This research was funded by Washington College's Provost's Office, Middendorf Fund, Hodson Trust, and Franklin and Marshall's Hackman Fund and College of Grants. We thank E. Counihan, S. Giordano, F. Rauh, and A. Roth for assistance in the field. We thank M. Conner, R. Fleegle, and D. Startt at Chesapeake Farms, and Chino Farms for permission and access. The Washington College GIS Program helped with the preparation of maps.

All procedures involving animal subjects were approved by the Institutional Animal Care and Use Committees of Franklin and Marshall and Washington Colleges and followed all local, state, and federal regulations.
1. Capture and Handling
<ol>
	<li>Place hoop traps in the target body of water that is known to contain turtles. Identify water depth ensuring that 4 - 5 inches of the trap remains above water to allow trapped turtles to surface and breathe. Be certain to expand and secure hoop traps to their maximum length with the cross beams to prevent trap collapse. Stake traps into the bed of the body of water to prevent drift.</li>
	<li>Bait traps with several dead fish, chicken livers, or chicken necks, a can of cat food, and/or a can of vegetables25.</li>
	<li>Check traps twice daily and remove turtles. When removing turtles from traps, hold animal by the side and use caution to avoid injury from claws or beak.
	<ol>
		<li>Release by-catch. Reset traps if continued trapping is desired. Assess state of bait. If the bait had been consumed, add more. Pull traps to shore if additional trapping is not desired.</li>
	</ol>
	</li>
	<li>Determine the sex and age of turtles if desired26, 27.</li>
	<li>Place turtle in a holding bag and measure body mass with a spring scale to the nearest g.</li>
	<li>Transport turtles to the lab in climate-controlled transport boxes with a small amount of water. House animals singly in aquaria with untreated pond water held at approximately 25 &#176;C and deep enough to cover the head only.</li>
</ol>
2. Affixing Radio Transmitter
<ol>
	<li>To maximize transmitter life and output, choose the largest radio transmitter possible that does not exceed 5% of the animal&#39;s body mass28.</li>
	<li>Identify the location of transmitter placement on the carapace approximately halfway between the midline and lateral edge of the carapace approximately 1/3 of the length up from the rear edge of the carapace.</li>
	<li>Prepare area by removing mud, debris, and algal growth with a dry cloth. Swab area with 70% isopropanol.</li>
	<li>Attach transmitter with a small amount of 5 min epoxy. Orient the transmitter to maximize contact with the surface of the carapace. Position the antenna so that it trails behind the animal parallel to the long axis of the body.</li>
	<li>Once suitably positioned, cover the entire transmitter and approximately 1 cm of the surrounding carapace surface with 5 min epoxy.</li>
	<li>Return the turtle to the aquarium and allow the epoxy to cure overnight.</li>
</ol>
3. Pharmacological Preparation
Caution: Scopolamine hydrobromide and scopolamine methylbromide are potent acetylcholine antagonists. When working with these drugs, consult the Materials Safety Data Sheet, use proper personal protective equipment (e.g., gloves, fume hood), and follow laboratory safety protocols to avoid accidental contact.
<ol>
	<li>Using sterile and antipyrogenic supplies, compound a stock solution of scopolamine hydrobromide. Weigh out the desired quantity of the drug on an analytical balance. Mix the scopolamine with injection saline in a conical tube to the concentration of 1 mg/ml. Vortex solution until dissolved. Ensure that chemical purity of the base chemicals meets or exceeds United States Pharmacopeia (USP) formulation when possible11.</li>
	<li>Repeat step 3.1 with scopolamine methylbromide.</li>
	<li>Process solution through a 0.22 &#181;m pore nylon or mixed cellulose esters syringe filter into a sterile sealed serum vial.</li>
