Introduction
The social system distinguished by postovipositional parental care in invertebrates, called subsociality, is known to have arisen several times in insects (Eickwort Reference Eickwort1981). In terrestrial Hemiptera, simple subsociality limited to guarding of eggs and/or nymphs is recognised broadly throughout the taxonomic group (Tallamy and Wood Reference Tallamy and Wood1986). Specifically, numerous species from at least nine families in the infraorder Pentatomorpha display egg and/or nymph guarding (Schuh and Slater Reference Schuh and Slater1995). Seven of these families are in the superfamily Pentatomoidea, one is in the Coroidea (Coreidae), and one is in the Aradoidea (Aradidae). Guarding of eggs has been found to be an extremely effective defense against pedestrian predators (see Costa Reference Costa2006 for an extensive review). However, all species within the Pentatomorpha that are known to display the complex cares beyond egg and nymph guarding, i.e., production of trophic eggs and progressive provisioning, are confined to a handful of genera in the subfamily Sehirinae (Hemiptera: Cydnidae): Adomerus Mulsant and Rey (Nakahira Reference Nakahira1994; Mukai et al. Reference Mukai, Hironaka, Baba, Yanagi, Inadomi and Filippi2010), Canthophorus Mulsant and Rey (Filippi et al. Reference Filippi, Baba, Inadomi, Yanagi, Hironaka and Nomakuchi2009), and Sehirus Amyot and Serville (Sites and McPherson Reference Sites and McPherson1982; Kight Reference Kight1997; Agrawal et al. Reference Agrawal, Brodie and Brown2001, Reference Agrawal, Combs and Brodie2005; Kolliker et al., Reference Kolliker, Chuckalovcak, Haynes and Brodie2006), and Parastrachia japonensis (Scott) (Filippi-Tsukamoto et al. Reference Filippi-Tsukamoto, Nomakuchi, Kuki and Tojo1995; Filippi et al. Reference Filippi, Hironaka and Nomakuchi2001). The subfamily containing the species P. japonensis was recently elevated to family status (Parastrachiidae) from the Cydnidae (Sweet and Schaefer Reference Sweet and Schaefer2002), but some sources still classify it as a subfamily within Cydnidae (Costa Reference Costa2006; Grazia et al. Reference Grazia, Schuh and Wheeler2008). Because this level of complex subsociality occurs with such phylogenetically clustered manifestation, a very interesting and fundamental biological question arises as to whether the common ancestor to these genera obtained the traits once in the past or whether some or all of the behaviours evolved independently several times among a few taxonomic groups. However, at present there is insufficient information available to answer this question, primarily because of the very small number of species that have actually been investigated for subsociality in Cydnidae and Parastrachiidae.
Of the nine genera in the Sehirinae, Sehirus is the only common representative in the Palaearctic (Schuh and Slater Reference Schuh and Slater1995), and the parental care behaviours of Sehirus cinctus Palisot, which inhabits North America from Canada to Mexico, have been extensively studied (Sites and McPherson Reference Sites and McPherson1982; Kight Reference Kight1997; Agrawal et al. Reference Agrawal, Brodie and Brown2001, Reference Agrawal, Combs and Brodie2005; Kolliker et al., Reference Kolliker, Chuckalovcak, Haynes and Brodie2006). Although there has been no report on whether S. cinctus females produce trophic eggs, they do guard and provision nests with nutlets of several Lamium Linnaeus (Lamiaceae) species. A very early report by Southwood and Hine (Reference Southwood and Hine1950) indicates that Sehirus bicolor mothers also care for eggs and young nymphs, however, there is no specific mention of provisioning. Several cydnids in Japan have already been characterised with regard to their extensive maternal care. Canthophorus niveimarginatus Scott guards eggs and nymphs, provides pre and posthatch trophic eggs, and provisions nests (Filippi et al. Reference Filippi, Baba, Inadomi, Yanagi, Hironaka and Nomakuchi2009). Three species of the genus Adomerus are distributed in Japan. In this genus, Adomerus triguttulus (Motschulsky) and Adomerus variegatus (Signoret) have already been found to show complex subsociality with provisioning behaviour. Mothers of A. triguttulus and A. variegatus display egg and nymph guarding, production of trophic eggs, and progressive provisioning (Nakahira Reference Nakahira1994; Mukai et al. Reference Mukai, Hironaka, Baba, Yanagi, Inadomi and Filippi2010). However, there is currently no information about trophic egg production or the provisioning behaviour of the third Japanese Adomerus species, Adomerus rotundus (Hsiao), other than a brief mention that the behaviours exist (Mukai et al. Reference Mukai, Hironaka, Tojo and Nomakuchi2012), which makes it ideal as the next target for investigation of the phylogenetic pattern of occurrence of complex maternal care in this cluster. Therefore, in the present study we characterised the manifestations of trophic egg production and progressive provisioning in A. rotundus.
