histological criteria to classify different developmental ...

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BIOJOURNAL, JUNE - 2017 BIOJOURNAL Vol.12 No.: 1

ISSN - 0970-9444

Page No. : 04 - 26

HISTOLOGICAL CRITERIA TO CLASSIFY DIFFERENT DEVELOPMENTAL PROGLOTTID STAGES OF A CESTODE, MONIEZIA EXPANSA RUDOLPHI, 1810, DURING GROWTH AND DIFFERENTIATION G. Johnrose1, S. Rajan2 and P. Vivek Raja3, 1,2

PG and Research Department of Zoology, Pachaiyappas College, Chennai-600030, India. 3

PG and Research Department of Zoology, Presidency College, Chennai-600005, India.

ABSTRACT Strobilated cestodes exhibit continuous embryonic development, producing new segments in anterior end and usually shedding gravid segments in posterior end by apolysis. As a result of growth, varying degrees of developmental stages are established along the length of the strobila. Previous investigations made in different regions along the length of the strobila of tapeworms revealed regional differences in morphological and anatomical features. In the present investigation, different developmental proglottid stages of M. expansa were assessed based on the criteria developed by Bell and Smyth (1958) and Smyth et al.,(1967) with suitable modification based on histology of developing organs in the proglottids. It is classified in to six different developmental proglottid stages: 1. Scolex and neck stage (stage I-pre-proglottid stage), 2. Segmentation stage (stage II), 3. Organogeny stage (stage III), 4. Gametogeny stage (stage IV), 5. Egg-shell formation and uterus dilation stage (stage V) , and 6. Gravid stage (stage VI). Morphometric aspects of M. expansa revealed that there was a significant increase in length and weight of different developmental proglottid stages during the growth and differentiation and suggested that the growth was very significant, rapid and continuous, without any break till the gravid proglottids were

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detached continually by apolysis from the posterior end of the worm. The mean number of proglottids in M. expansa decreased gradually from 729 in stage II to 271 in stage VI, whereas the length of the worm increased continuously during growth and differentiation. This result suggested that the formation of new proglottids in segmentation stage (Stage II) exceeded apolytic loss of proglottids from the posterior end of strobila in stage VI. Key Words: Cestode, Moniezia expansa, organogeny, gametogeny, proglottid, reproduction, scolex, segmentation.

INTRODUCTION Moniezia expansa is an intestinal parasite of sheep Ovis aries, grows to about 5-6 meters in length. It is well established that metazoan organisms during growth and differentiation from fertilization to adult, pass through a long succession of stages which are characterized by gradual morphological and biochemical changes associated with them (Needham, 1942). Unlike other metazoan organisms, individuals of strobilated cestodes exhibit continuous embryonic development, producing new segments in anterior end and usually shedding gravid segments in posterior end by apolysis. As a result of growth, varying degrees of developmental stages are established along the length of the strobila (Bell and Smyth, 1958: Smyth et al.,1967 and Barrett et al.,1982). This unique phenomenon of growth provides unusual possibilities for studies on cyto-differentiation which is a central problem in biology (Lee and Campbell, 1964; Smyth, 1969; Gustafsson, 1977; Smyth and McManus, 2007). Previous investigations made in different regions along the length of the strobila of tapeworms revealed regional differences in morphological and anatomical features (Race et al., 1966; Berger and Mettrick, 1971; Andersen, 1975; Thompson et al., 1980; Barrett et al., 1982), biochemical composition (Hopkins and Hutchison, 1960; Fairbairn et al., 1961; Roberts, 1961; Lee and Campbell, 1964; Mettrick and Cannon, 1970; Rani et al., 1987a, b; Bhure et al., 2014; Johnrose and Rajan, 2015a,b), physiological properties (Friedheim and Baer, 1933; Daugherty, 1957; Taylor and Thomas, 1968; Bolla and Roberts, 1971a,b), metabolic pattern (Coles and Simpkin, 1977; Dendinger and

