Decline of an isolated timber rattlesnake (Crotalus horridus) population: Interactions between climate change, disease, and loss of genetic diversity

Extinction of populations from anthropogenic forces rarely has a single cause. Instead, population declines result from a variety of factors, including habitat loss, inbreeding depression, disease, and climate change. These impacts often have synergistic effects that can lead to rapid decline in isolated populations, but case studies documenting such processes are rare. Here, we describe the recent decline of the last known population of timber rattlesnakes (Crotalus horridus) in the state of New Hampshire. We used polymorphic nuclear DNA markers to compare genetic diversity of this population to other populations in the region that are not isolated. We also compare results from ongoing field monitoring of these populations. Genetic analyses reveal that the New Hampshire population lacks genetic diversity and exhibits signs of a recent bottleneck. New Hampshire snakes also exhibited high levels of morphological abnormalities (unique piebald coloration, amelanistic tongues) indicative of inbreeding depression. Furthermore, after a year with exceptionally high summer rainfall, a skin infection of unknown etiology caused significant mortality in the New Hampshire population, whereas other surveyed non-inbred populations were unaffected. This case study demonstrates how different anthropogenic impacts on natural environments can interact in unexpected ways to drive threatened populations toward extinction.     

Combination tibial plateau leveling osteotomy and transverse corrective osteotomy of the proximal tibia for the treatment of complex tibial deformities in 12 dogs

Objective: To describe a surgical technique, and outcome, for treatment of proximal tibial deformity (varus, valgus, excessive tibial plateau angle [eTPA], tibial torsion and patellar luxation) by combined tibial plateau leveling osteotomy (TPLO) and transverse corrective osteotomy. Study Design: Cases series. Animals: Dogs (n=12; 19 stifle joints). Methods: Medical records of dogs that had combination TPLO and transverse corrective osteotomy, were reviewed. Pre‐ and postoperative tibial angulation, tibial torsion, tibial plateau angle (TPA), corrective osteotomy technique, method of fixation, and complications were recorded. In hospital re‐evaluation of limb function and alignment and length of time to radiographic healing were reviewed. Long‐term outcome was assessed by visual analog scale (VAS) questionnaire and owner telephone interview. Results: Proximal tibial varus or valgus was present in 68.4%; 73.7% had eTPA; and 47.4% had both. Medial patellar luxation (MPL) was present in 57.9%, of which 47.4% had tibial tuberosity displacement. Severe tibial torsion was present in 68.4%. Mean pre‐ and postoperative TPA was 37.5° and 5.7°, respectively. The mean postoperative mechanical medial proximal tibial angle (mMPTA) and mechanical medial distal tibial angle (mMDTA) were 92.2° (range, 88–96°) and 96.1° (range, 94–101°), respectively. Postoperative surgical complications were documented in 21.0%, which included implant loosening or breakage (5.3%), seroma (5.3%), septic arthritis (5.3%), and infection of the proximal tibia (5.3%). All complications were considered major because they required additional surgery. Mean time to document radiographic healing was 10.4 weeks. In‐hospital re‐evaluation of lameness was obtained at the same time; 82.4% were not lame or had a mild lameness, 17.6% had severe lameness (2/3 with infection). The VAS evaluation revealed excellent results and owner satisfaction in all ten dogs in which long‐term follow‐up was obtained. Conclusions: Long‐term clinical outcome of combination TPLO and transverse corrective osteotomy was excellent, and had a high owner satisfaction. Healing times were comparable to standard TPLO with a similar complication rate.     

