Rapid nectar-meal effects on a predator's capacity to kill mosquitoes

Using Evarcha culicivora, an East African jumping spider (Salticidae), we investigate how nectar meals function in concert with predation specifically at the juvenile stage between emerging from the egg sac and the first encounter with prey. Using plants and using artificial nectar consisting of sugar alone or sugar plus amino acids, we show that the plant species (Lantana camara, Ricinus communis, Parthenium hysterophorus), the particular sugars in the artificial nectar (sucrose, fructose, glucose, maltose), the concentration of sugar (20%, 5%, 1%) and the duration of pre-feeding fasts (3 days, 6 days) influence the spider's prey-capture proficiency on the next day after the nectar meal. However, there were no significant effects of amino acids. Our findings suggest that benefits from nectar feeding are derived primarily from access to particular sugars, with fructose and sucrose being the most beneficial, glucose being intermediate and maltose being no better than a water-only control.

Using Evarcha culicivora, an East African jumping spider (Salticidae), we investigate how nectar meals function in concert with predation specifically at the juvenile stage between emerging from the egg sac and the first encounter with prey. Using plants and using artificial nectar consisting of sugar alone or sugar plus amino acids, we show that the plant species (Lantana camara, Ricinus communis, Parthenium hysterophorus), the particular sugars in the artificial nectar (sucrose, fructose, glucose, maltose), the concentration of sugar (20%, 5%, 1%) and the duration of prefeeding fasts (3 days, 6 days) influence the spider's prey-capture proficiency on the next day after the nectar meal. However, there were no significant effects of amino acids. Our findings suggest that benefits from nectar feeding are derived primarily from access to particular sugars, with fructose and sucrose being the most beneficial, glucose being intermediate and maltose being no better than a water-only control.

Introduction
There has been a longstanding interest in explaining the origins and adaptive significance of omnivory (i.e. feeding at more than one trophic level). Frequently considered hypotheses include minimizing overexposure to toxins associated with otherwise superior food and surviving periods when superior food is scarce by relying on inferior food sources [1][2][3]. Omnivory is especially interesting when animals traditionally envisaged as being simply predators are shown also to take nutrients directly from plants [4]. For example, spiders are widely regarded as being exclusively predators, but Bagheera kiplingi is a striking exception [5]. This Central American jumping spider (Salticidae)  into a hole in the bottom of the cage and positioned so that it extended into a water-filled plastic pot below the cage. There was a mesh-covered hole in the top of the cage for ventilation. A second hole in the top of the cage was plugged with a rubber stopper which could be removed when introducing prey. All holes were 8 mm in diameter.
We assigned spiders at random to one of 18 meal-type groups and, after a fast of a specified duration, we gave the spider access to a first meal corresponding to the meal-type group (table 1). The first meal was artificial nectar (i.e. a solution of sugar or sugar plus amino acids), a plant or a water-only control (table 1). For the control and all artificial nectar, we used distilled water. For plants, we used L. camara and R. communis, as well as Parthenium hysterophorus, a species that is common in the same habitat but not known to attract E. culicivora in olfactometer experiments (R. R. Jackson 2008, unpublished data).
For the water-only control group, the individuals used were derived from 29 sibships. For each other group, the individuals used were derived from 8-10 sibships. A 'sibship' is defined as the progeny of a particular male and female. No sibships contributed individuals to more than one group. The number of individuals from any one of the sibships was never more than eight or less than three. Owing to the way we took individuals from a range of sibships, we did not include sibship as a variable in our data analyses.
The sugar and amino acid content of L. camara nectar is known (cited in [51] as personal communication from Irene Baker to Alm et al.): sucrose 187.25 g l −1 , fructose 57.00 g l −1 , glucose 55.80 g l −1 , proline 0.256 g l −1 , glycine 0.178 g l −1 , serine 0.144 g l −1 , glutamine 0.136 g l −1 , threonine 0.080 g l −1 , alanine 0.064 g l −1 , asparagine 0.056 g l −1 , tyrosine 0.040 g l −1 , glutamic acid 0.048 g l −1 , arginine 0.032 g l −1 and valine 0.016 g l −1 . For our experiments, we made two artificial nectar blends based on the reported ratio of the three sugars and the four dominant amino acids in this plant's nectar.
The sugar content of R. communis and P. hysterophorus nectar is not known precisely, but the floral tissues of these plants contain sucrose, fructose, glucose and other sugars, including especially maltose [52]. We included maltose in our experiments as a sugar that may be in R. communis nectar, but is not known to be present in the nectar of L. camara or prevalent in the nectar of plants, in general. For each meal-type group, there were two fasting-duration subgroups (3 day and 6 day): spiders kept without access to food for 3 days or 6 days before being given access to the meal corresponding to the meal-type group (table 1) on the 4th or 7th day.

