Studies on the in vitro cultivation of ciliate protozoa from the kangaroo forestomach

Accepted Manuscript Title: Studies on the in vitro cultivation of ciliate protozoa from the kangaroo forestomach Author: Burk A. Dehority Andr´e-Denis G. Wright PII: DOI: Reference:

S0932-4739(14)00034-0 http://dx.doi.org/doi:10.1016/j.ejop.2014.04.001 EJOP 25332

To appear in: Received date: Revised date: Accepted date:

25-2-2014 4-4-2014 5-4-2014

Please cite this article as: Dehority, B.A., Wright, A.-D.G.,Studies on the in vitro cultivation of ciliate protozoa from the kangaroo forestomach, European Journal of Protistology (2014), http://dx.doi.org/10.1016/j.ejop.2014.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Studies on the in vitro cultivation of ciliate protozoa from the

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Burk A Dehoritya,∗, André-Denis G. Wrightb

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kangaroo forestomach

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CSIRO Livestock Industries, Centre for Environment and Life Sciences, Wembley, Western Australia 6913, Australia

a

Present Address: Department of Animal Sciences, Ohio Agricultural Research and Development

b

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Center,The Ohio State University, Wooster, OH 44691, United States Present Address: Department of Animal Science, University of Vermont, Burlington, VT

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05405,United States

_____________________________ ∗

corresponding author.Tel.: +1 330 263 3909; fax: +1 330 263 3949 E-mail address: [email protected] (B. Dehority)

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Abstract The methods used for culturing rumen protozoa were found to be unsatisfactory for

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growth of ciliate protozoa from the kangaroo forestomach. Based on published measurements of physical parameters in the marsupial forestomach, several modifications were incorporated into

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the procedure, i.e., an increase in % hydrogen in the gas phase, adjustment of initial pH of the

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medium to 6.9-7.0 range, feed only forage as a substrate and incubate at a lower temperature (3336ºC). Only incubation at the lower temperature increased survival time of the kangaroo

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protozoa. Two species of Bitricha were still viable after 28 days in culture. Cultures had to be terminated at that time. One of the species differed considerably in size and shape from

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previously described species and based on 18S rRNA data, may represent a new species of Bitricha. The second species, present in low numbers was identified as Bitricha oblata. In a

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had to be terminated.

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separate trial, Macropodinium yalanbense survived for 11 days, at which time these cultures also

Keywords: Bitricha; Cultivation; Forestomach; Marsupial; Protozoa

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Introduction In 1996, Dehority described a new family of ciliates present only in marsupials, the Macropodiniidae. It contained a new genus, Macropodinium, and five new species. More

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recently, Cameron and O’Donoghue (2002, 2003), have described two new ciliate families, the

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Amylovoracidae and the Polycostidae, both unique to the marsupial foregut.

Compared to ruminants, the macropodine marsupials (kangaroo and wallabies) consume

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less feed on a metabolic weight basis and have a basal metabolic rate which is about 70% of that in eutherian mammals (Hume 1982). With a presumably faster ingesta passage through the

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foregut, a question arises as to how the macropodine obtain sufficient energy from poor quality,

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high fiber feedstuffs. In addition to differences in intake and passage rate it is also possible that the microbes in the macropodine foregut have a faster rate of digestion. Bacteria, protozoa and

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fungi have all been observed in forestomach contents of the kangaroo at concentrations similar to

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those which occur in the rumen of domestic sheep (Dellow et. al. 1988, Hume 1982, Moir and Hungate, unpublished results from R.J. Moir). Baker et al. (1995) were unsuccessful in their

