ArticlePDF Available

Biochemical transformation of birch leaf phenolics in larvae of six species of sawflies

Authors:

Abstract and Figures

Summary. We investigated the biochemical transformation of individual phenolic compounds of mountain birch leaves in larvae of six birch-feeding sawfly species: Amauronematus amplus, Pristiphora alpestris, Nematus brevivalvis, Priophorus pallipes, Arge sp. and Nematus viridis by comparing the phenolic residues in larval faeces to those of their leaf diet. Partial hydrolysis of hydrolysable tannins, isomerisation of chlorogenic acid and glycosylation of flavonoid aglycones were observed in all studied species. Moreover, we found considerable among-species variation in the composition of phenolic compounds in larval faeces. In addition to foliar phenolics, seventeen non-foliar phenolic metabolites, including eight phenolic acids and nine flavonoid glycosides were detected from the faeces. Of the non-foliar phenolic acids, four were egested species-specifically and only two by all six sawfly species. We also detected differences in the ratios of chlorogenic acid isomers in the faeces of different species, which can indicate different physiological conditions in the guts of studied larvae. In addition to the qualitative differences, quantitative differences were detected in the egestion of chlorogenic acids, possible o-quinone precursors in the larvae. Detected differences, either qualitative or quantitative, could not be explained by seasonal changes in the content of compounds in the leaf diet.
Content may be subject to copyright.
Chemoecology 15:153–159 (2005)
0937–7409/05/030153–7
© Birkhäuser Verlag, Basel, 2005
DOI 10.1007/s00049-005-0307-7
CHEMOECOLOGY
Biochemical transformation of birch leaf phenolics in larvae
of six species of sawflies
Maria Lahtinen1, Lauri Kapari2, Vladimir Ossipov1,2, Juha-Pekka Salminen1, Erkki Haukioja2and Kalevi
Pihlaja1
1Laboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, University of Turku, FI-20014 Turku, Finland
2Section of Ecology, Department of Biology, University of Turku, FI-20014 Turku, Finland
Summary. We investigated the biochemical transformation
of individual phenolic compounds of mountain birch leaves in
larvae of six birch-feeding sawfly species: Amauronematus
amplus, Pristiphora alpestris, Nematus brevivalvis,
Priophorus pallipes, Arge sp. and Nematus viridis by com-
paring the phenolic residues in larval faeces to those of their
leaf diet. Partial hydrolysis of hydrolysable tannins, iso-
merisation of chlorogenic acid and glycosylation of flavo-
noid aglycones were observed in all studied species.
Moreover, we found considerable among-species variation
in the composition of phenolic compounds in larval faeces.
In addition to foliar phenolics, seventeen non-foliar pheno-
lic metabolites, including eight phenolic acids and nine
flavonoid glycosides were detected from the faeces. Of the
non-foliar phenolic acids, four were egested species-
specifically and only two by all six sawfly species. We also
detected differences in the ratios of chlorogenic acid isomers
in the faeces of different species, which can indicate differ-
ent physiological conditions in the guts of studied larvae.
In addition to the qualitative differences, quantitative differences
were detected in the egestion of chlorogenic acids, possible
o-quinone precursors in the larvae. Detected differences,
either qualitative or quantitative, could not be explained
by seasonal changes in the content of compounds in the
leaf diet.
Key words. Birch sawfly larvae biochemical transfor-
mation – phenolic compounds
Introduction
Birch leaves contain a wide variety of phenolic compounds,
such as flavonoids, tannins and phenolic acids, which can affect
consumption rates and pupal masses of herbivores consuming
the leaves (e.g. Haukioja 2003). Salminen et al. (2004) pro-
posed that understanding the true effects of particular phenolic
compounds on insect performance requires knowledge of how
phenolics are metabolized in the digestive tract of insects.
Recently, we have reported fates of individual birch (Betula
pubescens) leaf phenolics in larvae of the geometrid moth
Epirrita autumnata, the main defoliator of birch (Salminen &
Lempa 2002; Salminen et al. 2004). However, so far we have
not studied the fate of birch leaf phenolics in any other insect
species, although we know that birch is also attacked, for
example, by numerous species of sawflies; mountain birch
alone accommodates close to 40 sawfly species, most of which
are birch specialists (Hanhimäki et al. 1995). In the present
study, we expand our compound-specific birch-herbivore studies
to six species of birch-feeding sawflies: Amauronematus
amplus, Pristiphora alpestris, Nematus brevivalvis, Priophorus
pallipes, Arge sp. and Nematus viridis.
In our earlier studies with birch and flush-feeding
E. autumnata we have reported particular reactions of foliar
phenolics in larvae, such as partial hydrolysis of hydrolysable
tannins, isomerisation of chlorogenic acid and glycosylation
of flavonoid aglycones (Salminen & Lempa 2002; Salminen
et al. 2004).Because of drastic changes in the composition of
foliar phenolics during the growing season (e.g. Salminen
et al. 2001; Riipi et al. 2002; Haukioja 2003; Valkama et al.
2004), we supposed that sawflies, which mostly consume
mature leaves (Hanhimäki et al. 1995; Martel et al. 2001), may
have developed different ways for metabolizing phenolics
compared to flush feeding E. autumnata. Lahtinen et al.
(2004) showed that high levels of flavonoid aglycones on the
surface of young birch leaves can be harmful for first instar
E. autumnata larvae and Salminen et al. (2004) showed that
fifth instar E. autumnata larvae are able to detoxify aglycones
by glycosylating them. Since the flavonoid aglycone content
of birch leaves declines rapidly as leaves mature (Valkama
et al. 2004) and sawflies tend to consume these aglycone poor
leaves, we might expect that the ability to detoxify flavonoid
aglycones is more a property of E. autumnata than of sawfly
larvae. There is large seasonal variation also among the sawfly
species in the maturity of the leaves they feed on, the timing of
the last larval instar ranging from early July to early
September. Thus, differences in phenology might correspond
to varying metabolic fates of phenolics even among the
sawflies. Species- or even instar-specific ways to metabolize
ingested leaf material could be assumed, because Martel et al.
(2001) found that birch sawflies were poor in using leaves that
were phenologically earlier or later in development than the
leaves that they typically encounter.
Correspondence to: Maria Lahtinen, e-mail: makahe@utu.fi
307.qxd 7/25/05 7:29 PM Page 153
To test hypotheses mentioned above, the aims of the
present study were to document the biochemical transforma-
tion of individual birch leaf phenolic compounds in larvae of
six sawfly species by comparing the phenolic compounds in
larval faeces to those of their leaf diet and to examine
possible differences in phenolic metabolism between
species. The factors behind the detected differences are also
discussed.
2. Materials and methods
2.1. Experimental design
The experiments were conducted at the Kevo Subarctic Research
Station in Utsjoki, Northern Finland (69°45’N, 27°00’E) during
the summer (June-August) of 2001. Of the six sawfly species
tested (Amauronematus amplus Konow, Pristiphora alpestris
Konow, Nematus brevivalvis Thomson, Priophorus pallipes
Lepeletier, Arge sp. Schrank and Nematus viridis Stephens), Arge
sp. belongs to Argidae, the others to Tenthredinidae (subfamily
Nematinae). The original experimental design included 14 trees
with three shaded branches and three control branches in each tree.
Only the data from control branches is presented here. Of the three
branches per tree branch A was used for A. amplus and P. pallipes,
branch B for N. brevivalvis and N. viridis and branch C for
P. alpestris and Arge. We have previously demonstrated that, unlike
in primary metabolites, within-tree variation in foliar phenolics is
low compared to among-tree variation (Suomela et al. 1995).
