We thank Yuzhen Wen, Shiping Liang, Shengzhe Jian, and Yanjun Li (College of Forestry and Landscape Architecture, South China Agricultural University) for their help in the insect rearing and experimental set-up. This work was funded by the National Natural Science Foundation of China (Grant No. 31600516), the Guangdong Natural Science Foundation (Grant No. 2016A030310445), and the Science and Technology Planning Project of Guangdong Province (Grant No. 2015A020208010).

1. Moisture-choice Bioassays to Determine Pupation Preference of E. grisescens 
<ol>
	<li>Obtaining mature last-instar larvae of E. grisescens
	<ol>
		<li>Cut fresh shoots (30 - 40 cm in length) of tea plants (Camellia sinensis L.). Insert 25 - 30 shoots into a 250 mL triangular flask. Fill the flask with tap water. Put 3 - 4 flasks (with tea shoots) in a plastic basin (upper side: 51 cm in diameter; bottom side: 40 cm in diameter; height: 16 cm).</li>
		<li>Release 1,000 - 2...

<ol>
	<li>Dimou, I., Koutsikopoulos, C., Economopoulos, A. P., Lykakis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depth+of+pupation+of+the+wild+olive+fruit+fly,+Bactrocera+(Dacus)+oleae+(Gmel.)+(Dipt.,+Tephritidae),+as+affected+by+soil+abiotic+factors.">Depth of pupation of the wild olive fruit fly, Bactrocera (Dacus) oleae (Gmel.) (Dipt., Tephritidae), as affected by soil abiotic factors.</a> Journal of Applied Entomology. 127 (1), 12-17 (2003).</li><li>Chen, M., Shelton, A. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrates+preference+for+pupation+on+sawfly+Notofenusa+surosa+(Hymenoptera:+Tenthredinidae).">Substrates preference for pupation on sawfly Notofenusa surosa (Hymenoptera: Tenthredinidae).</a> Journal of Insect Behavior. 28 (3), 257-267 (2015).</li><li>Buitenhuis, R., Shipp, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+plant+species+and+plant+growth+stage+on+Frankliniella+occidentalis+pupation+behaviour+in+greenhouse+ornamentals.">Influence of plant species and plant growth stage on Frankliniella occidentalis pupation behaviour in greenhouse ornamentals.</a> Journal of Applied Entomology. 132 (1), 86-88 (2008).</li><li>Zheng, X. L., Cong, X. P., Wang, X. P., Lei, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pupation+behaviour,+depth,+and+site+of+Spodoptera+exigua.">Pupation behaviour, depth, and site of Spodoptera exigua.</a> Bulletin of Insectology. 64 (2), 209-214 (2011).</li><li>Wen, Y., 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=Effect+of+substrate+type+and+moisture+on+pupation+and+emergence+of+Heortia+vitessoides+(Lepidoptera:+Crambidae):+choice+and+no-choice+studies.">Effect of substrate type and moisture on pupation and emergence of Heortia vitessoides (Lepidoptera: Crambidae): choice and no-choice studies.</a> Journal of Insect Behavior. 29 (4), 473-489 (2016).</li><li>Wen, Y., 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=Soil+moisture+effects+on+pupation+behavior,+physiology,+and+morphology+of+Heortia+vitessoides+(Lepidoptera:+Crambidae).">Soil moisture effects on pupation behavior, physiology, and morphology of Heortia vitessoides (Lepidoptera: Crambidae).</a> Journal of Entomological Science. 52 (3), 229-238 (2017).</li><li>Wang, H., 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=Pupation+behaviors+and+emergence+successes+of+Ectropis+grisescens+(Lepidoptera:+Geometridae)+in+response+to+different+substrate+types+and+moisture+contents.">Pupation behaviors and emergence successes of Ectropis grisescens (Lepidoptera: Geometridae) in response to different substrate types and moisture contents.</a> Environmental Entomology. 46 (6), 1365-1373 (2017).</li><li>Wang, H., 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=No-substrate+and+low-moisture+conditions+during+pupating+adversely+affect+Ectropis+grisescens+(Lepidoptera:+Geometridae)+adults.">No-substrate and low-moisture conditions during pupating adversely affect Ectropis grisescens (Lepidoptera: Geometridae) adults.</a> Journal of Asia-Pacific Entomology. 21 (2), 657-662 (2018).</li><li>Ge, C. M., Yin, K. S., Tang, M. J., Xiao, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+characteristics+of+Ectropis+grisescens+Warren.">Biological characteristics of Ectropis grisescens Warren.