HDC incubation: detailed method by John M
Introduction
This article describes raising a H. diminuta (rat tapeworm) culture in the amateur laboratory, with the goal of harvesting HDC (H. diminuta cysticercoid larvae) for therapeutic use. When this material is used as a guide, the procedures may be divided into two stages.
The first stage of rearing H. diminuta from ova to the cysticerci (larvae) needed for therapy is simpler and less costly, and needs about six to seven weeks of light maintenance to complete. The novice experimenter is strongly encouraged to follow this procedure a few times before committing to the second stage.
The second stage of cultivating H. diminuta cysticerci to sexually mature adult and to next generation ova requires a greater initial investment of time and money and additional ongoing maintenance, and the much greater 4-5 year commitment to humane care for the rats that host this stage. Because of this great responsibility, this stage should not be undertaken before serious consideration.
WARNING: Although this article is the result of dozens of hours researching scholarly articles, I am a self-taught amateur in this subject, and have no formal training in biology. I am enough of a scientist to see many potential problems with the procedures. At best, one may be certain there are better methods than these. At worst, there could be serious safety issues that I am ignorant of. Please do your own research, and understand you are solely liable for the risks you take following this guide.
The H. Diminuta Life Cycle
H. diminuta larvae, like most all of the cestode (tapeworm) class, develop in an 'intermediate host'. The larvae become infective when ingested by a 'definitive host' where they transform into adults and become sexually mature. The cycle completes when ova are passed through the feces of the definitive host and ingested by the intermediate host. The below diagram illustrates this life cycle for H. diminuta.
First stage: ovum to cysticercoid
The first stage of raising oncosphere (commonly but incorrectly called 'ova') to cysticerci in this guide are depicted in items (1) through (4). In the laboratory, workers typically use some species of grain beetle for the intermediate host, either Tenebrio molitor (mealworm beetle) or Tribolium confusum (confused flour) beetle. T. molitor is convenient in the amateur laboratory for reasons described later. The first stage procedure may be completed in about three weeks (numbers refer to diagram):
- (1) Obtain ova in rat feces from Carolina Biological Supply
- Obtain mealworm beetles from Carolina or elsewhere; starve for four days
- (2) Feed feces with ova to beetles
- (3) Ova 'hatch' within infected beetles; beetles are transferred to wheat bran while larvae mature for one month when their therapeutic properties are useful to humans.
- (4) Larvae are ingested by definitive host, either rat or human
Second stage: cysticercoid to adult to ovum
Infecting the definitive host, cultivation of tapeworms to sexual maturity and production of next generation ova make up the second stage, completing the life cycle, depicted in (4) through (1). Although many vertebrates may serve as the definitive host, H. diminuta can only reproduce in the rat. Humans, mice and hamsters may be infected, but reject the parasite before its maturity. In the rat, however, the tapeworm's life span is limited only by that of its host.
The second stage procedure is completed in about a month (numbers refer to diagram):
- (4) Cysticercoid larvae are fed to rats
- (5) In the rat's small intestine, the larvae are excysted and attach to the intestinal mucosa with for muscular suckers on the scolex
- (6) Over 25 days, tapeworms mature to adults
- (7) Tapeworms produce ova; gravid proglottids (egg-carrying body segments) break off and are excreted with feces
- (1) Rat droppings are collected, examined for ova, and refrigerated before feeding to beetles
Tenebrio Molitor: Intermediate Host
In nature, a number of insects may be the intermediate host for H. diminuta. In the laboratory, grain beetles are the most often used, either Tribolium confusum ("confused flour beetle") or Tenebrio molitor ("mealworm beetle"). The mealworm beetle is convenient in some ways: mealworms, used as feeders for reptiles and chickens, are inexpensive and available from many sources; the large beetles, about the size of a gelcap, are easy to handle; and there are plentiful online instructions for rearing them.
Obtaining beetles
T. molitor beetles may be found at Carolina Biological Supply, and irregularly on eBay.
Beetles may also be raised from mealworms sold as feeders in about three weeks in ideal conditions. Use care when buying worms, and prefer organic or hormone-free. Avoid 'super worms', which look like mealworms but are another species. Also avoid 'giant' mealworms, given hormones to increase their size but prevent them from pupating.
Maintaining a mealworm culture provides a constant supply of beetles. It is inexpensive and not much more difficult than maintaining beetles, and there are many excellent guides online. The complete mealworm life cycle is several months, so ideally find a starter culture with a mixture of eggs, larvae, pupae and beetles to ensure the constant supply of new beetles needed for a constant supply of cysticercoids.
