02 Insect Biochemistry Molecular
02 Insect Biochemistry Molecular
02 Insect Biochemistry Molecular
1
0
8
2
1
0
7
7
which are universally conserved among SPs and
constitute the catalytic triad. The presence of a glycin
residue at position 189 has been found in insect
chymotrypsins, such as the re ant (Solenopsis invicta)
chymotrypsin (Botos et al., 2000). Therefore, the
SeCT34 was putatively classied as a chymotrypsin-like
protein.
The SeCT34 protein was aligned and compared with
representative SPs from different species (Fig. 1). Blast-
X analysis (Altschul et al., 1997) of SeCT34 against the
NCBI database did not show high homology to any
known lepidopteran SP. The highest homology was
found with a chymotrypsin from the cat ea, Ctenoce-
phalides felis and dipteran chymotrypsin-like proteins. A
blast search against the Bombyx mori Silkworm EST
database (Mita et al., 2003) revealed homology to a
cDNA fragment (mg0778), encoding a putative
chymotrypsin (BmCT0778 in this work). SeCT34 has
two insertions of approximately six amino acids relative
to bovine chymotrypsin, and to the dipteran chymo-
trypsins (Fig. 1). Insertions are also present in
BmCT0778 at these locations, although their size is
different. The regions around the catalytic-triad resi-
dues, which are conserved in most SPs (TAAHC
around His59, DIAL around Asp102), have also been
conserved in SeCT34. However, both SeCT34 and
BmCT0778 show a remarkable change in the conserved
GDSGGP region (around the catalytic Ser195) to
GDSGSA.
SeCT34 clearly forms a distinct group among the
lepidopteran SPs. This becomes obvious when the
protein is included in a phylogenetic tree with lepidop-
teran trypsins and chymotrypsins known to be expressed
in the midgut (Fig. 2). These proteinases, presumab-
ly involved in digestion of dietary protein, are
only distantly related to SeCT34, whereas the dipteran
chymotrypsins that came out of the homology
analysis appeared to be more closely related to it.
Only the B. mori protein BmCT0778 is located at the
same branch of the tree; this branch remains sepa-
rate when all known insect SP are included in the tree
(Fig. 2).
3.2. Expression analysis of SeCT34
Expression of SeCT34 was examined by RT-PCR on
RNA from S. exigua eggs, neonates, 2nd, 3rd and 4th
instar larvae, early and late 5th instar larvae, in midgut
tissue from 4th, early 5th and late 5th instar larvae and
from mature insects, and in hemocytes from larvae 1 day
into their 5th instar (Fig. 3). Expression was detected
exclusively in the midgut of late 5th instar larvae. Under
the conditions employed, we did not detect SeCT34
expression when RNA from the whole late 5th instar
larvae was used as a template, probably as a result of the
dilution of midgut RNA in the total body RNA.
3.3. Heterologous expression and purication of SeCT34
SeCT34 protein was expressed in Sf21 insect cells,
using the baculovirus-insect cells expression system. For
this purpose the SeCT34 gene was fused to the
polyhedrin promoter, and to a C-terminal 6xHis-tag
encoding DNA. The expression of the recombinant
SeCT34 (rSeCT34) protein was monitored by Western
blot analysis, using an antibody against the 6xHis tag.
At 48 h.p.i, rSeCT34 was detected in the medium as well
as in the cells (Fig. 4A). The rSeCT34 was puried from
the medium by 6xHis afnity chromatography, to a level
where less than 10% contaminant protein was detected
by silver-staining (Fig. 4B). Puried rSeCT34 protein
showed an estimated mobility of around 30 KDa, which
is close to the predicted molecular weight (29 KDa) of
the mature rSeCT34 protein on the basis of the gene
sequence, including the 6xHis-tag. The estimated yield
of puried rSeCT34 was around 100 mg/l of medium.
ARTICLE IN PRESS
0.1
100
100
94
100
SeCT34
BmCT0778
96
98
94
83
100
100
100 65
99
AiT (L)
PiT (L)
MsT (L)
HzT (L)
HaT (L)
HvCT (L) HaCT (L)
SfCT (L)
AeCT (L)
MsCT (L)
DsCT (D)
DmCT (D)
AaeCT (D)
AdCT (D)
AaCT (D)
Fig. 2. Unrooted phylogenetic tree derived from a ClustalX alignment
of selected insect trypsin and chymotrypsin-like proteins. Numbers on
the branches report the level of condence as determined by bootstrap
analysis (100 bootstrap replicates). T in the name indicates trypsin and
CT indicates chymotrypsin. Letter in parenthesis indicates the insect
order L for lepidopera, D for diptera. The proteins used in the tree are:
AdCT: Anopheles darlingi, AAD17494. AeCT: Anopheles aquasalis,
AAD17492. AaeCT: Aedes aegypti AAL93243. DmCT: Drosophila
melanogaster, NP_732210, Dp: Drosophila pseudoobscura, EAL27112.
HzT: Helicoverpa zea, AAF74742. PiT: Plodia interpunctella,
AAF24226. AiT: Agrotis ipsilon,, AAF74752. MsT: Manduca sexta,
T10109. HaT: Helicoverpa armigera, CAA72962. HaCT: Helicoverpa
armigera, CAA72966. SfCT: Spodoptera frugiperda, AAO75039.
HvCT: Heliothis virescens, AAF43709. MsCT: Manduca sexta,
AAA58743. AiCT: Agrotis ipsilon, AAF71516. BmCT0778: Bombyx
mori, mg-0778. The scale bar indicates an evolutionary distance of 0.1
amino acid substitutions per position in the sequence.
S. Herrero et al. / Insect Biochemistry and Molecular Biology 35 (2005) 10731082 1078
3.4. Characterization of the recombinant SeCT34
(rSeCT34) activity
Chymotrypsins are known to cleave peptide bonds
behind Phe, Tyr, Trp and Leu residues (Barrett et al.,
2003). To determine the substrate preference of
rSeCT34, the enzyme was incubated with ve different
synthetic substrates. Maximum activity was obtained
with SAAPFpNA, while hydrolysis of SAAPLpNA was
low (5% of the activity obtained with SAAPFpNA).
Under the conditions employed, rSeCT34 was not able
to hydrolyze SAAApNA (an elastase substrate), BAp-
NA (a trypsin substrate) and EFLpNA (a thiol-
proteinase substrate).
The pH optimum of activity was analyzed by
measuring the hydrolysis of SAAPFpNA in a pH range
from 5 to 11 (Fig. 5). rSeCT34 was more active at basic
pH values, with a maximum activity at pH 11. We could
not test higher pH values, as the substrate appeared to
be unstable at pH411.
Kinetic parameters were obtained at two different pH
values. Under our assay conditions (i.e. in the presence
of BSA, which may compete as a substrate but stabilizes
the enzyme), the Km value for the hydrolysis of
SAAPFpNA was around 4-fold higher at pH 11
(Km 6.2 mM) than at pH 8 (Km 1.6 mM). Substrate
turnover at pH 11 (Kcat 1.8 s
1
) was around 10-fold
higher than at pH 8 (Kcat 0.17 s
1
). Catalytic
efciency values (Kcat/Km) were around 3-fold higher
at pH 11 than at pH 8. The observed 3-fold higher
catalytic efciency at pH 11 is in agreement with the
differences previously found in the activity studies
performed at different pH range (Fig. 5).
Since lepidopteran midgut proteinases are targets for
plant proteinase inhibitors, rSeCT34 was also charac-
terized with regard to its sensitivity to different
proteinase inhibitors. Proteinaceous inhibitors such as
aprotinin and BBI almost fully inhibited the activity of
the rSeCT34 proteinase at all the concentrations tested.
The most active inhibitor was aprotinin, which showed
values of 100% inhibition at the lowest concentration
tested. In contrast, synthetic inhibitors such as PMSF
and Antipain could only inhibit around 50% of the
activity at the highest concentrations tested. Inhibitors
as TPCK and EDTA hardly affected the activity of
rSeCT34 even at the highest concentrations tested
(Table 1).
4. Discussion
4.1. Recombinant expression of lepidopteran gut
proteinases
In this study, we describe the characterization and
funtional expression of SeCT34, a novel chymotrypsin
expressed in the midgut of S. exigua. To our knowledge
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Fig. 3. RT-PCR of SeCT34 transcripts in different tissues and
development stages. rRNA refers to ribosomal RNA.
Fig. 4. Expression and purication of insect-cell expressed rSeCT34.
Panel A, detection of the 6xHis-tag from rSeCT34 by western analysis
in the medium and in the cell extract of Sf21 cells culture infected with
baculovirus expressing rSeCT34 (34) or infected with the control
baculovirus lacking rSeCT34 (C). Panel B, silver stained 12% SDS-
PAGE of rSeCT34 puried from the medium of cells infected with
baculovirus expressing rSeCT34 (34) or with a control baculovirus
lacking rSeCT34 (C). Crude, refers to crude protein extract loaded
onto the column and Pure, refers to the elution from the column of the
puried protein.
4 5 6 7 8 9 10 11 12
0
20
40
60
80
100
120
NaAc
Tris
Gly
pH
%
r
e
l
a
t
i
v
e
a
c
t
i
v
i
t
y
Fig. 5. Inuence of pH on the activity of rSeCT34. Different buffers
were employed to cover the whole pH range as indicated by different
marks (see also Section 2).
S. Herrero et al. / Insect Biochemistry and Molecular Biology 35 (2005) 10731082 1079
this is the rst report of the functional expression of a
recombinant midgut serine proteinase from insects,
despite the availability of many SP sequences from a
broad range of insect species. Sf21 cells, in which
rSeCT34 was expressed, are derived from S. frugiperda,
a lepidopteran species closely related to S. exigua Thus,
the applied recombinant protein expression system is
closely related to the insect species where SeCT34 was
isolated from. Another contribution to the successful
expression of rSeCT34 was possibly made by the use of
an improved baculovirus expression vector, from which
the viral cathepsin and chitinase genes had been removed
(Kaba et al., 2004). V-cathepsin is a cysteine proteinase
involved, in conjunction with the chitinase, in liquefac-
tion of the insect host cell at the end of the baculovirus
infection (Hawtin et al., 1997). Production and stability
of recombinant proteins has been described to be
enhanced when both genes had been eliminated (Kaba
et al., 2004; Berger et al., 2004). Therefore the system
used here may well be applicable to other lepidopteran
midgut SPs, though still some may proof difcult to
express without damage to the expression system.
However, in the case of SeCT34 we have not observed
any indications of such damage.
4.2. Features of SeCT34
SeCT34 is a representative of a novel subgroup of
lepidopteran chymotrypsins, which map in a distinct
branch of the lepidoptera SP phylogenetic tree (Fig. 2).
Characteristic for this sub-group seems to be the
substitution of GP-SA in the highly conserved
GDSGGP domain around the catalytic Ser195 residue.
These substitution occur in both SeCT34 and its
homologue in B. mori (BmCT0778). Some variation is
known to exist at these residues in the SP family from D.
melanogaster (Ross et al., 2003). Out of 148 members of
the SP family, 11 are different in either of these two
residues. Out of 6 members where the Gly197 position is
different, 4 have the change Gly-Ser. Similarly,
sequence analysis of human SP (subfamily S1A)
revealed that only two out of 79 proteins had changed
the Gly197 residue, both of them Gly-Ser, and from
the four members where Pro198 is different, three of
them have Pro-Ala (Yousef et al., 2004). These
observations suggest that the GP-SA substitution is
typical for the SeCT34 subgroup and is one of the very
rare variations allowed at this position that still yields a
functional protein. It is likely that phylogenetic compar-
ison of the SPs from other insect orders reveals the
presence of this sub-group in other orders. In the crystal
structure of bovine chymotrypsin and re ant chymo-
trypsin (Hynes et al., 1990; Botos et al., 2000) the
Gly197 residue (Ser in SeCT34) localizes adjacent to the
catalytic triad His57, Asp102, and Ser195, though it is
not in direct contact with the substrate. This suggests
that Gly197 may have a possible role in positioning of
the catalytic triad relative to the substrate, rather than in
positioning the substrate relative to the enzyme.
The rSeCT34 is a true chymotrypsin, as can be
deduced from its ability to hydrolyze SAAPFpNA.
However, in contrast to most vertebrate chymotrypsins
(Barrett et al., 2003) and invertebrate chymotrypsins
(Lee and Anstee, 1995; Valaitis, 1995), which hydrolyze
SAAPLpNA with similar efciency, rSeCT34 does not
digest the substrate having Leu at the P1 position very
well. This suggests that SeCT34 may have a specic
function, rather than being involved in general digestion
of dietary protein.
The activation site of SeCT34 is atypical. Most
proteinases that are expressed in the gut, both in man
and in insects, are activated by a trypsin-like activity,
which acts on an Arg residue at position 15 (bovine
chymotrypsin numbering) (Brown and Hartley, 1966).
This holds true for trypsins, carboxypeptidases and
chymotrypsins. SeCT34 is an exception to this rule, as it
carries a Phe in this position, suggesting that it is
activated by chymotrypsin rather than by a trypsin. Our
activity assays showed chymotrypsin activity for
SeCT34 without the need of pre-incubation with trypsin,
and the migration of rSeCT34 as a single band. This
suggests that the proteinase activates itself.
4.3. Physiological role of SeCT34
The role of SeCT34 in the midgut of S. exigua remains
unclear. The pH optimum of the recombinant enzyme
(at pH411) is very similar to that of the total gut
proteinase activity (Jongsma et al., 1996). This suggests
that it functions in a similar environment as the
proteinases involved in digestion of dietary protein, i.e.
the gut lumen. This is further supported by our
observation that both SeCT34 and the total gut
proteolytic activity of S. exigua are relatively sensitive
to proteinaceous inhibitors such as BBI and aprotinin
(Table 1; Jongsma et al., 1996).
The possible role of SeCT34 in insect gut physiology
can be inferred from the timing of its expression. The
SeCT34-encoding transcript could exclusively be de-
tected in the fth instar insect, just prior to pupation. To
gain further support for this apparent restricted expres-
sion of SeCT34, expression data for the B. mori
homologue, BmCT0778, available on the Internet were
searched (http://kaikocdna.dna.affrc.go.jp/page_pub.
html). BmCT0778 has only been identied in the
mg-B. mori cDNA library, which was obtained from
the midgut of larvae four days after molting to 5th
instar. No similar fragment has been found in 39 other
cDNA libraries obtained from different tissues and
developmental instars. Thus, both SeCT34 and
BmCT0778 seem to be exclusively expressed during the
transition from 5th instar larvae to pupae. Specic
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S. Herrero et al. / Insect Biochemistry and Molecular Biology 35 (2005) 10731082 1080
proteolytic events occur during this period, when the
midgut epithelium is replaced completely and the
material is recycled by the action of digestive proteinases
(Uwo et al., 2002). Expression levels of SeCT34 were
low in comparison with levels of other midgut protei-
nase genes (data not shown). The precise tuning of its
expression, its auto-activation and its relatively narrow
substrate specicity could mean that SeCT34 is involved
in the activation of other proteinases, which are
involved in midgut remodeling upon pupation. Knock-
out experiments should be carried out to conrm the
role of SeCT34 in this process. Although the specic role
of SeCT34 remains unclear, the functional expression of
SeCT34 in the baculovirus-insect cell system opens a
wide range of possibilities for the study of insect SPs.
Mutational studies could be applied to determine the
role of the different residues in the interaction with plant
proteinase inhibitors or in substrate specicity. Most
signicantly, we have demonstrated that the baculovirus
system is capable of expressing a lepidopteran midgut
SP, and this system should be tested with other
lepidopteran SP genes relevant to digestion of dietary
protein.
Acknowledgments
S. Herrero was supported by a Marie Curie fellowship
contract No. HPMF-CT-2002-01994 from the EU. R.
de Maagd was supported by Program subsidy 347 of the
Dutch Ministry of Agriculture, Nature Management
and Fisheries.
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Insect
Biochemistry
and
Molecular
Biology
Insect Biochemistry and Molecular Biology 35 (2005) 10831099
Acquisition, transformation and maintenance of plant pyrrolizidine
alkaloids by the polyphagous arctiid Grammia geneura
T. Hartmann
a,
, C. Theuring
a
, T. Beuerle
a
, E.A. Bernays
b
, M.S. Singer
c
a
Institut fur Pharmazeutische Biologie der Technischen Universitat Braunschweig, Mendelssohnstrasse 1, D-38106 Braunschweig, Germany
b
Department of Entomology, University of Arizona, P.O. Box 210088, Tucson, AZ 85721-0088, USA
c
Department of Biology, Wesleyan University, Hall-Atwater Labs, Rm. 259, Middletown, CT 06459, USA
Received 9 March 2005; accepted 6 May 2005
Abstract
The polyphagous arctiid Grammia geneura appears well adapted to utilize for its protection plant pyrrolizidine alkaloids of almost
all known structural types. Plant-acquired alkaloids that are maintained through all life-stages include various classes of macrocyclic
diesters (typically occurring in the Asteraceae tribe Senecioneae and Fabaceae), macrocyclic triesters (Apocynaceae) and open-chain
esters of the lycopsamine type (Asteraceae tribe Eupatorieae, Boraginaceae and Apocynaceae). As in other arctiids, all sequestered
and processed pyrrolizidine alkaloids are maintained as non-toxic N-oxides. The only type of pyrrolizidine alkaloids that is neither
sequestered nor metabolized are the pro-toxic otonecine-derivatives, e.g. the senecionine analog senkirkine that cannot be detoxied
by N-oxidation. In its sequestration behavior, G. geneura resembles the previously studied highly polyphagous Estigmene acrea.
Both arctiids are adapted to exploit pyrrolizidine alkaloid-containing plants as drug sources. However, unlike E. acrea, G. geneura
is not known to synthesize the pyrrolizidine-derived male courtship pheromone, hydroxydanaidal, and differs distinctly in its
metabolic processing of the plant-acquired alkaloids. Necine bases obtained from plant acquired pyrrolizidine alkaloids are re-
esteried yielding two distinct classes of insect-specic ester alkaloids, the creatonotines, also present in E. acrea, and the
callimorphines, missing in E. acrea. The creatonotines are preferentially found in pupae; in adults they are largely replaced by the
callimorphines. Before eclosion the creatonotines are apparently converted into the callimorphines by trans-esterication. Open-
chain ester alkaloids such as the platynecine ester sarracine and the orchid alkaloid phalaenopsine, that do not possess the unique
necic acid moiety of the lycopsamine type, are sequestered by larvae but they need to be converted into the respective creatonotines
and callimorphines by trans-esterication in order to be transferred to the adult stage. In the case of the orchid alkaloids, evidence is
presented that during this processing the necine base (trachelanthamidine) is converted into its 7-(R)-hydroxy derivative
(turneforcidine), indicating the ability of G. geneura to introduce a hydroxyl group at C-7 of a necine base. The creatonotines and
callimorphines display a striking similarity to plant necine monoesters of the lycopsamine type to which G. geneura is well adapted.
The possible function of insect-specic trans-esterication in the acquisition of necine bases derived from plant acquired alkaloids,
especially from those that cannot be maintained through all life-stages, is discussed.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: Grammia geneura (Lepidoptera; Arctiidae); Alkaloid sequestration; Alkaloid processing; Pyrrolizidine alkaloids; Insect alkaloids;
Creatonotines; Callimorphines; Chemical defense
1. Introduction
Among insects that sequester plant pyrrolizidine
alkaloids and utilize them for their own chemical defense,
the tiger moths (Lepidotpera: Arctiidae) represent an
impressive example. The ability to sequester pyrrolizidine
alkaloids from the larval diet is most parsimoniously
ARTICLE IN PRESS
www.elsevier.com/locate/ibmb
0965-1748/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibmb.2005.05.011
1
0
9
9
1
0
8
8
accumulate about 50% of the trace amounts of these
alkaloids present in their larval food. No toxic or
detrimental effects of senkirkine were observed in the
experiment during further larval development, indicat-
ing that the larvae are well adapted to tolerate otonecine
derivatives present in their alkaloid meals.
In all feeding experiments callimorphines (Fig. 2B)
could be recovered as insect alkaloids from adults but
not larvae. Creatonotines (Fig. 2A) were only detected
in trace amounts in larvae and males fed on S. jacobaea
alkaloids. Insects fed on S. congestus alkaloids con-
tained 1,2-dihydrocallimorphines indicating insect-spe-
cic esterication of platynecine obtained from the
plant-acquired platyphyllines (Fig. 3B).
Pyrrolizidine alkaloid-containing species of the Apoc-
ynaceae often possess unique macrocyclic triesters.
Examples are 14-deoxyparsonsianidine and 14-deoxy-
parsonsianine (Fig. 1A) the major alkaloids of Parsonsia
laevigata. Larvae are able to sequester and store these
alkaloids (Table 3). It is interesting to note that 14-
deoxyparsonsianine, the less abundant pyrrolizidine
alkaloid in the larval diet, accumulates in adults as the
major component. The two pyrrolizidine alkaloids differ
in just one carbon atom (Fig. 1A). In adults the
callimorphines represent a considerable portion (15 to
38%) of total pyrrolizidine alkaloids.
