Molecular Dissection of Nuptial Gifts in Divergent Strains of Ostrinia Moths

Physiological Entomology (2018) 43, 10–19 DOI: 10.1111/phen.12220

Molecular dissection of nuptial gifts in divergent strains of Ostrinia moths

NOORIA AL-WATHIQUI, SARA M. LEWIS and ERIK B. DOPMAN Biology Department, Tufts University, Medford, Massachusetts, U.S.A.

Abstract. Seminal fluid proteins (SFPs) produced in the male accessory glands and ejaculatory duct are subject to strong sexual selection, often evolve rapidly and therefore may play a key role in reproductive isolation and species formation. However, little is known about reproductive proteins for species in which males transfer ejaculate to females using a spermatophore package. By combining RNA sequencing and proteomics, we characterize putative SFPs, identify proteins transferred in the male spermatophore and identify candidate genes contributing to a one-way gametic incom- patibility between Z and E strains of the European corn borer moth Ostrinia nubilalis. We find that the accessory glands and ejaculatory duct secrete over 200 highly expressed gene products, including peptidases, peptidase regulators and odourant-binding proteins. A comparison between Ostrinia strains reveals that accessory gland and ejaculatory duct sequences with hormone degradation and peptidase activity are among the most extremely differentially expressed. However, most spermatophore peptides lack reproductive tissue bias or canonical secretory signal motifs and aproximately one-quarter may be produced elsewhere before being sequestered by the male accessory glands during spermatophore production. In addition, most potential gene candidates for postmating reproductive isolation do not meet standard criteria for predicted SFPs and almost three-quarters are novel, suggesting that both postmating sexual interactions and gametic isolation likely involve molecular products beyond traditionally recognized SFPs. Key words. Differential expression, Lepidoptera, seminal fluid proteins, transcriptome.

Introduction to drive the rapid evolution of such reproductive proteins in mammals (Makalowski & Boguski, 1998), marine gastropods Sexual selection can act as a powerful force shaping the evo- (Metz et al., 1998) and insects (Swanson et al., 2001; Swanson lutionary trajectory of traits and helping to drive speciation & Vacquier, 2002; Swanson, 2004). Rapid molecular evolution (Panhuis et al., 2001). Divergence in sexual traits may often increases the likelihood of male–female protein incompatibili- occur by intersexual selection and by positive or negative cor- ties between populations as a byproduct, increasing the potential related evolution of male and female traits (including sexual for failed reproduction and reduced fertilization success (Price conflict: Parker & Partridge, 1998; Arnqvist et al., 2000; Chap- et al., 2001; Larson et al., 2011). man et al., 2003; Gavrilets & Hayashi, 2005; Gavrilets, 2014). The evolution of postmating interactions between the sexes For example, after mating, male seminal fluid proteins from is recognized as an important factor for reproductive isolation. male ejaculate interact within the female reproductive tract For example, when Drosophila mojavensis females mate with D. with the ejaculates of other males and with female reproduc- arizonae males, there are multiple barriers to successful repro- tive proteins (Simmons, 2001; Ram & Wolfner, 2007; Sirot duction. These include failure to transfer sperm, store sperm and et al., 2009; Wolfner, 2009). These interactions are assumed degrade the insemination reaction (Kelleher & Markow, 2007). When D. simulans females mate with a D. mauritiana males, Correspondence: Nooria Al-Wathiqui, Biology Department, Tufts there is also a smaller number of sperm that get stored and University, 163 Packard Avenue, Medford, Massachusetts 02155, females lay fewer eggs than if they were to mate with a conspe- U.S.A. Tel.: +1 262 389 5160; e-mail: [email protected] cific male (Price et al., 2001). Although reproductive proteins