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Roberts, 1977; Goldberg and Nolf, 1954; Gao and Li, 2004), drug action (Becker et al., 1980; Pampori and Srivastava, 1987; Markoski et al., 2006), immunological properties (Duwe, 1967) and gene expression properties (Bo et al., 2012; Wang et al., 2015). Coles and Simpkin (1977) reported a metabolic gradient with regard to the distribution of some metabolites such as lactate, succinate, acetate and NAD/ NADH ratio from anterior to posterior end of the strobila of Hymenolepis diminuta. In view of these observations, they have suggested that the metabolic gradient in other species may be investigated before undertaking a study on the metabolism of the scolex region alone and designing drugs. This suggestion gained support from the subsequent studies of Becker et al. (1980) who had found that praziquantel, an anthelmintic agent with a broad spectrum of activity and a high therapeutic index, produced varying degree of vacuolization of tegument leading to disruption of the syncytial layer of the tegument of H. nana, and they reported that the anthelmintic property decreased with increasing distance from the neck region. Despite these reports, most of the studies on cestode biochemistry have been attempted using whole animal (Smyth, 1969 and 1976; von Brand, 1973). Different criteria were used by various researchers, such as two halves, three thirds, four quarters of the cestode (Read, 1956; Goodchild and Vilar, 1962); linear length such as 2, 5 and 10 cm fractions (Daugherty and Taylor, 1956; Read and Rothman, 1958) and morphological and anatomical features such as immature, mature and gravid stages (Fairbairn et al. 1961; Andersen, 1975; Roberts,1980; Thompson et al., 1980;). The results of these studies warranted further investigation to establish different growth stages of cestode, along the length of the strobila based on histology and reproductive organs, during growth and differentiation. For the assessment of developmental stages along the length of the strobila, Bell and Smyth (1958), Smyth et al.,(1967) and Barrett et al.,(1982) have pointed out that evaluation of criterion for the recognition of the stages is very essential and it may be related to the normal pattern of development, precisely definable, readily recognizable and covering the whole range of the maturation process. Although, the need for developing criterion for the assessment of stage has

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long been given importance, a common pattern in the assessment is yet to be developed. Bell and Smyth (1958) used cytological and histochemical changes as criteria demonstrated seven stages viz region of cell multiplication, segmentation, organogeny, early gemetogeny, late gametogeny, egg-shell formation and vitellogenesis and oviposition in a mature Diphyllobothrium sp. They were of the opinion that this kind of assessment was essential for studies relating to biochemistry and physiology. Taking all these factors into consideration, in the present investigation, different developmental proglottid stages of M. expansa were assessed based on the criteria developed by Bell and Smyth (1958) and Smyth et al.(1967) with suitable modification based on histology of developing organs in the proglottids. Hence, the present study is attempted to classify scolex and strobila of an adult M. expansa in to different developmental proglottid stages based on morphological, histological and anatomical features.

MATERIALS AND METHODS Specimens of Moniezia expansa Rudolphi, 1810 were collected from the intestines of sheep, Ovis aries. After careful removal of adult M. expansa from the intestines, they were washed twice in physiological saline solution to ensure the removal of the intestinal contents of sheep. HISTOLOGICAL PREPARATION For histological studies, one cm length bits of the worm M. expansa from the anterior end with scolex and neck as well as from different regions of the strobila containing different developmental proglottid stages were fixed in 10% neutral buffered formalin. The stages were ascertained by observation under microscope from fresh squash preparation, and whole mount preparation after staining with 3% aceto-carmine stain. T.S of different stages For histological studies, tissues of various stages (6 developmental proglottid stages) were fixed in 10% NBF for 24 hours. Tissue processing, sectioning and staining with Haematoxylin and Eosin were carried out as described in Bancroft and Stevens (1977).

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DIVISON OF DIFFERENT DEVELOPMENTAL PROGLOTTID STAGES The fully grown parasite with all six developmental proglottid stages were selected and used for the study (Plate I). Damaged and partially cut specimens were discarded. After washing, the parasite was cut at regular interval of about 10 cm lengths from the scolex to the posterior end. Each bit was serially placed on a graph sheet and length was measured in mm. The worm was divided into six different developmental proglottid stages based on tissues and organs present in the proglottids following the procedure of Bell and Smyth (1958)and Smyth et al. (1967) with some changes in the initial stages (Table 1 and 2; Plates II to IV). The following criteria were used to identify the different stages of the developing proglottids. Stage I : Scolex and neck stage: It is represented by the scolex and unsegmented part of the neck (Pre-proglottid stage). Stage II : Segmentation stage: Numerous transverse divisions are seen. These segments do not have any organs. Stage III: Organogeny stage: Appearance of various internal organs such as testis, ovaries and interproglottid glands. Stage IV: Gametogeny stage: Characterised by the presence of fully developed male and female reproductive system. Active sperm, vasdeferens, cirrus and cirrus pouch were clearly visible. Stage V: Egg shell formation and uterus dilation stage: It is characterized by the appearance of mesh like branched uterus and fully developed eggs in the uterus. Stage VI: Gravid stage: It is characterized by the appearance of embryonated eggs containing fully formed six hooked oncospheres in the uterus. Wherever necessary the segments were stained with aceto-carmine to confirm the stages. The length in mm, number of segments, the wet weight and dry weight in mg of each stage were recorded. The data are subjected to ANOVA analysis using SPSS Package (SPSS, 2015).