</a> Acta Agriculturae Zhejiangensis. 28 (3), 464-468 (2016).</li><li>Xi, Y., Yin, K. S., Tang, M. J., Xiao, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geographic+populations+of+the+tea+geometrid,+Ectropis+obliqua+(Lepidoptera:+Geometridae)+in+Zhejiang,+eastern+China+have+differentiated+into+different+species.">Geographic populations of the tea geometrid, Ectropis obliqua (Lepidoptera: Geometridae) in Zhejiang, eastern China have differentiated into different species.</a> Acta Entomologica Sinica. 57, 1117-1122 (2014).</li><li>Zhang, G. H., 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=Detecting+deep+divergence+in+seventeen+populations+of+tea+geometrid+(Ectropis+obliqua+Prout)+in+China+by+COI+mtDNA+and+cross-breeding.">Detecting deep divergence in seventeen populations of tea geometrid (Ectropis obliqua Prout) in China by COI mtDNA and cross-breeding.</a> PloS One. 9 (6), e99373 (2014).</li><li>Ma, T., 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=Analysis+of+tea+geometrid+(Ectropis+grisescens)+pheromone+gland+extracts+using+GC-EAD+and+GC&#215;+GC/TOFMS.">Analysis of tea geometrid (Ectropis grisescens) pheromone gland extracts using GC-EAD and GC&#215; GC/TOFMS.</a> Journal of Agricultural and Food Chemistry. 64 (16), 3161-3166 (2016).</li><li>Zhang, G. H., 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=Asymmetrical+reproductive+interference+between+two+sibling+species+of+tea+looper:+Ectropis+grisescens+and+Ectropis+obliqua.">Asymmetrical reproductive interference between two sibling species of tea looper: Ectropis grisescens and Ectropis obliqua.</a> Bulletin of Entomological Research. , (2016).</li><li>Luo, Z. X., Li, Z. Q., Cai, X. M., Bian, L., Chen, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+premating+isolation+between+two+sibling+moths:+Ectropis+grisescens+and+Ectropis+obliqua+(Lepidoptera:+Geometridae).">Evidence of premating isolation between two sibling moths: Ectropis grisescens and Ectropis obliqua (Lepidoptera: Geometridae).</a> Journal of Economic Entomology. 110 (6), 2364-2370 (2017).</li><li>Li, Z. Q., 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=Chemosensory+gene+families+in+Ectropis+grisescens+and+candidates+for+detection+of+Type-II+sex+pheromones.">Chemosensory gene families in Ectropis grisescens and candidates for detection of Type-II sex pheromones.</a> Frontiers in Physiology. 8, (2017).</li><li>Chen, L. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+on+structure+of+soil+particle+by+hydrometer+method.">Research on structure of soil particle by hydrometer method.</a> Environmental Science Survey. 29 (4), 97-99 (2010).</li><li>Kucera, M., Malmgren, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Logratio+transformation+of+compositional+data:+a+resolution+of+the+constant+sum+constraint.">Logratio transformation of compositional data: a resolution of the constant sum constraint.</a> Marine Micropaleontology. 34 (1-2), 117-120 (1998).</li><li>Hulthen, A. D., Clarke, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+soil+type+and+moisture+on+pupal+survival+of+Bactrocera+tryoni+(Froggatt)+(Diptera:+Tephritidae).">The influence of soil type and moisture on pupal survival of Bactrocera tryoni (Froggatt) (Diptera: Tephritidae).</a> Australian Journal of Entomology. 45 (1), 16-19 (2006).</li><li>Alyokhin, A. V., Mille, C., Messing, R. H., Duan, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+pupation+habitats+by+oriental+fruit+fly+larvae+in+the+laboratory.">Selection of pupation habitats by oriental fruit fly larvae in the laboratory.</a> Journal of Insect Behavior. 14 (1), 57-67 (2001).</li><li>Torres-Muros, L., H&#243;dar, J. A., Zamora, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+habitat+type+and+soil+moisture+on+pupal+stage+of+a+Mediterranean+forest+pest+(Thaumetopoea+pityocampa).">Effect of habitat type and soil moisture on pupal stage of a Mediterranean forest pest (Thaumetopoea pityocampa).</a> Agricultural and Forest Entomology. 19 (2), 130-138 (2017).</li></ol>

