Oecologia DOI 10.1007/s00442-014-3210-5
BEHAVIORAL ECOLOGY - ORIGINAL RESEARCH
Resource availability as a proxy for terminal investment in a beetle Indrikis A. Krams · Tatjana Krama · Fhionna R. Moore · Markus J. Rantala · Raivo Mänd · Pranas Mierauskas · Marika Mänd
Received: 1 July 2014 / Accepted: 15 December 2014 © Springer-Verlag Berlin Heidelberg 2015
Abstract Terminal investment hypothesis is a longstanding theoretical idea that organisms should increase their reproductive effort as their prospects for survival and reproduction decline. However, numerous attempts to test the terminal investment in reproduction have yielded contradictory results. This study reports an experimental confirmation of the terminal investment hypothesis. It was predicted that immune-challenged yellow mealworm beetles (Tenebrio molitor) are more likely to follow terminal investment strategy when their food resources are limited. Our results suggest the key role of food resources while making decisions to follow a terminal investment strategy. We found that male individuals invested in their sexual attractiveness Communicated by A. Brandl. I. A. Krams · T. Krama Institute of Systematic Biology, University of Daugavpils, Daugavpils, Latvia I. A. Krams (*) · R. Mänd Institute of Ecology and Earth Science, University of Tartu, Vanemuise 46, 51014 Tartu, Estonia e-mail:
[email protected] T. Krama · M. Mänd Department of Plant Protection, Institute of Agricultural and Environmental Sciences, Estonian University of Life Science, Tartu, Estonia F. R. Moore School of Psychology, University of Dundee, Dundee, UK M. J. Rantala Department of Biology, University of Turku, Turku, Finland P. Mierauskas Department of Environment Policy, Mykolas Romeris University, Vilnius, Lithuania
at the expense of immune response and survival when food was not available. In contrast, the beetles did not decrease their lifespan and did not invest in the attractiveness of their sex odours under conditions of food ad libitum. Our results show the importance of food availability and quality in understanding the evolution of reproductive strategies. Keywords Reproduction · Terminal investment · Resources · Sexual attractiveness · Tenebrio molitor
Introduction Key events in an organism’s lifetime are shaped by natural selection to produce the largest possible number of surviving offspring (Stearns 1992; Speakman 2008). Life history theory predicts that reproduction is costly and variations in life history characteristics reflect different allocations of an individual’s resources. However, resource acquisition is limited and so increased resource allocation to one trait is at the expense of other traits requiring the same resource (e.g. Bascunan-Garcia et al. 2010). Many factors can determine the evolution of an organism’s life history, especially under conditions of unpredictable environment containing predators, competitors and pathogens (e.g. Krams et al. 2012; Enriquez-Vara et al. 2012). After being attacked by parasites which substantially decrease future reproductive success of their host, the host individuals may allocate most of their resources in an effort to produce as many offspring as possible. In the context of reproductive costs, Williams (1966) suggested that reproductive investment should increase as the probability for future reproductive success decreases, which was termed ‘terminal investment’ by Clutton-Brock (1984). Although many studies have documented such trade-offs, many findings are
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contradictory even within the same study system (Javoiš 2013). This has led several authors to question the basis for the hypothesis that terminal investment is an important and common reproductive strategy (see Javoiš 2013 for review). Difficulty in revealing terminal investment in many animal species has been attributed to methodological problems associated with measuring reproductive effort (Clutton-Brock 1984). It was also proposed that individuals with low residual reproductive value cannot follow a terminal investment strategy due to physiological constraints (Langley and CluttonBrock 1998; Javoiš and Tammaru 2004; Isaac and Johnson 2005; Sadd et al. 2006; Hoffman et al. 2010; Cotter et al. 2011; Fisher and Blomberg 2011). Some studies showed that resources can be drawn away from reproduction to cover costs of improved immunity (Hanssen 2006; Reaney and Knell 2010; Prokkola et al. 2013), or to self-repair in order to survive parasite attack (Morrow et al. 2003; Uller et al. 2006; Marzal et al. 2008; Schwanz 2008; Kivleniece et al. 2010; Krams et al. 