Vulva Location Research Articles

Mating sub-behaviours in Bursaphelenchus xylophilus on agar and evidence for female cooperation

Summary The pine wood nematode, Bursaphelenchus xylophilus, is the cause of pine wilt disease and reproduces by copulation within host pine trees. Female mate selection in B. xylophilus mating behaviour has been reported but definitive evidence is lacking. Furthermore, while the mating behaviour of the free-living nematode Caenorhabditis elegans on agar has been well understood, the mating behaviour of B. xylophilus on agar has not been studied in detail, and the differences in their characteristics remain unclear. Here, we addressed these knowledge gaps by observing B. xylophilus mating behaviour on agar. We identified six primary mating sub-behaviours in B. xylophilus males. Comparative analysis with C. elegans revealed distinct differences in the sequence of mating stages. Notably, B. xylophilus males actively searched for the female’s vulva during the wrapping stage, whereas females exhibited back-and-forth locomotion to align the male spicule with the vulva location. Additionally, we conducted mating assays using anaesthetised female nematodes to evaluate the female’s role in the mating behaviours of B. xylophilus. The mating duration increased significantly when females were anaesthetised, and the probabilities of reaching the wrapping and vulva location stages were reduced. Therefore, B. xylophilus females actively cooperate in mating behaviours. Our findings highlight unique aspects of mating behaviours in B. xylophilus and provide insights concerning differences in mating behaviours between B. xylophilus and C. elegans.

Morphobiological analysis of Trichuris vulpis (Nematoda, Trichuridae), obtained from domestic dogs

The parasitic nematode Trichuris vulpis Frölich, 1789 is the pathogen of trichuriasis in domestic and wild carnivores, and humans. This helminth species is distributed world-wide in populations of domestic dog (Canis lupus familiaris). The prevalence of T. vulpis in dogs depends to a large degree on the morphofunctional and biological adaptations of the parasite which support its high survivability in various environmental conditions. The present study considers the species-specific peculiarities of morphology, and metric parameters of the mature and embryonic stages of Trichuris nematodes parasitizing in C. lupus familiaris. We studied the periods of stages of development and infectious egg formation, and their survivability under optimal conditions for T. vulpis in laboratory culture. The differential characteristics of both female and male adult T. vulpis nematodes include the metric characteristics of cuticular protrusions at the surface of the anterior body part. The male T. vulpis nematodes can be distinguished from males of other species by the specifics of spicule sheath ornamentation, the shape and size of spicule, and the width of spicule sheath at different sections. In identification of the female nematodes of this species, it is necessary to consider the presence and size of papillary processes in the vulval area and metric parameters of vulva location. Nine metric characters of sexual dimorphism are described for T. vulpis nematodes. In laboratory conditions, five embryonic stages were observed for T. vulpis: protoplast, blastomere cleavage, and formation of bean-like embryo, larva and mobile larva. These stages are characterized by specific morphological features. The egg develops to the infectious stage at 27 °С in 18 days of culture, and their survivability is up to 76.6%. The egg development is associated with changes in their metric characters, such as decreasing egg length and width of egg shell, and increasing egg width and egg plug width.

Cdc-25.4, a Caenorhabditis elegans Ortholog of cdc25, Is Required for Male Mating Behavior.

Cell division cycle 25 (cdc25) is an evolutionarily conserved phosphatase that promotes cell cycle progression. Among the four cdc25 orthologs in Caenorhabditis elegans, we found that cdc-25.4 mutant males failed to produce outcrossed progeny. This was not caused by defects in sperm development, but by defects in male mating behavior. The cdc-25.4 mutant males showed various defects during male mating, including contact response, backing, turning, and vulva location. Aberrant turning behavior was the most prominent defect in the cdc-25.4 mutant males. We also found that cdc-25.4 is expressed in many neuronal cells throughout development. The turning defect in cdc-25.4 mutant males was recovered by cdc-25.4 transgenic expression in neuronal cells, suggesting that cdc-25.4 functions in neurons for male mating. However, the neuronal morphology of cdc-25.4 mutant males appeared to be normal, as examined with several neuronal markers. Also, RNAi depletion of wee-1.3, a C. elegans ortholog of Wee1/Myt1 kinase, failed to suppress the mating defects of cdc-25.4 mutant males. These findings suggest that, for successful male mating, cdc-25.4 does not target cell cycles that are required for neuronal differentiation and development. Rather, cdc-25.4 likely regulates noncanonical substrates in neuronal cells.