Experimental procedure
The spider's first meal was placed in its cage at 8.00 and removed 60 min later (laboratory photoperiod 12 L : 12 D, lights on at 7.00). During this feeding period, the spider was observed continuously. For each plant meal, the plant used in an experiment was a cutting taken from a living plant in the field. In each instance, the plant was held in a closed plastic box under 100% carbon dioxide for 10 min and then examined under a microscope for any arthropods that might have remained (none were found). The cut end of the stem was the only incision or wound on the plant and it remained outside the cage (i.e. the stem, positioned alongside the cotton roll, went through the hole in the bottom of the cage so that the cut end was in the pot of water below the cage). The remainder of the plant (stems, flowers and leaves) was inside, and almost filled, the cage.
When the first meal was artificial nectar or the water-only control, a disc (diameter 5 mm, thickness 2 mm, cut from a clean kitchen sponge) was submerged in the specified solution (or water alone for the control) for 10 s at 7.30 [46]. The sponge disc was then attached by a pin to the centre top of a clean, dry cotton roll. The cotton roll that was providing water to the spider was removed at 8.00 and replaced with the clean cotton roll along with the attached solution-soaked sponge disc. The sponge disc was positioned horizontally at the top of the pin (25 mm above the cage floor, 30 mm below the cage ceiling and 22.5 mm from the side of the cage). During each trial, the cotton roll remained dry (i.e. there was no water in the pot below the cage).
We removed any spider that failed to feed while the first meal was on offer and replaced it with another spider. Our criterion for recording that a spider fed was seeing its mouthparts pressed against a plant (flower petal, leaf or stem), or against a sponge disc that had been soaked in artificial nectar [46].
On the following day at 7.30, each test spider was transferred to a testing cage. Testing cages were similar to maintenance cages, but larger (height 110 mm, diameter 60 mm). The larger size allowed sufficient space for mosquitoes to fly, making them harder for the spider to capture. At 8.00, 24 h after the first meal, we removed the stopper from the hole in the top of the cage and, using an aspirator, introduced four mosquitoes (Anopheles gambiae s.s.), after which the stopper was returned to the hole. The mosquitoes were taken from stock cultures and had fed on blood 4 h before being used in the experiments (for methods pertaining to mosquito culturing and feeding, see [53]).
The outcome of a trial was recorded as successful when the test spider attacked the mosquito, held on and then fed and it was recorded as unsuccessful when the spider attacked the mosquito, but failed to hold on and feed. Whenever 2 h elapsed without the test spider attacking a mosquito, the test ended and these spiders were excluded from further analysis (i.e. all data came from instances of a test spider attacking the prey and then being either successful or unsuccessful at capturing the prey; no instances of multiple attacks were considered).

Data analysis
The statistics package R [54] was used for all data analyses. We applied a logistic regression to preycapture data, with each instance of the spider capturing the prey being coded as 1 and each instance of the spider failing to capture the prey it attacked being coded as 0. Meal type was included as a factor in the model and pre-trial fast duration (3 or 6 days) was included as a standard variable. Using the 'glm' function in the stats package, we created logit models and compared them by likelihood-ratio testing from the 'anova' function in the stats package. We made pairwise comparisons of coefficients by using Wald tests (based on χ 2 ) with Holm-Bonferroni corrections from the aod package [55].

General
The best-fit logistic model was   where P (capture) is prey-capture success expressed as the probability that, after making an attack, the spider will hold on and eat the prey, e is the base of the natural logarithm, d is the pre-trial fast duration in days, −0.21 (z = 0.03, s.e. = −6.78, p < 0.001) is the coefficient for fast duration and β m is the coefficient for meal-type m (table 1). This model was a significantly better predictor of the data than a reduced (intercept only) model (likelihood-ratio testing, χ 2 = 410.81, p < 0.001), and it was not significantly different from an expanded model that included interaction terms (χ 2 = −11.26, p = 0.843). There were no significant interaction effects in the expanded model and Akaike's Information Criterion (AIC) was smaller for the best-fit model than for the reduced model ( AIC = 22.7) or the expanded model ( AIC = 374.8).
There was a significant effect of pre-trial fast duration (figure 1; Wald test, χ 2 1 = 40.8, p < 0.001) on the spider's success, with fewer spiders capturing prey after the longer fast (odds ratio = 0.81). We also found a significant effect of meal type (Wald tests, χ 2 18 = 332.4, p < 0.001).

