Experimental Cell Research

Cells in hybrid state of the epithelial-to-mesenchymal transition (EMT) have been shown to be responsible for tumor cell metastasis. However, the precise mechanisms underlying the morphological changes and acquisition of invasive phenotypes in hybrid EMT cells are still unknown. Here, we introduced the deletion of a proto-cadherin and well described oncogene, FAT1, in skin carcinoma cells to generate a hybrid state of EMT. Surprisingly, the FAT1 knock-out (KO) cells were less motile than the parental non-mutated cell line they were derived from. However, we observed that FAT1 KO cells secrete specific factors in the form of extra-cellular vesicles into their microenvironment, which promote the migration of surrounding non-mutant cells. When stimulated with these extracellular vesicles, groups of non-mutated parental cells collectively migrated faster and formed finger-like instabilities at the migrating front. Furthermore, we found that the actomyosin contractility of FAT1 KO cells in hybrid EMT states was much lower than the parental cells. It appeared that the factors secreted by FAT1 KO cells relaxed the traction forces in recipient cells. This force release likely fostered the scattering and migration of non-mutated cells surrounding FAT1 mutant cells. Thus, we characterized a non-autonomous promotion of cell invasiveness in the cancer cells surrounding FAT1-deficient cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/556588v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@104063eorg.highwire.dtl.DTLVardef@135f9fforg.highwire.dtl.DTLVardef@b0052eorg.highwire.dtl.DTLVardef@243864_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure:C_FLOATNO Schematic showing how FAT1 deletion stimulates the migration of neighboring non mutant cells. FAT1 KO cells secrete extracellular vesicles that carry factors that promote migration of non-mutant control cells, possibly through relaxation of traction forces in the recipient cells. C_FIG

To facilitate rapid changes in morphology without endangering cell integrity, each cell possesses a substantial amount of cell surface excess (CSE) that can be promptly deployed to cover cell extensions. CSE can be stored in different types of small surface projections such as filopodia, microvilli, and ridges, with rounded bleb-like projections being the most common and rapidly achieved form of storage. We demonstrate in this paper that cells migrating in 3D collagen use CSE to cover the developing protrusions. After retraction of a protrusion, the CSE this produces is stored over the cell body similar to the CSE produced by cell rounding. For the coordinated process of CSE storage and release, all cells should have specific mechanisms of regulation, and we hypothesize that microtubules (MT) play an important role in this mechanism. We show here that different effects of MT depolymerization on cell motility such as inhibiting mesenchymal motility and enhancing amoeboid, can be explained by the essential role of MT in CSE regulation and dynamics.

Human mesenchymal stem cells (hMSCs) are multipotent cells that can differentiate into adipocytes, chondrocytes and osteoblasts. Due to their differentiation potential, hMSCs are among the most frequently used cells for therapeutic applications in tissue engineering and regenerative medicine. However, the number of cells obtained through isolation alone is insufficient for hMSC-based therapies and basic research, necessitating their in-vitro expansion. Conventionally, this is often carried out on rigid surfaces such as tissue culture petriplates (TCPs). However, during in-vitro expansion, hMSCs lose their proliferative ability and multilineage differentiation potential, making them unsuitable for clinical use. Although multiple approaches have been tried to maintain hMSC stemness over prolonged expansion, finding a suitable culture system to achieve this remains an unmet need. Recently, few research groups including ours have shown that hMSCs maintain their stemness over long passages when cultured on soft substrate. In addition, it has been shown that hMSCs cultured on soft substrates have more condensed chromatin and lower levels of histone acetylation compared to those cultured on stiff substrates. It has also been shown that condensing/decondensing chromatin by deacetylation/acetylation can delay/hasten replicative senescence in hMSCs during long-term expansion on TCPs. However, how chromatin condensation/decondensation influences nuclear morphology and DNA damage - which are strongly related to the onset of senescence and cancer - is still not known. To answer this question, here we cultured hMSCs for long duration (P4-P11) in presence of epigenetic modifiers histone acetyltransferase inhibitor (HATi) which promotes chromatin condensation by preventing histone acetylation and histone deacetylase inhibitor (HDACi) which promotes chromatin decondensation and investigated their effect on various nuclear markers related to senescence and cancer. We have found that consistent acetylation causes severe nuclear abnormalities whereas chromatin condensation by deacetylation helps in safeguarding nucleus from damages caused by in-vitro expansion.

