A cholinergic-regulated circuit coordinates the maintenance and bi-stable states of a sensory-motor behavior during Caenorhabditis elegans male copulation

Penetration of a male copulatory organ into a suitable mate is a conserved and necessary behavioral step for most terrestrial matings; however, the detailed molecular and cellular mechanisms for this distinct social interaction have not been elucidated in any animal. During mating, the Caenorhabditis elegans male cloaca is maintained over the hermaphrodite's vulva as he attempts to insert his copulatory spicules. Rhythmic spicule thrusts cease when insertion is sensed. Circuit components consisting of sensory/motor neurons and sex muscles for these steps have been previously identified, but it was unclear how their outputs are integrated to generate a coordinated behavior pattern. Here, we show that cholinergic signaling between the cloacal sensory/motor neurons and the posterior sex muscles sustains genital contact between the sexes. Simultaneously, via gap junctions, signaling from these muscles is transmitted to the spicule muscles, thus coupling repeated spicule thrusts with vulval contact. To transit from rhythmic to sustained muscle contraction during penetration, the SPC sensory-motor neurons integrate the signal of spicule's position in the vulva with inputs from the hook and cloacal sensilla. The UNC-103 K(+) channel maintains a high excitability threshold in the circuit, so that sustained spicule muscle contraction is not stimulated by fewer inputs. We demonstrate that coordination of sensory inputs and motor outputs used to initiate, maintain, self-monitor, and complete an innate behavior is accomplished via the coupling of a few circuit components.     

Coordination of opposing sex-specific and core muscle groups regulates male tail posture during Caenorhabditis elegans male mating behavior

Background  

To survive and reproduce, animals must be able to modify their motor behavior in response to changes in the environment. We studied a complex behavior of Caenorhabditis elegans, male mating behavior, which provided a model for understanding motor behaviors at the genetic, molecular as well as circuit level. C. elegans male mating behavior consists of a series of six sub-steps: response to contact, backing, turning, vulva location, spicule insertion, and sperm transfer. The male tail contains most of the sensory structures required for mating, in addition to the copulatory structures, and thus to carry out the steps of mating behavior, the male must keep his tail in contact with the hermaphrodite. However, because the hermaphrodite does not play an active role in mating and continues moving, the male must modify his tail posture to maintain contact. We provide a better understanding of the molecular and neuro-muscular pathways that regulate male tail posture during mating.     

Characterization of a novel D2-like dopamine receptor with a truncated splice variant and a D1-like dopamine receptor unique to invertebrates from Caenorhabditis elegans

We have cloned two novel Caenorhabditis elegans dopamine receptors, DOP-3 and DOP-4. DOP-3 shows high sequence homology with other D2-like dopamine receptors. As a result of alternative splicing, a truncated splice variant of DOP-3, DOP-3nf, was produced. Because of the in-frame insertion of a stop codon in the third intracellular loop, DOP-3nf lacks the sixth and seventh transmembrane domains that are found in the full-length DOP-3 receptor. Reporter gene assay showed that DOP-3 attenuates forskolin-stimulated cAMP formation in response to dopamine stimulation, whereas DOP-3nf does not. When DOP-3 was coexpressed with DOP-3nf, the ability to inhibit forskolin-stimulated cAMP formation was reduced. DOP-4 shows high sequence homology with D1-like dopamine receptors unique to invertebrates, which are distinct from mammalian D1-like dopamine receptors. Reporter gene assay showed that DOP-4 stimulates cAMP accumulation in response to dopamine stimulation. These two receptors provide new opportunities to understand dopaminergic signaling at the molecular level.     

