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Pleomorphism as a Sequential Phenotypic Manifestation in Blood Flagellates of Different Vertebrate Groups

Neelima Gupta* and Gupta DK

Neelima Gupta, Centre of Excellence Laboratory, Department of Animal Science, MJP Rohilkhand University, Bareilly, Uttar Pradesh, India

Gupta DK, Department Biotechnology, Bareilly College, Bareilly, Uttar Pradesh, India

*Corresponding Author:
Neelima Gupta
Centre of Excellence Laboratory, Department of Animal Science, MJP Rohilkhand University, Bareilly 243006, Uttar Pradesh, India
E-mail: [email protected]

Received Date: October 21, 2016; Accepted Date: October 24, 2016; Published Date: October 31, 2016

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Abstract

Objective: The objective of the research work was to observe pleomorphism (more than one morphological phenotypic expression of a species) in flagellate parasites (Trypanosoma and Trypanoplasma). Methods: The blood of selected vertebrate hosts (fishes, amphibians, reptiles and mammals) was scanned for haemoflagellate infectivity by examination of fresh blood (hanging drop preparations and haematocrit) and stained smears. The flagellate parasites (Trypanosoma and Trypanoplasma) were morpho-taxonomically identified and the phenotypic manifestation expressed as pleomorphism recorded for each species. Outcomes: Twenty-four species of trypanosomes from 12 fish hosts were discovered (12 species monomorphic, 6 each dimorphic and polymorphic). From amphibians and mammals, only monomorphic forms were encountered. The reptiles (Hemidactylus flaviviridis, Calotes versicolor) were not susceptible to trypanosome infection. Conclusions: Host-wise analysis indicated that Heteropneustes fossilis was most susceptible to trypanosomes. Trypanoplasms from fish too exhibited pleomorphism (6 species monomorphic, 2 each dimorphic and polymorphic).

Keywords

Blood; flagellate; pleomorphism; Trypanosoma; Trypanoplasma

Introduction

Pleomorphism is the ability of organisms to alter their shape or size. It is one of the most remarkable feature of trypanosomes and is also apparent in trypanoplasms. Pleomorphism is the sequential phenotypic manifestation of a single genotype as it appears in the trypomastigote stage in the vertebrate host. The phenomenon is triggered by changes in the host antibody spectrum. There are a few fish trypanosomes which seem to be virtually monomorphic, displaying only variability in size and/or in length/width ratio. The developmental stages in their vertebrate host, if any, seem to be limited to other than morphological manifestations.

The blood flagellates, Trypanosoma and Trypanoplasma are widely reported to dwell in the blood of vertebrate hosts (Gupta, 1994., Woo, 2003., Gupta, 2006) but studies pertaining to the expression of pleomorphism are rare. The present study examines the morphology of the trypanosomes and trypanoplasms infecting different vertebrate hosts from Uttar Pradesh, India. Pleomorphism in these parasites based on intra specific morpho-variants is reported.

Materials and Methods

Live host samples were obtained from the local fishing grounds (fish), ponds or amphibious habitats (amphibians), and domestic habitats, godowns and laboratories (lizards and rats). They were acclimatized to laboratory conditions and maintained in the Animal House of the Department of Animal Science under adequate conditions of food and water. Diagnosis of infection of blood parasites was done by hanging drop preparation or microhaematocrit (4000 rpm) and parasites observed in the buffy layer. The parasites were identified by preparing at least 4-6 films of fresh blood on methanol cleaned slides by aseptic microtechniques. The films were air dried, fixed in methanol and stained in Giemsa/Leishman (Qualigens) and buffer (Hale, 1965) in the ratio of 1:7, mixed uniformly by a thin hair brush, stained for 40-50 mins and mounted in DPX (Gupta, 1986 and Gupta, 2010). The microphotographs were taken under oil immersion in LEICA-DMLB microscope. The forms were drawn to scale, measured, tabulated and morphotaxonomically identified.

