Surprisingly long body length of the lungworm Parafilaroides gymnurus from common seals of the Dutch North Sea.

Lungworms of the genera Parafilaroides and Otostrongylus are responsible for parasitic bronchopneumonia, the foremost disease of eastern Atlantic common seals (EACS, Phoca vitulina vitulina) in the Dutch North Sea. Recently, there have been increased reports of lungworm cases and observations of unusually long Parafilaroides sp. adults in this location. The initial aim of this study was to confirm the identity of the Parafilaroides species infecting this population. Parafilaroides are usually small and delicate, making them difficult to extract from host tissue, and there is often difficulty accessing fresh specimens for morphological study. The large size of the Dutch worms and the accessibility of specimens from numerous animals enabled the description and measurement of many intact specimens (N = 64) from multiple host animals (N = 20). Species identity was confirmed by targeted sequencing of ribosomal and mitochondrial DNA amplicons from a subset of worms. Worm morphology was consistent with descriptions for P. gymnurus, but the mature females were 1.9-fold and 3.4-fold longer than those recovered from French EACS (P ≤ 0.001) and Canadian western Atlantic common seals (Phoca vitulina concolor; P ≤ 0.0001). They were also significantly longer than mature female P. gymnurus described from other seal species, with the exception of those from harp seals of Les Escoumins, Quebec. We suggest that intraspecific genetic differences in P. gymnurus and the environment within the host could contribute to the variation reported here. This study is the first to describe P. gymnurus using morphological and molecular methods and should serve as a reference for identification of the species.

Selection on male sex pheromone composition contributes to butterfly reproductive isolation.

Selection can facilitate diversification by inducing character displacement in mate choice traits that reduce the probability of maladaptive mating between lineages. Although reproductive character displacement (RCD) has been demonstrated in two-taxa case studies, the frequency of this process in nature is still debated. Moreover, studies have focused primarily on visual and acoustic traits, despite the fact that chemical communication is probably the most common means of species recognition. Here, we showed in a large, mostly sympatric, butterfly genus, a strong pattern of recurrent RCD for predicted male sex pheromone composition, but not for visual mate choice traits. Our results suggest that RCD is not anecdotal, and that selection for divergence in male sex pheromone composition contributed to reproductive isolation within the Bicyclus genus. We propose that selection may target olfactory mate choice traits as a more common sensory modality to ensure reproductive isolation among diverging lineages than previously envisaged.

The plastic fly: the effect of sustained fluctuations in adult food supply on life-history traits.

Many adult traits in Drosophila melanogaster show phenotypic plasticity, and the effects of diet on traits such as lifespan and reproduction are well explored. Although plasticity in response to food is still present in older flies, it is unknown how sustained environmental variation affects life-history traits. Here, we explore how such life-long fluctuations of food supply affect weight and survival in groups of flies and affect weight, survival and reproduction in individual flies. In both experiments, we kept adults on constant high or low food and compared these to flies that experienced fluctuations of food either once or twice a week. For these 'yoyo' groups, the initial food level and the duration of the dietary variation differed during adulthood, creating four 'yoyo' fly groups. In groups of flies, survival and weight were affected by adult food. However, for individuals, survival and reproduction, but not weight, were affected by adult food, indicating that single and group housing of female flies affects life-history trajectories. Remarkably, both the manner and extent to which life-history traits varied in relation to food depended on whether flies initially experienced high or low food after eclosion. We therefore conclude that the expression of life-history traits in adult life is affected not only by adult plasticity, but also by early adult life experiences. This is an important but often overlooked factor in studies of life-history evolution and may explain variation in life-history experiments.

Footprints of selection in wild populations of Bicyclus anynana along a latitudinal cline.

One of the major questions in ecology and evolutionary biology is how variation in the genome enables species to adapt to divergent environments. Here, we study footprints of thermal selection in candidate genes in six wild populations of the afrotropical butterfly Bicyclus anynana sampled along a c. 3000 km latitudinal cline. We sequenced coding regions of 31 selected genes with known functions in metabolism, pigment production, development and heat shock responses. These include genes for which we expect a priori a role in thermal adaptation and, thus, varying selection pressures along a latitudinal cline, and genes we do not expect to vary clinally and can be used as controls. We identified amino acid substitution polymorphisms in 13 genes and tested these for clinal variation by correlation analysis of allele frequencies with latitude. In addition, we used two F(ST) -based outlier methods to identify loci with higher population differentiation than expected under neutral evolution, while accounting for potentially confounding effects of population structure and demographic history. Two metabolic enzymes of the glycolytic pathway, UGP and Treh, showed clinal variation. The same loci showed elevated population differentiation and were identified as significant outliers. We found no evidence of clines in the pigmentation genes, heat shock proteins and developmental genes. However, we identified outlier loci in more localized parts of the range in the pigmentation genes yellow and black. We discuss that the observed clinal variation and elevated population divergence in UGP and Treh may reflect adaptation to a geographic thermal gradient.

