Design and use of group-specific primers and probes for real-time quantitative PCR

Real-time quantitative polymerase chain reaction (qPCR) has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil, water, sediments, and sludge. Although qPCR is a very useful technique for nucleic acid quantification, accurately quantifying the target microbial group strongly depends on the quality of the primer and probe used. Many aspects of conducting qPCR assays have become increasingly routine and automated; however, one of the most important aspects, designing and selecting primer and probe sets, is often a somewhat arcane process. In many cases, failed or non-specific amplification can be attributed to improperly designed primer-probe sets. This paper is intended to provide guidelines and general principles for designing group-specific primers and probes for qPCR assays. We demonstrate the effectiveness of these guidelines by reviewing the use of qPCR to study anaerobic processes and biologic nutrient removal processes. qPCR assays using group-specific primers and probes designed with this method, have been used to successfully quantify 16S ribosomal Ribonucleic Acid (16S rRNA) gene copy numbers from target methanogenic and ammonia-oxidizing bacteria in various laboratory- and full-scale biologic processes. Researchers with a good command of primer and probe design can use qPCR as a valuable tool to study biodiversity and to develop more efficient control strategies for biologic processes.     

Development and environmental application of a genus-specific quantitative PCR approach for Pseudo-nitzschia species

Quantitative polymerase chain reaction (qPCR) for the identification and quantification of microbes has become a common tool for the study of harmful algal blooms (HABs). We developed a qPCR method for the diatom genus Pseudo-nitzschia. Several species of this genus form toxic blooms through the production of the neurotoxin domoic acid (DA). Outbreaks of toxicity attributed to DA along the US west coast have caused sickness and death of marine mammals and seabirds through food web contamination. The method developed here quantifies Pseudo-nitzschia spp. at low abundances in natural samples, thereby, providing a method to improve our understanding of the environmental conditions leading to blooms of these species. This has been accomplished previously by techniques for identification and quantification that are slow and laborious compared to qPCR. The approach was successfully tested and validated using eight species of Pseudo-nitzschia and 33 non-target organisms and employed to follow local bloom dynamics.     

Lipid content of some Caribbean corals in relation to depth and light

Three procedures for the extraction of coral lipids were compared and a rapid and effective method for future use is suggested. This method was used to measure the lipid content of the branching coral Porites porites (Pallas) and the massive corals Montastrea annularis (Ellis and Solander) and Siderastrea siderea (Ellis and Solander) during July and August 1991. P. porites and M. annularis were sampled on two fringing reefs, each characterised by different water quality affecting light transmission, and at depths of down to 30 m on a barrier reef on the west coast of the island. m. annularis contained, on average, 29% of dry weight as lipid, and there were no significant differences in lipid levels between corals sampled on either fringing reef at 3 and 6 m, or between fringing reefs and the barrier reef at 13, 20 or 30 m depth. Five samples were also taken from a single massive colony of S. siderea at 3 m on a fringing reef and at 13, 20 and 30 m depth on the barrier reef. Values for lipid ranged from 26 to 35% of dry tissue weight. P. porites from 3 and 6 m depth on both fringing reefs contained the same amount of lipid (11% of dry tissue weight). However, at 13 m depth on the barrier reef this coral contained significantly less lipid (8.5% of dry tissue weight). This difference may be attributable to a higher nutritional intake by P. porites on the fringing reefs.     

Three party symbiosis: acoelomorph worms,corals and unicellular algal symbionts in Eilat (Red Sea)

Epizoic worms were found to occur on certain coral colonies from reefs off the coast of Eilat (Red Sea). We identified 14 coral species infested by acoelomorph worms at a depth range of 2–50 m. The host corals were all zooxanthellate and included both massive and branching stony corals and a soft coral. Worms from all hosts were identified as belonging to the genus Waminoa and contained two distinct algal symbionts differing in size. The smaller one was identified as Symbiodinium sp. and the larger one is presumed to belong to the genus Amphidinium. Worm-infested colonies of the soft coral, Stereonephthya cundabiluensis, lacked a mucus layer and exhibited distinct cell microvilli, a phenotype not present in colonies lacking Waminoa sp. In most cases, both cnidarian and Acoelomorph hosts displayed high specificity for genetically distinctive Symbiodinium spp. These observations show that the epizoic worms do not acquire their symbionts from the “host” coral.     

