Ernest Williams

Responses of Nannochloropsis oceanica IMET1 to Long-Term Nitrogen Starvation and Recovery

Nannocloropsis oceanica IMET1 has the ability to recover from long periods of nitrate deprivation without apparent detriment to the culture. The Nannochloropsis genus contains oleaginous microalgae that have served as model systems for developing renewable biodiesel. Recent genomic and transcriptomic studies on Nannochloropsis species have provided insights into the regulation of lipid production in response to nitrogen stress. Previous studies have focused on the responses of Nannochloropsis species to short-term nitrogen stress, but the effect of long-term nitrogen deprivation remains largely unknown. In this study, physiological and proteomic approaches were combined to understand the mechanisms by which Nannochloropsis oceanica IMET1 is able to endure long-term nitrate deprivation and its ability to recover homeostasis when nitrogen is amended. Changes of the proteome during chronic nitrogen starvation espoused the physiological changes observed, and there was a general trend toward recycling nitrogen and storage of lipids. This was evidenced by a global down-regulation of protein expression, a retained expression of proteins involved in glycolysis and the synthesis of fatty acids, as well as an up-regulation of enzymes used in nitrogen scavenging and protein turnover. Also, lipid accumulation and autophagy of plastids may play a key role in maintaining cell vitality. Following the addition of nitrogen, there were proteomic changes and metabolic changes observed within 24 h, which resulted in a return of the culture to steady state within 4 d. These results demonstrate the ability of N. oceanica IMET1 to recover from long periods of nitrate deprivation without apparent detriment to the culture and provide proteomic markers for genetic modification.

Microarray analysis of the hyperthermophilic archaeon Pyrococcus furiosus exposed to gamma irradiation

The remarkable survival of the hyperthermophilic archaeon Pyrococcus furiosus to ionizing radiation was previously demonstrated. Using a time course study and whole-genome microarray analyses of mRNA transcript levels, the genes and regulatory pathways involved in the repair of lesions produced by ionizing irradiation (oxidative damage and DNA strand breaks) in P. furiosus were investigated. Data analyses showed that radA, encoding the archaeal homolog of the RecA/Rad51 recombinase, was moderately up regulated by irradiation and that a putative DNA-repair gene cluster was specifically induced by exposure to ionizing radiation. This novel repair system appears to be unique to thermophilic archaea and bacteria and is suspected to be involved in translesion synthesis. Genes that encode for a putative Dps-like iron-chelating protein and two membrane-bound oxidoreductases were differentially expressed following gamma irradiation, potentially in response to oxidative stress. Surprisingly, the many systems involved in oxygen detoxification and redox homeostasis appeared to be constitutively expressed. Finally, we identified several transcriptional regulators and protein kinases highly regulated in response to gamma irradiation.

Lactobacillus rhamnosus accelerates zebrafish backbone calcification and gonadal differentiation through effects on the GnRH and IGF systems.

Endogenous microbiota play essential roles in the host’s immune system, physiology, reproduction and nutrient metabolism. We hypothesized that a continuous administration of an exogenous probiotic might also influence the host’s development. Thus, we treated zebrafish from birth to sexual maturation (2-months treatment) with Lactobacillus rhamnosus, a probiotic species intended for human use. We monitored for the presence of L. rhamnosus during the entire treatment. Zebrafish at 6 days post fertilization (dpf) exhibited elevated gene expression levels for Insulin-like growth factors -I and -II, Peroxisome proliferator activated receptors -α and -β, VDR-α and RAR-γ when compared to untreated-10 days old zebrafish. Using a gonadotropin-releasing hormone 3 GFP transgenic zebrafish (GnRH3-GFP), higher GnRH3 expression was found at 6, 8 and 10 dpf upon L. rhamnosus treatment. The same larvae exhibited earlier backbone calcification and gonad maturation. Noteworthy in the gonad development was the presence of first testes differentiation at 3 weeks post fertilization in the treated zebrafish population -which normally occurs at 8 weeks- and a dramatic sex ratio modulation (93% females, 7% males in control vs. 55% females, 45% males in the treated group). We infer that administration of L. rhamnosus stimulated the IGF system, leading to a faster backbone calcification. Moreover we hypothesize a role for administration of L. rhamnosus on GnRH3 modulation during early larval development, which in turn affects gonadal development and sex differentiation. These findings suggest a significant role of the microbiota composition on the host organism development profile and open new perspectives in the study of probiotics usage and application.

