We thank our colleagues Vladimir Shilo, Vera Morozov&#1072;, Ulyana Rotskaya, Olga Polenogova, and Oksana Tomilova for their help with organizing and execution of the field and laboratory procedures.
The research was supported by the Russian Science Foundation, project No. 22-14-00309.

1. Description of the cages for hibernation
NOTE: Depending on the aims of the experiment, the number of cages varies. Use at least three cages per sampling date. To estimate the number of beetles that will emerge, prepare at least three additional cages, which will not be taken out of the soil until spring.
<ol>
	<li>Use cages made of a rigid wooden frame with a size of 25&#160;&#215; 25&#160;&#215; 40 cm (L&#160;&#215; W&#160;&#215; H).</li>
	<li>To build a frame for the cage, use wooden slats at least 2 cm thick and 4 cm wide.</li>
	<li>Cover the inside of the cage with a stainless-steel mesh having a size of openings no larger than 5 mm &#215; 3 mm. Use a wood stapler to fix the mesh.</li>
	<li>Fix the stainless-steel mesh to the outside of the bottom with the stapler.</li>
	<li>Line the inside of the cage with a black synthetic geotextile with a density of 60 g/m2. 
	NOTE: The geotextile serves as an additional barrier to prevent the beetles&#39; escape. Do not use it in experiments related to actively moving entomopathogens and parasitoids.</li>
	<li>Tightly attach a tube of synthetic translucent breathable fabric approximately 60 cm high to the top of the cage.</li>
	<li>Cross and fix two strong ropes to the bottom of the cage to pull it out from the soil when needed.</li>
</ol>
2. Installation of the cages
<ol>
	<li>Dig a hole 40 cm deep in the soil and place the cage inside.</li>
	<li>Lay dry grass or hay onto the hole.</li>
	<li>Place the cage inside so that the hay or dry grass is between the cage&#39;s walls and the soil.</li>
	<li>Fill the cages up with soil from the same potato field where the insects are collected.</li>
	<li>Install waterproof temperature and humidity data loggers into the cages at required depths. 
	NOTE: Data loggers from any manufacturer may be used and must be able to operate at low temperatures.</li>
	<li>Plant potato seedlings inside each cage 3-4 weeks prior to beetles&#39; introduction and water them moderately.</li>
	<li>Fix a tube of synthetic fabric vertically to a stick of any material installed on the outside of the cage.</li>
</ol>
3. Rearing of insects before overwintering
<ol>
	<li>Manually collect adult beetles in pesticide-free potato fields toward the end of potato vegetation. 
	NOTE: Adult beetles differ substantially from larvae and are characterized by striped elytra, whereas larvae are red.</li>
	<li>Keep the collected beetles in 15-20 L plastic buckets (200 individuals max per bucket) containing potato tops for feeding the insects before placing them into the cages.</li>
	<li>Cover the buckets with breathable fabric. 
	NOTE: Do not keep insects in buckets for more than 12 h. Use large enough potato tops to prevent the accumulation of beetles at the bottom of the buckets.</li>
	<li>Place no more than 200 CPB individuals on the potato plants covered with the synthetic fabric mesh.</li>
	<li>When the potato tops are consumed, add fresh ones set in a plastic jar containing water and change the potato tops daily afterward. 
	NOTE: To fix stems in a jar, use cotton wool and parafilm. Carefully check old stems for the beetles when removing them.</li>
	<li>Once all the beetles are burrowed into the soil for overwintering, untie the tube of synthetic fabric from the stick and lay the fabric down.</li>
</ol>
4. Collection of insects during the winter season
<ol>
	<li>Remove snow above the surface of the cage.</li>
	<li>Loosen the cage on each side with a strong shovel.</li>
	<li>Pull the cage out of the soil using the ropes.</li>
	<li>Bring the cage to the lab. 
	NOTE: Depending on the aims of the experiment, hibernating beetles may have to be inactive prior to the analysis. In this case, the temperature in the laboratory during the isolation of beetles from the soil should be ~2-5 &#176;C.</li>
	<li>Remove the soil from the box in small portions, carefully break up large pieces of soil, and isolate beetles using tweezers.</li>
	<li>Separate live beetles from cadavers. Live healthy beetles create compact soil around them, forming an air cavity (a so-called cradle), and are, therefore, easily separated from the soil. Beetles killed by fungi are mummified or have visible mycelium on the surface. Bacterially decomposing insects are dark.</li>
	<li>Sift the soil through a sieve to make sure all the beetles are isolated and not damaged.</li>
	<li>Place cadavers with symptoms of a fungal infection or bacterial decomposition in an individual sterile 15 mL centrifuge tube for future identification.</li>
	<li>Store live beetles in a refrigerator at a temperature of 0-2 &#176;C until analysis in a closed-ventilated container containing a damp cotton ball.</li>
</ol>
5. Preparation of organ and tissue samples
<ol>
	<li>To collect hemolymph, make a puncture in the lateral part of the abdomen under elytra using an insulin needle. 
	NOTE: During overwintering, the amount of hemolymph is significantly reduced, which makes it difficult to collect this liquid.</li>
	<li>To isolate the gut, cut off the head capsule, squeeze out all the contents into a Petri dish with phosphate buffer, separate the gut, and cleanse it of fat and Malpighian vessels.</li>
	<li>Separate a desired section of the gut, such as the foregut, midgut, or hindgut.</li>
	<li>To isolate the fat body, separate it from other tissues after the isolation of the gut. 
	&#8203;NOTE: The isolated tissues can be used for measuring the activity of antioxidant and detoxifying enzymes (an example: Supplementary Figure 1), analysis of regulation of immune signaling pathway genes (an example: Supplementary Figure 2), or metabarcoding of insect gut contents, etc.</li>
</ol>
6. Isolation of microorganisms from the cadavers
<ol>
	<li>To isolate entomopathogenic fungi from the cadavers, put the mummified insects into a sterile humidity chamber.</li>
	<li>Use aerial conidia (if available) or sclerotia from the internal contents of the beetles for plating on Sabouraud dextrose agar with 0.4% lactic acid. 
	NOTE: Use beetles with mycelium and conidia for plating immediately (without placing them in humidity chambers).</li>
	<li>Isolate bacteria from the cadavers with symptoms of bacterial decomposition.</li>
	<li>Cut off a beetle&#39;s head, squeeze out the internal contents, and collect them into tubes for subsequent plating on media for bacteria (Luria-Bertani agar, endo agar, and bile esculin agar). 
	NOTE: Use microscopy and molecular methods to identify genera and species of the pathogens. If necessary, an analysis for the presence of other parasites can be performed.</li>
</ol>

