Receptor activation alters inner surface potential during phagocytosis.

Abstract

The surface potential of biological membranes varies according to their lipid composition. We devised genetically encoded probes to assess surface potential in intact cells. These probes revealed marked, localized alterations in the charge of the inner surface of the plasma membrane of macrophages during the course of phagocytosis. Hydrolysis of phosphoinositides and displacement of phosphatidylserine accounted for the change in surface potential at the phagosomal cup. Signaling molecules such as K-Ras, Rac1, and c-Src that are targeted to the membrane by electrostatic interactions were rapidly released from membrane subdomains where the surface charge was altered by lipid remodeling during phagocytosis.

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Page 1

flow recorded by the longitudinal dunes of the
eastern Sand Hills to be one of those feedbacks.
Whensoilmoistureislow,diurnalsurfaceheating
is very strong, and the momentum from winds
aloft can be transferred to the land surface. Dry,
southwesterly surface winds across the eastern
Sand Hills were greatly enhanced after wetlands
weredesiccated(5), grasscoverwasbreached,and
sandy soils were exposed to direct solar radiation.
We used National Centers for Environmen-
tal Prediction/National Center for Atmospheric
Research (NCEP/NCAR) reanalysis data from
1949 to 2005 to determine if years of low
precipitation corresponded with years when
spring-summer surface flow was more wester-
ly. Circulation composites for wet, dry, and
normal years were constructed for May, June,
and July. Although the Plains suffered severe
droughts in the 1950s and 1970s, and experi-
enced numerous dry (individual) years, the
reanalyses do not show a westward wind
shift. The NCEP/NCAR reanalysis is model-
dependent and has coarse resolution, therefore
surface winds can be suspect. Further, by
considering only monthly means, strong winds
capable of sand transport may not be well-
represented. Therefore, daily and monthly wind
dataforindividualstationsincentralandwestern
Nebraska were also examined. Again, no
consistent westward shift was found, indicating
that modern droughts and the Medieval drought
cannot be explained by the same mechanism.
Droughts of the last 57 years were relatively
short and due more to diminished moisture
convergence than to diminished moisture trans-
port. The dunes record a historically unprec-
edented large-scale shift of circulation that
removed the source of moisture from the region
during the growing season. Eastward or south-
ward migration of the North Atlantic subtropical
ridge of high pressure (the BBermuda High[)
likely initiated the Medieval drought (32),
allowing midlevel southwesterly flow to de-
scend. The drought may then have been
enhanced and prolonged by reduced soil
moisture and related surface-heating effects.
The MWP was a time of warmth and aridity
throughout much of the western United States
(7, 8, 16); this suggests that the circulation
change indicated by dune morphology is part of
a larger climate anomaly (33). A switch in
Pacific sea-surface temperature (SST) to a
quasi-perennial BLa NiDa[ state may be an
important factor (33), because such an SST
regime has been associated with drought
throughout much of the western half of the
United States (8). This concept may also help
explain more pronounced episodes of aridity
during the mid-Holocene, and it has seen recent
support from climate modeling studies (34).
References and Notes
1. D. A. Wilhite, K. G. Hubbard, in An Atlas of the Sand
Hills, A. Bleed, C. Flowerday, Eds. (Univ. of Nebraska
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5. J. A. Mason, R. J. Goble, J. B. Swinehart, D. B. Loope,
Holocene 14, 209 (2004).
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7. C. A. Woodhouse, J. T. Overpeck, Bull. Am. Meteorol. Soc.
79, 2693 (1998).
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D. W. Stahle, Science 306, 1015 (2004).
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211 (2000).
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Facies and Stratigraphy, H. G. Reading, Ed. (Blackwell,
Oxford, ed. 3, 1996), pp. 125–153.
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E. D. McKee, Ed. (U.S. Geological Survey Professional
Paper, vol. 10521979), p. 137.
22. D. M. Rubin, R. E. Hunter, Sedimentology 32, 147
(1985).
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London, 1991), pp. 448–481.
25. R. F. Madole, Quat. Sci. Rev. 14, 155 (1995).
26. A. F. Arbogast, J. Arid Environ. 34, 403 (1996).
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(1993).
29. S. D. Schubert, M. J. Suarez, P. J. Pegion, R. D. Koster,
J. T. Bacmeister, Science 303, 1855 (2004).
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1, 26 (2000).
