SYSTEMATICS

Molecular and Morphological Characters Discriminate

Sitophilus oryzae and S. zeamais (Coleoptera: Curculionidae)
and Confirm Reproductive Isolation

PURNAMA HIDAYAT,1 THOMAS W. PHILLIPS,2- 3 AND RICHARD H. FFRENCH-CONSTANT1

Ann. Entomol. Soc. Am. 89(5): 645-652 (1996)

ABSTRACT The Rice Weevil, Sitophilus oryzae (L.), and the maize weevil, S. zeamais Mot-
schulsky, are sibling species of grain weevils that are usually distinguished by grain preferences
and subtle differences in morphology. Previous findings of successful laboratory hybridization,
genetic similarity of allozymes and chromosomes, and identity of aggregation pheromones
raised questions about the validity of S. oryzae and S. zeamais as reproductively isolated
biological species. We used molecular techniques to test the hypothesis that individuals as-
signed as S. oryzae or S. zeamais by morphological criteria represent members of 2 distinct
gene pools, and hence are reproductively isolated species. Weevils from 18 different localities,
which were collected from Africa, Australia, Asia, the south Pacific, and North America, were
studied. All individuals were scored for the presence or absence of morphological characters
that have been used historically for species determination. In most cases, determinations for
the same individual were not consistent, depending on the morphological character used. The
polymerase chain reaction (PCR) was used on the same specimens to analyze randomly am-
plified polymorphic DNA (RAPD-PCR) markers and to amplify selectively regions of mito-
chondrial DNA for analysis of restriction site polymorphisms with restriction endonucleases
(RFLP-PCR). Both methods yielded markers that were consistently associated with either
presence or absence of specific genitalic characters in both males and females. This correlation
of molecular markers with genitalic morphotypes was consistent in all specimens studied,
whether collected sympatrically from the same farms or from widely separated geographic
populations, and supports a model of 2 reproductively isolated species. Other morphological
characters involving pronotal punctures proved unreliable as correlates with genetic markers
and are not useful for species diagnosis.

