Secondary Compounds in Floral Rewards of Toxic Rangeland Plants: Impacts on Pollinators

Abstract

The study of plant secondary chemistry has been essential in understanding plant consumption by herbivores. There is growing evidence that secondary compounds also occur in floral rewards, including nectar and pollen. Many pollinators are generalist nectar and pollen foragers and thus are exposed to an array of secondary compounds in their diet. In this review we document secondary compounds in the nectar or pollen of poisonous rangeland plants of the Western United States, and the effects of these compounds on the behavior, performance, and survival of pollinators. Further, we discuss the biochemical, physiological, and behavioral mechanisms by which pollinators cope with secondary compound consumption, drawing parallels between pollinators and herbivores. Finally, we propose three avenues of future research on floral reward chemistry. Given that the majority of flowering plants require animals for pollination, understanding how floral reward chemistry affects pollinators has implications for plant reproduction in agricultural and rangeland habitats.

Full text

Secondary Compounds in Floral Rewards of Toxic Rangeland Plants:
Impacts on Pollinators
Rebecca E. Irwin,*
,†
Daniel Cook,
‡
Leif L. Richardson,
†
Jessamyn S. Manson,
§
and Dale R. Gardner
‡
†
Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755, United States
‡
Poisonous Plant Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Logan, Utah 84321, United
States
§
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada
ABSTRACT: The study of plant secondary chemistry has been essential in understanding plant consumption by herbivores.
There is growing evidence that secondary compounds also occur in floral rewards, including nectar and pollen. Many pollinators
are generalist nectar and pollen foragers and thus are exposed to an array of secondary compounds in their diet. This review
documents secondary compounds in the nectar or pollen of poisonous rangeland plants of the western United States and the
effects of these compounds on the behavior, performance, and survival of pollinators. Furthermore, the biochemical,
physiological, and behavioral mechanisms by which pollinators cope with secondary compound consumption are discussed,
drawing parallels between pollinators and herbivores. Finally, three avenues of future research on floral reward chemistry are
proposed. Given that the majority of flowering plants require animals for pollination, understanding how floral reward chemistry
affects pollinators has implications for plant reproduction in agricultural and rangeland habitats.
KEYWORDS: alkaloid, nectar, pollen, pollinator, secondary metabolite
■INTRODUCTION
Rangeland plants contain secondary compounds that may
influence the palatability and/or toxicity of the plant to cattle
and other herbivores. Plant secondary compounds are not
restricted to vegetative tissues but can also be found in plant
reproductive tissues and rewards, including nectar and
pollen.
1−3
Thus, like grazing animals, pollinators are exposed
to a diverse array of secondary compounds when they collect
and consume floral rewards. Studies have documented the
identities and concentrations of secondary compounds in
nectar and pollen
4
as well as proposed and tested hypotheses
for the existence of these compounds in floral rewards
1,3,5
and
their costs and benefits for plant fitness via changes in
pollinator behavior.
6−8
Moreover, a growing number of studies
are focusing on the dietary effects of secondary compounds in
nectar and pollen on the performance of pollinators and the
biochemical, physiological, and behavioral mechanisms that
pollinators utilize when confronted with these compounds.
However, no study to our knowledge has reviewed the effects
of secondary compounds on pollinators and the mechanisms
involved. As rangeland plants can contain toxic secondary
compounds, there may be deleterious consequences to
livestock as well as beneficial insects that forage on these plants.
Secondary compounds in nectar and pollen can have a range
of effects on pollinator preference and performance, from
negative to neutral to positive. Anecdotal reports of pollinators
suffering toxic or lethal effects of secondary compounds are
frequent;
1
however, few empirical studies have explicitly linked
pollinator mortality to secondary compounds in floral rewards.
9
More common are reports of pollinator deterrence due to
nectar secondary compounds, with evidence that compounds
from a number of chemical families elicit aversive responses in
bees, ants, and butterflies.
10−12
For pollinators that do consume
secondary compounds, studies have documented deleterious
consequences such as reduced mobility,
4,7
ovary develop-
ment,
13
and survivorship.
9,14−17
It is important to note that the
negative effects of secondary compounds on pollinators are not
universal, and in some cases, consuming nectar secondary
compounds can have indirect benefits for pollinators, as some
compounds found in nectar can reduce gut pathogen loads.
18
Moreover, low concentrations of nectar secondary compounds
can directly act as pollinator attractants in some cases,
increasing visitation rates
19
and even improving a pollinator’s
ability to remember floral cues.
20
The goal of this review is to highlight the identities and
concentrations of secondary compounds in the nectar and
pollen of toxic rangeland plants and their effects on pollinators.
