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Micronutrient consumption by female Argiope bruennichi affects offspring survival
Journal of Insect Physiology 100 (2017) 128–132
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Micronutrient consumption by female Argiope bruennichi affects offspring survival
MARK
⁎
Shawn M. Wildera,b, , Jutta M. Schneiderc a b c
School of Biological Sciences and Charles Perkins Centre, University of Sydney, Sydney, Australia Department of Integrative Biology, Oklahoma State University, OK, USA Department of Biology, Zoological Institute and Museum, University of Hamburg, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords: Micronutrients Survival Spider Argiope bruennichi
Sexual cannibalism has long been hypothesized to be a foraging decision in which females consume males for the nutrients in their bodies. While few studies have documented fecundity benefits of sexual cannibalism, several recent studies have documented benefits of cannibalism to egg hatching success or offspring survival. We tested if small supplements of dietary essential nutrients fed to female spiders, Argiope bruennichi, would result in increases in offspring survival similar to those seen following sexual cannibalism. All female spiders were prevented from cannibalizing their mates and subsequently fed either: a dead male spider, or a similarly-sized dead fly with one of four nutrient supplements (water control, dietary essential fatty acids, dietary essential amino acids, or nonessential amino and fatty acids). Females that consumed a small supplement of dietary essential amino acids produced offspring that survived simulated overwintering conditions significantly longer than offspring of other treatments. While a previous study found a significant effect of cannibalism on offspring survival using field-collected males as prey, the current study, which used lab-reared males as prey, found no effect of sexual cannibalism on offspring survival. Hence, our results suggest that dietary essential amino acids, which may be sequestered by males from their diet, could be valuable supplements that increase the success of the offspring of cannibalistic females. Further work is needed to determine the source and identity of these dietary essential amino acids and if other essential nutrients (e.g., trace elements, vitamins, etc.) may also be limiting in female diets and affect offspring success.
1. Introduction Sexual cannibalism, when females consume males in the context of mating, is widespread among spiders and occurs in a range of other invertebrates (Elgar and Crespi, 1992; Elgar and Schneider, 2004). This behavior is often described as a form of sexual conflict because it provides the female with a meal at the expense of future mating opportunities of the male (Schneider, 2014). However, this cost to the male is reduced in species with male monogyny or where consumption of the male body increases female fecundity (Fromhage et al., 2005; Barry et al., 2008). Many hypotheses have been proposed for the occurrence of sexual cannibalism including male sacrifice, spillover or genetic constraints on aggression, female choice, female control of copulation duration or paternity, mistaken identity, and female foraging (Elgar, 1992; Andrade, 1996; Arnqvist and Henriksson, 1997; Johnson, 2001; Schneider, 2014). Of the explanations for sexual cannibalism, the female foraging hypothesis has received the most empirical attention (Wilder et al.,
⁎
2009). Female foraging is one of the most parsimonious explanations for sexual cannibalism because many species of spiders are aggressive, generalist predators and because killing of males is almost always followed by consumption of his body. Support for the female foraging hypothesis is provided by studies demonstrating that hungry females are more likely to cannibalize males (Wilder et al., 2009) and that smaller males, which are presumably easier to capture, are killed more frequently (Wilder and Rypstra, 2008). However, one of the key predictions of the female foraging hypothesis, that cannibalistic females have higher fecundity, has seldom been supported (i.e., only 3 of 12 studies document a fecundity benefit of cannibalism; Barry et al., 2008; Wilder et al., 2009). Lack of a fecundity benefit of sexual cannibalism may be explained by the small size of males relative to females in many species and the imbalanced macronutrient content of male bodies relative to the nutrients females need for egg production (Barry et al., 2008; Wilder and Rypstra, 2010). While consumption of the male body rarely increases female fecundity, consuming male bodies could increase female fitness if it
Corresponding author at: Department of Integrative Biology, Oklahoma State University, OK, USA. E-mail address: [email protected] (S.M. Wilder).
http://dx.doi.org/10.1016/j.jinsphys.2017.06.007 Received 25 January 2017; Received in revised form 8 June 2017; Accepted 9 June 2017 Available online 11 June 2017 0022-1910/ © 2017 Elsevier Ltd. All rights reserved.
Journal of Insect Physiology 100 (2017) 128–132
S.M. Wilder, J.M. Schneider
are similar to those of other animals remains largely unknown. Amino acid treatments were sprayed onto the spiders web because these premixed solutions consisted of dilute amounts of amino acids in water (8.1–11.2 g/L). Spraying the web with amino acid solutions or water as a sham control consisted of three sprays from a small, handheld spray bottle directed at the hub of the web. Females could only consume any liquid that stuck to the web, which was a small proportion of the total amount sprayed at the web. The fatty acid treatments were injected into prey. The oil was not amenable to spraying onto the web. For the specific treatment manipulations, the webs of “Control” females were sprayed with water and were provided with two flies each injected with 0.01 mL water. The webs of “Cannibalism” treatment females were sprayed with water and provided with one fly injected with water and one freshly killed male. The webs of “Essential Amino Acid” treatment females were sprayed with a 7:3 mixture solution of dietary essential amino acids:non-essential amino acids (Sigma-Aldrich, catalog numbers B6766 and M7145, respectively) and provided with two flies each injected with 0.01 mL of macadamia oil, which is naturally low in polyunsaturated fatty acids. The webs of “Essential Fatty Acid” treatment females were sprayed with a solution of non-essential amino acids and provided with two flies, each injected with 0.01 mL of an oil solution containing macadamia oil supplemented with dietary essential fatty acids. This dietary essential fatty acid mixture was made by adding 1 g linoleic acid (Sigma-Aldrich, catalog number L1376), 500 mg linolenic acid (Sigma-Aldrich, catalog number L2376), and 2.5 mg cholesterol (Sigma-Aldrich, catalog number C3045) to 4 mL of macadamia oil. Finally, the webs of “Nonessential Amino and Fatty Acid” treatment females were sprayed with a solution of non-essential amino acids (Sigma-Aldrich, catalog number M7145) and provided with two flies, each injected with 0.01 mL of macadamia oil. Hence, for the three nutrient manipulation treatments, all females received the same amount of amino and fatty acids but differed in whether those nutrients were primarily non-essential or essential nutrients. For all treatments, any prey that were not immediately attacked by the female were stimulated with an electronic toothbrush until they were attacked and wrapped by the spider. After the mating and feeding trials, females were kept under standard maintenance conditions of 3 bottle flies twice per week. For the first two egg sacs, we recorded the date of egg sac production, mass of the egg sac and weight of the female after egg sac production. Females were sacrificed after they produced their second egg sac and we measured the tibia-patella length of the first pair of legs as a measure of female body size. Egg sacs were placed in individual cups with mesh lids in an incubator at 8 °C with an open container of water to maintain high humidity levels. Egg sacs were also periodically misted with water. We recorded the date of egg sac hatching and opened all egg sacs approximately one month after hatching to count the number of unhatched eggs. We placed 15 individuals from each egg sac into a petri dish and recorded the number of individuals alive once every two weeks. Individuals were not fed and did not grow during the survival trials. We observed no cases of cannibalism, which is similar to a previous study of offspring survival in this species (Welke and Schneider, 2012).