KEY WORDS stored products, grain, mitochondrial DNA, polymerase chain reaction

THE RICE WEEVIL, Sitophilus oryzae (L.), and the
maize weevil, S. zeamais Motschulsky, are sibling
species that are difficult to identify. Although S.
oryzae and S. zeamais are both internal grain feed-
ers with similar outward appearance, the species
have been considered distinct based on body size,
food preferences, mating incompatibility, and mor-
phology of the pronotum and genitalia (Floyd and
Newsom 1959). Of the morphological characters
proposed for distinguishing S. oryzae and S. zea-
mais, Kuschel (1961) and Halstead (1963) con-
eluded that only the male aedeagus and the female
Y-shaped sclerite (formed by the spiculum ventrale
and fused hemisternites) gave consistent separa-
tions (see also Kuschel 1978). Halstead (1963) as-
signed identifications to Sitophilus specimens in
the worldwide collections of his institution and of
'Department of Entomology, University of Wisconsin, Madison,
Wi 53706
2
Stored-Product Insect Research Unit, USDA-ARS, Depart-
ment of Entomology, University of Wisconsin, Madison, WI
53 6
3™ - , , , , , , ,, , T . . „ .f
J
To whom correspondence should be addressed: Tropical Fruit
and Vegetable Research Laboratory, USDA-ARS, P.O. Box 4459,
Hilo, HI 96720.
the British Museum, but no independent measure
was used to test the aedeagal characters against,
nor was any mention made of, the 2 morphotypes
being found in the same collections. Boudreaux
(1969) reported on characters associated with
punctation of the pronotum that purportedly sep-
arated S. oryzae and S. zeamais. Boudreaux (1969)
developed his study of characters from a single lab-
oratory culture each of S. zeamais and S. oryzae,
but he used his method to discriminate nearly
isOO specimens in the U.S. National Museum and
n o t e d that the 2 species occurred together in the
same accessions.
Studies on controlled matings, genetics, and
pheromones led us to question the validity of S.
oryzae a n d s zeamais a s rep roductively isolated
s p e c i e s i n n a t u r e F i hybrids were produced in
the laboratory by several researchers (Richards
, « , , ,7 . { inon n- A. ±. l i r»r»i \
1944, Yang et al. 1989, Pmtureau et al. 1991).
Smith (1953) reported that S. oryzae and S. zea-
mais had apparently identical karyotypes of 2N
= 22 (confirmed by Yang et al. 1989), whereas
. l i - n i - . n •/ T \ I I
the
morphologically distinct S. granariUS (L) had
a karyotype of 2N = 24. Beiras and Petitpierre
646 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA
S. oryzae
75\im
Fig. 1. Scanning electron micrograph (SEM) of dorsal surface of phallus (male aedeagus) in S. oryzae (left) and
S. zeamais (right); note longitudinal grooves on S. zeamais and smooth surface on S. oryzae.
(1981) analyzed 6 allozyme loci in 3 species of
Sitophilus and found for S. oryzae and S. zea-
mais that populations were monomorphic for the
same electromorphs at all loci except for ester-
ases, for which there were polymorphisms and
frequency differences. Pintureau et al. (1991)
successfully hybridized S. oryzae and S. zeamais
in ==10—20% of crosses from broadly different
geographic strains, and they demonstrated her-
etibility of esterase alleles in hybrid offspring.
Grenier et al. (1994) studied esterase polymor-
phism in 34 strains of S. oryzae and S. zeamais,
and grouped strains into species based on simi-
larity in allele frequencies; there were no diag-
nostic alleles that were fixed in one species and
not the other. All samples analyzed in the elec-
trophoretic studies described above were from
laboratory cultures of various origins, none were
field-collected in sympatry, and no a priori meth-
od of species determination (e.g., morphology)
was used before allozyme analyses. Thus, cyto-
logical and allozyme studies reveal a close ge-
netic relationship between S. oryzae and S. zea-
mais, and controlled mating studies suggest the
potential for hybridization in the field. Walgen-
Vol. 89, no. 5
S. zeamais
75|im
bach et al. (1983) reported that S. oryzae and S.
zeamais were mutually cross-attracted in labo-
ratory bioassays of a male-produced pheromone,
and males of both species were found to produce
the same pheromone compound (reviewed in
Burkholder 1990). Use of the same pheromone
by S. oryzae and S. zeamais suggests that there
may be limited behavioral barriers to interbreed-
ing in nature.
In the current study we used molecular mark-
ers to determine if weevils identified morpholog-
ically as either S. oryzae or S. zeamais are ge-
netically distinct. We aimed to collect
morphologically distinct individuals, represent-
ing each presumed species, in sympatry so that
the potential for hybridization would be high.
Throne and Cline (1989, 1991) documented the
seasonal flight activity and occurrence of S. ory-
zae and S. zeamais at the same field sites in
South Carolina, and made identifications of
trapped specimens using Halstead's (1963) gen-
italic characters. Therefore, we knew weevils
possessing morphological characters representa-
tive of each species could be collected sympat-
rically, and tests of reproductive isolation be-
September 1996 HlDAYAT ET AL.: DISCRIMINATION OF Sitophilus SPECIES 647

Table 1. Collection localities, numbers of specimens examined, and determinations of specimens as S. oryzae or
S. zeamais using morphological characters
Species determination based on morphological characters
Male aedeagus/
Locality Type n" female Y- Shape of Puncture-
shaped :sclerite punctures free zone No. punctures DNA samples"
O Z O Z O Z O Z RAPD RFLP
Australia, no. 1 L 20 0 20 0 20 6 14 5 15 5 5
Australia, no. 2 L 20 20 0 18 2 18 2 16 4 5 5
Australia, no. 3

