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Research Article
Revised

Variable effects of nicotine, anabasine, and their interactions on parasitized bumble bees

[version 2; peer review: 2 approved]
Previously titled: Variable effects of nicotine and anabasine on parasitized bumble bees
Lukas P. Thorburn1, Lynn S. Adler1, Rebecca E. Irwin2,3, Evan C. Palmer-Young1
Lukas P. Thorburn1, Lynn S. Adler1, Rebecca E. Irwin2,3, Evan C. Palmer-Young1
PUBLISHED 16 Dec 2015
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS

Abstract

Secondary metabolites in floral nectar have been shown to reduce parasite load in two common bumble bee species. Previous studies on the effects of nectar secondary metabolites on parasitized bees have focused on single compounds in isolation; however, in nature, bees are simultaneously exposed to multiple compounds. We tested for interactions between the effects of two alkaloids found in the nectar of Nicotiana spp. plants, nicotine and anabasine, on parasite load and mortality in bumble bees (Bombus impatiens) infected with the intestinal parasite Crithidia bombi. Adult worker bees inoculated with C. bombi were fed nicotine and anabasine diet treatments in a factorial design, resulting in four nectar treatment combinations:  2 ppm nicotine, 5 ppm anabasine, 2ppm nicotine and 5 ppm anabasine together, or a control alkaloid-free solution. We conducted the experiment twice: first, with bees incubated under variable environmental conditions (‘Variable’; temperatures varied from 10-35°C with ambient lighting); and second, under carefully controlled environmental conditions (‘Stable’; 27°C incubator, constant darkness). In ‘Variable’, each alkaloid alone significantly decreased parasite loads, but this effect was not realized with the alkaloids in combination, suggesting an antagonistic interaction. Nicotine but not anabasine significantly increased mortality, and the two compounds had no interactive effects on mortality. In ‘Stable’, nicotine significantly increased parasite loads, the opposite of its effect in ‘Variable’. While not significant, the relationship between anabasine and parasite loads was also positive. Interactive effects between the two alkaloids on parasite load were non-significant, but the pattern of antagonistic interaction was similar to that in the variable experiment. Neither alkaloid, nor their interaction, significantly affected mortality under controlled conditions. Our results do not indicate synergy between Nicotiana nectar alkaloids; however, they do suggest a complex interaction between secondary metabolites, parasites, and environmental variables, in which secondary metabolites can be either toxic or medicinal depending on context.

Keywords

Bumble bee, Bombus impatiens, parasites, Crithidia bombi, plant secondary metabolites, nicotine, alkaloids, tritrophic interactions

Corresponding author: Evan C. Palmer-Young
Competing interests: No competing interests were disclosed.
Grant information: This research was funded by the National Science Foundation under NSFDEB-1258096, NSF GRFP (Grant DGE-0907995 to ECPY), and NSF DDIG (Grant NSFDEB-1501907 to ECPY and LSA); by the National Research Initiative (NRI) Arthropod and Nematode Biology and Management Program of the USDA Cooperative State Research, Education, and Extension Service (CSREES) Grant no. USDA-AFRI 2013-02536; and by the Garden Club of America Centennial Pollinator Fellowship (ECPY).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Copyright:  © 2015 Thorburn LP et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).
How to cite: Thorburn LP, Adler LS, Irwin RE and Palmer-Young EC. Variable effects of nicotine, anabasine, and their interactions on parasitized bumble bees [version 2; peer review: 2 approved]. F1000Research 2015, 4:880 (https://doi.org/10.12688/f1000research.6870.2) First published: 21 Sep 2015, 4:880 (https://doi.org/10.12688/f1000research.6870.1) Latest published: 16 Dec 2015, 4:880 (https://doi.org/10.12688/f1000research.6870.2)

Revised Amendments from Version 1

We have revised Version 1 in response to the Reviewers’ comments, for which we are grateful. The major changes are as follows:

  1. We altered the title to emphasize interactions between multiple secondary metabolites, and have given additional attention to this novel aspect in the introduction. In addition, we have renamed the ‘Controlled’ experiment to the ‘Stable’ experiment to avoid confusion with the alkaloid-free ‘Control’ nectar treatment, which was included in each experiment.
     
  2. The revised version includes additional statistical analysis to clarify the effects of the alkaloids on mortality in the ‘Stable’ experiment. First, for the Cox proportional hazards model, we report death hazard ratios and confidence intervals comparing the ‘Control’ and ‘Nicotine + Anabasine’ treatment groups. Second, we report results of a binomial generalized linear mixed model in Table S2, to allow direct comparison with the results of the ‘Variable’ experiment.
     
  3. We added a paragraph to the discussion that contrasts our study with that of Baracchi et al. 2015, and removed the first paragraph of the introduction.
     
  4. We conducted an additional ‘Consumption experiment’, in which we measured nectar consumption at different temperatures. This experimented tested our hypothesis that temperature-induced increases in nectar consumption contributed to the stronger effects of alkaloids in the generally hot ‘Variable’ experiment. To our surprise, bees drank less than half of much artificial nectar at 33 °C than at 27 °C (Figure 5, Table 5). We have accordingly removed speculation that high temperatures alone would have been sufficient to elevate alkaloid consumption. Instead, we recommend future experiments to clarify how the medicinal and toxic effects of secondary metabolites are shaped by interactions with other factors, including combination with other secondary metabolites, climatic and rearing conditions, host species, and parasite strain.

We thank the reviewers for their improvement of the article.

See the authors' detailed response to the review by David Baracchi
See the authors' detailed response to the review by Michael Simone-Finstrom and Margarita Lopez-Uribe

Introduction

Throughout the past two decades, many wild and managed bee species have experienced severe declines (Allen-Wardell et al., 1998; Cameron et al., 2011; Potts et al., 2010). In many cases of bee decline, parasitism has been implicated as a potential cause (reviewed in Goulson et al., 2015 and Potts et al., 2010). Secondary metabolites – plant compounds that do not play a role in the plant’s primary metabolism – frequently have antimicrobial properties (Schmidt et al., 2012), and could offer a means of natural parasite control. Secondary metabolites are found in the floral nectar of many plant species (Heil, 2011).

The effects of secondary metabolites on insects, including bees and other pollinators, are context-dependent. A wide range of secondary metabolites, including terpenes, alkaloids, and phenolics, are toxic to insects (Detzel & Wink, 1993; Kumrungsee et al., 2014; Raffa et al., 1985; Singaravelan et al., 2006; Wink & Theile, 2002). Interaction with other stressors, such as infection or climatic stress, can exacerbate these toxic effects (Goulson et al., 2015; Holmstrup et al., 2010; Köhler et al., 2012a). However, under some circumstances, the antimicrobial properties of secondary metabolites can provide health benefits to infected insects. Insects have been shown to self-medicate with secondary metabolites in response to parasite infection (reviewed in Abbott, 2014). For example, Grammia incorrupta (wooly bear) caterpillars exhibited self-medication behavior in response to tachinid fly parasitism by increasing their consumption of pyrrolizidine alkaloids, which decreased the survival of unparasitized caterpillars but increased the survival of parasitized caterpillars (Singer et al., 2009).

Several recent studies have indicated that plant secondary metabolites, including those found in nectar, can benefit infected pollinators as well. Honey bees self-medicated in response to parasitism through increased foraging for resins, which are used in hive construction and have antimicrobial properties (Simone-Finstrom & Spivak, 2012), and through preferentially feeding on certain types of honey, such as sunflower honey, which reduced pathogen load (Gherman et al., 2014). Bumble bees (Bombus terrestris) infected with the intestinal parasite Crithidia bombi displayed increased preference for nicotine-containing artificial nectar, which also reduced parasite load (Baracchi et al., 2015). In another bumble bee species (B. impatiens), consumption of the alkaloid gelsemine significantly reduced C. bombi infection intensity (Manson et al., 2010), and in a separate study, four other nectar secondary compounds had significant medicinal effects, with an additional four compounds causing non-significant decreases in infection severity (Richardson et al., 2015a).

Previous studies of the effects of nectar secondary metabolites on pollinators have focused primarily on single compounds in isolation. Under natural conditions, however, pollinators would likely encounter several compounds at once, since many plant species produce multiple secondary metabolites. For example, many Nicotiana species contain both nicotine and anabasine in nectar (Adler et al., 2012), and Chelone glabra contains the iridoid glycocides aucubin and catalpol in nectar (Richardson et al., 2015b). This raises the possibility of interactions between nectar secondary metabolites.

Synergistic interactions between secondary metabolites from other plant tissues are well established. Among herbivores, the iridoid glycosides aucubin and catalpol had synergistic effects on the survival of common buckeye (Junonia coenia Hübner) caterpillars that specialize on plants with these compounds; caterpillars that consumed both iridoid glycosides had an increased rate of survival relative to caterpillars that consumed either glycoside alone (Richards et al., 2012). Amides in plants in the Piper genus had synergistic deterrent effects on herbivorous ants, while the same compounds were neutral or attractive in isolation (Dyer et al., 2003). Synergy between secondary metabolites can also alter antimicrobial effects. Carvacrol and thymol, for example, inhibited the growth of the bacterium Listeria innocua more effectively in combination than alone (García-García et al., 2011). Carvacrol was also more effective against the bacterium Vibrio cholerae when combined with cymene, although cymene alone had no antimicrobial activity (Rattanachaikunsopon & Phumkhachorn, 2010).

Antagonism between secondary metabolites has also been demonstrated. The deterrent effect of the amide piperine on the hemipteran Sibaria englemani is significantly reduced when piperine is combined with the amide piplartine, although piplartine alone had no effect on S. englemani feeding preference (Whitehead & Bowers, 2014). The linear furanocoumarins psoralen, bergapten, and xanthotoxin exhibited antagonistic interactions in their effects on insect mortality; the toxicity of psoralen combined with either or both of the other two compounds was significantly lower than would be predicted based on their toxicities in isolation (Diawara et al., 1993). If similar interactions, either synergistic or antagonistic, are present between secondary metabolites in nectar, they could exacerbate or ameliorate the effects of single compounds found in previous studies.

To evaluate interactions between secondary metabolites from the nectar of a single plant, we tested the effects of nicotine and anabasine alone and in combination on bumble bee resistance to the gut parasite Crithidia bombi. Nicotine and anabasine co-occur in the nectar of several species in the genus Nicotiana, which includes cultivated tobacco (Nicotiana tabacum) as well as several ornamental species (Adler et al., 2012). The effects of nicotine and anabasine in combination on bee disease have not previously been studied. In addition, this is the first study to our knowledge that explicitly tests for interactive effects of multiple secondary compounds on bumble bee disease.

We tested the effects of these compounds in two environmental contexts, variable and controlled conditions. Bumble bees in the wild encounter a wide range of environmental conditions, which could alter the effects of diet and parasitism. In general, temperature can decrease tolerance to environmental toxins, including secondary metabolites (Holmstrup et al., 2010), and exert unpredictable effects on insect-parasite interactions through modulation of host survival, host immune function, and parasite viability (Thomas & Blanford, 2003). Variable temperatures impose exceptional energetic costs on bumble bees by forcing them to actively regulate body temperature in order to fly (Heinrich, 1972). These costs might create caloric deficits that increase parasite virulence in Bombus (Brown et al., 2000). Alternatively, heightened energy needs could lead to increased consumption of plant foods, thereby elevating exposure to secondary metabolites. Globally, responses to environmental variability have implications for conservation: Bumble bee species with narrow climatic ranges are particularly vulnerable to decline (Williams et al., 2007; Williams et al., 2009), and projected climate change may further restrict these species’ distributions through increases in mean temperature and the frequency of extreme events (Diffenbaugh & Field, 2013).

Methods

Study system

Bombus impatiens is the most common bumble bee species in eastern North America, with a range extending from Ontario and Maine to southern Florida (Balaban et al., 2014). It is an important pollinator in agriculture, and commercial distribution of B. impatiens is becoming increasingly common (Colla et al., 2006).

Crithidia bombi is a common trypanosome parasite of bumble bees in Europe and North America (Colla et al., 2006; Lipa & Triggiani, 1988). Its range has been expanding within North America and into parts of South America, potentially due to spillover from commercial to wild bumble bee populations (Colla et al., 2006; Schmid-Hempel et al., 2014; but see Whitehorn et al., 2013). C. bombi is known to increase mortality in bumble bees under food stress conditions (Brown et al., 2000), and to reduce bumble bee foraging rate (Otterstatter & Thomson, 2006).

Nicotine is an agonist of the nicotinic acetylcholine receptor (nAChR), and therefore acts as both a stimulant drug and a toxin to many organisms (Benowitz, 1998). Nicotine is toxic to many insects, and has been historically used as an insecticide (Ujváry, 1999). Honey bees are deterred by nicotine in nectar (Köhler et al., 2012b), and both honey bees (Köhler et al., 2012b; Singaravelan et al., 2006) and bumble bees (Baracchi et al., 2015) are adversely affected by nicotine consumption when they are not infected by parasites. However, nicotine also has antimicrobial properties (Pavia et al., 2000), and recent studies have suggested that it can reduce parasite load in bumble bees infected with C. bombi (Baracchi et al., 2015; Richardson et al., 2015a), and may improve survival of diseased honey bee colonies (Köhler et al., 2012b). Anabasine, like nicotine, is a nAChR agonist, and has been used as an insecticide (MacBean, 2012). The behavioral effects of anabasine are similar to those of nicotine, although anabasine, unlike nicotine, does not have addictive effects (Caine et al., 2014). Anabasine in nectar deterred honey bees (Singaravelan et al., 2005), and reduced C. bombi load in infected bumble bees (Richardson et al., 2015a).

Secondary compound treatments

We inoculated bumble bees with C. bombi, and assessed the differences in pathogen load and mortality between adult bees fed nicotine (yes/no) and anabasine (yes/no) in a factorial design, resulting in four diet treatments: 2 ppm nicotine, 5 ppm anabasine, 2 ppm nicotine and 5 ppm anabasine together, or a control alkaloid-free solution. All diet treatments also contained 30% sucrose in distilled water. Chemicals ((-)-nicotine, cat. no. N3876; (+/-)-anabasine, cat. no. 284599) were purchased from Sigma-Aldrich (St. Louis, MO). Alkaloid concentrations were chosen to mimic the highest concentrations that would be found in Nicotiana nectar under natural conditions (Adler et al., 2006; Tadmor-Melamed et al., 2004).