Results

A. Trypanosome Infectivity and Pleomorphism

Flagellates discovered from fishes up to mammals could range from monomorphic to polymorphic forms. The facts given below are the compiled information of the entire work.

Twenty-four species of trypanosomes from 12 fish hosts have been discovered. Out of these, 12 species were monomorphic, 6 dimorphic and 6 polymorphic. Out of these, from Heteropneustes fossilis, 2 monomorphic (T. kargenensis Gupta and Gupta, 1994., T. karelensis Gupta et al. 2000), 2 dimorphic (T. artii Gupta et al. 2002., T. heteropneusti Gupta et al. 2006) and one polymorphic (T. singhii Gupta and Jairajpuri, 1981) species were discovered. From Channa punctatus, one monomorphic (T. rohilkhandae Gupta and Saraswat, 1991), 2 dimorphic (T. aligaricus Gupta and Jairajpuri, 1982., T. saulii Gupta et al. 2006) and one polymorphic (T. bareilliana Gupta et al. 1987) were discovered. From Colisa (Trichogaster) fasciata, 2 monomorphic (T. trichogasteri Gupta and Jairajpuri, 1981., T. piscidium Gupta et al. 2003), one new variety (T. trichogasteri var. nov. fasciatae ) and one polymorphic (T. colisi Gupta, 1986) species were discovered. From Mystus vittatus, one monomorphic (T. bagroides Gupta and Gupta, 1996) and one dimorphic (T. tengari n.sp., Gupta et al. 2015) whereas from Catla catla, one monomorphic (T. monomorpha Gupta and Jairajpuri, 1985) and one new variety (T. monomorpha var. nov. catlae Gupta et al., 2000) were discovered. Amongst the remaining species, 3 of them were monomorphic (T. notopteri Gupta and Jairajpuri, 1985 from Notopterus notopterus; T. ritae Gupta and Yadav, 1989 from Rita rita; T. danilewskyi Laveran and Mesnil, 1904 from Cyprinus carpio), one dimorphic (T. batrachi Gupta and Jairajpuri, 1983 from Clarias batrachus) and three polymorphic (T. attii Gupta and Jairajpuri, 1981 from Wallago attu; T. barbi Gupta et al. 1987 from Barbus barbus; T. ticti Gupta et al. 1998 from Puntius ticto) species have been put on record (Table 1).

In addition to the above, representative vertebrates from Amphibia (Rana tigrina, Bufo melanostictus), Reptilia (Hemidactylus flaviviridis, Calotes versicolor), Aves (Columba livia) and Mammals (Rattus rattus from India and Clethrionomys glareolus, Apodemus flavicollis and Microtus oceanus from Poland) were also examined for flagellate parasites. Amongst these, the infected hosts were Bufo melanostictus (T. bisalpurensis n. sp. unpublished), Rana tigrina (T. rotatorium Mayer, 1843), Rattus rattus [Trypanosoma (Herpetosoma) lewisi Laveran and Mesnil, 1901], Clethrionomys glareolus [Trypanosoma (Herpetosoma) evotomys Molyneux, 1969], Apodemus flavicollis [Trypanosoma (Herpetosoma) grosi kosewiense Karbowiak and Wita, 2004] and Microtus oeconomus [Trypanosoma (Herpetosoma) microti Laveran and Pettitt, 1909] (Table 1). It was interesting to conclude that amongst fishes, monomorphic, dimorphic as well as polymorphic forms were observed but from amphibians and mammals, only monomorphic forms were encountered. The reptiles (Hemidactylus flaviviridis, Calotes versicolor), however, were not prone to trypanosome infection. Host-wise analysis indicated that Heteropneustes fossilis was most susceptible to trypanosomes (Table 2).

