Power of a Snapshot Observation: Using iNaturalist To Teach Undergraduates about Ethograms in Animal Behavior.

Engaging undergraduate biology majors may present challenges for educators disseminating science concepts utilizing standard lecture classroom formats. Moreover, animal behavior courses teaching ethology may often require the use of live animals, field excursions, or students having to develop projects which can be both time-consuming or require financial investment, or that may not be well-suited to the flexibility of being taught online. Therefore, developing in-class activities which allow students to use self-discovery when generating their own observational data, work in groups, and practice hands-on science may serve to ameliorate these challenges facing faculty teaching animal behavior course content. To this end, I developed a straightforward, engaging in-class activity which allowed students to scan images available on the smartphone identifier iNaturalist to generate their own ethograms (catalogs of behaviors) for local state species. Students successfully described behaviors across a variety of animal taxa, reptiles, mammals, birds, and insects when generating their own ethograms and data, and they actively discussed how this activity enabled them to further understand both ethograms and their importance to animal behavior and overall how animals behave and allocate time performing a variety of behaviors. This activity can be modified for further use in both introductory and upper-level course work in organismal biology and can incorporate data analysis, graphing, or presentation skill sets for science majors.

Macroinvertebrate Communities and Body Condition of Larval Eastern Hellbender Salamanders (Cryptobranchus alleganiensis) in North Carolina.

Salamanders are important biological indicators of freshwater aquatic ecosystems. The Eastern Hellbender, Cryptobranchus alleganiensis, is found primarily in streams across the southeastern, midwestern, and eastern portions of the United States. However, this unique large aquatic salamander is facing numerous threats and declines across its geographic range, including in Appalachia. Moreover, little is known regarding the early life history stages (gilled larvae), particularly regarding food availability (aquatic insects present in streams) and body condition. In this study, we assessed the macroinvertebrate communities of streams sampled for larval C. alleganiensis and report on the body condition index within western North Carolina streams. We found varying levels of diversity across sample locations for macroinvertebrates (total sampled = 3,619, representing over 30 genera), with the most prevalent insects from Trichoptera order, with an overall high percent of Ephemeroptera, Plecoptera, and Trichoptera (EPT) in all streams, ranging from 68.5% to 90.7%. Functional Feeding Groups included 40.5% filterers, 24.3% predators, 17.6% gatherers, 10.7% shredders, and 6.9% scrapers. Shannon Diversity Index in sample streams ranged from 2.101 to 2.698. Body condition or SMI (scaled mass index) ranged from 1.5 to 3.3 2 across sites with a largely consistent and strong linear relationship between log mass and total length (r = 0.910). Our results add to the body of knowledge on the larval ecology of this North American salamander and may aid in future management of hellbender stream habitats.

Chronic exposure to low doses of ionizing radiation impacts the processing of glycoprotein N-linked glycans in Medaka (Oryzias latipes).

PURPOSE: Ionizing radiation is found naturally in the environment. Low doses of IR may have beneficial applications, yet there is also potential for detrimental long-term health effects. Impacts following exposure to low levels of IR have been refractory to identification and quantification. Glycoprotein glycosylation is vital to cell-cell communication and organismal function, and sensitive to changes in an organism's macro- and cellular environment. We investigated whether accumulated low doses of IR (LoDIR) affect the N-linked glycoprotein glycans using Medaka fish (Oryzias latipes). MATERIALS AND METHODS: State-of-the-art methods in radiation exposure and glycan analysis were applied to study N-glycan changes after 190 day exposure at three different rates of gamma irradiation (2.25, 21.01, and 204.3 mGy/day) in wild-type adult Medaka. Tissue N-glycans were analyzed following enzymatic release from extracted proteins. RESULTS: N-linked glycan profiles are dominated by complex type N-glycans modified with terminal sialic acid and core fucose. Fucosylation and sialylation of N-linked glycoprotein glycans are affected by LoDIR and a subset of N-glycans are involved in the organismal radio-response. CONCLUSION: This is the first indication that the glycome can be interrogated for biomarkers that report the impact of chronic exposure to environmental stressors, such as low-level IR.

Prevalence of Batrachochytrium dendrobatidis in immature eastern hellbenders Cryptobranchus alleganiensis from North Carolina, USA.

Batrachochytrium dendrobatidis (Bd) has been detected in wild hellbender Cryptobranchus alleganiensis populations, with rare instances of chytridiomycosis and Bd-induced mortality. To date, Bd surveillance in hellbender populations has been disproportionately focused on adult age classes. A lingering question is whether Bd might contribute to hellbender declines through disproportionate negative effects on immature age classes. The objective of this study was to quantify Bd prevalence and load in immature hellbenders in western North Carolina, USA. We conducted field surveys during 2018 and 2019 and collected 88 skin swabs from 84 hellbenders spanning 3 age classes. Bd was detected on 11% of individuals, including 8 larvae and 1 juvenile. We did not detect symptoms of chytridiomycosis or a decline in body condition in Bd-positive hellbenders. Load varied from approximately 1-153 zoospore equivalents for the 9 Bd-positive hellbenders and was not associated with size class of the individual. While hellbenders appeared to be abundant in each survey reach, more work is needed to determine whether Bd may increase the vulnerability of immature hellbenders to anthropogenic stressors.

An Effective Protocol for Proteome Analysis of Medaka (Oryzias latipes) after Acute Exposure to Ionizing Radiation.