	<li>Store at room temperature. Use within 24 hr.</li>
</ol>
4. Track Turtle Movements Using Radio Telemetry11, 12
<ol>
	<li>Move to the general location of the target animal, remaining at least 25 m from the animal. Using a directional Yagi antenna and setting the receiver gain at medium, scan the horizon to determine the rough direction and location of the animal. When receiving interference or a weak signal, find a new position. Increase elevation or hold Yagi aloft when possible to enhance signal.</li>
	<li>Once a suitable location is found to take a bearing, record your position with a GPS.</li>
	<li>Using the null/peak method28, determine the bearings of the left and right nulls.
	<ol>
		<li>Identify the direction of the strongest signal. Turn the gain down as far as possible while still receiving a detectable signal. Use the attenuator switch on the receiver if so equipped. Move the antenna to the left and record the compass bearing at which the signal is lost.</li>
		<li>Repeat the previous step for the right bearing.</li>
	</ol>
	</li>
	<li>Repeat step 4.3 from at least two additional locations for the same animal. 
	NOTE: These additional points should be taken in such a manner as to surround the animal.
	<ol>
		<li>When a set of bearings is taken from a great distance or with unavoidable interference, take at least two additional bearings to increase accuracy. Collect sets of bearings used to estimate a single location as quickly as possible, particularly if the animal is moving. 
		NOTE: Alternatively, multiple personnel can coordinate to take multiple independent bearings on the same animal simultaneously.</li>
	</ol>
	</li>
	<li>Record locations of animals digitally or by hand every 10 - 15 min using above steps.</li>
	<li>Manipulation of cognitive processing
	<ol>
		<li>Once the unmanipulated animal&#39;s path is documented (a within-subjects control), provide a dose of either scopolamine or methylscopolamine. Using the mass collected in step 1.5, calculate the amount of drug to be given to the animal to achieve a reptile-specific final dose of 6.4 mg/kg of scopolamine or 6.8 mg/kg of methylscopolamine19, 20.</li>
		<li>Deliver the drug directly into the peritoneum via the caudal peritoneal sinus using a 1.0 ml syringe with a 22 gauge needle. Ensure that the total volume delivered does not exceed 1 ml.</li>
		<li>Release animal as soon as possible at its site of capture.</li>
		<li>Continue monitoring the animal&#39;s movement every 10 - 15 min until it reaches its projected destination.</li>
	</ol>
	</li>
</ol>
5. Spatial Analysis
<ol>
	<li>Calculate estimates of animal location.
	<ol>
		<li>For each set of null bearings, bisect the angle (by hand or via software) to find the resultant transmitter bearing. Repeat for all bearings at a given time point.</li>
		<li>Using triangulation software according to manufacturer&#39;s protocol, estimate the animal&#39;s position and associated error with the multiple transmitter bearings. Convert position estimates to x/y coordinates if software provides different output.</li>
	</ol>
	</li>
	<li>Repeat steps 5.1 for all sets of bearings.</li>
	<li>Calculate the spatial precision of movement.
	<ol>
		<li>Calculate the cumulative geometric mean (i.e., mean path) of unmanipulated animals (negative control) as they move towards their goal12.</li>
		<li>For each individual in the treatment and positive control groups, calculate the cumulative number of points that overlap successively larger swaths from the geometric mean line in 5 m intervals until 100% of all points have been included11, 12.</li>
		<li>Calculate the mean and standard error at each swath distance for all individuals in each group.</li>
		<li>Statistically analyze the data, comparing among the treatment groups either for the interval to accommodate 100% of the points or the points of overlap at a given interval, depending upon the question.</li>
	</ol>
	</li>
</ol>