We tried to test all the species during the early days of the ulti-
mate instar except N. brevivalvis which was tested in the penultimate
instar. Due to variation in the number of instars and large variability
within broods, some penultimate instar larvae were included in the
tests with other species (most substantially in N. viridis). A. amplus
was tested on July 6, P. alpestris on July 10, N. brevivalvis on July 13,
P. pallipes on July 20, Arge sp. on July 26, and N. viridis on
August 15. The tested larvae were haploid males (produced by
unmated females), except in the experiment with P. alpestris which
included both males and females. The emerging adult sawfly females
were allowed to oviposit on foliage enclosed in mesh bags, and the
larvae were allowed to grow on these branches until taken into labo-
ratory approximately one week before the experiments. The larvae
were thus reared throughout the study on mountain birch leaves, but
during the early development the trees were not the same for differ-
ent species or broods within species. Approximately 18 hours before,
and during the experiments, the larvae of all species were fed in the
lab on leaves from the same 14 individual trees. Five (four for
P. pallipes) larvae belonging to different broods were tested in each
tree. The experiments were conducted in growth chambers at 13 ±1 °C
and with a 24 hour light cycle (typical for our high latitude study site).
Faeces samples were collected from the rearing vials once
during the 24 h experiment, which might have enabled post-intestinal
modification of the larval metabolites and the formation of
artefacts in the faeces. However, earlier Salminen et al. (2004)
showed that the only HPLC-detectable difference between fresh and
24 h old faeces of E. autumnata, was the appearance of uric acid
in the latter types of samples. On the basis of this study, we have
assumed that also in the faeces of sawfly larvae the phenolic compo-
sition stays relative constant during the 24 h experiment. The forma-
tion of uric acid was also detected from the faeces of sawfly larvae.
2.2. Sample preparation
Freeze-dried faeces from five (four for P. pallipes) larvae per tree
were pooled for tree-specific samples before the chemical analyses.
Preparation of leaf and faeces samples for HPLC-DAD/HPLC-ESI-
MS analyses of their vacuolar phenolics were conducted as previ-
ously described (Salminen et al. 1999, 2001; Salminen & Lempa
2002). Six leaves from each branch were collected for analyses, in
which the contents of individual phenolic compounds (hydrolysable
tannins, catechin, chlorogenic acids, coumaroylquinic acids, and
flavonoid glycosides) were determined. Flavonoid aglycones were
extracted with 95 % ethanol from two leaves of each branch as in
Valkama et al. 2003. The sampling dates of leaves were July 6, 13,
26 and August 16.
2.3. HPLC-DAD and HPLC–ESI-MS
Leaf and faeces extracts were analysed with HPLC-DAD at
280 nm, 315 nm and 349 nm. The HPLC system (Merck-Hitachi,
Tokyo, Japan) consisted of a pump L-7100, a diode array detector
L-7455, a programmable autosampler L-7250, and an interface
D-7000. Column and chromatographic conditions were as
described earlier (Salminen et al. 1999), except that 0.1M H3PO4
was replaced with 0.05M H3PO4. A selected set of samples was
analysed also with HPLC-ESI-MS as in Salminen et al. (1999)
except that the datasystem used was Analyst Software 1.1.
2.4. Compound identification and quantification
Compounds were identified on the basis of their UV and mass spectra
and retention times reported in the literature (Ossipov et al. 1995, 1996;
Salminen et al. 1999, 2001; Valkama et al. 2003; Salminen et al. 2004)
and by comparison to authentic standards (protocatechuic acid from
Sigma). Galloylquinic acids were identified by their UV and mass
spectra: Their UV spectra were similar to that of gallic acid and mass
spectra showed ions at m/z 343, m/z 191 and m/z 169, corresponding to
[M–H]of galloylquinic acid, [M–H]of quinic acid and [M–H]of
gallic acid.
Galloylglucoses were quantified as monogalloyl glucose,
pedunculagin and its derivative as pedungulacin, catechin as
catechin, chlorogenic acids (isomers and derivatives) as chlorogenic
acid, coumaroylquinic acids as coumaric acid, gallic acid and
galloylquinic acids as gallic acid, protocatechuic acid as protocate-
chuic acid and flavonoid glycosides as corresponding aglycones.
Unknown flavonoid glycosides were quantified as quercetins.
Quantification was done both in relative (mg/g dry weight) and
absolute values (mg per sample). Absolute values were used for the
calculation of egestion percentages of compounds. The ingested
amounts were calculated based on measurements of dry weight
consumption by the larvae during the growth trials. Initial and final
leaf areas of the leaves were measured from digital photographs
(Olympus C-2040/resolution 640 480) using the Sigma Scan Pro
4 (©SPSS Inc.) image analysis program. Partly consumed leaves
were vacuum dried and weighted. Dry weight in mg per area from
the leaves was used to calculate the initial dry weight of each
experimental leaf. Tree specific means for the dry weight consump-
tion and the dry weight of the faeces were used in calculation of
egestion percentages of compounds.
2.5. Statistical analyses
Tree-specific values for the amount egested in relation to the
amount ingested (%) were used as dependent variables in
statistical tests for differences between species in the relative
amounts of egested o-dihydroxylated compounds (chlorogenic
acids and quercetin glycosides) and new non-foliar flavonoid
glycosides. The differences between species in egestion percentages
were tested with non-parametric Kruskal-Wallis tests using SAS 8.2
statistical software (SAS institute inc. 1999–2001).
3. Results and discussion
Even though the phenolic composition in leaves stayed qualita-
tively constant during the experiment, the compound composi-
tion in larval faeces was found to differ significantly among the
sawfly species (Tables 1–3). All species egested non-foliar,
previously undetected products of larval phenolic metabolism.
Some metabolites were detected in faeces of all species while
some were characteristic to one or two species only.
154 Maria Lahtinen et al. CHEMOECOLOGY
307.qxd 7/25/05 7:29 PM Page 154
3.1. Transformation of hydrolysable tannins
Possible reactions for hydrolysable tannins in an insect’s gut
are their hydrolysis, oxidation, or adsorption on the peritrophic
envelope (Appel & Martin 1990; Appel 1993; Barbehenn &
Martin 1994; Barbehenn et al. 1996; Salminen & Lempa
2002). With the sawfly larvae studied here, the transformation
of hydrolysable tannins was similar among the species; of the
foliar tannins only pedunculagin could be detected in the
faeces (see Table 1). Although the leaf diet contained more
than 5.7 mg/g (seasonal average) of galloylglucoses, they were
not found in the faeces as such. Instead, 0.3-2.5 mg/g of gallic
acid, the hydrolysis product of galloylglucoses, was detected
in the faeces of all species, reflecting at least partial hydrolysis
of these tannins in studied larvae. Interestingly, although
pedunculagin was egested at different levels (1.6-2.3 mg/g),
the detected pedunculagin derivative was totally retained, oxi-
dized or degraded to undetectable catabolites in the digestive
tract of all larvae. Salminen and Lempa (2002) showed that
individual galloylglucoses differ in their fates within the gut of
E. autumnata larvae and here we observed the same to be true
with individual ellagitannins (pedunculagin and its derivative)
in the guts of six species of sawfly larvae. Different fates of
individual tannins in larvae might reflect their variable biologi-
cal activities. In addition to pedunculagin, P. pallipes also
produced catechin to the faeces, while the other species did not.
There are several factors affecting the biological activity
of hydrolysable tannins in plant-herbivore interactions. Tannin
oxidation products, such as quinones, and oxidation by-products
including different reactive oxygen species can cause enzyme
inactivation, membrane lipid peroxidation, and strand breaks
in DNA (Appel 1993) Reactive o-quinones can also form
covalent bonds to nucleophilic group of amino acids decreas-
ing their assimilation in herbivore digestive tract (Felton et al.
