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Chemoecology 15:251–260 (2005)
0937–7409/05/040251–10
© Birkhäuser Verlag, Basel, 2005
DOI 10.1007/s00049-005-0318-4
CHEMOECOLOGY
Chemistry of preen gland secretions of passerines: different
pathways to same goal? why?
Meena Haribal1, André A. Dhondt1, David Rosane3,4 and Eloy Rodriguez3
1
Laboratory of Ornithology, Cornell University, 159, Sapsucker Woods Road, Ithaca, NY 14850 U.S.A
Plant Sciences, Cornell University, Ithaca, NY 14853 U.S.A
4
Present address: Nurture New York’s Nature, Inc., 75 East 55th Street, New York, NY 10022 U.S.A
3
Summary. Passeriformes is the largest order of birds and
includes one third of the bird species of the world, living in
very diverse habitats. We investigated the chemistry of preen
gland secretions of some groups of passerines from temperate regions found in diverse microhabitats. Some of the
common components were mixtures of homologous
monoesters made up of long chain acids and alcohols.
Individual species had characteristic distribution of esters
and was unique to a given species, although there were some
individual variations. We compared the composition of acids
and alcohols that formed same molecular weight esters in
different species and we found that the combination of acids
and alcohols to arrive at same molecular compositions
varied distinctly between species. To compare compositions
of over all acids and alcohols that formed the esters, the
representative samples of secretions from the individual
species were transesterfied the produce methyl esters and
alcohols. We found that there were distinct differences in
number of acids and alcohols that produced the combination
of homologous mixtures of esters. Also they differed both
qualitatively and quantitatively. There were also seasonal
differences in the secretion components. Thus though the
intact mixtures of esters in individual species had some similarities, they were very complex mixtures and differed
characteristically for individual species. Here we discuss
possible causes for evolution of these variations. We suggest
that the evolution in variation of preen gland secretion is
probably due to selective pressures caused by ectosymbionts
such as feather-mites and feather-chewing lice that live on
feathers and probably feed on the secretions and surrounding environments
Key words. preen glands – secretion – birds – longchain
esters – Corvidae – Emberizidae – Fringillidae – Mimidae –
Passerines – Passeriformes – chemistry – ectosymbionts
Introduction
To keep their feathers clean and supple, birds spend 5-30 %
of their time preening whereby they cover the feathers with
Correspondence to: Meena Haribal, e-mail: [email protected]
secretion taken from their preen gland. Preening duration
varies among species and also depends on feather condition,
season and many other factors (Espino Barros & Baldassarre
1989; Stock & Hunt 1989; Walther 2003). Feathers are
hosts to various ectosymbionts such as feather degrading
bacteria, fungi, feather mites, feather-chewing lice, etc. (Burtt
& Ichida 1999; Clayton & Janice 1997; Loye & Zuk 1991;
Muza et al. 2000). Birds have evolved diverse mechanisms
to combat these organisms. Some of the physical methods
include scratching, preening, sunbathing, taking a mud or
water bath, etc. But birds also use chemicals to combat parasites and pathogens. Some chemicals are produced by the
birds themselves and some are sequestered or collected from
external sources. Direct or indirect use of external chemicals
examples include selective use of neem leaves (Azadirachta
indica) in the nests by House Sparrows (Sengupta 1981);
aromatic green plant material by European Starling (Clark &
Mason 1988) and Blue Tits (Petit et al. 2002) as protection
against ectoparasites; formic acid or other chemicals used
during anting (Revis & Waller 2004); use of Garlic snail and
Schinus fruits by Hawaiian Elepaio (VanderWerf 2005);
selecting specifically chemically rich pine trees for nesting
as by Red-cockaded Woodpecker (Conner et al. 2003) etc.
Pitohui birds and birds of the genus Ifrita of Papua New
Guinea contain neurotoxins in their feathers (Dumbacher
1999; Dumbacher et al. 2000). These neurotoxins have been
found to be effective against feather-lice (Dumbacher 1999). It
is speculated that the birds sequester these chemicals from
their food, presumably from a beetle (Dumbacher et al.
2004). But by the far most commonly used chemicals by
birds are those present in the oily secretions from the preen
glands.
Preen gland – also known as uropygial gland, an essential accessory to feathers is present in most bird species.
They vary greatly in shape and size, constituting between
0.05 % and 1.14 % of the birds’ body weight (Jacob &
Ziswiler 1982). Taxa whose plumages often get wet (grebes,
ducks, osprey) have relatively larger preen glands as compared to other taxa, although, preen gland of the forestliving European robin (Erithacus rubecula), for example,
makes up 1.11 % of its body mass (Jacob & Ziswiler 1982).
