For Review OnlyMature gonadal histology and gametogenesis of the golden

tortoise beetle, Aspidimorpha sanctaecrucis (Fabricius, 1792) (Coleoptera: Chrysomelidae: Cassidinae): Histological

observation

Journal: Songklanakarin Journal of Science and Technology

Manuscript ID SJST-2017-0465.R3

Manuscript Type: Original Article

Date Submitted by the Author: 05-Apr-2019

Complete List of Authors: Senarat, Sinlapachai; Chulalongkorn University, Department of Marine Science, Faculty of Science

Keyword: Germ cell, Golden tortoise beetle, Microanatomy, Reproductive system

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Author’s Response

No. Comments Reply to comments Please find the revised MS “Boonyoung et al_Revised.doc” Abstract1. wrong sentence: "Histologically, each seminiferous tubule is divided into two regions: vitellarium and germarium." : this sentence should be deleted

Because the vitellarium part are part of the ovaryol which consist the female reproductive system. Authors agreed. We already deleted

ResultsLine 114-115 2. wrong sentence: "The germarium can be classified into three zones: growth, maturation, and transformation". the germarium region is anterior part of testis

This sentence should be corrected as follows:

"The seminiferous tubules (testis folicules) can be classified into three zones: growth, maturation, and transformation". Authors agreed. We already added.

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1 Mature gonadal histology and gametogenesis of the ladybird beetle Aspidimorpha

2 sanctaecrucis (Fabricius, 1792) (Coleoptera: Cassidinae: Chrysomelidae): Histological

3 observation

4

5 Piyakorn Boonyoung1, Sinlapachai Senarat2,*, Jes Kettratad2, Wannee Jiraungkoorskul3,

6 Narit Thaochan4, Kong-Wah Sing5, Theerakamol Pengsakul6, Pisit Poolprasert7

7

8 1Department of Anatomy, Faculty of Science, Prince of Songkla University, Songkhla 90110,

9 Thailand

10 2Department of Marine Science, Faculty of Science, Chulalongkorn University, Bangkok

11 10330, Thailand

12 3Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400,

13 Thailand

14 4Department of Pest Management, Faculty of Natural Resources, Prince of Songkla

15 University, Hat Yai, Thailand. 90112.

16 Bio-Analysis Laboratory, Chemical Metrology and Biometry Department, National Institute

17 of Metrology (Thailand), Pathumthani, 10120, Thailand

18 5State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology,

19 Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming 650223, China

20 6Faculty of Medical Technology, Prince of Songkla University, Songkhla, 90110, Thailand

21 7Program of Biology, Faculty of Science and Technology, Pibulsongkram Rajabhat

22 University, Phitsanulok, 65000, Thailand

23

24

25 *Corresponding author

26 Dr. Sinlapachai Senarat

27 Department of Marine Science, Faculty of Science, Chulalongkorn University

28 Bangkok 10330, Thailand

29 Email: Senarat.S@hotmail.com

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30 Abstract

31 The gonadal structure of insects has been well-reported, but there is a lack of such

32 information from ladybird beetles that are an economically important insect. In this study, the

33 gonadal structure and differentiating stage of gametogenesis of ladybird beetles,

34 Aspidomorpha sanctaecrucis is examined using the histological technique. Based on light

35 microscopic level, our results revealed that A. sanctaecrucis male reproduction system has a

36 pair of testicular consisting of six seminiferous tubules. All zones contained various stages of

37 spermatogenesis, and can be divided into eleven stages depending on its shape and

38 histological organization (particularly chromatin organization), which was divided to

39 spermatogonia, primary spermatocytes (8 sub-steps; leptotene, zygotene, pachytene,

40 diplotene, diakinesis, metaphase, anaphase and telophase), secondary spermatocyte,

41 spermatids (two sub-steps) and spermatozoa. A pair of the ovarian was observed in female A.

42 sanctaecrucis. Each ovary is considered as a meroistic ovariole that existed with nurse cells.

43 The differentiating oogenic stages including previtellogenic and vitellogenic stages were

44 observed. Our study provides baseline data that will be useful for future work involved in the

45 ultrastructure and physiology of the reproductive tissue in ladybird beetles, especially the

46 differential characterizations of seminiferous tubule and ovarian type in beetles.

