extfieldsofstudy: Chemistry Medicine Biology

text:
Mitochondrial Ca2+ uptake by the voltage-dependent anion channel 2 regulates cardiac rhythmicity

Tightly regulated Ca2+ homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca2+ handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca2+ extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca2+ uptake and accelerates the transfer of Ca2+ from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca2+ sparks and thereby inhibits Ca2+ overload-induced erratic Ca2+ waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca2+ uptake in the regulation of cardiac rhythmicity. DOI: http://dx.doi.org/10.7554/eLife.04801.001

Introduction
During development, well-orchestrated cellular processes guide cells from diverse lineages to integrate into the primitive heart tube and establish rhythmic and coordinated contractions. While many genes and pathways important for cardiac morphogenesis have been identified, molecular mechanisms governing embryonic cardiac rhythmicity are poorly understood. The findings that Ca 2+ waves traveling across the heart soon after the formation of the primitive heart tube (Chi et al., 2008) and that loss of function of key Ca 2+ regulatory proteins, such as the L-type Ca 2+ channel, Na/K−ATPase and sodium-calcium exchanger 1 (NCX1), severely impairs normal cardiac function (Rottbauer et al., 2001;Shu et al., 2003;Ebert et al., 2005;Langenbacher et al., 2005), indicate an essential role for Ca 2+ handling in the regulation of embryonic cardiac function.
Ca 2+ homoeostasis in cardiac muscle cells is tightly regulated at the temporal and spatial level by a subcellular network involving multiple proteins, pathways, and organelles. The release and reuptake of Ca 2+ by the sarcoplasmic reticulum (SR), the largest Ca 2+ store in cardiomyocytes, constitutes the primary mechanism governing the contraction and relaxation of the heart. Ca 2+ influx after activation of the L-type Ca 2+ channel in the plasma membrane induces the release of Ca 2+ from the SR via ryanodine receptor (RyR) channels, which leads to an increase of the intracellular Ca 2+ concentration and cardiac contraction. During diastolic relaxation, Ca 2+ is transferred back into the SR by the SR Ca 2+ pump or extruded from the cell through NCX1. Defects in cardiac Ca 2+ handling and Ca 2+ overload, for example during cardiac ischemia/reperfusion or in long QT syndrome, are well known causes of contractile dysfunction and many types of arrhythmias including early and delayed afterdepolarizations and Torsade des pointes (Bers, 2002;Choi et al., 2002;Yano et al., 2008;Greiser et al., 2011).
Ca 2+ crosstalk between mitochondria and ER/SR has been noted in many cell types and the voltagedependent anion channel (VDAC) and the mitochondrial Ca 2+ uniporter (MCU) serve as primary routes for Ca 2+ entry through the outer and inner mitochondrial membranes, respectively (Rapizzi et al., 2002;Bathori et al., 2006;Shoshan-Barmatz et al., 2010;Baughman et al., 2011;De Stefani et al., 2011). In the heart, mitochondria are tethered to the SR and are located in close proximity to Ca 2+ release sites (García-Pérez et al., 2008;Boncompagni et al., 2009;Hayashi et al., 2009). This subcellular architecture exposes the mitochondria near the Ca 2+ release sites to a high local Ca 2+ concentration eLife digest The heart is a large muscle that pumps blood around the body by maintaining a regular rhythm of contraction and relaxation. If the heart loses this regular rhythm it works less efficiently, which can lead to life-threatening conditions. Regular heart rhythms are maintained by changes in the concentration of calcium ions in the cytoplasm of the heart muscle cells. These changes are synchronised so that the heart cells contract in a controlled manner. In each cell, a contraction begins when calcium ions from outside the cell enter the cytoplasm by passing through a channel protein in the membrane that surrounds the cell. This triggers the release of even more calcium ions into the cytoplasm from stores within the cell. For the cells to relax, the calcium ions must then be pumped out of the cytoplasm to lower the calcium ion concentration back to the original level.
Shimizu et al. studied a zebrafish mutant-called tremblor-that has irregular heart rhythms because its heart muscle cells are unable to efficiently remove calcium ions from the cytoplasm. Embryos of the tremblor mutant were treated with a wide variety of chemical compounds with the aim of finding some that could correct the heart defect.
A compound called efsevin restores regular heart rhythms in tremblor mutants. Efsevin binds to a pump protein called VDAC2, which is found in compartments called mitochondria within the cell. Although mitochondria are best known for their role in supplying energy for the cell, they also act as internal stores for calcium. By binding to VDAC2, efsevin increases the rate at which calcium ions are pumped from the cytoplasm into the mitochondria. This restores rhythmic calcium ion cycling in the cytoplasm and enables the heart muscle cells to develop regular rhythms of contraction and relaxation. Increasing the levels of VDAC2 or another similar calcium ion pump protein in the heart cells can also restore a regular heart rhythm.
Efsevin can also correct irregular heart rhythms in human and mouse heart muscle cells, therefore the new role for mitochondria in controlling heart rhythms found by Shimizu et al. appears to be shared in other animals. The experiments have also identified the VDAC family of proteins as potential new targets for drug therapies to treat people with irregular heart rhythms. DOI: 10.7554/eLife.04801.002 that is sufficient to overcome the low Ca 2+ affinity of MCU and facilitates Ca 2+ crosstalk between SR and mitochondria (García-Pérez et al., 2008;Dorn and Scorrano, 2010;Kohlhaas and Maack, 2013). Increase of the mitochondrial Ca 2+ concentration enhances energy production during higher workload and dysregulation of SR-mitochondrial Ca 2+ signaling results in energetic deficits and oxidative stress in the heart and may trigger programmed cell death (Brandes and Bers, 1997;Maack et al., 2006;Kohlhaas and Maack, 2013). However, whether SR-mitochondrial Ca 2+ crosstalk also contributes significantly to cardiac Ca 2+ signaling during excitation-contraction coupling requires further investigation.
In zebrafish, the tremblor (tre) locus encodes a cardiac-specific isoform of the Na + /Ca 2+ exchanger 1, NCX1h (also known as slc8a1a) (Ebert et al., 2005;Langenbacher et al., 2005). The tre mutant hearts Fractional shortening (FS) deduced from the line-scan traces. While cardiac contraction was not observed in tre, efsevin-treated wild type and tre hearts have similar levels of FS to those observed in control hearts. Ventricular FS of wild type v.s. wild type + efsevin vs tre + efsevin: 39 ± 0.6%, n = 8 vs 39 ± 1%, n = 10 vs 35 ± 3%, n = 6; and Atrial FS: 37 ± 1%, n = 11 vs 35 ± 2%, n = 11 vs 33 ± 2%, n = 15. (D) While efsevin restored a heart rate of 46 ± 2 beats per minute (bpm) in tre embryos, same treatment does not affect the heart rate in wild type embryos (126 ± 2 bpm in vehicle-treated embryos vs 123 ± 3 bpm in efsevin-treated wild-type embryos). ***, p < 0.001 by one-way ANOVA. (E) Dose-dependence curve for efsevin. The tre embryos were treated with various concentrations of efsevin from 24 hpf and cardiac contractions were analyzed at 48 hpf. (F-H) Representative time traces of local field potentials for wild type (F), tre (G) and efsevin-treated tre (H) embryos clearly display periods of regular, irregular, and restored periodic electrical activity. (I) In vivo optical maps of Ca 2+ activation represented by isochronal lines every 33 ms recorded from 36 hpf wild type (left), tre (center) and efsevin-treated tre (right) embryos. DOI: 10.7554/eLife.04801.003 lack rhythmic Ca 2+ transients and display chaotic Ca 2+ signals in the myocardium leading to unsynchronized contractions resembling cardiac fibrillation (Langenbacher et al., 2005). In this study, we used tre as an animal model for aberrant Ca 2+ handling-induced cardiac dysfunction and took a chemical genetic approach to dissect the Ca 2+ regulatory network important for maintaining cardiac rhythmicity. A synthetic compound named efsevin was identified from a suppressor screen due to its potent ability to restore coordinated contractions in tre. Using biochemical and genetic approaches we show that efsevin interacts with VDAC2 and potentiates its mitochondrial Ca 2+ transporting activity and spatially and temporally modulates cytosolic Ca 2+ signals in cardiomyocytes. The important role of mitochondrial Ca 2+ uptake in regulating cardiac rhythmicity is further supported by the suppressive effect of VDAC2 and MCU overexpression on cardiac fibrillation in tre.

