Name: protocol for assessing the relative effects of environment and genetics on antler and body growth for a long-lived cervid
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We thank the Mississippi Department of Wildlife, Fisheries and Parks (MDWFP) for financial support using resources from the Federal Aid in Wildlife Restoration Act (W-48-61). We thank MDWFP biologists W. McKinley, A. Blaylock, A. Gary, and L. Wilf for their extensive involvement in data collection. We also thank S. Tucker as facility coordinator and multiple graduate students and technicians for their help collecting data. This manuscript is contribution WFA427 of the Mississippi State University Forest and Wildlife Research Center.

Ethics Statement: The Mississippi State University Institutional Animal Care and Use Committee approved all capture, handling, and marking techniques under protocols 04-068, 07-036, 10-033 and 13-034.
1. Establish Capture Sites, Immobilize and Transport wild White-tailed Deer
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	<li>Identify public and private properties that are enrolled in the Deer Management Assistance Program22 and establish &#8805;29 capture sites throughout three source regions in Mississippi, USA.</stron...

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There are several steps associated with our protocol; however, there are four critical steps that must be taken to ensure success with this protocol. First, during capture of wild deer, there must be several capture locations throughout a single source region (step 1.1.1). Having multiple capture locations ensures that any genetic variability associated with the source region will be represented among deer. Second, deer must be kept separated by source region during breeding season (steps 1.4.9 and 5.2.1). Ensuring anima...

Environmental factors a mother experiences during gestation and lactation may influence her offspring&#39;s phenotype, independent of genotype1,2,3. Mothers who inhabit high-quality environments likely will produce offspring that exhibit a luxury phenotype (large antler and body size4), whereas mothers who inhabit a low-quality environment may produce offspring that exhibit an efficiency phenotype (small antler and body size4). Therefore, persisting in a high-quality environment may allow a mother to produce male offspring with large phenotypic characteristics, which may directly influence the offspring&#39;s reproductive opportunities5,6,7,8 and indirectly influence the mother&#39;s inclusive fitness.
Although nutrition directly influences phenotypic characteristics across taxa (Ursus americanus, Ursus arctos9; Liasis fuscusI10; Larus michahellis11), several factors may affect white-tailed deer phenotypes in Mississippi, USA. Antler and body size are about one-third larger for some populations compared to others12. This variation is strongly correlated with forage quality13,14; the largest males are found in areas with the greatest quality of forage. However, historic restoration efforts of white-tailed deer in Mississippi may have led to genetic bottlenecks and/or founder effects15,16, which may also partially explain some of the observed regional variation in white-tailed deer phenotype.
We provide the protocol we used to control nutritional quality of wild-caught white-tailed deer, which allowed us to assess whether male phenotype is restricted by population-level genetics. This protocol also allowed us to assess whether lagging maternal effects were present in our populations. Our controlled design is preferential to studies conducted on free ranging populations which are limited to using environmental variables as a proxy for nutritional restriction3,17. Our controlled design also allows for other variables such as potential chronic stress related to social interactions to be held constant as all individuals are subjected to similar housing and husbandry practices. Additionally, because nutrition directly influences other life history aspects ranging from reproduction to survival18,19, controlling nutrition allows for investigators to assess other variables that affect mammalian life history aspects. Similar protocols have been described to assess questions related to life history aspects for other ungulates across North America (e.g., 20,21).

