Identification, RNAi Knockdown, and Functional Analysis
of an Ejaculate Protein that Mediates a Postmating,
Prezygotic Phenotype in a Cricket
Jeremy L. Marshall1*, Diana L. Huestis1, Yasuaki Hiromasa2, Shanda Wheeler1, Cris Oppert3, Susan A.
Marshall1, John M. Tomich4, Brenda Oppert1,5
1Department of Entomology, Kansas State University, Manhattan, Kansas, United States of America, 2Biotechnology Core Laboratory, Kansas State University, Burt Hall,
Manhattan, Kansas, United States of America, 3Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, Tennessee, United States of America,
4Department of Biochemistry, Kansas State University, Manhattan, Kansas, United States of America, 5USDA ARS Grain Marketing and Production Research Center,
Manhattan, Kansas, United States of America
Postmating, prezygotic phenotypes, especially those that underlie reproductive isolation between closely related species,
have been a central focus of evolutionary biologists over the past two decades. Such phenotypes are thought to evolve
rapidly and be nearly ubiquitous among sexually reproducing eukaryotes where females mate with multiple partners.
Because these phenotypes represent interplay between the male ejaculate and female reproductive tract, they are fertile
ground for reproductive senescence – as ejaculate composition and female physiology typically change over an individual’s
life span. Although these phenotypes and their resulting dynamics are important, we have little understanding of the
proteins that mediate these phenotypes, particularly for species groups where postmating, prezygotic traits are the primary
mechanism of reproductive isolation. Here, we utilize proteomics, RNAi, mating experiments, and the Allonemobius socius
complex of crickets, whose members are primarily isolated from one another by postmating, prezygotic phenotypes
(including the ability of a male to induce a female to lay eggs), to demonstrate that one of the most abundant ejaculate
proteins (a male accessory gland-biased protein similar to a trypsin-like serine protease) decreases in abundance over a
male’s reproductive lifetime and mediates the induction of egg-laying in females. These findings represent one of the first
studies to identify a protein that plays a role in mediating both a postmating, prezygotic isolation pathway and
Citation: Marshall JL, Huestis DL, Hiromasa Y, Wheeler S, Oppert C, et al. (2009) Identification, RNAi Knockdown, and Functional Analysis of an Ejaculate Protein
that Mediates a Postmating, Prezygotic Phenotype in a Cricket. PLoS ONE 4(10): e7537. doi:10.1371/journal.pone.0007537
Editor: Pawel Michalak, University of Texas Arlington, United States of America
Received September 4, 2009; Accepted September 11, 2009; Published October 23, 2009
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This work was completed with funds to JLM from the National Science Foundation (DEB-0746316) and the Kansas Agricultural Experiment Station
(contribution #09-078-J), as well as support given to the Kansas State University Biotechnology Core Facility by a NSF MRI Program Grant (#0521587). The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Answers to many of evolutionary biology’s biggest questions lie
in understanding the production and interactions of sex-specific,
reproductive tract proteins. Insights into questions such as ‘what
determines successful fertilization?’, ‘what genes underlie sperm
competition and sexual conflict?’, ‘what mechanisms influence the
evolution of postmating, prezygotic isolation?’, and ‘why do
reproductive tract genes evolve more rapidly than non-reproduc-
tive genes?’ depend on an understanding of the functions,
interactions, and evolution of reproductive tract proteins [e.g.,
1–2]. In species with internal fertilization, a particularly important
group of reproductive proteins are those that are transferred from
the male to the female during copulation – i.e., ejaculate proteins.
The importance of ejaculate proteins is easy to understand, as
sperm and seminal fluid proteins not only mediate successful
sperm-egg interactions [e.g., 3–8] but often regulate physiological
processes such as sperm storage [9–11], a male’s probability of
paternity [12–13], induction of egg-laying [13–14], female
attractiveness , and even life span .
The advent of genetic tools such as RNAi  has enabled
researchers to identify the genetic mechanisms underlying a range
of physiological traits, including egg-production and sexual
receptivity in Drosophila [e.g., 14]. Studies on insect systems
especially have benefited from RNAi technology; indeed, injection
of dsRNA or siRNA into adult or juvenile insects has been a
successful strategy to knockdown gene transcripts in a diverse
array of taxa, including aphids [e.g., Acyrthosiphon pisum; 18], beetles
[Tribolium castaneum; 19], cockroaches [e.g., Blattella germanica; 20],
field crickets [e.g., Gryllus bimaculatus; 21], fruit flies [e.g., Drosophila
melanogastor; 22], grasshoppers [e.g., Schistocerca americana; 23],
honeybees [e.g., Apis mellifera; 24], moths [e.g., Spodoptera frugiperda;
21], mosquitoes[e.g., Anopheles gambiae; 25], and termites [e.g.,
Reticulitermes flavipes; 26]. Thus, RNAi technology, in combination
with studies on ejaculate and reproductive tract proteins in insects,
offers an opportunity to assess the function of individual proteins
and their role in mediating reproductive physiologies.