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attempts to establish kangaroo protozoa in either faunated or defaunated sheep. In an earlier unpublished study, using basal medium M (Dehority 1998) and either ground clover hay or oaten hay plus wheat as substrates, cultures were inoculated with filtered forestomach fluid (KFF) from three kangaroos. After two days, the protozoa numbers had declined in all cultures, so 0.5 ml of KFF was added. On day 3, new medium SP (Dehority 1998) plus 20% KFF was prepared and cultures were split and transferred into the two media (M and SP). By day 7, no viable cells were present in any of the culture tubes. Two additional kangaroo samples were obtained and used to inoculate tubes of each media (M & SP), but only clover hay plus wheat was used as the substrate. After three days, no protozoa were visible from one animal,

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and low numbers were present in cultures established from the second animal. These were transferred into fresh media with double the mineral concentration, to evaluate the possible effects of osmolarity. A few motile cells were observed on day 6 and all were dead by day 8.

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One last kangaroo sample was obtained, and the ingesta was dried and used as a substrate for the in vitro cultures. All protozoa were dead after three days. Thus, none of the modifications in the

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media used for rumen ciliates was successful in culturing protozoa from the kangaroo.

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A thorough search of the literature revealed that there are some subtle differences between the rumen environment and that in the marsupial forestomach (Dellow 1979, Dellow et

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al. 1988, Hume 1982, Hunton 1982, Moir and Hungate, unpublished results from R. J. Moir). These differences are outlined in Table 1 and were incorporated into the experimental design of

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the present study. Successful culture of these unique protozoa would allow a comparison of their

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Material and Methods

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generation time, pH tolerance and substrate utilization with those of the rumen ciliates

A licence (SF005174) to take wild fauna for scientific purposes was obtained from the Government of Western Australia Department of Conservation and Land Management (under the Wildlife Conservation Act 1950, Regulation 17). This experiment was reviewed and approved by the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Western Australia Animal Experimentation Ethics Committee. Forestomach contents were obtained from seven western grey kangaroos (Macropus fuliginosus), two male (K-1 and K-2) and five female (K-3 to K-7), shot at the Yalanbee Commonwealth Scientific Industrial Research Organization (CSIRO) Research Station near

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Perth, Western Australia. Ingesta was taken from both the sacciform and tubiform regions of the stomach. Media (medium M and medium SP), procedures for preparing media, sterilization,

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inoculation and transfer of cultures have all been described previously (Dehority 1998). All procedures were carried out under a stream of 100% CO2 (Dehority 1998). Rumen fluid or

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kangaroo forestomach fluid for use in media was obtained by filtering contents through a double

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layer of cheesecloth. This material was centrifuged at 1000 x g for 10 min and the supernatant was either added directly into the medium or frozen for use later. Results from preliminary

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studies indicated that using sheep rumen fluid (10%) in the medium allowed equal or better growth than kangaroo forestomach fluid. Unless noted otherwise, normal substrate for the

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protozoa cultures was a suspension containing 1% ground subterranean clover (Trifolium

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subterranean) plus 1.5% finely ground wheat grains (Triticum aestivum). A total of 0.15 ml of

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the suspension were added daily per 10.0 ml culture (0.15 mg clover and 0.225 mg wheat / ml of protozoal culture). In some experiments, a 2% suspension of ground oaten hay was added with

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the normal substrate (0.1 ml) or served as the only substrate (0.2 ml). Immediately after the animal was shot, the stomach was opened and the contents strained through cheesecloth and the strained kangaroo forestomach fluid (KFF) was transferred to a previously warmed thermos. Cultures were started in one of two ways: (1.) In the field, one ml of the KFF was added by syringe through a rubber septum into several Hungate tubes containing 9.0 ml of medium M plus substrate (Hungate tubes are a 16 x 125 mm screw thread style glass tube sealed with a flange type butyl stopper and screw cap, Bellco Glass, Inc., 340 Edrudo Rd., Vineland, NJ 08360). The tubes were placed into a warm insulated container and transported back to the lab. In the lab, five ml of the material in the Hungate tube was inoculated

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anaerobically under 100% CO2 into a conventional 16 x 150 mm culture tube closed with a rubber stopper; (2) In the lab, using anaerobic techniques, cultures were started by adding 0.5 ml of the KFF from the thermos to culture tubes containing 10 ml of medium M.

