WHOLE GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. (C. cuniculus, C. hominis, C. meleagridis, C. parvum, C. tyzzeri, and C. viatorum) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.

WHOLE GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. ( C. cuniculus , C. hominis , C. meleagridis , C. parvum , C. tyzzeri , and C. viatorum ) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.

Nephrotoxicity of epigenetic inhibitors used for the treatment of cancer.

This study determined the anti-neoplastic activity and nephrotoxicity of epigenetic inhibitors in vitro. The therapeutic efficacy of epigenetic inhibitors was determined in human prostate cancer cells (PC-3 and LNCaP) using the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza) and the histone deacetylase inhibitor trichostatin A (TSA). Cells were also treated with carbamazepine (CBZ), an anti-convulsant with histone deacetylase inhibitor-like properties. 5-Aza, TSA or CBZ alone did not decrease MTT staining in PC-3 or LNCaP cells after 48 h. In contrast, docetaxel, a frontline chemotherapeutic induced concentration-dependent decreases in MTT staining. Pretreatment with 5-Aza or TSA increased docetaxel-induced cytotoxicity in LNCaP cells, but not PC-3 cells. TSA pretreatment also increased cisplatin-induced toxicity in LNCaP cells. Carfilzomib (CFZ), a protease inhibitor approved for the treatment of multiple myeloma had minimal effect on LNCaP cell viability, but reduced MTT staining 50% in PC-3 cells compared to control, and pretreatment with 5-Aza further enhanced toxicity. Treatment of normal rat kidney (NRK) and human embryonic kidney 293 (HEK293) cells with the same concentrations of epigenetic inhibitors used in prostate cancer cells significantly decreased MTT staining in all cell lines after 48 h. Interestingly, we found that the toxicity of epigenetic inhibitors to kidney cells was dependent on both the compound and the stage of cell growth. The effect of 5-Aza and TSA on DNA methyltransferase and histone deacetylase activity, respectively, was confirmed by assessing the methylation and acetylation of the CDK inhibitor p21. Collectively, these data show that combinatorial treatment with epigenetic inhibitors alters the efficacy of chemotherapeutics in cancer cells in a compound- and cell-specific manner; however, this treatment also has the potential to induce nephrotoxic cell injury.

Assessing the microbiomes of scalder and chiller tank waters throughout a typical commercial poultry processing day.

The commercial poultry processing environment plays a significant role in reducing foodborne pathogens and spoilage organisms from poultry products prior to being supplied to consumers. While understanding the microbiological quality of these products is essential, little is known about the microbiota of processing water tanks within the processing plant. Therefore, the goal of this study was to assess the microbiomes of the scalder and chiller tanks during a typical commercial processing d, and determine how bacterial populations, including foodborne pathogens and spoilage organisms, change during the processing day in relation to the bacterial communities as a whole. Additionally, considering this is the first microbiomic analysis of processing tank waters, 2 water sampling methods also were compared. Results of this study show that Proteobacteria and Firmicutes represented over half of the sequences recovered from both tanks at the phylum level, but the microbiomic profiles needed to be analyzed at the genus level to observe more dynamic population shifts. Bacteria known to predominate in the live production environment were found to increase in the scalder tank and gram negative spoilage-related bacteria were found to decrease in the chiller tank throughout the processing day. Directly sampling the scalder water, as compared to analyzing filtered samples, resulted in significantly different microbiomic profiles dominated by Anoxybacillus species. While no sequences related to major foodborne pathogens were found, further sampling collection and processing optimization should provide researchers and the poultry industry a new tool to understand the ecological role of spoilage and pathogenic bacteria within processing tank waters.

Genetic status of the wood stork (Mycteria americana) from the southeastern United States and the Brazilian Pantanal as revealed by mitochondrial DNA analysis.

