Mate-guarding behavior enhances male reproductive success via familiarization with mating partners in medaka fish

[Background] Male-male competition and female mating preference are major mechanisms of sexual selection, which influences individual fitness. How male-male competition affects female preference, however, remains poorly understood. Under laboratory conditions, medaka (Oryzias latipes) males compete to position themselves between a rival male and the female (mate-guarding) in triadic relationships (male, male, and female). In addition, females prefer to mate with visually familiar males. In the present study, to examine whether mate-guarding affects female preference via visual familiarization, we established a novel behavioral test to simultaneously quantify visual familiarization of focal males with females and mate-guarding against rival males. In addition, we investigated the effect of familiarization on male reproductive success in triadic relationships. [Results] Three fish (female, male, male) were placed separately in a transparent three-chamber tank, which allowed the male in the center (near male) to maintain closer proximity to the female than the other male (far male). Placement of the wild-type male in the center blocked visual familiarization of the far male by the female via mate-guarding. In contrast, placement of an arginine-vasotocin receptor mutant male, which exhibits mate-guarding deficits, in the center, allowing for maintaining close proximity to the female, did not block familiarization of the far male by the female. We also demonstrated that the reproductive success of males was significantly decreased by depriving females visual familiarization with the males. [Conclusions] Our findings indicated that, at least in triadic relationships, dominance in mate-guarding, not simply close proximity, allows males to gain familiarity with the female over their rivals, which may enhance female preference for the dominant male. These findings focusing on the triadic relationships of medaka may contribute to our understanding of the adaptive significance of persistent mate-guarding, as well as female preference for familiar mates

Mate-guarding behavior enhances male reproductive success via familiarization with mating partners in medaka fish

Background: Male-male competition and female mating preference are major mechanisms of sexual selection, which influences individual fitness. How male-male competition affects female preference, however, remains poorly understood. Under laboratory conditions, medaka (Oryzias latipes) males compete to position themselves between a rival male and the female (mate-guarding) in triadic relationships (male, male, and female). In addition, females prefer to mate with visually familiar males. In the present study, to examine whether mate-guarding affects female preference via visual familiarization, we established a novel behavioral test to simultaneously quantify visual familiarization of focal males with females and mate-guarding against rival males. In addition, we investigated the effect of familiarization on male reproductive success in triadic relationships. Results: Three fish (female, male, male) were placed separately in a transparent three-chamber tank, which allowed the male in the center (near male) to maintain closer proximity to the female than the other male (far male). Placement of the wild-type male in the center blocked visual familiarization of the far male by the female via mate-guarding. In contrast, placement of an arginine-vasotocin receptor mutant male, which exhibits mate-guarding deficits, in the center, allowing for maintaining close proximity to the female, did not block familiarization of the far male by the female. We also demonstrated that the reproductive success of males was significantly decreased by depriving females visual familiarization with the males. Conclusions: Our findings indicated that, at least in triadic relationships, dominance in mate-guarding, not simply close proximity, allows males to gain familiarity with the female over their rivals, which may enhance female preference for the dominant male. These findings focusing on the triadic relationships of medaka may contribute to our understanding of the adaptive significance of persistent mate-guarding, as well as female preference for familiar mates

Na+/K+-ATPase α1 Identified as an Abundant Protein in the Blood-Labyrinth Barrier That Plays an Essential Role in the Barrier Integrity