Rearing conditions

We conducted two experiments under different environmental conditions. The first experiment (‘Variable’, conducted 26 February 2014 to 20 March 2014, Dataset 1) had a smaller sample size (n = 178 bees) and variable environmental conditions. In ‘Variable’, experimental bees and pupae were kept on the lab bench in a room where temperatures fluctuated between 10 and 35°C. This fluctuation was the result of a building steam leak. During most of the daytime when experimenters were present, the temperature was near 35°C, although on several weekends the heating system was shut down entirely for repairs, and temperatures as low as 10°C were reached. Bees in ‘Variable’ were also exposed to everyday stimuli and ambient fluorescent and window lighting (approximately 12 h photoperiod).

The second experiment (‘Stable’, conducted 20 May 2014 to 14 July 2014, Dataset 2) had a larger sample size (n = 339 bees) and carefully controlled environmental conditions (see sample sizes in Table S1). In ‘Stable’, experimental bees as well as pupae were kept in an incubator at 27°C in constant darkness to reduce mortality and more closely mimic conditions in a bumble bee hive.

Experimental bees were obtained from pupal clumps of commercial B. impatiens (Biobest, Leamington, Ontario, Canada). Pupal clumps were removed from colonies weekly and kept in 500 mL plastic containers, with each container containing the pupal clumps from a single colony that were collected on a specific date. In ‘Variable’, pupal clumps were incubated on the lab bench for the majority of the experiment until 2 d before the emergence of the final experimental bees, at which time the clumps were moved to a 30°C incubator (Percival Scientific, Perry, IA) due to excessive pre-experiment mortality under the variable lab conditions. Hence, all bees would have spent the majority of the pupation period under variable conditions. In ‘Stable’, pupal clumps were incubated at 27°C in darkness throughout the experiment. In both experiments, callow bees (newly emerged worker bees less than one day old) were collected upon emergence from pupal clumps. They were weighed and their mass at emergence, date of emergence, and colony of origin were recorded. Bees were then isolated in individual 20 mL vials. The lid of each vial was equipped with a 2 mL microcentrifuge tube with a cotton wick containing 500 μL artificial nectar (30% sucrose solution). Each day, bees were transferred to clean vials and given 500 μL fresh artificial nectar and a 10 mg piece of multifloral pollen (Koppert Biological Systems, Howell, MI) on which they fed ad libitum. For two days after emergence, bees were fed pollen and control nectar (30% sucrose solution), then were inoculated with C. bombi. They were starved for several hours to ensure that they would consume the inoculum, then fed 10 μL of C. bombi inoculum containing 6,000 C. bombi cells (see below). Following inoculation, bees were fed the appropriate nectar treatment and pollen ad libitum for 7 days. Bees were assigned systematically to secondary compound treatments in blocks of four, such that each block contained a bee in each treatment.

Inoculation

To inoculate experimental bees, inoculum (C. bombi cells in sucrose solution) was prepared from the gut tracts of bees taken from colonies infected with C. bombi. These colonies were obtained from the same supplier as the experimental colonies, and were infected with C. bombi from wild bees collected in Amherst, Massachusetts (September 2013). Infected bees were dissected and their gut tracts were macerated with a plastic pestle in microcentrifuge tubes containing 300 μL distilled water. Samples were incubated for 5 hours at room temperature to allow gut tissue to settle. C. bombi cell density was then assessed using a hemocytometer, and inoculum was prepared from the supernatant of the samples with sufficient concentrations of C. bombi cells. The supernatant was diluted to a concentration of 1200 cells/μL and had an equal volume of 50% sucrose solution added to result in a 25% sucrose solution. Each bee was fed 10 μL of inoculum, containing 6,000 C. bombi cells, using a 20 μL micropipette.

Bumble bee dissection and parasite quantification

Seven days after inoculation, bees were dissected to assess parasite loads. Gut tracts were extracted and crushed with a pestle in microcentrifuge tubes containing 300 μL distilled water. Samples were allowed to sit for 5 hours to allow gut tissue to settle. C. bombi cell concentrations in the gut extract were measured using a hemocytometer. C. bombi cells were counted in five cells of the hemocytometer and summed (0.004 µL each; 0.02 µL total).

Consumption experiment

To determine the effects of temperature on nectar consumption, we measured 24 h nectar consumption of bees incubated at 2 different temperatures, 27 or 33°C. Bees were removed from their natal colonies and starved in snap-cap vials 2 h before the experiment began. To begin the experiment, bees were given access to a 2 mL microcentrifuge feeding tube for 24 h. The feeding tube was filled with 1 mL artificial nectar (30% w/w sucrose) and punctured in the center of the lid with a 0.8 mm diameter sewing needle to allow bees access to the nectar. The upper half of the feeding tube was then inserted into a hole punched in the vial cap. Vials were incubated on their sides at a 10° angle in constant darkness. The feeding tube was weighed at the beginning and end of the 24 h experiment to estimate nectar consumption. The average mass loss of 6 control tubes, which were incubated identically in vials without bees, was subtracted from each bee’s estimated consumption to correct for leakage and evaporation. Bees were weighed post-experiment to allow use of bee mass as a model covariate. At each temperature, we ran 3 one-day trials of 24 bees (12 from each of 2 colonies), alternating between days of 27 and 33°C.

Statistics

Data were analyzed using R version 3.2.1 for Windows (R Core Team, 2014).

Mortality data

For ‘Variable’, for which exact dates of death were not recorded, mortality was analyzed using a generalized linear mixed model with binomial error distribution (Pinheiro et al., 2015). Probability of death was used as the response variable with nicotine treatment, anabasine treatment, and their interaction as predictor variables. Bee colony was included as a fixed predictor, and date of inoculation was included as a random factor. Wald tests (Lesnoff & Lancelot, 2012) were used to test the marginal significance of individual predictor variables (see Supplementary material script 1). Mortality data for ‘Stable’, in which we recorded time from inoculation to death to the nearest day, were analyzed using a Cox proportional hazards mixed-effects model (Therneau, 2015). Death hazard rate was used as the response variable; nicotine, anabasine, and their interaction as predictor variables; colony as a fixed predictor; and date of inoculation as a random factor (see Supplementary material script 3). To facilitate direct comparison between results of ‘Variable’ and ‘Stable’, the results of ‘Stable’ were also analyzed with the same binomial mixed model used for the ‘Variable’ experiment.

Parasite load

Parasite counts were found to best fit the log-normal distribution and were analyzed using generalized linear mixed models (Bates et al., 2015) with penalized quasi-likelihood parameter estimation (Venables & Ripley, 2002). Parasite counts were (x+1)-transformed for use as the response variable. Nicotine, anabasine, and their interaction were used as predictor variables. Bee colony was included as a fixed predictor, mass (at emergence from pupations) as a model covariate, and date of inoculation as a random factor. Marginal significance of individual terms was evaluated using Wald tests (Lesnoff & Lancelot, 2012). Code for analysis is given in Supplementary material script 2 (‘Variable’ experiment) and Supplementary material script 4 (‘Stable’ experiment).

Consumption experiment

Nectar consumption was analyzed using an analysis of variance with 24 h net nectar consumption as the response variable, temperature and colony as fixed predictors, and bee mass as a covariate. We excluded bees that died during the 24 h trial.