1989). The ability of tannins as such to precipitate proteins has
been suggested as an important factor reducing the suitability
Vol. 15, 2005 Transformation of phenolics in sawfly larvae 155
Table 1 Quantities of hydrolysable tannins and catechin (mg/g dry weight) present in both leaf and faeces samples. The values shown are
means ±SE of n =14 in both faeces and leaf samples
Faeces
July 6 July 10 July 13 July 20 July 26 August 15
Hydrolysable
tannins and Leaf diet
catechin July 6 July 13 July 26 August 16 A. amplus P. alpestris N. brevivalvis P. pallipes Arge sp. N. viridis
Monogalloyl
glucose 4.70 ±0.99 3.49 ±0.81 2.86 ±0.76 2.97 ±0.67 – – – – –
Digalloyl
glucose 0.91 ±0.21 1.05 ±0.21 0.88 ±0.25 1.26 ±0.24 – – – – –
Trigalloyl
glucose 1.38 ±0.37 1.40 ±0.26 1.04 ±0.27 1.04 ±0.25 – – – – –
Pedunculagin 2.14 ±0.67 2.09 ±0.57 3.25 ±1.86 1.99 ±0.61 2.24 ±0.83 2.30 ±0.78 1.87 ±0.50 1.60 ±0.50 1.82 ±0.68 1.79 ±0.61
Pedunculagin
derivative 4.05 ±0.78 3.03 ±0.62 2.19 ±0.45 2.76 ±0.59 – – – – –
Catechin 2.55 ±0.26 2.48 ±0.22 2.86 ±0.32 3.64 ±0.41 2.35 ±0.19 –
Table 2 Quantities of phenolic acids (mg/g dry weight) present in both leaf and faeces samples. The values shown are means ±SE of n =14 in
both faeces and leaf samples
Faeces
July 6 July 10 July 13 July 20 July 26 August 15
Leaf diet
Phenolic acid July 6 July 13 July 26 August 16 A. amplus P. alpestris N. brevivalvis P. pallipes Arge sp. N. viridis
Chlorogenic acid 11.06 ±1.43 9.76 ±1.23 8.44 ±1.29 8.79 ±1.24 4.39 ±0.74 4.06 ±0.83 4.45 ±0.74 5.68 ±0.83 5.28 ±0.73 3.61 ±0.75
Coumaroylquinic
acid 0.49 ±0.09 0.31 ±0.06 0.20 ±0.04 0.15 ±0.03 1.17 ±0.10 0.18 ±0.05 0.50 ±0.06 0.26 ±0.04 0.25 ±0.04 0.26 ±0.04
Neochlorogenic
acid 0.28 ±0.12 0.29 ±0.10 0.19 ±0.09 0.17 ±0.09 3.60 ±0.55 1.78 ±0.39 1.81 ±0.26 1.18 ±0.11 1.26 ±0.16 0.84 ±0.15
Chlorogenic acid
isomer 2.85 ±0.45 1.56 ±0.33 1.87 ±0.25 1.45 ±0.15 1.63 ±0.21 1.00 ±0.19
Chlorogenic acid
derivative I 0.96 ±0.10 –
Chlorogenic acid
derivative II 2.16 ±0.24 –
Chlorogenic acid
derivative III 0.94 ±0.13 –
Gallic acid 2.45 ±0.71 1.45 ±0.27 0.47 ±0.10 1.00 ±0.24 0.45 ±0.11 0.31 ±0.11
Galloylquinic
acid isomer I 0.14 ±0.05 0.69 ±0.17 0.13 ±0.07 0.83 ±0.21 0.22 ±0.08
Galloylquinic
acid isomer II 0.56 ±0.11 0.40 ±0.10 0.38 ±0.09 –
Protocatechuic
acid 3.02 ±0.16 – – –
307.qxd 7/25/05 7:29 PM Page 155
156 Maria Lahtinen et al. CHEMOECOLOGY
Table 3 Quantities of flavonoids (mg/g dry weight) present in both leaf and faeces samples. The values shown are means ±SE of n =14 in both faeces and leaf samples
Faeces
July 6 July 10 July 13 July 20 July 26 August 15
Leaf diet
Flavonoids July 6 July 13 July 26 August 16 A. amplus P. alpestris N. brevivalvis P. pallipes Arge sp. N. viridis
Flavonoid aglyconesa5.16 ±0.47 4.49 ±0.50 3.33 ±0.23 3.48 ±0.28 –
Kaempferol-3-O-glucoside 0.39 ±0.06 0.42 ±0.05 0.36 ±0.03 0.43 ±0.05 0.95 ±0.14 0.96 ±0.16 0.99 ±0.16 1.13 ±0.16 0.71 ±0.08 1.07 ±0.16
Kaempferol-3-O-rhamnoside 0.60 ±0.15 0.52 ±0.12 0.39 ±0.07 0.40 ±0.05 –
Kaempferol glycoside I 0.91 ±0.14 0.88 ±0.13 0.73 ±0.11 0.77 ±0.13 1.24 ±0.13 1.42 ±0.14 1.26 ±0.15 1.44 ±0.14 1.43 ±0.13 1.32 ±0.14
Kaempferol glycoside II 0.19 ±0.10 0.12 ±0.01 0.11 ±0.01 0.17 ±0.01 0.23 ±0.04 0.28 ±0.04 0.68 ±0.13 0.11 ±0.01
Kaempferol glycoside III 0.12 ±0.05 0.12 ±0.06 0.11 ±0.04 0.16 ±0.06 –
Myricetin-3-O-galactoside 2.03 ±0.24 1.81 ±0.25 1.62 ±0.26 1.70 ±0.27 1.68 ±0.23 1.88 ±0.28 1.16 ±0.19 2.03 ±0.28 1.66 ±0.24 1.11 ±0.24
Myricetin-3-O-glucuronoside 0.57 ±0.12 0.44 ±0.07 0.38 ±0.08 0.35 ±0.08 0.38 ±0.08 0.51 ±0.12 0.27 ±0.07 0.42 ±0.08 0.33 ±0.06 0.24 ±0.06
Myricetin glycoside 0.22 ±0.11 0.12 ±0.07 0.12 ±0.08 0.15 ±0.08 –
Quercetin-3-O-arabinoside 1.06 ±0.13 0.98 ±0.12 0.84 ±0.11 0.94 ±0.12 0.43 ±0.07 0.61 ±0.07 0.59 ±0.12 0.60 ±0.12 1.36 ± 0.11 –
Quercetin-3-O- galactoside 2.93 ±0.38 2.60 ±0.30 2.22 ±0.28 2.40 ±0.33 1.66 ±0.18 2.91 ±0.40 2.57 ±0.27 3.14 ±0.36 3.02 ±0.30 2.35 ±0.25
Quercetin-3-O-glucuronoside 7.27 ±0.92 7.10 ±0.91 6.45 ±0.91 7.20 ±0.86 9.17 ±0.84 10.24 ±0.88 9.77 ±0.92 9.64 ±0.69 9.31 ±0.95 10.35 ±0.98
Quercetin glycoside I 0.49 ±0.13 0.48 ±0.09 0.39 ±0.09 0.42 ±0.10 0.60 ±0.11 0.59 ±0.11 0.73 ±0.12
Quercetin glycoside II 0.27 ±0.10 0.25 ±0.10 0.24 ±0.09 0.29 ±0.11 –
Quercetin glycoside III 0.15 ±0.07 0.15 ±0.07 0.14 ±0.06 0.18 ±0.07 –
Quercetin glycoside IV 0.90 ±0.28 0.90 ±0.27 0.86 ±0.28 1.07 ±0.30 –
Acacetin-7-O-glucoside 0.58 ±0.06 0.65 ±0.05 0.68 ±0.09 0.62 ±0.08 0.58 ±0.06 0.51 ±0.05
Kaempferide-3-O-glucoside 0.13 ±0.02 0.08 ±0.03 –
Flavonoid glycoside I 0.35 ±0.03 0.30 ±0.02 0.30 ±0.03 0.28 ±0.03 0.18 ±0.02 0.23 ±0.03
Flavonoid glycoside II 0.87 ±0.07 0.78 ±0.05 0.88 ±0.08 0.83 ±0.08 0.65 ±0.04 0.69 ±0.06
Flavonoid glycoside III 0.18 ±0.02 0.23 ±0.02 0.14 ±0.03 0.08 ±0.01 0.11 ±0.02
Flavonoid glycoside IV 0.34 ±0.05 –
Flavonoid glycoside V 0.54 ±0.06 –
Flavonoid glycoside VI 0.06 ±0.02 0.13 ±0.01 0.12 ±0.01 0.07 ±0.01
Flavonoid glycoside VII 0.03 ±0.01 0.06 ±0.01
aA sum of 12 individual flavonoid aglycones (same compounds as in Salminen et al. 2004).