PSII activity and pigment dynamics of Symbiodinium in two Indo-Pacific corals exposed to short-term high-light stress

This study examined the capacity for photoprotection and repair of photo-inactivated photosystem II in the same Symbiodinium clade associated with two coexisting coral species during high-light stress in order to test for the modulation of the symbiont’s photobiological response by the coral host. After 4 days exposure to in situ irradiance, symbionts of the bleaching-sensitive Pocillopora damicornis showed rapid synthesis of photoprotective pigments (by 44 %) and strongly enhanced rates of xanthophyll cycling (by 446 %) while being insufficient to prevent photoinhibition (sustained loss in F _v/F _m at night) and loss of symbionts after 4 days. By contrast, Pavona decussata showed no significant changes in F _v/F _m, symbiont density or xanthophyll cycling. Given the association with the same Symbiodinium clade in both coral species, our findings suggest that symbionts in the two species examined may experience different in hospite light conditions as a result of different biometric properties of the coral host.     

Predominance of clade D <Emphasis Type="Italic">Symbiodinium</Emphasis> in shallow-water reef-building corals off Kish and Larak Islands (Persian Gulf,Iran)

Scleractinian coral species harbour communities of photosynthetic taxa of the genus Symbiodinium. As many as eight genetic clades (A, B, C, D, E, F, G and H) of Symbiodinium have been discovered using molecular biology. These clades may differ from each other in their physiology, and thus influence the ecological distribution and resilience of their host corals to environmental stresses. Corals of the Persian Gulf are normally subject to extreme environmental conditions including high salinity and seasonal variation in temperature. This study is the first to use molecular techniques to identify the Symbiodinium of the Iranian coral reefs to the level of phylogenetic clades. Samples of eight coral species were collected at two different depths from the eastern part of Kish Island in the northern Persian Gulf, and Larak Island in the Strait of Hormuz. Partial 28S nuclear ribosomal (nr) DNA of Symbiodinium (D1/D2 domains) were amplified by polymerase chain reaction (PCR). PCR products were analyzed using single stranded conformational polymorphism and phylogenetic analyses of the LSU DNA sequences from a subset of the samples. The results showed that Symbiodinium populations were generally uniform among and within the populations of eight coral species studied, and there are at least two clades of Symbiodinium from Kish and Larak islands. Clade D was detected from eight of the coral species while clade C was found in two of species only (one species hosted two clades simultaneously). The dominance of clade D might be explained by high temperatures or the extreme temperature variation, typical of the Persian Gulf. Publication of this article was held up owing to technical problems. The publisher apologizes sincerely for this lengthy delay.     

Chemoautotrophic symbionts and translocation of fixed carbon from bacteria to host tissues in the littoral bivalve Loripes lucinalis (Lucinidae)

Specimens of Loripes lucinalis (Lucinidae) living in reducing sediments were collected near a sewage outfall at low tide on the Moulin Blanc beach, Brest, France, from January to March 1987. Electron microscope studies revealed numerous Gram-negative-type bacteria in the gill cells. Ribulosebiphosphate carboxylase, a diagnostic enzyme of the Calvin-Benson cycle of CO₂-fixation was measured only in the gill extracts. Various tissues of L. lucinalis were examined for activity of APS reductase, (EC 1.8.99.2), ATP sulphurylase (EC 2.7.7.4) and rhodanese (EC 2.8.1.1), enzymes involved in sulphide oxidation. APS reductase was only found in symbiont-containing tissues, i.e., gills. These enzymatic studies characterise the symbionts as chemoautotrophic sulphide-oxidizing bacteria. Histoautoradiography demonstrated that part of the carbon dioxide fixed by symbiotic bacteria in the gills is translocated to symbiont-free tissues of the bivalve. The ultrastructure of the gill is detailed and a nomenclature based on established and new terminology is proposed to describe the various cellular types comprising the gill filament.     