benefitted offspring production in other ways. Recent studies have documented that cannibalistic females produce eggs with higher energy density, higher hatching success, and greater offspring survival (Johnson, 2001; Blamires, 2011; Welke and Schneider, 2012; Pruitt et al., 2014; Schwartz et al., 2016). For example, in Argiope bruennichi, the median survival of offspring of cannibalistic females was one to two weeks longer than the survival of offspring of control females (Welke and Schneider, 2012). The benefits of cannibalism for offspring success have been observed in species with extreme sexual size dimorphism in which males are only a fraction the size of females. Hence, if these effects are mediated by substances in the male body, then these substances are more likely to be micronutrients than bulk macronutrients. While micronutrient content of prey has long been suggested to be important for spider nutrition, very few studies have manipulated micronutrients in spider diets (Greenstone, 1979; Wilder, 2011). The goal of this study was to replicate the findings of Welke and Schneider (2012), which demonstrated higher survival of offspring from cannibalistic female Argiope bruennichi, and to test if similar increases in offspring survival could be achieved by providing mothers with small supplements of dietary essential amino or fatty acids. The five diet treatments consisted of feeding females either a dead male spider, or a similarly-sized dead fly with one of four nutrient treatments (water control, dietary essential fatty acids, dietary essential amino acids, or nonessential amino and fatty acids). 2. Materials and methods 2.1. Study animals All male and female A. bruennichi used in these experiments were the lab-reared offspring from egg sacs collected within 100 km of Hamburg, Germany. Spiders were maintained in overturned plastic cups (males: 250 mL; females: 330 mL) whose bottom had been replaced by mesh to allow ventilation and watering. Individually-housed juveniles were fed Drosophila melanogaster. The D. melanogaster were reared on Drosophila Instantmix WP (Birlin-Mühle, Germany). Males remained on a twice weekly feeding of D. melanogaster for their entire lives. Juvenile females were switched to a diet of 2–3 bottle flies (Calliphora sp.) twice per week as soon as they were large enough to capture these prey. Once females matured, they were fed three bottle flies, twice per week. All individuals were misted with water once per day, had the substrate below their webs replaced once every one to two weeks, and were kept under a natural daylight cycle coming through windows along one wall of the rearing room, which was approximately 17 h of light and 7 h darkness. 2.2. Experiments Females were transferred to Perspex web frames (36 × 36 × 6 cm) within one week of maturation. On the day of mating, a male was introduced to the periphery of the web and allowed to court and mate with the female. Males were only allowed one palpal insertion to standardize the number of insertions. One insertion is sufficient to fertilize eggs and some females kill males after the first insertion. If females attacked and wrapped males with silk after mating, then the male was immediately removed before the female could begin consumption. This study included five diet treatments: control (n = 20 females), cannibalism (n = 17), non-essential amino and fatty acids (n = 17), essential amino acids (n = 19), and essential fatty acids (n = 17). The treatment conditions were imposed directly after mating. Treatments were meant to be very small nutrient supplements given once, near the time of mating, analogous to females receiving nutrients from cannibalism. Classification of amino and fatty acids as essential versus nonessential was based on the standard use of these terms (Klowden, 2007). Whether or not the essential amino and fatty acids required by spiders
2.3. Analyses One-factor analysis of variance (ANOVA) was used to test if there were any initial differences in the size of males or females assigned to the treatments and to test if the treatments affected a range of reproductive traits (latency to produce an egg sac, egg sac mass, female mass after egg production, number of eggs, number hatching, hatching success, and mass per egg). Survival data are often analyzed using Kaplan-Meier analysis. However, such study designs treat all individuals identically in analyses. Our study design had 15 spiders per clutch and spiders from the 129
Journal of Insect Physiology 100 (2017) 128–132
S.M. Wilder, J.M. Schneider
Table 1 Comparison of the effects of diet treatments on response variables (mean ± 1 SE) for the first and second egg sacs produced by females. Control
Cannibalism
Nonessential
Essential Amino Acids
Essential Fatty Acids
df
Egg Sac 1 Latency to Produce (days) Egg Sac Mass (mg) Female Mass (mg)* Number of Eggs Number Hatching Hatching Success (%)** Mass per Egg (mg)
18.1 ± 0.5 147 ± 9 244 ± 9 278 ± 17 210 ± 23 68 ± 6 0.54 ± 0.03
18.8 ± 0.7 152 ± 10 259 ± 9 334 ± 16 238 ± 25 72 ± 7 0.49 ± 0.03
18.5 ± 0.6 160 ± 10 273 ± 9 297 ± 16 235 ± 23 78 ± 6 0.56 ± 0.03
18.6 ± 0.5 160 ± 9 271 ± 9 278 ± 17 195 ± 25 75 ± 7 0.60 ± 0.03
18.9 ± 0.6 149 ± 11 264 ± 10 268 ± 19 216 ± 28 79 ± 7 0.54 ± 0.03
4 4, 4, 4, 4, 4, 4,
Egg Sac 2 Latency to Produce (days)** Egg Sac Mass (mg)** Female Mass (mg)** Number of Eggs Number Hatching Hatching Success (%)** Mass per Egg (mg)
26.9 ± 0.8 151 ± 9 255 ± 12 309 ± 25 242 ± 37 74 ± 9 0.57 ± 0.04
27.8 ± 1.0 135 ± 12 246 ± 13 262 ± 27 178 ± 37 63 ± 9 0.52 ± 0.05
28.4 ± 0.8 140 ± 10 268 ± 13 278 ± 22 210 ± 30 76 ± 7 0.55 ± 0.04
27.8 ± 0.9 146 ± 11 260 ± 13 275 ± 27 248 ± 38 86 ± 9 0.57 ± 0.05
28.7 ± 0.8 119 ± 10 294 ± 13 300 ± 32 264 ± 49 87 ± 12 0.41 ± 0.05
4 4, 4, 4, 4, 4, 4,
Test Statistic
P
89 89 72 72 72 72
χ2 = 1.5 F = 0.39 F = 0.19 F = 2.46 F = 0.55 F = 0.54 F = 1.60
0.82 0.81 0.94 0.06 0.70 0.71 0.18
70 69 37 37 37 37
χ2 = 3.2 F = 1.61 F = 2.06 F = 0.49 F = 0.76 F = 0.98 F = 1.72
0.52 0.18 0.10 0.74 0.56 0.44 0.17
* Log transformed. ** Not from a normal distribution and Log transformation did not result in a normal distribution.