3.2. Sequestration and processing of pyrrolizidine
alkaloids of the lycopsamine type
Alkaloids of the lycopsamine type are characterized by
their unique necic acid moiety, 2-isopropyl-2,3-dihydrox-
ybutyric acid. At least three stereoisomers of this rare
acid are known to occur in alkaloids of the lycopsamine
type: (-)-trachelanthic acid with (2R)(3S)-conguration
in indicine; (-)-viridioric acid, (2
0
S)(3S), in lycopsamine
and echinatine and (+)-trachelanthic acid, (2S)(3R), in
intermedine and rinderine (Fig. 1B). Alkaloids of this
type are typical for pyrrolizidine alkaloid-containing
species of the Boraginaceae, Apocynaceae and the tribe
Eupatorieae of the Asteraceae. For example, indicine and
lycopsamine (from Heliotropium indicum) were seques-
tered and maintained without discrimination (Table 4). It
is notable that the concentration of 3acetylindicine, an
alkaloid that is only detectable in trace amounts in the
larval diet and larval extract, is considerably increased in
adults; it is accompanied by trace amounts of 3-
acetyllycopsamine which does not occur in the larval diet.
Feeding of a puried alkaloid extract from Eupator-
ium cannabinum gave more complex results (Table 4).
Rinderine as a major alkaloid in the larval diet was
found at already decreased levels in larvae and only in
traces in adults which instead contained lycopsamine
and echinatine as major alkaloids. Obviously, alkaloids
with a 3S-conguration (Fig. 1B) are preferentially
transferred to the adult life-stage. While for larvae the
changed alkaloid composition could be accomplished by
uptake discrimination, this explanation can be excluded
for adults. In particular, the strong increase in the
lycopsamine level indicates an insect-specic epimeriza-
tion of (3R)-congurated alkaloids, probably accom-
panied by the known (see Chapter 3.4) epimerization of
(7S)-congurated alkaloids (Fig. 1B).
In addition, like in the experiment with indicine small
amounts of acetyl derivatives are detectable, which were
not present in the larval diet and thus must have been
formed by the insect. Interestingly, besides 3-acetyl
derivatives, 7-acety esters are detected.
In both feeding experiments considerable amounts of
callimorphines are detectable. In the experiment with H.
indicum alkaloids the insect-specic alkaloids account
for 1012%, while in the E. cannabinum experiment, the
callimorphines add up to 27% (males) and 50%
(females) of total alkaloids (Table 4).
ARTICLE IN PRESS
Table 2
Pyrrolizidine alkaloid prole established by GCMS for G. geneura that as larvae (penultimate instar) had received about 1 mg senkirkine per
individual added to the articial diet
Pyrrolizidine alkaloids recovered from insects m/z [M
+
] RI Relative abundance (%)
Diet Larvae (n 2) Males (n 3) Females (n 4)
Plant acquired alkaloids
Senecivernine 335 2267 2 42.571.5 38.572.5 40.071.4
Senecionine 335 2275 1 28.071.0 33.571.5 32.070.9
Seneciphylline 333 2288 Tr 12.071.0 13.570.5 12.770.8
Integerrimine 335 2335 Tr 12.070 13.571.5 14.370.5
Senkirkine 365 2460 97 5.571.5
a
Nd Nd
Callimorphines
Homocallimorphine 311 2037 1.171.0 1.170.6
Total alkaloid (mg/individual 18.9710.8 14.371.3 12.872.8
Total alkaloid (mg/g dry wt) 0.0770.04 0.1670.02 0.0970.03
a
Most likely due to the gut content
T. Hartmann et al. / Insect Biochemistry and Molecular Biology 35 (2005) 10831099 1089
3.3. Sequestration and metabolism of open-chain
platynecine and trachelanthamidine esters
Feeding of a dietary alkaloid mixture that contained
the open-chain platynecine diester sarracine (containing
5% of its (E)(Z)-isomer sarracinine) (Fig. 3B) (Table 5).
In contrast, adults did not contain even traces of
the plant-derived pyrrolizidine alkaloids but instead
stored the respective platyphylline analogs of creatono-
tines and callimorphines, i.e. (1S)-1,2-dihydrocreatono-
tines and (1S)-1,2-dihdyrocallimorphines (Table 5).
Hydrolysis of the insects alkaloids recovered from
ARTICLE IN PRESS
Fig. 2. Retronecine and heliotridine are converted into insect-specic monoesters. (A) Creatonotines are found in pupae and probably synthesized at
early stages of pupation, (B) callimorphines are found in adults and probably are synthesized shortly before eclosion at the expense of creatonotines,
and (C) (7S)-Congurated heliotridine is partly epimerized yielding (7R)-congurated retronecine and partly converted into callimorphine derivatives
with (7S)-conguration.
T. Hartmann et al. / Insect Biochemistry and Molecular Biology 35 (2005) 10831099 1090
adults and GC-MS analysis of the necine base fraction
revealed the presence of platynecine as exclusive necine
base. The two chlorinated alkaloids are most likely
artifacts generated during treatment with dichloro-
methane.
Insects given the dietary mixture of T-phalaenopsine
(trachelanthamidine ester, 80%) and Is-phalaenopsine
(isoretronecanol ester, 20%) (Fig. 3C) did not, as
adults, contain even trace amounts of the dietary
pyrrolizidine alkaloids. Instead the respective 7-deso-
xy-1,2-dihydrocreatonotines and 7-desoxy-1,2-callimor-
phine were present (Table 6). Most interestingly
adults were found to contain as major alkaloids 1,2-
dihydrocallimorphine and 1,2-dihydrohomocallimor-
phine which account for more than 60% of total
pyrrolizidine alkaloids recovered from the insects.
The two compounds display mass fragmentation
patterns identical to those of the 1,2-dihydrocallimor-
phines identied after feeding of plant-acquired platy-
necine esters, i.e. S. congestus (Table 1) and sarracine
(Table 5) but show different RI values (Fig. 4).
Hydrolysis of the alkaloid mixtures recovered from
adults and analysis of the TMS-derivatives of the necine
base fraction revealed the presence a necine base with a
fragmentation pattern identical to that of platynecine
but with a different RI. It was identied as the
platynecine isomer turneforcidine with (1R)-congura-
tion like trachelanthamidine (Fig 3). Trachelanthami-
dine itself was identied in the same experiment
accompanied by only traces of its (1S)-congurated
isomer, i.e. isoretronecanol. This conrms, rstly, that
the alkaloids recovered from the insects have (1R)-
conguration (Table 6) and, secondly, that, G. geneura
must be able to hydroxylate the trachelanthamidine
moiety at C-7 (Table 6; Fig. 3B, C).
ARTICLE IN PRESS
Fig. 3. Formation of insect-specic necine esters with insect-specic
necic acids, i.e. creatonotic acids and callimorphic acids (A). (B)
Formation of 1,2-dihydro derivatives from plant-acquired platynecine,
and (C) formation of 7-deoxy-1,2-dihdyro derivatives from plant
acquired trachelanthamidine and insect-specic 7-hydroxylation of
trachelanthamidine yielding turneforcidine.
Table 3
Pyrrolizidine alkaloid proles established by GCMS for G. geneura that as larvae (penultimate instar) had received about 2 mg per individual of an
alkaloid mixture derived from in vitro cultivated Parsonsia laevigata plantlets added to the articial diet
Pyrrolizidine alkaloids recovered from insects m/z [M
+
] RI Relative abundance (%)
Diet Larvae (n 2) Males (n 4) Females (n 3)
Plant acquired alkaloids
14-Deoxyparsonsianine 425 2773 23 45.077.0 35.776.5 44.371.2
14-Deoxyparsonsianidine 439 2860 61 52.574.5 22.575.9 38.070.6
Heterophylline
a
453 2920 5 1.571.5
Parsonsianidine 455 2935 7
17-Methylparsonsianidine
a
469 2993 3
Creatonotines
Creatonotine B 269 1973 Tr 2.371.3 0.470.3
Callimorphines
Deacetylcallimorphine 255 1821 1.070.99 1.070.6
Callimorphine 297 1955 14.574.8 8.771.3
Homocallimorphine 341 2033 23.376.7 6.770.9
Total alkaloids (mg/individual) 37.2736.8 14.374.0 33.078.2
Total alkaloids (mg/g dry wt) 0.370.3 0.1170.07 0.270.06
a
Tentatively identied
T. Hartmann et al. / Insect Biochemistry and Molecular Biology 35 (2005) 10831099 1091
3.4. Metabolism of retronecine and heliotridine:
formation of creatonotines and callimorphines
To study the specicity and temporal sequence of the
formation of insect-specic necine esters, retronecine
and heliotridine were fed with larval diet to G. geneura.
The results are summarized in Table 7. Pupae of
individuals that as larvae received retronecine contain,
besides a small proportion of residual retronecine, the
full set of creatonotines (Fig. 2A) but not even traces of
ARTICLE IN PRESS
Table 4
Proles of the pyrrolizidine alkaloids established by GCMS for G. geneura that as larvae (penultimate instar) had received about 1 mg per individual
of the indicated plant derived alkaloid mixtures added to the articial diet
Alkaloids recovered m/z [M
+
] RI Relative abundance (%)
Alkaloid mixture from Eupatorium cannabinum Alkaloid mixture from Heliotropium indicum
Diet Larvae
n 4
Males
n 4
Females
n 2
Diet Larvae
n 2
Males
n 1
Females
n 5
Plant acquired alkaloids
Supinine 283 1967 8 5.070.4
Amabiline 283 1972 Tr 5.872.2
Indicine 299 2120 88 83.572.5 64 50.873.6
Intermedine 299 2131 3 1.870.6
Lycopsamine 299 2145 1 1.871.2 32.5713.9 3575 12 15.071.0 9 8.270.7
Rinderine 299 2151 60 36.576.6 Tr
Echinatine 299 2164 19 42.874.4 30.5711.2 2.570.5
3
0
-Acetylindicin 341 2182 Tr Tr 15 27.873.4
3
0
-Acetylrinderine 341 2210 9
7
0
-Acetyllycopsmaine 341 2210 5.071.8 0.670.2
7
0
-Acetylechinatine 341 2228 6.571.7 2.570.7 0.370.2
3
0
-Acetyllycopsamine 341 2239 Tr 2.370.5 7.570.5 Tr 1.570.4
3
0
-Acetylechinatine 341 2269 1.470.7 0.470.2
Creatonotines
Estigmine B 253 1830 Tr 0.870.3
Creatonotine A 255 1880 Tr
Creatonotine B 269 1973 Tr
Callimorphines
Isodeacetylcallimorphine 255 1814 0.370.1 1.071.0
Deacetylcallimorphine 255 1822 1.570.3 5.070
Callimorphine 297 1955 20.572.4 40.571.5 Tr 9 9.070.52
Homocallimorphine 5.372.4 5.573.5 3 1.670.4
Total alkaloid (mg/individual) 75.8716.5 47.378.5 58.5720.5 186759 105 165722
Total alkaloid (mg/g dry wt) 0.3370.09 0.3570.12 0.3570.15 1.1870.42 0.9 0.9870.09
Table 5
Pyrrolizidine alkaloid proles established by GCMS for G. geneura that as larvae (penultimate instar) had received about 1 mg per individual of
sarracine/sarracinine added to the articial diet
Pyrrolizidine alkaloids recovered from insects m/z [M
+
] RI Relative abundance (%)
Diet Larvae (n 2) Males (n 7) Females (n 1)
Plant acquired alkaloids
Sarracine 337 2390 95 56.072.0
Sarracinine 337 2401 5 10.1710.0
9-Angeloylplatynecine 239 1842 34.078.0
Creatonotines
(1S)-1,2-Dihydrocreatonotine A 257 1923 Tr Tr Tr
(1S)-1,2-Dihydrocreatonotine B 271 2032 Tr 11.974.2 Tr
Callimorphines
(1S)-1,2-Dihydrocallimorphine 299 2016 54.475.6 60
(1S)-1,2-Dihydrohomocallimorphine 313 2097 30.076.0 30
7-Chlormethoxy-(1S)-1,2-dihydrocallimorphine
a
347 2207 2.571.9 8
7-Chlormethoxy-(1S)-1,2-dihydrohomocallimorphine
a
361 2282 Tr 3
Total alkaloid (mg/individual) 7.775.3 6.872.5 27
Total alkaloid (mg/g dry wt) 0.03570.025 0.06170.023 0.17
a
Most likely artifacts generated during extraction with dichloromethane.
T. Hartmann et al. / Insect Biochemistry and Molecular Biology 35 (2005) 10831099 1092
callimorphines. In contrast male and female adults were
found to contain the full set of callimorphines (Fig 2B)
and a reduced level of creatonotines. A comparison of
the absolute amounts of the two classes of insect-specic
retronecine esters clearly conrms that the callimor-
phines in adults must have been synthesized at the
expense of the creatonotines (Fig. 5).
Feeding of heliotridine with the larval diet revealed
the full pattern of callimorphines in adult males and
females. However, in the case of callimorphine and
homocallimorphine, in addition to the respective retro-
necine esters, two isomers with different RIs but
identical mol masses and mass fragmentation pattern
were detected and tentatively identied as the respective
7(S)-congurated esters called 7(S)-callimorphines (Fig.
2C). In the case of deacetylcallimorphine that, however,
account for less than 10% of the total callimorphines
only a single peak with an RI identical to the 7(R)-
congurated compound was detected, indicating either
insufcient resolution or absence of 7(S)-deacetycalli-
morphine. In males and females 78% and 48%,
respectively, of total alkaloids accounted for 7(R)-
callimorphines. Hydrolysis of total callimorphines of
both male and females and GC-MS of the resulting
necine bases revealed 69% retronecine and 31%
heliotridine. These proportions are similar to the 66%
retronecine and 34% heliotridine calculated from the
GC-MS data documented in Table 7.
The total amount of insect-specic pyrrolizidine alka-
loids recovered from adults is approximately vefold
higher in the retronecine experiment (Table 7) indicating a
less efcient utilization of heliotridine. In both experiments
females accumulated somewhat higher total amounts than
males but due to their higher body weight the alkaloid
concentrations was almost the same for both sexes.
3.5. Pyrrolizidine alkaloid analysis in exuviae of eld
collected larvae
Exuviae from eld-collected caterpillars varied in
their pyrrolizidine alkaloid content (Table 8). At one
ARTICLE IN PRESS
Table 6
Pyrrolizidine alkaloid proles established by GCMS for G. geneura that as larvae (penultimate instar) had received about 1 mg per individual of a
puried alkaloid mixture derived from a Phalaenopsis hybrid added to the articial diet
Pyrrolizidine alkaloids recovered from insects m/z [M
+
] RI Relative abundance (%)
Diet Males n 2 Females n 1
Plant acquired alkaloids
T-Phalaenopsine (necine base trachelanthamidine, with 1(R)-conguration) 361 2522 81 Nd Nd
Is-Phalaenopsine (necine base isoretronecanol, with 1(S)-conguration) 361 2560 19 Nd Nd
Creatonotines
7-Deoxy-(1R)-1,2-dihydrocreatonotine A (necine base trachelanthamidine) 241 1674 5 8
7-Deoxy-(1R)-1,2-dihydrocreatonotine B (necine base trachelanthamidine) 255 1822 12.572.5 Tr
Callimorphines
7-Deoxy-(1R)-1,2-dihydrocallimorphine (necine base trachelanthamidine) 283 1833 20.578.5 31
7-Deoxy-(1R)-1,2-dihydrohomocallimorphine (necine base trachelanthamidine) 297 1913 Tr Tr
(1R)-1,2-Dihydrocallimorphine (necine base turneforcidine) 299 1975 39.579.5 37
(1R)-1,2-Dihydrohomocallimorphine (necine base turneforcidine) 313 2053 22.071.0 25
Total alkaloids (mg/individual) 8.571.5 5
Total alkaloids (mg/g dry wt) 0.1270.02 0.03
Nd not detected; Tr traces
Fig. 4. GCMS analysis of (1S)-1,2-dihydrocallimorphine (necine
base: platynecine) obtained from G. geneura adults that had received
sarracine with their larval diet (A). Analysis of (1R)-1,2-dihydrocalli-
morphine (necine base: turneforcidine) obtained from G. geneura
adults that had received the orchid alkaloid phalaenopsine with their
larval diet (B).
T. Hartmann et al. / Insect Biochemistry and Molecular Biology 35 (2005) 10831099 1093
eld site (A) all 10 caterpillars were devoid of alkaloids,
at two eld sides (E and F) alkaloid-containing and
alkaloid-free caterpillars were found, and at three sites
(B, C, D) all specimens were found to have pyrrolizidine
alkaloids. The characteristic alkaloid patterns of the
alkaloid-positive individuals clearly indicated the kind
of pyrrolizidine alkaloid source: either S. longilobus
(Asteraceae) or Plagiobothrys arizonicus (Boraginaceae)
(Hartmann et al., 2004b). In one case, eld site B, two
individuals with trace amounts of creatonotine B as
exclusive alkaloids were found. In addition to the
summer annual, Crotalaria pumila, which was not yet
present at the time of sampling (March-April), S.
longilobus and P. arizonicus were the only two pyrroli-
zidine alkaloid-containing species found at the sites of
sampling.
4. Discussion
4.1. Larvae of G. geneura are adapted to exploit any
potential plant pyrrolizidine alkaloid source
In a previous study we demonstrated that the arctiid
E. acrea is well adapted to recruit pyrrolizidine alkaloids
from almost any plant source. The ingested alkaloids are
detoxied by N-oxidation, stored and partially trans-
formed into insect-specic creatonotines, the female-
specic creatonotine diesters (i.e., platyphorines) and
the male-specic mating pheromone hydroxydanaidal
(Hartmann et al., 2005). G. geneura shows the same
ARTICLE IN PRESS
Table 7
Metabolism of retronecine and heliotridine by G. geneura. Each individual (penultimate instar) received 1 mg retronecine or heliotridine with the
larval diet. Pupae and adults were sexed before analysis; m males, fm females. Pupae were preserved within 48 h after begin of pupation
Alkaloid recovered m/z[M
+
] RI Relative abundance (%)
Retronecine Heliotridine
Pupae (m)
n 2
Pupae (fm)
n 3
Adults (m)
n 9
Adults (fm)
n 11
Adults (m)
n 5
Adults (fm)
n 3
Retronecine 155 1425 5.575.5 11.073.1 0.570.4
Heliotridine 155 1445
Creatonotines
Isocreatonotine A 255 1857 2.070.6
Creatonotine A 255 1878 3.770.9
Isocreatonotine B 269 1955 33.070 27.771.7 0.770.7 0.0670.05
Creatonotine B 269 1981 61.575.5 55.772.7 16.471.9 10.472.8
Total creatonotines 100 100 17.272.1 10.472.8
Callimorphines
Isodeacetylcallimorphine 255 1818 1.270.3 1.870.2 0.470.4 0.370.3
Deacetylcallimorphine 255 1825 5.970.5 6.970.8 3.271.5 3.372.4
Callimorphine 297 1956 69.871.6 75.272.7 41.4710.6 26.3711.3
(S)-Callimorphine 297 1986 16.276.1 46.7714.9
Homocallimorphine 311 2036 5.970.7 3.970.7 33.076.3 17.775.4
(S)-Homocallimorphine 311 2060 5.675.6 5.072.5
Total (R)-callimorphines 82.972.0 89.372.8 78.0710.2 48.3717.4
Total (S)-callimorphines 21.8710.4 51.7717.4
Total alkaloid (mg/individual) 32.075.0 70.674.5 56.377.7 97.0711.9 10.973.3 19.277.4
Total alkaloid (mg/g dry wt) 0.270 0.3770.03 0.6270.09 0.5670.09 0.1270.03 0.1270.05
Fig. 5. Recovery of creatonotines and callimorphines from sexed
pupae and adults of G. geneura that had received retronecine with their
larval diet. Pupae were preserved within 48 h after begin of pupation.
Within sexes the amounts of creatonotines were signicantly different
between pupae and adults, males P 0:00123, females P o0:0001
(t-test); the respective values of total insect pyrrolizidine alkaloids were
not signicantly different.
T. Hartmann et al. / Insect Biochemistry and Molecular Biology 35 (2005) 10831099 1094
general adaptations: (i) recognition of pyrrolizidine
alkaloid-containing plants through phagostimulatory
taste receptor neurons specically dedicated to the
perception of pyrrolizidine alkaloids (Bernays et al.,
2002b); (ii) detoxication of ingested alkaloids by
specic N-oxidation indicating the presence of senecio-
nine N-oxygenase which appears to be present in any
arctiid adapted to pyrrolizidine alkaloids (Lindigkeit et
al., 1997; Naumann et al., 2002); (iii) partial or complete
hydrolysis of the various types of plant-acquired
pyrrolizidine alkaloids and subsequent transformation,
sometimes modication, of the resulting necine bases
into insect-specic alkaloids (Fig. 6).
The specicity of uptake and biochemical processing
of plant acquired pyrrolizidine alkaloids by G. geneura
largely corresponds to the pattern established for E.
acrea but also shows distinctive differences. Macrocyclic
retronecine diesters and triesters (Fig. 1) as those found
in species of the Asteraceae (tribe Senecioneae), the
Fabaceae (Crotalaria) and the Apocynaceae are seques-
tered and transmitted to adults in the same manner as
shown for E. acrea. The same accounts for alkaloids of
the prominent lycopsamine type (Fig. 1) found in
alkaloid-containing species of the Asteraceae (tribe
Eupatorieae), the Boraginaceae and some Apocynaceae.
A difference between the two arctiid species exists in
their ability to epimerize heliotridine, the 7S-epimer of
retronecine. Adults that as larvae had received helio-
tridine contain between about 2050% as insect-specic
heliotridine esters (Table 7) while in E. acrea heliotridine
was always completely epimerized (Hartmann et al.,
2005) yielding exclusively retronecine esters. A simple
explanation for this difference could be that E. acrea
males need an efcient 7S-epimerization for a proper
courtship pheromone biosynthesis since hydroxydanai-
dal has 7R-conguration (Schulz et al., 1993) while this
requirement does not apply for G. geneura.