RESULTS AND DISCUSSION DIFFERENT DEVELOPMENTAL PROGLOTTID STAGES OF M. EXPANSA The body of Moniezia expansa consists of three regions namely the scolex, neck and strobila. 1. The scolex is located in the anterior end with four suckers. 2. The neck region present immediately posterior to the scolex, is unsegmented, poorly

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differentiated and narrower than the scolex and strobila. It is the differentiating zone that continuously gives rise to undifferentiated proglottids of the strobila. 3. The strobila is made of a chain of proglottids (Plate-I). The anteriorly situated proglottids are undifferentiated with no specific organs and it is followed by immature proglottids with non functional reproductive organs. Proglottids located posterior to the immature proglottids are sexually mature, whereas those located at the distal end are filled with mature embryonated oncospheres. The gravid proglottids shed off from the posterior end of the worm from time to time, thereby the oncospheres escape from the host sheep along with feces. In the present study, the scolex, neck and strobila were divided into six developmental proglottid stages based on the formation of reproductive organs and other related structural changes in proglottids of the strobila (Table 1 and 2). The scolex and unsegmented neck present in the anterior region were considered as stage I of M. expansa. This stage is also considered as pre-proglottid stage (Plate-II a). Stage –I : Scolex-neck stage. The remaining five stages were the proglottids present in the strobila, from the proximal end to the distal end. These stages were characterized based on the cytological and anatomical features and reproductive organs present in the proglottids. The different developmental proglottid stages were named as given below: Stage-II: Segmentation stage. Stage-III: Organogeny stage. Stage-IV: Gametogeny stage Stage-V: Egg-shell formation and uterus dilation stage, and Stage- VI: Gravid stage Stage -I : Scolex and Neck stage (pre-proglottid stage) Scolex is the anterior most part of the body which is followed by the neck. The scolex contains four muscular suckers (Plate-IIa) which aid for the attachment of the worm to its hosts’ (sheep) intestinal mucosa. The apex of the scolex is devoid of hooks. The neck region immediately posterior to scolex is devoid of any visible internal organs except for the four collecting tubules of osmoregulatory system. The section passing through the scolex region (Plate-IVa) showed the presence of four small osmoregulatory canals in the centre surrounded by four muscular suckers.

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The gland cells present in the scolex region were basophilic in nature, since they were stained deep blue with haematoxylin – eosin. The secretory granules exhibited intense basophilic reaction with haematoxylin. In the neck region, the medullary and cortical parenchyma could not be distinguished clearly. The mean length of the stage-I, the scolex with unsegmented neck region was 1.83 ± 0.14 cm (Table 3) and the mean total dry weight of this stage was 6.37 ± 0.77mg. (Table 5). Stage- II : Segmentation Stage The unsegmented neck is followed by the region of segmentation. Numerous transverse divisions are formed and each division is called proglottid. The shape of the proglottid changes as it was pushed farther away from the neck region. The width of each proglottid greatly exceeded its length. These proglottids were devoid of any organ. But the cell accumulation was seen at this stage. The mean length of this stage was 12.39 ± 3.5 cms (Table 3) and the mean number of segments was 729.3 ± 205 (Table 4). The total dry weight of this stage was 30.90 ± 13.61 mg (Table 5). The appearance of the proglottid of the developing strobila represented clearly recognizable stage of development, without any reproductive organs and the segments were arranged closely (Plate –IIb and IVb,c). The cross section passing through the stage II showed the presence of two pairs of collecting tubules of osmoregulatory system in the medullary region (Plate-IVb). The musculature which separated the medullary and cortical parenchyma was poorly developed. However, the cortical and medullary parenchyma could be distinguished clearly. The medullary parenchyma was loosely packed, whereas the cells in the cortical region were compactly arranged. Both medullary and cortical parenchyma cells were stained by eosin, whereas the parenchyma cells around the osmoregulatory canal were dark pinkish in colour by haematoxylin - eosin (Plate IVb). These deeply stained basophilic cells around the excretory canal may be due to the initial formation of anlagen through the aggregation of germinative cells at certain foci in inner parenchyma, partly by mitotic division and partly due to cell immigration of germinative cells from the surrounding panenchyma as demonstrated by Gustafsson (1977) in Diphyllobothrium dendriticum.