Dopamine Receptors Antagonistically Regulate Behavioral Choice between Conflicting Alternatives in C. elegans

Caenorhabditis elegans is a useful model to study the neuronal or molecular basis for behavioral choice, a specific form of decision-making. Although it has been implied that both D1-like and D2-like dopamine receptors may contribute to the control of decision-making in mammals, the genetic interactions between D1-like and D2-like dopamine receptors in regulating decision-making are still largely unclear. In the present study, we investigated the molecular control of behavioral choice between conflicting alternatives (diacetyl and Cu²⁺) by D1-like and D2-like dopamine receptors and their possible genetic interactions with C. elegans as the assay system. In the behavioral choice assay system, mutation of dop-1 gene encoding D1-like dopamine receptor resulted in the enhanced tendency to cross the Cu²⁺ barrier compared with wild-type. In contrast, mutations of dop-2 or dop-3 gene encoding D2-like dopamine receptor caused the weak tendency to cross the Cu²⁺ barrier compared with wild-type. During the control of behavioral choice, DOP-3 antagonistically regulated the function of DOP-1. The behavioral choice phenotype of dop-2; dop-1dop-3 triple mutant further confirmed the possible antagonistic function of D2-like dopamine receptor on D1-like dopamine receptor in regulating behavioral choice. The genetic assays further demonstrate that DOP-3 might act through Gα_o signaling pathway encoded by GOA-1 and EGL-10, and DOP-1 might act through Gα_q signaling pathway encoded by EGL-30 and EAT-16 to regulate the behavioral choice. DOP-1 might function in cholinergic neurons to regulate the behavioral choice, whereas DOP-3 might function in GABAergic neurons, RIC, and SIA neurons to regulate the behavioral choice. In this study, we provide the genetic evidence to indicate the antagonistic relationship between D1-like dopamine receptor and D2-like dopamine receptor in regulating the decision-making of animals. Our data will be useful for understanding the complex functions of dopamine receptors in regulating decision-making in animals.     

The Caenorhabditis elegans D2-like dopamine receptor DOP-2 physically interacts with GPA-14, a Gαi subunit

Dopaminergic inputs are sensed on the cell surface by the seven-transmembrane dopamine receptors that belong to a superfamily of G-protein-coupled receptors (GPCRs). Dopamine receptors are classified as D1-like or D2-like receptors based on their homology and pharmacological profiles. In addition to well established G-protein coupled mechanism of dopamine receptors in mammalian system they can also interact with other signaling pathways. In C. elegans four dopamine receptors (dop-1, dop-2, dop-3 and dop-4) have been reported and they have been implicated in a wide array of behavioral and physiological processes. We performed this study to assign the signaling pathway for DOP-2, a D2-like dopamine receptor using a split-ubiquitin based yeast two-hybrid screening of a C. elegans cDNA library with a novel dop-2 variant (DOP-2XL) as bait. Our yeast two-hybrid screening resulted in identification of gpa-14, as one of the positively interacting partners. gpa-14 is a G?? coding sequence and shows expression overlap with dop-2 in C. elegans ADE deirid neurons. In-vitro pull down assays demonstrated physical coupling between dopamine receptor DOP-2XL and GPA-14. Further, we sought to determine the DOP-2 region necessary for GPA-14 coupling. We generated truncated DOP-2XL constructs and performed pair-wise yeast two-hybrid assay with GPA-14 followed by in-vitro interaction studies and here we report that the third intracellular loop is the key domain responsible for DOP-2 and GPA-14 coupling. Our results show that the extra-long C. elegans D2-like receptor is coupled to gpa-14 that has no mammalian homolog but shows close similarity to inhibitory G-proteins. Supplementing earlier investigations, our results demonstrate the importance of an invertebrate D2-like receptor's third intracellular loop in its G-protein interaction.     

Regulation of distinct muscle behaviors controls the C. elegans male's copulatory spicules during mating.

We demonstrate through cell ablation, molecular genetic, and pharmacological approaches that during C. elegans male mating behavior, the male inserts his copulatory spicules into the hermaphrodite by regulating periodic and prolonged spicule muscle contractions. Distinct cholinergic neurons use different ACh receptors and calcium channels in the spicule muscles to mediate these contractile behaviors. The PCB and PCC sensory neurons facilitate periodic contraction through muscle-encoded UNC-68 ryanodine receptor calcium channels. The SPC motor neurons trigger prolonged contraction through EGL-19 L-type voltage-gated calcium channels. The male gonad then lengthens the duration of EGL-19-mediated prolonged muscle contraction. This regulation of muscle contraction provides a paradigm to explain how animals initiate, monitor, and maintain a behavioral motor program.     