i—i
L 25 25 0 21 4 24 1 24 5 5
Indonesia, Bogor L 57 12 45 1 56 22 35 39 18 0 4
Burma L 20 20 0 16 4 18 2 19 1 5 5
China, Chengdu F/L 20 0 20 0 20 7 13 10 10 17 5
China, Wuhan F/L 25 0 25 1 24 3 22 17 8 25 7
New Caledonia F 13 0 13 0 13 3 10 6 7 12 9
Pakistan, Kashmir F/L 22 0 22 0 22 3 19 10 12 19 18
Tanzania, no. 1 L 20 20 0 12 8 19 1 18 2 4 5
Tanzania, no. 2 L 20 0 20 0 20 7 13 11 9 5 5
USA, Nebraska, Butler County F/L 20 0 20 0 20 0 20 4 16 11 6
USA, KS, Pottawatomie County F/L 20 20 0 1 19 17 3 18 2 20 19
USA, Kentucky, Frankfort F 21 0 21 1 20 1 20 8 13 19 21
USA, S. Car., Barnwell Co. F 45 7 38 0 45 8 37 23 22 6 8
USA, S. Car., Bamberg Co. 1 F 20 20 0 7 13 12 8 20 0 17 10
USA, S. Car., Bamberg Co. 2 F 83 14 69 1 82 18 65 33 50 54 51
USA, Wisconsin, Dane Co. F 33 0 33 0 33 15 18 12 21 22 4
Totals 504 158 346 79 425 201 203 293 211 251 192

Types of collection with regard to breeding history: F, field collected and frozen immediately; F/L, recently field collected and
maintained in the laboratory no more than 1 yr; L, laboratory strain maintained for more than 1 yr, usually with limited collection
data, n, Total number of specimens subjected to morphological analysis and from which DNA samples were derived.
" Numbers of specimens from the sample used for morphology that were subjected to each of the DNA analyses; RAPD-PCR was
performed with primer UBC-431, and RFLP-PCR with Msp-1 restriction enzyme (see text for details).

