The author would like to thank Katsuyuki Takahashi, Hiroo Kobayashi, Fumihiro Sato, Daikichi Ogiso, Toshihiro Hayakawa, and Hisaki Imai for teaching him the traditional wasp hunt method. The author would like to offer special thanks to Kevin J. Loope and Davide Santoro for carefully proofreading the manuscript. The author is grateful to Masato Abe, Yasukazu Okada, Yuichiro Kobayashi, Masakazu Shimada, and Koji Tsuchida for their discussion. The author wants to thank Yuya Shimizu and Haruna Fujioka for their technical assistance with evaluating colony productivity. The author would like to thank Tsukechi black bee club for supporting the video shooting. The author wishes to thank three anonymous reviewers for their comments on an early version of this paper. This study was supported in part by Takeda Science Foundation, Fujiwara Natural History Foundation, Funding of the Nagano Society for The Promotion of Science, Shimonaka Memories Foundation, Takara Harmonist Fund, and the Dream Project by Come on UP, Ltd.

1. Evaluation of Colony Productivity
<ol>
	<li>Estimation of the number of cells per comb
	<ol>
		<li>Separate the combs one by one. Sweep away all adult wasps from the comb and pull out all larvae and pupae from the cells with tweezers.</li>
		<li>Measure the square measures of 10 randomly chosen cells per a comb, by using imaging software (e.g., Image J version 1.48, see http://imagej.nih.gov/ij/).
		<ol>
			<li>Take a picture with the scale reference so that all the cells are pictured from right above.</li>
			<li>Based on the actual length of the scale, convert all measured lengths to pixels.</li>
			<li>Measure the areas of the 10 cells in pixels and convert them to the actual areas.</li>
			<li>Calculate the average area of worker and queen cells.</li>
		</ol>
		</li>
		<li>Estimate the number of worker and queen cells by dividing the area of each comb by the average cell area per comb.</li>
	</ol>
	</li>
	<li>Counting the number of meconia for the evaluation of colony productivity
	<ol>
		<li>Count the number of meconia per 100 cells for each comb by carefully breaking the comb and examining meconia. 
		NOTE: This number of cells was determined here to be sufficient (the SE of the number of meconia per cell is within 0.05, see the representative results section). Meconia may have solidified into two or more layers in the cell (Figure 1).</li>
		<li>Calculate the average number of meconia per cell for these 100 cells.</li>
		<li>Calculate the total number of meconia for each comb (i.e., the number of individuals produced, the colony productivity), extrapolated from the estimated number of cells and the average number of meconia per cell for that comb.</li>
	</ol>
	</li>
</ol>
2. Finding Vespula Nests
<ol>
	<li>Baiting
	<ol>
		<li>Hang pieces of cuttlefish, freshwater fish, or chicken heart (approximately 10 g in total) on a tree branch at a height that can be easily reached by hand (Figure 2).</li>
		<li>Place these baits along a transect (e.g., along a road crossing a forest or along a river) at 50 to 100 stations, with at least 5 m between each station.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59044/59044fig2.jpg" /> 
Figure 2: Providing wasps with a flagged meat bait. (A) Baiting wasps with meat attached to the tip of a stick. (B) The piece of meat is tied with a thread to a plastic flag. (C) The wasp holds on the meat which is tied to the flag. Such &#8220;flagged&#8221; baits will increase the visibility of the flying forager. The photos in panels B and C were taken by Fumihiro Sato. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59044/59044fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Providing wasps with a &#8220;flagged&#8221; bait
	<ol>
		<li>Flag construction and attachment
		<ol>
			<li>Cut plastic (polyethylene) bags into strips of 3 - 5 mm in width and 15 cm in length by using a box cutter.</li>
			<li>Prepare 1.5 mm3 of chicken heart or cuttlefish on a bamboo skewer or thin branch (the diameter of the meat bait can be 1 - 2 mm, less than 15 mg for a V. shidai worker; Figure 2).</li>
			<li>Tie a thread to the flag (plastic strip, less than 10 mg) and then to meat bait, attaching it within 3 mm from the flag (this is called the &#8220;flagged&#8221; bait). Cut off the loose thread above the knot. 
			NOTE: Use extremely fine polyester thread normally used with sewing machines.</li>
		</ol>
		</li>
		<li>Presentation of the meat bait to a wasp 
		NOTE: A nest is found most efficiently by following wasps that return to the bait repeatedly within 4 min of leaving. This is because wasps that take the bait and return quickly have a nest nearby.
		<ol>
			<li>Paint a unique mark on each thorax to identify the wasps individually when they are biting the baits (preferable with water-based paint pens, see Table of Materials).</li>
			<li>Orient the flag with the thread under the wasp while it bites the flagged bait when presenting the bait to the wasp (place the flag so that it and the thread pass under the wasp&#8217;s abdomen from below its thorax).</li>
		</ol>
		</li>
		<li>Following a marked wasp
		<ol>
			<li>Gather the baits from the surrounding area, so that the returning wasp is more likely to return to the same spot, before following a wasp. 
			NOTE: Following marked wasps is best accomplished with a group of two or more people. At least one person stays on at the transect, providing the foraging wasps with flagged baits, while the other(s) follow the marked wasp. When more than one wasp is attracted to the same bait, mark and follow only wasps that fly away in the same direction.</li>
			<li>Follow a wasp with a flagged bait.</li>
			<li>When a followed wasp lands somewhere on the way to its nest, gently lift the wasp with a long stick (branch) or fishing rod and watch it until it resumes flight. 
			NOTE: Be gentle and do not strike the resting wasp because it will drop the bait and fly away.</li>