The Caenorhabditis elegans D2-like dopamine receptor DOP-2 physically interacts with GPA-14, a Gαi subunit

ABSTRACT: Dopaminergic inputs are sensed on the cell surface by the seven-transmembrane dopamine receptors that belong to a superfamily of G-protein-coupled receptors (GPCRs). Dopamine receptors are classified as D1-like or D2-like receptors based on their homology and pharmacological profiles. In addition to well established G-protein coupled mechanism of dopamine receptors in mammalian system they can also interact with other signaling pathways. In C. elegans four dopamine receptors (dop-1, dop-2, dop-3 and dop-4) have been reported and they have been implicated in a wide array of behavioral and physiological processes. We performed this study to assign the signaling pathway for DOP-2, a D2-like dopamine receptor using a split-ubiquitin based yeast two-hybrid screening of a C. elegans cDNA library with a novel dop-2 variant (DOP-2XL) as bait. Our yeast two-hybrid screening resulted in identification of gpa-14, as one of the positively interacting partners. gpa-14 is a Gα coding sequence and shows expression overlap with dop-2 in C. elegans ADE deirid neurons. In-vitro pull down assays demonstrated physical coupling between dopamine receptor DOP-2XL and GPA-14. Further, we sought to determine the DOP-2 region necessary for GPA-14 coupling. We generated truncated DOP-2XL constructs and performed pair-wise yeast two-hybrid assay with GPA-14 followed by in-vitro interaction studies and here we report that the third intracellular loop is the key domain responsible for DOP-2 and GPA-14 coupling. Our results show that the extra-long C. elegans D2-like receptor is coupled to gpa-14 that has no mammalian homolog but shows close similarity to inhibitory G-proteins. Supplementing earlier investigations, our results demonstrate the importance of an invertebrate D2-like receptor's third intracellular loop in its G-protein interaction.     

Inositol 1,4,5-trisphosphate signaling regulates mating behavior in Caenorhabditis elegans males

Complex behavior requires the coordinated action of the nervous system and nonneuronal targets. Male mating in Caenorhabditis elegans consists of a series of defined behavioral steps that lead to the physiological outcomes required for successful impregnation. We demonstrate that signaling mediated by inositol 1,4,5-trisphosphate (IP(3)) is required at several points during mating. Disruption of IP(3) receptor (itr-1) function results in dramatic loss of male fertility, due to defects in turning behavior (during vulva location), spicule insertion and sperm transfer. To elucidate the signaling pathways responsible, we knocked down the six C. elegans genes encoding phospholipase C (PLC) family members. egl-8, which encodes PLC-beta, functions in spicule insertion and sperm transfer. itr-1 and egl-8 are widely expressed in the male reproductive system. An itr-1 gain-of-function mutation rescues infertility caused by egl-8 RNA interference, indicating that egl-8 and itr-1 function together as central components of the signaling events controlling sperm transfer.     

Coexpressed D1- and D2-like dopamine receptors antagonistically modulate acetylcholine release in Caenorhabditis elegans

Dopamine acts through two classes of G protein-coupled receptor (D1-like and D2-like) to modulate neuron activity in the brain. While subtypes of D1- and D2-like receptors are coexpressed in many neurons of the mammalian brain, it is unclear how signaling by these coexpressed receptors interacts to modulate the activity of the neuron in which they are expressed. D1- and D2-like dopamine receptors are also coexpressed in the cholinergic ventral-cord motor neurons of Caenorhabditis elegans. To begin to understand how coexpressed dopamine receptors interact to modulate neuron activity, we performed a genetic screen in C. elegans and isolated mutants defective in dopamine response. These mutants were also defective in behaviors mediated by endogenous dopamine signaling, including basal slowing and swimming-induced paralysis. We used transgene rescue experiments to show that defects in these dopamine-specific behaviors were caused by abnormal signaling in the cholinergic motor neurons. To investigate the interaction between the D1- and D2-like receptors specifically in these cholinergic motor neurons, we measured the sensitivity of dopamine-signaling mutants and transgenic animals to the acetylcholinesterase inhibitor aldicarb. We found that D2 signaling inhibited acetylcholine release from the cholinergic motor neurons while D1 signaling stimulated release from these same cells. Thus, coexpressed D1- and D2-like dopamine receptors act antagonistically in vivo to modulate acetylcholine release from the cholinergic motor neurons of C. elegans.     