In so doing, this review links the chemistry of plants to the diet,
performance, and survival of pollinators, organisms critical to
the reproductive success of many agricultural and rangeland
species. First, we review the occurrence of secondary
compounds in nectar and pollen using a case-study approach
of select rangeland plants of the western United States. We
focused on these plants because of their well-studied chemistry
and because they are known to be poisonous to livestock; yet,
many of these plants are reliant on pollinators for pollination,
and a growing number of studies are documenting the
chemistry of their floral rewards and subsequent effects on
pollinators. Within the context of each plant case study, we
Special Issue: Poisonous Plant Symposium, Inner Mongolia
Received: January 29, 2014
Revised: April 17, 2014
Accepted: April 26, 2014
Published: April 26, 2014
Review
pubs.acs.org/JAFC
© 2014 American Chemical Society 7335 dx.doi.org/10.1021/jf500521w |J. Agric. Food Chem. 2014, 62, 7335−7344

explore how secondary compounds isolated from nectar and/or
pollen affect the behavior, reproduction, and/or survival of
pollinators. Second, because pollinators are often able to
survive the consumption of secondary compounds in nectar or
pollen, we review the biochemical, physiological, and behavioral
mechanisms by which pollinators cope with their consumption.
Given that bees are the dominant pollinators globally, we focus
most of our review on how bees cope with secondary
compound consumption and only touch on other types of
pollinators, such as Lepidoptera and birds. Finally, we propose
avenues for future research on the chemistry, biochemistry, and
ecology of secondary compounds in floral rewards from the
plant’s and the pollinator’s perspectives.
■SECONDARY COMPOUNDS IN NECTAR AND
POLLEN: CASE STUDIES AND THEIR EFFECTS ON
POLLINATORS
Several genera of poisonous plants are found in rangelands
throughout the world and the western United States. These
plants are typically visited by a suite of generalist pollinators or,
more rarely, by select specialists. Here we highlight eight genera
of plants (Figure 1), including their pollinators, secondary
compounds, and relevant literature describing the presence and
concentrations of these secondary compounds in floral rewards
as well as their effects on pollinators (Table 1). We focus on
alkaloid-containing plants as estimates suggest that 10−25% of
angiosperm species contain alkaloids, and alkaloids are well-
known to mediate plant−animal interactions.
21
Zigadenus spp. (= syn. Amianthium,Anticlea,Sta-
nanthium, and Toxicoscordion;
22
Death Camas; Melan-
thiaceae). Reports show that several Zigadenus species,
including Zigadenus nuttallii and Zigadenus paniculatus, are
visited by a single bee, Andrena astragali, or a subset of the bee
community in the family Halictidae, whereas others such as
Zigadenus elegans are visited by flies.
23−25
Zigadenus spp.
contain a group of cevanine-type steroidal alkaloids (Table 1)
including esters of germine, protoverine, and zygadenine.
26−28
Zygacine, 1(Figure 2), an ester of zygadenine, is the major
alkaloid in Z. paniculatus and Zigadenus venenosus.
27,28
These
neurotoxic alkaloids bind open voltage-sensitive calcium
channels
29
and are toxic to mice, sheep, and cattle.
30,31
Humans
have also been poisoned accidentally via consumption of death
camas bulbs.
30
Zygacine and related alkaloids can be detected
in all above-ground vegetative parts.
28
Moreover, preliminary
data suggest that zygacine can be detected in floral rewards,
including nectar and pollen of Z. paniculatus.
32
In an
experiment, adults of the generalist bee Osmia lignaria were
paralyzed and died soon after feeding on biologically relevant
doses of zygacine,
32
matching results suggesting toxicity of
nectar and pollen of Z. venenosus to honey bees (Apis
mellifera).
33
Larval progeny of O. lignaria also died when they
were fed provisions with zygacine.
32
The authors suggest that
the presence of zygacine in nectar and pollen may explain the
absence of generalist pollinators visiting this plant and that A.
astragali and pollinating flies may have adaptations to cope with
consumption of zygacine.
Veratrum spp. (Corn Lily; Melanthiaceae). The domi-
nant floral visitors of Veratrum spp., such as Veratrum album,
are ants, flies, and beetles.
34
In V. album there was also a single
record of a bee belonging to the Halictidae visiting the
flowers.
34
Veratrum spp. contain a series of steroid alkaloids
termed azasteroids (Table 1), such as cyclopamine, 2(Figure
2), of which there are different groups, including veratranine,
jervanine, and cevanine; the cevanine types are similar to those
found in Zigadenus spp.
35
These alkaloids are found in roots as
well as vegetative parts and, like zygacine, are also neurotoxins
that bind open voltage-sensitive calcium channels.
29
Consum-
ing alkaloids from V. album suppresses larval growth in house
flies (Musca vicina), an effect that dwindles as larvae mature and
has been reported in a number of nonpollinating insect
species,
36
suggesting negative post-ingestive consequences for
Figure 1. Floral images of select genera of poisonous plants found in
rangelands throughout the world and the western United States: (A)
Zigadenus elegans (= syn. Anticlea elegans); (B) Veratrum californicum
(= syn V. tenuipetalum); (C) Delphinium barbeyi; (D) Aconitum
columbianum; (E) Nicotiana attenuata; (F) Lupinus prunophilus (= syn.