31. S. D. Schubert, M. J. Suarez, P. J. Pegion, R. D. Koster,
J. T. Bacmeister, J. Clim. 17, 485 (2004).
32. S. L. Forman, R. Oglesby, R. S. Webb, Global Planet.
Change 29, 1 (2001).
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(2003).
34. S. Shin, P. D. Sardeshmukh, R. S. Webb, R. J. Oglesby,
J. J. Barsugli, J. Clim. 19, 2801 (2006).
35. We thank D. Wedin, A. Houston, and K. Hubbard for
helpful discussions and R. Goble and the Department of
Geosciences for support of OSL dating. Our work is part
of the Sand Hills Biocomplexity Project and was funded
by NSF (grant nos. DEB 0322067 and BCS 0352683).
Supporting Online Material
www.sciencemag.org/cgi/content/full/313/5785/345/DC1
Figs. S1 to S4
19 April 2006; accepted 14 June 2006
10.1126/science.1128941
Receptor Activation Alters Inner
Surface Potential During Phagocytosis
Tony Yeung,1,3Mauricio Terebiznik,2Liming Yu,1John Silvius,4Wasif M. Abidi,5Mark Philips,5
Tim Levine,6Andras Kapus,7Sergio Grinstein1,3*
The surface potential of biological membranes varies according to their lipid composition. We
devised genetically encoded probes to assess surface potential in intact cells. These probes revealed
marked, localized alterations in the charge of the inner surface of the plasma membrane of
macrophages during the course of phagocytosis. Hydrolysis of phosphoinositides and displacement
of phosphatidylserine accounted for the change in surface potential at the phagosomal cup.
Signaling molecules such as K-Ras, Rac1, and c-Src that are targeted to the membrane by
electrostatic interactions were rapidly released from membrane subdomains where the surface
charge was altered by lipid remodeling during phagocytosis.
T
cumulationofnegativechargescreatesanelectric
field,estimatedat105V/cm, that strongly attracts
cationic molecules, including peripheral mem-
brane proteins (1). This electrostatic interaction
has been best documented for the myristoylated
he plasma membrane of mammalian cells
contains about 20 mol % of anionic lipids
on the inner leaflet. The preferential ac-
alanine-rich C kinase substrate (MARCKS),
which interacts with the plasmalemma through
a polycationic domain, in conjunction with a
myristoyl anchor (2). The realization of this
charge-dependent anchorage led to the postula-
tion of an Belectro-static switch[ model (2),
which predicts that the formation and stability
of electrostatic associations can be regulated by
changes in the charge of either the cationic
protein complex or the anionic lipid layer.
Little is known about the regulation of the
electrostatic potential of the plasmalemma. It is
not clear whether the surface potential of the
cytoplasmic leaflet undergoes regulated changes
and, if so, whether such changes play a role in
modulating the association of cationic proteins.
This paucity of information is due to the absence
of methods to monitor thesurface potential of the
inner membranes of intact cells.
1Division of Cell Biology,
and Nutrition Department, Hospital for Sick Children,
Toronto, Ontario M5G 1X8, Canada.3Institute of Medical
Sciences, University of Toronto, Toronto, Ontario M5S 1A8,
Canada.
Montreal, Quebec H3G 1Y6, Canada.
Medicine, New York University School of Medicine, New
York, NY 10016, USA.6Division of Cell Biology, University
College, London EC1V 9EL, UK.
Research Institute, Toronto, Ontario M5B 1W8, Canada.
2Gastroenterology, Hepatology,
4Department of Biochemistry, McGill University,
5Department of
7St. Michael’s Hospital
*To whom correspondence should be addressed. E-mail:
sga@sickkids.ca
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Phagocytosis is associated with extensive
remodeling of the plasma membrane lipids
(3). Such lipid changes could potentially alter
the overall charge of the plasmalemma and
may therefore serve as an Belectrostatic switch[
to modulate the interaction with cationic lig-
ands, which could in turn affect the phagocytic
response. To investigate this possibility, we de-
veloped means to assess the electrostatic po-
tential of the inner aspect of the plasma
membrane in intact cells. We designed several
polycationic fluorescent probes that are selec-
tively targeted to the plasmalemma by virtue of
its unique negative surface charge. One probe
was modeled after the C terminus of K-Ras,
which was shown to associate with the mem-
brane in a charge-dependent manner (4). To
ensure that the probe was not phosphorylated,
we mutated all serine and threonine residues
to alanine to create a second probe (K-pre,
Fig. 1A). In a third probe, all lysines were sub-
stituted by arginines to avoid ubiquitination
(R-pre, Fig. 1A).