			<li>When the wasp is shaping another meat ball before flying back to its nest again, readjust the flag, if necessary. 
			NOTE: Wasps will sometimes land and chew through the thread, removing the flag from the meat bait. If this happens frequently, make the flags shorter to increase forager flying ability.</li>
			<li>When a wasp escapes detection while being followed, wait for the wasp to return to the bait station on the transect before resuming the chase. This time, while the wasp is biting the new bait, carry the bait stick (and wasp) to the point where it had last escaped detection. 
			NOTE: Foraging wasps do not let go of their baits easily, and do not sting if handled gently. Hence, the wasp with the flagged bait can be moved to the desired location by holding the flag, without the wasp escaping.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Transfer of the Nest
<ol>
	<li>Structure of the carrying box
	<ol>
		<li>Construct nest boxes of various sizes, from 10 to 20 cm in length and width and from 10 to 20 cm in height, to accommodate nests of various sizes. 
		NOTE: Boxes of this size are big enough to accommodate young nests of V. shidai (collected in Central Japan between mid-July and mid-August). Make a carrying box according to the nest size of each species, for each growth stage.</li>
		<li>Construct the bamboo grid and attach it to the inside of the box, about 2 cm above the bottom of the box, to facilitate the placement of the nest inside the carrying box.</li>
		<li>Cover the bottom of the carrying box with newspaper and paste it to a wooden, removable board (Figure 3). 
		NOTE: The newspaper will, later, allow the wasps to chew through it as they build additional combs below the carrying box when this is placed in a nest box (see section 3.2).</li>
	</ol>
	</li>
	<li>Excavation of the nest
	<ol>
		<li>Before the exposure of the whole nest 
		NOTE: Wear protective clothing to avoid being stung by the wasps defending their nest.
		<ol>
			<li>Once the wasp nest is found, excavate the nest.</li>
			<li>Vigorously stamp on the ground around the nest for about 10 to 20 min so that workers leaving and returning to the nest remain inside to protect it, to collect as many workers as possible. 
			NOTE: If the wasps continue to stay outside the nest, it is better to capture them using an insect net. Although the stamping is useful for V. flaviceps, V. shidai, and V. vulgaris, other species&#8217; workers from the nest may attack the individual performing the stamping. In the case, skip this step.</li>
			<li>Shine a light directly into the nest entrance to determine the direction in which the nest entrance runs. Use a finger to confirm the orientation of the nest hole, while gently excavated soil from around the nest.</li>
		</ol>
		</li>
		<li>After the exposure of the whole nest
		<ol>
			<li>When the whole nest is exposed, spread a cloth and place the nest on top of it to prevent wasps from escaping into the ground under the nest.</li>
			<li>Place the excavated nest into a wooden (carrying) box for transportation to the lab (Figure 3); then, cover it with branches and newspaper. Leave the top of the nest uncovered while it is in the box.</li>
			<li>Place the carrying box on a cloth for 5 to 10 min, until the wasps become calm.</li>
			<li>Collect any wasps in the vicinity with an insect net and transport them to the laboratory with the nest. 
			NOTE: As an alternative collection procedure, anesthetize the nest occupants by fanning celluloid smoke or diethyl ether into the nest before excavating it.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59044/59044fig3.jpg" /> 
Figure 3: Carrying box. (A) Box for carrying nests collected in the field. (B) A bamboo grid is placed at on the bottom of the box. The two boxes in the image on the right are upside down. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59044/59044fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Rearing Vespula
<ol>
	<li>Structure of the nest box 
	NOTE: The nest box is made of wood, with dimensions of 50 cm in length and width and 70 cm in height for rearing V. shidai (a mature nest is approximately 40 cm in diameter in the wild). Make a nest box according to the nest size of the species to be reared.
	<ol>
		<li>Provide the nest box with an entrance hole (usually placed in the upper part of the box) to allow wasps to leave the nest to forage.</li>
		<li>Fill about 1/3 of the nest box with soil like that occurring at the location where the nest was collected.</li>
		<li>Install a wire mesh (with a mesh size of 1.5 cm2) at the entrance of the nest box to prevent any intrusion by other wasps (predators, such as Vespa mandarinia and Vespa simillima).</li>
		<li>Place two wooden bars in the nest box that can bear the carrying box (Figure 4).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59044/59044fig4.jpg" /> 
Figure 4: Laboratory setup. (A) Setting a carrying box into a nest box used for a long-term study. Before placing the carrying box in the nest box, the wood board at the bottom of the carrying box was removed, leaving only the newspaper to cover the bottom of the nest. (B) A series of nest boxes with food resources hanging from a wire line. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59044/59044fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Transplantation of the carrying box into the nest box
	<ol>
		<li>Keep the nest box in a dry place while rearing wasps in the collected nests (i.e., somewhere not exposed to rain).</li>
		<li>Remove the wooden board at the bottom of the carrying box and put it in the nest box for a long-term study (Figure 4). 
		NOTE: Often, wasps will have bitten holes in the newspaper covering the bottom of the carrying box, and so, there is a danger of being stung by wasps escaping through the holes. Therefore, wear protective clothing when transplanting the nest.</li>