Maintaining beetles
Maintaining beetles is not difficult, but the insectarium requires ongoing monitoring and maintenance of food and moisture every 3-5 days. Temperature is also a concern.
Beetles should be fed oats. Although wheat bran is more nutritious, Dr. Parker said (in private conversation) that an overly healthy beetle's immune system may hurt H. diminuta development, and his protocols call for oats.
While precise humidity is not critical, the insect habitat must not be allowed to become too moist or too dry. Online mealworm culture guides recommend using certain vegetables such as carrot, potato or apple slices for moisture. The moisture source should be changed regularly to avoid growing mold, mites or flies. Many dead beetles and dead, blackened mealworms may signal too much moisture. While it is easy to overdo moisture, it is also possible to err toward overly dry conditions, which cause cannibalism. This balance may take time to learn, and mistakes should be expected.
An incubator will maintain ideal temperature, but is not essential if temperatures are moderate and the maturing larvae are not hurried to harvest. The H. diminuta cysticercoid takes over two weeks longer to develop at 20 deg. C (68 deg. F) compared to the ideal temperature of 30 deg. C (86 deg. F). Avoid exceeding this ideal temperature. (Arai, p. 144)
Rattus Norvegicus: Definitive Host
H. diminuta can only complete its life cycle in the rat. Mice reject the parasite after 10-15 days (Arai, 552). Experiments with humans failed to produce viable ova (personal communication). Workers have found hamsters infected with H. diminuta in pet shops (citation needed), but I found no accounts of hamster hosts in the laboratory.
Ethical Treatment of Rat Hosts
As said earlier, experimenters are strongly encouraged to raise H. diminuta from ova to cysticerci a few times before committing to complete the life cycle, which requires acquiring rats, which live two to three years. Professional laboratories must comply with certain standards of ongoing care for the animals, and an amateur laboratory should be no exception. Ideally, I hope that those caring for rats could value them as we do Norma and Rhonda, our family's intelligent, hilarious, fun and companionable pets. The JoinRats website provides complete information about how to care for rats, how to bond with them and how they can make wonderful pets. In any case, PLEASE make the decision responsibly.
Along these same lines, I ask myself whether I am inflicting suffering on our pets by burdening them with parasites. So far I have seen no reason to think so, but I hope to be corrected if wrong.
Aside from the use of the common term in the field, 'burden', there have been no hints in the literature that H. diminuta harms its host. Willis and Poulin found no ill effects on weight or reproduction on infected rats fed ad libitum, compared to control. Burden in the H. diminuta/rat system appears to self-regulate: heavier infections incur higher losses (Arai, p. 592), and worm size decreases as count increases with the well-known 'crowding effect' (Brant and Hanelt).
Most reassuring of all is Norma and Rhonda seem to be just as happy, healthy and energetic as ever since their infection. Perhaps H. diminuta has as few harmful effects in rats as it is reputed to have in humans.
Infecting beetles from Ova and Nurturing Cysticerci
In nature, grain beetles infesting the same food supply as rats would consume ova-infected droppings. The laboratory procedure is just as simple.
In ideal temperature conditions (see above), it takes eight days to form infective cysticercoids. Anecdotes say that larvae should mature for a month before therapeutic benefits become most effective.
There are various ways to expose beetles to feces; I don't know which is best. Some workers find that beetles starved for four days before exposure to feces will have higher levels of infection (Arai, p. 145). Brant's procedure doesn't call for starving beetles, but instead exposes beetles to feces for three days.
Carolina's ova in rat feces seem to be ready to use, already mixed with water and homologated [FIXME sp?]. If starting with rat droppings, see 'Harvesting Ova' below; feces should already have water and wheat bran added and homologated.
Materials list
Infecting beetles from ova:
- About 15 cc. rat feces containing H. diminuta ova, already moistened, baited and homologated
- 20-50 Mealworm beetles
- 1 Applicator stick
- Paper towels
- Dish to contain beetles
- Distilled or aged water; optionally in plant mister
Nurturing cysticerci:
- Infected beetles
- Inexpensive digital hygrometer/thermometer
- Wheat bran
- Vegetables for moisture source
- Optional: incubator
- Paint for container walls that insects can't climb
Obtaining materials
Carolina Biological Supply supplies rat feces containing ova. They ship a full 50 cc. centrifuge tube, enough to infect hundreds of beetles.
Beetles can also be purchased from Carolina, or other sources; see the 'Obtaining beetles' section, above.
The applicator stick may be any improvised, clean tool to spread feces on paper towels.
A dish for beetles should have steep, smooth sides. Many disposable plastic containers work. It should be partly covered to retain some moisture but also let in fresh air. Most simply, punch many small holes in the lid, or cover a larger hole with screen or cheese cloth.