2011a, b; Reaney and Knell 2010; Wilcoxen et al. 2011). Finally, a recent model suggests that, to explain terminal investment, an organism’s reproductive capacity must not be considered as a common pool of resources, but as a set of different resources, which are not easily convertible from one to another, and should be exhausted in balance (Javoiš 2013). Surprisingly, most of the previous studies paid little attention to food availability or predictability while testing the hypothesis of terminal investment, despite the fact that resource limitation is fundamental to a life-history trade-off. It has also been shown that these trade-offs might differ substantially under different conditions of resource availability (de Block and Stoks 2008). Since ad libitum food availability might be crucial while making decisions to invest in reproduction under the risk of parasitism, we tested whether immune-challenged mealworm beetles (Tenebrio molitor) are more likely to follow terminal investment strategy when provided with food ad libitum or when their food resources are limited. We predicted that male mealworm beetles should invest more in their sexual attractiveness, decrease their immune responses and die sooner under conditions of poor food availability. It was also predicted that, under ad libitum food conditions, tradeoffs between costs of reproduction and costs of self-repair mechanisms ensuring survival will be much less frequent.
was maintained. We weighed pupae and sexed these visually by examining genitalia on the eighth abdominal segment (Bhattacharya et al. 1970). The pupae and newly emerged males were kept individually in small, cylindrical, 48-ml, clear plastic containers (height 75 mm, diameter 45 mm), until the end of the experiments. Food availability We weighed 12-day-old males to the nearest 0.1 mg (n = 160; mean body weight ± SD = 105.01 ± 5.00 mg ). The beetles were randomly assigned to two groups: a food-deprived group (n = 80) and ad libitum food group (n = 80) (Fig. 1). At day 15 after imaginal eclosion, we provided only drinking water to individuals in the fooddeprived group, while the food-ad libitum group received 10 g of bran and wheat flour mix and fresh apple in each plastic canister. This was done because availability of food resources may affect survival and reproduction strategies (Javoiš 2013; Metcalfe and Monaghan 2013). The environments of the beetles were similar in terms of humidity. Odour collection and activation of immune system We collected two samples of sex pheromones from each male beetle. To collect the initial sample of male
Materials and methods Beetles The beetles were captured from natural populations in Latvia and reared in plastic containers (0.38 × 0.27 × 0.16 m) containing wheat bran, flour, fresh carrots and apples in the laboratory, where a controlled environment (24 ± 0.5 °C)
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Fig. 1 The experimental protocol used to study sexual attractiveness and survival of male mealworm beetles (Tenebrio molitor) in food-ad libitum and food-deprived groups
Oecologia
pheromones, we placed individual 18-day-old males in a small (Ø 37 mm) closed Petri dish containing a filter paper disc for 9 h (Fig. 1). This represents a reliable method of collecting sex pheromones of T. molitor (e.g. Vainikka et al. 2007; Pölkki et al. 2012). As soon as the odour discs were collected, we divided each group into treatment subgroup (n = 40) and control subgroup (n = 40). In the treatment subgroup, the males received one sterile nylon monofilament implant (2 mm length, 0.18 mm diameter, knotted at one end) through their pleural membrane between the third and fourth abdominal sternite (Rantala et al. 2002; Krams et al. 2011a, b; Daukšte et al. 2012) for 6 h at 22 ± 0.5 °C. Males of the control subgroup were handled similarly but their cuticle was not punctured and these animals were not implanted. We placed the males in individual small plastic containers containing no food, and, as soon as 6 h were over, we removed the implants from males of treatment subgroup (Fig. 1). After the implants were removed, we immediately placed all of the beetles of both groups and all subgroups back to the Petri dishes for another 9 h to collect the second sample of odours to see whether implantation affected the attractiveness of odours (Vainikka et al. 2007; Kivleniece et al. 2010). To avoid any time and agerelated effects on insect immunity and attractiveness, we performed all the above procedures during 24 h (Fig. 1). Survival After the second odour sample collection, the males (n = 40 in each group) were placed