10 © 2017 The Royal Entomological Society Nuptial gifts in Ostrinia moths 11 are likely mediators of both these barriers, their potential role in strains are visually similar and differ mainly with respect to reproductive isolation is only beginning to be explored. the composition of their sex pheromone and are estimated to To understand postmating sexual selection and how this may have diverged approximately 100 000 years ago (Malausa, 2005; contribute to species divergence, male reproductive proteins Dopman et al., 2010). Although visually similar, ECB moths need to be characterized for diverse organisms. In many insects, do differ in several reproductive barriers, including seasonal the male ejaculate is often complex and along with sperm isolation, temporal isolation, male orientation to females and can contains sugars, salts, defensive compounds and seminal female discrimination of males based on pheromone, etc. (Dop- fluid proteins (SFPs) (Chen, 1984; Simmons, 2001; Gillott, man et al., 2010). Of the seven strong reproductive isolating 2003; Poiani, 2006). SFPs are produced in and secreted by the barriers between strains, one acts during the postmating, prezy- male accessory glands and male ejaculatory duct and are then gotic (PMPZ) time point and consists of reduced and asym- transferred to the female in the male ejaculate (Chen, 1984). metric oviposition. Specifically, Z-strain females that mate with These proteins exert profound effects on female behaviour and E-strain males experience a 30% reduction in egg laying; how- physiology after mating. In D. melanogaster, SFPs decrease ever, the percentage of fertilized eggs is not affected (Dopman female receptivity to future mates, increase egg laying, decrease et al., 2010). This PMPZ reproductive barrier may arise through longevity, increase feeding and decrease sleep (Ram et al., 2005; dysfunctional interactions at any stages preceding egg deposi- Sirot et al., 2009; Wolfner, 2009). tion (i.e. sperm storage, egg maturation, fertilization and ovipo- In numerous insects, males transfer their sperm packaged sition) and could involve interactions between known female into a complex spermatophore manufactured by male accessory reproductive products (Al-Wathiqui et al., 2014) and SFPs or glands. The spermatophore adds an additional level of complex- other products produced by the male accessory glands. In the ity to male and female reproductive interactions and conflict present study, we compare males from these two ECB strains (Chapman, 2003; Lewis & South, 2012; Lewis et al., 2014). aiming to: (i) characterize seminal fluid proteins transferred to In Lepidoptera, where spermatophore transfer is ubiquitous females and (ii) identify candidate genes that may be involved (Wedell, 2005), the male spermatophore can act as a type of in the PMPZ isolating barrier acting in ECB. mating plug that temporarily prevents females from re-mating (Sugawara, 1979). During mating, females receive the spermatophore inside a muscular organ called the bursa copulatrix Materials and methods (henceforth, the bursa). Distension of the bursa stimulates stretch receptors, which cause females to become unreceptive Moth strains to additional mating (Sugawara 1979); therefore, a female will not remate until the spermatophore is partially degraded. Based on population surveys, Z and E strain moths can be Lepidopteran females have evolved several traits that function found living allopatrically, as well as sympatrically (Klun et al., in spermatophore degradation, including female reproductive 1975; Kochansky et al., 1975; Carde’ et al., 1978; Anglade & secretions (Al-Wathiqui et al., 2014; Meslin et al., 2015) and Stockel, 1984). In the U.S.A, ECB populations range from the a muscular reproductive tract containing a chitinized structure east coast to the Rocky Mountains. For the present study, Z called the signum (Galicia et al., 2008; Cordero, 2009; Sánchez and E strain ECB moths were collected as caterpillars, pupae et al., 2011). Because these traits are expected to evolve rapidly and adults from a Z strain population in Bouckville, New York, in response to sexual conflict, they may cause variation in and an E strain population in Geneva, New York (n = 500 males fertilization success and contribute to reproductive isolation and n = 500 females each from each location) (Dopman et al., between populations (Parker & Partridge, 1998; Chapman et al., 2010). Laboratory populations have been maintained by mass 2003; Gavrilets & Hayashi, 2005). rearing approximately 200 adults per generation and have been There are recent studies that investigate gene expression described previously (Dopman et al., 2005). in the female bursa and identify secretions contributing to degradation of the spermatophore (Al-Wathiqui et al., 2014; Meslin et al., 2015). However, specific male reproductive genes Sample preparation and RNA sequencing and proteins may interact with the bursa to accomplish or inhibit spermatophore degradation, although these products have yet to A total of 18 accessory glands and 18 ejaculatory ducts be fully characterized. One previous study examining seminal were dissected from 2-day-old virgin males for each strain proteins in two Heliconius butterfly species, Heliconius erato (Fig. 1A). Tissues were pooled from six individuals and used to and Heliconius melpomone, describes several secreted proteases create each of the six accessory gland and six ejaculatory duct and cysteine-rich proteins (Walters & Harrison, 2010). Based libraries (three per strain). Individuals were pooled because we on their similarity to D. melanogaster SFPs, these male proteins wanted to obtain a representative sample of reproductive gene may be responsible for mediating physiological and behavioural expression, which may not be represented in a single individual. changes in females after mating (Wolfner, 2009; LaFlamme & We also wanted to ensure that an appropriate amount of RNA Wolfner, 2013). could be obtained from each pooled sample for cDNA library In the present study, we characterize male reproductive pro- construction. In accordance with Al-Wathiqui et al. (2014), teins in two divergent strains of the European corn borer (ECB) tissues were stored in RNAlater at −80 ∘C prior to total RNA Ostrinia nubilalis, a species for which female reproductive extraction (RNeasy Midi kit, Qiagen, California) and cDNA genes are already characterized (Al-Wathiqui et al., 2014). Both library construction (Truseq RNA sample preparation kit v2;

