Samantha Castro, Zack Stamford and Michael Kunicki

Varying Concentration of Trichonympha in the Hind-Gut Fluid of the Soldier and Worker Castes of Reticulitermesflavipes

Samantha Castro, Zack Stamford and Michael Kunicki

Department of Biological Sciences[Anonymous1]

Saddleback College

Mission Viejo, CA 92692

The colonial structures of social insects has been [Anonymous2]an area of biology that has been studied and observed extensively. Insects such as an ants and bees are common examples if organism[Anonymous3] that sustain this kind of lifestyle and habitat. One species that has been studied less so[Anonymous4] is the termite, Reticulitermesflavipes. The mystery around these animals[Anonymous5] is the protosymbiontTrichonympha that lives in the digestive hind-gut of the termite and is found nowhere else[Anonymous6]. Their purpose,[Anonymous7] digest and break down cellulose from the substrate they eat with cellulose, an enzyme [Anonymous8]they produce. The termite and the Trichonympha have co-evolved and have a large influence on the activities of the termite, even its lifespan. This study looked at two castes of a single termite colony, worker(n=20) and soldier (n=20)[Anonymous9], and quantified the Trichonympha present in their hind-guts. Knowing that workers do most of the work such as building tunnels, chambers, feeding the colony, they would need to possess more of the protozoan compared to the soldier whose only purpose is to defend against intruders.[Anonymous10]The results indicated a significant difference (p=0.00194)[Anonymous11] between the number of Trichonympha present in 0.314µL of hind-gut fluid from the worker caste (mean=74.55±8.23) and those from the soldier caste (mean=47.0±3.45). These numbers can be used to do further study into Trichonympha’s effect on the caste determination with in a colony.

INTRODUCTION[Anonymous12]

In the United States, subterranean termites, Reticulitermes[Anonymous13],are mainly found in[Anonymous14] the southeast[Anonymous15] coast and California, but can be found in all[Anonymous16] states besides Alaska. They are wood destroying, substrate feeding insects that live in colonies, and are best suited for moist and warm environments. They are common recognized as pests due to their destructive nature to homes and other wooden structures. Since they live inside of their food source, they have mobile nest sites, which is most often dead soft wood that they excavate and inhabit. Subterranean termites are a fragile species thatcan easily succumb to the environment and their [Anonymous17]natural predators and competitors, such as ants and other termite colonies. They are social insects that require cooperation for rearing their young, sharing resources[Anonymous18] such as food, water and shelter. They produce overlapping generations, and [Anonymous19]have a division of labor performed by each caste, which are categorized into workers, soldiers, and reproductives. (Suiter, et al. 2009)

Unlike most other insect species, termites in each caste are comprised of males and females equallyandareboth physically and sexually immature except for the reproductive caste (Frédéric Legendre, et. al. 2013). The reproductive caste consists of alates, primary reproductives, secondary neotenic reproductives, and tertiary neotenic reproductives. Alates are winged termites with a set of compound eyes and have a dark brown pigment to their exoskeleton, which aids in the prevention of water loss when swarming for new food sources. Primary reproductives, or the kings and queens, are wingless, dark in color, have a set of compound eyes, and are typically the largest in a colony; queens being considerably larger than kings. Once the first set of eggs has hatched and matured into workers, colony growth beginsto accelerate. Since newly developed workers are able to attend to the young, primary reproductives cease to care for the eggs.Secondary and tertiary neotenic reproductivesare also large in size but possess lighter pigments. They mainly aid in accelerating egg production and gestation, determining how fast and how much a colony can grow (Frédéric Legendre, et. al. 2013). The most abundant in a colony is the worker caste. They are wingless, white, blind, and smaller than the reproductive caste. They have many tasks such as locating and colonizing food resources, excavating, building galleries and shelter tubes, feed[Anonymous20], groom, and care for young termites and other caste members. They will also [Anonymous21]participate in colony defense if needed, which is the primary function of the soldiers. Soldiers have enlarged yellowish to yellowish-brownish heads with elongated dark mandibles to ward off invaders. (Suiter, et al. 2009)

Each individual termite hatches from an egg and becomes a larva. After a few instars, larva becomes animmature adult worker. Workers can remain a worker for the remainder of its life, or develop into a soldier, nymph, or tertiary neotenic reproductives. If a worker develops into a nymph, they can either revert back into a worker or further develop to become an alate or secondary neotenic reproductive; unlike, [Anonymous22]a worker developing into a soldier or tertiary neotenic reproductive, which is a permanent change. If a nymph develops into alate and survives after swarming, in which most die, they may shed their wings and become either king or queen of a newly established colony. The majority of termites in a colony are workers, not only because every termite from every caste has to be a worker as an intermediate stage, but also because they’re processing most of the food that creates the environment that they live in and they perform many tasks that benefit the well being of the entire colony, with the help of a specific protozoan [Anonymous23]unique to termites. (Suiter,[Anonymous24] et al. 2009)

