Light and temperature induced shedding, encystment and excystment of avian fluke Philophthalmu sp. under laboratory conditions

doi:10.21203/rs.3.rs-2635267/v1

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Light and temperature induced shedding, encystment and excystment of avian fluke Philophthalmu sp. under laboratory conditions

https://doi.org/10.21203/rs.3.rs-2635267/v1

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Objective: Cercarial shedding, encystment and excystment of digeneans are complex processes, dependent on intrinsic and extrinsic factors. We reportsequential events occur during shedding, encystment and excystment of cercariae of Philophthalmus sp.,released by freshwater snail, Thiara scabra. Snails were exposed to direct sunlight in petri dishes containing dechlorinated tap water (pH~ 6.5, 27° C) under laboratory conditions and the shedding, encystment and excystment of cercariae were observed under dissecting microscope.

Results:Megarulous cercariae actively emerged from the snail exhibited vigorous movement prior to encystment. They attached to the petri dish with the adhesive gland at the tip of the tail,immobilized the body, shed the tail and formed a flask shapedcyst within 10−15 min. After 30−40 min, excystment started with sequential events; activation, breaching and final release of the metacercaria from the cyst, completed in another 10−15 min. The excessive heat emanating from the microscope light bulb may have induced this behaviour. This is the first report of Philophthalmus sp, infecting T.scabrain Sri Lanka.Experimental induction of the life cycle stages of digeneans may provide valuable insight to explore their life history and provide models for testing the efficacy of antihelmintics against trematodes of medical and veterinary importance.

Encystment

Excystment

Megarulous cercariae

Metacercariae

Philophthalmussp., belonging to Family Philophthalmidae, Looss, 1899 primarily infect the conjunctival sac of the eyes of birds and hire aquatic snails as their firstintermediate host[1]. Philophthalmins were recorded in many parts of the world, infecting different bird species such as geese, ducks, chickens, peacocks, turkeys, ostriches and rheas[2]as the definitive host while utilizing different aquatic snail species such as Melanoides tuberсulatus, Fagotia acicularis, Amphimelania holandri, Melanopsis praemorsa, Pleurocerca acuta and Tarebia granifera, as intermediate hosts[3]. Accidental cases of human philophthalmosis have also been reported [4–6].

Philophthalmus lucipetus (P. gralli, Mathis and Leger, 1910)with a wide geographical range reported a broad host spectrum, infecting at least 30 bird species as the definitive host [2] and numerous snail host species.Digenean species such as P. gralli favor adaptations that maximize the probability of transmission to the definitive host by encysting on hard substrata that are likely to be consumed by the definitive host. The encysted metacercariae then enter their vertebratehost in an inactive form. After the infection, excystmentoccurs in the host body under specific physicochemicalfactors of intrinsic or extrinsic innature. The intrinsic factors are internal factors of the parasites such as enzymes, bile, secretions, muscularactivities, etc. The extrinsic factors are features in the host body, includingenzymes, pH, temperature, bilesalt, CO₂ tension, reductants, etc. [7].

, very little data is available on theintrinsic and extrinsic factors that affect the encystment and the excystment processes of P. gralli. This study reports ectopic encystment followed by an immediate excystment of Philophthalmus sp occurred in dechlorinated tapwater medium under intense light and heat condition.

Freshwater snails (Thiara scabra) were collected from Sarasavi Oya in Peradeniya (KandyDistrict) and were individually placed in small plastic cups containing ~ 50 mL of dechlorinated tap water. The cups were placed under direct sunlight to induce shedding of cercariae. After 4 − 6 hours of exposure, the water in the cups was observed under a dissecting microscope and the cercariae were identified using standard keys [8]. Cercariae were isolated and transferred into petridishes containing dechlorinated tap water. The movements of the cercariae and the snail hosts were continuously observed under the dissecting microscope and the images were captured using a Magnus LiveUSB®viewer. Thirty cercariae (n = 30) were immobilized with 90% alcohol for obtaining the measurements.