directly enhanced offspring survival. Alternately, the dietary essential amino acid supplement may have affected female physiology in some way that changed the quality of eggs produced. For example, essential amino acids may have provided limiting nutrients that enhanced a particular metabolic pathway involved in allocating nutrients to eggs. Our data suggest some preliminary support for nutrients enhancing female physiology, given that the amino acid supplement was very small and appeared to enhance offspring survival in both egg sacs. However, analysis of the nutrient content of eggs or radiotracer studies of amino acids would be required to test these hypotheses. The potential importance of dietary essential nutrients for spiders was first highlighted by Greenstone (1979). This study showed that wolf spiders balanced their consumption of three prey items in the field to levels that allowed them to ingest amino acids in ratios that were similar to the ratios of these nutrients in the spider’s body (Greenstone, 1979). Hence, these data suggested that spiders were choosing prey to optimize amino acid balance (Greenstone, 1979). Since this study, very few studies have tested the effects of dietary essential nutrients on spider performance (Wilder, 2011). Higgins and Rankin (1999) demonstrated that choline was a dietary essential nutrient for webbuilding spiders, Nephila clavata, and that when choline is limited in their diet individuals face a tradeoff between allocating this nutrient to their body versus their web (Higgins and Rankin, 1999). Mayntz and Toft (2001) tested if supplementing the diet of prey with macronutrients, vitamins or methionine affected growth and survival of wolf spiders. Macronutrient (fat or protein) supplements resulted in higher growth but vitamins and methionine had no effect on growth or survival (Mayntz and Toft, 2001). Clearly, more research is needed before conclusions can be drawn about the importance of dietary essential nutrients for the growth, survival and reproduction of spiders. Supplements of specific nutrients provided at realistic concentrations and the use of a hierarchical approach may prove useful to identify which essential nutrients affect spider life history traits (Wilder, 2011). Several studies have shown that dietary essential amino acids can affect the lifespan of some animals. In D. melanogaster, individuals whose diet was supplemented with essential amino acids had shorter lifespans but higher reproductive output than individuals supplemented with nonessential amino acids (Grandison et al., 2009). In other studies of individual amino acids, restriction of methionine, a dietary essential amino acid, has been shown to extend lifespan (Orentreich et al., 1993; Lee et al., 2014). Hence, studies of the effects of essential amino acids on lifespan show patterns opposite to what we observed for survival of food-deprived juvenile spiders. However, differences between juveniles that are growing versus adults engaged in reproduction (e.g., gene
same clutch cannot be considered independent. As such, we used a repeated measures design to analyze how the number of spiders alive in each clutch changed over time among the different treatments, which treats clutch as the unit of replication. 3. Results There were no initial differences among treatments in female mass at maturation (F4,89 = 0.27, p = 0.89), female post-mating mass (F4,78 = 1.50, p = 0.21), female tibia-patella length (F4,84 = 0.70, p = 0.60), mass of the male mated to the female (F4,89 = 0.34, p = 0.34), or copulation duration (F4,85 = 0.46, p = 0.77), which were all measured prior to applying the treatments. We quantified a range of variables related to egg production in both first and second egg sacs to test if they were affected by cannibalism or micronutrient supplementation including: latency to produce an egg sac, egg sac mass, female mass after egg production, number of eggs, number hatching, hatching success, and mass per egg. There were no significant effects of cannibalism or micronutrient supplementation on any of the egg production measures (Table 1). In terms of survival, spiderlings of mothers whose diet was supplemented with dietary essential amino acids survived 2–4 weeks longer on average than spiderlings on the other treatments (Fig. 1A). For example, clutches of most treatments had declined to approximately 7 surviving spiderlings by 22 weeks, whereas clutches of spiders on the dietary essential amino acid treatment took approximately 26 weeks to reach similar levels. The higher survival of offspring whose mother was supplemented in amino acids was more pronounced in the first than the second egg sac (Fig. 1); although, there was no time by treatment interaction in offspring survival (Table 2). Higher survival of spiderlings whose mothers had been fed dietary essential amino acids appeared around weeks 20–22 and persisted until the end of the experiment for spiderlings from the first egg sac. 4. Discussion Supplementing the diet of female spiders with essential amino and fatty acids had no effect on female reproductive output (e.g., time to produce eggs, mass and number of eggs, etc.). Yet, a small supplement of dietary essential amino acids provided to a female resulted in a significant increase the survival of her offspring under simulated overwintering conditions. There are at least two potential mechanisms to explain this result. First, the dietary essential amino acids may have been transferred from the mother to eggs and provided nutrients that 130
Journal of Insect Physiology 100 (2017) 128–132
S.M. Wilder, J.M. Schneider
Number of Spiders Alive (Mean + 1 SE)
16 14 12 10 8 Control
6
Cannibalism 4
Nonessential Amino and Fatty Acids
2
Essential Amino Acids Essential Fatty Acids
0 2
4
6
8
10
12
14
Number of Spiders Alive (Mean + 1 SE)
-2
16
18
20
22
24
26
28
30
32
Weeks
16 14 12 10 8 Control
6
Cannibalism 4
Nonessential Amino and Fatty Acids Essential Amino Acids
2
Essential Fatty Acids 0 2
4
6
8
10
12
14
16 18 Weeks
20
22
24
26
28
30
32
Fig. 1. Effects of diet treatment on the number of spiderlings alive (mean ± 1 SE) over the course of the survival experiment for offspring from the first (A) and second (B) egg sacs.
