Future studies should compare survival rates following manual and automated injection at different parts of the body, e.g. injection into the thorax vs. the abdomen. The abdomen has been chosen as the injection site in different studies (e.g. Amdam et al., 2006; Schlüns and Crozier, 2007; Richard et al., 2008). The intersegmental membrane is soft between the tergites and can be easily punctured by the needle. Drugs are directly administered into the haemolymph and spread throughout the body with haemolymph circulation. However, if the worker has ingested a large meal prior to injection, one may puncture the full stomach with the needle. This may be avoided by injecting individuals immediately following emergence prior to the first ingestion of honey from the comb.
Survival rates may also differ between honey bees injected with or without anaesthesia. Workers handled without anaesthesia showed lower sucrose responsiveness 30 min after handling than immobilised individuals (Pankiw and Page, 2003), and a delayed onset of feeding may increase mortality. Control individuals should be subjected to the same handling times to control for stress effects.
Lastly, nutritional composition of the diet may also affect survival. Worker survival on sucrose-only solutions was drastically reduced following injection (Köhler et al., 2012), but adding protein to the diet may increase the production of immune system components (e.g. antibacterial peptides), which may help in fighting infections and improve survival (Alaux et al., 2010; DeGrandi-Hoffman et al., 2010); see also the BEEBOOK paper on ‘maintaining adult honey bees in vitro under laboratory conditions’ by Williams et al. (2013). Mortality rate following injection may depend on multiple factors, including injection technique, types and amounts of injected substances, type of needle, needle thickness and sharpness, age of the honey bees, handling stress, and type of anaesthesia (if any). It may prove valuable to assess the mortality following injection in a particular experimental setup to be able to adjust the sample size for the study (see the BEEBOOK paper on statistics (Pirk et al., 2013). The effects of injections should be considered when deciding on a technique for substance application and all parameters or injection methods should be described in detail.
Checklist for injections:
- Decide on the injection method (Hamilton syringe, Nanoject device, etc.).
- Decide on a suitable injection site (e.g. abdomen of adults, between tergites).
- Decide whether anaesthesia is required. Young workers do not sting or fly, older workers may need to be immobilised by cooling prior to injection.
- Make sure to have defined age-cohorts.
- Determine the dose and injection volume (ideally <5 µl for adult workers).
- Decide on a suitable buffer (saline, insect ringer).
- Reduce possibilities of unintended secondary infections: use new glass capillaries; disinfect the Hamilton syringe (e.g. ethanol, acetone) before and after use; the needle can be sterilised in a flame to avoid contamination between individuals.
- Take initial high mortality into account.
- A test run to practise the injection technique is recommended. This way survival rate following injection can also be determined.