Name: choice and no-choice bioassays to study the pupation preference and emergence success of ectropis grisescens
Uploaded: 2018-10-30T13:00:00.000+00:00
Duration: 5 min 19 s
Description: 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

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 - 2,000 larvae (second to fifth instar) of the laboratory colony of E. grisescens onto the leaves of the tea shoots in each basin. Maintain these larvae at controlled laboratory conditions [a photoperiod of 14 h of light followed by 10 h of dark (14:10 L:D), 60 - 90% relative humidity (RH), and 24 - 28 &#176;C]. Carefully transfer the larvae onto fresh leaves by hand every 1 - 2 d. Each day remove feces and debris from the bottom of the basins.</li>
		<li>Select mature last-instar larvae that fall from the leaves of the tea shoots and actively wander on the bottom of the basin. Obtain at least 240 mature larvae to ensure that enough larvae are available for the bioassays. 
		NOTE: Only select actively wandering larvae for the experiments. Do not select larvae that stay on the leaves, because these are not ready to pupate. Also, do not select prepupae with limited mobile activities because they will not actively search for the proper conditions after being released into the bioassay arenas.</li>
	</ol>
	</li>
	<li>Substrate Preparation
	<ol>
		<li>Collect and identify 4 types of substrate (e.g., sand, sandy loam 1, sandy loam 2, and silt loam) using the hydrometer method20. Sterilize the soil and the sand at an 80 &#176;C oven dryer for &#62; 3 d, and then completely dry the soil and the sand at 50 &#176;C for several weeks until the dry weight of the substrate samples does not change anymore over time.</li>
		<li>Ground the dry soil with wooden pestles and mortars. Sift the sand and the grounded soil through a 3 mm sieve and store them in sealable plastic bags.</li>
		<li>Calculate the different moisture contents of each substrate (sand, sandy loam 1, sandy loam 2, or silt loam) as follows2: 
		<img alt="Equation 1" src="/files/ftp_upload/58126/58126eq1.jpg" /></li>
		<li>Add the required amount of distilled water into the sealable plastic bags containing the dry soil or sand to prepare 5%-, 20%-, 35%-, 50%-, 65%-, and 80%-moisture substrate. Thoroughly mix the distilled water and the soil or sand.</li>
	</ol>
	</li>
	<li>Bioassay arena preparation
	<ol>
		<li>Equally divide the polypropylene containers (upper side: 20.0 cm in length x 13.5 cm in width, bottom side: 17.0 cm in length x 10.0 cm in width, height: 6.5 cm) into 6 chambers with waterproof polyvinyl chloride (PVC) sheets (height: 3.5 cm). Fix the PVC sheets and seal the cracks using hot glue. 
		NOTE: Completely seal any crack to prevent water permeation.</li>
		<li>For each test, fill the 6 chambers using the same type of substrate with different moisture contents (5%-, 20%-, 35%-, 50%-, 65%-, and 80%-moisture) (Figure 1a). 
		NOTE: Only use 1 type of substrate of different moisture contents in each test. Randomly assign the order of chambers that contain the substrate with the 6 moisture contents.</li>
		<li>Paste 4 - 6 pieces of fresh tea leaves using small pieces of tape to cover the inner surface of the lids of the polypropylene containers (Figure 1b).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58126/58126fig1.jpg" /> 
Figure 1: Examples of bioassay arenas for the choice tests. (a) Waterproof polyvinyl chloride (PVC) sheets are used to equally divide the polypropylene containers into 6 chambers. PVC sheets are fixed with hot glue, and any cracks are carefully sealed. In this example, sandy loam 2 with different moisture contents (5%, 20%, 35%, 50%, 65%, and 80% moisture) are used to fill the chambers in the randomly assigned orders. (b) Fresh tea leaves are pasted on the inner side of the lids where the mature Ectropis grisescens larvae will be released. (c) PVC sheets are used to equally divide the polypropylene containers into 4 chambers, which are filled with 4 types of substrates (sand, sandy loam 1, sandy loam 2, and silt loam) at 50% moisture. This figure has been modified from Wang et al.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58126/58126fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Bioassay setting and data recording
	<ol>
		<li>Release 30 mature last-instar larvae (obtained in step 1.1.3) onto the fresh tea leaves pasted on the lid of the polypropylene container. Carefully overturn the lid and tightly cover the polypropylene container.</li>
		<li>Repeat each test 8x. Maintain the bioassay arenas in an environmental chamber setting at a 14:10 (L:D) photoperiod and 26 &#176;C.</li>
		<li>On day 5, count the number of pupae on the surface of the soil in each chamber. Also, dismantle the bioassays and count the number of pupae within the substrate. 
		NOTE: Only count the live pupae on or within the substrate. Check the pupae viability by observing abdominal motions after touching the pupae using forceps.</li>
	</ol>
	</li>
	<li>Data analyses
	<ol>
		<li>For each test, calculate the percentage of pupae found in each chamber of each replicate. Transfer the percentage data to the log-ratio using the method provided by Kucera and Malmgren21.</li>
		<li>Compare the percentage of pupae (transformed data) in each chamber using a one-way analysis of variance (ANOVA). Set the significance levels at &#945; = 0.05 for each test.</li>
	</ol>
	</li>
</ol>
2. Substrate-Choice Bioassays to Determine the Pupation Preference of E. grisescens
<ol>
	<li>Repeat step 1.1 to obtain mature last-instar larvae, and step 1.2 to prepare the substrate with different moisture contents. This time, only 20%, 50%, and 80%-moisture substrate are needed.</li>