Plants and artificial nectar compared with the water-only control
We use the expression 'effect' for instances of spiders from a plant group or an artificial nectar group having significantly greater prey-capture success than spiders from the water-only control. We found an effect when spiders fed on each of the three plant species and when spiders fed on artificial L. camara nectar (table 2). When we used single-sugar solutions, we found an effect when the spiders fed on 20% and 5% solutions of sucrose, fructose and glucose. However, we found no effect for spiders that fed on 1% solutions of these sugars and no effect even at 5% or 20% when the sugar was maltose.

Plants compared
Prey-capture success was significantly higher when spiders fed on L. camara instead of R. communis

Lantana camara and artificial Lantana camara nectar compared
The prey-capture success of spiders that fed on L. camara was not significantly different from the success of spiders that fed on either type of artificial L. camara nectar (figure 2b): full (χ 2 = 0.71, p = 0.399), sugaronly (χ 2 = 0.05, p = 0.824). Spiders that fed on full and sugar-only artificial L. camara nectar were not significantly different from each other (χ 2 = 0.39, p = 0.530).

Discussion
Numerous studies have shown that sugars and amino acids acquired by feeding on nectar can have beneficial effects on the growth, survival and reproduction of insects (e.g. [56][57][58][59]), but our objective was different. We investigated rapidly expressed benefits that apply during a particular phase in a spider's life, namely the phase immediately after the spider emerges from egg sacs and before it has its first prey meal. The specific benefit we considered was plant meal derived improvement in prey-capture proficiency 1 day after the meal and the plant species we considered were L. camara, R. communis and P. hysterophorus. As predicted, we found that, compared with spiders from the water-only control, spiders that fed on these plants were significantly more successful at capturing prey.
The particular plant species from which the juvenile acquired its nectar meal also mattered. In our experiments, and in an earlier study [46], we never saw a spider enter or bite into flowers and instead we saw spiders feed by pressing their mouthparts against petals, leaves and stems of the plant and, when the plant was R. communis, drops of nectar from EFNs. Conspicuous EFNs are characteristic of R. communis [60,61], but not characteristic of L. camara or P. hysterophorus. However, many plants have EFNs and EFNs are not always conspicuous [62]. Even R. communis has, besides its large, conspicuous EFNs, additional EFNs that are evident only with magnification [63].
It is unlikely that the spider fed on phloem or plant tissue instead of nectar. Although nectar is derived primarily from phloem, fructose is characteristic of nectar, whereas phloem is dominated by sucrose alone [64]. Moreover, cold-anthrone testing from an earlier study [46] confirmed that E. culicivora ingests fructose when pressing its mouthparts against the surface of the three plant species we used.
It has been suggested that the volume of nectar provided by P. hysterophorus is especially low [65], and yet cold-anthrone testing showed that E. culicivora acquires fructose from this plant [46] and we have now shown that, after feeding on P. hysterophorus, spiders become significantly more successful than the control spiders at capturing prey, but not as successful as spiders that fed on L. camara. If nectar volume matters, then spiders from the L. camara group being significantly more successful than spiders from the P. hysterophorus group is as expected [65]. However, spiders from the L. camara group were also significantly more successful than spiders from the R. communis group, despite the copious secretion of nectar from EFNs being characteristic of R. communis. These findings suggest that, for the plant species we used, the primary influence on prey-capture success is something other than simply variation in the nectar volume available to E. culicivora. Our findings also suggest that the presence of amino acids (or at least the four dominant amino acids) in nectar was not a primary influence on prey-capture success, but that the particular sugars present in a solution, and their concentrations, did matter. The prey-capture success of spiders from each of our 1% single-sugar groups (sucrose, fructose, glucose and maltose) was not significantly different from the success of spiders from the water-only control, nor were there any significant differences between spiders that fed on the different sugars at 1%. However, findings from using 5% and 20% solutions revealed a ranking of the four sugars: maltose lowest, glucose intermediate, sucrose and fructose tied for highest.
Spiders that fed on sucrose and fructose at concentrations of 5% or 20% were significantly more successful than spiders that fed on glucose or maltose at the same concentrations. We also found that, when the single-sugar concentration was 5% or 20%, spiders that fed on sucrose and spiders that fed on fructose became significantly more successful than spiders from the water-only control, but there was no significant difference between the sucrose and fructose groups when concentration was 5% or 20%.