Results and discussion
Identification of a chemical suppressor of tre cardiac dysfunction Homozygous tre mutant embryos suffer from Ca 2+ extrusion defects and manifest chaotic cardiac contractions resembling fibrillation (Ebert et al., 2005;Langenbacher et al., 2005). To dissect the regulatory network of Ca 2+ handling in cardiomyocytes and to identify mechanisms controlling embryonic cardiac rhythmicity, we screened the BioMol library and a collection of synthetic compounds for chemicals that are capable of restoring heartbeat either completely or partially in tre embryos. A dihydropyrrole carboxylic ester compound named efsevin was identified based on its ability to restore persistent and rhythmic cardiac contractions in tre mutant embryos in a dose-dependent manner ( Figure 1A,E, and Videos 1-4). To validate the effect of efsevin, we assessed cardiac performance of wild type, tre and efsevin-treated tre embryos (Nguyen et al., 2009). Fractional shortening of efsevin treated tre mutant hearts was comparable to that of their wild type siblings and heart rate was restored to approximately 40% of that observed in controls ( Figure 1B-D). Periodic local field potentials accompanying each heartbeat were detected in wild type and efsevin-treated tre embryos using a microelectrode array ( Figure 1F-H). Furthermore, while only sporadic Ca 2+ signals were detected in tre hearts, in vivo Ca 2+ imaging revealed steady Ca 2+ waves propagating through efsevin-treated tre hearts ( Figure 1I, Videos 5-7), demonstrating that cardiomyocytes are functionally coupled and that efsevin treatment restores regular Ca 2+ transients in tre hearts.
Video 1. The video shows a heart of a wild-type zebrafish embryo at 2 dpf. Robust rhythmic contractions can be observed in atrium and ventricle. DOI: 10.7554/eLife.04801.004 Video 2. This video shows a heart of a tremblor embryo at 2 dpf. Embryos of the mutant line tremblor display only local, unsynchronized contractions, comparable to cardiac fibrillation. DOI: 10.7554/eLife.04801.005 Video 3. This video shows a heart of a tremblor embryo at 2 dpf treated with efsevin. Treatment of tremblor embryos with efsevin restores rhythmic contractions with comparable atrial fractional shortening compared to wild-type embryos and approximately 40% of wild-type heart rate. DOI: 10.7554/eLife.04801.006

Efsevin suppresses Ca 2+ overload-induced irregular contraction
We next examined whether efsevin could suppress aberrant Ca 2+ homeostasis-induced arrhythmic responses in mammalian cardiomyocytes. Mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) establish a regular contraction pattern with rhythmic Ca 2+ transients (Figure 2A,B,E,F). Mimicking Ca 2+ overload by increasing extracellular Ca 2+ levels was sufficient to disrupt normal Ca 2+ cycling and induce irregular contractions in mESC-CMs ( Figure 2C,E,F). Remarkably, efsevin treatment restored rhythmic Ca 2+ transients and cardiac contractions in these cells ( Figure 2D-F). Similar effect was observed in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) ( Figure 2G). Together, these findings suggest that efsevin targets a conserved Ca 2+ regulatory mechanism critical for maintaining rhythmic cardiac contraction in fish, mice and humans.

VDAC2 mediates the suppressive effect of efsevin on tre
To identify the protein target of efsevin, we generated a N-Boc-protected 2-aminoethoxyethoxyethylamine linker-attached efsevin (efsevin L ) ( Figure 3A,C). This modified compound retained the activity of efsevin to restore cardiac contractions in ncx1h deficient embryos ( Figure 3B,D) and was used to create efsevin-conjugated agarose beads (efsevin LB ). A 32kD protein species was detected from zebrafish lysate due to its binding ability to efsevin LB and OK-C125 LB , an active efsevin derivative conjugated to beads, but not to beads capped with ethanolamine alone or beads conjugated with an inactive efsevin analog (OK-C19 LB ) ( Figure 3A-E). Furthermore, preincubation of zebrafish lysate with excess efsevin prevented the 32kD protein from binding to efsevin LB or OK-C125 LB ( Figure 3E). Mass spectrometry analysis revealed that this 32kD band represents a zebrafish homologue of the mitochondrial voltage-dependent anion channel 2 (VDAC2) ( Figure 3F and VDAC2 is expressed in the developing zebrafish heart ( Figure 4A), making it a good candidate for mediating efsevin's effect on cardiac Ca 2+ handling. To examine this possibility, we injected in vitro synthesized VDAC2 RNA into tre embryos and found that the majority of these embryos had coordinated cardiac contractions similar to those subjected to efsevin treatment ( Figure 4B, Videos 8-11). In addition, we generated myl7:VDAC2 transgenic fish in which VDAC2 expression can be induced in the heart by tebufenozide (TBF) ( Figure 4C). Knocking down NCX1h in myl7:VDAC2 embryos results in chaotic cardiac movement similar to tre. Like efsevin treatment, induction of VDAC2 expression by TBF treatment restored coordinated and rhythmic contractions in myl7:VDAC2;NCX1h MO hearts ( Figure 4D, Videos 12,13). Conversely, knocking down VDAC2 in tre hearts attenuated the suppressive effect of efsevin ( Figure 4E, Videos 14-16). Furthermore, we generated VDAC2 null embryos by the Zinc Finger Nuclease gene targeting approach ( Figure 4G). Similar to that observed in morpholino knockdown embryos, homozygous VDAC2 LA2256 embryos do not exhibit noticeable morphological Video 4. The video shows a heart of a wild-type zebrafish embryo at 2 dpf treated with efsevin. Treatment of wild-type embryos with efsevin did not affect cardiac performance, indicated by robust, rhythmic contractions comparable to untreated wild-type embryos. DOI: 10.7554/eLife.04801.007 Video 5. Heat map of Ca 2+ transients recorded in 1 day old wild type heart. DOI: 10.7554/eLife.04801.008 defects, but the suppressive effect of efsevin was attenuated in homozygous VDAC2 LA2256 ; NCX1MO embryos ( Figure 4F). These findings demonstrate that VDAC2 is a major mediator for efsevin's effect on ncx1h deficient hearts.
VDAC2-dependent effect of efsevin on mitochondrial Ca 2+ uptake VDAC is an abundant channel located on the outer mitochondrial membrane serving as a primary passageway for metabolites and ions ( Figure 5A) (Rapizzi et al., 2002;Bathori et al., 2006;Shoshan-Barmatz et al., 2010). At its close state, VDAC favours Ca 2+ flux (Tan and Colombini, 2007). To examine whether efsevin would modulate mitochondrial Ca 2+ uptake via VDAC2, we transfected HeLa cells with VDAC2. We noted increased mitochondrial Ca 2+ uptake in permeabilized VDAC2 transfected and efsevin-treated cells after the addition of Ca 2+ and the combined treatment further enhanced mitochondrial Ca 2+ levels ( Figure 5B).
Mitochondria are located in close proximity to Ca 2+ release sites of the ER/SR and an extensive crosstalk between the two organelles exists (García-Pérez et al., 2008;Hayashi et al., 2009;Brown and O'Rourke, 2010;Dorn and Scorrano, 2010;Kohlhaas and Maack, 2013). We examined whether Ca 2+ released from intracellular stores could be locally transported into mitochondria through VDAC2 in VDAC1/VDAC3 double knockout (V1/V3DKO) MEFs where VDAC2 is the only VDAC isoform being expressed (Roy et al., 2009a). While treatments with ATP, an IP3-linked agonist, and thapsigargin, a SERCA inhibitor, stimulated similar global cytoplasmic [Ca 2+ ] elevation in intact cells, only ATP induced a rapid mitochondrial matrix [Ca 2+ ] rise (Figure 5-figure supplement 1). This finding is consistent with observations obtained in other cell types (Rizzuto et al., 1994;Hajnóczky et al., 1995) and suggests that Ca 2+ was locally transferred from IP3 receptors to mitochondria through VDAC2 at the close ER-mitochondrial associations. We next investigated whether this process could be modulated by efsevin. In permeabilized V1/V3DKO MEFs, treatment with efsevin increased the amount of Ca 2+ transferred into mitochondria during IP 3 -induced Ca 2+ release ( Figure 5C). Also, in intact V1/V3 DKO MEFs, efsevin accelerated the transfer of Ca 2+ released from intracellular stores into mitochondria during stimulation with ATP ( Figure 5D,E).