Pupation preferences responding to different soil variables have been studied in a few pests6,9,22,23. For example, to study the preference of mature larvae of Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) among different soil moisture conditions, Hulthen and Clarke22 set a 3 x 3 Latin-square design containing 9 containers filled with soil at either 0%, 75...

Compared to the larval and adult stages of insects, the pupal stage is highly vulnerable due to the limited mobile ability of pupae, which cannot rapidly escape from dangerous situations. Pupating below the ground is a common strategy used by diverse groups of insects (e.g., in the orders Diptera1,2,3,4, Coleoptera5, Hymenoptera6, Thysanoptera7, and Lepidoptera8,9,10,11,12) to protect them from above-ground predators and environmental hazards. Many of them are severe agricultural and forestry pests1,2,3,4,5,6,7,8,9,10,11,12. The mature larvae of these soil-pupating insects usually leave their hosts, fall on the ground, wander to find a proper site, burrow into the soil, and construct a pupal chamber for pupating8,10.
The tea looper, Ectropis grisescens Warren (Lepidoptera: Geometridae), is one of the most significant defoliator pests of the tea plant Camellia sinensis&#160;L.13. Although this species was first described in 1894, it has been mistakenly identified as Ectropis obliqua Prout (Lepidoptera: Geometridae) in the past decades14,15. The differences in morphology, biology, and geographic distribution between the two sibling species have been described in some recent studies14,15,16. For example, Zhang et al.15 reported that E. oblique mainly occurred on the borders of three provinces (Anhui, Jiangsu, and Zhejiang) of China, whereas E. grisescens has a much wider distribution compared to E. oblique. Therefore, the economic losses caused by E. grisescens are largely overlooked, and the knowledge of this pest needs to be extensively revised and renewed16,17,18,19. Our previous studies showed that E. grisescens prefer to pupate within soil but could also pupate when soil is not available (no-pupation-substrate conditions)11,12.
This paper provides a step-by-step procedure to (1) determine the pupation preference of E. grisescens in response to factors such as substrate type and moisture content by using multiple-choice bioassays, and (2) determine the impact of abiotic factors on the pupation behaviors and emergence success of E. grisescens by using no-choice bioassays. All of these bioassays are conducted under well-controlled laboratory conditions. Also, these bioassays are adapted to evaluate the influence of other factors on the pupation behaviors and survivorship of diverse soil-pupating insects.

<table>
	<tbody>
		<tr>
			<td>Triangular flask</td>
			<td>Bomex Chemical (Shanghai) Co., LTD</td>
			<td>99</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>Plastic basin</td>
			<td>Chahua, Fuzhou, China</td>
			<td>100</td>
			<td>upper side: 51 cm in diameter; bottom side: 40 cm in diameter; height: 16 cm</td>
		</tr>
		<tr>
			<td>Zip lock bags</td>
			<td>Glad, Guangzhou, China</td>
			<td>126/133</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene containers</td>
			<td>Youyou Plastic Factory, Taian, China</td>
			<td>139/155/160/161/190</td>
			<td>upper side: 20.0 cm [L] &#215; 13.5 cm [W], bottom side: 17.0 cm [L] &#215; 10.0 cm [W], height: 6.5 cm</td>
		</tr>
		<tr>
			<td>Waterproof polyviny chloride sheet</td>
			<td>Yidimei, Shanghai, China</td>
			<td>141</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>V-tech, Guangzhou, China</td>
			<td>VT-710</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven drier</td>
			<td>Kexi, Shanghai, China</td>
			<td>KXH-202-3A</td>
			<td></td>
		</tr>
		<tr>
			<td>Environmental chamber</td>
			<td>Life Apparatus, Ningbo, China</td>
			<td>PSX-280H</td>
			<td></td>
		</tr>
	</tbody>
</table>

choice and no-choice bioassays to study the pupation preference and emergence success of ectropis grisescens

