Selective bird predation on the peppered moth: the last experiment of Michael Majerus

Colour variation in the peppered moth Biston betularia was long accepted to be under strong natural selection. Melanics were believed to be fitter than pale morphs because of lower predation at daytime resting sites on dark, sooty bark. Melanics became common during the industrial revolution, but since 1970 there has been a rapid reversal, assumed to have been caused by predators selecting against melanics resting on today's less sooty bark. Recently, these classical explanations of melanism were attacked, and there has been general scepticism about birds as selective agents. Experiments and observations were accordingly carried out by Michael Majerus to address perceived weaknesses of earlier work. Unfortunately, he did not live to publish the results, which are analysed and presented here by the authors. Majerus released 4864 moths in his six-year experiment, the largest ever attempted for any similar study. There was strong differential bird predation against melanic peppered moths. Daily selection against melanics (s ≈ 0.1) was sufficient in magnitude and direction to explain the recent rapid decline of melanism in post-industrial Britain. These data provide the most direct evidence yet to implicate camouflage and bird predation as the overriding explanation for the rise and fall of melanism in moths.     

Impact of predation by a cave-dwelling bat, Rhinolophus ferrumequinum, on the diapausing population of a troglophilic moth, Goniocraspidum preyeri

The predation pressure of the greater horseshoe bat, Rhinolophus ferrumequinum, on the diapausing population of the noctuid moth, Goniocraspidum preyeri, was examined at an abandoned mine in central Japan. These bats did not prey on the moths in summer. The number of moths preyed on was largest in March. More than 60% of the diapausing moths were eaten by only three or four bats, which accounted for over 90% of all the moth deaths. This moth was an important source of food at the end of the hibernating period when the bat had already used most of its stored subcutaneous fat. The predation on G. preyeri may have raised the overwintering success rate of R. ferrumequinum.     

Taking the Peppered Moth with a Grain of Salt

H. B. D. Kettlewell's (1955, 1956) classic field experiments on industrial melanism in polluted and unpolluted settings using the peppered moth, Biston betularia, are routinely cited as establishing that the melanic (dark) form of the moth rose in frequency downwind of industrial centers because of the cryptic advantage dark coloration provides against visual predators in soot-darkened environments. This paper critiques three common myths surrounding these investigations: (1) that Kettlewell used a model that identified crypsis as the only selective force responsible for the spread of the melanic gene, (2) that Kettlewell's field experiments alone established that selection for crypsis was the most important factor in the spread of melanic forms, and (3) that Kettlewell's investigations in an unpolluted wood near Dorset constituted a control for his earlier Birmingham studies (contra Hagen 1993, 1996). This analysis further identifies two features that distinguish manipulative experiments in evolutionary biology from experiments in other contexts. First, experiments in evolutionary biology rest on a wealth of information provided by strictly observational ecological studies; in the absence of such information experiments in evolutionary biology make no sense. Second, there is a trade-off between how much control investigators have over the conditions being studied and how informative the results of the experiment will be with regard to natural populations.     

Changing views on melanic moths

The rapid rise in frequency of melanic morphs in several moth species, especially the peppered moth Biston betukria , in industrial regions during the 19th century, and the subsequent rapid decline, indicate the action of strong selection. There has recently been a tendency to criticise and question all aspects of research on industrial melanism, including the experiments which suggest that selective predation plays an important part in the changes. These experiments are reexamined, together with evidence for changes in appearance of tree surfaces and for relation of initial melanic frequency to subsequent rate of decline. It is suggested that intense pollution may have been required to drive the carbonaria morph to a high frequency, with frequency patterns over a mosaic environment smoothed by migration. Improvements in these extreme locations then triggered the decline, with litde indication of the environmental changes in areas of moderate pollution. Reasons for criticism of past work are discussed. Industrial melanism continues to provide an exceptional opportunity to analyse a pattern of selection and change in gene frequency.     

Industrial Melanism in the Peppered Moth, Biston betularia: An Excellent Teaching Example of Darwinian Evolution in Action

The case of industrial melanism in the peppered moth has been used as a teaching example of Darwinian natural selection in action for half a century. However, over the last decade, this case has come under attack from those who oppose Darwinian evolution. Here, the main elements of the case are outlined and the reasons that the peppered moth case became the most cited example of Darwinian evolution in action are described. Four categories of criticism of the case are then evaluated. Criticisms of experimental work in the 1950s that centered on lack of knowledge of the behavior and ecology of the moth, poor experimental procedure, or artificiality in experiments have been addressed in subsequent work. Some criticisms of the work are shown to be the result of lack of understanding of evolutionary genetics and ecological entomology on the part of the critics. Accusations of data fudging and scientific fraud in the case are found to be vacuous. The conclusion from this analysis of criticisms of the case is that industrial melanism in the peppered moth is still one of the clearest and most easily understood examples of Darwinian evolution in action and that it should be taught as such in biology classes.     

