The structure of testes and ovaries can be described in its simplest form by the number of follicles and ovarioles they contain. Sixty-five years after the last review of the internal reproductive systems in true bugs (Heteroptera), the data accumulated today on the number of testicular follicles and ovarioles in their gonads are summarized. In addition, data on the number and type (mesadenia/ectadenia) of accessory glands are given. The hemipteran suborder Heteroptera constitutes one of the most diverse groups of non-homometabolous (‘Hemimetabola’) insects, comprising more than 40,000 described species worldwide and approximately 100 families, classified into seven infraorders. Data are available for all infraorders; however, more than 90% of studied species belong to the largest and most evolutionarily derived infraorders Cimicomorpha and Pentatomomorpha. In true bugs, in general, the number of follicles varies from one to nine (in a testis), and the number of ovarioles varies from two to 24 (in an ovary). Seven follicles per testis and seven ovarioles per ovary prevail being found in approximately 43.5% (307 species) and 24.4% (367 species) of studied species, respectively. Such a structure of testes and ovaries is considered an ancestral character state in the Heteroptera. In the evolution of this group, the number of follicles and ovarioles both increased and decreased, but the trend towards a decrease clearly prevailed.

Accessory glands, ectadenia, evolution, mesadenia, ovarioles, phylogeny, testicular follicles, true bugs

The hemipteran suborder Heteroptera (or true bugs) displays remarkable morphological variation and comprises more than 40,000 described species worldwide in ~ 100 families classified into seven infraorders including Enicocephalomorpha, Dipsocoromorpha, Nepomorpha, Gerromorpha, Leptopodomorpha, Cimicomorpha, and Pentatomomorpha. The majority of true bug species are herbivorous but some are predators and blood-suckers (Štys and Kerzhner 1975; Henry 2009; Schuh and Weirauch 2020).

The internal anatomy of the true bug males and females has been investigated for nearly two centuries. The first report on this topic appeared in the thirties of the 19^th century (Dufour 1833). Substantial comparative research on the internal anatomy of true bugs had accumulated by the 1960s mostly as a consequence of publications by J. Carayon, D. Leston, S. Miyamoto, T.E. Woodward, and J.G. Pendergrast (see Pendergrast 1957 and Miyamoto 1957 for references). Pendergrast (1957) published the first and, up to the present time, the only review of data available by that time on the internal reproductive organs of true bugs. Overall, Pendergrast summarized the literature (~ 90 titles) and his own data on ~ 50 families of Heteroptera and also shortly commented on the value of different parts of the male and female reproductive organs as characters in the taxonomy and classification of the Heteroptera. Despite the fact that Pendergrast singled out some organs as potentially promising for these purposes (in particular, testes in terms of the number of follicles they contain), he concluded that there is still very little data to make any generalizations. A huge contribution was made by Miyamoto (1957), who summarized the literature and own data on the structure of the ovaries in almost 300 representatives of ~ 40 families (some of them no longer families and other subfamilies recently erected to the separate families). Since the Pendergrast’s overview and Miyamoto’s list, ~ 120 original articles concerning the internal reproductive organs of Heteroptera have been published. In most cases, studies concern single species, and comparative data across multiple species of a group are few (e.g., Kumar 1967: Aradidae; Leston 1961a; Leston and Gibbs 1968; Malipatil 1978: Lygaeidae; Akingbohungbe 1983: Miridae; Grozeva and Kuznetsova 1992: Aradidae, Piesmatidae, Berytidae, Lygaeidae, Pyrrhocoridae). Much valuable information on true bugs can be found in special monographic works devoted to the reproductive systems of insects in general (Matsuda 1976; Büning 1994a, b; Chapman et al. 2013; Klowden 2013).

Although there is considerable diversity in detail, the internal parts of the male and female reproductive systems are organized similarly in different insects. In males of true bugs, it is formed by a pair of testes consisting of a variable number of testicular follicles, two different ducts, a median ejaculatory duct, and accessory glands. Accessory glands may be ectodermal or mesodermal in origin being known as ectadenia or mesadenia, respectively. Ectadenia open into the ejaculatory duct, whereas mesadenia open into the vasa deferentia or the distal end of the ejaculatory duct. In some species, both ectadenia and mesadenia are present, while other species have no accessory glands at all (Pendergrast 1957; Suppl. material 1). In females, the reproductive system is formed by a pair of ovaries consisting of a variable number of ovarioles, two lateral oviducts, a median common oviduct, and a spermatheca. The ovarioles are known to be of the meroistic telotrophic type (Chapman et al. 2013). In some species, accessory glands may also be present.

