Anatomy of male and female reproductive organs of stink bugs pests (Pentatomidae: Heteroptera) from soybean and rice crops Anatomy of male and female reproductive organs of stink bugs (Pentatomidae: from soybean and

: Pentatomidae comprises a diverse group of stink bugs widely distributed in the Neotropical region. Many species are phytophagous and cause injuries to plants, and can thus be defined as agricultural pests. In this study, the anatomy of the female and male reproductive tracts of three important agricultural pests in Colombia is described: Piezodorus guildinii Westwood, 1837 and Chinavia ubica Rolston 1983, found on soybeans, and Oebalus insularis Stål, 1872, found in rice crops. For that, light microscopy techniques were used. The anatomy of the reproductive tract of sexually mature males of the three species studied consisted of a pair of testes, vas deferens , seminal vesicles, ejaculatory bulb, an ejaculatory duct that opens into an aedeagus, and paired accessory glands. The reproductive tract of females consisted of a pair of ovaries, each with seven telotrophic-meroistic ovarioles, a pair of lateral oviducts, common oviduct, spermatheca, and a genital chamber. Telotrophic ovarioles were comprised of terminal filament, tropharium, vitellarium, and pedicel. Differences in size, color, and position of structures along the reproductive tract were observed between the species examined. Reproductive biology of insects provides informative characters for behavioral and evolutionary studies, as well as useful data for pest control strategies.


Introduction
Stink bugs (Pentatomidae) comprise the fourth largest Heteroptera family, with approximately 4,700 species described (Grazia et al. 2015). Most pentatomids are phytophagous and generalists, feeding on sap and other plant-produced substances (Grazia et al. 2012). Pentatomids have piercing sucking mouthparts, and most of them are phytophagous, including several species that are severe pests of agricultural crops. However, some species, particularly in the subfamily Asopinae, are predatory and may be considered beneficial. Phytophagous species feed on a wide range of food resources and have an extensive global distribution, which leads many of them to be defined as important agricultural pests (Panizzi et al. 2000, Grazia et al. 2012, Li et al. 2017.
In the Neotropical region, pentatomids are one of the major insect groups that cause damage to different crops (McPherson & MacPherson 2000, Panizzi et al. 2000, Smaniotto & Panizzi 2015. In crops such as soybeans and rice, these stink bugs can cause physiological changes in plants, affecting their development, flowering, fruiting, and therefore, crop performance and grain and seed quality , Panizzi et al. 2000, Karban & Agrawal 2002, Possebom et al. 2020.
The stink bug Piezodorus guildinii (Westwood 1837) is widely distributed in the Neotropical region and is one of the most harmful pests to soybean crops in the Americas (Panizzi & Slansky 1985). If the attack strikes during grain development and pod filling, major losses might occur (Galileo & Heinrichs 1978). Chinavia ubica Rolston, 1983 is a minor pest of soybean crop (Silva et al. 2015); however, Chinavia Orian, 1965 -senior synonym of Acrosternum Fieber, 1860 -is polyphagous and causes damage to several crops, such as vegetables and fruits (Panizzi et al. 2000). The stink bug Oebalus insularis Stål, 1872 is a harmful pest to rice crops and is very common in Colombia, Central America, and the Caribbean. This species is responsible for significant economic losses in these regions due to its ability to suck grains during development, thus affecting grain amount and quality, reducing production by more than 50% (Gutiérrez et al. 1985, Rodriguez et al. 2006. The comparative morphology of the reproductive tract in male and female stink bugs has already been described in several Pentatomidae species, and males generally exhibit a pair of testes, deferent ducts, ejaculatory bulb complex, paired accessory glands, and an ejaculatory duct (Pendergrast 1956, Adams 2001, Lemos et al. 2001, Candan et al. 2010, Araújo et al. 2011, Özyurt et al. 2013a, 2013b, Cremonez et al. 2017). On the other hand, females exhibit a pair of ovaries connected to the lateral oviducts by the pedicels of ovarioles, a common oviduct, and a spermatheca (Lemos et al. 2005, Candan et al. 2010, Cremonez et al. 2017. Insect ovaries and ovarioles can be classified into two types according to the presence (or absence) of nurse cells. In panoistic ovaries, nurse cells are absent, usually observed in more basal groups. In meroistic ovaries, the nurse cells are associated with the development of eggs. There are two types of meroistic ovaries, the polytrophic ovariole ones, in which oocytes are closely associated with the nurse cells, and the telotrophic ones, typical of Hemiptera and some Coleoptera, in which the nurse cells are at the apex of the ovary, in the germinal region (Bonhag 1958). These studies indicate that there are interspecific differences in reproductive anatomy regarding size or number of structures, the absence of any of them, and their color or position along the reproductive tract. Hence, the anatomy of these insects provides important characters for systematics and expands knowledge on the reproductive biology of the group.
The anatomy of both male and female reproductive tracts of three important pest insects was described in the present study: P. guildinii and C. ubica, found on soybeans, and O. insularis, found in rice crops, in order to provide knowledge on their reproductive biology and new data to be used in the systematics of these important agricultural pests.

