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PROYECTO DE MAESTRÍA

TÍTULO: “EL ROL DE LAS HORMIGAS TEJEDORAS (OECOPHYLLA SMARAGDINA)

Y LA ESTRUCTURA DE SUS NIDOS EN SU REGULACIÓN MICROCLIMÁTICA”

GIOVANNI M. RAMÓN C.

UNIVERSIDAD JAMES COOK – ESCUELA DE BIOLOGÍA MARINA Y TROPICAL

TOWNSVILLE, QUEENSLAND, AUSTRALIA

2013

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Giovanni Ramón

Student No. 12632085

Minor project report

Word count: 6101

The role of green tree ants (Oecophylla smaragdina) and the structure of

their nests in the microclimatic regulation

Abstract

The climate of a region is fundamental in determining the local species

composition and distribution. On smaller scales, organisms experiment

microclimates different from the bigger scale macroclimate. The environmental

temperature and humidity are important in determining the life history,

distribution and abundance of organisms, including insects. The particularities of

this group make them more prone to respond to changes in these abiotic factors.

Ants, an ecologically important, diverse and widely distributed group of social

insects, are also influenced by temperature and humidity. The capacity of ants to

build nests, however, enables them to be active even when external conditions are

not favorable and it has allowed them to colonize many environments. There has

been a lack of research in tropical canopy-nesting ant species and how they

regulate the temperature and humidity inside their nests. In this study, the

microclimate inside the nests of Oecophylla smaragdina, a conspicuous arboreal

species, and the factors determining it are investigated. We installed data loggers

inside nests of O. smaragdina to record their temperature and humidity. Results

showed that high levels of humidity were maintained, regardless of the external

environmental conditions. The high levels of humidity were caused mainly by the

presence of the ants inside the nests, as confirmed by an experimental

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manipulation. Threshold temperatures, in which development stops, were not

reached although it is possible that these temperatures are reached during the dry

season. With base on our results and previous findings, a mechanism that O.

smaragdina would use to cope with low temperature and humidity is proposed.

Implications of our findings for the ecology of O. smaragdina and their nests are

discussed. Finally, we discuss the implications of our findings for other arboreal

ant species and at a bigger scale.

Introduction

The climate of a given region is fundamental in determining the species

composition and distribution of a given region. However, on a smaller scale

organisms experiment microclimates that can vary widely in time and space

(Kearney and Porter 2009; Sears et al. 2011) and differ from the general

macroclimate (Geiger et al. 2009). Ultimately the life history, distribution and

abundance of species reflect the operation of ecological interactions and metabolic

processes in the context of varying environments, especially temperature and

water availability (Andrewartha and Birch 1954). These two abiotic factors are

also fundamental in determining the abundance and distribution of a diverse

group: insects (Chown and Nicolson 2004), mainly because of two reasons. First,

because they are small they have a high surface area to volume ratio that makes

them more vulnerable than bigger animals to stressful environmental conditions.

They loss and gain heat more readily (Hood and Tschinkel 1990; Chown and

Nicolson 2004). Secondarily, because insects are ectotherms they rely on external

heat sources to perform their basic functions such as locomotion, growth and

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reproduction (Deutsch et al. 2008). Therefore the incorporation of the effects of

these two important factors is essential in the study of the ecology of insects.

Because most insects are poikilothermic ectotherms, they allow their temperature

to vary considerably and are not able to generate their own metabolic heat. Their

activities perform optimally only if their body temperatures fall within a range that

permits the necessary biochemical, developmental, physiological, and behavioral

functions (Harrison et al. 2012). This range is determined by individual age, size,

color, water reserves and other factors (Schowalter 2011) and for most insects

these ranges are narrow. For example, for the fire ant Solenopsis invicta its range

has been determined to go from 22 to 32°C (Porter 1988) outside which colony

development stops or, at extreme temperatures, ants die. Insects must respond,

physiologically or behaviorally, to changes in environmental temperature in order

to stay within their optimal temperature range (Chown and Nicolson 2004).

Because of these, insects are more vulnerable to environmental change in

temperature than bigger or endotherm organisms that can generate their own

heat.