During the years that have passed since the aforementioned overviews, the number of species and higher taxa of true bugs studied in relation to testes and ovaries has almost tripled, and it seems appropriate to publish an updated list. In this review article, all the data available today, including new data on 140 species obtained by the authors, are summarized in two tables. Suppl. material 1 includes all species studied to date in terms of the number of follicles and ovarioles. In some cases, additional information, e.g., on the number and type of accessory glands (ectadenia or mesadenia) in the male reproductive system, is also provided. Table 1 summarizes data presented in Suppl. material 1 and shows the variability of the number of follicles and ovarioles at different taxonomic levels, together with the modal values for each of the higher taxa explored. On Fig. 1, the internal reproductive organs of a male and a female of Arocatus longiceps Stål, 1872 (Lydaeidae) is given. In Figs 2 and 3, character states of testicular follicle and ovariole numbers, respectively, are mapped onto the phylogenetic tree of Heteroptera families taken from Weirauch et al. (2019). In the final section of the review, the diversity of the analyzed characteristics and main tendencies of their evolution in true bugs are discussed. We hope that our review will be of importance for future work on the diversity of reproductive morphology within the Hemiptera, and that the data may provide additional information for understanding relationships between the higher taxa of true bugs.

Figure 1. 

a Male (seven follicles) and b female (seven ovarioles) reproductive systems of Arocatus longiceps Stål, 1872 (Lygaeidae). Abbreviations: t – testis, vd – vas deferens, vs – vesicula seminalis, mg – mesadenia, b – bulbus, mt – malpighian tubule, d – ductus (spermatheca and accessory glands were not visible in females).

The current review article is part of a long-term research project dedicated to the study of true bugs, including the morphology of their reproductive system, cytogenetics, and evolution. The data we have obtained over years on the structure of testes and ovaries of true bugs in terms of the number of testicular follicles and ovarioles constitute at the present time a significant part of all such data currently available for the Heteroptera in general (Suppl. material 1). The material was collected during the expeditions of the authors (1986–2021) or provided by colleagues, mainly by D. Gapon, L. Hill, and H. Gunther. Material comes from different regions of Palaearctic (Europe: predominantly southern Europe and the Balkans, Central Asia, Caucasus) and some parts of the Old Tropics (Vietnam, Indonesia, Himalayas, India, Thailand, Tasmania). Species identification was made by authors or collectors (in case of donated material). Voucher samples are deposited in the insect collections of the National Museum of Natural History (NMNHS, Sofia, Bulgaria) and of the Institute of Biodiversity and Ecosystem Research (IBER, Sofia, Bulgaria) of the Bulgarian Academy of Sciences (BAS), and the Zoological Institute (ZIN) of the Russian Academy of Sciences (RAS), St. Petersburg, Russia.

Figure 2. 

Testicular follicle numbers mapped on the true bug phylogeny after Weirauch et al. (2019).

Our original data cover 140 species belonging to 30 families, six infraorders, and represent 13.8% of all data on the structure of the testes and ovaries of true bugs accumulated to date and presented in Suppl. material 1. Some of these data have been published previously, the other part is presented here for the first time. Our study was carried out on specimens fixed in Carnoy fixative (3:1 96% ethanol and glacial acetic acid) that allowed both morphological and chromosomal analyses of the same individual. Material fixed in Duboscq Brasil (alcoholic Bouin’s) and ethanol (95%) was also used. The gonads were dissected out of the abdomen in a drop of 45% acetic acid on a microscope slide. The testicular follicles of the male and the ovarioles of the female were carefully separated from each other and counted under a stereomicroscope. Other parts of the reproductive system have also been studied, most notably, the accessory glands in males.

Figure 3. 

Ovariole numbers mapped on the true bug phylogeny after Weirauch et al. (2019).

Classification used in this work follows Schuh and Weirauch (2020) except for Micronectidae which are treated as a separate family due to distinct cytogenetic features (Grozeva et al. 2008) and following Nieser (2002). All taxa are listed alphabetically.