Distribution of deep-water gorgonian corals in relation to benthic habitat features in the Northeast Channel (Atlantic Canada)

The distribution and abundance of deep-water gorgonian corals were investigated along 52 transects at 183–498 m depth in the Northeast Channel, between Georges Bank and Browns Bank in the northwest Atlantic, using a remotely operated vehicle and a towed video-camera system. Three species (Paragorgia arborea, Primnoa resedaeformis, and Acanthogorgia armata) were observed. Primnoa occurred on 35 transects below 196 m depth, with highest local abundance in stands of 104 colonies per 100 m². Paragorgia was present on 21 transects deeper than 235 m, with highest local abundance of 49 colonies per 100 m². Acanthogorgia was observed at only four transects between 231 m and 364 m, with a local maximum abundance of 199 colonies per 100 m². The maximum abundance averaged for whole transects was 19.2 and 6.2 colonies per 100 m² for Primnoa and Paragorgia, respectively. The corals were more common in the outer part of the channel along the shelf break and slope than on the shelf in the inner part. All three species showed a patchy distribution with no signs of competitive exclusion at any spatial scale. Transects with high abundance of corals were characterised by depths greater than 400 m, maximum temperatures less than 9.2°C, and a relatively high percentage coverage of cobble and boulder (more than 19% and 6%, respectively). High temperatures probably control the upper depth limit of the corals, and Primnoa seems to tolerate slightly higher temperatures than Paragorgia. Abundance of both species was negatively correlated with average temperature and positively with cobbles. Together, temperature, percentage cobble and salinity accounted for 38% of the variance of Primnoa. The comparable figure for Paragorgia was 15%. The observed distribution indicated that the abundance of coral is controlled by additional factors such as larger-scaled topographic features governing the current regimes and thus also the supply of food and larvae.Communicated by R. J. Thompson, St. Johns     

Seasonal and sex-specific variations in levels of photo-protecting mycosporine-like amino acids (MAAs) in soft corals

This study focusses on the nature and extent of variation in mycosporine-like amino acids (MAAs) in relation to annual cycles in solar radiation, seawater temperature, and reproduction in reef-flat populations of two soft coral species. The results show MAA tissue concentrations in shallow water colonies of Lobophytum compactum and Sinularia flexibilis to be significantly correlated to annual cycles in solar radiation (P<0.0006 and P<0.0005, respectively) and seawater temperature (P<0.0006 and P<0.0004, respectively). Evidence of seasonal cycles in MAA levels in the tissues of shallow-reef invertebrates positively correlating with annual cycles in solar radiation and temperature suggests that they are an integral component of the soft corals' biochemical defence system against high irradiance and/or temperature stress and thus bleaching. This is further corroborated by the higher production of MAAs in females than males prior to spawning (up to 67% and 56% for L. compactum and S. flexibilis, respectively), presumably to provide a high level of protection against irradiance stress for progeny.     

Functional autotrophy of Heteroxenia fuscescens (Anthozoa: Alcyonaria): carbon assimilation and translocation of photosynthates from symbionts to host

The association of the alcyonarian Heteroxenia fuscescens (Ehrb.) with its cytosymbiotic algae shows structural and physiological adaptations optimizing the living together of the two partners as one functional unit. To enhance the energetic contribution of the autotrophic partner, the organization of the heterotrophic partner bears typical plant-like imprints. Up to 20% of the inorganic C photosynthetically fixed was translocated to the host (=2 mg C mg d.w.^-1 d^-1). This net C gain by the host is used for anabolic purposes including the deposition of storage material. Especially the wax-esters and triglycerides of the host-in contrast to those of the symbionts-were intensively labelled. The in-vivo ¹⁴C-fixation of zooxanthellae is more than double the in-vitro fixation. In both symbionts and host, the lipids show the highest relative ¹⁴C-incorporation. In particular the polyol component was strongly labelled. After 120 min of continuous incubation, approximately 40 labelled intracellular metabolites were detectable in the ethanol/water soluble fractions of zooxanthellae. Glycerol is the main low-molecular weight carbohydrate being transferred. This is corroborated by the deacylation of lipids of the host.     