Fish Hosts
S.No. Parasites Hosts Forms Authors
1. T. trichogasteri Trichogasterfasciata Monomorphic Gupta andJairajpuri, 1981
2. T. singhii Heteropneustesfossilis Polymorphic Gupta andJairajpuri, 1981
3. T. attii Wallagoattu Polymorphic Gupta andJairajpuri, 1981
4. T. aligaricus Ophiocephaluspunctatus Dimorphic Gupta andJairajpuri, 1982
5. T. batrachi Clariasbatrachus Dimorphic Gupta andJairajpuri, 1983
6. T. monomorpha Catlacatla Monomorphic Gupta andJairajpuri, 1985
7. T. notopteri Notopterusnotopterus Monomorphic Gupta andJairajpuri, 1985
8. T. colisi Colisafasciata Polymorphic Gupta, 1986
9. T. bareilliana Channapunctatus Polymorphic Gupta et al.,1987
10. T. barbi Barbusbarbus Polymorphic Gupta et al.,1987
11. T. ritae Rita rita Monomorphic Gupta andYadav, 1989
12. T. rohilkhandae Channapunctatus Monomorphic Gupta andSaraswat, 1991
13. T. kargenensis Heteropneustesfossilis Monomorphic Gupta and Gupta, 1994
14. T. danilewskyiLaveran and Mesnil, 1904 Cyprinuscarpio Monomorphic Gupta andPilarczyk, 1994
15. T. bagroides Mystusvittatus Monomorphic Gupta and Gupta, 1996
16. T. trichogasterivar.nov. fasciatae Colisafasciatus Monomorphic Gupta et al.,1998
17. T. tictin.sp. Puntiusticto Polymorphic Gupta et al.,1998
18. T. monomorphavar.nov. catlae Catlacatla Monomorphic Gupta et al.,2000
19. T. karelensis Heteropneustesfossilis Monomorphic Gupta et al.,2000
20. T. artii Heteropneustesfossilis Dimorphic Gupta et al.,2002
21. T. piscidium Colisafasciata Monomorphic Gupta et al.,2003
22. T. saulii Channapunctatus Dimorphic Gupta et al.,2006
23. T. heteropneusti Heteropneustesfossilis Dimorphic Gupta et al.,2006
24. T. tengari Mystusvittatus Dimorphic Gupta et al.,2015
Amphibian hosts
1. T. bisalpurensisn.sp. Bufomelanostictus Monomorphic Unpublished
2. T. rotatorium Ranatigrina Monomorphic Mayer, 1843
Mammalian hosts
  1. Trypanosoma(Herpetosoma)lewisi Rattusrattus Monomorphic Laveran and Mesnil, 1901
2. Trypanosoma(Herpetosoma)evotomys Clethrionomysglareolus   Monomorphic Molyneux, 1969
  3. Trypanosoma(Herpetosoma)grosikosewiense Apodemusflavicollis Monomorphic KarbowiakandWita, 2004
  4. Trypanosoma(Herpetosoma) microti Microtusoeconomus Monomorphic LaveranandPettitt, 1909

Table 1: List of Trypanosoma Species.