Illumina, San Diego, CA) (Al-Wathiqui et al., 2014). Sample To conduct differential expression analysis, normalized reads quality was verified using an Advanced Analytical fragment were mapped to our transcriptome using bowtie 2 and the analyzer (Advanced Analytical, Ankeny, Iowa). Sample RIN ‘very-sensitive’ setting (Langmead et al., 2009; Langmead & scores ranged between 7.2 and 9.0, with two samples having RIN Salzberg, 2012) and then the normalized read counts for each scores below 8.0. The six accessory gland cDNA libraries and gene were tested for differential gene expression using edger six ejaculatory duct cDNA libraries were then sequenced using (Robinson & Smyth, 2007; Robinson et al., 2009; Robinson & an Illumina HiSeq2000 (50-bp single-end reads). To compare Oshlack, 2010; McCarthy et al., 2012). edger uses empirical patterns across reproductive and nonreproductive tissues, two Bayes methods to estimate gene-specific variation. We used male thorax libraries (one per strain) were also sequenced for a generalized linear model approach in which we assessed each strain. Male thorax cDNA libraries were created using the differential expression with ECB strain and tissue type as SMART cDNA library 6.7 protocol (Takara Bio, Japan) and factors. For all comparisons, genes were considered significantly sequenced using 40-bp paired-end reads on an Illumina GA IIx, differentially expressed if they had a false-discovery rate (FDR) < ≥ with a 200-bp insert length. of 0.01 and an expression log2 fold change (LFC) of 2. We applied two commonly used criteria to identify putative male SFPs (Sirot et al., 2008; Walters & Harrison, 2010). First, Sequence processing and assembly we looked for genes with up-regulated gene expression in ≥ reproductive tissue compared with the thorax (log2 FC 2). After sequencing, trimmomatic, version 0.17 (Usadel Lab- Second, we looked for the presence of secretory peptide motifs, oratory, Germany) was used to cleave Illumina sequencing which indicates that a protein is secreted from the cell that adapters and remove the first and last 5 bp from each read it is produced in and possibly transferred to females during because these usually have low sequencing quality scores (Lind- copulation. To identify secretory peptides, we created a protein green, 2012). After adapter and quality trimming, only reads database using estscan to estimate protein sequences from our ≥ 36 bp were retained. Subsequent to trimming, mtRNA and de novo transcriptome (Iseli et al., 1999). The resulting protein rRNA contamination was removed from each library using sequences were then analyzed using signalp, version 4.0 the short read aligner, bowtie 2 and the ‘very-sensitive’ set- (Petersen et al., 2011) to detect the presence of signal peptides. ting (Langmead et al., 2009; Langmead & Salzberg, 2012). We then used fastq/a collapser (http://hannonlab.cshl.edu/fastx_ toolkit) to reduce the number of identical sequences in our read Proteomics libraries without affecting read counts. After all quality control steps were completed, we used trin- A proteomics approach was used to identify reproductive ity, version r2013-02-25 to assemble the six male accessory proteins in Z and E strain male spermatophores. Spermatophores gland libraries, six male ejaculatory duct libraries and two were collected from females immediately after mating. Mating male thorax libraries into a single de novo transcriptome using trials consisted of placing two males and one female of the recommended settings (Grabherr et al., 2011). After assembly, same strain in a paper cup (diameter 10 cm). Mating cups redundancy was reduced at each locus by selecting the longest were kept in an incubator during the dark cycle and monitored transcript. To identify putative homologues to known proteins in every 15 min for signs of mating (LD 16 : 8 h at 27 ∘Cand other organisms, a blast search (http://www.ncbi.nlm.nih.gov/ 70% relative humidity). When mating occurred, the mating pair BLAST) was conducted between each sequence and the entire was monitored and the spermatophore was dissected out of the NCBI nonredundant protein database (https://www.ncbi.nlm female immediately after mating stopped. Sperm will begin .nih.gov/refseq/about/nonredundantproteins/). All sequences leaving the spermatophore within 30 min after mating; however, with significant blast hitse ( ≤ 10−3) were then mapped and it is unknown when SFPs will begin to leave the spermatophore. annotation scores were computed for all possible Gene Ontol- ECB females are unable to mate for 24 h after mating, and so ogy terms using blast2go (Conesa et al., 2005; Conesa & it is likely that this is a critical period when the spermatophore Götz, 2008; Götz et al., 2008). is processed and SFPs are released. Six spermatophores were obtained from males of each strain. After spermatophore collection, the sperm containing ampulla RNA-sequencing and the spermatophore tail were removed (Fig. 1B) and the remaining tissue was stored at −80 ∘C. All six spermatophores To account for differences in RNA-sequencing library prepa- were pooled to create a single sample per strain. Spermatophores ration between thorax tissue and male reproductive tissue, all were crushed in sodium dodecyl sulphate using a mortar and libraries were normalized prior to read mapping and differen- pestle and then centrifuged briefly. The supernatant was col- tial expression using edaseq (Risso et al., 2011). edaseq allows lected, and boiled for 5 min. Each sample was then run on a for within-lane normalization to account for differences in read 10% Tris-glycine gel just until the entire sample migrated out lengths and nucleotide bias, which could result from using dif- of the well and the resulting two bands (one per strain) were ferent preparation methods that differ in the number of poly- fixed and stained with Coomassie brilliant blue, cut fromthe merase chain reaction amplifications, as well as between-lane gel and sent to the Harvard Mass Spectrometry and Proteomics normalization to account for differences in sequencing depth Resource Laboratory (Harvard, Cambridge, MA). Samples were across sequencing lanes. enzymatically digested and analyzed using high-performance