An in vitro assay assessed the effect of
surface potential on the affinity of R-pre for
pure lipid bilayers (5). R-pre labeled with bi-
mane, a solvochromic dye, partitioned prefer-
entially to liposomes containing anionic lipids
(Fig. 1B). Inclusion of phosphatidylinositol 4,5-
bisphosphate (PIP2) or phosphatidylserine (PS),
at concentrations resembling those in the plasma-
lemma, or phosphatidic acid (PA) increased the
partition coefficient by factors of 10, 51, and
87, respectively, relative to liposomes containing
only phosphatidylcholine (PC) with or without
phosphatidylethanolamine (PE) (Fig. 1D). More-
over, progressive elevation of the ionic strength
reduced the interaction of R-pre with anionic
liposomes and minimized the affinity difference
relative to uncharged liposomes (Fig. 1, C and
D, and fig. S1B).
We next expressed a genetically encoded
form of R-pre conjugated to red fluorescent pro-
tein (RFP) in RAW264.7 macrophages. R-pre–
RFP associated almost exclusively with the
inner aspect of the plasma membrane (Fig. 1E),
as found for the tail of K-Ras (Fig. 1F) (6). We
also used polybasic constructs containing an
N-terminal myristoylated sequence (K-myr
and Nt-Src) or containing an amphiphilic helix
(KRf) instead of a farnesylation site (Fig. 1A)
(6, 7). Like R-pre, KRf partitions preferential-
ly to anionic liposomes in an ionic strength–
dependent manner (fig. S1, A and C). When ex-
pressed in macrophages, KRf (Fig. 1E), K-myr
(Fig. 1E), and Nt-Src (fig. S5A) localized to the
plasma membrane, which implies that the com-
mon feature of these constructs—namely their
positive charge—was a primary determinant of
their targeting. Accordingly, progressive elimi-
nation of cationic residues resulted in graded
detachment of K-Ras tail–derived mutants
from the plasmalemma (Fig. 1, F and G) (6).
Three approaches were used to show that
the probes responded to changes in the elec-
tric field at the inner surface of the plasma
membrane. First, cells were treated with an
ionophore to elevate cytosolic calcium, which
shields the surface charge of the membrane
and induces PIP2hydrolysis through activa-
tion of phospholipase C (PLC). The exten-
sive degradation of PIP2was verified with the
use of PH-PLCd-GFP, a green fluorescent pro-
tein (GFP)–tagged probe for this phospho-
inositide (Fig. 2A). Calcium also activates the
lipid scramblase, resulting in net translocation
of PS to the outer leaflet (Fig. 2A). In parallel
with the changes in anionic lipid composition
and distribution, ionomycin induced a pro-
nounced dissociation of R-pre, KRf, and K-myr
from the inner surface of the membrane (Fig.
2A and movie S1). Redistribution of the probes
was not due to wholesale remodeling or dis-
ruption of the membrane. This was estab-
lished with the use of three different genetically
encoded fluorescent markers retained at the
plasma membrane by hydrophobic interactions:
glycosylphosphatidylinositol (GPI)–linked GFP,
a transmembrane chimeric protein (GT46), and
a farnesylated and diacylated GFP termed Palm
(fig. S1D). When coexpressed with the cationic
probes, the distribution of these markers re-
mained essentially unaltered after treatment with
ionomycin (Fig. 2A), which confirmed the in-
tegrity of the plasmalemma. Quantitation of the
effects of ionomycin on the distribution of the
probes is shown in Fig. 2B.
