Temperature is probably the abiotic factor with the largest single influence on the distribution and abundance of insects (Andrewartha and Birch, 1954).  Predicted changes in climate suggest that many insect species will be exposed to higher median temperatures and extreme temperature events in the future, with consequences for their population dynamics, distribution and evolution (Walters, 2012; Van Velzen et al., 2013). Species involved in obligate mutualisms are likely to be particularly responsive to climate change, because of the strong interdependency between partners  (Kiers et al., 2010; Colwell et al., 2012; Gilman et al., 2012).

 

Fig trees (Ficus, Moraceae) are an example of obligate mutualists that have very wide ecological significance; more species vertebrates feed on their fruits than on those of any other plants (Shanahan et al., 2001). The approximately 800 described species of fig trees are found mainly in tropical and subtropical regions, in habitats ranging from rainforests to deserts (Berg and Corner, 2005).They are all pollinated exclusively by host specific fig wasps (Agaonidae), with each fig tree species dependent on one or a small number of fig wasp species.

 

Adult female fig wasps enter the figs in order to lay their eggs inside the numerous female flowers the inner surface of these specialized inflorescences. A single wasp larva develops inside each of the galled ovules of female flowers, which will usually also have been pollinated. Monoecious fig trees have figs where fig wasp offspring and seeds develop in the same figs. This contrasts with fig trees that have a dioecious breeding system, where there are distinct male and female plants, characterized by having mature figs that contain either the next generation of fig wasps or seeds, respectively. Fig wasps enter female figs, despite being unable to reproduce there, because of reciprocal mimicry displayed by male and female figs (Grafen and Godfray, 1991).  Female fig wasps escape from their natal figs via holes chewed through the fig wall by the males (Suleman et al., 2012). They are small (typically less than 2 mm in length), do not feed, and short lived – most probably die within 24 h of emergence if they fail to find a suitable fig to enter (Kjellberg et al., 2008). Fig wasps lose their wings when entering through the ostiole into a fig, preventing them from dispersing further to other trees.

 

Temperatures control many aspects of the biology of fig wasps. Variations in temperature have direct impacts on the insects and also act indirectly via their host plants. The fruiting and leafing phenologies of fig trees vary considerably between species, but most respond by initiating and maturing fewer crops at colder times of the year. Studies have shown strong seasonal effects on leaf phenology and fig initiation even under controlled conditions (Suleman et al., 2011). The time that fig crops take to reach maturity (and therefore the generation times of the fig wasps) is highly variable among those fig trees that grow in seasonal environments, with some winter crops taking months to complete development that is completed in weeks during the summer (Bronstein and Patel, 1992; Compton, 1993). Also, for some fig trees, more numerous figs have been recorded in summer thanin winter (Suleman et al., 2013).  Emergence times from natal figs differ between diurnal and nocturnal-flying species, and in the day-flying Elisabethiella baijnathi the time when this occurs is linked to temperature, with the wasps emerging earlier in the day during summer months (Ware and Compton, 1994). The minimum temperature at which emergence takes place may be particularly significant, because fig wasps maintained below this critical temperature can develop through to pupation, but then eventually die within their galls (Zavodna and Compton, unpublished).

 

Adult female fig wasps do not eat or drink. Outside of figs, they are rapidly incapacitated when exposed to air temperatures above 30°C (Patiño et al., 1994) and longevity declines above 25°C (Jevanandam et al., 2013). The more rapid deaths of adult female fig wasps in response to higher temperatures are likely to be the result of more rapid dehydration as well as heat stress, though Jevanandam et al. (2013) could not confirm this in their experiments. The darker coloration typical of day flying fig wasps, compared with night-flying species, suggests that they nonetheless display adaptations to reduce water losses (Compton et al., 1991) and among the other insects that inhabit figs, a non-pollinating fig wasp Walkerella sp. (Pteromalidae) has a specialist male morph adapted for mating outside the figs that is darker in color and more resistant to desiccation than typical males (Wang et al., 2010). Although darker fig wasps are not necessarily more resistant to dehydration (Warren et al., 2010), thermal tolerances may nonetheless limit the distributions of some fig wasps to sub-sets of the distributions of their host plants, with the day-flying Ceratosolen galili, a fig wasp that fails to pollinate its host figs, absent from the western desert areas of the range of F. sycomorus, unlike its night-flying congener C. arabicus (Compton et al., 1991; Warren et al., 2010). Differing thermal tolerances may also explain seasonal variation in the relative abundance of the pollinator of F. altissima and an undescribed congener that utilizes the same host plant, but fails to pollinate it (Peng et al., 2010).