Our ultimate long-term goal is to accumulate further information on the maternal behaviours of as many species in this group as possible. We expect that correlation of the different manifestations of these behaviours with the ecological constraints imposed on each of the species in this cluster should facilitate a truly meaningful discussion of the evolutionary history and phylogenetic origin of complex parental care behaviours in these species. Moreover, in combination with morphological and molecular data, these findings will contribute to our understanding of the phylogenetic relationships in the Cydnidae and its close relatives, which remain somewhat controversial (Grazia et al. Reference Grazia, Schuh and Wheeler2008; Pluot-Sigwalt and Lis Reference Pluot-Sigwalt and Lis2008).
Materials and methods
Adomerus rotundus is iteroperous, with females producing multiple broods over a few months. In addition, it is multivoltine: multiple generations are produced from April to September in the region of northern Kyushu, Japan. It feeds on seeds of the herb, Lamium amplexicaule Linnaeus (Lamiaceae). Adults were collected in Yobuko-cho, Karatsu, Japan (33°53′N; 129°9′E) and transferred to the laboratory. Females and males were reared together on host seeds in a clear, round plastic case (diameter: 12 cm, height: 6 cm) lined with moist Kimwipe in an incubator kept under a 12-hour light/12-hour dark photoregime at 20 °C. The container was sprayed with water daily to prevent desiccation. Half of the collected adults were used for successive rearing as the laboratory population, and the other half were used for direct observations. When gravid females were recognised in the observation group, half were transferred individually to smaller clear plastic cups (diameter: 6 cm, height: 3.5 cm) with host seeds and reared under the above conditions for the observation of possible egg guarding and trophic egg production behaviours (Group A, n > 32). The other half were reared individually in a rectangular plastic cage (16 × 12 × 6 cm), in which a nest site and a foraging site were set at opposite diagonal corners, under a 16-hour light/8-hour dark photoregime at 27 °C, and observed for possible provisioning behaviour (Group B, n > 13). The foraging site of the latter cage was checked daily, and seeds that had been removed by the female were replaced, such that 50 seeds were constantly available at the “foraging site”.
First we dissociated individual egg masses of a portion of the egg-guarding females from Group A (n = 16) in water just prior to hatch. Eggs were determined to be near hatching by the appearance of eyespots on fertile eggs. Eggs that did not have eyespots remained white in colour, similar to the colour of freshly laid eggs, and were considered to be prehatch trophic eggs. The numbers of prehatch and fertile eggs in each egg mass were counted. Because eggs do not hatch after being dissociated in this way, the eggs from these 16 females were then discarded.
Next, to examine egg-guarding behaviour, hatching behaviour, and posthatch trophic egg production, we then observed individual females from among the females remaining in Group A and their egg masses for about one hour daily for ∼12 days from oviposition to hatch under a binocular microscope (n = 16). On the day when hatching was predicted to occur, again, as indicated by the presence of eyespots in the eggs, we observed the females and their egg masses continuously by direct visual inspection supported by a video camera (HDR-HC3, Sony, Tokyo, Japan).
Finally, the behavioural patterns of females and nymphs in Group B after hatching were observed once a day, and the number of seeds provided by females into the nest was also checked daily by counting the number of seeds removed from the foraging site as well as the number of seeds in the nest (n = 13). Thus, because data for prehatch trophic eggs, posthatch trophic eggs, and provisioning were obtained from three different groups of females, no correlations between the rates of production of prehatch and posthatch trophic eggs and provisioning rate could be made.
