An Experimental Study on Colorado Potato Beetle Hibernation Under Natural Conditions

Summary

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.

Abstract

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 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.

Introduction

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 mortality^4,5.

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 shock^{7,8,9,10,11,12}; 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-1980s^{13,14,15,16,17,18}. 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.

Protocol

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.

1. Use cages made of a rigid wooden frame with a size of 25 × 25 × 40 cm (L × W × H).
2. To build a frame for the cage, use wooden slats at least 2 cm thick and 4 cm wide.
3. Cover the inside of the cage with a stainless-steel mesh having a size of openings no larger than 5 mm × 3 mm. Use a wood stapler to fix the mesh.
4. Fix the stainless-steel mesh to the outside of the bottom with the stapler.
5. 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' escape. Do not use it in experiments related to actively moving entomopathogens and parasitoids.
6. Tightly attach a tube of synthetic translucent breathable fabric approximately 60 cm high to the top of the cage.
7. Cross and fix two strong ropes to the bottom of the cage to pull it out from the soil when needed.

2. Installation of the cages

1. Dig a hole 40 cm deep in the soil and place the cage inside.
2. Lay dry grass or hay onto the hole.
3. Place the cage inside so that the hay or dry grass is between the cage's walls and the soil.
4. Fill the cages up with soil from the same potato field where the insects are collected.
5. 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.
6. Plant potato seedlings inside each cage 3-4 weeks prior to beetles' introduction and water them moderately.
7. Fix a tube of synthetic fabric vertically to a stick of any material installed on the outside of the cage.

3. Rearing of insects before overwintering

1. 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.
2. 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.
3. 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.
4. Place no more than 200 CPB individuals on the potato plants covered with the synthetic fabric mesh.
5. 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.
6. 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.

4. Collection of insects during the winter season

1. Remove snow above the surface of the cage.
2. Loosen the cage on each side with a strong shovel.
3. Pull the cage out of the soil using the ropes.
4. 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 °C.
5. Remove the soil from the box in small portions, carefully break up large pieces of soil, and isolate beetles using tweezers.
6. 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.
7. Sift the soil through a sieve to make sure all the beetles are isolated and not damaged.
8. Place cadavers with symptoms of a fungal infection or bacterial decomposition in an individual sterile 15 mL centrifuge tube for future identification.
9. Store live beetles in a refrigerator at a temperature of 0-2 °C until analysis in a closed-ventilated container containing a damp cotton ball.

5. Preparation of organ and tissue samples

1. 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.
2. 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.
3. Separate a desired section of the gut, such as the foregut, midgut, or hindgut.
4. To isolate the fat body, separate it from other tissues after the isolation of the gut.
   ​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.

6. Isolation of microorganisms from the cadavers

1. To isolate entomopathogenic fungi from the cadavers, put the mummified insects into a sterile humidity chamber.
2. 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).
3. Isolate bacteria from the cadavers with symptoms of bacterial decomposition.
4. Cut off a beetle'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.

Results

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 ± 0.1 °C (mean ± standard error). The lowest recorded temperature was minus 7.9 °C in mid-February.

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Agar-agar bacteriological purified	diaGene	1806.5000
Bile Esculin Agar	HiMedia	M972
Endo Agar	HiMedia	M029
Glucose monohydrate-D	PanReac Applichem	143140.1000Φ
Lactic acid	PanReac Applichem	141034.1211
Luria-Bertani liquid medium	HiMedia	G009
15 ml conical centrifuge tubes	Axygen	SCT-15ML-25-S
Peptone	FBIS SRCAMB	M030/O61
Phosphate buffered saline	Medigen	PBS500
Temperatutre and humidity datalogger Ecklerk-M-11	Relsib		Waterproof datalogger