if those nutrients affected energy provisioning to eggs. Blamires (2011) found that female A. keyserlingi that cannibalized males produced eggs with a higher energy density than females that did not cannibalize males. Furthermore, Blamires (2011) found that female spiders fed high protein but low energy flies also produced eggs that had higher energy density than females that fed on low protein but higher energy flies. Interestingly, females that produced more energy dense eggs also lost greater mass indicating that the consumption of males or high protein foods caused females to allocate more of their soma to eggs (Blamires, 2011). Unfortunately, we did not measure egg energy density in our experiment. But, if generalizations can be made across Argiope spp., then our results combined with those of Blamires (2011) might suggest that dietary essential amino acids in male bodies may stimulate females to produce eggs with higher energy density, which would then provide offspring with more resources to survive periods without food like those that occur during overwintering. Further work is needed to determine the extent to which generalizations can be made across Argiope species. These combined results have the potential to identify a previously unknown mechanism through which sexual cannibalism benefits female fitness, especially in species where males are tiny meals. We did not detect any effect of providing dietary essential fatty acids to females on reproductive output or the hatching success or
Table 2 Statistical results for the repeated measures analysis of variance testing how diet treatments affected offspring survival. DF
F
P
Between Subjects Treatment Sac Number Treatment * Sac
4, 99 1, 99 4, 99
3.04 1.29 0.28
0.02 0.25 0.89
Within Subjects Time Time * Treatment Time * Sac Time * Treatment * Sac
15, 60, 15, 60,
150.58 1.15 0.66 0.87
< 0.001 0.22 0.81 0.74
85 334 85 334
expression and metabolic pathways) and differences in the response measured (survival during starvation conditions vs. lifespan of fed adults) complicate any comparison of our results with these studies of amino acids and adult lifespan. Yet, these disparate results suggest that the amino acid composition of animal diets can have significant and complex effects on fitness-related traits throughout an animal’s life. Given that juvenile spiders were not provided food in our experiment, one way that nutrients could have increased offspring survival is 131
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raising spiders and monitoring spiderling survival for over 8 months, to Stephanie Zimmer for collecting and raising spiders, and to Lizzy Lowe and Shannon Wilder for assistance in the laboratory. Funding was provided by Australian Research Council DECRA (DE130100833) to SMW.
survival of offspring. Polyunsaturated fatty acids (PUFA) are dietary essential nutrients for many animals including fish, mammals and some invertebrates (Neuringer et al., 1988; Sargent et al., 1999; MartinCreuzburg and Von Elert, 2009; Martin-Creuzburg et al., 2010). These PUFAs can have an important role in the structure and function of cell membranes throughout the body, including those of neurons or cells of the visual system, and as precursors for important signaling molecules involved in reproduction and the immune system (Neuringer et al., 1988; Sargent et al., 1999; Martin-Creuzburg and Von Elert, 2009; Martin-Creuzburg et al., 2010). A deficiency in PUFA compounds can result in reduced growth, reduced reproduction or lack of proper vision or neural function (Neuringer et al., 1988; Sargent et al., 1999; MartinCreuzburg and Von Elert, 2009; Martin-Creuzburg et al., 2010). Several non-mutually exclusive hypotheses may explain the lack of an effect of PUFA supplementation in our study including 1) PUFAs may not be as important for spiders as they are for other animals such as fish and mammals, 2) the two PUFAs (linoleic and linolenic, which are both C18) that we manipulated may not be the most important PUFAs for spiders (e.g., C20, C22 or other PUFA may be important), 3) PUFAs may be more important for growth while our study only measured egg hatching and survival, and 4) spiders can biosynthesize PUFAs. Further work manipulating a wider range of PUFAs and more response variables (growth and behavior) are needed to evaluate the importance of PUFAs or other dietary essential fatty acids for spiders. In our study, the offspring of cannibalistic females did not survive longer than control females, unlike the results of Welke and Schneider (2012). One factor that could explain this difference is the stage at which males were collected from the field. Males in our study were hatched and raised in the laboratory their entire lives while males in Welke and Schneider (2012) were collected as subadults from the field and reared to maturity in the laboratory. If male bodies act as dietary essential nutrient supplements to females, then males need to acquire these dietary essential nutrients from prey since, by definition, they cannot be synthesized by males. Field collected males, like those used in Welke and Schneider (2012) were likely exposed to a greater diversity of dietary essential nutrients in their diverse diet in the field relative to the lab-reared males used in the current study, which were fed a monotypic diet of D. melanogaster. For example, D. melanogaster lack C20 and C22 PUFAs and, as a consequence, male spiders feeding only on these prey would also likely lack these PUFAs (Shen et al., 2010). This hypothesis has important implications for interpreting the results of studies aimed at testing the nutritional benefit of sexual cannibalism. If lab-reared males have fewer or lower concentrations of dietary essential nutrients in their bodies, then use of these males in studies of sexual cannibalism could decrease the probability of detecting a nutritional benefit of sexual cannibalism for females. Analysis of the fatty and amino acid profiles of lab-reared and field-collected males in A. bruennichi and other species would aid in testing if studies that use exclusively lab-reared animals are underestimating the potential nutritional benefit of the male body. In addition, more detailed studies of the biochemical composition of male spiders and the contribution of these chemicals to female reproductive output or the success of offspring would provide a better understanding of the potential nutritional benefits of sexual cannibalism in species with high sexual size dimorphism, which are the species in which cannibalism is most frequent (Wilder and Rypstra, 2008; Wilder et al., 2009).