Results
Egg-guarding behaviour and egg characteristics
Females laid eggs in a round, cohesive mass. They then assumed the egg-guarding posture: females held the egg mass under the abdomen with the legs. The egg-guarding posture continued until hatching. During the egg-guarding period, there was no foraging behaviour by females and no seeds were carried into the nests from the surrounding area. Further, when approached with forceps, females showed a defensive guarding behaviour presenting their dorsum to the forceps.
The colour of the egg mass changed from milk-white to pink as time progressed towards hatching. Most of the eggs in an egg mass developed eyespots, indicating that they were fertilised eggs, while in several egg masses, a few eggs distributed on the surface of the egg mass failed to develop eyespots. The latter were determined to be “trophic eggs” from the definition of Nakahira (Reference Nakahira1994), Mukai et al. (Reference Mukai, Hironaka, Baba, Yanagi, Inadomi and Filippi2010), and Hironaka et al. (Reference Hironaka, Nomakuchi, Iwakuma and Filippi2005), specifically, “prehatch trophic eggs” from the definition of Filippi et al. (Reference Filippi, Baba, Inadomi, Yanagi, Hironaka and Nomakuchi2009).
Hatching and posthatch trophic eggs
Hatching began about 12 days after oviposition. The hatching of nymphs was very synchronised. Although hatched nymphs initially aggregated on the egg mass shells, a number of them immediately began to climb and gather at the end of the female's abdomen while the female assumed a characteristic posture: she raised her body over the cluster of new first instar nymphs lifting her abdomen out and upward into the air. As soon as an egg-like object came out from the tip of the female's abdomen, nymphs rushed to cluster around it, appearing to pierce it with the proboscis (Fig. 1). The nymphs apparently fed on the expressed “egg” immediately because it was completely shriveled by the time it dropped to the floor of the container a few minutes later, and the nymphs then dispersed. These egg-like oval objects were unequivocally determined to be “posthatch trophic eggs” as per the definition of Filippi et al. (Reference Filippi, Baba, Inadomi, Yanagi, Hironaka and Nomakuchi2009). Every time a posthatch trophic egg was expressed, at intervals of several minutes, a number of nymphs gathered around it and then dropped to the floor of the container in a cluster. Expression of these posthatch trophic eggs continued sequentially for about two hours. Then, after feeding on the posthatch trophic eggs, most nymphs turned to feed on the prehatch trophic eggs left in the egg mass shell. The numbers of total eggs, prehatch trophic eggs (n = 16) and posthatch trophic eggs (n = 16) produced by females were 86.1 ± 13.9, 1.8 ± 2.0, and 12.8 ± 5.1 (mean ± standard deviation), respectively (Fig. 2).
First instar nymphs of Adomerus rotundus gather at their mother's abdomen to feed on posthatch trophic egg as it is expressed.
Numbers of total, prehatch trophic and posthatch trophic eggs produced by female Adomerus rotundus.
Provisioning behaviour
Females began to provision the nest with host seeds on the day after they expressed the posthatch trophic eggs. The daily changes in the provisioning frequency are shown in Fig. 3, as determined by the number of seeds provided by females (n = 13). The provisioning rate peaked late in the first nymphal stadium, after which it gradually declined and ceased late in the third stadium. As indicated in Fig. 3, the cessation of provisioning behaviour coincided with nymphal independence, i.e., most nymphs had departed from the nest and relocated to the foraging site when provisioning ceased. After nymphs became independent, females no longer carried seeds into the nest, but instead fed on seeds themselves in the foraging area.
Provisioning rate of female Adomerus rotundus, n = 16; error bars represent standard deviation from the mean. Horizontal arrows delineate duration of the first, second, and third nymphal stadia.
