Histological and electron microscopy observations on the testis and spermatogenesis of the butterfly Dione juno (Cramer, 1779) and Agraulis vanillae (Linnaeus, 1758) (Lepidoptera: Nymphalidae)

Accepted Manuscript Title: Histological and Electron Microscopy Observations on the Testis and Spermatogenesis of the Butterfly Dione juno (Cramer, 1779) and Agraulis vanillae (Linnaeus, 1758) (Lepidoptera: Nymphalidae) Authors: Isabelle Pereira Mari, Adriana Aparecida Sin´opolis Gigliolli, Satiko Nanya, Ana Luiza de Brito Portela Castro PII: DOI: Reference:

S0968-4328(17)30401-8 https://doi.org/10.1016/j.micron.2018.03.004 JMIC 2539

To appear in:

Micron

Received date: Revised date: Accepted date:

21-10-2017 28-2-2018 19-3-2018

Please cite this article as: Mari, Isabelle Pereira, Gigliolli, Adriana Aparecida Sin´opolis, Nanya, Satiko, Castro, Ana Luiza de Brito Portela, Histological and Electron Microscopy Observations on the Testis and Spermatogenesis of the Butterfly Dione juno (Cramer, 1779) and Agraulis vanillae (Linnaeus, 1758) (Lepidoptera: Nymphalidae).Micron https://doi.org/10.1016/j.micron.2018.03.004 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.

Histological and Electron Microscopy Observations on the Testis and Spermatogenesis of the Butterfly Dione juno (Cramer, 1779) and Agraulis vanillae (Linnaeus, 1758) (Lepidoptera: Nymphalidae) Isabelle Pereira Mari1, Adriana Aparecida Sinópolis Gigliolli1, Satiko Nanya1, Ana Luiza de Brito Portela Castro1.

Departamento de Biotecnologia, Genética e Biologia Celular, Universidade Estadual de

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Maringá, Avenida Colombo, 5790, 87020-900, Maringá-PR, Brasil.

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Corresponding author

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Isabelle Pereira Mari

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Departamento de Biotecnologia, Genética e Biologia Celular, Universidade Estadual de

Fone: +55 44 30114688/+55 44 9 98199809

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Maringá, Avenida Colombo, 5790, 87020-900, Maringá-PR, Brasil.

Highlights

Sperm dimorphism is observed in Dione juno and Agraulis vanillae. Spermatogonia begin the differentiation process in third larval instar. In both species testes begin a fusion process in prepupal stage. Lacinate appendages are not found in Dione juno eupyrene spermatozoa. Brazil is the world´s largest producer and consumer of passion fruit.

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[email protected].

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E-mails: [email protected]; [email protected]; [email protected];

Abstract

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Lepidopteran species present an interesting case of sperm polymorphism and testicular fusion. The study of these features are of great importance in understanding the reproductive biology of these insects, especially in the case of those considered pests. Dione juno and Agraulis vanillae stand out as the most important pests of passion fruit (Passiflora sp.) crops in Brazil. Therefore, the objective of the present study was to characterize the testes and germ cells of Dione juno and Agraulis vanillae at different life stages, using light microscopy and scanning and transmission electron microscopy, to understand the maturation mechanisms of the male gametes in these species. The study showed that the larvae of both species have a pair of brown kidney-shaped testes, covered by epithelial cells which divide the organ into four follicles. The testes are full of spermatogonia which begin to differentiate in the third larval instar. In the fifth larval instar, spermatozoa can be observed. When they enter the prepupal stage the testes begin a fusion process that is completed in the adult insects, where they present as spherical organs divided into eight follicles, containing all the cells of the germ line. Spermatogenesis occurs centripetally, and in both species, sperm dimorphism is observed, where two different types of spermatozoa are formed, eupyrene (nucleated) and apyrene (anucleate), which differ in morphology and function. Apart from contributing to scientific basic research on the reproductive biology of these insects, the present study provides important data that can aid in research on the physiology, systematics, and control of these species.

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Keywords: Spermatozoon, sperm dimorphism, insect reproduction, passion fruit.