Cercarial shedding and encystment

Megarulous cercariae, emerged from the snail host were relatively large, elongate pear-shaped (372 ± 20 µm long and 138 ± 12 µm wide) and distinctly granulose, with no eyespots (Fig. 1.0). The maximum width of the body is recorded near the ventral sucker. The tail is 285 ± 15 µm long and 46 ± 4 µm wide. There was an adhesive gland with a sucker-like projection at the tip of the tail containing glandular cells stained deeply pink with acetocarmine. The pharynx is large and extends into an esophagus that is bifurcating (Y-shape) into two blind ending intestinal caeca, distinctly visible due to granulated nature. The subterminal oral sucker was 37 µm in size. The ventral sucker is bigger than the oral sucker, reporting 46 µm in size.

The encystation is usually initiated by the attachment of the cercaria with its tail to asubstrate and the tail’s detachment from the body. The entire process was completed within 10–15 minutes. The flask shaped cyst was 230 ± 5 µm long and 115 ± 6 µm wide (Fig. 1.0 b-f).

Excystment of the metacercariae

Spontaneous excystment was observed after 30–40 minutes of the encystment (Fig. 2.0). The metacercariae were released from an aperture of the cyst. Acylindrical wormlike form (345 ± 15 µm long and 92 ± 4 µm wide)with two well defined suckers emerged out of the cyst (Fig. 2.0 d&e).This excystment was completed in 10 − 15min.

Release of mature sporocyst

Mature redia/sporocyst was emerged out of the foot area of the snail. This sporocyst was a sac-like elongated body that averaged 1200 ± 25 µm long and 250 ± 16 µm wide (Fig. 3.0). A well-developed oral sucker (98 µm long by 92 µm wide) was clearly visible. A muscular pharynx was measured 112 µmlong by 110 µmwide. Dark-colored intestinal caecum extended in the anterior half of the body. Germ balls and cercariae at different developmental stageswere observed inside the body. Annular constriction was visible at the posterior half of the body. Mature cercariae forced their way outthroughthebirth pore and started their vigorous swimming movement in water soon after the escape(Fig. 3c-e).

Morphological and biological characteristics of the cercariae, cyst stage and the sporocyst stages lead to identify these megarulous cercariae as belonging to the trematodes of the genus Philophthalmus. [1, 9].Our observations on the encytment process were similar to Shakarbaev and colleagues [3], who reported the encystment of Philophthalmus lucipetus. Similar to our study, the cercarie of P. lucipetushas attached tightly to thewalls of the petri dishes so that they immobilized and started the encystment [3]. Adhesive tip of the tail has allowed the cercariae to place thebody vertically and to make vigorous pendular or circular movements to facilitate the detachmentof the body from the tail, consequently, forming the flask shaped cyst [3]. Other studies also noted the use of tail of the Philophthamid cercariae as an anchoring organ [10–13].Other forms of free-living cercariae use the oral and ventralsuckers as the anchoring organs and thus, contain secretingglandular cells extending their ducts onto the surface of thesuckers. Unlikeother trematode cercariae, the distal end of the tail inPhilophthalmus sp. has an adhesive glandwith a sucker-like organ, capable of secreting adhesive substance the cercariaeuse to stick to a substrate [1–3, 14]. This might allow cercariae to restrict into a limited area on the surfaceof a substrate, aggregate in that area, accelerate the encystment and enhance the chances of infecting the definitive hosts [3].

According to West, 1961 [1] the metacercaria is infective immediately after encystment and undergoes no apparent development during the two weeks or longer period. Similar to our observations, the movements are stimulated by a rise in temperature. A sudden increase in temperature caused the worm to extend the body immediately into full length in a single movement, resulting the emergence of the fore body through the neck of the cyst, followed more slowly by the hind body [1]. This confirms that the heat stress caused by the bulb of the microscope may have given the required heat signal for the excystation.