47

48 Keywords: Germ cell, Ladybird beetle, Microanatomy, Reproductive system

49

50

51

52

53

54

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55 1. Introduction

56 The reproductive system of insect has received renewed interest because it is

57 important to understand the insect's biology and physiology that have important implications

58 such as biological control and outbreak of insect pest. The reproductive structures in insects

59 such as shield bug (Özyurt, Candan, Suludere, & Amutkan, 2013; Özyurt, Candan, &

60 Suludere, 2014); blister beetle (Gerber, Chltrc, & Rempel, 1971) and dock bug (Karakaya,

61 Özyurt, Candan, & Suludere, 2012) have been well studied and provided important

62 information on the anatomy, histology, and physiology of insects. Light microscopic studies

63 revealed that the reproductive tissue of insects consists of two distinct parts (gonadal tissue

64 and reproductive duct) based on the basic tissue and cell types in each region (Drecktrah,

65 1966; Özyurt et al., 2013). Studies revealed that a pair of gonadal organ and a reproductive

66 duct formed the reproduction system of insects such as Nezara viridula (Hemiptera:

67 Pentatomidae) (Linnaeus, 1758) (Pendergrast et al., 1956; Ramamurty, 1969) and Ostrinia

68 nubilalis (Lepidoptera: Crambidae) (Hübner, 1796) (Drecktrah, 1966), Lytta nuttalli

69 (Coleoptera: Meloidae) Say 1824 (Gerber, Chltrc, & Rempel, 1971), Abedus ovatus (Stål,

70 1862) (Hemiptera: Belostomatidae) (Lalitha, Shyamasundari, & Hanumantha, 1997), Coreus

71 marginatus (Linnaeus, 1758) (Heteroptera: Coreidae) (Karakaya et al., 2012), Graphosoma

72 lineatum (Linnaeus, 1758) (Hemiptera: Pentatomidae) (Özyurt et al., 2013; Özyurt et al.,

73 2014), and Catopsilia Pomona Fabricius, 1775 (Lepidoptera: Pieridae) (Tongjeen et al.,

74 2014). Although the description of the histological structure of the reproductive system has

75 been widely developed in insects as mentioned above, it has never been reported on ladybird

76 beetles.

77 One of the most important ladybird beetles, A. sanctaecrucis was chosen in this study,

78 which belongs to Coccinellidae. This insect is considered as one of the most harmful and

79 destroys the plant as an agricultural pest. Therefore, to provide key information on gonadal

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80 structure and gametogenic stages of A. sanctaecrucis in the histological images were

81 necessary.

82

83 2. Materials and Methods

84 Male and female of ladybird beetles (n = 10, each sex = 5), A. sanctaecrucis (n=10,

85 mean length 0.93±0.31 cm), were obtained from infected morning glory (Ipomea aquatica) -

86 an economically important vegetable in Southeast Asia, during the rainy season (October to

87 December 2016) at Chawang District, Nakhon Si Thammarat Province, Thailand (8°28’10"

88 N, 99°29’45" E). Ladybird beetles were euthanized by rapid cooling shock (Wilson, Bunte, &

89 Carty, 2009) and were intermediately fixed with Davidson's fixative solution (for ~48 h) for

90 histological investigation. Under histological investigation, the reproductive tissues were

91 processed under standard histological techniques (Presnell & Schreibman, 2013; Suvarna,

92 Layton, & Bancroft, 2013). All reproductive tissue blocks were cut at 4 µm thickness and

93 routinely stained with Harris's haematoxylin and eosin (H&E) and Masson's Trichrome

94 (Presnell & Schreibman, 2013; Suvarna et al., 2013). Histology of the gonadal structure and

95 gametogenesis of A. sanctaecrucis were determined and photographed under a light

96 microscope (10x and 40x; LM TE2000-Ua).

97

98 3. Results and Discussion

99 With the lack of gonadal histological information of ladybird beetles, our data is the first

100 study showed the reproductive information of A. sanctaecrucis in both histological images

101 and schematic diagrams (Figures 1-3).