Results

bee.IDsource.colonytreatmentNicotine.treatmentAnabasine.treatmentmassinoculation.dateinoculateddead.before.dissectiondissection.count
1L2C000.140628-Feb11NA
2L2N100.118528-Feb1031
3L1A010.125328-Feb11NA
4L2together110.081628-Feb100
5L1C000.15283-Mar1036
6L2N100.100133-Mar1026
7L3A010.12913-Mar0NANA
8L3together110.12843-Mar11NA
9L4C000.12173-Mar1024
10L5N100.09983-Mar11NA
11L5A010.11463-Mar0NANA
12L5together110.09713-Mar0NANA
13L5C000.09073-Mar1010
14L5N100.06093-Mar11NA
15L2A010.0964-Mar11NA
16L3together110.20024-Mar1014
17L3C000.17774-Mar0NANA
18L3N100.2234-Mar11NA
19L3A010.2164-Mar104
20L4together110.1714-Mar108
21L4C000.22394-Mar0NANA
22L4N100.17574-Mar1014
23L4A010.15544-Mar107
24L4together110.10574-Mar1014
25L5C000.1094-Mar1026
26L5N100.07224-Mar11NA
27L5A010.08844-Mar101
28L5together110.08754-Mar100
29L5C000.10094-Mar100
30L5N100.07464-Mar0NANA
31L5A010.10114-Mar105
32L5together110.06494-Mar11NA
33L5C000.09964-Mar1055
34L4N100.22025-Mar11NA
35L4A010.19875-Mar1020
36L4together110.17785-Mar11NA
37L5C000.12365-Mar11NA
38L5N100.12235-Mar11NA
39L2A010.11426-Mar109
40L2together110.08966-Mar0NANA
41L2C000.07876-Mar1016
42L2N100.08476-Mar1014
43L2A010.09976-Mar11NA
44L2together110.09296-Mar1016
45L3C000.20176-Mar0NANA
46L3N100.26026-Mar11NA
47L4A010.17086-Mar108
48L4together110.20116-Mar11NA
49L4C000.21716-Mar105
50L4N100.13026-Mar11NA
51L4A010.20696-Mar11NA
52L5together110.15026-Mar11NA
53L5C000.16626-Mar11NA
54L5N100.14466-Mar100
55L5A010.0956-Mar1025
56L5together110.15617-Mar11NA
57L2C000.17917-Mar101
58L2N100.13437-Mar11NA
59L4A010.21257-Mar100
60L4together110.22557-Mar11NA
61L4C000.1887-Mar0NANA
62L4N100.23417-Mar1095
63L4A010.17237-Mar101
64L4together110.247-Mar1NANA
65L2C000.1237-Mar102
66L2N100.14697-Mar100
67L2A010.12717-Mar1028
68L3together110.21467-Mar11NA
69L5C000.16967-Mar1013
70L5N100.15857-Mar11NA
71L4A010.20857-Mar11NA
72L4together110.20267-Mar1012
73L4C000.18297-Mar106
74L4N100.20217-Mar11NA
75L4A010.24217-Mar106
76L2together110.13047-Mar106
77L2C000.19877-Mar1032
78L2N100.17167-Mar1083
79L2A010.1837-Mar108
80L2together110.12247-Mar1015
81L4C000.19467-Mar11NA
82L4N100.21977-Mar101
83L4A010.23567-Mar104
84L2together110.266111-Mar11NA
85L2C000.190611-Mar11NA
86L2N100.27211-Mar101
87L2A010.246911-Mar11NA
88L2together110.183511-Mar11NA
89L2C000.205711-Mar11NA
90L4N100.265511-Mar103
91L4A010.231911-Mar107
92L4together110.196211-Mar11NA
93L5C000.190411-Mar100
94L5N100.110711-Mar104
95L5A010.269511-Mar100
96L5together110.188211-Mar1011
97L2C000.253410-Mar11NA
98L2N100.192810-Mar11NA
99L2A010.179110-Mar1063
100L5together110.239810-Mar1011
101L5C000.214510-Mar1012
102L5N100.127410-Mar11NA
103L5A010.241310-Mar1095
104L5together110.167610-Mar11NA
105L5C000.224510-Mar1036
106L5N100.168110-Mar11NA
107L5A010.165710-Mar109
108L5together110.134210-Mar102
109L5C000.265410-Mar11NA
110L5N100.17310-Mar11NA
111L4A010.180210-Mar11NA
112L2together110.208612-Mar11NA
113L4C000.238112-Mar103
114L5N100.178912-Mar103
115L5A010.231212-Mar100
116L2together110.080312-Mar11NA
117L5C000.188613-Mar101
118L5N100.144313-Mar100
119L4A010.179813-Mar100
120L4together110.143813-Mar107
121L4C000.257114-Mar103
122L5N100.184214-Mar11NA
123L5A010.255914-Mar11NA
124L5together110.18614-Mar0NANA
125L5C000.144714-Mar100
126L5N100.179214-Mar1012
127L5A010.115114-Mar1023
128L5together110.159514-Mar11NA
129L5C000.180214-Mar11NA
130L4N100.116714-Mar0NANA
131L5A010.227114-Mar104
132L4together110.225914-Mar103
133L4C000.225714-Mar103
134L4N100.14210NANA
135L4A010.174817-Mar1017
136together110.086717-Mar11NA
137C00NA17-Mar101
138L4N100.131224-Mar105
139L4A010.122824-Mar11NA
140L4together110.119225-Mar11NA
141L6C000.151726-Mar1036
142L6N100.107626-Mar103
143L7A010.115626-Mar1NANA
144L7together110.138826-Mar105
145L7C000.123226-Mar1011
146L7N100.139526-Mar11NA
147L7A010.179327-Mar1024
148L7together110.177727-Mar1019
149L7C000.139327-Mar1050
150L7N100.163327-Mar11NA
151L7A010.13827-Mar1046
152L7together110.140727-Mar1041
153L4C000.10427-Mar10116
154L4N100.101127-Mar11NA
155L4A010.103127-Mar1036
156L4together110.114327-Mar0NANA
157L4C000.123727-Mar0NANA
158L4N100.140127-Mar11NA
159L4A010.144827-Mar0NANA
160L6together110.093427-Mar11NA
161L6C000.125827-Mar11NA
162L6N100.170427-Mar0NANA
163L6A010.130427-Mar1041
164L6together110.128527-Mar0NANA
165L6C000.142227-Mar10106
166L4N100.142527-Mar1179
167L4A010.191127-Mar1022
168L4together110.178227-Mar11NA
169L4C000.112827-Mar10150
170L4N100.130227-Mar1018
171L4A010.077127-Mar1057
172L4together110.086627-Mar10118
173L4C000.067827-Mar10136
174L6N100.197227-Mar1NANA
175L6A010.139627-Mar100
176L6together110.204627-Mar0NANA
177L6C000.118927-Mar11NA
178L6N100.125727-Mar1074
179L6A010.194527-Mar11NA
180L6together110.130827-Mar11NA
181L6C000.140427-Mar10204
182L6N100.188927-Mar1027
183L6A010.130827-Mar11NA
184L6together110.133227-Mar10191
185L6C000.177227-Mar103
186L6N100.133227-Mar105
187L6A010.162727-Mar1NANA
188L6together110.155828-Mar0NANA
189L6C000.164728-Mar101
190L6N100.181128-Mar11NA
191L6A010.148928-Mar104
192L6together110.169128-Mar1045
193L6C000.137628-Mar11NA
194L6N100.174228-Mar101
195L6A010.169628-Mar0NANA
196L6together110.137628-Mar11NA
197L7C000.099128-Mar1NANA
198L7N100.190128-Mar11NA
199L7A010.139728-Mar10112
This is a portion of the data; to view all the data, please download the file.
Dataset 1.Data for ‘Variable’ experiment.
Abbreviations: bee.ID—unique number assigned to each experimental bee; source.colony—colony of origin; treatment—letter corresponding to one of four diet treatments: “C” = control, “N” = nicotine (2 ppm), “A”= anabasine (5 ppm), “together” = nicotine (2 ppm) with anabasine (5 ppm); Nicotine.treatment—binary variable for diet treatment indicating “0” for no nicotine or “1” for 2 ppm nicotine; Anabasine.treatment—binary variable for diet treatment indicating “0” for no anabasine or “1” for 5 ppm anabasine; mass—mass of bee at time of emergence from pupal clump; inoculation.date—date of inoculation; inoculated—binary variable indicating whether bee was successfully inoculated (“1”) or not (“0”); dead.before.dissection—binary variable indicating whether bee died (“1”) or survived (“0”) until the time of dissection at 7 days; dissection.count—number of C. bombi cells counted in 0.02 µL gut extract.
beecolonytreatmentInoc.DateNicotineAnabasinenicotineanabasinemassTime.To.DeathDead.Binarycount
1E25Nicotine5/22/2014YesNo100.13841NA
2E25Anabasine5/22/2014NoYes010.10357061
3E25Nic + Ana5/22/2014YesYes110.130941NA
4E25Control5/22/2014NoNo000.17197034
5E25Nicotine5/23/2014YesNo100.1042-417821NA
6E25Anabasine5/23/2014NoYes010.109270105
7E25Nic + Ana5/23/2014YesYes110.08067045
8E25Control5/23/2014NoNo000.1008-417821NA
9E25Nicotine5/23/2014YesNo100.071341NA
10E25Anabasine5/23/2014NoYes010.055821NA
11E25Nic + Ana5/23/2014YesYes110.0493-417821NA
12E25Control5/23/2014NoNo000.0589-417821NA
13E25Nicotine5/23/2014YesNo100.1357044
14E25Anabasine5/23/2014NoYes010.087251NA
15E25Nic + Ana5/23/2014YesYes110.0755-417821NA
16E25Control5/23/2014NoNo000.0797060
17E25Nicotine6/5/2014YesNo100.0429-417950NA
18E25Anabasine6/5/2014NoYes010.097029
19E25Nic + Ana6/5/2014YesYes110.08667031
20E25Control6/6/2014NoNo000.10047046
21E25Nicotine6/6/2014YesNo100.103670219
22E25Anabasine6/6/2014NoYes010.12697049
23E25Nic + Ana6/9/2014YesYes110.12427023
24E25Control6/9/2014NoNo000.10987124
25E25Nicotine6/9/2014YesNo100.08547035
26E25Anabasine6/9/2014NoYes010.123361NA
27E25Nic + Ana6/10/2014YesYes110.1538708
28E25Control6/10/2014NoNo000.15427020
29E25Nicotine6/10/2014YesNo100.11687057
30E25Anabasine6/10/2014NoYes010.12657035
31E25Nic + Ana6/11/2014YesYes110.153641NA
32E25Control6/11/2014NoNo000.19327052
33E25Nicotine6/11/2014YesNo100.1148709
34E25Anabasine6/11/2014NoYes010.1267012
35E25Nic + Ana6/11/2014YesYes110.10317046
36E25Control6/11/2014NoNo000.13027034
37E25Nicotine6/11/2014YesNo100.12557056
38E25Anabasine6/11/2014NoYes010.07937023
39E25Nic + Ana6/11/2014YesYes110.120641NA
40E25Control6/11/2014NoNo000.170441NA
41E25Nicotine6/11/2014YesNo100.1109709
42E25Anabasine6/12/2014NoYes010.19377050
43E25Nic + Ana6/12/2014YesYes110.14997128
44E25Control6/12/2014NoNo000.131741NA
45E25Nicotine6/12/2014YesNo100.15047023
46E25Anabasine6/12/2014NoYes010.216441NA
47E25Nic + Ana6/12/2014YesYes110.190821NA
48E25Control6/12/2014NoNo000.136341NA
49E25Nicotine6/12/2014YesNo100.13257017
50E25Anabasine6/12/2014NoYes010.11677053
51E25Nic + Ana6/12/2014YesYes110.18887051
52E25Control6/12/2014NoNo000.145351NA
53E25Nicotine6/12/2014YesNo100.138911NA
54E25Anabasine6/12/2104NoYes010.17627065
55E25Nic + Ana6/12/2014YesYes110.151541NA
56E25Control6/12/2014NoNo000.151651NA
57E25Nicotine6/12/2014YesNo100.13561NA
58E25Anabasine6/13/2014NoYes010.071261NA
59E25Nic + Ana6/13/2014YesYes110.2177028
60E25Control6/13/2014NoNo000.1837057
61E25Nicotine6/13/2014YesNo100.22367042
62E25Anabasine6/13/2014NoYes010.11187067
63E25Nic + Ana6/13/2014YesYes110.24157040
64E25Control6/13/2014NoNo000.1317021
65E25Nicotine6/16/2014YesNo100.1027-418061NA
66E25Anabasine6/19/2014NoYes010.0967028
67E25Nic + Ana6/19/2014YesYes110.14-418090NA
68E25Control6/19/2014NoNo000.08757079
69E25Nicotine6/19/2014YesNo100.08747022
70E25Anabasine6/19/2014NoYes010.08547026
71E25Nic + Ana6/20/2014YesYes110.13247023
72E25Control6/20/2014NoNo000.09187041
73E25Nicotine6/20/2014YesNo100.11027047
74E25Anabasine6/20/2014NoYes010.08017052
75E25Nic + Ana6/20/2014YesYes110.10187034
76E25Control6/20/2014NoNo000.16037035
77E25Nicotine6/20/2014YesNo100.13057026
78E25Anabasine6/20/2014NoYes010.14367030
79E25Nic + Ana6/20/2014YesYes110.211921NA
81E26Nicotine5/22/2014YesNo100.1138-417811NA
82E26Anabasine5/22/2014NoYes010.102970116
83E26Nic + Ana5/22/2014YesYes110.12741NA
84E26Control5/22/2014NoNo000.1452-417811NA
85E26Nicotine5/22/2014YesNo100.102151NA
86E26Anabasine5/22/2014NoYes010.0918-417811NA
87E26Nic + Ana5/23/2014YesYes110.12577096
88E26Control5/23/2014NoNo000.111161NA
89E26Nicotine5/23/2014YesNo100.1247-417821NA
90E26Anabasine5/23/2014NoYes010.10527093
91E26Nic + Ana5/23/2014YesYes110.15077022
92E26Control5/23/2014NoNo000.09027054
93E26Nicotine5/23/2014YesNo100.0139131NA
94E26Anabasine5/23/2014NoYes010.121921NA
95E26Nic + Ana5/23/2014YesYes110.1141-417821NA
96E26Control5/23/2014NoNo000.102541NA
97E26Nicotine5/23/2014YesNo100.12317128
98E26Anabasine5/26/2014NoYes010.1027-417850NA
99E26Nic + Ana5/26/2014YesYes110.130131NA
100E26Control5/26/2014NoNo000.09217028
101E26Nicotine5/27/2014YesNo100.0627073
102E26Anabasine5/27/2014NoYes010.10617080
103E26Nic + Ana5/28/2014YesYes110.087970168
104E26Control5/29/2014NoNo000.108371NA
105E26Nicotine5/30/2014YesNo100.11797037
106E26Anabasine5/30/2014NoYes010.05097042
107E26Nic + Ana5/30/2014YesYes110.095270152
108E26Control5/30/2014NoNo000.06367014
109E26Nicotine5/30/2014YesNo100.076670124
110E26Anabasine6/2/2014NoYes010.109941NA
111E26Nic + Ana6/2/2014YesYes110.054370136
112E26Control6/2/2014NoNo000.06361NA
113E26Nicotine6/3/2014YesNo100.0408-417930NA
114E26Anabasine6/4/2014NoYes010.07367055
115E26Nic + Ana6/4/2014YesYes110.1206-417940NA
116E26Control6/4/2014NoNo000.0635-417940NA
117E26Nicotine6/6/2014YesNo100.12567086
118E26Anabasine6/6/2014NoYes010.113270103
119E26Nic + Ana6/6/2014YesYes110.072970105
120E26Control6/6/2014NoNo000.11147025
121E26Nicotine6/6/2014YesNo100.1162-11NA
122E26Anabasine6/9/2014NoYes010.18321NA
123E26Nic + Ana6/9/2014YesYes110.17947039
124E26Control6/9/2014NoNo000.16337033
125E26Nicotine6/10/2014YesNo100.1979-418000NA
126E26Anabasine6/10/2014NoYes010.11667088
127E26Nic + Ana6/10/2014YesYes110.19947049
128E26Control6/10/2014NoNo000.13367026
129E26Nicotine6/10/2014YesNo100.1194-418000NA
130E26Anabasine6/10/2014NoYes010.10967060
131E26Nic + Ana6/10/2014YesYes110.11967050
132E26Control6/11/2014NoNo000.1263707
133E26Nicotine6/11/2014YesNo100.20227021
134E26Anabasine6/11/2014NoYes010.20027062
135E26Nic + Ana6/11/2014YesYes110.1341NA
136E26Control6/11/2014NoNo000.20470109
137E26Nicotine6/11/2014YesNo100.12827052
138E26Anabasine6/11/2014NoYes010.209331NA
139E26Nic + Ana6/11/2104YesYes110.12703
140E26Control6/11/2104NoNo000.1974-746730NA
141E26Nicotine6/12/2014YesNo100.195541NA
142E26Anabasine6/12/2014NoYes010.2237051
143E26Nic + Ana6/12/2014YesYes110.136731NA
144E26Control6/12/2014NoNo000.12547088
145E26Nicotine6/12/2014YesNo100.083941NA
146E26Anabasine6/13/2014NoYes010.16317018
147E26Nic + Ana6/13/2014YesYes110.129670126
148E26Control6/13/2014NoNo000.157841NA
149E26Nicotine6/13/2014YesNo100.107570104
150E26Anabasine6/13/2014NoYes010.114451NA
151E26Nic + Ana6/13/2014YesYes110.13057044
152E26Control6/13/2014NoNo000.10837038
153E26Nicotine6/13/2014YesNo100.10317076
154E26Anabasine6/16/2014NoYes010.17361NA
155E26Nic + Ana6/16/2014YesYes110.128761NA
156E26Control6/17/2014NoNo000.16647024
157E26Nicotine6/17/2014YesNo100.14627022
158E26Anabasine6/18/2014NoYes010.093251NA
159E26Nic + Ana6/18/2014YesYes110.1483-418080NA
160E26Control6/18/2014NoNo000.1227-418080NA
161E27Nicotine5/22/2014YesNo100.12047061
162E27Anabasine5/22/2014NoYes010.107531NA
163E27Nic + Ana5/23/2014YesYes110.165708
164E27Control5/23/2014NoNo000.122241NA
165E27Nicotine5/23/2014YesNo100.108661NA
166E27Anabasine5/23/2014NoYes010.15617010
167E27Nic + Ana5/23/2014YesYes110.140731NA
168E27Control5/23/2014NoNo000.09337038
169E27Nicotine5/23/2014YesNo100.11117044
170E27Anabasine5/23/2014NoYes010.06457038
171E27Nic + Ana5/23/2014YesYes110.09057012
172E27Control5/26/2014NoNo000.072670106
173E27Nicotine5/26/2014YesNo100.045331NA
174E27Anabasine5/27/2014NoYes010.105970151
175E27Nic + Ana5/27/2014YesYes110.150470123
176E27Control5/28/2014NoNo000.132770140
177E27Nicotine5/28/2014YesNo100.081170209
178E27Anabasine5/28/2014NoYes010.11367017
179E27Nic + Ana5/28/2014YesYes110.106570174
180E27Control5/28/2014NoNo000.08027076
181E27Nicotine5/28/2014YesNo100.098441NA
182E27Anabasine5/28/2014NoYes010.0743-417870NA
183E27Nic + Ana5/28/2014YesYes110.080670104
184E27Control5/28/2014NoNo000.053870143
185E27Nicotine5/29/2014YesNo100.10217131
186E27Anabasine5/29/2014NoYes010.0718-417880NA
187E27Nic + Ana5/30/2014YesYes110.042821NA
188E27Control6/3/2014NoNo000.073370NA
189E27Nicotine6/3/2014YesNo100.059961NA
190E27Anabasine6/4/2014NoYes010.09867050
191E27Nic + Ana6/4/2014YesYes110.0799-417940NA
192E27Control6/4/2014NoNo000.0771-417940NA
193E27Nicotine6/4/2014YesNo100.0639-417940NA
194E27Anabasine6/5/2014NoYes010.081501NA
195E27Nic + Ana6/5/2014YesYes110.09617070
196E27Control6/5/2014NoNo000.08347046
197E27Nicotine6/5/2014YesNo100.08757051
198E27Anabasine6/6/2014NoYes010.126521NA
199E27Nic + Ana6/6/2014YesYes110.09957054
200E27Control6/6/2014NoNo000.1015-417960NA
This is a portion of the data; to view all the data, please download the file.
Dataset 2.Data for ‘Stable’ experiment.
Abbreviations: bee—unique number assigned to each experimental bee; colony—colony of origin; treatment—describes one of four diet treatments: “Control” = control; “Nicotine” = nicotine (2 ppm), “Anabasine”= anabasine (5 ppm), “Nic + Ana” = nicotine (2 ppm) with anabasine (5 ppm); Inoc.Date—date of inoculation; Nicotine—column denoting whether nectar treatment contained (“Yes”) or did not contain (“No”) 2 ppm nicotine; Anabasine—column denoting whether nectar treatment contained (“Yes”) or did not contain (“No”) 5 ppm anabasine; nicotine—binary variable for diet treatment indicating “0” for no nicotine or “1” for 2 ppm nicotine; anabasine—binary variable for diet treatment indicating “0” for no anabasine or “1” for 5 ppm anabasine; mass—mass of bee at time of emergence from pupal clump; Time.To.Death—number of days from inoculation to death, with negative numbers denoting excluded bees that died before inoculation or escaped before dissection; Dead.Binary—binary variable indicating whether bee died (“1”) or survived (“0”) until the time of dissection at 7 days; count—number of C. bombi cells counted in 0.02 µL gut extract.

