Limb ablation and regeneration in Harmonia axyridis: costs for regenerators, but benefits for their progeny

Introduction

The ability of insects to regenerate a limb lost during an immature stage has been documented in a wide range of hexapod orders. These include Blattodea (Roberts, 1973; Tan et al., 2013), Coleoptera (Mitten et al., 2012; Lee et al., 2013), Hemiptera (Luscher, 1948; Knobloch & Steel, 1988), Lepidoptera (Yang et al., 2016), Mantodea (Karruppanan, 1998), Odonata (Parvin & Cook, 1968), Orthoptera (Maleville & Reggi, 1981; Luedke & Lakes-Harlan, 2008; Yang et al., 2016), and Phasmatodea (Chen et al., 1999; Maginnis, 2006a). In holometabolous insects, the pupal stage provides an opportunity for regeneration of a leg lost during larval development, as the entire body undergoes metamorphosis at this time. Apart from the obvious benefits of regeneration, the process involves physiological costs in addition to those associated with structure loss. The energetic burden of regeneration is termed the ‘regenerative load’ (Juanes & Smith, 1995) and it can have negative impacts on growth, reproduction, and behavior (Maginnis, 2006b). An improved understanding of the adaptive value of limb regeneration in specific ecological contexts could provide key insights into its evolution and maintenance.

Wigglesworth (1972) demonstrated that limb regeneration capacity is an exclusive property of the insect epidermis. In general terms, the base of each appendage is surrounded by a ‘regenerative field’ where epidermal cells are able to dedifferentiate following appendage removal, proliferate into a blastema, and then regenerate the appendage (Heming, 2003). The molecular genetic basis of insect limb development has been most studied in Drosophila melanogaster Meigen (e.g., Grubbs et al., 2013; Cordoba et al., 2016), beginning with the seminal work of Nüsslein-Volhard & Wieschaus (1980). The little work that does exist on appendage development in Coleoptera suggests some contrasts with the dipteran system (Angelini et al., 2009).

Limb regeneration ability could be subject to selection either directly, if the ability itself conferred a selective advantage, or indirectly, if the ability was linked to other traits that elevate fitness. Empirical evidence for direct natural selection of larval insect leg regeneration can be difficult to obtain, because it requires estimating the frequency of limb loss in natural populations of immature insects (e.g., Maginnis, 2008). However, physiological cascades associated with limb regeneration may have important consequences for development or life history that can be revealed by careful laboratory observations. For example, tail regeneration in the lizard Liolaemus nitidus (Wiegmann) is linked to an increase in metabolic rate (Naya & Bozinovic, 2006). Epigenetic factors appear to be involved in regulating the leg regeneration process in Gryllus bimaculatus DeGeer (Hamada et al., 2015). Wang et al. (2015) demonstrated that regenerating males of Harmonia axyridis Pallas (Coleoptera: Coccinellidae) were preferred as mates by females over non-ablated controls, even though they are visually indistinguishable to the observer, suggesting some linkage between regeneration and other traits that might have a chemical basis and thus be distinguishable by females. We infer that limb regeneration can evolve in response to either natural or sexual selection, and that the ability may be an indirect target of selection if it represents a pleiotropic side effect of other traits that are strongly correlated with fitness.

The recent discovery that many species of Coccinellidae possess varying levels of limb regeneration ability during pupation (Wang et al., 2015; Wu et al., 2015; Saxena et al., 2016) has raised various questions about the biological and ecological significance of limb regeneration within the family. If limb regeneration evolves in response to natural selection, we might expect its adaptive value to vary among ecological contexts. This could be tested by comparing the regenerative abilities of species with herbivorous or fungivorous habits to those that are predaceous, on the hypothesis that the latter will be a greater risk of limb loss due to development in more competitive environments. However, insights into the physiological consequences of regeneration for life history and fitness can be revealed by simple comparisons of regenerating beetles vs. unoperated controls, or vs. non-regenerating beetles when these are obtained. The latter can be especially informative if the decision to regenerate or not represents a significant bifurcation in developmental pathways, in which case it should result in distinct life-history consequences (Maginnis et al., 2015). Alternatively, if regeneration is the norm, a failure to regenerate may simply reflect a physiological constraint such as poor condition.

Working with Coccinella septempunctata L., Wu et al. (2015) found that complete regeneration was achieved by over 80% of individuals when complete ablation of a leg occurred in the second instar, with a declining proportion of individuals regenerating the leg completely when ablation occurred in subsequent instars; only about 38% of individuals regenerated legs ablated in the fourth instar, but it was not clear if these data included partially as well as fully regenerated individuals. Saxena et al. (2016) examined Menochilus sexmaculatus (Cheilomenes sexmaculata) (Fabricius) and found that only 32% of individuals fully regenerated limbs when they were severed proximal to the coxa in the fourth instar, and that the proportion regenerating was lower when ablation was conducted in the third instar. In contrast, Wang et al. (2015), working with a Chinese population of H. axyridis, observed 83% survival of larvae subjected to foreleg amputation in the fourth instar, with 75% of individuals regenerating the leg. These contrasting results regarding the effect of life stage on regenerative capacity indicate significant species-specific variation, not only in regenerative capacity, but also in associated life-history consequences.

