Notes on the bionomy of two spider wasp species in an urban forest fragment in Brazil

There are few studies dedicated to the natural history of Neotropical spider wasps (Pompilidae). When it comes to urban areas, the knowledge on these wasps is even scarcer. This study sheds light on the natural history of Auplopus cf. rufipes and A. cf. brasiliensis (Hymenoptera: Pompilidae: Ageniellini) from an urban Atlantic Forest fragment in the Jardim Botânico do Rio de Janeiro, Rio de Janeiro City, Brazil. Auplopus cf. rufipes displays a multivoltine pattern and frequently nests in bamboo canes scattered in the open areas, whereas A. cf. brasiliensis is a rare sight, with only a single nest collected from a hose trapnest. Females of the former species show aggressive behaviour, which suggests a solitary lifestyle. Their nests were partly enveloped in a complex structure, similar to other species that build free-standing, unconcealed nests and display some degree of sociality. Such behaviour could not be observed for A. cf. brasiliensis. Despite that, nests of both species were built using mud pellets, though the brood cells differed regarding arrangement and position in the trap. Two parasites were found attacking the cells: Caenochrysis crotonis (Chrysididae) in a cell of A. cf. rufipes, and Chaenotetrastichus neotropicalis (Eulophidae) in two cells of A. cf. brasiliensis, both recorded for the first time. Auplopus cf. rufipes was found to prey on Frigga cf. quintensis (Salticidae), the first record of this genus as a host of Auplopus. Key-Words. Ageniellini; Auplopodina; Auplopus; Nesting; Atlantic Forest.


INTRODUCTION
The pompilid wasps (Pompilidae) are cosmopolitan and most species are spider parasitoids, although some genera are known to be cleptoparasites of other pompilids as well. It is a highly diverse group comprising approximately 4,855 species worldwide (Aguiar et al., 2013), which challenges systematists (Waichert et al., 2015) and still lacks comprehensive studies on their biology.
Ageniellini presents several derived traits regarding nesting biology within Pompilidae (Shimizu et al., 2010). For instance, the species of this tribe nest above the ground, using a variety of sheltered locations, and build their nest prior to finding a host. In this tribe, the genus Auplopus (Auplopodina) is largely distributed throughout the Neotropical region and its species are known for building their nests using mainly mud (Wcislo et al., 1988;Dos Santos et al., 2017). This and other genera of Ageniellini, display levels of social behaviour, which is related with the nesting dynamic (Shimizu et al., 2010).
It is interesting to point out the importance of these data in the context of urban cities, especially regarding the rearing and management of these key organisms for the sake of ecosystemic functions. Wasps are very important control agents of several arthropod populations, like insects and spiders. We still need to fully understand how these interactions are taking place in biomes that have been ravaged by human activity such as the Atlantic Forest. Unfortunately, little data is available for these organisms, especially for wasps like the Pompilidae (Zanette et al., 2004;Buschini et al., 2007;Waichert & Pitts, 2012). This study represents a step towards filling this gap, presenting data on the natural history of Auplopus cf. rufipes and, to a lesser extent, A. cf. brasiliensis, from an urban forest fragment in the city of Rio de Janeiro, Brazil.

MATERIAL AND METHODS
Nesting activities of females were observed at the Jardim Botânico do Rio de Janeiro (22°58′14″S, 43°13′18″W) (JBRJ henceforth). The JBRJ is an urban forest fragment that belongs to the Atlantic Forest biome, and is located in the city of Rio de Janeiro, Brazil. It comprises two main areas: the arboretum, which has several native and exotic plant species; and a forest matrix contiguous with Parque Nacional da Tijuca and Parque da Cidade, two relatively well preserved fragments of the Atlantic Forest.
Trap-nests (referred to as traps, from now on) made of bamboo cane and hoses were placed in five transects, each of them with five spots approximately 125 m apart, to a total of 25 sample units in the area. Each sample unit had 30 traps, 15 bamboo cane traps (with diameters ranging from 0.4 cm to 2.4 cm) and 15 hose traps (split into three diameter categories of 0.79 cm, 0.95 cm and 1.27 cm -with five of each). Additionally, 96 traps made of plastic straw with 0.5 cm of diameter were set in three localities in the JBRJ, the forest matrix, the middle of the arboretum and the outermost point in the area, near the end of the park.
When the nests were built to completion, they were taken to the Laboratório de Hymenoptera of the Museu Nacional of Rio de Janeiro (HYMN), and replaced by empty traps of the same diameter. Nests were maintained in the laboratory with organza fabric attached to its open end, fastened with rubber string to stop emerging adults from leaving the nest. A single nest collected in September 2017 was opened to identify the hosts stored in the brood cells.
Images were recorded using a Leica DFC450 camera attached to a Leica M205C stereomicroscope and using the extended-focus software Leica Application Suite v4.8.0. Image stacking was performed by Combine ZP v.7.0.0.1 software. Voucher specimens ( Fig. 1) are deposited in the entomological collections of Museu Nacional/ Universidade Federal do Rio de Janeiro and Universidade Vila Velha, Espírito Santo, Brazil.