Unfortunately, it is often difficult to study ejaculate proteins in
non-vertebrate systems with internal fertilization. The primary
reason for this difficulty is the all-too-often inability to collect
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whole ejaculates just prior to insemination (i.e., an ejaculate that
does not suffer from being incomplete because it is still inside the
male or being ‘‘contaminated’’ by female proteins just after
insemination). However, there are insect species, like the ground
cricket Allonemobius socius, where males produce an external
ejaculate (Fig. 1A) that is surrounded by a protective protein coat
which not only encapsulates the ejaculate, but allows for internal
insemination during copulation via a small duct (see arrow in
Fig. 1B). This entire structure, including ejaculate, is called a
spermatophore (Fig. 1B) and provides a means by which
researchers can study the contents and variation in ejaculates just
prior to insemination. With this biological feature in hand,
ejaculate proteins can be studied both independently and in the
context of protein-protein interactions within the female’s
The A. socius complex of crickets (including the species A. socius,
A. fasciatus, and A. sp. nov. Tex) has been a model system within
ecology and evolutionary biology for nearly three decades and has
been at the forefront of studies assessing the importance of
postmating, prezygotic reproductive isolation [27–32]. Indeed,
research has shown that postmating, prezygotic phenotypes, such
as conspecific sperm precedence [CSP; 28–30] and the ability of a
male to induce a female to lay eggs [27,30,31], isolate species in
this complex, while phenotypes such as calling song , mating/
courtship behavior , phenology [35–37], and postzygotic
phenotypes  do not. Additionally, research on the effects of
male age on ejaculate composition and postmating, prezygotic
phenotypes has uncovered several patterns. Specifically, one of the
most abundant proteins in the ejaculate, initially called protein
‘‘X’’, decreases with male age (Fig. 2A, B). Older males are also
less able to induce females to lay eggs (Fig. 2C) – a form of
reproductive senescence. Together, these data suggest the
hypothesis that the abundance of protein ‘‘X’’ underlies a male’s
ability to induce a female to lay eggs. If confirmed, this protein
would not only be linked to male reproductive senescence, but also
as a critical player in one of the postmating, prezygotic phenotypes
that isolate species in this complex of crickets.
Here, using the A. socius complex of crickets, our goals were to
identify protein ‘‘X’’ using biochemical and genetic analyses,
sequence and clone the full length transcript that produces protein
‘‘X’’, assess tissue- and sex-specificity of this transcript, and use
RNAi technology to knockdown transcript expression and
evaluate the phenotypic effects. We found that protein ‘‘X’’ is a
male accessory gland-biased protein exhibiting the molecular
features of a trypsin-like serine protease. Additionally, we provide
evidence that this protein mediates a male’s ability to induce a
female to lay eggs – which is a phenotype that contributes to both
male reproductive senescence and postmating, prezygotic repro-
Identification of an abundant ejaculate protein
Protein ‘‘X’’ (Fig. 2), an approximately 29 kDa protein, was
excised from a 1D-SDS-PAGE gel, trypsin digested, and analyzed
via MALDI-TOF and MALDI-TOF/TOF-MS analyses. Seven
high intensity MS peaks of peptides were then subjected to MS/
MS analysis, with the resulting peptide sequences searched against
the male reproductive accessory gland EST database from A.
fasciatus (GenBank accession numbers: EG018565-EG019055)
using MASCOT (http://www.matrixscience.com). Four of the
seven peptides (Table 1) corresponded to a single transcript
derived from a contig of seven A. fasciatus ESTs (GenBank
EG018669, EG018803, EG018819, EG018935). The amino acid
sequences of these four peptides exhibited 100% sequence identity
to peptides from this EST contig and had significant MASCOT
MS/MS scores (Table 1).
From this male accessory gland EST contig, we developed
primers to amplify the entire coding region of this transcript (see
Methods below). Following PCR amplification of this transcript
from male accessory gland cDNA, we cloned and sequenced the
resulting product (NCBI accession #GQ911573). The gene
nucleotide sequence from these clones was identical to that of
the EST contig and was translated to a 313 amino-acid residue
sequence [Fig. 3; the four significant peptide matches are denoted
1 through 4 (as in Table 1) with the sequences being in bold and
underlined]. The translated sequence contains a signal peptide,
presumably cleaved between Ala45and Phe46(SignalP 3.0
probability =0.957), resulting in a mature protein of 268
amino-acid residues (Fig. 3). The calculated molecular mass of
this protein, 29.3 kDa, was in agreement with the estimated mass
based on migration in the 1D SDS-PAGE gel (Fig. 2A).