1. Introduction

Efficient reproductive mechanisms are one of the main factors that have contributed to the great success of insects. These mechanisms result in the production of a large number of gametes and consequently a high number of eggs and immature individuals during each cycle (Morais et al., 2009). In lepidopteran species, the male reproductive system comprises the testes (where spermatogenesis occurs), vas deferens, seminal vesicles, accessory glands,

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double and single ejaculatory ducts, and aedeagus (Duarte, 2012). In the larval period, individuals have a pair of testes, which can merges into a single organ in adults. Another

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important feature observed in Lepidoptera is the presence of a sperm polymorphism that produces two types of gametes (Mancini and Dolder, 2001; Pereira and Santos, 2015). Sperm polymorphism is not an exclusive feature of the order Lepidoptera. It can also be

observed in rotifers, turbellarians, mollusks, and other insects. This polymorphism can occur

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in different ways. For instance, in Diptera, the spermatozoa differs in length. Alternatively, in

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Coleoptera, the chromosome number differs or even morphology, which is also observed in Lepidoptera, Hemiptera, Heteroptera, and Hymenoptera (Mancini and Dolder, 2004).

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Lepidoptera exhibit another case of morphological polymorphism, where the two different

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gametes are produced. The presence or absence of a nucleus is the main feature that differentiates these two types. In addition to this marked difference, the nucleate (eupyrene)

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and the anucleate (apyrene) sperm can also differ in the presence or absence of other structures, such as the acrosome, glycocalyx, and extracellular appendages (Mancini and

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Dolder, 2001, 2004; Alves et al., 2006).

Eupyrene and apyrene spermatozoa also exhibit distinct functions. Eupyrene spermatozoa fertilize eggs, whereas the accepted function of the apyrene spermatozoa is to play a role in

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competition with sperm from other males (Silberglied et al., 1984; Friedlander et al., 2005). Apyrene differentiation appears to be regulated by a hemolymph apyrene-spermatogenesisinducing factor (ASIF), which becomes active close to, before, or after pupation, depending

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on the species. Differentiation occurs when these cells are still spermatocytes, i.e., before meiosis (Friedlander, 1997). This spermatic polymorphism was found in all butterfly and moth species studied so far, with the exception of those from the basal group Micropterygidae (Mancini and Dolder, 2004). The study of the testes and spermatogenesis are of great importance in understanding the reproductive biology of insects and can be used as a basis for related research, especially in controlling pests, such as the defoliating lepidopteran caterpillars of passion fruit (Passiflora

sp.) crops in Brazil. Brazil is the world’s largest producer and consumer of the fruit. In 2016, production reached 703,489 tons with a revenue of R$ 1,028.998 (Brazilian Institute of Geography and Statistics, 2016). Dione juno and Agraulis vanillae stand out for their potential to cause substantial economic damage, because an infestation may result in the loss of an entire crop (Peña et al., 2002; Joy and Sherin, 2013; Picanço, 2010). Both species belong to the Heliconiinae subfamily and are orange-colored butterflies with black spots on the wings, and a wingspan of approximately 6 cm. After copulation, the female

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of these species may lay up to 150 eggs either in a cluster or in an isolated fashion. After

hatching, the caterpillars begin to feed on the leaves of the host plant (Tavares et al., 2002;

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Picanço, 2010).

In the present study, we investigated the morphology and ultrastructure of the testes, along with the spermatogenesis process in D. juno and A. vanillae with a special focus on the organization and characterization of germ cells in the testes. To our knowledge, this is the

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first work to study these aspects of these two species of Lepidoptera.

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2. Materials and methods

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2.1. Insects

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Specimens of Dione juno and Agraulis vanillae (eggs and immature individuals) were obtained from passion fruit plants in the cities of Maringá (23º25’30’S and 51º56’20’O) and

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Terra Boa (23º76’87’S and 52º44’74’O) in Paraná state, Brazil, a map showing the location of the cities can be observed in Fig. 1, that was developed on the program QGIS 2.0. The specimens were kept in polypropylene boxes at room temperature and 70 ± 10% relative

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humidity with 12 h photoperiods, and fed with plant leaves. The larval instars were identified through cephalic capsule measurements according to Tavares et al. (2002) and Silva et al. (2006). Individuals were treated with cold anesthesia and dissected in saline solution (0.1 M

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NaCl, 0.1 M Na2HPO4, and 0.1 M KH2PO4).

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Fig. 1. Map showing the location of cities where specimens of Dione juno and Agraulis vanillae (eggs and immature individuals) were collected.

2.2. Light microscopy

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For light microscopy the testicles of 10 larvae in third instar, 5 larvae in fifth instar, 5

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prepupae and 10 adults of each species were dissected and fixed in aqueous Bouin’s solution

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(Picric acid 7.5%, formaldehyde 2.5%, acetic acid 0.5%) for 6 h. Samples were dehydrated in a series of increasing concentrations of alcohol (70%, 80%, 90%, and 100%), cleared in

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xylene, embedded in histological paraffin, and cut into 6µm thick sections with a Leica RM 2250 microtome. The sections were stained with hematoxylin and eosin (HE) (Junqueira and

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Junqueira, 1983) and periodic acid Schiff (PAS) (Pearse, 1961). The analyses were examined

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on an Olympus microscope and imaged with a Motican 2300 digital camera.

