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 (). 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 −80°C until used for protein analysis.
Prior to gel electrophoresis, spermatophores were ground and sonicated in 15–20 µL 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 spermatophore contained 30–80 µg of water-soluble protein.
For protein quantification, 25 µg of spermatophore proteins were separated on 4–12% Bis-Tris SDS-PAGE (using an Invitrogen NuPAGE system) following manufacturer's protocols (). 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 () was compared to a control protein band, which was the 31 kDa protein band just above EJAC-SP (, 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/MS
After staining gels with Coomassie G-250, the selected gel band (protein “X” in ) 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 µL 1
1 acetonitrile: water at 30°C. 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 55°C for 10 min followed by 100 mM iodoacetamide in the dark at 30°C for 60 min. The carboxymethylated gels were thoroughly washed and re-dried in vacuo, then incubated with sequencing grade trypsin (Trypsin Gold, Promega, Madison, WI), 20 ng/µL in 40 mM ammonium bicarbonate, in 20 µL. Upon rehydration of the gels, an additional 15 µL of 40 mM ammonium bicarbonate and 10% acetonitrile was added, and gel sections were incubated at 30°C 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 µl of 50% acetonitrile and 2% trifluoracetic acid (TFA) at 30°C 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 µL of 30 mg/mL 2,5-dihydroxylbenzonic acid (DHB) (Sigma, St. Louis, MO) dissolved in 33% acetonitrile/0.1% TFA and 2 µL of peptide/matrix solution was applied on a Bruker Massive Aluminum plate for MALDI-TOF and TOF/TOF analysis.
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, carbamidomethylation 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, EG018599, EG018669, 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 µL 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 µL of the total RNA solution, 5 µL 5X RT buffer, 1.3 µL dNTP's, 0.7 µL rRNasin, 1 µL M-MLV reverse transcriptase, 2 µL of poly-T primer and nuclease-free water to 20 µL - all reagents from Promega, Madison, WI)], and a thermocycler profile of 94°C for 2 mins, 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, and a final extension period of 72°C 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 Cloning® 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, CGGATGAGGGCGATGTCTTC). 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 5′RACE for each tissue within each sex. For 5′RACE, we utilized the FirstChoice® RLM-RACE kit from Ambion. Following 5′RACE 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 5′ to the conserved IVGG region.
Preparation and injection of dsRNA
Following the identification of a unique region 5′ 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 5′ end of each primer. Our final 5′ primer was TAATACGACTCACTATAGGGAGA TACTCATCTTGGTGGCCTG and our final 3′ primer was TAATACGACTCACTATAGGGAGAGTGTTGAGACACCGTCAGACA (with the underlined sections being the T7 region). These primers were used with standard PCR reaction chemistry for a 50 µL reaction (same as above), 1 µg of male accessory gland-specific first-strand cDNA (isolated as above), and a thermocycler profile of 94°C for 2 min, 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, and a final extension period of 72°C 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/µL, as measured by a ND-1000 spectrophotometer.
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 µL for a higher yield of dsRNA. We also used 1 µg of template DNA (i.e., our concentrated PCR product). The RNA transcription reaction was incubated at 37°C 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 µg/µL.
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 µL of either ejacsp-dsRNA or saline depending on the experimental treatment. To accomplish the injections, we anesthetized adult males with CO2 and 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 our target protein (EJAC-SP) in the spermatophore, we injected males (from a Texas population of A. sp. nov. Tex) with either ejacsp-dsRNA or saline (all males were 14 and 22 post-eclosion). Three 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 −80C following a successful copulation. Females were given seven days to lay eggs before being scored for possible female sterility (i.e., females were considered sterile if the total number of eggs 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 was run on a SDS-PAGE gel and scored for the relative abundance of 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 in the ejaculate and the age-specific egg-laying rate for each treatment (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 analysis.
Real-time quantitative PCR (qPCR)
Males were frozen immediately at −80°C 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; including the DNase I treatment to remove any DNA from the sample, and final RNA volume was 75 µL. 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/µL. 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
50, and 1
250 was conducted. The test-gene primers used for this test and the subsequent qPCR were 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 male accessory 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, β-actin
(sense primer, AACTGGGACGACATGGAGAAGAT
; anti-sense primer, GCCAAGTCCAGACGC AGGAT
), and similar dilutions were used for each gene in each tissue type. Primer efficiencies were acceptable with values between 90 and 103% for both genes in all tissues except for the serine protease primer in the digestive tract (efficiency
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 ΔCt was calculated as CtSP – Ctactin, resulting in trypsin-like serine protease values being corrected for by the amount of β-actin in the sample. Positive values mean the Ct value was higher for the trypsin-like serine protease and thus less transcript than β-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.