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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.

Representative 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.

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).

Figure 1: Soil temperature dynamics. Soil temperature dynamics as measured by a waterproof temperature data logger installed at a depth of 30 cm. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Figure 3: Infections in overwintering Colorado potato beetle cadavers. Please click here to view a larger version of this figure.

Supplemental Materials: Please click here to download the below Supplemental Files.

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's test, P < 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.

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 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 °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 (≤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'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 °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 fungi^14,15,16 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 20×20 cm to 90×90×90 cm (ref.¹³) or 180×60×30 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.

Materials

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