102

103 Testicular histology and spermatogenesis

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104 As an important feature from male A. sanctaecrucis was depicted a pair of testis

105 consisting of six seminiferous tubules that occurred in the abdominal region (Figure 1A). The

106 length of each tubule was about 800-850 µm. The vas efferent was jointed with the

107 seminiferous tubule. The size of vas efferent was ~100 µm. It was lined by a simple high

108 columnar epithelium. Each epithelial cell had a basophilic nucleus and was surrounded by a

109 thin muscular layer (Figure 1B).

110 Histologically, the testis is prominently surrounded by a capsule comprising of a

111 thick, loose connective tissue layer and few smooth muscle layers that support the germinal

112 cells. Each seminiferous tubule is covered by a thin layer of connective tissue (Figure 1C).

113 The structure of a seminiferous tubule is divided into two zones; (i) vitellarium and (ii)

114 germarium (Figure 1A), with the differential development of the male germ cells. At high

115 magnification, the vitellarium is distinctly seen at the outer rim of the tubule where a group of

116 the spermatogonia is arranged systematically. Seminiferous tubule is surrounded by spherical

117 cluster called the spermatocytes. The seminiferous tubules (testis folicules) can be classified

118 into three zones: growth, maturation, and transformation (Figure 1A). The zone of growth is

119 composed of the spermatocytes under mitosis, which differs from spermatogonia. The

120 maturation zone is found at the spermatids and is transferred under two meiotic divisions.

121 The transformation zone contained spermatozoa. This feature is similar to other insects such

122 as Podisus nigrispinus (Lemos, Ramalho, Serrao, & Zanuncio, 2005) and Dolycoris

123 baccarum (Özyurt et al., 2012). Hereby, we confirmed that the seminiferous tubule of A.

124 sanctaecrucis is responsible for sperm production, as similarly reported in Graphosoma

125 lineatum (Özyurt et al., 2013). The spermatogeneic stages of ladybird beetle in the

126 seminiferous tubules are classified into eleven stages depending on the shape and histological

127 organization of chromatin organization. The stages are spermatogonia, primary

128 spermatocytes (six sub-steps: leptotene, zygotene, pachytene, diplotene, diakinesis,

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129 metaphase, anaphase and telophase), secondary spermatocyte, spermatids (two sub-steps) and

130 spermatozoa, as similarly reported in some invertebrates (Sagi, Milner, & Cohen, 1988;

131 Polijaroen, 2010)

132

133 Spermatogonia

134 The spermatogonium is either of oval or spherical shapes with a cell diameter ~10-12

135 µm. It contains a large centrally nucleus, and the majority of the nuclei have the euchromatin

136 with one or two nuclei (Figures 1B-1C, 1G-1H). The spermatogonium is also found in

137 eosiophilic cytoplasm (Data not shown).

138

139 Primary spermatocytes

140 The primary spermatocytes (PSc) are cells with oval or spherical shape with a mean

141 cell diameter ~10 µm (A. sanctaecrucis) (Figures 1C-1E). The primary spermatocytes have

142 patchy heterochromatin throughout the basophilic nucleus. Hence, the structure of the

143 chromatin fiber and heterochromatin is used to classify the stages of the primary

144 spermatocyte, which could be divided into six sub-steps as follows. Leptotene spermatocyte

145 is in a spherical shape with ~8 µm diameter and contains small heterochromatin blocks

146 throughout the nucleus (Figure 1E). Zygotene spermatocyte have round nucleus, similar to

147 leptotene spermatocyte but with bigger size. The density of heterochromatin blocks with the

148 synaptonemal complexes is detected in the middle part of nucleus (Figure 1E). Pachytene

149 spermatocyte is still seen as in previous steps but its large blocks or cords of

150 heterochromatin are observed at the inner nuclear envelope (Figure 1E). Diplotene

151 spermatocyte is smaller than a PSc (~7 µm in diameter). The oval nucleus contains a large

152 chromatin block. It is arranged along the nuclear membrane and also seen as a cart-wheel

153 pattern. Diakinesis spermatocyte has smaller cell size compared to diplotene spermatocyte

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154 (Figure 1E). The major characteristics of this stage are identified by the large piece and

155 almost fully formed chromosomes. The nuclear membrane has disappeared. Metaphase

156 spermatocyte is completely condensed with the chromatin and arranged along the equatorial

157 region (Figure 1E). No nuclear membrane is seen in this stage. The chromatin of anaphase

158 spermatocyte moves to opposite poles of the cell (Figure 1E). The final stage is the

159 telophase spermatocyte where the cell membrane separates into two daughter cells.