 

Fig wasps are clearly highly vulnerable to dehydration when in transit between natal and receptive figs, but once they have found and entered receptive figs they are in a more benign environment. Humidities inside healthy figs will be consistently high, and larger figs also lose heat through transpiration, allowing foundress figs wasps to survive in figs where high ambient temperatures would otherwise quickly cause mortalities (Patiño et al., 1994). However, the survivorship of foundresses within figs at a wide range of temperatures does not necessarily mean that the number of flowers they pollinate, or the number of eggs that they manage to lay, are independent of temperature. Under warmer summer conditions, the pollinators of F. racemosa lay fewer eggs, but generate the same number of seeds as in the winter (Wang et al., 2005). Wang et al. (2009) argued that this results in contrasting summer and winter temperatures stabilising the mutualism between fig trees and fig wasps, because it results in seasonal variation in the balance between seed and pollinator offspring production. They related the performance of the foundresses to temperature and humidity by removing the females shortly after they had entered receptive figs and monitoring survivorship under varying conditions. As with experiments utilising females that had recently emerged from their natal galls, longevity was found to decline with temperature, suggesting a likely cause for the observed seasonal effects.

 

Objectives

 

Here we describe experiments that more directly relate temperature to the longevity, behavior and reproductive success of foundresses within figs maintained at different temperatures. Utilizing a small dioecious fig tree maintained under controlled conditions within three different temperature ranges, we addressed the following questions: Do ambient temperatures influence the behavior and survivorship of adult females after they have entered receptive male figs to oviposit? Do the numbers of their offspring vary according to temperatures at the time when eggs were laid? And are the numbers of seeds in female figs similarly modified by temperature at the time that pollinators are active inside receptive figs?

Natural history

 

Ficusmontana (subgenus Sycidium) is a small dioecious shrub growing up to 2 m tall, with a tropical distribution from Thailand and the Malay Peninsula southwards to Sumatra, Java and Borneo (Berg and Corner, 2005). It grows in rainforest understory, in clearings, and in riparian situations (Tarachai et al., 2012) and is pollinated by Kradibia (=Liporrhopalum) tentacularis Grandi, a daytime-flying fig wasp. The two species had been maintained at the experimental gardens of The University of Leeds, UK since 1995 (Moore, 2001). They originated from the Centre for International Forestry Research (CIFOR) Plantation, Bogor, West Java, Indonesia, and Rakata, Krakatau Islands, Indonesia.

 

The figs of F. montana are produced asynchronously by individual plants of both sexes throughout the year, resulting in figs at different developmental stages often being present at the same time (Suleman et al., 2011). Mature figs on male plants contain pollen and the female pollinator offspring that will transport it, whereas mature figs on female plants produce only seeds. K. tentacularis females actively pollinate the flowers in female figs, even though they cannot lay eggs there (Raja et al., 2008). The female flowers in its female figs have much longer styles and feathery stigmas that help prevent oviposition, but the mechanism preventing seed set in male figs is unclear (Raja et al., 2008). Figs at the receptive (B) stage (Galil and Eisikowitch, 1968) release volatile blends that are attractive to K. tentacularis females.  They squeeze through the ostiolar bracts, losing their wings and part of their antennae in the process (Suleman, 2007).  Once inside a male fig, they deposit a single egg in each galled ovule (Ghana et al., 2012). Larvae that develop in figs that have not been pollinated are less likely to survive (Tarachai et al., 2008). K. tentacularis foundresses often re-emerge from the receptive figs they enter, and walk to receptive figs nearby (Suleman et al., 2013). Offspring sex ratios in K. tentacularis are female-biased, but the extent of this bias depends on clutch size, because females lay mainly male eggs initially, then mainly female eggs (Raja et al., 2008a).

 

Glasshouse conditions

 

We used F. montana growing in three heated glasshouses with contrasting mean and maximum temperatures to provide low, medium and high temperature treatments. Temperatures were monitored during the periods when foundress fig wasps were ovipositing using ‘tiny tag’ data loggers (Meaco measurement and control, Newcastle under Lyme, England) situated within the plants, recording at 30 min intervals. Outside of these experimental periods, the plants and wasps were maintained under medium temperature conditions.

 

Foundress behavior and survival

 

Pre-receptive (A phase) figs on male plants were placed in cotton bags to exclude pollinator females. When the figs were receptive (B phase), single foundresses were placed at their ostioles using a fine paint brush. After the foundresses had entered, the bags were replaced to prevent entry by additional pollinators. The foundresses had emerged the same morning from mature (D phase) figs collected from other male plants that had been placed in netting-covered pots, with the wasps allowed to emerge naturally. The experimental figs were opened 3, 6, 12 or 24 h after foundress entry and we recorded how many of the fig wasps were dead (or moribund), alive and active, or had exited the figs. For each treatment, generally one experimental fig per plant were bagged, but this was not always possible.