2.3. Scanning electron microscopy

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The testicles of 5 larvae in third instar and 5 adults of each species were isolated and

fixed in aqueous Bouin’s solution for 24h at room temperature. The samples were post-fixed in 1% osmium tetroxide indistilled water for 30 min, dehydrated in a series of increasing

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concentrations of ethanol (7.5%, 15%, 30%, 50%, 70%, 90%, and 100%), dried with the Leica CPD030 Critical Point Dryer, and covered with gold dust using sputter coated with a thin layer of gold of a thickness of 60 nm on Shimadzu IC-50 metalizer. The analyses were performed

using a Quanta 200-Fei SEM at the Microscopy Centre of the Complex of Research Support Center (COMCAP) of the State University of Maringá, Paraná, Brazil (Gigliolli et al., 2015).

2.4. Transmission electron microscopy

The testicles of 5 adults of each species were isolated and fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) for 24 h at room temperature and post-fixed in 1% osmium tetroxide in the same buffer for 2 h. Samples were then washed in distilled water, contrasted with an aqueous solution of 0.5% uranyl acetate for 2 h, dehydrated in a series of increasing concentrations of acetone (50%,70%,

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90%, and 100%), and embedded in resin (Araldite®). Ultrathin sections were contrasted in a

saturated alcoholic solution of uranyl acetate and lead citrate and analyzed using a JEOL

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JEM-1400 TEM at the COMCAP of State University of Maringá, Paraná, Brazil (Gigliolli et al., 2015).

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3.1. Testicular morphology and histological analysis

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3. Results

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The testes and germ cells of Dione juno and Agraulis vanillae display common

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features; however, structural modifications have evolved for the two species (Figs. 2, 3 and 4).

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Larvae of D. juno and A. vanillae have a brown kidney-shaped pair of testes, aerated by several tracheoles and located laterodorsally in the median region of the body (Figs. 2a-c).

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In the prepupal stage, the testes begin a fusion process (Fig. 2d) and in adults, they form a unique, completely fused organ, located between the fourth and fifth abdominal segments (Fig. 2e). In this stage, it presents a rounded format, dark brown in color, is aerated by several

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tracheoles, and connects to the rest of the reproductive system by a pair of vas deferens (Figs.

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2e-g).

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Fig. 2. (a) Light microscopy from Dione juno larvae in third instar showing a pair of testes (arrows), digestive tube (dt) and fatty tissue (Ft). (b) Scanning electron microscopy of the testes from D. juno and (c) Agraulis vanillae larvae in third instar showing that the organ is divided into four follicles (Fo(1-4)) and is aerated by several tracheoles (tc). (d) Light microscopy from A. vanillae prepupae showing the begining of the testicular fusion (arrow) and fatty tissue (Ft). (e) Light microscopy from D. juno adult showing the comppletely fused testis (arrow) and fatty tissue (Ft). (f) Scanning electron microscopy of the testis from D. juno, conecting with a pair of vas deferens (vd), in detail, a tracheole, and (g) A. vanillae adult showing the spherical fused testes, tracheoles (tc) and vas deferens (vd). Bars: a= 1cm; d, e= 3 cm; b, c= 300 µm; f, g= 500 µm.

In the larval period, each testis is coated by an epithelium; this emits septa that divide the testis into four testicular follicles (Figs. 3a-d). The epithelial cells have PAS-positive granular cytoplasm, which can be indicative of the role of these cells in glycogen storage (Fig.

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3b).

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Fig. 3. (a and c) Histological sections of the testes from Dione juno larvae in third instar stained by HE showing the coating epithelium (arrow), testicular folicles (Fo(1-4)), apical cell (ac), spermatogonia (Eg) and spermatocytes (Et). (b) Histological section of the testis from Agraulis vanillae larvae in third instar stained by PAS showing the coating epithelium PAS positve (arrow) and apical cell (ac). (d) Histological section of the testis from D. juno larvae in fifth instar stained by HE showing coating epithelium (arrow), spermatogonia (Eg), spermatocyte (Et) and spermatozoa (Ez). (e and f) Histological sections of the testes from A. vanillae prepupae showing the beginning of the fusion process, germ (G), spermatozoa (Ez), coating epithelium (arrow), folicular septa (fs), spermatogonia (Eg), spermatocyte (Et) and spermatozoa (Ez). (g) Histological sections of the testis from D. juno adult stained by HE showing the fusion process complete and the organ divided into eight follicles ((Fo(1-8)). (h) Histological sections of the testis from A. vanillae adult stained by HE showing the germ (G) and spermatozoa (Ez). Bars: a,e,g,h= 200 µm; b,d,f= 100 µm; c= 20 µm.