Discussion
This study showed that females of A. rotundus display very similar complex postovipositional maternal cares to those of the North American cydnid, S. cinctus (Palisot de Beauvois) (Sites and McPherson; Kight Reference Kight1997; Agrawal et al. Reference Agrawal, Brodie and Brown2001, Reference Agrawal, Combs and Brodie2005; Kolliker et al., Reference Kolliker, Chuckalovcak, Haynes and Brodie2006) and the two other Japanese Adomerus species, A. triguttulus (Nakahira Reference Nakahira1994) and A. variegatus (Mukai et al. Reference Mukai, Hironaka, Baba, Yanagi, Inadomi and Filippi2010), another Japanese cydnid, C. niveimarginatus (Filippi et al. Reference Filippi, Baba, Inadomi, Yanagi, Hironaka and Nomakuchi2009), and a close Japanese relative, P. japonensis (Filippi-Tsukamoto et al. Reference Filippi-Tsukamoto, Nomakuchi, Kuki and Tojo1995; Filippi et al. Reference Filippi, Hironaka and Nomakuchi2001). In addition to egg and nymph guarding, female A. rotundus also produce both prehatch and posthatch trophic eggs and display progressive provisioning behaviour.
Although egg-guarding behaviour by A. triguttulus and S. cinctus females is relatively facultative because it is often interspersed with bouts of foraging behaviour (Sites and McPherson Reference Sites and McPherson1982; Nakahira Reference Nakahira1994), in A. rotundus egg guarding was constant, with no interruptions, as it is in A. variegatus (Mukai et al. Reference Mukai, Hironaka, Baba, Yanagi, Inadomi and Filippi2010), C. niveimarginatus (Filippi et al. Reference Filippi, Baba, Inadomi, Yanagi, Hironaka and Nomakuchi2009), and P. japonensis (Filippi et al. Reference Filippi, Hironaka and Nomakuchi2001). It is curious that only the North American species, S. cinctus, apparently does not produce trophic eggs, and this singular difference from the Asian species could shed light on the ecological factors under which the more complex behaviours arose. On the other hand, because none of the studies on S. cinctus make any reference to trophic eggs, perhaps S. cinctus does produce them, but this went unrecognised because that aspect of parental care was not investigated. Clearly, this requires further exploration. Among the other species, variations in the tenacity of egg guarding coincide with differences in trophic egg production patterns and could be related to differences in predation/cannibalism pressure (Filippi et al. Reference Filippi, Hironaka and Nomakuchi2001). Adomerus triguttulus females produce trophic eggs (prehatch trophic eggs) by adding them one at a time to the surface of an egg mass during the egg-guarding period (Nakahira Reference Nakahira1994). Apparently all resources acquired prior to oviposition are converted into fertile eggs, and females produce and add trophic eggs to the egg mass over the 10 or so days until hatching, using resources they acquire through foraging during short breaks from egg guarding. However, females of the other Japanese species, C. niveimarginatus, A. variegatus, and P. japonensis, as well as A. rotundus of the present study, may not need more nutrients during guarding, because both fertilised eggs and prehatch trophic eggs, are entirely laid at the time of oviposition. It has been speculated that posthatch trophic eggs could be retained in the ovarioles throughout oviposition and guarding to facilitate their reabsorption and conversion to fertile eggs should the initial mass fail for any reason: once the eggs are laid as trophic eggs, they cannot be fertilised (Filippi et al. Reference Filippi, Baba, Inadomi, Yanagi, Hironaka and Nomakuchi2009). It seems likely that species suffering high rates of egg failure due to predation would be more likely to produce posthatch trophic eggs, however, this hypothesis has not been tested. Alternatively, in the case of A. rotundus, females may have to devote themselves to guarding the egg mass without provisioning or feeding, because cannibalism pressure by surrounding conspecific nymphs is high in the field (S.N., personal observation). The egg defense behaviour itself was consistent with that reported for other subsocial Sehirinae and Parastrachiidae (Filippi et al. Reference Filippi, Hironaka and Nomakuchi2001): females present their dorsum to the disturbance.
