Structure and Function of Salivary Reservoirs of the Eastern Subterranean Termite, Reticulitermes Flavipes (Kollar)

Home , Anacanthotermes, Hodotermes, Prorhinotermes simplex, Reticulitermes, Reticulitermes hesperus, Reticulitermes speratus

STRUCTURE AND FUNCTION OF SALIVARY RESERVOIRS OF THE EASTERN SUBTERRANEAN TERMITE, RETICULITERMES FLAVIPES (KOLLAR)

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Nicola Tracey Gallagher, M.S.

Graduate Program in Entomology

The Ohio State University

2010

Dissertation Committee:

Dr. Susan C. Jones, Advisor

Dr. Woodbridge Foster

Dr. David J. Horn

Dr. David Shetlar

Nicola T. Gallagher

2010

Abstract

The eastern subterranean termite, Reticulitermes flavipes, is highly susceptible to desiccation and moisture is a critical factor for its survival. Termites possess a pair of salivary reservoirs (water sacs) that store water, which presumably are used to increase the humidity in unfavorable microclimates. The function and structure of salivary reservoirs in R. flavipes was investigated. This dissertation investigates the physiological mechanisms of water transport, specifically how water is maintained within the termite salivary reservoirs and how it is moved to new food sources.

The main objective of the first study was to measure the moisture change of a dry food source (cellulose pad) in the presence of R. flavipes workers during a 21-day period.

Termites rapidly transported water to a dry food source, with the mean moisture content of the cellulose pad reaching ~7% just 3 hours after termites entered the test arena; the cellulose pad subsequently attained a maximum moisture content of ~46% at the 21-day observation. Salivary reservoir volume was smallest for termites removed from the food source in comparison to termites removed from the moist sand throughout the entire study (except on day 7). These data indicate that termites indeed use their salivary reservoirs as “water sacs” to relocate water from moist areas to dry resources.

The objective of the second study was to evaluate the variability of salivary reservoir dimensions among the various castes, when collected from different locations in laboratory colonies and at field sites. Salivary reservoirs dimensions differed among

castes and stages when collected from food sources, shelter tubes and nurseries. Salivary reservoir volume was always larger for termites collected from food sources and nurseries. Salivary reservoirs were usually depleted in termites collected from shelter tubes. Soldiers always had the largest salivary reservoirs, followed by workers (which were categorized into three sizes based on head width). Alates and nymphs consistently had empty salivary reservoirs. These data demonstrate that salivary reservoir dimensions differ among stages and worker sizes; however this apparently is not related to any task specialization.

Current data are conflicting on how water enters the salivary reservoirs. This study investigated the route of water into the salivary reservoirs over a 6-hr period. When dehydrated workers were placed on moist sand, their crops were the first to fill (~80-85

% full) after 15 minutes. Crops remained near full capacity (≥75%) for 30–60 minutes before decreasing in size. An increase in salivary reservoir volume appeared to coincide with the decrease in crop volume. Salivary reservoir volumes were more than 50% full by

30-60 minutes and typically increased in size thereafter, though some colony variation was observed. Furthermore, water used in this study contained blue food coloring that was observed in the crop, but never in the salivary reservoirs. These data indicate that water reaches the salivary reservoirs via the hemolymph. Furthermore, preliminary gas chromatography and mass spectrometry results suggest the salivary reservoir contents may have a similar chemical profile to hemolymph. However, further analyses are required to determine if amino acids or sugars are present in the reservoirs.

An ultrastructural investigation was performed at the same time as the previous study to gather additional information on any mechanisms involved with water release

iii and/or retention in the salivary reservoirs. Salivary reservoirs were composed of one layer of epithelial cells and were surrounded by an inner cuticle layer. Secretory-like cells were found in the reservoir walls and a lipid-like secretion was found in the reservoir lumen. No evidence of structures associated with an ionic gradient or valve-like structures where observed in the reservoirs. Two types of secretory cells were found in the salivary glands of workers. Type I secretory cells contained electron lucent secretion material, while Type II secretory cells contained electron dense secretion material.

Furthermore, Type II could be separated into Type IIa and Type IIb, as Type IIa contained material of variable electron density and Type IIb was more uniform in density.

The salivary reservoirs are an important structure within the termite and the more information we gather on termite biology and colony dynamics, the better equipped we are to control them. Termite inspectors should not focus strictly on moisture-prone areas, as termites can relocate water to dry areas. This research also emphasizes the importance of educating homeowners on moisture reduction around homes. Furthermore, salivary reservoirs may be a candidate to target using RNA interference (RNAi)-based termiticides.

Dedication

Dedicated to my mother, who taught me all about perseverance

Acknowledgments

It is with gratitude and respect that I thank all the family members, friends, colleagues and mentors that provided support during my journey through graduate school. To Dr. Susan C. Jones, for her unwavering support and guidance while pursuing my doctorate degree. I am truly grateful for the many opportunities she has given me, especially collecting termites in Puerto Rico. I also credit Dr. Jones for her impeccable eye for detail and hard work editing this dissertation.

To my committee members, Drs. Woody Foster, Dave Horn and Dave Shetlar for their excellent input and support. I know I made the right choice when I can ask for advice and enjoy good beer at the same time. To my undergraduate advisor, Dr. Gene

Kritsky, for introducing me to the world of entomology. Tiger beetles will always have a special place in my heart (and Schmitt box). To Dr. Desouky Ammar and Towheda

Ammar, for help preparing samples, guidance using the microtome and TEM and advice analyzing results. To Drs. Matt Tarver and John Bland (USDA) for help with the GC-MS analysis. To Dr. Mariam Lekveishvili for translating Russian papers.

To the Department of Entomology (OSU), Ohio Pest Control Association, the

Root family, the LaFage family, Pi Chi Omega, Board Certified Entomologists (BCE) program, National Pest Management Association (NPMA) and Bayer Environmental

Science for financial support.

I have received endless love and support from so many friends and family members. In particular, Dr. Kyle Jordan, Mary Daniels, Ye Ye, Josh Bryant, Bobby

Aldridge, Bridget Behrmann, Megan Meuti, and Christina Kwapich. I am truly grateful to call you friends and colleagues. To my mother, Anne Gallagher, and brother, Jack

Gallagher, my two favorite scousers. I couldn’t ask for a better family. Most of all, I thank Dr. Rich Gary, for his endless support and love. I am lucky to have you.

…

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Vita

February 23, 1978 ...... Born - Liverpool, England

2000...... B.S. Biology, The College of Mount St. Joseph

2003...... M.S. Entomology, The Ohio State University

2000 - 2003 ...... Graduate Research Associate, The Ohio State University

2003 – 2008...... Research Associate, The Ohio State University

2008 to present...... Graduate Teaching Associate, The Ohio State University

Awards

2010...... Fred Hink Research Award, The Ohio State University, Department of Entomology

2009...... Jeffery P. LaFage Graduate Student Research Award, Entomological Foundation

2009...... 2nd place, Section SVPHS3 Oral Presentation Competition, Entomological Society of America Annual Meeting

2009...... Pi Chi Omega Scholarship

2008...... Bayer Environmental Science and National Pest Management Association “Young Scientist of the Year” Award (2nd runner up)

2008...... 3rd place, Student Paper Competition, National Conference on Urban Entomology

2008...... Delong Award, The Ohio State University, Department of Entomology

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2008...... 2nd place, Section SVPHS3 Oral Presentation Competition, Entomological Society of America Annual Meeting

2008...... Ohio Pest Control Association Scholarship

2006...... Student Certification Award, Entomological Society of America Award (sponsored by Springer Pest Solutions)

2000...... 2nd place, Undergraduate Competition, Beta Beta Beta, National Honors Biology Society

1999...... Best Student Paper, Entomology, Indiana Academy of Science

1998-2000 ...... Dean's list, College of Mount St. Joseph

1997...... 2nd place, Undergraduate Competition, Ohio Valley Entomological Association Research Competition

Publications

Gallagher, N. T., and S. C. Jones. Moisture Augmentation of Food Items by Reticulitermes flavipes (Isoptera: Rhinotermitidae). Sociobiology 55:735-747 (2010).

Gallagher, N. T., and S. C. Jones. Moisture on the move. Pest Control Technology. April 2010: 54-59.

Gallagher, N. T., and S. C. Jones. Termite-mediated alteration of food items, p.18-19 (Abstract). In Susan C. Jones [ed.], Proceedings of the 2008 National Conference on Urban Entomology, Tulsa, OK, 18-21 May (2008).

Jones, S. C., and N. T. Gallagher. Efficacy of the Exterra system with Labyrinth AC termite bait in Ohio field trials, p.44 (Abstract). In Susan C. Jones [ed.], Proceedings of the 2008 National Conference on Urban Entomology, Tulsa, OK, 18-21 May (2008).

Jenkins, T. M., S. C. Jones, C.-Y. Lee, B. T. Forschler, Z. Chen, G. Lopez- Martinez, N. T. Gallagher, G. Brown, M. Neal, B.Thistleton, and S. Kleinschmidt. Phylogeography illuminates maternal origins of exotic Coptotermes gestroi (Isoptera: Rhinotermitidae). Mol. Phylogenet. Evol. 42: 612-621. (2007)

Jones, S. C. and N. T. Gallagher. Efficacy of the Advance® termite bait system in Ohio, p.54-55 (Abstract). In Tamara Sutphin, Bob Cartwright, and Richard Houseman [eds.], Proceedings of the 2006 National Conference on Urban Entomology. 21- 24 May, Raleigh, NC. (2006).

Gallagher, N. T. and S. C. Jones. Seasonal variation in time to termite colony elimination using Recruit™ IV bait in Ohio, p.54 (Abstract). In Tamara Sutphin, Bob Cartwright, and Richard Houseman [eds.], Proceedings of the 2006 National Conference on Urban Entomology. 21-24 May, Raleigh, NC. (2006).

Gallagher, N. T. and S. C. Jones. Effects of resource availability on search tunnel construction by the eastern subterranean termite, Reticulitermes flavipes (Isoptera: Rhinotermitidae). Sociobiology 45(3): 553-564. (2005).

Gallagher, N. T. and S. C. Jones. Field efficacy of fipronil as an exterior perimeter treatment against Reticulitermes flavipes in Ohio, p. 120-121 (Abstract). In: T. Sutphin, D. Miller and R. Kopanic [eds.], Proceedings of the 2004 National Conference on Urban Entomology, 20-22 May, Phoenix, AZ (2004).

Kritsky, G., A.Watkins, J.Smith and N.T.Gallagher. Mixed assemblages of tiger beetles on sand piles of various ages (Coleoptera: Cicindelidae). Cicindela 31:73-80 (1999).

Kristky, G, N.T.Gallagher, J.Smith and A.Watkins. The decline of Cincindela hirticollis Say in Ohio (Coleoptera: Cicindelidae). Biological Notes of the Ohio Biological Survey. Ohio Biol. Survey Biol. Notes 2: 49-52 (1999).

Kritsky, G, N.T.Gallagher and J.Smith. The 1999 emergence of periodical cicadas in Ohio (Homoptera: Cicadidae: Magicicada spp. brood V). Biological notes of the Ohio Biological Survey. Ohio Biol. Survey Biol. Notes 2:43-48 (2000).

Field of Study

Major Field: Entomology Emphasis on urban entomology

Table of Contents

Abstract ...... ii Acknowledgements ...... vi Vita...... viii List of Tables ...... xiv List of Figures...... xv

Chapters: 1. Introduction...... 1 1.1 Biology of Reticulitermes flavipes...... 1 1.2 Termite developmental stages and castes ...... 2 1.2.1 Larva ...... 2 1.2.2 Worker ...... 2 1.2.3 Soldier...... 3 1.2.4 Nymph ...... 3 1.2.5 Alate...... 3 1.2.6 Neotenic reproductive ...... 4 1.3 Environmental requirements ...... 4 1.4 Salivary glands and salivary reservoirs ...... 6 1.4.1 Salivary reservoirs...... 7 1.4.2 Salivary glands ...... 9 1.5 Termite management...... 10 1.6 Objectives of this research ...... 11 References cited...... 12

2. Moisture augmentation of food items by Reticulitermes flavipes (Isoptera: Rhinotermitidae) ...... 16 2.1 Abstract...... 16 2.2 Introduction...... 17 2.3 Materials and methods...... 19 2.3.1 Experimental units...... 20 2.3.2 Termites ...... 20 2.3.3 Experimental design and observation periods...... 21 2.3.4 Consumption and moisture content of cellulose pads...... 21 2.3.5 Salivary reservoir measurements ...... 21 2.4 Statistical analysis...... 22 2.5 Results...... 22 2.5.1 Consumption...... 22 2.5.2 Moisture...... 23

2.5.3 Salivary reservoirs...... 23 2.6 Discussion...... 24 References cited...... 28

3. Variability of salivary reservoirs among developmental stages and castes of Reticulitermes flavipes (Isoptera: Rhinotermitidae)...... 38 3.1 Abstract...... 38 3.2 Introduction...... 39 3.3 Materials and methods...... 42 3.3.1 Termite collections ...... 42 3.3.2 Moisture readings ...... 43 3.4 Statistical analysis...... 44 3.5 Results...... 44 3.5.1 Salivary reservoir length and head width ...... 44 3.5.2 Salivary reservoir volume ...... 46 3.5.2.1 Caste and developmental stage ...... 46 3.5.2.2 Location of collected termites...... 46 3.5.2.3 Location and caste/developmental stage of collected termites...... 47 3.5.2.4 Workers ...... 47 3.5.2.5 Moisture readings and salivary reservoir volume ...... 48 3.6 Discussion...... 48 References cited...... 52

4. Water movement into the salivary reservoirs and chemical analysis of salivary glands and salivary reservoirs of Reticulitermes flavipes (Isoptera: Rhinotermitidae) ...... 67 4.1 Abstract...... 67 4.2 Introduction...... 68 4.3 Materials and methods...... 71 4.3.1 Water movement study...... 71 4.3.1.1 Termites...... 71 4.3.1.2 Dissections...... 72 4.3.2 Gas chromatography and mass spectrometry – conducted by USDA-ARS, New Orleans, LA...... 73 4.3.2.1 Termites...... 73 4.3.2.2 Gas chromatography and mass spectrometry...... 73 4.4 Statistical analysis...... 75 4.5 Results...... 75 4.5.1 Movement of food coloring within the termite ...... 75 4.5.2 Pathway of water after consumption ...... 76 4.5.3 Weight loss and gain ...... 77 4.5.4 Gas chromatography and mass spectrometry...... 78

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4.6 Discussion...... 78 References cited...... 82

5. Preliminary investigation into the ultrastructure of the salivary glands and salivary reservoirs of Reticulitermes flavipes workers (Isoptera: Rhinotermitidae) ...... 90 5.1 Abstract...... 90 5.2 Introduction...... 91 5.3 Materials and methods...... 93 5.3.1 Termites ...... 93 5.3.2 Preparation of samples for transmission electron microscopy (TEM) ...... 94 5.4 Results...... 95 5.4.1 Gross morphology of the salivary glands and reservoirs ...... 95 5.4.2 Ultrastructure of the salivary glands ...... 96 5.4.3 The salivary reservoir...... 96 5.5 Discussion...... 97 References cited...... 100

Conclusion ...... 113

Bibliography ...... 115

Appendix A: Analysis of mulch samples from three termite colonies (R. flavipes) and acetone control by mass spectrometry. Compound highlighted by the box was identified as pesticide lambda-cyhalothrin in Colony Stinson and A104 ...... 121

Appendix B: Analysis of the salivary reservoirs, salivary glands and hemolymph of termite workers and soldiers of R. flavipes (Colony A62) by mass spectrometry ...... 123

Appendix C: Analysis of the salivary reservoirs, salivary glands and hemolymph of termite workers and soldiers of R. flavipes (Colony Stinson) by mass spectrometry. Control was the same used in Colony A62...... 125

Appendix D: Analysis of the salivary reservoirs, salivary glands and hemolymph of termite workers and soldiers of R. flavipes (Colony A104) by mass spectrometry ...... 127

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List of Tables

Table Page

2.1 Mean (± S.E.) volume (μl) of the salivary reservoirs* of worker termites (Colony A39) removed from the food arena or the moist sand arena during a 36-h monitoring period...... 30

2.2 Mean (±S.E.) volume (μl) of worker salivary reservoirs during a 21-d period...... 31

3.1 Average salivary reservoir volume (μl) (±1.S.E.) of termite castes/stage collected at various locations ...... 55

4.1 List of compounds tentatively identified by GC-MS in termite salivary glands, salivary reservoirs and hemolymph from three colonies of R. flavipes ...... 83