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In each testicular follicle, the germ cells are organized in cysts formed of somatic cells. In third instar larvae, spermatogonia are observed, grouped around an apical cell, forming dense and homogeneous cysts (Figs. 3b, c). The spermatocytes formed from successive spermatogonial mitotic divisions can also be seen (Fig. 3c).

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In fifth instar larvae, the testes increase in size, because in addition to spermatogonia

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and spermatocytes, they contain spermatid cysts, cells originated from meiotic division of spermatocytes, and spermatozoa, formed by the spermiogenesis; process of modifications to

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the spermatids (Fig. 3d).

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In prepupae, the same cells are found (Figs. 3e, f); however, because in both species, each testis begins the fusion process (Figs. 2d; 3e), when it reaches adulthood it becomes a

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unique structure (Figs. 2e, f, g) containing eight follicles (Figs. 3g, h). The coating epithelium

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has two layers of cells (Figs. 4a, b), with PAS-positive granular cytoplasm (Figs. 4d, g, h).

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Fig. 4. (a, c, e, g ) Histological sections of the testes from Dione juno adult stained by HE showing the outer lining and the internal lining epithelium (arrows), spermatogonia (Eg), spermatocyte (Et), nucleus (n), folicular septa (fs), eupyrene spermatids (eEm), apyrene spermatids (aEm), eupyrene spermatozoa (eEz), apyrene spermatozoa (aEz), cyst cell (cc) and glycogen granules (gg). (b, f, h ) Histological sections of the testes from Agraulis vanillae adult stained by HE showing the caoting epithelium (arrow), spermatogonia (Eg), spermatocyte (Et), nucleus (n), eupyrene spermatozoa (eEz), apyrene spermatozoa (aEz), cyst cell (cc) and glycogen granules (gg). (d) Histological sections of the testis from A. vanillae stained by PAS showing apyrene spermatids (aEm), folicular septa (fs) PAS positive and glycogen granules (gg). Bars: 20 µm.

It is possible to observe that germ cells are organized in cysts in the testis follicular lumen, and in different positions according to degree of maturation. This maturation occurs

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centripetally, i.e., the more differentiated the cell, the more centrally in the testis it is located (Figs. 3g, h).

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In both species, spermatogonia can be observed surrounding the entire testis, and just below are the spermatocytes (Figs. 4a, b). It is also possible to observe two distinct cysts of spermatids, where the cells are elongating to form spermatozoa (Figs. 4c, d).

On spermatids that will form eupyrene spermatozoa, the elongation process occurs in

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the opposite direction from the nucleus (Fig. 4c), whereas on spermatids that will form

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apyrene spermatozoa, the elongation occurs randomly (Figs. 4c, d). The spermatogenesis is dimorphic in both species, with two types of spermatozoa

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(nucleated and anucleate) produced (Figs. 4e-h). Each cyst has just one type of spermatozoon

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and these can be distinguished by the presence of an elongated and strongly basophilic

not seen (Figs. 4g, h).

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nucleus in the eupyrene cysts (Figs. 4e, f), whereas in the apyrene cysts, this characteristic is

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3.2. Transmission electron microscopy analysis

The spermatogonia are relatively large cells with a well delimited plasma membrane.

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The nucleus is rounded with a regular nuclear membrane; this has visible euchromatin, and many heterochromatin nodules. In a cyst, some cells have a nucleolus whereas others do not. The cytoplasm contains a large amount of smooth endoplasmic reticulum, mitochondria, and

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the Golgi apparatus (Figs. 5a-d).

IP T SC R U N A M ED PT CC E A Fig. 5. Transmission electron microscopy from the male germ cells of Dione juno and Agraulis vanillae adults. Spermatogonia cysts from (a) D. juno and (b) A. vanillae showing nucleus (n), nucleolus (nu) and the cyst cell nucleus (ccn). Spermatogonia from (c) D. juno and (d) A. vanillae showing nucleus (n), mitochondria (mt), Golgi apparatus (ga) and endoplasmic reticulum (er). Spermatocytes from (e) D. juno and (f) A. vanillae showing the concentration of mitochondria (mt) and smooth endoplasmic reticulum (ser). Bars: a= 2 µm; b= 5 µm; c,d= 1 µm; e, f= 2 µm.