Fig. 1. Male reproductive structures of European corn borer moths. (A) The male accessory gland (upper left) shown with a small portion of the ejaculatory duct, as denoted by an asterisk (*). These structures secrete proteins transferred to the female in a sperm-containing package known as the spermatophore. (B) Male spermatophore constructed by a Z-strain European corn borer male, dissected from the female reproductive tract immediately after mating. The ampulla, indicated by the white box, contains sperm, whereas the composition of the remaining spermatophore is unknown. [Colour figure can be viewed at wileyonlinelibrary.com]. liquid chromatography coupled with nanoelectrospray were considered. This more stringent cut-off was used to focus tandem mass spectrometry on a LTQ-Orbitrap mass spec- our analysis to non-SFP genes that are highly differentially trometer (Thermo Fisher, Waltham, Massachusetts). The expressed between strains because these may be more likely to resulting spectra were annotated with proteome discoverer be involved in reproductive isolation. (Thermo Scientific, Waltham, Massachusetts) using the combined UniProt Swiss-Prot database (http://www.uniprot.org). Results For the remaining un-annotated peptide sequences, we used a blast search against our male reproductive tissue transcriptome Male transcriptome assembly to further characterize and annotate these sequences. From both Z and E strains, we obtained ∼400 000 000 raw Candidate speciation genes single-end reads from the accessory glands and ejaculatory ducts and ∼60 000 000 paired-end reads from male thorax To identify male reproductive genes that could be involved in tissue. On average, each replicate contained 15 247 378 reads. a reproductive isolating barrier, we looked for genes that were The resulting assembly contained 46 771 loci with a relatively significantly differentially expressed between ECB strains. large N50 score of 7351 bp, a minimum sequence length of First, we determined which of our putative SFP sequences were 201 bp and a maximum sequence length of 18 129 bp. We differentially expressed between E and Z strains (LFC ≥ 2, reduced the complexity of this assembly by selecting the longest FDR ≤ 0.01). We also characterized differential expression transcript at each locus, resulting in 44 285 sequences in the between strains for other sequences expressed in the accessory final transcriptome. Of these, 46.3% (23 761; e ≤ 0.001) showed glands and ejaculatory duct. We chose to look for differentially similarity to sequences in other organisms and 21.4% (9491) expressed sequences that are not predicted to be SFPs because were successfully annotated with Gene Ontology information. differentially expressed genes not specific to or secreted from the reproductive tract could still play a role in reproductive isola- Putative SFPs tion (Findlay et al., 2008). For this comparison, only sequences that were significantly up-regulated in male accessory gland Compared with the male thorax, 4877 and 5114 transcripts or ejaculatory duct tissue by a LFC ≥ 10andanFDR≤ 0.01 were significantly up-regulated in the ejaculatory duct and

