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Bruchins: Insect-derived plant regulators that stimulate neoplasm formation


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Pea weevil (Bruchus pisorum L.) oviposition on pods of specific genetic lines of pea (Pisum sativum L.) stimulates cell division at the sites of egg attachment. As a result, tumor-like growths of undifferentiated cells (neoplasms) develop beneath the egg. These neoplasms impede larval entry into the pod. This unique form of induced resistance is conditioned by the Np allele and mediated by a recently discovered class of natural products that we have identified from both cowpea weevil (Callosobruchus maculatus F.) and pea weevil. These compounds, which we refer to as "bruchins," are long-chain alpha,omega-diols, esterified at one or both oxygens with 3-hydroxypropanoic acid. Bruchins are potent plant regulators, with application of as little as 1 fmol (0.5 pg) causing neoplastic growth on pods of all of the pea lines tested. The bruchins are, to our knowledge, the first natural products discovered with the ability to induce neoplasm formation when applied to intact plants.
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Bruchins: Insect-derived plant regulators that
stimulate neoplasm formation
Robert P. Doss*
, James E. Oliver
, William M. Proebsting
, Sandra W. Potter
, SreyReath Kuy*, Stephen L. Clement
R. Thomas Williamson**, John R. Carney
, and E. David DeVilbiss
*Horticultural Crops Research Unit, United States Department of Agriculture, Agricultural Research Service, Corvallis, OR 97330; Departments of
Horticulture and Zoology, and **College of Pharmacy, Oregon State University, Corvallis, OR 97331; §Insect Chemical Ecology Laboratory, United States
Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705; Regional Plant Introduction Station, United States Department of
Agriculture, Agricultural Research Service, Pullman, WA 99164; and ††KOSAN Biosciences, Hayward, CA 94545
Edited by Clarence A. Ryan, Jr., Washington State University, Pullman, WA, and approved March 29, 2000 (received for review February 8, 2000)
Pea weevil (Bruchus pisorum L.) oviposition on pods of specific
genetic lines of pea (Pisum sativum L.) stimulates cell division at the
sites of egg attachment. As a result, tumor-like growths of undif-
ferentiated cells (neoplasms) develop beneath the egg. These
neoplasms impede larval entry into the pod. This unique form of
induced resistance is conditioned by the Np allele and mediated by
a recently discovered class of natural products that we have
identified from both cowpea weevil (Callosobruchus maculatus F.)
and pea weevil. These compounds, which we refer to as
‘‘bruchins,’’ are long-chain
-diols, esterified at one or both
oxygens with 3-hydroxypropanoic acid. Bruchins are potent plant
regulators, with application of as little as 1 fmol (0.5 pg) causing
neoplastic growth on pods of all of the pea lines tested. The
bruchins are, to our knowledge, the first natural products discov-
ered with the ability to induce neoplasm formation when applied
to intact plants.
Cell division in plants ordinarily occurs in meristems, and the
newly formed cells differentiate to form plant tissues and
organs (1, 2). In contrast, plant neoplasms, most commonly
represented by various galls (3–5), arise when cell division is
stimulated in nonmeristematic areas (1, 2). We study an inter-
esting phenomenon observed in lines of pea (Pisum sativum L.)
that exhibit the neoplastic pod phenotype, which is conferred by
the wild-type allele, Neoplastic pod (Np), often found in pea
germplasm (6, 7). This phenotype, first noted over 30 years ago,
is typified by the formation of large patches of callus tissue
(neoplasms) on the surface of pods grown under greenhouse
conditions. Greenhouse coverings filter out the UV wavelengths
from sunlight that ordinarily inhibit neoplasm formation (7).
Plants possessing the Np gene grown under unfiltered sunlight in
the field and plants homozygous for np do not form neoplasms.
The neoplastic pod trait remained a poorly understood botanical
curiosity until recently when it was reported that the Np gene
conferred responsiveness to oviposition by the pea weevil (Bru-
chus pisorum L.), an economically important insect pest of pea,
with neoplastic growth at the sites of egg attachment (8–10).
Herein, we show that this response is a previously unidentified
form of induced resistance to an insect, and we identify the
insect-derived chemical signal that stimulates cell division and
the resultant neoplastic growth.
Materials and Methods
Plant Material and Growing Conditions. Lines of pea (P. sativum)
homozygous for either the Np or np allele were used for all
studies. For the field study described below and most bioassays,
the lines used were selected from an F
heterozygote from a cross
between C887-332 (NpNp) and I
(npnp) (10). Pods to be used
for bioassay were obtained from plants grown in a greenhouse
with set points of 16°C and 21°C. Natural sunlight was supple-
mented with light from high-pressure sodium lamps that were on
from 0600 until 2200 and a bank of eight cool white f luorescent
tubes (F96T12CWSS) that were on continuously. Such sup-
plemental lighting reduced the incidence of spontaneous neo-
plasm formation relative to that ordinarily seen on greenhouse-
grown plants (7). A field study to compare weevil infestation on
the NpNp line with that on the npnp line was conducted in 1997
with five randomized blocks with 30 plants of each line per block.
Plants were grown in raised beds on the Oregon State University
Campus where they were exposed to a natural population of pea
weevil (B. pisorum), an insect common in this area (11).
Pea Pod Bioassays. Pods in the late flat pod stage (12) were split
along the suture, and the half pod was placed in a Petri dish on
moist filter paper with the outside surface exposed (10). Chro-
matographic fractions, appropriately diluted, and synthetic com-
pounds to be tested for neoplasm-inducing activity were applied
to the pod as 1-
l drops in 50% (volvol) ethanol. The bioassays
were conducted in a growth chamber with continuous f luores-
cent light (3040 microeinstein m
) and a temperature of
23°C. The neoplastic tissue formed in 1 week was removed with
a scalpel and weighed.
Histology and Microscopy. Tissue to be used for light microscopy
was fixed in 2–2.5% (volvol) glutaraldehyde in 0.1 M phosphate
buffer, dehydrated through an alcohol series, embedded in
paraffin, sectioned at 10
m, and mounted on slides. The
deparaffinized sections were stained with a Lillie–Mayer-type
hematoxylin-eosin procedure. Specimens prepared for scanning
electron microscopy were fixed as described above, critical point
dried, and coated with gold:palladium (60:40, wtwt).
Insect Collecting and Rearing. Adult pea weevils were collected
from experimental plantings of peas in eastern Washington,
frozen, and shipped on dry ice to Corvallis, OR. Before extrac-
tion, the male and female insects were separated (13). Sexually
mature female pea weevils were also obtained by collecting
adults as they emerged from infested seed, determining their sex,
and placing the females individually into Petri dishes containing
several detached pea flowers (to serve as a source of pea pollen).
As soon as egg deposition was noted (7–10 days), the insects were
frozen and stored at 20°C.