Light microscopy
Insects were cryoanesthetized at -5 0 C for 10 min and their reproductive tracts were dissected in 0.1 M sodium chloride. For anatomical analysis, the reproductive tracts, freshly fixed in 2.5% glutaraldehyde buffered with sodium cacodylate, 0.1 M, pH 7.2 for a few minutes, were placed on histological slides with drops of the same buffer. Their reproductive tracts were photographed unstained using a Leica M205 C light microscope and a Leica MC 170 HD digital camera.
All measurements were performed using Image Pro-Plus (Media Cybernetcs), and lengths were expressed as the mean value of ten dissected individuals from each species analyzed. Species measurements were compared using ANOVA and Tukey's test. Data were analyzed using Generalized Linear Models (GLM) in the R software (R Core Team 2016).

Results
The male reproductive tract of the three studied species consists of a pair of testes, a pair of vas deferens, a pair of ectodermal sacs, a complex ejaculatory bulb, accessory glands, and ejaculatory duct (Figures 2a,3a,4a). Each testis is comprised of testicular follicles, which are usually enclosed in a common sheath. Six testicular follicles were observed in O. insularis (Figure 2b). The testes and deferent ducts are externally covered by a red-pigmented peritoneal sheath . In P. guildinii, the basal region of the deferent ducts exhibited a yellowish peritoneal sheath (Figures 3a, 3c).
Testis length (F3;45 = 7.08; P = 0.001), testis width (F3;45 = 19.65; P = 0.003), and vas deferens length (F3;45 = 13.65; P = 0.002) varied significantly between the sampled species (Table 1 and Figures  2a, 3a-b, 4a-b). The vas deferens did not exhibit an enlarged and differentiated region along its length to be distinguished as a seminal vesicle (Figures 2a, 3a, 4a). Tubular accessory glands were observed in all three species studied (Figures 2a, 3a, 4a). In O. insularis, tubular accessory glands and ectodermal sac open in a single duct that converges into the ejaculatory duct near the proximal portion of the ejaculatory bulb ( Figure 2d). This species has a yellow pear-shaped ejaculatory bulb (Figures 2a, 2d). In C. ubica, tubular accessory glands open at the base of the deferent ducts, immediately above the ejaculatory bulb, which was ovoid and transparent in this species (Figures 3c-d).
In P. guildinii, tubular accessory glands and the ectodermal sac open in the region of a greenish oval-shaped ejaculatory bulb (Figure 4a-b). The ejaculatory duct of this species was long and folded (Figures 4a, 4c). The female reproductive tract of the three studied species is formed by a pair of ovaries, each one with seven telotrophic-meroistic ovarioles connected by a pair of lateral oviducts, a spermatheca, a common oviduct, and a vagina that opens into the genital chamber (Figures 2e,  3e, 4c). The telotrophic-meroistic ovarioles developed synchronously and exhibited four different regions: terminal filament, tropharium, vitellarium, and pedicel (Figures 2e-h).
O. insularis oocytes had greenish coloration and a well-developed spermatheca connecting to the common oviduct through a spermathecal duct (Figures 2e-h). Oocytes are white in C. ubica (Figure 3e) and yellowish in P. guildinii, with an elongated spermatheca and a brown spermathecal duct (Figures 4d-e). In all three species, female adults exhibited structural changes in the reproductive tract during oogenesis (Figures 2e-h, 3e, 4c-d).  Table 1. Averages measurements of total body length and width (mm) of testis, length of vas deferens and ratio of length of vas deferens to body in males of Oebalus insularis, Chinavia ubica and Piezodorus guildinii (n = 10).

Species
Total body length (mm)