	</ol>
	</li>
	<li>Feeding the wasps
	<ol>
		<li>Place various types of meat (squid, freshwater fish, chicken breast, or chicken heart) and a 1:3 solution of honey and water at approximately 3 m from the nest box.</li>
		<li>Provide enough food for the feeding requirements of 1 day. Replenish fresh food every day (Vespinae do not forage on old/rotting meat).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Davies, N. B., Krebs, J. R., West, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An introduction to Behavioural Ecology. , (2012).</li><li>Tibbetts, E. A., Reeve, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benefits+of+foundress+associations+in+the+paper+wasp+Polistes+dominulus:+increased+productivity+and+survival,+but+no+assurance+of+fitness+returns.">Benefits of foundress associations in the paper wasp Polistes dominulus: increased productivity and survival, but no assurance of fitness returns.</a> Behavioural Ecology. 14, 510-514 (2003).</li><li>Mattila, H. R., Seeley, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Diversity+in+Honey+Bee+colonies+Enhances+Productivity+and+Fitness.">Genetic Diversity in Honey Bee colonies Enhances Productivity and Fitness.</a> Science. 317, 362 (2007).</li><li>Weber, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gardening+Ants,+the+Attines.">Gardening Ants, the Attines.</a> American Philosophical Society. , (1972).</li><li>Baer, B., Schmid-Hempel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+influences+female+hibernation+success,+survival+and+fitness+in+the+bumble-bee+Bombus+terrestris.">Sperm influences female hibernation success, survival and fitness in the bumble-bee Bombus terrestris.</a> Proceedings: Biological Science. 272 (1560), 319-323 (2005).</li><li>Spradbery, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Wasps. An Account of the Biology and Natural History of Social and Solitary Wasps, with Particular Reference to Those of the British Isles. , (1973).</li><li>Matsuura, M., Yamane, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comparative Ethology of the Vespine Wasps. , (1984).</li><li>Greene, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+schedules+of+vespine+wasps:+an+empirical+test+of+the+bang-bang+optimization+model.">Production schedules of vespine wasps: an empirical test of the bang-bang optimization model.</a> Journal of Kansas Entomological Society. 57 (4), 545-568 (1984).</li><li>Cole, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+mating+and+the+evolution+of+social+behavior+in+the+Hymenoptera.">Multiple mating and the evolution of social behavior in the Hymenoptera.</a> Behavior Ecology Sociobiology. 12, 191-201 (1983).</li><li>Goodisman, M. A. D., Kovacs, J. L., Hoffman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+multiple+mating+in+the+social+wasps+Vespula+maculifrons.">The significance of multiple mating in the social wasps Vespula maculifrons.</a> Evolution. 61 (9), 2260-2267 (2007).</li><li>Greene, A., Ross, K. G., Matthews, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dolichovespula+and+Vespula.">Dolichovespula and Vespula.</a> The Social Biology of Wasps. , 263-305 (1991).</li><li>Yamane, S., Yamane, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+methods+of+dead+vespine+nests+(Hymenoptera,+Vespidae)+(Methods+of+taxonomic+and+bio-sociological+studies+on+social+wasps.+II).">Investigating methods of dead vespine nests (Hymenoptera, Vespidae) (Methods of taxonomic and bio-sociological studies on social wasps. II).</a> Teaching Materials for Biology. 12, 18-39 (1975).</li><li>Loope, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matricide+and+queen+sex+allocation+in+a+yellowjacket+wasp.">Matricide and queen sex allocation in a yellowjacket wasp.</a> The Science of Nature. 103 (57), 1-11 (2016).</li><li>Matsuura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Social Wasps of Japan in Color. , (1995).</li><li>Foster, K. R., Ratnieks, F. L. W., Gyllenstrand, N., Thoren, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colony+kin+structure+and+male+production+in+Dolichovespula+wasps.">Colony kin structure and male production in Dolichovespula wasps.</a> Molecular Ecology. 10 (4), 1003-1010 (2001).</li><li>Loope, K. J., Chien, C., Juhl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colony+size+is+linked+to+paternity+frequency+and+paternity+skew+in+yellowjacket+wasps+and+hornets.">Colony size is linked to paternity frequency and paternity skew in yellowjacket wasps and hornets.</a> BMC Evolutionary Biology. 14 (1), 1-12 (2014).</li><li>Nonaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultural+and+commercial+roles+of+edible+wasps+in+Japan.">Cultural and commercial roles of edible wasps in Japan.</a> Forest Insects as Food: Humans Bite Back. Proceedings of a workshop on Asia-Pacific resources and their potential for development. , 123-130 (2010).</li><li>Yamane, S., Funakoshi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unique+ecology+of+Vespula+shidai+amamiana+and+the+origin+of+distribution.">The unique ecology of Vespula shidai amamiana and the origin of distribution.</a> Ecological Society of Japan. Biodiversity of the Nansei Islands, its formation and conservation. , (2015).</li><li>. R: The R Project for Statistical Computing Available from: <a target="_blank" href="https://www.R-project.org/">https://www.R-project.org/</a> (2018)</li><li>Saga, T., Kanai, M., Shimada, M., Okada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutual+intra-+and+interspecific+social+parasitism+between+parapatric+sister+species+of+Vespula+wasps.">Mutual intra- and interspecific social parasitism between parapatric sister species of Vespula wasps.</a> Insectes Sociaux. 64 (1), 95-101 (2017).</li><li>Van Huis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Edible insects: future prospects for food and feed security. , (2013).</li></ol>