In contrast to all tested macrocyclic ester alkaloids
and open-chain esters of the lycopsamine type, various
other open-chain esters (i.e., 9-angeloylplatynecine,
sarracine and phalaenopsines) are sequestered by larvae
but only transmitted to the adult life-stage after trans-
esterication into insect-specic pyrrolizidine alkaloids.
In the course of this trans-esterication G. geneura was
shown to convert a major proportion of the trache-
lanthamidine moiety of the orchid alkaloids into its 7-
hydroyl derivative (e.g., turneforcidine moiety). Thus,
the insect is not only able to epimerize the 7-hydroxyl
group but even to introduce it into the molecule. The
mechanism of this hydroxylation awaits elucidation.
Interestingly, E. acrea is not able to catalyze this
reaction, although it utilizes platynecine esters as
pheromone precursors (Hartmann et al., 2005).
ARTICLE IN PRESS
Table 8
Pyrrolizidine alkaloids in the exuviae of eld caught larvae (penultimate instar) of G
Parameter A n 10 B n 2 C n 12 D n 11 E n 9 F n 12
Pyrrolizidine alkaloids
mg / individual Nd 0.4570.15 5.1670.85 1.8470.60 2.6270.87 0.7070.10
mg / g dry weight 0.04370.018 0.53670.078 0.41170.105 0.17770.060 0.06070,010
Individuals with traces of alkaloids 0 0 0 0 2 4
Individuals devoid of alkaloids 10 0 0 0 1 6
Type of alkaloid prole Creatonotines Senecio Senecio Plagiobothrys Plagiobothrys
The eld sites A to F in south-eastern Arizona and date of sampling are: A Santa Rita Mountains, Gardner Canyon (20 March 2002); B Santa
Rita Mountains, Box Canyon (20 March 2002); C Patagonia Mountains, Harshaw Canyon (29 March 2002); D Patagonia Mountains,
Harshaw Road (7 April 2002); E Santa Catalina Mountains, Oracle (3 April 2002); F Rincon, Happy Valley (5 April 2002). The alkaloid
proles of the exuviae indicate larval host-plants, i.e. Senecio S. longilobus and Plagiobothrys P. arizonicus; in one case (B) only creatonotines
were detectable.
Percent of total alkaloids
0 20 40 60 80 100 120
Phalaenopsis sp.
Sarracine/sarracinine
Parsonsia laevigata
Heliotropium indicum
Eupatorium cannabinum
Senecio congestus
Senecio vernalis
Senecio jacobaea
Males
Females 12.5
9.9
2.1
10.4
4.6
4.2
17.4
12.6
5.5
5.8
27
6.8
5
8.5
13.1
30.4
Fig. 6. The percentage of insect pyrrolizidine alkaloids (creatonotines
plus callimorphines) of total pyrrolizidine alkaloids recovered from
adult females and males that as larvae had received various
pyrrolizidine alkaloid mixtures as indicated. Notice: Adults that as
larvae had received sarracine or phalaenopsine contain exclusively
insect alkaloids. The numbers alongside the columns give the
respective absolute amounts (mg) of insect alkaloids.
T. Hartmann et al. / Insect Biochemistry and Molecular Biology 35 (2005) 10831099 1095
4.2. Is insect-specic trans-esterication the answer of
polyphagous arctiids to cope with the structural diversity
of plant acquired pyrrolizidine alkaloids?
Both E. acrea and G. geneura are able to specically
esterify a variety of necine bases derived from plant
acquired pyrrolizidine alkaloids. This led to the dis-
covery of at least two classes of insect-made pyrrolizi-
dine alkaloids, the callimorphines and the creatonotines.
The callimorphines contain 2-hydroxy-2-methylbuty-
ric acid as basic necic acid (Fig. 3A). This acid moiety
occurs either free or acetylated (dominating derivative)
or propionylated (Hartmann et al., 2004b). These three
callimorphic acids are only found as the ester moiety of
arctiid-specic pyrrolizidine alkaloids (Fig. 2B). Calli-
morphine, the retronecine-O
9
-ester with the acetylated
callimorphic acid was rst described as pyrrolizidine
alkaloid-metabolite from pupae of Tyria jacobaeae
(Aplin et al., 1968). Later its structure was elucidated
(Edgar et al., 1980) and the biosynthesis from plant-
derived retronecine demonstrated in T. jacobaeae
(Ehmke et al., 1990). Callimorphine has been identied
in a number of arctiids: Arctia caja (Aplin and Roths-
child, 1972), Callimorpha dominula (Edgar et al., 1980),
Gnophaela latipennis (LEmpereur et al., 1989), Hyalur-
ga syma (Trigo et al., 1993) and Creatonotos transiens
(Wink et al., 1988; Hartmann et al., 1990).
The creatonotines, which contain in place of calli-
morphic acids 2-hydroxy-3-methylbutanoic acid (creato-
notine A) or 2-hydroxy-3-methylpentanoic acid
(creatonotine B, the major compound) (Fig. 3A), were
rst identied as insect alkaloids in C. transiens adults
that with their larval diet had received retronecine or a
plant-derived pyrrolizidine alkaloid mixture (Hartmann
et al., 1990). Creatonotine A and B are usually
accompanied by their O
7
-esters (isocreatonotines) (Fig.
2A). In E. acrea exclusively creatonotines are found
(Hartmann et al., 2004b; 2005); in C. transiens they are
accompanied by trace amounts of callimorphine (Hart-
mann et al., 1990). In both species creatonotines are
considered direct pheromone precursors (Schulz et al.,
1993; Hartmann et al., 2003a). In E. acrea it has been
demonstrated that all plant-acquired pyrrolizidine alka-
loids that after hydrolysis yield retronecine or platynecine
are pheromone precursors (Hartmann et al., 2005). The
same is true for heliotridine esters after C-7 epimeriza-
tion. In any case esterication with creatonotic acids
appears to be the committed step. Pheromone formation
in males occurs at the expense of previously synthesized
creatonotines (Hartmann et al., 2003a; 2004a). G.
geneura, not known to produce hydroxydanaidal, synthe-
sizes creatonotines from retronecine like E. acrea.
However, in E. acrea the creatonotines are already
synthesized in the larval stage (Hartmann et al., 2004a)
while in G. geneura they are rst observed in the pupal
stage (Table 7) (Hartmann et al., 2004b). The most
intriguing difference between the two arctiid species is
that G. geneura transforms most of its creatonotines into
callimorphines during transition from the pupal to the
adult stage (Table 7, Fig. 5). In T. jacobaeae,which does
not form creatonotines, callimorphine is not detectable
before the pupal stage (Aplin et al., 1968; Aplin and
Rothschild, 1972). Its biosynthesis appears to be re-
stricted to the very early stages of pupation. Callimor-
phine is rst detectable in pre-pupae (Ehmke et al., 1990).
Since in G. geneura the creatonotines are found in young
pupae but not larvae, we assume that they are synthesized
at the early stages of pupation, like the callimorphines in
T. jacobaeae. The conversion of the creatonotines into the
callimorphines, the major insect alkaloids in adults, by
trans-esterication most likely occurs just before eclosion,
but this needs to be conrmed.
The present study together with the results of previous
work with E. acrea (Hartmann et al., 2003a; 2004b;
2005) provides the rst evidence on the functional
importance of the insect-specic pyrrolizidine alkaloids.
Both arctiid species sequester as larvae all kinds of plant
pyrrolizidine alkaloids. Apparently only macrocyclic
pyrrolizidine alkaloids and open-chain esters of the
lycopsamine type are maintained through all life-stages,
while other pyrrolizidine alkaloids need insect-specic
trans-esterication before transfer to the pupal and
adult stages (see 4.1.). With the exception of the
otonecine derivatives all tested classes of pyrrolizidine
alkaloids are subjected to partial or total trans-
esterication (Fig. 6). Thus, the insect-specic trans-
esterication provides a means to recover and salvage all
kinds of necine bases from plant acquired pyrrolizidine
alkaloids, especially those that cannot be transmitted to
later life-stages. Moreover, in E. acrea insect-specic
trans-esterication is the essential step to create creato-
notines as common precursor for the formation of the
male pyrrolizidine alkaloid-signal hydroxydanaidal
from all kinds of sequestered pyrrolizidine alkaloids
including retronecine, heliotridine and platynecine esters
(Hartmann et al., 2003a; 2004a, b; 2005).
The insect-specic creatonotines and callimorphines
appear to represent the only necine monoesters that, in
addition to the plant acquired pyrrolizidine alkaloids of
the lycopsamine type, are maintained through all life-
stages. This implies that the insect-made necic acids of
these alkaloids have common structural features allowing
their stable maintenance and transmission between life-
stages. Indeed, common structural features between plant
monoesters of the lycopsamine type and the insect-specic
monoesters exist (Fig. 7): (i) they all represent aliphatic
branched-chain 2-hydroxy acids; (ii) in the callimorphines
and the plant necic acids this hydroxyl groups is tertiary
hydroxyl (Fig. 7B); (iii) the branching of the carbon
skeletons of all three types of necic acids display
similarities. The most conspicuous difference between the
plant-specic and the insect-specic necic acids is the
ARTICLE IN PRESS
T. Hartmann et al. / Insect Biochemistry and Molecular Biology 35 (2005) 10831099 1096
second hydroxyl group (at the 3-carbon) in the plant
acids. The stereochemistry of the 3-hydroxyl appears to be
important in plant-acquired alkaloids since in G. geneura
adults only (3S)-congurated monoesters are maintained,
i.e. lycopsamine, echinatine and indicine (Fig. 1B).
Rinderine the major pyrrolizidine alkaloid in E. cannabi-
num has (3R), (7S)-conguration. It is epimerized in both
positions yielding lycopsamine (Fig. 1B). Since the
inversion of conguration at C-7 is not total in G. geneura
(Table 7), echinatine accumulates in addition to lycopsa-
mine (Table 4). Epimerization of (3R)- and (7S)-
congurated alkaloids of the lycopsamine type in arctiids
is not unique. It has also been demonstrated in ithomiine
butteries, which as adults imbibe pyrrolizidine alkaloids
of the lycopsamine type mainly from Eupatorium and
Heliotropium species (Trigo et al., 1996). Although the
butteries sequester all kinds of lycopsamine stereoisomers
(see Fig. 1B) they maintain almost exclusively lycopsa-
mine. The reason for this is their ability to efciently
epimerize (3R)- and (7S)-congurated alkaloids (Trigo et
al., 1994). Even leaf-beetles of the neotropical genus
Platyphora, which are specialized on pyrrolizidine alka-
loids of the lycopsamine type, were found to convert
rinderine into intermedine and lycopsamine (Hartmann et
al., 2001). A pyrrolizidine alkaloid-sequestering Platyphora
clade radiated on single species of the three plant families,
Asteraceae tribe Eupatorieae, Apocynaceae and Boragi-
naceae (Termonia et al., 2002), which represent the only
families with species that contain pyrrolizidine alkaloids of
the lycopsamine type (Hartmann and Witte, 1995). Six
Platyphora species sequester pyrrolizidine alkaloids of the
lycopsamine type and concentrate them in the secretions of
their exocrine defense glands and all synthesize creatono-
tine A and few related mono and O
9
,O
7
-diesters with
insect-specic 2-hydroxy acids, e.g. lactic acid (Hartmann
et al., 2001; 2003b). The common pressure to invent a
necic acid that most properly meets the structural demands
of the necic acids of alkaloids of the lycopsamine type, to
which both arctiids and leaf-beetles are adapted, could be
the explanation for this intriguing biochemical conver-
gence. These mimics allow adapted insects to attain,
transmit and recycle necine bases from all kinds of
otherwise lost plant pyrrolizidine alkaloids. More experi-
mental evidence is needed to evaluate this general
hypothesis. Particularly, a complete elucidation of the
stereochemistry of the insect-made necic acids is required
for a precise structure-function comparison between plant
and insect necic acids. Moreover, additional feeding
experiments are needed to corroborate the assumed role
of the insect alkaloids.
4.3. Ecological aspects
As discussed above, G. geneura appears well adapted to
encounter and exploit any plant containing pyrrolizidine
alkaloids. Like E. acrea, Grammia larvae exploit alkaloid
plants primarily as a source for obtaining their chemical
defenses rather than for their use as foodmost feeding
generally occurs on plants without pyrrolizidine alka-
loids. Previous work shows that G. geneura larvae gain
resistance to parasitoids by eating a diet dominated by
the alkaloid-containing Senecio longilobus (Singer et al.,
2004a). This anti-parasitoid resistance was positively
associated with the concentration of sequestered pyrro-
lizidine alkaloids (Singer et al., 2004a). However, the
defensive benet of a diet dominated by Senecio comes at
the cost of reduced larval growth efciency (Singer et al.,
2004a). This same trade-off is demonstrated more clearly
in similar experiments with E. acrea (Singer et al., 2004b),
for which pyrrolizidine alkaloids themselves do not
appear to reduce larval performance (Hartmann et al.,
2005). We therefore suspect that G. geneura performance
is not negatively affected by the pyrrolizidine alkaloids,
but by other characteristics of Senecio. If true, this would
echo the nding in E. acrea that these caterpillars are
adapted to use pyrrolizidine alkaloid plants more as a
source of drugs than of high quality food.
Pyrrolizidine alkaloid-containing plants, such as
Senecio, Crotalaria, and Plagiobothrys, may be relatively
uncommon in the habitat (Singer and Stireman, 2001).
As such, G. geneura caterpillars were expected to vary in
the type and concentration of pyrrolizidine alkaloids
obtained from host plants. Indeed, this expectation was
supported in the present study by the analysis of exuviae
from eld-collected larvae (Table 8). Little can be said
about the possible role of pyrrolizidine alkaloids in G.
geneura courtship because nothing is known about the
mating behavior of this species. However, due to the
ARTICLE IN PRESS
Lycopsamine Type Creatonotine A Creatonotine B
N
O
H
O
OH
HO
OH
N
O
H
O
OH
HO
N
O
H
O
OH
HO
N
O
H
O
HO
O
O
Homocallimorphine
N
O
H
O
OH
HO
Deacetylcallimorphine
N
O
H
O
HO
O
O
Callimorphine
N
O
H
O
OH
HO
OH
Lycopsamine Type
(A)
(B)
Fig. 7. Structural similarity between the necic acid moiety of
pyrrolizidine alkaloids of the lycopsamine type and the insect-made
necic acids of the creatonotines and callimorphines. Structural
congruence is given in (red). The stereochemistry of the necic acids is
not given since it is still unknown for the callimorphines and needs to
be conrmed for the creatonotines.
T. Hartmann et al. / Insect Biochemistry and Molecular Biology 35 (2005) 10831099 1097
uncertainty of acquiring pyrrolizidine alkaloids during
the larval stage, we expect the alkaloids to be transferred
from males to females during mating and incorporated
into eggs of the offspring as in E. acrea (Hartmann et
al., 2004a). This adult transfer of alkaloids allows a
female to gain pyrrolizidine alkaloids even if she did not
acquire them as a larva.
The present study suggests that a wide variety of
structural types of pyrrolizidine alkaloids are likely to be
functional in the ecological contexts described above.
1,2-Dihydropyrrolizidine alkaloids are assumed to be
non-toxic, nevertheless they are sequestered and main-
tained by G. geneura either per se (e.g., platyphylline) or
after insect-specic trans-esterication (e.g., sarracine).
E. acrea converts (aromatizes) the platynecine moiety to
hydroxydanaidal, whereas G. geneura even creates the
platynecine isomer turneforcidine (Fig. 3) by 7-hydro-
xylation. Obviously even the so-called non-toxic pyrro-
lizidine alkaloids are valuable for both insects. If we
speak of toxic pyrrolizidine alkaloids we restrict toxicity
to metabolic bioactivation of 1,2-unsaturated pyrrolizi-
dine alkaloids resulting in pyrrolic intermediates re-
sponsible for the well known cell toxicity, mutagenicity
and genotoxicity of pyrrolizidine alkaloids for verte-
brates and insects (Mattocks, 1986; Frei et al., 1992; Fu
et al., 2004; Hartmann et al., 2005). Probably pyrroli-
zidine alkaloids with 1,2-saturated necine bases possess
still unknown biological activities which are advanta-
geous for sequestering insects. There is only one report
indicating deterrent properties of 1,2-saturated pyrroli-
zidine alkaloids (Reina et al., 1997). In this context it is
important to recall that there are plant taxa, like
pyrrolizidine alkaloid-containing orchids (Hartmann
and Witte, 1995) or pyrrolizidine alkaloid-containing
Ipomoea species (Convolvulaceae) (Jenett-Siems et al.,
1998), that produce exclusively esters of 1,2-saturated
necine bases. If pyrrolizidine alkaloid-adapted larvae
sequester these pyrrolizidine alkaloids and specically
convert them into insect-specic pyrrolizidine alkaloids
by trans-esterication that can be maintained and
transmitted to all life-stages (see 4.2.) a functional
importance of these pyrrolizidine alkaloids is likely.
Acknowledgements
This work was supported by grants of the Deutsche
Forschungsgemeinschaft and Fonds der Chemischen
Industrie to T.H., and by the Center for Insect Science
(U. Arizona) through NIH Training Grant # 1 K12
Gm00708.
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tionary signicance. J. Chem. Ecol. 20, 28832899.
Trigo, J.R., Brown, K.S., Henriques, S.A., Barata, L.E.S., 1996.
Qualitative patterns of pyrrolizidine alkaloids in ithomiine
butteries. Biochem. Syst. Ecol. 24, 181188.
Weller, S.J., Jacobson, N.L., Conner, W.E., 1999. The evolution of
chemical defences and mating systems in tiger moths (Lepidoptera:
Arctiidae). Bot. J. Linn. Soc. 68, 557578.
Wink, M., Schneider, D., Witte, L., 1988. Biosynthesis of pyrrolizidine
alkaloid-derived pheromones in the arctiid moth, Creatonotos
transiens: stereochemical conversion of heliotrine. Z. Naturforsch.
43c, 737741.
Witte, L., Ehmke, A., Hartmann, T., 1990. Interspecic ow of
pyrrolizidine alkaloids; from plants via aphids to ladybirds.
Naturwissenschaften 77, 540543.
Witte, L., Rubiolo, P., Bicchi, C., Hartmann, T., 1993. Comparative
analysis of pyrrolizidine alkaloids from natural sources by gas
chromatography-mass spectrometry. Phytochemistry 32, 187196.
Yamamoto, R.T., 1969. Mass rearing of the tobacco hornworm II.
Larval rearing and pupation. J. Econ. Entomol. 62, 14271431.
ARTICLE IN PRESS
T. Hartmann et al. / Insect Biochemistry and Molecular Biology 35 (2005) 10831099 1099
Insect
Biochemistry
and
Molecular
Biology
Insect Biochemistry and Molecular Biology 35 (2005) 11001111
Molecular characterization and evolution of pheromone binding
protein genes in Agrotis moths
David Abraham, Christer Lo fstedt, Jean-Franc- ois Picimbon
g
/
m
i
n
)
V
(
n
m
o
l
/
g
/
m
i
n
)
9 5
3 7 11
pH
9 5
WT
D306N
E310Q
D306NE310Q
(B)
(C)
(A)
Fig. 3. pH proles of wt and mutant forms of BmChi-h. (A) Amino
acid sequences of active sites. The substituted amino acid residues are
shown in bold. (B and C) Comparison of pH proles for D306N (n),
E310Q (&), and D306NE310Q (x) with wt BmChi-h (K). Chitinase
activities were determined over the range of pH 4.011.0 using MU-
(GlcNAc)
2
(B) and MU-(GlcNAc)
3
(C) as substrates. The points
indicate the mean7SD (n 3).
T. Daimon et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11121123 1116
SMART RACE cDNA Amplication Kit (BD Bio-
sciences) following the manufacturers protocol. Clon-
ing of PCR products and DNA sequencing were
performed as described previously (Daimon et al., 2003).
2.7. Sequence comparison and phylogenetic analysis
The nucleotide sequences of the ORFs of bacterial-
type chitinase genes were aligned using the CLUSTALX
program (Thompson et al., 1997), and the nucleotide
differences (K) at both synonymous (K
S
) and nonsynon-
ymous sites (K
N
) were calculated using the MEGA
program version 2.1 (Kumar et al., 2001). Phylogenetic
analysis was conducted as previously described (Daimon
et al., 2003). Briey, the amino acid sequences of
catalytic domains were aligned with the CLUSTALX
program (Thompson et al., 1997), and phylogenetic
trees were constructed with the neighbor-joining meth-
ods using the MEGA program version 2.1 (Kumar
et al., 2001).
ARTICLE IN PRESS
Fig. 4. Western blot and immunohistochemistry of BmChi-h. (A) Western blot of BmChi-h. Proteins were extracted from fatbody (FB), midgut
(MG), integument (I), hemolymph (H), and molting uid (MF) of B. mori larvae at the feeding and molting stages and analyzed by Western blot. (B,
C, and D) Immunohistochemistry of BmChi-h. Cross sections at the feeding (B) and molting stages (C and D) were used. The bright-green
uorescence indicates the presence of BmChi-h, and the nucleus is counter-stained with DAPI. M, muscles; EP, epidermal cells; T, trachea; NC, new
cuticle; OC, old cuticle; MF, molting uid. The bar indicates 50 mm.