Digital hygrometer/thermometers cost $5-$10 on eBay. Check it has your preferred temperature scale, Centigrade or Fahrenheit. The device should be put into the dish where the beetles can not climb on it, and where it can be read. Mounting tape or a blob of hot glue might be helpful.
Wheat bran costs less than $2/lb. in the bulk section of the local health food store. It can also be found online. Age water by leaving tap water in an uncovered dish for two days, allowing chlorine to evaporate.
Many people think carrots are the best vegetables to provide moisture for beetles because they don't mold easily. Potato, apple and other vegetables also work. Avoid citrus. I like cactus, handy and free in Texas.
Infecting Beetles
If you choose to starve the beetles before infection for four days, [to be written].
Cover the bottom of the dish with a layer of paper towel, cutting it small enough that it lays flat. Smear some feces onto the towel with the applicator. Just a spoonful is enough, or two for a large number of beetles. Too much will go uneaten and quickly mold.
Cut one or two smaller pieces of towel and place on feces. These satisfy the beetles' urge for cover, as well as keep the feces moist longer.
Sprinkle or mist towels with water. Don't make them soggy.
Put beetles in dish and cover.
Put dish in incubator, if available, and set at 30 deg. C (86 deg. F).
If the beetles have been starved, they should quickly seek the feces and begin feeding.
If the beetles have not been starved, for the next few days, check the dish. If the feces have been consumed, add more. Sprinkle or mist water daily. If the hygrometer gets close to 50%, add more water.
Three days should be long enough.
Nurturing Cysticerci
After the beetles are infected, remove towels and feces from the dish or find a new one. Add beetles and about 1 cm. (1/2") wheat bran to dish.
Put a chunk or slice of carrot or other moisture-providing vegetable in the dish and cover.
Every day, check the hygrometer. If it gets close to 50%, add more moisture vegetables. Also watch for too high humidity, which can bring mold. Clean out old vegetables before they get moldy.
When using an incubator, cysticerci may develop within eight days. If not, it may take longer, sometimes two or three weeks. Harvest after one month.
When it seems likely that cysticerci might have developed, remove a beetle and check it for infective larvae; see the below procedure, 'Harvesting Cysticerci'.
Harvesting Cysticerci
In nature, the procedure for 'harvesting cysticerci' is simply to eat infected beetles. That's an alternative to the following procedure if you are brave, and are unwilling to spend the money and time to follow this procedure. In my estimation, one infected beetle from my experiments contains between 20 and 60 HDC.
This procedure gives the following benefits compared to nature:
- Certainty over the number of HDC ingested
- Fewer insect parts: more palatable to the squeamish
Materials list
- One or more infected beetles
- Small scissors, knife or scalpel
- Tweezers
- Watchglass or petri dish
- Saline solution, 0.6% - 0.9%
- 10 ul pipettor and tips
- 5 ml or smaller disposable pipettes
- 1.5 ml or 2 ml centrifuge tubes and rack
- Dissecting microscope
Obtaining materials
Most items can be purchased from Carolina Biological Supply or eBay (except for beetles, which are obtained from following previous procedures).
Scissors, knife or scalpel and tweezers may be purchased together in an inexpensive student dissection kit. If using household items, nail scissors, hobby knife or box cutter, and eyebrow tweezers may be substitutes.
Saline solution can be made by dissolving nine grams uniodized salt in one liter of distilled water.
A pipettor can be very expensive, but new bargain brand or older models may be found on eBay for under $30, as can a new box of tips. A used pipettor's history is unknown; consider the risk that it may be contaminated.
A dissecting microscope is the most expensive tool. I was recommended the OMAX model CS-G223E, and also looked at AmScope models, both under $150. Look for stereo or binocular models; looking through a single eyepiece for a long time is tiring. Also look for forward models; pipetting is much more difficult with cheaper models with reversed image. Another consideration is illumination: tungsten vs. LED, and top-only vs. top and bottom lighting.
Pipetting technique
To use the pipettor effectively, one must be familiar with the instrument. Pipettors might be thought of as high precision eyedroppers. Analogous to squeezing and releasing an eydropper bulb, one depresses a plunger on the end of the pipettor to expel liquid from the tip, and releases to draw liquid in.
The plunger has two ranges. The first range is the normal fill range. When depressing the plunger, one will feel light spring resistance. At the bottom of the fill range, the overfill range begins. The spring resistance becomes suddenly greater at this point. Become familiar with your pipette to identify these two ranges.
When drawing up liquid, first depress the plunger to the bottom of the fill range. Do not depress farther into the overfill range. Release the plunger to draw liquid into the pipette tip.