Efsevin modulates Ca 2+ sparks and suppresses erratic Ca 2+ waves in cardiomyocytes
We next examined the effect of efsevin on cytosolic Ca 2+ signals in isolated adult murine cardiomyocytes. We found that efsevin treatment induced faster inactivation kinetics without affecting the Video 6. Heat map of Ca 2+ transients recorded in 1 day old tremblor heart. DOI: 10.7554/eLife.04801.009 Video 7. Heat map of Ca 2+ transients recorded in 1 day old efsevin treated tremblor heart. DOI: 10.7554/eLife.04801.010 amplitude or time to peak of paced Ca 2+ transients ( Figure 6A). Similarly, efsevin treatment did not significantly alter the frequency, amplitude or Ca 2+ release flux of spontaneous Ca 2+ sparks, local Ca 2+ release events, but accelerated the decay phase resulting in sparks with a shorter duration and a narrower width ( Figure 6B). These results indicate that by activating mitochondrial Ca 2+ uptake, efsevin accelerates Ca 2+ removal from the cytosol in cardiomyocytes and thereby restricts local cytosolic Ca 2+ sparks to a narrower domain for a shorter period of time without affecting SR Ca 2+ load or RyR Ca 2+ release. Under conditions of Ca 2+ overload, single Ca 2+ sparks can trigger opening of neighbouring Ca 2+ release units and thus induce the formation of erratic Ca 2+ waves ( Figure 6C). Efsevin treatment significantly reduced the number of propagating Ca 2+ waves in a dosage-dependent manner ( Figure 6C,D), demonstrating a potent suppressive effect of efsevin on the propagation of Ca 2+ overload-induced Ca 2+ waves and suggesting that efsevin could serve as a pharmacological tool to manipulate local Ca 2+ signals. . After treatment with 10 mM Ca 2+ for 10 min, the EB showed an irregular pattern of Ca 2+ transients (C). Efsevin treatment restores regular Ca 2+ transients under Ca 2+ overload conditions in mESC-CMs (D). (E) Plotted intervals between peaks of Ca 2+ signals detected in mESC-CMs prior to treatment (control), in 10 mM Ca 2+ ext (Ca 2+ ) and in 10 mM Ca 2+ ext +10 μM efsevin (Ca 2+ +efsevin). (F, G) Plotted intervals of contractions detected in EBs prior to treatment (control), in 10 mM Ca 2+ ext (Ca 2+ ) and in 10 mM Ca 2+ ext + 10 μM efsevin (Ca 2+ + efsevin) for mouse ESC-CMs (F) and 5 mM Ca 2+ ext (Ca 2+ ) and in 5 mM Ca 2+ ext + 5 μM efsevin (Ca 2+ + efsevin) for human ESC-CMs (G). ***, p < 0.001 by F-test. DOI: 10.7554/eLife.04801.011 Mitochondrial Ca 2+ uptake modulates embryonic cardiac rhythmicity We hypothesize that efsevin treatment/VDAC2 overexpression suppresses aberrant Ca 2+ handlingassociated arrhythmic cardiac contractions by buffering excess Ca 2+ into mitochondria. This hypothesis predicts that activating other mitochondrial Ca 2+ uptake molecules would likewise restore coordinated contractions in tre. To test this model, we cloned zebrafish MCU and MICU1, an inner mitochondrial membrane Ca 2+ transporter and its regulator (Perocchi et al., 2010;Baughman et al., 2011;De Stefani et al., 2011;Mallilankaraman et al., 2012;Csordas et al., 2013). In situ hybridization showed that MCU and MICU1 were expressed in the developing zebrafish heart ( Figure 7A) and their expression levels were comparable between the wild type and tre hearts (Figure 7-figure  supplement 1). Overexpression of MCU restored coordinated contractions in tre, akin to what was observed with VDAC2 ( Figure 7B). In addition, tre embryos injected with suboptimal concentrations of MCU or VDAC2 had a fibrillating heart, but embryos receiving both VDAC2 and MCU at the suboptimal concentration manifested coordinated contractions ( Figure 7C), demonstrating a synergistic effect of these proteins. Furthermore, overexpression of MCU failed to suppress the tre phenotype in the absence of VDAC2 activity and VDAC2 could not restore coordinated contractions in tre without functional MCU ( Figure 7B,D). Similar results were observed by manipulating MICU1 activity ( Figure 7E,F). Together, these findings indicate that mitochondrial Ca 2+ uptake mechanisms on outer and inner mitochondrial membranes act cooperatively to regulate cardiac rhythmicity.

Conclusion
In summary, we conducted a chemical suppressor screen in zebrafish to dissect the regulatory network critical for maintaining rhythmic cardiac contractions and to identify mechanisms underlying aberrant

Research article
Ca 2+ handling-induced cardiac dysfunction. We show that activation of VDAC2 through overexpression or efsevin treatment potently restores rhythmic contractions in NCX1h deficient zebrafish hearts and effectively suppresses Ca 2+ overload-induced arrhythmogenic Ca 2+ events and irregular contractions in mouse and human cardiomyocytes. We provide evidence that potentiating VDAC2 activity enhances mitochondrial Ca 2+ uptake, accelerates Ca 2+ transfer from intracellular stores into mitochondria and spatially and temporally restricts single Ca 2+ sparks in cardiomyocytes. The crucial role of mitochondria in the regulation of cardiac rhythmicity is further supported by the findings that VDAC2 functions in concert with MCU; these genes have a strong synergistic effect on suppressing cardiac fibrillation and loss of function of either gene abrogates the rescue effect of the other in tre. Video 8. This video shows a heart of a wild-type zebrafish embryo at 1 dpf. Robust rhythmic contractions can be observed in atrium and ventricle. DOI: 10.7554/eLife.04801.015 The regulatory roles of mitochondrial Ca 2+ in cardiac metabolism, cell survival and fate have been studied extensively (Brown and O'Rourke, 2010;Dorn and Scorrano, 2010;Doenst et al., 2013;Kasahara et al., 2013;Kohlhaas and Maack, 2013;Luo and Anderson, 2013). Our study provides genetic and physiologic evidence supporting an additional role for mitochondria in regulating cardiac rhythmicity and reveals VDAC2 as a modulator of Ca 2+ handling in cardiomyocytes. Our findings, together with recent reports of the physical interaction between VDAC2 and RyR2 (Min et al., 2012) and the close proximity of outer and inner mitochondrial membranes at the contact sites between the mitochondria and the SR (García-Pérez et al., 2011), suggest an intriguing model. We propose that mitochondria facilitate an efficient clearance mechanism in the Ca 2+ microdomain, which modulates Ca 2+ handling without affecting global Ca 2+ signals in cardiomyocytes. In this model, VDAC facilitates mitochondrial Ca 2+ uptake via MCU complex and thereby controls the duration and the diffusion of cytosolic Ca 2+ near the Ca 2+ release sites to ensure rhythmic cardiac contractions. This model is consistent with our observation that efsevin treatment induces faster inactivation kinetics of cytosolic Ca 2+ transients without affecting the amplitude or the time to peak in cardiomyocytes and the reports that blocking mitochondrial Ca 2+ uptake has little impact on cytosolic Ca 2+ transients (Maack et al., 2006;Kohlhaas et al., 2010). Further support for this model comes from the observation of the Ca 2+ peaks on the OMM (Drago et al., 2012) and the finding that downregulating VDAC2 extends Ca 2+ sparks (Subedi et al., 2011;Min et al., 2012) and that blocking mitochondrial Ca 2+ uptake by Ru360 leads to an increased number of spontaneous propagating Ca 2+ waves (Seguchi et al., 2005). Future studies on the kinetics of VDAC2-dependent mitochondrial Ca 2+ uptake and exploring potential regulatory molecules for VDAC2 activity will provide insights into how the crosstalk between SR and mitochondria contributes to Ca 2+ handling and cardiac rhythmicity.
Aberrant Ca 2+ handling is associated with many cardiac dysfunctions including arrhythmia. Establishing animal models to study molecular mechanisms and develop new therapeutic strategies are therefore major preclinical needs. Our chemical suppressor screen identified a potent effect of efsevin and its biological target VDAC2 on manipulating cardiac Ca 2+ handling and restoring regular cardiac contractions in fish and mouse and human cardiomyocytes. This success indicates that fundamental mechanisms regulating cardiac function are conserved among vertebrates despite the existence of species-specific features and suggests a new paradigm of using zebrafish cardiac disease models for the dissection of critical genetic pathways and the discovery of new therapeutic approaches. Future studies examining the effects of efsevin on other Video 9. This video shows a heart of a wild-type zebrafish embryo injected with zebrafish VDAC2 mRNA at 1 dpf. Robust rhythmic contractions can be observed in atrium and ventricle. DOI: 10.7554/eLife.04801.016 Video 10. This video shows a heart of a tremblor embryo at 1 dpf. Tremblor embryos display only local, unsynchronized contractions, comparable to cardiac fibrillation. DOI: 10.7554/eLife.04801.017 Video 11. This video shows a heart of a tremblor embryo injected with zebrafish VDAC2 mRNA at 1 dpf. Overexpression of zebrafish VDAC2 mRNA restores rhythmic contractions in tremblor embryos. DOI: 10.7554/eLife.04801.018 arrhythmia models would further elucidate the potential for efsevin as a pharmacological tool to treat cardiac arrhythmia associated with aberrant Ca 2+ handling.

Molecular Biology
Full length VDAC2 cDNA was purchased from Open Biosystems (Huntsville, AL) and cloned into pCS2+ or pCS2+3XFLAG. Full length cDNA fragments of zebrafish MCU (Accession number: JX424822) and MICU1 (JX42823) were amplified from 2 dpf embryos and cloned into pCS2+. For mRNA synthesis, plasmids were linearized and mRNA was synthesized using the SP6 mMES-SAGE mMachine kit according to the manufacturers manual (Ambion, Austin, TX.).

Zebrafish injections
VDAC2 mRNA and morpholino antisense oligos (5′-GGGAACGGCCATTTTATCTGTTAAA-3′) (Genetools, Philomath, OR) were injected into one-cell stage embryos collected from crosses of tre tc318 heterozygotes. Cardiac performance was analyzed by visual inspection on 1 dpf. The tre mutant embryos were identified either by observing the fibrillation phenotype at 2-3 dpf or by genotyping as previously described (Langenbacher et al., 2005).

