Elytra are critical in tolerating a variety of environmental stresses in Tribolium

It is relatively intuitive to imagine the hardened tight-fitting elytra as a dorsal shield of beetles. A multitude of literature, including research papers and textbooks, often assume that elytra protect beetles from various environmental challenges1,3,4,6,7. However, studies experimentally testing the function of elytra as a protective shield are surprisingly scarce. In this study, we demonstrated that elytra are indeed crucial in protecting beetles from a variety of environmental stresses. Our work provides the first comprehensive overview demonstrating the functional significance of elytra, which furthers our understanding of the significance of this structure in the evolution of Coleoptera.

Hindwing damage

The importance of elytra in protecting the hindwings is often assumed to be the driving force for the successful radiation of Coleoptera, by allowing beetles to explore the niches where other winged insects have difficulty dwelling without damaging their flight wings. However, despite the popularity of this statement, to our knowledge, this function of elytra has never been empirically tested. In this study, we demonstrated that hindwings sustained significant damage without the protection of elytra, even though the flour in which Tribolium live is relatively soft. Therefore, the protection over hindwings provided by elytra should be quite significant for beetles living in more abrasive niches, such as under the bark of trees or inside the soil or sand.

Predation

Our predation assay revealed that elytra are also pivotal as a protective layer against predation. It is quite interesting that elytra provide an advantage at two levels; during the initial attack where the predator quickly gives up when elytra are intact, and at the feeding step where the predator exhibits difficulties killing (and likely consuming) beetles with elytra. There might be a link between the defensive function of elytra and another defense mechanism that Tribolium possess, a chemical defense. Tribolium have two pairs of ‘stink’ glands, one in the thorax and the other in the abdomen40, which secrete a quinone-based chemical cocktail to repel predators41. Since elytra are covered with numerous sensory bristles in most beetles (including Tribolium)13,20, it is possible that the elytra serve as a sensitive detector for predatory attacks. Removing elytra might have lowered the ability of the beetles to sense the attack, which may have caused a delay in secreting defensive chemicals in the ER beetles. This could explain the two levels of defense advantages we observed in our assay; (i) the large sensory surface of elytra allows sensitive detection of predatory attack, which is followed by a chemical defense to dampen predator’s motivation, and (ii) the exoskeletalized surface of the elytra provides mechanical protection, making it more difficult for a predator to consume the beetle. Analyzing the behavior of the spider when the defensive gland is removed from Tribolium (via a gene knockdown technique) will help further our understanding of interaction between the chemical defense and the protective function of elytra. It is also worth mentioning that beetles with extensive chemical defense systems (such as lycid net-winged beetles and meloid blister beetles) tend to have soft bodies and less sclerotized elytra42,43. Therefore, there might be a significant connection between the protective function of elytra (and the hardening of the entire body) and the chemical properties of beetles during coleopteran evolution.

Desiccation

A possible role of elytra in desiccation tolerance has previously been studied, showing that elytra (and subelytral space) have important roles in water retention, transpiration rates, and discontinuous gas exchange patterns, mainly in beetles from arid environments8,9,10,44. Our study demonstrated that elytra are essential even in a beetle living in a more temperate environment (though flour is a dry niche compared to other natural environments), and that the lack of elytra actually causes high mortality. We also showed that this high mortality was likely caused by desiccation, as providing higher humidity reduced mortality in beetles without elytra (Fig. 3). It is yet to be determined whether the lower desiccation tolerance in ER beetles is due to the exposure of the abdominal spiracles (usually hidden underneath the elytra) or the exposure of the thin thoracic and abdominal integument. In a majority of our desiccation assays, we did not detect any differences in sexes when virgin beetles were used (Supplementary Table S5 and Fig. 3). It will be interesting to see how fertilization will influence their ability to survive in a dry condition.

Cold shock

Insulator and the thermal buffer functions of elytra and subelytral space were initially proposed in desert beetles, in which subelytral space helps beetles withstand high temperature (ref. 45, though this idea was argued against in ref. 46). Our study showed that elytra are critical for beetles to withstand a brief cold-shock, suggesting that elytra (and subelytral space) might also be able to buffer low temperature. A caveat to our assay was the possibility of increased mortality due to rapid desiccation, as we did not control RH in our cold shock assay system. However, as shown in Fig. 4b, the water loss was far less in the cold shock condition compared to the loss at 0% RH 30 °C, even though the former condition caused more rapid mortality. Studies examining cold and desiccation stresses in other insects show that resistance to these two stresses is often acquired mutually via similar physiological responses (see ref. 47 for review). This cross-tolerance would suggest that mild desiccation could actually improve resistance to cold shock. In our assay, however, we saw that beetles lacking elytra are more susceptible to cold shock, despite the possible advantages provided by losing more water by desiccation compared to control beetles. The exact mechanism underlying this increased mortality is yet to be determined. Direct measurement of the temperature of beetles during cold shock may be insightful to assess if elytra (and subelytral space) are in fact acting as an insulator, allowing beetles to maintain their body temperature.