The glands generally consist of two lobes and the internal
structures of these lobes differ significantly between
species, suggesting different functions, and also probably
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M. Haribal et al.
differ in the mechanisms of secretion production and storage
in different species.
It is very clear that feather maintenance is one of the
important functions of preen glands. When preen glands
have been removed, the feathers have became brittle and
rough (Moyer et al. 2003).
A few studies support the idea that preen gland secretions protect the birds against bacteria (Louis-Jacques et al.
1999) or ectoparasites such as mites, ticks and lice (Clayton
1990; Clayton 1991; Jacob & Ziswiler 1982; Moyer et al.
2003). The effect of preen gland secretions on feather mites
is controversial and the results of different studies are contradictory (Proctor & Owens 2000).
Kolattukudy and his co-workers have shown sex-related
changes in the composition of the secretion during the
breeding season in mallard ducks (Anas platyrhynchos)
(Kolattukudy et al. 1985). Thus secretions may possibly also
serve as pheromones. Recently, Piersma (1999), speculated
that the secretions enhance the attractiveness of the birds,
but in their subsequent reports they believe it may not be the
only true reasons (Piersma et al. 1999; Sinninghe Damste
et al. 2000; Reneerkens et al. 2002).
The importance of the role played by preen gland
secretions in defense against parasites and pathogens, kin
recognition, sexual selection, as pheromones, or olfactory
conspicuousness is poorly understood.
The chemical compositions of secretions of species
belonging to diverse families have been described about more
than quarter century ago. Except for some recent studies on
intact secretions of Charadriformes (Reneerkens et al. 2002)
and the woodhoopoes (Burger et al. 2004), most of the chemical studies were on the hydrolyzed or esterified products of
the secretion and very little is known about their intact components (Gamo 1971; Gebauer et al. 2004; Jacob and Ziswiler
1982; Morr et al. 1992; Odham 1967). They consist mostly
of monoesters of saturated, unbranched and or mono methyl,
di-methyl and poly methyl branched carboxylic acids. Most
of these acids are esterified with straight chain or methyl
substituted monoalcohols. Some diesters of waxes containing
hydroxy acids (at 2 or 3 position) with mono alcohol and
diesters of dihydroxy alcohol (both threo and erythro) with
acids have also been reported (Jacob & Ziswiler 1982). In the
secretions of some species, triterpenoids and steroids like
squalene, cholestanol, cholesterol, and cholestanone have
been also identified (Haribal et al. unpublished; Jacob &
Ziswiler 1982; Louis-Jacques et al. 1999).
Using SPME extraction methods, recently, Burger et al.
(2004) and Douglas et al. (2004) have shown that the preen
secretions contain many volatile molecules that were not
reported in the past by other investigators and have possible
varied functions (Burger et al. 2004; Douglas et al. 2004).
Also, preliminary investigation of some of the secretions in
our laboratory using other techniques such as ESI-MS-MS
indicated the presence of compounds that are not observed
using GC-MS techniques (Haribal, unpublished).
Thus birds seem to produce an array of compounds of different chemical classes and polarities, which vary from species
to species. Individual components or groups of components
may serve as repellents or toxicants to one or more of the
aforementioned ectosymbionts. Some of the chemicals may be
even stimulants to some of the organisms birds encounter.
CHEMOECOLOGY
With very limited data available on the intact compositions of secretions of the order Passeriformes (Jacob and
Ziswiler 1982; Louis-Jacques et al. 1999), we decided to
focus on the evolution and functions of preen glands in different species of passerines living in diverse habitats and
microhabitats in a given geographical region. Accordingly,
our aim was to review intact components of passerines of
the temperate regions that live in different habitats and use
different ecological niches. We mainly focused on two
closely related families, Emberizidae and Fringillidae, that
included the subfamilies Emberizinae, Icterinae, Fringillidae
and Carduelinae, and two unrelated representative species of
widely different taxa that belonged to Corvidae and Mimidae
respectively, as outgroups. In this paper we present only the
results of analyses of preen gland secretions by GC-MS
techniques.