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Acknowledgements Many thanks to Tomma Dirks and Angelika Taebel-Hellwig for help
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Micronutrient consumption by female Argiope bruennichi affects offspring survival
MARK
⁎
Shawn M. Wildera,b, , Jutta M. Schneiderc a b c
School of Biological Sciences and Charles Perkins Centre, University of Sydney, Sydney, Australia Department of Integrative Biology, Oklahoma State University, OK, USA Department of Biology, Zoological Institute and Museum, University of Hamburg, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords: Micronutrients Survival Spider Argiope bruennichi
Sexual cannibalism has long been hypothesized to be a foraging decision in which females consume males for the nutrients in their bodies. While few studies have documented fecundity benefits of sexual cannibalism, several recent studies have documented benefits of cannibalism to egg hatching success or offspring survival. We tested if small supplements of dietary essential nutrients fed to female spiders, Argiope bruennichi, would result in increases in offspring survival similar to those seen following sexual cannibalism. All female spiders were prevented from cannibalizing their mates and subsequently fed either: a dead male spider, or a similarly-sized dead fly with one of four nutrient supplements (water control, dietary essential fatty acids, dietary essential amino acids, or nonessential amino and fatty acids). Females that consumed a small supplement of dietary essential amino acids produced offspring that survived simulated overwintering conditions significantly longer than offspring of other treatments. While a previous study found a significant effect of cannibalism on offspring survival using field-collected males as prey, the current study, which used lab-reared males as prey, found no effect of sexual cannibalism on offspring survival. Hence, our results suggest that dietary essential amino acids, which may be sequestered by males from their diet, could be valuable supplements that increase the success of the offspring of cannibalistic females. Further work is needed to determine the source and identity of these dietary essential amino acids and if other essential nutrients (e.g., trace elements, vitamins, etc.) may also be limiting in female diets and affect offspring success.
1. Introduction Sexual cannibalism, when females consume males in the context of mating, is widespread among spiders and occurs in a range of other invertebrates (Elgar and Crespi, 1992; Elgar and Schneider, 2004). This behavior is often described as a form of sexual conflict because it provides the female with a meal at the expense of future mating opportunities of the male (Schneider, 2014). However, this cost to the male is reduced in species with male monogyny or where consumption of the male body increases female fecundity (Fromhage et al., 2005; Barry et al., 2008). Many hypotheses have been proposed for the occurrence of sexual cannibalism including male sacrifice, spillover or genetic constraints on aggression, female choice, female control of copulation duration or paternity, mistaken identity, and female foraging (Elgar, 1992; Andrade, 1996; Arnqvist and Henriksson, 1997; Johnson, 2001; Schneider, 2014). Of the explanations for sexual cannibalism, the female foraging hypothesis has received the most empirical attention (Wilder et al.,
⁎
2009). Female foraging is one of the most parsimonious explanations for sexual cannibalism because many species of spiders are aggressive, generalist predators and because killing of males is almost always followed by consumption of his body. Support for the female foraging hypothesis is provided by studies demonstrating that hungry females are more likely to cannibalize males (Wilder et al., 2009) and that smaller males, which are presumably easier to capture, are killed more frequently (Wilder and Rypstra, 2008). However, one of the key predictions of the female foraging hypothesis, that cannibalistic females have higher fecundity, has seldom been supported (i.e., only 3 of 12 studies document a fecundity benefit of cannibalism; Barry et al., 2008; Wilder et al., 2009). Lack of a fecundity benefit of sexual cannibalism may be explained by the small size of males relative to females in many species and the imbalanced macronutrient content of male bodies relative to the nutrients females need for egg production (Barry et al., 2008; Wilder and Rypstra, 2010). While consumption of the male body rarely increases female fecundity, consuming male bodies could increase female fitness if it
Corresponding author at: Department of Integrative Biology, Oklahoma State University, OK, USA. E-mail address: [email protected] (S.M. Wilder).
http://dx.doi.org/10.1016/j.jinsphys.2017.06.007 Received 25 January 2017; Received in revised form 8 June 2017; Accepted 9 June 2017 Available online 11 June 2017 0022-1910/ © 2017 Elsevier Ltd. All rights reserved.
Journal of Insect Physiology 100 (2017) 128–132
S.M. Wilder, J.M. Schneider
are similar to those of other animals remains largely unknown. Amino acid treatments were sprayed onto the spiders web because these premixed solutions consisted of dilute amounts of amino acids in water (8.1–11.2 g/L). Spraying the web with amino acid solutions or water as a sham control consisted of three sprays from a small, handheld spray bottle directed at the hub of the web. Females could only consume any liquid that stuck to the web, which was a small proportion of the total amount sprayed at the web. The fatty acid treatments were injected into prey. The oil was not amenable to spraying onto the web. For the specific treatment manipulations, the webs of “Control” females were sprayed with water and were provided with two flies each injected with 0.01 mL water. The webs of “Cannibalism” treatment females were sprayed with water and provided with one fly injected with water and one freshly killed male. The webs of “Essential Amino Acid” treatment females were sprayed with a 7:3 mixture solution of dietary essential amino acids:non-essential amino acids (Sigma-Aldrich, catalog numbers B6766 and M7145, respectively) and provided with two flies each injected with 0.01 mL of macadamia oil, which is naturally low in polyunsaturated fatty acids. The webs of “Essential Fatty Acid” treatment females were sprayed with a solution of non-essential amino acids and provided with two flies, each injected with 0.01 mL of an oil solution containing macadamia oil supplemented with dietary essential fatty acids. This dietary essential fatty acid mixture was made by adding 1 g linoleic acid (Sigma-Aldrich, catalog number L1376), 500 mg linolenic acid (Sigma-Aldrich, catalog number L2376), and 2.5 mg cholesterol (Sigma-Aldrich, catalog number C3045) to 4 mL of macadamia oil. Finally, the webs of “Nonessential Amino and Fatty Acid” treatment females were sprayed with a solution of non-essential amino acids (Sigma-Aldrich, catalog number M7145) and provided with two flies, each injected with 0.01 mL of macadamia oil. Hence, for the three nutrient manipulation treatments, all females received the same amount of amino and fatty acids but differed in whether those nutrients were primarily non-essential or essential nutrients. For all treatments, any prey that were not immediately attacked by the female were stimulated with an electronic toothbrush until they were attacked and wrapped by the spider. After the mating and feeding trials, females were kept under standard maintenance conditions of 3 bottle flies twice per week. For the first two egg sacs, we recorded the date of egg sac production, mass of the egg sac and weight of the female after egg sac production. Females were sacrificed after they produced their second egg sac and we measured the tibia-patella length of the first pair of legs as a measure of female body size. Egg sacs were placed in individual cups with mesh lids in an incubator at 8 °C with an open container of water to maintain high humidity levels. Egg sacs were also periodically misted with water. We recorded the date of egg sac hatching and opened all egg sacs approximately one month after hatching to count the number of unhatched eggs. We placed 15 individuals from each egg sac into a petri dish and recorded the number of individuals alive once every two weeks. Individuals were not fed and did not grow during the survival trials. We observed no cases of cannibalism, which is similar to a previous study of offspring survival in this species (Welke and Schneider, 2012).