T. Daimon et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11121123 1117
2.8. Sequence deposition
The nucleotide sequences have been deposited in the
DDBJ/GenBank/EMBL database with the following
accession numbers: ApChi-h, AB201279; ScChi-h,
AB201280; OfChi-h, AB201281; AcChi-h, AB201282;
and PsChi-h, AB201283.
3. Results
3.1. Expression and purication of BmChi-h protein using
a baculovirus expression system
To characterize the substrate specicity, recombinant
His-tagged BmChi-h protein was expressed using a
baculovirus expression system (Fig. 1). The protein of
approximately 58 kDa was puried by nickel-chelating
chromatography (Fig. 1A). The presence of the His-tag
sequence was conrmed by Western blot (Fig. 1B). The
N-terminal sequence of this puried protein corre-
sponded to that of BmChi-h but started at the residues
of Gly 21, indicating that the signal peptide of the
puried protein was cleaved (Fig. 1C). Chitinase activity
was measured by zymogram analysis (Fig. 1D). After
electrophoresis in native polyacrylamide gels containing
the glycol chitin, the gels were incubated to allow
substrate hydrolysis by chitinases and then stained with
a uorescent dye that binds the substrate. Brightly
uorescent areas represent regions with no active
enzyme, whereas dark areas represent regions where
enzyme activity degraded the substrate. Chitinolytic
activity was observed in the puried protein and
S. marcescens chitinase (Sigma) whereas it was not
observed in the BSA (Fig. 1D), indicating that BmChi-h
possesses chitinolytic activity. Therefore, we used this
puried protein for further examination of the enzy-
matic properties.
3.2. Enzymatic properties of BmChi-h
The activity of BmChi-h was assessed with native
chitin oligosaccharide (GlcNAc)
16
and uorescent
chitin oligosaccharide MU-(GlcNAc)
13
(Fig. 2).
In the native chitin oligosaccharide assay (Fig. 2AC),
BmChi-h did not hydrolyze GlcNAc or (GlcNAc)
2
but
was able to hydrolyze longer substrates (GlcNAc)
36
(Fig. 2A and B). BmChi-h produced predominantly
(GlcNAc)
2
and very little GlcNAc (Fig. 2A and B).
Fig. 2C shows that BmChi-h primarily converts
(GlcNAc)
6
into (GlcNAc)
2
and (GlcNAc)
4
[subse-
quently converted to (GlcNAc)
2
]. These results indicate
that BmChi-h is an exochitinase. However, a small
amount of (GlcNAc)
3
was detected in the digestion
product of (GlcNAc)
6
(Fig. 2B and C), suggesting that
BmChi-h possesses some endochitinase activity.
The uorescent chitin oligosaccharide assay also
showed that BmChi-h did not have activity against
MU-(GlcNAc) but hydrolyzed MU-(GlcNAc)
23
(Fig. 2D). BmChi-h showed nine fold higher activity
against MU-(GlcNAc)
2
than MU-(GlcNAc)
3
, indicating
that BmChi-h is an exochitinase. This exo-type substrate
preference was similar to that of bacterial and baculo-
virus chitinases (Hawtin et al., 1995; Brurberg et al.,
1996; Shinoda et al., 2001) and different from BmChi-
tinase, which showed endo-type substrate preference
(Kramer and Muthukrishnan, 1997; Shinoda et al.,
2001; Merzendorfer and Zimoch, 2003).
The pH proles of BmChi-h were determined using
uorescent chitin oligosaccharide MU-(GlcNAc)
23
as
substrates (Fig. 3). To do this, the amino acid residues of
the chitinase active site were substituted as shown in
Fig. 3A. In the MU-(GlcNAc)
2
assay, it was shown that
wt BmChi-h kept 50% activity in a broad pH range
(4.08.0) with a pH optimum of 6.0 (Fig. 3B). In
contrast, the mutant D306N showed very low activity at
the acidic pH range and no activity at the neutral and
alkaline pH ranges. Neither E310Q nor D306NE310Q
showed any activity at any pH values. Similar results
were obtained in the MU-(GlcNAc)
3
assay (Fig. 3C).
3.3. Expression and localization of BmChi-h in vivo
Previously, Northern blot analysis demonstrated that
BmChi-h mRNA is expressed specically at the molting
stages and in chitin-containing tissues (Daimon et al.,
2003). To further investigate, the expression and
localization of the BmChi-h protein were examined at
the larval feeding stage and the larvallarval molting
stage. Western blot analysis using some tissues at the
ARTICLE IN PRESS
96
98
98
52
89 %
89 %
89 %
73 %
85 %
85 %
Homology
555
555
555
563
555
553
555
Size
(aa)
Difference (K)
K
N KS
0.102 0.681
0.108 0.749
0.0749 0.686
0.0642 0.739
0.0704 0.797
(Identity)
(vs. BmChi-h)
ApChi-h (Antheraea pernyi )
ApChi-h (Agrius convolvuli )
ScChi-h (Samia cynthia)
BmChi-h (Bombyx mori )
PsChi-h (Pseudaletia separata)
OfChi-h (Ostrinia furnacalis)
Serratia marcescens ChiA
Fig. 5. Sequence comparisons of bacterial-type chitinases from
lepidopteran insets and S. marcescens chiA. A phylogenetic tree was
constructed using the total protein sequences. The size of the predicted
protein (aa) and the sequence homology (%) to BmChi-h are shown.
The nucleotide differences at both synonymous (K
S
) and nonsynon-
ymous sites (K
N
) were calculated. Only the nucleotide differences to
BmChi-h are shown here.
T. Daimon et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11121123 1118
ARTICLE IN PRESS
Fig. 6. Alignment of the amino acid sequences of bacterial-type chitinases. BmChi-h orthologues were cloned from S. cynthia, A. pernyi, A.
convolvuli, P. separata, and O. furnacalis and designated as ScChi-h, ApChi-h, AcChi-h, PsChi-h, and OfChi-h, respectively. (A) Total amino acid
sequences were aligned with the ClustalX program and shaded using the Boxshade program. Serratia marcescens chiA (SwissProt: P07254) and
AcNPV chiA (SwissProt: P41684) were included for comparison. The positions at the degenerate primer are shown by arrow and the putative active
site is underlined.
T. Daimon et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11121123 1119
larval feeing stage and the larvallarval molting stage
showed that BmChi-h was expressed strongly in the
epidermis and molting uid during the molting stage but
was not expressed during the feeding stage (Fig. 4A). In
addition, an immunohistochemical study showed that
no signals were detected at the feeding stage (Fig. 4B),
whereas apparent signals were observed at the larval
larval molting stage (Fig. 4C and D). Strong signals for
BmChi-h were observed in old cuticle and molting uid,
and weak signals were observed in the cuticle of the
trachea. Taken together with the results of enzymolo-
gical studies, these results strongly indicated that
BmChi-h plays a role in chitin degradation at the
molting stages.
3.4. Cloning and sequence comparisons of the bacterial-
type chitinase genes from lepidopteran insects
To investigate whether bacterial-type chitinase genes
were present in the genome of lepidopteran insects other
than B. mori, we tried to clone the BmChi-h orthologues
from ve species of lepidopteran insects (Anthraea
pernyi, Saturniidae; Samia cynthia, Saturniidae; Agrius
convolvuli, Sphingidae; Pseudaletia separata, Noctuidae;
and Ostrinia furnacalis, Pyralidae). By 5
0
- and 3
0
-RACE
(A. convolvuli, P. separata, and O. furnacalis) or
genome-walking PCR (A. pernyi and S. cynthia),
bacterial-type chitinase genes were successfully cloned
from all ve lepidopteran species (Fig. 5).
Their predicted amino acid sequences shared exten-
sive identities with that of BmChi-h (85%89%) (Fig. 5).
The nucleotide differences at synonymous sites (K
S
)
were shown to be saturated ( 0.75) between chitinases
from different families. In contrast, the nucleotide
differences at non-synonymous sites (K
N
) were less than
0.1, suggesting that nucleotide substitutions at synon-
ymous sites have been limited by functional constraints
(Fig. 5). All of them were predicted to be a secretory
protein by the PSORT program (Nakai and Kanehisa,
1992). Domain structures including the active site
signature of family 18 chitinases (Henrissat, 1991,
1999) were also highly conserved (Fig. 6). Comparison
of domain structures showed that domain structures of
these bacterial-type chitinases were identical to that
of BmChi-h (Figs. 6 and 7). The genomic structure of
BmChi-h has shown that there was no intron in BmChi-
h, while three isoforms containing different 5
0
-UTR
generated from different promoters were present (Dai-
mon et al., 2003). The genome structures of all the
bacterial-type chitinase genes cloned in this report
seemed to be similar to that of BmChi-h. We found no
introns but identied several isoforms in 5
0
-UTR of
PsChi-h and OfChi-h (data not shown). Although the
biological signicance of 5
0
-UTR isoforms is not clear,
these data indicate that not only the encoded protein but
also the genome structure of the bacterial-type chitinase
gene are highly conserved among lepidopteran insects.
Phylogenetic analysis showed that these chitinase
genes of lepidopteran insects and baculoviruses be-
longed to bacterial lineages with a strong bootstrap
support, suggesting that they originated from bacteria
and have been maintained through evolution since they
transferred laterally (Fig. 8).
4. Discussion
In the silkworm genome project, a large-scale cDNA
database has been constructed (Mita et al., 2003), and
whole genome sequencing of B. mori has been
performed (Mita et al., 2004). An interesting nding is
that there are genes showing signicant homology with
bacterial or baculoviral genes (Mita et al., 2003;
Goldsmith et al., 2004). The BmChi-h gene, a bacter-
ial-type chitinase gene of B. mori, is one of them
(Daimon et al., 2003). In this study, we demonstrated
that BmChi-h encodes an exochitinase and plays a role
in chitin degradation at the molting stages.
Chitinases (EC 3.2.1.14) can be classied into two
major categories: endochitinases and exochitinases
(Robbins et al., 1988). Endochitinases cleave glycosidic
linkages randomly along the chitin chain and eventually
produce short oligomers of GlcNAc. Exochitinases, also
called chitobiosidase, cleave off chitobiose [(GlcNAc)
2
]
from (GlcNAc)
n
. The recombinant BmChi-h expressed
by a baculovirus expression system showed that BmChi-
h is a chitinolytic enzyme and a secretory protein
(Fig. 1). We propose that BmChi-h is an exochitinase for
the following reasons: (1) BmChi-h does not hydrolyze
(GlcNAc)
2
and MU-(GlcNAc), indicating that BmChi-h
is not a 14-b-N-acetylglucosaminidase (Fig. 2A, B
and D); (2) BmChi-h hydrolyzes MU-(GlcNAc)
2
faster
than MU-(GlcNAc)
3
(Fig. 2D); (3) when (GlcNAc)
6
is
used as a substrate, BmChi-h produces predominantly
(GlcNAc)
4
and (GlcNAc)
2
as an initial product
ARTICLE IN PRESS
Fig. 7. Schematic representation of the domain structures of bacterial-
type chitinases (BmChi-h and its orthologues) and endochitinases of
lepidopteran insects (BmChitinase and its orthologues).
T. Daimon et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11121123 1120
ARTICLE IN PRESS
OpNPV
AcNPV
XcGV
ApChi-h
ScChi-h
AcChi-h
BmChi-h
OfChi-h
PsChi-h
S. marcescens ChiA
E. agglomerans
A. hydrophila
Alteromonas sp.
V. cholerae
S. plicatus
B. circulans
S. marcescens ChiB
C. immitis
A. album
T. harzianum
O. aries
B. taurus
H. sapiens
M. musculus
B. malayi
C. elegans
A. aegypti
B. mori
M. sexta 100
100
100
100
42
90
69
100
69
100
96
86
46
99
100
100
100
80
95
82
93
99
100
76
96
65
0.1
Baculoviruses
Lepidopteran
insects
Bacteria
Fungi
Animals
Fig. 8. Phylogenetic analysis of family 18 chitinases. The amino acid sequences of the catalytic domain (about 400 aa) were extracted and aligned
with the ClustalX program, and pair-wise distances were calculated based on the blosum matrix. The phylogenetic tree was constructed by the
neighbor-joining method using the MEGA2 program package. Bootstrap values after 1000 replications are shown. Sequence used for analysis:
Autographa californica nuclear polyhedrosis virus (SwissProt: P41684), Orgyia pseudotsugata multicapsid polyhedrosis virus (SwissProt: O10363),
Xestia c-nigrum granulovirus (AF162221), Serratia marcescens ChiA (SwissProt: P07254), Enterobacter agglomerans (U59304), Aeromonas hydrophila
(AF251793), Alteromonas sp. (SwissProt: P32823), Vibrio cholerae (PIR: D82510), Streptomyces plicatus Chi63 (SwissProt: P11220), Bacillus circulans
ChiA1 (SwissProt: P20533), S. marcescens ChiB (SwissProt: P11797), Coccidioides immitis (SwissProt: P54196), Aphanocladium album (SwissProt:
P32470), Trichoderma harzianum (SwissProt: P48827), Caenorhabditis elegans CHT-1 (SwissProt: Q11174), Brugia malayi (SwissProt: P29030), Mus
musculus CHI3L3 (SwissProt: Q61362), Ovis aries (SwissProt: Q28542), Bos taurus (SwissProt: Q28042), Homo sapiens CHI3L2 (SwissProt: Q15782),
Aedes aegypti (AF026491), M. sexta (SwissProt: P36362), and B. mori (AB048355).
T. Daimon et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11121123 1121
(Fig. 2C). This exo-type substrate preference is the same
as those of S. marcescens chiA and baculovirus
chitinases (Hawtin et al., 1995; Brurberg et al., 1996;
Shinoda et al., 2001) but different from that of
BmChitinase, which showed endo-type substrate pre-
ference (Kramer and Muthukrishnan, 1997; Shinoda et
al., 2001; Merzendorfer and Zimoch, 2003). The TLC
pattern for the reaction products of (GlcNAc)
6
showed
minor (GlcNAc)
3
spots in the course of the enzyme
reaction (Fig. 2B and C). This suggests that BmChi-h is
an exochitinase with some endochitinase activity.
Similarly, both S. marcescens chiA and baculovirus
chitinase have been demonstrated to possess some
endochitinase activity (Brurberg et al., 1996; Hawtin et
al., 1995). The chitinase gene has been cloned from
many lepidopteran insects and showed endo-type sub-
strate preference in all cases (Kramer et al., 1993;
Kramer and Muthukrishnan, 1997; Kim et al., 1998;
Mikitani et al., 2000; Shinoda et al., 2001; Zheng et al.,
2002; Merzendorfer and Zimoch, 2003). Therefore, this
is the rst report to describe the molecular cloning and
characterization of exochitinase from lepidopteran
insects.
In our previous studies, BmChi-h mRNA was
observed in a tissue- and stage-specic manner (Daimon
et al., 2003). BmChi-h mRNA was expressed specically
at chitin-containing tissues during the molting stage, and
its expression pattern was almost identical to that of
BmChitinase (Daimon et al., 2003). In Western blot
analysis, the BmChi-h protein was observed in the
integument and molting uid at the molting stage
(Fig. 4A), consistently with the result of Northern blot
analysis (Daimon et al., 2003). Immunohistochemical
analysis revealed that BmChi-h localized in the old
cuticle, molting uid, and trachea (Fig. 4BD). This
spatial and temporal expression pattern seems to be
closely similar to that of CfChitinase, a BmChitinase
orthologue from Choristoneura fumiferana (Zheng et al.,
2003). These data strongly indicate that both BmChi-h
and BmChitinase degrade the chitin of the exoskeleton
during the molting stage. As a strong synergic effect was
observed when chitinolytic enzymes of different sub-
strate specicity were combined (Fukamizo and Kra-
mer, 1985a, b; Brurberg et al., 1996; Suzuki et al., 2002),
BmChi-h (exochitinase) may catalyze the native chitin by
a concerted mechanism with BmChitinase (endochiti-
nase) and b-N-acetylglucosaminidase (EC 3.2.1.30)
(Fukamizo and Kramer, 1985a, b). Taken together,
these ndings lead to the conclusion that the biological
role of the bacterial-type chitinases of lepidopteran
insects is chitin degradation for molting.
To see whether the bacterial-type chitinase is encoded
by lepidopteran insects other than B. mori, we cloned
the BmChi-h orthologues from ve species of lepidop-
teran insects. The BmChi-h orthologues were success-
fully cloned from all species examined (Fig. 5). Sequence
analysis strongly indicated that all of them encoded
functional enzymes belonging to the family of 18
chitinases (Figs. 5 and 6). Since BmChi-h orthologues
were cloned from species of the superfamily Noctuoidea
(P. separata) and Pyraloidea (O. furnacalis), which are
phylogenetically distant from Bombycoidea, it is likely
that the bacterial-type chitinase gene is encoded by
broad taxa of lepidopteran insects. In the phylogenetic
analysis, the bacterial-type chitinase genes of lepidop-
teran lineages were shown to be monophyletic (Fig. 7).
Therefore, an ancestral lepidopteran species might have
acquired the chitinase gene from a bacterium or a
baculovirus.
The nucleotide substitutions at the ORFs of the
bacterial-type chitinase gene of lepidopteran insects
have already been saturated (Fig. 6B). Therefore, it
seems difcult to obtain denitive evidence for hor-
izontal transfer at this point. One possible way would be
to investigate the species distribution of bacterial-type
chitinase genes in the genome of broader taxa of
arthropods and viruses. Accumulating genome data of
microbes and viruses and, especially, the complete
genome sequences of honeybee and Tribolium, which
will be available in a few years, would provide further
clues in this respect.
Acknowledgements
We are grateful to K. Kiguchi (Shinshu University,
Japan), S. Matsumoto (RIKEN, Japan), and S. Tatsuki
(Univ. of Tokyo) for providing specimens of
A. convolvuli, P. separata, and O. furnacalis, respec-
tively. We also thank N. Omuro (Univ. of Tokyo) for
her technical assistance. This work was nancially
supported by PROBRAIN (T.S.), the Insect Technology
Project, MAFF (T.S.), and Grants-in-Aid for Scientic
Research, MEXT/JSPS (T.S. and T.D.).
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ARTICLE IN PRESS
T. Daimon et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11121123 1123
Insect
Biochemistry
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Insect Biochemistry and Molecular Biology 35 (2005) 11241132
Effect of chloroquine on the expression of genes involved in the
mosquito immune response to Plasmodium infection
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Table 1 (continued )
NCBI Id
number and link
Seq. size SigP result Cleavage
position
Mature MW pI Best match to non-redundant NCBI protein
database
E-value Identication
51011570 189 SIG 2021 19.028 4.92 Putative 18.7 kDa secreted protein [I 3E043 Putative 19 kDa secreted protein [Ixodes
pacicus]
Group 6Sequences similar to I. scapularis 5.3 kDa peptideunknown function
51011582 61 SIG 1819 4.283 9.38 Putative 5.3 kDa secreted protein [Ix 0.015 Similar to I. scapularis 5.3 kDa peptide
51011582 72 SIG 2930 4.284 9.38 Putative 5.3 kDa secreted protein [Ix 0.014 Similar to I. scapularis 5.3 kDa peptide
51011556 66 SIG 2223 4.991 9.59 Putative 5.3 kDa secreted protein [Ix 0.033 Similar to I. scapularis 5.3 kDa peptide
51011562 79 SIG 2223 6.314 9.57 ENSANGP00000017973 [Anopheles gambi 0.34 Similar to I. Scapularis 5.3 kDa peptide
Group 7Sequences similar to I. scapularis 9.4 kDa peptideunknown function
51011580 103 SIG 2021 9.57 8.23 Putative 9.4 kDa secreted protein [Ix 3E019 Similar to I. scapularis 9.4 kDa peptide
51011396 101 SIG 2021 9.416 8.46 Putative 9.4 kDa secreted protein [Ix 6E035 Similar to I. scapularis 9.4 kDa peptide
51011434 79 SIG 1819 6.779 5.19 Putative 7 kDa secreted protein [Ixod 2E034 Similar to I. scapularis 9.4 kDa peptide
Group 8Metalloprotease familyputative anti-hemostatic
51011584 344 CYT Truncated secreted metalloprotease [I 0.0 Truncated secreted metalloprotease
Group 9Ixodegrin family: disintegrinsputative anti-hemostatic
51011476 60 SIG 2122 4.173 6.47 HL01481p [Drosophila melanogaster] 33 1.8 1.8 Cys-rich
Group 10Ixostatin family: short-coding cysteine-rich peptides similarly found in ADAMST-4 (Thrombospondins)putative anti-hemostatic
51011550 115 SIG 1819 10.688 5.6 Thrombospondin [Ixodes scapularis] 80 1e014 1E014 Kunitz protease inhibitor domain
51011596 114 SIG 1718 10.821 5.6 Putative secreted protein [Ixodes sca 1E036 Kunitz protease inhibitor domain
Group 11Histamine binding proteinsputative anti-hemostatic
51011424 313 SIG 1718 33.149 5.08 Putative secreted histamine binding p 1E101 Putative secreted histamine binding protein
51011558 191 SIG 1819 19.53 4.49 Putative secreted histamine binding p 5E030 Putative secreted histamine binding protein
51011480 196 SIG 1819 19.708 5.66 Putative 22.7 kDa secreted protein [I 1E093 Putative secreted histamine binding protein
51011422 265 CYT Putative secreted histamine binding p 1E142 Truncated histamine binding protein
51011604 244 SIG 2021 25.901 8.85 Putative protein [Ixodes scapularis] 331 7e090 7E090 Putative secreted histamine binding protein
51011586 215 SIG 2021 22.52 8.71 Putative protein [Ixodes scapularis] 333 2e090 2E090 Putative secreted histamine binding protein
51011498 160 CYT Histamine binding protein [Ixodes sca 4E017 Truncated histamine binding protein
51011532 210 SIG 1617 22.767 9.35 Serotonin and histamine binding prote 2E005 Putative secreted histamine binding protein
51011410 214 SIG 2728 21.262 6.12 Putative 22.5 kDa secreted protein [I 0.79 Putative secreted histamine binding protein
51011416 193 SIG 19-20 20.018 6.16 Putative secreted protein [Ixodes sca 4E027 Putative secreted histamine binding protein
Group 12Neuropeptide-like protein with GGY repeatsputative anti-microbial
51011444 73 SIG 2324 4.758 9.63 Neuropeptide-like protein (nlp-31) 6E017 Reference
51011404 78 SIG 2324 5.416 9.7 Neuropeptide-like protein (nlp-31) 6E020 Reference
Group 13Oxidant metabolism
51011420 116 CYT Plasma glutathione Peroxidase [Homo sa 5E027 Truncated glutathione peroxidase
51011588 189 SIG 2324 17.439 7.15 Mn superoxide dismutase [Melopsittacu 8E051 Mn superoxide dismutase
Group 14Similar to other Ixodid proteins
51011484 119 SIG 1617 11.293 9.01 15 kDa salivary gland protein [Ixodes 5E026 Salp15 family
51011390 124 SIG 2021 11.314 4.66 15 kDa salivary gland protein [Ixodes 0.001 Salp15 family
51011590 154 SIG 1819 14.408 5.14 Salivary gland 16 kDa protein [Ixodes 1E031 Salp15 family
51011394 123 SIG 1819 11.19 6.26 Salivary gland 16 kDa protein [Ixodes 4E010 Domain 8 of human ADAMS
51011564 108 SIG 2425 9.089 8 16 kDa salivary gland protein A [Ixod 4E004 Some similarity with factor VII
51011592 178 SIG 2223 17.328 4.38 20 kDa salivary gland protein [Ixodes 7E065 Anti-complement, ISAC
51011398 119 SIG 2627 9.778 7.76 Is3 [Ixodes scapularis] 36 0.13 0.13 Similar to is3 protein
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51011402 240 SIG 1819 21.295 9.03 Hypothetical protein [Ixodes ricinus] 267
2e070
2E070 Possible cuticle or salivary duct protein
Group 15Novelunknown
51011472 87 SIG 2324 6.298 4.13 Insecticidal neurotoxin Tx4(5-5 0.75 Cys-rich, weak similarity to neurotoxin
51011482 120 SIG 2021 11.203 4.36 Nematocyst outer wall antigen precurs 0.077
51011392 163 SIG 3031 14.98 8.58 Probable K5 antigen synthesis [Vibrio 3.4
51011602 117 SIG 1920 11.113 9.59 AGR_C_2052p [Agrobacterium tumefaci 0.13
51011598 78 SIG 2324 5.358 4.9 OSJNBa0086B14.5 [Oryza sativa (japon 0.35 polyGly tailglue?