	<li>Preparation of the bioassay arenas
	<ol>
		<li>Similar to step 1.3.1, equally divide the polypropylene containers into 4 chambers using PVC sheets. Fix the PVC sheets and seal the cracks using hot glue.</li>
		<li>For each test, fill the chambers with the 4 types of substrates (sand, sandy loam 1, sandy loam 2, and silt loam) that have the same moisture content (20%, 50%, or 80% moisture) with randomly assigned orders (Figure 1c). Repeat step 1.3.3 to prepare the lids.</li>
	</ol>
	</li>
	<li>Repeat step 1.4 to set the bioassays and record the data and step 1.5 to analyze the data.</li>
</ol>
3. No-choice Bioassays to Determine the Soil-burrowing Behavior and Emergence Success of E. grisescens
<ol>
	<li>Repeat step 1.1 to obtain the mature last-instar larvae, and step 1.2 to prepare the 4 substrates (sand, sandy loam 1, sandy loam 2, and silt loam) at 3 moisture contents (20%, 50%, and 80% moisture).</li>
	<li>Bioassay setting
	<ol>
		<li>Add the substrate into a plastic container (upper side: 11.5 cm in diameter; bottom side: 8.5 cm in diameter; height: 6.5 cm) to a depth of 3 cm. In total, ensure that there will be 12 treatments (the combinations of 4 substrate types and 3 moisture contents). Repeat each treatment 7x.</li>
		<li>Release 15 mature last-instar larvae onto the substrate of each bioassay arena. Seal the containers by tightly covering the lids. Maintain the bioassays in an environmental chamber setting at a 14:10 (L:D) photoperiod and 26 &#176;C. 
		NOTE: There will be no need to paste fresh tea leaves on the lids as mentioned in the choice bioassays.</li>
	</ol>
	</li>
	<li>Data recording and analyses
	<ol>
		<li>On day 3, count the number of pupae and any dead larvae on the surface of the substrate of each replicate. Calculate the percentage of E. grisescens individuals that burrowed into the substrate as follows: 
		<img alt="Equation 2" src="/files/ftp_upload/58126/58126eq2.jpg" /></li>
		<li>Record the number of emerging adults each day until no more adult emerged for 15 d. Calculate the emergence success as follows: 
		<img alt="Equation 3" src="/files/ftp_upload/58126/58126eq3v2.jpg" /></li>
		<li>Compare the percentage of burrowed individuals and the emergence success among the treatments using one-way ANOVA. Set the significance levels at &#945; = 0.05.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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%, or 100% field capacity, and 25 mature larvae were released onto the surface of each container. Alyokhin et al.23 placed 100 containers (filled with soil) in a wooden frame, with the 36 center containers (either dry or wet) arranged in a &#34;chessboard&#34; pattern, and 350 - 450 late third-instar larvae of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) were released onto the center of the 36 containers. These studies are suitable for B. tryoni and B. dorsalis larvae because most of them were recovered within the soil in the bioassay arenas22,23. However, these arenas were not covered. As a result, wandering larvae with a strong moving capacity may travel a long distance and escape from the arenas. Here, we provided a simple method to study the preference of soil-pupating insects regardless of their sizes and mobile abilities. Compared to previous studies, these bioassays are easy to set up. Also, multiple levels (e.g., &#62; 4) of soil variable can be studied in relatively small arenas.
It is worth noting that the data obtained from the choice tests described here cannot be directly analyzed using ANOVA because the percentage data are not independent (the sum of the percentage of pupae in each chamber always equals 1, and, therefore, the increase of the percentage of pupae in 1 chamber will cause the decrease of the percentage in the remaining ones). Here, we performed the log-ratio transformation because it is a simple procedure that &#34;effectively removes the CSC (constant-sum constraint) from any compositional data and simultaneously retains their true covariance structure&#34;21. In the present study, the survivorship of E. grisescens pupae was high, and we only recorded the percentage of live pupae in each chamber. However, some soil-pupating pests such as the pine processionary moth, Thaumetopoea pityocampa (Denis &#38; Schifferm&#252;ller) (Lepidoptera: Thaumetopoeidae), usually exhibit a high mortality during pupating24. In that case, it would be proper to count both live and dead pupae.
No-choice bioassays have been widely used to investigate the effect of soil variables on the emergence success of soil-pupation pests. The substrate types and moisture contents were the most frequently studied factors in these studies2,3,4,5,9,10,11,12. E. grisescens can either pupate within or on the substrates. As a result, we recorded the percentage of burrowed individuals. This result would be important to help understand the pupation patterns of E. grisescens.
Both the choice and the no-choice bioassays can be modified to investigate the impact of other soil factors (such as soil density, surface compactness, content of organic matters, etc.) on the preference and performance of various soil-pupating insects. In a recent work, we modified these bioassays to study (1) whether the soil treated with a chemical pesticide or biocontrol agent repel pupating E. grisescens (both live and dead pupae were counted), and (2) the effect of such treatments on the pupation behaviors (e.g., the percentage of burrowed individuals) and emergence success of E. grisescens.

