Prevalence of leg regeneration in damselflies reevaluated: A case study in Coenagrionidae

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Highlights

  • Odonates exhibit effective leg regeneration abilities that result in a fully functional limb.

  • Prevalence of leg regeneration in Vanuatubasis is reported as much higher than previously understood.

  • Sex and species do not explain the extent of regeneration suggesting environmental factors may play a larger role.

Abstract

The leg regeneration capabilities of damselflies are understudied. Here we present the first data of regenerated limbs across a genus of damselfly based on adult specimens collected in the field to illustrate the prevalence of limb loss among nymphs. We show that this phenomenon is much more prevalent than previously thought, as 42 percent of individuals were found with regenerated limbs. Furthermore, we test for patterns within these data to begin to unravel the potential causes of limb loss in nymphal damselflies, showing that intrinsic factors such as sex and species cannot explain the patterns of limb loss pointing to environmental factors as the probable cause. We argue that Odonata limb regeneration provides a potentially unique perspective into the nymphal stage of these organisms.

Introduction

The phenomenon of structural regeneration, the act of regrowing damaged body parts (e.g., limbs), has been fascinating scientists for decades (Alvarado and Tsonis, 2006). From lizards to crabs, this ethereal ability has been viewed by many as the key to unlocking similar regeneration capabilities in our own species (Alvarado and Tsonis, 2006; Maginnis, 2006a). Arthropods, in particular, are capable of extensive structural regeneration (Maruzzo et al., 2005; Bely and Nyberg, 2010; Suzuki et al., 2019), but this can vary between groups. For example, regeneration in insects is different from that of other arthropods as successive molting is often required to complete the process, and adults therefore cannot regenerate limbs (Truby, 1983; Khan et al., 2016; Das, 2015; Suzuki et al., 2019). The absence of regeneration in adults makes it more difficult to study the causes and effects of limb loss in insects than in other groups where injuries are more easily studied, such as crustaceans in which adults continue to molt and regenerate throughout their life (Suzuki et al., 2019). There is a distinction between hemimetabolous insects whose regenerative capabilities seem very similar to that of other arthropods, such as crabs, and holometabolous insects whose regeneration is complicated by a pupal life stage required for complete metamorphosis (Das, 2015). In holometabolous insects, such as Coleoptera (Shah et al., 2011; Abdelwahab et al., 2018), Lepidoptera (Yang et al., 2016), and Diptera (Bosch et al., 2010), it is likely that imaginal cells or discs play an important role in their regenerative abilities (Lee et al., 2013; Das, 2015). In hemimetabolous insects, such as Ephemeroptera (Almudi et al., 2019), Odonata, Orthoptera (Yang et al., 2016), Phasmatodea (Maginnis, 2006b), Blattodea (Tan et al., 2013), and Hemiptera (Knobloch and Steel, 1988), there is a “breakage point” on the limb that results in the limb easily falling off when subject to pressure, a mechanism called autotomy (Maruzzo et al., 2005; Maginnis, 2006b). It has been suggested that the ability to break off limbs and regenerate them allows these organisms to better survive predators and intraspecific aggressors (Bely and Nyberg, 2010). Furthermore, this common breaking point could enhance healing and prevent signaling potential predators to weakened individuals (Matsuoka and Ishihara, 2010; Maruzzo et al., 2005).

Odonates, a hemimetabolous order encompassing dragonflies and damselflies, have effective regeneration abilities with regard to both, legs and caudal lamellae (Maruzzo et al., 2005; Stoks and De Block, 2007). The loss and regeneration of caudal lamellae is relatively well-studied in damselfly nymphs and is largely attributed to interactions with other organisms such as conspecifics and predators (Robinson et al., 1991; Black et al., 2019). The loss of lamellae is also considered to occur frequently with natural populations having anywhere from 50% to 90% of individuals with lost lamellae (Robinson et al., 1991; Stoks, 1998; Stoks and De Block 2000). Leg regeneration, on the other hand, is poorly studied perhaps due to some evidence that it occurs infrequently, with nymphs largely relying on caudal lamellae autotomy rather than leg autotomy to escape predation (Robinson et al., 1991; Stoks and De Block, 2007). Perhaps owing to this assumption, little work on the leg regeneration capabilities of the order has been done beyond that completed in the early twentieth century. Furthermore, the underlying causes and impacts of limb regeneration on individuals are unknown. Although the majority of the research focuses on the caudal lamellae and limbs of damselflies (suborder Zygoptera), there is some evidence that similar regeneration occurs in dragonflies (suborder Anisoptera) (Przibram, 1909).