A BLASTP search identified a trypsin-like serine protease motif
between residues Ile66and Met300, beginning with the conserved
N-terminal motif IVGG (Fig. 3; box around IVGG motif). This
Figure 1. The spermatophore. The spermatophore is the sperm-
and accessory gland fluid-filled package that male crickets produce and
hold externally (A) prior to copulation. During copulation, the male
transfers the spermatophore (B) to the female via threading the tip of
the spermatophore duct (see arrow in B) into the reproductive tract of
the female. The white material inside the spermatophore in (B) is the
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protein contains the catalytic triad His106, Asp157, and Ser251
characteristic of serine proteases in the S1 family (numbering
based on the uncleaved protein; Fig. 3). These catalytic residues
were found in the highly conserved motifs TAGHC, DIAL, and
GDSGGP, respectively (Fig. 3; ). Based on the above features,
we propose that this protein is a functional trypsin-like serine
Given that this transcript appears to be a trypsin-like serine
protease and such proteases are common in many tissues, we
evaluated the tissue-and sex-specificity of this and other trypsin-
like serine proteases. To accomplish this, we developed conserved
primers in the IVGG and DIAL amino acid regions (see Methods
below). These primers amplified trypsin-like serine proteases from
the following tissues: male accessory gland, male testis, male
thorax, male digestive tract, female spermatheca, female ovaries,
female thorax, and female digestive tract. Next, we conducted
59RACE using primers in the IVGG and DIAL regions as the
inner and outer primers respectively (see Methods below), and
found considerable variation between different tissues and males
and females (Table 2). Specifically, from a total of 137 sequenced
clones, we identified 19 unique trypsin-like serine protease
sequences, and significantly, the male accessory gland yielded a
single transcript that was not found in the other male tissues nor in
any tissue from females (sequence #5, Table 2). This particular
transcript also has not been found in our testis and female
reproductive tract EST libraries, which consist of .30,000 EST
generated with 454 sequencing (unpubl. data). Although this
particular trypsin-like serine protease appears unique to the male
accessory gland, it is still possible that it is a rare transcript in these
other tissues and within females. Therefore, we suggest that this
transcript is at least male- and accessory gland-biased and
potentially uniquely expressed in this tissue.
As for amino-acid sequence similarity to proteins from other
organisms, our BLASTP search found a nearly identical transcript
Table 1. Peptides generated with MS/MS analyses and their percent match to an EST contig from the male reproductive accessory
Peptide #Peptide Sequence
% Match toEST
containing this peptide
1KEDLTVVLGLHDR13 100% 58 EG018587, EG018935, EG018803
2 GQDIYADQVAFVTGWGR17 100%150 EG018599, EG018669, EG018819
3VEQIGVVSWGIGCAR 15100% 70 EG018591, EG018669, EG018819
4PGMPGVYTTVSYYLDWIR 18100% 20EG018591, EG018669
Note: Individual ion scores from the MS/MS analysis that are .56 indicate significant homology (P,0.05).
Figure 2. Protein ‘‘X’’ and the effects of male age. The decrease in abundance of protein ‘‘X’’ in a male’s ejaculate is associated with increased
male age (A; each lane represents the protein from a single spermatophore), while (B) demonstrates the statistical significance of this relationship
(each point represents the data from a single spermatophore). (C) The association between male age and a male’s ability to induce a female to lay
eggs – suggesting that the relative abundance of protein ‘‘X’’ may be linked to a male’s ability to induce a female to lay eggs.
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in the field cricket (Gryllus firmus) EST library from the male
reproductive accessory gland (1e2144; Table 3). In G. firmus, this
appears to be a male-biased transcript (e.g., AG-0308F; Fig. 2 in
ref. ) that, like in Allonemobius, is produced in the male
reproductive accessory gland. This A. socius transcript also is
similar to S1 family serine proteases in other insects (Table 3).
Given the male-biased expression of this transcript in the accessory
gland and its presence in the male ejaculate, we name the gene
encoding this transcript ejaculate serine protease – with the protein and
gene being abbreviated EJAC-SP and ejac-sp, respectively.
RNAi knockdown and functional analysis of ejac-sp
We developed ejac-sp specific primers to amplify a 99 bp region
that is 59 to the conserved IVGG site (Fig. 3) and unique to this
transcript (see Methods below). We made ejacsp-dsRNA, following
standard protocols, and injected it into the abdomen of adult male
crickets (see below). We found that males injected with ejacsp-
dsRNA produced spermatophores with significantly reduced levels
of the EJAC-SP protein in their ejaculates, relative to the saline
controls (F2,45=22.1, P,0.00001; Fig. 4A&B). Significant knock-
down was seen 3 days post-injection and was still evident 6 days
post-injection (additional experiments, data not shown, suggest
that RNAi knockdown is effective for 12 days post-injection and
can last the entire reproductive life of the male). At 6 days post-
injection, ,79% (15 of 19 individuals) of saline-injected males
produced spermatophores with an EJAC-SP relative abundance of
1.1, while none (0 of 16 individuals) of the ejacsp-dsRNA injected
males produced spermatophores with relative abundances this
Figure 3. Amino acid sequence of protein ‘‘X’’. The features of the protein ‘‘X’’ sequence include a predicted signal peptide (the sequence 59 of
the arrow) and cleavage site (at the arrow), a trypsin-like N-terminal motif (open box around IVGG), the three catalytic residues (white letters) in the
three conserved motifs (gray boxes) of a serine protease, and the four peptides recovered from MS/MS analysis (labeled 1–4 and highlighted in bold
Table 2. BLASTP analysis of the full length transcript underlying protein ‘‘X’’.
