Reduced infectivity in Himasthla elongata (Trematoda, Himasthlidae) cercariae with deviant photoreaction.

Digenean larvae of hermaphroditic generation - cercariae - are known to be polymorphic at genetic and behavioural levels. Cercariae arise as a result of parthenogenetic reproduction of intramolluscan stages, and represent a clone if a snail was infected with a single miracidium. Here we investigated cercarial clones of Himasthla elongata - namely, the infectivity of cercariae with normal (negative) and deviant (positive) photoreaction. In our study, most H. elongata clones showed intraclonal variance in their response to light. The proportion of photopositive cercariae ranged between 0.2% and 60% in different H. elongata clones. Photopositive larvae demonstrated significantly reduced rates of encystment in Mytilus edulis haemolymph in vitro and in young mussels. We discuss the possible mechanisms behind intraclonal variations, such as non-specific genomic rearrangements.

Complex and interactive effects of ocean acidification and warming on the life span of a marine trematode parasite.

Human activities have caused an increase in atmospheric CO2 over the last 250 years, leading to unprecedented rates of change in seawater pH and temperature. These global scale processes are now commonly referred to as ocean acidification and warming, and have the potential to substantially alter the physiological performance of many marine organisms. It is vital that the effects of ocean acidification and warming on marine organisms are explored so that we can predict how marine communities may change in future. In particular, the effect of ocean acidification and warming on host-parasite dynamics is poorly understood, despite the ecological importance of these relationships. Here, we explore the response of one himasthlid trematode, Himasthla sp., an abundant and broadly distributed species of marine parasite, to combinations of elevated temperature and pCO2 that represent physiological extremes, pre-industrial conditions, and end of century predictions. Specifically, we quantified the life span of the free-living cercarial stage under elevated temperature and pCO2, focussing our research on functional life span (the time cercariae spend actively swimming) and absolute life span (the period before death). We found that the effects of temperature and pCO2 were complex and interactive. Overall, increased temperature negatively affected functional and absolute life span, e.g. across all pCO2 treatments the average time to 50% cessation of active swimming was approximately 8 h at 5 °C, 6 h at 15 °C, 4 h at 25 °C, and 2 h at 40 °C. The effect of pCO2, which significantly affected absolute life span, was highly variable across temperature treatments. These results strongly suggest that ocean acidification and warming may alter the transmission success of trematode cercariae, and potentially reduce the input of cercariae to marine zooplankton. Either outcome could substantially alter the community structure of coastal marine systems.

Temperature and pCO2 jointly affect the emergence and survival of cercariae from a snail host: implications for future parasitic infections in the Humboldt Current system.

Ocean warming and acidification are general consequences of rising atmospheric CO2 concentrations. In addition to future predictions, highly productive systems such as the Humboldt Current System are characterized by important variations in both temperature and pCO2 level, but how these physical-chemical ocean changes might influence the transmission and survival of parasites has not been assessed. This study experimentally evaluated the effects of temperature (14, 18 and 25 °C) and the combined effects of temperature (∼15 and 20 °C) and pCO2 level (∼500 and 1400 microatmospheres (µatm) on the emergence and survival of two species of marine trematodes-Echinostomatidae gen. sp. and Philophthalmidae gen. sp.-both of which infect the intertidal snail Echinolittorina peruviana. Snails were collected from intertidal rocky pools in a year-round upwelling area of the northern Humboldt Current System (23°S). Two experiments assessed parasite emergence and survival by simulating emersion-immersion tidal cycles. To assess parasite survival, 2 h old cercariae (on average) were taken from a pool of infected snails incubated at 20-25 °C, and their mortality was recorded every 6 h until all the cercariae were dead. For both species, a trade-off between high emergence and low survival of cercariae was observed in the high temperature treatment. Species-specific responses to the combination of temperature and pCO2 levels were also observed: the emergence of Echinostomatidae cercariae was highest at 20 °C regardless of the pCO2 levels. By contrast, the emergence of Philophthalmidae cercariae was highest at elevated pCO2 (15 and 20 °C), suggesting that CO2 may react synergistically with temperature, increasing transmission success of this parasite in coastal ecosystems of the Humboldt Current System where water temperature and pH are expected to decrease. In conclusion, our results suggest that integrating temperature-pCO2 interactions in parasite studies is essential for understanding the consequence of climate change in future marine ecosystem health.