<ol>
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The authors have nothing to disclose.

The protocol presented here allows the experimenter to document and quantify the role of cognition in navigation. Manipulating cognition in the field has proven difficult, as most approaches leave experimenters unable to know which specific aspects of the animal&#39;s behavior are being manipulated. However, the protocol presented here allows the experimenter to accurately manipulate and thus assess the role of cognition in navigation. The technique further allows experimenters to monitor animal navigation in real-time w...

Cognition (herein defined as &#34;all processes involved in acquiring, storing, and using information from the environment&#34; 1) is central to an array of complex navigation tasks2. For example, Sandhill cranes (Grus canadensis) show a marked improvement in migratory precision with experience3, and sea turtle species imprint on their natal beaches as hatchlings and return as adults4-6. Similarly, successful migration, dispersal, and foraging hinge on an animal&#39;s ability to gather information about their spatial environment7,8. Some animals appear to learn navigational routes in relation to specific landscape features9 and may use spatial cognition when moving between nesting and foraging areas10. Recent work on Eastern Painted turtles (Chrysemys picta) suggests a critical period in navigation, where successful navigation of upland habitat as adults hinges on juvenile experience within a narrow age range (&#60; 4 years old11-13). Though together these studies demonstrate the progress that has been made in understanding the role of learning in navigation4-6, 14-16, the mechanisms that underlie such behaviors and the full role of cognition in navigation remain enigmatic, especially in vertebrates8, 17, 18.
Field investigations into the role of cognition in navigation are rare2, 8, 18, due largely to the methodological difficulties involved in monitoring, manipulating, and tracking wild animals. For example, the large spatial and temporal scales on which many animals navigate often preclude investigating both type of information that those animals potentially learn and how that information is acquired. Experimenters often face the logistical difficulties of detecting and locating animals when monitoring behavior over such large areas and time frames, thereby limiting the type of data that can be collected and the conclusions that can be drawn. Although the use of animal-mounted global positioning system (GPS) recorders may improve the probability of detection of widely ranging animals, spatial data collected by these means are generally of very coarse resolution and lack a detailed behavioral component. Consequently, the data that can be collected under such circumstances are of limited value for examining subtle variation in behavior among different groups or experimental treatments. Similarly, the direct, controlled manipulation of target behaviors is often prohibited by the spatial and temporal scales typical of navigation behaviors, as well as by inherent logistical constraints of field studies. Finding animals in their natural habitat, catching and manipulating them, and then collecting behavioral data without inadvertently producing spurious behaviors are major challenges of working with animals in the field. Therefore, the design of experiments on free-ranging animals is often constrained and the ability to conduct rigorous, controlled field experiments on the role of cognition in navigation is limited.
The present study circumvents many of the previous difficulties of investigating the relationship between cognition and navigation in the field by using a novel combination of pharmacological manipulation and high-resolution tracking of freely navigating animals under field conditions. Scopolamine, a muscarinic acetylcholine receptor (mAChR) antagonist, has been shown to block spatial memory formation and recall by blocking cholinergic activity in the brains of a variety of vertebrate taxa18-24. Scopolamine can be used effectively on free-ranging animals under field conditions11, 18 and has a marked but temporary effect (e.g., 6 - 8 hr in reptiles). Methylscopolamine, a mAChR antagonist that does not cross the blood-brain-barrier19-21, can be used to control for the possible peripheral effects of scopolamine and for non-cognitive aspects of behavior11. Pharmacology allows for the precise manipulation of cognition by directly affecting receptors, and high-precision radio telemetry allows for the observation of the resulting effects on behavior. Measurements taken via remote triangulation with both high spatial (&#177; 2.5 m) and temporal (15 min) resolution allow for the precise documentation and quantification of animal behavior relative to the experimental manipulation of cognition.
This study11 was conducted between May and August 2013 and 2014 at Chesapeake Farms, a 3,300 acre wildlife management and agriculture research area in Kent Co., MD, USA (39.194&#176;N, 76.187&#176;W). The protocol involves five main steps: (1) capturing and handling animals (2) affixing radio transmitters (3) preparing the pharmacological agents (4) monitoring and manipulating animal movements, and (5) analyzing spatial data. The study described herein focused on the Eastern painted turtle (Chrysemys picta). Turtles in the focal population engage in annual overland movements in which they leave their home ponds and navigate to alternative aquatic habitats using one of four very precise (&#177; 3.5 m), complex, and highly predictable routes11, 12. Pharmacological manipulation of animals in this system paired with high-resolution radio telemetry sheds light on the role of cognition in freely navigating wild animals.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Scopolamine bromide</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;">S0929</td>
			<td style="width:167px;">USP</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Scopolamine methylbromide</td>
			<td>Sigma</td>
			<td>S8502, 1421009</td>
			<td style="width:167px;">USP and non USP versions</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Saline</td>
			<td>Hanna Pharmaceutical Supply Co., Inc.</td>
			<td>409488850</td>
			<td style="width:167px;">USP, formulated as an injectable&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Syringe filter</td>
			<td>Fisher</td>
			<td>09-720-004</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Syringe</td>
			<td>Fisher</td>
			<td>14-823-30</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Hypodermic needle</td>
			<td>Fisher</td>
			<td>14-823-13</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;">Antenna</td>
			<td>Wildlife Materials</td>
			<td>3 Element Folding Yagi</td>
			<td style="width:167px;">Antennae with additional elements are available, but can be cumbersome in the field.&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Radio Receiver</td>
			<td>Wildlife Materials</td>
			<td>TRX-2000S</td>
			<td style="width:167px;">Water resistant models are also available.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Compass</td>
			<td>Brunton&#160;</td>
			<td>Truarc 15</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="169">
			<td height="169" style="height:169px;">Radio transmitters</td>
			<td>Holohil Inc.</td>
			<td>BD-2, PD-2, RI-2B</td>
			<td style="width:167px;">Transmitter models vary in lifespan and signal output as a function of battery size and pulse rate settings, which can be customized based on the study question and organism.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">GPS</td>
			<td>Garmin</td>
			<td>eTrex Venture</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Coaxial cable</td>
			<td>newegg.com</td>
			<td>C2G 40026</td>
			<td style="width:167px;">BNC connections are necessary.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;">Hoop net</td>
			<td>Memphis Net and Twine&#160;</td>
			<td>TN325</td>
			<td style="width:167px;">Net mesh size should be chosen based on the minimum size of the target animal.&#160;</td>
		</tr>
	</tbody>
</table>

