Long-Term Storage of Ascosphaera Aggregata and Ascosphaera Apis, Pathogens of the Leafcutting Bee (Megachile Rotundata) and the Honey Bee (Apis Mellifera)

Journal of Invertebrate Pathology 101 (2009) 157–160

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Journal of Invertebrate Pathology

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Short Communication Long-term storage of Ascosphaera aggregata and Ascosphaera apis, pathogens of the leafcutting bee (Megachile rotundata) and the honey bee (Apis mellifera)

A.B. Jensen a,*, R.R. James b, J. Eilenberg a a Department of Ecology and Agriculture, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark b USDA-ARS Pollinating Insects—Biology, Management and Systematics Research Unit, Logan, UT, USA article info abstract

Article history: Survival rates of Ascosphaera aggregata and Ascosphaera apis over the course of a year were tested using Received 19 January 2009 different storage treatments. For spores, the storage methods tested were freeze–drying and ultra-low Accepted 24 March 2009 temperatures, and for hyphae, freeze–drying, agar slants, and two methods of ultra-low temperatures. Available online 28 March 2009 Spores of A. aggregata and A. apis stored well at À80 °C and after freeze–drying. A. aggregata hyphae did not store well under any of the methods tested while A. apis hyphae survived well using cryopreser- Keywords: vation. Spores produced from cryopreserved A. apis hyphae were infective. Long-term storage of these Ascosphaera aggregata two important fungal bee diseases is thus possible. Ascosphaera apis Ó 2009 Elsevier Inc. All rights reserved. Chalkbrood Bee diseases Storage

1. Introduction gal cultures. Cultures can be stored at temperatures above freezing, in a frozen state or by lyophilization (freeze–drying). Each method Leafcutting bees (Megachile rotundata) and honey bees (Apis has its advantages, shortcomings, different costs and requirements mellifera) are both important pollinators in various crops and nat- for special devices (Humber, 1997). Here, we test the effectiveness ural habitats, and are often subjected to chalkbrood diseases of different long-term storage methods on the survival of A. aggre- caused by two closely related fungi: Ascosphaera aggregata (Skou, gata and A. apis, including two spore treatments (freeze–drying and 1975) and Ascosphaera apis (Spiltoir, 1955), respectively. The isola- ultra-low temperatures) and four hyphal treatments (freeze–dry- tion of fungus strains pathogenic to bees is critical for certain kinds ing, cold storage on agar slants and two methods of cryopreserva- of studies such as population genetics, virulence comparisons, tion), methods available in most laboratories. understanding disease development, and ecological competition. Once strains are obtained they must be maintained by weekly to 2. Materials and methods monthly transfers to fresh growth media, since hyphae of A. aggregata and A. apis, in our experience, collapse after approxi- Hyphae of four in vitro strains for each fungus, A. aggregata (AR- mately 1–2 months of storage at temperatures between 5 and SEF 690, ARS-Wild, ARS-Dale and ARS-CMF) and A. apis (ARSEF 34 °C. However, frequent culture transfers are expensive, time con- 7405, ARSEF 7406, KVL 06-147 and KVL 06-154), were tested. A. suming and increase the risk for contamination. Furthermore, aggregata was grown on modiﬁed V8 agar or broth as described repeated passages through artiﬁcial media may lead to changes by James and Buckner (2004), and A. apis was grown on Sabouraud in fungal morphology, a loss in virulence and a loss of sporulation dextrose agar or broth. A. aggregata is not known to produce spores capacity (Butt et al., 2006). Such phenomena are known from in vitro; therefore, spores were collected from four sporulating al- insect pathogenic fungi from Hypocreales (Morrow et al., 1989; falfa leafcutting bee cadavers after verifying with PCR that Ascosph- Shah and Butt, 2005) and Entomophthorales (Dumas and Papierok, aera proliperda was not present (James and Skinner, 2006). Spores 1989; Hajek et al., 1990) and may interfere with studies of from each cadaver were handled as separate replicates. A. apis is virulence. heterothallic and spores are readily produced in vivo when two An alternative to repeated subculturing is using one of the sev- strains of each mating type are plated together. The four selected eral methods available for preserving viable insect pathogenic fun- strains included two of each mating type, thus allowing four different crosses. For spore treatments, sporocysts were collected and then ground in glass tissue homogenizers to separate the spores * Corresponding author. Fax: +45 3533 2670. E-mail address: [email protected] (A.B. Jensen). for easy enumeration.

