Seminal fluid protein genes of the brown planthopper, Nilaparvata lugens
Yu et al. BMC Genomics
Seminal fluid protein genes of the brown planthopper, Nilaparvata lugens
Bing Yu 0 1
Dan-Ting Li 0 1
Jia-Bao Lu 0 1
Wen-Xin Zhang 0 1
Chuan-Xi Zhang 0 1
0 State Key Laboratory of Rice Biology and Ministry of Agriculture Key Laboratory of Agricultural Entomology, Institute of Insect Science, Zhejiang University , Hangzhou 310058 , China
1 Abbreviations: BPH , Brown planthopper; CB, Copulatory bursa; CDS, Coding sequence; DGE, Digital gene expression; EGF, Epidermal growth factor; FRT, Female reproductive tract; ITPL, Ion transport peptide like; MADF, mesencephalic astrocyte-derived neurotrophic factor; MAG, Male accessory gland; MRT, Male reproductive tract; RNAi, RNA interference; RPKM, Reads Per Kilo bases per Million mapped Reads; RT-qPCR, Reverse-transcription quantitative PCR; SFPs, Seminal fluid proteins; SR, Seminal receptacle; TE, Testes; VD, vas deferen
Background: Seminal fluid proteins (SFPs) are produced mainly in the accessory gland of male insects and transferred to females during mating, in which they induce numerous physiological and post-mating behavioral changes. The brown plant hopper (BPH), Nilaparvata lugens, is an economically important hemipterous pest of rice. The behavior and physiology of the female of this species is significantly altered by mating. SFPs in hemipteran species are still unclear. Results: We applied high-throughput mass spectrometry proteomic analyses to characterize the SFP composition in N. lugens. We identified 94 putative secreted SFPs, and the expression levels of these proteins was determined from the male accessory gland digital gene expression database. The 94 predicted SFPs showed high expression in the male accessory gland. Comparing N. lugens and other insect SFPs, the apparent expansion of N. lugens seminal fluid trypsins and carboxylesterases was observed. The number of N. lugens seminal fluid trypsins (20) was at least twice that in other insects. We detected 6 seminal fluid carboxylesterases in N. lugens seminal fluid, while seminal fluid carboxylesterases were rarely detected in other insects. Otherwise, new insect SFPs, including mesencephalic astrocyte-derived neurotrophic factor, selenoprotein, EGF (epidermal growth factor) domain-containing proteins and a neuropeptide ion transport-like peptide were identified. Conclusion: This work represents the first characterization of putative SFPs in a hemipeteran species. Our results provide a foundation for future studies to investigate the functions of SFPs in N. lugens and are an important addition to the available data for comparative studies of SFPs in insects.
Nilaparvata lugens; Seminal fluid protein; Proteome; UPLC/MS/MS
Insect seminal fluid proteins (SFPs) are important for
fertilization and are weapons for males in sexual
competition, such as manipulating post-mating physiological
and behavioral changes in females [
]. SFPs have
complex structures and perform a diversity of functions [
Under natural selection and selection by females, and
under male competition [
], the rapid evolution of SFPs
has been observed [
]. The SFP compositions of insect
species exhibit significant diversity, presumably enabling
a variety of reproductive strategies. Week-long
refractoriness toward further copulation and enhanced egg
laying levels are generated by the seminal fluid sex
peptide (Acp70A) pathway in Drosophila melanogaster, and
sperm is required in this process [
]. An unknown
mechanism leads to life-long behavioral changes in
mosquitoes such as Anopheles gambiae, and sperm is not
]. The female behavior and physiology of
multiple mating social insects are apparently unaffected
by a single copulation, but SFPs may respond to the
long-term storage of sperm and sperm competition after
The brown planthopper (BPH), Nilaparvata lugens
Stål (Hemiptera: Delphacidae), is one of the most serious
insect pests of rice in Asia [
]. Asian countries have
continually experienced serious outbreaks of BPH
although new BPH-resistant rices, new insecticides, as
well as integrated pest management programs are used.
Mated BPH females display stimulated egg laying levels
] and almost life-long refractoriness to further
]. The sex peptide model, as used to describe
post-mating behavior in D. melanogaster, may not
provide a reasonable explanation for post-mating behavior
in N. lugens. At present, chemical control remains the
first choice for N. lugens management [
]. Seminal fluid
might play a part in the rapid establishment of drug
resistance. Insecticide (triazophos and
deltamethrin)treated male N. lugens had higher protein content than
untreated males; treated males also transferred more
SFPs to mated females [
Proteomic approaches to elucidating the function of
SFPs have been carried out on several insect species,
including Apis mellifera [
], D. melanogaster, D. simulans,
D. yakuba [
], Tribolium castaneum [
], Aedes aegypti
], Aedes albopictus [
], Teleogryllus oceanicus [
Heliconius erato, and H. melpomene [
research on SFPs has not been performed for any
hemipterous species to date, such as N. lugens. Furthermore,
the functions of insect SFPs have been poorly studied in
insects other than D. melanogaster, despite major
differences in reproductive physiology exist between species.
More seminal protein information from multiple insects
could provide more insights into the evolutionary
patterns of reproductive traits [
]. As more is learned
about the reproductive biology of specific arthropods,
their SFPs may provide tools or targets for the control of
disease vectors and agricultural pests [
]. The N. lugens
seminal fluid proteome could benefit research into the
reproductive physiology of N. lugens that uses tools such
as RNA interference (RNAi). Illustrating the molecular
interactions between SFPs and N. lugens females may
aid researchers in identifying molecular targets for pest
control, as the regulation of female behaviors after
mating appears to be long-lasting in N. lugens.
Recently, the whole genome sequences and gene
annotation information for N. lugens were described [
Gene expression information regarding developmental
stages, wing dimorphism, sex differences, and tissues
was collected using next-generation high-throughput
Illumina technology [
]. The male reproductive
tract (MRT) of N. lugens comprises two testes (TE), two
vas deferens (VD), two male accessory glands (MAGs),
and one ejaculatory duct (Fig. 1). Sperm are produced by
the TE, and SFPs are produced primarily by the MAGs.