Feeding behavior and ecology of shallow-water unstalked crinoids (echinodermata) in the Caribbean Sea

The feeding behavior, living position, and skeletal morphology of 8 species of reef-dwelling Caribbean comatulid crinoids are intimately related to the regime of water movement prevailing in the microhabitat. These adaptations are related to the dependence of the crinoid suspension-feeding mechanism on externally produced water movements for a continuous food supply. Greater numbers of co-occurring comatulid species (6 to 7) and larger populations have been found off Colombia and Panamá than off Curaçao and Jamaica (4 species). It is suggested that these differences may be related to increased or diversified primary productivity close to the larger land masses. Overal food availability as determined by primary productivity may, thus, be an important factor controlling the regional diversity and abundance of these species.     

Ultrastructure of the spirocyst tubule in black corals (Coelenterata: Antipatharia) and its taxonomic implications

Spirocyst tubules in five shallow-water antipatharian species Antipathes pennacea, Antipathes sp. cf. salix, A. fiordensis, A. galapagensis, and Cirrhipathes luetkeni contain an electron-opaque, helically arranged structure, composed of four distinct and separate bundles of microfibril-like material. Each bundle appears to be solid, rather than hollow, and is enveloped by a sheath of lesser electron-opacity that is continuous with the pleats. Tubules from a more distantly related, deep-water antipatharian, are similarly configured but are composed of only three distinct bundles, separated from one another by electron-opaque sheath material. The bundles are electron-lucent; their substructure was not preserved because this specimen was prepared for microscopy after preservation in alcohol for 27 yr, apparently without initial fixation. Most other details of the spirocyst tubule and capsule can be determined from this museum specimen. The arrangement within the tubule of multiple bundles, clearly defined by sheaths, is structurally distinct from other zoantharian spirocysts, and appears to be unique to the Antipatharia. The tubule structure of the anemone Aiptasia sp., examined for comparison, is consistent with that of spirocysts from other actiniarians and zoantharians generally, including the appearance of electron-opaque rodlets within the tubule lumen. No substructural information was obtained from Aiptasia sp. rodlets. It is unclear whether the microfibrillar bundles of antipatharian spirocysts differ from those of other zoantharians by being more amenable to resolution, or if their tubule substructure is fundamentally distinct from those of more typical zoantharians. In terms of overall structure, Aiptasia sp. spirocyst tubules contain a single bundle of rodlets without a clearly defined sheath, in contrast to antipatharians which have three or four bundles, each clearly defined. These structural differences may be useful in determining relationships both within the Antipatharia, and between antipatharians and allied orders.     

Use of pulse amplitude modulated (PAM) fluorometry for in situ measurements of photosynthesis in two Red Sea faviid corals

Measurements of the photosynthetic activity of symbiotic zooxanthellae in corals under natural growth conditions has been limited until recently, and this is one of the first reports on utilising a newly developed underwater pulse amplitude modulated (PAM) fluorometer (the Diving-PAM, Walz Gmbh, Germany) for such studies in situ. Photosynthetic responses to irradiance (photosynthetic photon flux, PPF) of the two faviid corals Favia favus (Forskål) and Platygyra lamellina (Ehrenberg) were measured while snorkelling or SCUBA diving (in August 1997), and we report here the results in terms of effective quantum yields of photosystem II (Y?) and estimated rates of photosynthetic electron transport (ETR, calculated as Y?×?0.5?× PPF?×?FA, where FA is the estimated fraction of light absorbed by the photosymbiont-containing tissue). Both species showed a reduction in Y with increasing actinic irradiances produced by the instrument above 500?μmol photons m^?2 s^?1, and the corresponding ETR values yielded apparently typical photosynthesis versus irradiance (P-I?) curves, which saturated between 1500 and 2000?μmol photons m^?2 s^?1. It was found that 30?s irradiation at each PPF level was sufficient to give optimal ETR values and, therefore, each P-I curve could be obtained within a few minutes. In situ point measurements from various areas of colonies under ambient light showed average ETR values within the range expected from the P-I curves. In order to test the Diving-PAM in an eco-physiologically relevant experiment, photosynthetic ETR versus PPF was measured for three sections of a large P. lamellina, each section of which received different natural irradiance levels. The results clearly demonstrated adaptations to the ambient light field in that vertical and downward-facing portions of the colony showed gradually lower maximal ETRs, steeper initial slopes of the P-I curves and, accordingly, lower light saturation points than upward-facing areas receiving higher light levels. Based on these trials, some evaluations are given as to the applicability of the Diving-PAM for photosynthetic measurements when monitoring similar corals.     