51011474 45 SIG 1920 2.496 4.84 COG3451: Type IV secretory pathwa 2.5 HEAHEAHEA protein
51011540 59 SIG 2223 4.117 7.92
51011566 122 BL 1920 11.717 9.83 Predicted protein [Ustilago maydis 521] 31 4.2 4.2 Unknown, possible Dopachrome tautomerase
precursor
51011384 124 SIG 2324 10.597 5.28 Keratin associated protein 18-7; ke 6E005 Very cys-richglue?
51011400 168 SIG 2122 15.407 6.54 Putative protein (4I100) [Caenorhab 0.003
51011594 47 SIG 3738 0.976 11 Hypothetical protein Tb927.2.4250 [ 1.5
51011478 99 SIG 1920 8.723 9.06 Hypothetical protein MGC63561 [Dani 4E025 Unknown conserved protein
51011426 54 SIG 2021 4.02 7.01 Hypothetical protein CBG15127 [Caeno 0.099
51011406 225 SIG 2324 22.54 4.92 Cytotoxin [Bacteriophage phi CTX] 4 6E004
51011600 205 SIG 1718 21.659 8.65 Similar to tenascin-N [Rattus norve] 5E020
51011418 196 SIG 2627 18.516 8.71 CG17035-PA [Drosophila melanogaster] 2E025 Group XIV secreted phospholipase A2
Group 16Probably housekeeping proteins
Other possible housekeeping proteins
51011496 74 CYT CG32446-PA [Drosophila melanogaster] 3E014 Copper transport protein
51011504 174 CYT CG3595-PA [Drosophila melanogaster] 5E080 Myosin regulatory light chain
51011578 147 CYT CG7013-PA [Drosophila melanogaster] 7E047 ARMET-like protein precursor, truncated
51011442 93 CYT CG7630-PA [Drosophila melanogaster] 0.030 Unknown
51011554 101 CYT Heat shock protein 10 [Gallus gallu 8E029 Heat shock protein 10
51011552 80 CYT Hypothetical protein Magn027998 [ 0.34 Unknown
51011386 265 CYT Isopentenyl-diphosphate delta-is 3E063 Isopentenyl-diphosphate delta-isomerase
51011506 232 CYT Similar to Shwachman-Bodian-Diamond 9E084 ShwachmanBodianDiamond syndrome
homolog
51011446 66 CYT Unnamed protein product [Tetraodon n 1.8
51011524 66 CYT Unnamed protein product [Tetraodon n 1.4
Kunitz-containing intracellular proteins
51011388 179 CYT Putative secreted protein [Ixodes sca 4E072 Truncated peptide with Kunitz protease
inhibitor domain
51011382 205 CYT Putative secreted protein [Ixodes sca 9E028 Truncated peptide with Kunitz protease
inhibitor domain
Ribosomal proteins
51011528 165 CYT CG3195-PA [Drosophila melanogaster] 1E068 Ribosomal protein
51011568 123 CYT Ribosomal protein L35 [Mus musculus 7E046 Ribosomal protein
51011502 100 CYT 60S Ribosomal protein L37 4gnl| 1E037 Ribosomal protein
51011508 105 CYT Ribosomal protein L44 [Chlamys farreri] 194
3e049
3E049 Ribosomal protein
51011522 268 CYT Ribosomal protein L6 [Gallus gallus 7E057 Ribosomal protein
51011548 90 CYT Ribosomal protein L7a [Argopecten irr 8E035 Ribosomal protein
51011530 112 CYT Ribosomal protein L30 [Argopecten irr 1E052 Ribosomal protein
51011534 269 CYT Unknown (protein for MGC:73183); wu 1E113 Ribosomal protein
51011526 151 CYT Ribosomal protein S14; wu:fa92e08 [ 8E072 Ribosomal protein
51011510 149 CYT Ribosomal protein S15 [Argopecten irr 2E068 Ribosomal protein
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Table 1 (continued )
NCBI Id
number and link
Seq. size SigP result Cleavage
position
Mature MW pI Best match to non-redundant NCBI protein
database
E-value Identication
51011516 25 CYT Ribosomal protein L41 [Mus musculus] 2E006 Ribosomal protein
51011518 81 CYT Ribosomal protein S4 [Argopecten irra 2E032 Ribosomal protein
51011520 114 BL Ribosomal protein, large P2 [Mus mu 3E037 Ribosomal protein
51011536 133 CYT FinkelBiskisReilly murine sarcoma 3E036 Ribosomal protein
51011494 209 CYT 40S ribosomal protein S5 [Dermacentor 1E110 Ribosomal protein
Oxidant metabolism
51011512 230 CYT Putative glutathione S-transferase [D 1E036 Glutathione S-transferase
51011500 220 CYT Glutathione S-transferase [Boophilus 1E080 Glutathione S-transferase
Vacuolar sorting protein?
51011408 222 CYT Neuroendocrine differentiation factor 1E073 Vacuolar sorting protein VPS24
Energy metabolism
51011608 109 CYT Cytochrome c 4gnl|BL_ORD_ID|145934 8E050 Cytochrome c
51011610 153 CYT Cytochrome c oxidase subunit Va [Rhyz 5E050 Cytochrome c oxidase subunit Va
51011612 73 CYT Cytochrome oxidase subunit VIIc [Mac 5E013 Cytochrome oxidase subunit VIIc
51011614 152 CYT ATP synthase c-subunit [Dermacentor v 1E065 ATP synthase c-subunit
51011616 134 CYT CG2140-PB [Drosophila melanogaster] 6E036 Cytochrome b5
51011618 69 CYT ENSANGP00000013087 [Anopheles gambi] 0.002 Cytochrome c oxidase polypeptide VIII
51011620 182 CYT CG9350-PA [Drosophila melanogaster] 2E010 Probable NADH-ubiquinone oxidoreductase
subunit
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On the other hand, some proteins from this family, such
as gi 22652868 and gi 22164158, display a negatively
charged tail composed of six glutamic acid (Glu, E)
anionic residues (Fig. 2A). The evolutionary relation-
ships of BTP were inferred by constructing the
phylogenetic tree using the NJ algorithm; a cladogram
is shown in Fig. 2B. Of interest, these proteins share
sequence similarities to exogenous anticoagulants such
as SALP 14 (gi 15428308) from I. scapularis. SALP 14 is
a FXa inhibitor that appears to interact with the
catalytic domain of FXa and with the so-called exosite
(Narasimhan et al., 2002). Exositesregions far from
the catalytic site and known to determine specicity and
afnity of blood coagulation factors toward sub-
stratesalso are critical for the assembly of the
prothrombinase, a multimolecular complex that leads
to thrombin generation (Krishnaswamy, 2005). Target-
ing these domains appears to be an effective strategy
evolved by blood-feeding arthropods to effectively
impair blood coagulation. In fact, we recently reported
that ixolaris, a FX(a) scaffold-dependent inhibitor
of Factor VIIa/tissue factor complex, specically
recognizes the FXa heparin-binding exosite (Monteiro
et al., 2005).
ARTICLE IN PRESS
(A)
gi|22652868| MGLTEIMLVL-VSLAFVATAAAHDCQNGTRPASEEKREGCDYYCWNTETKSWDKFFFGNGERCFYNNGDEGLCQNGECHLTTDSGVPNDTDAKIEETEEELEA-----------------------------------
gi|22164158| MGLTEIMLVL-VSLAFVATAAAHDCQNGTRPASEEKREGYDYYCWNTETKSWDKFFFGNGERCFYNNGDEGLCQNGECHLTTDSGVPNDTDAKIEETEEELEA-----------------------------------
gi|22164164| MGLTGTTLVL-VSLVFFGSAAAHNCKNGTRPASEENREGCDYYCWNDGTNSWDQFFFGNGEICFYNSGEKGICQNGECHLTNNSGGPNETDENTPATTEKPKQKKKKTKKPKKPKRKSKKDQ----------------
gi|22164176| MGLTGTTLVL-VSLAFFGSAAAHNCKNGTRPASEENREGCDYYCWNDGTNSWDQFFFGNGEICFYNSGEKGICQNGECHLTNNSGGPNETDDNTPAPTEKPKQKKKKPKKPKKPKRKSKKDH----------------
gi|15428308| MGLTGTMLVL-VSLAFFGSAAAHNCQNGTRPASEQDREGCDYYCWNAETKSWDQFFFGNGEKCFYNSGDHGTCQNGECHLTNNSGGPNETDDYTPAPTEKPKQKKKKTKKTKKPKRKSKKDQEKNL------------
gi|22164190| MGLTGTTLML-VCVAFFGTAAAHNCKNGTRPASEENREGCDYYCWNEVTNSWDQFFFGNGERCFYNTGENGKCQNGECHLTTNSDGPNETDDNTPPPTEKPK------------------------------------
gi|22164156| MGLTGTTLVL-VCVAFFGSAAAHNCQNGTRPASEENREGCDYYCWNEVTNSWDQFFFGNGERCFYNTGENGKCQNGECHLTTNSDGPNETDDNTPPPTEKPKQKKKKPKKPKKPKRKSKKDQ----------------
gi|22164184| MGLTGTTLVL-VCVAFFGTAAAHNCKNGTRPASEENREGCDYYCWNEVTNSWDQFFFGNGERCFYNTGENGKCQNGECHLTTNSGGPDDTDDNTPPPTEKPKQKKKKPKKPKKPKRKSKKDQ----------------
gi|22164186| MGLTGTTLVL-VCVAFFGSAAAHNCQNGTRPASEKNREGCDYYCWNAETKSWDQFFFGDGERCFYNTGENGTCRNGECHLTTSSGGPNETDDNTPPPTEKPKQKKKKPKKTKKPKRKSRKDQ----------------
gi|22164174| MGLTGTTLVL-VSLAFFGSAAAHNCQNGTRPTSEKNREGCDFYCWNADTNLWDKFFFGNGEKCFYNTGEKGTCLNGECHLTTSSGGPDDTGDNTPPPTEKPKQKKKKPKKTKKPKRKSKKDQKENF------------
gi|22164194| MGLTGTALVL-VSLAFFGSAAAHNCQNGTRPASEENREGCDYYCWNSETQSWDQYFFGDGERCFYNSGDRGICQNGECHLTTSSGGPDDTDENTPPPTEKPKQKKKKPKKTKEPKRKSKKD-----------------
IP_5_100_90_1_CLU MGLTGATLVL-VSLAFFGSAAAHNCKNGTRPASEENREGCDFYCWSTDTNSWEIFFFGNGEECFYNNGDRGTCQDGACHLTTHSGGPNETDDYTPAPTEKPKQKKKKPKKTKKPKRNSKKD-----------------
IP_clu3 MGLTGATLVQGVSLAFFGSAAAHNCKNGTRPASEENREGCDFYCWSTDTNSWEIFFFGNGEECFYNNGDRGTCQDGACHLTTHSGGPNETDDYTPAPTEKPEQKKKKPKKTKKPKRNTKKKKKKKKKKKNFLGPPGPH
IP_5_100_90_1_CLU1 MGLTGATLVL-VSLAFFGSAAAHNCQNGTRPASEENREGCDFYCWNTDTNSWDIFFFGNGEKCFYNNGDRGTCQDGACHLTTHSGGPNETDDYTPAPTEKPKQKKKKPKKTKKPKRKSKKD-----------------
IP_5_100_90_2A_CLU MGLTGITLVL-VSLAFFGSAAAHNCQNGTRPASEENREGCDFYCWNAGTNSWDIFFFGNGEKCFYNNGDRGTCQDGACHLTTHSGGPNETDDYTPAPTEKPKQKKKKPKKTKKPKRKSKKDKEG-----NF-------
IP_5_100_90_2_CLU MGLTGATLVL-VSLAFFGSAAAHNCKNGTRPASEETREGCDFYCWNTDTSSWDIFFFGNGEKCFYNNGDRGTCRDGACHLTTLSGGPNETDDYTSAPTEKPKQKKKKLKKTKKPKRKSKKD-----------------
gi|45593710| MGLTGTTLVL-ASLAFFGSAAAHNCKNGTRPASEEKREGCDFYCWNSDTSRCDQFFFRDGETCFYNNGDRGSCQNGECHLNTNSGVPTHNDDYTPSPTEKPKQKKKKPKKTKKPKRQSNKD-----------------
IP-7-60-92-2-CLU MGLTGITLVL-VSFAFFGSVAAHNCQNGTRPTSEQNREGCDYYCWNTDTKSWDQFFFGNGERCFYSNGDTGVCTNGECHLNTESSVPTETDVETPAPTKKPKQKKKKQKKTKKPKR----------------------
IP_5_100_90_25_CLU MGLTGITLVL-VSFAFFGSVAAHNCQNGTRPASEQNREGCDYYCWNTDTKSWDQFFFGNGERCFYSNGDTGVCTNGECHLNTESGVPTETDVETPAPTKKPKHKKKKQKKSKKPKG----------------------
IP_5_100_90_6_CLU MGLTGTTLVL-VSFAFFGSVAAHNCQNGTRPASEENREGCDYYCWNTDTRSWEQFFFGNGERCFYNTGEKGECKNGECHLTTESGVPTDTDVDTPAPTKKPKQKKKKQKKTKKPKR----------------------
gi|22164182| MEFTGITLVL-VSLTFFGSAAAETCRNGTRPGSQTQREGCDYYCWNSQTSSWDKYFFGDNEPCFYNTGLRGTCQNGECHLTSEGGVPTDPNQYPSEPTEKPKKNKKKSKKTKKPKKTKKPKDN---------------
gi|22164160| MEFTGITLLL-VSLAFFGSAAAETCRNGTRPASQTQREGCDYYCWNLQTSSWDKYFFGDNEPCFYNTGLRGTCQNGECHLTSEGGVPTDPNQYPSEPTEKPKKNKKKSKKTKKPKKTKKPKDN---------------
gi|22164178| MEFTGITLVL-VSVAFFGSAAAETCRNGTRPASQTDREGCDYYCWNTLTSSWDKYFFGDEEPCFYNTGLRGTCKNGGCHLTSEGNVPTDPDQYPSEPTEKPKKSKKKSKKTKKPKKTKKPKDN---------------
gi|22164180| MEFTGITLVL-VSVAFFGSAAAETCRNGTRPASQTDREGCDYYCWNTLTSSWDKYFFGDEEPCFYNTGLRGTCKNGECHLTSEGGVPTDPNQYPSEPTEKPKKNKKKSKKTKKPKKSKKPKDN---------------
gi|22164168| MELTGITLVL-VSLALFGSAAAETCRNGTRPASQTDREGCDYYCWNTLTSSWDKYFFGDEEPCFYNTGLKGTCKNGECHLTSEGGVPTDPHQYPSEPTEKPKKNKKKSKKTKKPKKSKKPKNN---------------
____________________
Poly K (lysine) tail
(B)
0.1
gi|22652868|
gi|22164158|
gi|22164182|
gi|22164160|
gi|22164168|
gi|22164178|
gi|22164180|
IP 5 100 90 6 CLU
IP-7-60-92-2-CLU
IP 5 100 90 25 CLU
gi|45593710|
IP 5 100 90 2A CLU
P 5 100 90 2 CLU
IP 5 100 90 1 CLU1
IP 5 100 90 1 CLU
IP clu3
gi|15428308|
gi|22164164|
gi|22164176|
gi|22164174|
gi|22164186|
gi|22164156|
gi|22164190|
gi|22164184|
gi|22164194|
Fig. 2. Group 1: Basic tail proteins (BTP). (A) Alignments of peptides from I. pacicus (Table 1) and I. scapularis BTP deduced from cDNA libraries.
Conserved amino acid residues are shown in black background. Lysine residues (K) are shown in bold (Poly K, lysine tail). (B) The bar represents the
degree of divergence among sequences.
I.M.B. Francischetti et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11421161 1149
The fact that BTP and SALP 14 contain a poly-Lys
tail adds an additional layer of anticoagulation, as it
directs the inhibitor to negatively charged membranes
(e.g., activated platelets) critical for productive blood
coagulation complex assembly (Broze, 1995). As a
result, the effective concentration of the inhibitor is
increased at sites that are predominantly pro-coagulant.
Also, we speculate that FXawhich is usually protected
from physiologic inhibitors (e.g., TFPI, heparin/ATIII)
when the prothrombinase is fully assembled (Mast and
Broze, 1996; Rezaie, 2001)would be more susceptible
to these bifunctional molecules. Demonstration that
proteins rich in positively charged residues effectively
block the coagulation cascade comes from studies
performed with a recombinant Rhodnius prolixus
salivary lipocalin (nitrophorin-7, NP-7). NP-7 contains
a cluster of positively charged residues in the N-terminus
and specically binds to anionic phospholipids, prevent-
ing thrombin formation by the prothrombinase (Ander-
sen et al., 2004). Finally, a bifunctional fusion protein
containing Kunitz and annexin domains was shown
recently to inhibit the initiation of blood coagulation
(Chen et al., 2005).
3.2. Group 2: Similar to Group 1, but without the basic
tail
These sequences contain a cysteine pattern identical to
Group 1 peptides except that, remarkably, the poly K
tail is missing. Many other amino acids also are not
conserved. Sequence alignment between the Group 1
peptides (containing poly K and poly E) and the
peptides similar to Group 1 is shown in Fig. 3A. Fig.
3B shows that these proteins come from a common
ancestor that appears to have evolved to display
different functions. The function of the peptides of
Group 2 deserves further investigation.
3.3. Group 3: Kunitz-containing proteins
Kunitz domains are about 60 residues and contain six
specically spaced cysteines (XnCX8CX15CX7CX12
CX3CXn) that form disulphide bonds typically repre-
sented by bovine pancreatic trypsin inhibitor (BPTI). In
most cases, they are reversible inhibitors of serine
proteases that bind the active site (Laskowski and Kato,
1980); however, Kunitz inhibitors such as the dendro-
toxins from Dendroaspis angusticeps snake venom block
K+ channel but display negligible protease inhibitory
properties (Harvey, 2001). Kunitz-containing proteins
also interact with protease exosites (Monteiro et al.,
2005) or platelets (Mans et al., 2002a). Of note,
sequencing the I. pacicus cDNA library yields a
number of proteins containing Kunitz-like domains.
