The authors thank the three anonymous reviewers for their helpful comments on the manuscript. This work was partly supported by Grant-in-Aid for JSPS Fellows (26.11613).

NOTE: This article is based on our previous work30. Some parts are reprinted with permission from The Botanical Society of Japan and Springer Japan.
1 . Observation of Floral Visitors
<ol>
	<li>Selection of the Observation Fields
	<ol>
		<li>Search the areas where the plant materials are distributed, and select the candidate study sites using reliable resources, such as picture books, academic journals, etc. Adjust the number of study sites to fit the research objectives (e.g., a wide range of locations for the comparison of flower visitors throughout Japan).</li>
		<li>Check the locations of selected candidate populations and the distances from relevant starting points, such as research institutes and accommodations, if long-term research is needed.</li>
		<li>Roughly estimate each population size by counting the number of plant individuals in a unit area. Select large populations for manipulated experiments using many plant individuals.</li>
		<li>Pre-observation of floral visitors
		<ol>
			<li>Decide which target individuals in each area will be observed during the same time interval, depending on the population densities. Select 5-10 freshly-opened flowers or breaking buds as target flowers, and reject degraded ones that have already opened.</li>
			<li>Start the visual observation for the period of an entire day, because the flowers could be visited by various pollinators in different time ranges31,32.</li>
			<li>On the recording sheets, record the species name of floral visitors and the time of each visitation per hr per flower.</li>
			<li>Closely observe whether floral visitors touch the anthers and/or stigmata; if they do, record the visitors as pollinators. If the floral visitors only visit the flowers and do not touch the reproductive organs, record them as visitors.</li>
			<li>Using an insect net or hand-made aspirator, capture the floral visitors to identify and preserve them as specimens (Figure 1f). Kill the trapped visitors quickly with ethyl acetate or finger pressure to the thorax, and preserve them in a soft bag or in a plastic tube or case with 100% ethanol.</li>
			<li>After the observation, identify the specific names of specimens using morphological features or, in case of difficulties in identification, request the help of experts for each taxonomic group.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Observation in the Field
	<ol>
		<li>Select research sites based on their accessibility, population size, and number of floral visitors. Choose time periods that include a representative sample of floral visitors in the flowering season, and determine observation periods based on research objectives (e.g., for research on visitation-frequency fluctuations by floral visitors, long time periods should be set).</li>
		<li>Prepare the appropriate recording equipment, such as digital video cameras (e.g., for night recording, video cameras with an infrared function should be used).</li>
		<li>Select the target flowers in the same way as for 1.1.4.</li>
		<li>Fix the video cameras to aluminum tripods. Set the cameras with tripods in front of the target individuals, approximately 50 cm away.</li>
		<li>Check and modify the quantity of light and the focus of the materials on the screen displays of the cameras before filming.</li>
		<li>Start the visual observation and video filming at the same time for the appropriate time zones estimated by the pre-observations (in the present case, observation periods were for more than six hours on average, from approximately 05:00 am to 13:00 pm).</li>
		<li>Identify the species names of the visitors and pollinators, and record their names and the times of their visits on the recording sheets.</li>
		<li>Capture and identify the visitors and pollinators in the same way as in 1.1.4.5.</li>
		<li>Repeat Steps 1.2.1 to 1.2.7 for each observation period. Set the observation periods based on the research objectives and pre-observation data.</li>
	</ol>
	</li>
	<li>Data Analyses After Observation
	<ol>
		<li>Identify the species names of captured specimens after pollinator observation in the same way as in 1.1.4.6.</li>
		<li>Check the video clips and note the species names and their visit times in the same way as for the visual observation.</li>
		<li>Calculate the visitation frequencies per flower per hour of each floral visitor from the visual observation and video recording data. Compare these frequencies statistically between the sites and years of each visitor using appropriate statistical methods based on fundamental statistics and using suitable software, such as R, SPSS, and/or SAS33-35 (e.g., a two-way analysis of variance (ANOVA) with Tukey&#39;s honest significant difference (HSD) test in R software).</li>
	</ol>
	</li>
</ol>
2 . Bagging and Cage Experiments
<ol>
	<li>Preparations for the Bagging and Cage Experiments
	<ol>
		<li>For the bagging experiments, prepare bags that have small mesh sizes (~0.5-1 mm) and use them to completely prevent flower visitation (e.g., non-woven fabric bags were used for the bagging experiments in 2011 and 2012).</li>
		<li>For the cage experiments, prepare wire or plastic mesh boards with a diameter that allows the target smaller visitors to pass through but excludes the larger ones, and connect these mesh boards to form a cage. Ensure that there are no gaps larger than the mesh diameter. Adjust the size and shape of cages based on the target plant species and individual numbers.</li>