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] &amp;times; 13.5 cm [W], bottom side: 17.0 cm [L] &amp;times; 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).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58126/58126fig2.jpg" /> 
Figure 2: Results from the moisture-choice bioassays. These panels show the percentages of live pupae found in each chamber containing different moisture contents (5%, 20%, 35%, 50%, 65%, and 80%-moisture) of (a) sand, (b) sandy loam 1, (c) sandy loam 2, or (d) silt loam. The data are presented as mean &#177; SE. The different letters indicate significant differences (P &#60; 0.05). This figure has been modified from Wang et al.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58126/58126fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The substrate-choice bioassays showed that sand was significantly more preferred by E. grisescens individuals compared to sandy loam (1 and 2) under the 20%-moisture condition (Figure 3a). There was no significant difference in percentages of pupae found in the chambers containing the 4 substrates at a 50%-moisture content (Figure 3b). Significantly more individuals pupated on or within the sand than on or within the other substrates under the 80%-moisture condition (Figure 3c).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58126/58126fig3.jpg" /> 
Figure 3: Results from the substrate-choice bioassays. These panels show the percentages of live pupae found in each chamber containing sand, sandy loam 1, sandy loam 2, or silt loam at (a) a 20%-, (b) a 50%-, or (c) an 80%-moisture content. The data are presented as mean &#177; SE. The different letters indicate significant differences (P &#60; 0.05). This figure has been modified from Wang et al.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58126/58126fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Different moisture contents of sand did not significantly affect the percentage of burrowed individuals and emergence success of E. grisescens (Figure&#160;4a and 4b). Significantly fewer E. grisescens burrowed into dry (20% moisture) or wet (80% moisture) soil for pupating (Figure 4a). Also, significantly fewer adults emerged from 20%-moisture sandy loam 2 and silt loam than those that had pupated in 50%- or 80%-moisture sandy loam 2 and silt loam (Figure 4b).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58126/58126fig4.jpg" /> 
Figure 4: Results from the no-choice bioassays. These panels show (a) the percentages of burrowed individuals and (b) the emergence success of Ectropis grisescens in response to different substrate types (sand, sandy loam 1, sandy loam 2, and silt loam) and moisture contents (20%, 50%, and 80%). The data are presented as mean &#177; SE. The different letters indicate significant differences (P &#60; 0.05). This figure has been modified from Wang et al.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58126/58126fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

choice-no-choice-bioassays-to-study-pupation-preference-emergence

Nanyang Technological University

Research

JoVE Journal

Environment

6.2K Views.  South China Agricultural University. Deze methode kan belangrijke informatie met betrekking tot de verpopting ecologie van theeblad rupsen, Ectropis grisescens, zoals hun verpoppen gedrag en de poppen overleven in reactie op verschillende omgevingsfactoren. Het belangrijkste voordeel van deze techniek, het stelt u in staat om een eenvoudige methode te gebruiken, zoals het meten van de pupation voorkeur en opkomst succes van Ectropis grisescens. Om te beginnen, snijd verse scheuten van theeplanten.Vul vervolgens een drieho...