4.2 Contaminants tentatively indentified by GC-MS in termite samples from A104 and Stinson colonies...... 85

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List of Figures

Figure Page

2.1 Test apparatus comprised of a moist sand arena connected via Tygon® tubing to a smaller food arena containing an oven-dried cellulose pad...... 32

2.2 A filled salivary reservoir (SR) and associated structures (C, crop; H, head; SG, salivary gland) of a worker termite, R. flavipes...... 33

2.3 Mean (± S.E.) consumption (%) of cellulose pads by R. flavipes from 3 colonies at 3, 7, 14, and 21 d. Four replicates, 250 termites per replicate. Within each daily observation, * indicates a significant difference between the treatments (P ≤ 0.05, Tukey’s HSD test)...... 34

2.4 Mean (± S.E.) water content (%) of cellulose pads in the presence or absence of termites at various times during a 36-h period. Six replicates with 250 termites (Colony A39) per replicate. Within each hourly observation, * indicates a significant difference between the treatments (P ≤ 0.05, Tukey’s HSD test)...... 35

2.5 Mean (± S.E.) water content (%) of cellulose pads in the presence or absence of termites during a 21-d period. Four replicates with 250 termites per replicate (3 colonies). Within each daily observation, * indicates a significant difference between the treatments (P ≤ 0.05, Tukey’s HSD test)...... 36

2.6 Linear relationship between the water content of cellulose pads versus cellulose pad consumption by R. flavipes during a 21-d period (confidence level .95)...... 37

3.1 Average length of largest salivary reservoir (mm) (±1 S.E.) for each caste/developmental stage. (All collections combined). Means with the same letter are not significantly different (P>0.05) ...... 56

3.2 Non-linear relationship between worker salivary reservoir length and head width (mm). R2 = 0.1676; R = 0.4094; y = -0.249431333 + 0.812847569*x ...... 57

3.3 Distribution of workers based on head width (mm)...... 58

3.4 Average length of salivary reservoirs (mm) (±1.S.E.) in large (LW), medium (MW) and small workers(SW). Means with the same letter are not significantly different (P>0.05)...... 59 xv

3.5 Non-linear relationship between salivary reservoir length and head width of nymphs (r2 = 0.0140; r = 0.1183, y = 0.357700761 + 0.169900795*x), nymphoid neotenics (r2 = 0.0285; r = -0.1688, y = 1.573783140 - 0.795487981*x) and soldiers (r2 = 0.0031; r = 0.0558, y = 0.509505803 + 0.331082151*x)...... 60

3.6 Average salivary reservoir volume (μl) (± 1 S.E.) of various termite castes/stages. Means with the same letter are not significantly different (P>0.05)..61

3.7 Average salivary reservoir volume (μl) (± 1 S.E.) of termites collected from various locations. Means with the same letter are not significantly different (P>0.05)...... 62

3.8 Average salivary reservoir volume (mm) (± 1 S.E.) of large (LW), medium (MW) and small workers (SW). Means with the same letter are not significantly different (P>0.05)...... 63

3.9 Average salivary reservoir volume (μl) (± 1 S.E.) of small, medium and large workers collected from various locations. Within the same location, means were not significantly different (P>0.05)...... 64

3.10 Average moisture content (%) (± 1 S.E.) of collection locations (shelter tubes and food sources) where active termites were collected from two field populations. Means with the same letter are not significantly different (P>0.05) ...... 65

3.11 Linear relationship between moisture level of collection site and termite salivary reservoir volume. Shelter tube site: r2 = 0.1419; r = 0.3767; y = -0.0291863785 + 0.0144608977*x. Food source site: r2 = 0.2086; r = 0.4567; y = -0.894144723 + 0.0508741023*x...... 66

4.1 Volume of salivary reservoirs a) <5% full, b) 5-25% full, c) ≥25-≤50% full and d) ≥50% full ...... 86

4.2 Volume of crop a) <5% full, b) 5-25% full, c) ≥25-≤50% full and d) ≥50% full ...... 87

4.3 Approximate volume of crop and salivary reservoirs prior to water ingestion and at different times thereafter for termites from (a) Colony C10 (n=62), (b) Colony A104 (n=76), and (c) Colony Paulding (n=68)...... 88

4.4 Mean body weight of termites (mg) (± 1 S.E.) before and after a 6-hr dehydration period from three colonies (A104, C10 and Paulding). Within each colony observation, * indicates a significant difference between the treatments (P ≤ 0.05, Mann Whitney U test)...... 89

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5.1 Dissection of a R. flavipes worker showing the head (H), salivary (labial) glands (SG), salivary reservoirs (SR), crop (C), midgut (MG) and hindgut (HG). Scale bar = 500μm...... 102

5.2 Paired salivary reservoirs near full capacity and highly translucent. Scale Bar = 1000μm...... 103

5.3 Salivary reservoirs depleted and folded. Scale = 1000μm...... 104

5.4 Ultrastructure of the worker salivary gland, showing secretory cells, Types I (I) and IIb (IIb), in addition to parietal cells (PC) with invaginated basal plasma membrane (PM) and extensive microvilli (MV) around their lumen and salivary duct cells (DC) and duct lumen (DL). FB, fat body cells (surrounding the salivary gland,); N, nucleus...... 105

5.5 Higher magnification of the worker salivary gland, showing secretory cell Types I (I), IIa (IIa) and IIb (IIb) which contain rough endoplasmic reticulum (RER) and secretory material (S) of various electron density,. The outer, parietal cells (PC), have highly invaginated basal plasma membrane (PM) and numerous microvilli (MV) around their lumen (L)...... 106

5.6 An acinus (A) of a worker, containing salivary gland secretory cells (SC) and surrounded by salivary ducts (SD)with microvilli (MV) and duct lumen (L)...... 107

5.7 Trachea (TR), lined with tanidea (T), along the outer margin of acini cells (AC) ...... 108

5.8 Salivary gland duct, lined with cuticle (C), within acini secretory cell Type I (I). DL, duct lumen, MF, myelin fiber...... 109

5.9 Ultrastructure of the salivary reservoir of a worker termite; reservoir lumen (RL) lined with cuticle (C) and a thin layer of epithelium (E), N nucleus...... 110

5.10 Close up of the salivary reservoir wall showing epithelial cells (EC), their nuclei (N), and secretory-like cells (SC) with their smaller nuclei (N) and microvilli-lined lumen (L), and cuticular ridges (CR)...... 111

5.11 Secretion material (S) inside the salivary reservoir lumen (RL); SC, secretory-like cell...... 112

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Chapter 1

General Introduction

1.1 Biology of Reticulitermes flavipes

Termites are social insects that belong to the order Isoptera. Termite classification has been modified since the original proposal of only four families suggested by Holmgren (1911, 1912). Today, it is widely accepted that there are a total of seven termite families and more than 2900 species worldwide (Synder 1949; Grasse

1949; Emerson 1965; Engel et al. 2009). The families Mastotermitidae, Kalotermitidae,

Termopsidae, Hodotermitidae, Rhinotermitidae, and Serritermitidae all possess hindgut protozoa that aid in the digestion of cellulose, and they are collectively called the lower

termites. The higher termites are comprised of only one family, the Termitidae. This is

the largest of all the termite families and the termitids do not possess hindgut protozoa

(Krishna and Weesner 1969).

All rhinotermitids have a subterranean lifestyle, as they live mostly in soil. The

family Rhinotermitidae contains three major pest genera: Coptotermes, Reticulitermes, and Heterotermes. Reticulitermes spp. are widespread throughout the United States, and both Reticulitermes flavipes and Reticulitermes virginicus are found in Ohio. R. flavipes is the most common and economically important termite in Ohio.

1 Termites feed primarily on cellulosic materials and colony members will frequently exchange both liquid food and feces with each other. When food is released from the crop it is stomodeal trophallaxis. Nutrients and hindgut protozoa are transferred via the anus which is proctodeal trophallaxis.

Subterranean termites are diploid social insects whose populations are organized into large, dynamic, poorly understood units called colonies. The division of labor among the several life forms (castes) in the colony enables an efficient, productive, and cooperative unit. Termite colonies typically include three main castes (reproductives, soldiers, and workers) as well as numerous developmental stages that can lead to the main castes.

1.2 Termite developmental stages and castes

1.2.1 Larva

Unlike the social Hymenoptera, the termites are hemimetabolous, therefore the term larva, as it is applied to immature termites is not equivalent to its use in holometabolous insects. The term is derived from the French, where “larva” refers to an immature developmental stage for all insect species (Thorne 1996). Termite larvae are the first and second instars of the termite colony. Termite larvae are non-functional with regards to colony tasks and require other colony members to feed them via anal or oral trophallaxis.

1.2.2 Worker

Worker termites (3rd instar and above) are renowned for eating wood, and they are responsible for feeding the dependent colony members (reproductives, soldiers,

2 maturing alates, nymphs, and larvae), tending eggs and larvae, grooming nest mates, tunneling through soil, and building aboveground shelter tubes (Thorne 1999). Workers have strong, serrated mandibles making them adept at chewing through wood.

1.2.3 Soldier

Soldiers are the defensive caste and may act aggressively toward invaders such as ants. Reticulitermes soldiers have large sclerotized heads and sickle-shaped mandibles.

Because they are unable to chew wood, they receive nutrients from workers via trophallaxis.

1.2.4 Nymph

Nymphs are immature individuals with wing pads that are progressing along a developmental pathway to become alates. Nymphal termites are also capable of regressive molts and may molt back into an undifferentiated worker form, known as a pseudergate (false worker). Nymphs’ bodies are typically larger than workers and have a higher fat content. Nymphs within the Termopsidae and Kalotermitidae are thought to aid in colony tasks (Noirot and Pasteels 1987; Crosland et al. 2004).

1.2.5 Alate

Alates are winged imagoes that are capable of founding new colonies. Alates are produced by mature colonies, but the minimum size or age of such colonies is not known.

The timing of alate flights or “swarms” varies according to species, season, and geographic location (Nutting 1969). After dealation and pairing, the male and female imago find an appropriate nest site, which is usually a small hole, crack, or other protected crevice within or adjacent to damp soil or wood, and they then mate (Weesner

3 1956, 1965). Reproductives derived from alates are known as primary reproductives. The

male and female primary pair that head a colony are called the king and queen, respectively.

1.2.6 Neotenic reproductive

Neotenic reproductives are individuals with juvenile characteristics that may

replace the primary reproductives. A colony may contain several types of neotenic

reproductives. Nymphoid neotenics develop from nymphs, and are therefore

characterized by the presence of short or long wing buds. Ergatoid reproductives develop

from workers and therefore lack wing buds.

Neotenic reproductives of either sex can be present in the hundreds within a

single colony. They are thought to allow for colony expansion (Myles 1999) and to

compensate for the loss of either of the primary reproductives.

1.3 Environmental requirements

Termites are highly susceptible to desiccation, and moisture is a critical factor in

their survival. The workers of most termite species are only lightly sclerotized and can

lose water through their cuticle and from external openings such as the mouth and

spiracles. Most insects obtain water metabolically, from food, from drinking and in some

cases through water vapor absorption (Chapman 1998).

Desiccation tolerance of rhinotermitid species is varied due to body size and

different water proofing mechanisms (Strickland 1950; Collins and Richards 1963).

When various caste members (workers, soldiers and reproductives) of Reticulitermes

4 tibialis were grouped together and placed in a desiccating environment they had an

average group survival time of 234 minutes, which was higher than the Reticulitermes arenincola group at 174 minutes and R. flavipes at 230 minutes (Strickland 1950).

Desiccation tolerance of R. flavipes is low, and these termites appear to be inefficient at retarding transpiration (Collins and Richards 1963). R. flavipes workers had a survival range of 3.0-6.2 h when exposed to a desiccating environment of 34-35°C and 0-4% RH

(Collins 1969). Similar findings were reported by Sponsler and Appel (1990) where R. flavipes workers only survived 5.1 h when exposed to 30°C and 0-2% RH.

Termite feeding behavior is influenced by the moisture content of a food substrate. In choice-feeding tests, groups of Coptotermes formosanus preferentially fed on wood blocks with the highest initial moisture content (~96%) (Delaplane and La Fage

1989). Nakayama et al. (2005) obtained similar results as C. formosanus workers consumed significantly more wood when wood blocks had a moisture content between

79-103% (based on dry weight). These researchers also reported that R. speratus consumed the most wood at both 79-103% and 140-182% moisture content. The authors of this study did not comment on how they calculated the moisture content of more than

100%, which would require the wood to contain more water than the dry wood substance.

The authors added moisture to the wood samples by dipping them in water for one day

and they subsequently vacuum impregnated water directly into the wood. The extra water

must have been stored in the cell cavities of the wood (free water) as well as the water

within the cell walls (bound water) (Reeb 1995).

5 Tunneling behavior can also be influenced by substrate moisture levels. A study of tunneling behavior of Reticulitermes spp. in a two-dimensional arena found that tunneling distance on days after the addition of water was higher than on days prior to adding water (Houseman and Gold 2003). Su and Puche (2003) found that C. formosanus and R. flavipes tunneled more in sand with a higher moisture content than in sand with a low moisture content, but tunneling activity was unaffected by moisture 7-10 days after termites were released into the arena. It was hypothesized that termites moved water into low moisture areas of the arena, thus negating the artificial moisture gradient. It has been suggested that termites transport water in their salivary reservoirs (Hewitt et al. 1971;

Watson et al. 1971; Grube and Rudolph 1999a).

1.4 Salivary glands and salivary reservoirs

Termites possess a pair of salivary glands, each of which consists of a series of lobed acini, connected by a branching salivary canal. Also arising from the salivary canal is a pair of thin walled translucent sacs, the salivary reservoirs (also called water sacs), which are connected posterior to the salivary glands via a salivary reservoir duct (Noirot

1969; Watson et al. 1971; Grube and Rudolph 1999b). It has been suggested that salivary glands perform a variety of functions, including digestion, communication, and defense, yet these aspect of termite biology are still poorly understood. Salivary reservoirs are thought to function in water storage.

6 1.4.1 Salivary reservoirs

Imbibition of free water is known in the alates of Hodotermes mossambicus

(Hodotermitidae) (Hewitt et al. 1971; Watson et al. 1971) and in workers of Macrotermes

michaelensi (Termitidae) where water was most frequently released onto the fungus

comb (Sieber and Leuthold 1981). Grube and Rudolph (1999b) demonstrated the ability

of R. santonensis workers to use water stored in the salivary reservoirs to raise the humidity in unfavorable microclimates. They also found that this stored water could not be used as an individual water source to hydrate themselves during high water evaporation loss.

Ultrastructural studies have typically focused on the salivary glands of termites

(Billen et al. 1989; Costa-Leonardo and da Cruz-Landim 1991), and the salivary

reservoirs are usually overlooked in these morphological studies. The only research that

has concentrated on the ultrastructure of water sacs includes that of Grube et al. (1997) on

the workers of R. santonensis and that of Šobotnik and Weyda (2003) on multiple stages

(castes) of the Cuban subterranean termite, Prorhinotermes simplex (Rhinotermitidae).

Grube et al. (1997) described the salivary reservoir wall as a “single-layered

epithelium, consisting of uniform flat cells with elongated nuclei and rare cytoplasmic

organelles throughout the organ”. The cuticular layer of the wall was not sclerotized and

less than 50 nm thick. The reservoir ducts were lined with cuticular ridges and were

nearly twice as thick as the reservoir wall. Grube et al. (1997) did not mention any

structure within the salivary reservoirs or ducts that could be attributed to the mechanism

of water uptake in R. santonensis workers.

7 The more recent study by Šobotnik and Weyda (2003) described the fine structure of the salivary glands and salivary reservoirs in P. simplex. They observed that the connection of the salivary reservoir with the salivary reservoir duct was simple in second instar larvae and presoldiers, as a “taenidium” occurred at the neck of the salivary reservoir. In soldiers and male neotenics the salivary reservoir duct was found to be immerged into the water sac, appearing as a valve-like structure. No muscles were found near the reservoirs and its ducts in any of these stages. Nerves were observed under the basement membrane and it was suggested that these nerves may act as receptors that controlled the degree of filling of the reservoirs. These observations were made only in larvae, soldiers, and reproductives, none of which contributes to colony maintenance.