	</ol>
	</li>
	<li>Bagging Experiments
	<ol>
		<li>Select 30 individuals per treatment that have no damage from herbivores or from a severe environment. For treatment, choose a single flower from each individual plant, or use individual flowers on a single plant.</li>
		<li>Bag the target flowers in the field after labeling them with tape (serial number and alphabet; Figure 1g). Be careful not to touch the anthers or the stigmata within the bags to avoid the possibility of self-pollination.</li>
		<li>Carefully fix bags to the flowers using a soft string or wire (Figure 1d).</li>
		<li>Carefully set individual flowers upright with supports using soft string or wire to support the individuals against tilt or collapse under the weight of the bags or the wind, as necessary. Twist or wind a string or wire softly around the stalk of a target individual, so as not to create damage.</li>
		<li>Treatments in the bagging experiments
		<ol>
			<li>For the &#34;Control&#34; treatment, attach the labels to the target flowers and conduct no treatment. Allow floral visitors to freely visit.</li>
			<li>For the &#34;Outcrossing&#34; treatment, cover the buds until they flower, and then remove the anthers of the bagged flowers. Put some pollen grains from multiple individuals on the stigmata.</li>
			<li>For the &#34;Selfing&#34; treatment, cover the buds until the flowers open, and put pollen from the same flowers onto their stigmata. Cover these treated flowers again.</li>
			<li>For the &#34;Auto-self&#34; treatment, cover the buds with bags until the end of the flowering season.</li>
			<li>For the &#34;Breaking bud&#34; treatment, identify the breaking buds that have been visited and observe the entrance or exit of small bees (Figures 1b, c). Remove the anthers of the breaking buds to prevent repeated pollen depositions on the same flower after visitation by small bees, and bag these buds quickly to prevent further visitations.</li>
			<li>For the &#34;Flowering&#34; treatment, bag the buds until the opening stage to avoid visitations at the breaking-bud stage. After that, remove the bags and allow the visitors to collect nectar and pollen.</li>
			<li>For the &#34;Bud&#34; treatment, remove the anthers from unopened buds and outcross them artificially. Bag these buds quickly to prevent flower visitations.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cage Experiments
	<ol>
		<li>Cover the target plant individuals with prepared cages. Adjust the positions of caged flowers by hand to prevent contact and pollen depositions between different stigmata (Figure 1e).</li>
		<li>For the &#34;Cage&#34; treatment, select the individuals with unopened buds and put labels on them for the identification of selected flowers. Cage the individuals with labeled buds to reject the influences from before the establishment of cages (i.e., floral insects cannot visit unopened buds, and only the effects of insects that visit after the placement of cages can be estimated).</li>
		<li>Attach the cages firmly to the ground using iron poles to prevent entry by visitors between the cage base and the ground.</li>
	</ol>
	</li>
	<li>Data Analyses After the Bagging and Cage Experiments
	<ol>
		<li>Collect all of the labeled flowers at the end of the flowering season (Figure 1g) by cutting and separating them from the maternal individuals. Preserve each labeled sample separately to prevent their contamination.</li>
		<li>Check for the presence or absence of fruit set on each labeled flower.Record the seed numbers of each fruit in case of setting fruit.</li>
		<li>Calculate the ratio of fruit numbers per flower (fruit-set ratio, defined as the number of fruits divided by the number of flowers) and of seed numbers per mature fruit (seed-set ratio, defined as the number of seeds divided by the number of ovules) using all of the recorded numbers.</li>
		<li>Statistically compare the fruit- and seed-set ratios between the treatments using appropriate methods and software, such as those listed in 1.3.3 (e.g., one-way ANOVA with Tukey&#39;s HSD or Fisher&#39;s exact test in R software33).</li>
		<li>Investigations of the materials using the results of the bagging experiments
		<ol>
			<li>To test the need for animal pollinators, statistically compare the results between &#34;Control&#34; and &#34;Auto-self&#34; treatments.</li>
			<li>To estimate the degree of pollen limitation, compare the &#34;Control&#34; and &#34;Outcrossing&#34; treatments.</li>
			<li>To test self-compatibility, compare the &#34;Outcrossing&#34; and &#34;Selfing&#34; treatments.</li>
		</ol>
		</li>
		<li>Evaluations of the influences of breaking-bud pollination
		<ol>
			<li>Compare the &#34;Control&#34; and &#34;Bud&#34; treatments to determine whether the stigmata of the breaking buds are reproductively mature and whether the value of the &#34;Control&#34; treatment can be used for the control at the breaking-bud stage.</li>
			<li>Compare the &#34;Auto-self&#34; and &#34;Breaking bud&#34; treatments to determine whether the reproductive success of flowers pollinated with small bees at the breaking-bud stage is statistically higher than that of plants performing autogamy (i.e., to test the existence of breaking-bud pollination). After that, compare these two treatments with the &#34;Control&#34; to estimate the pollination efficiencies of breaking-bud pollination.</li>
			<li>Compare the &#34;Breaking bud,&#34; &#34;Cage,&#34; and &#34;Flowering&#34; treatments to estimate the reproductive influences of breaking-bud pollination.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