A culture of cowpea weevils (Callosobruchus maculatus F.)
provided by W. E. Burkholder (Stored Products Insects Re-
search, U.S. Department of Agriculture, Agricultural Research
Service, Madison, WI) was increased and used to inoculate 24
20-liter containers, each holding 500 g of chickpeas (Cicer
arietinum L.). Cultures were held in the dark at room temper-
This paper was submitted directly (Track II) to the PNAS office.
Abbreviation: TMS, tetramethylsilane.
To whom reprint requests should be addressed. E-mail:
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073pnas.110054697.
Article and publication date are at www.pnas.orgcgidoi10.1073pnas.110054697
May 23, 2000
vol. 97
no. 11
ature. Adult insects were collected (by sieving) twice per week,
frozen, and stored at 80°C. Chickpeas were replenished as
required to maintain the cultures.
The vetch weevil, Bruchus brachialis F., was collected from
wild vetch, Vicia villosa Roth., in eastern Washington (10). The
bruchids Stator limbatus Horn and Stator pruininus Horn were
provided in seeds of cat claw acacia, Acacia greggii A., by C. W.
Fox (University of Kentucky, Lexington, KY).
Extraction and Isolation of Neoplasm-Inducing Compounds. The first
successful isolation began with a total lipid extraction procedure
(14) that yielded9gofaviscousyellow oil from 100 g of adult
cowpea weevils (about 22,000 insects). Flash chromatography
was conducted with2gofthis material with a 5 15-cm column
of Florisil (Sigma F-9127) and with conditions as described for
separation of simple lipid classes (15). Fractions were obtained
by stepwise elution with hexane; 5, 15, and 30% (volvol) diethyl
ether in hexane; diethyl ether; 2% (volvol) acetic acid in diethyl
ether; and methanol.
An active fraction, 0.28 g of yellow oil, eluted with diethyl
ether and acetic acid in diethyl ether, was passed over a 1
30-cm low-pressure, reversed-phase liquid chromatography col-
umn [C-18, J. T. Baker 7025-00, gradient elution starting with
85% (volvol) methanol in water and ending with 100% (volvol)
methanol]; 80 7.5-ml fractions were collected. Bioassay indicated
that activity was present in fractions 53– 67, which were pooled
(0.159 g of yellow oil).
Analysis of 10 mg of the pooled active fraction indicated that
it was comprised largely of free fatty acids that were themselves
inactive but were difficult to separate from the active materials.
Accordingly, the remaining sample was reacted with 2-bro-
moacetophenone (Sigma B3145) to form the phenacyl esters of
the fatty acids (16), thereby changing their chromatographic
behavior and facilitating their separation from the active
On repeating the low-pressure chromatographic separation
described above with the 2-bromoacetophenone-treated mate-
rial, two active samples were obtained, one eluting in fractions
5660 (2.2 mg) and the other in fractions 6467 (55 mg). The
2.2-mg fraction (light yellow oil) was subjected to TLC on a silver
nitrate-impregnated silica gel plate [Whatman K5F; layer thick-
ness 0.25 mm; plate soaked in 12.5% (wtvol) AgNO
activated at 80°C). After development, the plate edges were
removed, sprayed with H
:methanol (1:1, volvol), and
charred; 11 fractions were taken based on appearance of the
plate edges. Fraction 7 (white solid; R
0.380.42; 0.4 mg),
which was the most active on the basis of weight, was used for
structure determination.
A refined and scaled-up version of this procedure was used to
prepare additional active compounds starting with a 1,000-g
sample of cowpea weevils. A similar but much scaled-down
procedure was used to prepare active materials from a 4.96-g
sample of field-collected female pea weevils (500 insects). In
this case, HPLC was used instead of low-pressure liquid chro-
matography. The final active fractions obtained f rom pea weevils
contained too little material for accurate weighing but could be
analyzed by GC-MS.
Instrumentation. NMR spectra were obtained on a Bruker DRX
600 spectrometer (Billerica, MA). Spectra were obtained in
deuterochloroform. GC-MS was carried out with a Finnigan-
MAT (San Jose, CA) Incos-50 GC-MS with a short (15-m
0.25-mm i.d.) fused silica capillary column (DB-5,J&W
Scientific, Folsom, CA). Both electron ionization-MS (70 V;
block source temperature 150°C) and chemical ionization-MS
(ammonia or deuteroammonia as ionization gas; reagent gas
pressure 0.5 torr, 1 torr 133 Pa; block temperature 60°C)
were used.
Derivatizations and Degradation. Hydrolyses were achieved with
8% (wtvol) NaOH in 85% (volvol) methanol at 70°C for 30
min. After neutralization with 2 M HCl, solvent was evaporated,
and the residue was dissolved in ethyl acetate and passed through
a very small column of silica gel. The substances in the eluate
were analyzed by GC-MS or were derivatized for further anal-
ysis. Trimethylsilyl ethers were prepared by brief treatment with
N,O-bis[trimethylsilyl]trifluoroacetamide at 60°C. Exhaustive
hydrogenationhydrogenolysis of the hydrolysis product was
carried out over LiAlH
at 300°C (17). Ozonolyses
were conducted by collecting ozone in methylene chloride at
78°C and then treating cold methylene chloride solutions of the
substrates with a few microliters of the ozone solution. The
reactions were held outside the cooling bath for a few minutes
and then quenched with dimethylsulfide.
Synthesis. Unsaturated
-diols were synthesized by standard
routes involving acetylene alkylations and semihydrogenations
andor Wittig condensations. The (3-hydroxypropyl) esters were
initially prepared by oxidative desilylation of 3-(phenyldimeth-
ylsilyl)propanoates as described for 1,(Z)-9-docosene-1,22-diol,
1-(3-hydroxypropanoate)ester (bruchin A, see below); 9-decyn-
1-ol was deprotonated with butyllithium (two equivalents) in
tetrahydrofuran and alkylated with the tetrahydropyranyl ether
of 12-bromododecanol. The product was semihydrogenated
(Lindlar catalyst; cyclohexene as solvent), and the olefinic
alcohol was esterified with the acid chloride obtained by treating
3-(phenyldimethylsilyl) propanoic acid (18) with oxalyl chloride.
After removal of the tetrahydropyranyl group (methanol; tolu-
enesulfonic acid), the resulting monoester was treated with
fluoroboric acid etherate in dichloromethane (room tempera-
ture; 5 h). Flash chromatography on silica gel (increasing por-
tions of ethyl acetate in hexanes) was used to separate (Z)-9-
docosene-1,22-diol (from ester hydrolysis) from the desired
mono 3-(fluorodimethylsilyl)propanoate; the latter was then
stirred at room temperature in methanol-tetrahydrofuran solu-
tion containing sodium bicarbonate, potassium fluoride, and
30% (volvol) hydrogen peroxide. After work-up and f lash
chromatography [40% then 50% (volvol) ethyl acetate in
hexanes], 1(bruchin A) was obtained as a white solid (melting
point 47–48°C; shrink 45°C), after crystallization from heptane.