Materials and methods
Sources of birds for collection of preen gland secretion
We collected most of the preen gland samples from local species of
Emberizidae, and Corvidae in New York and a few samples
(Mimidae) from the Dominican Republic. Samples from the Blue
Jay – Cyanocitta cristatus, (Linnaeus) (BLJA), the Common
Grackle – Quiscalus quiscula (Linnaeus) (COGR), the Chipping
Sparrow – Spizella passerina (Bechstein) (CHSP), the Song
Sparrow – Melospiza melodia (Wilson) (SOSP), the American
Goldfinch – Carduelis tristis (Linnaeus) (AMGO) and the House
Finch – Carpodacus maxicana (Müller) (HOFI) were collected
from birds mist-netted in Ithaca, New York. White-throated
sparrow – Zonotrichia albicolis (Gmelin) (WTSP), White-crowned
sparrow – Zonotrichia leucophrys (Forster) (WCSP/EWCSP)
and Swamp Sparrow – Melospiza Georgiana, (Latham) (SWSP)
samples were collected from birds mist-netted during the
fall/spring migration banding at Braddock Bay Banding Station, in
New York. Samples of Rufous Collared Sparrows – Zonotrichia
capensis (Müller) (RCOSP) and Northern Mockingbirds – Mimus
polyglottus (Linnaeus) (NOMO) were obtained in the Dominican
Republic in July 2002.
Procedure for collection of preen samples
and sample preparation
Generally, preen gland secretion samples for analyses were
collected from 5 to 15 or more individuals of each species, except
for CHSP where only two samples were collected. Also, BLJA,
SOSP, WTSP, AMGO, HOFI were sampled in different seasons.
Samples of secretions from individual birds were collected by
gently pressing a sterile cotton swab over the gland, collecting
secretions from the opening several times. The swabs were transferred to individual vials and stored tightly capped at 4 °C till
used. The individual swabs were then extracted with 500 µl of a
1:1 mixture of dicholoromethane (DCM) and methanol (MeOH)
by warming for 30-40 sec at 45 °C. The resulting solution was
pipetted out into a GC vial and evaporated under a nitrogen
stream to 300-350 µl. Most of the GC runs were recorded at this
concentration.
Analyses of components of the secretions
We analyzed the samples by GC-MS on HP 6890 coupled to MSD
5972A mass detector.
All separations of secretions were carried out on fused silica column (30 m × 0.32 mm ID × 0.25 µ film) with a cross linked methyl
silicone bonded phase (HP-5 MS cross linked S-1, PH-Me Siloxane)
starting initially at 120 °C (held for 2 minutes), then a gradient of
10 °C/min to 300 °C, finally held at 300 °C for 10 min (Program I).
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Chemistry of preen glad secretions of passerines
EWCSP
SWSP
40
40
30
30
20
20
10
10
0
0
14
16
18
20
22
24
26
28
30
14
16
18
20
22
24
OCT WTSP
30
26
28
30
RCOSP
MAY WTSP
% TIC
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40
30
20
20
10
10
0
0
14
16
18
20
22
24
26
28
14
30
MAY SOSP
SEP SOSP
40
30
20
10
0
16
18
20
22
24
26
28
30
CHSP
253
Fig. 1 Characteristic distribution of components in the preen
gland secretions of Emberizidae
species. The larger gray diamonds in each graph represent
the component with molecular
ions of m/z 494 daltons. Preen
gland secretion samples Whitecrowned Sparrow (EWCSP), Whitethroated Sparrow (WTSP), Song
Sparrow (SOSP), Swamp Sparrow (SWSP) were collected in
May 2004. Rufous-collared Sparrow (RCOSP) was collected in
Dominican Republic in July 2002
and Chipping Sparrow (CHSP)
was collected in September.
Additionally graphs of WTSP
and SOSP also represent the
components for samples collected in October and September
2003 respectively to show seasonal variation in the secretions.
All samples were run on HP-5
MS cross-linked S-1, PH-Me
Siloxane column (30 m × 0.2 mm
ID × 0.25 µm film thickness)
using program I
40
30
20
10
0
14
16
18
20
22
24
26
28
30
14
16
18
20
22
24
26
28
30
Ret. Time (min)
We transesterified representative samples of a few species, to
further identify the acid and alcohol portions of the esters that were
recognized by GC-MS. We used Alltech’s standard Methprep
solution to transesterify longchain esters to their respective methyl
ester. We added 200 to 400 µl of this solution to the sample of
secretion, which was previously dried using slow stream of nitrogen gas. The mixture was warmed for about 5 min at 60 °C and left
overnight. Then most of the solvent was evaporated under nitrogen
stream and 200-300 µl of water was added and then extracted with
600-800 µl of DCM. The DCM solution was dried over anhydrous
sodium sulfate and then evaporated to 300 µl. 3 µl of sample was
injected into the GC. We used transesterified products of hexadecyl hexanoate and arachidyl stearate as standards for reference.