High Genetic Diversity and Implications for Determining Population Structure in the Blue Crab Callinectes sapidus

ABSTRACT Understanding the population structure of a commercially fished species and how those populations change over time is essential for proper management. Although the blue crab Callinectes sapidus (Rathbun, 1896) covers a large geographic range spanning two continents and several distinct ecosystems, there are no clear morphological characters and a paucity of genetic data that can be used to distinguish populations within and among management areas. In this study, diversity indices were calculated for four loci in the mitochondrial genome of C. sapidus using specimens collected from the Rhode River in Maryland during the summers of 2003 to 2005. The locus with the highest diversity, which occurs within the open reading frame of the nad2 mitochondrial gene, was then used as a marker to determine population structure for samples collected from the Atlantic locations of the Chesapeake Bay; Raleigh, North Carolina; Meadowlands and Tuckerton, New Jersey; and Massachusetts, as well as locations in the Gulf of Mexico spanning the waters of Florida, Alabama, Mississippi, and Texas from 2003 to 2008. For the entire dataset, the genetic variation within each sample site accounted for 99.5%of the total diversity. Also, the Atlantic sampling sites could not be distinguished from the Gulf of Mexico sites using Bayesian inference. Interestingly, the sampling sites from Massachusetts did not show a reduction in their level of genetic diversity despite its location at the northern limit of C. sapidus habitat. There were significant differences looking temporally, however, with the 2003 samples, the year of hurricane Isabel, differing from the 2004 and 2005 samples. Using the nad2 marker along with four microsatellite loci on a sample of 28 megalopae entering the mouth of Chesapeake Bay also demonstrated extremely high diversity at all loci, and the megalopae were significantly different from an adult population of the same year at two of the microsatellite loci. This indicates that genetic diversity is high in adult as well as larval populations and that offshore mixing may be playing a large role in the observed diversity shared among geographically distant habitats. Seasonal weather patterns during larval development followed by selection during the multiple subsequent recruitment events may play a large role in shaping local populations, and larval transport and settling models will provide much more understanding of the population dynamics of this species.

Extensive Heteroplasmy and Evidence for Fragmentation in the Callinectes sapidus Mitochondrial Genome

ABSTRACT Heteroplasmy is the presence of multiple genotypes in an individual and is commonly associated with mitochondria where thousands of copies of the genome can be present in a single organelle. Even in low frequencies, it can cause severe illness or death and strong selective pressures usually result in a low occurrence. Despite this, cloning and sequencing of a portion of the open reading frames of the nad2, nad4, and coI mitochondrial loci in the blue crab Callinectes sapidus (Rathbun, 1896) revealed abnormally high levels of heteroplasmy at all loci with as many as 24 haplotypes in a single individual and the dominant haplotype accounting for as little as 43.9% of the total sequences. A minimum spanning network comparing these individuals at each locus demonstrated an overlap of haplotypes between the female and themegalopa with almost no overlap of themale sequences. Illumina sequencing ofmuscle and testes froma single individual usingmultiple differentDNA-based amplificationmethods as well as cDNA amplification confirmed heteroplasmic circular mitochondrial DNA templates with portions of the reads containing unmapped highly repetitive regions at specific sequence positions in both tissue types. Thus, the blue crab appears to possess a highly degenerate and possibly fragmented mitochondrial genome that is present in germline tissue and linearly inherited. A general lack of fragmentation present in the cDNA sequences as well as site-specific sequence changes argues for mitochondrial editing as a way of maintaining sequence integrity. This is the first evidence for mitochondrial editing in a crustacean and a potential explanation for observations of extremely high population diversity when using blue crab mitochondrial markers.

Successful Identification and Discrimination of Hatchery-Reared Blue Crabs (Callinectes sapidus) Released into the Chesapeake Bay Using a Genetic Tag

ABSTRACT Before animals reared in captivity can be used as a model for study or as a supplement for wild populations, it is important to demonstrate that they can integrate with high success and without adversely affecting the natural population. For the blue crab Callinectes sapidus (Rathbun, 1896), coded wire tags have been used to positively discriminate hatchery-raised individuals from wild. This technique can be prohibitively expensive, however, due to high start-up costs and manpower needs during implantation, and mortality as a result of the tagging process is also a consideration. To reduce costs and increase the number of possible individuals monitored per unit effort, the suitability of the mitochondrial gene nad2 as a genetic tag to identify hatchery-produced crabs was tested. Batches of juvenile crabs approximately 20 mm in carapace width were implanted with wire tags and released into a variety of locations in the upper Chesapeake Bay. Juveniles were later recaptured from release sites and scanned for wire tags in the field. Tissue samples from recaptured crabs were also sequenced with nad2 specific primers. Comparison of sequences from recaptured individuals to batch mothers showed 95% congruence in positive identification through wire tagging and nad2 sequencing and an ability to discriminate hatchery batches from wild crabs with 97.8% success. Thus, use of an nad2 marker as a genetic tag is as successful as wire tags in C. sapidus with lower costs and mortality.