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

The moisture-choice bioassays showed that significantly more E. grisescens individuals pupated on or within the 5%- and 35%-moisture sand compared to the 80%-moisture sand (Figure 2a). However, significantly more individuals preferred to pupate on or within the soil (sandy loam 1 and 2 and silt loam) that had an intermediate moisture content (Figures 2b - 2d).
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Watch this Scientific Journal Video about Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens at JoVE.com

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

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

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

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

This method can provide important information related to the pupation ecology of tea leaf caterpillars, Ectropis grisescens, such as their pupation behaviors and the pupae survivorship in response to different environmental factors. The main advantage of this technique, it allows you to use a simple method such as measuring the pupation preference and emergence success of Ectropis grisescens. To begin, cut fresh shoots from tea plants.Then fill a 250 milliliter triangular flask with tap water and place the shoots into the flask. Transfer three to four flasks to a plastic basin. Next, release 1, 000 to 2, 000 larvae from a laboratory colony of E.grisescens onto the leaves of the tea shoots.Maintain the larvae in controlled laboratory conditions. Obtain the mature, last-instar larvae that fall from the leaves of the shoots and actively wander along the bottom of the basin. Sterilize soil and sand in an 80 degree Celsius oven dryer for three days.Then dry both the soil and sand at 50 degrees Celsius for several weeks. The substrate samples are sufficiently dried when the weight does not change over time. Use a wooden pestle and mortar to grind the dried soil.Then sift the sand and the ground soil through a three millimeter sieve. Add the required amount of distilled water to the sealed bags containing the substrates and mix thoroughly. Next, use PVC sheets to divide polypropylene containers into six equal chambers.Fix the PVC sheets and seal the cracks with hot glue. Next, fill the six chambers with the same type of substrate with different moisture contents. Use small pieces of tape to paste four to six pieces of fresh leaves to the inner surface of the lid of the polypropylene container.Next, release 30 mature last-instar larvae onto the leaves pasted to the lid of the chambered container. Carefully turn the lid over and cover the container. Maintain the bioassay arena in an environmental chamber with a 14 to 10 light/dark photoperiod at 26 degrees Celsius.On day five, count the number of pupae on the surface of the substrate in each chamber. Dismantle the bioassay and count the number of pupae. First, use PVC sheets to divide the polypropylene containers into four chambers.Then, fix and seal the PVC sheets with hot glue. Fill the chambers of the container with four types of substrate with the same moisture content and randomly assigned orders. Then, use tape to paste fresh tea leaves to the lid before turning it over onto the container.Next, transfer larvae to the leaves and record and analyze the data as outlined in the text protocol. After preparing the 12 substrate moisture content treatments, add the substrate to plastic containers. Then release 15 mature last-instar larvae into the substrate of each arena.Maintain each of the bioassay arenas in an environmental chamber with a 14 to 10 light/dark photoperiod at 26 degrees Celsius. On day three, count the number of pupae and any dead larvae on the surface of the substrate of each arena. Finally, record the number of emerging adults each day until no new adults emerge for 15 days.In this protocol, moisture and substrate choice bioassays were used to study the pupation preference of E.grisescens. The moisture choice bioassays showed that more individuals pupated on or within the 5%and 35%moisture sand compared to the 80%moisture sand. However, significantly more individuals preferred to pupate on or within the soil with intermediate moisture content.Substrate choice bioassays revealed that sand was most preferred by E.grisescens under 20%moisture conditions, and under 80%moisture conditions. However, no pupation preference was found among the four substrates under 50%moisture conditions. After development, this technique paved the way for researchers in the field of entomology to explore the pupation ecology of many soil-pupating insects.

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6.1K visualizações.  South China Agricultural University. Este método pode fornecer informações importantes relacionadas à ecologia de pupation de lagartas de folhas de chá, ectropis grisescens, como seus comportamentos de pupação e a sobrevivência da pupae em resposta a diferentes fatores ambientais. A principal vantagem dessa técnica, permite que você use um método simples, como medir a preferência de pupação e o sucesso de emergência do Ectropis grisescens. Para começar, corte brotos frescos de plantas de chá.Em seguida, e...