Cell excitability necessary for male mating behavior in Caenorhabditis elegans is coordinated by interactions between big current and ether-a-go-go family K(+) channels

Variations in K(+) channel composition allow for differences in cell excitability and, at an organismal level, provide flexibility to behavioral regulation. When the function of a K(+) channel is disrupted, the remaining K(+) channels might incompletely compensate, manifesting as abnormal organismal behavior. In this study, we explored how different K(+) channels interact to regulate the neuromuscular circuitry used by Caenorhabditis elegans males to protract their copulatory spicules from their tail and insert them into the hermaphrodite's vulva during mating. We determined that the big current K(+) channel (BK)/SLO-1 genetically interacts with ether-a-go-go (EAG)/EGL-2 and EAG-related gene/UNC-103 K(+) channels to control spicule protraction. Through rescue experiments, we show that specific slo-1 isoforms affect spicule protraction. Gene expression studies show that slo-1 and egl-2 expression can be upregulated in a calcium/calmodulin-dependent protein kinase II-dependent manner to compensate for the loss of unc-103 and conversely, unc-103 can partially compensate for the loss of SLO-1 function. In conclusion, an interaction between BK and EAG family K(+) channels produces the muscle excitability levels that regulate the timing of spicule protraction and the success of male mating behavior.     

The C. elegans D2-Like Dopamine Receptor DOP-3 Decreases Behavioral Sensitivity to the Olfactory Stimulus 1-Octanol

We previously found that dopamine signaling modulates the sensitivity of wild-type C. elegans to the aversive odorant 1-octanol. C. elegans lacking the CAT-2 tyrosine hydroxylase enzyme, which is required for dopamine biosynthesis, are hypersensitive in their behavioral avoidance of dilute concentrations of octanol. Dopamine can also modulate the context-dependent response of C. elegans lacking RGS-3 function, a negative regulator of Gα signaling. rgs-3 mutant animals are defective in their avoidance of 100% octanol when they are assayed in the absence of food (E. coli bacterial lawn), but their response is restored when they are assayed in the presence of food or exogenous dopamine. However, it is not known which receptor might be mediating dopamine''s effects on octanol avoidance. Herein we describe a role for the C. elegans D2-like receptor DOP-3 in the regulation of olfactory sensitivity. We show that DOP-3 is required for the ability of food and exogenous dopamine to rescue the octanol avoidance defect of rgs-3 mutant animals. In addition, otherwise wild-type animals lacking DOP-3 function are hypersensitive to dilute octanol, reminiscent of cat-2 mutants. Furthermore, we demonstrate that DOP-3 function in the ASH sensory neurons is sufficient to rescue the hypersensitivity of dop-3 mutant animals, while dop-3 RNAi knockdown in ASH results in octanol hypersensitivity. Taken together, our data suggest that dopaminergic signaling through DOP-3 normally acts to dampen ASH signaling and behavioral sensitivity to octanol.     

Sexual conflict over mating in red-sided garter snakes (Thamnophis sirtalis) as indicated by experimental manipulation of genitalia

Sexual conflict over mating can result in sex-specific morphologies and behaviours that allow each sex to exert control over the outcome of reproduction. Genital traits, in particular, are often directly involved in conflict interactions. Via genital manipulation, we experimentally investigated whether genital traits in red-sided garter snakes influence copulation duration and formation of a copulatory plug. The hemipenes of male red-sided garter snakes have a large basal spine that inserts into the female cloaca during mating. We ablated the spine and found that males were still capable of copulation but copulation duration was much shorter and copulatory plugs were smaller than those produced by intact males. We also anaesthetized the female cloacal region and found that anaesthetized females copulated longer than control females, suggesting that female cloacal and vaginal contractions play a role in controlling copulation duration. Both results, combined with known aspects of the breeding biology of red-sided garter snakes, strongly support the idea that sexual conflict is involved in mating interactions in this species. Our results demonstrate the complex interactions among male and female traits generated by coevolutionary processes in a wild population. Such complexity highlights the importance of simultaneous examination of male and female traits.     