Scientific NameCommon NameGenBank accession #E-valueGene Identity
Gryllus firmussand field cricket ABG758401e-142 hypotheical Acp
Gryllus pennsylvanicus fall field cricket ACD695158e-117 trypsin-like serine protease
Nasonia vitripennisjewel wasp XP_0016062678e-76hypotheical protein
Bombyx mori domestic silkworm NP_0011536758e-76male reproductive organ serine protease 2
Tribolium castaneumred flour beetleXP_9741135e-50 similar to oviductin
Drosophila melanogasterfruit flyNP652645 2e-46 CG18735
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high (Fig. 4A). Conversely, ,56% (9 of 16 males) of ejacsp-dsRNA
injected males produced spermatophores with EJAC-SP relative
abundances of less than one, while none of the saline-injected
males had abundances this low (0 of 19 males; Fig. 4A).
The above data suggest that our RNAi approach was successful
in knocking down the abundance of EJAC-SP protein in the male
ejaculate. To evaluate knockdown in the male accessory gland,
testes, digestive tract, and thorax, we conducted quantitative real-
time PCR (qPCR) using conserved trypsin-like serine protease
primers for the target gene and actin primers for the housekeeping
gene (see Methods below). We found significant knockdown of
trypsin-like serine proteases in the male accessory gland of ejacsp-
dsRNA injected males (t=3.52, P=0.0022, n=11 per treatment
for all tissues; Fig. 5A). This significant difference translated into a
63-fold average difference between the ejacsp-dsRNA and saline
injected treatments. However, there were no significant differences
between the ejacsp-dsRNA- and saline-injected treatments for the
remaining three tissues (Fig. 5B–D; testis: t=1.44, P=0.1643;
digestive tract: t=1.63, P=0.1184; thorax: t=1.84, P=0.0810).
The phenotypic effect of knocking down ejac-sp expression,
yielding reduced amounts of EJAC-SP protein in the male
ejaculate, was a significantly reduced ability to induce a female
to lay eggs [number of eggs laid per day: msaline=16.93 (n=10),
mejacsp-dsRNA=12.73 (n=11); tm1.m2=1.925, P=0.0346; Fig. 6].
This knockdown represented an approximately 25% reduction in
egg-laying rates relative to the saline control. Overall, there was a
significant, linear relationship between the relative abundance of
EJAC-SP in the male ejaculate and the number of eggs laid per
day by females (F1,19=13.06, P=0.0018; r=0.638; Fig. 6). These
results are consistent with the hypothesis that the abundance of
protein ‘‘X’’ (identified here as EJAC-SP) mediates a male’s ability
to induce a female to lay eggs.
Our primary goals for conducting this research were to identify
protein ‘‘X’’, which we now refer to as EJAC-SP (a product of the
gene ejaculate serine protease), and determine if the correlation
between decreased levels of EJAC-SP in the male ejaculate and
decreased ability of males to induce females to lay eggs are causally
linked or each independently related to male age (Fig. 2). With
regard to protein identity, we found that EJAC-SP has molecular
characteristics of a trypsin-like serine protease and that the ejac-sp
transcript exhibits a male, reproductive accessory gland-biased
pattern of expression (Table 3). Indeed, ejac-sp appears to be
exclusively expressed in the accessory gland of males, but further
research is needed to distinguish between ‘‘biased’’- and
‘‘exclusive’’- expression. Such sex-biased expression is not
uncommon, especially for reproductive tract genes [e.g., 40–42].
To address the question of ejac-sp function, we evaluated the
utility of a common RNAi protocol. We found that injecting gene-
specific dsRNA (i.e., ejacsp-dsRNA) into the abdomen of adult male
crickets resulted in a significant, 63-fold decrease in transcript
abundance of trypsin-like serine proteases in the male accessory
gland, but no significant knockdown in other male tissues (Fig. 5).
This significant knockdown of transcript expression resulted in a
significant decrease of EJAC-SP protein levels in the male
ejaculate (Fig. 4) – even though all males in the saline and ejacsp-
dsRNA treatments were between 14 and 22 days post-eclosion
(i.e., reproductively young males given sexual maturity is usually
Table 3. The occurrence of specific sequences encoding putative trypsin-like serine protease in various male and female cricket
Male TissuesFemale Tissues
ACGTestes ThoraxDig. TrackSpmthca.OvariesThorax Dig. Track
#Sequence (20 bp 59 of IVGG)N(%)N(%)N(%)N(%)N(%)N (%)N (%)N(%)
1 GAAGACATCGCCCTCATCCG5 38.516.7940.93 18.816.3
2 TGTATTCCCCGATGTGTCTT2 13.3
ACG = reproductive accessory gland; Dig. Tract = digestive tract; Spmthca. = spermatheca.
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reached 10 to 14 days post-eclosion). In terms of RNAi
effectiveness, at least 56% of dsRNA-injected males exhibited
significant knockdown of EJAC-SP, as these males had EJAC-SP
relative abundances lower than any saline-injected male. Togeth-
er, these results suggest that injecting gene-specific dsRNA into the
abdomen of adult Allonemobius crickets can knockdown the
abundance of a targeted transcript within a specific tissue,
indicting the occurrence of a systemic RNAi pathway. Once
again, this is not unexpected given that a systemic RNAi response
has been found in other Orthopterans [e.g., crickets  and
grasshoppers ] and basal insects [e.g., cockroaches  and
As for phenotype, we found a significant, linear relationship
between EJAC-SP abundance in a male’s ejaculate and a male’s
ability to induce a female to lay eggs (Fig. 6). This relationship
parallels the hypothesized relationship derived from naturally-
occurring variation linked to male age (Fig. 2). Moreover, RNAi
knockdown of ejac-sp expression, and the resulting reduction in
EJAC-SP protein, yields the predicted decrease in a male’s ability
to induce a female to lay eggs (Fig. 6). These results support a
causal link between EJAC-SP abundance in the male ejaculate and
a male’s ability to induce a female to lay eggs.
