Single-cell analysis of peptide expression and electrophysiology of right parietal neurons involved in male copulation behavior of a simultaneous hermaphrodite.

Male copulation is a complex behavior that requires coordinated communication between the nervous system and the peripheral reproductive organs involved in mating. In hermaphroditic animals, such as the freshwater snail Lymnaea stagnalis, this complexity increases since the animal can behave both as male and female. The performance of the sexual role as a male is coordinated via a neuronal communication regulated by many peptidergic neurons, clustered in the cerebral and pedal ganglia and dispersed in the pleural and parietal ganglia. By combining single-cell matrix-assisted laser mass spectrometry with retrograde staining and electrophysiology, we analyzed neuropeptide expression of single neurons of the right parietal ganglion and their axonal projections into the penial nerve. Based on the neuropeptide profile of these neurons, we were able to reconstruct a chemical map of the right parietal ganglion revealing a striking correlation with the earlier electrophysiological and neuroanatomical studies. Neurons can be divided into two main groups: (i) neurons that express heptapeptides and (ii) neurons that do not. The neuronal projection of the different neurons into the penial nerve reveals a pattern where (spontaneous) activity is related to branching pattern. This heterogeneity in both neurochemical anatomy and branching pattern of the parietal neurons reflects the complexity of the peptidergic neurotransmission involved in the regulation of male mating behavior in this simultaneous hermaphrodite.

The distinction between retractor and protractor muscles of the freshwater snail's male organ has no physiological basis.

Many animals are equipped with organs that can be everted, a notable example being male copulatory organs. The ability to protrude or evert an organ generally requires protractor and retractor muscles. Male copulatory behaviour of the pond snail Lymnaea stagnalis (L.) involves eversion (protraction) and retraction of the relatively large penis-carrying organ. For this preputium, protractor and retractor muscle bands have been defined, which implies eversion and retraction through the activity of these muscle bands. However, no physiological data are available that confirm that the terms protractor and retractor are appropriate. To test whether eversion and retraction are possible without protractor and/or retractor muscle bands, lesion experiments were performed. The results show that with either one or several muscle bands lesioned, snails were still capable of everting their preputium and using it for copulation. However, the majority of animals that had six or more muscle bands lesioned were unable to retract its preputium. Hence, retractor muscle bands serve their designated function whereas protractor muscle bands do not. We therefore suggest that a different terminology is used in which all muscle bands are retractors and, based on their location, are either called distal or proximal retractors. The findings furthermore indicate that the preputium muscle bands are normally contracted, possibly in a catch state, retaining the organ inside without high-energy expenditure.

"Allohormones": a class of bioactive substances favoured by sexual selection.

During close bodily contact, many species transfer substances that influence the behaviour or physiology of conspecifics. Such transfer is especially common during courtship and copulation. When this is the case the involved bioactive substances are favoured by sexual selection because their effects include increased egg production, inhibited remating, and changed sperm transport or storage in the partner. The direct mode of action of these substances is fundamentally different from that of pheromones and nuptial gifts. Therefore, the term allohormone is introduced here. An allohormone is defined as a substance that is transferred from one individual to another free-living member of the same species and that induces a direct behavioural or physiological response, bypassing external sensory organs. Although we emphasise the importance of allohormones in reproductive processes, allohormones may also have important functions outside of copulation.

A conserved location for the central nervous system control of mating behaviour in gastropod molluscs: evidence from a terrestrial snail.

We have investigated the role of the right mesocerebrum in the expression of mating behaviour in the garden snail Helix aspersa. Using an in vivo stimulation and recording technique, we provide evidence for both sensory and motor functions in the mesocerebral neuronal population. Some neurones were specifically sensitive to tactile stimuli delivered to the skin on the superior tentacles and around the genital pore. Electrical stimulation of the right mesocerebrum evoked genital eversion and, in combination with tactile stimulation, dart-shooting and penial eversion. Genital eversions were also elicited by injections of APGWamide. During courtship, one recorded unit increased its activity only in correlation with penial eversion, while six other units increased their activity only during dart-shooting. Three additional units increased their activity during both types of behaviour. In addition, most of the recorded units showed increased neuronal activity during times of contact with a partner. Comparison of our results with available data from other molluscs leads us to conclude that the right anteromedial region of the cerebral ganglion is an evolutionarily conserved region of the gastropod brain specialised for the control of male mating behaviour. It is striking to find such functional conservation in the central nervous system of phylogenetically distant gastropods given the large differences in behaviour during mating.

Changes in the reproductive system of the snail Helix aspersa caused by mucus from the love dart.

The function of the love dart in certain species of terrestrial snails is unknown. In Helix aspersa, the dart is a sharp calcareous structure that is used to pierce the partner's skin during courtship. When expelled, the dart is covered with a thick mucus. The hypothesis tested here is that the mucus contains a biologically active substance. Extracts of the digitiform glands that produce this mucus were applied to parts of the reproductive system in vitro. The extracts triggered an initial reconfiguration of the copulatory canal that caused the bursa tract diverticulum to become more accessible to the spermatophore. The reconfiguration of the copulatory canal also closed off the tract leading to the bursa copulatrix, a sperm-digesting organ. A few minutes after the initial contraction, the peristaltic contractions in the diverticulum became significantly more frequent. This latter effect continued for at least 1 h, provided that the mucus extract remained in the saline bath. The minimum effective dosage was less than the 2.2 mg of mucus transferred with the dart. Sperm competition is expected in Helix aspersa since multiple matings occur before eggs are laid. By influencing the female organs involved in the processing of foreign sperm, the dart shooter may increase the chance that his sperm will fertilise eggs.