Myosin-X (MYO10) is an actin-based motor protein involved in cytoskeletal dynamics, membrane interactions, and integrin-mediated adhesion. To investigate MYO10s cellular roles, we generated MYO10 knockdown (MYO10KD) HeLa and COS7 cell lines using lentiviral shRNA. Compared to wild-type cells, both MYO10KD lines showed reduced proliferation and impaired cell migration in wound assays. There were fewer edge filopodia in HeLa cells. Additionally, MYO10KD cells demonstrated increased spreading on laminin-coated substrates, suggesting altered integrin activation and cytoskeletal linkage. Our results reinforce MYO10s importance in cell proliferation, adhesion, and migration; these MYO10KD lines provide an accessible cell culture model for further study of MYO10.

Cellular regeneration, which relies on extensive restructuring of cytoplasmic materials, is an essential process to restore tissues and organs lost during aging, degenerative diseases and injury. At early stages of Drosophila spermatogenesis, when cellular constituents are intensely remodeled, there are two different populations of stem cells, the somatic stem cells and the germline stem cells (GSCs). GSCs divide by asymmetric division to give rise two distinct daughter cells. One of them will leave the stem cells niche and differentiate into spermatogonial cells (SCs). Both aging and cellular stress can lead to the loss of GSCs. Lost GSCs can be restored by dedifferentiation of SCs into functional GSCs. In other tissues, macrophages provide specific conditions for cellular transformation. Here we examined the potential role of immune surveillance cells called hemocytes during dedifferentiation of SGs into GSCs. We found an elevated number of hemocytes during this dedifferentiation process. Immune depletion of hemocytes decreased the regeneration capacity of germline. We also show that autophagy, which plays a pivotal role in cellular differentiation by eliminating unwanted, superfluous parts of the cytoplasm, becomes upregulated in dedifferentiating SCs upon JAK-STAT signaling emitted by hemocytes. Furthermore, these immune cells regulate expression of Omi/HtrA2, a key regulator of apoptosis in early spermatogenesis. Together, we suggest that hemocytes have important functions in the dedifferentiation process of GSCs.

Alaph-1 antitrypsin overexpressing mesenchymal stromal/stem cells (AAT-MSCs) showed improved innate properties with a faster proliferation rate when studied for their protective effects in mouse models of diseases. Here, we investigated the potential mechanism(s) by which AAT gene insertion increases MSC proliferation. Human bone marrow-derived primary or immortalized MSCs (iMSCs) or AAT-MSCs (iAAT-MSCs) were used in the study. Cell proliferation was measured by cell counting and cell cycle analysis. Possible pathways involved in the pro-proliferation effect of AAT were investigated by measuring mRNA and protein expression of key cell cycle genes. Interval cell counting showed increased proliferation in AAT-MSCs or iAAT-MSCs compared to their corresponding MSC controls. Cell cycle analysis revealed more cells progressing into the S and G2/M phases in iAAT-MSCs, with a notable increase in the cell cycle protein, Cyclin D1. Moreover, treatment with Cyclin D1 inhibitors showed that the increase in proliferation is due to Cyclin D1 and that the AAT protein is upstream and a positive regulator of Cyclin D1. Furthermore, AATs effect on Cyclin D1 is independent of the Wnt signaling pathway as there were no differences in the expression of regulatory proteins, including GSK3{beta} and {beta}-Catenin in iMSC and iAAT-MSCs. In summary, our results indicate that AAT gene insertion in an immortalized MSC cell line increases cell proliferation and growth by increasing Cyclin D1 expression and consequently causing cells to progress through the cell cycle at a significantly faster rate.

