# WikiJournal Preprints/Moisture Content as a Proximate Factor in Nest Site Attractiveness for Temnothorax rugatulus

Open access • Publication charge free • Public peer review

<meta name='citation_doi' value=>

## Article information

Author: Andrew Z. Colvin[i]

Andrew Z. Colvin, "Moisture Content as a Proximate Factor in Nest Site Attractiveness for Temnothorax rugatulus", WikiJournal preprints, Wikidata Q104711952

## Introduction

Temnothorax rugatulus specimen

April Nobile from AntWeb.org, CC-BY-4.0

Species of the genus Temnothorax are valuable subjects for colony emigration research. Temnothorax rugatulus colonies typically nests in small natural crevices such as hollowed seeds and branches or rock cracks, often comprising less than several hundred individuals (Pratt 2005). When nests are disturbed, colonies are adept at emigrating to new nests (Sasaki et al. 2015). Disturbances arise from a number of proximate factors, many of which likely play a critical role in collective nest site decision making. These factors may include threats such as predation; elemental risks such as wind, water, light, or heat; and conspecific or heterospecific competition. These threats and risks correspond directly to preferences of small nest entrances, small and thin nest cavities, and low light conditions. Other proximate factors may include proximity to food or water sources as a means for survival, or moisture content of a nest site to reduce desiccation, as insects are often susceptible to this condition (Karlik et al., 2016).

These various nest-attractiveness factors may influence a colony's decision to emigrate. It has been shown that T. curvispinosus can collectively compare qualities of nest sites, distinguishing between different proximate factors (Pratt, 2005). Other studies have manipulated proximate factors differentiated by nest cavity size, cavity thickness, entrance size, and light levels in Temnothorax (Mallon & Franks, 2000, Mallon et al., 2001; Pratt & Pierce, 2001; Franks et al., 2003). It has also been shown that Temnothorax will select nests that are closer to food sources (Cao & Dornhaus, 2012); however, little work has been done when considering water.

This paper examines moisture content as a single proximate factor in nest site attractiveness. As water plays a central role in the survival of individuals, a colony must necessitate proximity to water for two reasons: to consume and to minimize desiccation risk. Here, I investigate T. rugatulus' emigration preferences to low-quality nests with differing moisture contents.

## Methods

Three experiments were conducted to test the hypothesis that T. rugatulus prefers nests with high moisture content. The experiments allowed for colonies of T. rugatulus to emigrate to alternate nests of their choice after disturbance or removal of their “home” nest. The experiments were run on different days and structured like the following:

### Subjects and Design

Eleven colonies of T. rugatulus were housed in an artificial nest made of 2.4 mm thick balsa wood slats. Each rectangular nest (50 x 75 mm) contained a 38 mm diameter circular cutout sandwiched between two 50 x 75 mm glass microscope slides. The entrance of the nests were small, 2 mm cut notches. Before and between experiments, the colony’s housings were placed within their “home base” (11 x 11 cm plastic arenas coated in ethylene tetrafluoroethylene to prevent escape). When located in their “home base”, they were provided water and an agar-based protein-rich diet based on Bhatkar & Whitcomb (1970).

The experiments were conducted in 25 x 25 cm plastic arenas, the edges coated with ethylene tetrafluoroethylene to prevent escape. Each arena contained two identical empty nests placed on the left and right sides. These nests consisted of two 2.4 mm think balsa wood slats cut into 50 x 13 mm rectangles that acted as legs to hold a 50 x 75 mm glass microscope slide. Underneath these nests was placed a thin rectangular slice of hydrophilic foam (roughly 7 x 24 mm, with a thickness of 2.4 mm or less).

After the emigration timeframe was completed, photographs were taken off each nest and the number of ants at each nest were counted and recorded (the “home” nest, the wet hydrophilic foam nest, and the dry hydrophilic foam nest). The presence of brood and queens were noted as well.

#### Experiment A: Nest disturbance

Nest disturbance

Two trials of n=10 colonies each were conducted over a 24 hour period to allow for emigration. Ten colonies were placed into the experimental arenas and the top slides of the “home” nests removed. Under each alternate nest choice, one side had wet hydrophilic foam and the other dry. Each arena alternated the position of the wet foam from A (left) to B (right). The lids were placed on top of the arenas and stacked. The stack was then surrounded by a cover with the top and bottom open. This was done to reduce the amount of light that could potentially interfere with nest choice decision, but was not eliminated, as T. rugatulus relies heavily on visual sense for navigation (Bowens et al, 2013). The colonies experienced standard laboratory florescent lighting along with minimal ambient sunlight. The alternating wet foam location was designed to account for potential light gradients, as T. rugatulus will invariably choose nest sites with lower light conditions (Sasaki & Pratt, 2013). For trial one, after 24 hours, the number of ants could be counted at each nest (“home”, wet, and dry) for all ten colonies. For trial two, after 24 hours, the number of ants could be counted at each nest for all ten colonies. Experiencing a disturbed nest, under an equal likelihood probability, T. rugatulus will choose to emigrate to the nest with the highest moisture content, even if it is of low-quality.