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Adding hydrogen to gaseous phase

Upon return to the lab, two Hungate tubes per animal (K-1,K-2 and K-3), inoculated in

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the field, were subdivided by transferring 5.0 ml by syringe into two Hungate tubes containing

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5.0 ml of medium M plus substrate. One tube from each pair was kept under 100% CO2 gas and the headspace gas in the second tube was replaced by flushing with a gas mixture containing

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80% H2 – 20% CO2. Increase medium pH to 6.9-7.0 range

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Medium MPH, a modification of medium M in which an equal volume of 12% Na2CO3 was

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substituted for the 6.0% NaHCO3. This was done to raise the initial pH of the medium and

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provide buffering capacity to maintain this higher pH. In order to determine the effectiveness of the MPH medium, cultures were established with sheep protozoa and followed over 19 days.

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After 13 days which included 3 transfers, pH in regular medium M was 6.45, compared to 6.89 in medium MPH. The cultures were transferred and maintained an additional 6 days at which time pH values were 6.41 in medium M and 6.90 in medium MPH. Feed only forage

The normal substrate of 0.15ml of subterranean clover and wheat was replaced with 0.2 ml of 2% oaten hay. Incubate at lower temperature To simulate the lower body temperatures previously observed in the marsupials, in addition to incubation at 39ºC, cultures were also incubated at either 36ºC or 33ºC.

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Counting procedure Protozoa were counted in a Sedgewick-Rafter counting chamber (Thomas Scientific, no. 9851 C20, Swedesboro, NJ) following the procedures described by Dehority (1984, 1998).

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Cryopreservation and recovery

Cryopreservation was accomplished by adding dimethyl sulfoxide (DMSO) to the culture

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to give a final concentration of 4% DMSO and after incubating at 39ºC for 15 min, 1.8 ml

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aliquots were pipetted anaerobically into 2.0 ml plastic screw-cap tubes. The tubes were flushed with CO2 and closed. The tubes were placed into a freezing container containing isopropanol

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(Thermo Scientific Cryogenic freezing container, Cole-Parmer, 625 East Bunker Court, Vernon Hills, IL 60061) and the container placed into an ultra-low freezer ay -72ºC. Procedure for

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revival of cultures was removal from the freezer, placing into the 39ºC water bath for a few

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minutes until thawed and then transferring anaerobically into a 16 x 150 mm culture tube

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containing 5 ml of medium M plus substrate. Silver carbonate impregnation

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Protozoa were stained using the silver carbonate method of Augustin et al. (1984).

Results

Adding H2 to the headspace gas did not appear to benefit the in vitro longevity of kangaroo protozoa. After 14 hours, no viable protozoa could be observed in tubes established with KFF from animals K-2 and K-3. Small numbers of protozoa were still viable after 36 hours in tubes inoculated from kangaroo K-1; however, they had disappeared by 62 hours. Results from incorporating the other three cultural changes into the in vitro cultures are summarized in Table 2. It was of interest that viability of the cultures was markedly improved

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when grown in conventional 16 x 150 mm culture tubes as compared to Hungate tubes used in the previous study of adding H2 to the headspace gas. Of the three different treatments, the only major improvement in survivability was incubation at 36ºC with animal K-2.

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Based on the results above, new cultures were initiated using forestomach contents from four additional kangaroos (K-4 thru K-7). Treatments were: (1) Control- feed 0.15 ml normal

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substrate (CW) and incubate at 36ºC; (2) Feed 0.2 ml of substrate containing 0.5% clover hay,

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0.75% ground wheat and 1.0% oaten hay (CW+OH). The reason for this treatment was to lower the readily available carbohydrate concentration which might slow bacterial growth which

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lowers pH; (3) Incubate at 33ºC to simulate the lower body temperatures previously reported from marsupials. Results of these treatments are summarized Table 3.