When transferring liquid into a centrifuge tube, which must be held stationary in a rack, place the tip against the inside of the centrifuge tube. Depress the plunger past the fill range to the bottom of the overfill range. In this way, the entire contents of the tip will be expelled into the tube.
If when drawing up liquid, the plunger is pressed into the overfill range, or when expelling liquid, the plunger is not pressed into the overfill range, a small amount of liquid will remain in the tip after the transfer.
Experiment transferring water into a centrifuge tube until you become familiar with pipettor operation.
Dissecting Microscope technique
(To be written)
Procedure
Select a beetle, and use scissors or knife remove the abdomen. Cut the abdomen open and scrape the insides out into the watchglass or petri dish. Dispose of the beetle's head, thorax, inner and outer wings, and shell of the abdomen. With a disposable pipette, dispense enough saline solution to cover the innards. Place the dish under the microscope.
Install a tip onto the pipettor. Place an open centrifuge tube into the rack.
Looking into the microscope, move the dish around to locate a cysticercoid. Cysticerci will have settled on the dish at the bottom of the pool of saline.
Depress the pipettor plunger to the bottom of the fill range and place the tip on the dish, next to and pointing toward the cysticercoid. Release the plunger to draw the cysticercoid into the tip.
Move the pipette tip into the centrifuge tube, touching the inside wall. Depress the plunger past the fill range to the bottom of the overfill range to expel the cysticercoid into the tube.
Continue transferring cysticerci into the centrifuge tube until the desired number have been collected. I have been able to collect 20 or more cysticerci from a single beetle with this technique.
When finished, ensure larvae will not dry out. Use a disposable pipette to add more saline solution to fill the tube and seal shut.
Larvae should be observable through the microscope in the bottom of the tube. These may be used immediately or stored in the refrigerator up to two weeks.
Infecting rats with cysticerci
Brant's parasitology lab guide describes a means to infect rats with a known number of cysticerci by harvesting (as described above) and feeding to anaesthetized rats. Nature's method, where rats eat insects containing infective H. diminuta cysticercoids, is simpler and arguably better suited for the simple purpose of producing ova in the amateur laboratory.
Brant's procedure requires anaesthetizing the rat to administer larvae using an eyedropper. Anaesthetization risks the rat's life, and the restricted chemicals are not easily obtainable in the amateur laboratory. Eyedropper administration of larvae to rats without anaesthetization is sure to be a struggle and destroy trust in humans. Knowing the exact count (above one) of larvae administered to the rat is unnecessary. A single mature worm produces thousands of ova each day.
Overburdening the rat with the parasite is also not a risk. The well-known 'crowding effect' (which Brant's procedure is designed to demonstrate) limits the burden from a single infection. Arai's Immunity and Hymenolepis diminuta discusses in great detail how following infections are rejected, probably by the rat's immune system.
Therefore, this guide suggests the rats be infected just as they are in nature, simply by feeding infected beetles to the rats, which they find a tasty treat. If greater surety of infection is desired, the beetle may be examined to verify presence of cysticerci, and then fed to the rat.
Harvesting Ova
Ova leave the definitive host in its feces. Before attempting to infect beetles, ova content should be verified, some bait should be added, and the droppings should be moistened and homologated for easy handling.
Materials list
- Rat droppings
- 50 cc. centrifuge tube or other small mixing container
- Wooden applicator stick or other clean tool for mixing feces
- Distilled or aged water
- Wheat bran
- Microscope
- Slide and cover glass
Obtaining materials
The rat droppings should be fresh as possible because moist droppings are easier to mix and manipulate. Find a small container and clean tool for mixing up feces. A 50 cc. centrifuge tube and wooden applicator stick is a great combination, and the tube can be capped for refrigeration.
Wheat bran costs less than $2/lb. in the bulk section of the local health food store. It can also be found online. Age water by leaving tap water in an uncovered dish for two days, allowing chlorine to evaporate.
Procedure
Put droppings in container. Fill centrifuge tube up to 3/4 with uncompressed droppings.
Add enough water to cover about 1/2 of droppings.
Add a three-finger pinch of wheat bran.
Compress droppings until covered by water. Optionally wait for them to soften, especially if dry and hard.
Thoroughly mix feces, water and bran to a pasty consistency. Add more water if needed.
Wipe tip of tool on slide to transfer a thin coating of just the liquid part of the mixture onto the slide. Wipe off any thicker pieces of solid matter. Try to cover an area about the size of the cover glass.
Add a drop of water and cover with cover glass.
Put slide on microscope and set magnification to around 100x.
Scan slide for ova.
If ova are present, cover the container, mark it with the date, and store in refrigerator for infecting beetles.