‘Variable’

In variable temperature conditions, the nicotine treatment significantly increased mortality (Table 1). Nearly half of bees fed nicotine-containing nectar died within 7 days of inoculation, which was nearly double the frequency of death in treatments without nicotine (Figure 1). Anabasine did not affect mortality, and there was no significant interaction between the two alkaloid treatments (Figure 1, Table 1).

Table 1. Effects of nicotine and anabasine consumption on mortality in ‘Variable’ experiment.

Table shows binomial mixed model results of χ2 tests for effects of predictor variables on probability of death during the 7 d experiment.

Sourceχ2DfP
Nicotine4.174910.041
Anabasine0.037410.85
Nicotine*Anabasine0.025610.87
Colony0.91140.92
033bd580-1aa3-4ade-8de7-f9360701b72f_figure1.gif

Figure 1. Effects of nicotine and anabasine on mortality in ‘Variable’ experiment.

Points show adjusted mean probability of death in each treatment group. Error bars represent ±1 standard error. Sample sizes: n=45 (Control), n=46 (Nicotine), n=46 (Anabasine), n=41 (Nicotine + Anabasine).

Nicotine (linear model β = -1.01 ± 0.295 standard error) and anabasine (β = -0.94 ± 0.31 S.E.) each significantly decreased parasite loads. However, nicotine and anabasine displayed antagonistic effects (Nicotine * Anabasine β = 1.96 ± 0.44 S.E.), such that bees consuming both alkaloids did not realize the medicinal effects of either compound (Figure 2, Table 2). Parasite load had a significant negative relationship with bee mass (β = -11.17 ± 3.60 S.E., Table 2), indicating that larger bees had lower parasite loads after controlling for other factors.

033bd580-1aa3-4ade-8de7-f9360701b72f_figure2.gif

Figure 2. Effects of nicotine and anabasine on parasite load in ‘Variable’ experiment.

Points show adjusted mean parasite count in each treatment group. Error bars represent ±1 standard error. Sample sizes: n=31 (Control), n=24 (Nicotine), n=33 (Anabasine), n=20 (Nicotine + Anabasine).

Table 2. Effects of nicotine and anabasine on parasite loads in ‘Variable’ experiment.

Results of Wald tests for marginal significance of terms in a generalized linear mixed model with penalized quasi-likelihood parameter estimation. “Mass” refers to bee mass at time of emergence.

Sourceχ2DfP
Nicotine10.05410.0025
Anabasine12.8431<0.001
Nicotine*Anabasine22.0451<0.001
Colony15.4840.0038
Mass10.51710.0012

‘Stable’

Under controlled conditions (27°C with constant darkness), neither alkaloid nor their interaction significantly affected mortality (Figure 3, Table 3, Table S2). Although the death hazard ratio was lowest in the Control group (estimate = 0.82, 95% CI = (0.56, 1.19)) and highest in the group that received both alkaloids (estimate = 1.19, 95% CI = (0.90, 1.78)), this difference did not approach statistical significance (pairwise comparison: z = 1.49, P = 0.45). However, nicotine significantly increased parasite loads (β = 0.28 ± 0.12 S.E., Table 4), while the effects of anabasine (β = 0.20 ± 0.12 S.E.) were also positive but not significant (Figure 4, Table 4). This was the opposite result of that observed in ‘Variable’, in which alkaloid ingestion decreased the severity of Crithidia infection. Although much weaker than in ‘Variable’, we found the same pattern of antagonistic interaction between the two alkaloids (Nicotine * Anabasine β = -0.26 ± 0.16 S.E., Figure 4), indicating that the deleterious effects of each compound were reduced in bees consuming the nicotine + anabasine combination (Figure 4). However, this interaction was not statistically significant (Table 4). Overall parasite loads in ‘Stable’ were much higher, with median parasite counts (37.5 cells * 0.02 µL-1) more than triple those observed in ‘Variable’ (11.0 cells * 0.02 µL-1). As in ‘Variable’, there was a significant negative relationship between mass at emergence and parasite count (β = -3.41 ± 1.32 S.E., Table 4). Average bee mass in ‘Stable’ (121 mg) was significantly lower than in ‘Variable’ (160 mg) (t(290) = -9.17, P<0.001), possibly reflecting elevated pre-experiment mortality of smaller bees under the fluctuating conditions of ‘Variable’. These bees might have been more likely to die during pupation or prior to inoculation.

033bd580-1aa3-4ade-8de7-f9360701b72f_figure3.gif

Figure 3. Effects of nicotine and anabasine on mortality in ‘Stable’ experiment.

Lines show survival curves for bees each treatment group. There were no significant effects of diet treatments on survival. Sample sizes: n=83 (Control), n=79 (Nicotine), n=91 (Anabasine), n=86 (Nicotine + Anabasine).

Table 3. Effects of nicotine and anabasine consumption on mortality in ‘Stable’ experiment.

Table shows marginal significance of individual terms in Cox proportional hazards test for effects of predictor variables on mortality hazard rate.

Sourceχ2DfP
Nicotine0.1410.71
Anabasine0.2110.65
Nicotine*Anabasine0.1910.66
Colony7.630.054

Table 4. Effects of nicotine and anabasine on parasite loads in ‘Stable’ experiment.

Results of Wald tests for marginal significance of terms in a generalized linear mixed model with penalized quasi-likelihood parameter estimation. “Mass” refers to bee mass at time of emergence.

Sourceχ2DfP
Nicotine5.8410.026
Anabasine2.7810.095
Nicotine*Anabasine2.5910.11
Colony6.7630.080
Mass6.9110.0086
033bd580-1aa3-4ade-8de7-f9360701b72f_figure4.gif

Figure 4. Effects of nicotine and anabasine on parasite load in ‘Stable’ experiment.

Points show adjusted mean parasite count in each treatment group. Error bars represent ±1 standard error. Sample sizes: n=60 (Control), n=59 (Nicotine), n=66 (Anabasine), n=61 (Nicotine + Anabasine).

Consumption experiment

Contrary to our expectation that increased temperature would increase nectar consumption, bees drank significantly less when incubated at 33°C than at 27°C (Table 5). Mass-adjusted mean consumption at 33°C (117 mg) was less than half that at 27°C (256 mg) (Figure 5). Bee mass was also a significant predictor of consumption, but colony of origin was not (Table 5).

Table 5. Anova results for effects of incubation temperature on nectar consumption at 27°C and 33°C.

SourceSSDfFP
Temperature0.64164.04<0.001
Colony0.0110.980.33
Mass0.24123.61<0.001
Residuals1.38138
033bd580-1aa3-4ade-8de7-f9360701b72f_figure5.gif

Figure 5. Effects of incubation temperature on nectar consumption in uninfected bees.

Points show adjusted mean nectar consumption at each incubation temperature. Error bars represent ±1 standard error. Sample sizes: n=69 (27°C), n=74 (33°C).