Comparative species richness,composition, and abundance of invertebrates in Caribbean seagrass (Thalassia testudinum) meadows (Panamá)

The results of a year-long study in which epibenthic invertebrates were collected monthly from seagrass (Thalassia testudinum) meadows along the Caribbean coast of Panamá and the Panamá Canal Zone are described in this paper. Differences in species composition and abundance among sites were primarily due to the proximity of surrounding habitats, especially coral reefs, which contain a number of species that utilize the seagrass meadows. In contrast to many previous characterizations of tropical marine habitats, important seasonal fluctuations in both species number and abundance took place at each of the sites. Data on breeding activity among several species of decapod crustaceans indicate year-round reproduction, although considerable seasonal differences occur in the percentage of ovigerous females. These interspecific differences in observed reproductive output may be explained by differences in life-cycle length, a factor not often considered in discussions of seasonal breeding patterns in tropical marine invertebrates. Overall species composition was qualitatively similar to that reported in comparable studies of tropical and subtropical seagrass meadows elsewhere, although caridean shrimp and xanthid orab species were reduced in number and total abundance were much lower than in previous studies.     

Molecular characterization of symbiotic algae (Symbiodinium spp.) in soritid foraminifera (Sorites orbiculus) and a scleractinian coral (Orbicella annularis) from St John,US Virgin Islands

The exchange of Symbiodinium symbionts among scleractinian and soritid hosts could facilitate acclimatization to changing conditions by establishing novel symbiotic unions better tuned to prevailing conditions. In this study, we compare the communities of Symbiodinium spp. in neighboring populations of Orbicella annularis and Sorites orbiculus from St. John, US Virgin Islands, using operational taxonomic unit (OTU) clustering of cloned internal transcribed spacer 2 (ITS-2) rDNA sequences. We tested for partitioning of Symbiodinium OTUs by host and depth within and between two sites to explore the potential for symbiont exchange between hosts and light-dependent microhabitat specialization. An apparent lack of overlap in Symbiodinium communities (13 OTUs representing 7 clades) hosted by O. annularis and S. orbiculus suggests that exchange among these hosts does not occur. A low number of novel clade G ITS-2 sequences were found in one O. annularis and one S. orbiculus. A phylogenetic analysis of these sequences revealed them to be sub-clade G2 Symbiodinium, which are most commonly hosted by excavating clionid sponges. A permutational MANOVA revealed within host differences in the partitioning of Symbiodinium OTUs by site but not depth. This finding highlights the potential roles of either dissimilar environmental conditions between sites, or at least partial separation between populations, in determining the types of Symbiodinium contained in different hosts on a spatial scale of a few kilometers.     

Serial patterns of biodiversity change in corals across shallow reef flats in Ko Phuket,Thailand, due to the effects of local (sedimentation) and regional (climatic) perturbations

A 17-year monitoring programme of reef flats at Ko Phuket, Thailand afforded an opportunity to evaluate both univariate and multivariate measures of environmental stress in an assessment of change on coral reef ecosystems. The sites at Ko Phuket suffered the effects of dredging in 1986-1987 and then anomalously low sea levels in 1997-1998 as a result of climate-related events in the Indian Ocean. Univariate measures of species diversity and taxonomic distinctness ((*) increased across the reef flat, reflecting the greater effects of physical stresses on the inner reef flats (compared with outer reef flats) at all sites, with more congeneric species present on the inner reef and more confamilial species on the outer reef. Multivariate measures showed a clear breakdown in seriation patterns at all sites during the dredging in 1987 and in 1998, as a result of earlier negative sea-level anomalies. Recovery from environmental disturbances was obvious within 12 months in each case. The domination of the reefs by massive coral species, which are physiologically adapted to intertidal living and which display partial rather than total colony mortality, may be a factor contributing to the apparent resilience of the reef flats together with continued recruitment and survival of juvenile corals during adverse environmental conditions. Elevated sea temperatures and extensive bleaching of corals in 1991, 1995, and 1998 had no effect upon coral community measures, with many corals recovering their zooxanthellae numbers within 3-5 months of the bleaching events.     

