Reproductive Apparatus, Gonadic Maturation, and Allometry of Cyclocephala barrerai Martínez (Coleoptera: Melolonthidae: Dynastinae)

Simple Summary Cyclocephala barrerai larvae feed on the roots of ornamental and commercial grasses. Most of their biology is still unknown, and there are no tools to manage their populations. The objective of this work was to study the reproductive system, gonadic maturation, and allometry of C. barrerai. This is the first report into the Melolonthidae family that characterizes the reproductive apparatus, gonadic maturation of both sexes, and allometric relationships of virgin F1 specimens. Adults do not feed, so gonadic maturation depends on larvae reserves. Females present sclerotized accessory glands and a genital chamber with muscular fibers and bacteria. The morphology of the parameres and the antennae are under sexual selection. This is the first report of two main differences among sexes: females are heavier while males have longer antennae. Males and females exhibit allometric relationships that can be used to predict the bodyweight of field-collected specimens. Abstract The Order Coleoptera provides good examples of morphological specializations in the reproductive apparatus, gonadic maturation, and allometry differing between the sexes. The female and male reproductive apparatus has been modified to ensure reproduction between individuals of the same species. The genus Cyclocephala has more than 500 species distributed in America, and Cyclocephala barrerai Martínez is an economically important species in the central part of Mexico. The objective of this work was to study the reproductive system, gonadic maturation, and allometry of C. barrerai. We used light, scanning electron, and laser scanning confocal microscopy to describe the reproductive apparatus and gonadic maturation of females and males. The relationship between adult weight and different parts of the body was established by linear regression. Regardless, the reproductive apparatuses of C. barrerai are like those of other Melolonthidae: the genital chamber, the type II accessory glands, and the ventral plaques of the female and the ejaculator bulb and genital capsule of the males are specific to C. barrerai. The gonads are fully developed when 18 d old. The weight of adult C. barrerai has a positive linear relationship with distinct parts of its body, while the antennae of males are larger than those of the females.

Insects were reared according to Sanchez-Cruz et al. (in preparation). In short, we setup 98 mating triads (a mature female and two mature males); each one was allocated to a 50 mL cylinder plastic container filled with 25 mL of soil and covered with a cheesecloth secured by a rubber band. Eggs were collected every 24 h and each one allocated to a 50 mL plastic container (the same one mentioned above) with a nutritious substrate (25 mL) until pupation. The substrate was kept humid by spraying tap water as needed. The nutritious substrate was replaced every month.
A single pupa was allocated to a 35 mL plastic container with 29 mL of humid soil. Four of these containers were kept in a 15 cm length by 5 cm height and 10 cm width squared container whose top had four holes for ventilation. To prevent pupae dehydration, 50 mL of water was added into the container. Newly emerged adults were collected 24 h after emergence and allocated to a 50 mL cylinder plastic container filled with 25 mL of soil in the above-mentioned plastic containers with the nutritious substrate.

Reproductive Apparatus Description
The reproductive apparatus (RA) of 20 virgin insects (10 for each gender) with no deformations or apparent injuries and bright cuticles of 18 d old were dissected in saline solution and stained for 5 min with 10% Giemsa. The RA was rinsed with tap water prior to observation on a Zeiss Discovery-V20 (Thornwood, NY, USA, EE. UU) stereoscopic microscope. Several papers [8,9,14] were used to identify the reproductive structures. Drawing and image processing were performed with Krita 4.4.2 software (Stichting Krita Foundation, Deventer, The Netherlands).

Gonadic Maturation
To study the male gonadal maturation, 30 males were killed at 0, 10, and 18 d after emergence (n = 10), and their RA (accessory glands, vasa deferentia, and testes) were dissected and cleaned in saline solution. The RA were stained with 10% Giemsa for 5 min and observed under the stereoscope previously mentioned. The sperm were obtained from vasa efferentia and allocated to 1.5 mL Eppendorf tubes plus 200 µL of distilled water before manual stirring and observation was conducted under an ESEM.
To study the female gonadic maturation, the RA (ovaries and oocytes) of 10 females of 0, 6, 12, and 18 d old were dissected in saline solution, stained for 5 min with 10% Giemsa, and observed under the stereoscopic microscope; they were classified according to Grodowitz and Brewer [39]. The ovaries of 12 d old females were dissected to separate the ovarioles. The ovarioles were dehydrated at ethanol series (70%, 96%, and 100%) prior observation under the above-mentioned LSCM.