The route by which water might reach the salivary reservoirs remains unclear as there is conflicting data from different research groups. Watson et al. (1971) traced the movement of water by allowing termites to drink water from cellulose pads marked with radioactive colloidal gold. They found that the majority of the radioactivity occurred in the gut and not in the salivary reservoirs. They hypothesized that water reached the salivary reservoirs via the gut to hemolymph route. A similar finding was reported for

Anacanthotermes ahngerianus (Hodotermitidae) with the use of methyl blue to trace the movement of water after ingestion (Mednikova 1988). The water in the salivary reservoirs always remained clear, while methyl blue was found in the gut and Malpighian tubules. It was suggested that water was transported into the salivary reservoirs from the hemolymph, possibly by an ionic gradient.

8 In contrast, Grube et al. (1997) concluded that R. santonensis refilled their salivary reservoirs by oral water uptake wherein water passed directly from the oral cavity into the reservoir lumen via the reservoir ducts. When they placed salivary reservoirs in solutions of different osmotic gradients for up to six hours, no decrease or increase of reservoir volume was detected, indicating that there was no net water flow into the reservoir lumen via the reservoir wall. Grube et al. (1997) interpreted this to mean that the salivary reservoirs were not filled via the hemolymph route. They observed that the osmotic gradient of the salivary reservoir fluid and the hemolymph were not conducive for movement of fluid through the salivary reservoir wall. The osmotic concentration of the salivary reservoir fluid was low and comparable to tap water, but the hemolymph had a significantly higher ionic concentration. Therefore osmotic water flow into the salivary reservoirs from the hemolymph could be excluded. Furthermore, these researchers discovered that the salivary reservoir walls or ducts do not contain any structures associated with water transport against an ionic gradient (basal invaginations penetrated with mitochondria and apical microvilli); similar to what is found for cockroach rectal pads (Wall et al., 1970; Berridge and Oschman, 1972).

1.4.2 Salivary glands

Salivary glands are a complex set of glands that differ in size and function depending on caste member and size (Noirot 1969, Mednikova 1995). Salivary glands are known to produce digestive enzymes such as endo-β-1,4-glucanase and β- glucosidases

(Tokuda et al. 2009, Inoue et al.1997). Benzoquinones can be produced by the salivary reservoirs of soldiers in order to protect the colony when under attack (Olagbemiro et al.

9 1988). Salivary glands may also be used to provide nutrition to other colony members such as reproductives and immature members (Hinze et al. 2002). Secretions from worker labial glands can also stimulate a communal exploitation of food sources (Kaib and

Zeissman 1992, Reinhart and Kaib 2004).

The morphology of the salivary glands differs depending on the caste members

(Noirot 1969, Billet et al. 1989, Kaib and Zeissman 1992). The acini of salivary glands typically consist of secretory and cannicular cells. Workers have three types of secretory cells (Type I, II and III). Soldiers and alates only have Type I and III secretory cells.

1.5 Termite management

When structures are built near, over, or in potential subterranean termite territories the termites eventually locate and attempt to feed on the structure (Forschler

1999). Termite control measures protect wooden structures and products, thus conserving wood supply by prolonging the life of structural wood (Kard 2002). Termites cause an estimated $5 billion per year for costs associated with their prevention and treatment, making them the most economically important wood-destroying insect in the United

States. Furthermore, many thousands of hectares of timber are harvested yearly to replace structural wood destroyed by termites (Su and Scheffrahn 1990).

Integrated pest management (IPM) for termites consists of a variety of strategies, such as complete and thorough inspections to determine the extent of the termite infestations, the use of baiting systems, soil-applied termiticides, wood preservatives, physical barriers, non-cellulose building components, sanitation, elimination of

10 conducive conditions, building practices, steel frame construction, fumigation, and

biological agents (Kard 2002). However, one of the primary strategies should be to

recognize and alter conditions around one’s home so as to reduce the termites’

environmental requirements for moisture, food (wood), and shelter (Jones 2000).

1.6 Objectives of this research

While we know that moisture plays an integral role in termite survival, the

ability of termites to conserve and transport water requires further research. This dissertation investigates the physiological mechanisms of water transport, specifically how water is maintained within the termite salivary reservoirs and how it is moved to new food sources. If the physiological and behavioral mechanisms involved with water maintenance can be manipulated, this information may eventually be used to develop or improve non-chemical approaches to termite prevention and control.

This dissertation addresses the following questions: 1) How do termites transport water?; 2) How much water do termites transport?; 3) Is there a division of labor associated with the use of salivary reservoirs?; 4) How does water reach the salivary reservoirs?; 5) Does the ultrastructure of the salivary reservoirs reveal any mechanisms involved with water ingestion or release?; and 6) Do the salivary reservoirs contain only water?

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Chapter 2

Moisture Augmentation of Food Items by Reticulitermes flavipes (Isoptera: Rhinotermitidae)

(Published in Sociobiology Vol 55:735-747 (2010)

2.1 Abstract

Subterranean termites are highly susceptible to desiccation and moisture is a

critical factor in their survival. Termites possess a pair of salivary reservoirs that store

water, which presumably is used to increase the humidity in unfavorable microclimates.

The main objective of this study was to measure the moisture change of a dry food source

(cellulose pad) in the presence of the eastern subterranean termite, Reticulitermes

flavipes, over a 21-day period. The volume of the workers’ salivary reservoirs was

determined concomitantly as an indicator of the amount of stored water. The test

apparatus consisted of two cylindrical plastic containers connected by a 95 cm-length of

Tygon® tubing; the larger container held moist sand and the smaller food arena held an oven-dried cellulose pad. This test set-up demonstrated that termites rapidly transported water to the dry food source, with the mean moisture content of the cellulose pad reaching ~7% after just 3 hours and subsequently attaining a maximum of ~46% at the

21-day observation. Over time, moisture levels continuously increased in the presence of termites and moisture levels were highly correlated with consumption of the cellulose

16 pad. Salivary reservoir volume was smallest for termites removed from the food chamber

in comparison to termites removed from the moist sand throughout the entire study

(except on day 7); however this difference was significant only at the 3-h and 18-h

observations. The termites’ salivary reservoirs were observed to be at their maximum

volume (~0.7 ul) when workers were removed at 3 hours from the moist sand arena, and

they were at their minimum volume (~0.1 ul) for workers removed at 9 h from the food

arena. These data indicate that termites indeed use their salivary reservoirs as “water

sacs” to relocate water from moist areas to dry resources.

2.2 Introduction

The workers of most termite species are only lightly sclerotized, making them

highly vulnerable to desiccation (Delaplane and La Fage 1989). Consequently, water

relations govern many facets of termite biology (Collins and Richards 1963, Delaplane

and La Fage 1989; Sponsler and Appel 1990). Desiccation tolerance of Reticulitermes

flavipes (Rhinotermitidae) is low, and these termites appear to be inefficient at retarding

transpiration (Collins and Richards 1963). R. flavipes workers survived an average of 292

minutes in a desiccating environment; they died after they lost ~59% of their wet weight

(Collins 1969).

Termite feeding behavior is influenced by the moisture content of a food

substrate. In choice-feeding tests, groups of Coptotermes formosanus (Rhinotermitidae)

preferentially fed on wood blocks with the highest initial moisture content (~96%)

(Delaplane and La Fage 1989). As termites consumed these high moisture wood blocks,

17 wood moisture decreased, which contrasts to the lower moisture wood blocks (~14%)

where food removal was accompanied by wood moisture gain. These researchers

proposed that this was indirect evidence that termites perhaps were actively relocating

water. However, they cautioned that it was uncertain whether moisture gain in the low

moisture wood blocks was due to equilibration of relative humidity or a termite-mediated

event.

Termite foraging behavior also is influenced by substrate moisture. Su and

Puche (2003) reported that foragers of C. formosanus and R. flavipes tunneled significantly more in sand with a higher moisture content versus a lower moisture content in their studies involving an artificial tunnel arena with a moisture gradient. Because the moisture gradient had been negated after 7-8 days, they hypothesized that subterranean termites were able to transfer water to areas of low moisture after tunneling for several days.

Termites possess a pair of salivary glands, each of which consists of a series of lobed acini, connected by a branching salivary canal. Each of these salivary glands is connected to a thin walled translucent sac, the salivary reservoir, sometimes also called a water sac (Noirot 1969, Watson et al. 1971, Grube and Rudolph 1999b). Salivary reservoirs are thought to function in water storage. Alates of Hodotermes mossambicus

(Hodotermitidae) imbibe free water which is stored in two large “water-sacs” (salivary reservoirs) (Hewitt et al. 1971, Watson et al. 1971). The water is thought to aid the alates during the hot and dry period, which occurs shortly after the nuptial flight. Workers of

18 Macrotermes michaelensi (Termitidae) also imbibe free water; this water is most frequently released onto the fungus comb (Sieber and Leuthold 1981).

Grube and Rudolph (1999a, b) demonstrated that Reticulitermes santonensis workers were able to use water stored in the salivary reservoirs to moisten building material and raise the humidity in unfavorable microclimates. The average moisture content of sand clod samples was ~5.1% and increased to ~44.6% after termites added water. However, the stored water could not be used as an individual water source during high water evaporation loss. While we know that moisture plays an integral role in termite survival, the ability of termites to conserve and transport water requires further research.

Our research focuses on the mechanisms of water transport, specifically how termites move water to new food sources. It is predicted that termites will use their salivary reservoirs as “water sacs” to relocate water to dry resources. Furthermore, it is predicted that termites will acclimate their food source to a moisture level of approximately 20%, which often is an indicator of termite activity during home inspections (pers. obs.).

2.3 Materials and methods

This experiment evaluated moisture change of a dry food item in the presence of termites, and the control assessed background change in moisture due to water absorption from the environment in the absence of termites. Another control containing termites, but

19 no water source, was determined to be unfeasible as termites succumbed to desiccation

within 48 hours.

2.3.1 Experimental units

Each test unit (Fig. 2.1) consisted of two cylindrical plastic containers connected

by a 95 cm-length of Tygon® tubing (0.5 cm diameter). The larger arena (5 cm diameter,

3.5 cm height) (Pioneer Plastics, Inc., Dixon, KY) held 30 g of sand (QUIKRETE®

premium play sand) moistened with 6 ml of dyed water. The dye, 0.05% methyl blue,

served as a visual aid for assessing whether termites were ingesting and/or transferring water. The smaller food arena (5 cm diameter, 1 cm height) (Pall Life Sciences, East

Hills, NY) held an oven-dried, pre-weighed cellulose pad (4.7 cm diameter) (Pall Life

Sciences). A total of 250 termites (workers of the 3rd instar or above), 2% of which were soldiers, were placed into the larger arena. In order to minimize water loss, Parafilm

(Parafilm M, Pechiney Plastic Packaging, Menasha, WI) was wrapped around the lids of both containers and the connection between the Tygon® tubing and the containers.

Controls were similarly established, but without termites. Test units were placed in an environmental chamber (Percival Intellus Environmental Controller, Percival Scientific,

Iowa, USA) in complete darkness at 26.7°C and 85% RH.

2.3.2 Termites

Four laboratory colonies of R. flavipes were used for this study. Each of these

colonies had been established during spring 2002 by pairing a single male and female de-

20 alate. Colonies were ~5 years old and each contained approximately 4000-6000 termites

at the time of this study.

2.3.3 Experimental design and observation periods

Hourly observations: An experiment was conducted with one Colony (A39) to

evaluate initial time periods (3, 9, 18, and 36 h) and was replicated six times.

Daily observations: Four experimental units were established using termites from

three colonies (A38, A54, and A78) for each time period (3, 7, 14, and 21 d) (i.e., 12

units per time period).

2.3.4 Consumption and moisture content of cellulose pads

Cellulose pads were oven dried for a minimum of 24 h at 54°C in an Isotemp

500 series oven (Fisher Scientific) to obtain an initial dry weight. At each evaluation

period, each cellulose pad was weighed as soon as it was removed from the food arena to

determine its moisture content, then it was again oven dried for a minimum of 24 h and

weighed to assess weight loss due to consumption by termites.

2.3.5 Salivary reservoir measurements

At each observation, 3 to 5 workers were removed from each of the paired sand

and food arenas and immediately stored at -80 °C. Dissections then were performed

under the microscope to extract the termites’ salivary reservoirs in an isotonic solution

(Ringer’s solution). Auto-Montage Pro software (Synoptics Ltd. Version 5.01, 2004) was

used to measure the length and width of the largest salivary reservoir. The volume of the

4 2 reservoir was then determined using the formula for an ellipsoid, V = /3 x π x L x W , as this shape closely resembles that of a salivary reservoir, which is depicted in Fig.2.1.

2.4 Statistical analyses

Statistica was used to conduct all analyses (StatSoft Inc. 2001). An analysis of variance was performed to test for any significant differences over time in the consumption and moisture gain of cellulose pads when exposed to termites from different colonies. Tukey's studentized range test (HSD) compared the treatments and the control, over time, for significant differences (alpha level = 0.05). For each observation period, salivary reservoir volume was compared for termites from the food arena versus those from the moist sand arena by using a Mann-Whitney U test (alpha = 0.05). Statistica was also used to perform regression analyses to test for any correlation of moisture gain and consumption of the cellulose pad over time.

2.5 Results

2.5.1 Consumption

Cellulose consumption significantly increased over time for all three colonies. All colonies consumed a similar amount of cellulose until day 14 when a significant colony and day interaction in cellulose consumption was evident (F = 5.7; df = 6, 35; P =

0.0003) (Fig. 2.3). This was due to Colony A54 exhibiting a larger appetite and consuming more than twice as much cellulose on day 14 (65%) as compared to day 7

(31%). By the end of the 21-d experiment, Colony A54 had consumed the greatest amount of food with only about 10% of the cellulose pad remaining. There were significant differences in consumption among all colonies at this final time period, with

22 Colony A38 having consumed 75% of the pad and Colony A78 having consumed the

least amount of food (61%).

2.5.2 Moisture

The moisture content of cellulose pads was significantly greater (F = 7.32; df = 3,

36; P = 0.0006) when termites were present compared to when they were absent

(controls) during all hourly observations except 3 h (P = 0.146) (Fig.2.4). This alteration

occurred within 9 h, with cellulose pads averaging ~12% moisture when termites were

present but only ~4% when they were absent. At 36 h, termites had altered their food source to an average of ~23% moisture, which was significantly greater than the controls, which averaged 6% (P = 0.0001). Similarly, moisture content was significantly greater

(F = 6.5; df = 3, 55; P = 0.0007) during all daily observations when termites were present compared to when they were absent, and no colony differences were evident (Fig.2.5).

Moisture levels of cellulose pads were highly correlated with consumption (R2 =

0.68) during the 21-d period (Fig.2.6). Moisture levels were the highest at day 21 with an

average of 46% in the presence of termites and 9% in controls (Fig.2.5).

2.5.3 Salivary reservoirs

Termites in the food arena had smaller salivary reservoirs compared to those in the moist sand arena during the initial hours of observation (Table 2.1). However, this difference was significant only at the 3 h and 18 h observations (P = 0.017 and P = 0.01, respectively). Salivary reservoirs were at their largest, approximately 0.7 µl, for termites removed at 18 h from the moist sand arena, whereas the smallest volume was approximately 0.1 µl for termites removed at 9 h from the food arena (Table 2.1). The

23 difference between salivary reservoir volumes became less notable during the daily

observations (Table 2.2); in fact the salivary reservoir volumes were similar from day 3

through day 14 regardless of the test arena (food or moist sand) in which the termites

were located. At the final observation (21 d), the salivary reservoir volumes in the two

arenas were significantly different (P = 0.03) (Table 2.2).

2.6 Discussion

Our research demonstrates that termites can relocate free water to dry resources,

as previously postulated by Delaplane and La Fage (1989). R. flavipes altered their food source to a moisture level of ~20% after only 36 h. At the end of the 21 d study, cellulose pads averaged 46% moisture, which may be a preferred moisture level for R. flavipes.

This is consistent with the data reported by Green et al. (2005) wherein R. flavipes occurred more frequently in arenas at 55% moisture compared to lower moisture levels.

However, moisture levels possibly could have increased even more if our study had continued beyond a 21-d monitoring period.

As previous research has suggested, termites apparently use their salivary reservoirs to relocate water (Grube and Rudolph 1999a, b). In our study, R. flavipes workers sampled from food arenas had smaller salivary reservoirs as compared to those from moist sand arenas, thereby indicating that termites were releasing their reservoir’s contents onto their food. This was especially evident during the initial hours of

investigation.