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The authors declare that there are no competing interests.

This study shows that the proposed method for studying the overwintering of CPBs enables us to obtain a sufficient number of insects in different periods of hibernation. The success of the presented technique depends on several independent factors, the most important of which is weather conditions. In a cold, snowless winter, the soil may freeze to the entire depth of the cage. In this case, the risk of death of all beetles goes up significantly18. The survival of the beetle depends on a combination of many factors, which can vary significantly from year to year6.
In the experiment, the soil temperature inside the cages during winter did not fall below minus 7.9 &#176;C. No ice was observed at a depth greater than 25 cm, and the soil remained loose even during the period of the greatest cooling (January-February). Most beetles accumulated in the lower part of each cage, at a depth of 30-40 cm. The beetles might have burrowed deeper with a greater depth of the cages. On the other hand, increasing the depth of the cage would lead to an increase in weight, making it challenging to extract the cage from the soil, especially in winter. Moreover, according to our observations in potato fields of the region under study, the CPB does not burrow deeper than 35 cm for overwintering. This finding can be explained by the clay soil layer at the depth of 30-35 cm, which the beetles cannot overcome. In our experiment, sandy, loamy soil from the upper 30 cm horizon was used. This is probably the reason why the beetles were able to burrow deeper than under natural conditions. The depth at which the CPB hibernates is typically 10-25 cm (ref. 1), but this may vary among geographic regions. For example, in the northeastern United States (New Jersey), most of the beetles hibernate at a depth of 10-13 cm (ref. 13). Similar overwintering depths of beetles (&#8804;15 cm) have also been documented in Wisconsin, USA18. In the southern Urals (Russia), the depth at which the CPB burrows for overwintering is in the range of 5-30 cm (ref. 20). It should be noted that the survival rate of insects does not always positively correlate with an increase in overwintering depth1. Indeed, in a field overwintering experiment in Estonia6, it was shown that the survival rate of the CPB was higher at a depth of 30 cm than at a depth of 50 cm. Those authors propose that this finding may be due to the lack of oxygen. Similar data were obtained in a field experiment in Wisconsin, USA18: the highest survival rate (51.5%) of overwintering CPBs was recorded at a depth of 15-25 cm. At the same time18, 100% mortality of the beetle was noted at a depth of 25-35 cm. We believe that a depth of 40 cm is sufficient for experiments in the temperate zone because the percentage of surviving beetles was high, and the soil freezing did not extend to the entire depth of the cage. The presence of snow cover contributes to the lesser cooling of the soil. If necessary, the thickness of the snow cover above the surface of the cages can be adjusted.
Another key point in the protocol is the possible insufficient readiness of the CPB for overwintering owing to a small amount of stored nutrients. Some adult CPBs remained on the soil surface after the mass burrowing of the beetles into the soil. It is possible that they did not store enough fat because the success of overwintering depends also on the amount of accumulated nutrients21. Additionally, when the cages were removed from the soil in the winter, some of the beetles were in a frozen state on the soil surface or in the near-surface layer. Perhaps these were the beetles that did not have enough energy to burrow due to malnutrition, infections, or other damaging factors. Lashomb et al.13 noted in experiments with CPB overwintering that ~15% of adults did not burrow into the soil for overwintering. Those authors did not discuss the reasons. In any case, it is necessary to provide beetles with enough food.
Depending on a study&#39;s objectives, it may be necessary to keep beetles in a hibernation state after they are collected from the soil. To this end, the laboratory temperature should be cool, and the beetles should be immediately placed into conditions of 0-2 &#176;C after the extraction from the soil. It was observed in our work that in autumn and spring, CPBs almost immediately start locomotor activity after being excavated from the soil; this process takes place much more slowly in the middle of winter.
This study did not take into account actively moving entomopathogens and parasitoids. We used a geotextile as an additional barrier against the spread of the CPB. Note that a geotextile should not be used in research on actively moving entomopathogens (e.g., entomopathogenic nematodes), predators, or parasitoids because it will impede their movement through it.
It is important to point out that studies on the immunity and diseases of the CPB are mostly conducted during active feeding stages. Resting stages are less investigated and have been examined mainly under laboratory conditions. Under these conditions, however, it is difficult to simulate the fluctuations of temperature, humidity, and aeration that occur at natural breeding sites. Thus, field experiments are preferable22. To determine the causes of CPB winter mortality in the field in different hibernation periods, it is necessary to excavate overwintering beetles from the soil. Studies on CPB overwintering under natural conditions were actively conducted in the 1970s-1980s. The methods described in those papers mainly consist of collecting and counting individuals that emerge in spring13, evaluating the effectiveness of using entomopathogenic fungi14,15,16 or entomopathogenic nematodes15 before overwintering, and collecting overwintering beetles from the soil in spring and autumn to determine natural winter mortality17. At the same time, sizes and shapes of the cages used in those experiments varied from 20&#215;20 cm to 90&#215;90&#215;90 cm (ref.13) or 180&#215;60&#215;30 cm (ref.18). It should be pointed out that the aforementioned studies were not aimed at developing the methodology for CPB overwintering with the possibility of collecting insects in winter. Unlike existing methods, the technique described in this article makes it possible to investigate natural CPB populations in a snow period.
In conclusion, the proposed method enables researchers to obtain the desired number of overwintering CPB individuals under natural conditions with relatively low mortality of the insects and at a low cost. Investigation of various aspects of CPB hibernation is essential from both basic-research and applied points of view: for improving approaches to the control of this pest. This technique can be adapted to other insect species overwintering in soil. Future researchers may apply this method to study general physiology and biochemistry-including immunity of overwintering phases-of various insect species. In addition, this method can be used for predicting the abundance of pests of interest, on the basis of their winter mortality.