All GC-MS analyses of transesterfication products of the secretions were carried out on fused silica column (30 m × 0.25 mm
ID × 0.25 µm film thickness) with a cross linked methyl
silicone bonded phase (HP-5 MS cross linked S-1, PH-Me Siloxane)
initially starting at 70 °C (held for 2 minutes), then the gradient of
10 °/min to 270 °C , finally held at 270 °C for 5 min (Program II).
TMS derivatives of the transesterified samples were prepared
by evaporating solvent using slow nitrogen stream, to the residue
300 µl of Trisil ‘Z’ was added and warmed at 60-70 °C for 5 min.
The sample was then directly injected into GC. The GC-MS
analyses were carried out using program II as described for
transesterified products.
Standards and Mass Spectral Library
Standards of hexadecyl hexanoate, arachidyl stearate, stearyl
arachidate, and n-octacosanol were obtained from Sigma-Aldrich
Inc USA. Methprep for transesterfication was purchased from
Alltech, USA. Trisil ‘Z’ was purchased from Pierce Chemicals
USA. Wiley 600 k Mass Spectral Library of Pallisade Corp. Ithaca,
New York was used for library search.
Results and discussion
Preen gland secretions of individual species for a given
month showed characteristic distribution of compounds in
GC-MS chromatograms, though there was some variation in
concentrations of individual components within a species
(Fig. 1 and 2). Mass spectral analyses of components showed
that most species consisted of homologous series of wax
monoesters and there was some similarity in patterns of
esters in the species we report here. Most of our compounds
did not give any match, as these compounds are not found in
the library. Most species showed a unimodal distribution of
homologous esters of varying chain length, except for the
NOMO, which had a very complex pattern with highest
number of components (Fig. 2). The overall chain lengths of
esters and the concentrations of esters in each species varied
considerably. (Figs 1 and 2; Table 1). The species that were
sampled more than once a year showed seasonal variation in
quantities and qualities of the components (see WTSP and
SOSP Fig. 1 and BLJA, HOFI and AMGO in Fig. 2).
Generally around a given retention time, for example,
around 19-20 min most samples showed presence of
monoesters with molecular composition of C33H66O2 (MW 494)
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CHEMOECOLOGY
COGR
25
20
15
10
5
0
Fig. 2 Characteristic distribution of components in the preen
gland secretions of Fringillidae
and outgroups Mimidae and
Corvidae species The larger
gray diamonds in each graph
represent the component with
molecular ions of m/z 494 da.
Preen gland secretion samples
of House Finch (HOFI) and
American Goldfinch (AMGO)
and Common Grackle (COGR),
Blue Jay (BLJA) were collected
in May 2004 and Northern
Mockingbird (NOMO) was
collected in July 2002. Additionally graphs of BLJA and
HOFI and AMGO also distribution of components of samples
collected in October 2003 to
show seasonal variation in the
secretions. All samples were
run on HP-5 MS cross linked
S-1, PH-Me Siloxane column
(30m × 0.2 mm ID × 0.25 µm
film thickness) using program I
MAY AMGO
OCT AMGO
25
20
15
14
16
18
20
22
24
26
28
30
10
5
6
% TIC
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NOMO
0
14
4
16
18
20
22
24
26
28
30
2
0
14
16
18
20
22
24
26
28
30
MAY HOFI
OCT HOFI
25
20
MAY BLJA
SEP BLJA
60
40
15
10
5
20
0
0
14
16
18
20
22
24
26
28
30
14
16
18
20
22
24
26
28
30
Ret. Time (min)
(Figs 1 and 2). Close elution time suggested that the esters
were very similar in nature. All esters give characteristic m/z
ions due to CH3(CH2)n-CO fragment ions. Further, mass
spectral analyses of each chromatographic peak that showed
molecular ion for m/z 494 in different species, based on the
acetyl fragment ions observed in mass spectrum, was made
up of more than one compound that also had very close elution times. For example, in the House Finch peak at 19.99
min showed presence acyl ions corresponding to C14- C17
acid moieties and the intensity for each acyl ion differed
suggesting that the esters due to these acyl moieties were
of not equal quantities (Fig. 3). Therefore, we could conclude that the esters were made up of combination of homologous series of acids and alcohols to produce the esters of
same molecular composition and their quantities differed.