The spermatocytes are larger cells with an irregular plasma membrane. It was observed that the mitochondria, present in large numbers, start to organize themselves in a specific region in the cytoplasm. The Golgi apparatus is evident and the smooth endoplasmic reticulum is developed. In the nucleus, both euchromatin and heterochromatin are evident and the nucleolus is well developed (Figs. 5e, f). The eupyrene spermatids still have abundant cytoplasm with a bulky and spherical nucleus, composed largely of euchromatin. At this stage, the mitochondria are seen to merge,

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giving rise to a large spherical structure called the nebenkern, composed of an electron-dense matrix and electrolytic cisterns (Figs. 6a-c). As they mature, spermatids undergo compaction

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and the nucleus elongates with the cell morphology (Fig. 6a). Cytoplasm is lost, and at the

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end of spermiogenesis the spermatozoa are formed (Figs. 6d-i).

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Fig. 6. Transmission electron microscopy of eupyrene germ cells from Agraulis vanillae and Dione juno adults. (a and b) Eupyrene spermatids from A. vanillae and (c) D. juno showing the beginning of the elongation process, nucleus (n) and nebenkern (nk). Cross section of the anterior region from (d) A. vanillae and (e) D. juno spermatozoa showing nucleus (n), acrosome (a), reticular appendage (r) and lacinate appendage (l). Cross section of the spermatozoa tails from (f and h) A. vanillae and (g) D. juno showing mitochondrial derivatives (md), axoneme (ax), reticular appendage (r) and lacinate appendage (l). (i) Eupyrene spermatozoa cyst from A. vanillae. Bars: a= 2 µm; b,e= 1 µm; c,d= 0,5 µm; f,g= 0,2 µm; h= 0,1 µm; i= 2 µm.

The eupyrene spermatozoa have a nucleus with condensed chromatin, and the

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acrosome appears in transverse sections in the form of a “v,” covering the anterior face of the

nucleus (Fig. 6d). In both the head and tail regions, there is a structure called the reticular

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appendage, a rounded and electron-dense tube formed from extensions of the plasma membrane (Figs. 6d-h).

In A. vanillae, another type of appendage is found; these are also formed from extensions of the plasma membrane and are called lacinate appendages. They appear in the

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form of rays, and their quantity varies for each spermatozoon (Figs. 6d, f). Sections of the tail

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show the axoneme organized in 9+9+2 form: nine accessory, nine peripheral, and two central microtubules. Yet, for all the extension of the tail, there are two adjacent mitochondrial

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derivatives from the division of nebenkern during spermiogenesis (Figs. 6f-h). In both

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species, the eupyrene gametes remain inside cysts, where they are inserted in invaginations of the plasma membrane, and this is how they leave the testis (Fig. 6i).

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The apyrene spermatids lose their nuclei to form the anucleate spermatozoa and, in this way, the modifications that follow and the result of the spermiogenesis process are

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different. Spermatids have an irregular shape, a larger volume of cytoplasm, and the presence of several micronuclei originating from the fragmentation of the initial nucleus, which are

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wrapped in vacuoles and eliminated from the cell by exocytosis (Figs. 7a, b).

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Fig. 7. Transmission electron microscopy of apyrene germ cells from Agraulis vanillae and Dione juno adults. Spermatids from (a) A. vanillae and (b) D. juno showing the presence of several micronuclei (mn) and the axoneme (ax). (c) Longitudinal section of the anterior region from A. vanillae spermatozoa showing the eletron-dense layer (c). (d) Cross section of the anterior region from D. juno spermatozoa showing the eletron-dense layer (c) and the axoneme (ax). Cross section of the spermatozoa tails from (e and g) A. vanillae and (f) D. juno showing the axoneme (ax), two mitochondrial derivatives "shoe sole" shaped (md) and microtubules (black arrows). (h) Apyrene spermatozoa cyst from D. juno. Bars: a,b,d= 1 µm; c= 0,5 µm; e= 0,2 µm; f,g= 0,1 µm; h= 1 µm.

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Apyrene spermatozoa have an anterior region composed of an electron-dense cape (Figs. 7c, d), and, just like in the eupyrene gametes, the tail is formed by the axoneme organized in

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9+9+2 form (Figs. 7e-g). Two mitochondrial derivatives extend parallel to the axoneme and

they appear "shoe sole" shaped, with an electron-dense region at one end (Figs. 7e-g). Similar to eupyrene gametes, the apyrene gametes remain inside cysts, where they are also inserted in

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invaginations of the plasma membrane, and this is how they leave the testis (Fig. 7h).