, 600 MHz) d 5.34 (2H, m, olefinic), 4.11 (2H,
t,J4.4 Hz, CH
R), 3.86 (2H, t, J 3.9 Hz,
OH), 3.63 (2H, t, J 4.6 Hz, alkyl-CH
OH), 2.70
(2H, t J 3.8 Hz, -O
OH), 2.09 (4H, m, allylic), 1.78
(br. s., OH). Electron ionization-MS, mz(in percentages): 412
(0.4), 124 (14), 123 (16), 121 (14), 111 (11), 109 (28), 97
(25), 96 (59), 95 (56), 94 (21), 93 (12), 91 (58), 83 (34), 82 (74),
81 (69), 80 (36), 79 (18), 73 (83), 71 (11), 69 (51), 68 (36), 67 (67),
57 (19), 56 (14), 55 (100), 54 (31), 45 (13), 43 (42), 42 (14), 41
(58). 1-bis(Trimethylsilyl) ether, electron ionization-MS mz(in
percentages): 556 [M]
(0.2), 541 (0.2), 235 (10), 219 (22),
163 (31), 149 (10), 147 (100), 109 (13), 105 (28), 103 (79), 97 (12),
96 (14), 95 (20), 91 (12), 83 (17), 82 (14), 81 (23), 75 (58), 73 (57),
69 (24), 67 (23), 55 (41), 43 (15), 41 (16).
Provides Resistance to Pea Weevil. In a field trial with near-
isogenic pea lines exposed to a natural population of pea weevil
in Corvallis, OR, the rate of infestation of npnp seed was 85.4%
versus 62.2% for NpNp seed (P0.0017; blocks 5). Neo-
plastic growth resulting from oviposition on NpNp pods was
clearly evident (Fig. 1 aand b).
Bruchids Contain Neoplasm-Inducing Activity. Application of crude
extracts, prepared with either freshly killed or frozen pea weevil
adults, to Np pea pods resulted in neoplastic growth (Fig. 1c; ref.
10). Extracts of either pea weevil eggs or accompanying fluid
Doss et al. PNAS
May 23, 2000
vol. 97
no. 11
were also mitogenic (10). The cowpea weevil, a bruchid that was
reared more easily than the pea weevil, also yielded extracts that
induced formation of neoplasms. In contrast to the pea weevil,
where earlier work had demonstrated that sexually mature
female insects were much richer sources of neoplasm-inducing
activity than were males or immature females (10), newly
emerged female or male cowpea weevils were equally good
sources of activity. Consequently, this insect was used for initial
isolation of neoplasm-inducing compounds. The sensitive and
reliable Np pod assay (10) was used to guide fractionation of
weevil extracts.
Characterization of Neoplasm-Inducing Compounds in Cowpea Weevil.
The mass spectrum of the first compound isolated (compound 1;
Fig. 2) contained a barely detectable (0.4%) molecular ion. In
many respects, it resembled the spectrum of oleyl alcohol with
the addition of the prominent ions mz73 and 91. Chemical
ionization with ammonia produced an ion with mz430, indi-
cating a molecular weight of 412. Chemical ionization-MS with
deuteroammonia demonstrated the presence of two exchange-
able hydrogens, and formation of a bis-tetramethylsilane (bis-
TMS) derivative on treatment with N,O-bis[trimethylsilyl]trif lu-
oroacetamide was consistent with this conclusion.
On alkaline hydrolysis, 1gave a new compound with mo-
lecular weight 340 whose mass spectrum lacked the ions at mz
73 and 91. Exhaustive hydrogenationhydrogenolysis (17)
yielded n-docosane, n-heneicosane, and n-eicosane, indicating
that the hydrolysis product was a docosene-1,22-diol. Indeed,
catalytic hydrogenation (PdC; 1 atm; 1 atm 101.3 kPa)
provided docosane-1,22-diol. Ozonolysis gave rise to C
hydroxyaldehydes, indicating that the diol possessed a 9-10
double bond.
The fact that the intact natural product contained two active
hydrogens, one of which must have been present in the fragment
lost during hydrolysis, suggested that the original compound
could be a monoester of either lactic acid or the much less
common 3-hydroxypropanoic acid. Published spectra of lactic
acid esters did not contain the prominent mz73 and 91 ions, and
spectra of esters of 3-hydroxypropanoic acid were not available.
Accordingly, a small sample of the hydroxypropanoate of 1-de-
canol was prepared as a model compound. Its mass spectrum
contained prominent mz73 and 91 ions, suggesting that the
natural product was a mono 3-hydroxypropyl ester of the 9-do-
A 1-(3-hydroxypropanoate) ester with a (Z) double bond at
the 9 position was prepared as illustrated in Fig. 3; a key
element in the synthesis was the use of Fleming’s masked
hydroxy technology (19) for the construction of the 3-hy-
droxypropanoate group. We initially had no way of knowing
which of the two OHs was esterified, nor did we have any
information on the geometry of the double bond. We felt,
however, that the natural product was of lipid origin and that
the double-bond geometry would, accordingly, be (Z). (More
detailed synthetic details appear in J.E.O., R.P.D., R.T.W.,
J.R.C., and E.D.D., unpublished work.) The synthetic 1ex-
hibited the same mass spectrum as the natural product, and GC
retention times of the TMS derivatives were identical. Subse-
quent NMR experiments also confirmed the identity of the
natural and synthetic compounds and supported the (Z)
geometry assigned to the double bond. Synthetic 1was as
active in the pea pod bioassay as was the isolated compound.
Fig. 1. Stimulation of cell division on pods of an Np pea line (a derivative of
C887-332; ref. 10) by several treatments. (a) Scanning electron micrograph
showing the response of a pod from an Np pea line to oviposition by a pea
weevil. The pod was obtained from a field-grown plant several days after
oviposition. E, egg; N, neoplastic tissue formed in response to oviposition. (b)
Cross section through a pod showing neoplastic tissue formed in response to
pea weevil oviposition. Pod was harvested and fixed 8 days after oviposition.
(c) Neoplasms present 1 week after application of various amounts of 2
(bruchin B; Fig. 4a), a compound present in extracts from the cowpea and pea
weevil. Amounts applied as 1-
l drops in 50% (volvol) ethanol were (from
left) 10, 5, 1, 0.5, and 0.0 pg. (Bars 100
Fig. 2. Bruchin A, a monoester bruchin isolated from a lipid extract of
cowpea weevils.
Fig. 3. Synthesis scheme for (Z)-9-docosene-1,22-diol, 1-(3-hydroxypropano-
ate)ester (bruchin A), 1.