Flower observations and bagging experiments were employed in this study to reveal the visitation frequencies and the female reproductive success of plants, respectively. In Dafni (1992)38, the videotape method was effective because it could record the timing and duration of visitors for analysis and prevent observer bias. However, at the time, this method required expensive equipment, and the observation times were limited by battery life. Recently, the cost of equipment for producing video records has decline...

Plant-pollinator interactions are prime examples for the study of evolutionary biology and ecology. The mutualistic relationships between flowers and pollinators are thought to have promoted the diversification of angiosperm1,2 as a result of natural selection, although other biotic and abiotic factors have also exerted influence3,4,5. It is also thought that floral traits have changed to adapt to the most effective pollinators and to produce more fruit and seeds6. These beliefs have been constructed though large studies based on different indices, such as pollination effectiveness, that involve various interpretations7.
Flowering plants that have generalized pollination systems are visited by various types of pollinators8. Herein, a flower visitor was defined as an animal species that visited to get a floral reward, and pollinators were defined as floral visitors that pollinate. Some of these visitors carry conspecific pollen grains to the stigmata of the flowers visited and can be classified as pollinators. Other visitors may also have some intraspecific pollen; they might conduct less pollination due to behavioral or morphological mismatches between the pollinators and the flowers. These comparable differences in the contribution to plant reproduction could produce varying degrees of selective pressure on floral traits9 and could cause the adaptive divergence of flowering plants. Therefore, although the composition of the pollinator community and the relative species abundance are important10, the accurate evaluation of each visitor&#39;s effectiveness is also critical to determine the adaptive and/or evolutionary processes of the plants.
In this study, quantitative estimations of pollinator efficiencies, defined as the fruit and seed production per visitation frequency, were determined11. The species and frequency of each floral visitor were observed, and reproductive effects on the visited flowers were estimated. The recording of floral visits through human observations is a classical method in pollination ecology. However, this method imposes a large burden on observers, who are required to remain in front of the plants and to take careful, long-term measurements. Recently, the technologies of filming and recording have rapidly developed, and low-cost digital video cameras have enabled the introduction of video recording to pollinator observations12,13. These methods can facilitate the gathering of basic information on floral visitors and could help to develop an understanding of the pollination ecology of a target plant species.
However, the visitation frequencies of the pollinators are not necessarily correlated to their pollination effectiveness7,14, and it is important to evaluate the qualitative effects of each pollinator on flower fitness. Pollination effectiveness has been estimated through the number of pollen grains on the stigmata15,16, pollen tube growth17,18and fruit and/or seed production19,20. Bagging experiments, conducted using visitor-exclusive bags, are the typical methods for testing self-compatibility, autogamy21,22, and the presence of apomixes23. Additionally, the evaluation of pollination effectiveness for a certain pollinator in the visitor assemblage has been frequently conducted in environments where other floral visitors have been restricted (i.e., a wire cage, net, or bag with a mesh small enough to exclude larger pollinators that is set on flowering plants). For example, bagging experiments with small mesh bags were conducted to reveal the pollination ability of ants or thrips24,25. Moreover, bird exclusion experiments using a wire cage or net have shown the effective pollinators of the Aloe taxa26-28.