All terrestrial organisms are subject to evolutionary pressures associated with natural sources of ionizing radiation (IR). The legacy of human-induced IR associated with energy, weapons production, medicine, and research has changed the distribution and magnitude of these evolutionary pressures. To date, no study has systematically examined the effects of environmentally relevant doses of radiation exposure across an organismal proteome. This void in knowledge has been due, in part, to technological deficiencies that have hampered quantifiable environmentally relevant IR doses and sensitive detection of proteomic responses. Here, we describe a protocol that addresses both needs, combining quantifiable IR delivery with a reliable method to yield proteomic comparisons of control and irradiated Medaka fish. Exposures were conducted at the Savannah River Ecology Laboratory (SREL, in Aiken, SC), where fish were subsequently dissected into three tissue sets (carcasses, organs and intestines) and frozen until analysis. Tissue proteins were extracted, resolved by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and each sample lane was divided into ten equal portions. Following in-gel tryptic digestion, peptides released from each gel portion were identified and quantified by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to obtain the most complete, comparative study to date of proteomic responses to environmentally relevant doses of IR. This method provides a simple approach for use in ongoing epidemiologic studies of chronic exposure to environmentally relevant levels of IR and should also serve well in physiological, developmental, and toxicological studies.

Development and characterization of 33 novel polymorphic microsatellite markers for the brown tree snake Boiga irregularis.

BACKGROUND: Boiga irregularis is a widespread invasive species on Guam and has led to extirpation of most of the island's native avifauna. There are presently no microsatellite markers for this invasive species, hence we developed highly polymorphic microsatellite markers to allow for robust population genetic studies on Guam. FINDINGS: We isolated and characterized 33 microsatellite loci for the brown tree snake, B. irregularis. The loci were screened across 32 individuals from Guam. The number of alleles per locus ranged from three to ten, with an average of 4.62. The expected (He) and observed heterozygosity (Ho) ranged from 0.294 to 0.856 and from 0.031 to 0.813, with an average of 0.648 and 0.524, respectively. Significant deviations from Hardy-Weinberg equilibrium were detected at seven loci after Bonferoni correction. Probability of identity values ranged from 0.043 to 0.539. CONCLUSIONS: These genetic markers are useful for understanding a suite of post-invasion population genetic parameters, sources of invasions, and effectiveness of management strategies for this invasive species.

Genetic analysis reveals multiple parentage in captive reared eastern hellbender salamanders (Cryptobranchus alleganiensis).

Information on the parentage of captive reared clutches is vital for conservation head-starting programs. Molecular methods, such as genotyping individuals with hyper-variable markers, can elucidate the genealogical contribution of captive-reared, reintroduced individuals to native populations. In this study, we used 12 polymorphic microsatellite loci to infer parentage of a clutch of 18 eastern hellbenders collected from a single nest from Buffalo Creek, West Virginia, subsequently reared in captivity, and used for translocations in Indiana. Collectively, these markers successfully detected the presence of multiple parentage for this species of conservation concern presently used in captive management programs in zoos across many states. This study highlights the need for genetic analysis of captive reared clutches used in translocations to minimize the loss of genetic diversity and potential for genetic swamping at release sites.

Population genetics of the Eastern Hellbender (Cryptobranchus alleganiensis alleganiensis) across multiple spatial scales.

Conservation genetics is a powerful tool to assess the population structure of species and provides a framework for informing management of freshwater ecosystems. As lotic habitats become fragmented, the need to assess gene flow for species of conservation management becomes a priority. The eastern hellbender (Cryptobranchus alleganiensis alleganiensis) is a large, fully aquatic paedamorphic salamander. Many populations are experiencing declines throughout their geographic range, yet the genetic ramifications of these declines are currently unknown. To this end, we examined levels of genetic variation and genetic structure at both range-wide and drainage (hierarchical) scales. We collected 1,203 individuals from 77 rivers throughout nine states from June 2007 to August 2011. Levels of genetic diversity were relatively high among all sampling locations. We detected significant genetic structure across populations (Fst values ranged from 0.001 between rivers within a single watershed to 0.218 between states). We identified two genetically differentiated groups at the range-wide scale: 1) the Ohio River drainage and 2) the Tennessee River drainage. An analysis of molecular variance (AMOVA) based on landscape-scale sampling of basins within the Tennessee River drainage revealed the majority of genetic variation (∼94-98%) occurs within rivers. Eastern hellbenders show a strong pattern of isolation by stream distance (IBSD) at the drainage level. Understanding levels of genetic variation and differentiation at multiple spatial and biological scales will enable natural resource managers to make more informed decisions and plan effective conservation strategies for cryptic, lotic species.

Health and habitat quality assessment for the eastern hellbender (Cryptobranchus alleganiensis alleganiensis) in Indiana, USA.

The eastern hellbender (Cryptobranchus alleganiensis alleganiensis) has experienced precipitous population declines throughout its range. Numerous factors are speculated to be involved, but no empirical evidence has been presented for any. We implemented a population-wide health assessment in Indiana, USA, examining both the physical well-being of individuals and the quality of their habitat. Physicochemical parameters were analyzed directly in the field and later in the laboratory, when appropriate. Samples were collected June 2008-October 2008 and June 2009-September 2009 for reproductive analysis, blood screening, and disease prevalence. Of 27 chemicals screened in water samples, three were found in the study site, including atrazine. Atrazine was found at levels reported to cause reproductive problems in other amphibians. Vitellogenin was detected only in females and proved a reliable indicator of sex. Sperm parameters were generally of high quality and similar to other populations. Most plasma parameters were similar between sexes, although there were significant differences in calcium and potassium concentrations. Abnormalities were common, occurring in 68% of individuals. No hemoparasites were found, but amphibian chytrid fungus (Batrachochytrium dendrobatidis) was detected on one individual. Our findings establish a baseline for hematology and water-quality parameters that can be used as a model for evaluating population health throughout the hellbender range.