Name: visually sexing loggerhead shrike (lanius ludovicianus) using plumage coloration and pattern
Uploaded: 2020-03-08T12:30:00
Description: Watch this Scientific Journal Video about Visually Sexing Loggerhead Shrike Lanius Ludovicianus Using Plumage Coloration and Pattern at JoVE.com

Funding for fieldwork during which our methodologies were developed was provided by Environment Canada&#39;s Canadian Wildlife Service, Environment Canada&#39;s Strategic Technologies Application of Genomics in the Environment Research Fund, the Endangered Species Recovery Fund, the Interdepartmental Recovery Fund, the Natural Sciences and Engineering Research Council and the Ontario Ministry of Training, Colleges and Universities (scholarships to A.A.C.), Queen&#39;s University (Duncan and Urlla Carmichael Fellowship to A.A.C.) and Wildlife Preservation Canada. We wish to thank the Editor and four anonymous reviewers for their comments, which greatly improved this manuscript. Thanks to Wildlife Preservation Canada staff and the members of the North American Loggerhead Shrike Working Group for discussions that assisted in development of this methodology. We extend our thanks to all the Citizen Scientists and staff at African Lion Safari, Cambridge, Ontario, for their assistance in completing the survey. We especially thank Erin Sills, Marketing and PR Coordinator, African Lion Safari, for her assistance in producing the on-line survey and summarizing results.

The research protocol presented herein complies with African Lion Safari&#39;s Animal Care Committee guidelines.
1. Sexing Loggerhead Shrikes by Color and Patern of the Sixth Primary Wing Feather
NOTE: Shrikes can be sexed in hand based on the coloration and pattern in the sixth primary wing feather (P6). In brief, the technique requires the observer to visually extrapoloate a line along the lower edge of the primary wing coverts, and then to assess how far the brown ...

Herein, we describe a simple and efficient method whereby loggerhead shrike can be sexed based only on visual cues, and provide an assessment of the method&#39;s accuracy. Our simple method is easily and quickly undertaken, with results indicating a high accuracy rate that increases with a small amount of training. Our results support those of previous work13 that indicated the method originally developed for use in northern shrike12 had utility for sexing adult loggerhead ...

The loggerhead shrike (Lanius ludovicianus) is a North American passerine with a broad geographical range encompassing most of North America and a variety of habitats that can generally be described as grassland1. It is one of only two species of shrikes (Order Passeriformes) that occur in North America. Shrikes are best known for the unique raptor-like bill, which allows them to take vertebrate prey, and their unique behavior of impaling food items on thorns or other sharp objects. The loggerhead shrike is the only species of &#39;true shrike&#39; (Family Laniidae) endemic to the continent. Shrikes breeding above 40&#176;N are generally obligate migrants1,2,3, with wintering grounds almost entirely encompassed within that of non-migratory conspecifics1,4.
North American Breeding Bird Survey data5 for loggerhead shrike indicate a significant (3.18% yr-1) range-wide population decline. The loggerhead shrike is one of Partners in Flight&#39;s 24 &#34;Common Birds in Steep Decline&#34;-i.e., those that have lost more than 50% of their populations over the past 40 years, but which lack other elevated vulnerability factors that would warrant higher &#34;Watch List&#34; status6. Habitat loss due to succession and human development likely contributed to the initial declines4,7, but continued population declines are outpacing habitat loss in the breeding season, suggesting other limiting factors, in particular in areas where the species is an obligate migrant4,8. Results of a population viability analysis conducted for the critically endangered population of loggerhead shrike in Ontario suggests that over-wintering success of birds in their first year of life is a driver of the population trends9,10. Results further indicate that the conservation breeding effort, which is augmenting the wild population, has kept the species from extirpation in this area9,10.
Understanding sex differences is an important component of both ecological and evolutionary hypotheses. The plumage of loggerhead shrikes (Laniusludovicianus) is sexually monochromatic and therefore individuals cannot reliably be sexed in the hand. However, based on a method applicable to the northern shrike (Lanius excubitor)11,12, it has been shown to be possible to sex at least some populations of adult loggerhead shrikes using the coloration pattern in the sixth primary wing feather13. We have revised this methodology13 to include consideration of a second variable, specifically the extent of pigmentation in the rachis of the sixth primary, which allows reliable identification of sex in the majority of individuals in eastern populations, and tested its application (previously only applied to adult birds) to fledged young of the year. The method requires no specialized equipment or costly lab assays, and no measurements are required that would be subject to observer bias. Based on our results, the method is easily learned and, once mastered, highly accurate. Here, we present detailed instructions on how to sex shrike in the hand using our method and discuss the wider implications of including sex assessment in future research and conservation efforts for this unique and enigmatic species of conservation concern.

visually sexing loggerhead shrike (lanius ludovicianus) using plumage coloration and pattern

The loggerhead shrike is a small sexually monomorphic passerine bird using grassland habitats across North America. Based on Breeding Bird Survey data, the species has undergone a drastic decline since the mid-1960s. The cause of decline is unknown, and research is actively underway to address this knowledge gap. These efforts are hindered by an inability to sex the species in hand, which to date was only possible using molecular markers. Here, we present a protocol to sex loggerhead shrikes by visually analyzing the coloration and pattern in the sixth primary feather. The application of the method will facilitate our ability to identify threats on a finer scale than has been possible to date and to address various ecological and evolutionary hypotheses. The methodology is simple and results reliable&#8211;we encourage including this method for research of both in situ and ex situ populations.

