Isopod Experiment: A. Vulgare Choice Chamber Tests

[ohaple]

I have recently decided to start an isopod breeding project. While I quite enjoy keeping mantises, I am not terribly interested in their breeding. Other invertebrates are much easier to breed since they are kept as a culture rather than individuals. Species that breed and live more quickly and have higher brood sizes are easier to conduct breeding projects on. Isopods are great because many types are polymorphic, most are easy to breed, and they take minimal maintenance. I am particularly fond of armadillidium vulgare since they are widespread, critical to many ecosystems, common, reasonably large, and can volvate (roll into a ball). My breeding project will seek to isolate a morph of A. vulgare starting with wild specimens from the Colorado front range. I am going to choose the morph based on what I can find locally.

Prior to starting the breeding project I am interested to run some tests to determine the preferred conditions for A. vulgare. Those tests are what this thread is about.

Special thanks to @Orin for writing the wonderful Isopods in Captivity book, and to other scholars like Isabel Caseiro, J.P. Sousa, and S. Santos (Optimization of Culture Conditions of Porcellio dilatatus for Laboratory Test Development, 1999) who have worked on studies that I have reviewed to help me figure out what I want to do. I will do my best to cite where possible. I intend for this to be a pseudo-scientific test; not up to true scientific standards, but following basic requirements like controlling other variables and ensuring the results are statistically significant. I understand there may be a significant difference between the conditions A. vulgare prefer, and the conditions under which they breed best/fastest, but I will only be testing for preference here. I will seek to conduct the test in an ethical fashion by not testing conditions that are known to damage isopods and only collecting specimens from areas where there is already an abundance.

Anyone with knowledge, corrections, links to existing studies, or suggestions, please chime in. 

To conduct the study I will be using a choice chamber. Choice chamber experiments are generally conducted by placing specimens into a container with multiple chambers. Each chamber has different conditions. After a period of time, the specimens are counted in each chamber, and a chi-squared analysis is conducted to determine whether the results are statistically significant. For instance, if you place 10 specimens in a two-chamber choice chamber apparatus and each chamber has 5 specimens after a period of time, that would not be statistically significant because we would expect such a result by chance. If you found 10 in one side and 0 in the other, that would be statistically significant. If the results are statistically significant, the isopods prefer the condition where more of them appear. If not, they don't have a strong preference either way. The bigger the sample, the more granular and accurate the results will be.

To conduct the test I have designed a modular choice chamber. It is a 10x10x2.5" box made of white opaque acrylic. The modular dividers allow the box to be divided into 2 connected chambers, 4 connected chambers, or two pair of connected chambers. When I transition the project to breeding, The dividers can separate the box into non-connected chambers as well by inverting them. The lid is made from clear acrylic with narrow vents cut into the lid to allow some ventilation without excessive moisture loss (and hopefully no gnats). Each corner of the lid is labeled with a laser-engraved letter to allow me to easily keep track of each over time. Here are photos of the apparatus:

2

The variables I am interested most in testing are food, substrate, and temperature.

I plan to test dried shrimp, Hikari crab cuisine, Shrimp King Complete, Shrimp King Yummy Gum, Snowflake (a common shrimp food made from soybean shells), dried leaves, dried mealworms, cofee grounds, a molt from a mantis, roach frass, mushrooms, potato, carrot, and other veggies.

Next step is to gather the specimens. That will be easier said than done in Colorado this time of year. Our weather just exited many near 0 degree days, and has been in the 40s for a week or two. The survive the winter, but I believe their populations will be small and inactive. I may have to wait for a while before they are easily found.

 

[Orin]

Cooked or fresh veggies? lichen? 

 

[ohaple]

Cooked or fresh veggies? lichen? 

