Current address: Department of Biology, College of Wooster, Wooster, Ohio, United States of America
Current address: Center for Vector Biology and Zoonotic Diseases, The Connecticut Agricultural Experiment Station, New Haven, Connecticut, United States of America
Current address: AAAS-NSF S&T Policy Fellow, American Association for the Advancement of Science, Washington, D. C., United States of America
Conceived and designed the experiments: LKS MEHH MFW LCH. Performed the experiments: LKS MEHH MK. Analyzed the data: LKS MCH JMCR PD. Contributed reagents/materials/analysis tools: MFW LCH. Wrote the paper: LKS MCH MEHH JMCR MFW LCH. Generated databases of predicted peptides for protein discovery, and generated output for each of these proteins on predicted protein domains, homologs, and other features: JMCR.
The authors have declared that no competing interests exist.
No commercially licensed vaccine or treatment is available for dengue fever,
a potentially lethal infection that impacts millions of lives annually. New
tools that target mosquito control may reduce vector populations and break
the cycle of dengue transmission. Male mosquito seminal fluid proteins
(Sfps) are one such target since these proteins, in aggregate, modulate the
reproduction and feeding patterns of the dengue vector,
Using a stable-isotope labeling method coupled with proteomics to distinguish
male- and female-derived proteins, we identified Sfps and sperm proteins
transferred from males to females. Sfps were distinguished from sperm
proteins by comparing the transferred proteins to sperm-enriched samples
derived from testes and seminal vesicles. We identified 93 male-derived Sfps
and 52 predicted sperm proteins that are transferred to females during
mating. The Sfp protein classes we detected suggest roles in protein
activation/inactivation, sperm utilization, and ecdysteroidogenesis. We also
discovered that several predicted membrane-bound and intracellular proteins
are transferred to females in the seminal fluids, supporting the hypothesis
that
This is the first study to directly identify Sfps transferred from male
Dengue is a potentially lethal infection that impacts millions of humans
annually. This disease is caused by viruses transmitted by infected female
Male seminal fluid proteins (Sfps) influence female reproductive and feeding
behaviors in a range of insects studied to date (reviewed in
Understanding mosquito reproductive biology is critical to developing effective
vector control methods. Previous research on
Previously, we identified over 250 proteins from male
In the process of identifying the Sfps, we also identified a subset of 52 putative
In order to distinguish male- and female-derived proteins in the mated female
reproductive tract, we adapted a stable-isotope labeling technique that had
been developed for
The Liverpool strain of
15N-labeled virgin females (3–5 days after emergence) were individually introduced into a 5 L bucket container that contained 20–40 unlabeled males (4–6 days after emergence). If a successful mating event was observed, both the female and male were removed from the bucket at the termination of mating as the pair began to separate. After mating, each female was placed individually in a test-tube and stored on ice for dissection. Females that did not mate within approximately 5 min were removed from the male cage and discarded; a new female was introduced into the male cage for mating. Dissections of the 15N-labeled females mated to unlabeled males were conducted within 30 min of mating.
Female lower reproductive tracts (i.e., spermathecae, bursa, common oviduct) were dissected in MOPS buffer (80 mM NaCl, 10 mM KCl, 1 mM CaCl2, 0.2 mM MgCl2,10 mM MOPS) on ice and homogenized in 20 µl Dulbecco's PBS (DPBS) with protease inhibitors (Roche Complete Protease Inhibitor Tablets, Indianapolis, IN). Reproductive tracts from 20 females were pooled for each biological replicate. Samples were centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was transferred to a separate tube, the pellet was resuspended in 20 µl DPBS with protease inhibitors, and 20 µl of 2× SDS sample buffer (125 mM Tris–HCl pH 6.8, 20% glycerol, 4% SDS, 10% β-mercaptoethanol, 0.001% bromophenol blue) was added to each sample. The samples were boiled for 4 min and stored at −80°C.
