Towards in situ Single Cell Systems Biology

Systems-level studies of biological systems rely on observations taken at a resolution lower than the essential unit of biology, the cell. Recent technical advances in DNA sequencing have enabled measurements of the transcriptomes in single cells excised from their environment, but it remains a daunting technical problem to reconstruct in situ gene expression patterns from sequencing data. In this thesis I develop methods for the routine, quantitative in situ measurement of gene expression using fluorescence microscopy.

The number of molecular species that can be measured simultaneously by fluorescence microscopy is limited by the pallet of spectrally distinct fluorophores. Thus, fluorescence microscopy is traditionally limited to the simultaneous measurement of only five labeled biomolecules at a time. The two methods described in this thesis, super-resolution barcoding and temporal barcoding, represent strategies for overcoming this limitation to monitor expression of many genes in a single cell. Super-resolution barcoding employs optical super-resolution microscopy (SRM) and combinatorial labeling via-smFISH (single molecule fluorescence in situ hybridization) to uniquely label individual mRNA species with distinct barcodes resolvable at nanometer resolution. This method dramatically increases the optical space in a cell, allowing a large numbers of barcodes to be visualized simultaneously. As a proof of principle this technology was used to study the S. cerevisiae calcium stress response. The second method, sequential barcoding, reads out a temporal barcode through multiple rounds of oligonucleotide hybridization to the same mRNA. The multiplexing capacity of sequential barcoding increases exponentially with the number of rounds of hybridization, allowing over a hundred genes to be profiled in only a few rounds of hybridization.

The utility of sequential barcoding was further demonstrated by adapting this method to study gene expression in mammalian tissues. Mammalian tissues suffer both from a large amount of auto-fluorescence and light scattering, making detection of smFISH probes on mRNA difficult. An amplified single molecule detection technology, smHCR (single molecule hairpin chain reaction), was developed to allow for the quantification of mRNA in tissue. This technology is demonstrated in combination with light sheet microscopy and background reducing tissue clearing technology, enabling whole-organ sequential barcoding to monitor in situ gene expression directly in intact mammalian tissue.

The methods presented in this thesis, specifically sequential barcoding and smHCR, enable multiplexed transcriptional observations in any tissue of interest. These technologies will serve as a general platform for future transcriptomic studies of complex tissues.

The impact of molecular biology on assessment of water quality: advantages and limitations of current techniques

The advent of molecular biology has had a dramatic impact on all aspects of biology, not least applied microbial ecology. Microbiological testing of water has traditionally depended largely on culture techniques. Growing understanding that only a small proportion of microbial species are culturable, and that many microorganisms may attain a viable but non-culturable state, has promoted the development of novel approaches to monitoring pathogens in the environment. This has been paralleled by an increased awareness of the surprising genetic diversity of natural microbial populations. By targeting gene sequences that are specific for particular microorganisms, for example genes that encode diagnostic enzymes, or species-specific domains of conserved genes such as 16S ribosomal RNA coding sequences (rrn genes), the problems of culture can be avoided. Technical developments, notably in the area of in vitro amplification of DNA using the polymerase chain reaction (PCR), now permit routine detection and identification of specific microorganisms, even when present in very low numbers. Although the techniques of molecular biology have provided some very powerful tools for environmental microbiology, it should not be forgotten that these have their own drawbacks and biases in sampling. For example, molecular techniques are dependent on efficient lysis and recovery of nucleic acids from both vegetative forms and spores of microbial species that may differ radically when growing in the laboratory compared with the natural environment. Furthermore, PCR amplification can introduce its own bias depending on the nature of the oligonucleotide primers utilised. However, despite these potential caveats, it seems likely that a molecular biological approach, particularly with its potential for automation, will provide the mainstay of diagnostic technology for the foreseeable future.

Freshwater biology and water supply in Britain

This paper is designed to give a general account of freshwater biology as it bears on waterworks practice. Most water that is used for consumption will commonly go through a storage reservoir. Here special reference is given to the biological relations in standing waters, the biological control of water supplies, methods of plankton estimation, the biology of slow sand filtration and the use of algicides.