In this study, transcriptomic analysis of the N. lugens
MRT was performed, and gene expression information
concerning the MAG was obtained using a tag-based
digital gene expression (DGE) system. We used UPLC/
MS/MS to identify the transferred SFPs of N. lugens.
The N. lugens strain was originally collected from a rice
field located in the Huajiachi Campus of Zhejiang
University in Hangzhou, China. The insects used in this
experiment were the offspring of a single female. Insects
were reared on rice seedlings at 28 °C (Xiu shui 128)
under a 12:12 h light: dark photoperiod.
Preparation of N. lugens MRT transcriptome database
N. lugens males were anesthetized on ice for 20 min and
dissected under a Leica S8AP0 stereomicroscope. The
whole MRT (including the TE, VD, and MAGs) (Fig. 1)
were isolated and quickly washed in a diethylpyrocarbonate
(DEPC)-treated phosphate-buffered saline (PBS) solution
(137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, and
1.47 mM KH2PO4 at pH 7.4) and were immediately frozen
at −80 °C. The MRT sample was used for transcriptome
and DGE sequencing, and the MAG sample was used for
Total RNA was isolated from N. lugens MRT and
MAG using TRIzol reagent (Invitrogen, Carlsbad, CA,
USA) following the manufacturer’s instructions.
Sequencing and assembly of transcriptome reads, including
DGE library reads, was performed using Illumina
HiSeq™2000 and Trinity (v2012-10-05), respectively, and
the annotation of unigenes were performed as described
]. The longest assembled transcripts of
each gene were taken as unigenes. The readcount of
each unigenes was normalized to RPKM (Reads Per Kilo
bases per Million mapped Reads) to display the
expression level of each unigene. The coding sequence (CDS)
of each unigene was analyzed using blastx and estscan
(3.03). The generated peptide database was used to
support the proteomic analysis.
Seminal fluid protein sample preparation
Seminal fluid samples were collected from males and
mated females, and soluble protein samples were
collected from unmated females (all individuals were 4–7
days post-eclosion). Mated females were obtained by
placing one female in a glass tube containing a rice
seedling with one male for 2 h. The female copulatory bursa
(CB) and seminal receptacle (SR) (Fig. 1) were dissected
in PBS solution and squeezed using grinding rod in
100 μl PBS with 1 % protease inhibitor cocktail
(Thermo, USA). Reproductive tracts from ≈ 50 females
were pooled for each biological replicate. MAGs (Fig. 1)
were dissected using the same method, and MAGs
from ≈ 50 males were collected for each biological
replicate. Samples were centrifuged at 12,000 rpm for 20 min
at 4 °C. The supernatant was transferred to a separate
tube and stored at −80 °C. Three replicates were
prepared for each kind of sample including the MAG, the
mated-female reproductive tract (FRT) and the
A filter aided sample preparation (FASP) method was
used for the preparation of samples [
]. Samples were
added to 3 kD ultrafiltration centrifuge tubes (Millipore),
and centrifuged at 14000 g for 20 min. 100 μl of UA
solution [8 M Urea (Sigma), 0.1 M Tris/HCl pH 8.5, 1 %
EDTA (Thermo), 1 % protease inhibitor Cocktail
(Thermo)], and centrifuged at 14000 g for 20 min; this
step was repeated twice. 2 μl DTT (Sigma) (200 mM)
was added. Samples were vortexed for 1 min and
incubated at 37 °C for 1 h. 20 μl iodoacetamide (Sigma)
(200 mM) was then added. Samples were vortexed for
1 min and incubated at 25 °C for 1 h in the dark.
Samples were centrifuged at 14000 g for 20 min. To
each sample, 100 μl UA was added. Samples were then
centrifuge at 14000 g for 20 min; this step was repeated
once. 200 μl NH4HCO3 (Sigma) (0.05 M) was added,
and samples were centrifuged at 14000 g for 20 min; this
step was repeated twice. The remaining sample was
moved into a 10 kD ultrafiltration centrifuge tube, and
40 μl NH4HCO3 (0.05 M) and trypsin (Promega) (5 μg
in total) were added. Samples were incubated at 30 °C
for 12 h, and then centrifuged at 14000 g for 20 min.
40 μl NH4HCO3 (0.05 M) was added; then samples were
centrifuged at 14000 g for 30 min. Filtered liquid was
removed into a 1.5 ml centrifuge tube and dehydrated in
a vacuum freeze-drying device. Dehydrated samples were
dissolved in 25 μl 0.1 % formic acid (Sigma). The
concentrations of the dissolved peptide solutions were
analyzed by A280 absorption using a NanoDrop UV–vis
spectrophotometer (Thermo Fisher Scientific, Waltham,
UPLC/MS/MS methods and data analyses
The peptide mixtures were injected onto the trap
column at a flow rate of 10 μl/min for 2 min (2 μg) using a
Thermo Scientific Easy nanoLC 1000. The trap was
equilibrated at a maximum pressure of 500 bar for 12 μl,
followed by column equilibration at a maximum of
500 bar for 3 μl before beginning the gradient elution of
the column. The samples were subsequently eluted using
the following five-step linear gradient (A: ddH2O with
0.1 % formic acid,B: ACN with 0.1 % formic acid): 0–
10 min, 3–8 % B; 10–120 min, 8–20 % B; 120–137 min,
20–30 % B; 137–143 min, 30–90 % B; and 143–150 min,
90 % B. The column flow was maintained at 250 nL/min.
The chromatographic system was composed of a trapping
column (75 μm × 2 cm, nanoviper, C18, 3 μM, 100 Å) and
an analytical column (50 μm × 15 cm, nanoviper, C18,
2 μM, 100 Å). Data collection was performed using a
Thermo LTQ-Orbitrap Velos Pro equipped with a
Nanospray Flex ionization source and a FTMS (Fourier
transform ion cyclotron resonance mass spectrometry)
analyzer combined with a Thermo LTQ-Orbitrap Elite
equipped with an ion trap analyzer. The parameters for
FTMS were as follows: Data collection at 60 K for the full
MS scan, positive polarity, data type profile, and then
proceeded to isolate the top 20 ions for MS/MS by CID
(1.0 m/z isolation width, 35 % collision energy, 0.25
activation Q, 10 ms activation time). The scan range was set as
300 m/z first mass and 2000 m/z last mass. The
parameters for the ion trap analyzer were the normal mass range,
rapid scan rate, and centroid data type.