Evolution of elytra and subsequent evolutionary changes in other tissues

Elytra are not simply hardened forewings. In addition to the highly sclerotized and exoskeletalized surface, the shape and size of elytra have also been evolutionarily modified, allowing the elytra to fit exactly to the dorsal surface of the abdominal segments. The vein-derived patterns of elytra are also significantly modified, from a regular parallel pattern of impressed striae and elytral veins in some, to no trace of vein-derived structures in others4. Furthermore, unlike typical flight wings of insects, elytra are usually covered with numerous sensory bristles (setae), both on the veins and in the intervein regions. Considering these diverse evolutionary modifications of the beetle forewing, it is reasonable to assume that many different types of environmental stress have contributed to shaping the beetle elytra. The data presented here demonstrate that the four categories of environmental stress we tested (damage to hindwings, desiccation, predation, and cold shock) can be valid stresses that have facilitated the evolution of elytra.

The sequence of evolutionary events giving rise to the beetle forewing is still largely elusive. Fossils of protocoleopteran beetles provide a hint to a transitional state during the evolution of beetle elytra. Protocoleopteran elytra had several primitive (i.e. flight-wing like) traits, such as the lack of heavy exoskeletalization in the inter-vein regions, a larger overall size, and a more typical wing vein pattern48,49. Some of these primitive traits (such as the lack of inter-vein exoskeletalization) are still retained in Archostemata4, a suborder of Coleoptera, suggesting that the evolutionary changes on elytra happened in a stepwise manner. We have previously shown that at least three independent co-option events have occurred to achieve the overall exoskeletalization of the Tribolium elytra21, while neofunctionalization of a wing gene (abrupt) has facilitated the evolution of elytron-unique shape24. These evo-devo findings further support a stepwise evolution of beetle elytra.

The acquisition of elytra has triggered several secondary changes to the beetle anatomy, possibly making beetles more dependent on elytra. This poses a significant caveat to our study. For example, beetle hindwings are thinner and more fragile than the flight wings of other insects and folded into a small subelytral space4, suggesting that these are secondary traits due to the acquisition of elytra. Because of these characteristics, beetle hindwings are likely more susceptible to damage compared to wings of other insects. Therefore, the dependency of the hindwing on elytra to maintain its integrity is likely exaggerated in our assay system using a modern beetle (Tribolium) as a model. Likewise, compared to the ancestral state of Coleoptera, the very thin dorsal abdominal surface typical of modern beetles has likely caused more water loss through the dorsal cuticle and less protection against predation to beetles when elytra are removed, possibly resulting in exaggerated outcomes in our predation and desiccation assay systems. Nonetheless, we believe that the advantages of elytra in Tribolium that we demonstrated in our assays reflect the evolutionary advantages provided by ‘proto-elytra’ (albeit less efficiently compared to the modern elytra) in the lineage leading to beetles, thus helping us further understand how the evolution of elytra has contributed to the remarkable success of beetle radiation.

Tribolium as a model to study stress tolerance

With a variety of genetic and genomic tools available, Tribolium is becoming a valuable model system for genetic and developmental studies25. Here, we show that Tribolium can also be an excellent model to study stress responses. In addition, we have established unique desiccation and predation assay systems, which allow us to study the response of beetles (and the spider in the predation assay) to environmental challenges with various parameters precisely controlled. The data presented here will serve as a baseline to further investigate how beetles handle desiccation and predation stresses.

Combining the assay systems we developed in this study with gene knockdown techniques, such as RNA interference (RNAi), will be quite powerful for exploring the molecular basis behind the organismal stress responses. For instance, several membrane pore molecules, such as Aquaporins, have been implicated in desiccation tolerance in insects50, however the detailed involvement of these molecules is still unclear. We have identified 6 Aquaporin genes in the Tribolium genome (DL and YT unpublished data). It will be insightful to perform the desiccation assay on beetles with the Aquaporin genes knocked down. Recently, Noh et al., showed that the yellow-e gene is important for desiccation tolerance in Tribolium, further supporting the benefit of using RNAi in Tribolium to study the molecular basis of insect stress responses51. RNAi can also be used to ablate specific tissues, which will be useful to determine their importance in a stress response. As discussed above, the stink gland may play a significant role in the elytron-dependent defense mechanism. RNAi for a stink gland specific gene41 can be used to ablate the gland, allowing us to test the protective function of elytra against predation when the chemical-based defense mechanisms are absent. Although, caution must be taken for the latter type of study, as the targeted gene can have important functions in more than one tissue (i.e. pleiotropic). For our study, we did not use RNAi-based ablation to remove elytra, as RNAi for the previously identified wing genes (e.g. vestigial, apterous, and abrupt) result in abnormalities in several tissues in addition to elytra and hindwings21,22,24. Nevertheless, Tribolium offers a key opportunity to study physiological, behavioral, and molecular mechanisms on how organisms deal with various environmental challenges.