First Report of the Lesion Nematode, Pratylenchus agilis, Parasitizing Wheat in China

Wheat (Triticum aestivum L.) fields in Zhengzhou, Henan Province that exhibited reduced height and number of tillers were surveyed in 2008, and samples were collected for analyses. Fifteen of thirty-five samples contained a nematode suspected of causing small, brown lesions in roots. Both lateral roots and root hairs were reduced in infected plants. Nematode presence was associated with enlarging lesions and necrotic roots. We also observed lesion nematode infestation in samples with no symptoms in the roots. During the growing season, wheat yield increased by 14% with nematocide treatments in Zhengzhou suburb fields. The morphological and molecular analyses of the nematodes isolated from soil samples established the identity of the species as the lesion nematode, Pratylenchus agilis (2,3). Morphological characteristics that were used for identification included female body, lip annules, tail terminus, number of lines in the lateral field, stylet knobs, stylet length, and vulva location in relation to body length. Females were cylindrical, measured 452 to 811 μm long, and contained two lip annules, a smooth tail terminus, four lateral lines, a vulva at 72.64 to 79.97%, a stylet of 16.20 to 17.55 μm, basal esophageal lobes elongated less than twice the body width, and stylet knobs rounded posteriorly. Males were rare and the spermatheca was empty. Molecular analysis was conducted by amplifying the internal transcribed spacer (ITS) regions ITS1 and 2. Sequence of ITS regions from this population (GenBank Accession No. JQ039330) showed highest sequence homology to P. agilis isolate PagKL5 (GenBank Accession No. FJ71289.1) with the identity of 90%. High variability at species level has been found for Pratylenchus species (1). On the basis of the morphological traits and molecular analyses, the nematodes were identified as P. agilis. To our knowledge, this is the first report of P. agilis parasitizing wheat.

The CIL-1 PI 5-Phosphatase Localizes TRP Polycystins to Cilia and Activates Sperm in C. elegans

C. elegans male sexual behaviors include chemotaxis and response to hermaphrodites, backing, turning, vulva location, spicule insertion, and sperm transfer, culminating in cross-fertilization of hermaphrodite oocytes with male sperm. The LOV-1 and PKD-2 transient receptor potential polycystin (TRPP) complex localizes to ciliated endings of C. elegans male-specific sensory neurons and mediates several aspects of male mating behavior. TRPP complex ciliary localization and sensory function are evolutionarily conserved. A genetic screen for C. elegans mutants with PKD-2 ciliary localization (Cil) defects led to the isolation of a mutation in the cil-1 gene. Here, we report that a phosphoinositide (PI) 5-phosphatase, CIL-1, regulates TRPP complex ciliary receptor localization and sperm activation. cil-1 does not regulate the localization of other ciliary proteins, including intraflagellar transport (IFT) components, sensory receptors, or other TRP channels in different cell types. Rather, cil-1 specifically controls TRPP complex trafficking in male-specific sensory neurons and does so in a cell-autonomous fashion. In these cells, cil-1 is required for normal PI(3)P distribution, indicating that a balance between PI(3,5)P2 and PI(3)P is important for TRPP localization. cil-1 mutants are infertile because of sperm activation and motility defects. In sperm, the CIL-1 5-phosphatase and a wortmannin-sensitive PI 3-kinase act antagonistically to regulate the conversion of sessile spermatids into motile spermatozoa, implicating PI(3,4,5)P3 signaling in nematode sperm activation. Our studies identify the CIL-1 5-phosphatase as a key regulator of PI metabolism in cell types that are important in several aspects of male reproductive biology.

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

BackgroundTo 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.ResultsGenetic and laser ablation analysis, in conjunction with behavioral assays were used to determine neurotransmitters, receptors, neurons and muscles required for the regulation of male tail posture. We showed that proper male tail posture is maintained by the coordinated activity of opposing muscle groups that curl the tail ventrally and dorsally. Specifically, acetylcholine regulates both ventral and dorsal curling of the male tail, partially through anthelmintic levamisole-sensitive, nicotinic receptor subunits. Male-specific muscles are required for acetylcholine-driven ventral curling of the male tail but dorsal curling requires the dorsal body wall muscles shared by males and hermaphrodites. Gamma-aminobutyric acid activity is required for both dorsal and ventral acetylcholine-induced curling of the male tail and an inhibitory gamma-aminobutyric acid receptor, UNC-49, prevents over-curling of the male tail during mating, suggesting that cross-inhibition of muscle groups helps maintain proper tail posture.ConclusionOur results demonstrated that coordination of opposing sex-specific and core muscle groups, through the activity of multiple neurotransmitters, is required for regulation of male tail posture during mating. We have provided a simple model for regulation of male tail posture that provides a foundation for studies of how genes, molecular pathways, and neural circuits contribute to sensory regulation of this motor behavior.