L. polyphyllus); (G) Senecio serra. Photographs provided courtesy and
copyright of Al Schneider (www.swcoloradowildflowers.com).
Table 1. Ecology and Secondary Chemistry of Select Poisonous Plants of the Western United States and Impacts on Floral
Visitors
plant genus secondary chemistry primary pollinators effects on floral visitors
Zigadenus cevanine-type steroidal alkaloids flies, specialized bee Andrena astragali, some bees
in the Halictidae zygacine causes mortality in Osmia lignaria
32
Veratrum azasteroid-type steroidal
alkaloids ants, flies, beetles, rarely bees induces mortality in house flies and honey bees
36,38,39
Delphinium lycoctonine-type C19
norditerpene alkaloids bees (especially bumble bees), hummingbirds dose-dependent deterrence and reductions in bee activity
4,7
Aconitum aconitine-type C19 diterpene
alkaloids bees (especially bumble bees), hummingbirds detected at trace concentrations in bumble bee tissues but
effects on bees unknown
48
Nicotiana piperidine and pyridine alkaloids bees, hawkmoths, hummingbirds dose-dependent deterrence and reductions in bee health and
survival
9,15,19
Lupinus piperidine and quinolizidine
alkaloids solitary and social bees sparteine has both deterrent and toxic effects on honey bees
9
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pollinator development. Moreover, alkaloids of V. album are
sequestered by the specialist sawflyRhadinoceraea nodicornis,
reducing predation by ants, spiders, and bush crickets.
37
However, no studies to our knowledge have documented the
identities and concentrations of alkaloids in Veratrum spp. floral
rewards, although reports exist of Veratrum californicum toxicity
to honey bees foraging in the field.
38,39
The structural similarity
between some of the Veratrum spp. alkaloids and those found
in Zigadenus spp. leads to speculation of the possibility that
these compounds may deter generalist bee pollinators from
visiting these plants, as has also been speculated in Zigadenus
spp. This hypothesis warrants further investigation.
Delphinium spp. (Larkspur; Ranunculaceae). The
dominant floral visitors of Delphinium spp. are generalists,
including a variety of bees, especially bumble bees, as well as
hummingbirds.
40,41
The nectar of Delphinium flowers is
recessed within a nectar spur, so only bees and hummingbirds
with a long proboscis or bill can reach the nectar rewards.
Delphinium spp. contain a series of C19 norditerpene alkaloids
mainly of the lycoctonine type (Table 1); several of these
alkaloids are usually found in any given species. These
lycoctonine C19 norditerpene alkaloids are divided in two
structural classes, the N-(methylsuccinimido)anth-
ranoyllycoctonine (MSAL) type, 3(Figure 2), and the 7,8-
methylenedioxylycoctonine (MDL) type, the former being
more toxic and the latter less toxic.
42
These alkaloids bind to
acetylcholine receptors and are toxic to mammals and
insects.
43−45
Consumption of these plants causes significant
cattle losses on an annual basis throughout the western United
States.
42
Delphinium barbeyi and Delphinium nuttallianum
contain alkaloids in vegetative and floral parts as well as the
floral rewards, including nectar and pollen.
4
Alkaloid concen-
trations differ between floral rewards, vegetative, and floral parts
(Figure 3), with alkaloid concentrations in the nectar being at
least 450 times less than any vegetative or floral part in both
species. Alkaloid concentrations in Delphinium pollen are likely
to be intermediate to concentrations in pollen loads and
anthers as anthers contain other tissues that may overestimate
actual pollen concentrations, whereas pollen loads contain
nectar that may underestimate pollen concentrations.
4
The
alkaloid profile observed in floral rewards is qualitatively similar
to the profile found in vegetative parts in both species. Bees fed
sugar water supplemented with Delphinium alkaloids exhibited
Figure 2. Chemical structures of zygacine, 1, a steroidal alkaloid from
Zigadenus spp.; cyclopamine, 2, a steroidal alkaloid from Veratrum
spp.; methyllycaconitine, 3, a norditerpene alkaloid from Delphinium
spp.; aconitine, 4, a diterpene alkaloid from Aconitum spp.; nicotine, 5,
a pyridine alkaloid from Nicotiana spp.; lupanine, 6, a quinolizidine
alkaloid from Lupinus spp.; and senecionine, 7, a pyrrolizidine alkaloid
from Senecio spp.
Figure 3. Total alkaloid amounts in different plant parts of (A)
Delphinium barbeyi and (B) Delphinium nuttallianum. Bars are means +
SE.
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reduced activity, but only at concentrations at least 50 times
higher than those detected in floral nectar.
4,7
D. barbeyi flowers
supplemented with alkaloids had fewer visits by bee pollinators,
and bees spent less time per visit, but the effect was
concentration dependent.
7
Whereas D. barbeyi nectar alkaloid
concentrations within the natural range had no effect on bee
behavior, alkaloid concentrations higher than reported in nature
strongly deterred pollinators, suggesting an adaptive benefitto
low nectar alkaloid concentrations to encourage pollination.