160

161 Secondary spermatocyte (SSc)

162 Secondary spermatocyte appear under the first meiotic division from primary

163 spermatocyte but it is rarely observed because of the rapid development. The cell size during

164 this step is about 5 µm in diameter. The chromatin is of highly condensed cord-like structures

165 (Figure 1F).

166

167 Spermatids (St)

168 The differentiation of the spermatids occurs during spermiogenesis. Under light

169 microscopic level, this stage could be divided into two sub-steps according to the chromatin

170 condensation; early and late spermatids (Figures 1G-1H). Early spermatid is oval in shape

171 and the cell size is smaller than the secondary spermatocytes, about 4 µm in diameter. The

172 chromatin of this stage is condensed into interconnecting thick cords throughout the nucleus.

173 The late spermatid is completely condensed of the heterochromatin and in the beginning to

174 produce the tail (Figure 1H).

175

176 Spermatozoa (Sz)

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177 Under light microscopic level, mature spermatozoon has two distinct regions: a head

178 and tail region (flagellum). Based on our observation, the head of the spermatozoon was an

179 elongated shape (about 15-20 µm) and thin (Figure 1I).

180

181 Ovarian histology and oogenesis

182 A pair of the ovary of A. sanctaecrucis is observed. Each ovary contains several

183 ovarioles (Figures 2A-2B). All ovarioles are surrounded by the peritoneal membrane (or

184 sheath). Histological studies of the ovarian duct show the jointed ovaries and indicate that it

185 is covered by a simple columnar epithelium (Figure 2C). The function of this epithelium

186 concerns to produce the secretion with composting of carbohydrate and protein, which

187 supports the production of the egg membrane (Gerber et al., 1971). Both circular muscular

188 and longitudinal muscular layers are also surrounded by this duct (Figure 2C).

189 The ovary is embedded by the differential stages of oocytes (Figure 2D), which is also

190 considered as a meroistic telotrophic type due to containing the nurse cells in the terminal

191 filament (Figure 1A). This type was similarly reported in Perillus bioculatus (Adams, 2000),

192 Perillus bioculatus (Adams, 2001), Podisus maculiventris (Wittmeyer, Coudron, & Adams,

193 2001) and Brontocoris tabidu (Lemos et al., 2005). The nurse cell is normally seen in the

194 terminal filament (Figures 1E-1F). The function of the nurse cell involves supporting the

195 nutrition for the oocyte (Klowden, 2007). Two distinct zones are divided according to

196 histological features including germarium and vitellarium. It was similarly observed in

197 coleopteran (Buning, 2006).

198

199 Germarium

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200 Oogonium mainly contains in this zone and was mitotically processed; however, this

201 stage is much differed to clarify under light microscopic level. Ultrastructural observation is

202 required to support this problem.

203

204 Vitellarium

205 This zone contains the differentiate oocytes in the mature ovaries including

206 previtellogenic (Pv) and vitellogenic (Vg) stages in a linear arrangement as follows (Figures

207 3A-3F);

208 The oval shape of Pv with size about 300 µm in diameter is observed (Figures 3A-3B). It

209 contains the central nucleus with about 70 µm in diameter with containing a large

210 heterochromatin blocks (Figure 3B). No nucleoli are detectable close to the nuclear

211 membrane. The strong basophilic staining of the ooplasm also appears (Figures 1A-3B). At

212 the same time, the single layer of the elongated cuboidal follicle cells surrounding the oocyte

213 is seen (Figures 3A-3D). At the end of this stage, the nucleus is of irregular shape and

214 obviously decreases in size (about 10 µm), on the other hand the increasing of follicular cell

215 layer is noted as simple high columnar cell (Figure 3D).

