We are grateful to Drs. Aravinthan Samuel and Marc Gershow who helped develop the initial version of the behavioral apparatus and Matthew Batie who modified the behavioral apparatus. This research was supported by Trustee Grant from Cincinnati Children&#8217;s Hospital, JST/PRESTO, March of Dimes and NIH R01 GM107582 to F.N.H.

1. Preparation of Flies
<ol>
	<li>Light Dark (LD) Experiments
	<ol>
		<li>Raise flies in incubators (25 &#176;C/40-60%relative humidity (RH)) under light 12 hr/dark 12 hr (LD) cycles. The light intensity of the incubators is ~500-1,000 lux.</li>
		<li>Two incubators are necessary to complete the behavior assays over a 24 hr period. Both incubators should have a programmable light with ON OFF functions. They should also have solid doors that are not permeable to light (i.e. no glass or plexiglass). 
		Note: One incubator should be designated a &#8220;day&#8221; incubator and set to a LD cycle of 12 hr&#160;light and 12 hr&#160;dark. The second incubator should be designated a &#8220;night&#8221; incubator and set to the inverse of the first with 12 hr&#160;dark followed by 12 hr&#160;light. The night incubator must be placed in the room that is accessible in the dark, in such a way flies experiencing night conditions can be easily accessed for the experiments.</li>
		<li>Place the fly vials in either day or night incubators. Collect newly hatched flies in a fresh vial, 20-30 per assay and keep in the same incubator for 2-3 days.</li>
		<li>After 2-3 days, use flies for the temperature preference behavioral assay.
		<ol>
			<li>For day (Zeitgeber time (ZT) 0-12) experiments, collect flies from the day incubator.</li>
			<li>Right before the behavioral experiments, take the collected fly vials out of the day incubator.</li>
			<li>For night (ZT 13-24) experiments, collect flies from the night incubator.</li>
			<li>Right before the behavioral experiments, take the collected fly vials out of the night incubator, wrap with aluminum foil and place in a box in the dark room under a red lamp. 
			Note: Since the temperature preference behavioral assay is performed under darkness for the night experiments, light exposure to the flies must be prevented until the end of the behavioral experiments. 
			Note: The flies should not be exposed to carbon dioxide on the day the experiments are to take place.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Constant Darkness (DD) Experiments
	<ol>
		<li>DD Day
		<ol>
			<li>An extra incubator is necessary for DD day experiments,&#160;which we refer to as a &#34;transition&#34; incubator for the rest of the manuscript. The transition incubator must be placed in the room that is accessible in the dark, in such a way flies experiencing DD conditions can be easily accessed for the experiments. An example light schedule for a transition incubator would have the light ON from 1 pm-7 pm and the light OFF at 7 pm-1 pm (Figure&#160;1).</li>
			<li>Collect the flies which have been raised in the day incubator. Place the fly vials in the transition incubator between 1 pm-7 pm, when the light is ON. In this way, the flies are properly exposed to light until 7 pm, at which time the light shuts OFF.</li>
			<li>The next day, before 1 pm, under dark conditions, take the fly vials out of the transition incubator, wrap them with aluminum foil and place them in a box. Keep the box in any incubator for one more days.</li>
		</ol>
		</li>
		<li>DD Night
		<ol>
			<li>Collect the flies which have been raised in the night incubator in the dark under a red lamp. Or additionally collect the flies when the night incubator&#39;s light is ON.</li>
			<li>Wrap collected vials with aluminum foil in the dark anytime the lights are OFF and place the vials in a box. Keep the box in any incubator for two more days (Figure&#160;1B). 
			Note: Since the temperature preference behavioral assay is performed under darkness for the night experiments, light exposure to the flies must be prevented until the end of the behavioral experiments.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Constant Light (LL) Experiments
	<ol>
		<li>LL Day
		<ol>
			<li>An extra incubator is necessary for LL day experiments. This incubator maintains the LL condition (25 &#176;C, 800 lux), with the light ON continuously.</li>
			<li>Collect flies which have been raised in the day incubator. Place the fly vials in the LL incubator anytime during their &#34;day&#34;.</li>
		</ol>
		</li>
		<li>LL Night
		<ol>
			<li>Collect flies which have been raised in the night incubator. Transfer the fly vials from the night incubator to the LL incubator during the time the light in the night incubator is ON. 
			Note: For example, the light turns OFF at 7 am in the night incubator. Transfer the fly vials from the night incubator to the LL incubator before 7 am and keep the vials in the LL incubator for 4 more days. 
			Note: At day 4 in LL conditions, the oscillation of the locomotor activity is abolished15,16, while the TPR is still sustained&#160;3.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. The Apparatus for the Temperature Preference Behavioral Assay
<ol>
	<li>Place a plexiglass cover (29 cm x 19.2 cm) (Figure&#160;4) on an aluminum plate.</li>
	<li>Monitor the air temperature between the plate and the cover. Six temperature probes are attached at various positions on the inside of the cover within one of the lanes (Figure&#160;2). 