307.qxd 7/25/05 7:29 PM Page 156
of plants for herbivores (Feeny 1970; Hagermann & Robbins
1987). On the other hand, some hydrolysable tannins could act
as antioxidants, thus protecting proteins and amino acids in the
insects (Hagerman et al. 1998). The hydrolysis products of
individual tannins and their metabolism within the insect
digestive tract have been shown to differ, resulting negative,
neglible or even positive effects for the insect (Klocke et al.
1986, Bernays et al. 1989).
3.2. Transformation of phenolic acids
Earlier we have shown that chlorogenic acid, the main
phenolic acid of birch leaves, was isomerised in the alkaline
gut of lepidopteran E. autumnata (Salminen et al. 2004).
Similar isomerisation occurred also in guts of all sawflies
tested, but there were differences in the ratios of the egested
isomers between the species. For instance, while E. autumnata
larvae excreted the three chlorogenic acid isomers
(neochlorogenic acid, chlorogenic acid and the third
non-foliar chlorogenic acid isomer) in approx. ratio of 1:1:1
(Salminen et al. 2004, unpublished data), these ratios
varied in the present study from 1:1:1 (A. amplus) to 1:2:1
(N. brevivalvis) and 1:3:1 (P. alpestris,P. pallipes, Arge sp.,
N. viridis). In the leaf diet the ratio of chlorogenic acid
to neochlorogenic acid varied from 30:1 (first sample) to
50:1 (last sample). In addition to variable metabolism, the
observed differences could suggest different pH conditions
in digestive tracts of larvae, because the isomerisation of
chlorogenic acid has also been observed to happen in vitro
under basic conditions (Nagels et al. 1980).
Due to the potential role of chlorogenic acids in the
production of o-quinones through auto-oxidation, or the
action of the polyphenol oxidases, we also tested quantitative
differences in the egestion of chlorogenic acids between
studied sawfly species. Significant differences between
species in relative amounts of egested chlorogenic acids
(ChiSq =26.8, DF =5, p <0.0001) were found and
egestion percentages varied from 55 % in N. brevivalvis
to 120 % in P. pallipes. The high egestion percentage of
P. pallipes might be due to their specific way of consuming
leaves; P. pallipes do not eat leaf veins and thus the concen-
tration of compounds can be higher in faeces than in their
leaf diet. Detected differences in relative amounts of egested
chlorogenic acids may indicate differences in oxidation
of chlorogenic acids and hence in amount of produced
o-quinones in larvae.
In addition to chlorogenic acid isomers, gallic acid and
low amount of coumaroylquinic acid were detected from
faeces of all studied sawflies (Table 2). Moreover, previously
undetected non-foliar phenolic acids, i.e., galloylquinic acid,
protocatechuic acid and chlorogenic acid derivatives were
detected. At least one of the two isomers of galloylquinic
acid was detected in the faeces of all species, while protocat-
echuic acid was characteristic of P. alpestris and chlorogenic
acid derivatives of P. pallipes only. Interestingly, gallic and
quinic acids would be needed as building blocks for the non-
foliar galloylquinic acids; the latter is directly available for
larvae from the foliage, where free gallic acid is found only
in traces. This suggests that the formation of galloylquinic
acids in sawfly larvae is possible only after gallic acid is
released via hydrolysis of galloylglucoses (see above).
Protocatechuic acid in turn could originate, e.g., from the
side chain reduction of caffeic acid (Waterman & Mole
1994). However, caffeic acid is not found in birch leaves, and
hence would first have to be cleaved from chlorogenic acid
(i.e., caffeoylquinic acid) isomers or derivatives. Alternatively,
some micro-organisms are known to utilize flavonoids, and
the first step in flavonoid metabolism is their conversion into
different hydroxybenzoic acids, such as protocatechuic acid
(Pillai & Swarup 2002).
3.3. Transformation of flavonoids
The transformations of flavonoids in sawfly larvae resembled
those observed in the lepidopteran E. autumnata (Salminen
et al. 2004). The main pattern was the disappearance
of flavonoid aglycones and the simultaneous appearance of
new flavonoid glycosides into the faeces (Table 3). Since the
flavonoid aglycone levels in birch leaves decline rapidly
as leaves mature (Valkama et al. 2004), we would have
expected that the ability to detoxify flavonoid aglycones
is more a property of E. autumnata than of sawfly larvae.
Apparently the seasonal decline in foliar levels of aglycones
does not significantly reduce the ability of sawflies to metab-
olize aglycones, since even the autumn-feeder N. viridis
excreted detectable levels of six glycosylation products. We
also tested statistical differences in egestion of non-foliar
flavonoid glycosides transformed from foliar flavonoid agly-
cones, but there were no significant differences in relative
amounts of egested glycosides (ChiSq =4.7, DF =5,
p<0.4474). The egestion percentages varied from 35 % in A.
amplus to 44 % in P. alpestris with no indication of seasonal
trends. In spite of similar egestion percentages, the faecal
profiles of non-foliar flavonoid glycosides did vary: three of
the nine compounds (acacetin-3-O-glucoside and flavonoid
glycosides I and II) were found in the faeces of all sawflies
while two compounds (flavonoid glycosides IV and V) were
specific to P. alpestris only. Other non-foliar flavonoid
glycosides showed diverse patterns, and none of them was
found in the faeces of all sawfly species.
Similar to the above mentioned chlorogenic acids, the
quercetin flavonoids are o-dihydroxylated phenolics. Thus, in
principle, they also could auto-oxidize or react with polyphe-
nol oxidases to produce harmful o-quinones. However, we did
not find statistically significant differences between species
in the relative amounts of egested quercetin glycosides
(ChiSq =10.3, DF =5, p <0.0674). Overall, the egestion
percentage was rather high in all studied species (76-117 %).
Similarly E. autumnata has reported to egest quercetin
glycosides mostly unaltered (Salminen et al. 2004).
3.4. Possible factors behind the detected transformations
There are lots of factors which can influence on the fates of
foliar phenolic compounds in larvae. As mentioned above, we
know that isomerisation of chlorogenic acid is, at least partly,
due to the alkaline conditions in the larvae of E. autumnata
(Salminen et al. 2004). On the basis of the results of the present
study, we suggest that pH-conditions in studied sawfly larvae
are also alkaline. The varying ratios of isomers between the
species could indicate differences in gut pH-conditions, but this
preliminary conclusion needs further studies. In addition to
Vol. 15, 2005 Transformation of phenolics in sawfly larvae 157
307.qxd 7/25/05 7:29 PM Page 157
physiological factors, enzymes, either foliar or larval, can cause
some of the detected transformations. E.g., glycosylation of
flavonoids is probably due to the enzymatic activity. Glycosyl
transferases should be part of birch leaf chemistry, because of
number of flavonoid glycosides present in leaves (Ossipov et al.
1995) and after ingestion these enzymes might also act in larvae.
The role of symbiotic micro-organisms in the food
metabolism of sawfly larvae is unknown to us, but their
potential for creating both among- and within species varia-
tion could be substantial. Furthermore, as well as being
indigenous (maternally transmitted to the offspring) the
microbiota inhabiting the digestive tract may also be derived
from the surrounding environment, including the host plant
(Douglas & Beard 1996; Dillon & Dillon 2004). Although
at least part of the observed differences between species
could be explained by exogenous factors, such as the non-
indigenous micro-organisms, the differences may simply
reflect the evolutionary histories of the species. Pairwise
coevolution (Janzen 1980; Thompson 1994) between a host
plant and its herbivores can in theory produce herbivore
species specific adaptations in food utilization. Furthermore,
host shifts are common in many externally feeding insect
herbivore taxa (e.g. Powell et al. 1998; Farrell & Sequeira
2004) which might increase the current variation between
herbivore species sharing a host plant.