The alignment of BPTI, snake venom, and I.
scapularis and I. pacicus single Kunitz-like proteins is
shown in Fig. 4A. Some I. pacicus proteins contain
one-Kunitz-like domain, here named the Monolaris-1
family (or similar to 6.58.4-kDa proteins from I.
scapularis). These molecules display the following
cysteine pattern: XnCX8CX18CX5CX12CX3CXn.
Other single-Kunitz sequences present in I. scapularis
belong to the Monolaris-2 family (or similar to 7.98.7-
kDa proteins from I. scapularis) and display the
sequence pattern XnCX8CX15CX8CX11CX3CXn. We
could not, however, nd members of the Monolaris-2
family sequences in our I. pacicus cDNA library. Fig.
4A also shows that the well-known tick anticoagulant
peptide from the soft tick Ornithodoros moubata (Wax-
man et al., 1990) has Kunitz-like folding with the
sequence pattern XnCX9CX17CX5CX15CX3CXn. At
present, the functions of Monolaris-1 and -2 are
unknown, but they may target specic proteases. The
phylogenetic tree shown in Fig. 4B suggests that snake
venom peptides containing Kunitz domains (non-
neurotoxic or neurotoxic) and the tick families of
Monolaris and basic tail peptides have diverged into
two different main groups from a commom ancestor,
suggesting that these proteins have evolved to perform
different functions.
Additionally, cDNAs were sequenced coding for
proteins containing two- or ve-Kunitz domains. These
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Fig. 3. Group 2: Similar to Group 1, without basic tail. (A) Alignment
of Group 2 peptides (Table 1). Conserved amino acid residues are
shown in gray background. (B) The unrooted cladogram of all
sequences. The bar represents the degree of divergence among
sequences.
I.M.B. Francischetti et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11421161 1150
proteins share sequence similarity to ixolaris (Fran-
cischetti et al., 2002b) and penthalaris (Francischetti
et al., 2004a), two I. scapularis TFPI salivary proteins that
prevent initiation of blood coagulation through specic
inhibition of the Factor VIIa/tissue factor complex. It is
possible that these proteins block other proteases (Ruf,
2004) or affect angiogenesis (Hembrough et al., 2004).
Fig. 4C depicts the predicted secondary folding of I.
scapularis and I. pacicus Kunitz-like-containing pro-
teins based on the crystal structure determined for BPTI
(Huber et al., 1974).
3.4. Group 4: Proline-rich proteins
Group 3 cDNA sequences code for short peptides of
mature molecular mass ranging from 3.5 to 4.8 kDa of
both basic and acidic nature (Table 1). Alignments and
cladograms (presented in Fig. 5A and B, respectively),
show that all sequences are relatively glycine and proline
rich in both I. pacicus and I. scapularis salivary glands.
Some sequences display weak matches to proteins
annotated as collagen in the NR database; these possess
two conserved cysteine residues in the mature peptide
and remarkable conservation of the secretory signal
peptide (Fig. 5). Most amino acids of the predicted
signal secretory peptide are conserved, versus few on the
mature peptide, suggesting functional diversity. The
possible function of these peptides remains to be
characterized, but taking into account its similarity to
collagen, it may somehow affect vascular biology
through inhibition of cellcell, cellmatrix, or cellli-
gand interactions. These peptides may also function
as adhesive molecules to cement the tick into their
hosts skin.
ARTICLE IN PRESS
Fig. 4. Group 3: Kunitz-containing proteins. (A) Alignment of Group 3 peptides (Table 1) with single Kunitz-containing protein from snake venoms.
Conserved amino acid residues are shown in gray background. (B) The phylogram was constructed using protein from snake venom single-kunitz
(neurotoxic or non-neurotoxic from Elapidae and Viperidae families) and tick salivary gland, plus BPTI (all accession numbers are depicted). The bar
represents the degree of divergence among sequences. (C) Predicted secondary folding of Kunitz-containing proteins from Ixodidae sp. based on
BPTI folding (Huber et al., 1974).
I.M.B. Francischetti et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11421161 1151
3.5. Group 5: Similar to I. scapularis 18.7-kDa protein
This group of proteins (Table 1) is similar to
orthologs described in I. scapularis and code for an
acidic putative protein of unknown function. Only low
e-values have been found when compared with proteins
in the NR database including coagulation factor X (gi
9837158, e-value 0.069), venom metalloprotease acur-
hagin precursor (gi 4689408; e-value 3.8), and pro-
protein convertase subtilin (gi 51771463, e-value 8.4).
Accordingly, this family of proteins may have evolved
from a protease precursor; however, any functional
assignment will be possible only after testing the
recombinant protein in screening assays.
3.6. Groups 6 and 7: Similar to I. scapularis 5- and 9.4-
kDa protein
Groups 6 and 7 code for basic proteins of 5 and
9.4 kDa that also are present in I. scapularis. No protein
motif was identied for either protein; accordingly, the
function of these proteins is not evident.
3.7. Group 8: Metalloprotease
These enzymes are capable of hydrolyzing various
components of the extracellular matrix including bri-
nogen and bronectin and reportedly affect endothelial
cells, leading to apoptosis. These enzymes are organized
ARTICLE IN PRESS
(A)
gi|22164234| MKATLVAICFLATVAFSMGESWGSSTPCPGAPGEPCQNGQPSQPPA---------PGSGPNVHPPENTSPPGRK--------------------------
gi|22164232| MKATLVAICFLAAVAFSMGESWGSSTPCPGAPGEPCQNGNPSKPPA---------PGSGPNVHPPRNTSPPGRK--------------------------
gi|22164236| MKATFMAICFLAAVAFSMGESWGSSTPCPGAPGEPCQNGNPSQPPA---------PGSGPNVRPPQNTSPPGRK--------------------------
gi|22164240| MKATFMVICFLAAVAFSMGESWGSSTPCPGAPGEPCQNGQPSQPPA---------PGSGPNVHPPQSTSPPGRK--------------------------
gi|22164230| MKATLIAICFLAAVAFSMGESWGSSTPCPGAPGEPCGNGNSPGGPS---------NPPGPSQPGMRDPSPPGRK--------------------------
gi|22164226| MKATLIAICFLAAVAFSMGESWGSSTPCPGAPGEPCGNGNSPGGPS---------NSPGPSQPGTRDPSPPGRK--------------------------
gi|22164224| MKATLLAICFLAAVTLTMGESHGSTTPCPGAPGQDCHPGNGPQGPS---------GPSGPQQPGTRGPNPPGTK--------------------------
gi|22164228| MKATLLAICLLAAVTLTMGESHGSTTPCPGAPGEACNPGNGPQGPS---------NSPGLRQPGTSDPSPPGTK--------------------------
gi|22164242| MKATLLAICFLAAVTLTMGESHGSTTPCPGAPGQPCNPLQGSQGPS---------NSPGPNQPGTRGSYAPGNSRK------------------------
gi|22164244| MKATLLAICFLAAVTLTMGESHGSTTPCPGAPGQPCNPLQGSQGPS---------NSPRPNQPGTRGSYAPGNSRK------------------------
gi|22164222| MKATLIAICFLAAVTFSMGETHGSSTPCPTAPGEDCNPGSRLQGPS---------NPSGPNQP-------------------------------------
gi|22164238| MKATLIAICFLAAVTFSMGETHGSSTPCPGAPGEDCNPGSRLQGPS---------NPSGPNHPGTRDTSPPGRK--------------------------
IP_7_60_92_16_CLUB MKATLIAICFLAAVTFSMGESIGSSTPCPNPPGQPCGPGQGPQGP-----------PSGPSHQPGKSPNAPGSSSKVSSRWLPPETCDGG----------
IP_5_100_90_27_CLU MKATLIAICFLAAVTFSMGESIGSSTPCPNPPG-----------P-----------PSGPIHQPGRSPHAPGSSSK------------------------
IP_5_100_90_30_CLU MKATLIAICFLAAVTFSMGESIGSSTPCPNPPGQPCGPGQGPQGP-----------PSGPSHQPGKSPSAPGSSTK------------------------
IP_clu28 MKATLIAICFLAAVTFSMGDTYGSSTPCPNAPGTPCGPGQGPQGP-----------PSAPSHQPDRSPNAPGGSSK------------------------
IP_clu28A MKATLIAICFLAAVTFSMGDTYGSSTPCPNAPGTPCGPGQGPQGP-----------PSAPSHQPDRSPNAPGGSSKAVGGCLQRRAMADRILPRCSKFSE
IP_5_100_90_29_CLU2 MKATLIAICFLAAVAFSMGESWGSSTPCPNPPGQPCGPGQPPQGPS---------QNPGPSPQPPRDTSAPGRK--------------------------
IP_5_100_90_32_CLU MKATLIAICFLAGVAFSMGESWGKLPPCPNPPGQPCGPGQPPQGPS---------QNPGPSPQPPRDTSAPGRK--------------------------
IP_5_100_90_29_CLU4 MKATLIAICFLAGVTFSMGETWGKPPPCSSPPGQPCPPGEGPQGPSSSPRPYPQPEGPSPSPQPPRDQSPPGTR--------------------------
IP_5_100_90_29_CLU5 MKATLIAICFLAGVTFSMGETWGKPPPCSSPPGKPCPPGEGPQGPSSSPRPYPQPEGPSPSPQPPRDQSPPGTR--------------------------
IP-7-60-92-15-CLU MKATLIAICFLAGVAFSMGETYGKPPPCSSPPGKPCPPGEGPQGPSSSPRPYPQPQGPAPSPQPPKDQSPPGTR--------------------------
IP_5_100_90_29_CLU3 MKATLIAICFLAGVAFSMGETYGQPPPCTSPPGEPCPPGEGPQGPPSSPRPYPRPHGPAPSPQPPKDQSPPGTR--------------------------
IP_5_100_90_29_CLU6 MKSTLIAICFLAAVTFTMGETWGNPPPCSSPPGQPCPPGEGPQGPSSSPRPYPQPQGPAPNPQPPRDQSPPGTRT-------------------------
**:*::.**:** *:::**:: *. .**. .** * .
(B)
0.1
gi|22164230|
IP clu28
IP 5 100 90 30 CLU
IP 5 100 90 27 CLU
IP 5 100 90 32 CLU
IP 5 100 90 29 CLU6
IP 5 100 90 29 CLU4
IP-7-60-92-15-CLU
IP 5 100 90 29 CLU3
gi|22164224|
gi|22164242|
gi|22164228|
gi|22164244|
gi|22164222|
gi|22164238|
gi|22164234|
gi|22164236|
gi|22164240|
Fig. 5. Group 4: Proline-rich peptides. (A) Alignment of Group 4 peptides (Table 1). Signal peptide is shown in gray background, and conserved
amino acid residues are shown in black background. (B) The unrooted cladogram of all sequences. The bar represents the degree of divergence
among sequences.
I.M.B. Francischetti et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11421161 1152
into four classes, PI through PIV, according to size and
domain composition (Bjarnason and Fox, 1995).
Our library contains a truncated cDNA that codes for
a mature metalloprotease similar to one described in I.
scapularis (gi 31322779) (Francischetti et al., 2003) and
I. ricinus (gi 5911708). The alignment of the mature
metalloproteases from I. pacicus, I. scapularis, and I.
ricinus, where the zinc-binding motif HExxHxxGxxH
common to these enzymes was identied, is shown in
Fig. 6A. Fig. 6B compares the PIII class of metallopro-
teases from snake venom and the I. pacicus, I.
scapularis and I. ricinus enzymes. It is clear that enzymes
from both genera have pre-, pro-, metalloprotease,
disintegrin-like, and cysteine-rich-like domains; how-
ever, the Ixodidae disintegrin-like and cysteine-rich like
domains are signicantly shorter in the number of
amino acid residues when compared with the corre-
sponding domains of metalloproteases from the repro-
lysin family (Bjarnason and Fox, 1995). We suggest that
this pattern of cysteines confer a different specicity for
these enzymes. This family of proteins also appears to
account for the a-brinogenase and brinolytic activity
recently reported for I. scapularis saliva (Francischetti
et al., 2003). Degradation of brinogen and brin are
associated with inhibition of platelet aggregation and
clot formation. Metalloproteases also may interact with
endothelial cell integrins, leading to apoptosis and
inhibition of angiogenesis (Francischetti et al, 2005).
3.8. Group 9: GPIIB/IIIa antagonists from the short
neurotoxin family
Inhibitors of platelet aggregation that targets the
brinogen receptor (GPIIbIIIa, integrin aIIbb3) have
been described in the hard tick Dermacentor variabilis
(variabilin) and the soft ticks, Ornithodoros moubata
(disagregin) and O. savignyi (savignygrin) (Karczewski
et al., 1994; Wang et al., 1996; Mans et al., 2002a).
Savignygrin belongs to the Kunitz-BPTI family and
presents the integrin RGD-recognition motif on the
substrate binding loop of the Kunitz fold (Mans et al.,
2002a). In contrast, variabilin possesses an RGD-motif
in its C-terminal region that is not anked by cysteines
(Wang et al., 1996). A search for possible GPIIb/IIIa
antagonists with RGD-motifs and anking cysteines,
termed the Ixodegrins, identied one candidate in I.
pacicus and several homologs in I. scapularis (Table 1).
It is clear that the Ixodegrins are related to variabilin,
but do possess anking cysteines. Variabilin probably
possesses a anking disulphide motif too, but was
missed due to the technical difculties in identifying
cysteines correctly during N-terminal sequencing. Data-
base searches using SAM-T99 (Karplus et al., 1998), a
program that utilizes hidden Markov models to nd
remote homologous sequences, identied dendroaspin
as the highest hit. Dendroaspin, also known as mambin,
is part of the short neurotoxin family found in elapid
snakes (McDowell et al., 1992; Williams et al., 1993;
Sutcliffe et al., 1994). Strikingly, the RGD-active site
loop (loop3) is conserved between snake and tick
integrin antagonists (Fig. 7A). This includes the anking
cysteines involved in a disulphide bond that constricts
the RGD-loop conformation and the anking prolines
that was shown to be important for presentation of the
RGD sequence (Lu et al., 2001). The tick inhibitors
maintain loops 2 and 3 of the short neurotoxin fold, but
do not possess the N-terminal loop 1 and the C-terminal
extension (Fig. 7A). This makes them the shortest
members of the short neurotoxin family described to
date, with only 39 amino acids forming the core active
fold. Phylogenetic analysis of the neurotoxin family
indicates that dendroaspin and tick inhibitors group
within one clade to the exclusion of the other short
neurotoxins (Fig. 7B). This suggests either an extreme
form of convergent evolution, where ticks and elapid
snakes used the same protein fold to evolve the same
function or raises the possibility that ticks or snakes
acquired the ancestral protein via a horizontal gene
transfer event or that there is a true evolutionary
relationship between the ixodegrins and short neurotox-
ins. The fact that orthologs of the Ixodegrins are present
in both Ixodes (prostriate) and Dermacentor (metastri-
ate) ticks, suggests that this inhibitor was present in the
last common ancestor of hard ticks. Snakes evolved
most of their venom properties approximately 6080
million years ago (Fry, 2005), whereas most hard tick
genera diverged at least 110 million years ago or earlier
(Klompen et al., 1996). If tick and snake proteins are
related, then the ancestral gene may have a platelet
antagonist function and the neurotoxic properties (and
the rest of the short neurotoxin foldloop1 and the C-
terminal extension) evolved later. In contrast, soft ticks
in the genus Ornithodoros evolved integrin antagonists
from the BPTI-fold which suggests that hard and soft
ticks evolved different strategies to obtain a blood meal
(Mans et al., 2002b; Mans and Neitz, 2004). Accord-
ingly, ixodegrin may affect platelet or neutrophil
integrin function or neutrophil function.
3.9. Group 10: Ixostatin family, or short-coding cysteine-
rich peptides (thrombospondin)
The two sequences in Group 11 match a sequence
deposited in the NR database from I. scapularis;
alignments are shown in Fig. 8A. These sequences have
been annotated thrombospondin (gi 15428290), but
thrombospondin motifs are lacking. On the contrary,
these short coding region cysteine-rich peptideshere
named ixostatinsare remarkably similar to the cy-
steine-rich domain of ADAMTS (Fig. 8B). Of note,
ADAMTS-4 (a disintegrin and metalloproteinase with
thrombospondin motifs), also known as aggrecanase,
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I.M.B. Francischetti et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11421161 1153
ARTICLE IN PRESS
Fig. 6. Group 8: Metalloproteases. (A) Alignment of metalloproteases from I. pacicus (Ip) (Table 1), I. scapularis (Is), and I. ricinus (Ir). The
characters in bold represent the conserved Zn binding motif present in the catalytic domain. Asterisks, colons, and stops below the sequences indicate
identity, high conservation, and conservation of the amino acids, respectively. (B) Diagram comparing the protein motifs (pre, pro, catalytic,
disintegrin-like, and cysteine rich-like domains) of class III metalloproteases from snake venoms and tick class III-like metalloproteases.
I.M.B. Francischetti et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11421161 1154
ARTICLE IN PRESS
Fig. 7. Group 9: Ixodegrin: disintegrins. (A) Alignment of the ixodegrins from I. pacicus (Ixodegrin_Ip), I. scapularis (Ixodegrin_Sc1/2/3), variabilin
and dendroaspin. Shadowed in gray are conserved cysteine regions and the RGD motif. Also indicated are the loops and disulphide bond pattern of
the short neurotoxin fold and the inferred disulphide bond patterns of the ixodegrins. (B) A neighbor-joining tree of the short neurotoxin family.
Indicated are different functional clades found for the family. Snake proteins are referred to by their SwissProt name. Black circles indicate
condence levels 470% from 10 000 bootstraps.
I.M.B. Francischetti et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11421161 1155
are enzymes involved in cartilage cleavage (Flannery
et al., 2002). The role of the cysteine-rich domain of
ADAMTS proteases is unknown, but it is postulated to
interact with integrins and/or other attachment motifs
of cells and matrix proteins (Porter et al., 2005).
Accordingly, the ixostatin family of peptides could be
involved in disruption of platelet aggregation or
neutrophil function, cellmatrix interactions, or inhibi-
tion of angiogenesis (Porter et al., 2005). The protein
modules of ixostatin and of ADAMST-4 are compared
in Fig. 8C.
3.10. Group 11: Histamine-binding proteins (lipocalins)
Group 11 contains sequences with similarities to
histamine-binding proteins discovered in the saliva of
R. appendiculatus ticks (Paesen et al., 1999). The
alignments of these sequences (Fig. 9A) reveal that they
do not display a highly conserved signal peptide which
suggest that they may not share a common ancestor. In
addition, the mature proteins contain few consensus
sequences indicating that they may have diverged to
perform distinct functions (Fig. 9B). This contention is
also supported by the cladogram presented in Fig. 9B. It
is likely that these proteins function by binding small
ligands such as histamine, serotonine, and adrenaline
(Andersen et al., 2005). Fig. 9C shows a predicted 3-D
model for sequence gi 51011604 that has an e-value of
768 for HBP from R. appendiculatus. The gure shows
amino acid side chains of the histamine-binding protein
from R. appendiculatus (red) surrounding the bound
histamine ligand with the corresponding residues for gi
51011604 shown in cyan. In the histamine-binding
protein, the imidazole ring of the ligand is stabilized
by surrounding aromatic residues, while in the
I. scapularis protein the binding pocket remains hydro-
phobic and fewer aromatic residues are present,
suggesting different ligand specicity. Polar residues
(Tyr 36 and Glu 135) forming electrostatic interactions
with the aliphatic amino group of histamine in the
ARTICLE IN PRESS
Fig. 8. Group 10: Ixostatin: short coding cysteine-rich peptides. (A) Alignment of Group 9 peptides from I. pacicus (Table 1) and I. scapularis.
Conserved amino acid residues are shown in black background. (B) Alignment between ixostatin and the cysteine-rich domain of ADAMST-4
(aggrecanase). (C) Diagram comparing the protein motifs (pre, pro, catalytic, disintegrin-like, cysteine-rich-like, and spacer domains) of ADAMST-4
(Flannery et al., 2002) and ixostatin.
I.M.B. Francischetti et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11421161 1156
histamine-binding protein are conserved in gi 51011604
suggesting the possibility of a similar role in this protein.
3.11. Group 12: Neuropeptide-like protein (npl-31) with
GGY repeat
A cDNA coding for a protein that shows remarkable
sequence homology to a neuropeptide-like protein (npl-
21) described in Caenorhabditis elegans (Nathoo et al.,
2001). This family of peptides displays a potent
antimicrobial activity toward Drechmeria coniospora,
Neurospora crassa, and Aspergillus fumigatus (Couillault
et al., 2004). Identication of these peptides in ticks
reinforces the notion that saliva contains a cocktail of
antimicrobial peptides. These peptides may prevent
growth of yeast and bacteria that, per se, can elicit an
inammatory/immune response that may be detrimental
to the feeding behavior of the attached ticks. Expression
of these molecules is particularly important vis-a` -vis the
remarkably immunosupressive property of the saliva
(Wikel, 1999) that helps ticks to feed for days but
otherwise creates an appropriate environment for
pathogen overgrowth. The sequence alignments for C.
elegans npl-21 and I. pacicus npl-21-like proteins are
presented in Fig. 10A and the cladogram in Fig. 10B.
This is the rst time that this family of antimicrobial
peptides has been identied in the salivary gland of a
blood-sucking arthropod.
3.12. Group 13: Oxidant metabolism
Proteins with similarity to glutathione peroxidase and
a putative secreted superoxide dismutase were found
(Table 1). These sequences categorize the prominent
ARTICLE IN PRESS
Fig. 9. Group 11: Histamine-binding proteins (lipocalins). (A) Alignment of Group 10 peptides from I. pacicus (Table 1). Conserved amino acid
residues are shown in black background. (B) The unrooted cladogram of all sequences. The bar represents the degree of divergence among sequences.