216 The size of the Vg has obviously increased about 450-500 µm than that in previous

217 stages (Figures 1E-1F). Association to the major characterization showed that the large yolk

218 granules are firstly distributed, which is positively stained as a deeply acidophilic stain in

219 ooplasm (Figure 1F). The follicular cell remains a single layer; however, it changes to low

220 cuboidal cell (Figure 3F).

221

222 4. Conclusion

223 Although information of the gonadal structure and gametogenesis from our

224 observation is a basic information, it has put forward to understand about the

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225 histopathology/status of reproductive histology. We hope to follow up this initial research

226 and explore a reproductive system with relation to cellular association patterns, the

227 requirement for yolk accumulation, the involvement of gonad-inhibiting hormone (GIH) and

228 the neurotransmitters, dopamine and serotonin, which are known to be involved in the

229 regulation of gonadal maturation.

230

231 Acknowledgement

232 The authors are thankful to the Department of Anatomy, Faculty of Science, Prince of

233 Songkla University.

234

235 References

236 Adams, T.S. (2000). Effect of diet and mating status on ovarian development in a predaceous

237 stink bug Perillus bioculatus (Hemiptera: Pentatomidae). Annals of the Entomological

238 Society of America, 93, 529-535.

239 Adams, T.S. (2001). Morphology of the internal reproductive system of the male and female

240 two spotted stink bug Perillus bioculatus (F.) (Heteroptera: Pentatomidae) and the

241 transfer of products during mating. Invertebrate Reproduction and Development, 39,

242 45-53.

243 Buning, J. (2006) Ovariole structure supports sistergroup relationship of Neuropterida and

244 Coleopteran. Arthropod Systematics and Phylogeny, 64, 115-126.

245 Drecktrah, H.G. (1966). Morphology and histology of the internal reproductive systems of

246 the European corn borer Ostrinia nubilalis (Hübner). Retrospective Thesis and

247 Dissertations, Paper 5310, Iowa State University of Science and Technology, Iowa,

248 USA.

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249 Gerber, G.H., Chltrc, N.S., & Rempel, J.G. (1971). The anatomy, histology, and physiology

250 of the reproductive systems of Lytta nuttalli Say (Coleoptera: Meloidae): The internal

251 genitalia. Canadian Journal of Zoology, 49, 523-533.

252 Karakaya, G., Özyurt, N., Candan, S., & Suludere, Z. (2012). Structure of the male

253 reproductive system in Coreus marginatus (L.) (Hemiptera: Coreidae). Turkish Journal

254 of Entomology, 36, 193-202.

255 Klowden, M.J. (2007). Physiology System in Insects. Elsevier, Inc., San Diego.

256 Lalitha, T.G., Shyamasundari, K., & Hanumantha, K.R. (1997). Morphology and histology of

257 the female reproductive system of Abedus ovatus Stal (Belostomatidae: Hemiptera:

258 Insecta). Memorias do Instituto Oswaldo Cruz 92: 129-135.

259 Lemos, W.P., Ramalho, F.S., Serrao, J.E., & Zanuncio, J.C. (2005). Morphology of female

260 reproductive tract of the predator Podisus nigrispinus (Dallas) (Heteroptera:

261 Pentatomidae) fed on different diets. Brazilian Archives of Biology and Technology,

262 48, 129-138.

263 Özyurt N., Candan S., & Suludere Z. (2012). The morphology and histology of the male

264 reproductive system in Dolycoris baccarum Linnaeus 1758 (Heteroptera:

265 Pentatomidae). Light and scanning electron micoscope studies. Micron, 44, 101-106.

266 Özyurt, N., Candan, S., Suludere, Z., & Amutkan, D. (2013). Morphology and histology of

267 the male reproductive system in Graphosoma lineatum (Heteroptera: Pentatomidae)

268 Based on optical and scanning electron microscopy. Journal of Entomology and

269 Zoology Studies, 1, 40-46.

270 Özyurt N., Candan S., & Suludere Z. (2014). Structure of the male reproductive system and

271 spermatogenesis of Codophila varia (Fabricius, 1787) (Heteroptera: Pentatomidae) by