Acknowledgements
Sinikka Hanhimäki’s supervision in rearing of the larvae and
execution of the bioassays was invaluable. We would also
like to thank Juha Järvenpää who extracted and analysed the
flavonoid aglycones and our numerous field assistants for
their input. This work was supported in part by grants from
the Emil Aaltonen foundation (to M.L.) and from the
Academy of Finland (V.O. grant no. 201073 and E.H. grants
nos. 202165 and 204209).
References
Appel HM (1993) Phenolics in ecological interactions: the
importance of oxidation. J Chem Ecol 19: 1521–1552
Appel HM, Martin MM (1990) Gut redox conditions in herbivorous
lepidopteran larvae. J Chem Ecol 16: 3277–3290
Barbehenn RV, Martin MM (1994) Tannin sensitivity in larvae of
Malacosoma disstria (lepidoptera): roles of peritrophic envelope
and midgut oxidation. J Chem Ecol 20: 1985–2001
Barbehenn RV, Martin MM, Hagerman AE (1996) Reassessment
of the roles of the peritrophic envelope and hydrolysis in pro-
tecting polyphagous grasshoppers from ingested hydrolysable
tannins. J Chem Ecol 22: 1901–1919
Bernays EA, Driver GC, Bilgener M (1989) Herbivores and plant
tannins. Adv Ecol Res 19: 263–302
Dillon RJ, Dillon VM (2004) The gut bacteria of insects: non
pathogenic interactions. Annu Rev Entomol 49: 71–92
Douglas AE, Beard CB (1996) Microbial symbioses in the
midgut of insects. Pp 419–431 in Lehane MJ, Billingsley PF
(eds) Biology of the Insect Midgut. GB-London: Chapman &
Hall
Farrell BD, Sequeira AS (2004) Evolutionary rates in the adaptive
radiation of beetles on plants. Evolution 58: 1984–2001
Felton GW, Donato K, Del Vecchio RJ, Duffey SS (1989) Activation
of plant foliar oxidases by insect feeding reduces nutritive
quality of foliage for noctuid herbivores. J Chem Ecol 15:
2667–2694
Feeny P (1970) Seasonal changes in oak leaf tannins and nutrients
as a cause of spring feeding by winter moth caterpillars.
Ecology 51: 565–581
Hagerman AE, Riedl KM, Jones GA, Sovik KN, Ritchard NT,
Hartzfeld PW, Riechel TL (1998) Tannins, plant phenolics,
antioxidant, total antioxidant activity, oxidative damage,
dietary antioxidant. J Agric Food Chem 46: 1887–1892
Hagermann AE, Robbins CT (1987) Implications of soluble tannin-
protein complexes for tannin analysis and plant defence
mechanisms. J Chem Ecol 13: 1243–1259
Hanhimäki S, Senn J, Haukioja E (1995) The convergence in
growth of foliage-chewing insect species on individual
mountain birch trees. J Anim Ecol 64: 543–552
Haukioja E (2003) Putting the insect into the birch-insect interaction.
Oecologia 136: 161–168
Janzen, DH (1980) When is it coevolution? Evolution 34: 611–12
Klocke JA, van Wagenen B, Balandrin MF (1986) The ellagitannin
geraniin and its hydrolysis products isolated as insect growth
inhibitors from semi-arid land plants. Phytochemistry 25: 85–91
Lahtinen M, Salminen J-P, Kapari L, Lempa K, Ossipov V, Sinkkonen
J, Valkama E, Haukioja E, Pihlaja K (2004) Defensive effect of
surface flavonoid aglycones of Betula pubescens leaves against
first instar Epirrita autumnata larvae. J Chem Ecol 30: 2257–2268
Martel J, Hanhimäki S, Kause A, Haukioja E (2001) Diversity of
birch sawfly responses to seasonally atypical diets across a
seasonal gradient of larval feeding periods. Entomol Exp Appl
100: 301–309
Martin JS, Martin MM (1983) Precipitation of ribulose-1,6-
bis-phosphate carboxylase/oxygenase by tannic acid, quebracho,
and oak foliage extracts. J Chem Ecol 9: 285–294
Nagels L, van Dongen W, de Brucker J, de Potter J (1980)
High-performance liquid chromatographic separations of
naturally occurring esters of phenolic acids. J Chromatogr
187: 181–187
Ossipov V, Nurmi K, Loponen J, Prokopiev N, Haukioja E, Pihlaja
K (1995) HPLC isolation and identification of flavonoids from
white birch Betula pubescens leaves. Biochem Syst Ecol
23: 213–222
Ossipov V, Nurmi K, Loponen J, Haukioja E, Pihlaja K (1996)
High-performance liquid chromatographic separation and
identification of phenolic compounds from leaves of Betula
pubescens and Betula pendula. J Chromatogr A 721: 59–68
Pillai PVS, Swarup S (2002) Elucidation of the flavonoid catabo-
lism pathway in Pseudomonas putida PML2 by comparative
metabolic profiling. Appl Environ Microbiol 68: 143–151
Powell JA, Mitter C, Farrell B (1998) Evolution of larval food
preferences in Lepidoptera. Pp 403–422 in Kristensen NP (ed)
Handbook of zoology. Lepidoptera, Vol. 1. Systematics and
evolution. DE-Berlin: De Gruyter
Riipi M, Ossipov V, Lempa K, Haukioja E, Koricheva J, Ossipova S,
Pihlaja K (2002) Seasonal changes in birch leaf chemistry:
are there trade-offs between leaf growth and accumulation of
phenolics? Oecologia 130: 380–390
Salminen J-P, Lahtinen M, Lempa K, Kapari L, Haukioja E, Pihlaja K
(2004) Metabolic modifications of birch leaf phenolics by an
herbivorous insect: detoxification of flavonoid aglycones via
glycosylation. Z Naturforsch 59C: 437–444
Salminen J-P, Lempa K (2002) Effects of hydrolysable tannins
on an herbivorous insect: fate of individual tannins in insect
digestive tract. Chemoecology 12: 203–211
Salminen J-P, Ossipov V, Haukioja E, Pihlaja K (2001) Seasonal
variation in the content of hydrolysable tannins in the leaves of
Betula pubescens. Phytochemistry 57: 15–22
Salminen J-P, Ossipov V, Loponen J, Haukioja E, Pihlaja K (1999)
Characterisation of hydrolysable tannins from leaves of Betula
pubescens by high-performance liquid chromatography – mass
spectrometry. J Chrom A 864: 283–291
Salminen J-P, Ossipov V, Pihlaja K (2002) Distribution of
hydrolysable tannins in the foliage of Finnish birch species.
Z Naturforsch 57C: 248–256
Suomela, J, Ossipov V, Haukioja E (1995) Variation among and
within mountain birch trees in foliage phenols, carbohydrates
and amino acids, and in growth of Epirrita autumnata larvae.
J Chem Ecol 21: 1421–1446
158 Maria Lahtinen et al. CHEMOECOLOGY
307.qxd 7/25/05 7:29 PM Page 158
Thompson JN (1994) The Coevolutionary Process. USA-Chicago:
University of Chicago Press
Valkama E, Salminen J-P, Koricheva J, Pihlaja K (2003) Comparative
analysis of leaf trichome structure and composition of epicuticu-
lar flavonoids in Finnish birch species. Ann Bot 91: 643–655
Valkama E, Salminen J-P, Koricheva J, Pihlaja K (2004) Changes
in leaf trichomes and epicuticular flavonoids during leaf
development in three birch taxa. Ann Bot 94: 1–10
Waterman PG, Mole S (1994) Pp. 28 in Analysis of plant phenolic
metabolites. Oxford, Blackwell scientific publications
Vol. 15, 2005 Transformation of phenolics in sawfly larvae 159
Received 9 December 2004; accepted 7 April 2005.