The combination of acids and alcohols to produce the
required compound of molecular composition C33H66O2 differed in different species (Fig. 3). Some species such as
BLJA and AMGO consisted of fewer acid moieties, while
COGR and RCOSP had many more combinations of acid
and alcohol components to arrive at the same molecular
composition. Moreover, using SIM (Single Ion Monitoring)
technique we monitored presence of components that
produced molecular ions m/z 494 da (molecular ions for
ester of MF C33H66O2) in the mixture at different retention
times. Most of the samples showed at least two distinct
peaks corresponding to molecular composition C33H66O2
and each peak further consisted of different combinations of
homologous mixtures of acids and alcohols, although in
some species like NOMO and HOFI many molecular ions
for m/z 494 da were observed (Fig. 4). This further suggested that each acid and alcohol component of esters were
structural isomers with different amounts of branching. The
elution times of peaks of the components with m/z 494 were
lower than that of the corresponding straight chain ester
(Fig. 5). We could not achieve any further separation of
these peaks because of their close structural resemblance
and elution time.
The above techniques provided us with information
about number of carbon atoms in acid moieties but we could
not know if the acid moieties were branched, and also no
information could be obtained about alcohol moieties. Thus
transesterfication was one of the methods to provide further
insight into the complexity of these wax esters.
Results of transesterification products were analyzed by
mass spectral studies. Some species such as BLJA and
AMGO had fewer numbers of total acid and alcohol moieties while species like COGR and NOMO showed enormous complexities, and consisted of large number of both
acids and alcohols; other species were of intermediate
nature (Fig. 5). Among sparrows, SWSP had alcohols of
lower molecular compositions and SOSP had alcohols with
higher molecular compositions. Methyl esters of mono
methyl substituted acids (at 2, 3 or 4 position) produce characteristic fragment ions (Jacob 1975; Jacob 1978). Further
di and tri methyl substitutions can be possibly recognized by
characteristic fragment ions and the ratios of some of these
characteristic fragment ions. But it was not possible to confirm these components in our complex mixture as we did not
have access to GC-MS-MS, but we could only speculate
(Table 2). Most of the fringillids, icterids and emberyzids
contained monomethyl 3-methyl branched acids but some
were further branched (Fig. 6). Many species also showed
presence of small quantities of straight chain and 2-methyl
branched acids. The BLJA chad mostly 2-methyl branched
acids and small quantities of straight chain components
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Chemistry of preen glad secretions of passerines
255
Table 1 Carbon contents of esters and composition of acids and alcohols that produce the esters in the
species we investigated
Species*
Ester composition
(Ester of highest
concentration)
Acids as methyl
eaters
Alcohols as TMS
derivatives
BLJA
NOMO
WTSP
SOSP
EWCS
SWSP
CHSP
RCOSP
HOFI
AMGO
COGR
C28-C37 (C32)
C25->C45 (none)
C24-C36 (C30)
C27-C37 (C32)
C28-C35 (C32)
C26-C36 (C29)
C30-C40 (C35/C36)
C28-C37 (C30)
C26-C40 (C36)
C23-C35 (C30)
C25-C34 (C29)
C19-C23
C12-C17
C13-C17
C13-C19
C12-C18
C12-C18
C11-C19
C12-C19
C15-C21
C11-C16
C11-C20
C10-C17
C13-C32
C13-C20
C14-C24
C14-C17
C12-C22
C19-C21
C13-C19
C11-C19
C14-C19
C13-C22
*
Secretions of most species were collected in May 2004 except for NOMO and RCOSP were collected in
Jul 2002 and SOSP in Sep 2003. Note: These results are for these given months only and should not be
generalized for the whole year, as there is variation in both quality and quantity of the secretions
RCSP 20.27
SOSP 19.9
100
CHSP 19.81
80
SWSP 19.95
60
WTSP 19.8
40
EWCSP 19.5
20
0
C13
C14
C15
C16
C17
C18
C19
C20
C21
100
% Composition
318.qxd
C22
C23
AMGO 19.91
80
HOFI 19.99
60
40
20
0
C13
C14
C15
C16
C17
C18
C19
C20
C21
100
C22
C23
C24
BLJA 19.81
NOMO 19.94
COGR 19.56
80
60
40
20
0
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
Composition of acyl fragment ions
Fig. 3 Compositions of acids (as acyl group) for the gas chromatogram peaks that showed molecular ion at m/z 494 daltons that
eluted around 19.5-20 min in the secretions of different species
(Table 2). In most species, the MS analyses of individual
peaks of methyl esters of poly methyl substituted acids,
based on the characteristic fragment ions produced due to
branching and their ratios, indicated they were probably
mixtures of several isomers of same molecular weights and
were difficult to characterize as an individual component
(Table 2).