Fig. 1. Design and characterization of surface potential probes. (A) Structure of surface potential–
sensitive probes. Amino acid abbreviations: A, Ala; C, Cys; D, Asp; F, Phe; G, Gly; I, Ile; K, Lys; L,
Leu; M, Met; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp. (B) Transfer of R-pre from donor
liposomes (100 mol % PC) to acceptor liposomes containing PC (98 mol %), PC/PE (78/20 mol %),
PC/PS (78/20 mol %), PC/PA (78/20 mol %), or PC/PIP2(96/2 mol %). F/Fo, fluorescence intensity
ratio. (C) Transfer of R-pre to PC (98 mol %; navy) or PC/PS (78/20 mol %; other colors) acceptor
liposomes in medium supplemented with the indicated NaCl concentrations. (D) Partition
coefficient (Kp; relative to PC) of R-pre binding to liposomes containing PC (100 mol %), PE, PS, or
PA (20 mol % each), or PIP2(2 mol %) in the presence (red) or absence (blue) of 1 M NaCl. (E)
Macrophages transfected with R-pre, KRf, or K-myr. (F) Macrophages transfected with K-Ras tail
containing þ8, þ4, and þ2 charges. Scale bars, 2 mm. (G) Effect of charge on the membrane/cytosol
ratio of K-Ras tail variants.
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An alternative means of reducing surface
charge is to inhibit the ongoing formation of
polyphosphoinositides by depleting cellular
adenosine triphosphate (ATP). Simultaneous
impairment of glycolysis and mitochondrial
respiration was accompanied by dissociation
of PH-PLCd-GFP from the membrane, indicat-
ing loss of PIP2(Fig. 2A). All three cationic
probes detached from the plasma membrane of
ATP-depleted cells, whereas GPI-GFP, GT46,
and Palm remained unaltered (Fig. 2, A and B).
Flipping of PS can be induced by the an-
esthetic dibucaine, as indicated by the marked
increase in annexin-V binding (Fig. 2A). Loss of
PS from the inner leaflet, together with the
positive charges contributed by dibucaine it-
self, sufficed to displace R-pre, KRf, and K-myr
from the membrane (Fig. 2A). Because the three
probes responded similarly to all treatments,
regardless of their mode of hydrophobic inter-
action with the membrane, we conclude that
their dissociation occurred in response to changes
in surface charge. These probes are therefore
suitable for monitoring the surface potential of
the inner aspect of the plasma membrane.
We next applied the cationic probes to study
membrane remodeling during phagocytosis. Con-
focal imaging was used to monitor ingestion of
immunoglobulin G–opsonized latex particles by
macrophages expressing the surface potential
probes. The hydrophobically anchored markers
wereusedalongwiththecationicprobestocontrol
forremodelingcausedbyfusionandfissionevents.
Thecationicandhydrophobicallyanchoredprobes
localized to the nascent phagocytic cup (Fig. 3A
and fig. S2, B and C). Both probes were also
present in pseudopods as they progressed along
the sides of the particle, although the cationic
probes were often partially depleted from the base
Fig. 2. Effect of manipulating surface poten-
tial on probe distribution. (A) Distribution of
probes in cells treated with ionomycin (top),
antimycin/deoxyglucose (middle), or dibucaine
(bottom). Insets: cells before ionomycin. Scale
bars, 2 mm. (B) Quantification of effect of
treatments on probe distribution (ratio of
membrane fluorescence of probes specified).
Data are means T SE (n 9 20).
Fig. 3. Charge and lipid changes during phagocy-
tosis.(A) Time course of R-pre/GPI-GFP redistribution.
Asterisks indicate latex beads. (B to D) Fluorescence
ratio of probes in phagosomal membrane/unengaged
plasma membrane, calculated at onset of phago-
cytosis (0 s) and after 180 s. Data are means T SE
(n 9 20); *P G 0.001. (E) Redistribution of PH-
PLCd and GPI-GFP during phagocytosis. (F) Distribu-
tion of PS (white) during phagocytosis. Green, total
beads; red, extracellular beads. (G) Fluorescence
ratio of PH-PLCd, GPI-GFP, or annexin-V in phago-
somal membrane/unengaged plasma membrane.
Data are means T SE (n Q 16); *P G 0.001. (H)
Distribution of R-pre (red) and PH-PLCd (green) in
absence (left) or presence (right) of LY294002. In
(A), (E), and (H), insets show separately the
fluorescence of RFP (red) and GFP (green) of the
area boxed in the main panels. In (F), insets show
(from left to right) PS, external beads, and total
beads. Scale bars, 2 mm.
