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plants. Occasionally, microsporidia are noticed as important mortality factors in outbreak populations of the gypsy moth, Lymantria dispar. Reports of high prevalence of infections by these pathogens exist e.g. from Sardinia, Poland, the Ukraine (McManus and Solter, 2003, Solter et al., 2012). More typically, however, they seem to occur at lower enzootic level and can be found in most populations when a thorough screening is done (Novotny, 1989; Hoch et al., 2001; Pilarska et al., 1998; 2006). Other examples of microsporidian species that have significant adverse effects on forest insect pests are Nosema fumiferanae in the spruce budworm, Choristoneura fumiferana, and Nosema tortricis in Tortrix viridana (Solter et al., 2012).
For over 20 years, the gypsy moth, Lymantria dispar, which is a major pest in the broadleaf forests of Europe, Asia, Northern Africa, and its invaded area in North America, was the focus of a group of insect pathologists from Europe (Germany, Austria, Slovakia, Czech Republic, Bulgaria, Georgia) and the United States. As a result of this cooperation, three species of microsporidia and more than 25 microsporidian isolates were recovered from European populations of the gypsy moth (Pilarska et al., 1998; Solter et al., 2000; Vavra et al., 2006; Solter et al., 2010). The life history, morphology, host tissue specificity, virulence and persistence, and biology of several of these microsporidia have been intensively studied to elucidate interactions with the host and to facilitate decisions regarding use in biological control programs (Solter et al., 2000; 2002; Goertz et al., 2004; Vavra et al., 2006; Pilarska et al., 2006; Solter et al., 2010; Pilarska et al., 2017; Hoch and Solter, 2018).
The necessity for in-vivo production in host insects and the slow progress of disease put constraints on the use as biological pesticides applied in inundative releases. Only one microsporidian species, Paranosema (Nosema) locustae, is commercially produced for control of grasshoppers and crickets (Bjørnson and Oi, 2014). Inoculative releases to augment epizootics or into naïve host populations to initiate establishment – also in classical biological control programs – appear more promising for a wider variety of insects (Hoch and Solter, 2018). There have been experimental releases against the gypsy moth in Slovakia, Bulgaria and in the United States using the microsporidia Nosema lymantriae and Vairimorpha disparis (Weiser and Novotny, 1987, Jeffords et al., 1988; Jeffords et al., 1989; Solter et al., 2010). Results from a 3–year monitoring of two experimental populations in Bulgaria showed that N. lymantriae was established after introduction and its prevalence varied from 3.5 to 33.0% (Pilarska et al., 2010).
An important issue for both, studies of microsporidia and production of material for biological control, is the storage of viable material for extended periods. Maddox and Solter (1996) showed that storage of microsporidian spores from terrestrial insect hosts in liquid nitrogen reduces the loss of infectivity and recommend such storage. Consequently, many studies of pathogen biology and host-parasite interaction became possible because all collected microsporidian isolates from gypsy moth were available in liquid nitrogen storage in several laboratories in the US and Europe. For applied research, like experimental infections or for the use in biological pest control applications, a collection of isolates with infective spores is essential.
In this paper we present data about the viability of different microsporidian isolates from L. dispar after long-term storage in liquid nitrogen for periods between 7 to 18 years and 9 months.
Material and Methods
MATERIJALI I METODE
a) Microsporidia – Mikrosporidije
Microsporidian spores from the microsporidia collection at the laboratory of A. Linde, Eberswalde University of Sustainable Development, Germany were used in our experiments. All spores had been stored in a liquid nitrogen dewar flask for at least 7 years. The filling level of the storage tank is checked every 14 days to ensure that all samples are covered with liquid nitrogen at all times. A refill with liquid nitrogen is necessary 3 to 4 times per year. Stored microsporidian spores had been harvested from laboratory infected L. dispar larvae following a standard protocol: For production of clean material, the infested tissues, such as fat body or silk glands, were dissected out of larvae. The tissue was homogenized in distilled water in a tissue grinder, filtered through cellulose tissue and centrifuged. The spore pellet was re-suspended in distilled water and mixed 1:1 with glycerol. 1 ml of the suspension was filled into a cryo vial and submerged in liquid nitrogen in the dewar. The spore suspensions tested in this study had never been removed from the dewar throughout the storage period until used in the experiment.
b) Surface contamination experiment – Test kontaminacije hranjivog supstrata
Eight microsporidian isolates from L. dispar were tested for their infectivity against L. dispar larvae: Vairimorpha disparis, Nosema lymantriae, Nosema portugal, Nosema sp. (Poland), Nosema sp. (Ebergassing), Nosema sp. (Germany), Nosema sp. (Schweinfurt) and Nosema sp. (Veslec). Vials containing spore suspensions of the test isolates in concentrations higher than 1x105 spores/µl were removed from liquid nitrogen and thawed at room temperature. The spores were cleaned from the glycerol by repeated centrifugation and re-suspension in distilled water. Then, 1 ml of spore suspension was evenly spread on the surface