Thermodynamics of egg production, development and hatching in trematodes.

Temperature is a key factor influencing the rate of biological processes of ectothermic animals and is intrinsically linked to climate change. Trematode parasites may be potentially susceptible to temperature changes and, in order to develop a predictive framework of their response to climate change, large-scale analyses are needed. In particular, the biology of the egg of all species is at some time influenced by environmental conditions. The present study uses Arrhenius activation energy (E*), a common measure of temperature-mediated reaction rates, to analyse experimental data from the scientific literature on the effects of temperature on the production, development and hatching of trematode eggs. Egg production declines at high temperatures, with habitat-specific climatic factors determining the optimal thermal range. Egg development, as is typical of invertebrates, shows a simple response to temperature, with minimal differences between mid- (35-60°) and low-latitude (<35°) species. Egg hatching demonstrates variable thermodynamics with high E* values at low temperature ranges and thermostability at mid-temperatures, before declining at high temperature ranges, with wide thermostable zones being a common feature. Comparisons between development and hatching indicate that these two parameters demonstrate different thermodynamical responses. The significance of these results in furthering our understanding of trematode egg biology under natural conditions is discussed.

Natural light influences cercarial emergence of Calicophoron daubneyi but not that of Haplometra cylindracea from temperature-challenged Galba truncatula.

Laboratory investigations on Galba truncatula experimentally infected with Calicophoron daubneyi were carried out to study the influence of natural light and sky nebulosity on cercarial emergence in snails subjected every week outdoors to a thermal shock (a mean of 12 °C for 3 h) during the patent period. The same study was also performed in G. truncatula naturally infected with Haplometra cylindracea according to the same protocol. Compared to infected controls always reared indoors at 20 °C, the number of cercariae emerging from snails subjected outdoors to a thermal shock was significantly greater, whatever parasite species. Natural light had an effect on snails releasing C. daubneyi cercariae after the thermal shock because their numbers were significantly higher between 601 and 1,200 lx and for the highest nebulosity values (7-8 octas). In contrast, the type of light used during thermal shock did not influence cercarial emergence of H. cylindracea because the numbers of cercariae per shedding snail noted under natural light or a 3,000-lx artificial light did not significantly differ from each other. Most snails releasing H. cylindracea cercariae were significantly more numerous for light levels higher than 3,000 lx. As for Fasciola hepatica cercariae, natural light had a significant influence on cercarial emergence of C. daubneyi, and this behavioural particularity for cercariae of both digeneans might be due to the fact that both species develop in the same ruminants and the same snail host.

Analysis of trait mean and variability versus temperature in trematode cercariae: is there scope for adaptation to global warming?

The potential of species for evolutionary adaptation in the context of global climate change has recently come under scrutiny. Estimates of phenotypic variation in biological traits may prove valuable for identifying species, or groups of species, with greater or lower potential for evolutionary adaptation, as this variation, when heritable, represents the basis for natural selection. Assuming that measures of trait variability reflect the evolutionary potential of these traits, we conducted an analysis across trematode species to determine the potential of these parasites as a group to adapt to increasing temperatures. Firstly, we assessed how the mean number of infective stages (cercariae) emerging from infected snail hosts as well as the survival and infectivity of cercariae are related to temperature. Secondly and importantly in the context of evolutionary potential, we assessed how coefficients of variation for these traits are related to temperature, in both cases controlling for other factors such as habitat, acclimatisation, latitude and type of target host. With increasing temperature, an optimum curve was found for mean output and mean infectivity, and a linear decrease for survival of cercariae. For coefficients of variation, temperature was only an important predictor in the case of cercarial output, where results indicated that there is, however, no evidence for limited trait variation at the higher temperature range. No directional trend was found for either variation of survival or infectivity. These results, characterising general patterns among trematodes, suggest that all three traits considered may have potential to change through adaptive evolution.

Cercarial survival in an intertidal trematode: a multifactorial experiment with temperature, salinity and ultraviolet radiation.

