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(ᖪ 24: 137-145 (2004ٿέ៉ Formosan Entomol. 24: 137-145 (2004)

Prevalence and Observation of Intestine-Dwelling Gregarines in the corallinus (: Pachybolidae) Collected from Shoushan, Kaohsiung, Taiwan

Wei-Luen Chang, Chih-Ya Yang, Ya-Chi Huang and David Chao* Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung Ting-Wen Chen Department of Biology, National Cheng Kung University, Tainan, Taiwan, R.O.C.

ABSTRACT

The intestine of a Formosan millipede , Trigoniulus corallinus, was noted to be heavily infected with gregarine parasites. used in this study were randomly collected from Shoushan, Kaohsiung from April 1999 to October 2002. Among 63 millipedes examined, our results showed that the total prevalence rate for cephaline gregarines was 87.30%. The infection in the male millipedes (91.67%) was higher than that in the females (84.67%). In some cases, more than two species of different gregarines were found to have simultaneously parasitized a single millipede. Distribution of gregarines was more concentrated in the anterior and middle parts of the intestine than in the posterior part. The predominant form observed in the millipede’s intestine was free sporadins. The shape, size, and movement of these gregarines are described. This is the first report of the occurrence of gregarines as intestinal parasites in T. corallinus in Taiwan.

Key words: Trigoniulus corallinus, millipede, gregarine

Introduction studying physiology (Marcus et al., 1987). However, their own phy- Millipedes are a major group of siological conditions might be distorted if detritivores in tropical and temperate they were to be infected by gregarine broadleaf forests. They are important in parasites. nature as they play the ecological role of Gregarines are protozoan parasites deposit feeders that enhance the rate of found in the intestinal tracts of many decomposition for other smaller deposit invertebrates, especially and feeders as well as decomposers (Hopkin et annelids, which often show extremely al., 1985; Price, 1988). They are also heavy infections (Manwell, 1977). ideal models in the laboratory for Although unimportant economically as

*Correspondence address e-mail: [email protected] ! Prevalence and Observation of Intestine-Dwelling Gregarines in the millipede 137 parasites, their generalized life cycle to these. Since there are many species of makes them significant objects for study millipedes as well as gregarines, the as aids to understanding the important present study focuses on the gregarines sporozoan parasites of humans, domestic parasitizing the alimentary canal of the mammals, and birds (Schmidt, 1992). rusty millipede, Trigoniulus corallinus, They are frequently found in mosquitoes collected from Shoushan, Kaohsiung. in Taiwan (Yen et al., 1994; Chen and Yang, 1996; Chen et al., 1997). Infection Materials and Methods by cephaline gregarines is common and widespread in millipedes (Crawford et al., Millipede collection and maintenance 1987). They belong to the family Trigoniulus corallinus (Gervais, 1842), Gregarinidae, suborder Septatina, order the rusty millipede, was collected ran- Eugregarinida, class Conoidasida, and domly from the southern Shoushan area phylum Apicomplexa (Margulis et al., especially on the campus of National Sun 1993). Levine (1970) depicted the life Yat-sen University from April 1999 to cycle of gregarines and described as October 2002 without regard to age, sex, many as 120 species of gregarines from or reproductive status. They were either the eight genera that can infect dissected directly after they were brought millipedes, Amphoroides, Cnemidospora, back to the laboratory or housed in Fonsecaia, Hyalosporina, Monoductus, plastic boxes filled with dead leaves Phleobum, Spirosoma, and Stenoductus. collected from their habitats until use. However, their shape and internal structure vary so much that it is very Observation of gregarines from difficult to select a form that can be millipedes considered typical. In fact, the life cycles Millipedes used in this study were of gregarines present problems of special distinguished by their sex and anesthe- interest, for many are correlated with tized by low-temperature knock-down at both the life cycle and the external 0oC. After careful dissection, the in- environment of the host (Smyth, 1994). testines were taken out from millipedes Due to Taiwan’s tropical and sub- and divided into three parts, the anterior, tropical climates, millipedes here are middle, and posterior, with each part abundant and widespread (Wang, 1961, comprising one-third of the intestines. 1964). However, proper investigations After pounding each part of the have been lacking for years, and we are intestine into pieces in saline, the stages currently attempting to bridge systematic or forms of the gregarines in the gaps in the study of the millipede species intestine were studied in fresh prepa- which Wang mentioned in the past. A rations after extractory or in fixed and preliminary morphological description of stained thin smears. Gregarines were some Formosan millipedes of the order isolated from the residues of intestines by Spirobolida was made in Chinese by Y. C. micropipettes, placed in a depression slide, Liu (pers. commun.). A database of covered with a cover glass and examined gregarines parasitic in Formosan milli- with a compound microscope under 40 × pedes has not been established, nor is and 100 × magnifications. much known about these millipedes All different stages of gregarines in themselves which possess a great deal of the gut were observed and counted. The variation. This study attempts to begin length and width of the protomerites and building up databases of parasitic grega- deutomerites were measured. The types rines found in Formosan millipedes so of movement exhibited by the gregarines that further studies can continue to add were also observed and recorded. Gre-