Initiation of male sperm-transfer behavior in Caenorhabditis elegans requires input from the ventral nerve cord

Background  

The Caenorhabditis elegans male exhibits a stereotypic behavioral pattern when attempting to mate. This behavior has been divided into the following steps: response, backing, turning, vulva location, spicule insertion, and sperm transfer. We and others have begun in-depth analyses of all these steps in order to understand how complex behaviors are generated. Here we extend our understanding of the sperm-transfer step of male mating behavior.     

Expression and function of dopamine in odontoblasts

Dopamine (DA) is produced from tyrosine by tyrosine hydroxylase (TH). A recent study has reported that DA promotes the mineralization of murine preosteoblasts. However, the role of DA in odontoblasts has not been examined. Therefore, in this investigation, we researched the expression of TH and DA in odontoblasts and the effects of DA on the differentiation of preodontoblasts (KN-3 cells). Immunostaining showed that TH and DA were intensely expressed in odontoblasts and preodontoblasts of rat incisors and molars. KN-3 cells expressed D1-like and D2-like receptors for DA. Furthermore, DA promoted odontoblastic differentiation of KN-3 cells, whereas an antagonist of D1-like receptors and a PKA signaling blocker, inhibited such differentiation. However, antagonists of D2-like receptors promoted differentiation. These results suggested that DA in preodontoblasts and odontoblasts might promote odontoblastic differentiation through D1-like receptors, but not D2-like receptors, and PKA signaling in an autocrine or paracrine manner and plays roles in dentinogenesis.     

Cuticular hydrocarbons and their role in copulatory behavior in Phormia regina (Meigen)

The cuticular hydrocarbons from adult Phormia regina (Meigen) were characterized by gas chromatography-mass spectrometry. Both sexes had similar components in nearly identical quantities, consisting of complex mixtures of saturated n-, monomethyl- and dimethylalkanes from 23 to 33 total carbons. Although no diet-, age-, or sex-specific differences were observed, cuticular hydrocarbons were shown to be involved in copulatory behavior. Hydrocarbon profiles of wild, compared to laboratory reared flies, showed no major differences. Behaviorally, males responded the same to dead decoys of either sex. Removal of the hydrocarbons, using hexane, from either male or female decoys, did not affect the number of mating strikes, but markedly reduced the number of copulatory attempts and the amount of time males spent mounted on either decoy. House fly, Musca domestica L., males when paired with a female M. domestica decoy produced copulatory attempts: whereas, when P. regina males were placed with M. domestica female decoys, there were no copulatory attempts. It is concluded that the cuticular hydrocarbons of P. regina function as species-specific but not sex-specific mating cues and elicit species-specific copulatory behavior in males.     

Challenging the Aggressive Spillover Hypothesis: Is Pre‐Copulatory Sexual Cannibalism a Part of a Behavioural Syndrome?