Parasite transmission takes place in the context of a multitude of simultaneously fluctuating environmental factors. As a particularly vulnerable step in the transmission, trematode cercariae are directly exposed to ambient conditions during their search for a host. Here, we investigated the survival of cercariae of the intertidal trematode Maritrema novaezealandensis in a multifactorial experiment (2 × 2 × 2 design) with temperature (20 and 30 °C), salinity (35 and 40 practical salinity units (psu)) and ultraviolet radiation (UVR; exposed and not exposed) as main factors. All factors had significant effects, with cercariae dying faster at the higher temperature, increased salinity and when exposed to UVR. Full activity ceased within ~6 h in all treatments at 30 °C, except at 35 psu under no exposure to UV; in all other treatments full activity was maintained for >6 h. Several factor interactions were identified, of which the interactive negative effect of temperature and UVR was the most important. These results imply that conditions during the main transmission window of M. novaezealandensis are highly challenging for cercariae. Our findings highlight the importance of considering multiple environmental factors in the study of parasite transmission to gain a more ecologically relevant understanding of transmission dynamics.

Effects of ultraviolet radiation on an intertidal trematode parasite: an assessment of damage and protection.

Trematode parasites are integral components of intertidal ecosystems which experience high levels of ultraviolet radiation. Although these parasites mostly live within hosts, their life cycle involves free-living larval transmission stages such as cercariae which are directly exposed to ambient conditions. UV has previously been shown to considerably reduce the survival of cercariae. Here, we investigated potential mechanisms of protection and damage related to UV in the intertidal trematode Maritrema novaezealandensis. Firstly, the presence of sunscreen compounds (i.e. mycosporine-like amino acids) was quantified in the parasite tissue producing cercariae within a snail host, as well as in the free-swimming cercariae themselves. Secondly, levels of oxidative stress in cercariae after exposure to UV were investigated (i.e.protein carbonyls, catalase and superoxide dismutase). Thirdly, the DNA damage (i.e. cyclobutane­pyrimidine dimers) was compared between cercariae exposed and not exposed to UV. Lastly, functional aspects(survival and infectivity) of cercariae were assessed, comparing cercariae under light conditions versus dark after exposure to UV. We confirmed the presence of my cosporine-like amino acids in cercariae-producing tissue from within snail hosts, but were unable to do so in cercariae directly. Results further suggested that exposure to UV induced high levels of oxidative stress in cercariae which was accompanied by a reduction in the levels of protective antioxidant enzymes present. We also identified higher levels of DNA damage in cercariae exposed to UV, compared with those not exposed. Moreover, no clear effect of light condition was found on survival and infectivity of cercariae after exposure to UV. We concluded that cercariae are highly susceptible to UV damage and that they have very little scope for protection against or repair of UV-induced damage.

Effects of ultraviolet radiation on the transmission process of an intertidal trematode parasite.

The transmission of parasites takes place under exposure to a range of fluctuating environmental factors, one being the changing levels of solar ultraviolet radiation (UVR). Here, we investigated the effects of ecologically relevant levels of UVR on the transmission of the intertidal trematode Maritrema novaezealandensis from its first intermediate snail host (Zeacumantus subcarinatus) to its second intermediate amphipod host (Paracalliope novizealandiae). We assessed the output of parasite transmission stages (cercariae) from infected snail hosts, the survival and infectivity of cercariae, the susceptibility of amphipod hosts to infection (laboratory experiments) and the survival of infected and uninfected amphipod hosts (outdoor experiment) when exposed to photo-synthetically active radiation only (PAR, 400-700 nm; no UV), PAR+UVA (320-700 nm) or PAR+UVA+UVB (280-700 nm). Survival of cercariae and susceptibility of amphipods to infection were the only two steps significantly affected by UVR. Survival of cercariae decreased strongly in a dose-dependent manner, while susceptibility of amphipods increased after exposure to UVR for a prolonged period. Exposure to UVR thus negatively affects both the parasite and its amphipod host, and should therefore be considered an influential component in parasite transmission and host-parasite interactions in intertidal ecosystems.

Localization of photoreceptors in the cercariae of Proterometra macrostoma (Trematoda: Azygiidae).

The swimming behavior of the cercaria of the digenetic trematode Proterometra macrostoma changes in response to light. However, this cercaria does not possess obvious eyes or eyespots. Using behavioral assays, we were able to show that both intact and distome-removed cercariae swim significantly greater vertical distances under dim, red light than under brighter, white light. Electrophysiological experiments confirmed this result and further showed that the transverse band of the tail, known to control cercariae swimming behavior, was necessary and sufficient for the display of the light-dependent swimming behavior. Together, these data show that the distome is not required for light-dependent swimming behavior in P. macrostoma cercariae and indirectly demonstrates the presence of photoreceptors in the transverse band of the cercaria tail.