The objectives of this study were: 1) to introduce the methods used in a previous paper and 2) to improve these methods for general use in other studies on floral visitors, their visiting frequencies, and their effects on plant fitness. Lycoris sanguinea var. sanguinea is one of the species included in the genus Lycoris, which is distributed broadly throughout Japan and narrowly in Korea29and has funnel-shaped reddish-orange flowers (Figure 1a). A previous study revealed that L. sanguinea var. sanguinea was visited by multiple insect species, including an unidentified small bee species and the larger species Amegilla florea29. However, the visitation frequencies and pollination effectiveness of these visitors were not identified. Pollinator observations for the identification of floral visitors were performed first. Visitation by small bees was observed on flowers that had not completely opened yet (breaking buds; Figures 1b, c). Small bees moved hurriedly around the undehisced anthers in the breaking buds and collected pollen using their mandibles. The hypothesis was that the small bees could be pollinators at the breaking-bud stage because the gaps between the anthers and the stigmata in the flowers were smaller than the body length of the bees. Therefore, bagging experiments were conducted to test the pollination ability of small bees at the breaking-bud stage, and additionally to examine the reproductive strategies of L. sanguinea var. sanguinea. These buds were bagged after the small bees visited, which allowed an estimation of the pollination ability of the bees. The individuals were caged with unopened buds. A small-mesh cage was used, through which only small bees could pass, allowing an estimation of the pollination efficiencies of small bees throughout the entire flowering stage.

<table border="0" cellpadding="0" cellspacing="0" width="535">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:200px;">recording sheet</td>
			<td style="width:159px;">any</td>
			<td style="width:112px;">NA</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">insect net</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pooter</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ethyl acetate</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% Ethanol</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">plastic tube</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">plastic case</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">soft bag</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">digital video camera(s)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tripod(s)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">bags</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">wire or plastic mesh boards</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iron wires</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">labeling tape</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">stick supporters</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">soft strings or wire</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pincette(s)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

field experiments of pollination ecology: the case of lycoris sanguinea var. sanguinea

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify the pollination effectiveness of each pollinator for visited flowers. Pollinator observations have been one of the most common methods to identify pollinators, and bagging and cage experiments have been conducted to show the effectiveness of specific pollinators. In a previous study of Lycoris sanguinea var. sanguinea, its effective pollinators, the visitation frequencies of each floral visitor, and its reproductive strategies were not identified. This study reports the observation that small bees visited flowers that were partially opened (breaking buds). To the best of our knowledge, this phenomenon has not been reported previously. Further, this study investigates the hypothesis that small bees can pollinate at that flowering stage. This study demonstrates the basic methods of field experiments in pollination ecology using L. sanguinea var. sanguinea. Pollinator observations and digital video showed the visitation frequencies of each floral visitor. Bagging and cage experiments revealed that these flowers could be pollinated fully and that breaking-bud pollination could be important for the pollination of this plant species. The advantages and disadvantages of each method are discussed, and recent developments, including laboratory experiments, are described.