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			<td height="20" style="width:416px;height:20px;">Shelled Corn</td>
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			<td style="width:64px;"></td>
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			<td height="20" style="height:20px;">Elevated Stand</td>
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			<td height="20" style="height:20px;">Safety Harness</td>
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			<td height="20" style="height:20px;">Ground Blind</td>
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			<td height="20" style="height:20px;">Model 196 Projector</td>
			<td style="width:316px;">Pneu-Dart, Pennsylvania, USA</td>
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			<td height="20" style="height:20px;">3cc Radio-Telemetry Darts</td>
			<td style="width:316px;">(Pneu-Dart, Pennsylvania, USA)</td>
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			<td height="20" style="height:20px;">Various Sized Darts&#160;</td>
			<td style="width:316px;">(Pneu-Dart, Pennsylvania, USA)</td>
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			<td height="20" style="height:20px;">Teletamine HCl&#160;</td>
			<td style="width:316px;">(Telazol, Fort Dodge Animal Health, Iowa, USA)</td>
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			<td height="20" style="height:20px;">Xylazine HCl&#160;</td>
			<td style="width:316px;">(West Texas Rx Pharmacy, Amarillo, Texas, USA)</td>
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			<td height="20" style="height:20px;">Yhoimbine HCl</td>
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			<td height="20" style="height:20px;">Tolazoline HCl</td>
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			<td height="20" style="height:20px;">Military Style Gurney</td>
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			<td height="20" style="height:20px;">Rectal Thermometer</td>
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			<td height="20" style="height:20px;">Shade Cloth</td>
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		<tr height="20">
			<td height="20" style="height:20px;">20% Crude Protein Deer Pellets&#160;</td>
			<td style="width:380px;">(Purina AntlerMax Professional High Energy Breeder 59UB, Purina, Missouri, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trough Style Feeders</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Commercial Clover&#160;</td>
			<td style="width:380px;">(Durana Clover, Pennington Seed Co., Georgia, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Commercial Fescue&#160;</td>
			<td style="width:380px;">(Max-Q Fescue, Pennington Seed Co., Georgia, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Blankets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice Packs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Broadleaf Weed Control (2, 4-DB Herbacide, Butyrac 200)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Grass Control&#160;</td>
			<td style="width:316px;">(Poast Herbacide, BASF Co.)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pelleted Wormer</td>
			<td>Safeguard Co.,&#160;</td>
			<td></td>
			<td style="width:227px;">active ingredient fenbendazole</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Parasite Pour-on Treatment&#160;</td>
			<td>(Ivomec, Merial Co.)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insecticide</td>
			<td style="width:316px;">Riptide, McLaughlin Gormley King Co.)&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Medium and Large Plastic Ear Tags&#160;</td>
			<td>(Allflex, Texas, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Remote site that assigned parentage</td>
			<td style="width:380px;">DNA Solutions Animal Solutions Manager (DNA Solutions, Oklahoma, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital Hanging Scale&#160;</td>
			<td style="width:316px;">(Moultrie, EBSCO Industries, Inc.)&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tape Measure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clostridium Perfringens Types C and D Toxoid Essential 3&#160;</td>
			<td>(Colorado Serum Co.)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clostridium Perfringens Types C and D Antitoxin Equine Origin</td>
			<td>(Colorado Serum Co.)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ivermectin in propylene glycol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic</td>
			<td style="width:380px;">(Nuflor, Schuering-Plough Animal Health Corp., New Jersey, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ivermectin&#160;</td>
			<td style="width:380px;">(Norbrook Labratories, LTD., Down, Northern Ireland, UK)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clostidrial vaccine</td>
			<td style="width:316px;">(Vision 7 with SPUR, Ivesco LLC, Iowa, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leptospirosis vaccine&#160;</td>
			<td style="width:316px;">(Leptoferm-5, Pfizer, Inc., New York, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trailer for transport</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reciprocating saw&#160;</td>
			<td style="width:316px;">(DEWALT, Maryland, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scientific Digital Scale&#160;</td>
			<td style="width:316px;">(Global Industrail, Global Equipment Company Inc)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antler Measuring Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fogger</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic Ear Tags&#160;</td>
			<td>(Allflex, Texas, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic Ear Tagger</td>
			<td>(Allflex, Texas, USA)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

protocol for assessing the relative effects of environment and genetics on antler and body growth for a long-lived cervid

