2.2.6.2. Infection of heterologous lepidopteran cell lines

Most of the commercially available, permanent insect cell lines are derived from Lepidoptera. Several lepidopteran cell lines are described to support propagation of homologous microsporidia (where the source species of the cell line is the original host), as well as microsporidia originally infecting other hosts (Jaronski, 1984). Likewise, several lepidopteran cell lines have proved to be susceptible to N. apis and N. ceranae infection. Susceptibility could recently be demonstrated for the following cell lines (Gisder et al., 2010b): MB-L2 (Mamestra brassicae), Sf-158 and Sf-21 (Spodoptera frugiperda), SPC-BM-36 (Bombyx mori), and IPL-LD-65Y (Lymantria dispar), and BTI-Tn-5B1-4 (Trichoplusia ni) which can all be obtained through national cell culture collections together with protocols how to maintain and passage the cell lines. It is recommended to maintain the cell lines for routine culture in 75 cm2 cell culture flasks (e.g. Roth) in a cooling incubator (e.g. Thermo Fisher) at 27°C, and supply them with their individual medium composition (Table 3). Approximately 2E+05 cells per ml should be used to establish the next passage (Table 3). 

    For infection of these insect cell lines with germinating Nosema spp. spores (after Gisder et al., 2010b):

  1. Prepare a pre-culture
  2. Incubate the cells to their exponential growth phase.
  3. Harvest the cells growing in exponential phase
  4. Centrifuge at 210xg for 5 min. (Eppendorf 5810 R, rotor F34-6-38).
  5. Remove medium by aspiration
  6. Wash the cell pellet twice with 1 ml of freshly prepared 0.1 M sucrose in PBS-buffer.
  7. Dilute the cells to a final concentration of 2E+06 cells per ml in 0.1 M sucrose solution.
  8. Transfer approximately 5E+07 freshly prepared spores (see above), diluted in AE-buffer (Qiagen), were into a chamber slide (VWR, 4 chambers each slide) for infection
  9. air dry for 3 hours at room temperature.
  10. Initialize infection by adding 100 µl 0.1 M sucrose in PBS buffer to the dried spores, so that spore germination is triggered.
  11. Immediately add 50 µl of cell suspension (2.5E+05 cells) to the germinating spores.
  12. Resuspend the spore-cell suspension (150 µl) thoroughly
  13. Incubate at room temperature for 5 minutes
  14. Add 350 µl medium with 250 µg ml-1 penicillin/streptomycin (Roth) and 125 µl antimycotic/antibiotic solution (Sigma-Aldrich) to the spore-cell suspension to a final volume of 500 µl
  15. Incubate the cells at 27°C up to 10 days

For in situ-hybridization (ISH) or fluorescence in situ-hybridization (FISH) medium was aspirated and cells were fixed on glass slides with 4 % formalin (e.g. Roth) up to 24 hours. ISH or FISH were performed according to recently published protocols (Gisder et al., 2010b; Yue et al., 2007, see also the BEEBOOK paper on molecular methods (Evans et al., 2013)).

Six lepidopteran cell lines could be infected successfully (Fig. 8) as demonstrated by in situ-hybridization performed 72 hours post infection. For a detailed analysis of the life cycle of Nosema spp. in infected cells, IPL-LD-65Y cells were chosen and analysed by fluorescence in situ-hybridization (FISH, see the BEEBOOK paper on molecular methods (Evans et al., 2013)). Although these newly developed cell culture models are valuable tools for studying pathogen-host interactions on cellular and molecular level (Troemel, 2011), the protocol does not yet allow the continuous propagation of Nosema

spp. in cell culture and, hence, is not yet suitable to replace infection of bees for the production of spore suspensions.

Table 3. Maintained insect cell lines which can be infected with Nosema spp. FCS= fetal calf serum; w/o= without.

cell line

cell growth

Medium

FCS

time of passage

MB-L2

mostly adherent

Insect-Xpress (Lonza)

w/o

every 5 days

Sf-158

adherent

Insect-Xpress (Lonza)

5 %

every 6 days

Sf-21 

adherent

Insect-Xpress (Lonza)

5 %

every 6 days

SPC-BM-36

mostly adherent

TC-100 (Invitrogen)

12%

every 7 days

IPL-LD-65Y

suspension

TC-100 (Invitrogen)

11%

every 7 days

BTI-Tn-5B1-4

adherent

Sf-900 II (Invitrogen)

w/o

every 5 days

 



Fig. 8. In situ-hybridization (ISH) of Nosema spp. infected lepidopteran cell lines 72 hours post infection. Infected cells are stained dark blue. Specific hybridization was performed with 16S rRNA (SSU) probes coupled with digoxigenin and subsequent colour reaction to detect hybridized probes.

Figure 8