ௐ˟ഇסᖪௐ˟˩αٿέ៉ 138 Fig. 1. Rusty millipede, Trigoniulus corallinus.

Table 1.! Infection rate of gregarines in Trigoniulus corallinus Sex No. examined No. positive Percentage (%) Male 24 22 91.67 Female 39 33 84.67 Total 63 55 87.30

garines were identified according to their single millipedes greatly varied (Table 2). characteristic features as described by One individual millipede might be Clopton (2002). infected by as many as 535 gregarines or be free of any infection. In some cases, Results more than two species of gregarines were found to simultaneously be parasitizing a In total, 63 rusty millipedes were single millipede host. Most of the randomly collected and studied. Fig. 1 gregarines were found in the anterior and shows the host of the gregarines, middle parts of the alimentary canal Trigoniulus corallinus. Our results show known as the midgut of the millipede that T. corallinus collected from the (Table 3). We also observed up to at least Shoushan area was highly infected with six types of gregarines with different gregarines with a total prevalence rate of morphological characters parasitizing the 87.30% (Table 1). The infection rate in rusty millipede, T. corallinus (Fig. 2). The males (91.67%) was higher than that in shapes of the gregarines appeared to be females (84.67%). round, egg-like, or stick-like. Lengths The number of gregarines infecting ranged from 30 to 1227 µm. The body of

Prevalence and Observation of Intestine-Dwelling Gregarines in the millipede 139 Table 2.! Estimated species and number of gregarines from each millipede Size No. of gregarine species found Average no. of gregarines Sex* (cm) In each millipede per millipede Large Female (2) 3 274 ± 185 (≥ 5.0) Male (1) 4 519 Medium Female (5) 2 99 ± 40 (4.1 ~ 4.9) Male (5) 2 17 ± 7 Small Female (3) 2 133 ± 102 (≤ 4.0) Male (0) - - * Numbers in parentheses are the number of millipedes examined.

Table 3.! Distribution of gregarines in the intestines of Trigoniulus corallinus (n = 10) Part of the intestine Total Anterior Middle Posterior Number 291 263 7 561 Percentage (%) 51.87 46.88 1.25 100

a complete trophozoite consists of two length with dark cytoplasm and had a major parts, a short anterior protomerite tadpole-like deutomerite. TcS2 ranged and a long posterior deutomerite con- from 250 to 400 µm in length with a taining the nucleus. Their epimerites stick-shaped body, and a crescent-shaped were usually not detected. The two parts nucleus with dark cytoplasm. TcS3 are divided by a transverse, membranous ranged from 100 to 250 µm in length septum. The shape of the nuclei was with an egg-like shape and light round, crescent, or irregular. The color of cytoplasm. TcS4 ranged from 100 to 200 the cytoplasm was dark or light under µm in length with a short stick body the light microscopes and white or shape and dark cytoplasm. TcS5 ranged colorless under the dissecting microscope. from 100 to 250 µm in length with a In the deutomerite, there was usually one round body and light cytoplasm. TcS6 nucleus located near the center or pos- was less than 150 µm in length, with a terior end of the parasite body. However, short stick-shaped or rounded body and the location of the nucleus varied in dark cytoplasm. Species identification of certain species (Fig. 2A). The shape of fixed specimens is now in progress. the nucleus was either round or crescent Most of time the gregarines remained (Fig. 2C). motionless, but two types of gregarine According to the morphometric mea- movements were observed. One was a surements and the position of the nucleus creepy gliding movement, in which as described by Clopton (2002), two gregarines moved forward a rather long families of gregarines could be identified distance without changing their shapes. from our collections (Table 4). The family The other was a rapid springing move- Monoductidae includes one suspected ment, in which the cytoplasm flow species, TcM1, that ranged from 300 to produced a protomerite extrusion toward 500 µm in size with a stick body shape the deutomerite, then they returned to and dark cytoplasm. The family Steno- their original shape along with a slight phoridae included six forms (TcS1 to 6). movement forward (Fig. 3). Only few TcS1 measured more than 700 µm in gregarines exhibited this springing move-