2.1. Spore storage methods overnight in 1 ml deionized water and then 100 ll of spore suspen- sion was transferred onto agar plates. Pieces of hyphae stored on To freeze–dry spores, spore concentrations were adjusted to agar slants were transferred onto new agar plates. Frozen hyphal 1 Â 107 spores per ml in a 50:50 mixture (by volume) of water plugs or 100 ll of hyphal mass from the broth were transferred and skim milk, then allowed to cure overnight at 5–7 °C before ra- to agar plates. Agar plates were all incubated at 32 °C until growth pid freezing at À80 °C followed by freeze–drying in a lyophilizer was observed, for up to 2 weeks. (HetosiccÒ CD53-1 [HETO Lab Equipment, Birkerød, DK] or a Dura Viability of spores and hyphae was tested before treatment, and Dry [FIS Systems Inc., Stoneridge, NY]) overnight, and then stored then 1 day, 1 week, 1 month, 3 months, 6 months and 1 year after at 5 °C. storage began. Each time, three samples were examined per isolate The method for ultra-low temperature storage of spores was and per treatment. The effects of storage methods on the survival adopted after Lopez Lastra et al. (2001). One ml aliquots of spores of the fungi over time were analyzed using logistic regression (Proc (1 Â 107 spores per ml of 10% glycerol in water) were placed in GENMOD; SAS ver 9.1) with a Poisson distribution and a log link cryogenic vials in a 5100 Cryo 1 °C Freezing Container, ‘Mr. Frosty’ function. The dependent variable (growth or no growth on a given

(Nalgene Co., Rochester, NY) to produce À1 °C/min cooling rate. plate) was binomial and the independent variable was the log10 of First, the spores were conditioned by placing the Mr. Frosty con- days in storage. The slopes of the regression lines were compared tainers at 5 °C for 4 h, then in an ultra-low freezer (À80 °C). After for each storage treatment, with spores and hyphae being analyzed 24 h the vials were transferred to standard freezer storage boxes separately. The number of plates with growth for each strain and stored at À80 °C. (weighted by the total number of plates tested), on each date, Viability of spores was determined based on the ability of a was considered a separate point in the estimate for the regression sample to form colonies on agar plates. Freeze–dried spores were lines. The two fungal species were analyzed separately because A. soaked overnight in 1 ml deionized water and 100 ll aliquots were aggregata was treated and assessed in Logan, UT, USA and A. apis transferred onto agar plates. Plates were incubated at 32 °C with in Copenhagen, Denmark.

P20% CO2 for 2 days to stimulate spore germination, and then To test the pathogenicity of A. apis spores produced from hy- up to 2 weeks at ambient CO2. phae that were stored at À80 °C for more than one year, spores from a cross between previously stored isolates ARSEFF 7405 and 2.2. Hyphal storage methods ARSEF 7406 were produced, and a dose of 500 spores were fed to in vitro reared honeybee larvae as described in Jensen et al. (in Hyphae were freeze–dried by first growing each strain in 50 ml press). In short, 1-day-old larvae were grafted into 48-well tissue of broth on a shaker (170 rpm) for 4 days at 22–25 °C. Hyphae were culture plates (one larva per well) and reared on a royal jelly diet. removed from the broth through a filter, then resuspended in skim The larvae were fed daily, and 3-days-old larvae were fed diet trea- milk and cured overnight at 5–7 °C before rapid freezing at À80 °C, ted with the A. apis spores. Larval mortality and A. apis infection in followed by freeze–drying in a lyophilizer overnight and storage at laboratory bioassays was recorded nine days after the spore expo- 5 °C. sure. The entire bioassay was replicated on three different dates Agar slant storage treatments were comprised of 5 ml agar in using 48 control and 48 treated larvae, all from a single hive, for 15 ml centrifuge tubes with hyphae grown at 32 °C for 4–7 days. each replicate time. The mortalities were corrected using Abbott’s Sterile deionized water (5 ml) was added to avoid dehydration of formula (Abbott, 1925). the hyphae. The tubes were sealed with parafilm and stored at 5 °C. Two ultra-low temperature storage methods were tested. For 3. Results and discussion one, hyphal lawns were grown on agar, then three 0.2 Â 0.2 cm plugs were placed in 1 ml of 10% glycerol in cryogenic vials and fro- The spore viability of A. aggregata differed significantly between zen in Mr. Frosty containers following the procedure above. For the storage treatments (v2 = 13.09; df =1; P = 0.0003), while storage other, hyphae were cultured in broth on a shaker (170 rpm) for time had no significant effect (v2 = 0.09; df =1; P = 0.7672). The 4 days at 22–25 °C, then 0.5 ml of strained hyphae were trans- spores survived freeze–drying and at ultra-low temperatures very ferred to cryogenic vials with 0.5 ml 20% glycerol and frozen in well during the whole experiment; the slopes were not signifi- Mr. Frosty containers as above. cantly different from zero (Table 1) and the viability declined only Viability of hyphae was determined based on the ability of a between 93.1% and 96.9% after one year (Fig. 1A). Hence, both sample to grow on agar plates. Freeze–dried hyphae were soaked spore treatments provided very good storage over the course of

Table 1 Effect of storage time on the survival of Ascosphaera aggregata and A. apis spores and hyphae subjected to different long-term storage treatments, as determine by logit regression analysis. Parameters for each of the individual regression lines are presented.