Five populations were selected for pollinator observations. In the pre-observation phase, visitations of various insect species to opening flowers and small bees to breaking buds were confirmed. Floral visitor observations revealed that most of the visitors to all five study sites were individuals of the small bee species Lasioglossum japonicum. The total visitation record showed that the visitation ratios of this species were more than 90% at three sites (Figure 2</stron...

Watch this Scientific Journal Video about Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea at JoVE.com

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

To reveal the pollinator effectiveness of a given plant species, multiple methods of field experiments have been developed. This study demonstrates the basic methods of field experiments for pollination ecology using the case study of Lycoris sanguinea var. sanguinea and the novel pollination mechanism, breaking-bud pollination.

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify ...

field-experiments-pollination-ecology-case-lycoris-sanguinea-var

Research

JoVE Journal

Environment

11.4K Views.  Chiba University. To reveal the pollinator effectiveness of a given plant species, multiple methods of field experiments have been developed. This study demonstrates the basic methods of field experiments for pollination ecology using the case study of Lycoris sanguinea var. sanguinea and the novel pollination mechanism, breaking-bud pollination.

Video: Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

Modifying the Bank Erosion Hazard Index (BEHI) Protocol for Rapid Assessment of Streambank Erosion in Northeastern Ohio

Understanding the source of pollution in a stream is vital to preserving, restoring, and maintaining the stream&#8217;s function and habitat it provides. Sediments from highly eroding streambanks are a major source of pollution in a stream system and have the potential to jeopardize habitat, infrastructure, and stream function. Watershed management practices throughout the Cleveland Metroparks attempt to locate and inventory the source and rate the risk of potential streambank erosion to assist in formulating effect stream, riparian, and habitat management recommendations. The Bank Erosion Hazard Index (BEHI), developed by David Rosgen of Wildland Hydrology is a fluvial geomorphic assessment procedure used to evaluate the susceptibility of potential streambank erosion based on a combination of several variables that are sensitive to various processes of erosion. This protocol can be time consuming, difficult for non-professionals, and confined to specific geomorphic regions. To address these constraints and assist in maintaining consistency and reducing user bias, modifications to this protocol include a &#8220;Pre-Screening Questionnaire&#8221;, elimination of the Study Bank-Height Ratio metric including the bankfull determination, and an adjusted scoring system. This modified protocol was used to assess several high priority streams within the Cleveland Metroparks. The original BEHI protocol was also used to confirm the results of the modified BEHI protocol. After using the modified assessment in the field, and comparing it to the original BEHI method, the two were found to produce comparable BEHI ratings of the streambanks, while significantly reducing the amount of time and resources needed to complete the modified protocol.

Modifying the Bank Erosion Hazard Index BEHI Protocol for Rapid Assessment of Streambank Erosion in Northeastern Ohio

Streambank erosion potential can be evaluated and ranked using David Rosgen&#8217;s Bank Erosion Hazard Index (BEHI), however this protocol has significant limitations. Here we present protocol modifications to address time constraints, allow nonprofessionals to complete accurate assessments, and account for non-alluvial stream conditions in Northeast Ohio.

Understanding the source of pollution in a stream is vital to preserving, restoring, and maintaining the stream&#8217;s function and habitat it provides. ...