Life cycle truncation in a trematode: does higher temperature indicate shorter host longevity?

The typical three-host life cycle of most trematodes creates transmission challenges for which a variety of adaptations have evolved to increase the probability of transmission. Some species can abbreviate their life cycle via progenesis, the precocious maturation of the parasite in the second intermediate host resulting in the production of eggs through self-fertilisation without requiring a definitive host. Adoption of the progenetic life cycle may be a conditional strategy in response to different environmental cues related to low probability of transmission to the definitive host. Using high water temperature and/or limited diet as experimental stressors, we tested the effect of body condition and life span of the fish second intermediate host on facultative truncation of the typical three-host life cycle by progenesis in Stegodexamene anguillae. The results suggest that environmental cues, such as temperature and encystment site, may signal transmission opportunities to the parasite so that it may adjust its developmental strategy accordingly. Indeed, a greater proportion of worms became progenetic at higher temperatures, and progenesis was more common among worms encysted in the gonads or body cavity of their fish hosts than among those in other host tissues. These findings highlight the often unrecognised plasticity in parasite developmental and transmission strategies.

Encystment and excystment of the paramphistomid trematode Zygocotyle lunata.

This study reports further observations on encystment and excystment of the paramphistomid trematode Zygocotyle lunata. Of numerous substrates tested in the laboratory for cercarial encystment, i.e. plastic and glass dishes, Styrofoam™, aluminium foil, snail shells, and cheesecloth, all but cheesecloth allowed for 100% encystment within 4 h of cercarial release at 28°C. Numerous physical and chemical factors were tested to kill cysts, and their capacity to do so was determined by the subsequent inability of cysts to excyst within a chemical excystment medium. Vinegar, Italian salad dressing, soy sauce and 10% solutions of sucrose, acetic acid and sodium chloride were the most detrimental. Freezing, boiling and storage of cysts for more than 2 years also killed them. Several marinades such as lemon juice, Worcestershire sauce, and various concentrations of potassium permanganate, did not kill the majority of cysts tested. Since Z. lunata is a potential foodborne trematode for humans and animals, it is important to determine suitable ways to control or kill these cysts.

Effects of temperature, salinity, and pH on the survival and activity of marine cercariae.

Alterations of abiotic factors due to global climate change are predicted to impact disease dynamics, particularly for pathogens with complex life cycles involving free-living infectious stages, such as the cercariae of trematode parasites. Previous investigations of cercarial output, longevity, and infectivity suggest an overall increase in trematode transmission in response to elevated temperature. However, while increased temperature will likely be accompanied by changes in salinity and pH in marine ecosystems, little is known regarding their influence on cercariae. We investigated the response of trematode cercariae of the intertidal horn snail Cerithidea californica to altered temperature, salinity, and pH. The survival and activity of one trematode species, Euhaplorchis californiensis (Heterophydae), appears to be largely unaffected by increased temperature, while that of a second species, Acanthoparyphium spinulosum (Echinostomatidae), decreased at the warmer temperature (25 degrees C). Cercariae of E. californiensis generally fared best at the highest salinity (40 ppt), whereas A. spinulosum showed the opposite effect. Neither species was affected by pH alone although there were interactions with salinity and time. These results may reflect different emergence patterns of the two species and demonstrate that trematode parasitism in intertidal zones may be impacted by alterations of the marine environment resulting from climate change.

Impact of several control measures on the encysted metacercariae of heterophyids.

The present study evaluated the effect of temperature, chemicals and microwave irradiation on the viability and infectivity of encysted metacercariae (EMC) of Pygidiopsis genata in fish. Flesh of infected fish was divided into two groups; control (GI) and experimental (GII). GII was subdivided into IIa: exposed to -4 degrees C & -20 degrees C for different periods, IIb: marinated in sodium chloride, acetic and citric acid at different concentrations and exposure times and IIc: subjected to microwave irradiation for 20 seconds. Assessment of the effect of these factors was by studying the viability and infectivity of EMC after isolation from the fish. The ultrastructure change was studied by scanning electron microscope (SEM), in comparison with control. The results showed that the highest significant reduction in the EMC viability and infectivity was detected in those exposed to microwave irradiation (100%). SEM examination showed distortion in the shape of microwave-treated EMC with change in the surface nature. Freezing of EMC at -4 degrees C & -20 degrees C for 10 days at either temperature was sufficient to abolish their infectivity, although some were remained viable. Soaking of flesh in salt or acids reduced EMC viability and infectivity. So, microwave irradiation proved prophylactic for EMC.