Published Online First 10 June 2005.
307.qxd 7/25/05 7:29 PM Page 159
307.qxd 7/25/05 7:29 PM Page 160
... 5-CQA can be a substrate for PPOs and is oxidatively active at alkaline pH. In addition, it was shown that 5-CQA isomerizes into a mixture of 3-CQA, 4-CQA and 5-CQA in alkaline conditions and that different insect species produce various isomer ratios of CQAs in the frass after feeding on 5-CQA-rich foliage (Salminen et al. 2004;Lahtinen et al. 2005). If the observed isomer ratios reflect the pH differences of the midguts of the species, studies on 5-CQA isomerization could be used to estimate the alkaline oxidative conditions of the larval midgut. ...
... We reanalyzed the frass data we had earlier obtained for eight species of insect herbivores (Salminen and Lempa 2002;Salminen et al. 2004;Lahtinen et al. 2005;Salminen 2018) fed on birch leaf diet that contained 5-CQA, but very little 3-CQA and no 4-CQA. These studies included two species of lepidopteran larvae (Epirrita autumnata and Agriopis aurantiaria) together with larvae of six sawfly species (Amauronematus amplus, Nematus brevivalvis, Pristiphora alpestris, Priophorus pallipes, Arge sp. and Nematus viridis). ...
... All leaf and frass samples were freeze-dried, ground into fine Laccase enzymes are versatile, and are able to oxidize e.g. o-, m-and pdiphenols and triphenols to quinones powder, extracted and analyzed by HPLC-DAD as reported in Salminen and Lempa (2002), Salminen et al. (2004), Lahtinen et al. (2005) and Salminen (2018). ...
Article
Full-text available
We developed a combination of methods to estimate the alkaline oxidative conditions of the midgut of insect larvae and to reveal the alkaline and enzymatic oxidative activities for individual phenolic compounds present in the larval host plants. First, we monitored the in vitro isomerization of 5-O-caffeoylquinic acid (5-CQA) into 3-CQA, 4-CQA and 5-CQA at pH 9.0–11.0. Then we calculated the isomer ratios of 3-CQA, 4-CQA and 5-CQA from the frass of eight species of insect herbivores fed on foliage containing 5-CQA. The isomer ratios suggested that the midgut pH of these larvae ranged from 9.4 to around 10.1. Second, we developed an in situ enzymatic oxidation method that enabled oxidation of phenolics in a frozen plant sample at 30 °C by species- and tissue-specific enzymes. Then we measured the alkaline and enzymatic oxidative activities of the individual phenolics in 20 plant species by quantifying the proportion of the compound concentration lost due to the auto-oxidation of a plant extract at pH 10 and due to the enzymatic oxidation of the frozen plant sample at 30 °C. Our results showed that both of the oxidative activity types depended primarily on the type of phenolic compound, but the enzymatic oxidative activity depended also on the plant species and tissue type. This combination of methods offers an approach to characterize a wide array of phenolics that are susceptible to oxidation by the plant enzymes and/or by the alkaline conditions estimated to prevail in the insect midgut. We propose that these kinds of compound-specific results could guide future studies on specific plant-herbivore interactions to focus on the phenolics that are likely to be active rather than inactive plant phenolics.
... Some of these beetles can even sequester salicylates and produce salicylaldehyde, which their larvae use as a defence against invertebrate predators (Rowell-Rahier & Pasteels, 1986). Faster growth rates on salicylate hosts have been also recorded in sawflies that probably use salicylates as oviposition cues (Lahtinen et al., 2005). ...
... Green lines indicate mean DSI* specialisation relative to salicylates and flavonoids was not different from the one towards overall chemical β-diversity. Both metabolite groups are involved in host recognition and are used as feeding or oviposition cues by sawflies or female lepidopterans (Kolehmainen et al., 1994;Lahtinen et al., 2005;Roininen et al., 1999;Vihakas, 2014). ...
Article
• Plants produce multiple specialised metabolites to defend themselves against insect herbivores. Phytochemical diversity plays important roles in plant–insect interactions, but specific roles of its various dimensions are poorly known. Interspecific chemical β-diversity represents variation in presence of species-specific metabolites or quantitative variation in concentrations of metabolites common to several plant species. • We hypothesised that qualitative and quantitative variation in plant chemistry can have differential effects on herbivores from various insect orders. • We linked phytochemical variation in willow salicylates (Salicaceae-specific metabolites) and flavonoids (widespread metabolites) to a standardised distance-based specialisation index (DSI*) in three orders of leaf-chewing insects: sawfly larvae, beetles, and caterpillars. • In beetles, average DSI* accounting for host chemical β-diversity did not differ from DSI* disregarding host chemistry. Levels of chemical specialisation did not differ among beetle species feeding only on Salicaceae and those using other plant families, suggesting that both can overcome willow chemistry by alternative physiological or behavioural adaptations. Contrastingly, sawflies and caterpillars responded to willow chemistry, with their DSI* corresponding mainly to quantitative differences in willow metabolites. The DSI* accounting for salicylates did not differ from the one accounting for flavonoids in either of the two orders. • Our results suggest that β-diversity in plant chemistry has differential effects on insect herbivores depending on their order and chemical β-diversity measurement used. Our results emphasise the importance of quantitative variation in plant chemical composition, suggesting that it does not always have to be rare or species-specific metabolites that drive host-choice of leaf-chewing insects.
... Besides oxidation, a chemically plausible modification of hydrolyzable tannins is hydrolysis, catalyzed by extremes of pH or by esterases, and yielding gallic acid and/or ellagic acid. For example, hydrolyzable tannins are hydrolyzed in vivo in lepidopterans, beetles, and sawflies (Gross et al. 2008;Lahtinen et al. 2005;Salminen and Lempa 2002). In our study, ellagic acid was found after in vitro incubation of ellagitannin-containing plant extracts (e.g., A. castaneifolia and T. catappa in Figs. ...
... This new compound was possibly derived from caffeoylquinic acids in the host plant. Protocatechuic acid earlier was found in the frass of sawflies feeding on birch leaves (Lahtinen et al. 2005). In other studies, monocaffeoylquinic acid (i.e., chlorogenic acid) and other phenolic acids were suggested to be oxidized in the guts of lepidopteran larvae (Salminen et al. 2004), and to cause lipid peroxidation and protein oxidation in larvae (Summers and Felton 1994). ...
Article
Lepidopteran larvae encounter a variety of phenolic compounds while consuming their host plants. Some phenolics may oxidize under alkaline conditions prevailing in the larval guts, and the oxidation products may cause oxidative stress to the larvae. In this study, we aimed to find new ways to predict how phenolic compounds may be modified in the guts of herbivorous larvae. To do so, we studied the ease of oxidation of phenolic compounds from 12 tropical tree species. The leaf extracts were incubated in vitro in alkaline conditions, and the loss of total phenolics during incubation was used to estimate the oxidizability of extracts. The phenolic profiles of the leaf extracts before and after incubation were compared, revealing that some phenolic compounds were depleted during incubation. The leaves of the 12 tree species were each fed to 12 species of lepidopteran larvae that naturally feed on these trees. The phenolic profiles of larval frass were compared to those of in vitro incubated leaf extracts. These comparisons showed that the phenolic profiles of alkali-treated samples and frass samples were similar in many cases. This suggested that certain phenolics, such as ellagitannins, proanthocyanidins, and galloylquinic acid derivatives were modified by the alkaline pH of the larval gut. In other cases, the chromatographic profiles of frass and in vitro incubated leaf extracts were not similar, and new modifications of phenolics were detected in the frass. We conclude that the actual fates of phenolics in vivo are often more complicated than can be predicted by a simple in vitro method.