Fish Hosts
Hosts Parasites Forms Authors
  Heteropneustesfossilis T. singhii Polymorphic Gupta andJairajpuri, 1981
T. kargenensis Monomorphic Gupta and Gupta, 1994
T. karelensis Monomorphic Gupta et al., 2000
T. artii Dimorphic Gupta et al.,2002
T. heteropneusti Dimorphic Gupta et al.,2006
  Channapunctatus T. aligaricus Dimorphic Gupta andJairajpuri, 1982
T. bareilliana Polymorphic Gupta et al.,1987
T. rohilkhandae Monomorphic Gupta andSaraswat, 1991
T. saulii Dimorphic Gupta et al.,2006
  Colisafasciata T. trichogasteri Monomorphic Gupta andJairajpuri, 1981
T. colisi Polymorphic Gupta, 1986
T. trichogasteri var.
 nov. fasciatae
Monomorphic Gupta et al.,1998
T. piscidium Monomorphic Gupta et al.,2003
Mystusvittatus T. bagroides Monomorphic Gupta and Gupta, 1996
T. tengari Dimorphic Gupta et al.,2015
  Catlacatla T. monomorpha Monomorphic Gupta andJairajpuri, 1985
T. monomorpha var.
nov.catlae
Monomorphic Gupta et al.,2000
Wallagoattu T. attii Polymorphic Gupta andJairajpuri, 1981
Clariasbatrachus T. batrachi Dimorphic Gupta andJairajpuri, 1983
Notopterusnotopterus T. notopteri Monomorphic Gupta andJairajpuri, 1985
Barbusbarbus T. barbi Polymorphic Gupta et al.,1987
Rita rita T. ritae Monomorphic Gupta andYadav, 1989
Cyprinuscarpio T. danilewskyi Laveran andMesnil, 1904 Monomorphic Gupta andPilarczyk, 1994
Puntiusticto T. ticti Polymorphic Gupta et al.,1998
  Amphibian hosts
Bufomelanostictus T. bisalpurensisn.sp. Monomorphic Unpublished
Ranatigrina T. rotatorium Monomorphic Mayer, 1843
  Mammalian hosts
Rattusrattus Trypanosoma
(Herpetosoma)lewisi
Monomorphic Laveran andMesnil,
1901
Clethrionomysglareolus Trypanosoma
(Herpetosoma)evotomys
Monomorphic Molyneux, 1969
Apodemusflavicollis Trypanosoma
(Herpetosoma)grosi
kosewiense
Monomorphic Karbowiak
andWita, 2004
Microtusoeconomus Trypanosoma(Herpetosoma)microti Monomorphic Laveran andPettitt,
1909.

Table 2: Host-Wise List of Trypanosomes Discovered

B. Trypanoplasm Infectivity and Pleomorphism

Trypanoplasms are characteristic of fishes. Ten species of this parasite have been recorded during this entire period. These parasites too exhibited pleomorphism: 6 forms being monomorphic, 2 dimorphic and 2 polymorphic. Mystus vittatus was infected with T. guptii Gupta and Gupta, 1987 (polymorphic) and T. tengari Gupta et al. 1988 (monomorphic), Cyprinus carpio with T. golysiana Gupta and Pilarczyk, 1994 (monomorphic) and T. polygolysiana Gupta and Pilarczyk, 1994 (polymorphic); Clarias batrachus with T. (Cryptobia) maguri Gupta and Gupta, 1997 (dimorphic), T. haematalis Gupta and Gupta, 2010 (monomorphic) and T. monomorpha. n.sp (unpublished) (monomorphic). Puntius ticto was infected with T. cyprinoides Gupta et al. 1998 (dimorphic) and a new variety, T. sarnae var. nov. sophorae Gupta and Gupta, 2014 and Catla catla with T. catli Gupta et al. 1999 (monomorphic) (Table 3). Host-wise analysis for trypanoplasm infectivity indicated that Clarias batrachus was the most prone species (Table 4).

S.No. Parasites Hosts Forms Authors
1. T. guptii Mystusvittatus Polymorphic Gupta and Gupta, 1987
2. T. tengari Mystusvittatus Monomorphic Gupta et al.,1988
3. T. golysiana Cyprinuscarpio Monomorphic Gupta and Pilarczyk, 1994
4. T. polygolysiana Cyprinuscarpio Polymorphic Gupta and Pilarczyk, 1994
5. T. (Cryptobia) maguri Clariasbatrachus Dimorphic Gupta and Gupta, 1997
6. T. cyprinoides Puntiusticto Dimorphic Gupta et al.,1998
7. T. catli Catlacatla Monomorphic Gupta et al.,1999
8. T. haematalis Clariasbatrachus Monomorphic Gupta and Gupta, 2010
9. T. monomorpha. n.sp. Clariasbatrachus Monomorphic Unpublished
10. T. sarnae var. nov.sophorae Puntiusticto Monomorphic Gupta and Gupta, 2014