Chemical screen
Chemicals from a synthetic library (Castellano et al., 2007;Choi et al., 2011;Cruz et al., 2011) and from Biomol International LP (Farmingdale, NY) were screened for their ability to partially or completely restore persistent heartbeat in tre embryos. 12 embryos collected from crosses of tre tc318 heterozygotes were raised in the presence of individual compounds at a concentration of 10 µM from 4 hpf (Choi et al., 2011). Cardiac function was analyzed by visual inspection at 1 and 2 dpf. The hearts of tre tc318 embryos manifest a chaotic movement resembling cardiac fibrillation with intermittent contractions in rare occasion (Ebert et al., 2005;Langenbacher et al., 2005). Compounds that elicit persistent coordinated cardiac contractions were validated on large number of tre mutant embryos and NCX1h morphants (>500 embryos).
Video 12. This video shows a heart of a 2 dpf Tg-VDAC2 embryo injected with a morpholino targeting NCX1h. Morpholino knock-down of NCX1h results in a fibrillating heart. DOI: 10.7554/eLife.04801.019 Video 13. This video shows a heart of a 2 dpf NCX1h morphant in the Tg-VDAC2 genetic background. TBF treatment induces VDAC2 expression and restores coordinated cardiac contractions. DOI: 10.7554/eLife.04801.020 Video 14. This video shows a heart of a 2 dpf wild type zebrafish embryo injected with a morpholino targeting VDAC2. Morpholino knockdown of VDAC2 did not have obvious effects on cardiac performance. DOI: 10.7554/eLife.04801.021

Zebrafish cardiac imaging
Videos of GFP-labelled myl7:GFP hearts were taken at 30 frames per second. Line-scan analysis was performed along a line through the atria or the ventricles of these hearts (Nguyen et al., 2009). Fraction of shortening was deduced from the ratio of diastolic and systolic width and heart rate was determined by beats per minute. Cardiac parameters were analyzed in tremblor tc318 and VDAC2 LA2256 at 2 dpf.
Zebrafish optical mapping 36 hpf myl7:gCaMP4.1 embryos were imaged at a frame rate of 30 ms/frame. Electromechanical isolation was achieved by tnnt2MO (Milan et al., 2006). The fluorescence intensity of each pixel in a 2D map was normalize to generate heat maps and isochronal lines at 33 ms intervals were obtained by identifying the maximal spatial gradient for a given time point (Chi et al., 2008).

Mouse and human embryonic stem cells
The mouse E14Tg2a ESC and human H9 ESC line were cultured and differentiated as previously described (Blin et al., 2010;Arshi et al., 2013). At day 10 of differentiation, beating mouse EBs were exposed to external solution containing 10 mM CaCl 2 for 10 min before DMSO or efsevin (10 μM) treatment. Human EBs were differentiated for 15 days and treated with 5 mM CaCl 2 for 10 min before DMSO or efsevin (5 μM) treatment. Images of beating EBs were acquired at a rate of 30 frames/s and analyzed by motion-detection software. For calcium recording, the EBs were loaded with 10 μM fluo-4 AM in culture media for 30 min at 37°C. Line-scan analysis was performed and fluorescent signals were acquired by a Zeiss LSM510 confocal microscope.

Microelectrode array measurements
2-day-old wild type, tre, and efsevin-treated tre embryos were placed on uncoated, microelectrode arrays (MEAs) containing 120 integrated TiN electrodes (30 μm diameter, 200 μm interelectrode spacing). Local field potentials (LFPs) at each electrode were collected for three trials per embryo type over a period of three minutes at a sampling rate of 1 kHz using the MEA2100-HS120 system (Multichannel Systems, Reutiligen, Germany). Raw data was low-pass filtered at a cutoff frequency of 10 Hz using a third-order Butterworth filter. Data analysis was carried out using the MC_DataTool (Multichannel Systems) and Matlab (MathWorks).

Ca 2+ imaging
Murine ventricular cardiomyocytes were isolated as previously described (Reuter et al., 2004). Cells were loaded with 5 µM fluo-4 AM in external solution containing: 138.2 mM NaCl, 4.6 mM KCl, 1.2 mM MgCl, 15 mM glucose, 20 mM HEPES for 1 hr and imaged in external solution supplemented with 2, 5 or 10 mM CaCl 2 . For the recording of Ca 2+ sparks and transients, the external solution contained 2 mM CaCl 2 . For Ca 2+ transients, cells were field stimulated at 0.5 Hz with a 5 ms pulse at a voltage of 20% above contraction threshold. For all measurements, efsevin was added 2 hr prior to the actual experiment. Images were recorded on a Zeiss LSM 5 Pascal confocal microscope. Data analysis was carried out using the Zeiss LSM Image Browser and ImageJ with the SparkMaster plugin (Picht et al., 2007). Cells were visually inspected prior to and after each recording. Only those recordings from Video 15. This video shows a heart of a 2 dpf tremblor mutant embryo injected with a morpholino targeting VDAC2. DOI: 10.7554/eLife.04801.022 Video 16. This video shows a heart of a 2 dpf tremblor mutant embryo injected with a morpholino targeting VDAC2. Efsevin treatment cannot restore coordinated cardiac contractions in the absence of VDAC2. DOI: 10.7554/eLife.04801.023 Research article

Research article
Rhod2. Arrows denote the addition of Ca 2+ . Mitochondrial Ca 2+ uptake was assessed when VDAC2 was overexpressed (left), cells were treated with 1 µM efsevin (middle) and combination of both at suboptimal doses (right). Control-traces with ruthenium red (RuRed) show mitochondrial specificity of the signal. (C) Representative traces of cytosolic [Ca 2+ ] ([Ca 2+ ] c ) changes upon the application of 7.5 µM IP 3 in the presence (+) or absence (−) of RuRed. Mitochondrial Ca 2+ uptake was assessed by the difference of the -and + RuRed conditions normalized to the total release (n = 4; mean ± SE). (D) MEFs overexpressing zebrafish VDAC2 (polycistronic with mCherry) were stimulated with 1 μM ATP in a nominally Ca 2+

Biochemistry
For pull down assays mono-N-Boc protected 2,2'-(ethylenedioxy)bis(ethylamine) was attached to the carboxylic ester of efsevin and its derivatives through the amide bond. After removal of the Boc group using TFA, the primary amine was coupled to the carboxylic acid of Affi-Gel 10 Gel (Biorad, Hercules, CA). 2-day-old zebrafish embryos were deyolked by centrifugation before being lysed with Rubinfeld's lysis buffer (Rubinfeld et al., 1993). The lysate was precleaned by incubation with Affi-Gel 10 Gel to eliminate non-specific binding. Precleaned lysate was incubated with affinity beads overnight. Proteins were eluted from the affinity beads and separated on SDS-PAGE. Protein bands of interest were excised. Gel plugs were dehydrated in acetonitrile (ACN) and dried completely in a Speedvac. Samples were reduced and alkylated with 10 mM dithiotreitol and 10 mM TCEP solution in 50 mM NH 4 HCO 3 (30 min at 56°C) and 100 mM iodoacetamide (45 min in dark), respectively. Gel plugs were washed with 50 mM NH 4 HCO 3 , dehydrated with ACN, and dried down in a Speedvac. Gel pieces were then swollen in digestion buffer containing 50 mM NH 4 HCO 3 , and 20.0 ng/μl of chymotrypsin (25°C, overnight). Peptides were extracted with 0.1% TFA in 50% ACN solution, dried down and resuspended in LC buffer A (0.1% formic acid, 2% ACN).

Mass spectrometry analyses and database searching
Extracted peptides were analyzed by nano-flow LC/MS/MS on a Thermo Orbitrap with dedicated Eksigent nanopump using a reversed phase column (New Objective, Woburn, MA). The flow rate was 200 nl/min for separation: mobile phase A contained 0.1% formic acid, 2% ACN in water, and mobile phase B contained 0.1% formic acid, 20% water in ACN. The gradient used for analyses was linear from 5% B to 50% B over 60 min, then to 95% B over 15 min, and finally keeping constant 95% B for 10 min. Spectra were acquired in data-dependent mode with dynamic exclusion where the instrument selects the top six most abundant ions in the parent spectra for fragmentation. Data were searched against the Danio rerio IPI database v3.45 using the SEQUEST algorithm in the BioWorks software program version 3.3.1 SP1. All spectra used for identification had deltaCN>0.1 and met the following Xcorr criteria: >2 (+1), >3 (+2), >4 (+3), and >5 (+4). Searches required full cleavage with the enzyme, <4 missed cleavages and were performed with the differential modifications of carbamidomethylation on cysteine and methionine oxidation.

In situ hybridization
In situ hybridization was performed as previously described (Chen and Fishman, 1996). DIG-labeled RNA probe was synthesized using the DIG RNA labeling kit (Roche, Indianapolis, IN).
Mitochondria Ca 2+ uptake assay in VDAC1/VDAC3 double knockout (V1/V3 DKO) MEFs V1/V3 DKO MEFs were cultured as previously described (Roy et al., 2009a). Efsevin-treated (15 μM for 30 min) or mock-treated MEFs were used for measurements of [Ca 2+ ] c in suspensions of permeabilized cells or imaging of [Ca 2+ ] m simultaneously with [Ca 2+ ] c in intact single cells. Permeabilization of the plasma membrane was performed by digitonin (40 μM/ml). Changes in [Ca 2+ ] in the cytoplasmic buffer upon IP 3 (7.5 μM) addition in the presence or absence of ruthenium red (3 μM) was measured by fura2 in a fluorometer (Csordás et al., 2006;Roy et al., 2009b). To avoid endoplasmic reticulum Ca 2+ uptake 2 μM thapsigargin was added before IP 3 . For imaging of [Ca 2+ ] m and [Ca 2+ ] c , MEFs were co-transfected with plasmids encoding polycistronic zebrafish VDAC2 with mCherry and mitochondria-targeted inverse pericam for 40 hr. Cells were sorted to enrich the transfected cells and attached to glass coverslips. In the final 10 min, of the efsevin or mock-treatment, the cells were also loaded with fura2AM (2.5 μM) and subsequently transferred to the microscope stage. Stimulation with 1 μM ATP was carried out in a norminally Ca 2+ free buffer. Changes in [Ca 2+ ] c and [Ca 2+ ] m were imaged using fura2 (ratio of ex:340 nm-380 nm) and mitochondria-targeted inverse pericam (ex: 495 nm), respectively (Csordas et al., 2010).