24 Temporal differences in salivary reservoir volume were observed in this study.

During the first 36 h of investigation, the termites’ salivary reservoirs were always smaller on the food side as compared to the moist sand side of the test apparatus, albeit not significantly so except at the 3 h and 18 h observations (Table 2.1). Significant differences in salivary reservoir volumes were not noted thereafter until the final observation at day 21 (Table 2.2). This may provide insight into subterranean termite behavior since adding moisture to a dry environment or food source apparently is an important task of worker termites during the initial hours of investigation. Grube et al.

(1999a) observed R. santonensis workers releasing droplets of water from their salivary reservoirs when they entered a glass tube. The released droplets were thought to temporally increase the humidity of the microhabitat.

During our initial hourly observations (3 – 18 h), salivary reservoirs exhibited an average difference of 0.35 µl for termites removed from the moist sand arena as compared to those from the food arena. This difference became much smaller during the period from 36 h to 14 d, when regardless of the test arena from which termites were removed, the volume of their salivary reservoirs was quite similar, with an the average difference of only 0.03 µl. However, by 21-d, the difference in salivary reservoir volume once again increased, perhaps partly due to the fact that only a small amount of cellulose pad remained in each arena. With an increasing edge effect attributable to termite feeding behavior, the cellulose pad likely began to dry out, thereby stimulating the termites to add more water.

25 Termites may not always fill their salivary reservoirs to capacity or release all

contents once a food resource reaches a certain moisture level, which we observed to be

~20%. There may be an advantage to retaining water reserves for other colony functions

such as communication or digestion. The variation in salivary reservoir volume observed

during our study may be due to innate colony differences, or it may be an artifact of the

experimental design given that individual termites were not monitored and some may not

have been in an arena long enough to either fully imbibe or release water.

A subterranean life style offers protection from the elements and prevents water

loss, but because subterranean termites explore above ground and construct tunnels in

unfavorable habitats, their salivary reservoirs may be an important morphological feature,

allowing them to expand their foraging territory. This study is the first to report the

amount of water transported to dry food resources by R. flavipes, as well as the average

volume of their salivary reservoirs during this process. The largest salivary reservoir

volume for R. flavipes in this study was 0.7 µl (range 0.1 – 0.9 µl), which is larger than

other termite species reported so far. Grube and Rudolph (1999b) reported that R.

santonensis released 0.15 µl of water on average from their salivary reservoirs (range

0.08-0.25 µl) onto dry sand clods, and Kaib and Ziess (1992) reported a similar quantity for Schedorhinotermes lamanianus (range 0.05 – 0.25 µl).

Based on the results of our research which demonstrates that areas of ≥20%

moisture are strong indicators of termite activity, we recommend that moisture meters should be used during termite inspections. Furthermore, inspectors should not focus strictly on moisture-prone areas, as termites can relocate water to dry areas. A holistic

26 approach to termite control is important and homeowners should be educated on the importance of reducing food and moisture sources around their home.

References Cited

Chapman, R. F. 1998. The Insects: Structure and Function. Cambridge University Press, Cambridge, England

Collins, M. S., and G. A. Richards. 1963. Studies on water relations in North American termites. I. Eastern species of the genus Reticulitermes (Isoptera: Rhinotermitidae). Ecology: 44: 600-604.

Collins, M. S. 1969. Water relations in termites, pp. 433-458. In: K. Krishna and F.M. Weesner [eds.], Biology of Termites, vol. I. Academic Press, New York.

Delaplane, K. S., and J. P. La Fage. 1989. Preference for moist wood by the Formosan subterranean termite (Isoptera: Rhinotermitidae). J Econ Entomol 82: 95-100.

Green, J. M., M. E. Scharf, and G. W. Bennett. 2005. Impacts of soil moisture level on consumption and movement of three sympatric subterranean termites (Isoptera: Rhinotermitidae) in a laboratory assay. J Econ Entomol 98:933-937.

Grube, S., and D. Rudolph. 1999b. Water supply during building activities in the subterranean termite Reticulitermes santonensis De Feytaud (Isoptera, Rhinotermitidae). Insectes Soc 46: 192-193.

Hewitt, P. H., J.J.C. Nel, and I. Schoeman. 1971. Influence of group size on water imbibition by Hodotermes mossambicus alate termites. J Insect Physiol 17: 587- 600.

Kaib, M., and J. Ziesmann. 1992. The labial gland in the termite Schedorhinotermes lamanianus (Isoptera: Rhinotermitidae): morphology and function during communal food exploitation. Insectes Soc 39: 373-384.

Noirot, C. 1969. Glands and secretions, pp. 89-123. In K. Krishna and F.M. Weesner [eds.], Biology of Termites, vol. I. Academic Press, New York.

28 Sieber, R., and R. H. Leuthold. 1981. Behavioral elements and their meaning in incipient colonies of the fungus growing termite Macrotermes michaelseni (Isoptera: Macrotermitinae). Insectes Soc 28 (4): 371-382.

Sponsler, R. C., and A. G. Appel. 1990. Aspects of the water relations of the Formosan and eastern subterranean termites (Isoptera: Rhinotermitidae). Environ Entomol 19: 15-20.

StatSoft Inc. 2001. STATISTICA for Windows computer program manual. Tulsa, OK: StatSoft Inc.

Su, N. Y., and H. Puche. 2003. Tunneling activity of subterranean termites (Isoptera: Rhinotermitidae) in sand with moisture gradients. J Econ Entomol 96: 88-93.

Watson, J.A.L., P. H. Hewitt, and J.J.C. Nel. 1971. The water-sacs of Hodotermes mossambicus. J Insect Physiol 17: 1705-1709.

Table 2.1. Mean (± S.E.) volume (µl) of the salivary reservoirs* of worker termites (Colony A39) removed from the food arena or the moist sand arena during a 36-h monitoring period

Hour

Test Arena 3 9 18 36

Food 0.28 ± 0.07a 0.14 ± 0.04a 0.23 ± 0.08a 0.23 ± 0.06a

Moist Sand 0.61 ± 0.1b 0.43 ± 0.09a 0.66 ± 0.13b 0.30 ± 0.05a

* Six replicates, 3-5 termites per replicate Values followed by the same letter within each column are not significantly different (Mann-Whitney U-test, P ≥ 0.05).

Table 2.2. Mean (±S.E.) volume (µl) of worker salivary reservoirs during a 21-d period*

Day

Test Arena 3 7 14 21

Food 0.29 ± 0.05a 0.27 ± 0.03a 0.22 ± 0.03a 0.31 ± 0.05a

Moist Sand 0.30 ± 0.05a 0.25 ± 0.04a 0.19 ± 0.03a 0.43 ± 0.04b

*For each of 3 colonies (A38, A78, A54), 4 replicates per time period, 3-5 termites per replicate Values followed by the same letter within each column are not significantly different (Mann-Whitney U-test, P ≥ 0.05).

Figure 2.1. Test apparatus comprised of a moist sand arena connected via Tygon® tubing to a smaller food arena containing an oven-dried cellulose pad.

32 H

C SR

Figure 2.2. A filled salivary reservoir (SR) and associated structures (C, crop; H, head; SG, salivary gland) of a worker termite, R. flavipes.

33 100

90 *

70 *

30 S.E.)consumption (%) ofcellulose pad 20

Mean (+Mean 10

0 Colony: A38 3 5 7 9 11 13 15 17 19 21 Colony: A54 Colony: A78 Day

Figure 2.3. Mean (± S.E.) consumption (%) of cellulose pads by R. flavipes from 3 colonies at 3, 7, 14, and 21 d. Four replicates, 250 termites per replicate. Within each daily observation, * indicates a significant difference between the treatments (P ≤ 0.05, Tukey’s HSD test).

34 28

26 *

18 16 * 14 * 12

8 S.E.) water content (%) ofcellulose pad 6

4 Mean (+ 2 Termites: Present 0 Termites: Absent 0 5 10 15 20 25 30 35 40 Hour

Figure 2.4. Mean (± S.E.) water content (%) of cellulose pads in the presence or absence of termites at various times during a 36-h period. Six replicates with 250 termites (Colony A39) per replicate. Within each hourly observation, * indicates a significant difference between the treatments (P ≤ 0.05, Tukey’s HSD test).

35 50 * 45 *

40 *

30 *

15 S.E.) water content (%) of cellulose pad 10

Mean (+Mean 5

Termites: Present 0 Termites: Absent 3 5 7 9 111315171921 Day

Figure 2.5. Mean (± S.E.) water content (%) of cellulose pads in the presence or absence of termites during a 21-d period. Four replicates with 250 termites per replicate (3 colonies). Within each daily observation, * indicates a significant difference between the treatments (P ≤ 0.05, Tukey’s HSD test).

36 65

35 Water content (%) content Water

r2 = 0.6833 25 r = 0.8266 y = 25.8762005 + 0.293244261*x 20

15 0 102030405060708090100 Consumption (%)

Figure 2.6. Linear relationship between the water content of cellulose pads versus cellulose pad consumption by R. flavipes during a 21-d period (confidence level .95).

Chapter 3

Variability of Salivary Reservoirs among Developmental Stages and Castes of Reticulitermes flavipes (Isoptera: Rhinotermitidae)

3.1 Abstract

Termites are soft bodied and lightly sclerotized making them vulnerable to water loss. Due to this susceptibility, moisture is a critical factor in their survival. Termites possess a pair of salivary reservoirs that function in water storage. The stored water presumably is used to raise the humidity in unfavorable microclimates. The main objective of this study was to evaluate the variability of salivary reservoir dimensions among the various castes of the eastern subterranean termite, Reticulitermes flavipes, when collected from different locations in laboratory raised colonies and at field sites.

Termites were collected from different locations, including food sources, shelter tubes

and the nursery. Termites were dissected and the volume of salivary reservoirs was determined. Salivary reservoirs were always largest from termites collected in food

sources and the nursery. Salivary reservoirs were usually depleted in termites collected

from shelter tubes. Soldiers always had the largest salivary reservoirs, followed by

workers. Alates and nymphs consistently had empty salivary reservoirs. These data

demonstrate that salivary reservoir dimensions differ among stages and worker size;

however this does not appear to be directly linked to task specialization.

3.2 Introduction

Division of labor, polyethism, is a behavioral specialization that can be based on temporal and/or physical features making individual colony members adept at specialized roles within the colony (Oster and Wilson 1978). Through polyethism, efficiency of the colony is greatly increased which is an ecological benefit to eusocial insects.

Polyethism in the Hymenoptera has been well studied (reviewed by: Wilson 1971;

Oster and Wilson 1978; Robinson 1992; Beshers and Fewell 2001). Members of the social Hymenoptera are holometabolous and immature colony members rely on the adults for nutrition and survival. Adult worker honey bees, Apis mellifera, are well known to exhibit age polyethism (Seeley 1982). The younger workers remain in the safety of the nest while tending to the larvae and queen. The older workers do more dangerous tasks such as foraging for food and defending the nest.

In contrast, information on polyethism among the Isoptera is lacking. Termites are hemimetabolous and hence many immature stages are capable of performing colony tasks. Worker age and size are interlinked as workers undergo multiple molts and typically increase with size at each molt. It has been suggested that termites should

exhibit a full discretized caste system (Oster and Wilson 1978). Therefore, workers of the

same age should perform similar tasks.

A review of temporal polyethism in the Isoptera conducted by McMahan (1978)

indicated that older workers of most species conduct tasks related to foraging and nest

39 repair, while young workers remain close to the nest. Furthermore, development of exocrine and endocrine glands is correlated with task activities.

Rosengaus and Traniello (1993) found that temporal polyethism was absent in the dampwood termite, Zootermopsis angusticollis (Termopsidae). The first and second instars were mainly inactive and only allogroomed other nestmates and reproductives.

Instars III-VII performed 64-100% of all the tasks within the colony. Furthermore, no correlation between age and task was found suggesting that age-based polyethism did not occur in this termite species.

Crosland et al. (1997) also found that division of labor in Reticulitermes fukienensis (Rhinotermitidae) did not follow a similar pattern to the Hymenoptera.

Workers of all sizes performed overlapping tasks, but larger workers performed all investigated tasks at a higher frequency. It was hypothesized that the youngest workers

(small workers) did not perform colony tasks because they were too fragile. If these fragile young workers did too much work it may be disadvantageous to the colony. A second hypothesis suggested that young workers may be selfish and it may be easier for them to switch to the reproductive developmental line.

Watson and McMahan (1978) observed polyethism in the Australian harvester termites, Drepanotermes perniger and Drepanotermes rubriceps (Termitidae). Workers of these species have 5 stages/instars and only the oldest workers (instar 4-5) initiate repairs to damaged nests. The younger workers only enter the area after approximately 24 hours when the threat of danger had decreased. Furthermore, these researchers determined that more than 75% of the foragers were in stages 4 and 5.

Food processing is related to age polyethism and caste in some species of higher

termites. In colonies of Macrotermes subhyalinus, the older workers (≥30 days) forage

outside of the nest, while the younger workers remain in the nest tending the fungus

garden and feeding the dependent caste members (Badertscher et al. 1983). Researchers

studying Macrotermes bellicosus obtained similar results with 70% of major workers

foraging for food, while 81% of minor workers were found within the queen cell (Gerber

et al. 1988).

Hinze et al. (2002) also suggested that polyethism was related to food processing

in M. bellicosus. This phenomenon was evaluated by investigating the gut contents of termites from three different areas including foraging sites, the fungus comb and the queen cell. The dry weight and protein content of the salivary glands were also investigated. It was discovered that the mean percent of major workers feeding on primary food (plant litter) was significantly higher than the minor workers in both the fungus comb and the queen cell. Minor and major workers consumed secondary food

(fungus comb) at all three locations, with 100% of termites collected in foraging sites having consumed secondary food. However, an average of 76.2% minor workers consumed secondary food in the queen cell compared to 42.8% major workers. A similar difference was found for termites collected in the fungus comb as 86.2% of minor workers consumed secondary food compared to 64.3% of major workers. Furthermore, the salivary glands of major and minor workers within the queen cell had a significantly higher protein content compared to workers collected from foraging sites. Also, termites

41 in the queen cell that fed on the secondary food source (fungus comb) had salivary glands with significantly higher protein content as compared to termites that fed on the primary food source (plant litter). Termite queens require a high protein diet and it was hypothesized that workers within the queen cell provided extra protein through their saliva.

While salivary glands play an important role in colony tasks, the closely associated salivary reservoirs are typically disregarded. Salivary reservoirs are thought to be important for water storage, but are poorly understood (Watson et al. 1971; Grube and

Rudolph 1999 a,b). Salivary reservoirs are thin-walled translucent sacs, sometimes also called water sacs and are attached to the salivary glands. Salivary reservoirs were overlooked by Hinze et al. (2002) in their studies of M. bellicosus salivary glands. Due to their close association with the salivary glands, I investigated whether the salivary reservoirs play a role in polyethism in the eastern subterranean termite, Reticulitermes flavipes. It was hypothesized that salivary reservoir dimensions would differ among termite castes. Furthermore, salivary reservoirs were expected to be larger in castes performing tasks associated with high moisture (i.e., feeding).

3.3 Materials and methods

3.3.1 Termite collections

R. flavipes were collected from three laboratory colonies and seven field populations in Columbus, Ohio. Each of the laboratory colonies had been established during spring 2002 by pairing a single male and female de-alate. Colonies were ~7 years

42 old and each contained approximately 4000-6000 termites at the time of this study. For

the laboratory colonies, termites were collected from shelter tubes, food sources and

nurseries and they were immediately placed in a -80°C freezer. A nursery was defined as

any area where groups of eggs and 1st and 2nd instar termites, with or without reproductives, were found. For the field populations, termites were collected from various locations including shelter tubes, food sources, nurseries and swarms, and they were immediately placed in a container with dry ice for transport to the laboratory, with subsequent storage in the -80°C freezer.

Termites subsequently were examined using a dissecting microscope as necessary and were categorized into various castes and developmental stages. These included workers (3rd instar or greater), nymphs, soldiers, nymphoid neotenic reproductives and alates. Approximately 5-10 termites of each caste/stage per location (n=338) were randomly selected and dissections were performed with the aid of the microscope to extract the termites’ salivary reservoirs in an isotonic solution.

Auto-Montage Pro software (Synoptics Ltd. Version 5.01, 2004) was used to measure the length and width of the largest of each pair of salivary reservoirs. The

4 volume of the reservoir was then determined using the formula for an ellipsoid, V = /3 x

π x L x W2, as this shape closely resembles that of a salivary reservoir. At the same time, head capsule width was measured for each termite (Roonwal 1969).

