Nesting Biology of the Solitary Wasp Pisoxylon amenkei (Hymenoptera, Crabronidae, Trypoxylini) in a Neotropical Hotspot of Southern Brazil

© 2021 Academia Sinica, Taiwan Open Access Nesting Biology of the Solitary Wasp Pisoxylon amenkei (Hymenoptera, Crabronidae, Trypoxylini) in a Neotropical Hotspot of Southern Brazil Jean Pablo Alves de Deus1,* , Caroline Nepomuceno Queiros1, and Maria Luisa Tunes Buschini1 1Alameda Élio Antonio Dalla Vecchia, 838, Vila Carli, Campus CEDETEG, Universidade Estadual do Centro Oeste - UNICENTRO, Guarapuava - PR, 85040-167. *Correspondence: E-mail: [email protected] (Deus). Tel: +5542999509852. E-mail: [email protected] (Queiros); [email protected] (Buschini) Received 27 June 2020 / Accepted 31 December 2020 / Published 8 March 2021 Communicated by John Wang Pisoxylon amenkei is a specie of solitary wasp that builds its nests in pre-existing cavities; it has only been recorded nesting in the Araucaria forest, one of the Atlantic forest phytophysiognomies and a biodiversity hotspot. So far, the only information on the biology of the genus Pisoxylon is based on one species. In addition, the genus has many similarities to Trypoxylon, showing an increased need for studies on the biology of these species of wasps. In the present study, we introduce unpublished information about Pisoxylon amenkei nesting biology and described many aspects of its natural history. Research was carried out between August 2017 and August 2019, in a rural area of Guarapuava municipality, Paraná, Brazil. Pisoxylon amenkei nested only in fragmented parts of Araucaria forest, during summer and autumn. It is a multivoltine species, and thus remains in diapause, in the pre-pupa phase during winter and spring. Their nests had an interior design similar to that of Pisoxylon xanthosoma and some Trypoxylon species, such as Trypoxylon lactitarse and Trypoxylon agamenon. Moreover, the sex ratio of P. amenkei was 1: 1, which can be linked to an equal cost in the production of females and males. Like other species of Pisoxylon, P. amenkei nests primarily in forest areas. In southern Brazil, it nests in fragments of Araucaria forest, which are threatened. Therefore, more efforts are needed to preserve these remaining fragments; a failure to do so could have devastating results, considering the number of threatened species that these forests house. We conclude that further studies should focus on the phylogeny of the group and use molecular analyses to clarify the hypothesis of Antropov (1998), that Pisoxylon should be classified as a subgenus of Trypoxylon. Key words: Hymenoptera, Crabronidae, Diapause, Multivoltine, Conservation. Citation: Deus JPAd, Queiros CN, Buschini MLT. 2021. Nesting biology of the solitary wasp Pisoxylon amenkei (Hymenoptera, Crabronidae, Trypoxylini) in a Neotropical Hotspot of Southern Brazil. Zool Stud 60:5. doi:10.6620/ZS.2021.60-05. BACKGROUND Pisoxylon, described in 1968 by Arnold S. Menke, is a genus of solitary wasp that builds its nests in preexisting cavities in forest areas (Morato and Campos 1994; Iantas et al. 2017; Nether et al. 2019). This genus has three described species: Pisoxylon roosevelti Antropov 1998, occurring in Bolivia; Pisoxylon xanthosoma Menke, 1968, occurring in Brazil and Peru; and Pisoxylon amenkei Antropov 1998, occurring only in Brazil, in Paraná and Rio de Janeiro states (Antropov 1998; Amarante 2002). In southern Brazil, P. amenkei Zoological Studies 60: 5 (2021) doi:10.6620/ZS.2021.60-05 1 © 2021 Academia Sinica, Taiwan has been recorded as a rare species, occurring with low frequency in trap nests (Iantas et al. 2017; Nether et al. 2019) and nesting only in Araucaria forests (Mixed Ombrophylous Forest), one of the phytophysiognomies of the Atlantic forest, and one of the most threatened biomes due to the reduction of its vegetation cover, which has left only isolated fragments (Ribeiro et al. 2011). The Atlantic forest is currently among the 35 biodiversity hotspots in the world, and among the biomes most prioritized for conservation, due to its high levels of richness and endemism and the destruction that it has suffered in the past. Current data indicate that only 28% of its original area remains (Rezende et al. 2018). It is the fifth richest hotspot in endemism (Mittermeier et al. 2004), but many of its species have not yet been sampled or even described. Studies have indicated that its coastal areas have maintained populations of species from the late Quaternary to the present day, making these zones ecological refuges for biodiversity (Carnaval et al. 2009). Despite this, knowledge about many groups of animals, particularly insects, that live