Cortex glia in Drosophila central nervous system forms a niche around neural cells for necessary signals to establish cross-talk with their surroundings. These cells grow and expand their thin processes around neural cell bodies. Although essential for the development and function of the nervous system, how these cells make extensive and intricate connected networks remain largely unknown. Here we show that Cut, a homeodomain transcription factor, directly regulates the fate of the cortex glia, impacting NSC homeostasis. Focusing on the thoracic ventral nerve cord (tVNC), we found that Cut is required for normal growth and development of cortex glia and timely increase in DNA content to undergo endomitosis. Knockdown of Cut in cortex glia significantly reduces the growth of cellular processes, the network around NSCs, and their progenys cell bodies. Conversely, overexpression of Cut induces overall growth of the main processes at the expense of side ones. Whereas the Cut knockdown slowdown the timely increase of DNA, Cut overexpression results in a significant increase in nuclear size and volume and a threefold increase in DNA content of cortex glia. Further, we note that constitutively high Cut also interfered with nuclei separation during endomitosis. Since cortex glia form syncytial networks around neural cells, the finding identifies Cut as a novel regulator of glial growth and endomitosis to support a functional nervous system. Article SummaryCut homeodomain transcription factor is crucial for cortex glia growth and the formation of complex cellular processes around neural cells. This regulation ensures a timely increase in DNA content, allowing the cells to enter endomitosis. Constitutively high Cut levels increase the DNA content of these cells to several folds. The finding emphasizes the need to investigate if activated CUX1, the human homolog of Cut, in glioma enhances chromosomal instability and, in conjunction with other mutations, enhances their tumorigenic potential.

Sarcopenia is a degenerative condition characterized by the atrophy and functional decline of myofibers, resulting in disability. While the clinical risk factors are known, there is no validated in vitro model to understand the molecular mechanisms and identify therapeutics. To tackle this challenge, we generated an in vitro post-mitotic muscular system by differentiating mouse myoblast cells, namely C2C12. After 12 days of differentiation, cells were expressing physiological markers of myotubes and became self-contracting. Importantly, transcriptomic analyses demonstrated high similarity (r=0.70) when compared to primary human myotubes (HSkMC) providing evidence of resemblance to human cells. Next, we starved and incubated cells with dexamethasone and observed myotube shrinkage, oxidative stress, modification of anabolic, inflammatory, and catabolic markers recapitulating sarcopenia. Conversely, cell refeeding resulted in a recovery in the model with nutrient deprivation but not when incubated also with dexamethasone. In conclusion, we present a model of sarcopenia due to nutrient deprivation and corticosteroids. This model may allow more efficient and effective future research to identify therapeutics against sarcopenia in humans.

Primordial germ cells (PGCs) are lineage restricted precursor cells of sperm and eggs. Whilst avian PGCs from chicken can be cultured and modified to produce genome-edited chickens, the long-term culture of PGCs from other bird species has not been achieved. Here, we explored the effects of a cell-matrix interaction on the in vitro propagation of chicken PGCs. Blocking integrin signaling severely reduced the self-renewal of the PGC, indicating that a PGC-matrix interaction is an essential process for self-renewal. We investigated the properties of somatic cell differentiated PGCs and expression analyses suggested that the PGCs undergo a partial epithelial-to-mesenchymal transition caused by excess PGC-matrix interactions. Finally, we conducted a long-term culture of chicken PGCs with matrix components, resulting in significant induction of their somatic conversion. These results suggested that a high level of PGC-matrix interaction can cause somatic conversion, whilst a moderate level is essential for their proliferation. Overall, we identified a molecular aspect of self-renewal and maintaining undifferentiated states of avian PGCs.

Myoblast fusion is a crucial step in myogenesis during embryogenesis and adulthood. In Drosophila, the scaffold protein Antisocial (Ants)/Rols7 plays is essential in myoblast fusion by connecting the cell adhesion surface proteins to the cytoskeleton. Most molecular pathways governing fusion are evolutionary conserved between mammals and flies, but the relative contributions of Tanc1 and Tanc2, mammalian orthologs of Ants/Rols7, in myoblast fusion have not been established. We used the myoblast C2C12 cell line as a model for differentiation and fusion to assess the contributions of Tanc1 and Tanc2 in fusion. We found that Tanc1 and Tanc2 expressions are not modulated during differentiation, but that both proteins are enriched at the cell cortex in proliferating myoblasts. The knockdown of either Tanc1 or Tanc2 in either of the fusing myoblasts impaired fusion. Notably, the expression of human Tanc1 or Tanc2 restored fusion defects observed in Tanc1- or Tanc2-depleted cells. We found that neither Tanc1 nor Tanc2 could substitute for Ants/Rols7 during Drosophila myoblast fusion. We conclude that both Tanc1 and Tanc2 play a role in mammalian myoblast fusion, but there may be some mechanistic differences with the functions of the Drosophila orthologous protein.

