0.659 in flies from the Grand Canyon. These frequencies were intermediate to those of the low (Baja California peninsula, Mexico) and high (Sonora, Mexico and southern Arizona) frequency Adh-2S populations of D. mojavensis that utilize different species of host cacti.

Changes in lipid composition during metamorphosis of bonefish (Albula sp.) leptocephali.

During metamorphosis of bonefish (Albula sp.) larvae (leptocephali) all energy requirements are provided by breakdown of endogenous compounds, with lipid catabolism accounting for about 80% of total energy production. The principal objective of the present study was to characterize the lipid classes and fatty acids utilized. Analysis of whole-body lipid content indicated that larvae lost about half (3.6 mg) of their total lipid during the 10-d period. Percentages of neutral and polar lipid in early metamorphosing larvae were 64.2 and 35.8%, respectively; these values showed little change during metamorphosis, indicating that both lipid classes were catabolized. Triacylglycerols, the principal neutral lipid of all metamorphic stages, decreased by 1.8 mg, accounting for half of the decrease in total lipid. Levels of phosphatidylethanolamine, the principal polar lipid in early larvae, decreased by more than 50% during metamorphosis; levels of phosphatidylcholine, which was not detected in early larvae, increased. Fatty acids showing the largest net decreases, presumedly used as energy sources, were 16:0 (30.4%), 14:0 (13.8%), 16:1n-7 (12.2%), 20:5n-3 (7.7%), 18:1n-9 (7.4%), and 18:4n-3 (6.9%). Most of 22:6n-3, the second most abundant fatty acid of early larvae, was conserved.

Energetics of metamorphosis in bonefish (Albula sp.) leptocephali: Role of keratan sulfate glycosaminoglycan.

Energy budget calculations, based on published values for changes in biochemical composition and oxygen consumption, have confirmed that metamorphosing bonefish (Albula sp.) larvae from the Gulf of California receive all of their metabolic energy requirements from the breakdown of endogenous reserves. The calculations showed that during metamorphosis, about 80% of the energy was supplied by lipid, and most of the remainder was provided by keratan sulfate glycosaminoglycan. These results support the suggestion that keratan sulfate can function as a storage polysaccharide, a novel function for glycosaminoglycans in vertebrates.

Metabolic Rates in Early Life History Stages of Elopomorph Fishes.

The respiratory electron transport system (ETS) assay was used to estimate metabolic rates in four species of eel (Anguilliformes: Ophichthidae and Congridae) leptocephali (Myrophis punctatus, Ophichthus sp., Hildebrandia flava, and one unidentified congrid) and the bonefish (Albuliformes: Albulidae: Albula sp.). Wet-weight-specific ETS values in whole-body homogenates, assayed at physiological temperatures, ranged from 4-20 {mu}-at O h-1 (g wet wt)-1. Arrhenius activation energies (Ea) ranged from 11.0-15.7 kcal mole-1. Both wet-weight-specific ETS activity and oxygen consumption rate increased approximately fivefold during metamorphosis of leptocephali of Albula sp. Wet-weight-specific ETS activity showed little change as leptocephali of M. punctatus transformed into glass eels, but increased about fivefold as glass eels metamorphosed into elvers. No significant difference was found in ETS activity measured in fresh early metamorphic leptocephali of Albula sp. and leptocephali that had been stored frozen at -70{deg}C for up to 15 months. The data suggest that metabolic rates are low in leptocephali, which implies that the demand for nutrients is also relatively low. We argue that the apparent diet of these larvae seems capable of providing a sufficient supply of nutrients under these conditions.

Characterization and distribution of undersulfated chondroitin sulfate and chondroitin in leptocephalous larvae of elopomorph fishes.

Undersulfated chondroitin sulfate (i.e., chondroitin sulfate with sulfate: hexosamine molar ratios ranging from 0.06-0.17) has been identified as a principal glycosaminoglycan (GAG) in whole-body extracts of leptocephalous larvae of four species of marine teleost fishes, representing two orders and three families. These include the eels (Anguilliformes)Ariosoma balearicum (Congridae),Rhechias dubia (Congridae) and Ophichthus sp. (Ophichthidae) and the ladyfish (Elopiformes: Elopidae: Elops saurus). A second GAG found in relatively large amounts in A. balearicum has been identified as chondroitin. Chondroitin and undersulfated chondroitin sulfate formed distinctive precipitates during extraction and were easily separated. Undersulfated chondroitin sulfate also was extracted and purified from early metamorphosing bonefish (Albuliformes: Albulidae: Albula sp.) leptocephali, where it is a minor GAG component, and was shown to be similar in composition to the compounds obtained from eels and ladyfish.

Glycosidase and sulfatase activities and their possible role in keratan sulfate degradation in metamorphosing bonefish (Albula sp.) leptocephali.

During metamorphosis of leptocephalous larvae of the bonefish (Albula sp.), keratan sulfate, the principal glycosaminoglycan of the extracellular gelatinous body matrix, is degraded. Artificial substrates have been utilized to demonstrate the presence of ß-N-acetylglucosaminidase, ß-galactosidase and sulfatase activities in whole-body homogenates of early metamorphosing leptocephali. The concerted action of these enzymes has been shown to degrade the keratan sulfate polymer in other tissues. This paper describes the extraction, partial purification and some of the physical and kinetic properties of these enzymes. Additionally, starch gel electrophoresis was used to follow glycosidase activities in early, intermediate and advanced metamorphosing larvae. No differences were observed in electrophoretic migration or banding pattern of either ß-N-acetylglucosaminidase or ß-galactosidase during metamorphosis.