272 Light and Scanning Electron Microscopy. Journal of the Entomological Research

273 Society, 16, 101-110.

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274 Pendergrast, J.G. (1956). The male reproductive organs of Nezara viridula with a preliminary

275 account of their development (Heteroptera: Pentatomidae). Transactions of the Royal

276 Society of New Zealand, 84, 139-146.

277 Polijaroen, J. (2010). Spermatogenesis and spermiogenesis in the giant freshwater prawn,

278 Macrobrachium rosenbergii. PhD Thesis, Mahidol University, Bangkok, Thailand.

279 Presnell, J.K. & Schreibman, M.P. (2013). Humason’s Animal Tissue Techniques. 5th ed.

280 US: Johns Hopkins University Press.

281 Ramamurty, P.S. (1969). Histological studies of the internal organs of reproduction in Nezara

282 viridula Fabr. Pentatomidae: Heteroptera, Insecta. Zoologischer Anzeiger, 183, 119-

283 139.

284 Sagi, A., Milner, Y., & Cohen, D. (1988). Spermatogenesis and sperm storage in the testes of

285 the behaviorally distinctive male morphotypes of Macrobrachium rosenbergii

286 (Decapoda, Palaemonidae). Biologicl Bulletin, 174, 330–336.

287 Suvarna, K.S., Layton, C., & Bancroft, J.D. (2013). Bancroft’s Theory and Practice of

288 Histological Techniques. 7th ed. Canada, Elsevier.

289 Tongjeen, P., Kinrot, S., Poolprasert, P., Yenchum, W., Senarat, S., & Chantarasawat, N.

290 (2014). Histology and histochemistry of Catopsilia pomona (Fabricius, 1758)

291 reproductive system. Thammasat Journal of Science and Technology, 22, 23-33.

292 Wilson, J.M., Bunte, R.M., & Carty, A.J. (2009). Evaluation of rapid cooling and tricaine

293 methanesulfonate (MS222) as methods of euthanasia in zebrafish (Danio rerio).

294 American Association for Laboratory Animal Science, 48, 785-789.

295 Wittmeyer, J.L., Coudron, T.A., & Adams, T.S. (2001). Ovarian development, fertility and

296 fecundity in Podisus maculiventris (Say) (Heteroptera: Pentatomidae): an analysis of

297 the impact of nymphal, adult, male and female nutritional source on reproduction.

298 Invertebrate Reproduction and Development, 39, 9-20.

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299

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1

2 Figure legends

3 Figure 1A-I. Light photomicrograph of the testicular structure and spermatogenesis in

4 Aspidomorpha sanctaecrucis. Anp = anaphase, Dsc = diplotene primary spermatocyte, Disc =

5 diakinesis, Ep = epithelium, Est = early spermatid, Fb = fat body, Ger = germarium, He =

6 head, Hn = heterochromatin condensation, Lsc = leptotene, Lst = late spermatid, MI = thin

7 muscular layer, Mp = metaphase, Psc = pachytene primary spermatocyte, PSc = primary

8 spermatocytes, SSc = secondary spermatocytes, St = seminiferous tubules, Sz = spermatozoa,

9 Ta = tail, Te = telophase, Tlc = Tunica abuginea, Ve = vas efferent, Vit = vitellarium, Zg =

10 growth, Zm = zone of maturation, Zt = zone of transformation, Zsc = zygotene.

11 Scale Bar: A, B, E, F, G, H, I = 20 µm; C, D = 50 µm.

12

13 Figure 2A-F. Light photomicrograph of the ovarian structure and differential stage of

14 oocytes (Oc) in Aspidomorpha sanctaecrucis. Ep = epithelial layer, Mul = muscular layer, Nc

15 = nurse cells, Od = ovarian duct, Ova = ovarioles.

16

17 Figure 3A-F. Light photomicrograph of the differential stage of oocytes in Aspidomorpha

18 sanctaecrucis. Ep = epithelial layer, Fc = follicle cell, Hb = large heterochromatin block, Nu

19 = nucleus, Pn = previtellogenic stage, Vg = vitellogenic stage, Yg = yolk granule.

20

21

22

23

24

25

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26

27

28 Figure 1

29

30

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31

32

33 Figure 2

34

35

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36

37

38 Figure 3

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