Industrial melanism: Do we have the answers?

Entomologists from the late 19th century onwards recognized the evolutionary interest of the association of black forms of the peppered moth with industrialization. They developed a qualitative explanation of the phenomenon involving a change in relative crypsis of the phenotypes due to the blackening of the moth's resting background by air pollution. More recently, ecological geneticists have obtained some estimates of predation by birds and of population parameters such as migration rate. Models incorporating these estimates have explored the ways in which natural selection influences spatial variation and the maintenance of polymorphism. Studies on the peppered moth and some of the many other insects exhibiting industrial melanism have concentrated on the variability and dynamics of adult populations. Recent work which has begun to examine the ecology and behaviour of individuals, complete life cycles, and gene-phenotype relationships, is refining our understanding of this adaptation and also of present-day declines in melanic frequencies in response to falling air pollution.     

High duty cycle pulses suppress orientation flights of crambid moths

Bat-and-moth is a good model system for understanding predator–prey interactions resulting from interspecific coevolution. Night-flying insects have been under predation pressure from echolocating bats for 65 Myr, pressuring vulnerable moths to evolve ultrasound detection and evasive maneuvers as counter tactics. Past studies of defensive behaviors against attacking bats have been biased toward noctuoid moth responses to short duration pulses of low-duty-cycle (LDC) bat calls. Depending on the region, however, moths have been exposed to predation pressure from high-duty-cycle (HDC) bats as well. Here, we reveal that long duration pulse of the sympatric HDC bat (e.g., greater horseshoe bat) is easily detected by the auditory nerve of Japanese crambid moths (yellow peach moth and Asian corn borer) and suppress both mate-finding flights of virgin males and host-finding flights of mated females. The hearing sensitivities for the duration of pulse stimuli significantly dropped non-linearly in both the two moth species as the pulse duration shortened. These hearing properties support the energy integrator model; however, the threshold reduction per doubling the duration has slightly larger than those of other moth species hitherto reported. And also, Asian corn borer showed a lower auditory sensitivity and a lower flight suppression to short duration pulse than yellow peach moth did. Therefore, flight disruption of moth might be more frequently achieved by the pulse structure of HDC calls. The combination of long pulses and inter-pulse intervals, which moths can readily continue detecting, will be useful for repelling moth pests.     

A proposed selective mechanism based on metal chelation in industrial melanic moths

Industrial melanism, a phenomenon observed in some moths and especially in the case of the peppered moth (Biston betularia), has received much attention as an example of Darwinian evolution in action. The rapid rise in the proportion of the darker melanic form of the adult moth coincided with the advent of atmospheric pollution resulting from industrialization, and was ascribed to the improved camouflage of the melanotic insects against a background blackened by soot, which conferred a selective advantage in the avoidance of predation by birds. The topic of the increase in melanization during the initial period of industrial expansion and the reversal of the process after the introduction of the Clean Air Act has received much attention. Although there is sound experimental evidence to support selective avian predation as a major mechanism to account for the changes in the relative frequency of melanics, it is not clear that this is the only selective factor involved in industrial melanism. It is possible that other processes may have made a contribution to the preponderance of melanic variants. In the present study, the hypothesis is advanced that melanization may have conferred a selective advantage by protecting the insects from the toxic effects of metals by virtue of the strong metal chelating action of melanin. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 298–301.     

Insect melanism: the molecules matter

Insect melanism, especially in the peppered moth Biston betularia, has long been a textbook case of evolution in action. Hypotheses of the role of natural selection in maintaining melanic polymorphisms have implicated a wide range of explanations in various species, but to understand fully the ecology of melanism, we need to understand its molecular and developmental genetic basis. Because developmental genes often affect more than one trait, identifying the genes responsible for melanism is crucial for a thorough understanding of the fitnesses and selective responses of melanic alleles in nature. Molecular genetic information is also vital for elucidating the evolutionary history and possible mechanistic diversity of melanism among species. Recent studies of the developmental genetics of melanin pigmentation in Drosophila, and of the genetics of pigmentation differences among other insect species, have provided valuable insights into the underpinnings of this important source of polymorphism throughout the Insecta.     