Allometry
All C. barrerai pupae were weighed using an analytical balance (Ohasus Explorer, 0.0001 g precision, Zurich, Switzerland). All newly emerged adults were weighted on the To study the relationship between body parts and size, we separated the hind-tibia, middle-tibia, and the external-lamella of the antennae of both sexes and the genital chamber and bursa copulatrix in females and the tarsal claw (width and length) of the front legs of the male. All the structures were measured and photographed under a stereoscope Zeiss Discovery-V20 (Thornwood, NY, USA). The photos were analyzed with ImageJ software (Scion Corporation, Chicago, IL, USA) [40].

Statistical Analysis
The relationship between the body parts and the size or the length of the structures was analyzed by linear regression. We compared the measurements of the different body parts between sexes with a t-test. Unless stated otherwise, all reported data are the mean ± Standard Error of the Mean (SEM). All analyses were carried out on.

Male Reproductive Apparatus
The male RA consists of two accessory glands; two glandular ducts; two testes, each with six follicles; six vasa efferentia; two vasa deferentia; two seminal vesicles; one ejaculatory duct; one ejaculatory bulb; one genital capsule; and one aedeagus ( Figure 1). The accessory glands are long ducts with thick walls. The distal portion is long and narrow, and secretion is not observed in its lumen. The proximal portion is thicker and showed luminal secretion. The subsequent zone is thick with a fluid. The accessory glands are connected to the distal end of the ejaculatory duct ( Figure 1). Each testis is composed of six free follicles, lobed, dorsoventrally flattened, in form of a "flower", and every testis follicle is connected to vasa efferentia. The vasa efferentia are short and thin ducts connected to one vasa deferentia. same scale and sexed according to the claw of the first legs where males present a larger claw than females. A total of 31 males and 36 females, 18 d old, were sacrificed by freezing.
To study the relationship between body parts and size, we separated the hind-tibia, middle-tibia, and the external-lamella of the antennae of both sexes and the genital chamber and bursa copulatrix in females and the tarsal claw (width and length) of the front legs of the male. All the structures were measured and photographed under a stereoscope Zeiss Discovery-V20 (Thornwood, NY, USA). The photos were analyzed with ImageJ software (Scion Corporation, Chicago, IL, USA) [40].

Statistical Analysis
The relationship between the body parts and the size or the length of the structures was analyzed by linear regression. We compared the measurements of the different body parts between sexes with a t-test. Unless stated otherwise, all reported data are the mean ± Standard Error of the Mean (SEM). All analyses were carried out on.

Male Reproductive Apparatus
The male RA consists of two accessory glands; two glandular ducts; two testes, each with six follicles; six vasa efferentia; two vasa deferentia; two seminal vesicles; one ejaculatory duct; one ejaculatory bulb; one genital capsule; and one aedeagus ( Figure 1). The accessory glands are long ducts with thick walls. The distal portion is long and narrow, and secretion is not observed in its lumen. The proximal portion is thicker and showed luminal secretion. The subsequent zone is thick with a fluid. The accessory glands are connected to the distal end of the ejaculatory duct ( Figure 1). Each testis is composed of six free follicles, lobed, dorsoventrally flattened, in form of a "flower", and every testis follicle is connected to vasa efferentia. The vasa efferentia are short and thin ducts connected to one vasa deferentia.