#### Experiment B: Nest removal

Nest removal

Under the same conditions, n=9 colonies were forced to choose a nest. This was done by collecting the entire colony and brood with a pooter. The “home” nest was not placed in the arena. The collected colony was extracted from the pooter collector and placed into the arena in the space that would normally house the “home” nest. When forcing a nesting decision (by complete nest removal) to emigrate to either option of two equally low-quality nests, T. rugatulus will choose a nest with high moisture content.

A control to determine statistical significance in nest preference was conducted by replicating the previous experiment except with n=10 colonies and both nest options containing wet foam. The expected outcome here was that T. rugatulus will choose nests A and B with an equal distribution.

### Statistical Analysis

The preference criteria to determine colony nest choice consisted of calculating the percentage of ants located at the wet nest out of the total number of ants present at each nest. For experiment B, the “home” nest was not present, so preference was determined by calculating the percentage of ants present at the wet nest over the total number of ants in the arena. Noting the presence of brood and queens was done to make judgments in cases of splits (where the colony chooses both nests, or even all three nests). A judgment was made when the number of ants at the wet nest over the total was >50%.

Preference criteria were set to determine judgment for purposes of creating a binary “yes or no” wet decision. These preferences were input in a chi square goodness-of-fit contingency table with equal likelihood distributions. To find a statistical preference for experiment A, the chi square was calculated in conjunction with a paired, two-tailed ${\displaystyle t}$-test comparing the number of ants at the wet nest versus the dry nest. For experiment B, and the control, paired ${\displaystyle t}$-tests were used. An additional paired ${\displaystyle t}$-test was calculated for experiment A comparing the number of ants at the wet nest versus the number at the “home” nest. This was done to determine if there was preference for alternative nests entirely, regardless of the presence of moisture.

## Results

A summary of the data expressing the mean number of ants at each nest
Mean number of ants
WET DRY "HOME"
Experiment A 78.9 2.6 99.75
Experiment B 150 3.9 N/A
Control A (WET) B (WET)
47.9 83.9

A chi square goodness-of-fit test was calculated using the binary preference judgments in experiment A. Ten out of twenty colonies chose the wet nests and zero chose the dry nests. The expected values were ten and ten for the wet and dry respectively. This resulted in χ2=10; df=1; ${\displaystyle p}$=0.002. A statistically significant result, in such that, more ants chose the nests with wet foam than with dry foam. Note that ten chose wet and zero chose dry, equalling less than the sample size of twenty. This resulted due to ten colonies not choosing either nest, but preferring to not emigrate at all. This unexpected result (${\displaystyle t}$=0.74962; df=19; ${\displaystyle p}$=0.4581) will be discussed below. A paired ${\displaystyle t}$-test comparing the number of ants that preferred the wet nest versus the dry nest was found to be highly significant (${\displaystyle t}$=4.27649; df=19; ${\displaystyle p}$=0.000123). Experiment B also resulted in a statically significant difference in the number of ants that preferred the wet nest versus the dry nest (${\displaystyle t}$=4.43932; df=8; ${\displaystyle p}$=0.000412). Nine out of nine that chose to emigrate, did so in favor of the wet nest, with one colony not emigrating to any nest at all. The control for experiment B was analyzed with a paired ${\displaystyle t}$-test, finding no significance in preference for nest A or nest B (${\displaystyle t}$=-1.23442; df=9; ${\displaystyle p}$=0.23292).