3.3.2 Moisture readings

For two field populations, moisture readings were recorded using a non-

destructive moisture meter (Tramex Ltd., Littleton, CO) at as many collection locations

43 as possible. The moisture readings were only recorded for collections that came from

shelter tubes and food sources with active termites.

3.4 Statistical analyses

Statistica was used to conduct all analyses (StatSoft Inc. 2001). An analysis of variance (ANOVA) was performed to test for any significant differences in the salivary reservoir dimensions among collections locations (shelter tubes, food sources, etc) and caste/developmental stage. Tukey’s HSD test for unequal sample size was used to differentiate any significance between collection locations and caste/developmental stage

(alpha level = 0.05). A Mann Whitney U test (alpha level = 0.05) was used to compare moisture levels from two locations (shelter tubes and food sources). Statistica was also used to perform regression analyses to test for any correlation of head width and salivary reservoir dimensions. A regression analysis was also performed to test for any correlation of salivary reservoir volume and moisture level at collection sites.

3.5 Results

3.5.1 Salivary reservoir length and head width

A significant difference in salivary reservoir length was observed among termite stages (F=31.6; df = 4; P< 0.0001) (Fig. 3.1). Soldiers had the longest salivary reservoirs, with an average length of 0.90 ± 0.03 mm (S.E.); their salivary reservoirs were significantly longer than all other stages (P ≤ 0.01). Nymphs, neotenics and alates salivary reservoirs had similar lengths ( x = 0.54 ± 0.04 mm [S.E.]; x = 0.67 ± 0.05 mm

44 [S.E.] and x = 0.51 ± 0.02 mm [S.E.], respectively). Workers had an average salivary reservoir length of 0.66 ± 0.01 mm (S.E.) which was significantly larger than alates

(P<0.0001), but similar to neotenics and nymphs.

Salivary reservoir length and head width were not strongly correlated for any of the stages. For workers, a correlation of r = 0.41 was observed for head width and salivary reservoir length (Fig 3.2). Though this correlation was not very strong, it was evident that as workers’ head sizes increased, the length of their salivary reservoirs tended to increase. The increase in head width and salivary reservoir length most likely represents the different worker instars. These instars include workers of 3rd, 4th and 5th or

greater instars. Workers were subsequently grouped into three sizes: small workers (SW),

medium workers (MW) and large workers (LW) (Fig 3.3).

The salivary reservoir length of these worker groups was significantly different (F

= 10.1; df = 2; P<0.0001). SW had a significantly shorter salivary reservoir length ( x =

0.49 ± 0.04 mm [S.E.]) compared to both MW ( x = 0.67 ± 0.02 mm [S.E.]) and LW ( x

=0.73 ± 0.03 mm [S.E.]) (Fig. 3.4). However, variability in salivary reservoir length was quite evident among the different worker group sizes (Fig 3.2), indicating salivary reservoir dimensions were not the same within groups of similar sized workers.

There was not a strong correlation of head width and salivary reservoir length for nymphs (r = 0.12) or nymphoid neotenics (r = - 0.16). The majority of nymphs had an average salivary reservoir length of 0.54 ± 0.03 mm (S.E.), indicating little variation (Fig.

3.5). The average salivary reservoir length of neotenics salivary reservoirs was 0.68 ±

0.05 mm (S.E.), but some variability among this group was evident. However, the sample

45 size is small since only a total of 12 neotenics were collected, all of which were

nymphoid.

There was little variation in soldier head width, with average of 1.2 ± 0.02 mm

(S.E.), but there was a large amount of variability in soldier salivary reservoir length with

a range of 0.5 to 0.9 mm (Fig. 3.5). The correlation of soldier head width and salivary reservoir length was not strong (r = 0.06).

3.5.2 Salivary reservoir volume

3.5.2.1 Caste and developmental stage

Significant differences were found between termite caste/developmental stage and salivary reservoir volume (F= 22.6; df =4; p <0.0001) (Fig. 3.6). Soldiers had the largest salivary reservoir volume and were significantly different (p ≤ 0.00001) from all

other termites with an average of 0.55 ± 0.05 μl (S.E.). Workers had an average salivary

reservoir volume of 0.23 ± 0.02 μl (S.E.), which was significantly different from soldiers

and alates (P<0.0001), but similar to nymphs and neotenics. Nymphs (0.05 ± 0.06 μl

[S.E.]), neotenics (0.05 ± 0.08 μl [S.E.]) and alates (0.03 ± 0.03 μl [S.E.]) all shared a

similar low salivary reservoir volume.

3.5.2.2 Location of collected termites

Significant differences in salivary reservoir volumes were found depending on

location of the collected termites (excluding swarms) (F= 4.5; df = 2; P=0.01) (Fig.3.7).

The average salivary reservoir volume was smaller for termites in shelter tubes ( x = 0.16

± 0.03 μl [S.E.]) which was significantly different (P=0.037) when compared to termites

46 collected from food sources ( x = 0.29 ± 0.04 μl [S.E.]) and nurseries ( x = 0.30 ± 0.04 μl

[S.E.]).

3.5.2.3 Location and caste/developmental stage of collected termites

No significant interaction was found among termite stages and location based on volume of the salivary reservoirs. The average volume of worker salivary reservoirs was similar on food sources ( x = 0.25 ± 0.04 μl [S.E.]) and nurseries ( x = 0.28 ± 0.05 μl

[S.E.]), but was smallest within shelter tubes ( x = 0.17 ± 0.04 μl [S.E.]), albeit not

significant. Salivary reservoirs of soldiers collected from food sources were usually full

to capacity with an average volume of 0.72 ± 0.1 μl (S.E.), followed by 0.64 ± 0.1 μl

(S.E.) in nurseries and the lowest volume was in the shelter tubes with an average of 0.28

± 0.1 μl (S.E.). Nymphoid neotenics were only collected in shelter tubes ( x = 0.06 ± 0.12

μl [S.E.]) and nurseries ( x = 0.02 ± 0.14 μl [S.E.]) and had consistently small salivary

reservoirs, regardless of the location. Salivary reservoir volumes of nymphs were also

consistently small when collected from food sources ( x = 0.04 ± 0.1 μl [S.E.]), shelter tubes ( x = 0.06 ± 0.1 μl [S.E.]) and nurseries ( x = 0.01 ± 0.23 μl [S.E.]) (Table 3.1).

3.5.2.4 Workers

Salivary reservoir volume was significantly different among workers grouped

according to head width (SW, MW and LW) (F=4.9; df=2; P=0.008) (Figure 3.8). Small

workers had a significantly smaller average salivary reservoir volume ( x = 0.09 ± 0.07 µl

[S.E.]) (P=0.015), followed by medium workers ( x =0.22 ± 0.03 µl [S.E.]) then large

workers ( x =0.34 ± 0.06 µl [S.E.]). Large workers and medium workers had similarly

47 large salivary reservoir volume at all locations (Fig. 3.9). No significant differences were

found among these workers based on collection locations

3.5.2.5 Moisture readings and salivary reservoir volume

The average moisture content of shelter tube sites ( x = 12.73 ± 1.0 % [S.E.]) and

food sites ( x = 23.94 ± 1.0 % [S.E.]) was significantly different (Mann -Whitney U: P ≤

0.0000) (Fig. 3.10). However, the moisture level at collection sites and salivary reservoir

volume were not strongly correlated, though this correlation was slightly higher at food

source sites (r = 0.46) than shelter tubes (r = 0.38) (Fig. 3.11). Nonetheless, when wood moisture levels were quite low (~<5%), salivary reservoirs tended to be depleted, whereas termites in areas with moisture levels of ≥20% had fuller reservoirs.

3.6 Discussion

My investigation provides information on the variability of salivary reservoir

dimensions of R. flavipes castes/developmental stages. There is scant data to suggest that

R. flavipes workers perform discrete tasks based on instar as hypothesized by Oster and

Wilson (1978). However, large and medium workers had the largest salivary reservoirs at

all locations and most likely perform the majority of tasks associated with salivary

reservoir use. These results are consistent with Crosland et al. (1997) where an array of

tasks could be performed by workers of all sizes, but large workers conducted colony tasks more frequently and at a faster rate. In my study, large and medium workers were collected more frequently than small workers. This may be because younger workers tend to be less active than older workers (Crosland et al. 1997) and are less likely to be

48 encountered in areas were termites build, forage and tend to the very early instars and

reproductives.

No evidence of polyethism in salivary reservoir use among the worker stages was

evident in my study. No differences were found in the salivary reservoir dimensions of R.

flavipes workers among the different collection sites. This may indicate that the salivary reservoirs are used only for water storage, as initially suggested by Watson et al. (1971), and therefore do not play a role in providing nutrients. However, termites collected from food sources had large salivary reservoirs. Termites on food sources may benefit from having water reserves inside the reservoirs to dispense onto wood to soften it and keep it moist. Furthermore, moisture levels of food sources may have been sufficiently high

(~20%) for termites to keep salivary reservoirs full. In contrast, termites in shelter tubes often had depleted reservoirs, indicating that shelter tubes may be a desiccating environment and termites have to forage away from the tubes to obtain water (Grube et al. 1997).

R. flavipes alates always had empty salivary reservoirs. This indicates that alates forgo filling their salivary reservoirs until they have flown. If alates had full salivary reservoirs, the extra weight likely would make flight difficult. Watson et al. (1971) observed that Hodotermes mossambicus alates filled their salivary reservoirs after they had flown. Such water likely is used to help maintain the newly established colony during periods of drought (Hewitt et al., 1971). However, Watson et al. (1971) did not determine if the salivary reservoirs were empty prior to flight.

49 Nymphs and neotenic salivary reservoirs were consistently small in volume, regardless of where the collection occurred. This may suggest that these termites do not contribute to colony maintenance, especially as salivary reservoirs are thought to provide moisture for building and food consumption (Grube et al.1999b; Gallagher and Jones

2010).

Soldiers are specialized for defense, and salivary glands are known to produce chemical substances that are used when the colony is under attack (Stuart 1969). Soldiers in my study had salivary reservoirs that were usually full to capacity. Previous research has suggested that soldiers of some species use salivary reservoirs in defense. Mukerji and Raychaudhuri (1943) described the soldier salivary reservoirs of Termes redemanni to be more developed than the workers and to contain a creamy substance. In my study of

R. flavipes, the contents of the soldiers’ salivary reservoirs appeared similar to other stages in that the translucent reservoirs contained a clear liquid with a consistency similar to water and with no obvious odor. R. flavipes soldiers have a cephalic gland that resides close to the salivary reservoirs and holds a yellow liquid that contains sesquiterpenes; however it is not known to be used in defense (Zalkow et al. 1981). R. flavipes soldiers may be using chemicals from the frontal gland for defense (Prestwich 1979).

The function of the salivary reservoirs in R. flavipes soldiers remains unclear.

Soldiers likely contribute to colony maintenance by providing water for workers in shelter tubes as soldiers’ salivary reservoirs were not full to capacity at these collection sites. Su and La Fage (1988) discovered that Coptotermes formosanus workers solicit soldiers for regurgitated food, regardless of the soldiers’ nutritional status. It is possible

50 that workers solicit water from soldiers in the same manner. An additional possibility is

that the water stored in the salivary reservoirs acts as a solvent or carrier for any chemical

produced by the salivary glands, as suggested by Kaib and Ziesmann (1992) for

Schedorhinotermes lamanianus.

In summary, these finding do not suggest that salivary reservoirs are an indicator of polyethism among R. flavipes workers. However, it is likely that older (and larger) workers are more adept at colony functions related to water as they have the largest salivary reservoirs. As salivary reservoirs originate from the ectoderm, the cuticle from the salivary reservoirs must be shed during molting and allow for the reservoirs to increase in size with each progressive molt. However, salivary reservoir dimensions did differ among termite castes and further research is needed to understand if they have any function beyond water storage. However, it is likely that salivary reservoirs of most termite castes and stages of R. flavipes do function in water storage, highlighting the importance of water relations in termite biology. Moisture is a critical factor in termite survival and if this vulnerability can be manipulated via genetics or desiccants it may lead to new forms of termite control.

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53 Watson, J. A. L. and E. A. McMahan. 1978. Polyethism in the Australian harvester termite Drepanotermes (Isoptera: Termitinae). Insects Soc 5: 97-128.

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Table 3.1. Average salivary reservoir volume (μl) (±1.S.E.) of termite castes/stages collected at various locations

Location of collected termites

Termite Food Shelter Nursery caste/stage Tube

Worker 0.25 ± .04 0.17 ± 0.04 0.28 ± 0.05

(n) (62) (70) (46)

Soldier 0.72 ± 0.10 0.28 ± 0.10 0.64 ± 0.11

(n) (11) (10) (8)

Nymph 0.04 ± 0.10 0.06 ± 0.1 0.01 ± 0.23

(n) (10) (11) (2)

Neotenic n/a 0.06 ± 0.12 0.02 ± 0.14

(n) (7) (5)

55 1.00 B 0.95

0.90 N=29 1S.E.)

0.85

0.80

0.75 A, C

0.70 A N=12 0.65 N=178

0.60 A, C 0.55 C N= 23

Salivary Reservoir Length (mm) (+ Length (mm) Reservoir Salivary 0.50 N=95

0.45 Worker Nymph Soldier Neotenic Alate Mean ±SE Termite Caste/Developmental Stage

Figure 3.1. Average length of largest salivary reservoir (mm) (±1 S.E.) for each caste/developmental stage. (All collections combined). Means with the same letter are not significantly different (P>0.05).

56 1.40

1.22

1.12

1.00 0.90

0.80 0.71 0.61

0.51

0.42

0.32

0.20 Salivary Reservoir Length(mm) Reservoir Salivary Small Workers 0.00 Medium Workers 0.70 0.85 0.97 1.06 1.16 1.25 1.40 Large Workers 0.80 0.90 1.01 1.11 1.20 1.30

Head Width (mm)

Figure 3.2. Non-linear relationship between worker salivary reservoir length and head width (mm). R2 = 0.1676; R = 0.4094; y = -0.249431333 + 0.812847569*x

57 120

100

40 Number of Workers Number

20 Small Workers Medium Workers Large Workers 0 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4

Head Width (mm)

Figure 3.3. Distribution of workers based on head width (mm).

58 0.80 A

0.75 N=31 1 S.E.)

0.70 A N=127 0.65

0.60

0.55 B

0.50 N=20 Salivary Reservoir Length ( + (mm) Reservoir Salivary

0.45 LW MW SW Mean ±SE Termite

Figure 3.4. Average length of salivary reservoirs (mm) (±1.S.E.) in large (LW), medium (MW) and small workers (SW). Means with the same letter are not significantly different (P>0.05).

59 1.6

1.4

1.2

1.0

0.8

0.6

Salivary Reservoir Length (mm) Reservoir Salivary 0.4 Nymph Neotenic Soldier 0.2 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 Head Width (mm)

Figure 3.5. Non-linear relationship between salivary reservoir length and head width of nymphs (r2 = 0.0140; r = 0.1183, y = 0.357700761 + 0.169900795*x), nymphoid neotenics (r2 = 0.0285; r = -0.1688, y = 1.573783140 - 0.795487981*x) and soldiers (r2 = 0.0031; r = 0.0558, y = 0.509505803 + 0.331082151*x).

60 0.7 B

0.6

1 S.E.) N=29 0.5

0.4

0.3 A N=178 0.2

0.1

Salivary Reservoir Volume (ul) (+ Volume Reservoir Salivary A,C A,C C N= 23 N=12 N=95 0.0 Worker Nymph Soldier Neotenic Alate Mean ±SE Termite Caste/Developmental Stage

Figure 3.6. Average salivary reservoir volume (μl) (± 1 S.E.) of various termite castes/stages. Means with the same letter are not significantly different (P>0.05).

61 0.36

0.34 A A

0.32

1S.E.) 0.30 N=61 0.28 N=83 l) ( + u 0.26

0.24

0.22

0.20

0.18 B 0.16 N=98 0.14 Salivary Reservoir Volume ( Volume Reservoir Salivary 0.12

0.10 Food Shelter Tube Nursery Mean ±SE Location

Figure 3.7. Average salivary reservoir volume (μl) (± 1 S.E.) of termites collected from various locations. Means with the same letter are not significantly different (P>0.05).

62 0.45 A 0.40

1S.E.) 0.35 N= 31

0.30

0.25 A N= 127 0.20

0.15 B 0.10 N= 20

Salivary Reservoir Volume (ul) ( Volume + Reservoir Salivary 0.05

0.00 LW MW SW Mean ±SE Worker

Figure 3.8. Average salivary reservoir volume (mm) (± 1 S.E.) of large (LW), medium (MW) and small workers (SW). Means with the same letter are not significantly different (P>0.05).