In order to distinguish Sfps from sperm proteins, we conducted proteomic analyses of sperm-enriched samples derived from the seminal vesicles or the testes. Seminal vesicles or testes from 40 males were dissected in DPBS with protease inhibitors, on ice. The tissues were then placed in a fresh droplet of DPBS, teased apart with a needle, swirled in the buffer to release sperm, and then removed from the droplet. The buffer droplet containing the sperm was transferred to a microcentrifuge tube containing 500 µl DPBST (DPBS with 0.1% Tween-20). The samples were spun at 20,800×g for 5 min at 4°C. The supernatant was discarded and the pellet was washed twice in DPBST. After the final wash, the pellet was resuspended in 2× SDS sample buffer. We considered the resulting samples as “sperm-enriched” as they contained not only sperm, but likely also some tissue, tissue secretions, and Sfps.
Both the supernatant and the pellet samples were analyzed using two
independent biological replicates of each sample type, except for the virgin
females for which only one sample each was used for verification of our
techniques. Protein separation and identification was conducted as
previously described
Protein identifications were based on a significance threshold of <0.05.
Additionally, we only considered proteins to be high confidence hits if
either two different peptides from the same sample exceeded the significance
threshold or if one peptide hit exceeded the significance threshold in two
independent biological replicates (single or multiple peptide hits are
reported in
All of the databases are derived from sequencing of the Liverpool strain. The
Vectorbase database is based on 8× coverage and includes 4,758
supercontigs, 1.3 gigabases, 15,988 genes, and 17, 402 predicted peptides
(
By using BLASTP, we identified homologs of the
In order to identify male-derived proteins that are transferred to females during
mating, we used a whole-organism isotope labeling method. The principle of this
method is to mate males to females whose proteins are labeled with the stable
isotopes so as to exclude the female proteins from proteomic identification.
Specifically, female proteins are labeled with 15N, which shifts
their masses upward such that the masses of female-derived peptides do not match
those expected in a standard search (uncorrected for 15N) of a
predicted protein database. The method was developed by Krijgsveld et al.
In our search against the Vectorbase database, we identified 128 proteins in the
reproductive tracts of labeled females after mating with unlabeled males (
Molecular function | Predicted protein class | Molecular function (cont.) | Predicted protein class | ||
Annexin | 11302 | Protease | 01588 | ||
Calcyphosine | 08489 | 02000 | |||
Dipeptidase | 08893 | 06403 | |||
Fibrinogen | 01713 | 06414 | |||
Kakapo | 02829 | 06421 | |||
Lectin | Supp4872 | 06429 | |||
04679 | 10725 | ||||
Mitochondrial brown fat uncoupling protein | 07046 | 11558 | |||
Moesin | 07915 | 12217 | |||
Mucin | 00718 | 15386 | |||
Odorant-binding | AaegSfp1 | Protease inhibitor | 02715 | ||
Phosphatidylethanol- binding protein | 11263 | AaegSfp2 | |||
AaegSfp3 | |||||