One of the earliest experiments was that of Child and Young (1903) who performed a series of experiments on damselfly nymphs (Agrionidae) to determine the effects of certain segments of the legs being removed. They provided an in-depth study of what the regenerated limb looked like after being removed from each section of the leg, as well as what they believed to be the breakage plane on odonates, between the femur and trochanter (Child and Young, 1903). They found that regeneration resulted in different levels of a “normal” leg depending on where the leg was cut. An attempt was made to summarize their findings (Table 1), however, it should be noted that there was variability within each of these scenarios and we only portray the most common results. Furthermore, depending on the age of the nymph, the time left during development that the leg had to regenerate was also a factor in some of these variable results. For example, when segments of the tarsus were removed from nymphs that were 27 days from their next molt, they had more developed claws and spines when compared to nymphs 18 days from their next molt (Child and Young, 1903). Furthermore, in conjunction with other work, Child and Young (1903) noted that when the leg was broken off at the articulation point between the femur and trochanter, there was less bleeding making this point a seemingly adaptive place to break off. Legs that regenerated at this breaking point, as well at the less-adaptive trochanter, form highly uneven non-articulated claws that appear as a single tarsal claw, as opposed to a normally formed pair of claws (Fig. 1) (Parvin and Cook, 1968; Forbes and Baker, 1989; McPeek, 1995). It appears that regenerated legs never regain a normal tarsal claw, even when the leg is broken off very early in nymph development (Forbes and Baker, 1989). This unusual tarsal claw led some researchers, without the background knowledge on regeneration, to wrongly hypothesize that the presence of abnormal tarsi in 35 percent of their study population was due to some sort of genetic mutation (Hilton, 1985). The idea that these limbs may represent genetic abnormalities was quickly refuted by Forbes and Baker (1989), however, it further highlights the limited scope of literature on this topic.

The unusual tarsal claw resulting from regenerated limbs broken off at this articulation point provides a unique opportunity to study the prevalence and possible factors affecting limb loss in nymphs within this order. This observable physical abnormality persisting through the often more readily collected and effectively preserved adult stage allows the investigation into the prevalence and patterns associated with nymphal leg loss with a much larger dataset. Prior work investigating this phenomenon has looked at how often nymphs of different Coenagrionidae species are wounded, finding a correlation between wound frequency and nymph dispersal rather than density as the authors had hypothesized (Baker and Dixon, 1986). More recently, some researchers have suggested that this ability should be utilized in non-lethal DNA sampling techniques within this order (Monroe et al., 2010; Ožana et al., 2020). However, more work needs to be done on the best method to cut the limb as well as the energetic and fitness costs that limb loss has on an individual before this method should be fully incorporated into conservation plans, especially when working with threatened species. Overall, the possible insights that limb regeneration may provide into different aspects of odonate ecology and behavior have yet to be fully employed.

Vanuatubasis Ober and Staniczeck, are a group of endemic damselflies found on the islands of Vanuatu (Ober and Staniczek, 2009). There are currently only three species assigned to this genus; however, recent fieldwork in the region has resulted in the discovery of several undescribed species, each endemic to a single island in the archipelago (Marinov et al., 2019). While working on the taxonomy of this genus we noticed that many of the specimens had the abnormal tarsal claw described and illustrated by Forbes and Baker (1989). Further, the abnormal claw was found more often than expected based on the literature available. Here we present the first data looking at the prevalence of limb regeneration across a genus of damselfly. We attempt to both, rule out intrinsic factors related to leg loss, such as sex and species, and to provide insight into just how prevalent loss and regeneration of legs can be in odonate nymphs. Specifically, we look at the pervasiveness of limb loss between sexes and look at patterns of regeneration between islands (here used as a proxy for species due to the high levels of endemism) and test whether the loss of a specific pair of legs is correlated with either of these two factors.

Section snippets

Material and methods

We examined 202 Vanuatubasis specimens collected using aerial nets during the months of April, May and June across several field seasons spanning 2017–2019. Specimens were first sorted into six island groups using both Leica GZ6 and Olympus SZ51 stereo microscopes. Only specimens with at least five visible legs were included in the analysis. For each specimen we recorded the number and location of the abnormal tarsal claws (appearing as a single claw), indicating that the leg had likely been

Results

Of the 202 specimens examined across six islands, there were a total of 85 individuals (42%) with a leg matching that of Fig. 1B, indicating that regeneration had previously occurred from the articulation point (Table 2). In Vanuatubasis this regenerated limb has dense, small spines along the arch of the apical tarsal segment and ends in a sclerotized point (Fig. 1B). Of these 85 individuals, there were a total of 109 abnormalities. These were most commonly observed on the metathoracic legs

Discussion

Our results show that leg loss and regeneration in odonate nymphs likely is significantly more widespread than has been assumed in the past, due to the observed prevalence among Vanuatubasis. The distinctly regenerated leg with a single tarsal claw and the reduced number of tarsal segments in the adults make the study of regeneration in damselfly nymphs easily observable. Our estimate most closely matches that of Hilton (1985) who found it in 35% of a single population of Amphiagrion Selys,

Author statement

NAS: Conceptualization, Data Curation, Writing-Original Draft, Writing-Review and Editing, Validation, Visualization. GSP: Conceptualization, Data Curation, Formal Analysis, Writing-Original Draft, Writing-Review and Editing, Validation, Visualization. SMB: Resources, Writing-Review and Editing, Validation, Funding Acquisition.

Declaration of competing interest

None declared.

Acknowledgements

The authors would like to thank Donna Kalfatakmoli for helping to secure collecting permits for Vanuatu. We also appreciate field assistance provided by Brigham Young University undergraduates. The authors also appreciate advice on statistical analyses from Alexandra Duffy and the use of images taken by Colin Jensen. Lastly, the authors thank the reviewers and editorial staff that greatly improved this work. The study was funded by Brigham Young University, and NSF DEB-1265714.

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