A SEQUEST HT search engine configured with a
Proteome Discoverer 1.4 workflow (Thermo Fischer
Scientific, Bremen, Germany) was used for mass
spectrometer data analyses. An N. lugens MRT peptide
database generated from transcriptome unigene sequences
database containing 17902 sequences were configured
with SEQUEST HT for dataset searches. The search
parameters included 10 ppm and 0.8 Da mass tolerances
for MS and MS/MS, respectively, trypsin as the proteolytic
enzyme with two allowed missed cleavages, oxidation and
deamidated as dynamic modifications, and
carbamidomethyl as a static modification. Furthermore, the peptides
were extracted using high peptide confidence. 1 % FDR
(False discovery rate) was calculated using a decoy
database by searching both the MRT peptide sequence and
the decoy database.
Identification of seminal fluid proteins of N. lugens
High confidence proteins were identified with the
following standards: 1) Proteins identified from more than
two samples (proteins derived from at least two MAG
samples, two unmated-FRT samples and two mated-FRT
samples) were predicted to be “true” detected proteins.
2) Seminal fluid proteins must have been identified from
both MAG and mated-FRT samples. 3) We tested for
predicted secretion signal sequences of detected proteins
using SignalP 4.1 (www.cbs.dtu.dk/services/SignalP/).
Some sequences had a “bad” coding sequence CDS
prediction (Lost in N-terminal), in which the signal peptide
was not be predicted from the sequence. We
repredicted CDS sequences for proteins with no signal
peptide using ESTScan (http://myhits.isb-sib.ch/cgi-bin/
estscan) from unigene nucleotide sequences, and
performed Signalp detection with new predicted CDS
sequences for improved signal peptide detection.
Proteins possessing a signal peptide were considere d to be
secreted proteins. 4) Some proteins did not possess a
signal peptide. Proteins without signal peptides that were
not detected in unmated-FRT samples and showed
male-specific expression (an analysis of the male-specific
expression of unigenes was performed as described
]) were also predicted to be secreted. 5) In
addition, other proteins that were not predicted to be
secreted and had homologues in the SFPs of other
insects were classied as unconfirmed SFPs.
Annotation of seminal fluid proteins and comparison with other insects
In addition to machine annotation, we performed a
manual annotation for the sequences detected. Blast
results from NCBI, conserved domains, and GO terms
were used in combination to annotate proteins. Brief
descriptions from NCBI, SMART
(http://smart.emblheidelberg.de/) descriptions of conserved domains, and
functional descriptions of gene names from UniProtKB
(http://www.uniprot.org/help/uniprotkb) were used to
classify the functions of each sequence. Based on these
matches, proteins were classified into one of the
following categories: cell structure (including cell
structure proteins and their binding proteins), metabolism,
protein modification machinery, proteolysis regulators
(proteases and protease inhibitors), signal transduction
(including hormones), transporters and protein export
machinery, and RNA and protein synthesis
(transcription factors, transcription machinery, and protein
synthesis enzymes). Proteins that were classified into
different categories were classified as “other” (including
salivary proteins, chitin binding proteins, binding
proteins, proteasome machinery, protein kinases,
ubiquitination pathway proteins, protein phosphatases, and
oxidoreductases). Proteins that were not assigned a
function were classified as “unknown”.
Seminal fluid proteome sequences of D. melanogaster,
A. aegypti, A. albopictus, A. mellifera and Homo sapien
] were chosen for comparison with SFPs of N. lugens.
SFP sequences of D. melanogaster were extracted from
Flybase (http://flybase.org/) using IDs given from
]. SFPs sequences of A. aegypti were extracted
from Ensembl Metazoa (http://metazoa.ensembl.org/
info/website/ftp/index.html) using IDs given from
]. SFP sequences of A. albopictus were directly
given by reference [
]. SFPs sequences of A. mellifera
were extracted from NCBI (http://www.ncbi.nlm.nih.
gov/sites/batchentrez) using IDs given from reference
]. Signal peptides of these proteins were identified as
mentioned in 2.5. And prediction of conserved domains
of predicted protein domains was using the Batch Web
CD-Search Tool (http://www.ncbi.nlm.nih.gov/Structure/
bwrpsb/bwrpsb.cgi). N. lugens SFPs possessing the same
conserved domain with other insect SFPs were marked
as “Domain”. The rest proteins with blastp (Evalue < 10−5)
hits with other insect SFPs were marked as “Blast”. The
same method was used for comparison of SFPs between
To locate the detected proteins in the N. lugens
genome scaffold sequences, we run a megablast with Evalue
< 10−20, and indentity > 95 % between detected proteins
and scaffold sequences.
The functional serine protease domains of the N. lugens
seminal fluid trypsins were aligned with seminal fluid
trypsins of other insect species using the ClustalX
program. The phylogenetic tree was constructed by the
maximum likelihood (ML) method using the program
Mega 5.05 (http://www.megasoftware.net/). Homologous
relationships were determined using bootstrap analysis
with 1000 replications.
Reverse-transcription quantitative PCR (RT-qPCR) analysis
MRT, FRT, and dissections of MRT (including testes, vas
deferens, and male accessory glands) (Fig. 1) were
dissected from males (4–7 days post-eclosion). As the
mRNA quantity of an individual tissue is extremely low,
tissues dissected from 40 individuals were pooled into
each tissue sample, respectively. RT-qPCR was
performed according to the method of [
]. Primers used
in RT-qPCR for the tissue specific expressions of
seminal fluid protein genes are given in Additional
file 1: Table S1.
