For nesting site choice of Aculeata, and accordingly for the design of suitable nest boxes, the nest tube diameter is an important factor.

For cavity-nesting Aculeata the choice of diameter can have an effect on various nesting parameters which, on the other hand, can affect reproductive success, like number of offspring (e.g. Pinilla‐Gallego et al. 2022) or sex ratio (e.g. O’Neill and O’Neill 2003; Coville et al. 2015; Pinilla‐Gallego et al. 2022). In principle, for most solitary Aculeata, females seem to emerge more often from nests with a larger diameter (e.g. O’Neill and O’Neill 2003; Coville et al. 2015; Pinilla‐Gallego et al. 2022). Even when small diameters are accepted for nesting, it is possible that only male offspring emerge (e.g. Buschini and Woiski 2006; Coville et al. 2015). In addition, Aculeata offspring from larger diameters are often heavier (e.g. Rust 1998) and larger (e.g. Buschini and Woiski 2006; Coville et al. 2015) than offspring from smaller diameters. Larger females, on the other hand, can be more successful hunters, for instance due to better flight performance, as shown for the crabronid species Philanthus triangulum (Strohm and Linsenmair 1997). Additionally, diameter can influence provisioning efficiency itself. For example, bees collect more pollen and nectar per cell when nesting in tubes with large diameters (Stephen and Oscood 1965). However, diameter preferences vary between species (Staab et al. 2018; Qu et al. 2022; Torretta et al. 2023) and the largest diameter is not necessarily always chosen (e.g. Budrienė et al. 2004).

Compared to cavity-nesting species, pith-nesting species excavate their own nest tunnels within twigs of a given diameter. Consequently, it remains unclear to what extent findings on the effect of diameter on fitness parameters in cavity-nesting Aculeata apply to pith-nesting species. Nevertheless, far fewer studies have investigated diameter preferences and their effects on fitness parameters in pith-nesting species.

One example of a pith-nesting species is the digger wasp Pemphredon lethifer Shuckard, 1837 (Hymenoptera: Pemphredonidae). P. lethifer is a solitary, holarctic species (Blösch 2000), which prefers nesting in sunny sites (Danks 1971a). One to several generations can appear, depending on region and climate (Janvier 1961; Danks 1971a). Nests are often dug in cut stems of Rubus spp. (e.g. Borries 1897; Benno 1957; Danks 1971a), but also other plants are used (e.g. Goureau 1855; Benno 1958; Janvier 1961; Danks 1971b; McIntosh 1996) and seldom the full pith diameter is used. The nest structure can be linear or branched (Oehlke 1970) and the cells of a nest are separated by pith (Borries 1897; Janvier 1961). As females of P. lethifer prey exclusively on aphids to provision their nests, thus completely removing the aphids from the plant (Danks 1971b), P. lethifer could furthermore be a new biocontrol agent for aphid control.

In field experiments with pithy twigs, Danks (1971a) observed that a greater proportion of Aculeata used stems with a large pith diameter, which matches findings for Exoneura bees (Coates et al. 2022). Also P. lethifer were found to prefer a larger diameter in the field (McIntosh 1996). However, the study focused primarily on the outer diameter and not the pith diameter of twigs. In addition, the influence of different pith diameters on various fitness parameters remains unknown and there are currently no studies under laboratory conditions excluding external factors such as inter- and intraspecific competition or parasitism.

This knowledge, on the other hand, is also needed to design suitable nest boxes to promote P. lethifer and to allow its efficient rearing and use as biocontrol agent. A nest box should i) be attractive for P. lethifer for nesting and ii) support factors which lead to optimized biocontrol efficacy. For this, a nest box should on one hand produce a high number of offspring. On the other hand, as mainly the females function as biocontrol agents by collecting aphids to supply their nests (Danks 1971b), a female focussed sex ratio should be supported.

Taken together, studies on nesting preferences and their effect on fitness parameters are essential for a better understanding of pith-nesting wasps in general, their promotion and for their use as biocontrol agents. Therefore, we carried out field trials with trap nests and a laboratory choice experiment with single twigs in 2022 and 2023. Answering the following questions was a main aim: i) What pith diameter does Pemphredon spp., in particular P. lethifer, prefer in nature and under controlled conditions? and ii) How does pith diameter affect number of offspring and sex ratio in P. lethifer?

In this study we showed that P. lethifer prefers large pith diameters and that this choice has an impact on multiple fitness parameters important for its rearing, promotion and the design of suitable nest boxes.

In both study years Pemphredon spp. was the most abundant genus (2022: 52%; 2023: 41%) and P. lethifer (2023: 26%) was the most abundant species in trap nests made of Sambucus spp. Our findings were similar to other studies (e.g. Tormos et al. 2005). In contrast to the present cage study, Danks (1971b) observed that females tended to build multiple nests in the field as well as in the laboratory; however no average number of nests per females was reported. In the present cage study, many females only built one nest, which might indicate suboptimal conditions, but could also mean the opposite: The long straight twigs in various diameters offered here are likely ideal nesting sites serving for more nest cells, which might be rare in nature. Furthermore, parasitism and usurpation were excluded under laboratory condition. While in nature building several nests might reduce the risk of parasitism (Rust 1998), parasitoids were not present in the cages. Both factors might explain that P. lethifer built fewer nests here. As the digging of further nests is energy-intensive (Ostwald et al. 2021), it might be energy-saving to extend existing nests. However, the number of offspring in the cage experiment was not representative and targeted experiments would be needed to further investigate these hypotheses.

There are few studies focussing on diameter preferences of pith-nesting Aculeata. In our field trials, Pemphredon spp. resp. P. lethifer favoured twigs of the largest PD, which is consistent with other studies for this species that did however not limit the largest pith diameter (> 5 mm (Danks 1971a, 1971c); > 6.6 mm (McIntosh 1996)). McIntosh (1996) also suggested that there might be a preference for large diameters, but not for a specific category. This hypothesis cannot be confirmed by the results of our study, because larger PDs, including larger defined categories as compared to both former studies, were significantly preferred under field conditions.

In our cage experiment, the smallest PD was also used least frequently, but in contrast to the field studies, no differences were found between larger PDs. There are various potential reasons for the latter. For cavity-nesting bees, it has been suggested that they prefer specific diameters to avoid competition (Budrienė et al. 2004). As there was no competition in the laboratory, this may have played a subordinate role here. Another explanation for the differences could be the experimental set up. P. lethifer is known to circle the twigs before nesting, likely in order to analyse the diameter of the twig (McIntosh 1996), bark structure, branches or cracks. While this behaviour was possible in the cage experiment, it was not always possible with bundled twigs in the field. Therefore, maybe PD was more relevant as stimulus for the choice of twigs in the field. The diameter of the nest site was also suggested by Kunjwal et al. (2021) to be important for attractiveness for cavity-nesting bees. However, unlike as, for example, in cavity-nesting Osmia lignaria propinqua (Tepedino and Torchio 1994), in our cage experiment P. lethifer’s preference for diameters was not influenced by the PD of the previous nest. Generally, the choice of a small diameter could have multiple disadvantages. For instance, the wasp may no longer be able to dig sufficiently wide tunnels and turning could therefore be a problem (Coville et al. 2015). This could make provisioning more difficult and slower. Tunnel diameter of P. lethifer has to be large enough for moving and turning (Janvier 1961) and based on the findings of our study, this may not be possible below a pith diameter of 4.5 mm, which therefore likely is the minimum suitable diameter for P. lethifer. Nevertheless, when designing a nest box, it is important to consider that using twigs with a larger diameter entails a trade-off: within a given nest box size, fewer twigs can be installed, which in turn reduces the number of potential nests. Therefore, the expected occupancy rate and number of offspring should always be taken into account.

In line with some studies on cavity-nesting Aculeata, PD did not have an influence on the probability of parasitism (Michołap et al. 2020) or mortality (Drapeau Picard et al. 2024). Nevertheless, Rinehart et al. (2024) should also be considered. However, in contrast to McIntosh (1996), when assessed in the 2023 field trial, head width was correlated with PD and larger females preferred larger diameters for nesting.

In both years in the field, number of offspring per nest was higher at the beginning of the nesting season, which was also shown for other Aculeates (Torchio and Tepedino 1980). Mortality increased at the end of the season. Together, our results support the hypothesis that the fitness of P. lethifer is higher at the early season. As mortality also increased with nest length, similar to other Aculeates (e.g. Vieira et al. 2022), offspring may not always find their way out or the path may be blocked by other cells in long nests (Stephen and Oscood 1965). As nest length is in turn limited by the twig or tube length, length should be considered when developing a nest box for P. lethifer to limit mortality.