The author has nothing to disclose.

The colony productivity of bees, ants, and wasps has been estimated previously by the number of workers and cells in nests or by the weight of the nests3,9,10. This study shows that the estimate of the number of meconia provides a better estimate of the overall number of individuals produced (i.e., a better indicator of colony productivity). In fact, it was found that, for both worker and queen combs, the number of meconia ranged from 0.53 to 1.96 times the number of larval cells in the comb. These findings quantify how inaccurate the determination of the number of workers and queens produced can be when it is based on the number of cells in a comb. Despite being more labor-intensive, estimating the number of meconia in a nest seems to guarantee a more precise evaluation of colony productivity. On the other hand, in this study, it was not evaluated how accurately the number of meconia represents the number of individuals produced.
This paper shows how many cells of a V. shidai nest should be examined to estimate colony productivity, based on the results of a bootstrap simulation approach using sample data on the number of meconia in the nest. Based on these results, it would be appropriate to investigate 100 cells per comb of both worker and queen cells. The method for counting meconia can also be applied to a nest after it has collapsed (i.e., is inactive), which can be advantageous for researchers: the reproductive period of vespine wasp colonies is quite long8 and studying a nest after it has collapsed means that the total number of adults produced over the entire reproductive period can be estimated. Such colonies are also easier to collect.
To collect nests of V. shidai, some researchers have followed either marked (e.g., coated with fluorescent powder) or unmarked wasps21. The nest location method presented here (feeding the wasps &#34;flagged&#34; meat) facilitates following wasps to their nests. This approach is also helpful if a tracked wasp is lost because the same wasp will eventually return to the bait along the transect. Provide new flagged bait to this wasp and carry it to the point where it was last lost, thus allowing chasers to resume the chase from that point onward (closer to the nest). Some of the flags brought to the nest are dislodged at the nest entrance, which also facilitates finding ground nests. However, this method is not suitable for rainy days because markers tend to stick to branches and leaves when they get wet. Although chasing flagged wasps is useful for V. shidai, V. flaviceps, and V. vulgaris in Japan, this method could not be applied to Vespula rufa because these wasps do not come to the bait and do not grab flagged bait. The nest location method can probably not be used for some Vespula wasps.
More sustainable diets are needed by an ever-increasing global population. In addition, the demand for edible insects increases daily. Many edible insects, which are being consumed locally and traditionally throughout the world, have been identified by the Food and Agriculture Organization of the United Nations21 as a promising alternative protein source for overcoming food insecurity worldwide. Larvae and pupae of Vespula have traditionally been used as food in mountainous areas of Japan16, and so, they could be used to provide a source of protein elsewhere in the world. The set of protocols developed in this study is likely applicable to locating nests of other yellowjacket species. Therefore, the protocols outlined in this paper will be useful for collecting yellowjackets as an edible resource and studying wasp behavior.