The occurrence of ‘‘egg-laying induction’’ proteins in the
ejaculate of male insects is expected, as several have been
identified in Drosophila [reviewed in 43]. The fact that a serine
protease was identified as an important part of the male’s egg-
Figure 4. RNAi and the knockdown of EJAC-SP protein in the
male ejaculate. The relative abundance of EJAC-SP protein in saline-
and ejacsp-dsRNA-injected males at three and/or six days post-injection
(A; a and b refer to statistically different groups based on post-hoc tests,
P,0.00001). The gel (B) is a representative 1D-SDS PAGE gel of proteins
from individual spermatophores from males in both the saline- and
ejacsp-dsRNA treatments at $6 days post-injection. The EJAC-SP
protein and the protein used as a control are outlined in boxes.
Figure 5. Real-time PCR of trypsin-like serine protease in each
tissue for each injection treatment. The expression, as quantified
by DCt via qPCR, of trypsin-like serine protease relative to b-actin (the
control gene) in the saline- and ejacsp-dsRNA-injected males (gray and
open bars, respectively) for the male reproductive accessory gland (A),
testis (B), digestive tract (C), and thorax (D). Significant knockdown only
occurred in the male accessory gland. Sample size equals 11 in both
treatments for all tissue types.
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laying induction pathway is also consistent with previous research,
as serine proteases and serine protease inhibitors play critical roles
in many postmating, prezygotic phenotypes [reviewed in 44].
Although more work is needed to identify the function and
biochemical target of EJAC-SP, we hypothesize that its function is
to cleave a specific protein in the female reproductive tract that
initiates the egg-laying pathway; we are currently working to assess
the function and biochemical target of EJAC-SP.
The natural decrease in EJAC-SP abundance as males get older
not only helped identify this protein as an egg-laying induction
candidate, but also represents a form of reproductive senescence.
Such senescence is widespread among animals and typically results
in decreased fitness of older males [e.g., 45–47]. The form of
reproductive senescence seen in Allonemobius, that of a changing in
the quality or size of ejaculate as males age, can be found in animals
ranging from insects [e.g., 48,49] to mammals [e.g., 50,51] and
would appear to be a common form of male reproductive
senescence. Unfortunately, except for the literature on the genetic
basis of reproductive senescence in human males [e.g., 51], relatively
few genes and proteins that underlie male reproductive senescence
have been identified and functionally evaluated. Therefore, to our
knowledge, EJAC-SP is one of the first non-mammalian proteins to
be directly linked with male reproductive senescence.
The link between EJAC-SP abundance to a male’s ability to
induce a female to lay eggs provides the first insight into the genes,
and resulting proteins, that mediate one of the main postmating,
prezygotic phenotypes that reproductively isolate species in the
Allonemobius socius complex of crickets. The proposed serine
protease activity of EJAC-SP may or may not contribute to the
species-specific ability of conspecific males to induce females to lay
eggs – although we are currently evaluating this possibility. As
stated above, we are in the process of identifying the function and
target of EJAC-SP and once identified, we will be in a position to
evaluate the degree to which species-specificity of this postmating,
prezygotic phenotype is controlled by structural variation in the
EJAC-SP protein. However, regardless the role played by EJAC-
SP in reproductively isolating species, it does play a role in this
postmating, prezygotic phenotype and is a part of the molecular
pathway that underlies a component of reproductive isolation – a
pathway that through the ‘‘magic’’ of biochemistry we will be able
to identify the species-specific components.
In conclusion, the above results indicate that RNAi can be a
useful technique to manipulate the ejaculate proteins of adult male
crickets. This tool enables researchers to assess the function and
importance of individual ejaculate proteins in determining
fertilization success, the outcome of sperm competition, and the
genes that underlie postmating, prezygotic isolation. Moreover,
given that non-genetic model insects, particularly crickets,
butterflies, and water striders [reviewed in 52&53], are key model
systems for addressing questions about sexual selection and sexual
conflict, the success of this technique demonstrates the power of
combining proteomic/biochemical techniques and RNAi technol-
ogy to address these types of questions in systems that have been
studied for decades, yet currently lack a sequenced genome.
Materials and Methods
Spermatophores: sample preparation, SDS-PAGE, and
estimates of abundance
Spermatophores were collected from males (both A. socius and A.
sp. nov. Tex were used for all analyses) non-destructively, thus
allowing multiple spermatophores to be collected from individual
males throughout their reproductive lives. Specifically, a single
male was paired with a single female and allowed to proceed
through the normal courtship ritual [outlined in 54]. During this
courtship ritual, males produce a spermatophore and hold it
externally with their genitalia (Fig. 1). Once produced, it takes
approximately 12 minutes for the outer protein coat of the
spermatophore to harden and become structurally sufficient for
successful ejaculate transfer. Following this time period, males will
initiate copulation via a ritualized calling song and copulation
dance. Just prior to copulation, we would disturb the courtship,
anesthetize the male with CO2, remove the spermatophore, and
store it at 280uC until used for protein analysis.