Subsequent excystment of larval trematodes soon aftertheir encystment appears to be unusual in the absence ofextrinsicenzymes. However, studies have shown thatfor some digeneans temperature is the major extrinsic factor for their excystment particularly the trematodes of homeothermic vertebrate definitive hosts[7]. Many complex cysts formed on vegetation require strong stimuli such as temperature as high as 39⁰ C, low redox potential, CO₂ with the presence of bile[15] for the excystment. However, most of the studies suggested that acid-pepsin is detrimental for the survival of excysted larvae of the digeneans[16]. Theexcysted larvae were unable to move freely in the mediumand may have died soon after the excystment process due to lack of suitable physicochemical surrounding. According to West [1] the excystment of the metacercariae of P.gralli occurs immediately upon reachingthe mouth or crop of the 'bird and not in the stomach or intestineas in many other digenetic trematodes. Thus, the presence of extrinsic enzymes may not be an essential factor for the excystment of Philophthalmus sp.

Rajapakse et al [6] reported an accidental infection of human by Philophthalmus sp, after bathing from an in an irrigation canal in the dry zone of Sri Lanka. As a common freshwater species in the dry zone, Thiara scabra [17] may have served as the intermediate host to complete the life cycle of Philophthalmus sp. Furthermore, T.scabracollected from the same habitat was reported to serve as hosts for many trematode species causing malformations in some amphibians of Sri Lanka [18–21]. Thus, as coexisting species with T.scabra, the potential of Philophthalmid infections in different vertebrates should be investigated.

Studies on invitroencystment and excystment are important for testing the efficacy of anti-helminthic drugsof digenetic trematodes of medical and veterinary importance.

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Acknowledgements

The authors acknowledge the Department of Zoology, Faculty of Science, University of Peradeniya for providing facilities and the technical assistance.

Author’s contributions

UAJ carried out the study under the guidance of PHA and RSR. UAJ drafted themanuscript and PHA and RSR reviewed it before the initial submission. Allauthors read and approved the final manuscript.

Funding

The study was conducted with the existing facilities of the Department of Zoology, Faculty of Science, University of Peradeniya. Thus, no other form of funding involved in the study.

Availability of data and materials

The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

Ethics approval and consent to participate

Ethical approval was obtained from the Research Ethics committee of the Postgraduate Institute of Science, University of Peradeniya, Sri Lanka. Authorsdeclare that the experiments conducted are complied with the standardanimal care guidelines and the current laws of Sri Lanka.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Department of Zoology, Faculty of Science, University of Peradeniya, Sri Lanka, ²Department of Zoology, Faculty of Natural Sciences, The Open University of Sri Lanka, ³International Water Management Institute, 127, Sunil Mawatha, Pelawatte, Battaramulla, Colombo, Sri Lanka