using pharmacological manipulation and high-precision radio telemetry to study the spatial cognition in free-ranging animals

An animal&#39;s ability to perceive and learn about its environment plays a key role in many behavioral processes, including navigation, migration, dispersal and foraging. However, the understanding of the role of cognition in the development of navigation strategies and the mechanisms underlying these strategies is limited by the methodological difficulties involved in monitoring, manipulating the cognition of, and tracking wild animals. This study describes a protocol for addressing the role of cognition in navigation that combines pharmacological manipulation of behavior with high-precision radio telemetry. The approach uses scopolamine, a muscarinic acetylcholine receptor antagonist, to manipulate cognitive spatial abilities. Treated animals are then monitored with high frequency and high spatial resolution via remote triangulation. This protocol was applied within a population of Eastern painted turtles (Chrysemys picta) that has inhabited seasonally ephemeral water sources for ~100 years, moving between far-off sources using precise (&#177; 3.5 m), complex (i.e., non-linear with high tortuosity that traverse multiple habitats), and predictable routes learned before 4 years of age. This study showed that the processes used by these turtles are consistent with spatial memory formation and recall. Together, these results are consistent with a role of spatial cognition in complex navigation and highlight the integration of ecological and pharmacological techniques in the study of cognition and navigation.

Using the above protocol, the role of cognition in navigation was assessed in a population of Eastern painted turtles (Chrysemys picta) that has experienced seasonal ephemeral water sources for ~100 years. This population inhabits a mix of ephemeral (drained annually and rapidly - in several hr) and permanent aquatic habitats (Figure 1). Previous studies suggest that after their ponds are drained, resident turtles navigate to alternative water sources with high p...

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Using Pharmacological Manipulation and High-precision Radio Telemetry to Study the Spatial Cognition in Free-ranging Animals

This paper describes a novel protocol that combines the pharmacological manipulation of memory and radio telemetry to document and quantify the role of cognition in navigation.

An animal&#39;s ability to perceive and learn about its environment plays a key role in many behavioral processes, including navigation, migration, ...

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6.7K Views.  Franklin and Marshall College. This paper describes a novel protocol that combines the pharmacological manipulation of memory and radio telemetry to document and quantify the role of cognition in navigation.

Video: Using Pharmacological Manipulation and High-precision Radio Telemetry to Study the Spatial Cognition in Free-ranging Animals

The Use of an Automated System (GreenFeed) to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during storage. These processes are the major sources of greenhouse gas (GHG) emissions from animal production systems. Techniques for measuring enteric CH4 vary from direct measurements (respiration chambers, which are highly accurate, but with limited applicability) to various indirect methods (sniffers, laser technology, which are practical, but with variable accuracy). The sulfur hexafluoride (SF6)&#160;tracer gas method is commonly used to measure enteric CH4 production by animal scientists and more recently, application of an Automated Head-Chamber System (AHCS) (GreenFeed, C-Lock, Inc., Rapid City, SD), which is the focus of this experiment, has been growing. AHCS is an automated system to monitor CH4 and carbon dioxide (CO2) mass fluxes from the breath of ruminant animals. In a typical AHCS operation, small quantities of baiting feed are dispensed to individual animals to lure them to AHCS multiple times daily. As the animal visits AHCS, a fan system pulls air past the animal&#8217;s muzzle into an intake manifold, and through an air collection pipe where continuous airflow rates are measured. A sub-sample of air is pumped out of the pipe into non-dispersive infra-red sensors for continuous measurement of CH4 and CO2 concentrations. Field comparisons of AHCS to respiration chambers or SF6 have demonstrated that AHCS produces repeatable and accurate CH4 emission results, provided that animal visits to AHCS are sufficient so emission estimates are representative of the diurnal rhythm of rumen gas production. Here, we demonstrate the use of AHCS to measure CO2 and CH4 fluxes from dairy cows given a control diet or a diet supplemented with technical-grade cashew nut shell liquid.