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4. Discussion

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The anatomies observed in D. juno and A. vanillae are similar to those cited for other

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lepidopteran species. Table 1 compares the main features observed in the testes of the studied

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species with those from other Lepidoptera.

Table 1. Main features observed in the testes of some lepidopteran species.

FUSION

COLOR

LOCATION

NUMBER OF FOLLICLES IN LARVAE

NUMBER OF FOLLICLES IN ADULTS

Agraulis vanillae

Prepupae

Dark brown

Median region of the body

Four

Eight

Atrophaneura alcinous (Kubo-Irie et al., 1998)

Pupae

Reddish

Median region of the body

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Boarmia selenaria (Scheepens and Wysoki, 1984)

Prepupae

White

5th abdominal segment

Four

-

Bombyx mori (Ômura, 1936)

Does not occurs

-

5th abdominal segment

Four

Four

Diatraea saccharalis (Bilha et al., 2012)

Pupae

White

5th abdominal segment

Four

Ten or Fourteen

Dione juno

Prepupae

Dark brown

Median region of the body

Four

Eight

Ephesia kuehniella (Garbini and Imberski, 1977)

Prepupae

-

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Four

Galleria mellonella (Polanska et al., 2005)

5th larval instar

-

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Four

Grapholita molesta (Morais et al., 2009)

Prepupae

Yellow to violet.

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Heliothis virescens (LaChance and Olstad, 1988 )

Prepupae

-

-

Trichoplusia ni (Holt and North, 1969)

Prepupae

-

-

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SPECIES

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Eight

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The presence of one pair of kidney-shaped testes in larvae and unique round testes in adults, has been already described in several species, such as Trichoplusia ni (Holt and North,

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1969), Ephesia kuehniella (Garbini and Imberski, 1977), Boarmia selenaria (Scheepens and Wysoki, 1984), Heliothis virescens (LaChance and Olstad, 1988), Atrophaneura alcinous

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(Kubo-Irie et al., 1998), Galleria mellonella (Polanska et al., 2005), Grapholita molesta (Morais et al., 2009) and Diatraea saccharalis (Bilha et al., 2012).

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In most of these species, testes fusion occurs during the pupal stage, except in G. mellonella, where the testes are already fused in the last larval instar. However, testes fusion

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is not found in all lepidopteran species; it does not occur in the primitive groups such as Hepialidae family and Zeugloptera suborder, of which Bombyx mori is included (Ômura, 1936; Friedlander et al., 2005). Testes coloration varies according to species. In Diatraea saccharalis, they are whitish; in

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Atrophaneura alcinous, reddish; whereas in G. molesta, they vary from yellow to violet. However, the dark brown to black coloration observed in D. juno and A. vanillae has not been described previously in any other species. Both in larvae and adults, the testis localization and the presence of many tracheoles is conserved, as is seen in B. mori (Ômura, 1936), Ostrinia nubilalis (Drecktrah, 1966), Atrophaneura alcinous (Kubo-Irie et al., 1998) and Diatraea saccharalis (Bilha et al., 2012).

Histological analyses demonstrated that both in larvae and adults, the testes are enveloped by an outer lining epithelium also called the outer layer, connective membrane, peritoneal sheath, or outer tunic. An internal lining epithelium is also observed, which is called the lobular capsule, inner layer, follicular epithelium or inner tunic. The tracheoles, tracheal ramifications, are inserted in these epithelia and are responsible for testes aeration (Ômura, 1936; Caspari and Blomstrand, 1958; Pereira and Santos, 2015). In D. juno and A. vanillae larvae, the follicular epithelium produces septa that divide the

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testis into four follicles or lobes, whereas eight follicles are observed in adults, resulting from

testis fusion during the pupal stage. The same configuration is described in Diatraea

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saccharalis, whereas in other species such as Euptoieta hegesia, Ephesia kuehniella, and T. ni, the presence of four follicles is observed in larvae, whereas in adults, the number is variable (Holt and North, 1969; Mancini and Dolder, 2004; Pereira and Santos, 2015). According to King and Akai (1982), the cells comprising the coating epithelia and septa

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contain large amounts of glycogen particles, which can be observed by PAS staining. The

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function of this storage is still unknown for these insects; however, it is already well established that spermatogonia of rats can use glucose as the main energy substrate