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of the cup at this intermediate stage (Fig. 3A and
fig. S2, A to C). At more advanced stages of
internalization, a further reduction of the cationic
probes was observed, which was invariably
followed by near-total depletion shortly after
completion of particle engulfment (Fig. 3A; fig.
S2, B and C; and movies S2 and S3). By
contrast, a substantial fraction of the hydropho-
bically anchored probes remained associated with
the phagosome even after internalization was
completed (Fig. 3A and fig. S2, B and C). When
calculated 3 min after initiation of phagocytosis,
the phagosome-to-bulk (unengaged) plasma
membrane ratios for R-pre (0.15 T 0.02), KRf
(0.20 T 0.03), and K-myr (0.20 T 0.02) were
significantly (P G 0.001) lower than those for
GPI (0.58 T 0.04), GT46 (0.59 T 0.04), and Palm
(0.52T0.03)(Fig.3, B to D). The depletion of the
cationic probes was more profound than expected
on the basis of remodeling, suggesting alterations
in the surface potential of forming phagosomes.
We investigated whether changes in anionic
lipid composition or distribution account for the
alterations in surface potential of the phago-
somal membrane. PIP2was markedly depleted
from forming phagosomes (Fig. 3, E and G,
and movie S4). Moreover, dissociation of the
cationic probe (R-pre) could be prevented when
the loss of PIP2was impaired (Fig. 3H).
Because PS contributes È30% of the charge
on the plasma membrane, its fate during phago-
cytosis was also studied. Annexin-V was used to
monitor the distribution of PS at the onset of
phagocytosisand3minthereafter.Innonpermea-
bilized cells, there was no discernible binding of
annexin-V; this result implies that little PS is
present on the outer monolayer before, during, or
after phagocytosis. To gain access to PS in the
inner leaflet, we fixed cells and gently permeabi-
lized them with saponin. PS was clearly detect-
able at the base of nascent phagosomes but
appeared greatly depleted from formed phago-
somal vacuoles (Fig. 3, F and G). Similar results
were obtained with a PS-specific antibody (fig.
S4). Jointly, the metabolism of PS and phos-
phoinositides could account for the changes in
surface potential during phagocytosis.
Could the change in surface potential have
physiological consequences? Molecules attracted
to the membrane by its negative potential are
anticipated to dissociate, possibly altering signal
transduction and cytoskeletal structure. The fact
thatcertainmembersoftheRassuperfamily(e.g.,
K-Ras, Rac1) contain a polybasic domain gives
credencetothisconcept(8). K-Ras constitutively
associates with the plasma membrane by both
prenylation and a polycationic domain in its
hypervariable region (Fig. 4A) (8). The impor-
tance of the positive charges in this region was
validated by introduction of three negatively
charged residues, which resulted in partial
dissociationoftheproteinfromtheplasmalemma
(Fig. 4A). Moreover, K-Ras responded to
changes in surface potential induced by ionomy-
cin, whereas H-Ras, which is dually palmitoy-
lated, was unaffected (Fig. 4A). As anticipated,
H-Ras was retained in sealed phagosomes to an
extent comparable to that of the GPI-anchored
marker (Fig. 4, A and B). In sharp contrast,
K-Ras was virtually absent from formed phago-
somes (Fig. 4, A and B, and movie S6).
Like K-Ras, Rac1 also contains a polybasic
domain(9). Recombinantprenylated Rac1bound
preferentially to anionic (PS/PC-coated) beads
relative to beads coated with PC only (Fig. 4C).