Treatment group Storage method Intercept Wald 95% conﬁdence limits Slope Wald 95% conﬁdence limits Slope P-values Scaled person (v2/df) Spores A. aggregata Freeze–dried À1.26 À1.69 to À0.83 À0.03 À0.27 to 0.21 0.81 0.016 Ultra-low temp À1.90 À2.46 to À1.34 À0.03 À0.34 to 0.29 0.86 0.042 A. apis Freeze–dried À1.98 À2.55 to À1.42 À0.21 À0.53 to 0.11 0.21 0.739 Ultra-low temp À1.78 À2.24 to À1.34 À0.14 À0.41 to 0.13 0.30 0.278 Hyphae A. aggregata Freeze–dried À2.64 À3.34 to À1.94 À0.26 À0.69 to 0.18 0.25 1.136 Agar slant À2.46 À2.90 to À2.02 À1.50 À2.04 to À0.95 <.0001 0.797 Ultra-low plug À2.65 À3.11 to À2.18 À0.99 À1.41 to À0.57 <.0001 1.657 Ultra-low broth À2.52 À3.20 to À1.84 À0.50 À0.98 to À0.03 0.04 1.742 A. apis Freeze–dried À3.43 À4.57 to À2.29 À1.04 À2.11 to 0.03 0.04 1.263 Agar slant À1.57 À2.01 to À1.13 À0.79 À1.15 to À0.44 <.0001 0.801 Ultra-low plug À1.90 À2.44 to À1.37 0.00 À0.30 to 0.30 1.00 0.000 Ultra-low broth À1.99 À2.55 to À1.44 0.03 À0.28 to 0.35 0.83 0.081 A.B. Jensen et al. / Journal of Invertebrate Pathology 101 (2009) 157–160 159