tween morphotypes using genetic markers were gitudinal puncture-free zone on the pronotum, and
possible. we counted the number of punctures longitudi-
nally along the midline of the pronotum (<20 for
S. oryzae and >20 for S. zeamais). The shape of
Materials and Methods pronotal punctures, either elliptical for S. oryzae
In total, 504 specimens from 18 different field or circular for S. zeamais, were also recorded. All
collections or laboratory strains were analyzed morphological characters were determined using a
morphologically, and a subset of each of these was dissecting microscope, and each specimen was
subjected to molecular analysis. Field collections uniquely identified for cross-referencing with mo-
were made from in-grain samples or grain/bait lecular data.
bags (Throne and Cline 1991) on farms or other DNA Markers. The polymerase chain reaction
facilities with grain storages. Weevils were received (PCR) was used for 2 techniques to obtain genetic
in our laboratory alive, determined to be either S. markers for this study. Total genomic DNA was
oryzae or S. zeamais (and not the potentially coin- extracted from single specimens using previously
festing S. granarius), analyzed for pronotal mor- described methods (Ashburner 1989). We used the
phology, dissected for genitalia, and then frozen in technique of randomly amplified polymorphic
individual containers at — 80°C for subsequent DNA (RAPD-PCR; Williams et al. 1990) and
DNA extraction. screened over 50 randomly designed 10-base prim-
Morphology. For each specimen we recorded ers from the University of British Columbia RAPD
the morphological characters described by Hal- Primer Synthesis Project. One primer, having the
stead (1963) for the genitalia and Boudreaux sequence 5'-CTGCGGGTCA-3' and referred to
(1969) for the pronotum, and we assigned species here as UBC-431, was subsequently selected and
determinations based on each character set. The used routinely because it produced consistently
presence, in S. zeamais, or absence, in S. oryzae, clear and interpretable banding patterns. For the
of ridges on the dorsum of the male aedeagus (Fig. PCR reaction, =10 ng of sample DNA was com-
1), and the shape of the tips on the invaginated Y- bined with 0.2 [iM of the primer, 0.5 U of Taq
shaped 8th sclerite of the female, which are pur- polymerase enzyme (Promega, Madison, WI), IX
ported to be rounded and thick on S. oryzae or Taq buffer provided by the manufacturer, 2 mM
pointed and thin on S. zeamais (Fig. 2), were de- of MgCl2) and 0.2 mM of dNTPs in a total reaction
termined following dissection. For the pronotal volume of 25 ;ul PCR was conducted for 40 cycles
with an initial temperature of 94°C for 2 min, then
characters (Fig. 3), we scored the presence, in S.
denaturation at 94°C for 1 min, cooled to 36°C for
oryzae, or absence, in S. zeamais, of a median lon-
648 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA
5. oryzae
250 |im
Fig. 2. Y-shaped sclerite of invaginated 8th sternite (spiculum ventrale) from female S. oryzae and S. zeamais;
note shape (rounded or pointed) and thickness of distal processes.
annealing, then extension at 72°C for 1 min. PCR
products were separated by electrophoresis on 2%
agarose gels at 90 V for —90 min, then visualized
under UV light following ethidium bromide stain-
ing.
Restriction fragment-length polymorphisms
(RFLP-PCR, Simon et al. 1993) were analyzed us-
ing genomic DNA from the same individuals used
for morphology and RAPD-PCR. In RFLP-PCR a
specific sequence is amplified with PCR, digested
with a specific restriction endonuclease, then elec-
trophoresed to score fragments and the presence
or absence of restriction sites. We amplified a mi-
tochondrial DNA (mtDNA) sequence of =1,635
bp spanning the cytochrome oxidase subunits I and
II (COI, COII) genes, corresponding to the loca-
tion at 2,188-3,771 bp in the sequence of Dro-
sophila yakuba Burla (Clary and Wolstenholme
1985), using primers from conserved regions, 5'-
CAACATTTATTTTGATTTTTTGG-3' and 5'-GA-
GACCATTACTTGCTTTCAGTCATCT-3'. These
2 primers have been used successfully in other re-
cent insect studies (e.g., Sperling and Hickey 1994,
Langor and Sperling 1995). The PCR reaction mix-
ture consisted of 5 /u-1 of 10 X Taq buffer, 5 /u-1 of
Vol. 89, no. 5
S. zeamais
250
MgCl2 (25 mM), 4 /il of dNTPs (2 mM), 31.6 fi\
of water, 2 fi\ of each primer (10 fiM), and 0.4 fx\
of Taq polymerase (5 U//U.1). PCR was conducted
for 35 cycles of 94°C for 1 min of denaturation,
45°C for 1 min of annealing, and extension at 72°C
for 1 min. Two microliters of the PCR product
were digested with 0.5 fi\ of a given restriction en-
zyme mixed with 10 /AI of water and 1.5 fjd of the
manufacturer's buffer for 2 h at temperatures rec-
ommended by the manufacturer. Seven restriction
enzymes (below) were screened based on cost,
availability, and the likelihood that they might di-
gest our samples. The digests were visualized un-
der UV light after electrophoresis on 2% agarose
gels at 90 V for 90 min and staining with ethidium
bromide.
Results
Determinations of specimens as either S. oryzae
or S. zeamais using morphological characters were
not consistent for any of the populations studied
(Table 1). Of the 504 specimens examined, 31%
were determined to be S. oryzae based on male
genitalia or female Y-shaped sclerite; but use of
September 1996 HIDAYAT ET AL.: DISCRIMINATION OF Sitophilus SPECIES 649

S. oryzae S. zeamais

0.5 mm 0.5 mm
Fig. 3. Morphology of pronotal punctures used for diagnosis (Boudreaux 1969) of S. oryzae and S. zeamais; note
the puncture-free zone near the midline of pronotum on S. oryzae and larger number of predominantly circular
punctures in S. zeamais; punctures on the pronotum of S. oryzae are more elliptical.