The vasa deferentia are long, with thicker walls, and the diameter of its anterior part is smaller than the posterior. Like the accessory glands, they open into the anterior end of the ejaculatory duct ( Figure 1). One end of the ejaculatory duct is connected to the accessory glands and the vasa deferentia while the other connects to the ejaculatory bulb ( Figure 1). The ejaculatory bulb is a membranous sac with fluted-like muscular tissue ( Figure 2).  The vasa deferentia are long, with thicker walls, and the diameter of its anterior part is smaller than the posterior. Like the accessory glands, they open into the anterior end of the ejaculatory duct ( Figure 1). One end of the ejaculatory duct is connected to the accessory glands and the vasa deferentia while the other connects to the ejaculatory bulb ( Figure 1). The ejaculatory bulb is a membranous sac with fluted-like muscular tissue ( Figure 2).  The endophallus is a membranous and large structure that connects to the ejaculatory duct (Figure 2A), and it is surrounded by the genital capsule ( Figure 2B). The genital capsule is a symmetric structure formed of three parts ( Figure 3): (1) The connecting membrane ( Figure 3A) is in the ventral zone, connecting the phallobase to parameres. (2) The parameres ( Figure 3A,B) are at the end of the genital capsule; they are symmetric, with a curvilinear triangle shape, and ornamented with small cones and pits ( Figure  3C). Some areas at the proximal tip of the parameres have some pallid fine lines between the pits ( Figure 3D).  The endophallus is a membranous and large structure that connects to the ejaculatory duct (Figure 2A), and it is surrounded by the genital capsule ( Figure 2B). The genital capsule is a symmetric structure formed of three parts ( Figure 3): (1) The connecting membrane ( Figure 3A) is in the ventral zone, connecting the phallobase to parameres. (2) The parameres ( Figure 3A,B) are at the end of the genital capsule; they are symmetric, with a curvilinear triangle shape, and ornamented with small cones and pits ( Figure 3C). Some areas at the proximal tip of the parameres have some pallid fine lines between the pits ( Figure 3D). (3) Posterior phallobase and anterior phallobase ( Figure 3E). The posterior phallobase is a sub-cylindric structure that involves the aedeagus and connects to the parameres. It is made up of one piece and presents a high diversity of ultrastructure ( Figure 3F). The posterior phallobase has four types of ultrastructure: (1) large needle, (2) small needle, (3) pits ( Figure 3F), and (4) small seta with a circular base ( Figure 3G).   When males are 18 d old, the distal section of the accessory glands are filled with a white content, and the mature sperm is in the proximal portion of the vasa deferentia. Mature sperm has a filamentary shape ( Figure 4B).  When males are 18 d old, the distal section of the accessory glands are filled with a white content, and the mature sperm is in the proximal portion of the vasa deferentia. Mature sperm has a filamentary shape ( Figure 4B).  When males are 18 d old, the distal section of the accessory glands are filled with a white content, and the mature sperm is in the proximal portion of the vasa deferentia. Mature sperm has a filamentary shape ( Figure 4B).