The maintenance of physiologically adequate water levels is also a constant

challenge for small organisms. As noted by Harrison et al. (2012) dehydration can

increase ion concentrations affecting membrane potentials and causing the

denaturation of proteins. Other insects face the opposite problem (e.g. aquatic

insects, sap feeders) of having too much water, in which case blood dilution can

destroy cells, change ion concentration and increase the insects volume and weight

(Benoit and Denlinger 2010). Insects acquire water by drinking, from food and by

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the aerobic respiration and they loss it by excretion, via the cuticle and through the

spiracles (Buckley et al. 2010). As with temperature, insects must maintain

adequate water levels of humidity in their bodies in order to perform their

activities (Chown 2002). For example, a study by Zachariassen (1991) in the

routes of water loss in the beetle Phrynocolus petrosus, that inhabits African dry

savannas, found that cuticular water permeability was considerably lower than

related species that live in more benign habitats. Similar results have been found

for many other insect species (Zachariassen 1996; Klok and Chown 1998; Addo-

Bediako et al. 2001; Chown and Nicolson 2004), showing the importance of

maintaining adequate levels of humidity for this group.

Enclosed in one of the biggest insect orders, Hymenoptera, the family of ants

represents the most diverse and ecologically important group of social insects

(Hölldobler and Wilson 1990; Grimaldi and Engel 2005). With 12500 species

described (Bolton et al. 2007), ants have colonized almost all terrestrial habitats.

Since their first appearance about 140 million years ago (Brady et al. 2006;

Moreau et al. 2006), ants have played important ecological roles as soil turners,

scavengers and predators of many plants and animals. They also engage in a

myriad of symbiotic relationships with other insects, plants and bacteria

(Hölldobler and Wilson 1990; Ness et al. 2010). The understanding of the

adaptations this insects have developed to become such a cosmopolitan group is

essential to understand their biology.

Ants as most insects are poikilothermic ectotherms, although there are a few

species that can generate their own metabolic heat (e.g. Rosengren et al. 1987;

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Franks 1989). Some ant species have managed to adapt to habitats with extreme

temperature conditions. In Western Australia, for example, the ant Melophorus

bagoti ants start foraging when temperatures reach 50°C and can survive

temperatures of up to 70°C (Christian and Morton 1992). On the other extreme of

the spectrum Camponotus obscuripes workers can survive temperatures of -20°C

for short periods of time (Ohyama and Asahina 1972). Most ant species, however,

are comfortable at temperatures ranging from 10 to 40°C (Kaspari 2000) and they

have evolved many adaptations to maintain themselves within their optimal

temperature ranges (Hölldobler and Wilson 1990). Temperature then, is one of the

most important factors limiting the distribution and abundance of ants at large and

small scales.

The maintenance of physiologically adequate water levels is also a constant

challenge for ants. As with temperature, different ant species have different

moisture preferences. For example, a study of carpenter ant queens, Camponotus

modoc and C. vicinus, found that colony founding (the establishment of a new

colony) in one species was less sensitive to wood moisture content, suggesting that

this is better adapted to low moisture conditions (Mankowski and Morrell 2011).

Brood development is also strongly dependent on nest temperature and humidity,

with an ideal range between 20 to 35°C outside which colony growth is inhibited

(Crozier et al. 2010). On the other extreme high air humidity, fog, rain and

waterlogging may limit foraging activities of ants and even destroy their colonies

(Brown 1959; Skinner 1980; Milford 1999; Kaspari 2000; Ballinger et al. 2007).

That is why it is important for ants to keep adequate levels of humidity, not only

when they are foraging for food but also inside their nests.

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The ecological success of ants is principally due to their capacity to build nests that

allow them to be active when other non-social insects need to seek refuge and also

to raise their brood under stable conditions. While foragers are directly affected by

the pre-existing ambient temperature (Malsch et al. 2008) and humidity (Kaspari

and Weiser 2000), ants in the colony can control the microenvironment by

modifying the structure or location of the nest (e.g. digging deeper nests) or by

clustering together to retain metabolic heat and moisture (Hölldobler and Wilson

1990). At high latitudes most ant species nest under the ground and create

networks of underground galleries that allow them to create a gradient of

microclimates (Mikheyev and Tschinkel 2004). While we approach to the tropics

we see that as conditions become milder and more stable more ants species nest

and forage above ground and over the vegetation (Hood and Tschinkel 1990; Ward

2000). Most studies however, have focused on the regulation of temperature and

humidity in ground-nesting high-latitude species (see Jones and Oldroyd 2007 and

references therein) and invasive species (e.g. Hartley and Lester 2003; Xu et al.

2009). More research is needed in tropical canopy-nesting ant species in order to

understand how they cope with changes in temperature and humidity at this

microhabitat in which they reach high densities (Davidson and Patrell-Kim 1996).

The green tree ant, Oecophylla smaragdina, is widely distributed in India, across

tropical Asia, the Solomon Islands and northern Australia (Holldobler 1983). They

are known among entomologists for the conspicuous nests they build at treetops,

by gluing leaves together with larval silk, and by their aggressive behavior (Crozier

et al. 2010; Newey et al. 2010). They form large colonies that are usually spread

among many nests across their territories, can dominate up to 50 to 75% of the

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trees and host more than half a million workers (Lokkers 1990). Lokkers

(1986,1990) proposed that the distribution of this species in Australia is primarily

determined by temperature and humidity and even produced a formula to predict

the likeliness of this species presence at a given site. However, there have not been

studies of the microclimatic conditions inside the nests of this conspicuous species,

or of any other ant species, and the main factors determining it.