Marine biology of the Sierra Leone River Estuary. 1. The physical environment

The Sierra Leone River Estuary is a relatively young drowned river valley, it is shallow except for a deep channel which passes close to the Freetown shoreline. The upper reaches merge into a network of creeks and channels fringed by large areas of mangrove swamps. It is a tidal estuary of the semi-mixed type with the saline oceanic water entering it on a diurnal cycle. The climate of Sierra Leone is marked by a very distinct change between a very wet rainy season and a dry season. The tidal range of the Estuary (spring 3.03m; neap 2.28m) does not impede normal use of the harbour. The tidal variations can be felt as far as 42 miles inland along the water courses of the Sierra Leone River and its tributaries. The volume of fresh water entering the Estuary is large during the rainy season and greatly reduced during the dry season. Consequently there is a marked fall in salinity during the rainy season and higher salinities due to the marine influence prevailing during the dry season. The nature of the shores and bottom, the hydrography and chemistry of the estuarine system have been outlined in relation to the prevailing climatic conditions.

A review of IATTC research on the early life history and reproductive biology of scombrids conducted at the Achotines Laboratory from 1985 to 2005

English: For nearly a century, fisheries scientists have studied marine fish stocks in an effort to understand how the abundances of fish populations are determined. During the early lives of marine fishes, survival is variable, and the numbers of individuals surviving to transitional stages or recruitment are difficult to predict. The egg, larval, and juvenile stages of marine fishes are characterized by high rates of mortality and growth. Most marine fishes, particularly pelagic species, are highly fecund, produce small eggs and larvae, and feed and grow in complex aquatic ecosystems. The identification of environmental or biological factors that are most important in controlling survival during the early life stages of marine fishes is a potentially powerful tool in stock assessment. Because vital rates (mortality and growth) during the early life stages of marine fishes are high and variable, small changes in those rates can have profound effects on the properties of survivors and recruitment potential (Houde 1989). Understanding and predicting the factors that most strongly influence pre-recruit survival are key goals of fisheries research programs. Spanish: Desde hace casi un siglo, los científicos pesqueros han estudiado las poblaciones de peces marinos en un intento por entender cómo se determina la abundancia de las mismas. Durante la vida temprana de los peces marinos, la supervivencia es variable, y el número de individuos que sobrevive hasta las etapas transicionales o el reclutamiento es difícil de predecir. Las etapas de huevo, larval, y juvenil de los peces marinos son caracterizadas por tasas altas de mortalidad y crecimiento. La mayoría de los peces marinos, particularmente las especies pelágicas, son muy fecundos, producen huevos y larvas pequeños, y se alimentan y crecen en ecosistemas acuáticos complejos. La identificación los factores ambientales o biológicos más importantes en el control de la supervivencia durante las etapas tempranas de vida de los peces marinos es una herramienta potencialmente potente en la evaluación de las poblaciones. Ya que las tasas vitales (mortalidad y crecimiento) durante las etapas tempranas de vida de los peces marinos son altas y variables, cambios pequeños en esas tasas pueden ejercer efectos importantes sobre las propiedades de los supervivientes y el potencial de reclutamiento (Houde 1989). Comprender y predecir los factores que más afectan la supervivencia antes del reclutamiento son objetivos clave de los programas de investigación pesquera.

Biology and Management of the American Shad and Status of the Fisheries, Atlantic Coast of the United States, 1960

This paper summarizes current information on the American shad, Alosa sapidissima, and describes the species and its fishery. Emphasis is placed on (1) life history of the fish, (2) condition of the fishery by State and water areas in 1960 compared to 1896 when the last comprehensive description was made, (3) factors responsible for decline in abundance, and (4) management measures. The shad fishery has changed little over the past three-quarters of a century, except in magnitude of yield. Types of shad-fishing gear have remained relatively unchanged, but many improvements have been made in fishing techniques, mostly to achieve economy. In 1896 the estimated catch was more than 50 million pounds. New Jersey ranked first in production with about 14 million pounds, and Virginia second with 11 million pounds. In 1960 the estimated catch was slightly more than 8 million pounds. Maryland ranked first in production with slightly more than 1.5 million pounds, Virginia second with slightly less than 1.4 million pounds, and North Carolina third with about 1.3 million pounds. Biological and economic factors blamed for the decline in shad abundance, such as physical changes in the environment, construction of dams, pollution, over-fishing, and natural cycles of abundance, are discussed. Also discussed are methods used for the rehabilitation and management of the fishery, such as artificial propagation, installation of fish-passage facilities at impoundments, and fishing regulations. With our present knowledge, we can manage individual shad populations; but, we probably cannot restore the shad to its former peak of abundance.

