The Colorado potato beetle is one of the most serious pests of potato in temperate zone. Studies of the immunity and pathogenesis of this pest are mainly carried out at the active stage. However, in temperate zone, this pest spend most of its lifecycle in a state of hibernation.
In this study, we present a method for hibernation of Colorado potato beetle and a technique for collecting beetles in winter. The main advantage of this method is that it allows obtaining any number of overwintering individuals for virus analysis at any period of hibernation. Use cages made of a rigid wooden frame with a size of 25 by 25 by 40 centimeters.
To build a frame for the cage, use wooden slats at least two centimeters thick and four centimeters wide. Cover the inside of the cage with a stainless steel mesh with a size of no larger than five by three millimeters. Fix the mesh with a wood stapler.
Line the inside of the cage with a black synthetic geotextile with a density of 60 grams per square meter. Tightly attach a tube of synthetic translucent breathable fabric, approximately 60 centimeters high to the top of the cage. Cross and fix two strong ropes to the bottom of the cage to pull it out from the soil when needed.
Dig a hole in the soil 40 centimeters deep. Lay dry grass or hay onto the hole. Place the cage inside so that the hay or dry grass is between the cage's walls and the soil.
Fill the cages up with soil from the same potato field where the insects are collected. Install waterproof temperature and humidity data loggers into the cages at required depths. Plant potato seedlings inside the cage three to four weeks prior to beetle introduction and water them moderately.
Fix a tube of synthetic fabric vertically to a stick of any material installed on the outside of the cage. Manually collect adult beetles in the pesticide-free potato fields toward the end of potato vegetation. Keep the collected beetles in 20 liter plastic buckets containing potato tops for feeding the insects before placing in the cages.
Cover the buckets with a breathable fabric. Place no more than 200 individuals of the beetles onto the potato plants covered by the synthetic fabric mesh. When the potato tops are consumed, add fresh ones set in a plastic jar containing water and change the potato tops daily afterwards.
Once all the beetles burrowed into the soil for overwintering, untie the tube of synthetic fabric from the stick and lay the fabric down. Remove the snow above the surface of the cage. Loosen the cage on each side with a strong shovel.
Pull the cage out the soil using ropes. Bring the cage to the lab. Remove the soil from the cage in small portions, carefully break up large pieces of soil and isolate beetles using tweezers.
Separate live beetles from cadavers. Live, healthy beetles compact soil around them forming an air cavity, a so-called cradle, and therefore are easily separated from the soil. Beetles killed by fungi are mummified or have visible mycelium on the surface.
Bacterially decomposing insects are dark. Sift the soil through a sieve to make sure all the beetles are isolated and not damaged. Place cadavers with symptoms of a fungal infection or bacterial decomposition in an individual sterile Falcon tube for future identification.
Store live beetles in a refrigerator at a temperature of 2 degrees Celsius until analysis in closed ventilated containers containing a damp cotton ball. To collect hemolymph, make a puncture in the lateral part of the abdomen under aorta with an insulin needle. 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 from fat and Malpighian vessels. Separate a desired section of the gut, such as the foregut, midgut or hindgut. To isolate the fat body, separate it from the other tissues after the isolation of the gut.
To isolate entomopathogenic fungi from the cadavers, put the mummified cadavers into a sterile humidity chamber. Use aerial conidia or sclerotia from the internal contents of the beetles for plating on Sabouraud dextrose agar with 0.4%lactic acid. Isolate bacteria from the cadavers with symptoms of bacterial decay.
Cut off a beetle's head, squeeze out the internal contents and collect into tubes for subsequent plating on media for bacteria. Temperatures below zero in the cages at a depth of 30 centimeters, were registered from the end of November to the beginning of April. The average temperature during this period was 3.3 0.1 Celsius.
The lowest registered temperature was 7.9 Celsius in mid-February. Insect mortality increased during the hibernation and reached a maximum in spring before the emergence. The initial number of beetles was 2, 000, of which 1, 470 individuals survived.
Thus, the survival rate of the beetles during the hibernation was 61%An analysis of 530 cadavers showed that during the hibernation period 53%of them were characterized by symptoms of bacterial decomposition and 25%were characterized by symptoms of fungal infection. Beauveria dominated among the isolated cultures of entomopathogenic fungi. Metarhizium, Cordyceps and Lecanicillium were much less common.
Among the bacteria isolated from the cadavers with symptoms of bacterial decomposition, species belonging to genera Bacillus, Sphingobacterium, Peribacillus, Pseudomonas, Serratia, Rahnella and Glutamicibacter were identified. Using this method can solve the problem of obtaining Colorado potato beetles from the field in winter. It allows getting as much individuals as you need with the low mortality rate of the insects.
The study of various aspects of the beetles hibernation is important from both fundamental and applied points of view for improving approach to the control of this pest. And of course, this technique can be adapted for other insect species overwintering in the soil.