Statistics
All values are expressed as mean ± SEM, unless otherwise specified. Significance values are calculated by unpaired student's t-test unless noted otherwise. JS, Designed, performed, analyzed, and interpreted experiments, Wrote the manuscript, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article; JH, Designed, performed, analyzed, and interpreted experiments, Conception and design, Acquisition of data, Analysis and interpretation of data; KL, Designed and synthesized the compound library and all efsevin used for experiments, Conception and design, Acquisition of data; SN, AE, Examined mitochondrial Ca 2+ uptake in V1/V3KO MEFs, Acquisition of data, Analysis and interpretation of data; FL, KW, CT, Performed, analyzed, and interpreted experiments, Acquisition of data, Analysis and interpretation of data; SF, Designed and performed the mass-spec analysis, Acquisition of data, Analysis and interpretation of data; HDGF, Designed and synthesized the compound library and all efsevin used for experiments, Acquisition of data, Analysis and interpretation of data; HZ, Designed and performed MEA analysis, Acquisition of data, Analysis and interpretation of data; BL, Performed, analyzed, and interpreted experiments, Acquisition of data; HN, Designed and performed hESC-CM experiments, Acquisition of data, Analysis and interpretation of data; JN, Provided gCaMP construct, Contributed unpublished essential data or reagents; AZS, JKG, Designed and interpreted MEA analysis, Analysis and interpretation of data; AN, Designed and performed hESC-CM experiments, Analysis and interpretation of data; JIG, Supervised physiological analysis, Analysis and interpretation of data; TMV, Designed and performed the mass-spec analysis, Analysis and interpretation of data; GH, Examined mitochondrial Ca 2+ uptake in V1/V3KO MEFsDesigned and formulated hypothesis, Designed and synthesized the compound library and all efsevin used for experiments, Analysis and interpretation of data; OK, Designed and synthesized the compound library and all efsevin used for experiments, Conception and design, Analysis and interpretation of data; J-NC, Designed and formulated hypothesis, Performed, analyzed, and interpreted experiments, Wrote the manuscript, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article Author ORCIDs Adam Z Stieg, http://orcid.org/0000-0001-7312-9364 Ethics Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of the University of California, Los Angeles and the Cedars-Sinai Hospital. The protocols were approved by the Cedars-Sinai Institutional Animal Care and Use Committee (#003574 for the use of mouse cardiomyocytes), the Office of Animal Research Oversight that oversees the Ethics of Animal Experiments (ARC# 2000-051-43B for the use of zebrafish) and Embryonic Stem Cell Research Oversight (#2009-006-06 for the use of ES cells) of the University of California, Los Angeles. Every effort was made to minimize suffering.


extfieldsofstudy: Biology Medicine

text:
Transcriptome-Based Identification of Highly Similar Odorant-Binding Proteins among Neotropical Stink Bugs and Their Egg Parasitoid

Olfaction plays a fundamental role in insect survival through resource location and intra and interspecific communications. We used RNA-Seq to analyze transcriptomes for odorant-binding proteins (OBPs) from major stink bug pest species in Brazil, Euschistus heros, Chinavia ubica, and Dichelops melacanthus, and from their egg parasitoid, Telenomus podisi. We identified 23 OBPs in E. heros, 25 OBPs in C. ubica, 9 OBPs in D. melacanthus, and 7 OBPs in T. podisi. The deduced amino acid sequences of the full-length OBPs had low intraspecific similarity, but very high similarity between two pairs of OBPs from E. heros and C. ubica (76.4 and 84.0%) and between two pairs of OBPs from the parasitoid and its preferred host E. heros (82.4 and 88.5%), confirmed by a high similarity of their predicted tertiary structures. The similar pairs of OBPs from E. heros and C. ubica may suggest that they have derived from a common ancestor, and retain the same biological function to bind a ligand perceived or produced in both species. The T. podisi OBPs similar to E. heros were not orthologous to any known hymenopteran OBPs, and may have evolved independently and converged to the host OBPs, providing a possible basis for the host location of T. podisi using E. heros semiochemical cues.

In insects, it is well known that olfaction occurs in olfactory sensilla, mostly found in the antenna, through a complex of proteins, in which the odorant binding proteins (OBPs) have been the most studied because of their key role in the first step of olfaction [29,30]. They act as a semiochemical solubilizer, transporter, and ligand-specific selector for mediating the activation of the olfactory receptors enabling insect olfactory perception [30][31][32][33][34][35][36][37][38][39]. They were first obtained through biochemical protein isolation from crude antennal extracts (e.g., [40]) and were characterized as small globular water-soluble acidic proteins (about 120-150 amino acids) with a signal peptide in the N-terminal region, six cysteine (Cys) residues in conserved positions, and a predominant alpha-helical secondary structure (e.g., [40,41]). With the application of cDNA libraries (EST-based) and massive DNA parallel sequencing (e.g., [42,43]) OBPs had been found to comprise a multigene family found in 10 different orders, including 32 hemipteran and 43 hymenopteran species (National Center for Biotechnology Information-NCBI). Since then, OBPs have been classified into subfamilies based on the number of conserved Cys: classical OBPs (six), plus-C (more than six and a conserved proline, and according to Ji et al. [44] with a C-terminal extension), minus-C (less than six), and OBP dimers (two complete OBP domains each with six conserved Cys) [37,41,43,[45][46][47][48][49][50].
The pattern of six conserved cysteines (Cys motif) has been used as a signature for OBP identification (e.g., [37,43,48,51,52]). The Cys motif forms three disulphide bridges [47], which, in combination with the amino acid sequence and the physicochemical environment, generate the tertiary structure with a binding pocket that reversibly binds a particular ligand. This OBP domain comprises more than 70% of the amino acid sequence, with a general pattern of low similarity within and among species [41]. Exceptions were observed in some cases intraspecifically, such as in splice variants [53], and interspecifically in some closely related species with known similar ligands [33,54,55]. Such OBP diversity suggests selective binding of odorants in a given species (e.g., [34,55,56]), and may reflect the diversity of chemosensory behavior among the insects [56].
In this work, we analyzed transcriptomes from the main stink bug pest species of soybean in Brazil (E. heros, C. ubica and D. melacanthus), and from one of their main natural enemies, the egg parasitoid T. podisi to identify and characterize OBP transcripts. Finally, the similarity and phylogeny among these and other available OBPs were analyzed to determine if closely related species share similar OBPs, and if hosts and their natural enemy also share similar OBPs, suggesting they may respond to similar chemical signals/cues.

Insects
The three stink bugs species, E. heros, C. ubica and D. melacanthus, and the parasitoid T. podisi were obtained from laboratory colonies in Embrapa Genetic Resources and Biotechnology (Brasília, Distrito Federal, Brazil), and reared according to Borges et al. [57] and Moraes et al. [7] on raw peanuts (Arachis hypogaea), soybeans (Glycine max), sunflower (Helianthus annuus), fresh green beans (Phaseolus vulgaris), and water. Insects were kept in 8 L plastic containers (50-60 insects/container) with the food supply renewed three times per week. Unmated male and female adults were placed in separate cages, and after 12 days when the sex pheromone is produced at a consistently high level [58], their antennae were removed using entomological scissors, and immediately used for RNA extraction. Telenomus podisi was reared according to Borges et al. [11] in 25 cm 3 plastic cages, fed with pure honey and provided E. heros eggs to be used as hosts. When the parasitoids were 20 days-old, they were used for RNA extraction.

RNA-Seq Construction and Sequencing
A total of 50 pairs of antennae from stink bug males and females (1:1) from each species and 300 T. podisi, mostly females, were ground in TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, California, USA) and total RNA was obtained according to the method described by Chomczynski and Sacchi [59]. Immediately after extraction, 1 μg of the total RNA of each sample was individually placed in RNAstable tubes (Biomatrica, San Diego, California, USA) and shipped to Beijing Genomics Institute (BGI, China) for sequencing. The integrity/quality of the RNA samples was evaluated using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA) and as the RNA Integrity Numbers were 7.0, the RNA samples were used for RNA-Seq library construction and sequencing using the Illumina GAIIx platform (one lane/sample, 100 bp paired-end).

De novo Transcriptome Assembly and Annotation
The four transcriptome datasets were received in Fastq format. Quality control was performed by Trimmomatic (http://etal.usadellab.org/cms/?page = trimmomatic) using leading 3, trailing 3, sliding window 4:15 and minimum length 36. Before assembly, the high quality paired and unpaired reads were run through VelvetOptimiser 2.2.5 (http://bioinformatics.net.au/software. velvetoptimiser.shtml) to find the best k-mer per dataset in the interval 15 to 47 k-mers. The assembly was first made using Velvet (version 1.2.0.7), and then Oases (version 0.2.0.8) was applied to the output from Velvet (both K-mers). CEGMA software (Core Eukaryotic Genes Mapping Approach) was used (default parameters) to assess the completeness of the transcriptome assemblies, and identify the presence of a core consisting of 248 highly conserved proteins that are found in a wide range of eukaryotes, mostly housekeeping genes, that can be expected to be expressed [60].
To search for related olfaction transcripts, the assembled transcripts (contigs) were annotated using a BLASTx search against a non-redundant protein database from NCBI (E-value 1e-5) and the results were loaded into Blast2GO software version 2.7.0 [61]. The Blast2GO annotation and mapping steps added the gene ontology terms and the transcripts were categorized into biological processes and molecular functions. For each species, the 15 most common biological processes or molecular functions were identified and combined, resulting in a total of 24 biological processes or molecular functions for the four species. Then, the number of annotated sequences in each process or function was normalized by the total number of sequences for each species and the normalized proportions were ranked for statistical analyses with the Cochran-Mantel-Haenszel (CMH) test (Chi-square statistic) on ranks (SAS version 9.3). The species distribution charts of the four species were compared with the Kruskal-Wallis (H) test for independence of ranks.
The putative OBP contigs were screened for open reading frames (ORFs) using the software ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) to identify the coding region. In addition, the OBP domain (SM00708) and gene family (PF01395) were checked by InterProScan 5 [62]. We have named the mined putative OBP ORFs by the first letter of the genus followed by the first three letters of the species name followed by a number in increasing scaffold order. The only exception is for the putative OBP found for D. melacanthus, which we named using the first two letters of the genus, so that it is not confused with Drosophila melanogaster OBPs. The raw sequence transcriptome data were deposited in the Sequence Read Archive (SRA) at DDBJ/EMBL/GenBank database and can be accessed under BioProject PRJNA246320. The assembly results were deposited in the Transcriptome Shotgun Assembly (TSA) and can be accessed as first versions: GBER01000000 for E. heros, GBFA01000000 for C. ubica, GBES01000000 for D. melacanthus, and GBEU01000000 for T. podisi.