Skeletal muscle stem cells (satellite cells) are well known to participate in regeneration and maintenance of the tissue over time. Studies have shown increases in the number of satellite cells after exercise, but their functional role in endurance training remains unexplored. Here, we found that injured muscles from endurance-exercised mice display improved regenerative capacity, demonstrated through higher densities of newly formed myofibers compared to controls, as well as lower inflammation and fibrosis. Enhanced myogenic function was accompanied by an increased fraction of satellite cells expressing self-renewal markers. Control satellite cells had morphologies suggestive of early differentiation, while endurance exercise enhanced myogenic colony formation. The beneficial effects of endurance exercise were associated with satellite cell metabolic reprogramming, including reduced mitochondrial respiration (O2 consumption) under resting conditions (absence of muscle injury) and increased stemness. During proliferation or activated states (three days after injury), O2 consumption was equal in control and exercised cells. Surprisingly, inhibition of mitochondrial O2 consumption was sufficient to enhance muscle stem cell self-renewal characteristics in vitro. Moreover, transplanted muscle satellite cells from exercised mice or cells with reduced mitochondrial respiration promoted a significant reduction in inflammation compared to controls. We propose that endurance exercise promotes self-renewal and inhibits differentiation in satellite cells, an effect promoted by metabolic reprogramming and respiratory inhibition, and which is associated with a more favorable muscular response to injury.

ARID1A, a subunit of SWI/SNF, has been shown to play a major role in recruitment of the chromatin remodeler to enhancers for transcriptional regulation. Mutations inARID1A have been found in various cancers, many of which form solid tumors. Recent studies have revealed vulnerabilities in cells lacking ARID1A, specifically ARID1B, an ortholog and mutually exclusive subunit, in 2D cell culture. However, identification of vulnerabilities within SWI/SNF for loss of ARID1A in a multicellular tumor spheroid, that mimic in vivo condition within tumors, has not been explored. Here we show in the absence of ARID1A in a MTS model, ARID1B continues to be a vulnerability but we have identified PBRM1 as a new vulnerability within SWI/SNF. Levels of ARID1B and PBRM1 are elevated on loss of ARID1A. Further, reduction of ARID1B and PBRM1 protein levels, decreases cell survival and reduces induction of several hypoxia regulated genes in ARID1A deficient MTSs. Our studies have identified PBRM1 as a new vulnerability in ARID1a deficient cancers and which provides a new target for therapeutic strategies.

In culture system, environmental factors, such as increasing exogenous growth factors and adhesion to type I collagen (Col-I) induce epithelial-to-mesenchymal transition (EMT) in cells. Col-I molecules maintain a non-fibril form under acidic conditions, and they reassemble into fibrils under physiological conditions. Col-I fibrils often assemble to form three-dimensional gels. The gels and non-gel-form of Col-I can be utilized as culture substrates and different gel-forming state often elicit different cell behaviors. However, gel-form dependent effects on cell behaviors, including EMT induction, remain unclear. EMT induction in lung cancer cell line A549 has been reported via adhesion to Col-I but the effects of gel form dependency are unelucidated. This study investigated the changes in EMT-related behaviors in A549 cells cultured on Col-I gels. We examined cell morphology, proliferation, single-cell migration and expression of EMT-related features in A549 cells cultured on gels or non-gel form of Col-I and non-treated dish with or without transforming growth factor (TGF)-{beta}1. On Col-I gels, some cells kept cell-cell contacts and formed clusters, others maintained single-cell form. In cell-cell contact regions, E-cadherin expression was downregulated, whereas that of N-cadherin was upregulated. Vimentin and integrins 2 and {beta}1 expression were not increased. In TGF-{beta}1-treated A549 cells, cadherin switched from E- to N-cadherin. Their morphology changed to a mesenchymal form and cells scattered with no cluster formation. Vimentin, integrins 2 and {beta}1 expression were upregulated. Thus, we concluded that culture on Col-I fibrous gels induced E- to N-cadherin switching without other EMT-related phenotypes in A549 cells.