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which promotes growth of large weaponry and body size. Populations of the same species display each phenotype depending on environmental conditions. Although antler and body size of male white-tailed deer (Odocoileus virginianus) varies by physiographic region in Mississippi, USA and is strongly correlated with regional variation in nutritional quality, the effects of population-level genetics from native stocks and previous re-stocking efforts cannot be disregarded. This protocol describes how we designed a controlled study, where other factors that influence phenotype, such as age and nutrition, are controlled. We brought wild-caught pregnant females and six-month-old fawns from three distinct physiographic regions in Mississippi, USA to the Mississippi State University Rusty Dawkins Memorial Deer Unit. Deer from the same region were bred to produce a second generation of offspring, allowing us to assess generational responses and maternal effects. All deer ate the same high-quality (20% crude protein deer pellet) diet ad libitum. We uniquely marked each neonate and recorded body mass, hind foot, and total body length. Each subsequent fall, we sedated individuals via remote injection and sampled the same morphometrics plus antlers of adults. We found that all morphometrics increased in size from first to second generation, with full compensation of antler size (regional variation no longer present) and partial compensation of body mass (some evidence of regional variation) evident in the second generation. Second generation males that originated from our poorest quality soil region displayed about a 40% increase in antler size and about a 25% increase in body mass when compared to their wild harvested counterparts. Our results suggest phenotypic variation of wild male white-tailed deer in Mississippi are more related to differences in nutritional quality than population-level genetics.

Individual age, nutritional quality, and genetics influence male white-tailed deer phenotype. Our study design allowed us to control the quality of nutrition deer were consuming and allowed us to identify the age of each deer for valid comparisons within year classes. By controlling nutrition and age with our study design, we were better able to understand whether population-level genetics were restricting the phenotype of males from two study populations. Improved nutrition had a positiv...

Watch this Scientific Journal Video about Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid at JoVE.com

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Phenotypic differences among cervid populations may be related to population-level genetics or nutrition; discerning which is difficult in the wild. This protocol describes how we designed a controlled study where nutritional variation was eliminated. We found that phenotypic variation of male white-tailed deer was more limited by nutrition than genetics.

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

The overall goal of this experimental design is to assist whether population level genetics restrict white-tailed deer, odocoileus virginianus antler and body size by controlling for other variables that also influence antler and body size such as age and nutrition. This methodology can really help us understand the true cause of morphological variation in wildlife species across their range. And this is important because morphology is the basis for historical designation of sub-species and with Endangered Species Act codifying those sub-species differences it's important to understand are those differences really due to genetic differences that are meaningful and need to be preserved or are they just nutritional habitat quality differences that are associated with the habitat where those sub-species occur?The main advantage of this technique is that it allows us in a controlled environment to focus on a specific characteristic of the environment that the animals were raised in naturally and in this case we're removing the animals from their natural habitat and controlling for nutrition to see if nutrition is the cause for the morphological differences. This technique can actually improve the implementation of the Endangered Species Act because it's gonna allow state resource agencies with limited resources to identify meaningful genetic variation. Demonstrating the technique today will be Dan Morina.A graduate student here at the MSU Deer Lab. Begin by insuring that side fencing has shade cloth to act as a visual and physical barrier between pens. Install elevated box blinds at one end of each pen to facilitate darting events during data collection.Keep two trough style feeders at separate ends of each pen to reduce competition for food among deer. Finally, provide deer with a high quality diet add Lupitum. Begin by walking the technician who'll be darting to the end of the pen where the elevated blinds are located.Post a single technician into a blind. Then have the individual who walked the technician to the blind walk back to the opposite end of the pen. After confirming sedation of the deer by checking for eye reflexes apply ophthalmic ointment to the eyes.And blindfold deer to reduce stress. Load the deer on a military-style gurney. Next use a rectal thermometer to assess body temperature after recovery.Warm deer with heated blankets if the animals temperature is below 37.7 degrees Celsius. Or cool the deer with ice packs if the animal's temperature is above 40 degrees Celsius. And transport it via utility task vehicle to a predetermined data collection area.Once transported, measure body mass to the nearest hundredth of a kilogram with a digital hanging scale. As well as hindfoot length and total body length to the nearest millimeter. Then administer size-appropriate amounts of antibiotic ivermectin, a custodial vaccine and a leptospirosis vaccine.Next, while the animal is sedated take three antler measurements from adult males using an antler measuring tape. Measure the inside spread, basal circumference and the main beam length of antlers prior to antler removal. Then remove antlers about three centimeters above the burr using a reciprocating saw.After the data is collected place the deer into the appropriate pen. And administer either 125 milligrams per kilogram of yohimbine hydrochloride or 4.0 milligrams per kilogram of tylosin hydrochloride to reverse the effects of xylazine hydrochloride. Finally, monitor the deer to ensure they remain in a sternal position until they come out of sedation and are fully alert.After acquiring the antler samples measure each individual tine protruding from the main beam and additional abnormal points by aid of wire. Wrap the wire around where the tine intersects the main beam and mark that point for reference. Then measure from this reference point to the tip of the tine and repeat for each tine.Next collect the remaining circumference measurements by identifying the smallest point between the G1 and G2 tines the G2 and G3 tines and the G3 and G4 tine if present. If the G4 tine is not present measure the distance between the mid-point of the G3 tine at the end of the main beam. And measure the H4 circumference at the midway point.Finally, weigh antlers to the nearest tenth of a gram using a scientific digital scale. And design a minimal, critical antler mass of one gram for first year animals with antler shorter than three centimeters. Results from this study indicated that improved nutrition had a positive effect on all morphometric measurements on a 3.5 year old male white-tailed deer from each source region.Antler mass and antler size did not vary among the three source regions after two generations of improved nutrition. Which suggest that antler size is not restricted by population level genetics. Further, body mass greatly increased from first to second generation.Suggesting there is a nutritional limitation in the wild for all populations. However, there were still regional variation among second generation males. Once mastered, this technique only takes about an hour per animal.But sample size is really important so you wanna have as many animals in your study as possible so you could end up spending several days if needed to process all of your animals. While attempting this procedure it's really important that everything within the deer pens is set up for success. The blind needs to be properly placed at the correct distance from the fence.Shooting lanes need to be cut if there are branches in the way. And everything that you can control needs to be controlled in advance because the one thing you cannot control is how the animal will behave under the particular stressful situation you're putting them into. Using this procedure you could also modify the nutritional components of the experiment.And look at how protein or energy intake might alter that genetic potential and the morphological expression of the animal. This same methodology can be used by other researchers to vary the type of treatment that's supplied to the animals and look at other things beside morphological development. You could look at how nutritional intake affects life history traits such as reproductive traits and recovery from the demand of gestation and lactation.After watching this video you should have a good idea of how to control nutrition and age to see how genetics and nutrition interact in the morphological development of an animal within a captive facility. Don't forget that whenever you sedate a wild animal even in a captive facility there's a chance for injury to the animal so we have to take care and monitor them closely.