SNP genotyping for detecting the 'rare allele phenomenon' in hybrid zones.

Hybrid zones are regions where genetically distinct populations meet, mate and produce offspring. In such zones, genetically less compatible gene combinations are usually generated, resulting in reduced fitness, and hybrid zones are often maintained because of continuous removal of unfit genotypes, balanced by gene flow into the zone from the parental populations (and are then referred to as 'tension zones'). Tension zones often display unexpectedly high frequencies of gene variants that are rare outside the zone. Previous work has shown that this 'rare allele phenomenon' is not the result of intragenic recombination or increased mutation rates. Further understanding of the population genetics of the phenomenon requires an approach in which both the numbers of individuals and the numbers of loci is increased. Here, we report an approach using a combination of Illumina next-generation sequencing and mass spectrophotometer genotyping to identify markers that may be used for genome-wide investigations of the rare allele phenomenon. We test this approach on a hybrid zone in the land snail Albinaria hippolyti from Greece.

Environmentally induced dispersal-related life-history syndrome in the tropical butterfly, Bicyclus anynana.

Dispersal is a key process for understanding the persistence of populations as well as the capacity of organisms to respond to environmental change. Therefore, understanding factors that may facilitate or constrain the evolution of dispersal is of crucial interest. Assessments of phenotypic variation in various behavioural, physiological and morphological traits related to insect dispersal and flight performance are common, yet very little is known about the genetic associations among these traits. We have used experiments on the butterfly Bicyclus anynana to estimate genetic variation and covariation in seven behavioural, physiological and morphological traits related to flight potential and hence dispersal. Our goal was to characterize the heritabilities and genetic correlations among these traits and thus to understand more about the evolution of dispersal-related life-history syndromes in butterflies. Using a version of the animal model, we showed that all of the traits varied between the sexes, and most were either positively or negatively (phenotypically and/or genetically) correlated with body size. Heritable variation was present in most traits, with the highest heritabilities estimated for body mass and thorax ratio. The variance in flight activity among multiple measurements for the same individual was high even after controlling for the prevailing environmental conditions, indicating the importance of behavioural switching and/or inherent randomness associated with this type of movement. A number of dispersal-related traits showed phenotypic correlations among one another, but only a few of these were associated with significant genetic correlations indicating that covariances between these traits in Bicyclus anynana are mainly environmentally induced.

Patterns of stranding and mortality in common seals (Phoca vitulina) and grey seals (Halichoerus grypus) in The Netherlands between 1979 and 2008.

Pathological examination of stranded marine mammals provides information on the causes of mortality in their populations. Patterns of stranding and causes of death of dead-stranded seals on the Dutch coast were analyzed over a 30-year period (1979-2008). Stranding data (n=1,286) and post-mortem data (n=379) from common seals (Phoca vitulina) and grey seals (Halichoerus grypus) found dead, or that died before admission to rehabilitation, were obtained from the Seal Rehabilitation and Research Centre database. Data for the years 1988 and 2002, when mass mortality occurred due to phocine distemper virus epidemics, were excluded. Common seal stranding increased from one to nearly 100 per year over this period. This coincides with the increase in the number of common seals in Dutch waters over recent decades. Grey seal stranding increased gradually from one to about 40 per year over the period, reflecting recolonization of Dutch waters by this species. For both species, the trend in stranding of dead seals was found to be in line with that of seals observed in Dutch waters during aerial surveys and did not provide any indications of a relative change in the stranding rate of dead seals. The total monthly stranding rates peaked at more than 120 in June and July for common seals and at nearly 60 in January for grey seals. This coincides with the pupping periods of the two species. Besides phocine distemper, the most common causes of death in investigated common seals (n=286) were by-catch (confirmed and inferred) (19%), pup starvation (7%), intestinal volvulus (7%) and parasitic bronchopneumonia (6%). The most common causes of death in investigated grey seals (n=93) were by-catch (confirmed and inferred) (15%), pup starvation (11%) and trauma (5%). The relative occurrence of by-catch significantly decreased over time for grey seals, but not for common seals. Common seals were affected by infectious disease significantly more often than grey seals, mainly because of a higher occurrence of parasitic pneumonia. Phocine distemper caused mass mortalities among common seals, but not among grey seals. These findings in dead-stranded seals differ in part from those reported elsewhere in live-stranded seals, for which pup starvation and parasitic bronchopneumonia were the main causes of stranding. A substantial proportion of seals in Dutch waters die from causes related to human activity. Continued monitoring of stranding patterns and causes of death is warranted for early detection of changes and the possibility of taking timely management actions.