benefitted offspring production in other ways. Recent studies have documented that cannibalistic females produce eggs with higher energy density, higher hatching success, and greater offspring survival (Johnson, 2001; Blamires, 2011; Welke and Schneider, 2012; Pruitt et al., 2014; Schwartz et al., 2016). For example, in Argiope bruennichi, the median survival of offspring of cannibalistic females was one to two weeks longer than the survival of offspring of control females (Welke and Schneider, 2012). The benefits of cannibalism for offspring success have been observed in species with extreme sexual size dimorphism in which males are only a fraction the size of females. Hence, if these effects are mediated by substances in the male body, then these substances are more likely to be micronutrients than bulk macronutrients. While micronutrient content of prey has long been suggested to be important for spider nutrition, very few studies have manipulated micronutrients in spider diets (Greenstone, 1979; Wilder, 2011). The goal of this study was to replicate the findings of Welke and Schneider (2012), which demonstrated higher survival of offspring from cannibalistic female Argiope bruennichi, and to test if similar increases in offspring survival could be achieved by providing mothers with small supplements of dietary essential amino or fatty acids. The five diet treatments consisted of feeding females either a dead male spider, or a similarly-sized dead fly with one of four nutrient treatments (water control, dietary essential fatty acids, dietary essential amino acids, or nonessential amino and fatty acids). 2. Materials and methods 2.1. Study animals All male and female A. bruennichi used in these experiments were the lab-reared offspring from egg sacs collected within 100 km of Hamburg, Germany. Spiders were maintained in overturned plastic cups (males: 250 mL; females: 330 mL) whose bottom had been replaced by mesh to allow ventilation and watering. Individually-housed juveniles were fed Drosophila melanogaster. The D. melanogaster were reared on Drosophila Instantmix WP (Birlin-Mühle, Germany). Males remained on a twice weekly feeding of D. melanogaster for their entire lives. Juvenile females were switched to a diet of 2–3 bottle flies (Calliphora sp.) twice per week as soon as they were large enough to capture these prey. Once females matured, they were fed three bottle flies, twice per week. All individuals were misted with water once per day, had the substrate below their webs replaced once every one to two weeks, and were kept under a natural daylight cycle coming through windows along one wall of the rearing room, which was approximately 17 h of light and 7 h darkness. 2.2. Experiments Females were transferred to Perspex web frames (36 × 36 × 6 cm) within one week of maturation. On the day of mating, a male was introduced to the periphery of the web and allowed to court and mate with the female. Males were only allowed one palpal insertion to standardize the number of insertions. One insertion is sufficient to fertilize eggs and some females kill males after the first insertion. If females attacked and wrapped males with silk after mating, then the male was immediately removed before the female could begin consumption. This study included five diet treatments: control (n = 20 females), cannibalism (n = 17), non-essential amino and fatty acids (n = 17), essential amino acids (n = 19), and essential fatty acids (n = 17). The treatment conditions were imposed directly after mating. Treatments were meant to be very small nutrient supplements given once, near the time of mating, analogous to females receiving nutrients from cannibalism. Classification of amino and fatty acids as essential versus nonessential was based on the standard use of these terms (Klowden, 2007). Whether or not the essential amino and fatty acids required by spiders
2.3. Analyses One-factor analysis of variance (ANOVA) was used to test if there were any initial differences in the size of males or females assigned to the treatments and to test if the treatments affected a range of reproductive traits (latency to produce an egg sac, egg sac mass, female mass after egg production, number of eggs, number hatching, hatching success, and mass per egg). Survival data are often analyzed using Kaplan-Meier analysis. However, such study designs treat all individuals identically in analyses. Our study design had 15 spiders per clutch and spiders from the 129
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Table 1 Comparison of the effects of diet treatments on response variables (mean ± 1 SE) for the first and second egg sacs produced by females. Control
Cannibalism
Nonessential
Essential Amino Acids
Essential Fatty Acids
df
Egg Sac 1 Latency to Produce (days) Egg Sac Mass (mg) Female Mass (mg)* Number of Eggs Number Hatching Hatching Success (%)** Mass per Egg (mg)
18.1 ± 0.5 147 ± 9 244 ± 9 278 ± 17 210 ± 23 68 ± 6 0.54 ± 0.03
18.8 ± 0.7 152 ± 10 259 ± 9 334 ± 16 238 ± 25 72 ± 7 0.49 ± 0.03
18.5 ± 0.6 160 ± 10 273 ± 9 297 ± 16 235 ± 23 78 ± 6 0.56 ± 0.03
18.6 ± 0.5 160 ± 9 271 ± 9 278 ± 17 195 ± 25 75 ± 7 0.60 ± 0.03
18.9 ± 0.6 149 ± 11 264 ± 10 268 ± 19 216 ± 28 79 ± 7 0.54 ± 0.03
4 4, 4, 4, 4, 4, 4,
Egg Sac 2 Latency to Produce (days)** Egg Sac Mass (mg)** Female Mass (mg)** Number of Eggs Number Hatching Hatching Success (%)** Mass per Egg (mg)
26.9 ± 0.8 151 ± 9 255 ± 12 309 ± 25 242 ± 37 74 ± 9 0.57 ± 0.04
27.8 ± 1.0 135 ± 12 246 ± 13 262 ± 27 178 ± 37 63 ± 9 0.52 ± 0.05
28.4 ± 0.8 140 ± 10 268 ± 13 278 ± 22 210 ± 30 76 ± 7 0.55 ± 0.04
27.8 ± 0.9 146 ± 11 260 ± 13 275 ± 27 248 ± 38 86 ± 9 0.57 ± 0.05
28.7 ± 0.8 119 ± 10 294 ± 13 300 ± 32 264 ± 49 87 ± 12 0.41 ± 0.05
4 4, 4, 4, 4, 4, 4,
Test Statistic
P
89 89 72 72 72 72
χ2 = 1.5 F = 0.39 F = 0.19 F = 2.46 F = 0.55 F = 0.54 F = 1.60
0.82 0.81 0.94 0.06 0.70 0.71 0.18
70 69 37 37 37 37
χ2 = 3.2 F = 1.61 F = 2.06 F = 0.49 F = 0.76 F = 0.98 F = 1.72
0.52 0.18 0.10 0.74 0.56 0.44 0.17
* Log transformed. ** Not from a normal distribution and Log transformation did not result in a normal distribution.