BACKGROUND:The endothelial-blood/tissue barrier is critical for maintaining tissue homeostasis. The ear harbors a unique endothelial-blood/tissue barrier which we term "blood-labyrinth-barrier". This barrier is critical for maintaining inner ear homeostasis. Disruption of the blood-labyrinth-barrier is closely associated with a number of hearing disorders. Many proteins of the blood-brain-barrier and blood-retinal-barrier have been identified, leading to significant advances in understanding their tissue specific functions. In contrast, capillaries in the ear are small in volume and anatomically complex. This presents a challenge for protein analysis studies, which has resulted in limited knowledge of the molecular and functional components of the blood-labyrinth-barrier. In this study, we developed a novel method for isolation of the stria vascularis capillary from CBA/CaJ mouse cochlea and provided the first database of protein components in the blood-labyrinth barrier as well as evidence that the interaction of Na(+)/K(+)-ATPase α1 (ATP1A1) with protein kinase C eta (PKCη) and occludin is one of the mechanisms of loud sound-induced vascular permeability increase. METHODOLOGY/PRINCIPAL FINDINGS:Using a mass-spectrometry, shotgun-proteomics approach combined with a novel "sandwich-dissociation" method, more than 600 proteins from isolated stria vascularis capillaries were identified from adult CBA/CaJ mouse cochlea. The ion transporter ATP1A1 was the most abundant protein in the blood-labyrinth barrier. Pharmacological inhibition of ATP1A1 activity resulted in hyperphosphorylation of tight junction proteins such as occludin which increased the blood-labyrinth-barrier permeability. PKCη directly interacted with ATP1A1 and was an essential mediator of ATP1A1-initiated occludin phosphorylation. Moreover, this identified signaling pathway was involved in the breakdown of the blood-labyrinth-barrier resulting from loud sound trauma. CONCLUSIONS/SIGNIFICANCE:The results presented here provide a novel method for capillary isolation from the inner ear and the first database on protein components in the blood-labyrinth-barrier. Additionally, we found that ATP1A1 interaction with PKCη and occludin was involved in the integrity of the blood-labyrinth-barrier

Chemoproteomics reveals Toll-like receptor fatty acylation

Partial funding for Open Access provided by The Ohio State University Open Access Fund.Background: Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells. Results: A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands. Conclusions: This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. Spalmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.This work was supported by funding from the NIH/NIAID (grant R00AI095348 to J.S.Y.), the NIH/NIGMS (R01GM087544 to HCH), and the Ohio State University Public Health Preparedness for Infectious Diseases (PHPID) program. NMC is supported by the Ohio State University Systems and Integrative Biology Training Program (NIH/NIGMS grant T32GM068412). BWZ is a fellow of the National Science Foundation Graduate Research Fellowship Program (DGE-0937362)

An Essential Role of the Arginine Vasotocin System in Mate-Guarding Behaviors in Triadic Relationships of Medaka Fish (<i>Oryzias latipes</i>)

<div><p>To increase individual male fitness, males of various species remain near a (potential) mating partner and repel their rivals (mate-guarding). Mate-guarding is assumed to be mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. The genetic/molecular mechanisms underlying how mate presence affects male competitive motivation in a triadic relationship has remained largely unknown. Here we showed that male medaka fish prominently exhibit mate-guarding behavior. The presence of a female robustly triggers male-male competition for the female in a triadic relationship (2 males and 1 female). The male-male competition resulted in one male occupying a dominant position near the female while interfering with the other male's approach of the female. Paternity testing revealed that the dominant male had a significantly higher mating success rate than the other male in a triadic relationship. We next generated medaka mutants of arginine-vasotocin (<i>avt</i>) and its receptors (<i>V1a1</i>, <i>V1a2</i>) and revealed that two genes, <i>avt</i> and <i>V1a2</i>, are required for normal mate-guarding behavior. In addition, behavioral analysis of courtship behaviors in a dyadic relationship and aggressive behaviors within a male group revealed that <i>avt</i> mutant males displayed decreased sexual motivation but showed normal aggression. In contrast, heterozygote <i>V1a2</i> mutant males displayed decreased aggression, but normal mate-guarding and courtship behavior. Thus, impaired mate-guarding in <i>avt</i> and <i>V1a2</i> homozygote mutants may be due to the loss of sexual motivation toward the opposite sex, and not to the loss of competitive motivation toward rival males. The different behavioral phenotypes between <i>avt</i>, <i>V1a2</i> heterozygote, and <i>V1a2</i> homozygote mutants suggest that there are redundant systems to activate V1a2 and that endogenous ligands activating the receptor may differ according to the social context.</p></div

Possible model of AVT system in mate-guarding, aggressive and courtship behaviors.

