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 ...

Institute of Systematics and Ecology of Animals of Siberian Branch of Russian Academy of Sciences

The interaction between the entomopathogenic fungus Metarhizium robertsii and natural avermectin metabolites of the actinomycete Streptomyces avermitilis were investigated on Colorado potato beetle larvae. A synergy in the mortality of larvae was detected after simultaneous treatment with half-lethal doses of avermectins (commercial name actarophit) 0.005% and fungus (5×10conidia/ml). The treatment with avermectins led to rapid fungal colonization of the hemolymph. The defense strategies of insects infected by fungus and treated with avermectins and untreated insects were compared to investigate the mechanisms of this synergy. We have shown an increase in hemocytes, especially immunocompetent cells - plasmatocytes and granular cells in the initial stages of mycosis (third day post inoculation). In contrast, avermectins suppressed cellular immunity in hemolymph. Specifically, avermectins dramatically decreased the count of granular cells in larvae infected and uninfected with fungus. Apoptosis inducement and hemocyte necrosis under the influence of avermectins has been shown in vitro as one of the possible reasons for hemocyte mortality. In addition, avermectins enhanced the activity of phenoloxidases in integuments and hemolymph and increased the activity of glutathione-S-transferases activity in the fat body and hemolymph of infected and uninfected larvae, thereby intensifying the development of fungal infection by M. robertsii in Colorado potato beetle larvae. The combination of fungal infection and avermectins constitutes a new perspective for developing multicomponent bioinsecticides.

Immune-physiological aspects of synergy between avermectins and the entomopathogenic fungus Metarhizium robertsii in Colorado potato beetle larvae.

The synergistic effect between the entomopathogenic fungus Metarhizium robertsii and a sublethal dose of the bacterium Bacillus thuringiensis ssp. morrisoni var. tenebrionis was studied in terms of immune defense reactions and detoxification system activity of the Colorado potato beetle, Leptinotarsa decemlineata, fourth instar larvae. Bacterial infection led to more rapid germination of fungal conidia on integuments. We found a significant decrease of cellular immunity parameters, including total hemocyte count and encapsulation response, under the influence of bacteria. Phenoloxidase activity in integuments was increased under bacteriosis, mycosis and combined infection compared to controls. However, phenoloxidase activity in the hemolymph was enhanced under bacteriosis alone, and it was decreased under combined infection. Activation of both nonspecific esterases and glutathione-S-transferases in the hemolymph was shown at the first day of mycosis and third day of bacteriosis. However, inhibition of detoxification enzymes was detected under combined infection. The suppression of cellular immunity and detoxification reactions in Colorado potato beetle larvae with a sublethal dose of bacteria is discussed as a reason for synergy between B. thuringiensis and M. robertsii.

Immunological mechanisms of synergy between fungus Metarhizium robertsii and bacteria Bacillus thuringiensis ssp. morrisoni on Colorado potato beetle larvae.

Thirty-four isolates of Metarhizium spp. from Russian collections were genotyped using 5' EF-1α gene sequence analysis. Four species were identified, of which M. robertsii and M. brunneum were the most frequent, whereas M. anisopliae and M. pemphigum were sporadic. Radial growth studies in the temperature range of 10-40°C revealed that growth at high temperatures (35-37.5°C) was inherent for M. robertsii isolates but not for M. brunneum isolates. In contrast, M. brunneum isolates were more active at cold temperatures (10°C) compared to M. robertsii. Virulence was evaluated against larvae of the Colorado potato beetle (CPB), Leptinotarsa decemlineata Say, under two regimes: humid (21°C, 80% relative humidity (RH)) and arid (31°C, 55% RH). M. brunneum isolates were less virulent compared to M.robertsii under both regimes. M. robertsii activity did not differ under the two regimes, but M. brunneum was less virulent under the arid regime compared to the humid one. A field experiment under natural conditions (steppe zone of Western Siberia) with daily ranges of 10-43°C and 13-98% RH showed that M. robertsii was significantly more active than M. brunneum against CPB larvae.

Ecological preferences of Metarhizium spp. from Russia and neighboring territories and their activity against Colorado potato beetle larvae.

Entomopathogenic fungi form different strategies of interaction with their insect hosts. The influence of fungal infection on insect physiology has mainly been studied for generalists (Metarhizium, Beauveria), but studies of specialized teleomorphic species, such as Cordyceps militaris, are rare. We conducted a comparative analysis of the immune reactions of the wax moth Galleria mellonella after injection with blastospores of C. militaris (Cm) and Metarhizium robertsii (Mr) in two doses (400 and 4000 per larva). Cm-injected insects died more slowly and were more predisposed to bacterial infections than Mr-injected insects. It was shown that Cm infection led to a predominance of necrotic death of hemocytes, whereas Mr infection led to apoptotic death of cells. Cm-infected insects produced more dopamine and reactive oxygen species compared to Mr-infected insects. Moreover, Cm injection led to weak inhibition of phenoloxidase activity and slight enhancement of detoxification enzymes compared to Mr-injected insects. Blastospores of Cm that were cultivated in artificial medium (in vitro) and proliferated in wax moth hemolymph (in vivo) were characterized by equal intensity of fluorescence after staining with Calcofluor White. In contrast, Mr blastospores that proliferated in the wax moth had decreased fluorescence intensity compared to Mr blastospores grown in medium. The results showed that insects combat Cm infection more actively than Mr infection. We suggest that Cm uses fewer universal tools of killing than Mr, and these tools are available because of specific interactions of Cm with hosts and adaptation to certain host developmental stages.

Comparative analysis of the immune response of the wax moth Galleria mellonella after infection with the fungi Cordyceps militaris and Metarhizium robertsii.

Olga N. Yaroslavtseva

Institute of Systematics and Ecology of Animals of Siberian Branch of Russian Academy of Sciences

An Experimental Study on Colorado Potato Beetle Hibernation Under Natural Conditions