directly enhanced offspring survival. Alternately, the dietary essential amino acid supplement may have affected female physiology in some way that changed the quality of eggs produced. For example, essential amino acids may have provided limiting nutrients that enhanced a particular metabolic pathway involved in allocating nutrients to eggs. Our data suggest some preliminary support for nutrients enhancing female physiology, given that the amino acid supplement was very small and appeared to enhance offspring survival in both egg sacs. However, analysis of the nutrient content of eggs or radiotracer studies of amino acids would be required to test these hypotheses. The potential importance of dietary essential nutrients for spiders was first highlighted by Greenstone (1979). This study showed that wolf spiders balanced their consumption of three prey items in the field to levels that allowed them to ingest amino acids in ratios that were similar to the ratios of these nutrients in the spider’s body (Greenstone, 1979). Hence, these data suggested that spiders were choosing prey to optimize amino acid balance (Greenstone, 1979). Since this study, very few studies have tested the effects of dietary essential nutrients on spider performance (Wilder, 2011). Higgins and Rankin (1999) demonstrated that choline was a dietary essential nutrient for webbuilding spiders, Nephila clavata, and that when choline is limited in their diet individuals face a tradeoff between allocating this nutrient to their body versus their web (Higgins and Rankin, 1999). Mayntz and Toft (2001) tested if supplementing the diet of prey with macronutrients, vitamins or methionine affected growth and survival of wolf spiders. Macronutrient (fat or protein) supplements resulted in higher growth but vitamins and methionine had no effect on growth or survival (Mayntz and Toft, 2001). Clearly, more research is needed before conclusions can be drawn about the importance of dietary essential nutrients for the growth, survival and reproduction of spiders. Supplements of specific nutrients provided at realistic concentrations and the use of a hierarchical approach may prove useful to identify which essential nutrients affect spider life history traits (Wilder, 2011). Several studies have shown that dietary essential amino acids can affect the lifespan of some animals. In D. melanogaster, individuals whose diet was supplemented with essential amino acids had shorter lifespans but higher reproductive output than individuals supplemented with nonessential amino acids (Grandison et al., 2009). In other studies of individual amino acids, restriction of methionine, a dietary essential amino acid, has been shown to extend lifespan (Orentreich et al., 1993; Lee et al., 2014). Hence, studies of the effects of essential amino acids on lifespan show patterns opposite to what we observed for survival of food-deprived juvenile spiders. However, differences between juveniles that are growing versus adults engaged in reproduction (e.g., gene
same clutch cannot be considered independent. As such, we used a repeated measures design to analyze how the number of spiders alive in each clutch changed over time among the different treatments, which treats clutch as the unit of replication. 3. Results There were no initial differences among treatments in female mass at maturation (F4,89 = 0.27, p = 0.89), female post-mating mass (F4,78 = 1.50, p = 0.21), female tibia-patella length (F4,84 = 0.70, p = 0.60), mass of the male mated to the female (F4,89 = 0.34, p = 0.34), or copulation duration (F4,85 = 0.46, p = 0.77), which were all measured prior to applying the treatments. We quantified a range of variables related to egg production in both first and second egg sacs to test if they were affected by cannibalism or micronutrient supplementation including: latency to produce an egg sac, egg sac mass, female mass after egg production, number of eggs, number hatching, hatching success, and mass per egg. There were no significant effects of cannibalism or micronutrient supplementation on any of the egg production measures (Table 1). In terms of survival, spiderlings of mothers whose diet was supplemented with dietary essential amino acids survived 2–4 weeks longer on average than spiderlings on the other treatments (Fig. 1A). For example, clutches of most treatments had declined to approximately 7 surviving spiderlings by 22 weeks, whereas clutches of spiders on the dietary essential amino acid treatment took approximately 26 weeks to reach similar levels. The higher survival of offspring whose mother was supplemented in amino acids was more pronounced in the first than the second egg sac (Fig. 1); although, there was no time by treatment interaction in offspring survival (Table 2). Higher survival of spiderlings whose mothers had been fed dietary essential amino acids appeared around weeks 20–22 and persisted until the end of the experiment for spiderlings from the first egg sac. 4. Discussion Supplementing the diet of female spiders with essential amino and fatty acids had no effect on female reproductive output (e.g., time to produce eggs, mass and number of eggs, etc.). Yet, a small supplement of dietary essential amino acids provided to a female resulted in a significant increase the survival of her offspring under simulated overwintering conditions. There are at least two potential mechanisms to explain this result. First, the dietary essential amino acids may have been transferred from the mother to eggs and provided nutrients that 130
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Number of Spiders Alive (Mean + 1 SE)
16 14 12 10 8 Control
6
Cannibalism 4
Nonessential Amino and Fatty Acids
2
Essential Amino Acids Essential Fatty Acids
0 2
4
6
8
10
12
14
Number of Spiders Alive (Mean + 1 SE)
-2
16
18
20
22
24
26
28
30
32
Weeks
16 14 12 10 8 Control
6
Cannibalism 4
Nonessential Amino and Fatty Acids Essential Amino Acids
2
Essential Fatty Acids 0 2
4
6
8
10
12
14
16 18 Weeks
20
22
24
26
28
30
32
Fig. 1. Effects of diet treatment on the number of spiderlings alive (mean ± 1 SE) over the course of the survival experiment for offspring from the first (A) and second (B) egg sacs.