back in their plastic containers. All beetles in all the groups were provided with bran and flour mix and with fresh apple, and we checked for their survival at day 23 and at day 85 after imaginal eclosion (Fig. 1). We compared survival rates of the treatment and control males in each group and between groups. Immune assays To quantify the strength of encapsulation response to a foreign body, we analysed the lightness of each nylon insert. It is widely appreciated that insect immune systems respond to the insert as though it were a parasitoid or fungal invasion by attempting to encapsulate the foreign body (e.g. Rantala et al. 2000; Contreras-Garduño et al. 2012; Dubovskiy et al. 2013a, b). The resulting melanisation correlates with the level of immune system response (e.g. Sadd et al. 2006; Krams et al. 2011a, 2013a, b; Rantala et al. 2000, 2002). It has also been found that the ability to encapsulate a synthetic substrate is positively related to the encapsulation of parasites (Paskewitz and Riehle 1994; Gorman et al. 1998) and to the ability to resist an entomopathogenic fungal disease (Rantala and Roff 2007; Dubovskiy et al. 2013a, b). Overall, high levels of melanization or darkening of the inserts
indicates increased levels of immune system activity and response (Yourth et al. 2001, 2002; Krams et al. 2013a, b). To quantify lightness, we photographed each of the removed inserts from two directions under constant light conditions using a Zeiss Lumar V12 Stereo (Carl Zeiss, Jena, Germany) microscope. We then analysed the digital images using image analysis software (Image J; Abramoff et al. 2004). We marked the area of that portion of the insert that had been within the beetle’s body and the program calculated the lightness value. Since increasing melanization indicated a stronger immune response in this study, we calibrated reflectance of an implant before the insertion to zero level. Female preference tests Two tests were performed by using the collected odour discs (Worden et al. 2000; Rantala et al. 2002; Kivleniece et al. 2010): one test before the immune system of males of treatment subgroups was activated and a second test was done immediately following exposure to the filter paper disc (for 9 h) on removal of implants (Fig. 1). During both tests, we tested the attractiveness of male odours in a pairwise design offering the females a pair of odour discs collected from two different males. Odour discs were cut into two pieces. One half was used for comparisons within each of the groups to see possible differences between control and treatment subgroups, while a second piece was used for intergroup comparisons. In this case, we compared odours of control subgroups between the food-ad libitum group and the food-deprived group, and also female choice between odours of treatment male subgroups from the food-ad libitum group and males of the food-deprived group. For the female preference tests, the odour discs collected were presented to 14- to 17-dayold virgin females. The disc halves for the preference tests were prepared simultaneously for each pair. The arena for preference trials consisted of a 9-cm-diameter glass dish inverted over a new filter paper on which the odour samples were placed. Preceding the trial, a stock originated, virgin female was placed under a small (Ø 37 mm) Petri dish in the centre of the circular arena to acclimatize for 5 min. At the start of the trial, the dish that restricted the beetle was removed, and a glass dish was placed over the entire arena. Each trial lasted for 10 min, during which time the beetle’s movements were recorded. Trials were conducted under red light. Preference was measured as the total time that the female spent on each half of filter paper. Statistics The strength of encapsulation response was distributed normally in the both groups (Kolmogorov–Smirnov tests, all
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Fig. 2 The strength of encapsulation response of treatment male mealworm beetles in food-deprived and food-ad libitum groups. The lightness of an implant was calibrated before the insertion to zero level (0: no melanization). Bars mean ± SE. Implant photographs are shown as examples of melanization responses of mealworm beetles in food-deprived and food-ad libitum groups
P > 0.05). Attractiveness of odours was analysed using a nested design analysis of variance. All statistical tests used in this study were two-tailed.
Results Encapsulation response We found a significant heterogeneity in encapsulation response between food-deprived and food-ad libitum groups (one-way ANOVA: F1,78 = 176.84, P