Multiple mid-Atlantic field experiments show no economic benefit to fungicide application when fungal disease is absent in winter wheat

Strobilurin fungicides produce intensified greening and delayed senescence in plants, and have been claimed to enhance yields of field crops in the absence of disease. To help evaluate this claim, available publicly sponsored tests of fungicides on soft red winter wheat in Virginia and North Carolina (n = 42) were analyzed for the period 1994 to 2010. All tests were replicated and had a randomized complete block, split-plot, or split-block design. Each test included 1 to 32 cultivars and one to five fungicides (two strobilurins, one triazole, and two strobilurin-triazole mixtures). There was a total of 311 test-cultivar-fungicide treatment comparisons, where a comparison was the reported yield difference between sprayed and unsprayed treatments of a given cultivar in a given test. Parameters used to calculate the economic benefit or loss associated with fungicide application included a grain price range of $73.49 to 257.21 Mg(-1) ($2 to 7 bu(-1)), a total fungicide application cost of $24.71 to 74.13 ha(-1) ($10 to 30 acre(-1)), and a 0.14 to 0.21 Mg ha(-1) (2.3 to 3.4 bu acre(-1)) loss in yield from driving over wheat during application (with a sprayer 27.4 or 18.3 m [90 or 60 feet] wide, respectively). The yield increase needed to pay for a fungicide application at each combination of cost and price was calculated, and the cumulative probability function for the fungicide yield-response data was modeled. The model was used to predict the probability of achieving a break-even yield, and the probabilities were graphed against each cost-price combination. Tests were categorized as "no-disease" or "diseased" based on reports of the researchers rating the tests. Subsets of the data were analyzed to assess the profitability of the triazole fungicide and the strobilurin-containing fungicides separately in no-disease versus diseased experiments. From the results, it was concluded that, with routine fungicide application based solely on wheat growth stage, total fungicide application costs had to be <$24.71 ha(-1) ($10 acre(-1)) in order to average a ≥ 50% probability of breaking even or making a profit (compared with not spraying). By contrast, if fungicides were applied when fungal disease was present, total application costs of ≤ $47 ha(-1) ($19 acre(-1)) for strobilurins and ≤ $72 ha(-1) ($29 acre(-1)) for propiconazole alone were associated with a ≥ 50% probability of breaking even or making a profit at a wheat price of $184 Mg(-1). The results do not support the application of strobilurin or triazole fungicides to mid-Atlantic wheat crops for "plant health" in the absence of disease. Rather, they support basing the decision to apply fungicide on observation of disease, if an economic return for the input is desired.     

Bioactive contaminants leach from disposable laboratory plasticware

Disposable plasticware such as test tubes, pipette tips, and multiwell assay or culture plates are used routinely in most biological research laboratories. Manufacturing of plastics requires the inclusion of numerous chemicals to enhance stability, durability, and performance. Some lubricating (slip) agents, exemplified by oleamide, also occur endogenously in humans and are biologically active, and cationic biocides are included to prevent bacterial colonization of the plastic surface. We demonstrate that these manufacturing agents leach from laboratory plasticware into a standard aqueous buffer, dimethyl sulfoxide, and methanol and can have profound effects on proteins and thus on results from bioassays of protein function. These findings have far-reaching implications for the use of disposable plasticware in biological research.     

Analysis of bulked and redundant accessions of Brassica germplasm using assignment tests of microsatellite markers

This study was conducted to determine if Brassica germplasm bulks created and maintained by the USDA-ARS North Central Plant Introduction Station (NCRPIS) were made with genetically indistinguishable component accessions and to examine newly identified putative duplicate accessions to determine if they can be bulked. Using ten microsatellite primer pairs, we genotyped two bulks of B. rapa L. ssp. dichotoma (Roxb.) Hanelt comprising four accessions and three bulks of B. rapa L. ssp. trilocularis (Roxb.) Hanelt comprising fourteen accessions, as well as four pairs of putatively duplicate accessions of B.␣napus L. Assignment tests on ten individual plants per accession were conducted using a model-based clustering method to arrive at probabilities of likelihood of accession assignment. The assignment tests indicated that one of the two bulks of B. rapa ssp. dichotoma involves genetically heterogeneous accessions. It was observed in the B. rapa ssp. trilocularis bulks that the component accessions could be differentiated into groups, with misassignments observed most frequent within groups. In B. napus, only one of the four pairs of putative duplicates showed significant genetic differentiation. The other three pairs of putative duplicates lack differences and support the creation of bulks. The results of the assignment tests were in agreement with cluster analyses and tests of population differentiation. Implications of these results in terms of germplasm management include the maintenance and/or re-creation of some Brassica germplasm bulks by excluding those accessions identified as being unique in this study.     