Female Reproductive Apparatus
The female RA has a genital chamber, two pairs of genital plaques, two pairs of ventral plaques, a spermatheca with a spermathecal gland, a common oviduct, and two ovaries with six ovarioles each ( Figure 5). The spermatheca is a tubular, sickle-shaped structure with a spermatheca muscle and spermatheca gland. The spermatheca gland is balloonshaped and is larger than the spermatheca. Both structures join the spermathecal duct ( Figure 5). The bursa copulatrix is a bimembranous and striated bag arising from the genital chamber. It is bigger than the common oviduct ( Figure 5). The common oviduct is a large tubular structure connecting posteriorly to the anteroventral area of the genital chamber and bifurcating anteriorly to the paired lateral oviducts that connect to the paired ovaries ( Figure 5).

Female Reproductive Apparatus
The female RA has a genital chamber, two pairs of genital plaques, two pairs of ventral plaques, a spermatheca with a spermathecal gland, a common oviduct, and two ovaries with six ovarioles each ( Figure 5). The spermatheca is a tubular, sickle-shaped structure with a spermatheca muscle and spermatheca gland. The spermatheca gland is balloon-shaped and is larger than the spermatheca. Both structures join the spermathecal duct ( Figure 5). The bursa copulatrix is a bimembranous and striated bag arising from the genital chamber. It is bigger than the common oviduct ( Figure 5). The common oviduct is a large tubular structure connecting posteriorly to the anteroventral area of the genital chamber and bifurcating anteriorly to the paired lateral oviducts that connect to the paired ovaries ( Figure 5). The genital chamber is cylindric, in which the common oviduct, bursa copulatrix, and the spermatheca duct arrive ( Figure 6A,B). The latest is made of muscular fibers rolled up over each other ( Figure 6C,D). In a cross-section view ( Figure 6D), the tissue is striated. The distal part, close to the genital plaques of the genital chamber, of 18 d old females has  The genital chamber is cylindric, in which the common oviduct, bursa copulatrix, and the spermatheca duct arrive ( Figure 6A,B). The latest is made of muscular fibers rolled up over each other ( Figure 6C,D). In a cross-section view ( Figure 6D), the tissue is striated. The distal part, close to the genital plaques of the genital chamber, of 18 d old females has bacteria. These have a circular shape and are in the chamber inside the muscular tissue ( Figure 6E,F). They can be found alone, aggregated, or in couples or triples. The bacteria size varied from 3 to 14 µm. The genital chamber has two types of genital plates: (1) Dorsal plates are sclerotized rectangular shape structures. They insert near the accessory glands, around the genital chamber. (2) Ventral plates are oval with many setae and inserted in the distal part of the genital chamber ( Figure 7A). The setae interact with the parameres of the male during mating. The ventral part has pits ( Figure 7B). bacteria. These have a circular shape and are in the chamber inside the muscular tissue ( Figure 6E,F). They can be found alone, aggregated, or in couples or triples. The bacteria size varied from 3 to 14 μm. The genital chamber has two types of genital plates: (1) Dorsal plates are sclerotized rectangular shape structures. They insert near the accessory glands, around the genital chamber. (2) Ventral plates are oval with many setae and inserted in the distal part of the genital chamber ( Figure 7A). The setae interact with the parameres of the male during mating. The ventral part has pits ( Figure 7B).  We observed two types of accessory glands: (1) Type I is oval-shaped, and it inserts in the genital chamber ( Figure 5).
(2) Type II is sclerotized ( Figure 8A) and has a spherical shape. It is formed by acellular triangular plates ( Figure 8B-D). We observed two types of accessory glands: (1) Type I is oval-shaped, and it inserts in the genital chamber ( Figure 5).
(2) Type II is sclerotized ( Figure 8A) and has a spherical shape. It is formed by acellular triangular plates ( Figure 8B-D).
The ovarian development of C. barrerai ( Figure 10) exhibits three nulliparous stages and a parous (with ovulation) one ( Figure 10). On day 0 (emerged from pupae), the ovary is in nullipara 1 stage; germarium and vitellarium are not differentiated and are without follicles. On day six, the ovary is in nullipara 2 stage: well-defined ovarioles with multiple follicles, the proximal follicle transparent indicating that it had not completed maturation. On day 12, the ovary is in nullipara 3 stage: proximal follicle opaquer and closer to ovulation. On day 12, the ovary is in the parous stage. The ovarian development of C. barrerai ( Figure 10) exhibits three nulliparous stages and a parous (with ovulation) one ( Figure 10). On day 0 (emerged from pupae), the ovary is in nullipara 1 stage; germarium and vitellarium are not differentiated and are without follicles. On day six, the ovary is in nullipara 2 stage: well-defined ovarioles with multiple follicles, the proximal follicle transparent indicating that it had not completed maturation. On day 12, the ovary is in nullipara 3 stage: proximal follicle opaquer and closer to ovulation. On day 12, the ovary is in the parous stage.

Discussion
The male reproductive apparatus of C. barrerai has a similar arrangement and organization of the Melolonthidae: testes, accessory glands, and vasa efferentia and vasa deferentia [9, 11,13]. Like the six Cyclocephala species described by Martínez et al. [9], the male reproductive apparatus lacks a glandular reservoir, and the ejaculator duct and ejaculatory bulb present well-developed muscular fibers. The spermatophore is formed at the two latter structures by fusing the sperm and the content of the accessory glands before it is transferred to the females [2].
Due to their taxonomic relevance, the parameres of Cyclocephala have been extensively described. Breeschoten et al. [44] analyzed and compared the parameres of 48 species identifying three different types: (1) symmetric, (2) minor asymmetry, and (3) asymmetric. The same authors indicated that species subject to higher interspecific selection pressure, such as sympatric species of the same genera, have asymmetric parameres. Males of C. barrerai have a genital capsule type 1 formed for three plates with symmetric