Subterranean termites are not actually capable of digesting wood and cellulose themselves. Among other endosymbiotic bacteria such as, Trichomonads and Streblomastix, [Anonymous25]Trichonympha is found in the hindgut of termites. These organism are responsible for breaking down cellulose materials in the termite’s diet (Yamin, 1981). It was thought that these organisms were incapable of processing cellulose [Anonymous26]on their own and that they required the help of bacteria that produced cellulose; enzymes capable of depolymerizing cellulose. Further study has indicated that when isolated in a culture, Trichonympha will continue to break down cellulose into acetate, carbon dioxide, and hydrogen (Yamin, 1981). Little is known about the protozoans on an evolutionary scale, but studies on their general physiology show that they possess an esophagus-like structure and a gut. (Cleveland, 1925).This will be the organism observed in this [Anonymous27]study. Trichonympha does has various external effects on the termite’s ability to eat, do work, andprocess plant material. This study will simply be looking at the differences in Trichonympha counts in a known volume of sample from the hind-gut of 40 termites, 20 from the worker caste and 20 from the soldier caste. Knowing these numbers[Anonymous28] may give the foundation for further study into direct and indirect effects that Trichonympha have on the termite and on the entire colony.Since workers perform more of the duties that require a rigorous and efficient digestive process, the amount of Trichonympha present in the hindgut of termites would be greater in the worker caste than in the soldier caste.

METHODS AND MATERIALS[Anonymous29]

Fifty subterranean termites (Reticulitermesflavipes) were purchased from Carolina Biological Supply. Upon delivery, the termites were inspected and transferred to a terrarium, which replicated their ideal environmental conditions. Termites were contained at a temperature of 20 degrees Celsius and in an area that was[Anonymous30] dark and damp. The termites were given adequate food, which originated from their place of extraction, along with a damp paper towel that helped regulate humidity. The sample group was purchased during the Fall of 2015 when worker counts are[Anonymous31] relatively low. The termites were differentiated and separated according to their caste. They were then extracted from the terrarium and analyzed one day after their delivery, to ensure healthy specimen samples.

Forty termites, 20 of each caste, were individually selected and analyzed. 10 termites were spared in case of procedural error. The amount of Trichonympha in each termite was determined by smearing the hindgut onto a hemocytometer. Termites were weighed and identified before the smearing process. A scalpel was used to separate the head region from the abdomen. The abdomen was then isolated using forceps and placed onto the hemocytometer. Two microliters of 0.9% saline solution was pipetted onto the sample and a probe was used to break down the visceral tissue of the abdomen and expose the Trichonympha. A glass cover slip was placed over the sample and Newton’s rings were observed to ensure the cover slip adhered properly. The Trichonympha were counted at 100X magnification under a Zeiss upright light microscope and an Olympus CK2 inverted microscope. The region of the highest Trichonympha concentration was located and a total count was recorded. As general rules, the symbiotic protist must have been completely visible within the field of view to be counted, identified accurately and in good definition, and counted at the point of highest concentration. These rules were applied throughout the entire study. [Anonymous32]

The area of the confined counting field was determined by recognizing the diameter of the field of view at a specific [Anonymous33]magnification (100X). A known depth of .10 millimeters was provided by the hemocytometer. A volume was calculated using both the known depth and area. The data collected was expressed as the number of Trichonympha counted per volume, for each termite. The average amount of Trichonympha/volume was then calculated for each sample group; twenty termites of each caste. The data collected will be transferred to Microsoft Excel (Microsoft Corporation, Redmond, Washington) [Anonymous34]where an unpaired two[Anonymous35]-tailed t-test will be used to determine if a significant difference is observed between the two castes.

RESULTS

Termites were dissected and their hind-gut fluid was extracted, using a consistent smearing method and 0.9% isotonic sodium chloride solution. These samples were then analyzed using a CK2 dissection microscope and a compound microscope. The specimens were counted under various magnifications including magnification CK 20 on the dissection microscope – equivalent to 20x on a compound scope – as well as 10x and 40x magnification on the compound microscope. The weights of each worker and soldier were recorded and theTrichonympha within each area of view under the microscope was counted.

[Anonymous36]The mean Trichonympha count for 20 termites from the worker caste was 74.55 ± 8.23 in 0.314µL of fluid. The mean Trichonympha count for 20 termites from the soldier caste was 47.00 ± 3.45 in 0.314µL of fluid. After performing an one-tailed, unpaired t-test of the all the data from each of the termites from the separate castes, indicating there [Anonymous37]was a significant difference in the Trichonympha counts in 0.314µL of fluid between termites from the worker caste and the soldiers (p=0.00194). This data is graphed for visual comparison in Figure 1.

Analysis was also done on the individual weights of each termites and a comparison between the weight and Trichonympha count was made (Figure 2 and 3). There was no consistent or significant correlation between the weight of the termites from the separate castes and the

Trichonympha count in their hind-gut. Although some termites’, particularly from the soldier[Anonymous38] caste, higheror lower protist counts did match with a similarly higher or lower body weight (Figure 3).

