Distributed under Creative Commons Cc-by 4.0 Fine Structure of the Spermatozoon in Three Species of Cambaridae (arthropoda: Crustacea: Decapoda) Cambarus Robustus, Orconectes Propinquus and Orconectes Rusticus: a Comparative Biometrical Study

The ultrastructure of spermatozoa in three species of cambarid crayfish, Cambarus robustus, Orconectes propinquus, and Orconectes rusticus, were studied and compared with eight previously studied species from different crayfish families using morphological features and biometrical data. The ultrastructure of spermatozoa show a generally conserved pattern including an acrosome and nucleus in the anterior and posterior parts of the cell, respectively, radial arms that wrap around the nucleus, and the whole cell is enclosed by an extracellular capsule. The most outstanding morphological feature in spermatozoa of three studied cambarid crayfish is the crest-like protrusions in the anterior part of the acrosome that can be used as one of the features for distinguishing the members of this family. Results of biometrical data reveal that acrosome size in the representatives of Parastacidae are the smallest, while representatives of Astacidae show the biggest acrosome. The acrosome size in species belonging to Cambaridae occupy an intermediate position between the two other families of freshwater crayfish. In conclusion, a combination of morphological features and biometrical data of spermatozoa can help distinguishing different species of the freshwater crayfish.


INTRODUCTION
Non-motile spermatozoa of decapods are very diverse in their morphology and that makes them suitable cases for phylogenetic studies (Jamieson, 1991;Jamieson & Tudge, 2000;Tudge, 2009;Klaus & Brandis, 2011;Braga et al., 2013). Currently, studies investigating decapod crustacean sperm morphology cover 100% of the decapod infraorders, 50% of the families, approximately 10% of the extant genera, but only 2% of the described, extant species (Tudge, 2009). Freshwater crayfish are highly diverse and commercially and ecologically important animals currently comprising 3 families, 33 genera, and over 640 known species (Crandall & Buhay, 2007). In crayfish, aflagellate spermatozoa bear a relatively large acrosome in the anterior part and a nucleus in the posterior containing extensions of microtubular radial arms (Moses, 1961a;Moses, 1961b;Dudenhausen & Talbot, 1982). Spermatozoa of the freshwater crayfish have already been the subject of many ultrastructural studies (Table 1). The different dimensions of the most prominent organelle in crayfish spermatozoa, the acrosome, have been used for taxonomic studies, as well as the presence of some morphological features, especially in the anterior part of the spermatozoon, such as the spike, apical zone and crest can help distinguishing spermatozoa from different species (Yasuzumi & Lee, 1966;Anderson & Ellis, 1967;Niksirat et al., 2013a;Niksirat et al., 2013b;Kouba, Niksirat & Bláha, 2015).
The objective of the present study is to compare spermatozoal ultrastructure in three cambarid crayfish with other members of crayfish via morphological features and biometrical data.

Morphological features
The acrosome complex, as the most prominent organelle, is located in the anterior part of the spermatozoon. It consists of two distinct components including the main body of the acrosome vesicle and the subacrosomal zone (

Morphological features
The results of the present study show that the general morphology of spermatozoa in studied members of Cambaridae is similar to other crayfish including a relatively large acrosome, nucleus and radial arms that are enclosed by an extracellular capsule (Jamieson & Tudge, 2000;Vogt, 2002). Radial arms are usually the extensions of the nucleus that wrap around the acrosome vesicle. These arms are present in Astacidae and Cambaridae, but not in studied Cherax species (Beach & Talbot, 1987;Kouba, Niksirat & Bláha, 2015). The radial arms in decapod spermatozoa may be composed of microtubules, nuclear material, or both (Tudge, 2009). Molecular studies identified tubulin proteins, as major units of microtubules in the proteomic profile of the crayfish male gamete that confirms the microtubular nature of radial arms (Niksirat et al., 2014a;Niksirat et al., 2015b) and as seen in the TEM images in this study. Although, microtubular radial arms undergo protein tyrosine phosphorylation during spermatophore post-mating storage on the body surface of female crayfish (Niksirat et al., in press), the exact role(s) of radial arms in fertilization is yet to be determined. The extracellular capsule seems to be an envelope for tight compaction of long organelles such as radial arms. This hypothesis is further supported by the absence of a capsule in the studied Cherax spermatozoa, where radial arms are not present (Beach & Talbot, 1987;Vogt, 2002;Kouba, Niksirat & Bláha, 2015). The membranous lamella is an organelle that has been reported in spermatozoa of several crayfish (Jamieson & Tudge, 2000). It has been observed that some mitochondria lose their internal matrix and are transformed into membranous lamellae during the early spermatid stage of the crayfish Cambarus sp. which is still able to provide energy to the cell (André, 1962;Anderson & Ellis, 1967). A positive staining of Janus green B, an indicator of active mitochondria, in the same area of the crayfish spermatozoon has been reported (André, 1962). Several proteins related to metabolism and energy production were identified in the protein profile of the crayfish male gamete that may confirm the presence of an energy supply center in the sperm cell (Niksirat et al., 2014a;Niksirat et al., 2015b). A set of electron lucent pores, a unique morphological feature, have only been reported at the margins of the main body of the acrosome in P. leniusculus (Niksirat et al., 2013b). The anterior-most margin of the main body of the acrosome vesicle in different crayfish species showed a diversity in shape that can be used as an important morphological feature for distinguishing different species of freshwater crayfish (Niksirat et al., 2013a;Niksirat et al., 2013b;Kouba, Niksirat & Bláha, 2015;present study). For example, in Cambaridae, a horn-like spike was observed in the anterior part of the fully developed spermatozoon of Cambaroides japonicus (Yasuzumi & Lee, 1966). A similar spike-shaped structure has been reported in spermatozoa of Cambarus sp. (Anderson & Ellis, 1967) and Procambarus leonensis (Felgenhauer & Abele, 1991). While several other studies on spermatozoal ultrastructure and spermatogenesis in Procambarus (Moses, 1961a;Moses, 1961b;Hinsch, 1992;Hinsch, 1993a;Hinsch, 1993b) did not report an acrosomal spike, development of a spike in the anterior part of the acrosome vesicle has been observed in Procambarus clarkii when the spermatozoa are inside the vas deferens (Niksirat et al., 2013a). An apical zone, an area filled with bundles of curled filaments has been reported in A. astacus, A. leptodactylus, A. torrentium, P. leniusculus (Pochon-Masson, 1968;López-Camps et al., 1981;Niksirat et al., 2013a;Niksirat et al., 2013b). Those filaments and some material originating from the acrosome are released outside the spermatozoon and form a new formation called filament-droplet structure that could facilitate egg-spermatozoon binding during fertilization in crayfish (Niksirat, Kouba & Kozák, 2014b). In the present study, crest-like protrusions observed in the anterior part of the acrosome vesicle of spermatozoa can be used as one of the morphological features for distinguishing cambarids from other species of freshwater crayfish.

Biometrical data
Results of acrosome measurement in the spermatozoa of eleven species of freshwater crayfish show that despite some similarities, a combination of different acrosome dimensions (length, width, and length:width ratio) can be useful for distinguishing different species of crayfish. The length:width ratio of the acrosome vesicle has been applied to divide crustaceans into three different categories: depressed (<1), spherical (1) and elongated (>1). The eleven species of crayfish fall into the depressed acrosome category sharing this position with a few thoracotreme and heterotreme brachyurans, all investigated podotreme brachyurans, some astacid, palinurid and enoplometopid lobsters (Jamieson, 1991), and Pylocheles (Bathycheles) from the Anomura (Tudge, Scheltinga & Jamieson, 2001).