The Male Reproductive Structure and Spermatogenesis of Trypophloeus Klimeschi Eggers (Coleoptera: Curculionidae: Scolytinae)

Background Trypophloeus Klimeschi Eggers (Coleoptera: Curculionidae: Scolytinae) is one of the most destructive pests of Populus alba var. pyramidalis (Bunge), resulting in significant losses in economic, ecological and social benefits in China’s northwest shelter forest. But research of reproductive system, spermiogenesis and spermatozoon ultrastructure of T. klimeschi that is basis of phylogeny, reproductive biology and controlling is still black. The male reproductive organ of T. klimeschi is composed of testis, seminal vesicle, strand shaped accessory gland containing long branch of strand shaped accessory gland and short branch of strand shaped accessory gland, curly accessory gland, vas deferens and a common ejaculatory duct. The number of sperm per cyst is 350~512. Its spermatozoon is slender, measuring about 75 μm in length and 0.5 μm in wide and composed of a 3-layred acrosomal complex, a nucleus with two different states of aggregation, two mitochondrial derivatives with dark crystal, a 9+9+2 axoneme that run more or less parallel to mitochondrial derivatives, two crystalline accessory bodies with a big compact “puff”-like expansion. Especially in the seminal vesicle, its long flagella folded into several turns and the whole sperm is wrapped in a film. The general male spermatozoa T. most similar Curculionidae. some distinct differences were found: electron-3

3 dense band in the cytoplasm of spermatocytes; two different aggregation states of spermatozoon nucleus; especially the stored way of T. klimeschi spermatozoa.

Background
Curculionidae includes 60000 species that indicate it is difficult to provide a systematic arrangement based on phylogenetic considerations. Many characteristics of insects are used to study phylogeny, but undoubtedly comparative spermatology can significantly advance this work [1][2][3][4]. In addition, the strong reproductive capacity of insects is the key to its widespread transmission. Understanding the reproductive mechanisms of insects is the key to controlling the population of certain pests, and the study of spermatology lays a physiological foundation for understanding its reproductive mechanisms.
Previous research showing that the male reproductive system of Curculionidae composed of a pair of testes, seminal vesicle, vasa efferentia, vas deferens, accessory glands and ejaculatory duct [5]. The spermatozoa of Curculionidae display the classic structure, which is characterized by a head region formed by a 3layered acrosome and nucleus; two enormous accessory bodies and a large and crystallized mitochondrial derivatives support the tail region; a 9 + 9 + 2 axoneme pattern is the most common pattern within the Curculionidae [1,2,6,7], only the Rynchiitidae exhibit an axoneme pattern of 9 + 9 + 0 [1].
Trypophloeus Klimeschi Eggers (Coleoptera: Curculionidae: Scolytinae) is one of the most destructive pests of Populus alba var. pyramidalis (Bunge). T. klimeschi was first recorded in the Kyrgyz Republic, which borders Xinjiang Uygur Autonomous Region in China [8]. Following an outbreak in 2003 in Xinjiang Uygur Autonomous Region, T. klimeschi spread rapidly to the adjacent areas. The widespread outbreak of this beetle has caused huge economic, ecological, and social losses in China's northwest shelter forest [9][10][11][12]. To protect P. alba var. pyramidalis from succumbing to the insect pest, populations of T. klimeschi must be maintained at manageable levels below the threshold. But so far, only a few aspects such as breeding preferences, frequency of oviposition, and the mating cycle of T. klimeschi have been studied [13,14]. So, it is necessary to study the reproductive system, spermiogenesis and spermatozoon ultrastructure of T. klimeschi to explore their reproductive potential.

Gross Morphology of the Male Reproductive Tract
The male internal reproductive tract in T. klimeschi is composed of two units. Each unit is formed by a bilobed testis comprising about 200 cysts, a seminal vesicle which becomes thicker as the maturation of sperm (Fig. 1B) and inserted in the testis depression, two accessory glands (curled gland and strand shaped gland) and vas deferens (Fig. 1A). The strand shaped accessory gland connected to the seminal vesicle while the curly accessory gland surrounded the vas deferens (Fig. 1A). Two units fuse at their ends flowing into an ejaculatory duct (Fig. 1A).
The cysts are full of testes (Fig. 1C), within which spermatogenesis occurs. The cyst cell with a big nucleus ( Fig. 2A)  Longitudinal section showing (Fig. 6D) that the first to insert into the nucleus is the large mitochondrial derivative, followed by the small mitochondrial derivative, axoneme and two accessory bodies. The cross section indicates that the axoneme (Figs. 6F, G) has a diameter of 277.89 ± 10.58 nm with 9 + 9 + 2 microtubules, the large mitochondrial derivatives with dark crystals accounted for about 60% of its circular cross section is always thinner than the axoneme and will become thinner along the axoneme until disappearing completely (Figs. 6F, H). The dark crystals accounted for about 40% of the drop shape cross section of the small mitochondrial derivative (Fig. 6F). The longitudinal section indicates that mitochondrial derivatives are comb-like (Figs. 5C, 6E). The axoneme and mitochondrial derivatives of T.
klimeschi are not embracing each other, but rather run more or less parallel to each other (Fig. 5B).
Two accessory bodies are different (Fig. 6F). The completely crystalline crescent accessory body is smaller than a quarter of axoneme and close to the small mitochondrial derivative. The other consists of a small crystal that was mostly triangular and sometimes crescent and a big compact "puff"-like expansion.
At the end of the sperm, the first to disappear is the two accessory bodies, followed by small mitochondrial derivatives, large mitochondrial derivatives and finally is the disintegration and disappearance of axoneme (Fig. 6H).
In this study, bi-and multi-flagellate spermatids and spermatozoa were observed at a low rate (Figs. 6I, J).
The spermatozoa of T. klimeschi individually stored in the seminal vesicle, its long 8 flagella folded into several turns and the whole sperm is wrapped in a film (Figs. 5D, E). Compare to slender spermatozoon, this state spermatozoon is more like a tadpole. The head of the "tadpole" is a circle surrounded by the middle of spermatozoon, in some circle form an "S" shape, and the tail is juxtaposed by the two ends of the spermatozoon.