Based on the mass spectral fragmentation patterns,
alcohol moieties were mostly straight chain alcohols. Many
of them did not give molecular ions as they were of low
intensity or were masked by the fragments observed by the
Fig. 4 SIM (Single Ion Monitored) gas chromatogram of
Northern Mockingbird (NOMO) and House Finch (HOFI) that
show peaks due to molecular ion at m/z 494 da
co-eluting peaks. Therefore we derivatized the transesterified
mixture with Trisil ‘Z’ to produce TMS derivatives of alcohols. Again the analyses of TMS derivatives of alcohols
showed presence of n-alcohols as major components in most
species, but also consisted of small variable percentages of
other alcohols such as branched and secondary alcohols
(Table 2) in some species. Thus the original esters in each
species may be more complex than we can perceive. In
this paper we have refrained from identifying individual
components, as there are no standard compounds available
in the market; a very limited number of compounds are
available in the known Mass Spectrometry libraries. In order
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CHEMOECOLOGY
COGR
BLJA
MOBI
srandard acids
70
60
50
40
30
20
10
0
70
60
50
40
30
20
10
0
9
14
19
24
WCSP
SOSP
srandard acids
COGR
BLJA
MOBI
std alcohols
9
29
14
19
WCSP
SOSP
std alcohols
SWSP
RCOSP
WSTP
70
60
50
40
30
20
10
0
24
SWSP
RCOSP
WSTP
70
60
50
40
30
20
10
0
11
13
15
17
19
21
23
11
16
21
HOFI
std alcohols
AMGO
HOFI
srandard acids
AMGO
70
60
50
40
30
20
10
0
Fig. 5 Figures a-c represent the
composition and distribution of
acids (as methyl esters) that are
involved in the formation of esters
in different species. Standard
acids were n-hexadecanoic acid
and n-octadecanoic methyl esters
and an impurity in octadecanoic
acid respectively. Figures d-f represent alcohol products of the
esters components in the preen
gland secretions after transesterfication with Methprep. Standard
alcohols were n-hexacosanol and
n-octacosanol. All samples were
run on HP-5 MS cross-linked S-1,
PH-Me Siloxane (30 m × 0.2 mm
ID × 0.25 µm film thickness) using
program II
70
60
50
40
30
20
10
0
11
13
15
17
19
R4
21
R2
R3
Longchain esters
R1, R2, R3, R4, R5 = H st chain
R1 = CH3 and R2, R3, R4, R5 = H
R2 = CH3 and R1, R3, R4, R5 = H
R1, R3 = CH3 and R2, R4, R5 = H
R2, R4 = CH3 and R1, R3, R5 = H
11
R5
O
O
m
23
n
R1
2- methyl acids
3- methyl acids
2, x- di-methyl acids
3, x- di-methyl acids and so on
to identify individual components that we would need to
conduct some GC-MS-MS studies and undertake syntheses
of model compounds, and also run multi-dimensional NMRs
to identify the components. This was beyond the scope of our
current project. Nevertheless, our results are of significant
importance in understanding the evolution and possible
functions of preen gland secretions.
16
21
Different combinations of alcohols and acids that produce compounds of same molecular weight have different
physical properties though they may have similar chemical
properties. Their viscosity, melting point, boiling point,
refractive index etc. vary. Further, the presence of multiple
branching produces diastereomers and optical isomers that
again differ in both biological and physical properties
(Kulkarni and Sawant 2002). Therefore one can have at least
as many as M (number of acids) × N (number of alcohols)
numbers of esters of different molecular weights as possible
components in the secretions, with all of them occurring in
varying concentrations and in some case all combinations
may not be formed. Also, there is considerable variation in
the amounts as well as quality and mixing components in
different ratios can further change their physical properties
(Kulkarni and Sawant 2002) and biological functions. This
result suggests that even though the basic components and
final results of the mixtures are similar, individual species
are chemically diverse and further, may have diverse physical and biological properties.