... Exceptions were fractions that contained chlorogenic acid (stem fraction 1B, leaf fraction 3); instead of hydrolysis, chlorogenic acid (5-O-caffeoylquinic acid) produced new isomers, so that at the end of the measurement it had three isomers 3-O-, 5-O-and 4-O-caffeoylquinic acid in the ratio of 1:1:1 respectively. The isomerization of chlorogenic acid under alkaline conditions into these isomers has been observed before in vitro (Nagels et al., 1980), and during in vivo feeding tests with herbivorous insects (Salminen et al., 2004;Lahtinen et al., 2005). ...
... The glycosylation of the 3-OH group observed in most of the flavonoids of G. sylvaticum may explain this (Tuominen et al., 2013, part 1 in the same issue) as the 3-OH group has a crucial role in the enzymatic oxidation process of flavonoids (El-Safi et al., 2007). In addition, the results at pH 10 are consistent with the previous in vivo studies where flavonoid glycosides excreted mostly unaltered in all studied sawfly species (Lahtinen et al., 2005) and E. autumnata (Salminen et al., 2004). ...
Article
Geranium sylvaticum is a common herbaceous plant in Fennoscandia, which has a unique phenolic composition. Ellagitannins, proanthocyanidins, galloylglucoses, gallotannins, galloyl quinic acids and flavonoids possess variable distribution in its different organs. These phenolic compounds are thought to have an important role in plant-herbivore interactions. The aim of this study was to quantify these different water-soluble phenolic compounds and measure the biological activity of the eight organs of G. sylvaticum. Compounds were characterized and quantified using HPLC-DAD/MS, in addition, total proanthocyanidins were determined by BuOH-HCl assay and total phenolics by the Folin-Ciocalteau method. Two in vitro biological activity measurements were used: the prooxidant activity was measured by the browning assay and antioxidant activity by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Organ extracts were fractionated using column chromatography on Sephadex LH-20 and the activities of fractions was similarly measured to evaluate which polyphenol groups contributed the most to the biological activity of each organ. The data on the activity of fractions were examined by multivariate data analysis. The water-soluble extracts of leaves and pistils, which contained over 30% of the dry weight as ellagitannins, showed the highest pro-oxidant activity among the organ extracts. Fraction analysis revealed that flavonoids and galloyl quinic acids also exhibited high pro-oxidant activity. In contrast, the most antioxidant active organ extracts were those of the main roots and hairy roots that contained high amounts of proanthocyanidins in addition to ellagitannins. Analysis of the fractions showed that especially ellagitannins and galloyl quinic acids have high antioxidant activity. We conclude that G. sylvaticum allocates a significant amount of tannins in those plant parts that are important to the fitness of the plant and susceptible to natural enemies, i.e. pistil and leaf tannins protect against insect herbivores and root tannins against soil pathogens.
Article
Full-text available
The paper studies various groups of substances of secondary metabolism of phenolic nature in the leaves of model trees of English oak growing in the field-protective forest belts of the agroforestry complex Kamennaya Steppe. A physiological and biochemical analysis was carried out on samples of oak leaves from each tree in two stages - in the first decade of June and in early August. From each model tree 4-6 shoots of the lower tier of the southern exposure were selected. Significant biochemical diversity of the control and experimental groups of model oak trees was revealed. It is shown that the most significant fluctuations in the level of phenolic substances occur at the beginning of the growing season and then their content is stabilized. The informativeness of biochemical monitoring studies in the complex assessment of the current state of oak stands was confirmed. It is proved that the content of substances of secondary metabolism of phenolic nature and their combination can serve as a criterion of potential energy efficiency.
Article
Afrofittonia silvestris Lindau, commonly known as the hunter's weed, is aprocumbent herb trailing on moist ground. The leaves of the plant are used to heal sorefeet, skin infections and as laxative. The leaves were macerated in 50 % ethanol and theliquid extract concentrated to dryness. The dry extract was evaluated for antibacterialactivity by adopting agar diffusion method. The extract was partitioned between water,ethyl acetate and butanol successively and further subjected to antibacterial testing. Themost active extract, ethyl acetate extract, was purified through various chromatographicmethods to obtain pure compounds identified by spectroscopic methods as kaempferide3-O-ß-D-glucopyranoside and kaempferol 5,4'-dimethoxy-3,7-O- α-L-dirhamnoside.These compounds produced significant antibacterial effects, while the minimuminhibitory concentrations of the fractions and the pure compounds ranged between 25 and250 μg/mL. These flavonoids are reported for the first time in this plant, whilekaempferol 5,4'-dimethoxy-3,7-O- α-L-dirhamnoside is a new compound.
Article
Full-text available
Flavonoids are a group of secondary metabolites found in most families. They are known to have important physiological functions in plants by protecting them against biotic stresses. Liquid chromatography (HPLC) was used to determine the flavonoid profiles, especially apigenin glycosides, their total concentration, as well as changes in the amount of six flavones found in the aerial parts of alfalfa (Medicago sativa L.) (Fabaceae) Radius cv. for three vegetative stages, uninfested and infested by the pea aphid (Acyrthosiphon pisum Harris) (Homoptera: Aphididae). It has been shown that both control and infested green aerial parts of alfalfa plants had similar flavonoid profiles. The dominant flavonoid of alfalfa was compound 7-O-[2-O-feruloy1-ß-D- glucuronopyranosyl(l-→2)-O-β-D-glucuronopyranosyl]-4'-O-β-D- glucuronopyranosideapigenin. Compound 4'-O-β-D-glucuronopyranosideapigenin was present in the smallest amounts. The total concentration of flavones was rather high and ranged from 10.32 to 12.28 mg/g d.m., but there were no significant differences between uninfested and infested alfalfa plants. There was a negative correlation between the concentration of total apigenin glycosides in the alfalfa plants and pea aphid abundance and phloem sap ingestion. This finding may indicate the importance of apigenin glycoside forms as nutritional compounds.
Article
Previous studies of purified phenolic compounds have revealed that some phenolics, especially ellagitannins, can autoxidise under alkaline conditions, which predominate in the midgut of lepidopteran larvae. To facilitate screening for the pro-oxidant activities of all types of phenolic compounds from crude plant extracts, we developed a method that combined our recent spectrophotometric bioactivity method with an additional chromatographic step via UPLC-DAD-MS. This method allowed us to estimate the total pro-oxidant capacities of crude extracts from 12 plant species and to identify the individual phenolic compounds that were responsible for the detected activities. It was found that the pro-oxidant capacities of the plant species (i.e., the concentrations of the easily-oxidised phenolics) varied from 0 to 57 mg/g dry wt, representing from 0% to 46% of the total phenolics from different species. UPLC-DAD-MS analysis revealed that most flavonol and flavone glycosides were only slightly affected by alkaline conditions, thus indicating their low pro-oxidant activity. Interestingly, myricetin-type compounds differed from the other flavonoids, as their concentrations decreased strongly due to alkaline incubation. The same effect was detected for hydrolysable tannins and prodelphinidins, suggesting that a pyrogallol sub-structure could be a key structural component that partially explains their easy oxidation at high pH. Other types of phenolic compounds, such as hydroxycinnamic acids, were relatively active, as well. These findings demonstrate that this method displays the potential to identify most of the active and inactive pro-oxidant phenolic compounds in various plant species.
Article
The effects of artificially added flavonoid aglycones to birch leaf surfaces on the larval performance of six species of leaf-chewing sawflies were investigated. Significantly negative effects of increased contents of both total flavonoid and individually fed flavonoid compounds were found for the larval performance of certain mid to late and late, but not early season, sawfly species. Species-specific variations in the quantity of faecal flavonoid glycosides, which were examined to investigate whether effective glycosylation of foliar flavonoid aglycones in larvae correlated with varying tolerance to these compounds, also yielded significant species-specific differences between early and late season species. The results suggest seasonal adaptations in host plant use by sawflies feeding on mountain birch, such that phenologically earlier species are better adapted to coping with leaf surface flavonoid aglycones, which occur in the highest concentrations in young leaves.