Table 3: List of Trypanoplasma Discovered from Fish Hosts

S.No. Hosts Parasistes Forms Authors
1. Mystusvittatus T. guptii Polymorphic Gupta and Gupta, 1987
2. T. tengari Monomorphic Gupta et al.,1988
3. Cyprinuscarpio T. golysiana Monomorphic Gupta andPilarczyk, 1994
4. T. polygolysiana Polymorphic Gupta andPilarczyk, 1994
5.   Clariasbatrachus T. (Cryptobia) maguri Dimorphic Gupta and Gupta, 1997
6. T. haematalis Monomorphic Gupta and Gupta, 2010
7. T.monomorpha. n.sp. Monomorphic Unpublished
8. Puntiusticto T. cyprinoides Dimorphic Gupta et al.,1998
9. Catlacatla T. catli Monomorphic Gupta et al.,1999
10. Puntiusticto T. sarnaevar. nov.sophorae Monomorphic Gupta et al., 2014

Table 4: Host-Wise List of Trypanoplasms Discovered from Fish Hosts

Discussion

It has been observed in the past that during the course of infection, most fish trypanosomes display forms successively differing conspicuously in their shape and size; they were designated by early authors (Laveran, 1912) as the pleomorphic forms, “var. parva ” or “var. magna ” and sometimes these forms were erroneously credited with separate taxonomic status (Fantham, 1919), as even reported in later times (Bauer, 1984). However, it soon became clear that these are but extremes in the sequence of forms occurring during the course of infection with one species (Minchin, 1909., Briendl, 1915., Laird, 1951., Khan, 1976). Out of the large number of piscine Trypanosomes, only in a few has the full sequence been established.

In such pleomorphic species, the first trypanosomes to appear in the blood of the infected host are the “young” forms-rather small and slender with a few, shallow waves of the undulating membrane and nucleus revealing no distinct karyosome. After some time, intermediate forms appear until, eventually, “adult” forms prevail to persist during the chronic phase of infection. They are large, stout, sometimes very wide, and usually have a richly spiraling undulating membrane with numerous bends, a nucleus with a prominent karyosome and sub-surface striation. They often have more chromatic granules in their cytoplasm and free flagellum is shorter than in the young forms. Depending on the phase of infection at which the fish is examined, one finds young, slender or large adult forms only, or intermediate ones. A mixture of forms is probably the result of sequential leech feeds.

The existence of a sequence of different morphological types of trypanoplasms during the course of infection in carps was firmly established early (Keysselitz, 1906). Although he misinterpreted numerous stages both in the blood and in the leech as ‘gametes’, his reproduction of polymorphism is the most complete to be found in existing reports. The sequence of stages was confirmed in goldfish (Robertson, 1911), in loaches (Briendl, 1915) and in tench (Kipp, 1968) trypanoplasms. Trypanoplasma catostomi in parasite-free fingerlings of the host fish were noted (Bower, 1977) and the authors observed a remarkable polymorphism including the size as well as differing body structure coupled with the progress of the infection originally started by the introduction of a single flagellate into the host. In view of the paucity of information, sequential pleomorphism in blood-stream stages of trypanoplasms may or may not be a generally distributed phenomenon (Lom, 1979).

Pleomorphism is expressed in the following features:

1. Size changes.

2. Changes in length/width ratio i.e. slender or broad forms.

3. The number, width and depth of the waves of the undulating membrane.

4. The presence or absence of a distinct karyosome in the nucleus by light microscopy.

5. Presence and number of stainable cytoplasmic granules.

6. Length of the free end of the flagellum.

7. Distance of the kinetoplast from the posterior end.

8. Shifts in the position of the nucleus in the body.

9. Presence of subsurface pellicular striation (“myonemes”) in stained preparations by light microscopy (longitudinal or spirally oriented striations, probably sub-pellicularly located ribbons of mitochondrial systems.

Separate morphological stages may be defined by the combination of the above characters; marked changes of points 1 to 5 are proper to truly polymorphic species while changes in points 1, 2, 6, 7, 8, 9 may also be found in “monomorphic” species.