6220 Doss et al.
A second, somewhat refined, bioassay-guided fractionation of
a 1,000-g sample of insects yielded three major active compounds
(24, Fig. 4). All possessed the mz73 and 91 ions diagnostic for
3-(hydroxypropyl) esters. All formed bis-TMS ethers whose mass
spectra contained ions with mzof 103, 145, 147, and 163.
[Although the first three of these ions are not uncommon in
spectra of TMS ethers, particularly of polyfunctional compounds
(20), collectively the four proved useful for selected ion moni-
toring-based identifications of TMS derivatives of both mono-
and bis-3-hydroxypropyl esters.] Molecular weights from chem-
ical ionization-MS were 628, 656, and 654. Hydrolyses were
conducted as described above, and the molecular weight 628
compound (2) provided the same C
diol characterized earlier.
Hydrolysis of 3and 4gave monosaturated and diunsaturated C
diols, respectively. These data, collectively, suggested that the
active compounds were bis-3-(hydroxypropyl) diesters of
diols. Ozonolyses of the C
diols demonstrated that the mono-
unsaturated compound, as was the case with the C
analyzed earlier, possessed a double bond at C
, whereas the
diunsaturated diol had double bonds at C
and C
The C
each with (Z) double-bond geometry, were also synthesized with
standard acetylene alkylations andor Wittig condensations.
Their NMR spectra confirmed the structural assignments, in-
cluding the (Z) configurations for all double bonds. These three
diesters were more abundant than the monoester characterized
from the first isolation, and we believe that the bis-3-
hydroxypropyl esters are the principal neoplasm-inducing
agents. A more detailed discussion of the characterization and
synthesis of 24(bruchins B, C, and D, respectively) is included
elsewhere (J.E.O., R.P.D., R.T.W., J.R.C., and E.D.D., unpub-
lished work).
Neoplasm-Inducing Compounds in Pea Weevil. GC-MS analysis of
fractionated whole-body extracts of adult female pea weevils
identified the same three bis-3-(hydroxypropyl) esters (24)
identified from the cowpea weevil. Monoester 1, if present, was
far less abundant.
Neoplasm Formation in Response to Synthetic Compounds. Synthetic
versions of all three diesters (J.E.O., R.P.D., R.T.W., J.R.C., and
E.D.D., unpublished work) were as active as the natural products
in the pea pod bioassays (Fig. 4). As little as 1 fmol, 0.5 pg,
when applied to an Np pea pod, resulted in neoplasms large
enough to remove from the pods and weigh after 1 week.
Physiological Activity of Bruchins. We propose the name, ‘‘bruchins’’
to refer to these neoplasm-inducing long-chain diols esterified at
one or, more commonly, both oxygen atoms with 3-hydroxypro-
panoic acid. Bruchin application to Np pods causes browning of
tissue at the treatment site within 3– 6 h after initial exposure to
the chemical. Swelling, resulting from mitosis in cells underlying
the epidermis, is visible within 2448 h (Fig. 5; ref. 10).
Neoplasms, weighing from several hundred micrograms to sev-
eral milligrams, are formed after 5–7 days (10). Bruchins do not
cause callus formation when applied to leaves or stems of pea.
Bruchin application also stimulates browning and swelling on
pods homozygous for the np allele; however, in this case, much
of the swelling results from cell enlargement rather than cell
division, and neoplasms are usually too small to remove and
weigh (Fig. 5). Pods of npnp plants typically fail to respond to
pea weevil oviposition, although barely detectable swelling may
occur under some eggs.
It has been suggested, without direct evidence, that Np is a
source of resistance to the pea weevil, a monophagous bruchid
(8, 9, 21) that is one of the most serious insect pests of peas
worldwide (22). Our use of isogenic lines confirmed such
resistance even with large weevil populations. Neoplastic
growth seems to provide resistance by reducing larval sur vival
(8, 9, 21). Ordinarily, pea weevil larvae burrow through the
ventral surface of the egg and directly into the pod to reach the
immature seed (11). In contrast, eggs on Np pods are displaced
from the pod surface by a mound of neoplastic tissue (Fig. 1
aand b). This mound causes the larvae to wander about before
Fig. 4. Structures and activities of the principal bruchins present in sexually
mature female pea weevils. Structures of bruchins B, C, and D are shown in a,
b, and c, respectively. Neoplasms (calli) resulting from application of the
indicated amounts of the bruchins were removed from pods with a scalpel and
weighed 1 week after treatment (10). Each bar represents the mean SEM for
six pods (blocks). Pea plants (derived from line C887-332; ref. 10) homozygous
for the Np gene were grown in a greenhouse with light provided by a
combination of high-pressure sodium lamps and fluorescent tubes. Bruchins
were applied as 1-
l drops in 50% (volvol) ethanol to pods at the late flat pod
stage (12).
Doss et al. PNAS
May 23, 2000
vol. 97
no. 11
attempting to burrow through the pod wall, thus exposing them
to environmental hazards including predators, parasites, and
desiccation (9, 21). Moreover, the neoplasms with attached
eggs are sometimes sloughed off of the pod before larval
emergence (8).
We originally attempted to isolate neoplasm-inducing com-
pounds from pea weevil but were unable to obtain sufficient
numbers of this insect to provide enough active material for
chemical characterization. Newly emerged female pea weevils
must ingest fresh pollen to become sexually mature (10, 13), and
only limited numbers of egg-bearing females could be collected
from the field. Fortunately, the easily reared cowpea weevil
provided a suitable alternative. Purified fractions were obtained
by using this insect, and the distinctive mass spectrum of
3-(hydroxypropanoic) esters provided the key to identifying the
Among the four bruchins described in this report, the mo-
noester (Fig. 2) was the first identified, not because it was more
abundant, but because it was the first active material obtained in
a sufficiently pure state to allow characterization. In fact,
subsequent isolations demonstrated that the diester bruchins
(Fig. 4) were more abundant than the monoester.
Thus far, bruchins have been identified in adult insects of
two bruchid genera. We have also detected strong neoplasm-
inducing activity in crude extracts of the three other bruchid
species that have been tested, namely: the vetch weevil, B.
brachialis (10); S. limbatus; and S. pruininus. Moreover, we
found that application of bruchin B (compound 2of Fig. 4) to
pods of Lathyrus tingitanus (Tangier peavine; PI 493288;
‘‘Raiano’’) results in neoplasm formation. L. tingitanus is one
of three legume species for which neoplastic growth in re-
sponse to bruchid oviposition has been reported (8 –10, 23).