Prior to gel electrophoresis, spermatophores were ground and
sonicated in 15–20 mL of purified water and then centrifuged for
30 sec at 12,000 rpm. The protein content of the resulting
supernate was quantified with a ND-1000 (NanoDrop Industries,
Wilmington, DE) spectrophotometer by A280. A typical spermato-
phore contained 30–80 mg of water-soluble protein.
For protein quantification, 25 mg of spermatophore proteins
were separated on 4–12% Bis-Tris SDS-PAGE (using an
Invitrogen NuPAGE system) following manufacturer’s protocols
(Fig. 2A). After staining with Coomassie Blue and de-staining with
water, gel images were digitized with a Kodak Gel Logic 200. The
amount of EJAC-SP (Fig. 4B) was compared to a control protein
Figure 6. Phenotypic effects of ejac-sp knockdown. The abundance of EJAC-SP protein in a spermatophore (taken from a male just prior to
being allowed to copulate successfully with a virgin female) predicts a male’s ability to induce a female to lay eggs. The difference between saline-
and ejacsp-dsRNA injected males was also significant (P=0.0346).
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band, which was the 31 kDa protein band just above EJAC-SP
(Fig. 4B, see box; which MALDI TOF/TOF MS indicates is
sperm specific – data not shown). Protein bands were quantified
by densitometry using ImageJ 1.37v software from NIH
(rsbweb.nih.gov/ij/). Specifically, the amount of protein was
estimated as maximum gray value for each protein band. The
relative amount of EJAC-SP for each spermatophore was
calculated as ‘‘EJAC-SP abundance/control protein abundance’’.
Protein identification with MALDI-TOF/TOF MS and MS/
After staining gels with Coomassie G-250, the selected gel band
(protein ‘‘X’’ in Fig. 2A) was excised as 1–2 mm diameter pieces
and transferred to a 1.5 mL Eppendorf tube. A protein-free region
of the gel was also excised as background control. The control and
test gel sections were destained using three 30 min washes of
60 mL 1:1 acetonitrile: water at 30uC. Gel pieces were then dried
for 10 min under vacuum. The gel sections were subjected to
reduction and alkylation using 50 mM Tris (2-carboxyethyl)
phosphine (TCEP) at 55uC for 10 min followed by 100 mM
iodoacetamide in the dark at 30uC for 60 min. The carbox-
ymethylated gels were thoroughly washed and re-dried in vacuo,
then incubated with sequencing grade trypsin (Trypsin Gold,
Promega, Madison, WI), 20 ng/mL in 40 mM ammonium
bicarbonate, in 20 mL. Upon rehydration of the gels, an additional
15 mL of 40 mM ammonium bicarbonate and 10% acetonitrile
was added, and gel sections were incubated at 30uC for 17 h in
sealed Eppendorf tubes. The aqueous digestion solutions were
transferred to 1.5 mL clean Eppendorf tubes, and tryptic
fragments remaining within the gel sections were recovered by a
single extraction with 50 ml of 50% acetonitrile and 2%
trifluoracetic acid (TFA) at 30uC for 1 h. The acetonitrile fractions
were combined with previous aqueous fractions and the liquid was
removed by speed vacuum concentration. The dried samples were
resuspended in 10 mL of 30 mg/mL 2,5-dihydroxylbenzonic acid
(DHB) (Sigma, St. Louis, MO) dissolved in 33% acetonitrile/0.1%
TFA and 2 mL of peptide/matrix solution was applied on a Bruker
Massive Aluminum plate for MALDI-TOF and TOF/TOF
MS and MS/MS analysis - Mass spectra and tandem mass spectra
were obtained on a Bluker Ultraflex II TOF/TOF mass
spectrometer. Positively charged ions were analyzed in the
reflector mode. MS and MS/MS spectra were analyzed with
Flex analysis 3.0 and Bio Tools 3.0 software (Bruker Daltonics).
Measurements were externally calibrated with 8 different peptides
ranging from 757.39 to 3147.47 (Peptide Calibration Standard I,
Bruker Daltonics) and internally re-calibrated with peptides from
the autoproteolysis of trypsin. Peptide ion searches were
performed with EST_others_20080308 in NCBInr database (as
well as an EST database specific to these crickets) using MASCOT
software (Matrix Science). The following parameters were used for
the database search: MS and MS/MS accuracies were set to
,0.6Da, trypsin/P as an enzyme, missed cleavages 1, carbami-
domethylation of cysteine as fixed modification, and oxidation of
methionine as a variable modification. Homology of the predicted
protein sequence was searched in NCBI database with Blast 2.0.