The Use of an Automated System GreenFeed to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Accuracy and precision of the techniques used to measure methane emissions from ruminant animals are critically important for the success of greenhouse gas mitigation efforts. This manuscript describes the principles and operation of an automated system to monitor methane and carbon dioxide mass fluxes from the breath of ruminant animals.

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Experimental Study of the Relationship Between Particle Size and Methane Sorption Capacity in Shale

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard for evaluating the mining value of shale gas. Currently, studies on the correlation between particle size and methane adsorption are controversial. In this study, an isothermal adsorption apparatus, the gravimetric sorption analyzer, is used to test the adsorption capacity of different particle sizes in shale to determine the relationship between the particle size and the adsorption capacity of shale. Thegravimetric method requires fewer parameters and produces better results in terms of accuracy and consistency than methods like the volumetric method. Gravimetric measurements are performed in four steps: a blank measurement, preprocessing, a buoyancy measurement, and adsorption and desorption measurements. Gravimetric measurement is presently considered to be a more scientific and accurate method of measuring the amount of adsorption; however, it is time-consuming and requires a strict measuring technique. A Magnetic Suspension Balance (MSB) is the key to verify the accuracy and consistency of this method. Our results show that adsorption capacity and particle size are correlated, but not a linear correlation, and the adsorptions in particles sieved into 40 - 60 and 60 - 80 meshes tend to be larger. We propose that the maximum adsorption corresponding to the particle size is approximately 250 &#181;m (60 mesh) in the shale gas fracturing.

We use an isothermal adsorption apparatus, the gravimetric sorption analyzer, to test the adsorption capacity of different particle sizes of shale, in order to find out the relationship between particle size and the adsorption capacity of shale.

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard ...

Measuring Stolons and Rhizomes of Turfgrasses Using a Digital Image Analysis System

Length and diameter of stolons or rhizomes are usually measured using simple rulers and calipers. This procedure is slow and laborious, so it is often used on a limited number of stolons or rhizomes. For this reason, these traits are limited in their use for morphological characterization of plants. The use of digital image analysis software technology may overcome measurement errors due to human mistakes, which tend to increase as the number and size of samples also increase. The protocol can be used for any kind of crop but is particularly suitable for forage or grasses, where plants are small and numerous. Turf samples consist of aboveground biomass and an upper soil layer to the depth of maximum rhizome development, depending on the species of interest. In studies, samples are washed from the soil, and stolons/rhizomes are cleaned by hand before analysis by digital image analysis software. The samples are further dried in a laboratory heating oven to measure dry weight; therefore, for each sample, the resultant data are total length, total dry weight, and average diameter. Scanned images can be corrected before analysis by excluding visible extraneous parts, such as remaining roots or leaves not removed with the cleaning process. Indeed, these fragments normally have much smaller diameters than stolons or rhizomes, so they can be easily excluded from analysis by fixing the minimum diameter below which objects are not considered. Stolon or rhizome density per unit area can then be calculated based on sample size. The advantage of this method is quick and efficient measurement of the length and average diameter of large sample numbers of stolons or rhizomes.

A software-based image analysis system provides an alternative method to study the morphology of stoloniferous and rhizomatous species. This protocol permits measurement of the length and diameter of stolons and rhizomes and can be applied to samples with a large amount of biomass and to a wide variety of species.

Length and diameter of stolons or rhizomes are usually measured using simple rulers and calipers. This procedure is slow and laborious, so it is often ...