pronotal puncture shape, presence or absence of a
pronotal puncture-free zone, and the number of
punctures counted along the midline of the pro-
notum determined 16, 40, and 58%, respectively,
of the same group as S. oryzae. Thus, in many
cases the same specimen could be determined as
either S. oryzae with one character or S. zeamais
using another character.
DNA analysis revealed diagnostic markers that
were consistently associated with weevils possess-
ing particular morphological characters. When
subjected to RAPD-PCR using the UBC-431
primer, all male weevils with a smooth dorsal sur-
face on the aedeagus (Fig. 1) or females with
rounded lobes on the spiculum ventrale (Fig. 2)
also had a bright band at —1,400 bp that was lack-
ing in individuals with grooved aedeagi or pointed
lobes on the Y-shaped sclerite (Fig. 4). Therefore,
all weevils we studied with genital morphology
consistent with Halsteads (1963) concept of S. ory-
zae possessed the RAPD-PCR product at 1,400 bp,
and those with S. zeamais genital morphology
lacked this product. Several smaller bands were of-
ten produced with the UBC-431 primer, but they
were polymorphic and none was clearly associated
with a particular morphotype. None of the pron-
otal characters was correlated with any RAPD-
PCR markers or with other morphological char-
acters.
Analysis with RFLP-PCR also provided diagnos-
tic markers that were consistently associated with
the character states of the male genitalia or the
female Y-shaped sclerite, but not with pronotal
characters. Amplification of mtDNA using specific
primers spanning the COI and COII genes yielded
a single product of «1,635 bp for the 192 speci-
mens studied (Table 1). When this product was
digested with Mspl enzyme all specimens possess-
ing S. oryzae genitalic (male) or Y-shaped sclerite
(female) morphology revealed 2 digest products of
=1,325 and 310 bp, indicating a single restriction
site, whereas weevils possessing the alternate mor-
phological character states of S. zeamais retained
the undigested 1,635 bp product (Fig. 5). Four ad-
ditional restriction enzymes also provided diagnos-
tic markers correlated with genital morphology of
a subsample of specimens when they were reacted
with mtDNA from the COI and COII genes.
650 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 89, no. 5

S. oryzae S. zeamais
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1,635 bp

1,018 bp

516 bp

Fig. 4. Agarose gel with RAPD-PCR products from Sitophilus weevils with primer UBC-431. Individuals are
grouped according to their possession of genital morphology (aedeagus of males, Y-shaped sclerite of females) of S.
oryzae or S. zeamais. Lanes 1-4 from Kansas collection, lanes 5-8, and 9-12 were collected from the same farm in
Bamberg County, South Carolina, lanes 13-16 from Kashmir, Pakistan, collection. Lane 17 is a control PCR reaction
with no added template DNA, and the unlabeled lane to the far right is a 1-kb ladder for molecular weight estimation.

EcoRI did not digest mtDNA from S. oryzae spec- these field sites contained single breeding popu-
imens, but had 1 restriction site in S. zeamais spec- lations of weevils, and successful matings occurred
imens with a product at —1,500 and 100 bp. Rsal between weevils with different genitalic morphol-
had a single restriction site for each species, pro- ogy, then we would have expected to see a random
ducing products of 1,500 and 100 bp for S. oryzae association of molecular markers with weevils of
and 1,300 and 300 bp for S. zeamais. Apal also different morphotypes. The correlated molecular
had a single restriction site for each species, with and morphological markers were consistent across
products of =1,000 and 600 bp for S. oryzae mor- widely separated geographic populations, and in 1
photypes and 1,100 and 500 bp for those with S. mixed laboratory colony (Bogor, Indonesia). A sin-
zeamais morphology. Finally, Ddel had 2 restric- gle character, whether morphological or molecular,
tion sites in S. oryzae specimens, yielding products would be inadequate for concluding the presence
at 900, 400, and 300 bp, but did not digest mtDNA of 2 species because an alternate hypothesis of
from S. zeamais. The restriction enzymes EcoRV polymorphism within a single species could be
and Dral were also studied, but they did not pro- made. Our data are particularly strong in support-
vide diagnostic markers. ing a 2-species model because we have 3 presum-
ably independent character sets, 1 morphological,
and 2 molecular, that are associated nonrandomly
Discussion in 2 distinct groups of specimens.
Our results show that S. oryzae and S. zeamais Although our data indicate that S. oryzae and S.
exist as genetically distinct species. Specimens can zeamais are reproductively isolated species, the
be identified using genitalic characters, and these mechanism or mechanisms that maintain isolation
species are reproductively isolated when they oc- are unknown. Although earlier studies generated a
cur sympatrically. In 2 of our field collections from low level of presumed interspecific hybrids in the
South Carolina (Barnwell County and Bamberg laboratory, it is likely that postmating isolation is
County 2), weevils of both genital morphotypes not complete. Although S. oryzae and S. zeamais
with correlated molecular markers were found. If use the same aggregation pheromone, it is possible
September 1996 HlDAYAT ET AL.: DISCRIMINATION OF Sitophilus SPECIES 651