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

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.

Plant resistance to chewing insect herbivores can be tested in several ways. Here, we demonstrate how to set-up a choice and a no-choice experiment with the model plant Arabidopsis thaliana to identify resistance against the pest species Pieris rapae.

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which ...

Biology

Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;s innate behaviors (positive photo-taxis) as a means to lure the insect into a population control or monitoring trap. Light emitting diodes (LEDs) have created customizable lighting options with specific wavelengths (colors), intensities, and bandwidths, all of which can be customized to the target insects. Photo-attraction behavioral bioassays can use LEDs to optimize the attractive color(s) for an insect species down to specific life history stages or behaviors (mating, feeding, or seeking shelter). Researchers must then confirm the bioassay results in the field and understand the limited attractive distance of the visual attractants.
The cloverleaf bioassay arena is a flexible method to assess photo attraction while also assessing a range of natural insect behaviors such as escape and feeding responses. The arena can be used for terrestrial or aerial insect experiments, as well as diurnal, and nocturnal insects. Data collection techniques with the arena are videotaping, counting contact with the lights, or physically collecting the insects as they are attracted towards the lights. The assay accounts for insects that make no-choice and the arenas can be single (noncompetitive) color or multiple (competitive) colors. The cloverleaf design causes insects with strong thigmotaxis to return to the center of the arena where they can view all the options in a competitive LED tests. The cloverleaf arena presented here has been used with mosquitoes, bed bugs, Hessian fly, house flies, biting midges, red flour beetles, and psocids. Bioassays are used to develop accurate and effective insect traps to guide the development and optimization of insect traps used to monitor pest population fluctuations for disease vector risk assessments, the introduction of invasive species, and/or be used for population suppression.

Photo-attraction bioassay arenas are used to determine the optimal light color(s) to maximize insect attraction; however bioassays and methods are specific to target insect behaviors and habitats. Customizable equipment and modifications are explained for nocturnal or diurnal and terrestrial or aerial insects.

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;s innate behaviors (positive photo-taxis) as a means ...

Behavior

Vertical T-maze Choice Assay for Arthropod Response to Odorants

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, various technologies are being developed as control tools. A subset of these tools focuses on modifying the behavior of arthropods by attraction or repulsion. Therefore, arthropods are often the focus of behavioral investigations. Various tools have been developed to measure arthropod behavior, including wind tunnels, flight mills, servospheres, and various types of olfactometers. The purpose of these tools is to measure insect or arachnid response to visual or more often olfactory cues. The vertical T-maze oflactometer described here measures choices performed by insects in response to attractants or repellents. It is a high throughput assay device that takes advantage of the positive phototaxis (attraction to light) and negative geotaxis (tendency to walk or fly upward) exhibited by many arthropods. The olfactometer consists of a 30 cm glass tube that is divided in half with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the test subjects to make a choice between two potential odor fields in assays involving attractants. In assays involving repellents, lack of normal response to known attractants can also be measured as a third variable.

A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device.

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, ...

Neuroscience

Exploring Life History Choices: Using Temperature and Substrate Type as Interacting Factors for Blowfly Larval and Female Preferences

Blowflies (Diptera: Calliphoridae) present a wide range of larval lifestyles, typically classified as obligate parasitism, facultative parasitism, and complete sapro-necrophagy. Several parasitic species, both obligate and facultative, are considered to be of sanitary and economic importance, as their larvae can cause myiasis (maggot infestation in live tissue). However, it is noteworthy that the adult female plays a decisive role as she chooses the oviposition site, and, therefore, largely determines the feeding habit and developmental conditions of the larvae. In this study, two protocols are proposed to test larval feeding preference and female oviposition site preference considering two interacting factors: meat substrate type and temperature. The setups presented here allowed to test Lucilia cuprina larvae and gravid females in a four-choice assay with two temperatures (33 &plusmn; 2 &#176;C and 25 &plusmn; 2 &#176;C) and two types of meat substrates (fresh meat supplemented with blood and 5-day-old rotten meat). Larvae or gravid females can choose to burrow or lay their eggs, respectively, in either of the following: rotten meat at 25 &#176;C (simulating a necrophagous species condition), fresh meat supplemented with blood at 33 &#176;C (simulating a parasitic species condition), and two controls, rotten meat at 33 &#176;C, or fresh meat supplemented with blood at 25 &#176;C. The preference is assessed by counting the number of larvae or eggs laid in each option for each replicate. Comparing the observed results to a random distribution allowed for the estimation of the statistical significance of the preference. The results indicated that L. cuprina larvae have a strong preference for the rotten substrate at 25 &#176;C. Conversely, oviposition-site preference by females was more varied for the meat type. This methodology can be adapted to test the preference of other insect species of similar size. Other questions can also be explored by using alternative conditions.