MRT transcriptome sequencing and assembly
Illumina sequencing produced 2.59 GB nucleotides. The
quality of this transcriptome sequence was high, with a
Q20 percentage (the percentage of sequences with a
sequencing error rate of 0.03 %) and GC content of
98.21 % and 40.08 %, respectively. These short reads
were assembled into 57568 transcripts with a mean
length of 741 bp. Ultimately, we obtained 37443
unigenes with a mean size of 641 bp and lengths ranging
from 201 to 9670 bp. Annotation of these sequences
revealed that 13089 (35 %) sequences were annotated in
the NR database, 10156 (27 %) sequences were
annotated in the SwissProt database, 10589 (28 %) sequences
were annotated in the GO database, and 8301 (22 %)
sequences were annotated in the KOG database. Of
these, 523 sequences showed male-specific expression. A
peptide database with 17902 sequences was generated as
a query database for the raw data of the proteome
Two DGE libraries from the MRT and MAG were
also sequenced, generating approximately 0.64 GB
clean tags for each library. Among the clean tags,
approximately 92 % of sequences could be mapped to
unigenes in each library. A total of 33798 unigenes
expressed in the MRT had a RPKM value > 0.3; 19772
unigenes expressed in the MAG had a RPKM value >
0.3 (Table 1). The MRT transcriptome yielded a
peptide database used in proteome sequencing, and the
The longest assembled transcripts of each gene were taken as unigenes. Only
unigenes with RPKM value larger than 0.3 were counted as expressed. Owing
to the accomplishment of the transcriptomic sequencing of the differences
between the N. lugens development and sex genes in our previous study [
we are able to use the predicted SFP genes as reference sequences to map
the transcriptomic datasets and to analyze the expression sex-specific genes.
The coding sequence (CDS) of each unigene was analyzed using blastx and
estscan (3.03). The generated peptide database was used in proteome query
MRT and MAG DGE databases provided the
expression levels of detected protein-coding genes in the
MRT and MAG.
Proteins transferred to females during mating
We identified a total of 218 putative SFPs from both the
MAG and mated-female reproductive tract (FRT)
samples. Of these, 65 sequences were not detected in
unmated-FRT samples and showed male-specific
expression. Fifty-five of the 65 sequences had a signal peptide;
the remaining 10 proteins showed high expression in the
MAG. In addition, 29 proteins that were detected in
both mated- and unmated-FRT samples also had a signal
peptide. These proteins were predicted to be secreted in
the MAG. Eventually, a total of 94 proteins were
predicted secreted SFPs (Table 2).
One hundred and twenty-four of the 218 proteins had
identical conserved domains or were aligned in blastp
results with SFPs detected from other insects; this latter
class of proteins contained no signal peptide and did not
show male-specific expression (Additional file 2: Table
S2). Homologues of these proteins had been detected in
the SFPs of D. melanogaster, A. aegypti, A. albopictus, or
Apis mellifera previously and were classified as
unconfirmed SFPs. Whether these proteins are “true”
transmitted seminal proteins in N. lugens could not be
confirmed due to technical limitations (transmitted SFPs
could not be distinguished from FRT proteins).
Through sequence annotation, the 218 proteins were
classified into different functional groups. By combining
protein annotation and the expression patterns of these
proteins in the MAG and MRT, we found that predicted
secreted SFPs and unconfirmed SFPs exhibited large
differences in expression patterns and functional group
classifications. MAGs are the main production center
for SFPs. The proteins predicted to be secreted showed
high expression in the MAG; predicted secreted proteins
accounted for 91.10 % of the accumulated RPKM value
of the 218 genes. Some proteases, two apolipoprotein D
proteins, two cysteine-rich secretory proteins, two
peritrophin A–type chitin-binding proteins, three dumpy
proteins, a nucleoside diphosphate kinase, a
chemosensory protein, and four proteins with unknown function
showed extremely high expression in the MAG (RPKM
value > 1000). When analyzing the numbers of genes in
each functional group, we found that proteolysis
regulators represented the largest percentage of secreted
proteins (37, 16.97 %); this was followed by protein
modification machinery (12, 5.50 %), proteins with other
functions (10, 4.59 %), and lipid metabolism proteins
(10, 4.59 %) (Additional file 3: Table S3 and Fig. 2).
Most of the unconfirmed SFPs showed very low
expression levels in the MAG; these genes accounted
for only 9.01 % of the accumulated RPKM value of
D. melanogaster A. aegypti A. albopictus A. mellifera
Numbers after the gene names stand for the number of proteins detected. Conserved domains of insect SFPs were identifed using the Batch Web CD-Search Tool
(http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). A local blastp analyse (evalue = 1e-5) were performed between BPH SFPs with other insect SFPs. “T”
stands for the sequences possess the same conserved domain or show blastp (1e-5) results with SFPs with other insect SFPs. D. melanogaster, Drosophila
melanogaster; A. aegypti, Aedes aegypti; A. albopictus, Aedes. albopictus; A. mellifera, Apis mellifera
the 218 genes. Only several genes encoding cell
structure proteins (such as actin, tubulin, myosin, profilin,
and tropomyosin), proteins involved in RNA and
protein synthesis (such as ribosomal proteins and
translation initiation factors), energy metabolism proteins
(such as cytochrome c, ATP synthase, and malate
dehydrogenase), and other proteins (two thioredoxin
genes in particular) (Fig. 2 and Additional file 3:
Table S3) had relatively higher expression levels in
the MAG. Most of these proteins are ubiquitous
within tissues and throughout the developmental
stages. We could not distinguish these proteins from
One hundred and ninety-six of the 218 proteins had
homologues in the SFPs in four other insect species; 73,
91, 114, and 77 homologous proteins were identified in
D. melanogaster, A. aegypti, A. albopictus, and Apis
mellifera, respectively. Among the 196 N. lugens
proteins, only trypsins, cysteine-rich secretory proteins, a
low-density lipoprotein receptor, and an aminopeptidase
were found in all insect species. In N. lugens, the
lowdensity lipoprotein receptor and the aminopeptidase
were not predicted to be secreted; these two proteins
also showed very low expression levels in the MAG.
