X-ray microtomography (microCT) of male genitalia of Nothybus kuznetsovorum (Nothybidae) and Cothornobata sp. (Micropezidae)

Abstract The results of manual dissection of the musculature of the male genitalia in Nothybus kuznetsovorum are fully confirmed by the modern methods of Micro-CT. A comparative analysis of Neria commutata and Cothornobata sp. shows that an increase in the flexion in the genitalia of males and the displacement of syntergosternite VII to the ventral side in Cothornobata sp. caused the disappearance of the muscles ITM6–7r and ITM7–8r. In addition, this increase in flexion apparently caused the fusion of the M18 muscles into one bundle. The muscle ISM5-6c goes on to moving the second segment of the forcipate appendages of sternite V.


Introduction
Micropezidae is an average-sized family of acalyptrate flies (Diptera). It comprises approximately 700 described species in 50 genera (Marshall 2010(Marshall , 2012Ferro and Carvalho 2014). The family is globally distributed, with the greatest species richness found in tropical regions (Steyskal 1968). The body of adult flies is 3.5 to 20.0 mm long (Steyskal 1987).
The family Nothybidae includes only one genus, Nothybus Rondani, 1875, distributed in the Oriental Region. These acalyptrate flies are 5.5 to 15.0 mm body long (Lonsdale and Marshall 2016).
Study of the musculature is helpful not only for specifying the functions of genital sclerites, but also for revealing the homology of some poorly traced structures (Ovtshinnikova 1989, 1994, Ovtshinnikova and Yeates 1998Galinskaya and Ovtshinnikova 2015).
Among the Diptera the X-ray micro-computed tomography (Micro-CT) was used for revealing the morphology of the feeding apparatus of Philoliche rostrata and Ph. gulosa (Tabanidae) (Karolyi et al. 2014); for the characterization of the morphology of the venom system of Eutolmus rufibarbis (Asilidae) (Drukewitz et al. 2018); for revealing the morphology of the proboscis, the food canal and suction pump muscles of Prosoeca sp. (Nemestrinidae) (Karolyi et al. 2013); for analysis of the Drosophila larvae central nervous system (Drosophilidae) (Mizutani et al. 2007); for revealing the morphology of the late pupa of Calliphora vicinia (Calliphoridae) (Metscher 2009); for performing qualitative and quantitative analyses of the morphological changes taking place during the intra-puparial period of Calliphora vicina and Lucilia sericata (Calliphoridae) (Richards et al. 2012;Hall et al. 2017;Martín-Vega et al. 2016;Martín-Vega et al. 2017a, b); and for the visualization of the three-dimensional movements of flight muscles and thoracic sclerites of Calliphora vicina (Calliphoridae) (Walker et al. 2014).
In this study X-ray micro-computed tomography (micro-CT) was utilizzed for revealing genital sclerites and muscles of Nothybus kuznetsovorum (Nothybidae), comparing it with results of manual dissection (Galinskaya et al. 2016). The sclerites and muscles of Cothornobata sp. (Micropezidae) are also revealed. Males of Micropezidae are characterized by specialized forceps-like processes of variable size, shape, and ornamentation arising from sternite V and by sternite VI sometimes with specialized processes (these processes are secondarily reduced in some spp). The examined species is characterized by elongated forcipate appendages of sternite V, and the musculature of these appendages was investigated.