The wood stork (Mycteria americana) is a colonial wading bird that inhabits the Neotropical region from the southeastern United States (US) to northern Argentina. The species is considered to be endangered in the US due to degradation of its foraging and breeding habitat. In other parts of its range, such as in the Brazilian Pantanal region, breeding populations of this species appear to be stable. We compared the levels of genetic variability and population structuring of the US and the Pantanal breeding populations using mitochondrial DNA (mtDNA) control region sequences. Twenty-seven haplotypes were identified among 88 wood stork samples collected from eight breeding colonies in the US and eight in the Pantanal. Patterns indicative of heteroplasmy were observed in 35.3% of the mtDNA sequences that were examined. Significantly higher levels of haplotype diversity were observed in the Pantanal samples compared to those from the US, suggesting that during the last century, demographic declines or a recent evolutionary bottleneck reduced the levels of mtDNA variability of the US population. Analyses of genetic structuring revealed non-significant genetic differentiation between these regions, indicating that either the populations were only recently separated or that gene flow continues to occur at low levels. Haplotype network analysis indicated low current levels of gene flow between populations that were closely related in the past.

Comparative phototoxicity of nanoparticulate and bulk ZnO to a free-living nematode Caenorhabditis elegans: the importance of illumination mode and primary particle size.

The present study evaluated phototoxicity of nanoparticulate ZnO and bulk-ZnO under natural sunlight (NSL) versus ambient artificial laboratory light (AALL) illumination to a free-living nematode Caenorhabditis elegans. Phototoxicity of nano-ZnO and bulk-ZnO was largely dependent on illumination method as 2-h exposure under NSL caused significantly greater mortality in C. elegans than under AALL. This phototoxicity was closely related to photocatalytic reactive oxygen species (ROS) generation by the ZnO particles as indicated by concomitant methylene blue photodegradation. Both materials caused mortality in C. elegans under AALL during 24-h exposure although neither degraded methylene blue, suggesting mechanisms of toxicity other than photocatalytic ROS generation were involved. Particle dissolution of ZnO did not appear to play an important role in the toxicity observed in this study. Nano-ZnO showed greater phototoxicity than bulk-ZnO despite their similar size of aggregates, suggesting primary particle size is more important than aggregate size in determining phototoxicity.

QTL mapping for two commercial traits in farmed saltwater crocodiles (Crocodylus porosus).

The recent generation of a genetic linkage map for the saltwater crocodile (Crocodylus porosus) has now made it possible to carry out the systematic searches necessary for the identification of quantitative trait loci (QTL) affecting traits of economic, as well as evolutionary, importance in crocodilians. In this study, we conducted genome-wide scans for two commercially important traits, inventory head length (which is highly correlated with growth rate) and number of scale rows (SR, a skin quality trait), for the existence of QTL in a commercial population of saltwater crocodiles at Darwin Crocodile Farm, Northern Territory, Australia. To account for the uncommonly large difference in sex-specific recombination rates apparent in the saltwater crocodile, a duel mapping strategy was employed. This strategy employed a sib-pair analysis to take advantage of our full-sib pedigree structure, together with a half-sib analysis to account for, and take advantage of, the large difference in sex-specific recombination frequencies. Using these approaches, two putative QTL regions were identified for SR on linkage group 1 (LG1) at 36 cM, and on LG12 at 0 cM. The QTL identified in this investigation represent the first for a crocodilian and indeed for any non-avian member of the Class Reptilia. Mapping of QTL is an important first step towards the identification of genes and causal mutations for commercially important traits and the development of selection tools for implementation in crocodile breeding programmes for the industry.

Multiyear multiple paternity and mate fidelity in the American alligator, Alligator mississippiensis.

We examined multiple paternity during eight breeding events within a 10-year period (1995-2005) for a total of 114 wild American alligator nests in Rockefeller Wildlife Refuge in south-west Louisiana. Our goals included examining (i) within population variation in multiple paternity among years, (ii) variation in multiple paternity in individual females and (iii) the potential for mate fidelity. To accomplish this, in the current study, eggs were sampled from 92 nests over 6 years and analysed along with 22 nests from a previous 2-year study. Genotypes at five microsatellite loci were generated for 1802 alligator hatchlings. Multiple paternity was found in 51% of clutches and paternal contributions to these clutches were highly skewed. Rates of multiple paternity varied widely among years and were consistently higher in the current study than previously reported for the same population. Larger females have larger clutches, but are not more likely to have multiply sired nests. However, small females are unlikely to have clutches with more than two sires. For 10 females, nests from multiple years were examined. Seven (70%) of these females exhibited long-term mate fidelity, with one female mating with the same male in 1997, 2002 and 2005. Five females exhibiting partial mate fidelity (71%) had at least one multiple paternity nest and thus mated with the same male, but not exclusively. These patterns of mate fidelity suggest a potential role for mate choice in alligators.