(comp22488_c0_seq1) and sphingomyelin phosphodiesterase (comp8052_c0_seq1). In the Z strain, we identified eight peptidases and one peptidase regulator that were transferred to the female in the spermatophore. These consisted of two sequences (comp237626_c0_seq1 and comp20483_c0_seq1) showing homology to a serine peptidase called vitellin-degrading protease, which is responsible for degrading vitellin found in insect eggs (Ikeda et al., 1990; Chapman, 2003). The Z strain spermatophore also contained a sequence with homology to a cysteine protease, cathepsin L (comp311204_c0_seq1). Cathepsin L is a lysosomal endopeptidase that can be secreted and interacts with structural proteins, such as collagen and fibronectin (Ishidoh & Kominami, 1995). Fig. 2. Gene ontology category distribution for putative seminal fluid proteins in Ostrinia nubilalis. Three proteins in the Z strain spermatophore showed homology to proteins involved in insect immunity. These included sequences showing homology to a lysozyme accessory gland, respectively (LFC ≥ 2andFDR≤ 0.01) (see (comp22897_c0_seq1), defence protein hdd11 (comp25125_ Supporting information, Figure S1). Of transcripts with repro- c0_seq1) and two phenoloxidase subunits (comp22426_c0_ ductive tissue bias, 217 also contained a secretory signal and seq1, subunit 1 and comp21505_c0_seq1, subunit 2; see thus were considered putative SFPs (Fig. 2; see also Sup- Supporting information, Table S2). Both lysozymes and porting information, Table S1). More than half (119/217) defence protein hdd11 are antibacterial proteins. Lysozymes are were expressed in both the accessory gland and ejacula- glycoside hydrolases that damage bacterial cell walls (Ellison, tory duct. Of those showing tissue specificity, approximately 1991); however the precise function of defence protein hdd11 23% (50/217) were primarily expressed in the accessory is unknown (Bao, 2003). Prophenoloxidases are a key compo- gland and 22% (47/217) were primarily expressed in the nent of the insect melanogenesis defence response, in which ejaculatory duct. pathogens are encapsulated by melanin (González-Santoyo & We were able to identify annotation terms for approximately Cordoba-Aguilar, 2011). two-thirds (146/217) of putative SFPs. Gene products included In general, fewer proteins were identified in the E strain peptidases and peptidase regulators (Fig. 2; see also Supporting spermatophore (see Supporting information, Table S2). How- information, Table S1). Peptidases differed in their hydrolysis ever, similar to Z strain spermatophores, we identified proteins mechanisms (Polgar, 1989) and were identified as metallopepti- showing homology to proteases and defence proteins that are dases, cysteine peptidases, and serine peptidases and regulators transferred to the female. These included a sequence showing (Fig. 2; see also Supporting information, Table S1). We were homology to cathepsin-L (comp311204_c0_seq1) and two also able to identify genes with activities associated with bind- proteins that were homologous to proteins involved in the ing ligands such as pheromones and odor molecules (Leal, insect immune response. One sequence (comp22426_c0_seq1) 2013). We identified two putative SFPs with odourant-binding showed homology to phenoloxidase, which was also found activity (comp7771_c0_seq1 and comp21759_c0_seq1). We in Z-strain spermatophores, and another sequence (P81605) also found one pheromone binding protein that was up-regulated showed homology to dermcidin, an anionic antimicrobial in the accessory glands compared with the male thorax protein in humans (Schittek et al., 2001; Narayana & Chen, (comp18104_c0_seq1). 2015).

Proteomics Differential expression analysis

We identified 93 Z-strain spermatophore proteins and24 Our analysis of between-strain expression patterns first E-strain spermatophore proteins (see Supporting information, focused on the 217 genes that we identified as putative Table S2). The difference in the number of proteins identified SFPs using RNA sequencing and bioinformatics tools. In between strains is likely a rersult of how the proteomics anal- the accessory gland, we found four sequences that were ysis was conducted, which made it easier to identify highly differentially expressed (LFC ≥ 2) between males of the abundant proteins but more difficult to identify proteins in Z and E strains (see Supporting information, Table S3). low abundance. E strain males may transfer more proteins None of these sequences were successfully annotated. In that are low in abundance in the spermatophore. Further test- the ejaculatory duct, three sequences were differentially ing will need to be conducted to confirm this difference in expressed between strains (LFC ≥ 2) and one was func- spermatophore protein abundance. Four putative SFPs with tionally annotated: comp1865_c0_seq1 showed homology various activities had peptide counterparts: peptidyl-prolyl to an ionotropic glutamate receptor that was up-regulated cis-trans isomerase 5 (comp17755_c0_seq1), protein disul- in the E-strain (LFC =−3.8; see Supporting information, fide isomerase (comp20773_c0_seq1), a carboxypeptidase Table S3).