63 0.8

0.7

0.6 1 S.E.)

0.5

0.4

0.3

0.2 Salivary Reservoir Volume (ul) ( + 0.1 Small Workers Medium Workers Large Workers 0.0 Food Shelter Tube Nursery Location

Figure 3.9. Average salivary reservoir volume (µl) (± 1 S.E.) of small, medium and large workers collected from various locations. Within the same location, means were not significantly different (P>0.05).

64 26 B 24 N=38 22 1S.E.)

16 A 14 Moisture Content (%) ( + (%) Content Moisture

12 N=37 10 ST Food Mean Shelter tube Food ±SE Location of Termites

Figure 3.10. Average moisture content (%) (± 1 S.E.) of collection locations (shelter tubes and food sources) where active termites were collected from two field populations. Means with the same letter are not significantly different (P>0.05).

65 2.4756 l) u

1.4507

1.1836

0.9772

0.6696

0.4436

0.2022 Salivary Reservoir Volume ( Volume Reservoir Salivary 0.0024

5193051930

Shelter tube site Food source site Moisture (%)

Figure 3.11. Linear relationship between moisture level of collection site and termite salivary reservoir volume. Shelter tube site: r2 = 0.1419; r = 0.3767; y = -0.0291863785 + 0.0144608977*x. Food source site: r2 = 0.2086; r = 0.4567; y = -0.894144723 + 0.0508741023*x.

Chapter 4

Water Movement into the Salivary Reservoirs and Chemical Analysis of Salivary Glands and Salivary Reservoirs of Reticulitermes flavipes

4.1 Abstract

The eastern subterranean termite, Reticulitermes flavipes, possesses a pair of salivary reservoirs that have been suggested to aid in water storage. However, there is conflicting evidence as how water reaches the salivary reservoirs. The main objective of this study was to investigate the route of water into the salivary reservoirs during a 6-hr period. Dehydrated workers were placed on moist sand and the volume of the salivary reservoirs and crop was determined over a 6-hr period. Crops were the first to fill (~ 80 -

85 % full) after 15 minutes. Crops remained near full capacity (≥75%) for 30 – 60

minutes before decreasing in size. The increase in salivary reservoir volume appeared to

coincide with the decrease in crop volume. Salivary reservoir volumes were more than

50% full by 30-60 minutes and typically increased in size thereafter, though some colony

variation was observed. Furthermore, water used in this study contained blue food

coloring that was observed in the crop, but never in the salivary reservoirs. These data

indicate that water reaches the salivary reservoirs via the hemolymph.

In addition, the contents of the salivary reservoirs (and salivary glands) were

analyzed using gas chromatography and mass spectrometry. These preliminary results

67 indicate that salivary reservoir contents are similar to hemolymph; however additional

analyses are required to determine the presence of amino acids and sugars.

4.2 Introduction

Water relations govern many facets of termite biology (Collins and Richards

1963, Delaplane and La Fage 1989; Sponsler and Appel 1990). A subterranean lifestyle

reduces the amount of contact with the desiccating environment. If the search for food

takes termites above ground, they build shelter tubes that protect them from desiccating

factors as well as predators (Becker 1972; Holway 1941). Termites are also known to

time alate flights according to rainfall (Nutting 1969; Henderson 1996; Neoh and Lee

2009).

The workers of most termite species are highly susceptible to desiccation. The

cuticle of termite workers is lightly sclerotized, making it difficult to retain water. Insects

lose water through their cuticle and from external openings such as the mouth and

spiracles. Water can be obtained metabolically, from food, from drinking, and in some

insects through water vapor absorption (Chapman 1998).

Termites possess a pair of salivary glands, each of which consists of a series of

lobed acini, connected by a branching salivary canal. Also arising from the salivary canal

is a pair of thin walled translucent sacs, the salivary reservoirs (also called water sacs),

which are connected posterior to the salivary glands via a salivary reservoir duct (Noirot

1969; Watson et al. 1971; Grube and Rudolph 1999 a,b). It has been suggested that

salivary glands perform a variety of functions, including digestion, communication, and

68 defense (Reinhard and Kaib 2001). Salivary reservoirs have been suggested to function in

transport of water (Hewitt et al. 1971, Watson et al. 1971, Grube and Ruldolph 1999a).

The route by which water might reach the salivary reservoirs remains unclear as there is conflicting data from different research groups. Watson et al. (1971) traced the

movement of water by allowing Hodotermes mosambicus alates to drink water from

cellulose pads marked with radioactive colloidal gold. The majority of the radioactivity

occurred in the gut and not in the salivary reservoirs, and they hypothesized that water

reached the salivary reservoirs via the gut to hemolymph route. They suggested that water

reached the reservoirs via an ionic gradient and entered the reservoirs either directly or

thru the acini of the salivary glands.

A similar finding was reported for Anacanthotermes ahngerianus with the use of

methyl blue to trace the movement of water after ingestion (Mednikova 1988). The water

in the salivary reservoirs always remained clear, while methyl blue was found in the gut

and Malpighian tubules. It was observed that water first moved into the crop which filled

to capacity in 1 hour. In comparison, the salivary reservoirs remained empty until the

crop had begun to empty, after ~1.5 hr, and were full to capacity 2 hours after the

termites had drunk suggesting that water was transported into the salivary reservoirs from

the hemolymph, possible by an ionic gradient. Sieber and Kokwaro (1982) allowed

major workers of Macrotermes michaelensi to dehydrate for 15 hours and then allowed

termites to imbibe water. It was assumed that capillary forces produced by hairs on the

hypopharynx aided the movement of water into the oral cavity. Imbibed water was stored

in the foregut and then transferred into the salivary reservoirs after approximately 4

69 hours. Sieber and Kokwaro (1982) were in agreement with Watson et al. (1971) that water reached the salivary reservoirs via the gut to hemolymph route.

Grube et al. (1997) findings differed as they reported that Reticulitermes santonensis refilled their salivary reservoirs by oral water uptake wherein water passed

directly from the oral cavity into the reservoir lumen via the reservoir ducts. When

salivary reservoirs were placed in solutions of different osmotic gradients for up to 6

hours, no decrease or increase of reservoir volume was detected, indicating that there was

no net water flow from the reservoir lumen via the reservoir wall. Furthermore, it was

noted that the salivary reservoir and reservoir duct tissues of R. santonensis workers did

not show epithelial structures that would allow ion transport into the reservoir or the

recycling of ions out of the reservoir lumen. It was also found that the ionic concentration

of the salivary reservoir contents was similar to tap water, hence reinforcing their

suggestion that water reached the reservoirs via the reservoir duct and not from the

hemolymph.

Social insects rely on diverse communication systems to perform coordinated

colony tasks. During food exploitation, termites form feeding aggregations due to a

feeding stimulant that is released from the salivary glands and placed onto food via saliva

(Kaib and Zeismann 1992). When extracts from the salivary glands or saliva of

Schedorhinotermes lamanianus workers were placed on filter paper, termites readily

gnawed on the cellulose. However, when extracts from the salivary reservoirs were

placed on filter paper, there was no evidence of termite gnawing. It was suggested that

the contents of the salivary reservoirs are used as a carrier or solvent for the salivary

70 gland products. Furthermore, Kaib and Zeismann (1992) suggested that the salivary reservoirs contents may help humidify the food and make it easier to tear. Although the source of the salivary reservoir contents was not specified, the authors suggested that the salivary reservoirs did not hold the contents of the salivary glands.

My study provides information on water intake in the eastern subterranean termite, Reticulitermes flavipes. In addition, the contents of the salivary reservoirs were investigated using gas chromatography and mass spectrometry.

4.3 Materials and methods

4.3.1Water movement study

4.3.1.1Termites

Three colonies of R. flavipes were used for this study. One colony was a field collection from Paulding Co., OH in 2007 that was maintained in the laboratory thereafter. Colonies A104 and C10 had been established during spring 2002 by pairing a single male and female de-alate from swarm sites in Columbus, OH. All colonies contained approximately 4000-6000 termites at the time of this study.

Workers of the third instar or greater from the three colonies were starved by placing them, individually, on moist sand for 24 hours to ensure that their crops were empty of food. Termites then were placed individually into a cell of a multi-well plate

(Linbro®, Flow Laboratories, McLean, Virginia) without food or water for a 6-hour desiccation period. Termites were maintained at room temperature (~25°C). A relative

71 humidity of 17% was maintained by placing the multiwall plate in a larger plastic

container that held anhydrous CaSO4 (Drierite®, Rochester NY).

In order to access water loss, termites were individually weighed immediately before and after the desiccation period using an analytical scale (Sartorius AG,

Goettingen, Germany). Weighted termites subsequently were placed into another multi-

well plate containing moist sand (QUIKRETE® premium play sand, Atlanta, GA) dyed with blue food coloring (Kroger®, Cincinnati, OH). Termites were kept on the blue moist

sand for 30 minutes and then they were transferred to sand moistened with red food

coloring for the duration study, to visually determine if termites continued to drink.

One group of 4-6 termites from each of the three colonies was removed at each of

fourteen time intervals (0, 15, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330 and 360

mins). Termites were individually weighed and immediately stored in a -80оC freezer

(n=217).

4.3.1.2 Dissections

Termites were placed in an isotonic solution and their salivary reservoirs and crop

were removed with the aid of dissecting microscope (Zeiss Stemi SV II, Oberkochen,

Germany). The approximate volume of the salivary reservoirs and crop was visually

estimated using Auto-Montage Pro software (Synoptics Ltd. Version 5.01, 2004) and a

rated scale of 0-3. A value of ‘0’ was assigned to reservoirs and crops that were less than

5% full (i.e. empty or nearly empty); ‘1’ for >5 % to ≤25% full; ‘2’ for >25% to ≤ 50%;

and ‘3’ for >50% full (Figs. 4.1 and 4.2). The crop appeared deflated when empty (or

nearly so) and took on a spherical shape as it filled. Empty salivary reservoirs also

72 appeared deflated and their walls were deeply enfolded. As the salivary reservoir filled, it took on an ellipsoid shape and the walls appeared smoother. The color of the salivary reservoir contents and crop contents was visually assessed in order to determine whether either organ contained blue or red dye from the moist sand.

4.3.2 Gas chromatography and mass spectrometry -- conducted by USDA-ARS, New

Orleans, LA.

4.3.2.1 Termites

Workers and soldiers from three laboratory colonies of R. flavipes were used.

Colony A104 and Colony A62 had been established during spring 2002 by pairing a single male and female de-alate from swarms in Columbus, OH. Each of these colonies contained approximately 4000-6000 termites at the time of this study. Colony Stinson was established during spring 2005 by pairing a single male and female de-alate from

Columbus, OH and contained approximately 2000-4000 at the time of this study.

4.3.2.2 Gas chromatography and mass spectrometry

Salivary reservoir contents were collected from 10 workers and 10 soldiers from each of the three colonies by piercing the salivary reservoir wall and collecting the contents with a stretched glass micro-pipette (tip diameter <10 µm). Samples were placed in 100 μl of acetone (AR grade) and separated by caste and colony. Salivary glands were also collected from workers and soldiers from each of the three colonies, keeping them separated by caste and colony. An additional set of samples that contained whole salivary reservoirs was collected from 10 workers and 10 soldiers from Colony A104. Samples collected from Colony A104 were placed in plastic vials (due to lack of glass vials at time

73 of collection) and samples from Colony A62 and Stinson were placed in glass vials. The

mulch that housed the termites was also tested to determine if any compounds produced by the termites were de novo. When salivary glands and salivary reservoir samples were collected, termites were dissected in a drop of double-distilled water (HPLC grade).

Controls consisted of acetone (AR grade), double-distilled water and hemolymph collected from the legs of the same termites that were dissected.

Gas chromatography and mass spectrometry (GC-MS) was conducted with an HP

6890 GC and 5973N MS (Agilent Technologies, Santa Clara, CA) equipped with a modular accelerated column heater (MACH) (Gerstel/Agilent) containing a DB-5ms column (20 m x 0.18 mm, 0.18 μm dp). Helium gas was used at 1.0 ml/min constant

flow. The split/splitless inlet was capped with a Merlin Microseal® and heated to 270°C with samples injected under splitless mode. The GC temperature gradient was programmed with a one min hold at 40°C followed by a 50°C/min gradient to 250°C and then a 200°C/min gradient to 320°C with a final 4 min hold. A QuickSwap was installed

between the column and MSD transfer line, with a 4 psi Helium back-pressure. The

MSD transfer line heater was set at 280°C. An electron ionization source was used at 70

eV. The MS source and quad temperatures were 230°C and 150°C, respectively. Spectra

were recorded for m/z 35-450 in fast-scan mode. One microliter of samples was injected

using an HP 6793 autosampler (Agilent). Compounds were identified by comparison to

the Wiley/NIST 2008 library.

74 4.4 Statistical analysis

Statistica was used to conduct all analyses (StatSoft Inc. 2001). A Kruskal-

Wallis ANOVA was conducted to determine if any significant differences were evident among colonies (P<0.05). A Mann Whitney U t-test (P<0.05) was performed to determine any significant differences between termite weight before and after a 6-hour

dehydration period for each colony.

4.5 Results

4.5.1 Movement of food coloring within the termite

Upon contact with the moist sand, the majority of the dehydrated termites immediately placed their heads against the moist sand and began opening and closing their mandibles. Termites kept their head close to the sand and appeared to drink continuously for approximately 2 minutes. When a termite was finished, it pulled its head away with a sharp jerk as if the head was stuck.

Within the first 15 minutes, blue dye was observed in the crop for all termites and also in the midgut of some termites. Some termites did continue to drink once they were moved onto the red sand at the 30-minute interval as this color was observed in the crop of termites at the 60-minute interval and beyond. The blue dye was observed in the hindgut and feces at 240 minutes. Red dye was found in the hindgut of termites at the

330-minute interval, but only for Colony A104. Neither blue nor red dye was observed in the salivary reservoirs. Full reservoirs always appeared to contain a clear liquid.

75 4.5.2 Pathway of water after consumption

Significant differences in water consumption over time were found among the three colonies (P<0.0001), therefore data was graphed and analyzed separately by colony.

Salivary reservoirs and crops of termites were empty or close to empty after the dehydration period, however some variability was evident (Fig. 4.3a-c). A few termites reserved some contents in the salivary reservoirs, but this was uncommon and their reservoirs were not full.

For all colonies, once termites were given access to a water source, the crop was the first to increase in size (Fig. 4.3a-c). After 15 minutes, all termites had crops that were approximately 50% or more full. For Colony C10 (Fig.4.3a), crops remained

≥50% full into the 120-minute interval, but crop volume decreased at all later time intervals, becoming nearly empty or empty at 300 min and thereafter. For Colony A104

(Fig. 4.3b), crop volume decreased to <50% at 90-min and became nearly empty at 150- min. However, at 180-min crop volume increased again to ~40% and fluctuated close to this volume for all later time intervals. For Colony Paulding County (Fig. 4.3c) crop volume was nill at 60-min, but crop volume increased thereafter, reaching ~50% at 180 min and 210 min. Crop volume decreased again thereafter for all remaining time intervals.

A 6-hr dehydration period was not sufficient to ensure that all termite salivary reservoirs were 100% empty at time zero. A few individuals were able to conserve some contents within their salivary reservoirs, with an average volume of 20% for all three colonies (Fig.4.3a-c). However, a longer dehydration period would render termites

76 incapable of a healthy recovery (pers. obs.). Salivary reservoirs began to increase at 30

mins for colonies C10 and A104 (Fig.4.3a,b), while reservoirs began to increase at 60

mins for Paulding Co. (Fig.4.3c). This corresponded closely to when crops began to

decrease in volume. Salivary reservoir volume tended to remain at 50% or greater in

volume throughout the remainder of the study, though the salivary reservoir volume of

termites from Paulding Co. fluctuated more than for the other colonies.

4.5.3 Weight loss and gain

The initial wet weight of termites was significantly different depending on the

colony (Kruskal-Wallis H=111.26; df = 2; P< 0.0001). Colony A104 had the largest

starting body weight with an average of 3.1 ± 0.041mg, n= 78, followed by Colony C10

with an average of 2.5 ± 0.044 mg, n= 67, then Paulding Co., with an average of 2.3 ±

0.042 mg, n= 72.

