Laboratory Cultivation of Vairimorpha (Nosema) ceranae (Microsporidia: Nosematidae) in Artificially Infected Worker Bees

Abstract

Nosemosis type C is a dangerous and widespread disease of the adult European honey bee Apis mellifera and is caused by the spore-forming intracellular parasite Vairimorpha (Nosema) ceranae. The search for new ways of therapy for this disease is complicated due to the seasonal availability of V. ceranae-infected insects as well as the lack of a developed system for the pathogen's cultivation. By carrying out trials which used different infectious dosages of the parasite, spore storage protocols, host age, and incubation temperatures, we present a simple, safe, and efficient method of V. ceranae propagation in artificially infected worker bees in the laboratory. The method is based on feeding the groups of adult worker bees with microsporidian spores and insect maintenance in plastic bottles at 33 °C. The source of the spores originated from the cadavers of infected insects from the previous round of cultivation, in which the infective spores persist for up to six months. An analysis of five independent cultivation rounds involving more than 2500 bees showed that the proposed protocol exploiting the dosage of one million spores per bee yielded over 60 million V. ceranae spores per bee, and most of the spore samples can be isolated from living insects.

Keywords: Apis mellifera; Nosema ceranae; Vairimorpha ceranae; artificial infection; cultivation; nosemosis.

Figure 1

Laboratory maintenance of the worker honey bees for Vairimorpha ceranae cultivation. Insects were kept in plastic bottles with 40% sugar syrup poured into 10 mL glass vials closed with a cotton pad and inserted into the bottleneck.

Figure 2

Dynamics of Vairimorpha ceranae development in adult honey bees experimentally infected with either 0.3 (“300,000”) or 1 million spores/bee (“1,000,000”) and maintained at 25 °C. Spores were isolated from dried honey bee cadavers stored at room temperature. The curves show cumulative insect mortality, and different letters indicate statistically significant differences at a given time point at p < 0.05 (*) or p < 0.01 (**), estimated using Pearson’s chi-square. Higher probability data are not indicated for clarity. The bars show the mean spore production, calculated as the ratio of the total number of spores observed in cadavers to the total number of bees that perished up to a given time point. The asterisks below bars indicate pairs of values that are significantly different from each other at p < 0.01 (**) or p < 0.001 (***) at a given time point, as inferred from the Kruskal–Wallis one-way analysis of variance on ranks and Mann–Whitney rank sum tests. The data which this scheme was created from are found in Table S1.

Figure 3

Dynamics of Vairimorpha ceranae development in newly emerged honey bee adults, maintained at 25 °C and experimentally infected with 1 million spores/bee; the spores were isolated from dried honey bee cadavers stored at room temperature. The indications are as in Figure 2. The data which this scheme was created from are found in Table S2.

Figure 4

Dynamics of Vairimorpha ceranae development in honey bee adults, maintained at 25 °C and experimentally infected with 1 million spores/bee. Spores were either isolated from dried honey bee cadavers stored at room temperature (“cadavers/RT”) or isolated from live bees and frozen at −80 °C (“spores/−80”) prior to the experiments. The indications are as in Figure 2. The data which this scheme was created from are found in Table S3.

Figure 5

Dynamics of Vairimorpha ceranae development in honey bee adults, maintained either at +25 °C (“RT”) or at +33 °C (“+33”) and experimentally infected with 1 million spores/bee (“inf”). Spores were isolated from dried honey bee cadavers stored at room temperature. The indications are as in Figure 2. The data which this scheme was created from are found in Table S4.

Figure 6

Dynamics of Vairimorpha ceranae development in adult honey bees, maintained at 33 °C and experimentally infected with 1 million spores/bee. Spores were isolated from dried honey bee cadavers stored at room temperature. The mean number of spores per bee was calculated using estimates of the total amount of bees from the beginning of the experiment (a) or within a given time interval (b). The indications are as in Figure 2. The data which this scheme was created from are found in Table S5.

Figure 7

Light microscopy of artificially infected worker bees. (a) Segment of infected bee intestine after 30 days of V. ceranae cultivation; (b) homogenized infected bee intestine after 30 days of V. ceranae cultivation. Scale bars: 20 mkm.