Moreover, mutation of the cationic residues in the
polybasic region to glutamine resulted in dissoci-
ationofconstitutivelyactiveRac1(Q61L)fromthe
plasmalemma (Fig. 4D), and Rac1(Q61L) local-
ization was sensitive to changes in surface
potential, whereas a mutant with the polybasic
Fig. 4. Surface potential modulates guanosine triphosphatase localization. (A) Top: Distribution of
full-length K-Ras, K-Ras-3E, and H-Ras before and after ionomycin treatment. K-Ras 3E is a mutant
form of K-Ras with three additional negative charges (see fig. S6 for structure). Bottom:
Distribution of H-Ras and GPI-GFP (left) or K-Ras and H-Ras (right) during phagocytosis. (B)
Phagosome/bulk membrane ratio of GPI-GFP, H-Ras, or K-Ras at onset of phagocytosis (0 s) or after
180 s. Data are means T SE (n 9 20); *P G 0.01. (C) Full-length prenylated Rac1 partitions
preferentially (by a factor of 2.3 T 0.7; n 0 8) to beads coated with PC/PS (80/20 mol %) relative to
beads coated with PC (100 mol %). (D) Distribution of Rac1(Q61L), Rac1(Q61L)-6Q, or
Rac1(Q61L)–H-Ras tail before and after ionomycin treatment. From left to right: plain Rac1(Q61L);
Rac1(Q61L)-6Q; plain Rac1(Q61L), treated with ionomycin; Rac1(Q61L)–H-Ras tail, treated with
ionomycin [see fig. S6 for structures of Rac1(Q61L)-6Q and Rac1(Q61L)–H-Ras tail]. (E)
Redistribution of Rac1 and Palm during phagocytosis. (F) Phagosome/cytosol ratio of wild-type
Rac1 at 0 and 180 s. Data are means T SE (n 9 20); *P G 0.001. (G) Redistribution of Rac1(Q61L)
and Palm during phagocytosis. (H) Phagosome/membrane ratio of Rac1(Q61L) at 0 and 180 s. Data
are means T SE (n 9 20); *P G 0.01. Scale bars, 2 mm.
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domain substituted by the hydrophobic tail of
H-Ras was not (Fig. 4D). Rac1 is of particular
importance to Fc receptor-mediated phagocytosis
and accumulates at the base of forming phago-
somes, detaching rapidly upon sealing (Fig. 4, E
and F, and fig. S7A) (10). Rac1(Q61L) also de-
tached from sealing phagosomes with kinetics
indistinguishable from those of wild-type Rac1
(Fig. 4, G and H, and movie S7). Because
Rac1(Q61L) is constitutively bound to guanosine
triphosphate (GTP),its dissociation from phago-
somes was not due to nucleotide hydrolysis or
cessation of nucleotide exchange. Instead, release
was likely mediated by termination of its electro-
static association with the plasmalemma. Accord-
ingly, the C-terminal tail of Rac1 containing the
polybasic domain behaved similarly (fig. S7B).
Our data indicate that the surface potential of
theinnerleafletofthemembranedecreaseslocally
during phagosome formation. The change is at-
tributable primarily to depletion of PIP2and PS,
but depletion of phosphatidylinositol 4-phosphate
was also observed (fig. S3 and movie S5). Activa-
tion of inositide lipases, kinases, and phosphatases
occurs during phagocytosis and bacterial invasion
(3), readily accounting for the changes in PIP2. PS
could be converted to PE by decarboxylation or
could be externalized during phagocytosis by
scramblases and/or efflux pumps.
Our results also indicate that the anchor-
age of important signaling molecules, includ-
ing K-Ras and Rac1, can be modulated focally
by localized changes in surface potential. Other
proteins anchored electrostatically to the mem-
brane, such as MARCKS, are equally suscepti-
ble to the charge alterations that accompany
lipid remodeling. Indeed, we also obtained
evidence for localized detachment of the
tyrosine kinase c-Src (fig. S5, B and C).
The consequences of altered surface charge in
other important biological phenomena must be
considered. Activation of phosphoinositide metab-
olism, elevation in cytosolic calcium, and PS
flipping occur after stimulation of multiple
receptorsandchannelsaswellasduringapoptosis.
The effect of such responses on inner surface
potential may be measurable with the use of
approaches like the one described here. Cycles of
membrane dissociation/reassociation may add a
layer of functional control to complement the
traditional biochemical mode of regulation of sig-
naling proteins.
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10. A. D. Hoppe, J. A. Swanson, Mol. Biol. Cell 15, 3509
(2004).
11. We thank E. Pick for providing Rac1 and D. Russell for
providing Nucleosil beads. Supported by the Canadian
Institutes for Health Research and an NIH grant, by a
Canadian Institutes of Health Research studentship (T.Y.),
and by the Pitblado Chair in Cell Biology (S.G.).