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% fine dust formation within the EU15. But NH3 emissions also mean a considerable loss of nutrients. Many studies on NH3 emission from organic and mineral fertilizer application have been performed in recent decades. Nevertheless, research related to NH3 emissions after application fertilizers is still limited in particular with respect to relationships to emissions, fertilizer type, site conditions and crop growth. Due to the variable response of crops to treatments, effects can only be validated in experimental designs including field replication for statistical testing. The dominating ammonia loss methods yielding quantitative emissions require large field areas, expensive equipment or current supply, which restricts their application in replicated field trials. This protocol describes a new methodology for the measurement of NH3 emissions on many plots linking a simple semi-quantitative measuring method used in all plots, with a quantitative method by simultaneous measurements using both methods on selected plots. As a semi-quantitative measurement method passive samplers are used. The second method is a dynamic chamber method (Dynamic Tube Method) to obtain a transfer quotient, which converts the semi-quantitative losses of the passive sampler to quantitative losses (kg nitrogen ha-1). The principle underlying this approach is that passive samplers placed in a homogeneous experimental field have the same NH3 absorption behavior under identical environmental conditions. Therefore, a transfer co-efficient obtained from single passive samplers can be used to scale the values of all passive samplers used in the same field trial. The method proved valid under a wide range of experimental conditions and is recommended to be used under conditions with bare soil or small canopies (&#60;0.3 m). Results obtained from experiments with taller plants should be treated more carefully.

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

Ammonia emissions are a major threat to the environment by eutrophication, soil acidification and fine particle formation and stem mainly from agricultural sources. This method allows ammonia loss measurements in replicated field trials enabling statistical analysis of emissions and of relationships between crop development and emissions.

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% ...

Spotting Cheetahs: Identifying Individuals by Their Footprints

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges widely over sub-Saharan Africa and in parts of the Middle East. Cheetah conservationists face two major challenges, conflict with landowners over the killing of domestic livestock, and concern over range contraction. Understanding of the latter remains particularly poor2. Namibia is believed to support the largest number of cheetahs of any range country, around 30%, but estimates range from 2,9053 to 13,5204. The disparity is likely a result of the different techniques used in monitoring.
Current techniques, including invasive tagging with VHF or satellite/GPS collars, can be costly and unreliable. The footprint identification technique5 is a new tool accessible to both field scientists and also citizens with smartphones, who could potentially augment data collection. The footprint identification technique analyzes digital images of footprints captured according to a standardized protocol. Images are optimized and measured in data visualization software. Measurements of distances, angles, and areas of the footprint images are analyzed using a robust cross-validated pairwise discriminant analysis based on a customized model. The final output is in the form of a Ward's cluster dendrogram. A user-friendly graphic user interface (GUI) allows the user immediate access and clear interpretation of classification results.
The footprint identification technique algorithms are species specific because each species has a unique anatomy. The technique runs in a data visualization software, using its own scripting language (jsl) that can be customized for the footprint anatomy of any species. An initial classification algorithm is built from a training database of footprints from that species, collected from individuals of known identity. An algorithm derived from a cheetah of known identity is then able to classify free-ranging cheetahs of unknown identity. The footprint identification technique predicts individual cheetah identity with an accuracy of &gt;90%.

The cheetah (Acinonyx jubatus) is an iconic, endangered species, but conservation efforts are challenged by habitat shrinkage and conflict with commercial farmers. The footprint identification technique, a robust, accurate and cost-effective image classification system, is a new approach to monitoring cheetahs.

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges ...

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

Construction of a Compact Low-Cost Radiation Shield for Air-Temperature Sensors in Ecological Field Studies