Every species is thought to develop an optimal strategy for survival and reproduction among a vast array of possible strategies. In natural selection, individuals with traits that maximize an individual&#39;s reproductive success will leave more offspring (and genes) to the next generation. Therefore, the number of offspring produced by an individual can be used as an indicator of the individual&#39;s relative evolutionary fitness. In a given ecological context, the comparison of the number of offspring produced relative to alternative behavioral strategies can help researchers predict the best strategy for optimizing fitness1.
Social Hymenoptera (such as wasps, bees, and ants) have a system of three different castes, which are workers (sterile females), queens (gynes), and males1. Only new queens (gynes) and males count toward fitness in social Hymenoptera. Worker production does not directly contribute to fitness since the worker is infertile. On the other hand, a queen that can produce a higher colony productivity (such as a higher number of total cells or a heavier nest) is considered to have a higher fitness in social Hymenoptera, regardless of the number of actually produced new queens and males (see, e.g.,Tibbetts and Reeve2 and Mattila and Seeley3). In general, it is difficult to precisely count the number of offspring produced by a colony of social Hymenoptera. In fact, the queens of many social insects live for more than 1 year (e.g., leaf-cutter ant queens can live &#62;20 years4 and honeybee queens may live for 8 years5). In addition, one queen may produce thousands of reproductive offspring over the course of several weeks or months, even in annual species of genera Vespa and Vespula6,7,8. Furthermore, the lifespan of workers is shorter than that of their mother queen, and workers often die away from their nests. Hence, even if one could accurately count all adults in a nest at any given point in time, such a count would not accurately depict the number of offspring produced. Therefore, the number of offspring produced has been roughly estimated from the size of the nest, the number of workers in the nest, or the weight of the nest at a given point in time3,9,10. The number of larval cells could result in an overestimation of the offspring production when some cells are empty. The same method could also result in a potential underestimation of the offspring production because combs of small cells that contain worker brood can produce two or three cohorts of larvae6,7,11.
The first aim of this work is to provide an improved method for estimating vespine wasp colony productivity in terms of the number of adults produced. Yamane and Yamane suggested that the best way to estimate the number of offspring produced by a colony is to count the meconia in the nest12. The meconia is the fecal pellet comprising larval cuticle, gut, and gut contents that a larva leaves in its cell when pupating (Figure 1A). The total number of meconia produced per comb is calculated by multiplying the total number of cells present by the average number of meconia per cell. There are often multiple layers of meconia in a cell, and each meconia indicates that an individual successfully pupated in that cell6,11 (Figure 1B). When estimating the mean number of meconia per cell, if the number of cells examined is small (a small sample size), the standard error (SE) increases, and as a result, the error for the total number of meconia per comb becomes higher than if the sample size was larger. The SE of the mean (SEM) is a measure of the dispersion of sample means around the population mean. Therefore, in this study, I&#160;focus on the SEM of the number of meconia per cell to estimate the population (the number of adults produced) from the sample mean (the average number of meconia per cell). This study attempts to determine how many samples are required to obtain an SE rate of less than 0.05 per cell. To do this, a numerical simulation is performed with real data on the number of meconia per comb, to determine the minimum sample size (for both worker and queen combs) needed to estimate this value accurately within the defined SE of 0.05.
Vespine wasp colonies live in concealed nests (underground or aerial) composed of multiple horizontal combs, built in series from top to bottom6,7,11. The average size of the cells increases from the first (top) to the last (bottom) comb. In the bottom combs, a sudden shift in the average cell size can be seen. These wider cells are built for the development of new queens. Hence, a more accurate estimate of colony productivity (i.e., the number of individuals produced) can be obtained when the total number of meconia in the worker cells (small cells) and queen cells (large cells) are considered. In order to estimate fitness at the colony level, researchers could estimate the number of queens produced and focus on the meconia in the queen cells alone. As for reproductive males, these are reared either in worker or queen cells, depending on the species. Thus, it may be difficult to estimate the male production of a colony, except in species where males have a third, unique cell size13 (e.g., Dolichovespula arenaria).
The second aim of this work is to present a useful technique for locating wild vespine wasp colonies in the field and transplanting them into laboratory nest boxes. Although some researchers obtain wasp nests from pest control calls (i.e., people reporting them as pests14,15), this method is not always possible or desirable. Researchers might need to collect nests in wild and inhabited areas where pest controllers do not operate, or to conduct their research by more flexibly obtaining nests at specific times. Interestingly, people living in the mountainous areas of central Japan traditionally collect and rear wasps (Vespula shidai, Vespula flaviceps, and Vespula vulgaris) for food. Therefore, collecting and artificial rearing techniques for these wasps are well developed in those areas17.
This paper also summarizes the methods employed to rear Vespula wasps. The experimental organism for this study was V. shidai, a social, ground-nesting wasp inhabiting western Asia and Japan. V. shidai possesses the largest colony size among all Japanese vespine wasps, with a total of 8,000 to 12,000 cells per nest, with a maximum of 33,400 cells14,18. Workers of V. shidai have an average wet weight of 67.62 &#177; 9.56 mg. Males are usually reared in worker cells; in contrast, new queens are reared in specially constructed, wider queen cells14.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59044/59044fig1.jpg" /> 
Figure 1: Meconium in a larval cell. (A) Cross section of a comb of Vespula shidai. Meconia is indicated by red arrows. (B) Two meconia are layered. Each blue arrow indicates one meconium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59044/59044fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cuttlefish</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">fresh/ as a bait</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dace</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td>fresh/ as a bait</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chichken heart</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td>fresh/ as a bait</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic bag (polyethylene)</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td>as a flag</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bamboo skewer</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">industrial sewing thread</td>
			<td style="width:71px;">FUJIX Ltd.</td>
			<td style="width:119px;">King polyester, No.100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">paint marker pen</td>
			<td style="width:71px;">Mitsubishi pencil</td>
			<td style="width:119px;">UNI, POSCA, PC5M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fishing rod</td>
			<td style="width:71px;">ANY</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">carrying box</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>made of wood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">nest box</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>made of wood</td>
		</tr>
	</tbody>
</table>