Pre‐copulatory cannibalism – females devouring males during courtship – may bring no benefit to either sex. The ‘aggressive spillover hypothesis’ (ASH) posits that pre‐copulatory cannibalism represents a spillover of female aggressiveness from the juvenile foraging context, when aggressiveness is advantageous, to the adult mating context, when aggressiveness may be non‐adaptive or maladaptive. The ASH suggests that individuals exhibit limited plasticity in aggressive behaviours because they are genetically canalised for indiscriminate aggressiveness towards prey and conspecifics, including males. Hence, a tendency to employ pre‐copulatory cannibalism is a part of the female aggression syndrome, an assertion generally accepted in the personality field. We here re‐evaluate the previous findings in the light of personality criteria, which we propose for ASH validation: between‐individual differences, repeatability and heritability in tendency for pre‐copulatory attacks (and pre‐copulatory cannibalism) and voracity towards prey, and their correlation. To re‐evaluate ASH and to allow for additional or alternative explanations, we ask whether pre‐copulatory cannibalism depends on female hunger, mating status, size and/or male quality. Finally, we ask whether cannibalistic females have a reduced reproductive success as predicted by the ASH. While repeatability and heritability in voracity towards prey and its correlation with the tendency to engage in pre‐copulatory cannibalism were found in certain systems, we lack any evidence for repeatability and heritability in pre‐copulatory cannibalistic attempts and for its maladaptiveness. Rather than only resorting to the ASH, foraging and mate choice hypotheses may also explain pre‐copulatory cannibalism. We suggest clarifying the use of the terms sexual cannibalism (effect) and female aggressiveness or tendency to attack and devour males (cause), and argue that male strategies to avoid cannibalism should be considered. We propose testing the ASH as the explanation for pre‐copulatory cannibalism in those cases where female tendency to devour males correlates with actual pre‐copulatory cannibalism and when all the above criteria are fulfilled. Finally, we propose future directions for studying the ASH.     

昆虫多巴胺及其受体的研究进展及展望

多巴胺(dopamine,DA)是一种重要的神经递质,通过特异地结合其相关的多巴胺受体(dopamine receptors,DARs)发挥作用。昆虫DARs可分为D1-like DARs,D2-like DARs和多巴胺/蜕皮激素受体(dopamine/ecdysteroid receptor,DopEcR)。D1-like DARs包含两种亚型即DOP1和DOP2,都能偶联G s蛋白引起胞内cAMP上升,且DOP2还能偶联G q蛋白引起胞内Ca 2+浓度升高;D2-like DARs只有一种亚型DOP3,偶联G i蛋白导致胞内cAMP下降;DopEcR可以同时被DA和蜕皮激素激活。本文综述了近年来关于昆虫DA的调控、多巴胺神经元、DARs的药理学特性及生理功能等方面的研究进展。DA合成、转运和降解过程中的基因调控昆虫的多种表型,如表皮黑化、翅的颜色和图案等。DA在多巴胺神经元中合成和释放,不同类型的多巴胺神经元参与调控不同的功能。随着近年来单细胞测序和DA实时成像技术的兴起,这将有利于进一步探讨特异神经元的功能。不同昆虫DARs的激动剂和拮抗剂活性存在很大异同,这些药理学差异将为以昆虫DARs为作用靶标开发高效选择性杀虫剂提供重要依据。DARs参与调控昆虫的多种生理与行为过程,如取食、学习、记忆、遗忘、求偶、交配、睡眠及觉醒等。随着CRISPR/Cas9技术在不同昆虫中成功地应用,以及结合模式昆虫黑腹果蝇中丰富的遗传学操作手段,这些都将有利于精准解析DARs的功能。     

Dopamine induces inhibitory effects on the circular muscle contractility of mouse distal colon via D1- and D2-like receptors

Dopamine (DA) acts as gut motility modulator, via D1- and D2-like receptors, but its effective role is far from being clear. Since alterations of the dopaminergic system could lead to gastrointestinal dysfunctions, a characterization of the enteric dopaminergic system is mandatory. In this study, we investigated the role of DA and D1- and D2-like receptors in the contractility of the circular muscle of mouse distal colon by organ-bath technique. DA caused relaxation in carbachol-precontracted circular muscle strips, sensitive to domperidone, D2-like receptor antagonist, and mimicked by bromocriptine, D2-like receptor agonist. 7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390), D1-like receptor antagonist, neural toxins, L-NAME (nitric oxide (NO) synthase inhibitor), 2′-deoxy-N₆-methyl adenosine 3′,5′-diphosphate diammonium salt (MRS 2179), purinergic P2Y1 antagonist, or adrenergic antagonists were ineffective. DA also reduced the amplitude of neurally evoked cholinergic contractions. The effect was mimicked by (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide (SKF-38393), D1-like receptor agonist and antagonized by SCH-23390, MRS 2179, or L-NAME. Western blotting analysis determined the expression of DA receptor proteins in mouse distal colon. Notably, SCH-23390 per se induced an increase in amplitude of spontaneous and neurally evoked cholinergic contractions, unaffected by neural blockers, L-NAME, MRS 2179, muscarinic, adrenergic, or D2-like receptor antagonists. Indeed, SCH-23390-induced effects were antagonized by an adenylyl cyclase blocker. In conclusion, DA inhibits colonic motility in mice via D2- and D1-like receptors, the latter reducing acetylcholine release from enteric neurons, involving nitrergic and purinergic systems. Whether constitutively active D1-like receptors, linked to adenylyl cyclase pathway, are involved in a tonic inhibitory control of colonic contractility is questioned.     

