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><tbody><tr><td>cuttlefish</td><td>Any</td><td></td><td>fresh/ as a bait</td></tr><tr><td>dace</td><td>Any</td><td></td><td>fresh/ as a bait</td></tr><tr><td>chichken heart</td><td>Any</td><td></td><td>fresh/ as a bait</td></tr><tr><td>plastic bag (polyethylene)</td><td>Any</td><td></td><td>as a flag</td></tr><tr><td>bamboo skewer</td><td>Any</td><td></td><td></td></tr><tr><td>industrial sewing thread</td><td>FUJIX Ltd.</td><td>King polyester, No.100</td><td></td></tr><tr><td>paint marker pen</td><td>Mitsubishi pencil</td><td>UNI, POSCA, PC5M</td><td></td></tr><tr><td>fishing rod</td><td>ANY</td><td></td><td></td></tr><tr><td>carrying box</td><td></td><td></td><td>made of wood</td></tr><tr><td>nest box</td><td></td><td></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

8.3K Views.  Tajimi Senior High School. Aangezien Vespula wespenkolonies niet kunnen worden gelokt uit bustes, moeten nesten worden verzameld uit het wild. Hier demonstreer ik traditionele wespenjacht zoals is ontwikkeld in de Japanse cultuur. Bij het volgen van wespen naar hun nesten, kan deze techniek helpen bij het hervatten van de nestlocatie vanaf het punt waarop een wesp is ontsnapt detectie.Om vespula aan te trekken, hang 10-gram stukken van unflagged aas op een boomtak op een hoogte die gemakkelijk met de hand langs ...

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

A Push-pull Protocol to Reduce Colonization of Bird Nest Boxes by Honey Bees

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. These bees select cavities that are suitable nest sites for birds, resulting in competition. The difficulty of removing bees and their defensive behavior makes a prevention protocol necessary. Here, we describe a push-pull integrated pest management protocol to deter bees from inhabiting bird boxes by applying a bird safe insecticide, permethrin, to repel bees from nest boxes, while simultaneously attracting them to pheromone-baited swarm traps. Shown here is an example experiment using Barn Owl nest boxes. This protocol successfully reduced colonization of Barn Owl nest boxes by Africanized honey bees. This protocol is flexible, allowing adjustments to accommodate a wide range of bird species and habitats. This protocol could benefit conservation efforts where AHB are located.

Preventing colonization of bird nest boxes by invasive Africanized honey bees is important for conservation efforts of nest-site limited birds. We provide an integrated pest management approach to &#34;push&#34; bees away from nest boxes with a repellant insecticide, permethrin, and &#34;pull&#34; them toward pheromone baited swarm traps.

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

Genetics

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Immunology and Infection

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model organism. However, little is known about the sex-determination mechanisms found in other non-model hymenopteran taxa, such as ants. Because of the complex nature of the life cycles that have evolved in hymenopteran species, it is difficult to maintain and conduct experimental crosses between these organisms in the laboratory. Here, we describe the methods for conducting inbreeding crosses and for evaluating the success of those crosses in ant Vollenhovia emeryi. Inducing inbreeding in the laboratory using V. emeryi, is relatively simple because of the unique biology of the species. Specifically, this species produces androgenetic males, and female reproductives exhibit wing polymorphism, which simplifies identification of the phenotypes in genetic crosses. In addition, evaluating the success of inbreeding is straightforward as males can be produced continuously by inbreeding crosses, while normal males only appear during a well-defined reproductive season in the field. Our protocol allow for using V. emeryi as a model to investigate the genetic and molecular basis of the sex determination system in ant species.

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

In this protocol, methods for conducting inbreeding crosses, and for evaluating the success of those crosses, are described for the ant Vollenhovia emeryi. These protocols are important for experiments aimed at understanding the genetic basis of sex determination systems in Hymenoptera.

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ingestion of provisions contaminated with pesticides may increase the mortality rate of the larvae, decrease the collection rate and the population of adult solitary bees in the next year from a demographic perspective. But there are limited studies on the effects of pesticides on the larvae of solitary bees. Therefore, understanding how pesticides influence the larvae of solitary bees should be considered an integral part of pesticide ecological risk assessment. This study presents a method to expose the larvae of solitary bee, Osmia excavata, to lethal or sublethal doses of pesticide, tracking larval weight gain, developmental duration, eclosion ability, and food consumption efficiency conversion of ingested food. To demonstrate the effectiveness of this method, the larvae of O. excavata were fed with provisions containing acute lethal and sublethal doses of chlorpyrifos. Then, the above indexes of the treated larvae were investigated. This technique helps to predict and mitigate the risk of pesticides to pollinators.

The present protocol explains a method to feed pesticide-contaminated provisions to the larvae of the solitary bees, Osmia excavata. The procedure examines the ecotoxicity of the pesticide to the larvae of the solitary bees.

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ...