In silico Characterization
The full-length putative OBPs were submitted to a series of in silico analyses to predict: the deduced amino acid sequences (Expasy Translate Tool, http://web.expasy.org/translate/); the isoeletric point (pI) and molecular weight (MW) (Compute pI/Mw, http://web.expasy.org/ compute_pi/); the signal peptide presence (SignalP 4.1, http://etal.cbs.dtu.dk/services/SignalP/) [63]; as well as tertiary structure and alignment (I-TASSER server 4.2) [64]. To predict the tertiary structure, a query sequence was first threaded by LOMETS through the nr Protein Data Bank (PDB) library to identify structural templates, which were adopted for further structural reassembly, model selection and refinement, following the I-TASSER Suite pipeline [65], which was especially designed for proteins with unknown structure and with low identity (25%) with known structures (Table A in S2 File).

Similarity Analyses
The deduced amino acid sequences of the putative full-length OBPs were aligned with the OBPs from other species available at GenBank using MAFFT 7.017 [66] with the algorithm auto, scoring matrix BLOSUM62, gap opening penalty 1.53, and offset value of 0.123 using Geneious 7.0.5. The OBP sequence orthologs were selected by BLASTx using the criteria of: minimum similarity of E-value< 10e-20 with identity higher than 35% in query coverage of at least 70% for the alignment with the parasitoid putative OBPs, and E-value< 10e-7 with identity higher than 26% in query coverage of at least 77% for the alignment with the target stink bug putative OBPs. We also constructed phylogenetic trees with the mined OBPs from the target stink bugs and the 185 hemipteran OBPs, and with the mined OBPs from the target parasitoid and the 215 hymenopteran OBPs, all available at DDBJ/EMBL/GenBank. First we used the MEGA 6.06 program [67] to find the best Maximum Likelihood (ML) model using the BIC and AICc. Both phylogenetic trees were constructed using the LG+G+I model with 1,000 bootstrap replications, partial deletion of gaps, coverage cutoff 95%, and Nearest-Neighbor Interchange (NNI) ML heuristic method, with strong branch swap filter, to generate an unrooted tree.

Library Statistics and Transcriptome Assembly, Annotation and Completeness
Transcriptome data of four important economic species in soybean in Brazil were generated and compared for the first time: the phytophagous stink bugs E. heros, C. ubica and D. melacanthus, and their biological control agent the egg parasitoid T. podisi. The total number of reads obtained after quality control in the stink bug and parasitoid libraries is presented in Table 1, as well as the number and maximum length of contigs. The total number of reads before quality control for the four libraries was similar (data not showed). However, after quality control, the number was uneven, resulting in uneven assembly with 19,697 contigs for E. heros (3,812 transcripts annotated), 41,860 for C. ubica (4,594 annotated), 8,647 for D. melacanthus (1,680 annotated), and 51,106 for T. podisi (5,540 annotated) (Table B in S2 File). As the RNA samples had similar quality scores (RIN 7.0) and the total number of reads was similar in the four libraries before quality control, the lower number of high quality reads for D. melacanthus could have occurred due to some bias, possibly in library construction or sequencing. Similarly, regarding the transcriptome completeness assessments, we identified 223 out of 248 constitutive genes indicated by CEGMA for T. podisi, i.e. 89.9% completeness, while for the stink bugs it was 50.0% for C. ubica, 44.35% for E. heros, and 14.9% for D. melacanthus (Table C in S2 File). One factor that may have influenced the completeness was that the parasitoid T. podisi transcriptome was extracted from the whole body, while the transcriptomes from the stinkbugs were from a specialized tissue (antennae). The uneven number of annotated contigs obtained from the four target species reflected both the BLASTx results and annotation process: 5,540 annotations out of 22,926 BLASTx results for T. podisi, 4,594 annotations out of 7,370 for C. ubica, 3,812 out of 5,142 for E. heros, and 1,680 out of 2,346 for D. melacanthus. The predicted olfaction related transcripts are indicated in Tables D to G in S2 File. The total number of putative olfaction related transcripts was similar for the stink bugs E. heros (23 OBPs, with four full-length) and C. ubica (25 OBPs, with four full-length), but lower for the stink bug D. melacanthus (9 OBPs, with one full-length) and for the parasitoid T. podisi (7 OBPs, with three full-length). We previously identified two other putative full-length E. heros OBPs (EherOBP1 and EherOBP2, GenBank codes ADJ18275.1 and ADO24165.1, respectively) [68] through antennal cDNA library screening, so the total number of putative OBPs for E. heros is actually 25, the same as found for C. ubica. The lower number of putative olfaction transcripts from D. melacanthus compared to the other stink bugs might be related to its lower transcriptome coverage, although it could also be related to a biological reason. Studies on D. melacanthus have shown that they produce a similar blend of defensive compounds identified in other stink bugs [58,69,70], but there is currently no report identifying its sex or aggregation pheromone. It is not yet possible to determine if this species has or does not have similar chemical communication as other stink bugs, i.e, this species may also use non-chemical cues to locate host and partners, such as vibrational or visual signals, which could explain why they have fewer putative OBPs [7]. Regardless, the number of OBPs identified could have been higher by using a higher performance sequencing or increasing the number of lanes used.
Gu et al. [71] constructed cDNA libraries from the male and female antennae of the lucerne plant bug, Adelphocoris lineolatus (Hemiptera: Miridae), and selecting 2,915 ESTs they obtained 1,423 unigenes (with 215 contigs) with an average length of 532 bp and 549 bp for ESTs from the male and the female libraries, respectively, from which were annotated 14 putative OBPs. In the antennal cDNA library from the green plant bug Apolygus lucorum (Hemiptera: Miridae), Ji et al. [44] obtained 5,018 ESTs, which assembled into 3,881 unigenes (with 783 contigs) with an average length of 144 bp, from which were annotated 12 putative OBPs that also had a molecular function of putative binding proteins. The number of putative OBPs found in the antennae of the stink bugs E. heros and C. ubica was higher than that found in the lucerne and green plant bugs, however, the plant bug studies produced the transcriptome through Sanger sequencing instead of high throughput sequencing and had a much smaller number of unigenes/contigs from which to search for putative OBP transcripts. The heterogeneity of OBPs mined from the Neotropical stink bugs E. heros and C. ubica was also higher than that identified for other hemipterans, including the aphids Acyrthosiphon pisum, with 15 putative OBPs identified through its genome sequence [41], and Aphis gossypii, with 10 OBPs identified using a high throughput sequencing platform (from total of 54,547 unigenes) [72], the human body louse, with five genes obtained from its genome project [73], and the white-backed planthopper Sogatella furcifera, with 12 OBPs obtained from a transcriptome analysis [74]. The only exception was the tarnished plant bug Lygus lineolaris, from which 33 putative OBPs were found using a high throughput sequencing platform; however, instead of a single life stage, the researchers sequenced all developmental stages to generate the libraries [53]. No comparison was possible with other Pentatomidae species because we were the first group to identify putative OBPs in this family [68].
In the parasitoid wasp, Cotesia vestalis, using transcriptome pyrosequencing of antennae, Nishimura et al. [75] obtained 17,238 contigs (average size 549 bp) and 31,921 singletons, which had a similar species distribution as that found for the target parasitoid T. podisi. They annotated 22 putative OBPs, a higher number of OBPs than mined from the whole body of T. podisi. The number of OBPs from T. podisi was also smaller than that obtained from genome analyses of other hymenoptera, e.g., 12 putative OBPs mined from the fire ant Solenopsis invicta [76], 21 from Apis mellifera [48], and 90 from the parasitoid Nasonia vitripennis [50]. Despite the high transcriptome coverage of T. podisi (51,106 contigs), the smaller number of OBPs mined from it compared to these other hymenopteran species might be related to having analyzed the whole body, instead of a tissue specialized in olfaction, such as the antenna. However, in the solitary bee Osmia cornuta, Yin et al. [77] annotated only six putative OBPs from a transcriptome analysis of the antennae using high throughput sequencing, which assembled 85,143 contigs (average length of 914.5 bp).
The retained 21 match species were ranked among the three target stink bugs and parasitoid data ( Table 2). The distribution of species matches was significantly different among the three target stink bug hosts and their parasitoid (H = 146.803, df = 60, P< 0.0001). As expected, most matches were with species in the same order as the target species. For the stink bugs the main species were Hemiptera, and for the parasitoid the main species were Hymenoptera. Six Identification of Similar OBPs through Transcriptome Analyses species were significantly different among the four target species. Acyrthosiphon pisum, Tri. castaneum, P. humanus, and Bombyx mori were similarly ranked among the three stink bugs, but ranked significantly lower in the parasitoid. The first three species were also the most similar to the antennal transcriptome of the 3 day-old adult hemipteran A. lineolatus (Miridae) [71]. Bombus terrestris and N. vitripennis were significantly higher ranked in the parasitoid compared to the stink bugs, and the parasitoid top-hits species distribution was similar to that found for the antennal transcriptome of 24 h adult wasps Co. vestalis (Braconidae) [75]. Based on this initial result, data were analyzed for the three stink bugs only. There was no difference in match species among these three species (H = 5.994, df = 40, P = 1.000).
Overall, comparing the BLASTx results between the stink bug species and the parasitoid, the number of hits found for the parasitoid was more than twice that for the stink bug species. This may reflect the difference in the starting number of contigs (more from T. podisi) or indicate that the transcriptome of a specific tissue (antenna) may have poorer representation in the public databases. The list of matching species should be considered carefully, as it could be related to the proportion and representation of species in the public databases (and the particular physiologic condition of the insects from which the transcriptomes were obtained) rather than having a phylogenetic meaning. The relative abundance of the transcripts was also analyzed based on Gene Ontology (GO) classifications for biological process and molecular function (Fig 2). Some of the most common biological processes differed quantitatively among the target species (CMH = 58.34, df = 21, P< 0.0001). The three most common of these were oxidation-reduction processes, regulation of transcription DNA-dependent, and translation. Of the 24 most common biological processes in the four target species, C. ubica had the highest richness and differed from the others in ribosome biogenesis, electron transport, serine family amino acid metabolic process, signal transduction, sodium ion transport and proton transport. Telenomus podisi differed from the others in ion transport, and all four species differed from each other in microtubule-based movement.
The molecular functions had a rich representation of binding activities (such as to ATP, nucleic acids and ions). The most common molecular functions also differed quantitatively among the species (CMH = 81.67, df = 21, P< 0.0001). Of the 24 most common molecular functions in the four target species, D. melacanthus differed from the others in RNA binding; T. podisi differed from the others in catalytic activity, and sequence-specific DNA binding (GO:0043565 and GO:0003700). All four species differed from each other in protein serine/ threonine kinase activity.
The biological processes diverged more among the target species than the molecular functions, as can be observed in Fig 3. More biological processes were identified in the antennae of C. ubica (n = 160, 70 exclusive), than in T. podisi (n = 121, 43 exclusive), E. heros (n = 87, 13 exclusive) and D. melacanthus (n = 39, 1 exclusive). In total, 32 biological processes were shared among the four species, but only two were exclusive to the three stink bug species. More molecular functions were identified in the whole body of T. podisi (n = 121, 39 exclusive), than in the stink bugs C. ubica (n = 107, 14 exclusive), E. heros (n = 99, 7 exclusive), and D. melacanthus (n = 49, none exclusive). The stink bugs shared more molecular functions among themselves than with the parasitoid T. podisi.
Overall, the transcriptome composition of the stink bugs and the parasitoid was similar to other hemipteran and hymenopteran species. In the antenna of 3 day-old adult hemipterans A. lineolatus [71] and Ap. lucorum [44], the relative abundance of the transcripts also had higher representation of binding and catalytic activity in the molecular function ontology, and cellular process in the biological process ontology. In the 24 h emerged adult wasps Co. vestalis, the genes expressed in the antennae were also mostly associated with molecular binding activity or categorized as catalysts [75].