In this study I investigate how temperature and humidity vary inside green tree

ant nests, in comparison with the external environment, to determine if the extent

to which green tree ants nests may buffer the colony against changes in

temperature and humidity. I also examine how any microclimate buffering may be

influenced by the elements that comprise the nest itself: the nest walls made of

living leaves, nest walls made of dead leaves and/or the presence of living ants.

Methods

Study Site

The study was conducted at the campus of James Cook University, Townsville,

Australia (19°19’40’’S, 146°45’31’’E; 39m). A total of 52 green ant, Oecophylla

smaragdina, colonies were located at trees and bushes around the university

campus. From these, 36 were randomly selected. A temperature/humidity Ibutton

(model DS1923-F5, Maxim Integrated ™) enclosed by a rubber cover was placed

inside each nest, along with a second Ibutton within 30 cm outside of the nest

opening (Figure 1).

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Thirty-six Ibuttons were deployed to record temperature and relative humidity at

20 minutes intervals from February 25th to March 19th. Because of some

eventualities (e.g. lost Ibuttons on the field, data corrupted in Ibuttons, nests

abandoned by ants during the study period, nests withering and falling off) the

number of effective nests usable for our analysis was reduced to 28 (Active Green

treatment=9, Empty Green=5, Empty Withered=4, Displacement=10, see

explanation of treatments below). This period coincided with the late wet season

(Lokkers 1990).

Nest selection and field study design

To test the whether nests buffered the ants from variations in temperature and

humidity 12 nests with green leaves that contained active colonies were randomly

selected (Active Green nests, n=9) (Fig. 2A). Previous studies have shown that

sometimes these ants abandon their nests and that the leaves that form them keep

the nest shape (Lokkers 1990). Our experimental design took advantage of this

phenomenon. To explore further how much of the variation in temperature and

humidity was explained either by the presence of the ants or the leaves that

formed the nest 6 nests with green leaves but without ants were selected (Empty

Green nests, n=5) (Fig. 2B). 6 nests with withered leaves and without ants as a

control were also selected (Empty withered nests, n=4) (Fig.2C).

To explore how the environmental buffering effect changes within a nest after ants

abandon it and understand better the role of ants in determining the internal

microclimate, an experimental manipulation was performed. Twelve nests were

selected with active colonies and green leaves and were given a treatment after the

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first week of data recording (Displacement nests, n=10) in order to make the ants

abandon them. Many treatments to displace ants were tested initially: inserting

naphthalene pellets inside the nests, spraying the nests with insect repellant,

poking the nests, etc. None of these treatments effectively displaced ants out.

Naphthalene returned mostly mixed results, repellant too, with the disadvantage

that it killed the plants leaves, and poking was the most ineffective of all as it just

triggered a defensive response by ants. Using smoke allowed us to get ants to

evacuate the nest. To produce the smoke 3 to 4 A4 paper printing sheets were

burned inside a small cylindrical metallic container (13*11 cm) and then some dry

vegetation from the ground was added to produce smoke. The nests were poked,

while holding the container about 20 cm away from the nest, to make sure that

ants left. This process went for 4 to 7 minutes in each nest.

To explore the influence of some relevant environmental factors on the variations

of temperature and humidity the length and width of every nest were measured,

across their longest two axis. These two measures were added in order to obtain

an approximation of the nest size. As canopy cover is important in determining the

microclimate in the understory (Marquis et al. 1986) 5 measures of the canopy

cover were taken, one just above the nest and other four two meters apart from

the center of the nest forming a cross with 90° angles. To take these measures a 10

cm. diameter tube with a mirror attached to the bottom and a square grid at the

top, a densiometer, was used. All the measurements were taken by the same

person and at the same height above ground (≈ 1.76 cm).

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Statistical analysis

Because they were taken along time, data points at each nest were not

independent (e.g. they were auto correlated). To avoid this drawback we

calculated the mean temperature and humidity in every nest and comprised all the

data points into a single number (e.g. mean temperature and humidity values for

each nest were calculated). Also, because sometimes data inside data loggers was

corrupted for one of the variables the total number of colonies available was not

the same for each variable (Temperature=24 colonies, AG=7, EG=5, EW=4, D=8;

Humidity=18, AG=3, EG=4, EW=4, D=7). Shapiro tests were performed to

determine if data was normally distributed and used analysis of variances, if data

was normally distributed, or non-parametric Kruskall-Wallis test to determine if

there were differences in temperature and humidity between the treatments and

between the treatments and the outside. Because sample size was unbalanced

between treatments type-III sum of squares were used in the ANOVAs.