Standardized reference ideogram for physical mapping in the saltwater crocodile (Crocodylus porosus).

Basic cytogenetic data, such as diploid number and general chromosome morphology, are available for many reptilian species. Here we present a detailed cytogenetic examination of the saltwater crocodile (Crocodylus porosus) karyotype, including the creation of the first fully annotated G-band standard ideogram for any crocodilian species. The C. porosus karyotype contains macrochromosomes and has a diploid number of 34. This study presents a detailed description of each chromosome, permitting unambiguous chromosome identification. The fully annotated standardized C. porosus ideogram provides the backbone to a standard nomenclature system which can be used to accurately identify specific band locations. Seven microsatellite containing fosmid clones were fluorescently labeled and used as fluorescent in situ hybridization (FISH) probes for physical localization. Chromosome locations for each of these FISH probes were successfully assigned, demonstrating the utility of the fully annotated ideogram for genome mapping.

Radiation-induced untargeted germline mutations in Japanese medaka.

Radiation has been shown to increase mutation frequencies at tandem repeat loci by indirect interactions of radiation with DNA. We studied germline mutations in chronically exposed Japanese medaka (Oryzias latipes) using microsatellite loci. After screening 26 randomly selected loci among unirradiated parents and their 200 offspring, we selected seven highly mutable loci (0.5-1.0 x 10(-2) mutants per locus per gamete) and two bonus loci for further study. To determine if radiation exposure increases mutation frequencies in these loci, medaka were chronically irradiated from subadults through maturation at relatively low dose rates of 68 mGy/d. Total doses for males and females were 10.4 and 3 Gy, respectively. The mean number of mutations for the offspring of exposed families (0.149+/-0.044) was significantly higher (P=0.018) than for control families (0.080+/-0.028), indicating induction of germline mutations from chronic irradiation. This increase in the microsatellite mutation rate is greater than expected from direct interaction of radiation with DNA, suggesting indirect, untargeted mechanism(s) for mutations. This study identified microsatellite loci with a high mutational background in medaka, variation among loci and families as important variables, and demonstrated the usefulness of this fish model for studying radiation-induced germline mutations.

Comparative overview of brain perfusion imaging techniques.

Numerous imaging techniques have been developed and applied to evaluate brain hemodynamics. Among these are: Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), Xenon-enhanced Computed Tomography (XeCT), Dynamic Perfusion-computed Tomography (PCT), Magnetic Resonance Imaging Dynamic Susceptibility Contrast (DSC), Arterial Spin-Labeling (ASL), and Doppler Ultrasound. These techniques give similar information about brain hemodynamics in the form of parameters such as cerebral blood flow (CBF) or volume (CBV). All of them are used to characterize the same types of pathological conditions. However, each technique has its own advantages and drawbacks. This article addresses the main imaging techniques dedicated to brain hemodynamics. It represents a comparative overview, established by consensus among specialists of the various techniques. For clinicians, this paper should offers a clearer picture of the pros and cons of currently available brain perfusion imaging techniques, and assist them in choosing the proper method in every specific clinical setting.

Assessment of cerebral blood flow by means of blood-flow-volume measurement in the internal carotid artery: comparative study with a 133xenon clearance technique.

