Quantifying spore viability of the honey bee pathogen Nosema apis using flow cytometry

Abstract

Honey bees are hosts to more than 80 different parasites, some of them being highly virulent and responsible for substantial losses in managed honey bee populations. The study of honey bee pathogens and their interactions with the bees' immune system has therefore become a research area of major interest. Here we developed a fast, accurate and reliable method to quantify the viability of spores of the honey bee gut parasite Nosema apis. To verify this method, a dilution series with 0, 25, 50, 75, and 100% live N. apis was made and SYTO 16 and Propidium Iodide (n 5 35) were used to distinguish dead from live spores. The viability of spores in each sample was determined by flow cytometry and compared with the current method based on fluorescence microscopy. Results show that N. apis viability counts using flow cytometry produced very similar results when compared with fluorescence microscopy. However, we found that fluorescence microscopy underestimates N. apis viability in samples with higher percentages of viable spores, the latter typically being what is found in biological samples. A series of experiments were conducted to confirm that flow cytometry allows the use of additional fluorescent dyes such as SYBR 14 and SYTOX Red (used in combination with SYTO 16 or Propidium Iodide) to distinguish dead from live spores. We also show that spore viability quantification with flow cytometry can be undertaken using substantially lower dye concentrations than fluorescence microscopy. In conclusion, our data show flow cytometry to be a fast, reliable method to quantify N. apis spore viabilities, which has a number of advantages compared with existing methods. V C 2013International Society for Advancement of Cytometry Key terms Nosema apis; viability; fluorescence microscopy; flow cytometry; microsporidia; SYTO 16; SYBR 14; Propidium Iodide; SYTOX Red UNDERSTANDING host-parasite interactions depends on experimental methods that can estimate life history traits of parasites related to fitness. Quantification of these is often crucial to gain detailed insights into the intimate interactions between the parasite and its host and consequently determine the virulence or the damage caused by a parasite. Pathogens can avoid detection or overcome attacks by the host's immune system, so their survival and viability within the host is an important factor to understand the dynamics of initial establishment and consequent progression of an infection. The ability to quantify the viability of specific parasites can have a range of experimental applications to assess the effect of host defences on parasite survival. However, such a technique has applied benefits as well, for example to quantify the effectiveness of treatments on parasites or as biosecurity tools to monitor and limit the spreading of infections. Flow cytometry (FCM) is a technique that has been used in the past to detect and monitor the viability of parasites, for example, to quantify Mycoplasm spore survival in goats and sheep after antibacterial treatment (1)....

Seminal fluid of honeybees contains multiple mechanisms to combat infections of the sexually transmitted pathogen Nosema apis

“…For each treatment and incubation time, we used three independent biological replicates of seminal fluid that we collected prior to the experiment as described above. Spore viability was quantified using a flow cytometry method developed earlier [36]. In brief, we used fluorescent nucleic acid dyes and stained spore samples for 90 min in the dark on ice with 5 mM SYTO (16) green and 0.02 mM SYTOX red (Invitrogen, USA), which allowed us to distinguish live and dead spores [36].…”

“…Spore viability was quantified using a flow cytometry method developed earlier [36]. In brief, we used fluorescent nucleic acid dyes and stained spore samples for 90 min in the dark on ice with 5 mM SYTO (16) green and 0.02 mM SYTOX red (Invitrogen, USA), which allowed us to distinguish live and dead spores [36]. To increase spore concentration in samples, we centrifuged them at 20 800g for 10 min at 48C and discarded 680 ml of the supernatant before resuspending the spores in the remaining supernatant.…”

Flight behaviour of honey bee (Apis mellifera) workers is altered by initial infections of the fungal parasite Nosema apis

“…We then fed bees with either 2 μl of a 50% (w/v) sucrose solution (control) or 2 μl of a 50% sucrose solution containing 20,000 Nosema apis spores (infected). Spores used for inoculations originated from several colonies and were collected prior to the experiment using established protocols63. In brief, we collected 100 bees from a total of 4 colonies, freeze-killed them at −20 °C and macerated their abdomens with a mortar and pestle.…”

“…Final spore concentration was determined using an Improved Neubauer haemocytometer and spores were kept at −80 °C prior to any further experiments. These procedures do not affect spore viability63.…”

“…The gut tissue was gently macerated using a small pestle and vortexed for 2 minutes and a 7 μl subsample was loaded onto an improved Neubauer haemocytometer to count the number of spores in five squares using a light microscope (Leica DM 1000) at 400x magnification. Final spore concentrations were calculated by multiplying the total number of spores counted by a factor of 50,00063.…”