	Note: Make sure the probes do not touch either the aluminum plate or the plexiglass cover. The air temperature should be set to a gradient from 18-32 &#176;C.</li>
	<li>Place the apparatus in an environmental room maintained at 25 &#176;C/65-75% RH. This room needs to be sealed off from any outside light. An environmental room is normally equipped with fans to maintain a certain temperature and humidity. 
	Note: The air from the fan likely disturbs a stable temperature gradient on the apparatus. To prevent this, we use a transparent sheet that covers the area surrounding the apparatus.</li>
	<li>Prepare a thermometer and hygrometer to check the temperature and humidity in the environmental room. 
	Note: Light influences the temperature preference of Drosophila3. The same intensity of light should be supplied on the apparatus uniformly. The intensity of our environmental room lights is ~800 lux. 
	Note: When the behavioral experiments are done, place a tube connected from the CO2 tank or supply near the hole of the top of the apparatus in order to anesthetize and get rid of the flies.</li>
</ol>
3. Preparation of Apparatus for Use
<ol>
	<li>Turn on the apparatus for at least 30 min in order to establish the temperature gradient properly on the surface of the plate (Figure&#160;3).</li>
	<li>Coat the cover of the behavior apparatus with water repellent to prevent flies from climbing the walls or ceiling of the cover. Wipe the excess water repellent off and leave the cover for 25-30 min to dry.</li>
	<li>Remove any condensation on the aluminum plate. Place the plexiglass cover on the aluminum plate and secure with six C-clamps (Figure&#160;2). 
	Note: It is very important that the cover is sealed well, if necessary double stick tape may be used.</li>
	<li>Leave the cover for at least 15 min. The air temperature gradient between the aluminum plate and the cover is created from 18-32 &#176;C.</li>
</ol>
4. Temperature Preference Behavior Assay
<ol>
	<li>Load the flies into the space between the aluminum plate and the plexiglass cover of the apparatus through small holes in the center of each lane of the cover (Figures&#160;2 and 4). Cover the holes with cover slips to prevent the flies from escaping.</li>
	<li>For the dark conditions, turn OFF all the lights in the environmental room. A red lamp can be used when the flies are placed into the apparatus. Make sure the flies are not exposed to any lights except a red lamp until the behavioral experiments are done.</li>
	<li>For each trial, use 20-25 adult flies, which are not to be reused in subsequent trials. The behavioral assay is performed for 30 min. Take a few pictures with or without a flash. Be careful not to make much noise or any sudden movements during the experiments.</li>
	<li>Record the temperature of all six probes on the apparatus. Take note of the room temperature as well as the humidity.</li>
	<li>Anesthetize the flies in the apparatus with carbon dioxide gas, loosen the clamps, remove the plexiglass cover, and remove the flies from the plate. After each experiment, the flies are discarded. Wipe any condensation or moisture off the plate. Replace the cover on the plate and tighten with the clamps in preparation for the next experiment.</li>
	<li>In order to have a representation of the temperature preference throughout the entire day, the 24 hr period is split into eight time zones, four during the day and four at night. For example, we use these ZT or CT 1-3, 4-6, 7-9, 10-12, 13-15, 16-18, 19-21 and 22-24. 
	Note: Since phenotype variations caused by masking effects are expected right after the light is turned ON (ZT0) or OFF (ZT12), we do not examine the temperature preference behavior during these times (ZT or CT 0-1, 11.5-13 and 23.5-24). At least five trials must be done in each time zone in order for the results to be statistically sound.</li>
</ol>
5. Data Analysis
<ol>
	<li>Calculate the temperature gradient as follows: Determine where the temperature probes are placed based on the two rulers that are placed on the top and bottom side of the plexiglass cover along the edges (Figure&#160;2A).</li>
	<li>The temperature gradient between the temperature probes is estimated to be linear. Based on the locations of the temperature probes, as well as their corresponding recorded temperatures, draw lines representing each degree of temperature in the appropriate position on the pictures. Count the number&#160;of flies located in each degree&#160;interval. Exclude any flies on the walls or the ceiling of the cover.</li>
	<li>Calculate the percentage of the flies in each temperature range of each lane. A mean preferred temperature is calculated by summing the products of each interval&#39;s percentage of flies and temperature, as shown below: 
	% of flies x 18.5 &#176;C + % of flies x 19.5 &#176;C + % of flies x 20.5 &#176;C &#8230;.+ % of flies x 31.5 &#176;C + % of flies x 32.5 &#176;C.</li>
	<li>Calculate the average preferred temperature in each time zone: Temperature preference behavior is performed &#62;5 times during each time zone (ZT 1-3, 4-6, 7-9, 10-12, 13-15, 16-18, 19-21, and 22-24). To calculate the average preferred temperature in each time zone, the mean preferred temperature of each trial are averaged together. The s.e.m. error bars are equal to the error between the trials.</li>
</ol>

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There is nothing to disclose.