DayTempColonyVialTypeDateStart.timeLid.mass.0hcomment.0hEnd.timeLid.mass.24hBee.masscomment.24hconsumption.grossconsumption.net
One33PD21Bee12-Nov14:152.327613:552.08450.13880.24310.1796
One33PD22Bee12-Nov14:152.272213:552.07630.11930.19590.1324
One33PD23Bee12-Nov14:152.303713:552.05250.11620.25120.1877
One33PD24Bee12-Nov14:152.185713:551.8030.1411Moist0.38270.3192
One33PD25Bee12-Nov14:152.348513:551.97750.1034Dead-Exclude0.371
One33PD26Bee12-Nov14:152.281813:552.01160.0952Wet- Keep0.27020.2067
One33PD27Bee12-Nov14:152.244613:551.4230.2056Wet- Exclude0.8216
One33PD28Bee12-Nov14:152.27813:551.52070.1753Wet- Exclude0.7573
One33PD29Bee12-Nov14:152.32713:551.99270.18750.33430.2708
One33PD210Bee12-Nov14:152.334813:552.09890.11890.23590.1724
One33PD211Bee12-Nov14:152.365413:552.19390.08880.17150.108
One33PD212Bee12-Nov14:152.271513:551.98150.12580.290.2265
One33PD313Bee12-Nov14:152.149613:551.67390.1974Wet- Possible Exclude0.47570.4122
One33PD314Bee12-Nov14:152.226613:551.37090.4516Wet- Exclude0.8557
One33PD315Bee12-Nov14:152.321713:551.26480.239Wet- Exclude1.0569
One33PD316Bee12-Nov14:152.348213:551.29580.1372Very Wet- Exclude1.0524
One33PD317Bee12-Nov14:152.308813:551.96720.25270.34160.2781
One33PD318Bee12-Nov14:152.305113:551.91760.30520.38750.324
One33PD319Bee12-Nov14:152.345513:552.12230.28010.22320.1597
One33PD320Bee12-Nov14:152.314213:551.86820.2022Possible Spill- Keep0.4460.3825
One33PD321Bee12-Nov14:152.336713:551.99240.11520.34430.2808
One33PD322Bee12-Nov14:152.320413:551.99660.25240.32380.2603
One33PD323Bee12-Nov14:152.303413:552.14770.10950.15570.0922
One33PD324Bee12-Nov14:152.278113:551.96580.20650.31230.2488
One3325Control12-Nov14:152.336213:552.27181 Drop Leaked0.0644
One3326Control12-Nov14:152.321913:552.25241 Drop Leaked0.0695
One3327Control12-Nov14:152.249713:552.16211 Drop Leaked0.0876
One3328Control12-Nov14:152.32513:552.28741 Drop Leaked0.0376
One3329Control12-Nov14:152.321313:552.25291 Drop Leaked0.0684
One3330Control12-Nov14:152.217713:552.16421 Drop Leaked0.0535
Two27PD231Bee14-Nov12:002.364611:502.05820.12430.30640.269533333
Two27PD232Bee14-Nov12:002.365611:502.03180.14180.33380.296933333
Two27PD233Bee14-Nov12:002.344111:501.85730.16430.48680.449933333
Two27PD234Bee14-Nov12:002.362611:502.34030.0853DEAD
Two27PD235Bee14-Nov12:002.350311:502.04630.19430.3040.267133333
Two27PD236Bee14-Nov12:002.347411:502.08050.09720.26690.230033333
Two27PD237Bee14-Nov12:002.342211:501.93310.13210.40910.372233333
Two27PD238Bee14-Nov12:002.354611:502.03630.13030.31830.281433333
Two27PD239Bee14-Nov12:002.349811:502.11260.1350.23720.200333333
Two27PD240Bee14-Nov12:002.352311:502.3230.0782DEAD
Two27PD241Bee14-Nov12:002.343511:502.12570.0970.21780.180933333
Two27PD242Bee14-Nov12:002.324811:502.00390.16360.32090.284033333
Two27PD443Bee14-Nov12:002.354411:502.14580.12360.20860.171733333
Two27PD444Bee14-Nov12:002.363911:501.76650.1690.59740.560533333
Two27PD445Bee14-Nov12:002.326811:502.10770.11520.21910.182233333
Two27PD446Bee14-Nov12:002.309511:502.29110.1216DEAD
Two27PD447Bee14-Nov12:002.328411:502.10930.10660.21910.182233333
Two27PD448Bee14-Nov12:002.3211:501.90980.15870.41020.373333333
Two27PD449Bee14-Nov12:002.33211:502.22470.11720.10730.070433333
Two27PD450Bee14-Nov12:002.331211:502.10370.13870.22750.190633333
Two27PD451Bee14-Nov12:002.359111:502.11680.13940.24230.205433333
Two27PD452Bee14-Nov12:002.35411:501.95960.17560.39440.357533333
Two27PD453Bee14-Nov12:002.313911:501.87070.17070.44320.406333333
Two27PD454Bee14-Nov12:002.358911:502.05580.12970.30310.266233333
Two2755Control14-Nov12:002.35911:502.33271 Drop Leaked0.0263
Two2756Control14-Nov12:002.350411:502.3041 Drop Leaked0.0464
Two2757Control14-Nov12:002.332211:502.28041 Drop Leaked0.0518
Two2758Control14-Nov12:002.358911:502.321 Drop Leaked0.0389
Two2759Control14-Nov12:002.331511:502.31141 Drop Leaked0.0201
Two2760Control14-Nov12:002.254411:502.21671 Drop Leaked0.0377
Three33PD261Bee15-Nov12:102.399912:002.27560.140.12430.044733333
Three33PD262Bee15-Nov12:102.386112:002.09740.13530.28870.209133333
Three33PD263Bee15-Nov12:102.425612:002.31120.11890.11440.034833333
Three33PD264Bee15-Nov12:102.361412:002.16860.09670.19280.113233333
Three33PD265Bee15-Nov12:102.373312:002.18910.08520.18420.104633333
Three33PD266Bee15-Nov12:102.368512:002.16220.1180.20630.126733333
Three33PD267Bee15-Nov12:102.344812:002.22340.10380.12140.041833333
Three33PD268Bee15-Nov12:102.395712:002.27980.07090.11590.036333333
Three33PD269Bee15-Nov12:102.415212:002.32750.09550.08770.008133333
Three33PD270Bee15-Nov12:102.411112:002.26620.1050.14490.065333333
Three33PD271Bee15-Nov12:102.374512:001.99240.20030.38210.302533333
Three33PD272Bee15-Nov12:102.419312:002.30980.09070.10950.029933333
Three33PD473Bee15-Nov12:102.406112:002.21620.13720.18990.110333333
Three33PD474Bee15-Nov12:102.431512:002.15890.12870.27260.193033333
Three33PD475Bee15-Nov12:102.426612:002.15520.12480.27140.191833333
Three33PD476Bee15-Nov12:102.394212:002.15390.14130.24030.160733333
Three33PD477Bee15-Nov12:102.424112:002.17240.13480.25170.172133333
Three33PD478Bee15-Nov12:102.398712:002.24020.10330.15850.078933333
Three33PD479Bee15-Nov12:102.412312:002.15130.16450.2610.181433333
Three33PD480Bee15-Nov12:102.432412:002.23760.1370.19480.115233333
Three33PD481Bee15-Nov12:102.359712:002.02480.12430.33490.255333333
Three33PD482Bee15-Nov12:102.398612:002.16990.12960.22870.149133333
Three33PD483Bee15-Nov12:102.420112:002.13710.1950.2830.203433333
Three33PD484Bee15-Nov12:102.393812:002.18530.13750.20850.128933333
Three3385Control15-Nov12:102.40812:002.33671 Drop Leaked0.0713
Three3386Control15-Nov12:102.41112:002.33121 Drop Leaked0.0798
Three3387Control15-Nov12:102.418812:002.3431 Drop Leaked0.0758
Three3388Control15-Nov12:102.415812:002.33351 Drop Leaked0.0823
Three3389Control15-Nov12:102.383812:002.29941 Drop Leaked0.0844
Three3390Control15-Nov12:102.341612:002.25781 Drop Leaked0.0838
Four27PD291Bee16-Nov13:302.399213:201.96890.15570.43030.379566667
Four27PD292Bee16-Nov13:302.403913:202.30580.12930.09810.047366667
Four27PD293Bee16-Nov13:302.419213:202.23530.12280.18390.133166667
Four27PD294Bee16-Nov13:302.427613:202.32310.14310.10450.053766667
Four27PD295Bee16-Nov13:302.407213:202.23080.16240.17640.125666667
Four27PD296Bee16-Nov13:302.419213:202.21030.10910.20890.158166667
Four27PD297Bee16-Nov13:302.419413:202.3120.1140.10740.056666667
Four27PD298Bee16-Nov13:302.332713:202.13370.09720.1990.148266667
Four27PD299Bee16-Nov13:302.312613:201.86240.15330.45020.399466667
Four27PD2100Bee16-Nov13:302.375713:202.11980.09850.25590.205166667
Four27PD2101Bee16-Nov13:302.405613:202.22160.11860.1840.133266667
Four27PD2102Bee16-Nov13:302.375213:201.92120.15040.4540.403266667
Four27PD4103Bee16-Nov13:302.33813:202.19180.12930.14620.095466667
Four27PD4104Bee16-Nov13:302.37213:201.77730.21890.59470.543966667
Four27PD4105Bee16-Nov13:302.261513:201.9690.18110.29250.241766667
Four27PD4106Bee16-Nov13:302.423413:202.08520.24990.33820.287466667
Four27PD4107Bee16-Nov13:302.426613:202.04010.20240.38650.335766667
Four27PD4108Bee16-Nov13:302.321813:202.00680.14580.3150.264266667
Four27PD4109Bee16-Nov13:302.290513:201.89450.1610.3960.345266667
Four27PD4110Bee16-Nov13:302.428113:202.10350.16950.32460.273866667
Four27PD4111Bee16-Nov13:302.351213:202.03340.14160.31780.267066667
Four27PD4112Bee16-Nov13:302.298413:201.63660.14350.66180.611066667
Four27PD4113Bee16-Nov13:302.373813:202.18360.14460.19020.139466667
Four27PD4114Bee16-Nov13:302.401313:202.14340.13660.25790.207166667
Four27115Control16-Nov13:302.344613:202.29160.053
Four27116Control16-Nov13:302.330813:202.27420.0566
Four27117Control16-Nov13:302.41713:202.37580.0412
Four27118Control16-Nov13:302.38313:202.34380.0392
Four27119Control16-Nov13:302.405413:202.35810.0473
Four27120Control16-Nov13:302.392213:202.32510.0671
Five33PD2121Bee19-Nov13:452.385813:352.25240.09760.13340.0212
Five33PD2122Bee19-Nov13:452.382113:352.27010.10210.112-0.0002
Five33PD2123Bee19-Nov13:452.384713:352.24620.08520.13850.0263
Five33PD2124Bee19-Nov13:452.386813:352.26180.10260.1250.0128
Five33PD2125Bee19-Nov13:452.3713:352.1540.15620.2160.1038
Five33PD2126Bee19-Nov13:452.379713:352.28460.0996DEAD- Exclude0.0951
Five33PD2127Bee19-Nov13:452.382113:352.24780.07190.13430.0221
Five33PD2128Bee19-Nov13:452.384313:352.1250.10260.25930.1471
Five33PD2129Bee19-Nov13:452.383213:352.14790.10680.23530.1231
Five33PD2130Bee19-Nov13:452.346813:352.06550.11240.28130.1691
Five33PD2131Bee19-Nov13:452.347413:352.19890.11730.14850.0363
Five33PD2132Bee19-Nov13:452.380713:352.13880.1150.24190.1297
Five33PD4133Bee19-Nov13:452.388813:352.18660.1340.20220.09
Five33PD4134Bee19-Nov13:452.380813:352.07950.15950.30130.1891
Five33PD4135Bee19-Nov13:452.38713:352.0850.15750.3020.1898
Five33PD4136Bee19-Nov13:452.400513:352.11220.14330.28830.1761
Five33PD4137Bee19-Nov13:452.397913:352.12560.12370.27230.1601
Five33PD4138Bee19-Nov13:452.40313:352.19340.13010.20960.0974
Five33PD4139Bee19-Nov13:452.34413:352.20230.1620.14170.0295
Five33PD4140Bee19-Nov13:452.400313:352.3160.11810.0843-0.0279
Five33PD4141Bee19-Nov13:452.402213:352.29540.13490.1068-0.0054
Five33PD4142Bee19-Nov13:452.390413:352.18560.15620.20480.0926
Five33PD4143Bee19-Nov13:452.357113:352.23210.09390.1250.0128
Five33PD4144Bee19-Nov13:452.397113:352.25170.08350.14540.0332
Five33145Control19-Nov13:452.384813:352.25830.1265
Five33146Control19-Nov13:452.338213:352.21990.1183
Five33147Control19-Nov13:452.376513:352.23790.1386
Five33148Control19-Nov13:452.378913:352.23530.1436
Five33149Control19-Nov13:452.383113:352.3760.0071
Five33150Control19-Nov13:452.37313:352.23390.1391
Six27PD2151Bee21-Nov11:452.346411:352.10260.10450.24380.176766667
Six27PD2152Bee21-Nov11:452.3676drone11:352.24120.09280.12640.059366667
Six27PD2153Bee21-Nov11:452.336711:352.10260.09880.23410.167066667
Six27PD2154Bee21-Nov11:452.39211:351.90270.14170.48930.422266667
Six27PD2155Bee21-Nov11:452.375Bee is sluggish, sugar on lid, use lidx211:352.06430.12680.31070.243666667
Six27PD2156Bee21-Nov11:452.385211:352.24350.10340.14170.074666667
Six27PD2157Bee21-Nov11:452.305711:351.98770.10310.3180.250966667
Six27PD2158Bee21-Nov11:452.409811:352.09510.10320.31470.247666667
Six27PD2159Bee21-Nov11:452.408311:351.81130.09080.5970.529966667
Six27PD2160Bee21-Nov11:452.3575drone/ didn�t drink anything11:352.06650.14350.2910.223966667
Six27PD2161Bee21-Nov11:452.39911:352.05170.10680.34730.280266667
Six27PD2162Bee21-Nov11:452.3618drone11:352.19560.15590.16620.099166667
Six27PD4163Bee21-Nov11:452.503811:352.10950.15670.39430.327266667
Six27PD4164Bee21-Nov11:452.349311:352.04720.13390.30210.235066667
Six27PD4165Bee21-Nov11:452.345211:351.91160.13080.43360.366566667
Six27PD4166Bee21-Nov11:452.356311:351.90530.20370.4510.383966667
Six27PD4167Bee21-Nov11:452.348711:352.16520.15750.18350.116466667
Six27PD4168Bee21-Nov11:452.387511:351.71630.15960.67120.604166667
Six27PD4169Bee21-Nov11:452.322411:352.0520.16270.27040.203366667
Six27PD4170Bee21-Nov11:452.328411:352.10240.08670.2260.158966667
Six27PD4171Bee21-Nov11:452.320511:351.66050.14890.660.592966667
Six27PD4172Bee21-Nov11:452.373511:352.08940.13070.28410.217066667
Six27PD4173Bee21-Nov11:452.353511:351.79470.13770.55880.491766667
Six27PD4174Bee21-Nov11:452.334711:351.97130.20620.36340.296366667
Six27175Control21-Nov11:452.363511:352.29850.065
Six27176Control21-Nov11:452.323511:352.2470.0765
Six27177Control21-Nov11:452.333511:352.26820.0653
Six27178Control21-Nov11:452.366211:352.32440.0418
Six27179Control21-Nov11:452.336711:352.28110.0556
Six27180Control21-Nov11:452.332711:352.23470.098
Seven33PD2181Bee22-Nov11:402.347411:402.11130.17440.23610.135383333
Seven33PD2182Bee22-Nov11:402.352511:402.0750.11480.27750.176783333
Seven33PD2183Bee22-Nov11:402.319211:402.16060.10270.15860.057883333
Seven33PD2184Bee22-Nov11:402.360611:402.22670.07590.13390.033183333
Seven33PD2185Bee22-Nov11:402.35911:402.17450.08790.18450.083783333
Seven33PD2186Bee22-Nov11:402.358211:402.14330.15020.21490.114183333
Seven33PD2187Bee22-Nov11:402.350511:402.22190.09010.12860.027883333
Seven33PD2188Bee22-Nov11:402.335411:402.17270.10980.16270.061983333
Seven33PD2189Bee22-Nov11:402.326511:402.0460.15570.28050.179783333
Seven33PD2190Bee22-Nov11:402.340911:402.21260.14330.12830.027583333
Seven33PD2191Bee22-Nov11:402.334411:402.16830.11880.16610.065383333
Seven33PD2192Bee22-Nov11:402.317811:402.0340.13950.28380.183083333
Seven33PD4193Bee22-Nov11:402.27811:402.01370.14660.26430.163583333
Seven33PD4194Bee22-Nov11:402.360411:402.17890.13390.18150.080783333
Seven33PD4195Bee22-Nov11:402.364111:402.13560.19260.22850.127783333
Seven33PD4196Bee22-Nov11:402.370611:402.12020.13580.25040.149683333
Seven33PD4197Bee22-Nov11:402.330311:402.00040.12090.32990.229183333
Seven33PD4198Bee22-Nov11:402.333311:402.15220.09650.18110.080383333
Seven33PD4199Bee22-Nov11:402.392411:402.15970.14690.23270.131983333
This is a portion of the data; to view all the data, please download the file.
Dataset 3.Data for Consumption experiment.
Day ‘One’ was omitted from analysis due to use of a different style of feeder tube from that used on subsequent days. Abbreviations: Day: day of experiment; Temp: temperature of incubator; Colony: hive of origin; Vial: unique identifier for each sample; Type: “Bee” or evaporation “Control” (i.e., empty vial without a bee); Date: Date of start of trial; Start.time: time at start of trial; Lid.mass.0h: mass of full tube with nectar at start of trial; comment.0h: observations at start time; End.time: clock time on following day at end of trial; Lid.mass.24h: mass of nectar tube after 24h of bee feeding; Bee.mass: mass of bee following the trial; comment.24h: observations at end of trial; consumption.gross: Difference between Lid.mass.0h and Lid.mass.24h; consumption.net: consumption.gross minus average mass loss of bee-free control tubes on same date, which estimates amount of nectar consumed. Incubator: location of trial.

Discussion

Nicotine increased mortality under variable temperature conditions

Nicotine consumption increased mortality in ‘Variable’, but did not affect mortality in ‘Stable’. The climatic differences between the two experiments may be responsible for this context-dependent response. In ‘Variable’, the incubation temperature of the experimental bees was not controlled: Experimental bees, pupal clumps, and colonies were kept in a room with temperatures that ranged from 10 to 35°C, with the temperature usually at the high end of that range during the day. The stressful nature of the conditions was evidenced the overall higher masses of bees in ‘Variable’, which we believe reflects pre-experiment mortality of many smaller and weaker bees. In ‘Stable’, by contrast, bees were incubated at a constant temperature of 27°C.

We hypothesize that the toxic effects of nicotine in the ‘Variable’ experiment may have been exacerbated by exposure to heat and fluctuating temperatures. Interaction between heat stress and secondary metabolites has been documented in several other species (reviewed in Holmstrup et al., 2010), including some insects and related arthropods. For example, Li et al. (2014) found synergistic interaction between heat stress and avermectin toxicity in the western flower thrips (Frankliniella occidentalis), which led to reduced survival and increased upregulation of heat shock proteins. Mercury exposure reduced heat tolerance in springtails (Folmosia candidia) (Slotsbo et al., 2009), and high temperature increased uptake and toxicity of organophosphate insecticides to the midge Chironomus tentans (Lydy et al., 1999). Our results suggest that interaction between heat stress and toxins may occur in B. impatiens as well. An experiment in which temperature and secondary metabolite consumption are manipulated in a factorial design would more definitively test for such interaction.