Similarly, oviposition on pods of certain lines of common bean,
Phaseolus vulgaris L., by the bean-pod weevil, Apion godmani
Wagner, a nonbruchid Coleopteran, results in callus formation
that inhibits insect infestation (24). Hence, several legume
species possess similar resistance mechanisms. However,
bruchins have been identified only from pea weevil and
cowpea weevil, and it is possible that unrelated compounds
could induce the same response with peas as well as with other
It is noteworthy that pods of all pea lines tested, both Np and
np, responded to bruchin application. The response of the np
lines was attenuated, however. Only slight swelling occurred,
even when large amounts of bruchin were applied (Fig. 5). We
assume that the failure of np pods to react strongly to pea
weevil oviposition is a result of this weak response and the low
bruchin dose delivered with the egg and accompanying f luid.
We believe the bruchins to be the first regulators isolated from
natural sources that stimulate neoplasm formation in intact
plants. In peas possessing the Np allele, bruchins mediate a
sensitive and efficient form of induced resistance wherein mi-
tosis is stimulated and neoplasms form only in tissue in contact
with the insect egg. Because bruchins are synthesized by the pea
weevil at the expense of increased lar val mortality, they must be
‘‘. . . the focus of intense selection pressure. . . ’’ (25). Their
presence in the insect despite this pressure suggests that they play
an important, but as yet undefined, role in the bruchid life cycle.
There have been several reports of host-marking pheromones
associated with the Bruchidae, including C. maculatus (26).
However, preliminary tests indicate that bruchins do not play
such a role in this insect.
The bruchins are relatively stable, low-melting, white solids,
sparingly soluble in common solvents. Structurally, these
-diols, monoesterified or diesterified with 3-hy-
droxypropanoic acid, represent a previously unknown class of
compounds. In contrast to 3-hydroxybutanoic acid (27), its
relatively important higher homolog, 3-hydroxypropanoic
acid, was previously unknown as an element of natural prod-
ucts. The free acid is a hygroscopic liquid that is normally
found only as a hydrate (28) and is mentioned only infre-
quently in the literature.
Preliminary results indicate that at least one 3-hydroxypro-
panoate functionality is required for activity. For example, the
mono- and bis-(3-hydroxypropanoate) esters, bruchins A and B
(1and 2) have approximately equivalent activity, but the
itself is inactive. Although unsaturation in the diol chain is not
required and the bis (3-hydroxypropanoate) ester of docosane-
1,22-diol is fully active, the bis-propanoate, bis-lactate, and
bis-(3-hydroxybutanoate) esters of this diol are virtually inactive.
Further investigation will be necessary to identify the structural
requirements for activity.
The ability of insects to manipulate lipids chemically for their
own purposes is well established (29). It is noteworthy that the
four bruchins characterized thus far resemble the common
unsaturated fatty acids in possessing a (Z)-9 double bond.
Despite the widely separated double bonds in bruchin D (4) that
are in contrast to the usual 1,3 arrangement seen in polyunsat-
urated fatty acids, it seems likely that the bruchins are offshoots
of fatty acid synthesis or metabolism. If so, the bruchins would
join a slowly growing group of insect-produced difunctional fatty
acid derivatives with unprecedented structures such as the
defensive nitrogen-containing macrocycle from Epilachna
varivestis recently reported by Attygalle et al. (30) and the
remarkable volicitin from Pieris brassicae caterpillars. Volicitin,
like the bruchins, initiates a complex plant-signaling sequence
(31) that ultimately has a negative effect on the insect that
produced the chemical.
We thank J. K. Christian and H. Throop for technical support and John
Fellman for review of the manuscript. This work is technical paper 11642
of the Agricultural Experiment Station, Oregon State University.
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Doss et al. PNAS
May 23, 2000
vol. 97
no. 11
... Similar responses have been reported in other plant species. For example, egg deposition by B. pisorum induces neoplasia formation in Pisum sativum L. (Fabaceae) [41]. It has also been suggested that oviposited eggs can trigger the biosynthesis of plant specialized metabolites, with detrimental effects on the eggs. ...
... The molecular mechanism related to the ability to form a neoplasia and encapsulate single eggs or egg masses (clutches) in an oviposition-induced plant response is almost unknown, except for what has been reported in the pea P. sativum. The pea plant senses the oviposition fluid of B. pisorum, which contains bruchin that induces neoplastic growth, precluding the development of the larvae [41,114]. Recently, three genomic sites associated with HR-type cell death induced by eggs were reported in Brassica rapa; these regions contain cell surface receptors, intracellular receptors, and genes related to the immune response [115]. ...
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Citation: Aluja, M.; Vázquez-Rosas-Landa, M.; Cerqueda-García, D.; Monribot-Villanueva, J.L.; Altúzar-Molina, A.; Ramírez-Vázquez, M.; Velázquez-López, O.; Rosas-Saito, G.; Alonso-Sánchez, A.G.; Ortega-Casas, R.; et al. Assessment of the Molecular Responses of an Ancient Angiosperm against Atypical Insect Oviposition: The Case of Hass Avocados and the Tephritid Fly Anastrepha ludens. Int. J. Mol. Sci. 2023, 24, 2060. https:// (M.A.); (E.I.-L.) † These authors contributed equally to this work and share first authorship. Abstract: Anastrepha spp. (Diptera: Tephritidae) infestations cause significant economic losses in commercial fruit production worldwide. However, some plants quickly counteract the insertion of eggs by females by generating neoplasia and hindering eclosion, as is the case for Persea americana Mill., cv. Hass (Hass avocados). We followed a combined transcriptomics/metabolomics approach to identify the molecular mechanisms triggered by Hass avocados to detect and react to the oviposition of the pestiferous Anastrepha ludens (Loew). We evaluated two conditions: fruit damaged using a sterile pin (pin) and fruit oviposited by A. ludens females (ovi). We evaluated both of the conditions in a time course experiment covering five sampling points: without treatment (day 0), 20 min after the treatment (day 1), and days 3, 6, and 9 after the treatment. We identified 288 differentially expressed genes related to the treatments. Oviposition (and possibly bacteria on the eggs' surface) induces a plant hypersensitive response (HR), triggering a chitin receptor, producing an oxidative burst, and synthesizing phytoalexins. We also observed a process of cell wall modification and polyphenols biosynthesis, which could lead to polymerization in the neoplastic tissue surrounding the eggs.
... The active compounds, bruchins, were characterized as C22-C24 long chain α,ω-diols esterified at one or both ends with 3-hydroxypropanoic acid. [25] Oviduct secretions from the pine sawfly Diprion pini and the elm leaf beetle Xanthogaleruca luteola induce emission of volatile terpenoids that attract egg parasitoids. Recently, the nature of the defense-eliciting activity in D. pini was elucidated by fraction-Remarkably, Manduca sexta moth prefers to oviposit on Datura wrightii that is already infested by the potato beetle Lema daturaphila, to the detriment of larval performance. ...