A preliminary experiment with our locally invasive population of H. axyridis indicated that almost 100% of fourth instars regenerated forelegs that were amputated proximal to the coxa. This result denied us the opportunity to contrast regenerating with non-regenerating individuals in subsequent experiments, as the only contrasts possible were with non-ablated controls. We hypothesized that processes associated with the recovery from ablation and limb regeneration would require the up-regulation of many developmental genes in the pupal stage, with the potential for a cascade of secondary effects on subsequent physiology and life history. As parental effects in coccinellids are quite sensitive to parental condition (e.g., Vargas et al., 2012a,2012b, 2013, 2014), we also hypothesized that physiological and pleiotropic effects associated with regeneration would be transgenerational and that regenerative costs would be evident in the life histories of progeny.

Materials and methods

Results

Regeneration

Of the 104 larvae ablated, nine died in the pupal stage (8.7%), significantly more than the two (of 104, 1.9%) in the control treatment (test of proportions: P = 0.030). Two males in the amputation treatment did not regenerate the limb completely and their data were excluded from analysis, but the remaining 93 beetles regenerated a limb that was normal in appearance. Interestingly, one female individual appeared to overcompensate for the treatment and regenerated a slightly longer than normal limb. The sex ratio was 42.2% female in the control treatment, not significantly different from 44.2% in the ablation treatment (test of proportions: P = 0.78).

Larvae surviving amputation spent longer in pupation than control larvae (F_1,193 = 48.17, P<0.001; Figure 1A). The fresh weight of regenerating males at emergence was not different from that of control males (F_1,108 = 0.73, P = 0.40), but control females weighed more than regenerating females (F_1,83 = 4.97, P = 0.028; Figure 1B).

Development of progeny

Among the progeny of the four reciprocal crosses derived from mating regenerated and control beetles, a total of five larvae underwent a supernumerary fifth instar (two females in T2, one male and one female in T3, and one male in T4) that required 3 days to complete in one case, and 4 days in the others. These periods were included in the total larval development time for these individuals. Larvae in the control treatment suffered 19.1% mortality, compared to 4.4–5.6% in other treatments, but the differences among treatments were not significant (F_3,40 = 2.04, P = 0.12).

Treatment did not affect the time required for eggs to hatch (F_3,50 = 2.81, P = 0.10), but did decrease total larval development time (F_3,392 = 6.14, P<0.001; Table 2). Differences among treatments were also significant for duration of the first instar (F_3,392 = 6.74), second instar (F_3,392 = 6.26), third instar (F_3,392 = 6.56, all P<0.001), fourth instar (F_3,392 = 5.61, P = 0.001), and pupation time (F_3,392 = 2.84, P = 0.038; Figure 2). In most cases treatment led to a decrease in developmental time compared to the control. Male fresh weight at emergence was unaffected by treatment (F_3,191 = 0.50, P = 0.68) but female weight was enlarged (F_3,197 = 4.82, P = 0.003).

Table 2. Mean (± SE) developmental parameters of Harmonia axyridis progeny from four crosses between regenerated and control individuals. Beetles each regenerated a right foreleg during pupation after having it severed at the base of the coxa within 24 h of molting to the fourth instar (regen), or were unoperated (control)

Dependent variable	Cross
	♀REGEN × ♂REGEN	♀REGEN × ♂CONTROL	♀CONTROL × ♂REGEN	♀CONTROL × ♂CONTROL
Egg incubation period (days)	3.48 ± 0.06	3.47 ± 0.04	3.53 ± 0.05	3.45 ± 0.04
Total larval period (days)	11.18 ± 0.08ab	11.22 ± 0.12ab	10.88 ± 0.08b	11.58 ± 0.14a
Pupation period (days)	6.16 ± 0.05	6.32 ± 0.06	6.26 ± 0.05	6.13 ± 0.06
Male fresh weight (mg)	29.36 ± 0.44	29.01 ± 0.42	29.15 ± 0.41	28.58 ± 0.52
Female fresh weight (mg)	33.28 ± 0.60a	33.28 ± 0.54a	33.66 ± 0.47a	31.09 ± 0.48b

• Means within a row followed by different letters were significantly different among crosses (Bonferroni test: P<0.05).