A steep cline in ladybird melanism has decayed over 25 years: a genetic response to climate change?

A cline in the frequency of melanic morphs of the two-spot ladybird, Adalia bipunctata, was first surveyed in 1980 along a transect extending inland from the coast in the Netherlands. At that time, the frequency of melanics increased over some 40 km from 10% near the coast to nearly 60% inland. Additional surveys made in 1991 and 1995 demonstrated some progressive change in cline shape. New samples from 1998 and 2004 confirm these dynamics, and show that over a period of about 50 generations for the beetle, the cline had decayed rapidly to yield rather uniform frequencies of melanic morphs at around 20% along the whole transect by 2004. Climate data and evidence for thermal melanism in this species support our contention that these dynamics reflect a dramatic example of a rapid genetic response within populations to climate change and local selection.

Evolutionary dynamics of multilocus microsatellite arrangements in the genome of the butterfly Bicyclus anynana, with implications for other Lepidoptera.

The sequences flanking microsatellites isolated from the butterfly Bicyclus anynana display high levels of similarity among different loci. We examined sequence data for evidence of the two mechanisms most likely to generate these similarities, namely recombination mediated events, such as unequal crossing over or gene conversion and through transposition of mobile elements (MEs). Many sequences contained tandemly arranged microsatellites, lending support to recombination as the multiplication mechanism. There is, however, also support for ME-mediated multiplication of microsatellites and their flanking sequences. Homology with a known Lepidopteran ME was found in B. anynana microsatellite regions, and polymorphic microsatellite markers with partial similarities in their flanking sequences were passed on to the next generation independently, indicating that they are not linked. Therefore, the rise of these similarities appears to be mediated through both processes, either as an interaction between the two, or by each being responsible for part of the observations. A large proportion of microsatellites embedded in repetitive DNA is representative for most studied butterflies and moths, and a BLAST survey of the B. anynana sequences revealed four short microsatellite-associated sequences that were present in many species of Lepidoptera. The similarities usually start to deviate beyond these sequences, which suggests that they define the extremes of a repeated unit. Further study of these conserved sequences may help to understand the mechanism underlying the multiplication events, and answer the question of why these redundancies are predominantly found in this insect group.

Extraordinary long life spans in fruit-feeding butterflies can provide window on evolution of life span and aging.

Information on the life span of organisms in the field is essential for elucidating the evolution of life span and aging. We present mark-recapture data (>30,000 marked individuals, >4000 recaptured at least once) on 47 species of fruit-feeding butterflies in a tropical forest in Uganda. The data reveal adult life spans in the field for several species that are significantly longer than previously recorded in Lepidoptera (butterflies and moths). Longevity records for species of which more than 100 individuals were recaptured ranged from 67 (Bicyclus auricruda) to 293 days (Euphaedra medon). In contrast to the majority of Lepidoptera which are short-lived, these all show exceptionally long life spans, and may thus help to better identify factors that affect aging, particularly when combined with information on temporal patterns in reproduction, strategies to avoid predation, and nutritional ecology. These key traits are readily measurable in butterflies and thus studies on fruit-feeding butterflies have much potential for gaining insight into the evolution of life span and aging, especially given the tradition of field-research on butterflies.

Realized correlated responses to artificial selection on pre-adult life-history traits in a butterfly.

We use artificial selection experiments targeted on egg size, development time or pupal mass within a single butterfly population followed by a common-garden experiment to explore the interactions among these life-history traits. Relationships were predicted to be negative between egg size and development time, but to be positive between development time and body size and between egg size and body size. Correlated responses to selection were in part inconsistent with these predictions. Although there was evidence for a positive genetic correlation between egg and body size, there was no support for genetic correlations between larval development time and either egg size or pupal mass. Phenotypic correlations among the three target traits of selection gave comparable results for the relationships between egg mass and development time (no association) as well as between egg mass and pupal mass (positive association), but not for the relation between development time and pupal mass (negative phenotypic correlation). In summary, correlated responses to selection as well as phenotypic correlations were rather unpredictable. The impact of variation in acquisition and allocation of energy as well as of the benign conditions used deserve further investigation.

Exploring evolutionary constraints is a task for an integrative evolutionary biology.