Reef-basel herbivores and the distribution of two seagrasses (Syringodium filiforme and Thalassia testudinum) in the San Blas Islands (Western Caribbean)

Two seagrasses, manatee grass (Syringodium filiforme) and turtle grass (Thalassia testudinum), predominated in the areas bordering Ukkup Tupo, San Blas Islands, Western Caribbean. These seagrasses occupied the following three concentric zones extending outward from the reef: a near-reef turtle grass zone, an intermediate manatee grass zone and an off-reef turtle grass zone. Feeding experiments between January and March 1980 indicate that the absence of manatee grass close to the reef resulted from grazing by reef-associated herbivores, mainly day-active fishes and night-active sea urchins (Diadema antillarum). Grazing on manatee grass by fishes was approximately six times greater than grazing by sea urchins; thus, it appears that herbivorous fishes restrict the near-reef distribution of manatee grass at the study area. Where grazing was heaviest, the inner boundary of the manatee grass zone was farthest from the reef. The volume of manatee grass grazed during experiments was five times the volume of turtle grass consumed, strongly suggesting that the former species is a preferred food item. This is the first evidence for selective grazing on seagrasses.     

Development of the nasal olfactory organs in the larvae, settlement-stages and some adults of 14 species of Caribbean reef fishes (Labridae, Scaridae, Pomacentridae)

Larval fishes likely use a variety of settlement cues to locate and navigate toward the habitats they will inhabit as juveniles. Information about the morphology and state of development of the sensory organs of larval stages of fishes provides insight into their capabilities at the time of settlement. The peripheral olfactory organ of wild-caught late-stage larvae and early juveniles and some adults of 14 species of the Caribbean reef fishes wrasses (Labridae), parrot fishes (Scaridae) and damselfish (Pomacentridae) were examined using scanning electron microscopy and compared in terms of settlement specificity. Ages in days after hatching and days post-settlement were determined from the otoliths. Morphology of the nares and the olfactory epithelium are described for these species by stage. The separation of the anterior and posterior nares occurred before settlement in the labrids but in some specimens of scarids this separation was not complete by the time of settlement. Densities of ciliated and microvillous receptor cells and non-sensory ciliated epithelial cells were calculated. Densities of ciliated receptor cells ranged from 0.389 μm⁻² in a specimen of Thallasoma bifasciatum to 0.0057 μm⁻² in Bodianus rufus and of microvillous receptor cells from 0.038 μm⁻² in a Clepticus parrae juvenile to 0.266 μm⁻² in a juvenile Doratonotus megalepis. Densities of non-sensory cilia, also associated with high olfactory ability, were also high. The olfactory organ in wrasses is well developed prior to settlement and is comparable to that of adult fishes. The possible role of olfaction in larval schooling, reef cue detection and orientation toward habitat at settlement is discussed.

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Polimetrics: the quantitative study of urban systems (and its applications to atmospheric and hydro environments)

The rapid urbanization of the Earth has led to highly populated cities that act as concentrated centers of anthropogenic stressors on the environment. Much of these stressors originate from nonlinear coupling between man-made urban elements (i.e., networks of engineering and socio-economic infrastructures) and the natural environment. An urban area can be treated as a system of urban elements (or elemental systems), and simple interactions between the latter may lead to complex outcomes, known as emergent properties, in the former. Emergent properties of urban systems play an important role in determining their resilience and sustainability, studies on which require an in-depth knowledge of dynamics, interdependencies and feedbacks of urban elements as well as development of quantitative holistic models to integrate such knowledge to predict the overall system response. To facilitate this demanding effort, it is proposed to initiate a new sub-discipline within the discipline of Earth System Engineering, with the portmanteau of Polimetrics, to deal with quantitative scientific aspects of urban systems. This paper discusses two examples of polimetrics related to urban fluid flows. The first deals with the urban heat island and the other with storm surges in coastal cities.