evaluation of the productivity of social wasp colonies (vespinae) and an introduction to the traditional japanese vespula wasp hunting technique

For vespine wasps, colony productivity is typically estimated by counting the number of larval cells. This paper presents an improved method that enables researchers to estimate more accurately the number of adults produced, counting the number of meconia (the stools left in the cells by wasp larvae when pupating into adults, per 100 cells) in each comb. This method can be applied before or after colony collapse (i.e., in active or inactive nests). The paper also describes how to locate wild Vespula wasp colonies by &#34;flagging&#34; wasp baits and chasing the wasp collecting them, using a method traditionally performed by local people in central Japan (as illustrated in the associated video). The Vespula chasing method described has several advantages: it is easy to reinitiate the chase from a point where the forager flying back to the nest was lost, and it is easy to pinpoint the nest location as marked wasps often lose their flag at the nest entrance. These methods for estimating colony productivity and collecting nests can be valuable for researchers studying social wasps.

One goal of this study was to determine how many samples are required to obtain an SEM of the number of meconia per cell which is less than 0.05. In this study, a comb with an average cell size of &#60;20 mm2 was defined as a worker comb, whereas larger combs were defined as queen combs. I&#160;counted the number of cells for queen combs and worker combs (in this study, counts were made of six queen combs and six worker combs from five V. shidai colonies). The actual number of cells per comb was estimated from these data via extrapolation (Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ID</td>
			<td>State</td>
			<td>Collection date</td>
			<td>Area (mm2)</td>
			<td>Estimated number of cells (ENC)</td>
			<td>Actual number of cells (ANC)</td>
			<td>Actual number of meconium (ANM)</td>
			<td>Mean number of meconium in a cell</td>
			<td>ANM /ENC</td>
		</tr>
		<tr>
			<td>WW-Kb01</td>
			<td>Alive</td>
			<td>18-Oct-16</td>
			<td>27756.7</td>
			<td>1599.9</td>
			<td>1433</td>
			<td>2430</td>
			<td>1.70</td>
			<td>1.52</td>
		</tr>
		<tr>
			<td>WW-Kb02</td>
			<td>Alive</td>
			<td>18-Oct-16</td>
			<td>4098</td>
			<td>381.9</td>
			<td>347</td>
			<td>494</td>
			<td>1.42</td>
			<td>1.29</td>
		</tr>
		<tr>
			<td>WW-Kb02</td>
			<td>Alive</td>
			<td>18-Oct-16</td>
			<td>22439.3</td>
			<td>1118.9</td>
			<td>986</td>
			<td>1317</td>
			<td>1.34</td>
			<td>1.18</td>
		</tr>
		<tr>
			<td>WR-Ksb</td>
			<td>Collapse</td>
			<td>3-Nov-16</td>
			<td>19094.9</td>
			<td>1098.6</td>
			<td>1,181</td>
			<td>974</td>
			<td>0.82</td>
			<td>0.89</td>
		</tr>
		<tr>
			<td>WR-Ksc</td>
			<td>Collapse</td>
			<td>27-Nov-16</td>
			<td>38,933.40</td>
			<td>2,198.70</td>
			<td>2,455</td>
			<td>4,321</td>
			<td>1.76</td>
			<td>1.96</td>
		</tr>
		<tr>
			<td>WR-Kb05</td>
			<td>Collapse</td>
			<td>29-Nov-16</td>
			<td>10970</td>
			<td>860</td>
			<td>763</td>
			<td>1315</td>
			<td>1.72</td>
			<td>1.53</td>
		</tr>
		<tr>
			<td>QW-Kb01</td>
			<td>Alive</td>
			<td>18-Oct-16</td>
			<td>29186.2</td>
			<td>1094.4</td>
			<td>1095</td>
			<td>759</td>
			<td>0.69</td>
			<td>0.69</td>
		</tr>
		<tr>
			<td>QW-Kb01</td>
			<td>Alive</td>
			<td>18-Oct-16</td>
			<td>36920.5</td>
			<td>1361.6</td>
			<td>1341</td>
			<td>1075</td>
			<td>0.80</td>
			<td>0.79</td>
		</tr>
		<tr>
			<td>QW-Kb02</td>
			<td>Alive</td>
			<td>18-Oct-16</td>
			<td>37295.9</td>
			<td>1047.2</td>
			<td>1080</td>
			<td>1068</td>
			<td>0.99</td>
			<td>1.02</td>
		</tr>
		<tr>
			<td>QR-Ksb</td>
			<td>Collapse</td>
			<td>3-Nov-16</td>
			<td>24811.2</td>
			<td>1011.9</td>
			<td>893</td>
			<td>701</td>
			<td>0.78</td>
			<td>0.69</td>
		</tr>
		<tr>
			<td>QR-Ksc</td>
			<td>Collapse</td>
			<td>27-Nov-16</td>
			<td>33352.8</td>
			<td>1384.5</td>
			<td>1241</td>
			<td>1069</td>
			<td>0.86</td>
			<td>0.77</td>
		</tr>
		<tr>
			<td>QR-Kb05</td>
			<td>Collapse</td>
			<td>29-Nov-16</td>
			<td>25157.6</td>
			<td>1071.4</td>
			<td>922</td>
			<td>572</td>
			<td>0.62</td>
			<td>1.97</td>
		</tr>
		<tr>
			<td colspan="9">WW = a worker comb from a wild nest, WR = a worker comb from a rearing nest, QW = a queen comb from a wild nest, QR = a queen comb from a rearing nest. Alive = viable wasp larvae in cells, Collapse = no viable larvae in cells.</td>