Our results indicate that nicotine can be toxic to bumble bees even at very low concentrations when bees are parasitized. Another recent study (Baracchi et al., 2015) also found that chronic consumption of low concentrations (2.5 ppm) of nicotine increased mortality in uninfected B. terrestris, although not in those infected with Crithidia. These findings contrast with previous studies of honey bees, which did not find significant effects of naturally occurring nicotine concentrations on mortality in bees of unknown parasite status. Detzel & Wink (1993) determined the honey bee LD50 for nicotine to be 2000 ppm, far higher than any concentration that occurs in nectar. Singaravelan et al. (2006) found that larval survival of honey bees was not affected by naturally occurring concentrations of nicotine (up to 5 ppm), even when consumed consistently for several days, although a much higher concentration of nicotine (50 ppm) did significantly reduce survival. The discrepancy between their results and ours may be due to bumble bees having a greater sensitivity to nicotine than do honey bees. Further study is needed to compare the tolerance of a range of pollinator species to secondary metabolites consumed alone and in combination.

Nicotine had stronger effects on mortality in the ‘Variable’ than in the ‘Stable’ experiment. This result suggests that sensitivity of bumble bees to nicotine is further increased under temperature-stressed conditions that are nonetheless realistic in many habitats. Drastic temperature variation similar to that experienced by bees in ‘Variable’ is common in continental climates, where bumble bees are abundant. For example, in Amherst, MA, where this study was conducted, daily temperature swings of over 15°C are common, and temperatures as low as 10°C and as high as 30°C are frequently experienced within a few days of each other, or even within a single day (Menne et al., 2012a; Menne et al., 2012b). Wild bees, therefore, are likely to experience temperature conditions under which nicotine could be significantly toxic.

Under variable temperature conditions, nicotine and anabasine—but not their combination—decreased infection

To our surprise, the ‘Variable’ conditions under which nicotine consumption increased mortality were also the conditions under which alkaloid consumption reduced parasitism. In ‘Variable’, bees that consumed either alkaloid alone had significantly lower parasite counts than control bees; in contrast, bees that consumed both alkaloids had similar parasite counts to controls. Our results are consistent with the results of recent studies that found reduced parasite loads under nicotine and anabasine consumption (Baracchi et al., 2015; Richardson et al., 2015a). The reduction in parasite load may be due to alkaloid-induced increases in gut motility. Both nicotine and anabasine have been demonstrated to reduce gut transit time in the Palestinian sunbird Nectarinia osea (Tadmor-Melamed et al., 2004). Although their effect on gut transit time in insects has not been studied, rapid excretion is known to be part of some insects’ physiological response to alkaloids (Wink & Theile, 2002). It is therefore plausible that consumption of nicotine and anabasine could cause an increased rate of excretion in bees, thus clearing C. bombi cells from the gut and leading to the observed reduction in parasite load.

The lack of effect of the combined alkaloids on parasite load is more puzzling. The concentrations of the individual alkaloids may have been within the medicinal window of concentration at which antiparasitic effects were dominant. However, the combined effects of both alkaloids may have weakened bees’ ability to fight infection through excessive stimulatory, laxative, and/or immunosuppressive effects. These combined toxic effects could have offset the medicinal effects realized at lower concentrations in the single-alkaloid treatments.

In a controlled temperature environment, nicotine increased parasite loads without affecting mortality

When bees were kept under constant conditions in ‘Stable’, alkaloid ingestion had different effects on mortality and parasitism from those found in ‘Variable’. In contrast with the antiparasitic effects of alkaloids in ‘Variable’, in ‘Stable’, nicotine consumption significantly increased parasite counts, while anabasine also increased parasite loads, although not significantly. This result is consistent with a growing body of research demonstrating that neonicotinoids, a class of insecticides chemically similar to nicotine, have immunosuppressant effects on bees (reviewed in Goulson et al., 2015). While the effects of nicotine are not necessarily the same as those of neonicotinoids, both nicotine and neonicotinoids function as nAChR agonists, (Jeschke et al., 2011), suggesting similar pharmacological activity.

The immunosuppressant effects of neonicotinoids have been most well studied in honey bees. Under lab conditions, sub-lethal colony-level exposure to imidacloprid increased levels of Nosema infection (Pettis et al., 2012). In the field, colonies that had foraged on corn treated with thiabendazole had significantly elevated levels of black queen cell virus and Varroa mites (Alburaki et al., 2015). At the molecular level, clothianidin and imidacloprid induced increased transcription of a gene coding for a negative modulator of NF-Kβ immune signaling in honey bees, causing decreased immune function and increased viral replication (Di Prisco et al., 2013). Additional studies on bumble bees are needed to compare the effects of neonicotinoids and naturally occurring alkaloids, as well as to evaluate how the effects of these compounds vary across species, genera, and dietary contexts.

Interactive effects of abiotic conditions, alkaloids, and parasites on bees

We hypothesized that the stronger medicinal and toxic effects of the alkaloids in ‘Variable’ may have resulted from increased total alkaloid consumption due to elevated nectar intake under the generally hotter conditions of ‘Variable’. However, our Consumption experiment showed that increasing incubator temperature from that used in ‘Stable’ (27°C) to one characteristic of ‘Variable’ (33°C) significantly decreased the volume of nectar consumed. Hence, it appears that the warmer temperatures of ‘Variable’ would have curtailed nectar consumption rather than promoting it. Still, we cannot exclude the possibility that warmth-related decreases in consumption would have been offset by other factors that promoted nectar intake, including increased activity, energy expenditure, and stress-related defecation in the comparatively stimulating experimental environment. In addition, the Consumption experiment used uninfected bees, whereas bees in both ‘Variable’ and ‘Stable’ were inoculated with parasites. Further investigation is needed to define how the thermal environment, external stimuli, nectar alkaloid content, and infection interact to influence patterns of consumption.

The stronger effects of alkaloids in ‘Variable’ may therefore reflect complex interactions between alkaloids, temperature stress, environmental stimuli, and immunity rather than simple differences in alkaloid consumption. Under the fluctuating conditions of ‘Variable’, bees may have been more susceptible to the effects of the alkaloids, both in the form of increased toxicity and increased gut motility, accounting for both the higher mortality and decreased C. bombi counts in ‘Variable’. For example, the hot conditions of ‘Variable’ would appear to have increased evaporative moisture losses while decreasing nectar intake, leaving bees more susceptible to dehydration. Nicotine's agonistic effects on intestinal peristalsis may have helped to clear parasites from the gut, but also have contributed to further dehydration. In addition, bees in ‘Variable’ were exposed to external stimuli in the lab environment, including light and vibration, which may have synergized with stimulatory effects of the alkaloids to increase defecation. In the lab, bees often defecate explosively when startled—sometimes tens of centimeters into the air (ECPY, personal observation). Bees on the lab bench in ‘Variable’ would have been agitated more frequently than bees in the incubator in ‘Stable’, and their sensitivity to agitation may have been increased by alkaloid consumption.

The higher temperatures of ‘Variable’ may have additionally functioned as an externally imposed fever that reversed the immunosuppressive effects of nicotine. Febrile amelioration of infection has been shown in many animals (reviewed in Kluger, 1978), including honey bees (Campbell et al., 2010) and other insects (Stahlschmidt & Adamo, 2013). The lower absolute parasite counts relative to ‘Stable’ may reflect heat-related inhibition of C. bombi, which grows best at 27°C (Salathé et al., 2012). Stimulatory effects of nicotine and anabasine, enhanced by exposure to everyday disturbance in ‘Variable’, could have increased activity level and metabolic rate, thereby further raising body temperature and slowing parasite growth, albeit at the expense of increasing heat stress on bees. The effects of a given increase in body temperature would have been more pronounced under the hot conditions of ‘Variable’, which may have approached the parasite’s thermal tolerance limit.

Our results in ‘Stable’ contrast with those of a recent study (Baracchi et al., 2015), which showed that nicotine consumption reduced parasite loads in Bombus. There were a number of differences between that study and ours that may explain the differing results. First, the two experiments used different bee species: Baracchi and colleagues used the larger B. terrestris, which may be more resistant to potential immunosuppressive effects of nicotine. Even within our experiments, which used bees of a single species from a single supplier, we found highly significant effects of mass and colony, indicating strong effects of both size and genetic variation on parasitism that are consistent with previous findings (Otterstatter & Thompson, 2006). Interspecific variability could be tested by comparing effects of secondary metabolites on parasitism in a range of Bombus species. Second, Baracchi et al. employed different bee housing materials. Bees were housed in petri dishes, which may have been less confining than our snap-cap vials, as suggested by the overall lower rates of mortality in the experiments of Baracchi et al. compared to ours. This extra space could have allowed bees to take advantage of alkaloid-induced increases in gut motility by expelling Crithidia-rich feces in less-frequented parts of their arenas, whereas our bees would have had close and persistent contact with their own waste. It would be intriguing to investigate how the effects of alkaloids differ depending on levels of crowding, including in densely populated hive environments. A third explanation could be that the antiparasitic effects of alkaloids depend on other environmental cues, including daylight. Both our study and that of Baracchi et al. showed medicinal effects of nicotine when bees were housed on the lab bench (as in ‘Variable’), whereas nicotine increased parasitism for bees housed in constant darkness in ‘Stable’. Future experiments are needed to explicitly test the roles of environmental conditions and stimuli on bee and parasite tolerance to alkaloids.

Our results also contrast with those of Richardson et al. (2015a), which employed the same bee species and unlit rearing conditions as our ‘Stable’ experiment, but found that both nicotine and anabasine significantly reduced C. bombi parasite load in B. impatiens. The differing effects of nicotine may be due to our use of the (-)-enantiomer of nicotine, whereas Richardson et al. (2015a) used a +/- enantiomeric mixture (Sigma N0267, personal communications). (-)-Nicotine, which is far more common in nature (Armstrong et al., 1998), appears to be more pharmacologically active than (+)-nicotine in vertebrates, aquatic invertebrates (Barlow & Hamilton, 1965; Gause & Smaragdova, 1939), and insects including honey bees (Tomizawa & Yamamoto, 1992). Interestingly, Gause & Smaragdova (1939) found the two enantiomers to be isotoxic to protozoa. If (-)-nicotine is more toxic to bees than is (+)-nicotine, but both enantiomers are equally toxic to C. bombi, then (-)-nicotine might have lesser medicinal value.

Another possible explanation for our differing results relates to the C. bombi itself. C. bombi is known to be genetically diverse; Salathé & Schmid-Hempel (2011) identified 213 strains infecting bumble bees in Switzerland. Multiple strains are often present in a single host. Tognazzo et al. (2012) found that 67% of infected workers and 54% of infected queens carried mixed-genotype infections, with queens harboring up to 29 different genotypes. In addition, it is possible that not all supposed C. bombi infections in fact represent a single Crithidia species. Schmid-Hempel & Tognazzo (2010) identified two genetically and morphologically distinct lineages within the C. bombi complex, which they classified as cryptic species. They retained the name C. bombi for the lineage which more closely matches Lipa & Triggiani’s (1988) original description of C. bombi, and proposed the name C. expoeki for the other lineage. Both lineages are present in both Europe and North America, suggesting an old divergence. If our C. bombi cultures and those used by Richardson et al. (2015a) and (Baracchi et al., 2015) represent different strains, or different species, it is possible that they vary in alkaloid tolerance. In addition, these strains might vary in virulence and pathogenicity. The medicinal effects of alkaloids in ‘Variable’, where absolute parasite loads were lower overall, suggests that alkaloid ingestion might be most therapeutic against relatively mild infections.

Implications of secondary metabolites for pollinator health in a changing landscape

Our results represent an important first step towards understanding the interactive effects of multiple secondary metabolites on pollinators. We did not find evidence for synergy between Nicotiana nectar alkaloids, although we did find some evidence for antagonism. To elucidate the potential role of interactions between compounds in the plant-pollinator-parasite system, it will be necessary to test for interactions between other sets of compounds. Within Nicotiana, the wild tobacco N. attenuata contains at least 35 nectar secondary compounds, including sesquiterpenes (Kessler & Baldwin, 2007); many terpenoids have potent trypanocidal activity, yet are relatively benign to animal cells (Otoguro et al., 2011). Among other plant families, Asclepias species are pollinated by bumble bees and contain several cardenolides in their nectar (Manson et al., 2012) that could be tested for interactive effects. Another plant species to investigate is Chelone glabra, which has high concentrations of the iridoid glycosides aucubin and catalpol in its nectar (Richardson et al., 2015b). Synergy between these glycosides has been demonstrated in their effect on Junonia coenia caterpillars (Richards et al., 2012).

Overall, the effects of nectar alkaloids on parasitized pollinators may represent a tradeoff between toxicity to the parasite and toxicity to the host. In the case of nicotine, bees appear to be more sensitive to alkaloid toxicity than parasites are. While nicotine inhibits the growth of many microbial pathogens, significant antimicrobial effects require concentrations between 100 and 250 ppm (Pavia et al., 2000). By contrast, Singaravelan et al. (2006) found that nicotine was toxic to bees at 50 ppm, and our own results suggest that nicotine can have toxic effects at concentrations as low as 2 ppm. However, the studies establishing toxicity of nicotine in bees have all focused on chronic consumption of a diet high in nicotine. In nature, bumble bees are generalist pollinators, and are known to forage on several plant species within a narrow time frame and even within a single foraging trip (Free, 1970). In landscapes with varied floral resources, they might be able to avoid chronic nicotine toxicity by exploiting a range of plant species.