Discussion

The body mass of females was reduced following ablation/regeneration of a foreleg in the fourth instar, but this effect was not evident in males. This result is consistent with coccinellid females having higher resource requirements than males due to the demands of egg maturation, which renders them more sensitive to resource limitation. Interestingly, female body size was increased relative to controls by parental effects from an ablated/regenerated parent, regardless of which parent was treated. So a significant cost of ablation/regeneration borne by mothers resulted in a benefit for their daughters.

Another immediate cost of ablation/regeneration was an extension of pupation time by a little more than half a day, but remarkably, there was no apparent impact of the procedure on any measure of reproductive success. Although there are usually significant physiological trade-offs associated with limb regeneration (Maginnis, 2006b), this is not always the case. For example, Maginnis & Redmond (2009) failed to find any somatic cost of limb regeneration in either males or females of the twostriped walkingstick, Anisomorpha buprestoides (Stoll), although leg regeneration in the phasmid Sipyloidea sipylus (Westwood) resulted in disproportionately smaller wings (Maginnis, 2006a).

Wang et al. (2015) performed similar amputations on fourth instar H. axyridis from Beijing, China, and reported that regenerating beetles extended their pupation time by 4–5 h, relative to controls, whereas our beetles were delayed by about 12 h. The slightly lower temperature employed in our present experiment may account for some of this difference. Furthermore, Wang et al. (2015) observed that pupae of regenerating beetles were heavier than control pupae; we did not weigh pupae, but regenerating females weighed less at emergence than did controls. Progeny of the Chinese population that were sired by amputated males, whether they regenerated or not, had slower development in larval stages, and in total, compared to those sired by control males. In the present study, progeny of control females sired by regenerated males of the Kansas population (T3) developed faster than controls in the first and fourth instars, and in total larval development. Thus, a negative paternal effect in the indigenous population emerged as a positive paternal effect in the invasive population, contrary to our initial hypothesis. Collectively, these observations suggest some divergence between the indigenous and native H. axyridis populations with respect to the developmental responses to limb loss and the process of regeneration.

The daughters of ablated/regenerated parents all emerged at heavier weights than those of control parents in our experiment, and it did not matter which parent regenerated. Thus, another transgenerational impact of ablation/regeneration was positive, again contradicting our initial hypothesis. Coccinellids are known to exert maternal and paternal effects on their progeny, probably to raise their own fitness (Vargas et al., 2012a,2012b, 2014; Mirhosseini et al., 2014). If limb regeneration in H. axyridis cues an epigenetic cascade that involves the activation of many genes, some could be responsible for pleiotropic enhancement of parental effects. This is notable because parental stress or trauma normally translates into negative, rather than positive, effects for developing offspring, as was observed in the Chinese H. axyridis population (Wang et al., 2015). For example, restriction of larval diet diminishes the maternal effects normally conferred by female Hippodamia convergens Guerin-Meneville to their progeny (Vargas et al., 2012a). However, there is evidence that insects challenged by bacterial infection can use epigenetic mechanisms to improve the immunological resistance of their progeny, for example in tobacco hornworm (Rosengaus et al., 2017), although immunological challenges resulting in negative maternal effects have been also observed in mosquitoes (Vantaux et al., 2014).

Limb regeneration itself may not be a direct target of selection in H. axyridis, and the ability per se may not confer any direct fitness benefit in this species. Because larval development requires only 10–12 days, and larvae missing legs are not normally observed in the field (JP Michaud, pers. obs.), limb loss followed by survival to pupation is probably a rare event in nature. In contrast, limb loss in aquatic Crustacea can be common for various reasons, thus providing a selective advantage for limb regeneration (Maginnis et al., 2014). Similarly, in stick insects, leg loss during molting is fairly common, as is autotomy to escape predation (Maginnis, 2008), again providing selective advantages to regenerators. However, the campodeiform larvae of coccinellids possess legs that are far more robust than those of phasmids, and more strongly attached to thoracic segments; any molting complications almost invariably result in death. We speculate that the ability to regenerate limbs in H. axyridis is a side effect of other physiological traits expressed by high-fitness genotypes, possibly mediated by pleiotropic mechanisms, and that these traits are the true targets of selection. Interestingly, invasive populations of H. axyridis also have been shown to differ in larval egg cannibalism behavior relative to native populations, and those mass-reared commercially for biological control purposes (Tayeh et al., 2014), the former being the most cannibalistic. Similarly, the greater regenerative abilities observed in our beetles compared to the indigenous Chinese population (Wang et al., 2015) may reflect an association of both cannibalism and limb regeneration with generally higher fitness, and thus with successful invasion of novel environments, whether the traits themselves are direct targets of selection. Invasive H. axyridis populations appear to provide a good model system in which to explore the epigenetic mechanisms of limb regeneration in holometabolous insects, and their life-history consequences.