Aedeagus Structure of Cassida prasina Illiger, 1798 (Coleoptera: Chrysomelidae: Cassidinae) in Scanning Electrone Microscope (SEM)

It is accepted that male genitalia are not diagnostic and spermathecae are partly diagnostic within the genus Cassida Linnaeus, 1758 (Coleoptera: Chrysomelidae: Cassidinae). However, studies on genitalia have been based on only stereo microscope up to now. Ultrastructures of genitalia have never been studied except for a few works. The aim of this study was to determine whether the ultrastructural works are efficient or not. In the present work, male genitalia from two specimens of Cassida prasina Illiger, 1798 collected from Düzce and Karabük provinces in 2001 and 2003, which have been examined by electron microscope for the first time. New diagnostic characters were obtained and it revealed that it was diagnostic from the previously worked species in others subgenus. Photos of aedeagus in both SEM and stereo microscope are also given in the text. Research Article Article History Received : 08.10.2019 Accepted : 02.01.2020

The Cassidinae fauna of Turkey includes 51 species and 6 genera. The genus Cassida has 41 species arranged in 11 subgenera (Ekiz et al., 2013;. However, a total of 5 species in 5 subgenera namely; Cassida (Cassida) Kısmalı&Sassi (1994), Warchalowski (2003Warchalowski ( , 2010 and Borowiec and Sekerka (2010) from Turkey without any exact locality, but only as Asian Turkey (Anatolia). With this reason, the occurrence of these species in Turkey needs confirmation. Consequently, it can be said that the fauna of Turkish Cassida includes 36 species of 9 subgenera on the base of exact localities in Turkey.
The nominotypical subgenus Cassida (Cassida) Linnaeus, 1758 includes 46 species distributed around the world (45 Palaearctic species including the type species Cassida nebulosa Linnaeus, 1758 and one native species from North America) (Borowiec, 2007). The nominotypical subgenus is represented by 21 species in Turkey. Cassida prasina Illiger, 1798 is one of the species in the nominotypical subgenus.
According to Bordy and Doguet (1987), Borowiec and Świętojańska (2001) and Borowiec (2007), male genitalia are not diagnostic within the genus Cassida Linnaeus, 1758. Spermathecae are partly diagnostic. However, studies on genitalia have been based on only stereo microscope up to now. Ultrastructures of genitalia have never been studied except for a few recent works (Ataş et al., 2019a(Ataş et al., , 2019b. For this reason, ultrastructural investigations of aedeagi and spermathecae are very important in the genus Cassida.
Yet, we think that arrangement of the subgeneric classification in the genus Cassida on the base of aedeagal and especially spermathecal morphologies was overlooked due to this acceptance and approval. However, we believe that ultrastructural and detailed investigations of aedeagi and spermathecae could be very important in the genus Cassida with regard to subgeneric classification. The aim of this study was to present detailed investigations on aedeagus of C. prasina Illiger, 1798 from Turkey by using scanning electron microscope. Obtained data are presented below.

MATERIALS and METHODS
The available specimens (a total of 2 specimens) for the present work were collected from Düzce and Karabük provinces in Turkey in 2001 and2003. The specimens were deposited at Gazi University (Turkey, Ankara).
The aedeagus was dissected from abdomen, remaining tissue were removed with fine tweezers. For microscopic examination after cleaning, the samples were kept in 70% ethanol and examined with Olympus SZX7 stereomicroscope.
For scanning electron microscopy (SEM), cleaned samples were dehydrated using an ascending series of ethanol (70%, 80%, 90%, and 100%) and then air dried. After that the specimens were mounted onto SEM stubs using a double-sided adhesive tape, coated with gold using a Polaron SC 502 Sputter Coater, and examined with a JEOL JSM 6060 Scanning Electron Microscope (SEM) at 5 kV. The median lobe is completely light brown.
In lateral view, median lobe is generally elliptical or semicircular, from the median foramen to the apex, prominent and more or less regularly curved towards the ventral. The median lobe gradually narrows slightly from the base to the apical portion. The apex of the median lobe is thinner in the short section and is flat and pointed.
In dorsal view, the median lobe is slightly enlarged only in the apical portion. The apex is more or less prolonged and clearly truncated. In this section, the apex is curved towards the dorsal, and thus the apex appears to be truncated. The ratio of the truncated portion of the apex to the width of the apical portion of the median lobe is about 26%, more than about 1/4. Upper and lateral margins of orifice are more or less KSU J. Agric Nat 23 (3) In ventral view, median lobe slightly widened only apical part and the apex more or less prolonged and clearly truncated. Ventral surface of median lobe from median foramen to near apex with a median flattened area that broadly and longitudinally.
Median lobe is especially in anterior half with scattered, irregular and sparsely ultrastructural pits. The pits on ventral parts of median lobe are much more than on dorsal parts. The pits are located only in lateral parts of terminal part of median lobe in dorsal view. Dorsal plate and flattened area are behind it without ultrastructural pits in dorsal view. Also, the terminal area from upper margin of orifice to aedeagal apex is without ultrastructural pits in dorsal view. Apex of median lobe is folded to dorsal surface and so appears like a truncated-shaped, but not cut. Apex of median lobe is gradually narrowed, not additionally prolonged.   Ölçek çubuk boyutu 200 μm.   (Bordy, 2009'dan).
In lateral view, median lobe is distinctly and regularly curved from median foramen to apex in general. Median lobe is gradually, but slightly narrowed from the base to the apex. The apex of median lobe is almost abruptly sharpened and pointed.
In dorsal view, median lobe is barely widened from the median part to the apex, and the apex is more or less prolonged and clearly truncated. Upper and lateral margins of orifice are more or less rounded. Dorsal plate is distinct and almost covered with basal half of orifice. Median lobe is in lateral parts and the fore part of orifice is thickened. Thickening in lateral parts is smaller than the fore part. Median lobe is more or less V-shaped behind the orifice joined. -. Median lobe varies from dark brown to brown, dark brown in 3/4 of its length basally, apical part (apical fourth) is clearly brown or light brown. In lateral view, median lobe is distinctly and almost regularly (elliptically or semicircularly) curved from median foramen to apex in general; the apex of median lobe gradually narrowed and pointed; the projection of apex is longer and right. In dorsal view, median lobe is slightly widened in only apical part………...………………Cassida prasina Illiger, 1798 CONCLUSION As mentioned above, 10 aedeagal characters for Cassida nebulosa, which is the type species of the subgenus Cassida (Cassida), were determined. The type species is clearly distiguished fromCassida prasina by 4 determined aedeagal characters.
Accordingly, it could be said that the type species Cassida nebulosa and Cassida prasina are probably not congeneric.
According to the results of the present work, we believe that ultrastructural and detailed investigations of aedeagi and spermathecae will be very important in the genus Cassida with regard to subgeneric classification.