Tubulin β-chain | 02848 | Thioesterase | 03569 | ||
None | 00479 | Transferase | 03746 | ||
08274 | None | 02793 | |||
09201 | 13559 | ||||
Catalase | 13407 | 17451 | |||
Decarboxylase | 05790 | 17460 | |||
Dehydrogenase | 04338 | Actin | 01928 | ||
05308 | 05964 | ||||
06928 | Vitellogenin | 05815 | |||
10464 | None | 03348 | |||
12014 | Cytochrome c oxidase subunit | 00929 | |||
NADH-ubiquinone oxidoreductase subunit | 05946 | 13751 | |||
Peroxidase | 04112 | Glutamate receptor | 09813 | ||
Aminopeptidase | 02399 | Mitochondrial glutamate carrier | 11276 | ||
02978 | Sodium/calcium exchanger | 12480 | |||
07201 | Niemman-Pick Type C-2 | 09760 | |||
Asparaginase | 02796 | Rab GDP-dissociation inhibitor | 12904 | ||
ATP synthase subunit | 06516 | Venom allergen | 09239 | ||
07777 | None | 04944 | |||
11025 | 05219 | ||||
12035 | 10824 | ||||
Supp3543 | |||||
12819 | Supp4095 | ||||
ATPase | 14053 | AaegSfp5 | |||
Dehydrogenase | 04294 | AaegSfp6 | |||
Dynein | 11478 | AaegSfp7 | |||
Gamma glutamyl transpeptidase | 10935 | AaegSfp8 | |||
14580 | AaegSfp9 | ||||
Glutathione transferase | 11741 | AaegSfp10 | |||
Hydrolase | 03666 | AaegSfp11 | |||
06485 | AaegSfp12 | ||||
Kinase | 12359 | AaegSfp13 | |||
12731 | AaegSfp14 | ||||
Lipase | 07063 | ||||
Mannosidase | 05763 |
5-digit numbers are the Vectorbase database identification numbers
without the proceeding “AAEL0”. Numbers with
“Supp” prefix refer to proteins from the Supplementary
predicted peptide database from AaegL1.1 Gene Build. Numbers with
the prefix “AaegSfp” refer to proteins from either the
6-frame translation or the small peptides databases. The amino acid
sequences for all of the “Supp” and
“AaegSfp” predicted proteins are given in
Molecular function | Predicted protein class | Molecular function (cont.) | Predicted protein class | ||
Aminopeptidase | 06975 | Protease inhibitor | AaegSp1 | ||
Heat shock protein 70 | Supp4130 | None | 06509 | ||
Histone | 00490 | 10754 | |||
15674 | 17349 | ||||
Reticulocalbin | 14589 | Actin | 01673 | ||
Tubulin α-chain | 06642 | 11197 | |||
13229 | Myosin | 12543 | |||
None | 00637 | Tubulin β-chain | 02851 | ||
08779 | 05052 | ||||
10149 | ADP, ATP carrier | 04855 | |||
10882 | Cytochrome c | 04457 | |||
14231 | Cytochrome c oxidase subunit | 05170 | |||
Dehydrogenase | 00454 | Ubiquinol-cytochrome c reductase unit | 03675 | ||
02881 | 05269 | ||||
03757 | Voltage-dependent anion-selective channel | 01872 | |||
08166 | None | 17508 | |||
NADH-ubiquinone oxidoreductase | 12552 | Netrin receptor | 07195 | ||
Aconitase | 03734 | None | 09707 | ||
Aminopeptidase | 00108 | 12282 | |||
ATP synthase subunit | 02827 | 17096 | |||
05173 | Supp4104 | ||||
05610 | Supp7141 | ||||
05798 | AaegSp2 | ||||
08787 | AaegSp3 | ||||
08848 | |||||
12175 | |||||
Kinase | 06042 | ||||
Protease | 03308 |
5-digit numbers are the Vectorbase database identification numbers
without the proceeding “AAEL0”. Numbers with
“Supp” prefix refer to proteins from the Supplementary
predicted peptide database from AaegL1.1 Gene Build. Numbers with
the prefix “AaegSp” refer to proteins from either the
6-frame translation or the small peptides databases. The amino acid
sequences for all of the “Supp” and “AaegSp”