Planned post-hoc comparisons were performed on ANOVA results when

significant differences were found. The Fisher least significant differences method

was used to compare treatments with the conditions outside used as a control and

using the ‘comparison-wise’ error type, which gives the narrowest confidence

intervals for the difference between the two means. Displacement colonies were

not included in this analysis.

Maximum and minimum temperature and humidity values were not used, as a

more conservative approach, and to avoid the influence of outliers, we calculated

the 5th and 95th percentiles for each colony and each variable. We used this values

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to test if there were differences in maximum and minimum temperature and

humidity using the same procedure described above.

To verify the changes in the microclimatic conditions inside the displacement

nests, after the ants were forced to abandon them, the mean humidity inside each

nest at the start (from the second to the third day) and at the end of the study

period (from the twentieth to the twenty-second day) were calculated and then a

two-sample equal variances t-Test was performed. Finally, Pearson´s correlation

tests were performed between each colony mean temperature, humidity and

colony size and mean canopy cover, all colonies were included in these analyses.

All analyses were performed on S-Plus 8.2 (TIBCO Software Inc.).

Results

The temperature inside the nests varied from 18.4°C to 45.3°C with a mean of

26.96±3.32°C. Outside nests mean temperature was 26.77±3.02°C with a minimum

of 18.3°C and a maximum of 37°C (Table 1). No differences could be detected in

mean temperature neither between treatments nor between inside and outside

nests (ANOVA, F2,30=1.16; p=0.34). Similar results were found regarding the

minimum temperature (e.g. 5th temperature percentile)(ANOVA, F2,24=1.54;

p=0.23). However, significant differences in maximum temperature (e.g. 95th

temperature percentile) between treatments were found (ANOVA, F2,24=4.04;

p=0.018). Fisher comparisons showed that EW nests had significantly higher

maximum temperatures in comparison to the other treatments and conditions

outside (Fig. 3A).

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Relative humidity inside nests varied from 24.6% to a 100%, with a mean of

81.78±13.08%. Outside nests mean humidity was 78.11±14.02% with a minimum

of 29% and reaching 100% (Table 2). No differences could be detected in

maximum humidity (e.g. 95th humidity percentile) between treatments or between

inside and outside nests (Kruskal-Wallis rank test, X2=3.42, df=3, p=0.33).

However, significant differences in minimum (e.g. 5th humidity percentile)

(ANOVA, F2,24=14.9; p=0.00001) and mean humidity (ANOVA, F2,25=15.14;

p=8.05*10-6) between treatments were found (Figure3B). Both minimum and

mean humidity were significantly higher inside active green nests. To explore how

the differences in humidity varied in each treatment the difference between the

95th and 5th humidity percentiles for each colony was calculated. Significant

differences were found (Kruskal-Wallis rank test, X2=14.32, df=3, p=0.0025, Fig. 4).

From the visual inspection of the temperature and humidity data along time it was

found that temperature inside nests during the day did not differ considerably

from the other treatments, and in the night and noon these differences disappear

completely (Fig. 5A). Additionally, it was observed that active green nests heated

earlier in the morning than the other treatments (Fig. 5A). Temperature tended to

decrease for all treatments as the study progressed (Fig. 5B). Humidity was clearly

higher inside active green nests at every hour of the day (Fig. 5C) when compared

to the other treatments and it also varied less when comparing the cold hours of

the day with the warmer ones. A similar pattern could be seen along all the study

period (Fig 5D).

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In the displacement colonies, the internal microclimate at beginning of the

experiment was similar to that of active green nests and after the smoking

treatment that displaced the ants, conditions inside became similar to those of

empty green nests (t2,34=-3.07, p=0.004, Fig. 6). The average nest length was

26.08±9.27 cm., the average width 19.34±5.64 cm. and the mean canopy cover was

76.62±18.38% for all the 28 colonies measured. A significant (negative)

correlation was found between temperature inside nests and canopy cover

(Pearson’s correlation, r=-0.47, t2,23 =-2.5282, p=0.0019, Fig. 7), no significant

correlation was found between nest humidity and canopy cover (Spearman’s rank

correlation, rs=0.07, z=0.34, p=0.73). No significant correlation was found between

temperature (r=0.24, t(2)23 =1.17, p=0.25) or humidity (r=-0.12, t(2)20 =-0.54,

p=0.59) and nest size.