We identified 1361 ejaculatory duct transcripts and 1550 ECB male seminal fluid proteins accessory glands transcripts that were highly up-regulated in these tissues compared with the male thorax (LFC ≥ 10 and Proteases and protease inhibitors. Peptidases and peptidase FDR ≤ 0.01), although they lacked canonical secretory motifs so regulators are a conserved class of proteins found in the seminal were not classified as SFPs (Fig. 3; see also Supporting infor- fluid of many taxa, from humans to arthropods (Gillott, 2003; mation, Table S3). Of these, 48 sequences were significantly Avila et al., 2011; LaFlamme & Wolfner, 2013). In insects, they differentially expressed between strains in the ejaculatory duct, are known to play important roles in postmating processes. Ser- 41 were highly differentially expressed between strains in the ine peptidases and inhibitors in D. melanogaster are responsible accessory gland and three were differentially expressed in both for SFP transfer (aquarius) (Findlay et al., 2014), full induc- tissues (LFC ≥ 2, FDR ≤ 0.01). Genes that were up-regulated in tion of egg laying (seminase) and semen liquefaction (protein C the Z-strain showed homology to proteases, odourant-binding inhibitor) (Pilch & Mann, 2006). Metallopeptidases present in proteins and odourant receptors (Fig. 3). There were five serine fruit flies are also involved in the induction of egg-laying, sperm peptidases that were significantly up-regulated in the Z-strain storage (Ram et al., 2006) and the immune response subsequunt compared with the E-strain in the male reproductive tissues with to bacterial infection (Mueller et al., 2007). LFC between 10.0 and 12.5. Genes that were up-regulated in In ECB, we find that males transfer serine peptidases, metal- the E-strain showed homology to proteins related to juvenile lopeptidases and serine peptidase inhibitors to females in their hormone degradation (Fig. 3; see also Supporting information, ejaculate. Our list of putative SFPs includes nine genes that are Table S3). In both strains, the majority (59/99; aproximately annotated as serine peptidases and five genes annotated as ser- 60%) of differentially expressed genes were novel and lacked ine peptidase inhibitors (Fig. 2; see also Supporting information, hits to known genes. Table S1). Although we are not able to identify serine peptidases and inhibitors in the male spermatophore, we do identify a zinc metalloprotease and vitellin-degrading proteases that are Discussion transferred to females in the Z strain spermatophore (see Sup- porting information, Table S2). A zinc metallopeptidase in D. The female reproductive tract is the primary venue for post- melanogaster, encoded by CG11864, processes two other male mating sexual selection and gametic isolation, although our SFPs, sex peptide and Acp36DE, after all three proteins enter the understanding of the specific mechanisms underlying postmat- female reproductive tract (Ram et al., 2006). Vitellin-degrading ing interactions is still limited. Much research emphasizes the proteases are characterized in the silk moth Bombyx mori as transfer of male SFPs to females for these interactions and, cleaving two different yolk proteins during embryogenesis and, because many SFPs rapidly evolve, they make good candidates in sea squirts and sea urchins, the acrosome reaction is assisted for reproductive isolation. However, male ejaculates are com- by two vitellin envelope degrading proteases (Kim et al., 2008). plex and known to contain diverse compounds in addition to In ECB, these proteins may be transferred in the spermatophore SFPs (Poiani, 2006; Perry et al., 2013). To disentangle this com- to mediate the acrosome reaction between male sperm and the plexity, in the present study, we profile the transcriptome of female egg. SFP-producing tissues, as well as the proteome of ejaculate In humans and fruit flies, males also transfer cysteine pro- that males transfer to females. We find that a large number teases to females in their ejaculate (LaFlamme and Wolfner, of putative SFPs are produced by ECB males (217) and that 2013). We identify cathepsin L, a cysteine peptidase, in the sper- they are similar in number and composition to other species matophores of both Z and E-strain males (see Supporting infor- (Avila et al., 2011; Bonilla et al., 2015). However, most SFPs mation, Table S2). The function of cathepsins in reproduction is are not identified in the male spermatophore (4/217; approxi- poorly understood; however, these proteins play important roles mately 2%). ECB spermatophores contain an additional 108 pro- in both digestion and egg maturation. For example, in the cow- teins, including peptidases, peptidase regulators, anti-microbial pea weevil Callosobruchus maculatus, cathepsin L is respon- proteins and odourant-binding proteins. Some of these sper- sible for digestive proteolysis in the gut (Pedra et al., 2003). In matophore proteins appear to be derived from nonreproductive mice, cathepsin L is up-regulated in pre-ovulatory egg follicules, tissue (28% lack matches to reproductive tissue transcripts) or where it is predicted to play a role in follicular rupture (Robker are possibly broadly expressed (16% are biased towards non- et al., 2000). Cathepsins in the ECB spermatophore may poten- reproductive tissue), whereas another 20% show reproductive tially act to mediate male–female sexual conflict by cleaving tissue bias yet lack classical SFP secretory signals. Further- and disabling the female reproductive proteins or by aiding in more, although most of the 99 candidate genes for postmat- postmating egg maturation. ing isolation between ECB strains are novel and thus may be We also identify two SFPs that are annotated as odourant- rapidly evolving like SFPs (59/99; approximately 60%), only binding proteins (comp7771_c0_seq1 and comp21759_c0_seq1). seven gene candidates are actually recognized as SFPs. More Odourant-binding proteins are mainly associated with olfaction; work is needed to confirm these broad trends, although our work however, they are also found in the male ejaculate of many dif- on moths joins recent findings in grasshoppers (Bonilla et al., ferent insects (Chapman, 2008; Findlay et al., 2008; Sirot et al., 2015) and flies (Findlay et al. 2008) in suggesting a nuanced 2008; Kelleher et al., 2009; South et al., 2011). These proteins and possibly limited role of traditionally defined SFPs as medi- are small, soluble proteins that are found in high concentra- ators of postmating sexual selection and gametic reproductive tions around olfactory-receptor neurons and are considered isolation. to bind odours and to shuttle them to neurones for sensing

Fig. 3. Reproductive genes that are significantly differentially expressed between Ostrinia nubilalis strains and their Gene Ontology categories. The heat map depicts normalized read counts averaged across replicates for each tissue type and strain. Lighter shading indicates higher expression level. Gene Ontology pie charts summarize functional terms for genes up-regulated in at least one reproductive tissue in the Z-strain (top) or the E-strain (bottom).