predicted proteins are given in
Nine of the identified proteins share identical amino acid sequence with other
Of the 145 transferred proteins we identified using the whole-organism isotope
labeling method, 123 are newly-recognized components of
In order to distinguish Sfps from sperm proteins among those transferred to
females, we conducted a proteomics analysis of sperm-enriched samples from the
seminal vesicles (SVs) and testes of virgin males. Sperm-enriched samples were
obtained by releasing sperm from these organs, pelleting the sperm by
centrifugation, and washing them repeatedly, as in Dorus
Of the 145 total transferred proteins (see section “Proteins transferred to
females during mating”), 16 were isolated from only one of the
sperm-enriched tissues (SV: 5; testes: 11) and therefore were considered SV- or
testes-derived Sfps, respectively, although we recognize that these could be
sperm proteins. Additionally, 77 of the transferred proteins did not overlap
with either sperm-enriched sample. Together, the 5 SV-derived Sfps, 11
testes-derived Sfps, and 77 of the 145 total transferred proteins that did not
overlap with the sperm-enriched samples comprise a total of 93 proteins assigned
with high-confidence as
The
A. Seminal fluid proteins; B. Putative sperm proteins.
As might be expected, the genes encoding the Sfps identified in this study tend
to have highly male-biased expression when gene expression of whole males is
compared to gene expression of whole females (
Below, we discuss (i) the unannotated, newly-identified predicted proteins
from the 6-frame translation and small peptides database, (ii) new insights
into the mode of
Based on comparisons to the 6-frame translation and small peptides databases,
we discovered 14 previously unannotated predicted Sfps (
Sixty-two of the Sfps that we identified are predicted intracellular or
membrane-bound proteins (e.g., ATPases, dipeptidyl peptidase, gamma glutamyl
transpeptidase, glutathione S-transferase, angiotensin converting enzyme).
Predicted intracellular proteins also have been reported in the seminal
fluid of other organisms including bed bugs (
Importantly, our results emphasize that studies of Sfps in other species
should not exclude proteins whose sequence (alone) suggest that they are
intracellular and membrane-bound proteins. For species in which
intracellular and/or membrane-bound proteins are found in the seminal fluid
(e.g., bed bugs and honey bees), further research should be conducted on the
mode of secretion of the male reproductive gland cells. Furthermore,
secretion by
Across a wide range of organisms, seminal fluid is rich in proteolysis
regulators (e.g.,
PCSK4s can activate precursors of membrane receptors, peptide hormones,
antibacterial peptides and neuropeptides through proteolytic processing
(
One of the Sfps we identified (AAEL009760) is a predicted sterol carrier in
the Niemann-Pick type C-2 (NPC2) family. In insects, sterol carriers are
essential for the production of ecdysteroids (ECDs) (e.g.,
From
We discovered three previously unannotated predicted sperm proteins from the
6-frame translation database (
The likely biological functions of putative
Secretions of the reproductive glands of male
The Sfps identified in this study suggest roles in protein
activation/inactivation, ecdysteroidogenesis, and sperm utilization.
Furthermore, our discovery that many predicted intracellular and membrane-bound
proteins are transferred to females in the seminal fluid indicates that findings
of such proteins in the seminal fluid of other species (e.g.,
Genes encoding Sfps showed higher male-biased expression than the genome average.
On the one hand, this is not unexpected because Sfps are made in the male
reproductive tract and are then transferred to females. On the other hand, it is
not necessarily predicted
Together, our results provide a foundation for functional analyses to associate
individual Sfps with their function in the mated female. Once functions are
identified for individual proteins, investigations of the pathways by which they
induce effects on male and female reproductive biology could identify novel
targets for control of
Predicted seminal fluid proteins transferred in
(0.18 MB DOC)
Predicted sperm proteins transferred in
(0.11 MB DOC)
Proteins identified from unlabeled unmated
(0.07 MB DOC)
Amino acid sequences of unannotated predicted sperm and seminal fluid
proteins from
(0.07 MB DOC)
(0.05 MB DOC)
Putative
(0.08 MB DOC)
We thank D. Severson, University of Notre Dame, for providing us with material to start the Liverpool colony. We thank O. Marinotti for providing insights and sharing data regarding gene expression patterns of the predicted proteins we report. We thank G. Findlay and W. Swanson for helpful discussions regarding experimental design and data interpretation. L. Cator, G. Findlay, E. Kelleher, M. Sirot, and three anonymous reviewers provided insightful feedback on an earlier draft of this manuscript. We thank S. Zhang, S. Baumgart, R. Sherwood, W. Chen, and A. Ptak of the Cornell University Life Sciences Core Laboratories Center, and S. Pitcher for technical help.