ௐ˟ഇסᖪௐ˟˩αٿέ៉ 140 Fig. 2. Morphology of gregarines from Formosan millipedes. A, stick-like gregarines; B, a large gregarine with round nuclei and dark cytoplasm; C, a gregarine with a crescent nucleus (n); D, a small egg-shaped gregarine; E, a small short-stick-like gregarine; F, a round gregarine which appears to have light cytoplasm; G, a small gregarine with dark cytoplasm. dm, deutomerite; n, nucleus; pm, protomerites; ts, transverse septum. Scale bar = 50 µm

ment after a long period of inactivity morphologically described by Clopton during the observation. (2002). TcS3 and TcS5 were similar in size and in the color of their cytoplasm. Discussion More-detailed studies on their morpho- logy and life history are required to The shape, size, color, and intra- clarify their taxonomic positions. This is cellular structures of the sporonts are the first report of the millipede gre- important characteristics for species garines in Taiwan. No comparable survey identification of gregarines. The ratios of has been conducted, and no museum certain parts of gregarines are also collection of millipede gregarines cur- helpful in identification (Clopton, 1999). rently exists in Taiwan. Thus, this Our results show that at least seven survey initiates descriptions and speci- forms in two families could be detected in men collections for the study of grega- T. corallinus. Among them, TcM1 was rines and their arthropod hosts. likely to be Monoductus lunatus as According to the intestinal location

Prevalence and Observation of Intestine-Dwelling Gregarines in the millipede 141 Table 4.! Measurements of gregarines found in the intestines of Trigoniulus corallinus Morphometric measures* Family Code L W L/W PL PW PL/PW DL DW DL/DW Monoductidae TcM1 368 49 7.51 36 53 0.68 332 49 6.78 Stenophoridae TcS1 1,227 409 3.00 45 75 0.60 1152 409 2.82 TcS2 254 60 4.23 40 38 1.05 214 60 3.57 TcS3 199 100 1.99 23 37 0.62 176 100 1.76 TcS4 155 49 3.16 35 35 1.00 120 49 2.45 TcS5 121 86 1.41 17 32 0.53 104 86 1.21 TcS6 104 55 1.89 27 35 0.77 77 55 1.40 * L, total length; W, maximum width; PL, protomerite length; PW, protomerite width; DL, deutomerite length; DW, deutomerite width. All measurements other than ratios are in micrometers.

Fig. 3. Springing movement exhibited by some of the gregarines. Cytoplasmic flow causes the protomerite of a gregarine to extrude toward the deutomerite (A and B) and when returning to the original shape, the gregarine moves one step forward (C). The arrows indicate the directions of body movement exhibited by the gregarine. Scale bar = 50 µm

of gregarines parasitizing millipedes and cells of the midgut and initiate infection. findings of the life cycles of species After a period of developmental growths, described, the life history of millipede they become trophozoites and later leave gregarines may be similar (Jahn, 1979; the host cell as sporonts, which wander Janardanan and Ramachandran, 1982, about the coelom looking for partners. 1983a, b). When a potential host ingests Eventually, oocysts are formed after the oocysts of gregarines, the oocysts are syzygy of negative and positive sporonts usually ruptured by digestive fluids, (Manwell, 1977; Smyth, 1994). However, which releases the sporozoites into the if we are able to breed millipedes without gut. The sporozoites then enter epithelial natural infection in the laboratory, iso-