Article
Full-text available
The foliage and fruit of the tomato plantLycopersicon esculentum contains polyphenol oxidases (PPO) and peroxidases (POD) that are compartmentally separated from orthodihydroxyphenolic substrates in situ. However, when leaf tissue is damaged by insect feeding, the enzyme and phenolic substrates come in contact, resulting in the rapid oxidation of phenolics to orthoquinones. When the tomato fruitwormHeliothis zea or the beet army-wormSpodoptera exigua feed on tomato foliage, a substantial amount of the ingested chlorogenic acid is oxidized to chlorogenoquinone by PPO in the insect gut. Additionally, the digestive enzymes of the fruitworm have the potential to further activate foliar oxidase activity in the gut. Chlorogenoquinone is a highly reactive electrophilic molecule that readily binds cova-lently to nucleophilic groups of amino acids and proteins. In particular, the -SH and -NH2 groups of amino acids are susceptible to binding or alkylation. In experiments with tomato foliage, the relative growth rate of the fruitworm was negatively correlated with PPO activity. As the tomato plant matures, foliar PPO activity may increase nearly 10-fold while the growth rate of the fruitworm is severely depressed. In tomato fruit, the levels of PPO are highest in small immature fruit but are essentially negligible in mature fruit. The growth rate of larvae on fruit was also negatively correlated with PPO activity, with the fastest larval growth rate occurring when larvae fed on mature fruit. The reduction in larval growth is proposed to result from the alkylation of amino acids/protein byo-quinones, and the subsequent reduction in the nutritive quality of foliage. This alkylation reduces the digestibility of dietary protein and the bioavailability of amino acids. We believe that this mechanism of digestibility reduction may be extrapolatable to other plant-insect systems because of the ubiquitous cooccurrence of PPO and phenolic substrates among vascular plant species.
Article
Full-text available
Large interspecific differences in redox potential exist among herbivorous lepidopteran larvae. Reducing conditions occur in the midguts ofManduca sexta (Sphingidae) andPolia latex (Noctuidae), whereas oxidizing conditions prevail in the midguts ofLymantria dispar (Lymantriidae),Danaus plexippus (Danaidae), andPapilio glaucus (Papilionidae). The epithelium of the posterior midgut ofM. sexta fed a diet containing bismuth subnitrate accumulates bismuth sulfide, suggesting that sulfide might be one of the reducing agents responsible for the maintenance of reducing conditions in this species. We propose that the effects of plant allelochemicals in insect herbivores will be strongly affected by gut redox conditions and that the regulation of gut redox conditions is an important adaptation of insect herbivores to the chemical defenses of plants. The redox state of the gut is yet another insect trait that must be included in the analysis of plant-insect interactions.
Article
Full-text available
The ecological activities of plant phenolics are diverse and highly variable. Although some variation is attributable to differences in concentration, structure, and evolutionary history of association with target organisms, much of it is unexplained, making it difficult to predict when and where phenolics will be active. I suggest that our understanding is limited by a failure to appreciate the importance of oxidative activation and the conditions that influence it. I summarize examples of oxidative activation of phenolics in ecological interactions, and argue that physicochemical conditions of the environment that control phenolic oxidation generate variation in ecological activity. Finally, I suggest that measurements of oxidative conditions can improve our predictions of phenolic activity and that experiments must be designed with conditions appropriate to the biochemical mode of phenolic action.
Article
1. In two subsequent years and under standardized experimental conditions, we bioassayed the leaf quality of the same 20 subarctic mountain birches Betula pubescens ssp. tortuosa with 14 sawfly and one lepidopteran species to study the effects of annual and seasonal leaf quality variation on herbivore growth, and potential species interactions. The larval periods of the 15 species covered the whole growth season of birch. 2. Insect growth rates were higher in 1987 than in 1988, except for two late-season species. In spite of the large annual variation in foliage quality, the relative ranking of individual trees for growth of the herbivore species remained constant between the 2 years, Differences in insect growth between the years seemed to be explained by plant vigour. 3. Birch leaf quality decreases rapidly within a season. The early species had two to three times higher relative growth rates (RGR) than species feeding in mid- and late season. Growth rates of the early season species also varied more than those of late season, both among insect species and among individual trees. 4. We compared tree-specific herbivore growth between pairs of species to investigate similarity of the trees for herbivores. All significant correlations within a year between the RGR of the herbivore species were positive, but significant correlations were found between early and mid-season species only, i.e. at the time when foliage quality was temporally best and most variable among trees. When split into years, these positive correlations were more common (44% of cases) in 1988, when foliage quality was poor, than in 1987 (1%). 5. Growth of several insect species differed significantly among the 20 trees in 1987 and in the early season of 1988. This happened when larvae were consuming high-quality foliage, and indicated that the herbivore species were able to adjust their growth performance to take advantage of high-quality diets. But they did this in species-specific ways, as indicated by the fewer significant correlations in growth between herbivore species on high-quality than on low-quality diets. Accordingly, growth of different herbivore species converged under conditions of poor leaf quality, and herbivore species showed some specialization when leaf quality was high.
Article
Factors which establish whether tannin and protein interact to form soluble complexes or precipitates were identified. The ratio of tannin to protein in the reaction mixture influenced solubility of the tannin-protein complexes. At protein-to-tannin ratios larger than the optimum ratio, or equivalence point, soluble tannin-protein complexes apparently formed instead of insoluble complexes. Several other factors influenced the amount of protein precipitated by tannin-containing plant extracts, including the length of the reaction time and the conditions of the tannin extraction. The analytical and ecological significances of soluble complexes were considered. A titration method which allows simultaneous determination of the equivalence point and assessment of the protein-precipitating capacity of any plant extract was developed. It was postulated that in vivo, tannin and protein may not only form insoluble complexes with antinutritional effects, but may also form soluble complexes which have unknown metabolic effects.
Article
High-performance liquid chromatographic (HPLC) data of cinnamic and benzoic acid derived phenols, esterified with quinic acid at the C(5) hydroxyl, and with glucose at the C(1) hydroxyl (β anomeric form), are presented for the first time. These naturally occurring compounds have been obtained synthetically. They are chromatographed on reversed-phase and on diol HPLC systems. The four chlorogenic acid isomers are also chromatographed on these columns.
Article
Leaf quality of the mountain birch (Betula pubescens ssp.tortuosa) for herbivores was studied at several hierarchical levels: among trees, among ramets within trees, among branches within ramets, and among short shoots within branches. The experimental units at each level were chosen randomly. The indices of leaf quality were the growth rate of the larvae of a geometrid,Epirrita autumnata, and certain biochemical traits of the leaves (total phenolics and individual phenolic compounds, total carbohydrates and individual sugars, free and protein-bound amino acids). We also discuss relationships between larval growth rate and biochemical foliage traits. Larval growth rates during two successive years correlated positively at the level of tree, the ramet, and the branch, indicating that the relationships in leaf quality remained constant between seasons both among and within trees. The distribution of variation at different hierarchical levels depended on the trait in question. In the case of larval growth rate, ramets and short shoots accounted for most of the explained variation. In the case of biochemical compounds, trees accounted for most of the variance in the content of total phenolics and individual low-molecular-weight phenolics. In the content of carbohydrates (total carbohydrates, starch, fructose, glucose, and sucrose) and amino acids, variation among branches was generally larger than variation among trees. Variation among ramets was low for most compounds. No single leaf trait played a paramount role in larval growth. Secondary compounds, represented by phenolic compounds, or primary metabolites, particularly sugars, may both be important in determining the suitability of birch leaves for larvae. If phenols are causally more important, genet-specific analyses of foliage chemistry are needed. If sugars are of primary importance, within-genet sampling and analysis of foliage chemistry are necessary.