Radioresistance of Anisakis simplex third-stage larvae and the possible role of superoxide dismutase.

The radioresistance of Anisakis simplex third-stage larvae and the possible role of sublethal radiation on superoxide dismutase (SOD) were investigated. Larvae were isolated from the viscera of the sea eel Anago anago; irradiated with 10, 100, 200, 500, or 1,000 Gy; and then given orally to rats. Worms were recovered at 16 hr postinoculation. Most larvae were found to have invaded the gastric wall, omentum, and abdominal cavity, suggesting that their viability and infectivity were not controlled by irradiation with the doses used. To determine the relationship between SOD activities in parasites and their radiosensitivities, the larvae of A. simplex and the metacercariae of Neodiplostomum seoulense (a radiosensitive control) were irradiated with 0, 30, 100, or 500 Gy, and parasite SOD levels were measured. In nonirradiated A. simplex larvae, the average SOD level was 38.9 U/mg, and this increased to 51.3 U/mg at 500 Gy. However, at all radiation doses applied, SOD activities of N. seoulense metacercariae were significantly (P < 0.05) lower than those of A. simplex larvae. Our results demonstrate that A. simplex third-stage larvae are radioresistant, and suggest that SOD plays a role in this radioresistance.

Effects of gamma-irradiation on the survival and development of Gymnophalloides seoi in C3H mice.

An experimental study was carried out to observe the effects of radiation on the infectivity of metacercariae of Gymnophalloides seoi to C3H mice. Oysters, the second intermediate host, were collected from an endemic area, and non-irradiated control, metacercaria-irradiation, and oyster-irradiation groups were prepared. One hundred metacercariae were infected orally to each mouse, and worm recovery rate was compared by groups at 7th day post-infection. In the metacercaria-irradiation group, the worm recovery rate was significantly reduced at radiation doses higher than 200 Gy, and the number of intrauterine eggs significantly reduced at doses over 50 Gy. In the oyster-irradiation group, 50 Gy significantly reduced both the worm recovery rate and number of uterine eggs. In the two groups, no worm was recovered at 1,000 Gy irradiation. Conclusively, irradiation of oysters with 200-1,000 Gy was effective to control infectivity of metacercariae to C3H mice, and could be adopted as a control measure for gymnophalloidiasis.

A response to light in an adult encotyllabine (capsalid) monogenean from the pharyngeal tooth pads of some marine teleost fishes.

A response to light in an adult monogenean is reported for the first time. Living encotyllabine (capsalid) monogeneans, identified tentatively as Encotyllable caballeroi Velasquez, 1977, attached to pharyngeal tooth pads excised from two species of teleost fish, contracted rapidly when the intensity of the incident illumination was suddenly increased. Contracted individuals were immobile and largely concealed in crevices in the tooth pads. Parasites habituated to the higher light intensity after 60-100 s and re-extended their bodies and resumed activity. Contracted, immobile individuals re-extended prematurely if the light intensity was reduced during the habituation period. Detached parasites responded in the same way to abrupt changes in the illumination intensity. The possible significance of this response in relation to the biology of the hosts is briefly discussed.

Studies on amphistomiasis in Andhra Pradesh (India) VI. Immunization of lambs and kids with gamma irradiated metacercariae of Cercariae indicae XXVI.

The life span and the pathogenicity of amphistomes developing from Cercariae indicae XXVI (Paramphistomum epicilitum) were considerably reduced when the metacercariae were gamma-irradiated at 2 krad or 3 krad. The higher irradiation dose resulted in the complete absence of the flukes in infected animals. Single vaccination of lambs and kids with 3000 metacercariae irradiated at 2 krad or 3 krad stimulated a significant degree of resistance against challenge with 5000 non-irradiated metacercariae. The resistance was more pronounced in the group vaccinated with metacercariae irradiated at 3 krad.