Discussion
The general morphology of the male reproductive tract of T. klimeschi is similar to that reported for other Curculionidae [5,[15][16][17][18]. T. klimeschi have two testes like others; each testis is bilobed like Dendroctonus. armandi [ 18], some are single lobed like Sibinia arenariae [ 19], or multilobed like one of Nemonychidae [5]; each lobe consists of a single large spherical follicle like Nanophyes nigrovarius [ 5], while Allaeometrus breviceps has no visible follicular structure and most have 3-50 follicles [5]. Each follicle of T. klimeschi has about 100 cysts that at various stages of development. Spermatozoa of a cyst are synchronous development like Rhynchophorus ferrugineus [ 3]. The number of sperm per cyst is varies among different species, while that in T. klimeschi is between 350-512 that means this species is a primitive Coleopteran species [20].
Before spermiogenesis, the characters of T. klimeschi is the low electron-dense band comprised by smooth endoplasmic reticulum in the cytoplasm of spermatocytes, compared to Euptoieta hegesia [ 24], Agraulis vanillae [ 25], Tribolium castaneum [ 23] and Timema poppensis [ 26]. In addition, its spermatogonia has a large and irregular nucleus, that is similar to T. poppensis [ 26] but different from A. vanillae [ 25]. In the process of spermiogenesis, the nuclear 9 chromatin undergoes different course of condensation in all species; sometimes the central zone, and other times the periphery, is the first to condense [2], both of these situations exist in this study. With the condensation of nuclear chromatin, two regions could be distinguished in this study that also be mentioned in other research [3], one showing the chromatin more homogeneously condensed and another showing the chromatin with a fibrillar aspect. In addition, homogeneous [27] and honeycombed [2,17] nuclear chromatin were discovered. The centriolar adjunct of T. klimeschi appear in the spermatid stage but disappear in the mature sperm like Rhynchophorus ferrugineus [ 3,17], D. armandi [ 18], Sitophilus zeamais and Sitophilus oryzae [ 2]. These researchers believe that the reason is that the centriolar adjuncts stabilize axoneme during spermiogenesis. Our research also shows a more detailed process of mitochondrial derivatives formation that is similar to previous research [28].
The spermatozoa of T. klimeschi are linear and slender and are similar to the general description for other Curculionidae sperm [2,3,17,18,29,30]. Its length is about 75 µm and is very short compared to other Curculionidae that spermatozoa are 110 to 300 µm in length [1,17]. But its structure is similar to most Curculionidae [1,2,4,17,18,[29][30][31]: 3-layered acrosome with a cup-like acrosome vesicle, but its roundly hooked extra acrosomal layer is similar to few of Curculionidae in which most is conical; a 9 + 9 + 2 axoneme; two comb-like mitochondrial derivatives of different sizes and crystallization; two accessory bodies and one big compact "puff"-like expansion. Of course, these differences, which will be mentioned below, deserve our attention.
At present, there are two storage method of spermatozoon in seminal vesicle: in bundles or individually [32][33][34]. The bundled spermatozoa are in cyst and their heads are inserted into the extracellular matrix. But there seems no description of how individually spermatozoon to store. In this study, we find that in seminal vesicle T. klimeschi spermatozoa folded their long flagella into several turns and wrapped in a film. By this way spermatozoa may make it not easy to knot and increase its survival rate.
Nuclear chromatin of most insect spermatozoa is dense and uniform, only few are unhomogeneous. The cross section of Dryomyia lichtensteini (Cecidomid: Lasiopteridi) spermatozoa showing that elliptical high electron density chromatin envelops round circular low electron density chromatin [35]. The cross section of Blaptostethus pallescens (Heteroptera: Anthocoridae) spermatozoa [36] showing irregular lower density chromatin. There are similar phenomena in Hypanthidium foveolatum (Hymenoptera: Apidae: Megachilinae) [21]. In this study, the nucleus of T. klimeschi spermatozoa is characterized by the two different states. Obviously, this feature of insect spermatozoa nucleus is not extensive, and its formation should be related to different living habits of different species, but the specific reasons need to be explored in subsequent experiments.

Conclusions
The general morphology of male reproductive tract, the spermatogenesis and the spermatozoa of T. klimeschi are, for the most part, similar to the majority of the Curculionidae. However, some distinct differences were found: the low electron-

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The authors declare that they have no competing interests.