A much larger number of trypsins (20) were detected
in N. lugens seminal fluid than in other insects (D.
melanogaster, 10; A. aegypti, 4; A. albopictus, 11; Apis
mellifera, 1) (see the phylogenetic tree in Fig. 3). As the
phylogenetic tree shows, all N. lugens seminal fluid
trypsins were on the same branch. Although the SFP genes
evolve rapidly, one D. melanogaster trypsin showed a
closer relationship with N. lugens seminal fluid trypsins,
and two D. melanogaster trypsin showed a closer
relationship with mosquito seminal fluid trypsins. The
seminal fluid trypsins from the A. aegypti and A. albopictus
were all mixed together. The duplication of N. lugens
seminal fluid trypsins (including other N. lugens SFPs) in
the same scaffold was observed (Additional file 2: Table
S2). Furthermore, the number of carboxylesterases was
also expanded in N. lugens. Only one carboxylesterase
was identified in the D. melanogaster SFPs, and
carboxylesterase was not detected in other insects.
Seven proteins detected in N. lugens had not been
previously detected in other insect SFPs. These included
lysosomal Pro-X carboxypeptidase, carboxypeptidase E,
carboxypeptidase Q, prolylcarboxypeptidase,
mesencephalic astrocyte–derived neurotrophic factor (MANF),
selenoprotein, EGF (epidermal growth factor)-domain
containing proteins, and an ion transport peptide–like
Considering SFPs may undergo enzymatic digestion or
other alternations in the female reproductive system,
thus, they may not be detected from female samples.
Accordingly, we analyzed the signal peptide of the
proteins that were only detected from MAG samples. We
identified 14 proteins with signal peptides, and their
information, including their sequences, is provided in
Additional file 4: Table S8.
Expression profile analysis of SFP genes by RT-qPCR
We tested the expression profiles of 34 genes in the
MRT, FRT, and dissections of the MRT using RT-qPCR.
Thirty-one genes showed MRT-specific or -biased
expression (Additional file 5: Figure S1). Of these 31 genes,
we have previously reported that 9 trypsins are
specifically expressed in the MRT [
] (not shown in figure).
When analyzing the expression of these sequences
within the MRT, most of the MRT-specific or -biased
genes also showed MAG-specific or -biased expression.
Two exceptions were apolipophorin III, which showed
the greatest expression in the VD, and a
carboxylesterase, which showed high expression in both the VD and
the MAG (Additional file 5: Figure S1). The qPCR
results were consistent with the gene expression level
results of the transcriptome.
The presence of a signal peptide is the classic method of
predicting whether a protein is secreted. In D.
melanogaster, 142 SFPs were detected; of these, 112 had a signal
peptide. Seminal fluid proteome studies in other insects
have reported a much lower proportion of proteins with
signal peptides (29 of 93 detected proteins in A. aegypti,
57 of 198 detected proteins in A. albopictus, 20 of 53
detected proteins in Apis mellifera). These intracellular
or membrane-bound proteins are predicted to be
secreted via “apocrine secretion”; cells in the posterior
portion of the glands are thought to secrete proteins
through granules and/or via the rupture of the cell
], though some unsolved problems exist
concerning “apocrine secretion”: 1. The biological
importance of these proteins in the female reproductive
tract remains to be demonstrated; 2. Proteins may be
randomly included in seminal fluid during “apocrine
secretion”; 3. The inclusion in the database of peptide
sequences lacking an N-terminus may have complicated
the prediction of signal peptides. The high proportion of
proteins with a signal peptide in the D. melanogaster
SFP proteome may have been due to the high quality of
the D. melanogaster protein database. In this study, we
combined the proteome and expression levels of
detected proteins in the MAG and found that proteins
with more reliable evidence were secreted (84 proteins
with a signal peptide and 10 proteins not detected in
unmated female MRT) and showed much higher
expression in the MAG than other proteins did. The high
expression of these proteins in the MAG is in
accordance with the theoretical SFP expression profile.
In D. melanogaster, four trypsins are required in the
sex peptide pathway, and C-type lectins are also needed
in this pathway. In N. lugens, although a large number of
trypsins was detected, we did not find C-type lectin in
N. lugens seminal fluid. Otherwise, astacin family zinc
], a protein required in fly ovulin (a
prohormone-like SFP stimulating ovulation) [
not detected in N. lugens seminal fluid. In addition, we
performed a tblastn using D. melanogaster ovulin and
sex peptide against N. lugens MRT transcriptome
(See figure on previous page.)
Fig. 3 Phylogenetic analysis of insect seminal fluid trypsins. The phylogenetic tree was constructed based on the deduced amino acid sequences
of the conserved domains of seminal fluid trypsin genes by maximum likelihood using Mega 5.05 (http://www.megasoftware.net/). The
JonesTaylor-Thornton (JTT) model for amino acid substitution was used, while a test of phylogeny was carried out using bootstrap analysis with 1000
replications. Sequences starting with “comp” stand for transcriptome unigene IDs of seminal fluid trypsins in N. lugens. Sequences starting with
“FBpp” stand for D. melanogaster genome peptide IDs of seminal fluid trypsins (http://flybase.org/). Sequences starting with “AAEL” stand for A.
aegypti genome peptide IDs of seminal fluid trypsins (http://www.vectorbase.org/). Sequences starting with “GB” stand for Apis mellifera genome
peptide IDs of seminal fluid trypsins (http://www.ncbi.nlm.nih.gov/). Sequences starting with “Aa” stand for A. albopictus seminal fluid trypsins IDs
unigene sequences, genome coding sequences, and
genome DNA sequences, but no homologous sequences
were identified. This indicates that the typical sex
peptide and ovulin pathway are may not be present in N.
lugens. The molecular mechanisms behind the
postmating phenomena of D. melanogaster and N. lugens
An angiotensin-converting enzyme was detected in N.