Low cost temperature sensors are increasingly used by ecologists to assess climatic variation and change on ecologically relevant scales. Although cost-effective, if not deployed with proper solar radiation shielding, the observations recorded from these sensors will be biased and inaccurate. Manufactured radiation shields are effective at minimizing this bias, but are expensive compared to the cost of these sensors. Here, we provide a detailed methodology for constructing a compact version of a previously described custom fabricated radiation shield, which is more accurate than other published shielding methods that attempt to minimize shield size or construction costs. The method requires very little material: corrugated plastic sheets, aluminum foil duct tape, and cable ties. One 15 cm and two 10 cm squares of corrugated plastic are used for each shield. After cutting, scoring, taping and stapling of the sheets, the 10 cm squares form the bottom two layers of the solar radiation shield, while the 15 cm square forms the top layer. The three sheets are held together with cable ties. This compact solar radiation shield can be suspended, or placed against any flat surface. Care must be taken to ensure that the shield is completely parallel to the ground to prevent direct solar radiation from reaching the sensor, possibly causing increased warm biases in sun-exposed sites in the morning and afternoon relative to the original, larger design. Even so, differences in recorded temperatures between the smaller, compact shield design and the original design were small (mean daytime bias = 0.06 &#176;C). Construction costs are less than half of the original shield design, and the new design results in a less conspicuous instrument that may be advantageous in many field ecology settings.

With the advent of small, low-cost environmental sensors, it is now possible to deploy high-density networks of sensors to measure hyper localized temperature variation. Here, we provide a detailed methodology for constructing a compact version of a previously described custom-fabricated radiation shield for use with inexpensive thermochrons.

Low cost temperature sensors are increasingly used by ecologists to assess climatic variation and change on ecologically relevant scales. Although ...

Visualizing Efficacy of Pesticides Against Disease Vector Mosquitoes in the Field

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform across ecological zones. To best protect public and veterinary health from these insects, the environmental limitations of pesticides need to be investigated to inform effective use of the most appropriate pesticide formulations and techniques. We have developed a research program to evaluate combinations of pesticides, pesticide application equipment, and application techniques in hot-arid desert, hot-humid tropical, warm and cool temperate, and urban locations to derive pesticide use guidelines specific to target insect and environment. To these ends we designed a system of protocols to support efficient, cost-effective, portable, and standardized evaluation of a diverse range of pesticides and equipment across multiple environments. At the core of these protocols is the use of an array of small cages with colony-reared sentinel mosquitoes (adults and immatures) and sand flies (adults), strategically arranged in natural habitats and exposed to pesticide spray. Spatial and temporal patterns of pesticide efficacy are derived from percent mortality in sentinel cages, then mapped and visualized in a geographic information system. Maps of sentinel mortality data may be statistically compared to evaluate relative efficacy of a pesticide across multiple environments, or to study multiple pesticides in a single environment. Protocols may be modified to accommodate a variety of scenarios, including, for example, the vertical orientation of sentinels in canopy habitats or simultaneous testing of ground and aerial application methods.

The efficacy of public health pesticides targeting nuisance and disease-vector insects is not uniform across different ecological zones. Here we present a system of techniques using captive vector insects as sentinels for pesticide efficacy to derive electronic maps supporting the standard evaluation of pesticides across multiple environments.

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform ...

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 ...

Standard Operating Procedure for Lyssavirus Surveillance of the Bat Population in Taiwan

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are natural reservoirs of most lyssaviruses, a lyssavirus surveillance program of bats has been conducted in Taiwan since 2008 to understand the ecology of these viruses in bats. In this program, non-governmental bat conservation organizations and local animal disease control centers cooperated to collect dead bats or bats dying of weakness or illness. Brain tissues of bats were obtained through necropsy and subjected to direct fluorescent antibody test (FAT) and reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus antigens and nucleic acids. For the FAT, at least two different rabies diagnosis conjugates are recommended. For the RT-PCR, two sets of primers (JW12/N165-146, N113F/N304R) are used to amplify a partial sequence of the lyssavirus nucleoprotein gene. This surveillance program monitors lyssaviruses and other zoonotic agents in bats. Taiwan bat lyssavirus is found in two cases of the Japanese pipistrelle (Pipistrellus abramus) in 2016&#8211;2017. These findings should inform the public, health professionals, and scientists of the potential risks of contacting bats and other wildlife.

This protocol introduces a standard laboratory operating procedure for diagnostic testing of lyssavirus antigens in bats in Taiwan.

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are ...