After the 6-hr dehydration period, which resulted in empty or near empty crops and salivary reservoirs, termites did lose weight. Colony C10 lost the greatest amount of weight (20 ± 0.5%, n= 67) followed by Paulding Co. (17 ± 0.5%, n= 72) then Colony

A104 (15 ± 0.5%, n= 78). While all three colonies increased their weight after having access to water, the weight regained for Colony Paulding Co. (2.1 ± 0.046 mg, n= 66) was significantly less than the starting weight (p=0.000012) (Fig.4.4). Only Colony A104

(3.0 ± 0.040 mg, n= 72) and Colony C10 recovered all of their weight (2.5 ± 0.047 mg, n=62).

77 4.5.4 Gas chromatography and mass spectrometry

Gas chromatography and mass spectrometry analyses identified a variety of peaks in the samples from workers, soldiers and mulch (Appendix A-D). There were no peaks of significant interest in the salivary reservoir samples for workers and soldiers, which may indicate a similar chemical profile to hemolymph. However, evidence of hydrocarbons was found in the salivary glands, salivary reservoirs, and hemolymph of workers and soldiers (Table 4.1). Squalene was found in the salivary reservoirs and salivary glands of workers and soldiers from the colonies Stinson and A62 (Table 4.1). It did appear to be in higher abundance in soldiers (Colony A62); however no external standard was included in the analysis. Furthermore, almost all samples did contain some level of squalene. Evidence of plastic contamination, most likely from the lid of vials for

Colony C10 and Paulding Co., or from the plastic vials used for Colony A104, was found in some of the samples and controls (Table 4.2). The pesticides lambda-cyhalothrin and dichlorobenzene were found in the mulch and termites (Table 4.1).

4.6 Discussion

This investigation confirms the findings of Watson et al. (1971), Mednikova

(1988) and Sieber and Kokwaro (1982) that ingested water travels to the crop and then is transferred to the salivary reservoirs. Sieber and Kokwaro (1982) suggested that water reaches the salivary reservoirs via the foregut, and this may have been the case in this study as salivary reservoirs began increasing in size approximately 30-60 minutes after water ingestion. During this time, the crop typically was full and water was moving into

78 the midgut. Blue dye was not yet observed in the hindgut, indicating that water may not have been transferred into the hemolymph via the Malpighian tubules. In contrast, my data do not support the findings of Grube et al. (1997) who proposed that water reaches the salivary reservoirs via the oral cavity and then to the salivary reservoir ducts.

Gas chromatography and mass spectrometry results are preliminary; however results would seem to indicate that the salivary reservoir contents were similar in composition to hemolymph. Hence, salivary reservoirs may be used for water storage as first suggest by Watson et al. (1971). However, additional analyses are required to determine whether proteins and sugars are present in the reservoirs.

Evidence of hydrocarbons was present in salivary reservoirs, salivary glands, and hemolymph of workers and soldiers. This is to be expected since hydrocarbons produced on the outer cuticle of termites are species-specific and hydrocarbon blends can be unique enough to be used for colony recognition (Howard et al. 1978; Blomquist 1979; Haverty et al. 2005; Nelson et al. 2008).Though unlikely, hydrocarbons present on the outer cuticle may have mixed with the samples during the collection process. However, cuticle is present in the salivary glands and salivary reservoirs, and the hydrocarbons most likely originated from these structures. Furthermore, the salivary glands produce hydrocarbons that may be used in defense, communication and other colony tasks (Noirot 1969). It is not clear if the hydrocarbons found in the salivary reservoirs have a specific function.

Squalene is a hydrocarbon and a triterpene and was first discovered in the liver of sharks (Tsujimoto 1916). It is a precursor to cholesterol and other steroids. Squalene was found in the salivary reservoirs and glands of soldiers and workers from Colony Stinson

79 and A62. The match rate was only 64%, therefore the squalene may be a contaminant, however it was not observed in controls. This compound may lead to production of cholesterol and ultimately ecdysone, which would be necessary for workers to molt, but soldiers are a terminal stage. Termites may also be using squalene to generate defensive secretions (Yoder et al.1993). It may be possible that the glands are producing the squalene and it is being transferred into the salivary reservoirs. However, only a small amount of squalene was found in the salivary reservoirs and glands, and further investigation is needed to determine what role it might play in termite behavior.

Recent research has focused on the importance of aquaporins which are proteins that facilitate the rapid movement of water molecules across cell membranes. They belong to the major intrinsic protein (MIP) family and are found in mammals, amphibians, insects, plants and bacteria (Agre et al. 1993, Chrispeels and Agre 1994,

Campbell et al. 2008, Nishihara et al. 2008). While aquaporins have not yet been shown to be responsible for the transport of water into the salivary reservoirs from the hemolymph, it has been verified that aquaporins are present in the termite Coptotermes formosanus (Kambara et al. 2009). Their study found aquaporins in the midgut, which may contribute to water absorption from food. Aquaporins are also present in the

Malpighian tubules indicating their function in water recycling. These researchers are also the first to report the presence of aquaporins in the foregut, including the salivary reservoirs. This is further indication that water is transported into the salivary reservoirs via the hemolymph route, as suggested by Watson et al. (1971).

80 Termites are one of the most important wood-destroying insects in the United

States, but they cannot survive without moisture. If the salivary reservoirs could be manipulated to prevent termites from transporting water, the termites likely would be unable to forage and consume wood effectively and eventually would die. Researchers

have suggested that RNAi is a potential tool to target and knockdown genes to control

termites (Zhou et al. 2008; Itakura et al. 2009). However, knockdown of AQP genes with

RNAi has not been successful in ticks or aphids. Ixodes ricinus ticks were able to

compensate for the loss of IrAQP1 which is found in the salivary glands (Cambell et al.

2010). Knockdown of ApAQP1 in pea aphids (Acyrthosiphon pisum) disrupted osmoregulation; however it did not affect body weight or mortality (Shakesby et al.

2009).

In conclusion, the contents of the salivary reservoirs appear to be most similar to hemolymph; however additional analyses are required to confirm if it is appropriate to continue calling them “water sacs”, as first suggested by Watson et al. (1971). It also likely that “water” reaches the salivary reservoirs via the hemolymph through aquaporins; research is needed to investigate the possibility of targeting termite aquaporins as a form of control.

References Cited

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82 Grube, S., and D. Rudolph. 1999a. The labial gland reservoirs (water sacs) in Reticulitermes santonensis (Isoptera: Rhinotermitidae): studies of the functional aspects during microclimatic moisture regulation and individual water balance. Sociobiology 33: 307-323.

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83 Nelson, L.J., L.G. Cool, C. W. Solek and M.I. Haverty. 2008. Cuticular hydrocarbons and soldier defense secretions of Reticulitermes in southern California: a critical analysis of the taxonomy of the genus in North America. J Chem Ecol 34:1452– 1475.

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85 Table 4.1. List of compounds tentatively identified by GC-MS in termite salivary glands, salivary reservoirs and hemolymph from three colonies of R. flavipes.

Colony Caste Compound, time Found in Type Match (%)

salivary reservoirs, salivary A62, worker, 1-decene,4.22 min glands and hemolymph Hydrocarbon 93 Stinson soldier

salivary reservoirs, salivary A62, worker, 1-tetradecene, 4.76 min glands and hemolymph Hydrocarbon 97 Stinson soldier

salivary reservoirs, salivary A62, worker, 1-hexadecene, 5.21 min glands and hemolymph Hydrocarbon 95 Stinson soldier

salivary reservoirs, salivary A62, worker, 83 1-octadecene, 5.55 min glands and hemolymph Hydrocarbon 95 Stinson soldier

Salivary reservoirs, worker, salivary glands and A62 1-heptadecene, 5.83 min Hydrocarbon 95 soldier hemolymph

worker, salivary reservoirs, salivary A62 Eicosane, 6.54 min Hydrocarbon 93 soldier glands and hemolymph

Continued Table 4.1 continued

Colony Caste Compound, time Found in Type Match (%)

2,2,4,6,6-

Stinson worker pentamethylheptane, hemolymph Hydrocarbon 86 3.58 min

Stinson worker Tritriacontane, 6.53 min hemolymph Hydrocarbon 93

84 worker, A62 Squalene, 7.07 min salivary reservoir Triterpene 64 soldier

salivary reservoirs, salivary Stinson worker Oleamide, 6.34 and 7.04 Amide 96 and 87 glands and hemolymph

salivary glands and A62, worker, Lambda-cyhalothrin, 6.68 hemolymph Pesticide 93 Stinson soldier min

1,3-dichlororbenzene, hemolymph Pesticide Stinson worker 96 3.79 min

Table 4.2. Contaminants tentatively indentified by GC-MS in termite samples from A104 and Stinson colonies.

Colony Compound, time Type Match (%) Stinson cyclohexasiloxane, dodecamethyl, 4.48 min Plasticizer 83

Stinson cycloheptasiloxane, tetradecamethyl, 4.89 min Plasticizer 91

Stinson siloxane, 5.24 min Plasticizer 50

Stinson cyclononasiloxane, octadecamethyl, 5.49 min Plasticizer 80

Stinson diloxane, 5.70 min Plasticizer 83

A104 7,9 di-tert-butyl-1-oxaspiro[4,5]Deca-6,9 Plasticizer 97 Diene-2,8 Dione, 5.76 min 85

A104 dioctyl hexanediate, 6.35 min Plasticizer 59

a b c d

Figure 4.1. Volume of salivary reservoirs a) <5% full, b) 5-25% full, c) ≥25-≤50% full and d) ≥50% full

a b c d

Figure 4.2. Volume of crop a) <5% full, b) 5-25% full, c) ≥25-≤50% full and d) ≥50% full

120 100 a b

-20 S.E.) 0 30 90 150 210 270 330 0 30 90 150 210 270 330

Colony: C10 Colony: A104 120

100 c

80 Volume (%) (1+ (%) Volume

88 40

-20 Crop Status 0 30 90 150 210 270 330 SR status Colony: Paulding Time (mins)

Figure 4.3. Approximate volume of crop and salivary reservoirs prior to water ingestion and at different times thereafter for termites from (a) Colony C10 (n=62), (b) Colony A104 (n=76), and (c) Colony Paulding (n=68).

3.4

3.2

3.0 S.E.) 2.8

2.6

2.4 89

2.2 * Termite Body Weight (mg) (1+ (mg) Weight Body Termite

2.0

1.8 Starting weight A104 C10 Paulding Final weight Colony

Figure 4.4. Mean body weight of termites (mg) (± 1 S.E.) before and after a 6-hr dehydration period from three colonies (A104, C10 and Paulding). Within each colony observation, * indicates a significant difference between the treatments (P ≤ 0.05, Mann Whitney U test).

Chapter 5

Preliminary Investigation into the Ultrastructure of the Salivary Glands and Salivary Reservoirs of Reticulitermes flavipes Workers (Isoptera: Rhinotermitidae)

5.1 Abstract

Termite salivary glands produce secretions that function in colony communication, defense and digestion. The salivary reservoirs are thought to aid in water storage. Water most likely enters the salivary reservoirs from the hemolymph via water channels in the reservoir wall. However, previous research has suggested that a valve-like mechanism at the junction of the salivary reservoir and the reservoir duct may facilitate water intake and release. An ultrastructural investigation was conducted to determine any possible mechanism involved with water intake and release into the reservoirs. The salivary glands were also investigated as they have not been previously described for

Reticulitermes flavipes. Two types of secretory cells were found in the salivary glands of workers. Type I secretory cells contained electron lucent secretion material, while Type II secretory cells contained electron dense secretion material. Furthermore, Type II could be distinguished into Type IIa and Type IIb, as Type IIa contained material of variable electron density and Type IIb was more uniform in density. Salivary reservoirs were composed of one layer of epithelial cells and were surrounded by an inner layer of cuticle. No valve-like mechanisms involved with water movement were discerned.

Secretory-like cells were found in the reservoir walls and a lipid-like secretion was found in the reservoir lumen. The lipid-like secretion may aid with water movement or retention in the salivary reservoirs. Salivary reservoirs likely function to store water.

5.2 Introduction

Termites possess a pair of salivary or labial glands and salivary reservoirs which share a somewhat similar structure to those found in cockroaches (Noirot 1969). The salivary glands are located in the thorax and consist of a series of lobes, or acini, connected by a branching salivary canal (duct). Each of these salivary glands is connected via a duct to a thin-walled translucent sac, the salivary reservoir, sometimes also called a water sac (Noirot 1969, Watson et al. 1971, Grube et al.1999). The salivary reservoir ducts run parallel to the salivary gland ducts and join together inside the head forming with the hypopharynx.

While the general organization of salivary glands and salivary reservoirs remains uniform among the termites, their development can be different between species and even between castes of the same species (Noirot 1969). Depending on caste and species, the salivary gland acini are capable of producing secretory products containing lipids, glycoproteins and potentially mucopolysaccharides. It has been suggested that salivary glands perform a variety of functions, including digestion, communication, and defense (Reinhard et al. 2004), yet this aspect of termite biology remains poorly understood. It is thought that salivary reservoirs of termites aid in water storage (Watson et al. 1971, Grube et al.1999), though there is conflicting evidence how water reaches the

reservoirs (Watson et al. 1971; Mednikova 1988; Sieber and Kokwaro 1982; Grube et al.

1997). Grube et al. (1997) supports the movement of water from the oral cavity, directly

into the salivary reservoir duct and into their reservoir lumen. In contrast, research on

Reticulitermes flavipes (Gallagher and Jones, unpublished) supports the movement of

water from the hemolymph into the salivary reservoir lumen route via the reservoir wall

as has been proposed by numerous other researchers including Watson et al. (1971),

Mednikova (1988) and Sieber and Kokwaro (1982). The mechanism termites use to

release water from the salivary reservoirs is not understood; however Sutherland and

Chillseyzn (1968) found that cockroaches use specialized salivary reservoir muscles that

act as compressors. It is not yet confirmed if termites use muscles to force water out of

the salivary reservoirs, but it does seem a likely scenario as water is easily ejected from

the reservoirs when pressure is applied during dissection (pers. obs).

Ultrastructural studies typically have focused on the salivary glands of termites

(Billen et al. 1989, Costa-Leonardo and da Cruz-Landim 1991), and the salivary reservoirs are usually overlooked or information is limited in these morphological studies. The only research that concentrated on the ultrastructure of salivary reservoirs includes that of Grube et al. (1997) on the workers of Reticulitermes santonensis

(Rhinotermitidae) and that of Šobotnίk and Weyda (2003) on multiple stages (castes) of the Cuban subterranean termite, Prorhinotermes simplex (Rhinotermitidae). Grube et al.

(1997) did not observe any epithelial structures within the salivary reservoirs or ducts that could be attributed to the mechanism of water uptake in R. santonensis workers. The more recent study by Šobotnίk and Weyda (2003) described the fine structure of the

salivary gland and salivary reservoirs in P. simplex. They observed that the connection of

the salivary reservoir with its duct was made by a purported “taenidia” or a valve-like structure at the neck of the salivary reservoir, but no muscles were evident. This connection was observed only in second instar larvae, pre-soldiers, soldiers and male neotenic reproductives, none of which contribute to colony maintenance. The reservoir duct was immersed into the reservoir in soldiers and male neotenic, potentially acting as a valve-like structure. The junction between the salivary reservoir and the duct appeared simple in the pre-soldier and second instar larvae, a “taenidium” was found at the neck of the reservoir. In addition, no muscles were found associated with the salivary reservoirs.

Termite workers are responsible for conducting the majority of the work within a colony, and they also are the stage that causes damage to wood. Hence, there is a significant gap in the literature as there are no data on the mechanisms involved with water retention and release within termite worker salivary reservoirs. Furthermore, if a duct connection or valve-like structure is present, a thorough description is needed. The objective of this study was to investigate the ultrastructure of the salivary reservoirs and salivary glands of workers of the eastern subterranean termite, R. flavipes, and to reveal possible mechanisms involved with water transport into these reservoirs.

5.3 Materials and methods

5.3.1 Termites

Termites used in this study originated from three laboratory-reared colonies of

R. flavipes maintained at Ohio State University, Entomology Extension at 27oC and 85%

relative humidity (Percival, Intellus Environmental Controller), and then subsequently held at room temperature. Each colony was initiated during spring 2002 with a single

male and female de-alate collected from various field sites throughout Ohio. Each colony

was confined to an enclosed plastic container periodically re-provisioned with wood

mulch, wood pieces, and water. At the time of this study, colonies were 8 years old and contained thousands of individuals and all representative castes, except alates, which

occur in mature colonies and are seasonally produced. Termites typically had full salivary

reservoirs in these conditions (personal observations).