		</tr>
	</tbody>
</table>
Table 1:&#160;The actual and estimated numbers of cells in six worker combs and six queen combs and the number of meconia per comb. WW = a worker comb from a wild nest, WR = a worker comb from a rearing nest, QW = a queen comb from a wild nest, QR = a queen comb from a rearing nest. Alive = viable wasp larvae in cells, Collapse = no viable larvae in cells.
An analysis of the relationship between the sample size and the SEM of the number of meconia per cell demonstrated that the sample size should be established using a bootstrap approach based on the number of meconia counted (from real data). Using real data, the mean and standard deviation (SD) of the number of meconia per cell were calculated, with the number of samples set at 1,000 for each sample size (the number of cells to be examined were 1 to 500; Figure 5). I&#160;did not allow for an iterative extraction from the data at sampling. The SEM for the number of meconia per cell was calculated for each sample size for each set of real data. Then, the sample size at which the SEM was less than 0.05 was examined. All calculations were made using software R.3.2.4.19 This analysis showed that the SEM was &#60;0.05 when the sample size was 100 cells (for both worker and queen combs) (Figure 5). Therefore, the following results are based on examining the number of meconia per 100 cells per comb.
The actual and estimated numbers of cells in six worker combs and six queen combs and the number of meconia per comb are shown in Table 1. The estimates of the number of cells in the worker combs, based on comb area measurements, were both higher and lower than the true count. The mean number of meconia in the cells of worker combs, which represents the number of workers produced, ranged from 1.96 times more than the number of estimated larval cells to 0.89 times less than the estimated number of cells (Table 1). In the queen combs, the actual number of cells was often less than the estimated number of cells. The number of meconia in the queen combs, which can represent a component of fitness (i.e., a part of the reproductive success of the founding queen), was 0.53 to 1.02 times the estimated number of cells.
All cells and meconia were counted in six randomly selected worker combs and six randomly selected queen combs from the five nests (Table 1). The total number of cells counted in the worker combs was 7,165, whereas the number of meconia counted in the worker combs was 10,851. The average number of cells per comb was 1,194.2 &#177; 720.3 (average &#177; SD), whereas the average number of meconia in the worker combs was 1,808.5 &#177; 1,368.2. In the queen combs, the total number of all cells was 6,572, whereas the number of all meconia was 5,244. The average number of cells per comb in the queen combs was 1,095.3 &#177; 174.820, whereas the average number of meconia was 874.0 &#177; 223.8. Meconium layers in worker cells ranged from zero to three, whereas the queen cells had either one or no meconium layer.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59044/59044fig5.jpg" /> 
Figure 5: The relationship between the sample size and the standard error (SE) relative to the number of meconia counts. (a) Meconia per cell in worker combs. (b) Meconia per cell in queen combs. Each circle depicts an SE relative to the number of meconia per cell obtained via simulation with actual data. Color differences represent the data from each sampled nest. Simulating the SE for the number of meconia per cell in comb WWkb02 (worker comb) was accomplished with a sample size of 300 because that comb only had 347 cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59044/59044fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of the Productivity of Social Wasp Colonies Vespinae and an Introduction to the Traditional Japanese Vespula Wasp Hunting Technique at JoVE.com

Evaluation of the Productivity of Social Wasp Colonies Vespinae and an Introduction to the Traditional Japanese Vespula Wasp Hunting Technique

This methodological paper evaluates the productivity of a social wasp colony by examining the number of meconia per 100 cells of comb, to estimate the total number of adults the wasps produced. The associated video describes how to search for Vespula wasp nests, a method developed by amateur wasp chasers.