7
Aconitum spp. (Monkshood; Ranunculaceae). Aconitum
spp. are typically visited by generalist pollinators, including
bumble bees and hummingbirds, and in rare instances by
specialists.
46−48
The flowers of Aconitum spp. are characterized
by one of their petaloid sepals forming a hood that holds two
nectar spurs on stalks; thus, like Delphinium spp., nectar is only
accessible to visitors with a long proboscis. Aconitum spp.
contain a series of C19 diterpene alkaloids mainly of the
aconitine type, 4(Table 1; Figure 2). Aconitine-type alkaloids
are neurotoxic and cardiotoxic to insects and mammals,
30,49
including livestock; however, there are few livestock morbidity
reports from consumption of Aconitum as it is seldom eaten in
sufficient quantity to cause mortality.
30
Gosselin et al.
48
reported alkaloid concentrations in different plant parts as
well as floral rewards in Aconitum septentrionale. Alkaloid
concentrations in pollen were similar to leaf and flower
concentrations, whereas concentrations in nectar were
significantly lower. Lappaconitine was the dominant alkaloid
in all plant parts investigated including the floral rewards,
although some differences were observed in the rank order of
abundance of other alkaloids between other tissues and floral
rewards. A. septentrionale pollen contains alkaloids in concen-
trations above the LD50 concentrations for most alkaloids
tested by Detzel and Wink
9
on honey bees, suggesting the
potential for post-consumptive effects on pollinator perform-
ance. However, effects of A. septentrionale alkaloids on bumble
bee pollinator larval survival and performance remain unknown.
Additionally, Gosselin et al.
48
detected the alkaloid lappaconi-
tine in the tissues of the specialist bumble bee Bombus
consobrinus but not the generalist Bombus wurflenii. Whether
this observation has any biological significance or results in a
fitness benefit is not known.
Nicotiana spp. (Tobacco; Solanaceae). Nicotiana spp. are
visited by a suite of generalist pollinators, including bees,
hawkmoths, and hummingbirds. Nicotiana spp. contain several
piperidine and pyridine alkaloids (Table 1), including nicotine,
5(Figure 2), that can be toxic to insects and mammals.
50,51
Nicotine acts as a stimulant and binds as an agonist to nicotinic
acetylcholine receptors. Nicotine is synthesized in the root and
transported to all above-ground plant parts, including floral
rewards,
50,52,53
with nicotine concentrations in leaves and
flowers being greater than that in nectar. In general, species that
contain greater concentrations of nicotine in leaves have greater
amounts of nicotine in their nectar.
53
Nicotine can reduce the
amount of time spent per flower, as well as the amount of
nectar removed per visit, by hawkmoth and hummingbird
pollinators.
8,54
However, the deterrent effects of nicotine in
nectar can be dose-dependent
15,55
and dependent on nectar-
sugar concentration,
15
and even at the highest concentrations of
nicotine in artificial nectar, there is not complete deterrence.
15
Moreover, Singaravelan et al.
19
found that nectar with low
nicotine concentrations is preferred by honey bees over nectar
that contains no nicotine, suggesting that low nicotine
concentrations may act as attractants or feeding cues. Nicotine
consumption also has dose-dependent effects on honey bee
health and survival.
9,14,15
Lupinus spp. (Lupine; Fabaceae). The pea-like flowers of
Lupinus spp. have an upper banner petal and two lower petals
fused into a keel. Pollinators must be strong enough to push
down the keel to gain access to the pollen, and in some cases
nectar, with bees being the most common pollinators. Lupinus
spp. contain piperidine and quinolizidine alkaloids (Table 1),
such as lupanine, 6(Figure 2), that bind to nicotinic and
muscarinic acetylcholine receptors. These alkaloids are toxic to
livestock and are thought to provide protection against natural
enemies, including insects.
56−58
Detzel and Wink
9
documented
alkaloids in the pollen of Lupinus polyphyllus, with pollen
alkaloid concentrations being 5 times less than that in flowers
and 27 times less than that in leaves. Sparteine, identified in L.
polyphyllus pollen, has both deterrent and toxic effects on honey
bees in the laboratory.
9
Other Genera. There are several other toxic rangeland
plant genera of the western United States, including Senecio
spp. (Asteraceae) and Echium spp. (Boraginaceae). Senecio spp.
are visited by a suite of generalist pollinators; Echium spp. such
as Echium vulgare can be visited by generalist species of bumble
bees in their introduced range in North America but are also
the host plants of several oligolectic bees in their native range.
59
Senecio and Echium spp. both contain pyrrolizidine alka-
loids.
30,60
Pyrrolizidine alkaloids, such as senecionine, 7(Figure
2), are hepatotoxic to livestock and are thought to provide
protection to the plants against natural enemies, including
insects.
61
These alkaloids have been detected in the pollen and/
or flowers (or florets) of different species within each genus.