FMRFamide-Like Neuropeptides and Mechanosensory Touch Receptor Neurons Regulate Male Sexual Turning Behavior inCaenorhabditis elegans

Caenorhabditis elegans male mating provides a powerful model to study the relationship between the nervous system, genes, and innate sexual behaviors. Male mating is the most complex behavior exhibited by the nematode C. elegans and involves the steps of response, backing, turning, vulva location, spicule insertion, and sperm transfer. Because neuropeptides are important neural regulators of many complex animal behaviors, we explored the function of the FMRFamide-like neuropeptide (flp) gene family in regulating male copulation. We found that peptidergic signaling mediated by FMRF-amide like neuropeptides (FLPs) FLP-8, FLP-10, FLP-12, and FLP-20 is required for the sensory transduction involved in male turning behavior. flp-8, flp-10, flp-12, and flp-20 mutant males significantly increase repetition of substep(s) of turning behavior compared with wild-type males. Genes controlling neuropeptide processing and secretion in general, including egl-3, egl-21, ida-1, and unc-31, are also required for inhibiting repetitive turning behavior. Neuropeptidergic signaling adjusts the repetitiveness of turning independently of serotonergic modulation of the timing of turning. Surprisingly, the mechanosensitive touch receptor neurons are found to be part of the neural circuitry regulating male turning behavior, indicating the existence of functional dimorphisms in the nervous system with regard to sex-specific behaviors.

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

BackgroundThe 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.ResultsBased on observation of wild-type males and on genetic analysis, we have divided the sperm-transfer step of mating behavior into four sub-steps: initiation, release, continued transfer, and cessation. To begin to understand how these sub-steps of sperm transfer are regulated, we screened for ethylmethanesulfonate (EMS)-induced mutations that cause males to transfer sperm aberrantly. We isolated an allele of unc-18, a previously reported member of the Sec1/Munc-18 (SM) family of proteins that is necessary for regulated exocytosis in C. elegans motor neurons. Our allele, sy671, is defective in two distinct sub-steps of sperm transfer: initiation and continued transfer. By a series of transgenic site-of-action experiments, we found that motor neurons in the ventral nerve cord require UNC-18 for the initiation of sperm transfer, and that UNC-18 acts downstream or in parallel to the SPV sensory neurons in this process. In addition to this neuronal requirement, we found that non-neuronal expression of UNC-18, in the male gonad, is necessary for the continuation of sperm transfer.ConclusionOur division of sperm-transfer behavior into sub-steps has provided a framework for the further detailed analysis of sperm transfer and its integration with other aspects of mating behavior. By determining the site of action of UNC-18 in sperm-transfer behavior, and its relation to the SPV sensory neurons, we have further defined the cells and tissues involved in the generation of this behavior. We have shown both a neuronal and non-neuronal requirement for UNC-18 in distinct sub-steps of sperm-transfer behavior. The definition of circuit components is a crucial first step toward understanding how genes specify the neural circuit and hence the behavior.

Inositol 1,4,5-Trisphosphate Signaling Regulates Mating Behavior inCaenorhabditis elegansMales

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.

Distinct roles of transcription factors EGL-46 and DAF-19 in specifying the functionality of a polycystin-expressing sensory neuron necessary for C. elegans male vulva location behavior.

Caenorhabditis elegans polycystins LOV-1 and PKD-2 are expressed in the male-specific HOB neuron, and are necessary for sensation of the hermaphrodite vulva during mating. We demonstrate that male vulva location behavior and expression of lov-1 and pkd-2 in the ciliated sensory neuron HOB require the activities of transcription factor EGL-46 and to some extent also EGL-44. This EGL-46- regulated program is specific to HOB and is distinct from a general ciliogenic pathway functioning in all ciliated neurons. The ciliogenic pathway regulator DAF-19 affects downstream components of the HOB-specific program indirectly and is independent of EGL-46 activity. The sensory function of HOB requires the combined action of these two distinct regulatory pathways.

Spauligodon aloisei n. sp. (Nematoda: Pharyngodonidae) parasite of Podarcis sicula (Reptilia: Lacertidae) from Italy.

A new species of an oxyurid nematode is described from the large intestine of the lacertid lizard Podarcis sicula (Rafinesque. 1810) (Reptilia: Lacertidae) from Italy. Spauligodon aloisei n. sp. differs from other species of the genus by a combination of morphological and metrical characters, including a spined tail in males and females, absence of a spicule in males, arrangement of the last pair of the male caudal papillae, vulva location, and egg morphology. The species most similar to S. aloisei n. sp. is Spauligodon caspius Annayev, 1987, described from Tenuidactylus (=Gymnodactylus) caspius in Turkmenistan. Tail length and location of the excretory pore and vulvar opening separate S. caspius from S. aloisei n. sp. This article increases the number of species of this genus from reptiles in the Paleartic Realm to 19.