BACKGROUND AND PURPOSE: We sought to evaluate a new, angle-independent ultrasonic device for assessment of blood flow volume (BFV) in the internal carotid artery (ICA). METHODS: Nineteen patients and 4 healthy volunteers were enrolled in a comparative study conducted in the Care Unit of the Division of Neurosurgery at UCLA Medical Center. All patients had been admitted because of severe brain injury: 15 patients with severe head trauma (Glasgow Coma Scale score< or =8) and 4 patients with subarachnoid hemorrhage due to aneurysm rupture. In all patients and subjects, cerebral blood flow (CBF) values obtained with the 133xenon-clearance technique were compared with BFV measurements in the ipsilateral ICA. RESULTS: Hemispheric CBF values showed a close and linear correlation with BFV measurements (r=0.76, P<0.0001). Global CBF values showed a higher correlation with the total BFV value obtained from both ICAs (r=0.84, P<0.0001). With 37 mL x min(-1) x 100 g(-1) as a cutoff value for the ischemic range, a BFV value of 220 mL/min would yield a positive predictive value of 91.7% and a negative predictive value of 82.6% (sensitivity 73.3%, specificity 95%). Conversely, BFV sensitivity and specificity were 60% and 96%, respectively, for the hyperemic range defined by a CBF value >55 mL x min(-1) x 100 g(-1) (positive predictive value of 85.7% and negative prediction value of 85.7%). CONCLUSIONS: BFV measurements with this new technology proved to accurately correlate with CBF values evaluated by the 133xenon-clearance technique. These results support the implementation of this technique for bedside assessment of cerebral hemodynamics in critically ill neurosurgical patients.

PET investigation of post-traumatic cerebral blood volume and blood flow.

Hemodynamic changes following traumatic brain injury (TBI) may reflect cellular damage leading to secondary injury. The purpose of this study was to investigate the regional hemodynamic parameters acutely after TBI among regions in and around contusions. Sixteen patients (11 male, 5 female) showing evidence of contusion on CT and 18 normal volunteers (12 male, 6 female) underwent positron emission tomography (PET) with O-15 CO and O-15 H2O to estimate cerebral blood volume (CBV) and cerebral blood flow (CBF), respectively. A flow to volume ratio (FVR = CBF/CBV) was also calculated as an index of vasodilatation. The hemodynamic parameters were compared among contusion, pericontusion, and remote areas. Globally, hemodynamic parameters did not differ between patients and normal volunteers, and did not correlate with intracranial pressure (ICP). Regionally, contusional and pericontusional areas showed significantly lower CBF and FVR compared with normal volunteers, while CBV did not differ significantly. The correlation between CBF and CBV was significant (r = 0.37, p < 0.01). Remote areas did not show a significant difference in any of the PET parameters. In conclusion, regional brain edema is likely to occur in contusion and pericontusion areas, while some of the contusional tissue may show vascular engorgement.

Metabolic suppressive therapy as a treatment for intracranial hypertension--why it works and when it fails.

Thirty years after its first description metabolic suppressive therapy is still controversial in patients with intractable intracranial hypertension. In this study high dose propofol was used to induce metabolic suppression. The effects on intracranial pressure (ICP) and the cerebral metabolic rates for oxygen and glucose (CMRO2 and CMRGlc) are reported. A total of 28 studies were performed on 14 head injured patients. A Xenon133 cerebral blood flow (CBF) and a CO2-reactivity (CO2R) test were performed prior to induction of metabolic suppression. The following parameters were continuously monitored: EEG, etCO2, SjvO2, ICP, MAP and bilateral MCA flow velocity (VMCA). PCO2 was obtained before and during propofol-induced EEG burst-suppression in arterial and jugular-venous blood. CMRO2, CMRGlc and Metabolic Ratio (MR = CMRO2/CMRGlc) were calculated. MR < 0.6 was defined as relative hyperglycolysis. ICP decreased by 24.1 +/- 29.0% during burst-suppression. Arterial, jugular-venous and etCO2 also decreased. Multiple regression analysis revealed that CO2 was the strongest predictor for ICP. Lower baseline ICP and normal CO2 reactivity were predictors for normal metabolic suppression reactivity. In studies with normal metabolic ratio, ICP reduction was associated with a reduction in CMRO2. In studies with hyperglycolysis, ICP reduction was poor but CMRGlc decreased significantly. In conclusion, intact CO2R, normal or only moderately elevated ICP and normal MR are predictive of ICP reduction with high dose propofol after head injury.

Is CPP therapy beneficial for all patients with high ICP?