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A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

A Protocol for Collecting and Constructing Soil Core Lysimeters

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the landscape has complex and varied soil morphology, an accompanying disparity in flow paths for leachate through the soil macropore and matrix structure is present. The rate of flow through these paths is further affected by antecedent soil moisture. Lysimeters are used to quantify flow rate, volume of water and concentration of nutrients leaching downward through soils. While many lysimeter designs exist, accurately determining the volume of water and mass balance of nutrients is best accomplished with bounded lysimeters that leave the natural soil structure intact. 
Here we present a detailed method for the extraction and construction of soil core lysimeters equipped with soil moisture sensors at 5 cm and 25 cm depths. Lysimeters from four different Coastal Plain soils (Bojac, Evesboro, Quindocqua and Sassafras) were collected on the Delmarva Peninsula and moved to an indoor climate controlled facility. Soils were irrigated once weekly with the equivalent of 2 cm of rainfall to draw down soil nitrate-N concentrations. At the end of the draw down period, poultry litter was applied (162 kg TN ha-1) and leaching was resumed for an additional five weeks. Total recovery of applied irrigation water varied from 71% to 85%. Nitrate-N concentration varied over the course of the study from an average of 27.1 mg L-1 before litter application to 40.3 mg L-1 following litter application. While greatest flux of nutrients was measured in soils dominated by coarse sand (Sassafras) the greatest immediate flux occurred from the finest textured soil with pronounced macropore development (Quindocqua).

A detailed method for extraction and assembly of intact soil core lysimeters and their use for study of leachate and associated loss of nutrients from surface applied poultry litter is demonstrated.

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Comprehensive monitoring of coffee berry borer and host plant dynamics is essential for aggregating landscape-level data to improve management of this invasive pest. Here, we present a protocol for scientific monitoring of coffee berry borer movement, infestation, mortality, coffee plant phenology, weather, and farm management via a mobile electronic data recording application.

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...