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Control cultures

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After 5 days it was observed that one of the K-6 cultures in the control group was growing

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extremely well, but differed from the others in that the total volume was only 5 ml. This suggested that perhaps an increase in level of feed might be advantageous. Thus feed level in

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control cultures was doubled on day 5. However, concentrations were visibly lower than those cultures incubated at 33ºC and the control cultures were shifted to the lower temperature on day 6. Viable cells were still present on day 11, when the experiment had to be terminated because the Fellowship was completed.

Clover hay, wheat and oaten hay (CW+OH ) cultures Concentrations in cultures from K-5 and K-6 were extremely low and they were terminated on day 8. Although there were protozoa in cultures from animals K-4 and K-7 on day 11, numbers were very low. Incubation at 33ºC

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Cultures K-4 and K-7 were growing extremely well and were cryopreserved on day 8. Low numbers of protozoa were still viable in cultures from animals K-5 and K-6 on day 11, when they were terminated.

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Cryopreserved cultures

The cryopreserved cultures were returned to The Ohio State University packed with dry

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ice and stored in an ultra-low temperature freezer (-60ºC). Unfortunately, all attempts to recover

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viable cells were unsuccessful. Molecular data

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All seven kangaroos were positive for protozoa and a pooled ingesta sample was used to prepare a small 18S rRNA gene clone library following previously published methods (Wright et

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al. 2004). The 18S rRNA gene was sequenced for 14 clones, of which 13 were of eukaryotic

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origin. The 13 sequences produced 7 different phylotypes (Table 4) that were assigned to 4

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operational taxanomic units (OTU). OTU-1 appears to be a new species of Bitricha, OTU-2 is a fungal species belonging to the genus Cryptococcus and OTU-3 and OTU-4 may be new species

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of Macropodinium.

The nucleotide sequences reported in this paper have been deposited in the GenBank database under accession numbers KJ476735 to KJ476741 Microscopic study of culture from K-2, grown 27 days in vitro at 36ºC The culture which survived 28 days (Table 2) was sampled on day 27 and the sample fixed with an equal volume of formaldehyde. Two species of Bitricha were observed. The predominant species was quite different from previously described species and based on the molecular data may represent a new species (Figure 1). Figure 2 is focused on the anterior end of the cell showing the unique cilia pattern for the genus Bitricha. The second species was present

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in very low numbers and identified as Bitricha oblata. To date, only two species of Bitricha have been described, Bitricha oblata and Bitricha tasmaniensis (Cameron et al. 2000), both of which were observed in forestomach contents from several of the kangaroos in this study. Table 5

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compares measurements of these two species as given in their original description and as

measured in the present samples, with the measurements for the unknown species cultured in this

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study.

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Concentrations of protozoa were determined in forestomach contents from two kangaroo (K-1 and K-2) and are shown in Table 6. Concentrations of protozoa in culture are shown after 8,

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11 and 27 days. Cultures were terminated at the indicated times because the Fellowship ended and the author returned home. Those cultures started from animals K-5, K-6 and K-7 all

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contained M. yalanbense (Fig. 3). Mean size of 10 cells: L=57.0; W=30.8; L/W=1.85.

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However, many of the cells appeared empty and probably would not survive.

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As mentioned above, culture K-2 at 27 days primarily contained an unidentified species of Bitricha (83.3%) and Bitricha oblata (16.7%). Unfortunately, the starting sample of

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forestomach contents from animal K-2 did not contain any Macropodinium sp. The concentration of protozoa in culture at 27 days was only 5.3 % of that present in vivo.