Recent history of melanism in American peppered moths

Industrial melanism in peppered moths has been studied most intensively in Britain. The first melanic phenotype (effectively solid black) was recorded near Manchester in 1848. By 1895 about 98% of the specimens near Manchester were melanic, and this once rare phenotype had spread across regions of the country blackened by industrial soot. In rural, unpolluted regions, well away from industrial centers, the pale phenotype (peppered with white and black scales) remained the predominant form. During the latter half of the 20th century, following legislation designed to improve air quality, melanics began to decline in frequency and are now rare where once they had been common. Similar evolutionary changes have occurred elsewhere, but records from outside Britain are fragmentary. We have extended previous surveys of American peppered moth populations and present a composite picture of the recent decline in melanism in northern industrial states-Michigan and Pennsylvania-where melanic phenotypes decreased from more than 90% in 1959 to 6% by 2001. We contrast these changes to the near absence of melanism in a southern state-Virginia-during that same period. As in Britain, the decline in melanism in American peppered moths followed clean air legislation.     

Retelling Experiments: H.B.D. Kettlewell's Studies of Industrial Melanism in Peppered Moths

H. B. D. Kettlewell's field experiments on industrial melanism in the peppered moth, Biston betularia, have become the best known demonstration of natural selection in action. I argue that textbook accounts routinely portray this research as an example of controlled experimentation, even though this is historically misleading. I examine how idealized accounts of Kettlewell's research have been used by professional biologists and biology teachers. I also respond to some criticisms of David Rudge to my earlier discussions of this case study, and I question Rudge's claims about the importance of purely observational studies for the eventual acceptance and popularization of Kettlewell's explanation for the evolution of industrial melanism.     

Acoustic Aposematism and Evasive Action in Select Chemically Defended Arctiine (Lepidoptera: Erebidae) Species: Nonchalant or Not?

Tiger moths (Erebidae: Arctiinae) have experienced intense selective pressure from echolocating, insectivorous bats for over 65 million years. One outcome has been the evolution of acoustic signals that advertise the presence of toxins sequestered from the moths’ larval host plants, i.e. acoustic aposematism. Little is known about the effectiveness of tiger moth anti-bat sounds in their natural environments. We used multiple infrared cameras to reconstruct bat-moth interactions in three-dimensional (3-D) space to examine how functional sound-producing organs called tymbals affect predation of two chemically defended tiger moth species: Pygarctia roseicapitis (Arctiini) and Cisthene martini (Lithosiini). P. roseicapitis and C. martini with intact tymbals were 1.8 and 1.6 times less likely to be captured by bats relative to those rendered silent. 3-D flight path and acoustic analyses indicated that bats actively avoided capturing sound-producing moths. Clicking behavior differed between the two tiger moth species, with P. roseicapitis responding in an earlier phase of bat attack. Evasive flight behavior in response to bat attacks was markedly different between the two tiger moth species. P. roseicapitis frequently paired evasive dives with aposematic sound production. C. martini were considerably more nonchalant and employed evasion in fewer interactions. Our results show that acoustic aposematism is effective at deterring bat predation in a natural context and that this strategy is likely to be the ancestral function of tymbal organs within the Arctiinae.     

Female greater wax moths reduce sexual display behavior in relation to the potential risk of predation by echolocating bats

Female greater wax moths Galleria mellonella display by wing fanning in response to bursts of ultrasonic calls produced bymales. The temporal and spectral characteristics of these callsshow some similarities with the echolocation calls of batsthat emit frequency-modulated (FM) signals. Female G. mellonellatherefore need to distinguish between the attractive signalsof male conspecifics, which may lead to mating opportunities,and similar sounds made by predatory bats. We therefore predictedthat (1) females would display in response to playbacks of male calls; (2) females would not display in response to playbacksof the calls of echolocating bats (we used the calls of Daubenton'sbat Myotis daubentonii as representative of a typical FM echolocatingbat); and (3) when presented with male calls and bat callsduring the same time block, females would display more whenperceived predation risk was lower. We manipulated predationrisk in two ways. First, we varied the intensity of bat callsto represent a nearby (high risk) or distant (low risk) bat.Second, we played back calls of bats searching for prey (lowrisk) and attacking prey (high risk). All predictions weresupported, suggesting that female G. mellonella are able todistinguish conspecific male mating calls from bat calls, andthat they modify display rate in relation to predation risk.The mechanism (s) by which the moths separate the calls ofbat and moth must involve temporal cues. Bat and moth signalsdiffer considerably in duration, and differences in durationcould be encoded by the moth's nervous system and used in discrimination.     

Trinucleotide microsatellite loci for the peppered moth (Biston betularia)

Fourteen polymorphic trinucleotide microsatellites were isolated in the peppered moth, Biston betularia, using an enrichment protocol. Moderate to high allelic diversity (three to 22 alleles per locus) was found in a sample from northern England. We are currently using these markers to study the population structure of B. betularia in northern England, where a cline for melanism still exists.     