## Discussion

Experiment A lends support to the prediction, in such that, all of the colonies that chose to emigrate, did so in favor of the wet nest (ten out of ten, with one favoring the dry nests). However, upon completion of both trials, an unexpected result arose. Many of the ants did not chose either the low-quality wet nests or dry nests, but instead remained in their “home” colony despite missing a “roof”. The colonies that remained, were exposed and lacked the typical crevice-like nest they typically prefer (Pratt, 2005). Upon counting the individuals that remained, a ${\displaystyle t}$-test was performed comparing the number of ants present at the wet nests versus the “home” nest (${\displaystyle t}$=0.74962; df=19; ${\displaystyle p}$=0.4581). There was no statistical difference in nest preference for their “home” nest or the wet nest. As discussed previously, the total that chose nests equaled less than the sample size of 20 colonies (in binary, ten chose the wet nest, zero chose the dry nest, and ten chose their original “home” nest). This result suggests that the nest choices may have been of low enough quality to compel T. rugatulus to not emigrate. For this reason, experiment B (and its control) was devised in conjunction, simply to see if, when forced, T. rugatulus would move at all. This contributed to the confidence that the conditions of the arenas, lighting, or other unknown factors were not inhibiting colony emmigration.

Furthermore, this result prompted the scoring of the presence of queens. When identified, all colonies (out of the 20) that did not emigrate to a suitable nest, contained at least one queen. This may support the conclusions of Doering and Pratt (2016), that split colonies of T. rugatulus will often reunite at a nest that contains the queen. In this study, continual observation or recording was not done, so it is possible that in a few cases, during the 24 hour period allowed for emigration, some of the colony emigrated to one of the nests and subsequently emigrated back to the “home” nest due to the presence of the queen.

Experiment B supports the prediction that T. rugatulus, when faced with a forced decision between two low-quality nests, will choose the nest with the highest moisture content. This was supported by both the statistically significant ${\displaystyle p}$-value when comparing the number of ants at each nest and by the fact that 100 percent of colonies that chose to emigrate (eight out of a sample size of nine), emigrated to the nest housing the wet foam. The one colony that did not emigrate, despite the absence of a “home” nest, was a colony of less than 25 individuals, and as such, are granted greater flexibility in nest choice as their nest site can be extremely small. In this particular case, 18 individuals emigrated to the corner of the arena. Furthermore, the control for experiment B supported the prediction of an insignificant ${\displaystyle p}$-value, in such that, colonies did not show a specific preference for either nest A or B—establishing at A, B, both locations (colony split), or no location at all. This result was expected from the control, as both nests were of equal low-quality and both contained wet foams.

These results contrast with Karlik et al. (2016) who showed that the related species T. curvispinosus showed no preference for moisture in tests of colony organization and position within a nest. This also contrasts to the idea by Hood and Tschinkel (1990) that above ground ants are more tolerant to drier conditions than those of soil-living ants (in Karlik et al. 2016). T. rugatulus appears to favor high-moisture nests despite living outside of soil. Furthermore, this suggests that T. rugatulus may have a greater sensitivity to desiccation than previously thought. This may also correspond to the established preferences (in T. albipennis) for highly compacted spaces and small nest entrances, as these factors have been suggested to increase defense abilities, decrease predation, and allow for space to tend to their brood (Franks et al., 2003). In T. rugatulus, this same nest site characterization may act to confer a fitness advantage, as a compact space will naturally increase the humidity within, thereby decreasing the risk of desiccation.

## Supplemental materials

 Colony WET location (Left = L, right = R) Number at WET nest Brood (B) or Queen (Q) Number at DRY nest Brood (B) or Queen (Q) Number at OLD nest Brood (B) or Queen (Q) Percent at WET nest Binary Preference Experiment A (Trial 1) G15 L 102 BQ 0 62 BQ 62.195 WET G6 R 274 BQ 2 55 BQ 82.780 WET G1 L 21 4 160 BQ 11.351 OLD H19 R 2 4 312 BQ 0.629 OLD H6 L 12 4 288 BQ 3.947 OLD G19 R 101 BQ 0 2 98.058 WET G9 L 75 BQ 2 62 BQ 53.957 WET G14 R 3 3 109 BQ 2.609 OLD G20 L 1 0 22 BQ 4.348 OLD H10 R 31 B 2 198 BQ 13.420 OLD Experiment A (Trial 2) H10 L 212 BQ 6 16 90.598 WET H6 R 143 BQ 4 67 B 66.822 WET H19 L 41 B 1 264 BQ 13.399 OLD G7 R 3 3 77 BQ 3.614 OLD G14 L 5 1 107 BQ 4.425 OLD G6 R 161 BQ 7 45 BQ 75.587 WET G9 L 17 6 92 BQ 14.783 OLD G19 R 94 BQ 0 0 100.0 WET G1 L 164 BQ 0 30 B 84.536 WET G15 R 116 BQ 3 27 B 79.452 WET Colony WET location (Left = L, right = R) Number at WET nest Brood (B) or Queen (Q) Number at DRY nest Brood (B) or Queen (Q) Number at relocation site Brood (B) or Queen (Q) Percent at WET nest Binary Preference Experiment B (ants relocated manually) G1 R 126 BQ 1 68 B 64.615 WET G19 L 91 BQ 0 0 100.0 WET G9 R 107 BQ 6 31 B 74.306 WET G7 L 60 BQ 21 B 0 74.074 WET G14 R 245 BQ 0 0 100.0 WET G20 L 1 1 18 BQ 5.0 OLD H19 R 313 BQ 2 1 99.051 WET H6 L 196 BQ 3 0 98.492 WET H10 R 211 BQ 1 1 99.061 WET Control Colony WET location (Left = L, right = R) Number at A Brood (B) or Queen (Q) Number at B Brood (B) or Queen (Q) Number at relocation site Brood (B) or Queen (Q) H10 LR 117 BQ 8 108 B H6 LR 14 151 BQ 68 BQ G7 LR 2 61 BQ 7 H19 LR 4 261 BQ 41 G14 LR 77 BQ 21 BQ 55 BQ G6 LR 13 120 BQ 79 BQ G19 LR 23 BQ 72 BQ 0 G1 LR 128 BQ 33 B 31 B G15 LR 17 98 BQ 28 B G9 LR 84 BQ 14 2