I was intending to use blanched vegetables for things that spoil more easily and raw for root vegetables. Blanching is nice for a few reasons. First, it allows things like zucchini to last longer so that spoiling and nutrient loss is held off for a while longer in the enclosure. Second, while blanching removes some nutrition value it also helps stored vegetables to keep their nutrients longer, which is important for convenience since a whole zucchini can be cut, blanched, dried, and refrigerated or frozen for storage. Third, it allows those vegetables to sink in water. This is important for feeding aquatic snails and shrimp, which we also keep.

I also was planning to avoid vegetables like spinach that are known to bind calcium with oxalates. Calcium is important for most inverts and I do not want to bind calcium and potentially cause failed molts. I have read some on how isopods are unique in their storage and utilization of calcium in highly soluble stores, but I must admit those journal articles are mostly over my head. (see. Microscopical and functional aspects of calcium-transport and deposition in terrestrial isopods). This method of storage may prevent binding from oxalates but since I am not confident, I intend to avoid oxalates.

Lichen is a very good idea. I believe it is primarily fungus, but may be processed differently by the isopods than other types. It will be difficult to collect around here since we primarily only see it in the higher-altitude regions. I may be able to order some online. I like it as an idea because it could operate as food, shelter, and decoration for a display enclosure.

I also have access to a couple of types of algae. While terrestrial isopods usually won't be working with much algae or aquatic biofilm (as far as I know), I wonder if those would be good food sources since they are utilized by asellus aquaticus and other aquatic detritivores (see. Feeding and growth of Asellus aquaticus (Isopoda) on food items from the littoral of Windermere, including green leaves of Elodea canadensis).

Another variable that I intend to test, but expect non-significant results, is water quality used for moisture. Distilled or RO water may be less beneficial than high gH/TDS water. High gH water will have more magnesium and calcium ions. This may be insignificant on its own since many nutrients will leech from the substrate anyways, and high-calcium foods are likely to be better for isopod intake. Distilled water is preferred for display enclosures to cut down on hard water spots on the glass that will inhibit viewing.

I have a little background in statistics from my undergraduate studies, but no formal biology training. Every time I search a new subject I am surprised at the depth and specificity of research that has already been conducted in the field. Unfortunately most of it is hidden in journals that the public usually doesn't look at.

 

[ohaple]

With the recent weather and some more research I think that I am being overly optimistic with my expectation that I can gather enough wild isopods in Colorado. The populations will not grow and become active for several months, in which time I could have already tested many variables on the choice chamber.

So, I am changing plans a little. I will be buying a set of A. Vulgare to run most of the tests. I still intend to find wild-caught individuals as I can in the coming months to work on some breeding projects.

The other reason is sample size. I am investigating the best type of statistical analysis to use. Chi squared is poor with small sample sizes, where Fisher's exact test should be used instead. While Chi squared is pretty easy to run, I will need to re-research Fisher's exact test since I cannot even remember how the test is completed. I am worried that I will have difficulty finding more than 10-20 wild caught specimens, while I can buy however many are needed.

 

[hysteresis]

brooooother

You're taking me waaaay back with teh stats.

 

[ohaple]

brooooother

You're taking me waaaay back with teh stats.

Lol. Yeah. I feel that too. Math used to be my strongest subject, but now its been 8 years since my last statistics class so I am very rusty.

I put in the order for the isopods and will have 50-60 wild type A. Vulgare coming next week. That should be plenty to allow me to begin the choice chamber tests. I also ordered a powder mix because I couldn't resist, but those won't be part of this test.

 

[hysteresis]

@ohaple I graduated engineering in 1996. My head hurts. 

 

[ohaple]

They arrived yesterday. A nice mix of larger juveniles. A few DOA due to failed molts in transit. I separated out those displaying different morphology (brown body or yellow spotted). There are about 40 left of the wild type. One thing I neglected to research was alternatives to traditional substrate. For ease of counting and eliminating nutrition from the substrate, I hope to use damp paper towel or similar. Will need to research it first to ensure they will not suffer on such a substrate. Going to be a busy couple of days but I am hoping to initiate the first choice chamber tests on Sunday.