Judging by the volume of writings about evolutionary constraints, they are an important topic in evolutionary biology. However, their involvement in shaping patterns of evolutionary change from morphological stasis to adaptive radiation remains contentious. This is at least in part because of the paucity of robust analyses of potential examples of constraints, whether of a more absolute or a relative nature. Here, we argue that what is needed to explore the type of constraints and bias on evolutionary change that may emerge from the way in which phenotypic variation is generated is an integrative approach applied to systems that can be tackled at different levels of biological organization. This is illustrated using research on the evolution of patterns in butterfly wing eyespots that has applied a combination of evolutionary genetics and evo-devo to an emerging model species with the beginnings of a comparative approach to describe patterns of variability among the extant taxa of two species-rich genera.

Methuselah life history in a variety of conditions, implications for the use of mutants in longevity research.

The laboratory has yielded many long-lived mutants of several model-organisms in the past few years. Many of the resulting claims for extended longevity have been nuanced or shown to be restricted to specific conditions, including environments and genetic backgrounds. Here, we test whether the long-lived mutant fruit fly methuselah (mth(1)) displays its apparent superiority in longevity and stress resistance in different environments, at different ages and in correlated traits. The results demonstrate that stress resistance at different times in life is not consistently higher in the mutant relative to its progenitor strain (w(1118)). Furthermore, the mth(1) genotype only leads to an increase in longevity in an environment where reproduction is not stimulated. Also, virgin and mated life span were compared and showed that mating negatively affects life span, especially in the mth(1) individuals. This reduced the life span enhancing effect of the mutation to zero. This apparent environment and mating dependent trade-off between longevity and reproduction supports the disposable soma theory of ageing. We conclude that these data can only provide limited information on natural variation. The data show the need to uncover the full complexity of variation in such traits in natural environments.

Do mothers producing large offspring have to sacrifice fecundity?

We artificially selected on egg size in a butterfly to study the consequences for fecundity, reproductive effort and offspring fitness. Correlated responses in either pupal mass, larval or pupal development time were virtually absent. Offspring size was positively related to fitness, but only partly traded off against fecundity. Rather, total reproductive effort (measured as fresh mass), egg water content and the decline of egg size with female age increased in the large-egg selected lines compared to either small-egg or control lines. Accounting for these effects showed that reproductive investment (in dry mass) was in fact similar across lines. Such mechanisms may enable increased investment in (early) offspring without a reduction in their number, revealing a much more complex picture than a simple trade-off between offspring size and number. Substantial variation among replicates suggests that there are different underlying mechanisms for change, rather than any single, unitary pathway.

Sexual functionality of Leptopilina clavipes (Hymenoptera: Figitidae) after reversing Wolbachia-induced parthenogenesis.

Females infected with parthenogenesis-inducing Wolbachia bacteria can be cured from their infection by antibiotic treatment, resulting in male production. In most cases, however, these males are either sexually not fully functional, or infected females have lost the ability to reproduce sexually. We studied the decay of sexual function in males and females of the parasitoid Leptopilina clavipes. In western Europe, infected and uninfected populations occur allopatrically, allowing for an investigation of both male and female sexual function. This was made by comparing females and males induced from different parthenogenetic populations with those from naturally occurring uninfected populations. Our results indicate that although males show a decay of sexual function, they are still able to fertilize uninfected females. Infected females, however, do not fertilize their eggs after mating with males from uninfected populations. The absence of genomic incompatibilities suggests that these effects are due to the difference in mode of reproduction.

The evolutionary genetics of egg size plasticity in a butterfly.

Abstract The evolution of phenotypic plasticity requires that it is adaptive, genetically determined, and exhibits sufficient genetic variation. For the tropical butterfly Bicyclus anynana there is evidence that temperature-mediated plasticity in egg size is an adaptation to predictable seasonal change. Here we set out to investigate heritability in egg size and genetic variation in the plastic response to temperature in this species, using a half-sib breeding design. Egg size of individual females was first measured at a high temperature 4 days after eclosion. Females were then transferred to a low temperature and egg size was measured after acclimation periods of 6 and 12 days respectively. Overall, additive genetic variance explained only 3-11% of the total phenotypic variance, whereas maternal effects were more pronounced. Genotype-environment interactions and cross-environmental correlations of less than unity suggest that there is potential for short-term evolutionary change. Our findings strengthen the support for the adaptive nature of temperature-mediated plasticity in egg size.

The effects of larval density on adult life-history traits in three species of Drosophila.

