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

2007

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Saxton et al. (2008) recently described the use of a trypsinbile salts-cysteine (TBC) medium to excyst the metacercarial cysts of Echinostoma caproni and Echinostoma trivolvis. The medium was prepared as described by Irwin et al. (1984) to excyst Himasthla leptosoma, except that bile salts were substituted for sodium taurocholate. The medium was used fresh by combining two solutions, A and B, just before use. Solution A consisted of trypsin and bile salts, whereas solution B consisted of the reductant L-cysteine. Details of the formulation and use of this medium were described by Saxton et al. (2008). A frozen and thawed TBC medium has not been used previously for work on E. caproni, although brief mention of this medium for the excystation of E. trivolvis was given by Saxton et al. (2008).The life cycle of this echinostome is maintained in at least six laboratories (see Toledo and Fried 2005), and encysted metacercariae of this echinostome are usually available for teaching and research purposes. One of us (BF) supplies colleagues with encysted metacercariae of this echinostome, and other workers mentioned in Toledo and Fried (2005) also provide this echinostome to colleagues. Some workers request BF to supply a medium for excystation of cysts of E. caproni. In the past, a frozen trypsin and bile (TB) medium of Fried and Roth (1974) was sent to investigators on dry ice to be used for the excystation of E. caproni. As explained by Saxton et al. (2008), the inability to get certain batches of the original trypsin stocks made it difficult to prepare and supply an effective TB medium. The newer TBC medium described in Saxton et al. (2008) has now replaced the old TB medium.Because of the advantages of making up large amounts of medium, e.g. 100 ml, and freezing it for long times, e.g., up to 2 years (see Fried 1994), we recently initiated work to describe the efficacy of a frozen and thawed TBC medium. The purpose of this report is to provide the results of our studies on excysting E. caproni metacercarial cysts in a frozen and thawed TBC medium.Cysts of E. caproni were obtained from the kidney/ pericardium of experimentally infected Biomphalaria glabrata snails and used within 1 to 14 days after removal from the snails (Fried and Huffman 1996). Cysts were placed, 25 to 30, in a 3.5-cm diameter Petri dish containing 3 ml of the frozen and thawed medium. The TBC medium was prepared as described in Saxton et al. (2008), and then 5 ml of solution A and 5 ml of solution B were frozen individually at either −10°C or −60°C for 24 h, 1 week, or 4 weeks before defrosting the medium and then combining solutions A and B. Defrosting of the medium was done under tap water at 22-24°C. Just before use, the two solutions were combined, and since no particulate matter was seen in the thawed medium, filtration was not necessary. The Petri dish containing the cysts was placed in an incubator at 41 ± 0.5°C for up to 60 min. In preliminary trials, observations made at 60 min revealed a very high (approaching 100%) percentage of excyst...

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mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Excystation of the metacercarial cysts of Echinostoma caproni in a frozen and thawed trypsin–bile salts–cysteine medium

Saxton

2007

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The effects of various chemical and physical factors on encystment and excystment of Zygocotyle lunata

Peoples

2008

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The effects of various chemical and physical treatments on the cercariae and metacercariae of Zygocotyle lunata were studied. Cercariae that actively emerged from Helisoma trivolvis snails were examined in 4 ml of test solution in a 3-cm diameter Stender dish. Observations were usually made on encystment and excystment from 2 to 24 h after cercariae were placed in the Stender dishes. Preferences for certain substrates for encystment were noted, such as smooth glass rather than rough glass, the macrophyte Eleocharis acicularis, and plastic Petri dishes. Many of the solutions tested allowed for encystment, i.e., artificial spring water (ASW), 10% artificial ocean water (AOW), and saline (0.85% NaCl). Certain hypertonic solutions allowed for both in vitro encystment and subsequent excystment, i.e., Locke's solution, 25% AOW, and Locke's 1:1. Serial dilutions of ammonium hydroxide, neutral buffered formalin, potassium permanganate, hydrochloric acid, and acetic acid killed cercariae at all concentrations. Ethanol was effective in killing cercariae at all concentrations except at 3.1%, which allowed for encystment. Cercariae were able to encyst at a low temperature of 16 degrees C and a high temperature of 41 degrees C. Morphometric analysis was performed on both live and fixed cercariae of Z. lunata as well as metacercarial cysts formed in Locke's, Locke's 1:1, and ASW. Live cercariae had larger body dimensions than cercariae fixed in hot 5% neutral buffered formalin and mounted in glycerin jelly. Cysts formed in ASW had very similar length and width measurements. Cyst dimensions became less similar to one another as the tonicity of the medium increased. The phenomenon of spontaneous excystation in Z. lunata after in vitro encystment in a hypertonic medium appears to be reported for the first time in digeneans.

An update on metacercarial excystment of trematodes

Saxton

2009

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This review updates an earlier one by Fried (Adv Parasitol 33:91-144, 1994) on metacercarial excystation of trematodes. Our coverage of the salient literature is mainly from 1995 to 2008 and includes significant studies on echinostomatid and non-echinostomatid trematodes. Most of the echinostomatid studies are on species of 37-collar spined Echinostoma, although one study is concerned with a species of Echinochasmus. The non-echinostomatid coverage includes work on species of Paragonimus, Haplorchis, Apatemon, Microphallus, Clonorchis, Riberoia, Maritema, Opisthorchis, and Zygocotyle. Most of the studies were concerned with methods used to excyst the metacercariae of the species being investigated. However, some studies were concerned with metacercarial excystation in order to use the excysted larvae for in vitro cultivation or morphometric studies. Other studies used the excysted larvae for physiological, biochemical, or behavioral work. Several studies were also concerned with elucidating both intrinsic and extrinsic factors which mediate chemical excystation of the metacercarial stage of digenetic trematodes.