ௐ˟ഇסᖪௐ˟˩αٿέ៉ 142 late some gregarines oocysts of a certain sitism. Further studies will be carried out species from millipede feces, and infect on these host-parasite relationships, and those millipedes with oocysts, the life the possibility of using them as biological cycles could be explored. markers for local millipede distribution In the millipedes Trigoniulus goesi will be assessed. and Xenobolus acuticonus, infections by the gregarine Stenoductus trigoniuli References caused cytopathological effects (Janar- danan and Ramachandran, 1982). Fat Chen, W. J., and C. S. Yang. 1996. globules were produced in midgut Developmental synchrony of Ascogre- epithelial cells of millipedes by the garina taiwanensis (Aapicomplexa: intracellular stage of gregarines and Lecudinidae) within Aedes albopictus accumulated in the host-cell cytoplasm, (Diptera: Culicidae). J. Med. Entomol. causing a fatty degeneration that 33: 212-215. facilitated the release of intracellular Chen, W. J., C, Y. Chow, and S. T. Wu. gregarines into the nutrition-rich gut 1997. Ultrastructure of infection, lumen. According to Hopkin and Read development and gametocyst for- (1992), heavy infections of gregarines mation of Ascogregarina taiwanensis may cause the development of gangrene (Apicomplexa: Lecudinidae) in its in the legs of millipedes, and the legs mosquito host, Aedes albopictus may decay leaving stumps. In our study, (Diptera: Culicidae). J. Eukary. we observed that the legs of a heavily Microbiol. 44: 101-108. infected millipede exhibited unusual Clopton, R. E. 1999. Revision of the impaired motion on the central and genus Xiphoceohalus and decription posterior body parts, and that the of Xiphocephalus ellisi n. sp. millipede eventually died of infection. (Apocomplexa: Eugregarinida: Sty- This is why we consider millipede locephalidae) from Eleodes opacus gregarines to be parasitic rather than (Coleoptera: Tenebrionidae) in the symbiotic. western Nebraska Sandhills. J. Two types of movement were noticed Parasitol. 85: 84-89. in this study, although no special orga- Clopton, R. E. 2002. Phylum Apicomplexa nelle for movement was observed in these Levine, 1970: Order Eugregarinoride gregarines. As a matter of fact, they Leger, 1900. pp. 205-288. In: J. J. showed few activities. Some cell biologists Lee, G. Leedale, D. Patterson, and P. have reported the gliding movement in C. Bradbury, eds. Illustrated Guide the early stage (Mackenzie, 1980), but to the Protozoa, 2nd ed. Society of many questions remain. However, a Protozoologists, Lawrences, KS. model of the gliding movement in Crawford, C. S., K. Bercovitz, and M. R gregarines was later proposed, and the Warburg. 1987. Regional environ- gliding rate was estimated to be 1~10 ments, life-history patterns and µm/s (King, 1988). habitat use of spirostreptid milli- The results indicated that T. pedes in arid regions. Zool. J. Linn. corallinus collected from the Shoushan Soc. Lond. 89:63-88. area was highly infected with gregarines. Hopkin, S. P., K. Watson, M. H. Martin, It may possibly become a laboratory and M. L. Mould. 1985. The model for the study of host-parasite assimilation of heavy metals by relationships. This study raises questions Lithobius variegates and Glomeris with respect to the effects of host age, sex, marginata (Chilopode; Diplopode). and reproductive status upon the para- Bijd. Dierkund. 55: 88-94.