Discussion

The results in this preliminary study would indicate that body temperature might explain

the previous inability to establish kangaroo forestomach ciliates in the rumen of ten defaunated sheep (Baker et al. 1995). Six of the ten defaunated sheep inoculated with kangaroo forestomach digesta were free of protozoa 18 h after inoculation and no ciliate protozoa from kangaroos were observed in any of the sheep after 14 days. 10 Page 10 of 24

In general, none of the modifications in the procedure for in vitro cultivation of protozoa from the kangaroo forestomach permitted growth of all species in very high concentrations. Lowering the incubation temperature did allow continued growth of several species beyond that

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observed at 39ºC. On day 11, many of the surviving protozoa belonged to the genus

Macropodinium; however, most of the cells appeared empty. In contrast, the two species which

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grew for 28 days in vitro (those entirely covered with cilia) became opaque after feeding and

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transparent prior to the next feeding, similar to the cycle observed in the rumen species Isotricha and Dasytricha (Dehority 2003).

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The predominant Bitricha species which grew in culture for 28 days appears to differ from the two previously described species in this genus, based on size and L/W ratio. In support,

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molecular data indicated the presence of an additional unknown species of Bitricha in the

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combined sample of forestomach contents from all 7 kangaroo.

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Based on these preliminary results, further studies on in vitro culture of kangaroo protozoa should probably start by determining the optimum lower temperature for growth, i.e.,

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between 33 and 39º C. With that established, re-examination of the parameters tested in this study, plus possible modifications in the medium composition might provide changes needed for continuous culture of the kangaroo ciliates in vitro. In an earlier study on the in vitro culture of rumen protozoa, growth response to the reduction of the substrate particle size was reduced for Entodinium caudatum and Epidinium caudatum. However, in contrast, growth of Eudiplodinium impalae was increased (Dehority 2010). This parameter should also be studied in future work on kangaroo ciliates. These studies were carried out while Dr. Dehority was on a two month CSIRO Sir Frederick McMaster Fellowship in the co-author’s laboratory (CSIRO Livestock Industries,

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Floreat, Western Australia). For this reason, at the end of the two months, viable cultures had to be terminated. Several cultures were cryopreserved or fixed with formaldehyde and transported

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back to Dr. Dehority’s laboratory in Ohio for microscopic study.

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Acknowledgements

Salary and research support in Ohio were provided by State and Federal funds

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appropriated to the Ohio Agricultural Research and Development Center, The Ohio State

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Fellowship.

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University. In Australia, Dr. Dehority was supported by a CSIRO Sir Frederick McMaster

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References Augustin, H., Foissner, W. Adams, H., 1984. An improved pyridinated silver carbonate method which needs few specimen and yields permanent slides of impregnated ciliates (Protozoa,

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Ciliophora). Mikroskopie 41, 134-137.

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Baker, S. K., Dehority, B. A., Chamberlain, N. L., Purser D. B., 1995. Inability of protozoa from the kangaroo forestomach to establish in the rumen of sheep. Ann Zootech 44, Suppl. 143.

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Cameron, S. L., O’Donoghue, P. J., 2002. The ultrastructure of Amylovorax dehorityi comb. Nov. and erection of the family Amylovoracidae fam. Nov. (Ciliophora Trichostomatia).

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Europ. J. Protistol. 38, 29-44.

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Cameron, S. L., O’Donoghue, P. J., 2003. Trichostome ciliates from Australian marsupials. II. Polycosta gen. nov. (litostomata: Polycostidae fam. Nov.). Europ. J. Protistol. 39, 83-100.

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Cameron, S. L., O’Donoghue, P. J., Adlard, R. D., 2000. Novel isotrichid ciliates endosymbiotic

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in Australian macropodid marsupials. Systematic Parasitology. 46, 45-57. Dehority, B. A., 1996. A new family of entodiniomorph protozoa from the marsupial

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forestomach with descriptions of a new genus and five new species. J. Eukaryot. Microbiol. 43, 285-295.