 

Reproductive success

 

Figs on male and female plants were bagged and entered by single foundresses, as before. The bags were then returned around the figs to prevent entry of further pollinators, and subsequently to prevent oviposition by non-pollinating fig wasps, that lay their eggs from outside the figs. After 48 h (when all foundresses had died), the plants from the three temperature regimes were all housed together in the medium temperature greenhouse, where they remained for several weeks until the figs had matured. The contents of the figs (total female and male flowers, seeds, failed empty galled ovules (‘bladders’) and pollinator offspring were then recorded.

 

Analysis

 

Numbers of galls, empty galls and unpollinated flowers in male figs, and seed set in female figs, were expressed as proportions of the female flowers present in the figs. Temperature effects were analysed by mixed-effects logistic regression, with temperature included as a continuous explanatory variable. To account for the non-independence of data from flowers in the same figs and figs on the same plant, plant and fig were included as random effects. Three male figs which produced no offspring (1 in each temperature group) were excluded from the analysis. Analysis was carried out in Stata 11.2 (StataCorp, College Station, Texas).

Glasshouse conditions

 

Maximum temperatures during the periods when foundress behavior and survival were monitored inside the figs were 22.7, 27.9 and 34.5°C in the low, medium and high temperature regimes respectively. Temperatures during the 48 h after pollinators were introduced into figs and offspring plus seed production were monitored are summarized in Table 1.

 

 

Foundress behavior and survival

 

No active foundress females remained in the figs 12 h after single individuals had been introduced, irrespective of temperature (Table 2). Some females died within the figs, but the majority re-emerged to seek out oviposition opportunities in other figs. The overall proportion of wasps that were dead or moribund varied from 1 of 32 wasps at medium temperatures to 5 of 32 wasps at low temperatures, but there was no significant variation across temperatures (Fisher’s exact test, p=0.29).

 

 

Reproductive success

 

The proportion of female flowers in male F. montana figs that K. tentacularis females managed to exploit successfully varied between about 40 and 60% in different temperature treatments (Table 3), but there was considerable variation between individual figs within treatments and no significant relationship between the proportion of galls (galls/total flowers) and temperature treatment (Wald c² = 1.77, df = 1, p = 0.18). Empty galls in F. montana figs are likely to be ovules where pollinators laid eggs, but their larvae failed to develop successfully (Ghana et al., 2012). The proportion of unsuccessful galls (empty galls/total flowers) decreased as temperature increased, but not significantly (Wald c²=3.44, df=1, p=0.064). In female figs, where K. tentacularis cannot lay eggs (Table 4), there was no significant relationship between the proportion of seeds (seeds/total flowers) and temperature treatment (Wald c² =0.13, df=1, p=0.72).

 

 

 

Our results narrow down the situations during the life cycle of fig wasps when heat stress is likely to be significant, but do not reduce the likely significance of increasing temperatures for the mutualism. Our experimental temperature range reflected temperatures that F. montana figs are likely to experience, but not extremes of temperature (Fetcher et al., 1985). Even if brief, extreme events when the temperatures inside figs rise above 30°C are highly likely to be detrimental to the fig wasps (Patino et al., 1994). Some fig wasp species are more likely to be subject to heat stress than others, because habitat and the location of figs on plants will influence the temperatures to which female fig wasps will be exposed once they enter the figs. Understory plants, for example, experience less diurnal variation in temperatures than in the rainforest canopy, or plants growing in gaps or forest edges (Bazzaz and Pickett, 1980; Fetcher et al., 1985), and figs located on tree trunks may be less exposed that those located among the leaves.

 

Longer term, persistence of fig tree and fig wasp populations is linked to habitat fragmentation (Mawdsley et al., 1998) and the dispersal abilities of the pollinators (Bronstein et al., 1990). Host-finding among fig wasps is surprisingly effective, and some fig wasps can disperse more than 100 km between natal trees and receptive figs (Ahmed et al., 2009), but the longer that females are in the air, the more susceptible they will be to dehydration and the effects of increased ambient temperatures. Some species of fig wasps disperse during the day, others at night, with fig wasps that disperse at night more common in tropical than sub-tropical/temperate latitudes. Any increases in temperature are more likely to have a negative impact on day-flying species, and night-fliers may even benefit, if low temperatures limit when they can fly.

The authors have no potential conflicts of interest to declare. SGC holds joint appointments at the University of Leeds, UK and Rhodes University, Grahamstown, South Africa.

Authors are grateful to Martin Lappage for invaluable help with maintaining the glasshouse populations of fig trees and fig wasps, and managing the temperatures to which they were exposed.