DISCUSSION

After data analysis, it was found that the worker caste does have significantly more of the protozoan symbiontTrichonympha in a known volume than termites from the soldier caste (p=0.00194). The hypothesis was accepted[Anonymous39]. This was also to be expected. Worker termites are the members of the colony responsible for building the physical components of the colony, tunnels, chambers, exits and entrances and therefore are ingesting most of the wood. With the increased amount of wood and materials containing cellulose entering the hind-gut of worker termites, they must need more of the protozoan to digest and process these materials [Anonymous40](Ohkuma Moriya 413). Soldiers on the other hand, are responsible for defending the colony from invaders, the most common being ants. They are not responsible for digging into the wood and are also physiology hindered by their comparatively massive mandibles. However they were observed to have Trichonympha present in their gut. This can suggest two things about the effect of caste change on the protozoan. Once a[Anonymous41] worker develops into a soldier, a caste that is incapable of reverting back to a worker, the high concentration of Trichonympha becomes unnecessary, being that the termite is no longer digging tunnels and entire colonial chambers out of wood[Anonymous42]. With no cellulose to be digested[Anonymous43], the Trichonympha may die off, lowering their counts with in the gut of soldiers. There was no significant correlation between weight and the amount of Trichonympha in the hind-gut fluid but there were samples of higher Trichonympha counts that correlated with an increase in body weight, especially with the soldier caste.

There are improvements that could be made in the case of future study. A consistent smearing method was used yet some smears yielded better results than others (Lewis, J. L. et. Al 1244). This could potentially effects counts made when being observed under a microscope. Also for more substantial and definitive data, the total volume of the gut sample should be made prior to observation. This was attempted in this study with a P200 pipette, measuring 1.0µL accurately and consistently was difficult and often destroyed the sample by scrambling the microbes. [Anonymous44]Trichonympha can be anywhere from 0.021mm to 0.090mm wide (Carpenter, Chow, Keeling, 306), very large in comparison to many of the other microbes present in the hind-gut material, making them fragile and susceptible to blunt force tearing and bursting. Some of the Trichonympha observed looked shriveled. This could be due to the isotonic sodium chloride solution used to dilute the hind-gut material. A lower percentage of the sodium chloride [Anonymous45]may be used to observe theseprotist[Anonymous46]so as to not cause dehydration or a “nitrogen-sparged saline solution that extends cell life under microscopic examination”. (Lewis, J. L. et. Al 1244). The counting method also followed three rules, organism must be in complete focus, organism’s body must be incomplete view under a specific magnification, and location to be counted was the area on the slide with the highest special concentration of Trichonympha. These rules were necessary but could be affected by an inconsistent smearing method.

In regards to the average amounts of Trichonympha seen between soldiers and workers, it is unclear where the protist originates. Two possibilities could be the distribution of Trichonympha through proctodealtrophallaxis based on the need for more digestive symbionts or growth within the termite’s gut in coordination with the growth of the individual termite. It is understood that the microbial community is conserved through vertical transmission from reproductives to offspring and also transported to newly formed colonies. The real question is when do these symbiotic organisms begin to accumulate and become functional within each caste and if a morphological change requires less or more Trichonympha, where are those lost or gained? This is a question that can be answered through further analysis during a future study. A possible method being; forming a new observable colony and extracting Trichonympha samples during each phase of the termite growth cycle, including the pathways of different terminal castes.

There is speculation on various aspects of a termite’s development and pre-destination, the complete role Trichonympha plays in the colony as a whole, and the source of Trichonympha itself. What is known is how the colony is regulated. The kings and queen are responsible for the majority of reproduction as well as regulating colonial position determination (Eggleton, Paul). [Anonymous47]All larvae in a termite colony receive pheromones[Anonymous48] by the kings and queen that activate or deactivate certain hormones. In the case of larva the majority of the hormonal activation is dedicated to growth, juvenile hormone (JH) (Howard and Haverty 269). Worker termites are an intermediate between larva and all other caste in the colony. Since workers are the most needed within a colony most remain in the intermediate stage of workers which are physically and sexually immature. The speculation is why the pheromones are released by the kings and queen. Many of the theories involve colonial need. The kings and queen will release these chemical signals to certain members of the colony based on whether or not a soldier or reproductive, for example, are needed for a colonies survival[Anonymous49]. We are suggesting[Anonymous50] an alternate theory that may work in conjunction with the theory previously stated. It has been observed that termites can undergo proctodealtrophallaxis, when one termite can transfer gut fluid to another based on need of symbiotic organisms. This study has shown that soldiers possess considerably less Trichonympha than soldiers and we believe that this could suggest the symbiont’s indirect involvement in termite development. Knowing that development in[Anonymous51] directly influenced by pheromones activating certain hormones in a termite to remain or change castes, it is possible that the reason for this is not completely dependent on colonial need. It could be a result of individual need. For example, if a worker termite is low on Trichonympha, which is a reality of some termites, especially during the molting process, instead of undergoing proctodealtrophallaxis (Ohkuma420) from another termite, the kings or queen may determine that worker (via pheromones) to become a soldier, tertiary reproductive or a nymph. This is speculation but certainly an area for future study.