5.3.2 Preparation of samples for transmission electron microscopy (TEM)

Live worker termites were removed from the main colony and immobilized by

placing them in a -80 oC freezer for about thirty seconds. Termites were then placed in

0.1M potassium phosphate buffer (pH 7.4) and viewed using a stereomicroscope (Carl

Zeiss MicroImaging Inc., Thornwood, NY). A subset of the termites was dissected in

order to extract the salivary reservoirs and associated salivary glands using fine forceps.

For others, the tip of both the head and abdomen were removed with a sharp razor blade

to facilitate faster penetration of the fixative and other reagents used for TEM preparation.

Termite tissues were then quickly immersed for fixation in cold 2.5% gluteraldehyde in the buffer for 3 hours at 4°C, washed 3 times in phosphate buffer (30 min. each), post-fixed for 2 hours in 1% OsO4 at 4°C, stained overnight in uranyl acetate

(at 4°C) , dehydrated in an ethanol- acetone series and embedded in Spurr’s medium.

Embedded termite tissues were sectioned serially using an Ultratome II (Leica

microsystems, Wetzlar, Germany). Identification of the salivary glands and reservoirs

was aided by cutting semi-thick (1-2 um) sections between each group of ultrathin

sections (90-99 nm). Semi-thick sections were stained with toluidine blue and examined

by light microscopy. Ultrathin sections were stained with uranyl acetate and lead citrate

and examined in a Tecnai G2 Spirit transmission electron microscope (TEM) (FEI,

Hillboro, OR). Sections from approximately 20 specimens were examined.

5.4 Results

5.4.1 Gross morphology of the salivary glands and salivary reservoirs

The paired salivary glands resemble clusters of grapes as they are composed of multiple lobes (acini). They are more white compared to the translucent salivary

reservoirs (Fig. 5.1). The diameter of the acini ranged from 60μm to 104μm.

The paired salivary reservoirs are ellipsoid in shape and appear as translucent

sacs. The reservoir walls are thin and they can be easily ruptured upon dissection

particularly with pressure on surrounding tissues. The reservoirs are positioned dorsal-

lateral to the foregut (Fig. 5.1). They typically reside in the thorax next to the crop, but

can extend posteriorly into the abdomen. Depending on worker size and fullness of the

reservoir, the range in salivary reservoir length was 0.5-1.0 mm and the range in width

was 0.1- 0.5 mm. When the reservoirs are full, they are completely extended and the

walls appear smooth and highly translucent (Fig. 5.2). When the reservoirs are depleted,

they resemble a deflated balloon, with many folds (Fig. 5.3). The salivary reservoir duct

and the salivary gland duct separate from each other at the base of the head, and run

parallel to each other into the thorax.

5.4.2 Ultrastructure of the salivary glands

Each lobe (acini) of the paired salivary glands is composed of outer parietal cells

and inner secretory cells (Figs. 5.4-5.6). The parietal cells have deeply invaginated basal

plasma membranes, and extensive microvilli surrounding a lumen (Figs. 5.4-5.5). Worker

salivary glands contained two types of secretory cells both with large nuclei, an elaborate endoplasmic reticulum and numerous Golgi apparatus. Type I cells were filled with electron lucent round to irregular shaped secretion material, whereas Type II cells contained more electron dense secretions (Figs. 5.4-5.5). In some cases, Type II cells can be distinguished into IIa and IIb, in which IIa contained secretion material of variable electron density, whereas IIb contain more uniformly dense material (Fig. 5.5). Large intracellular space appeared between acinar cells, most likely representing the acini lumen (Fig. 5.6). Rough endoplasmic reticulum (RER), nuclei, microvilli and Golgi apparatus were present in the cytoplasm of both cell types. Salivary ducts were observed running between acinar cells, and trachea was found intersecting one of these ducts (Fig.

5.7). Ducts were also observed within acinar cells, usually close to the Type I and Type II secretory cells (Fig. 5.8).

5.4.3 The salivary reservoir

The salivary reservoirs are composed of one layer of thin epithelial cells surrounding a cuticle-lined lumen (Figs. 5.9-5.10). The cuticular lining is folded into cuticular ridges which appear “taenidia-like” (Fig. 5.10). The cuticle layer varies in how

close it resides to the epithelium (Fig. 5.9). The cuticular layer of the reservoirs was

approximately 0.05 μm thick and the epithelium 1μm thick. The epithelium consists of uniform flat cells, with elongated nuclei of various sizes, mitochondria and RER.

Smaller, more electron dense cells that seem to have secretory function were also found

in the reservoir walls (Figs. 5.10-5.11). These secretory-like cells contained more

extensive RER as well as smaller lumens surrounded with microvilli (Fig. 5.10), typical

of secretory cells. The reservoir lumen in one sample did contain secretions that appeared

lipid-like (Fig.5.11). Evidence of a valve-like mechanism at the salivary reservoir and reservoir duct juncture was not observed.

5.5 Discussion

These preliminary data are similar to those previously reported (Noirot 1969;

Grube et al. 1997; Šobotnίk and Weyda 2003). Salivary glands of workers had Type I and

Type II cells. The products of these cells are thought to aid in colony tasks such as

digestion, communication and defense (Noroit 1969; Kaib and Ziesmann 1992; Reinhard et al. 1997).

The structure of the salivary reservoirs was similar to that reported by Grube et al.

(1997) and Šobotnίk and Weyda (2003), except I also observed secretory-like cells in the

reservoir walls. Šobotnίk and Weyda (2003) did observe lipid-like secretions and

vacuoles in salivary reservoirs (mostly in workers), but secretion cells were not

described. The function of this lipid-like secretion is not clear. The salivary reservoirs are

thought to function in water storage and the fat secretions may act as protection against

pathogens or to modify the physical properties of water as suggested by Šobotnίk and

Weyda (2003). The fat may ensure the reservoir is hydrophobic and allow all water to empty when needed for colony functions (Šobotnίk and Weyda 2003).

Another possibility is that the secretion in the salivary reservoir may be a

glycerol-like product. Aquaporins are known to transport water across cell membranes

and are present in the salivary reservoirs of termites (Kambara et al. 2009); however

aquaglyceroporins are known to transport glycerol across cell membranes (Agre et al.

2002). Aquaglyceroporins have not yet been identified in termites and glycerol is thought

to function in cryoprotection when insects reside in colder climates (Philip et al. 2008).

The termites used in the current study were not exposed to cold temperatures, therefore if

the secretion is glycerol it may function in preventing water loss. If the secretion in the

salivary reservoir is lipid or glycerol, further investigations are required to elucidate its

function.

Similar to Grube et al. (1997) and Šobotnίk and Weyda (2003) no structures

associated with ion or water transport across cell membranes were observed in the

salivary reservoirs. However, it should not be inferred that water cannot reach the reservoir lumen via the hemolymph route. My research has shown that the hemolymph

route is the most likely scenario (Gallagher and Jones, unpublished). Aquaporins, which are proteins that facilitate the rapid movement of water across membranes, have recently been discovered in the salivary reservoirs of Coptotermes formosanus (Kambara et al.

2009) and further research is needed to confirm that they are responsible for facilitating water movement into the reservoir.

My preliminary investigation did not find any valve-like structure in the salivary reservoir ducts of R. flavipes workers. Workers are the stage responsible for tasks associated with water use, such as adding moisture to food (Gallagher and Jones 2010), and a valve in the reservoir duct may inhibit the flow of water.

Šobotnίk and Weyda (2003) found a valve-like structure only in non-worker stages of P. simplex. They termed the valve-like structure a “taenidium” because of its purported function in providing support for the reservoir duct. Given that the definition of taenidium is “the band or chitinized fibre forming a part of the spiral thread in the trachea of insects” (Torre-Bueno 1978), the term “taenidium” is not warranted when describing the salivary reservoirs. It is likely that the deep enfolding of the dehydrated salivary reservoir wall was mistaken for taenidia, therefore, “cuticular ridges” or “taenidia-like” are more appropriate terms (Grube et al. 1997; Gallagher, unpublished). Hence, the non- worker stages of P. simplex should be re-examined for the presence or absence of a valve-like structure.

The mechanisms involved with water release are still not understood, however it is likely that liquid in the salivary reservoir is forced out by contracting the thorax and abdomen. Sutherland and Chillseyzn (1968) found that cockroaches have specific muscles for emptying their salivary reservoirs, but this has not been confirmed for termites. Therefore it is hypothesized that termites are using muscles residing in the thorax and abdomen to squeeze the salivary reservoirs and force the contents into the oral cavity. Furthermore, the contents of salivary reservoirs can be easily ejected when pressure is applied to them during dissection (pers. obs).

References Cited

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Billen, J., L. Joye and R.H. Leuthold. 1989. Fine structure of the labial gland in Macrotermes bellicosus (Isoptera, Termitidae). Acta Zool. Stockh: 70: 37-45.

Costa-Leonardo, A. M. and C. Cruz-Landim. 1991. Morphology of salivary gland acini in Grigiotermes bequaerti (Isoptera: Termitidae). Entomol Gen 16: 13–21.

Gallagher, N.T., and S.C. Jones. 2010. Moisture augmentation of food items by Reticulitermes flavipes (Isoptera: Rhinotermitidae). Sociobiology. 55: 735-747.

Grube, S., D. Rudolph and I. Zerbst-Boroffka. 1997. Morphology, fine structure, and functional aspects of the labial glands of the subterranean termite Reticulitermes santonensis De Feytaud (Isoptera: Rhinotermitidae). Int J Insect Morphol & Embryol 26: 49-53

Grube, S. and D. Rudolph. 1999. The labial gland reservoirs (water sacs) in Reticulitermes santonensis (Isoptera: Rhinotermitidae): studies of the functional aspects during microclimatic moisture regulation and individual water balance. Sociobiology 33: 307-323.

Kaib, M. and J. Zeismann. 1992. The labial gland in the termite Schedorhinotermes lamanianus (Isoptera: Rhinotermitidae): Morphology and function during communal food exploitation. Insectes Soc 39:373–384.

100

Mednikova, T.K. 1988. The role of salivary gland reservoirs in water exchange in the termite Anacanthotermes ahngerianus Jacobson. Nauchnye doklady vysshei shkoly 6: 32-38.

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Šobotnίk, J., and F, Weyda. 2003. Ultrastructural ontogeny of the labial gland apparatus in termite Prorhinotermes simplex (Isoptera, Rhinotermitidae). Arthro Struct & Dev 31: 255-270.

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101

H SR

Figure 5.1. Dissection of a R. flavipes worker showing the head (H), salivary (labial) glands (SG), salivary reservoirs (SR), crop (C), midgut (MG) and hindgut (HG). Scale bar = 500μm.

102

Figure 5.2. Paired salivary reservoirs near full capacity and highly translucent. Scale bar = 1000um.

103

Figure 5.3. Salivary reservoir depleted and folded. Scale = 1000um.

104

L DL IIb

PC N DC I MV PM II FB

MV IIb PC

N N

Figure 5.4. Ultrastructure of the worker salivary gland, showing secretory cells Types I (I) and IIb (IIb), in addition to parietal cells (PC) with invaginated basal plasma membrane (PM) and extensive microvilli (MV) around their lumen (L) and salivary duct cells (DC) and duct lumen (DL). FB, fat body cells (surrounding the salivary gland,); N, nucleus.

105

L PC RER MV PM

I RER S

IIa IIb

S IIa

Figure 5.5. Higher magnification of the worker salivary gland, showing secretory cells,Types I (I), IIa (IIa) and IIb (IIb), which contain rough endoplasmic reticulum (RER) and secretory material (S) of various electron density. The outer, parietal cells (PC) have highly invaginated basal plasma membrane (PM) and numerous microvilli (MV) around their lumen (L).

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SC SC SD L SD MV

Figure 5.6. An acinus (A) of a worker, containing salivary gland secretory cells (SC) and surrounded by salivary ducts (SD) with microvilli (MV) and duct lumen (L).

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TR T

Figure 5.7. Trachea (TR), lined with tanidea (T), along the outer margins of acini cells (AC). 108

DL MF C

Figure 5.8. Salivary gland duct, lined with cuticle (C), within acini secretory cell Type I (I). DL, duct lumen, MF myelin fiber.

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RL C

E N

Figure 5.9. Ultrastructure of the salivary reservoir of a worker termite; reservoir lumen (RL) lined with cuticle (C) and a thin layer of epithelium (E), N nucleus.

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L SC CR

Figure 5.10. Close up of the salivary reservoir wall showing epithelial cells (EC), their nuclei (N), and secretory-like cells (SC) with their smaller nuclei (N) and microvilli-lined lumen (L), and cuticular ridges (CR).

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Figure 5.11. Secretion material (S) inside the salivary reservoir lumen (RL); SC, secretory-like cell.

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Conclusion

Subterranean termites are one of the most important economic pests in the United

States, yet we do not clearly understand all aspects of their biology. My dissertation was

an attempt to provide new, insightful information on the important aspect of moisture

within the termite colony by investigating their salivary reservoirs, which are also known as ‘water-sacs”. Without water termites quickly succumb to desiccation, therefore it is important that they keep their food and microhabitat moist.

Results from moisture augmentation of food by termites indicate that termites

use their salivary reservoirs to transport water and they increase dry food sources to

≥20% moisture within 3 days. This information is important for the pest management industry, especially as a simple moisture meter would be a vital tool for inspecting

termites. It also emphasizes the importance of educating homeowners on the importance of moisture reduction around the home.

Results from salivary reservoir dimensions among R. flavipes castes/stages

shows that salivary reservoir size is variable; indicating there may be polyethism among

salivary reservoir use. However, while all termite castes/stages possess a pair of salivary reservoirs, they may not all contribute to colony tasks associated with moisture. It is most

likely that the older workers, which have large salivary reservoirs, perform the majority

of tasks within the colony, including moisture movement. The role of salivary reservoirs

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in other castes/stages warrants further investigation as we do not understand the use of

salivary reservoirs in these stages.

Water likely reaches the salivary reservoirs from the hemolymph, entering

through the salivary reservoir wall. Termites may be using muscles in the thorax and/or

abdomen to force water out of the salivary reservoirs and into the oral cavity as TEM results did not show any structure within the reservoir associated with release. It also appears that the salivary reservoirs of workers do store water, and the “water sacs” are an

appropriate name. Gas chromatography and mass spectrometry analysis determined that the chemical profile of salivary reservoir contents of workers was similar to hemolymph.

Soldiers did have a slightly elevated level of squalene in the salivary reservoirs, which may indicate a defensive use. If soldiers are storing defensive products it may be advantageous to have large salivary reservoirs, as seen in my study. However, further analyses are required.

These data provide insight into the salivary reservoirs of R. flavipes termites.

These results suggest that R. flavipes termites use their salivary reservoirs for colony tasks involving water. While this is most likely to moisten food, salivary reservoirs also appeared to be used in shelter tubes and nests as termite salivary reservoir volumes were in various stages of depletion. Salivary reservoirs are an important structure within the termite and the more information we gather on termite biology and colony dynamics, the better equipped we are to control them. Hopefully, this research will provide information and inspire future studies on the “water-sacs” of termites.

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Acetone - control Colony Stinson Colony A62 Colony A104

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Appendix B. Analysis of the salivary reservoirs, salivary glands and hemolymph of termite workers and soldiers of R. flavipes (Colony A62) by mass spectrometry.

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Acetone - control Soldier hemolymph Soldier hemolymph Worker hemolymph Worker hemolymph Soldier salivary glands Worker salivary glands Soldier salivary reservoir reservoir Soldier salivary Worker salivary reservoir reservoir Worker salivary

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Appendix C. Analysis of the salivary reservoirs, salivary glands and hemolymph of termite workers and soldiers of R. flavipes (Colony Stinson) by mass spectrometry. Control was the same used in Colony A62.

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Soldier hemolymph Soldier hemolymph Worker hemolymph Worker hemolymph Soldier salivary gland Worker salivary gland Soldier salivary reservoir reservoir Soldier salivary Worker salivary reservoir reservoir Worker salivary

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Appendix D. Analysis of the salivary reservoirs, salivary glands and hemolymph of termite workers and soldiers of R. flavipes (Colony A104) by mass spectrometry.

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Continued Acetone Water Worker hemolymph Worker salivary reservoir contents contents reservoir Worker salivary Worker salivary reservoir -whole reservoir Worker salivary

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Soldier hemolymph Soldier hemolymph Soldier salivary reservoir -whole reservoir Soldier salivary Soldier salivary reservoir contents reservoir Soldier salivary Appendix D continued

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