The biological and behavioural basis of host selection in the transmission of Gyrodactylus (Monogenea)

Abstract

The ectoparasitic monogenean fluke, Gyrodactylus salaris, is a parasite known to be highly pathogenic to Atlantic salmon (Salmo salar). Although present in the environment of several neighbouring European countries, the UK is thought to be G. salaris-free, but, if national contingency plans to control this parasite are to be effective, it is vital that we understand the factors underlying its transmission from host to host. This study demonstrates that the majority of parasites transferring to new hosts are mature parasites that have reproduced at least once. Since, exploration and host transfer strategies pose a risk to survival; the parasite will endeavour to pass on its genes before attempting to transfer from one host to another. This study has also shown that when pregnant parasites are forced to leave their hosts, their offspring are aborted prematurely to ensure the survival of the mature parasite. Gyrodactylids do not possess a free-swimming stage in their life cycle, which allows for their migration between hosts. In spite of this, they are able to rapidly colonise naïve hosts, even in non-shoaling populations of fish. This study investigates the transmission strategies employed by detached parasites in the colonisation of new hosts. Observations of gyrodactylids collected from 3-spine sticklebacks, Gasterosteus acuelatus, suggest that their activity increases as a stickleback approaches, alerting the host to its presence. The parasite is then ingested directly by the prospective host. A time series of experimental exposures and specimens prepared for Scanning Electron Microscopy (SEM) suggest that once ingested, the parasites attach to the lining of the buccal cavity and then migrate out to their preferred colonisation site on the outer surface of the fish. It is proposed that this may be an alternative route for host infection. Similarly, direct ingestion by the scavenging on infected hosts by 3-spine sticklebacks suggests another route of infection of new hosts. Although these routes of transmission may be of lesser significance, infections in the buccal cavity may be an important indicator for detection of infection and those personnel involved in screening fish for gyrodactylids should be aware that this is an area in which infections can occur. This study also demonstrated that the use of the anaesthetic 2-phenoxyethanol does not affect the number of gyrodactylids which leave the host to colonise a new host. Additionally, observations of the transmission process suggest that turbulence produced by the movement of the fish’s fins may facilitate the transfer of detached parasites from the substrate. While this hypothesis appears to be supported by video evidence and photographic stills gathered throughout the duration of this study, further work should be conducted using particle tracking techniques to determine the efficacy of using a vortex effect as a means of colonising new hosts. Field sampling processes may have an effect on this type of research, giving rise to problems with the accurate diagnosis, management and control of gyrodactylids in a variety of fish. Gyrodactylus infected specimens of 3-spine stickleback (Gasterosteus aculeatus L.), minnows (Phoxinus phoxinus L.) and stone loach (Barbatula barbatula L.) from one Scottish river were cohabited. The study found that small numbers of Gyrodactylus do transfer to atypical hosts. This study highlights that personnel involved in fish disease surveillance programmes should be aware of the consequences of transporting multiple species in the same transport vessel as gyrodactylids may infect species previously thought to be resistant. Equally, diagnosticians should be aware of the fact that atypical species may act as temporary hosts and that their gyrodactylid fauna should not be assumed. Non-feeding life-cycle stages, such as the dispersal stages of parasites, are dependant for survival upon finite energy reserves gathered during feeding phases. Thus, those individuals with more limited reserves will die sooner and consequently have less time available to find a new host once detached. At this stage, the principal energy reserves in gyrodactylids are stored as large lipids droplets. Confocal laser scanning microscopy (CLSM) has been used to investigate the distribution of lipid droplets in Gyrodactylus, which have migrated off their fish host, testing the hypothesis that these droplets function as a proxy for the nutritional state. This study, demonstrated that the lipid droplets were particularly associated with the gut and that there is a significant variability in the volume of stored lipid carried out by each individual. Transmission Electron Microscopy (TEM) showed that gyrodactylids carry lipid droplets at all stages of their life cycle, including at release from the birth pore. It is likely that transferring worms require stored energy reserves to survive in the event of failure to establish contact with a new host. These reserves could allow the parasite to survive without a host for several days. As gyrodactylids appear to respond to a range of stimuli including vibration and chemicals released from the host, the presence or absence of such cues may have consequences on the rates of Gyrodactylus transmission. If these chemical stimuli can be identified and then mimicked or blocked, then this may offer potential opportunities for the control of gyrodactylid behaviour and for disrupting their transmission to new hosts. Baseline gyrodactylid behaviour, in the absence of a host, was determined under white light and infrared. This was achieved using a specially constructed arena and purpose written image analysis software to analyse parasite movement under different lighting conditions. The study found that gyrodactylids were more active in the dark than in light conditions, typically displaying longer, more sinuous tracks under red light than under white light. To begin investigating the effect of chemical presence on gyrodactylid behaviour, the activity of octopaminergic agonists and antagonist which bind to muscle receptors and alter muscle activity, were assessed. The impact of octopamine hydrochloride, clonidine hydrochloride, amitraz and, a toxic reference, chlordimeform, over a range of concentrations (0.2 to 3.2µM/L) were assessed on gyrodactylid behaviour. All of the four chemicals affected Gyrodactylus and produced muscle tetanus, causing muscle spasms when extension was attempted. Prolonged exposure resulted in death. Only the highest concentration of chlordimeform, the toxic reference, affected 100% of Gyrodactylus after 24 hours. After 48 hours, all of the Gyrodactylus treated with chlordimeform were either affected, moribund or dead. Amitraz was more toxic than chlordimeform with 80% of Gyrodactylus being dead after 24 hours at the highest concentration. After 48 hours 100% of Gyrodactylus exposed to 3.2 µm/L amitraz were dead, and up to 80% were dead in those exposed to lower concentrations; with no parasites being left unaffected. Although these particular compounds are toxic to fish, the effect of these agonistic chemicals on Gyrodactylus behaviour and survival is interesting and suggests that a closely related compound that is safe for use against fish may offer a potential treatment for the control of G. salaris infections in rivers. An ultrastructure study was undertaken to contribute to the current understanding of gyrodactylid ultrastructure. The findings of this research require broad understanding of gyrodactylid behaviour for their interpretation. Photographic evidence was gathered using transmission and electron microscopy. From these results, it is clear that Gyrodactylus gasterostei on a three-spine stickleback host will respond to a range of stimuli (i.e. vibration or chemical cues released from the host) in their assessment of host suitability. This study illustrated for the first time a chemical sensory structure found in Gyrodactylus gasterostei, located close to the cephalic lobe. It also identified apparently ciliated photoreceptors; as well mechanoreceptors in this species.