Growth and Enzyme Production in Blue Crabs (Callinectes sapidus) Fed Cellulose and Chitin Supplemented Diets

ABSTRACT The blue crab [Callinectes sapidus (Rathbun, 1896)] is a benthic decapod with a varied diet. The diet includes invertebrates and detrital material that can have relatively large amounts of chitin and cellulose, both of which can be difficult to digest for many organisms and often require the aid of specific bacteria in the gut microbiome. In this study, juvenile blue crabs were fed an optimized defined pelleted diet with a 20% replacement of wheat flour filler with either chitin, cellulose, or a 14%/6% mix of both, followed by a diet switch to the opposing ingredient. Crabs had increasing growth performance with increasing amounts of cellulose in the diet versus chitin and had an additional molt in most cases. This occurred during the initial phase and following the switch, indicating that performance can be recovered. Subsequently, cellulose and chitin digestion assays were used to show that the foregut, midgut, and hindgut were all able to significantly digest more cellulose than chitin with the majority of activity in the foregut and midgut. Implications for rearing and diet formulations as well as the role of cellulose and chitin digestion in the natural diet are discussed.

Transcriptome Analysis of Core Dinoflagellates Reveals a Universal Bias towards “GC” Rich Codons

Although dinoflagellates are a potential source of pharmaceuticals and natural products, the mechanisms for regulating and producing these compounds are largely unknown because of extensive post-transcriptional control of gene expression. One well-documented mechanism for controlling gene expression during translation is codon bias, whereby specific codons slow or even terminate protein synthesis. Approximately 10,000 annotatable genes from fifteen “core” dinoflagellate transcriptomes along a range of overall guanine and cytosine (GC) content were used for codonW analysis to determine the relative synonymous codon usage (RSCU) and the GC content at each codon position. GC bias in the analyzed dataset and at the third codon position varied from 51% and 54% to 66% and 88%, respectively. Codons poor in GC were observed to be universally absent, but bias was most pronounced for codons ending in uracil followed by adenine (UA). GC bias at the third codon position was able to explain low abundance codons as well as the low effective number of codons. Thus, we propose that a bias towards codons rich in GC bases is a universal feature of core dinoflagellates, possibly relating to their unique chromosome structure, and not likely a major mechanism for controlling gene expression.

Multiple Paternity in the Blue Crab (Callinectes sapidus) Assessed with Microsatellite Markers

ABSTRACT In the blue crab Callinectes sapidus (Rathbun, 1896), double ejaculations have been observed; however, it is not known if these multiple inseminations are the products of different males or if they lead to effective multiple paternities. In this study, 75 mature females and their spermathecae were genotyped for five polymorphic microsatellite loci. The incidence of multiple paternity was analyzed by comparing the genotypes of either spermathecae or individual ejaculates with the corresponding mother, and the male contribution was then deduced. Females contained double ejaculates (5.3%),which genotyping confirmed as the products of different males. In addition, a routine screening of 68 crabs from a single hatchery brood at two loci indicated more than two parents. Mitochondrial analysis confirmed that the brood shares the same mother; therefore, the extra alleles must be due to different fathers. Our findings indicate that female blue crabs are not only capable of mating with more than one male but that multiple inseminations can lead to multiple paternity within a brood.

Assimilation efficiency of sediment‐bound PCBs ingested by fish impacted by strong sorption

Uptake of polychlorinated biphenyls (PCBs) by fish is controlled by the bioavailability of ingested PCBs in the gut and the freely dissolved concentration in the water moving across the gills. The prediction of bioaccumulation in fish relies on models that account for these exposure routes; however, these models typically do not account for incidental ingestion of sediment by fish, which is not well studied. The literature values for the PCB assimilation efficiency in the gut have been reported for compounds in food matrices and not associated with sediment particles. It is also unclear how mitigation strategies that alter PCB bioavailability in sediments affect predictions made by the bioaccumulation models when sediment ingestion is involved. To test the bioavailability of PCBs from treated and untreated sediments, dietary assimilation efficiencies were measured for 16 PCB congeners in mummichogs (Fundulus heteroclitus) that were fed 4 experimental diets. Diets consisted of PCB-spiked earthworms, spiked untreated sediment mixed with earthworms, spiked activated carbon-treated sediment mixed with earthworms, and spiked activated carbon mixed with earthworms. Assimilation efficiencies were determined by calculating the ratio of PCB mass in the fish tissue to the PCB mass in the food after a pulse feeding experiment. Assimilation efficiencies of PCBs associated with earthworm diet were similar to the values reported in the literature. Fish that were fed the PCB-spiked untreated sediment and activated carbon particles exhibited the highest and lowest assimilation efficiencies, respectively, over a wide KOW range. Assimilation efficiencies of sediment-bound PCBs were significantly reduced (31-93% reduction for different congeners) after amendment with activated carbon. The present study indicates that assimilation of PCBs can be reduced by sorption to black carbon. Environ Toxicol Chem 2017;36:3480-3488.