Discussion

Previous studies by Lokkers (Lokkers 1986,1990) showed that temperature and

humidity are the main factors that determine the distribution of green tree ants.

However, studies of the microclimate inside the nests of this species were lacking.

In this study we found that throughout the wet season the nests of Oecophylla

smaragdina maintain high levels of humidity, regardless of the external

environmental conditions. These high levels of humidity are caused by the

presence of the ants inside the nests, although the transpiration of the leaves

partially contributes to increase the humidity. In contrast, inside empty withered

nests, humidity varies in correspondence to environmental humidity. This shows

that humidity is not only important in determining the big scale distribution of this

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species but that it is also important at the microhabitat scale inside green tree ant

nests.

Not only did ants maintained high levels of humidity inside their nests throughout

the whole period but also the variation in humidity (the difference between

maximum and minimum humidity) was less pronounced than the other

treatments. Although studies on the maintenance of stable humidity conditions are

scarce in comparison to those studying variations in temperature, some have

found similar results. The distribution of the Argentine ant Linepithema humile, for

example, is limited primarily by the soil moisture content (Walters and Mackay

2003) because it builds its nests near the surface and it can not cope with

variations in humidity (Newell and Barber 1913). Neotropical leaf-cutting ants,

which maintain several fungus inside their nests as a food source, need to maintain

an appropriate climate inside their nest to maximize the growth of the fungus

(Farji-Brener 2000). Interestingly a study that investigated the responses of

workers of the leaf-cutting ant Acromyrmex heyeri to the conflicting demands of

temperature and humidity conditions inside their nests found that they traded off

the response related to thermoregulation (i.e. they tolerated higher temperatures)

for the maintenance of appropriate minimum levels of internal nest humidity

necessary for the growth of their fungus and brood (Bollazzi and Roces 2010).

Similar results have been found for Holarctic ants (Banschbach et al. 1997). Our

results, in addition to those from previous studies, show that the maintenance of

high and stable levels of humidity might be also important for ant species that nest

over the vegetation and in the canopy of tropical regions.

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During the sampling period the minimum threshold temperatures were not

reached. Temperature fluctuated from 18.9°C to 41.4°C. Previous studies by

Lokkers (1990) determined that one of the most important factors limiting the

growth of green tree ant colonies is minimum temperature. Specifically, in

laboratory trials he determined the minimum growth temperature of the different

developmental stages and found that while eggs and pupae can withstand

relatively low temperatures (10 and 7°C respectively), larvae would stop growing

at temperatures lower to 17°C. While this temperature was not reached at any

time during our study, it has been observed that during the dry season

temperatures can go below 12°C in this area (Lokkers 1990). Lokkers (1990) also

determined that larvae are produced during all year, so ants can build and repair

the nests; more studies on how larvae are protected from low temperatures during

the dry season are needed although a possible mechanism is discussed in following

paragraphs.

There are several potential explanations for the high levels of humidity recorded

inside green tree ant nests. While the evapotranspiration of the leaves that are

used to build the nest account for a good proportion of the humidity present inside

nests, it was the presence of the ants that significantly increased these levels. The

source of this humidity from ants could be the loss through their respiratory water

loss. It is known that the need to obtain oxygen from the air posses a problem for

insects, as they need to prevent water loss to the usually drier atmosphere (Chown

2002). Studies on North American dessert ant species, adapted to low humidity

conditions, have shown that respiratory transpiration ant cuticular water loss

account only for a small proportion of total water loss (Lighton and Feener Jr

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1989; Lighton 1992; Quinlan and Lighton 1999). However in species like green

tree ants, which have a thinner cuticle and are not adapted to live in low humidity

conditions, respiratory transpiration and cuticular water loss might be more

significant. Not only would workers contribute to increase the humidity but eggs

and larvae would also contribute to increase these levels, mainly because they

have a higher surface area to volume ratio (Jones and Oldroyd 2007). Another

possible source of humidity would be that produced from the respiratory water

loss and cuticular transpiration of the associated insects that live inside green tree

ant nests (see discussion below). Mores studies are needed to identify the most

important sources for the humidity inside green tree ant nests.