Cloning the full-length transcript of ejac-sp
Using a contig of ejac-sp ESTs derived from the male
reproductive accessory gland EST library (GenBank accession
numbers: EG018587, EG018591,
EG018803, EG018819, EG018935), we developed primers to
sequence the entire coding region (forward primer: Ovi-Full-F2,
CGCTTCTGACAGCCATGC; reverse primer, Ovi-R-985a,
CGCTACTCCTTATCCGTACCTTGCT). These primers were
used with standard PCR reaction chemistry for a 50 mL reaction
(outlined in 31), 100 ng of male accessory gland-specific first-
strand cDNA [generated by isolating RNA with an Ambion
RNAqueous-4PCR kit and standard protocols for 1st-strand
cDNA synthesis (i.e., using 8 mL of the total RNA solution, 5 mL
5X RT buffer, 1.3 mL dNTP’s, 0.7 mL rRNasin, 1 mL M-MLV
reverse transcriptase, 2 mL of poly-T primer and nuclease-free
water to 20 mL - all reagents from Promega, Madison, WI)], and a
thermocycler profile of 94uC for 2 mins, 30 cycles of 94uC for
30 sec, 55uC for 30 sec, 72uC for 1 min, and a final extension
period of 72uC for 7 min. The resulting PCR product was run on
a 1% agarose gel and gel extracted using a Qiagen QIAquick Gel
Extraction Kit. The cleaned PCR product was cloned using a TA
CloningH Kit (Invitrogen) and sequenced with standard M13
forward and reverse primers.
Tissue- and sex-specificity of ejac-sp and other trypsin-
like serine proteases
To determine the presence of trypsin-like serine protease
transcripts in a variety of male and female cricket tissues (i.e.,
male accessory gland, male testis, male thorax, male digestive
tract, female spermatheca, female ovaries, female thorax, and
female digestive tract), we developed nucleotide primers in the
conserved amino-acid motifs IVGG and DIAL (forward primer in
IVGG region, ovi F 230con, ATCGTCGGGGGCACAATC;
reverse primer in DIAL region, ovi R 500con, CGGATGAG-
GGCGATGTCTTC). These primers yield a ,290 bp fragment
that was present in all tissues sampled from both sexes. Next, we
utilized the ovi R 500con primer (i.e., the reverse primer in the
DIAL region) and the reverse complement of the ovi F 230con
primer (i.e., ovi R 230con, GATTGTGCCCCCGACGAT, which
is in the IVGG region) as the gene-specific outer and inner
primers, respectively, for 59RACE for each tissue within each sex.
For 59RACE, we utilized the FirstChoiceH RLM-RACE kit from
Ambion. Following 59RACE on all eight samples, we cloned (using
the TA Cloning Kit) and sequenced the resulting products. We
sequenced 10 to 30 clones per sample with an average of 17 (i.e.,
137 sequenced clones in total). The resulting sequences were
analyzed for the occurrence of unique sequence 59 to the
conserved IVGG region.
Preparation and injection of dsRNA
Following the identification of a unique region 59 of the IVGG
site for the ejac-sp transcript, we developed ejac-sp specific primers
to amplify a 99 bp fragment in this unique region (forward primer,
ovi F 140, TACTCATCTTGGTGGCCTG; reverse primer, Ovi
R 209, GTGTTGAGACACCGTCAGACA). We added the T7
promoter sequence (TAATACGACTCACTATAGGGAGA) to
the 59 end of each primer. Our final 59 primer was TAATAC-
and our final 39 primer was TAATACGACTCACTATAGGGA-
GAGTGTTGAGACACCGTCAGACA (with the underlined
sections being the T7 region). These primers were used with
standard PCR reaction chemistry for a 50 mL reaction (same as
above), 1 mg of male accessory gland-specific first-strand cDNA
(isolated as above), and a thermocycler profile of 94uC for 2 min,
30 cycles of 94uC for 30 sec, 55uC for 30 sec, 72uC for 1 min, and
a final extension period of 72uC for 7 min. The resulting PCR
product was isolated from a 1% agarose gel using a Qiagen
QIAquick Gel Extraction Kit. The cleaned PCR product was
subjected to a standard ethanol precipitation to yield a final
concentration of greater than 111 ng/mL, as measured by a ND-
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PLoS ONE | www.plosone.org8October 2009 | Volume 4 | Issue 10 | e7537
The Ambion T7 MEGAscript kit was used to generate ejacsp-
dsRNA via RNA transcription. We followed the specified
manufacturer’s protocol, except that we increased reactions to
30 mL for a higher yield of dsRNA. We also used 1 mg of template
DNA (i.e., our concentrated PCR product). The RNA transcrip-
tion reaction was incubated at 37uC for 14–16 h. Following
incubation, the reaction was subjected to DNase treatment
following T7 MEGAscript kit guidelines. The dsRNA was then
cleaned using Ambion’s MEGAclear Kit, resulting in a ready-to-
inject dsRNA solution. The concentration of dsRNA was checked
with a ND-1000 spectrophotometer and the volume adjusted to a
final concentration of 1 mg/mL.
For injections, we used a manual injection system consisting of a
syringe and disposable glass needles. We injected adult males in
the abdomen with 1 mL of either ejacsp-dsRNA or saline depending
on the experimental treatment. To accomplish the injections, we
anesthetized adult males with CO2and performed the injections
under a dissection microscope. After injection, males were placed
in individual cages with ample water, food, and cover. No increase
in mortality was observed following injection of saline or dsRNA.