lugens: this SFP is critical for increasing female egg
laying in T. castaneum [
] and is related to female
fecundity in Anopheles stephensi [
]. Two additional
proteins associated with angiotensin were also detected
in N. lugens seminal fluid. One is a renin receptor
(detected in N. lugens and Apis mellifera) that
induces the conversion of angiotensinogen to
angiotensin I [
]. The second protein is a lysosomal
Pro-X carboxypeptidase (only detected in N. lugens)
that can cleave C-terminal amino acids linked to
proline in peptides such as angiotensin II in response to
Some new insect SFPs were identified in this proteome
research, including new seminal fluid proteases,
selenoprotein, secreted proteins containing EGF domains, a
secreted neurotrophic factor MANF, and a
neuropeptide ITPL protein. Selenoproteins were only recently
detected in human seminal fluid; they are likely
important for protecting sperm during storage [
blastp analysis, secreted proteins containing EGF
domains detected in N. lugens seminal fluid aligned
with dumpy from Drosophila. Dumpy is a huge
protein with an EGF domain repeat predicted to be a
membrane-anchored fiber almost a micrometer in
length; the EGF domain is involved in cell
]. A secreted protein with an EGF domain
was found to bind to the surface of sperm and was
important in sperm–egg binding in mice [
protects and repairs the dopaminergic neurons. It is
up-regulated in response to misfolded proteins, and it
protects against various forms of endoplasmic
reticulum stress in non-neuronal cells [
Most interesting is the discovery of the new
seminal fluid neuropeptide, ITPL. IPTs were originally
identified in Schistocerca gregaria and are regulators
of ion and fluid transport across the ileum. However,
ITPL lacks this activity due to C-terminal disparity
(Fig. 4). ITP/ITPLs are highly conserved
neuropeptides in insects and crustaceans and are grouped into
the crustacean hyperglycemic hormones (CHH) [
]. Several insect studies have suggested that ITP
functions in ecdysis in Manduca sexta  and in D.
melanogaster clock neurons [
]. RNAi of ITPL in T.
castaneum led to significant reduction in egg
numbers due to failure in ovarian maturation and
reduced survival of offspring after dsRNA injections at
the pupal stage [
]. In N. lugens, ITPL was
identified as a SFP transferred to females after mating.
The function of ITPL as an SFP is a topic worth
From the proteomic analysis, we identified 94 putative
secreted SFPs of N. lugens and the expression level of
these proteins in the MAG was yielded by the DGE
database. We found that proteins with more reliable
evidence predicted to be secreted showed much higher
expression in the MAG than other proteins, lending
credibility to the detected SFPs. Comparative analyses
revealed duplication and expansion of SFPs in N. lugens
and the identification of novel SFPs in this species. Our
results provide a foundation for future studies to
investigate the functions of SFPs in N. lugens and are an
important addition to the available data for comparative
studies of SFPs in insects.
Additional file 1: Table S1. Primers used in this artical. This file lists the
sequences of the primers used in the RT-qPCR analysis, as described in
Methods. (XLSX 11 kb)
Additional file 2: Table S2. Detailed annotation of detected proteins.
This file gives the detailed annotation of detected proteins including the
high confident SFPs and unconfirmed SFPs. The annotation process was
described in Methods. (XLSX 134 kb)
Additional file 3: Table S3. Statistics of function groups. This file gives
a statistics analysis of the detected proteins of each function groups, as
described in Methods. (XLSX 12 kb)
Additional file 4: Table S8. Proteins only detected in MAG samples
possessing a signal peptide. We analyzed the signal peptide of the
proteins that were only detected from MAG samples. We identified
14 proteins with signal peptides, and their information, including their
sequences, is provided in this file. (XLSX 31 kb)
Additional file 5: Figure S1. Analysis of the expression profiles of
seminal fluid protein genes in MRT dissections, MRT, and FRT by qRT-PCR.
This file gives the RT-qPCR results of the detected proteins. (PDF 613 kb)
Additional file 6: Table S4. Sequences of detected proteins. This file
gives the nucleotide sequences and amino acid sequences of the
detected proteins including the high confident SFPs and unconfirmed
SFPs. (XLSX 236 kb)
Additional file 7: Table S5. Comparison of SFPs between different
insect species. This file gives the comparison results of SFPs between
different insect species, as described in Methods. (XLSX 39 kb)
Additional file 8: Table S6. Proteome data of each samples. This file
gives the proteome raw data of each proteins samples. (XLS 1675 kb)
Additional file 9: Table S7. Sequences of insect SFPs. This file gives
the SFP sequences of other insect species used in the comparison with
N. lugens SFPs. (XLSX 502 kb)
This work was supported by the National Natural Science Foundation of
China (grant no.31471765, 31272374).
Availability of data and materials
The N. lugens transcriptomic dataset and gene expression profile datasets
used in the male specific expression analysis of SFPs are available in the
Sequence Read Archive (SRA) database (http://www.ncbi.nlm.nih.gov/sra).
The accession number of the N. lugens transcriptomic dataset is SRX023419.
The accession numbers of the N. lugens gene expression profile datasets are
as follows: macropterous female adults (MFA) (SRX023495) and macropterous
male adults (MMA) (SRX023496). The nucleotide sequences of detected
proteins using UPLC/MS/MS (including the predicted secreted SFPs and
unconfirmed SFPs) were submitted to National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/). The accession number of these
sequences are as follows: KU932205 - KU932419. We also provide the
nucleotide and protein sequences of SFPs as an Additional file 6: Table S4
(3 genes with nucleotide sequences length shorter than 200 bp can be
found here, they can’t be submitted to GenBank due to short length). The
phylogenetic data was uploaded to Dryad (http://purl.org/phylo/treebase/
phylows/study/TB2:S19675). The annotation and gene expression profile
in MRT and MAG of detected proteins were provided in Additional file 2:
Table S2. Comparison of SFPs between different insect species were
provided in Additional file 7: Table S5. The proteome raw data of each
proteins samples were provided as Additional file 8: Table S6. The SFP
sequences of other insect species used in the comparison with N. lugens
SFPs were provided as Additional file 9: Table S7.