There is evidence that longevity and starvation resistance are determined by a common genetic mechanism. Starvation resistance in Drosophila strongly correlates with both fat content and longevity, and is affected by density during rearing. In this study, we examine how three species, Drosophila melanogaster, Drosophila ananassae and Drosophila willistoni, respond to three larval density treatments. Starvation resistance after adult eclosion, and after 2 days of feeding, and longevity were examined in each sex. D. willistoni reacted differently to larval density than the other two species. This species showed an effect of density on longevity whilst D. ananassae and D. melanogaster showed no such effects. The results also indicate that starvation resistance is not solely determined by fat content. Resistance to starvation at two time points after eclosion differed among species. This may reflect differences in resource acquisition and allocation, and we discuss our findings in relation to how selection may operate in the different species.

Genetic basis and fitness consequences of winglessness in the two-spot ladybird beetle, Adalia bipunctata.

The genetic basis and fitness consequences of winglessness were investigated in the two-spot ladybird beetle, Adalia bipunctata. By breeding lines from a wingless individual found at The Uithof, Utrecht in The Netherlands, the wingless condition was confirmed to be under the control of a major allele, recessive to the wild type. Wingless individuals, on average, had a longer developmental period, a lower egg production and a shorter lifespan than the wild type with wings, suggesting that the expression of the wingless allele has functionally interrelated gene actions involving a wide range of fitness components. While the wingless allele influences various traits, significant among-family variation in the degree of winglessness suggests that its phenotypic expression is also dependent on the genetic background and modifier loci. Furthermore, there was a consistent pattern of correlation between the degree of winglessness and life history traits; the most extreme wingless individuals showed the lowest fitness while those with more fully developed wings tended to have the highest fitness. This correlation suggests that the modifier genes influence both wing formation and fitness components. The significance of such epistatic effects to the evolution of flightlessness in insects is discussed.

Effectiveness of the defence mechanism of the turnip sawfly, Athalia rosae (Hymenoptera: Tenthredinidae), against predation by lizards.

The turnip sawfly, Athalia rosae Linnaeus, is a pest on cruciferous crops. Larvae sequester secondary plant compounds, namely glucosinolates, in their haemolymph. When attacked, their integument is easily disrupted and a droplet of haemolymph is exuded ('easy bleeding'). This has been shown to be an effective, chemical-based, defence against invertebrate predators. The efficiency of this proposed defence was tested against a vertebrate predator, using groups of the iguanid lizard Anolis carolinensis Voigt as a model predator. Caterpillars of Pieris rapae Linnaeus and Pieris brassicae Linnaeus served as control prey species that do not sequester glucosinolates. Lizards attacked far fewer sawfly larvae than pierid caterpillars. Several of the sawfly larvae were rejected after an initial attack, demonstrating unpalatability to the lizards, while the Pieris larvae were not rejected. However, P. rapae larvae topically treated with extracts of haemolymph of A. rosae had no deterrent effect on the lizards and no avoidance learning occurred over a period of two weeks. Adult sawflies do not easy bleed but have glucosinolates carried over from the larval stage. Lizards attacked them at a higher rate than larvae and they were never rejected. The results suggest that for the defensive effectiveness of the pest sawfly species against vertebrates the chemical cue is not necessarily sufficient. Movement and colour may be important additional factors triggering the behaviour of vertebrate predators.

On the neutrality of molecular genetic markers: pedigree analysis of genetic variation in fragmented populations.

Many studies employ molecular markers to infer ecological and evolutionary processes, assuming that variation found at genetic loci offers a reliable representation of stochastic events in natural populations. Increasingly, evidence emerges that molecular markers might not always be selectively neutral. However, only a few studies have analysed how deviations from neutrality could affect estimates of genetic variation, using populations with known genealogy. We monitored changes in allozyme variation over eight generations in captive metapopulations of the butterfly Bicyclus anynana. Population demography was recorded by individually marking 35 000 butterflies and constructing pedigrees. We designed a computer program that simulated the inheritance of founder allozyme alleles in butterfly pedigrees. We thus tested whether the observed transmission of allozyme alleles could be explained by random genetic drift alone, or whether there was evidence for positive or negative selection. This analysis showed that in the smallest metapopulations the loss of allozyme variation exceeded the neutral rate. Possibly, linkage disequilibria between deleterious mutations and marker alleles resulted in background selection and a faster erosion of allozyme variation. In larger metapopulations, one locus (MDH) showed a significant heterozygote excess and smaller than expected loss in heterozygosity, observations consistent with (associative) overdominance. This study demonstrates that the neutrality of molecular markers cannot always be assumed, particularly in small populations with a high mutation load.