To determine if gene-specific dsRNA could knockdown the
abundance of ourtarget protein(EJAC-SP) in the spermatophore, we
injected males (from a Texas population of A. sp. nov. Tex) with either
days post-injection, we began collecting spermatophores from each
male (as outlined above). Between days six and eight we allowed both
ejacsp-dsRNA- and saline-injected males to mate once with a virgin
female (as above; all females were 17 to 27 days post-eclosion). Males
were frozen at 280C following a successful copulation. Females were
given seven days to lay eggs before being scored for possible female
in her abdomens and those laid was less than 20; most females have
.80 eggs). Sterile females were removed from all analyses. Based on
the number of eggs laid, we scored females as either having been
induced to lay eggs or not. Many times a successful copulation and
ejaculate transfer does not result in a female laying any eggs (i.e., a
successful copulation with the female having laid few eggs despite
many in her abdomen). To remove the effects of these females from
our analyses, females had to lay more than 5 eggs per day – which is
about the maximum number of unfertilized eggs a virgin female will
lay per day.
Protein from each spermatophore for males from each treatment
EJAC-SP (as described above). The relative abundance of EJAC-SP
between the saline and ejacsp-dsRNA treatments was evaluated with
an ANOVA. Also, quantitative real-time PCR (qPCR) was used to
evaluate the degree of trypsin-like serine protease knockdown in the
accessory gland, testis, digestive tract, and thorax (see below). Finally,
we analyzed the relationship between relative expression of EJAC-SP
(i.e., the number of eggs laid per day by females). The difference in
egg-laying rates between treatments were analyzed with a one-tailed
t-test, as we were specifically interested in the question: does
knockdown of EJAC-SP protein levels in the male ejaculate result
in females laying fewer eggs than expected from matings with saline-
injected males? The overall relationship between the relative amount
of EJAC-SP and egg-laying rate was assessed with a regression
Real-time quantitative PCR (qPCR)
Males were frozen immediately at 280uC after obtaining the final
spermatophore. Accessory glands, testes, the digestive tract, and the
thorax were dissected from individual males (i.e., the males that
induced females to lay eggs and were used in the phenotype analysis)
and total RNA was isolated using the RNAqueous 4-PCR kit from
Ambion.Kit manual instructions were followed;includingtheDNase
I treatment to remove any DNA from the sample, and final RNA
volume was 75 mL. cDNA synthesis was performed on each sample
using standard protocols (see above). The concentration of cDNA
resulting from these reactions was measured using a NanoDrop ND-
1000. Nuclease-free water was added to obtain a concentration of
,800 ng/mL. qPCR reactions were carried out using a BioRad
iCycler iQ multicolor Real-Time PCR detection system using
standard protocols, including three technical replicates per reaction.
To determine the correct dilution of cDNA for each tissue type for
qPCR a dilution series of 1:10, 1:50, and 1:250 was conducted. The
conserved ovi F 230con and ovi R 500con mentioned above (gene
abbreviated as SP in subsequent analyses). These conserved primers
were used because they amplify trypsin-like serine proteases in all
tissue types, not just the maleaccessory gland. Moreover, given ejac-sp
is a male accessory gland biased transcript, primers specific to this
transcript would not amplify products in the other tissues, thus
eliminating our ability to test if ejacsp-dsRNA’s effect was specific to
the trypsin-like serine proteases in the male accessory gland or all
tissues. We found that a dilution of 1:10 worked for all tissue types
except the male accessory gland where a dilution of 1:250 was used.
This was repeated for the control gene, b-actin (sense primer,
AACTGGGACGACATGGAGAAGAT; anti-sense primer, GCC-
AAGTCCAGACGC AGGAT), and similar dilutions were used for
values between 90 and 103% for both genes in all tissues except for
theserine proteaseprimerinthedigestive tract (efficiency =115.8%).
As for analyses, we conducted qPCR on each of the four tissues
from 11 individuals in each of the two treatments (22 individuals in
total; saline- and ejacsp-dsRNA-injected males at $6 days post-
injection). A Ct value (i.e., the PCR cycle number where
amplification causes the amount of product to cross a set threshold)
was calculated for each gene and tissue for each individual
(important note: a higher Ct value means a lower amount of gene
product in the sample). A DCt was calculated as CtSP– Ctactin,
resulting in trypsin-like serine protease values being corrected for by
the amount of b-actin in the sample. Positive values mean the Ct
value was higher for the trypsin-like serine protease and thus less
transcript than b-actin in the sample; the converse is the case if the
value was negative. A two-tailed t-test was used to compare this
metric between the treatments for each tissue type, as any potential
bias in protocol or primer efficiencies would be similar within each
tissue type. For significant differences, the formula 2n, where n is the
average number of cycles different between treatments, was used to
estimate the fold difference between treatments.
The authors thank S. Kambhampati and four anonymous reviewers for
reviewing an earlier version of this manuscript, as well as technical help
from the Kansas State University Biotechnology Core Facility. Mention of
trade names or commercial products in this publication is solely for the
purpose of providing specific information and does not imply recommen-
dation or endorsement by the U.S. Department of Agriculture.
Conceived and designed the experiments: JLM. Performed the experi-
ments: JLM DLH YH SW CO SAM. Analyzed the data: JLM YH.
Contributed reagents/materials/analysis tools: JLM JMT BO. Wrote the
paper: JLM DLH JMT BO.
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