Numbers of testicular follicles and ovarioles were treated as unordered states of two hidden characters and optimized on the Bayesian total-evidence tree of Heteroptera published by Weirauch et al. (2019) with all terminals of the original topology collapsed to family level. The phylogenetic reconstruction of Weirauch et al. (2019) was chosen for character mapping as the most recent analysis of the entire Heteroptera employing comprehensive coverage of true bug families. The data recorded in this publication comes from all available literature (Dufour 1833; Gross 1901; Marshall and Severin 1903; Bowen 1922a, b; Kerkis 1926; Ludwig 1926; Poisson 1927; Sarel-Whitfield 1929; Wille 1929; Hagan 1931; Payne 1934; Baumgartner and Witherspoon 1937; Kirkpatrick 1937; Larsen 1938; Slack 1938; Schneider 1940; Kullenberg 1947; Carayon 1949, 1950, 1951, 1954, 1955, 1972; Woodward 1949, 1950, 1977; Bonhag and Wick 1953; Davis 1955, 1956, 1966,1970; Pendergrast 1956, 1957; Miyamoto 1957, 1959; Scudder 1957; Barth 1958; Usinger and Matsuda 1959; Drake and Davis 1960; Schrader 1960a; Leston 1961a, b; Kumar 1962, 1964, 1965, 1967, 1969; Slater and Miyamoto 1963; Bhargava 1967; Leston and Gibbs 1968; Brunt 1971; Kauffman 1971; Huebner and Anderson 1972; Louis and Kumar 1973; Wightman 1973; Jansson and Scudder 1974; Malipatil 1978; Lung and Goeden 1982; Akingbohungbe 1983; Heming-van Battum and Heming 1986, 1989; Gonçalves et al. 1987; Ma and Ramaswamy 1987; Postle and Woodward 1988; Grozeva and Kuznetsova 1989, 1992; Socha et al. 1988; Papáček and Gelbič 1989; Biliński et al. 1990; Jawale and Ranade 1990; Gelbič et al. 1991; Hunt and Stebbing 1992; Papáček and Soldán 1992, 2008; Lent et al. 1994; Grozeva 1995, 2003, 2007; Grozeva and Nokkala 1996, 2001; Simiczyjew et al. 1996, 1998; Lalitha et al. 1997; Papáček et al. 1997; Štys et al. 1998; Simiczyjew 1999; Adams 2001; Couturier et al. 2002; Lis 2003; Kuznetsova et al. 2004, 2007; Lemos et al. 2005a, b, 2010; Santos et al. 2005; Grozeva et al. 2006, 2007, 2008, 2009, 2013; Jahnke et al. 2006; Kugler et al. 2006; Freitas et al. 2007a, b, 2008, 2010, 2012; Mróz 2007, 2012; Ogorzałek 2007; Pires et al. 2007; Grozeva and Simov 2008; Esquivel 2009; Marchini et al. 2009; Ogorzałek and Trochimczuk 2009; Souza et al. 2009; Chiang 2010, 2013; Van Doesburg et al. 2010; Horton and Lewis 2011; Mróz and Wojciechowski 2011; Souza et al. 2011, 2014; Uceli et al. 2011; Chiang et al. 2012; Kaur and Patial 2012, 2016; Soto et al. 2012; Gomes et al. 2013; Özyurt et al. 2013a, b, 2014a, b, 2015; Pluot-Sigwalt and Chérot 2013; Jyoti et al. 2015; Pereira et al. 2015; Cremonez et al. 2017, 2019; Elelimy et al. 2017a, b; Novais et al. 2017; Candan et al. 2018; Mahmood et al. 2018; Grodowitz et al. 2019, 2020; Khandelwal et al. 2019; Koçakoğlu et al. 2019; Xia 2019; Araújo et al. 2020a, b, 2021; Munhoz et al. 2020, 2021; Vélez et al. 2020; Gapon 2021; Oliveira et al. 2021; Bugaj-Nawrocka et al. 2022; Koutsogeorgiou et al. 2022; Samanta et al. 2022), and original observations (see Suppl. material 1).

The coding strategy aimed at reflecting diversity in numbers of follicles per testis (hereafter p.t.) and ovarioles per ovary (hereafter p.o.) observed within a given family. Therefore, states were coded as polymorphic in cases of large variability of follicle and ovariole numbers within a given family. This was the case for large and comparatively well studied families lacking widely accepted phylogenetic resolution e.g., Aradidae, Miridae, or Pentatomidae. However, in groups with a robust backbone phylogeny in place, e.g., Reduviidae (Weirauch 2008; Weirauch and Munro 2009; Hwang and Weirauch 2012), there was no doubt that the testis follicle number of seven is not only most common across the family, but may be treated as ancestral, and was coded as such. Within Veliidae, Halovelia septentrionalis Esaki, 1924 remains the only veliid species having two ovarioles p.o. Phylogeny of Gerromorpha by Damgaard (2008) rendered Haloveliinae as a sister group to Microveliinae which in turn forms a sister group to Gerridae. All veliid taxa including the second studied haloveliine species have four ovarioles p.o. and therefore we consider this number as an ancestral state for all veliids. History of both characters was traced using Mesquite 3.6.1 (Maddison and Maddison 2018) with the parsimony ancestral state reconstruction method (Figs 2, 3).

In this work, we have compiled ~ 1200 records of the numbers of testicular follicles and ovarioles in 1008 true bug species from across 63 families and seven infraorders, including Enicocephalomorpha (one family, two species), Dipsocoromorpha (three families, twelve species), Nepomorpha (eleven families, 50 species), Gerromorpha (five families, 50 species), Leptopodomorpha (two families, five species), Cimicomorpha (12 families, 474 species), and Pentatomomorpha (29 families, 437 species). Below we will comment on the available data for each infraorder, with emphasis on those families in which such data are more or less representative. Data on male accessory glands, their numbers, and origins (mesadenia/ectadenia), although provided in Suppl. material 1, are not specifically discussed since they are still very few.