Fig. 1. Viability of spores of (A) Ascosphaera aggregata and (B) A. apis after storage Fig. 2. Viability of hyphae of (A) Ascosphaera aggregata and (B) A. apis after storage for up to a year under two different conditions (see text for details). Viability is the for up to a year under four different conditions (see text for details). Viability is the proportion of stored samples showing fungal growth on nutrient agar averaged over proportion of stored samples showing fungal growth on nutrient agar, averaged three to four plates per strain, and using four replicate strains per fungal species. over three to four plates per strain, and using four replicate strains per fungal species. the experiment. Since A. aggregata does not produce spores in vitro, 54% and 78%, respectively, over the year (Fig. 1B). This decline oc- long-term storage of A. aggregata spores becomes even more curred almost entirely in the first couple of weeks, which probably important. explains the lack of significance in the slope. This early drop in via- Viability of A. aggregata hyphae differed significantly between bility means that the spores were affected by the storage process in storage treatments (v2 = 12.45; df =3; P = 0.0060), and storage particular the freeze–drying, but remained stable afterwards. time also had a significant effect on viability (v2 = 55.55; df =1; Toumanoff (1951) found spores of A. apis to be rather persistent P 6 0.0001), except for freeze–dried A. aggregata hyphae, where and infective for at least 15 years when stored at ambient tempera- time had no significant effect on the viability when analyzed inde- tures. Other than an initial decline in viability, the spore treatments pendently from the other treatments (Table 1). Since the freeze– provided good storage conditions over the course of the experiment. drying procedure had a huge impact on the viability (the initial de- Viability of A. apis hyphae, on the other hand, differed signifi- 2 cline in Fig. 2A), this storage method is still suboptimal. Thus none cantly between storage treatments (v = 77.22; df =3;P < 0.0001) of the hyphal storage methods worked satisfactorily for A. aggre- and storage time had significant effects on the viability 2 gata, for which viability declined to between 2.5% and 25% after (v = 6.71; df =1; P = 0.0096). The freeze–drying and agar slant one year (Fig. 2A). A. aggregata is fastidious and hyphae only grow storage methods were not successful, with slopes significantly dif- well on specific media (Youssef and McManus, 1991; James and ferent from zero (Table 1) and with viability declining to 1.5% and Buckner, 2004), and even though we used a suitable growth med- 18.5%, respectively (Fig. 2B). The viability of A. apis hyphae during ium, the A. aggregata hyphae often collapsed during handling. Our cryogenic storage did not, however, decline significantly over time experience has been that if hyphae are left on a growth plate for (Table 1). In other words, A. apis hyphae stored very well at À80 °C too long (i.e. several weeks), the colonies collapse and die. Thus, over the course of a year with viability remaining as high as 98.5% maintenance of A. aggregata in vitro in standard labs requires con- and 100% (for the cultures grown on agar or in broth, respectively) tinuous monthly transfers, and for long-term storage, we recom- (Fig. 2B). An alternative for A. apis is to store cultures on rice ker- mend liquid nitrogen, which requires more specialized nels (Ruffinengo et al., 2000). Palacio et al. (2007) found viability equipment than utilized in this study. to be 73% for A. apis stored in this way for one year. Thus, the via- The decline in A. apis spore viability over time did not differ sig- bility over time was not as stable with this method as for the cryo- nificantly between storage treatments (v2 = 2.14; df =1;P = 0.1436) genic storage used in our study. and storage time had no effect on the viability of the spores We were able to produce spores from A. apis hyphae that were (v2 = 2.49; df =1;P = 0.1146). Despite this lack of a statistically sig- stored for one year at À80 °C. We found, as did Palacio et al. (2007), nificant effect of time on spore viability, we did see a decline in that such spores were still infective to honey bee larvae. In our bio- viability of freeze–dried and cryogenically stored A. apis spores to assays, 65% (SE = 0.1064) of the larvae exposed to the 500-spore 160 A.B. Jensen et al. / Journal of Invertebrate Pathology 101 (2009) 157–160 dosages were killed by A. apis infections. The control mortality was Grundschober, A., Freimoser, F.M., Tuor, U., Aebi, M., 2001. In vitro spore formation 31% (SE = 0.0592). Jensen et al. (in press) estimate the LD of A. and completion of the asexual life cycle of Neozygites parvispora, an obligate 50 biotrophic pathogen of thrips. Microbiol. Res. 56, 247–257. apis spores harvested directly from mummies collected from a Hajek, A.E., Humber, R.A., Griggs, M.H., 1990. Decline in virulence of Entomophaga honey bee hive to be between 55 and 905 spores. Thus, even maimaiga (Zygomycetes: Entomophthorales) with repeated in vitro subculture. though we did not test whether spores produced from stored hy- J. Invertebr. Pathol. 56, 91–97. Hajek, A.E., Shimazu, M., Humber, R.A., 1995. Instability in pathogenicity of phae differed in virulence from the original culture, the virulence Entomophaga maimaiga after long-term cryopreservation. Mycologia 87, 483– we found was within the expected range. 489. In conclusion, spores of both A. aggregata and A. apis stored well Humber, R.A., 1997. Fungi-preservation. In: Lacey, L. (Ed.), Manual of Techniques in Insect Pathology. Academic Press, London, pp. 269–279. using either cryopreservation or freeze–drying. A. apis hyphae James, R.R., Buckner, J.S., 2004. Lipids stimulate spore germination in the stored very well using cryopreservation, whereas A. aggregata hy- entomopathogenic ascomycete Ascosphaera aggregata. Mycopathology 158, phae did not store well in any of the four treatments tested. There 293–302. James, R.R., Skinner, J.S., 2006. PCR diagnostic methods for Ascosphaera infections in seems to be a general pattern between the degree of fastidiousness bees. J. Invertebr. Pathol. 90, 98–103. of a pathogen and difficulties in finding suitable methods for long- Jensen, A.B., Pedersen, B.V., Eilenberg, J., in press. Differential susceptibility across term preservation of living cultures (Sandskär and Magalhães, honey bee colonies in larval chalkbrood resistance. Apidologie. 1994; Hajek et al., 1995; Grundschober et al., 2001). This study is Lopez Lastra, C.C.L., Hajek, A.E., Humber, R.A., 2001. Effects of two cryopreservation techniques on viability and pathogenicity of entomophthoralean fungi. Can. J. another example of such a positive correlation since isolation Bot. 79, 861–864. and hyphal growth of A. aggregata is more difficult than A. apis. Morrow, B.J., Boucias, D.G., Heath, M.A., 1989. Loss of virulence in an isolate of an entomopathogenic fungus, Nomuraea rileyi after serial in vitro passage. J. Econ. Acknowledgments Entomol. 82, 404–407. Palacio, M.A., Peña, N., Clemente, G., Ruffinengo, S., Escande, A., 2007. Viability and pathogenicity of Ascosphaera apis preserved in integral rice cultures. Spanish J. The authors wish to thank Ellen Klinger and Christina Wolsted Agricul. 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