62
Moreover, pyrrolizidine alkaloids have been detected in honey
from Echium
62−65
and Senecio;
66
whether these alkaloids in
honey come from pollen, nectar, or both reward sources
remains to be determined. 1,2-Unsaturated tertiary pyrrolizi-
dine alkaloids of Senecio vernalis were toxic to honey bees at a
concentration of 2% in the laboratory, and honey bees were
deterred by pyrrolizidine-N-oxides at concentrations >0.2%.
17
How common these effects are on the preference and
performance of other pollinators remains to be investigated.
Synthesis. Three general patterns emerge from examination
of the case studies. First, secondary compound concentrations
in nectar tend to be lower than in pollen or leaves. Second,
pollen secondary compound concentrations can, in some cases,
be equal to or even higher than those concentrations found in
leaves. Third, secondary compounds in floral rewards can act as
filters on the community of species visiting flowers, sometimes
discouraging visitation by nonspecialist pollinators or nectar
robbers or thieves, which could increase the chance of pollen
transfer for plants. The ecological and evolutionary implications
of these patterns for both plants and pollinators remain to be
explored in most cases, and the biochemical and physiological
mechanisms behind secondary compounds in nectar are an
active area of research.
3
■HOW DO POLLINATORS COPE WITH PLANT
SECONDARY COMPOUNDS?
Given that pollinators are exposed to secondary compounds in
their diet and frequently survive after consuming these
compounds, pollinators, like herbivores, may have mechanisms
to cope with the consumption of secondary compounds.
Indeed, pollinators and herbivores likely use some of the same
biochemical, physiological, and behavioral mechanisms. We
focus our review primarily on how bee pollinators survive the
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consumption of secondary compounds, but we note that the
frugivory literature
67
may also provide testable predictions for
how vertebrate pollinators cope with the consumption of not
only fruit but also nectar or pollen with secondary compounds.
Moreover, we broaden this portion of the review beyond
alkaloids to include other classes of secondary compounds that
can occur in floral rewards and how pollinators cope with their
consumption, including phenolics and terpenoids.
Biochemical and Physiological Mechanisms. There are
a variety of biochemical and physiological mechanisms that may
help bees survive the consumption of secondary compounds,
including detoxification, conjugation, target-site insensitivity,
sequestration, rapid excretion, and endosymbionts. Although
these mechanisms have been studied in greater depth in
herbivores, there is some evidence that bees and/or other
pollinators also use some of the same types of mechanisms,
with the most literature documenting detoxification.
Detoxification. A common way that animals cope with a
variety of potentially toxic compounds is through enzymatic
conversion to less toxic forms that can then be eliminated. One
of the most prominent superfamilies of enzymes is the
cytochrome P450 superfamily of monooxygenases that catalyze
the oxidation of organic substances. P450s are present in a wide
diversity of prokaryotic and eukaryotic taxa, including
pollinating bees. For bees, the diversity and activity of P450s
have been studied in the most depth in honey bees and to a
lesser extent in bumble bees. Honey and bumble bees contain
significantly fewer P450 genes than other insect genomes, such
as flies;
68
for example, the honey bee genome contains only 46
P450 genes, almost half that contained in other insect
genomes.
69
Why bee genomes are relatively depauperate in
detoxification genes relative to other insects remains an active
area of research, especially given the generalized foraging
strategy of most solitary and social bees. However, one relevant
hypothesis is that because nectar and sometimes pollen have
lower concentrations of secondary compounds than leaves,
4,6,9
natural selection for detoxification genes in bees may be weaker
than that in phytophagous insects,
68
the adults of which may
nectar-feed but larvae feed on leaf material.
Nonetheless, a number of P450s in bees have been
implicated in the metabolism of secondary compounds found
in nectar and pollen as well as xenobiotics, such as acaricides
used to control Varroa mites,
70
and the CYP6 family of P450s
has shown expansion relative to other insect genomes and may
be involved in the metabolism of phytochemicals found in
nectar, pollen, and honey.
69,71
For example, quercetin, a
flavonoid found in honey and pollen, is metabolized by five
enzymes in the CYP6AS and CYP9Q subfamilies of P450s.
70,71
In a similar vein, phytochemicals found in pollen and honey,
such as p-coumaric acid, result in the up-regulation of
detoxification genes.
72
p-Coumaric acid is ubiquitous in pollen
and nonfiltered honey and, although unlikely to be toxic to
bees, it may serve as a predictable cue for the up-regulation of
detoxification genes against a wide variety of secondary
compounds. Honey bees are also exposed to plant secondary
compounds when they gather resins from vegetation to
produce propolis, a sticky substance used in hive maintenance
and architecture.
73
A set of propolis-derived phenolic
compounds, such as pinocembrin and pinobanksin, are present
in most honeys, where they are considered minor contami-
nants.
74
These compounds have also been shown to up-regulate
P450 detoxification genes.