in these forests is still only incipient (Freitas and Marini-Filho 2011). Little is known about the nesting biology of wasps that occur in this biome. As their habitats are modified, the populations of predatory wasps, which nest in branches or tree trunks, have also been altered (Tylianakis et al. 2006) due to a lack of suitable places to build their nests. Consequently, the rate of predation by these insects has decreased, causing changes in the populations of their prey and their natural enemies (Holzschuh et al. 2009). These three functional guilds remain connected in the ecosystem where they live (LaSalle and Gauld 1993), therefore, make them relevant indicators of ecological changes, due to their participation in the three types of interaction (Tscharntke et al. 1998), which makes them bioindicators (Gerlach et al. 2013). Pisoxylon, as well as several species of the Crabronidae family, prey on spiders (Buschini et al. 2008 2010a b; Pitilin et al. 2012; Moura et al. 2019; Musicante and Salvo 2010) and are parasitized mainly by parasitoid wasps (Nether et al. 2019). These wasps capture a wide range of spider taxa, including orbweaving spiders (e.g., Araneidae, Tetragnathidae, Nephilidae), araneida sheet web weavers (e.g., Theridiidae), and even several taxa that usually do not use webs to capture prey (e.g., Lycosidae, Salticidae, Oxyopidae, Clubionidae) (Coville and Coville 1980; Camillo and Brescovit 1999; Blackledge et al. 2003, Moura et al. 2019; Buschini et al. 2010a b; Pitilin et al. 2012). Knowledge about the biology of this genus is based only on what has been documented for P. xanthosoma, with nests containing from one to four cells organized linearly, which are separated by clay partitions (Morato and Campos 1994; Antropov 1998). However, there is no information about their mortality rate, time of development, number of generations and sex ratio, structure and organization of the cocoons and disposition of the sexes in the nests (Antropov 1998). In addition to the lack of information on the biology of these species, another factor that increases the need for studies on this group is the similarities between the genera Pisoxylon and Trypoxylon (both from the Trypoxylini tribe), mainly in the way they build their nests, although the Trypoxylon species can be distinguished because they build their nest in preexisting cavities (Antropov 1998; Amarante 2002; Coville 1987). Moreover, Menke (1968) considered the compact abdomen of Pisoxylon to be an autopomorphy; however, with the description of T. monjuba Amarante 1995, it was discovered that this species has the first abdomen segment compact, similar to that of Pisoxylon, making the relationship between these genera even closer. So much so that, according to Antropov (1998), the genus Pisoxylon should be included as a subgenus of Trypoxylon. Recently, a study by Sann et al. (2018), which compiled a comprehensive set of phylogenomic data on the main lineages of Apoid wasps and bee families, confirmed that the Crabronidae family is polyphyletic, which reinforces the need for a general review of this group. Although many studies have been carried out on the biological aspects of Trypoxylon species (Buschini et al. 2006 2010a b; Buschini and Wolff 2006; Santoni et al. 2009; Buschini et al. 2008; Buschini and Fajardo 2010; Musicante and Salvo 2010; Buschini and Donatti 2012; Moura et al. 2019), there is only one article on the biology of Pisoxylon (Morato and Campos 1994). Therefore, more research is needed on the species of this genus, since it helps systematists elucidate aspects about the phylogeny of these animals. In the present study, we introduce unpublished information about P. amenkei nesting biology and describe many aspects of its natural history. MATERIALS AND METHODS Study site This study was carried out between August 2017 and August 2019 in a rural area in the municipality of Guarapuava, Paraná, Brazil. According to the Köppen classification (Kottek et al. 2006), this municipality has a Subtropical Humid Mesothermal climate, with no dry season, severe frosts, an average annual temperature around 22°C and average annual precipitation of 1961 mm. The months that have the highest temperatures are page 2 of 11Zoological Studies 60:05 (2021) © 2021 Academia Sinica, Taiwan from September to April, with average temperatures of 23.5°C, and the lowest temperatures extends from May to August, with average temperatures of 12.8°C and records of absolute minimums below 0°C (IAPAR 2014). We collected in four forest areas and four open grassland areas (where the forest was removed) in two rural areas (Jordão neighborhood and locality of Guabiroba). The forest areas were denominated FJ1 (25°26'58.0"S 51°27'02.1"W), FJ2 (25°26'06.2"S 51°26'34.0"W), FG1 (25°23'56.1"S 51°25'34.4"W) and FG2 (25°23'48.2"S 51°24'49.3"W). The open grassland areas were denominated OJ1 (25°26'50.6"S 51°27'10.9"W), OJ2 (25°25'48.5"S 51°26'50.9"W), OG1 (25°23'43.1"S 51°25'27.7"W) and OG2 (25°24'15.4"S 51°23'44.0"W). All forest fragments used for collection were native and had sizes of FJ1 with 32.9ha, FJ2 with 113ha, FG1 with 256ha and FG2 with 153ha. All these fragments are linked to other nearby fragments, making them even bigger. In addition, all sampling stations (see the next session) located in the forest were at least 50 m away from the edge. All properties had plantations in their vicinity (Fig. 1). Rural areas FJ1, FJ2, OJ1 and OJ2 were monitored from 2017 to 2019 and areas FG1, FG2, OG1 and OG2 from 2018 to 2019. Trap nests We used trap nests—pre-existing artificially built cavities—to describe the biology of P. amenkei. Both nests were made out of wood and bamboo. The wooden nests were standardized to 2 × 2 × 12 cm. The cavities measured 0.5, 0.7, 1.0 and 1.3 cm in diameter and were 8 cm deep. Bamboo nests, on the other hand, could not be standardized due to the natural range of sizes; therefore, we used nests 8 to 14 cm long with cavities ranging from 0.4 to 1.5 cm in diameter. Both the wooden and bamboo trap nests were cut longitudinally and joined with adhesive tape so that their interior could be seen. In each collection area, six sampling stations were arranged, separated from each other by at least 50 m, totaling 48 sampling stations and 1,536 trap nests. Each sampling station contained two blocks with 16 trap nests each: one block had wooden trap nests and the other had bamboo trap nests. The arrangement of trap nests in the blocks was random. They were placed 1.5 m from the ground, on a wooden stake and covered with a roofing tile to prevent the accumulation of rain water. Collections were carried out every 15 days for 3 years. Trap nests containing wasp or bee nests that were already finished were removed and replaced with a new Fig. 1. P. amenkei nest collection areas. F: Forest fragment, O: Open grassland, J: Jordão and G: Guabiroba. N page 3 of 11Zoological Studies 60:05 (2021) © 2021 Academia Sinica, Taiwan nest of the same diameter or similar size for bamboo, and taken to the laboratory, where they were placed in PET (Polyethylene terephthalate), their bottles were capped with cotton for later emergence and adults were identified. Afterwards, adults were killed under Ethyl acetate, then weighed and tagged. The nests that were identified as belonging to P. amenkei, were separated to study their biology. All individuals were deposited into the collection of the Laboratório de Ecologia e Biologia de Vespas e Abelhas da UNICENTRO, Guarapuava, Paraná. Nest contents A digital caliper was used to measure brood cells, vestibular and intercalary cells, and the thickness of nest closure. The brood cells had wasp prey, larvae or cocoons. Vestibular and intercalary cells are the empty cells located immediately after the nest closure, or between two brood cells. Unfortunately, for nests with diameters of 0.7, 1 and 1.3 cm, it was possible to measure the length of only one cell because the delimitation of each cell was not sufficiently clear. As the cocoons of P. amenkei have a cylindrical shape, their size was calculated by the volume occupied inside the nest, using the formula of the volume of a cylinder: V = π.r2.h. Where: V = volume; π (pi) = 3.14; r2 = radius (half the diameter of the cocoon); h = cocoon length. The weight of the newly emerged adults was recorded using a Bel Mark precision scale with four decimal places. Using a Leica MZ6 stereomicroscope, the width of their heads (greater distance between the surfaces of the compound eyes in frontal view) and intertergular distance ITD (distance between the inner margins of the tegules) were measured. The development time of these wasps was calculated as the number of days from the collection of the nests to the emergence of adults in the laboratory. The number of adult generations of this species was calculated considering the month that they started to build their nests, the time that the immature ones took to complete development, and the month in which the adults stopped nesting entered diapause. All data collected were tested for normality by the Shapiro-Wilk test, where everyone has failed and the data are therefore non-normal. So, to verify if there was a significant difference between the study variables, the Mann-Whitney non-parametric test was used. Due to the difficulty of manipulating individuals within the laboratory, the measures used varied in sample number. Only the weight of those individuals that emerged and shortly afterwards were killed under ethyl acetate were used; therefore, individuals that were dead inside nests or inside PET bottles were not measured. Likewise, measurements of intertegular distance, head width, cocoon volume and cell size were taken only when the object of interest was not damaged (Table 1). Sex ratio and position The sex ratio was calculated by the ratio between the number of females and the number of males, and the binomial test was used to verify whether the calculated sex ratio was significantly different from the expected 1: 1 ratio. All statistical analyses were performed using R version 3.6.2 (R development core team 2020). Aiming to check the position of the sexes inside the nest, each cocoon found was removed from its nest and separated into transparent plastic bottles and capped with cotton. The bottles were labeled with information such as the number and diameter of the nest, the day it was collected, the collection area and which cell nest Table 1. Explanation of the sample numbers used Measures Adults Eggs Larvae Pupa Parasitoids total cells 74 10 5 12 5 106 54 alive 20 dead Weight (g) 52 - Only individuals who emerged and were killed under ethyl acetate and that could be sexed were weighed. Intertegular distance (mm) 71 - of the total number of individuals (living and dead) 71 did not have a damaged thorax and that could be sexed were measured. Head width (mm) 70 - from total number of individuals (living and dead) 70 did not have their heads damaged and that could be sexed were measured. Cocoon volume (cm³) 66 - of the total number of individuals (living and dead) 66 did not have their cocoons damaged and that the adults could be sexed were measured. Cell length (cm) 66 - of the total number of individuals (living and dead) 66 did not have the margins of their cells damaged and that the adults could be sexed were measured. page 4 of 11Zoological Studies 60:05 (2021) © 2021 Academia Sinica, Taiwan Fig. 2. Number of nests of P. amenkei obtained monthly from August 2017 to August 2019. it was in. The cocoons stayed in those bottles until the adults emerged. Mortality and natural enemies All deaths were counted in five categories: death of eggs, death of larvae, death of pupae, death of adults from unknown causes and death from natural enemies. RESULTS During the two years of collection, and a total of 1,536 trap nests were constructed 59 of which were founded by P. amenkei, and 106 cells with individuals were constructed. Seventy-four of these cells contained adult individuals (54 alive and 20 dead), 10 eggs, 5 larvae, 12 pupae and 5 parasitoids (Table 1). One nest was built in the same trap-nest as Michantidium sp., a bee in the Megachilidae family. All nesting occurred in the forest fragments, which was on average 138.7 ha. In addition, the nests were constructed only in the hottest months of the year, from January to May in 2018, entering diapause from May and restarting activities in December of the same year, nesting until June 2019. The greatest frequency nesting took place in January both years (Fig. 2). Nest contents Pisoxylon amenkei preferred building in wooden trap nests, and only one nest was established in a bamboo trap. The most used diameter was 0.5 cm (39 nests), followed by 0.7 (14 nests), 1.3 (4 nests) and 1.0 cm (1 nest). Regardless of the diameter used to nest, the interior design of the nests was similar (Fig. 3). In all nests, the cells and cocoons were arranged linearly. The number of cells ranged from 1 to 7 (Table 2). The partitions between the cells were built with brown clay, which were convex on the outside and concave on the inside. The average cell size was 1.97 cm. In the closure plug, the wasps used organic matter containing resin and vegetable pieces (tiny pieces of leaves and wood), in addition to using clay in the innermost part of the closure plug, providing considerable resistance to the entry of the nests (average thickness of 0.305 cm) (Table 2, Fig. 3). Most nests (50 nests) had vestibular cells soon after this closure, with an average of two vestibular cells per nest. In addition, 21 nests had intercalary cells with an average of 1.4 cells per nest (Fig. 3). The cocoon of P. amenkei has the shape of a cylinder with its concave ends. The posterior end is more tapered than the previous one regarding the entrance of the nest. These cocoons are smooth on the inside and outside and have a light brown and opaque color, becoming beige at the ends. All cocoons are covered and fixed to the sides of the nest by silk produced by the larva. To feed the larvae, this wasp uses spiders immobilized with venom (average of 5 per cell), so that they remain alive for consumption. page 5 of 11Zoological Studies 60:05 (2021) © 2021 Academia Sinica, Taiwan Sex ratio and position Pisoxylon amenkei have two different times of development times: either they pupated and emerged as adults later in the same season (direct development), or they entered diapause, overwintering and pupating the following spring (delayed development) (Table 3). Considering the average time of direct development of the species (37.4 days), there may have been up to four P. amenkei generations in 2018, and up to six generations in 2019. The first nest collection of P. amenkei nests was in January 2018, which means that first generation adults completed their development and nest in February (2018), second generation adults in March (2018), third generation adults in April (2018) and fourth generation adults in May (2018). In addition, this same reasoning, from December 2018 to June 2019, these wasps had up to six generations since they entered diapause later. The sex ratio of P. amenkei was 39 females and 32 males, which did not differ significantly from the expected ratio of 1: 1, considering only adult males and females, not including pupae. Moreover, when we calculated the sex ratio from the two development times separate (direct and delayed development), they did not differ significantly either. For this analysis of the sex ratio for the two development times, we used only the individuals from which these times could be measured: Fig. 3. Nest of P. amenkei. A1: a vestibular cell; A2: cell partition; B3: intercalary cells; and C4: closure plug. Table 2. Measurements of P. amenkei trap nests Diameter Number of nests Thickness of closure plug (cm) Number of cells provisioned Length of cells (cm) 0.5 39 x = 0.305 ± 0.106 x = 1.769 ± 0.776 x = 1.973 ± 0.434 md = 0.300 md = 2 md = 1.927 0.7 14 x = 0.306 ± 0.095 x = 2 ± 0.960 2.765* md = 0.311 md = 2 1 2 x = 0.306 ± 0.087 x = 1.5 ± 0.707 2.100* md = 0.291 md = 1.5 1.3 4 x = 0.287 ± 0.129 x = 1.5 ± 0.577 1.800* md = 0.274 md = 1.5 TOTAL 59 x = 0.305 ± 0.106 x = 1.830 ± 0.854 x = 1.972 ± 0.428 md = 0.305 md = 2 md = 1.927 x indicates the mean with the standard deviation; md indicates the median. * it was possible to obtain the length of just one cell. page 6 of 11Zoological Studies 60:05 (2021) © 2021 Academia Sinica, Taiwan five males and 13 females for direct development and 18 males and 17 females for delayed development (Table 4). Of the 51 nests from which the sexes could be identified, the most of them (n = 43) were occupied by only one sex, with an average of 1.256 ± 0.539 individuals per nest. In the other eight nests, males and females emerged, all of whom had two individuals. In these nests, with the exception of one, the males were found in the anterior cells and the females in the posterior cells. Females were significantly larger than males, both in weight and in the intertegular distance and width of head. The cells where the females developed and their cocoon, were also significantly larger than those of males (Table 5, Fig. 4). Mortality and natural enemies Of the 106 total cells that contained individuals supplied by nests of P. amenkei, 52 had mortality (49.05%). Of these, 10 deaths were eggs (~20%), most likely due to dehydrated spider prey; five cells had dead larvae (~10%); 12 cells had dead pupae (~24%); 19 cells had adults that died from unknown causes (~36%). Messatoporus sp. and Photochryptus sp., parasitoids of the Ichneumonidae family, caused mortality in four cells (~8%) and one cell (~2%), respectively. Table 3. Development time of P. amenkei N Sample Male Female Mann-Whitney test (U) p Direct development (days) M (5) x = 34.2 ± 4.7 x = 32 ± 3.7 21.5 0.294 F (13) md = 33 md = 31 Delayed development (days) M (18) x = 246.2 ± 58.9 x = 262.5 ± 52.4 109 0.381 F (17) md = 251 md = 287.5 x indicates the mean with the standard deviation; md indicates the median. M: Male; F: Female. Table 4. Binomial test for P. amenkei sex ratio Males Females Probability of success p Direct development 5 13 0.722 0.096 Delayed development 18 17 0.472 0.868 Total 32* 39* 0.549 0.477 * These values represent both individuals who had their development times measured, as well as those who could not have that time measuring. Table 5. Adult size and cocoon dimensions of P. amenkei N Sample Male Female Mann-Whitney test (U)p Weight (g) M (22) x = 0.011 ± 0.005 x = 0.023 ± 0.005 49.5 2.141 e-07 F (30) md = 0.010 md = 0.025 Intertegular distance (mm) M (32) x = 1.311 ± 0.099 x = 1.672 ± 0.137 29 5.122 e-12 