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Chemistry of preen glad secretions of passerines
257
Table 2 Contents of branched, straight chain acids and alcohols in the transesterification products of the species we investigated
Species
Emberizidae
WTSP
EWCSP
SWSP
RCOSP
SOSP
COGR
Fringillidae
HOFI
AMGO
Outgroups
NOMO
BLJA
Acids*
Alcohols
n, m #
2-m
3-m
2,x-dim
3,x-dim
2,x,y poly m
1
1
1
2
1
1
1
1
1
1
1
5
6
6
6
6
6
1
1
1
1
1
1
1
1
1
1
6
6
1
1
1
2
6
4
1
1
1
1
3,x,y poly m
Other ##
n
1
4
6
4
6
3
1
4
1
1
1
4
4
1
1
1
1
1
Branched
Other ##
5
6
1
1
1
5
5
1
2
2
*The abundance of branched acids and alcohols are ranked as follows: 6 = >80 %, 5 = 60-80 %, 4 = 40-60 %, 3 = 20-1 % 2 = 20-1 % and
1 = <1 %
#
Both n- acids and acids branched beyond C4 produce fragment ions similar to that of straight chain acids but their ratio differ. Most peaks
we analyzed seemed to be mixture of more than one compound
2-m = 2-methyl; 2,x di-m = 2, x dimethyl substituted (x- anywhere in the chain); 2 x,y, poly m = 2, x, y, poly methyl substituted and same
is true for 3 substituted
##
In other, some species such as HOFI showed presence of 2-hydroxy acids, and in other cases we could not identify the alcohols, but based
on retention times they were different from straight chain alcohols
The excellent review by Jacob and Ziswiler on preen
gland histology, secretion chemistry, and biosynthetic
pathway is the baseline for understanding the evolutionary
history (Jacob & Ziswiler 1982). But most chemical studies
were carried out on either hydrolyzed or transesterfied products and not on intact components. They found that most of
species of fringillids they investigated to contain esters made
up of very high percentage of 3-methyl branched acids. For
example, Pyrrhula pyrrhula and Carduelis cannabina were
characterized by esters made up of 100 % 3-methyl branched
acids (Jacob and Ziswiler 1982).
The samples from the same species collected at different
time of the year showed variation in quantities and qualities
of the components. The detailed studies of preen gland secretions of House Finches (Haribal et al. unpublished) and a few
other studies (Dekker et al. 2000) have shown that through
out the year there is significant variation in the components.
The occurrence and seasonal variation is important both
for understanding the origin of the components (e.g. whether
synthesized de novo and/or sequestered from food) and for
understanding their possible functions. There is some evidence and speculation that at least some of the compounds
are sequestered by the birds through their diets (Dumbacher
et al. 2004; Rodriguez 1999). The diets of the birds we
investigated vary a fair amount and also depend on season
and their biology. During the breeding season their diet is
mainly insectivorous and in non-breeding season it is mostly
plant oriented. There is no detailed information available on
the specific diets for most of the species (Poole 2005). It
seems therefore impossible to speculate that they derive
these compounds from the diet unless we learn more about
their feeding ecology. A few studies have shown that birds
can synthesize some of these compounds de novo via several
pathways to produce even and odd numbered, and branched
acids and alcohols (Buckner & Kolattukudy 1975; Buckner
et al. 1978; Hiremath et al. 1992; Kolattukudy & Rogers
1978; Kolattukudy & Rogers 1987). Thus it appears probable
that birds have evolved to produce different mixtures to
arrive at similar types compounds to perform similar types of
functions in response to the ectosymbionts they encounter,
but the organisms may be different for different species.
The birds we investigated occupy different microhabitats
and have diverse ecologies. For example, Swamp Sparrows
(SWSP) spend most of their life in the swampy areas while
Chipping Sparrows (CHSP) use both forest edges and man
made habitats. White throated sparrows nest exclusively in
the coniferous forests and White crowned sparrows (EWCS)
throughout their range use different habitats in different
regions, while other species like Blue Jays (BLJA), Common
Grackles (COGR) and Northern Mockingbirds (NOMO) all
seem to occupy disturbed habitats and forest edges. Except
for the Rufous-collared Sparrow (RCOSP), which is found at
higher altitudes and southern latitudes in South America, all
of the species we investigated are restricted to North and
Central America. There is also some separation of the geographical regions they occupy during breeding and nonbreeding season, some of them having a breeding range as far
north as the Arctic while others breed south almost as far as
Florida and Texas. Most of these species are to some extent
migratory and some of them may spend winters as far south
as the southern US and Mexico (Poole 2005).
So if everything is so variable why do these bird species
produce very similar components to arrive at such complex
composition?
We do know now that birds may produce an array of
chemicals and a few experiments have shown that the total
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preen gland extracts are effective in combating some of
the ectosymbionts (Louis-Jacques et al. 1999; Moyer et al.
2003; Shawkey et al. 2003). But it is most likely that different classes of chemicals are active against different kinds of
organisms. Further, birds preen their feathers several times
a day, suggesting that some components either evaporate or
degrade over the course of a day and therefore need to be
replenished. While components such as longchain esters
will persist on the wing for a longer time that will eventually
evaporate or degrade over time. The studies by Sweeny et al.
(2004) confirm that in museum specimens some of the
feather waxes persist for decades (Sweeney et al. 2004).