72
Along with P450s, other enzyme systems may also play a role
in metabolic detoxification, including the carboxylesterases
(COEs). COEs catalyze the hydrolysis of esters into amino
acids and alcohol. For example, albeit to a lesser extent than
P450s, COEs may also be responsible for the detoxification of
pyrethroids,
75
insecticides derived from pyrethrins naturally
occurring in Tanacetum spp. daisies. Like the P450s, there are
only about half as many COE protein-coding genes compared
to other insect genomes.
69
Additionally, the expression and
thus activity of the various detoxification genes may vary as a
function of bee sex and/or exposure at a particular life history
stage.
68
Conjugation. Conjugation, another form of detoxification,
involves the production of a compound that binds to the
secondary compound, which is thus rendered harmless and
excreted.
76,77
For example, the superfamily of glutathione S-
transferases (GSTs) can catalyze the conjugation of exogeneous
toxins, including pyrethroids (originally derived from plants)
and other xenobiotics.
78,79
Treatment with the pyrethroid
flumethrin increased GST activity in larvae, pupae, and nurse
honey bees.
80
However, in another study exploring honey bee
tolerance to pyrethroids, Johnson et al.
75
found that GSTs did
not contribute significantly to honey bee pyrethroid tolerance.
To our knowledge, the role of conjugation as a countermeasure
for secondary compound consumption is not well explored in
bees and warrants further research.
Target-Site Insensitivity. Target-site insensitivity occurs
when there is a change at a neuroreceptor site for a particular
secondary compound that subsequently inhibits or excludes
binding, or when there is a change in the number of receptors
for a particular compound, and is a primary mechanism by
which herbivores can become resistant to pesticides.
81,82
Although the role of target-site insensitivity to secondary
compounds is relatively unexplored in bees, it may play a role in
other types of pollinators consuming secondary compounds.
For example, target-site insensitivity to milkweed (Asclepias
spp.) cardenolides occurs in a number of herbivores, including
the monarch butterfly(Danus plexippus; Lepidoptera) and four
other insect orders (Heteroptera, Orthoptera, Coleoptera, and
Diptera).
83
Because D. plexippus adults can also act as
pollinators of milkweed and milkweed nectar can contain
toxic cardenolides,
84
it is plausible that target-site insensitivity
may also play a role in allowing adults to cope with the
consumption of secondary compounds. We should note,
however, that target-site insensitivity is not universal in
herbivores that feed on cardenolides; for example, arctiid
moths feeding on cardenolides may use the perineurium
surrounding the nervous tissue as a barrier, preventing
cardenolides from reaching the Na+/K+-ATPase in the central
nerve cord.
85
Nonetheless, adults sometimes nectar feed on the
same host plants as their folivorous larvae. These cases may
provide insights into the biochemical and physiological
mechanisms that adult pollinators use to cope with these
compounds.
86
Sequestration. Some foliar herbivores concentrate and
retain secondary compounds from their diets, a phenomenon
known as sequestration.
87
As most pollinators consume plant
material, it is possible that they too sequester secondary
metabolites, but this has been studied for only a few pollination
systems. Several species of male ithomiine butterflies
(Nymphalidae: Danainae) avidly seek and consume floral
nectars containing pyrrolizidine alkaloids, which they seques-
ter.
88
These alkaloids deter predators,
89,90
serve as pheromone
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precursors, and operate in a sexual selection mating system.
91
In another example, Southeast Asian Bulbophyllum orchids
attract their fruit fly (Tephritidae) pollinators with phenyl-
propanoid secretions including zingerone and methyleugenol;
male flies sequester these compounds, converting them to sex
pheromones.
92−94
It is possible that bees might also sequester secondary
compounds, because their diet consists almost entirely of nectar
and pollen that often contain those compounds, and other
herbivorous Hymenoptera such as sawflies commonly sequester
compounds such as iridoid glycosides and glucosinolates.
87
In
the only study to our knowledge that has investigated
sequestration by bees, Gosselin et al.
48
reported trace amounts
of six alkaloids in the tissues of a bumble bee species foraging
on Aconitum septentrionale, including the aconitine-type alkaloid
lappaconitine, the major alkaloid in pollen and nectar; however,
there was no evidence that bees concentrated the alkaloids.
Sequestration is most often found in insects with specialized
diets, and given that ∼20% of bees specialize in their pollen
collections, the possibility of sequestration of secondary
compounds by bees is a research avenue that deserves further
attention.
Rapid Excretion. Rapid excretion of secondary compounds
either with or without detoxification may serve as another
mechanism by which pollinators cope with the consumption of
these compounds. One striking example is the reduced gut
retention time following consumption of secondary compounds
involving Palestine sunbirds (Nectarinia osea) feeding on the
nectar of tree tobacco (Nicotiana glauca). The nectar of N.
glauca contains the pyridine alkaloids anabasine and nicotine.
Gut transit time of a sugar solution containing either of the
pyridine alkaloids was significantly lower when compared to
that of birds fed a sugar solution without the alkaloids.
55
The
authors suggest that consuming pyridine alkaloids may induce a
laxative effect, as is observed in mammals that consume plant
material containing pyridine alkaloids, which results in
accelerated gut transit time due to increased tone and activity
of the intestinal smooth muscles.