(C) The gure shows amino acid side chains of the histamine-binding protein from R. appendicuatus (red) surrounding the bound histamine ligand.
The corresponding residues for gi 51011604 are shown in cyan. In the histamine-binding protein, the imidazole ring of the ligand is stabilized by
surrounding aromatic residues. In the I. scapularis protein the binding pocket remains hydrophobic, fewer aromatic residues are present, suggesting a
different ligand specicity.
I.M.B. Francischetti et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11421161 1157
salivary gland proteins in I. pacicus and demonstrate
the presence of a potent antioxidant in tick saliva. Of
interest, cluster F12_IPL_P23 has sequence similarity to
SALP 25, a protein that catalyzes the reduction of
hydrogen peroxide in the presence of reduced glu-
tathione and glutathione reductase (Das et al., 2001).
The functions of these proteins are likely related to
maintenance of the physiologic redox of cellular
intracellular milieu or to modulation of the extracellular
levels of pro-oxidants often associated with inamma-
tory events.
3.13. Group 14: Similar to other ixodid proteins
A number of sequences show sequence homology to
proteins from Ixodidae described before. We have found
sequences similar to SALP 15, a immunodominat
protein in I. scapularis (Das et al., 2001), and to ISAC,
the anti-complement from I. scapularis (Valenzuela et
al., 2002). We also have found sequences similar to
domain 8 of human ADAMS and Factor VII.
3.14. Group 15: Novel, unknown
Some sequences containing a signal peptide and a
stop codon and with a clear open reading frame were
without database hits and were characterized as
unknown-function proteins. Assignment of function
for these proteins will only be possible after expressing
and screening for testable biologic activities.
3.15. Group 16: Housekeeping cDNA
Thirty-seven sequences with homology to housekeep-
ing protein are given in Table 1. They assign to
ribosomal, glutathione S-transferase, vacuolar assorting
proteins, cytochrome, ATP synthase subunit, and
NADH-ubiquinone oxidoreductase, among other mole-
cules. In addition, housekeeping proteins may be useful
in phylogenetic studies (Black and Piesman, 1994).
3.16. I. pacicus salivary gland protein diversity:
modulators of vascular biology and candidates for an anti-
saliva experimental vaccine
We describe the set of cDNA present in the salivary
glands of I. pacicus salivary gland. Our library contains
a remarkably large degree of redundancy, as shown by
the many related mRNAs. It appears that the long
evolutionary history of ticks may be responsible for the
complexity of transcripts reported here. Also, many
protein families we have identied were found pre-
viously in I. scapularis salivary glands (Valenzuela et al.,
2002) which conrms the diverse nature of these
secretions compared with the salivary composition of
fast feeders such as sand ies (Charlab et al., 1999) and
mosquitoes (Francischetti et al., 2002a). This variability
ARTICLE IN PRESS
Fig. 10. Group 12: Neuropeptide-like (npl-31) peptides. (A) Alignment of Group 11 peptides from I. pacicus (Table 1) and I. scapularis. Conserved
amino acid residues are shown in gray background. (B) The unrooted cladogram of all sequences. The bar represents the degree of divergence among
sequences.
I.M.B. Francischetti et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11421161 1158
in the tick salivary gland is consistent with the high
polymorphism of salivary proteins among individual
ticks analyzed by SDS-PAGE (Wang et al., 1999). Also,
the diversity across and within species could reect the
range of host species and the need to have modulators of
specic pathways that differ in distinct host species. The
adaptive role of this diversity appears to be explained at
least in part by a gene-duplication phenomenon. This
contention is supported by the diversity of sequences
containing Kunitz-like domains in addition to a weak
similarity observed among members of the lipocalin
family of proteins reported here. It may be that these
inhibitors have evolved to inhibit different proteases or
to bind to different ligands (Andersen et al., 2005). It is
also plausible that gene duplication may help ixodid
ticks to evade the immune system. If so, this may help to
explain why hard ticks can remain attached to many
hosts for days without apparent detrimental effects
(Ribeiro and Francischetti, 2003). Finally, the possible
closer association of I. persulcatus with I. pacicus
makes the former an interesting species for future
salivary gland transcriptome analysis and phylogenetic
studies.
The functions of many tick sequences described in this
paper are unknown. Cloning and expressing select
cDNAs will help in the identication of molecule
specicity and to nd potential targets for gene silencing
(Sanchez-Vargas et al., 2004), and accordingly, our
understanding of how ticks successfully feed on blood.
It also may provide tools to understand vascular biology
ARTICLE IN PRESS
Fig. 11. Negative modulators of vascular biology are present in I. pacicus and I. scaularis saliva. Vascular injury is accompanied by vasoconstriction
and activation of the extrinsic and intrinsic pathways of blood coagulation (Broze, 1995). Vasoconstriction is mediated by molecules such as
serotonine that may be removed by salivary protein with a lipocalin folding (Andersen et al., 2005). The extrinsic pathway is initiated by tissue factor/
factor VIIa complex and effectively blocked by ixolaris (Francischetti et al., 2002b) and penthalaris (Francischetti et al., 2004a). FXa generated by
the intrinsic or extrinsic Xnase may be inhibited by Group 1 peptides containing a basic tail that may prevent productive prothrombinase complex
assemble (Rezaie, 2000; Narasimhan et al., 2002; Andersen et al., 2004, Monteiro et al., 2005). Platelet, neutrophil, and endothelial cell function may
be affected by Ixodegrins (disintegrins) or Ixostatins (short-coding cysteine-rich peptides). Metalloproteases appear to cleave brinogen and brin,
therefore inhibiting platelet aggregation and clot formation (Francischetti et al., 2003). Metalloproteases also may affect endothelial cell function and
angiogenesis (Francischetti et al., 2005). The intrinsic pathway that is activated by contact leads to bradykinin formation, a peptide that increases
vascular permeability and induces pain. Bradykinin is degraded by a salivary kinininase, thus preventing its pro-inammatory effects (Ribeiro and
Francischetti, 2003).
I.M.B. Francischetti et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11421161 1159
and the immune system. A diagram with the putative
targets of salivary proteins and how they may affect
vascular biology is shown in Fig. 11. Finally, dening
the most abundant antigens or those that may effec-
tively help ticks to feed or transmit Borrelia could
be an effective approach to develop a protective
vaccine directed toward tick salivary molecules (Lane
et al., 1999).
Acknowledgements
We thank Drs. Thomas E. Wellems, Robert W.
Gwadz, and Thomas J. Kindt for encouragement and
support. We are thankful to Brenda Rae Marshal for
editorial assistance.
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Received 15 March 2005; received in revised form 23 May 2005; accepted 25 May 2005
Abstract
Teratocytes derived from the embryonic membrane (serosa) of parasitoids are released into the host hemocoel when the parasitoid
eggs hatch, where they perform several functions during the post-embryonic stage. A full-length cDNA encoding a putative
carboxylesterase was isolated from the teratocytes of Dinocampus coccinellae and was designated as teratocyte-specic
carboxylesterase (TSC). It contained an open reading frame of 2571 bp coding for a protein of 857 amino acids with a calculated
molecular mass of 89 kDa. The deduced amino acid sequence had many structural features that are highly conserved among serine
hydrolases including Ser, Glu and His as a catalytic triad, carboxylesterase type-B (FGGNPNSVTLLGYSAG)/ lipase-serine
(VTLLGYSAGA) active sites, and six N-glycosylation sites. Interestingly, the mRNA encoding the TSC gene was expressed
exclusively in teratocytes but not in the parasitoid larva or in the non-parasitized host. Most notably, the TSC protein was
distinguished by an insertion of 294 amino acids towards the N-terminal region and was anked by carboxylesterase domains.
Furthermore, sequence alignment and homology search revealed these additional amino acids to be unique to TSC and the insertion
contributed signicantly to its molecular mass resulting in a larger protein than other esterases. In addition to sequence analysis, the
possible role of TSC in relation to the host (Coccinella septempunctata) and parasitoid (D. coccinellae) system is discussed.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: Dinocampus coccinellae; Coccinella septempunctata; Host-parasitoid relationship; Teratocytes; Carboxylesterase
1. Introduction
Parasitoids have evolved a high degree of nutritional,
physiological, and behavioral interactions with their
hosts. For survival in their hosts, parasitoid wasp-
derived components such as venom, polydnavirus and
teratocytes play important roles by manipulating the
hosts physiology (Beckage and Gelman, 2004). Terato-
cytes are unique cells originated from the serosal
membrane of some endoparasitoid embryos which
become dissociated after hatching (Dahlman, 1990;
Lawrence, 1990; Buron and Beckage, 1997). They have
nutritive and immunosuppressive roles in some species
and can also regulate host growth and development in
others (Kitano et al., 1990; Strand and Wong, 1991;
Pennacchio et al., 1992; Dahlman and Vinson, 1993).
Although several studies on teratocytes are available in
literature, only one teratocyte-specic gene has been
characterized at molecular level (Rana et al., 2002;
Dahlman et al., 2003).
The braconid wasp, Dinocampus coccinellae parasi-
tizes several aphidophagous lady beetle species. In a host
beetle Coccinella septempunctata, teratocytes increase in
size and decrease in number during parasitism indicating
that D. coccinellae teratocytes primarily provide nutri-
tion for developing parasitoid larvae in the host
(Kadono-Okuda et al., 1995). The teratocytes synthesize
a great amount of teratocyte-specic protein of 540 kDa,
with a major subunit of 94 kDa, a hexamerin and
is shown to be nutritive in function (Okuda and
ARTICLE IN PRESS
www.elsevier.com/locate/ibmb
0965-1748/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibmb.2005.05.010
Fig. 3. An alignment using ClustalX program of the inferred amino acid sequence of TSC of D. coccinellae (AY498693) with ve insect
carboxylesterases: M. persicae (P35502); D. melanogaster (A28022); N. lugens (AAG40239); L. cuprina (AAB67728); and A. calandrae (AF064523).
GenBank accession numbers are given in parentheses. Functionally important residues (catalytic triad) are indicated by arrows. Identical amino acid
residues are shown by asterisks. The : and indicate strong and weak group of amino acids that are conserved respectively. Amino acids from
position 111 to 404 were removed from TSC sequence to avoid a long gap in the alignment (see text for details).
R. Gopalapillai et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11711180 1176
esterase assay, together with the lipophorin (Lp) of the
host beetle.
As in many insects the lipophorin (HDLp) of the host
coccenellids is yellow in color due to carotenoids and its
density was 1.078 g/ml. This HDLp formed a clear
yellow band upon KBr density gradient centrifugation.
The density of the Lp increased to 1.14 g/ml when it was
incubated with teratocytes and analyzed after density
gradient centrifugation. This indicates the conversion of
HDLp to very high-density lipophorin (VHDLp)
resulting in the band shift (Fig. 6). Increased density
of Lp may be caused by the hydrolysis of lipids by the
teratocytes. Lp incubated with bacterial lipase also
showed a similar result and the disappearance of the
yellow band was noticed when increased quantities of
lipase or teratocyte extracts were used (data not shown).
We have observed a marked decrease in triacylglycer-
ide (TAG) in the fat body of parasitized host compared
to the TAG in the fat body of non-parasitized host
(Fig. 7B). Further, TAG in the teratocytes showed a
gradual increase from day 7 and reached a maximum on
day 12 after parasitization. After this, the TAG content
either reached a plateau or slightly decreased (Fig. 7A),
with decrease in number of the teratocytes (Kadono-
Okuda et al., 1995).
4. Discussion
Carboxylesterases are a group of serine esterases that
catalyze the hydrolysis of a wide variety of ester
and amide containing endogenous and xenobiotic
compounds (Heyman, 1980). Some of them hydrolyze
palmitoyl CoA, acyl-carnitine, and mono-and diacylgly-
cerols and are thought to be involved in lipid
metabolism (Satoh, 1987) while others are able to
hydrolyze a broad range of substrates (Oakeshott
et al., 1999). Furthermore, carboxylesterases are
glycoproteins characterized by high mannose and
N-glycosylation may play an important role in their
catalytic activity (Kroetz et al., 1993). Previous work
shows that teratocytes of D. coccinellae synthesize a
teratocyte-specic polypeptide (Okuda and Kadono-
Okuda, 1995), a high mannose containing glycoprotein,
produced as a hexamer, and the molecular mass of its
major subunit is 94 kDa (Kadono-Okuda et al., 1998).
This is the most prominent protein in teratocytes
observed when analyzed on a SDS-PAGE with Coo-
massie blue staining indicating its abundance (Okuda
and Kadono-Okuda, 1995). Moreover, it exhibits a
strong esterase activity but is devoid of juvenile
hormone esterase activity (Kadono-Okuda et al.,
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Fig. 5. Western blot analysis of TSC expressed in a cell- free protein
expression system on a 7.5% SDS-PAGE gel. The TSC cDNA was
cloned into pIVEX2.3d plasmid and expressed using RTS100 E. coli
kit (Roche). TSC, TSC protein of molecular mass 90.1 kDa containing
His
6
-tag at C-terminal. C, negative control (plasmid without insert).
Five microlitre of each reaction product was loaded. Protein markers
(Bio-Rad) on the left.
Fig. 4. RT-PCR analysis demonstrating TSC gene expression. RNA
was isolated from teratocytes (TC), parasitoid larva (L) and fat bodies
of non-parasitized host (NPH) reverse transcribed, amplied by PCR
with TSC-specic primers and the amplication products were
analyzed on a 0.8% agarose gel. Molecular size markers (M)
(Invitrogen) are indicated at left.
R. Gopalapillai et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11711180 1177
1998). The deduced amino acid sequence of the
teratocyte cDNA has the most characteristics of an
active carboxylesterase enzyme. These include a cataly-
tic triad, a conserved esterase/lipase motif, two carbox-
ylesterase type-B (involved in lipid metabolism) motifs,
a lipase-serine active site, and six N-glycosylation sites.
Several lines of evidence indicate that we have cloned the
major gene from the teratocytes of D. coccinellae. First,
the cDNA sequence obtained would encode peptides
similar to that of fragments obtained from Edman
degradation of the puried teratocyte protein. Second,
regions of the inferred amino acid sequence of TSC have
esterase motifs and third, the predicted molecular mass
of the protein (89 kDa) is in good agreement with that of
its native protein (94 kDa). This difference in molecular
mass is mainly due to glycosylation of the native protein
and it is further supported by the presence of six
N-glycosylation sites in the TSC sequence. The presence
of a signal peptide and the absence of a consensus
C-terminal endoplasmic reticulum retention signal,
HXEL may indicate that TSC protein is secretory.
Interestingly, TSC has a clear HDEL tetrapeptide
(753756), albeit 101 amino acids away from the
C-terminal. Additionally, a secretory signal TEHT
(Medda and Proia, 1992) found at the C-terminal of
some carboxylesterases is absent in TSC. The PSORT II
program predicts that TSC can be both cytoplasmic and
extracellular in equal probability. Retention of rat liver
carboxylesterase in the endoplasmic reticulum is still
possible with a signal peptide and the absence of a
retention sequence (Takagi et al., 1988). Taken together
these data may thus agree with our previous report
(Kadono-Okuda et al., 1998) that teratocyte protein largely
accumulates in teratocyte cells with a little secretion.
While most insect carboxylesterases are implicated in
insecticide resistance (Field et al., 1988; Karunaratne et
al., 1993; Newcomb et al., 1997), many vertebrate
carboxylesterases are reported to be involved in the
hydrolysis of several lipids (Satoh, 1987). Our data do
not seem to support a detoxication or xenobiotic
degradation by TSC. The host beetles have a defensive
alkaloid Coccinellin which is bitter in taste and indeed
toxic (Tursch et al., 1971). However, the alkaloid does
not contain ester or amide bonds to be digested by
carboxylesterase.
ARTICLE IN PRESS
Fig. 7. Triacylglyceride content (in mg per animal) in teratocytes and
parasitoid larvae (A), fat bodies of parasitized and non-parasitized
host (B) after different days of parasitization. (A) Open circle: total
teratocytes in a host beetle, closed circle: a parasitoid larva (B) Open
circle: fat bodies from a non-parasitized host beetle, closed circle: fat
bodies from a parasitized host beetle, Values are mean7SE of four
animals.
Fig. 6. Hydrolysis of lipids by teratocyte extract. HDLp incubated in
the absence (Control) and in the presence of teratocyte extract (TC).
Subsequent density gradient ultracentrifugation showing Lp band shift
in the right tube (TC) due to the formation of VHDLp from HDLp.
R. Gopalapillai et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11711180 1178
As mentioned above, TSC is marked by the presence
of a lipase serine active site and two carboxylesterase
type-B motifs that are all thought to be involved in lipid
metabolism. TSC may be involved in the break down of
host lipids following lipid accumulation in teratocytes
(Fig. 7) as part of the utilization of fat as an energy
source for the growing parasitoid larva. This is
consistent with a nutritional role of D. coccinellae
teratocytes (Okuda and Kadono-Okuda, 1995). Terato-
cytes serving as a nutrient source for developing
parasitoid larvae are reported in several species such
as Aphidius ervi (Falabella et al., 2000), Microplitis
mediator (Quin et al., 2000), Microctonus aethiopoides
(Barratt and Sutherland, 2001) and Cotesia kariyai
(Nakamatsu et al., 2002) parasitoid systems.
Although the functions of teratocytes are not
completely well understood, there is considerable
published evidence to support their role in parasitoid
development (reviewed by Beckage and Gelman, 2004).
Microplitis croceipes teratocytes produce a 13.9 kDa
protein (monomer, TSP14) that inhibits host protein
synthesis that is linked to larval growth and develop-
ment (Zhang and Dahlman, 1989; Dahlman, 1990).
TSP14 appears to be the only teratocyte-specic gene
cloned and characterized. It encodes a protein of 129
amino acids (including a signal sequence of 22 amino
acids) and carries a cysteine-rich motif similar to that
described from Campoletis sonorensis polydnavirus
(Dahlman et al., 2003). Like native protein, the
recombinant TSP14 inhibited host protein synthesis
selectively in a dose dependent manner (Rana et al.,
2002). However, there is no signicant sequence
similarity between TSP14 and TSC of the present study
suggesting the existence of host-specic differences in
the primary structure of major teratocyte-specic genes.
Likewise, the biological role of teratocytes in host-
parasitoid relationships may differ from one system to
another (Dahlman, 1990).
It is interesting to note the distinct sequence difference
of TSC and other carboxylesterases. Although TSC
shares homologies with the members of the carboxyles-
terases and exhibits domain structures that are typical
for this group, it signicantly differs from them by a
unique insertion of 294 amino acids. A NCBI conserved
domain search (rpsblast) reveals a clear demarcation in
the TSC protein domain structure. While the amino acid
positions 27110 and positions 405829 display domain
similarity (with E value ranging from 0.008 to 4e-77) to
the members of the carboxylesterase family, the inter-
vening amino acids (111404) do not show any putative
domain. In addition, an alignment of the complete
amino acids of TSC with other insect carboxylesterases
shows that the inserted 294 amino acids leaves a long
gap in the alignment, indicating that this non-aligned
sequence is unique to TSC and that they are anked by
carboxylesterase domains. The physiological signi-
cance of this insertion is uncertain. Interestingly this
region is enriched with glutamine and glycine. BLAST
search (protein-protein) of these unique amino acids
alone shows no known protein domains but reveals
similarity to immunoglobulin binding proteins. Since
insects have no antigenic immunity, this non-aligned
part of the sequence may function in host immune
suppression through other means or be involved in other
function(s) which warrants further investigation. While
the size range of most esterases is 6070 kDa (Cygler et
al., 1993), TSC is 94 kDa and this relatively large
molecular size invites speculation that in addition to a
possible ester bond hydrolysis, TSC may also perform
other function(s). Although the precise physiological
functions of TSC remain unclear, our data on the
cloning and sequence analysis clearly indicate the
presence of a unique carboxylesterase in the teratocytes
of a parasitic wasp.
Acknowledgements
G.R. is thankful to Japan Science and Technology
Agency for a postdoctoral fellowship. We thank Dr.
Kazuo Masaki for his help in the peptide sequencing,
Drs. Takahiro Kikawada, Takahiro Shiotsuki, Yuichi
Nakahara, and Yusuke Kato for their kind help in
various ways. We also thank Dr. Barbara Barratt for
her critical reading of the manuscript.
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ARTICLE IN PRESS
R. Gopalapillai et al. / Insect Biochemistry and Molecular Biology 35 (2005) 11711180 1180
Insect
Biochemistry
and
Molecular
Biology
Insect Biochemistry and Molecular Biology 35 (2005) 11811188
The extensible alloscutal cuticle of the tick, Ixodes ricinus
Svend Olav Andersen
a,
, Peter Roepstorff
b
a
August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen O, Denmark
b
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
Received 28 October 2004; received in revised form 19 May 2005; accepted 25 May 2005
Abstract
The proteins in the distensible alloscutal cuticle of the blood-feeding tick, Ixodes ricinus, have been characterized by
electrophoresis and chromatography, two of the proteins were puried and their total amino acid sequence determined. They show
sequence similarity to cuticular proteins from the spider, Araneus diadematus, and the horseshoe crab, Limulus polyphemus, and to a
lesser extent to insect cuticular proteins. They contain a conserved sequence region, which is closely related to the chitin-binding
RebersRiddiford consensus sequence present in many insect cuticular proteins.
