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Research Article

Variable effects of nicotine and anabasine on parasitized bumble bees

[version 1; peer review: 2 approved with reservations]
PUBLISHED 21 Sep 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 synergistic 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); and second, under carefully controlled environmental conditions (‘Controlled’; 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 ‘Controlled’, 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

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). The parasitic mite Varroa destructor has been linked to honey bee declines in Ontario, Canada (Guzmán-Novoa et al., 2010). Infection with the microsporidian pathogen Nosema ceranae may be responsible for cases of honey bee colony collapse in Spain (Higes et al., 2009). Bumble bee species that have experienced recent declines had significantly higher levels of Nosema bombi than the species whose populations were stable (Cameron et al., 2011). Finding means of mitigating the effects of parasitism on bees would therefore be beneficial to the agricultural community as well as natural ecosystems.

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). Consumption of the alkaloid gelsemine significantly reduced infection intensity in bumble bees (Bombus impatiens) infected with the intestinal parasite Crithidia bombi (Manson et al., 2010), and four other nectar secondary compounds had significant medicinal effects in the same bee-parasite system, 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 secondary metabolites in nectar. Synergistic interactions between secondary metabolites from other plant tissues are well established. 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.

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 Bomubs (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 has been found to deter honey bees (Singaravelan et al., 2005), and reduced C. bombi load in infected bumble bees (Richardson et al., 2015a).

Diet 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. The first experiment (‘Variable’, conducted 26 February 2014 to 20 March 2014, Dataset 1) had a smaller sample size (n = 178 bees) and less strictly controlled environmental conditions, while the second experiment (‘Controlled’, 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 ‘Variable’, experimental bees were kept on the lab bench (temperature range 10–35°C due to a steam leak, approximately 12 h photoperiod). In ‘Controlled’, experimental bees were incubated at 27°C in constant darkness to 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 initially incubated on the lab bench, but were later incubated at 30°C in an incubator (Percival Scientific, Perry, IA) due to excessive pre-experiment mortality under the variable lab conditions. In ‘Controlled’, pupal clumps were incubated at 27°C throughout the experiment. 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 assigned systematically to diet treatments in blocks of four, such that each block contained a bee in each treatment. 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, bees were fed pollen and control nectar (30% sucrose solution). Bees were inoculated with C. bombi two days after emergence. They were starved for several hours to ensure that they would consume the inoculum, and then fed 10 μL of C. bombi inoculum containing 6,000 C. bombi cells (see below). Bees were then fed pollen and the appropriate nectar treatment ad libitum for 7 days.

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).

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 ‘Controlled’, 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).

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 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 (‘Controlled’ experiment).

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 ‘Controlled’ 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).

f09927b9-49bd-4a80-a5ce-f4340f0ae41d_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.

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

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).

f09927b9-49bd-4a80-a5ce-f4340f0ae41d_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.

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.

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

‘Controlled’

Under controlled conditions (27°C with constant darkness), neither alkaloid nor their interaction significantly affected mortality (Figure 3, Table 3). 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 ‘Controlled’ were much higher, with median parasite loads more than double those observed in ‘Variable’ (compare Figure 2 and Figure 4).

f09927b9-49bd-4a80-a5ce-f4340f0ae41d_figure3.gif

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

Lines show survival curves for bees each treatment group. There were no significant effects of diet treatments on survival.

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

Results of Wald tests for marginal significance of terms in a generalized linear mixed model with penalized quasi-likelihood parameter estimation.

Sourceχ2DfP
Nicotine5.8410.026
Anabasine2.7810.095
Nicotine*Anabasine2.5910.11
Colony6.7630.080
Mass6.9110.0086
f09927b9-49bd-4a80-a5ce-f4340f0ae41d_figure4.gif

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

Points show adjusted mean parasite count in each treatment group. Error bars represent ±1 standard error.

Discussion

Nicotine increased mortality under variable temperature conditions

Nicotine consumption increased mortality in ‘Variable’, but did not affect mortality in ‘Controlled’. The difference in temperature between the two experiments may be responsible for this context-dependent response. In ‘Variable’, the incubation temperature of the experimental bees was not controlled. Bees were kept on lab benches, in a room with temperatures that ranged from 10 to 35°C. In ‘Controlled’, by contrast, bees were incubated at a constant temperature of 27°C. One hypothesis to explain these divergent responses is that bees in the first experiment may have experienced heat stress that could have exacerbated toxic effects of nicotine, and may have consumed larger quantities of the alkaloid-rich artificial nectar to compensate for evaporative water loss. However, we did not measure nectar consumption, and so cannot be certain that consumption increased under heat stress. 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 bees even at very low concentrations when bees are parasitized. This contrasts with previous studies, which did not find significant toxic effects of nicotine at natural 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. These studies focused on honey bees, while our study used bumble bees, so the discrepancy between their results and ours may be due to bumble bees having a greater sensitivity to nicotine than honey bees. However, our results suggest that the toxic effects of nicotine are greater under temperature-stressed conditions; use of optimal incubation conditions could account for the lack of toxic effects observed in these previous studies. Drastic temperature variation similar to that experienced by bees in ‘Variable’ is common in continental climates. 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

In ‘Variable’, bees that consumed either alkaloid alone had significantly lower parasite counts than control bees, but this effect was not present in bees that consumed both alkaloids. This is 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

In ‘Controlled’, 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. Sub-lethal colony-level exposure to the neonicotinoid imidacloprid has been shown to lead to increased levels of Nosema infection in honey bees (Pettis et al., 2012). A field study by Alburaki et al. (2015) found significantly higher levels of infection by both brood queen cell virus and Varroa mites in honey bee colonies that had foraged on corn treated with the neonicotinoid thiabendazole than in colonies that had foraged on untreated corn. The neonicotinoid pesticides 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).