... [49] Similarly, bruchins stimulated neoplasm formation on pea pods that physically impede larval entry. [25] In addition, oviposition by bruchids on the black gram pod triggered HR-like and accumulation of ROS but whether this impacted egg development was not tested. [65] Also, treatment of pea pods with bruchin B led to the accumulation of pisatin, a known antimicrobial isoflavone, although the role of such defense compound against eggs was not evaluated (Fig. 4). ...
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Insect eggs deposited on plants constitute a threat that has led to the evolution of sophisticated defenses. The interactions between insect eggs and plants are governed by a diverse variety of chemicals that inform butterflies about suitable hosts, repel gravid females, alert plants about the presence of an egg, act as signal molecules to induce defenses, directly impair egg development, and indirectly attract egg parasitoids. In recent years, significant progress has been made on the chemical identification, perception and role of compounds associated with oviposition. Knowledge on the genetic basis of oviposition-induced responses is also accumulating. An emerging theme is that insect eggs are not passive structures on leaves but induce complex responses that result from million years of coevolution.
... While it is admitted that nonmalignant tumors do not harm the organism in most cases, less attention has been devoted to exploring the hypothesis that they could sometimes be beneficial to their host. In plants, for example, some lineages of pea (Pisum sativum L) have developed resistance to the pea weevil (Bruchus pisorum L) by developing neoplasia under egg-laying sites, which block the larva's entry into the pod [181]. Physalis sp. and Solanum dulcamara can even kill eggs deposited by parasitic Lepidoptera (Heliothis subflexa and Spodoptera exigua respectively) by inducing specific neoplasm formations that induce egg detachment and/or poisoning through toxic chemicals [182,183]. ...
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Tumors form as a result of abnormal and uncontrolled proliferation of cells within a multicellular organism, which can lead to cancer. Although this phenomenon exists in all metazoans, most research has focused on malignancies in humans and domestic animals. Thus, the evolutionary ecology of host-tumor interactions and their consequences for ecosystem functioning is a virtually untouched area of research. To address these scientific questions, the biological model used in this thesis is a freshwater cnidarian, the brown hydra hydra oligactis, some of whose laboratory lines harbor tumors with notable specificities. In addition to the fact that the benign/malignant status of these tumors is not clear, they are capable of vertical transmission, during asexual reproduction of their host by budding. Furthermore, tumorous hydras have an increased number of tentacles compared to healthy hydras. This thesis is organized in 5 sections, an introduction with two synthesis articles, three chapters presenting the research done and a general discussion. The first synthesis deals with the comparison between benign and malignant tumors, the second one deals with the costs of anticancer defenses in host organisms. Our research on H. oligactis first described spontaneous tumors within several wild-type lineages (Chapter 1). This work shows that brown hydra tumors always appear to be of germline origin. Moreover, we show that the presence of a bacterial species of the order Chlamydiales, could play a role in the initiation and/or maintenance of these tumor processes. We then studied (chapter 2) the impact of the tumor-bacterial transmissible complex (i.e. tumor cells and bacteria) on the life history traits of the hydra host. This work shows that polyps derived from tumorous parents, prior to becoming tumoral themselves, intensify their sexual and asexual reproductive e�orts. Moreover, these tumorous polyps subsequently have a reduced survival compared to healthy ones. The adaptive nature of these life history trait changes, for the host and/or for the transmissible tumor cells, is discussed. This chapter also focuses on the origin of the increased number of tentacles in tumor polyps. By transplanting di�erent tumors into hydras of varying genetic background, we showed that polyps developing supernumerary tentacles after transplantation were always those that had received tumor tissue from hydras lines harboring transmissible tumors, and already associated with the appearance of supernumerary tentacles in their original host. Rather than a compensatory response initiated by the host, the growth of supernumerary tentacles in some tumor-bearing hydras would therefore be induced by transmissible tumor cells. Finally, in order to improve our understanding of the ecological consequences of host-tumor interactions on ecosystem functioning, we experimentally explored (Chapter 3) the relationships that tumor-bearing hydras have with other animal species living in aquatic environments. We demonstrated that, compared to healthy hydras, tumorous ones had an increased risk of predation by fish, a higher rate of colonization by commensal ciliates, and their ability to capture prey was superior due to their increased number of tentacles. Taken together, this work argues for a better consideration of tumor processes in evolutionary ecology, both in terms of the ecological and evolutionary trajectory of host species and the consequences of these interactions on ecosystem functioning.
... Apart from its acaricidal activity, Bis (dimethylethyl)-Phenol also has repellent, and oviposition deterrent properties [31]. Insect derived plant regulators, i.e., Docosene [32], were detected in corresponding strains of Bb8 (3.54%), Bb12 (8.81%), Bb15 (4.30%) and Bb21 (7.90%). Among these strains, Bb12 produced a higher amount of long chain hydrocarbons, which might have helped penetrate the outer coverage of mites. ...
... As the egg surface and egg-exterior associated secretions are in direct contact with the leaves, it could be expected that plants evolve to detect elicitors in egg-enveloping secretions. It resembles the natural situation of leaf-egg interaction, more so than, for example, crushing of eggs (Little et al., 2007;Bruessow et al., 2010) or crushing of adults (Doss et al., 2000;Yang Y. et al., 2014). Previously, a male-derived anti-aphrodisiac compound transferred during mating to the female ARG, benzyl cyanide, was suggested as potential elicitor (Fatouros et al., 2008). ...
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Plants perceive and respond to herbivore insect eggs. Upon egg deposition on leaves, a strong hypersensitive response (HR)-like cell death can be activated leading to egg desiccation and/or dropping. In Brassica spp., including many crops, the HR-like mechanism against eggs of cabbage white butterflies ( Pieris spp.) is poorly understood. Using two Brassica species, the crop B. rapa and its wild relative B. nigra , we studied the cellular and molecular plant response to Pieris brassicae eggs and characterized potential insect egg-associated molecular patterns (EAMPs) inducing HR-like cell death. We found that eggs of P. brassicae induced typical hallmarks of early immune responses, such as callose deposition, production of reactive oxygen species and cell death in B. nigra and B. rapa leaf tissue, also in plants that did not express HR-like cell death. However, elevated levels of ethylene production and upregulation of salicylic acid-responsive genes were only detected in a B. nigra accession expressing HR-like cell death. Eggs and egg wash from P. brassicae contains compounds that induced such responses, but the eggs of the generalist moth Mamestra brassicae did not. Furthermore, wash made from hatched Pieris eggs, egg glue, and accessory reproductive glands (ARG) that produce this glue, induced HR-like cell death, whereas washes from unfertilized eggs dissected from the ovaries or removal of the glue from eggs resulted in no or a reduced response. This suggests that there is one or multiple egg associated molecular pattern (EAMP) located in the egg glue a that teresponse in B. nigra is specific to Pieris species. Lastly, our results indicate that the EAMP is neither lipidic nor proteinaceous. Our study expands the knowledge on the mechanism of Brassica - Pieris -egg interaction and is a step closer toward identification of EAMPs in Pieris egg glue and corresponding receptor(s) in Brassica.