(Hekmat-Scafe et al., 2002). The role of these molecules in ejaculatory duct (see Supporting information, Table S3). Pep- reproduction remains poorly understood but, in Helicoverpa tidases and peptidase regulators are particularly attractive tar- armigera and Helicoverpa assulta, odourant-binding proteins gets for further characterization as mediators of reproductive that are transferred to females in the male ejaculate and found in isolation because of their involvement in multiple postmating fertilized eggs act as an oviposition deterrent for other females interactions between the sexes (LaFlamme & Wolfner, 2013). (Sun et al., 2012). These proteins may serve a similar function In the Allonemobius complex of crickets, one trypsin-like serine in ECB, where larval cannibalization is common and such protease called Ejac-SP mediates a postmating, prezygotic bar- deterrence could enhance survival (Breden & Chippendale, rier, in which heterospecific males fail to induce egg-laying after 1989). mating (Marshall et al., 2009). Proteases that are differentially expressed between ECB strains may play a similar role in the induction of egg laying. Specifically, the 30% reduction in egg Divergence of genes between European corn borer strains laying (Dopman et al., 2010) when Z-strain females mate with E-strain males could result from an inability of E-strain males In the presence of gene flow, regions of the genome involved to activate relevant pathways controlling egg-laying in Z-strain in reproductive isolation or recurrent local adaptation will tend females. to remain differentiated, whereas other gene regions become In Z-strain male accessory glands, we find up-regulation homogenized (Ting et al., 2000; Dopman et al., 2005; Harrison, of a gene annotated as Juvenile Hormone (JH) diol kinase 2012). Hence, if genes encoding reproductive proteins have (comp16883_c0_seq1; see Supporting information, Table S3), played an important role in speciation, greater sequence and/or a protein involved in the JH degradation pathway. JH is an regulatory differentiation is expected compared with genes essential insect hormone with respect to controlling a myriad encoding other proteins expressed in the same tissue. of processes, including inhibiting pheromone production after In the present study, we focus on identifying candidate genes mating and stimulating production and uptake by oocytes of that could be involved in postmating reproductive isolation the egg yolk precursor, vitellogenin (Cusson & McNeil, 1989; between ECB strains based on patterns of differential reg- Hartfelder, 2000). In Aedes mosquitoes, a reduction in female ulation and find that five serine peptidases are up-regulated JH titre is required for the completion of oogenesis (Shapiro in the Z-strain ejaculatory duct compared with the E-strain et al., 1986). Thus, the up-regulation of JH diol kinase in the