Discussion
The anatomy of the male reproductive tract maintains a general pattern in Pentatomidae, with some differences in size, shape, color, and location of structures (Pendergrast 1956, Adams 2001, Lemos et al. 2005, Rodrigues-Agna et al. 2008, Esquivel et al. 2009, Kaur & Patial 2012, 2016, Özyurt et al. 2013a, Jyoti et al. 2015. Among Pentatomidae, the number of testicular follicles can vary from three, e.g. in Aeliomorpha lineaticollis Westwood, 1837 (Kaur & Patial 2016) Six testicular follicles were observed in Oebalus insularis Stål, 1872, as well as in Nezara viridula Linnaeus, 1758 (Esquivel et al. 2009), Halys dentatus Fabricius, 1775 (Jyoti et al. 2015), and Podisus nigrispinus Dallas, 1851 (Lemos et al. 2005) -ranging from 4 to 6 follicles. Furthermore, the presence of four follicles in Oebalus ypsilongriseus De Geer, 1773  demonstrates that differences concerning the number of follicles occur even within the same genus, and therefore, does not seem to be a potential character for the identification at taxonomic levels above genus.
Of nineteen revised Pentatomidae species, including the three studied here, seventeen exhibited a peritoneal sheath covering the testes and part of deferent ducts with a red tinge (Pendergrast 1956, Biota Neotrop., 20(4) Lemos et al. 2005, Kaur & Patial 2012, Ozyurt et al. 2013a, Jyoti et al. 2015, whereas only two species -N. viridula (Esquivel et al. 2009) and Thyanta perditor Fabricius, 1794 ) -exhibited a yellow-orange peritoneal sheath. The basal region of deferent ducts with a yellowish color in P. guildinii is one of the major anatomical differences between the studied species. The color of the peritoneal sheath does not follow a pattern among Hemiptera (Gomes et al. 2013). However, the sheath is transparent in the studied aquatic Heteroptera families (Castanhole et al. 2008(Castanhole et al. , 2010Munhoz et al. 2020), as occurs in the subfamily Triatominae (Alevi et al. 2014).
Lipophorin is one of the major insect lipoproteins (Van Hoof et al. 2005), particularly involved in lipid transport from absorption or synthesis to storage and uptake, such as the fat body, ovary, and testis (Jung & Yun 2007). Differences in diet and metabolism may be related to differences in pigment color of the peritoneal sheath of the reproductive tract. Elongated testes have also been described for the pentatomids Tropicoris punctipes Stål, 1876, Erthesina fullo Thurnberg, 1783 (Kaur & Patial 2012), and T. perditor ). Among Pentatomidae, species can have elongated-ovoid (Esquivel et al. 2009, Kaur & Patial 2012, Ozyurt et al. 2013a), cylindrical (Ozyurt et al. 2014, and kidney-shaped testes (Jyoti et al. 2015). The shape of testicles highly varies among Pentatomidae. However, this characteristic is conserved at the species level and may be a character of taxonomic potential.
In most Pentatomidae, spermatozoa migrate from the testes after spermatogenesis and are stored along the deferent ducts (Kaur & Patial 2012, 2016, which was observed in the three studied species. The presence of a seminal vesicle has been described in some pentatomid species, N. virdula Pendergrast, 1956and Brachymena cincta Fieber, 1861(Abbasi 1973), Dolycoris indicus Stål, 1876(Santos et al. 2003, and E. ventralis (Ozyurt et al 2015). Testis follicles with gem cells at different stages of spermatogenesis suggest that the species studied here have continuous spermatozoa production, which may allow multiple mating during adulthood, as reported in other Hemiptera (Moreira et al. 2008).
Females of P. guildinii lay an egg mass that ranges from 3 to 37 eggs (Panizzi et al 2000), similar to that observed in figures 4d-e from the present study, which indicates an approximate load of 42 eggs. Zachrisson et al (2014) demonstrated that O. insularis can deposit masses containing 17 to 24 eggs, yet figures 2e-g in the present study show a laying potential of up to 56 eggs in a short period. For this reason, the number of eggs deposited may depend primarily on the type of host plant, food quality, and defensive behavior against natural enemies (Panizzi et al 2000, Panizzi & Silva 2012, Zachrisson et al 2014.
The spermatheca is a structure typically present in the reproductive tract of female heteropterans, and is responsible for storing and maintaining viable spermatozoa until oocyte fertilization (Pendergrast 1957, Singh 1968, Kumar 1969a, 1969bAhmad & McPherson 1998, Rider & Chapin 1991. Interspecific variations in size, spermathecal shape, and number of associated glands are important characters for the systematics of this group (Kumar 1971, Rider & Chapin 1991, Ahmad & McPherson 1998. The spermathecas in the three species studied have different shapes and coloration, similar to O. insularis, in which this organ is quite developed with an evident spermathecal duct. According to Panizzi & Silva (2012) the longevity of females from several stink bug species, including P. guildinii and C. ubica, is sharply reduced according to the reproductive behavior associated with sexual activity and copulation. This finding shows how data on reproductive morphophysiology can provide important information for strategies aiming at population control of pest insects.
Reproductive parameters of pentatomids can be affected by growthregulating insecticides (Castro et al. 2012) via chemical communication, e.g. pheromones (McBrien & Millar 1999), and substrate vibrations (Laumann et al. 2018). These control alternatives should be monitored and analyzed through possible morphological changes in the reproductive tracts of female and male stink bugs.
The reproductive tract anatomy of female and male O. insularis, C. ubica and P. guildinii stink bugs in this study were described as the major characters that differentiate these species, as is the size of the testes and deferent ducts, and insertion sites of the accessory glands. The morphology of the reproductive tract generates important characters that can contribute to elucidate reproductive strategies as useful tools in agricultural pest management plans.