Investigating cat predation as the cause of bat wing tears using forensic DNA analysis

Cat predation upon bat species has been reported to have significant effects on bat populations in both rural and urban areas. The majority of research in this area has focussed on observational data from bat rehabilitators documenting injuries, and cat owners, when domestic cats present prey. However, this has the potential to underestimate the number of bats killed or injured by cats. Here, we use forensic DNA analysis techniques to analyze swabs taken from injured bats in the United Kingdom, mainly including Pipistrellus pipistrellus (40 out of 72 specimens). Using quantitative PCR, cat DNA was found in two‐thirds of samples submitted by bat rehabilitators. Of these samples, short tandem repeat analysis produced partial DNA profiles for approximately one‐third of samples, which could be used to link predation events to individual cats. The use of genetic analysis can complement observational data and potentially provide additional information to give a more accurate estimation of cat predation.     

Acoustic courtship communication inSyntomeida epilais Wlk. (Lepidoptera: Arctiidae,Ctenuchinae)

The polka-dot wasp moth,Syntomedia epilais Wlk. (Lepidoptera, Arctiidae, Ctenuchinae), engages in extensive acoustic signaling during courtship. The signals, which are produced by both sexes, consist of sexually dimorphic trains of ultrasonic clicks. Field and laboratory behavioral experiments demonstrated that (1) sound production from both the male and the female is required for mating success, (2) femaleS. epilais show a higher level of acoustic responsiveness to male acoustic signals than to female signals, and (3) males use the female acoustic signals to locate their potential mates. It is suggested that the existence of the system is made possible by a reduction in bat predation pressure, perhaps the result of extreme distastefulness combined with the distinctive acoustic image that flying ctenuchines present to foraging bats.     

Tut-tut Tutt, not so fast. Did Kettlewell really test Tutt's explanation of industrial melanism?

Over the past fifty years a legend has developed surrounding H.B.D. Kettlewell's pioneering research on the phenomenon of industrial melanism as illustrated in the peppered moth, Biston betularia. This legend is particularly pernicious in that it unfairly diminishes Kettlewell's intellectual involvement in the work for which he is most famous (Rudge 2006). One of the most persistent allegations made is that Kettlewell's published work neglects or ignores his debt to J.W. Tutt, identified as the first to propose the explanation of industrial melanism at issue in Kettlewell's original investigations (Owen 1997). The following essay first examines whether J.W. Tutt was indeed the first to explain the phenomenon of industrial melanism in terms of differential bird predation and the long term effects of air pollution, before considering further whether J.W. Tutt's explanation was, in point of fact, the explanation at issue in Kettlewell's investigations.     

Listening for bats: pulse repetition rate as a cue for a defensive behavior inCycnia tenera (Lepidoptera:Arctiidae)

Summary The tympanate, arctiid moth,Cycnia tenera responds to pulsed, 30 kHz acoustic stimuli resembling bat echolocation signals by emitting trains of clicks. This phonoresponse was used to determine that this moth is maximally sensitive to stimulus pulse repetition rates of 30–50 pulses/s, rates typically emitted by bats shortly before they close with their targets. At rates both above and below this optimum moths exhibit higher thresholds and reduced responsiveness. These data suggest thatC. tenera is capable of using the repetition rate emitted by an approaching bat as a cue in determining the relative proximity of the bat. The use of repetition rate information should allow this moth both an unambiguous indication of a bat at very close range as well as the ability to distinguish sources of nocturnal, high-frequency sounds not emitted by predators.     

Predation by squirrel monkeys and double-toothed kites on tent-making bats

Central American squirrel monkeys (Saimiri oerstedi) appear to recognize the modified leaves that phyllostomid bats utilize for diurnal roost sites. The monkeys visually and manually search these bat tents for both bats and insects. Adult males are the most successful at capturing bats. Nonvolant juvenile bats are more vulnerable to monkey predation than are adults. Bats that escape monkey predation frequently are captured by doubletoothed kites (Harpagus bidentatus) that tend foraging troops of monkeys. Predation by squirrel monkeys, coupled with that of double-toothed kites, may be a significant source of mortality for tent-making bats.     

The peppered moth and industrial melanism: evolution of a natural selection case study

From the outset multiple causes have been suggested for changes in melanic gene frequency in the peppered moth Biston betularia and other industrial melanic moths. These have included higher intrinsic fitness of melanic forms and selective predation for camouflage. The possible existence and origin of heterozygote advantage has been debated. From the 1950s, as a result of experimental evidence, selective predation became the favoured explanation and is undoubtedly the major factor driving the frequency change. However, modelling and monitoring of declining melanic frequencies since the 1970s indicate either that migration rates are much higher than existing direct estimates suggested or else, or in addition, non-visual selection has a role. Recent molecular work on genetics has revealed that the melanic (carbonaria) allele had a single origin in Britain, and that the locus is orthologous to a major wing patterning locus in Heliconius butterflies. New methods of analysis should supply further information on the melanic system and on migration that will complete our understanding of this important example of rapid evolution.