 

[hysteresis]

Dr. Ohaple

 

[RebelleSinner]

How exciting!

 

[ohaple]

Test 1

I set up the first test last Friday evening. I decided to test a few fish foods to start. Hikari Crab Cuisine, Hikari Micro Pellets, Tetra River Shrimp, and PE Pellets. This is an initial test to start fine tuning the setup and learning some initial hypotheses to test in more detail later.

For "substrate" I used four layers of paper towel, lightly sprayed so that each layer was damp, but not wet. I sprayed all four compartments at once with the bottle held about 18" from the container to reduce chances of uneven moisture. I placed 7 isopods in each compartment to start (except one that had six due to a counting error), along with a small portion of each food in the center of each compartment. Then I placed identically-cut portions of egg crate in each section as a hide so the isopods would be more willing to eat. Then I put the lid on and put it on the shelf.

After about 24 hours, the Crab Cuisine (A) had 11 individuals, the Tetra River Shrimp (B) had 14 individuals, the PE Pellets (C) had 1 individual, and the Micro Pellets (D) had 1 individual.

After these results I suspected that the shelf they were on was causing the results to be skewed. A and B were in the back and presumably receive less light. Isopods are light-averse, so you would expect them to gather in an area with lower light if all else is equal. I turned the box 180 degrees to see if it had an impact.

After about 36 hours, the Crab Cuisine (A) had 4 individuals, the Tetra River Shrimp (B) had 10 individuals, the PE Pellets (C) had 9 individuals, and the Micro Pellets (D) had 3 individuals.

At 36 hours I noticed that all three varieties of processed food were molding. I inspected each food to see what had actually been eaten. It appears they ate some of the shrimp's head, and a little bit of the Crab Cuisine. I didn't notice any real change with the other two. I removed the isopods and cleaned out the enclosure by changing the top towel. Due to the results likely being skewed by light, I will not be running a statistical test for this one, but you can see the trends.

This gave me a few hypotheses and observations:

1. dIsopods generally care more about moisture and light than available food. This will make the tests difficult because if these factors aren't kept constant with hides, light, and moisture, the results regarding food could be considerably skewed.
   
2. Isopods appreciate a source of chitin. Chitin is what the exoskeleton is made of. You will notice that your isopods eat their own molt to obtain the chitin to make a new exoskeleton. The dried shrimp likely had the highest percentage of chitin of the foods offered, and has not been cooked or processed, which may break down some components. Chitin is also important to invertebrate immune systems (see p71 here). It does not appear that the high protein content (60%) was the primary driving force for this preference since the PE Pellets had 42% protein and the Micropellets had 43%, and both didn't test great.
   
3. Isopods need a course of calcium. Their natural diet of decaying leaves contains calcium. Calcium content of many trees can be found in this report. The Crab Cuisine was the only food with appreciable calcium content (still less than leaves). This likely contributed to the success of the Crab Cuisine prior to molding.
   
4. The boost of chitin or some other factor may have jump-started a molting response. We know that diet impacts the molting frequency of isopods, so this is possible. See, Drobne, Damjana & Strus, Jasna. (1996). Moult frequency of the isopod Porcellio scaber, as a measure of zinc‐contaminated food. Environmental Toxicology and Chemistry. 15. 126 - 130. 10.1002/etc.5620150209. At the end of 36 hours about 1/5 of the population had a half color change, which is characteristic of the beginning of isopod biphasic molting.
   
5. @Orin recommends "brown, old leaves that have been on the ground at least a few months" as the primary food for isopods in "Isopods in Captivity" p. 29. I think this is a good and well-supported recommendation since it is their natural diet. That said, I believe that we may be able to make a colony more productive if isopods have more access to the nutrients they need through supplemental food. I plan to keep a close eye on the content of food offered and compare it with hardwood leaf nutrition to see if we can find any correlation.
   
6. Regardless of nutrition, the ability to resist mold is a valuable trait in isopod food since they will be left in a mold-prone environment. The shrimp showed no mold, and no sign of degradation at all, though its texture became somewhat softer from being partially rehydrated. This leads me to want to test other dried natural foods like mealworms and bloodworms. My small p. dilitatus culture (7 adult, 50-100 young juveniles) devoured an entire shrimp in three days, in which time the shrimp didn't even begin to mold.
   
7. Results may be overstated due to the pheromone communication used by isopods. When one finds conditions it likes, it will signal to others. This might lead to increased grouping even when two foods are largely identical, and may make differences seem greater than they actually are. Unfortunately this can only be reduced by isolating isopods and running tests with separate choice chamber setups for each individual.
   

This test taught me a few important things to change for future tests:

1. Make sure to start with a number of isopods equal to the possible choices. I should have used 28 instead of 27, but messed up counting. This is just to keep as many controls as possible, but the isopods move so much over 24 hours that I do not believe initial position has any effect on the final distribution.
   
2. Use much less food, and try to keep the food a little more dry. Where possible, foods that don't mold easily should be used.
   
3. Pin down the corners of paper towel. The isopods try to dig into the corners to get in-between the layers. That isn't a major problem for their health, but effectively takes them out of the test.
   

Test 2

I am adding additional controls for test 2 to allow more reliable results. I now am keeping the container in a large drawer that receives no light. With all specimens in darkness, differences in light do not exist. I do not believe this is cruel since isopods have only rudimentary eyes and typically choose to be in low-light environments when given the chance. They will still get light during observation periods when I count them. I also added small magnets to each corner to pin the paper towel down so isopods couldn't dig. I added one more isopod to make the numbers even. Now there are 28.

This time I am testing Tetra RiverShrimp, Aqua Culture Freeze Dried Mealworms, Aquatic Foods California Blackworm Co Freeze Dried Bloodworms and Aqueon Shrimp Pellets.

I hope that these foods will not mold as quickly. I also used smaller portions. Bloodworms have chitin in their headplates and spine, and otherwise have a soft outer that I believe is primarily protein. Mealwoms also have chitin. I am hopeful that the shrimp pellets underperform the other types of food, because it would imply that the isopods are seeking chitin sources, which is much lower in the shrimp pellets. I expect that the dried shrimp will perform best, and I will pay attention to the part of the shrimp that is consumed. It would be interesting if the head is consumed first again, implying that there are some other nutrients in the head that the isopods seek.

The test was setup around noon today, so the first observation period will be about 32 hours after. Ideally I would keep these periods consistent, but I have to work around work and other responsibilities. I do not believe that there would be any difference at longer time intervals, but will keep reading to obtain more data points.

 

[ohaple]

The results for Test 2 are in after 24 hours, and I am more concerned about the viability of this experiment moving forward.

Tetra RiverShrimp had 1 individual, Aqua Culture Freeze Dried Mealworms had 12, Aquatic Foods California Blackworm Co Freeze Dried Bloodworms had 15 and Aqueon Shrimp Pellets had 0.

Here is the issue. None of the food appears to have been eaten at all. I inspected the mealworm that had the second most specimens, and found that there were no bite marks observable. The bloodworms may have been eaten some, but it is too difficult to tell due to the way they shrivel when they rehydrate. The RiverShrimp was not visibly eaten either. Good news, no mold.

I intend to observe again tonight at 48 hours to see if anything has changed. The next test I am going to vary the food more. I had originally intended to begin by testing "classes" of food (i.e. different fish food, different vegetables, different leaves, etc) and then test to determine which class of food was preferred. It seems now that the foods selected are too similar, and that the isopods are likely grouping due to pheromone response and not due to the food available. Next test will be 1 vegetable, 1 leaves, 1 dried natural food, and one fish food. It is also possible that the isopods are uncomfortable enough in the paper towel substrate that they are simply prioritizing shelter and moisture over food available. I will need to continue troubleshooting to determine whether the test is useful and worth continuing.