Female response to predation risk alters conspecific male behaviour during pre‐copulatory mate guarding

Mating behaviour often increases predation risk, but the vulnerability within mating pairs differs between the sexes. Such a sex difference is expected to lead to differences in responses to predation risk between the sexes. In the two‐spotted spider mite Tetranychus urticae, males engage in pre‐copulatory mate guarding because only the first mating results in fertilisation. We investigated (i) whether pre‐copulatory pairs are more conspicuous to the predatory mite Phytoseiulus persimilis than solitary females, (ii) whether the vulnerability to the predator differs between sexes within the pre‐copulatory pair, (iii) whether each sex of T. urticae responds to predation risk during pre‐copulatory mate guarding and (iv) whether T. urticae's response to predation risk affects predator behaviour. Because T. urticae females are immobile during pre‐copulatory mate guarding, we observed male behaviour to evaluate effects of predation risk. We found that the predators detect more pre‐copulatory pairs than solitary females and that more females than males of the pre‐copulatory pairs are preyed upon by the predators. The preference of spider mite males for pre‐copulatory pairs versus solitary females was affected by whether or not the female had been exposed to predators during development. Male T. urticae exposed to predation risk did not alter their behaviour. These results suggest that only the most vulnerable sex, that is the female, responds to predation risk, which modifies male behaviour. Regardless of T. urticae females’ experience, however, P. persimilis detected more T. urticae pre‐copulatory pairs than solitary females, suggesting that pre‐copulatory mate guarding itself is dangerous for T. urticae females when these predators are present. We discuss our results in the context of sex‐dependent differences in predation risk.     

Spiders,microbes and sex: Bacterial exposure on copulatory organs alters mating behaviour in funnel‐web spiders

Environmental microbes have the potential to be involved in nearly all behavioural processes. For example, mating systems where males use intromittent organs to transfer sperm to females represent a means by which environmental microbes collected by males can breach entry into females' body cavities during mating. However, the degree to which the acquisition of environmental microbes onto important sex structures alters courtship behaviours remains unknown. Here, we collected bacteria from the copulatory organs of Agelenopsis pennsylvanica funnel‐weaving spiders in situ to test whether exposure to bacteria on copulatory organs can alter hosts' courtship behaviour, reproductive success and survival. We used a standardized assay to repeatedly measure each spider's aggressiveness, a behavioural component of both male courtship and female sexual receptivity. Then, we experimentally altered the bacteria present on male and female spiders' copulatory organs with an application of either (a) a mixture of bacteria collected from conspecifics to increase bacterial presence, (b) an antibiotic to reduce bacterial presence or (c) a procedural control. Each spider was paired with a size‐matched spider of the opposite sex whose copulatory organs were unaltered, and we measured the latency until the onset and the duration of courtship. Spiders were then isolated, and we measured each individual's time until death and female fecundity over the next 40 days. We found that female exposure to bacteria had multiple effects on mating dynamics. Males took over four times longer to begin courting females that had been exposed to bacteria compared to unexposed and antibiotic‐treated females. Only when courting these bacteria‐exposed females, males began courtship sooner when females were more aggressive. Lastly, females whose mate had been exposed to bacteria experienced reduced survival. These data suggest that bacteria present on animals' copulatory organs can alter courtship behaviours, female survivorship, and may potentially play a role in mating dynamics.