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Stage –III: Organogeny stage Proglottids of organogeny stage were characterized by the appearance of various internal organs such as testes, ovaries and inter-proglottidal glands (Plate IIc and IVd). The proglottids of this stage were larger than the proglottids of the previous stage. The cross section passing through a proglottid of this stage showed well developed parenchymal muscles. In the medullary parenchyma cells, granules were densely packed whereas in cortical parenchyma the granules were loosely packed. In this stage the three layers of tegument were clearly seen but the sub-tegumental cells were more closely packed than the previous stage (stage II). The sub–tegumental muscles were more distinct than the previous stage. The medullary parenchyma islets of the testicular cells were clearly distinguished by dark blue colour with haematoxylin-eosin stain (Plate IVd). The mean length of this stage was 21.79 ±7.85 cm (Table 3) and the total dry weight of this stage was 62.22 ±9.30 mg (Table 5). The mean number of segments present in this stage was 424.00 ± 114.4 (Table 4). Stage –IV: Gametogeny stage Proglottids of gametogeny stage followed the region of organogeny stage, and were characterized by the presence of fully developed male and female reproductive systems in which active sperm, vas deferens, cirrus and cirrus pouch were clearly visible (Plate IIIa,b and IVe). Various stages of spermatogenesis could be seen in the section as well as in squash preparations stained with aceto- carmine stain. Ovary, vitelline gland, Mehli’s gland, seminal receptacle and vagina were also present. Loosely packed oocytes and vitelline cells in various developmental stages were present. In the mature vitelline cells, nucleus was not readily visible, since they were masked by vitelline globules. The cells of the Mehli’s gland were visible in section around the ootype. The cells of Mehli’s gland were flask shaped with a long stalk which opened in the ootype. The ootype was followed by the uterine rudiment. The mean length of this stage was 34.43 ± 10.72cm (Table 3) and the total dry weight was 159.54 ±30.19 mg (Table 5). The mean number of the proglottids present in this stage was 376.00 ± 76.2; ( Table 4).

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Stage –V: Egg - shell formation and Uterus dilation stage Proglottids of egg - shell formation and uterus dilation stage were characterized by the appearance of mesh like branched uterus and fully developed eggs in the uterus (Plate IIIc, d and IVf). The egg-shell formation began in the ootype region and they passed into the uterus. The secretory activity of the Mehli’s gland was very high at this stage. The ootype region possessed many coalescing vitelline cells and eggs. The uterus was fully packed with developing eggs and early embryos. The organs of male reproductive system were not conspicuous at this stage. The size and weight of each proglottid attained its maximum size than all the other previous stages. The mean length of this stage was 47. 11 ± 10.60 cm (Table 3) and the mean total dry weight was 388.12 ± 77.52 mg (Table 5). The mean number of the proglottids present in this stage was 336.90 ± 85.3 ( Table 4). Stage –VI: Gravid Stage Proglottids of gravid stage occupied the posterior region of the strobila where shedding of segments have been taking place. It was characterized by the appearance of six hooked embryonated eggs called oncospheres in the disintegrated uterus. In this stage, the disintegrated uterus and embyonated eggs were clearly visible (Plate IIIf and IVg). All other reproductive organs like testes, ovary, vitelline gland and Mehli’s gland were absent. This stage possessed the largest proglottids. However, the proglottids of the distal end were in the disintegrated form due to apolysis. The posterior most proglottid or a group of a few proglottids, detach from the posterior end of the strobila, shed off periodically by the process of apolysis. The mean length of this stage was 69.25 ± 30.81 cm (Table 3) and the mean total dry weight was 706.80 ± 85.70 mg (Table5). The mean number of the proglottids present in this stage was 271.10 ± 105.7 (Table 4). 4.2. MORPHOMETRICAL FEATURES OF M. EXPANSA Length of different developing proglottid stages The measurement of length of each stage showed that length increased from stage I to VI (Table 3). The length of first stage was 1.83 ± 0.14 cm, followed by 12.39 ±

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3.71cm in stage II, 21.84± 7.94cm, in stage III, 34.43 ± 10.72 cm in stage IV, 47.11 ± 10.60 cm in stage V and the maximum length of 69.25 ± 30.81 cm in stage VI. ANOVA test revealed that the difference in length between stages was highly significant (P