Conclusion

Our results emphasize the importance of interactions between stressors in pollinator health, and demonstrate that the effect of any single factor can vary greatly depending on the other factors involved. Research on pollinator health often focuses on single factors in isolation; however, in natural conditions, pollinators are often exposed to several stressors simultaneously (Goulson et al., 2015). Previous research has demonstrated both medicinal and toxic effects of secondary metabolites such as nicotine and anabasine. Our results suggest that the predominant effect can vary with environmental and dietary context. In order to better elucidate the role of secondary metabolites in pollinator health, future research should explicitly address the role of these complex interactions.

Data availability

F1000Research: Dataset 1. Data for ‘Variable’ experiment, 10.5256/f1000research.6870.d101937 (Thorburn et al., 2015a).

F1000Research: Dataset 2. Data for ‘Stable’ experiment, 10.5256/f1000research.6870.d101940 (Thorburn et al., 2015b).

F1000Research: Dataset 3. Data for Consumption experiment, 10.5256/f1000research.6870.d109062 (Thorburn et al., 2015c).

Author contributions

All authors conceived the study. ECPY and LPT designed and conducted the experiments using methods developed by REI. ECPY analyzed the data. LPT and ECPY prepared the first draft of the manuscript. All authors revised the manuscript and have agreed upon the final content.

Competing interests

No competing interests were disclosed.

Grant information

This research was funded by the National Science Foundation under NSFDEB-1258096, NSF GRFP (Grant DGE-0907995 to ECPY), and NSF DDIG (Grant NSFDEB-1501907 to ECPY and LSA); by the National Research Initiative (NRI) Arthropod and Nematode Biology and Management Program of the USDA Cooperative State Research, Education, and Extension Service (CSREES) Grant no. USDA-AFRI 2013-02536; and by the Garden Club of America Centennial Pollinator Fellowship (ECPY).

I confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgments

The authors are grateful to Biobest for supplying bee colonies; and to Ali Hogeboom, Melissa Ha, Caitlin McAllister, and lab assistants for help with ‘Stable’ experiment. We thank our reviewers Drs David Baracchi, Michael Simone-Finstrom, and Margarita Lopez-Uribe for their improvement of the paper.

Supplementary material

Script 1. R script for analysis of C. bombi parasite loads in ‘Variable’ experiment.

Click here to access the data.

http://dx.doi.org/10.5256/f1000research.6870.s101933

Script 2. R script for analysis of mortality in ‘Variable’ experiment.

Click here to access the data.

http://dx.doi.org/10.5256/f1000research.6870.s101934

Script 3. R script for analysis of C. bombi parasite loads in ‘Stable’ experiment.

Click here to access the data.

http://dx.doi.org/10.5256/f1000research.6870.s101935

Script 4. R script for analysis of mortality in ‘Stable’ experiment.

Click here to access the data.

http://dx.doi.org/10.5256/f1000research.6870.s101936

Script 5. R script for analysis of ‘Consumption experiment’.

Click here to access the data.

http://dx.doi.org/10.5256/f1000research.6870.s109063

Table S1. Sample sizes for the 2 experiments, ‘Variable’ and ‘Controlled’.

TreatmentExperiment
VariableStable
Control4583
Nicotine4679
Anabasine4691
Nicotine + Anabasine4186
Total 178339

Table S2. GLMM for effects of nicotine and anabasine consumption on mortality in ‘Stable’ experiment.

Table shows binomial mixed model results of χ2 tests for effects of predictor variables on probability of death during the 7 d experiment.

Sourceχ2DfP
Nicotine0.11710.7323
Anabasine0.044410.8331
Nicotine*Anabasine0.545610.4601
Colony6.853630.0767

References

  •  Abbott J: Self-medication in insects: current evidence and future perspectives. Ecol Entomol. 2014; 39(3): 273–280. Publisher Full Text
  •  Adler LS, Seifert MG, Wink M, et al.: Reliance on pollinators predicts defensive chemistry across tobacco species. Ecol Lett. 2012; 15(10): 1140–1148. PubMed Abstract | Publisher Full Text
  •  Adler LS, Wink M, Distl M, et al.: Leaf herbivory and nutrients increase nectar alkaloids. Ecol Lett. 2006; 9(8): 960–967. PubMed Abstract | Publisher Full Text
  •  Alburaki M, Boutin S, Mercier PL, et al.: Neonicotinoid-Coated Zea mays Seeds Indirectly Affect Honeybee Performance and Pathogen Susceptibility in Field Trials. PLoS One. 2015; 10(5): e0125790. PubMed Abstract | Publisher Full Text | Free Full Text
  •  Allen-Wardell G, Bernhardt P, Bitner R, et al.: The potential consequences of pollinator declines on the conservation of biodiversity and stability of food crop yields. Conserv Biol. 1998; 12(1): 8–17. Publisher Full Text
  •  Armstrong DW, Wang X, Ercal N: Enantiomeric composition of nicotine in smokeless tobacco, medicinal products, and commercial reagents. Chirality. 1998; 10(7): 587–591. Publisher Full Text
  •  Balaban J, Balaban J, Moisset B, et al.: Species Bombus impatiens - common eastern bumble bee. 2014. Reference Source
  •  Baracchi D, Brown MJF, Chittka L: Behavioural evidence for self-medication in bumblebees? [version 2; referees: 3 approved]. F1000Res. 2015; 4: 73. Publisher Full Text
  •  Barlow RB, Hamilton JT: The stereospecificity of nicotine. Br J Pharmacol Chemother. 1965; 25(1): 206–212. PubMed Abstract | Publisher Full Text | Free Full Text
  •  Bates D, Maechler M, Bolker B, et al.: R package {lme4}: Linear mixed-effects models using {Eigen} and {S4}. 2015. Reference Source
  •  Benowitz NL: Nicotine safety and toxicity. Oxford University Press, New York, New York. 1998. Reference Source
  •  Brown MJF, Loosli R, Schmid-Hempel P: Condition-dependent expression of virulence in a trypanosome infecting bumblebees. Oikos. 2000; 91(3): 421–427. Publisher Full Text
  •  Caine SB, Collins GT, Thomsen M, et al.: Nicotine-like behavioral effects of the minor tobacco alkaloids nornicotine, anabasine, and anatabine in male rodents. Exp Clin Psychopharmacol. 2014; 22(1): 9–22. PubMed Abstract | Publisher Full Text
  •  Cameron SA, Lozier JD, Strange JP, et al.: Patterns of widespread decline in North American bumble bees. Proc Natl Acad Sci U S A. 2011; 108(2): 662–667. PubMed Abstract | Publisher Full Text | Free Full Text
  •  Campbell J, Kessler B, Mayack C, et al.: Behavioural fever in infected honeybees: parasitic manipulation or coincidental benefit? Parasitology. 2010; 137(10): 1487–1491. PubMed Abstract | Publisher Full Text
  •  Colla SR, Otterstatter MC, Gegear RJ, et al.: Plight of the bumble bee: Pathogen spillover from commercial to wild populations. Biol Conserv. 2006; 129(4): 461–467. Publisher Full Text
  •  Detzel A, Wink M: Attraction, deterrence or intoxication of bees (Apis mellifera) by plant allelochemicals. Chemoecology. 1993; 4(1): 8–18. Publisher Full Text
  •  Di Prisco G, Cavaliere V, Annoscia D, et al.: Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees. Proc Natl Acad Sci U S A. 2013; 110(46): 18466–71. PubMed Abstract | Publisher Full Text | Free Full Text
  •  Diawara MM, Trumble JT, White KK, et al.: Toxicity of linear furanocoumarins to Spodoptera exigua: Evidence for antagonistic interactions. J Chem Ecol. 1993; 19(11): 2473–2484. PubMed Abstract | Publisher Full Text
  •  Diffenbaugh NS, Field CB: Changes in ecologically critical terrestrial climate conditions. Science. 2013; 341(6145): 486–92. PubMed Abstract | Publisher Full Text
  •  Dyer LA, Dodson CD, Stireman JO, et al.: Synergistic effects of three Piper amides on generalist and specialist herbivores. J Chem Ecol. 2003; 29(11): 2499–2514. PubMed Abstract | Publisher Full Text
  •  Free JB: The flower constancy of bumblebees. J Anim Ecol. 1970; 39(2): 395–402. Publisher Full Text
  •  García-García R, López-Malo A, Palou E: Bactericidal action of binary and ternary mixtures of carvacrol, thymol, and eugenol against Listeria innocua. J Food Sci. 2011; 76(2): M95–100. PubMed Abstract | Publisher Full Text
  •  Gause GF, Smaragdova NP: On the killing action of optically isomeric nicotines in relation to problems of evolution of the nervous system in animals. Physiol Zool. 1939; 12(3): 238–255. Reference Source
  •  Gherman BI, Denner A, Bobiş O, et al.: Pathogen-associated self-medication behavior in the honeybee Apis mellifera. Behav Ecol Sociobiol. 2014; 68(11): 1777–1784. Publisher Full Text
  •  Goulson D, Nicholls E, Botías C, et al.: Bee declines driven by combined stress from parasites, pesticides, and lack of flowers. Science. 2015; 347(6229): 1255957. PubMed Abstract | Publisher Full Text
  •  Heil M: Nectar: generation, regulation and ecological functions. Trends Plant Sci. 2011; 16(4): 191–200. PubMed Abstract | Publisher Full Text
  •  Heinrich B: Temperature Regulation in the Bumblebee Bombus vagans: A Field Study. Science. 1972; 175(4018): 185–187. PubMed Abstract | Publisher Full Text
  •  Holmstrup M, Bindesbøl AM, Oostingh GJ, et al.: Interactions between effects of environmental chemicals and natural stressors: a review. Sci Total Environ. 2010; 408(18): 3746–62. PubMed Abstract | Publisher Full Text
  •  Jeschke P, Nauen R, Schindler M, et al.: Overview of the status and global strategy for neonicotinoids. J Agric Food Chem. 2011; 59(7): 2897–2908. PubMed Abstract | Publisher Full Text
  •  Kessler D, Baldwin IT: Making sense of nectar scents: the effects of nectar secondary metabolites on floral visitors of Nicotiana attenuata. Plant J. 2007; 49(5): 840–54. PubMed Abstract | Publisher Full Text
  •  Kluger MJ: The evolution and adaptive value of fever. Am Sci. 1978; 66(1): 38–43. PubMed Abstract
  •  Köhler A, Pirk CW, Nicolson SW: Simultaneous stressors: interactive effects of an immune challenge and dietary toxin can be detrimental to honeybees. J Insect Physiol. 2012a; 58(7): 918–923. PubMed Abstract | Publisher Full Text
  •  Köhler A, Pirk CW, Nicolson SW: Honeybees and nectar nicotine: deterrence and reduced survival versus potential health benefits. J Insect Physiol. 2012b; 58(2): 286–292. PubMed Abstract | Publisher Full Text
  •  Kumrungsee N, Pluempanupat W, Koul O, et al.: Toxicity of essential oil compounds against diamondback moth, Plutella xylostella, and their impact on detoxification enzyme activities. J Pest Sci. 2014; 87(4): 721–729. Publisher Full Text
  •  Lesnoff M, Lancelot R: R package aod: Analysis of overdispersed data. 2012. Reference Source
  •  Li HB, Zheng YT, Sun DD, et al.: Combined effects of temperature and avermectins on life history and stress response of the western flower thrips, Frankliniella occidentalis. Pestic Biochem Physiol. 2014; 108: 42–48. PubMed Abstract | Publisher Full Text
  •  Lipa J, Triggiani O: Crithidia bombi sp. n. a flagellated parasite of a bumble-bee Bombus terrestris L. (Hymenoptera, Apidae). Acta Protozoologica. 1988; 27: 287–290. Reference Source
  •  Lydy MJ, Belden JB, Ternes MA: Effects of temperature on the toxicity of m-parathion, chlorpyrifos, and pentachlorobenzene to Chironomus tentans. Arch Environ Contam Toxicol. 1999; 37(4): 542–547. PubMed Abstract | Publisher Full Text
  •  MacBean C: A world compendium: The pesticide manual. 16th edition. British Crop Production Council, Alton, UK. 2012. Reference Source
  •  Manson JS, Otterstatter MC, Thomson JD: Consumption of a nectar alkaloid reduces pathogen load in bumble bees. Oecologia. 2010; 162(1): 81–89. PubMed Abstract | Publisher Full Text
  •  Manson JS, Rasmann S, Halitschke R, et al.: Cardenolides in nectar may be more than a consequence of allocation to other plant parts: a phylogenetic study of Asclepias. Funct Ecol. 2012; 26(5): 1100–1110. Publisher Full Text
  •  Menne MJ, Durre I, Korzeniewski SM, et al.: Global Historical Climatology Network - Daily (GHCN-Daily), Version 3. NOAA National Climatic Data Center. 2012a; 3. . Publisher Full Text
  •  Menne MJ, Durre I, Vose RS, et al.: An overview of the global historical climatology network-daily database. J Atmos Oceanic Technol. 2012b; 29: 897–910. Publisher Full Text
  •  Otoguro K, Iwatsuki M, Ishiyama A, et al.: In vitro antitrypanosomal activity of plant terpenes against Trypanosoma brucei. Phytochemistry. 2011; 72(16): 2024–30. PubMed Abstract | Publisher Full Text
  •  Otterstatter MC, Thomson JD: Within-host dynamics of an intestinal pathogen of bumble bees. Parasitology. 2006; 133(Pt 6): 749–61. PubMed Abstract | Publisher Full Text
  •  Pavia CS, Pierre A, Nowakowski J: Antimicrobial activity of nicotine against a spectrum of bacterial and fungal pathogens. J Med Microbiol. 2000; 49(7): 675–6. PubMed Abstract | Publisher Full Text
  •  Pettis JS, vanEngelsdorp D, Johnson J, et al.: Pesticide exposure in honey bees results in increased levels of the gut pathogen Nosema. Naturwissenschaften. 2012; 99(2): 153–158. PubMed Abstract | Publisher Full Text | Free Full Text
  •  Pinheiro J, Bates D, DebRoy S, et al.: {nlme}: Linear and nonlinear mixed effects models. 2015. Reference Source
  •  Potts SG, Biesmeijer JC, Kremen C, et al.: Global pollinator declines: trends, impacts and drivers. Trends Ecol Evol. 2010; 25(6): 345–53. PubMed Abstract | Publisher Full Text
  •  R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2014. Reference Source
  •  Raffa KF, Berryman AA, Simasko J, et al.: Effects of grand fir monoterpenes on the fir engraver, Scolytus ventralis (Coleoptera: Scolytidae), and its symbiotic fungus. Environ Entomol. 1985; 14(5): 552–556. Publisher Full Text
  •  Rattanachaikunsopon P, Phumkhachorn P: Assessment of factors influencing antimicrobial activity of carvacrol and cymene against Vibrio cholerae in food. J Biosci Bioeng. 2010; 110(5): 614–619. PubMed Abstract | Publisher Full Text
  •  Richards LA, Lampert EC, Bowers MD, et al.: Synergistic effects of iridoid glycosides on the survival, development and immune response of a specialist caterpillar, Junonia coenia (Nymphalidae). J Chem Ecol. 2012; 38(10): 1276–1284. PubMed Abstract | Publisher Full Text
  •  Richardson LL, Adler LS, Leonard AS, et al.: Secondary metabolites in floral nectar reduce parasite infections in bumblebees. Proc Biol Sci. 2015a; 282(1803): 20142471. PubMed Abstract | Publisher Full Text | Free Full Text
  •  Richardson LL, Bowers MD, Irwin RE: Nectar chemistry mediates the behavior of parasitized bees: consequences for plant fitness. Ecology. 2015b. Publisher Full Text
  •  Salathé RM, Schmid-Hempel P: The genotypic structure of a multi-host bumblebee parasite suggests a role for ecological niche overlap. PLoS One. 2011; 6(8): e22054. PubMed Abstract | Publisher Full Text | Free Full Text
  •  Salathé R, Tognazzo M, Schmid-Hempel R, et al.: Probing mixed-genotype infections I: extraction and cloning of infections from hosts of the trypanosomatid Crithidia bombi. PLoS One. 2012; 7(11): e49046. PubMed Abstract | Publisher Full Text | Free Full Text
  •  Schmid-Hempel R, Eckhardt M, Goulson D, et al.: The invasion of southern South America by imported bumblebees and associated parasites. J Anim Ecol. 2014; 83(4): 823–837. PubMed Abstract | Publisher Full Text
  •  Schmid-Hempel R, Tognazzo M: Molecular divergence defines two distinct lineages of Crithidia bombi (Trypanosomatidae), parasites of bumblebees. J Eukaryot Microbiol. 2010; 57(4): 337–45. PubMed Abstract | Publisher Full Text
  •  Schmidt TJ, Khalid SA, Romanha AJ, et al.: The potential of secondary metabolites from plants as drugs or leads against protozoan neglected diseases - part II. Curr Med Chem. 2012; 19(14): 2176–2228. PubMed Abstract | Publisher Full Text
  •  Simone-Finstrom MD, Spivak M: Increased resin collection after parasite challenge: a case of self-medication in honey bees? PLoS One. 2012; 7(3): e34601. PubMed Abstract | Publisher Full Text | Free Full Text
  •  Singaravelan N, Inbar M, Ne’eman G, et al.: The effects of nectar-nicotine on colony fitness of caged honeybees. J Chem Ecol. 2006; 32(1): 49–59. PubMed Abstract | Publisher Full Text
  •  Singaravelan N, Nee’man G, Inbar M, et al.: Feeding responses of free-flying honeybees to secondary compounds mimicking floral nectars. J Chem Ecol. 2005; 31(12): 2791–2804. PubMed Abstract | Publisher Full Text
  •  Singer MS, Mace KC, Bernays EA: Self-medication as adaptive plasticity: increased ingestion of plant toxins by parasitized caterpillars. PLoS One. 2009; 4(3): e4796. PubMed Abstract | Publisher Full Text | Free Full Text
  •  Slotsbo S, Heckmann LH, Damgaard C, et al.: Exposure to mercury reduces heat tolerance and heat hardening ability of the springtail Folsomia candida. Comp Biochem Physiol C Toxicol Pharmacol. 2009; 150(1): 118–123. PubMed Abstract | Publisher Full Text
  •  Stahlschmidt ZR, Adamo SA: Context dependency and generality of fever in insects. Naturwissenschaften. 2013; 100(7): 691–696. PubMed Abstract | Publisher Full Text
  •  Tadmor-Melamed H, Markman S, Arieli A, et al.: Limited ability of Palestine sunbirds Nectarinia osea to cope with pyridine alkaloids in nectar of tree tobacco Nicotiana glauca. Funct Ecol. 2004; 18(6): 844–850. Publisher Full Text
  •  Therneau TM: R package coxme: Mixed effects cox models. 2015. Reference Source
  •  Thomas MB, Blanford S: Thermal biology in insect-parasite interactions. Trends Ecol Evol. 2003; 18(7): 344–350. Publisher Full Text
  •  Thorburn LP, Adler LS, Irwin RE, et al.: Dataset 1 in: Variable effects of nicotine, anabasine, and their interactions on parasitized bumble bees. F1000Research. 2015a. Data Source
  •  Thorburn LP, Adler LS, Irwin RE, et al.: Dataset 2 in: Variable effects of nicotine, anabasine, and their interactions on parasitized bumble bees. F1000Research. 2015b. Data Source
  •  Thorburn LP, Adler LS, Irwin RE, et al.: Dataset 3 in: Variable effects of nicotine, anabasine, and their interactions on parasitized bumble bees. F1000Research. 2015c. Data Source
  •  Tognazzo M, Schmid-Hempel R, Schmid-Hempel P: Probing mixed-genotype infections II: high multiplicity in natural infections of the trypanosomatid, Crithidia bombi, in its host, Bombus spp. PLoS One. 2012; 7(11): e49137. PubMed Abstract | Publisher Full Text | Free Full Text
  •  Tomizawa M, Yamamoto I: Binding of nicotinoids and the related compounds to the insect nicotinic acetyicholine compounds receptor. J Pestic Sci. 1992; 17(4): 231–236. Publisher Full Text
  •  Ujváry I: Nicotine and other insecticidal alkaloids. In Yamamoto I, and Casida JE, editors. Nicotinoid insecticides and the nicotinic acetylcholine receptor. Springer-Verlag, Tokyo. 1999: 29–69. Publisher Full Text
  •  Venables WN, Ripley BD: Modern applied statistics with S. Fourth edition. Springer, New York, 2002. Publisher Full Text
  •  Whitehead SR, Bowers MD: Chemical ecology of fruit defence: Synergistic and antagonistic interactions among amides from Piper. Funct Ecol. 2014; 28(5): 1094–1106. Publisher Full Text
  •  Whitehorn PR, Tinsley MC, Brown MJF, et al.: Investigating the impact of deploying commercial Bombus terrestris for crop pollination on pathogen dynamics in wild bumble bees. J Apicult Res. 2013; 52(3): 149–157. Publisher Full Text
  •  Williams PH, Araújo MB, Rasmont P: Can vulnerability among British bumblebee (Bombus) species be explained by niche position and breadth? Biol Conserv. 2007; 138(3–4): 493–505. Publisher Full Text
  •  Williams P, Colla S, Xie Z: Bumblebee vulnerability: common correlates of winners and losers across three continents. Conserv Biol. 2009; 23(4): 931–940. PubMed Abstract | Publisher Full Text
  •  Wink M, Theile V: Alkaloid tolerance in Manduca sexta and phylogenetically related sphingids (Lepidoptera: Sphingidae). Chemoecology. 2002; 12(1): 29–46. Publisher Full Text