Evaluation of the Productivity of Social Wasp Colonies (Vespinae) and an Introduction to the Traditional Japanese Vespula Wasp Hunting Technique

For vespine wasps, colony productivity is typically estimated by counting the number of larval cells. This paper presents an improved method that enables ...

evaluation-productivity-social-wasp-colonies-vespinae-an-introduction

Nanyang Technological University

Research

JoVE Journal

Environment

8.0K Views.  Tajimi Senior High School. This methodological paper evaluates the productivity of a social wasp colony by examining the number of meconia per 100 cells of comb, to estimate the total number of adults the wasps produced. The associated video describes how to search for Vespula wasp nests, a method developed by amateur wasp chasers.

Video: Evaluation of the Productivity of Social Wasp Colonies Vespinae and an Introduction to the Traditional Japanese Vespula Wasp Hunting Technique

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2 facility was constructed which consists of 24 individual 33.6 m2 field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4-P, K+, Mg2+, and Ca2+ had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

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 ...

A Video Surveillance System to Monitor Breeding Colonies of Common Terns (Sterna Hirundo)

Many waterbird populations have faced declines over the last century, including the common tern (Sterna hirundo), a waterbird species with a widespread breeding distribution, that has been recently listed as endangered in some habitats of its range. Waterbird monitoring programs exist to track populations through time; however, some of the more intensive approaches require entering colonies and can be disruptive to nesting populations. This paper describes a protocol that utilizes a minimally invasive surveillance system to continuously monitor common tern nesting behavior in typical ground-nesting colonies. The video monitoring system utilizes wireless cameras focused on individual nests as well as over the colony as a whole, and allows for observation without entering the colony. The video system is powered with several 12 V car batteries that are continuously recharged using solar panels. Footage is recorded using a digital video recorder (DVR) connected to a hard drive, which can be replaced when full. The DVR may be placed outside of the colony to reduce disturbance. In this study, 3,624 h of footage recorded over 63 days in weather conditions ranging from 12.8 &#176;C to 35.0 &#176;C produced 3,006 h (83%) of usable behavioral data. The types of data retrieved from the recorded video can vary; we used it to detect external disturbances and measure nesting behavior during incubation. Although the protocol detailed here was designed for ground-nesting waterbirds, the principal system could easily be modified to accommodate alternative scenarios, such as colonial arboreal nesting species, making it widely applicable to a variety of research needs.

A Video Surveillance System to Monitor Breeding Colonies of Common Terns Sterna Hirundo

This paper describes a protocol that uses a remote video monitoring surveillance system to continuously monitor breeding colonies of ground-nesting waterbirds. The system includes five cameras monitoring individual nests and one camera monitoring the colony as a whole, and is powered by car batteries that are recharged via solar panels.

Many waterbird populations have faced declines over the last century, including the common tern (Sterna hirundo), a waterbird species with a widespread ...

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to bacteria control the amount that will accumulate in staple food crops. The bioavailability of these metals is dependent on their speciation in solution, but more particularly under anoxic conditions where Hg is methylated to toxic monomethylmercury (MeHg) and Cd persists in the rhizosphere. Whole-cell microbial biosensors give a quantifiable signal when a metal enters the cytosol and therefore are useful tools to assess metal bioavailability. Unfortunately, most biosensing efforts have so far been constrained to oxic environments due to the limited ability of existing reporter proteins to function in the absence of oxygen. In this study, we present our effort to develop and optimize a whole-cell biosensor assay capable of functioning anaerobically that can detect metals under anoxic condition in quasi-real time and within hours. We describe how the biosensor can help assess how chemical variables relevant to the environmental cycling of metals affect their bioavailability. The following protocol includes methods to (1) prepare Hg and Cd standards under anoxic conditions, (2) prepare the biosensor in the absence of oxygen, (3) design and execute an experiment to determine how a series of variable affects Hg or Cd bioavailability, and (4) to quantify and interpret biosensor data. We show the linear ranges of the biosensors and provide examples showing the method's ability to distinguish between metal bioavailability and toxicity by utilizing both metal-inducible and constitutive strains.

Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

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, ...

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...

Evaluation of the Productivity of Social Wasp Colonies (Vespinae) and an Introduction to the Traditional Japanese Vespula Wasp Hunting Technique

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Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

Design and Construction of an Urban Runoff Research Facility

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Spotting Cheetahs: Identifying Individuals by Their Footprints

A Video Surveillance System to Monitor Breeding Colonies of Common Terns (Sterna Hirundo)

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings