A Study of the Effect of a Compromised Immune System on Tenebrio molitor Exposed to Environmental Bacteria and Fungi at Increasing Temperatures

Olivia Silva Massachusetts Academy of Math and Science Abstract The effects were investigated of culturing the common mealworm (Tenebrio molitor) with a compromised immune system in soil at different temperatures containing environmental bacteria and fungi. It was hypothesized that with increased temperatures, the rate of movement of T. molitor will decrease and the rate of mortality will remain the same. Three groups of T. molitor larvae with varying simulated injuries were cultured in soil at 19 C, 21C, 23C, and 25C. With increasing temperatures, it was found that the rate of movement decreased and the rate of mortality remained constant. By increasing the soil temperatures, the effects of global warming on the effects of potential microbial pathogens were simulated in T. molitor cultures. The collected data suggests a trend that may aid in predicting future insect population fluctuations based on environmental temperatures. Introduction In 2006, 34.6 million people were discharged from non-federal short stay hospitals filled with potential hosts for Vancomycin-resistant Enterococcus (VRE) bacteria (National Hospital Discharge Survey, 2006). The human immune system is vulnerable to multidrug resistant organisms (MDROs), such as VRE bacteria. While researchers spent decades studying new antibiotics, some strains of bacteria evolved enough to become resistant and were classified as MDROs. VRE bacteria are resistant to vancomycin-based antibiotics, creating multiple complications in the treatment process because most antibiotics contain vancomycin. In the hospital setting, new pathogens can infect patients who already have a compromised immune system. When discharged, patients will occasionally return home with a new infection, resulting in the patient returning to the hospital. Having a compromised immune system can be dangerous, especially when a potential host is subjected to new pathogens. In nature, organisms must rely solely on their immune systems to fight pathogens. The insect immune system is stronger and more efficient than that of humans, but it is still vulnerable to pathogens. Studying the relationship between pathogens and their insect host elucidates aspects of evolutionary biology (Indikris Krams, personal communication). Insect immunology aids in studying pest control and some aspects of human disease. Global warming has a significant impact on insects environment, and potentially affects their bodily systems and evolution. The study of insect immunology can provide parameters for predicting insect population sizes, life span, and evolution. Global warming affects microorganisms in the same manner, and their interactions with hosts may be elucidated by means of studying immunology. Researchers have used

Immunity of Tenebrio molitor

Tenebrio molitor multiple times to conduct studies focusing on the immune system, but the researchers usually conducted studies to discover links between the immune system and other bodily systems, such as the reproductive system. Few studies have been conducted to test for a correspondence between environmental temperature and immune function. Literature Review The Anatomy and Life Cycle of Tenebrio molitor The metamorphic life cycle of the Tenebrio molitor comprises four phases: egg, larvae, pupa, and beetle, as seen in Figure 1. Completion of the entire life cycle takes an average of five to six months. In warm temperatures, the insect will take an average of one to two weeks to complete the gestation period, but in cooler temperatures, the gestation period lasts up to three weeks. The larval stage takes an average of two months, during which the insect will grow to two centimeters in length. During the larval stage the insect will go through ten to fourteen instars, each represented by the formation of a sternite. The insect will then pupate and remain a pupa for two to three weeks, and then the insect will develop into its final state, which is the darkling beetle. As a beetle, the T. molitor will live for an average of two to three months (Greenberg, 1996). The term mealworm refers only to the larval stage of the Tenebrio molitor. During this phase, T. molitor has a segmented body, each of the thirteen segments representing a separate instar. On the larva, the head is located on the first instar. In the head, there is the larval eye, labrum, antenna, claws, and mouth, as shown in Figure 2. The prothorax, mesothorax, and metathorax regions are posterior to the head, each having one pair of legs. Posterior to the thorax, the abdomen makes up the majority of the larva. Unlike male larvae, female larvae have a genital swelling on the seventh instar posterior to the head. The diet of Tenebrio molitor consists of leaves, grain, fruits, vegetables, and waste. Larvae inhabit areas in close proximity to a food source, in either soil or grain stores. Adult T. molitor inhabit dry, dark climates. T. molitor are photophobic; therefore, they migrate away from light (Hurd, 1991).

Immunity of Tenebrio molitor

Figure 1. Life cycle of Tenebrio molitor. The T. molitor completes four growth phases in its life. (Smith life science, 2012)

Figure 2. Basic anatomy of Tenebrio molitor. The T. molitor body has three main sections: head, thorax, and abdomen. (Enchanted learning, 2012)

Functions of the Tenebrio molitor Immune System During infection, parasites attack an organisms immune system. The infection is detrimental to its normal habits; therefore, organism can no longer function properly. To eliminate the pathogen, the organisms immune system will attack the parasite using one

Immunity of Tenebrio molitor

of many defense strategies specific to that organism. The insect immune system functions in two steps, the humoral and cellular defenses. With this system, the insects will entirely rid themselves of the pathogen. When the T. molitor is infected, the immune system releases antimicrobial peptides, taking one to three hours to commence and will last an average of two weeks (Haine, 2008). These peptides work to eliminate 99.5% of the pathogens in the first phase of the immune response, and in the second phase, the insect will undergo the melanization-encapsulation response (Schneider, 2008). During the encapsulation and melanization phase, the insects hemocytes will recognize the pathogen and surround it. Hemocytes are mesodermally formed blood cells in invertebrates, such as insects (Lavine, 2002). A reaction then occurs which hardens the capsule of hemocytes and breaks down melanin. The pathogen enclosed in the capsule will die of either suffocation or exposure to necrotizing oxygen and nitrogen compounds. This immunological technique is far more effective than that of humans with a compromised immune system, who have a high risk of contracting a pathogen. Humans will purchase antibiotics to aid antibodies in the elimination of a pathogen, but only because they are potent and inexpensive, not because they are fully effective. When fighting off a single pathogen, Tenebrio molitor has a large chance of eliminating it; however, researchers have not conducted studies with more than one pathogen attacking the insect. Fungi, Bacteria, and Parasites in Temperate Soil Tenebrio molitor thrive in a temperate climate zone with moderately dry soil; they are native to Africa but have become naturalized in North America. T. molitor commonly live under rocks, in logs, animal burrows, and in stored grains. Because the organisms live in many different parts of the Earth, they are exposed to many disparate types of soil and multiple different strains of pathogens. Soil in temperate climates varies greatly in chemical composition; strains of bacteria, parasites, and fungi; and insect inhabitants. When testing soil, researchers examine a pedon of soil, a unit of measure that specifies the minimum amount of soil that researchers use to examine its qualities and composition in the classification process. A pedon commonly has a minimal horizontal area of one m2, but may be as large as 10 m2 (Soil Survey Staff, 1999). Researchers classify soil into multiple different categories and types using a hierarchy based on moisture levels, salt levels, seed content, deposits from running water, and the types of rock, among many other criteria. Soil on the horizon may be composed of different elements and organisms than soil found centimeters below the surface. The two major categories in soil classification are mineral soil and organic soil. These categories are composed of numerous subcategories including anthropic epipedon, folistic epipedon, histic epipedon, melanic epipedon, and mollic epipedon (Soil Survey Staff, 1999).

Immunity of Tenebrio molitor Parasites That Attack Tenebrio molitor

Hymenolepis diminuta, commonly known as the rat tapeworm, is a parasite that commonly infects Tenebrio molitor. In the beginning of the Hymenolepis diminuta lifecycle, the intermediate host, the Tenebrio molitor, ingests the eggs. The host T. molitor must then be ingested by another host in order for H. diminuta to mature to the adult stage. H. diminuta will progress to the haemocoel where it will develop into a larva in T. molitor. Hymenolepis diminuta will remain dormant and encapsulated in the juvenile stage until the definitive host eats both T. molitor and H. diminuta. In the definitive host, H. diminuta will attach itself to the intestinal mucosa and grow into an adult. As seen in Figure 3, the adult contains male and female sex organs in each segment and is able to fertilize over 250,000 eggs per day, but most do not reach reproductive maturity (Dewey, 2001). Gregarina niphandrodes is a parasite that also commonly infects Tenebrio molitor, among other arthropods. The lifecycle of G. niphandrodes begins when adult T. molitor consumes G. niphandrodes as a haploid sporozite. In the intestine, the sporozite grows into a trophozoite, which has three sections, a protomerite, epimerite, and deutomerite (Janvoy, 2001). G. niphandrodes is at the reproductive stage when it attaches its protomerite to the base of its mates deutomerite. The trophozoites then become gamonts, which encase themselves in a gametocyst. T. molitor expels the gamonts with the frass, and the gamonts undergo cell divisions and differentiation. The gamonts are then male and female gametes, and when they are mixed, fertilization occurs. The zygote undergoes more cell division, which results in the gametocyst releasing oocytes that contain haploid sporozoites (Toso, 2007).

Figure 3. Reproduction in of the Hymenolepis diminuta. The H. diminuta attaches to its host at the scolex, where each new segment is formed. (Thomas, 2002)

Immunity of Tenebrio molitor

Figure 4. Gregarina niphandrodes reproducing in Tenebrio molitor host. In stage a the G. niphandrodes is a trophozoite, and in stage e it is attached to its mates deutomerite. ( Leander, 2003)

Effects of Global Climate Change on Insects Because environmental temperature is an important ecological element for ectothermic organisms such as insects, global climate change challenges the normal function of bodily systems. Temperature differences affect developmental rate, reproduction, survival, and various other life functions. In the past century, global surface temperature has increased 0.6 C, which has subsequently caused a growth in population sizes in tropical insects (Mandrioli, 2012). In moderate and continental climates, increasing temperatures will result in a longer reproductive season; therefore, the number of generations of insects will increase per year, accelerating the rate of evolution. There have been several studies concerning reproduction and growth, but few relating to the insect immune system. Researchers have conducted studies on the effects of increased temperatures on development and reproduction in Myzus varians. A small temperature increase (3.7 C) resulted in M. varians never fully completing the lifecycle, stopping before adulthood (Mandrioli, 2012). There have been studies regarding fitness level in adult Bicyclus anynana in warmer environments, and because the hemocyte level decreased, researchers concluded that the B. anyana immune system would be impaired with climate warming. However, no studies have shown a direct correlation between immune system functionality and climate warming. Another study showed that in M. varians large levels of CO2 resulted in a delayed pheromone response, and large levels of O3 resulted in an exaggerated pheromone response. Both gases relate to climate warming; in the past century, there has been a 30% increase in both gases (Bienen, 2004). Researches have shown that the increase in gases to cause disruptions in insect behavior because of the changes in pheromone release. Insects rely on pheromones for interaction purposes, such as signaling an alarm or sexual communication.

Immunity of Tenebrio molitor

Genes have responded to the effects of global warming. Researchers have mathematically proven a relationship between an increase in global mean surface temperature and the size of cells, metabolic rate, and lifespan using allometry and Einfinity theory (He, 2006). Because of the shortened lifespan, the rate of evolution accelerates. The mathematical proof supported results of a previous study, which showed that the average masses of the ants decreased up to 31% per colony, and the population size increased (Kaspari, 2005). Previous Studies on Tenebrio molitor Recent studies on the Tenebrio molitor have focused on the relationship between the immune system and the pheromones in the reproductive system, as well as the twostep process of the immune response. Researchers found that there is a correlation between the reproductive selection and the fitness of the immune system. If a parasite infects the insect, the level of attractiveness to potential mates will decrease. In 91 of the 114 trials, female Tenebrio mated with the healthy uninfected beetles (Krams, 2010). In addition, the Tenebrio were able to kill all Staphylococcus aureus 14 days after becoming infected. However, there have not been any studies on the reaction of the immune system to two induced pathogens. Studying the reaction of the immune system to a pathogen while already infected with a parasite has the potential for understanding the roles Tenebrio molitor play in their ecosystem, as well as understanding the efficiency of the insect immune system and its application to humans. Tenebrio molitor are capable of fighting VRE bacteria without creating new strains, which is something humans cannot do with the aid of antibiotics. Recently, a common topic of study has been the effect of global warming on insects and insect populations. As mentioned before, increased temperatures cause shorter life spans and larger populations, which accelerates the rate of evolution. There is also some evidence that increased temperatures cause a decrease in the average size of insects, a slower metabolism, and a higher overall fitness. However, there have only been a few studies focusing on the impact of global warming on the insect immune system.

Research Proposal Researchable question How does inserting a nylon monofilament into the abdomen of Tenebrio molitor and removing it after melanization begins affect the insect mortality rate and movement when cultured in soil of different temperatures that contains environmental fungi and bacteria? Hypothesis At room temperature, the mortality rate will remain the same, but rate of movement will decrease when Tenebrio molitor are cultured in garden soil containing

Immunity of Tenebrio molitor

environmental fungi and bacteria. As soil temperatures increase, the movement rate will decelerate and mortality rate will increase. Methods The mealworms will be massed and measured. Two thirds of them will then be placed on ice until they become immobile, which will numb their bodies. Using a 33gauge syringe, a hole will be punctured between the third and fourth sternite, and a 2 mm by 0.18 mm length of knotted nylon monofilament will be inserted into the hole in one third of the mealworms. The nylon will be scratched with sandpaper to imitate a parasite inserting eggs into its host. All the mealworms will be kept at a specific temperature in individual containers containing soil and wheat grain. The mealworms will be kept away from direct sunlight and any artificial light. After three hours, the nylon will be removed from the mealworms and they will be placed back in the soil. The remaining mealworms will be kept in individual containers under the same conditions. The dead mealworms will be counted daily. Each day, a flashlight will be shined on the mealworms as they get recorded for five minutes. The data will then be analyzed using LoggerPro. The entire process will be repeated at four different temperatures: (19, 21, 23, 25). A black heating light bulb and spotlight will be used to heat the environment to specific temperatures for testing. Methodology The mealworms (60 count, mixed size, PetCo) were divided into three groups, the control group (Group A), the treatment group (Group B), and the experimental group (Group C), and each group was placed in a separate container (11.5 cm x 19 cm x 6.5 cm, Lock-Top model, Food Storage brand, purchased at Dollar Tree) containing natural garden soil and half a small carrot (Stop and Shop brand, purchased at Super Stop and Shop), and were kept at standard room temperature, as seen in Figure 6. After waiting twelve hours, the insects in Groups B and C were immobilized on ice and were held down using two emery boards (11.7 cm x 1cm, Sally Hansen brand, purchased at Walgreens). A hole was punctured in the insects between the fifth and seventh sternite in their plural membrane using a needle (sterile, 30 gauge, Kendall Monojet). For the insects in Group C, a piece of nylon monofilament (two mm x 0.17 mm, Megathin model, Stren brand) was inserted in each hole using forceps (Medical Action Industries Inc. brand), as seen in Fig. 5, and was kept for three hours. The insects were returned to their respective containers. After three hours, the Group C insects were immobilized on ice and the nylon monofilament was removed from the insects using forceps (Medical Action Industries Inc. brand), and the insects were returned to their container. The Group A mealworms were then removed from their containers and placed in a cup (Solo brand, purchased at Wal-Mart) and then were poured onto a paper plate (Solo brand, purchased at Wal-Mart). A camera (Mino HD model, Flip Video brand) was positioned 46 cm over the plate with an LED flashlight (LED XL50 model, Mag-Lite brand) positioned 46 cm over the plate shining on the insects. The insects were then recorded for one minute and returned to their container. The recording process was repeated for the two remaining groups. Four days later, the mealworms were recorded again. Data was then gathered from the recordings using Logger Pro. The entire process was completed at four different

Immunity of Tenebrio molitor

temperatures (19 C, 21 C, 23 C, 25 C) using a heat lamp with a 75-watt incandescent light bulb (Zilla brand, purchased at PetCo). The data was then analyzed.

Figure 5. Inserting monofilament into Tenebrio molitor. The specimen was punctured with a needle and a piece of nylon monofilament was inserted into the hole.

Immunity of Tenebrio molitor

Fig 6. Tenebrio molitor habitat. The organisms have nylon monofilament inserted into their abdomens and were returned to their tank. A half of a small carrot was placed in the tank.

Immunity of Tenebrio molitor Results

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Figure 7. A Logger Pro file. This image depicts the path a mealworm took during a span of 60 seconds at 19 degrees on day 1.

Table 1. Summarized speed data for each variable.

Average Speed,

Day 1 Temp Avg. Speed cm/s 0.17 0.10 0.10 0.15 0.16 0.13 0.15 0.11 0.16 0.06 0.11 0.15 STDEV 0.10 0.04 0.06 0.07 0.09 0.07 0.06 0.06 0.08 0.02 0.07 0.06

Average Speed,

Day 4 Temp Avg. Speed cm/s 0.20 0.11 0.10 0.06 0.11 0.09 0.12 0.07 0.17 0.07 0.15 0.05 STDEV 0.07 0.05 0.05 0.05 0.06 0.05 0.05 0.05 0.08 0.07 0.11 0.05

Control Control Control Control Puncture Puncture Puncture Puncture Monofilament Monofilament Monofilament Monofilament

19 21 23 25 19 21 23 25 19 21 23 25

Control Control Control Control Puncture Puncture Puncture Puncture Monofilament Monofilament Monofilament Monofilament

19 21 23 25 19 21 23 25 19 21 23 25

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Average Speeds, Day 1

0.30 Puncture Control Monofilament 0.25

0.20

19 degrees C 21 degrees C 23 degrees C 25 degrees C

0.15

0.10

0.05

0.00
Figure 7. Average speeds, day 1 of experimentation. A graphical model of the average speeds of T. molitor at different temperatures on the first day of experimentation.

Average Speeds, Day 4

0.30 Control Monofilament Punctured 0.25

0.20

19 degrees C 21 degrees C 23 degrees C 25 degrees C

0.15

0.10

0.05

0.00
Figure 8. Average speeds, day 4 of experimentation. A clear trend is apparent in the graphical model of the average speeds of T. molitor at different temperatures on the fourth day of experimentation.

Immunity of Tenebrio molitor

Table 2. Number of deaths per setting.

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Injury Control Control Control Control Puncture Puncture Puncture Puncture Monofilament Monofilament Monofilament Monofilament

Deaths Temp. Deaths 19 0 21 0 23 0 25 0 19 0 21 1 23 0 25 0 19 0 21 0 23 1 25 0

Living T. molitor
12 10 8 6 4 2 0
Figure 9. Living T. molitor during experimentation for each setting. There is a clear trend between the temperatures.

Control

Punctured

Monofilament

19 degrees C 21 degrees C 23 degrees C 25 degrees C

Data Analysis and Discussion There are few significant trends in the Day 1 average speeds. Because the data was collected on the first day of experimentation, T. molitor did not have a sufficient

Immunity of Tenebrio molitor

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amount of time to adjust to the temperature and environment; therefore, anomalies were expected. However, the speeds at 19 degrees were consistently higher than the others, which correlates with the hypothesis. The data was significant (p=0.02), but did not fully support the hypothesis; in the control and monofilament groups, the average speed at 25 degrees was higher than the average speeds at 21 and 23 degrees. The data collected on Day 4 was more pertinent to the study because T. molitor had a four-day period to stabilize and react to the temperatures of their environments. The trend apparent in the control group perfectly supports the hypothesis; the average speed decreases as temperature increases. At 19 and 23 degrees T. molitor seem to move the fastest. This anomaly may be explained by male and female temperature preferences. However, the speeds at 25 degrees are consistently the lowest, which supports the hypothesis. The overall speeds of punctured T. molitor are significantly lower than the speeds of the control group and the monofilament group. Statistical analysis reveals that the data from Day 4 is statistically significant (p<0.0001) and partially supports the hypothesis. The mortality rate of T. molitor during the entire experiment is so low (2% died) that it is considered insignificant. Two out of a total of 120 organisms died during the entire experimentation. Because there werent enough to form a trend that corresponds with temperature or injury, these deaths are considered anomalies. The hypothesis was fully supported where the mortality rate was unaffected. Previous studies have showed a decrease in the metamorphosis rate and immune function in insects cultured in high temperatures. This experiment followed in the same trend; with increased temperatures, the rate of movement decreased. However, there have been few studies focusing on the effects of temperature on the insect immune system. Conclusions The decrease in the average speeds of T. molitor that are injured suggests a correlation between the immune system and environmental temperature. The punctured organisms were significantly slower than both the control and monofilament groups and were more susceptible to infection with environmental bacteria. This suggests that when the organisms immune systems are activated their speed decreases, as evidenced by the values for 19 degrees in the control and punctured group (Figure 8). Considering rate of movement as a measurement of fitness, the data reveals that the insect immune system handles more than one pathogen better than it does a single pathogen. Inserting the monofilament simulated a parasite entering the insects bodies, and therefore activating the immune systems. The monofilament was removed after the immune systems were activated and the organisms were placed in soil containing environmental bacteria. The specimens with the monofilament had higher overall average speeds than those with just the puncture. The miniscule mortality rate suggests that the immune system of T. molitor is able to handle and eliminate multiple pathogens at one time with a low risk of death. Thus, as evidenced by the data, temperature affects both the immune system and rate of movement of Tenebrio molitor, but being exposed to multiple pathogens will not increase the rate of mortality.

Immunity of Tenebrio molitor Limitations and Assumptions

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The experiment had limitations that include time constraints and testing space. Because of the short allotment of time between the start of testing and the completion of the study, only 15 trials were completed. The area in the room in which the testing was completed only allowed for two trials to be completed at one time. Some of the constants were based on several assumptions. One of the principle assumptions was that T. molitor behave in captivity as they do out of captivity. T. molitor were assumed to be of the same health status, which was accounted for by culturing recently purchased organisms. It was also assumed that the temperature in the tank remained constant between data collections, and that the thermometer was functioning properly. There are several potential sources of error. Handling the organisms may have harmed them and later affected their rate of movement. In the video analysis, there may have been human error in accurately plotting the points. Applications and Future Experiments

Discoveries in insect immunology are seminal in studying the immune system in other organisms. Insects can serve as valuable models because the fundamental features of the immune system are the same in most animals. Examining the interactions between the Tenebrio molitor host and the pathogen has potential for discoveries in ecology and evolutionary biology (Indikris Krams, personal communication). Researchers may be able to find new ways to combat pathogens and manipulate the immune system. From an ecological standpoint, examining the functions of T. molitor immune system in increasing temperatures will elucidate the effects of global warming on its population sizes, thus affecting the ecosystem. T. molitor is a critical food source for various organisms. Thus being able to predict how the T. molitor populations will fluctuate in increased temperatures may enable researchers to extrapolate information about population sizes of other organisms in the ecosystem. To further this experiment, research could be conducted to study how the immune system of T. molitor functions when they are given foods with varied nutritious elements, to examine the effects of T. molitor being exposed to a higher or lower temperature range, or to investigate the rate of reproduction at higher temperatures. Information gathered from these experiments can further illuminate the functions of the immune system, how it interacts with other bodily systems, and the effects of global warming on insect populations. Literature Cited Buie V.C., Owings M.F., DeFrances C.J., and Golosinskiy A. (2010). National Hospital Discharge Survey: 2006 summary. National Center for Health Statistics. Vital Health Stat 13(168). 1-70.

Immunity of Tenebrio molitor Bienen, L. (2004, November). Global warming and insect pheromones. Frontiers in econology and the environment, 2(9), 455. Dewey, S. (2001). Hymenolepis diminuta. Retrieved December 2, 2012 from http://animaldiversity.ummz.umich.edu Greenberg, S., and Ar, A. (1996). Effects of chronic hypoxia, normoxia, and hyperoxia on larval development in the beetle Tenebrio molitor. Journal of Insect Physiology, 42, 991-996. Haine, E. R., Moret, Y., Siva-Jothy, M. T., and Rolff, J. (2008). Antimicrobial defense and persistent infection in insects. Science, 322(5905), 1257-1259.

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He, J. (2007). Shrinkage of body size of small insects: a possible link to global warming?. Chaos, Solitons & Fractals, 34(3), 727729. Hurd, H., Fogo, S. (1991). Changes induced by Hymenolepis diminuta (Cestoda) in the behaviour of the intermediate host Tenebrio molitor (Coleoptera). Canadian Journal of Zoology, 69(9), 2291-2294. Janvoy Jr., J, J., and Schawang, J. E. (2001). The response of Gregarina niphandrodes (Apicomplexa: Eugregarinida: Septatina) to host starvation in Tenebrio molitor (Cleoptera: Tenebrionidae) adults. Journal of Parasitology, 87(3), 600-605. Kasapari, M. (2008). Global energy gradients and size in colonial organisms: Worker mass and worker number in ant colonies. Proceedings of the National Academy of Sciences of the United States of America, 102(14), 50795083 Krams, I., Kaukste, J., Kivlenience, I., Krama, T., Rantala, M. J., Ramey, G., and Sausa, L. (2011). Female choice reveals terminal investment in male mealworm beetles, Tenebrio molitor, after a repeated activation of the immune system. Journal of Insect Science, 11(56). Lavine, M. D., and Strand, M. R. (2002). Insect Hemocytes and their role in immunity. Insect Biochemistry and Molecular Biology, 32(10), 295-309. Leander, B. S., Clopton, R. E., and Keeling, P. J. (2003). Phylogeny of gregarines (Apicomplexa) as inferred from small-subunit rDNA and -tubulin. International Journal of Systematic and Evolutionary Microbiology, 53(1), 345-354. Mandrioli, M. (2012). Someone like it hot? Effects of global warming on insect immunity and microbiota. Retrieved December 2, 2012 from http://www.isj.unimo.it

Immunity of Tenebrio molitor Mealworm Diagram. (2012, December 2). Retrieved from http://www.smithlifescie nce.com Mealworm Lifecycle. (2012, December 2). Retrieved from http://www.enchantedlea rning.com Schneider, D. S., and Chambers, M. C. (2008). Rogue Insect Immunity. Science, 322(5905), 1199-1200.

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Soil Survey Staff. (1999). Soil taxonomy, a basic system of soil classification for making and interpreting soil surveys. Washington, DC: United States Department of Agriculture. Thomas, F., Brown, S. P., Sukhdeo, M., and Renaud, F. (2002). Understanding parasite strategies: a state-dependent approach?. Trends in Parasitology, 18(9), 387-390. Toso, M. A., and Omoto, C. K. (2007). Ultrastructure of Gregarina niphandrodes nucleus through stages from unassociated trophozoites to gamonts in syzygy and the syzygy junction. Journal of Parasitology, 93(3), 479-484.

Acknowledgements The author wishes to thank several mentors who assisted her throughout the project and, without whom, the project couldnt have happened. Dr. Judith Sumner and Mrs. Shari Weaver provided extraordinary amounts of support and guidance in the experimental design and presentation of the data. Dr. Judith Sumner also provided instruction for writing and assisted in editing the research paper. The author would also like to thank her parents, who provided both monetary and emotional support.