Supporting Online Material
www.sciencemag.org/cgi/content/full/313/5785/347/DC1
Materials and Methods
Figs. S1 to S7
Movies S1 to S7
4 May 2006; accepted 5 June 2006
10.1126/science.1129551
Parallel Declines in Pollinators and
Insect-Pollinated Plants in
Britain and the Netherlands
J. C. Biesmeijer,1* S. P. M. Roberts,2M. Reemer,3R. Ohlemu ¨ller,4M. Edwards,5T. Peeters,3,6
A. P. Schaffers,7S. G. Potts,2R. Kleukers,3C. D. Thomas,4J. Settele,8W. E. Kunin1
Despite widespread concern about declines in pollination services, little is known about the
patterns of change in most pollinator assemblages. By studying bee and hoverfly assemblages in
Britain and the Netherlands, we found evidence of declines (pre- versus post-1980) in local bee
diversity in both countries; however, divergent trends were observed in hoverflies. Depending on
the assemblage and location, pollinator declines were most frequent in habitat and flower
specialists, in univoltine species, and/or in nonmigrants. In conjunction with this evidence,
outcrossing plant species that are reliant on the declining pollinators have themselves declined
relative to other plant species. Taken together, these findings strongly suggest a causal connection
between local extinctions of functionally linked plant and pollinator species.
A
vertebrate taxa (1), and evidence has been ac-
cumulating that insect biodiversity is at risk as
well (2). Of particular concern is the possibility
of community-level cascades of decline and
extinction (3), whereby decline of some ele-
ments of the biota lead to the subsequent loss of
other species that directly or indirectly rely upon
them. Here we examine sets of pollinators and
the plants that they pollinate to test (i) whether
species that are linked to one another within
communities show coincident declines and (ii)
whether species with more links within com-
munities are more robust to change because of
nthropogenic changes in habitats and
climates have resulted in substantial re-
ductions in biodiversity among many
the availability of alternative links, if an inter-
acting species is lost.
Any loss in biodiversity is a matter of public
concern, but losses of pollinating insects may
be particularly troubling because of the poten-
tial effects on plant reproduction. Many agricul-
tural crops and natural plant populations are
dependent on pollination and often on the ser-
vices provided by wild, unmanaged, pollinator
communities. Substantial concerns have been
raised about the decline or loss of these services
E(4) but see (5)^, culminating in formal rec-
ognition within the Convention on Biological
Diversity (6) in the S,o Paulo Declaration (7)
and the International Initiative for the Conser-
vation and Sustainable Use of Pollinators (8).
However, the evidence for such declines re-
mains scanty (5).
To adequately demonstrate a decline in pol-
linator services, one would need to document
(i) overall declines in pollinator density; and/or
(ii) reductions in species diversity or substantial
shifts in the species composition of pollinator
communities, combined with changes in the
distribution of traits represented in those com-
munities (thus indicating that the loss of some
pollinators has not been compensated by the
rise of functionally equivalent species); and (iii)
declines in either the reproductive success or
abundance of plant species dependent on these
pollinators. No suitable data are available to
address overall pollinator density, but here we
provide evidence for the remaining points,
using data for bees, hoverflies, and plants from
Britain and the Netherlands.
We compiled almost 1 million records for
bee (all native species except the largely
1Institute of Integrative and Comparative Biology and Earth
and Biosphere Institute, University of Leeds, Leeds, LS2 9JT,
UK.2Centre for Agri-Environmental Research, University of
Reading, Reading, RG6 6AR, UK.
Survey–Netherlands/National Museum of Natural History
Naturalis, Postbus 9517, 2300 RA Leiden, Netherlands.
4Department of Biology, University of York, York, YO10 5YW,
UK.
Sussex, UK.
Foundation, Radboud University of Nijmegen, Postbox 9010,
6500 GL Nijmegen, Netherlands.7Nature Conservation and
Plant Ecology Group, Wageningen University and Research
Centre, Bornesteeg 69, 6708 PD Wageningen, Netherlands.
8Umweltforschungszentrum–Centre for Environmental Re-
search Leipzig-Halle, Community Ecology (Biozo ¨nosefor-
schung), Theodor-Lieser-Strasse 4, 06120 Halle, Germany.
3European Invertebrate
5Lea-side, Carron Lane, Midhurst, GU29 9LB, West
6Department of Animal Ecology, Bargerveen
*To whom correspondence should be addressed. E-mail:
j.c.biesmeijer@leeds.ac.uk
www.sciencemag.org SCIENCEVOL 31321 JULY 2006
351
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