Name: an experimental study on colorado potato beetle hibernation under natural conditions
Uploaded: 2023-11-17T13:00:00
Description: Watch this Scientific Journal Video about An Experimental Study on Colorado Potato Beetle Hibernation Under Natural Conditions at JoVE.com

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 c...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+internal+and+external+lipids+of+nondiapausing+and+diapause+initiation+phase+adult+Colorado+potato+beetles,+Leptinotarsa+decemlineata.">A comparison of internal and external lipids of nondiapausing and diapause initiation phase adult Colorado potato beetles, Leptinotarsa decemlineata.</a> Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 159 (3), 163-170 (2011).</li><li>Nikonorov, Y. M., Syrtlanova, L. A., Kitaev, K. A., Benkovskaya, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+activity+of+genes+involved+in+diapause+regulation+in+the+Colorado+Potato+beetle+and+its+change+under+a+fipronil+impact.">Transcription activity of genes involved in diapause regulation in the Colorado Potato beetle and its change under a fipronil impact.</a> Russian Journal of Genetics: Applied Research. 8, 80-86 (2018).</li><li>Govaere, L., 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=Transcriptome+and+proteome+analyses+to+investigate+the+molecular+underpinnings+of+cold+response+in+the+Colorado+potato+beetle,+Leptinotarsa+decemlineata.">Transcriptome and proteome analyses to investigate the molecular underpinnings of cold response in the Colorado potato beetle, Leptinotarsa decemlineata.</a> Cryobiology. 88, 54-63 (2019).</li><li>Lehmann, P., Westberg, M., Tang, P., Lindstr&#246;m, L., K&#228;kel&#228;, 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+diapause+lipidomes+of+three+closely+related+beetle+species+reveal+mechanisms+for+tolerating+energetic+and+cold+stress+in+high-latitude+seasonal+environments.">The diapause lipidomes of three closely related beetle species reveal mechanisms for tolerating energetic and cold stress in high-latitude seasonal environments.</a> Frontiers in Physiology. 11, 576617 (2020).</li><li>Lebenzon, J. E., Torson, A. S., Sinclair, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diapause+differentially+modulates+the+transcriptomes+of+fat+body+and+flight+muscle+in+the+Colorado+potato+beetle.">Diapause differentially modulates the transcriptomes of fat body and flight muscle in the Colorado potato beetle.</a> Comparative Biochemistry and Physiology Part D: Genomics and Proteomics. 40, 100906 (2021).</li><li>Lashomb, J. H., Ng, Y. S., Ghidiu, G., Green, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+of+spring+emergence+by+the+Colorado+potato+beetle,+Leptinotarsa+decemlineata+(Say)+(Coleoptera:+Chrysomelidae),+in+New+Jersey.">Description of spring emergence by the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), in New Jersey.</a> Environmental entomology. 13 (3), 907-910 (1984).</li><li>Cantwell, G. E., Cantelo, W. W., Schroder, R. F. <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+Beauveria+bassiana+on+underground+stages+of+the+Colorado+potato+beetle,+Leptinotarsa+decemlineata+(Coleoptera:+Chrysomelidae).">Effect of Beauveria bassiana on underground stages of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).</a> The Great Lakes Entomologist. 19 (2), 6 (1986).</li><li>Fedorko, A., Bajan, C., Kmitowa, K., Wojciehowska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+the+use+of+selected+microorganisms+to+control+the+Colorado+beetle+during+hibernation.">Effectiveness of the use of selected microorganisms to control the Colorado beetle during hibernation.</a> Ecological Studies. 3 (2), 127-134 (1977).</li><li>Gaugler, R., Costa, S. D., Lashomb, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+and+efficacy+of+Beauveria+bassiana+soil+inoculations.">Stability and efficacy of Beauveria bassiana soil inoculations.</a> Environmental Entomology. 18 (3), 412-417 (1989).</li><li>Bajan, C., Kmitowa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+entomopathogenic+fungi+to+the+natural+winter+reduction+of+Colorado+beetle+adults.">Contribution of entomopathogenic fungi to the natural winter reduction of Colorado beetle adults.</a> Polish Ecological Studies. 3 (2), 107-114 (1977).</li><li>Milner, M., Kung, K. S., Wyman, J. A., Feldman, J., Nordheim, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+overwintering+mortality+of+Colorado+potato+beetle+(Coleoptera:+Chrysomelidae)+by+manipulating+the+temperature+of+its+diapause+habitat.">Enhancing overwintering mortality of Colorado potato beetle (Coleoptera: Chrysomelidae) by manipulating the temperature of its diapause habitat.</a> Journal of Economic Entomology. 85 (5), 1701-1708 (1992).</li><li>Do&#287;an, C., 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=Characterization+of+calcium+signaling+proteins+from+the+fat+body+of+the+Colorado+Potato+Beetle,+Leptinotarsa+decemlineata+(Coleoptera:+Chrysomelidae):+Implications+for+diapause+and+lipid+metabolism.">Characterization of calcium signaling proteins from the fat body of the Colorado Potato Beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae): Implications for diapause and lipid metabolism.</a> Insect Biochemistry and Molecular Biology. 133, 103549 (2021).</li><li>Benkovskaya, G. V., Udalov, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorado+potato+beetle+investigations+in+the+south+Urals.">Colorado potato beetle investigations in the south Urals.</a> Nova Science Publishers. , (2011).</li><li>Short, C. A., Hahn, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+enough+for+the+winter?+Does+nutritional+status+affect+diapause.">Fat enough for the winter? Does nutritional status affect diapause.</a> Journal of Insect Physiology. , 104488 (2023).</li><li>McDonald, J. R., Bale, J. S., Walters, K. F. 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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 combinati...

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.
<p class="jo...

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 ...

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