Name: choice and no-choice bioassays to study the pupation preference and emergence success of ectropis grisescens
Uploaded: 2018-10-30T13:00:00
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...

<ol>
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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. 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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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Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

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Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

A Protocol for Conducting Rainfall Simulation to Study Soil Runoff

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The objective of this study was to characterize the effects of antecedent soil moisture content on the fate and transport of surface applied commercial urea, a common form of nitrogen (N) fertilizer, following a rainfall event that occurs within 24&#160;hr&#160;after fertilizer application. Although urea is assumed to be readily hydrolyzed to ammonium and therefore not often available for transport, recent studies suggest that urea can be transported from agricultural soils to coastal waters where it is implicated in harmful algal blooms. A rainfall simulator was used to apply a consistent rate of uniform rainfall across packed soil boxes that had been prewetted to different soil moisture contents. By controlling rainfall and soil physical characteristics, the effects of antecedent soil moisture on urea loss were isolated. Wetter soils exhibited shorter time from rainfall initiation to runoff initiation, greater total volume of runoff, higher urea concentrations in runoff, and greater mass loadings of urea in runoff. These results also demonstrate the importance of controlling for antecedent soil moisture content in studies designed to isolate other variables, such as soil physical or chemical characteristics, slope, soil cover, management, or rainfall characteristics. Because rainfall simulators are designed to deliver raindrops of similar size and velocity as natural rainfall, studies conducted under a standardized protocol can yield valuable data that, in turn, can be used to develop models for predicting the fate and transport of pollutants in runoff.

A rainfall simulator was used to apply a consistent rate of uniform rainfall to packed soil boxes in a study of the fate and transport of urea, a nonpoint source environmental contaminant. Under uniform soil and rainfall conditions, antecedent soil moisture content exerted strong control over urea loss in surface runoff.

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Using Pharmacological Manipulation and High-precision Radio Telemetry to Study the Spatial Cognition in Free-ranging Animals

An animal&#39;s ability to perceive and learn about its environment plays a key role in many behavioral processes, including navigation, migration, dispersal and foraging. However, the understanding of the role of cognition in the development of navigation strategies and the mechanisms underlying these strategies is limited by the methodological difficulties involved in monitoring, manipulating the cognition of, and tracking wild animals. This study describes a protocol for addressing the role of cognition in navigation that combines pharmacological manipulation of behavior with high-precision radio telemetry. The approach uses scopolamine, a muscarinic acetylcholine receptor antagonist, to manipulate cognitive spatial abilities. Treated animals are then monitored with high frequency and high spatial resolution via remote triangulation. This protocol was applied within a population of Eastern painted turtles (Chrysemys picta) that has inhabited seasonally ephemeral water sources for ~100 years, moving between far-off sources using precise (&#177; 3.5 m), complex (i.e., non-linear with high tortuosity that traverse multiple habitats), and predictable routes learned before 4 years of age. This study showed that the processes used by these turtles are consistent with spatial memory formation and recall. Together, these results are consistent with a role of spatial cognition in complex navigation and highlight the integration of ecological and pharmacological techniques in the study of cognition and navigation.

This paper describes a novel protocol that combines the pharmacological manipulation of memory and radio telemetry to document and quantify the role of cognition in navigation.

An animal&#39;s ability to perceive and learn about its environment plays a key role in many behavioral processes, including navigation, migration, ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Experimental Study of the Relationship Between Particle Size and Methane Sorption Capacity in Shale

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard for evaluating the mining value of shale gas. Currently, studies on the correlation between particle size and methane adsorption are controversial. In this study, an isothermal adsorption apparatus, the gravimetric sorption analyzer, is used to test the adsorption capacity of different particle sizes in shale to determine the relationship between the particle size and the adsorption capacity of shale. Thegravimetric method requires fewer parameters and produces better results in terms of accuracy and consistency than methods like the volumetric method. Gravimetric measurements are performed in four steps: a blank measurement, preprocessing, a buoyancy measurement, and adsorption and desorption measurements. Gravimetric measurement is presently considered to be a more scientific and accurate method of measuring the amount of adsorption; however, it is time-consuming and requires a strict measuring technique. A Magnetic Suspension Balance (MSB) is the key to verify the accuracy and consistency of this method. Our results show that adsorption capacity and particle size are correlated, but not a linear correlation, and the adsorptions in particles sieved into 40 - 60 and 60 - 80 meshes tend to be larger. We propose that the maximum adsorption corresponding to the particle size is approximately 250 &#181;m (60 mesh) in the shale gas fracturing.

We use an isothermal adsorption apparatus, the gravimetric sorption analyzer, to test the adsorption capacity of different particle sizes of shale, in order to find out the relationship between particle size and the adsorption capacity of shale.

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard ...

Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is an accumulating evidence showing that the expressions of certain miRNAs are altered when grafting, an agricultural practice commonly used by farmers to improve crop tolerance to biotic and abiotic stresses. Bottle gourd is an inherently climate-resilient crop compared to many other major cucurbits, including watermelon, rendering it one of the most widely used rootstocks for the latter. The recent advancement of high-throughput sequencing technologies has provided great opportunities to investigate cold-responsive miRNAs and their contributions to heterograft advantages; yet, adequate experimental procedures are a prerequisite for this purpose. Here, we present a detailed protocol for efficiently generating homo- and heterografts between the cold-susceptible watermelon and the cold-tolerant bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis. The presented methods are also useful for other plant-grafting systems, to interrogate miRNA regulations under various environmental stresses, such as heat, drought, and salinity.

Here we present a detailed protocol for efficiently making homo- and heterografts between watermelon and bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis, for the investigation of cold-responsive microRNAs.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is ...

A Precise and Autonomous System for the Detection of Insect Emergence Patterns

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of emerging insects they can detect. In order to obtain precise measurement of insect emergence, it is necessary for systems to be semi-automated and able to measure large numbers of emerging insects. We addressed these issues by designing and building a system that is automated and can measure emergence of up to 1200 insects. We modified the existing &#34;falling-ball&#34; system using Arduino microcontrollers to automate data collection and expand the sample size through multiple data channels. Multiple data channels enable the user to not only increase their sample size, but also allows for multiple treatments to be run simultaneously in a single experiment. Furthermore, we created an R script to automatically visualize the data as a bubble plot, while also calculating the median day and time of emergence. The current system was designed using 3D printing so the user can modify the system to be adjusted for different species of insects. The goal of this protocol is to investigate important questions in chronobiology and stress physiology, using this precise and automated system to measure insect emergence patterns.

Measurement of insect emergence patterns requires precision. Existing systems are only semi-automated and sample size is limited. We addressed these issues by designing a system using microcontrollers to precisely measure the time of emergence of large numbers of emerging insects.