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

The apparent contradiction between the results of our first and second experiments may be due to a complex multi-directional interaction between alkaloid consumption, heat stress, and immunity. Under the variable conditions of ‘Variable’, bees may have consumed more liquid, causing them to ingest greater amounts of alkaloids. This increased alkaloid consumption could lead to stronger 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’. Bees in ‘Variable’ were also exposed to external stimuli in the lab environment, including light and vibration, which may have further promoted alkaloid consumption by increasing energetic requirements and synergized with stimulatory effects of the alkaloids to promote intestinal peristalsis.

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 ‘Controlled’ 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. 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 contrast with the results of a recent study by Richardson et al. (2015a). Richardson et al., found that both nicotine and anabasine significantly reduced C. bombi parasite load in bumble bees without affecting mortality. In ‘Variable’, we similarly found that both alkaloids reduced pathogen load, but we also found that nicotine increased mortality. This discrepancy may be due to a simple difference in study design: we used the (-)-enantiomer of nicotine, whereas Richardson et al. (2015a) used a +/- enantiomeric mixture (Sigma N0267, personal communications). (-)-Nicotine is far more common in nature, comprising between 99.77% and 99.83% of the nicotine in tobacco (Armstrong et al., 1998), and is more pharmacologically active than (+)-nicotine. In vertebrates, (-)-nicotine was 2.4–3.1 times more toxic to vertebrates than was (+)-nicotine (Gause & Smaragdova, 1939) and had stronger effects on the peripheral nervous system, particularly on muscle contraction (Barlow & Hamilton, 1965). In aquatic invertebrates known to use acetylcholine as a neurotransmitter, (-)-nicotine was again on average 2.6-fold more toxic that the (+)-enantiomer (Gause & Smaragdova, 1939). In insects, the (-)-enantiomer had stronger affinity for the nAChR in housefly and honey bee head membranes (Tomizawa & Yamamoto, 1992). Interestingly, Gause & Smaragdova (1939) found the two enantiomers to be isotoxic to the protozoans they tested. If this is the case for C. bombi, it suggests an explanation for our difference in results. If (-)-nicotine is more toxic to bees than (+)-nicotine, but both enantiomers are equally toxic to C. bombi, than (+)-nicotine could reduce parasite count without significantly affecting bee mortality, while (-)-nicotine could reduce parasite count but also be toxic to bees.

Another possible explanation for our differing results is a difference in 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) represent different strains, or different species, it is possible that they vary in their alkaloid tolerance.

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).

The effect 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. This suggests that bees are less tolerant of nicotine than microbes are. Despite their significant toxicity, nectar secondary metabolites such as nicotine are unlikely to pose a health risk to bees in the wild. The studies establishing toxicity of nicotine in bees have all focused on chronic consumption of a diet high in nicotine. This is an artificial condition that bees would be unlikely to encounter 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). They would therefore be unlikely to consume enough nicotine from nectar to experience toxic effects, although exposure to pharmacologically similar, agriculturally ubiquitous neonicotinoid insecticides appears concerning (Goulson et al., 2015).

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 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 ‘Controlled’ experiment, 10.5256/f1000research.6870.d101940 (Thorburn et al., 2015b).

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Thorburn LP, Adler LS, Irwin RE and Palmer-Young EC. Variable effects of nicotine and anabasine on parasitized bumble bees [version 1; peer review: 2 approved with reservations]. F1000Research 2015, 4:880 (https://doi.org/10.12688/f1000research.6870.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Open Peer Review

Current Reviewer Status: ?
Key to Reviewer Statuses VIEW
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 1
VERSION 1
PUBLISHED 21 Sep 2015
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21
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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
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
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CITE
HOW TO CITE THIS REPORT
Baracchi D. Reviewer Report For: Variable effects of nicotine and anabasine on parasitized bumble bees [version 1; peer review: 2 approved with reservations]. F1000Research 2015, 4:880 (https://doi.org/10.5256/f1000research.7396.r10860)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • 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
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
Views
25
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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
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 and anabasine on parasitized bumble bees [version 1; peer review: 2 approved with reservations]. F1000Research 2015, 4:880 (https://doi.org/10.5256/f1000research.7396.r10403)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • 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
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

Comments on this article Comments (0)

Version 2
VERSION 2 PUBLISHED 21 Sep 2015
Comment
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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