Dehority, B. A., 1984. Evaluation of subsampling and fixation procedures used for counting rumen protozoa. Appl. Environ. Microbiol. 48, 182-185. Dehority, B. A., 1998. Generation times of Epidinium caudatum and Entodinium caudatum determined in vitro by transferring at various time intervals. J. Anim. Sci.76, 1189-1196. Dehority, B. A., 2003. Rumen Microbiology. Nottingham University Press. Nottingham, UK. Dellow, D. W., 1979. Physiology of digestion in the macropodine marsupials. PhD Thesis, The University of New England, Armidale, N.S.W., Australia.

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Dellow, D. W., Hume, I. D., Clarke, R. T. J., Bauchop, T., 1988. Microbial activity in the forestomach of free-living macropodid marsupials: comparisons with laboratory studies. Aust. J. Zool. 36, 383-395.

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Hume, I. D. 1982. Digestive Physiology and Nutrition of Marsupials. Cambridge University Press, Cambridge, UK.

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Hunton, J. R., 1982. The microbial ecology of the quokka (Setonix brachyurus: Quoy and

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Gaimard) forestomach. BS Thesis. University of Western Australia, Perth, Australia. Wright, A.-D.G., Williams, A.J., Winder, B., Christophersen, C.T., Rodgers, S.L., Smith, K.D.

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2004. Molecular diversity of rumen methanogens from sheep in Western Australia. Appl.

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te

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Environ. Microbiol. 70, 1263-1270.

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Figures Fig. 1. Predominant species of kangaroo forestomach protozoa cultured in vitro after 28 days.

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Stained with silver carbonate. Bar = 20 µm.

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Fig. 2. Enlargment of cell in Fig. 1 showing orientation of cilia in pattern of the genus Bitricha. Stained with silver carbonate. Bar = 5 µm.

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Fig. 3. Macropodinium yalanbense in culture after 11 days (from animal K-7). Stained with

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methylene blue. Bar = 20 µm.

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Tammar

Eastern grey

pademelom

Wallaby

kangaroo

Quokka

Cattle

pH

7.0-7.4

7.3-7.4

6.4-6.9

5.8-8.0

5.5-7.0

VFA, mM

99-165

72-109

77-136

101-117

94-137

% Acetic acid

74

75

71

87

49-77

% H2 in gasb

-

-

1.1-2.8

12-18

0.2

11.8-17.1

-

16.1-18.7

-

10-13

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% DM

Sheep &

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Red-necked

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Table 1. Physical parameters in the marsupial forestomach environmenta.

Temperature,ºC --------------------------- 33.8-36.4 -------------------------------------

Dellow 1979 (Physiology of digestion in the macropodine marsupials. PhD Thesis, The

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a

38-40

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University of New England, Armidale, N.S.W., Australia), Hume, 1982, Hunton 1982 (The

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microbial ecology of the quokka (Setonix brachyurus: Quoy and Gaimard) forestomach. BS Thesis. University of Western Australia, Perth, Australia), Dellow et al. 1988, Moir and Hungate

b

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(unpublished results from R.J. Moir).

In additional reports from free-living red-necked wallaby and the wallaroo, H2 concentrations

ranged from 0.8-2.2%, and from 9.8-11% in the swamp wallaby.

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Table 2. Effect of several different changes in culture parameters on the in vitro survivability of

Survival time, days K-2

7 to < 10

7 to < 10

0.6 to < 1.5

< 0.6

High pHc

< 0.6

< 0.6

0.6 to < 1.5

Incubate at 36º C

6<7

28+d

0.6 to < 1.5

Oaten hayb

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Nonea

K-3

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K-1

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Culture change

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ciliate protozoa from the kangaroo forestomach.

a

< 0.6

0.6 to < 1.5

Medium M, 100% CO2, clover hay plus ground wheat substrate, incubated at 39ºC. Only substrate added, 0.2 ml of a 2% suspension of oaten hay.

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b c

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Cultures were still viable in low numbers, but had to be terminated at that time.