Induced blood pressure elevation has become a popular treatment for intracranial hypertension. However, there remains a concern that in some patients blood pressure elevation will further elevate ICP. This study was conducted to test the hypothesis that increasing MAP decreases ICP. A total of 47 studies were performed on 23 intubated patients with head injury. MAP and SjvO2 were continuously monitored. MAP was raised significantly by 13.8 (5.9) mmHg (t-test; p < 0.0001) using phenylephrine infusion. The percent change ICP per mmHg increase in MAP (% delta ICP/mm Hg MAP) was calculated. Pearson correlation coefficient, t-test and logistic regression analysis were used for statistical evaluation. Increasing MAP resulted in a decrease in ICP in 38.3% and in an increase in ICP in 61.7% out of 47 studies. The following characteristics were seen in patients in whom a decrease in ICP was associated with an increase in MAP: High GCS (r = -0.61; p = 0.004) and low SjvO2 ((2 = 4.89; p = 0.027). In patients with lower GCS and high SjvO2 an increase in MAP resulted in an increase in ICP. We concluded that in the majority of studies increasing MAP was followed by an increase in ICP. CPP therapy has a selective indication in patients with high GCS, low SjvO2 and increased ICP.

Can hyperventilation improve cerebral microcirculation in patients with high ICP?

Gosling's pulsatility index (PI) is generally thought to reflect cerebrovascular resistance. Hyperventilation and increased intracranial pressure (ICP) usually increase PI. In this study, the effect of hyperventilation on PI was assessed in head injured patients with and without elevated ICP. A total of 73 hyperventilation studies were conducted on 20 intubated TBI patients. EtCO2, ICP, MCA flow velocity and MCA PI bilaterally were recorded simultaneously. Hemispheric CO2-Reactivity (hCO2R) was calculated. Only studies with a baseline ICP < 15 or > 30 mm Hg were included for analysis. With hyperventilation pCO2 decreased on average from 33.54.2 to 26.84.8 mmHg. PI increased significantly in patients with baseline ICP < 15 mmHg (11.8%; p < 0.0001) but decreased significantly if baseline ICP was > 30 mm Hg (12.21%; p = 0.0013). High baseline ICP, low initial GCS and impaired hCO2R were associated with the decrease of PI. Hyperventilation unexpectedly reduced PI in patients with high ICP. Because decreased PI suggests decreased CVR, it is postulated that hyperventilation in the setting of raised ICP improves cerebral microcirculation.

Development and use of microsatellite DNA loci for genetic ecotoxicological studies of the fathead minnow (Pimephales promelas).

The development of new DNA technologies has increased the opportunities to study the effect of genetic variation as a factor in selection, resistance, and fitness of individuals and populations. Genetic composition affects the evolutionary potential of an organism; however, whether genetic variation has a role in adaptation and survival due to chemical stress is still under debate. The strategy of measuring such variation should be based on the desired number of loci needed and degree of polymorphism at each locus. Microsatellite DNA loci (also known as Simple Tandem Repeats, STRs) have among the highest levels of variation within most eukaryotic genomes. Methods to determine the high levels of variation in individuals and populations have become more refined, making DNA-based studies in ecotoxicology more feasible. Herein, we describe the general characteristics of microsatellite DNA loci, their isolation, and their applications to toxicological studies using the fathead minnow (Pimephales promelas) as a focal species.

Transcranial Doppler pulsatility indices as a measure of diffuse small-vessel disease.

