Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

The overall goal of these procedures is to establish, maintain, and refresh a captive population of Eristalis tenax in a laboratory setting. This method allows for the long-term maintenance of the hoverfly, Eristalis tenax. Hoverflies are emerging as useful models for investigating a range of scientific questions including fly behavior, pollination efficiency, and the neural mechanisms underlying motion vision.

While large-scale commercial breeding for agriculture already occurs in Europe, there is no standardized protocol for maintaining captive populations for scientific studies. Using our methods, Eristalis tenax can be successfully maintained for over a year in captivity, and both eggs and collected larvae can be reared to establish, or refresh, captive populations. To start a captive colony, either adult hoverflies or mature larvae need to be collected.

Second and third instar larvae have perviously been found in the manure pits of cattle farms. They are easiest to find during the beginning of their migratory phase as they are actively seeking a dark, dry environment to pupate. This tends to be near the borders of the manure pits where damp manure is close to dry areas containing large amounts of straw.

Collected larvae can then be reared to adulthood in the laboratory. Adult hoverflies can be collected by netting in the field, usually from botanic gardens and parks where there is an abundance of flowering plants. Hoverflies are easiest to catch when they are stationary, for instance, when they are feeding on flowers or sunning themselves.

Either an insect cage or plastic bag should be used for housing. This is dependent on the number of flies being housed. Less than 20 hoverflies can be stored in a bag, whilst numbers over 20 should be stored in an insect cage.

Flies are fed a mixture of bee pollen and honey, which is placed on top of damp cotton balls. Soak the cotton balls, remove any excess liquid, and place the honey and bee pollen on top. For housing in plastic bags, it is important that the cotton balls are moist, but not overly saturated, as any accumulation of water inside the bag can be detrimental to survival rates.

Conversely, for housing in an insect cage, the mesh sides allow for significant evaporation to occur. As such, cotton balls can be placed in a shallow container and need to be fully saturated to prevent over-drying. After allowing hoverflies to feed for a minimum of four hours at room temperature, they were placed in the fridge at eight to 10 degrees Celsius, and kept in complete darkness.

This temperature range induces artificial hibernation. To ensure longevity, our protocol requires a strict maintenance routine, which involves breaking the artificial hibernation and allowing hoverflies to feed and groom. Every three to four days, hoverflies were brought to room temperature, transferred into a new plastic bag or clean insect cage, provided with fresh food and water, and kept at room temperature for six to eight hours.

This allowed for both feeding and grooming to occur. The transferring of hoverflies was either done manually for small numbers, as being demonstrated here, or by utilizing phototaxis for larger numbers. This step should ideally be done within the first few minutes of removing the hoverflies from the fridge as they will still be in a state of hibernation and much easier to handle.

To utilize the the phototactic response of Eristalis tenax and efficiently move large numbers of hoverflies, first connect the old cage to a new, clean insect cage, and prepare the fresh food and place it inside the cage. Clip the two cages together whilst ensuring there is enough room for hoverflies to crawl through. Cover the old cage with opaque fabric being careful to tuck it around the sides and avoid light seeping in.

Hoverflies will quickly move towards the light and into the clean insect cage. Oviposition can occur either in the fridge, or at feeding time, at room temperature. Immediately after eggs have been identified, place them in a small Petri dish with tap water.

Eggs typically hatch over the course of two to three days. Movement will be very apparent amongst the newly hatched first instar larvae. Movement is also an indication that the eggs are ready to be transferred into the rearing set-up.

Hatched eggs should be transferred into a slurry of rabbit feces and water. To create the slurry, line a bucket with a plastic bag to ensure easy disposal after use. Three quarter fill the lined bucket with rabbit feces.

Pour water over the remaining space until the bucket is almost full. The slurry will need to be checked every two to three days to ensure the mixture is still moist. Top up the slurry with additional tap water if the mixture appears dry.

Place the bucket inside a larger plastic tub filled with wood shavings. The wood shavings should completely surround the bucket and extend up to the rim, thus allowing the third instar larvae to crawl into the sawdust to pupate. The set-up is now ready for the newly-hatched first instar larvae to be transferred.

Once this is done, cover the set-up with a double-layered mosquito net to prevent any larvae or newly-emerging hoverflies from escaping. Eristalis tenax larvae are commonly known as rat-tailed maggots. This is due to the tail-like structure at their posterior end.

This is actually a specialized breathing tube called a siphon. This can extend several times the length of the body to allow a larvae to breath air even while remaining completely submerged. The siphon piercing the surface, or the occasional larvae feeding on the top, are often visible within the slurry.

Also note that there is a change in the consistency of the slurry. This is because the larvae have developed and assisted in the decomposition of the rabbit feces mixture. We can also see the telltale signs that the third instar larvae have been crawling out of the slurry and into the wood shavings to pupate.

This is apparent as there are lots of trails visible on the edge of the bucket and a significant discoloration of the wood shavings. We would expect to see these changes 15 to 20 days after the newly-hatched larvae were placed in the slurry. These signs are a good indication that it is time to start looking for pupae.

Remove the bucket and sift through the wood shavings removing any pupae. Place the pupae into a dry container with some wood shavings, then into an insect cage equipped with food and water. Within six to 10 days of pupation, hatching will occur, and newly-emerging hoverflies can be seen.

Upon hatching, the wings will not be fully functional. However, within a few hours, the wings will unfurl and dry. Hoverflies will soon be capable of flight.

Once this has occurred and the newly-emerged adults have had a minimum of six hours to feed and groom, they can be placed into clean housing with food and water and put into the fridge to induce the artificial hibernation cycle. Our techniques to rear eggs laid by captured gravid females have proved to be successful with three batches of eggs reared to completion, resulting in an average of 163 plus or minus 33.65 emerged hoverflies with no observed gender bias. The health of these lab-reared hoverflies was then determined by a comparison of the weight and locomotor activity of female hoverflies compared to field-caught individuals.

No significant differences in weight or activity were observed between lab-reared and wild-caught hoverflies. Additionally, the effect of long-term maintenance using our described methods was further assessed by a comparison of weights over time for both sexes of lab-reared hoverflies. We observed a significant increase in weight over a period of four months for both sexes, with females consistently weighing more than their male counterparts.

We have developed a three-way strategy that maintains a healthy population for both visual and behavioral studies by capturing and successfully maintaining adult hoverflies, harvesting mature larvae from cattle farms, and rearing the offspring of wild, mated females.