if those nutrients affected energy provisioning to eggs. Blamires (2011) found that female A. keyserlingi that cannibalized males produced eggs with a higher energy density than females that did not cannibalize males. Furthermore, Blamires (2011) found that female spiders fed high protein but low energy flies also produced eggs that had higher energy density than females that fed on low protein but higher energy flies. Interestingly, females that produced more energy dense eggs also lost greater mass indicating that the consumption of males or high protein foods caused females to allocate more of their soma to eggs (Blamires, 2011). Unfortunately, we did not measure egg energy density in our experiment. But, if generalizations can be made across Argiope spp., then our results combined with those of Blamires (2011) might suggest that dietary essential amino acids in male bodies may stimulate females to produce eggs with higher energy density, which would then provide offspring with more resources to survive periods without food like those that occur during overwintering. Further work is needed to determine the extent to which generalizations can be made across Argiope species. These combined results have the potential to identify a previously unknown mechanism through which sexual cannibalism benefits female fitness, especially in species where males are tiny meals. We did not detect any effect of providing dietary essential fatty acids to females on reproductive output or the hatching success or
Table 2 Statistical results for the repeated measures analysis of variance testing how diet treatments affected offspring survival. DF
F
P
Between Subjects Treatment Sac Number Treatment * Sac
4, 99 1, 99 4, 99
3.04 1.29 0.28
0.02 0.25 0.89
Within Subjects Time Time * Treatment Time * Sac Time * Treatment * Sac
15, 60, 15, 60,
150.58 1.15 0.66 0.87
< 0.001 0.22 0.81 0.74
85 334 85 334
expression and metabolic pathways) and differences in the response measured (survival during starvation conditions vs. lifespan of fed adults) complicate any comparison of our results with these studies of amino acids and adult lifespan. Yet, these disparate results suggest that the amino acid composition of animal diets can have significant and complex effects on fitness-related traits throughout an animal’s life. Given that juvenile spiders were not provided food in our experiment, one way that nutrients could have increased offspring survival is 131
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raising spiders and monitoring spiderling survival for over 8 months, to Stephanie Zimmer for collecting and raising spiders, and to Lizzy Lowe and Shannon Wilder for assistance in the laboratory. Funding was provided by Australian Research Council DECRA (DE130100833) to SMW.
survival of offspring. Polyunsaturated fatty acids (PUFA) are dietary essential nutrients for many animals including fish, mammals and some invertebrates (Neuringer et al., 1988; Sargent et al., 1999; MartinCreuzburg and Von Elert, 2009; Martin-Creuzburg et al., 2010). These PUFAs can have an important role in the structure and function of cell membranes throughout the body, including those of neurons or cells of the visual system, and as precursors for important signaling molecules involved in reproduction and the immune system (Neuringer et al., 1988; Sargent et al., 1999; Martin-Creuzburg and Von Elert, 2009; Martin-Creuzburg et al., 2010). A deficiency in PUFA compounds can result in reduced growth, reduced reproduction or lack of proper vision or neural function (Neuringer et al., 1988; Sargent et al., 1999; MartinCreuzburg and Von Elert, 2009; Martin-Creuzburg et al., 2010). Several non-mutually exclusive hypotheses may explain the lack of an effect of PUFA supplementation in our study including 1) PUFAs may not be as important for spiders as they are for other animals such as fish and mammals, 2) the two PUFAs (linoleic and linolenic, which are both C18) that we manipulated may not be the most important PUFAs for spiders (e.g., C20, C22 or other PUFA may be important), 3) PUFAs may be more important for growth while our study only measured egg hatching and survival, and 4) spiders can biosynthesize PUFAs. Further work manipulating a wider range of PUFAs and more response variables (growth and behavior) are needed to evaluate the importance of PUFAs or other dietary essential fatty acids for spiders. In our study, the offspring of cannibalistic females did not survive longer than control females, unlike the results of Welke and Schneider (2012). One factor that could explain this difference is the stage at which males were collected from the field. Males in our study were hatched and raised in the laboratory their entire lives while males in Welke and Schneider (2012) were collected as subadults from the field and reared to maturity in the laboratory. If male bodies act as dietary essential nutrient supplements to females, then males need to acquire these dietary essential nutrients from prey since, by definition, they cannot be synthesized by males. Field collected males, like those used in Welke and Schneider (2012) were likely exposed to a greater diversity of dietary essential nutrients in their diverse diet in the field relative to the lab-reared males used in the current study, which were fed a monotypic diet of D. melanogaster. For example, D. melanogaster lack C20 and C22 PUFAs and, as a consequence, male spiders feeding only on these prey would also likely lack these PUFAs (Shen et al., 2010). This hypothesis has important implications for interpreting the results of studies aimed at testing the nutritional benefit of sexual cannibalism. If lab-reared males have fewer or lower concentrations of dietary essential nutrients in their bodies, then use of these males in studies of sexual cannibalism could decrease the probability of detecting a nutritional benefit of sexual cannibalism for females. Analysis of the fatty and amino acid profiles of lab-reared and field-collected males in A. bruennichi and other species would aid in testing if studies that use exclusively lab-reared animals are underestimating the potential nutritional benefit of the male body. In addition, more detailed studies of the biochemical composition of male spiders and the contribution of these chemicals to female reproductive output or the success of offspring would provide a better understanding of the potential nutritional benefits of sexual cannibalism in species with high sexual size dimorphism, which are the species in which cannibalism is most frequent (Wilder and Rypstra, 2008; Wilder et al., 2009).
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Acknowledgements Many thanks to Tomma Dirks and Angelika Taebel-Hellwig for help
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