BACKGROUND AND PURPOSE: Elevation in pulsatility indices (PIs) as measured by transcranial Doppler (TCD) have been postulated to reflect downstream increased vascular resistance caused by small-vessel ischemic disease. METHODS: The authors retrospectively compared TCD PIs and magnetic resonance imaging (MRI) manifestations of small-vessel disease in 55 consecutive patients who underwent TCD studies and brain MRI within 6 months of each other during a 2-year period. RESULTS: Correlations between TCD middle cerebral artery PIs and MRI measures were as follows: periventricular hyperintensity (PVH) = 0.52 (P < .0001), deep white matter hyperintensity (DWMH) = 0.54 (P < .0001), lacunar disease = 0.31 (P = .02), and combined PVH/DWMH/lacunes = 0.54 (P < .0001). Correlation between pontine ischemia and vertebrobasilar PIs was 0.46 (P = .0004). Univariate analysis showed that age, elevated PI, and hypertension strongly correlated with white matter disease measures. After adjusting for these factors in a multivariate Poisson regression analysis, PI remained an independent predictor of white matter disease. Receiver operator curve analyses identified PI cut points that allowed discrimination of PVH with 89% sensitivity and 86% specificity and discrimination of DWMH with 70% sensitivity and 73% specificity. CONCLUSIONS: Elevation in PIs as measured by TCD shows strong correlation with MRI evidence of small-vessel disease. TCD may be a useful physiologic index of the presence and severity of diffuse small-vessel disease.

Investigation of the radioadaptive response in brain and liver of pUR288 lacZ transgenic mice.

The radioadaptive response, where a small priming dose of ionizing radiation can lessen the effects of subsequent exposure to a higher radiation challenge dose, was investigated in brain and liver within transgenic mice. Although it is well characterized in models in vitro, current radioadaptive response research has focused on particular cell types (i.e., lymphocytes) and does not provide comparative data for responses of multiple tissues within an organism. Transgenic animals are useful for such comparisons, because the transgene is integrated into all cells in the body. The pUR288 lacZ plasmid-based transgenic mouse model utilizes a plasmid vector allowing highly efficient recovery of mutational targets, including large size-change mutations that result from radiation exposure. Female C57BI/6 pUR288 lacZ mice were exposed to priming doses of 0.075- to 0.375-Gy x-rays over a 3-d period. After 3 wk, they received an acute challenge dose of 2.5-Gy x-rays. Spontaneous mutant frequencies in lacZ were significantly higher in liver than in brain (6.62 x 10(-5) vs. 3.51 x 10(-5)). In the absence of a priming dose, the 2.5-Gy challenge doubled the mutant frequency of both liver and brain (13.38 x 10(-5), and 7.63 x 10(-5) respectively). Priming doses of 0.15, 0.225, and 0.375 Gy significantly reduced (by 40%) the mutagenic effects of the 2.5-Gy challenge in brain. Restriction enzyme analysis of rescued mutant plasmids revealed a decrease in large size-change mutations at the three priming doses in brain. This study demonstrates the utility of this model for the investigation of radiological processes of large size-change mutations, as well as showing a radioadaptive response in brain, but not liver, of mice in vivo.

Multiple paternity and mating patterns in the American alligator, Alligator mississippiensis.

Eggs were sampled from 22 wild American alligator nests from the Rockefeller Wildlife Refuge in south-west Louisiana, along with the females guarding the nests. Three nests were sampled in 1995 and 19 were sampled in 1997. Females and offspring from all clutches were genotyped using five polymorphic microsatellite loci and the three nests from 1995 were also genotyped using one allozyme locus. Genotypes of the hatchlings were consistent with the guarding females being the mothers of their respective clutches. Multiple paternity was found in seven of the 22 clutches with one being fathered by three males, and the remaining six clutches having genotypes consistent with two males per clutch. Paternal contributions of multiply sired clutches were skewed. Some males sired hatchlings of more than one of the 22 clutches either as one of two sires of a multiple paternity clutch, as the sole sire of two different clutches, or as the sole sire of one clutch and one of two sires of a multiply sired clutch. There was no significant difference between females that had multiple paternity clutches and those that had singly sired clutches with respect to female total length (P = 0.844) and clutch size (P = 0.861). Also, there was no significant correlation between genetic relatedness of nesting females and pairwise nest distances (r2 = 0.003, F1,208 = 0.623, P = 0.431), indicating that females in this sample that nested close to one another were no more related than any two nesting females chosen at random. Eleven mutations were detected among hatchlings at the five loci over the 22 clutches. Most of these mutations (eight of 11) occurred at Ami(mu)-17, the only compound microsatellite locus of the five used in this study, corresponding to a mutation rate of 1.7 x 10-3. Finally, most of the mutations (82%) were homoplasious, i.e., mutating to an allelic state already present in this Louisiana population.