95
A laxative effect of
secondary compounds may be common in other plant-
pollinator systems as well but remains to be tested widely.
Endosymbionts. Many species of invertebrates harbor
endosymbiotic microbes in their guts, which may play a role
in the detoxification of secondary compounds.
96,97
Bees interact
with specific suites of horizontally transmitted bacteria
98,99
that
mediate immune responses to gut pathogens
100
and may play a
role in digestion of plant foods.
101
Some phytophagous beetles
interact with endosymbiotic gut fungi that assimilate carbon
from their hosts’diets, sometimes detoxifying alkaloids and
other potentially toxic compounds to do so.
96,102
Although a
detoxifying effect has not been demonstrated for endosymbiotic
fungi of pollinators such as bees, the possibility is worth
exploring given that bees harbor a diversity of gut fungi.
103
Behavioral Mechanisms. Behavioral mechanisms may also
help pollinators cope with secondary compounds. Here we
highlight three strategies: avoidance, diet mixing, and storage.
Avoidance. Biochemical and physiological mechanisms to
cope with secondary compounds usually occur or are induced
following compound ingestion. In contrast, behavioral mech-
anisms such as avoidance (or deterrence) can prevent
pollinators from consuming or collecting nectar or pollen
with secondary compounds prior to tasting their food (i.e., via
olfactory or visual cues) as well as reduce feeding once a
foraging choice has been made (i.e., via reduced per-flower visit
length or the number of flowers visited per plant). For example,
the darkly colored nectar of the South African aloe Aloe
vryheidensis may serve as a warning to some floral visitors of the
bitter-tasting phenolic nectar, albeit other visitors might find
such coloration attractive.
104
In comparison, high concen-
trations of nectar alkaloids in Gelsemium sempervirens have no
effect on whether or not a plant is visited, but do affect floral
visitor behavior once pollinators have tasted the nectar,
reducing the proportion of flowers visited per plant and the
time spent per flower.
6
Secondary compounds in floral rewards may not only affect
adult foragers but also their offspring when bees collect nectar
and pollen to provision to their larvae. Just as adult
phytophagous insects can avoid exposing their larvae to toxic
plants by not ovipositing on them,
105
adult bees could similarly
limit larval exposure to secondary compounds by reduced
nectar and pollen gathering from sources with floral secondary
compounds. In Lepidoptera, the link between oviposition
preference and offspring performance can be genetically
controlled.
106
The degree to which such preference−perform-
ance matches occur and are genetically determined in bees is
less explored. One strong difference, however, between
phytophagous insects and bees is that, in some cases, adult
phytophagous insects are unable to determine which plants are
suitable for their larval offspring,
107
but larvae can move
between plant species if poor oviposition choices are made.
108
In contrast, bee larvae typically cannot use movement as a
behavior to avoid feeding on unsuitable provisions, including
those that may have secondary compounds, and thus are forced
to eat provisions that may be distasteful and/or toxic.
Diet Mixing. Like many mammalian herbivores, most bees
are dietary generalists and are thus exposed to a great diversity
of plant secondary compounds. Mixing the products of many
plants could dilute the negative effects of any one compound,
thereby allowing generalists to exploit a food base that includes
nectar and pollen containing secondary compounds.
109
Research with solitary bees (Osmia spp.: Megachilidae)
demonstrates that foragers will incur greater energetic costs
than necessary to assemble pollen loads from a variety of plant
species, rather than a single species near the nest,
110
and that
bees may develop normally on pollen mixtures that contain up
to 50% of a toxic pollen that is lethal when consumed alone.
111
This increased energy expenditure to collect pollen from a
variety of plant species may be a mechanism to prevent larval
toxicity and/or to balance nutrient acquisition, although such
hypotheses warrant further investigation.
Storage. There is evidence that pollinators can avoid
negative consequences of secondary compound consumption
through social behaviors such as honey production and storage.
Honey is the product of enzymatic conversion and dehydration
of floral nectar by honey bees and related Apidae.
112
Glucose
oxidase, a product of the hypopharyngeal gland of nurse honey
bees, may deactivate (or suppress induction of) alkaloids
113
and
lower the secondary compound content of plant nectar within
the hive.
14,114
Moreover, honey bees maintain a nest homeo-
stasis that is high in temperature and low in CO2relative to the
environment, which promotes the modification and/or
degradation of phenolic compounds.
114
However, toxic honeys
have long been known
115
and may also contain unadulterated
secondary compounds such as the grayanotoxins of Rhododen-
dron (Ericaceae).
116
Although there are reports of nectars that
are toxic to bees
117
and some honey is toxic to humans, we do
not know whether such “toxic honeys”are toxic to the bees that
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produce them. Future research should examine the role of
apparently toxic honey in the ecology of social bees.
■FUTURE DIRECTIONS
Exciting challenges remain in the study of secondary
compounds in floral rewards. Throughout this review we
have highlighted a diversity of topics warranting further
investigation. Here we highlight three areas that we believe
are particularly promising for future research.