## References

• Bhatkar, A. P. and Whitcomb, W. H. (1970). "Artificial diet for rearing various species of ants". Florida Entomologist 53: 229–232. doi:10.2307/3493193.
• Bowens, S. R., Glatt, D. P., and Pratt, S. C. (2013). "Visual navigation during colony emigration by the ant Temnothorax rugatulus". PLOS One 8 (5): e64367. doi:10.1371/journal.pone.0064367.
• Cao, T. T. and Dornhaus, A. (2012). "Ants use pheromone markings in emigrations to move closer to food-rich areas". Insectes Sociaux 59: 87–92. doi:10.1007/s00040-011-0192-8.
• Doering, G. N., and Pratt, S. C. (2016). "Queen location and nest site preference influence colony reunification by the ant Temnothorax rugatulus". Insectes Sociaux 63: 585-591. doi:10.1007/s00040-016-0503-1.
• Franks, N. R., Mallon, E. B., Bray, H. E., Hamilton, M. J. and Mischler, T. C. (2003). "Strategies for choosing between alternatives with different attributes: exemplified by house-hunting ants". Animal Behavior 65: 215–223. doi:10.1006/anbe.2002.2032.
• Hood, W. G. and Tschinkel, W. R. (1990). "Desiccation resistance in arboreal and terrestrial ants". Physiological Entomology 15: 23–35. doi:10.1111/j.1365-3032.1990.tb00489.x.
• Karlik, J., Epps, M. J., Dunn, R. R., and Penick, C. A. (2016). "Life inside an acorn: how microclimate and microbes influence nest organization in Temnothorax ants". Ethology 122: 790–797. doi:10.1111/eth.12525.
• Mallon, E. B. and Franks, N. R. (2000). "Ants estimate area using Buffon's needle". Proceedings of the Royal Society of London 267B: 765–770. doi:10.1098/rspb.2000.1069.
• Mallon, E. B., Pratt, S. C. and Franks, N. R. (2001). "Individual and collective decision-making during nest site selection by the ant Leptothorax albipennis". Behavioral Ecology and Sociobiology 50: 352–359. doi:10.1007/s002650100377.
• Pratt, S. C. (2005). "Behavioral mechanisms of collective nest-site choice by the ant Temnothorax curvispinosus". Insectes Sociaux 52: 383–392. doi:10.1007/s00040-005-0823-z.
• Pratt, S. C. and Pierce, N. E. (2001). "The cavity-dwelling ant Leptothorax curvispinosus uses nest geometry to discriminate between potential homes". Animal Behavior 62: 281–287. doi:10.1006/anbe.2001.1777.
• Sasaki, T., Colling, B., Sonnenschein, A., Boggess, M. M., and Pratt, S. C. (2015). "Flexibility of collective decision making during house hunting in Temnothorax ants". Behavioral Ecology and Sociobiology 69: 707–714. doi:10.1007/s00265-015-1882-4.
• Sasaki, T. and Pratt, S. C. (2013). "Ants learn to rely on more informative attributes during decision-making". Biology Letters 9: 20130667. doi:10.1098/rsbl.2013.0667.