Females of A. rotundus produced two types of trophic eggs, prehatch trophic eggs and posthatch trophic eggs, as does C. niveimarginatus (Filippi et al. Reference Filippi, Baba, Inadomi, Yanagi, Hironaka and Nomakuchi2009). Therefore, to the best of our knowledge, this is the second report of two types of trophic eggs being produced by one female. In the case of A. rotundus, the numbers of prehatch and posthatch trophic eggs are fewer than reported for C. niveimarginatus (mean ± SE: 6.2 ± 1.2 and 26.1 ± 0.4, respectively; Filippi et al. Reference Filippi, Baba, Inadomi, Yanagi, Hironaka and Nomakuchi2009). Moreover, in the present study, there were frequently no prehatch trophic eggs in an egg mass (Fig. 2), suggesting prehatch trophic eggs might be less important to offspring success in A. rotundus than they are in C. niveimarginatus. First stadium nymphs of C. niveimarginatus require prehatch and, particularly, posthatch trophic eggs from mothers to survive (Baba et al. Reference Baba, Hironaka, Hosokawa, Mukai, Nomakuchi and Ueno2011), and trophic eggs dramatically enhance survivorship of P. japonensis nymphs (Hironaka et al. Reference Hironaka, Nomakuchi, Iwakuma and Filippi2005); how essential trophic eggs are to the survival of A. rotundus nymphs remains to be determined.
Thus far, all species that have already been investigated for maternal care in Sehirinae and Parastrachiidae indicated progressive provisioning behaviour, and most produce trophic eggs. The present study revealed that A. rotundus also displays complex maternal care involving trophic egg production and progressive provisioning. The timing for females to initiate provisioning varies among the subsocial bugs discussed here (Mukai et al. Reference Mukai, Hironaka, Baba, Yanagi, Inadomi and Filippi2010). Provisioning began at the second stadium in C. niveimarginatus, but at the first stadium in S. cinctus, A. triguttulus, A. variegatus, and P. japonensis (Nakahira Reference Nakahira1994; Filippi-Tsukamoto et al. Reference Filippi-Tsukamoto, Nomakuchi, Kuki and Tojo1995; Kight Reference Kight1997; Filippi et al. Reference Filippi, Baba, Inadomi, Yanagi, Hironaka and Nomakuchi2009). In A. rotundus, provisioning began at the first stadium. This variation may reflect variation in the hardness of the different host seeds. In the case of C. niveimarginatus, the seed coat of the host plant, Thesium chinense Turcz (Santalaceae), is apparently too tough for first stadium nymphs to pierce with the proboscis, thus first stadium nymphs cannot feed on it (Baba et al. Reference Baba, Hironaka, Hosokawa, Mukai, Nomakuchi and Ueno2011). Consistent with this observation, females of C. niveimarginatus start the provisioning behaviour at the second stadium, when nymphs are able to feed on the hard seeds. On the other hand, in all other species, including A. rotundus, females apparently begin provisioning earlier because first stadium nymphs can feed on seeds of the host plant, genus Lamium for Adomerus species and drupes of the host tree, Schoepfia jasminodora Siebold and Zuccarini (Schoepfiaceae), for P. japonensis. Provisioning also peaks at different developmental stages across the species. For example, it was greatest near the end of the first stadium for A. rotundus, but provisioning peaked late in the second stadium in A. variegatus (Mukai et al. Reference Mukai, Hironaka, Baba, Yanagi, Inadomi and Filippi2010) and in the early third stadium in P. japonensis (Filippi et al. Reference Filippi, Hironaka and Nomakuchi2005).