Herein, two protocols for assessing food source and oviposition preferences in larvae and females of blowflies are detailed. These comprise four choices with two interacting factors: substrate type and temperature. The assays enable the determination of the food source preference of the larvae and the oviposition site preference for the females.

Blowflies (Diptera: Calliphoridae) present a wide range of larval lifestyles, typically classified as obligate parasitism, facultative parasitism, and ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

At-Risk Butterfly Captive Propagation Programs to Enhance Life History Knowledge and Effective Ex Situ Conservation Techniques

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. Research on such captive populations can also yield valuable data to address key information gaps about the behavior, life history, and ecology of the target taxa. We describe a protocol for captive propagation of the federally endangered Cyclargus thomasi bethunebakeri that can be used as a model for other at-risk butterfly ex situ programs, especially those in the family Lycaenidae. We further provide a simple and straightforward protocol for recording various life history metrics that can be useful for informing ex situ methodologies as well as adapted for laboratory studies of other lepidoptera.

Here, we present protocols for 1) the laboratory captive propagation of the federally endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri), and 2) assessing basic life history information such as immature development time and number of larval stadia. Both methods can be adapted for use with other ex situ conservation programs.

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. ...

Non-invasive Electropenetrography to Study Mosquito Probing Behaviors

AC-DC Electropenetrography for the Study of Probing and Ingestion Behaviors of Culex tarsalis Mosquitoes on Human Hands

Mosquitoes transmit pathogens that negatively affect human and animal health. A greater understanding of their blood-feeding biology and interactions with hosts and pathogens could be exploited to develop new targets for controlling mosquito-borne diseases. Unfortunately, probing (i.e., biting) behaviors of mosquitoes are poorly understood because they occur inside host tissues. Here, a non-invasive procedure is described for using AC-DC electropenetrography (EPG) to indirectly visualize and quantify mosquito feeding behaviors by recording changes in electrical signals generated when probing and ingesting on human hands. Thin gold wires are attached to the mosquitoes using conductive silver glue and connected to the EPG instrument. The human host holds a substrate voltage probe in their hand. Probing of the host by the wired mosquito completes the electrical circuit, and electrical signals are recorded on a computer as &#34;waveforms&#34; that can then be measured and enumerated for analysis. This methodology has been used to characterize the probing and ingestion behaviors of Aedes aegypti and Culex tarsalis mosquitoes and can be applied to other mosquito species. EPG can be used to study the effects of pathogens, insecticides, and other factors on mosquito feeding behaviors.

Procedures are described for studying the probing and ingestion behaviors of Culex tarsalis&#160;mosquitoes on human hands using AC-DC electropenetrography.

Mosquitoes transmit pathogens that negatively affect human and animal health. A greater understanding of their blood-feeding biology and interactions with ...

Insect Puparium Removal: A Method for Pupal Investigation

Source: Fukutomi, Y., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56935/methods-for-staging-pupal-periods-measurement-wing-pigmentation">Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of&#160;Drosophila guttifera.</a>&#160;J. Vis. Exp. (2018).
This article describes a technique to remove an insect&#8217;s puparium while keeping the inside pupa intact and therefore fit for further investigation. The example protocol shows the procedure with Drosophila guttifera; however, this technique is applicable to other Drosophila species.

Source: Fukutomi, Y., et al. Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of&#160;Drosophila guttifera.&#160;J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Pupa

Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans

Source: Lee, S. Y. and Kang, K. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60419/measuring-effects-bacteria-chemicals-on-intestinal-permeability">Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp. (2017).
C. elegans egg-laying behavior can provide important information in developmental, reproductive, and anatomical studies.&#160; This video introduces the important steps of an egg laying assay and shows a sample experiment in which a drug is tested for reproductive toxicity.

Source: Lee, S. Y. and Kang, K. Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans.&#160;J. Vis. Exp. 
...

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

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