The Colorado potato beetle Leptinotarsa decemlineata Say (CPB) is an important pest of Solanaceae plants, predominantly potato Solanum tuberosum L. The geographic range of this species is more than 16 million km2 and constantly expands1. The CPB has facultative winter diapause, and hibernation is obligatory in the temperate zone. The diapause is induced by a short-day photoperiod and modulated by temperature1. These beetles overwinter in the adult stage by burrowing into soil. With increasing latitudes, the duration of the hibernation period extends. In the temperate zone, especially on northern territories of its range, the overwintering lasts up to 9 months: from August-September until May-June (Noskov et al., personal observations). During this period, the CPB-just like any other insect in the temperate zone-is exposed to unfavorable winter conditions and must increase its cold tolerance. At the same time, contact of the beetles with soil increases the risk of infection by various opportunistic and pathogenic microorganisms2. Therefore, these beetles need to maintain a certain level of immune-system activity during hibernation, which is also energetically costly. Nonetheless, even if the insect survives an infection, the disease may reduce its cold hardiness3. It should be noted that low temperature is not the only reason for winter mortality of the CPB. An important role is also played by the lack of oxygen, and under some conditions, it could be the main factor of winter mortality4,5.
It is known that natural winter mortality of the CPB can be very high, reaching 100% in clay loam soils6. Thus, overwintering is one of the most crucial periods in the CPB life cycle. Nevertheless, data on the physiology, immune-system activity, survival, and other parameters of CPB hibernation under natural conditions are still limited. There are studies on differential gene expression and various physiological parameters in CPB adults during the diapause and in response to cold shock7,8,9,10,11,12; however, these analyses have mainly been carried out by induction of diapause or cold stress under laboratory conditions without natural fluctuations of temperature, humidity, and native pathogen load. Nonetheless, research on the physiology of these beetles collected by excavation from soil under natural conditions is important. Different aspects of CPB overwintering under natural conditions were actively studied in the 1970s-1980s13,14,15,16,17,18. On the other hand, these studies did not involve CPB excavation from the soil in winter. In addition, a technique for controlled hibernation of the CPB and a description of the cages are not provided in detail. Thus, investigation into the physiology of CPBs overwintering in natural settings is needed19.
The aim of this study was to develop and test a method for controlled hibernation of CPB adults under natural conditions. The proposed method allows to obtain a desired number of CPB individuals for microbiological, immunological, and other assays during hibernation under field conditions of a continental climate. This method can be adapted and applied to other insect species overwintering in soil under snow.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar-agar bacteriological purified</td>
			<td>diaGene</td>
			<td>1806.5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bile Esculin Agar</td>
			<td>HiMedia</td>
			<td>M972</td>
			<td></td>
		</tr>
		<tr>
			<td>Endo Agar&#160;</td>
			<td>HiMedia</td>
			<td>M029</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose monohydrate-D</td>
			<td>PanReac Applichem</td>
			<td>143140.1000&#934;</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactic acid&#160;</td>
			<td>PanReac Applichem</td>
			<td>141034.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria-Bertani liquid medium</td>
			<td>HiMedia</td>
			<td>G009</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical centrifuge tubes</td>
			<td>Axygen</td>
			<td>SCT-15ML-25-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>FBIS SRCAMB</td>
			<td>M<img alt="Equation 1" src="/files/ftp_upload/65862/65862eq01v2.jpg" style="margin: auto;" />030/O61</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Medigen</td>
			<td>PBS500</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperatutre and humidity datalogger Ecklerk-M-11</td>
			<td>Relsib</td>
			<td></td>
			<td>Waterproof datalogger</td>