Sensory systems and behavior of premetamorphic and metamorphic leptocephalous larvae.

The superorder Elopomorpha (orders Anguilliformes, Elopiformes and Notacanthiformes) is characterized by the presence of a unique larval stage termed the leptocephalus. The basic morphology, chemical composition, developmental pattern, and behavior of premetamorphic and metamorphic leptocephali are reviewed. The eyes, olfactory organs and pores of the lateral-line system are well developed, suggesting that these sensory structures play an important role in larval behavior. Premetamorphic larvae are pelagic and in some elopomorphs subsequent development is associated with migration to coastal waters where metamorphosis takes place. The factors controlling onshore migration, as well as the metamorphic trigger(s), are unknown. The possible relationships between sensory systems and behavioral changes during the different phases of larval development are presented.

Isolation and partial characterization of a novel keratan sulfate proteoglycan from metamorphosing bonefish (Albula) larvae.

Proteoglycans (PGs) were isolated from leptocephalous larvae of the bonefish (Albula sp.), which were in the early stages of metamorphosis, using both associative and dissociative conditions in the presence of protease inhibitors. The procedure was rapid and resulted in an extraction efficiency of 75% (associative) and 85-90% (dissociative). The majority of co-extracted protein could be effectively separated from the PGs by utilizing either Sepharose CL-2B or CL-6B gel chromatography. Sepharose CL-2B chromatography of extracted PGs after treatment with bacterial keratan sulfate-endo-ß-galactosidase (keratanase) showed that most of the high molecular weight (M r) carbohydrate was degraded. Free keratan sulfate (KS) chains were prepared from whole-larva extracts (which also contain small amounts of chondroitin sulfate) by both chondroitinase ABC treatment and ethanol fractionation. Sepharose CL-6B chromatography under dissociative conditions showed that larval KS chains were much larger (M r∼55,000) than those from cornea. These chains tended to aggregate when chromatographed under associative conditions. Larval KS was degraded by keratanase and resistant to chondroitinase, ABC and testicular hyaluronidase. Differences were also noted in the oligosaccharides produced by keratanase treatment of the two preparations. However, biochemical composition of larval and corneal KS was similar.

Free amino acids in metamorphosing bonefish (Albula sp.) leptocephali.

Metamorphosing leptocephalous larvae of the bonefish (Albula sp.) were analyzed for total ninhydrinpositive substances (NPS) and free amino acids. Total NPS content showed little change during metamorphosis. The average NPS value (±S.E.) for 16 larvae was 1.8 (±0.1) mg×larva(-1), which represents approximately 4% and 8% of the total dry weight of early and advanced larvae, respectively. Taurine was the most abundant free amino acid in whole-larva extracts, accounting for 36% and 59% of the total by weight in early and advanced larvae, respectively. The essential amino acids, leucine, isoleucine, phenylalanine, histidine, valine, methionine, lysine and arginine, accounted for about half (47%) of the total in early larvae but were reduced to about 23% of the total in advanced larvae. All of the component essential amino acids decreased during metamorphosis, but the greatest effect was seen with the first five. The remaining non-essential amino acids comprised less than 20% of the total in early larvae and, although the overall value changed little during metamorphosis, certain components such as glycine and glutamic acid showed large increases whereas others such as tyrosine and serine were reduced. Increases in amino acid content after acid hydrolysis of whole-larva extracts indicated that trichloroacetic acid-soluble, low molecular weight peptides were present in both early and advanced leptocephali.

Inhibition of gill (Na+ + K+)-ATPase in rainbow trout (Salmo gairdneri) by a purinedisulfide analog of adenosine triphosphate.

The effect of the adenosine triphosphate analog, 6,6'-dithiobis(inosinyl imidodiphosphate), (sIMP-PNP)2, was tested on the ouabain-sensitive (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and the ouabain-insensitive Mg2+ - ATPase in microsomes prepared from gill tissue of sea water-adapted rainbow trout, Salmo gairdneri. The (Na+ + K+)-ATPase was completely inhibited by low concentrations of (sIMP-PNP)2 (6 micrometer) but the Mg2+ - ATPase was unaffected by the inhibitor at concentrations as high as 28 micrometer, supporting the suggestion that the two activities represent separate enzymes. The specificity of inactivation could be demonstrated both at a physiological temperature (13 degrees C) and at 37 degrees C. The rates of inactivation were similar at both temperatures. Inactivation of the (Na+ + K+)-ATPase by (sIMP-PNP)2 was reversed by dithiothreitol, suggesting that the inhibitor forms a mixed disulfide with sulfhydryl groups on the enzyme. The inability of substrate (either ATP or its analog, adenyl-5'-yl imidodiphosphate) to protect against inactivation suggests that (sIMP-PNP)2 is reacting with sulfhydryl groups which are not associated with the active site.