... Plant responses to insect oviposition include strategies focused on eliminating eggs, and the juveniles that hatch from those eggs. Direct mechanisms include defenses that remove or kill insect eggs, such as production of ovicides, necrosis, neoplasm formation and egg crushing or extrusion (Doss et al., 2000;Desurmont and Weston, 2011;Fatouros et al., 2012;Yang et al., 2013). Indirect mechanisms are based on egg or larval parasitoids attracted to the host plant through emission of oviposition-induced plant volatiles (OIPVs; Meiners and Hilker, 2000;Bruce et al., 2010). ...
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IntroductionInsect oviposition can enhance plant defenses and decrease plant quality in response to future feeding damage by hatched larvae. Induced resistance triggered by egg deposition and its negative effect on insect herbivore performance is known for several annual plants but has been much less studied in woody perennials, such as species of the Salicaceae. Here we studied the response of the willow Salix babylonica to oviposition by the specialist willow sawfly Nematus oligospilus and its impact on insect performance.Methods We measured the effect of oviposition on larval feeding and pupa formation and evaluated its influence on plant phytohormones and volatile emission profile.ResultsWe showed that oviposition reduced neonate larval growth and increased the proportion of prepupae that delayed their transition to pupae, thus extending the length of the sawfly cocoon phase. Oviposited willows increased jasmonic acid levels and changed their volatile profile through enhanced concentrations of the terpenoids, (E/E)-α-farnesene, (Z)- and (E)-β-ocimene. Volatile profiles were characteristic for each type of insect damage (oviposition vs. feeding), but no priming effect was found.DiscussionWe demonstrated that willows could perceive sawfly oviposition per se as a primary factor activating defense signaling via the jasmonic acid pathway. This induced response ultimately determined changes in pupation dynamics that may affect the whole insect population cycle.
... Apart from its acaricidal activity, Bis (dimethylethyl)-Phenol also has repellent, and oviposition deterrent properties [31]. Insect derived plant regulators, i.e., Docosene [32], were detected in corresponding strains of Bb8 (3.54%), Bb12 (8.81%), Bb15 (4.30%) and Bb21 (7.90%). Among these strains, Bb12 produced a higher amount of long chain hydrocarbons, which might have helped penetrate the outer coverage of mites. ...
Full-text available
A desirable substitute for chemical pesticides is mycopesticides. In the current investigation, rDNA-ITS (Internal transcribed spacer) and TEF (Transcriptional Elongation Factor) sequencing were used for molecular identification of six Beauveria bassiana strains. Both, leaf discs and potted plant bioassaye were carried out to study their pathogenicity against the cassava mite, Tetranychus truncatus . LC 50 and LC 90 values of potential B . bassiana strains were estimated. We also discovered a correlation between intraspecific B . bassiana strains pathogenicity and comprehensive metabolome profiles. Bb5, Bb6, Bb8, Bb12, Bb15, and Bb21 strains were identified as B . bassiana by sequencing of rDNA-ITS and TEF segments and sequence comparison to NCBI (National Center for Biotechnology Information) GenBank. Out of the six strains tested for pathogenicity, Bb6, Bb12, and Bb15 strains outperformed against T . truncatus with LC 50 values 1.4×10 ⁶ , 1.7×10 ⁶ , and 1.4×10 ⁶ and with a LC 90 values 7.3×10 ⁷ , 1.4×10 ⁸ , and 4.2×10 ⁸ conidia/ml, respectively, at 3 days after inoculation and were considered as potential strains for effective mite control. Later, Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the above six B . bassiana strains was done on secondary metabolites extracted with ethyl acetate revealed that the potential B . bassiana strains (Bb6, Bb12, and Bb15) have higher levels of acaricidal such as Bis(dimethylethyl)-phenol: Bb6 (5.79%), Bb12 (6.15%), and Bb15 (4.69%). Besides, insecticidal ( n -Hexadecanoic acid), and insect innate immunity overcoming compound (Nonadecene) were also identified; therefore, the synergistic effect of these compounds might lead toa higher pathogenicity of B . bassiana against T . truncatus . Further, these compounds also exhibited two clusters, which separate the potential and non-potential strains in the dendrogram of Thin Layer Chromatography. These results clearly demonstrated the potentiality of the B . bassiana strains against T . truncatus due to the occurrence of their bioactive volatile metabolome.
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The interactions between plants and herbivorous insects are complex and involve multiple factors, driving species formation and leading to the beginning of co-evolution and diversification of plant and insect molecules. Various molecular processes regulate the interactions between plants and herbivorous insects. Here, we discuss the molecular patterns of plant perception of herbivorous insect feeding through activation of early signaling components, crosstalk of plant defense network composed of multiple plant hormones, and various adaptive changes in insect responses to plant defenses. Both plant defenses and insect counter-defenses are molecular adaptation processes to each other. Molecular models of plant-herbivorous insect interactions can more intuitively help us to understand the co-evolutionary arms race between plants and herbivorous insects. These results will provide detailed evidence to elucidate and enrich the interaction network of plant-herbivorous insects.
The dried bean beetle, Acanthoscelides obtectus , is an economically important pest of stored legumes worldwide. Tracking the human‐aided dispersion of its primary hosts, the Phaseolus vulgaris beans, it is now widespread in most bean‐growing areas of the tropics and subtropics. In temperate regions where it can only occasionally overwinter in the field, A. obtectus proliferates in granaries, having multiple generations a year. Despite its negative impact on food production, no sensitive detection or monitoring tools exist, and the reduction of local populations still relies primarily on inorganic insecticides as fumigating agents. However, in the quest to produce more nutritious food more sustainably and healthily, the development of environmentally benign crop protection methods is vital against A. obtectus . For this, knowledge of the biology and chemistry of both the host plant and its herbivore will underpin the development of, among others, chemical ecology‐based approaches to form an essential part of the toolkit of integrated bruchid management. We review the semiochemistry of the mate‐ and host‐finding behaviour of A. obtectus and provide new information about the effect of seed chemistry on the sensory and behavioural ecology of host acceptance and larval development. This article is protected by copyright. All rights reserved.
This datasheet on Bruchus pisorum covers Identity, Overview, Distribution, Dispersal, Hosts/Species Affected, Diagnosis, Biology & Ecology, Natural Enemies, Impacts, Prevention/Control, Further Information.