		</tr>
	</tbody>
</table>

an experimental study on colorado potato beetle hibernation under natural conditions

One of the major pests of potato Solanum tuberosum L. in the temperate zone is the insect Colorado potato beetle (CPB). Most studies on the immunity and diseases of the CPB are conducted during active feeding stages. Nonetheless, there are fewer studies on resting stages, although these beetles spend most of their life cycle in a state of winter diapause (hibernation). In this work, a method for investigating CPB hibernation under natural conditions was developed and tested, offering an opportunity to collect a sufficient number of individuals in winter. In this article, CPB survival was assessed, and infectious agents at different stages of hibernation were identified. CPB mortality increased during the hibernation, reaching a maximum in April-May. Entomopathogenic fungi (Beauveria, Isaria, and Lecanicillium) and bacteria Bacillus, Sphingobacterium, Peribacillus, Pseudomonas, and Serratia were isolated&#160;from the dead insects. The survival rate of the beetles for the entire hibernation period was 61%. No frozen or desiccated beetles were found, indicating the success of the presented method.

The results below on overwintering CPBs show soil temperature, survival, and infections.
Soil temperature dynamics. 
Temperatures below zero in the cages at a depth of 30 cm were registered from the end of November to the beginning of April (Figure 1). The average temperature during this period was minus 3.3 &#177; 0.1 &#176;C (mean &#177; standard error). The lowest recorded temperature was minus 7.9 &#176;C in mid-February.
Survival of overwintering CPBs. 
Insect mortality was seen during hibernation and reached a maximum in spring before emergence. The initial number of beetles was 2000, of which 1470 individuals survived by the end of May. The survival rate of the beetles during the hibernation was 61% (Figure 2).
Infections in overwintering CPBs. 
An analysis of 530 dead beetles showed that during the hibernation period, 53% of them had symptoms of bacterial decomposition, and 25% had symptoms of fungal infections (Figure 3). Beauveria dominated (45 isolates) among the isolated cultures of entomopathogenic fungi. Metarhizium, Cordyceps (=Isaria), and Lecanicillium were much less common (two isolates each). Among the bacteria isolated from the cadavers with symptoms of bacterial decomposition (n = 30), species belonging to genera Bacillus, Sphingobacterium, Peribacillus, Pseudomonas, Serratia, Rahnella and Glutamicibacter were identified (Supplementary Table 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65862/65862fig01.jpg" /> 
Figure 1: Soil temperature dynamics. Soil temperature dynamics as measured by a waterproof temperature data logger installed at a depth of 30 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65862/65862fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65862/65862fig02.jpg" /> 
Figure 2: Survival of overwintering Colorado potato beetles in different periods of hibernation. The cages were dug out, and the surviving and dead beetles were counted in November, January, April, and May. Bars represent the number of surviving beetles. Whiskers indicate standard error. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65862/65862fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65862/65862fig03.jpg" /> 
Figure 3: Infections in overwintering Colorado potato beetle cadavers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65862/65862fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Materials:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65862/ESM.zip" target="_blank">Please click here to download the below Supplemental Files.</a>
Supplementary Figure 1. Activity of nonspecific esterases in the midgut of the CPB during the hibernation. Whiskers denote standard error. Different letters indicate significant differences between time points (Dunn&#39;s test, P &#60; 0.05).
Supplementary Figure 2: Alterations of the expression of transcription factor NFkB (IMD pathway) in the gut and fat body of the CPB during hibernation. Data are presented as fold changes relative to an August time point. Rp4, Rp18, and Arf19 were used as reference genes. Whiskers show standard error.
Supplementary Table 1: Putative identification of 16S rRNA (~800 bp) gene sequences of bacteria isolated from a dead CPB during hibernation.