Cancer (tumorigenic) cells exhibit cellular properties such as tumorigenicity, evasion of anti-tumor immunity and cell death, metastasis, etc. We showed previously that the core property of cancer cells is neural stemness, which represents general stemness and confers cancer cells with tumorigenicity and pluripotency. Immunogenicity of cancer cells plays the key role in anti-tumor immunity. While tumorigenicity is defined by neural stemness and its regulatory networks, how immunogenicity of cancer cells is determined and how tumorigenicity is related with immunogenicity remained unknown. In the present study, we show that genes conferring tumorigenicity and genes conferring immunogenicity are inversely correlated with differentiation status of cells. Cancer cells can be induced to differentiate into different types of cells either by lineage specific differentiation factors, e.g., muscle differentiation factor MYOD1 or adipocyte differentiation factor PPARG, or by blocking cell-intrinsic oncofactors, e.g., SETDB1. Induced differentiation reprograms both cellular properties and transcriptomes of cancer cells, leading to suppression of genes conferring tumorigenicity and upregulation of genes conferring immunogenicity, and ultimately, leading to loss of neural stemness, suppression of tumorigenicity and enhancement of immunogenicity in cancer cells after differentiation. Vice versa, dedifferentiation leads to the opposite effect. The results identified the cellular mechanism that tumorigenicity and immunogenicity of cancer cells are inversely connected and determined by differentiation status. Such a mechanism, together with our previous studies, reinforces that cancer cell properties should be understood by understanding neural stemness and the principle of embryonic cell/tissue differentiation, and novel cancer therapies can be developed by employing differentiation effect of cancer cells induced by differentiation factors. As a proof of concept test, we showed that muscle differentiation factor MYOD1 suppresses tumorigenesis in a mouse model of hepatocellular carcinoma.

The presomitic mesoderm (PSM) is initially an unsegmented structure localized on each side of the neural tube of the developing embryo, which progressively segments to form the somites. The somites will segregate and partition to generate the dorsal dermomyotome and the ventral sclerotome. Endothelial and myogenic cells of both the trunk and limbs are derived from the somites. There is a lack of efficient reporter mouse models to label and trace the PSM derivatives, despite their crucial contribution to many developmental processes. In this study, we generated a tamoxifen inducible transgenic Tbx6 mouse line, Tg(Tbx6_Cre/ERT2)/ROSA-eYFP, to tag and follow PSM-derivatives from early embryonic stages until adulthood. After induction, endothelial and myogenic cells can be easily identified within the trunk and limbs with proper expression patterns. Since our Tg(Tbx6_Cre/ERT2)/ROSA-eYFP model allows to permanently label the PSM-derived cells, their progeny can be studied at long-term, opening the possibility to perform lineage tracing of stem cells upon aging.

Metastatic tumor cell invasion into interstitial tissue is a mechanochemical process that responds to tissue cues and further involves proteolytic remodeling of the tumor stroma. How matrix density, tissue guidance and the ability of proteolytic tissue remodeling cooperate and determine decision-making of invading tumor cells in complex-structured three-dimensional (3D) tissue remains unclear. We here developed a collagen-based invasion assay containing a guiding interface of low collagen density adjacent to randomly organized 3D fibrillar lattice and examined the invasion of melanoma cells from multicellular spheroids in response to matrix density, guidance cues and collagenolysis. After 48 hours of culture, two invasion niches developed, (i) sheet-like collective migration along the interface and (ii) single cell- and strand-like invasion into randomly organized 3D matrix. High collagen density impeded migration into the random matrix, whereas migration along a high-density collagen interface was increased compared to the low-density matrix assay. In silico analysis predicted that facilitated interface migration in high-density matrix depended on physical guidance without collagen degradation, whereas migration into randomly organized matrix was strongly dependent on collagenolysis. When tested in 3D culture, inhibition of matrix metalloprotease (MMP)-mediated collagen degradation compromised migration into random matrix in dependence of density, whereas interface-guided migration remained effective. In conclusion, with increasing tissue density, matrix cues bordered by dense matrix, but not randomly organized matrix, support effective MMP-independent migration. This identifies the topology of interstitial tissue a primary determinant of switch behaviors between MMP-dependent and MMP-independent cancer cell invasion.