© 2017 The Royal Entomological Society, Physiological Entomology, 43, 10–19 Nuptial gifts in Ostrinia moths 17 accessory glands of Z-strain ECB males may facilitate oogenesis References by reducing female JH titres. Additional studies are needed to determine how these differentially expressed genes influence Al-Wathiqui, N., Lewis, S.M. & Dopman, E.B. (2014) Using RNA ECB reproduction. sequencing to characterize female reproductive genes between Z and E strains of European corn borer moths (Ostrinia nubilalis). BMC Finally, we identify an odourant-binding protein secreted from Genomics, 15, 1–13. the male ejaculatory duct (comp11571_c0_seq1; see Supporting Anglade, P. & Stockel, J. (1984) Intraspecific sex-pheromone variability infromation, Table S3) that is up-regulated in Z-strain compared in the European corn borer, Ostrinia nubilalis (Lepidoptera, Pyrali- with E-strain males. However, additional work is needed to dae). Agronomie, 4, 183–187. clarify the function of odourant-binding proteins in reproduction Arnqvist, G., Edvardsson, M., Friberg, U. & Nilsson, T. (2000) Sex- and species divergence. ual conflict promotes speciation in insects. Proceedings of the To understand how reproductive proteins contribute to sex- National Academy of Sciences of the United States of America, 97, ual selection and reproductive isolation, we must character- 10460–10464. ize various organisms across the speciation continuum, from Avila, F.W., Sirot, L.K., Laflamme, B.A. et al. (2011) Insect seminal diverged populations to species. Here, we find evidence for pro- fluid proteins: identification and function. Annual Review of Entomol- ogy, 56, 21–40. teases and genes encoding proteins involved in egg develop- Bao, Y. (2003) cDNA cloning and expression of bacteria-induced ment and egg production acting as possible molecular drivers Hdd11 gene from eri-silkworm, Samia cynthia ricini. Comparative of postmating isolation in diverged pheromone strains of Biochemistry and Physiology Part C: Toxicology & Pharmacology, moths. Peptidases, peptidase regulators and odourant-binding 136, 337–342. proteins are found among the more than 200 gene prod- Bonilla, M.L., Todd, C., Erlandson, M. & Andres, J. (2015) Combining ucts that show traditional signals of SFPs in male accessory RNA-seq and proteomic profiling to identify seminal fluid proteins glands and the ejaculatory duct. Finally, a proteomic anal- in the migratory grasshopper Melanoplus sanguinipes (F). BMC ysis of male spermatophores reveals similar functions with Genomics, 16, 1–15. respect to reproductive tissue, despite a limited overlap of Breden, F. & Chippendale, G.M. (1989) Effect of larval density and can- constituent SFPs. Overall, the results of the present study nibalism on growth and development of the southwestern corn borer, Diatraea grandiosella, and the European corn borer, Ostrinia nubi- suggest that gametic isolation and postmating sexual selec- lalis (Lepidoptera: Pyralidae). Journal of the Kansas Entomological tion may involve molecular products beyond the traditionally Society, 62, 307–315. recognized SFPs. Carde’, R.T., Roelofs, W.L., Harrison, R.G. et al. (1978) European corn borer: pheromone polymorphism or sibling species. Science, 199, 555–556. Supporting Information Chapman, R.F. (2003) The Insects: Structure and Function. Cambridge University Press, U.K. Additional Supporting Information may be found in the Chapman, T. (2008) The soup in my fly: Evolution, form and function online version of this article under the DOI reference: of seminal fluid proteins. PLoS Biology, 6, e179. DOI: 10.1111/phen.12220 Chapman, T., Liddle, L.F., Kalb, J.M. et al. (2003) Sexual conflict. Trends in Ecology & Evolution, 18, 41–47. Figure S1. Biological coefficient of variation analysis of gene Chen, P.S. (1984) The functional-morphology and biochemistry of abundance (normalized read counts) across all male tissue insect male accessory glands and their secretions. Annual Review of libraries and replicate samples. Entomology, 29, 233–255. Conesa, A. & Götz, S. (2008) Blast2GO: a comprehensive suite for Table S1. Putative seminal fluid proteins secreted from the functional analysis in plant genomics. International Journal of Plant Ostrinia nubilalis male accessory glands and ejaculatory duct. Genomics, 2008, 1–12. Conesa, A., Götz, S. & García-Gómez, J.M.et al. (2005) Blast2GO: a Table S2. O. nubilalis spermatophore proteins and their func- universal tool for annotation, visualization and analysis in functional tions identified using LC-MS/MS. genomics research. Bioinformatics, 21, 3674–3676. Table S3. Sequences that are significantly differentially Cordero, C. (2009) On the function of cornuti, sclerotized structures of the endophallus of Lepidoptera. Genetica, 138, 27–35. expressed between strains of European corn borer males in Cusson, M. & McNeil, J.N. (1989) Involvement of juvenile hormone in the accessory gland and the ejaculatory duct. the regulation of pheromone release activities in a moth. Science, 243, 210–212. Dopman, E.B., Pérez, L., Bogdanowicz, S.M. & Harrison, R.G. (2005) Acknowledgements Consequences of reproductive barriers for genealogical discordance in the European corn borer. Proceedings of the National Academy of We thank Rick Harrison and Steve Bogdanowicz for ECB male Sciences of the United States of America, 102, 14706–14711. thorax library preparation, as well as Charlie Linn and Callie Dopman, E.B., Robbins, P.S. & Seaman, A. (2010) Components of reproductive isolation between North American pheromone strains of Musto for ECB specimens. This work was supported by a the European corn borer. Evolution, 64, 881–902. USDA-NIFA (grant number 2010-65106-20610 to E.B.D.) and Ellison, R.T. (1991) Killing of gram-negative bacteria by lactoferrin and NSF (grant number DEB-1257521 to E.B.D.). All sequences lysozyme. Journal of Clinical Investigation, 88, 1080–1091. reads have been deposited in the NCBI short-read archive and Findlay, G.D., Yi, X., Maccoss, M.J. & Swanson, W.J. (2008) Pro- can be accessed using the accession numbers (PRJNA401440): teomics reveals novel Drosophila seminal fluid proteins transferred will be included in published manuscript. at mating. PLoS Biology, 6, e178.

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