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d

d

pH measured after 86 hours, values ranged from 6.82-6.87.

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Table 3. Effect of lower temperature, increased fiber content and increased substrate

Days

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concentration on survivability of kangaroo forestomach ciliate protozoaa.

K-5

K-6

K-7

36ºC (6d)→ 33ºC (CW)b

11

8d

<8

11d

36ºC (CW+OH)c

11

<8

33ºC (CW)

8d

11

<8

11

11

8d

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a

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K-4

b

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Experiment terminated on day 11(Fellowship time completed).

CW = 1.0% clover hay + 1.5% ground wheat. Increased to 0.3 ml on day 5.

c

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d

Culture cryopreserved on that day.

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d

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CW+OH = 0.5% clover hay + 0.75% ground wheat + 1.0% oaten hay.

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Table 4. Full-length 18S rRNA clone sequence similarity to validly described eukaryotes OTUa

18S Clonesb

Phylotype

Accession

Nearest Taxon

% Seq

No. #

ID

1

KP04, KP05, KP06

KJ476735

Bitricha tasmaniensis

98.5

2

2

KP07, KP70, KP80

KJ476736

Cryptococcus cellulolyticus

99.8

3

3

KP13, KP15

KJ476737

Macropodinium ennuensis

97.3c

3

4

KP18

KJ476738

Macropodinium ennuensis

97.3

3

5

KP31, KP32

KJ476740

Macropodinium ennuensis

97.4

3

6

KP41

KJ476741

Macropodinium ennuensis

97.5

4

7

KP27

KJ476739

Macropodinium ennuensis

96.4c

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cr

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1

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a. OTU-1 is a putative new species of Bitrichia; OTU-2 is a fungus; OTU-3 is a

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Macropodinium.

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putative new species of Macropodinium; and OTU-4 is a putative new species of

b. More than one clone per row indicates identical sequences. c. Distance between Macropodinium ennuensis and Macropodinium yalanbense is 97.9% which is greater than the distance between OTU-3 and Macropodinium

ennuensis, between OTU-4 and Macropodinium ennuensis, and between OTU-3 and OTU-4 (97.8%).

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Table 5. Dimensions of Bitricha species in µm. Bitricha spa.

Bitricha tasmanienis

Bitricha oblata

Observedb

Descriptionc

Observedb

Length (L)

113.1

92.1

103.3

59

79

Width (W)

34.2

40.4

55.3

36

39

L/W

3.31

2.29

1.7

Ma (L)d

26.6

22.6

24.3

b

cr -

2.0 -

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Bitricha species which grew in the in vitro cultures.

1.6

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a

Descriptionc

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Observedb

Measurements taken in the present study.

c

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d

Macronucleus length.

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Measurements given in the original species description.

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Table 6. Protozoa concentrations. Days in

or

temp.

culture

Culture

ºC

Species

Protozoa/ml X103

Forestomach

-

-

Macropodinium sp.

16.5

“

“

-

-

Othera

4

K-2

Forestomach

-

_

Macropodinium sp.

0

“

“

-

-

Other

4.5

K-5

In vitro

36 (6d)→33

8

M. yalanbense

0.28

Bitricha sp.

0.14

8

M. yalanbense

0.82

11

M. yalanbense

1.79

27

Bitricha oblata

0.04

Bitricha sp.

0.2

36

K-7

In vitro

36 (6d)→33

Ac ce p In vitro

36

us

an

M

d

In vitro

te

K-6

cr

K-1

K-2

a

Sample

ip t

Incubation

Animal

Not identified, but most cells covered in cilia, included Bitricha.

21 Page 21 of 24

Ac ce p

te

d

M

an

us

cr

ip t

Figure

Page 22 of 24

Ac ce p

te

d

M

an

us

cr

ip t

Figure

Page 23 of 24

Ac ce p

te

d

M

an

us

cr

ip t

Figure

Page 24 of 24