Burger et al. (2004) have suggested that these long chain
waxes act as slow releasers of active components that
degrade very quickly once released (Burger et al. 2004).
The long chain esters of preen gland secretions of
different species have same molecular weights and elute
closely, suggest that they may have some similar physical
properties such as boiling and melting point, viscosity, opacity etc., although the properties of their mixtures vary
minutely because the proportions of individual components
in the mixtures vary. Thus, they may all have some common
functions, such as waterproofing feathers and keeping them
supple and malleable. The species we investigated had a fair
overlap of habitats in various seasons, although their
microenvironments may vary. Appropriate experiments are
necessary to determine if variation in composition has an
affect on the feathers’ physical properties. To our knowledge
no such experiments have been conducted so far. Although
Piersma (Piersma et al. 1999) suggested that the change in
preen gland secretion in breeding season may serve as
“avian makeup” to enhance the attractiveness of the mate, in
their subsequent papers they reported similar compounds in
both sexes, and in other closely related species, therefore
they further concluded that experiments are needed to confirm this function of preen gland secretions (Reneerkens
et al. 2002; Sinninghe Damste et al. 2000).
However, additional possibility is that the slow evaporating
waxes may be interacting with those organisms that remain on
the feather for longer times. Feather mites and feather-chewing
lice thus seem to be ideal ectosymbionts that are affected by
either presence or absence of these waxes. Bird-feather mite
relationship is poorly understood. Almost nothing is known
about the biology and ecology of these organisms.
Traditionally, feather mites have been thought to be
detrimental to the health of the bird and several studies indicate that the presence of these organisms may compromise
their fitness (Blanco et al. 1999; Booth et al. 1993; Clayton
et al. 2003; Figuerola et al. 2003; Hamilton & Zuk 1982;
Harper 1999). But others have argued they may be either
beneficial or have no affect on birds (Dubinin 1951; Proctor
2003). Dubinin has shown that feather mites feed on the feather
waxes and other microbial organisms encountered on the birds.
Also, prevalence of mites vary through the season and their
breeding biology which involves several nymphal stages before
they reach adult stages also vary (Haribal el al. unpublished).
It may be that mites if present in smaller numbers they are
beneficial but in larger numbers they may be detrimental to
their hosts. Therefore, it is necessary to conduct appropriate
bioassays in order to determine the functions and effects of
preen gland secretions on the ectosymbionts.
CHEMOECOLOGY
Furthermore, mites have co-evolved with birds and
have co-speciated with their hosts and some of them are very
specific to some species while some of them may be oligophagous or polyphagous (Atyeo & Braasch 1966; Gaud &
Atyeo 1996; Mironov 1990). For example, Proctophyllodes
polyxenes occurs on many species of sparrows and some nonsparrow species, while P. tristis so far has been exclusively
reported on Carduelis tristis (AMGO). Also some studies
have shown that the mites might be even specific to particular
populations (Gaud & Atyeo 1996), Gaud and Atyeo have suggested that the feather mites from one species may occur on
the other species but will not survive (Gaud & Atyeo 1996).
Unfortunately, there are no detailed systematic studies of the
species of mites that occur on the birds.
The fact that there is similarity among the secretions of
the sparrows reflects that they may have to combat similar
or same species of ectosymbionts. But yet the components
of secretions show finer and minute differences in their composition indicate that this could be owing to the fact that to
combat the mites, the birds altered the pathways to these
chemicals to produce newer components and in turn, the
mites evolve to overcome these changes and resist the new
chemicals. It is this co-evolution that has probably led to
such variation in preen gland ester chemistry. The recent
reports by Mironov et al. and Clayton et al. corroborates the
concept of evolution and co-speciation in bird-ectoparasites
interactions (Clayton et al. 2003; Mironov 1990). Further
studies are needed to understand the interactions between
bird-feather mites and the preen gland secretions, after taking into accounts of variation in mites and their life history
and the variation in secretions.
Acknowledgements
This work was funded by the grant from Biogeochemistry
and Biocomplexity Initiative awarded to AAD and ER and
by NIH grants no. NIH-T37MD00142608 to ER. We are
grateful to Alan Renwick and BTI for the donation of equipments, also AR reviewed the previous version of this manuscript. We thank Elizabeth Brooks and David Bonter and
their crew at Braddock Bay Banding Station for helping
with collections of samples. Also we would like to thank
Elliot Swarthrout, Melanie Driscoll, Tom Muscat, Keila
Sydenstricker and others bird banders in House Finch project helping with collecting samples in Ithaca area.
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