Biochemistry

Artificial Host Parasitization by Trichogramma dendrolimi and Venom Extraction

Extracting Venom from the Parasitoid Wasp Trichogramma dendrolimi Using an Artificial Host

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, parasitoid wasps inject venom into their hosts to suppress their hosts&#39; immunity, modulate hosts&#39; development, metabolism, and even behavior. With over 600,000 estimated species, the diversity of parasitoid wasps surpasses that of other venomous animals, such as snakes, cone snails, and spiders. Parasitoid wasp venom is an underexplored source of bioactive molecules with potential applications in pest control and medicine. However, collecting parasitoid venom is challenging due to the inability to use direct or electrical stimulation and the difficulty in dissection because of their small size. Trichogramma is a genus of tiny (~0.5 mm) egg parasitoid wasps that are widely used for the biological control of lepidopteran pests in both agriculture and forests. Here, we report a method for extracting venom from T. dendrolimi using artificial hosts. These artificial hosts are created with polyethylene film and amino acid solutions and then inoculated with Trichogramma wasps for parasitism. The venom was subsequently collected and concentrated. This method enables the extraction of large amounts of Trichogramma venom while avoiding contamination from other tissues caused by dissection, a common issue in venom reservoir dissection protocols. This innovative approach facilitates the study of Trichogramma venom, paving the way for new research and potential applications.

Author Spotlight: Efficient Venom Extraction Method from Trichogramma Parasitoid Wasps 

Here, we present a protocol for extracting venom from Trichogramma dendrolimi using an artificial host created with polyethylene film and amino acid solution.

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

Biology

Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii

Native to East Asia, the spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has established widely in the Americas, Europe, and parts of Africa over the last decade, becoming a devastating pest of various soft-skinned fruits in its invaded regions. Biological control, especially by means of self-perpetuating and specialized parasitoids, is expected to be a viable option for sustainable area-wide management of this highly mobile and polyphagous pest. Ganaspis brasiliensis Ihering (Hymenoptera: Figitidae) is a larval parasitoid that is widely distributed in East Asia, and has been found to be one of the most effective parasitoids of D. suzukii.
Following rigorous pre-introduction evaluations of its efficacy and potential non-target risks, one of the more host-specific genetic groups of this species (G1 G. brasiliensis) has been approved recently for introduction and field release in the United States and Italy. Another genetic group (G3 G. brasiliensis), which was also commonly found to attack D. suzukii in East Asia, may be considered for introduction in the near future. There is currently enormous interest in rearing G. brasiliensis for research or in mass-production for field release against D. suzukii. This protocol and associated video article describe effective rearing methods for this parasitoid, both on a small scale for research and a large scale for mass-production and field release. These methods may benefit further long-term research and use of this Asian-native parasitoid as a promising biological control agent for this global invasive pest.

Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii

Ganaspis brasiliensis-a larval parasitoid of Drosophila suzukii (a global invasive fruit crop pest)-has been approved or is considered for introduction into Europe and the United States for biological control of this pest. This article provides protocols for both small-scale and large-scale rearing of this parasitoid.

Native to East Asia, the spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has established widely in the Americas, Europe, ...

Rearing Germ-Free Nasonia Wasps

Optimizing the Rearing Procedure of Germ-Free Wasps

Aseptic rearing technology is a method of culturing insects under sterile or almost sterile conditions, which can effectively eliminate the influence of external microorganisms on insect microbiota and thus promote the rapid development of insect microbiota research. Nasonia (wasp genus) is a parasitic wasp insect that has many advantages, such as a short lifespan, high genetic variation, easy operation, etc., and is widely used as an insect model system. Unlike antibiotic treatment, which can only reduce the number of microorganisms in animals, aseptic rearing techniques can control both the composition and quantity of microorganisms in animals, further facilitating the study of host-microbe interactions. However, previous versions of Nasonia rearing medium (NRM) have some defects and problems, such as a complex and time-consuming preparation process, easy contamination by bacteria or fungi, and short storage time. Therefore, this study solves these problems by optimizing the tools used in the NRM preparation process, storage conditions, and component ratios. The optimized medium could allow storage at -20 &#176;C for at least 3 months and eliminate the possibility of NRM contamination during feeding sterile wasps. This further improves the survival rate and health level of aseptic Nasonia, which is important for using Nasonia as a model for microbial research.

Author Spotlight: Optimizing the Rearing Procedure of Germ-Free Wasps  

Nasonia wasp embryos were dissected from Lucillia sericata pupae after parasitization for 12-24 h and washed with alcohol and 10% sodium hypochlorite solution to obtain germ-free embryos. After rearing the germ-free embryos&#160;and supplying them with Nasonia rearing medium to grow and develop in vitro, germ-free Nasonia adults were obtained.

Aseptic rearing technology is a method of culturing insects under sterile or almost sterile conditions, which can effectively eliminate the influence of ...

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

Samenvatting

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