<p>(A) Mate-guarding requires two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex, while courtship and aggressive behaviors require social motivation to either the opposite (red arrow) or same (blue arrow) sexes. When mate-guarding emerges in a triadic relationship, the presence of a potential mating partner may engage competitive motivation toward rival males via AVT system, leading to male-male competition (orange arrow). (B) The homozygote <i>avt</i> mutant males exhibited fewer courtship displays than wild-type males (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005009#pgen.1005009.g004" target="_blank">Fig. 4B</a>), whereas the mutant males exhibited normal aggression (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005009#pgen.1005009.g004" target="_blank">Fig. 4A</a>). Thus <i>avt</i> mutants normally have sexual motivation, but not competitive motivation toward the same sex. Decreased sexual motivation toward the opposite sex may cause low dominance of male-male competition in mate-guarding. <i>avt</i> was required just for dominance of mate-guarding. <i>avt</i> was not required for either normal aggressive behaviors or elicitation of mate-guarding, suggesting the presence of redundant system activating V1a2 receptor. (C) The heterozygote <i>V1a2</i> mutant males exhibited normal courtship displays (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005009#pgen.1005009.g004" target="_blank">Fig. 4B</a>), whereas they exhibited low aggression (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005009#pgen.1005009.g004" target="_blank">Fig. 4A</a>). Thus heterozygote <i>V1a2</i> mutants normally have competitive motivation, but not sexual motivation toward the opposite sex. Decreased competitive motivation towards the same sex has no effect on male-male competition in mate-guarding, because sexual motivation may dominantly drive the motivation for mate-guarding. The single functional <i>V1a2</i> allele may not produce enough of a gene product, leading to attenuated aggression. (D) The homozygote <i>V1a2</i> mutant males exhibited decreased courtship displays and low aggression (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005009#pgen.1005009.g004" target="_blank">Figs. 4A and 4B</a>). Thus homozygote <i>V1a2</i> mutants did not normally have social motivation to either the same sex or opposite sex. <i>V1a2</i> was required for elicitation of mate-guarding (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005009#pgen.1005009.g003" target="_blank">Fig. 3C</a>).</p

Effect of AVT-related genes mutations on aggressive behavior (intrasexual interaction) and courtship behavior (intersexual interaction).

<p>(A) <i>avt</i> mutant males exhibited normal aggression, whereas <i>V1a2</i> heterozygote and homozygote mutant males exhibited low aggression. Mean ± SEM. Each n = 8, Dunnett’s test: *<i>P</i><0.05, **<i>P</i><0.01 VS wild-type. (B) <i>avt</i> and <i>V1a2</i> mutant males showed lower motivation to mate than wild-type males. Mean ± SEM. Each n = 11, Dunnett’s test: *<i>P</i><0.05, ***<i>P</i><0.001 VS wild-type.</p

Effect of the AVT system on mate-guarding behavior.

<p>(A) Manning compound injection decreased the guarding index. Mean ± SEM. Each n = 12, one-way repeated measures ANOVA: Bonferroni correction, ***<i>P</i><0.001. (B) Saline injection didn’t change the guarding index. Mean ± SEM. Each n = 12, one-way repeated measures ANOVA. (C) Effect of the AVT system on emergence of mate-guarding behavior (guarding test). <i>avt</i> (<i>avt</i>^{<i>M1R/M1R</i>}) and <i>V1a1</i> (<i>V1a1</i>^{<i>F93Y/F93Y</i>}) mutants exhibited mate-guarding behavior, whereas <i>V1a2</i> (<i>V1a2</i>^{<i>N68I/N68I</i>}) mutants did not. Mean ± SEM. Each n = 12, Student’s t-test: *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. (D) Effect of the AVT system on dominance of mate-guarding behavior (dominance test). <i>avt</i> homozygote mutants (<i>avt</i>^{<i>M1R /M1R</i>}) and <i>V1a2</i> homozygote mutants (<i>V1a2</i>^{<i>N68I/N68I</i>}) tended to be subordinate males in the dominance test. Mean ± SEM. Each n = 12, Student’s t-test: ***<i>P</i><0.001.</p