ZCX conceived the experiments. BY designed the experiments and wrote
the manuscript. DTL, JBL and ZWX helped to perform the experiments.
All authors discussed the results and commented on the manuscript.
All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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1. LaFlamme BA , Wolfner MF . Identification and function of proteolysis regulators in seminal fluid . Mol Reprod Dev . 2013 ; 80 ( 2 ): 80 - 101 .
2. Poiani A . Complexity of seminal fluid: a review . Behav Ecol Sociobiol . 2006 ; 60 ( 3 ): 289 - 310 .
3. Andres JA , Maroja LS , Bogdanowicz SM , Swanson WJ , Harrison RG . Molecular evolution of seminal proteins in field crickets . Mol Biol Evol . 2006 ; 23 ( 8 ): 1574 - 84 .
4. Gillott C . Male accessory gland secretions: Modulators of female reproductive physiology and behavior . Annu Rev Entomol . 2003 ; 48 : 163 - 84 .
5. Ram KR , Wolfner MF . Seminal influences: Drosophila Acps and the molecular interplay between males and females during reproduction . Integr Comp Biol . 2007 ; 47 ( 3 ): 427 - 45 .
6. Ram KR , Wolfner MF . Sustained post-mating response in Drosophila melanogaster requires multiple seminal fluid proteins . Plos Genet . 2007 ; 3 : 2428 - 38 .
7. Thailayil J , Magnusson K , Godfray HCJ , Crisanti A , Catteruccia F . Spermless males elicit large-scale female responses to mating in the malaria mosquito Anopheles gambiae . Proc Natl Acad Sci U S A . 2011 ; 108 ( 33 ): 13677 - 81 .
8. Baer B , Heazlewood JL , Taylor NL , Eubel H , Millar AH . The seminal fluid proteome of the honeybee Apis mellifera . Proteomics . 2009 ; 9 ( 8 ): 2085 - 97 .
9. den Boer SPA , Baer B , Boomsma JJ . Seminal Fluid Mediates Ejaculate Competition in Social Insects . Science . 2010 ; 327 ( 5972 ): 1506 - 9 .
10. Xue J , Zhou X , Zhang C , Yu L , Fan H , Wang Z , Xu H , Xi Y , Zhu Z , Zhou W , et al. Genomes of the rice pest brown planthopper and its endosymbionts reveal complex complementary contributions for host adaptation . Genome Biol . 2014 ; 15 : 521 .
11. Long Y , Hou ML , Yang X , Shi BK . Effects of delayed mating on reproduction in brachypterous females of the brown planthopper, Nilaparvata lugens . Plant Prot . 2010 ; 6 : 36 - 9 .
12. Ichikawa T. Studies on the mating behavior of the four species of auchenorrhynchous Homoptera which attack the rice plant . Memoirs of Faculty of Agriculture Kagawa University. 1979 .
13. Xue J , Bao Y , Li B , Cheng Y , Peng Z , Liu H , Xu H , Zhu Z , Lou Y , Cheng J , et al. Transcriptome Analysis of the Brown Planthopper Nilaparvata lugens . Plos One . 2010 ; 5 ( 12 ):e14233. doi: 10 .1371/journal.pone. 0014233 .
14. Ge L , Wu J , Zhao K , Chen Y , Yang G . Induction of Nlvg and suppression of Nljhe gene expression in Nilaparvata lugens (Stal) (Hemiptera: Delphacidae) adult females and males exposed to two insecticides . Pestic Biochem Phys . 2010 ; 98 ( 2 ): 269 - 78 .
15. Wang L , Shen J , Ge L , Wu J , Yang G , Jahn GC . Insecticide-induced increase in the protein content of male accessory glands and its effect on the fecundity of females in the brown planthopper Nilaparvata lugens Stal (Hemiptera: Delphacidae) . Crop Prot . 2010 ; 29 ( 11 ): 1280 - 5 .
16. Findlay GD , Yi X , MacCoss MJ , Swanson WJ . Proteomics reveals novel Drosophila seminal fluid proteins transferred at mating . Plos Biol . 2008 ; 6 ( e1787 ): 1417 - 26 .
17. Xu J , Baulding J , Palli SR . Proteomics of Tribolium castaneum seminal fluid proteins: Identification of an angiotensin-converting enzyme as a key player in regulation of reproduction . J Proteomics . 2013 ; 78 : 83 - 93 .
18. Sirot LK , Hardstone MC , Helinski MEH , Ribeiro JMC , Kimura M , Deewatthanawong P , Wolfner MF , Harrington LC . Towards a Semen Proteome of the Dengue Vector Mosquito: Protein Identification and Potential Functions . Plos Neglect Trop D. 2011 ; 5 ( 3 ): e989 .
19. Boes KE , Ribeiro JMC , Wong A , Harrington LC , Wolfner MF , Sirot LK . Identification and Characterization of Seminal Fluid Proteins in the Asian Tiger Mosquito, Aedes albopictus . Plos Neglect Trop D. 2014 ; 8 ( 6 ): e2946 .
20. Simmons LW , Tan YF , Millar AH . Sperm and seminal fluid proteomes of the field cricketTeleogryllus oceanicus: identification of novel proteins transferred to females at mating . Insect Mol Biol . 2013 ; 22 ( 1 ): 115 - 30 .
21. Walters JR , Harrison RG . Combined EST and Proteomic Analysis Identifies Rapidly Evolving Seminal Fluid Proteins in Heliconius Butterflies . Mol Biol Evol . 2010 ; 27 ( 9 ): 2000 - 13 .
22. Avila FW , Sirot LK , LaFlamme BA , Rubinstein CD , Wolfner MF . Insect Seminal Fluid Proteins: Identification and Function . Annu Rev Entomol . 2011 ; 56 : 21 - 40 .
23. Bao Y , Wang Y , Wu W , Zhao D , Xue J , Zhang B , Shen Z , Zhang C. De novo intestine-specific transcriptome of the brown planthopper Nilaparvata lugens revealed potential functions in digestion, detoxification and immune response . Genomics . 2012 ; 99 ( 4 ): 256 - 64 .