Male and female plumage is, overall, monomorphic in the loggerhead shrike. However, it has been established that sex can be discerned based on the pattern of coloration in the 6th primary in the population that occurs on mainland California13 and northern shrike12. We tested Sustaita et al.&#39;s (2014) protocol13 to determine if it was applicable to northeastern populations of loggerhead shrike and to youn...

Watch this Scientific Journal Video about Visually Sexing Loggerhead Shrike Lanius Ludovicianus Using Plumage Coloration and Pattern at JoVE.com

Visually Sexing Loggerhead Shrike Lanius Ludovicianus Using Plumage Coloration and Pattern

We present a protocol to characterize the sex of loggerhead shrike visually based on the coloration and pattern of the sixth primary wing feather.

Visually Sexing Loggerhead Shrike (Lanius Ludovicianus) Using Plumage Coloration and Pattern

The loggerhead shrike is a small sexually monomorphic passerine bird using grassland habitats across North America. Based on Breeding Bird Survey data, ...

Loggerhead shrike has lost more than 50%of the population across North America over the past 40 years. The ability to determine sex in loggerhead shrike has given us another tool to help understand what is causing the decline and how conservation breeding can best help offset the threats. The plumage of loggerhead shrike is sexually monomorphic so birds can not be reliably sexed in hand, using physical characteristics as in the case for many other species.DNA sexing is possible but is expensive, time delayed and requires special equipment. Our simple method is easy and quickly undertaken, with results indicating a high accuracy rate, that increases with a small amount of training. Shrike's can be sexed, in hand, by examining the sixth primary wing feather.However, the pattern varies amongst individuals. The most difficult part of our method is deciding how far the brown in the rachis extends through the white and only going on to additional steps if the sex cannot be determined based on this characteristic alone. We suggest that anyone who hasn't used this method before, review the video and become familiar with the characteristics they are looking at, as well as the order in which to assess them.Feel free to contact us and we can provide sample photos used in our citizen science survey. Begin by firmly holding the bird using the banders grip and carefully extending one wing to allow the sixth primary feather to be viewed, making sure to not overextend the wing. Locate the P6 feather by counting forward from the first primary.Assess the brown in the rachis and determine if it extends at least halfway through the white and touches the distal end of the primary covert feathers. If it does, the bird is female. If unsure of the sex, based on the color of the rachis, assess the symmetry of the brown to white transition on each side of the shaft.If the brown meets at the same spot on either side of the shaft, the bird is male. If the line meets asymmetrically at the shaft, creating a step or notch, the bird is a female. If there is still uncertainty, examine the angle of the transition between the brown and the white coloration.If the brown has a steep angle where it meets the white, forming an upside down V, the bird is a female. If the pattern looks more like an M, the bird is male. This sexing protocol was tested on 120 participants, who read a brief instruction on how to sex using feather patterns and then attempted to sex a series of 26 shrikes based on a photograph.Most participants ranked themselves as having little or no experience with birds. The participants averaged a 77%correct assessment for both females and males. Ten volunteers received in person training on this method and attempted to sex the same series of 26 photos which they had not seen before.The individuals trained one on one, correctly assessed 12 out of 13 photos of females and 11 out of 13 photos of males. When attempting this method, the most critical step is determining how far the brown in the rachis extends through the white, only if there is question about whether or not the brown touches the primary coverts, should the observer continue on to assess other characteristics. Out method of sexing shrike in hand, can be done when handling wild shrike's during banding, and included with other data collection like age and wing cord length.For captive birds, sexing can coincide with the need to handle birds for vaccination, banding or other routine management. To date, more than 1000 young birds have been released into the wilds of Ontario. A rapid and inexpensive method for sexing young birds makes it possible to quantify how many birds of each sex are released and if sex biased mortality is occurring.

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Research

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Biology

Understanding Cerebellar Pattern Formation

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Fruit Volatile Analysis Using an Electronic Nose

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit aroma. Maturity at harvest is one of the key factors influencing the flavor quality of fruits and vegetables1. The validation of robust methods that rapidly assess fruit maturity and aroma quality would allow improved management of advanced breeding programs, production practices and postharvest handling. 
Over the last three decades, much research has been conducted to develop so-called electronic noses, which are devices able to rapidly detect odors and flavors2-4. Currently there are several commercially available electronic noses able to perform volatile analysis, based on different technologies. The electronic nose used in our work (zNose, EST, Newbury Park, CA, USA), consists of ultra-fast gas chromatography coupled with a surface acoustic wave sensor (UFGC-SAW). This technology has already been tested for its ability to monitor quality of various commodities, including detection of deterioration in apple5; ripeness and rot evaluation in mango6; aroma profiling of thymus species7; C6 volatile compounds in grape berries8; characterization of vegetable oil9 and detection of adulterants in virgin coconut oil10. 
This system can perform the three major steps of aroma analysis: headspace sampling, separation of volatile compounds, and detection. In about one minute, the output, a chromatogram, is produced and, after a purging cycle, the instrument is ready for further analysis. The results obtained with the zNose can be compared to those of other gas-chromatographic systems by calculation of Kovats Indices (KI). Once the instrument has been tuned with an alkane standard solution, the retention times are automatically converted into KIs. However, slight changes in temperature and flow rate are expected to occur over time, causing retention times to drift. Also, depending on the polarity of the column stationary phase, the reproducibility of KI calculations can vary by several index units11. A series of programs and graphical interfaces were therefore developed to compare calculated KIs among samples in a semi-automated fashion. These programs reduce the time required for chromatogram analysis of large data sets and minimize the potential for misinterpretation of the data when chromatograms are not perfectly aligned.
We present a method for rapid volatile compound analysis in fruit. Sample preparation, data acquisition and handling procedures are also discussed.

A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...