Prevalence and Observation of Intestine-Dwelling Gregarines in the millipede 143 Hopkin, S. P., and H. J. Read. 1992. Manwell, R. D. 1977. Gregarines and Predators, parasites, and defenses. pp. haemogregarines. pp. 1-32. In: J. P. 142-149. In: S. P. Hopkin, and H. J. Kreier, ed. Parasitic Protozoa. Vol. III. Read, eds. The Biology of Millipedes. Academic Press, London, UK. Oxford University Press, Oxford, UK. Marcus E. R., M. Baransky, de G. M. Jahn, T. L., E. C. Bovee, and F. F. Jahn. Kooker, and C. M. Knox. 1987. 1979. Phylum Sporozoa. pp. 170-181. Haemolymph composition of the In: T. L. Jahn, E. C. Bovee, and F. F. Kalahari millipede Triaenostreptus Jahn, eds. How to Know the Protozoa, triodes (Attems). Comp. Biochem. 2nd ed. Wm. C. Brown Company Physiol. 87:603-606. Publishers, Dubuque, IA. Margulis, L., H. I. McKhann, and L. Janardanan, K. P., and P. Ramachandran. Olendzenski. 1993. p. 276. Illustrated 1982. Studies on a new cephaline Glossary of Protoctista. Jones and gregarine, Stenoductus trigoniuli n. Bartlett Publishers, London, UK. sp. with a note on its cytopathology. Price, P. W. 1988. An overview of Arch. Protistenk. 125: 249-256. organismal interactions in ecosys- Janardanan, K. P., and P. Ramachandran. tems in evolutionary and ecological 1983a. Observations on 2 new species time. Agric. Ecosys. Environ. 24: 369- of cephaline gregarines (Protozoa: 377. Sporozoa) of the genus Stenoductus Schmidt, G. D. 1992. Phylum Apicom- Ramachandran, 1976 from sphae- plexa. pp. 18-35. In: G. D. Schmidt, rotherid millipedes in Kerala, India. ed. Essentials of Parasitology. Wm. C. Arch. Protistenk. 127: 405-412. Brown Publishers, Dubuque, IA. Janardanan, K. P., and P. Ramachandran. Smyth, J. D. 1994. Sporozoea: gregarines 1983b. On a new septate gregarine, and coccidian. pp. 88-108. In: J. D. Stenoductus polydesmi sp. n. (Spo- Smyth, ed. Introduction to rozoa: Cephalina) from the millipede, Parasitology. 3th ed. Cambridge Uni- Polydesmus sp. with observations on versity Press, London, UK. host-specificity of gregarines infecting Wang, Y. M. 1961. Millipedes of Taiwan — the millipedes in Kerala, India. Acta. a new species of Family Spirobolidae. Protozool. 22: 87-94. Q. J. Taiwan Mus. 14: 141-142. King, C. A. 1988. Cell motility of Wang, Y. M. 1964. Wallacea and insular sporozoan protozoa. Parasitol. Today fauna of millipedes. Q. J. Taiwan 4: 315-319. Mus. 17: 67-76. Levine, N. D. 1970. Phylum Apicomplexa. Yen, M. L., C. S. Chen, and K. C. Liu. 1994. pp. 322-374. In: J. J. Lee, S. H. Experimental infection of mosquitoes Hutner, and E. C. Bovee, eds. 1985. with two Ascogregarina species An Illustrated Guide to the Protozoa, (Eugregarinida: Lecudinidae). Bull. Society of Protozoologists, Lawrence, Tung-Hai Univ. Taiwan ROC 35: 87- KS. 106. Mackenzie, C. 1980. Gliding in Gregarina garnhami - movement without actin. Received: Feb. 3, 2004 Cell Biol. Intl. Reports 4: 769. Accepted: June 10, 2004

ௐ˟ഇסᖪௐ˟˩αٿέ៉ 144 ၅៍ވгડ᎑ࡓݓϥ੺ౙབ྽૙Ϡᒭᖪ̝አߤᄃ၁រ̋ု

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ώࡁտ੫၆᎑ࡓݓϥ੺ౙ (Trigoniulus corallinus) བ྽̰૙Ϡ۞ᒭᖪซҖአ ! ! ߤĂٙѣ੺ౙӮࠎ 1999 ѐ 4 ͡Ҍ 2002 ѐ 10 ͡ҋ੼ฯု̋гડͽᐌ፟͞ёଳ଀Ąд ᅬ੺ౙٺٙᑭߤ۞ 63 ੸੺ౙ̚Ăᓁຏߖதࠎ 87.30%Ăฯ੺ౙ۞ຏߖத (91.67%) ੼ ຏߖத (84.67%)Ąਕ૙Ϡд᎑ࡓݓϥ੺ౙབ̰۞ᒭᖪΞਕк྿˛჌ĂͷдТ˘੸੺۞ πܧౙ࣎វ۞བ྽̚ΞТॡజҌ͌׌჌ͽ˯̙Т۞ᒭᖪٙ૙ϠĄᒭᖪд੺ౙབ྽֭̚ ߱Ą੺ౙវ̰΍ன۞ᒭᖪͽдབ̚ҋϤޢٺ߱ᄃ߱̚۞ᇴϫᅈᅈ݈̂ٺӮ̶ҶĂ΍ன ă̂̈̈́ྻજଐԛү៍၅ͧېഫજ۞ sporadin ࠎ౵кĂࡁտ̚ӈͽѩॡഇᒭᖪ۞ԛ Ąώࡁտࠎдέ៉᎑ࡓݓϥ੺ౙབ྽૙Ϡᒭᖪ۞ௐ˘ቔಡӘĄྵ

ᜰᗤຠȈ᎑ࡓݓϥ੺ౙă੺ౙăᒭᖪ

Prevalence and Observation of Intestine-Dwelling Gregarines in the millipede 145