Team:HZAU-China/Project/Methods for rearing flea

• Overview
• The innovation of HZAU-iGEM Team
• The design of experiment
• Rabies
• Flea and Yersinia pestis
• Bacterial Spores as Vaccine Vehicles
• Bacillus subtilis as Probiotics
• Antimicrobial Peptides
• Methods for rearing flea

For sharing, we make a conclusion on the methods for rearing flea. We guess other team may use flea in the future because of some advantages. We hope iGEMers can solve the potential safety risk of flea together. Fleas play an important role in our project, but they may carry bacteria and parasites, so we don’t use the fleas until we find a safe method to rear and handle it. Considering the completeness of our project, we gathered the methods for rearing and handling fleas to prove our idea is possible.

Generally, there are four methods to catch fleas^[1]:

1.Collection from animal: The habits and environment of fleas’ host must be taken into consideration when we choose the proper method to catch fleas. Usually a mouse cage is used to catch small rodents and Insectivores. The wild medium sized mammals, such as fox, weasel, marmot, squirrel and mole, must be killed by shotgun before they are caught. As for bat, in most cases, they should be caught in bat cave. The captured animals must be put into a sack as soon as possible to avoid the fleas leaving their host. We can drown the captured animal to make the fleas escape from their host. Then, the fleas will float on the water. We can use tweezers to collect fleas and then put them into a tube.

2. Collection from nest: Nest is the main place where fleas multiply. The amount and species number of fleas here are far more than on the host. But not all caves are easy to dig, so this way only applies to few animals and some underground nests. This method is usually used to catch fleas that live in the nests of voles. We can dig out a vole nest from a cave (Figure 1) and put it (including the soil bottom of the nest) into a bag. For the hopping ability of a general nest flea is weak, we can put the nest that we collected into an enamel basin and collect the fleas from it. The fleas living in the henhouse are usually Pulex irritans that have strong jumping ability so their nest must be put into a higher aquarium and the one who picks the flea should wear long latex gloves. The collected nest has not only adults but also many larvae and cocoons, so we will get more adults after a few days.

3.Collection from the channel between the entrance and nest: This method needs an elastic branch about 2m long with one end twined by gauze. When collecting the flea, we insert the branch into a cave and move (Figure 1). After a few seconds, take out the tool and then catch the fleas from it. This way is applicable to the cave of marmot and similar ones, but not the general cases.

4.Collection indoors: This method has a trap to catch fleas. For example, Caleb H. Creswell has made a device to catch fleas by using materials commonly found in home."A 15-watt bulb lamp, ideally with gooseneck design, is placed in the center of a shallow basin (5- to 10-cm high) of water with a thin layer of vegetable oil on the top. The bulb lamp is 20 cm to 40 cm high off the ground. The fleas jump from the floor toward the light source and some land in the basin directly below. Once the fleas fall into the basin, the oil reduces the surface tension enough so they cannot escape and will be captured.^[2]

Fleas are holometabolous insects, going through the four life cycle stages of egg, larva, pupa, and imago (adult)^[3]. The device for rearing larva is simple, while adult fleas must be reared with blood. The methods can be divided into two types according to the ways of rearing adult flea. One method uses animals as the fleas’ host and the other one doesn’t.

Smith and Eddy^[4] published a classical method that uses animals as the host of fleas, which is covered next. The adult fleas (Oriental Rat Flea) are fed on white rats, and the larva rearing medium is a mixture of dried blood, yeast powder and sand (sifted through 16 to 40 mesh screens), mixed in proportion of 1 : 1 : 10, baked at 60℃ for 24 hours^[5]. The rat will turn back to bite fleas. In order to prevent fleas from being killed, we can confine rat in a small cage or fix its head by a particular tool like cangue. A pan, slightly bigger than the cage, with some bibulous papers absorbing urine of rat and feces, is placed under the cage. The bucket should be high enough to prevent the fleas from escaping from the bucket . (Figure 2)

The larva medium is 0.5~1.0cm high and the bucket should be covered by cotton yarn. The bucket is kept in an air-conditioned room and a temperature of 27℃ and at a relative humidity of 80%. The rat is fed a piece of apple and some rat-food every day. About 500 adult fleas are put on the rat. 21 days later, take out the rat from the bucket and then catch fleas on it.

After all fleas are caught, the rat is placed in a clean cage to rest for several weeks. By this time the second generation adults are almost ready to emerge. The larva medium that contains eggs, larva and pupae is removed from the bucket and sifted through 8 and 16 mesh screens. The coarser screen removes large debris, and the finer removes the cocoons.

The remaining larva medium that is containing eggs and larva is placed in a pupation pan covered with bleached muslin. Once a week, the larva medium is sifted through a 16 mesh screen to remove the cocoons. The cocoons are placed in the device (Figure 3) that is designed for collecting the adult fleas. The device consists of a basket, a three wire hooks for immobilizing the basket, a collecting jar with a disk of paper placed in the bottom, a jar from which the bottom has been cut (used as funnel), and a collar which is made of two of the ring portions of fruit-jar caps, soldered together. The cocoons are put in the basket. After the cocoons become fleas, they will jump out the basket and fall into the collecting jar. There are many other methods to rear fleas with a host. For example, W. HUDSON el^[6] published a method for large-scale rearing of the cat flea, this method is suitable for the production of the a large number of fleas required by the allergy study.

The method above is cumbersome and there is another alternative method. The way of rearing larva is the same as above method , but the adult fleas are not fed on animal but the artificial flea-feeding apparatus. We use Figure 3 to introduce this apparatus.^[7]

The membrane is the most important part of this method. The method simulates the way that fleas take blood from animal. The membrane simulates the skin of an animal and is made of parafilm. The blood is under the membrane, and a flea will impale the membrane to take blood. The blood needs be heated by warm water to reach a certain temperature. There are some other artificial flea-feeding apparatus which have been patented. For example, Susan E. Wade^[8] published an apparatus (Figure 5) for cat flea rearing in 1998 and Hong-Jie ZHANG ^[9] published another device for xenopsylla cheopis in 2004. Compared with the flea rearing methods on animal hosts, these methods have less steps and smaller risk of flea escape.

Fleas often need to be transfer from one place to another, during which the fleas may escape. For this reason, it is important to take some measures. Fleas can be immobilized when exposed to 4℃ for 30 min. This processing will reduce the risk of flea escape but not enough. Lian-Fang BI ^[10] has published a device for transferring fleas. We made a similar device (Figure 5) to demonstrate the working principles. This device consists of a vacuum pump, a rubber tube, a straw, a pipette tip and a veil of gauze. The vacuum pump provides power to suck the fleas. Because the rubber tube is soft, this device can catch flea from different container. The gauze can prevent the fleas from being sucked into the vacuum pump and leave the fleas in the pipette tip.

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