MSI-1, a neural RNA-binding protein, is involved in male mating behaviour in Caenorhabditis elegans.

Neural RNA-binding proteins are thought to play important roles in neural development and the functional regulation of postmitotic neurones by mediating post-transcriptional gene regulation. RNA-binding proteins belonging to the Musashi family are highly expressed in the nervous system; however, their roles are poorly understood. We identified a Caenorhabditis elegans Musashi homologue, MSI-1, whose RNA-recognition motifs show extensive similarity to those of Drosophila and vertebrate Musashi proteins. We isolated a msi-1 mutant and found males with this mutation to have a mating defect. C. elegans male mating behaviour includes a distinct series of steps: response to contact, backing, turning, vulva location, spicule insertion, and sperm transfer. msi-1 is required for the turning and vulva location steps. Like other Musashi family members, MSI-1 is expressed specifically in neural cells, including male-specific neurones required for turning and vulva location. However, msi-1 was not expressed in proliferating neural progenitors in C. elegans, unlike the Musashi family genes in other systems. Our results suggest that msi-1 is expressed specifically in postmitotic neurones in C. elegans. msi-1 is required for full development of male mating behaviour, possibly through regulation of msi-1 expressing neurones.

A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans.

The stereotyped mating behaviour of the Caenorhabditis elegans male is made up of several substeps: response, backing, turning, vulva location, spicule insertion and sperm transfer. The complexity of this behaviour is reflected in the sexually dimorphic anatomy and nervous system. Behavioural functions have been assigned to most of the male-specific sensory neurons by means of cell ablations; for example, the hook sensory neurons HOA and HOB are specifically required for vulva location. We have investigated how sensory perception of the hermaphrodite by the C. elegans male controls mating behaviours. Here we identify a gene, lov-1 (for location of vulva), that is required for two male sensory behaviours: response and vulva location. lov-1 encodes a putative membrane protein with a mucin-like, serine-threonine-rich amino terminus followed by two blocks of homology to human polycystins, products of the autosomal dominant polycystic kidney-disease loci PKD1 and PKD2. LOV-1 is the closest C. elegans homologue of PKD1. lov-1 is expressed in adult males in sensory neurons of the rays, hook and head, which mediate response, vulva location, and potentially chemotaxis to hermaphrodites, respectively. PKD-2, the C. elegans homologue of PKD2, is localized to the same neurons as LOV-1, suggesting that they function in the same pathway.

The LIN-29 Transcription Factor Is Required for Proper Morphogenesis of theCaenorhabditis elegansMale Tail

TheCaenorhabditis elegansgenelin-29encodes a zinc-finger transcription factor that is required for hypodermal cell terminal differentiation and proper vulva morphogenesis. Here we demonstrate thatlin-29is also required in males for productive mating. We show thatlin-29males can perform the early mating behaviors including response to hermaphrodite contact and vulva location, but they do not perform the subsequent steps of vulva attachment via spicule insertion and sperm transfer. Consistent with this observation, we found thatlin-29mutant spicules are on average 43% shorter than wild-type spicules while other male mating structures appear unaltered. Inlin-29mutants, spicule development goes awry after the generation of spicule cells, when spicule morphogenesis occurs in wild-type males. We show that LIN-29 accumulates in many cells of the wild-type male tail, including those that form the spicules. We demonstrate, through analysis of genetic mosaics, that the formation of wild-type-length spicules requireslin-29(+)in the AB.p lineage, the lineage that gives rise to the spicules and other male copulatory structures. Our mosaic analysis also reveals a role forlin-29(+)in the P1 lineage, which mainly produces sex muscles, cells of the somatic gonad, and body wall muscles.

Sensory regulation of male mating behavior in Caenorhabditis elegans.

C. elegans male mating behavior comprises a series of steps: response to contact with the hermaphrodite, backing along her body, turning around her head or tail, location of the vulva, insertion of the two copulatory spicules into the vulva, and sperm transfer. By ablation of male-specific copulatory structures and their associated neurons, we have identified sensory structures and neurons that participate in each of these steps: the sensory rays mediate response to contact and turning; the hook, the postcloacal sensilla, and the spicules mediate vulva location; the spicules also mediate spicule insertion and regulate sperm transfer. Generally, successful completion of each step places the male in a position to receive a cue for the next step in the pathway. However, the high degree of sensory regulation allows the male to execute some steps independently.