A combination of findings implies that glucose was intermediate between maltose and sucrosefructose. Spiders that fed on 20% glucose alone were significantly less successful than spiders that fed on 20% sucrose or 20% fructose, but they were significantly more successful than spiders that fed on 20% maltose or spiders from the water-only control. However, the success of spiders that fed on 5% glucose, although significantly better than the success of spiders from the control group, was not significantly different from the success of spiders that fed on 5% maltose.
Fructose and glucose are monosaccharides, but sucrose and maltose are disaccharides. The hydrolysis of sucrose releases fructose and glucose, but maltose hydrolysis releases only glucose. Spiders from the 20% glucose group were significantly more successful than spiders from the 20% maltose group and, at all concentrations, the success of spiders that fed on maltose alone was not significantly different from the success of spiders from the water-only control. This combination of findings suggests that the spider has little capacity for maltose hydrolysis and also suggests that acquiring glucose in addition to fructose from sucrose is of little or no advantage over solely acquiring the fructose (i.e. there was no significant difference between 20% sucrose group and the 20% fructose group).
Sucrose, fructose and glucose are known to be present in roughly comparable ratios in the floral nectar of L. camara (Irene Baker cited in [51]) and the EFN of R. communis [60], and the same sugars in similar ratios might be expected for the nectar of P. hysterophorus [49,52]. The explanation for L. camara being ranked best, R. communis intermediate and P. hysterophorus worst might have more to do with interspecific variation in sugar concentration instead of interspecific variation in the ratios of the available sugars, but testing this hypothesis will probably be especially difficult. The concentration of sugar in nectar is known to be sensitive to relative humidity, time of day and other environmental factors [66][67][68], and estimating the sugar concentration encountered by a spider when it presses its mouthparts on the surfaces of the different plant species might be especially difficult. However, we can propose how the spider's access to sucrose and especially fructose might vary across the plant species we used in our experiments.
Finding no significant difference between the success of spiders that fed on 20% sucrose or 20% fructose and the success of spiders that fed on L. camara suggests that, on L. camara, E. culicivora juveniles can gain access to one or both of these sugars at an optimal concentration. That the spiders we let feed on 20% sucrose or 20% fructose were significantly more successful at prey-capture than the spiders we let feed on R. communis or P. hysterophorus suggests that, on these two plant species, E. culicivora juveniles cannot readily gain access to either of these sugars at the concentration available from L. camara. However, there are alternative hypotheses we cannot rule out at this stage. For example, we cannot rule out a hypothesis that unknown non-sugar compounds from R. communis and P. hysterophorus, but not L. camara, had negative effects on prey-capture success [69].
Spiders were less successful at capturing prey after a 6 day fast than after a 3 day fast, suggesting that longer fasting weakened the spider. Yet, the distribution of success rates across groups followed much the same pattern irrespective of fasting duration. These findings suggest that, although hungrier spiders benefit more from plant-derived nutrients, the benefit-related ranking of the nutrient sources is stable across hunger level.
Nectar and other plant-derived nutrients may often be important in the natural diets of spiders and predatory insects and, when the predators kill agricultural pests, there is an impetus to determine whether ensuring the availability of plant meal sources might make predators more effective in the biological control of the pest species [70,71]. When discussing agricultural systems, the most frequently considered beneficial effects of nectar meals include the sustaining of predator populations during periods of prey scarcity, giving predators access to nutrients not available from prey and reducing the level of competition between predators that target the same prey (e.g. [72]). These benefits would normally be expressed over a considerable timespan and comparable long-term benefits may apply to E. culicivora. However, the benefits implied by our findings are expressed the next day after a nectar meal and it might be of interest to investigate whether similar rapid benefits apply to other predators, including predators that target agricultural pests.
Ethics statement. All required approvals and permits were included in R.R.J.'s successive visiting scientist contracts (1994 to present) with icipe.