_______________________________________________
Got another set of results last night for Test 2 after 48 hours
Tetra RiverShrimp had 3 individuals, Aqua Culture Freeze Dried Mealworms had 2, Aquatic Foods California Blackworm Co Freeze Dried Bloodworms had 16 and Aqueon Shrimp Pellets had 7.
_________________________________________________
Another set of results for Test 2 after 72 hours.
Tetra RiverShrimp had 11 individuals, Aqua Culture Freeze Dried Mealworms had 6, Aquatic Foods California Blackworm Co Freeze Dried Bloodworms had 8 and Aqueon Shrimp Pellets had 2.

Here they are side by side for three observation periods.

24 Hours: A 1, B 12, C 15 and D 0.
48 Hours: A 3, B 2, C 16 and D 7.
72 Hours: A 11, B 7, C 8 and D 2.
Avg: A 5, B 7, C 13 and D 3

With a sample of 28 and 4 choices, the null is an average of 7 each. While 28 is a low sample for Chi-Squared, the results (using the calculator here to save time) are:

Chi squared equals 8.000 with 3 degrees of freedom.
The two-tailed P value equals 0.0460
By conventional criteria, this difference is considered to be statistically significant.

This means that something besides chance is likely the cause for the distribution of specimens. Our primary variable is food, but moisture levels, pheromone grouping, and even small folds in the paper towel could also contribute to the distribution. On observation it does appear that the bloodworms were consumed slightly, while I could not observe any consumption of the other food types. The data is not as satisfying as one might hope. Again, the next test will seek to test significantly different foods so that we can hopefully see a clear distribution.

I plan to put the isopods in a traditional substrate and enclosure for a few days now to give them a break and relieve any stress they may be exhibiting due to the non-traditional substrate and staple food like decaying leaves.

 

[Charoozz520]

@ohaple excited to see your results! I know this is a controlled experiment but as you mention their need for chitin/calcium. What I have done is added crushed cuttlefish bones for this requirement. 

 

[Orin]

One thing to keep in mind is a desire to eat something does not equal suitability. Imagine if you tried the same experiment with humans and had one room with candy, another with red meat, a third with tofu and a fourth with alcohol and drugs.

 

[hysteresis]

... and a fourth with alcohol and drugs.

ZOMG 

 

[ohaple]

One thing to keep in mind is a desire to eat something does not equal suitability. Imagine if you tried the same experiment with humans and had one room with candy, another with red meat, a third with tofu and a fourth with alcohol and drugs.

This is a good point, and I definitely agree. I tried to acknowledge it in my original post when I wrote "I understand there may be a significant difference between the conditions A. vulgare prefer, and the conditions under which they breed best/fastest, but I will only be testing for preference here" but I know this is a wall of text so things like that get lost easily. Testing for best conditions is largely anecdotal based on keepers who have years of experience. Their experience is useful even though they don't have hard numbers. The only such study I know of is "Optimization of Culture Conditions of Porcellio dilatatus for Laboratory Test Development, 1999"

Choice will determine what they prefer but not necessarily what is best for them. There is some correlation between preference and suitability due to evolution, but it breaks down somewhat when scarcity isn't present. Candy and fatty meats are great for humans when food isn't plentiful. In fact, lean meats like rabbit have been known to cause "rabbit starvation" since they don't have enough fat. On the contrary, our plentiful western society can see fatty food lead to heart disease etc. I am going on the assumption that isopods are somewhat the same.

Protein and chitin heavy foods may have benefits in small quantities, but should not be a meal replacement. I advocate for a balance of foods with leaves being always available and supplemental food being rotated so that the isopods can get a variety of nutrients. The purpose of the choice experiment is to help narrow down what sort of supplemental foods the isopods crave because there is likely a correlation between what they crave and nutrients that benefit them.