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less attention has been paid on how environmental factors affect these insects when they pupate within the soil. The tea looper, Ectropis grisescens Warren (Lepidoptera: Geometridae), is a severe pest of tea plants and has caused huge economic losses in South China. The protocols described here aim to investigate, through multiple-choice bioassays, whether mature last-instar E. grisescens larvae can discriminate soil variables such as the substrate type and moisture content, and determine, through no-choice bioassays, the impact of the substrate type and moisture content on pupation behaviors and the emergence success of E. grisescens. The results would enhance the understanding of the pupation ecology of E. grisescens and may bring insights into soil-management tactics for suppressing E. grisescens populations. In addition, these bioassays can be modified to study the influences of various factors on the pupation behaviors and survivorship of soil-pupating pests.

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Here, we present a protocol to investigate the pupation preference of mature larvae of Ectropis grisescens in response to soil factors (e.g., substrate type and moisture content) using choice bioassays. We also present a protocol of no-choice bioassays to determine the factors that affect the pupation behaviors and survivorship of E. grisescens.

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less ...

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to individually track cohoused animals. We tested the tracking routine with four cohoused Norway lobsters (Nephrops norvegicus) under light-darkness conditions for 5 days. The lobsters had been individually tagged. The experimental setup and the tracking techniques used are entirely based on the open source software. The comparison of the tracking output with a manual detection indicates that the lobsters were correctly detected 69% of the times. Among the correctly detected lobsters, their individual tags were correctly identified 89.5% of the times. Considering the frame rate used in the protocol and the movement rate of lobsters, the performance of the video tracking has a good quality, and the representative results support the validity of the protocol in producing valuable data for research needs (individual space occupancy or locomotor activity patterns). The protocol presented here can be easily customized and is, hence, transferable to other species where the individual tracking of specimens in a group can be valuable for answering research questions.

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster Nephrops norvegicus

Here we present a protocol to individually track animals over a long period of time. It uses computer vision methods to identify a set of manually constructed tags by using a group of lobsters as case study, simultaneously providing information on how to house, manipulate, and mark the lobsters.

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Automated 3D Optical Coherence Tomography to Elucidate Biofilm Morphogenesis Over Large Spatial Scales

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm morphogenesis, that is the structural diversification of biofilms during community assembly, represents a remarkable challenge across spatial and temporal scales. Here, we present an automated biofilm imaging system based on optical coherence tomography (OCT). OCT is an emerging imaging technique in biofilm research. However, the amount of data that currently can be acquired and processed hampers the statistical inference of large scale patterns in biofilm morphology. The automated OCT imaging system allows covering large spatial and extended temporal scales of biofilm growth. It combines a commercially available OCT system with a robotic positioning platform and a suite of software solutions to control the positioning of the OCT scanning probe, as well as the acquisition and processing of 3D biofilm imaging datasets. This setup allows the in situ and non-invasive automated&#160;monitoring of biofilm development and may be further developed to couple OCT imaging with macrophotography and microsensor profiling.

Microbial biofilms form complex architectures at interphases and develop into highly scale-dependent spatial patterns. Here, we introduce an experimental system (hard- and software) for the automated acquisition of 3D optical coherence tomography (OCT) datasets. This toolset allows the non-invasive and multi-scale characterization of biofilm morphogenesis in space and time.

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

Manipulation of Color Patterns in Jumping Spiders for Use in Behavioral Experiments

In the field of behavioral ecology, many experiments are designed to investigate the evolutionary purposes of colorful traits in the context of sexual selection and predation. Methods are various but mostly consist of modifying the color patterns of individuals with diverse colorants. Such techniques have been used across many vertebrate taxa, particularly in birds, but have remained underdeveloped for invertebrates because of the difficulty of effectively manipulating color in small organisms. Instead, to manipulate the appearance of invertebrates, scientists have usually modified the lighting environment to filter out certain wavelengths. However, such a method affects not only the phenotypic trait of interest but the entire appearance of the individual and its surrounding. Here, scaling down the techniques previously used on colorful birds, we present ways of manipulating the colors of small arthropods, using equally emblematic but understudied species: the colorful jumping spiders.

The goal of this protocol is to manipulate the color patterns of jumping spiders and other very small arthropods with paint in order to study questions related to sexual selection, sexual cannibalism, predation, aposematism, or any other field of animal coloration.

In the field of behavioral ecology, many experiments are designed to investigate the evolutionary purposes of colorful traits in the context of sexual ...