Discussion
The male reproductive apparatus of C. barrerai has a similar arrangement and organization of the Melolonthidae: testes, accessory glands, and vasa efferentia and vasa deferentia [9, 11,13]. Like the six Cyclocephala species described by Martínez et al. [9], the male reproductive apparatus lacks a glandular reservoir, and the ejaculator duct and ejaculatory bulb present well-developed muscular fibers. The spermatophore is formed at the two latter structures by fusing the sperm and the content of the accessory glands before it is transferred to the females [2].
Due to their taxonomic relevance, the parameres of Cyclocephala have been extensively described. Breeschoten et al. [44] analyzed and compared the parameres of 48 species identifying three different types: (1) symmetric, (2) minor asymmetry, and (3) asymmetric. The same authors indicated that species subject to higher interspecific selection pressure, such as sympatric species of the same genera, have asymmetric parameres. Males of C. barrerai have a genital capsule type 1 formed for three plates with symmetric parameres, indicating that this attribute is not under interspecific selection pressure, as C. barrerai is the only one inhabiting the collecting area.
The ultrastructure found at the phallobase and parameres are like the trichoid and basiconic sensilla, respectively; both sensilla have been reported on the antennae of the Melolonthidae [45,46]. Ultrastructures similar to those found in C. barrerai have been reported in the genital capsule of species of the Chrysomelidae, but no explanation of their diversity or function has been proposed so far [47][48][49].
We propose that the ultrastructure at the genital capsule of C. barrerai has a mechanoreceptor function facilitating sexual recognition and coupling, as proposed for Drosophila melanogaster Meigen [50]. This hypothesis also explains the function of the setae of the ventral genital plaques found on C. barrerai females as the genital plaques and the genital capsule have an active role during mating [12]. The parameres of the male provide support during mating and in conjunction with the female genital plaques, facilitate the insertion of the penis and sperm transfer [51].
The reproductive apparatus of C. barrerai females has the general arrangement of the Melolonthidae [8,13,14,29]. The bursa copulatrix and the spermatheca connect to the genital chamber, storing and nourishing the sperm inside the female body until oviposition [52]. The genital chamber presents muscular fibers as mentioned for C. lunulata (Benítez-Herrera, unpublished data), and searching for pheromone glandular cells is mandatory.
The female genital chamber has muscular fibers, and its static positive allometry indicates a female cryptic sexual selection process. This provides the female some control over sperm transfer to avoid mating to unfit individuals and to reduce the risk of hybridization [21,53]. The parameres' morphology is the result of the coevolution of the male and female genitalia [12,21,42,54].
The presence of microorganisms in the female genital chamber agrees with a previous report [30] that identified Klebsiella oxytoca and Citrobacter freundii in C. barrerai wild females. The presence of microorganisms in lab-reared females indicates that these might be transferred by the mother or acquired during the larval period and not transferred by the male during mating. The architecture of the genital chamber and the presence of microorganisms suggest that they are relevant during attractant production [14,30].
Cyclocephala barrerai and C. jalapensis have type II accessory glands. These are highly sclerotized superimposed plaques [29] whose function is unknown. This contrasts with those observed in Phyllophaga [14], Macrodactylus [8], and C. zealandica [16], where the type II accessory glands are semi-sclerotized, and the cells form conduits to transport pheromones.
Cyclocephala barrerai female and male gonadic maturation initiate once the adults emerge from the pupae, and they surface and search for mates when they are sexually mature: similar behavior to that reported for Holotrichia titanis Reitter, Holotrichia trichopora Fairmaire, and Anomala exoleta Faldermann [15]. C. barrerai adults from the lab colony were not fed, and their digestive system was filled with a dark liquid. Most Coleoptera adults depend (partially or completely) on food for gonadic maturation [55][56][57][58]. C. barrerai must obtain and store all the nutrients needed for metamorphosis and gonadic maturation as larvae, explaining their voracious appetite as larvae [58].
There is a strong relationship between the hind-tibia length and the body weight in C. barrerai and can be used to assess their bodyweight [59]. C. barrerai females are heavier than males; however, in C. borealis, it is the other way around: males are heavier than females [25]. This could be related to the life history of each species. In addition, males' lamellae have a larger surface and are longer than those of females. According to the drawings of the males' lamellae for Cyclocephala borealis Arrow by Johnson [28] and for Cyclocephala signaticollis Burmeister by Carne [60], males' are larger than those of females. Unfortunately, there is no quantitative information on lamella length and surface area reported for any other Cyclocephala.
When mate location relies on the prompt detection of pheromones, those individuals with larger antennae or more sensitive sensilla have a clear advantage over the rest of the sexually active population. Males of C. barrerai present static positive allometry due to their strong relationship between antenna length and body size [61] indicating a strong sexual selection process [21,62]; therefore, heavier males have an adaptative advantage over lighter males during mate location and mating [63,64]. Mate location on Cyclocephala depends on the prompt detection of receptive females in the vicinity, as a contest between males is rare.
It is possible to observe delicate ultrastructure under the advance Life Science microscopes. Samples do not require extensive handing or manipulation, so results are relievable, replicable, and comparable among studies [65,66]. The ultrastructure observed in the RA helps to explain their possible role during pairing.

Conclusions
The reproductive apparatuses of C. barrerai show the same arrangement and organization as the Melolonthidae, with ultrastructures in the phallobase and the parameres. The genital chamber has sclerotized ventral genital plates and type II accessory glands. Male and female gonadic maturation is achieved 18 d after emergence, and no food intake is required. The hind-tibia length and lamella length can be used to assess female and male bodyweight. The positive relationship between the size of the first-leg claw and antennae and the weight suggests that those structures had been formed under sexual selection. The use of new microscopy techniques offered evidence of the ultrastructure of the reproductive apparatus relevant during mating, providing useful information for the development of behavioral control techniques.