It has been observed that the size and density of green tree ant nests vary

seasonally in the wet tropics. During the wet season, when temperature and

humidity are high, green tree ant colonies nests tend to be dispersed in many small

nests across the whole territory. During the dry season on the other hand, when

temperature and humidity are lower, colonies tend to contract into the core areas

of their territories into fewer but bigger nests (Lokkers 1990). Although this

phenomenon might be partially explained by the deciduousness of some trees, it

suggests that during the dry season bigger nests, which would have a lower

surface area to volume ratio, might buffer ants from lower temperature and

humidity. The modification of the nest architecture for microclimatic regulation

has also been observed for Solenopsis invicta (Cassill et al. 2002). The migration of

ants between nests has been observed in ant species of the Palearctic region (Brian

and Brian 1951; Pontin 1960) that relocate their nests into sites with greater

thermal isolation. In the tropics, this movement would also be related to the

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conservation of water in the colder, but also dryer, seasons. However, other factors

like vegetation density and the degree of connectedness of the canopies, limit the

distribution of green tree ants at local scales (Lokkers 1990; Peng et al. 1997). The

study of the interaction of these factors on the distribution and size of the nests of

green tree ants and how they vary across seasons represent a promising field of

study.

A similar behavior has been observed for the sister species of green tree ants: O.

longinoda. In cocoa trees in Zanzibar, nests concentrated in the southern side of

the trees during winter and in the northern side during summer. This migration

was related to the position of the sun. Vanderplank (1960) suggested that this

might help ants to increase insolation levels throughout the seasons, while Way

(1954) proposed that this might also help ants to shelter from the desiccating

prevailing winds. A similar behavior in which workers move larvae and pupa

through the nest in order to rear them under appropriate conditions has been

observed in many ant species (Jones and Oldroyd 2007). Fire ants, Solenopsis

invicta for example, build mounds over their nests with tunnels and galleries inside

(Hölldobler and Wilson 1990). On cool mornings S. invicta ants move their brood

up into the mound to the side receiving the most sunlight. When temperature

exceeds optimal they move their brood inner into the nest (Penick and Tschinkel

2008). For green tree ants it is known that brood is moved from and to different

nests at night (Lokkers 1990), probably to avoid environmental stress. The

movement of brood from one portion of the nest to another to sites with an

adequate microclimate is a widespread behavior in ants, and it is also very likely to

be used by green tree ants.

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Interestingly, it was found that temperatures reached their higher values inside

empty withered nests. Many factors could account for this (e.g. the loss of the

impermeable waxy cuticle, the loss of protective pubescence) but probably the two

most important factors are the complete loss of water from the leaves and the

change into darker colors, that might increase the heat absorbance of the nest

surface (Aston and Van Bavel 1972). Interestingly, even after withering some nests

continued to host major workers. In the field it was also noticed that these nests

might also serve as a refugee for other insects after ants abandon them,

particularly cockroaches (Blattaria). With base on these observations it seems like

even after the nests of green tree ants wither and lost their buffering capacity they

might still provide green tree ants on the periphery of the territory, and probably

other insects, with a refugee from other factors (e.g. predators, parasites).

Lokkers (1990) found that daily activity patterns are correlated to temperature at

the end of the wet season and during the dry season. This circadian pattern tends

to disappear as wet season starts and conditions are more benign. This has also

been observed in other arboreal ant species (Medeiros and Oliveira 2009; Camargo

and Oliveira 2012). Although a significant buffering effect of temperature inside

nests was not found, the activity of foragers outside nests is influenced by

temperature. Foraging activity is reduced by extreme temperatures and reaches a

maximum peak between 28 and 33° (Lokkers 1990), which falls within the range

of temperature recorded inside the nests. The activity of workers inside green tree

ant nests therefore should resemble that outside nests. Our results also showed

that while during at night and dawn temperature inside green active nests is not

different when compared with the outside, during the first hours of the mornings

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active green nests heat faster than empty green or withered nests (Fig. 5A). This

would probably allow ants to start performing their activities earlier although only

studies correlating the activity of ants with the temperatures inside the nests

would allow to verify this.

Non-significant correlations were found for most of the covariables studied. No

significant correlation was found between canopy cover and nest humidity. This

can be explained by the fact that, as previously mentioned, green tree ants prefer

to build their nests under thick vegetation (Peng et al. 1997) where moisture levels

would be expected to be high in comparison to open areas. The lack of significance

between nest size and any of the two environmental variables could be due to the

fact that most of the nests would have been small in size. It is probable that during

the dry season, when nests tend to be bigger (Lokkers 1990), an effect of nest size

over temperature or humidity would be significant. It is also possible, however,

that this is just a sampling artifact due to our sample size that was not big enough

to detect an effect. Future studies must try to include bigger sample sizes to

improve the statistical power and verify these observations.