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TheNp mutant of pea (Pisum sativum L.) is characterized by two physiological responses: growth of callus under pea weevil (Bruchus pisorum L., Coleoptera: Bruchidae) oviposition on pods, and formation of neoplastic callus on pods of indoor-grown plants. Although these two responses are conditioned byNp, they are anatomically and physiologically distinguishable, based on sites of origin, distribution pattern, and sensitivity to plant hormones. Further characterization of the response to extracts of pea weevil showed that response of excised pods, measured by callus formation, was log-linear, and treatment with as little as 10−4 weevil equivalents produced a detectable response. Mated and unmated females contained similar amounts of callus-inducing compound(s), and immature females contained significantly less of the compound(s). Female vetch bruchids (Bruchus brachialis F., Coleoptera: Bruchidae), a related species, contained callus-inducing compound(s), but usually less than pea weevils on a per weevil basis. Males of both species contained less than 10% of the activity of the mature females. Extracts of female black vine weevils, a nonbruchid species, did not stimulate callus formation. Based on partitioning and TLC analysis, the biologically active constitutent(s) was stable and nonpolar. Thus, theNp allele probably conditions sensitivity to a nonpolar component of pea weevil oviposition as a mechanism of resistance to the weevil.
Lipid decomposition studies in frozen fish have led to the development of a simple and rapid method for the extraction and purification of lipids from biological materials. The entire procedure can be carried out in approximately 10 minutes; it is efficient, reproducible, and free from deleterious manipulations. The wet tissue is homogenized with a mixture of chloroform and methanol in such proportions that a miscible system is formed with the water in the tissue. Dilution with chloroform and water separates the homogenate into two layers, the chloroform layer containing all the lipids and the methanolic layer containing all the non-lipids. A purified lipid extract is obtained merely by isolating the chloroform layer. The method has been applied to fish muscle and may easily be adapted to use with other tissues.
The bean-pod weevil (BPW), Apion godmani Wagner, often causes heavy losses in crops of common bean (Phaseolus vulgaris L.). Farmers need resistant bean cultivars to minimize losses, cut production costs, stabilize seed yield, and reduce pesticide use and consequent health hazards. To design effective breeding methods, breeders need new and better sources of resistance and increased knowledge of their modes of inheritance. We therefore: (1) compared sources of resistance to BPW, (2) studied the inheritance of resistance, and (3) determined whether the sources possess similar or different genes for BPW resistance. The following sources of resistance, originating from the Mexican highlands, were evaluated for 3 years at INIFAP-Santa Lucía de Prias, Texcoco, Mexico: 'Amarillo 153', 'Amarillo 169', 'Hidalgo 58', 'J 117', 'Pinto Texcoco', 'Pinto 168', and 'Puebla 36'. All except 'Puebla 36' were crossed with the susceptible cultivar 'Jamapa'. 'Amarillo 153' and 'Puebla 36' were crossed with another susceptible cultivar, 'Bayo Mex'. The parents, F1 hybrids, and F2 populations were evaluated for BPW damage in 1992. Backcrosses of the F1 of Jamapa/Pinto 168 to the respective susceptible and resistant parents were also evaluated in 1992. All seven resistant accessions were crossed in all possible combinations, excluding reciprocals. The resulting 21 F1 hybrids and 21 F2 populations were evaluated for BPW damage in 1994. 'J 117' had the highest level of resistance to BPW. 'Pinto Texcoco' and 'Puebla 36' had the highest mean damage score of all seven sources of resistance. The F1 hybrids between susceptible parents and resistant sources were generally intermediate. Two genes segregating independently controlled the BPW resistance in each accession. One gene, Agm, has no effect when present alone, whereas the other gene, Agr, alone conferred intermediate resistance. When both genes were present, resistance to BPW was higher. Based on mean BPW damage scores, all 21 F1 hybrids and their F2 populations, derived from crosses among seven resistant accessions, were resistant. However, data from individual plant damage scores in F2 populations of Amarillo 169/Pinto 168 and Pinto Texcoco/Pinto 168 suggested that at least one gene in each of the three accessions was non-allelic. Data also indicated that 'Amarillo 169' had a dominant gene that conferred high levels of BPW resistance, irrespective of the alleles at the other locus; and that 'Pinto Texcoco' and 'Pinto 168' possessed two different genes for intermediate resistance.
A procedure is described for the rapid preparation of phenacyl and naphthacyl derivatives of fatty acids. These derivatives of saturated, monounsaturated, and polyunsaturated fatty acids were analyzed by high-performance liquid chromatography (HPLC) on a C18 reversed-phase column at nanogram sensitivity. Standard mixtures of fatty acids gave quantitative mole percentages. HPLC analyses of phenacyl and naphthacyl derivatives of fatty acids from several seed oils compared well with values obtained by gas—liquid chromatography (GLC). Oleic and elaidic, geometrical octadecenoate isomers, were well resolved. Three of the four Δ 9,12-octadecadienoic acids, geometrical isomers of linoleic acid, were also resolved. The rapid quantitative analysis of fatty acid phenacyl derivatives by HPLC hold some advantages over GLC, especially with samples that contain heat labile and short-chain fatty acids.Phenacyl derivatives of monoenoic fatty acids were also shown to undergo cis-trans isomerization when exposed to ultraviolet light.
A stereogenic centre carrying a silyl group, a carbon substituent, and a hydrogen atom adjacent to a double bond (1) induces highly diastereoselective attack by electrophiles. The reasons for the effectiveness of this combination and its limitations are discussed, and illustrated by electrophilic substitution reactions (3) and the hydroboration (4) of allylsilanes, and enolate alkylations of esters having a β-silyl group (5). Studies directed towards the synthesis of ebelactone-a (17) illustrate how well and with what versatility the silyl group can control relative stereochemistry.
In the war between plants and plant-eating insects, the rules of conflict can get complex. In this issue, Alborn et al. (p. 945) identify a chemical signal volicitin, produced by the beet armyworm when it consumes maize plants. As discussed by Farmer in his Perspective, volicitin undoubtedly serves an important, but presently unknown, function for the insect. At the same time, however, it also elicits release of volatile molecules from the plant that attract a parasitic wasp, which consumes the beet armyworm, thus defending the plant.
In laboratory and greenhouse tests, we investigated the effects of access to sources of pollen on oögenesis, spermatogenesis, and copulation of post-hibernation pea weevils, Bruchus pisorum (L.). Also, we described the internal reproductive systems of the male and female weevils. The male weevil is sexually mature but the female is sexually immature when leaving hibernation. Females given pea, Pisum sativum L., pollen matured sexually in ca. 18 days. Females provided a mixture of alfalfa, Medicago sativa L., and other pollen for 14 and 21 days had significantly larger ovarioles than those provided with water only. Adults copulated regardless of whether they were provided or denied access to pollen, but the frequency of copulation was about twice as great when pollen was available. Females removed from hibernation in cold storage should be provided access to pea pollen for ca. 15 days before they are used in tests of host plant resistance.