In silico Characterization
The full-length putative OBPs (Table 3) revealed OBPs with physicochemical properties similar to other OBPs [31,33,78]. They are small (136-212 residues and 14,627.8-23,783.1 Da), have an amino terminal signal peptide (from position 1 to around 20) followed by the OBP domain until a few residues prior to the carboxyl terminal end, and have an acidic pI< 6, except for DimelOBP1 with a pI of 6.57 and TpodOBP3 with an unusual basic pI of 9.41. The predicted tertiary structures had five to seven alpha-helixes (Fig 4), held together by three disulfide bridges between Cys1-Cys3, Cys2-Cys5, and Cys4-Cys6, as expected for an OBP structure [38].
Regarding the C-pattern, the putative OBPs from the target stink bugs and parasitoid had six cysteines (Cys) in conserved positions, as in a classical OBP, except for DimelOBP1 with five Cys (minus C6), which would be classified as a minus-C OBP [43]. In this work, all putative OBPs have the strictly conserved three residues between C2-C3, and most of them (9 of 14) have the expected Cys motif-pattern for Hemiptera (C1-X 22-32 -C2-X 3 -C3-X 36-46 -C4-X 8-14 -C5-X 8 -C6) [52], except for EherOBP1 and EherOBP5 with C1-X 41 -C2, EherOBP5 and Eher-OBP6 and CubiOBP1 with C4-X 22 -C5, EherOBP6 and CubiOBP1 with C5-X 9 -C6, and Tpo-dOBP2 with C3-X 46 -C4. In addition, EherOBP6 and CubiOBP1 have additional conserved Cys: C1a, C1b, C1c, C6a, C6b and C6c, a proline (P) after the C6a, and an extended C-terminal end. These characteristics indicate that EherOBP6 and CubiOBP1 are OBP Plus-C [43].  Therefore, of the six putative OBPs found for E. heros, five are classical OBPs and one is a Plus-C OBP, and of the four found for C. ubica, three are classical OBPs and one is a Plus-C OBP. The number of Plus-C OBPs found was less than that from other Hemiptera species, e.g., two from A. lineolatus [71]; four-six from Ap. lucorum [44,71]; 12 from L. lineolaris [53]; two from pea aphid A. pisum [41] and three from S. furcifera [74]. For the parasitoid, all three fulllength putative OBPs had the classical C-pattern, no other OBP category was found, similar to the parasitoid N. vitripennis, with a large number (90) of putative OBPs identified [50], and for Co. vestalis [75].