A significant negative correlation, however, was found between mean canopy

cover and the temperature inside nests. In tropical regions with seasonal climates

it has been generally found that the increase in vegetation cover, with the

consequent reduction in temperature, generally result in a local increase in the

relative abundance of the most common species (Retana and Cerdá 2000). In other

ecosystems, however, increasing habitat complexity and canopy cover may lead to

high diversity and reduced dominance in ants (Perfecto and Snelling 1995;

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Perfecto and Vandermeer 1996). At our study site, however, the threshold

temperature below which the development of green tree ant stops (Lokkers 1990),

was never reached and, therefore, low temperatures would not limit the growth of

this species in the wet season. Moreover, it has been noted that O. smaragdina

prefers sites with a thick vegetation cover (Lokkers 1990). These results however

cannot be interpolated to the dry season when there are lower temperatures that

could potentially limit the growth of this species.

Another interesting field that needs to be researched is how the microclimatic

conditions inside green tree ant nests influence the arthropods associated with

them. It is well known now that ant nests represent environmentally buffered

habitats for many organisms (Hughes et al. 2008). Only for the neotropical Army

ant Eciton burchellii for example, 80 obligate associate organisms have been

described at their temporary nests (Rettenmeyer et al. 2011). Similarly, inside the

complex nests of leaf-cutting ants many organisms have been described; only for

the group of beetles for example 411 species have been found (Navarrete-Heredia

2001). Green tree ants with their woven nests are considered one of the great

achievements of organic evolution and many organisms living in association with

their nests have been described (e.g. salticid spiders, Allan and Elgar 2001; Nelson

et al. 2005; lycaenid butterflies, Saarinen 2006; beetles, Bily et al. 2008; moths and

flies, Leong and D'rozario 2012). More research is needed to determine the

influence of the microclimate inside green tree ant nests and how it influences

these organisms.

22

Green tree ants are also known for establishing mutualisms with sap-sucking

hemipterans (e.g. Blüthgen and Fiedler 2003; Schwartz and Cassis 2003; Crozier et

al. 2010) in which ants protect them in return for honeydew, a carbohydrate-rich

substance. Green tree ants have been found to build leaf nests around these insects

(Pers. Obs.). Besides benefiting from the protection from predators, hemipterans

might benefit from the humid microclimate inside green tree ant nests. Indirect

evidence for this comes from observations in Aphis clerodenroni in Australia. This

aphid is known for producing galls that deform the leaves of its host plant; where

the shoots are galled the leaves close around each other forming an almost

completely concealed refugee (Carver et al. 2003) that to a certain degree

resemble the leaf nests of green tree ants . This aphids may receive some of the

benefits that other hemiptera receive inside nests of green tree ants (e.g. humid

microclimate, protection from predators, etc.). Verifying this observation would

add another function of adaptive value to the already remarkable nests of green

tree ants.

During the field study we notice there were variations in the proportion of larval

silk used to build the nests. The physical properties of the silk of Oecophylla have

been described by Crewe and Thompson (1979); they found that the silk is

designed to withstand the numerous mechanical stresses to which it is subject in

holding the leaves of the nest together. It has also been proposed that the larval

silk may provide an hygienic substrate from which to build nests and that it might

even have antimicrobial properties that benefit weaving ants (Fountain and

Hughes 2011). In this study and on previous studies by Lokkers (1990), it has been

noticed that nests vary in the proportion of larval silk they have and in some cases

23

it can represent a very high proportion of the nest walls. It is possible that this

affects the isolating capacities of green tree ant nests and ultimately their interior

microclimate.

Overall, the results reported here show that high levels of humidity are maintained

inside nests of green tree ants, Oecophylla smaragdina, throughout the wet season.

Our study design allowed us to discriminate between the effects of the structure of

the nest itself (empty withered nests), the nest and the green leaves (empty green

nests) and the combined effects of all the factors plus the presence of the ants

inside (active green nests). Additionally, an experimental manipulation that

displaced ants out of their nests showed that after ants left, conditions inside

became similar to those of empty green nests, with lower levels of humidity.

Therefore, it can be confidently conclude that the high levels of humidity observed

inside nests are caused mostly by the presence and activity of ants inside the nests.

These would be another of the adaptations (Hölldobler and Wilson 1990; Crozier

et al. 2010) that enable green tree ants to rear their brood all year round and

dominate up to 50 to 70% of the trees of a given area, depending on the season

(Lokkers 1990). Not only are nests of green tree ants remarkable for the way in

which they are constructed but also because they represent environmental

‘fortresses’ (Wilson 1968) for their occupants.

The nests of social insects have been described as one of the pinnacles of biological

evolution. Many studies in high latitudes, have shown that nests have an important

function of thermal buffering and that this is the main factor that has allowed

many ant species to colonize those ecosystems (e.g. Farji-Brener 2000; Mikheyev

24

and Tschinkel 2004; Bollazi et al. 2008). In the tropics however, and specially for

arboreal ants, where species live in ecosystems with generally stable temperatures

(Diamond et al. 2012) humidity probably represents a more important factor at a

local scale. The nests of the so-called weaver ants, a complex of ants that includes

green tree ants and species from several genera that use larval silk to build their

nests over vegetation (Hölldobler and Wilson 1990), probably help them to

maintain high levels of humidity to perform their activities. This needs to be tested

for other species and in different seasons especially because arboreal ants have

been identified as one of the most vulnerable groups to changes due to climate

change (Diamond et al. 2012).