Here, we illustrate the details of the temperature preference behavioral apparatus and analysis of the TPR behavior. Drosophila exhibit the salient, robust, and reproducible features of clock-controlled TPR. However, our data suggests that at least two factors, ambient light and age, significantly disturb the TPR behavioral phenotypes.
We observe that light significantly affects temperature preference in Drosophila. It is consistent with the fact that w1118</em...

Temperature is a ubiquitous environmental cue. Animals exhibit a variety of behaviors in order to avoid harmful temperatures and seek comfortable ones. Drosophila exhibit a robust temperature preference behavior6,7. When flies are released into a temperature gradient from 18-32 &#176;C, the flies avoid both warm and cold temperatures and finally choose a preferred temperature of 25 &#176;C in the morning3. The warm temperature sensors are a set of thermosensory neurons, AC neurons, which express Drosophila transient receptor potential (TPR) channel, TRPA16,9. The cold temperature sensors are located in the 3rd antennal segments, since ablating the 3rd antennal segments causes the lack of cold temperature avoidance6. Recently, the TRPP protein Brivido (Brv) was identified10. Since Brv is expressed in the 3rd antennal segments and mediates cold detection, Brv is a possible cold sensing molecule, which is critical for the temperature preference behavior. In sum, the flies use these two temperature sensors to avoid the warm and cold temperatures and find a preferred temperature.
While mammals generate heat to regulate their body temperature, ectotherms generally adapt their body temperatures to the ambient temperature11. Some ectotherms are known to exhibit a daily TPR behavior which is believed to be a strategy for the ectotherms to regulate their BTR12. To determine whether the flies exhibited TPR, we repeated the temperature preference behavioral analysis at various points during a span of 24 hr. We found that Drosophila exhibit a daily TPR, which is low in the morning and high in the evening and follows a pattern similar to that of BTR in humans13.
In Drosophila, there are ~150 clock neurons in the brain. The clock neurons that regulate locomotor activity are called&#160;M and E oscillators. However, interestingly, M and E oscillators do not regulate TPR, instead, we showed that DN2 clock neurons in the brain regulate TPR but not locomotor activity. These data indicate that TPR is regulated independently from locomotor activity. Notably, mammalian BTR is also independently regulated from locomotor activity. Ablation studies in rats show that BTR is controlled through specific SCN neurons that target a different subset of subparaventricular zone neurons than those that control locomotor activity14. Therefore, our data considers the possibility that the mammalian BTR and the fly TPR are evolutionally conserved3, since both fly TPR and mammalian BTR exhibit circadian clock-dependent temperature rhythms, which are independently regulated from locomotor activity.
Here, we describe the details of how to analyze the TPR behavioral assay in Drosophila. This method allows for the investigation of not only the molecular mechanism and neural circuits of TPR, but also how the brain integrates different environmental cues and inner biological clocks.

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	</colgroup>
	<tbody>
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			<td style="width:144px;">Water repellent: Apply the plexiglass cover</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:172px;">C-Clamp</td>
			<td style="width:109px;">Home Depot</td>
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</table>

design and analysis of temperature preference behavior and its circadian rhythm in drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

An example of the temperature preference rhythm is shown in Figure 5. If the behavior procedure is successfully done, the flies should exhibit a TPR in which they prefer a low temperature in the morning and higher temperature in the evening. The ~1-1.5 &#176;C increase during the daytime in temperature preference should be observed during the course of the day, regardless of the genetic background, since we showed that w1118, yw and&#160;Canton S flies exhibit a simi...

Watch this Scientific Journal Video about Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila at JoVE.com

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

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8.0K Views.  Cincinnati Childrens Hospital Medical Center. We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here we describe the design and analysis of TPR in Drosophila.

Video: Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

Circadian Entrainment of Drosophila Melanogaster

Nearly universal among organisms, circadian rhythms coordinate biological activity to earth&#39;s orbit around the sun. To identify factors creating this rhythm and to understand the resulting outputs, entrainment of model organisms to defined circadian time-points is required. Here we detail a procedure to entrain many Drosophila to a defined circadian rhythm. Furthermore, we detail post-entrainment steps to prepare samples for immunofluorescence, nucleic acid, or protein extraction-based analysis.

Here, we detail how to synchronize Drosophila to a circadian day. This is the first, and most important step necessary for studying biological rhythms and chronobiology.

Nearly universal among organisms, circadian rhythms coordinate biological activity to earth&#39;s orbit around the sun. To identify factors creating this ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...