24. Ji R , Yu H , Fu Q , Chen H , Ye W , Li S , Lou Y. Comparative Transcriptome Analysis of Salivary Glands of Two Populations of Rice Brown Planthopper, Nilaparvata lugens That Differ in Virulence . Plos One . 2013 ; 8 : e7961211 .
25. Tanaka Y , Suetsugu Y , Yamamoto K , Noda H , Shinoda T . Transcriptome analysis of neuropeptides and G-protein coupled receptors (GPCRs) for neuropeptides in the brown planthopper Nilaparvata lugens . Peptides . 2014 ; 53 : 125 - 33 .
26. Yu H , Ji R , Ye W , Chen H , Lai W , Fu Q , Lou Y. Transcriptome Analysis of Fat Bodies from Two Brown Planthopper (Nilaparvata lugens) Populations with Different Virulence Levels in Rice . Plos One . 2014 ; 9 : e885282 .
27. Wisniewski JR . Quantitative Evaluation of Filter Aided Sample Preparation (FASP) and Multienzyme Digestion FASP Protocols . Anal Chem . 2016 ; 88 ( 10 ): 5438 - 43 .
28. Pilch B , Mann M . Large-scale and high-confidence proteomic analysis of human seminal plasma . Genome Biol . 2006 ; 7 : R405 .
29. Bao Y , Qin X , Yu B , Chen L , Wang Z , Zhang C . Genomic insights into the serine protease gene family and expression profile analysis in the planthopper Nilaparvata lugens . BMC GENOMICS . 2014 ; 15 : 507 .
30. Ravi Ram K , Sirot LK , Wolfner MF . Predicted seminal astacin-like protease is required for processing of reproductive proteins in Drosophila melanogaster . Proc Natl Acad Sci U S A . 2006 ; 103 ( 49 ): 18674 - 9 .
31. Heifetz Y , Lung O , Frongillo EA , Wolfner MF . The Drosophila seminal fluid protein Acp26Aa stimulates release of oocytes by the ovary . Curr Biol . 2000 ; 10 ( 2 ): 99 - 102 .
32. Ekbote U , Looker M , Isaac RE . ACE inhibitors reduce fecundity in the mosquito Anopheles stephensi . Comp Biochem Phys B . 2003 ; 134 ( 4 ): 593 - 8 .
33. Oshima Y , Morimoto S , Ichihara A . Roles of the (pro)renin receptor in the kidney . World J Urol . 2014 ; 3 : 302 - 7 .
34. Cousin C , Bracquart D , Contrepas A , Corvol P , Muller L , Nguyen G . Soluble Form of the (Pro) Renin Receptor Generated by Intracellular Cleavage by Furin Is Secreted in Plasma . Hypertension . 2009 ; 53 ( 6 ): 1077 - 82 .
35. Xu S , Lind L , Zhao L , Lindahl B , Venge P. Plasma Prolylcarboxypeptidase (Angiotensinase C) Is Increased in Obesity and Diabetes Mellitus and Related to Cardiovascular Dysfunction . Clin Chem . 2012 ; 58 ( 7 ): 1110 - 5 .
36. Tan F , Morris PW , Skidgel RA , Erdös EG . Sequencing and cloning of human prolylcarboxypeptidase (angiotensinase C) . Similarity to both serine carboxypeptidase and prolylendopeptidase families . J Biol Chem . 1993 ; 268 : 26032 - 8 .
37. Michaelis M , Gralla O , Behrends T , Scharpf M , Endermann T , Rijntjes E , Pietschmann N , Hollenbach B , Schomburg L. Selenoprotein P in seminal fluid is a novel biomarker of sperm quality . Biochem Bioph Res Co . 2014 ; 443 ( 3 ): 905 - 10 .
38. Wilkin MB , Becker MN , Mulvey D , Phan I , Chao A , Cooper K , Chung HJ , Campbell ID , Baron M , MacIntyre R. Drosophila Dumpy is a gigantic extracellular protein required to maintain tension at epidermal-cuticle attachment sites . Curr Biol . 2000 ; 10 ( 10 ): 559 - 67 .
39. Shur BD , Ensslin MA , Rodeheffer C. SED1 function during mammalian sperm-egg adhesion . Curr Opin Cell Biol . 2004 ; 16 ( 5 ): 477 - 85 .
40. Yang W , Shen Y , Chen Y , Chen L , Wang L , Wang H , Xu S , Fang S , Fu Y , Yu Y , et al. Mesencephalic astrocyte-derived neurotrophic factor prevents neuron loss via inhibiting ischemia-induced apoptosis . J Neurol Sci . 2014 ; 344 ( 1-2 ): 129 - 38 .
41. Audsley N , McIntosh C , Phillips JE . Isolation of a neuropeptide from locust corpus cardiacum which influences ileal transport . J Exp Biol . 1992 ; 173 : 261 - 74 .
42. Meredith J , Ring M , Macins A , Marschall J , Cheng NN , Theilmann D , Brock HW , Phillips JE . Locust ion transport peptide (ITP): Primary structure, cDNA and expression in a baculovirus system . J Exp Biol . 1996 ; 199 : 1053 - 61 .
43. Drexler AL , Harris CC , Dela Pena MG , Asuncion-Uchi M , Chung S , Webster S , Fuse M. Molecular characterization and cell-specific expression of an ion transport peptide in the tobacco hornworm Manduca sexta . Cell Tissue Res . 2007 ; 329 ( 2 ): 391 - 408 .
44. Hermann-Luibl C , Yoshii T , Senthilan PR , Dircksen H , Helfrich-Foerster C. The Ion Transport Peptide Is a New Functional Clock Neuropeptide in the Fruit Fly Drosophila melanogaster . J Neurosci . 2014 ; 34 ( 29 ): 9522 - 36 .
45. Begum K , Li B , Beeman RW , Park Y. Functions of ion transport peptide and ion transport peptide-like in the red flour beetle Tribolium castaneum . Insect Biochem Molec . 2009 ; 39 ( 10 ): 717 - 25 .