F (39) md = 1.344 md = 1.667 Head width (mm) M (32) x = 2.214 ± 0.119 x = 2.744 ± 0.196 56 6.436 e-11 F (38) md = 2.210 md = 2.812 Cocoon volume (cm³) M (30) x = 0.089 ± 0.027 x = 0.174 ± 0.059 91 7.661 e-09 F (36) md = 0.089 md = 0.179 Cell length (cm) M (30) x = 1.813 ± 0.341 x = 2.106 ± 0.451 333.5 0.008 F (36) md = 1.812 md = 2.079 x indicates the mean with the standard deviation; md indicates the median. M: Males; F: Females. page 7 of 11Zoological Studies 60:05 (2021) © 2021 Academia Sinica, Taiwan DISCUSSION This study was developed in a mosaic landscape composed by Araucaria forest fragments, grassland and monocultures. P. amenkei nested only in forest fragments characterized by the presence of Araucaria angustifolia (Bertolini) (Coniferae; Araucariaceae), which has been classified as vulnerable according to IUCN standards (Hilton-Taylor 2000) and is protected by law (National Forest Code 4771/65); however, the conservation status of the forest itself is critical (Fonseca et al. 2009). In view of this scenario, we believe that species such as P. amenkei, which have been classified as rare and nest only in these forests (Nether et al. 2019; Iantas et al. 2017), are at serious risk of becoming extinct. It is possible that one of the factors that influence the distribution of P. amenkei in Araucaria forest, in addition to the abiotic conditions, is the availability of spiders and holes to construct its nests, which will undoubtedly affect the distribution of its parasitoids. For example, according to Araújo et al. (2018), that study Trypoxylon lactitarse (Saussure, 1867), and Buschini and Fajardo (2010) studying Trypoxylon agamenon Richards, 1934, which also nested only in these forest fragments and using pre-existing cavities, and spiders to provision, native forests have a microenvironment with lower temperatures and luminosities, and higher relative air humidity (Buschini 2006; Buschini and Wolff 2006; Buschini and Woiski 2008), in addition to presenting a higher density of prey, or even offering more cavities for nests (Buschini et al. 2006 2008; Morato and Martins 2006). The vegetation structure of a given habitat is generally considered an important factor for spider diversity (Baldissera et al. 2004; Corcuera et al. 2008). The three-dimensional structure generated by plants on a given forest can have a strong influence on the number and variety of spiders that can live in that environment, be they web spinners or hunters (Rodrigues and Mendonça 2012), although anthropogenic changes in the ecosystem such as urbanization and habitat fragmentation in particular (Miyashita et al. 1998, Bolger et al. 2000) have had a great influence on spider communities in general (Whitehouse et al. 2002). We are losing many species of arthropods that are vulnerable to habitat fragmentation. The study by Sánchez-Bayo and Wyckhuys (2019) revealed that the extinction rates of the world’s insect species in the coming decades could reach 40%, with Hymenoptera being one of the most affected taxa in terrestrial ecosystems. According to these authors, this decline may occur both with specialist species, which occupy particular ecological niches, and many common and generalist species. On parasitoids, the impact of habitat fragmentation is further exacerbated by the fact that they often occur at low densities, in populations that are therefore more likely to be vulnerable to changes (Kruess and Tscharntke 2000; Ewers and Didham 2006). Perhaps this explains the fact that our study recorded for the first time the presence of parasitoids in the nests of P. amenkei in southern Brazil. Messatoporus and Photochryptus, of the Ichineumonidae family, were the ones that emerged from its nest and in low abundance. According to Audusseau et al. (2020), the persistence of the parasitoid population at a site is therefore the result of the interplay between local habitat suitability, species’ capacity to disperse between patches and the distribution in time and space of its potential hosts in the landscape. The interior design of wasp nests can also influence the rate of parasitism (Krombein 1967) by Fig. 4. P. amenkei female. General body coloration dark brown, in frontal view, head somewhat widened dorsally. Median clypeal lobe convex in the middle and strongly protruding, trapeziform; laterally with frontal depression and bent frontally along anterior margin (Antropov 1998). page 8 of 11Zoological Studies 60:05 (2021) © 2021 Academia Sinica, Taiwan parasitoids such as Ichneumonidae, which has been seen introducing its ovipositor through the closure plug of the Trypoxylon nests (Buschini personal information). In addition to the clay in closure plug, P. amenkei uses an outer layer of dark and resistant organic matter, which is mixed with vegetable resin, beyond the presence of intercalary and vestibular cells that hinder parasitism by natural enemies (Assis and Camillo 1997). Their nests were very similar to the nests of Trypoxylon species such as T. opacum, T. agamenon and T. lactitarse, with a linear arrangement of the cells, partitions between them built out of mud, the presence of vestibular and interlayer cells and provision of cells with spiders (Buschini and Wolff 2006; Buschini and Fajardo 2010; Buschini et al. 2006). What differentiates them is the shape and color of the cocoons, and the closing of the nests (Buschini and Wolff 2006; Buschini 2007; Buschini et al. 2010b). The disposition of the sexes in P. amenkei nests also differs from those of the Trypoxylon species that nest in pre-existing cavities. In most P. amenkei nests, females and males were raised in separate nests. In shared nests, females were raised in the innermost cells and males in the outermost cells. In T. lactitarse (Buschini et al. 2006), T. opacum (Buschini and Wolff 2006) and T. agamemnon (Buschini and Fajardo 2010), the position of the sexes was random and they laid together, regardless of the diameter of the cavity used for nesting. The position of the sexes of P. amenkei was similar to that registered for P. xanthosoma, which also had oviposited males and females in separate nests, and in those with shared sexes, in two of them the females emerged from the innermost cells (Morato and Campo 1994). Its sex ratio was 1: 1, which means that the cost of producing males and females is equal. According to Fisher (1930), an equal investment in sons and daughters, in most species, is the result of frequency dependence. If the cost of producing sons and daughters is equal, then this investment also translates into equal numbers of males and females (Fisher 1930; Seger 1983; Buschini 2007). However, considering that P. amenkei females are larger than males, it can be observed that, although the sex ratio was 1: 1, parental investment was higher during female production (Buschini 2007). CONCLUSIONS Results like those here are part of so-called basic research, which is a fundamental part of science. This type of research can be very important because it can present results that are often unexpected, thus generating new research fields (Courchamp et al. 2015). In addition, basic research is the basis for future applied research, which has more specific objectives, such as species conservation and Systematics (Courchamp et al. 2015; Freitas et al. 2020). We conclude that P. amenkei presented the interior design of nests similar to the Trypoxylon species that nest in cavities, but differed in relation to the shape and color of the cocoons, the closing of the nests and the disposition of the sexes in the nests. Like other species of Pisoxylon, P. amenkei nests primarily in forest areas. In southern Brazil it nests in fragments of Araucaria forest, which are threatened. Therefore, more efforts are needed for these remaining fragments to be preserved, otherwise our responsibility will be even greater considering the list of extinct species. Therefore, further studies focusing on the phylogeny of the group and the use of molecular analyses are needed to clarify the hypothesis of Antropov (1998), that Pisoxylon should be classified as a subgenus of Trypoxylon. Acknowledgments: We thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and Instituto Nacional de Ciência e Tecnologia (INCT dos Hymenoptera Parasitóides) for the scholarship and the Graduate Program in Evolutionary Biology of Universidade Estadual do Centro-Oeste (UNICENTRO), Paraná state, Brazil and the Dra Angélica Maria Penteado Martins Dias for her help with the project. We also thank Prof. Dr. David Barros Muniz (Universidade Federal do Paraná) and Prof. Dr. Eduardo F. dos Santos (Universidade Estadual Paulista, Brazil), for the identification of P. amenkei. Authors’ contributions: Jean Pablo Alves de Deus carried out field research, drafted the manuscript, and performed the analyses. Caroline Nepomuceno Queiroz helped collect field data and with all methodology carried out in the laboratory. Maria LuisaTunes Buschini supervised this study. Competing interests: All authors declare that they have no conflicts of interests. Availability of data and materials: The data generated and analyzed during the current study are available from the corresponding author. Consent for publication: Not applicable. Ethics approval consent to participate: Not applicable. page 9 of 11Zoological Studies 60:05 (2021)