Only a fraction of the alloscutal proteins can be readily dissolved, and the dissolved proteins are difcult to separate by
electrophoresis and column chromatography. The insoluble fraction can only be dissolved after degradation to smaller peptides. The
mixture of extractable proteins as well as hydrolysates of the insoluble fraction are uorescent when exposed to ultraviolet light, and
the uorescence corresponds in excitation and emission maxima to the uorescence of the rubber-like arthropodan protein, resilin,
and to the amino acid dityrosine. Small amounts of dityrosine were obtained from ticks in the early phase of a blood meal when the
cuticle weighs less than 4 mg; increasing amounts were obtained from animals in the initial period of feeding, during which the
cuticular weight increases from 4 to 11 mg, whereas little increase in dityrosine content was observed during the nal period of
engorgement. Cuticle from fully distended ticks contains about 6080 nmole dityrosine per tick, corresponding to 23 mg/mg cuticle.
It is suggested that the major part of the cuticular proteins is made inextractable by cross-linking by dityrosine residues, and that
dityrosine plays a role in stabilizing the cuticular structure during the extensive distension occurring during a blood meal.
Small amounts of 3-monochlorotyrosine and 3,5-dichlorotyrosine were obtained from the distended tick cuticle, corresponding to
chlorination of between 0.5% and 1.5% of the tyrosine residues. It is suggested that the chlorotyrosines are a side-product of
oxidative processes in the cuticle.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: Cuticle; Ticks; Amino acid sequence; Dityrosine; Chlorotyrosine
1. Introduction
The adult female sheep tick, Ixodes ricinus, sucks
blood from its host for a period of 810 days. During
the feeding period the tick ingests a large volume of
blood; its body weight increases from about 2 mg to
more than 250 mg, and the alloscutal cuticle is distended
to allow for the necessary volume increase (Lees, 1952).
The epicuticular layer of the alloscutal cuticle is deeply
folded in unfed ticks, and during feeding the folds are
attened and the apparent surface area of the alloscu-
tum is increased to about 15 times the initial value,
although the area of the epicuticle is not increased (Lees,
1952; Hackman and Filshie, 1982). The underlying
procuticle is not sclerotized, it has been classied as
endocuticle, and it is extensively stretched during
feeding. The endocuticle of unfed female ticks is thin;
it increases in thickness during the feeding period due to
addition of more material and at the same time a layer
of inner endocuticle is deposited, which has a more open
structure than the initial layer of endocuticle. After
ARTICLE IN PRESS
www.elsevier.com/locate/ibmb
0965-1748/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibmb.2005.05.009
m
o
l
/
g
f
r
e
s
h
w
e
i
g
h
t
)
0
2
4
6
8
Age of larva (days)
8 10 12 14 16 18 20
8 10 12 14 16 18 20
G
l
u
c
o
s
i
n
o
l
a
t
e
(
m
o
l
/
l
a
r
v
a
)
0.00
0.05
0.10
0.15
(A)
(B)
Fig. 3. Glucosinolate levels in Athalia rosae over development. Larvae
were reared on Sinapis alba. The glucosinolate amount of a larva
reects almost exclusively the glucosinolate sequestered in the
hemolymph (see text). Glucosinolate amount in the larvae (A), and
glucosinolate concentration (B) are shown in dependence of age. Black
bars: sinalbin; grey bars: glucotropaeolin. Results of one out of four
independent experiments are shown. Each bar represents the mean7se
of four individual larvae. Arrows indicate molting event to next larval
instar.
C. Muller, U. Wittstock / Insect Biochemistry and Molecular Biology 35 (2005) 11891198 1193
3.4. Long-term changes of glucosinolate levels
When transferring larvae from S. alba to B. stricta,
the glucosinolate from the original host, sinalbin,
dropped to less than 6% after 24 h (Fig. 5A), and was
not detectable after 24 days (Fig. 5A, B). Also over a
longer time period, sinalbin and glucobarbarin were
mainly located in the hemolymph while the indole
glucosinolate glucobrassicin was detectable in bled body
tissue only. When transferring larvae from B. stricta to
S. alba, the original major glucosinolate, glucobarbarin,
dropped less drastically than sinalbin, after 24 h to a
relative abundance of 1527% (Fig. 5C). It could still be
detected after 4 days in all larvae. Glucobrassicin was
detectable for up to 23 days (Fig. 5C, D). The amounts
of glucosinolates that were taken up from the new
host did not steadily increase over the period of 5 days
but uctuated around the level they reached at the
second day after transfer (Fig. 5B, D).
3.5. Glucosinolate turn-over: feeding versus injection of
glucotropaeolin
In larvae that were injected into their hemolymph
with 250 nmol of glucotropaeolin, a rapid decrease of
this glucosinolate down to about 10% of the injected
amount was found after 24 h (Fig. 6A). The turn-over
rate of glucotropaeolin was on average only slightly
lower in larvae that were injected with glucotropaeolin
than in larvae that had taken up the same amount orally
(Fig. 6B), but did not differ signicantly (P40:8 for
both experiments, MannWhitney U-test). The same
pattern was found in four independent experiments,
however, levels of glucosinolate recovered varied (in-
dependently of the volumes used to dissolve glucotro-
paeolin).
3.6. Fate of [
14
C]glucotropaeolin
To study, whether glucosinolates and their metabo-
lites are stored in the body or excreted, larvae were fed
leaf discs with [
14
C]glucotropaeolin. After 14 h they were
dissected and analyzed for radioactivity. The radio-
activity in guts and feces accounted for 84% of the total
radioactivity recovered from larvae and feces.
3.7. Search for glucosinolate-metabolizing enzymes in
larvae
To test, whether A. rosae possesses a sulfatase to
convert glucosinolates to desulfoglucosinolates, two
approaches were taken. On the one hand, larval parts
of A. rosae reared on S. alba were analyzed for the
presence of desulfoglucosinolates. Desulfosinalbin was
detectable in hemolymph and bled larvae (0.370.05 and
0.0570.01 mmol/g fw, respectively), however, the
ARTICLE IN PRESS
(A)
(B)
(C)
Fig. 4. Short-term metabolism of glucosinolates in larvae of Athalia
rosae. Relative abundance of glucobarbarin (A), glucobrassicin (B),
and sinalbin (C) in percent of the total glucosinolate content in
complete larvae (hemolymph and bled body) of A. rosae. While the
aromatic glucosinolates were mainly found in the hemolymph, the
indol glucosinolate was present in the bled body only. Larvae fed on
Barbarea stricta after a transfer from Sinapis alba for periods between
0 and 20 h. Concentration of glucobarbarin and glucobrassicin (in
parenthesis) in leaf discs of B. stricta after 3 h: replicate 1: 0.43
(0.07), repl. 2: 2.53 (0.33), repl. 3: 0.07 (0.18) and repl. 4: 1.40
(0.38) mmol/g fw.
C. Muller, U. Wittstock / Insect Biochemistry and Molecular Biology 35 (2005) 11891198 1194
concentration was about twenty times lower than that of
intact sinalbin (6.670.8 and 1.970.2 mmol/g fw, respec-
tively, n 6). In feces, desulfosinalbin was present in
low concentrations between 0.05 and 0.15 mmol/g fw,
while intact sinalbin was present only in traces
(0.0270.01 mmol/g fw).
On the other hand, sulfatase activity was evaluated by
incubation of protein extracts of larval tissue using
glucotropaeolin or sinalbin as substrates. No desulfo-
glucosinolates were formed within 1.5 h. In control
assays, which contained authentic H. pomatia sulfatase,
in addition to the A. rosae protein extracts, desulfoglu-
cosinolates were formed.
To test for myrosinase activity, protein extracts were
incubated with benzylglucosinolate and formation of
hydrolysis-products was followed. Myrosinase activity
was not detectable in extracts of hemolymph, gut tissue
and the remaining body. When external myrosinase was
present in addition to the protein extracts, benzyl
isothiocyanate and traces of benzylcyanide were formed
as hydrolysis products.
No 4-hydroxybenzylcyanide sulfate was detectable in
samples of larvae fed on S. alba or their feces, indicating
that the metabolism of sinalbin in A. rosae occurs via a
different route than in P. rapae.
4. Discussion
Larvae of A. rosae sequester intact glucosinolates,
except for indole glucosinolates, probably more or less
exclusively in the hemolymph. The small amounts of
glucosinolates found in bled bodies are likely due to
the fact, that the hemolymph cannot be collected
ARTICLE IN PRESS
(A) (B)
(C) (D)
Fig. 5. Long-term metabolism of glucosinolates in larvae of A. rosae. Content of specic glucosinolates expressed as relative abundance in percent of
the total glucosinolate content (A, C) and as amount in larvae (B, D) of A. rosae. Larvae were transferred from Sinapis alba to Barbarea stricta (A,
B), and B. stricta to S. alba (C, D) and were analyzed after different numbers of days. While the aromatic glucosinolates were mainly found in the
hemolymph, the indol glucosinolate was present in the bled body only.
C. Muller, U. Wittstock / Insect Biochemistry and Molecular Biology 35 (2005) 11891198 1195
exhaustively from the larva. In lled guts and feces only
traces of intact glucosinolates were detected. Therefore,
the glucosinolate content of complete larvae was used in
this study as an approximate measure for hemolymph
glucosinolate content.
The larvae of A. rosae sequester glucosinolates from
their host plant as soon as they hatch from the egg,
which is laid into leaf tissue. Along the development, the
glucosinolate concentration of larvae varied in a
uctuating manner around 5 mmol/g fw. Fluctuation in
uptake of allelochemicals might be a common feature
for sequestering insect species. For example, it has been
reported previously for caterpillars of Utethesia ornatrix
(L.) (Lepidoptera: Arctiidae) feeding on diet containing
pyrrolizidine alkaloids (Kelley et al., 2002).
The decrease in glucosinolate concentration and
amount at the last feeding instar of A. rosae is not
related to an incorporation of glucosinolates in the
molted exuvia. In these, only traces of glucosinolate
were found. Adults still contained some of the glucosi-
nolates, although in a ten times lower concentration
than the larvae. This is in contrast to a previous study
where we showed similar concentrations of sinalbin in
larvae and adults (Mu ller et al., 2001). In the current
development experiments carried out in winter, the
pupal stage took about three times as long as in the
previously reported experiment. Within this long pupal
period some of the glucosinolates might have been
metabolized. The high variation of glucosinolate con-
centration in A. rosae samples of different experiments is
due to a high variation in plant glucosinolate levels
(which can vary 10100 times) but also possible
variation in sequestration ability between the larvae
(Mu ller et al., 2003b).
The sequestration ability of A. rosae larvae is very
efcient. After feeding of only about 2 mg leaf within
30 min, a newly offered glucosinolate is already detect-
able in the hemolymph (Fig. 4). While the amount of a
new glucosinolate increased within the rst 20 h in A.
rosae, it remained (or uctuated) at about a certain level
over the next four days of continuous feeding, for
glucobarbarin below 0.12 mmol/larva and for sinalbin
below 0.17 mmol/larva in this experiment (Fig. 5).
Within the rst 20 h the amount of old glucosinolates
decreased rapidly and then was slowly further diluted.
This indicates an equilibrium between a continuous and
efcient transport of glucosinolates into the hemo-
lymph, as well as a constant degradation. Glucobrassi-
cin was not found in the hemolymph, however, it is also
not excreted as intact glucosinolate (Mu ller et al., 2001).
Thus, this glucosinolate must be metabolized elsewhere.
The hemolymph is not only the main storage place for
intact glucosinolates (except the indole glucosinolate).
Hemolymph glucosinolates must also be the principle
source for glucosinolate degradation, as the degradation
rate was similar in larvae that fed on a leaf disc treated
with glucotropaeolin and larvae that were injected with
glucotropaeolin (Fig. 6B). It is not known yet, whether
glucosinolates are degraded in the hemolymph or
whether they are excreted in the hind gut and degraded
there. The slightly higher mean amount of glucosino-
lates recovered in larvae that took up glucosinolates
orally in replicates 2 and 3 is probably only due to the
fact that injection happened at time-point zero, while
feeding was continuous over 23 h.
The feeding experiment with labeled [
14
C]glucotro-
paeolin revealed that more than 80% of the radio-
activity were excreted within 14 h. As only traces of
intact glucosinolates were detectable in lled guts and
feces in experiments with unlabeled glucosinolates (see
above), the radioactivity in guts and feces must be due to
the product(s) of glucosinolate metabolism. The [
14
C]-
feeding experiment indicates furthermore that metabo-
lite(s) of the glucosinolates are not stored in the body.
The storage of glucosinolates (and their metabolites) is
thus not very efcient. In contrast, among others
several highly adapted Longitarsus spp. (Coleoptera:
ARTICLE IN PRESS
(A)
(B)
Time after injection (h)
Fig. 6. Turn-over of injected versus fed glucosinolate. Percentage of
glucotropaeolin recovered in larvae at 0, 1, 5 and 24 h after injection of
250 nmol into hemolymph (mean7se of n 8 per time point) (A) and
of injected (black bars) versus fed (gray bars) glucotropaeolin (50 or
250 nmol) after 5 h (n.s.not signicant, MannWhitney U-test,
number of replicates is given above each column (B). (1), (2): 50,
250 nmol dissolved in a volume of 5 ml, (3) 250 nmol in a volume of 2 ml.
C. Muller, U. Wittstock / Insect Biochemistry and Molecular Biology 35 (2005) 11891198 1196
Chrysomelidae) show an efcient storage of pyrrolizi-
dine alkaloids for at least 2 weeks without major losses
(Narberhaus et al., 2004).
To investigate possible turn-over mechanisms of
glucosinolates, it was investigated whether A. rosae
might possess glucosinolate-metabolizing enzyme acitiv-
ities known from other insects specialized on Brassicaceae
(Fig. 2). Although small amounts of desulfoglucosino-
lates were detected in larvae and feces, a sulfatase activity
could not be veried. No conversion of sinalbin and
glucotropaeolin into the corresponding desulfo-deriva-
tives by larval protein extracts occurred within an
incubation time of up to 1.5 h, while a clear formation
of desulfoglucosinolate was shown for protein extracts of
P. xylostella already within 3 min (Ratzka et al., 2002).
However, we cannot exclude that the sulfatase assay has
to be optimized to enable detection of sulfatase activity in
A. rosae. In contrast to P. xylostella larvae that need a
high glucosinolate sulfatase activity to deplete the plant
myrosinase of substrate (Ratzka et al., 2002), a low
sulfatase activity might be sufcient for A. rosae. It can be
assumed that the intact glucosinolates are harmless
compounds for A. rosae as long as they circulate in the
hemolymph separately from myrosinase. Thus, the small
amounts of desulfoglucosinolates in A. rosae larvae and
feces might be an intermediate in the glucosinolate turn-
over pathway or a by-product of a minor degradation
pathway.
Caterpillars of P. rapae possess a nitrile-specier
protein that converts glucosinolates into their corre-
sponding nitriles which are excreted (Wittstock et al.,
2004). When feeding on sinalbin-containing plant
material, the caterpillars excrete 4-hydroxybenzylcya-
nide sulfate (Mu ller et al., 2003a). In contrast, in feces of
the sawy larvae fed on S. alba this metabolite was not
found. Thus, at least the metabolism of sinalbin in A.
rosae does not result in the formation of the same
metabolite as in P. rapae.
Also, there was no myrosinase activity detectable in
protein extracts of hemolymph, gut tissue or the
remaining body of A. rosae. Volatile repellent products
such as isothiocyanates are not involved in the bleeding
defense mechanism of this sawy (Mu ller and Brake-
eld, 2003). In contrast to glucosinolates, isothiocya-
nates have frequently been reported to be toxic to insects
and other animals (Newman et al., 1992; Li et al., 2000).
Some specialist aphids produce their own myrosinases
which are stored in crystalline microbodies, apart from
the sequestered glucosinolates (Pontoppidan et al., 2001;
Bridges et al., 2002). In one predator of these aphids, the
hovery Episyrphus balteatus De Geer (Diptera: Syrphi-
dae), glutathione S-transferases are induced as detox-
ication enzymes when feeding on B. brassicae
(Vanhaelen et al., 2001).
To summarize, the following scenario for A. rosae can
be proposed. When feeding, most glucosinolates are
transported rapidly into the hemolymph probably at the
beginning of the digestive tract. A transporter must be
present, as glucosinolates are rather polar and can thus
not diffuse passively via the gut membrane. Further-
more, there has to be a continuous turn-over of
glucosinolates in the larval hemolymph to keep a
steady-state level. Plant myrosinases are either inhibited
or out-competed by larval enzymes that produce a
transport form. Alternatively, transport of intact
glucosinolates into the hemolymph is so fast that the
activity of plant myrosinases is severely impaired due to
a lack of substrate. The specialist A. rosae offers a
convenient system to characterize the sequestration and
metabolism as it is possible to switch larvae between
plants with different glucosinolate proles. Further
feeding experiments with labeled compounds might help
to identify the metabolite(s) of glucosinolates in A.
rosae. Nevertheless, we were able to exclude degradation
strategies that are known from other specialists on
Capparales for glucosinolate metabolism in A. rosae.
Thus, during evolution specialists have obviously found
various ways to handle the mustard oil bomb and A.
rosae must proceed yet another route.
Acknowledgements
The authors thank N. Martens and M. Hoffstadt for
technical assistance; M. Riederer for access to the HPLC
equipment in Wu rzburg and discussions; J. Gershenzon
for discussions and supporting the stay of C. Mu ller at
the MPI; B. Halkier (Royal Veterinary and Agricultural
University, Copenhagen) for transgenic lines; and N.
Agerbirk for useful comments on an earlier draft of this
manuscript. This research was in part funded by the
Sonderforschungsbereich 567 Interspezische Interak-
tionen of the Deutsche Forschungsgemeinschaft. Fi-
nancial support by the Max Planck Society is gratefully
acknowledged.
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ARTICLE IN PRESS
C. Muller, U. Wittstock / Insect Biochemistry and Molecular Biology 35 (2005) 11891198 1198
Insect
Biochemistry
and
Molecular
Biology
Insect Biochemistry and Molecular Biology 35 (2005) 11991207
Mutant Mos1 mariner transposons are hyperactive in Aedes aegypti
David W. Pledger
a,b
, Craig J. Coates
b,
a
Department of Biology (MSC-158), Texas A&M University, Kingsville, TX 78363, USA
b
Department of Entomology (MS-2475), Texas A&M University, College Station, TX 77843, USA
Received 2 April 2005; received in revised form 23 May 2005; accepted 10 June 2005
Abstract
The development of genetic strategies to control the spread of mosquito-borne diseases through the use of class II transposons has
been hampered by suboptimal rates of transformation and the absence of post-integration mobility for all transposons evaluated to
date. Two Mos1 mariner transposase mutants were produced by the site-directed mutagenesis of amino acids, E137 and E264, to K
and R, respectively. The effects of these mutations on the transpositional activities of Mos1-derived transposon constructs were
evaluated by interplasmid transposition assays in Escherichia coli and Aedes aegypti. The transpositional activities of two Mos1
transposons, one with imperfect wild type inverted terminal repeats (ITRs) and another that contained two perfectly matched 3
0
ITRs, were increased when the mutant transposases were supplied in trans in E. coli. The use of the perfect repeat transposon with
wild type transposase did not result in an increase in transposition frequency in Ae. aegypti. However, an improvement in the
integrity of the transposition process did occur, as evidenced by a lower rate of recombination events in which the transgene was
transferred. An increase in the transpositional activity of the perfect repeat transposon was observed in the mosquito in the presence
of either mutant transposase, and in the case of the E264R transposase, the observed increase in transposition frequency was also
accompanied by a further improvement in the integrity of transposition. We discuss the possible contributions of these mutant
residues to the transposition of the perfect repeat Mos1 transposon, the implications of these results with respect to the molecular
evolution of Mos1, and the potential uses of the perfect repeat transposon and mutant transposases for the improvement of Mos1
mediated germ line transformation of Ae. aegypti.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: Mos1; Mariner; Aedes aegypti; Transposon; Transposable element; Transposition assay
1. Introduction
In an attempt to stem the global rise in the incidence of
mosquito-borne diseases, a variety of avenues are being
explored in an effort to develop an efcient method of
altering the ability of a mosquito species to harbor and/
or transmit a specic disease causing pathogen (Ito et al.,
2002; Moreira et al., 2002; James, 2003; Dean and
Dobson, 2004; Kim et al., 2004; Travanty et al., 2004).
The production of transgenic mosquitoes expressing
refractory genes represents one approach, and the use of
plasmid-borne transposons to produce transgenic insects
has been well documented (for a review see OBrochta
and Atkinson, 2004). The mariner family of transposons
are class II transposable elements that have been
identied in a variety of insects, with a partial list
including Drosophila mauritiana (Jacobson and Hartl,
1985), Haematobia irritans, Oncopeltus fasciatus, Hyalo-
fora cecropia, Apis melifera, Anopheles gambiae, Ephestia
cautella (Robertson, 1993), Bactrocera tryoni (Green and
Frommer, 2001), Bombyx mori (Kumaresan and Matha-
van, 2004), Musca domestica and Blattella germanica
(Liu et al., 2004). Other mariner elements have identied
in non-arthropod invertebrates (Garcia-Fernandez et al.,
1995), as well as vertebrates (Morgan, 1995) and plants
(Jarvik and Lark, 1998).
ARTICLE IN PRESS
www.elsevier.com/locate/ibmb
0965-1748/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibmb.2005.06.002