Chemistry and Biochemistry of Secondary Com-
pounds in Floral Rewards. Plant secondary compounds
have been quintessential in the study of plant−herbivore
interactions, and there is growing recognition that these
compounds occur in floral rewards.
1,3
A historical survey of
plants suggests the commonality of secondary compounds in
nectar,
2
with 55% of plants surveyed containing nonprotein
amino acids in their nectar, 36% containing phenolics, and 9%
containing alkaloids. However, few studies have identified and
measured actual concentrations of secondary compounds in
nectar and/or pollen rewards and how they relate to secondary
compound expression in a whole-plant context.
4,9,48,53,84,118,119
Measuring secondary compounds in floral rewards within a
whole-plant context provides an ideal opportunity to test
predictions about compound expression related to optimal
defense theory.
84,120
A variety of analytical tools are now
available to measure both within-species and among-species
variation in floral reward chemistry, including large-scale,
nontargeted metabolomics approaches.
There is a dearth of information not only on the identities
and concentrations of secondary compounds in floral rewards
but also of the origin of these reward components. Especially
within the context of nectar, where nectar secondary
compounds are produced and how they are added to the
nectar remain mysteries in most cases. Studies that measure
nectar secondary compounds within a whole-plant context
should also consider measuring their identities and concen-
trations in the phloem sap. Doing so may help to identify those
compounds that are manufactured by the nectary itself versus
those that come from the phloem sap and thus may be a
pleiotropic consequence of plant defense against herbivores.
Physiological and molecular research into the secretion
mechanisms of secondary compounds may also help to identify
compounds that serve an adaptive function.
Costs and Benefits of Secondary Compound Con-
sumption for Pollinators. Studies that have focused on the
costs and benefits of secondary compounds in floral rewards
have in many cases been phytocentric, measuring their effect on
plant fitness via changes in pollinator behavior.
6,119
However, as
consumers of nectar and pollen, pollinators such as bees are
exposed to a diverse array of secondary compounds in their
diet. Thus, there is nearly unlimited opportunity to assess how
secondary compounds in a pollinator’s diet affect performance
and fitness and to assess the mechanisms involved. Since the
1800s, there has been recognition that honeys can become
contaminated with floral products from nectar and pollen that
are harmful to human health, and so it is logical to extend these
effects to natural consumers of nectar and pollen. The handful
of studies conducted thus far show variable effects of secondary
compounds on pollinator performance, even when the same
compound is explored. For example, the nectar alkaloid
gelsemine reduced oocyte width in subordinate but not
dominant bumble bees of Bombus impatiens,
13
whereas the
same nectar alkaloid had no effect on larval survival or weight of
the solitary bee Osmia lignaria.
121
Future research should
consider that secondary compounds in floral rewards may have
not only costs for pollinator preference, performance, and
fitness but also potentially benefits and that the relative costs
versus benefits may be dependent on the identify and
concentration of secondary compound consumed. Moreover,
more mechanistic studies are needed from a diversity of
approaches, including molecular, biochemical, physiological,
and behavioral, that explore how pollinators cope with and
benefit from secondary compound consumption and the
potential consequences for interactions with other factors,
including xenobiotics and parasites. Given widespread concerns
over pollinator declines, a mechanistic exploration into how
pollinators survive secondary compound consumption may
provide insights into how pollinators handle chemical exposure
in rangeland and agricultural habitats.
Role of Secondary Compounds in Pollen. Whereas the
costs and benefits of nectar secondary compounds are
becoming more widely explored,
1,3
research on secondary
compounds in pollen has lagged behind. One challenge with
the study of pollen is its dual role, both as a gamete for plants
and as a reward for pollen-harvesting bees. Nonetheless,
research to date suggests that pollen secondary compound
concentrations can sometimes mirror or even be higher than
those in flowers or leaves, albeit sometimes less chemically
diverse.
4,48,118
Whether pollen secondary compounds are
simply a pleiotropic consequence of plant defense and
development or whether they serve an adaptive function
remains to be tested in most cases. Studies that explore pollen
secondary compounds using a comparative phylogenetic
approach and that incorporate research both from the plant’s
and the floral visitor’s perspective will provide important
scientific insights.
■AUTHOR INFORMATION
Corresponding Author
*(R.E.I.) Phone: (603) 646-3688. Fax: (603) 646-1347. E-mail:
Rebecca.Irwin@Dartmouth.edu.
Funding
L.L.R. was funded by a grant from the National Science
Foundation during the writing of this review (DEB-1256817).
Any opinions, findings, and conclusions or recommendations
expressed in this material are those of the authors and do not
necessarily reflect the views of the National Science
Foundation.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
We thank J. Andicoechea, Z. Gezon, R. Molyneux, G. Pardee,
R. Schaeffer, C. Urbanowicz, and two anonymous reviewers for
comments on the manuscript and J. Cane for permission to cite
unpublished data.
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