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(Villarroel-Espíndola et al., 2015), which supports the theory of Ômura (1936) that this

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glycogen storage functions as a source of nutrients for germ cells during spermatogenesis. Within the testicular follicles of D. juno and A. vanillae are the germ cells, organized in

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cysts formed by somatic cells of mesoderm origin. Cell maturation happens in a synchronous way inside each cyst, and in a centripetal way inside each follicle, that is, towards the center,

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where the vas deferens are located and where the mature sperm leave the testis as observed in E. hegesia and Diatraea saccharalis (Mancini and Dolder, 2004; Pereira and Santos, 2015). According to Friedlander et al. (2005), Polanska et al. (2005) and Simmons, (2013)

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spermatogenesis can be divided into: growing zone, where spermatogonia dividing and increasing in size to form spermatocytes; maturation and reduction zone, where each spermatocyte undergoes two meiotic divisions to form the spermatids; and finally, the

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transformation zone, where spermatids pass through the spermiogenesis process, producing the mature spermatozoa. Both D. juno and A. vanillae larvae have a region known as the germarium, located inside the testicular follicles, in which an apical cell is surrounded by innumerable spermatogonia, as observed in E. kuehniella (Garbini and Imberski, 1977). The apical cell seems to originate from the follicular epithelium, and functions to provide support and nutrition to spermatogonia. These cells are observed not only in the order Lepidoptera, but also in insects

of the orders Orthoptera, Blattodea, and Homoptera (Garbini and Imberski, 1977; Simmons, 2013). Spermatogonia are relatively large cells, and as the insect develops, their frequency decreases and they occupy a peripheral position in the testis. They have many mitochondria in their cytoplasm and the heterochromatin is distributed evenly within the nucleus (Lai-Fook, 1982; Mancini and Dolder, 2004; Pereira and Santos 2015). According to Lai-Fook (1982) and Friedlander et al. (2005) spermatogonia proliferate

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throughout the life of Lepidoptera, and this can be observed in the studied species; however,

according to Holt & North (1969), these cells stop their proliferation when individuals reach

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adulthood. Despite these differences, it is a consensus among the authors that each

spermatogonium will undergo six consecutive mitotic divisions, creating 64 spermatocytes, and only then will undergo two meiotic divisions, creating 256 spermatids that will develop into 256 mature spermatozoa.

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As spermatogonia proliferate, they leave the germarium and associate themselves with

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somatic cells originating the spermatocyte cysts, which can be observed in the third larval instar. Spermatocytes have a great volume of cytoplasm, a developed nucleus with dispersed

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chromatin, heterochromatin positioned close to the nuclear envelope, and quite an evident

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nucleolus. In the cytoplasm, the smooth endoplasmic reticulum and the Golgi apparatus are very well developed because they play an important role in the acrosome formation of

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spermatozoa. Mitochondria are present in large quantities grouped in a specific region; this happens because during spermiogenesis, these organelles fuse, giving rise to a large spherical

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structure called the "nebenkern" (Pratt, 1968; Mancini and Dolder, 2004; Simmons, 2013; Pereira and Santos, 2015).

The nebenkern is observed in all lepidopteran species already studied, as well as in other

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insect orders, such as Diptera and Hemiptera. The nebenkern extends adjacent to the axonema, and in the spermatozoa, they divide, giving rise to two mitochondrial derivatives, which provide energy for flagellar movement (Pratt, 1968; Mancini and Dolder, 2004;

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Simmons, 2013; Pereira and Santos, 2015). According to Gattardo et al. (2016), the fusion of the mitochondria into mitochondrial derivates is a typical feature of Hexapoda, and two are the number found in most groups of insects. According to Friedlander et al. (2005), even before the first meiotic division occurs, it is possible to identify whether a cyst will develop eupyrene or apyrene gametes. This differentiation appears to be related with the active of a haemolymph apyrene-spermatogenesisinducing factor produced by the somatic cells of the cyst close to pupation, before or after,

according to the species. So the cells would already be programmed to give rise to eupyrene or apyrene sperm before going through the meiosis process (Friedlander, 1997). Although, in D. juno and A. vanillae, this differentiation is only visually possible after the cells undergo the two meiotic divisions, i.e., when they are already considered spermatids, which is also observed in Calpodes ethlius (Lai-Fook, 1982). At this stage, identification is possible because the chromatin is reorganized into a dense and typical nucleus in eupyrene spermatids,

are eliminated from the cell during spermiogenesis (Lai-Fook, 1982).