S. oryzae S. zeamais
1 2 3 4 5 6 7 8 9 10 11 12 13 14

1,635 bp

1,018 bp

516 bp

Fig. 5. Restriction digest of 1,635 bp PCR product from mtDNA of Sitophilus weevils using the enzyme Mspl.
Species designations refer to determinations from genital morphology. Digestion products only occur for S. oryzae,
the undigested product is shown in all S. zeamais lanes. Lanes 1-3 are from Australia, lanes 4-6, and 7-9 were
collected from the same farm in Barnwell County, South Carolina, and lanes 10-12 are from New Caledonia; lanes
13 and 14 represent undigested PCR product from S. oryzae and S. zeamais, respectively. The unlabeled lane to the
far right is a 1-kb ladder for molecular weight estimation.

there are many other behavioral mechanisms that
prevent interspecific matings in nature. From our
18 widely distributed collections it appears that the
2 species rarely cooccur. Even in the 2 South Car-
olina populations that harbored both species, each
was dominated by S. zeamais, suggesting the pos-
sibilities of competitive displacement or niche par-
titioning not detected by our sampling. There is
some evidence from South Carolina for seasonal
differences in activity between the species. The
Bamberg County 1 population was collected on 6
November 1992, and all 20 weevils were S. oryzae.
Bamberg County 2 was collected from the same
farm on 23 June 1993, >7 mo later, and contained
a mix of the 2 species predominated by S. zeamais.
Throne and Cline (1991) also found a predomi-
nance of S. zeamais at this same site over a 3-yr
period, but did report periods of several weeks
when S. oryzae predominated. Mechanisms main-
taining reproductive isolation and distribution of S.
oryzae and S. zeamais require further study.
This study shows that the genital characters of the
male aedeagus and the female Y-shaped sclerite are
sufficient for determining specimens as either S.
oryzae or S. zeamais. Although we did not examine
type material of these species to aid in assigning
names to specimens with correlated morphological
and molecular characters, we based our determi-
nations on the findings of Kuschel (1961, 1978),
who referred to type material, and Halstead (1963).
The pronotal characters of Boudreaux (1969) are
clearly inadequate and should not be used for spe-
cies determinations. Host affinities are also not re-
liable to aid in species determinations. Although dif-
ferences in host preferences may occur between the
species (e.g., Coombs and Porter 1986), and our
own collections (Table 1) included populations of S.
oryzae infesting wheat and populations of S. zea-
mais from corn, we also had collections of S. zea-
mais that were infesting wheat. Current findings
(P.H., unpublished data) suggest that body size is
also of no use in making species determinations. S.
oryzae and S. zeamais are among the most serious
stored grain pests worldwide, and proper identifi-
cation of specimens is critical. Use of the reliable
genitalic characters studied here will be important
for future studies of ecology, behavior, and pest
management in this group.
Acknowledgments
We | ;reatly appreciate field collection of weevils by
Wendel 1 Burkholder (USDA-ARS in Madison, WI), John
652 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 89, no. 5

Sedlacek (Kentucky State University, Frankfort, KY), and
David Weaver (USDA-ARS in Gainesville, FL). James
Baker (USDA-ARS, Manhattan, KS), Isham Harahap
(Bogor Agricultural University, Bogor, Indonesia), and
Jane Wright (Commonwealth Scientific and Industrial
Research Organisation, Canberra, NSW, Australia) kindly
provided various laboratory strains. We are particularly
grateful to David Langor (Canadian Forestry Service, Ed-
monton, AB, Canada) for suggesting the analyses of
mtDNA and for providing us with his methods-. We thank
David Langor, Felix Sperling (University of California,
Berkeley, CA), and James Throne (USDA-ARS, Man-
hattan, KS) for providing reviews of an earlier draft of
the manuscript. This work was funded in part by a Re-
search Support Agreement between USDA-ARS and the
University of Wisconsin at Madison, and by a USDA
grant to R.H.f-C.
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Received for publication 21 February 1996; accepted
12 June 1996.