Canine multifocal retinopathy caused by a BEST1 mutation in a Boerboel

A 7‐month‐old male intact Boerboel presented to the Cummings School of Veterinary Medicine at Tufts University for the evaluation of ocular discharge. Bilateral multifocal serous retinal detachments were noted on fundus examination as an incidental finding. Genetic testing confirmed the dog to be homozygous for a mutation in the BEST1 gene, where a C₇₃T/R₂₅X change results in premature termination codon. Further testing, including electroretinography and optical coherence tomography (OCT), demonstrated that there was no evidence of retinal photoreceptor dysfunction and confirmed that observed lesions were characteristic of canine multifocal retinopathy. No progression of the lesions was noted 3 months after the initial examination. To the authors’ knowledge, this is the first report of canine multifocal retinopathy in the Boerboel breed.     

Effect of Photoperiod Manipulation on Broodstock Spawning, Fertilization Success, and Egg Developmental Abnormalities in Atlantic Cod, Gadus morhua

Spawning activity in two captive Atlantic cod broodstock groups previously captured from the wild stock fishery was monitored over three spawning seasons. Both groups spawned under ambient photoperiod (PP) in the first year, after which photomanipulation was applied to compress the PP cycle of one broodstock group, while the second group was maintained on natural PP. Increased total egg volumes, number of egg batches, total egg production, and duration of the spawning season were observed in both ambient PP and advanced PP groups in the second and third spawning seasons. Compared to ambient controls, the photoadvanced group commenced spawning earlier, had a longer spawning season, produced a lower total volume of eggs over the entire spawning season, and had lower mean daily batch volumes of eggs, and the eggs were of smaller mean diameter. Fertilization success was >90% in all years, increased in both groups in the first year after photomanipulation but declined to prior levels in the following year. No consistent difference in fertilization success was observed between advanced PP and ambient PP groups. Rates of cell development abnormalities were low (<10%) in both groups over the three spawning seasons. Abnormalities of cell symmetry and clarity increased in both ambient PP and advanced PP groups over the three spawning seasons. However, no consistent differences in rate of cell abnormalities were observed between advanced PP and ambient PP groups within years. We conclude that PP advancement is an effective technique to compress the time between successive spawning periods in Atlantic cod and does not negatively affect fertilization success or rates of cell developmental abnormalities. However, it has a negative effect on both volume and size of eggs produced.     

Insights and Ideas Garnered from Marine Metabolites for Development of Dual-Function Acetylcholinesterase and Amyloid-β Aggregation Inhibitors

Due to the diversity of biological activities that can be found in aquatic ecosystems, marine metabolites have been an active area of drug discovery for the last 30 years. Marine metabolites have been found to inhibit a number of enzymes important in the treatment of human disease. Here, we focus on marine metabolites that inhibit the enzyme acetylcholinesterase, which is the cellular target for treatment of early-stage Alzheimer’s disease. Currently, development of anticholinesterase drugs with improved potency, and drugs that act as dual acetylcholinesterase and amyloid-β aggregation inhibitors, are being sought to treat Alzheimer’s disease. Seven classes of marine metabolites are reported to possess anti-cholinesterase activity. We compared these metabolites to clinically-used acetylcholinesterase inhibitors having known mechanisms of inhibition. We performed a docking simulation and compared them to published experimental data for each metabolite to determine the most likely mechanism of inhibition for each class of marine inhibitor. Our results indicate that several marine metabolites bind to regions of the acetylcholinesterase active site that are not bound by the clinically-used drugs rivastigmine, galanthamine, donepezil, or tacrine. We use the novel poses adopted for computational drug design of tighter binding anticholinesterase drugs likely to act as inhibitors of both acetylcholinesterase activity and amyloid-β aggregation inhibition.