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ŠUMARSKI LIST 7-8/2019 str. 36     <-- 36 -->        PDF

storage: None of the 48 tested larvae was infected after ingestion of 100 spores. However, dosages of 1000 spores per larva led to infections of 4.1 % of the tested larvae becoming infected; and when applying 10 000 spores per larva, 68.8% were infected (Table 2). In all cases, the infections at the end of the incubation period were heavy and the infected fat body was filled with spores.
Maddox and Solter (1996) investigated the viability of 31 species of microsporidia, isolated from terrestrial insects and stored in liquid nitrogen for up to 25 years and reported that all species were able to produce infections in hosts from six insect orders. In their experiments, the only microsporidium isolated from Lymantria dispar was Endoreticulatus schubergi which was stored for 9 years in liquid nitrogen. This species is very different from the Nosema and Vairimorpha species used in our study in many aspects, such as spore survival under winter conditions (Goertz and Hoch 2008). We demonstrated that V. disparis and N. lymantriae can survive 7 years of storage in liquid nitrogen.
Nosema sp. (Ebergassing) survived for even more than 18 years. However, spore viability was clearly reduced. Dosages of 1000 spores per larva caused infections in only 4.1 %. The same dosage of spores stored in liquid nitrogen no longer than 2 months caused 100% infections in experiments using the same method of inoculation (G. Hoch, unpublished data). When spores of Nosema sp. (Ebergassing) in concentrations of 1x103 spores/µl and 1x105 spores/µl stored for less than a year in liquid nitrogen were used for surface contamination of diet fed to L. dispar larvae, 95% of the larvae fed with the lower concentration spores and 100% of the larvae fed with the higher concentration were infected (Hoch, 1999).
Hoch et al. (2004; 2009) also conducted individual inoculations of L. dispar larvae with  1x104 spores of Nosema portugal stored in liquid nitrogen for no longer than 2 months. The results showed high larval mortality (almost 100%) of the larvae caused by this microsporidium. In our experiments, even dosages of 1x105 spores or high concentrations for surface contamination did not cause any infection, which shows that the spores of Nosema portugal spores lost their viability infectivity after 18-year long-term storage in liquid nitrogen.
Goertz et al. (2004) performed infection experiments with spores of Nosema sp. (Veslec) stored in liquid nitrogen for less than 7 years. The authors used five spore dosages per larva (2x102, 1x103, 5x103, 1x104 and 5x104) for individual infections. The experimental infection rates ranged between 97–100%, when third instar larvae were fed in dosages ranging from 1x103, to 5x104 spores. The low dosage of 2x102 spores resulted in an infection rate of 77%.
The results from our experiments with spores used in surface contamination showed that Nosema sp. (Veslec) lose their viability and infectivity after storage for 11 years in liquid nitrogen. Spores of Nosema sp. (Schweinfurth), stored for 7 years in liquid nitrogen, also did not cause infections when fed to L. dispar larvae via surface contamination.
Our study confirms that storage in liquid nitrogen is a suitable option for long-term storage of Nosema and Vairimorpha species from lepidopteran hosts. Spores of some isolates survived for more than 18 years; however, the experiments show that there is a significant loss of viability. Spores of some isolates had lost viability already after 7 years in liquid nitrogen. Therefore, it is recommended to produce fresh material every 5 years to maintain collections. Based on our experiments or field application, no material should be used that had been stored in liquid nitrogen for period longer than five years. Liquid nitrogen storage offers the opportunity to produce and maintain large quantities of homogenous microsporidian inoculum for experiments and inoculative biocontrol releases.
The authors would like to thank to the German Academic Exchange Service (DAAD) for supporting this research.
References - LITERATURA
Bjornson, S., D. Oi, 2014: Microsporidia biological control agents and pathogens of beneficial insects. In: (Eds: L.M. Weiss and J.J. Becnel), Microsporidia: Pathogens of Opportunity, 1. ed., 635–67, John Wiley & Sons, Inc.
Goertz, D., D. Pilarska, M. Kereselidze, L.F. Solter, A. Linde, 2004: Studies on the impact of two Nosema isolates from Bulgaria on the gypsy moth (Lymantria dispar L.). Journal of Invertеbrate Pathology, 87: 105-113. 
Goertz, D., G. Hoch, 2008: Vertical transmission and overwintering of microsporidia in the gypsy moth, Lymantria dispar. Journal of Invertebrate Pathology, 99: 43–48.
Hoch, G., 1999: Wechselwirkungen zwischen einer ento­mo­pathogenen Mikrosporidie und dem Endoparasitoiden Glyptapanteles liparidis in ihrem gemeinsamen Wirt, der Lymantria dispar Larve. Ph.D. thesis, Universität für Bodenkultur, Wien, 64 p. 
Hoch, G., M. Zubrik, J. Novotny, A. Schopf, 2001: The natural enemy complex of the gypsy moth, Lymantria dispar (Lep., Lymantriidae) in different phases of its population dynamics in eastern Austria and Slovakia – a comparative study. Journal of Applied Entomology 125, 217-227.
Hoch, G., L.F. Solter, A. Schopf, 2004: Hemolymph melanization and alterations in hemocyte numbers in Lymantria dispar