Aspects of the biology and population dynamics of freshwater mussels in Lake Kariba and Lake McIlwaine

The essential aim of this study was to provide a broad foundation of biological knowledge upon which a programme of mussel utilization and management could be built. Results of the study are presented in three main sections. Part 1 describes the stock of Lake Kariba and Lake McIlwaine; part 2 describes various aspects of the breeding biology of the three species; and part 3 presents the results of morphological, biochemical and age analyses - aspects which are used for initial standing crop and production calculations. The final discussion concludes the thesis with a general examination in ecological terms of the factors which have influenced the development and nature of the mussel faunas of the two lakes under consideration.

Reproductive biology of blue marlin (Makaira nigricans) in the western Pacific Ocean

The reproductive biology of blue marlin (Makaira nigricans) was assessed from 1001 fish (ranging from 121 to 275 cm in eye-to-fork length; EFL) caught by Taiwanese offshore longliners in the western Pacific Ocean from September 2000 to December 2001 and from 843 gonad samples from these fish, The overall sex ratio of the catch was approximately 1:1 dur ing the sampling period, but blue marlin are sexually dimorphic; females are larger than males. Reproductive activity (assessed by histology), a gonadosomatic index, and the distribution of oocyte diameters, indicated that spawning occurred predominantly from May to September. The estimated sizes-at-maturity (EFL50) were 179.76 ±1.01 cm (mean ±standard error) for females and 130 ±1 cm EFL for males. Blue marlin are multiple spawners and oocytes develop asynchronously. The proportion of mature females with ovaries containing postovulatory follicles (0.41) and hydrated oocytes (0.34) indicated that the blue marlin spawned once every 2–3 days on average. Batch fecundity (BF) for 26 females with the most advanced oocytes (≥1000 μm), but without postovulatory follicles, ranged from 2.11 to 13.50 million eggs (6.94 ± 0.54 million eggs). The relationships between batch fecundity (BF, in millions of eggs) and EFL and round weight (RW, kg) were BF = 3.29 × 10 –12 EFL5.31 (r2 = 0.70) and BF = 1.59 × 10–3 RW 1.73 (r2= 0.67), respectively. The parameters estimated in this study are key information for stock assessments of blue marlin in the western Pacific Ocean and will contribute to the conservation and sustainable yield of

Reproductive biology, spawning season, and growth of female rex sole (Glyptocephalus zachirus) in the Gulf of Alaska

Rex sole (Glyptocephalus zachirus) have a wide distribution throughout the North Pacific, ranging from central Baja California to the western Bering Sea. Although rex sole are an important species in the commercial trawl fisheries off the U.S. West Coast, knowledge of their reproductive biology is limited to one study off the Oregon coast where ovaries were analyzed with gross anatomical methods. This study was initiated to determine reproductive and growth parameters specific to rex sole in the Gulf of Alaska (GOA) stock. Female rex sole (n=594) ranging in total length from 166 to 552 mm were collected opportunistically around Kodiak Island, Alaska, from February 2000 to October 2001. All ovaries were analyzed by using standard histological criteria to determine the maturity stage. Year-round sampling of rex sole ovaries confirmed that rex sole are batch spawners and have a protracted spawning season in the GOA that lasts at least eight months, from October to May; the duration of the spawning season and the months of spawning activity are different from those previously estimated. Female rex sole in the GOA had an estimated length at 50% maturity (ML50) of 352 mm, which is greater than the previously estimated ML50 at southern latitudes. The maximum age of collected female rex sole was 29 years, and the estimated age at 50% maturity (MA50) in the GOA was 5.1 years. The von Bertalanffy growth model for rex sole in the GOA was significantly different from the previously estimated model for rex sole off the Oregon coast. This study indicated that there are higher growth rates for rex sole in the GOA than off the Oregon coast and that there are differences in length at maturity and similarity in age at maturity between the two regions.