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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cold Spring Harb Protoc. Author manuscript; available in PMC 2010 October 1.
Published in final edited form as:
PMCID: PMC2916737
NIHMSID: NIHMS197920

Strain Maintenance of Nasonia vitripennis (Parasitoid Wasp)

Abstract

This protocol describes standard rearing of N. vitripennis strains on Sarcophaga bullata hosts. By using incubators at different temperatures, Nasonia’s development rate can be adjusted to conform to the investigator’s schedule. Nasonia will produce diapause larvae when reared at 18C with a short-day light cycle (Saunders 1965); these can be archived at 4C for over a year. Diapausing Nasonia may lose their Wolbachia infections (Perrot-Minnot et al. 1996). N. longicornis, N. giraulti and N. oneida can be reared under similar conditions, with the caveat that N. giraulti and N. oneida lay all-diapause broods more frequently and are therefore best reared at 25C.

MATERIALS

REAGENTS

  • Sarcophaga bullata pupae (see Protocol 2 – fly rearing) or other calliphorid fly pupae (stored at 4C)
  • 25mm diameter Drosophila vials and plugs (cotton, foam, rayon etc.)
  • 12mm diameter plastic vials and cotton plugs
  • honey water (40% honey : 60 % H2O aliquots in 1.5mL tubes)

EQUIPMENT

  • Incubators: 25C and 21C (constant light), 18C (8h light : 16h dark)
  • Refrigerator: 4C (preferably two for redundancy).
  • Small paintbrushes (number 2 or similar)
  • Rubber or foam pad

METHOD

  1. 25C and 21C strain maintenance
    1. Transfer 20 mated females and 4 or fewer males to new 25mm Drosophila vial. If transferring from same sized vial, place the new vial upside-down over the old vial. Mated females will climb up into the new vial. Otherwise, use a brush.
    2. Cover vial opening with two fingers and knock vial bottom against rubber pad. This will make most Nasonia fall to the bottom.
    3. Add 20 Sarcophaga hosts (or fill the vial 1/4 full if hosts are small).
    4. Plug with foam or cotton plugs and incubate new vial in 25C or 21C incubator. Used foam plugs can be autoclaved and reused.
    5. Backup emerged cultures in 4C refrigerator. Mated females will survive and remain fertile for approximately 1–3 weeks.
    6. Adults will emerge approximately 14 days later (25C) or 21 days later (21C). Nasonia females will mate immediately upon emergence.
  2. 18C strain maintenance
    1. Follow the same procedure as for 25C maintenance, except incubate at 18C using a 8h light : 16h dark cycle. Nasonia reared at 18C will emerge in approximately one month and live for several weeks.
    2. If no Nasonia have emerged after six weeks, or after all emerged Nasonia have died, check the vial for diapause larvae. Crack open all hosts at the midline using thumbnail or a probe. Diapause larvae are as large or larger than the average Nasonia and are distinguished by prominent white fat cells visible through the cuticle.
    3. Transfer hosts containing diapause larvae to a new 12mm plastic vial, plug with a cotton ball, label, and store in a 4C refrigerator for 6–24 months. Store in box with air holes, such as empty pipet tip box. Record strain and deposit time in a diapause log. If possible, split tubes between two 4C refrigerators to reduce the impact of equipment failure.
  3. Recovery of diapause larvae
    1. Remove tubes of diapausing larvae from 4C refrigerator. Strains are most likely to recover after refrigeration for 6–18 months.
    2. Remove cotton plug. Take new cotton plug and gently moisten with one or two drops of distilled water (do not saturate or mold will grow). Cover all tubes with a sheet of aluminum foil to reduce loss of humidity.
    3. Incubate recovering diapause larvae at 25C until wasps pupate, eclose and mate (about 14 days). If greater than 50% males emerge, separate approximately 20 females into a new vial using a paintbrush, add 2–4 males, paint a dot of honey water on the wall of the tube, and allow mating to occur for 4h or overnight.
    4. For long-term strain maintenance, pull 2–3 full 12mm vials from diapause at 18–20 months. After rebuilding the population for a generation or two at 25C, return the strain to 18C culture to produce more diapause larvae.
    5. Remove from 18C culture only when several batches of diapause larvae have been collected and stored (generally >6 vials worth).

TROUBLESHOOTING

Problem: low sex ratio (mostly males)

Solution: Crowding by multiple males may prevent some females from mating, who then produce all male broods and repeat the cycle. Remove as many females as possible using paintbrush, or, if necessary, knock out with CO2 and sort. Separate up to 20 females into a new vial, add 2–4 males, paint a dot of honey water on the wall of the tube, and allow mating to occur for 4h or overnight. All-male broods means no female mated. Recover strains from a culture backed up at 4C.

Acknowledgments

JHW and DL acknowledge support from the NIH 1 R24 GM084917-01 and assistance from Rachel Edwards, Jon Giebel, Michael Clark and Rhitoban Raychoudhury.

References

  • Perrot-Minnot MJ, Guo LR, Werren JH. Single and Double Infections with Wolbachia in the Parasitic Wasp Nasonia vitripennis: Effects on Compatibility. Genetics. 1996;143:961–972. [PubMed]
  • Saunders DD. Larval Diapause of Maternal Origin: Induction of Diapause in Nasonia vitripennis (Walk.) (Hymenoptera: Pteromalidae) Journal of Experimental Biology. 1965;42:495.