OBP Similarity
The similarity analysis of the mined hypothetical OBPs from the target stink bugs and parasitoid (Fig 4) with other orthologous OBPs from different insect species is represented in Figs 5 and 6 and in Table H in S2 File. The predicted tertiary structure of the stinkbug and parasitoid full-length putative OBPs are individually presented in Figure A in S2 File. In all the alignments the most conserved regions were around the conserved Cys. The sequence similarity among EherOBPs ranged from 5.1 to 25.2%, among CubiOBPs from 9.3 to 25.3%, and among Tpo-dOBPs from 16.3 to 27.5%.
The highest sequence similarities were obtained from four interspecific comparisons. Euschistus heros and C. ubica had two cases of high similarity across their OBPs: between Eher-OBP6 and CubiOBP1 with 84% similarity, and between EherOBP3 and CubiOBP3 with 76.4% similarity. The most interesting findings of this work were the high similarities of OBPs between the preferred stink bug host and its natural enemy: TpodOBP1 & EherOBP2 (88.5%) and TpodOBP2 & EherOBP1 (82.4%). TpodOBP1 also had similarity to OBPs from the Coleoptera, Hemiptera, Orthoptera, and Thysanoptera (ranging from 29.4 to 49.3%), and Tpo-dOBP2 only with hemipteran OBPs (ranging from 28.6 to 44.2%). TpodOBP3 was the only one similar to hymenopteran OBPs, although at no more than 30% similarity. The alignment of the predicted tertiary structure between these four pairs also presented high similarity, especially among the α-helixes (Fig 4). As expected, the loops were the most variable regions.
Previous studies have generally found low OBP similarities among species, typically < 40% [71], with a few exceptions among related species. Lepidopteran Pheromone Binding Proteins (PBPs) and general OBPs have 31-91% and 47-100% similarities respectively [33]. The OBPs from 12 aphid species all cluster to 10 groups with 81.2% similarity within groups compared to an overall similarity of only 20.0% [72]. High interspecific similarity was not common within the Heteroptera and only two pairs of mirid OBPs had high similarity, 89% and 74% [44,71]. The high similarities observed between EherOBP6 & CubiOBP1 and EherOBP3 & CubiOBP3 are consistent with these reports. Only Yin et al. [77] report similarities of OBPs from insects of different families, and the highest observed similarity was 65%. The observed similarity across insect orders between TpodOBP1 & EherOBP2, and TpodOBP2 & EherOBP1 was much higher than previously reported for phylogenetically unrelated species. Similarity is scored by matrix Blosum62 where the black color indicates 100% identity, darker grey 100% > identity 80%, lighter grey 80% > identity 60% and white color identity <60%. The sequence logo is at the top of the alignment. The amino acid percentage identity matrix is presented in Table H in S2 File. The conserved Cys are indicated by sequence logo. The species names are abbreviated with four letters, and their full names with all accession numbers of the OBP amino acid sequences are provided in Table I   (c) TpodOBP3. Similarity is scored by matrix Blosum62 where the black color indicates 100% identity, darker grey 100% > identity 80%, lighter grey 80% > identity 60% and white color identity <60%. The sequence logo is at the top of the alignment. The amino acid percentage identity matrix is presented in Table H in S2 File. The conserved Cys are indicated by sequence logo. The species names are abbreviated with four letters, and their full names with all accession numbers of the OBP amino acid sequences are provided in Table I  The phylogenic analysis (Fig 7) for the target stink bug OBPs with the other 185 hemipteran sequences from 28 species confirmed the intraspecific divergence of their putative OBPs. The OBP phylogeny did not correspond to the known hemipteran phylogeny. The intraspecific OBPs did not form a single clade, and for both E. heros and C. ubica, their OBPs mapped to different clades. At the same time, the phylogeny showed the convergence or shared derived character of the two pairs of similar putative OBPs across E. heros and C. ubica. EherOBP6 and CubiOBP1 were sisters with high (99) bootstrap support. They had three mirid OBPs as their closest outgroup with high (92) bootstrap support, but the next closest outgroup was unclear (bootstrap support 12). EherOBP3 and CubiOBP3 were sisters with high (98) bootstrap support, but they were not clearly linked to any outgroup (bootstrap support 0).
The phylogenetic relations of the other stink bug OBPs revealed two patterns. The first was monophyly with early divergence from other OBPs. EherOBP1 was monophyletic with one reduviid and 7 mirid OPBs with high (96) bootstrap support (Fig 7B), EherOBP5 was monophyletic with four mirid, one delphacid, and seven aphidid OBPs (bootstrap support 93), and DimelOBP1 was monophyletic with three mirid and one reduviid OBPs (bootstrap support 94). The EherOBP6 & CubiOBP1 pair mentioned above was also a part of a monophyletic group of three mirids (bootstrap support 92). As the database is enriched with more species of Heteroptera, these clades are likely to remain monophyletic, but the phylogenetic pattern within the clade will become clarified.
The second pattern was exhibited by EherOBP2, EherOBP4, CubiOBP2, CubiOBP4, and the EherOBP3/CubiOBP3 pair, which had uncertain phylogenetic position, probably because there were no similar OBPs among the Hemiptera in the database. EherOBP2 was distantly related to two mirids and delphacids (bootstrap support 45). CubiOBP2 was distantly related to other hemipteran OBPs (bootstrap support 23). EherOBP4 and CubiOBP4 may be related to three reduviid OBPs (bootstrap support 50 and 69, respectively). The EherOBP3/CubiOBP3 pair mentioned above also had uncertain phylogenetic position (bootstrap support 0). Database enrichment will clarify the phylogenetic relationships of these OBPs.
These results support the hypothesis that within the Heteroptera there has been extensive duplication and differentiation of OBPs [50] especially compared to the conservative evolution in the aphids [69] and Lepidoptera [43]. We found no intraspecific orthologues in the stink bugs, unlike the mirid studies [44,53,71], but a slightly higher rate of orthologues between related species. The absence of intraspecific orthologues might be related to the comparatively lower number of full-length OBPs characterized in the three stink bugs species studied. Additionally, as indicated by the alignment and phylogenetic analyses, the stink bug OBPs are probably not splice variants, which according to Hull et al. [53] can generate intraspecific orthologous OBP clades.
The phylogenetic relation of the target parasitoid putative OBPs was made with 215 hymenopteran putative OBPs from 36 species, with a rich representation of Solenopsis spp. (18 species), N. vitripennis putative OBPs (90 units), and Apis spp. putative OBPs (42 units) (Fig 8). The OBP phylogeny did not correspond to the known hymenopteran phylogeny. The three TpodOBPs mapped to different parts of the OBP phylogeny and probably are products of different genes for recognition of different odors. None of them are closely related to any of the other hymenopteran OBPs (bootstrap support < 17), and all diverged early from their closest sister OBP. TpodOBP1 and TpodOBP3 were related to OBPs in different hymenopteran families, but with low (5) bootstrap support. TpodOBP2 is most closely related to five other OBPs with low (17) bootstrap support. The phylogenetic pattern for the T. podisi OBPs differs from other hymenopteran OBPs, which generally have more intra-and interspecific orthologues [50,77], suggesting that evolutionary history of the T. podisi OBP genes may have been independent of the other identified hymenopteran OBPs.  Table I Table I  The phylogenetic analyses support these inferences. The monophyly between EherOBP3 and EherOBP6 with CubiOBP3 and CubiOBP1, respectively, may imply that they detect similar odors, and that these are different from the odors recognized by the other hemipteran OBPs presently in GenBank. The corresponding parasitoid OBPs, TpodOBP2 and TpodOBP1, have low relatedness to other hymenopteran OBPs, suggesting that they have evolved independent of phylogenetic constraint, and therefore may have evolved to capture host semiochemical cues.
In 2009, Qiao et al. [79] demonstrated that different aphid species shared some highly conserved OBPs, for instance OBP3, which has affinity to sequisterpene (E)-β-farnesene (EβF), an aphid alarm pheromone that is released by most aphid species. In addition, lepidopteran PBPs are also similar and monophyletic [43] supporting a hypothesis that OBPs with shared semiochemical ligands are conserved through evolution [54]. Steinbrecht [80] tested the reactivity of an antiserum against the PBP of Antheraea polyphemus in pheromone-sensitive sensilla tricodea in nine moth species belonging to six families and three superfamilies of Lepidoptera and concluded that the observed cross-reactivity was related to the chemical relatedness of the pheromones used by the tested species rather than their taxonomic relatedness. The similar OBPs in the two closely related stink bug species, which have broadly overlapping host plant ranges and have similar ecological niches, could suggest that these species can recognize a pheromone component of the other species, such as defense compounds [7,81], to avoid resource competition with the other species, or use it as a cue to locate host plants for mating, feeding and laying eggs. Borges et al. [21,22,82] have shown in field studies that E. heros sex pheromone baited traps captured not only E. heros, but also other pentatomids, such as Edessa meditabunda and Piezodorus guildinii, and platygastrid egg parasitoids. The shared OBP would not be species-specific but rather ligand-specific, which would imply that species sharing a similar ecological niche (such as host plants, natural enemies, defence compounds), could have a similar OBP, no matter how those species are phylogenetically related.
The similar OBPs in the parasitoid and its host could suggest that the parasitoid can recognize a host pheromone, or some other scent that the stink bug uses in its chemical communication. Laboratory bioassays and field tests showed that T. podisi uses the sex pheromone of E. heros to locate its hosts [21,22,82]. Therefore, TpodOBP1 and TpodOBP2 might be involved in recognizing a pheromone of E. heros. Vandermoten et al. [83] found an OBP in the aphid Sitobion avenae (SaveOBP3), and two of its predators, the syrphid fly Episyrphus balteatus (Eba-lOBP3) and the ladybird beetle Harmonia axyridis (HaxyOBP3), with amino acid sequence similarities 94%. Both SaveOBP3 and EbalOBP3 recognized a component of the aphid alarm pheromone EβF, which was acting as a kairomone for the natural enemies.
The high primary and tertiary structure similarities between the two pairs of similar OBPs in the stink bugs E. heros and C. ubica, may indicate that they have derived from a common ancestor, retaining the same biological function to bind to a ligand perceived or produced in both species. In the case of the parasitoid and host, the parasitoid OBPs may have evolved to become similar to the host OBPs to recognize host semiochemicals. Hence the parasitoid may be eavesdropping on stink bug chemical communication to find suitable hosts to parasitize. Such convergent evolution of a parasitoid OBP would be expected in the tertiary structure, specifically in the OBP domain. However, the OBP domain in insects comprises a uniquely large portion of the protein, so convergent evolution must also act on the OBP primary structure. Overall, these results reinforce that OBP similarity could be more related to specificity to a ligand than to species relatedness. Highly similar OBPs among species might occur via multiple evolutionary processes and provide one pathway to a refined and selective interspecific semiochemical signaling via allomones, kairomones and synomones.

Conclusion
The transcriptomes of E. heros, C. ubica and D. melacanthus stink bugs were similar to that of other hemipteran species and the transcriptome of T. podisi parasitoid was similar to other hymenopteran species. Overall, the biological processes and molecular functions were similar among the four species. The most common biological process was related to oxidation-reduction, and the most common molecular function was related to ATP-binding, which have been commonly reported for other insect transcriptomes. Transcriptome analyses enabled the mining of putative OBPs, with a similar number identified from E. heros and C. ubica stink bugs, which was higher than from other Hemiptera in the Miridae, Aphididae and Delphacidae families. The lone exception was the mirid L. lineolaris. Fewer OBPs were identified from D. melacanthus compared to the other stink bugs, and fewer were from their parasitoid compared to most other Hymenoptera. The full-length putative OBPs had physicochemical properties expected of OBPs, except for an unusual basic pI for one T. podisi OBP. They largely exhibited the classical Cys-motif pattern, except one minus-C in D. melacanthus and one plus-C in E. heros and C. ubica. High similarity (> 75%) in the 1D and 3D predicted structures were found between two pairs of E. heros and C. ubica OBPs, and also between two pairs of E. heros and its egg parasitoid OBPs, for which the latter had much lower similarity (< 28%) to other hymenopteran OBPs. The 1D and 3D alignments and phylogenetic analyses suggest that these highly similar OBPs might bind to similar semiochemicals. For species sharing a similar ecological niche, such as the stink bugs, this might function for finding shared host plants or recognizing alarm pheromones to shared threats (e.g., natural enemies). For the parasitoid, this might function for recognizing host semiochemical cues to help find hosts.