Auplopus cf. rufipes
Nests of this species were sparsely collected in the studied area. They were built in bamboo cane traps that ranged from 12 to 16 mm in diameter and were approximately 15 cm long. Five nests were collected between April/2017 and February/2019 (Table 1). The nest collected in April/2017 spawned two specimens, a male  Table 2. Nests collected and spawning of adults at the Jardim Botânico do Rio de Janeiro. Nests 2 and 3 of Auplopus cf. rufipes lack information because of the fire that hit the Museu Nacional on September 3 rd , 2018. The immatures were diapausing by the time the Museu Nacional suffered the fire accident, so that their mortality was displayed as unknown.   (Tables 1 and 2). Additionally, one larva died due to handling and another one for unknown reasons. In 2018, only one nest was collected in November, from which emerged one male and a specimen of cuckoo wasp Caenochrysis crotonis (Ducke) (Hymenoptera: Chrysididae) emerged. Other four immatures died from unknown causes. Direct observations of female nesting activities were frustrated given their aggressive behaviour whenever attempts to record the foundress activity closely were made, causing them to stop working on the nests. In such events, the females would face the unwelcome observer, flapping their wings against the trap floor and extending their metasoma, as if preparing to sting. This menacing posture behaviour was only witnessed twice, since the observed nests were unattended most of the time. Prey carrying behaviour, unfortunately, could not be seen.

Nest Nest building Nest collected Emergence Sex
The nests of this species were made of mud. Moist mud droplets were stacked, creating a papillated outer surface for the nest cells. After some time, the mud dried and became cemented. Measurements for brood cells are summarised in Table 3. Most nests were built near the bottom of the trap, except for one that was built detached from the trap bottom by about 4 mm, leaving a small empty space. The first cells were built sequentially, occupying only half of the trap's width. As the nest grew, other brood cells were added next to the first row, occupying the remaining space. Nests with more than two cells show a typical "V" formation when seen from above ( Fig. 2A). The second set of nests, embedded between the first ones, had their entrances directed upward, filling all the space. When the two halves of the trap were separated, part of the mud that formed the nest was adhered to its superior half. In addition, the cells were tightly glued together, in a way that the entire nest may be removed from the trap.
Empty spaces of the nests were covered by one layer of mud, which partially enveloped all the brood cells (Fig. 2B) creating an overlay. Although most of the exposed surface of each brood cell was papillated (Fig. 2C), the regions in contact with the trap walls or other brood cells were smooth (Fig. 2D). The inner surface of each brood cell was smooth, with almost no wrinkle. Brood cells were cylindric-oblong and they were tilted about 80° related to the substrate (Fig. 2B). Unfinished cells had a structure binding the entrance orifice, similar to a "lip" (Fig. 2C). Brood cells colour was uniform.
One of the nests collected was opened to identify the prey hunted by the foundress wasp. All spiders stocked as food for the larvae belonged to the species Frigga cf. quintensis (Tullgren) (Salticidae). Six cells were analysed which contained two males, three females and a juvenile, which could not be sexed. Figure 3 depicts the different stages of the immatures from the innermost (Fig. 3A) to the outermost cell (Fig. 3F) of the opened nest. The spiders were placed with their abdomen facing the posterior part of the brood cell, the cephalothorax facing the opening of the cell, and only the posterior-most pair of legs were amputated near the coxa-trochanter joint (Fig. 3C). The egg was laid on the ventral surface of the spider's opisthosoma, dislocated to the right (Figs. 3D-F). The larva started to feed in the region where the egg was laid, sucking the prey's internal fluids. Upon reaching the third instar, the larva consumed the prey entirely and, after the fourth instar, it pupated, but did not spin a cocoon. Subsequently, the adult emerged by chewing through the cell operculum, making a small hole.

Auplopus cf. brasiliensis
Only one nest of this species was collected in July/2017 (Table 1). This species was the only Hymenoptera species to build its nest in a hose trap. The nest had three brood cells: two of them were attacked by the parasitoid wasp Chaenotetratischus neotropicalis Marinho et al., 2019 (Eulophidae) and the third cell had a male specimen (Tables 1 and 2). One cell was opened (the broken cell in Fig. 4; the same brood cell is displayed in Marinho et al. (2019), Figs. 14-15, with the cocoon of the host full of several parasitoid pupae). The parasitoids were found inside the thick whitish cocoon, suggesting that they attacked the hosts right after it spun the cocoon and pupated. No specimens emerged from the second parasitized cell. This cell was opened after the first two specimens emerged; the specimens inside were also identified as C. neotropicalis. Due to the small size and fragile complexion, all of them were dried by the time the cell was opened.
The brood cells were arranged horizontally to the substrate, two cells side by side on the trap's floor and one built on top of the first two cells (Fig. 4). The cell's surface was papillated and had a rough aspect, whereas the inner surface was smooth. The anterior part of the cell was truncated, conferring it a jug-shaped form. The measurements taken from this nest are shown in Table 3.

DISCUSSION
Auplopus cf. rufipes and A. cf. brasiliensis are two species with no previous data on their natural history. Based on their emergence pattern, A. cf. rufipes is multivoltine and its nesting activity occurs from May to November. No pattern of nesting could be inferred for A. cf. brasiliensis, since only one nest was caught. The multivoltinism in the genus Auplopus was already observed for other species by Zanette et al. (2004) for A. militaris (Lynch-Arribalzaga) and by Buschini et al. (2007) for five unidentified species of Auplopus, with evidence that those species are multi-voltine with at least three generations per year, a pattern that matched the observed for A. cf. rufipes in this study. Shimizu et al. (2010) correlated nesting behaviour with the development of some degree of sociality, reaching parasociality in some species of the tribe Ageniellini. Auplopus cf. rufipes seems to be a solitary species. Nest architecture, however, indicates apomorphic behaviour, according with Shimizu et al. (2010), which consider soil nesting the ancestral condition in Ageniellini and plastering soil with regurgitated water, a derived condition. Particularly in this case, females always built the nests by themselves. Also, extending and contracting their abdomen when an unwelcome observer approaches may be seen as a defensive or aggressive posture, suggesting lack of communal nesting, as observed by other authors for Ageniellini (Wcislo et al., 1988;Dos Santos et al., 2017). Nevertheless, communality can be present even when such aggressive behaviour exists, and the imbalance of maintaining this behaviour is thought to be on other traits, such as nest protection (Wcislo et al., 1988). The lack of a resolved phylogeny for this tribe, however, hinders placing these behaviours on an evolutionary perspective.
Based on field observations, A. cf. rufipes follows the general nest-construction pattern observed for other Auplopus species: building the cell, provisioning, oviposition, closing the cell, repeating the process all over until nest construction is finished (Evans, 1953). Auplopus cf. rufipes left its nests unattended for several hours, and for many occasions one unfinished cell was seen open and empty. The "lip" that these cells had before oviposition is probably used for cell closure right after it stores the prey and oviposits, adding water from its crop to reshape this lip-like structure into an operculum, as observed on the cell of A. semialatus Dreisbach (Wcislo et al., 1988). It could be hypothesised that this lip-shaped structure may be a defence against parasites, eliminating the need for the female to leave the nest unattended during the process of closing brood cells. Nest parasites usually succeed at oviposition when the female is away to search for materials for cell closure after provisioning. Since females of Auplopus species are able to carry water in the crop, this pre-made lip-shaped structure allows the wasp to seal the nest without getting away from it.
Regarding egg-laying behaviour, A. cf. rufipes differed from those species of Pompilidae, for which the position of the egg laid in the prey is documented. Two closely related species of Anoplius studied by Evans (1953), An. apiculatus atumnalis (Banks) and An. semirufus (Cresson) oviposit and place the egg in rather different ways from one another upon their prey. Since great variation is observed on the exact spot where the egg is laid in the spider among Pompilidae species, this character may be species specific (Evans, 1953;Evans & Matthews, 1973). The larval feeding behaviour of A. cf. rufipes suggests a strong pumping musculature, similar to other Pepsinae larvae (Evans, 1959) that feed by sucking the fluid contents of the stocked prey. After development of the head capsule and mandibles, the larva may consume all that is left of the prey and pupate. Interestingly, Figure 4. Nest of Auplopus cf. brasiliensis. The third cell broke during handling. Note the arrangement of the cells forming a cluster, the papillated surface suggesting the employment of several mud pellets to construct the cells. Scale bar = 0.5 cm.