Cells exhibit pathological behaviors in response to increased extracellular matrix (ECM) stiffness, including accelerated cell proliferation and migration [1-9], which are correlated with increased intracellular stiffness and tension [2, 3, 10-12]. The biomechanical signal transduction of ECM stiffness into relevant molecular signals and resultant cellular processes is mediated through multiple proteins associated with the actin cytoskeleton in lamellipodia [2, 3, 10, 11, 13]. However, the molecular mechanisms by which lamellipodial dynamics regulate cellular responses to ECM stiffening remain unclear. Previous work described that lamellipodin, a phosphoinositide- and actin filament-binding protein that is known mostly for controlling cell migration [14-21], promotes ECM stiffness-mediated early cell cycle progression [2], revealing a potential commonality between the mechanisms controlling stiffness-dependent cell migration and those controlling cell proliferation. However, i) whether and how ECM stiffness affects the levels of lamellipodin expression and ii) whether stiffness-mediated lamellipodin expression is required throughout cell cycle progression and for intracellular stiffness have not been explored. Here, we show that the levels of lamellipodin expression in cells are significantly increased by a stiff ECM and that this stiffness-mediated lamellipodin upregulation persistently stimulates cell cycle progression and intracellular stiffness throughout the cell cycle, from the early G1 phase to M phase. Finally, we show that both Rac activation and intracellular stiffening are required for the mechanosensitive induction of lamellipodin. More specifically, inhibiting Rac1 activation in cells on stiff ECM reduces the levels of lamellipodin expression, and this effect is reversed by the overexpression of activated Rac1 in cells on soft ECM. We thus propose that lamellipodin is a critical molecular lynchpin in the control of mechanosensitive cell cycle progression and intracellular stiffness.

Sertoli cells are the major nurse cells in the testis which regulate the division and differentiation of germ cell by secreting various factors like inhibin B, transferrin and lactate. In this study, we demonstrated that inhibition of extracellular signal-regulated kinase (ERK) leads to an increase in the basal level of lactate. This increase in lactate production was due to the rise in lactate dehydrogenase (LDH) activity and glucose uptake, simultaneously. Moreover, an increase in lactate production wasnot due to the change in mRNA level of GLU1, GLUT3 and LDHA. Importantly, the lactate production was independent of the level of cAMP suggesting that the tonic signaling of ERK is important for the growth factor mediated lactate production. These findings suggest that ERK signaling inhibits lactate production in the absence of any stimulant in rat Sertoli cells to maintain metabolic homeostasis.

Mitochondrial networks in eukaryotic cells are dynamic, undergoing fusion, fission, biogenesis and transfer between cells to manage cellular requirements for energy production and biosynthetic components. Mitochondrial transfer has been observed in response to cellular injury and can happen by a variety of mechanisms. To examine whether mitochondria could be restored to injured astrocytes, we established a model of discrete astrocyte-specific, mitochondrial-specific injury in a mixed culture model. Immortalized {rho}0 astrocytes were generated, then put under lethal metabolic stress by withdrawal of pyruvate and uridine. When cells stressed in this way were grown in the presence of mesenchymal stromal cells, enhanced survival of the {rho}0 cells was seen, accompanied by the rapid acquisition of an entire mitochondrial networks. Gap-fill ligation and rolling circle amplification for cell-specific mitochondrial genome SNP confirmed that the mitochondrial networks were derived from the mesenchymal stromal donor cells, and both mitochondrial DNA and mitochondrial function were restored to the p0 cells long-term. The presence of two nuclei in the {rho}0 cells in the early stages of mitochondrial transfer and recovery was observed. This demonstrated that in this instance, the mechanism of mitochondrial rescue was fusion of the {rho}0 astrocyte and the mesenchymal stromal cell. In support of this, other neural cells were similarly shown to fuse with stressed {rho}0 astrocytes, suggesting that whole organelles may be donated to restore critical mitochondrial function.

The replenishment of specialized cells depends on the activity of stem cells. Recent advances in stem cell research have shown that the germline stem cells (GSCs) in Drosophila melanogaster can increase their mitotic activity in response to mating. Here, we show that this ability to respond to mating is eliminated if the males are mutant for the ABC transporter, White, the genetic background for a plethora of fly lines. Furthermore, we were not able to reproduce previous findings that female flies increase their GSC numbers and mitotic activity upon mating. Our findings underline the importance of careful experimental design and control specimen.