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Thorburn LP, Adler LS, Irwin RE and Palmer-Young EC. Variable effects of nicotine, anabasine, and their interactions on parasitized bumble bees [version 2; peer review: 2 approved]. F1000Research 2015, 4:880 (https://doi.org/10.12688/f1000research.6870.2)
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 2
VERSION 2
PUBLISHED 16 Dec 2015
Revised
Views
10
Cite
Reviewer Report 29 Jan 2016
Michael Simone-Finstrom, Honey Bee Breeding, Genetics, and Physiology Research, USDA Agricultural Research Service, Baton Rouge, LA, USA 
Margarita Lopez-Uribe, Department of Entomology, North Carolina State University, Raleigh, NC, USA 
Approved
VIEWS 10
https://doi.org/10.5256/f1000research.8151.r11592
Overall we feel that the authors fully addressed our concerns and those of the other reviewer and significantly improved their manuscript. The fact that they were able to conduct a short experiment adds to the paper and to their conclusions, ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Simone-Finstrom M and Lopez-Uribe M. Reviewer Report For: Variable effects of nicotine, anabasine, and their interactions on parasitized bumble bees [version 2; peer review: 2 approved]. F1000Research 2015, 4:880 (https://doi.org/10.5256/f1000research.8151.r11592)
The direct URL for this report is:
https://f1000research.com/articles/4-880/v2#referee-response-11592
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Report a concern
Views
10
Cite
Reviewer Report 29 Dec 2015
David Baracchi, School of Biological and Chemical Sciences, Queen Mary, University of London, London, UK 
Approved
VIEWS 10
https://doi.org/10.5256/f1000research.8151.r10398
The authors responded appropriately to my concerns and other important points raised by Dr.’s Simone-Finstrom and Lopez-Uribe. I appreciated that authors conducted an additional ‘Consumption experiment’ as required. Despite the new results are puzzling and did not provide a clear ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Baracchi D. Reviewer Report For: Variable effects of nicotine, anabasine, and their interactions on parasitized bumble bees [version 2; peer review: 2 approved]. F1000Research 2015, 4:880 (https://doi.org/10.5256/f1000research.8151.r10398)
The direct URL for this report is:
https://f1000research.com/articles/4-880/v2#referee-response-10398
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Report a concern
Version 1
VERSION 1
PUBLISHED 21 Sep 2015
Views
21
Cite
Reviewer Report 19 Oct 2015
David Baracchi, School of Biological and Chemical Sciences, Queen Mary, University of London, London, UK 
Approved with Reservations
VIEWS 21
https://doi.org/10.5256/f1000research.7396.r10860
The authors investigated how the consumption of nectar alkaloids, either in isolation or combination, affect survival and pathogen load in a pollinator species. The main goal of the paper is to test the synergistic effects of two alkaloids on bee ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Baracchi D. Reviewer Report For: Variable effects of nicotine, anabasine, and their interactions on parasitized bumble bees [version 2; peer review: 2 approved]. F1000Research 2015, 4:880 (https://doi.org/10.5256/f1000research.7396.r10860)
The direct URL for this report is:
https://f1000research.com/articles/4-880/v1#referee-response-10860
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Report a concern
  • Author Response 16 Dec 2015
    Evan Palmer-Young, Department of Biology, University of Massachusetts at Amherst, Amherst, USA
    16 Dec 2015
    Author Response
    Dear Dr. Baracchi,
    Many thanks for your thorough critical review of our article. We feel that the revised version is now improved in response to your comments. We address each point ... Continue reading
    Report a concern
  • Respond or Comment
COMMENTS ON THIS REPORT
  • Author Response 16 Dec 2015
    Evan Palmer-Young, Department of Biology, University of Massachusetts at Amherst, Amherst, USA
    16 Dec 2015
    Author Response
    Dear Dr. Baracchi,
    Many thanks for your thorough critical review of our article. We feel that the revised version is now improved in response to your comments. We address each point ... Continue reading
    Report a concern
Views
25
Cite
Reviewer Report 07 Oct 2015
Michael Simone-Finstrom, Honey Bee Breeding, Genetics, and Physiology Research, USDA Agricultural Research Service, Baton Rouge, LA, USA 
Margarita Lopez-Uribe, Department of Entomology, North Carolina State University, Raleigh, NC, USA 
Approved with Reservations
VIEWS 25
https://doi.org/10.5256/f1000research.7396.r10403
Thorburn et al. describe an experiment where they test the dual effect of two secondary plant compounds on bumble bee mortality and parasite load after infections with the pathogen Crithidia bombi. This paper addresses an interesting question that follows up ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Simone-Finstrom M and Lopez-Uribe M. Reviewer Report For: Variable effects of nicotine, anabasine, and their interactions on parasitized bumble bees [version 2; peer review: 2 approved]. F1000Research 2015, 4:880 (https://doi.org/10.5256/f1000research.7396.r10403)
The direct URL for this report is:
https://f1000research.com/articles/4-880/v1#referee-response-10403
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Report a concern
  • Author Response 16 Dec 2015
    Evan Palmer-Young, Department of Biology, University of Massachusetts at Amherst, Amherst, USA
    16 Dec 2015
    Author Response
    Dear Dr.’s Simone-Finstrom and Lopez-Uribe,
    Many thanks for your careful review of our research. We have revised the manuscript to incorporate your suggestions, and hope that you find the new version ... Continue reading
    Report a concern
  • Respond or Comment
COMMENTS ON THIS REPORT
  • Author Response 16 Dec 2015
    Evan Palmer-Young, Department of Biology, University of Massachusetts at Amherst, Amherst, USA
    16 Dec 2015
    Author Response
    Dear Dr.’s Simone-Finstrom and Lopez-Uribe,
    Many thanks for your careful review of our research. We have revised the manuscript to incorporate your suggestions, and hope that you find the new version ... Continue reading
    Report a concern

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Version 2
VERSION 2 PUBLISHED 21 Sep 2015
Comment

Open Peer Review

Reviewer Status

Alongside their report, reviewers assign a status to the article:

Approved
The paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations
A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved
Fundamental flaws in the paper seriously undermine the findings and conclusions

Reviewer Reports

Invited Reviewers
1 2
Version 2
(revision)
 16 Dec 15 		read read
Version 1
21 Sep 15 		read read
  1. Michael Simone-Finstrom, USDA Agricultural Research Service, Baton Rouge, USA
    Margarita Lopez-Uribe, North Carolina State University, Raleigh, USA
    Margarita Lopez-Uribe, North Carolina State University, Raleigh, USA
  2. David Baracchi, Queen Mary, University of London, London, UK

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    Alongside their report, reviewers assign a status to the article:
    Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
    Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
    Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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