Watch this Scientific Journal Video about An Experimental Study on Colorado Potato Beetle Hibernation Under Natural Conditions at JoVE.com

An Experimental Study on Colorado Potato Beetle Hibernation Under Natural Conditions

Here we present a method for studying Colorado potato beetle hibernation under the natural conditions of the temperate zone as well as a technique for collecting beetles in winter. This method allows to obtain a desired number of overwintering individuals for various analyses at any stage of hibernation.

One of the major pests of potato Solanum tuberosum L. in the temperate zone is the insect Colorado potato beetle (CPB). Most studies on the immunity and ...

an-experimental-study-on-colorado-potato-beetle-hibernation-under

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4.1K Views.  Institute of Systematics and Ecology of Animals of Siberian Branch of Russian Academy of Sciences. Here we present a method for studying Colorado potato beetle hibernation under the natural conditions of the temperate zone as well as a technique for collecting beetles in winter. This method allows to obtain a desired number of overwintering individuals for various analyses at any stage of hibernation.

Video: An Experimental Study on Colorado Potato Beetle Hibernation Under Natural Conditions

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.
Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.

Current HIV-1 strategies act to suppress the viral life cycle but do not effectively eradicate infection. Here, we demonstrate that an engineered recombinase can efficiently excise integrated HIV-1 proviral DNA from the genome of infected cells.

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies ...

Processing the Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. 
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips&trade; are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48&deg;C in high humidity conditions. After hybridization, a series of standard washes are done at 53&deg;C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract3,4. In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins5-9. The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms8,10-12. As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media.
Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods13. Secreted mucins and lipids are extracted from the tissue during the paraffin-embedding clearing step, which requires prolong incubation with organic solvents (xylene or Citrisolv). Therefore this approach is sub-optimal for studies focusing on the nature and distribution of mucins and mucus in vivo.
In contrast, freezing tissues in Optimal Cutting Temperature (OCT) embedding medium avoids dehydration and clearing of the sample, and maintains the sample hydration. This allows for better preservation of the hydrated mucus layer, and thus permits the study of the numerous roles of mucins in epithelial biology. As this method requires minimal processing of the tissue, the tissue is preserved in a more natural state. Therefore frozen tissues sections do not require any additional processing prior to staining and can be readily analyzed using immunohistochemistry methods.
We demonstrate the preservation of micrometers-thick secreted mucus layer in frozen colon samples. This layer is drastically reduced when the same tissues are embedded in paraffin. We also demonstrate immunofluorescence staining of glycan epitopes presented on mucins using plant lectins. The advantage of this approach is that it does not require the use of special fixatives and allows utilizing frozen tissues that may already be preserved in the laboratory.

Unfixed frozen tissue samples embedded in Optimal Cutting Temperature medium (OCT) can be used to study natural distribution and glycosylation of secreted mucus. In this approach tissue processing is minimal and the natural presentation of glycolipids, mucins and glycan-epitopes is preserved. Tissue sections can be analyzed by immunohistochemistry using fluorescence or chromogenic detection.

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...

An Experimental Study on Colorado Potato Beetle Hibernation Under Natural Conditions

Summary

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Chapters in this video

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Computer-Generated Animal Model Stimuli

Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase

Processing the Loblolly Pine PtGen2 cDNA Microarray

Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions