In the course of evolution, animals and particularly insects, have developed efficient and complex mechanisms for survival. Biomimetics aims to find applications for these features of organisms (or organs) in industry, agriculture, and medicine. One of these features is the thin, flexible, and mobile insect ovipositor, which is also capable of carrying substances and drilling various substrates, usually of plant origin. Despite the well-studied structure of the ovipositor, the principles of its operation and real possibilities remain poorly understood. In our study, we first discovered an unusual behavioral pattern of oviposition of the female parasitoid Eupelmus messene Walker (Hymenoptera: Eupelmidae): she drilled with her ovipositor through the wall of a polystyrene Petri dish and laid her egg outside the dish. Due to the transparency of the plastic, we described the technique of ovipositor movement and studied its structure using scanning electron microscopy. Our research may contribute to developing minimally invasive guided probes and various other instruments.

Chalcidoidea, gall, oviposition, ovipositor structure, parasitoid

Insects inspire researchers to develop new biomimetic substances for industry and biomedicine (Elvin et al. 2005); to improve mobility, strength, and control systems of robots; to develop micro-electro-mechanical devices (MEMS) for supersensitive sensory systems in medicine, etc. (Gorb 2011); to produce new materials (Scarangella et al. 2020).

One of the examples of structures that may be beneficial for bionics is the ovipositor which in parasitoid wasps is a flexible and thin egg-laying organ used to drill holes in various natural materials, for example, host puparia, plant buds, stems, and galls (Askew 1971; Peters and Abraham 2010; Polidori et al. 2013). Studying the drilling process in nature is challenging due to the dense structure of plant substrates (for ex., fig syconia) (Kundanati and Gundiah 2014). Transparent gel-like substrates on the other hand, allow a detailed investigation of ovipositor insertion and steering (Gouache et al. 2010; Quicke 2014; Cerkvenik et al. 2017; van Meer et al. 2020); however, the mechanisms may differ due to the differences in stiffness of natural and artificial substrates.

Here we present the first observation of drilling and egg laying by the parasitoid wasp Eupelmus messene Walker (Hymenoptera: Eupelmidae) in a transparent and solid substrate – wall of a polystyrene Petri dish.

Eupelmus messene, which was recently separated from its synonym Eupelmus vesicularis Retzius (Hymenoptera: Eupelmidae) by Fusu (2017), is a 2–3 mm wasp. Among other hosts, it is a parasitoid of the gall wasp Aulacidea hieracii Linnaeus (Hymenoptera: Cynipidae). The latter forms a gall on the stems of the hawkweed Hieracium × robustum Fr. (Anikin and Nikelshparg 2017). The female of E. messene drills the walls of the gall with her ovipositor in search of a gall wasp larva and, upon finding it, lays an egg next to it (Gokhman and Nikelshparg 2021). Study of the drilling the gall is challenging due to its opaque and dense structure, whereas the transparency of the polystyrene made it possible to describe the drilling process and oviposition in detail.

Out of 18 females that were placed in Petri dishes without host galls, only 5 started drilling the wall of the Petri dish (Fig. 1A). Also, 3 wasps drilled the plastic in the other group of 38 females despite having the host galls inside the Petri dish. Each drilled between one and five holes (mean±SE = 1.75±0.5). The process of drilling each perforation in the polystyrene wall took more than two hours. Periodically the process was interrupted by feeding, drinking, or washing, after which the female found the same place in the Petri dish and continued drilling. In the end, the insect managed to complete the perforation in the plastic wall and lay one egg on the outside of the Petri dish (Fig. 1B, See Suppl. material 1). This process was repeated 4 more times, by the same female. After the female drilled 3 holes, it was transferred to a new Petri dish, where she drilled 2 more times. We recorded the drilling of four perforations out of five (See Suppl. material 1).

Several strictly sequential movement patterns for drilling a polystyrene dish can be distinguished:

1. Pushing movements resembling shaking, with a frequency of 5.7 ± 0.5 Hz and small amplitude, 0.21 ± 0.07 mm;
2. Rotational movements – the insect rotates the ovipositor in both directions up to 360 degrees – such movements were often combined with pushing movements;
3. Ejection movements, jerky movements of the ovipositor towards the insect body with a frequency of 0.8 Hz and an amplitude of 0.58 ± 0.07 mm. To move upwards, E. messene straightened its legs and partially retracted the ovipositor inside the body but never completely removed it from the drilled perforation.
4. Cementing step, this is the longest uninterrupted step in oviposition, lasting about 6–7 minutes, resulting in dome-shaped insulation at the entrance of the perforation, consisting of the UV fluorescent biological substance (Fig. 1C).

Drilling of natural substance (a plant gall) differs from drilling the polystyrene substrate. We studied in detail the drilling by 9 E. messene females out of 38 at six winter galls on H. × robustum. The frequency of pushing movements was 4.66 ± 0.34 Hz while the amplitude was 0.24 ± 0.03 mm. Thus, such movements in galls are less frequent than in polystyrene. Rotational movements were also observed, but we never observed ejection movements while the wasp was drilling a gall.

The structure of the ovipositor and the drilled perforation in the Petri dish was examined using a scanning electron microscope. The diameter of the drilled perforation is about 30 µm (Fig. 2A). As in all apocritans the ovipositor consists of paired ovipositor sheaths (3^rd valvulae), paired 2^nd valvulae that enclose a pair of 1^st valvulae. The 1^st and 2^nd valvulae form the ovipositor stylets. The exerted part of the stylets in the measured specimen has a total length of 2 mm and diameter of 20 µm (Fig. 2B–E). The apical part of 1^st valvula has a sawtooth structure (Fig. 2D). The length of this section is 70 µm, with about 6 teeth in a row; height of each tooth is 1 to 4.5 µm. We assume that with the help of the multiple teeth on the stylet, the E. messene not only cuts the perforation but also ejects excess material.

Figure 2. 

Ovipositor structure of the Eupelmus messene and perforation drilled in plastic, SEM A perforation in the Petri dish made by E. messene B–E structure of the E. messene ovipositor, lateral view: B apex of gaster C ovipositor D apex of 1^st valvifer E apex of 2^nd valvifer. ovs – ovipositor sheath, 2vl – 2^nd valvula, 1vl – 1^st valvula.

We described an interesting case of E. messene spontaneous drilling in a polystyrene Petri dish wall for oviposition in the absence of a host stimulus. The reason for such behavior of the parasitoid wasp remains unknown. Since plant gall is opaque, the mechanism of egg-laying behavior has never been described in detail. The unusual behavior of the female allowed us to characterize the drilling patterns. We distinguished four steps of drilling: pushing movements, rotational movements, ejection movements, as well as the cementing step. However, in natural gall, we never observed ejection movements. We suppose that such a type of movement is required to rake out plastic particles, which is unnecessary for more elastic plant gall substrate. Also, we suggest that pushing movements in galls are less frequent than in plastic as gall has other material properties than polystyrene. Importantly, fresh summer galls are suggested to be less solid than winter ones used here. Thus, we assume that the frequency of pushing movements would be decreased even more. The process of drilling the polystyrene and gall tissue may differ due to the difference in stiffness, however, a more detailed study of the gall and polystyrene material properties should be conducted. To mimic the drilling of wasps, Cerkvenik et al. (2017) applied gellan gel. However, we suggest that polystyrene is a more relevant model material to study drilling and oviposition in natural substrates, especially autumn firm galls.

The transparency of the polystyrene allows for describing the features of drilling in a solid substrate invisible in natural materials. Studying in details the drilling behavior of parasitic mycrohymenopterans can be useful in medicine for the creation of minimally invasive guided probes in neurosurgery (Frasson et al. 2010; Ramadi et al. 2019), the development of orthopedic surgical instruments (Nakajima and Schwarz 2014), needle biopsies using functionally graded tools (Kundanati and Gundiah 2014). Due to the flexibility, potential hardness, and effective types of movement, ovipositor structures can be used to develop methods of vertical and directional drilling, whereas ultra-low mass and energy efficiency make it possible to use ovipositor features for studying space structures and celestial bodies, extraterrestrial drilling or sampling (Gouache et al. 2010). Some eupelmid species (such as Eupelmus vuilleti Craw (Hymenoptera: Eupelmidae) and Eupelmus microzonus Förster (Hymenoptera: Eupelmidae)) provide a protective web-like coating for their eggs (Leveque et al. 1993; Gokhman and Nikelshparg 2021), but E. messene has not previously shown any ability to protect its offspring. Due to the transparency of the plastic Petri dish, we found that after laying the egg, the female carefully cements the drilled perforation with a yet unknown biological substance. We suggest that this substance plays a role in isolation from temperature fluctuations, water, microorganisms, etc. It should be noted that it is practically impossible to detect such perforation cementing under natural conditions.

We revealed the phenomenon of E. messene drilling the plastic Petri dish using three types of ovipositor movements: pushing, rotational, and ejection. In the natural gall, the ejection movements were absent, and the pushing movements were less frequent in the gall than in plastic. Additionally, we provided the first evidence that E. messene isolates the perforation after the oviposition. The structure of the ovipositor was described with SEM. The proposed analysis may contribute to developing minimally invasive guided probes for medicine or methods of vertical and directional drilling of rocks.

We acknowledge Lucian Fusu (Faculty of Biology, Alexandru Ioan Cuza University, Iasi, Romania) and V. E. Gokhman (Botanical Garden, Moscow State University, Moscow, Russia) for the identification of the parasitoid. We would like to express our gratitude to Lucian Fusu and the anonymous reviewer for their meticulous review of the manuscript. We are grateful to Gary A.P. Gibson (Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada) for depositing the voucher specimen of the E. messene in the Canadian National Collection of Insects, Arachnids, and Nematodes. The work was supported by the Russian Science Foundation (project No. 22–14–00028, https://rscf.ru/en/project/22-14-00028/ to A.A.P., morphological part of the study). The authors have declared that no competing interests exist.