We demonstrated that this species of Japanese Adomerus, A. rotundus, shows complex postovipositional maternal care including production of two types of trophic eggs and provisioning behaviour. From Lis (Reference Lis1999), it is known that there are six species of Old World Adomerus. With the results of this study, all three species of Japanese Adomerus have now been investigated to some extent, and all have been shown to display complex maternal care including guarding, trophic egg production, and provisioning behaviour. Considering these species, as well as S. cinctus, C. niveimarginatus, and P. japonensis, the patterns of production of trophic eggs show more variation across these species than the provisioning behaviour. It also seems likely that trophic egg production could evolve fairly easily, and initially might result from a failure to fertilise some eggs. However, the progressive provisioning behaviour seen in Sehirinae, repeatedly dragging seeds into a nest containing nymphs, is a more complex behaviour, one that we suspect arose from a preadaptation in a single common ancestor. We regularly see at our field sites other cydnid species in the genus Macroscytus Fieber dragging seeds on the ground (S.N., L.F., personal observation), and others have reported similar observations for other species (Tallamy and Wood Reference Tallamy and Wood1986; Takeuchi and Tamura Reference Takeuchi and Tamura2000; Panizzi and Hirose Reference Panizzi and Hirose2002). Jacobs (Reference Jacobs1989) reported females of a thaumestellid species, Thaumastella elizabethae Jacobs, dragging seeds into cavities (Schuh and Slater Reference Schuh and Slater1995). Dragging a seed to a sheltered site to minimise exposure while feeding would be a reasonable preadaptation for progressively provisioning a nest. Another preadaptation would be ovipositing a cohesive mass of eggs that could be guarded and provisioned upon hatching (Tallamy and Wood Reference Tallamy and Wood1986). The final behavioural prerequisite would be nest fidelity, which should coincide with the evolution of proximal mechanisms for returning repeatedly to a designated nest site. Hironaka et al. (Reference Hironaka, Filippi, Nomakuchi, Horiguchi and Hariyama2006) have done much to clarify these mechanisms in P. japonensis.
Tallamy and Schaefer (Reference Tallamy and Schaefer1997) present a compelling argument for the possibility that parental care in the Hemiptera, and in insects, overall, represents a plesiomorphy. They argue that because parental care is the rule, rather than the exception, in most arthropod relatives of insects, it is likely that the common ancestor of the arthropods had parental care. They further propose that parental care was likely lost in insect lineages as eggs became more robust and insects adapted to terrestrial habitats. It appears that some uncertainties remain regarding the precise phylogenetic relationships within the Pentatomoidea (Grazia et al. Reference Grazia, Schuh and Wheeler2008; Pluot-Sigwalt and Lis Reference Pluot-Sigwalt and Lis2008), but egg and nymph guarding are common throughout this group. Therefore, it seems reasonable to consider that these behaviours could be plesiomorphies. What about the origin of the most complex behaviour of progressive provisioning? Morphological and molecular data consistently suggest that the Cydnidae is most likely a paraphyletic grouping, and Parastrachia placement remains controversial (Grazia et al. Reference Grazia, Schuh and Wheeler2008; Pluot-Sigwalt and Lis Reference Pluot-Sigwalt and Lis2008). Nonetheless, exclusive of Parastrachia, the genera comprising the cluster currently identified to show complex parental care that includes progressive provisioning are all from the cydnid subfamily Sehirinae and appear to consistently be identified as each other's closest relatives. It seems plausible that they share an ancestor that provisioned its nest (i.e., displayed seed-dragging behaviour and nest fidelity), thus, we consider provisioning as a homologous derived state across the representatives in the Sehirinae. Whether Parastrachia truly belongs in its own family (Parastrachiidae, Sweet and Schaefer Reference Sweet and Schaefer2002), or as a subfamily (Parastrachiinae) within the Cydnidae, it seems likely that provisioning arose independently in this lineage, as no representatives of the other cydnid subfamilies show this behaviour. This distinction is consistent with some of the life history constraints imposed on P. japonensis that are not seen in the other species, such as obligate semelparity (Filippi et al. Reference Filippi, Hironaka and Nomakuchi2001). We hope to examine European Sehirinae representatives to further explore this hypothesis. Also, an important aspect of clarifying the evolutionary pathway to emergence of these complex behaviours requires detailed understanding of the ecological factors that make them adaptive. Thus, we also intend to investigate how the species in this cluster vary in the respective ecological constraints experienced over evolutionary time.
Acknowledgements
The authors thank the members of the laboratory of Animal Behavioral Ecology at Saga University for their help with the experiments and for their continued support, and Dr. Shinichi Kudo for his valuable discussions and encouragement. This research was supported in part by a Grant-in-Aid for scientific research (18570025 and 21370011) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and Faculty Research and Development Grant and Presidential Research Awards Program grants to L.F. from Hofstra University.