Materials and methods
One specimen of Nothybus kuznetsovorum and one specimen of Cothornobata sp. were collected in Northern Vietnam, PhuTho Province, Thanh Son District, Xuan Son National Park; 21°8.333'N; 104°56.25'W; h = 300-900 m, 23 October 2014 by T.V. Galinskaya. The specimens were fixed in 70% ethanol. They were prepared for X-ray micro-computed tomography (micro-CT) analysis by contrasting with iodine as outlined in Gignac and Kley (2014) and by critical point drying. Specimens were glued with the thorax pointing downwards on the tip of a small pin of 1 mm diameter, with the tip of the male abdomen as close to the rotation axis as possible. Micro-CT scans were produced under phase contrast (40 KV, 8 W), using a 4× detector (10 s; 4.15 μm pixel size) and 10× detector (30 s; 1.89 μm pixel size).
The male genital muscles were classified into several groups (muscles of the epandrial complex, muscles of the hypandrial complex, tergosternal muscles, and pregenital muscles) and described according to the system of Ovtshinnikova and Galinskaya (Ovtshinnikova 1989(Ovtshinnikova , 2000Ovtshinnikova and Galinskaya 2017). Male genital sclerites were described according to the Sinclair (2000). Figure 1 All sclerites and muscles and their places of attachments revealed using manual dissection and decribed by us previously (Galinskaya et al. 2016) have been confirmed by micro-CT. The only questionable muscle after manual dissection was M7 (Galinskaya et al. 2016). Here the presence and attachment sites of attachment of M7 are confirmed ( Figure 1B).
Muscles. The muscles are grouped by the site of insertion of their proximal parts. Thin paired muscles ISM4-5 attached to basal area of sternite V ( Figure 3B). Tergosternal muscle TSM4 poorly developed ( Figure 4A).
Muscles of segments V and VI. Two pairs of muscles ISM5-6 lying between sternites V and VI: proximal retractors of sternite VI ISM5-6a broadly fan-shaped, extending from basal area of sternite V to lateral surface of sternite VI (Figures 3C, 4C); median flexors of forcipate appendages ISM5-6c broadly fan-shaped, extending from lateral outgrowths of sternite V to central part of sternite VI (Figures 3B, 4B, G). Contraction of muscles ISM5-6 powers the forcipate appendages of sternite V that participate in the fixation of the female's abdomen during copulation. Tergosternal muscles TSM5 and TSM6 in segments V-VI poorly developed ( Figure 4A). Muscles associated with segment VII are asymmetrical. Left intersegmental tergal muscle ITM6-7l wide, extending from distal area of tergite VI to left lateral margin of syntergosternite VII ( Figure 4G). No muscles TSM6-7 present in sternal area between segments VI and VII. Left muscle ISM7-8l wide, extending from basal margin of syntergosternite VII to syntergosternite VIII ( Figure 4G). Tergosternal muscles of segment VII absent, probably due to fusions or reductions.
Tergosternal muscles of segment VIII absent, probably due to fusions or reductions. Long and powerful symmetrical tergosternal abductors M5 extending from laterobasal margin of epandrium to latero-distal margin of hypandrium.
Muscles of hypandrial complex symmetrical. Short and powerful paired phallic retractors M1 connecting distal part of phallapodeme to distal inner part of hypandrium   ( Figure 4C). Long and powerful paired phallic protractors M2 connecting distal part of phallapodeme to lateral and median parts of hypandrium ( Figure 4B) and lying above M1. Paired retractors of pregonites M42 extending from inner distal part of hypandrium to pregonites ( Figure 4B). Compressors of ejaculator M23 well developed ( Figure 4G).

Discussion
Since the results of the study of the male genitalia in Nothybus kuznetsovorum using micro-CT completely coincide with the results of manual dissection, we conclude that the method of manual anatomy has not lost its significance. However, the micro-CT takes much more time than manual anatomy. The undoubted advantage of micro-CT is its higher accuracy and the fact that only one specimen is needed for the study, while manual anatomy usually needs 4-5 specimens.
We assume that the presence of the elongated appendages of sternite V in the species Cothornobata sp. can cause the presence of additional muscles ISM5-6 (due to the complication of sclerites in Cothornobata sp. comparing with Neria commutata). However, no muscle going from the basal to the distal segments of the appendages of V sternite was discovered. Additionally, we did not find the muscles ISM5-6b and ISM5-6d. Neria commutata has the lateral flexors of forcipate appendages ISM5-6d broad and short, extending from inner vanes of sternite V to those of sternite VI and occupying a considerable part of the surfaces of both sclerites; the distal retractors of sternite VI ISM5-6b are narrow, extending from the distal parts of the outgrowths of sternite V to the distal area of sternite VI. Apparently, the muscle ISM5-6c goes on to moving the distal half of the forcipate appendages of sternite V.
In Cothornobata sp., unlike Neria commutata, no muscles ITM6-7r and ITM7-8r have been detected. Neria commutata has the right muscle ITM6-7r narrow, conical, extending from the median part of tergite VI to the membrane in front of syntergosternite VII, and the right muscle ISM7-8r small and short, extending from the membrane at syntergosternite VII to syntergosternite VIII (Ovtshinnikova and Galinskaya 2017). We assume that in Cothornobata sp. these muscles are absent due to a stronger flexion of syntergosternite VII to the ventral side. Increased flexion of syntergosternite VII in Cothornobata sp. caused the disappearance of muscles ITM6-7r and ITM7-8r. Also, an increase of the flexion apparently caused the fusion of the M18 muscles into one bundle.