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whereas in the apyrene spermatids, the chromatin is organized in several micronuclei, which

The sperm dimorphism found in D. juno and A. vanillae can be observed in almost every

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lepidopteran species, even in those from older groups. Exceptions are found in species of the

genus Micropterix within the suborder Zeugloptera (Friedlander et al., 2005). Dichotomous spermatogenesis seems to be a feature which developed independently within the order, because apyrene spermatozoa do not occur in the Trichoptera, or in related orders like Diptera

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and Siphonaptera (Friedlander et al., 2005).

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Anucleate spermatozoan function is not yet fully understood; however, many hypotheses have been developed to try to explain the apparent costly production of these gametes,

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amongst them, nutrition, transport and competition, this latter being the most accepted.

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Apyrene spermatozoa features such as reduced size, faster maturation, high production, and motility in the male reproductive system, support this hypothesis; it is also proven that these

Friedlander et al., 2005).

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are the first to reach the spermathecae (Silberglied et al., 1984; Swallow and Wilkinson, 2001;

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Sperm formation through the spermiogenesis process follows different events for the different types of gametes. During the apyrene spermiogenesis, the nucleus is lost, the nebenkern is formed and, subsequently, mitochondrial derivatives form, an electron-dense

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layer develops in the anterior region, and the tail elongates (Mancini and Dolder, 2004). In D. juno and A. vanillae, the micronuclei appear in a range of sizes, which is also observed in E. hegesia (Mancini and Dolder, 2004), however in Alabama argillacea (Medeiros, 1997), the

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micronuclei present only two distinct forms. In D. juno and A. vanillae, the micronucleus elimination occurs by exocytosis, which is

also seen in E. hegesia (Mancini and Dolder, 2001). In B. mori, peristaltic contractions are responsible for eliminating these structures (Yamashiki and Kawamura, 1997). Other features that seem to remain in the Lepidoptera include the presence of an electron-dense layer on the anterior region of the spermatozoa; this is formed from the basal body, from which the

axoneme extends posteriorly (Medeiros, 1997; Yamashiki and Kawamura 1997; Lai-Fook, 1982; Mancini and Dolder, 2004). In eupyrene spermatogenesis, modifications occur in nuclear morphology, nebenkern formation and mitochondrial derivatives, the appearance of extracellular appendages, and tail elongation (Mancini and Dolder, 2004). In the nucleus, the euchromatin concentrates in the center, whereas the heterochromatin is distributed around the periphery. In spermatozoa, the nucleus becomes elongated, the chromatin becomes dense and homogeneous, as can be

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observed in E. hegesia (Mancini and Dolder, 2004) and C. ethlius (Lai-Fook, 1982). The

lacinate and reticular appendages are structures exclusive to eupyrene gametes and order

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Lepidoptera, being found in almost all the studied species, with the exception of the genera Micropterix and Korscheltellus (Alves et al., 2006).

The lacinate appendages are a peculiar type of glycocalyx, which take different forms depending on the species, and vary in number in a single individual, it is recording for some

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members of Lepidoptera, however is absent in Micropterix (Phillips, 1970; Gattardo et al.,

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2016). One of the appendages is visibly different to the others, and forms a dense tube; this is known as the reticular appendage. The appendages disappear when the spermatozoa leave the

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testis, coinciding with the appearance of an amorphous material around the gametes, raising

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the hypothesis that the appendages contribute to the formation of this material, that keeps the eupyrene spermatozoa together in the vas deferens (Phillips, 1970).

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Transmission microscopy analyses have not shown lacinate appendages in D. juno; this fact can be attributed either to the tissue sectioned, which would indicate that these

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appendages are not evenly distributed throughout the extension from the gamete, or even that they are absent in this species.

To our knowledge, this is the first study to describe testes morphology and

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spermatogenesis in D. juno and A. vanillae. Anatomical and morphological aspects observed were similar for the two species, and follow the patterns previously described for the order Lepidoptera. In the present work it was possible know important features about the

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reproduction process in these species, such the maturation stages of the germ cells, presence of testicular fusion and sperm polymorphism. In addition to contributing to basic scientific research related to insect reproductive biology, the present study provides important data that can assist in research on the physiology, systematics, and control of these species.

Acknowledgements

We thank of the Centro de Microscopia Eletrônica (CME) at the Universidade Estadual Paulista (UNESP), Botucatu, SP and Centro de Microscopia (CMI) of Complexo de Centrais de Apoio à pesquisa (COMCAP) at the Universidade Estadual de Maringá (UEM), Maringá, PR in processing material used and assistance inhandling the equipment. This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

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(CAPES).

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