Pleomorphism in Trypanosoma is well marked in having a markedly variegated shape and five different stages in varying combinations at various periods of the digenetic life cycle (vertebrate and invertebrate hosts) may occur (trypomastigote, amastigote, promastigote, sphaeromastigote and metacyclic stages). However, trypomastigotes are the predominant stages reported from fish blood, but in the vertebrate blood too, pleomorphism may be visible as seen above.

Pleomorphism in fish trypanosomes has been reported by some authors (Becker 1979 Joshi 1979) without assigning any names whereas others have given specific names to the various forms. ‘Large’ and ‘small’ forms of T. remarki (Laveran, 1907); Type I, II and III (Tanabe, 1925); small, medium and large (Dutton 1907); small and large forms of T. striati (Qadri, 1955); large and stumpy forms of T. batrachi (Qadri, 1962); slender and broad forms of T. occidentalis (Becker, 1967); short, elongated and stumpy forms of T. aori (Joshi, 1982) have been distinguished in different species of Trypanosoma by various authors.

The form that predominates depends on the course of infection, which again in its turn is subject to many factors. Although this time sequence of morphs has been verified in some species only (e.g. T. barbatulae Breindl, 1915., T. murmanensis Khan, 1976), it most probably holds true for all of the polymorphic species. The most marked examples of pleomorphism can be drawn from among marine species, e.g. those from skates-T. giganteum, T. rajae, T. gargantua. The freshwater examples are T. granulosum, T. percae and T. remarki.

In fish blood flagellates, a condition similar to trypanosomatids of other host groups exists. Pleomorphism is presumed to be sequential and is regarded to be a phenotypic expression of the genotype (Lumsden, 1976). However, as aptly stated (Bardsley, 1973), only observation of the parasite in its vertebrate host when it is subjected to a complete variety of naturally-occurring conditions and factors can reveal whether it really is monomorphic. In experimental transfers among various hosts (carps, goldfish, gudgeon, pike, Barbus conchonius), varying temperature regimes or study of various stages of infection failed to reveal any true pleomorphism (Lom, 1979).

Bloodstream forms of the initial stages of infection are always rather small and of a regular shape (young stages have commalike or crescent-like shapes). In more advanced infections, bigger, very irregular forms prevail, reflecting in their shape wave-like undulatory movements of the whole body. In the chronic, often long lasting, period of infection, the number of trypanoplasms was reduced; assuming a large size and an irregular shape, sometimes with numerous large bends of the undulating membrane; there was a nucleus with a conspicuous endosome not seen in young stages (Lom, 1979). The length of flagellum relative to the body length may also greatly vary during the cycle, as well as the number of stainable granules in the body.

Given the extreme metaboly of the cell shape, it is difficult to characterize the shape differences in young and advanced infection stages in more precise terms but they are quite conspicuous as are the differences in size. For example, in Trypanoplasma sp. from pikes (T. guerneyorum), the average size of a young form is 20 X 4 μm (excluding flagella), while in the chronic stage form it is 38 × 9 μm.

The large forms of trypanosomes and trypanoplasms found in late infection in the circulating blood were considered to be “adult” forms and were conveniently used as reference stages for taxonomic comparisons (Lom, 1979). While it seems certain that Trypanosoma murmanensis is competent to initiate growth in the vector (Khan, 1978), there is no evidence as to whether it is this or some cryptic, tissue-inhabiting, persisting, stage which is responsible for relapses of parasitaemia.

Conclusion

In this report we describe the morphological polymorphism of trypanosomes and trypanoplasms, which includes the different trypomastigote phenotypes from the blood of different vertebrates of Uttar Pradesh, India.

The survey indicates that pleomorphism amongst fish hosts appears to be a regular feature. However, amongst amphibian and mammalian hosts, monomorphism was dominant suggestive of chronic trypanosomiasis. But even in fish hosts, it is safe to consider that monomorphism dominates (12 species) as compared to dimorphism (6 species) and polymorphism (6 species) as visualized above.

Acknowledgment

Thanks are expressed to the Uttar Pradesh Council of Agricultural Research (UPCAR), Lucknow for providing financial assistance by awarding a major research project.

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