West AF. Studies on the biology of Philophthalmus gralli Mathis and Leger, 1910 (Trematoda: Digenea). American Midland Naturalist. 1961 Oct 1:363-83.
Literák I, Heneberg P, Sitko J, Wetzel EJ, Callirgos JM, Čapek M, Basto DV, Papoušek I. Eye trematode infection in small passerines in Peru caused by Philophthalmus lucipetus, an agent with a zoonotic potential spread by an invasive freshwater snail. Parasitology international. 2013 Aug 1;62(4):390-6.
Shakarbaev UA, Akramova FD, Azimov DA. Philophthalmus Lucipetus (Trematoda, Philophthalmidae) Cercariae in Molluscs Melanoides Kainarensis. Int J Cur Res Rev| Vol. 2020 Jul;12(14):100.
Lamothe-Argumedo R, Diaz-Camacho SP, Nawa Y. The firsthuman case in Mexico of conjunctivitis caused by an avianparasite Philophthalmus lacrimosus. J Parasitol 2003;89: 183–5.
Waikagul J, Dekumyoy P, Yoonuan T, Praevanit R. Conjunctiva philophthalmosis: a case report in Thailand. The American Journal of Tropical Medicine and Hygiene. 2006; 74:848–9.
Rajapakse RD, Wijerathne KM, Wijesundera M. Ocular infection with an avian trematode (Philophthalmus sp). Ceylon Medical Journal. 2009 Dec 11;54(4).
Fried B. Metacercarial excystment of trematodes. Advances in Parasitology. 1994 Jan 1;33:91-144.
Yamaguti S. Synoptical review of life histories of digenetic trematodes of vertebrates with special reference to the morphology of their larval forms.
Pinto HA, Melo AL. Melanoides tuberculata as intermediate host of Philophthalmus gralli in Brazil. Revista do Instituto de Medicina Tropical de São Paulo. 2010;52:323-7.
Chernogorenko MI. Trematodes larvae in molluscs of the Dnieper and its water reservoirs. Kyiv: Naukova Dumka. 1983.
Abdul-Salam J, Sreelatha BS, Ashkanani H. Digenea: Philophthalmidae) in Kuwait Bay. Kuwait J. Sci. Eng. 2004;31(1):119-33.
Alicata JE. Life cycle and developmental stages of Philophthalmus gralli in the intermediate and final hosts. Journal of Parasitology. 1962;48(1).
Ching HL. The development and morphological variation of Philophthalmus gralli Mathis and Leger, 1910 with a comparison of species of Philophthalmus Looss, 1899. Proceedings of the Helminthological Society of Washington. 1961 Jan 1;28(2):130-8.
Nollen PM, Murray HD. Philophthalmus gralli: identification, growth characteristics, and treatment of an oriental eyefluke of birds introduced into the continental United States. The Journal of parasitology. 1978 Feb 1;64(1):178-80.
Fried B, Butler MS. Infectivity, excystation, and development on the chick chorioallantois of the metacercaria of Echinostoma revolutum (Trematoda). The Journal of parasitology. 1978 Feb 1;64(1):175-7.
Saxton TM, Fried B, Peoples RC. Excystation of the encysted metacercariae of Echinostoma trivolvis and Echinostoma caproni in a trypsin-bile salts-cysteine medium and morphometric analysis of the excysted larvae. Journal of Parasitology. 2008 Jun;94(3):669-71.
Jayawardena U, Rajakaruna R, Amerasinghe P. Cercariae of trematodes in freshwater snails in three climatic zones in Sri Lanka. Ceylon Journal of Science (Biological Sciences). 2011 Apr 21;39(2).
Rajakaruna RS, Piyatissa PM, Jayawardena UA, Navaratne AN, Amerasinghe PH. Trematode infection induced malformations in the common hourglass treefrogs. Journal of Zoology. 2008 May;275(1):89-95.
Jayawardena UA, Rajakaruna RS, Navaratne AN, Amerasinghe PH. Monostome cercariae induced malformations in amphibians: effect of infection at the pre-limb-bud stage tadpoles of Polypedates cruciger Blyth. Journal of the National Science Foundation of Sri Lanka. 2010 Dec 30;38(4):241-8.
Jayawardena UA, Rohr JR, Navaratne AN, Amerasinghe PH, Rajakaruna RS. Combined effects of pesticides and trematode infections on hourglass tree frog Polypedates cruciger. EcoHealth. 2016 Mar 1;13(1):111-22.
Jayawardena UA, Rohr JR, Amerasinghe PH, Navaratne AN, Rajakaruna RS. Effects of agrochemicals on disease severity of Acanthostomum burminis infections (Digenea: Trematoda) in the Asian common toad, Duttaphrynus melanostictus. BMC Zoology. 2017 Dec;2(1):1-0.

No competing interests reported.

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Light and temperature induced shedding, encystment and excystment of avian fluke Philophthalmu sp. under laboratory conditions

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Abstract

Figures

Introduction

Methods

Results

Cercarial shedding and encystment

Excystment of the metacercariae

Release of mature sporocyst

Discussion

Abbreviations

Declarations

References

Additional Declarations

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