In our study, sex ratio was influenced by several factors, as assumed by Danks (1983). Similar to the findings for other Aculeata (e.g. Torchio and Tepedino 1980; Wittmann et al. 2023), sex ratio was higher at the beginning of the nesting season. In addition, sex ratio was higher for offspring emerging without a cooling interval. Other Aculeata also produced more males in the overwintering generation (Longair 1981; Wittmann et al. 2023) and this was also observed for P. lethifer in other studies (Danks 1971b). Since P. lethifer can have several generations per year (Janvier 1961), this may explain why sex ratio was lower in nests built late in the season, as those nests are more likely to overwinter (Danks 1983). Additionally, PD did have an effect on sex ratio. In contrast to several studies on cavity-nesting Aculeata (e.g. Longair 1981; O’Neill and O’Neill 2003), sex ratio was not the highest for nests with the largest diameter, but rather for nests with a diameter of > 6.5 to 8.5 mm. This indicates there could be a diameter optimum for increasing sex ratio. A study with the cavity-nesting bee Megachile rotundata showed similar results, as it was not the largest diameter offered which led to the highest sex ratio (Rinehart et al. 2024). Consequently, with regard to potential use as biocontrol agent, a medium pith diameter can be chosen to optimize sex ratio in nest boxes for P. lethifer, as only females collect aphids for supplying their nests. However, the findings are contradicted Danks' (1983) hypothesis, according to which larger twigs do not automatically result in more female offspring, as P. lethifer can control the size of its nest by digging. In general, however, it is difficult to determine sex ratio precisely – and thus also the factors influencing it – as it is also affected by losses due to mortality and parasitism (Danks 1983).

In our study, similar to Danks (1971b), the most abundant parasitoids were Chrysididae, followed by Chalcidoidea and Ichneumonidae. The fact that not a single Ichneumonidae was found in 2022 can be explained by the short sampling interval. As Ichneumonidae parasitise particularly finalized, plugged nests of Aculeata (Mader 2010) the non-occurrence of Ichneumonidae supports the hypothesis that nesting was not finished in 2022. At 13% (2022) and 15% (2023), the proportion of parasitized cells was lower than the value observed by Danks (1971c) (21 to 30%). However, our study did not account for gregarious parasitoids, and differences in the sampling intervals of trap nests could also explain these discrepancies. In the 2023 field trial with a longer sampling interval, further factors influenced the probability for parasitism. Here, branched nests were more frequently parasitised than linear nests. This effect occurred regardless of the number of offspring. No known studies have yet observed or investigated this, but a likely explanation is that parasitoids can reach several cells in branched nests more easily than in linear ones even after nesting is finished and probably can more easily hide in unfinished nests.

In the field trial in both years as well as in the laboratory, larger females built significantly longer nests and built less frequently branched than linear nests when compared with smaller females. This result is contrary to McIntosh (1996), who suggested that nests of P. lethifer are always branched after a few days if the diameter is large enough and can also not confirm the suggestions from Tolstova (1962) and Danks (1971b) that nests are only linear in thin twigs. Following the previous argumentation, linear nests are preferable to avoid parasitism. However, the construction of long linear nests may cost more energy per cell and may in turn be more affordable for larger females, which often show a higher fitness (e.g. Honěk 1993; Strohm and Linsenmair 1997). This suggests that in pith-nesting Aculeata, the head width may have a more decisive influence on the nest structure than the pith diameter. Apart from that, species nesting in pithy stems may behave differently from cavity-nesters, which often choose twig diameters according to body size to balance brood cell size (MacIvor 2016), energy requirements for nesting (Mariano et al. 2020) and to reduce parasitism (MacIvor 2016). In contrast, species that nest in pithy twigs can influence the nest structure themselves by excavating the nest tunnel. However, because the 2023 field trial also showed that larger females often preferred larger diameters for nesting, larger diameters likely have additional advantages such as increased isolation or greater overall stability.

Our study sheds light on the importance of pith diameter preference for various nesting parameters of P. lethifer. Nevertheless, there is still little known about preferences and effects on fitness concerning other parameters like angle (Coates et al. 2022), height or material (Collins and Jennings 1987; MacIvor 2016). Finding out more about such parameters remains important in order to promote digger wasps and to design an effective nest box for P. lethifer, in particular with regard to rearing success and its potential use as biocontrol agent.

Our study showed that, P. lethifer prefers large pith diameters and that nest structure is influenced by female size with larger females building more linear nests. Cells in branched nests where more often parasitized than those in linear nests. Nests produced in the early season produced more offspring with a higher percentage of females and a lower mortality rate. Regarding its usage in biocontrol, a nest box for P. lethifer should contain pith diameters between 6.5 and 8.5 mm in order to achieve a more female based sex ratio and pith diameters smaller than 4.5 mm are not suitable for nesting. A shorter tube length could reduce mortality. Efficacy of P. lethifer can be expected to be highest in the early season. For continuous rearing, laboratory conditions and their effects on the fitness of P. lethifer need to be optimized.