Acknowledgements

Simon Robson and Justin Welbergen helped to design this study, analyze the

results and provided valuable comments that helped to improve the quality of this

manuscript. Ben Phillips, Betsy Roznik and David Pike kindly loaned their Ibuttons.

An Australian Research Council Discovery Grant founded this study.

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34

Tables and Figures

Position Treatment Number of

colonies Mean SD Maximum Minimum

Inside Active Green 7 26.97 3.24 39.7 19.9

Empty Green 5 26.78 2.95 37 20.3

Empty Withered 4 27.18 3.51 41.4 18.9

Displacement 8 26.89 3.57 45.3 18.4

Total/Mean 24 26.96 3.32 40.85 19.375

Outside Active Green 7 26.81 3.00 35.6 19.8

Empty Green 5 26.72 2.78 35.5 20.2

Empty Withered 4 26.82 3.13 37 18.6

Displacement 8 26.73 3.18 36.5 18.3

Total/Mean 24 26.77 3.02 36.15 19.225

Grand Total/Mean 24 26.86 3.17 38.50 19.30

Table 1. Summary of temperatures (°C) recorded inside and outside nests across

the different treatments. Underlined numbers represent maximum and minimum

values. SD=Standard deviation.

Position Treatment Number of

colonies Mean SD Maximum Minimum

Inside Active Green 3 88.00 8.70 100 43.1

Empty Green 4 82.21 15.10 100 28.8

Empty Withered 4 80.62 13.16 100 32

Displacement 7 76.31 15.35 100 24.6

Total/Mean 18 81.78 13.08 100 32.1

Outside Active Green 3 79.18 13.73 100 35.8

Empty Green 4 77.83 14.83 100 30.9

Empty Withered 4 78.05 13.37 100 31.4

Displacement 7 77.38 14.15 100 29

Total/Mean 18 78.11 14.02 100 31.8

Grand Total/Mean 18 79.76 14.37 100 24.6

Table 2. Summary of the relative humidity (%) recorded inside and outside nests

across the different treatments. Underlined numbers represent maximum and

minimum values. SD=Standard deviation.

35

Figure 1. Picture of an active green nest on the field, the square shows the Ibutton

used to record temperature and humidity (A). Detail of the Ibutton inside the nest.

Notice how ants incorporated it into their nest (B). Picture of an Ibutton inside the

protective cases. Notice the plastic mesh used to protect the surface of the Ibutton

(C).

36

Figure 2. Photographs of the nests used for the study. Active Green nests were

defined as those formed by green leaves and occupied by ants (A). Empty green

nests were formed by green leaves but contained no ants (B) and empty withered

nests were those with withered brown leaves and containing no ants (C).

37

Figure 3. Boxplots showing differences between treatments and conditions

outside. Maximum temperature, calculated as the 95th percentile, was higher inside

empty withered nests (ANOVA, F2,24=4.04; p=0.018) (Sample size=16 colonies,

AG=7, EG=5, EW=4) (A). Relative humidity was higher inside active green nests

(ANOVA, F2,25=15.14; p=8.05*10-6) (Sample size=11 colonies, AG=3, EG=4, EW=4)

38

(B). Letters over the boxplots (a,b) represent groupings of significantly different

treatments as revealed by Fisher LSD comparisons.

Figure 4. Maximum (95th percentile) minus minimum (5th percentile) relative

humidity differences for each treatment. A Kruskal-Wallis test found significant

differences between treatments (Kruskal-Wallis rank test, X2=14.32, df=3,

p=0.0025).

39

Figure 5. Mean temperature and relative humidity in the different treatments across each hour of the day (A and C), and across each day

of the study period (B and D).

40

41

Figure 6. Mean humidity for each treatment inside nests at the start and at the end

of the study period (A). Mean relative humidity data for active green, empty green

and displacement treatments across the study period. Arrow signals the date of

nests smoking (B). A two-sample equal-variances t-test revealed significant

differences in humidity at the start of the study period in comparison with

humidity at the end (t2,34=-3.07, p=0.004, Fig. 6). After displacement nests were

smoked relative humidity became similar to that in empty green nests.

Figure 7. Scatter plot showing the correlation between temperature inside nests

and canopy cover (r=-0.47, t2,23 =-2.5282, p=0.019).